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WO2025241665A1 - Lymphocyte t amélioré et son utilisation - Google Patents

Lymphocyte t amélioré et son utilisation

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Publication number
WO2025241665A1
WO2025241665A1 PCT/CN2025/082078 CN2025082078W WO2025241665A1 WO 2025241665 A1 WO2025241665 A1 WO 2025241665A1 CN 2025082078 W CN2025082078 W CN 2025082078W WO 2025241665 A1 WO2025241665 A1 WO 2025241665A1
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WIPO (PCT)
Prior art keywords
cells
car
protein
cell
variant
Prior art date
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Pending
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PCT/CN2025/082078
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English (en)
Chinese (zh)
Inventor
郭志刚
陈正亮
张照
黄庭晖
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing Miracle Biotechnology Co Ltd
Original Assignee
Nanjing Miracle Biotechnology Co Ltd
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Priority claimed from CN202410626978.2A external-priority patent/CN121046318A/xx
Application filed by Nanjing Miracle Biotechnology Co Ltd filed Critical Nanjing Miracle Biotechnology Co Ltd
Publication of WO2025241665A1 publication Critical patent/WO2025241665A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Definitions

  • This invention relates to the fields of genetic engineering and cell biology, particularly to T cells with reduced immunogenicity, their preparation methods, and related applications.
  • Chimeric antigen receptor T-cell (CAR-T) therapy has achieved great success in anti-tumor treatment, especially for hematologic malignancies.
  • CAR-T therapies require "personalized customization,” have long preparation times, and are extremely expensive, often costing millions, making them daunting for ordinary people.
  • Universal CAR-T therapies are considered the key to making CAR-T therapy accessible to the general public.
  • Universal CAR-T cells derived from healthy donor T cells, can generate a large number of universal CAR-T cells, providing patients with readily available cell therapy. This makes it a potentially ideal approach for the commercialization of cell therapy.
  • Theoretically, universal CAR-T cells could perfectly solve the problems of high cost and lack of availability associated with autologous CAR-T cells.
  • the objective response rate was 55.8%, with 34.9% of patients achieving a good partial response or better, and a median duration of response of 9.2 months.
  • ORR objective response rate
  • Bristol-Myers Squibb's BCMA CAR-T therapy Abecma which also targets MM, achieved an ORR of 72%, a sCR of 28%, and a median duration of response of 11 months.
  • Legend Biotech's BCMA CAR-T therapy Carvykti achieved an even higher ORR of 98% and a sCR of 82.5%, but the median progression-free survival was not reached during the 28-month follow-up period.
  • T cells especially CAR-T cells
  • This invention enhances the proliferative capacity of T cells, especially CAR-T cells, by overexpressing a truncated IL7Ra mutant on T cells. Combined with the knockout of specific endogenous genes to reduce immunogenicity, this provides more favorable conditions for the survival of T cells, particularly universal CAR-T cells, in the host. Furthermore, this invention further reduces the immunogenicity of T cells by overexpressing a fusion protein of the truncated IL7Ra mutant and CD47 protein (also known as SAP (self-activating and protective motif) protein).
  • SAP self-activating and protective motif
  • the fusion protein provides universal CAR-T cells with sustained basal anti-apoptotic survival signals and weak proliferative signals, and can resist attacks from macrophages and NK cells, effectively enhancing the long-term in vivo and in vitro survival capacity of universal CAR-T cells and reducing in vivo attrition rates.
  • the present invention provides a T cell engineered to express an interleukin-7 receptor ⁇ (IL7R ⁇ ) variant comprising a transmembrane region and an intracellular region.
  • the IL7R ⁇ variant comprises a mutated transmembrane region in the amino acid sequence shown in SEQ ID NO: 6 or 7, and a functional variant having high homology, for example, at least 85% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) to the intracellular region in the amino acid sequence shown in SEQ ID NO: 6 or 7.
  • the IL7R ⁇ variant comprises an amino acid sequence shown in SEQ ID NO: 6 or 7 or a functional variant having at least 90% homology to SEQ ID NO: 6 or 7.
  • the T cells also have the gene encoding one or more proteins selected from the following knockout or inactivation: T cell receptor (TCR), HLA class I molecules and HLA class II molecules, optionally, the gene encoding one or more proteins selected from the following knockout or inactivation: TCR ⁇ , TCR ⁇ , ⁇ -2 microglobulin (B2M) and HLA-DRA.
  • TCR T cell receptor
  • B2M ⁇ -2 microglobulin
  • HLA-DRA HLA-DRA
  • the cells have knocked-out or inactivated genes encoding TCR ⁇ , B2M, and HLA-DRA.
  • the T cells also overexpress CD47 protein or an extracellular region fragment thereof.
  • the CD47 protein or an extracellular region fragment thereof comprises the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof or a functional variant having at least 90% homology with SEQ ID NO:8.
  • the T cells are engineered to express a fusion protein (i.e., a SAP protein) of CD47 protein or its extracellular region with an IL7R ⁇ variant.
  • the fusion protein preferably comprises the CD47 protein or its extracellular region operatively linked to the N-terminus of the IL7R ⁇ variant. This operative linking can be direct or via a linker.
  • the linker can be any peptide linker known in the art suitable for linking two peptides, such as the GS series linkers.
  • the fusion protein comprises the amino acid sequence of SEQ ID NO: 9 or 10.
  • the T cells are CAR-T cells, optionally CAR-T cells obtained by modifying donor-derived T cells with a CAR construct.
  • the expressed CAR polypeptide may be in the form of a single-chain polypeptide containing VHH or scFv as an extracellular antigen-binding region.
  • the present invention provides a method for preparing modified T cells, the method comprising: engineering the T cells to overexpress an interleukin-7 receptor ⁇ (IL7R ⁇ ) variant, wherein the variant comprises an amino acid sequence as shown in SEQ ID NO:6 or 7 or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology with SEQ ID NO:6 or 7.
  • INF7R ⁇ interleukin-7 receptor ⁇
  • the engineering is performed by transforming or transfecting the T cells with an expression vector encoding the IL7R ⁇ variant.
  • the expression vector further comprises a nucleotide sequence encoding a CD47 protein or an extracellular region thereof, the CD47 protein or the extracellular region thereof comprising the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof or a functional variant having at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) homology with SEQ ID NO:8.
  • the expression vector comprises a nucleotide sequence encoding a fusion protein of the CD47 protein or its extracellular region with the IL7R ⁇ variant.
  • the fusion protein comprises the CD47 protein or its extracellular region operatively linked to the N-terminus of the IL7R ⁇ variant.
  • the fusion protein comprises the amino acid sequence of SEQ ID NO: 9 or 10.
  • the method further includes engineering the T cells to express a chimeric antigen receptor (CAR), thereby preparing CAR-T cells.
  • CAR chimeric antigen receptor
  • the CAR and the IL7R ⁇ variant may be encoded by the same expression vector or by separate expression vectors.
  • the expression vector simultaneously comprises a nucleotide sequence encoding a CAR and a nucleotide sequence encoding a fusion protein of the CD47 protein or its extracellular region with the IL7R ⁇ variant.
  • the nucleotide sequence encoding the CAR is operatively linked to the nucleotide sequence encoding the fusion protein via a nucleotide sequence encoding a self-cleaving adapter sequence (such as a 2A adapter), a furin cleavage site, or an internal ribosome entry site (IRES).
