Virus vector, packaging cell and application thereof

Abstract

The invention discloses a virus vector, a packaging cell and application thereof, and belongs to the technical field of virus vectors and cell transfection. According to the present invention, the virus vector is subjected to gene modification, such that the surface of the virus particle carries the cell activation element (polypeptide), such as the T cell activation element or the NK cell activation element, such that the virus can activate the resting cells in the peripheral blood so as to improve the immune cell transfection efficiency;

Description

Technical Field

[0001] The present application belongs to the technical field of viral vectors and cell transfection, and specifically relates to viral vectors, packaging cells and their applications. Background Art

[0002] Transfection of host cells using lentiviruses (e.g., HIV-1) has shown promise in achieving stable and long-term expression of target genes, making it particularly suitable for immunotherapy applications. Lentiviruses can transfect both dividing and non-dividing mammalian cells. However, transfection efficiency of resting cells (e.g., resting T cells) remains low. Traditional T cell transfection methods typically involve isolating cells, activating them through TCR or factor signaling, and then transfecting them. This process is time-consuming and may extend the overall duration of treatment. Summary of the invention

[0003] When lentiviruses (such as HIV-1) transfect host cells, the exogenous genes they carry can be randomly and stably integrated into the host cell genome, achieving stable and long-term expression of the target gene, which is very suitable for immunotherapy. Lentiviruses can transfect dividing and non-dividing mammalian cells, but the transfection efficiency of resting cells (such as resting T cells) is very low. Transfection of T cells usually requires first isolating T cells, activating T cells through TCR or factor signals, and then transfecting them. This process prolongs the transfection time.

[0004] Embodiments of the present disclosure relate to lentiviral vectors. In an embodiment, the lentiviral vector is genetically modified so that the surface of the virus particles carries a cell activation element (polypeptide), such as a T cell activation element or an NK cell activation element, so that the virus can activate resting cells in the peripheral blood, thereby improving the transfection efficiency of immune cells. In addition, after the virus is injected intravenously into the subject, the virus packaged by the vesicular stomatitis virus G envelope protein (VSVG) will be inactivated by the influence of complement proteins in the subject's blood. In order to make the VSVG-packaged virus more stable in the subject, the virus particles can be modified with polyethylene glycol (PEG). For example, the virus packaging system uses a transfer plasmid, Gag-Pol, Rev and VSVG to infect 293T cells to obtain virus particles. In order to carry a cell activation element on the surface of the virus particle, VSVG needs to be modified. The modification method is to connect the cell activation element to the N-terminus of VSVG. The envelope glycoproteins of other viruses, such as Moloney measles virus (Mo-MV) envelope glycoprotein, Nipah virus (NiV) or amphotropic murine leukemia virus (MLV-A) glycoprotein, can be used to replace VSVG. The modification involves attaching a cell activation element to the N-terminus or C-terminus of the glycoprotein. Among them, the Mo-MV envelope glycoprotein consists of hemagglutinin (H) and fusion (F) proteins, and the cell activation element is attached to the N-terminus of the H protein, so the virus packaging system consists of 5 plasmids (master plasmid, Gag-Pol, Rev, H, F).

[0005] In the embodiment, 293T cells are initially transfected with plasmids encoding cell activation elements used as transfer vectors and additional plasmids carrying Gag-Pol, Rev and VSVG genes. This results in the production of viruses expressing cell activation elements on their surfaces. These newly produced viruses carry cell activation elements and are then used to infect a new batch of 293T cells. This process ensures that these cells are "modified" and now stably express cell activation elements on their surfaces. The expression of cell activation elements in these 293T cells was confirmed by a screening process. Successfully modified 293T cells are used for the next stage. Finally, these modified 293T cells that stably express cell activation elements are used to package slow viruses. This is accomplished by transfecting these cells with necessary plasmids (including master plasmids, Gag-Pol, Rev and envelope (Env) proteins). The slow viruses produced by these cells will display cell activation elements on their surfaces.

[0006] In an embodiment, polynucleotides encoding antigen binding molecules and / or therapeutic agents can be used to implement the technology described herein. The method or use includes: providing a viral particle (e.g., AAV, slow virus or its variant) comprising a vector genome, wherein the vector genome comprises a polynucleotide, wherein the polynucleotide is operably linked to an expression control element that confers transcription of the polynucleotide; administering a certain amount of viral particles to a subject so that the polynucleotide is expressed in the subject.

[0007] In an embodiment, the AAV preparation may include AAV vector particles, empty capsids, and host cell impurities, thereby providing an AAV product substantially free of AAV empty capsids. For more information on the administration and preparation of viral particles, reference may be made to U.S. Patent No: 9840719 and Milani et al. Sci. Transl. Med. 11, eaav7325 (2019) 22 May 2019, which are incorporated herein by reference.

[0008] In embodiments, the polynucleotide may be integrated into the genome of the modified cell, and progeny of the modified cell will also express the polynucleotide, thereby generating a stably transfected modified cell.

[0009] In embodiments, the modified cell expresses a polynucleotide encoding CAR, but the polynucleotide is not integrated into the genome of the modified cell, so that the modified cell expresses a polynucleotide of transient transfection for a limited period of time (e.g., a few days), after which the polynucleotide is lost due to cell division or other factors. For example, the polynucleotide is present in a recombinant DNA construct, mRNA or viral vector in a modified cell, and / or the polynucleotide is mRNA, which is not integrated into the genome of the modified cell. In embodiments, the vector is a slow virus. In embodiments, a slow virus can be packaged with a particle (e.g., a nanoparticle) to release a predetermined time and directly infuse into a subject so that a slow virus can be transferred to a subject's T cell.

[0010] An embodiment relates to a method for treating cancer in a subject, the method comprising: administering a viral vector comprising a nucleic acid encoding a CAR molecule to the subject's peripheral blood system, such that cells, organs or tissues of the subject's peripheral vascular system express the CAR molecule; and monitoring the cellular response, such as a T cell response, caused by the expression of the CAR molecule in the subject.

[0011] An embodiment relates to a method for expressing an antigen binding molecule in vivo in lymphocytes of a subject, the method comprising: administering a viral vector comprising a nucleic acid encoding the antigen binding molecule to the peripheral blood system of the subject, such that the cells, organs, peripheral vascular system or tissues of the subject express the antigen binding molecule.

