Method for reducing immunogenicity of t cells

Abstract

Provided are T cells with reduced immunogenicity and a preparation method therefor. The T cells have reduced immunogenicity, which enhances the resistance thereof to NK cells and macrophages, thereby improving the survival capability of T cells, particularly CAR-T cells, within a host.

Description

A method of reducing T cell immunogenicity

[0001] Related applications

[0002] This application claims priority to Chinese patent application 202410629544.8, filed May 20, 2024, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to a T cell with reduced immunogenicity, a preparation method thereof and related applications. BACKGROUND

[0004] T cells, especially chimeric antigen receptor T-cell immunotherapy (CAR-T) therapy, have achieved great success in anti-tumor treatment, especially for hematological malignancies. However, some limitations in autologous CAR-T cell therapy have prevented its large-scale clinical application, such as expensive and lengthy production processes and limited cell sources.

[0005] To overcome these shortcomings, allogeneic universal CAR-T cells have emerged. Existing universal CAR-T cells can reduce cell immunogenicity and avoid attacks from allogeneic cells on the host by knocking out human leukocyte antigen (HLA) type I and T cell receptor (TCR), thus providing support for more patients to effectively treat through CAR-T therapy.

[0006] This method can avoid universal CAR-T cells from escaping attacks from most host myeloid cells, however, knocking out HLA class I molecules can cause natural killer cells (NK cells) to be more reactive to universal CAR-T cells, and also cannot avoid attacks from myeloid cells including macrophages on universal CAR-T cells.

[0007] Therefore, there is a need in the art for improved T cells (particularly CAR-T cells) to further reduce immunogenicity, especially that caused by NK cells and macrophages. SUMMARY

[0008] The present application achieves the effect of reducing the immunogenicity of T cells by simultaneously overexpressing CD47 and CD24 or expressing a fusion protein of CD47 and CD24 in T cells, and further reduces the immunogenicity by combining with the knockout of specific endogenous genes, thus providing more favorable conditions for the survival of T cells, especially universal CAR-T cells, in the host.

[0009] In one aspect, the present application provides a T cell engineered to overexpress a CD47 protein and a CD24 protein. In some embodiments, the CD47 protein comprises the amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 5. In some embodiments, the CD24 protein comprises the amino acid sequence of SEQ ID NO: 9 or a fragment thereof, e.g., the fragment set forth in SEQ ID NO: 6. In some embodiments, the CD24 protein comprises the amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 6.

[0010] In one aspect, the present application provides a T cell engineered to express a fusion protein of CD47 and CD24, wherein the fusion protein comprises a CD47 protein operably linked (directly or through a flexible linker) to a CD24 protein, the CD47 protein comprises the amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 5, and the CD24 protein comprises the amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 6. In the fusion protein, the CD47 protein can be at the N-terminus or C-terminus of the CD24 protein. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 7. The linker can be a peptide linker, e.g., a GS series linker, e.g., (GGGGS) n series linker, wherein n = 1-5, or a peptide linker consisting of glycine.

[0011] In some embodiments, the fusion protein further comprises a transmembrane region, e.g., a transmembrane region derived from a CD28 protein. The transmembrane region can be derived from a native cell surface receptor molecule, such as a costimulatory receptor molecule, to anchor CD47 and CD24 to the cell surface. For example, the transmembrane region can be derived from CD28, CD18, CD2, CD7, CD27, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), DAP10, etc. In some embodiments, the fusion protein comprises a CD47 protein comprising the amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 5, a CD24 protein comprising the amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 6, and a transmembrane region. In a particular embodiment, the fusion protein comprises the amino acid sequence set forth in SEQ ID NO: 8.

[0012] In some embodiments, the T cell further has a knockout or inactivation of a gene encoding one or more proteins selected from the group consisting of: T cell receptor (TCR), HLA class I molecule, and HLA class II molecule, optionally, a knockout or inactivation of a gene encoding one or more proteins selected from the group consisting of: TCRa, TCRp, beta-2 microglobulin (B2M), and HLA-DRA.

[0013] In some further embodiments, the cell has a knockout or inactivation of genes encoding TCRa, B2M, and HLA-DRA.

[0014] In some embodiments, the T cell is a CAR-T cell, optionally a CAR-T cell obtained by engineering a donor-derived T cell with a CAR construct.

[0015] In one aspect, the present disclosure provides a method of preparing a T cell with reduced immunogenicity, the method comprising: engineering the T cell to overexpress a CD47 protein and a CD24 protein, wherein the CD47 protein comprises an amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% homology to SEQ ID NO: 5, and the CD24 protein comprises an amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% homology to SEQ ID NO: 6.

[0016] In some embodiments, the engineering is performed by transforming or transfecting the T cell with one or more expression vectors. In some embodiments, the expression vector comprises both a nucleotide sequence encoding the CD47 protein and a nucleotide sequence encoding the CD24 protein (and optionally, a nucleotide sequence encoding the transmembrane region). The nucleotide sequence encoding the CD47 protein and the nucleotide sequence encoding the CD24 protein can be operably linked by a nucleotide sequence encoding a linker sequence, such as a glycine serine series linker. In some embodiments, the linker is (G4S)n, where n = 1-4.

[0017] In some embodiments, the method further comprises engineering the T cell to express a chimeric antigen receptor (CAR), thereby preparing a CAR-T cell.

[0018] In some embodiments, the CAR, the CD47 protein, and the CD24 protein are encoded by the same expression vector or by separate expression vectors.

[0019] In some embodiments, the expression vector comprises a nucleotide sequence encoding the CAR, a nucleotide sequence encoding the CD47 protein, and a nucleotide sequence encoding the CD24 protein (optionally, together with the transmembrane region), optionally, the three nucleotide sequences are operably linked by a nucleotide sequence encoding a linker sequence, wherein the nucleotide sequence encoding the CAR is operably linked to the nucleotide sequence encoding the CD47 protein or to the nucleotide sequence encoding the CD24 protein by a nucleotide sequence encoding a self-cleaving linker sequence, such as a 2A linker, a furin cleavage site, or an internal ribosome entry site (IRES); wherein the nucleotide sequence encoding the CD47 protein is directly linked to the nucleotide sequence encoding the CD24 protein or operably linked by a nucleotide sequence encoding a linker sequence, such as a GS linker or other flexible linker sequence known to one skilled in the art. The CAR is preferably a single chain polypeptide, such as a CAR comprising a VHH or a scFv as the antigen recognition region.

[0020] In some particular embodiments, the expression vector comprises a nucleotide sequence encoding a CD47 protein operably linked to a nucleotide sequence encoding a CD24 protein, which is operably linked to a nucleotide sequence encoding the CAR; or, the expression vector comprises a nucleotide sequence encoding a CD24 protein operably linked to a nucleotide sequence encoding a CD47, which is operably linked to a nucleotide sequence encoding the CAR.

[0021] In some embodiments, the expression vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a transposon vector, and a viral vector; optionally, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0022] In some embodiments, the method further comprises knocking out or inactivating a gene in the T cell that encodes one or more proteins selected from the group consisting of a T cell receptor (TCR), an HLA class I molecule, and an HLA class II molecule. Optionally, this step can be performed before or after engineering the T cell to express the CAR, CD47, and CD24.

