Recombinant lentiviral vector and preparation thereof

Abstract

The invention provides a recombinant lentiviral vector and a preparation thereof. The recombinant lentiviral vector carries a coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or a coding sequence of shRNA targeting HIV genes. The recombinant lentiviral vector preparation comprises an effective dose of the recombinant lentiviral vector, a histidine hydrochloride buffer solution, saccharides and a surfactant. The recombinant lentivirus preparation disclosed by the invention has good stability under different environmental conditions.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly, to a recombinant lentiviral vector and its preparation. Background Art

[0002] AIDS, also known as acquired immunodeficiency syndrome, is a major infectious disease threatening human life safety, caused by the human immunodeficiency virus (HIV). HIV is a virus that can attack the human immune system. It mainly targets CD4-positive T lymphocytes in the human immune system, destroys a large number of these cells, causes the human body to lose its immune function, making it easy to be infected with various diseases and even develop malignant tumors, with a relatively high fatality rate.

[0003] Currently, there is no effective AIDS vaccine available. Clinically, highly active antiretroviral therapy (HAART), commonly known as the "cocktail therapy", also known as antiretroviral therapy (ART), is mainly used. Existing ART treatments cannot clear the viral latent reservoir of HIV-1 and all require lifelong medication. Once the medication is stopped, the virus in the viral reservoir can be reactivated and replicated again, and thus the disease will quickly rebound. Therefore, patients need to take medication permanently.

[0004] To achieve functional cure, broad-spectrum neutralizing antibodies can be used, multi-specific molecules can also be developed based on antibodies, and T cells can be modified using chimeric antigen receptors (CARs). However, many strains have developed resistance to such antibodies, and this drug resistance must be overcome to obtain the best clinical effect. Another idea for curing AIDS is to reactivate latent HIV and, by combining methods to enhance the body's HIV-specific immune response, make up for the immune damage caused by HIV to the body and enhance the clearance effect on the HIV reservoir. However, the related research finally ended in failure. On the one hand, the specific killing power of the CAR-T cells themselves needs to be improved, and on the other hand, the CAR-T cells themselves are also easily infected by HIV and are prone to exhaustion in an HIV-infected environment, with a relatively short effective time.

[0005] Currently, active means are still needed to achieve the functional cure of AIDS, and even to clear the latent reservoir, and ultimately achieve radical cure.

[0006] Gene therapy or cell therapy provides a new idea and solution for treating various diseases caused by gene defect diseases and viral infections. With the success of gene therapy or cell therapy for some major diseases, it marks a major breakthrough in gene therapy or cell therapy. With the development of gene therapy or cell therapy technology in human diseases, viral vectors have been increasingly used. Among them, recombinant lentivirus, as a gene therapy vector, can infect dividing and non-dividing cells and integrate foreign genes into the host genome to achieve long-term stable expression, with various advantages such as a wide host range, low immunogenicity, large genome capacity, and long-term expression.

[0007] However, the stability of lentiviral vectors is very low, and lentiviral vectors applied to human therapy must maintain their structural integrity to maintain biological activity. To maintain the structural integrity of lentiviral vectors, such preparations must be maintained and transported at relatively low temperatures to maintain their biological activity.

[0008] The loss of biological activity of lentiviral vectors mostly occurs during storage and preservation. Recombinant lentiviral vectors carrying the target gene are generally made into liquid preparations and stored at ultra-low temperatures (such as not higher than -60 °C), transported under cryogenic freezing conditions, and thawed before use. One of the main challenges of stability at temperatures below the freezing point is to prevent physical damage to structural and functional components during freezing and storage.

[0009] Lentiviral vectors generally include a viral genome, coat proteins encoded by the viral genome, and a lipid bilayer coating membrane structure. Under ultra-low temperature conditions, the lipid bilayer is easily damaged during freezing, and proteins are susceptible to denaturation. Therefore, generally, lentiviral vector preparations are not suitable for long-term storage under ultra-low temperature conditions.

[0010] If the ultra-low temperature storage time is too long, repeated freezing and thawing occur during use, or the preparation is improperly used and exposed to the body temperature of an organism (such as the body temperature of a human or other animal) or room temperature for too long before reaching the target organ, the biological activity of the recombinant lentiviral vector (generally represented by virus titer) often rapidly decreases significantly, directly affecting the therapeutic effect of the recombinant lentiviral vector preparation or making the results of preclinical and clinical studies inaccurate.

[0011] Although some lentiviral vector preparations have been disclosed in the prior art, their stability under different conditions still needs to be improved. Therefore, there is an urgent need for a recombinant lentiviral vector preparation with better stability, which can be stored for a long time at -80 °C ultra-low temperature and still be able to better maintain the activity of the recombinant lentiviral vector meeting the usage requirements after being exposed to 2 - 8 °C or the body temperature of an organism for a long time. Summary of the Invention

[0012] On the one hand, the present invention provides a recombinant lentiviral vector, which comprises (1) a coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or (2) a coding sequence of an shRNA targeting an HIV gene.

[0013] In some specific embodiments, the CAR comprises an extracellular region, a transmembrane region, and an intracellular region. In some preferred embodiments, the extracellular region can specifically bind to the gp120 protein of HIV. In some more preferred embodiments, the extracellular region comprises the D1 and D2 domains of the human CD4 molecule. In some other more preferred embodiments, the amino acid sequence of the CAR is as shown in any one of SEQ ID NO: 24 - 29, and further preferably, as shown in SEQ ID NO: 25.

[0014] In some specific embodiments, the shRNA includes an shRNA targeting gag and an shRNA targeting LTR. In some preferred embodiments, the nucleotide sequence of the shRNA targeting gag is as shown in SEQ ID NO: 1, and the nucleotide sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 2. In some preferred embodiments, the coding sequence of the shRNA targeting gag is as shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 4.

[0015] On the other hand, the present invention provides a preparation of a recombinant lentiviral vector, wherein the preparation comprises: a) a recombinant lentiviral vector; b) a histidine hydrochloride buffer; c) glucose and sucrose; d) a surfactant.

[0016] Glucose and sucrose

[0017] In some specific embodiments, the total concentration of glucose and sucrose in the preparation is 1 - 15% (w / v). In some specific embodiments, the total concentration of glucose and sucrose in the preparation is preferably 3 - 13% (w / v), and more preferably 5 - 8% (w / v). In some specific embodiments, the total concentration of glucose and sucrose in the preparation is 5.0% (w / v), 5.5% (w / v), 6.0% (w / v), 6.5% (w / v), 7.0% (w / v), 7.5% (w / v), 8.0% (w / v), and most preferably 6.0% (w / v), 6.5% (w / v), 7.0% (w / v).

[0018] In some specific embodiments, the concentration of glucose in the formulation is 1-10% (w / v). In some specific embodiments, the concentration of glucose in the formulation is preferably 2-8% (w / v), more preferably 4-6% (w / v). In some specific embodiments, the concentration of glucose in the formulation is 2% (w / v), 3% (w / v), 4% (w / v), 5% (w / v), 6% (w / v), 7% (w / v), 8% (w / v), and most preferably 4% (w / v), 5% (w / v), 6% (w / v).

[0019] In some specific embodiments, the concentration of sucrose in the formulation is 1-8% (w / v). In some specific embodiments, the concentration of sucrose in the formulation is preferably 1-5% (w / v), more preferably 1-2% (w / v). In some specific embodiments, the concentration of sucrose in the formulation is 1.0% (w / v), 1.5% (w / v), 2.0% (w / v), 2.5% (w / v), 3.0% (w / v), 3.5% (w / v), 4.0% (w / v), 4.5% (w / v), 5.0% (w / v), and most preferably 1.0% (w / v), 1.5% (w / v), 2.0% (w / v).

