Viral vectors selectively targeting immune cells, methods of making and use thereof

Abstract

The application provides a virus carrier selectively targeting immune cells, a preparation method and application thereof. Specifically, the application provides a lentivirus envelope protein mutant with substantially removed LDL-R binding capacity and retained cell fusion function, and uses a targeting molecule specifically targeting immune cells, so that a lentivirus carrier precisely targeting T / NK cells is constructed, and the lentivirus carrier can be used for generating CAR-T cells in vivo. The application has application prospects in the field of tumor immunotherapy.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to viral vectors that selectively target immune cells, their preparation methods, and their applications. Background Technology

[0002] Chimeric antigen receptor T-cell (CAR-T) therapy has demonstrated remarkable clinical efficacy in hematologic malignancies, particularly in the treatment of B-cell tumors and multiple myeloma. Furthermore, multiple clinical studies have shown that CAR-T therapy also holds great promise in the field of autoimmune diseases (AIDs), potentially achieving deep remission or even functional cure by precisely eliminating pathogenic B-cell subsets and restoring immune homeostasis.

[0003] While CAR-T therapy has shown transformative efficacy in hematological malignancies and autoimmune diseases, the inherent limitations of its traditional in vitro preparation methods severely restrict its widespread application. Furthermore, the technology relies on complex, personalized manufacturing processes, with an average "vein-to-vein" time of 2-4 weeks (including leukocyte apheresis, T-cell activation, lentiviral transduction, amplification, and quality control), causing some patients to lose their treatment opportunities due to disease progression. To overcome these bottlenecks, the CAR-T cell therapy industry is accelerating the exploration of next-generation technological pathways.

[0004] In vivo CAR technology is a technique that directly delivers the CAR-encoding gene into the patient's body. Using viral or non-viral vectors, the CAR gene is transduced into the patient's own T cells or other immune cells, programming them into CAR-T cells or CAR-modified immune cells to achieve therapeutic goals. In vivo CAR technology skips the in vitro T cell isolation, activation, transduction, and expansion steps of traditional CAR-T processes, fundamentally shortening the treatment cycle. Because the vectors can be mass-produced and standardized, treatment costs are significantly reduced. Furthermore, CAR-T cells generated naturally in the body reduce the depletion problems that may occur with in vitro culture and avoid the lymphocyte clearance pretreatment required before reinfusion in traditional CAR-T therapy. Therefore, in vivo CAR technology holds the promise of becoming a truly revolutionary next-generation immune cell therapy, opening up new frontiers in gene therapy for cancer and autoimmune diseases.

[0005] The development of in vivo CAR technology mainly relies on two major technical routes: viral vectors and non-viral vectors. The non-viral vector route, represented by lipid nanoparticles (LNPs), achieves transient CAR expression through mRNA delivery. The LNP-mRNA platform avoids the risk of insertional mutations due to the non-integration of mRNA into the genome, and has lower production costs, making it compatible with existing mRNA vaccine production systems. However, the transient expression characteristic of LNP-mRNA technology means that in vivo CAR-T therapies based on it have poor sustained efficacy, requiring multiple doses and potentially triggering adaptive immune responses.

[0006] Currently, lentiviral vectors are the primary representative of viral vector technology. By modifying lentiviral vectors, precise targeting of immune cells (especially T cells and NK cells) can be achieved, enabling highly efficient transduction. Lentiviral vector technology can integrate genes into the host genome, achieving long-term stable expression, potentially allowing for sustained responses with a single dose. Furthermore, the lentiviral vector production process is mature and can be mass-produced in accordance with GMP standards. However, due to their gene integration characteristics, lentiviral vectors may carry a certain risk of gene insertion mutations. Modified lentiviral vectors may also exhibit some immunogenicity, limiting repeated dosing. Therefore, compared to LNP-mRNA technology, lentiviral vectors have advantages in efficiency and durability, but overcoming safety bottlenecks remains.

[0007] Lentiviral vectors are the most commonly used vector form in traditional CAR-T preparation processes, and most approved CAR-T products use lentiviral vector technology. The commonly used lentiviral vector system is the third-generation system, which includes four types of plasmids: (1) "transfer plasmid" which encodes the nucleotide sequence delivered to the target cell by the lentiviral vector, such as the coding sequence of the CAR molecule; (2) two "packaging plasmids", one of which encodes Gag-Pol and the other encodes Rev; (3) "envelope plasmid" which encodes the envelope protein (Env), among which VSV-G (vesicular stomatitis virus glycoprotein) is the most commonly used lentiviral vector envelope protein.

[0008] VSV-G envelope protein pseudotyping endows lentiviral vectors with broad tropism and stability, making them resistant to centrifugation concentration and freeze-thaw treatment. VSV-G mediates viral adsorption by binding to low-density lipoprotein receptor (LDL-R) family proteins on the host cell surface. Extensive expression of LDL-R allows VSV-G pseudotyped lentiviral vectors to infect various cell types, including T cells. After adsorption, the lentiviral vector enters the cell via clathrin-mediated endocytosis, forming endosomes. With endosome acidification (pH 5.0-6.0), VSV-G undergoes a conformational change, exposing a fusion peptide, inserting into the endosome membrane, and inducing fusion of the viral envelope with the endosome membrane, thereby releasing the viral core (containing viral RNA and reverse transcriptase, etc.) into the cytoplasm. The reverse transcriptase in the viral core reverse transcribes the RNA into DNA, which then enters the cell nucleus and integrates into the host genome, completing the transduction process of the lentiviral vector. Therefore, receptor binding and mediating membrane fusion are two important biological functions of VSV-G envelope glycoprotein.

[0009] Traditional lentiviral vectors, due to their broad tropism, cannot selectively transduce T cells or other immune cells in vivo. Therefore, to achieve in vivo application, it is first necessary to modify the envelope glycoproteins of lentiviral vectors to precisely target target cells and avoid transduction to non-target cells. Lentiviral vectors for in vivo application may also require surface modifications to reduce recognition and clearance by the immune system, such as avoiding complement-mediated inactivation or innate immune responses. Given the complexity of the in vivo environment, it is also necessary to optimize the in vivo delivery efficiency of lentiviral vectors to ensure stability in the bloodstream, resistance to serum protein adsorption and non-specific clearance, and effective infection of target cells.