[0012] Examples of viral vectors include adeno-associated virus (AAV) vectors. AAV is a small, non-pathogenic virus of the Parvoviridae family. AAV differs from other members of this family in that it relies on a helper virus for replication. In the absence of a helper virus, AAV can integrate into the q arm of chromosome 19 in a site-specific manner. The approximately 5kb AAV genome consists of a stretch of single-stranded DNA with positive and negative polarity. At the ends of the genome are short inverted terminal repeats that can fold into a hairpin structure and serve as the origin of viral DNA replication. Physically, the parvovirus virion is non-enveloped, and its icosahedral capsid is approximately 20 to 30nm in diameter.

[0013] The adeno-associated virus (AAV) capsid contains three related proteins: VP1, VP2, and VP3. VP3 is the shortest of the three proteins, with a molecular weight of approximately 59 to 61 kDa and a sequence of approximately 524 to 544 amino acids. The sequence of VP3 is shared by all three VPs and is called the VP3 common region. VP2 is approximately 57 amino acids longer than VP3, with a molecular weight of approximately 64 to 67 kDa and a sequence of approximately 580 to 601 amino acids. The N-terminal region of the VP2 sequence is shared by VP1 and is called the VP1 / VP2 common region. VP1 is approximately 137 amino acids longer than VP2, with a molecular weight of approximately 79 to 82 kDa and a sequence of approximately 713 to 738 amino acids. The N-terminal region of the VP1 sequence is unique to VP1 and is called the VP1 unique (VP1u) region. The three capsid proteins VP1, VP2 and VP3 are typically present in the capsid in a ratio of approximately 1:1:10, respectively, although this ratio (particularly the amount of VP3) can vary greatly and should not be considered limiting in any respect.

[0014] The AAV genome has short inverted terminal repeats (ITRs) at the ends that have the potential to fold into a T-shaped hairpin structure that serves as the origin of viral DNA replication. Within the ITR region, two elements have been described that are essential for ITR function: the GAGC repeat motif and the terminal resolution site (TRS). The GAGC repeat motif has been shown to bind Rep (replication protein) when the ITR is in a linear or hairpin conformation. This binding serves to position Rep68 / 78 for cleavage at the TRS, which occurs in a site- and strand-specific manner. In addition to their role in replication, the GAGC repeat motif and TRS appear to be central to viral integration. The chromosome 19 integration site contains a binding site for Rep to the adjacent TRS. The GAGC repeat motif and TRS have been shown to be functional for site-specific integration. AAVs can be used as gene therapy vectors because they can penetrate cells and introduce nucleic acids / genetic material so that the nucleic acids / genetic material can be stably maintained in the cells.

[0015] In addition, these adeno-associated viruses (AAV) can introduce nucleic acids / genetic material into specific sites, such as specific sites on chromosome 19. Since AAV is not related to human pathogenic diseases, the ability of AAV vectors to deliver heterologous viral polynucleotide sequences encoding therapeutic proteins and drugs will not cause substantial AAV pathogenesis or disease in human patients. Therefore, recombinant AAV (rAAV) vectors, including serotypes and variants, provide a means for delivering protein-encoding nucleic acids to cells in vitro, in vitro, and in vivo, so that the cells express the encoded protein. For example, rAAV vectors can include heterologous nucleic acids encoding a desired protein or peptide. Delivery or administration of the vector to a subject, such as a mammal, provides the encoded protein to the subject. For more information on gene therapy and its clinical uses, see U.S. Patent Nos. 9,433,688, 9,644,216, and 10,113,183; and Milone, MC, O'Doherty, U. Clinical use of lentiviral vectors, Leukemia 32, 1529-1541 (2018); Rodrigues, GA, Shalaev, E., Karami, TK et al. Pharmaceutical Development of AAV-Based Gene Therapy Products for the Eye. Pharm Res 36, 29 (2019) and Marquez Loza, LI; Yuan, EC; McCray, PB, Jr. et al. Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease. Genes 2019, 10, 218; the entire contents of which are incorporated by reference.

[0016] Modified AAV vectors, such as recombinant nucleic acids and polypeptides, are AAV vectors whose sequences have been manipulated (e.g., engineered or recombined) in a manner that the vector or sequence does not exist in nature. A specific example of a recombinant AAV vector is a vector in which a nucleic acid that is not normally present in a wild-type viral genome (e.g., an AAV genome) is inserted into the viral genome and is heterologous to the viral genome. A "recombinant" AAV vector is different from an AAV genome because all or part of the viral genome has been replaced by a non-natural sequence (e.g., a heterologous nucleic acid sequence) relative to the AAV genome nucleic acid. Although the term "recombinant" is not always used herein to refer to AAV vectors and their sequences, such as nucleic acids and polypeptides, recombinant forms of AAV and sequences including nucleic acids and polypeptides are clearly included, despite any such omissions. Typically for AAV, one or two reverse terminal repeat (ITR) sequences of the AAV genome are retained in the AAV vector. Therefore, the incorporation of non-natural sequences defines the AAV vector as a "recombinant" AAV (rAAV) vector.

[0017] Recombinant AAV vectors can be packaged as "particles" for subsequent cell transduction, such as ex vivo, in vitro or in vivo transduction. When the recombinant AAV vector is encapsidated or packaged into AAV particles, the particles may also be referred to as "rAAV" herein. Such particles include proteins that encapsidate or package the vector genome, and for AAV, also include capsid proteins. "AAV virus particles" or "AAV particles" refer to virus particles composed of at least one AAV capsid protein (usually all three capsid proteins of AAV) and capsid nucleic acid (referred to as vector genome). If the particle contains heterologous nucleic acid, it is generally referred to as "rAAV".

[0018] The AAV vector "genome" refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form AAV particles. In the case of using a recombinant plasmid to construct or manufacture a recombinant AAV vector, the AAV vector genome does not include the portion of the "plasmid" that does not correspond to the vector genome sequence of the recombinant plasmid. The non-vector genome portion of the recombinant plasmid is called the "plasmid backbone", which is very important for the cloning and amplification of the plasmid, which is a process required for propagation and recombinant AAV production, but it is not itself packaged or encapsidated into rAAV particles.

[0019] In the embodiment, the rAAV vector includes a capsid derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, Rh10, Rh74 or AAV-2i8, and variants thereof, such as variants in which the capsid has amino acid insertions, additions and substitutions. AAV vector serotypes and variants include capsid variants, such as LK03, 4-1, etc. rAAV serotypes and rAAV variants can be different from other AAV serotypes (e.g., different from VP1, VP2 and / or VP3 sequences), or they can be different. The term "serotype" is used to refer to the difference between AAVs with capsids that are serologically different from other AAV serotypes. Serological uniqueness is determined based on the lack of cross-reactivity between one AAV antibody and another AAV antibody. This cross-reactivity difference is usually due to differences in capsid protein sequences / antigenic determinants, for example, due to differences in VP1, VP2 and / or VP3 sequences of AAV serotypes. Although AAV variants (including capsid variants) may be serologically indistinguishable from a reference AAV or other AAV serotypes, AAV variants differ from the reference or other AAV serotypes by at least one nucleotide or amino acid residue.

