Modified immune cells and uses thereof

Abstract

The present application relates to immune cells (e.g., T cells such as CAR-T cells, NK cells such as CAR-NK cells) modified to have no or reduced expression and / or function of one or more target proteins selected from the group consisting of signal peptide peptidase-like 3 (SPPL3), FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B (e.g. SPPL3), uses thereof, and methods of their production. Also provided are uses of one or more target proteins (e.g., SPPL3) as biomarkers.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This patent application claims priority to international application PCT / CN2022 / 124003 filed on October 12, 2022, and international application PCT / CN2023 / 110694 filed on August 2, 2023, the entire contents of which are incorporated herein by reference.

[0003] Reference Electronic Sequence Listing

[0004] The contents of the electronic sequence listing (165392001442seqlist.xml; size: 6407 bytes; creation date: October 10, 2023) are incorporated herein by reference in their entirety. Technical Field

[0005] The present application relates to immune cells (e.g., T cells such as CAR-T cells, NK cells such as CAR-NK cells), which are modified to not have or reduce the expression and / or function of one or more target proteins, the target proteins being selected from: signal peptide peptidase-like 3 (SPPL3), FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (e.g., immune cells modified to not have or reduce the expression and / or function of SPPL3 protein), uses thereof, and methods for producing the same. Also provided is the use of one or more target proteins (e.g., SPPL3) as biomarkers. Background Art

[0006] Immunotherapy approaches, including adoptive immune cell therapy (e.g., CAR-T therapy, CAR-NK therapy), are playing an increasingly important role in the treatment of cancer, viral infections, and other pathophysiological autoimmune diseases. Compared to autologous immune cell therapy (which often requires a lengthy and expensive custom manufacturing process and is not suitable for all patients), allogeneic immune cell therapy (e.g., universal CAR-T or CAR-NK therapy) has become a more attractive approach, in which immune cells are derived from healthy donors, providing an off-the-shelf product that is suitable for many patients rather than just one individual.

[0007] Universal CAR-T or CAR-NK therapy has the following advantages: i) it can be pre-made and supplied directly from stock, rather than prepared as needed, which takes weeks; ii) the raw material T cells can be obtained from healthy donors and screened using rigorous procedures, such as having strong activity and not contaminating diseased cells (such as tumor cells); iii) it has a wide range of adaptability, and patients will not lose treatment opportunities due to formulation failure or non-compliance with formulation standards; iv) product quality is controllable, such as compliance with good manufacturing practice (GMP) standards; and v) it is suitable for industrial manufacturing, which can effectively reduce production and processing costs.

[0008] The poor persistence of modified immune cells (e.g., allogeneic CAR-T or CAR-NK cells) limits their therapeutic efficacy (L. Jafarzadeh et al., Front Immunol. 2020; 11: 702) and remains one of the biggest challenges to their application. First, the patient's immune system (e.g., host T cells, NK cells) recognizes the infused non-HLA-matched allogeneic immune cells as foreign immune cells and rejects them, i.e., host to graft (HvG) response. To overcome this problem, researchers used clustered regularly interspaced short palindromic repeats (CRISPR) / Cas9 (CRISPR-associated protein 9) (CRISPR / Cas9) system to knock out the beta-2 microglobulin (B2M) required for human leukocyte antigen (HLA) class I expression in CAR-T cells to prevent host TCRαβ cells from recognizing donor CAR-T cells as foreign cells through HLA class I (Ren et al., Clin. Cancer Res. 2017; 23: 2255–2266). However, T cells with reduced HLA class I expression are also targeted by host natural killer (NK) cells, representing an obstacle to preventing allogeneic T cell rejection (Liu et al. Curr. Res. Transl. Med. 2018; 66: 39-42; Ludigs et al. Nat Commun. 2016; 7: 10554). Second, in addition to targeting T cells with reduced HLA class I expression, host NK cells can also kill activated T cells (e.g., allogeneic CAR-T cells) due to the expression of ligands on activated T lymphocytes, such as those that can be recognized by NKG2D or NKp46 expressed on NK cells (Waggoner et al. Nature. 2011; 481(7381): 394-398; Cerboni et al. Blood. 2007; 110(2): 606-15). Third, allogeneic immune cells (such as CAR-T or CAR-NK cells) can undergo activation-induced cell death (AICD), which is a programmed cell death process caused by the interaction of Fas receptor (Fas, CD95) and Fas ligand (FasL, CD95 ligand). Generally, FasL is a killer effector molecule expressed and / or secreted by immune cells (such as T cells and NK cells), Fas is an apoptosis receptor expressed on the surface of tumor cells, and the interaction between Fas and Fas ligand is the mechanism by which immune cells kill tumor cells. However, activated immune cells (such as T cells, B cells, and NK cells) express Fas in addition to FasL, and therefore will be killed by themselves or each other. See, for example, Huan et al., Hum Cell.2022; 35(2): 441-447. All of the above mechanisms result in poor persistence of allogeneic immune cells in vivo.

[0009] Adoptive immunotherapy can also be limited in patients due to graft-versus-host disease (GvHD). For example, CAR-T cells expressing endogenous T cell receptors (TCRs) can recognize major and minor histocompatibility antigens after administration to allogeneic patients, leading to nonspecific effects and the development of GvHD in patients. To overcome this problem, researchers have knocked out the endogenous TCR in allogeneic T cells (such as CAR-T cells) through gene editing such as CRISPR / Cas9.

[0010] The activity of NK cells is regulated by a complex interaction of various cell surface inhibitory and activating receptors. Inhibitory receptors include killer immunoglobulin-like receptors (KIR) and CD94 / NKG2A, which recognize major histocompatibility complex (MHC) or HLA class I molecules, allowing NK cells to recognize autologous cells and prevent them from attacking host tissues. When there is no matching MHC class I molecule, the inhibition of NK cell toxicity is released, and the balance is shifted to NK cell activation by activating receptor binding. During viral infection or malignant transformation, transformed cells reduce the expression of cell surface MHC class I antigens to avoid being recognized by T cells. However, NK cells can recognize such transformed cells as “altered self”, and the abnormal level of its MHC class I expression leads to a reduction in the participation of inhibitory KIR to provide effector responses and cytotoxic killing of transformed cells (Nayyar et al., Front Oncol. 2019; 9: 51).

[0011] Activated NK cells can kill target cells through the following pathways: i) perforin and granzyme, which are released from the cytoplasmic granules of NK cells to lyse or activate apoptosis of target cells; ii) TNFα and IFNγ, which are released by activated NK cells to induce apoptosis of tumor cells, including changing the stability of target cell lysosomes, affecting target cell membrane phospholipid metabolism, changing target cell pH, and activating target cell endonucleases to degrade genomic DNA; and iii) FasL and TNF-related apoptosis-inducing ligand (TRAIL), which are secreted / expressed by activated NK cells, interact with death receptors (Fas, DR4 / TRAIL-R1, and DR5 / TRAIL-R2) on target cells, induce conformational changes in death receptors, promote the binding of the adaptor protein Fas-associated death domain protein (FADD), and then bind to the caspase-8 precursor protein to initiate the caspase cascade reaction, activate caspase-3, lead to the degradation of intracellular structural and functional proteins, and ultimately target cell apoptosis.

[0012] The CRISPR / Cas9 system is capable of editing targeted genomic sites with high efficiency and specificity. One of its widespread applications is to identify the functions of coding genes, noncoding RNAs, and regulatory elements through high-throughput combined screening combined with next-generation sequencing ("NGS") analysis. By introducing a pooled library of single guide RNAs ("sgRNAs") or paired guide RNAs ("pgRNAs") into cells expressing Cas9 or catalytically inactive Cas9 (dCas9) fused to an effector domain, researchers can perform a variety of genetic screens by generating different mutations, large genomic deletions, transcriptional activation, or transcriptional repression.

[0013] In order to generate high-quality gRNA cell libraries for any given pooled CRISPR screen, a low multiplicity of infection ("MOI") must be used during the cell library construction process to ensure that each cell carries, on average, less than one sgRNA or pgRNA, thereby minimizing the false discovery rate (FDR) of the screen. To further reduce the FDR and increase the reproducibility of the data, in-depth coverage of gRNAs and multiple biological replicates are often required to obtain hit genes with high statistical significance, thereby increasing the workload. Additional difficulties may arise when performing large numbers of whole-genome screens, when cell material for library construction is limited, or when performing more challenging screens (i.e., in vivo screening) because it is difficult to obtain experimental replicates or control MOI. The "internal barcode" ("iBAR") method previously developed by the applicant (see WO2020125762, the contents of which are incorporated herein by reference in their entirety) provides a reliable and efficient screening strategy for large-scale target identification in eukaryotic cells, with much lower false positive and false negative rates, and allows the generation of cell libraries using high MOI. For example, compared to traditional CRISPR / Cas screening with a low MOI of 0.3, the iBAR method can reduce the starting cell number by more than 20-fold (e.g., at an MOI of 3) to more than 70-fold (e.g., at an MOI of 10) while maintaining high efficiency and accuracy. The iBAR system is particularly suitable for cell-based screening with limited cell numbers or in vivo screening where it is difficult to control viral infection of specific cells or tissues at low MOIs.

[0014] The disclosures of all publications, patents, patent applications, and published patent applications mentioned herein are incorporated by reference in their entirety. Summary of the Invention

[0015] In one aspect, the present invention provides an immune cell, wherein the immune cell is modified to lack or reduce the expression and / or function of one or more target proteins selected from the group consisting of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B. In some embodiments, the immune cell is modified to lack or reduce the expression and / or function of the SPPL3 protein. In some embodiments, the immune cell has at least about 10% less activation-induced cell death (AICD) compared to a reference immune cell that does not have the modification that reduces or eliminates the expression and / or function of the SPPL3 protein. In some embodiments, the expression of the SPPL3 protein is reduced or inhibited by antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding the SPPL3 protein. In some embodiments, the immune cell is modified to express a dominant-negative SPPL3 protein variant or a dominant-negative fragment thereof. In some embodiments, the immune cell is genetically modified at the SPPL3 locus or SPPL3 RNA. In some embodiments, the SPPL3 locus is modified by gene editing, or the SPPL3 RNA is modified by RNA editing. In some embodiments, gene editing or RNA editing is mediated by non-homologous end joining (NHEJ), homology-directed repair (HDR), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), or CRISPR / Cas. In some embodiments, gene editing comprises gene knockout (KO). In some embodiments, gene editing or RNA editing comprises base editing. In some embodiments, gene editing or RNA editing is mediated by CRISPR / Cas. In some embodiments, the gene editing or RNA editing comprises contacting a precursor immune cell with i) a guide RNA (gRNA) construct and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under conditions that allow introduction of the gRNA construct and, optionally, the Cas component into the precursor immune cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to a target site in the SPPL3 locus or SPPL3 RNA. In some embodiments, the precursor immune cell expresses a Cas protein. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein comprising i) a dead Cas protein (dCas) and ii) adenine deaminase (ADA) or cytidine deaminase (CDA) or a functional fragment thereof. In some embodiments, the Cas protein is Cas9. In some embodiments, the guide sequence is encoded by a nucleic acid sequence comprising a sequence of SEQ ID NO: 1.

[0016] In some embodiments according to any of the above immune cells, the immune cell lacks or is further modified to lack or reduce the expression and / or function of one or more other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and ligands of NKp46.

[0017] In some embodiments according to any of the above immune cells, the immune cell expresses or is further modified to express an engineered receptor, such as a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen conjugate (TAC). In some embodiments, the engineered receptor is a CAR comprising: i) an extracellular antigen binding domain that specifically recognizes a target antigen; ii) a transmembrane domain; and iii) an intracellular signal transduction domain. In some embodiments, the modification of the expression and / or function of one or more target proteins is reduced or eliminated: i) the expression and / or function of the engineered receptor is not lowered or eliminated; or ii) the expression and / or function of the engineered receptor is lowered by up to about 30%.

[0018] In some embodiments according to any of the above immune cells, the immune cell is a T cell, a B cell, or a natural killer (NK) cell. In some embodiments, the immune cell is a T cell, for example, selected from helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, natural killer T (NKT) cells, mucosal-associated invariant T (MAIT) cells, double negative T (DNT) cells, and γδT cells. In some embodiments, the expression and / or function of one or more target proteins are reduced or eliminated by: i) reducing at least about 10% of the cell surface expression of one or more of Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligands; ii) reducing at least about 10% of the killing of allogeneic T cells; and / or iii) reducing at least about 10% of the killing of autologous or allogeneic NK cells.

[0019] In some embodiments of the immune cell according to any of the above, the immune cell has at least about 10% longer persistence in vivo compared to a reference immune cell without the modification that reduces or eliminates expression and / or function of one or more target proteins.

[0020] In some embodiments of the immune cell according to any of the above, the immune cell is autologous or allogeneic.

[0021] In another aspect, the present invention provides a method for identifying an individual as a suitable donor of immune cells having prolonged in vivo persistence, comprising examining the expression and / or function of one or more target proteins selected from the group consisting of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B in the individual, wherein the expression and / or function of the one or more target proteins is reduced or abolished compared to a reference, and the individual is identified as a suitable donor. In some embodiments, the method comprises examining the expression and / or function of SPPL3 protein in the individual, wherein the expression and / or function of SPPL3 protein is reduced or abolished compared to a reference, and the individual is identified as a suitable donor. In some embodiments, the method further comprises examining the expression and / or function of one or more other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and ligands for NKp46, wherein identification of a reduction or elimination of expression and / or function of one or more other proteins further identifies the individual as a suitable donor. In some embodiments, the reference is the average expression and / or function of one or more target proteins in a population of individuals. In some embodiments, the reference is the average expression and / or function of SPPL3 protein in a population of individuals. In some embodiments, examining the expression and / or function of one or more target proteins and / or one or more other proteins comprises examining the sequence of a nucleic acid encoding the one or more target proteins and / or one or more other proteins, wherein identification of a mutation in the nucleic acid that reduces expression and / or function of the one or more target proteins and / or the one or more other proteins identifies the individual as a suitable donor. In some embodiments, examining the expression and / or function of the SPPL3 protein and / or one or more other proteins comprises examining the sequence of a nucleic acid encoding the SPPL3 protein and / or one or more other proteins, wherein identification of a mutation in the nucleic acid that reduces expression and / or function of the SPPL3 protein and / or the one or more other proteins identifies the individual as a suitable donor.

[0022] In another aspect, the present invention provides a method for excluding an individual as a suitable donor of immune cells with prolonged in vivo persistence, comprising examining the expression and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B in the individual, wherein if no reduction or abolition of expression and / or function of the one or more target proteins is identified as compared to a reference, the individual is excluded as a suitable donor. In some embodiments, the method comprises examining the expression and / or function of SPPL3 protein in the individual, wherein if no reduction or abolition of expression and / or function of SPPL3 protein is identified as compared to a reference, the individual is excluded as a suitable donor. In some embodiments, the reference is the average expression and / or function of the one or more target proteins in a population of individuals. In some embodiments, the reference is the average expression and / or function of SPPL3 protein in a population of individuals.

[0023] In another aspect, the present invention provides a method for i) extending the in vivo persistence of immune cells, ii) reducing AICD in immune cells, and / or iii) reducing host versus graft (HvG) responses in immune cells, comprising modifying the immune cells to reduce or eliminate the expression and / or function of one or more target proteins selected from the group consisting of: SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B. In some embodiments, the method comprises modifying the immune cells to reduce or eliminate the expression and / or function of SPPL3 protein. In some embodiments, the expression of SPPL3 protein is reduced or inhibited by antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding the SPPL3 protein. In some embodiments, the immune cells are modified to express a dominant-negative SPPL3 protein variant or a dominant-negative fragment thereof. In some embodiments, the immune cells are genetically modified at the SPPL3 locus or SPPL3 RNA. In some embodiments, the SPPL3 locus is modified by gene editing, or the SPPL3 RNA is modified by RNA editing. In some embodiments, gene editing or RNA editing is mediated by NHEJ, HDR, ZFN, TALEN, or CRISPR / Cas. In some embodiments, gene editing includes gene KO. In some embodiments, gene editing or RNA editing includes base editing. In some embodiments, wherein the gene editing or RNA editing is mediated by CRISPR / Cas. In some embodiments, the method comprises contacting the precursor immune cell with i) a gRNA construct and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under conditions that allow introduction of the gRNA construct and optional Cas components into the precursor immune cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to a target site in the SPPL3 locus or SPPL3 RNA. In some embodiments, the precursor immune cell expresses a Cas protein. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein comprising i) dCas and ii) ADA or CDA or a functional fragment thereof. In some embodiments, the Cas protein is Cas9. In some embodiments, the guide sequence is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 1.

[0024] In some embodiments of any one of the methods of i) extending the in vivo persistence of immune cells, ii) reducing the AICD of immune cells, and / or iii) reducing the HvG response of immune cells, the method further comprises modifying the immune cells to reduce or eliminate the expression and / or function of one or more other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligands. In some embodiments, the immune cells are genetically modified at one or more loci or RNA encoding one or more other proteins.

[0025] In some embodiments of any of the above methods of i) extending the in vivo persistence of immune cells, ii) reducing AICD of immune cells, and / or iii) reducing HvG responses of immune cells, the immune cells express an engineered receptor. In some embodiments, the method further comprises introducing a nucleic acid encoding the engineered receptor into the immune cells. In some embodiments, the nucleic acid encoding the engineered receptor, the nucleic acid encoding a gRNA targeting SPPL3, and / or the nucleic acid encoding the Cas protein are on different vectors. In some embodiments, the engineered receptor is a CAR, an engineered TCR, or a TAC. In some embodiments, the engineered receptor is a CAR comprising: i) an extracellular antigen-binding domain that specifically recognizes a target antigen; ii) a transmembrane domain; and iii) an intracellular signaling domain. In some embodiments, the modification that reduces or eliminates the expression and / or function of one or more target proteins: i) does not downregulate or eliminate the expression and / or function of the engineered receptor; or ii) downregulates the expression and / or function of the engineered receptor by up to about 30%.

[0026] In some embodiments of any one of the methods of i) extending the in vivo persistence of immune cells, ii) reducing AICD of immune cells, and / or iii) reducing HvG responses of immune cells, the immune cells are T cells, B cells, or NK cells. In some embodiments, the immune cells are T cells, for example, selected from helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, NKT cells, MAIT cells, DNT cells, and γδ T cells. In some embodiments, the modification that reduces or eliminates the expression and / or function of one or more target proteins: i) reduces at least about 10% of the cell surface expression of one or more of Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligand; ii) reduces killing by allogeneic T cells by at least about 10%; and / or iii) reduces killing by autologous or allogeneic NK cells by at least about 10%.

[0027] In some embodiments, according to any one of the above methods of i) extending the in vivo persistence of immune cells, ii) reducing the AICD of immune cells and / or iii) reducing the HvG response of immune cells, the modification that reduces or eliminates the expression and / or function of one or more target proteins: i) extends the in vivo persistence of immune cells by at least about 10%; and / or ii) reduces the AICD of the immune cells by at least about 10% compared to the same immune cells that have not been modified to reduce or eliminate the expression and / or function of one or more target proteins.

[0028] On the other hand, the present invention provides a method for identifying a target gene in an immune cell whose mutation increases resistance to AICD, comprising: a) providing an immune cell library comprising a plurality of immune cells, wherein each of the plurality of immune cells has a mutation at a hit gene (hit gene mutation), wherein the hit genes of at least two immune cells in the plurality of immune cells are different from each other; wherein under conditions allowing the introduction of sgRNA constructs and Cas components into an initial population of immune cells and generating mutations at the hit gene, the initial population of immune cells is introduced by combining i) a guide RNA (sgRNA) comprising a plurality of sgRNA constructs; gRNA) library and ii) contacting a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein and producing the immune cell library, wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence complementary to a target site in a corresponding hit gene; b) contacting the immune cell library with Fas ligand (FasL); c) obtaining an AICD-resistant immune cell population from the immune cell library; and d) identifying the target gene based on the difference between the profiles of sgRNA or hit gene mutations in the AICD-resistant immune cell population and the control immune cell population.

[0029] In some embodiments of any of the above methods for identifying a target gene, the control immune cell population is i) a subpopulation of the immune cell pool prior to step b); or ii) the same immune cell pool cultured under the same conditions and not exposed to FasL. In some embodiments, the method further comprises i) obtaining a control immune cell population prior to step b); or ii) the same immune cell pool cultured under the same conditions and not exposed to FasL.

[0030] In some embodiments according to any one of the above methods of identifying a target gene, identification of the target gene is based on a difference between the profiles of the sgRNA in the AICD-resistant immune cell population and the control immune cell population.

[0031] In some embodiments of any of the methods for identifying target genes described above, the profiles of sgRNAs in the AICD-resistant immune cell population and the control immune cell population are identified by next generation sequencing (NGS). In some embodiments, the method comprises comparing the counts of sgRNA sequences obtained from the AICD-resistant immune cell population with the counts of sgRNA sequences obtained from the control immune cell population, wherein the hit genes whose corresponding sgRNA guide sequences are identified as enriched in the AICD-resistant immune cell population compared to the control immune cell population with a false discovery rate (FDR) ≤ 0.2 are identified as target genes whose mutations increase resistance to AICD (AICD resistance genes).

[0032] In some embodiments of any one of the methods for identifying target genes described above, the sgRNA library and the Cas components are sequentially introduced into an initial population of immune cells. In some embodiments, the initial population of immune cells expresses the Cas protein before the introduction of the sgRNA library. In some embodiments, the Cas protein is Cas9. In some embodiments, each sgRNA includes a guide sequence fused to a second sequence, and wherein the second sequence includes a repeat-anti-repeat stem loop that interacts with Cas9. In some embodiments, the second sequence of each sgRNA further includes stem loop 1, stem loop 2, and / or stem loop 3.

[0033] In some embodiments according to any one of the above methods for identifying a target gene, each sgRNA further comprises an internal barcode (iBAR) sequence (sgRNA iBAR ), where each sgRNA iBAR Can be operated with Cas proteins to modify hit genes. In some embodiments, each sgRNA iBAR In the 5' to 3' direction, the first stem sequence and the second stem sequence are included, wherein the first stem sequence hybridizes with the second stem sequence to form a double-stranded RNA (dsRNA) region that interacts with the Cas protein, and wherein the iBAR sequence is arranged from the 3' end of the first stem sequence to the 5' end of the second stem sequence. In some embodiments, the Cas protein is Cas9, and wherein each sgRNA iBAR In some embodiments, each guide sequence comprises about 17 to about 23 nucleotides. In some embodiments, the sgRNA library is a sgRNA iBAR library, in which the sgRNA iBAR The library contains multiple sets of sgRNAs iBAR Construct, where each set of sgRNA iBAR The construct contains four sgRNAs iBAR Construct, each sgRNA iBAR Constructs containing or encoding sgRNA iBAR , where four sgRNAs iBAR The guide sequences of the constructs are identical, wherein the four sgRNAs iBAR The iBAR sequence of each construct was different from the other, and each set of sgRNAs iBAR The guide sequence of the construct is complementary to a different target site in the hit gene. In some embodiments, the immune cell library has a different target site for each sgRNA. iBAR Having a coverage of at least about 500-fold.

[0034] In some embodiments according to any one of the above methods of identifying a target gene, at least about 95% of the sgRNA constructs in the sgRNA library are introduced into the starting population of immune cells.

[0035] In some embodiments according to any one of the above methods of identifying target genes, the immune cell library has at least about 2000-fold coverage for each sgRNA.

[0036] In some embodiments according to any one of the above methods of identifying a target gene, the sgRNA library comprises at least about 2000 sgRNA constructs.

[0037] In some embodiments according to any one of the above methods of identifying a target gene, each sgRNA construct in the sgRNA library is a plasmid.

[0038] In some embodiments according to any of the above methods for identifying a target gene, each sgRNA construct in the sgRNA library is a viral vector, such as a lentiviral vector. In some embodiments, the sgRNA library is contacted with the initial population of immune cells at a multiplicity of infection (MOI) of at least about 2.

[0039] In some embodiments of any of the methods for identifying target genes described above, step c) comprises using fluorescence activated cell sorting (FACS) or centrifugation to obtain an AICD-resistant immune cell population. In some embodiments, step c) further comprises contacting the immune cell pool with a viability indicator, such as one or more of propidium iodide (PI), DAPI, 7-AAD, and Annexin V. In some embodiments, the AICD-resistant immune cell population is Annexin V-negative and DAPI-negative.

[0040] In some embodiments according to any one of the methods of identifying a target gene above, step b) comprises culturing the immune cell pool in the presence of FasL for about 16 hours.

[0041] In some embodiments of any of the methods for identifying target genes described above, the sgRNA sequence counts are normalized to median ratios and then subjected to mean variance modeling. In some embodiments, the sgRNA library is a sgRNA iBAR library, and based on sgRNA corresponding to the guide sequence iBAR The variance of each guide sequence is adjusted based on the data consistency between the iBAR sequences in the sequence. In some embodiments, the sgRNA corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence. iBAR Data consistency between iBAR sequences in a sequence, where the variance of the guide sequence increases if the iBAR sequences are in different orientations relative to each other.

[0042] In some embodiments of any of the above methods for identifying a target gene, the method comprises: a) providing an immune cell library; b1) contacting the immune cell library with a first FasL (a first FasL treatment step); c1-i) obtaining a first AICD-resistant immune cell population from the immune cell library (a first obtaining step); c1-ii) optionally culturing the first AICD-resistant immune cell population for about 6 days (an optional first recovery step); b2) optionally contacting the first AICD-resistant immune cell population with a second FasL (an optional second FasL treatment step); c2-i) optionally obtaining a second AICD-resistant immune cell population from step b2) (an optional second FasL treatment step); c2-ii) optionally culturing the second AICD-resistant immune cell population for about 6 days (optional second recovery step); b3) optionally contacting the second AICD-resistant immune cell population with a third FasL (optional third FasL treatment step); c3-i) optionally obtaining a final AICD-resistant immune cell population from step b3) (optional third acquisition step); c3-ii) optionally culturing the final AICD-resistant immune cell population for about 6 days (optional third recovery step); and d) identifying the target gene based on the difference between the profiles of sgRNA or hit gene mutations in the final AICD-resistant immune cell population and the control immune cell population. In some embodiments, the first, second, and third FasLs have the same concentration. In some embodiments, the first, second, and third FasLs have different concentrations.

[0043] In some embodiments of any of the methods for identifying target genes described above, the method comprises: a) providing an immune cell library; b1) contacting the immune cell library with FasL (a first FasL treatment step); c1-i) centrifuging the immune cell library after step b1) to obtain a first AICD-resistant immune cell population (a first acquisition step); c1-ii) culturing the first AICD-resistant immune cell population for about 6 days (a first recovery step); b2) contacting the first AICD-resistant immune cell population with FasL (a second FasL treatment step); c2-i) centrifuging the first AICD-resistant immune cell population after step b2) to obtain a second AICD-resistant immune cell population (a second acquisition step); c2-ii) culturing the second AICD-resistant immune cell population for about 6 days (a second recovery step); b) contacting the second AICD-resistant immune cell population with the FasL (third FasL treatment step); c3-i) centrifuging the second AICD-resistant immune cell population after step b3) to obtain a final AICD-resistant immune cell population (third obtaining step); c3-ii) culturing the final AICD-resistant immune cell population for about 6 days (third recovery step); and d) comparing the sgRNA sequence counts obtained from the final AICD-resistant immune cell population with the sgRNA sequence counts obtained from the control immune cell population, wherein the hit genes whose corresponding sgRNA guide sequences are identified as enriched in the final AICD-resistant immune cell population compared to the control immune cell population with an FDR≤0.2 are identified as target genes (AICD resistance genes) whose mutations increase resistance to AICD.

[0044] In some embodiments of any of the methods for identifying target genes described above, the method further comprises ranking the identified target genes, wherein the target gene ranking is based on the enrichment of sgRNA guide sequences in the AICD-resistant immune cell population compared to a control immune cell population. In some embodiments, the sgRNA library is a sgRNA iBAR library, and wherein the sgRNA corresponding to the guide sequence of the target gene iBAR In some embodiments, the method further adjusts the target gene ranking based on the data consistency between the iBAR sequences in the sequence. In some embodiments, the method further includes assigning an AICD resistance score to the identified target gene, wherein the target genes whose mutations increase AICD resistance are ranked from high to low based on the enrichment fold of the sgRNA guide sequence in the AICD-resistant immune cell population compared to the control immune cell population, and each target gene is assigned an AICD resistance score from high to low accordingly.

[0045] Also provided is a method for producing immune cells with increased resistance to AICD, which includes inactivating an AICD resistance gene identified by any method of identifying the above-mentioned target genes in a precursor immune cell. In some embodiments, the immune cell is a T cell, a B cell, or a NK cell. In some embodiments, the immune cell is a T cell, for example, selected from helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, NKT cells, MAIT cells, DNT cells, and γδT cells. In some embodiments, the precursor immune cell expresses an engineered receptor. In some embodiments, the method further includes introducing a nucleic acid encoding the engineered receptor into the precursor immune cell. In some embodiments, the engineered receptor is a CAR.

[0046] Also provided are immune cells obtained by any of the methods described above, such as methods for generating immune cells with increased resistance to AICD, or methods for i) extending the in vivo persistence of immune cells, ii) reducing AICD in immune cells, and / or iii) reducing HvG responses in immune cells. In some embodiments, the immune cells are autologous or allogeneic.

[0047] Also provided are pharmaceutical compositions comprising any of the above-described immune cells and, optionally, a pharmaceutically acceptable excipient.

[0048] On the other hand, the present invention provides a method for treating an individual disease, comprising administering to the individual an effective amount of any of the above-mentioned immune cells or any of the above-mentioned pharmaceutical compositions. In some embodiments, the disease is associated with the expression of a target antigen, and wherein the immune cells express an engineered receptor that specifically recognizes the target antigen, such as a CAR. In some embodiments, the disease is cancer, infection, inflammation, autoimmune disease, or an immune-related disease characterized by exhaustion of effector cells.

[0049] Also provided are kits and articles of manufacture useful in the methods described herein, such as kits for generating modified immune cells (e.g., modified T cells, such as modified CAR-T cells) with increased resistance to AICD. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 An exemplary process for screening AICD / FasL resistance genes in immune cells (eg, T cells) is shown.