[0023] In some embodiments, the method comprises knocking out or inactivating an endogenous gene in the T cell that encodes one or more proteins selected from the group consisting of TCRa, TCRp, B2M, and HLA-DRA, optionally, it comprises knocking out or inactivating endogenous genes in the cell that encode TCRa, B2M, and HLA-DRA.

[0024] The knocking out or inactivation of the gene can be performed by contacting the T cell with a gene editing system, such as a CRISPR / Cas9 gene editing system, and introducing components of the system into the cell. In some embodiments, the gene editing system comprises:

[0025] any combination of one or more of a sgRNA targeting a TCRa constant region-encoding gene, a sgRNA targeting a TCRp constant region-encoding gene, a sgRNA targeting a B2M gene, and a sgRNA targeting a HLA-DRA gene, or a combination of one or more vectors expressing the sgRNAs; and

[0026] a nuclease or a nucleic acid encoding the nuclease.

[0027] In some further embodiments, the gene editing system comprises: an sgRNA targeting a TCRa constant region-encoding gene, an sgRNA targeting a B2M gene, and an sgRNA targeting a HLA-DRA gene. The nucleases are capable of inactivating the respective targeted genes under the guidance of each sgRNA, optionally the nucleases are Cas nucleases, such as Cas9 nucleases or Cas12 nucleases, more specifically spCas9 nucleases.

[0028] In some embodiments, the method comprises mixing and incubating the sgRNAs with nucleases before contacting the T cells with the gene editing system. The sgRNAs can be mixed and incubated with nucleases to form RNP complexes before the contacting. In some specific embodiments, the method comprises mixing and incubating equimolar amounts of an sgRNA targeting a TCRa constant region-encoding gene, an sgRNA targeting a B2M gene, and an sgRNA targeting a HLA-DRA gene together with a Cas9 nuclease to form RNP complexes.

[0029] In some embodiments, the components of the system are introduced into the T cells by electroporation.

[0030] In some embodiments, the sgRNA targeting a TCRa constant region-encoding gene has a recognition sequence as set forth in SEQ ID NO: 1. In some embodiments, the sgRNA targeting a B2M gene has a recognition sequence as set forth in SEQ ID NO: 2. In some embodiments, the sgRNA targeting a HLA-DRA gene has a recognition sequence as set forth in SEQ ID NO: 3. The sgRNAs further comprise a constant sequence as a frame, such as the sequence set forth in SEQ ID NO: 4. In some embodiments, the frame sequence is located at the 3’ end of the recognition sequence. The sgRNA targeting a TCRa constant region-encoding gene, the sgRNA targeting a B2M gene, and the sgRNA targeting a HLA-DRA gene are not limited to these disclosed herein, as long as it is capable of effectively knocking out or inactivating the respective genes in T cells. Thus, the sgRNAs can be sgRNAs known to target the respective genes. For example, can be those sgRNAs disclosed in Chinese patent application numbers 202410488220.7, 202410494575.7, and 202410493983.0.

[0031] In one aspect, the present application provides T cells, such as CAR-T cells, prepared by the methods disclosed herein. Such T cells have reduced immunogenicity.

[0032] In one aspect, the present application provides a vector comprising a nucleotide sequence encoding CD47 and a nucleotide sequence encoding CD24, which are operably linked directly or through a nucleotide sequence encoding a linker sequence, such as a glycine serine linker.

[0033] In some embodiments, the vector comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a fusion protein of CD47 and CD24, which are operably linked through a nucleotide sequence encoding a self-cleaving linker sequence or an internal ribosome entry site (IRES).

[0034] In some embodiments, the self-cleaving linker sequence is a 2A sequence, such as a P2A, T2A sequence.

[0035] In some embodiments, the vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a transposon vector, and a viral vector. Optionally, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or a retroviral vector.

[0036] In one aspect, the present application provides a pharmaceutical composition comprising the T cell and a pharmaceutically acceptable carrier.

[0037] In one aspect, the present application provides a T cell (e.g., a CAR-T cell) prepared by the method of the present application for use as a medicament. In one aspect, the present application provides use of a T cell (e.g., a CAR-T cell) prepared by the method of the present application in the manufacture of a medicament for treating a cancer, an autoimmune disease, or an inflammatory disease in a subject, optionally wherein the T cell is allogeneic or autologous to the subject. In one aspect, the present application provides a method of treating a cancer, an autoimmune disease, or an inflammatory disease in a subject, wherein a T cell (e.g., a CAR-T cell) prepared by the method of the present application is administered. In one aspect, the present application provides a T cell (e.g., a CAR-T cell) prepared by the method of the present application for use in treating a cancer, an autoimmune disease, or an inflammatory disease in a subject. The cancer can be any cancer suitable for cell therapy, such as CAR-T therapy, for example, leukemia including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), lymphoma, multiple myeloma (MM), glioma, breast cancer, kidney cancer, liver cancer, esophageal cancer, stomach cancer, pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, head and neck cancer, gallbladder cancer, etc. The autoimmune disease or inflammatory disease includes, but is not limited to, graft versus host disease, inflammatory bowel disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, aplastic anemia, celiac disease, type 1 diabetes, Graves' disease, psoriasis, scleroderma, etc.

[0038] In one aspect, the present application provides use of T cells prepared by the method of the present application in the manufacture of a medicament for organ transplantation. The cells can be CAR-T cells.

[0039] BRIEF DESCRIPTION OF DRAWINGS

[0040] Figure 1 shows that the constructed CAR-T cells express both CD47 and CD24.

[0041] Figure 2 shows the different survival of CAR-T cells expressing or not expressing CD47, CD24 after co-incubation with NK cells.

[0042] Figure 3 shows the different CD107a expression of NK cells after co-incubation, reflecting the degree of degranulation of NK cells after co-incubation.

[0043] Figure 4 shows the different survival of CAR-T cells expressing or not expressing CD47, CD24 after co-incubation with macrophages.

[0044] DETAILED DESCRIPTION

[0045] DEFINITIONS

[0046] As used herein, “CD47”, also known as integrin-associated protein, is a cell membrane protein belonging to the immunoglobulin superfamily, which is almost widely expressed on the surface of all normal cells and highly expressed on the surface of tumor cells. CD47 sends a “don’t eat me” signal by binding to the signal regulatory protein alpha (Sirpa) on the surface of macrophages, thereby protecting tumor cells from being phagocytosed by macrophages and causing tumor immune escape. NK cell Sirpa is up-regulated under IL-2 stimulation, interacts with target cell CD47 in a threshold-dependent manner, and counteracts other stimulatory signals, including IL-2, CD16 or NKG2D. The elevated expression of CD47 protects tumor cells against SIRPa+ primary NK cells. CD47 as used herein encompasses native human CD47 protein, an extracellular domain fragment thereof, or a functional variant thereof.