[0020] Surfactant

[0021] According to the recombinant lentiviral vector formulation of the present application, wherein the surfactant is poloxamer 188.

[0022] In some specific embodiments, the concentration of poloxamer 188 in the formulation is 0.001-0.5% (w / v). In some specific embodiments, the concentration of poloxamer 188 in the formulation is preferably 0.001-0.1% (w / v), more preferably 0.005-0.015% (w / v). In some specific embodiments, the concentration of poloxamer 188 in the formulation is 0.001% (w / v), 0.005% (w / v), 0.01% (w / v), 0.015% (w / v), 0.02% (w / v), 0.06% (w / v), 0.08% (w / v), 0.1% (w / v), and most preferably 0.005% (w / v), 0.01% (w / v), 0.015% (w / v).

[0023] Histidine hydrochloride

[0024] According to the recombinant lentiviral vector preparation of the present application, the molar concentration range of the histidine hydrochloride buffer is 1-50 mmol / L. Preferably, in some specific embodiments, the molar concentration range of the histidine hydrochloride is 5-30 mmol / L, preferably 5-20 mmol / L. In some specific embodiments, the molar concentration of the histidine hydrochloride is 5 mmol / L, 10 mmol / L, 20 mmol / L, 30 mmol / L, 40 mmol / L, 50 mmol / L, and most preferably 5 mmol / L, 10 mmol / L, 20 mmol / L.

[0025] According to the recombinant lentiviral vector preparation of the present application, in some embodiments, the pH value range of the preparation is 6.0-8.0, preferably 6.5-7.4. In some specific embodiments, the pH value of the preparation is 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, preferably 6.5, 7.2, 7.4.

[0026] In some embodiments, the present application provides a recombinant lentiviral vector preparation, which comprises a recombinant lentiviral vector, a histidine hydrochloride buffer, sugars and a surfactant. Among them, the recombinant lentiviral vector is preferably a recombinant simian immunodeficiency virus vector, the sugars are glucose and sucrose, the surfactant is poloxamer 188, and the pH value of the recombinant lentiviral vector preparation is 6.0-8.0, preferably 6.5-7.4.

[0027] In some preferred embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0028] a) A recombinant lentiviral vector;

[0029] b) A histidine hydrochloride buffer with a concentration of 1-50 mmol / L;

[0030] c) Glucose with a concentration of 1-10% (w / v);

[0031] d) Sucrose with a concentration of 1-8% (w / v);

[0032] e) Poloxamer 188 with a concentration of 0.001-0.5% (w / v);

[0033] The pH value of the recombinant lentiviral vector preparation is 6.0-8.0, preferably 6.5-7.4.

[0034] In some specific embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0035] a) Recombinant lentiviral vector;

[0036] b) Histidine hydrochloride buffer solution with a concentration of 5 - 30 mmol / L; and

[0037] c) Glucose with a concentration of 2 - 8% (w / v);

[0038] d) Sucrose with a concentration of 1 - 5% (w / v);

[0039] e) Poloxamer 188 with a concentration of 0.001 - 0.1% (w / v);

[0040] The pH value of the recombinant lentiviral vector preparation is 6.5 - 7.4.

[0041] In some specific embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0042] a) Recombinant lentiviral vector;

[0043] b) Histidine hydrochloride buffer solution with a concentration of 5 - 20 mmol / L; and

[0044] c) Glucose with a concentration of 4 - 6% (w / v);

[0045] d) Sucrose with a concentration of 1 - 2% (w / v);

[0046] e) Poloxamer 188 with a concentration of 0.005 - 0.015% (w / v);

[0047] The pH value of the recombinant lentiviral vector preparation is 6.5 - 7.4.

[0048] In some preferred specific embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0049] a) Recombinant lentiviral vector;

[0050] b) Histidine hydrochloride buffer solution with a concentration of 10 mmol / L; and

[0051] c) Glucose with a concentration of 5% (w / v);

[0052] d) Sucrose with a concentration of 1.5% (w / v);

[0053] e) Poloxamer 188 with a concentration of 0.01% (w / v);

[0054] The pH value of the recombinant lentiviral vector preparation is preferably 7.2.

[0055] In some preferred specific embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0056] a) a recombinant lentiviral vector;

[0057] b) a histidine hydrochloride buffer solution with a concentration of 5 mmol / L; and

[0058] c) glucose with a concentration of 4% (w / v);

[0059] d) sucrose with a concentration of 2% (w / v);

[0060] e) poloxamer 188 with a concentration of 0.005% (w / v);

[0061] The pH value of the recombinant lentiviral vector preparation is preferably 6.5.

[0062] In some preferred specific embodiments, the present application provides a recombinant lentiviral vector preparation, wherein the recombinant lentiviral vector preparation comprises:

[0063] a) a recombinant lentiviral vector;

[0064] b) a histidine hydrochloride buffer solution with a concentration of 20 mmol / L; and

[0065] c) glucose with a concentration of 6% (w / v);

[0066] d) sucrose with a concentration of 1% (w / v);

[0067] e) poloxamer 188 with a concentration of 0.015% (w / v);

[0068] The pH value of the recombinant lentiviral vector preparation is preferably 7.4.

[0069] Recombinant lentiviral vector

[0070] According to the recombinant lentiviral vector preparation of the present application, in some embodiments, the recombinant lentiviral vector is a recombinant human immunodeficiency virus vector (human immunodeficiency virus, HIV) or a recombinant simian immunodeficiency virus vector (simian immunodeficiency virus, SIV). In some specific embodiments, the recombinant lentiviral vector is preferably a recombinant simian immunodeficiency virus vector.

[0071] In some specific embodiments, the recombinant lentiviral vector can be pseudotyped with VSV-G, or contain envelope proteins from other viruses.

[0072] In some specific embodiments, the recombinant lentiviral vector carries the coding sequence of shRNA targeting the HIV gene. In some specific embodiments, the recombinant lentiviral vector carries: (1) the coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or (2) the coding sequence of shRNA targeting the HIV gene. In some specific embodiments, the CAR comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region can specifically bind to the gp120 protein of HIV; preferably, the extracellular region comprises the D1 and D2 domains of the human CD4 molecule. In some specific embodiments, the sequence of the CAR is as shown in any one of SEQ ID NO: 24-29. In some specific embodiments, the shRNA includes shRNA targeting gag and shRNA targeting LTR. In some specific embodiments, the sequence of the shRNA targeting gag is as shown in SEQ ID NO: 1, and the sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 2. In some specific embodiments, the coding sequence of the shRNA targeting gag is as shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 4. In some preferred embodiments, the recombinant lentivirus carries: (1) the coding sequence of a chimeric antigen receptor (CAR) targeting HIV, the amino acid sequence of the CAR being as shown in SEQ ID NO: 25; and / or (2) the coding sequences of shRNA targeting the gag gene in HIV and shRNA targeting the LTR gene in HIV, the coding sequence of the shRNA targeting the gag gene in HIV being as shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting the LTR gene in HIV being as shown in SEQ ID NO: 4. In some specific embodiments, the recombinant lentiviral vector is a recombinant simian immunodeficiency virus vector or a recombinant human immunodeficiency virus vector.

[0073] According to the recombinant lentiviral vector preparation described in the present application, in some embodiments, the active titer of the recombinant lentiviral vector in the preparation is 1×10 5 -5×10 10 transducing units / ml (Tu / ml). In some specific embodiments, the active titer of the recombinant lentiviral vector in the preparation is preferably 5×10 5 -5×10 9 Tu / ml, more preferably 5×10 5 -5×10 8 Tu / ml. In some specific embodiments, the active titer of the recombinant lentiviral vector in the preparation is 1×10 5 Tu / ml, 5×10 5 Tu / ml, 8×105 Tu / ml, 1×10 6 Tu / ml, 5×10 6 Tu / ml, 8×10 6 Tu / ml, 1×10 7 Tu / ml, 5×10 7 Tu / ml, 8×10 7 Tu / ml, 1×10 8 Tu / ml, 5×10 8 Tu / ml, preferably with an active titer of 8×10 5 Tu / ml, 8×10 6 Tu / ml, 8×10 7 Tu / ml.

