WO2024000223A1

WO2024000223A1 - Modified viral envelope protein and use thereof

Info

Publication number: WO2024000223A1
Application number: PCT/CN2022/102194
Authority: WO
Inventors: 蔡珍珍; 唐国发; 欧阳学农; 葛均友
Original assignee: 成都科伦精准生物科技有限公司
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2024-01-04

Abstract

Provided are a modified viral envelope protein and the use thereof, and a pseudotype virus-like particle containing the modified viral envelope protein and the use thereof. The modified viral envelope protein can improve the infection efficiency of the pseudotype virus-like particle with regard to immune cells (e.g., B cells, T cells, and NK cells).

Description

Modified viral envelope proteins and their uses

Technical field

This application relates to the field of biomedicine, specifically to a modified viral envelope protein, and pseudotyped virus-like particles containing the modified viral envelope protein. The modified viral envelope protein can enhance pseudotyping. The infection efficiency of virus-like particles on immune cells (e.g., B cells, T cells, NK cells). Furthermore, the present application relates to the use of the engineered viral envelope proteins and the use of the pseudotyped virus-like particles.

Background technique

Natural killer cells (hereinafter abbreviated as "NK cells") are a unique group of lymphocytes in the human body that can recognize and destroy virus-infected cells or cancerous cells (also known as tumor cells). When NK cells come into contact with tumor cells, they can directly kill tumor cells in an MHC-independent manner, and at the same time produce and secrete related cytokines to promote killing functions and recruit other immune cells in the body to jointly play an anti-tumor role. These functional characteristics make NK cells a very attractive cancer treatment drug in clinical practice. With the rapid development of cell therapy technology, methods such as in vitro isolation, expansion and culture of NK cells have become increasingly mature. Moreover, whether in theory or in current clinical trials, it has been shown that NK cells can be allogeneic and achieve off-the-shelf supply of cell therapy drugs (May Daher et al., 2020).

Genetic modification of immune cells can effectively improve their tumor targeting and killing functions, such as T cells expressing chimeric antigen receptor (CAR) (referred to as CAR-T), which have been shown in clinical trials, especially in the treatment of hematomas. Remarkable therapeutic effect. Therefore, the use of immune cells expressing chimeric antigen receptors (CARs) is a promising therapeutic option in cancer treatment. From the perspective of production cost and off-the-shelf supply, CAR-NK cells are a good substitute for CAR-T cells (Elizabeth L Siegler et al., 2018). However, compared with primary T cells, genetic modification of primary NK cells faces significant technical challenges (Klingemann et al., 2014). Judging from current research reports, on the one hand, transfection methods such as electroporation and lipofection can achieve effective target gene delivery, but the expression of the target gene is transient and cannot be used in clinical applications. In addition, electroporation and transfection can cause higher cell death (Tae Kyung Kim, 2010). On the other hand, viral vector-based transduction can be an option for persistent genetic modification of NK cells. Retrovirus can achieve transduction of primary NK cells, but the transduction efficiency is low and requires a high virus titer (Stephan Müller, 2020). In addition, since retroviral vectors contain heterologous envelope proteins from foreign viral species within the retroviral membrane. Safety issues such as insertional mutations of retroviral vectors limit their clinical application.

In the retroviridae family, lentiviral vectors are currently reported to be less genetically toxic and widely used in clinical applications, such as the application of CAR-T (Sara Ghorashian et al., 2019). However, although lentiviral vectors have high transduction efficiency on primary T cells, they have poor transduction efficiency on primary NK cells, and only 10% of NK can be transduced even with higher lentiviral titers. cells, which may be related to the natural antiviral mechanism of NK cells (Sutlu et al., 2012). In addition, the use of higher titers of lentivirus will cause certain toxic side effects on NK cells themselves, resulting in a decrease in NK cell viability. Therefore, there is an urgent need to develop viral vectors that can stably transduce NK cells and sustain expression.

In WO2013/045639A1, a method of transducing quiescent HSCs as well as quiescent T and B cells using viral vectors pseudotyped with modified baboon endogenous retrovirus (BaEV) envelope glycoproteins is disclosed. Compared with lentivirus pseudotyped with VSV-G protein, viral vectors pseudotyped with BaEV envelope glycoprotein significantly improved the transduction efficiency in HSCs. In WO2019/121945, a viral vector pseudotyped with modified baboon endogenous retrovirus (BaEV) envelope glycoprotein was disclosed. Compared with lentivirus pseudotyped with VSV-G protein, primary NK cells can Achieve higher efficiency transduction. However, the disadvantage is that this modified baboon endogenous retrovirus (BaEV) envelope glycoprotein pseudotyped viral vector lacks the inhibitory R peptide, causing the viral envelope to produce severe cytotoxicity to packaging cells. , it is especially easy to produce syncytia, which is not conducive to the large-scale production of virus particles (Hélio A. Tomás et al., 2019).

Therefore, it is necessary to develop durable, effective, safe and stable viral vectors for lymphocytes that are difficult to genetically modify, such as primary NK.

Contents of the invention

After extensive experiments and repeated explorations, the inventors of the present application prepared a modified viral envelope protein, and further prepared pseudotyped virus-like particles containing such viral envelope proteins. The modified viral envelope protein improves the infection efficiency of pseudotyped virus-like particles on immune cells (eg, B cells, T cells, NK cells). Such pseudotyped virus-like particles are particularly useful, for example, for the efficient introduction of exogenous nucleic acids of interest (eg, exogenous nucleic acids encoding chimeric antigen receptors (CARs)) into cells (especially immune cells, e.g. B cells, T cells, NK cells), and allow them to express the product encoded by the target exogenous nucleic acid (such as chimeric antigen receptor (CAR)). The cells thus obtained (especially immune cells) may have good application prospects in many aspects (for example, in cancer treatment).

Thus, in the first aspect of the present application, a modified viral envelope protein is provided, which includes the transmembrane domain and extracellular structure of the baboon endogenous virus (BaEV) envelope glycoprotein. domain, and a modified cytoplasmic tail domain comprising amino acids 1-13 of the cytoplasmic tail domain of murine leukemia virus (MLV) envelope glycoprotein residues, the cleavage sequence of the lentiviral protease, and the fusion inhibitory R peptide.

The BaEV envelope glycoprotein includes the wild type of BaEV envelope glycoprotein or has at least 85%, preferably at least 90%, more preferably at least 95%, still more preferably at least 99% of the wild type BaEV envelope glycoprotein. Identity mutants of the wild-type BaEV envelope glycoprotein, the mutant glycoprotein retains the ability of the wild-type glycoprotein to bind to and fuse with the cell membrane.

In certain embodiments, the engineered cytoplasmic tail domain is composed of amino acid residues 1-13 of the cytoplasmic tail domain of the MLV envelope glycoprotein, a cleavage sequence of a lentiviral protease, and a fusion inhibitory R peptide composition.

In certain embodiments, the lentiviral protease is selected from the group consisting of proteases of human immunodeficiency virus HIV (eg, HIV-1, HIV-2). In certain embodiments, the lentiviral protease is HIV-1 protease.

In certain embodiments, the cleavage sequence of the lentiviral protease is a natural HIV-1 protease cleavage sequence or a synthetic HIV-1 protease cleavage sequence.

In certain embodiments, the lentiviral protease is able to cleave polyproteins such as Gag and Gag-Pro-Pol at the site it recognizes, thereby converting unique morphologically immature virions into mature viruses. By analyzing the sequence of the lentiviral protease cleavage site, the cleavage sequences of natural lentiviral proteases (e.g., HIV-1 protease) are divided into two types: cleavage sequences with aromatic residues and proline residues. ; and, a cleavage sequence having hydrophobic residues but excluding proline residues. A detailed description of the cleavage sequences of lentiviral proteases can be found, e.g., József

Comparative Studies on Retroviral Proteases:Substrate Specificity[J]. Viruses, 2010, 2(1):147-165. Without being limited by theory, in the embodiments of the present application, various lentiviral protease cleavage sequences can be used, including natural HIV-1 protease cleavage sequences, as well as artificially designed or artificially synthesized lentiviral proteases (e.g., HIV-1 protease ) cleavage sequence.

Thus, in certain embodiments, the cleavage sequence of the lentiviral protease is a cleavage sequence having aromatic residues and proline residues. In certain embodiments, the cleavage sequence of the lentiviral protease is a cleavage sequence having hydrophobic residues but excluding proline residues.

In certain preferred embodiments, the cleavage sequence of the lentiviral protease is SEQ ID NO: 1 or SEQ ID NO: 3.

In certain embodiments, the fusion-inhibiting R peptide is selected from the group consisting of fusion-inhibiting R peptides of MLV envelope glycoprotein. In certain embodiments, the fusion-inhibiting R peptide has the amino acid sequence set forth in SEQ ID NO: 6.

In certain embodiments, the cytoplasmic tail domain of the MLV envelope glycoprotein has the amino acid sequence set forth in SEQ ID NO: 5.

In certain embodiments, the engineered cytoplasmic tail domain has the amino acid sequence set forth in SEQ ID NO: 42 or SEQ ID NO: 43.