  • a self-cleaving adapter sequence such as a 2A adapter
  • a furin cleavage site such as a 2A adapter
  • IRS internal ribosome entry site
  • the expression vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, and viral vectors; optionally, the vector is a viral vector, such as a lentiviral vector, adenovirus vector, or retroviral vector.
  • the method further includes knocking out or inactivating a gene in the T cell encoding one or more proteins selected from: T cell receptor (TCR), HLA class I molecules, and HLA class II molecules.
  • TCR T cell receptor
  • HLA class I molecules HLA class II molecules
  • HLA class II molecules HLA class II molecules
  • the method includes knocking out or inactivating an endogenous gene encoding one or more proteins selected from TCR ⁇ , TCR ⁇ , B2M, and HLA-DRA in the T cells, optionally including knocking out or inactivating an endogenous gene encoding TCR ⁇ , B2M, and HLA-DRA in the cells.
  • the knockout may be performed after engineering to express the IL7R ⁇ variant.
  • the knockout or inactivation of the gene is carried out by contacting the T cell with a gene editing system, such as the CRISPR/Cas9 gene editing system, and introducing components of the system into the cell.
  • a gene editing system such as the CRISPR/Cas9 gene editing system
  • the gene editing system comprises:
  • sgRNA targeting the TCR ⁇ constant region gene sgRNA targeting the TCR ⁇ constant region gene
  • sgRNA targeting the TCR ⁇ constant region gene sgRNA targeting the B2M gene
  • sgRNA targeting the HLA-DRA gene a combination of one or more vectors expressing said sgRNA
  • nuclease or nucleic acid encoding the nuclease are provided.
  • the gene editing system comprises: sgRNA targeting the TCR ⁇ constant region encoding gene, sgRNA targeting the B2M gene, and sgRNA targeting the HLA-DRA gene.
  • the sgRNA targeting the TCR ⁇ constant region coding gene has a recognition sequence as shown in SEQ ID NO:1.
  • the sgRNA targeting the B2M gene has a recognition sequence as shown in SEQ ID NO:2.
  • the sgRNA targeting the HLA-DRA gene has a recognition sequence as shown in SEQ ID NO:3.
  • the sgRNA also includes a constant sequence as a frame, such as the sequence shown in SEQ ID NO:4. In some embodiments, the frame sequence is located at the 3' end of the recognition sequence.
  • the sgRNA targeting the TCR ⁇ constant region coding gene, the sgRNA targeting the B2M gene, and the sgRNA targeting the HLA-DRA gene are not limited to those disclosed herein, as long as they can effectively knock out or inactivate the corresponding target gene in T cells. Therefore, the sgRNA can be a known sgRNA targeting the corresponding gene. For example, it could be those sgRNAs disclosed in Chinese patent applications 202410488220.7, 202410494575.7 and 202410493983.0.
  • the nuclease is capable of inactivating the corresponding target gene under the guidance of each sgRNA, optionally the nuclease is a Cas nuclease, such as Cas9 or Cas12 nuclease, more specifically spCas9 nuclease.
  • the sgRNA is mixed with and incubated with a nuclease before the T cells are brought into contact with the gene editing system.
  • the sgRNA can be mixed with and incubated with the nuclease before contact to form an RNP complex.
  • the method includes mixing equimolar amounts of sgRNA targeting the TCR ⁇ constant region gene, sgRNA targeting the B2M gene, and sgRNA targeting the HLA-DRA gene together with a Cas9 nuclease and incubating to form an RNP complex.
  • components of the system are introduced into the T cells via electrotransfection.
  • the present invention provides T cells, particularly CAR-T cells, prepared by the methods disclosed herein. These CAR-T cells possess improved in vivo or in vitro survival and proliferation capabilities.
  • the present invention provides a vector comprising a nucleotide sequence encoding a CD47 protein or an extracellular region thereof operably linked to a nucleotide sequence encoding an IL7R ⁇ variant, wherein the CD47 protein or the extracellular region thereof comprises the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof or a functional variant having at least 90% homology to SEQ ID NO:8, said IL7R ⁇ variant comprising the amino acid sequence as shown in SEQ ID NO:6 or 7.
  • the operably linked connection is a direct connection or a connection via a linker.
  • the present invention provides a vector comprising a nucleotide sequence encoding a CAR, a nucleotide sequence encoding a CD47 protein or its extracellular region, and a nucleotide sequence encoding an IL7R ⁇ variant, wherein the nucleotide sequence encoding the CAR is operatively linked to the nucleotide sequence encoding the CD47 protein or its extracellular region or the nucleotide sequence encoding the IL7R ⁇ variant via a nucleotide sequence encoding a self-cleaving adapter sequence or an internal ribosome entry site (IRES).
  • IRS internal ribosome entry site
  • the CD47 protein or its extracellular region contains the amino acid sequence of SEQ ID NO:8 or a functional fragment thereof or a functional variant having at least 90% homology with SEQ ID NO:8, and the IL7R ⁇ variant contains the amino acid sequence shown in SEQ ID NO:6 or 7.
  • the vector comprises a nucleotide sequence encoding the CD47 protein or its extracellular region directly linked to a nucleotide sequence encoding an IL7R ⁇ variant.
  • the self-cutting connector sequence is a 2A connector sequence, such as a P2A connector sequence.
  • the vector is selected from DNA vectors, RNA vectors, plasmids, transposon vectors, and viral vectors; optionally, the vector is a viral vector, such as a lentiviral vector, adenovirus vector, or retrovirus.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the T cells and a pharmaceutically acceptable carrier.
  • the present invention provides the use of T cells (e.g., CAR-T cells) prepared by the method of the present invention as a medicament.
  • the present invention provides the use of T cells (e.g., CAR-T cells) prepared by the method of the present invention in the preparation of a medicament for treating cancer, autoimmune diseases, or inflammatory diseases in a subject, optionally wherein the T cells are allogeneic or autologous to the subject.
  • the present invention provides a method for treating cancer, autoimmune diseases, or inflammatory diseases in a subject, wherein T cells (e.g., CAR-T cells) prepared by the method of the present invention are administered.
  • the present invention provides T cells (e.g., CAR-T cells) prepared by the method of the present invention for treating cancer, autoimmune diseases, or inflammatory diseases in a subject.
  • the cancer can be any cancer suitable for cell therapy such as CAR-T therapy, such as leukemia including acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL), lymphoma, multiple myeloma (MM), glioma, breast cancer, kidney cancer, liver cancer, esophageal cancer, gastric cancer, pancreatic cancer, prostate cancer, lung cancer, ovarian cancer, colorectal cancer, head and neck cancer, gallbladder cancer, etc.
  • AML acute myeloid leukemia
  • ALL acute lymphoblastic leukemia
  • MM multiple myeloma
  • glioma breast cancer
  • kidney cancer liver cancer
  • esophageal cancer multiple myeloma
  • gastric cancer pancreatic cancer
  • prostate cancer lung cancer
  • ovarian cancer
  • the autoimmune or inflammatory diseases mentioned include, but are 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.
  • the present invention provides the use of T cells prepared by the method of the present invention in the preparation of medicaments for organ transplantation.
  • the cells may be CAR-T cells.
  • Figure 1 shows the CAR-T positivity rate test results.
  • FIG. 2 shows the results of the phosphorylated STAT5 assay.