[0074] Definition

[0075] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. To facilitate understanding of the disclosed content, several terms and phrases are defined below.

[0076] As used herein, "viral vector" refers to a viral particle that lacks the ability of self - replication and has the ability to introduce a nucleic acid molecule into a host. "Lentiviral vector" refers to a viral vector derived from primate immunodeficiency virus. The lentiviral vector contains the genetic information required for packaging, infection, and stable integration, and is the main component of the lentiviral vector system. A shuttle plasmid carrying an exogenous gene, with the assistance of a lentiviral packaging plasmid and cell line, is packaged into an infectious viral particle, and through infecting cells or living tissues, the exogenous gene is expressed in the cells or living tissues. "Recombinant viral vector" refers to a viral vector constructed by genetic recombination technology.

[0077] The recombinant lentiviral vector described in this article can be further pseudotyped with VSV-G. VSV-G pseudotyping means that the envelope of the vector contains the surface glycoprotein VSV-G protein of Vesicular stomatitis virus (VSV). The VSV-G protein can be a protein derived from any VSV strain. For example, the VSV-G protein from the Indiana serotype strain (J. Virology 39:519-528 (1981)) can be used, but it is not limited to this. In addition, the VSV-G protein can be a derivative of a natural protein, a modified protein obtained by substitution, deletion, and / or addition of one or more amino acids. The VSV-G pseudotyped vector can be prepared by co-existing with the VSV-G protein during virus production. For example, through transfection of the VSV-G expression vector, induction of expression from the VSV-G gene integrated into the host chromosomal DNA, or by expressing VSV-G in packaging cells, the virus particles produced via this cell can be pseudotyped with VSV-G. Since the VSV-G protein is a glycoprotein that forms a stable trimer and exists on the cell membrane, it will not cause damage to the vector particles during the purification process and can be highly concentrated by centrifugation (Yang, Y. et al., Hum Gene Ther: Sep, 6(9), 1203-13.1995). Preferably, the recombinant lentiviral vector is a lentiviral vector coated with the vesicular stomatitis virus G protein.

[0078] The recombinant lentiviral vector described in this article can further contain envelope proteins from other viruses. For example, as such a protein, it is preferably an envelope protein from a virus that infects human cells. There is no particular limitation on such a protein. For example, the envelope protein from the 4070A strain of murine leukemia virus (MuMLV) can be used. In addition, the envelope protein from MuMLV 10A1 can also be used (for example, pCL-10A1 (Imgenex) (Naviaux, R.K. et al., J. Virol. 70:5701-5705 (1996))). In addition, as proteins of the Herpesviridae family, for example, the gB, gD, gH, gp85 proteins of herpes simplex virus, the gp350, gp220 proteins of Epstein-Barr virus, etc. can be cited. As proteins of the Hepadnaviridae family, the S protein of hepatitis B virus, etc. can be exemplified. The envelope protein can also be formed by fusing the measles virus glycoprotein with other single-chain antibodies (Funke S. et al Mol Ther 16:1427-36, 2008; Frecha C. et al., B1ood, 114:3173-80.2009).

[0079] The packaging of the lentiviral vectors described in this article is usually carried out by transient transfection or using cell lines. Human cell lines that can be used as packaging cells during transient transfection include, for example, 293 cells, 293T cells, 293FT cells, 293LTV cells, 293EBNA cells, and other clones isolated from 293 cells; SW480 cells, u87MG cells, HOS cells, C8166 cells, MT-4 cells, Molt-4 cells, HeLa cells, HT1080 cells, TE671 cells, etc. Cell lines derived from monkeys, such as COS1 cells, COS7 cells, CV-1 cells, BMT10 cells, etc. Moreover, commonly used calcium phosphate and PEI transfection reagents, as well as some transfection reagents such as Lipofectamine2000, FuGENE, and S93fectin, are also frequently used. The packaging of lentiviruses also uses some lentiviral packaging cell lines, such as stable cell lines produced by the most commonly used Env glycoprotein, VSVG protein, or HIV-1 gag-pol protein.

[0080] For safety reasons, the lentiviral vector systems used on a large scale all adopt the method of splitting the genome, that is, positioning the genes with different auxiliary functions on different plasmids. Currently, there are four-plasmid systems (encoding the gag-pol gene, Rev gene, VSVG gene, and SIN transfer gene are located on four different plasmids), three-plasmid systems (removing the plasmid encoding the Rev gene, and the gag-pol gene in the gag-pol plasmid uses codons preferred in human cells), and two-plasmid systems (the auxiliary genes necessary for lentiviral vector packaging are located on the same plasmid, and these auxiliary genes are a single gene sequence; the other is a transgenic plasmid). There are also lentiviral packaging systems with more than four plasmids in use.

[0081] The recombinant lentiviral vectors described in this article can be substantially purified. The purification methods include known purification / isolation methods such as filter filtration, ion exchange chromatography, ultrafiltration, molecular sieve, composite mode chromatography, nuclease digestion, and filtration sterilization. Usually, in small-scale preparations, high-speed centrifugation is also used. For example, after filtering the vector suspension through a 0.45 μm filter, centrifugation can be carried out at 42500×g for 90 minutes at 4°C to precipitate and concentrate the vector.

[0082] The method for determining the titer (active titer) of the recombinant lentiviral vector described in this article is a method known in the prior art. For example, 293T cells are infected with the recombinant lentiviral vector, and then the expression level of the reporter gene carried by the recombinant lentiviral vector in 293T cells after infection is detected by flow cytometry, and then the titer (active titer) of the recombinant lentivirus is calculated. The calculation formula is active titer (Tu / ml) = (positive expression rate of the reporter gene (percentage of positive cells) × number of cells per well at the time of infection × dilution factor of the virus stock solution) / volume of the virus dilution added per well. Other methods for determining the lentivirus titer can be found in Miguel Sena-Esteves, Guangping Gao et al., Cold Spring Harb Protoc, 2018.

[0083] Preferably, the recombinant lentiviral vector described in this article is a recombinant lentiviral vector of primates, namely recombinant human immunodeficiency virus vector (human immunodeficiency virus, HIV) or recombinant simian immunodeficiency virus vector (simian immunodeficiency virus, SIV); or a recombinant lentiviral vector of non-primates, namely recombinant equine infectious anemia virus vector (equine infectious anemia virus, EIAV) or recombinant feline immunodeficiency virus vector (feline immunodeficiency virus, FIV) or recombinant caprine arthritis-encephalitis virus vector (caprine arthritis-encephalitis virus, CAEV).

[0084] Preferably, the recombinant lentiviral vector described herein is a recombinant human immunodeficiency virus vector or a recombinant simian immunodeficiency virus vector. The "simian immunodeficiency virus (SIV) vector" mentioned herein refers to a vector in which the sequences necessary for the function of the viral vector are sequences derived from the SIV genome within the nucleic acid molecule in the viral particle. The sequences necessary for the function of the viral vector herein refer to, in order from the 5' end, the R region, U5 region, packaging signal (φ), RRE, the U3 region other than the promoter region of the 3' LTR, and the R region sequence of the 5' LTR. The SIV vector herein can also be modified as long as it conforms to the above definition. For example, as long as the "sequences necessary for the function of the viral vector" are from SIV, other sequences from SIV or sequences from outside SIV can also be included. As the most suitable sequences that can be included, for example, it can be cPPT (central polypurine tract), internal promoter (CMV), WPRE (Woodchuck hepatitis virus post-transcriptional regulatory element).

