Engineered immune cells with the CIITA gene knocked out and their applications

Abstract

This invention provides a method for manipulating the CIITA gene to reduce or avoid immune rejection of CD4+ T cells in CAR-T cell immunotherapy. [Solution] The present invention provides a method for knocking out the CIITA gene by introducing a spacer sequence, a nucleic acid encoding the sgRNA, a vector containing the nucleic acid, and a Cas9 nuclease into cells, which specifically targets the CIITA gene.

Description

[Technical Field]

[0001] <Cross-reference of related applications> This invention claims priority to a Chinese patent application filed with the Chinese National Patent Office on April 28, 2022, with application number CN202210460793.X and title "Manipulated immune cells with knockout of the CIITA gene and their uses," the entirety of which is incorporated into this invention by reference.

[0002] This invention belongs to the field of biopharmaceuticals. More specifically, this invention relates to engineered immune cells in which the CIITA gene has been knocked out and their uses. [Background technology]

[0003] In recent years, cancer immunotherapy technologies have developed rapidly, and in particular, immunotherapy related to chimeric antigen receptor T cells (CAR-T) has achieved excellent clinical efficacy in treating hematological malignancies. CAR-T cell immunotherapy achieves the goal of tumor treatment by genetically modifying T cells in vitro to enable them to recognize tumor antigens, amplifying them to a certain number, and then returning them to the patient's body to kill cancer cells.

[0004] In 2017, two autologous CAR-T therapies received FDA approval and were launched in the United States: one for B-cell acute leukemia and the other for diffuse B-cell non-Hodgkin lymphoma. While these two CAR-T cells demonstrate excellent clinical efficacy, their high cost, relatively long manufacturing cycle, and large-scale deployment are extremely difficult. Therefore, there is a need to develop a general-purpose CAR-T product that can be used for allogeneic transplantation to address these issues. General-purpose CAR-T cells can be manufactured using T cells isolated from the peripheral blood of healthy donors, thereby enabling allogeneic transplantation and significantly reducing the waiting time for patients to receive treatment. Furthermore, the vitality and function of T cells obtained from healthy donors are superior to those of patient-derived T cells, which can increase the CAR infection rate and improve therapeutic efficacy.

[0005] However, the development of general-purpose CAR-T cells still faces two problems: (1) Manipulated and modified CAR-T cells may cause graft-versus-host disease (GvHD) by entering the patient's body, proliferating to a certain extent, and then attacking the patient's normal cells or tissues; and (2) the patient's normal immune system may reject the heterologous CAR-T cells, potentially causing host-versus-graft disease (HvGD). Currently, the risk of transplantation is reduced mainly by knocking out TCRs, MHC class I molecules, and / or class II molecules.

[0006] CIITA is the primary regulator of MHC class II molecule expression. CIITA consists of an N-terminus rich in acidic amino acids, a PST region rich in Pro, Ser, and Thr, a central GTP-binding region, and a C-terminus rich in Leu repeat sequences (LRRs). The N-terminal acidic region and the PST region are transcriptional activation regions. [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide an sgRNA that specifically targets the CIITA gene, engineered immune cells in which the CIITA gene has been knocked out using this sgRNA, and its use in the prevention and / or treatment of disease and / or in the diagnosis and treatment of cancer, infection, or autoimmune diseases. [Means for solving the problem]

[0008] According to a first aspect, the present invention provides an sgRNA comprising a spacer sequence shown in any one of SEQ ID NO: 1-6, 10, 11, 15, 16, 19, 21, 23. Preferably, the spacer sequence is as shown in any one of SEQ ID NO: 4, 6, 11, 15, 21, 23. More preferably, the spacer sequence is as shown in any one of SEQ ID NO: 4, 6, 11, 15.

[0009] According to a second aspect, the present invention provides a nucleic acid encoding the above-mentioned sgRNA and a vector containing the above-mentioned nucleic acid.

[0010] According to a third aspect, the present invention provides a method for in vitro knockout of the CIITA gene, the method comprising introducing a Cas nuclease and the sgRNA into cells.

[0011] In one embodiment, the Cas nuclease is Cas9, Cpf1, or Cas13a, and exists in the form of a protein, its coding nucleic acid, or a vector. Preferably, the Cas nuclease is Cas9.

[0012] In one embodiment, the sgRNA exists in the form of RNA, its coding nucleic acid, or a vector.

[0013] In one embodiment, the cells are immune cells, and the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells. Preferably, the immune cells are T cells, NK cells or NKT cells. More preferably, the T cells are CD4 + CD8 + T cells, CD4 + T cells, CD8 + These include T cells, memory T cells, immature T cells, γδ-T cells, and / or αβ-T cells.

[0014] In one embodiment, the method further includes introducing a chimeric antigen receptor or a T cell receptor or a nucleic acid encoding both into the immune cells.

[0015] In one embodiment, the chimeric antigen receptor comprises a ligand-binding domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain.

