WO2022012531A1 - Method for preparing modified immune cell - Google Patents

Abstract

A method for preparing a modified immune cell. The modification comprises the following steps: a) reducing the expression and/or activity of a Fas protein in the immune cell, and b) increasing the expression and/or activity of a FasL protein in the immune cell. Thus, compared to an immune cell which is not subjected to the modification, the expression and/or activity of the Fas protein in the modified immune cell are reduced or eliminated, and the expression and/or activity of the FasL protein in the modified immune cell are increased.

Description

A kind of preparation method of modified immune cells technical field

The present application relates to the field of biomedicine, in particular to a method for preparing a universal chimeric antigen T cell, including knocking out Fas protein and transferring FasL protein.

Background technique

Immune cells play an important role in antitumor immunity. The biggest challenge facing immune cell therapy is the allogeneic rejection, including the rejection of the infused cells to the patient's cells, namely GvHD, and the rejection of the patient's cells to the infused cells, that is, HvGR. Methods to avoid HvGR are still in the exploratory stage, and if some strategies are not employed to inhibit the allogeneic rejection of killer cells in the patient, the infused immune cells will be quickly eliminated. Cells that perform killing effect on foreign cells in the human body mainly include T cells or NK cells.

Therefore, it is necessary to adopt strategies to inhibit the killing of the imported immune cells by immune cells in vivo, so as to exert the therapeutic effect of the imported immune cells.

SUMMARY OF THE INVENTION

The present application provides a method for preparing a modified immune cell, the modification comprising the steps of: a) down-regulating the expression and/or activity of the Fas protein in the immune cell, b) up-regulating the FasL protein in the immune cell expression and/or activity. The modified immune cells prepared according to the method can have higher Fas protein knockout efficiency and higher FasL protein expression efficiency, and can effectively inhibit allogeneic rejection. For example, can resist the killing effect of T cells and/or PBMC cells.

In one aspect, the application provides a method for preparing a modified immune cell, the modification comprising the steps of:

a) down-regulating the expression and/or activity of the Fas protein in the immune cells, b) up-regulating the expression and/or activity of the FasL protein in the immune cells, such that, compared to the immune cells, the modified immune cells The expression and/or activity of Fas protein in the cells is reduced or eliminated, and the expression and/or activity of FasL protein is increased in the modified immune cells.

In certain embodiments, the step a) comprises down-regulating the expression and/or activity of a nucleic acid molecule encoding a Fas protein in the immune cell.

In certain embodiments, the down-regulation manner of step a) comprises down-regulation by gene knockout (knock out), gene knockdown (knock down), gene mutation, gene deletion, gene silencing or any combination of the above.

In certain embodiments, the down-regulation of step a) comprises administering to the immune cell one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas system, RNA editing Systems such as RNA adenosine deaminase (ADAR), RNA-guided endonucleases, zinc finger nucleases (ZFN), Mega-TAL nucleases, transcription activator-like effector nucleases (TALEN), meganucleases (Meganuclease), base editing, CRISPR interference, and, Zinc finger gene repressor and/or transcription activator-like effector (TALE) gene repressor-mediated transcriptional repression.

In certain embodiments, the step a) comprises administering to the immune cell a guide RNA targeting a nucleic acid molecule encoding a Fas protein.

In certain embodiments, the step a) comprises targeting any one or more of exons 1-5 of the nucleic acid molecule encoding the Fas protein in the immune cell.

In certain embodiments, the Fas protein comprises the amino acid sequence set forth in SEQ ID NO:46.

In certain embodiments, the nucleic acid molecule encoding the Fas protein comprises the nucleotide sequence shown under gene ID: 355 in the NCBI database or under ENSG00000026103 in the Ensembl database.

In certain embodiments, the guide RNA of the nucleic acid molecule targeting the Fas protein comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15.

In certain embodiments, the guide RNA targeting the nucleic acid molecule encoding the Fas protein is a single-stranded guide RNA.

In certain embodiments, the guide RNA targeting the nucleic acid molecule encoding the Fas protein is a double-stranded guide RNA comprising crRNA and tracrRNA.

In certain embodiments, the crRNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15.

In certain embodiments, the method comprises down-regulating T cell receptor (TCR), T cell receptor alpha constant region protein, T cell receptor beta constant region protein and/or PD-1 protein in the immune cell expression and/or activity.

In certain embodiments, the method comprises down-regulating the expression of a nucleic acid molecule encoding the TCR, T cell receptor alpha constant region protein, T cell receptor beta constant region protein and/or the PD-1 protein and/or / or activity.

In certain embodiments, the method comprises administering to the immune cell a guide RNA targeting a nucleic acid molecule encoding the T cell receptor alpha constant region protein (TRAC).

In certain embodiments, the T cell receptor alpha constant region protein comprises the amino acid sequence set forth in SEQ ID NO:49.

In certain embodiments, the guide RNA targeting the nucleic acid molecule encoding the T cell receptor alpha constant region protein (TRAC) comprises the nucleotides set forth in any one of SEQ ID NOs: 31-33 sequence.

In certain embodiments, the method comprises administering to the immune cell a guide RNA targeting a nucleic acid molecule encoding the PD-1 protein.

In certain embodiments, the method comprises targeting exons 1-3 of the nucleic acid molecule encoding the PD-1 protein in the immune cell.

In certain embodiments, the PD-1 protein comprises the amino acid sequence set forth in SEQ ID NO:48.

In certain embodiments, the guide RNA targeting the nucleic acid molecule encoding the PD-1 protein comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 16-30.

In certain embodiments, the guide RNA targeting the nucleic acid molecule encoding the Fas protein, targeting the nucleic acid molecule encoding the PD-1 protein, and/or targeting the nucleic acid molecule encoding the TCR Contains chemical modifications.

In certain embodiments, the method comprises administering a Cas protein to the immune cell.

In certain embodiments, the Cas protein is a Cas9 protein.

In certain embodiments, the step b) comprises up-regulating the expression and/or activity of the nucleic acid molecule encoding the FasL protein in the immune cell.

In certain embodiments, the step b) comprises administering to the immune cell a nucleic acid molecule encoding a FasL protein.

In certain embodiments, the method comprises administering to the immune cell a vector comprising a nucleic acid molecule encoding a FasL protein.

In certain embodiments, the FasL protein includes an extracellular domain.

In certain embodiments, the extracellular domain of the FasL protein comprises the amino acid sequence set forth in SEQ ID NO:52.

In certain embodiments, the FasL protein includes a hinge region.

In certain embodiments, the hinge region is derived from the tumor necrosis factor superfamily.

In certain embodiments, the hinge region is derived from FasL, TNFSF10 or OX40L.

In certain embodiments, the FasL protein includes an intracellular domain

In certain embodiments, the FasL protein comprises the amino acid sequence set forth in any of SEQ ID NOs: 50-52 and 63-64.

In certain embodiments, the nucleic acid molecule encoding the FasL protein comprises the nucleotide sequence set forth in SEQ ID NOs: 53-55.

In certain embodiments, the method further comprises up-regulating the expression and/or activity of PD-L1 and/or CD24 protein in the immune cells.

In certain embodiments, the method comprises administering to the immune cell a nucleic acid molecule encoding a PD-L1 protein and/or a nucleic acid molecule encoding a CD24 protein.

In certain embodiments, the method comprises administering to the immune cell a vector comprising a nucleic acid molecule encoding a PD-L1 protein and/or a nucleic acid molecule encoding a CD24 protein.

In certain embodiments, the PD-L1 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 56-57

In certain embodiments, the nucleic acid molecule encoding the PD-L1 protein comprises the nucleotide sequence set forth in SEQ ID NO:58.

In certain embodiments, the CD24 protein comprises the amino acid sequence set forth in SEQ ID NO:59.

In certain embodiments, the nucleic acid molecule encoding the CD24 protein comprises the nucleotide sequence set forth in SEQ ID NO:60.

In certain embodiments, the vector comprises a viral vector.

In certain embodiments, the immune cells include T cells, NK cells, NKT cells, monocytes, macrophages, B cells, plasma cells, granulocytes, dendritic cells, lymphocytes, leukocytes, stem cells and /or peripheral blood mononuclear cells.

In certain embodiments, the step b) is performed after the step a).

In certain embodiments, the step b) is performed after the down-regulation in the step a) is successful.

In certain embodiments, the successful down-regulation comprises that the expression and/or activity of Fas protein in the immune cells after the step a) is reduced by at least as compared to the immune cells before the step a). 50%.

In certain embodiments, the successful down-regulation comprises the occurrence of gene editing.

In certain embodiments, step b) is performed at least 6 hours after step a). In certain embodiments, the method further comprises the step of: causing the modified immune cells to comprise a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric autoantibody receptor (CAAR) ) and/or at least one synthetic receptor.

In certain embodiments, the method further comprises the step of: causing the modified immune cells to include expression of a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric autoantibody receptor ( CAAR) and/or nucleic acid molecules of at least one synthetic receptor.

In another aspect, the present application provides a modified immune cell prepared according to the method, the expression and/or activity of Fas protein in the modified immune cell compared to an immune cell without the modification is reduced or eliminated, and the expression and/or activity of Fas protein is increased in the modified immune cells.

In certain embodiments, the immune cells include a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric autoantibody receptor (CAAR), and/or at least one synthetic receptor.

In another aspect, the present application provides a population of cells comprising said immune cells, wherein at least 20% of the immune cells in said cell population do not substantially express Fas and at least 5% of the immune cells overexpress FasL.

In certain embodiments, at least 15% of the immune cells in the population of cells do not substantially express PD-1.

In certain embodiments, at least 5% of the immune cells in the population of cells overexpress PD-L1.

In certain embodiments, at least 5% of the immune cells in the population of cells overexpress CD24.

In another aspect, the present application provides a pharmaceutical composition comprising said immune cells and/or said cell population, and a pharmaceutically acceptable carrier.

In another aspect, the present application provides the use of the immune cells, the cell population and/or the pharmaceutical composition in the preparation of a medicament for treating a tumor.

In another aspect, the application provides a guide RNA targeting a nucleic acid molecule encoding a Fas protein, wherein the guide RNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15.

In another aspect, the application provides a guide RNA targeting a nucleic acid molecule encoding a PD-1 protein, wherein the guide RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16-30.

In another aspect, the application provides a CRISPR/Cas system comprising the guide RNA targeting a nucleic acid molecule encoding a Fas protein and a Cas protein.

In certain embodiments, the Cas protein comprises a Cas9 protein.

In another aspect, the present application provides a method of allogeneic cell transplantation comprising administering an effective amount of the immune cells, the cell population and/or the pharmaceutical composition.

In another aspect, the present application provides a method of treating a tumor, comprising administering an effective amount of the immune cells, the cell population, and/or the pharmaceutical composition.

Other aspects and advantages of the present application can be readily appreciated by those skilled in the art from the following detailed description. Only exemplary embodiments of the present application are shown and described in the following detailed description. As those skilled in the art will recognize, the content of this application enables those skilled in the art to make changes to the specific embodiments disclosed without departing from the spirit and scope of the invention to which this application relates. Accordingly, the drawings and descriptions in the specification of the present application are only exemplary and not restrictive.

Description of drawings

The invention to which this application relates is set forth with particularity characteristic of the appended claims. The features and advantages of the inventions involved in this application can be better understood by reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the drawings is as follows:

Figure 1 shows the knockout efficiency of the Fas-targeting guide RNA.

Figure 2 shows the knockout efficiency of Fas and the expression efficiency of FasL in the modified immune cells described in the present application.

Figure 3 shows the expansion efficiency of the modified immune cells described herein.

Figure 4 shows the expansion fold of the modified immune cells described in the present application after co-culture with allogeneic T cells.

Figure 5 shows the fold expansion of the modified immune cells described in the present application and allogeneic T cells co-cultured on day 6.

Figure 6 shows the expression efficiency of FasLF, FasL-M1 and FasL-M2 in the modified immune cells described herein.

Figure 7 shows the expansion fold of the modified immune cells described in the present application after co-culture with allogeneic T cells.

detailed description

The embodiments of the invention of the present application are described below with specific specific examples, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the contents disclosed in this specification.

Definition of Terms

In this application, the term "modification" generally refers to a change in the state or structure of a molecule or cell. Molecules can be modified in a variety of ways, including chemical, structural, and functional modifications. Cells can be modified by changing nucleotide or protein sequences. In the context of proteins, modifications can refer to fragments in which at least one amino acid residue has been substituted, deleted or added within the reference molecule. In the context of nucleic acids, a modification can refer to a fragment in which at least one nucleic acid residue has been replaced, deleted or added within the reference molecule. The modifications result in altered expression and/or activity of the protein in the cell.