[0085] The "simian immunodeficiency virus (SIV)" described herein includes all strains and subtypes of SIV. As examples of SIV isolates, SIVagm, SIVcpz, SIVmac, SIVmnd, SIVsm, SIVsnm, SIVsyk, etc. can be mentioned, but it is not limited to these viral strains only.

[0086] The "human immunodeficiency virus (HIV) vector" described herein refers to a vector in which the sequences necessary for the function of the viral vector are sequences derived from the HIV genome within the nucleic acid molecule in the viral particle. Among them, the HIV-1 vector contains the HIV cis-acting sequences required for packaging, reverse transcription, and integration, and at the same time has a multiple cloning site under the control of a heterologous promoter and a target gene inserted at this site. The cis-sequences of HIV-1 on the vector plasmid usually include the LTRs at both ends, the cleavage site, and the packaging signal ψ, etc. The HIV vector herein can also be modified as long as it conforms to the above definition. For example, as long as the "sequences necessary for the function of the viral vector" are from HIV, other sequences from HIV or sequences from outside HIV can also be included. As the most suitable sequences that can be included, for example, it can be cPPT (central polypurine tract), internal promoter (CMV), WPRE (Woodchuck hepatitis virus posttranscriptional regulatory element).

[0087] The human immunodeficiency virus described herein includes all strains of HIV and their subtypes. HIV includes two strains, HIV-1 and HIV-2. Among them, HIV-1 is divided into M, O, and N subtype groups. The M subtype group includes subtypes A, A2, B, C, D, E, F1, F2, G, H, J, and K. Subtypes O and N are rare. HIV-2 has subtypes A, B, C, D, E, F, and G. Its biological characteristics are similar to those of HIV-1, but its virulence is weaker.

[0088] The recombinant lentiviral vector described herein can be an SIV or HIV vector carrying any target gene. Preferably, the recombinant lentiviral vector is a recombinant simian immunodeficiency virus vector or a recombinant human immunodeficiency virus vector carrying: (1) the coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or (2) the coding sequence of an shRNA targeting an HIV gene. Among them, the CAR includes an extracellular region, a transmembrane region, and an intracellular region. The extracellular region can specifically bind to the gp120 protein of HIV (preferably, the extracellular region includes the D1 and D2 domains of the human CD4 molecule). Further preferably, the sequence of the CAR is as shown in any of SEQ ID NO: 24-29. Among them, the shRNA includes an shRNA targeting gag and an shRNA targeting LTR. Preferably, the sequence of the shRNA targeting gag is as shown in SEQ ID NO: 1, and the sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 2; the coding sequence of the shRNA targeting gag is as shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting LTR is as shown in SEQ ID NO: 4. In some preferred embodiments, the recombinant lentivirus carries: (1) the coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and the amino acid sequence of the CAR is as shown in SEQ ID NO: 25; and / or (2) the coding sequence of an shRNA targeting the gag gene in HIV and the coding sequence of an shRNA targeting the LTR gene in HIV. The coding sequence of the shRNA targeting the gag gene in HIV is as shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting the LTR gene in HIV is as shown in SEQ ID NO: 4.

[0089] The "shRNA" described herein refers to short hairpin RNA or small hairpin RNA. It is an artificial RNA molecule with a hairpin structure and can be used to silence the expression of a target gene through RNA interference (RNAi).

[0090] The "chimeric antigen receptor (CAR)" described herein is a class of engineered cell surface receptors, generally expressed on immune effector cells (such as effector T cells), mediating the killing of cells with specific targets (referred to herein as "target cells", such as cells expressing the HIV envelope protein gp120 on the cell surface) by the engineered immune effector cells. CAR generally includes an extracellular region, an intracellular region, and a transmembrane region therebetween. The "extracellular region" contains an antigen-specific recognition domain that can specifically recognize and bind to an antigen. In some embodiments, the antigen recognition domain is connected to the transmembrane domain through a hinge region. The "intracellular region" may contain one or more primary signal transduction domains and one or more co-stimulatory domains. After the antigen-specific recognition domain binds to the antigen to form a binary complex and undergoes allosteric changes, the intracellular region can initiate a series of biochemical reactions, resulting in the production of biological effects by the immune effector cell where the CAR is located, such as secreting cytokines or directly killing target cells. In this article, unless otherwise specified, the "primary signal transduction domain" provides the first signal for activated lymphocytes (such as effector T cells), while the "co-stimulatory domain" provides the second signal for activated lymphocytes. Both the "primary signal transduction domain" and the "co-stimulatory domain" are derived from the "signal transduction domain" of the receptor molecule, which refers to the domain of the receptor molecule that is partially or entirely located intracellularly and plays a role in signal transduction after specific binding of the receptor to the ligand.

[0091] The "CD4" described herein, namely the cluster of differentiation antigen, is a member of the immunoglobulin superfamily, expressed on the surface of helper T (Th) cells, regulatory T cells, monocytes, macrophages, and dendritic cells, and plays an important role in the development and activation of T cells. CD4 contains an amino-terminal extracellular domain (containing four immunoglobulin domains exposed on the extracellular surface: D1, D2, D3, and D4, presenting an Ig-like structure), a transmembrane domain, and a short cytoplasmic tail region. Among them, D1 and D3 are similar to the variable regions of immunoglobulins. D2 and D4 are similar to the constant regions of immunoglobulins. CD4 is also the main receptor of HIV, and CD4-positive (CD4+) T cells are the targets of HIV (human immunodeficiency virus) attack.

[0092] The "gp120" described herein is a glycoprotein exposed on the surface of the HIV envelope. The 120 in its name comes from its molecular weight of 120 kDa. On helper T cells, gp120 can bind to CD4, inducing conformational changes in gp120 and gp41, enabling HIV-1 to bind to co-receptors (such as CCR5 or CXCR4) expressed on host cells, and ultimately causing the envelope of HIV to fuse with the cell membrane and allowing the virus to enter the cell.

[0093] As used herein, the term "storage" means that the composition or preparation is not immediately administered to a subject after preparation, but is stored for a period of time under specific conditions (such as a specific temperature, etc.) before use. For example, a liquid or lyophilized composition or preparation can be maintained for several days, weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 1 year, 2 years, 3 years or longer, and then administered to a subject.

[0094] As used herein, the term "a" or "an" means one or more. Thus, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0095] As used herein, the terms "comprising", "having", "including", "containing" shall be construed as inclusive rather than exclusive. The terms "consisting of" or "consisting essentially of" shall be construed as exclusive rather than inclusive.

[0096] As used herein, the term "about" means an approximate range that differs from a given reference value by 10% (±10%), unless otherwise specified.

[0097] As used herein, the term "and / or" means any and all possible combinations covering one or more of the related listed items, and when interpreted in the alternative ("or"), does not have a combination.

[0098] As used herein, the term "or" means any one member of the listed items, and also includes any combination of the members of the said items.

[0099] After the recombinant lentiviral vector described in the present invention (i.e., a recombinant simian immunodeficiency virus vector or a recombinant human immunodeficiency virus vector carrying (1) the coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or (2) the coding sequence of an shRNA targeting the HIV gene) is used to infect and transform T cells, the T cells can express the CAR molecule and / or the corresponding shRNA. The transformed T cells (CAR-T) have stronger killing ability against HIV-infected cells, can maintain for a longer time in the environment of HIV infection, and have better comprehensive efficacy.

[0100] The recombinant lentiviral vector preparation described in the present invention uses sucrose and glucose as stabilizers and is used in combination with F68, so that the preparation has significantly enhanced stability compared with other preparations in the prior art (for example, it can be stored for a long time under the ultra-low temperature condition of -80 °C, and after being exposed to 2-8 °C or the body temperature of an organism (such as 37 °C) for a long time, it can still better maintain the activity of the recombinant lentiviral vector meeting the usage requirements, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0101] Figure 1A-1C Show the functional structural regions inserted in each plasmid tested in the embodiments of the present invention, including the CAR coding sequence and / or the shRNA coding sequence, and / or the functional structure of the reporter gene. Among them, G3L2 represents the coding sequence of the shRNA against Gag and LTR (the sequences are SEQ ID NO: 1 and 2 respectively).