[0051] Figure 2 An exemplary process for obtaining control or treatment samples to screen for the AICD / FasL resistance gene in immune cells (eg, T cells) is shown.

[0052] Figure 3An exemplary procedure for testing NK cell killing frequency in a mixture of SPPL3-KO T cells and control (NT-KO) T cells is shown.

[0053] Figure 4 Showing Cas9 + sgRNA iBAR An exemplary target gene identification workflow for immune cell (e.g., T cell) repertoires.

[0054] Figure 5 Shown for identification of Cas9 + sgRNA iBAR Testing of the appropriate FasL concentration for the AICD / FasL resistance gene in a Jurkat cell bank. Annexin V and DAPI staining indicated cell death.

[0055] Figure 6 Shown in Cas9 + sgRNA iBAR Comparison of Robust Rank Aggregation (RRA) Scores and Gene Ranking for AICD / FasL Resistance Screening in Jurkat Cell Banks. Ten exemplary AICD / FasL resistance genes with high scores were identified by name.

[0056] Figure 7A and 7B showed that 100 ng / mL FasL treatment resulted in 25.7% cell viability in control (NT-KO) Jurkat T cells (Annexin V - and DAPI - ), which was statistically significantly lower than the cell viability of 63.1% in SPPL3-KO Jurkat T cells. Figure 7C It was shown that SPPL3-KO Jurkat T cells were much more resistant to FasL-mediated AICD at various FasL concentrations compared to control (NT-KO) Jurkat T cells.

[0057] Figure 8A CellTrace treated with various concentrations of FasL TM Purple (CTV)-labeled SPPL3-KO primary T cells and CellTrace TM The ratio of control (NT-KO) primary T lymphocytes labeled with carboxyfluorescein succinimidyl ester (CFSE). The higher the FasL concentration, the higher the percentage of SPPL3-KO primary T cells labeled with CFSE. Figure 8B Cumulative data demonstrating results obtained using three T cell donors are shown. Figure 8C Anti-Fas monoclonal antibody and SPPL3 KO and AAVS1KO Comparison of T cell binding. Staining controls are indicated by black lines. The data in Figures A and C represent three independent experiments. The data in Figures A and B are reported as mean ± SD (n = 8). The horizontal line represents the mean. Statistical analysis was performed using the Mann-Whitney U test, ***p < 0.001.

[0058] Figure 9 SPPL3-KO Jurkat T cells showed lower expression of NK cell ligands (B7-H6, NKG2D ligand, NKp46 ligand) and lower expression of HLA-A, B, and C compared with control (NT-KO) Jurkat T cells.

[0059] Figure 10 The results showed that SPPL3-KO primary T cells had lower expression of Fas, NKG2D ligands, and HLA-A, B, and C compared with control (NT-KO) primary T cells.

[0060] Figure 11 Cell Trace showed that when treated with NK cells, as the E:T ratio increased, TM The frequency of violet (CTV)-labeled SPPL3-KO Jurkat T cells was increased relative to control (NT-KO) Jurkat T cells.

[0061] Figure 12A It is shown that when treated with NK cells, the frequency of CTV-labeled SPPL3-KO primary T cells increased relative to CFSE-labeled control (NT-KO) primary T lymphocytes as the E:T ratio increased. Figure 12B Cumulative results of T cells from three donors treated with NK cells are shown: In a mixture of SPPL3-KO and control primary T cells, the percentage of CTV-labeled SPPL3-KO primary T cells increased significantly with increasing E:T ratios. *** indicates statistical significance.

[0062] Figure 13 SPPL3-KO anti-CD19 CAR Jurkat T cells and control (NT-KO) anti-CD19 CAR Jurkat T cells showed CD19 + RAJI cells had similar killing efficiency and higher killing efficiency (lower CD19 + The percentage of RAJI cells) was consistently associated with a higher E:T ratio.

[0063] Figure 14ACFSE-labeled PBMCs cultured alone (negative control) showed no proliferating CD8+ T cells (upper panel; percentage of CFSE-low cells was 0.48), whereas CFSE-labeled PBMCs cultured with irradiated allogeneic primary T cells stimulated CD8+ T proliferation (lower panel; percentage of CFSE-low cells was 33.6). Figure 14B We show that irradiated SPPL3-KO primary T lymphocytes induced significantly reduced allogeneic T cell proliferation (CFSE low CD8+) compared to irradiated control primary T cells. Figure 14B Data from 2 different T cell donors are shown. *Indicates statistical significance. Figure 14C Anti-HLAABC monoclonal antibodies showed that they interacted with SPPL3 KO and AAVS1 KO Comparison of T cell binding. Staining control (IgG2a) is indicated by a black line. The graph shown is representative of three independent experiments.

[0064] Figure 15A and Figure 15B showed that SPPL3 ablation in primary T cells attenuated killing by alloantigen-specific T cells. Figure 15A Schematic diagram showing the NY-ESO-1 TCR-T cell killing assay. Figure 15B Display SPPL3 KO T cells were more resistant to killing by New York esophageal squamous cell carcinoma 1 (NY-ESO-1)-specific TCR-T cells. Figure 15B Data from 2 different T cell donors are also shown. *** indicates statistical significance.

[0065] Figure 16A Evaluation of allogeneic NK cell killing of activated T cells. KO and AAVS1 KO T cells were incubated with NaP overnight. KO T cells and an equal amount of control AAVS1 KO T cells were cultured and treated or not with purified NK cells for 6 hours. Flow cytometry was used to measure SPPL3 KO The percentage of T cells. Figure 16B Cumulative data obtained using different sgRNAs targeting SPPL3 are shown. The data provided represent three donors. Bars with an E:T ratio greater than 0 include NK cells that are effector (E) cells. Figure 16C Schematic diagram showing the experimental setup for in vivo evaluation of T cell killing by NK cells. Figure 16D Evaluation of NK cell killing of T cells in the spleen and liver is shown. Bars marked with + include NK cells. Figure 16E showed that NKG2D-Fc is expressed in SPPL3 KO and AAVS1 KO Comparison of binding on T cells. Staining controls are indicated by black lines. The graphs shown are representative of three independent experiments. Figure 16B (n=6) and Figure 16D Data in (n=4) are reported as mean ± SD. The horizontal line represents the mean. Statistical analysis was performed using the Mann-Whitney U test, *p < 0.05; **p < 0.01; ***p < 0.001. Figure 16F showed that anti-HLAABC monoclonal antibodies could be effective in SPPL3 KO and AAVS1 KO Binding on Jurkat T cells, staining control is indicated by a black line. Figure 16G The analysis strategy for human T cells from NSG mice is shown. Figure 16H showed that anti-B7-H6 antibodies, NKG2D-Fc and NKp46-Fc were expressed in SPPL3 KO and AAVS1 KO -Binding on Jurkat T cells, staining control is shown as a black line. Figure 16F and 16H The data in are representative of three independent experiments.

[0066] Figure 17A Display SPPL3 KO Primary T cells and controls (AAVS1 KO )CAR-T cells have similar proliferation capabilities. Figure 17B Display SPPL3 KO / TCR KO / anti-CD19 CAR-T cells (top line) and control AAVS1 KO / TCR KO / Anti-CCD19 CAR-T cells (bottom line) against CD19 + NALM6 cells had similar killing efficiency. Figure 17C Display SPPL3 KO / TCR KO Anti-CD19 CAR-T cells (top line in both graphs) showed superiority to the control AAVS1 during repeated CD19+NALM6 cell stimulation KO / TCR KO / Growth advantage of anti-CCD19 CAR-T (bottom line in both graphs). Figure 17D Display SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells eliminate tumors in vivo. Figure 17E Shows Figure 17D Kaplan-Meier plot of overall survival of all mouse groups in the study. The dotted line represents the no T cell group, and the overlapping light gray solid line represents the mock T cell group (T cells without CAR). By day 15, the survival rate of both groups was 0%. Figure 17E The markers represent cells that received anti-CD19 CAR-T cells (without other gene editing), SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells (SPPL3 KO ) or B2MKO / TCRKO / Anti-CD19 CAR-T cells (B2M KO ) of the group. Figure 17F Shown Figure 17D Graph of bioluminescence imaging (BLI) signals of animals in the indicated groups. The dotted line represents the no T cell group and the overlapping solid line represents the mock T cell group (T cells without CAR). Figure 17F The markers indicate the recipients of anti-CD19 CAR-T cells (without other gene editing), SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells (SPPL3 KO ) or B2M KO / TCR KO / Anti-CD19 CAR-T cells (B2M KO ) of the group. Figure 17E and Figure 17F Display SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells inhibited tumor growth and improved animal survival for up to 120 days. Figure 17G It shows that SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells showed a higher expression of CAR-T cells than control CAR-T cells (AAVS1 KO / TCR KO / Anti-C19 CAR-T and B2M KO / TCR KO / anti-CCD19 CAR-T) improved survival rate. Figure 17G Data from two different T cell donors and five different PBMC donors are shown. ** and *** indicate statistical significance. "ns" indicates not statistically significant.

[0067] Figure 18A The method used to determine SPPL3 is shown. KO / TCR KO / Anti-CD19 CAR-T cells replicate CD19 in vivo+ Experimental design to determine whether NALM6 cells exhibit enhanced expansion after stimulation. + NALM6 cells in mice, and then treated with CD19 + NALM6 cells were restimulated. Figure 18B Kaplan-Meier plot showing the overall survival of all groups. The dotted line represents the no T cell group, and the overlapping solid line represents the mock T cell group (T cells without CAR). All mice in the no T cell group and the mock T cell group died around day 18. Figure 18B The middle markers indicate cells that received anti-CD19 CAR-T cells (without other gene editing) or SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells (SPPL3 KO ) of the group. Figure 18C Quantification of tumor burden from BLI images is shown. The dotted line represents the no T cell group, while the overlapping gray solid line represents the mock T cell group (T cells without CAR). Figure 18C The middle markers indicate cells that received anti-CD19 CAR-T cells (without other gene editing) or SPPL3 KO / TCR KO / Anti-CD19 CAR-T cells (SPPL3 KO ) of the group. Figure 18D Show given SPPL3 KO / TCR KO / The percentage of anti-CD19 CAR-T-positive cells in the blood of mice that received anti-CD19 CAR-T cells or anti-CD19 CAR-T cells without other gene editing. Figures 18C-18D The dashed lines in the figure indicate the days on which mice were restimulated with tumor cells. Figure 18A As shown (n=6 per group). *** indicates statistical significance. "ns" indicates not statistically significant.

[0068] Figure 19A Display for evaluating SPPL3 KO / TCR KO / Experimental design for the cytotoxic function of anti-CD19 CAR-T cells. Figure 19B BLI images showing tumor burden in mice (n=6 per group). Figure 19C show Figure 19BKaplan-Meier plot of overall survival of all mouse groups in the study. The dotted line represents the group without T cells, while the light gray solid line (almost overlapping with the dotted line) represents the mock T cell group (T cells without CAR). KO / TCR KO / Anti-CD19 CAR-T cells or B2M KO / TCR KO / Anti-CD19 CAR-T cell group Figure 19C shown. Figure 19D Show from Figure 19B Quantification of tumor burden in BLI images. The dotted line represents the group without T cells, and the solid gray line that almost overlaps with the dotted line represents the mock T cell group (T cells without CAR). KO / TCR KO / Anti-CD19 CAR-T cells or B2M KO / TCR KO / Anti-CD19 CAR-T cell group Figure 19D shown. DETAILED DESCRIPTION

[0069] The poor persistence of modified immune cells (such as CAR-T or CAR-NK cells) is one of the biggest challenges for their effective application in immunotherapy (such as cancer immunotherapy). The present invention provides immune cells (e.g., allogeneic CAR-T or CAR-NK cells) with improved persistence (e.g., in vivo), such as reducing (e.g., reducing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) or eliminating one or more activation-induced cell death (AICD) and host-to-graft (HvG) reactions, such as being attacked by host T cells and / or NK cells. Also provided are methods for identifying target genes that regulate the persistence of immune cells (e.g., by high-throughput screening of NGS), methods for preparing such immune cells, and uses thereof, such as biomarkers for selection or exclusion of immune cell donors, or markers for quality control of modified immune cells with improved persistence.

[0070] The inventors of the present application have discovered that immune cells (e.g., T cells such as CAR-T cells, NK cells such as CAR-NK cells) are modified to have no or reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression (RNA and / or protein expression) and / or function of one or more target proteins selected from the group consisting of: SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (e.g., modified to have no or reduced SPPL3 protein). and / or functional immune cells) having one or more of the following characteristics: i) having at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) less AICD; ii) having at least about 10% (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) less cell surface expression of one or more ligands or receptors involved in immune cell-induced killing (e.g., T cells, NK cells), and / or AICD, for example such as a ligand for Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, or NKp46, iii) is killed by at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) less than the allogeneic T cells; iv) is killed by at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) less than the autologous or allogeneic NK cells. v) expression and / or function (e.g., mediating cytotoxicity) of an engineered receptor (e.g., CAR) of the immune cell is not downregulated (e.g., downregulated by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more), or has expression and / or function of the engineered receptor downregulated by at most about 30% (e.g., at most about 25%, 20%, 10%, 5%, 3%, 1% or less) as compared to a reference immune cell that does not have a modification that reduces or eliminates expression and / or function of the one or more target proteins;vi) conferring greater anti-tumor activity by reducing tumor size in animals provided with the modified immune cells by at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) relative to animals provided with reference immune cells that do not have the modification that reduces or eliminates the expression and / or function of one or more target proteins; vii) conferring greater anti-tumor activity by reducing tumor size in animals provided with the modified immune cells by at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%, 110%, 120%, 130%, 140%, or 150%) relative to animals provided with reference immune cells that do not have the modification that reduces or eliminates the expression and / or function of one or more target proteins. viii) in an allogeneic immune environment, the survival rate of the modified immune cells is increased by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100%) relative to the reference immune cells that do not have the modification that reduces or eliminates the expression and / or function of one or more target proteins; and ix) relative to the reference immune cells that do not have the modification that reduces or eliminates the expression and / or function of one or more target proteins, the immune cells that have the modification that reduces or eliminates the expression and / or function of one or more target proteins are enhanced by at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%) after repeated stimulation of the target cells of the immune cells. These characteristics contribute to the persistence of the modified immune cells while retaining their therapeutic function, for example, the therapeutic function mediated by the engineered receptor (e.g., CAR) expressed on the modified immune cells.

[0071] In one aspect, the present invention provides immune cells (e.g., CAR-T cells or CAR-NK cells) that are modified to have no or reduced expression (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (RNA and / or protein expression) and / or function. In some embodiments, immune cells (e.g., CAR-T or CAR-NK cells) modified to have no or reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) SPPL3 protein expression (RNA and / or protein expression) and / or function are provided, such as knockout (KO) SPPL3, for example, knockout (KO) SPPL3 mediated by CRISPR / Cas. Pharmaceutical compositions comprising the modified immune cells described herein and optionally a pharmaceutically acceptable excipient are also provided.

[0072] In another aspect, the present invention provides a method for treating a disease (e.g., cancer) in an individual, comprising administering to the individual an effective amount of any modified immune cell described herein (e.g., target protein-modified CAR-T or CAR-NK cell) or a pharmaceutical composition thereof.

[0073] In another aspect, the present invention provides a method for identifying an individual (e.g., a human) as a suitable donor of prolonged-persistence immune cells (e.g., T cells, NK cells) in vivo, comprising examining the expression (RNA and / or protein expression) and / or function (e.g., examining the expression and / or function of SPPL3 protein) of one or more target proteins selected from the group consisting of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B in the individual, wherein identification of reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) or eliminated expression and / or function of the one or more target proteins as compared to a reference (e.g., the average expression and / or function of the one or more target proteins (such as SPPL3) in a population of individuals) identifies the individual as a suitable donor.

[0074] In another aspect, the present invention provides a method for excluding an individual (e.g., a human) as a suitable donor of prolonged-persistence immune cells (e.g., T cells, NK cells) in vivo, comprising examining the expression (RNA and / or protein expression) and / or function (e.g., examining the expression and / or function of SPPL3 protein) of one or more target proteins selected from the group consisting of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B in the individual, wherein the individual is excluded as a suitable donor if the expression and / or function of the one or more target proteins is not identified as being reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) or abolished as compared to a reference (e.g., the average expression and / or function of the one or more target proteins (e.g., SPPL3) in a population of individuals).

[0075] In another aspect, the present invention provides a method of i) extending the in vivo persistence of immune cells, ii) reducing the AICD of immune cells, and / or iii) reducing the HvG response of immune cells (e.g., T cells such as CAR-T cells, NK cells such as CAR-NK cells), comprising modifying the immune cells to reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) or eliminate the expression (RNA and / or protein expression) and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B, for example, by CRISPR / Cas-mediated KO of SPPL3 to modify the immune cells to reduce or eliminate the expression and / or function of SPPL3 protein.

[0076] In another aspect, the present invention provides a method for identifying a target gene in an immune cell whose mutation increases resistance to AICD, comprising: a) providing an immune cell library comprising a plurality of immune cells (e.g., T cells, B cells, NK cells), wherein each of the plurality of immune cells has a mutation at a hit gene (hit gene mutation), wherein the hit genes of at least two immune cells in the plurality of immune cells are different from each other; wherein under conditions allowing the introduction of sgRNA constructs and Cas components into an initial population of immune cells and generating mutations at the hit gene, the initial population of immune cells is subjected to the treatment by combining the initial population of immune cells with i) a guide RNA comprising a plurality of sgRNA constructs; The immune cell library is produced by contacting a library of (sgRNA) and ii) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9), wherein each sgRNA construct comprises or encodes an sgRNA, and wherein each sgRNA comprises a guide sequence complementary to a target site in a corresponding hit gene; b) contacting the immune cell library with Fas ligand (FasL); c) obtaining an AICD / FasL-resistant immune cell population from the immune cell library; and d) identifying the target gene based on the difference between the profiles of sgRNA or hit gene mutations in the AICD-resistant immune cell population and a control immune cell population. In some embodiments, the control immune cell population is i) a subpopulation of the immune cell library before step b); or ii) the same immune cell library cultured under the same conditions and not contacted with FasL.

[0077] Also provided are methods of producing any of the modified immune cells described herein, comprising reducing (e.g., reducing by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminating expression (RNA and / or protein expression) and / or function (e.g., reducing or eliminating expression and / or function of SPPL3 protein) of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B, or one or more target proteins identified using any of the screening methods described herein, e.g., by NHEJ. , HDR, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) or CRISPR / Cas to genetically modify one or more DNA loci encoding one or more target proteins, or to modify one or more RNA encoding one or more target proteins, for example, by using antisense RNA, siRNA, shRNA, programmable editing of RNA using endogenous ADARs ("LEAPER"; see, for example, WO2020074001 and Qu et al. (Nat Biotechnol. 2019; 37(9): 105-9-1069), or RNA editing for programmable A to I substitutions ("REPAIR"; see, for example, Cox et al., "RNA editing with CRISPR-Cas13", Science. 2017; 358(6366): 1019-1027), etc., the contents of each of which are incorporated herein by reference in their entirety,

[0078] Also provided are modified immune cells obtained by any of the methods described herein.

[0079] In one aspect, the present invention provides a method for identifying a target gene in an immune cell (e.g., T cell, B cell or NK cell, such as a CAR-T cell) whose mutation increases resistance to AICD, comprising: a) providing an immune cell library comprising a plurality of immune cells, wherein each of the plurality of immune cells has a mutation (e.g., an inactivating mutation) at a hit gene ("hit gene mutation"), wherein the hit genes of at least two immune cells in the plurality of immune cells are different from each other; b) contacting the immune cell library with FasL; c) obtaining an AICD-resistant immune cell population from the immune cell library; and d) identifying the target gene based on the difference between the profiles of the hit gene mutations in the AICD-resistant immune cell population and the control immune cell population. In some embodiments, the control immune cell population is i) a subpopulation of the immune cell library prior to step b); or ii) the same immune cell library cultured under the same conditions and not contacted with FasL.

[0080] gRNA or sgRNA or sgRNA is also provided iBAR Also provided are molecules, constructs, panels or libraries that can be used to perform the screening methods described herein. Also provided are panels comprising gRNA or sgRNA or sgRNA iBAR Modified immune cells (e.g., CAR-T or CAR-NK cells) of molecules, constructs, groups or libraries and methods for producing the same. Target genes are also provided whose aberrations (e.g., inactivation, such as knockout, or reduction or elimination of expression) increase the resistance of immune cells such as SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (e.g., SPPL3) to AICD. gRNA or sgRNA or sgRNA directed against the AICD resistance genes identified herein are also provided. iBAR In some embodiments, a gRNA, sgRNA or sgRNA encoding a target of SPPL3 is provided. iBAR A nucleic acid comprising the sequence of SEQ ID NO: 1. In some embodiments, a gRNA, sgRNA, or sgRNA targeting SPPL3 is provided. iBAR , wherein the gRNA, sgRNA or sgRNA iBAR Comprising a guide sequence encoded by a nucleic acid comprising the sequence of SEQ ID NO: 1.

[0081] I. Definition

[0082] The present invention will be described with reference to specific embodiments and with reference to certain drawings, but the invention is not limited thereto. Any reference signs in the claims should not be interpreted as limiting the scope. In the accompanying drawings, for illustrative purposes, the sizes of some elements may be exaggerated rather than drawn to scale. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art. In the event of a conflict, this document (including definitions) shall prevail. Preferred methods and materials are described below, although methods and materials similar or equivalent to the methods and materials described herein may be used in the practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated herein by reference in their entirety. The materials, methods and embodiments disclosed herein are illustrative only and are not restrictive.

[0083] As used herein, an "internal barcode" or "iBAR" refers to an index inserted into or appended to a molecule that helps track its identity and performance. As exemplified herein, an iBAR can be, for example, a short nucleotide sequence inserted into or appended to a guide RNA in a CRISPR / Cas system. Multiple iBARs can be used to track the performance of a single guide RNA sequence in an experiment, thereby providing replicated data for statistical analysis without having to repeat the experiment.

[0084] "CRISPR system" or "CRISPR / Cas system" refers collectively to transcripts and other elements that participate in the expression of CRISPR-associated ("Cas") genes and / or direct their activity. For example, a CRISPR / Cas system can include sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active partial tracrRNA), tracr partner sequences (e.g., in endogenous CRISPR systems, including "direct repeats" and partial direct repeats processed by tracrRNA), guide sequences (also referred to as "spacers" in endogenous CRISPR systems), and other sequences and transcripts derived from the CRISPR locus.

[0085] In the context of CRISPR complex formation, a "target sequence" refers to a sequence designed to have complementarity with a guide sequence, wherein hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Perfect complementarity is not necessarily required as long as there is sufficient complementarity to cause hybridization and promote the formation of the CRISPR complex. The target sequence can include any polynucleotide, such as a DNA or RNA polynucleotide. The CRISPR complex can include a guide sequence that hybridizes to the target sequence and complexes with one or more Cas proteins.

[0086] The term "guide sequence" refers to a contiguous sequence of nucleotides in a guide RNA that has partial or complete complementarity with a target sequence in a target polynucleotide and can hybridize to the target sequence through base pairing facilitated by the Cas protein. In the CRISPR / Cas9 system, the target sequence is adjacent to the PAM site. The PAM sequence and its complementary sequence on the other strand together constitute the PAM site.

[0087] The term "guide RNA" is used interchangeably with gRNA herein and refers to a nucleic acid-based molecule, including but not limited to, a nucleic acid-based molecule that can form a protein-RNA complex with a Cas protein and comprises a sequence (e.g., a guide sequence or a spacer) that is sufficiently complementary to the target sequence to hybridize with the target sequence and guide the Cas protein-RNA complex to specifically bind to the target sequence. In some embodiments, the gRNA includes crRNA or is crRNA. In some embodiments, the gRNA comprises two RNA chains, wherein the spacer sequence and the direct repeat sequence (DR) are in different RNA chains, such as in the crRNA chain and the tracrRNA chain. In some embodiments, the gRNA is an RNA chain, such as an sgRNA.

[0088] The terms "single guide RNA", "synthetic guide RNA" and "sgRNA" are used interchangeably to refer to a polynucleotide sequence comprising a guide sequence and any other sequence required for sgRNA function and / or interaction of the sgRNA with one or more Cas proteins to form a CRISPR complex. In some embodiments, the sgRNA comprises a guide sequence fused to a second sequence comprising a tracr sequence derived from a tracr RNA and a tracr-partner sequence derived from a crRNA. The tracr-sequence may comprise all or part of the sequence of a tracrRNA from a naturally occurring CRISPR / Cas system. The term "guide sequence" refers to the nucleotide sequence that specifies the target site in a guide RNA and is used interchangeably with the terms "guide" or "spacer". The term "tracr partner sequence" may also be used interchangeably with the term "direct repeat sequence(s)". As used herein, "sgRNA iBAR ” refers to a single guide RNA with an iBAR sequence.

[0089] The term "Cas protein operable" means that the guide RNA can interact with the Cas protein to form a CRISPR complex.

[0090] As used herein, the term "wild type" is a term understood by those skilled in the art to refer to the typical form of an organism, strain, gene or trait occurring in nature, as distinguished from mutant or variant forms.

[0091] As used herein, the term "variant" shall be considered a display having a quality that deviates from the pattern occurring in nature.

[0092] "Complementarity" refers to the ability of one nucleic acid to form hydrogen bonds (one or more) with another nucleic acid sequence through traditional Watson-Crick base pairing or other non-traditional types. Percent complementarity represents the percentage of residues in a nucleic acid molecule that can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary). "Fully complementary" means that all consecutive residues of a nucleic acid sequence can hydrogen bond with the same number of consecutive residues in a second nucleic acid sequence. As used herein, "substantially complementary" refers to a degree of complementarity of at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50 or more nucleotides, or to two nucleic acids that hybridize under stringent conditions.

[0093] As used herein, "stringent conditions" for hybridization refer to conditions under which a nucleic acid having complementarity with a target sequence primarily hybridizes to the target sequence and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary according to many factors. Generally, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology - Hybridization With Nucleic Acid Probes Part 1, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, NY.

[0094] "Hybridization" refers to the reaction of one or more polynucleotide reactions to form a complex that is stabilized by hydrogen bonds between the nucleotide residue bases. Hydrogen bonds can occur by Watson-Crick base pairing, Hoogstein binding, or in any other sequence-specific manner. The complex can include two chains forming a double-stranded structure, three or more chains forming a multi-chain complex, a single self-hybridizing chain, or any combination of these. The hybridization reaction can constitute a more extensive process such as the start of PCR, or a step in the cutting of polynucleotides by an enzyme. A sequence that can hybridize to a given sequence is referred to as the "complement" of a given sequence.

[0095] As used herein, "doubling time" or "population doubling time" (PDT) refers to the time required for a cell population to double in size. Cell doubling time = ln(2) / (growth rate). Growth rate (gr) refers to the amount that doubles in one time unit. Where N(t) is the number of cells at time t, N(0) is the number of cells at time 0, and t is time (usually in hours). When the cell population is exponentially growing, that is, each individual cell doubles in each cell cycle, the growth rate depends only on the length of the cell cycle.

[0096] As used herein, "construct" refers to a nucleic acid molecule (such as DNA or RNA), or a vector capable of delivering this nucleic acid molecule. For example, when used in the context of gRNA or sgRNA, construct refers to a gRNA or sgRNA molecule, a nucleic acid molecule encoding gRNA or sgRNA (e.g., isolated DNA or viral vector) or a vector capable of delivering nucleic acid molecules encoding gRNA and sgRNA, such as a slow virus carrying a nucleic acid molecule encoding gRNA or sgRNA. When used for protein, construct refers to a nucleic acid molecule comprising a nucleotide sequence that can be transcribed into RNA or expressed as a protein. Construct can include necessary regulatory elements operably linked to a nucleotide sequence that, when present in a host cell, allows transcription or expression of the nucleotide sequence.

[0097] As used herein, "operably linked" means that the expression of a gene is controlled by a regulatory element (such as a promoter) that is spatially connected to it. The regulatory element can be located 5' (upstream) or 3' (downstream) of the gene under its control. The distance between the regulatory element (such as a promoter) and the gene can be approximately the same as the spacing between the regulatory element (such as a promoter) and the gene that it naturally controls and from which the regulatory element is derived. As is known in the art, changes in this distance can be accommodated without loss of function in the regulatory element (such as a promoter).

[0098] The term "vector" is used to describe a nucleic acid molecule that can be engineered to contain a cloned polynucleotide or a polynucleotide that can be propagated in a host cell. Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (e.g., circular); nucleic acid molecules comprising DNA, RNA, or both; and other types of polynucleotides known in the art. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and additional mammalian vectors having bacterial replication origins). Other vectors (e.g., non-additional mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, thereby replicating together with the host genome. In addition, certain vectors are capable of directing the expression of genes operably linked to them. Such vectors are referred to herein as "expression vectors." A recombinant expression vector may comprise a nucleic acid of the present invention suitable for expressing nucleic acid in a host cell, meaning that the recombinant expression vector comprises one or more regulatory elements that can be selected based on the host cell for expression, i.e., operably linked to the nucleic acid sequence to be expressed.

[0099] "Host cell" refers to a cell that can be or has been a recipient of a vector or isolated polynucleotide. The host cell can be a prokaryotic cell or a eukaryotic cell. In some embodiments, the host cell is a eukaryotic cell that can be cultured in vitro and modified using the methods described herein. The term "cell" includes the primary subject cell and its progeny.

[0100] As used herein, the term "autologous" refers to any material derived from the same individual that is to be later reintroduced into that individual.

[0101] "Allogeneic" refers to a transplant derived from different individuals in the same species. "Allogeneic T cells" refers to T cells from a donor that have a tissue human leukocyte antigen (HLA) type that matches the recipient. Typically, matching is based on the variability of three or more loci of the HLA gene, and is preferably a complete match at these loci. In some cases, allogeneic transplant donors are related (usually siblings with close HLA matching), syngeneic (monozygotic "identical" twins of the patient), or unrelated (donors with no relatedness and a very close HLA matching degree). The HLA gene is divided into two categories (Type I and Type II). Typically, the mismatch of Type I genes (i.e., HLA-A, HLA-B, or HLA-C) increases the risk of graft rejection. The mismatch of HLA type II genes (i.e., HLA-DR or HLA-DQB1) increases the risk of graft-versus-host disease (GvHD).

