WO2023125822A1 - 靶向hiv感染细胞的嵌合抗原受体t细胞 - Google Patents

靶向hiv感染细胞的嵌合抗原受体t细胞 Download PDF

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WO2023125822A1
WO2023125822A1 PCT/CN2022/143440 CN2022143440W WO2023125822A1 WO 2023125822 A1 WO2023125822 A1 WO 2023125822A1 CN 2022143440 W CN2022143440 W CN 2022143440W WO 2023125822 A1 WO2023125822 A1 WO 2023125822A1
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cells
sequence
seq
shrna
recombinant cell
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French (fr)
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朱卫军
王宏伟
杨英
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北京三诺佳邑生物技术有限责任公司
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Priority to CN202280007881.2A priority Critical patent/CN116917313A/zh
Publication of WO2023125822A1 publication Critical patent/WO2023125822A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/17—Lymphocytes; B-cells; T-cells; Natural killer cells; Interferon-activated or cytokine-activated lymphocytes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70514—CD4
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues

Definitions

  • the present application relates to the technical field of immunotherapy for infectious diseases, in particular to an engineered immune effector cell for HIV immunotherapy.
  • HIV Human Immunodeficiency Virus
  • HIV is a virus that can attack the human immune system. It takes CD4-positive T lymphocytes in the human immune system as the main attack target, destroys the cells in large quantities, and makes the human body lose immune function, so that it is easy to infect various diseases, and even Malignant tumors have a high mortality rate.
  • ART Highly Active Antiretroviral Therapy
  • ART Antiretroviral Therapy
  • ART has transformed HIV infection into a chronic infection.
  • the current unmet clinical needs mainly include: a long-acting, safer and tolerable regimen; about 10% of patients are resistant to existing ART and require treatment with a new mechanism Plan; it is necessary to aim at the function of the virus reservoir to achieve functional cure, and even remove the latent reservoir to achieve eradication and cure.
  • broad-spectrum neutralizing antibodies can be used, multi-specific molecules can be developed based on antibodies, and T cells can be engineered using chimeric antigen receptors (CAR).
  • CAR chimeric antigen receptors
  • broad-spectrum neutralizing antibodies can also bind macrophages, NK cells, and CD8+ T cells through Fc to eliminate cells displaying viral proteins.
  • broad-spectrum antibodies including 3BNC117 have been produced against gp120 Neutralizing antibodies (Scheid et al Science 2011).
  • virus strains have developed resistance to this type of antibody, and this resistance must be overcome to obtain the best clinical effect (Qian Wang et al., Front Med. 2020 Feb; 14(1):30-42).
  • NCT03240328 showed that the use of CAR-T was safe, but during the 6-month observation period, the HIV load of patients who stopped HAART treatment rebounded at an average of 5.3 weeks, and the rebounded virus escaped the neutralizing antibody VRC01; based on N6 There is no official result of the CAR-T clinical trial published yet.
  • CAR-T cells Another idea to cure AIDS is to reactivate the latent HIV, and through the method of jointly enhancing the body's HIV-specific immune response, it can compensate for the immune damage caused by HIV to the body and enhance the effect of clearing the HIV reservoir.
  • Roberts et al. tried to use CAR-T cells to treat HIV infection. They selected the complete CD4 sequence as the antigen-specific recognition domain to bind the envelope protein gp120 on the surface of HIV-infected cells, although it has a partial killing effect. The cells function, but after years of trying, it ended in failure. The reason is that on the one hand, the specific lethality of the CAR-T cells themselves needs to be improved; shorter.
  • the outer membrane glycoprotein gp120 of the virus first binds to the CD4 molecule on the cell surface and binds to co-receptors CCR5 or CXCR4, etc., thereby mediating the entry of the virus core into the cell.
  • CD4 on the cell surface is the main binding target of gp120, and they have high binding affinity.
  • the dissociation constant Kd of CD4 and several gp120 ranges from 2.2 ⁇ 10 - 8 to 8 ⁇ 10 -10 M (Lasky et al Cell 1987; Zhang Biochemistry et al 2000; Myszka et al PNAS 2000).
  • CD4 or CD8 with specific MHC-polypeptide complexes is only 10 -4 -10 -6 M (ZeNan L. Chang and Yvonne Y. Chen Trends in Molecular Medcine 2017).
  • affinity of CD4 to several typical HIV-1 gp120s is not high.
  • Weaker affinity for these HIV-1 gp120 than scFv derived from a typical broadly neutralizing antibody (3BNC117) (Scheid et al Science 2011; van Dorsten et al J Viral 2020).
  • the present application provides, on the one hand, a cell with stronger lethality to HIV-infected cells, which can be maintained for a longer period of time in an HIV-infected environment, and exerts Recombinant cells with longer potency (eg, CAR-T cells).
  • the present application also provides a chimeric antigen receptor (CAR) against HIV, the cells comprising the chimeric antigen receptor have efficient and specific killing activity against HIV-infected cells, and the above-mentioned CAR Combination with at least one shRNA/shRNA coding sequence/vector comprising a shRNA coding sequence.
  • the recombinant cells provided herein or cells comprising the chimeric antigen receptors are not prone to cytokine storms.
  • the present application relates to a recombinant cell, specifically:
  • a recombinant cell comprising the following functional structures:
  • CAR chimeric antigen receptor
  • said CAR comprises an extracellular region, a transmembrane region and an intracellular region, wherein said extracellular region can specifically bind to the gp120 protein of HIV;
  • shRNA short hairpin RNA
  • shRNA short hairpin RNA
  • the shRNA targets and inhibits any one or more hosts or HIV genes involved in the HIV life cycle selected from the following group: NF- ⁇ B , CCR5, TSG101, CXCR4, P-TEFb, tat, rev, nef, env, LTR, and gag.
  • the recombinant cells are derived from humans or primates. In other embodiments, the recombinant cells are derived from HIV-infected patients, or from healthy individuals. In some embodiments, the recombinant cells are derived from HIV recipient cells or peripheral blood mononuclear cells. In a further preferred embodiment of the present invention, said recombinant cells are derived from lymphocytes. In some embodiments, the recombinant cells are derived from T cells. In other embodiments, the recombinant cells are derived from naive T cells ( T), memory T cells, and effector T cells. In other embodiments, the recombinant cells are derived from cytotoxic T cells, helper T cells, and regulatory T cells.
  • T naive T cells
  • T naive T cells
  • the recombinant cells are derived from cytotoxic T cells, helper T cells, and regulatory T cells.
  • the T cells are derived from CD4+ T cells, CD8+ T cells.
  • the recombinant cells are derived from NKT cells.
  • the recombinant cells are derived from ⁇ T cells.
  • the recombinant cells are derived from NK cells.
  • the recombinant cells are derived from antigen-presenting cells, such as macrophages and dendritic cells.
  • said recombinant cells are derived from progenitor cells or stem cells.
  • the progenitor or stem cells comprise hematopoietic stem cells (eg, CD34+ cells) or hematopoietic progenitor cells.
  • the stem cells include memory T stem cells (memory T stem cells), such as central memory T cells (central memory T cells), effector memory T cells (effector memory T cells) or stem cell-like Memory T cell (stem cell memory T cell).
  • the stem cells can be induced to differentiate into any T cells as described above.
  • the recombinant cells are derived from an HIV-infected patient. In some embodiments, the recombinant cells are derived from healthy humans.
  • shRNA targets the gag gene and the LTR gene of HIV.
  • shRNA targets the gag and nef genes of HIV.
  • nucleic acid sequence of the shRNA comprises the sequence shown in SEQ ID NO: 1, 2, 5 and/or 6 or comprises the same sequence as SEQ ID NO: 1, 2, 5 and/or Or a nucleic acid sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity to the sequence shown in 6.
  • the promoters used for the expression of SEQ ID NO: 1 and 2 are strong promoters, such as the H1 promoter and the U6 promoter. In some embodiments, the same promoter is used for expression of said SEQ ID NO: 1 and 2. In some embodiments, the expression of said SEQ ID NO: 1 and 2 use different promoters. In some embodiments, the expression of said SEQ ID NO: 1 and 2 uses the H1 promoter and the U6 promoter, respectively.
  • the promoters used for the expression of SEQ ID NO: 5 and 6 are strong promoters, such as the H1 promoter and the U6 promoter. In some embodiments, the same promoter is used for expression of said SEQ ID NO: 5 and 6. In some embodiments, the expression of said SEQ ID NO: 5 and 6 use different promoters. In some embodiments, the expression of said SEQ ID NO: 5 and 6 uses the H1 promoter and the U6 promoter, respectively.
  • the nucleic acid sequences of the H1 promoter and the U6 promoter are shown in SEQ ID NO: 9 and 10, respectively, or have at least 85%, 90%, 93%, 95%, 97%, 98%, 99% sequence identity.
  • the extracellular region comprises a structure selected from the group consisting of the D1 domain of a human CD4 molecule, the D1 and D2 domains of a human CD4 molecule, the D1 to D3 domains, D1-D4 domains of a human CD4 molecule, a gp120-specific antibody or an antigen-binding fragment thereof (eg, the sequence shown in SEQ ID NO: 15).
  • the extracellular region comprises the D1 and D2 domains of the human CD4 molecule but does not comprise the D3 and D4 domains.
  • the extracellular region comprises an amino acid sequence as shown in SEQ ID NO: 13 or comprises at least 85%, 90%, 93%, 95%, 97%, 98% of the amino acid sequence , Amino acid sequences with more than 99% identity.
  • transmembrane region is derived from or comprises a transmembrane domain of one or more protein molecules selected from the group consisting of ⁇ , ⁇ or ⁇ of T cell receptors chain, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1(CD11a, CD18) , ICOS (CD278), 4-1BB (CD137), GITR, CD40, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4 , CD49D4, ITGA6, VLA-6, CD49f, ITGAD, CD11d, IT
  • transmembrane region is from or comprises the transmembrane domain of the CD8 ⁇ protein molecule
  • transmembrane region comprising the amino acid sequence shown in SEQ ID NO: 18 or comprising at least 85%, 90%, 93%, 95%, 97% thereof , 98%, 99% identity amino acid sequence.
  • the intracellular region comprises a primary signal transduction domain derived from or comprising one selected from the following Signal transduction domains of one or more protein molecules: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FCER1G, Fc ⁇ R1b, CD79a, CD79b, Fc ⁇ RIIa, DAP10 and DAP12
  • the intracellular region further comprises a co-stimulatory domain derived from or comprising signal transduction of one or more protein molecules selected from the following Domains: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM -1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB
  • the intracellular region further comprises a connecting peptide between the primary signal transduction domain and each co-stimulatory domain, wherein the connecting peptide is capable of connecting the primary The signal transduction domain and each co-stimulatory domain, and maintain a certain relative spatial distance so as to ensure their respective functional integrity, and any natural or synthetic peptide sequence for signal transduction therebetween.
  • the connecting peptide is a short peptide formed by G and S, such as (GS)n or (G)n(S)m, wherein n or m is any positive integer between 1-20 , such as (GGGGS)1,2,3.
  • the hinge region is from or comprises a human Ig hinge region, a GS linker, a KIR2DS2 hinge or a hinge region of CD8 ⁇ .
  • the hinge region is from or comprises the hinge region of human CD8 ⁇ .
  • the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 20 or comprises at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity thereto amino acid sequence.
  • the CAR is further linked to a signal peptide
  • the signal peptide can localize the CAR on the cell membrane of the recombinant cell.
  • the signal peptide is from or comprises the signal peptide of any secreted or membrane protein.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 35 or comprises A sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity thereto.
  • the recombinant cell comprises encoding the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 35 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 3 and 4 (or SEQ ID NO: 7 and 8) and the amino acid sequence shown in encoding SEQ ID NO: 35 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 36 or comprises A sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity thereto.
  • the recombinant cell comprises encoding the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 36 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 3 and 4 (or SEQ ID NO: 7 and 8) and the amino acid sequence shown in encoding SEQ ID NO: 36 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 43 or comprises A sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity thereto.
  • the recombinant cell comprises encoding the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 43 nucleic acid sequence.
  • the recombinant cell comprises the nucleotide sequence shown in SEQ ID NO: 3 and 4 (or SEQ ID NO: 7 and 8) and the amino acid sequence shown in encoding SEQ ID NO: 43 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 46 or comprises A sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity thereto.
  • the recombinant cell comprises encoding the nucleic acid sequence shown in SEQ ID NO: 1 and 2 (or SEQ ID NO: 5 and 6) and the amino acid sequence shown in SEQ ID NO: 46 nucleic acid sequence.
  • the recombinant cell comprises the nucleic acid sequence shown in SEQ ID NO: 3 and 4 (or SEQ ID NO: 7 and 8) and the amino acid sequence shown in encoding SEQ ID NO: 46 nucleic acid sequence.
  • the recombinant cell further comprises a reporter and/or a safety switch.
  • the safety switch is selected from one or more of: iCaspase-9, iCaspase-1, iCaspase-8, thymidine kinase (eg, HSV-TK, VZV-TK) , cytosine deaminase (CD), CD20, tEGFR, FR806 and RQP8.
  • the safety switch is tEGFR, preferably, the amino acid sequence of the tEGFR is shown in SEQ ID NO: 49 or 50.
  • a vector comprising a polynucleotide sequence encoding the CAR and/or shRNA of the recombinant cell according to any one of items 1-19.
  • the vector of item 20 selected from a plasmid, a viral vector or a linear nucleic acid molecule.
  • the vector is a retroviral shuttle plasmid vector.
  • the vector is a SIV viral vector.
  • a pharmaceutical composition comprising the recombinant cell according to any one of items 1-19 or the carrier according to any one of items 20-22.
  • the pharmaceutical composition can be used to treat patients infected with HIV.
  • the HIV-infected patient is on long-term antiretroviral therapy.
  • a method for treating and/or preventing HIV infection or AIDS comprising administering the recombinant cell according to any one of items 1-19 or any one of items 20-22 to a subject infected with HIV or suffering from AIDS A carrier for an item.
  • the present application also provides a method for preparing the recombinant cell according to any one of items 1-19, which comprises introducing the vector according to any one of items 20-22 into the cell for expression.
  • it comprises packaging the SIV virus shuttle plasmid vector into SIV virus particles and using the virus particles to infect the cells.
  • the present application also relates to a chimeric antigen receptor (CAR), specifically:
  • a chimeric antigen receptor comprising an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region is from or comprises the extracellular region of a human CD4 molecule, and the transmembrane region is from or comprises a CD8 ⁇ transmembrane domain.
  • chimeric antigen receptor according to item 1, wherein the chimeric antigen receptor further comprises a hinge region.
  • the extracellular region and the transmembrane region are connected by a hinge region.
  • said hinge region is derived from or comprises a human Ig hinge region, a GS linker, a KIR2DS2 hinge or a hinge region of CD8 ⁇ , preferably a hinge region of CD8 ⁇ .
  • a chimeric antigen receptor as described in item 3 said hinge region comprising an amino acid sequence as shown in SEQ ID NO: 20 or comprising at least 85%, 90%, 93%, 95% of the sequence , 97%, 98%, 99% identical amino acid sequences.
  • said intracellular region comprises a primary signal transduction domain and a co-stimulatory domain
  • said primary signal transduction domain being derived from Or comprise a signal transduction domain of one or more protein molecules selected from: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FCER1G, Fc ⁇ R1b, CD79a, CD79b, Fc ⁇ RIIa, DAP10 and DAP12, and the co-stimulatory domain
  • Signal transduction domains from or comprising one or more protein molecules selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL
  • chimeric antigen receptor as described in item 8, wherein said primary signal transduction domain is from or comprises a signal transduction domain of CD3 ⁇ or DAP10, and said co-stimulatory domain is from or comprises CD137, One or a combination of signal transduction domains of CD28, 2B4 or DAP10.
  • chimeric antigen receptor according to any one of items 1-9, wherein the intracellular region comprises:
  • chimeric antigen receptor according to any one of item 8 or 9, wherein a linker peptide is further included between the primary signal transduction domain and the co-stimulatory domain in the intracellular region.
  • sequence of the intracellular region comprises the amino acid sequence shown in any one of SEQ ID NO: 28-34 or comprises at least one of any sequence Amino acid sequences of 85%, 90%, 93%, 95%, 97%, 98%, 99% identity.
  • chimeric antigen receptor according to any one of items 1-13, wherein the chimeric antigen receptor is further linked to a signal peptide derived from or comprising a signal peptide of any secreted or membrane protein.
  • chimeric antigen receptor according to any one of items 1-15, wherein the chimeric antigen receptor comprises an amino acid sequence as shown in any one of SEQ ID NO: 35, 36, 43-46, Or comprising an amino acid sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity to any of these sequences.
  • a vector comprising the polynucleotide sequence as described in item 17.
  • a recombinant cell comprising a chimeric antigen receptor as described in any one of items 1-16, or a polynucleotide sequence as described in item 17, or a vector as described in item 18 or 19 .
  • the present application also relates to the combination of the above-mentioned chimeric antigen receptor and at least one shRNA/shRNA coding sequence/vector comprising the shRNA coding sequence, specifically:
  • a chimeric antigen receptor as described in items 1-16, or a polynucleotide sequence as described in claim 17, a vector as described in claim 18 or 19, or as described in claim 20 or 21 Combination of recombinant cells of at least one HIV-targeting shRNA or its coding sequence or a vector comprising its coding sequence.
  • said at least one shRNA targets to inhibit any one or more host or HIV genes involved in the HIV life cycle selected from the group consisting of NF- ⁇ B, CCR5, TSG101, CXCR4, P-TEFb, tat, rev, nef, env, LTR and gag.
  • shRNA targeting gag comprises the sequence shown in SEQ ID NO: 1 or 5 or comprises at least 85% identity with the sequence shown in SEQ ID NO: 1 or 5
  • sequence of the shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 2 or a sequence having at least 85% identity with the sequence shown in SEQ ID NO: 2
  • shRNA targeting nef comprises the sequence shown in SEQ ID NO: 6
  • sequence shown may comprise a sequence having at least 85% identity to the sequence shown in SEQ ID NO:6.
  • shRNA targeting gag comprises the sequence shown in SEQ ID NO: 1
  • shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 2;
  • shRNA targeting gag comprises the sequence shown in SEQ ID NO: 5
  • shRNA targeting nef comprises the sequence shown in SEQ ID NO: 6.
  • the coding sequence of the gag-targeting shRNA comprises a sequence shown in SEQ ID NO: 3 or 7 or comprises at least 85% of the sequence shown in SEQ ID NO: 3 or 7 %, 90%, 93%, 95%, 97%, 98%, 99% identical sequence
  • the coding sequence of the shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 4 or comprises the sequence shown in SEQ ID NO: 4
  • the sequence shown has a sequence of at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity
  • the coding sequence of the shRNA targeting nef comprises the sequence shown in SEQ ID NO: 8 or comprises A sequence having at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identity to the sequence shown in SEQ ID NO:8.
  • the coding sequence of the shRNA targeting gag comprises the sequence shown in SEQ ID NO: 3
  • the coding sequence of the shRNA targeting LTR comprises A sequence as shown in SEQ ID NO: 4, or a vector comprising the above coding sequence
  • the coding sequence of the shRNA targeting gag comprises the sequence shown in SEQ ID NO: 7
  • the coding sequence of the shRNA targeting nef comprises Sequence as shown in SEQ ID NO: 8, or a vector comprising the above coding sequence.
  • a pharmaceutical composition comprising the recombinant cell as described in item 20 or 21, or the recombinant cell as described in any one of items 22-29 and at least one shRNA or its coding sequence or a vector comprising its coding sequence combination, and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition further includes other anti-HIV drugs, including but not limited to reverse transcriptase inhibitors, protease inhibitors, HIV vaccines, broad-spectrum neutralizing antibodies and/or CAR-T cells.
  • the pharmaceutical composition of the present invention is used for treating and/or preventing HIV infection or AIDS.
  • the chimeric antigen receptor of item 1-16, or the polynucleotide sequence of item 17, or the vector of item 18 or 19, or the cell of item 20 or 21, or any of items 22-29 Use of a combination in the preparation of a medicament for treating and/or preventing HIV infection or AIDS.
  • a method for treating and/or preventing HIV infection or AIDS comprising administering the cell described in item 20 or 21, or the cell described in any one of items 22-29, to a subject infected with HIV or suffering from AIDS
  • the recombinant cell and at least one The shRNA or its coding sequence or a vector comprising its coding sequence can be administered sequentially or simultaneously.
  • the method further includes administering other anti-HIV drugs to the subject, including but not limited to other nucleoside inhibitors, HIV vaccines, broad-spectrum neutralizing antibodies and/or CAR -T cells.
  • Figures 1A-1C show the functional structural regions inserted between XbaI and BamHI restriction sites in each plasmid tested in the examples herein, including the CAR coding sequence and/or shRNA coding sequence, and/or the functional structure of the reporter gene.
  • G3L2 represents the coding sequence of the shRNA (sequences are respectively SEQ ID NO: 1 and 2) for Gag and LTR
  • G2N represents the coding sequence of the shRNA (sequence is respectively SEQ ID NO: 5 and 6) for Gag and Nef.
  • Figures 2A-2C show the structures of three representative plasmids herein, wherein Figure 2A is the original pGTV-PEDF plasmid, Figure 2B is the Z09 plasmid, and Figure 2C is the Z01 plasmid.
  • Figure 3 shows the results of flow cytometric detection of CAR molecules or intracellular reporter genes after transduction of 293T cells by recombinant lentivirus SIV carrying different CAR molecules.
  • CD4+ refers to the CD4 positive cells obtained by flow cytometric detection and analysis of the transduced 293T by an anti-human CD4 antibody
  • RFP+ refers to the monomeric red fluorescent protein mRFP positive cells co-expressed with CAR molecules by direct flow cytometry.
  • CTRL is 293T cells not transduced with recombinant lentivirus SIV.
  • Figure 4 shows the results of immunofluorescent staining and flow cytometric detection of CAR molecules or the extracellular reporter gene tEGFR co-expressed with CAR molecules after the recombinant lentivirus SIV-Z09 transduced 293T cells.
  • A is the result of double staining with two antibodies
  • B is the result of single staining with anti-CD4 antibody
  • C is the result of single staining with anti-EGFR antibody
  • D is the unstained control.
  • CD4+ refers to the CD4+ positive cells obtained by immunofluorescence staining of transduced 293T with anti-human CD4 antibody and flow cytometry analysis
  • EGFR+ refers to immunofluorescence staining of transduced 293T with anti-human EGFR antibody and flow cytometry detection The obtained EGFR+ positive cells were analyzed.
  • Figure 5 shows the flow cytometry results of CD3+T cells activated by CD3/CD28 magnetic beads and cytokines for 48 hours.
  • the ratio of activated cells refers to the ratio of CD3+CD25+ cells among CD3+ T cells.
  • the control group refers to CD3+T cells not activated by magnetic beads and cytokines, and the experimental group refers to CD3+T cells activated by magnetic beads and cytokines.
  • Figure 6 shows the results of the cell number and cell viability of CD3+T cells from activation to 12 days after activation. Forty-eight hours after activation, CD3+ T cells were transduced with recombinant lentivirus SIV.
  • the solid line in the figure indicates the number of cells, corresponding to the left Y-axis.
  • the dotted line indicates cell viability, corresponding to the right Y-axis.
  • EGFP refers to CD3+ T cells transduced by recombinant lentivirus SIV-EGFP
  • CD3 represents CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • the names of the CAR molecules carried by the recombinant lentivirus SIV are respectively marked on the top of Figure 6A- Figure 6Q.
  • Fig. 7 shows the results of flow cytometric detection and analysis of the surface markers and intracellular reporter genes of the transformed CD3+ T cells.
  • Figure 7A shows the proportion of positive cells on day 13 after activation
  • Figure B shows the proportion of positive cells on day 19 after activation.
  • CTRL refers to CD3+ T cells transduced with recombinant lentivirus SIV-EGFP
  • CD3 refers to CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • Figure 8 shows the results of flow cytometric detection and analysis of surface markers of untransduced CD3+ T cells.
  • Figure 8A is the ratio of CD3+T cells at different time points
  • Figure 8B is the ratio of CD4+T cells and CD8+T cells at different time points.
  • Figure 9 shows the results of flow cytometric detection and analysis of the surface markers of transduced CD3+ T cells.
  • Figure 9A shows the proportions of CD4+T cells and CD8+T cells after transduction of CD3+T cells by recombinant lentivirus SIV-Z09 (expressing CD4 extracellular region), and
  • Figure 9B shows the ratio of recombinant lentivirus SIV-Z14 (not expressing CD4 After the transduction of CD3+T cells, the proportions of CD4+T cells and CD8+T cells were detected respectively.
  • Figure 10 shows the results of flow cytometric detection and analysis of the modified CD3+ T cells mixed with target cells at a fixed ratio of 3:1.
  • All target cells were 293F cells, which were obtained by transfecting pAcGFP plasmids expressing the membrane protein gp120 of different HIV strains and screening with G418.
  • the original green fluorescent protein AcGFP in the plasmids was co-expressed with gp120 through P2A.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • the X-axis is marked with the corresponding CAR molecular structure in the transformed CD3+ T cells
  • CTRL refers to the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • Figure 11 shows the results of detecting and analyzing the cytokine IFN- ⁇ in the supernatant 24 hours after the modified CD3+ T cells were mixed with the target cells at a fixed ratio of 3:1.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • the X-axis is marked as the corresponding CAR molecular structure in the transformed CD3+T cells
  • CTRL indicates the CD3+T cells transduced with the recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+T cells not transduced with any recombinant lentivirus SIV.
  • Figure 12 shows the results of acute killing of target cells after the modified CD3+ T cells are mixed with target cells in different ratios.
  • CytoTox 96 non-radioactive cytotoxicity detection kit the content of lactate dehydrogenase LDH in the supernatant 4 hours after mixing was detected and calculated.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses human leukocyte antigen DR0401.
  • Icons Z09, Z10 and CTRL indicate the structure corresponding to the transformed cells, where Z09 and Z10 are CD3+ T cells transduced by recombinant lentivirus SIV-Z09 and SIV-Z10 respectively, and CTRL is CD3+T cells transduced by recombinant lentivirus SIV-EGFP +T cells.
  • Figure 13 shows the results of the inhibitory effect on target cells after the modified CD3+ T cells were mixed with target cells in different ratios for 24 hours.
  • 24 hours after mixing through flow cytometric detection and analysis, the flow cytometric detection results of GFP+CD3- 293F cells were used to calculate and detect the number of target cells mixed for 24 hours, and then calculate the relative ratio of target cells in the samples of each experimental group to 1:1
  • the reduction ratio of target cells in the CTRL control of the effect-to-target ratio was used as the inhibition rate of each sample on the target cells.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses human leukocyte antigen DR0401.
  • CTRL is CD3+ T cells transduced by recombinant lentivirus SIV-EGFP.
  • Figure 14 shows the results of multifactorial detection of the culture supernatant after the modified CD3+ T cells were mixed with target cells in different ratios. 24 hours after mixing, the levels of 12 cytokines in the supernatant were detected simultaneously by Luminex (FIG. 14A-FIG. 14L).
  • the HIV virus gp120 corresponding to the target cell is shown in the icon, and the corresponding structure and effector-target ratio of the transformed cell is shown on the X-axis.