In the present application, the term "down-regulated" generally refers to the level and/or level of a nucleic acid molecule (eg, RNA or DNA) encoding one or more proteins or functional fragments thereof in immune cells after modification as described herein. or activity, or the level and/or activity of one or more proteins or functional fragments thereof is reduced or eliminated from levels in immune cells that have not been modified as described herein.

In this application, the term "up-regulation" generally refers to the level and/or level of a nucleic acid molecule (eg, RNA or DNA) encoding one or more proteins or functional fragments thereof in an immune cell after the modification described herein. or activity, or the level and/or activity of one or more proteins or functional fragments thereof is increased above the level in immune cells that have not been modified as described herein. The nucleic acid molecules (eg, RNA or DNA) encoding one or more proteins or functional fragments thereof, or the nucleic acid molecules encoding one or more proteins or functional fragments thereof, may also be absent at levels in immune cells that have not been modified as described herein. Nucleic acid molecules (eg, RNA or DNA) of a protein or functional fragment thereof are not expressed.

In this application, the term "knock out" generally refers to genetic engineering techniques that result in the deletion or function elimination of a gene.

In this application, the term "knock down" generally refers to genetic engineering techniques that cause partial deletion of a gene or partial loss or reduction of function.

In this application, the term "gene mutation" generally refers to an insertion, substitution, deletion, frameshift mutation or missense mutation of one or more nucleotides in the nucleotide sequence of a gene, usually resulting in a reduction in the function of the gene or missing.

In this application, the term "gene deletion" generally refers to the loss of one or more nucleotides in the nucleotide sequence of a gene, eg, the loss of a fragment of nucleotides, and also includes the loss of the entire gene encoding a protein.

In this application, the term "gene silencing" generally refers to the inhibition of the expression of a gene, which can be inhibited by blocking the transcription or translation of the gene.

In this application, the term "Fas" generally refers to a cell surface death receptor, also known as the Fas receptor, APT1, CD95, FAS1, APO-1, FASTM, ALPS1A or the tumor necrosis factor receptor superfamily Member 6 (TNFRSF6). Mature Fas proteins include extracellular domains, transmembrane domains and/or intracellular domains. Exons 1 to 5 encode the extracellular domain, exon 6 encodes the transmembrane domain, and exons 7-9 encode the intracellular domain. The term includes full-length unprocessed Fas, precursors, mature forms, and any form of Fas or its variants, derivatives, analogs, homologues, fragments and functionalities thereof that are processed or artificially modified within cells Variants, eg, alternative splice variants and/or allelic variants. Amino acid sequences of exemplary human Fas proteins can be found in NCBI database accession numbers NP_000034.1 (variant 1), NP_001307548.1 (variant 4), NP_690610.1 (variant 2) and/or NP_690611.1 (variant 2) 3). Nucleic acid molecules encoding Fas proteins can be found in the nucleotide sequences shown in the NCBI database under gene ID: 355 or the Ensembl database under ENSG00000026103.

In this application, the term "FasL" generally refers to a type II transmembrane protein belonging to the tumor necrosis factor (TNF) family. The Fas-FasL interaction plays an important role in regulating the immune system and cancer progression, also known as Fas ligand, TNFSF6, CD178, CD95L, FASLG, TNLG1A or APT1LG1. Its receptors may include the Fas receptor and/or DcR3. Apoptosis triggered by Fas-FasL binding plays a fundamental role in the regulation of the immune system. The term includes full-length unprocessed FasL, precursors, mature forms, as well as any form of FasL or its variants, derivatives, analogs, homologues, fragments and functionalities thereof that are processed or artificially modified within cells Variants, eg, alternative splice variants and/or allelic variants. Another example is a variant in which the hinge region is mutated or substituted. FasL protein can include intracellular domain, transmembrane region, hinge region and/or extracellular domain; each region can be modified by mutation, substitution, insertion, or deletion, without affecting Fas-FasL binding, and still functioning Resistance to allograft rejection. The amino acid sequences of exemplary human FasL proteins can be found in NCBI database accession numbers NP_001192172.1 (variant 2) and/or NP_034307.1 (variant 1).

In this application, the term "PD-1" generally refers to programmed cell death 1, also known as PDCD1, CD279, PD1 or SLEB2, a type I transmembrane protein that is associated with BTLA, CTLA-4, ICOS and CD28 together constitute the CD28 family of T cell costimulatory receptors. "PD-1" includes complete PD-1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of human PD-1, and Analogues of common epitopes. Exemplary human PD-1 coding region sequences can be found under NCBI GenBank Accession No. NM_005018.3.

In this application, the term "PD-L1" generally refers to programmed cell death 1 ligand 1, also known as B7 homolog 1, B7-H1 or CD274, which downregulates T cell activation upon binding to PD-1 and cytokine secretion. "PD-L1" includes intact PD-L1 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs of human PD-1, and Analogues of common epitopes. Exemplary human PD-L1 sequences can be found under NCBI GenBank accession numbers NM_001267706.1, NM_014143.4 or NM_001314029.2.

In this application, the term "CD24" generally refers to a cell surface receptor protein, also known as thermostable antigen 24 (HSA). "CD24" includes intact CD24 and fragments thereof, as well as functional variants, isoforms, species homologues, derivatives, analogs, and analogs having at least one common epitope with CD24. Exemplary human CD24 sequences can be found under NCBI GenBank Accession Nos. NM_001291737, NM_001291738, NM_001291739, NM_013230, or NM_001359084.

In this application, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding an antigen and at least one intracellular domain. CAR is the core component of chimeric antigen receptor T cells (CAR-T), which can include targeting moieties (eg, moieties that bind tumor-associated antigens (TAAs)), hinge regions, transmembrane regions, and cells internal domain.

In this application, the term "T cell surface receptor (TCR)" may generally include the alpha beta form or the gamma delta form of TCR. According to the type of T cell surface receptor (TCR), T cells can be divided into αβT cells and γδT cells. αβ T cells express αβ TCR. There are many isoforms of αβTCR, which are expressed on the surface of αβT cells and are responsible for the recognition of specific antigens in a major histocompatibility complex (MHC)-dependent manner. γδT cells refer to T cells whose TCR is composed of γ chain and δ chain, and their immune function is between innate immunity and adaptive immunity. and has a broad antitumor spectrum.

In this application, the term "chimeric autoantibody receptor (CAAR)" generally refers to a protein that contains autoantigens and can be recognized by autoantibodies, and the English name is chimeric autoantibody receptors. CAAR can direct immune cells genetically modified to express CAAR to attack B cells expressing antibodies capable of recognizing the antigen (see Science.2016 Jul 8;353(6295):179-84.doi:10.1126/science.aaf6756.Epub 2016 Jun 30. Reengineering Chimeric Antigen Receptor T Cells for Targeted Therapy of Autoimmune Disease Christoph T Ellebrecht et al.).

In this application, the term "synthetic receptor" generally refers to an engineered cell surface protein or protein complex that contains (1) a target binding domain that can specifically bind a target molecule, and (2) can activate a signaling pathway functional domain. In the engineering unit, the target binding domain may comprise an extracellular domain and the functional domain may comprise an intracellular domain. Synthetic receptors may also include transmembrane sequences. Synthetic receptors can be protein complexes comprising proteins expressed from exogenous nucleic acids. The synthetic receptor can also be a protein complex comprising at least one exogenously expressed protein and at least one endogenously expressed protein. In some embodiments, the engineered cells can be immune cells, such as T cells, natural killer (NK) cells, B cells, macrophages, etc., and the functional domains can directly or indirectly activate the immune cells. In certain embodiments, the synthetic receptor may be selected from the group consisting of: Chimeric Antigen Receptor ("CAR"), T Cell Receptor ("TCR"), TCR Receptor Fusion Construct ("TRuC"), T Cell Antigen Conjugating Agents ("TACs"), Chimeric Autoantibody Receptors ("CAARs"), Antibody TCR Receptors ("AbTCRs"), and Chimeric CD3ε Receptors. In some embodiments, the synthetic receptor can be a CAR. In some embodiments, the synthetic receptor can be a TCR. In some embodiments, the synthetic receptor can be TRuC. In some embodiments, the synthetic receptor can be TAC. In some embodiments, the synthetic receptor can be an AbTCR. In some embodiments, the synthetic receptor can be a chimeric CD3ε receptor.

In this application, the term "CRISPR/Cas system" or "CRISPR-Cas system" generally refers to a nuclease system consisting of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-related proteins (i.e. Cas proteins), It can cut almost all genomic sequences adjacent to the protospacer-adjacent motif (PAM) in eukaryotic cells. The "CRISPR/Cas system" may be used to collectively refer to transcripts involving CRISPR-associated ("Cas") genes, as well as other elements involved in their expression or directing their activity, and may include sequences encoding Cas genes, tracr (transactivating CRISPR) sequences (e.g. tracrRNA or an active portion thereof), tracr partner sequence (in the context of endogenous CRISPR/Cas systems, encompassing "direct repeats" and processed partial direct repeats), guide sequences (in the context of endogenous CRISPR/Cas systems) Also known as "spacer"), or other sequences and transcripts from CRISPR loci. Five types of CRISPR systems have been identified (eg, Type I, Type II, Type III, Type U, and Type V).

In the present application, the term "Cas protein" also referred to as "CRISPR-associated protein" generally refers to a class of enzymes complementary to a CRISPR sequence, capable of using the CRISPR sequence as a guide, thereby recognizing and cleaving a specific DNA strand. Non-limiting examples of Cas proteins include: Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csf1 , Csf2, Csf3, Csf4, and/or their homologs, or modified forms thereof. In some embodiments, the Cas protein is a Cas9 protein.

In this application, the term "Cas9 protein" or "Cas9 nuclease", also known as Csn1 or Csx12, generally refers to a class of proteins in the Type II CRISPR/Cas system that are involved in both crRNA biosynthesis and destruction of invading DNA. Cas9 proteins typically include a RuvC nuclease domain and an HNH nuclease domain, which cleave two different strands of a double-stranded DNA molecule, respectively. It has been tested in different bacterial species such as S. thermophiles, Listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and Streptococcus pyogenes The Cas9 protein is described in (S. Pyogenes) (Deltcheva, Chylinski et al. 2011). For example, Streptococcus pyogenes Cas9 protein, the amino acid sequence of which can be found in SwissProt database accession number Q99ZW2; Neisseria meningitides Cas9 protein, whose amino acid sequence can be found in UniProt database number A1IQ68; Streptococcus thermophilus (Streptococcus thermophilus) Cas9 protein, its amino acid sequence is shown in UniProt database number Q03LF7; Staphylococcus aureus Cas9 protein, its amino acid sequence is shown in UniProt database number J7RUA5.

In this application, the term "guide RNA" generally refers to the RNA component contained in CRISPR, which may also be referred to as guide RNA (gRNA). Guide RNAs generally contain a guide sequence and a backbone sequence, which may be in the same molecule or in different molecules. The role of the guide RNA can be to instruct the Cas9 protein to cleave the DNA site complementary to the guide sequence, that is, the target sequence. In general, a guide sequence is any polynucleotide sequence that is sufficiently complementary to a target sequence to hybridize to the target sequence and to direct the specific binding of the CRISPR complex to the target sequence. The degree of complementarity between the guide sequence and its corresponding target sequence is about or more than about 50% or more. Typically a guide sequence is about or more than about 12 nucleotides in length. The backbone sequence is necessary for the guide RNA, and the remaining sequences except the guide sequence generally include the tracr sequence and the tracr partner sequence, and these sequences generally do not change due to changes in the target sequence. The term includes single-stranded guide RNA (sgRNA) as well as double-stranded guide RNA consisting of crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA).

In this application, the term "CRISPR interference", also referred to as CRISPR interference or CRISPRi, generally refers to a Cas protein (eg, Cas9) lacking endonuclease activity, which inactivated Cas protein (eg, Cas9) produces the gene of interest specific silencing or reduction.

In this application, the term "siRNA" may also be referred to as siRNA oligonucleotides," "RNAi oligonucleotides," or "short interfering RNAs," and generally refers to oligonucleotides that function through post-transcriptional gene splicing , also known as RNA interference (RNAi).

In this application, the term "shRNA" may also be referred to as short hairpin RNA, and generally refers to artificial single-stranded interfering RNA molecules that contain the sense and anti-siRNA duplexes in a stem-loop or hairpin structure. sense chain.