[0102] The term "donor subject" or "donor" refers to a subject whose cells are being obtained for further in vitro engineering. A donor subject can be a patient to be treated with a cell population produced by the methods described herein (i.e., an autologous donor), or can be an individual who donates a blood sample (e.g., a lymphocyte sample) that will be used to treat a different individual or patient when a cell population is produced by the methods described herein (i.e., an allogeneic donor). These subjects who receive modified cells (e.g., modified immune cells described herein or produced by the present methods) can be referred to as "recipients" or "recipient subjects."

[0103] "Multiplicity of infection" or "MOI" are used interchangeably herein to refer to the ratio of a pathogen (e.g., a phage, virus, or bacterium) to its target (e.g., a cell or organism) for infection. For example, when referring to a group of cells inoculated with viral particles, the multiplicity of infection or MOI is the ratio between the number of viral particles (e.g., viral particles comprising a sgRNA library) and the number of target cells present in the mixture during viral transduction.

[0104] As used herein, the "phenotype" of a cell refers to an observable characteristic or property of a cell, such as its morphology, development (such as growth, proliferation, differentiation or death), steady state, biochemical or physiological characteristics, phenology or behavior. The phenotype can be caused by the expression of genes in the cell, the influence of environmental factors or the interaction between the two. In some embodiments, the phenotype is growth, differentiation and / or maturation. In some embodiments, the phenotype is the inhibition of growth or proliferation. In some embodiments, the phenotype is persistence in the body. In some embodiments, the phenotype is death. In some embodiments, the phenotype is the effector function (such as cytokine release and / or cytotoxic killing) of an immune cell, or the effector function is reduced or absent. In some embodiments, the phenotype is exhaustion of an immune cell (e.g., T cell).

[0105] "Immune cell exhaustion" refers to a decrease in immune cell function, such as decreased proliferation, decreased effector function, and upregulation of immunosuppressive molecules. "T cell exhaustion" refers to a decrease in T cell function, and "NK cell exhaustion" refers to a decrease in NK cell function, which is a result of infection (such as chronic infection) or disease (such as cancer). T cell exhaustion is associated with increased expression of PD-1, TIM-3, TIGIT, and / or LAG-3, apoptosis, decreased cytotoxicity, and / or decreased cytokine secretion. Therefore, the terms "improving T cell exhaustion", "inhibiting T cell exhaustion", "reducing T cell exhaustion", etc. refer to conditions in which T cell function is restored, characterized by one or more of the following: reduced expression and / or levels of one or more of PD-1, TIM-3, TIGIT and / or LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased production and / or secretion of antigen-induced cytokines (e.g., IFN-γ, IL-2 and / or TNF-α); enhanced cytotoxicity / killing capacity; enhanced recognition of tumor targets with low surface antigens; and enhanced proliferative response to antigens. NK cell exhaustion is associated with increased expression of PD-1, TIGIT, TIM-3, LAG-3, and / or NKG2A, downregulation of activating receptors (e.g., NKG2D, CD16, NCRs (NKp30, NKp44, and NKp46), CD226, 2B4), cell apoptosis, decreased cytotoxicity, and / or reduced secretion of cytokines (e.g., IFN-γ and / or TNF-α). Therefore, the terms "improving NK cell exhaustion", "inhibiting NK cell exhaustion", "reducing NK cell exhaustion", etc. refer to conditions for restoration of NK cell function, characterized by one or more of the following: reduced expression and / or levels of one or more of PD-1, TIM-3, TIGIT, LAG-3 and / or NKG2A; prevention of cell apoptosis; increased production and / or secretion of cytokines (such as IFN-γ and / or TNF-α); enhanced cytotoxicity / killing ability; enhanced recognition of tumor targets with low surface antigen; enhanced proliferative response to antigens (such as MHC class I molecules); enhanced expression of one or more activating receptors (such as NKG2D, CD16, NCR (NKp30, NKp44 and NKp46), CD226, 2B4).

[0106] As used herein, the term "stimulation" refers to a primary response induced by ligating a cell surface moiety. For example, in the case of a receptor, this stimulation requires ligation of the receptor and subsequent signal transduction events. With regard to stimulation of T cells, this stimulation refers to the ligation of a T cell surface moiety, which in one embodiment subsequently induces a signal transduction event, such as binding to a TCR / CD3 complex. In addition, the stimulation event can activate cells and increase or decrease the expression or secretion of molecules, such as by decreasing TGF-β. Therefore, even in the absence of a direct signal transduction event, the ligation of the cell surface moieties can result in the reorganization of the cytoskeleton structure, or in the aggregation of the cell surface moieties, each of which can enhance, modify or alter subsequent cellular responses.

[0107] As used herein, the term "activated" refers to a state of a cell after sufficient ligation of cell surface moieties to induce significant biochemical or morphological changes. In the context of T cells, such activation refers to a state in which the T cell has been sufficiently stimulated to induce cell proliferation. Activation of T cells can also induce cytokine production and expression of regulatory or cytolytic effector functions. In the context of other cells, this term infers the upregulation or downregulation of specific physicochemical processes. The term "activated T cell" refers to a T cell that is currently undergoing cell division, cytokine production, expression of regulatory or cytolytic effector functions, and / or has recently undergone an "activation" process.

[0108] A "dominant negative" gene product or protein is one that interferes with the function of another gene product or protein. The other gene product affected may be the same as or different from the dominant negative protein. The dominant negative gene product may take a variety of forms, including truncations, full-length proteins or fragments thereof with point mutations, or fusions of full-length wild-type or mutant proteins or fragments thereof with other proteins.

[0109] As used herein, an "isolated" nucleic acid molecule refers to a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it was produced. Preferably, the isolated nucleic acid is free from all components associated with the production environment. The isolated nucleic acid molecule encoding the polypeptide herein is in a form that is different from the form or environment in which it is found in nature. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide herein that is naturally present in a cell.

[0110] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The term "nucleotide sequence encoding a protein or RNA" may also include introns, such that a nucleotide sequence encoding a protein may contain intron(s) in some versions.

[0111] As used herein, the term "transfection" or "transformation" or "transduction" refers to the process of transferring or introducing exogenous nucleic acid into a host cell (such as an immune cell). A "transfected" or "transformed" or "transduced" cell is a cell that has been transfected, transformed, or transduced with an exogenous nucleic acid. Such cells include primary subject cells and their progeny.

[0112] As used herein, "treatment" is a method for obtaining beneficial or desired results including clinical results. For the purposes of the present invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, alleviating the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for treating the disease, delaying the progression of the disease, improving the quality of life, and / or prolonging survival. "Treatment" also encompasses reducing the pathological consequences of cancer or immune diseases.

[0113] The term "effective amount" as used herein refers to an amount sufficient to treat a particular condition, illness or disease such as to improve, alleviate, mitigate and / or delay one or more symptoms thereof (e.g., cancer, infectious disease, GvHD, transplant rejection, autoimmune disease or radiation sickness) of a medicament (e.g., a modified immune cell as described herein or its pharmaceutical composition). With regard to cancer, an effective amount includes an amount sufficient to shrink a tumor and / or reduce tumor growth rate (e.g., inhibit tumor growth) or prevent or delay other unwanted cell proliferation. In some embodiments, an effective amount is an amount sufficient to delay development. In some embodiments, an effective amount is an amount sufficient to prevent or delay recurrence. An effective amount can be administered in one or more administrations. An effective amount of an agent (e.g., a modified immune cell) or composition can: (i) reduce the number of cancer cells; (ii) reduce tumor size; (iii) inhibit, hinder, slow down, and preferably terminate the infiltration of cancer cells into peripheral organs to a certain extent; (iv) inhibit (i.e., slow down and preferably terminate to a certain extent) tumor metastasis; (v) inhibit tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; and / or (vii) alleviate one or more symptoms associated with cancer to a certain extent. In the case of infectious diseases such as viral infections, a therapeutically effective amount of the modified immune cells described herein or their compositions can reduce the number of cells infected by pathogens; reduce the production or release of antigens derived from pathogens; inhibit (i.e., slow down and preferably terminate to a certain extent) the spread of pathogens to uninfected cells; and / or alleviate one or more symptoms associated with infection to a certain extent. In some embodiments, a therapeutically effective amount is an amount that prolongs patient survival.

[0114] As used herein, "individual" or "subject" refers to a mammal, including but not limited to humans, cows, horses, cats, dogs, rodents, or primates. In some embodiments, the individual is a human.

[0115] As used herein, "patient" includes any person suffering from a disease (eg, cancer). The terms "subject," "individual," and "patient" are used interchangeably herein.

[0116] Where the term "comprising" is used in the present description and claims, it does not exclude other elements or steps.

[0117] It should be understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0118] Reference herein to "about" a value or parameter includes (and describes) variations with respect to that value or parameter itself. For example, a description referring to "about X" includes a description of "X."

[0119] As used herein, reference to a "non" value or parameter generally refers to and describes an "other" value or parameter. For example, the method is not used to treat type X cancer, which means that the method is used to treat cancers other than type X.

[0120] As used herein, the term "about XY" has the same meaning as "about X to about Y"

[0121] For describing the numerical range of Nucleotide herein, each intermediate value therebetween is explicitly considered. For example, for the range of 19-21nt, the number 20nt is also considered in addition to 19nt and 21nt, and for the range of MOI, each intermediate value therebetween is explicitly considered, no matter it is an integer or a decimal.

[0122] As used herein and in the appended claims, the singular forms "a," "or," and "the" include plural references unless the context clearly dictates otherwise.

[0123] One of ordinary skill in the art will understand that both uracil and thymine can be represented by "t", rather than using "u" to represent uracil and "t'" to represent thymine; in the context of RNA, unless otherwise indicated, it should be understood that "t" is used to represent uracil.

[0124] II. Modified Immune Cells

[0125] In some embodiments, immune cells (e.g., T cells such as CAR-T cells, or NK cells such as CAR-NK cells) are provided, which are modified to have no or reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B. In some embodiments, the target protein is selected from one or more of ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B and SPPL3. In some embodiments, the target protein is SPPL3 protein. Thus, in some embodiments, immune cells (e.g., T cells such as CAR-T cells, or NK cells such as CAR-NK cells) are provided that are modified to lack or reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) SPPL3 protein expression (RNA and / or protein expression) and / or function. In some embodiments, the immune cells are further modified to lack or reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) TCR protein (e.g., one or more of TCRα, TCRβ, TCRγ, TCRδ) expression (RNA and / or protein expression) and / or function. In some embodiments, the modification is mediated by gene editing (e.g., CRISPR / Cas). In some embodiments, a gene encoding a SPPL3 knockout (KO) gene ("SPPL3 KO ”) immune cells (e.g., T cells such as CAR-T cells, or NK cells such as CAR-NK cells). In some embodiments, there is provided a method for producing an immune cell having SPPL3 gene KO and TCR gene KO (“SPPL3 KO / TCR KO ”) is an immune cell (e.g., a T cell such as a CAR-T cell, or a NK cell such as a CAR-NK cell). In some embodiments, the immune cell is a T cell, such as a CAR-T cell (e.g., an anti-CD19 CAR-T cell).

[0126] In some embodiments, the modified immune cell has at least about 10% less (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), such as at least about 20% less activation-induced cell death (AICD), compared to a reference immune cell that does not have a modification that reduces or eliminates expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein). In some embodiments, the modified immune cell has at least about 10% less (e.g., at least about any one of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), such as at least about 20% less AICD, compared to a reference immune cell that does not have a modification that reduces or eliminates expression (RNA and / or protein expression) and / or function of SPPL3 protein.

[0127] In some embodiments, expression of one or more target proteins (e.g., SPPL3 protein) is reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or inhibited by an antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding the one or more target proteins.

[0128] In some embodiments, the function of one or more target proteins (e.g., SPPL3 proteins) is reduced (e.g., reduced by at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) or inhibited by chemically modified mRNA, such as a chemically modified mRNA encoding a dominant negative inhibitor (e.g., a dominant negative variant or fragment thereof, or a dominant negative binding partner) of one or more target proteins (e.g., SPPL3 proteins). In some embodiments, immune cells are modified to express a dominant negative SPPL3 protein variant or a dominant negative fragment thereof. In some embodiments, immune cells are modified to express a dominant negative binding partner of a SPPL3 protein.

[0129] In some embodiments, the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) is reduced (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or inhibited by a small molecule compound, nucleic acid (or a vector comprising the same), lipid and / or protein molecule.

[0130] In some embodiments, immune cells are genetically modified at a DNA locus encoding one or more target proteins. In some embodiments, immune cells are genetically modified at the SPPL3 locus. In some embodiments, a DNA locus (e.g., the SPPL3 locus) is modified with a mutagen. Mutagens can be divided into three categories: physical (e.g., gamma rays, ultraviolet (UV) radiation), chemical (e.g., ethyl methanesulfonate (EMS)), and transposable elements (e.g., transposons, retrotransposons, T-DNA, retroviruses). In some embodiments, the mutagen or condition is any of ionizing radiation (IR), UV radiation, an alkylating agent (e.g., nitrogen mustard, methyl methanesulfonate (MMS), EMS, N-ethyl-N-nitrosourea (ENU)), an aromatic amine (e.g., 2-aminofluorene), a polycyclic aromatic hydrocarbon (PAH; e.g., dibenzo[a,l]pyrene, naphthalene, anthracene, pyrene), cross-linking, insertional mutagenesis (e.g., mediated by a transposon or virus), or other toxins (e.g., aflatoxin, N-nitrosamines). In some embodiments, a DNA locus (e.g., an SPPL3 locus) is modified by gene editing. In some embodiments, gene editing is mediated by site-directed mutagenesis (SDM). In some embodiments, gene editing is mediated by random and extensive mutagenesis (REM). In some embodiments, gene editing is accomplished by PCR method(s). In some embodiments, gene editing is accomplished by non-PCR method(s). Any known gene editing method can be used herein, including but not limited to non-homologous end joining (NHEJ)-mediated, homology-directed repair (HDR)-mediated, zinc finger nuclease (ZFN)-mediated, transcription activator-like effector nuclease (TALEN)-mediated or CRISPR / Cas-mediated gene editing. HDR can occur either non-conservatively or conservatively. In some embodiments, HDR is mediated by a single-strand annealing (SSA) pathway. In some embodiments, HDR is mediated by a classical double-strand break repair (DSBR) pathway, a synthesis-dependent chain annealing (SDSA) pathway, or a break-induced repair (BIR) pathway. In some embodiments, the cell modification method described herein further comprises introducing a nucleic acid template (e.g., comprising a desired mutation), such as inserting a double-strand break (DSB) to modify the target genomic sequence (e.g., by HDR). Gene editing can introduce one or more mutations into a DNA locus encoding one or more target proteins (e.g., SPPL3 protein), including but not limited to insertions, deletions, substitutions (e.g., non-synonymous substitutions), truncations, translocations, point mutations, and the like. In some embodiments, the mutation is a frameshift mutation, a loss of function (LOF) mutation, a dominant negative mutation, a missense mutation, or a nonsense mutation. In some embodiments, gene editing includes gene knockout (KO). In some embodiments, gene editing includes base editing (e.g., introducing a non-synonymous substitution).In some embodiments, base editing introduces a stop codon, which can reduce the expression of functional RNA and / or protein. In some embodiments, base editing introduces mutations that affect RNA and / or protein function. In some embodiments, gene editing is mediated by CRISPR / Cas. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein comprising i) a dead Cas protein (dCas) and ii) an adenine base editor (ABE) or adenine deaminase (ADA), or a cytidine base editor (CBE) or a cytidine deaminase, or a functional fragment thereof. The cytidine base editor can convert the target C:G base pair into a T:A base pair, and the adenine base editor can convert the A:T base pair into a G:C base pair. In short, these two types of base editors can be targeted to set all possible conversion mutations (C- to -T, G- to -A, A- to -G, T- to -C, C- to -U and A- to -U).

[0131] In some embodiments, immune cells are genetically modified at RNA encoding one or more target proteins. In some embodiments, immune cells are genetically modified at SPPL3 RNA. In some embodiments, RNA encoding one or more target proteins (e.g., SPPL3 protein) is modified by RNA editing. RNA editing can introduce one or more mutations in RNA encoding one or more target proteins (e.g., SPPL3 protein), including but not limited to insertion, deletion, substitution (e.g., non-synonymous substitution), truncation, point mutation, etc. In some embodiments, the mutation is a frameshift mutation, a LOF mutation, a dominant negative mutation, a missense mutation, or a nonsense mutation. In some embodiments, RNA editing includes base editing (e.g., introducing non-synonymous substitutions, such as C to U, A to I). Any known RNA editing method can be used herein (see, e.g., Guillermo Aquino Jarquin, “Novel Engineered Programmable Systems for ADAR Mediated RNAediting”, Mol Ther Nucleic Acids).2020;19:1065-1072; the contents of which are incorporated herein by reference in their entirety), including, but not limited to, programmable editing of RNA using endogenous ADARs ("LEAPER"; see, e.g., WO2020074001 and Qu et al. (Nat Biotechnol. 2019;37(9):1059-1069), or RNA editing for programmable A to I substitutions ("REPAIR"; see, e.g., Cox et al., "RNA editing with CRISPR-Cas13," Science. 2017;358(6366):1019-1027), recruiting endogenous ADARs to specific transcripts for oligonucleotide-mediated RNA editing ("RESTORE"; see, e.g., Merkle et al., "Acytosine deaminase for programmable single-base RNA editing,” Science. 2019; 365(6451): 382-386), Methods Mol Biol. 2021; 2181: 331-349), CRISPR-Cas inspired RNA targeting system (“CIRTS”; see, e.g., Rauch et al., “Programmable RNA-Guided RNAEffector Proteins Built from Human Parts,” Cell. 2019; 178(1): 122-134.e12), RNA editing with specific C to U exchange (“RESCUE”; see, e.g., Abudayyeh et al., “A cytosine deaminase for programmable single-base RNA editing,” Science. 2019; 365(6451): 382-386), or CLUSTER (see, e.g., P. Reautschnig et al., “CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo,” Nat Biotechnol. 2022 May; 40(5): 759-768), the contents of each of which are incorporated herein by reference in their entirety. In some embodiments, RNA editing is mediated by LEAPER.In some embodiments, RNA editing is mediated by CRISPR / Cas, for example by fusing an adenine base editor (ABE) or adenine deaminase (ADA), or a cytidine base editor (CBE) or cytidine deaminase (CDA), or functional fragments thereof, to a dead Cas (dCas, e.g., dCas13) protein.

[0132] Methods for preparing CRISPRs that recognize predetermined DNA or RNA sites are known in the art. Any known CRISPR / Cas system suitable for gene editing or RNA editing can be used herein. So far, based on the system's excellent functionality and evolutionary modularity, two types (Class 1 and Class 2) and six types (I-VI) of CRISPR-Cas systems have been characterized. See, for example, Nidhi et al., "Novel CRISPR–Cas Systems: An Updated Review of the Current Achievements, Applications, and Future Research Perspectives," Int J Mol Sci. 2021; 22(7): 3327, the contents of which are incorporated herein by reference in their entirety. Among the Class 2 CRISPR-Cas systems, the Type II Cas9 system and the Type VA / B / E / J Cas12a / Cas12b / Cas12e / Cas12j system have been used for genome editing and provide broad prospects for biomedical research. Cas13a (C2c2) is a Type VI-A RNA-guided CRISPR effector targeting RNA that can be used for RNA editing as described herein. The CRISPR / Cas system used herein can produce double-strand breaks (DSBs) or single-strand breaks at predetermined nucleic acid sites. In some embodiments, the CRISPR / Cas system used herein is a CRISPR / Cas9 system.

[0133] In some embodiments, gene editing or RNA editing includes contacting a precursor immune cell (e.g., a precursor T cell or a precursor NK cell) with i) a guide RNA (gRNA) construct under conditions allowing the introduction of a gRNA construct and optional Cas components into a precursor immune cell; and optionally ii) a Cas component construct comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9), wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to a target site in a DNA locus or RNA encoding one or more target proteins (e.g., SPPL3 protein). In some embodiments, the precursor immune cell has expressed the Cas protein prior to the introduction of the gRNA construct. In some embodiments, the precursor immune cell does not express the Cas protein prior to the introduction of the gRNA construct and Cas components. In some embodiments, the precursor immune cell expresses an engineered receptor (e.g., CAR, engineered TCR, or TAC) prior to the introduction of the gRNA construct and / or Cas components. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein, for example, a fusion protein comprising i) dCas (e.g., dCas13a, dCas9) and ii) ADA (e.g., TadA, e.g., TadA8e) or CDA or a functional fragment thereof. In some embodiments, the Cas protein is Cas9, e.g., dCas9. In some embodiments, the gRNA is an sgRNA. In some embodiments, the gRNA includes crRNA (or consists essentially of crRNA, or consists of crRNA). In some embodiments, the gRNA includes crRNA and tracrRNA (or consists essentially of crRNA and tracrRNA, or consists of it).

[0134] In some embodiments, immune cells (e.g., T cells such as CAR-T cells) that are modified to not have or reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the expression (RNA and / or protein expression) and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B do not have or are further modified to not have or reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the expression (RNA and / or protein expression) and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B. or more) is selected from the group consisting of: TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, SPPL3, FaS, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and ligands for NKp46. In some embodiments, the immune cells that are modified to lack or have reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression (RNA and / or protein expression) and / or function of the SPPL3 protein lack or are further modified to lack or have reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression (RNA and / or protein expression) of one or more proteins selected from ) and / or function: ligands of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46.In some embodiments, immune cells (e.g., T cells such as CAR-T cells) that are modified to have no or reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) SPPL3 protein expression (RNA and / or protein expression) and / or function do not have or are further modified to have no or reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) TCR protein (e.g., one or more of TCRα, TCRβ, TCRγ, TCRδ) expression (RNA and / or protein expression) and / or function. In some embodiments, the immune cells modified to lack or reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the expression (RNA and / or protein expression) and / or function of the SPPL3 protein lack or are further modified to lack or reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the expression (RNA and / or protein expression) and / or function of one or more additional proteins selected from the group consisting of Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B.

[0135] In some embodiments, immune cells (e.g., T cells) express or are further modified to express engineered receptors. In some embodiments, immune cells have expressed engineered receptors before being modified to reduce or eliminate the expression and / or function of one or more target proteins (e.g., SPPL3 proteins). In some embodiments, immune cells are modified to reduce or eliminate the expression and / or function of one or more target proteins (e.g., SPPL3 proteins), and are further modified to express engineered receptors. In some embodiments, reducing or eliminating the expression and / or function of one or more target proteins (e.g., SPPL3 proteins) and the modification of expressing engineered receptors occur simultaneously. In some embodiments, reducing or eliminating the expression and / or function of one or more target proteins (e.g., SPPL3 proteins) and the modification of expressing engineered receptors occur sequentially. Any engineered receptor that can be used to transduce signals to immune cells (e.g., to induce cell proliferation, cytokine production and / or regulation or cytolysis effector function) and / or recognize target antigens can be used herein. In some embodiments, the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen conjugate (TAC). In some embodiments, the engineered receptor is monovalent. In some embodiments, the engineered receptor is multivalent. In some embodiments, the engineered receptor is monospecific, such as monovalent and monospecific, or multivalent and single specificity. In some embodiments, the engineered receptor is multispecific (e.g., bispecific). Any CAR, engineered TCR or TAC can be used herein.

[0136] In some embodiments, CAR includes: i) an extracellular antigen binding domain that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); ii) a transmembrane domain; and iii) an intracellular signal transduction domain. In some embodiments, the extracellular antigen binding domain is selected from the extracellular domain of a ligand, a single domain antibody (sdAb), a single-chain Fv (scFv), and a Fab. In some embodiments, the transmembrane domain is derived from a molecule selected from the following: TCR α, TCR β, TCR γ, TCR δ, CD3 ζ, CD3 ε, CD3 γ, CD3 δ, CD4, CD5, CD6, CD7, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD47, CD52, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154, CISH, and PD-1. In some embodiments, the transmembrane domain is derived from CD8α. In some embodiments, the intracellular signaling domain comprises a primary intracellular signaling domain derived from a molecule selected from the group consisting of CD3ζ, CD3γ, CD3ε, CD3δ, FcRγ, FcRβ, CD5, CD22, CD79a, CD79b, CD66d, FcγRIIa, DAP10, and DAP12. In some embodiments, the primary intracellular signaling domain is derived from CD3ζ. In some embodiments, the intracellular signaling domain further comprises a costimulatory signaling domain derived from a costimulatory molecule selected from the group consisting of CARD11, CD2, CD4, CD7, CD19, CD27, CD28, CD30, CD40, CD160, ICAM-1, OX40, 4-1BB, SELPLG, LIGHT, HVEM, B7-H3, ICOS, PD-1, SLAMF7, LFA-1, NKG2C, CDS, GITR, BAFFR, NKp80, IPO-3, SLAMF8, LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46, NKG2D, CD83, SLAMF1, CTLA-4, LAG-3, PD-L2, PD-L1, DAP10, TRIM, ZAP70, a ligand that specifically binds to CD83, and any combination thereof. In some embodiments, the costimulatory signal transduction domain is derived from 4-1BB. In some embodiments, CAR also includes a hinge domain between the C-terminus of the extracellular antigen binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α or CD28. In some embodiments, CAR comprises from N' to C': an extracellular antigen binding domain (e.g., scFv)-optional hinge domain-transmembrane domain-primary intracellular signal transduction domain.In some embodiments, CAR comprises, from N' to C': an extracellular antigen binding domain (eg, scFv) - an optional hinge domain - a transmembrane domain - a co-stimulatory signaling domain - a primary intracellular signaling domain.

[0137] In some embodiments, the CAR is an anti-CD19 CAR. In some embodiments, the anti-CD19 CAR is derived from tisagenleucel CAR-T cells (CTL019, e.g. ) anti-CD19 CAR. See, for example, US9499629, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the anti-CD19 CAR comprises i) an extracellular antigen binding domain that is a scFv that specifically recognizes CD19; ii) a transmembrane domain derived from CD8α; and iii) an intracellular signaling domain comprising a primary intracellular signaling domain derived from CD3ζ. In some embodiments, the anti-CD19 CAR comprises, from N' to C': anti-CD19 scFv-CD8α hinge domain-CD8α transmembrane domain-4-1BB costimulatory signaling domain-CD3ζ primary intracellular signaling domain.

[0138] In some embodiments, T cells are provided that are modified to have no or reduced expression (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of SPPL3 protein (RNA and / or protein expression) and / or function, wherein the T cells express a CAR or are further modified to express a CAR. In some embodiments, CAR-T cells are provided that are modified to have no or reduced expression (e.g., at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) of SPPL3 protein (RNA and / or protein expression) and / or function. In some embodiments, a T cell is provided that is modified to have no or reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) SPPL3 protein and TCR protein (e.g., any one of one or more of TCRα, TCRβ, TCRγ, TCRδ) expression (RNA and / or protein expression) and / or function, wherein the T cell expresses a CAR or is further modified to express a CAR. In some embodiments, a CAR-T cell is provided that is modified to have no or reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) SPPL3 protein and TCR protein (e.g., one or more of TCRα, TCRβ, TCRγ, TCRδ) expression (RNA and / or protein expression) and / or function. In some embodiments, T cells (e.g., CAR-T cells) are genetically modified at the SPPL3 locus by gene editing (e.g., by CRISPR / Cas). In some embodiments, T cells (e.g., CAR-T cells) are genetically modified at the TCR locus by gene editing (e.g., by CRISPR / Cas). In some embodiments, T cells (e.g., CAR-T cells) are genetically modified at both the SPPL3 locus and the TCR locus by gene editing (e.g., by CRISPR / Cas). In some embodiments, gene editing of T cells (e.g., CAR-T cells) includes contacting precursor T cells with i) gRNA constructs; and optionally ii) Cas components comprising Cas proteins (e.g., Cas9) or nucleic acids encoding Cas proteins, wherein the gRNA constructs comprise or encode gRNAs comprising guide sequences complementary to the target sites in the SPPL3 locus. In some embodiments, the guide sequence is encoded by a nucleic acid sequence comprising a sequence of SEQ ID NO: 1.In some embodiments, precursor T cells express Cas proteins. In some embodiments, CAR includes: i) an extracellular antigen binding domain that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes) (e.g., anti-CD19 scFv or sdAb); ii) a transmembrane domain (e.g., CD8α transmembrane domain); and iii) an intracellular signal transduction domain (e.g., CD3ζ primary intracellular signal transduction domain). In some embodiments, CAR is an anti-CD19 CAR, for example, derived from tisagenleucel (CTL019, e.g., Ky. In some embodiments, a method is provided wherein the SPPL3 KO Anti-CD19 CAR-T cells (“SPPL3 KO / In some embodiments, a method for treating a patient with SPPL3 is provided. KO and TC R KO Anti-CD19 CAR-T cells ("SPPL3 KO / TCR KO / anti-CD19CAR-T cell”).

[0139] In some embodiments, the engineered TCR comprises: (a) an extracellular ligand binding domain comprising an antigen binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) an optional first linker; (c) an optional extracellular domain of a first TCR subunit (e.g., Cα, Cβ, Cδ, Cγ, CD3ε) or a portion thereof; (d) a transmembrane domain of a second TCR subunit (e.g., TCRα, TCRβ); and (e) an intracellular signaling domain comprising an intracellular signaling domain of a third TCR subunit (e.g., TCRα, TCRβ); wherein the first, second, and third TCR subunits are independently selected from TCRα, TCRβ, TCRγ, TCRδ, CD3ε, CD3γ, CD3δ, and CD3ζ. In some embodiments, the first, second, and third TCR subunits are identical (e.g., all are CD3ε, all are TCRα, or all are TCRβ). In some embodiments, the first, second, and third TCR subunits are different. In some embodiments, the engineered TCR further comprises a hinge domain between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain. In some embodiments, the hinge domain is derived from CD8α.