  • UCAR-T-tIL7Ra*-1 and UCAR-T-tIL7Ra*-2 represent UCAR-T cells connected to the intracellular domain of IL7R ⁇ via different mutant IL7R ⁇ transmembrane domains, respectively.
  • mock CAR-T represents CAR T cells that do not overexpress IL7R ⁇ or CD47 and have not undergone gene knockout.
  • UCAR-T represents a universal CAR-T cell control that does not overexpress IL7R ⁇ or CD47.
  • Figure 3 shows the results of the cytokine withdrawal experiment in the presence of IL-2.
  • Figure 4 shows the results of the cytokine withdrawal experiment in the absence of IL-2.
  • Figure 5 shows an example of a CAR construct (CD8 signal peptide + anti-CD70 scFv + CD8 hinge + CD8 ⁇ TM + CD28 + CD3 ⁇ ) linked to an SAP fusion protein, wherein the CAR peptide coding sequence targeting CD70 is linked to the SAP fusion protein coding sequence, which comprises a CD47 ECD protein fused to an IL7Ra* variant protein.
  • a CAR construct CD8 signal peptide + anti-CD70 scFv + CD8 hinge + CD8 ⁇ TM + CD28 + CD3 ⁇
  • Figure 6 shows the expression of CAR on CD47 UCAR-T cells as detected by flow cytometry.
  • Figure 7 shows the expression of CAR(A) and CD47(B) in SAP UCAR-T cells as detected by flow cytometry.
  • Figure 8 shows the intracellular factor levels of CAR-T cells as detected by intracellular flow cytometry: (A) pZAP70; (B) pSTAT5. The four bars from left to right correspond to the captions from top to bottom.
  • Figure 9 shows the results of various UCAR-T cells in the cytokine withdrawal experiment: (A) with cytokines; (B) without cytokines.
  • Figure 10 shows the tumor-killing ability of CD47 UCAR-T cells in the presence of NK cells or M1 cells: (A) experimental procedure; (B) cell lysis percentage results.
  • Figure B the four bars on the horizontal axis for each case correspond to the captions from top to bottom.
  • Figure 11 shows the results of NK cell immune rejection of various UCAR-T cells by apoptosis detection: (A) upregulation of CD107a, a marker of NK cell degranulation; (B) NK cell cytotoxicity results. The three bars from left to right correspond to the captions from top to bottom.
  • Figure 12 shows the results of immune rejection of various UCAR-T cells by macrophages as detected by apoptosis: (A) the phagocytic rate; (B) the cytotoxicity results of macrophages.
  • Figure 13 shows the phosphorylation levels of STAT5 and BCL2 proteins in various CAR-T cells in the presence (A) or absence of cytokines (B).
  • Figure 14 shows the survival time of various UCAR-T cells in immunodeficient mouse models.
  • CD47 also known as integrin-associated protein, is a cell membrane protein belonging to the immunoglobulin superfamily. It is widely expressed on the surface of almost all normal cells and is highly expressed on the surface of tumor cells. CD47 binds to macrophage signaling regulator ⁇ (Sirp ⁇ ) to send a "don't eat me” signal, thereby protecting tumor cells from phagocytosis by macrophages and enabling tumor immune escape.
  • NK cell SIRP ⁇ is upregulated upon IL-2 stimulation, interacting with target cell CD47 in a threshold-dependent manner and counteracting other stimuli, including IL-2, CD16, or NKG2D. Increased CD47 expression protects tumor cells against SIRP ⁇ + primary NK cells.
  • the CD47 used in this article encompasses the natural human CD47 protein, its extracellular domain, or functional variants thereof.
  • the term "functional variant” for a protein refers to a protein variant that has sequence variations relative to the protein, typically retains at least 85% homology, and substantially preserves the biological function of the protein.
  • the human "B2M gene,” or " ⁇ 2-microglobulin-encoding gene,” is located on human chromosome 15 (15q21.1) and contains four exons.
  • ⁇ 2M protein exists as a membrane protein and free ⁇ 2M.
  • the membrane protein ⁇ 2M as a constant light chain, non-covalently associates with the heavy chain of MHC class I molecules (i.e., human leukocyte antigen class I molecules, HLA-I) on the cell surface.
  • B2M participates in the recognition of lymphocytes and target cell surface antigens; therefore, B2M is closely related to tissue compatibility. Almost all nucleated cells in the body can synthesize ⁇ 2-microglobulin, which attaches to the cell surface. The absence of ⁇ 2-microglobulin leads to abnormal aggregation of HLA-I molecules, thus preventing the formation of complete functional molecules.
  • HLA-DRA also known as “MHC class II DR ⁇ ” refers to the ⁇ chain of the HLA class II histocompatibility antigen DR.
  • HLA-DR is a subtype of MHC class II molecules, a heterodimer composed of ⁇ and ⁇ chains anchored in the cell membrane.
  • HLA-DRA is a polymorphic cell surface glycoprotein that plays a crucial role in intercellular interactions during the immune response.
  • the ⁇ chain protein is approximately 33-35 kDa, and its encoding gene contains five exons: 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 imported or transplanted allogeneic cells, leading to transplant rejection.
  • mismatches of HLA-II molecules highly expressed on activated T cells can activate the recipient's allogeneic reactive CD4 + T cells. Therefore, reducing or knocking out HLA-DRA expression in donor cells can reduce HLA-II mismatches, making it an attractive target for cell therapy.
  • Cas9 nuclease or “Cas9” refers to an RNA-directed nuclease belonging to the CRISPR/Cas9 gene editing system, including wild-type Cas9 protein or variants or fragments thereof, such as proteins containing the active DNA-cutting domain and/or the gRNA-binding domain of Cas9.
  • Cas9 is a component of the CRISPR/Cas gene editing system, and wild-type Cas9, guided by gRNA, targets and cleaves DNA target sequences to form DNA double-strand breaks (DSBs).
  • the DNA-cutting activity of Cas9 depends on two domains: RuvC and HNH, which are responsible for cleaving the two strands of DNA, respectively.
  • Activity of the RuvC domain cleaves the complementary strand of the guiding RNA
  • activity of the HNH domain cleaves the non-complementary strand.
  • guide RNA refers to an RNA sequence containing a guide sequence (also referred to herein as a recognition sequence) and optionally a tracrRNA.
  • Common guide RNAs consist of crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) sequences that form a complex through partial complementarity, wherein the recognition sequence contained in the crRNA is sufficiently complementary to the target sequence to hybridize and target the CRISPR complex to the specifically binding target sequence.
  • CRISPR RNA crRNA
  • tracrRNA trans-activating crRNA
  • the term also includes single guide RNA (sgRNA), which combines the characteristics of both crRNA and tracrRNA.
  • the guide sequence of the sgRNA is complementary to the target nucleic acid sequence and is responsible for the initial guide RNA/target base pairing.
  • the guide sequence of the sgRNA is intolerant of mismatches.
  • specific recognition of a target site by a guide sequence or recognition sequence means that the base complementarity between the guide sequence or recognition sequence in the sgRNA and the target site sequence is at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably 100%.
  • CAR-T cell refers to a T cell that expresses any CAR construct or has been introduced with nucleic acid or a vector encoding a CAR construct.
  • Polynucleotides encoding CAR construct peptides can be introduced into cells using various methods, and CAR construct peptides can also be synthesized in situ within cells. Methods for 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 cell's genome.