In this application, the term "RNA adenosine deaminase (ADAR)" generally refers to an enzyme useful in RNA editing that converts adenosine in RNA to inosine.

In this application, the term "base editing" generally refers to gene editing techniques that cause single base changes on the genome.

In this application, the term "complementary" generally refers to a nucleic acid (eg, RNA) comprising a nucleotide sequence that enables it to bind non-covalently (eg, Watson-Crick base pairing), at appropriate in vitro and/or in vivo temperatures "Hybridizes" or "complements" to another nucleic acid in a sequence-specific, anti-parallel manner (ie, nucleic acid specifically binds to a complementary nucleic acid) under conditions of ionic strength and solution ionic strength. As known in the art, standard Watson-Crick base pairings include: adenine (A) pairing with thymidine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C) Pairing.

In this application, the terms "polypeptide", "peptide", "protein" and "protein" are used interchangeably and generally refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified. These modifications may include: disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation (eg, binding to labeling components). The term "amino acid" includes natural and/or non-natural or synthetic amino acids, including glycine and D and L optical isomers, as well as amino acid analogs and peptidomimetics.

The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid" and "oligonucleotide" are used interchangeably and generally refer to a polymeric form of nucleotides of any length, Such as deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of genes or gene fragments, multiple loci (one locus) defined by ligation analysis, exons, introns, messenger RNA (mRNA), Transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short hairpin RNA (shRNA), micro-RNA (miRNA), ribozyme, cDNA, recombinant polynucleotide, branched polynucleotide, plasmid, vector, any sequence of isolated DNA, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may contain one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modification of the nucleotide structure can be performed before or after polymer assembly. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation to labeled components.

In the present application, the "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host for transfer of the inserted nucleic acid molecule into and/or between host cells. The vectors may include vectors primarily for the insertion of DNA or RNA into cells, vectors primarily for replication of DNA or RNA, and vectors primarily for expression of transcription and/or translation of DNA or RNA. The carrier also includes a carrier having a variety of the above-mentioned functions. The vector may be a polynucleotide capable of being transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce the desired expression product by culturing a suitable host cell containing the vector.

In this application, the term "plasmid" generally refers to DNA molecules other than chromosomes or nucleoids in organisms such as bacteria and yeast, which exist in the cytoplasm and have the ability to autonomously replicate, enabling them to maintain a constant copy in daughter cells number and express the genetic information it carries. Plasmids are used as carriers of genes in genetic engineering research.

In this application, the term "retroviral vector" generally refers to a virus particle that can controllably and express foreign genes, but cannot self-package into proliferative virus particles. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, the genes that contain the proteins that make up the center and structure of the virus; pol, the genes that make up the reverse transcriptase, and env, the genes that make up the viral coat. Through retroviral transfection, retroviral vectors can randomly and stably integrate their own genome and the foreign genes they carry into the host cell genome, for example, CAR molecules can be integrated into host cells.

In this application, the term "lentiviral vector" generally refers to a diploid RNA viral vector that is a retrovirus. The lentiviral vector is based on the genome of the lentivirus, and many sequence structures related to the virus activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome backbone. and expression constructs, and prepared into vectors. Through lentiviral vector transfection, retroviral vectors can randomly and stably integrate their own genomes and the foreign genes they carry into the host cell genome, for example, CAR molecules can be integrated into host cells.

In addition to the specific proteins and nucleotides mentioned herein, the present application may also include functional variants, derivatives, analogs, homologues, and fragments thereof.

The term "functional variant" refers to a polypeptide having substantially the same amino acid sequence or encoded by substantially the same nucleotide sequence as the naturally occurring sequence and capable of possessing one or more activities of the naturally occurring sequence. In the context of this application, a variant of any given sequence refers to one in which a particular sequence of residues (whether amino acid or nucleotide residues) has been modified such that the polypeptide or polynucleotide substantially retains at least one Sequence of endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, substitution and/or variation of at least one amino acid residue and/or nucleotide residue present in a naturally occurring protein and/or polynucleotide, so long as the The original functional activity is sufficient.

In the present application, the term "derivative" generally refers to the polypeptide or polynucleotide of the present application including any substitution, variation, modification, substitution, deletion and /or addition, so long as the resulting polypeptide or polynucleotide substantially retains at least one of its endogenous functions.

In this application, the term "analog" generally refers to a polypeptide or polynucleotide and includes any mimetic of the polypeptide or polynucleotide, ie possessing at least one endogenous function of the polypeptide or polynucleotide that the mimetic mimics chemical compounds.

Generally, amino acid substitutions, such as at least 1 (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid substitutions, can be made, so long as the modified sequence remains substantially as desired activity or ability. Amino acid substitutions can include the use of non-naturally occurring analogs.

The proteins or polypeptides used in the present application may also have deletions, insertions or substitutions of amino acid residues that produce silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions can be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilic nature of the residues, so long as endogenous function is preserved. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids containing uncharged polar headgroups with similar hydrophilicity values include amino acids Paraparagine, Glutamine, Serine, Threonine and Tyrosine.

In this application, the term "homologue" generally refers to an amino acid sequence or nucleotide sequence that has some homology to a wild-type amino acid sequence and a wild-type nucleotide sequence. The term "homology" may be equivalent to sequence "identity". Homologous sequences can include amino acid sequences that can be at least 80%, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the subject sequence . Typically, a homologue will contain the same active site, etc., as the subject amino acid sequence. Homology can be considered in terms of similarity (ie, amino acid residues with similar chemical properties/functions), or it can be expressed in terms of sequence identity. In the present application, a reference to a sequence having a percent identity to any one of the SEQ ID NOs of an amino acid sequence or a nucleotide sequence refers to that percent identity over the entire length of the referenced SEQ ID NO. the sequence of.

To determine sequence identity, sequence alignments can be performed by various means known to those skilled in the art, eg, using BLAST, BLAST-2, ALIGN, NEEDLE or Megalign (DNASTAR) software and the like. Those skilled in the art can determine appropriate parameters for alignment, including any algorithms needed to achieve optimal alignment among the full-length sequences being compared.

In this application, the term "and/or" should be understood to mean either or both of the alternatives.

In this application, the term "comprising" generally means including the expressly specified features, but not excluding other elements.

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 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%.

Detailed description of the invention

In one aspect, the present application provides a method for preparing a modified immune cell, the modification comprising the steps of: a) down-regulating the expression and/or activity of Fas protein in the immune cell, b) up-regulating the expression and/or activity of the Fas protein in the immune cell FasL protein expression and/or activity such that Fas protein expression and/or activity in the modified immune cells is reduced or eliminated compared to immune cells without the modification, and the modified Elevated expression and/or activity of FasL protein in immune cells.

down

In the present application, the above method may comprise down-regulating the expression and/or activity of Fas protein in the immune cells. There can be various methods to downregulate the expression and/or activity of the Fas protein in the immune cells. For example, Fas protein expression and/or activity can be down-regulated at the gene or protein level. For example, by altering the structure and/or sequence of the Fas protein, transcription, translation and/or cellular trafficking of the Fas protein encoding the Fas protein gene.

In certain instances, the method can include down-regulating the expression and/or activity of a nucleic acid molecule encoding a Fas protein in the immune cell. The down-regulation includes the reduction or elimination of the functional expression of the gene encoding the Fas protein in the immune cell. In certain instances, reduction or elimination may be achieved by gene knockout, gene knockdown, gene mutation, gene deletion, gene silencing, or any combination of the foregoing. In some cases, the down-regulation effect can be achieved by altering the genome of the immune cell.

In the present application, the expression and/or activity of Fas protein in the immune cells can be down-regulated by gene knockout. Gene knockout is a genetic technique that renders a gene ineffective by disrupting its function. For example, the insertion of coding sequences into nucleic acids disrupts gene function. In addition, the entire gene encoding the Fas protein or a portion thereof can be deleted so that the modified immune cell does not express the Fas protein or does not express a functional Fas protein. Another possible approach is to introduce one or more knockout mutations into the gene sequence encoding the Fas protein, which provide a non-functional or less functional expression product. For example, one or more frameshift mutations can be introduced which result in a non-functional or less functional protein. Alternatively or additionally, one or more stop codons can be introduced into the coding sequence to obtain a truncated, non-functional or less functional Fas protein. The method may also include, but is not limited to, introducing one or more mutations in the promoter, 5'UTR, 3'UTR and/or other regulatory elements of the gene encoding the Fas protein. The method for realizing gene deletion to inhibit or eliminate the functional expression of target gene is well known to those skilled in the art, and will not be described in detail herein.

In certain instances, the gene encoding the Fas protein can be functionally knocked out by genetic engineering. Examples include, but are not limited to, gene editing, such as engineered nuclease-mediated gene editing (GEEN). Gene editing generally refers to the use of artificially engineered nucleases, or "molecular scissors," to insert, replace or remove DNA within the genome. The nucleases generate specific double-strand breaks (DSBs) at desired genomic locations, using endogenous cellular mechanisms to repair the induced breaks through the natural processes of homologous recombination (HR) and non-homologous end joining (NHEJ). Methods for knocking out a gene or reducing the expression level of a gene may include performing gene knockout, conditional gene knockout (eg, using the Cre/LoxP and/or FLP-frt system), inducible gene knockout (eg, using Cre/LoxP and/or FLP-frt systems). loxp system-based knockout, including tetracycline-induced, interferon-induced, hormone-induced, adenovirus-induced, etc.), gene knockout by random insertion mutagenesis (e.g., gene trapping method), gene knockout by RNAi, zinc Refers to gene editing technology mediated by endonuclease (zinc finger nucleases, ZFN), gene editing technology mediated by transcription activator-like effector nucleases (TALEN), clustering regularly spaced short loops Gene editing technology mediated by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) (CRISPR/Cas) system and/or NgAgo-gDNA gene editing technology.

In certain instances, the method can include administering to the immune cell one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas systems, RNA editing systems such as RNA adenosine deaminase (ADAR), RNA-Guided Endonuclease, Zinc Finger Nuclease (ZFN), Mega-TAL Nuclease, Transcription Activator-Like Effector Nuclease (TALEN), Meganuclease, Base Editing, CRISPR interference (or CRISPRi), and, Zinc finger gene repressor and/or transcription activator-like effector (TALE) gene repressor-mediated transcriptional repression. In the present application, the method may include administering to immune cells using inhibitory proteins, which may include substances capable of inhibiting the activity or function of the Fas protein, eg, inhibitory ligands, receptors, antibodies of the Fas protein and/or enzymes.

In the present application, one or more mutations may be included in the exons of the nucleic acid sequence encoding the Fas protein. In the present application, exons of the nucleic acid sequence encoding the Fas protein may be deleted in whole or in part. The exons may include any one or more of exons 1-5, for example, one of exon 1, exon 2, exon 3, exon 4, and exon 5 or Multiple (eg, 1, 2, 3, 4, or 5). Mutations or deletions in any one or more of exons 1-5 can encompass several different functional splice variants.

In the present application, the Fas protein may include the amino acid sequence shown in SEQ ID NO:46. For example, the Fas protein described herein may comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%) of the amino acid sequence set forth in SEQ ID NO:46 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity

In the present application, the nucleic acid molecule encoding Fas protein can be found in the nucleotide sequence shown under gene ID: 355 in the NCBI database or under ENSG00000026103 in the Ensembl database. For example, a nucleic acid molecule encoding a Fas protein described herein may comprise at least 80% (e.g., at least 85%, at least 90%, at least 80%, at least 90%, at least 80%, at least 90%, at least 80%, at least 80%, at least 90%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity nucleotide sequences.

In the present application, the method may comprise down-regulating the expression of T cell receptor (TCR), T cell receptor alpha constant region protein, T cell receptor beta constant region protein and/or PD-1 protein in the immune cell and/or activity.

In certain instances, the method can include targeting exons 1-3 of a nucleic acid molecule encoding the T cell receptor alpha constant region protein (TRAC) in the immune cell.

For example, the T cell receptor alpha constant region protein can include the amino acid sequence set forth in SEQ ID NO:49. For example, the T cell receptor alpha constant region protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%) of the amino acid sequence set forth in SEQ ID NO:49 %, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

In certain instances, the method can include targeting exons 1-3 of a nucleic acid molecule of the PD-1 protein in the immune cell.