  • CTRL refers to the CD3+ T cell transduced by the recombinant lentivirus SIV-EGFP.
  • the dotted line parallel to the X-axis is the detection threshold.
  • Figure 15 shows the results of flow cytometry on day 19 after activation of CD3+ T cells.
  • the ratio of activated cells refers to the ratio of CD3+CD25+ cells in CD3+T cells
  • the control group refers to unstained CD3+T cells
  • the experimental group refers to CD3+T cells detected after staining.
  • Figure 16 shows the results of flow cytometric detection and analysis of the modified CD3+ T cells after prolonged culture and target cells mixed at a fixed ratio of 3:1.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • the X-axis is marked with the corresponding CAR molecular structure in the transformed CD3+ T cells
  • CTRL refers to the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • Figure 17 shows the results of detecting and analyzing the culture supernatant after the modified CD3+ T cells and target cells were mixed at a fixed ratio of 3:1 after extended culture. 24 hours after mixing, the cytokine IFN- ⁇ in the supernatant was detected and analyzed by ELISA.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • CTRL refers to the CD3+ T cells transduced with recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+ T cells not transduced with any recombinant lentivirus SIV .
  • Figure 18 shows the results of flow cytometric detection and analysis of the target cells after the prolonged culture of the modified CD3+ T cells mixed with target cells in different ratios for 24 hours.
  • the flow cytometric detection results of GFP+CD3- 293F cells were calculated to detect the number of target cells mixed for 24 hours, and then the recombination of the target cells in the samples of each experimental group relative to the 1:1 effect target ratio was calculated.
  • the reduction ratio of target cells in CD3+T cell samples transduced by lentivirus SIV-EGFP was used as the inhibition rate of each sample on target cells.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses human leukocyte antigen DR0401( MHCII).
  • CTRL refers to CD3+ T cells transduced with recombinant lentivirus SIV-EGFP
  • CD3 refers to CD3+ T cells not introduced with recombinant lentivirus SIV.
  • Figure 19 shows the results of multifactorial detection of the supernatant after the engineered CD3+ T cells were mixed with target cells at a fixed ratio of 3:1. 24 hours after mixing, the levels of 12 cytokines in the supernatant were simultaneously detected by Luminex (FIG. 21A-FIG. 21L).
  • the HIV virus gp120 corresponding to the target cell is shown in the icon, and the corresponding structure and effector-target ratio of the transformed cell is shown on the X-axis.
  • CTRL refers to the CD3+ T cell transduced by the recombinant lentivirus SIV-EGFP.
  • the dotted line parallel to the X-axis is the detection threshold.
  • Figure 20 shows the results of flow cytometric detection and analysis of the modified cells after the modified CD3+ T cells were mixed with the target cells at a fixed ratio of 3:1 and cultured for an extended period of time.
  • the modified CD3+ T cells are distinguished from target cells based on CD3+, and CAR expression is detected by a fluorescent protein reporter gene.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • CTRL refers to the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • Figure 21 shows the results of flow cytometric detection and target cell analysis after the remodeled CD3+ T cells were mixed with target cells at a fixed ratio of 3:1 and cultured for an extended period of time.
  • Target cells are differentiated based on CD3- versus engineered CD3+ T cells.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses only AcGFP.
  • the X-axis is marked with the corresponding CAR molecular structure in the transformed CD3+ T cells
  • CTRL refers to the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP
  • CD3 refers to the CD3+ T cells not transduced with any recombinant lentivirus SIV.
  • Fig. 22 shows the results of flow cytometric detection and analysis of the modified cells after the modified CD3+ T cells were mixed with target cells in different ratios and cultured for an extended period of time.
  • the different structures and effect-target ratios are marked on the X-axis, where CTRL represents the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP.
  • the modified CD3+ T cells are distinguished from target cells based on CD3+, and CAR expression is detected by a fluorescent protein reporter gene.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses human leukocyte antigen DR0401.
  • Fig. 23 shows the results of flow cytometric detection and target cell analysis after the modified CD3+ T cells were mixed with target cells in different ratios and cultured for an extended period of time. See the X-axis label for different structures and effect-to-target ratios.
  • Target cells were differentiated based on CD3- and engineered CD3+ T cells.
  • the target cell in A expresses gp120 of HIV strain AC10.29
  • the target cell in B expresses gp120 of HIV strain NL4-3
  • the target cell in C expresses gp120 of HIV strain SF162
  • the target cell in D expresses human leukocyte antigen DR0401.
  • Icons Z09, Z10 and CTRL indicate the CAR molecular structure corresponding to the transformed cells, where CTRL represents the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP.
  • CTRL represents the CD3+ T cells transduced by the recombinant lentivirus SIV-EGFP.
  • Figure 24 shows the results of ELISA detection of HIV virus products collected at different time points in the culture supernatant of HIV patient cells after transformation, and the results of flow cytometry detection and analysis of cells at the final time point.
  • A is the virus p24 concentration in the culture supernatant
  • CTRL is the modified cell control transfected with the G3L2 shRNA-encoding plasmid only
  • CD3 is the modified cell control not transfected with any recombinant lentivirus SIV.
  • B is the result of flow cytometric detection and analysis of the cells at 24 days after activation.
  • the present application provides, on the one hand, a recombinant cell (for example, CAR-T cell) that is more lethal to HIV-infected cells, can be maintained in an HIV-infected environment for a longer period of time, and has a longer duration of efficacy and nucleic acids and vectors encoding the CAR and other functional structures of the recombinant cells, pharmaceutical compositions and therapies for treating HIV infection.
  • a chimeric antigen receptor molecule CAR
  • cells containing the CAR molecule have high-efficiency and specific killing activity against HIV-infected cells.
  • shRNA means short hairpin RNA or small hairpin RNA. It is an artificial RNA molecule with a hairpin structure that can be used to silence target gene expression through RNA interference (RNAi).
  • RNAi RNA interference
  • a "chimeric antigen receptor (CAR)” is a class of engineered cell surface receptors, generally expressed on immune effector cells (such as effector T cells), that mediate the targeting of immune effector cells with specific targets. Killing of cells at the point of interest (in this application, referred to as “target cells”, such as cells expressing the HIV envelope protein gp120 on the cell surface).
  • CAR usually includes an extracellular region, an intracellular region, and a transmembrane region between them.
  • the "extracellular region” contains an antigen-specific recognition domain, which can specifically recognize and bind to an antigen.
  • the antigen recognition region and the transmembrane domain are connected by a hinge region.
  • An “intracellular region” may comprise one or more primary signaling domains and one or more co-stimulatory domains. After the antigen-specific recognition domain binds to the antigen to form a binary complex and is allosteric, the intracellular region can initiate a series of biochemical reactions, causing the immune effector cells where the CAR is located to produce biological effects, such as secretion of cytokines or direct killing target cells.
  • a "primary signal transduction domain” provides a first signal for activated lymphocytes (eg, effector T cells), and a "co-stimulatory domain” provides a 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 means that the receptor molecule is partially or completely located in the cell and has a The domain that plays a role in signal transduction after sex binding.
  • the "co-stimulatory domain” is mainly used to provide co-stimulatory signals to enhance the ability of immune cells, including, for example, enhancing the proliferation, survival and/or development of memory cells.
  • the "co-stimulatory domain” is selected from the signal transduction domains of CD28, 4-1BB (CD137), OX40 (CD134), and the like.
  • the "transmembrane region” refers to a thermodynamically stable protein structural region anchored in the cell membrane.
  • Transmembrane domains can be obtained from natural proteins, such as those derived from the T cell receptor (TCR).
  • TCR T cell receptor
  • the transmembrane domain is selected from the group consisting of transmembrane domains of CD4, CD8 ⁇ , CD28, and CD3 ⁇ .
  • the "hinge region” is a peptide chain connecting the antigen-specific recognition domain or the extracellular region and the transmembrane domain, usually elastic.
  • the hinge region is derived from the hinge of IgG or the hinge region of CD8 ⁇ /CD28.
  • the “hinge” of IgG refers to the region between the CH1 and CH2 functional regions of IgG, which usually contains a large amount of proline.
  • CAR-T refers to chimeric antigen receptor T cells, which are T cells expressing chimeric antigen receptor molecules on the cell surface, and can recognize antigens on the surface of target cells.
  • CAR-T has been developed to the fourth generation.
  • the CAR molecule of the first-generation CAR-T is composed of the CD3 ⁇ chain or the signal transduction domain of Fc ⁇ RI ⁇ and the fusion of the antigen recognition region, and does not contain a co-stimulatory domain.
  • the first-generation CAR-T has limited proliferation ability in vivo and is prone to apoptosis.
  • the second-generation CAR adds a co-stimulatory domain, such as CD28 or 4-1BB (CD137).
  • CD28 has strong anti-tumor activity, and the advantage of 4-1BB is that it can prolong the survival time of T lymphocytes and maintain its anti-tumor effect.
  • the second-generation CAR has stronger proliferation ability than the first-generation CAR, and can secrete more cytokines and anti-apoptotic proteins.
  • the third-generation CAR-T can not only express two co-stimulatory signal molecules at the same time, but also secrete more IFN- ⁇ , which has higher anti-tumor cytotoxicity.
  • the fourth-generation CAR-T can also secrete specific cytokines (such as IL-12) in tumors, thereby changing the tumor microenvironment, and influencing and activating other immune cells to generate an immune response.
  • linker peptide refers to an oligopeptide or polypeptide region of about 1 to 100 amino acids in length that links together any domains/regions of the CAR of the invention.
  • Linker peptides can be composed of flexible residues such as glycine and serine so that adjacent protein domains are free to move relative to each other. Longer linker peptides can be used when it is desired to ensure that two adjacent domains do not sterically interfere with each other.
  • GS linker refers to a flexible connecting peptide composed of glycine (G) and serine (S).
  • G glycine
  • S serine
  • the most common GS linker is (GGGGS)n.
  • CD4 is Cluster of Differentiation, 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 consists of an amino-terminal ectodomain (containing four immunoglobulin domains exposed on the extracellular surface: D1, D2, D3, and D4, exhibiting an Ig-like structure), a transmembrane domain, and a short cytoplasmic tail.
  • D1 and D3 are similar to immunoglobulin variable regions.
  • D2 and D4 are analogous 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.
  • gp120 is a glycoprotein exposed on the surface of the HIV envelope.
  • the 120 in its name comes from its molecular weight of 120 kDa.
  • helper T cells gp120 can bind to CD4, induce gp120 and gp41 conformational changes, enable HIV-1 to bind to co-receptors (such as CCR5 or CXCR4) expressed on host cells, and finally fuse the HIV envelope with the cell membrane allowing the virus to enter the cell.
  • co-receptors such as CCR5 or CXCR4
  • the term "antibody” includes full-length antibodies and antigen-binding fragments thereof.
  • Full-length antibodies include two heavy chains and two light chains.
  • the variable regions of the light and heavy chains are responsible for antigen binding.
  • the variable regions in both chains usually include 3 hypervariable loops called complementarity determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2 and LC-CDR3, heavy chain (HC ) CDRs include HC-CDR1, HC-CDR2 and HC-CDR3).
  • CDRs complementarity determining regions
  • LC light chain
  • HC heavy chain
  • the three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold to support the hypervariable loops.
  • FRs framework regions
  • the constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions.
  • Antibodies are classified based on the amino acid sequence of the constant region of their heavy chains.
  • the five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG and IgM, characterized by heavy chains of the alpha, delta, epsilon, gamma and mu types, respectively.
  • major antibody classes are divided into subclasses such as IgG1 ( ⁇ 1 heavy chain), IgG2 ( ⁇ 2 heavy chain), IgG3 ( ⁇ 3 heavy chain), IgG4 ( ⁇ 4 heavy chain), IgA1 ( ⁇ 1 heavy chain) or IgA2 ( ⁇ 2 heavy chain).
  • antigen-binding fragment includes antibody fragments, for example, diabodies, Fab, Fab', F(ab')2, Fv fragments, disulfide bond-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide bond stabilized diabody (ds diabody), single chain Fv (scFv), scFv dimer (bivalent diabody), Multispecific antibodies, single domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragments that are capable of binding to an antigen but do not contain the complete antibody structure, consisting of antibody fragments containing one or more CDRs .
  • An antigen-binding fragment is capable of binding the same antigen as a parent antibody or a fragment of a parent antibody (eg, a parent scFv).
  • Antigen-binding fragments also include fusion proteins comprising the antibody fragments described above.
  • the term "specific binding” refers to contact between an antibody and an antigen, a receptor and a ligand, or a specific antigen recognition domain and an antigen with a binding affinity of at least 10 ⁇ 6 M.
  • the affinity is at least about 10 ⁇ 7 M, preferably 10 ⁇ 8 M, 10 ⁇ 9 M, 10 ⁇ 10 M, 10 ⁇ 11 M or 10 ⁇ 12 M binding affinity.
  • polynucleotide or “nucleic acid” and “nucleic acid molecule” are used interchangeably, including but not limited to DNA, RNA, cDNA (complementary DNA), mRNA (messenger RNA), rRNA (ribosomal RNA) , shRNA (small hairpin RNA), snRNA (small nuclear RNA), snoRNA (short nucleolar RNA), miRNA (microRNA), genomic DNA, synthetic DNA, synthetic RNA and/or tRNA.
  • RNA complementary DNA
  • mRNA messenger RNA
  • rRNA ribosomal RNA
  • shRNA small hairpin RNA
  • snRNA small nuclear RNA
  • snoRNA short nucleolar RNA
  • miRNA miRNA
  • vector refers to a polynucleotide sequence (such as a foreign gene) that can be introduced into a host cell to transform the host and facilitate the expression (such as transcription and expression) of the introduced sequence. translation) carrier.
  • Vectors include plasmids, phages, viruses, and the like.
  • immune effector cells refer to cells capable of achieving immune effects and immune responses against target antigens or target cells, such as immune killing effects and immune response effects, such as T cells and the like.
  • the "signal peptide” refers to a short peptide chain, usually 5 to 30 amino acids in length, that directs the transfer of newly synthesized proteins to the secretory pathway.
  • the signal peptide is a membrane localization signal peptide, that is, an amino acid sequence used to direct transmembrane transfer and/or localization of a protein. In most cases, the signal peptide is located at the N-terminus of the amino acid sequence.
  • the coding sequence of the signal peptide is usually located after the initiation codon, which is an RNA region encoding a hydrophobic amino acid sequence. After the signal peptide guides the protein to complete its positioning, it is usually excised under the action of signal peptidase.
  • the "safety switch” refers to an engineered protein designed to prevent potential toxicity or otherwise prevent adverse effects of a cell therapy.
  • the expression of the safety switch is conditionally controlled to address the safety of the transplanted engineered/recombinant cells.
  • the cell can permanently incorporate the gene encoding the safety switch into its genome.
  • the safety switch is capable of mediating induction of apoptosis, inhibition of protein synthesis, DNA replication, growth arrest, transcriptional and post-transcriptional gene regulation, and/or antibody-mediated depletion (e.g., by ADCC, CDC etc.).
  • activation of the safety switch results in the death of the cell in which the safety switch has been activated.
  • activation of the safety switch results in downregulation of the activity of the cell in which the safety switch has been activated, wherein the cell can be reactivated in the future. While in other embodiments, the activity of the cell is downregulated by default and activation of the safety switch results in an upregulation of the activity of the cell, wherein the cell can be deactivated again in the future.
  • the safety switches described herein include receptors capable of being targeted by antibodies or antibody fragments, examples of such receptors include EPCAM, VEGFR, integrins (e.g., integrins ⁇ v ⁇ 3, ⁇ 4, ⁇ 13 /4 ⁇ 3, ⁇ 4 ⁇ 7, ⁇ 5 ⁇ 1, ⁇ 3, ⁇ ), TNF receptor superfamily members (eg TRAIL-R1, TRAIL-R2), PDGF receptor, interferon receptor, folate receptor, GPNMB, ICAM-1, HLA- DR, CEA, CA-125, MUC1, TAG-72, IL-6 receptor, 5T4, GD2, GDB, CD2, CD3, CD4, CD5, CD11, CD11a/LFA-1, CD15, CD18/ITGB2, CD19, CD20, CD22, CD23/IgE receptor, CD25, CD28, CD30, CD33, CD38, CD40, CD41, CD44, CD51, CD52, CD62L, CD74, CD80, CD125, CD147/bas
  • Safety switches suitable for use in the present application are known in the art, preferred examples of which include, but are not limited to, e.g., iCaspase-9 (iCaspase-9), iCaspase-1, iCaspase-8, thymidine kinase (e.g. , HSV-TK, VZV-TK), cytosine deaminase (CD), CD20, truncated EGFR (tEGFR), FR806, RQP8, or any combination thereof.
  • iCaspase-9 iCaspase-9
  • iCaspase-1 iCaspase-1
  • iCaspase-8 thymidine kinase
  • thymidine kinase e.g. , HSV-TK, VZV-TK
  • cytosine deaminase CD
  • CD20 truncated EGFR (tEGFR)
  • the safety switch is activated by an exogenous molecule (eg, a prodrug), which when activated triggers apoptosis and/or cell death in the therapeutic cell.
  • the safety switch is recognized by a molecule (eg, an antibody) capable of inducing cell death (eg, ADCC or complement-induced cell death).
  • a dimerization drug that results in iCaspase-9 activation and apoptosis can be administered.
  • the iCaspase-9 molecule contains a dimerization process chemical inducer (CID) domain that mediates dimerization in the presence of CID, which leads to both inducible and selective depletion of CAR-expressing cells.
  • CID chemical inducer
  • tEGFR for recombinant cells comprising truncated EGFR, although tEGFR lacks signaling ability, it retains epitopes recognized by molecules capable of inducing ADCC (e.g., cetuximab), thereby contributing to recombinant cells. Administration of cetuximab to cells results in ADCC and subsequent depletion of the recombinant cells.
  • exogenous molecules capable of activating and/or recognizing safety switches include guanosine (GCV), acyclovir (ACV), bromodeoxyuridine (BVDU), 6- Methoxypurine arabinoside (AraTP), 5-fluorocytosine (5-Fc), AP20187, AP1903, tamoxifen, tacrolimus, etc.
  • reporter gene refers to a nucleotide sequence encoding a reporter molecule.
  • Reporter molecule refers to a substance detectable in any of a variety of detection systems that can be used to identify potentially transfected cells.
  • reporter molecules include, but are not limited to, luciferase (Luciferase), ⁇ -galactosidase ( ⁇ -galactosidase), chloramphenicol acetyltransferase (Chloramphenicol Acetyltransferase, CAT), alkaline phosphatase ( alkaline pkosphatase (AKP), fluorescent protein (fluorescent protein), such as green fluorescent protein (GFP or EGFP), yellow fluorescent protein (YFP), red fluorescent protein (RFP), or other reporter molecules known in the art.
  • tEGFR can also serve as a reporter molecule.
  • Identity of sequences as used herein refers to the degree of similarity between amino acid sequences or between nucleotide sequences as determined by sequence alignment software, such as BLAST. For example, in this application, at least 85% sequence identity includes, for example: at least 90%, 93%, 95%, 97%, 98%, 99% sequence identity.
  • the term "effective amount” or “therapeutically effective amount” refers to the amount of a pharmaceutical composition comprising one or more peptides, proteins, nucleic acids or mutants, variants, analogs or derivatives thereof disclosed in the present application. amount, and the patient or subject receiving the "effective amount” or “therapeutically effective amount” of the pharmaceutical composition can obtain a reasonable benefit/risk ratio of medical treatment, thereby alleviating or preventing at least one or more of the diseases or conditions A variety of symptoms, to achieve the desired therapeutic or preventive effect.
  • the term “about” refers to the usual error range for the respective value readily known to those skilled in the art. Reference to “about” a value or parameter herein includes (and describes) embodiments that are directed to that value or parameter per se. As used herein, the term “about” when preceding a numerical value means within 10% above or below the numerical value. For example, “about 100” encompasses 90 and 110.
  • the application provides a recombinant cell comprising the following functional structure:
  • a chimeric antigen receptor (CAR) or its coding sequence the CAR comprises an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region comprises D1 and D2 domains of a human CD4 molecule;
  • the recombinant cells are derived from HIV recipient cells or peripheral blood mononuclear cells. In other embodiments, the recombinant cells are derived from lymphocytes. In some embodiments, the recombinant cells are derived from T cells. In other embodiments, the recombinant cells are derived from naive T cells memory T cells, effector T cells. In other embodiments, the recombinant cells are derived from cytotoxic T cells, helper T cells, and regulatory T cells. In other embodiments, the T cells are derived from CD4+ T cells, CD8+ T cells. In other embodiments, the recombinant cells are derived from NKT cells.
  • the recombinant cells are derived from ⁇ T cells. In other embodiments, the recombinant cells are derived from NK cells. In other embodiments, the recombinant cells are derived from antigen-presenting cells, such as macrophages and dendritic cells.
  • the recombinant cells are derived from progenitor or stem cells.
  • the progenitor or stem cells comprise hematopoietic stem cells (eg, CD34+ cells) or hematopoietic progenitor cells.
  • the stem cells include memory T stem cells (memory T stem cells), such as central memory T cells (central memory T cells), effector memory T cells (effector memory T cells) or stem cell-like Memory T cell (stem cell memory T cell).
  • the stem cells can be induced to differentiate into any T cells as described above.
  • the recombinant cells are derived from an HIV-infected patient.
  • the recombinant cells are derived from healthy humans.
  • the genes involved in the life cycle of HIV include its structural and regulatory genes, regulatory elements, and some genes of the host cell. Interfering or inhibiting any link in the life cycle of HIV can affect its replication and proliferation. However, it is difficult to predict which gene or genes among them can better achieve this effect by interfering or inhibiting, and better balance the anti-HIV effect and the normal physiological function of the host cell. For example, it is difficult to know from the prior art how to minimize the infection of HIV to the cells, reduce and delay the exhaustion of recombinant cells while satisfying the recombinant cell killing ability of the present application to the greatest extent.
  • the HIV genome is known to consist of at least seven regulatory elements (LTR, TAR, RRE, PE, SLIP, CRS, and INS) and nine genes (gag, pol, env, tat, rev, nef, vif, vpr, vpu, and sometimes There is a tenth gene, tev, which is a fusion of tat, env and rev) and encodes a total of 19 proteins. Three of these genes, gag, pol and env, contain the information needed to make the structural proteins of new virus particles.
  • tat, rev, nef, vif, vpr, and vpu are regulatory genes for proteins that control HIV's ability to infect cells, make new copies of the virus, replicate, or cause disease. Regulatory elements interact with proteins regulating gene expression, participate in genome transcription, translation and other processes, or interfere with normal physiological activities of host cells.
  • genes involved in HIV life cycle also include some host genes, such as NF- ⁇ B, CCR5, TSG101, CXCR4, P-TEFb, etc. Therefore, the introduction of shRNA that can target and inhibit the above genes has the opportunity to interfere with the replication and proliferation of HIV and reduce its damage to host cells.
  • the shRNA targeted to suppress one or more host or HIV genes involved in the HIV life cycle comprised by the recombinant cell is an shRNA targeted to the gag gene and the LTR gene of HIV.
  • the shRNA is an shRNA targeting gag and nef of HIV.
  • the nucleic acid sequence of the shRNA targeting gag comprises a sequence as shown in SEQ ID NO: 1, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence of the shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 2, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence encoding the shRNA targeting gag comprises the sequence shown in SEQ ID NO: 3, or has at least 85%, 90%, 93% of the sequence shown in SEQ ID NO: 3 , 95%, 97%, 98%, 99% identical nucleic acid sequences.
  • the nucleic acid sequence of the shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 2, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence encoding the shRNA targeting LTR comprises the sequence shown in SEQ ID NO: 4, or has at least 85%, 90%, 93% of the sequence shown in SEQ ID NO: 4 , 95%, 97%, 98%, 99% identical nucleic acid sequences.
  • the nucleic acid sequence of the shRNA targeting gag comprises a sequence as shown in SEQ ID NO: 5, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence of the shRNA targeting nef comprises a sequence as shown in SEQ ID NO: 6, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence encoding the shRNA targeting gag comprises the sequence shown in SEQ ID NO: 7, or has at least 85%, 90%, 93% of the sequence shown in SEQ ID NO: 7 , 95%, 97%, 98%, 99% identical nucleic acid sequences.
  • the nucleic acid sequence of the shRNA targeting nef comprises a sequence as shown in SEQ ID NO: 6, or has at least 85%, 90%, 93%, Nucleic acid sequences with 95%, 97%, 98%, 99% identity.
  • the nucleic acid sequence encoding the shRNA targeting nef comprises the sequence shown in SEQ ID NO: 8, or has at least 85%, 90%, 93% of the sequence shown in SEQ ID NO: 8 , 95%, 97%, 98%, 99% identical nucleic acid sequences.
  • the H1 promoter is selected as the promoter of shRNA targeting gag, while the U6 promoter is selected as the promoter of shRNA targeting LTR or nef, and good transcription and stability of each shRNA are achieved sex.
  • the nucleotide sequences of the H1 promoter and the U6 promoter are respectively shown in SEQ ID NO: 9 and 10, or have at least 85% sequence identity with SEQ ID NO: 9 and 10, respectively .
  • the extracellular region of the CAR in the recombinant cell comprises a structure selected from the group consisting of the D1 domain of a human CD4 molecule, the D1 and D2 domains of a human CD4 molecule, the D1 to D3 structures of a human CD4 molecule Domain, D1-D4 domain of human CD4 molecule, antibody or antigen-binding fragment thereof that specifically binds to HIV.
  • the term "antigen-binding fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. Examples of antigen-binding fragments include Fab, Fab', F(ab')2, Fv fragments, diabodies, linear antibodies, scFv.
  • examples of the HIV-specific antibody or antigen-binding fragment thereof include gp120-binding specific antibody or antigen-binding fragment.
  • scFv single-chain antibody variable fragment
  • VH variable regions of the heavy (VH) and light (VL) chains, wherein VH and VL can be linked via a linker peptide connect.
  • scFv can be expressed as a single chain polypeptide.
  • scFvs retain the specificity of the intact antibody from which they were derived.
  • the light and heavy chains can be in any order, eg, VH-linker-VL or VL-linker-VH, as long as the specificity of the scFv for the target antigen is preserved.
  • linkers can also be omitted.
  • the scFv of the gp120 monoclonal antibody is NAb-scFv (the construction method thereof is shown in patent CN103797029B).
  • the NAb-scFv comprises the sequence shown in SEQ ID NO: 15.
  • the extracellular region comprises D1 and D2 domains of human CD4 molecule but does not comprise D3 and D4 domains.
  • the extracellular region comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 85% identity with the amino acid sequence.
  • HIV can enter host T cells through the specific combination of its envelope protein gp120 and CD4. Using this specific binding, CD4 or its part with specific binding ability to gp120 can be used as the specific antigen recognition domain of CAR.