In certain embodiments, the extracellular domain of the BaEV envelope glycoprotein has the amino acid sequence set forth in SEQ ID NO: 35.

In certain embodiments, the transmembrane domain of the BaEV envelope glycoprotein has the amino acid sequence set forth in SEQ ID NO: 45.

In certain embodiments, the engineered viral envelope protein has the amino acid sequence set forth in SEQ ID NO: 14 or 16.

In another aspect, the present application relates to a recombinant protein comprising an engineered viral envelope protein as described above, and additional peptide segments linked to the engineered viral envelope protein .

In the recombinant protein of the present application, the additional peptide segment can be connected to the modified viral envelope protein in various ways. For example, in certain preferred embodiments, the additional peptide segments are directly linked to the engineered viral envelope protein. In other words, the additional peptide segments are directly connected to the modified viral envelope protein through peptide bonds. In certain preferred embodiments, the additional peptide segments are linked to the engineered viral envelope protein via a linker. Suitable prior art linkers may consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₄ can be used, but variants thereof can also be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448). In addition, other linkers can also be used, such as Alfthan et al. (1995), Protein Eng. 8:725-731; Choi et al. (2001), Eur. J. Immunol. 31:94-106; Hu et al. (1996) , Cancer Res. 56:3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.

In the recombinant protein of the present application, the additional peptide segment can be connected to any end of the modified viral envelope protein. For example, in certain preferred embodiments, the additional peptide is linked to the N-terminus of the engineered viral envelope protein. In certain preferred embodiments, the additional peptide is linked to the C-terminus of the engineered viral envelope protein.

Recombinant proteins according to the invention may contain one or more additional peptide segments. For example, in certain preferred embodiments, the recombinant protein according to the present invention may comprise at least 1, at least 2, at least 3, at least 5 or more additional peptide segments. It is readily understood that each of these peptide segments independently can be linked to either terminus (N-terminus or C-terminus) of the engineered viral envelope protein in various ways. For example, in certain preferred embodiments, the recombinant protein of the present invention may comprise two additional peptide segments, wherein one additional peptide segment is connected to the modified viral envelope protein through a linker or not through a linker. N-terminus, and another additional peptide segment is connected to the C-terminus of the engineered viral envelope protein with or without a linker. In certain preferred embodiments, the recombinant protein of the present invention may comprise two or more additional peptide segments, wherein each of the two or more additional peptide segments independently passes through a linker or does not pass through a linker. Attached to the N-terminus or C-terminus of the engineered viral envelope protein. In certain preferred embodiments, when two or more additional peptide segments are linked to the N-terminus of the engineered viral envelope protein, the two or more additional peptide segments can be in any order. in series, and then connected to the N-terminus of the modified viral envelope protein through a linker or without a linker. Similarly, in certain preferred embodiments, when two or more additional peptide segments are linked to the C-terminus of the engineered viral envelope protein, the two or more additional peptide segments may concatenated in any order and then connected to the C-terminus of the engineered viral envelope protein with or without a linker.

Appropriate additional peptides can be selected according to actual needs. For example, in certain preferred embodiments, the additional peptide segments may be signal peptides. Without being bound by any theory, it is generally believed that the use of signal peptides can promote the secretion of recombinant proteins, thus facilitating the recovery of recombinant proteins. Typically, such signal peptides can be linked to the N-terminus of the engineered viral envelope protein. Additionally, during or after secretion, the signal peptide can be cleaved to produce the desired engineered viral envelope protein or recombinant protein.

In another aspect, the present application provides a polynucleotide comprising a nucleotide sequence encoding a modified viral envelope protein as described above, or a nucleotide sequence of a recombinant protein as described above.

In another aspect, the present application provides a vector comprising the polynucleotide as described above or the recombinant protein as described above.

In certain preferred embodiments, the vector is selected from plasmids, phagemids, cosmids, viral vectors (eg, retroviral vectors (eg, lentiviral vectors), adenoviral vectors).

Generally speaking, lentivirus is a type of virus that can deliver large amounts of viral nucleic acid into host cells. Lentiviruses are characterized by their unique ability to infect/transduce non-dividing cells and, after transduction, are able to integrate the nucleic acids they carry into the chromosomes of the host cell. Thus, in certain preferred embodiments, the vector is a lentiviral vector.

Lentiviral vectors are well known to those skilled in the art and are described inter alia in Naldini et al. (2000) Adv. Virus. Res. 55:599-609 and Negre et al. (2002) Biochimie 84:1161-1171. Lentivirus usually contains three major genes gag, pol and env that encode virulence proteins. In some cases, the lentivirus also includes two regulatory genes, tat and rev. Depending on the specific virus type, lentiviruses may also have additional genes encoding proteins involved in the regulation, synthesis, and/or processing of viral nucleic acid, as well as other replication functions.

Therefore, in certain preferred embodiments, the vector also contains genes required for the formation of lentiviral particles, such as the gag gene or functional variants thereof and/or the pol gene or functional variants thereof. In certain preferred embodiments, the vector also contains regulatory genes, such as tat genes or functional variants thereof and/or rev genes or functional variants thereof.

In certain preferred embodiments, the vector further comprises a nucleotide sequence encoding a lentiviral protease.

In certain preferred embodiments, the lentiviral protease is selected from the group consisting of proteases of HIV (eg, HIV-1, HIV-2).

In certain preferred embodiments, the lentiviral protease is HIV-1 protease.

In certain preferred embodiments, the lentiviral protease is capable of recognizing and cleaving the cleavage sequence of the lentiviral protease in the engineered cytoplasmic tail domain.

In another aspect, the present application provides a host cell comprising the polynucleotide as described above or the vector as described above. Such host cells include, but are not limited to, prokaryotic cells such as E. coli cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The host cell of the invention can also be a cell line, such as 293T cells. Without being bound by any theory, it is generally believed that the use of eukaryotic cells helps maintain the correct conformation of proteins and promotes protein folding. Thus, in certain preferred embodiments, the host cells of the invention are eukaryotic cells, such as human cells. In certain embodiments, host cells of the invention can be immune cells, such as B cells, T cells, NK cells.

In another aspect, the present application provides a method for preparing the modified viral envelope protein as described above, which includes culturing the host cell as described above under conditions that allow protein expression. In certain preferred embodiments, the method further includes recovering the engineered viral envelope protein expressed by the host cell.

In another aspect, the present application provides a pseudotyped virus-like particle comprising a modified viral envelope protein as described above.

In certain preferred embodiments, the pseudotyped virus-like particles further comprise biological material.

In certain preferred embodiments, the biomaterial involves one or more compounds that tend to alter the structure and/or function of the cell. Preferably, the biological material is one or more nucleic acids, which in the case of lentiviral vector particles may be contained within the genome of the vector particle. The genome typically contains one or more nucleic acids, preferably linked to genetic elements required for its expression in target cells, such as promoters and terminators, flanked by, for inclusion in the core element, the genome, its reverse transcription to deoxyribose The nucleic acid (DNA) and the reverse-transcribed genome are imported into the nucleus of the target cell and the cis-acting elements required for the integration of the reverse-transcribed genome into the genome of the target cell.

In certain preferred embodiments, the pseudotyped virus-like particles are lentiviral virus-like particles (e.g., HIV virus-like particles or SIV virus-like particles).

In certain preferred embodiments, the pseudotyped virus-like particles further comprise (eg, are packaged with) a nucleic acid molecule of interest.

In certain preferred embodiments, the nucleic acid molecule of interest comprises a gene of interest. In certain preferred embodiments, the gene of interest encodes a protein of interest (eg, a chimeric antigen receptor).

In certain preferred embodiments, the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane region and an intracellular region.

In certain preferred embodiments, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region capable of specifically binding an antigen, such as a tumor antigen, such as a B cell maturation antigen (BCMA); preferably, The antigen binding domain is a scFv.

In certain preferred embodiments, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 20.

In certain preferred embodiments, the amino acid sequence of the light chain variable region is SEQ ID NO: 22.

In certain preferred embodiments, the transmembrane region comprises the transmembrane domain of CD8.

In certain preferred embodiments, the intracellular region comprises the intracellular signaling domain of CD137 (also known as 4-1BB) and/or the intracellular signaling domain of CD3ζ.

In certain preferred embodiments, the chimeric antigen receptor comprises an anti-BCMA scFv, a hinge region of CD8, a transmembrane domain of CD8, an intracellular signaling domain of CD137 and an intracellular signaling domain of CD3ζ. In certain preferred embodiments, the chimeric antigen receptor further comprises IL-15, which can be linked to the intracellular signaling domain of CD3ζ, for example, via the self-cleaving polypeptide P2A. In certain preferred embodiments, the chimeric antigen receptor has the amino acid sequence shown in SEQ ID NO: 40.

In some embodiments, the pseudotyped virus-like particles described herein are capable of expressing interleukin 15 (IL-15). Of course, the pseudotyped virus-like particles described herein may also contain other cytokines, including, for example, IL-2, IL-18, IL-23, and IL-36.