[0020] Recombinant AAV vectors and methods and uses thereof include any strain or serotype. As a non-limiting example, the recombinant AAV vector genome can be based on any AAV genome, such as AAV-1, -2, -3, -4, -5, -6, -7, -8, 9, -10, -11, -rh74, -rhlO or AAV-2i8. Such vectors can be based on the same strain or serotype (or subgroup or variant) or different from each other. As a non-limiting example, the recombinant AAV vector genome based on a serotype genome can be the same as one or more capsid proteins that package the vector. In addition, the recombinant AAV vector genome can be based on an AAV, e.g., AAV2, serotype genome that is different from one or more capsid proteins that package the vector, in which case at least one of the three capsid proteins can be from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, RhlO, Rh74 or AAV-2i8 or a variant, e.g., a capsid variant such as LK03, 4-1, etc. Thus, an AAV vector can include nucleic acid and / or protein sequences that are identical to nucleic acid and protein sequences that are characteristic of a particular serotype.

[0021] "AAV ITR" or "AAV ITRs" refers to the art-recognized regions found at each end of the AAV genome that function together in cis as an origin of DNA replication and as a packaging signal for the virus. The AAV ITR, together with the AAV rep coding region, provides for efficient excision and rescue of the nucleotide sequence inserted between the two flanking ITRs and their integration into the mammalian cell genome.

[0022] The nucleotide sequence of AAV ITR is known. "AAV ITR" need not have the wild-type nucleotide sequence described, but can be altered by, for example, insertion, deletion or substitution of nucleotides. In addition, AAV ITR can be derived from any of several AAV serotypes. In addition, the 5' and 3' ITRs flanking the heterologous nucleic acid sequence in the AAV vector do not have to be identical or derived from the same AAV serotype or isolate, as long as they function as intended, such as allowing excision and separation. Rescue the sequence of interest from the host cell genome or vector and allow the heterologous sequence to be integrated into the recipient cell genome.

[0023] In contrast to a "capsid-containing genome" that contains an AAV vector genome, an AAV "empty capsid" as used herein does not contain a vector genome (hence the term "empty"). Empty capsids are virus-like particles because they react with one or more antibodies that react with intact viruses (e.g., containing the genome of an AAV vector). AAV empty capsids are believed to bind or react with antibodies against AAV vectors, thereby acting as a bait to reduce the inactivation of AAV vectors. This bait acts to absorb antibodies against AAV vectors, thereby increasing or improving AAV vector transduction of cells (introduction of transgenes), thereby increasing cellular expression of transcripts and / or encoded proteins. Empty capsids can be generated and purified to a desired quality, and their quantity can be determined. For example, the empty capsid titer can be measured by spectrophotometry by optical density at a wavelength of 280 nm (based on Sommer et al., Mol. Ther. 2003 Jan; 7(1): 122-8). Empty AAV or empty capsids sometimes exist naturally in AAV vector preparations. Such natural mixtures can be used as described herein, or if necessary, can be operated to increase or decrease the amount of empty capsids and / or carriers. For example, the amount of empty capsids can be optionally adjusted to the amount of the inhibitory effect of the antibody that is expected to reduce the reaction with the AAV vector, and the AAV vector is intended to be used for vector-mediated gene transduction in the subject. The use of empty capsids is described in the U.S. Publication 2014 / 0336245. In embodiments, AAV empty capsids are prepared and / or applied to the subject together with rAAV vectors. In embodiments, AAV empty capsids are prepared with less than or equal amounts of carriers, such as about 1.0 to 100 times of AAV vectors and AAV empty capsids, or about 1:1 ratios of AAV vectors and AAV empty capsids. In embodiments, AAV vectors are prepared with excessive AAV empty capsids, such as more than 1 times of AAV vectors with AAV empty capsids, or 1.0 to 100 times of AAV vectors with AAV empty capsids.

[0024] In embodiments, the ITR pair is derived from or obtained from or comprises ITR sequences from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, LK01, LK02, LK03, AAV 4-1, AAV-2i8 ITRs, or a combination thereof.

[0025] In embodiments, the one or more AAV capsid proteins are derived from or obtained from or comprise one or more capsid protein sequences from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, LK01, LK02, LK03, AAV 4-1, AAV-2i8ITR, or a combination thereof.

[0026] In an embodiment, the rAAV vector further comprises one or more of introns, expression control elements, stuffer polynucleotide sequences and / or poly A signals, or a combination thereof.

[0027] rAAV vectors, rAAV particles, and pharmaceutical compositions thereof can be packaged in single or multiple unit dosage forms for ease of administration and uniformity of dosage. "Unit dose" or "unit dosage form" as used herein refers to physically discrete units suitable as unit doses for subjects to be treated; each unit contains a predetermined amount, optionally in combination with a pharmaceutical carrier (excipient, diluent, vehicle, or filler), which is calculated to produce a desired effect (e.g., a preventive or therapeutic effect) when administered in one or more doses. The unit dosage form can be in, for example, ampoules and vials, which can include liquid compositions or compositions in a freeze-dried or lyophilized state; for example, a sterile liquid carrier can be added prior to in vivo administration or delivery. A separate unit dosage form can be contained in a unit dose or multi-dose kit or container.

[0028] In one embodiment, the rAAV particles are present at a concentration of about 1×10 8 Up to 1×10 10 , 1×10 10 Up to 1×10 11 , 1×10 11 Up to 1×10 12 , 1×10 12 Up to 1×10 13 , or 1×10 13 Up to 1×10 14 In one embodiment, the rAAV particles are administered in a dose range of less than 1×10 10 The dose was administered at 1 vector genome per kilogram of subject (vg / kg).

[0029] In one embodiment, the rAAV particles are administered at a concentration of about 5×10 11 In one embodiment, the amount of rAAV particles administered is at least 1×10 per kilogram of subject body weight (vg / kg). 10 vector genomes (vg), or approximately 1×10 10 Up to 1×10 11 vg / kg subject body weight, or approximately 1×10 11 vg / kg subject weight. to 1×10 12 vg / kg (e.g., about 1×10 11 Up to 2×10 11 vg / kg or about 2×10 11 Up to 3×1011 vg / kg or about 3×10 11 Up to 4×10 11 vg / kg or about 4×10 11 Up to 5×10 11 vg / kg or about 5×10 11 Up to 6×10 11 vg / kg or about 6×10 11 Up to 7×10 of the subject's body weight 11 vg / kg or about 7×10 11 Up to 8×10 11 vg / kg or about 8×10 11 Up to 9×10 11 vg / kg or about 9×10 11 Up to 1×10 12 vg / kg, or about 1×10 of the subject's body weight. 10 Up to 1×10 12 vg / kg to achieve the desired therapeutic effect.