[0047] As used herein, “CD24”, also known as heat-stable antigen (HSA), is a highly glycosylated glycosylphosphatidylinositol-anchored membrane protein. CD24 can act as an “eat-me” signal molecule, interacting with the molecule Siglec-10 on macrophages, delivering an inhibitory signal, suppressing destructive inflammatory responses, reducing macrophage phagocytosis, delivering an “eat-me” signal. CD24 / Siglec-10 interaction is associated with NK cell dysfunction in hepatocellular carcinoma, which can provide another pathway for CD24-mediated immune evasion. CD24 as used herein encompasses native human CD24 protein, an extracellular domain fragment thereof, or a functional variant thereof. In some embodiments, the CD24 protein comprises the amino acid sequence of SEQ ID NO: 9 (MGRAMVARLGLGLLLLALLLPTQIYSSETTTGTSSNSSQSTSNSGLAPNPTNATTKAA) or a fragment thereof.

[0048] As used herein, the “B2M gene” or “beta 2-microglobulin-encoding gene” in humans is a gene located on human chromosome 15 (15q21.1) comprising 4 exons. The beta 2M protein exists in a membrane protein and a free beta 2M form, the membrane protein beta 2M non-covalently associated with the heavy chain of MHC class I molecules (i.e., human leukocyte antigen class I molecules, HLA-I) on the cell surface as a constant light chain. B2M is involved in the recognition of lymphocytes with target cell surface antigens, and thus B2M is closely related to histocompatibility. Almost all nucleated cells in the body can synthesize beta 2-microglobulin, which is attached to the cell surface. The absence of beta 2-microglobulin will cause abnormal polymerization of HLA class I molecules, so that a complete functional molecule cannot be formed.

[0049] As used herein, “HLA-DRA”, also known as “MHC class II DR alpha”, refers to the alpha chain of the HLA class II histocompatibility antigen DR. HLA-DR is a subtype of MHC class II molecules, which is a heterodimer composed of an alpha chain and a beta chain anchored in the cell membrane. HLA DRA is a polymorphic cell surface glycoprotein that plays an important role in the intercellular interactions of the immune response. The alpha chain protein is about 33-35 kDa, and its encoding gene contains 5 exons, of which exon 1 encodes a leader peptide, exons 2 and 3 encode two extracellular domains, and exon 4 encodes a transmembrane domain and a cytoplasmic tail. The host immune system recognizes HLA mismatches on incoming or transplanted allogeneic cells, thus producing graft rejection. For example, mismatch of HLA-II molecules highly expressed on activated T cells can activate the recipient's alloreactive CD4 + T cells. Therefore, by reducing or knocking out the expression of HLA-DRA in donor cells, HLA-II class mismatch can be reduced, making it an attractive target in the field of cell therapy.

[0050] As used herein, the term "Cas9 nuclease" or "Cas9" is an RNA-guided nuclease belonging to the CRISPR / Cas9 gene editing system, including wild-type Cas9 protein or variants or fragments thereof, e.g. proteins comprising the active DNA cleavage domain of Cas9 and / or the gRNA binding domain of Cas9. As known in the art, Cas9 is a component of the CRISPR / Cas gene editing system, wild-type Cas9 is able to target and cleave a DNA target sequence under the guidance of a gRNA to form a DNA double-strand break (DSB). The DNA cleavage activity of Cas9 depends on two domains: RuvC and HNH, which are responsible for cleaving the two strands of DNA, respectively, wherein the complementary strand of the guide RNA is cleaved by RuvC domain activity, while the non-complementary strand is cleaved by HNH domain activity. Both domains can be artificially mutated to inactivate either for single- or double-strand cleavage, as desired.

[0051] As used herein, the term "guide RNA" or "gRNA" refers to an RNA sequence comprising a guide sequence (also referred to herein as a recognition sequence) and, optionally, a tracrRNA. A common guide RNA is composed of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) sequences that form a complex through partial complementarity, wherein the recognition sequence comprised by the crRNA is sufficiently complementary to the target sequence to hybridize and target the CRISPR complex to the specifically bound target sequence. The term also includes a single guide RNA (sgRNA), which contains features of both crRNA and tracrRNA. Typically, the guide sequence of the sgRNA is complementary to the target nucleic acid sequence, responsible for the initial guide RNA / target base pairing. Preferably, the guide sequence of the sgRNA does not tolerate mismatches.

[0052] As used herein, a guide sequence or recognition sequence "specifically recognizes" a target site means that the base complementarity of the guide sequence or recognition sequence in the sgRNA to the sequence of the target site is to the extent of at least 85%, preferably at least 90%, more preferably at least 95%, most preferably 100%.

[0053] As used herein, the term "CAR-T cell" refers to a T cell expressing any one of the CAR constructs, or into which a nucleic acid or vector encoding a CAR construct has been introduced. The polynucleotide encoding the CAR construct polypeptide can be introduced into the cell using a variety of methods, or the CAR construct polypeptide can be synthesized in situ in the cell. Methods of introducing polynucleotide constructs into cells are known in the art. In some embodiments, stable transformation methods can be used to integrate the polynucleotide construct into the genome of the cell. In other embodiments, transient transformation methods can be used to transiently express the polynucleotide construct, and the polynucleotide construct is not integrated into the genome of the cell. In other embodiments, viral-mediated methods can be used. The polynucleotide can be introduced into the cell by any suitable method, such as a recombinant viral vector (e.g., retrovirus, adenovirus), liposome, etc. Transient transformation methods include, for example, but are not limited to, microinjection, electroporation, or microprojectile bombardment. The polynucleotide can be included in a vector, such as a plasmid vector or a viral vector.

[0054] As used herein, the term "immunogenicity" refers to the ability of a CAR-T cell to induce an anti-CAR immune response by the humoral and cellular immune system against the non-self components of the CAR construct or the residual proteins of the gene transfer vector due to their inherent immunogenicity. The cellular immune response can arise from the processing and cross-presentation of the foreign (mouse, viral, or non-self human) sequences of the CAR molecule in the context of the major histocompatibility complex (MHC), and CAR-specific T cells will clear and inactivate the CAR-T cells. The humoral immune response can be initiated by the presentation of the CAR protein in apoptotic bodies by follicular dendritic cells to B cells; anti-CAR antibodies can induce CAR-T cell death through various mechanisms, including antibody-dependent cellular cytotoxicity, i.e., the interaction between the CAR-bound antibody and the Fc receptor (FcR) domain of innate immune cells (e.g., NK cells or macrophages) results in cytotoxicity through the release of perforin and / or granzymes or phagocytosis, promoting apoptosis of the CAR-T cells. Immunogenicity limits the efficacy and persistence of CAR-T cells in vivo, and thus reducing the immunogenicity of CAR-T cells facilitates better application of CAR-T cells in clinical treatment.

[0055] CAR-T cells

[0056] The basic principle of chimeric antigen receptor T cell technology (CAR-T) is to obtain T cells from donors and genetically engineer the T cells to express specific tumor antigen receptors in vitro. After recognizing tumor-associated antigens or tumor-specific antigens, the T cells can be efficiently activated and proliferate in large numbers, releasing anti-tumor active molecules, thereby exerting a strong tumor-killing effect. After the modified T cells are proliferated in vitro, the CAR-T cells are injected into the patient's body, which in turn attacks cancer cells expressing specific antigens.