For example, the PD-1 protein can include the amino acid sequence shown in SEQ ID NO:48. For example, the PD-1 protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%) of the amino acid sequence set forth in SEQ ID NO:48 %, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

CRISPR systems and guide RNAs

The expression and/or activity of the Fas protein can be down-regulated using the CRISPR system. In the present application, the method includes administering to the immune cell a CRISPR/Cas system, which may include an RNA component, sometimes referred to as a guide RNA (gRNA).

The guide RNAs described herein can include single-stranded guide RNAs (sgRNAs) as well as double-stranded guide RNAs consisting of crRNA (CRISPR RNA) and tracrRNA (transactivating crRNA). In certain embodiments, the guide RNA is a double-stranded structure consisting of a crRNA and a tracrRNA. CrRNA generally contains a guide sequence and a tracr partner sequence, and tracrRNA generally contains a tracr sequence. In certain embodiments, the guide RNA can be a single-stranded molecule that can comprise a guide sequence, a tracr partner sequence and a tracr sequence, this single-stranded molecule is also referred to as a chimeric single-stranded guide RNA (sgRNA). When a tracr sequence and a tracr partner sequence are included in a single transcript, hybridization between the two results in a transcript with secondary structure such as a hairpin. A sequence for use in a hairpin structure may be a loop forming sequence, eg, a sequence that may be four nucleotides in length, eg, a sequence for use in a hairpin structure may have the sequence GAAA. Longer or shorter loop sequences, such as alternative sequences, can also be used. In some cases, these sequences may include triplets (eg, AAA), as well as other nucleotides (eg, C or G). Examples of loop-forming sequences may include CAAA and AAAG. In certain instances, the transcript or transcribed polynucleotide sequence can have at least two or more hairpins. In some specific cases, the transcript may have two, three, four or five hairpins. In other cases, the transcript can have up to five hairpins. In certain instances, the single transcript may also include a transcription termination sequence, eg, a Poly-U sequence, or, for example, a six U nucleotide sequence.

In certain instances, the guide RNAs described herein can be complementary to the target nucleic acid. In other cases, the guide RNA can be the same as the target nucleic acid (when speaking of the same, the "U" in RNA corresponds to the thymine "T" in DNA due to the difference in the bases encoding RNA and DNA ). In other cases, the nucleic acid sequence (eg, DNA) encoding the guide RNA can be the same or complementary to the target nucleic acid. The guide RNA can be transcribed or replicated from the sequence encoding it, eg, the guide RNA can be transcribed from the DNA sequence encoding it. In this application, the terms "target nucleic acid", "target nucleic acid" and "target region" are used interchangeably, and usually refer to a nucleic acid sequence that can be recognized by a gRNA. The target nucleic acid can refer to a double-stranded nucleic acid or a single-stranded nucleic acid.

The guide RNA can hybridize to the target sequence and complex with one or more Cas proteins to form a CRISPR complex. The formation of the CRISPR complex results in the target sequence at or near (eg, within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs) ) of one or both strands of cleavage. tracr sequence (which may comprise or consist of all or part of a wild-type tracr sequence (e.g., about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides)) can also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or part of a tracr partner sequence operably linked to the guide sequence. In certain instances, the tracr sequence is sufficiently complementary to a tracr partner sequence to hybridize and participate in the formation of a CRISPR complex. In certain embodiments, the tracr sequence has at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr partner sequence when aligned sex. In some embodiments, one or more vectors that drive expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system directs CRISPR complex at one or more target sites formation of things.

In general, a tracr mate sequence includes a tracr mate sequence that is sufficiently complementary to a tracr sequence to facilitate formation of a CRISPR complex at the target sequence, wherein the CRISPR complex comprises a tracr mate sequence that hybridizes to the tracr sequence. Typically, the degree of complementarity is in terms of optimal alignment of the tracr mate sequence with the tracr sequence along the length of the shorter of the two sequences. Optimal alignment can be determined by any suitable alignment algorithm, and can further take into account the effects of secondary structure, such as self-complementarity within the tracr sequence or the tracr mate sequence. In optimal alignment, the degree of complementarity between the tracr sequence and the tracr mate sequence along the length of the shorter of the two is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. The tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides.

Complementary hybridization between the target sequence and the guide sequence promotes the formation of the CRISPR complex. Perfect complementarity is not required, as long as there is sufficient complementarity to cause hybridization and facilitate the formation of a CRISPR complex. The target polynucleotide of the CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide that resides in the nucleus of a eukaryotic cell. The target polynucleotide can be a sequence encoding a gene product (eg, a protein) or a non-coding sequence (eg, a regulatory polynucleotide or unwanted DNA). Without wishing to be bound by theory, the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex.

In some cases, the percent complementarity between the guide RNA and the target nucleic acid can be at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 65%, at least about 70%, at least about 75% %, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, at least about 99%, or 100%. In some cases, the percent complementarity between the guide RNA and the target nucleic acid can be at most about 30%, at most about 40%, at most about 50%, at most about 60%, at most about 65%, at most about 70%, at most about 75% %, up to about 80%, up to about 85%, up to about 90%, up to about 95%, up to about 97%, up to about 98%, up to about 99%, or 100%.

The methods of the present application include providing nucleic acid targeting the genome of an immune cell that can direct a relevant active polypeptide (eg, a Cas protein) to a specific target sequence (eg, a gene encoding a Fas protein) within the target nucleic acid. The nucleic acid targeted to the genome can be RNA.

In the present application, the guide RNA may comprise a variable length spacer sequence having 17-30 nucleotides at the 5' end of the guide RNA sequence. In other cases, the guide RNA may comprise a variable length spacer sequence having 17-24 nucleotides at the 5' end of the guide RNA sequence. For example, the guide RNA can comprise a 21 nucleotide sequence. For example, the guide RNA may comprise a sequence of 20 nucleotides. For example, the guide RNA may comprise a 19 nucleotide sequence. For example, the guide RNA may comprise a sequence of 18 nucleotides. For example, the guide RNA can comprise a 17 nucleotide sequence. For example, the guide RNA can comprise a 22 nucleotide sequence. For example, the guide RNA may comprise a sequence of 23 nucleotides. For example, the guide RNA may comprise a sequence of 24 nucleotides.

In the present application, the guide RNA may be a guide RNA targeting a nucleic acid molecule encoding a Fas protein, and the guide RNA targeting a nucleic acid molecule encoding the Fas protein may comprise any of SEQ ID NOs: 1-15 One of the nucleotide sequences shown. For example, the guide RNA described herein targeting a nucleic acid molecule encoding the Fas protein may comprise at least 80% (eg, at least 85%) of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15 , at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) of sequence identity Nucleotide sequence.

For example, the guide RNA described herein targeting the nucleic acid molecule encoding the Fas protein may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 2-4, 7-10 and 12. For example, the guide RNA described herein targeting a nucleic acid molecule encoding the Fas protein may comprise at least 80% of the nucleotide sequence set forth in any one of SEQ ID NOs: 2-4, 7-10 and 12 (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100 %) nucleotide sequences of sequence identity.

In the present application, the guide RNA may be a guide RNA targeting a nucleic acid molecule (TRAC) encoding a T cell receptor alpha constant region protein in an immune cell, and the target RNA targeting a T cell receptor alpha constant region protein encoding The guide RNA of the nucleic acid molecule (TRAC) may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 31-33. For example, a guide RNA targeting a nucleic acid molecule encoding a T cell receptor alpha constant region protein (TRAC) described herein may comprise a nucleotide sequence having at least 80 % (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.

In the present application, the guide RNA may be a guide RNA targeting a nucleic acid molecule encoding PD-1, and the guide RNA targeting a nucleic acid molecule encoding PD-1 may comprise any of SEQ ID NOs: 16-30 One of the nucleotide sequences shown. For example, a guide RNA targeting a PD-1-encoding nucleic acid molecule described herein may comprise at least 80% (eg, 85%, 90%) of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-30 %, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) sequence identity of nucleotide sequences.

In some cases, the guide RNA may comprise a backbone sequence that does not affect the recognition of the target sequence by the sgRNA, and thus, the backbone sequence may be any sequence available in the art. The backbone sequence typically includes a tracr mate sequence and a tracr sequence. The structure of the backbone sequence can be found in A and B in Figure 1 (Figure 1), A, B, C in Figure 3 (Figure 3), and Figure 4 ( Parts other than the spacer sequence described in A, B, C, D, and E in Fig. 4).

The backbone sequence of the present application can be derived from the backbone sequence described in PCT application publication WO2019011118A1, for example, can be the nucleotide sequence shown in any one of SEQ ID NOs: 34-45 (listed as 5' to 3') , where the first region in lowercase font represents the tracr mate sequence, and the second region in lowercase font represents the tracr sequence, and the last poly-U sequence represents the transcription terminator. The number of U in the poly-U is not limited to what is shown in this example, and can be increased or decreased. In some cases, poly-U can be removed without affecting activity. In certain instances, backbone sequences that are about or more than about 50%, 60%, 70%, 80%, 90%, or more similar to the following sequences can be used. In some cases, the tracr sequence can be a separate transcript from the transcript comprising the tracr partner sequence. In some cases, a chimeric single-stranded guide RNA (sgRNA) design can incorporate at least a 12 bp duplex structure between the direct repeat and the tracrRNA. In some cases, designs that contain critical RNA secondary structures for binding to the Cas9 protein, but whose sequences are mutated, or contain inserts, can also be used, as has been published in Nowak et al. Nucleic Acids Research 2016.44:9555 -9564 and Adamson et al. Cell 2016.167:1867-1882.

Table 1 Exemplary backbone sequences

The backbone sequence described herein may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 34-45, for example, the backbone sequence may comprise the nucleotide sequence set forth in SEQ ID NO:35.

In the present application, the guide RNA may be a double-stranded guide RNA targeting a nucleic acid molecule encoding a Fas protein, which may comprise crRNA and tracrRNA. The crRNA targeting the nucleic acid molecule encoding the Fas protein may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 1-15. For example, a crRNA targeting a nucleic acid molecule encoding a Fas protein described herein may comprise at least 80% (eg, at least 85%, at least 90%) of the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15 %, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotides of sequence identity sequence.

For example, the crRNA described herein targeting the nucleic acid molecule encoding the Fas protein may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 2-4, 7-10 and 12. For example, the crRNA described herein targeting a nucleic acid molecule encoding the Fas protein may comprise at least 80% ( For example, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% ) nucleotide sequences of sequence identity.

In the present application, the guide RNA may be a double-stranded guide RNA targeting a nucleic acid molecule encoding a T cell receptor, which may comprise crRNA and tracrRNA. The guide RNA targeting the nucleic acid molecule encoding the T cell receptor may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 31-33. For example, a guide RNA targeting a nucleic acid molecule encoding a T cell receptor described herein can comprise at least 80% (eg, 85%, 90%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) sequence identity of nucleotide sequences.

In the present application, the guide RNA may be a double-stranded guide RNA targeting a nucleic acid molecule encoding PD-1, which may comprise crRNA and tracrRNA. The crRNA targeting the nucleic acid molecule encoding PD-1 may comprise the nucleotide sequence shown in any one of SEQ ID NOs: 16-30. For example, the crRNA described herein targeting a nucleic acid molecule encoding PD-1 may comprise at least 80% (eg, 85%, 90%) of the nucleotide sequence set forth in any one of SEQ ID NOs: 16-30 , 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9%) nucleotide sequences of sequence identity.

In the present application, the Cas protein, sgRNA and/or tracr partner sequence and tracrRNA sequence are operably linked to the same promoter and can be expressed. In certain instances, the Cas enzyme, sgRNA, and/or tracr partner sequence and tracrRNA sequence can each be operably linked to separate regulatory elements located on separate vectors. The guide RNA and Cas protein can form a CRISPR complex.

The guide RNAs described herein for use in the CRISPR system can be synthesized by chemical methods, eg, high performance liquid chromatography. For example, two or more RNA molecules are linked together. RNAs of longer lengths, such as those encoding Cas9, can be obtained by enzymatic reactions.