[0140] Typically, a TAC comprises (i) an antigen binding domain, (ii) a TCR binding domain (e.g., scFv), and (iii) a co-receptor domain (e.g., a hinge, a transmembrane, and / or a cytoplasmic region). For example, see Helsen et al. Nat Commun. 2018; 9(1):3049. In some embodiments, a TAC comprises: (a) an extracellular ligand binding domain comprising an antigen binding fragment (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes (e.g., tumor epitopes); (b) an optional first linker; (c) an extracellular TCR binding domain (e.g., sdAb, scFv, Fab, DARPin) that specifically recognizes the extracellular domain of a TCR subunit (e.g., CD3ε); (d) an optional second linker; (e) a first TCR co-receptor ( (e.g., CD4, CD8) or a portion thereof; (f) a transmembrane domain comprising a transmembrane domain of a second TCR co-receptor (e.g., CD4, CD8); and (g) an optional intracellular signal transduction domain comprising an intracellular information transduction domain of a third TCR co-receptor (e.g., CD4, CD8); wherein the TCR subunit is selected from CD3ε, CD3δ, CD3γ, TCRα, TCRβ, TCRγ, and TCRδ; and wherein the first, second, and third TCR co-receptors are each independently selected from CD4, CD8, and CD28. In some embodiments, the first, second, and third TCR co-receptors are the same. In some embodiments, the first, second, and third TCR co-receptors are different. In some embodiments, the TAC further comprises a hinge domain (e.g., derived from CD8α) located between the C-terminus of the extracellular ligand binding domain and the N-terminus of the transmembrane domain.

[0141] In some embodiments, the modification that reduces or eliminates the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 proteins) does not downregulate or eliminate the expression (RNA and / or protein expression) and / or function of an engineered receptor (e.g., CAR). In some embodiments, the modification that reduces or eliminates the expression and / or function of one or more target proteins (e.g., SPPL3 proteins) downregulates the expression (RNA and / or protein expression) and / or function of the engineered receptor by up to about 30% (e.g., up to about 25%, 20%, 15%, 10%, 5%, 1% or any one of less).

[0142] In some embodiments, the immune cell is a T cell, for example, selected from helper CD4+ T cells, cytotoxic CD8+ T cells, memory T cells, regulatory CD4+ T cells, NKT cells, MAIT cells, DNT cells and γδ T cells. In some embodiments, the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) is reduced or eliminated: i) the cell surface expression of one or more of the ligands for Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and NKp46 is reduced by at least about 10% (e.g., by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 0%, 70%, 80%, 90%, 95% or 100%); ii) reducing killing by allogeneic T cells by at least about 10% (e.g., reducing killing by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%); and / or iii) reducing killing by autologous or allogeneic NK cells by at least about 10% (e.g., reducing killing by at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%).

[0143] In some embodiments, the modified immune cell has at least about 10% (e.g., at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1-fold, 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, or more) longer persistence in vivo compared to a reference immune cell that does not have a modification that reduces or eliminates expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein).

[0144] Immune cells modified to reduce or eliminate the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR protein) and / or modified to express engineered receptors (e.g., CAR) can be autologous or allogeneic.

[0145] immune cells

[0146] The term "immune cell" as referred to herein includes cells of hematopoietic origin and that play a role in an immune response. "Immune cell" used herein includes unmodified immune cells (e.g., parental immune cells, such as primary immune cells, immune cells from cell lines), reference immune cells and modified immune cells, such as modified to reduce or eliminate one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCR) expression (RNA and / or protein expression) and / or function and / or modified to express engineered receptors (e.g., CAR) immune cells. Immune cells include lymphocytes, such as B cells and T cells; Natural killer cells; Myeloid cells, such as monocytes, macrophages, dendritic cells, eosinophils, mast cells, basophils and granulocytes. In one embodiment, immune cells are immune effector cells. The term "immune effector cell" used herein refers to cells that participate in an immune response, such as cells that promote immune effector reactions. The example of immune effector cells includes T cells, B cells, NK cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes. The immune cells described herein can be autologous or allogeneic.

[0147] "Immune effector function" or "immune effector response" as used herein refers to a function or reaction that enhances or promotes an immune attack on a target cell, such as a function or reaction of an immune effector cell. For example, an immune effector function or reaction refers to a characteristic of a T cell or NK cell that promotes killing or growth or proliferation of a target cell (such as a tumor cell). In the case of a T cell, primary stimulation and co-stimulation are examples of immune effector functions or reactions.

[0148] The term "stimulation" refers to a primary response induced by the binding of a stimulatory molecule (e.g., a TCR / CD3 complex or CAR) to its cognate ligand (or tumor antigen in the case of a CAR), thereby mediating a signal transduction event such as, but not limited to, signal transduction through the TCR / CD3 complex or signal transduction through an appropriate NK receptor or signal transduction domain of a CAR. Stimulation can mediate changes in the expression of certain molecules.

[0149] In some embodiments, immune cells are recombinant or modified immune cells.The term "recombinant" includes cells modified by introducing heterologous nucleic acids, or cells derived from cells modified in this way, but does not include naturally occurring events (such as spontaneous mutations, natural transformations, natural transductions, natural transpositions) such as those that occur without human intervention to changes in cells. Recombinant immune cells can be non-naturally occurring cells. Recombinant immune cells can also be engineered cells. In one embodiment, recombinant immune cells are engineered immune cells, such as engineered T cells (such as CAR-T cells) or engineered NK cells (for example, CAR-NK cells). In some embodiments, recombinant immune cells are separated immune cells.

[0150] In some embodiments, the immune cell is a hematopoietic stem cell or a pluripotent stem cell. In some embodiments, the immune cell is a T cell, a B cell or a NK cell. In some embodiments, the immune cell is a modified T cell (e.g., SPPL3-KO T cell, CAR-T or SPPL3-KO CAR-T), a modified B cell (e.g., SPPL3-KO B cell) or a modified NK cell (e.g., SPPL3-KO NK cell, CAR-NK or SPPL3-KO CAR-NK).

[0151] In some embodiments, the immune cell is a T cell, such as a modified T cell, such as a CAR-T cell. In some embodiments, the T cell is selected from helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, NKT cells, mucosal-associated invariant T (MAIT) cells, double negative T (DNT) cells and γδT cells. In some embodiments, the T cell is a naive T cell, a memory stem T cell, a central memory T cell, an effector memory T cell, an effector T cell, a Th1 cell, a Tc1 cell, a Th2 cell, a Tc2 cell, a Th3 cell, a Th9 cell, a Th17 cell, a Th22 cell or a T (regulatory) Treg cell.

[0152] DNT cells, also known as CD3 + CD4 - CD8 - T cells, or TCRαβ + CD4 - CD8 - T cells, which also lack iNKT cell markers (such as CD56), are a subset of mature T lymphocytes. + or CD8 +Compared with T cells, DNT cells have lower Fas expression; however, FasL expression was found to be higher in DNT cells than in other T cell populations (K. Okamura et al. “The potential target of double-negative T cells in cancer immunotherapy,” conference abstract e15180 | 2020 ASCO Annual Meeting I).

[0153] γδ T cells are usually double negative (CD4 - CD8 - γδ T cells do not require antigen processing or major histocompatibility complex (MHC) molecules for epitope presentation. Some γδ T cells recognize MHC class Ib molecules or butyrophilin molecules. Due to their non-MHC-restricted antigen recognition and high cytokine (e.g., IL-17, IFN-γ) secretion, γδ T cells are suitable for effective cancer treatment.

[0154] For examples and sources of immune cells, see the “Isolation and Culture of Immune Cells” subsection below.

[0155] Pharmaceutical composition

[0156] Also provided are pharmaceutical compositions comprising any of the immune cells described herein (e.g., SPPL3-KO immune cells) and, optionally, a pharmaceutically acceptable excipient. The pharmaceutical composition can be prepared by mixing any of the modified immune cells described herein with a suitable pharmaceutically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) in the form of a lyophilized formulation or an aqueous solution.

[0157] Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers, antioxidants including ascorbic acid, methionine, vitamin E, sodium metabisulfite; preservatives, isotonicity agents, stabilizers, metal complexes (such as zinc protein complexes); chelating agents such as EDTA and / or nonionic surfactants.

[0158] Buffers are used to control the pH within a range that optimizes the therapeutic effect, particularly in situations where stability is dependent on pH. Suitable buffers for use in the present invention include organic and inorganic acids and their salts. For example, citrates, phosphates, succinates, tartrates, fumarates, gluconates, oxalates, lactates, acetates. Additionally, buffers may include histidine and trimethylamine salts, such as Tris.

[0159] Preservatives are added to inhibit microbial growth, typically in an amount of 0.2% to 1.0% (w / v). Suitable preservatives for use in the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

[0160] Tonicity agents, sometimes referred to as "stabilizers," are used to adjust or maintain the tonicity of the liquid in the composition. Taking into account the relative amounts of the other ingredients, the tonicity agent may be present in any amount from 0.1% to 25% by weight, preferably from 1% to 5% by weight. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

[0161] Suitable nonionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), poloxamer (184, 188, etc.), polyols, Polyoxyethylene sorbitan monoether ( TWEEN TM -80, etc.), laurin 400, polyoxyethylene 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50, and 60, glyceryl monostearate, sucrose fatty acid esters, methylcellulose, and carboxymethylcellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyl sodium sulfosuccinate, and sodium octyl sulfosuccinate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

[0162] In order for pharmaceutical compositions to be used for in vivo administration, they must be sterile.The pharmaceutical compositions herein are typically placed in a container having a sterile access port, for example, an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle.

[0163] Activation-induced cell death (AICD)

[0164] AICD is a programmed cell death caused by the interaction of Fas receptors (such as Fas, CD95) with Fas ligands (such as FasL, CD95 ligand), which helps maintain peripheral immune tolerance and / or kill tumor cells. AICD effector cells (e.g., T cells and NK cells) express FasL and induce apoptosis in cells expressing Fas receptors (e.g., tumor cells, immune cells such as T cells, B cells, and NK cells). AICD is a negative regulator of activated T lymphocytes caused by repeated stimulation of their T cell receptors (TCRs). Alterations in this process can lead to autoimmune diseases (Zhang J, et al. (2004) Cell Mol Immunol. 1(3):186-92).

[0165] Mechanistically, the binding of Fas ligand to the Fas receptor triggers the trimerization of the Fas receptor, which then binds to the death domain of the adaptor protein FADD (Fas-associated protein with a death domain). Procaspase 8 binds to the death effector domain of FADD and is proteolytically activated to form a signal transduction complex that induces death. Activated caspase 8 is released into the cytosol, where it activates the caspase cascade that initiates apoptosis (Nagata S. (1997) Cell. 88(3):355-65s).

[0166] The balance of proliferation and death induced by effector cell activation is the key to the steady-state expansion of immune cells. AICD is extremely important for the development of lymphocytes and can remove autoreactive immature lymphocytes. Antigen receptors strongly bind to self-antigens and produce immature lymphocytes with strong activation signals that are eliminated by apoptosis (DR Green et al., "Activation-Induced Apoptosis in Lymphoid Systems," Sem. Immunol. 4: 379-388 (1992)). When lymphocytes mature, productive activation requires not only the primary stimulation of cell antigen receptors, but also co-stimulatory signals. In T cells, the combination of CD28 and its B7 family ligands plays this co-stimulatory role, and in B cells, co-stimulation occurs by the combination of CD40 and its ligand gp39 (EA Clark & ​​J.A. Ledbetter, "How B and T Cells Talk to Each Other," Nature 367: 425-428 (1994)). In the absence of co-stimulation, lymphocytes that receive antigen receptor stimulation alone become anergic or inactivated, and their growth is inhibited (EA Clark & ​​J.A. Ledbetter (1994), supra).

[0167] Because activated immune cells (e.g., T cells, B cells, and NK cells) express Fas in addition to FasL, they can be killed by themselves or each other that express FasL. See, for example, Huan et al., Hum Cell. 2022; 35(2): 441-447. Therefore, AICD leads to poor persistence of therapeutic immune cells (such as CAR-T, CAR-NK) in the body and their limitations in clinical applications.

[0168] SPPL3

[0169] Signal peptide peptidase-like 3 (SPPL3; also known as IMP2, PSH1 or PSL4) is a multiluminal protein located in the membranes of Golgi-associated vesicles, the plasma membrane and the rough endoplasmic reticulum. SPPL3 has aspartic endopeptidase activity, intramembrane cleavage and protein homodimerization activity. Many of its substrates are localized in the Golgi apparatus and are involved in N- and O-linked glycan modification and glycosaminoglycan biosynthesis. SPPL3 is essential for the cleavage and extracellular release of the lumenal domains of glycosyltransferases and glycosidases. Shedding of glycan-modifying enzymes impairs their activity in the Golgi apparatus. Increased SPPL3 expression is associated with decreased glycosylation of many secretory and membrane proteins; decreased SPPL3 expression is associated with highly glycosylated proteins. See also Mentrup et al., “Latest emerging functions of SPP / SPPL intramembrane proteases,” Eur J Cell Biol. 2017; 96(5): 372-382, the contents of which are incorporated herein by reference in their entirety. In the absence of SPPL3, researchers noted upregulation of the neolactose series of glycosphingolipids (GSLs) on the cell surface, which in turn sterically hindered the interaction of antibodies and receptors with HLA class I (HLA-I) glycoproteins and reduced CD8+ T cell activation (Jongsma et al., Immunity. 2021;54(1):132-150.e9). SPPL3 also has non-proteolytic functions, including interaction with stromal interaction molecule 1 (STIM1) and Orai1, enhancing TCR signaling to greatly induce calcium influx and NFAT activation, which is critical for lymphocyte signaling (Mentrup et al., supra).

[0170] In some embodiments, the SPPL3 protein is a human SPPL3 protein. In some embodiments, the human SPPL3 protein comprises the sequence of SEQ ID NO:4.

[0171] SEQ ID NO:4

[0172] MAEQTYSWAYSLVDSSQVSTFLISILLIVYGSFRSLNMDFENQDKEKDSNSSSGSFNGNSTNNSIQTIDSTQALFLPIGASVSLLVMFFFFDSVQVVFTICTAVLATIAFAFLLLPMCQYLTRPCSPQNKISFGCCGRFTAAELLSFSLSVLVLIWVLTGHWLLMDALAMGLCVAMIAFVRLPSLKVSCLL LSGLLIYDVFWVFFSAYIFNSNVMVKVATQPADNPLDVLSRKLHLGPNVGRDVPRLSLPGKLVFPSSTGSHFSMLGIGDIVMPGLLLCFVLRYDNYKKQASGDSCGAPGPANISGRMQKVSYFHCTLIGYFVGLLTATVASRIHRAAQPALLYLVPFTLLPLLTMAYLKGDLRRMWSEPFHSKSSSSRFLEV

[0173] III. Methods for Producing Modified Immune Cells

[0174] One aspect of the present invention provides a method for producing any immune cell described herein, for example, an immune cell (e.g., CAR-T or CAR-NK cell) modified to reduce or eliminate the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR protein). In some embodiments, the method for producing a modified immune cell comprises inactivating or reducing (e.g., reducing at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) the expression (RNA and / or protein expression) of one or more target genes identified by any target gene identification method described herein, for example, SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B (e.g., SPPL3) in an immune cell (e.g., an AICD / FasL resistance gene). In some embodiments, the method of producing a modified immune cell comprises expressing an engineered receptor (e.g., CAR) in an immune cell (e.g., a primary immune cell, or an immune cell modified to reduce or eliminate the expression and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR)).

[0175] In some embodiments, a method is provided for: i) extending (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of an immune cell, ii) reducing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the AICD of an immune cell (or increasing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) resistance to AICD), and / or iii ) reduces (e.g., reduces any one of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) the host versus graft (HvG) response of an immune cell (e.g., CAR-T or CAR-NK cell), comprising modifying the immune cell to reduce (e.g., reduces any one of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate the expression (RNA and / or protein expression) and / or function of one or more target proteins identified herein (e.g., AICD / FasL resistance gene-encoded protein), for example, selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B. In some embodiments, the method comprises modifying an immune cell to reduce or eliminate the expression and / or function of an SPPL3 protein.Thus, in some embodiments, methods are provided for: i) extending (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of an immune cell, ii) decreasing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the AICD of an immune cell (or increasing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 100% or more) the AICD of an immune cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is a T cell. In some embodiments, the immune cell is further modified to express a CAR. In some embodiments, immune cells (e.g., T cells such as CAR-T) are further modified to reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate the expression (RNA and / or protein expression) and / or function of the TCR protein.

[0176] In some embodiments, a method is provided for producing an immune cell (e.g., CAR-T or CAR-NK) having increased resistance to AICD (e.g., an increase of at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more), comprising inactivating an AICD / FasL resistance gene identified using any of the target gene screening methods described herein, e.g., a gene selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B, in a precursor immune cell. In some embodiments, a method for producing an immune cell (e.g., CAR-T or CAR-NK) is provided, wherein the resistance of the immune cell to AICD is increased (e.g., an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more), the method comprising inactivating the SPPL3 gene in the precursor immune cell. In some embodiments, the immune cell is a T cell, a B cell or a NK cell. In some embodiments, the immune cell is a T cell, for example, selected from helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, NKT cells, MAIT cells, DNT cells and γδT cells. In some embodiments, the precursor immune cell expresses an engineered receptor (e.g., CAR, TAC, engineered TCR). In some embodiments, the method further comprises introducing a nucleic acid encoding the engineered receptor into the precursor immune cell. In some embodiments, the engineered receptor is a CAR. In some embodiments, a method for producing an immune cell (e.g., a T cell) is provided, wherein the resistance of the immune cell to AICD is increased (e.g., an increase of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1 time, 2 times, 5 times, 10 times, 20 times, 50 times, 100 times or more), including inactivating the SPPL3 gene in the precursor immune cell expressing CAR (e.g., anti-CD19 CAR). In some embodiments, the method further includes inactivating TCR genes (e.g., one or more of TCRα, TCRβ, TCRγ, TCRδ) in the precursor immune cell.

[0177] In some embodiments, the method for producing a modified immune cell comprises generating one or more mutations (e.g., inactivating mutations) at one or more target genes identified by any target gene identification method described herein, for example, at one or more target genes encoding one or more target proteins described herein (e.g., SPPL3 protein) (e.g., AICD / FasL resistance gene). In some embodiments, the method comprises contacting an initial population of immune cells (e.g., CAR-T cells or CAR-NK cells) with a mutagen and selecting modified immune cells comprising one or more mutations (e.g., inactivating mutations) at one or more target genes identified herein, for example, encoding one or more target proteins described herein (e.g., SPPL3 protein). In some embodiments, the method comprises generating one or more mutations (e.g., inactivating mutations) at one or more target genes identified herein, for example, encoding one or more target proteins described herein (e.g., SPPL3 protein) in precursor immune cells (e.g., CAR-T or CAR-NK cells) by gene editing, such as any gene editing method known in the art or described herein. For example, non-homologous end joining (NHEJ) or HDR (e.g., homologous recombination)-mediated genetic modification or destruction, or ZFN-, TALEN-, or CRISPR / Cas-mediated genetic modification or destruction. Methods for detecting such mutations are well known in the art, such as by PCR. In some embodiments, the method includes generating one or more mutations (e.g., inactivating mutations) in one or more target gene products identified herein, such as encoding one or more target proteins described herein (e.g., SPPL3 proteins), or one or more target proteins described herein, by RNA editing (e.g., LEAPER, REPAIR) or protein editing in precursor immune cells.

[0178] In some embodiments, the method of generating a modified immune cell further comprises modifying the immune cell (e.g., a target protein modified immune cell) to reduce (e.g., reduce at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate (RNA and / or protein expression) and / or function of other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA -C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, SLLP3, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and NKp46 ligands. In some embodiments, the method further comprises modifying the immune cell to reduce or eliminate the expression and / or function of one or more other proteins selected from Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B. In some embodiments, the method further comprises modifying immune cells to reduce or eliminate the expression and / or function of one or more other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46. In some embodiments, the method further comprises modifying immune cells to reduce or eliminate the expression and / or function of TCR proteins (e.g., one or more of TCRα, TCRβ, TCRγ, and TCRδ).

[0179] In some embodiments, the expression of one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs) is reduced or inhibited by antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs). In some embodiments, the method for producing a modified immune cell comprises introducing a nucleic acid (e.g., a vector) encoding an antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs) into a precursor immune cell (e.g., a CAR-T or CAR-NK cell). In some embodiments, the method comprises introducing an antisense RNA, siRNA, or shRNA that specifically recognizes RNA encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs) into an immune cell (e.g., a CAR-T or CAR-NK cell).

[0180] In some embodiments, the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is reduced or inhibited by small molecule compounds, nucleic acids (or vectors comprising the same), lipids, and / or protein molecules (e.g., dominant negative binding partners of target proteins or other proteins). In some embodiments, the method for producing modified immune cells comprises introducing or contacting nucleic acids (or vectors comprising the same), lipids, and / or protein molecules (or nucleic acids encoding protein molecules) into precursor immune cells (e.g., CAR-T or CAR-NK cells). In some embodiments, the method comprises contacting precursor immune cells (e.g., CAR-T or CAR-NK cells) with small molecule compounds. In some embodiments, the contact is in vivo, in vitro, or ex vivo. In some embodiments, in vivo contact of immune cells is by administering small molecule compounds, nucleic acids (or vectors comprising the same), lipids, and / or protein molecules to individuals (e.g., humans) having immune cells. Any suitable route of administration may be used herein, including but not limited to intravenous, subcutaneous, intratumoral, intramuscular, or oral administration.

[0181] In some embodiments, the function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is reduced or inhibited by chemically modified mRNA, such as a chemically modified mRNA encoding a dominant negative inhibitor (e.g., a dominant negative variant or fragment thereof, or a dominant negative binding partner thereof) of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR). In some embodiments, the method of generating a modified immune cell comprises introducing the chemically modified mRNA into a precursor immune cell (e.g., CAR-T or CAR-NK cell).

[0182] In some embodiments, immune cells are modified to express dominant negative variants (e.g., catalytically inactive, binding inactive, activity-deficient, or inactive) of one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs) or dominant negative fragments thereof. In some embodiments, immune cells are modified to express dominant negative binding partners or fragments thereof of one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs). In some embodiments, the method of producing modified immune cells comprises introducing nucleic acids (e.g., vectors or RNA) encoding dominant negative variants or fragments thereof of one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs), or dominant negative binding partners or fragments thereof into precursor immune cells (e.g., CAR-T or CAR-NK cells). In some embodiments, the DNA loci or RNA encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs) in immune cells are modified (e.g., by gene editing or RNA editing, or mutagen) to express dominant negative variants or dominant negative fragments thereof.

[0183] In some embodiments, immune cells are genetically modified at a DNA locus encoding one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR). In some embodiments, immune cells are genetically modified at the SPPL3 locus. In some embodiments, the DNA locus (e.g., SPPL3 locus) is modified with a mutagen. Mutagens can be divided into three categories: physical (e.g., gamma irradiation, ultraviolet light), chemical (e.g., ethyl methanesulfonate or EMS), and transposable elements (e.g., transposons, retrotransposons, T-DNA, retroviruses). In some embodiments, the method of generating modified immune cells comprises contacting a precursor immune cell (e.g., CAR-T or CAR-NK cell) with a mutagen to reduce or eliminate the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR). In some embodiments, the DNA loci encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCR) are modified by gene editing, including but not limited to NHEJ, HDR, ZFN, TALEN, or CRISPR / Cas-mediated gene editing. In some embodiments, gene editing includes gene KO. In some embodiments, gene editing includes base editing (e.g., introducing non-synonymous substitutions). In some embodiments, base editing is mediated by CRISPR / Cas, for example, by fusing ADE or adenine deaminase (ADA), or CBE or cytidine deaminase (CDA), or a functional fragment thereof to a dead Cas (e.g., dCas9) protein. In some embodiments, the method includes introducing one or more nucleic acids (e.g., vectors or RNA) of ZFN, TALEN, or CRISPR / Cas systems encoding target sequences that specifically recognize genes encoding one or more target proteins (e.g., SPPL3) and / or other proteins (e.g., TCR) into precursor immune cells (e.g., CAR-T or CAR-NK cells). In some embodiments, two or more nucleic acids encoding the different components of ZFN, TALEN or CRISPR / Cas systems are introduced into immune cells simultaneously. In some embodiments, two or more nucleic acids encoding the different components of ZFN, TALEN or CRISPR / Cas systems are introduced into immune cells in sequence. In some embodiments, nucleic acid templates (for example, with desired HDR modification and homology arms) and ZFN, TALEN or CRISPR / Cas systems are further introduced into immune cells in sequence or simultaneously. In some embodiments, the method for producing modified immune cells also includes introducing one or more nucleic acids encoding CAR into precursor immune cells or modified immune cells (for example, SPPL3 modified immune cells).

[0184] In some embodiments, immune cells are genetically modified at RNA encoding one or more target proteins (e.g., SPPL3 proteins) and / or other proteins (e.g., TCR), for example, by RNA editing (e.g., LEAPER, REPAIR, RESTORE, CIRTS, RESCUE, CLUSTER). In some embodiments, RNA editing includes base editing (e.g., introducing non-synonymous substitutions). In some embodiments, RNA editing is mediated by CRISPR / Cas, for example, by fusing adenine deaminase (ADA) or cytidine deaminase (CDA) or its functional fragment to a dead Cas (e.g., dCas13a) protein. In some embodiments, the method for producing modified immune cells includes introducing one or more nucleic acids (e.g., vectors or RNA) of a CRISPR / Cas or LEAPER system encoding a target sequence of RNA specifically encoding one or more target proteins (e.g., SPPL3) and / or other proteins (e.g., TCR) into a precursor immune cell (e.g., CAR-T or CAR-NK cell). In some embodiments, two or more nucleic acids encoding different components of the CRISPR / Cas system are simultaneously introduced into immune cells. In some embodiments, two or more nucleic acids encoding different components of the CRISPR / Cas system are sequentially introduced into immune cells. In some embodiments, the method for producing modified immune cells includes introducing adenosine deaminase recruiting RNA (arRNA) constructs into precursor immune cells (eg, CAR-T or CAR-NK cells), the constructs comprising or encoding arRNA for editing target sequences of RNA encoding one or more target proteins (eg, SPPL3) and / or other proteins (eg, TCR), wherein the target sequence includes target adenosine (a), wherein the arRNA includes a complementary RNA sequence (eg, gRNA) hybridized with the target sequence of the RNA, and wherein the arRNA is capable of recruiting adenosine deaminase (ADAR) (eg, endogenous ADAR1) acting on RNA to deaminate the target A in the target sequence. In some embodiments, the method for producing modified immune cells also includes introducing one or more nucleic acids encoding CAR into precursor immune cells or modified immune cells (eg, SPPL3 modified immune cells).