  • transient transformation methods can be used for transient expression of the polynucleotide construct, and the polynucleotide construct is not integrated into the cell's genome.
  • virus-mediated methods can be used.
  • Polynucleotides can be introduced into cells by any suitable method, such as recombinant viral vectors (e.g., retroviruses, adenoviruses), liposomes, etc.
  • Transient transformation methods include, for example, but not limited to, microinjection, electroporation, or particle bombardment.
  • Polynucleotides can be included in vectors, such as plasmid vectors or viral vectors.
  • CAR-T chimeric antigen receptor T-cell
  • CAR-T therapy is the engineering of T cells using chimeric antigen receptor (CAR) constructs.
  • CAR constructs typically contain extracellular antigen-binding domains, transmembrane domains, and intracellular signal transduction domains.
  • Antigen-binding domains often originate from antigen-binding fragments capable of recognizing and binding specific antigens, such as the variable region of a single-chain antibody (scFv, VHH, or Fab). By selecting appropriate antigen-binding domains, cell surface markers associated with specific disease states, such as tumors, can be identified.
  • Intracellular signal transduction domains are used to transduce effector and functional signals and guide cells to perform specific functions (e.g., cytolytic or co-activating activities, including cytokine secretion). These domains typically contain primary signal transduction domains and co-stimulatory signal transduction domains.
  • Primary signal transduction domains are protein portions that can regulate the primary activation of the TCR complex in a stimulatory or inhibitory manner. Stimulatory primary signal transduction domains often contain signal transduction motifs known to be based on the tyrosine-based activation motif of the immune receptor (ITAM).
  • Co-stimulatory signal transduction domains are intracellular signal transduction domains derived from co-stimulatory molecules. Co-stimulatory molecules are cell surface molecules, other than antigen receptors or Fc receptors, that provide a second signal required for the efficient activation and function of T lymphocytes after binding to an antigen.
  • the antigens bound to the CAR constructs can be selected from a variety of tumor-associated antigens, tumor-specific antigens, or antigens related to other immune diseases.
  • the CAR construct may be designed to recognize any of the following antigens: CD70, CD3, CD19, CD20, 4.1BB (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
  • the CAR construct comprises an antigen-binding domain in the form of scFv. In some other embodiments, the CAR construct comprises an antigen-binding domain in the form of VHH. In some specific embodiments, the CAR construct comprises the amino acid sequence of SEQ ID NO:11.
  • the primary signal transduction domain of the CAR construct contains an ITAM derived from TCR ⁇ , FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, and CD66d.
  • the co-stimulatory signal transduction domain of the CAR construct is derived from co-stimulatory molecules selected from CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54 (ICAM), CD83, CD134 (OX40), CD137 (4-1BB), CD150 (SLAMF1), CD270 (HVEM), CD278 (ICOS), and DAP10.
  • the CAR construct further includes a linker domain or adapter sequence between the antigen-binding domain and the transmembrane domain and/or between the transmembrane domain and the intracellular signal transduction domain.
  • Engineered CAR-T cells can be obtained by transfecting T cells with a virus containing a CAR coding sequence.
  • the virion used for transfection is generated by transfecting cells with a plasmid encoding the CAR construct and a viral packaging plasmid.
  • engineered CAR-T cells are obtained by transfecting immune cells with an expression vector of the CAR construct.
  • CAR-T cells that can be used for CAR construct modification are T lymphocytes, including thymocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes.
  • T cells can be T helper (Th) cells, such as T helper 1 (Th1) or T helper 2 (Th2) cells.
  • T cells can be helper T cells (HTL; CD4 T cells), CD4 T cells, cytotoxic T cells (CTL; CD8 T cells), CD4CD8 T cells, or any other T cell subset.
  • T cells may include primordial T cells and memory T cells.
  • the T cells used for engineering are isolated from peripheral blood mononuclear cells (PBMCs). Methods for isolating various cell fractions from PBMCs are well known to those skilled in the art.
  • the peripheral blood mononuclear cells are isolated from a subject requiring CAR-T cell therapy.
  • the T cells include any type of T cell, such as cytotoxic T lymphocytes and regulatory T cells.
  • Organisms typically reject xenografts or allogeneic grafts, including allogeneic T cells such as CAR-T cells. This is primarily because the donor's genes encode antigens not present in the recipient, which are recognized by the recipient's immune cells, triggering a rejection response known as host-graft reaction. Universal CAR-T (UCAR-T) cells have been developed, exhibiting reduced cellular immunogenicity, thus supporting more patients in receiving effective treatment through CAR-T therapy.
  • UAR-T Universal CAR-T
  • the general design principle of universal CAR-T cells is to generate CAR-T cells from an allogeneic donor and then use gene editing to destroy the TCR gene, HLA-I gene and/or HLA-II gene of the allogeneic T cells.
  • the resulting T cells cannot recognize allogeneic antigens, thereby effectively eliminating graft-versus-host disease.
  • T cell membranes express T cell receptors (TCRs), which are responsible for recognizing antigens presented by the major histocompatibility complex (MHC, also known as leukocyte antigens, or HLA molecules). Unlike B cell receptors, TCRs do not recognize free antigens but rather antigenic peptide fragments presented by MHC molecules. Normally, TCRs are glycoproteins on the cell membrane surface existing as heterodimers of ⁇ / ⁇ or ⁇ / ⁇ chains. The specific binding of T cell receptors to MHC-presented peptides triggers a series of biochemical reactions, activating T cells through numerous co-receptors, enzymes, and transcription factors, promoting their division and differentiation. In allogeneic transplantation, lymphocytes in the graft recognize antigens from the recipient cells, initiating an immune response that attacks the recipient cells, resulting in graft-versus-host disease.
  • MHC major histocompatibility complex
  • the gene encoding the endogenous TCR in universal CAR-T cells is disrupted or knocked out. For example, by targeting the genomic sequence of the constant region of the endogenous ⁇ or ⁇ subunit of the TCR, TCR expression can be eliminated, and the resulting T cells cannot recognize allogeneic antigens.
  • the gene expression of endogenous HLA class I antigens in universal CAR-T cells is disrupted or knocked out.
  • Cells expressing allogeneic major histocompatibility complex (MHC)-I can be recognized and eliminated by CD8 + T cells.
  • HLA class I genes HLA-A, HLA-B, HLA-C
  • HLA-A, HLA-B, HLA-C encode MHC-I; therefore, knocking out HLA class I molecules helps allogeneic T cells survive better in the host.
  • ⁇ 2-microglobulin (B2M) is a ⁇ -light chain of human leukocyte antigen class I (HLA-I), and its main function is to participate in the recognition of lymphocytes and target cell surface antigens; therefore, B2M is closely related to histocompatibility.
  • the expression of the B2M gene can be knocked out or disrupted. Knocking out the B2M gene can prevent the expression of HLA-I molecules in cells, reduce cellular immunogenicity, and effectively reduce the risk of host anti-graft reaction and immune rejection.
  • HLA class II molecules on the surface of T cells are important targets for CD4 + T helper cell recognition.
  • HLA class II molecules include HLA-DR, HLA-DP, HLA-DQ, HLA-DM, and HLA-DO, which are also targets for host cells to recognize allogeneic cells.
  • the expression of the gene encoding HLA-DRA can be knocked out or disrupted.
  • HLA-DRA is one of the paralogs of the HLA class II ⁇ chain.