  • the transmembrane region contained in the recombinant cell is derived from or comprises a transmembrane domain of one or more protein molecules selected from the group consisting of ⁇ , ⁇ or ⁇ chains of T cell receptors, CD28, CD3 ⁇ , CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD154, KIRDS2, OX40, CD2, CD27, LFA-1 (CD11a, CD18), ICOS (CD278) , 4-1BB(CD137), GITR, CD40, BAFFR, HVEM(LIGHTR), SLAMF7, NKp80(KLRF1), CD160, CD19, IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA1, VLA1, CD49a, ITGA4, IA4, CD49D4, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, I
  • the transmembrane region is derived from or comprises a transmembrane domain of a CD8 ⁇ protein molecule. In some specific embodiments, the transmembrane region comprises the amino acid sequence shown in SEQ ID NO: 18 or comprises an amino acid sequence having at least 85% identity thereto.
  • the intracellular region of the recombinant cell comprises a primary signaling domain.
  • the primary signal transduction domain comprises immunoreceptor tyrosine-based activation motifs (immune-receptor tyrosine-based activation motifs, ITAM, the basic composition of which is: YXXL/V. Wherein Y is Tyrosine, L/V refers to leucine or valine, X can be any amino acid).
  • the primary signal transduction domain is derived from or comprises a signal transduction domain of one or more protein molecules selected from: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FCER1G, Fc ⁇ R1b, CD79a, CD79b, FcyRIIa, DAP10 and DAP12.
  • the primary signaling domain is derived from or comprises a signaling domain of CD3 ⁇ .
  • the primary signaling domain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the intracellular region further comprises a co-stimulatory domain derived from or comprising a signal transduction domain of one or more protein molecules selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands specifically binding to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE , CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TNFR2,
  • the intracellular region comprises the signaling domains of CD3 ⁇ and CD137. In some embodiments, the intracellular region comprises the signaling domains of CD3 ⁇ , CD28, and CD137. In some embodiments, the intracellular region comprises the signaling domains of CD3 ⁇ , CD137, and DAP10. In some embodiments, the intracellular region comprises the signaling domains of CD28, DAP10, and CD3 ⁇ . In some embodiments, the intracellular region comprises the signaling domains of 2B4, DAP10, and CD3 ⁇ . In some embodiments, the intracellular region comprises the signaling domains of CD137, 2B4, and CD3 ⁇ .
  • the primary signal transduction domain and co-stimulatory domain in the intracellular region as well as linking peptides between each co-stimulatory domain.
  • the connecting peptide can be selected from any natural or synthetic peptides that can connect the various structural domains in the intracellular region and maintain a certain relative spatial distance between the structural domains to ensure their respective functional integrity and signal transduction therebetween. peptide sequence.
  • Alternative linker peptides are known to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.
  • the connecting peptide is a GS linker, such as (GGGGS)n, wherein n is selected from any natural number.
  • the connecting peptide is GGGGS.
  • the intracellular region comprises an amino acid sequence as shown in any one of SEQ ID NO: 28-34 or comprises at least 85%, 90%, 93 %, 95%, 97%, 98%, 99% identical amino acid sequences.
  • the extracellular region and the transmembrane region are connected by a hinge region.
  • the hinge region described in this application can be selected from hinges contained in natural proteins, and can also be selected from artificially synthesized hinge regions, such as GS linkers.
  • the hinge region is from or comprises a human Ig hinge region, a GS linker, a KIR2DS2 hinge, or a CD8 hinge region.
  • the hinge region is the hinge region of CD8 ⁇ .
  • the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 20 or comprises at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR in the recombinant cell of the present application further comprises a signal peptide, and the signal peptide locates the CAR on the cell membrane of the recombinant cell.
  • the linking of a signal peptide described in the present application includes the linking of a signal peptide to the CAR at any time of the cell life cycle.
  • the signal peptide is from or comprises the signal peptide of any secreted or membrane protein.
  • the signal peptide is from or comprises a signal peptide of CD4 or CD8.
  • the signal peptide comprises an amino acid sequence as shown in SEQ ID NO: 16 or 17 or comprises at least 85%, 90%, 93%, 95%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR intracellular region comprises an amino acid sequence selected from the following: any one of SEQ ID NO: 28-34 or comprises at least 85%, 90%, 93% of any sequence , 95%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR comprises an amino acid sequence selected from the following: any one of SEQ ID NO: 35-46 or comprises at least 85%, 90%, 93%, 95% of any of the sequences , 97%, 98%, 99% identical amino acid sequences.
  • the recombinant cells comprise shRNA targeting gag and LTR or coding sequences thereof, signal peptide of CD4, D1 and D2 domains of CD4, hinge region of CD8 ⁇ , transmembrane region of CD8 ⁇ , CD137 signaling domain and CD3 ⁇ signaling domain.
  • the recombinant cells comprise shRNA targeting gag and LTR or coding sequences thereof, signal peptide of CD4, D1 and D2 domains of CD4, hinge region of CD8 ⁇ , transmembrane region of CD8 ⁇ , Signal transduction domain of CD28, signal transduction domain of CD137, and signal transduction domain of CD3 ⁇ .
  • the recombinant cells comprise shRNA targeting gag and LTR or coding sequences thereof, signal peptide of CD4, D1 and D2 domains of CD4, hinge region of CD8 ⁇ , transmembrane region of CD8 ⁇ , The signal transduction domain of CD28, the signal transduction domain of CD137, and the signal transduction domain of CD3 ⁇ , and the signal transduction domain of CD28, the signal transduction domain of CD137, and the signal transduction domain of CD3 ⁇ They are connected by GS connecting peptide.
  • the recombinant cells comprise shRNA targeting gag and LTR or coding sequences thereof, signal peptide of CD4, D1 and D2 domains of CD4, hinge region of CD8 ⁇ , transmembrane region of CD8 ⁇ , Signal transduction domain of CD28, signal transduction domain of CD137, signal transduction domain of SLAMF4(2B4) and/or DAP10, and signal transduction domain of CD3 ⁇ .
  • the recombinant cell further comprises a reporter molecule and/or a safety switch.
  • the safety switch is selected from one or more of: iCaspase-9, iCaspase-1, iCaspase-8, thymidine kinase (eg, HSV-TK, VZV-TK) , cytosine deaminase (CD), CD20, tEGFR, FR806 and RQP8.
  • the safety switch is tEGFR, preferably, the amino acid sequence of the tEGFR is shown in SEQ ID NO: 49 or 50.
  • the reporter molecule is selected from one or more of the following: luciferase, ⁇ -galactosidase, CAT, AKP, GFP or EGFP, YFP, RFP, tEGFR.
  • the reporter molecule is preferably RFP or tEGFR, preferably, the amino acid sequence of the RFP is shown in SEQ ID NO: 48, and the amino acid sequence of the tEGFR is shown in SEQ ID NO: 49 or 50 shown.
  • the present application provides a chimeric antigen receptor comprising an extracellular region, a transmembrane region and an intracellular region, wherein the extracellular region is derived from or comprises the extracellular region of a human CD4 molecule, the transmembrane region From or comprising the transmembrane domain of CD8 ⁇ .
  • the chimeric antigen receptor further includes a hinge region, and the extracellular region and the transmembrane region are connected by a hinge region.
  • the hinge region is from or comprises a human Ig hinge region, a GS linker, a KIR2DS2 hinge or a hinge region of CD8 ⁇ , more preferably a hinge region of CD8 ⁇ .
  • the hinge region comprises an amino acid sequence as shown in SEQ ID NO: 20 or comprises an amino acid sequence having at least 85% identity with this sequence.
  • the extracellular region comprises D1 and D2 domains of human CD4 molecule but does not comprise D3 and D4 domains.
  • the extracellular region comprises the amino acid sequence shown in SEQ ID NO: 13 or an amino acid sequence having at least 85% identity with the amino acid sequence.
  • the transmembrane region comprised by the CAR comprises the amino acid sequence shown in SEQ ID NO: 18.
  • the intracellular region of the CAR comprises a primary signaling domain.
  • the primary signal transduction domain comprises immunoreceptor tyrosine-based activation motifs (immune-receptor tyrosine-based activation motifs, ITAM, the basic composition of which is: YXXL/V. Wherein Y is Tyrosine, L/V refers to leucine or valine, X can be any amino acid).
  • the primary signal transduction domain is derived from or comprises a signal transduction domain of one or more protein molecules selected from: CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , FCER1G, Fc ⁇ R1b, CD79a, CD79b, FcyRIIa, DAP10 and DAP12.
  • the primary signaling domain is derived from or comprises a signaling domain of CD3 ⁇ .
  • the primary signaling domain comprises the amino acid sequence shown in SEQ ID NO: 23.
  • the intracellular region further comprises a co-stimulatory domain derived from or comprising a signal transduction domain of one or more protein molecules selected from: CD27, CD28, 4-1BB, OX40, CD30, CD40, PD-1, ICOS, LFA-1, CD2, CD7, LIGHT, NKG2C, B7-H3, ligands that specifically bind to CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE , CD103, ITGAL, CD11a, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, ITGB7, TN
  • the intracellular region comprises the signaling domains of CD3 ⁇ and CD137. In some embodiments, the intracellular region comprises the signaling domains of CD3 ⁇ , CD28, and CD137. In some embodiments, the intracellular region comprises the signaling domains of CD3 ⁇ , CD137, and DAP10. In some embodiments, the intracellular region comprises the signaling domains of CD28, DAP10, and CD3 ⁇ . In some embodiments, the intracellular region comprises the signaling domains of 2B4, DAP10, and CD3 ⁇ . In some embodiments, the intracellular region comprises the signaling domains of CD137, 2B4, and CD3 ⁇ .
  • the primary signal transduction domain and co-stimulatory domain in the intracellular region as well as linking peptides between each co-stimulatory domain.
  • the connecting peptide can be selected from any natural or synthetic peptides that can connect the various structural domains in the intracellular region and maintain a certain relative spatial distance between the structural domains to ensure their respective functional integrity and signal transduction therebetween. peptide sequence.
  • Alternative linker peptides are known to those skilled in the art and may be used in conjunction with alternative embodiments of the invention.
  • the connecting peptide is a GS linker, such as (GGGGS)n, wherein n is selected from any natural number.
  • the connecting peptide is GGGGS.
  • the intracellular region comprises an amino acid sequence as shown in any one of SEQ ID NO: 28-34 or comprises at least 85%, 90%, 93 %, 95%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR comprises an amino acid sequence selected from the following: any one of SEQ ID NO: 35, 36, 43-46 or comprises at least 85%, 90%, 93 %, 95%, 97%, 98%, 99% identical amino acid sequences.
  • the CAR is further linked to a signal peptide
  • the signal peptide is derived from or comprises a signal peptide of any secreted protein or membrane protein, preferably, the signal peptide is derived from or comprises a signal peptide of CD4 or CD8.
  • the present application also provides a recombinant cell comprising the aforementioned chimeric antigen receptor.
  • the present application further provides the chimeric antigen receptor or a recombinant cell comprising the chimeric antigen receptor and at least one HIV-targeting shRNA described in the present application or its coding sequence or a vector containing its coding sequence The combination.
  • the present application also provides a nucleic acid or vector, which comprises a polynucleotide sequence encoding the CAR and/or shRNA contained in the aforementioned recombinant cell, or the aforementioned CAR molecule.
  • the vector is selected from a plasmid, a viral vector, or a linear nucleic acid molecule.
  • the vector is a retroviral shuttle plasmid vector, such as a Simian Immunodeficiency Virus (SIV) shuttle plasmid vector.
  • the vector is a packaged SIV viral vector.
  • the CAR and shRNA are expressed or transcribed under the same promoter.
  • the CAR and shRNA are expressed or transcribed under the drive of different promoters.
  • the polynucleotide sequences encoding the CAR and shRNA are contained in different vectors. In some embodiments, the polynucleotide sequences encoding the CAR and shRNA are contained in the same vector. In some embodiments, the polynucleotide sequences encoding shRNA are contained in the same or different vectors.
  • the present application also provides a composition comprising the nucleic acid or vector described in the present application.
  • the present application also provides liposomes comprising the nucleic acid, vector or composition.
  • the present application also provides a pharmaceutical composition, which comprises the above-mentioned recombinant cells, CAR molecules, nucleic acids or vectors, or the above-mentioned combination.
  • the pharmaceutical composition can be used to treat patients infected with HIV.
  • the HIV-infected patient is on long-term antiretroviral therapy.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient, carrier, and/or stabilizer.
  • suitable excipients, carriers, and/or stabilizers are selected from antioxidants, preservatives, pyrogen-free water, isotonic saline, and phosphate buffer, etc., or combinations thereof.
  • Such pharmaceutically acceptable carriers, excipients or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include, for example: buffers, such as phosphate, citrate or acetate, pH Usually 5.0 to 8.0, optionally 6.0 to 7.0; salts to achieve isotonicity, such as sodium chloride, potassium chloride, etc.; antioxidants; preservatives; low molecular weight polypeptides; proteins; hydrophilic polymers, such as polysorbate Alcohol esters 80; amino acids, such as glycine; carbohydrates; chelating agents; sugars; and other standard ingredients known to those skilled in the art (Remington: The Science and Practice of Pharmacy, 22nd Ed., Loyd V. Allen et al. eds. , Pharmaceutical Press (2012)).
  • buffers such as phosphate, citrate or acetate, pH Usually 5.0 to 8.0, optionally 6.0 to 7.0
  • salts to achieve isotonicity such as sodium chloride, potassium chloride, etc.
  • the pharmaceutical composition of the present application can be made into various dosage forms according to needs, and can be administered by a doctor according to the patient's type, age, body weight and general disease condition, administration method and other factors to determine the dose beneficial to the patient.
  • the mode of administration can be adopted, for example, parenteral administration (such as injection) or other therapeutic modes.
  • the present application further provides a method for treating HIV infection, which comprises administering to a subject infected with HIV an effective amount of the aforementioned recombinant cells, the aforementioned nucleic acid or vector, or the aforementioned combination, or a pharmaceutical composition as described above.
  • the administration is intravenous injection or arterial infusion.
  • the effective amount is determined according to the disease severity, age and physical condition of the patient.
  • the method for treating HIV infection further comprises administering to an HIV-infected subject an effective amount of the aforementioned recombinant cells, nucleic acids, or vectors, the aforementioned combination or the aforementioned The pharmaceutical composition, and combined administration of other nucleoside inhibitors, HIV vaccine, broad-spectrum neutralizing antibody against HIV and/or CAR-T cells.
  • the present application also provides a method for preparing the above-mentioned recombinant cells, which comprises introducing the above-mentioned vectors into the above-mentioned cells for expression.
  • the vector is a SIV shuttle plasmid
  • the preparation method comprises packaging the SIV shuttle plasmid into SIV virus particles, and using the virus particles to infect the cells.
  • the preparation method comprises packaging the SIV shuttle plasmid into SIV virus particles, using the virus particles to infect the progenitor cells or stem cells, and further stimulating the progenitor cells or stem cells by means of cytokine stimulation or the like. Stem cells differentiate into mature effector T cells.
  • Embodiment 1 vector construction
  • the plasmid carrying the exogenous gene expression cassette used in the examples of the present application is constructed based on the shuttle plasmid in the SIV third-generation lentiviral vector packaging system.
  • the schematic diagrams of the important functional structures of this series of plasmids inserted into the shuttle plasmids in the SIV third-generation lentiviral vector packaging system are shown in Figures 1A-1C. Wherein, the meanings of each structure in Fig.
  • G3L2 represents the coding sequence of the shRNA (sequence shown in SEQ ID NO:1 and 2 respectively) of targeting conserved sequence Gag and LTR in the HIV genome (respectively containing independent promoter
  • G2N represents the coding sequence (each containing an independent promoter) of the shRNA (sequences shown in SEQ ID NO: 5 and 6) targeting the conserved sequences Gag and Nef in the HIV genome
  • CD4 signal represents the signal peptide of CD4
  • D1D2 represents the D1 and D2 domains of CD4
  • CD8h represents the hinge region of CD8 ⁇
  • CD8a TM and CD28TM represent the transmembrane regions of CD8 ⁇ and CD28, respectively
  • CD28, CD137, 2B4, CD3 ⁇ and DAP10 represent CD28, CD137, 2B4, respectively , the signal transduction domain of CD3 ⁇ and DAP10
  • Nab-scFv means the neutralizing antibody scFv (3BNC117 is used in this example
  • Reporter represents a reporter gene, and the reporter gene used in the examples of this application is enhanced green fluorescent protein (EGFP), red fluorescent protein (RFP) or truncated epidermal growth factor receptor (tEGFR).
  • EGFP enhanced green fluorescent protein
  • RFP red fluorescent protein
  • tEGFR truncated epidermal growth factor receptor
  • L represents the connecting peptide GGGGS
  • L3 represents the connecting peptide (GGGGS) 3 .
  • Related sequences are shown in Table 2.
  • the plasmid carrying the Z09 structure was obtained by nucleic acid fragment synthesis, seamless connection, transformation and other methods (Fig. 2B). After that, construct plasmids carrying Z10-Z15, Z17, Z34-Z37 and other structures on the basis of the plasmid.
  • the series of plasmids carrying the Z01-Z07 structure are constructed based on the series of plasmids carrying the Z09-Z15 structure, where Z01 corresponds to Z09, Z02 corresponds to Z10, Z03 corresponds to Z11, Z04 corresponds to Z12, Z05 corresponds to Z13, Z06 corresponds to Z14, and Z07 corresponds to Z15.
  • the difference between the plasmids carrying the Z01-Z07 structure and their respective corresponding plasmids is that the former does not contain polynucleotide sequences encoding shRNA.
  • Fig. 2C shows the map of the plasmid carrying the Z01 structure.
  • the construction of the plasmid carrying the Z08 structure and the Z16 structure refers to the US patent application US 9,833,480B2.
  • Plasmid construction steps and methods such as nucleic acid fragment synthesis, seamless connection, and transformation are conventional techniques in the art, and can be referred to, for example, edited by J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the manufacture Operate according to the manufacturer's instructions.
  • Cell line 293T cells (ATCC, CRL-11268) were inoculated according to each T225 culture flask (Coring, Cat#431082) 9 ⁇ 10 6 cells, in 20 ml of D-MEM medium containing 10% fetal bovine serum (Thermofisher, After culturing in Cat#11995-065) for 48 hours, the medium was replaced with 10 ml of preheated OPTI-MEM medium (Thermofisher, Cat#31985-070), which was used as cells to be transfected.
  • the solution was added to the above-mentioned culture flask inoculated with cells, cultivated in a 37°C, 5% CO 2 incubator for 3 hours, and then each bottle was supplemented with DMEM medium containing 20% FBS, and the final concentration of FBS was Adjust to 10%, and continue culturing overnight in a 37°C, 5% CO 2 incubator.
  • the reporter gene was tEGFR
  • immunofluorescent staining was performed using an anti-tEGFR antibody (R&D, FAB9577G-100). Subsequently, the immunofluorescence signal was detected by flow cytometry, and the proportion of positive cells was obtained.
  • the part of the CAR molecule containing CD4 and the reporter gene are connected through P2A in the CAR molecule, it can also be confirmed by the detection of CD4.
  • Use the anti-human CD4 antibody (BD, Cat#562424) to perform immunofluorescence staining on the above DPBS resuspended cells according to the relevant instructions, and then use flow cytometry to detect the fluorescent signal conjugated to the human CD4 antibody to obtain the proportion of positive cells .
  • F represents the proportion of positive cells
  • N represents the number of cells per well at the time of infection
  • D represents the multiple of the diluted stock solution
  • V represents the volume of diluted virus actually added to each well.
  • the virus titers obtained in the three wells were combined and calculated to obtain an average value, and the average value was used as the infection titer of the 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 using the reporter gene detection.
  • Example 1 Each plasmid in Example 1 was directly transfected into 293T cells, and the corresponding cells were collected 48 hours after transfection. Cells were lysed by adding lysis buffer. After centrifugation, a supernatant was obtained. The protein concentration in the supernatant was quantitatively detected by BCA method. After appropriately diluting the supernatant according to the desired concentration, the protein of interest was detected by Western blot. In the Western blot detection method, anti-CD3 ⁇ antibody (Santacruz sc-166275) was used as the primary antibody, and horseradish peroxide-labeled goat anti-mouse IgG (H+L) (Beyond A0216) was used as the secondary antibody.
  • anti-CD3 ⁇ antibody Santacruz sc-166275
  • H+L horseradish peroxide-labeled goat anti-mouse IgG
  • Each recombinant lentivirus SIV obtained in Example 2 was used to infect 293T cells in the same volume according to the original infection titer. After 48 hours, refer to the method in Example 2 to detect the proportion of positive cells. That is, after immunofluorescent staining of 293T cells with an anti-human CD4 antibody (BD, Cat#562424), flow cytometry was used to detect coupled fluorescent signals to obtain the proportion of positive cells. Or directly use the flow cytometer (BD Celesta) to detect the signal of the fluorescent protein to obtain the proportion of positive cells.
  • BD Celesta flow cytometer
  • the results of detecting the reporter gene-red fluorescent protein (RFP) for its expression are shown in the black column in Figure 3, and the positive signal of the reporter gene can be detected in the 293T cells infected by these recombinant lentiviruses, and from Figure 3 It can be found that in the cells carrying the Z09-Z13, Z16-Z17, Z34-Z37, Z01-Z05, Z38-Z41 structures, the proportion of positive cells shown by the two colors is consistent.
  • the above results show that under the given detection conditions, the cells that can detect two positive signals (CD4 and reporter gene expression) at the same time belong to the same group of cells, and their proportions are also equivalent. Therefore, the expression of fluorescent protein can indicate the presence of CAR structure in cells. surface expression.
  • Figure 4 When tEGFR is selected as the reporter gene, use anti-human CD4 antibody and/or anti-tEGFR antibody to perform flow cytometry detection after staining. Taking Z09 as an example, positive signals can also be detected in cells carrying the above structure ( Figure 4 ).
  • Figure 4A is the result of double-staining with two antibodies
  • Figure 4B is the result of single-staining with anti-CD4 antibody
  • Figure 4C is the result of single-staining with anti-EGFR antibody
  • Figure 4D is the result of unstained recombinant lentivirus SIV-Z09 transduced 293T cells, as control.
  • CD4+ refers to the proportion of positive cells obtained by flow cytometric detection and analysis of the 293T transduced by the recombinant lentivirus SIV-Z09 using a fluorescent-coupled anti-human CD4 antibody
  • EGFR+ refers to the detection of a fluorescent-coupled anti-human EGFR antibody against the recombinant lentivirus
  • Ficoll-Paque PLUS (GE, Cat#17-1440-02) was used to separate PBMC cells from EDTA anticoagulated whole blood (blood donation from healthy volunteers) according to the kit instructions. Specifically, using a high-speed refrigerated centrifuge (Thermo Scientific Sorvall ST40R), use a horizontal rotor to centrifuge at 800g for 30min, set the centrifugation speed up to 3, speed down to 0, and the temperature to 20°C. After centrifugation, gently aspirate the PBMC cell layer and place in a new centrifuge tube. The PBMCs were washed with 3-5 times the volume of DPBS aspirated from the cell layer, and then centrifuged at 20° C. and 300 g for 10 min.
  • erythrocyte lysate (Tianjin Haoyang NH4CL2009)
  • pre-cooled DPBS was added to make the total volume 40-45ml, the cells were mixed evenly by pipetting up and down, and centrifuged at 300g at 4°C for 10min to obtain PBMC cells.
  • CD3+ T cells were purified using Dynabeads untouched Human T cells kit (Thermofisher 11344D) according to the manufacturer's instructions. Specifically, the PBMC cells obtained in the previous step were resuspended in a final volume of 300 ⁇ l in 4°C pre-cooled separation buffer (DPBS buffer containing 0.1% BSA and 2 mM EDTA), mixed by pipetting several times, and statically placed on ice. 10 ⁇ l was used for live cell counting. According to the number of viable cells, adjust the PBMC cells to 1 ⁇ 10 8 cells/ml by using pre-cooled separation buffer at 4°C, and calculate and add the corresponding volume of the required reagents according to the instructions of the Dynabeads kit.
  • DPBS buffer containing 0.1% BSA and 2 mM EDTA 4°C pre-cooled separation buffer
  • PBMC cells Take 5 ⁇ 107 PBMC cells as an example: First, take a 15ml centrifuge tube, add 0.5ml of PBMC cells at the above concentration, add 100 ⁇ l of pre-cooled FBS, mix well, then add 100 ⁇ l of pre-cooled Antibody Mix, pipette Mix several times and incubate at 4°C for 20 min in the dark. Subsequently, 4ml of pre-cooled separation buffer was added, mixed by pipetting several times, centrifuged at 350g for 8min at 4°C, and the supernatant was discarded.
  • T cells were activated using magnetic beads coated with anti-CD3 antibody and anti-CD28 antibody.
  • kit Dynabeads Human T-Activator CD3/CD28 for T-Cell Expansion and Activation Thermofisher, 11131D.
  • the cells and the washed CD3/CD28 magnetic beads were mixed according to the original concentration of 25ul magnetic beads corresponding to 1 ⁇ 10 6 cells, and IL-2 (R&D 202- IL-010), IL-7 (R&D 207-IL-025) and IL-15 (R&D 247-ILB-025), human serum albumin or human AB serum or CD3+ T cells at a final concentration of 3-5%
  • IL-2 R&D 202- IL-010
  • IL-7 R&D 207-IL-025
  • IL-15 R&D 247-ILB-025
  • human serum albumin or human AB serum or CD3+ T cells at a final concentration of 3-5%
  • the corresponding autologous serum was thoroughly mixed (recorded as day 0 after activation), and incubated at 37°C for 48 hours.
  • the mixture of cells and magnetic beads was taken into a 15ml centrifuge tube, shaken at 2000rpm for 30s, and placed on a magnetic stand for 1 minute. Aspirate the supernatant into a new centrifuge tube, resuspend the cells with X-vivo 15 medium, centrifuge at 300g for 5 minutes, discard the supernatant after centrifugation, resuspend the cell pellet with fresh X-vivo15 medium, and take a part for counting and Flow detection (day 2 after activation).
  • the CD3+, CD3+CD4+ double positive, CD3+CD8+ double positive, CD3+CD25+ double positive cell subsets and proportions were determined by immunofluorescence staining and flow cytometry. Specifically, according to the counting results, 1 ⁇ 10 6 cells were collected, centrifuged at 300g for 5 minutes at room temperature, resuspended in DPBS, and added multicolor staining buffer (BD Horizon Brilliant Stain Buffer Plus, 566385), and conjugated with PE- A combination of CY7 mouse anti-human CD3 antibody (BD, 557851), BV421-conjugated mouse anti-human CD4 antibody (BD, 562424) and PE-conjugated mouse anti-human CD8 antibody (BD, 555367), or Combination of mouse anti-human CD3 antibody conjugated to PE-CY7 (BD, 557851) and mouse anti-human CD25 antibody conjugated to BV421 (BD, 562442).
  • the ratio of activated cells refers to the ratio of CD3+CD25+ cells among CD3+ T cells, and CD3+ T cells not activated by magnetic beads and cytokines were used as a control.
  • the proportion of activated cells in the living CD3+ T cells reached over 91.4% ( FIG. 5 ).
  • X-vivo 15 medium (Lonza BE02-060F) to resuspend the cells, adjust the cell density to 1 ⁇ 10 6 /ml, and supplement IL-2 (R&D 202-IL-010), IL-7 (R&D 207- IL-025) and IL-15 (R&D 247-ILB-025), the final concentration is 3-5% human serum albumin or human AB serum or autologous serum corresponding to CD3+ T cells, according to cell density and growth time, etc. The concentration of cytokines is adjusted according to the situation.