[0185] In some embodiments, a method of generating an immune cell (e.g., a CAR-T or CAR-NK cell) that is modified to reduce (e.g., reduce at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate the expression and / or function of one or more target proteins identified herein (AICD / FasL resistance gene encoded protein) or selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is provided, including allowing the introduction of a gRNA construct and optionally a Cas component into a precursor immune cell. The precursor immune cell (e.g., a precursor T cell or a precursor NK cell) is contacted with i) a gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding a gRNA; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9) under conditions that are suitable for the production of an immune cell. The gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary) to a target site in a DNA locus or RNA encoding one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR).In some embodiments, methods are provided for: i) extending (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of immune cells; ii) reducing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the AICD of immune cells; or increasing (e.g., by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% the AICD of immune cells. For example, increasing resistance to AICD by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more, and / or iii) reducing (e.g., reducing any of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) the HvG response of immune cells (e.g., CAR-T or CAR-NK cells), including in allowing the gRNA construct to be The precursor immune cell (e.g., a precursor T cell or a precursor NK cell) is contacted with i) a gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding a gRNA) under conditions in which the gRNA construct and optionally a Cas component are introduced into the precursor immune cell; and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9), wherein the gRNA construct comprises or encodes a gRNA that comprises complementarity (e.g., at least about 60%, 70%, 80%, 90%, or more) with a target site in a DNA locus. , 95%, 96%, 97%, 98%, 99% or 100% complementary to a guide sequence, or RNA encoding one or more target proteins identified herein (e.g., AICD / FasL resistance gene encoding protein) or selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B (e.g., SPPL3 protein), thereby reducing or eliminating the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein). In some embodiments, the precursor immune cell already expresses the Cas protein before the introduction of the gRNA construct. In some embodiments, the precursor immune cell does not express the Cas protein before the introduction of the gRNA construct and the Cas component. In some embodiments, the method further comprises introducing a vector (e.g., a viral vector, such as a lentiviral vector) carrying a nucleic acid encoding a Cas protein (e.g., Cas9) or Cas (e.g., Cas9) mRNA into the precursor immune cell or the precursor immune cell comprising the gRNA construct.In some embodiments, the method further includes introducing a nucleic acid template (for example, including mutations) into immune cells for modifying the target site of the DNA locus encoding one or more target proteins (for example, SPPL3 proteins) and / or other proteins (for example, TCR) by HDR. In some embodiments, the Cas protein has endonuclease activity. In some embodiments, the Cas protein is a fusion protein, for example, a fusion protein comprising i) dCas (for example, dCas13, dCas9) and ii) adenine base editor (ABE) or ADA (for example, TadA, for example TadA8e), or cytidine base editor (CBE) or CDA, or a functional fragment thereof. In some embodiments, the Cas protein is a dCas13-ADAR fusion protein, for example, dCas13a-ADAR1, dCas13a-ADAR2, dCas13b-ADAR1, dCas13b-ADAR2 and a functional variant thereof capable of targeted RNA editing (for example, C to U, A to I). In some embodiments, the Cas protein is Cas9, for example, dCas9. In some embodiments, the Cas protein is a dCas9-ADA (e.g., TadA) or dCas9-CDA fusion protein. In some embodiments, the precursor immune cells (e.g., before modification of the target protein) have reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression (RNA and / or protein expression) and / or function of one or more other proteins selected from the following or do not have expression (RNA and / or protein expression) and / or function of one or more other proteins selected from the following: TCRα, TCRβ, TCRγ, TCRδ, HL A-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, SPPL3, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and NKp46 ligands. In some embodiments, the precursor immune cells (e.g., target protein modified immune cells) are further modified, for example, at one or more loci or RNA encoding one or more other proteins to reduce or eliminate the expression and / or function of one or more other proteins (e.g., TCR).In some embodiments, the method further comprises contacting the precursor immune cell (e.g., target protein modified immune cell) with i) gRNA construct; and optionally ii) Cas components comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9), under conditions allowing the introduction of a gRNA construct and optional Cas components into the precursor immune cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to the target site in the DNA locus or an RNA encoding one or more other proteins (e.g., Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B), thereby reducing or eliminating the expression (RNA and / or protein expression) and / or function of one or more other proteins. In some embodiments, the precursor immune cell (e.g., target protein modified immune cell) already has a Cas component, and only a gRNA construct targeting one or more other proteins is further introduced. In some embodiments, RNA encoding one or more target proteins (e.g., SPPL3 protein) and / or other proteins is modified by LEAPER. Thus, in some embodiments, a method is provided for producing an immune cell (e.g., a CAR-T or CAR-NK cell) that is modified to reduce (e.g., reduce by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate the expression (functional protein expression) and / or function of one or more target proteins identified herein or selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B (e.g., SPPL3 protein) and / or other proteins (e.g., TCR), comprising: introducing an arRNA construct into a precursor immune cell (e.g., a precursor immune cell) under conditions that allow for the introduction of an arRNA construct into the precursor immune cell. T cells or precursor NK cells) are contacted with an arRNA construct (e.g., vector, RNA) comprising or encoding an arRNA for editing an RNA encoding one or more target proteins (e.g., SPPL3) and / or other proteins (e.g., TCR), wherein the target sequence comprises a target adenosine (A), wherein the arRNA comprises a complementary (e.g., any of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary) RNA sequence (e.g., gRNA) that hybridizes to the target sequence of the RNA, and wherein the arRNA is capable of recruiting an ADAR (e.g., endogenous ADAR1) to deaminate the target A in the target sequence, wherein deamination of the target A in the target sequence reduces or eliminates expression and / or function of the one or more target proteins and / or other proteins.In some embodiments, the following methods are provided: i) extending (e.g., extending by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of immune cells, ii) reducing (e.g., reducing by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) the AI ​​of immune cells 100% or more) of the immune cells (e.g., CAR-T or CAR-NK cells) The method comprises contacting a precursor immune cell (e.g., a precursor T cell or a precursor NK cell) with an arRNA construct (e.g., a vector, RNA) comprising or encoding an arRNA under conditions that allow for introduction of the arRNA construct into the precursor immune cell for editing a target RNA encoding one or more target proteins identified herein or selected from the group consisting of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B (e.g., SPPL3 protein). Sequence, wherein the target sequence comprises a target adenosine (A), wherein the arRNA comprises a complementary (e.g., at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or any one of 100% complementary) RNA sequence (e.g., gRNA) that hybridizes to the target sequence of the RNA, and wherein the arRNA is capable of recruiting ADAR (e.g., endogenous ADAR1) to deaminate the target A in the target sequence, wherein the deamination of the target A in the target sequence reduces or eliminates the expression and / or function of one or more target proteins. In some embodiments, the precursor immune cell expresses an engineered receptor (e.g., CAR, engineered TCR, or TAC) prior to the introduction of a gRNA construct and / or Cas component or arRNA construct. In some embodiments, the method further comprises introducing a nucleic acid encoding the engineered receptor into a precursor immune cell (e.g., a target protein-modified immune cell). In some embodiments, the engineered receptor is a CAR comprising: i) an extracellular antigen binding domain (e.g., sdAb, scFv, Fab) that specifically recognizes one or more target antigens (e.g., tumor antigens) or target epitopes, ii) a transmembrane domain; and iii) an intracellular signaling domain.In some embodiments, the method for modifying one or more target proteins and / or other proteins is the same, for example, all by CRISPR-Cas mediated gene editing.In some embodiments, the method for modifying one or more target proteins and / or other proteins is different, for example, some by CRISPR-Cas mediated gene editing, some by LEAPER mediated RNA editing.In some embodiments, gRNA constructs or arRNA constructs targeting one or more target proteins (such as SPPL3 protein), targeting one or more other proteins (such as Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, one or more of HIST1H1B), Cas components, and / or nucleic acids encoding engineered receptors (for example, CAR) are simultaneously introduced into precursor immune cells. In some embodiments, gRNA constructs or arRNA constructs targeting one or more target proteins (e.g., SPPL3 protein), targeting one or more other proteins (e.g., one or more of Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B), Cas components, and / or nucleic acids encoding engineered receptors (e.g., CAR) are introduced into precursor immune cells sequentially, for example, i) first the gRNA constructs targeting one or more target proteins and / or other proteins + Cas components, followed by nucleic acids encoding engineered receptors; ii) first the Cas components, followed by gRNA constructs targeting one or more target proteins and / or other proteins, followed by nucleic acids encoding engineered receptors; or iii) first the nucleic acids encoding engineered receptors, followed by gRNA constructs targeting one or more target proteins and / or other proteins + Cas components, etc. In some embodiments, nucleic acids encoding engineered receptors, nucleic acids encoding gRNAs or arRNAs for one or more target proteins (e.g., SPPL3 proteins), nucleic acids encoding gRNAs or arRNAs for one or more other proteins, and / or nucleic acids encoding Cas proteins are on different vectors. In some embodiments, nucleic acids encoding engineered receptors, nucleic acids encoding gRNAs or arRNAs for one or more target proteins (e.g., SPPL3 proteins), nucleic acids encoding gRNAs or arRNAs for one or more other proteins, and / or nucleic acids encoding Cas proteins are on the same vector under the control of the same promoter or under the control of a separate promoter. In some embodiments, nucleic acids encoding engineered receptors, nucleic acids encoding gRNAs or arRNAs for one or more target proteins (e.g., SPPL3 proteins), nucleic acids encoding gRNAs or arRNAs for one or more other proteins, and / or nucleic acids encoding Cas proteins are connected by one or more IRES linkers and are under the control of the same promoter.In some embodiments, the modification of the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins is reduced or eliminated without downregulating or eliminating the expression (ribonucleic acid and / or protein expression) and / or function of the engineered receptor (e.g., CAR). In some embodiments, the modification of the expression and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is reduced or eliminated by downregulating the expression (RNA and / or protein expression) and / or function of the engineered receptor by up to about 30% (e.g., up to about 25%, 20%, 15%, 10%, 5%, 1% or any one of less). In some embodiments, the immune cell is a T cell, a B cell, or a NK cell. In some embodiments, the immune cell is a T cell, for example, selected from helper CD4+T cells, cytotoxic CD8+T cells, memory T cells, regulatory CD4+T cells, NKT cells, MAIT cells, DNT cells, and γδT cells. In some embodiments, the modification of expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is reduced or eliminated by: i) at least about 10% reduction (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100%) of one or more selected from Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, UL BP3, ULBP4, ULBP5, ULBP6, and NKp46; ii) reducing killing by allogeneic T cells by at least about 10% (e.g., by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%); and / or iii) reducing killing by autologous or allogeneic NK cells by at least about 10% (e.g., by at least about any of 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%).In some embodiments, the modification that reduces or eliminates the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) is i) prolonged compared to a reference immune cell (e.g., a precursor immune cell, such as a CAR-T or CAR-NK cell) that does not have the modification that reduces or eliminates the expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR). In some embodiments, the guide sequence is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO: 1.

[0186] In some embodiments, a method is provided for producing a T cell (e.g., a CAR-T cell, e.g., an anti-CD19 CAR-T cell) modified to reduce (e.g., reduce at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate expression (RNA and / or protein expression) and / or function of an SPPL3 protein, comprising contacting a precursor T cell (e.g., a precursor CAR-T cell) with a) a gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding the gRNA; and optionally b) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9) under conditions that allow introduction of the gRNA construct and, optionally, a Cas component into the precursor T cell. In some embodiments, a method for producing a T cell (e.g., a CAR-T cell, e.g., an anti-CD19 A method of producing a CAR-T cell modified to reduce (e.g., reduce at least about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminate expression (RNA and / or protein expression) and / or function of an SPPL3 protein and a TCR protein, comprising contacting the precursor T cell (e.g., a precursor CAR-T cell) with a) a first gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding the gRNA); b) a second gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding the gRNA); and optionally c) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9) under conditions that permit introduction of the gRNA construct and, optionally, the Cas component into the precursor T cell. In some embodiments, the present invention provides a guide sequence that is complementary (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a DNA locus, wherein the second gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary (e.g., at least about any of 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a TCR DNA locus.In some embodiments, methods are provided for i) extending the in vivo persistence of T cells (e.g., CAR-T cells, e.g., anti-CD19 CAR-T cells), ii) reducing the proliferation of T cells (e.g., CAR-T cells, e.g., anti-CD19 A method of iii) reducing AICD (or increasing resistance to AICD) in a T cell (e.g., a CAR-T cell, e.g., an anti-CD19 CAR-T cell), and / or iii) reducing an HvG response in a T cell (e.g., a CAR-T cell, e.g., an anti-CD19 CAR-T cell), comprising contacting a precursor T cell (e.g., a precursor CAR-T cell) with a) a gRNA construct (e.g., a gRNA or a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding the gRNA; and optionally b) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9) under conditions that allow introduction of the gRNA construct and, optionally, the Cas component into the precursor T cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary (e.g., any of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary) to a target site in the SPPL3 DNA locus. In some embodiments, i) extending the in vivo persistence of T cells (e.g., CAR-T cells, e.g., anti-CD19 CAR-T cells), ii) reducing AICD (or increasing resistance to AICD) of T cells (e.g., CAR-T cells, e.g., anti-CD19 CAR-T cells), and / or iii) reducing T cells (e.g., CAR-T cells, e.g., anti-CD19 The method comprises contacting a precursor T cell (e.g., a precursor CAR-T cell) with a) a first gRNA construct (e.g., a gRNA or a vector carrying a nucleic acid encoding a gRNA (e.g., a viral vector such as a lentiviral vector)) under conditions that allow for introduction of a gRNA construct and, optionally, a Cas component into the precursor T cell; b) a second gRNA construct (e.g., a gRNA or a vector carrying a nucleic acid encoding a gRNA (e.g., a viral vector such as a lentiviral vector)); and optionally c) a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., Cas9), wherein the first gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary (e.g., any of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a SPPL3 DNA locus, wherein the second gRNA construct comprises or encodes a gRNA comprising a guide sequence that is complementary to a target site in a TCR A guide sequence that is complementary (e.g., any of at least about 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% complementary) to a target site in a DNA locus.In some embodiments, the first gRNA construct (e.g., DNA) encoding the first gRNA and the second gRNA construct (e.g., DNA) encoding the second gRNA are on 2 vectors. In some embodiments, the first gRNA construct (e.g., DNA) encoding the first gRNA and the second gRNA construct (e.g., DNA) encoding the second gRNA are on the same vector. In some embodiments, the precursor CAR-T cell has expressed Cas protein before the introduction of gRNA construct (one or more). In some embodiments, the precursor CAR-T cell does not express Cas protein before the introduction of gRNA construct (one or more) and Cas components. In some embodiments, the method further includes introducing a vector (e.g., a viral vector, such as a lentiviral vector) carrying nucleic acid encoding Cas protein (e.g., Cas9) or Cas (e.g., Cas 9) mRNA into the precursor T cell or the precursor T cell comprising the gRNA construct (one or more). In some embodiments, the nucleic acid encoding the Cas protein and the gRNA construct (one or more) encoding the gRNA are on different vectors. In some embodiments, the nucleic acid encoding the Cas protein and the gRNA construct (one or more) encoding the gRNA are on the same vector. In some embodiments, Cas components and gRNA constructs (one or more) are introduced at the same time. In some embodiments, Cas components and gRNA constructs (one or more) are introduced in sequence. In some embodiments, the guide sequence targeting SPPL3 is encoded by a nucleic acid sequence comprising a sequence of SEQ ID NO: 1. In some embodiments, the precursor T cell is a CAR-T cell, i.e., CAR has been expressed before the introduction of gRNA constructs (one or more) and / or Cas components. In some embodiments, the precursor T cell does not express CAR before the introduction of gRNA constructs (one or more) and / or Cas components. In some embodiments, the method further comprises introducing a vector (e.g., a viral vector such as a lentiviral vector) carrying a nucleic acid encoding CAR or an mRNA encoding CAR into the precursor T cell or a precursor T cell comprising the gRNA construct (one or more) and / or the Cas component. In some embodiments, nucleic acid encoding CAR (one or more), Cas components and / or gRNA constructs (one or more) are introduced simultaneously (e.g., on the same vector or different vectors). In some embodiments, nucleic acid encoding CAR (one or more), Cas components and / or gRNA constructs (one or more) are introduced in sequence.

[0187] In some embodiments, when a population of precursor immune cells (e.g., CAR-T or CAR-NK cells) is used to produce a modified immune cell as described herein, the method further comprises one or more separation and / or enrichment steps, for example, isolating and / or enriching immune cells comprising one or more mutations (e.g., inactivating mutations) in a target gene or target RNA, gRNA construct or arRNA construct, Cas component, and / or engineered component (e.g., CAR) from an immune cell population contacted with any modifier described herein, or having reduced expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR) or lacking expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins (e.g., TCR). Such separation and / or enrichment steps can be performed using any known technique in the art and described herein, such as FACS or magnetic activated cell sorting (MACS), or a reporter gene based on a target gene (e.g., by expressing or not expressing a reporter gene of a target gene). See also the methods described in the subsection "Optional Enrichment Step" below.

[0188] In some embodiments, the method further comprises selecting from the population of modified immune cells a population of modified immune cells that: i) has reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) expression and / or function of one or more target proteins described or identified herein (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs), or lacks expression and / or function of one or more target proteins described or identified herein (e.g., SPPL3 proteins) and / or other proteins (e.g., TCRs); and / or ii) does not downregulate or downregulates by at most about 30% (e.g., by at most about 25%, 20%, 10%, 5%, 1% or less) an engineered receptor expressed in the modified immune cells.

[0189] In some embodiments, the engineered receptor (e.g., CAR), gRNA construct or arRNA construct and / or Cas components are introduced into the precursor immune cell by transduction / transfection of nucleic acid (DNA or RNA) or a vector encoding the same (e.g., a non-viral vector or a viral vector such as a lentiviral vector) or a virus (e.g., a lentivirus) comprising the nucleic acid encoding the same. In some embodiments, the engineered receptor (e.g., CAR), gRNA construct or arRNA construct and / or Cas components are introduced into the precursor immune cell by inserting the protein into the cell membrane while passing the cell through a microfluidic system, such as a CELL Cas components (e.g., Cas9 protein) are introduced into precursor immune cells (see, e.g., U.S. Patent Application Publication No. 20140287509).

[0190] Methods for introducing vectors (such as viral vectors) or isolated nucleic acids into mammalian cells are known in the art. The nucleic acids or vectors described herein can be transferred to immune cells by physical, chemical or biological methods.

[0191] Physical methods for introducing vectors (e.g., viral vectors) into cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York. In some embodiments, vectors (e.g., viral vectors) are introduced into cells by electroporation.

[0192] Biological methods for introducing vectors into cells include the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian (e.g., human) cells.

[0193] Chemical methods for introducing vectors (e.g., viral vectors) into cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as an in vitro delivery vehicle is a liposome (e.g., an artificial membrane vesicle).

[0194] In some embodiments, RNA molecules (e.g., gRNA, arRNA, or mRNA encoding Cas) can be prepared by conventional methods (e.g., in vitro transcription) and then introduced into immune cells by known methods such as mRNA electroporation. See, e.g., Rabinovich et al., Human Gene Therapy 17: 1027-1035.

[0195] In some embodiments, a viral vector (lentiviral vector) or virus (e.g., lentivirus) comprising a nucleic acid encoding any of the engineered receptors (e.g., CARs), gRNAs or arRNAs and / or Cas proteins described herein is contacted with a precursor immune cell at an MOI of at least about 1, e.g., at least about any of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 8, 9, or 10, e.g., at an MOI of about 3.

[0196] In some embodiments, the transduced / transfected immune cells are propagated ex vivo after introduction of the vector or isolated nucleic acid. In some embodiments, the transduced / transfected immune cells are cultured to propagate for at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, or 14 days, for example, 7 days. In some embodiments, the transduced / transfected immune cells are further evaluated or screened to select the desired modified immune cells described herein.

[0197] Reporter gene can be used for identifying the cell of potential transfection / transduction and for evaluating the function of regulatory sequence.Usually, reporter gene is not present in the gene of receptor organism or tissue or not expressed by receptor organism or tissue, and its coded polypeptide, the expression of this polypeptide is shown by some easily detectable characteristics such as enzymatic activity.The expression of reporter gene is measured at the appropriate time after DNA / RNA is introduced into the receptor cell.Suitable reporter gene can comprise the gene of coding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase or green fluorescent protein (GFP) gene (for example, Ui-Tei et al. FEBS Letters 479:79-82 (2000)).Suitable expression system is well-known, and can use known technology to prepare or commercially available.Antibiotic selection marker can also be used for identifying the cell of potential transfection / transduction.

[0198] Other methods for confirming the presence of any nucleic acid described herein (e.g., a gRNA construct or an arRNA construct) or the presence of a mutation (e.g., an inactivating mutation) in a target gene in a modified immune cell include, for example, molecular biological assays well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, PCR, DNA-seq, or RNA-seq; biochemical assays, such as detecting the presence or absence of a specific peptide by immunological methods (e.g., ELISA and Western blotting), fluorescence activated cell sorting (FACS), or magnetic activated cell sorting (MACS).

[0199] Also provided are modified immune cells produced by any of the immune cell production / modification methods described herein. In some embodiments, the modified immune cells are autologous. In some embodiments, the modified immune cells are allogeneic.

[0200] Source and culture of immune cells

[0201] Immune cells (e.g., T cells, NK cells, B cells) as described herein can be obtained from a variety of sources, including peripheral blood mononuclear cells (PBMCs), bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some embodiments, any number of immune cell lines available in the art can be used. In some embodiments, any number of techniques known to those skilled in the art, such as FICOLL TM Immune cells are obtained by separation from a unit of blood collected from a subject. In some embodiments, cells from the circulating blood of an individual are obtained by apheresis. Apheresis products typically contain lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets. In some embodiments, the cells collected by apheresis can be washed to remove the plasma portion, and the cells are placed in an appropriate buffer or culture medium for subsequent processing steps. In some embodiments, the cells are washed with phosphate buffered saline (PBS). In some embodiments, the wash solution lacks calcium and may lack magnesium, or may lack many (if not all) divalent cations. After washing, the cells can be resuspended in a variety of biocompatible buffers, for example, Ca-free 2+ , Mg-free 2+ Alternatively, the aliquot can be stripped of undesirable components and the cells resuspended directly in culture medium.

[0202] In some embodiments, immune cells are provided by umbilical cord blood banks, peripheral blood banks, or are derived from induced pluripotent stem cells (iPSCs), multipotent and pluripotent stem cells or human embryonic stem cells. In some embodiments, immune cells are derived from cell lines. In some embodiments, immune cells are obtained from xenogeneic sources, for example, from mice, rats, non-human primates and pigs. In some embodiments, immune cells are human cells. In some aspects, immune cells are primary cells, for example, directly separated from the subject and / or separated and frozen from the subject. In some embodiments, after blood collection, PBMCs are separated from donor blood samples, and then, for example, T cells, B cells or NK cells are separated from PBMCs using an immunomagnetic bead method. In some embodiments, cells include one or more subsets of immune cells. Those skilled in the art will recognize that multiple rounds of selection (for example, positive selection or negative selection) can also be used. For example, for T cells, it can be full T cell populations, CD4+ cells, CD8+ cells and subgroups thereof, such as those defined by function, activation state, maturity, differentiation potential, amplification, recirculation, positioning and / or lasting ability, antigen specificity, antigen receptor type, presence in a particular organ or chamber, marker or cytokine secretion profile and / or degree of differentiation. With regard to the subject to be treated, the cell can be allogeneic and / or autologous. In some cases, relative to one or more expected receptors, immune cells are allogeneic. In some cases, immune cells are suitable for transplantation, such as not inducing GvHD in the receptor. In some embodiments, immune cells are allogeneic CAR-T or CAR-NK cells. In some embodiments, immune cells (such as allogeneic T cells) are modified to express engineered receptors, such as CAR or engineered TCR. In some embodiments, T cells (such as allogeneic T cells) are modified to knock out endogenous TCR.

[0203] Among the subtypes and subsets of T cells and / or CD4+ and / or CD8+ T cells are naive T (T N ) cells, effector T cells (T EFF ), memory T cells and their subtypes, such as stem cell memory T (TSC M ), central memory T(TC M ), effect memory T(T EM ) or terminally differentiated effector memory T cells, tumor infiltrating lymphocytes (TIL), immature T cells, mature T cells, helper T cells, cytotoxic T cells (CTL), mucosal-associated invariant T (MAIT) cells, naturally occurring and adaptive regulatory T cells (Treg), helper T cells, such as TH1 cells, TH2 cells, TH3 cells, TH17 cells, TH9 cells, TH22 cells, follicular helper T cells, α / β T cells and δ / γ T cells.

[0204] Immune cells for stimulation can also be frozen after the washing step. Without wishing to be bound by theory, the freezing and subsequent thawing steps provide a more homogeneous product by removing granulocytes and, to a lesser extent, monocytes from the cell population. Following the washing steps to remove plasma and platelets, the cells can be suspended in a freezing solution. Many freezing solutions and parameters are known in the art and are useful herein. Any controlled freezing method can be used, as can immediate uncontrolled freezing at -20°C or in liquid nitrogen.

[0205] In some embodiments, cryopreserved cells are thawed and washed as described herein and allowed to rest at room temperature for one hour prior to activation.

[0206] It is also contemplated in this application that a blood sample or single collection product is collected from a subject some time before the amplified cells described herein are needed. In one embodiment, the blood sample or single collection product is taken from a generally healthy subject. In certain embodiments, the blood sample or single collection product is taken from a generally healthy subject who is at risk of developing the disease but has not yet developed the disease, and the target cells are separated and frozen for later use. In certain embodiments, the immune cells can be expanded, frozen, and used later. In certain embodiments, as described herein, a sample is collected from a patient shortly after a specific disease is diagnosed but before any treatment. In another embodiment, cells are isolated from a blood sample or single collection product from a subject before any number of related treatment modalities.

[0207] In some embodiments, immune cells are obtained directly from the patient after treatment. In this respect, it has been observed that after certain cancer treatments, particularly after treatment with drugs that damage the immune system, in the period when the patient is usually recovered from treatment, soon after treatment, the quality of the immune cells obtained is optimal or improves the ability of its in vitro expansion. Similarly, after using the methods described herein to perform ex vivo operation, these cells are in a preferred state for enhancing implantation and in vivo amplification. Therefore, within the scope of the present invention, it is contemplated that blood cells, including immune cells, can be collected in this recovery phase. In addition, in certain embodiments, mobilization and conditioning regimens can be used to produce conditions that are conducive to the repopulation, recycling, regeneration and / or amplification of specific cell types in the subject, particularly during the limited time window after treatment.

[0208] Activation and expansion of immune cells

[0209] In some embodiments, before genetic engineering or in association with genetic engineering, cells are hatched and / or cultured. The incubation step may include cultivating, stimulating, activating and / or breeding or amplifying. In some embodiments, compositions or cells are hatched in the presence of stimulating conditions or stimulants. These conditions include those designed to induce the proliferation, amplification, activation and / or survival of cells in the group, simulated antigen exposure and / or conditions that make cells carry out genetic engineering. The conditions may include one or more specific culture media, temperature, oxygen content, carbon dioxide content, time, reagents, such as nutrients, amino acids, antibiotics, ions and / or stimulating factors, such as cytokines, chemokines, antigens, binding partners, fusion proteins, recombinant soluble receptors and any other reagents designed for activating cells.

[0210] Whether before or after immune cell modification, immune cells can be activated and expanded, typically using methods such as, for example, U.S. Patent Nos. 6,352,694; 6,534,055; 6,905,680; 6,692,964; 5,858,358; 6,887,466; 6,905,681; 7,144,575; 7,067,318; 7,172,869; 7,232,566; 7,175,843; 5,883,223; 6,905,874; 6,797,514; 6,867,041; 9,938,498; US20060121005; and Magee et al. (“Chapter Nine - Isolation, culture and propagation of natural killer cells,” Natural Killer Cells, Basic Science and Clinical Application, 2010, pp. 125-135).

[0211] Typically, T cells can be amplified by contacting with the surface of a reagent that is attached to a signal related to the CD3 / TCR complex and a ligand that stimulates the co-stimulatory molecules on the T cell surface. In particular, T cell groups can be stimulated as described herein, for example, by contacting with anti-CD3 antibodies or their antigen-binding fragments, or anti-CD2 antibodies fixed on the surface, or by contacting with protein kinase C activators (such as moss proteins) in conjunction with calcium ion carriers to stimulate T cell groups. In order to costimulate the auxiliary molecules on the T cell surface, a ligand that combines the auxiliary molecules is used. For example, under conditions suitable for stimulating T cell proliferation, T cell groups can be contacted with anti-CD3 antibodies and anti-CD28 antibodies. In order to stimulate the proliferation of CD4+T cells or CD8+T cells, anti-CD3 antibodies and anti-CD28 antibodies are used. Examples of anti-CD28 antibodies include 9.3, B-T3, XR-CD28 (Diaclone, Besancon, France), and other methods known in the art may also be used (Berg et al., Transplant Proc. 30(8):3975-3977, 1998; Haanen et al., J. Exp. Med. 190(9):1319-328, 1999; Garland et al., J. Immunol Meth. 227(1-2):53-63, 1999). In some embodiments, paramagnetic beads can be used for expansion / activation, for example M-450 CD3 / CD28T paramagnetic beads.

[0212] NK cells express characteristic NK cell surface receptors and lack TCR rearrangement and T cell, B cell, monocyte and / or macrophage surface markers. Human NK cells are characterized by the presence of cell surface markers CD16 and CD56 and the absence of T cell receptor (CD3). Human bone marrow-derived NK cells are further characterized by a CD2+CD16+CD56+CD3 phenotype, further containing a T cell receptor ζ chain [ζ(Q-TCR]), and are typically characterized by NKp46, NKp30 or NKp44. Inhibitory NK cell receptors include HLA-E (CD94 / NKG2A); HLA-C (Group 1 or Group 2), KIR2DL; KIR3DL (HLA-Bw4) and HLA-A3 or A4+ peptides. Activating NK cell receptors include HLA-E (CD94 / NKG2C); KIR2DS (HLA-C) and KIR3DS (HLA-Bw4). Other receptors include NK cell receptor protein-1 (known as NK1.1 in mice) and the low-affinity receptor for the Fc portion of IgG (FcγRIII; CD16).

[0213] In some embodiments, immune cells are expanded by adding feeder cells, such as non-dividing peripheral blood mononuclear cells (PBMC) to the culture starting composition (e.g., so that the resulting cell population comprises at least about 5, 10, 20 or 40 or more PBMC feeder cells for each immune cell (such as T cell) in the initial population to be expanded); and incubating the culture (e.g., a time sufficient to expand the number of immune cells (e.g., T cells). In some aspects, non-dividing feeder cells can include γ-irradiated PBMC feeder cells. In some embodiments, PBMC are irradiated with gamma rays in the range of about 3000 to 3600 rads to prevent cell division. In some aspects, before adding an immune cell population (e.g., T cell), feeder cells are added to the culture medium.

[0214] In some embodiments, immune cells are combined with reagent-coated beads, and beads and cells are subsequently separated and then cultured. In alternative embodiments, before culture, the reagent-coated beads and cells are not separated, but cultured together. In another embodiment, beads and cells are first concentrated by applying a force (e.g., magnetic force), resulting in an increase in the connection of cell surface markers, thereby inducing cell stimulation.

[0215] Suitable conditions for culturing immune cells (e.g., T cells) include an appropriate culture medium (e.g., Minimum Essential Medium or RPMI medium 1640 or X-vivo 15 (Lonza)) containing factors required for proliferation and survival, including serum (e.g., fetal bovine or human serum), interleukin-2 (IL-2), insulin, IFN-γ, IL-4, IL-7, GM-CSF, IL-10, IL-12, IL-15, TGFβ and TNF-α, or any other additives for cell growth known to those skilled in the art. Other additives for cell growth include, but are not limited to, surfactants, plasma substitutes, and reducing agents, such as N-acetylcysteine ​​and 2-mercaptoethanol. The culture medium may include RPMI 1640, AIM-V, DMEM, MEM, α-MEM, F-12, X-Vivo 15 and X-Vivo 20, Optimizer, and added amino acids, sodium pyruvate and vitamins, serum-free or supplemented with an appropriate amount of serum (or plasma) or a specified hormone group, and / or cytokines (one or more) sufficient for the growth and expansion of T cells. Antibiotics, such as penicillin and streptomycin, are included only in experimental cultures and not in cell cultures to be infused into a subject. The target cells are maintained under conditions required to support growth, such as an appropriate temperature (e.g., 37°C) and atmosphere (e.g., air plus 5% CO2).

[0216] In some embodiments, the method includes evaluating the expression of one or more markers on the surface of the modified cell or cell to be engineered. In one embodiment, the method includes, for example, evaluating the surface expression of TCR or CD3ε by an affinity-based detection method, such as by flow cytometry. In some aspects, where the method discloses surface expression of an antigen or other marker, the gene encoding the antigen or other marker is destroyed or otherwise, for example, expressed in an inhibited manner using the methods described herein.

[0217] Isolation and enrichment of immune cells (e.g., modified immune cells)

[0218] In some embodiments, the methods described herein further include separating or enriching immune cells, and the immune cells include mutations (e.g., inactivating mutations) and / or some other features (e.g., expression of CAR) in a target gene or target RNA. In some embodiments, the methods described herein further include separating or enriching immune cells comprising Cas components, gRNA constructs or arRNA constructs and / or engineered receptors (e.g., CAR) as described herein, or having one or more target proteins as described herein (e.g., SPPL3 proteins) and / or other proteins. Expression (RNA and / or protein expression) and / or function or without one or more target proteins as described herein (e.g., SPPL3 proteins) and / or other proteins. Expression (RNA and / or protein expression) and / or function.