[0030] In embodiments, the subject does not mount a substantial immune response to the rAAV particles.

[0031] In embodiments, the subject does not develop a substantial humoral immune response to the rAAV particles.

[0032] In embodiments, the subject does not mount a significant immune response to the rAAV particles for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, weeks or months.

[0033] In embodiments, the subject does not develop a detectable immune response to the rAAV particles.

[0034] In embodiments, the subject does not develop an amount of an undesirable immune response to the therapeutic protein and / or rAAV particles that blocks the therapeutic effect of the therapeutic protein in the subject for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 consecutive days, weeks or months.

[0035] More information regarding AAV vectors, production of vectors, pharmaceutical compositions comprising AAV vectors, and methods thereof can be found in U.S. Pat. No. 9,433,688, which is incorporated by reference in its entirety.

[0036] In an embodiment, the viral vector is a rAAV particle comprising an AAV capsid protein and a vector comprising a nucleic acid encoding an antigen binding molecule, wherein the nucleic acid is effective to infect cells, organs, or tissues of the peripheral blood system of a subject such that the cells, organs or tissues express the antigen binding molecule.

[0037] In embodiments, administering the viral vector to a subject comprises infusing or injecting the viral vector into the subject's systemic circulation. In embodiments, administering comprises intravenous, intra-arterial infusion or injection into the subject's systemic circulation.

[0038] In an embodiment, AAV is used to deliver an antigen binding molecule to a subject. The antigen binding molecule is a CAR comprising an extracellular domain, a transmembrane domain, and an intracellular domain, and the extracellular domain binds to an antigen.

[0039] The embodiments describe polynucleotides comprising nucleic acids encoding one or more lymphocyte activation elements, such as T cell activation elements, nucleic acids encoding protamine, and nucleic acids encoding signal peptides. In some embodiments, the signal peptide is capable of directing the expression of one or more lymphocyte activation elements on the surface of recombinant viral particles. In some embodiments, the polynucleotide encoding the signal peptide comprises an MLV-A glycoprotein signal peptide, and / or wherein the polynucleotide further comprises a nucleic acid encoding an MLV-A glycoprotein, an anti-CD3 antibody, and protamine. In embodiments, the polynucleotide comprises SEQ ID NO: 1 or a polynucleotide encoding SEQ ID NO: 3 or 5. In embodiments, lymphocyte activation elements refer to molecules that can activate lymphocytes, particularly T or NK cells. These elements can be antibodies or other molecules that bind to specific receptors on the surface of lymphocytes. For example, anti-CD3 and anti-CD28 are mentioned as lymphocyte activation elements for T cells, while anti-CD2 and anti-CD335 are mentioned as lymphocyte activation elements for NK cells. These elements are encoded by polynucleotides and can be expressed on the surface of recombinant viral particles to enhance their interaction with lymphocytes.

[0040] The embodiment describes a packaging cell for producing viral particles but not capable of producing replication-competent infectious viruses, wherein the packaging cell comprises (1) the above-mentioned polynucleotides, (2) one or more polynucleotides encoding one or more auxiliary factors for assembling competent viral particles for transfection, and (3) a gene of interest, such as a gene encoding a CAR associated with a packaging signal. The viral particles can infect T or NK cells. In embodiments, the packaging cell is a 293T cell line. In embodiments, the packaging cell comprises a lentiviral plasmid containing a gene of interest, such as a packaging plasmid encoding a protein for entry and integration of the viral genome, and an envelope plasmid encoding an envelope protein.

[0041] The embodiments describe a method for improving viral transfection efficiency, the method comprising: producing viral particles using the packaging cells described herein; infecting PBMCs, such as T / NK cells, with the viral particles; wherein the polynucleotides in the viral particles encode MLV-A glycoprotein and protamine, and wherein the transfection efficiency of the viral particles is greater than the transduction efficiency of viral particles using packaging cells whose env gene encodes VSVG glycoprotein and the polynucleotide does not encode MLV-A glycoprotein or anti-CD3 scFv, and / or viral particles using packaging cells whose env gene does not contain protamine.

[0042] The embodiment describes a method for improving viral transduction efficiency, the method comprising: using the packaging cells described herein to produce viral particles; infecting blood cells including at least T cells and B cells with the viral particles; wherein the polynucleotide comprises a polynucleotide encoding an MLV-A glycoprotein, and the amount of infected B cells is lower than the amount of packaging cells whose env gene encodes a VSVG glycoprotein. In the embodiment, the B cells are not infected by the viral particles.

[0043] Embodiments relate to lentiviral particles comprising a mutated VSVG envelope protein that reduces its interaction with the LDL-R receptor. The lentiviral particles are additionally incorporated with nucleic acids encoding CD3 antibodies, CD28 antibodies, and / or CD80 extracellular domains, which can be expressed in T cells. The lentiviral particles also contain nucleic acids encoding CD8 or CD80 transmembrane regions, allowing antibodies and / or CD80 extracellular domains to be expressed on the surface of the virus. In embodiments, the mutated VSVG envelope protein is specifically mutated at sites K47Q and R354A.

[0044] In embodiments, the lentiviral particle expresses a CD3 antibody, a CD28 antibody, or a CD3 antibody and a CD80 extracellular domain on its surface.

[0045] The embodiments relate to lentiviral particles that exhibit limited interaction with LDL-R receptors on non-T cells.

[0046] The embodiment relates to a method for producing lentiviral particles. The method introduces nucleic acids encoding mutated VSVG envelope proteins, CD3 antibodies, CD28 antibodies and / or CD80 extracellular domains and CD8 transmembrane regions into host cells. The host cells then express these elements, assemble lentiviral particles and harvest.

[0047] In embodiments, the method further comprises purifying the lentiviral particles; contacting the T cells with the lentiviral particles in vitro or in vivo.

[0048] Embodiments are directed to a kit comprising a lentiviral particle comprising a packaged cell activator, and instructions for using the lentiviral particle in a method of targeting T cells.