[0010] Therefore, there is a need in this field to develop a viral vector that can selectively target immune cells. Summary of the Invention

[0011] The purpose of this invention is to provide a viral vector that selectively targets immune cells, its preparation method, and its application.

[0012] In a first aspect of the invention, a viral envelope protein mutant is provided, wherein the extracellular domain of the mutant has mutations at positions 352, 354, 47, and / or 48 relative to the extracellular domain of the wild-type envelope protein.

[0013] The amino acid sequence of the extracellular domain of the wild-type envelope protein is shown in SEQ ID NO:1.

[0014] In another preferred embodiment, the mutation at positions 352, 354, 47 and / or 48 relative to the wild-type envelope protein means that, after a global alignment with SEQ ID NO:1, the position of the mutation site in the extracellular domain of the envelope protein mutant corresponds to positions 352, 354, 47 and / or 48 of SEQ ID NO:1.

[0015] In another preferred embodiment, the mutation is an insertion, deletion, or substitution.

[0016] In another preferred embodiment, the extracellular domain of the envelope protein mutant has one or more amino acid mutations selected from the group consisting of, relative to the extracellular domain of the wild-type envelope protein:

[0017] T352V, R354Q, K47Q, T48S.

[0018] In another preferred embodiment, the extracellular domain of the envelope protein mutant has an amino acid mutation selected from the group consisting of, relative to the extracellular domain of the wild-type envelope protein:

[0019] 1) T352V and R354Q;

[0020] 2) K47Q and T48S;

[0021] 3) K47Q, T48S, and R354Q; and

[0022] 4)R354Q.

[0023] In another preferred embodiment, the envelope protein mutant comprises the amino acid sequence shown in SEQ ID NO:1, or an amino acid sequence having at least 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in SEQ ID NO:1.

[0024] In another preferred embodiment, apart from the mutation described above, the remaining amino acids of the extracellular domain of the envelope protein mutant differ from the amino acid sequence shown in SEQ ID NO:1 by one, two, three, four, or five amino acids.

[0025] In another preferred embodiment, apart from the mutation described above, the remaining amino acids of the extracellular domain of the envelope protein mutant are identical to the sequence shown in SEQ ID NO:1.

[0026] In another preferred embodiment, the envelope protein mutant comprises an amino acid sequence having at least 95%, 96%, 97%, 98%, or 99% identity with the sequence shown in SEQ ID NO: 6, 7, 8, or 9.

[0027] In another preferred embodiment, the envelope protein mutant comprises the amino acid sequence shown in SEQ ID NO:6, 7, 8 or 9.

[0028] In another preferred embodiment, the amino acid sequence of the extracellular domain of the said envelope protein mutant is shown in SEQ ID NO:6, 7, 8 or 9.

[0029] In another preferred embodiment, the envelope protein mutant, relative to the wild-type envelope protein, eliminates or substantially eliminates LDL-R binding function.

[0030] In another preferred embodiment, the mutant of the envelope protein retains the function of mediating cell membrane fusion relative to the wild-type envelope protein.

[0031] In another preferred embodiment, the envelope protein is an envelope glycoprotein of the Cocal virus.

[0032] In another preferred embodiment, the envelope protein mutant further comprises a transmembrane region and an intracellular domain.

[0033] In another preferred embodiment, the transmembrane region of the said envelope protein mutant is wild-type, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the wild-type transmembrane region.

[0034] In another preferred embodiment, the intracellular domain of the envelope protein mutant is wild-type, or has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the wild-type intracellular domain.

[0035] In a second aspect of the invention, an isolated polynucleotide is provided, said polynucleotide encoding a viral envelope protein mutant as described in the first aspect of the invention.

[0036] In a third aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the second aspect of the invention.

[0037] In another preferred embodiment, the vector is a plasmid.

[0038] In a fourth aspect of the invention, an antibody targeting CD7 or an antigen-binding fragment thereof is provided, wherein the heavy chain variable region of the antibody includes HCDR1, HCDR2, and HCDR3, wherein:

[0039] The amino acid sequence of HCDR1 is shown in SEQ ID NO:13;

[0040] The amino acid sequence of HCDR1 is shown in SEQ ID NO:14;

[0041] The amino acid sequence of HCDR1 is shown in SEQ ID NO:15; and

[0042] The light chain variable region of the antibody includes LCDR1, LCDR2, and LCDR3, wherein:

[0043] The amino acid sequence of LCDR1 is shown in SEQ ID NO:16;

[0044] The amino acid sequence of LCDR1 is shown in SNS;

[0045] The amino acid sequence of LCDR1 is shown in SEQ ID NO:17.

[0046] In another preferred embodiment, the antigen-binding fragment is a single-chain antibody.

[0047] In another preferred embodiment, the single-chain antibody amino acid sequence is as shown in SEQ ID NO:12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0048] The present invention also provides a polynucleotide encoding the antibody or an antigen-binding fragment thereof, and a vector containing the polynucleotide.

[0049] In a fifth aspect of the invention, a recombinant viral particle is provided, the recombinant viral particle comprising a viral envelope protein mutant as described in the first aspect of the invention.

[0050] In another preferred embodiment, the recombinant viral particle further includes a targeting portion.

[0051] In another preferred embodiment, the recombinant viral particles are pseudo-lentiviruses.

[0052] In another preferred embodiment, the viral particles further comprise a target nucleic acid molecule.

[0053] In another preferred embodiment, the target nucleic acid molecule is selected from the group consisting of: nucleic acids encoding therapeutic proteins, siRNA, shRNA, non-coding RNA (e.g., guide RNA of the CRISPR system), or combinations thereof.

[0054] In another preferred embodiment, the target nucleic acid molecule is a nucleic acid encoding a chimeric antigen receptor.

[0055] In another preferred embodiment, the chimeric antigen receptor includes an extracellular binding domain that specifically binds to tumor cell surface antigens.

[0056] In another preferred embodiment, the tumor cell surface antigens include: CD20, CD19, BCMA, CD22, CD33, CD123, CLL-1 (CLEC12A), FLT3 HER2, EGFR, MUC1, GD2, CEA, GUCY2C, GPC3, Claudin 18.2, TIM3, DLL1, CD70, Siglec-6, MSLN, B7-H3, and / or MUC1.

[0057] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:22, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0058] In another preferred embodiment, the viral particle further includes a targeting portion.