[0219] In some embodiments, the separation method includes separating different cell types based on the absence or presence of one or more specific molecules in the cell, such as surface markers, such as surface proteins (such as CAR), intracellular markers or nucleic acids (such as sgRNA, arRNA and / or nucleic acids encoding Cas). In some embodiments, any known separation method based on such markers can be used. In some embodiments, the separation is based on the separation of affinity or immunoaffinity. For example, in some aspects, separation includes the expression or expression level of one or more markers (typically cell surface markers) based on cells, such as by incubating with antibodies or binding partners that specifically bind to such markers, separating cells and cell groups, followed by washing steps and separating cells bound to antibodies or binding partners from those cells that are not bound to antibodies or binding partners. In some embodiments, separation includes separating cells and cell groups based on the expression of cell-based selective marker genes (such as antibiotic resistance genes such as puromycin or fluorescent protein encoding genes). Such separation steps can be based on positive selection and / or negative selection, in which cells that have bound the reagent, are resistant to antibiotics, or express a fluorescent protein are retained for further use, and in which cells that have not bound the antibody or binding partner or do not express the fluorescent protein are retained for further use. In some instances, both fractions are retained for further use. In some aspects, negative selection is particularly useful when antibodies that specifically identify cell types in a heterogeneous population are not available, making separation based on markers expressed by cells outside the desired population the best approach.

[0220] Isolation need not result in 100% enrichment or removal of a particular cell population or cells expressing a particular marker. For example, positive selection or enrichment of a particular type of cell (e.g., cells expressing a marker) refers to increasing the number or percentage of such cells, but need not result in the complete absence of cells that do not express the marker. Similarly, negative selection, removal, or depletion of a particular type of cell (e.g., cells expressing a marker) refers to reducing the number or percentage of such cells, but need not result in the complete removal of all such cells.

[0221] In some examples, multiple rounds of separation steps are performed, wherein the positively selected or negatively selected fractions from one step are subjected to another separation step, such as a subsequent positive selection or negative selection. In some examples, a single separation step can deplete cells that simultaneously express multiple markers, such as by incubating cells with multiple antibodies or binding partners, each specific for a marker that is targeted for negative selection. Similarly, multiple cell types can be positively selected simultaneously by incubating cells with multiple antibodies or binding partners that are expressed on various cell types.

[0222] For example, in some aspects, specific subpopulations of T cells, such as those that are positive for or express high levels of one or more surface markers, such as CD28, are isolated by positive or negative selection techniques. + 、CD62L + 、CCR7 + 、CD27 + 、CD127 + 、CD4 + 、CD8 + 、CD45RA + and / or CD45RO + In some embodiments, T cells are isolated that do not express certain markers (e.g., exhaustion markers). In some embodiments, T cells are isolated that do not express or express reduced markers, such as one or more of SPPL3, Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligands.

[0223] In some embodiments, isolation is performed by enriching a particular cell population by positive selection or depleting a particular cell population by negative selection. In some embodiments, positive or negative selection is achieved by incubating the cells with one or more antibodies or other binding agents that specifically bind to a marker expressed or expressed at relatively high levels on the positively or negatively selected cells, respectively. 高 ) expression (marker + )'s one or more surface markers.

[0224] In some aspects, a sample or composition of cells to be separated is incubated with a small, magnetizable or magnetically responsive material, such as a magnetically responsive particle or microparticle, such as paramagnetic beads (e.g., Dynabeads or MACS beads). The magnetically responsive material (e.g., particle) is typically attached directly or indirectly to a binding partner, such as an antibody, that specifically binds to a molecule, such as a surface marker, present on a cell, a plurality of cells, or a population of cells from which separation (e.g., negative or positive selection) is desired.

[0225] The incubation is typically performed under conditions such that the antibody or binding partner or molecule, such as a secondary antibody or other reagent, which specifically binds to such antibody or binding partner attached to the magnetic particles or beads, specifically binds to the cell surface molecule if present on cells within the sample.

[0226] In some embodiments, the sample is placed in a magnetic field, and those cells that have magnetically responsive or magnetizable particles attached thereto will be attracted to the magnet and separated from unlabeled cells.

[0227] In some embodiments, the magnetically responsive particles are attached to cells that are subsequently incubated, cultured, and / or engineered; in some aspects, the particles are attached to cells for administration to a patient. In some embodiments, the magnetizable or magnetically responsive particles are removed from the cells, for example, by using a competing unlabeled antibody, a magnetizable particle, or an antibody conjugated to a cleavable linker.

[0228] In some embodiments, affinity-based selection is performed by magnetic activated cell sorting (MACS) (Miltenyi Biotec, Auburn, CA). The magnetic activated cell sorting (MACS) system is capable of selecting cells attached to magnetized particles with high purity. In certain embodiments, MACS operates in a mode that sequentially washes out non-target species and target species after applying an external magnetic field. That is, the cells attached to the magnetized particles are fixed in place, and the unattached species are washed away. Then, after completing this first elution step, the species that were captured in the magnetic field and prevented from washing away are released in a certain manner so that they can be washed away and recovered. In certain embodiments, non-target cells are labeled and depleted from a heterogeneous cell population.

[0229] In certain embodiments, the separation or separation is performed using a system, device, or apparatus that performs one or more of the separation, cell preparation, separation, processing, incubation, cultivation, and / or formulation steps of the method. In some aspects, the system is used to perform each of these steps in a closed or sterile environment, e.g., to minimize error, user handling, and / or contamination.

[0230] In some embodiments, the system or device performs one or more, for example, all, separation, processing, engineering, and formulation steps in an integrated or self-contained system and / or in an automated or programmable manner. In some aspects, the system or device includes a computer and / or computer program in communication with the system or device that allows a user to program, control, evaluate results, and / or adjust various aspects of the processing, separation, engineering, and formulation steps.

[0231] In some embodiments, cell mass as described herein is collected and enriched (or depleted) by flow cytometry, wherein cells dyed for multiple cell surface markers are carried in fluid stream. In some embodiments, cell mass as described herein is collected and enriched (or depleted) by preparation scale (FACS) sorting. In certain embodiments, cell mass as described herein is collected and enriched (or depleted) by using a microelectromechanical system (MEMS) chip in combination with a FACS-based detection system (see, e.g., WO 2010 / 033140, Cho et al. (2010) Lab Chip 10, 1567-1573; and Godin et al. (2008) J Biophoton. 1 (5): 355-376.

[0232] IV. Methods of Treating Diseases and / or Selecting Donors

[0233] In another aspect, the present invention provides methods for treating a disease (e.g., cancer or an immune-related disease, such as infection or immune cell exhaustion) in an individual (e.g., a human), and methods for selecting or excluding an individual as a suitable donor of immune cells based on any target gene or target protein described herein (e.g., SPPL3 protein) or based on one or more target genes identified using any target gene identification method described herein (e.g., AICD / FasL resistance gene).

[0234] "Aberration" of a gene (e.g., a target gene) refers to a genetic and / or epigenetic aberration of a gene, an abnormal expression level and / or abnormal activity level, and / or abnormal modification level of a gene (or gene product, such as RNA or protein) that may result in abnormal loss of function or reduced function and / or abnormal expression (e.g., reduction or deletion) of the RNA and / or protein encoded by the gene. In some embodiments, the aberration may occur at the gene level, RNA level, and / or protein level. In some embodiments, the genetic aberration includes changes in nucleic acid (e.g., DNA or RNA) or protein sequence (i.e., mutation) or abnormal epigenetic features associated with the gene, including but not limited to coding, non-coding, regulatory, enhancer, silencer, promoter, intron, exon, and untranslated regions of the gene. In some embodiments, the aberration of a gene includes mutations of the gene, including but not limited to deletions, frameshifts, insertions, indels, missense mutations, nonsense mutations, point mutations, silent mutations, splice site mutations, splice variants, and translocations. In some embodiments, the mutation may be a loss or deletion of a gene. In some embodiments, the mutation is a deleterious mutation. In some embodiments, the distortion of a gene includes abnormal (for example, reduction or deletion) expression (for example, mRNA or protein) of a gene compared to a control level. In some embodiments, the distortion on a gene includes abnormal (for example, reduction or elimination) activity of a gene product (for example, RNA or protein) compared to a control level, such as activation or inhibition of a downstream target. In some embodiments, the distortion on a gene includes abnormal modification (for example, increase, decrease or mismodification) of a gene (for example, at DNA level or histone level) or a gene product (for example, RNA or protein) compared to a control level, such as post-translational modification (for example, phosphorylation, ubiquitination). In some embodiments, the distortion of a gene includes a change in the copy number of a gene. In some embodiments, the change in the copy number of a gene is caused by structural rearrangement (including deletion, duplication, inversion and translocation) of the genome. In some embodiments, the distortion of a gene includes abnormal epigenetic features of a gene, including but not limited to DNA methylation, hydroxymethylation, histone binding increase or decrease, histone methylation, tissue protein acetylation, chromatin remodeling, etc. In some embodiments, the aberration is determined compared to a control or reference (e.g., a reference sequence (e.g., a nucleic acid sequence or protein sequence), a control expression (e.g., RNA or protein expression) level, a control activity (e.g., activation or inhibition of a downstream target) level, or a control modification (e.g., a post-translational modification or epigenetic modification) level. In some embodiments, the aberrant expression level or aberrant activity level of a gene can be lower than the control level (e.g., any one of about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or more lower than the control level).In some embodiments, the abnormal modification level of a gene (e.g., modification of DNA, nucleosomes, RNA, or protein) may be lower than a control level (e.g., lower than about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or more of the control level), or higher than a control level (e.g., higher than about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90% or more of the control level). In some embodiments, the abnormal modification in a gene is an incorrect modification, such as ubiquitination instead of phosphorylation. In some embodiments, the control level (e.g., expression level or activity level or modification level) is the average or median level (e.g., expression level or activity level or modification level) of a control group. In some embodiments, the control group is a healthy group that does not have a disease (e.g., a disease to be treated) and optionally has demographic characteristics (e.g., sex, age, race, etc.) comparable to those of the individual being treated / to be treated. In some embodiments, the control level (e.g., expression level, activity level, or modification level) is the level (e.g., expression level, activity level, or modification level) or average level of healthy tissue from the same individual. In some embodiments, the control level (e.g., expression level, activity level, or modification level) is the level (e.g., expression level, activity level, or modification level) or average level of the same cell (e.g., from the same individual) without the aberration (e.g., target gene mutation). In some embodiments, the control or reference level is the average or median expression (RNA and / or protein expression) and / or function of one or more target proteins (e.g., SPPL3 protein) and / or other proteins in a population of individuals. Aberrations in a gene can be determined by comparison with a reference sequence (including the epigenetic pattern of a reference sequence in a control sample). In some embodiments, the reference sequence is a sequence (DNA, RNA, or protein sequence) corresponding to a fully functional allele of the corresponding gene, such as an allele (e.g., prevalent allele) of the corresponding gene present in an unmodified immune cell (e.g., a precursor immune cell).

[0235] Aberrations of target genes are also referred to herein as "target gene aberrations," including but not limited to target gene mutations. Aberrations of AICD resistance genes, or FasL resistance genes, or AICD / FasL resistance genes (used interchangeably herein) are also referred to herein as "AICD resistance aberrations," "FasL resistance aberrations," or "AICD / Fas resistance aberrations," including but not limited to AICD or FasL resistance mutations that increase the resistance of immune cells to death induced by AICD and / or FasL. In some embodiments, the AICD / FasL resistance gene is selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B.

[0236] The "state" of a gene aberration can refer to the presence or absence of a gene aberration, or an abnormal level of a gene (expression level or activity level or modification level). In some embodiments, the presence of aberrations (such as LOF mutations) in one or more AICD / FasL resistance genes (or RNA / proteins encoded therein) as compared to a control indicates that i) such immune cells may have higher resistance to AICD, may not induce or induce less host-to-graft (HvG) response (e.g., by host T cells and / or NK cells attaching thereto), and / or have better in vivo persistence and are therefore ideal for cell-based immunotherapy (e.g., CAR-T or CAR-NK); and / or ii) such individuals may be suitable for autologous or allogeneic immune cell therapy or for donating immune cells for immune cell therapy. In some embodiments, the absence of aberrations (e.g., mutations) in one or more AICD / FasL resistance genes (or RNA / proteins encoded therein) compared to a control indicates that i) such immune cells have low resistance to AICD, can induce a strong HvG response, and / or may have poor in vivo persistence, and are therefore less suitable for cell-based immunotherapy; and / or ii) the individual is less suitable for autologous or allogeneic immune cell therapy, or the individual is not an ideal donor for immune cells. In some embodiments, abnormal levels (e.g., expression levels, activity levels, or modification levels) of one or more AICD / FasL resistance genes are associated with i) the degree of resistance to AICD and / or HvG response and / or the degree of in vivo persistence; and / or ii) the likelihood that the individual will be an ideal immune cell donor. For example, a greater deviation in the level (e.g., expression level or activity level or modification level) of one or more AICD / FasL resistance genes in the direction of reducing or eliminating gene expression and / or function indicates that the immune cells are more resistant to AICD, induce less HvG response, and / or have longer in vivo persistence; and / or ii) the individual is more likely to respond to autologous or allogeneic immune cell therapy, or is more suitable as an immune cell donor.

[0237] In some embodiments, a method of treating a disease (e.g., cancer or an immune-related disease, such as infection or immune cell exhaustion) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of any modified immune cell described herein (e.g., SPPL3-KO CAR-T or CAR-NK cell) or a pharmaceutical composition thereof. In some embodiments, a method for treating a disease (e.g., cancer or an immune-related disease, such as infection or immune cell exhaustion) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of immune cells (or a pharmaceutical composition thereof) that have been modified to reduce (e.g., reduce at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more of any one) or eliminate one or more target proteins identified herein (e.g., AICD / FasL resistance gene encoding protein) or described herein (e.g., SPPL3 protein), such as SPPL3, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and / or HIST1H1B expression (RNA and / or protein expression) and / or function. In some embodiments, the modified immune cells are allogeneic. In some embodiments, the modified immune cells are autologous. In some embodiments, the modified immune cells further express engineered receptors (e.g., CAR, engineered TCR or TAC). In some embodiments, the disease is related to the expression of a target antigen (such as a tumor antigen), and wherein the immune cell expresses or is modified to express an engineered receptor (such as CAR) that specifically recognizes the target antigen. Also provided is the use of modified immune cells or their pharmaceutical compositions for treating an individual (such as a human) disease. Also provided is the use of modified immune cells or their pharmaceutical compositions in the preparation of a medicine for treating an individual (such as a human) disease. In some embodiments, the disease is cancer, infection, inflammation, autoimmune disease, or an immune-related disease characterized by exhaustion of effector cells.

[0238] In some embodiments, a method of treating cancer (eg, CD19+ cancer) in an individual (eg, a human) is provided, comprising administering to the individual an effective amount of SPPL3 KO / TCR KO CAR-T (e.g., SPPL3 KO / TCR KO In some embodiments, a method of treating cancer (e.g., CD19+ cancer) in an individual (e.g., a human) is provided, comprising administering to the individual an effective amount of SPPL3 KO CAR-T (e.g., SPPL3 KOAnti-CD19 CAR-T) cells or pharmaceutical compositions thereof. In some embodiments, the cancer is a B cell cancer, including but not limited to acute myeloid leukemia (AML), chronic myeloid leukemia (CML), myelodysplastic syndrome (MDS), acute B lymphocytic leukemia (B-ALL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma, chronic lymphocytic leukemia (CLL), hairy cell leukemia (HCL), blastic plasmacytoid dendritic cell neoplasm (BPDCN), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma, systemic mastocytosis and Burkitt lymphoma.

[0239] In some embodiments, a method of identifying an individual (e.g., a human) as a suitable donor of immune cells with prolonged in vivo persistence is provided, comprising examining the expression and / or function of one or more target proteins identified herein (e.g., AICD / FasL resistance gene-encoded proteins) in an individual (e.g., in an immune cell sample obtained from the individual), the target protein being selected from, for example, SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B, wherein the expression and / or function of the one or more target proteins is reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) or eliminated compared to a reference (e.g., an immune cell sample obtained from a reference) identifies the individual as a suitable donor. In some embodiments, the reference is the average or median expression and / or function of the one or more target proteins in a population of individuals (e.g., healthy individuals and / or patients). In some embodiments, a method of identifying an individual (e.g., a human) as a suitable donor of immune cells with prolonged in vivo persistence is provided, comprising examining the expression and / or function of a SPPL3 protein in the individual (e.g., in a sample of immune cells obtained from the individual), wherein identification of reduced or abolished expression and / or function of the SPPL3 protein compared to a reference identifies the individual as a suitable donor. In some embodiments, the reference is the average or median expression and / or function of the SPPL3 protein in a population of individuals. In some embodiments, the method further comprises examining the expression and / or function (e.g., in an immune cell sample obtained from the individual) of one or more additional proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, M Identification of a ligand for ICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 (e.g., selected from Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B), wherein the expression and / or function of one or more additional proteins is reduced (e.g., reduced by at least about any of 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) or eliminated further identifies the individual as a suitable donor.In some embodiments, examining the expression and / or function of one or more target proteins (e.g., SPPL3 proteins) and / or one or more other proteins comprises examining the sequence of a nucleic acid (DNA or RNA) encoding the one or more target proteins and / or the one or more other proteins, wherein identification of an aberration (e.g., a mutation such as a LOF mutation) in the nucleic acid that reduces the expression and / or function of the one or more target proteins and / or the one or more other proteins identifies the individual as a suitable donor. In some embodiments, identification of an aberration (e.g., a mutation such as a LOF mutation) in the nucleic acid that reduces the expression and / or function of the one or more target proteins identifies the individual as a suitable donor. In some embodiments, a method of identifying an individual (e.g., a human) as a suitable donor of immune cells with prolonged in vivo persistence is provided, comprising detecting in an immune cell sample from the individual one or more AICD / FasL resistance aberrations (e.g., AICD / Fas L resistance mutations such as LOF mutations) in one or more AICD / FasL resistance genes (e.g., selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B genes) identified using any of the target gene identification methods described herein, wherein the presence of the one or more AICD / FasL resistance aberrations (e.g., AICD / FasL resistance mutations, such as SPPL3-LOF mutation or KO) in the immune cell sample identifies the individual as a suitable donor. In some embodiments, the method further comprises detecting in the immune cell sample aberrations (e.g., mutations such as LOF mutations) in one or more additional genes selected from Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, PD-1, TIM-3, LAG-3, CTLA-4, and CISH, wherein the presence of the aberrations in the immune cell sample further identifies the individual as a suitable donor.

[0240] In some embodiments, a method for excluding an individual (e.g., a human) as a suitable donor of immune cells for prolonged in vivo persistence is provided, comprising examining the expression and / or function of one or more target proteins identified herein (e.g., AICD / FasL resistance gene-encoded proteins) in an individual (e.g., in an immune cell sample obtained from the individual), the target protein being selected from, for example, SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B, wherein if the expression and / or function of one or more target proteins is not identified as reduced (e.g., reduced by at least about 10%, 20%, 30%, 40%, 60%, 70%, 80%, 90%, 95% or more) or eliminated compared to a reference (e.g., an immune cell sample obtained from a reference), then excluding the individual as a suitable donor. In some embodiments, the reference is the average or median expression and / or function of one or more target proteins in a population of individuals (e.g., healthy individuals and / or patients). In some embodiments, a method of excluding an individual (e.g., a human) as a suitable donor of immune cells with prolonged in vivo persistence is provided, comprising examining the expression and / or function of a SPPL3 protein in the individual (e.g., in a sample of immune cells obtained from the individual), wherein if no reduction or abolition of the expression and / or function of the SPPL3 protein is identified as compared to a reference, the individual is excluded as a suitable donor. In some embodiments, the reference is the average or median expression and / or function of the SPPL3 protein in a population of individuals. In some embodiments, examining the expression and / or function of one or more target proteins (e.g., SPPL3 protein) comprises examining the sequence of a nucleic acid (DNA or RNA) encoding the one or more target proteins, wherein if no aberration (e.g., a mutation such as a LOF mutation) that reduces the expression and / or function of the one or more target proteins is identified in the nucleic acid, the individual is excluded as a suitable donor. In some embodiments, a method is provided for excluding an individual (e.g., a human) as a suitable donor of immune cells for prolonged persistence in vivo, comprising detecting in an immune cell sample from the individual one or more AICD / FasL resistance aberrations (e.g., AICD / Fas L resistance mutations such as LOF mutations) in one or more AICD / FasL resistance genes (e.g., selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B genes) identified using any of the target gene identification methods described herein, wherein the individual is excluded if no AICD / FasL resistance aberration (e.g., AICD / FasL resistance mutation, such as SPPL3-LOF mutation or KO) is identified in the immune cell sample.

[0241] In some embodiments, the method of treating a disease or selecting or excluding an individual as an immune cell donor further comprises detecting one or more AICD / FasL resistance aberrations (e.g., AICD / FasL resistance mutations) in an immune cell sample from the individual (e.g., by NGS), or detecting the expression and / or function of one or more target proteins identified or described herein (e.g., SPPL3 protein). In some embodiments, when the detection is at the genetic level (e.g., examination of mutations), the sample can be from any tissue of the individual. In some embodiments, the method further comprises identifying one or more AICD / FasL resistance genes. In some embodiments, the method further comprises detecting abnormal (e.g., reduced or absent) expression (e.g., RNA or protein) of one or more AICD / FasL resistance genes or their expression products (e.g., SPPL3) compared to a control / reference level, for example, by qPCR, RNA-seq, mass spectrometry, Western blot, or any other RNA or protein expression level detection method. In some embodiments, the method further comprises detecting abnormal modification of one or more AICD / FasL resistance genes or their expression products (e.g., SPPL3) compared to a control / reference level, such as epigenetic modification (e.g., DNA methylation, histone methylation, histone acetylation) or post-translational modification (e.g., phosphorylation, glycosylation, ubiquitination, nitrosation, methylation, acetylation, lipidation, and proteolysis). Any known method for detecting DNA, nucleosome, RNA, or protein modification(s) can be used herein, such as ChIP-seq, ChIP-qPCR, DNase-seq, MNase-seq, mass spectrometry, Western blot, etc. In some embodiments, the method further comprises detecting abnormal (e.g., reduced or absent) activity of expression products (e.g., RNA or protein) of one or more AICD / FasL resistance genes (e.g., SPPL3) compared to a control / reference level. Any suitable gene / protein function / activity assay can be used herein, such as assays for signal transduction, activation state of downstream pathway molecules (e.g., phosphorylation state), glycosylation state of interacting partner(s), protein-protein binding affinity and / or specificity, metabolism, cell behavior (e.g., cell proliferation, differentiation, activation, persistence, death, cell cycle), effector function (e.g., factor release or cytotoxicity), etc. In some embodiments, the reference is the average or median mutation frequency, expression, and / or function of the corresponding gene / protein in a population of individuals (e.g., healthy individuals and / or patients).

[0242] In some embodiments, a method for selecting immune cells for immune cell therapy is provided, comprising isolating and / or enriching from an initial population of immune cells (e.g., precursor immune cells, CAR-T cells, or CAR-NK cells) immune cells having reduced expression and / or function (e.g., a reduction of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of any one) of one or more target proteins identified herein (e.g., AICD / FasL resistance gene-encoded protein) or as described herein (e.g., SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, or HIST1H1B protein; such as SPPL3 protein) or not having the expression and / or function of the one or more target proteins. In some embodiments, a method of selecting immune cells for immune cell therapy is provided, comprising isolating and / or enriching immune cells having reduced expression and / or function of SPPL3 protein (e.g., reduced by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or lacking SPPL3 protein expression and / or function from a population of primary immune cells (e.g., precursor immune cells, CAR-T cells, or CAR-NK cells), or having a mutation (e.g., LOF mutation) in the SPPL3 gene that reduces (e.g., reduces at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more) or eliminates its expression (RNA and / or protein) and / or function. In some embodiments, the primary population of immune cells expresses an engineered receptor (e.g., CAR, TAC, engineered TCR). In some embodiments, the method also includes introducing nucleic acids encoding engineered receptors (e.g., CAR, TAC, engineered TCR) into isolated / enriched immune cells. In some embodiments, the method also includes separating and / or enriching those cells expressing engineered receptors from immune cells (e.g., immune cells modified by target genes / proteins, or an initial population of immune cells). Based on the reporter gene expressed by modified immune cells, any cell separation / enrichment method, such as FACS, can be used herein. For suitable methods, see above "Separation and enrichment of immune cells (such as modified immune cells)". In some embodiments, immune cells are autologous. In some embodiments, immune cells are allogeneic.

[0243] Diseases and conditions

[0244] Any disease that can be treated with immune cell-based therapy (e.g., CAR-T or CAR-NK therapy) can be treated using any of the methods described herein and / or modified immune cells. In some embodiments, the disease is selected from cancer, infection, inflammation, autoimmune disease, and immune-related diseases characterized by exhaustion of immune cells (e.g., effector cells).

[0245] The methods described herein are applicable to the treatment of a variety of cancers, including solid cancers and blood cancers, and cancers of all stages, including early-stage cancers, non-malignant cancers, primary cancers, advanced cancers, locally advanced cancers, metastatic cancers, or cancers in remission. In some embodiments, the solid cancer or blood cancer can be any of stages I, II, III, and IV according to the American Joint Committee on Cancer (AJCC) staging groups.

[0246] In some embodiments, the cancer is selected from the group consisting of colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophageal cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head or neck cancer, malignant melanoma of the skin or eye, uterine cancer, breast cancer, ovarian cancer, rectal cancer, anal cancer, stomach cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, Hodgkin's disease, non-Hodgkin's lymphoma (NHL), cutaneous T-cell lymphoma (CTL), and ovarian cancer. TCL), endocrine system cancers, thyroid cancer, parathyroid cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, solid tumors in children, bladder cancer, kidney or ureter cancer, renal pelvis cancer, central nervous system (CNS) tumors, primary CNS lymphoma, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid cancer, squamous cell carcinoma, T-cell lymphoma, environmentally induced cancers, combinations of said cancers, and said cancer metastases.

[0247] In some embodiments, the cancer is a hematological cancer selected from one or more of acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphocytic leukemia (ALL), B-cell acute lymphocytic leukemia, T-cell acute lymphocytic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell prolymphocytic leukemia, blastic plasmacytoid dendritic cell neoplasm (BPDCN), Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoproliferative disorders, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple myeloma, myelodysplasia and myelodysplastic syndrome, non-Hodgkin lymphoma, Hodgkin lymphoma, plasmablastic lymphoma, plasmacytoid dendritic cell neoplasm, Waldenstrom macroglobulinemia, or preleukemia.

[0248] In some embodiments, the disease is an infection or infectious disease, such as an infection or infectious disease caused by a pathogen. In some embodiments, the pathogen is any one of a virus, bacterium, fungus and parasite or its fragment. In some embodiments, the pathogen includes pathogen-associated molecular patterns (PAMPs). In other embodiments, PAMPs are selected from pathogen fragments, pathogen fragments, pathogen nucleic acids, pathogen lipoproteins, pathogen surface glycoproteins, pathogen membrane components and components released from pathogens. In some embodiments, the components released from pathogens include toxins. In other embodiments, toxins are selected from endotoxins, lipopolysaccharides (LPS), lipoteichoic acid (LTA), teichoic acid and ricin.

[0249] In some embodiments, the bacteria are selected from the group consisting of Acinetobacter baumanii, Burkholderia cepacia, Bacterioides fragilis, Chlamydia trachomatis, Citrobacter freundii, Campylobacter jejuni, Escherichia coli, Enterobacter aerogenes, Enterobacter cloacae, Haemophilus influenzae type b, Helicobacter pylori, Klebsiella oxytoca, K. pneumonia (MDR / CRE), Legionella pneumophila, Neisseria meningitidis, Neisseria gonorrhoeae, and Streptococcus aureus. gonorrhoeae), Pseudomonas aeruginosa, Salmonella typhi, paratyphi, typhimurium, Serratia marcescens, Shigella flexneri, Stenotrophomonas maltophilia, Yersinia pseudotuberculosis, Bacillus subtilis, Clostridium neoformans, C. difficile, C. perfringens, Corynebacterium spp., Enterococcus faecalis, Enterococcus faecium, vancomycin-resistant Enterococci Enterococci (VRE), Listeria monocytogenes, Mycobacterium avium, Mycobacterium tuberculosis (M.tuberculosis), Mycobacterium leprae, Nocardia farcinica, P. acnes, Staphylococcus aureus, methicillin-susceptible Staphylococcus aureus (MSSA), methicillin-resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus Group A, Streptococcus Group B (Agalactiae), and Streptococcus Group C. In some embodiments, the bacteria are antibiotic-resistant. In some embodiments, the bacteria are multidrug-resistant.

[0250] In some embodiments, the fungus is selected from Aspergillus spp., Blastomyces, Candida albicans, Candida glabrata, Candida guilliermondii, Candida krusei, parapsilosis, tropicalis Cryptococcus, Fusarium spp., Mucor spp., Saccharomyces, and Pneumocystis jirovecii (carinii).

[0251] In some embodiments, the virus is from any one of the families Orthomyxoviridae, Filoviridae, Flaviviridae, Coronaviridae, and Poxviridae. In some embodiments, the virus is selected from dengue virus, Ebola virus, EBV, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, HIV, HSV 1, HSV 2, cytomegalovirus (CMV), influenza A virus (e.g., H1N1, H5N1), influenza B virus, influenza C virus, Marburg virus, human respiratory syncytial virus (RSV), SARS-CoV, MERS-CoV, human papillomavirus (HPV), human rhinovirus (HRV), and Zika virus.

[0252] In some embodiments, the parasite is selected from Cryptosporidium, Leishmania, Malaria, Schistosoma, Trichomonas, and Trypanosoma.