The guide RNAs of the present application may also comprise modifications (eg, chemical modifications), eg, deletions, insertions, translocations, inactivation and/or activation of nucleotides. Such modifications may include introducing one or more mutations (including single or multiple base pair changes), increasing the number of hairpins, cross-linking, breaking specific stretches of nucleotides, and other modifications. Modifications can include inclusion of at least one non-naturally occurring nucleotide, or a modified nucleotide, or an analog thereof. The guide RNA may be modified at ribose, phosphate and/or base moieties. Modified guide RNAs can include 2'-O-methyl analogs, 2'-deoxy analogs, or 2'-fluoro analogs. The nucleic acid backbone of the guide RNA can be modified, for example, a phosphorothioate backbone can be used. Locked nucleic acid (LNA) or bridged nucleic acid (BNA) can also be used. Examples of modifications in guide RNAs may also include, but are not limited to: 2-aminopurine, 5-bromo-uridine, pseudouridine, inosine, 7-methylguanosine. These modifications can be applied to any component of the CRISPR system of the present application. In certain instances, these modifications can be made to the RNA component (eg, the guide RNA or chimeric polynucleotide sequence). For example, chemical modification of the guide RNA can include 2'-methoxy and/or 3'-phosphorothioate modifications. In some cases, designs that contain critical RNA secondary structures for binding to the Cas9 protein, but whose sequences are mutated, or contain inserts, can also be used, as has been published in Nowak et al. Nucleic Acids Research 2016.44:9555 -9564 and Adamson et al. Cell 2016.167:1867-1882.

In certain embodiments, the methods of the present application can comprise administering one or more of the Cas proteins to a host cell (e.g., an immune cell).

In another aspect, the application provides a CRISPR enzyme. In some cases, the CRISPR enzyme can be a Type II CRISPR system enzyme. In certain instances, the CRISPR enzyme can be a Cas9 protein. In certain embodiments, the Cas9 protein can be a Streptococcus pneumoniae, Streptococcus pyogenes, or Streptococcus thermophilus Cas9 protein, and can include mutated Cas9 proteins derived from these organisms. The Cas protein can also be a homolog or ortholog of a Cas9 protein. The systems described herein may include the Cas protein. The Cas protein may comprise any other protein, and optionally a linker sequence between any two domains. Examples of proteins that can be fused to Cas proteins include, but are not limited to, epitope tags, reporter genes, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.

The CRISPR system of the present application can be transferred into immune cells by methods known in the art, such as calcium phosphate transfection, protoplast fusion, electroporation, lipofection, microinjection, viral infection (eg, baculovirus, vaccinia virus) , adenovirus and other viruses) etc. For example, the CRISPR system (eg, Cas protein and/or guide RNA) can be introduced into immune cells using a point-of-care approach. For the electrotransfer method, please refer to the literature Schumann et al. PNAS 2015.112:10437-10442.

up

The methods described herein may further comprise up-regulating the expression and/or activity of the FasL protein in the immune cells. There can be various methods to upregulate the expression and/or activity of the FasL protein in the immune cells. Up-regulation can be manifested in enhancing or increasing the expression level of the FasL protein or increasing or increasing the activity of the FasL protein, or a combination of the two. FasL protein expression and/or activity can be up-regulated at the gene or protein level. For example, FasL protein expression and/or activity can be increased by altering the structure and/or sequence of the FasL protein, transcription, translation of the gene encoding the Fas protein, and/or cellular trafficking of the FasL protein.

In certain instances, the method can include upregulating the expression and/or activity of a nucleic acid molecule encoding a FasL protein in the immune cell. The up-regulation includes enhanced or increased functional expression of the gene encoding the FasL protein in the immune cell. In certain instances, a nucleic acid molecule encoding a FasL protein can be administered to the immune cell.

In the present application, the FasL protein may include an extracellular domain. For example, the extracellular domain of the FasL protein can include the amino acid sequence shown in SEQ ID NO:52. For example, the extracellular domain of the FasL protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%) of the amino acid sequence set forth in SEQ ID NO. , at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

In the present application, the FasL protein may comprise the amino acid sequence shown in SEQ ID NO:52. For example, the FasL protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 93%, at least 94%, at least 80%) of the amino acid sequence set forth in SEQ ID NO. amino acid sequences of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity.

In the present application, the FasL protein may also comprise a hinge region. The hinge region may contain fewer protease cleavage sites than the hinge region of the native FasL protein. For example, the hinge region may be a hinge region derived from a transmembrane protein other than FasL. For example, the hinge region may be a hinge region derived from the tumor necrosis factor superfamily. For example, the hinge region can be a hinge region derived from TNFSF10 or OX40L.

In the present application, the FasL protein may include an intracellular domain.

In the present application, the FasL protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 50-51 and 63-64. For example, the FasL protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%) of the amino acid sequence set forth in any of SEQ ID NOs. 50-51 and 63-64 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

In the present application, the FasL protein may comprise the amino acid sequence shown in any one of SEQ ID NOs: 50-52 and 63-64. For example, the FasL protein can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%) of the amino acid sequence set forth in any of SEQ ID NOs. 50-52 and 63-64 %, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

For example, the nucleic acid molecule encoding FasL may comprise the nucleotide sequence set forth in any one of SEQ ID NOs: 53-55. For example, the nucleic acid molecule encoding FasL may comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 80%) of the nucleotide sequence set forth in any of SEQ ID NOs. 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.

In certain instances, the method can include up-regulating the expression and/or activity of a nucleic acid molecule encoding a PD-L1 protein in the immune cell. The up-regulation includes enhanced or increased functional expression of the gene encoding the PD-L1 protein in the immune cell.

In certain instances, a nucleic acid molecule encoding a PD-L1 protein can be administered to the immune cell. For example, the PD-L1 protein can comprise the extracellular domain and/or the transmembrane domain of the full-length PD-L1 protein. For example, the PD-L1 protein can include the amino acid sequence set forth in any one of SEQ ID NOs: 56-57. For example, the PD-L1 protein may comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 85%, at least 90%, at least 91%, at least 92%) of the amino acid sequence set forth in any of SEQ ID NOs: 56-57, amino acid sequences of at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) sequence identity.

For example, the nucleic acid molecule encoding the PD-L1 protein can include the nucleotide sequence shown in SEQ ID NO:58. For example, the nucleic acid molecule encoding PD-L1 can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 92%, at least 80%) of the nucleotide sequence set forth in SEQ ID NO:58 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.

In certain instances, the method can include upregulating the expression and/or activity of a nucleic acid molecule encoding CD24 in the immune cell. The up-regulation includes enhanced or increased functional expression of the gene encoding CD24 in the immune cells. In certain instances, a nucleic acid molecule encoding CD24 can be administered to the immune cell.

For example, the CD24 can comprise the amino acid sequence set forth in SEQ ID NO:59. For example, the CD24 can comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 94%, at least 94%) of the amino acid sequence set forth in SEQ ID NO:59 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) amino acid sequences of sequence identity.

For example, the nucleic acid molecule encoding CD24 may comprise the nucleotide sequence set forth in any one of SEQ ID NO:60. For example, the nucleic acid molecule encoding CD24 may comprise at least 80% (eg, at least 85%, at least 90%, at least 91%, at least 92%) of the nucleotide sequence set forth in any one of SEQ ID NO:60 , at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%) nucleotide sequences of sequence identity.

The modifications may also include up- or down-regulating the expression and/or activity levels of other viable immune-related molecules. The immune-related molecules include, but are not limited to, major histocompatibility complex (MHC) (eg, MHC I, MHC II, and/or MHC III), cytokines (IL2, IL10, and/or IL12), and/or immune checks Point inhibitors (eg, CTLA-4, LAG-3, TIM-3 and/or TIGIT).

Nucleic acid molecules encoding the FasL protein (or, PD-L1 and/or CD24) can be obtained using recombinant methods known in the art, such as, for example, by screening libraries from cells expressing the gene, by The gene can be obtained in a vector, or by direct isolation from cells and tissues containing the gene using standard techniques, or the nucleic acid molecule can be produced synthetically.

The application also provides vectors for nucleic acid molecules comprising the FasL protein. Briefly summarized, the encoding of the polypeptide of interest can usually be achieved by operably linking a nucleic acid encoding the polypeptide of interest or a portion thereof (eg, FasL, PD-L1 and/or CD24) downstream of the promoter and incorporating the construct into an expression vector. Expression of natural or synthetic nucleic acids. The vector may be suitable for replication and integration in eukaryotic cells. A typical vector may contain transcriptional and translational terminators, initial sequences and promoters that can be used to regulate the expression of the desired nucleic acid sequence.

The nucleic acid molecules described herein can also be linked to many types of vectors. For example, the nucleic acid can be ligated to, including but not limited to, plasmids, phagemids, phages, viruses and/or cosmids. Particular vectors of interest can include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

The viral vector can be administered directly to the patient (in vivo) or can be administered in an indirect form, eg, by treating cells with the virus in vitro, and then administering the treated cells to the patient (ex vivo). Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks of virology and molecular biology. Conventional virus-based systems can include retroviral, lentiviral, adenoviral, adeno-associated, and herpes simplex vectors for gene transfer. In some cases, retroviral, lentiviral, and adeno-associated virus methods can be used to integrate gene transfer into the host genome, allowing long-term expression of the inserted gene. Lentiviral vectors are retroviral vectors capable of transducing or infecting non-dividing cells and typically producing higher viral titers. Lentiviral vectors may comprise long terminal repeats 5'LTR and truncated 3'LTRs, RREs, rev response elements (cPPT), central termination sequences (CTS) and/or post-translational regulatory elements (WPRE).

The methods of the present application may comprise introducing the vectors described herein into immune effector cells. For example, the vectors described herein can be introduced into the immune effector cells, such as T lymphocytes, B cells, macrophages, or natural killer (NK) cells. In certain embodiments, each or each cell may comprise one or one of the vectors described herein. In certain embodiments, each or each cell may comprise a plurality (eg, 2 or more) or more (eg, 2 or more) of the vectors described herein. For example, the vectors described herein can be introduced into the cells. For example, immune effector cells can be transfected with a retroviral vector, and the viral genome with the nucleic acid encoding the fusion protein can be integrated into the host genome to ensure long-term and stable expression of the target gene. For another example, using a transposon, a plasmid carrying a nucleic acid encoding the fusion protein and a plasmid carrying a transposase are introduced into target cells. In the present application, the vector with the nucleic acid encoding the fusion protein described in the present application can be introduced into the cell by methods known in the art, non-limiting examples include viral transduction, electroporation transfection , liposome delivery, polymer carriers, chemical carriers, lipid complexes, polymer complexes, dendrimers, nanoparticles, emulsions, native endocytic or phagocytic pathways, cell penetrating peptides, microinjection, microinjection Needle delivery method, particle bombardment method, etc.

For example, electroporation transfection can be employed, and non-limiting examples of electroporation instruments that can be used include: Neon Transfection System (Thermo Fisher Scientific), Gemini Instruments, and AgilePulse/CytoPulse Instruments (BTX-Harvard apparatus), 4D- Nucleofector system, Amaxa Nucleofector II, Nucleofector 20 2b instrument (Lonza), CTX-1500A instrument (Celetrix), MaxCyte GT or VLX instrument (MaxCyte), Gene Pulser Xcell (Biorad). Based on the manufacturer's instructions, the pulse duration, intensity, interval between pulses, and number of pulses can be modified to achieve optimal conditions for high transfection efficiency and low mortality.

In the present application, first, the expression and/or activity of Fas protein in the immune cells is down-regulated by step a), and the down-regulation can be performed using any method available in the art such as those mentioned above.

Then, after step a) successfully down-regulating the expression and/or activity of the Fas protein in the immune cells, step b) can be performed. The successful down-regulation can include a reduction in the activity or level of Fas protein by at least 50% (eg, at least 51%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or more). For example, before performing step a) or during performing step a), the expression and/or activity of Fas protein in immune cells is 100%, and after a certain period of time, when the expression and/or activity of Fas in the immune cells Or when the activity gradually decreases to 50% of the initial stage, it can be considered that the down-regulation of step a) is successful, and step b) can be performed. For example, after step a) is performed, it is possible to determine the relative level of Fas protein relative to Fas protein by detecting the content and/or activity of Fas protein, the content and/or activity of Fas gene, the degree of transcription of Fas gene (eg, mRNA content) in immune cells, etc. The extent to which the activity or level has decreased prior to performing step a).

Gene or protein expression levels can be detected by a variety of methods, including methods at the nucleic acid level (including by reverse transcriptase polymerase chain reaction (RT-PCR) or by Southern blotting, in situ hybridization for mRNA quantification, next-generation sequencing ) and methods at the protein level (including immunofluorescence labeling and analysis by flow cytometry, histochemistry, immunoblot analysis, and in vitro binding studies). In addition, protein expression levels can be quantified by ELISA techniques well known to those skilled in the art. Quantitative measurements can be done using a number of standard assays. For example, transcript levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis. Immunohistochemical staining and flow cytometry, Western blot analysis can also be used to assess whether and how much Fas protein and/or Fas gene is present.