In another aspect, the present application provides a method for preparing pseudotyped virus-like particles as previously described, comprising:

(a) Transfect host cells with:

(1) The first nucleic acid molecule encoding the modified viral envelope protein as described above;

(2) One or more accessory nucleic acid molecules containing genes or elements required for the formation of lentiviral virus-like particles (e.g., HIV virus-like particles or SIV virus-like particles); and

(3) Optionally, a target nucleic acid molecule containing a target gene;

(b) Culturing the host cell under conditions permissive for protein expression to produce pseudotyped virus-like particles.

In certain preferred embodiments, the one or more accessory nucleic acid molecules may comprise a gag gene and a pol gene.

In certain preferred embodiments, the gene of interest encodes a protein of interest (eg, a chimeric antigen receptor). It will be readily understood that various features described above for chimeric antigen receptors also apply here.

In certain preferred embodiments, the cells are mammalian (eg, human) cells; preferably, the cells are 293T cells.

Pseudotyped virus-like particles can be readily prepared by one skilled in the art according to the state of the art, for example by following the general guidance provided by Sandrin et al. (2002) Blood 100:823-832. In certain preferred embodiments, one or more genes or elements in the auxiliary nucleic acid molecules can be carried in the same or different vectors, for example, 1, 2, 3, or more vectors.

In another aspect, the application provides an immune cell comprising or infected with a pseudotyped virus-like particle as described above.

In certain preferred embodiments, the immune cells are mammalian (eg, human) immune cells (eg, T cells, B cells, NK cells).

In certain preferred embodiments, the immune cells are NK cells.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and a modified viral envelope protein selected from the group consisting of those described above.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and a polynucleotide selected from the group consisting of those described above.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and a carrier selected from the group consisting of those described above.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and pseudotyped virus-like particles selected from the group consisting of those described above.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and immune cells selected from the group consisting of those described above.

In another aspect, the present application provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and a plurality selected from the following:

(1) The modified viral envelope protein as described above;

(2) Polynucleotide as described above;

(3) The carrier as mentioned above;

(4) Pseudotyped virus-like particles as described above; and

(5) Immune cells as described above.

In another aspect, the present application provides a kit comprising a first nucleic acid molecule encoding the modified viral envelope protein as described above.

In certain preferred embodiments, the kit further comprises one or more accessory nucleic acid molecules containing genes or elements required for the formation of lentiviral virus-like particles (eg, HIV virus-like particles or SIV virus-like particles). In certain preferred embodiments, the one or more auxiliary nucleic acid molecules comprise the gag gene or functional variants thereof and/or the pol gene or functional variants thereof. In certain preferred embodiments, the one or more accessory nucleic acid molecules comprise gag genes and/or pol genes. In certain preferred embodiments, the one or more auxiliary nucleic acid molecules further comprise a tat gene or a functional variant thereof and/or a rev gene or a functional variant thereof. In certain preferred embodiments, the one or more accessory nucleic acid molecules further comprise a nucleotide sequence encoding a lentiviral protease. In certain preferred embodiments, the lentiviral protease is capable of recognizing and cleaving the cleavage sequence of the lentiviral protease in the engineered cytoplasmic tail domain.

In certain preferred embodiments, the kit further contains a nucleic acid molecule of interest comprising a gene of interest. In certain preferred embodiments, the gene of interest encodes a protein of interest (eg, a chimeric antigen receptor).

In certain preferred embodiments, the kit further comprises reagents for transferring the nucleic acid molecule into the host cell, and/or, a vector for inserting the nucleic acid molecule. In certain preferred embodiments, the host cell is a mammalian (eg, human) cell. In certain preferred embodiments, the host cell is a 293T cell. In certain preferred embodiments, the kit further comprises a host cell.

In certain preferred embodiments, the one or more accessory nucleic acid molecules are contained in the same vector or different vectors, for example, 1, 2, 3, or more vectors.

In certain preferred embodiments, the one or more auxiliary nucleic acid molecules and the first nucleic acid molecule are contained in the same vector or different vectors, for example, 1, 2, 3, or more in the carrier.

On the other hand, the present application provides a modified viral envelope protein as described above, or a polynucleotide as described above, or a vector as described above, or a pseudotyped virus as described above The use of similar particles, or immune cells as described above, in the preparation of medicaments for preventing and/or treating diseases in a subject.

In certain preferred embodiments, the disease is a disease that can be prevented or treated by the nucleic acid molecule of interest. In certain preferred embodiments, the nucleic acid molecule of interest contained (packaged) in the pseudotyped virus-like particles has a preventive or therapeutic effect on the disease. Therefore, the nucleic acid molecules of interest contained (packaged) in pseudotyped virus-like particles can be selected according to the disease to be prevented or treated. For example, when the disease to be prevented or treated is a tumor, the nucleic acid molecule of interest may be an anti-tumor gene, such as a gene encoding a chimeric antigen receptor (CAR). Similarly, when the disease to be prevented or treated is an inflammatory disease, the nucleic acid molecule of interest may be an anti-inflammatory gene, such as a gene encoding an anti-inflammatory factor (eg, interleukin).

In certain preferred embodiments, the disease is selected from viral infections, bacterial infections, tumors, inflammatory diseases, and autoimmune diseases.

In certain preferred embodiments, the medicament also contains an additional pharmaceutically active agent. Additional pharmaceutically active agents may be selected depending on the disease to be prevented or treated. For example, when the disease to be prevented or treated is a tumor, the additional pharmaceutically active agent is a drug with anti-tumor activity.

In certain preferred embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine Kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.

In certain preferred embodiments, the subject is a mammal, such as a human.

In certain preferred embodiments, the medicament further comprises a pharmaceutically acceptable carrier, excipient, stabilizer or other agent capable of providing beneficial properties for administration of the medicament (eg, to a human subject). Suitable pharmaceutical carriers include, for example, sterile water, saline, dextrose, condensation products of castor oil and ethylene oxide, liquid acids, lower alcohols (e.g., C1-4 alcohols), oils (e.g., corn oil, peanut oil, sesame oil; Optionally also included are emulsifiers such as mono- or diglycerides of fatty acids or phospholipids such as lecithin), ethylene glycol, polyalkylene glycols, sodium alginate, poly(vinylpyrrolidone) and the like. The carrier optionally may also contain adjuvants, preservatives, stabilizers, wetting agents, emulsifiers, penetration enhancers and the like.

In another aspect, the present application provides the use of an envelope protein as previously described for preparing pseudotyped virus-like particles.

In certain preferred embodiments, the pseudotyped virus-like particles are capable of infecting immune cells (eg, T cells, B cells, NK cells).

In certain preferred embodiments, the pseudotyped virus-like particles are lentiviral virus-like particles (eg, HIV virus-like particles or SIV virus-like particles).

In another aspect, the present application provides the use of pseudotyped virus-like particles as previously described for transferring nucleic acid molecules of interest to immune cells (e.g., T cells, B cells, NK cells) or for preparing The use of drugs that transfer nucleic acid molecules of interest to immune cells (e.g., T cells, B cells, NK cells).

In certain preferred embodiments, the nucleic acid molecule of interest is contained in a vector.

In certain preferred embodiments, the nucleic acid molecule of interest encodes a protein of interest, such as a chimeric antigen receptor.

In another aspect, the present application provides a method of transferring a nucleic acid molecule of interest into immune cells (e.g., T cells, B cells, NK cells), which method includes using pseudotyped virus-like particles as described above. The immune cell is infected, wherein the pseudotyped virus-like particle contains (packages) the nucleic acid molecule of interest.

In certain preferred embodiments, the methods are performed in vitro, ex vivo or in vivo.

In another aspect, the present application provides a method of preventing or treating a disease in a subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of a pseudotyped virus-like particle as described above Or the immune cells as mentioned above or the pharmaceutical composition as mentioned above or the kit as mentioned above.

In certain preferred embodiments, the subject is a mammal, such as a human.

In certain preferred embodiments, the methods further comprise administering to the subject an additional pharmaceutically active agent. Additional pharmaceutically active agents may be selected depending on the disease to be prevented or treated. For example, when the disease to be prevented or treated is a tumor, the additional pharmaceutically active agent is a drug with anti-tumor activity. In certain preferred embodiments, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitosis inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine Kinase inhibitors, radionuclide agents, radiosensitizers, anti-angiogenic agents, cytokines, molecularly targeted drugs, immune checkpoint inhibitors or oncolytic viruses.

Definition of Terms

In the present invention, unless otherwise stated, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Moreover, the procedures used in this article such as molecular genetics, nucleic acid chemistry, chemistry, molecular biology, biochemistry, cell culture, microbiology, cell biology, genomics and recombinant DNA are routine procedures widely used in the corresponding fields. . Meanwhile, in order to better understand the present invention, definitions and explanations of relevant terms are provided below.

As used herein, the terms "wild" or "natural" are used interchangeably. When these terms are used to describe a nucleic acid molecule, polypeptide or protein, it means that the nucleic acid molecule, polypeptide or protein exists in nature, is found in nature, and has not undergone any artificial modification or processing.