[0253] In some embodiments, disease is an inflammatory disease.Term " inflammatory disease ", " inflammatory disorder " or " inflammatory condition " refer to any disease with inflammation as a sign, which can be caused by a variety of stimulating events, including radiation, machinery, chemistry, infection and immune stimulation.The inflammatory condition can be identified by the expression of well-known inflammation-related molecules secreted by clinical and pathological characteristics and / or tissues and / or cells.Inflammatory disease can refer to any disease in which there is an inflammatory condition.Non-limiting examples of inflammatory diseases treatable with the present method, modified immune cells, medicine or pharmaceutical composition include cardiovascular disease, arthritis, asthma, psoriasis, inflammatory bowel disease, organ transplant rejection, lupus, autoimmune disease, radiation-induced damage, cancer, burns, trauma, rheumatic disease, nephropathy, allergic disease, infectious disease, eye disease, skin disease, gastrointestinal disease, liver disease, cerebral edema, sarcoidosis, thrombocytopenia and spinal cord injury.

[0254] In some embodiments, the inflammatory disease is an autoimmune disease. An autoimmune disease, or autoimmunity, is a condition in which an organism fails to recognize its own components (down to the submolecular level) as "self," resulting in an immune response against its own cells and tissues. Any disease caused by this abnormal immune response is known as an autoimmune disease. Prominent examples include celiac disease, type 1 diabetes mellitus (IDDM), systemic lupus erythematosus (SLE), Sjögren's syndrome, multiple sclerosis (MS), Hashimoto's thyroiditis, Graves' disease, idiopathic thrombocytopenic purpura, and rheumatoid arthritis (RA). Examples of autoimmune diseases include, but are not limited to, acute idiopathic thrombocytopenic purpura, chronic idiopathic thrombocytopenic purpura, dermatomyositis, Sydenham's chorea, myasthenia gravis, systemic lupus erythematosus, lupus nephritis, rheumatic fever, multinodular syndrome, bullous pemphigoid, diabetes mellitus, Henoch-Schonlein purpura, erythema nodosum, Takayasu's arteritis, Addison's disease, rheumatoid arthritis, multiple sclerosis, sarcoidosis, ulcerative colitis, polymyositis, leukemia, leukemia, schizoaffective disorder ... Erythema nodosum, IgA nephropathy, polyarteritis nodosa, ankylosing spondylitis, Goodpaste syndrome, thromboangiitis vasculitis, Sjögren's syndrome, primary biliary cirrhosis, Hashimoto's thyroiditis, thyrotoxicosis, scleroderma, chronic active hepatitis, polymyositis / dermatomyositis, polychondritis, Panfege disease, Wegener's granulomatosis, membranous nephropathy, amyotrophic lateral sclerosis, latissimus dorsi, giant cell arteritis / polymyalgia, and fibrosing alveolitis are the most common treatments, which are highly toxic.

[0255] In some embodiments, immune-related diseases are diseases caused by excessive activation and / or proliferation of various immune cells (e.g., effector T cells or cytotoxic T cells and inflammatory cells), and may include but are not limited to autoimmune diseases; graft-versus-host disease; organ transplant rejection; asthma atopy; or acute or chronic inflammatory diseases. In some embodiments, immune-related diseases can be prevented, improved, or treated by interfering with the intracellular signal transduction pathways necessary for the activation and / or proliferation of immune cells (e.g., T cells).

[0256] In some embodiments, immune-related diseases are associated with or are characterized by immune cell (e.g., T cell) dysfunction disorders. In certain embodiments, immune cell dysfunction disorders are characterized by immune cell exhaustion (e.g., T cell exhaustion or NK cell exhaustion). In certain embodiments, immune-related diseases are selected from unresolved acute infections, chronic infections, and tumor immunity.

[0257] As used herein, "tumor immunity" refers to the process by which tumors evade immune recognition and clearance. Therefore, as a therapeutic concept, tumor immunity is "treated" when this evasion is reduced, allowing the tumor to be recognized and attacked by the immune system. Examples of tumor recognition include tumor binding, tumor shrinkage, and tumor clearance.

[0258] During the resolution of an acute inflammatory response, a group of activated effector T cells differentiate into long-lived memory cells. In contrast, in patients with chronic infections or cancer, T cells can undergo pathological differentiation and enter a dysfunctional state, which is called T cell exhaustion. The root cause of T cell exhaustion is persistent antigen exposure leading to persistent TCR signaling. "T cell exhaustion" refers to a decline in T cell function as a result of infection (such as chronic infection) or disease. T cell exhaustion is associated with increased expression of PD-1, TIM-3, and LAG-3, decreased apoptosis, and cytokine secretion. Therefore, the terms "improving T cell exhaustion," "inhibiting T cell exhaustion," "reducing T cell exhaustion," and the like refer to conditions in which T cell function is restored, characterized by one or more of the following: reduced expression and / or levels of one or more inhibitory immune checkpoint molecules such as PD-1, TIM-3, and LAG-3; increased memory cell formation and / or maintenance of memory markers (e.g., CD62L); prevention of apoptosis; increased production and / or secretion of antigen-induced cytokines (e.g., IL-2); enhanced cytotoxicity / killing capacity; enhanced recognition of tumor targets with low surface antigen; and enhanced proliferative response to antigens.

[0259] Immune cell exhaustion (such as T cell exhaustion) can be characterized by significant changes in metabolic function, transcriptional programming, apoptosis, loss of effector function (such as cytokine secretion, killing ability) and the co-expression of multiple surface inhibitory receptors. Immune cell exhaustion (e.g., T cell exhaustion) can be associated with a reduced ability of the immune system to control tumor growth and control chronic infection. In some embodiments, the exhaustion of NK cells has one or more of the following characteristics: reduced or eliminated expression of IFN-γ and / or GM-CSF, reduced or eliminated cytotoxic or cytolytic activity (e.g., reduced expression of cytolytic molecules such as granzymes, perforin, FasL and TRAIL), and reduced or eliminated expression of surface activation receptors such as NKG2D, CD16, NCRs, CD226 (DNAM-1) and 2B4. See also Bi and Tian, ​​("NK Cell Exhaustion," Front Immunol. 2017; 8: 760). In some embodiments, the exhaustion of B cells is associated with a reduction or loss of expression of CD21, CD95 and / or CD27. Therefore, the terms "improving immune cell exhaustion," "inhibiting immune cell exhaustion," "reducing immune cell exhaustion," and the like refer to conditions in which immune cell function is restored, characterized by one or more of the following: reduced expression and / or levels of one or more inhibitory immune checkpoint molecules; increased memory cell formation and / or maintenance of memory markers; prevention of apoptosis; increased antigen-induced cytokine production and / or secretion; enhanced cytotoxicity / killing capacity; enhanced recognition of tumor targets with low surface antigen; enhanced proliferative response to antigens; and reduced exhaustion marker(s).

[0260] In the case of cancer, the treatment methods, modified immune cells or pharmaceutical compositions described herein can achieve one or more of the following: (i) reduce the number of cancer cells; (ii) reduce (e.g., reduce by at least about 10%) tumor size; (iii) inhibit, hinder, slow down (e.g., by at least about 10%), and preferably prevent cancer cell infiltration into peripheral organs to some extent; (iv) inhibit (i.e., slow down (e.g., by at least about 10%), and preferably stop) tumor metastasis to some extent; (v) inhibit (e.g., inhibit at least about 10%) tumor growth; (vi) prevent or delay the occurrence and / or recurrence of tumors; (vii) alleviate to some extent one or more symptoms associated with cancer; (viii) prolong (e.g., prolong by at least about 10%) patient survival; and / or (ix) reduce (e.g., reduce by at least about 10%) CRS.

[0261] In the case of infectious diseases such as viral infections, the therapeutic methods, modified immune cells or pharmaceutical compositions described herein can achieve one or more of the following: i) reduce (e.g., reduce by at least about 10%) the number of cells infected by the pathogen; ii) reduce (e.g., reduce by at least about 10%) the production or release of pathogen-derived antigens; iii) inhibit (i.e., slow down to some extent (e.g., at least about 10%) and preferably stop) the spread of the pathogen to uninfected cells; iv) alleviate to some extent one or more symptoms associated with the infection; v) prolong (e.g., prolong by at least about 10%) patient survival; and / or (vi) reduce (e.g., reduce by at least about 10%) CRS.

[0262] In the case of immune-related diseases associated with excessive immune response (e.g., inflammation, autoimmune diseases), the treatment methods, modified immune cells or pharmaceutical compositions described herein can achieve one or more of the following: i) control, ameliorate (e.g., reduce by at least about 10%) and / or prevent infiltration of inflammatory cells (e.g., NK cells, cytotoxic T cells); ii) control, ameliorate (e.g., reduce by at least about 10%) and / or prevent tissue and / or organ damage and / or failure; iii) control, ameliorate (e.g., reduce by at least about 10%) and / or prevent inflammation, CRS, sepsis, systemic inflammatory response syndrome (SIRS), septic shock and / or multiple sclerosis; organ dysfunction syndrome (MODS); iv) controlling, improving (e.g., reducing by at least about 10%) and / or preventing cell necrosis; v) reducing (e.g., reducing by at least about 10%) inflammatory markers, such as IL-6, IL-8, IL-10, IL1B, IL-12, IL-15, IL-17, CCL2, IL-1α, IL-2, IL-5, IL-9, CCL4, M-CSF, MCP-1, GCSF, MIP1A, CRP, TNFα, TNFβ, IFNγ, IP10, MCP1, SAA-1; and / or vi) prolonging (e.g., prolonging by at least about 10%) patient survival.

[0263] In the case of immune-related diseases associated with a reduced / eliminated immune response (e.g., immune cell depletion), the therapeutic methods, modified immune cells, or pharmaceutical compositions described herein can modulate the subject's immune response, such as inducing, activating, promoting, increasing, enhancing, or prolonging (e.g., by at least about 10%) the subject's immune response. For example, the proliferation / activation / persistence, cytokine release, and / or cytolytic or cytotoxic activity of the modified immune cells are enhanced.

[0264] In some embodiments, the treatment methods, modified immune cells, or pharmaceutical compositions described herein can i) extend (e.g., extend by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of immune cells (e.g., CAR-T or CAR-NK cells), ii) reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of immune cells (e.g., CAR-T or CAR-NK cells), iii) reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) the in vivo persistence of immune cells (e.g., CAR-T or CAR-NK cells), iv) reduce (e.g., reduce by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, or 100% or more) of the AICD of immune cells (e.g., CAR-T or CAR-NK cells), or increase (e.g., increase by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more) the resistance of immune cells (e.g., CAR-T or CAR-NK cells) to AICD, iii) decrease (e.g., decrease by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold or more) the resistance of immune cells (e.g., CAR-T or CAR-NK cells) to AICD, The invention also provides a method for improving the host versus graft (HvG) response of autologous or allogeneic immune cells (e.g., CAR-T or CAR-NK cells) by increasing any of: 10%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of the host versus graft (HvG) response of autologous or allogeneic immune cells (e.g., CAR-T or CAR-NK cells), such as reducing at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of the killing by allogeneic T cells, or reducing at least about 10% (e.g., at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%) of the killing by autologous or allogeneic NK cells. iv) does not downregulate or eliminate the expression and / or function (e.g., mediate cytotoxicity) of an engineered receptor (e.g., CAR) of an immune cell (e.g., CAR-T or CAR-NK cell), or has up to about 30% (e.g., up to about any of 25%, 20%, 10%, 5%, 3%, 1% or less) downregulated expression and / or function of an engineered receptor of an immune cell (e.g., CAR-T or CAR-NK cell).

[0265] The administration of the modified immune cell or its pharmaceutical composition can be carried out in any convenient manner, including by injection, infusion, implantation or transplantation. The modified immune cell or its pharmaceutical composition can be administered to the patient via artery, subcutaneous, intradermal, intratumoral, intramedullary, intramuscular, intravenous or intraperitoneal. In some embodiments, the modified immune cell or pharmaceutical composition is administered systemically. In some embodiments, the modified immune cell or its pharmaceutical composition is administered to an individual by infusion (e.g., intravenous infusion). The infusion technology of immunotherapy is known in the art (see, e.g., Rosenberg et al., New Eng. J. of Med. 319: 1676 (1988)). In some embodiments, the modified immune cell or its pharmaceutical composition is administered to an individual by intradermal or subcutaneous injection. In some embodiments, the modified immune cell or its pharmaceutical composition is administered by intravenous injection. In some embodiments, the modified immune cell or its pharmaceutical composition is directly injected into a tumor or lymph node. In some embodiments, the modified immune cell or its pharmaceutical composition is locally administered to the tumor site, for example, directly administered to the tumor cell, or administered to a tissue with a tumor cell.

[0266] The dosage of the modified immune cells of the present invention or their pharmaceutical compositions may vary depending on the specific use envisioned. The determination of an appropriate dosage or route of administration is well within the skill of those of ordinary skill. Animal experiments provide reliable guidance for determining effective dosages for human treatment. Interspecies scaling of effective dosages can be performed according to the principles set forth in Mordenti, J. and Chappell, W. "The Use of Interspecies Scaling in Toxicokinetics," In Toxicokinetics and New Drug Development, Yacobi et al., eds., Pergamon Press, New York 1989, pp. 42-46. Within the scope of this application, different formulations will be effective for different treatments and different conditions, and administration intended to treat a specific organ or tissue may need to be delivered in a manner different from the manner in which it is delivered to another organ or tissue.

[0267] In some embodiments, the modified immune cells described herein or pharmaceutical compositions thereof are expressed in an amount of at least about 10 4 , 10 5 , 10 6 , 10 7 , 10 8 or 10 9 In some embodiments, the modified immune cells or pharmaceutical compositions thereof are administered at a dose of about 10 cells / kg of individual body weight. 4 to about 10 5About 10 5 to about 10 6 About 10 6 to about 10 7 About 10 7 to about 10 8 About 10 8 to about 10 9 About 10 4 to about 10 9 About 10 4 to about 10 6 About 10 6 to about 10 8 or about 10 5 to about 10 7 cells / kg of individual body weight.

[0268] In some embodiments, the immune cell of modification as herein described or its pharmaceutical composition is by single administration.In some embodiments, the immune cell of modification or its pharmaceutical composition is applied repeatedly (for example, any one of 2,3,4,5,6 or more).In some embodiments, the immune cell of modification or its pharmaceutical composition is applied weekly, once in 2 weeks, once in 3 weeks, once in 4 weeks, once in every 1 month, once in every 2 months, once in every 3 months, once in every 4 months, once in every 5 months, once in every 6 months, once in every 7 months, once in every 8 months, once in every 9 months or once in a year.In some embodiments, the interval between application is any one of about 1 week to 2 weeks, 2 weeks to 1 month, 2 weeks to 2 months, 1 month to 2 months, 1 month to 3 months, 3 months to 6 months or 6 months to 1 year.The optimal dose of specific patient and treatment regimen can be easily determined by the technical staff of medical field by monitoring the disease signs of patient and adjusting treatment accordingly.

[0269] The dosage can be administered by one or more separate administrations or continuous infusions. In some embodiments, the modified immune cells or their pharmaceutical compositions are administered in divided doses, for example, in any one of about 2, 3, 4, 5 or more doses. In some embodiments, the divided doses are administered within about a week. In some embodiments, the dosage is equally distributed. In some embodiments, the divided doses are about 20%, about 30%, about 40% or about 50% of the total dose. In some embodiments, the intervals between consecutive divided doses are about 1 day, 2 days, 3 days or longer. For repeated administration over several days or longer, depending on the condition, treatment is continued until the desired disease symptoms are suppressed. However, other dosage regimens are useful. The progress of this treatment is easily monitored by traditional techniques and assays.

[0270] V. Methods for Identifying Target Genes in Immune Cells with Mutations Contributing to AICD Resistance

[0271] The present application provides a method for identifying a target gene that regulates (e.g., increases) resistance to AICD in immune cells. Any of the methods described in US20220064633 and WO2022143783 (e.g., immune cell library construction, sgRNA or sgRNA iBAR The target genes identified herein are particularly useful for patient selection / exclusion in disease treatment, such as cancer, infection, inflammation, immune-related diseases characterized by effector cell depletion, or immune cell donor selection / exclusion or adoptive cell therapy optimization / quality control. For example, individuals who carry a mutation (e.g., inactivation) of an AICD resistance gene identified herein (e.g., one or more of SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, and HIST1H1B, such as SPPL3), and / or whose AICD resistance gene expression (e.g., mRNA or protein) is reduced or absent compared to another individual (patient or healthy individual), and / or whose activity of the expression product (e.g., mRNA or protein) of the AICD resistance gene is reduced or eliminated compared to another individual (patient or healthy individual) are particularly suitable for autologous or allogeneic CAR-T or CAR-NK cell therapy, or for donating immune cells.

[0272] In some embodiments, a method is provided for identifying a target gene in an immune cell (e.g., a T cell, a B cell, or a NK cell) whose mutation increases (e.g., increases by at least about any of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 1-fold, 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more) resistance to AICD, the method comprising: a) providing an immune cell library comprising a plurality of immune cells, wherein each of the plurality of immune cells has a mutation (e.g., an inactivating mutation) at a hit gene ("hit gene mutation"), wherein the hit genes of at least two immune cells in the plurality of immune cells are different from each other; b) contacting the immune cell library with Fas ligand (FasL); c) obtaining an AICD-resistant immune cell population from the immune cell library; and d) identifying the target gene based on the difference between the profiles of the hit gene mutations in the AICD-resistant immune cell population and a control immune cell population. In some embodiments, the control immune cell population is a subpopulation of the immune cell library before step b). In some embodiments, the method also includes obtaining a subpopulation of the immune cell library before step b). In some embodiments, the control immune cell population is the same immune cell library cultured under the same conditions and not in contact with FasL. In some embodiments, the method also includes culturing the same immune cell library under the same conditions and not in contact with FasL. In some embodiments, the atlas of the hit gene mutations in the AICD-resistant immune cell population and the control immune cell population are identified by next generation sequencing (NGS). In some embodiments, the method comprises comparing sequence counts of sequences comprising hit gene mutations obtained from an AICD-resistant immune cell population with sequence counts of sequences comprising hit gene mutations obtained from a control immune cell population, wherein the hit genes whose corresponding hit gene mutation sequences are identified as enriched in the AICD-resistant immune cell population compared to the control immune cell population with a false discovery rate (FDR) ≤ 0.2 (and / or at least about 2-fold enrichment) are identified as target genes whose mutations increase resistance to AICD ("AICD resistance genes" or "FasL resistance genes"). In some embodiments, the immune cell library has at least about 100-fold (e.g., at least about 200-fold, 300-fold, 600-fold, 1000-fold, 2000-fold, 4000-fold, 6000-fold, 8000-fold, 10000-fold, 15000-fold or more) coverage for each hit gene, for example, about 1000-fold to about 12000-fold coverage for each hit gene. In some embodiments, each hit gene is targeted by at least 2 (e.g., 2, 3, 4, 5, 6, or more, such as 3 or 6-12) different hit gene mutations in the immune cell repertoire (e.g., targeting different target sites of the hit gene).In some embodiments, step c) includes using fluorescence activated cell sorting (FACS) or centrifugation (e.g., low-speed centrifugation) to obtain an AICD-resistant immune cell population. In some embodiments, step c) also includes contacting the immune cell library with a viability indicator, such as one or more of propidium iodide (PI), DAPI, 7-AAD, and annexin V. In some embodiments, the AICD-resistant immune cell population is annexin V-negative and DAPI-negative. In some embodiments, step c) includes obtaining annexin V-negative and DAPI-negative cells from the immune cell library using FACS. In some embodiments, step b) includes culturing the immune cell library for about 16 hours in the presence of FasL, for example, at 37°C, 5% CO2. In some embodiments, the sequence counts of the sequences including the hit gene mutations are normalized to the median ratio, and then mean variance modeling is performed. In some embodiments, the variance of each sequence including the hit gene mutation (e.g., inactivating mutation) is adjusted based on the data consistency between the same genes. In some embodiments, the data consistency between different hit gene mutation (e.g., inactivating mutation) sequences corresponding to the same hit gene is determined based on the direction of the fold change of each hit gene mutation sequence, wherein if for the same hit gene, the fold changes of different hit gene mutation sequences are in different directions relative to each other (e.g., increase and decrease, increase and unchanged, or decrease and unchanged are all considered different directions), then the variance of the hit gene mutation sequence increases. In some embodiments, the immune cell library comprises at least about 100 million cells.

[0273] In some embodiments, a library of immune cells is generated by contacting a starting population of immune cells (e.g., T cells such as CAR-T cells, or NK cells such as CAR-NK cells) with a mutagenic agent.

[0274] In some embodiments, immune cell libraries are produced by gene editing (for example, full genome or gene subset) to the initial group of immune cells (for example, T cells such as CAR-T cells, or NK cells such as CAR-NK cells).In some embodiments, under conditions where gRNA constructs and Cas components are allowed to be introduced into the initial group of immune cells and mutations are generated at hit genes, immune cell libraries are produced by contacting the initial group of immune cells with i) gRNA (such as sgRNA) libraries comprising multiple gRNA constructs, and ii) Cas components comprising Cas proteins or nucleic acids encoding Cas proteins, wherein each gRNA construct (such as a lentiviral vector or a lentivirus) includes or encodes gRNA, and wherein each gRNA includes a guide sequence complementary to the target site in the corresponding hit gene (for example, at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary).In some embodiments, gRNA (such as sgRNA) library and Cas components are simultaneously introduced into the initial group of immune cells. In some embodiments, gRNA library and Cas components are introduced into the initial group of immune cells in sequence.In some embodiments, the initial group of immune cells includes Cas components (such as Cas9).In some embodiments, gRNA constructs are allowed to be introduced into the initial group of immune cells comprising Cas (such as Cas9) and under conditions of generating mutations at hit genes, immune cell libraries are produced by contacting the initial group of immune cells comprising Cas components (such as Cas9) with gRNA (such as sgRNA) libraries comprising multiple gRNA constructs, wherein each gRNA construct (such as lentiviral vector or lentivirus) includes or encodes gRNA, and wherein each gRNA includes a guide sequence complementary to the target site in the corresponding hit gene (such as at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary).In some embodiments, Cas components are introduced into the initial group of immune cells before introducing gRNA library. In some embodiments, the immune cell pool is generated by i) contacting the initial population of immune cells with a Cas component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., a lentivirus or lentiviral vector encoding Cas9 or Cas9 mRNA) under conditions that allow for the introduction of the Cas component into the initial population of immune cells; ii) optionally obtaining a population of immune cells comprising the Cas component ("Cas9"). + iii) allowing the introduction of gRNA constructs into immune cells (e.g., Cas9-encoding markers on vectors); + immune cells) and produce mutations at the hit gene, so that the Cas +The immune cell population is contacted with a gRNA (e.g., sgRNA) library comprising a plurality of sgRNA constructs, wherein each gRNA construct (e.g., a lentiviral vector or lentivirus) comprises or encodes a gRNA, and wherein each gRNA comprises a guide sequence that is complementary to the target site in the corresponding hit gene (e.g., at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary). In some embodiments, the Cas protein is Cas9. In some embodiments, each gRNA (e.g., crRNA) includes a guide sequence fused to a second sequence, wherein the second sequence includes a direct repeat sequence (DR) that interacts with the Cas protein. In some embodiments, each gRNA (e.g., sgRNA) includes a guide sequence fused to a second sequence, wherein the second sequence includes a repeat anti-repeat stem loop that interacts with the Cas protein (e.g., Cas9). In some embodiments, the second sequence of each gRNA (e.g., sgRNA) further includes stem loop 1, stem loop 2, and / or stem loop 3. In some embodiments, each sgRNA further comprises an iBAR sequence ("sgRNA iBAR ”), where each sgRNA iBAR Can operate with Cas proteins to modify (e.g., cut or regulate expression) hit genes. In some embodiments, each sgRNA iBAR In some embodiments, the Cas protein is Cas9, and each sgRNA is a dsRNA. iBAR The iBAR sequence of the guide sequence is inserted into the loop region of the repeat anti-repeat stem loop. In some embodiments, each guide sequence includes about 17 to about 23 nucleotides. In some embodiments, at least about 95% (e.g., at least about 96%, 97%, 98%, 99% or more any one) of the gRNA library, for example, at least about 99% of the gRNA (e.g., sgRNA) constructs are introduced into the initial population of immune cells. In some embodiments, each hit gene in the immune cell library or gRNA (e.g., sgRNA) library is targeted by at least about 3 (e.g., about 6 to about 12) different gRNA constructs in at least about 3 (e.g., about 6 to about 12) different target sites of the hit gene. In some embodiments, the immune cell library targets each gRNA (e.g., sgRNA) or sgRNA iBARIn some embodiments, the immune cell library has a coverage of at least about 500 times (e.g., about 600 times to about 2000 times). In some embodiments, the immune cell library has a coverage of at least about 1000 times for each gRNA (e.g., sgRNA). In some embodiments, the immune cell library has a coverage of at least about 1500 times for each hit gene, for example, each hit gene has a coverage of about 2000 times to about 6000 times (e.g., 5000 times). In some embodiments, the gRNA (e.g., sgRNA) library comprises at least about 2000 gRNA constructs. In some embodiments, each gRNA (e.g., sgRNA) construct in the gRNA library is a plasmid. In some embodiments, each gRNA (e.g., sgRNA) construct in the gRNA library is a viral vector (e.g., a lentiviral vector). In some embodiments, the gRNA (e.g., sgRNA) library contacts the initial group of immune cells with an MOI of at least about 2 (e.g., 3).

[0275] In some embodiments, the sgRNA is allowed to iBAR Under conditions where the construct and Cas components are introduced into a primary population of immune cells and mutations are generated at the hit gene, the immune cell library is prepared by combining the primary population of immune cells with i) a plurality of sets of sgRNAs iBAR sgRNA constructs iBAR and ii) a Cas (e.g., Cas9) component comprising a Cas protein or a nucleic acid encoding a Cas protein (e.g., a lentiviral vector or lentivirus encoding Cas9 or Cas9 mRNA) to produce a library, wherein each set of sgRNA iBAR - The construct contains three or more (e.g., four) sgRNAs iBAR -Construct (e.g. lentiviral vector or lentivirus), each sgRNA iBAR -Construct contains or encodes sgRNA iBAR , where each sgRNA iBAR comprising a guide sequence and an iBAR sequence, wherein the three or more (e.g., four) sgRNAs iBAR The guide sequences of the constructs are identical and complementary to the same target site of the hit gene (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary), wherein the three or more (e.g., four) sgRNAs iBAR The iBAR sequence of each construct was different from each other, wherein each set of sgRNA iBAR The guide sequence of the construct is complementary to different target sites of the hit gene (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA iBARCan be operated with Cas (e.g., Cas9) proteins to modify target sites. In some embodiments, the initial population of immune cells includes a Cas component (e.g., Cas9). In some embodiments, the sgRNA is allowed to iBAR The construct is introduced into an initial population of immune cells containing Cas (e.g., Cas9) and a mutation is generated at the hit gene, by combining the initial population of immune cells containing Cas (e.g., Cas9) components with multiple sets of sgRNAs. iBAR sgRNA constructs iBAR The immune cell library is generated by contacting the library, in which each set of sgRNA iBAR The construct contains three or more (e.g., four) sgRNAs iBAR Construct (e.g., lentiviral vector or lentivirus), each sgRNA iBAR Constructs containing or encoding sgRNA iBAR , where each sgRNA iBAR comprising a guide sequence and an iBAR sequence, wherein the three or more (e.g., four) sgRNAs iBAR The guide sequences of the constructs are identical and complementary to the same target site of the hit gene (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary), wherein three or more (e.g., four) sgRNAs iBAR The iBAR sequence of each construct was different from each other, where each set of sgRNA iBAR The guide sequence of the construct is complementary to different target sites of the hit gene (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA iBAR Can be operated with Cas (e.g., Cas9) proteins to modify target sites. In some embodiments, under conditions that allow the introduction of Cas components into an initial population of immune cells, a library of immune cells is generated by i) contacting the initial population of immune cells with Cas components comprising Cas proteins or nucleic acids encoding Cas proteins (e.g., lentiviral vectors or lentiviruses encoding Cas9 or Cas9 mRNA); ii) optionally obtaining a population of immune cells comprising Cas components ("Cas9"). + iii) allowing the sgRNA to be expressed in the presence of a marker on a Cas-encoding vector; iBAR Constructs are introduced into immune cells (e.g., Cas9 + immune cells) and produce mutations at the hit gene, so that the Cas + Immune cell populations and multiple sets of sgRNAs iBAR sgRNA constructs iBARLibrary contacts, where each set of sgRNA iBAR The construct contains three or more (e.g., four) sgRNAs iBAR Construct (e.g., lentiviral vector or lentivirus), each sgRNA iBAR Constructs containing or encoding sgRNA iBAR , where each sgRNA iBAR comprising a guide sequence and an iBAR sequence, wherein the three or more (e.g., four) sgRNAs iBAR The guide sequences of the constructs are identical and complementary to the same target site of the hit gene (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary), wherein three or more (e.g., four) sgRNAs iBAR The iBAR sequence of each construct was different from each other, where each set of sgRNA iBAR The guide sequence of the construct is complementary to different target sites of the hit gene (e.g., different hit genes, or different sites within the same hit gene), and wherein each sgRNA iBAR Can operate with Cas (e.g., Cas9) proteins to modify target sites. In some embodiments, the sgRNAiBAR constructs and Cas (e.g., Cas9) components are introduced into a primary population of immune cells and generate mutations at the hit gene under conditions that allow the sgRNAiBAR constructs and Cas (e.g., Cas9) components to be introduced into a primary population of immune cells and generate mutations at the hit gene, by inducing the primary population of immune cells to generate mutations at the hit gene; i) sgRNAs comprising multiple sets of sgRNAiBAR constructs; iBAR The immune cell library is produced by contacting a Cas (eg Cas9) component comprising a Cas (eg Cas9) protein or a nucleic acid encoding a Cas (eg Cas9) protein, wherein each set of sgRNAs iBAR The construct contains three or more (e.g., four) sgRNAs iBAR Construct, each sgRNA iBAR Constructs containing or encoding sgRNA iBAR , a second sequence and an iBAR sequence, wherein three or more (e.g., four) sgRNAs iBAR The guide sequences of the constructs are identical and complementary to the same target site of the hit gene (e.g., at least about any of 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% complementary), wherein the three or more (e.g., four) sgRNAs iBARThe iBAR sequence of each construct is different from each other, wherein the guide sequence is fused to a second sequence, wherein the second sequence comprises a repeat-inverse-repeat stem-loop that interacts with a Cas (e.g., Cas9) protein, wherein the iBAR sequence is inserted into the loop region of the repeat-inverse-repeat stem-loop, wherein each set of sgRNAs iBAR The guide sequence of the construct is complementary to different target sites of the hit gene (e.g., different hit genes, or different target sites of the same hit gene), and wherein each sgRNA iBAR Can be operated with Cas (e.g., Cas9) proteins to modify target sites. In some embodiments, upon introduction of sgRNA iBAR In some embodiments, the sgRNA is introduced into the immune cell before the Cas component (e.g., Cas9) is introduced into the immune cell. iBAR In some embodiments, the Cas component (e.g., Cas9) and sgRNA are introduced into immune cells. iBAR In some embodiments, each iBAR sequence comprises about 1 to about 50 (e.g., 6) nucleotides. In some embodiments, each set of sgRNAs iBAR The construct includes four sgRNAs iBAR construct, and four sgRNAs iBAR The iBAR sequence of each construct is different from each other. In some embodiments, the sgRNA iBAR The library contains at least about 100 (e.g., 2000) sets of sgRNAs iBAR Construct. In some embodiments, the sgRNA iBAR The library contains at least about 2000 sgRNAs iBAR In some embodiments, different sets of sgRNAs iBAR At least two sgRNAs in the construct iBAR The iBAR sequences of the constructs were identical (e.g., the first and second sets of sgRNAs iBAR Constructs in two sets of sgRNA iBAR In some embodiments, at least two sets of sgRNAs have at least 1, 2, 3, 4, or more common iBAR sequences between constructs. iBAR The iBAR sequences of the constructs are identical. In some embodiments, the sgRNA iBAR Each sgRNA in the library iBAR The construct is a plasmid. In some embodiments, the sgRNA iBAR Each sgRNA in the library iBAR The construct is a viral vector (e.g., a lentiviral vector). In some embodiments, the sgRNA iBARThe library is contacted with the initial population of immune cells at an MOI greater than about 2 (e.g., at least about 3, 5, or 10), such as 3. In some embodiments, the library comprises multiple sgRNAs. iBAR sgRNA constructs iBAR The library contains or encodes sgRNAs with guide sequences complementary to target sites of hit genes associated with one or more of AICD, GvHD, HvG, immune cell proliferation, differentiation, maturation, activation, persistence, homeostasis, and effector function iBAR In some embodiments, the sgRNA iBAR At least about 95% (e.g., at least about any of 96%, 97%, 98%, 99%, or more), such as at least about 99% of the sgRNAs in the library iBAR In some embodiments, the immune cell library or sgRNA iBAR Each hit gene in the library was targeted by three different sgRNAs at three different target sites of the hit gene. iBAR Construct targeting. In some embodiments, the immune cell library targets each sgRNA iBAR With at least about 500-fold coverage, for example, for each sgRNA iBAR With a coverage of about 1000 to about 1500 times. In some embodiments, the immune cell library has a coverage of about 1000 to about 1500 times for each sgRNA iBAR In some embodiments, the immune cell library has at least about 1000-fold coverage for each set of sgRNAs. iBAR With at least about 2000-fold coverage, for example, for each set of sgRNAs iBAR About 4000 to about 6000-fold coverage. In some embodiments, the immune cell library has at least about 2000-fold coverage for each hit gene, for example, about 4000-fold to about 12000-fold coverage for each hit gene.