In the present application, when down-regulation is performed using gene editing (eg, application of the CRISPR/Cas system), step b) can be performed after the gene editing has occurred. The success of gene editing can be judged by various methods, for example, including but not limited to, immunofluorescence labeling of the protein encoded by the edited gene and detection of genome editing by flow cytometry analysis, next-generation sequencing (NGS), agarose gel, Gene editing detection by capillary electrophoresis, TIDE assays (using gRNA sequences and amplicon sequencing data of unmodified and edited DNA to determine the contribution of curves of different indel lengths to the mixed curve) or digital PCR (dPCR). For example, after detecting that the target gene is knocked out by technical means in the art, step b) can be performed.

In the present application, step b) is at least 6 hours after step a) (eg, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks afterward) or later).

In the present application, the method may comprise a) down-regulating the expression and/or activity of the Fas protein in the immune cells, b) up-regulating the expression and/or activity of the FasL protein in the immune cells. In certain instances, step b) follows (eg, 6 hours, 8 hours, 12 hours, 24 hours, 2 days, 3 days, 4 days, 1 week, 2 weeks or later) implementation.

modified immune cells

In another aspect, the application provides a modified immune cell. The immune cells may include T cells, B cells, natural killer (NK) cells, macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, leukocytes and/or peripheral blood mononuclear cells . In certain instances, the immune cells can include T lymphocytes. The T lymphocytes may include thymocytes, naive T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes. The T cells may be helper T cells (Th), such as T helper 1 (Th1) or T helper 2 (Th2) cells. The T lymphocytes can be CD4+ helper T cells (HTL; CD4 ⁺ T cells), cytotoxic T cells (CTL; CD8 ⁺ T cells), tumor-infiltrating cytotoxic T cells (TIL; CD8 ⁺ T cells), CD4+/ CD8 ⁺ T cells, CD4- / CD8 ^- T cells or any other subtypes of T lymphocytes. In certain instances, the modified T cells are human T cells.

In certain instances, the immune cells can include B cells. In certain instances, the B cells can include effector B cells (plasma cells), memory B cells. The B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, regulatory B cells. In certain instances, the immune cells can include macrophages. The B cells may include type I macrophages (M1), type II macrophages (eg, M2a, M2B, M2c). In certain instances, the immune cells can include NK cells. In certain instances, the NK cells can include CD56bright and CD56dim. In certain instances, the NK cells can include NK1 and NK2. In certain instances, the NK cells can include A-NK and NA-NK.

The expression and/or activity of T cell receptor, T cell receptor alpha constant region protein and/or T cell receptor beta constant region protein in the immune cells is down-regulated. In the present application, the operation of down-regulating the expression and/or activity of T cell receptor, T cell receptor alpha constant region protein and/or T cell receptor beta constant region protein may be performed before, simultaneously or after step a), It can also be carried out before, simultaneously or after step b). In certain instances, the down-regulation can include down-regulation of the expression and/or activity of a nucleic acid molecule encoding the T cell receptor, T cell receptor alpha constant region protein, and/or T cell receptor beta constant region protein; and /or, comprising down-regulating the expression and/or activity of said T cell receptor, T cell receptor alpha constant region protein and/or T cell receptor beta constant region protein.

The modified immune cells of the present application can be activated and expanded before or after any modification steps. Immune cells can be expanded in vitro or in vivo. In general, the immune cells of the present application can be expanded, for example, by contact with an agent that stimulates CD3-TCR complexes and co-stimulatory molecules on the surface of immune cells to generate immune cell activation signals. For example, an immune cell population can be contacted with an anti-CD3 antibody and an anti-CD28 antibody under conditions suitable for stimulating immune cell proliferation. Conditions suitable for culturing immune cells include suitable media that may contain factors necessary for proliferation and viability. Cells can be maintained under conditions necessary to support growth, such as an appropriate temperature (eg, 37°C) and environment (eg, air plus 5 %CO ₂ ).

In the modified immune cells described in the present application, the expression and/or reduction or elimination of the Fas protein in the modified immune cells, and the expression and/or expression of the FasL protein in the modified immune cells compared with the non-modified immune cells increased activity.

Modified immune cells obtained using the methods described herein can have good expansion efficiency. For example, the expansion efficiency of modified immune cells obtained using the methods described herein can be increased by at least 20% compared to modified immune cells obtained by first performing step b) followed by step a). (eg, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or higher). For example, flow cytometry can be used to detect the expansion efficiency of the immune cells.

The Fas protein in the modified immune cells obtained using the method described in this application can have good knockout efficiency. For example, the knockout efficiency of Fas protein in the modified immune cells obtained using the method described in this application can be compared to the modified immune cells obtained by first performing step b) followed by step a). Increase by at least 10% (eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99% or higher) . For example, the knockout efficiency of Fas protein can be tested using flow cytometry.

In the present application, the modified immune cells have comparable or higher expansion capacity and similar or higher immune properties, and anti-tumor activity, especially inhibition or killing of tumors, as compared to unmodified immune cells cell activity.

In the present application, the immune cells may be universal T cells knocked out of T cell receptor (TCR), or the T cells may be T cells containing chimeric antigen receptor (CAR) (CAR-T cells) .

Prior to expanding and genetically modifying the cells of the present application, a source of cells can be obtained from a subject, eg, a patient, by various non-limiting methods. Immune cells can be obtained from a number of non-limiting sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue at the site of infection, ascites, pleural effusion, spleen tissue, and tumors. In some cases, any number of immune cell lines available and known to those of skill in the art can be used. In other cases, the cells can be derived from a healthy donor, from a patient diagnosed with cancer, or obtained from a patient diagnosed with an infection. In other cases, the cells are part of a mixed population of cells with different phenotypic properties. In certain instances, the immune cells can be autologous cells derived from the subject. As used herein, "autologous" generally refers to a cell, cell line or population of cells used to treat a subject derived from the subject. In certain instances, the immune cells can be derived from allogeneic cells, eg, from a donor compatible with the subject's human leukocyte antigen (HLA). Cells from the donor can be transformed into non-aloreactive cells using standard protocols and replicated as needed, resulting in cells that can be administered to one or more patients.

In another aspect, the present application provides a cell population, which can comprise the immune cells, and at least 20% (eg, at least 30%, at least 40%, at least 50%, at least 20%) of the cell population 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) immune cells that do not substantially express Fas protein. Substantially no expression can mean that the expression of the Fas protein in the modified immune cell is reduced by at least about 50% (eg, about 50%, about 60%, about 70%) compared to the unmodified immune cell. %, about 80%, about 90%, or 99% or more), alternatively, the expression of the Fas protein is undetectable using routine techniques in the art.

In the present application, at least 15% (eg, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%) of the cell population %, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more) of immune cells substantially PD-1 protein is not expressed. Substantially no expression may refer to the expression of PD-1 protein being reduced by at least about 50% (eg, about 50%) in the down-regulated PD-1-modified immune cells compared to the non-modified immune cells , about 60%, about 70%, about 80%, about 90%, or 99% or more), or, the expression of the PD-1 protein cannot be detected using conventional techniques in the art.

In the present application, at least 5% (eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%) of the cell population %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more) of the immune cells overexpress FasL protein.

In the present application, at least 5% (eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%) of the cell population %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more) of the immune cells overexpress PD-L1 protein.

In the present application, at least 5% (eg, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%) of the cell population %, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or more) of the immune cells overexpress the CD24 protein.

Overexpression can refer to an increase in the level of expression of a protein in the modified immune cell by at least about 10% (eg, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 99% or more).

Pharmaceutical compositions and uses

In another aspect, the present application provides a pharmaceutical composition comprising said immune cells and/or said cell population, and a pharmaceutically acceptable carrier.

"Pharmaceutically acceptable carrier" as used herein can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In certain instances, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (eg, by injection or infusion). The "effective amount" generally refers to an amount of a substance, compound, material or cell that is at least sufficient to produce a therapeutic effect after administration to a subject. Thus, it is an amount necessary to prevent, cure, ameliorate, retard or partially retard the symptoms of a disease or disorder.

The method still further includes administering to the subject radiation and/or chemotherapy and/or additional tumor-targeting drugs (eg, antibodies or small molecule compounds targeting other antigens).

For example, an "effective amount" of the cell or pharmaceutical composition itself results in a reduction in the severity of symptoms of a disease, an increase in the frequency and duration of asymptomatic periods of the disease, or prevention of injury or disability due to the suffering of the disease. In practice, the dosage level of cells in the pharmaceutical compositions of the present application may be varied to obtain an amount of active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without toxicity to the patient . The dose level selected depends on a variety of pharmacokinetic factors, including the activity of the immune cells and/or pharmaceutical compositions of the present application employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, and the duration of treatment. , other drugs, compounds and/or materials used in combination with the particular composition being used, the age, sex, weight, condition, general health and medical history of the patient being treated, and similar factors well known in the medical arts.

In another aspect, the present application provides the use of the immune cells, the cell population and/or the pharmaceutical composition in the preparation of a medicament for treating a tumor.

The present application also provides a method for treating a tumor, the method comprising administering to a subject in need thereof a therapeutically effective amount of the modified immune cells, cell populations and/or the pharmaceutical composition.

The present application also provides a method of allogeneic cell transplantation, the method comprising administering to a subject in need thereof a therapeutically effective amount of the modified immune cells, cell populations and/or the pharmaceutical composition. The method can reduce the killing of the infused allogeneic cells by the immune system in the subject.

Without being bound by any theory, the following examples are only for illustrating the modified immune cells, preparation methods, uses, etc. of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Isolation and activation of human primary T cells

Human peripheral blood mononuclear cells (PBMCs, purchased from Shanghai Miaoshun Biotechnology) were diluted to 2×10 ⁶ /ml, and anti-human CD3/CD28 magnetic beads (Thermo Fisher Scientific) were used according to the ratio of cells to magnetic beads 1:3. activate T cells.

Example 2 Design and preparation of gRNA

According to the gene sequence of Fas, 15 spacers of guide RNA (gRNA) were designed (nucleotide sequences are SEQ ID NO: 1-15), and PCR primers containing these sequences and gRNA backbone sequence (SEQ ID NO: 35) were synthesized . The synthetic transcription template was amplified by PCR, the reaction system was 20 μl, as shown in Table 2 below, and the reaction conditions were shown in Table 3 below. Guide RNA1 to guide RNA20 (referred to as gRNA1 to gRNA20, respectively) were synthesized.

Table 2 PCR reaction system

reagent	Volume (μl)
Q5 hot start HF 2X mix	10
Forward primer (10 μM): sgRNA primer	1
Reverse primer (10 μM): universal primer	1
ddH 2 O	8

Table 3 PCR reaction conditions

Then the gRNA was transcribed in vitro, and the reaction system was 20 μl, see Table 4. Add reagents and operate on ice, then react at 37°C for 2-4 hours, add 1 μl of DNAase enzyme after completion, and react at 37°C for 15 minutes. For specific operation steps, please refer to PCT Patent Application Publication WO2019011118A1.

Table 4 Transcription reaction system

reagent	Volume (μl)
PCR product from the previous step	8
Transcription buffer	10
T7 transcriptase	2

Next, to purify the gRNA, the magnetic beads (Beckman, A63987) were taken out of the refrigerator and turned upside down 8-10 times and shaken several times until all the magnetic beads were fully suspended. Magnetic beads were then added to the gRNA product in 1:1.8 volumes and incubated at room temperature for 5-10 minutes. After incubation, place on the magnetic separator for 2 minutes, aspirate the supernatant, and leave the EP tube on the magnetic separator. Add 200 μl of 80% ethanol to each tube to wash, no need to shake and keep the EP tube on the magnetic separator, aspirate the supernatant, repeat this step once, and finally suck up the supernatant as much as possible. Air-dry at room temperature for 5 minutes, add 100 μl RNAase free water and mix well, let stand for 2-5 minutes at room temperature, put the EP tube on the magnetic separator for 1-2 minutes until the magnetic beads are adsorbed by the magnet, put the supernatant with gRNA Transfer the liquid to a new EP tube. Finally, the purified gRNA concentration was determined using Nanodrop according to the instructions.

Example 3 Preparation of Fas knockout T cells

After removing the magnetic beads from the activated T cells, CRISPR/Cas9 electroporation was used to knock out Fas in the T cells. The cell processing procedure is carried out with reference to patent WO2019011118A1. Briefly, gRNA from Example 2 was mixed with Cas9 protein purchased from IDT in T buffer provided by Neon kit (Thermo, MPK1096), incubated at room temperature for 5 to 10 minutes, and then T cells were prepared according to the manufacturer's instructions. ^{Electroporation guides were mixed with 6} μl T cells resuspended in T buffer at a quantity of 0.2×10 6 /ml and electroporated in a Neon’s 10 μl pipette tip. For the electrotransfer method, please refer to the literature Schumann et al. PNAS 2015.112:10437-10442.