[0057] A key to CAR-T therapy is the engineering of T cells with a chimeric antigen receptor, or CAR, which generally comprises an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain. The antigen binding domain is often derived from an antigen binding fragment capable of recognizing and binding a specific antigen, such as in the form of a scFv, VHH, or Fab, by selecting a suitable antigen binding domain to recognize a cell surface marker of a target cell associated with a particular disease state, such as a tumor. The intracellular signaling domain serves to transmit effector function signals and direct the cell to perform specialized functions (such as cytolytic activity or helper activity, including secretion of cytokines), which generally comprises a primary signaling domain and a costimulatory signaling domain. The primary signaling domain refers to a protein portion that is capable of modulating the primary activation of the TCR complex in a stimulatory manner or in an inhibitory manner, with the primary signaling domain acting in a stimulatory manner generally containing a signaling motif known as an immunoreceptor tyrosine-based activation motif (ITAM). The costimulatory signaling domain refers to an intracellular signaling domain derived from a costimulatory molecule. Costimulatory molecules are cell surface molecules other than antigen receptors or Fc receptors that provide a second signal required for efficient activation and function of T lymphocytes following binding to antigen.

[0058] The present application is not particularly limited as to the CAR construct for engineering T cells and the antigen to which it is designed to bind. The antigen to which the CAR construct binds can be selected from a variety of tumor-associated antigens or tumor-specific antigens or other immune disease-associated antigens. By way of example and not limitation, the CAR construct can be designed to recognize any one of the antigens selected from the group consisting of CD70, CD3, CD19, CD20, 4.1 BB (CD137), OX40 (CD134), CD16, CD47, CD22, CD33, CD38, CD123, CD133, CEA, cdH3, EpCAM, epidermal growth factor receptor (EGFR), EGFRvIII, HER2, HER3, dLL3, BCMA, Sialyl-Lea, 5T4, ROR1, mesothelin, folate receptor 1, VEGF receptor, EpCAM, HER2 / neu, HER3 / neu, G250, CEA, MAGE, VEGF, FGFR, alphaVbeta3-integrin, HLA, HLA-DR, ASC, CD1, CD2, CD4, CD5, CD6, CD7, CD8, CD11, CD13, CD14, CD21, CD23, CD24, CD28, CD30, CD37, CD40, CD41, CD44, CD52, CD64, c-erb-2, CALLA, MHCII, CD44v3, CD44v6, p97, gangliosides GM1, GM2, GM3, GD1a, GD1b, GD2, GD3, GT1b, GT3, GQ1, NY-ESO-1, NFX2, SSX2, SSX4 Trp2, gp100, tyrosinase, Muc-1, telomerase, survivin, G250, p53, CA125 MUC, Lewis Y antigen, HSP-27, HSP-70, HSP-72, HSP-90, Pgp, MCSP, EpHA2, GC182, GT468 or GT512, IL-17, IL-20, IL-13, and IL-4.

[0059] In some embodiments, the CAR construct comprises an antigen binding domain in the form of a scFv. In some other embodiments, the CAR construct comprises an antigen binding domain in the form of a VHH.

[0060] In some embodiments, the primary signaling domain of the CAR construct contains an ITAM derived from a member selected from the group consisting of TCR zeta, FcR gamma, FcR beta, CD3 gamma, CD3 delta, CD3 epsilon, CD3 zeta, CD22, CD79a, CD79b, and CD66d.

[0061] In some embodiments, the co-stimulatory signaling domain of the CAR construct is derived from a co-stimulatory molecule selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), DAP10.

[0062] In some embodiments, the CAR construct further comprises a linker sequence between the antigen binding domain and the transmembrane domain and / or between the transmembrane domain and the intracellular signaling domain.

[0063] Engineered CAR-T cells can be obtained by transfecting T cells with a virus comprising a CAR coding sequence. In some embodiments, the viral bodies used for transfection are produced by transfecting cells with a plasmid encoding the CAR construct and a viral packaging plasmid. In some other embodiments, engineered CAR-T cells are obtained by transfecting immune T cells with an expression vector of the CAR construct. CAR-T cells that can be used for CAR construct engineering are T lymphocytes, including thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes. The T cells can be T helper (Th) cells, such as T helper 1 (Thl) or T helper 2 (Th2) cells. The T cells can be helper T cells (HTL; CD4 T cells), cytotoxic T cells (CTL; CD8 T cells), CD4 CD8 T cells, or any other T cell subpopulation. In some embodiments, the T cells can include naive T cells and memory T cells.

[0064] In some embodiments, the T cells used for engineering are isolated from peripheral blood mononuclear cells (PBMCs). Isolation of various cell fractions from PBMCs is well known to those skilled in the art. In some embodiments, the peripheral blood mononuclear cells are isolated from a subject in need of administration of a CAR-T cell therapy. In some embodiments, the peripheral blood mononuclear cells are isolated from an individual that is allogeneic to a subject in need of administration of a CAR-T cell therapy. The T cells include any type of T cells, such as cytotoxic T lymphocytes and regulatory T cells.

[0065] Universal CAR-T cells

[0066] Biological organisms generally reject xenografts or allografts, including allogeneic CAR-T cells, mainly because the donor's genes encode antigens that the recipient does not have, which are recognized by the recipient's immune cells and cause rejection, i.e., host versus graft reaction. Based on allogeneic CAR-T cells, universal CAR-T (UCAR-T) cells have been further improved, which have reduced cellular immunogenicity, thereby providing support for more patients to be effectively treated by CAR-T therapy.

[0067] The general design principle of universal CAR-T cells is to destroy the TCR genes, HLA-I class genes and / or HLA-II class genes of allogeneic T cells by gene editing after generating CAR-T cells from allogeneic donors, so that the resulting T cells cannot recognize allogeneic antigens, thereby effectively eliminating graft versus host disease.

[0068] T cells express T cell receptors (TCRs) on their cell membranes, which are responsible for recognizing antigens presented by major histocompatibility complexes (MHCs, also known as human leukocyte antigen, HLA molecules). Unlike B cell receptors, TCRs cannot recognize free antigens, but can recognize antigenic peptide fragments presented by MHC molecules. Generally, TCRs are glycoproteins on the cell membrane in the form of heterodimers of α / β chains or γ / δ chains. The specific binding of TCRs to polypeptides presented by MHCs triggers a series of biochemical reactions and activates T cells through numerous co-receptors, enzymes and transcription factors, promoting their division and differentiation. In the process of allogeneic transplantation, the lymphocytes in the graft recognize the antigens of the recipient cells, trigger an immune response, attack the recipient cells, and cause graft versus host disease.

[0069] In some embodiments, the endogenous TCR of the universal CAR-T cell is disrupted or knocked out. For example, by targeting the genomic sequence of the constant region of the endogenous α or β subunit of TCR, the expression of TCR can be eliminated, so that the resulting T cells cannot recognize allogeneic antigens.