[0059] In another preferred embodiment, the targeting portion binds to immune cells, T cells, B cells, NK cells, dendritic cells, neutrophils, macrophages, and cancer cells.

[0060] In another preferred embodiment, the target portion specifically binds to molecules on the surface of immune cells.

[0061] In another preferred embodiment, the target portion specifically binds to CD7, CD2, CD3, CD4, CD5, CD8, CD58, CD80, and CD86.

[0062] In another preferred embodiment, the target portion specifically binds to CD7.

[0063] In another preferred embodiment, the targeting portion comprises an antibody or an antigen-binding fragment thereof as described in the fourth aspect of the invention.

[0064] In another preferred embodiment, the targeting portion is a single-chain antibody.

[0065] In another preferred embodiment, the targeted amino acid sequence is as shown in SEQ ID NO:12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0066] In another preferred embodiment, the recombinant viral particles do not bind to or substantially do not bind to LDL-R.

[0067] In another preferred embodiment, the recombinant viral particles have the function of mediating cell membrane fusion.

[0068] In a sixth aspect of the invention, a transfection composition is provided, the transfection composition comprising:

[0069] 1) An envelope plasmid containing polynucleotides as described in the second aspect of the present invention;

[0070] 2) Packaging plasmid, which is used for packaging lentiviral vectors.

[0071] In another preferred embodiment, the packaging plasmid includes a plasmid containing the Gag-Pol encoding gene and a plasmid containing the Rev encoding gene.

[0072] In another preferred embodiment, the reagent further comprises a transfer plasmid containing a target nucleic acid molecule.

[0073] In another preferred embodiment, the target nucleic acid molecule is selected from the group consisting of: nucleic acids encoding therapeutic proteins, siRNA, shRNA, non-coding RNA (e.g., guide RNA of the CRISPR system), or combinations thereof.

[0074] In another preferred embodiment, the target nucleic acid molecule is a nucleic acid encoding a chimeric antigen receptor.

[0075] In another preferred embodiment, the chimeric antigen receptor includes an extracellular binding domain that specifically binds to tumor cell surface antigens.

[0076] In another preferred embodiment, the tumor cell surface antigens include: CD20, CD19, BCMA, CD22, CD33, CD123, CLL-1 (CLEC12A), FLT3 HER2, EGFR, MUC1, GD2, CEA, GUCY2C, GPC3, Claudin 18.2, TIM3, DLL1, CD70, Siglec-6, MSLN, B7-H3, and / or MUC1.

[0077] In another preferred embodiment, the amino acid sequence of the chimeric antigen receptor is as shown in SEQ ID NO:22, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0078] In another preferred embodiment, the reagent further comprises a targeting plasmid containing a polynucleotide encoding a targeting moiety.

[0079] In another preferred embodiment, the targeting portion binds to immune cells, T cells, B cells, NK cells, dendritic cells, neutrophils, macrophages, and cancer cells.

[0080] In another preferred embodiment, the target portion specifically binds to molecules on the surface of immune cells.

[0081] In another preferred embodiment, the target portion specifically binds to CD7, CD2, CD3, CD4, CD5, CD8, CD58, CD80, and CD86.

[0082] In another preferred embodiment, the target portion specifically binds to CD7.

[0083] In another preferred embodiment, the targeting portion comprises an antibody or an antigen-binding fragment thereof as described in the fourth aspect of the invention.

[0084] In another preferred embodiment, the targeting portion is a single-chain antibody.

[0085] In another preferred embodiment, the targeted amino acid sequence is as shown in SEQ ID NO:12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0086] In a seventh aspect of the invention, a host cell is provided, which is obtained by transfection with the transfection composition as described in the sixth aspect of the invention.

[0087] In another preferred embodiment, the host cell is a eukaryotic cell or a prokaryotic cell.

[0088] In another preferred embodiment, the host cell is a plant cell, an insect cell, or an animal cell, preferably a mammalian cell.

[0089] In another preferred embodiment, the host cell is a HEK-293T cell.

[0090] In an eighth aspect of the present invention, a method for preparing recombinant virus particles as described in the fifth aspect of the present invention is provided, comprising the steps of:

[0091] (a) Culturing host cells as described in the seventh aspect of the invention under suitable conditions; and

[0092] (b) Isolate the recombinant virus particles from the culture.

[0093] In another preferred embodiment, the method further includes purifying or concentrating the recombinant viral particles.

[0094] In a ninth aspect of the present invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0095] (a) the recombinant viral particles as described in the fifth aspect of the invention; and

[0096] (b) Pharmaceutically acceptable carriers.

[0097] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral / nasal spray and nebulized inhalation.

[0098] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid, solid, or gel.

[0099] In another preferred embodiment, the formulation is a liquid formulation.

[0100] In another preferred embodiment, the dosage form of the preparation is an injection.

[0101] In a tenth aspect of the invention, a method for delivering a target nucleic acid molecule to a target cell is provided, comprising the steps of contacting a recombinant viral particle containing the target nucleic acid molecule, as described in the fifth aspect of the invention, with the target cell thereby delivering the target nucleic acid molecule.

[0102] In an eleventh aspect of the invention, the use of recombinant viral particles as described in the fifth aspect of the invention, transfection compositions as described in the sixth aspect of the invention, host cells as described in the seventh aspect of the invention, or pharmaceutical compositions as described in the ninth aspect of the invention in the preparation of medicaments for the prevention and / or treatment of diseases is provided.

[0103] In another preferred embodiment, the disease or symptom is selected from: cancer, autoimmune diseases.

[0104] In another preferred embodiment, the cancers include leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, stomach cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, and angiosarcoma.

[0105] In another preferred embodiment, the cancer is a hematologic malignancy.

[0106] In another preferred embodiment, the hematologic malignancy includes plasma cell malignancies and B-cell malignancies.

[0107] In another preferred embodiment, the hematologic malignancy includes multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma, marginal zone B-cell lymphoma, and / or B-cell chronic lymphocytic leukemia (CLL).

[0108] In another preferred embodiment, the autoimmune disease includes rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, ANCA-associated vasculitis (AAV), systemic lupus erythematosus (SLE), lupus nephritis, nephrotic syndrome, thrombocytopenia, graft-versus-host disease, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0109] In a twelfth aspect of the invention, a method for preventing and / or treating a disease is provided, the method comprising: administering to a desired subject recombinant viral particles as described in the fifth aspect of the invention, a transfection composition as described in the sixth aspect of the invention, a host cell as described in the seventh aspect of the invention, or a pharmaceutical composition as described in the ninth aspect of the invention, or a combination thereof.