  • the final concentration of IL-2 is 30 IU/mL
  • the final concentration of IL-7 is 5 or 10 ng/mL
  • the final concentration of IL-15 is 5 or 10 ng/mL.
  • the passages were counted every other day, and the cell density was adjusted to 1 ⁇ 10 6 /ml with fresh X-vivo 15 medium (containing IL-2, IL-7 and IL-15, the final concentration was as above). From then on 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 (EGFP) transduced with recombinant lentivirus SIV-EGFP, and CD3+ T cells (CD3) not transduced with recombinant lentivirus were used as controls.
  • the transduced cells (as used herein, "modified cells”, “transduced cells” and “modified T cells” were interchangeable using the same antibody combination as in Section 4.1.4). Both are used to refer to the CD3+ T cells or their mixtures that have successfully expressed the CAR structure transduced by the recombinant lentivirus SIV) for staining, and the expression of CD3, CD4, CD8, and CD25 was detected by flow cytometry, and at the same time A reporter gene co-expressed with the CAR was also tested.
  • Figure 7 shows the proportion of CAR-positive cells among CD3+ cells at two time points after activation, 13 days ( Figure 7A) and 19 days (Figure 7B). This result suggests that the proportion of CAR-positive cells can remain relatively stable over time.
  • Figure 8 shows the proportion of CD3+ cells in total cells ( Figure 8A) and the proportion of each cell subpopulation in CD3+ cells ( Figure 8B) without recombinant lentivirus SIV transduction.
  • Figure 8A shows the ratio of T cells at three different time points on 19 days of continuous culture, and the ratio of T cells is calculated based on CD3+T positive cells.
  • Figure 8B shows the subpopulations of T cells at three different time points on 19 days of continuous culture
  • the proportion of CD4+ T cells is decreasing, while the proportion of CD8+ cells is increasing. This is consistent with the results reported in the literature, under a condition that favors the growth of CD8+ T cells, the number of CD8+ T cells is constantly increasing.
  • Figure 9 shows the ratio of each cell subpopulation at different time points after CD3+ T cells were transduced with two recombinant lentiviruses (SIV-Z09 and SIV-Z14) carrying different CAR structures. It can be seen from the figure that over time, the proportion of CD4+ T cells in CD3+ cells transduced by recombinant lentivirus SIV-Z09 (Figure 9A) is higher than that of CD4+ T cells in CD3+ cells transduced by recombinant lentivirus SIV-Z14 ratio ( FIG. 9B ), which was also higher than that in CD3+ cells without any transduction ( FIG. 8B ). The above results further confirmed that the imported CAR molecules were correctly expressed on the surface of CD3+ cell membranes.
  • 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.
  • the target cells were seeded in a 24-well plate with a quantity of 1 ⁇ 10 4 cells/well.
  • the stimulation of cytokines to the modified T cells was stopped 24 hours before the experiment.
  • the modified T cells were added to different wells at a given effector-target ratio (i.e., the ratio of the number of effector cells to target cells, 10:1, 3:1 or 1:1) , mixed with the target cells, the final volume of the liquid is 200 ⁇ l/well.
  • ELISA kits Biolegend 430204, 431804 and 430104 were used to detect the release of cytokines TNF- ⁇ , IL2 or IFN- ⁇ , respectively.
  • R&D's Luminex Performance Human XL Cytokine Discovery Magnetic Panel R&D FSCTM18-12 was used to detect and analyze on the luminex200 instrument to simultaneously detect the expression levels of multiple factors.
  • the results of the acute killing activity of CD8+ T lymphocytes on target cells in the modified T cells in each experimental group showed that after 4 hours of mixing the effector target cells, the modified T cells (take the modified T cells carrying the Z09 or Z10 structure as an example) to express
  • the gp120 proteins of different HIV strains AC10.29, NL4-3, SF162
  • have specific killing effects on target cells and have a concentration-gradient dependent relationship (Fig. 12A-12C).
  • Figure 13A- Figure 13D are the results of the inhibitory activity of the modified T cells on the target cells at different effect-to-target ratios (10:1, 3:1 and 1:1), and the inhibitory effect has a concentration gradient dependence, the results It was further confirmed that target cells expressing gp120 can be specifically inhibited by CAR-T cells that specifically recognize antigens using the extracellular region of CD4, while engineered T cells that do not contain the intracellular signal transduction domain CD3 ⁇ (ie, Z17, without a complete CAR structure) Cells have no inhibitory effect on target cells.
  • the killing effect of modified T cells carrying the Z14 or Z34 structure was weaker than that of the modified T cells carrying the Z09 or Z10 structure; the killing effect of the modified T cells carrying the Z12 and Z13 structures was also weaker than that of the modified T cells carrying the Z09 structure,
  • the killing effect of the modified T cells carrying the Z11 or Z15 structure is weaker than that of the modified T cells carrying the Z10 structure; the killing effect of the modified T cells carrying the Z04 structure is weaker than that of the modified T cells carrying the Z01 structure.
  • the modified T cells carrying the Z09 structure have a higher cytostatic effect on the target cells expressing gp120 than those carrying the Z14 structure ( Figure 13A-C) , because the difference between the Z14 and Z09 structures is that the antigen-specific recognition domain contained in the extracellular region of the Z14 structure is the scFv of 3BNC117, while the antigen-specific recognition domain of Z09 is the D1-D2 segment of CD4, suggesting that the use of CD4 Compared with the CAR-T cells using the scFv of 3BNC117 to recognize the antigen, the CAR-T cells that recognize the antigen by the D1-D2 segment have a stronger inhibitory effect on the target cells expressing gp120.
  • the T cells were activated, transformed and cultured using the method described above, and the cytokines were stopped for 24 hours on the 13th day after activation, and the cell density was counted and adjusted. Then continue to use cytokines to culture until the 18th day, stop cytokine stimulation for 24 hours, use the method in 4.1.4 to measure the expression level of CD25 on the surface of T cells (day 19), confirm that after this stage of culture, the transformed T cells have Return to the inactive state (Figure 15).
  • the modified T cells were mixed with the target cells using the same method as in section 4.4, so as to detect and evaluate the inhibitory effect of the modified T cells on the target cells.
  • Twenty-four hours after the mixing of engineered T cells and target cells that is, the first day after mixing, some cells were collected, and CD3- (target cell-based) cells expressing different HIV strains (AC10) were analyzed by antibody immunostaining and cell flow cytometry. .29, NL4-3, SF162) gp120 protein cells or the proportion of control cells expressing AcGFP empty vector, and the proportion of transformed T cells in CD3+ T cells, to determine the inhibitory effect of transformed T cells on target cells and cytokines release level.
  • FIG. 16A-16C The results of the inhibition of target cells by engineered T cells showed that engineered T cells using CD4 or its fragments as the antigen-specific recognition domain of CAR could specifically inhibit target cells expressing gp120 at an effective-to-target ratio of 3:1 (Fig. 16A-16C), the target cells introduced with AcGFP empty vector were not significantly affected ( FIG. 16D ).
  • Figure 18A- Figure 18D shows the inhibitory effect of modified T cells on target cells at different effect-to-target ratios (10:1, 3:1 and 1:1), and the results show that modified T cells (carrying Z09 or Z10 structure) have The inhibition of target cells expressing HIV virus gp120 was concentration-gradient dependent (Fig. 18A-18C), while there was no obvious inhibitory effect on target cells expressing MHCII even at the most efficient target ratio of 10:1 (Fig. 18D).
  • the ELISA assay results of cytokine release using IFN- ⁇ as an example show that the specific stimulation from antigen can make the engineered T cells release cytokines related to killing ( FIG. 17 ).
  • the results of multifactor detection further confirmed the release of killing-related factors (Figure 19A-19J and 19L), and for the modified T cells carrying the Z09 or Z10 structure, no IL-6 associated with cytokine storm was detected ( Figure 19K ) and other factors are obviously released.
  • the proportion of T cells carrying a CAR structure that has a specific effect on gp120 that is, the percentage of T cells in CD3+ T cells
  • its ratio were detected. Inhibitory effect on target cells.
  • the modified 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 within 12 days thereafter, protease inhibitors Darunavir ethanolate (SelleckChem s1620) or Saquinavir mesylate (Sigma s8451) were added to the culture medium at a final concentration of 10uM for culture, and the rest were cultured Conditions are as described in Section 4.2. After 12 days, the addition of protease inhibitors was stopped and the culture was continued.
  • protease inhibitors Darunavir ethanolate
  • Saquinavir mesylate Sigma s8451
  • the day when the activated patient T cells were isolated and cultured was regarded as day 0, and the patient-derived CD3+ T cells were transformed according to the aforementioned method on the second day. From the 2nd day to the 24th day, collect the culture supernatant at different time points, use the Lenti-p24 rapid titer kit (Takara 632200), detect the p24 concentration in the supernatant according to its instructions, and indirectly quantify the HIV virus particles.
  • the killing and inhibiting effect of patient T cells modified with complete structure such as Z09, Z10, Z34, Z38-Z41, etc.
  • the ratio of the modified T cells themselves was determined to evaluate the protective effect of the introduced shRNA on the modified T cells.
  • the modified T cells carrying both shRNA and CAR structures showed no abnormalities in the supernatant during the whole preparation process and subsequent culture without protease inhibitors.
  • Virus p24 has not been detected in the solution or the concentration of virus p24 is near the detection threshold, and the modified T cells carrying the Z10, Z12, Z34, Z38-Z41 structure have better inhibitory effect on p24 than the modified T cells carrying the Z16 structure, which shows that this
  • the modified T cells of the carrier in the application have better killing and inhibiting effect on HIV than the modified T cells of Z16, and the effect time is longer.
  • the modified T cells carrying the CAR structure of the application can persistently and specifically kill and inhibit HIV virus-infected cells
  • the shRNA in the application has the advantage of protecting and maintaining the modified T cells.
  • the CAR-T of the present application when compared with the engineered T cells carrying the Z16 structure, the CAR-T of the present application has a stronger specific killing effect on HIV virus-infected cells, and can be maintained for a longer period of time under the condition of HIV infection.
  • the T cells carrying Z09 in the present invention , Z10, Z34, Z38-Z41 structure of the modified T cells have a relatively better comprehensive efficacy, has a more significant advantage.
  • Simultaneously introducing shRNA that can specifically inhibit the conserved sequence of the HIV genome and a CAR structure that targets HIV viral proteins into effector T cells can construct more durable and efficient CAR-T cells that target and kill HIV-infected cells.
  • the D1 and D2 domains of CD4 are selected as the antigen-specific recognition domain of CAR, compared to other antigen-specific recognition domains in the prior art or D1 of CD4
  • the -D4 domain has a better effect.
  • choosing shRNAs targeting G3L2 (for gag and LTR respectively) or G2N (for gag and nef respectively), compared with other shRNA options in the prior art can make CAR-T cells have better comprehensive efficacy.

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Abstract

提供了一种重组细胞，其包含嵌合抗原受体以及靶向抑制HIV生命周期的shRNA 。还提供了一种嵌合抗原分子，其胞外区来自或包含人CD4分子的胞外区，跨膜区来自或包含CD8α的跨膜结构域，以及包含上述嵌合抗原分子的重组细胞。上述重组细胞可用于治疗HIV感染，在HIV感染的环境中，可拥有更强的综合杀伤效力，更长的效用时间，以及更小的引起细胞因子风暴的风险。

Description

靶向HIV感染细胞的嵌合抗原受体T细胞 技术领域
本申请涉及感染性疾病免疫治疗技术领域，具体涉及一种用于HIV免疫治疗的工程化免疫效应细胞。
背景技术
艾滋病，亦称为获得性免疫缺陷综合征，是一种威胁人类生命安全的重大传染病，由人类免疫缺陷病毒(HIV)引起。HIV是一种能攻击人体免疫系统的病毒，它以人体免疫系统中的CD4阳性的T淋巴细胞作为主要攻击目标，大量破坏该细胞，使人体丧失免疫功能，从而易于感染各种疾病，甚至发生恶性肿瘤，病死率较高。至2019年底，全世界约有3800万人感染HIV，当年新发感染者约170万人，68万人因HIV而死亡(UNAIDS/WHO)。发现第一例艾滋病人已约40年，艾滋病仍为严重威胁公众健康的重要公共卫生问题。
目前尚无有效的艾滋病疫苗问世，临床主要使用高效联合抗反转录病毒治疗(Highly Active Antiretroviral Therapy，HAART)，俗称“鸡尾酒疗法”，也称抗逆转录病毒治疗(Antiretroviral Therapy，ART)。至2017年底，全世界约有2170万人接受过ART(UNAIDS/WHO)。现有的ART治疗并不能清除HIV-1的病毒潜藏库，均需终身服药，一旦停药病毒储藏库中的病毒可再度激活、复制，于是疾病会迅速反弹，因此需要患者永久用药。
迄今为止，全球仅有两例患者确认治愈，即“柏林病人”和“伦敦病人”。他们均通过移植来自缺乏多数HIV病毒株感染细胞所需的正常受体基因(CCR5)的捐赠者骨髓而获得治愈。然而，骨髓移植本身死亡率就较高，并且对于大多数人来说，具有这种遗传特征的匹配骨髓几乎不存在，这使得这种方法无法广泛应用于临床。
ART已经将HIV感染转变为慢性感染，目前未满足的临床需求主要包括：长效作用的、更安全耐受的方案；约有10％的病人对现有的ART耐药，需要新机制的治疗方案；需要针对病毒潜藏库作用，实现功能性治愈，乃至清除潜伏库，实现根除治愈。
而要实现功能性治愈，可以利用广谱中和性抗体，还可以基于抗体开发多特异性的分子以及利用嵌合抗原受体Chimeric antigen receptors(CAR)改造T细胞。广谱中和抗体除了可以直接中和病毒，也可以通过Fc结合巨噬细胞、NK细胞以及CD8+T细胞，清除展示病毒蛋白的细胞，例如针对gp120已产生出包括3BNC117在内的很多广谱性中和抗体(Scheid et al Science 2011)。但很多毒株已对这类抗体产生抗性，必须克服这种耐药性才能获得最佳的临床效果(Qian Wang et al.,Front Med.2020 Feb；14(1):30-42)。另一方面，通过广谱中和抗体或者病毒受体特异识别病毒，直接改造CAR-T细胞，可以在体内体外裂解被HIV感染的细胞，两项使用广谱中和抗体的3代CAR-T临床试验正在进行中，均在中国开展：一项为广州第八人民医院与中山大学合作，基于VRC01，改造CD8+T细胞，针对治疗有效病人开展(NCT03240328；Liu et al_J Clin Invest_2021)；另一项为武汉云谷与武汉金银潭医院合作，基于N6，针对初治病人，联合HAART方案(ChiCTR-OPN-17013068)。NCT03240328披露的结果显示CAR-T使用安全，但是在6个月观察期间内，停止HAART治疗的病人的HIV载量平均在5.3周时出现反弹，而且反弹的病毒都逃逸中和抗体VRC01；基于N6的CAR-T临床实验尚无正式结果公开。