As used herein, the terms "virus-like particles," "pseudovirus particles," and "virus-like particles" have the same meaning and may be used interchangeably. The term "virus-like particle" specifically refers to a virus-like particle formed by the self-assembly of viral proteins, which does not encapsulate nucleic acid or encapsulates other nucleic acids. Therefore, although the virus-like particle can infect host cells, it cannot infect host cells. Have the ability to replicate autonomously. Therefore, its biosafety is high compared to real viruses.

In the present application, the virus-like particles may be selected from virus-like particles of oncoviruses, such as murine leukemia virus (MLV), avian leukemia virus (ALV), respiratory syncytial virus (RSV) or Mason-Pfizer monkey virus (MPMV). Virus-like particles; virus-like particles of a lentivirus, such as human immunodeficiency virus (HIV, e.g., HIV-1 or HIV-2), simian immunodeficiency virus (SIV), feline immunodeficiency virus (FIV), or equine infectious anemia virus (EIAV) virus-like particles; and virus-like particles of foamy viruses, such as human foamy virus (HFV) virus-like particles.

Lentiviral virus-like particles are well known to those skilled in the art and are described in particular in Naldini et al. (2002) Adv.Virus.Res.55:599-609 and Negre et al. (2002) Biochimie 84:1161-1171 describe. Typically, lentiviral virus-like particles comprise at least the following components: (i) an envelope component consisting of a phospholipid bilayer bound to an envelope protein surrounding (ii) a core composed of bound gag proteins components, the core itself surrounding (iii) a genomic component usually consisting of ribonucleic acid (RNA) and (iv) an enzyme component (pol).

Lentiviral virus-like particles can be readily prepared by one skilled in the art, for example, by following the general guidance provided by Sandrin et al. (2002) Blood 100:823-832. Briefly, lentiviral virus-like particles can be formed by co-expressing in host cells (such as 293T cells) the components required to form lentiviral virus-like particles (including envelope components, core components, genome components and enzyme groups). points) to generate. Generally, 3 to 4 plasmids can be used to prepare lentiviral virus-like particles, but the number of plasmids can vary according to actual needs (such as the permutation and combination of lentiviral components).

For example, in an exemplary embodiment of the present invention, 3 plasmids are used to prepare lentiviral virus-like particles, wherein one plasmid contains a nucleotide sequence encoding an envelope protein (referred to as an "envelope plasmid"), and the other plasmid One plasmid contains the genomic component (called the "core plasmid") carrying the exogenous target gene (target nucleic acid molecule), and the other plasmid contains the nucleotide sequence (e.g., gag protein and pol protein) encoding other essential components (e.g., gag protein and pol protein). called "helper plasmid"). Cells are co-transfected with the above-mentioned core plasmid, envelope plasmid and helper plasmid, and then the pseudoviral particles are packaged in the cells, and then the packaged pseudoviral particles are secreted into the extracellular culture medium, thus producing high droplet fake virus particles.

Such core plasmids, envelope plasmids or auxiliary plasmids are well known to those skilled in the art. For example, auxiliary plasmids constructed based on HIV-1 include, but are not limited to, pSG3.Δenv (Wei X et al., Antibody neutralization and escape by HIV-1.Nature 422:307-312, 2003) and NL4-3.Fluc.R-.E-(Connor RI, et al., Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes.Virology 206:935–944, 1995).

As used herein, the expression "one or more accessory nucleic acid molecules containing genes or elements required for the formation of lentiviral virus-like particles" will include, except for genes encoding envelope proteins, all necessary genes or elements, For example, genome components, genes encoding gag proteins, genes encoding pol proteins, etc.

As used herein, the term "pseudotyped virus-like particles" refers to virus-like particles that contain foreign viral envelope glycoproteins. Typically, virus-like particles according to the invention are pseudotyped with engineered viral envelope proteins as defined above.

Baboon endogenous virus (also known as "baboon endogenous retrovirus", BaEV) is a type C retrovirus that exists as multiple proviral copies in baboon DNA. BaEV envelope glycoproteins are described inter alia in Benveniste et al. (1974) Nature 248: 17-20 and Todaro et al. (1974) Cell 2: 55-61.

As used herein, the term "BaEV envelope glycoprotein" is intended to include the wild-type form of the BaEV envelope glycoprotein or to be at least 85% identical to said wild-type BaEV envelope glycoprotein, preferably at least 90%, more preferably at least A mutant of the wild-type BaEV envelope glycoprotein that is 95%, still more preferably at least 99%, identical and retains the ability of the wild-type glycoprotein to bind to and fuse with the cell membrane.

The sequence of the natural BaEV envelope glycoprotein can be found in public databases, for example, the amino acid sequence shown in SEQ ID NO: 44. As used herein, when referring to the amino acid sequence of the BaEV envelope glycoprotein, it is described using the sequence shown in SEQ ID NO: 44. For example, the expression "amino acids 530 to 564 of BaEV envelope glycoprotein" refers to amino acid residues 530-564 of the polypeptide shown in SEQ ID NO: 44. However, those skilled in the art understand that in the amino acid sequence of the BaEV envelope glycoprotein, mutations or variations (including, but not limited to, substitutions, deletions and/or additions) can occur naturally or be artificially introduced without affecting its biological function. . Therefore, in the present invention, the term "BaEV envelope glycoprotein" shall include all such sequences, including for example the sequence shown in SEQ ID NO: 44 and its natural or artificial variants. Moreover, when describing the sequence fragment of the BaEV envelope glycoprotein, it includes not only the sequence fragment of SEQ ID NO: 44, but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid residues 530-564 of BaEV envelope glycoprotein" includes amino acid residues 530-564 of SEQ ID NO: 44, and corresponding fragments in its variants (natural or artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" means that when the sequences are optimally aligned, that is when the sequences are aligned to obtain the highest percent identity, equivalent positions are located in the sequences being compared. fragment.

As known to those skilled in the art, the BaEV envelope glycoprotein consists of a cytoplasmic tail domain, a transmembrane domain and an extracellular domain. The regions in the BaEV envelope glycoprotein sequence corresponding to the cytoplasmic tail domain, the transmembrane domain and the extracellular domain can be easily determined by the skilled person. Typically, the cytoplasmic tail domain includes amino acids 530 to 564 of the BaEV envelope glycoprotein; the transmembrane domain includes amino acids 507 to 529 of the BaEV envelope glycoprotein; and the extracellular domain includes amino acids 1 to 506 of the BaEV envelope glycoprotein. . In an exemplary embodiment, the transmembrane domain of the BaEV envelope glycoprotein includes or consists of the amino acid sequence SEQ ID NO: 45. In an exemplary embodiment, the wild-type extracellular domain of the BaEV envelope glycoprotein includes or consists of the amino acid sequence SEQ ID NO: 35.

As used herein, the term "MLV envelope glycoprotein" is intended to include the wild-type form of the MLV envelope glycoprotein or to be at least 85% identical to said wild-type MLV envelope glycoprotein, preferably at least 90%, more preferably at least A mutant of the wild-type MLV envelope glycoprotein that is 95%, still more preferably at least 99%, identical and retains the ability of the wild-type glycoprotein to bind to and fuse with the cell membrane.

The sequence of the natural MLV envelope glycoprotein can be found in public databases, for example, the amino acid sequence shown in SEQ ID NO: 34. As used herein, when referring to the amino acid sequence of the MLV envelope glycoprotein, it is described using the sequence set forth in SEQ ID NO: 34. For example, the expression "amino acids 623 to 655 of MLV envelope glycoprotein" refers to amino acid residues 623 to 655 of the polypeptide shown in SEQ ID NO: 34. However, those skilled in the art understand that in the amino acid sequence of the MLV envelope glycoprotein, mutations or variations (including, but not limited to, substitutions, deletions and/or additions) can occur naturally or be artificially introduced without affecting its biological function. . Therefore, in the present invention, the term "MLV envelope glycoprotein" shall include all such sequences, including for example the sequence shown in SEQ ID NO: 34 and its natural or artificial variants. Moreover, when describing the sequence fragment of the MLV envelope glycoprotein, it includes not only the sequence fragment of SEQ ID NO: 34, but also the corresponding sequence fragment in its natural or artificial variants. For example, the expression "amino acid residues 623 to 655 of MLV envelope glycoprotein" includes amino acid residues 623 to 655 of SEQ ID NO: 34, and variants thereof (natural or the corresponding fragment in artificial). According to the present invention, the expression "corresponding sequence fragment" or "corresponding fragment" means that when the sequences are optimally aligned, that is when the sequences are aligned to obtain the highest percent identity, equivalent positions are located in the compared sequences. fragment.