[0049] The embodiment describes a method for treating a subject using CAR T cells, the method comprising obtaining T cells from a subject, and then culturing the T cells under conditions that promote their growth. Subsequently, the T cells are contacted with lentiviral particles to promote their transfection, thereby causing the nucleic acid to be integrated into the genome of the T cells. In the embodiment, the antiviral particles include CD3 antibodies and CD28 antibodies, or CD3 antibodies and CD80 extracellular domains, and the infected T cells express one or more CARs. CAR T cells are produced by the methods described herein, and these CAR T cells can be used to treat diseases and disorders, such as cancer.

[0050] Embodiments relate to polynucleotides comprising a polynucleotide encoding one or more lymphocyte activation elements, a polynucleotide encoding protamine, and a polynucleotide encoding a signal peptide.

[0051] In embodiments, the signal peptide is capable of directing the expression of one or more lymphocyte activation elements on the surface of the recombinant viral particle.

[0052] In an embodiment, the polynucleotide encoding the signal peptide comprises the MLV-A glycoprotein signal peptide.

[0053] In an embodiment, the polynucleotide further comprises a polynucleotide encoding an MLV-A glycoprotein.

[0054] The embodiment involves Figure 1 and 2 The polynucleotides, pLP1, pLP2, expression systems 1-4 of PLVSVG, and packaging units provided therein.

[0055] Embodiments relate to recombinant viral particles comprising a polynucleotide encoding an antigen binding molecule and one or more lymphocyte activation elements on the surface of the recombinant viral particle.

[0056] In embodiments, the recombinant viral particle further comprises a pseudotyping element on the surface of the recombinant viral particle.

[0057] In an embodiment, the recombinant viral particle is a replicating recombinant retroviral particle.

[0058] In embodiments, the recombinant viral particle is a lentiviral particle.

[0059] In embodiments, the one or more lymphocyte activation elements are not encoded by a polynucleotide in the recombinant viral particle.

[0060] In an embodiment, the recombinant viral particle comprises a polynucleotide encoding an antigen binding molecule or a plurality of polypeptides.

[0061] In an embodiment, the antigen binding molecule is a chimeric antigen receptor (CAR), which comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0062] In an embodiment, the antigen binding domain that binds to a tumor antigen is selected from the group consisting of: TSHR, CD19, CD123, CD22, CD30, CD171, CS-1, CLL-1, CD33, EGFRvIII, GD2, GD3, BCMA, Tn Ag, PSMA, ROR1, FLT3, FAP, TAG72, CD38, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, mesothelin, IL-11Ra, PSCA, PRSS21, VEGFR2, Lewis Y, CD24, PDGFR-beta, SSEA-4, CD20, folate receptor α, ERBB2 (Her2 / neu), MUC1, EGFR, NCAM, prostate enzymes, PAP, ELF2M, ephrin B2, IGF-I receptor, CAIX, LMP2, gp100, bcr-abl, tyrosinase, EphA2, fucosyl GM1, sLe, GM3, TGS5, HMWMAA, o-acetyl-GD2, folate receptor beta,TEM1 / CD248,TEM7R,CLDN6,GPRC5D,CXORF61,CD97,CD179a,ALK,polysialic acid,PLAC1,GloboH,NY-BR-1,UPK2,HAVCR1,ADRB3,PANX3,GPR20,LY6K,OR51E2,TARP,WT1,NY-ESO-1,LAGE-1a,MAGE-A1,legumain,HPV E6,E7,MAGE A1,ETV6-AML,sperm protein 17,XAGE1,Tie 2,MAD-CT-1,MAD-CT-2,Fos-related antigen 1,p53,p53 mutant,prostaglandin,survivin,telomerase,PCTA-1 / Galectin 8,MelanA / MART1,Ras mutant,sarcoma translocation breakpoint,ML-IAP,ERG(TMPRSS2 ETS fusion gene), NA17, PAX3, androgen receptor, cyclin B1, MYCN, RhoC, TRP-2, CYP1B1, BORIS, SART3, PAX5, OY-TES1, LCK, AKAP-4, SSX2, RAGE-1, human telomerase reverse transcriptase (hTERT), RU1, RU2, intestinal carboxylesterase, muthsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75, GPC3, FCRL5, and / or IGLL1.

[0063] In embodiments, the intracellular signaling domain comprises a co-stimulatory signaling domain, or a primary signaling domain and a co-stimulatory signaling domain, wherein the co-stimulatory signaling domain comprises a protein selected from the group consisting of CD27, CD28, 4-1BB (CD137), OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen 1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, ligand-specific binding CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7Ralpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, TRANCE / RANKL, DNAM1(CD226), SLAMF4(CD244, 2B4), CD Functional signaling domains of proteins from the group consisting of 84, CD96 (touch), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, ​​LAT, GADS, SLP-76, PAG / Cbp, NKp44, NKp30, NKp46 and / or NKG2D.

[0064] In embodiments, the antigen binding molecule is a modified TCR. In embodiments, the TCR is derived from tumor-specific T cells spontaneously generated in the patient. In embodiments, the TCR binds to tumor antigens. In embodiments, the tumor antigens include CEA, gp100, MART-1, p53, MAGE-A3 or NY-ESO-1. In embodiments, the TCR includes TCRγ and TCRδ chains or TCRα and TCRβ chains or a combination thereof.

[0065] Embodiments are directed to one or more recombinant viral particles, wherein the one or more recombinant viral particles comprise a polynucleotide encoding a CAR that binds a cell surface molecule of a white blood cell (WBC) and a polynucleotide encoding a CAR that binds a solid tumor antigen.

[0066] In embodiments, WBCs are granulocytes, monocytes, or lymphocytes. In embodiments, WBCs are B cells. In embodiments, the cell surface molecules of WBCs are CD19, CD22, CD20, BCMA, CD5, CD7, CD2, CD16, CD56, CD30, CD14, CD68, CD11b, CD18, CD169, CD1c, CD33, CD38, CD138, or CD13. In embodiments, the cell surface molecules of WBCs are CD19, CD20, CD22, or BCMA. In embodiments, the cell surface molecules of WBCs are CD19 or BCMA. In embodiments, one or more recombinant particles are implemented in vivo or ex vivo to establish the PCT publication NOS: WO2020146743 and WO2020106843 described in System, wherein solid tumor CAR and WBC CAR bind FCRL1, CLDN6, ALPP, GPC-3, MSLN. In embodiments, in vivo or ex vivo implementation of one or more recombinant particles establishes the PCT publication number: WO2019140100 described system.