[0070] In some embodiments, the gene expression of the endogenous HLA class I antigen of the universal CAR-T cell is disrupted or knocked out. Cells expressing allogeneic major histocompatibility complex (MHC)-I can be recognized by CD 8 +T cells recognize and eliminate, HLA class I genes (HLA-A, HLA-B, HLA-C) encode MHC-I, so knocking out HLA class I molecules helps allogeneic T cells to survive better in the host body. Beta 2 microglobulin (B2M) is a beta light chain of human leukocyte antigen class I molecules (HLA-I), its main function is to participate in the recognition of lymphocytes and target cell surface antigens, so B2M is closely related to histocompatibility. Almost all nucleated cells in the body can synthesize beta 2 microglobulin, which is attached to the cell surface. The absence of beta 2 microglobulin will cause abnormal polymerization of HLA class I molecules, so that complete functional molecules cannot be formed. In some embodiments, the endogenous B2M gene of the universal CAR-T cell is disrupted or knocked out. By knocking out the B2M gene, the HLA-I class molecules in the cell can no longer be expressed, reducing the immunogenicity of the cell, effectively reducing the risk of host versus graft reaction and immune rejection.

[0071] In some embodiments, the gene expression of the endogenous HLA class II antigen of the universal CAR-T cell is disrupted or knocked out. The HLA-II class molecules on the surface of T cells are CD4 + The important target of T helper cell recognition, HLA-II class molecules include HLA-DR, HLA-DP, HLA-DQ, HLA-DM, HLA-DO, which are also a class of targets for host cells to recognize allogeneic cells. In some embodiments, the endogenous HLA DRA gene of the universal CAR-T cell is disrupted or knocked out. HLA DRA is one of the HLA II class alpha chain paralogs, and by knocking out HLA-DRA, the immunogenicity of the transplanted cell can be reduced, effectively reducing the risk of host versus graft reaction and immune rejection.

[0072] In some embodiments, the universal CAR-T cell is a CAR-T cell that does not express or low expresses one or more proteins selected from the group consisting of TCR, B2M, HLA class I, and HLA class II molecules.

[0073] In some embodiments, the universal CAR-T cell is a CAR-T cell that does not express or low expresses one or more proteins selected from the group consisting of TCR, B2M, and HLA-DRA.

[0074] In some embodiments, the universal CAR-T cell is a CAR-T cell that does not express or low expresses TCR, B2M, and HLA-DRA.

[0075] T cells overexpressing CD47 and CD24

[0076] The present application also further reduces the immunogenicity of T cells by overexpressing CD47 and CD24 in T cells. Allogeneic T cells, such as universal CAR-Ts, have the potential to increase the risk of being attacked by NK cells while evading the attack of host T cells, and also do not take into account the rejection of myeloid cells to allogeneic cells. On one hand, the reaction of natural killer cells (NK cells) to T cells is enhanced due to the possible knock-out of HLA class I molecules in T cells, and on the other hand, T cells cannot avoid the attack of myeloid cells, including macrophages, to T cells. The present application overcomes these shortcomings by overexpressing CD47 and CD24 in T cells.

[0077] CD47 is highly expressed in tumor cells, which can interact with signal regulatory protein alpha (Sirpa) on the surface of macrophages or NK cells to cause immune escape of tumor cells. CD24 can interact with Siglec-10 on macrophages to reduce macrophage phagocytosis. The present application unexpectedly found that by overexpressing CD47 and CD24 in universal CAR-T cells, the immunogenicity caused by the reaction with NK cells and the phagocytosis of macrophages can be reduced.

[0078] In some embodiments, the T cells are engineered by expressing a fusion protein of CD47 and CD24. The fusion protein can comprise a fusion of a human CD47 protein, a functional fragment or variant thereof, and a human CD24 protein, a functional fragment or variant thereof. For example, the fusion protein can comprise a human CD47 protein operably linked to a functional fragment of a human CD24 protein. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 5 or a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 5 as the CD47 portion. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 6 or a sequence having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology to SEQ ID NO: 6 as the CD24 portion.

[0079] Overexpression of CD47 and CD24 can be achieved by placing the coding sequences of CAR, CD47 and CD24 in the same or different expression vectors. In some embodiments, the nucleotide sequence encoding CAR, the nucleotide sequence encoding CD47 and the nucleotide sequence encoding CD24 are operably linked in an expression vector, such as a lentiviral vector. The nucleotide sequence encoding CAR and the nucleotide sequence encoding the fusion protein of CD47 and CD24 can be linked by a cleavable linker, in particular a 2A sequence (e.g. P2A or T2A) or an IRES sequence. Thereby, the mRNA after transcription is cleaved upon translation at the ribosome to generate the CAR polypeptide and the fusion protein of CD47 and CD24.

[0080] In some embodiments, the nucleotide sequence encoding CAR is operably linked to the nucleotide sequence encoding CD47 via a nucleotide sequence encoding a first linker, and the nucleotide sequence encoding CD47 is operably linked to the nucleotide sequence encoding CD24 via a nucleotide sequence encoding a second linker. In some other embodiments, the nucleotide sequence encoding CAR is operably linked to the nucleotide sequence encoding CD24 via a nucleotide sequence encoding a first linker, and the nucleotide sequence encoding CD24 is operably linked to the nucleotide sequence encoding CD47 via a nucleotide sequence encoding a second linker. The first linker can be selected from the group consisting of 2A sequences (e.g. P2A and T2A) and IRES sequences, and the second linker can be selected from the group consisting of flexible linker sequences, such as glycine serine linker sequences (e.g. (GGGGS)3) and glycine linker sequences (e.g. (Gly)8).

[0081] Methods of making T cells with reduced immunogenicity

[0082] In one aspect, the present application provides a method of making a T cell, comprising:

[0083] (a) providing a donor-derived T cell;

[0084] (b) engineering the T cell into a CAR-T cell expressing a CAR, optionally, into a CAR-T cell expressing a CAR, CD24 and CD47; and

[0085] (c) genetically editing the CAR-T cell.

[0086] A variety of methods known in the art can be employed to construct universal CAR-T cells with TCR deficiency, HLA class I and / or HLA class II gene deficiency. The most commonly used gene editing methods include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR / Cas9, etc. In some embodiments, the T cells with TCR, HLA class I and / or HLA class II gene knockout are constructed by CRISPR / Cas gene editing system, in particular, CRISPR / Cas9 gene editing system.

[0087] In step (b), the T cells can be transfected or transformed with one or more vectors encoding CAR, CD24 and CD47 simultaneously. The nucleotide sequence encoding CAR, the nucleotide sequence encoding CD24 and the nucleotide sequence encoding CD47 can be present in the same vector or in separate vectors. In some other embodiments, the T cells can be first transfected or transformed with a vector encoding CAR to obtain CAR-T cells, and then the CAR-T cells are further transfected or transformed with a vector encoding CD24 and CD47 fusion protein, or the T cells are first transfected or transformed with a vector encoding CD24 and CD47 fusion protein, and then the T cells are further transfected or transformed with a vector encoding CAR to obtain CAR-T cells.

[0088] In some embodiments, the endogenous beta-2 microglobulin (B2M) gene in the CAR-T cells is knocked out or inactivated in step (c). The modified CAR-T cells have reduced cellular immunogenicity due to the knockout of HLA class I molecules. After the target locus in B2M is cleaved by Cas9 guided by specific sgRNA, DNA sequence insertion or base deletion is introduced to make the B2M gene lose biological function. In some embodiments, the endogenous B2M gene is inactivated by deletion of nucleotides, making B2M not express at all or almost not express. Preferably, each allele of B2M in the genome (e.g., diploid genome) is inactivated.