As is known to those skilled in the art, the MLV envelope glycoprotein consists of a cytoplasmic tail domain, a transmembrane domain, and an extracellular domain. The regions of the MLV envelope glycoprotein sequence corresponding to the cytoplasmic tail domain, transmembrane domain and extracellular domain can be readily determined by the skilled person. Typically, the cytoplasmic tail domain includes amino acids 623 to 655 of the MLV envelope glycoprotein. Therefore, amino acid residues 1-13 of the cytoplasmic tail domain of the MLV envelope glycoprotein generally correspond to amino acids 623 to 635 of the MLV envelope glycoprotein. In an exemplary embodiment, the cytoplasmic tail domain of the MLV envelope glycoprotein consists of the amino acid sequence SEQ ID NO: 5. In an exemplary embodiment, amino acid residues 1-13 of the cytoplasmic tail domain of the MLV envelope glycoprotein are composed of amino acid residues 1-13 of the amino acid sequence SEQ ID NO: 5.

In the context of the present invention, the term "fusion-inhibitory R peptide" refers to the C-terminal part of the cytoplasmic tail domain of the envelope glycoprotein, which normally carries the tyrosine endocytic signal -YXXL and is involved in viral particle maturation It is cleaved by viral protease during the process, thereby enhancing the membrane fusion of envelope glycoproteins. Fusion-inhibiting R peptides in various envelope glycoproteins can be readily determined by, and are known to, those skilled in the art. For example, fusion-inhibiting R peptides of the BaEV envelope glycoprotein typically include amino acids 547 to 564 of the BaEV envelope glycoprotein. The fusion-inhibiting R peptide of the MLV envelope glycoprotein includes amino acids 640 to 655 of the MLV envelope glycoprotein, an exemplary sequence of which is shown in SEQ ID NO: 6.

In the context of the present invention, the term "cleavage sequence" refers to a sequence recognized and cleaved by the lentiviral protease. Typically, viral polyproteins (e.g., Gag and Gag-Pro-Pol polyproteins) are recognized and cleaved into multiple functional proteins (e.g., Pol protein can produce reverse transcriptase after cleavage) by lentiviral proteases at the final stage of replication , protease, ribonuclease H and integrase). These cleavage sequences can be divided into two types, natural cleavage sequences and synthetic cleavage sequences. The native cleavage sequences of lentiviral proteases are well known to those skilled in the art, see e.g. József

Viruses 2010, 2, 147-165; doi:10.3390/v2010147.

As used herein, the term "chimeric antigen receptor (CAR)" refers to a receptor having one or more intracellular signaling domains that engage an immune cell (e.g., NK cell, B cell, T cell) Engineered immune cell receptors with extracellular antibody-derived targeting domains (e.g., scFv). T cells that express such chimeric antigen receptors are called CAR-T cells. Typically, an immune cell capable of expressing a CAR will have an antigen specificity determined by the targeting domain of the CAR. Methods of constructing and manufacturing CARs (e.g., for cancer treatment) are known in the art, see, e.g., Park et al., Trends Biotechnol., 29:550-557, 2011; Grupp et al., N Engl J Med., 368:1509-1518, 2013; Han et al., J. Hematol Oncol., 6:47, 2013; PCT patent publications WO2012/079000, WO2013/059593; and US patent publication 2012/0213783, all of which are incorporated by reference in their entirety Incorporated herein.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell. Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40). A vector can contain a variety of expression-controlling elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication site.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, which includes, but is not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, etc. Insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.

Those skilled in the art will understand that the design of the expression vector may depend on factors such as the choice of host cell to be transformed, the desired level of expression, and the like. A vector can be introduced into a host cell to thereby produce transcripts, proteins, or peptides, including proteins, fusion proteins, isolated nucleic acid molecules, and the like as described herein.

As used herein, the term "codon degeneracy" refers to the property of the genetic code that allows nucleotide sequence variation without affecting the amino acid sequence of the encoded polypeptide. The skilled artisan is familiar with the "codon bias" exhibited by a particular host cell in the use of nucleotide codons to specify certain amino acids. Therefore, when synthesizing a gene with the goal of improving expression in a host cell, it is ideal to design the gene so that its codon usage frequency is close to that of the host cell's preferred codon usage.

As used herein, the term "codon optimization," when referring to the coding region of a gene or nucleic acid molecule used to transform various organisms, refers to changing the codons in the coding region of the gene or nucleic acid molecule to reflect that of the host organism. Typical codon usage without changing the polypeptide encoded by the DNA. Such optimization involves replacing at least one or more than one or a significant number of codons with one or more codons that are more frequently used in the genes of the organism.

As used herein, the terms "transformation," "transduction," or "transfection" mean the process of introducing a "foreign" (ie, heterologous) gene, DNA, or RNA sequence into a host cell. In some cases, transformation, transduction or transfection of a foreign gene or sequence will enable the host cell to express the introduced gene or sequence and produce the desired substance. Methods for transformation, transduction or transfection are known and include, for example, calcium phosphate transformation, polybrene transformation, protoplast fusion, electroporation, sonication methods (e.g., sonoporation), liposome transformation, visualization. Microinjection, naked DNA, plasmid vectors, viral vectors, gene gun (microparticle bombardment), silicon carbide-mediated transformation, aerosol beam, or PEG transformation, etc.

According to the present invention, the term "effective amount" means an amount sufficient to obtain, or at least partially obtain, the desired effect. For example, a disease-preventing effective amount refers to an amount sufficient to prevent, prevent, or delay the occurrence of a disease; a disease-treating effective amount refers to an amount sufficient to cure or at least partially prevent the disease and its complications in patients who already suffer from the disease. Determining such effective amounts is well within the capabilities of those skilled in the art. For example, the amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall status of the patient's own immune system, the patient's general condition such as age, weight and gender, the manner in which the drug is administered, and other treatments administered concurrently etc.

Beneficial effects of the invention

The inventor of the present application prepared a modified virus envelope protein through extensive experiments, and further prepared pseudotyped virus-like particles containing it. Compared with the prior art, the modified viral envelope protein improves the infection efficiency of virus-like particles on immune cells (eg, B cells, T cells, NK cells). In addition, it has been found that the toxicity of the obtained virus-like particles to ordinary host cells (eg, 293T cells) is significantly reduced.

Such pseudotyped virus-like particles are particularly useful, for example, for the efficient introduction of exogenous nucleic acids of interest (eg, exogenous nucleic acids encoding chimeric antigen receptors (CARs)) into cells (especially immune cells, e.g. B cells, T cells, NK cells), and allow them to express the product encoded by the target exogenous nucleic acid (such as chimeric antigen receptor (CAR)). The cells (especially immune cells) thus obtained can have good application prospects in many aspects, such as preventing and/or treating viral infections, bacterial infections, cancer, tumors, inflammatory diseases or autoimmune diseases.

The embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present invention and do not limit the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the accompanying drawings and the following detailed description of preferred embodiments.

Description of drawings

Figure 1 shows a schematic diagram of the connection of six BaEV envelope proteins, in which SP is the signal peptide, BaEVwt is the wild-type BaEV envelope structure, and BaEVwt contains the extracellular domain (Ectodomain) and transmembrane domain of the BaEV envelope glycoprotein. (TM) and cytoplasmic tail domain (Cytoplasm); BaEVRless contains the extracellular domain (Ectodomain) of the BaEV envelope glycoprotein, the transmembrane domain (TM) and the cytoplasmic tail domain lacking the fusion inhibitory R peptide ( Cytoplasm/R-); BaEVTR contains the extracellular domain (Ectodomain) and transmembrane domain (TM) of the BaEV envelope glycoprotein, and the MLV cytoplasmic tail domain (MLV Cytoplasm); BaEVTRless contains the BaEV envelope glycoprotein The extracellular domain (Ectodomain) and transmembrane domain (TM), as well as the MLV cytoplasmic tail domain (MLV Cytoplasm/R-) lacking the fusion inhibitory R peptide; BaEVTR-pro contains the extracellular domain of the BaEV envelope glycoprotein. Structural domain (Ectodomain) and transmembrane domain (TM), as well as the MLV cytoplasmic tail domain (MLV Cytoplasm-Pro) that replaces the pro sequence (natural HIV-1 protease cleavage sequence); BaEVTR-synt contains the BaEV envelope The extracellular domain (Ectodomain) and transmembrane domain (TM) of the glycoprotein, as well as the MLV Cytoplasm-synt that replaces the synt sequence (synthetic HIV-1 protease cleavage sequence).

Figure 2 shows a schematic diagram of the connection of two chimeric antigen receptors. BCMA CAR contains SP, BCMA scFv, CD8Hinge, CD8TM, 4-1BB and CD3ζ; BCMA CAR-sIL15 contains SP, BCMA scFv, CD8Hinge, CD8TM, 4 -1BB, CD3ζ, P2A and sIL15. Among them, SP is the signal peptide, BCMA scFv is the single chain antibody against B cell maturation antigen, CD8Hinge is the CD8 hinge region, CD8TM is the CD8 transmembrane region, 4-1BB is the costimulatory factor, and CD3ζ is the intracellular signaling domain of CD3ζ. , P2A is a self-cleaving polypeptide, and sIL15 is secreted interleukin 15.

Figure 3 shows the results of flow cytometry analysis of Jurkat and NK92 cells after transfection of virus-like particles constructed with five envelope proteins respectively.

Figure 4 shows the results of flow cytometry analysis of primary NK cells after they were transfected with viruses constructed from five envelope proteins.