[0016] In one embodiment, the ligand-binding domain is found to be CD7, TSHR, CD19, CD123, CD22, BAFF-R, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, GPRC5D, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, LewisY, CD24, PDGFR-β, SSEA-4, CD20, Folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, Claudin18.2, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gploo, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, Folate receptor β, TEM1 / CD248, TEM7R, CLDN6, GPRC5D, CXORF61, CD97, CD 179a, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, legumain, HPV E6, E7, MAGE Al, ETV6-AML, spermidine 17, XAGE1, Tie 2, MAD-CT-1, MAD-CT-2, Fos-related antigen 1, p53, p53 mutant, prostate-specific protein, survivor and telomerase, PCTA-I / Galectin 8, MelanA / MARTI, Ras mutant, hTERT, sarcoma translocation breakpoint, ML-IAP, ERG (TMPRSS2ETS fusion gene), NA17, PAX3, androgen receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut The target is hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, or any combination thereof. Preferably, the target is CD7, CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2(Her2 / neu), MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2. Selected from NKG2D or any combination thereof. Those skilled in the art can design the appropriate chimeric antigen receptor by determining the antigens that need to be targeted depending on the disease to be treated.

[0017] In one embodiment, the ligand-binding domain includes an antibody or antigen-binding fragment targeting CD19 and / or CD22. Preferably, the ligand-binding domain of the present invention includes a light chain variable region sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:28, and a heavy chain variable region sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:29.

[0018] In one embodiment, the transmembrane domain is selected from the transmembrane domains of a protein, such as TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof. Preferably, the transmembrane domain is derived from the CD8α chain and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:31, or the coding sequence of the CD8α transmembrane domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:32.

[0019] In one embodiment, the chimeric antigen receptor further includes a hinge region located between the ligand-binding domain and the transmembrane domain. Preferably, the hinge region includes a CD8α chain, an FcγRIIIα receptor, IgG4, or IgG1 hinge region, more preferably a CD8α hinge region having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:33, or the coding sequence of the CD8α hinge has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:34.

[0020] In one embodiment, the primary signaling domain is selected from the protein signaling domains FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof. Preferably, the primary signaling domain includes the CD3ζ primary signaling domain, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 35 or 37, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the nucleotide sequence shown in SEQ ID NO: 36 or 38.

[0021] In one embodiment, the co-stimulatory domains are TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18 (LFA-1), CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD152 (CTLA4), The costimulatory signaling domain includes, but is not limited to, a protein-derived costimulatory signaling domain such as CD223(LAG3), CD270(HVEM), CD272(BTLA), CD273(PD-L2), CD274(PD-L1), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70, or any combination thereof. Preferably, the costimulatory domain is derived from 4-1BB, CD28, or 4-1BB+CD28, and more preferably, the 4-1BB costimulatory domain. The 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:39, or the coding sequence of this co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:40.

[0022] In one embodiment, the chimeric antigen receptor further comprises a signal peptide, the signal peptide having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:41, or the coding sequence of the signal peptide having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:42.

[0023] In one embodiment, the T cell receptor targets one or more of HBV, HPV E6, NYESO, mNY-ESO, WT1, MART-1, MAGE-A3, MAGE-A4, P53, Thyroglobulin, and Tyrosinase.

[0024] In one embodiment, the chimeric antigen receptor or T cell receptor can be introduced into a host cell via a vector. Specifically, the vector is selected from plasmids, retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyomaviruses, and adeno-associated virus (AAV), phages, phagemids, cosmids, or artificial chromosomes. In some embodiments, this vector further includes elements such as an origin of autonomous replication in the host cell, a selection marker, a restriction enzyme cleavage site, a promoter, a polyadenylic acid tail (polyA), 3'UTR, 5'UTR, an enhancer, a terminator, an insulator, an operon, a selection marker, a reporter gene, a target sequence, and / or a protein purification tag. In one specific embodiment, the vector is a plasmid, a lentiviral vector, an AAV vector, an adenoviral vector, or a retroviral vector.

[0025] According to a fourth aspect, the present invention provides an engineered immune cell produced using the method for knocking out the CIITA gene as described above.

[0026] In one embodiment, the manipulated immune cells include GR, dCK, TCR / CD3 genes (e.g., TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC-related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes, e.g., PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, This further includes a system in which the expression of at least one gene selected from CD96, CRTAM, TNFRSF10B, TNFRSF10A, CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT, FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3 is inhibited or silenced. Preferably, the manipulated immune cells further include those in which the expression of at least one gene selected from TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, and more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, and CIITA is inhibited or silenced. More preferably, CIITA and TRAC in the manipulated immune cells are knocked out, and such inactivation renders the TCR-CD3 complex non-functional in the cell, and this strategy is particularly useful in avoiding graft-versus-host disease (GvHD).