The transfected T cells after power is placed at 37 degrees Celsius incubator with an atmosphere of 5% CO ₂ for 48 hours, shaken and charge suspension cells, the cells were stained with an antibody recognizing the Fas, and using a flow cytometer (Beckman Coulter CytoFLEX) analysis.

Figure 1 and Table 5 show the results of gRNA splicing efficiency. The results showed that all guide RNAs could cleave Fas, and the knockout efficiencies of SEQ ID NOs: 2-4, 7-10 and 12 were all greater than 30%. Subsequent experiments were performed with gRNAs with knockout efficiency greater than 30%.

Table 5 Knockout efficiency of Fas gRNA

Example 4 Lentiviral packaging expressing FasL

The lentiviral vector plasmid pELPs (for the sequence, refer to Sequence1 in patent US9499629B2) was obtained by a total synthesis method, and FasL and a truncated sequence FasLF were synthesized. Wherein, the amino acid sequence of FasL is shown in SEQ ID NO:50, the nucleotide sequence encoding FasL is shown in SEQ ID NO:53, the amino acid sequence of FasLF is shown in SEQ ID NO:51, and the nucleotide sequence encoding FasL is shown in SEQ ID NO:51 The sequence is shown in SEQ ID NO:54.

Among them, the FasL sequence refers to GenBank ID AY225406.1, but the 376th-390th nucleic acid sequence is mutated, and the corresponding amino acid sequence is changed so that it will not be secreted into the extracellular environment.

The nucleic acids encoding FasL and FasLF were ligated to the downstream of the EF-1α promoter in pELPs to obtain plasmids pELPs-FasL and pELPs-FasLF, and lentiviral packaging was performed.

Example 5 Fas knockout and/or transfection virus

Using the conditions shown in Table 6, the PBMC cells were treated respectively, and the recovery time of the cryopreserved PBMC was recorded as the 0th day, and the Fas of the T cells was knocked out using the method of Example 3 or FasL was transferred into the cells using the virus of Example 4. . After Fas knockout and addition of FasL or FasLF-expressing virus, the cells were placed in _{a cell incubator at 37°C, 5% CO 2} to continue the culture. On days 3 and 6, antibodies that recognize Fas and FasL were used. The cells were stained and detected by flow cytometry (Beckman Coulter CytoFLEX), as well as the efficiency of Fas knockout and FasL expression.

Table 6 Treatment conditions

The results are shown in Figures 2 and 3. If a virus containing a nucleic acid molecule encoding FasL/FasLF is added first, and then Fas is knocked out (conditions 1-2), the cell expansion efficiency is low and the Fas knockout effect is poor. By knocking out Fas first and then expressing FasL/FasLF (conditions 3-4), FasL/FasLF-expressing T cells with better Fas knockout effect and higher amplification efficiency can be prepared.

Example 6 FasL-expressing T cells are resistant to the killing of allogeneic T cells

In order to prove that T cells expressing FasL/FasLF can resist the killing of allogeneic T cells, in this example, the method of mixed culture of cells in vitro was used to compare the change trend of cell number of cells expressing or not expressing FasL/FasLF in the presence of allogeneic T cells. .

(1) Prepare T cells expressing FasLF according to condition 3 of Example 5, activate T cells during cell recovery, and add gRNA targeting Fas and TRAC to the step of electroporation of Cas9/gRNA on day 3 to achieve simultaneous knockdown Except for Fas and TCR purposes. Wherein, the gRNA sequences targeting TRAC are shown in SEQ ID NOs: 31-33. FasL/FasLF expressing virus was then added on day 4.

In order to ensure that the experimental results reflect the killing effect of TCR-knockout target cells by allogeneic T cells, MACS magnetic bead sorting was used to remove CD3-positive cells in the target cells, and TCR-deficient T cells could not be killed by allogeneic T cells. Activation of effector cells leads to expansion.

(2) T cells from the same source (donor 1) and different sources (donor 2) as FasLF-expressing T cells were then prepared: PBMCs activated with CD3/CD28 magnetic beads were cultured for 10 days and then sorted with MACS magnetic beads The method removes CD56-positive cells and excludes the interference of NK cells.

(3) As shown in Table 7, the effector cells from different sources prepared according to step (2) and the target cells from donor 1 from step (1) were mixed in a ratio of 5:1, wherein groups 1 and 2 As a blank control group, target cells were cultured alone. Effector cells and target cells were counted on the day after mixing (day 0), day 2, day 4, and day 6, respectively.

Table 7 Mixing conditions

group	effector cells	target cell
1	none	Donor 1 (knockout TCR)
2	none	Donor 1 (knockout TCR, Fas, express FasLF)
3	Donor 1	Donor 1 (knockout TCR)
4	Donor 1	Donor 1 (knockout TCR, Fas, express FasLF)
5	Donor 2	Donor 1 (knockout TCR)
6	Donor 2	Donor 1 (knockout TCR, Fas, express FasLF)

Cells were then stained with antibodies recognizing Fas, FasL, CD3 and analyzed by flow cytometry (Beckman Coulter CytoFLEX). Since the target cells are CD3-negative, and more than 98% of the effector cells are CD3-positive, CD3 staining can be used to distinguish the target cell population from the effector cell population, and the two populations of cells can be counted separately, and then converted to each sample by volume. The total cell number of effective target cells in the well. When counting the cell expansion fold, the cell number on day 0 was set to 1, and the total cell number at other time points was divided by the cell number on day 0 to obtain the cell expansion fold.

The results showed that on the 6th day, the differences in the expansion folds of target cells in each group of samples could be clearly seen (Fig. 4). In control groups 1 and 2 cultured alone (control group A in Figure 5), there was little difference in expansion folds, with 2 expanding slightly faster than 1, demonstrating that Fas knockout and FasLF-expressing treatments did not affect cell growth. In control groups 3 and 4 co-cultured with autologous cells (ie, control group B in Figure 5), the cell growth status was also not significantly different. However, in experimental groups 5 and 6 co-cultured with allogeneic cells (ie, the experimental group in Figure 5), the experimental group 5 without Fas knockout and FasLF expression showed obvious slow expansion, while the Fas knockout and FasLF expression group 5 showed obvious slow expansion. Experimental group 6 still expanded normally. Experiments show that Fas knockout and FasL-expressing T cells can resist the killing of allogeneic T cells when co-cultured with allogeneic T cells.

Example 7 T cells expressing FasL and PDL1/CD24 are resistant to the killing of allogeneic PBMCs

In order to prove that T cells expressing FasL and PDL1, or FasL and CD24, or FasL and PDL1, CD24 at the same time can resist the killing effect of allogeneic PBMC cells, this example adopts the method of in vitro mixed culture of cells, compared with not expressing exogenous protein, and The changing trend of cell number of cells expressing FasL, PDL1 and CD24 in the presence of allogeneic PBMC cells. Truncated proteins were used in all experiments in this example. In addition, PD-1 was also knocked out in this experiment.

(1) PD-1 gRNA screening: 15 gRNAs were designed and synthesized according to the gene sequence of PD-1 (the nucleic acid sequences are SEQ ID NOs: 16-30, respectively). After mixing the gRNAs with Cas9 protein, Jurkat cells were tested for Electric turn. After culturing Jurkat cells for 24 hours, anti-CD3 antibody was added to the medium, and the expression of PD-1 was detected after 30 hours of culture. The results are shown in Table 8. All gRNAs can successfully knock out PD-1.

Table 8 Knockout efficiency of PD-1 gRNA

(2) Construct a lentiviral vector expressing FasLF+PDL1F, FasLF+CD24, FasLF+PDL1F+CD24 at the same time and package the lentivirus: On the basis of Example 4, synthesize P2A-PDL1F (SEQ ID NO: 61), CD24 (SEQ ID NO: 62), the gene fragment was constructed on the plasmid pELPs-FasLF by the method of Gibson homologous recombination, and the lentiviral vectors pELPs-FasLF-P2A-PDL1F and pELPs-FasLF-P2A-CD24 were obtained. In addition, CD24-P2A-PDL1F-IRES-FasLF (SEQ ID NO: 47) was synthesized, the plasmid pELPs and the gene fragment were treated with restriction enzymes at the same time, and the synthesized gene fragment was ligated to the downstream of the EF-1α promoter in pELPs to obtain Lentiviral vector pELPs-CD24-P2A-PDL1F-IRES-FasLF. Lentiviral packaging was performed separately with the obtained lentiviral vectors.

(3) Preparation of T cells expressing FasLF, FasLF+PDL1F, FasLF+CD24, FasLF+PDL1F+CD24: Using condition 3 in Example 5, when adding lentivirus, the corresponding samples were respectively added into the lentivirus containing the nucleic acid molecule encoding FasLF. Virus and three lentiviruses obtained in step (2).

(4) As shown in Table 9, the target cells prepared according to step (3) were stained with CellTrace pigment. PBMCs and target cells from different sources were mixed at a ratio of 20:1, and effector cells and target cells were counted on the day after mixing (day 0), day 2, day 4, and day 6, respectively.

Table 9 Mixing conditions

group	effector cells	target cell
1	none	Donor 1 (knockout TCR)
2	none	Donor 1 (knockout TCR, Fas, express FasLF)
3	none	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F)
4	none	Donor 1 (knockout TCR, Fas, express FasLF+CD24)
5	none	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F+CD24)
6	Donor 1	Donor 1 (knockout TCR)
7	Donor 1	Donor 1 (knockout TCR, Fas, express FasLF)
8	Donor 1	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F)
9	Donor 1	Donor 1 (knockout TCR, Fas, express FasLF+CD24)
10	Donor 1	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F+CD24)
11	Donor 2	Donor 1 (knockout TCR)
12	Donor 2	Donor 1 (knockout TCR, Fas, express FasLF)
13	Donor 2	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F)
14	Donor 2	Donor 1 (knockout TCR, Fas, express FasLF+CD24)
15	Donor 2	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F+CD24)
16	Donor 3	Donor 1 (knockout TCR)
17	Donor 3	Donor 1 (knockout TCR, Fas, express FasLF)
18	Donor 3	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F)
19	Donor 3	Donor 1 (knockout TCR, Fas, express FasLF+CD24)
20	Donor 3	Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F+CD24)

Cells were stained with antibodies that recognize CD3, Fas, FasL, PD-1, PDL1, and CD24, and flow cytometry (Beckman Coulter CytoFLEX) was used to analyze the expansion fold of target cells as an indicator to detect the ability to resist killing. Target cells can be distinguished from effector PBMCs with CellTrace dyes.

As shown in Table 10, FasLF has a certain resistance to PBMC cell killing. This resistance is stronger when PDL1F or CD24 is simultaneously expressed, and when FasLF, PDL1F and CD24 are simultaneously expressed, the effect of resisting PBMC killing is good. , the three proteins function synergistically in resisting PBMC killing.

Table 10 Anti-PBMC killing ability

target cell	Resist PBMC killing ability
Donor 1 (knockout TCR)	--
Donor 1 (knockout TCR, Fas, express FasLF)	+
Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F)	++
Donor 1 (knockout TCR, Fas, express FasLF+CD24)	++
Donor 1 (knock out TCR, Fas, PD-1, express FasLF+PDL1F+CD24)	+++

Example 8 Variants with replacement of FasL hinge region also have resistance to killing of allogeneic T cells

The FasL hinge region was replaced to obtain two FasL variants, FasL-M1 (amino acid sequence shown in SEQ ID NO: 63) and FasL-M2 (amino acid sequence shown in SEQ ID NO: 64), and the extracellular region was unchanged . Lentiviral packaging was performed according to the methods of Example 4 and Example 5, and T cells were transfected. As shown in Figure 6, both FasL-M1 and FasL-M2 were expressed in T cells.

Then, according to Example 6, the method of in vitro cell mixed culture was used to compare the cell number change trend of T cells expressing FasL, FasL-M1, FasL-M2 and those that do not express foreign proteins in the presence of allogeneic T cells.

(1) Control T cells and T cells expressing FasL, FasL-M1 and FasL-M2 were prepared from T cells from Donor 1 according to the method of Example 6(1), and CD3-negative cells were sorted by MACS.

(2) According to the method of Example 6 (2), prepare T cells from a different source (donor 2) from the T cells used in (1), and remove CD56-positive cells by MACS sorting.