  • the universal CAR-T cells are CAR-T cells that do not express or express at low levels one or more proteins selected from the following: TCR, B2M, HLA-I and HLA-II molecules.
  • the universal CAR-T cells are CAR-T cells that do not express or express at low levels one or more proteins selected from the following: TCR, B2M, and HLA-DRA.
  • the universal CAR-T cells are CAR-T cells that do not express or express low levels of TCR, B2M, and HLA-DRA.
  • the cells that can be applied to this invention are not limited to CAR-T cells, but can be various T cells.
  • the improved CAR-T cells of this invention address the following issues: 1) the significant decrease in in vivo and in vitro proliferation and survival time of universal CAR-T cells after TCR knockout; and 2) the susceptibility of universal CAR-T cells to clearance by the host immune system in vivo.
  • Universal CAR-T cells e.g., SAP UCAR-T
  • obtained using the method of this invention exhibit significantly increased in vivo and in vitro proliferation capacity, as well as long-term survival and anti-apoptotic ability in vivo after transplantation.
  • the present invention provides a method for improving the survival and/or proliferation of T cells (such as CAR-T cells) by enabling T cells to co-express IL-7R ⁇ or a variant thereof.
  • IL-7R is the receptor for IL-7 and is composed of the IL-7R ⁇ chain (also known as CD127) and the common cytokine receptor ⁇ chain (also known as CD132, IL-2R ⁇ ).
  • the T cells are engineered to express an IL-7R ⁇ variant, particularly a truncated IL-7R ⁇ variant.
  • the truncated IL-7R ⁇ variant comprises a truncated and mutated extracellular domain and cytoplasmic region.
  • the IL-7R ⁇ variant comprises an amino acid sequence as shown in SEQ ID No: 6 or 7.
  • Co-expression of IL-7R ⁇ or its variants can be achieved by placing the coding sequences of CAR and IL-7R ⁇ or variants in the same or different expression vectors.
  • the nucleotide sequence encoding CAR and the nucleotide sequence encoding IL-7R ⁇ or its variants are operatively linked in an expression vector, such as a lentiviral vector.
  • These nucleotide sequences can be linked via adapters, particularly 2A adapter sequences (e.g., P2A or T2A) or IRES sequences.
  • 2A adapter sequences e.g., P2A or T2A
  • IRES sequences e.g., IRES sequences.
  • this invention further reduces the immunogenicity of T cells by overexpressing CD47.
  • Allogeneic T cells such as universal CAR-T cells, may have an increased risk of being attacked by NK cells when evading host T cell attack, and may also be subject to rejection by myeloid cells.
  • the knockout of HLA-I molecules in T cells may lead to increased responsiveness of natural killer (NK) cells to T cells.
  • NK natural killer
  • T cells are also unable to avoid attack by myeloid cells, including macrophages.
  • This invention addresses this problem by overexpressing CD47 in T cells, particularly by overexpressing a fusion protein of a truncated IL-7R ⁇ variant and CD47.
  • CD47 is highly expressed in tumor cells and can enable tumor cells to escape the immune system through interaction with the signal regulatory protein ⁇ (Sirp ⁇ ) on the surface of macrophages or NK cells.
  • This invention unexpectedly discovered that by expressing a fusion protein of a truncated IL-7R ⁇ variant and CD47 in universal CAR-T cells, immunogenicity caused by reactivity with NK cells and phagocytosis by macrophages can be further reduced.
  • the fusion protein (also referred to herein as the SAP protein) comprises a CD47 protein, an extracellular region thereof, or a functional variant thereof having a highly homologous sequence, operably linked to a truncated IL-7R ⁇ variant, wherein the CD47 protein or its extracellular region comprises the amino acid sequence of SEQ ID NO:8 or a functional variant having at least 90% homology with SEQ ID NO:8, and the truncated IL-7R ⁇ variant comprises the amino acid sequence shown in SEQ ID NO:6 or 7.
  • the CD47 protein, its extracellular region, or a functional variant thereof having a highly homologous sequence is located at the N-terminus of the truncated IL-7R ⁇ variant.
  • the operable link can be a direct link or a link via a peptide linker, provided that the peptide linker does not affect the function of the two peptide parts.
  • the fusion protein has the amino acid sequence shown in SEQ ID NO:9 or 10.
  • the CAR-T cells are engineered to overexpress a fusion protein of CD47 and a truncated IL7R ⁇ variant or both.
  • Overexpression of the CD47 and fusion protein can be achieved by placing the coding sequence of the CAR and the coding sequence of the fusion protein in the same or different expression vectors.
  • the nucleotide sequences encoding the CAR and the nucleotide sequences encoding the fusion protein are operatively linked in an expression vector, such as a lentiviral vector.
  • the nucleotide sequences can be linked via cleavable linkers, particularly 2A sequences (e.g., P2A or T2A) or IRES sequences. Consequently, the transcribed mRNA is cleaved during ribosome translation to generate two polypeptide chains: the CAR polypeptide and the fusion protein.
  • the nucleotide sequence encoding a CAR is operatively linked to a nucleotide sequence encoding a fusion protein via a nucleotide sequence encoding a first adapter, said nucleotide sequence comprising a nucleotide sequence encoding a CD47 protein, its extracellular region, or a functional variant thereof having a highly homologous sequence, directly linked to a nucleotide sequence encoding a truncated IL7R ⁇ variant.
  • the nucleotide sequence encoding a CAR is operatively linked to a nucleotide sequence encoding CD47 via a nucleotide sequence encoding a first adapter
  • the nucleotide sequence encoding CD47 is operatively linked to a nucleotide sequence encoding a truncated IL7R ⁇ variant via a nucleotide sequence encoding a second adapter.
  • the nucleotide sequence encoding a CAR is operatively linked to a nucleotide sequence encoding a truncated IL7R ⁇ variant via a nucleotide sequence encoding a first adapter
  • the nucleotide sequence encoding a truncated IL7R ⁇ variant is operatively linked to a nucleotide sequence encoding CD47 via a nucleotide sequence encoding a second adapter.
  • the first adapter may be selected from self-cleaving adapters such as 2A sequences (e.g., P2A and T2A) and IRES sequences.
  • the second connector can be a non-self-cutting connector, such as the GS series peptide connector.
  • the present invention provides a method for preparing T cells, comprising:
  • the present invention provides a method for preparing T cells, comprising:
  • T cells with TCR, HLA class I, and/or HLA-II gene knockouts are constructed using CRISPR/Cas gene editing systems, particularly the CRISPR/Cas9 gene editing system.
  • the present invention provides a method for preparing modified CAR-T cells, wherein the endogenous ⁇ -2 microglobulin (B2M) gene in the CAR-T cells is knocked out or inactivated.
  • the modified CAR-T cells exhibit reduced cellular immunogenicity due to the knockout of HLA-I class molecules.
  • the B2M gene can be rendered biologically inactive by cleaving a target locus in B2M via Cas9 guided by specific sgRNA, followed by the introduction of DNA sequence insertion or base deletion.
  • the endogenous B2M gene is inactivated by nucleotide deletion, resulting in complete non-expression of B2M.
  • each allele of B2M in the genome e.g., a diploid genome
  • the present invention provides a method for preparing modified CAR-T cells, wherein the gene encoding the ⁇ or ⁇ subunit constant region of an endogenous TCR in the CAR-T cells is knocked out or inactivated.