治愈艾滋病的另一个思路，是重新激活潜伏的HIV，并通过联合增强机体HIV特异性免疫应答的方法，弥补HIV对机体造成的免疫损伤，增强对HIV储藏库的清除效果。早在1994年，Roberts等人尝试用CAR-T细胞治疗HIV感染，他们选取完整的CD4序列作为抗原特异性识别结构域，用于结合HIV感染细胞表面的包膜蛋白gp120，虽然具有部分杀伤感染细胞功能，但经过多年的努力，最终以失败而告终。其原因一方面是由于所述CAR-T细胞本身特异性杀伤力有待提高，另一方面是所述CAR-T细胞本身亦容易受到HIV感染，在HIV感染的环境中易于耗竭，发挥效力的时间较短。
当HIV感染细胞时，病毒的外膜糖蛋白gp120首先与细胞表面的CD4分子结合并与辅助受体CCR5或CXCR4等结合，从而介导病毒核心进入细胞。细胞表面的CD4是gp120的主要结合靶点，它们之间具有很高的结合亲和力，CD4与几种gp120的解离常数Kd范围为2.2×10 - 8-8×10 -10M(Lasky et al Cell 1987；Zhang Biochemistry et al 2000；Myszka et al PNAS 2000)。相比之下，CD4或CD8与特定的MHC-多肽复合物的解离常数Kd仅为10 -4-10 -6M(ZeNan L.Chang and YvonneY.Chen Trends in Molecular Medcine 2017)。而且CD4对几种典型的HIV-1gp120(例如，利用CCR5作为辅助受体的AC10.29，利用CCR5作为辅助受体的NL4-3及同时可使用两种辅助受体的SF162)的亲和力并不弱于典型的广谱中和抗体(3BNC117)来源的scFv对这些HIV-1gp120的亲和力(Scheid et al Science 2011；van Dorsten et al J Viral 2020)。
美国基因技术公司AGT等应用针对CCR5的shRNA组合2个及以上针对HIV的shRNA，体外显示良好效果；AGT期望通过体内预先多肽免疫诱导HIV特异性CD4细胞，然后体外导入shRNA后再回输到体内，期望约占人体总CD4细胞0.3％的被保护的CD4细胞能在体内增殖维持，该项 临床试验正在进行中，目前只完成一例参与者的回输，但尚无结果。
目前仍需要积极手段来实现艾滋病的功能性治愈，乃至清除潜伏库，最终实现根除治愈。
发明概述
为进一步改良针对HIV感染的细胞治疗方法(例如，CAR-T细胞)，本申请一方面提供了一种对HIV感染细胞杀伤力更强，可在HIV感染的环境中维持更长时间，且发挥效力时间更长的重组细胞(例如，CAR-T细胞)。另一方面，本申请还提供了一种针对HIV的嵌合抗原受体(CAR)，包含所述嵌合抗原受体的细胞针对HIV感染细胞具有高效的、特异性的杀伤活性，以及上述CAR与至少一种shRNA/shRNA编码序列/包含shRNA编码序列的载体的组合。此外，本申请提供的重组细胞或包含所述嵌合抗原受体的细胞不易引起细胞因子风暴。
在一个方面，本申请涉及一种重组细胞，具体为：
1.一种重组细胞，其包含下述功能结构：
1)嵌合抗原受体(CAR)或其编码序列，所述CAR包含胞外区、跨膜区及胞内区，其中所述胞外区可特异性结合HIV的gp120蛋白；
2)至少一种短发夹RNA(short hairpin RNA，shRNA)或其编码序列，该shRNA靶向抑制选自下组的任一种或多种参与HIV生命周期的宿主或HIV基因：NF-κB、CCR5、TSG101、CXCR4、P-TEFb、tat、rev、nef、env、LTR和gag。
在一些实施方案中，所述重组细胞来源于人或灵长类动物。在另一些实施方案中，所述重组细胞源自感染HIV的患者，或者源自健康人群。在一些实施方案中，所述重组细胞来源于HIV受体细胞或外周血单核细胞。在本发明的进一步优选实施方案中，所述重组细胞来源于淋巴细胞。在一些实施方式中，所述重组细胞来源于T细胞。在另一些实施方式中，所述重组细胞来源于幼稚T细胞(
Figure PCTCN2022143440-appb-000001
T)、记忆性T细胞、效应性T细胞。在另一些实施方式中，所述重组细胞来源于细胞毒T细胞、辅助性T细胞、调节性T细胞。在另一些实施方式中，所述T细胞来源于CD4+T细胞，CD8+T细胞。在另一些实施方式中，所述重组细胞来源于NKT细胞。在另一些实施方案中，所述重组细胞来源于γδT细胞。在另一些实施方案中，所述重组细胞来源于NK细胞。在另一些实施方案中，所述重组细胞来源于抗原提呈细胞，例如巨噬细胞、树突状细胞。
在本发明的进一步优选实施方案中，所述重组细胞来源于祖细胞或干细胞。在本发明的一些实施方案中，所述祖细胞或干细胞包括造血干细胞(例如，CD34+细胞)或造血祖细胞。在本 发明的另一些实施方案中，所述干细胞包括记忆性T干细胞(memory T stem cell)，例如中枢记忆T细胞(central memory T cell)、效应记忆T细胞(effector memory T cell)或干细胞样记忆T细胞(stem cell memory T cell)。在本发明的另一个实施方案中，所述干细胞经过诱导可分化为如前所述的任一种T细胞。
在一些实施方案中，所述重组细胞源自感染HIV的患者。在一些实施方案中，所述重组细胞源自健康人群。
2.项1的重组细胞，其中所述shRNA靶向HIV的gag基因和LTR基因。在一些实施方案中，所述shRNA靶向HIV的gag基因和nef基因。
3.项1或2的重组细胞，其中所述shRNA的核酸序列包含如SEQ ID NO：1、2、5和/或6所示的序列或包含与SEQ ID NO：1、2、5和/或6所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。
在一些实施方案中，SEQ ID NO：1和2的表达所使用的启动子为强启动子，例如H1启动子和U6启动子。在一些实施方案中，所述SEQ ID NO：1和2的表达使用相同的启动子。在一些实施方案中，所述SEQ ID NO：1和2的表达使用不同的启动子。在一些实施方案中，所述SEQ ID NO：1和2的表达分别使用H1启动子和U6启动子。
在一些实施方案中，SEQ ID NO：5和6的表达所使用的启动子为强启动子，例如H1启动子和U6启动子。在一些实施方案中，所述SEQ ID NO：5和6的表达使用相同的启动子。在一些实施方案中，所述SEQ ID NO：5和6的表达使用不同的启动子。在一些实施方案中，所述SEQ ID NO：5和6的表达分别使用H1启动子和U6启动子。
在一些实施方案中，所述H1启动子和U6启动子核酸序列分别如SEQ ID NO：9和10所示，或分别与SEQ ID NO：9和10具有至少85％、90％、93％、95％、97％、98％、99％的序列同一性。
4.项1-3中任一项的重组细胞，其中所述胞外区包含选自下述的结构：人CD4分子的D1结构域、人CD4分子的D1和D2结构域、人CD4分子的D1至D3结构域、人CD4分子的D1-D4结构域、gp120特异性抗体或其抗原结合片段(例如SEQ ID NO：15中所示的序列)。
在一些实施方案中，所述胞外区包含人CD4分子的D1和D2结构域而不包含D3和D4结构域。在一些特定的实施方案中，所述胞外区包含如SEQ ID NO：13所示的氨基酸序列或包含与该氨基酸序列具有至少85％、90％、93％、95％、97％、98％、99％以上同一性的氨基酸序列。
5.项1-4中任一项的重组细胞，其中所述跨膜区源自或包含选自以下一种或多种蛋白分子 的跨膜结构域：T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D4、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、NKG2C。
6.项5的重组细胞，其中所述跨膜区来自或包含CD8α蛋白分子的跨膜结构域
7.项1-6中任一项的重组细胞，所述跨膜区包含如SEQ ID NO：18所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
8.项1-7中任一项所述的重组细胞，其中所述胞内区包含一级信号转导结构域，所述一级信号转导结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12
9.项8所述的重组细胞，其中所述胞内区还进一步包含共刺激结构域，所述共刺激结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。
10.项8或9的重组细胞，其中所述一级信号转导结构域源自或包含CD3ζ的信号转导结构域。
11.项9或10的重组细胞，其中所述共刺激结构域源自或包含CD137、CD28、2B4或DAP10的信号转导结构域之一或其组合。
12.项9或11的重组细胞，其中所述胞内区中一级信号转导结构域和各共刺激结构域之间还 包含连接肽，其中，所述连接肽为可连接所述一级信号传导结构域和各共刺激结构域，并维持一定相对空间距离从而保证其各自的功能完整性，和其间信号传导的任意天然的或合成的肽序列。在一些特定的实施方案中，所述连接肽为G和S形成的短肽，例如(GS)n或(G)n(S)m，其中n或m为1-20之间的任何正整数，例如(GGGGS)1,2,3。
13.项1-12中任一项的重组细胞，其中所述胞内区包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与他们中任一序列有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
14.项1-13中任一项的重组细胞，其中所述胞外区与所述跨膜区之间由铰链区相连。
15.项14的重组细胞，其中所述铰链区来自或包含人Ig铰链区、GS接头、KIR2DS2铰链或CD8α的铰链区。在一些实施方案中，所述铰链区来自或包含人CD8α的铰链区。在一些实施方案中，所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与其有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
16.项1-15中任一项的重组细胞，其中所述CAR包含选自以下的氨基酸序列：SEQ ID NO：35-46中任一项或包含与他们中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
17.项1-16中任一项的重组细胞，其中所述CAR进一步连接有信号肽，所述信号肽可将所述CAR定位于重组细胞的细胞膜上。在一些实施方案中，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽。
18.项17的重组细胞，其中所述信号肽来自或包含CD4或CD8的信号肽。
19.项1-18中任一项的重组细胞，所述信号肽包含如SEQ ID NO：16或17中所示的氨基酸序列或与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：35中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列。在一些具体的实施方案中，所述重组细胞包含编码SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：35中所示的氨基酸序列的核酸序列。在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：3和4(或SEQ ID NO：7和8)中所示的核酸序列以及编码SEQ ID NO：35中所示的氨基酸序列的核酸序列。
在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：1和2(或SEQ ID NO：5和6)中 所示的核酸序列以及SEQ ID NO：36中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列。在一些具体的实施方案中，所述重组细胞包含编码SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：36中所示的氨基酸序列的核酸序列。在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：3和4(或SEQ ID NO：7和8)中所示的核酸序列以及编码SEQ ID NO：36中所示的氨基酸序列的核酸序列。
在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：43中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列。在一些具体的实施方案中，所述重组细胞包含编码SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：43中所示的氨基酸序列的核酸序列。在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：3和4(或SEQ ID NO：7和8)中所示的核酸序列以及编码SEQ ID NO：43中所示的氨基酸序列的核酸序列。
在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：46中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列。在一些具体的实施方案中，所述重组细胞包含编码SEQ ID NO：1和2(或SEQ ID NO：5和6)中所示的核酸序列以及SEQ ID NO：46中所示的氨基酸序列的核酸序列。在一些具体的实施方案中，所述重组细胞包含SEQ ID NO：3和4(或SEQ ID NO：7和8)中所示的核酸序列以及编码SEQ ID NO：46中所示的氨基酸序列的核酸序列。
在另一些实施方案中，所述重组细胞还包含报告分子和/或安全开关。在一些具体的实施方案中，所述安全开关选自下述中的一种或多种：iCaspase-9、iCaspase-1、iCaspase-8、胸苷激酶(例如，HSV-TK、VZV-TK)、胞嘧啶脱氨酶(CD)、CD20、tEGFR、FR806和RQP8。在另一优选的实施方案中，所述安全开关为tEGFR，优选地，所述tEGFR的氨基酸序列如SEQ ID NO：49或50所示。
20.一种载体，其包含编码如项1-19中任一项的重组细胞的CAR和/或shRNA的多核苷酸序列。
21.项20的载体，选自质粒、病毒载体或线性核酸分子。在一些特定的实施方案中，所述载体为逆转录病毒穿梭质粒载体。在一些实施方案中，所述载体为SIV病毒载体。
22.项20或21的载体，其中所述嵌合抗原受体和shRNA在相同启动子或不同启动子的驱动下表达。
23.一种药物组合物，其包含如项1-19中任一项的重组细胞或如项20-22中任一项的载体。所述药物组合物，可用于治疗感染HIV的患者。在一些实施方案中，所述感染HIV的患者经过了 长期的抗逆转录病毒治疗。
24.一种治疗和/或预防HIV感染或艾滋病的方法，其包含向感染HIV或患有艾滋病的受试者施用如项1-19中任一项的重组细胞或如项20-22中任一项的载体。
25.如项1-19中任一项的重组细胞或如项20-22中任一项的载体在制备用于治疗和/或预防HIV感染或艾滋病的药物中的应用。
此外，本申请还提供了一种制备如项1-19中任一项的重组细胞的方法，其包含将如项20-22中任一项的载体导入细胞中进行表达。在一些实施方案中，其包含将SIV病毒穿梭质粒载体包装成SIV病毒颗粒，并使用所述病毒颗粒感染所述细胞。
在另一方面，本申请还涉及一种嵌合抗原受体(CAR)，具体为：
1.一种嵌合抗原受体(CAR)，包含胞外区、跨膜区及胞内区，其中所述胞外区来自或包含人CD4分子的胞外区，跨膜区来自或包含CD8α的跨膜结构域。
2.如项1所述的嵌合抗原受体，其中所述嵌合抗原受体还包括铰链区。优选地，所述胞外区与所述跨膜区之间由铰链区相连。
3.如项2中所述的嵌合抗原受体，所述铰链区来自或包含人Ig铰链区、GS接头、KIR2DS2铰链或CD8α的铰链区，优选CD8α的铰链区。
4.如项3中所述的嵌合抗原受体，所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与该序列有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
5.如项1-4中任一项所述的嵌合抗原受体，其中人CD4分子的胞外区由CD4分子的D1和D2结构域组成。
6.如项5中所述的嵌合抗原受体，其中胞外区包含如SEQ ID NO：13所示的氨基酸序列。
7.如项1-6中任一项所述的嵌合抗原受体，其中所述跨膜区包含如SEQ ID NO：18所示的氨基酸序列。
8.如项1-7中任一项所述的嵌合抗原受体，其中所述胞内区包括一级信号转导结构域和共刺激结构域，所述一级信号转导结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12，且所述共刺激结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、 B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。
9.如项8中所述的嵌合抗原受体，其中所述一级信号转导结构域来自或包含CD3ζ或DAP10的信号转导结构域，且所述共刺激结构域来自或包含CD137、CD28、2B4或DAP10的信号转导结构域之一或其组合。
10.如项1-9中任一项所述的嵌合抗原受体，其中胞内区包含：
(i)CD137和CD3ζ的信号转导结构域；
(ii)CD28、CD137和CD3ζ的信号转导结构域；
(iii)CD137、CD3ζ和DAP10的信号转导结构域；
(iv)CD28、DAP10和CD3ζ的信号转导结构域；
(v)2B4、DAP10和CD3ζ的信号转导结构域；或，
(vi)CD137、2B4和CD3ζ的信号转导结构域。
11.如项8或9中任一项所述的嵌合抗原受体，其中所述胞内区中的一级信号转导结构域和共刺激结构域之间还包含连接肽。
12.如项11中所述的嵌合抗原受体，其中所述连接肽的序列包含SEQ ID NO：22所示的氨基酸序列。
13.如项1-10中所述的嵌合抗原受体，其中胞内区的序列包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与其中任一序列有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
14.如项1-13中任一项所述的嵌合抗原受体，其中所述嵌合抗原受体进一步连接信号肽，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽。
15.如项14中所述的嵌合抗原受体，其中所述信号肽来自或包含CD4或CD8分子的信号肽，优选的，所述信号肽包含SEQ ID NO：16或17所示氨基酸序列。
16.如项1-15中任一项所述的嵌合抗原受体，其中所嵌合抗原受体包含如SEQ ID NO：35、36、43-46中任一项所示的氨基酸序列，或者包含与它们中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
17.一种多核苷酸序列，其编码如项1-16中任一项所述的嵌合抗原受体。
18.一种载体，其包含如项17中所述的多核苷酸序列。
19.如项18所述的载体，其选自质粒、病毒载体或线性核酸分子。
20.一种重组细胞，其包含如项1-16中任一项所述的嵌合抗原受体，或如项17中所述的多核苷酸序列，或项18或19中所述的载体。
21.如项20所述的重组细胞，所述重组细胞来源于人或灵长类动物。进一步优选的，所述重组细胞源自感染HIV的患者，或者源自健康人群。进一步优选的，所述重组细胞来源于HIV受体细胞、外周血单核细胞或淋巴细胞；进一步优选的，所述重组细胞来源于T细胞(例如，幼稚T细胞、记忆性T细胞、效应性T细胞、细胞毒T细胞、辅助性T细胞、调节性T细胞、CD4+T细胞、CD8+T细胞、NKT细胞、γδT细胞等)、NK细胞、抗原提呈细胞(例如，巨噬细胞、树突状细胞等)；或者所述重组细胞来源于祖细胞或干细胞，包括但不限于造血干细胞、造血祖细胞、记忆性T干细胞(例如中枢记忆T细胞、效应记忆T细胞或干细胞样记忆T细胞)。
进一步的，本申请还涉及上述嵌合抗原受体与至少一种shRNA/shRNA编码序列/包含shRNA编码序列的载体的组合，具体为：
22.如项1-16中所述的嵌合抗原受体，或如权利要求17中所述的多核苷酸序列，权利要求18或19中所述的载体，或权利要求20或21所述的重组细胞与至少一种靶向HIV的shRNA或其编码序列或包含其编码序列的载体的组合。
23.如项22中所述的组合，其中所述至少一种shRNA靶向抑制选自下组的任一种或多种参与HIV生命周期的宿主或HIV基因：NF-κB、CCR5、TSG101、CXCR4、P-TEFb、tat、rev、nef、env、LTR和gag。
24.如项23中所述的组合，其中所述组合包括：
(i)靶向gag的shRNA和靶向LTR的shRNA，或者
(ii)靶向gag的shRNA和靶向nef的shRNA。
25.如项23或24中所述的组合，其中靶向gag的shRNA包含如SEQ ID NO：1或5所示序列或包含与SEQ ID NO：1或5所示序列具有至少85％同一性的序列，靶向LTR的shRNA包含如SEQ ID NO：2所示序列或包含与SEQ ID NO：2所示序列具有至少85％同一性的序列，靶向nef的shRNA包含如SEQ ID NO：6所示序列或包含与SEQ ID NO：6所示序列具有至少85％同一性的序列。
26.如项25中所述的组合，其中所述组合包括：
(i)靶向gag的shRNA和靶向LTR的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：1所示序列，靶向LTR的shRNA包含如SEQ ID NO：2所示序列；
(ii)靶向gag的shRNA和靶向nef的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：5所示序列，靶向nef的shRNA包含如SEQ ID NO：6所示序列。
27.如项23中所述的组合，其中所述组合包括：
(i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，或包含上述编码序列的载体，或者
(ii)靶向gag的shRNA的编码序列和靶向nef的shRNA的编码序列，或包含上述编码序列的载体。
28.如项23或27中所述的组合，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：3或7所示序列或包含与SEQ ID NO：3或7所示序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列，靶向LTR的shRNA的编码序列包含如SEQ ID NO：4所示序列或包含与SEQ ID NO：4所示序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列，靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示序列或包含与SEQ ID NO：8所示序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的序列。
29.如项28中所述的组合，其中所述组合包括：
(i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：3所示序列，靶向LTR的shRNA的编码序列包含如SEQ ID NO：4所示序列，或包含上述编码序列的载体；或者
(ii)靶向gag的shRNA的编码序列和靶向nef的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：7所示序列，靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示 序列，或包含上述编码序列的载体。
30.一种药物组合物，其包含如项20或21所述的重组细胞，或项22-29中任一项所述重组细胞与至少一种shRNA或其编码序列或包含其编码序列的载体的组合，以及药学上可接受的载体。在一个具体实施方式中，所述药物组合物进一步包括其他的抗HIV药物，包括但不限于逆转录酶抑制物、蛋白酶抑制剂、HIV疫苗、广谱中和抗体和/或CAR-T细胞。在另一个具体的实施方式中，本发明所述的药物组合物用于治疗和/或预防HIV感染或艾滋病。
31.项1-16的嵌合抗原受体，或者项17的多核苷酸序列，或者项18或19中所述的载体，或者项20或21所述的细胞，或者项22-29中任一项的组合在制备用于治疗和/或预防HIV感染或艾滋病的药物中的用途。
32.一种治疗和/或预防HIV感染或艾滋病的方法，包含向感染HIV或患有艾滋病的受试者施用项20或21所述的细胞，或者项22-29中任一项所述的重组细胞与至少一种shRNA或其编码序列或包含其编码序列的载体的组合，或者项30中所述的药物组合物。在本发明的一个具体实施方式中，施用项22-29中任一项所述的重组细胞与至少一种shRNA或其编码序列或包含其编码序列的载体的组合时，重组细胞和至少一种shRNA或其编码序列或包含其编码序列的载体可顺序施用或同时施用。在本发明的一个具体实施方式中，所述方法进一步包括向受试者施用其他的抗HIV药物，包括但不限于其他的核苷类抑制剂、HIV疫苗、广谱中和抗体和/或CAR-T细胞。
附图说明
图1A-1C显示了本文实施例测试的各质粒中XbaI至BamHI酶切位点之间插入的功能结构区域，包括CAR编码序列和/或shRNA编码序列、和/或报告基因的功能结构。其中G3L2表示针对Gag及LTR的shRNA(序列分别为SEQ ID NO：1和2)的编码序列，G2N表示针对Gag及Nef的shRNA(序列分别为SEQ ID NO：5和6)的编码序列。
图2A-2C显示了本文中的3个代表性质粒的结构，其中图2A为原始的pGTV-PEDF质粒，图2B为Z09质粒，图2C为Z01质粒。
图3显示了携带不同CAR分子的重组慢病毒SIV转导293T细胞后，针对CAR分子或者胞内报告基因进行流式检测的结果。CD4+指通过抗人CD4的抗体对转导后的293T进行流式检测并分析获得的CD4阳性细胞，RFP+指直接流式检测与CAR分子共表达的单体红色荧光蛋白mRFP阳性细胞。CTRL为未转导重组慢病毒SIV的293T细胞。
图4显示了重组慢病毒SIV-Z09转导293T细胞后，针对CAR分子或者与CAR分子共表达的胞 外报告基因tEGFR进行免疫荧光染色和流式检测的结果。其中A为两种抗体双染结果，B为抗CD4抗体单染结果，C为抗EGFR抗体单染结果，D为未染色对照。CD4+指通过抗人CD4的抗体对转导后的293T进行免疫荧光染色并流式检测分析获得的CD4+阳性细胞，EGFR+指通过抗人EGFR抗体对转导后的293T进行免疫荧光染色并流式检测分析获得的EGFR+阳性细胞。
图5显示CD3+T细胞通过CD3/CD28磁珠及细胞因子激活48小时之后的流式检测结果。活化细胞的比例指CD3+CD25+细胞在CD3+T细胞中的比例。对照组指未经磁珠及细胞因子激活的CD3+T细胞，实验组指接受磁珠及细胞因子激活的CD3+T细胞。
图6显示CD3+T细胞从活化至活化后12天期间的细胞数量及细胞活率的结果。活化后48小时，重组慢病毒SIV转导CD3+T细胞。图中实线表示细胞数量，对应于左侧Y轴。虚线表示细胞活率，对应于右侧Y轴。EGFP指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3代表没有转导任何重组慢病毒SIV的CD3+T细胞。重组慢病毒SIV携带的各CAR分子的名称分别标注于图6A-图6Q的上方。
图7显示对改造后的CD3+T细胞的表面标记及细胞内报告基因进行流式检测分析的结果。图7A为活化后第13天阳性细胞的比例，图B显示活化后第19天阳性细胞的比例。改造阳性细胞的比例通过针对报告基因的流式检测及分析获得，CTRL指用重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。
图8显示针对未转导的CD3+T细胞的表面标记进行流式检测并分析的结果。图8A为不同时间点CD3+T细胞的比例，图8B为不同时间点CD4+T细胞及CD8+T细胞的比例。
图9显示对转导后的CD3+T细胞的表面标记进行流式检测并分析的结果。图9A为重组慢病毒SIV-Z09(表达CD4胞外区)转导CD3+T细胞后分别检测CD4+T细胞、CD8+T细胞的比例，图9B为重组慢病毒SIV-Z14(不表达CD4胞外区)转导CD3+T细胞后分别检测CD4+T细胞、CD8+T细胞的比例。
图10显示改造后的CD3+T细胞与靶细胞以固定3：1比例混合后对细胞进行流式检测并分析的结果。所有靶细胞均为293F细胞，分别通过转染表达不同HIV病毒株的膜蛋白gp120的pAcGFP质粒并通过G418筛选获得，质粒中原有绿色荧光蛋白AcGFP通过P2A与gp120共表达。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。基于对GFP阳性CD3-的293F细胞的流式检测结果计算检测混合24小时的靶细胞数量，再分别计算各实验组样品中靶细胞相对于重组慢病毒SIV-Z17转导的CD3+T细胞样品中靶 细胞的减少比例，作为各样品对靶细胞的抑制率。
图11显示改造后的CD3+T细胞与靶细胞以固定3：1比例混合后24小时对上清中细胞因子IFN-γ进行检测并分析的结果。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL表示用重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。
图12显示改造后的CD3+T细胞与靶细胞以不同比例混合后对靶细胞的急性杀伤结果。利用CytoTox 96非放射性细胞毒性检测试剂盒，对混合后4小时的上清中乳酸脱氢酶LDH的含量进行检测并计算。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞表达人白细胞抗原DR0401。图标Z09、Z10及CTRL指示改造细胞所对应的结构，其中Z09、Z10分别为重组慢病毒SIV-Z09、SIV-Z10转导的CD3+T细胞，CTRL为重组慢病毒SIV-EGFP转导的CD3+T细胞。
图13显示改造后的CD3+T细胞与靶细胞以不同比例混合24小时后对靶细胞抑制作用的结果。混合后24小时，通过流式检测并分析，对GFP+CD3-的293F细胞的流式检测结果计算检测混合24小时的靶细胞数量，再分别计算各实验组样品中靶细胞相对于1：1效靶比的CTRL对照中靶细胞的减少比例，作为各样品对靶细胞的抑制率。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞表达人白细胞抗原DR0401。CTRL为重组慢病毒SIV-EGFP转导的CD3+T细胞。
图14显示改造后的CD3+T细胞与靶细胞以不同比例混合后对培养上清进行多因子检测的结果。混合后24小时用Luminex同时检测上清中12种细胞因子的水平(图14A-图14L)。靶细胞对应的HIV病毒gp120见图标，改造细胞对应的结构及效靶比见X轴，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞。平行于X轴的虚线为检测的阈值。