Figure 5 shows the cell morphology after transfection of 293T cells with vectors encoding different envelope proteins. Figure 5A shows the cell morphology after transfection of 293T cells with vectors encoding BaEVRless. Figure 5B shows the cell morphology after transfection with vectors encoding BaEVTRless. The cell morphology of 293T cells after transfection with a vector encoding BaEVTR. Figure 5C shows the cell morphology of 293T cells after transfection with a vector encoding BaEVTR. Figure 5D shows the cell morphology of 293T cells after transfection with a vector encoding BaEVTR-pro. Figure 5E shows the cell morphology after transfection with a vector encoding BaEVTR-pro. Cell morphology after transfection of 293T cells with synt vector.

Figure 6 shows the results of flow cytometry analysis of human T lymphocytes after transfecting virus-like particles constructed with five envelope proteins respectively.

sequence information

Information on partial sequences involved in the present invention is provided in Table 1 below.

Table 1. Sequence information

Detailed ways

The invention will now be described with reference to the following examples which are intended to illustrate but not to limit the invention.

Unless otherwise indicated, the experiments and methods described in the examples were performed essentially according to conventional methods well known in the art and described in various references. For example, for conventional techniques such as immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA used in the present invention, see Sambrook, Fritsch ) and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd ed. (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY ) (edited by F.M. Ausubel et al., (1987)); "METHODS IN ENZYMOLOGY" series (Academic Publishing Company): "PCR 2: A PRACTICAL APPROACH" ) (edited by M.J. MacPherson, B.D. Hames, and G.R. Taylor (1995)), and "ANIMAL CELL CULTURE" (R.I. Frecheni) Edited by R.I. Freshney (1987)).

In addition, if the specific conditions are not specified in the examples, the conventional conditions or the conditions recommended by the manufacturer shall be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially. Those skilled in the art will appreciate that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed. All publications and other references mentioned herein are incorporated by reference in their entirety.

Example 1. Construction of vector

1. Construction of envelope plasmid

The connection schematic diagram of the five BaEV envelope glycoproteins is shown in Figure 1. Among them, BaEVRless (the amino acid sequence is shown in SEQ ID NO: 8) is a BaEV envelope glycoprotein that lacks the fusion inhibitory R peptide and contains the BaEV envelope. The glycoprotein's extracellular domain (Ectodomain), transmembrane domain (TM) and cytoplasmic tail domain (Cytoplasm/R-) lacking the fusion inhibitory R peptide;

BaEVTR (the amino acid sequence is shown in SEQ ID NO: 10) is a BaEV envelope glycoprotein that replaces the MLV cytoplasmic tail domain. It contains the extracellular domain (Ectodomain) and transmembrane domain (Ectodomain) of the BaEV envelope glycoprotein. TM), and MLV cytoplasmic tail domain (MLV Cytoplasm);

BaEVTRless (the amino acid sequence is shown in SEQ ID NO:12) is based on BaEVTR and deletes the fusion inhibitory R peptide, which contains the extracellular domain (Ectodomain) and transmembrane domain (TM) of the BaEV envelope glycoprotein, and MLV cytoplasmic tail domain lacking fusion inhibitory R peptide (MLV Cytoplasm/R-);

BaEVTR-pro (amino acid sequence shown in SEQ ID NO: 14) is a modified BaEV envelope glycoprotein, which contains the extracellular domain (Ectodomain) and transmembrane domain (TM) of the BaEV envelope glycoprotein, and The MLV cytoplasmic tail domain (MLV Cytoplasm-Pro, the amino acid sequence is shown in SEQ ID NO: 42) that replaces the pro sequence (natural HIV-1 protease cleavage sequence);

BaEVTR-synt (amino acid sequence shown in SEQ ID NO: 16) is a modified BaEV envelope glycoprotein, which contains the extracellular domain (Ectodomain) and transmembrane domain (TM) of the BaEV envelope glycoprotein, and The MLV cytoplasm tail domain (MLV Cytoplasm-synt, the amino acid sequence is shown in SEQ ID NO: 43) that replaces the synt sequence (synthetic HIV-1 protease cleavage sequence).

The nucleotide sequences encoding five envelope proteins were synthesized respectively (the nucleotide sequence encoding BaEVRless shown in SEQ ID NO:9, the nucleotide sequence encoding BaEVTR shown in SEQ ID NO:11, SEQ ID NO The nucleotide sequence encoding BaEVTRless shown in SEQ ID NO:13, the nucleotide sequence encoding BaEVTR-pro shown in SEQ ID NO:15, and the nucleotide sequence encoding BaEVTR-synt shown in SEQ ID NO:17).

The above five nucleotide sequences were constructed into the pCMV vector respectively, single clones were selected for culture and seed conservation, and the plasmids were finally extracted for sequencing. The correctly sequenced bacterial liquid was frozen and stored for later use.

2. Construction of core plasmid

This example constructed a total of 2 chimeric antigen receptors (as shown in Figure 2). Among them, BCMA CAR contains SP, BCMA scFv, CD8Hinge, CD8TM, 4-1BB and CD3ζ; BCMA CAR-sIL15 contains SP, BCMA scFv , CD8Hinge, CD8TM, 4-1BB, CD3ζ, P2A and sIL15. Among them, SP is the signal peptide (the amino acid sequence is shown in SEQ ID NO:18), BCMA scFv is the single-chain antibody against B cell mature antigen (the VH amino acid sequence is shown in SEQ ID NO:20, and the VL amino acid sequence is shown in SEQ ID NO:22), CD8Hinge is the hinge of the chimeric receptor (the amino acid sequence is shown in SEQ ID NO:26), CD8TM (the amino acid sequence is shown in SEQ ID NO:28) is the transmembrane region of the chimeric receptor, 4 -1BB is a co-stimulatory factor (the amino acid sequence is shown in SEQ ID NO:30), CD3ζ is an intracellular signal peptide (the amino acid sequence is shown in SEQ ID NO:32), and P2A is a connecting peptide (the amino acid sequence is shown in SEQ ID NO: 38), sIL15 is super interleukin 15 (the amino acid sequence is shown in SEQ ID NO: 36).

The nucleic acid molecules encoding the above two chimeric antigen receptors were constructed into Lenti-3 lentiviral vector (Aikangde Biotechnology (Suzhou) Co., Ltd.). Select single clones for culture and conservation, and finally extract the plasmid for sequencing, and freeze the correctly sequenced bacterial liquid for later use.

Example 2. Packaging of lentivirus

293T cells were inoculated into a 15cm culture dish, and plasmid transfection was performed the next day when the cell confluence reached 80%. The BCMA CAR-sIL15 plasmid constructed in Example 1, envelope plasmid and helper plasmid pGP (Aikand Biotechnology (Suzhou) Co., Ltd.), mix thoroughly with the transfection reagent PEImax (purchased from Polyscience, Cat. No. 24765-1), and incubate at room temperature for 15 minutes. Then add the plasmid mixture dropwise to the 293T cells, shake the culture dish gently, and incubate at room temperature for 15 minutes. Mix the culture medium thoroughly and place the culture dish in a 37°C, 5% _CO2 incubator; after 6-8 hours of culture, remove the culture medium containing the transfection reagent and replace it with fresh high-glucose DMEM containing fetal bovine serum. culture medium. Harvest the virus supernatant on the third day and add fresh complete culture medium; harvest the virus supernatant on the fourth day and mix it with the virus supernatant harvested on the third day, transfer it to a centrifuge tube, and centrifuge it at 2000 × g at 4°C for 10 minutes to remove it. Cell fragments were then centrifuged at 4000 g of virus supernatant at 4°C overnight. After centrifugation, in a biological safety cabinet, carefully suck away the liquid in the centrifuge tube, add virus supernatant containing 2% HSA, aliquot and store the virus at -80°C for later use.

Example 3. Comparison of virus infectivity on Jurkat and NK92 cells

1. Refer to the steps described in Example 1 and Example 2, package the virus under the same virus packaging system and the same plasmid ratio, collect the virus stock solution and store it at 4°C for later use.

2. Centrifuge Jurkat and NK92 cells, resuspend them in culture medium, adjust the cell density to 3×10^ ⁶ cells/ml, and then plate 100 μL/well into a 24-well plate with DEAE 5 μg/ml.

3. Then add 1 ml of virus stock solution according to the corresponding wells, and centrifuge at room temperature for 90 minutes.

4. After centrifugation, place the cells at 37°C for 48 hours and then use BCMA-PE antigen (purchased from ACRO, Cat. No. BCA-HP2H2) to detect the positive rate of cells expressing CAR protein by flow cytometry.

The nucleic acid sequence encoding CAR is expressed under the drive of a promoter. Jurkat and NK92 cells transfected with lentivirus are labeled using BCMA-PE antigen and measured by flow cytometry, reflecting the expression level of CAR on the surface of Jurkat and NK92 cells. The FACS test results are shown in Table 2 and Figure 3. The results showed that under the same conditions (using the same packaging plasmid and the same core plasmid), the infection rate of the pseudovirus prepared using the BaEVTR-pro envelope plasmid reached 92.17% and 96.05% on NK92 and Jurkat cells; using BaEVTR- The infection rates of pseudoviruses prepared from synt envelope plasmids on NK92 and Jurkat cells reached 78.55% and 94.32% respectively; significantly better than pseudoviruses prepared using other envelope plasmids.