[0027] According to a fifth aspect, the present invention further provides compositions comprising the manipulated immune cells, and uses of the immune cells or compositions in the manufacture of drugs for treating / preventing / diagnosing cancer, infection, or autoimmune diseases.

[0028] In one embodiment, the composition further comprises one or more pharmaceutically acceptable excipients.

[0029] In one embodiment, the manipulated immune cells or composition of the present invention may be further used in combination with one or more additional chemotherapeutic agents, biologics, drugs, or therapies. In this embodiment, the chemotherapeutic agents, biologics, drugs, or therapies are selected from radiotherapy, surgery, antibody reagents, small molecules, or any combination thereof. [Effects of the Invention]

[0030] An advantage of the present invention is that the screened sgRNA efficiently knocks out the CIITA gene, thereby reducing the CD4 threshold against heterozygous CAR-T cells. + The ability to reduce or avoid the immune rejection reaction of T cells and simultaneously knock out the TRAC gene allows for the production of general-purpose CAR-T cells.

[0031] The present invention will be described in detail below, with reference to the drawings and examples. It should be noted that those skilled in the art will understand that the drawings and embodiments of the present invention are for illustrative purposes only and do not limit the invention in any way. To the extent that they do not contradict each other, the embodiments and features described herein can be combined with each other. [Brief explanation of the drawing]

[0032] [Figure 1] This shows the knockout efficiency of the TRAC / CIITA gene in TRAC / HLA-II double knockout CD19 CAR-T cells (UCAR19). [Figure 2] This shows the expression level of CAR in TRAC / HLA-II double knockout CD19 CAR-T cells (UCAR19). [Figure 3]This shows the IL2 and IFNγ secretion levels of TRAC / HLA-II double knockout CD19 CAR-T cells (UCAR19). [Modes for carrying out the invention]

[0033] Details of the invention Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. For the sake of easier understanding of this application, some terms are defined below.

[0034] sgRNA The CRISPR / Cas system is a natural immune system found in prokaryotes. Some bacteria, after viral invasion, can store small fragments of viral genes in a storage space called CRISPR within their own DNA. Upon subsequent viral invasion, the bacteria can recognize the virus based on the stored fragments and cleave and inactivate the viral DNA. The CRISPR system has three types: type I, type II, and type III. CRISPR / Cas9 is the most widely studied type II, and in addition to the Cas9 nuclease, Cas12a (also known as Cpf1) and Cas13a (also known as C2c2) are also commonly used Cas nucleases. CRISPR / Cas9-mediated gene editing technology can be used for the creation of transgenic models, transcriptional regulation, and epigenetic control.

[0035] The CRISPR / Cas9 system is mainly composed of the Cas9 protein and single-stranded guide RNA (sgRNA). Here, the Cas9 protein has the function of breaking double-strand DNA, and the sgRNA acts as a guide. The principle of operation of this system is that CRISPR RNAs (crRNAs) bind to tracrRNA (trans-activating RNA) via base pairing to form a tracrRNA / crRNA complex. This complex induces the nuclease Cas9 protein to break double-strand DNA at a sequence target site that pairs with a spacer sequence in the crRNA.

[0036] As used herein, the term “sgRNA” is also called “single guide RNA” and refers to an artificially engineered RNA that fuses crRNA and tracrRNA molecules into a “single guide RNA.” crRNA may contain a nucleic acid target segment (e.g., a spacer sequence) of the guide nucleic acid and a single-segment nucleotide, which can form half of the double-stranded Cas protein-binding segment of the guide nucleic acid. tracrRNA may contain a single-segment nucleotide, which can form the other half of the double-stranded Cas protein-binding segment of the sgRNA. The nucleotide of one segment of crRNA is complementary to the nucleotide of one segment of tracrRNA and can hybridize to form the double-stranded Cas protein-binding domain of the guide nucleic acid. When sgRNA binds to Cas9 nuclease, it can recognize and cleave the specific DNA target of the guide RNA. The part of the sgRNA responsible for complementarity with the target DNA is the spacer sequence it contains. In one embodiment, the sgRNA includes a spacer sequence shown in any one of SEQ ID NO: 1-6, 10, 11, 15, 16, 19, 21, or 23.

[0037] As used herein, “PAM” is an abbreviation for protospacer adjacent motif, referring to a short nucleotide sequence adjacent to a target sequence recognized (targeted) by the sgRNA / Cas nuclease system. If the target DNA sequence is not adjacent to a suitable PAM sequence, the Cas nuclease may not be able to successfully recognize the target DNA sequence. The sequence and length of the PAM as used herein may be determined by the Cas protein or Cas protein complex used, and may be AGG, TGG, CGG, or GGG, etc.