图15显示CD3+T细胞活化后第19天的流式检测结果。活化细胞的比例指CD3+CD25+细胞在CD3+T细胞中的比例，对照组指未染色的CD3+T细胞，实验组指进行染色后检测的CD3+T细胞。
图16显示延长培养之后的改造后的CD3+T细胞与靶细胞以固定3：1比例混合后对细胞进行流式检测并分析的结果。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。基于对GFP+CD3-的293F细胞的流式检测结 果计算检测混合24小时的靶细胞数量，再分别计算各实验组样品中靶细胞相对于重组慢病毒SIV-Z17转导的CD3+T细胞样品中靶细胞的减少比例，作为各样品对靶细胞的抑制率。
图17显示延长培养之后的改造后的CD3+T细胞与靶细胞以固定3：1比例混合后对培养上清进行检测并分析的结果。混合后24小时，通过ELISA对上清中细胞因子IFN-γ进行检测并分析。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL指转导重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。
图18显示延长培养之后的改造后的CD3+T细胞与靶细胞以不同比例混合24小时对靶细胞进行流式检测及分析结果。通过流式检测并分析，对GFP+CD3-的293F细胞的流式检测结果计算检测混合24小时的靶细胞数量，再分别计算各实验组样品中靶细胞相对于1：1效靶比的重组慢病毒SIV-EGFP转导的CD3+T细胞样品中靶细胞的减少比例，作为各样品对靶细胞的抑制率。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞表达人白细胞抗原DR0401(MHCII)。CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指未导入重组慢病毒SIV的CD3+T细胞。
图19显示改造后的CD3+T细胞与靶细胞以固定3：1比例混合后对上清进行多因子检测的结果。混合后24小时用Luminex同时检测上清中12种细胞因子的水平(图21A-图21L)。靶细胞对应的HIV病毒gp120见图标，改造细胞对应的结构及效靶比见X轴，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞。平行于X轴的虚线为检测的阈值。
图20显示改造后的CD3+T细胞与靶细胞以固定3：1比例混合并延长培养后进行流式检测并对改造细胞分析的结果。改造后的CD3+T细胞基于CD3+与靶细胞区分，CAR表达通过荧光蛋白报告基因检测。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。
图21显示改造后的CD3+T细胞与靶细胞以固定3：1比例混合并延长培养后进行流式检测并对靶细胞分析的结果。靶细胞基于CD3-与改造后的CD3+T细胞区分。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞只表达AcGFP。X轴标注为改造的CD3+T细胞中对应的CAR分子结构，CTRL指重组慢病毒SIV-EGFP转导的CD3+T细胞，CD3指没有转导任何重组慢病毒SIV的CD3+T细胞。基于对GFP+CD3-的293F细胞的流式检测结果计算检测混合24小时后的靶细胞数量，再分别计算 各实验组样品中靶细胞相对于CD3对照组样品中靶细胞的减少比例，作为各样品对靶细胞的抑制率。
图22显示改造后的CD3+T细胞与靶细胞以不同比例混合并延长培养后进行流式检测并对改造细胞分析的结果。不同结构及效靶比见X轴标注，其中CTRL代表重组慢病毒SIV-EGFP转导的CD3+T细胞。改造后的CD3+T细胞基于CD3+与靶细胞区分，CAR表达通过荧光蛋白报告基因检测。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞表达人白细胞抗原DR0401。
图23显示改造后的CD3+T细胞与靶细胞以不同比例混合并延长培养后进行流式检测并对靶细胞分析的结果。不同结构及效靶比见X轴标注。靶细胞细胞基于CD3-与改造后的CD3+T细胞区分。A中靶细胞表达HIV病毒株AC10.29的gp120,B中靶细胞表达HIV病毒株NL4-3的gp120，C中靶细胞表达HIV病毒株SF162的gp120，D中靶细胞表达人白细胞抗原DR0401。图标Z09、Z10及CTRL指示改造细胞所对应的CAR分子结构，其中CTRL代表重组慢病毒SIV-EGFP转导的CD3+T细胞。基于对GFP+CD3-的293F细胞的流式检测结果计算检测混合24小时的靶细胞数量，再分别计算各实验组样品中靶细胞相对于1：1效靶比的CTRL样品中靶细胞的减少比例，作为各样品对靶细胞的抑制率。
图24显示对HIV患者细胞改造后的培养上清中不同时间点收集的HIV病毒产物进行ELISA检测，对最终时间点的细胞进行流式检测及分析的结果。A为培养上清中病毒p24浓度，CTRL为仅转染有编码G3L2shRNA质粒的改造细胞对照，CD3指未转导任何重组慢病毒SIV的改造细胞对照。B为活化后24天时对细胞进行流式检测及分析后的结果。
发明详述
现有技术中，针对HIV感染细胞的细胞治疗方法(例如，CAR-T细胞)还存在诸多局限。一方面，其本身的特异性杀伤能力不够，另一方面，其本身容易受到HIV感染，易于耗竭，从而发挥效力的时间较短。针对以上问题，本申请一方面提供了一种对HIV感染细胞杀伤力更强，可在HIV感染的环境中维持更长时间，且发挥效力时间更长的重组细胞(例如，CAR-T细胞)；以及编码所述重组细胞的CAR及其他功能结构的核酸及载体、治疗HIV感染的药物组合物及疗法。本申请另一方面提供了一种嵌合抗原受体分子(CAR)，包含该CAR分子的细胞针对HIV感染细胞具有高效的、特异性的杀伤活性。
定义
为了解释本说明书的目的，将应用以下定义，并且在适当时，单数形式使用的术语也将包括复数形式，反之亦然。如果以下提出的任何定义与通过引用并入本文的任何文件相冲突，则以提出的定义为准。
如本文所用，“shRNA”即短发夹RNA或小发夹RNA。是一种具有发夹结构的人工RNA分子，可用于通过RNA干扰(RNAi)使目标基因表达沉默。
如本文所用，“嵌合抗原受体(CAR)”是一类工程化的细胞表面受体，一般表达在免疫效应细胞(例如效应T细胞)上，介导工程化免疫效应细胞针对具有特定靶点的细胞(在本申请中，称为“靶细胞”，例如在细胞表面表达HIV包膜蛋白gp120的细胞)的杀伤。CAR通常包括胞外区、胞内区，以及他们之间的跨膜区。“胞外区”包含抗原特异性识别结构域，可特异性识别抗原，并与之结合。在一些实施方案中，所述抗原识别区与跨膜结构域之间通过铰链区相连接。“胞内区”可包含一个或多个一级信号转导结构域和一个或多个共刺激结构域。在抗原特异性识别结构域与所述抗原结合形成二元复合物并变构后，胞内区可启动一系列生化反应，导致CAR所在的免疫效应细胞产生生物效应，例如分泌细胞因子或直接杀伤靶细胞。在本文中，如非特别指出，“一级信号转导结构域”提供活化的淋巴细胞(例如效应T细胞)的第一信号，而“共刺激结构域”提供活化淋巴细胞的第二信号。“一级信号转导结构域”与“共刺激结构域”均源自受体分子的“信号转导结构域”，其指受体分子部分或全部位于胞内并在受体与配体特异性结合后起信号转导作用的结构域。
如本文所用，所述“共刺激结构域”主要用于提供共刺激信号来增强免疫细胞的能力，包括例如增强记忆细胞的增殖、存活和/或发育。在一些实施方案中，所述“共刺激结构域”选自CD28、4-1BB(CD137)、OX40(CD134)等的信号转导结构域。
如本文所用，所述“跨膜区”，是指锚定在细胞膜内具有热力学稳定的蛋白质结构区域。跨膜结构域可以从天然蛋白质中获得，例如来源于T细胞受体(TCR)的跨膜结构域。在一些实施方案中，所述跨膜结构域选自CD4，CD8α，CD28和CD3ζ的跨膜结构域。
如本文所用，所述“铰链区”是连接抗原特异性识别结构域或胞外区及跨膜结构域的一段肽链，通常具有弹性。在一些实施方案中，所述铰链区由IgG的铰链或CD8α/CD28的铰链区衍变而来。其中IgG的“铰链”是指IgG的CH1和CH2功能区之间的区域，通常含大量脯氨酸。
如本文所用，“CAR-T”即嵌合抗原受体T细胞，是在细胞表面表达有嵌合抗原受体分子的T细胞，可识别靶细胞表面的抗原。CAR-T目前已开发到第4代。其中第1代CAR-T的CAR分子由CD3ζ链或FcεRIγ的信号转导结构域与抗原识别区连接融合而成，不含有共刺激结构域。第1代 CAR-T在体内的增殖能力有限，容易凋亡。第2代CAR增加了1个共刺激结构域，例如CD28或4-1BB(CD137)。CD28具有较强的抗肿瘤活性，而4-1BB的优点是能延长T淋巴细胞的存活时间并维持其抗肿瘤作用。第2代CAR增殖能力较第1代强，且能分泌更多细胞因子、抗凋亡蛋白。第3代CAR-T不仅可以同时表达2个共刺激信号分子，而且能够分泌更多的IFN-γ，抗肿瘤细胞毒作用更高。第4代CAR-T还能够在肿瘤中分泌特定的细胞因子(如IL-12)，从而改变肿瘤微环境，并影响和激活其他免疫细胞产生免疫反应。
如本文所用，术语“连接肽”是指长度为约1至100个氨基酸的寡肽或多肽区，其将本发明的CAR的任何结构域/区连接在一起。连接肽可由柔性残基(如甘氨酸和丝氨酸)组成，以便相邻的蛋白质结构域相对于彼此自由移动。当期望确保两个相邻结构域在空间上不相互干扰时，可使用较长的连接肽。
在本申请中，“GS接头”作为连接肽的一种，是指由甘氨酸(G)和丝氨酸(S)组合而成的柔性连接肽。最常见的GS接头是(GGGGS)n，通过改变n的大小，可以将结构域之间的距离放大或减小。
如本文所用，“CD4”即分化抗原簇，是免疫球蛋白超家族的成员，表达于辅助T(Th)细胞，调节性T细胞、单核细胞、巨噬细胞和树突细胞的表面，并在T细胞的发育和激活方面起着重要作用。CD4包含一个氨基末端胞外域(含有四个暴露在细胞外表面的免疫球蛋白结构域：D1、D2、D3和D4，呈现出Ig样结构)、一个跨膜域以及一个短胞质尾区。其中，D1和D3类似于免疫球蛋白可变区。D2和D4类似于免疫球蛋白的恒定区。CD4也是HIV的主要受体，CD4阳性(CD4+)的T细胞是HIV(人类免疫缺陷病毒)攻击的对象。
如本文所用，“gp120”是一种暴露在HIV包膜表面的糖蛋白。其名称中的120来自其120kDa的分子量。在辅助T细胞上，gp120可与CD4结合，诱导gp120和gp41构象变化，使HIV-1能够与宿主细胞上表达的共受体(例如CCR5或CXCR4)结合，最终使HIV的包膜与细胞膜融合而使病毒进入细胞。
如本文所述，术语“抗体”包括全长抗体及其抗原结合片段。全长抗体包括两条重链和两条轻链。轻链和重链的可变区负责抗原的结合。两条链中的可变区通常包括3个高变的环，被称为互补决定区(CDRs)(轻链(LC)CDRs包括LC-CDR1、LC-CDR2和LC-CDR3，重链(HC)CDRs包括HC-CDR1、HC-CDR2和HC-CDR3)。重链或轻链的3个CDR区插入到被称为框架区(FRs)的侧翼区段之间，所述框架区比CDR区具有更高的保守性，并形成支撑高变环的支架。重链和轻链的恒定区并不参与抗原结合，但展示出多种效应功能。抗体是基于它们重链恒定区的氨基酸序列进行分类的。抗体的五种主要类别或同种型是IgA、IgD、IgE、IgG和IgM，其特征在于分别具有α、δ、ε、γ和μ型重链。几种主要的抗体类别被分为亚类，如IgG1(γ1重链)、IgG2(γ2重链)、 IgG3(γ3重链)、IgG4(γ4重链)、IgA1(α1重链)或IgA2(α2重链)。
如本文所述，术语“抗原结合片段”包括抗体片段，例如，双链抗体(diabody)、Fab、Fab’、F(ab’)2、Fv片段、二硫键稳定的Fv片段(dsFv)、(dsFv)2、双特异性dsFv(dsFv-dsFv’)、二硫键稳定的双链抗体(ds双链抗体)、单链Fv(scFv)、scFv二聚体(二价双链抗体)，由包含一个或多个CDRs的抗体片段组成的多特异性抗体、单域抗体、纳米抗体(nanobody)、域抗体、二价域抗体或者能够与抗原结合但不包含完整抗体结构的任何其他抗体片段。抗原结合片段能够与亲本抗体或亲本抗体片段(如亲本scFv)结合相同的抗原。抗原结合片段还包括包含上述抗体片段的融合蛋白。
如本文所用，术语“特异性结合”是指结合亲和力为至少10 -6M的抗体与抗原，受体与配体，或特异性抗原识别结构域与抗原之间的接触。在某些方面，所述亲和力为至少约10 -7M，优选10 - 8M、10 -9M、10 -10M、10 -11M或10 -12M的结合亲和力。
本申请中，术语“多核苷酸”或“核酸”、“核酸分子”可互换使用，包括但不限于DNA、RNA、cDNA(互补DNA)、mRNA(信使RNA)、rRNA(核糖体RNA)、shRNA(小发夹RNA)、snRNA(小核RNA)、snoRNA(短核仁RNA)、miRNA(微小RNA)、基因组DNA、合成DNA、合成RNA和/或tRNA。
如本文所用，“载体”、“克隆载体”和“表达载体”是指通过其可以将多核苷酸序列(例如外来基因)引入宿主细胞中，以转化宿主并促进引入序列的表达(例如转录和翻译)的载体。载体包括质粒、噬菌体、病毒等。
如本文所用，“免疫效应细胞”是指能够针对靶抗原或靶细胞实现免疫效应和免疫反应，例如免疫杀伤效应、免疫应答效应的细胞，例如T细胞等。
如本文所用，所述“信号肽”是指引导新合成的蛋白质向分泌通路转移的短肽链，长度通常为5至30个氨基酸。在一些实施方案中，所述信号肽为膜定位信号肽，即用于指导蛋白质的跨膜转移和/或定位的氨基酸序列。在多数情况下，信号肽位于氨基酸序列的N端。在mRNA中，信号肽的编码序列通常位于起始密码子后，是一段编码疏水性氨基酸序列的RNA区域。在信号肽引导蛋白质完成定位后，通常会在信号肽酶的作用下被切除。
如本文所用，所述“安全开关”是指经设计以防止细胞疗法的潜在毒性或以其它方式防止不良效应的工程化蛋白质。在一些实施方案中，安全开关的表达受到有条件的控制，以解决所移植的工程化细胞/重组细胞的安全问题。在一些实施方案中，所述细胞可以将编码安全开关的基因永久性地并入其基因组中。在一些实施方案中，安全开关能够介导细胞凋亡的诱导、蛋白质合成的抑制、DNA复制、生长阻滞、转录和转录后基因调控和/或抗体介导的耗竭(例如，通过ADCC、 CDC等途径)。在一些实施方案中，安全开关的激活导致其中安全开关已被激活的细胞的死亡。在一些实施方案中，安全开关的激活导致其中安全开关已被激活的细胞的活性下调，其中该细胞将来可以被重新激活。而在另一些实施方案中，默认情况下细胞的活性被下调并且安全开关的激活导致细胞活性的上调，其中该细胞将来可以被重新失活。
在一些实施方式中，本文所述的安全开关包括能够被抗体或抗体片段靶向的受体，这类受体的例子包括EPCAM、VEGFR、整联蛋白(例如，整联蛋白ανβ3、α4、αΙ3/4β3、α4β7、α5β1、ανβ3、αν)、TNF受体超家族成员(例如TRAIL-R1、TRAIL-R2)、PDGF受体、干扰素受体、叶酸受体、GPNMB、ICAM-1、HLA-DR、CEA、CA-125、MUC1、TAG-72、IL-6受体、5T4、GD2、GDB、CD2、CD3、CD4、CD5、CD11、CD11a/LFA-1、CD15、CD18/ITGB2、CD19、CD20、CD22、CD23/lgE受体、CD25、CD28、CD30、CD33、CD38、CD40、CD41、CD44、CD51、CD52、CD62L、CD74、CD80、CD125、CD147/basigin、CD152/CTLA-4、CD154/CD40L、CD195/CCR5、CD319/SLAMF7和EGFR及其截短形式(例如，保留一个或多个胞外表位但缺少胞质结构域内部一个或多个区域的形式)等。可适用于本申请的安全开关是本领域已知的，其优选示例包括但不限于，例如，半胱天冬酶9(iCaspase-9)、iCaspase-1、iCaspase-8、胸苷激酶(例如，HSV-TK、VZV-TK)、胞嘧啶脱氨酶(CD)、CD20、截短型EGFR(tEGFR)、FR806、RQP8或其任何组合。
如本领域所已知的，不同类型的安全开关可由不同的外源分子活化和/或识别。在一些实施方案中，所述安全开关被外源分子(例如前药)活化，其活化时触发治疗细胞发生细胞凋亡和/或细胞死亡。在另一些实施方案中，所述安全开关由能够诱导细胞死亡(例如，ADCC或补体诱导的细胞死亡)的分子(例如，抗体)所识别。在一些具体实施方式中，当安全开关是iCaspase-9时，可施用导致iCaspase-9激活和细胞凋亡的二聚化药物。iCaspase-9分子含有在CID存在下介导二聚化的二聚化过程化学诱导物(CID)结构域，这导致表达CAR的细胞诱导性耗竭和选择性耗竭。在另一些具体实施方式中，对于包含截短型EGFR的重组细胞，虽然tEGFR缺少信号传导能力，但是保留由能够诱导ADCC的分子(例如，西妥昔单抗)识别的表位，从而向重组细胞施用西妥昔单抗会引起ADCC和该重组细胞的后续耗竭。对于包含CD20的重组细胞，施用利妥昔单抗能够破坏该重组细胞。在另一些具体实施方式中，对于包含HSV-TK的重组细胞，当与更昔洛韦(gancyclovir)接触时其引起细胞死亡。在另外一些其他的实施方式中，能够活化和/或识别安全开关的外源分子包括还丙氧鸟苷(GCV)、无环鸟苷(ACV)、溴乙烯脱氧尿苷(BVDU)、6-甲氧基嘌呤阿拉伯核苷(AraTP)、5-氟胞嘧啶(5-Fc)、AP20187、AP1903、他莫昔芬、他克莫司等。
如本文所述，术语“报告基因”是指编码报告分子的核苷酸序列。“报告分子”是指可在多种检测系统中的任一种中检测到的物质，可用于鉴定潜在转染的细胞。在一个实施方案中，报告分子包括但不限于，荧光素酶(Luciferase)、β-半乳糖苷酶(β-galactosidase)、氯霉素乙酰 转移酶(Chloramphenicol Acetyltransferase，CAT)、碱性磷酸酶(alkaline pkosphatase，AKP)、荧光蛋白(fluorescent protein)，例如绿色荧光蛋白(GFP或EGFP)、黄色荧光蛋白(YFP)、红色荧光蛋白(RFP)，或本领域已知的其他报告分子。在另一个实施方案中，tEGFR也可作为报告分子。
如本文所用序列的“同一性”是指氨基酸序列之间或核苷酸序列之间通过序列比对软件，例如BLAST，确定的相似程度。例如，在本申请中，至少85％的序列同一性，包含例如：至少90％、93％、95％、97％、98％、99％的序列同一性。
如本文所用，术语“有效量”或“治疗有效量”是指包含一种或多种本申请公开的肽、蛋白、核酸或其突变型、变体、类似物或衍生物的药物组合物的量，且接受所述“有效量”或“治疗有效量”的药物组合物的患者或受试者可获得医学治疗的合理益处/风险比，从而减轻或预防疾病或病症的至少一种或更多种症状，达到期望的治疗或预防效果。
如本文所用，术语“约”是指本技术领域的技术人员容易知道的各个值的通常误差范围。提及“约”值或参数在本文中包括(及描述)针对该值或参数本身的实施方案。如本文所使用的，当术语“约”在数值之前时，表示该数值上或下10％的范围内。例如，“约100”涵盖90和110。
如本文所用，除非另外指出，否则单数形式“一个”，“一种”和“该”包括复数形式。
除非本文另外定义，否则本文使用的所有技术和科学术语具有与本发明所属领域的普通技术人员通常所理解的相同含义。
工程化细胞或重组细胞
一方面，本申请提供了一种重组细胞，其包含下述功能结构：
1)嵌合抗原受体(CAR)或其编码序列，所述CAR包含胞外区、跨膜区及胞内区，其中所述胞外区包含人CD4分子的D1和D2结构域；
2)靶向抑制选自下组的任一种或多种参与HIV生命周期的宿主或HIV基因的shRNA或其编码序列：NF-κB、CCR5、TSG101、CXCR4、P-TEFb、tat、rev、nef、env、LTR和gag。
在一些实施方案中，所述重组细胞来源于HIV受体细胞或外周血单核细胞。在另一些实施方案中，所述重组细胞来源于淋巴细胞。在一些实施方式中，所述重组细胞来源于T细胞。在另一些实施方式中，所述重组细胞来源于幼稚T细胞
Figure PCTCN2022143440-appb-000002
记忆性T细胞、效应性T细胞。在 另一些实施方式中，所述重组细胞来源于细胞毒T细胞、辅助性T细胞、调节性T细胞。在另一些实施方式中，所述T细胞来源于CD4+T细胞，CD8+T细胞。在另一些实施方式中，所述重组细胞来源于NKT细胞。在另一些实施方案中，所述重组细胞来源于γδT细胞。在另一些实施方案中，所述重组细胞来源于NK细胞。在另一些实施方案中，所述重组细胞来源于抗原提呈细胞，例如巨噬细胞、树突状细胞。
在一些实施方案中，所述重组细胞来源于祖细胞或干细胞。在本发明的一些实施方案中，所述祖细胞或干细胞包括造血干细胞(例如，CD34+细胞)或造血祖细胞。在本发明的另一些实施方案中，所述干细胞包括记忆性T干细胞(memory T stem cell)，例如中枢记忆T细胞(central memory T cell)、效应记忆T细胞(effector memory T cell)或干细胞样记忆T细胞(stem cell memory T cell)。在本发明的另一个实施方案中，所述干细胞经过诱导可分化为如前所述的任一种T细胞。在一些实施方案中，所述重组细胞源自感染HIV的患者。在一些实施方案中，所述重组细胞源自健康人群。
本领域技术人员应当知晓，参与HIV生命周期的基因包括其结构及调节基因、调控元件、以及宿主细胞的部分基因，干扰或抑制HIV生命周期的任意环节均可以对其复制、增殖产生影响。但干扰或抑制其中的哪个或哪几个基因可以更好地达到这一作用，并更好地平衡抗HIV作用及宿主细胞的正常生理功能，是难以预测的。例如，如何在最大限度满足本申请的重组细胞杀伤能力的同时，尽量减少HIV对所述细胞的感染，减少及延缓重组细胞耗竭是难以从现有技术中获知的。
已知HIV基因组由至少七个调控元件(LTR、TAR、RRE、PE、SLIP、CRS和INS)和九个基因(gag、pol、env、tat、rev、nef、vif、vpr、vpu，有时还存在第十个基因tev，其为tat、env和rev的融合)组成，共编码19种蛋白质。其中三个基因，gag、pol和env，包含制造新病毒颗粒的结构蛋白所需的信息。tat、rev、nef、vif、vpr和vpu(在HIV-2的情况下是vpx)是蛋白质的调节基因，这些基因控制HIV感染细胞的能力，产生新的病毒拷贝、复制，或引起疾病。调控元件则与调节基因表达蛋白相互作用，参与基因组的转录、翻译等过程，或干扰宿主细胞的正常生理活动。此外，参与HIV生命周期的基因还包括部分宿主基因，例如NF-κB、CCR5、TSG101、CXCR4、P-TEFb等。因此，引入可靶向抑制上述基因的shRNA，便有机会干扰HIV的复制和增殖，减少其对宿主细胞的损害。
因此，在一些实施方案中，所述重组细胞包含的靶向抑制一种或多种参与HIV生命周期的宿主或HIV基因的shRNA是靶向HIV的gag基因和LTR基因的shRNA。在一些实施方案中，所述shRNA为靶向HIV的gag和nef的shRNA。
在一些实施方案中，靶向gag的shRNA的核酸序列包含如SEQ ID NO：1中所示的序列，或与SEQ ID NO：1中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸 序列。在一些实施方案中，靶向LTR的shRNA的核酸序列包含如SEQ ID NO：2中所示的序列，或与SEQ ID NO：2中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，编码靶向gag的shRNA的核酸序列包含如SEQ ID NO：3中所示的序列，或与SEQ ID NO：3中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，靶向LTR的shRNA的核酸序列包含如SEQ ID NO：2中所示的序列，或与SEQ ID NO：2中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，编码靶向LTR的shRNA的核酸序列包含如SEQ ID NO：4中所示的序列，或与SEQ ID NO：4中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。
在一些实施方案中，靶向gag的shRNA的核酸序列包含如SEQ ID NO：5中所示的序列，或与SEQ ID NO：5中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，靶向nef的shRNA的核酸序列包含如SEQ ID NO：6中所示的序列，或与SEQ ID NO：6中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，编码靶向gag的shRNA的核酸序列包含如SEQ ID NO：7中所示的序列，或与SEQ ID NO：7中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，靶向nef的shRNA的核酸序列包含如SEQ ID NO：6中所示的序列，或与SEQ ID NO：6中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。在一些实施方案中，编码靶向nef的shRNA的核酸序列包含如SEQ ID NO：8中所示的序列，或与SEQ ID NO：8中所示的序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的核酸序列。
在本申请的一些实施方案中，选择H1启动子作为靶向gag的shRNA的启动子，而选择U6启动子作为靶向LTR或nef的shRNA的启动子，并实现了各shRNA良好的转录和稳定性。在一些实施方案中，所述H1启动子和U6启动子的核苷酸序列分别如SEQ ID NO：9和10所示，或分别与SEQ ID NO：9和10具有至少85％的序列同一性。
在一些实施方案中，所述重组细胞中CAR的胞外区包含选自下述的结构：人CD4分子的D1结构域、人CD4分子的D1和D2结构域、人CD4分子的D1至D3结构域、人CD4分子的D1-D4结构域、特异性结合HIV的抗体或其抗原结合片段。如本申请所用，术语“抗原结合片段”是指抗体的一个或多个片段，其保留与抗原特异性结合的能力。抗原结合片段的实例包括Fab、Fab'、F(ab')2、Fv片段、双体抗体、线性抗体、scFv。在本申请中，所述特异性结合HIV的抗体或其抗原结合片段的示例包括结合gp120的特异性抗体或抗原结合片段。
如本文所用，术语“scFv”(单链抗体可变片段)是指包含仅重链(VH)和轻链(VL)的可变 区的蛋白结构域或蛋白，其中VH和VL可通过接头肽连接。scFv能够被表达为单链多肽。scFv保留了其所源自的完整抗体的特异性。轻链和重链可以为任何顺序，例如，VH-接头-VL或VL-接头-VH，只要保留scFv对靶抗原的特异性即可。在一些特殊的实施方案中，也可以省去接头。在一些实施方案中，所述gp120单克隆抗体的scFv为NAb-scFv(其构建方法如专利CN103797029B所示)。在一些实施方案中，所述NAb-scFv包含如SEQ ID NO：15中所示的序列。
在本申请的一些优选实施方案中，所述胞外区包含人CD4分子的D1和D2结构域而不包含D3和D4结构域。在一些特定的实施方案中，所述胞外区包含如SEQ ID NO：13所示的氨基酸序列或与该氨基酸序列具有至少85％以上同一性的氨基酸序列。HIV可通过其包膜蛋白gp120与CD4的特异性结合进入宿主T细胞。利用这种特异性结合，可将CD4或其与gp120具有特异性结合能力的部分作为CAR的特异性抗原识别结构域。
在一些实施方案中，所述重组细胞包含的跨膜区源自或包含选自以下一种或多种蛋白分子的跨膜结构域：T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D4、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、NKG2C。在一些特定的实施方案中，所述跨膜区来自或包含CD8α蛋白分子的跨膜结构域。在一些特定的实施方案中，所述跨膜区包含如SEQ ID NO：18所示的氨基酸序列或包含与其具有至少85％同一性的氨基酸序列。
在一些实施方案中，所述重组细胞的胞内区包含一级信号转导结构域。在一些实施方案中，所述一级信号转导结构域包含免疫受体的酪氨酸活化基序(immune-receptor tyrosine-based activation motifs，ITAM，其基本组成是：YXXL/V。其中Y为酪氨酸，L/V指亮氨酸或缬氨酸，X可为任意氨基酸)。在一些实施方案中，所述一级信号转导结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12。在一些实施方案中，所述一级信号转导结构域源自或包含CD3ζ的信号转导结构域。在一些实施方案中，所述一级信号转导结构域包含如SEQ ID NO：23中所示的氨基酸序列。
在一些实施方案中，所述胞内区还进一步包含共刺激结构域，所述共刺激结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。在一些实施方案中，所述共刺激结构域包含CD137、CD28+CD137、或CD137+DAP10的信号转导结构域。
在一些实施方案中，所述胞内区包含CD3ζ和CD137的信号转导结构域。在一些实施方案中，所述胞内区包含CD3ζ、CD28和CD137的信号转导结构域。在一些实施方案中，所述胞内区包含CD3ζ、CD137和DAP10的信号转导结构域。在一些实施方案中，所述胞内区包含CD28、DAP10和CD3ζ的信号转导结构域。在一些实施方案中，所述胞内区包含2B4、DAP10和CD3ζ的信号转导结构域。在一些实施方案中，所述胞内区包含CD137、2B4和CD3ζ的信号转导结构域。
在一些实施方案中，所述胞内区中一级信号转导结构域和共刺激结构域，以及各共刺激结构域之间还包含连接肽。其中，所述连接肽可选自能够连接胞内区中的各个结构域，维持各结构域之间一定的相对空间距离以保证其各自的功能完整性和其间信号传导的任意天然的或合成的肽序列。可选的连接肽是本领域技术人员已知的，可以结合本发明的替代性实施方案使用。在一些特定的实施方案中，所述连接肽为GS接头，例如(GGGGS)n，其中n取自任意自然数。在一些特定的实施方案中，所述连接肽为GGGGS。
在本申请的一些特定的实施方案中，所述胞内区包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与其中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些实施方案中，所述胞外区与所述跨膜区之间由铰链区相连。本申请所述的铰链区可以选自天然蛋白中包含的铰链，也可以选自人工合成的铰链区，例如GS接头。在本申请的一些实施方案中，所述铰链区来自或包含人Ig的铰链区、GS接头、KIR2DS2铰链或CD8的铰链区。在一些实施方案中，所述铰链区为CD8α的铰链区。在一些特定的实施方案中，所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些实施方案中，本申请的重组细胞中的CAR还进一步包含信号肽，所述信号肽将所述 CAR定位于重组细胞的细胞膜上。在一些实施方式中，在信号肽引导蛋白质完成定位后，在信号肽酶的作用下被切除。因此，本申请所述的连接有一段信号肽，包含在细胞生命周期的任何时候所述CAR连接有一段信号肽。在一些实施方案中，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽。在一些实施方案中，所述信号肽来自或包含CD4或CD8的信号肽。在一些实施方案中，所述信号肽包含如SEQ ID NO：16或17中所示的氨基酸序列或包含与其具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述CAR胞内区包含选自以下的氨基酸序列：SEQ ID NO：28-34中任一项或包含与其中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述CAR包含选自以下的氨基酸序列：SEQ ID NO：35-46中任一项或包含与其中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述重组细胞包含分别靶向gag和LTR的shRNA或其编码序列、CD4的信号肽、CD4的D1和D2结构域、CD8α的铰链区、CD8α的跨膜区、CD137信号转导结构域和CD3ζ的信号转导结构域。