Table 2: BCMA CAR positive rate detection results of NK92 and Jurkat cells

Example 4. Comparison of virus infectivity on primary NK cells

1. Refer to the steps of Example 1 and Example 2 to prepare BCMA CAR virus concentrates with different virus envelope structures. All virus concentrates are packaged using the same packaging conditions, and the same concentration multiples are used for virus concentration, and the resulting virus is concentrated. The solution was stored at -80°C for later use.

2. Resuscitate cord blood CBMC, use magnetic beads to remove T lymphocytes, culture with MACS basic medium + 10% FBS + 500IU IL2 + 1% NK MACS supplement, and adjust the cell density to 1×10^ ⁶ /mL , add K562 feeder cells at a ratio of 1:1, and place them in a 37°C, 5% CO ₂ incubator for static culture.

3. After 3-4 days of culture, mix the transfer agent Novonectin with the virus concentrate, incubate at 37°C for 30 minutes, add umbilical cord blood cells (including NK cells), mix and place in a 37°C, 5% CO ₂ incubator Standing transduction.

4. After 24 hours of transduction, centrifuge and change the medium, resuspend the NK cells in fresh MACS basal medium + 10% FBS + 500IU IL2 + 1% NK MACS Supplement, and adjust the cell density to 1-2 × 10^ ⁶ /mL. After continuing to culture for 48 hours, BCMA-PE antigen was used to detect the positive rate of cells expressing CAR protein by flow cytometry.

The results of BCMA CAR positive rate detection of umbilical cord NK cells by FACS are shown in Figure 4. In the virus stock solution obtained under the same packaging conditions, the infection efficiency of the virus packaged by the BaEVTR-pro envelope plasmid on umbilical cord NK cells reached 82%, and the infection efficiency of the virus packaged by the BaEVTR-synt envelope plasmid on the umbilical cord NK cells reached 82%. The infection rate of viruses packaged by BaEVTR-pro envelope plasmid and BaEVTR-synt envelope plasmid on umbilical cord NK cells was significantly better than that of other envelope structures (the infection rate of BaEVTR envelope structure was 56% and that of BaEVTRless envelope structure infection rate 36%).

Example 5. Effects of different envelope structures on the cell status of packaging cells 293T

Referring to Example 2, during the plasmid transfection step of the virus packaging process, the packaging cells 293T were photographed 48 hours after plasmid transfection to observe the cytotoxicity of lentiviruses with different envelope structures to the packaging cells.

The results are shown in Figure 5. The BaEVRless (Figure 5A) and BaEVTRless (Figure 5B) enveloped viruses produced obvious cytotoxicity to the packaging cells 293T. The adherent cells were reduced and the cells fell off in clumps. However, BaEVTR (Figure 5C), BaEVTR-pro (Figure 5D) and BaEVTR-synt (Figure 5E) enveloped viruses caused less damage to packaging cells 293T, and the cells adhered well. In general, regardless of whether the cytoplasmic tail is replaced or not, removal of the R peptide of the BaEV envelope protein will increase the toxicity to the packaging cells and is not conducive to stable and effective toxin production by the packaging cells; and by replacing the cytoplasmic tail of the HIV protease, Sequences, such as BaEVTR-pro and BaEVTR-synt, not only reduce the cytotoxicity of the envelope protein, but also increase the toxin production efficiency of the virus (as shown by the results of Example 3 and Example 4).

Example 6. Virus titer detection

1. Cell plating: Jurkat cells were seeded at a density of 1×10 ⁵ cells/well, and the DEAE working concentration was 5 μg/ml. The cells were seeded in a 24-well plate at a density of 300 μl/well;

2. Virus dilution: Use cell culture medium as virus dilution, prepare 8 EP tubes, add 0.95ml medium to the first tube, and add 0.5ml medium to each of the others; follow the dilution ratio of 1:1, 1:2, 1: 4, 1:8, 1:16, 1:32, 1:64, 1:128 for dilution; add 50μl virus concentrate to the first tube, mix well, take 500μl into the second tube, mix well, take 500μl into the second tube 3 tubes, and so on. Then add the corresponding 200 μl virus diluent to each well of the 24-well plate with cells spread, and mix well.

3. Centrifuge at 800g for 90 minutes at room temperature; after centrifugation, gently blow the cells evenly and place the 24-well plate in a cell culture incubator.

4. After 24 hours of culture, aspirate the infection medium, add 1 ml of fresh complete culture medium to each well, and continue to culture for 2 days;

5. 72 hours after infection, take out the 24-well plate, aspirate some cells for antibody incubation, and flow cytometric detection;

6. Use cell samples not infected with the virus as the background signal, read the positive percentage of each sample, select the sample with a positive percentage closest to 20% in the 20%-50% range, and record the corresponding virus dilution factor.

Sample virus titer TU/ml = [(sample positive rate - background well positive rate) × total cell number]/virus volume ml.

The lenti-BCMA CAR-sIL15 virus titer test results showed that the virus titer of BaEVTR-pro envelope reached 2.16×10^ ⁸ TU/mL; the virus titer of BaEVTR-synt envelope reached 8.08×10^ ⁷ TU/mL. , second only to BaEVTR-pro; the virus titers of both are significantly higher than the other three virus envelopes, that is, the virus titer of the BaEVRless envelope is 4.45×10 ⁷ TU/mL, and the virus titer of the BaEVTRless envelope is 2.28 ×10 ⁷ TU/mL, the virus titer of BaEVTR envelope is 2.85 × 10 ⁷ TU/mL. Therefore, by replacing the HIV protease cleavage sequence of the BaEVTR cytoplasmic tail with pro or synt, the activity titer of the virus can be significantly increased.

Example 7. Transduction efficiency of modified envelope lentivirus in other immune cells

1. Transduction Efficiency in Human T Lymphocytes

Resuscitate human PBMC, use anti-CD3/CD28 magnetic beads to separate T lymphocytes from PBMC, use X-VIVO+10% FBS+200IU IL-2 to adjust the cells to 1-2×10^ ⁶ /mL, Place in a 37°C, 5% _CO2 incubator for 48h. Centrifuge the T cells, resuspend them in culture medium, adjust the cell density to 2×10^ ⁶ cells/ml, plate 1mL/well on a 24-well plate, add 100μL of virus concentrate, add DEAE 5μg/ml; centrifuge and transduce, liter 5 minus 1, and centrifuged at room temperature for 90 minutes. After centrifugation, the cells were cultured at 37°C for 48 hours, and BCMA-PE antigen was used to detect the positive rate of cells expressing CAR protein by flow cytometry.

The results are shown in Table 3 and Figure 6. The virus packaged by BaEVTR-pro envelope plasmid has an infection efficiency of 87.56% on human T lymphocytes, which is significantly higher than other envelope structures.

Table 3. Transduction efficiency of human T lymphocytes

Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details based on all teachings that have been published, and these changes are within the protection scope of the present invention. . The full scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

A modified viral envelope protein that contains the transmembrane domain and extracellular domain of the baboon endogenous virus (BaEV) envelope glycoprotein, as well as a modified cytoplasmic tail domain. The modified cytoplasmic tail domain includes amino acid residues 1-13 of the cytoplasmic tail domain of murine leukemia virus (MLV) envelope glycoprotein, the cleavage sequence of lentiviral protease, and fusion inhibition Sexual R peptide.
The viral envelope protein of claim 1, wherein the modified cytoplasmic tail domain is composed of amino acid residues 1-13 of the cytoplasmic tail domain of the MLV envelope glycoprotein and the cleavage sequence of lentiviral protease, And composed of fusion inhibitory R peptide.
The viral envelope protein of claim 1, the lentiviral protease is selected from the protease of human immunodeficiency virus HIV (for example, HIV-1, HIV-2); preferably, the lentiviral protease is HIV-1 protease .
The viral envelope protein of claim 1, wherein the cleavage sequence of the lentiviral protease is a natural HIV-1 protease cleavage sequence or a synthetic HIV-1 protease cleavage sequence.
The viral envelope protein of any one of claims 1-4, wherein the cleavage sequence of the lentiviral protease has the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.
The viral envelope protein of claims 1-5, wherein the fusion inhibitory R peptide is selected from the fusion inhibitory R peptide of MLV envelope glycoprotein; preferably, the fusion inhibitory R peptide has a value such as SEQ ID The amino acid sequence shown in NO:6.
The viral envelope protein of claims 1-6, wherein the cytoplasmic tail domain of the MLV envelope glycoprotein has the amino acid sequence shown in SEQ ID NO: 5.
The viral envelope protein of claims 1-7, wherein the modified cytoplasmic tail domain has an amino acid sequence as shown in SEQ ID NO: 42 or SEQ ID NO: 43.
The viral envelope protein of claims 1-8, wherein the extracellular domain of the BaEV envelope glycoprotein has the amino acid sequence shown in SEQ ID NO: 35;