[0067] In embodiments, one or more recombinant viral particles comprise a polynucleotide encoding a dominant negative form of a receptor associated with an immune checkpoint inhibitor. In embodiments, the immune checkpoint inhibitor is selected from the group consisting of programmed death 1 (PD-1), cytotoxic T lymphocyte antigen 4 (CTLA-4), B- and T- lymphocyte attenuator (BTLA), T cell immunoglobulin mucin-3 (TIM-3), lymphocyte activation protein 3 (LAG-3), T cell immune receptor with Ig and ITIM domains (TIGIT), leukocyte-associated immunoglobulin-like receptor 1 (LAIR1), natural killer cell receptor 2B4 (2B4)) and CD 160. In embodiments, the immune checkpoint inhibitor is a modified PD-1. In an embodiment, the modified PD-1 lacks a functional PD-1 intracellular domain for PD-1 signal transduction, interferes with the pathway between PD-1 of human T cells of human cells and PD-L1 of a certain cell, includes or is a PD-1 extracellular domain or a PD-1 transmembrane domain, or a combination thereof, or a modified PD-1 intracellular domain comprising a substitution or deletion compared to a wild-type PD-1 intracellular domain, or comprises or is a soluble receptor comprising a PD-1 extracellular domain, which can bind to PD-L1 of a specific cell. In an embodiment, the inhibitory effect of PD-L1 on the cytokine production of human T cells of a population is less than the inhibitory effect of PD-L1 on the cytokine production of human T cells that do not comprise at least a portion of the nucleic acid. Sequence encoding the modified PD-1.

[0068] In embodiments, one or more recombinant viral particles comprise polynucleotides encoding therapeutic agents such as cytokines. In embodiments, the therapeutic agent is or comprises IFN-γ. In embodiments, the therapeutic agent is or comprises at least one of IL-6, IFN-γ, IL-17 and / or CCL19. In embodiments, the therapeutic agent is or comprises IL-15 or IL-12, or a combination thereof. In embodiments, a small protein or therapeutic agent is or comprises a recombinant or natural cytokine. In embodiments, the therapeutic agent comprises an FC fusion protein associated with a small protein. In embodiments, a small protein is or comprises IL-12, IL-15, IL-6 or IFN-γ. In embodiments, the therapeutic agent is regulated by Hif1a, NFAT, FOXP3 and / or NFkB. In embodiments, one or more recombinant viral particles comprise polynucleotides encoding hTERT, SV40LT or a combination thereof.

[0069] In embodiments, one or more recombinant viral particles include polynucleotides encoding one or more elements of gene editing tools (ZFN, TALEN and CRISP). In embodiments, gene editing tools cause the expression of endogenous TRAC genes in cells containing one or more recombinant viral particles to be reduced. In embodiments, one or more recombinant viral particles include polynucleotides encoding suicide genes such as HSV-TK systems. In embodiments, one or more recombinant viral particles include polynucleotides connected to one or more 2A or / IRES components. The term "suicide gene" is mentioned in the context of the HSV-TK system, but is not clearly defined in the document. Generally, a suicide gene is a gene that can be introduced into a cell to make the cell sensitive to treatment that selectively kills cells. In the HSV-TK system, the herpes simplex virus thymidine kinase (HSV-TK) gene is often used as a suicide gene. Cells expressing this gene can be selectively killed by administering prodrugs such as ganciclovir, which becomes toxic only when phosphorylated by the HSV-TK enzyme.

[0070] In embodiments, the lymphocytes are T or NK cells. In embodiments, the one or more lymphocyte activation elements comprise anti-CD3 and anti-CD28, and the lymphocytes are T cells. In embodiments, the one or more lymphocyte activation elements comprise anti-CD2 and anti-CD335, and the lymphocytes are NK cells.

[0071] Embodiments relate to cells comprising one or more recombinant viral particles.

[0072] Embodiments relate to methods of performing or enhancing gene therapy comprising: administering to a subject having a form of cancer an effective pharmaceutical composition comprising one or more recombinant viral particles; and allowing the viral vector to be introduced into T or NK cells of the subject.

[0073] Embodiments relate to packaging cells for producing viral particles, the packaging cells comprising a polynucleotide encoding SEQ ID NO:3 or SEQ ID NO:5 or comprising a polynucleotide of SEQ ID NO:1.

[0074] In an embodiment, it further comprises one or more polynucleotides encoding one or more accessory factors for assembling transfection-competent viral particles.

[0075] In an embodiment, further comprising a polynucleotide encoding a chimeric antigen receptor (CAR), wherein the CAR comprises an antigen binding domain, a transmembrane domain, and an intracellular signaling domain.

[0076] In an embodiment, the viral particles produced by the packaging cells are capable of infecting T cells.

[0077] In an embodiment, the viral particles produced by the packaging cells are incapable of producing replication-competent infectious virus.

[0078] In an embodiment, wherein the one or more accessory factors are selected from the group consisting of Gag, Pol, Rev and Env proteins.

[0079] An embodiment relates to a polynucleotide encoding SEQ ID NO: 3 or SEQ ID NO: 5, for use in the packaging cell described above.

[0080] Embodiments relate to viral particles produced by the above-described packaging cells, wherein the viral particles are capable of infecting T cells.

[0081] In an embodiment, the viral particle is incapable of producing replication-competent infectious virus.

[0082] Embodiments relate to a method of producing viral particles, the method comprising placing the above-described packaging cells under conditions suitable for producing viral particles and harvesting the viral particles produced by the packaging cells.

[0083] In an embodiment, the harvested viral particles are further purified to obtain transduction-active viral particles.

[0084] The embodiment relates to a polynucleotide sequence having a specific base pair sequence (SEQ ID NO: 1), which functionally encodes a protein for virus particle assembly. This polynucleotide sequence may have ATCGGCTA (for example only) as its specific base pair sequence.

[0085] According to the previously described polynucleotide sequence, wherein the polynucleotide sequence further functionally encodes one or more auxiliary factors selected from the group consisting of Gag, Pol, Rev and Env proteins. In this example, the auxiliary factor may be Gag protein.

[0086] A polynucleotide functionally encoding a specific amino acid sequence (SEQ ID NO: 3). This polynucleotide may encode a protein having an amino acid sequence of Ala-Gly-Ser (for example only).

[0087] A polynucleotide functionally encoding a specific amino acid sequence (SEQ ID NO: 5). This polynucleotide may encode a protein having an amino acid sequence of Cys-Thr-Leu (for example only).

[0088] According to the previously described polynucleotide functionally encoding SEQ ID NO: 3 or SEQ ID NO: 5, wherein the polynucleotide further functionally encodes one or more auxiliary factors for assembling transfection-competent viral particles. In this example, the auxiliary factor may be Env protein.

[0089] According to the previously described polynucleotide, wherein the one or more auxiliary factors are selected from the group consisting of Gag, Pol, Rev and Env proteins. In this example, the auxiliary factors may be Gag and Env proteins.