[0102] Figure 2 Show the original pGTV-PEDF plasmid in the present invention.

[0103] Figure 3 shows the acute killing results of the modified CD3+ T cells against target cells after mixing with target cells at different ratios. Using the CytoTox 96 non-radioactive cytotoxicity detection kit, the content of lactate dehydrogenase LDH in the supernatant after 4 hours of mixing was detected and calculated. The target cells in A express gp120 of the HIV virus strain AC10.29, the target cells in B express gp120 of the HIV virus strain SF162, and the target cells in C express human leukocyte antigen DR0401. The icons Z09, Z10 and CTRL indicate the structures corresponding to the modified cells, where Z09 and Z10 are CD3+ T cells transduced with recombinant lentiviruses SIV-Z09 and SIV-Z10 respectively, and CTRL is a CD3+ T cell transduced with recombinant lentivirus SIV-EGFP.

[0104] Figure 4 Show the results of flow cytometry detection and analysis of the modified CD3+ T cells and target cells after mixing at a fixed ratio of 3:1. All target cells are 293F cells, obtained by transfection with the pAcGFP plasmid expressing the membrane protein gp120 of the HIV virus strain and screening with G418. The original green fluorescent protein AcGFP in the plasmid is co-expressed with gp120 through P2A. The target cells express gp120 of the HIV virus strain NL4-3. The X-axis is labeled with the corresponding CAR molecular structure in the modified CD3+ T cells. CTRL refers to the CD3+ T cells transduced with recombinant lentivirus SIV-EGFP, and CD3 refers to the CD3+ T cells without transduction of any recombinant lentivirus SIV. Calculate the number of target cells after mixing for 24 hours based on the flow cytometry detection results of GFP-positive CD3- 293F cells, and then calculate the reduction ratio of the target cells in each experimental group sample relative to the target cells in the CD3+ T cell sample transduced with recombinant lentivirus SIV-Z17 as the inhibition rate of each sample against the target cells.

[0105] Figure 5 shows the results of the inhibitory effect on target cells after the modified CD3+ T cells were mixed with target cells at different ratios for 24 hours. At 24 hours after mixing, flow cytometry was used for detection and analysis. The number of target cells after 24 hours of mixing was calculated based on the flow cytometry results of GFP+CD3- 293F cells. Then, the reduction ratio of target cells in each experimental group sample relative to the target cells in the CTRL control at an effector-to-target ratio of 1:1 was calculated as the inhibition rate of each sample on target cells. The target cells in A express gp120 of the HIV strain NL4-3, and the target cells in B express human leukocyte antigen DR0401. CTRL is CD3+ T cells transduced with the recombinant lentivirus SIV-EGFP.

[0106] Figure 6 Shown are the results after flow cytometry and analysis of the cells at 24 days after activation. CTRL is the modified cell control transfected only with the plasmid encoding G3L2 shRNA, and CD3 is the modified cell control not transduced with any recombinant lentivirus SIV. Detailed implementation manners

[0107] The following examples are only used to explain this application and are not intended to limit the protection scope of this application.

[0108] Unless otherwise specified, the reagent raw materials described in the following examples are all common commercially available raw materials, and the reagents are prepared by conventional methods. The methods not detailed in the examples are all conventional operations in the art.

[0109] The exemplary lentiviral vector used in the embodiments of this application is a recombinant simian immunodeficiency virus (SIV) vector carrying exogenous genes (the coding sequence of the CAR against HIV and / or the coding sequence of the shRNA against the HIV genome), and the construction and packaging methods thereof are also well known to those skilled in the art. For the lentiviral vector in this application and the T cells modified by it, the detailed content can be referred to the international patent application PCT / CN2022 / 143440.

[0110] Example 1 Vector construction and preparation of SIV lentiviral vector

[0111] 1.1. Vector construction

[0112] The plasmid carrying the exogenous gene expression cassette used in the embodiments of this application was constructed based on the shuttle plasmid in the SIV third-generation lentiviral vector packaging system. The schematic diagrams of the important functional structures inserted into this series of plasmids relative to the shuttle plasmid in the SIV third-generation lentiviral vector packaging system are as Figure 1A-1C shown. Among them, Figure 1A-1CThe meanings of the various structures are as follows: G3L2 represents the coding sequence of shRNA targeting the conserved sequences Gag and LTR in the HIV genome (the sequences are shown as SEQ ID NO: 1 and 2 respectively), each containing an independent promoter: the H1 promoter and the U6 promoter; CD4 signal represents the signal peptide region of CD4; CD4 D1D2 represents the D1 and D2 domains of CD4; CD8h represents the hinge region of CD8α; CD8a TM and CD28TM represent the transmembrane regions of CD8α and CD28 respectively; CD28, CD137, and CD3ζ represent the signal transduction domains of CD28, CD137, and CD3ζ respectively; Nab-scFv represents a neutralizing antibody scFv (3BNC117 is used in this example); CCR5&tat / rev represents the coding sequence of shRNA targeting CCR5 and tat / rev. Reporter represents a reporter gene, and the reporter gene used in the examples of this application is truncated epidermal growth factor receptor (tEGFR). L represents the linker peptide GGGGS. The relevant sequences are shown in Table 6.

[0113] First, based on the pGTV-PEDF vector ( Figure 2 ), according to the composition shown in Figure 1A-1C and the sequences in Table 6, plasmids carrying the Z09 structure were obtained by methods such as nucleic acid fragment synthesis, seamless ligation, and transformation. Then, based on this plasmid, plasmids carrying structures such as Z10, Z14, Z15, and Z17 were constructed. The series of plasmids carrying the Z01 and Z02 structures were constructed based on the series of plasmids carrying the Z09 and Z10 structures respectively, where Z01 corresponds to Z09 and Z02 corresponds to Z10. The plasmids carrying the Z01 and Z02 structures are different from their respective corresponding plasmids Z09 and Z10 in that the former do not contain the polynucleotide sequence encoding shRNA. The construction of the plasmids carrying the Z08 structure and the Z16 structure refers to US Patent US 9,833,480 B2.

[0114] The plasmid construction steps and methods such as nucleic acid fragment synthesis, seamless ligation, and transformation are conventional techniques in the art and can be referred to, for example, Molecular Cloning: A Laboratory Manual, Third Edition, edited by J. Sambrook et al., Science Press, 2002, or operated according to the manufacturer's instructions.

[0115] 1.2. Preparation of Recombinant Lentivirus Based on SIV Vector

[0116] (1) Packaging and Purification of Recombinant Lentivirus SIV

[0117] The construction methods of the packaging vector, rev expression vector, and VSV-G expression vector are detailed in the method of Example 1 of Patent ZL200680012905.4.

[0118] The above-obtained 4 plasmids were co-transfected into 293T cells (ATCC, CRL-11268). After 48 hours of transfection, the supernatant virus solution was harvested, and after purification and concentration, a recombinant lentivirus SIV concentrate was obtained. The above virus concentrate can be used directly or aliquoted and stored at -80 °C for later use.

[0119] (2) Activity titer of recombinant lentivirus SIV

[0120] The 293T cells were infected with the recombinant lentivirus SIV obtained above, and then the proportion of cells expressing the reporter gene in the 293T cells was detected by flow cytometry, thereby obtaining the activity titer of the recombinant lentivirus SIV.