(3) As shown in Table 11, the effector T cells in (2) and the target cells in (1) are mixed in a ratio of 5:1.

Table 11 Mixing conditions

group	effector cells	target cell
1	none	Donor 1 (knockout TCR, Fas)
2	none	Donor 1 (knockout TCR, Fas, express FasLF)
3	none	Donor 1 (knockout TCR, Fas, express FasL-M1)
4	none	Donor 1 (knockout TCR, Fas, express FasL-M2)

5	Donor 2	Donor 1 (knockout TCR, Fas)
6	Donor 2	Donor 1 (knockout TCR, Fas, express FasLF)
7	Donor 2	Donor 1 (knockout TCR, Fas, express FasL-M1)
8	Donor 2	Donor 1 (knockout TCR, Fas, express FasL-M2)

Cells were then stained with antibodies recognizing Fas, FasL, CD3 and analyzed by flow cytometry (Beckman Coulter CytoFLEX). Since the target cells are CD3-negative, and more than 98% of the effector cells are CD3-positive, CD3 staining can be used to distinguish the target cell population from the effector cell population, and the two populations of cells can be counted separately, and then converted to each sample by volume. The total cell number of effective target cells in the well. When counting the cell expansion fold, the cell number on day 0 was set to 1, and the total cell number at other time points was divided by the cell number on day 0 to obtain the cell expansion fold.

As shown in Figure 7, in groups 1-4 cultured alone, target cells expanded normally, and the folds of cell expansion were similar in the four groups. In contrast, in groups 5-8 co-cultured with allogeneic T cells, group 5 expanded significantly worse, and groups 6-8 expanded similarly. It can be seen that FasL and its variants FasL-M1 and FasL-M2 can protect allogeneic T cells from killing, so that T cells expressing FasL, FasL-M1 and FasLF-M2 can survive more under the killing effect of allogeneic T cells. .

Claims (66)

1. A method of preparing a modified immune cell, the modification comprising the following steps.

   a) down-regulating the expression and/or activity of Fas protein in said immune cells,

   b) up-regulating the expression and/or activity of FasL protein in said immune cells,

   such that the expression of Fas protein and/or in the modified immune cells compared to the unmodified immune cells

   Either the activity is reduced or eliminated, and the expression and/or activity of the FasL protein is increased in the modified immune cells.
2. The method of claim 1, wherein the step a) comprises down-regulating the expression and/or activity of a nucleic acid molecule encoding a Fas protein in the immune cell.
3. The method according to any one of claims 1-2, wherein the down-regulation of the step a) comprises by gene knock out (knock out), gene knock down (knock down), gene mutation, gene deletion, gene Silence or any combination of the above to down-regulate.
4. The method of any one of claims 1-3, wherein the down-regulation of step a) comprises administering to the immune cell one or more substances selected from the group consisting of antisense RNA, siRNA, shRNA, CRISPR/Cas systems, RNA editing systems such as RNA adenosine deaminase (ADAR), RNA-guided endonucleases, zinc finger nucleases (ZFN), Mega-TAL nucleases, transcription activator-like effector nucleic acids Enzyme (TALEN), meganuclease (Meganuclease), base editing, CRISPR interference, and, Zinc finger gene repressor and/or transcription activator-like effector (TALE) gene repressor mediated Transcriptional repression.
5. The method of any one of claims 1-4, wherein step a) comprises administering to the immune cell a guide RNA targeting a nucleic acid molecule encoding a Fas protein.
6. The method of any one of claims 1-5, wherein step a) comprises targeting any one or more of exons 1-5 of a nucleic acid molecule encoding a Fas protein in the immune cell.
7. The method of any one of claims 1-6, wherein the Fas protein comprises the amino acid sequence shown in SEQ ID NO:46.
8. The method of any one of claims 2-7, wherein the nucleic acid molecule encoding the Fas protein comprises the nucleotide sequence shown under gene ID: 355 in the NCBI database or under ENSG00000026103 in the Ensembl database.
9. The method of any one of claims 5-8, wherein the guide RNA targeting the nucleic acid molecule encoding the Fas protein comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15.
10. The method of any one of claims 5-9, wherein the guide RNA targeting the nucleic acid molecule encoding the Fas protein is a single-stranded guide RNA.
11. The method of any one of claims 5-10, wherein the guide RNA targeting the nucleic acid molecule encoding the Fas protein is a double-stranded guide RNA comprising crRNA and tracrRNA.
12. The method of claim 11, wherein the crRNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 1-15.
13. The method of any one of claims 1-12, comprising downregulating T cell receptor (TCR) complexes, T cell receptor alpha constant region proteins, T cell receptor beta constant region proteins in the immune cell , CD3-epsilon, CD3-gamma, CD3-delta, CD3-zeta, CD3-eta, and/or PD-1 protein expression and/or activity.
14. The method of claim 13, comprising down-regulating the TCR complex, T cell receptor alpha constant region protein, T cell receptor beta constant region protein, CD3-epsilon, CD3-gamma, CD3-delta, CD3 - expression and/or activity of nucleic acid molecules of zeta, CD3-eta, and/or said PD-1 protein.
15. The method of any one of claims 13-14, comprising administering to the immune cell a guide RNA targeting a nucleic acid molecule (TRAC) encoding the T cell receptor alpha constant region protein.
16. 16. The method of claim 15, comprising targeting exons 1-3 of a nucleic acid molecule encoding the T cell receptor alpha constant region protein (TRAC) in the immune cell.
17. The method of any one of claims 13-16, wherein the T cell receptor alpha constant region protein comprises the amino acid sequence set forth in SEQ ID NO:49.
18. The method of any one of claims 15-17, wherein the guide RNA targeting a nucleic acid molecule encoding the T cell receptor alpha constant region protein (TRAC) comprises as set forth in SEQ ID NOs: 31-33 The nucleotide sequence shown.
19. The method of any one of claims 13-18, comprising administering to the immune cell a guide RNA targeting a nucleic acid molecule encoding the PD-1 protein.
20. 19. The method of claim 19, comprising targeting exons 1-3 of a nucleic acid molecule encoding the PD-1 protein in the immune cell.
21. The method of any one of claims 13-20, wherein the PD-1 protein comprises the amino acid sequence shown in SEQ ID NO:48.
22. The method of any one of claims 19-21, wherein the guide RNA of the nucleic acid molecule targeting the PD-1 protein comprises a nucleoside as shown in any one of SEQ ID NOs: 16-30 acid sequence.
23. The method of any one of claims 5-22, wherein the guide RNA comprises a chemical modification.
24. The method of any one of claims 1-23, comprising administering a Cas protein to the immune cell.
25. The method of claim 24, wherein the Cas protein is a Cas9 protein.
26. The method of any one of claims 1-25, wherein the step b) comprises up-regulating the expression and/or activity of a nucleic acid molecule encoding a FasL protein in the immune cell.
27. The method of any one of claims 1-26, wherein step b) comprises administering to the immune cells a nucleic acid molecule encoding a FasL protein.
28. The method of any one of claims 1-27, comprising administering to the immune cell a vector comprising a nucleic acid molecule encoding a FasL protein.
29. The method of any one of claims 1-28, wherein the FasL protein comprises an extracellular domain.
30. The method of claim 29, wherein the extracellular domain of the FasL protein comprises the amino acid sequence shown in SEQ ID NO:52.
31. The method of any one of claims 1-30, wherein the FasL protein comprises a hinge region.
32. The method of claim 31, wherein the hinge region is derived from the tumor necrosis factor superfamily.
33. The method of any one of claims 31-32, wherein the hinge region is derived from FasL, TNFSF10 or OX40L.
34. The method of any one of claims 1-33, wherein the FasL protein comprises an intracellular domain.
35. The method of any one of claims 1-34, wherein the FasL protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 50-52 and 63-64.
36. The method of any one of claims 26-35, wherein the nucleic acid molecule encoding the FasL protein comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 53-55.
37. The method of any one of claims 1-36, further comprising up-regulating the expression and/or activity of PD-L1 protein and/or CD24 protein in the immune cells.
38. The method of claim 37, comprising administering to the immune cell a nucleic acid molecule encoding the PD-L1 and/or a nucleic acid molecule encoding a CD24 protein.
39. The method of any one of claims 37-38, comprising administering to the immune cell a vector comprising the nucleic acid molecule encoding the PD-L1 protein and/or the nucleic acid molecule encoding the CD24 protein.
40. The method of any one of claims 37-39, wherein the PD-L1 protein comprises the amino acid sequence set forth in any one of SEQ ID NOs: 56-57.
41. The method of any one of claims 38-40, wherein the nucleic acid molecule encoding the PD-L1 protein comprises the nucleotide sequence shown in SEQ ID NO:58.
42. The method of any one of claims 37-41, wherein the CD24 protein comprises the amino acid sequence shown in SEQ ID NO:59.
43. The method of any one of claims 38-42, wherein the nucleic acid molecule encoding the CD24 protein comprises the nucleotide sequence set forth in SEQ ID NO:60.
44. The method of any one of claims 28-43, wherein the vector comprises a viral vector.
45. The method of any one of claims 1-44, wherein the immune cells comprise T cells, NK cells, NKT cells, monocytes, macrophages, B cells, plasma cells, granulocytes, dendritic cells cells, lymphocytes, leukocytes, stem cells and/or peripheral blood mononuclear cells.
46. The method of any of claims 1-45, wherein step b) is performed after step a).
47. The method of any one of claims 1-46, wherein step b) is performed after successful down-regulation in step a).
48. The method of claim 47, wherein the successful down-regulation comprises the expression of Fas protein and/or the expression of Fas protein in the immune cells after the step a) compared to the immune cells before the step a). Activity is reduced by at least 50%.
49. 48. The method of any one of claims 47-48, wherein the successful down-regulation comprises the occurrence of gene editing.
50. The method of any one of claims 1-49, wherein step b) is performed at least 6 hours after step a).
51. The method of any one of claims 1-50, further comprising the step of: causing the modified immune cells to include a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric self Antibody receptor (CAAR) and/or at least one synthetic receptor.
52. The method of any one of claims 1-51, further comprising the step of: causing the modified immune cells to include expression of a chimeric antigen receptor (CAR), a T cell receptor (TCR), a chimeric Nucleic acid molecules of autoantibody receptors (CAARs) and/or at least one synthetic receptor.
53. The modified immune cells prepared by the method according to any one of claims 1-52, compared with the non-modified immune cells, the expression of Fas protein in the modified immune cells and/or Activity is reduced or eliminated, and FasL protein expression and/or activity is increased in the modified immune cells.
54. The immune cell of claim 53, comprising a chimeric antigen receptor (CAR), a T cell surface receptor (TCR), a chimeric autoantibody receptor (CAAR) and/or at least one synthetic receptor.
55. A cell population comprising the immune cells of any one of claims 53-54, wherein at least 20% of the immune cells in the cell population do not substantially express Fas and at least 5% of the immune cells overexpress FasL.
56. The cell population of claim 55, wherein at least 15% of the immune cells do not substantially express PD-1.
57. The cell population of any one of claims 55-56, wherein at least 5% of the immune cells overexpress PD-L1.
58. The cell population of any one of claims 55-57, wherein at least 5% of the immune cells overexpress CD24.
59. A pharmaceutical composition comprising the immune cell of any one of claims 53-54 and/or the cell population of any one of claims 55-58, and a pharmaceutically acceptable carrier.
60. Use of the immune cell according to any one of claims 53-54, the cell population according to any one of claims 55-58 and/or the pharmaceutical composition according to claim 59 in the preparation of a medicament, the Medicines are used to treat tumors.
61. A guide RNA targeting a nucleic acid molecule encoding a Fas protein, wherein the guide RNA comprises the nucleotide sequence set forth in any one of SEQ ID NOs: 1-15.
62. A guide RNA targeting a nucleic acid molecule encoding a PD-1 protein, wherein the guide RNA comprises the nucleotide sequence shown in any one of SEQ ID NOs: 16-30.
63. A CRISPR/Cas system comprising the guide RNA of any one of claims 61-62 and a Cas protein.
64. The system of claim 63, wherein the Cas protein comprises a Cas9 protein.
65. A method of allogeneic cell transplantation, comprising administering an effective amount of the immune cell of any one of claims 53-54, the cell population of any one of claims 55-58, and/or the described pharmaceutical composition.
66. A method of treating a tumor comprising administering an effective amount of the immune cell of any one of claims 53-54, the cell population of any one of claims 55-58, and/or the immunocyte of claim 59 pharmaceutical composition.

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