[0090] Embodiments relate to a viral particle encoded by a polynucleotide as previously described, wherein the viral particle is functionally capable of infecting a T cell. In this example, the viral particle may be capable of infecting a CD4+ T cell.

[0091] An embodiment relates to a method for producing a viral particle according to the previous description, the method comprising: culturing a cell comprising a polynucleotide as previously described under suitable conditions, and harvesting the produced viral particles. In this example, the cell may be a HEK293T cell.

[0092] According to the previously described method, the method further comprises purifying the harvested viral particles to obtain viral particles with transfection ability. In this example, the purification step may comprise centrifugation and filtration.

[0093] In the implementation scheme, functionally encoding: Functional encoding is a term used to describe how a polynucleotide sequence encodes a specific protein or amino acid sequence. The term generally covers various stages of gene expression, including transcription, splicing, translation, etc., as well as various regulatory mechanisms that may be involved, such as enhancers, inhibitors, etc. Transfection capability: Transfection capability is used to describe the ability of viral particles to enter host cells and deliver genetic material. It is usually measured using parameters such as infection rate, viral replication cycle, and stability of viral particles in host cells. Cofactors: Cofactors are used to describe other proteins or molecules involved in the assembly of viral particles. These factors generally include, but are not limited to, structural proteins, enzymes, and other small molecules. Optimal conditions are used to describe the optimal environmental conditions for culturing cells to produce viral particles. This generally includes temperature, pH, nutrients, oxygen concentration, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0094] Figure 1 Schematic representation of helper plasmids for producing lentiviral particles, namely pLP1, pLP2, pLP-VSVG.

[0095] Figure 2 The figure shows the GCC CAR expression of T cells in single blood samples detected by flow cytometry on day 8 after infection with various lentiviral vectors.

[0096] Figure 3 It shows that after infection with a single blood sample, a single blood sample was taken on the 8th day and co-cultured with T84 cells for 24 hours, and the expression of T cells was activated.

[0097] Figure 4 The release of cytokines after co-culture is shown.

[0098] Example 1

[0099] The plasmid carrying the exogenous gene is inserted into the auxiliary plasmid (lentiviral packaging plasmid, envelope plasmid, such as Figure 1 With the assistance of pLP1, pLP2 and pLP-VSVG) and cell lines shown in the figure, the virus is packaged into infectious virus particles (lentiviral vectors), which can be directly infect host cells or animal models after being collected and concentrated, so as to achieve the expression of exogenous genes in cells or living tissues. The ordinary lentiviral vector packaging system is a 4-plasmid system, including a transfer plasmid, a packaging plasmid and an envelope plasmid, wherein: the transfer plasmid is a vector plasmid carrying an exogenous gene (for example, a gene expressing CAR), and in this embodiment, it carries a gene encoding GCC CAR; the packaging plasmid contains PLP1 and PLP2, PLP1 carries the basic structural gene gag / pol of the lentivirus, and PLP2 carries the regulatory gene rev of the lentivirus; the envelope plasmid PLP-VSVG carries the gene encoding the lentiviral envelope protein vsv-g.

[0100] In this embodiment, the lentiviral vector (viral particles) is prepared by transfecting cells with the packaging system of this embodiment and culturing them. In the packaging system of this embodiment, in addition to the plasmids of the common packaging system, it also contains plasmids carrying genes encoding activation elements of immune cells (such as T cells, NK cells, DC cells, etc.), constituting a 5-plasmid lentiviral packaging system, and the specific steps are as follows:

[0101] Preparation of vector plasmid:

[0102] Transfer plasmid 6701 carries a gene encoding GCC CAR, the nucleotide sequence of which is shown in SEQ ID NO: 40 of PCT International Patent Application Publication WO2018064921;

[0103] The plasmid carrying the gene encoding the immune cell T cell activation element may be 2457, whose nucleotide sequence is shown in SEQ ID NO: 1; or 2752, whose amino acid sequence of the activation element is shown in SEQ ID NO: 3, and whose nucleotide sequence encoding the activation element gene is shown in SEQ ID NO: 2; or

[0104] 2756, the amino acid sequence of the activation element is shown in SEQ ID NO:5, and the nucleotide sequence encoding the activation element gene is shown in SEQ ID NO:4;

[0105] The envelope plasmid PLP-VSVG carries a gene encoding the lentiviral envelope protein VSVG or VSVG mutant 2596 (ModifiedVSVG), the amino acid sequence of VSVG is shown in SEQ ID NO:9, and the nucleotide sequence encoding VSVG is shown in SEQ ID NO:8; the amino acid mutation sites of VSVG mutant 2596 (Modified VSVG) are K47Q and R354A, the amino acid sequence of 2596 (Modified VSVG) is shown in SEQ ID NO:7, and the nucleotide sequence encoding 2596 (Modified VSVG) is shown in SEQ ID NO:6.

[0106] Transformation and Amplification:

[0107] The prepared vector plasmid is transformed into bacterial cells, in this embodiment, the bacterial cells are Escherichia coli cells. These bacterial cells reproduce and replicate plasmid DNA, thereby producing a large amount of vector plasmids, which are then extracted and purified to obtain vector plasmids for lentiviral packaging.

[0108] Lentiviral packaging:

[0109] Transfer plasmid, packaging plasmid (pLP1 and pLP2), envelope plasmid (pLP-VSVG or Modified VSVG 2596), T cell activation element plasmid (2457, 2752 or 2756) are co-transfected into the packaging cell line. In this embodiment, the packaging cell line is HEK293T cells. The molar ratio for co-transfection is specified as follows: transfer vector (6701 vector): VSVG or 2596: pLP1: pLP2: T cell activation element plasmid (2457, 2752 or 2756 vector), the molar ratio is 10:2:3:2:2.

[0110] Lentivirus Harvest:

[0111] Typically 48 to 72 hours later, the supernatant enriched with lentiviral particles is collected and then centrifuged and filtered to eliminate cell debris.

[0112] Lentivirus concentration and purification:

[0113] The filtered supernatant is subjected to ultracentrifugation to concentrate the viral particles, and the concentrated lentiviral particles are resuspended in a suitable buffer for use.

[0114] Titer and Testing:

[0115] The functional titer of the lentivirus was determined. In addition, the vector was tested for sterility, endotoxin levels, and bioassay performance.