[0110] In another preferred embodiment, the disease or condition is selected from: cancer, autoimmune diseases.

[0111] In another preferred embodiment, the cancers include leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, stomach cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, and angiosarcoma.

[0112] In another preferred embodiment, the cancer is a hematologic malignancy.

[0113] In another preferred embodiment, the hematologic malignancy includes plasma cell malignancies and B-cell malignancies.

[0114] In another preferred embodiment, the hematologic malignancy includes multiple myeloma, follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma, marginal zone B-cell lymphoma, and / or B-cell chronic lymphocytic leukemia (CLL).

[0115] In another preferred embodiment, the autoimmune disease includes rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, ANCA-associated vasculitis (AAV), systemic lupus erythematosus (SLE), lupus nephritis, nephrotic syndrome, thrombocytopenia, graft-versus-host disease, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0116] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0117] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0118] Figure 1 A schematic diagram of the lentiviral vector structure for in vivo CAR-T is shown.

[0119] Figure 2 The expression of CD7 and LDL-R on the surface of supT1 and Raji cells was shown by flow cytometry.

[0120] Figure 3A The flow cytometry results of lentiviral vector samples transduced with different envelope mutants in supT1 / Raji cells are shown (linear axes display GFP percentage values).

[0121] Figure 3B The flow cytometry results (logarithmic axis shows GFP percentage values) of lentiviral vector samples transduced into supT1 / Raji cells using different envelope mutants are shown.

[0122] Figure 4 The flow cytometry results of lentiviral vector samples transduced with different CD7 targeting molecules in supT1 / Raji cells are shown (GFP% values ​​are displayed on linear (A) or logarithmic (B) axes, respectively).

[0123] Figure 5 The results of flow cytometry analysis of virus samples transduced into supT1 or Raji cells are shown.

[0124] Figure 6 The flow cytometry results of the PBMC sample before viral transduction are shown.

[0125] Figure 7A and 7B The results of flow cytometry analysis of PBMC samples 3 days after viral transduction are shown.

[0126] Figure 8 The flow cytometry results of PBMC samples were shown 7 days after viral transduction. Detailed Implementation

[0127] Through extensive and in-depth research, the inventors have developed for the first time a viral vector that selectively targets immune cells, its preparation method, and its applications. This invention involves screening and constructing a lentiviral envelope protein mutant that essentially removes LDL-R binding ability while retaining cell fusion function. Combined with a targeting molecule specifically targeting immune cells, a lentiviral vector capable of precisely targeting T / NK cells was constructed, which can be used to generate CAR-T cells in vivo. Based on this, the invention was completed.

[0128] the term

[0129] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0130] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0131] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0132] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0133] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0134] The “identity” or “percentage of identity” of an amino acid sequence or nucleic acid sequence refers to the percentage of amino acid residues / nucleotides in the candidate sequence that are identical to the specific sequence shown in this specification after the candidate sequence is aligned with the specific sequence shown in this specification and, if necessary, vacancy is introduced to achieve the maximum percentage of sequence identity, without considering any conserved substitutions as part of the sequence identity, and the amino acid residues / nucleotides in the candidate sequence are identical to those in the specific sequence shown in this specification.

[0135] Viral envelope protein mutant

[0136] As used herein, the terms "viral envelope protein mutant," "enveloped protein mutant," and "enveloped protein variant" all refer to the viral envelope protein mutant obtained by mutation of the extracellular domain of the Cocal wild-type viral envelope protein shown in SEQ ID NO:1, provided in the first aspect of this invention.

[0137] In a preferred embodiment, the extracellular domain of the envelope protein mutant of the present invention has an amino acid mutation selected from the group consisting of, relative to the extracellular domain of the wild-type viral envelope protein:

[0138] 1) T352V and R354Q;

[0139] 2) K47Q and T48S;

[0140] 3) K47Q, T48S, and R354Q; and

[0141] 4)R354Q.

[0142] The viral envelope protein mutant of the present invention removes the LDL-R binding ability of the wild-type envelope protein while retaining its function of fusing target cells.

[0143] Targeted portion

[0144] The viral particles of the present invention may include a targeting portion that specifically binds to cell surface molecules, thereby enabling the viral particles to specifically bind to and fuse with the cell.

[0145] The targeting portion of this invention can be any element with specific binding capability, including but not limited to antibodies, antigen-binding fragments of antibodies (such as scFv, Fab), natural ligands, and other peptides. In one embodiment, the targeting portion of this invention is a single-chain antibody (scFv).

[0146] The targeting portion of the present invention preferably binds to molecules on the surface of immune cells (such as T cells or NK cells), thereby specifically delivering a target nucleic acid molecule (e.g., a chimeric antigen receptor-encoded nucleic acid) for immunotherapy into the cell. In one embodiment, the targeting portion of the present invention specifically binds to CD7.

[0147] Recombinant viral particles

[0148] As used herein, the terms "recombinant viral particle," "recombinant viral vector," "viral vector of the present invention," and "viral particle of the present invention" all refer to the viral particle containing the envelope protein mutant of the present invention provided in the fifth aspect of the present invention.

[0149] The recombinant viral particles of this invention remove the LDL-R binding ability of the wild-type envelope protein by mutating the envelope protein. Simultaneously, the recombinant viral particles of this invention may contain a targeting portion, thereby enabling the viral particles to specifically bind to target cells and deliver the target nucleic acid molecule, thus achieving the purpose of disease treatment. For example, the recombinant viral particles of this invention can be used in vivo to specifically deliver chimeric antigen receptor-expressing nucleic acids to CD7-positive T cells, thereby realizing the production of CAR-T cells in vivo.

[0150] Transfection Composition and Preparation Method

[0151] This invention also provides transfection compositions and methods for preparing the viral particles of this invention. Many methods are known in the art for packaging and producing lentiviral particles, such as four-plasmid co-transfection systems, and those skilled in the art can make appropriate selections as needed. The terms "transfection" or "transduction," etc., refer to the introduction of nucleic acids into cells via a vector.