在一些具体的实施方案中，所述重组细胞包含分别靶向gag和LTR的shRNA或其编码序列、CD4的信号肽、CD4的D1和D2结构域、CD8α的铰链区、CD8α的跨膜区、CD28的信号转导结构域、CD137的信号转导结构域、以及CD3ζ的信号转导结构域。
在一些具体的实施方案中，所述重组细胞包含分别靶向gag和LTR的shRNA或其编码序列、CD4的信号肽、CD4的D1和D2结构域、CD8α的铰链区、CD8α的跨膜区、CD28的信号转导结构域、CD137的信号转导结构域、以及CD3ζ的信号转导结构域，并且CD28的信号转导结构域、CD137的信号转导结构域、以及CD3ζ的信号转导结构域之间通过GS连接肽相连。
在一些具体的实施方案中，所述重组细胞包含分别靶向gag和LTR的shRNA或其编码序列、CD4的信号肽、CD4的D1和D2结构域、CD8α的铰链区、CD8α的跨膜区、CD28的信号转导结构域、CD137的信号转导结构域、SLAMF4(2B4)和/或DAP10的信号转导结构域，以及CD3ζ的信号转导结构域。
在一些的实施方案中，所述重组细胞还包含报告分子和/或安全开关。
在一些具体的实施方案中，所述安全开关选自下述中的一种或多种：iCaspase-9、iCaspase-1、iCaspase-8、胸苷激酶(例如，HSV-TK、VZV-TK)、胞嘧啶脱氨酶(CD)、CD20、tEGFR、 FR806和RQP8。在另一优选的实施方案中，所述安全开关为tEGFR，优选地，所述tEGFR的氨基酸序列如SEQ ID NO：49或50所示。
在一些具体的实施方案中，所述报告分子选自下述中的一种或多种：荧光素酶、β-半乳糖苷酶、CAT、AKP、GFP或EGFP、YFP、RFP、tEGFR。在另一优选的实施方案中，所述报告分子优选RFP或tEGFR，优选地，所述RFP的氨基酸序列如SEQ ID NO：48所示，所述tEGFR的氨基酸序列如SEQ ID NO：49或50所示。
嵌合抗原受体(CAR)及组合
另一方面，本申请提供了一种嵌合抗原受体，其包含胞外区、跨膜区及胞内区，其中所述胞外区来自或包含人CD4分子的胞外区，跨膜区来自或包含CD8α的跨膜结构域。
在本申请的一个优选实施方案中，所述嵌合抗原受体还包括铰链区，所述胞外区与所述跨膜区之间由铰链区相连。优选地，所述铰链区来自或包含人Ig铰链区、GS接头、KIR2DS2铰链或CD8α的铰链区，更优选CD8α的铰链区。在本申请的一个具体实施方案中，所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与该序列有至少85％同一性的氨基酸序列。
在本申请的一个优选实施方案中，所述胞外区包含人CD4分子的D1和D2结构域而不包含D3和D4结构域。在一些特定的实施方案中，所述胞外区包含如SEQ ID NO：13所示的氨基酸序列或与该氨基酸序列具有至少85％以上同一性的氨基酸序列。
在一些实施方案中，所述CAR包含的跨膜区包含如SEQ ID NO：18所示的氨基酸序列。
在一些实施方案中，所述CAR的胞内区包含一级信号转导结构域。在一些实施方案中，所述一级信号转导结构域包含免疫受体的酪氨酸活化基序(immune-receptor tyrosine-based activation motifs，ITAM，其基本组成是：YXXL/V。其中Y为酪氨酸，L/V指亮氨酸或缬氨酸，X可为任意氨基酸)。在一些实施方案中，所述一级信号转导结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12。在一些实施方案中，所述一级信号转导结构域源自或包含CD3ζ的信号转导结构域。在一些实施方案中，所述一级信号转导结构域包含如SEQ ID NO：23中所示的氨基酸序列。
在一些实施方案中，所述胞内区还进一步包含共刺激结构域，所述共刺激结构域源自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、 ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。在一些实施方案中，所述共刺激结构域包含CD137、CD28+CD137、或CD137+DAP10的信号转导结构域。
在一些实施方案中，所述胞内区包含CD3ζ和CD137的信号转导结构域。在一些实施方案中，所述胞内区包含CD3ζ、CD28和CD137的信号转导结构域。在一些实施方案中，所述胞内区包含CD3ζ、CD137和DAP10的信号转导结构域。在一些实施方案中，所述胞内区包含CD28、DAP10和CD3ζ的信号转导结构域。在一些实施方案中，所述胞内区包含2B4、DAP10和CD3ζ的信号转导结构域。在一些实施方案中，所述胞内区包含CD137、2B4和CD3ζ的信号转导结构域。
在一些实施方案中，所述胞内区中一级信号转导结构域和共刺激结构域，以及各共刺激结构域之间还包含连接肽。其中，所述连接肽可选自能够连接胞内区中的各个结构域，维持各结构域之间一定的相对空间距离以保证其各自的功能完整性和其间信号传导的任意天然的或合成的肽序列。可选的连接肽是本领域技术人员已知的，可以结合本发明的替代性实施方案使用。在一些特定的实施方案中，所述连接肽为GS接头，例如(GGGGS)n，其中n取自任意自然数。在一些特定的实施方案中，所述连接肽为GGGGS。
在本申请的一些特定的实施方案中，所述胞内区包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与其中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述CAR包含选自以下的氨基酸序列：SEQ ID NO：35、36、43-46中任一项或包含与其中任一序列具有至少85％、90％、93％、95％、97％、98％、99％同一性的氨基酸序列。
在一些具体的实施方案中，所述CAR进一步连接信号肽，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽，优选地，所述信号肽来自或包含CD4或CD8的信号肽。
进一步的，本申请还提供了包含如前所述的嵌合抗原受体的重组细胞。
本申请还进一步提供了所述嵌合抗原受体或包含该嵌合抗原受体的重组细胞与至少一种本申请中所述的靶向HIV的shRNA或其编码序列或包含其编码序列的载体的组合。
核酸、载体和组合物
本申请还提供了一种核酸或载体，其包含编码如前所述的重组细胞所包含的CAR和/或shRNA的多核苷酸序列，或者如前所述的CAR分子。在一些实施方案中，所述载体选自质粒、病毒载体或线性核酸分子。在一些特定的实施方案中，所述载体为逆转录病毒穿梭质粒载体，例如猴免疫缺陷病毒(SIV)的穿梭质粒载体。在一些实施方案中，所述载体为包装后的SIV病毒载体。在一些实施方案中，所述CAR和shRNA在相同启动子驱动下表达或转录。在一些实施方案中，所述CAR和shRNA在不同启动子的驱动下表达或转录。在一些实施方案中，编码所述CAR和shRNA的多核苷酸序列包含在不同的载体中。在一些实施方案中，编码所述CAR和shRNA的多核苷酸序列包含在同一个载体中。在一些实施方案中，所述编码shRNA的多核苷酸序列包含在同一个或不同的载体中。
本申请还提供了一种组合物，其包含本申请所述的核酸或载体。在一些实施方案中，本申请还提供了包含所述核酸、载体或组合物的脂质体。
药物组合物及治疗方法
本申请还提供了一种药物组合物，其包含如前所述的重组细胞，CAR分子，核酸或载体，或如前所述的组合。所述药物组合物，可用于治疗感染HIV的患者。在一些实施方案中，所述感染HIV的患者经过了长期的抗逆转录病毒治疗。在一些实施方案中，所述药物组合物还进一步包含药学上可接受的赋形剂、载剂、和/或稳定剂。在一些实施方案中，合适的赋形剂、载剂、和/或稳定剂选自抗氧化剂、防腐剂、无热原水、等渗盐溶液、和磷酸盐缓冲液等或其组合。所述药学上可接受的载剂、赋形剂或稳定剂在所采用的剂量和浓度下对接受者无毒，并且包括例如：缓冲液，诸如磷酸盐、柠檬酸盐或乙酸盐，pH通常为5.0至8.0，任选地6.0至7.0；实现等渗的盐，诸如氯化钠、氯化钾等；抗氧化剂；防腐剂；低分子量多肽；蛋白；亲水性聚合物，诸如聚山梨醇酯80；氨基酸，诸如甘氨酸；碳水化合物；螯合剂；糖；以及本领域的技术人员已知的其他标准成分(Remington:The Science and Practice of Pharmacy,第22版,Loyd V.Allen等人编,Pharmaceutical Press(2012))。
本申请的药物组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对患者有益的剂量进行施用。给药方式可以采用，例如肠胃外给药(如注射)或其它治疗方式。本申请进一步还提供了一种治疗HIV感染的方法，其包含向感染HIV的受试者施用有效量的如前所述的重组细胞、如前所述的核酸或载体，或如前所述的组合， 或如前所述的药物组合物。在一些实施方案中，所述施用为静脉注射或动脉灌注。在一些实施方案中，所述有效量根据患者疾病严重程度、年龄和身体状况等确定。在一些实施方案中，所述治疗HIV感染的方法，还包含向感染HIV的受试者施用有效量的如前所述的重组细胞、核酸或载体，如前所述的组合或如前所述的药物组合物，并联合施用其他核苷类抑制剂、HIV疫苗、针对HIV的广谱中和抗体和/或CAR-T细胞。
制备方法
此外，本申请还提供了一种制备如项前所述重组细胞的方法，其包含将如前所述的载体导入上述细胞中进行表达。在一些实施方案中，所述载体为SIV穿梭质粒，所述制备方法包含将所述SIV穿梭质粒包装成SIV病毒颗粒，并使用所述病毒颗粒感染所述细胞。在一些实施方案中，所述制备方法包含将所述SIV穿梭质粒包装成SIV病毒颗粒，使用所述病毒颗粒感染所述祖细胞或干细胞，并进一步通过细胞因子刺激等方式使所述祖细胞或干细胞分化为成熟的效应T细胞。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于此。在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，包括各个技术特征以任何其它的合适方式进行组合，这些简单变型和组合同样应当视为本发明所公开的内容，均属于本发明的保护范围。
应当理解，以上描述以及随后的实施例旨在说明而不是限制本发明的范围。在本发明范围内的其他方面，优点和修改对于本发明所属领域的技术人员将是显而易见的。
实施例
实施例1：载体构建
本申请实施例中所使用的携带外源基因表达框的质粒是基于SIV三代慢病毒载体包装系统中的穿梭质粒构建而成的。该系列质粒相对于SIV三代慢病毒载体包装系统中的穿梭质粒分别插入的重要功能结构示意图如图1A-1C所示。其中，图1A-1C中各结构的含义如下：G3L2表示靶向HIV基因组中保守序列Gag和LTR的shRNA(序列分别如SEQ ID NO：1和2所示)的编码序列(各自含有独立的启动子)；G2N表示靶向HIV基因组中保守序列Gag和Nef的shRNA(序列分别如SEQ ID NO：5和6所示)的编码序列(各自含有独立的启动子)；CD4 signal表示CD4的信号肽区域；CD4 D1D2表示CD4的D1和D2结构域；CD8h表示CD8α的铰链区；CD8a TM和CD28TM分别表示CD8α和CD28的跨膜区；CD28、CD137、2B4、CD3ζ和DAP10分别表示CD28、CD137、2B4、CD3ζ和DAP10的信号转导结构域；Nab-scFv表示中和抗体scFv(本实施例中采用3BNC117)； CCR5&tat/rev表示靶向CCR5及tat/rev的shRNA编码序列。Reporter表示报告基因，本申请实施例中使用的报告基因是增强型绿色荧光蛋白(EGFP)、红色荧光蛋白(RFP)或截短型表皮生长因子受体(tEGFR)。L表示连接肽GGGGS；L3表示连接肽(GGGGS) 3。相关的序列如表2所示。
首先，基于pGTV-PEDF载体(图2A)，根据图1A-1C中所示的组成和表2中的序列，通过核酸片段合成、无缝连接、转化等方法，获得携带Z09结构的质粒(图2B)。之后，再在该质粒的基础上构建携带Z10-Z15，Z17，Z34-Z37等结构的质粒。携带Z01-Z07结构的系列质粒是分别基于携带Z09-Z15结构的系列质粒构建而成，其中Z01对应Z09，Z02对应Z10，Z03对应Z11，Z04对应Z12，Z05对应Z13，Z06对应Z14，Z07对应Z15。携带Z01-Z07结构的质粒相对于其各自对应的质粒，差异在于前者不包含编码shRNA的多核苷酸序列。另外，Z01/Z02/Z09/Z10与Z11-Z15/Z03-Z07相比，差异在于前者的胞外区、铰链区、跨膜区分别为CD4的D1D2结构域、CD8α的铰链区和CD8α的跨膜区。为更直观地显示所述质粒与其对应质粒的差异，图2C示出了携带Z01结构的质粒的图谱。携带Z08结构和Z16结构的质粒的构建参照美国专利申请US 9,833,480B2。
核酸片段合成、无缝连接、转化等质粒构建步骤及方法是本领域常规技术，可参照例如J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002，或按照制造厂商说明书进行操作。
实施例2基于SIV载体的重组慢病毒制备
2.1重组慢病毒SIV的包装和纯化
包装载体、rev表达载体、VSV-G表达载体的构建方法详见专利ZL200680012905.4实施例1中的方法。
将细胞株293T细胞(ATCC,CRL-11268)按照每一个T225培养瓶(Coring，Cat#431082)9×10 6细胞进行接种，在20ml含有10％胎牛血清的D-MEM培养基(Thermofisher，Cat#11995-065)中培养48小时后，将培养基置换为10ml已预热的OPTI-MEM培养基(Thermofisher,Cat#31985-070)，作为待转染细胞备用。
为每一个T225培养瓶分别配置转染体系。首先，将实施例1中获得的穿梭质粒60μg与包装载体30μg、rev表达载体12μg、及VSV-G表达载体12μg共同溶解于2.25ml的HBSS缓冲液中，形成质粒混合物。然后，配制167nM的CaCl 2溶液，将上述2.25ml质粒混合物加入2.25ml的上述CaCl 2溶液中，立即涡旋5s混匀，并在室温条件下孵育10～15min，获得约4.5ml含有质粒DNA-CaCl 2复合物的溶液。随后，将所述溶液加入到上述接种有细胞的培养瓶中，在37℃、5％CO 2培养箱中培养 3小时，然后每瓶补加含20％FBS的DMEM培养基，将FBS终浓度调整为10％，继续在37℃、5％CO 2培养箱中培养过夜。
转染后20小时,将每个T225培养瓶中的培养基置换为20ml新鲜的已预热的DMEM培养基。转染后48小时，回收上清，并用0.45μm的过滤器过滤上清以获得初滤液，随后利用高速离心机进行浓缩操作。具体而言，将初滤液置于高速离心管中，于4℃，40000g条件下离心2小时。将离心管内的上清液完全移除后，向每个离心管内加入DPBS以覆盖沉淀，随后，重悬沉淀，获得重组慢病毒SIV浓缩液。上述病毒浓缩液可直接使用或者分装后于-80℃冻存备用。
2.2重组慢病毒SIV的感染滴度
用2.1中获得的重组慢病毒SIV感染293T细胞，随后通过流式细胞术检测所述293T细胞中表达报告基因或CAR结构胞外区的细胞所占的比例，从而得出重组慢病毒SIV的感染滴度(例如，参见Kunter et al，Nature Protocol，2009)。
具体而言，在感染前1天，将2×10 4/孔的293T细胞接种于24孔板中。感染当天，将各孔中的培养基置换为0.5ml已预热的DMEM培养基(Thermofisher,Cat#11995-065)。对于每种重组慢病毒SIV，各准备3个孔，分别加入0.5、5或50μl的已被DMEM培养基稀释100倍的同种重组慢病毒SIV浓缩液，并放入37℃，5％CO 2培养箱中过夜培养。感染后第2天补液：每孔按照1:1体积补加含20％血清的完全培养基，在37℃，5％CO 2培养箱中继续培养。
感染后第3天，移除孔内培养基，每孔用0.5ml DPBS洗一次，再加入0.1ml/孔TrypLE Express Enzyme(Thermofisher,Cat#12605028)消化细胞，每孔加入1ml DPBS，反复吹吸细胞使其悬浮于DPBS后，转移细胞至流式管中，以300g离心5分钟。弃去上清，利用DPBS重悬细胞。当报告基因为EGFP或RFP时，可直接通过流式细胞仪(BD Celesta)检测荧光蛋白的信号，获得阳性细胞的比例。当报告基因为tEGFR时，则利用抗tEGFR抗体(R&D，FAB9577G-100)进行免疫荧光染色。随后，利用流式细胞仪检测免疫荧光信号，获得阳性细胞的比例。
同时，由于CAR分子中是通过P2A连接包含CD4的CAR分子部分与报告基因，也可依靠针对CD4的检测来确认。利用抗人CD4的抗体(BD,Cat#562424)按照相关说明对上述DPBS重悬的细胞进行免疫荧光染色后，再利用流式细胞仪检测人CD4抗体偶联的荧光信号，获得阳性细胞的比例。
利用公式IU/ml＝(F×N×D×1000)/V来计算重组慢病毒SIV的感染滴度。其中，F代表阳性细胞的比例；N代表感染时每孔细胞的数量；D，代表稀释原液的倍数；V代表每孔实际加入的稀释后病毒的体积。对于同一种重组慢病毒，将3个孔中获得的病毒滴度合并计算以获取平均值，并将所述平均值作为该种重组慢病毒的感染滴度。由携带EGFP的穿梭质粒pGTV-EGFP包装得到 的重组慢病毒SIV-EGFP作为对照。表1所示为利用报告基因检测的结果。
表1重组慢病毒SIV的感染滴度。
质粒名称 重组慢病毒SIV名称 感染滴度(IU/ml)
Z01 SIV-Z01 2.70E+07
Z02 SIV-Z02 3.95E+07
Z03 SIV-Z03 1.25E+07
Z04 SIV-Z04 9.05E+06
Z05 SIV-Z05 3.30E+07
Z06 SIV-Z06 2.43E+07
Z07 SIV-Z07 1.25E+07
Z08 SIV-Z08 3.23E+07
Z09 SIV-Z09 5.41E+07
Z10 SIV-Z10 1.35E+07
Z11 SIV-Z11 8.93E+06
Z12 SIV-Z12 3.70E+07
Z13 SIV-Z13 3.18E+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
Z34 SIV-Z34 3.96E+07
Z35 SIV-Z35 2.54E+07
Z36 SIV-Z36 2.52E+07
Z37 SIV-Z37 1.34E+07
Z38 SIV-Z38 5.72E+07
Z39 SIV-Z39 1.78E+07
Z40 SIV-Z40 4.23E+07
Z41 SIV-Z41 1.15E+07
pGTV-EGFP SIV-EGFP 3.09E+08
实施例3重组蛋白的表达
3.1使用Western blot方法检测目的蛋白的表达
分别将实施例1中的各个质粒直接转染293T细胞，转染48小时后，收集相应的细胞。在细胞中加入裂解液裂解细胞。离心后，获得上清液。使用BCA法定量检测所述上清液中的蛋白浓度。根据所需浓度适当稀释所述上清液后，使用Western blot检测目的蛋白。Western blot检测方法中以抗CD3ζ抗体(Santacruz sc-166275)作为一抗，以辣根过氧化物标记山羊抗小鼠IgG(H+L)(碧云天A0216)作为二抗。
除携带无CD3ζ的Z17结构的质粒及对照pGTV-EGFP以外，转染其他质粒的所有细胞裂解液均可在目标条带位置检测到阳性斑点(结果未显示)。这表明，所有质粒表达框均构建成功。
3.2流式检测细胞表面或内部的蛋白
分别将实施例2中获得的各重组慢病毒SIV按原始感染滴度以相同体积感染293T细胞。48小时后，参考实施例2中的方法检测阳性细胞的比例。即利用抗人CD4的抗体(BD，Cat#562424)对293T细胞免疫荧光染色后，再利用流式细胞仪检测偶联的荧光信号，获得阳性细胞的比例。或者直接利用流式细胞仪(BD Celesta)检测荧光蛋白的信号，获得阳性细胞的比例。
利用抗人CD4的抗体对感染后的293T细胞进行免疫荧光染色并经流式检测，结果如图3灰色柱子所示：Z09-Z13、Z17、Z34-Z37、Z01-Z05、Z38-Z41由于含有CD4胞外区的结构，因此在携 带上述结构的细胞中均可检测到阳性信号。而Z06-07、Z14-Z15由于不含CD4胞外区的结构，因此抗人CD4的抗体染色时均检测不到阳性信号。
针对其表达的报告基因-红色荧光蛋白(RFP)进行检测的结果如图3黑色柱子所示，在这些重组慢病毒SIV感染的293T细胞中均可以检测到报告基因的阳性信号，而且从图3中可发现在携带Z09-Z13、Z16-Z17、Z34-Z37、Z01-Z05、Z38-Z41结构的细胞中，两种颜色所示的阳性细胞比例是一致的。上述结果表明，在给定的检测条件下，能够同时检测到两种阳性信号(CD4及报告基因表达)的细胞为同一群细胞，其比例也相当，因此荧光蛋白的表达可以指示CAR结构在细胞表面的表达。
当选择tEGFR作为报告基因时，利用抗人CD4的抗体和/或抗tEGFR的抗体染色后进行流式检测，以Z09作为示例，在携带上述结构的细胞中亦都可以检测到阳性信号(图4)。其中图4A为两种抗体双染结果，图4B为抗CD4抗体单染结果，图4C为抗EGFR抗体单染结果，图4D为未染色的重组慢病毒SIV-Z09转导的293T细胞，作为对照。CD4+指利用荧光偶联的抗人CD4的抗体对重组慢病毒SIV-Z09转导后的293T进行流式检测并分析获得的阳性细胞比例，EGFR+指荧光偶联的抗人EGFR抗体对重组慢病毒SIV-Z09转导后的293T细胞进行流式检测并分析获得的阳性细胞比例。结果显示，在给定的检测条件下，双染阳性的细胞为同一群细胞。上述结果表明，CD4胞外区及tEGFR都在细胞表面正确表达，因此tEGFR的表达可以指示CAR结构在细胞表面的表达。
实施例4原代细胞改造后的功能验证
4.1原代细胞制备
4.1.1获得PBMC细胞：
利用Ficoll-Paque PLUS(GE，Cat#17-1440-02)，按照试剂盒说明书分离EDTA抗凝全血(健康人志愿献血)中的PBMC细胞。具体地，利用高速冷冻离心机(Thermo Scientific Sorvall ST40R)，使用水平转子以800g离心30min，设置离心升速为3，降速为0，温度为20℃。离心结束后，轻轻地吸出PBMC细胞层，并置于新的离心管中。使用所吸出细胞层的3-5倍体积的DPBS清洗PBMC，随后，在20℃，300g条件下，离心10min。离心结束后移除上清，向细胞沉淀中加入预冷的红细胞裂解液(天津灏洋NH4CL2009)，4℃放置2分钟以裂解红细胞。随后，加入预冷的DPBS，使总体积为40-45ml，上下吹打使细胞混匀，在300g，4℃条件下离心10min，从而获得PBMC细胞。
4.1.2分选CD3+T细胞：
根据厂家说明书，利用Dynabeads untouched Human T cells试剂盒(Thermofisher 11344D) 纯化CD3+T细胞。具体地，将上一步获得的PBMC细胞重悬于终体积为300μl的4℃预冷的分离缓冲液(含0.1％BSA及2mM EDTA的DPBS缓冲液)中，吹打数次混匀，冰上静置，取10μl用于活细胞计数。根据活细胞数，利用4℃预冷的分离缓冲液，将PBMC细胞调整至1×10 8个/ml，参照Dynabeads试剂盒说明书，计算并加入所需试剂相应的体积。
以5×10 7个PBMC细胞为例：首先取1支15ml的离心管，先加入0.5ml上述浓度的PBMC细胞，加入100μl预冷的FBS，混匀，再加入100μl预冷的Antibody Mix，吹打数次混匀，4℃避光孵育20min。随后，加入4ml预冷的分离缓冲液，吹打数次混匀，4℃条件下350g离心8min，弃上清。加入500μl分离缓冲液，重悬细胞，并加入预先清洗好的500μl Dynabeads，在Hula混合仪上(18-25℃，角度89，转速10，振动2-3)孵育15min。孵育结束后，加入5ml分离缓冲液，轻柔吹打5次，充分混匀。将装有该混合物的15ml的离心管置于磁力架上，静置2min后，将上清吸至1个新的15ml离心管中。
在原来装有Dynabeads的15ml离心管中，再次加入5ml的分离缓冲液，重复上面操作，充分混匀后，将该15ml离心管置于磁力架上，静置2min后，将上清吸出，并与上一步获得的上清在室温条件下合并混匀，获得CD3+T细胞，计数。将CD3+T细胞离心，重悬于X-vivo 15培养基(Lonza BE02-060F)中。
4.1.3活化T细胞：
利用包被了抗CD3抗体和抗CD28抗体的磁珠来激活T细胞。按照试剂盒Dynabeads Human T-Activator CD3/CD28 for T-Cell Expansion and Activation(Thermofisher，11131D)的说明书来操作。首先根据获得的CD3+T细胞的计数结果，将细胞和已清洗好的CD3/CD28磁珠按照1×10 6个细胞对应25ul原始浓度的磁珠进行混合，并补充IL-2(R&D 202-IL-010)、IL-7(R&D 207-IL-025)和IL-15(R&D 247-ILB-025)，终浓度为3-5％的人血清白蛋白或人AB血清或CD3+T细胞对应的自体血清，充分混匀后(记为活化后第0天)，37℃培养48小时。
将细胞与磁珠混合物取至15ml离心管中，2000rpm振荡30s后，放置于磁力架上1分钟。吸取上清至新的离心管中，利用X-vivo 15培养基重悬细胞，300g离心5分钟，离心后弃上清，用新鲜X-vivo15培养基重悬细胞沉淀，并取部分进行计数及流式检测(活化后第2天)。
4.1.4确定细胞比例：
利用免疫荧光染色和流式细胞检测来确定CD3+、CD3+CD4+双阳性、CD3+CD8+双阳性、CD3+CD25+双阳性的细胞亚群和比例。具体而言，根据计数结果，取1×10 6个细胞，300g室温离心5分钟，重悬至DPBS，并加入多色染色缓冲液(BD Horizon Brilliant Stain Buffer Plus，566385)，及偶联PE-CY7的小鼠抗人CD3抗体(BD，557851)、偶联BV421的小鼠抗人CD4抗体(BD，562424) 和偶联PE的小鼠抗人CD8抗体(BD，555367)的组合，或者偶联PE-CY7的小鼠抗人CD3抗体偶联(BD，557851)和偶联BV421的小鼠抗人CD25抗体(BD，562442)的组合。上机流式检测，并统计CD3+、CD3+CD4+双阳性、CD3+CD8+双阳性、CD3+CD25+双阳性的细胞亚群等比例。活化细胞的比例指CD3+CD25+细胞在CD3+T细胞中的比例，以未经磁珠及细胞因子激活的CD3+T细胞作为对照。
根据检测和统计分析结果，在活的CD3+T细胞中活化细胞的比例达到91.4％以上(图5)。
4.2病毒转导及细胞维持
将上述已验证活率及活化的CD3+T细胞接种于用RetroNectin(Takara T100B，5μg/cm 2)包被的培养板或培养瓶中。用实施例2中获得的系列重组慢病毒SIV(SIV-Z01、SIV-Z04、SIV09-SIV17、SIV34-SIV37)分别来感染所述CD3+T细胞(感染复数MOI不超过5)。感染48或72小时后，离心，去除原培养液。利用新鲜X-vivo 15培养基(Lonza BE02-060F)重悬细胞，调整细胞密度为1×10 6/ml，并补充IL-2(R&D 202-IL-010)、IL-7(R&D 207-IL-025)和IL-15(R&D 247-ILB-025)，终浓度为3-5％的人血清白蛋白或人AB血清或CD3+T细胞对应的自体血清，根据细胞密度及生长时间等情况调整细胞因子浓度，通常情况下所述IL-2的终浓度为30IU/mL、IL-7的终浓度为5或者10ng/mL、IL-15的终浓度为5或者10ng/mL。隔天计数传代，利用新鲜X-vivo 15培养基(含IL-2、IL-7及IL-15，终浓度如上所述)将细胞密度调整为1×10 6/ml。之后至活化后12天的期间，检测记录细胞的密度及活率，计算细胞总数。用重组慢病毒SIV-EGFP转导的CD3+T细胞(EGFP)，以及未转导重组慢病毒的CD3+T细胞(CD3)作为对照。
结果表明：在目前的培养条件下，在活化后第12天，细胞的数量为起始数量的140-440倍，其活率均大于80％(图6A-6Q)。
4.3转导效率及细胞亚群的鉴定
活化后第13和19天，利用与4.1.4部分相同的抗体组合，对转导后的细胞(如本文所用，“改造细胞”、“转导后的细胞”与“改造T细胞”可互换使用，均用于指代经重组慢病毒SIV转导成功表达CAR结构的CD3+T细胞或其混合物)进行染色，并使用流式细胞仪检测CD3、CD4、CD8、以及CD25的表达，同时也针对与CAR同时表达的报告基因进行了检测。
图7所示为在活化后两个时间点13天(图7A)和19天(图7B)时CAR阳性细胞在CD3+细胞中的比例。该结果表明，随着时间推移，CAR阳性细胞的比例可保持相对稳定。
图8所示为未经重组慢病毒SIV转导的情况下，CD3+细胞在总细胞中的比例(图8A)及CD3+细胞中各细胞亚群的比例(图8B)。图8A为在连续培养19天3个不同时间点T细胞的比例，T细胞 比例是依赖CD3+T阳性细胞来计算的，图8B为在连续培养19天3个不同时间点T细胞各亚群的比例，CD4+T细胞比例是下降的，CD8+细胞的比例是不断上升的。这与文献中报道的结果一致，在一个有利于CD8+T细胞生长的条件下，CD8+T细胞的数量是不断增加的。
图9所示为分别用两种携带不同CAR结构的重组慢病毒(SIV-Z09和SIV-Z14)转导CD3+T细胞后，在不同时间点时各细胞亚群的比例。由图可见，随着时间推移，重组慢病毒SIV-Z09转导的CD3+细胞中CD4+T细胞的比例(图9A)高于重组慢病毒SIV-Z14转导的CD3+细胞中CD4+T细胞的比例(图9B)，也高于未进行任何转导的CD3+细胞中的比例(图8B)。上述结果进一步证实了导入的CAR分子在CD3+细胞膜表面正确表达。
4.4改造T细胞与靶细胞混合后的作用
构建不同HIV毒株(AC10.29、NL4-3或SF162)gp120蛋白或人的MHCII(DR0401)分别与AcGFP共表达的重组质粒，将该系列质粒分别转染293F细胞，并通过G418筛选获得，其中，质粒中绿色荧光蛋白AcGFP通过P2A与gp120或MHCII共表达。以转染AcGFP空载体的293F细胞作为阴性对照。转染共表达MHCII(DR0401)和绿色荧光蛋白AcGFP的293F细胞作为与CD4结合的功能对照，由于CD4与MHCII可天然结合，通过该功能对照可评估本申请实施例中的改造T细胞对表达MHCII的细胞的影响。
本实验将一系列改造T细胞与上述293F靶细胞混合，通过检测一系列的指标来确定改造T细胞的作用。具体而言，将靶细胞接种于24孔板中，数量为1×10 4个/孔。在实验前24小时停止对改造T细胞进行细胞因子的刺激。在T细胞活化后第14天，将改造T细胞以给定的效靶比(即效应细胞和靶细胞数量的比值，10：1，3：1或1：1)分别加入到不同的孔中，与靶细胞进行混合，液体终体积为200μl/孔。