[0116] Lentiviral vector transfection host cells and testing:

[0117] On day 0, the frozen single blood was thawed. After cell counting, the single blood was infected with various lentiviral vectors at an MOI of 20 for 3 minutes, and the infected single blood was washed 3 times and then adjusted to 1×10 using Texmacs medium. 6 / mL density and placed in a 37°C incubator. The Texmacs medium was replaced on the second day and every day thereafter until day 8. On day 8, samples were taken and the expression of GCC CAR was detected by flow cytometry; and co-cultured with colorectal cancer target cells (T84) at a ratio of 3:1 (E:T) according to the GCC CAR ratio. On the second day after co-culture, cell expression activation and factor release were evaluated by flow cytometry.

[0118] Figure 2 The figure shows the GCC CAR expression of T cells in single blood samples detected by flow cytometry on day 8 after infection of single blood samples with various lentiviral vectors. In the figure: NC is a single blood sample that has not been infected with a lentiviral vector; the 6701 packaging system is a transfer plasmid 6701-pPLP1-pLP2-pLPVSVG; the 2457-VSVG packaging system is a transfer plasmid 6701-pPLP1-pLP2-pLPVSVG-2457; the 2457Modified VSVG packaging system is a transfer plasmid 6701-pPLP1-pLP2-2596-2457; the 2752Modified VSVG packaging system is a transfer plasmid 6701-pPLP1-pLP2-2596-2752; the 2756Modified VSVG packaging system is a transfer plasmid 6701-pPLP1-pLP2-2596-2756.

[0119] As can be seen from the figure,

[0120] The infection efficiency of 6701 directly infecting T cells in a single blood draw is significantly lower than that of 2457-VSVG directly infecting T cells in a single blood draw, that is, the infection efficiency of ordinary lentiviral vectors on resting cells is low. Adding T cell activation elements to the packaging system can improve the infection efficiency of lentivirus on resting cells. Similarly, T cell activation elements 2752 and 2756 can also improve the infection efficiency of lentivirus on resting cells.

[0121] In addition, VSVG can bind to the low-density lipoprotein receptor (LDLR) on the cell surface. 2596 encodes a mutant of VSVG, which can no longer bind to the LDLR on the cell surface after mutation, and thus cannot infect cells. However, T cell activation genes 2457, 2752, and 2756 are added during the lentiviral vector packaging process, hoping that under the conditions of VSVG mutants, T cells can be specifically infected without infecting other immune cells, thereby improving safety. Experimental results show that the use of VSVG mutant 2457Modified VSVG significantly reduces the infection rate of T cells in single blood collection to less than 1%, and using 2752Modified VSVG or 2756Modified VSVG instead of 2457VSVG can achieve infection of T cells in single blood collection.

[0122] Figure 3 It shows that after single blood infection, single blood was collected on day 8 and co-cultured with T84 cells for 24 hours, and the expression of T cells was activated. These results show that compared with the control group NC, after co-culture with target cells T84, the expression of CD137 in T cells and CART cells in single blood was upregulated, indicating activation.

[0123] Figure 4 The results showed that the release of cytokines after co-culture was significantly increased after co-culture of single blood with target cells T84 compared with the control group NC.

[0124] In summary, the three lentiviral vectors 2457-VSVG, 2752Modified VSVG, and 2756Modified VSVG can all infect T cells and retain normal T cell function. The infection rate of 2457-Modified VSVG (VSVG mutant) on T cells was greatly reduced to an almost undetectable level, indicating that 2457-Modified VSVG is infected through the LDL-R pathway. In order to ensure safety while retaining the effectiveness of the new CAR T therapy, plasmid 2457 was replaced with 2752 or 2756, and 2596 (VSVG mutant) was retained to enable it to infect T cells through CD3.

[0125] Example 2

[0126] This embodiment provides another method for generating virus particles, comprising the following steps:

[0127] Cell culture: First, select an appropriate cell line (e.g., HEK293T cells) and culture them in an appropriate culture medium. Cells should be maintained at 37°C and 5% CO. 2 cultured under the conditions of .

[0128] Polynucleotide introduction: Use an appropriate method (e.g., liposome-mediated transfection) to introduce a target polynucleotide (comprising SEQ ID NO: 1, a polynucleotide encoding SEQ ID NO: 3 or SEQ ID NO: 5) into a cell. In an embodiment, the target polynucleotide also includes one or more auxiliary factors encoding for assembling viral particles with transfection ability, such as auxiliary factors selected from the group consisting of Gag, Pol, Rev and Env proteins.

[0129] Virus particle collection: At appropriate time points, the culture fluid is collected and the virus particles are initially purified by centrifugation and filtration steps.

[0130] Virus Particle Purification: Virus particles are further purified using more advanced purification techniques (e.g., gradient centrifugation).

[0131] Functional validation: Use appropriate experimental methods (e.g., transfection efficiency test) to verify the transfection ability of the virus particles. For example, HEK293T cells are selected as host cells and cultured in DMEM medium containing 10% fetal bovine serum and 1% antibiotic-antimycotic. Plasmid DNA containing the previously described polynucleotides is introduced into HEK293T cells by a liposome-mediated transfection method. 48 hours after transfection, the culture fluid is collected and preliminarily filtered through a 0.45 μm filter membrane. Further, the virus particles are separated from the culture fluid by centrifugation, and the virus particles are further purified by gradient centrifugation, and then flow cytometry and fluorescent labeling are used to evaluate the transfection ability of the purified virus particles.

Claims

1. A packaging cell for producing viral particles, the packaging cell comprising SEQ ID NO: 1 or a polynucleotide encoding SEQ ID NO: 3 or SEQ ID NO:

5.

2. The packaging cell of claim 1, further comprising one or more polynucleotides encoding one or more auxiliary factors for assembling transfection-competent viral particles.

3. The packaging cell according to claim 1 or 2, further comprising a polynucleotide encoding a chimeric antigen receptor, wherein the chimeric antigen receptor comprises an antigen binding domain, a transmembrane domain and an intracellular signaling domain.

4. The packaging cell according to claim 3, wherein the viral particles produced by the packaging cell are capable of infecting T cells.

5. The packaging cell according to claim 3 or 4, wherein the viral particles produced by the packaging cell are incapable of producing replication-competent infectious virus.

6. The packaging cell of claim 5, wherein the one or more accessory factors are selected from the group consisting of Gag, Pol, Rev and Env proteins.

7. A viral particle produced by the packaging cell of any one of claims 1-6, wherein the viral particle is capable of infecting T cells.

8. A method for producing viral particles, the method comprising: Cultivating the packaging cell of any one of claims 1 to 6 under conditions suitable for producing viral particles, and; Harvest viral particles produced by packaging cells.

9. The method according to claim 8, further comprising purifying the harvested viral particles to obtain viral particles with transfection ability.

Images