Preferably, the transmembrane domain of the BaEV envelope glycoprotein has the amino acid sequence shown in SEQ ID NO: 45.
The viral envelope protein of claims 1-9, wherein the modified viral envelope protein has an amino acid sequence as shown in SEQ ID NO: 14 or 16.
A polynucleotide comprising a nucleotide sequence encoding the engineered viral envelope protein of any one of claims 1-10.
A vector comprising the polynucleotide of claim 11;

Preferably, the vector is selected from plasmids, phagemids, cosmids, viral vectors (such as retroviral vectors (such as lentiviral vectors), adenoviral vectors);

Preferably, the vector also contains genes required to form lentiviral particles, such as gag genes and/or pol genes;

Preferably, the vector further comprises a nucleotide sequence encoding a lentiviral protease;

Preferably, the lentiviral protease is selected from the protease of HIV (e.g., HIV-1, HIV-2);

Preferably, the lentiviral protease is HIV-1 protease;

Preferably, the lentiviral protease is capable of recognizing and cleaving the cleavage sequence of the lentiviral protease in the engineered cytoplasmic tail domain.
A host cell comprising the polynucleotide of claim 11 or the vector of claim 12.
The method for preparing the modified viral envelope protein of any one of claims 1-10, which includes culturing the host cell of claim 13 under conditions that allow protein expression.
A pseudotyped virus-like particle comprising the modified viral envelope protein according to any one of claims 1-10;

Preferably, the pseudotyped virus-like particles are lentivirus virus-like particles (such as HIV virus-like particles or SIV virus-like particles);

Preferably, the pseudotyped virus-like particles further comprise (e.g., are packaged with) a nucleic acid molecule of interest;

Preferably, the nucleic acid molecule of interest contains a gene of interest; preferably, the gene of interest encodes a protein of interest (for example, a chimeric antigen receptor);

Preferably, the chimeric antigen receptor comprises an antigen-binding domain, a transmembrane region and an intracellular region;

Preferably, the antigen-binding domain comprises a heavy chain variable region and a light chain variable region capable of specifically binding an antigen, such as a tumor antigen, such as B-cell maturation antigen (BCMA); preferably, the antigen-binding structure Domain is scFv;

Preferably, the amino acid sequence of the heavy chain variable region is SEQ ID NO: 20;

Preferably, the amino acid sequence of the light chain variable region is SEQ ID NO: 22;

Preferably, the transmembrane region includes the transmembrane domain of CD8;

Preferably, the intracellular region comprises the intracellular signaling domain of CD137 (also known as 4-1BB) and/or the intracellular signaling domain of CD3ζ;

Preferably, the chimeric antigen receptor includes an anti-BCMA scFv, a hinge region of CD8, a transmembrane domain of CD8, an intracellular signaling domain of CD137 and an intracellular signaling domain of CD3ζ; preferably, the chimeric antigen receptor The chimeric antigen receptor also includes IL-15, which can, for example, be connected to the intracellular signaling domain of CD3ζ through self-cleaving polypeptide P2A; Preferably, the chimeric antigen receptor has the structure shown in SEQ ID NO: 40 The amino acid sequence shown.
An immune cell comprising or infected with the pseudotyped virus-like particle of claim 15;

Preferably, the immune cells are mammalian (eg, human) immune cells (eg, T cells, B cells, NK cells);

Preferably, the immune cells are NK cells.
A pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent, or excipient, and one or more selected from the following:

(1) The modified viral envelope protein of any one of claims 1-10;

(2) The polynucleotide of claim 11;

(3) The carrier of claim 12;

(4) The pseudotyped virus-like particle of claim 15; and

(5) The immune cell of claim 16.
A kit comprising a first nucleic acid molecule encoding the modified viral envelope protein of any one of claims 1-10; optionally, it further comprises one or more selected from the following:

(1) One or more auxiliary nucleic acid molecules containing genes or elements required to form lentiviral virus-like particles (such as HIV virus-like particles or SIV virus-like particles); preferably, the one or more auxiliary nucleic acid molecules comprise gag gene and/or pol gene; preferably, the one or more auxiliary nucleic acid molecules also include a nucleotide sequence encoding a lentiviral protease; preferably, the lentiviral protease can recognize and cleave the modified cell The cleavage sequence of the lentiviral protease in the plasmoidal domain;

(2) A nucleic acid molecule of interest containing a gene of interest; preferably, the gene of interest encodes a protein of interest (for example, a chimeric antigen receptor);

Optionally, the kit further includes reagents for transferring nucleic acid molecules into host cells, and/or vectors for inserting nucleic acid molecules; preferably, the host cell is a mammal (e.g., human) cells; preferably, the host cell is 293T cell; optionally, the kit also contains a host cell;

Preferably, the one or more auxiliary nucleic acid molecules are contained in the same vector or different vectors;

Preferably, the one or more auxiliary nucleic acid molecules and the first nucleic acid molecule are comprised in the same vector or in different vectors.
A method for preparing the pseudotyped virus-like particles of claim 15, comprising:

(a) Transfect host cells with:

(1) The first nucleic acid molecule encoding the modified viral envelope protein of any one of claims 1-10;

(2) One or more auxiliary nucleic acid molecules containing genes or elements required to form lentiviral virus-like particles (such as HIV virus-like particles or SIV virus-like particles); preferably, the one or more auxiliary nucleic acid molecules comprise gag gene and pol gene; and

(3) Optionally, a nucleic acid molecule of interest comprising a gene of interest; preferably, the gene of interest encodes a protein of interest (for example, a chimeric antigen receptor);

(b) culturing the host cell under conditions that allow protein expression to produce pseudotyped virus-like particles;

Preferably, the cells are mammalian (eg, human) cells; preferably, the cells are 293T cells.
The modified viral envelope protein of any one of claims 1 to 10, or the polynucleotide of claim 11, or the vector of claim 12, or the pseudotyped virus-like particle of claim 15, or the polynucleotide of claim 16 The use of immune cells in the preparation of medicaments for preventing and/or treating diseases in a subject;

Preferably, the disease is a disease that can be prevented or treated by the target nucleic acid molecule; or, the target nucleic acid molecule contained (packaged) in the pseudotyped virus-like particles has a preventive or therapeutic effect on the disease;

Preferably, the disease is selected from viral infections, bacterial infections, tumors, inflammatory diseases or autoimmune diseases;

Preferably, the medicament also contains an additional pharmaceutically active agent;

Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses;

Preferably, the subject is a mammal, such as a human.
Use of the envelope protein of any one of claims 1 to 10 for preparing pseudotyped virus-like particles;

Preferably, the pseudotyped virus-like particles are capable of infecting immune cells (e.g., T cells, B cells, NK cells);

Preferably, the pseudotyped virus-like particles are lentivirus virus-like particles (eg, HIV virus-like particles or SIV virus-like particles).
Use of the pseudotyped virus-like particles of claim 15 for transferring nucleic acid molecules of interest to immune cells (e.g., T cells, B cells, NK cells) or for preparing transfer of nucleic acid molecules of interest to immune cells (e.g., The use of drugs in T cells, B cells, NK cells);

Preferably, the nucleic acid molecule of interest is contained in a vector;

Preferably, the nucleic acid molecule of interest encodes a protein of interest, such as a chimeric antigen receptor.
A method of transferring a nucleic acid molecule of interest into immune cells (e.g., T cells, B cells, NK cells), which includes infecting the immune cells with the pseudotyped virus-like particles of claim 15, wherein, Pseudotyped virus-like particles contain (package) the nucleic acid molecule of interest;

Preferably, the nucleic acid molecule of interest is contained in a vector;

Preferably, the nucleic acid molecule of interest encodes a protein of interest, such as a chimeric antigen receptor;

Preferably, the method is performed in vitro, ex vivo or in vivo.
A method of preventing or treating a disease in a subject, the method comprising administering to the subject a prophylactically or therapeutically effective amount of the pseudotyped virus-like particle of claim 15 or the immune cell of claim 16 or the immune cell of claim 16 The pharmaceutical composition of 17 or the kit of claim 18;

Preferably, the disease is a disease that can be prevented or treated by the target nucleic acid molecule; or, the target nucleic acid molecule contained (packaged) in the pseudotyped virus-like particles has a preventive or therapeutic effect on the disease;

Preferably, the disease is selected from viral infections, bacterial infections, tumors, inflammatory diseases and autoimmune diseases;

Preferably, the subject is a mammal, such as a human;

Preferably, the method further comprises administering to the subject an additional pharmaceutically active agent;

Preferably, the additional pharmaceutically active agent is a drug with anti-tumor activity, such as alkylating agents, mitotic inhibitors, anti-tumor antibiotics, antimetabolites, topoisomerase inhibitors, tyrosine kinase inhibitors, radioactive Nuclides, radiosensitizers, anti-angiogenic agents, cytokines, molecular targeted drugs, immune checkpoint inhibitors or oncolytic viruses.