[0089] In some embodiments, the coding gene for the alpha or beta subunit constant region of the endogenous TCR in the CAR-T cell is knocked out or inactivated in step (c). The modified CAR-T cell has reduced cellular immunogenicity due to the knockout of the TCR molecule. The target site in the coding gene for the alpha or beta subunit constant region of the TCR can be cleaved by Cas9 after the guidance of specific sgRNA, and then a DNA sequence insertion or base deletion is introduced to make it lose biological function. In some embodiments, the coding gene for the alpha or beta subunit constant region of the endogenous TCR is inactivated by deletion of nucleotides, making the TCR completely or almost completely not expressed. Preferably, each allele of the TCR in the genome (e.g., diploid genome) is inactivated.

[0090] In some embodiments, the endogenous HLA-DRA gene in the CAR-T cell is knocked out or inactivated in step (c). The modified CAR-T cell has reduced cellular immunogenicity due to the knockout of the HLA-II class molecule. The target locus in HLA-DRA can be cleaved by Cas9 after the guidance of specific sgRNA, and then a DNA sequence insertion or base deletion is introduced to make the HLA-DRA gene lose biological function. In some embodiments, the endogenous HLA-DRA gene is inactivated by deletion of nucleotides, making B2M completely or almost completely not expressed. Preferably, each allele of HLA-DRA in the genome (e.g., diploid genome) is inactivated.

[0091] In some embodiments, one or more genes selected from the following are knocked out or inactivated in the CAR-T cell in step (c): HLA-DRA coding gene, coding gene for the alpha or beta subunit constant region of the TCR, B2M coding gene. For example, multiple target sites in the corresponding target genes can be cleaved by Cas9 after the guidance of multiple specific sgRNAs, and then a DNA sequence insertion or base deletion is introduced to make the multiple target genes lose biological function. The multiple sgRNAs can include: sgRNAs targeting the alpha or beta subunit constant region of the TCR, sgRNAs targeting the B2M gene, sgRNAs targeting HLA-DRA, or any combination thereof. The sgRNAs can be those disclosed in Chinese Patent Application 202410624572.0.

[0092] The sgRNAs of the application are particularly suitable for CRISPR / Cas mediated oligonucleotide binding and / or editing, wherein the oligonucleotide binding and / or editing is mediated by a complex comprising the sgRNA of the application and a Cas enzyme, which can comprise any suitable Cas enzyme. In certain embodiments, the Cas enzyme is selected from the group consisting of Cas9, Casl2a, Casl2e, Casl2b, Casl2i, Casl2h, Casl2c, Casl2d, Casl2f, Casl2g, Casl2k, Casl2j, Casl3a, Casl3b, Casl3c, Casl3d and Casl4, including any recombinant variant thereof, in particular selected from the group consisting of Cas9, including any recombinant variant thereof. The Cas9 enzyme can be a Streptococcus, e.g. S. pyogenes or Lactobacillus Cas9 enzyme, as described in Briner et al. (2014), the contents of which are incorporated herein by reference, including any recombinant variant thereof. In some embodiments, the Cas nuclease is selected from the group consisting of a Cas9 nuclease and a Casl2 nuclease. The skilled person will understand that the sgRNAs of the application can be used in conjunction with a variety of Cas proteins, and thus for a variety of CRISPR / Cas systems, such as a CRISPR / Cas9 system, a CRISPR / Casl2 system, a CRISPR / nCas9 system, a CRISPR / dCas9 system. The Cas9 protein is a multifunctional protein, with a protein structure comprising a recognition region (REC) consisting of alpha-helices, a nuclease region consisting of a HNH domain and a RuvC domain, and a PAM binding region at the C-terminus. The two important nuclease domains RuvC and HNH can cleave the DNA-complementary and non-complementary strand of the gRNA, respectively, resulting in a blunt-ended DNA double-strand break. The Cas9 protein can be mutated as desired to result in a single-stranded DNA break. Recognition of Cas9 to the target DNA depends on the tracrRNA:crRNA complex and the PAM sequence located downstream of the target site. In some embodiments, the Cas9 protein is a wild-type Cas9. In some embodiments, the Cas9 protein is derived from a S. pyogenes Cas9 protein or a S. aureus Cas9 protein. Preferably, the Cas9 protein can induce a double-strand break at the target site in the target gene.

[0093] Disabling an endogenous target gene can be performed by introducing a Cas nuclease and one or more sgRNAs into a CAR-T cell. In some embodiments, a complex of a Cas nuclease and sgRNAs is introduced into a CAR-T cell. For example, a Cas nuclease and one or more sgRNAs can be incubated before being introduced into a CAR-T cell. The introduction can be performed by electroporation. Optionally, the various sgRNAs can be in equimolar proportions. In some other embodiments, a plasmid encoding one or more sgRNAs and a plasmid encoding a Cas nuclease are introduced into a CAR-T cell.

[0094] The sgRNA comprises a recognition sequence for a target site in a target gene. The recognition sequence is typically designed to be 20 nt. In addition to the recognition sequence, the sgRNA comprises a constant portion as a framework, which can comprise part of crRNA and tracrRNA sequences. The sgRNA has the role of accurately recognizing the sequence of a target gene in the CRISPR / Cas9 gene editing system, the effect of which can affect the efficiency of editing, whether off-target occurs, etc., and even play a decisive role in the final effect of gene editing. Therefore, designing a reasonable and effective sgRNA is an important basis for achieving gene editing, and selecting a suitable recognition sequence is the core work of sgRNA design. For the designed sgRNA, the best sgRNA can be selected based on specificity score, cleavage efficiency score, potential off-target situation and off-target site information, etc.

[0095] The sgRNAs targeting TCR, HLA class I, and / or HLA class II genes are not limited to those specifically used herein, but any sgRNA known in the art that can be used to knock out or disrupt TCR, HLA class I, and / or HLA class II gene expression can be used to perform gene editing to prepare universal CAR-T cells.

[0096] In some embodiments, an sgRNA targeting the constant region of the alpha subunit of TCR (also referred to as a TRAC sgRNA) is used. In some embodiments, the TRAC sgRNA comprises a recognition sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the TRAC sgRNA comprises a recognition sequence as set forth in SEQ ID NO: 1 and a framework sequence as set forth in SEQ ID NO: 4.

[0097] In some embodiments, an sgRNA targeting B2M (also referred to as a B2M sgRNA) is used. In some embodiments, the B2M sgRNA comprises a recognition sequence that is a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the TRAC sgRNA comprises a recognition sequence as set forth in SEQ ID NO: 2 and a frame sequence as set forth in SEQ ID NO: 4.

[0098] In some embodiments, an sgRNA targeting HLA-DRA (also referred to as a HLA-DRA sgRNA) is used. In some embodiments, the HLA-DRA sgRNA comprises a recognition sequence that is a nucleotide sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the TRAC sgRNA comprises a recognition sequence as set forth in SEQ ID NO: 3 and a frame sequence as set forth in SEQ ID NO: 4.