[0038] The design of sgRNA spacer sequences is generally as follows: (1) The length should generally be about 20 nt, (2) Regarding the base composition, the seed sequence (bases 1 to 12 closest to the PAM region) should be avoided as much as possible if it ends in four or more T's, and the GC% content should preferably be 40% to 60%, (3) The number of matches between the seed sequence and the off-target site should be as low as possible, (4) Examine whether SNPs are present in the target binding site genome sequence, (5) This is based on the principle that in whole-gene off-target effect analysis, it is necessary to consider the maximum number of mismatched bases that off-target sites can tolerate.

[0039] As will be seen, designing an sgRNA spacer requires considering many factors, such as length, GC content, target gene binding site, binding rate to non-target sites, whether or not it contains SNPs, and secondary structure. Currently, sgRNAs can be designed using various online tools. However, because the Cas enzyme can cleave any target sequence adjacent to the PAM site, the editing efficiency of a large number of sgRNAs designed with online tools differs, and even differs significantly, for a particular target gene. For example, the editing efficiency when the PAM site is NGG is generally higher than when it is NGA or NAG. sgRNA design is directly related to the gene editing efficiency of the CRISPR system. Highly specific sgRNA targets result in higher gene editing efficiency and more positive clones, and in subsequent screening and identification, twice the results can be achieved with half the effort. Therefore, screening for highly specific sgRNAs is extremely important for improving the editing efficiency of the CRISPR system. Generally, after designing sgRNAs, it is necessary to screen for cell activity in vitro to screen for highly specific sgRNAs and use them in subsequent experiments.

[0040] nucleic acids, vectors As used herein, the term “nucleic acid” may be DNA or RNA.

[0041] As used herein, the term “vector” refers to a mediating nucleic acid molecule used to transfer genetic material into a cell, in which the nucleic acid molecule may be replicated and / or expressed.

[0042] In one embodiment, the vector of the present invention includes, but is not limited to, linear nucleic acids (e.g., DNA or RNA), plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma viruses (RSV, polyomaviruses, and adeno-associated viruses (AAV)), phages, phagemids, cosmids, and artificial chromosomes (including BAC and YAC). The vector itself is generally a nucleotide sequence, and generally consists of a DNA sequence containing an insert (genetic recombination) and a relatively large sequence forming the vector "backbone".

[0043] Chimeric antigen receptor As used herein, the term “chimeric antigen receptor” (CAR) includes a ligand-binding domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain. “Ligand-binding domain” refers to any structure or functional variant thereof capable of ligand binding, and includes an antibody or antigen-binding fragment. The selection of the ligand-binding domain is determined by a cell surface marker on a target cell to be recognized, related to a specific disease state, such as a tumor-specific antigen or tumor-associated antigen. In one embodiment, the ligand-binding domain includes an antibody or antigen-binding fragment targeting CD19.

[0044] As used herein, the term “antibody” includes monoclonal antibodies (including complete antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments or synthetic polypeptides having one or more CDR sequences capable of exhibiting desired biological activity.

[0045] Generally, a complete antibody consists of two heavy chains and two light chains linked by disulfide bonds, with each light chain linked to its respective heavy chain by disulfide bonds, forming a "Y" shape. Both the heavy and light chains contain variable and constant regions, with each containing three CDRs, separated from the CDRs by more conserved framework regions (FRs). The variable regions are responsible for recognition and binding to the antigen, while the constant regions can mediate the binding of the antibody to host tissue or factors.

[0046] As used herein, the term “antigen-binding fragment” includes only a portion of a complete antibody, and generally includes the antigen-binding site of a complete antibody, and thus retains the ability to bind to an antigen. Examples of antigen-binding fragments in the present invention include, but are not limited to, Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, scFv, disulfide-bonded Fv(sdFv), heavy chain variable region (VH) or light chain variable region (VL) of an antibody, linear antibodies, “diabodies” having two antigen-binding sites, single-domain antibodies, nanoantibodies, natural ligands of the antigen or functional fragments thereof.

[0047] As used herein, the term “transmembrane domain” refers to a polypeptide structure that can express a chimeric antigen receptor on the surface of immune cells (e.g., lymphocytes, NK cells, or NKT cells) and induce a cellular response of the immune cells to the target cell. The transmembrane domain may be native or synthetic and may be derived from any membrane-bound or transmembrane protein. When the chimeric antigen receptor binds to the target antigen, the transmembrane domain can transmit signals. In one embodiment, the transmembrane domain is derived from the CD8α chain.

[0048] As used herein, the term “hinge region” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to a ligand-binding domain. Specifically, hinge regions are used to provide greater flexibility and reachability for the ligand-binding domain. A hinge region may contain up to 300 amino acids, preferably 10 to 100 amino acids, and most preferably 25 to 50 amino acids. A hinge region may be derived all or partly from a native molecule, for example, all or partly from the extracellular region of CD8, CD4, or CD28, or all or partly from the antibody constant region. In one embodiment, the hinge region includes a CD8α hinge region.