[0152] The recombinant viral particles of the present invention can be packaged and produced by transfecting host cells with the transfection composition of the present invention. A suitable system for producing recombinant viral particles includes one or more of the following components: 1) host cells, including, for example, human cell lines such as HEK-293T cells, or insect cell lines; 2) nucleic acid or plasmid encoding a viral envelope protein mutant; 3) Gag-Pol-encoded nucleic acid, Rev-encoded nucleic acid, or plasmid; 4) target portion-encoded nucleic acid or plasmid; 5) target nucleic acid molecule, preferably driven by an operably linked promoter; and 6) a suitable culture system to support the production of recombinant viral particles.

[0153] In some implementations, the method further includes separating recombinant virus particles from the culture supernatant.

[0154] In some embodiments, the method further includes lysing the host cell and isolating recombinant viral particles from the cell lysis products.

[0155] In some implementations, the method further includes concentrating or purifying recombinant viral particles.

[0156] Therefore, the present invention also relates to a host cell for producing recombinant viral particles, said host cell comprising nucleic acid encoding a mutant viral envelope protein described in the present invention.

[0157] Target nucleic acid molecules

[0158] As used herein, the term "target nucleic acid molecule" refers to a foreign nucleic acid molecule delivered into target cells using the recombinant viral particles of the present invention. The target nucleic acid molecule can be any nucleic acid molecule for therapeutic, diagnostic, or other purposes. In some embodiments, the target nucleic acid molecule can be a nucleic acid molecule encoding a therapeutic polypeptide, such as a therapeutic polypeptide for treating tumors. In one embodiment, the target nucleic acid molecule encodes a chimeric antigen receptor.

[0159] Chimeric antigen receptors (CARs) comprise extracellular domains, transmembrane domains, and intracellular domains. The extracellular domains include target-specific binding elements (also known as antigen-binding domains). As used herein, "antigen-binding domain" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, single Fv fragment, or heavy chain variable region of a single-domain antibody with antigen-binding activity. In a preferred embodiment of the invention, the extracellular domain of the CAR provided by the invention comprises an antigen-binding domain targeting CD20, CD19, or BCMA, etc.

[0160] Pharmaceutical Composition

[0161] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned recombinant viral particles and a pharmaceutically acceptable carrier. Typically, these substances can be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0162] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0163] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0164] The main advantages of this invention include:

[0165] 1) This invention provides a mutated lentiviral envelope protein that has been screened and has had its LDL-R binding ability removed while retaining its cell fusion function, which can be used for the development of viral particles that selectively target cells.

[0166] 2) This invention provides an antibody that specifically binds to CD7 and scFv, which can be used as the targeting portion of lentiviral particles that selectively target T cells.

[0167] 3) The present invention also provides recombinant viral particles comprising the lentiviral envelope protein mutant and the CD7 targeting portion, and transfection compositions for preparing the recombinant viral particles, thereby enabling in vivo production of CAR-T cells.

[0168] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0169] Example 1. Screening for envelope protein mutants

[0170] Experimental objective and principle:

[0171] Because the COCV-G receptor LDL-R is widely expressed on the surface of various cells, the COCV-G pseudolentiviral vector exhibits broad tropism and is not suitable for the specific generation of CAR-T cells in vivo. To enable the COCV-G pseudolentiviral vector to precisely target target cells, it is necessary to mutate and remove COCV-G's LDL-R binding ability while retaining its fusion function, and then combine it with a targeting molecule anchored to the viral envelope to achieve precise targeting of target cells.

[0172] This embodiment screened the nine COCV-G mutants listed in Table 1. Since the ideal COCV-G mutant cannot bind to LDL-R, lentiviral vectors using only COCV-G mutants will not transduce any cells. However, using a lentiviral vector packaged with a COCV-G mutant and a CD7-targeting molecule simultaneously can transduce CD7-positive cells (e.g., supT1 cells) but not CD7-negative cells (e.g., Raji cells).

[0173] As an example, the lentiviral vector was prepared by transiently transfecting HEK293T cells with 4 or 5 plasmids. The 5 plasmids were: (1) pMDLg / pRRE encoding Gag-pol (plasmid number 1267, sequence source Addgene#12260), (2) pRSV-Rev encoding Rev (plasmid number 1266, sequence source Addgene#12253), (3) pMD2.G encoding VSV-G (plasmid number 1265, sequence source Addgene#12259), or the plasmids encoding COCV-G or its mutants listed in Table 1, (4) the plasmids encoding CD7 target molecules listed in Table 2, and (5) the plasmids encoding CAR molecules listed in Table 3 as examples.

[0174] Table 1 COCV-G and its mutants

[0175]

[0176] Table 2 CD7 Target Molecules

[0177] Seq.No. plasmid numbering name 12 4469 scFv25.CD7-binder 18 4472 muMT701.CD7-binder 19 4473 huMT701.CD7-binder 20 4463 CD7-binder

[0178] Table 3 shows examples of CAR molecules.

[0179]

[0180] Experimental methods:

[0181] 1. Preparation of lentiviral vectors

[0182] In this embodiment, lentiviral vectors were prepared using plasmid 1267 encoding Gag-pol, plasmid 1266 encoding Rev, plasmid 4220 encoding GCC-CAR.eGFP(Transfer), plasmid 4463 encoding a CD7 targeting molecule, and plasmids listed in Table 1 encoding COCV-G and its mutant (Env).

[0183] One day in advance, seed HEK293T cells in logarithmic growth phase into T75 flasks to achieve 80-90% cell confluence for virus packaging. Mix 4 or 5 plasmids in an appropriate ratio, for example, a 4-plasmid system 1267(Gag-pol):1266(Rev):4220(Transfer):4462(Env) = 1:3:4:2, or a 5-plasmid system 1267(Gag-pol):1266(Rev):4220(Transfer):4462(Env):4463(CD7-binder) = 1:3:4:2:2, for a total of 10-12 μg of plasmid. Then mix with 3 times the mass of PEI and incubate for 20 minutes before transfecting HEK293T cells.

[0184] The supernatant was harvested 48 hours after plasmid transfection, filtered through a 0.45 μm filter, and then concentrated using a 100 kD Amicon Ultra centrifuge tube (approximately 20–40 times volumetric concentration). Virus samples can be flash-frozen in liquid nitrogen and then stored at -80°C.