[0099] In some embodiments, one or more of a TRAC sgRNA, a B2M sgRNA, and a HLA-DRA sgRNA is used, e.g., a combination of a TRAC sgRNA and a B2M sgRNA, a combination of a B2M sgRNA and a HLA-DRA sgRNA, a combination of a TRAC sgRNA and a HLA-DRA sgRNA, or a combination of all three of a TRAC sgRNA, a B2M sgRNA, and a HLA-DRA sgRNA is used. In some embodiments, a combination of all three of a TRAC sgRNA, a B2M sgRNA, and a HLA-DRA sgRNA is used to disrupt or knock out a TCR gene, a B2M gene, and a HLA-DRA gene in a CAR-T cell. A dual or multiple knock-out universal CAR-T cell will have lower immunogenicity compared to a single knock-out universal CAR-T cell, further reducing the likelihood of developing graft versus host disease.

[0100] Pharmaceuticals and formulations comprising T cells

[0101] In one aspect, provided herein is a pharmaceutical composition comprising any of the modified T cells provided herein and a pharmaceutically acceptable carrier.

[0102] In one aspect, provided herein is a pharmaceutical composition comprising any of the modified T cells provided herein and a pharmaceutically acceptable carrier.

[0103] Advantages of the Invention

[0104] 1) By corresponding modification, the immunogenicity of T cells is greatly reduced.

[0105] 2) By co-overexpressing CD47 and CD24 on T cells, allogeneic T cells (e.g., CAR-T cells) have resistance to macrophages and NK cells, which helps to improve their survival in the host. Examples

[0106] The following examples are provided to better illustrate the present application and are not intended to limit the scope of the application.

[0107] Materials and reagents

[0108] Example 1: Construction of CD47-CD24-CAR-T cells

[0109] Construct the fusion gene of CD47-CD24-CAR, in which the coding sequence of the fusion protein of CD47 and CD24 plus the transmembrane region is connected with the coding sequence of CAR polypeptide. The prepared CAR-T cells are made to co-overexpress CD47 and CD24 by lentiviral transfection.

[0110] Add 1 x 10 7 T75 bottles and incubate overnight. Prepare 2 sterile 1.5 ml centrifuge tubes, and prepare the plasmid and transfection reagent culture medium in the following order, respectively.

[0111] Mix well, incubate the transfection reagent medium (centrifuge tube 1) for 5 minutes. Add the incubated transfection reagent medium (centrifuge tube 1) to the plasmid solution (centrifuge tube 2) to form a transfection system, mix well, and incubate for 15 minutes. Discard the old medium in the culture bottle of the 293T cells incubated overnight, add 9 ml of new serum-free DMEM medium, then add 1 ml of the incubated transfection system, and incubate the cells in the incubator for 6 hours. Discard the medium in the culture bottle, and add 15 ml of new serum-free DMEM medium.

[0112] Collect the virus liquid (cell culture supernatant) in the culture bottle 48 hours after transfection, and add 15 ml of new serum-free DMEM medium. Collect the cell culture supernatant in the culture bottle again 72 hours after transfection. Put the total 30 ml of cell culture supernatant obtained in a 50 ml sterile syringe, filter through a filter membrane with a pore size of 0.45 nm, and transfer to a sterile ultracentrifuge tube.

[0113] Use the ultracentrifuge to centrifuge at 21000G for 2 hours. Use a pipette to aspirate the supernatant and discard it. Resuspend the virus precipitate with 200 μl of X-VIVO medium, and store it at 4°C overnight.

[0114] Isolate 2 x 10 6 T cells from the recovered PBMCs, and incubate overnight. Add 100 μL of the virus resuspension to the T cells after overnight incubation, and incubate in a 37°C incubator for 24 hours. Aspirate all the cells in the culture bottle, centrifuge at 500G for 5 minutes, and continue to culture after changing the medium.

[0115] Detect the obtained CD47-CD24-CAR-T cells by flow cytometry analysis, and obtain CD47-CD24-CAR-T cells with a high positive rate, as shown in FIG. 1.

[0116] Example 2: Gene knockout of CAR-T cells

[0117] TRAC-sgRNA, B2m-sgRNA, HLA-DRA-sgRNA were dissolved into 100 nmol / ul solution respectively. cas9 and each sgRNA were mixed in the amount of cas9:TRAC-sgRNA:B2m-sgRNA:HLA-DRA-sgRNA=60pmol:75pmol:75pmol:75pmol, and incubated for 10 min. sgRNA consists of a target gene recognition sequence and a constant sequence at the 3' end as a scaffold. The constant sequence is shown in SEQ ID NO: 4: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU. The recognition sequence of each sgRNA used is as follows (T is U in RNA):

[0118] 1 x 10 7 CD47-CD24-CAR-T cells were centrifuged and resuspended using 200 ul of electrotransformation solution. 20 ul of resuspended cell solution was added to each RNP. The cell solution was transferred to a strip. The electrotransformation instrument was turned on and the strip option was selected. The T cell editing program EN113 was used for electrotransformation knockout. The well into which the cell solution had been added was selected, and the T cell editing option was selected. The start button was pressed, and after the electrotransformation was completed, the mixture was transferred to the previously prepared culture medium and cultured in the incubator. The resulting cells were TRAC-KO / B2m-KO / HLADRA-KO triple knockout CAR-T cells (also referred to as tKO UCAR-T cells).

[0119] Example 3: Resistance experiment of CD47-CD24-CAR-T cells to NK cells

[0120] The resistance of CAR-T cells overexpressing CD47 and CD24 and triple knockout (47-24T) to NK cells was detected by CFSE staining. As controls, CD24 UCAR-T cells overexpressing only CD24 and triple knockout, CD47 UCAR-T cells overexpressing only CD47 and triple knockout, CAR-T cells triple knockout without overexpression of CD24 and CD47 (tKO T), and CAR-T cells single knockout of TRAC without overexpression of CD24 and CD47 (sKO T) were also tested for their resistance to NK cells.

[0121] CD47-CD24-CAR-T cells or controls were added as target cells in a 96-well plate, 1 x 10 5Each cell was tested in triplicate. NK cells as effector cells were added to the wells with T cells at an effector to target ratio of 1:1, and put into a 37°C incubator for co-incubation. After 24 hours of co-incubation, the co-incubated cells were stained using an apoptosis kit, and apoptosis was detected by flow cytometry.

[0122] As shown in FIG. 2, the killing rate of NK cells on CAR-T cells overexpressing CD47 and CD24 and triple knockout was significantly decreased compared to control tKO T, CD24 UCAR-T and CD47 UCAR-T cells.

[0123] CD107a can be used as an indicator to evaluate the killing function of NK cells. As shown in FIG. 3, the killing function of NK cells was significantly decreased when incubated with CD47-CD24-U CAR-T compared to incubation with U CAR-T overexpressing neither CD47 nor CD24 or only one of them. “*” indicates p<0.05, “**” indicates p<0.01, “***” indicates p<0.001, and “****” indicates p<0.0001.

[0124] Example 4: Resistance experiment of CD47-CD24-CAR-T cells on macrophages

[0125] The resistance of CD47-CD24-CAR-T cells was detected by CFSE staining. As a control, the resistance of CD24-CAR-T cells overexpressing only CD24 and triple knockout (CD24 UCAR-T), unknocked allogeneic T cells (allo CAR-T) to macrophages (M) was also tested.