[0276] In some embodiments, the sgRNAs described herein are used iBARLibrary screening methods can improve target identification and data reproducibility through statistical analysis and reduce FDR. In traditional CRISPR / Cas-based screening methods using mixed sgRNA libraries, low MOI is used during the cell library construction process to generate high-quality cell libraries expressing gRNAs to ensure that each cell contains, on average, less than one sgRNA or paired guide RNA ("pgRNA"). Because the sgRNA molecules in the library are randomly integrated into the transfected cells, a sufficiently low MOI ensures that each cell expresses a single sgRNA, thereby minimizing the FDR of the screening. To further reduce the FDR and increase the reproducibility of the data, in-depth coverage of gRNAs and multiple biological replicates are often required to obtain hit genes with high statistical significance. When large numbers of whole-genome screens are required, when the cell material for library construction is limited, or when more challenging screens (i.e., in vivo screening) are performed, traditional screening methods face difficulties in arranging experimental replicates or controlling MOI. The use of sgRNAs described herein iBAR The library screening method overcomes this difficulty by including an iBAR sequence in each sgRNA, which enables the collection of internal duplicates within each sgRNA group with the same guide sequence but different iBAR sequences. This iBAR approach can reduce experimental noise. For example, as shown in WO2020125762 (the contents of which are incorporated herein by reference in their entirety), an iBAR with four nucleotides per sgRNA can provide sufficient internal replication to evaluate different sgRNAs targeting the same genomic locus. iBAR Data consistency between constructs. The high consistency between the two independent experiments in WO2020125762 suggests that one experimental replicate is sufficient for CRISPR / Cas screening using the iBAR method. Because library coverage increases significantly with high MOI during viral transduction of host cells, the number of cells in the initial cell population can be reduced by more than 20-fold to achieve the same library coverage, as shown for the whole-genome human library constructed in WO2020125762. Similarly, using sgRNA iBARThe workload of each genome-wide screening can be proportionally reduced. Using sgRNAs with different iBAR sequences allows the performance of each guide sequence to be tracked multiple times in the same experiment by counting the guide sequence and the corresponding iBAR nucleotide sequence, significantly reducing the FDR and improving efficiency and reliability. Transduction efficiency and library coverage can be further improved by using a high viral titer in the viral transduction step, for example, using a high viral titer of MOI>1 (e.g., MOI>1.5, MOI>2, MOI>2.5, MOI>3, MOI>3.5, MOI>4, MOI>4.5, MOI>5, MOI>5.5, MOI>6, MOI>6.5, MOI>7, MOI>7.5, MOI>8, MOI>8.5, MOI>9, MOI>9.5, or MOI>10; for example, an MOI of about any of 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10).

[0277] In some embodiments, a method for identifying a target gene in an immune cell (e.g., a T cell such as a CAR-T cell) whose mutation (e.g., a LOF mutation) increases resistance to AICD is provided, comprising: a) providing a gRNA (e.g., sgRNA) or sgRNA comprising one or more hit genes as described herein; iBAR b) contacting the immune cell library with FasL; c) obtaining an AICD-resistant immune cell population from the immune cell library; and d) generating an immune cell library based on gRNA or sgRNA in the AICD-resistant immune cell population and the control immune cell population. iBAR or hit gene mutations to identify the target gene. In some embodiments, the control immune cell population is a subpopulation of the immune cell library before step b). In some embodiments, the control immune cell population is the same immune cell library cultured under the same conditions and not in contact with FasL. In some embodiments, the method further comprises obtaining a control immune cell population before step b). In some embodiments, the method further comprises culturing the same immune cell library under the same conditions and not in contact with FasL. In some embodiments, a method is provided for identifying a target gene whose mutation (e.g., LOF mutation) increases resistance to AICD in an immune cell (e.g., T cell such as CAR-T cell), comprising: a1) providing a gRNA (e.g., sgRNA) or sgRNA targeting one or more hit genes as described herein; iBARa2) optionally growing the immune cell library (e.g., at 37° C., 5% CO 2 for about 6 days); a3) obtaining a subpopulation of the immune cell library from a1) or a2) as a control immune cell population; b) contacting the immune cell library (from step a1) or a2) respectively) with FasL; c) obtaining an AICD-resistant immune cell population from the immune cell library from step b); and d) generating an immune cell library based on the gRNA (e.g., sgRNA) or sgRNA in the AICD-resistant immune cell population and the control immune cell population. iBAR In some embodiments, a method for identifying a target gene whose mutation (e.g., LOF mutation) increases resistance to AICD in an immune cell (e.g., a T cell such as a CAR-T cell) is provided, comprising: a1) providing a gRNA (e.g., sgRNA) or sgRNA targeting one or more hit genes as described herein; iBAR a2) culturing the same immune cell library under the same conditions as the control immune cell population and not undergoing step b); b) contacting the immune cell library from step a1) with FasL; c) obtaining an AICD-resistant immune cell population from the immune cell library from step b); and d) generating an AICD-resistant immune cell population based on gRNA (e.g., sgRNA) or sgRNA in the AICD-resistant immune cell population and the control immune cell population. iBAR or hit gene mutations to identify the target gene. In some embodiments, step c) includes using FACS or centrifugation (e.g., low speed) to obtain an AICD-resistant immune cell population. In some embodiments, step c) also includes contacting the immune cell library with a vitality indicator such as one or more of PI, DAPI, 7-AAD, and Annexin V after step b). In some embodiments, the AICD-resistant immune cell population is Annexin V-negative and DAPI-negative. In some embodiments, step b) includes culturing the immune cell library for about 16 hours in the presence of FasL. In some embodiments, a method is provided for identifying a target gene whose mutation (e.g., LOF mutation) increases resistance to AICD in an immune cell (e.g., T cell such as CAR-T cell), comprising: a1) providing a gRNA (e.g., sgRNA) or sgRNA targeting one or more hit genes as described herein iBAR a2) optionally growing the immune cell library (e.g., at 37° C., 5% CO 2 for about 6 days); b) contacting the immune cell library (respectively from a1) or a2)) with FasL; c) (after step b)) obtaining an AICD-resistant immune cell population from the immune cell library by centrifugation (e.g., at a low speed); and d) generating an immune cell library based on the gRNA (e.g., sgRNA) or sgRNA in the AICD-resistant immune cell population and the control immune cell population.iBAR or hit gene mutations to identify the target gene. In some embodiments, a control immune cell population is obtained by culturing the same immune cell pool under the same conditions and without contacting with FasL. In some embodiments, the sequence counts obtained from the AICD-resistant immune cell population are compared with the corresponding sequence counts obtained from the control immune cell population to provide a fold change (e.g., an actual fold change, or a derivative of the fold change, such as a log2 or log10 fold change). In some embodiments, the identification of the target gene is based on the difference between the gRNA (e.g., sgRNA) or sgRNA in the AICD-resistant immune cell population and the control immune cell population. iBAR In some embodiments, gRNA (e.g., sgRNA) or sgRNA in AICD-resistant immune cell populations and control immune cell populations is identified by NGS. iBAR In some embodiments, identifying the target gene in step d) comprises: obtaining a gRNA (e.g., sgRNA) or sgRNA from an AICD-resistant immune cell population. iBAR (or its guide sequence) sequence counts compared to gRNA (e.g., sgRNA) or sgRNA obtained from a control immune cell population iBAR (or its guide sequence) sequence count comparison, wherein: its corresponding gRNA (e.g., sgRNA) or sgRNA is compared to a control immune cell population with an FDR ≤ 0.2 (and / or at least about 2-fold enrichment) iBAR The hit genes whose guide sequences are identified as enriched in the AICD-resistant immune cell population are identified as target genes whose mutations increase resistance to AICD (AICD resistance genes). Therefore, in some embodiments, a method for identifying target genes in immune cells (e.g., T cells, such as CAR-T cells) whose mutations (e.g., LOF mutations) increase resistance to AICD is provided, comprising: a1) providing a gRNA (e.g., sgRNA) comprising one or more hit genes as described herein; iBAR ) library, and optionally growing the immune cell library (e.g., at 37° C., 5% CO 2 for about 6 days); a2) culturing the same immune cell library under the same conditions as the control immune cell library and without undergoing step b); b) contacting the immune cell library (from step a1)) with FasL; c) obtaining viable cells from the immune cell library using low speed centrifugation after step b) to obtain an AICD-resistant immune cell population; and d) determining the expression of gRNA (e.g., sgRNA) based on the expression of gRNAs (e.g., sgRNAs) in the AICD-resistant immune cell population and the control immune cell population. iBAR ) by comparing the difference between the profiles of the AICD-resistant immune cells (e.g., by NGS) to identify the target gene, wherein step d) comprises i) isolating a gRNA (e.g., sgRNA) obtained from the AICD-resistant immune cell population iBAR) sequence counts compared to gRNAs obtained from control immune cell populations (e.g., sgRNA iBAR ) sequence counts, wherein the corresponding gRNA (e.g., sgRNA) is enriched compared to a control immune cell population with an FDR ≤ 0.2 (and / or at least about 2-fold enrichment). iBAR ) guide sequence is identified as a hit gene enriched in the AICD-resistant immune cell population is identified as a target gene whose mutation increases resistance to AICD (AICD resistance gene). In some embodiments, gRNA (e.g., sgRNA) or sgRNA iBAR The library targets genes associated with one or more of AICD, GvHD, HvG, immune cell proliferation, differentiation, maturation, activation, persistence, homeostasis, and effector function. In some embodiments, the immune cell library targets one or more genes associated with each sgRNA. iBAR With coverage of about 100-fold to about 2000-fold, for example, for each sgRNA iBAR In some embodiments, the immune cell library has at least about 400-fold coverage for each hit gene, for example, about 1200-fold to about 12,000-fold coverage for each hit gene. In some embodiments, identifying the target gene in step d) includes: i) identifying a gRNA (e.g., sgRNA) or sgRNA in the AICD-resistant immune cell population; iBAR sequence; and ii) identifying a region corresponding to a gRNA (e.g., sgRNA) or sgRNA iBAR In some embodiments, identifying the target gene in step d) comprises: i) obtaining sgRNA in an AICD-resistant immune cell population; iBAR Sequence; ii) sgRNA based on sequence counts iBAR The sequence is sorted based on the corresponding guide sequence of the sequence, wherein the sorting includes based on the sgRNA corresponding to the guide sequence iBAR and iii) identifying hit genes corresponding to guide sequences that rank above a predetermined threshold level. In some embodiments, the method is a positive screen. In some embodiments, the gRNA (e.g., sgRNA) or sgRNA is screened for hits. iBAR Sequence counts were normalized to median ratios and then subjected to mean variance modeling. In some embodiments, the sgRNAs corresponding to the guide sequences were used to generate the sgRNAs. iBAR The variance of each guide sequence is adjusted based on the data consistency between the iBAR sequences in the sequence. In some embodiments, the sgRNA corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence. iBARIn some embodiments, the variance of each guide sequence is adjusted based on the data consistency between the same genes. In some embodiments, the data consistency between different guide sequences corresponding to the same hit gene is determined based on the direction of the fold change of each guide sequence, wherein for the same hit gene, if the fold change of different guide sequences is in different directions relative to each other (e.g., increase and decrease, increase and unchanged, or decrease and unchanged), the variance of the guide sequence is increased.

[0278] In some embodiments, the immune cell library undergoes at least two rounds (e.g., 2 rounds, 3 rounds, 4 rounds or more) of FasL treatment. In some embodiments, after obtaining the AICD-resistant immune cell population in step c), the AICD-resistant immune cell population is cultured under certain conditions (e.g., at 37° C., 5% CO 2 ) for about 5 to about 7 days, for example, 6 days, or about 3 PDT, to grow to an immune cell population that is about the same or similar (e.g., a variance of up to about 10%) as the number of immune cell libraries before FasL treatment (e.g., 100 million cells). In some embodiments, after contacting the immune cell library with FasL (e.g., culturing for about 16 hours in the presence of FasL), the FasL-treated immune cell library is contacted with a viability indicator, such as one or more of PI, DAPI, 7-AAD, and Annexin V, and the immune cell apoptosis rate or survival rate is detected. For example, Annexin-V negative and DAPI-negative cells are viable cells or cells that are more resistant to AICD / FasL treatment.

[0279] In some embodiments, a method for identifying a target gene in an immune cell (e.g., a T cell such as a CAR-T cell) whose mutation (e.g., a LOF mutation) increases resistance to AICD is provided, comprising: a) providing a gRNA (e.g., sgRNA) or sgRNA comprising one or more hit genes as described herein; iBARb1) contacting the immune cell library with a first FasL ("first FasL treatment step"); c1-i) obtaining a first AICD-resistant immune cell population from the immune cell library ("first obtaining step"; e.g., by low-speed centrifugation or FACS); c1-ii) optionally culturing the first AICD-resistant immune cell population for about 6 days ("optional first recovery step"); b2) optionally contacting the first AICD-resistant immune cell population with a second FasL ("optional second FasL treatment step"); c2-i) optionally obtaining a second AICD-resistant immune cell population from step b2) ("optional second obtaining step"; e.g., by low-speed centrifugation or FACS); FACS); c2-ii) optionally culturing the second AICD-resistant immune cell population for about 6 days ("optional second recovery step"); b3) optionally contacting the second AICD-resistant immune cell population with a third FasL ("optional third FasL treatment step"); c3-i) optionally obtaining a final AICD-resistant immune cell population from step b3) ("optional third obtaining step"; e.g., by low speed centrifugation or FACS); c3-ii) optionally culturing the final AICD-resistant immune cell population for about 6 days (optional third recovery step); and d) determining the expression of a gRNA (e.g., sgRNA) or sgRNA based on the expression of the gRNA in the final AICD-resistant immune cell population and the control immune cell population. iBAR or hit gene mutations to identify the target gene. In some embodiments, the first, second, and third FasLs have the same concentration. In some embodiments, the first, second, and third FasLs have different concentrations. In some embodiments, a method for identifying a target gene in an immune cell (e.g., a T cell such as a CAR-T cell) whose mutation (e.g., a LOF mutation) increases resistance to AICD is provided, comprising: a) providing a gRNA (e.g., sgRNA) or sgRNA comprising one or more hit genes as described herein; iBARb1) contacting the immune cell library with a first FasL ("first FasL treatment step"); c1-i) centrifuging the immune cell library after step b1) to obtain a first AICD-resistant immune cell population ("first obtaining step"); c1-ii) culturing the first AICD-resistant immune cell population for about 6 days ("first recovery step"); b2) contacting the first AICD-resistant immune cell population with the FasL ("second FasL treatment step"); c2-i) centrifuging the first AICD-resistant immune cell population after step b2) to obtain a second AICD-resistant immune cell population ( c2-ii) culturing the second AICD-resistant immune cell population for about 6 days ("second recovery step"); b3) contacting the second AICD-resistant immune cell population with FasL ("third FasL treatment step"); c3-i) centrifuging the second AICD-resistant immune cell population after step b3) to obtain a final AICD-resistant immune cell population ("third obtaining step"); c3-ii) culturing the final AICD-resistant immune cell population for about 6 days ("third recovery step"); and d) injecting a gRNA (e.g., sgRNA) or sgRNA obtained from the final AICD-resistant immune cell population. iBAR or sequence counts of sequences containing hit gene mutations compared to gRNAs (e.g., sgRNAs) or sgRNAs obtained from control immune cell populations iBAR The sequence counts of the hit gene mutations or the sequence counts of the hit gene mutations are compared, wherein the corresponding gRNA (e.g., sgRNA) or sgRNA is compared with the control immune cell population with an FDR ≤ 0.2 (and / or at least about 2-fold enrichment). iBAR The guide sequence or sequence comprising the hit gene mutation is identified as a hit gene that is enriched in the final AICD-resistant immune cell population and is identified as a target gene (AICD-resistance gene) whose mutation increases resistance to AICD. In some embodiments, the control immune cell population is the same immune cell library cultured under the same conditions (e.g., at 37°C, 5% CO2, for the same duration as the test immune cell library) and is not exposed to any FasL. In some embodiments, the method further comprises culturing the same immune cell library under the same conditions and not contacting with any FasL. In some embodiments, the immune cell library is cultured for each sgRNA iBAR With coverage of about 100-fold to about 2000-fold, for example, for each sgRNA iBAR In some embodiments, the immune cell library has at least about 400-fold coverage for each hit gene, for example, about 1200-fold to about 12,000-fold coverage for each hit gene. In some embodiments, the immune cell library has at least about 400-fold coverage for each hit gene, for example, about 1200-fold to about 12,000-fold coverage for each hit gene. iBARSequence counts were normalized to median ratios and then subjected to mean variance modeling. In some embodiments, the sgRNAs corresponding to the guide sequences were used to generate the sgRNAs. iBAR The variance of each guide sequence is adjusted based on the data consistency between the iBAR sequences in the sequence. In some embodiments, the sgRNA corresponding to each guide sequence is determined based on the direction of the fold change of each iBAR sequence. iBAR In some embodiments, the variance of each guide sequence is adjusted based on the data consistency between the same genes. In some embodiments, the data consistency between different guide sequences corresponding to the same hit gene is determined based on the direction of the fold change of each guide sequence, wherein for the same hit gene, if the fold change of different guide sequences is in different directions relative to each other (e.g., increase and decrease, increase and unchanged, or decrease and unchanged), the variance of the guide sequence is increased.

[0280] In some embodiments, any of the identification methods described herein further comprises ranking the identified target genes, wherein...

Claims

1. An immune cell, wherein the immune cell is modified to have no or reduced expression and / or function of one or more target proteins, wherein the target proteins are selected from: signal peptide peptidase-like 3 (SPPL3), FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B. 2 . The immune cell according to claim 1 , wherein the immune cell is modified to have no or reduced expression and / or function of SPPL3 protein.

3. The immune cell of claim 2, wherein the immune cell has at least about 10% less activation-induced cell death (AICD) compared to a reference immune cell that does not have a modification that reduces or eliminates expression and / or function of SPPL3 protein.

4. The immune cell according to claim 2 or 3, wherein: a) reducing or inhibiting the expression of SPPL3 protein by using antisense RNA, siRNA or shRNA that specifically recognizes RNA encoding SPPL3 protein; b) the immune cell is modified to express a dominant negative SPPL3 protein variant or a dominant negative fragment thereof; or c) The immune cell is genetically modified at the SPPL3 locus or SPPL3 RNA.

5. The immune cell of claim 4, wherein the immune cell is genetically modified at the SPPL3 locus or at the SPPL3 RNA, and wherein the SPPL3 locus is modified by gene editing, or wherein the SP PL3 RNA is modified by RNA editing.

6. The immune cell of claim 5, wherein the gene editing or RNA editing is mediated by CRISPR / Cas.

7. The immune cell of claim 6, wherein the gene editing or RNA editing comprises contacting the precursor immune cell with i) a guide RNA (gRNA) construct and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under conditions that allow the introduction of the gRNA construct and optionally the Cas component into the precursor immune cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to a target site in the SPPL3 locus or SPPL3 RNA.

8. The immune cell of claim 7, wherein the precursor immune cell expresses a Cas protein.

9. The immune cell according to claim 7 or 8, wherein the Cas protein has endonuclease activity.

10. The immune cell according to claim 7 or 8, wherein the Cas protein is a fusion protein comprising: i) a dead Cas protein (dCas), and ii) adenine deaminase (ADA) or cytidine deaminase (CDA) or a functional fragment thereof.

11. An immune cell according to any one of claims 7-10, wherein the Cas protein is Cas9.

12. The immune cell according to any one of claims 7 to 11, wherein the guide sequence is encoded by a nucleic acid sequence comprising a sequence of SEQ ID NO:

1.

13. The immune cell according to any one of claims 1-12, wherein the immune cell does not have or reduces the expression and / or function of one or more other proteins, or is further modified to not have or reduce the expression and / or function of one or more other proteins, wherein the other proteins are selected from: TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6 and ligands of NKp46.

14. The immune cell of any one of claims 1-13, wherein the immune cell expresses or is further modified to express an engineered receptor.

15. The immune cell of claim 14, wherein the engineered receptor is a chimeric antigen receptor (CAR), an engineered TCR, or a T cell antigen conjugate (TAC).

16. The immune cell of claim 15, wherein the engineered receptor is a CAR comprising: i) an extracellular antigen-binding domain that specifically recognizes the target antigen; ii) a transmembrane domain; and iii) Intracellular signal transduction domain.

17. The immune cell according to any one of claims 14-16, wherein the modification that reduces or eliminates the expression and / or function of the one or more target proteins: i) does not downregulate or eliminate the expression and / or function of the engineered receptor; or ii) downregulating the expression and / or function of said engineered receptor by up to about 30%.

18. The immune cell of any one of claims 1-17, wherein the immune cell is a T cell, a B cell or a natural killer (NK) cell, optionally wherein the immune cell is a T cell.

19. The immune cell of claim 18, wherein the modification that reduces or eliminates the expression and / or function of the one or more target proteins: i) reducing the cell surface expression of one or more of Fas, HLA-A, HLA-B, HLA-C, HLA-E, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and NKp46 ligand by at least about 10%; ii) reducing killing by allogeneic T cells by at least about 10%; and / or iii) reducing killing by autologous or allogeneic NK cells by at least about 10%.

20. The immune cell of any one of claims 1-19, wherein the immune cell has at least about 10% longer in vivo persistence compared to a reference immune cell that does not have a modification that reduces or eliminates expression and / or function of the one or more target proteins.

21. The immune cell of any one of claims 1-20, which is autologous.

22. The immune cell according to any one of claims 1-20, which is allogeneic.

23. A method for identifying an individual as a suitable donor of immune cells with prolonged in vivo persistence, comprising examining the expression and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B in the individual, wherein identification of reduced or abolished expression and / or function of the one or more target proteins compared to a reference identifies the individual as a suitable donor.

24. A method for excluding an individual as a suitable donor of immune cells with prolonged in vivo persistence, comprising examining the expression and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B in the individual, wherein if no reduction or elimination of expression and / or function of the one or more target proteins compared to a reference is identified, the individual is excluded as a suitable donor.

25. The method of claim 23 or 24, wherein the reference is the average expression and / or function of the one or more target proteins in a population of individuals.

26. The method of any one of claims 23-25, wherein examining the expression and / or function of the one or more target proteins comprises examining the sequence of a nucleic acid encoding the one or more target proteins, wherein identification of a mutation in the nucleic acid that reduces the expression and / or function of the one or more target proteins identifies the individual as a suitable donor.

27. A method for i) extending the in vivo persistence of immune cells, ii) reducing the AICD of immune cells, and / or iii) reducing the host versus graft (HvG) response of immune cells, comprising modifying immune cells to reduce or eliminate the expression and / or function of one or more target proteins selected from SPPL3, FADD, FAS, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2 and HIST1H1B.

28. The method of claim 27, wherein the method comprises modifying the immune cells to reduce or eliminate the expression and / or function of the SPPL3 protein.

29. The method of claim 28, wherein: a) reducing or inhibiting the expression of the SPPL3 protein by using antisense RNA, siRNA or shRNA that specifically recognizes the RNA encoding the SPPL3 protein; b) the immune cell is modified to express a dominant negative SPPL3 protein variant or a dominant negative fragment thereof; or c) The immune cell is genetically modified at the SPPL3 locus or SPPL3 RNA.

30. The method of claim 29, wherein the immune cell is genetically modified at the SPPL3 locus or at SPPL3 RNA, and wherein the SPPL3 locus is modified by gene editing, or wherein the SPPL3 RNA is modified by RNA editing.

31. The method of claim 30, wherein the gene editing or RNA editing is mediated by CRISPR / Cas.

32. The method of claim 31 , comprising contacting a precursor immune cell with i) a gRNA construct and optionally ii) a Cas component comprising a Cas protein or a nucleic acid encoding the Cas protein, under conditions that allow introduction of the gRNA construct and optionally the Cas component into the precursor immune cell, wherein the gRNA construct comprises or encodes a gRNA comprising a guide sequence complementary to a target site in the SPPL3 locus or SPPL3 RNA.

33. The method of claim 32, wherein the precursor immune cells express a Cas protein.

34. The method of claim 32 or 33, wherein the Cas protein has endonuclease activity.

35. The method of claim 32 or 33, wherein the Cas protein is a fusion protein comprising i) dCas and ii) ADA or CDA or a functional fragment thereof.

36. A method according to any one of claims 32-35, wherein the Cas protein is Cas9.

37. The method of any one of claims 32-36, wherein the guide sequence is encoded by a nucleic acid sequence comprising the sequence of SEQ ID NO:

1.

38. The method of any one of claims 27-37, further comprising modifying the immune cells to reduce or eliminate the expression and / or function of one or more other proteins selected from the group consisting of TCRα, TCRβ, TCRγ, TCRδ, HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, B2M, PD-1, TIM-3, LAG-3, CTLA-4, CISH, Fas, FADD, CASP8, ARID1A, BAK1, BID, ETS1, IKZF2, HIST1H1B, B7-H6, MICA, MICB, ULBP1, ULBP2, ULBP3, ULBP4, ULBP5, ULBP6, and ligands of NKp46.

39. The method of claim 38, wherein the immune cell is genetically modified at one or more loci or RNA encoding the one or more other proteins.

40. The method of any one of claims 27-39, wherein the immune cell expresses an engineered receptor.

41. The method of any one of claims 27-39, further comprising introducing a nucleic acid encoding an engineered receptor into the immune cell.

42. The method of claim 41, wherein the nucleic acid encoding the engineered receptor, the nucleic acid encoding the gRNA against SPPL3, and / or the nucleic acid encoding the Cas protein are on different vectors.

43. The method of any one of claims 40-42, wherein the engineered receptor is a CAR, an engineered TCR, or a TAC.

44. The method of claim 43, wherein the engineered receptor is a CAR comprising: i) an extracellular antigen-binding domain that specifically recognizes the target antigen; ii) a transmembrane domain; and iii) Intracellular signal transduction domain.

45. The method of any one of claims 40-44, wherein the modification that reduces or eliminates the expression and / or function of the one or more target proteins: i) does not downregulate or eliminate the expression and / or function of the engineered receptor; or ii) downregulating the expression and / or function of said engineered receptor by up to about 30%.

46. ​​The method of any one of claims 27-45, wherein the immune cell is a T cell, a B cell, or a NK cell.

47. The method of claim 46, wherein the immune cell is a T cell.

48. An immune cell obtained by the method of any one of claims 27-47.

49. A pharmaceutical composition comprising the immune cell according to any one of claims 1-22 or 48 and optionally a pharmaceutically acceptable excipient.

50. A method of treating a disease in an individual, comprising administering to the individual an effective amount of the immune cell according to any one of claims 1-22 and 48 or the pharmaceutical composition according to claim 49.

51. The method of claim 50, wherein the disease is associated with expression of a target antigen, and wherein the immune cell expresses an engineered receptor that specifically recognizes the target antigen.

52. The method of claim 51, wherein the engineered receptor is a CAR.

53. The method of any one of claims 50-52, wherein the disease is cancer, infection, inflammation, an autoimmune disease, or an immune-related disease characterized by effector cell exhaustion.

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