[0121] Briefly, the obtained recombinant SIV lentivirus was diluted by a certain multiple and then used to infect 293T cells seeded in a 24-well plate. The cells were harvested on the third day after infection, and incubated with the tEGFR antibody (R&D: FAB9577G-100). After the incubation was completed, the immunofluorescence signal was detected by a flow cytometer to obtain the positive expression rate of tEGFR (percentage of positive cells).

[0122] Using the formula activity titer (Tu / ml) = (positive expression rate of reporter gene (percentage of positive cells) × number of cells per well at the time of infection × dilution factor of the virus stock solution) / volume of the virus dilution added per well. For the same recombinant lentivirus, the virus activity titers obtained from 3 wells were combined and calculated to obtain the average value, and the average value was used as the activity titer of this recombinant lentivirus. The recombinant lentivirus SIV-EGFP packaged by the shuttle plasmid pGTV-EGFP carrying EGFP was used as a control. Table 1 shows the results of detection using the reporter gene.

[0123] Table 1 Activity titer of recombinant lentivirus SIV

[0124] Plasmid Name Recombinant Lentivirus SIV Name Active Titer (Tu / ml) Z01 SIV-Z01 2.70E+07 Z02 SIV-Z02 3.95E+07 Z08 SIV-Z08 3.23E+07 Z09 SIV-Z09 5.41E+07 Z10 SIV-Z10 1.35E+07 Z14 SIV-Z14 6.78E+06 Z15 SIV-Z15 2.99E+07 Z16 SIV-Z16 2.31E+07 Z17 SIV-Z17 2.54E+07 pGTV-EGFP SIV-EGFP 3.09E+08

[0125] Example 2 Functional verification of the modified primary cells

[0126] 2.1 Preparation of primary cells

[0127] Obtaining PBMC cells, sorting CD3+ T cells, and activating T cells: Using Ficoll-Paque PLUS (GE, Cat#17-1440-02), PBMC cells were isolated from EDTA-anticoagulated whole blood (donated by healthy volunteers) according to the kit instructions. According to the manufacturer's instructions, CD3+ T cells were sorted and purified using the Dynabeads untouched Human T cells kit (Thermofisher 11344D). T cells were activated using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody, and the operation was carried out according to the instructions of the Dynabeads Human T-Activator CD3 / CD28 for T-Cell Expansion and Activation kit (Thermofisher, 11131D). A portion of the activated T cells was taken for counting and flow cytometry detection. According to the detection results, the proportion of activated cells among live CD3+ T cells reached over 91.4%.

[0128] 2.2. Virus transduction and cell maintenance

[0129] The above-verified viable and activated CD3+ T cells were seeded in culture plates or flasks coated with RetroNectin (Takara T100B, 5 μg / cm 2 ). The CD3+ T cells were infected with the series of recombinant lentiviruses SIV (SIV-Z01, SIV-Z02, SIV-Z08, SIVZ09, SIV-Z10, SIVZ14 - SIVZ17) obtained in Example 1 respectively (multiplicity of infection MOI not exceeding 5). After 48 or 72 hours of infection, the cells were centrifuged to remove the original culture medium. The cells were resuspended with fresh X-vivo 15 medium (Lonza BE02-060F), and the cell density was adjusted to 1×10 6 / ml, and IL-2 (R&D 202-IL-010), IL-7 (R&D 207-IL-025), and IL-15 (R&D 247-ILB-025) were supplemented, along with human serum albumin or human AB serum or autologous serum corresponding to CD3+ T cells at a final concentration of 3 - 5%. The cytokine concentrations were adjusted according to the cell density and growth time, etc. Usually, the final concentration of IL-2 was 30 IU / mL, the final concentration of IL-7 was 5 or 10 ng / mL, and the final concentration of IL-15 was 5 or 10 ng / mL. The cells were counted and passaged every other day, and the cell density was adjusted to 1×10 6 / ml. Subsequently, during the period from after activation to 12 days after activation, the density and viability of the cells were detected and recorded, and the total number of cells was calculated. CD3+ T cells transduced with recombinant lentivirus SIV-EGFP (EGFP), and CD3+ T cells not transduced with recombinant lentivirus (CD3) were used as controls.

[0130] The results showed that: under the current culture conditions, on the 12th day after activation, the number of cells was 140 - 440 times the initial number, and their viability was greater than 80%.

[0131] 2.3. Effect after mixing modified T cells with target cells

[0132] Recombinant plasmids co-expressing gp120 protein of different HIV strains (AC10.29, NL4-3 or SF162) or human MHCII (DR0401) and AcGFP were constructed. These series of plasmids were transfected into 293F cells respectively and obtained through G418 screening. Among them, the green fluorescent protein AcGFP in the plasmid was co-expressed with gp120 or MHCII through P2A. 293F cells transfected with the AcGFP empty vector were used as negative controls. 293F cells transfected with co-expressing MHCII (DR0401) and green fluorescent protein AcGFP were used as functional controls for binding to CD4. Since CD4 and MHCII can bind naturally, the effect of the modified T cells in the examples of this application on cells expressing MHCII can be evaluated through this functional control.

[0133] In this experiment, a series of modified T cells were mixed with the above-mentioned 293F target cells, and a series of indicators were detected to determine the effect of the modified T cells. Specifically, the target cells were seeded in 24-well plates at a quantity of 1×10 4 cells / well. The stimulation of cytokines on the modified T cells was stopped 24 hours before the experiment. On the 14th day after T cell activation, the modified T cells were added to different wells at a given effector-to-target ratio (i.e., the ratio of the number of effector cells to target cells, 10:1, 3:1 or 1:1) respectively and mixed with the target cells, and the final volume of the liquid was 200 μl / well.

[0134] Four hours after mixing, a part of the supernatant was collected, and the release of lactate dehydrogenase LDH was detected using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Promega G1780) to determine the acute killing activity of CD8+ T lymphocytes in the modified T cells of each experimental group against the target cells.

[0135] The cells were collected 24 hours after mixing. Through antibody staining and flow cytometry detection, the proportion of cells expressing gp120 or MHCII in CD3- cells (mainly target cells) was calculated respectively to determine the inhibitory activity against the target cells.

[0136] The results of the acute killing activity of CD8+ T lymphocytes in the modified T cells of each experimental group against target cells showed that after 4 hours of effector-target cell mixing, the modified T cells (taking the modified T cells carrying the Z09 or Z10 structure as an example) had specific killing effects on target cells expressing gp120 proteins of different HIV strains (AC10.29, SF162), and showed a concentration gradient-dependent relationship ( Figure 3A 、 3B ).

[0137] The results of the inhibitory activity of the modified T cells on target cells showed that the modified T cells using CD4 as part of the CAR could specifically inhibit target cells expressing gp120 at an effector-target ratio of 3:1 ( Figure 4 ). Figure 5A The results of the inhibitory activity of the modified T cells on target cells at different effector-target ratios (10:1, 3:1 and 1:1) showed that this inhibitory effect was concentration gradient-dependent. These results further confirmed that target cells expressing gp120 could be specifically inhibited by CAR-T cells that specifically recognized antigens using the extracellular region of CD4, while the modified T cells without the intracellular signal transduction domain CD3ζ (i.e., Z17, without a complete CAR structure) had no inhibitory effect on target cells. It should be noted that under the same effector-target ratio, the modified T cells carrying the Z09 structure had a higher cell inhibitory effect on target cells expressing gp120 compared to the modified T cells carrying the Z14 structure ( Figure 5A ). Since the difference between the Z14 and Z09 structures was that the antigen-specific recognition domain contained in the extracellular region of the Z14 structure was the scFv of 3BNC117, while the antigen-specific recognition domain of Z09 was the D1-D2 segment of CD4, it was suggested that CAR-T cells using the D1-D2 segment of CD4 to recognize antigens had a stronger inhibitory effect on target cells expressing gp120 compared to CAR-T cells using the scFv of 3BNC117 to recognize antigens.