[0185] 2. Determination of P24 protein content in virus samples

[0186] In step 1, the harvested viral supernatant and viral concentrate samples were both detected using HTRF HIV P24 Detection Kits (Revvity, Cat. No. 64P24PEG). The specific experimental procedures were performed according to the product instructions. The brief steps are as follows: (1) Prepare a standard curve according to the instructions. The highest concentration of the standard curve is 10000 pg / ml (Std7), and then perform serial dilutions at a ratio of 1:2.4 (Std6-Std1); (2) Process the samples according to the instructions. If the P24 concentration is approximately 10000 pg / ml, pre-dilution is performed using culture medium; (3) After adding the standard and sample to the 96-well microplate, add 2 μl of d2 antibody and 2 μl of Eu Cryptate antibody to each well and gently shake to mix; (4) Cover with a sealing film and incubate at room temperature in the dark for 2 hours; (5) Detect the Eu Cryptate signal using a microplate reader, use four-parameter fitting to fit the standard curve, and calculate the P24 concentration of the sample.

[0187] 3. Transduction of viral samples into SupT1 and Raji cells

[0188] The steps are briefly as follows: (1) Thaw the concentrated virus sample from step 1 on ice and serially dilute it at a ratio of 1:3 (8 concentration gradients) using fresh culture medium. (2) Take supT1 and Raji cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in fresh culture medium to a suitable cell density, and then add 10,000 supT1 cells and 10,000 Raji cells to each well. (3) Then, add the serially diluted virus sample to the supT1 / Raji mixed cells at a volume of 280ul per well for virus transduction. (4) Three days after virus transduction, remove the cells for flow cytometry detection, use CD19 antibody to distinguish supT1 and Raji cells, and detect the virus transduction efficiency by GFP.

[0189] Experimental results:

[0190] like Figure 3A and 3B As shown in Figure ab, lentiviral vectors packaged with wild-type VSV-G or COCV-G can transduce supT1 (CD7-positive) and Raji (CD7-negative) cells, regardless of the presence or absence of CD7-targeting molecules. This is because both supT1 and Raji cells express LDL-R (such as...). Figure 2 As shown in the figure, this demonstrates that both wild-type VSV-G and COCV-G have normal LDL-R binding function, and that VSV-G or COCV-G dominates the binding and transduction function of lentiviral vectors to target cells.

[0191] like Figure 3A and 3B As shown in Figure c, using the VSV-G mutant encoded by plasmid 4462, the lentiviral vector could not effectively transduce supT1 or Raji cells when there was no CD7 targeting molecule; however, when there was a CD7 targeting molecule, the lentiviral vector could effectively transduce supT1 cells, but could not effectively transduce Raji cells. This is mainly because the VSV-G mutant lost its LDL-R binding function but retained its fusion function. The lentiviral vector only has the ability to bind to target cells and transduce when a CD7 targeting molecule is present.

[0192] like Figure 3A and 3B As shown in the middle dg, the COCV-G mutant mut01-04 could not effectively transduce supT1 or Raji cells, regardless of whether CD7 target molecules were present or absent. This may be because the mutation on COCV-G disrupted its fusion function, causing the lentiviral vector to lose its transduction ability. Even with CD7 target molecules providing binding ability to CD7-positive cells, it was still impossible to effectively transduce supT1 cells.

[0193] like Figure 3A and3B As shown in Figure 3c, the COCV-G mutant mut05-08 behaves similarly to the VSV-G mutant in Figure 3c. Without a CD7 targeting molecule, the lentiviral vector cannot effectively transduce supT1 or Raji cells; however, with a CD7 targeting molecule, the lentiviral vector can effectively transduce supT1 cells but cannot effectively transduce Raji cells. This indicates that the COCV-G mutant mut05-08 eliminates LDL-R binding ability while effectively preserving fusion function.

[0194] like Figure 3A and 3B As shown in Figure 1, high concentrations of the COCV-G mutant mut09 transduced supT1 cells in the absence of CD7 targeting molecules, which may be because the COCV-G mutant mut09 failed to completely eliminate the binding ability of LDL-R.

[0195] Therefore, in this example, the COCV-G mutant mut05-08 successfully eliminated LDL-R binding ability while retaining fusion function.

[0196] Example 2. Screening for CD7-targeting molecules

[0197] Experimental objective and principle:

[0198] In Example 1, mutations yielded COCV-G mutants that lost LDL-R binding ability but retained fusion function. Lentiviral vectors packaged with these COCV-G mutants require a target molecule to provide target cell binding function in order to possess full transduction capability. In this example, CD7 was selected as the cellular target for demonstration purposes. Suitable CD7 target molecules were screened to enable lentiviral vectors packaged with COCV-G mutants that had lost LDL-R binding ability to regain the ability to recognize target cells and restore transduction function.

[0199] Experimental methods:

[0200] 1. Preparation of lentiviral vectors

[0201] In this example, lentiviral vectors were prepared using plasmid 1267 encoding Gag-pol, plasmid 1266 encoding Rev, plasmid 4220 encoding GCC-CAR.eGFP (Transfer), plasmid 4462 encoding the VSV-G mutant (Env), and CD7 targeting molecules encoded by plasmids listed in Table 2.

[0202] One day in advance, seed HEK293T cells in logarithmic growth phase into T75 flasks to achieve 80-90% cell confluence for virus packaging. Mix 4 or 5 plasmids in an appropriate ratio, for example, a 4-plasmid system 1267(Gag-pol):1266(Rev):4220(Transfer):4462(Env) = 1:3:4:2, or a 5-plasmid system 1267(Gag-pol):1266(Rev):4220(Transfer):4462(Env):4469(CD7-binder) = 1:3:4:2:2, for a total of 10-12 μg of plasmid. Then mix with 3 times the mass of PEI and incubate for 20 minutes before transfecting HEK293T cells.

[0203] The supernatant was harvested 48 hours after plasmid transfection, filtered through a 0.45 μm filter, and then concentrated using a 100 kD Amicon Ultra centrifuge tube (approximately 20–40 times volumetric concentration). Virus samples can be flash-frozen in liquid nitrogen and then stored at -80°C.