[0049] As used herein, the term “primary signaling domain” refers to a protein portion that transmits effector functional signals and directs cells to perform a designated function. The primary signaling domain triggers the activation of immune cells and immune responses by carrying out intracellular primary signaling after the ligand-binding domain binds to an antigen. The primary signaling domain is the cytoplasmic sequences of the T cell receptor and co-receptor, and any derivatives or variants of these sequences, as well as any synthetic sequences having the same or similar function, which act together after the antigen receptor binds to trigger primary signaling. The primary signaling domain may include many immune receptor tyrosine activation motifs (ITAMs). In one embodiment, the primary signaling domain includes the CD3ζ primary signaling domain.

[0050] The chimeric antigen receptor of the present invention comprises one or more costimulatory domains. The costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, which comprises the entire intracellular portion of the costimulatory molecule or a functional fragment thereof. The “costimulatory molecule” refers to a homologous binding partner that mediates the costimulatory response (e.g., proliferation) of T cells by specifically binding to a costimulatory ligand on T cells. The costimulatory molecule includes, but is not limited to, class 1 MHC molecules, BTLA, and Toll ligand receptors. In one embodiment, the costimulatory domain is derived from 4-1BB, CD28, or 4-1BB+CD28, and more preferably, a 4-1BB costimulatory domain.

[0051] The chimeric antigen receptor of the present invention may further contain a signal peptide, which, when expressed in cells, such as T cells, induces the nascent protein in the endoplasmic reticulum and then on the cell surface. The core of the signal peptide may contain a long hydrophobic amino acid segment that tends to form a single α-helix. Signal peptides that can be used in the present invention are those well known to those skilled in the art, such as signal peptides derived from CD8α, IgG1, GM-CSFRα, etc.

[0052] Manipulated immune cells As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (e.g., cytotoxic cell killing activity, cytokine secretion, ADCC and / or CDC induction). For example, immune cells may be T cells, macrophages, dendritic cells, monocytes, NK cells and / or NKT cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells or hematopoietic stem cells, etc. Preferably, the immune cells are T cells. The T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats and their transgenic species. T cells can be obtained from many sources including peripheral blood monocytes, bone marrow, lymph node tissue, umbilical cord blood, thymus tissue, tissue from sites of infection, ascites, pleural effusion, spleen tissue and tumors. The T cells may be concentrated or purified. The T cells can be at any stage of development, CD4 + / CD8 + T cells, CD4 + helper T cells (e.g., Th1 and Th2 cells), CD8 + T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, γδ-T cells, αβ-T cells, etc., including but not limited to these. In one preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using many techniques well known to those skilled in the art, such as Ficoll apheresis.

[0053] By adopting the conventional methods known in the art (e.g., transduction, transfection, transformation, etc.), a nucleic acid sequence encoding a chimeric antigen receptor can be introduced into immune cells.

[0054] In one embodiment, in order to reduce the risk of graft-versus-host disease, the manipulated immune cells further include those in which the expression of at least one gene selected from TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, and more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, and CIITA is inhibited or silenced.

[0055] composition The present invention further provides compositions comprising the manipulated immune cell bodies described in the present invention. In one embodiment, one or more pharmaceutically acceptable excipients are further included.

[0056] As used herein, “pharmaceutically acceptable excipients” means carriers and / or excipients that are pharmacologically and / or physiologically compatible with the subject and the active ingredient (i.e., capable of producing the desired therapeutic effect without causing undesirable local or systemic effects).

[0057] The pharmaceutical compositions according to the present invention are applicable to administration via various routes. Generally, administration is completed parenterally. Parenteral delivery methods include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.

[0058] The pharmaceutical compositions according to the present invention may be manufactured in various forms, such as solid, liquid, gas or lyophilized forms, and in particular may be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or fluid extracts.

[0059] The compositions according to the present invention may also be administered in combination with one or more other agents applied to treat and / or prevent the disease to be treated. Preferred examples of agents to be applied in combination include well-known anticancer drugs such as cisplatin, meitansine derivatives, racelmycin, calichemycin, docetaxel, etoposide, gemcitabine, isocyclophosphamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium II, temozolomide, topotecan, trimethrexate glucuronide, auristatin E, vincristine, and doxorubicin, as well as lysine, diphtheria toxin, pseudomonas exotoxin A, DNA enzymes, and RNA enzymes. The present invention comprises peptide cytotoxins such as iodine-131, rhenium-186, indium-111, iridium-90, bismuth-210 and 213, actinium-225, and astatine-213, prodrugs such as antibody-targeting enzyme prodrugs, immunostimulants such as platelet factor 4 and melanoma growth-stimulating protein, and antibodies or fragments such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral / bacterial protein domains, and viral / bacterial peptides. The pharmaceutical compositions of the present invention may also be used in combination with one or more other therapeutic methods, such as chemotherapy and radiotherapy.

[0060] Treatment / prevention / diagnostic uses The manipulated immune cells and compositions in this invention can be used in drugs to treat / prevent / diagnose cancer, infection, or autoimmune diseases.