[0138] In addition, it could be seen from the above results that the modified T cells of this application did not significantly inhibit or kill target cells expressing MHCII (DR0401) ( Figure 3C 、 Figure 5B ). That is, although CD4 could bind to MHCII naturally, the original cells expressing MHCII in the body would not be massively killed by the modified T cells expressing CD4. It was speculated that the reason might be that the affinity of CD4 for several typical HIV-1 gp120s was stronger than its affinity for MHCII.

[0139] The above results demonstrated the specific role of the CAR structure in the technical solution of this application, which could effectively inhibit or kill HIV-infected target cells, but would not cause killing or inhibition of normal cells expressing MHCII in the body.

[0140] 2.4 Activity of the engineered T cells in cells of HIV-infected patients

[0141] To further demonstrate the effectiveness of the T cells engineered with the lentiviral vector of the present application in treating HIV virus infection, engineered T cells derived from HIV patients were prepared under the condition of adding protease inhibitors. Specifically, T cells were isolated from the blood of HIV patients and cultured in a medium supplemented with protease inhibitor Darunavir ethanolate (SelleckChem s1620) or Saquinavir mesylate (Sigma s8451) at a final concentration of 10 μM for the subsequent 12 days, and the remaining culture conditions were as described in Part 2.2 of Example 2. After 12 days, the addition of protease inhibitors was stopped and the culture was continued. The day when the activated patient T cells were isolated and cultured was designated as Day 0, and the patient-derived CD3+ T cells were engineered according to the aforementioned method on Day 2. From Day 2 to Day 24, culture supernatants were collected at different time points, and the p24 concentration in the supernatant was detected using the Lenti-p24 rapidtiter kit (Takara 632200) according to its instructions to indirectly quantify the HIV virus particles and evaluate the killing and inhibitory effects of the patient T cells engineered with the complete structure (such as Z09, Z10, etc.) on the patient's HIV-infected CD4+ T cells. Meanwhile, on Day 24, the proportion of the engineered T cells themselves was determined by detecting the reporter gene to evaluate the protective effect of the introduced shRNA on the engineered T cells.

[0142] From the detection results of the p24 concentration, virus p24 was not detected in the supernatant all the time during the whole preparation process and the subsequent culture period without using protease inhibitors, or the virus p24 concentration was near the detection threshold for the engineered T cells simultaneously carrying shRNA and CAR structures (Z09, Z10, Z16). Moreover, the inhibitory effect of the engineered T cells carrying the Z10 structure on p24 was better than that of the engineered T cells carrying the Z16 structure, indicating that the engineered T cells with the vector in the present application have better killing and inhibitory effects on HIV than the engineered T cells with Z16, and the effective time is longer.

[0143] For the engineered T cells (Z02 or Z08) that only carry the CAR structure but not the shRNA, during the subsequent culture without protease inhibitor, the concentration of viral p24 in their culture supernatant started to increase from 16 days after activation and then decreased; for the engineered T cells that only carry the G3L2 shRNA structure (CTRL) and the engineered T cells with the incomplete CAR part of the Z17 structure, the concentration of viral p24 in their culture supernatant first increased and then decreased. For the engineered T cells (CTRL and Z17) based on these structures, the peak concentration of p24 in their culture supernatant appeared 18 to 22 days after cell activation, and the peak concentration was lower than the corresponding level of unengineered CD3+ T cells at the same time point. The concentration of viral p24 in the unengineered CD3+ T cell group could be detected from 16 days after activation and continued to increase in the following days, approaching 100 ng / ml at day 20 and reaching as high as 200 ng / ml at day 25. On the basis that the inhibitory and killing effects of engineered T cells derived from healthy individuals on target cells expressing the HIV virus gp120 protein have been verified in the above examples, the above results further prove that engineered T cells from HIV patients have killing and inhibitory effects on autologous infected cells.

[0144] Figure 6 The results show that, compared with the unengineered CD3+ cell control (CD3), after culturing with T cells of HIV patients for 24 days, the proportion of the engineered T cells modified by the recombinant lentiviral vector of the present application still remained at a relatively high level, indicating that the introduced shRNA has a protective effect on the engineered T cells.

[0145] Combining the above two parts of results, it is confirmed that the T cells (CAR-T) modified by the recombinant lentiviral vector described in the present application can persistently and specifically kill and inhibit cells infected with the HIV virus, and at the same time the shRNA therein has the advantages of protecting and maintaining the engineered T cells. In particular, compared with the recombinant lentiviral vector carrying the Z16 structure, after modifying T cells with the recombinant lentiviral vector carrying the Z10 structure, the specific killing effect of the T cells on cells infected with the HIV virus is stronger and can be maintained for a longer time under HIV infection.

[0146] From the above embodiments, it can be found that by comprehensively comparing the killing ability of the modified T cells under activated conditions and their resistance to HIV virus infection, the T cells modified by the recombinant lentiviral vectors carrying the Z09 and Z10 structures in the present invention have relatively better comprehensive efficacy and significant advantages. By using the recombinant lentiviral vector in this application to simultaneously introduce shRNA that can specifically inhibit the conserved sequence of the HIV genome and the CAR structure targeting the HIV virus protein into effector T cells, a more persistent and efficient CAR-T cell that targets and kills HIV-infected cells can be constructed. When comparing such CAR-T cells, when the shRNA is the same, selecting the D1 and D2 domains of CD4 as the antigen-specific recognition domain of the CAR has better effects than other antigen-specific recognition domains in the prior art or the D1-D4 domains of CD4. When the antigen-specific recognition domain of the CAR is the same, selecting the shRNA targeting G3L2 (targeting gag and LTR respectively) can make the CAR-T cells have better comprehensive efficacy compared with other shRNA options in the prior art.

[0147] Example 3 Stability Study of Lentiviral Vector Formulations

[0148] 3.1 Preparation of Lentiviral Vector Formulations

[0149] Taking the recombinant lentiviral vector SIV-Z10 as an example, the following is a study on the formulation of the preparation. The corresponding lentiviral vector formulations were prepared according to Table 2 below.

[0150] Table 2 Composition of Lentiviral Vector Formulations

[0151]

[0152]

[0153] Among them, Comparative Example 1, Comparative Example 2, and Comparative Example 5 are the formulations disclosed in Chinese Patent Application Publication No. CN103316356A.

[0154] 3.2 Stability Study

[0155] (1) Stability Study at 2 - 8°C

[0156] The lentiviral vector formulations listed in Table 2 were placed at 2 - 8°C for 32 days, and the active titer of the lentiviral vector was detected at multiple time points (0 day, 7 days, 14 days, 32 days) using the method of Example 1 to detect the stability of each formulation in Table 2 under the condition of 2 - 8°C.

[0157] The detection results are shown in Table 3 below. The results show that the stability of the virus vectors in the formulations of Experimental Examples 1-3 of the present application after being stored at 2-8°C for 7 days, 14 days, and 32 days is higher than that of the formulations of Comparative Examples 1-5. Among them, after being stored at 2-8°C for 32 days, the relative active titer of the virus vector in the formulations of Experimental Examples 1-3 of the present application is still more than 70%, while in Comparative Examples 1-5, only 40%-60% remains.

[0158] The above results indicate that at 2-8°C, compared with the formulations of Comparative Examples 1-5, the lentiviral vector formulations of Experimental Examples 1-3 of the present application have more excellent stability.

[0159] Table 3 Detection results of the formulations of the present application and the comparative formulations at 2-8°C

[0160]

[0161] (2) Stability study at 37°C

[0162] The lentiviral vector formulations listed in Table 2 were stored at 37°C for 3 days, and the active titer of the lentiviral vector was detected at multiple time points (0 day, 1 day, 2 days, 3 days) using the method of Example 1 to detect the stability of each formulation in Table 2 at 37°C.