  • the modified CAR-T cells exhibit reduced cellular immunogenicity due to the knockout of the TCR molecule.
  • Biological function can be lost by introducing DNA sequence insertion or base deletion after Cas9 cleavage of the target site in the gene encoding the ⁇ or ⁇ subunit constant region of the TCR guided by specific sgRNA.
  • the gene encoding the ⁇ or ⁇ subunit constant region of the endogenous TCR is inactivated by nucleotide deletion, resulting in complete non-expression of the TCR.
  • each allele of the TCR in the genome e.g., a diploid genome
  • the present invention provides a method for preparing modified CAR-T cells, wherein an endogenous HLA-DRA gene in the CAR-T cells is knocked out or inactivated.
  • the modified CAR-T cells exhibit reduced cellular immunogenicity due to the knockout of HLA-II molecules.
  • the HLA-DRA gene can be rendered biologically inactive by cleaving a target locus in HLA-DRA with Cas9 guided by specific sgRNA, followed by the introduction of DNA sequence insertion or base deletion.
  • the endogenous HLA-DRA gene is inactivated by nucleotide deletion, resulting in complete non-expression of B2M.
  • each allele of HLA-DRA in the genome e.g., a diploid genome
  • the present invention provides a method for preparing modified CAR-T cells, wherein one or more genes selected from the following are knocked out or inactivated in the CAR-T cells: HLA-DRA encoding genes, genes encoding the constant regions of the ⁇ or ⁇ subunits of the TCR, and B2M encoding genes.
  • HLA-DRA encoding genes genes encoding the constant regions of the ⁇ or ⁇ subunits of the TCR
  • B2M encoding genes for example, guided by various specific sgRNAs, Cas9 can cleave the target sites in the corresponding target genes, introducing DNA sequence insertions or base deletions to render various target genes biologically inactive.
  • the various sgRNAs may include: sgRNAs targeting the constant regions of the ⁇ or ⁇ subunits of the TCR, sgRNAs targeting the B2M gene, sgRNAs targeting HLA-DRA, or any combination thereof.
  • the sgRNAs may be those disclosed in Chinese Patent Application 202410624572.0.
  • the sgRNA of the present invention is 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 present invention and a Cas enzyme, said complex may comprise any suitable Cas enzyme.
  • the Cas enzyme is selected from Cas9, Cas12a, Cas12e, Cas12b, Cas12i, Cas12h, Cas12c, Cas12d, Cas12f, Cas12g, Cas12k, Cas12j, Cas13a, Cas13b, Cas13c, Cas13d, and Cas14, including any recombinant variants thereof, particularly selected from Cas9, including any recombinant variants thereof.
  • the Cas9 enzyme may be streptococcal, such as Streptococcus pyogenes or Lactobacillus Cas9 enzyme, as described by Briner et al.
  • the Cas nuclease is selected from Cas9 nucleases and Cas12 nucleases.
  • the sgRNA of this invention can be used in conjunction with various Cas proteins for use in various CRISPR/Cas systems, such as the CRISPR/Cas9 system, CRISPR/Cas12 system, CRISPR/nCas9 system, and CRISPR/dCas9 system.
  • the Cas9 protein is a multifunctional protein whose protein structure includes a recognition region (REC) composed of an ⁇ -helix, a nuclease region composed of an HNH domain and a RuvC domain, and a PAM binding region located at the C-terminus. These two important nuclease domains, RuvC and HNH, can respectively cleave the complementary and non-complementary DNA strands of the gRNA, producing blunt-ended DNA double-strand breaks.
  • the Cas9 protein can be mutated as needed to form single-strand DNA breaks. Cas9 recognition of 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 wild-type Cas9.
  • the Cas9 protein is derived from Streptococcus pyogenes Cas9 protein or Staphylococcus aureus Cas9 protein.
  • the Cas9 protein can induce double-strand breaks at the target site in the target gene.
  • Inactivation of endogenous target genes can be achieved by introducing a Cas nuclease and one or more sgRNAs into CAR-T cells.
  • a complex of the Cas nuclease and sgRNA is introduced into CAR-T cells. For example, this can be done by incubating the Cas nuclease and one or more sgRNAs before introducing them into CAR-T cells. This introduction can be performed by electroporation.
  • a plasmid encoding one or more sgRNAs and a plasmid encoding the Cas nuclease are introduced into CAR-T cells.
  • the sgRNA contains a recognition sequence targeting a specific site in the target gene.
  • This recognition sequence is typically designed to be 20 nt in length.
  • the sgRNA also contains a constant frame, which may include partial crRNA and tracrRNA sequences.
  • sgRNA plays a crucial role in accurately recognizing target gene sequences. Its effectiveness can influence editing efficiency, off-target effects, and even determine the final gene editing outcome. Therefore, designing a reasonable and effective sgRNA is a fundamental aspect of gene editing, and selecting a suitable recognition sequence is the core task in sgRNA design. For designed sgRNAs, analysis can be performed based on specificity scores, splicing efficiency scores, potential off-target situations, and off-target site information to select the optimal sgRNA.
  • the sgRNAs targeting TCR, HLA class I and/or HLA class II genes are not limited to those specifically used in this article, but any sgRNA known in the art that can be used to knock out or disrupt the expression of TCR, HLA class I and/or HLA class II genes can be used for gene editing to prepare universal CAR-T cells.
  • an sgRNA (also known as a TRAC sgRNA) targeting the constant region of the ⁇ subunit of the TCR is used.
  • the TRAC 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 with SEQ ID NO:1.
  • the TRAC sgRNA comprises a recognition sequence as shown in SEQ ID NO:1 and a frame sequence as shown in SEQ ID NO:4.
  • a B2M-targeting sgRNA (also known as a B2M sgRNA) is used.
  • 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 with SEQ ID NO:2.
  • the TRAC sgRNA comprises a recognition sequence as shown in SEQ ID NO:2 and a frame sequence as shown in SEQ ID NO:4.
  • an HLA-DRA-targeting sgRNA (also known as HLA-DRA sgRNA) is used.
  • the HLA-DRA sgRNA includes 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 with SEQ ID NO:3.
  • the TRAC sgRNA includes a recognition sequence as shown in SEQ ID NO:3 and a frame sequence as shown in SEQ ID NO:4.
  • one or more of TRAC sgRNA, B2M sgRNA, and HLA-DRA sgRNA are used, such as a combination of TRAC sgRNA and B2M sgRNA, a combination of B2M sgRNA and HLA-DRA sgRNA, a combination of TRAC sgRNA and HLA-DRA sgRNA, or a combination of all three.
  • a combination of TRAC sgRNA, B2M sgRNA, and HLA-DRA sgRNA is used to disrupt or knock out the TCR gene, B2M gene, and HLA-DRA gene in CAR-T cells. Compared to universal CAR-T cells with single gene knockout, double or multiple knockout universal CAR-T cells will have lower immunogenicity, further reducing the likelihood of graft-versus-host disease.
  • this document provides modified T cells prepared by any of the methods provided herein. Specifically, it provides an improved universal CAR-T cell with reduced immunogenicity and enhanced survival in vivo.
  • the T cells provided by this invention effectively enhance their resistance to macrophages and NK cells and exhibit more durable effects in subjects receiving cell therapy, thereby improving the therapeutic efficacy of T cells.
  • this article provides pharmaceutical compositions comprising any of the modified T cells and pharmaceutically acceptable carriers provided herein.