[0061] In one embodiment, cancers that can be treated with the immune cells or composition of the present invention include, but are not limited to, acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), acute cerebrospinal cell leukemia (AML), breast cancer, lung cancer, colorectal cancer, gastric cancer, pancreatic cancer, ovarian cancer, metastatic adenocarcinoma, hepatic metastasis, sarcoma, osteosarcoma, esophageal cancer, eye cancer, head and neck cancer, biliary tract cancer, bladder cancer, bone cancer, neuroblastoma, melanoma, mesothelioma, glioblastoma, glioma, malignant glioma, liver cancer, non-small cell lung cancer (NSCLC), gangliocytoma, brain cancer, kidney cancer, and prostate cancer. Infections that can be treated with the immune cells or composition of the present invention include, but are not limited to, infections caused by viruses, bacteria, fungi, and parasites. Autoimmune diseases that can be treated with the immune cells or compositions of the present invention include, but are not limited to, type 1 diabetes, abdominal diseases, Graves' disease, inflammatory bowel disease, multiple sclerosis, psoriasis, rheumatoid arthritis, Addison's disease, Sjögren's syndrome, Hashimoto's thyroiditis, myasthenia gravis, vasculitis, pernicious anemia, and systemic lupus erythematosus.

[0062] Example 1: sgRNA design and CIITA gene knockout A spacer sequence was designed by selecting an appropriate PAM site on the CIITA gene. The spacer sequence targeted an exon of the CIITA gene, and the target sequence on the CIITA gene was unique. The crRNA containing the spacer sequence was fused with tracrRNA to obtain a single sequence of approximately 100 bp. This sequence was artificially synthesized to obtain the sgRNA product.

[0063] T cells were stimulated with DynaBeads CD3 / CD28 CTS™ (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 3 days. Then, using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX), 10 μg of Cas9 protein and 10 μg of sgRNA were electrically transfected into the activated T cells at 400 V and 0.7 ms to obtain T cells with CIITA knockout. Immediately after electrical transfection, the T cells were placed in 1 mL of preheated medium and cultured at 37°C and 5% CO2 in the presence of IL-2 (300 IU / mL). After 5 days, the knockout efficiency of the CIITA gene was determined by detecting HLA-II expression using a flow cytometer with APC anti-human HLA-DR, DP, and DQ Antibody (Biolegend, catalog number 361714). The spacer sequences in sgRNA and their knockout efficiencies are shown in Table 1.

[0064] Table 1. Spacer sequences of different sgRNAs and their knockout efficiency. JPEG2026136230000002.jpg232170

[0065] As can be seen from Table 1, different sgRNA spacer sequences differed in their knockout efficiency against the CIITA gene. Of the 20 sequences tested, SEQ ID NO: 1-6, 10, 11, 15, 16, 19, 21, and 23 showed knockout efficiencies of 80% or higher, while only SEQ ID NO: 4, 6, 11, and 15 showed knockout efficiencies of 90% or higher.

[0066] From the table above, A4, A11, and A15 were selected and sequence modified. See Table 2 for specific sequence information (horizontal lines indicate sequences complementary to the target sequence in sgRNA). T cells were stimulated with DynaBeads CD3 / CD28 CTS™ (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 3 days. Then, using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX), the amounts of Cas9 protein and sgRNA used were halved, and 5 μg of Cas9 protein and 5 μg of sgRNA were electrically transfected into activated T cells at 400V and 0.7 ms to obtain T cells with CIITA knockout. Knockout detection was performed according to the method described above, and the results are shown in Table 2.

[0067] Table 2 A4, A11, A15 and the spacer arrangement after sequence modification and its knockout efficiency JPEG2026136230000003.jpg163170

[0068] As can be seen from Table 2, after sequence modification of A4, A11, and A15, the spacer sequences of sgRNAs obtained using different modification methods also showed different CIITA gene knockout efficiencies. The inventors discovered that adding one or two additional Gs to the 5' end of sgRNAs that are perfectly complementary to the target sequence significantly improves the knockout efficiency. Specifically, A4-1 (18nt) and A4-2 (20nt) are perfectly complementary to the target sequence, and for A4, two additional Gs were added to the 5' end of A4-1. The knockout efficiency of A4 was significantly improved compared to A4-2 and A4-1. Similarly, the knockout efficiency of A11 was equivalent to A11-1 and was significantly higher than A11-2, and the knockout efficiency of A15 was significantly higher than A15-1 and A15-2. Depending on the knockout efficiency, A15 was selected and used in subsequent experiments.