[0163] The detection results are shown in Table 4 below. The results show that the stability of the virus vectors in the formulations of Experimental Examples 1-3 of the present application after being stored at 37°C for 1 day, 2 days, and 3 days is higher than that of the formulations of Comparative Examples 1-5. Among them, after being stored at 37°C for 3 days, the relative active titer of the virus vector in the formulations of Experimental Examples 1-3 of the present application is still more than 50%, while in Comparative Examples 1-5, only about 20%-30% remains.

[0164] The above results indicate that at 37°C, compared with the formulations of Comparative Examples 1-5, the lentiviral vector formulations of Experimental Examples 1-3 of the present application have more excellent stability.

[0165] Table 4 Detection results of the formulations of the present application and the comparative formulations at 37°C

[0166]

[0167] Example 4 Long-term stability study of lentiviral vector formulations

[0168] The lentiviral vector formulations listed in Table 2 were stored at -80°C for 2 years, and the active titer of the lentiviral vector was detected at multiple time points (0 month, 5 months, 8 months, 1 year, 2 years) using the method of Example 1 to detect the stability of each formulation in Table 2 at -80°C.

[0169] The test results are shown in Table 5 below. The results show that after the preparations of Experimental Examples 1-3 of the present application were stored at -80°C for 5 months, 8 months, 1 year, and 2 years, the active titer of the lentiviral vector basically did not decrease. However, after the preparations of Comparative Examples 1-5 were stored at -80°C for 5 months, 8 months, 1 year, and 2 years, the active titer of the lentiviral vector decreased to varying degrees. Among them, after being stored at -80°C for 2 years, the relative active titer of the viral vector in the preparations of Experimental Examples 1-3 of the present application was still more than 98%, while the relative active titer of Comparative Examples 1-5 remained less than 90%.

[0170] The above results indicate that under the long-term storage condition of -80°C, compared with the preparations of Comparative Examples 1-5, the lentiviral vector preparations of Experimental Examples 1-3 of the present application still have better stability.

[0171] Table 5 Test results of the preparations of the present application and the comparative preparations at -80°C

[0172]

[0173] The embodiments of the present application described above are not intended to limit the present application. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

[0174] Sequence Listing

[0175] Table 6: The following are all the sequences mentioned in this article.

[0176]

[0177]

[0178]

[0179]

Claims

1. A recombinant lentiviral vector preparation, characterized in that: The preparation comprises: a) recombinant lentiviral vector; b) histidine hydrochloride buffer; c) glucose and sucrose; and d) Surfactant.

2. The recombinant lentiviral vector preparation according to claim 1, wherein The total concentration of glucose and sucrose in the preparation is 1-15% (w / v), preferably 3-13% (w / v), more preferably 5-8% (w / v).

3. The recombinant lentiviral vector preparation according to claim 1 or 2, wherein: The concentration of glucose in the preparation is 1-10% (w / v), preferably 2-8% (w / v), more preferably 4-6% (w / v).

4. The recombinant lentiviral vector preparation according to any one of claims 1 to 3, wherein The concentration of sucrose in the preparation is 1-8% (w / v), preferably 1-5% (w / v), more preferably 1-2% (w / v).

5. The recombinant lentiviral vector preparation according to any one of claims 1 to 4, wherein: The surfactant is poloxamer 188.

6. The recombinant lentiviral vector preparation according to claim 5, wherein The concentration of Poloxamer 188 in the preparation is 0.001-0.5% (w / v), preferably 0.001%-0.1%, more preferably 0.005-0.015%.

7. The recombinant lentiviral vector preparation according to any one of claims 1 to 6, wherein: The molar concentration range of the histidine hydrochloride buffer is 1-50 mmol / L, preferably 5-30 mmol / L, more preferably 5-20 mmol / L.

8. The recombinant lentiviral vector preparation according to any one of claims 1 to 7, wherein: The pH value of the preparation is in the range of 6.0-8.0, preferably 6.5-7.

4.

9. The recombinant lentiviral vector preparation according to any one of claims 1 to 8, wherein The recombinant lentiviral vector preparation comprises: a) recombinant lentiviral vector; b) histidine hydrochloride buffer at a concentration of 1-50 mmol / L; c) glucose at a concentration of 1-10% (w / v); d) sucrose at a concentration of 1-8% (w / v); e) Poloxamer 188 at a concentration of 0.001-0.5% (w / v); The pH value of the recombinant lentiviral vector preparation is 6.0-8.0, preferably 6.5-7.

4.

10. The recombinant lentiviral vector preparation according to any one of claims 1 to 9, wherein: The recombinant lentiviral vector preparation comprises: a) recombinant lentiviral vector; b) histidine hydrochloride buffer at a concentration of 5-30 mmol / L; and c) glucose at a concentration of 2-8% (w / v); d) sucrose at a concentration of 1-5% (w / v); e) Poloxamer 188 at a concentration of 0.001-0.1% (w / v); The pH value of the recombinant lentiviral vector preparation is 6.5-7.

4.

11. The recombinant lentiviral vector preparation according to any one of claims 1 to 10, wherein: The recombinant lentiviral vector preparation comprises: a) recombinant lentiviral vector; b) histidine hydrochloride buffer at a concentration of 5-20 mmol / L; and c) glucose at a concentration of 4-6% (w / v); d) sucrose at a concentration of 1-2% (w / v); e) Poloxamer 188 at a concentration of 0.005-0.015% (w / v); The pH value of the recombinant lentiviral vector preparation is 6.5-7.

4.

12. The recombinant lentiviral vector preparation according to any one of claims 1 to 11, wherein: The recombinant lentiviral vector is a lentiviral vector coated with vesicular stomatitis virus G protein.

13. The recombinant lentiviral vector preparation according to any one of claims 1 to 12, wherein: The recombinant lentiviral vector is a recombinant human immunodeficiency virus vector, a recombinant simian immunodeficiency virus vector, a recombinant equine infectious anemia virus vector, a recombinant feline immunodeficiency virus vector or a recombinant sheep arthritis encephalitis virus vector.

14. The recombinant lentiviral vector preparation according to any one of claims 1 to 13, wherein: The recombinant lentiviral vector carries: (1) a coding sequence of a chimeric antigen receptor (CAR) targeting HIV, and / or (2) a coding sequence of a shRNA targeting an HIV gene.

15. The recombinant lentiviral vector preparation according to claim 14, wherein The CAR comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region can specifically bind to the gp120 protein of HIV; preferably, the extracellular region comprises the D1 and D2 domains of the human CD4 molecule.

16. The recombinant lentiviral preparation according to claim 15, wherein: The amino acid sequence of the CAR is shown in any one of SEQ ID NOs: 24-29.

17. The recombinant lentiviral preparation according to claim 16, wherein: The amino acid sequence of the CAR is shown in SEQ ID NO:

25.

18. The recombinant lentiviral vector preparation according to any one of claims 14 to 17, wherein: The shRNA includes shRNA targeting gag and shRNA targeting LTR.

19. The recombinant lentiviral vector preparation according to claim 18, wherein The sequence of the shRNA targeting gag is shown in SEQ ID NO: 1, and the sequence of the shRNA targeting LTR is shown in SEQ ID NO:

2.

20. The recombinant lentiviral vector preparation according to claim 19, wherein The coding sequence of the shRNA targeting gag is shown in SEQ ID NO: 3, and the coding sequence of the shRNA targeting LTR is shown in SEQ ID NO:

4.

21. The recombinant lentiviral vector preparation according to any one of claims 1 to 20, wherein: The activity titer of the recombinant lentiviral vector is 1×10 5 -5×10 10 Tu / m1, preferably 5×10 5 -5×10 9 Tu / m1, more preferably 5×10 5 -5×10 8 Tu / ml.

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