[0204] 2. Determination of P24 protein content in virus samples

[0205] In step 1, the harvested viral supernatant and viral concentrate samples were both detected using HTRF HIV P24 Detection Kits (Revvity, Cat. No. 64P24PEG). The specific experimental procedures were performed according to the product instructions. The brief steps are as follows: (1) Prepare a standard curve according to the instructions. The highest concentration of the standard curve is 10000 pg / ml (Std7), and then perform serial dilutions at a ratio of 1:2.4 (Std6-Std1); (2) Process the samples according to the instructions. If the P24 concentration is approximately 10000 pg / ml, pre-dilution is performed using culture medium; (3) After adding the standard and sample to the 96-well microplate, add 2 μl of d2 antibody and 2 μl of Eu Cryptate antibody to each well and gently shake to mix; (4) Cover with a sealing film and incubate at room temperature in the dark for 2 hours; (5) Detect the Eu Cryptate signal using a microplate reader, use four-parameter fitting to fit the standard curve, and calculate the P24 concentration of the sample.

[0206] 3. Transduction of viral samples into SupT1 and Raji cells

[0207] The steps are briefly as follows: (1) Thaw the concentrated virus sample from step 1 on ice and serially dilute it at a ratio of 1:3 (8 concentration gradients) using fresh culture medium. (2) Take supT1 and Raji cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in fresh culture medium to a suitable cell density, and then add 10,000 supT1 cells and 10,000 Raji cells to each well. (3) Then, add the serially diluted virus sample to the supT1 / Raji mixed cells at a volume of 280ul per well for virus transduction. (4) Three days after virus transduction, remove the cells for flow cytometry detection, use CD19 antibody to distinguish supT1 and Raji cells, and detect the virus transduction efficiency by GFP.

[0208] Experimental results:

[0209] like Figure 4 As shown, lentiviral vectors packaged with the VSV-G mutant (lacking LDL-R binding ability) cannot effectively transduce supT1 or Raji cells. When the lentiviral vector envelope carries a CD7 targeting molecule, the lentiviral vector can restore its transduction ability for CD7-positive supT1 cells to varying degrees, but it cannot transduce CD7-negative Raji cells. Among them, scFv25.CD7-binder can most effectively restore the transduction ability of the mutated lentiviral vector for supT1 cells.

[0210] Example 3. Specific transduction of T cells in PBMCs

[0211] Experimental objective and principle:

[0212] In Example 1, a COCV-G mutant was obtained by mutation, which removed LDL-R binding ability but retained fusion function; in Example 2, a CD7-binder with targeting function was obtained, which could effectively mediate the transduction ability of the lentiviral vector to CD7-positive cells. Therefore, in this example, the two were combined to prepare a lentiviral vector and its specific transduction ability was tested.

[0213] Experimental methods:

[0214] 1. Preparation of lentiviral vectors

[0215] In this example, HEK293T cells were transiently transfected with the five plasmid combinations listed in Table 4 to prepare the lentiviral vector.

[0216] Table 4. Plasmid combinations used to prepare lentiviral vectors

[0217] virus number Gag-pol Rev Transfer Env Binder Lv01 1267 1266 4358 4463 4462 Lv02 1267 1266 4358 4469 4521 Lv03 1267 1266 4220 4463 4462 Lv04 1267 1266 4220 4469 4521

[0218] One day in advance, HEK293T cells in logarithmic growth phase were seeded into T75 flasks to achieve 80-90% cell confluence for virus packaging. Five plasmids were mixed in an appropriate ratio, for example, 1267 (Gag-pol):1266 (Rev):4220 (Transfer):4462 (Env):4469 (CD7-binder) = 1:3:4:2:2, totaling 12ug of plasmid. This mixture was then incubated with 3 times the mass of PEI for 20 minutes before being used to transfect HEK293T cells.

[0219] The supernatant was harvested 48 hours after plasmid transfection, filtered through a 0.45 μm filter, centrifuged at 27000 g and 4 °C for 4 hours, and the virus precipitate was resuspended in an X-vivo 15 to obtain a concentrated virus sample (approximately 450-fold concentrated). The virus sample can be flash-frozen in liquid nitrogen and then stored at -80 °C.

[0220] 2. Determination of P24 protein content in virus samples

[0221] In step 1, the harvested viral supernatant and viral concentrate samples were both detected using HTRF HIV P24 Detection Kits (Revvity, Cat. No. 64P24PEG). The specific experimental procedures were performed according to the product instructions. The brief steps are as follows: (1) Prepare a standard curve according to the instructions. The highest concentration of the standard curve is 10000 pg / ml (Std7), and then perform serial dilutions at a ratio of 1:2.4 (Std6-Std1); (2) Process the samples according to the instructions. If the P24 concentration is approximately 10000 pg / ml, pre-dilution is performed using culture medium; (3) After adding the standard and sample to the 96-well microplate, add 2 μl of d2 antibody and 2 μl of Eu Cryptate antibody to each well and gently shake to mix; (4) Cover with a sealing film and incubate at room temperature in the dark for 2 hours; (5) Detect the Eu Cryptate signal using a microplate reader, use four-parameter fitting to fit the standard curve, and calculate the P24 concentration of the sample.

[0222] 3. Transduction of viral samples into SupT1 and Raji cells

[0223] The steps are briefly as follows: (1) Thaw the virus sample from step 1 on ice and serially dilute it at a ratio of 1:3 or 1:2 using fresh culture medium (8 concentration gradients). (2) Take supT1 and Raji cells in the logarithmic growth phase, centrifuge at 300g for 5 minutes at room temperature, resuspend the cells in fresh culture medium to a suitable cell density, and then add 20,000 supT1 or 20,000 Raji cells to each well. (3) Then, add the serially diluted virus sample to supT1 or Raji cells at a volume of 280ul per well for virus transduction. (4) Three days after virus transduction, remove the cells for flow cytometry detection and detect the virus transduction efficiency by staining with GFP or AF488-anti-EGFR antibody.

[0224] 4. Virus sample transduction PBMC

[0225] PBMCs were resuscitated, and samples were taken for flow cytometry analysis to detect markers such as CD3, CD8, CD56, CD19, and CD25. 1M PBMCs were added to each well of a 48-well plate, followed by the addition of concentrated virus for transduction. After 6 hours of transduction, the cells were centrifuged and the medium was changed to CTS. TM OpTmizer TM T cells were cultured in Gibco medium (Cat. No. A1048501). Samples were taken at 3 and 7 days after transduction for flow cytometry detection of biomarkers such as CD3, CD8, CD56, CD19, CD25, and GFP / tEGFR.