  • the survival and proliferation signals of universal CAR-T are increased, and it can resist the attack of macrophages and NK cells, effectively enhancing the long-term survival ability of universal CAR-T in vivo and in vitro and reducing the in vivo loss rate.
  • Lentiviral Vector 1 Gene Synthesis CAR-2A-tIL7Ra * fusion gene sequence, wherein the coding sequence of the CAR polypeptide chain is ligated to the coding sequence of the truncated IL-7R ⁇ variant tIL7Ra*-1 or tIL7Ra*-2 via a 2A cleavable adapter. Compared with the truncated sequence of natural IL-7R ⁇ SEQ ID NO:5, tIL7Ra*-1 or tIL7Ra*-2 has an additional amino acid inserted into the extracellular domain.
  • the fusion gene sequence is ligated into a PLV vector by enzyme digestion transformation, with the EF-1a promoter upstream of the gene.
  • the vector is transformed into DH5 ⁇ Escherichia coli strain, screened with ampicillin, positive clones are obtained, plasmids are extracted, clones are identified by enzyme digestion, and CD70 CAR-2A-tIL7Ra * lentiviral packaging vector is obtained.
  • the specific sequence of the truncated IL-7R ⁇ variant encoded in the fusion gene is as follows:
  • Lentiviral vector 2 The CAR polypeptide coding sequence is ligated to the extracellular coding sequence of the CD47 protein via a cleavable 2A adapter. The other end of the extracellular coding sequence of the CD47 protein is ligated to the coding sequence of the truncated IL-7R ⁇ variant tIL7Ra*-1 or tIL7Ra*-2.
  • a CD70 CAR-2A-CD47 ECD-tIL7Ra* fusion gene sequence is synthesized, transformed by enzyme digestion, and ligated into a PLV vector. The upstream of the gene is the EF-1a promoter.
  • the vector is transformed into DH5 ⁇ *Escherichia coli* strain, screened with ampicillin, and positive clones are obtained. Plasmids are extracted, and clones are identified by enzyme digestion to obtain the CD70 CAR-2A-CD47 ECD-tIL7Ra* lentiviral packaging vector.
  • Lentiviral vector 3 The CAR polypeptide coding sequence was ligated to the extracellular coding sequence of the CD47 protein via a cleavable 2A adapter.
  • a CD70 CAR-2A-CD47 ECD fusion gene sequence was synthesized, transformed by enzyme digestion, and ligated into a PLV vector. The upstream of the gene is the EF-1a promoter.
  • the vector was transformed into DH5 ⁇ *Escherichia coli* strain, screened with ampicillin, and positive clones were obtained. Plasmids were extracted, and clones were identified by enzyme digestion to obtain the CD70 CAR-2A-CD47 ECD lentiviral packaging vector.
  • 293T cells intended for virus packaging were cultured in a cell culture incubator at 37°C and 5% CO2 .
  • the culture medium used was DMEM containing 10% Gibco fetal bovine serum.
  • the day before the actual virus packaging the cultured 293T cells were passaged in T75 cell flasks at a rate of 1 ⁇ 107 cells. Lentiviral packaging began when the 293T cells reached 70-80% confluence and were evenly distributed in the culture flasks.
  • the lentiviral vector is lentiviral vector 1, 2, or 3 prepared above.
  • transfection reagent dilution (centrifuge tube 2) to the plasmid DNA solution (centrifuge tube 1) and mix thoroughly immediately. Note that the order of addition is very important. Incubate the transfection mixture at room temperature for 15–20 minutes. Discard the old culture medium in the culture flask containing the 293T cells, add 9 ml of fresh DMEM medium, then add 1 ml of the incubated transfection mixture, and gently aspirate to mix. Incubate the cells at 37°C in a 5% CO2 cell culture incubator for 6 hours, then discard the culture medium in the flask and add 15 ml of fresh culture medium.
  • cell culture supernatant was collected and 15 ml of fresh culture medium was added. 72 hours after transfection, a total of 30 ml of cell culture supernatant was collected. The supernatant was centrifuged at 1000 g for 8 min, filtered through a 0.45 nm pore size filter to remove cell debris, and then transferred to an ultracentrifuge tube.
  • TRAC-sgRNA/B2m-sgRNA/HLA-DRA-sgRNA Dissolve TRAC-sgRNA/B2m-sgRNA/HLA-DRA-sgRNA in a 100 nmol/ ⁇ l solution.
  • the sgRNA consists of a target gene recognition sequence and a constant 3' end sequence serving as a scaffold. This constant sequence is shown in SEQ ID NO:4: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU.
  • the recognition sequences of each sgRNA used are as follows (T is U in RNA):
  • CAR-T cells overexpressing tIL7Ra * and triple-knockout obtained using viral vector 1 are referred to as "UCAR-T-tIL7Ra*” cells.
  • CAR-T cells overexpressing CD47 and tIL7Ra * and triple-knockout obtained using viral vector 2 are referred to as "SAP UCAR-T” cells in this paper.
  • CAR-T cells overexpressing CD47 ECD and triple-knockout obtained using viral vector 3 are referred to as "CD47 UCAR-T” cells in this paper.
  • Figure 1 shows the CAR positivity rate detection results for mock T, UCAR-T-tIL7Ra*-1, and UCAR-T-tIL7Ra*-2, respectively.
  • Figure 6 shows the expression of CAR and CD47 in CD47 UCAR-T cells by flow cytometry 2 days after CD47 UCAR-T cell knockout.
  • Figure 7 shows the detection of CAR and CD47 expression in SAP UCAR-T cells by flow cytometry.
  • Transduced triple knockout (tKO) UCAR-T cells were collected, resuspended in cytokine-free complete medium at a density of 1.0 ⁇ 106 cells/ml, and seeded in 24-well cell culture plates. After 72 hours, the cells were harvested into FACS tubes and washed with pre-chilled flow cytometry buffer (PBS containing 5% FBS).
  • Immobilization and permeation reagent A (100 ⁇ L; Life Technologies) was added to the cells, gently swirled, and incubated at room temperature for 3 minutes. Then, while continuing to swirl, 3 mL of frozen methanol was slowly added to the tube. The cells were then incubated at 4°C for 10 minutes. Subsequently, the methanol was removed by centrifugation, and the cells were washed twice with pre-chilled flow cytometry buffer. Then, immobilization and permeation reagent B (100 ⁇ L; Life Technologies) and 5 ⁇ L of anti-STAT antibody were added to the cells. The cells were gently vortexed and incubated at room temperature in the dark for 30 minutes. The cells were then washed again with pre-chilled flow cytometry buffer and immediately analyzed.
  • Figure 3 shows the results in the presence of IL-2.
  • Figure 4 shows the results in the absence of IL-2.
  • the proliferation of UCAR-T-tIL7Ra* was still inferior to that of mock CAR-T in the presence of IL-2, the survival of UCAR-T-tIL7Ra* was significantly improved in the absence of the factor (the results for UCAR-T-tIL7Ra*-1 and UCAR-T-tIL7Ra*-2 were similar).
  • the level of intracellular phosphorylated Zap70 in various CAR-T cells was detected by intracellular flow cytometry.
  • the level of phosphorylated Zap70 in SAP UCAR-T cells did not change significantly compared with other groups, indicating that the SAP structure does not affect the Zap70-related signaling pathway.