[0069] Example 2: Production of CAR-T cells with TRAC / HLA-II knockout. 1. Production of CD19 CART cells The coding sequences for the following proteins were synthesized: CD8α signal peptide (SEQ ID NO: 41), anti-CD19scFv (SEQ ID NO: 30), CD8α hinge region (SEQ ID NO: 33), CD8α transmembrane region (SEQ ID NO: 31), 4-1BB costimulatory domain (SEQ ID NO: 39), and CD3ζ primary signaling domain (SEQ ID NO: 35). These sequences were then sequentially cloned into a pGEM-T Easy vector (Promega, catalog number A1360), and the precise insertion of the target sequences was confirmed by sequencing.

[0070] The plasmid was diluted in 3 mL of Opti-MEM (Gibco, catalog number 31985-070) in a sterile tube. Then, the packaging vector psPAX2 (Addgene, catalog number 12260) and the envelope vector pMD2.G (Addgene, catalog number 12259) were added in a plasmid:viral packaging vector:viral envelope vector ratio of 4:2:1. 120 μL of X-treme GENE HP DNA transfection reagent (Roche, catalog number 06366236001) was added and immediately mixed homogeneously. The mixture was incubated at room temperature for 15 minutes, and then the plasmid / vector / transfection reagent mixture was added dropwise to 293T cell culture flasks. Viruses were collected at 24 and 48 hours, combined, and then ultracentrifuged (25000 g, 4°C, 2.5 hours) to obtain concentrated lentivirus.

[0071] T cells were activated with DynaBeads CD3 / CD28 CTSTM (Gibco, catalog number 40203D) and cultured at 37°C and 5% CO2 for 1 day. Then, enriched lentivirus was added and the cells were cultured continuously for 3 days to obtain CAR-T cells targeting CD19.

[0072] Using a BTX Agile Pulse Max electroporator (Harvard Apparatus BTX), 10 μg of Cas9 protein, 5 μg of CIITA sgRNA (spacer sequence as shown in SEQ ID NO: 15), and 5 μg of TRAC sgRNA (spacer sequence as shown in SEQ ID NO: 27) were electrically transfected into activated CD19 CAR-T cells under conditions of 400 V and 0.7 ms to obtain TRAC / HLA-II double knockout CD19 CAR-T cells (UCAR19). Non-knockout CD19 CAR-T cells were used as positive controls (cCAR19). Wild-type T cells not transfected with CAR were used as negative controls (NT).

[0073] 2. Editing efficiency and CAR level detection cCAR19 and UCAR19 cells were cultured at 37°C and 5% CO2 for 11 days. Then, the gene editing efficiencies of TRAC and CIITA were detected by flow cytometry using PE-anti-human CD3 (BD ​​Pharmingen, catalog number 300456) and APC anti-human HLA-DR, DP, DQ (Biolegend, catalog number 361714) antibodies. The results are shown in Figure 1.

[0074] As can be seen from this, the double knockout efficiency of TRAC / HLA-II in UCAR19 was effective in both cases, indicating that the sgRNA selected in this invention can effectively knock out the TRAC and CIITA genes.

[0075] cCAR19 and UCAR19 cells were cultured at 37°C and 5% CO2 for 11 days. Then, Biotin-SP (long spacer) AffiniPure Goat Anti-Mouse IgG, F(ab') Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, catalog no. 115-065-072) was used as the primary antibody, and APC Streptavidin (BD Pharmingen, catalog no. 554067) or PE Streptavidin (BD Pharmingen, catalog no. 554061) was used as the secondary antibody. The expression levels of CAR on CAR T cells were detected by flow cytometry, and the results are shown in Figure 2.

[0076] As can be seen from this, the CAR expression level in cCAR19 corresponds to that of UCAR19, indicating that knockout of the TRAC and CIITA genes does not affect CAR surface expression.

[0077] Example 3, cytokine secretion levels of CAR-T cells When T cells kill target cells, the number of target cells decreases, and cytokines such as IL2 and IFN-γ are released. The levels of cytokine IL2 and IFNγ released when CART cells kill target cells were measured using an enzyme-linked immunosorbent assay (ELISA) according to the following steps.

[0078] (1) Collection of supernatant from cell co-culture 1 x 10 5 Target cells (Raji) were plated into 96-well plates, and three groups of cells were co-cultured with the target cells in a 1:1 ratio. The supernatant of the cell co-culture was collected after 18–24 hours.