[0226] Experimental results:

[0227] like Figure 5 As shown in Tables 5 and 6, both the harvested viral supernatant and concentrated sample could effectively transduce supT1 cells, while exhibiting very low transduction ability against Raji cells. High-speed centrifugation at 27000g collected most of the biologically active viral particles, and the post-centrifugation viral supernatant showed almost no transduction ability against supT1 or Raji cells. However, the recovery rate of P24 protein after high-speed centrifugation was very low, possibly because most of the P24 in the post-centrifugation viral supernatant was contained within biologically inactive particles. Simultaneously, the decrease in P24 protein content after high-speed centrifugation also indicates a reduction in the competitive inhibitory effect of inactive viral particles on transduction, which may explain why the TU recovery rate after high-speed centrifugation was much higher than 100%.

[0228] Table 5. Virus sample P24 detection data

[0229]

[0230] Table 6. Biotiters of virus samples detected on supT1 and Raji cells.

[0231]

[0232] Before viral transduction, such as Figure 6 As shown, the PBMC sample contained 67.3% CD3-positive T cells (CD4:CD8 ratio approximately 1.1), 4.10% CD19-positive B cells, and 8.68% NK cells (CD3-CD56+).

[0233] Three days after viral transduction, if Figure 7A As shown, approximately 4.90% of CD19-positive B cells survived in untransduced PBMCs. In the two virus-transduced samples lv03 and lv04 carrying GCC-CAR.eGFP, 8.44% and 7.75% of CD19-positive B cells were also detected, respectively. However, no CD19-positive cells were detected in the two virus-transduced samples lv01 and lv02 carrying CD20CAR.tEGFR. Figure 7B As shown, no significant expression of the activation marker CD25 was detected on T cells in PBMCs that had not undergone viral transduction. No significant CD25 expression was also detected in the two viral transduction samples (lv03 and lv04) carrying GCC-CAR.eGFP. However, in the two viral transduction samples (lv01 and lv02) carrying CD20CAR.tEGFR, significant CD25 expression was observed on CD3-positive T cells (CD4 and CD8 cells also partially expressed CD25). Although no significant CAR expression (detection of GFP or tEGFR signaling) was detected on T, B, and NK cells in any of the samples, B cell reduction and T cell activation occurred in the two viral transduction samples (lv01 and lv02) carrying CD20CAR.tEGFR. This indicates that lv01 and lv02 may have successfully transduced T cells in PBMCs, and that CD20CAR recognized B cells, providing T cell activation signals and mediating B cell killing.

[0234] 7 days after viral transduction, if Figure 8 As shown, no significant CD19-positive B cell survival (CTS) was detected in any of the samples. TM OpTmizer TM(The T cell culture medium does not support long-term B cell culture). In the two viral transduction samples lv01 and lv02 carrying CD20CAR.tEGFR, approximately 9.75% and 29.1% tEGFR expression were detected, respectively, indicating that both lv01 and lv02 successfully transduced T cells in PBMCs. A small amount of tEGFR expression also appeared on NK cells. In the two viral transduction samples lv03 and lv04 carrying GCC-CAR.eGFP, approximately 4.17% and 3.42% GFP expression were detected, respectively, primarily on CD4-positive cells. In the Lv04 transduction sample, 2.46% GFP expression was also detected on NK cells. The lower transduction efficiency of lv03 and lv04 may be because GCC-CAR lacks antigen stimulation and cannot provide T cell activation signals to promote CAR-positive cell growth.

[0235] Therefore, the lentiviral vector co-packaged with COCV-G mutant and CD7 targeting molecule can effectively transduce T and NK cells in resting PBMCs, and the CD20CAR molecule carried can exert B cell killing function.

[0236] The sequence of the present invention is shown in the table below:

[0237]

[0238]

[0239]

[0240]

[0241] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A viral envelope protein mutant, characterized in that, The extracellular domain of the mutant envelope protein has mutations at positions 352, 354, 47, and / or 48 relative to the extracellular domain of the wild-type envelope protein. The amino acid sequence of the extracellular domain of the wild-type envelope protein is shown in SEQ ID NO:

1.

2. The viral envelope protein mutant as described in claim 1, characterized in that, The extracellular domain of the mutant envelope protein, relative to the extracellular domain of the wild-type envelope protein, has one or more amino acid mutations selected from the group consisting of: T352V, R354Q, K47Q, T48S.

3. The viral envelope protein mutant as described in claim 1, characterized in that, The extracellular domain of the mutant envelope protein, relative to the extracellular domain of the wild-type envelope protein, has an amino acid mutation selected from the group consisting of: 1) T352V and R354Q; 2) K47Q and T48S; 3) K47Q, T48S, and R354Q; and 4)R354Q.

4. The viral envelope protein mutant as described in claim 1, characterized in that, The amino acid sequence of the extracellular domain of the said envelope protein mutant is shown in SEQ ID NO:6, 7, 8 or 9.

5. An isolated polynucleotide, characterized in that, The polynucleotide encodes a viral envelope protein mutant as described in any one of claims 1-4.

6. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 5.

7. An antibody or its antigen-binding fragment targeting CD7, characterized in that, The heavy chain variable region of the antibody includes HCDR1, HCDR2, and HCDR3, wherein: The amino acid sequence of HCDR1 is shown in SEQ ID NO:13; The amino acid sequence of HCDR1 is shown in SEQ ID NO:14; The amino acid sequence of HCDR1 is shown in SEQ ID NO:15; and The variable region of the light chain of the antibody includes LCDR1, LCDR2, and LCDR3, wherein: The amino acid sequence of LCDR1 is shown in SEQ ID NO:16; The amino acid sequence of LCDR1 is shown in SNS; The amino acid sequence of LCDR1 is shown in SEQ ID NO:

17.

8. A recombinant viral particle, characterized in that, The recombinant viral particles comprise a viral envelope protein mutant as described in any one of claims 1-4.

9. The recombinant virus particle as described in claim 8, characterized in that, The viral particles further comprise a target nucleic acid molecule, preferably a nucleic acid encoding a chimeric antigen receptor.

10. The recombinant virus particle as described in claim 8, characterized in that, The viral particle further includes a targeting portion, preferably, the targeting portion comprising the antibody or its antigen-binding fragment as described in claim 7.

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