  • Transduced tKO UCAR-T cells were collected, resuspended in cytokine-free complete medium at a density of 1.0 ⁇ 106 cells/ml, and seeded in 24-well cell culture plates. After 72 hours, the cells were harvested into FACS tubes and washed with pre-chilled flow cytometry buffer (PBS containing 5% FBS).
  • Immobilization and permeation reagent A (100 ⁇ L; Life Technologies) was added to the cells, gently swirled, and incubated at room temperature for 3 minutes. Then, while continuing to swirl, 3 ml of frozen methanol was slowly added to the tube. The cells were then incubated at 4°C for 10 minutes. Subsequently, the methanol was removed by centrifugation, and the cells were washed twice with pre-chilled flow cytometry buffer. Then, immobilization and permeation reagent B (100 ⁇ L; Life Technologies) and 5 ⁇ L of anti-STAT antibody were added to the cells. The cells were gently vortexed and incubated at room temperature in the dark for 30 minutes. The cells were then washed again with pre-chilled flow cytometry buffer and immediately analyzed.
  • UCAR-T cells refers to CAR-T cells that have undergone triple knockout but have not overexpressed CD47 or tIL7Ra*.
  • Figure 9A shows the results in the presence of IL-2.
  • Figure 9B shows the results in the absence of IL-2.
  • the SAP protein does not directly promote CAR-T cell proliferation, but rather increases CAR-T cell persistence, meaning that the survival ability of CAR-T cells is significantly improved.
  • tumor cells were seeded into 96-well plates at a rate of 2 ⁇ 104 cells/well, with a volume of 60 ⁇ L per well.
  • CAR-T cells were seeded into 96-well plates at three effector-to-target ratios of 1:1, 3:1, and 9:1, with a final culture medium volume of 200 ⁇ L per well. Blank wells and control wells with the highest sample value were also included.
  • the cell killing effect was observed under a microscope.
  • the 96-well plates were then centrifuged at 1000 ⁇ g for 10 min. 100 ⁇ L of culture medium was aspirated from the 96-well plates for subsequent cytokine detection. The remaining culture medium was discarded.
  • CD47 UCAR-T cells still exhibit good tumor-killing ability in the presence of NK cells and M1 cells.
  • Apoptosis was detected using the Annexin V-PE/7-AAD apoptosis assay kit.
  • One x 105 NK cells were harvested and stained with CellTrace CFSE.
  • NK cells and target cells were co-cultured at a 1:1 ratio in 200 ⁇ L of medium containing anti-CD107a antibody. After 1 h of co-culture, 100 ⁇ g/ml of monensin was added to the medium. After another 4 h of culture, cells were collected and washed twice with PBS. After washing, cells were resuspended in PBS for flow cytometry analysis. Degranulated NK cells were identified as the CFSE + CD107a + population.
  • overexpression of CD47 can inhibit the immune rejection of UCAR-T cells by NK cells.
  • Apoptosis was detected using the Annexin V-PE/7-AAD apoptosis assay kit.
  • One x 105 macrophages and target cells were stained with different fluorescent dyes (e.g., Celltrace CFSE or Celltrace Far Red).
  • Macrophages and target cells were co-cultured at a 1:1 ratio in 200 ⁇ L of medium. After 6 h of co-culture, cells were collected and washed twice with PBS. After washing, cells were resuspended in PBS for flow cytometry analysis. Cells phagocytosed by macrophages were identified as the portion positive for both fluorescent dyes.
  • overexpression of CD47 can inhibit the immune rejection of UCAR-T cells by M1 macrophages.
  • Antibody incubation Incubate the membrane with blocking buffer containing primary antibody overnight at 4°C. Wash the membrane three times with TBST for 5 min each time. Incubate the membrane with blocking buffer containing secondary antibody at room temperature for 2 h. Wash the membrane three times with TBST for 5 min each time.
  • Figure 13 shows the phosphorylation levels of STAT5 and BCL2 proteins in CAR-T cells with and without cytokines.
  • Western blot results indicate that, with cytokines, the expression levels of BCL2 protein in CAR-T cells across all groups were comparable; however, without cytokines, only SAP UCAR-T maintained a certain level of BCL2 expression, demonstrating its strong anti-apoptotic ability without cytokines.
  • C-NKG mice are a type of severely immunodeficient mouse independently developed by Cyagen Biosciences by knocking out the Il2rg gene in a NOD-Scid background strain.
  • C-NKG mice lack mature T, B, and NK immune cells, have reduced complement activity, and exhibit weak phagocytosis of human cells by macrophages. They can be efficiently transplanted with human hematopoietic stem cells (HSCs), peripheral blood mononuclear cells (PBMCs), patient-derived xenografts (PDXs), or adult stem cells and tissues. They are currently recognized as an excellent model for research on high levels of immunodeficiency and can be used in areas such as tumors, immunity, autoimmune diseases, immunotherapy vaccines, GvHD, transplantation, and safety assessment.
  • HSCs human hematopoietic stem cells
  • PBMCs peripheral blood mononuclear cells
  • PDXs patient-derived xenografts
  • adult stem cells and tissues are currently recognized as an
  • mice Six-week-old female C-NKG mice were purchased from Cyagen and housed at the Experimental Animal Center of Nanjing Normal University. Once the mice were stable, 1 ⁇ 107 PBMC cells (200 ⁇ L) were infused via the tail vein. From the day of infusion, 100 ⁇ L of blood was collected every 7 days via the tail vein and placed in an anticoagulant tube. Whole blood flow cytometry was used to detect the percentage of CD45+ cells in the blood using human CD45+ antibodies to assess the development of the human immune system in the mice.
  • mice 5 ⁇ 106 CAR-T cells were infused into the mice, namely the TRAC-/-CAR-T group, the UCAR-T group, the CD47 UCAR-T group, and the SAP UCAR-T group.
  • Collect 100 ⁇ L of blood from the mouse tail vein into an anticoagulant tube Transfer the blood sample from the anticoagulant tube to a 1.5 ml centrifuge tube, centrifuge at 1000 ⁇ g for 10 min, and discard the supernatant. Add 1 ml of erythrocyte lysis buffer to the centrifuge tube and wait 5-10 min until the liquid in the centrifuge tube becomes clear. Centrifuge at 1000 ⁇ g for 10 min and discard the supernatant. Resuspend the bottom pellet in 100 ⁇ L of PBS, and then add the desired flow cytometry antibody. After incubation for 15 min, without washing the cells, directly add 400 ⁇ L of PBS to resuspend the cells and analyze them using flow cytometry.
  • SAP UCAR-T cells exhibited the highest CAR-T cell persistence, indicating that they can prolong the survival of CAR-T cells.

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Abstract

L'invention concerne un lymphocyte T à immunogénicité réduite, en particulier une cellule CAR-T ayant des capacités d'auto-activation et de défense, et son procédé de préparation. Le lymphocyte T a une immunogénicité réduite, de telle sorte que sa résistance aux cellules NK et aux macrophages est améliorée, et les capacités de survie et de prolifération du lymphocyte T, en particulier de la cellule CAR-T, chez un hôte sont améliorées.
PCT/CN2025/082078 2024-05-20 2025-03-12 Lymphocyte t amélioré et son utilisation Pending WO2025241665A1 (fr)

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CN202410626978.2A CN121046318A (zh) 2024-05-20 一种改进的t细胞及其用途

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