[0079] (2) Detection of IL2 and IFNγ secretion levels in the supernatant by ELISA The secretion levels of cytokines IL-2 and IFN-γ in the supernatant were measured by ELISA. Purified anti-human IL-2 Antibody (Biolegend, catalog number 500302) and Purified anti-human IFN-γ Antibody (Biolegend, catalog number 506502) were coated onto 96-well plates and incubated overnight at 4°C. The antibody solution was then removed, and 250 μL of PBST (0.1% Zwein-containing 1XPBS) solution containing 2% BSA (sigma, catalog number V900933-1kg) was added, followed by incubation at 37°C for 2 hours. Finally, 50 μL of cell co-culture supernatant or standard was added to each well, and the mixture was incubated at 37°C for 1 hour. After washing the plate with 250 μL of PBST (1XPBS containing 0.1% Zweene), 50 μL each of the detection antibody Biotin anti-human IL-2 Antibody (Biolegend, catalog no. 517605) and Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, catalog no. ab25017) were added to each well, and incubated at 37°C for 1 hour. Then, HRP Streptavidin (Biolegend, catalog no. 405210) was added, and incubated at 37°C for 30 minutes. The supernatant was discarded, and the plate was washed with 250 μL of PBST (1XPBS containing 0.1% Zweene). 50 μL of TMB substrate solution was added to each well. The reaction was allowed to proceed at room temperature in the dark for 30 minutes, and then 50 μL of 1 mol / LH₂SO₄ was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, absorbance at 450 nm was detected using a plate reader, and cytokine content was calculated based on a standard curve (drawn based on readings and concentrations of the standard). The results are shown in Figure 3.

[0080] As can be seen, both cCAR19 and UCAR19 cells secreted large amounts of cytokines compared to the NT group. Furthermore, when killing target cells, the secretion levels of IL2 and IFN-γ were comparable between cCAR19 and UCAR19 cells. This indicates that TRAC and CIITA gene knockouts do not affect the killing function of CAR-T cells.

[0081] It should be noted that the above are merely preferred embodiments of the present invention and are not intended to limit it, and to those skilled in the art, the present invention is subject to various modifications and changes. As those skilled in the art should understand, any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should all be within the scope of protection of the present invention.

Claims

1. SEQ ID NO: sgRNA containing one of the spacer sequences shown in 1-6, 10, 11, 15, 16, 19, 21, or 23.

2. A nucleic acid encoding the sgRNA described in claim 1.

3. A vector comprising the nucleic acid described in claim 2.

4. A method for in vitro knockout of the CIITA gene, comprising introducing a Cas nuclease and sgRNA into a cell, wherein the sgRNA exists in the form of the sgRNA described in claim 1, the nucleic acid described in claim 2, or the vector described in claim 3.

5. The method according to claim 4, wherein the Cas nuclease is Cas9, Cas12a, or Cas13a.

6. The method according to claim 4, wherein the cells are immune cells, and the immune cells are T cells, B cells, macrophages, dendritic cells, monocytes, NK cells, or NKT cells.

7. Memo cells, CD4 ＋ CD8 ＋ T cells, CD4 ＋ T cells, CD8 ＋ T cells, memory T cells, immature T cells, γδ-T cells (also αβ-T cells), method of recording in request item 6.

8. The method according to claim 6, wherein the immune cells are introduced with nucleic acids encoding chimeric antigen receptors and / or T cell receptors.

9. The method according to claim 8, wherein the chimeric antigen receptor comprises a ligand-binding domain, a transmembrane domain, a costimulatory domain, and a primary signaling domain, and the ligand-binding domain targets one or more of the following: CD7, CD19, CD20, CD22, BAFF-R, CD33, EGFRvIII, BCMA, GPRC5D, PSMA, ROR1, FAP, ERBB2, MUC1, EGFR, CAIX, WT1, NY-ESO-1, CD79a, CD79b, GPC3, Claudin18.2, and NKG2D.

10. The method according to claim 9, wherein the ligand-binding domain comprises an antibody or antigen-binding fragment that targets CD19 and / or CD22.

11. The method according to claim 10, wherein the antibody or antigen-binding fragment targeting CD19 comprises a light chain variable region sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO: 28, and a heavy chain variable region sequence having at least 90%, 95%, 97%, 99%, or 100% sequence identity with the amino acid sequence shown in SEQ ID NO:

29.

12. The method according to claim 9, wherein the transmembrane domain is selected from the transmembrane domains of a protein, namely TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or any combination thereof.

13. The method according to claim 9, wherein the co-stimulatory domain is selected from the protein co-stimulatory signaling domains of TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134, CD137, CD150, CD152, CD223, CD270, CD272, CD273, CD274, CD276, CD278, CD357, DAP10, LAT, NKG2C, SLP76, PD1, LIGHT, TRIM, ZAP70, or any combination thereof.

14. The method according to claim 9, wherein the primary signaling domain is selected from the protein signaling domains FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, CD66d, or any combination thereof.

15. Engineered immune cells obtained by the method according to any one of claims 6 to 14, wherein the CIITA gene is knocked out.

16. The manipulated immune cells according to claim 15, further comprising those in which the expression of at least one gene selected from TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, or any combination thereof is inhibited or silenced.

17. The manipulated immune cells according to claim 16, wherein CIITA and TRAC in the manipulated immune cells are knocked out.

18. A composition comprising manipulated immune cells according to any one of claims 15 to 17.

19. Use of the manipulated immune cells according to any one of claims 15 to 17 or the composition according to claim 18 in the manufacture of a drug for treating / preventing / diagnosing cancer, infection or autoimmune disease.

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