[0126] CD47-CD24-CAR-T cells or controls were added as target cells in 12-well plates, 1 x 10 5 Each cell was tested in triplicate. Macrophages as effector cells were added to the wells with T cells at an effector to target ratio of 1:1, and put into a 37°C incubator for co-incubation. After 24 hours of co-incubation, the co-incubated cells were stained using an apoptosis kit, and apoptosis was detected by flow cytometry. Annexin V and 7AAD staining positive indicated apoptotic cells.

[0127] As shown in FIG. 4, the effect of macrophages on CD24 UCAR-T, CD47 UCAR-T and CD47-CD24 UCAR-T cells was not significantly increased. “ns” indicates no significance.

[0128] Those skilled in the art will appreciate that many modifications can be made to the disclosed embodiments and still obtain similar or similar results without departing from the spirit and scope of the disclosed subject matter in accordance with this disclosure.

Claims

1. A T cell engineered to overexpress a CD47 protein and a CD24 protein, wherein the CD47 protein comprises an amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% homology to SEQ ID NO: 5, and the CD24 protein comprises an amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% homology to SEQ ID NO:

6.

2. The T cell of claim 1, wherein the T cell overexpresses a fusion protein of the CD47 protein and the CD24 protein, the fusion protein comprising the CD47 protein operably linked to the CD24 protein, optionally, the operable linkage is through a flexible peptide linker, such as a GS series linker.

3. The T cell of claim 1 or 2, wherein the T cell further has a knockout or inactivation of a gene encoding one or more proteins selected from the group consisting of: a T cell receptor (TCR), an HLA class I molecule, and an HLA class II molecule, optionally, a knockout or inactivation of a gene encoding one or more proteins selected from the group consisting of: TCRa, TCRp, beta-2 microglobulin (B2M), and HLA-DRA.

4. The T cell of claim 3, wherein the cell has a knockout or inactivation of genes encoding TCRa, B2M, and HLA-DRA.

5. The T cell of any one of claims 1-4, which is a CAR-T cell, optionally, a CAR-T cell obtained by engineering a donor-derived T cell with a CAR construct.

6. A method of making T cells with reduced immunogenicity, the method comprising: the T cell is engineered to overexpress a CD47 protein and a CD24 protein, wherein the CD47 protein comprises an amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% homology to SEQ ID NO: 5, and the CD24 protein comprises an amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% homology to SEQ ID NO: 6, optionally, the T cell overexpresses a fusion protein of the CD47 protein and the CD24 protein, the fusion protein comprising the CD47 protein operably linked to the CD24 protein, for example, directly linked or linked through a peptide linker.

7. The method of claim 6, which performs the engineering by transforming or transfecting the T cell with an expression vector comprising a nucleotide sequence encoding the fusion protein.

8. The method of claim 6 or 7, which further comprises engineering the T cell to express a chimeric antigen receptor (CAR), thereby making a CAR-T cell.

9. The method of claim 8, wherein the CAR, the CD47 protein, and the CD24 protein are encoded by the same expression vector or by separate expression vectors.

10. The method of claim 9, wherein the expression vector comprises both a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a fusion protein, the nucleotide sequence encoding a CAR is operably linked to the nucleotide sequence encoding a fusion protein by a nucleotide sequence encoding a self-cleaving linker sequence (such as a 2A linker) or an internal ribosome entry site (IRES).

11. The method of any one of claims 7-10, wherein the expression vector is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a transposon vector, and a viral vector; optionally, the vector is a viral vector, such as a lentiviral vector, an adenoviral vector, or a retroviral vector.

12. The method of any one of claims 6-11, further comprising knocking out or inactivating a gene in the T cell encoding one or more proteins selected from the group consisting of: a T cell receptor (TCR), an HLA class I molecule, and an HLA class II molecule.

13. The method of claim 12, comprising knocking out or inactivating an endogenous gene in the T cell encoding one or more proteins selected from the group consisting of TCRa, TCRp, B2M, and HLA-DRA, optionally, comprising knocking out or inactivating endogenous genes in the cell encoding TCRa, B2M, and HLA-DRA.

14. The method of any one of claims 12-13, wherein the knocking out or inactivating of the gene is performed by contacting the T cell with a gene editing system, such as a CRISPR / Cas9 gene editing system, and introducing components of the system into the cell.

15. The method of claim 14, wherein the gene editing system comprises: any combination of one or more of an sgRNA targeting a TCRa constant region-encoding gene, an sgRNA targeting a TCRp constant region-encoding gene, an sgRNA targeting a B2M gene, and an sgRNA targeting an HLA-DRA gene, or a combination of one or more vectors expressing the sgRNAs; and a nuclease or a nucleic acid encoding the nuclease.

16. The method of claim 15, wherein the gene editing system comprises: an sgRNA targeting a TCRa constant region-encoding gene, an sgRNA targeting a B2M gene, and an sgRNA targeting an HLA-DRA gene.

17. The method of claim 15 or 16, wherein the nuclease is capable of inactivating the corresponding targeted gene under the guidance of each sgRNA, optionally the nuclease is a Cas nuclease, such as a Cas9 nuclease or a Casl2 nuclease, more specifically a spCas9 nuclease.

18. The method of any one of claims 15-17, wherein the sgRNAs are mixed and incubated with the nuclease prior to contacting the T cell with the gene editing system.

19. The method of any one of claims 14-18, wherein the components of the system are introduced into the T cell by electroporation.

20. A T cell prepared by the method of any one of claims 6-19.

21. A vector comprising a nucleotide sequence encoding a fusion protein of a CD47 protein and a CD24 protein, wherein the fusion protein comprises a CD47 protein operably linked to a CD24 protein, the CD47 protein comprising an amino acid sequence of SEQ ID NO: 5 or a functional variant having at least 90% homology to SEQ ID NO: 5, and the CD24 protein comprising an amino acid sequence of SEQ ID NO: 6 or a functional variant having at least 90% homology to SEQ ID NO:

6.

22. The vector of claim 21, wherein the CD47 protein is operably linked to the CD24 protein by a flexible peptide linker, such as a GS series linker.

23. The vector of claim 21 or 22, further comprising a nucleotide sequence encoding a CAR, wherein the nucleotide sequence encoding the CAR is operably linked to the nucleotide sequence encoding the CD47 or to the nucleotide sequence encoding the CD24 by a nucleotide sequence encoding a self-cleaving linker sequence or an internal ribosome entry site (IRES). Optionally, the self-cleaving linker sequence is a 2A linker sequence, such as a P2A linker sequence.

24. The vector of any one of claims 21-23, which is selected from the group consisting of a DNA vector, an RNA vector, a plasmid, a transposon vector, and a viral vector; optionally, the vector is a viral vector, such as a lentivirus vector, an adenovirus vector, or a retrovirus.

25. A pharmaceutical composition comprising the T cell of any one of claims 1-5 and 20 and a pharmaceutically acceptable carrier.

26. Use of the T cell of any one of claims 1-5 and 20 in the manufacture of a medicament for treating a cancer, an autoimmune disease, or an inflammatory disease in a subject.

27. Use of the T cell of any one of claims 1-5 and 20 in the manufacture of a medicament for allogeneic organ transplantation.

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