混合后4小时，收集部分上清，用CytoTox 96非放射性细胞毒性检测试剂盒(Promega G1780)检测乳酸脱氢酶LDH的释放，以确定每个实验组改造T细胞中CD8+T淋巴细胞对靶细胞的急性杀伤活性。
混合后24小时，收集上清，利用ELISA试剂盒(Biolegend 430204、431804及430104)分别检测细胞因子TNF-α、IL2或者IFN-γ的释放。此外，利用R&D的Luminex Performance Human XL Cytokine Discovery Magnetic Panel(R&D FSCTM18-12)在luminex200仪器上检测分析，以同时检测多因子的表达水平。
在混合后24小时也同时收集细胞，参照4.3部分的方案，通过抗体染色和流式检测，分别计算CD3-的细胞(靶细胞为主)中表达gp120或者MHCII的细胞的比例，确定对靶细胞的抑制活性。
每个实验组改造T细胞中CD8+T淋巴细胞对靶细胞的急性杀伤活性结果显示，效靶细胞混合4小时后，改造T细胞(以携带Z09或Z10结构的改造T细胞为例)对表达不同HIV毒株(AC10.29、NL4-3、SF162)gp120蛋白的靶细胞具有特异性杀伤作用，且具浓度梯度依赖关系(图12A-12C)。
对于细胞因子的释放，以IFN-γ为例，结果显示特异性刺激可以让改造T细胞释放与杀伤相关的细胞因子(图11A-图11D)。多因子检测的结果(图14A-14J及图14L)进一步证实了当本申请的改造T细胞与表达gp120的靶细胞共培养时，改造T细胞可被特异性激活并大量分泌细胞毒性相关的细胞因子，进而可强有力地介导靶细胞的裂解，并且对于例如携带Z09或Z10结构的改造T细胞，没有检测到与细胞因子风暴显著相关的IL-6(图14K)等因子的明显释放，表明将来体内发生细胞因子风暴等副作用的可能性较小。
改造T细胞对靶细胞抑制活性的结果显示：利用CD4作为CAR部分的改造T细胞在效靶比为3：1时可以特异性的抑制表达gp120的靶细胞(图10A-10C)，而对于转染AcGFP空载体的靶细胞(即不表达gp120的细胞)没有明显影响(图10D)。图13A-图13D为改造T细胞在不同的效靶比下(10：1，3：1和1：1)对靶细胞的抑制活性的结果，且该抑制作用具有浓度梯度依赖关系，该结果进一步证实表达gp120的靶细胞可被使用CD4胞外区特异性识别抗原的CAR-T细胞特异性抑制，而不含胞内信号转导结构域CD3ζ(即Z17，无完整CAR结构)的改造T细胞对靶细胞没有抑制作用。相较而言，携带Z14或Z34结构的改造T细胞杀伤作用弱于携带Z09或Z10结构的改造T细胞；携带Z12、Z13结构的改造T细胞杀伤作用同样弱于携带Z09结构的改造T细胞，携带Z11或Z15结构的改造T细胞杀伤作用弱于携带Z10结构的改造T细胞；携带Z04结构的改造T细胞杀伤作用弱于携带Z01结构的改造T细胞。值得注意的是，在效靶比相同的情况下，携带Z09结构的改造T细胞相较于携带Z14结构的改造T细胞对表达gp120的靶细胞具有更高的细胞抑制作用(图13A-C)，由于Z14与Z09结构的差异是Z14结构中胞外区包含的抗原特异性识别结构域为3BNC117的scFv，而Z09的抗原特异性识别结构域为CD4的D1-D2区段，提示利用CD4的D1-D2区段识别抗原的CAR-T细胞相较于利用3BNC117的scFv识别抗原的CAR-T细胞对表达gp120的靶细胞抑制作用更强。
此外，从上述结果中可以看出，本申请的改造T细胞均不会明显抑制或杀伤表达MHCII(DR0401)的靶细胞(图12D，13D)。即虽然CD4可与MHCII可天然结合，但体内原有的表达MHCII的细胞并不会被表达CD4的改造T细胞大规模杀伤，推测其原因可能是CD4对几种典型的HIV-1gp120的亲和力都强于其对MHCII的亲和力。
以上结果表明了本申请技术方案中CAR结构的特异性作用，并且预期其将来在体内应用时具有较低的细胞因子风暴风险，也不会对体内原有的表达MHCII的细胞造成杀伤或抑制。
4.5体外模拟改造T细胞长效抑制的效果
采用如前所述的方法对T细胞进行活化、改造和培养，在活化后第13天时停止细胞因子24小时，计数并调整细胞密度。随后继续使用细胞因子培养至第18天，停止细胞因子刺激24小时，采用4.1.4的方法通过对T细胞表面CD25表达水平的测定(第19天)，确认经过该阶段培养，改造T细胞已经恢复至非活化状态(图15)。
在活化后第20天时，利用与4.4部分相同的方法，将改造T细胞与靶细胞混合，以检测及评估改造T细胞对靶细胞的抑制作用。改造T细胞与靶细胞混合后24小时(即混合后第1天)，收集部分细胞，通过抗体免疫染色和细胞流式分析CD3-的(靶细胞为主)细胞中表达不同HIV毒株(AC10.29、NL4-3、SF162)gp120蛋白的细胞或者表达AcGFP空载体的对照细胞的比例，以及改造T细胞在CD3+T细胞中的比例，确定改造T细胞对靶细胞的抑制作用和细胞因子的释放水平。
改造T细胞对靶细胞的抑制结果显示：将CD4或其片段作为CAR的抗原特异性识别结构域的改造T细胞在效靶比为3：1下可以特异性地抑制表达gp120的靶细胞(图16A-16C)，导入AcGFP空载体的靶细胞不受明显影响(图16D)。图18A-图18D为改造T细胞在不同的效靶比下(10：1，3：1和1：1)对靶细胞的抑制作用，其结果显示改造T细胞(携带Z09或Z10结构)对于表达HIV病毒gp120的靶细胞的抑制具有浓度梯度依赖关系(图18A-18C)，而对于表达MHCII的靶细胞即使在最高效靶比10：1时都没有明显的抑制效应(图18D)。
以IFN-γ为例的细胞因子释放的ELISA测定结果表明来自抗原的特异性的刺激可以让改造T细胞释放与杀伤相关的细胞因子(图17)。多因子检测的结果进一步证实了与杀伤相关因子的释放(图19A-19J以及19L)，并且对于携带Z09或Z10结构的改造T细胞，没有检测到与细胞因子风暴相关的IL-6(图19K)等因子明显释放。
上述结果证明了本申请的CAR结构的长期效果，并且预期其将来在体内长期应用时具有较低的细胞因子风暴风险，也不会杀伤或抑制体内原有的表达MHC-II的细胞。
将上述的混合细胞继续培养9天后(即混合后第9天)，检测携带对gp120具有特异性作用的CAR结构的T细胞的比例(即所述T细胞占CD3+T细胞的百分比)和其对靶细胞的抑制作用。
结果显示，与靶细胞混合9天后(效靶比为3：1)，改造T细胞在CD3+T细胞中的比例维持在较高水平(图20A-C)，与混合第1天相比则可以继续维持甚至有所升高(结果未显示)。同时，与靶细胞混合9天后，改造T细胞仍显现出对表达不同HIV病毒gp120的靶细胞的持续抑制(图21A-C)，对只导入AcGFP空载体的靶细胞则没有明显的抑制作用(图21D)。
进一步地，对于携带CAR结构(例如Z09或Z10结构)的改造T细胞，不论效靶比(10：1，3：1或1：1)如何，在与靶细胞混合9天后均显示了自身比例的维持甚至升高(图22)，以及对表达不同HIV病毒gp120的靶细胞的持续抑制(图23A-C)，而最高至10：1的效靶比都没有对表达 MHCII的靶细胞产生明显的抑制作用(图23D)。
上述结果均进一步证实了基于本申请CAR结构的改造T细胞的体内长效抑制被感染细胞的潜能，同时对原有CD4+T细胞的功能不会产生明显干扰。
实施例5用患者细胞检测改造T细胞的活性
为了进一步证明携带本申请结构的改造T细胞对治疗HIV病毒感染的有效性，在添加蛋白酶抑制剂的条件下制备来源于HIV患者的改造T细胞。具体而言，从HIV患者血液中分离T细胞，在之后的12天内，在培养基内添加终浓度为10uM的蛋白酶抑制剂Darunavir ethanolate(SelleckChem s1620)或Saquinavir mesylate(Sigma s8451)进行培养，其余培养条件如4.2部分所述。12天之后停止添加蛋白酶抑制剂并继续培养。分离、并培养活化患者T细胞的当日记为第0天，在第2天时按照前述的方法改造患者来源的CD3+T细胞。从第2天开始至第24天，在不同时间点收集培养上清，利用Lenti-p24 rapid titer kit(Takara 632200)，根据其说明书检测上清中p24浓度，以间接定量HIV病毒颗粒，评估用完整结构(例如Z09、Z10、Z34、Z38-Z41等)改造的患者T细胞对患者感染HIV的CD4+T细胞的杀伤及抑制作用。同时，在第24天时通过检测报告基因，测定改造T细胞自身的比例，以评估导入的shRNA对改造T细胞的保护作用。
从图24A的结果看，同时携带shRNA及CAR结构(Z09、Z10、Z12、Z16、Z34、Z38-Z41)的改造T细胞在整个制备过程及后续未使用蛋白酶抑制剂的培养期间，在上清液中一直检测不到病毒p24或病毒p24浓度在检测阈值附近，并且携带Z10、Z12、Z34、Z38-Z41结构的改造T细胞对p24的抑制效果优于携带Z16结构的改造T细胞，说明本申请中的载体的改造T细胞对HIV的杀伤与抑制效果优于Z16的改造T细胞，且发挥效力时间较长。
仅携带CAR结构而未携带shRNA的改造T细胞(Z02或Z08)在后续不使用蛋白酶抑制剂培养期间，其培养上清中病毒p24浓度从活化后16天开始上升，随后又下降；对于只携带有G3L2shRNA结构的改造T细胞(CTRL)及携带CAR部分不完整的Z17结构的改造T细胞，其培养上清中病毒p24浓度先上升，随后又下降(图24A)。基于这些结构的改造T细胞(CTRL及Z17)，其培养上清中p24浓度的峰值出现在细胞活化后18至22天，且峰值浓度低于同时间点未经改造的CD3+T细胞(即附图中的CD3)的对应水平，而未改造的CD3+T细胞组中的病毒p24浓度从活化后16天开始即可以检测到并在随后的几天内持续上升(图24A)。在实施例4已经验证了来源于健康人的改造T细胞对表达HIV病毒gp120蛋白的靶细胞的抑制和杀伤作用的基础上，上述结果进一步证明来自HIV患者的改造T细胞对自体被感染细胞具有杀伤和抑制作用。
图24B的结果显示，与未经改造的CD3+细胞对照(CD3)相比，在与HIV患者的T细胞培养 24天之后，本申请的改造T细胞自身的比例仍维持在较高水平，表明导入的shRNA对于被改造T细胞具有保护作用。
综合上述两部分结果，证实携带本申请CAR结构的改造T细胞能够持久和特异地杀伤和抑制HIV病毒感染的细胞，同时本申请中的shRNA具有保护和维持改造T细胞的优势。尤其，当与携带Z16结构的改造T细胞相比时，本申请的CAR-T对HIV病毒感染的细胞的特异性杀伤作用更强，且可以在HIV感染的情况下维持更长时间。
通过以上实施例可以发现，综合比较改造T细胞在活化及长期作用情况下的杀伤能力、其本身对HIV病毒侵染的抵抗能力、以及其引起细胞因子风暴的风险性，本发明中的携带Z09、Z10、Z34、Z38-Z41结构的改造T细胞具有相对更优的综合效力，具有较为显著的优势。向效应T细胞中同时引入可针对性抑制HIV基因组保守序列的shRNA及靶向HIV病毒蛋白的CAR结构，可以构建出更加持久高效的，靶向杀伤HIV感染细胞的CAR-T细胞。而对比这样的CAR-T细胞，当所述shRNA相同时，选择CD4的D1及D2结构域作为CAR的抗原特异性识别结构域相对于现有技术中其他抗原特异性识别结构域或CD4的D1-D4结构域效果更优。而在CAR的抗原特异性识别结构域一致的情况下，选择针对G3L2(分别针对gag和LTR)或G2N(分别针对gag和nef)的shRNA，相较于现有技术中其他shRNA选项，可使CAR-T细胞具有更优的综合效力。
序列列表
表2：以下为本文提及的所有序列。
Figure PCTCN2022143440-appb-000003
Figure PCTCN2022143440-appb-000004
Figure PCTCN2022143440-appb-000005
Figure PCTCN2022143440-appb-000006
Figure PCTCN2022143440-appb-000007
Figure PCTCN2022143440-appb-000008
Figure PCTCN2022143440-appb-000009

Claims (67)

  1. 一种重组细胞，其包含下述功能结构：
    1)嵌合抗原受体(CAR)或其编码序列，所述CAR包含胞外区、跨膜区及胞内区，其中所述胞外区可特异性结合HIV的gp120蛋白；和
    2)至少一种shRNA或其编码序列，所述shRNA靶向抑制选自下组的任一种或多种参与HIV生命周期的宿主或HIV基因：NF-κB、CCR5、TSG101、CXCR4、P-TEFb、tat、rev、nef、env、LTR和gag。
  2. 权利要求1所述的重组细胞，其中所述shRNA包括：
    (i)靶向gag的shRNA和靶向LTR的shRNA，或者
    (ii)靶向gag的shRNA和靶向nef的shRNA。
  3. 权利要求1或2所述的重组细胞，其中所述靶向gag的shRNA的序列包含如SEQ ID NO：1或5中所示的序列或包含与SEQ ID NO：1或5所示序列具有至少85％同一性的序列；所述靶向LTR的shRNA的序列包含如SEQ ID NO：2所示序列或包含与SEQ ID NO：2所示序列具有至少85％同一性的序列；所述靶向nef的shRNA包含SEQ ID NO：6所示序列或包含与SEQ ID NO：6所示序列具有至少85％同一性的序列。
  4. 权利要求3所述的重组细胞，其中所述shRNA包括：
    (i)靶向gag的shRNA和靶向LTR的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：1所示序列，靶向LTR的shRNA包含如SEQ ID NO：2所示序列；
    (ii)靶向gag的shRNA和靶向nef的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：5所示序列，靶向nef的shRNA包含如SEQ ID NO：6所示序列。
  5. 如权利要求1所述的重组细胞，其中所述shRNA的编码序列包括：
    (i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，或者
    (ii)靶向gag的shRNA的编码序列和靶向nef的shRNA的编码序列。
  6. 如权利要求1或5中所述的重组细胞，其中所述靶向gag的shRNA的编码序列包含如SEQ ID NO：3或7所示的序列，或包含与SEQ ID NO：3或7所示序列具有至少85％同一性的序列；所述靶向LTR 的shRNA的编码序列包含如SEQ ID NO：4所示序列或包含与SEQ ID NO：4所示序列具有至少85％同一性的序列；所述靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示序列或包含与SEQ ID NO：8所示序列具有至少85％同一性的序列。
  7. 如权利要求6中所述的重组细胞，其中shRNA的编码序列包括：
    (i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：3所示序列，靶向LTR的shRNA的编码序列包含如SEQ ID NO：4所示序列；或者
    (ii)靶向gag的shRNA的编码序列和靶向nef的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：7所示序列，靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示序列。
  8. 权利要求1-7中任一项的重组细胞，其中所述CAR还包括铰链区，胞外区与所述跨膜区之间由铰链区相连。
  9. 权利要求8的重组细胞，其中所述铰链区来自或包含人Ig铰链区、GS接头、KIR2DS2铰链或CD8α的铰链区。
  10. 权利要求9的重组细胞，其中所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与该序列有至少85％同一性的氨基酸序列。
  11. 权利要求1-10中任一项的重组细胞，其中所述胞外区包含人CD4分子的胞外区，优选CD4分子D1-D4结构域，最优选CD4分子D1和D2结构域。
  12. 权利要求11的重组细胞，其中所述胞外区包含如SEQ ID NO：13所示的氨基酸序列或包含与该氨基酸序列具有至少85％以上同一性的氨基酸序列。
  13. 权利要求1-12中任一项的重组细胞，其中所述跨膜区来自或包含选自以下一种或多种蛋白分子的跨膜结构域：T细胞受体的α、β或ζ链、CD28、CD3ε、CD45、CD4、CD5、CD8、CD9、CD16、CD22、CD33、CD37、CD64、CD80、CD86、CD134、CD154、KIRDS2、OX40、CD2、CD27、LFA-1(CD11a、CD18)、ICOS(CD278)、4-1BB(CD137)、GITR、CD40、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、CD160、CD19、IL2Rβ、IL2Rγ、IL7Rα、ITGA1、VLA1、CD49a、ITGA4、IA4、CD49D4、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、PAG/Cbp、NKp44、 NKp30、NKp46、NKG2D、NKG2C。
  14. 权利要求13的重组细胞，其中所述跨膜区来自或包含CD8α蛋白分子的跨膜结构域。
  15. 权利要求14中的重组细胞，所述跨膜区包含如SEQ ID NO：18所示的氨基酸序列或包含与其具有至少85％同一性的氨基酸序列。
  16. 权利要求1-15中任一项所述的重组细胞，其中所述胞内区包含一级信号转导结构域和共刺激结构域，所述一级信号转导结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12，且所述共刺激结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。
  17. 权利要求16的重组细胞，其中所述一级信号转导结构域来自或包含CD3ζ的信号转导结构域，且所述共刺激结构域来自或包含CD137、CD28、2B4或DAP10的信号转导结构域之一或其组合。
  18. 如权利要求1-17中任一项所述的重组细胞，其中胞内区包含：
    (i)CD137和CD3ζ的信号转导结构域；
    (ii)CD28、CD137和CD3ζ的信号转导结构域；
    (iii)CD137、CD3ζ和DAP10的信号转导结构域；
    (iv)CD28、DAP10和CD3ζ的信号转导结构域；
    (v)2B4、DAP10和CD3ζ的信号转导结构域；或，
    (vi)CD137、2B4和CD3ζ的信号转导结构域。
  19. 权利要求16-18中任一项的重组细胞，其中在所述胞内区中一级信号转导结构域和共刺激结构域之间还包含连接肽，优选的，所述连接肽包含SEQ ID NO：22所示氨基酸序列。
  20. 权利要求1-19中任一项的重组细胞，其中所述胞内区包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与其中任一序列有至少85％同一性的氨基酸序列。
  21. 权利要求1-20中任一项的重组细胞，其中所述CAR包含选自以下的氨基酸序列：SEQ ID NO：35-46中任一项或包含与他们中任一序列具有至少85％同一性的氨基酸序列。
  22. 权利要求1-21中任一项的重组细胞，其中所述CAR进一步连接信号肽，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽。
  23. 权利要求22的重组细胞，其中所述信号肽来自或包含CD4或CD8的信号肽，优选的，所述信号肽包含SEQ ID NO：16或17所示氨基酸序列。
  24. 如权利要求1-23中任一项所述的重组细胞，其中所述重组细胞还包含报告分子和/或安全开关。
  25. 如权利要求24中所述的重组细胞，其中所述安全开关选自下述中的一种或多种：iCaspase-9、iCaspase-1、iCaspase-8、HSV-TK、VZV-TK、胞嘧啶脱氨酶(CD)、CD20、tEGFR、FR806和RQP8。
  26. 如权利要求25中所述的重组细胞，其中所述tEGFR的氨基酸序列如SEQ ID NO：49或50所示。
  27. 如权利要求1-26中任一项所述的重组细胞，所述重组细胞来源于HIV受体细胞、外周血单核细胞或淋巴细胞；优选的，所述重组细胞来源于T细胞(例如，幼稚T细胞、记忆性T细胞、效应性T细胞、细胞毒T细胞、辅助性T细胞、调节性T细胞、CD4+T细胞、CD8+T细胞、NKT细胞、γδT细胞等)、NK细胞、抗原提呈细胞(例如，巨噬细胞、树突状细胞等)；或者所述重组细胞来源于祖细胞或干细胞，包括但不限于造血干细胞、造血祖细胞、记忆性T干细胞(例如中枢记忆T细胞、效应记忆T细胞或干细胞样记忆T细胞)。
  28. 如权利要求27所述的重组细胞，其中所述重组细胞源自人或灵长类动物；优选的，所述细胞源自感染HIV的患者，或者源自健康人群。
  29. 一种载体，包含编码如权利要求1-28中任一项的重组细胞中的CAR、shRNA、报告分子和/或安全开关的多核苷酸序列。
  30. 权利要求29的载体，其选自质粒、病毒载体或线性核酸分子。
  31. 权利要求29或30的载体，其中所述嵌合抗原受体(CAR)和shRNA在相同启动子或不同启动子的驱动下表达，所述至少一种shRNA在相同或不同的启动子的驱动下表达。
  32. 一种嵌合抗原受体(CAR)，其包含胞外区、跨膜区及胞内区，其中所述胞外区来自或包含人CD4分子的胞外区，跨膜区来自或包含CD8α的跨膜结构域。
  33. 如权利要求32所述的嵌合抗原受体，其中所述嵌合抗原受体还包括铰链区，所述胞外区与所述跨膜区之间由铰链区相连。
  34. 如权利要求33中所述的嵌合抗原受体，所述铰链区来自或包含人Ig铰链区、GS接头、KIR2DS2铰链或CD8α的铰链区，优选CD8α的铰链区。
  35. 如权利要求34中所述的嵌合抗原受体，所述铰链区包含如SEQ ID NO：20中所示的氨基酸序列或包含与该序列有至少85％同一性的氨基酸序列。
  36. 如权利要求32-35中任一项所述的嵌合抗原受体，其中胞外区由CD4分子的D1和D2结构域组成。
  37. 如权利要求36中所述的嵌合抗原受体，其中胞外区包含如SEQ ID NO：13所示的氨基酸序列。
  38. 如权利要求32-37中任一项所述的嵌合抗原受体，其中所述跨膜区包含如SEQ ID NO：18所示的氨基酸序列。
  39. 如权利要求32-38中任一项所述的嵌合抗原受体，其中所述胞内区包括一级信号转导结构域和共刺激结构域，所述一级信号转导结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD3ζ、CD3γ、CD3δ、CD3ε、FCER1G、FcεR1b、CD79a、CD79b、FcγRIIa、DAP10和DAP12，且所述共刺激结构域来自或包含选自下述的一种或多种蛋白分子的信号转导结构域：CD27、CD28、4-1BB、OX40、CD30、CD40、PD-1、ICOS、LFA-1、CD2、CD7、LIGHT、NKG2C、B7-H3、特异性结合CD83的配体、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、DAP10。
  40. 如权利要求39中所述的嵌合抗原受体，其中所述一级信号转导结构域来自或包含CD3ζ或DAP10的信号转导结构域，且所述共刺激结构域来自或包含CD137、CD28、2B4或DAP10的信号转导结构域之一或其组合。
  41. 如权利要求32-40中任一项所述的嵌合抗原受体，其中胞内区包含：
    (i)CD137和CD3ζ的信号转导结构域；
    (ii)CD28、CD137和CD3ζ的信号转导结构域；
    (iii)CD137、CD3ζ和DAP10的信号转导结构域；
    (iv)CD28、DAP10和CD3ζ的信号转导结构域；
    (v)2B4、DAP10和CD3ζ的信号转导结构域；或，
    (vi)CD137、2B4和CD3ζ的信号转导结构域。
  42. 如权利要求39-41中任一项所述的嵌合抗原受体，其中所述胞内区中的一级信号转导结构域和共刺激结构域之间，或者各共刺激结构域之间还包含连接肽。
  43. 如权利要求42中所述的嵌合抗原受体，其中所述连接肽的序列包含如SEQ ID NO：22所示的氨基酸序列。
  44. 如权利要求32-43中任一项所述的嵌合抗原受体，其中胞内区的序列包含如SEQ ID NO：28-34中任一项所示的氨基酸序列或包含与其中任一序列有至少85％同一性的氨基酸序列。
  45. 如权利要求32-44中任一项所述的嵌合抗原受体，其中所述嵌合抗原受体进一步连接信号肽，所述信号肽来自或包含任意分泌蛋白或膜蛋白的信号肽。
  46. 如权利要求45中所述的嵌合抗原受体，其中所述信号肽来自或包含CD4或CD8分子的信号肽，优选的，所述信号肽包含SEQ ID NO：16或17所示氨基酸序列。
  47. 如权利要求32-46中任一项所述的嵌合抗原受体，其中所嵌合抗原受体包含如SEQ ID NO：35、36、43-46中任一项所示的氨基酸序列，或者包含与它们中任一序列具有至少85％同一性的氨基酸序列。
  48. 一种多核苷酸序列，其编码如权利要求32-47中任一项所述的嵌合抗原受体。
  49. 一种载体，其包含如权利要求48中所述的多核苷酸序列。
  50. 如权利要求49所述的载体，其选自质粒、病毒载体或线性核酸分子。
  51. 一种重组细胞，其包含如权利要求32-47中任一项所述的嵌合抗原受体，或如权利要求48中所述的多核苷酸序列，或权利要求49或50中所述的载体。
  52. 如权利要求51所述的重组细胞，所述重组细胞来源于人或灵长类动物；进一步优选的，所述细胞源自感染HIV的患者，或者源自健康人群的；进一步优选的，重组细胞来源于HIV受体细胞、外周血单核细胞或淋巴细胞；进一步优选的，所述重组细胞来源于T细胞(例如，幼稚T细胞、记 忆性T细胞、效应性T细胞、细胞毒T细胞、辅助性T细胞、调节性T细胞、CD4+T细胞、CD8+T细胞、NKT细胞、γδT细胞等)、NK细胞、抗原提呈细胞(例如，巨噬细胞、树突状细胞等)；或者所述重组细胞来源于祖细胞或干细胞，包括但不限于造血干细胞、造血祖细胞、记忆性T干细胞(例如中枢记忆T细胞、效应记忆T细胞或干细胞样记忆T细胞)。
  53. 如权利要求32-47中所述的嵌合抗原受体，或如权利要求48中所述的多核苷酸序列，权利要求49或50中所述的载体，或权利要求51或52所述的重组细胞与至少一种靶向HIV的shRNA或其编码序列或包含其编码序列的载体的组合。
  54. 如权利要求53中所述的组合，其中所述至少一种shRNA靶向抑制选自下组的任一种或多种参与HIV生命周期的宿主或HIV基因：NF-κB、CCR5、TSG101、CXCR4、P-TEFb、tat、rev、nef、env、LTR和gag。
  55. 如权利要求54中所述的组合，其中所述组合包括：
    (i)靶向gag的shRNA和靶向LTR的shRNA，或者
    (ii)靶向gag的shRNA和靶向nef的shRNA。
  56. 如权利要求54或55中所述的组合，其中靶向gag的shRNA包含如SEQ ID NO：1或5所示序列或包含与SEQ ID NO：1或5所示序列具有至少85％同一性的序列，靶向LTR的shRNA包含如SEQ ID NO：2所示序列或包含与SEQ ID NO：2所示序列具有至少85％同一性的序列，靶向nef的shRNA包含如SEQ ID NO：6所示序列或包含与SEQ ID NO：6所示序列具有至少85％同一性的序列。
  57. 如权利要求56中所述的组合，其中所述组合包括：
    (i)靶向gag的shRNA和靶向LTR的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：1所示序列，靶向LTR的shRNA包含如SEQ ID NO：2所示序列；
    (ii)靶向gag的shRNA和靶向nef的shRNA，其中靶向gag的shRNA包含如SEQ ID NO：5所示序列，靶向nef的shRNA包含如SEQ ID NO：6所示序列。
  58. 如权利要求54中所述的组合，其中所述组合包括：
    (i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，或包含上述编码序列的载体，或者
    (ii)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，或包含上述编码序列的载体。
  59. 如权利要求54或58中所述的组合，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：3或 7所示序列或包含与SEQ ID NO：3或7所示序列具有至少85％同一性的序列，靶向LTR的shRNA的编码序列包含如SEQ ID NO：4所示序列或包含与SEQ ID NO：4所示序列具有至少85％同一性的序列，靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示序列或包含与SEQ ID NO：8所示序列具有至少85％同一性的序列。
  60. 如权利要求59中所述的组合，其中所述组合包括：
    (i)靶向gag的shRNA的编码序列和靶向LTR的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：3所示序列，靶向LTR的shRNA的编码序列包含如SEQ ID NO：4所示序列，或包含上述编码序列的载体；或者
    (ii)靶向gag的shRNA的编码序列和靶向nef的shRNA的编码序列，其中靶向gag的shRNA的编码序列包含如SEQ ID NO：7所示序列，靶向nef的shRNA的编码序列包含如SEQ ID NO：8所示序列，或包含上述编码序列的载体。
  61. 一种药物组合物，其包含如权利要求1-28中任一项所述的重组细胞，权利要求29-31中任一项所述的载体，权利要求32-47中任一项所述的嵌合抗原受体，权利要求48所述的多核苷酸序列，权利要求49或50所述的载体，权利要求51或52所述的重组细胞，或权利要求53-60中任一项所述的重组细胞与至少一种shRNA或其编码序列或包含上述编码序列的载体的组合，以及药学上可接受的载体。
  62. 如权利要求61所述的药物组合物，其用于治疗HIV感染或艾滋病。
  63. 如权利要求61或62中所述的药物组合物，所述药物组合物进一步包括其他的抗HIV药物，包括但不限于核苷类抑制剂、HIV疫苗、广谱中和抗体和/或CAR-T细胞。
  64. 如权利要求1-28中任一项所述的重组细胞，权利要求29-31中任一项所述的载体，权利要求32-47中任一项所述的嵌合抗原受体，或如权利要求48中所述的多核苷酸序列，权利要求49或50中所述的载体，权利要求51或52所述的重组细胞，或权利要求53-60中任一项的组合在制备用于治疗和/或预防HIV感染或艾滋病的药物中的用途。
  65. 一种治疗HIV感染或艾滋病的方法，其包含向感染HIV或患有艾滋病的受试者施用如权利要求1-28中任一项的重组细胞，权利要求29-31中任一项所述的载体，权利要求32-47中任一项所述的嵌合抗原受体，权利要求48所述的多核苷酸序列，权利要求49或50所述的载体，权利要求51或52所述的重组细胞，权利要求53-60中任一项所述的重组细胞与至少一种shRNA或其编码序列或包含其编码序列的载体的组合，或权利要求61-63中任一项所述的药物组合物。
  66. 如权利要求65中所述的治疗方法，其中施用权利要求53-60中任一项所述的重组细胞与至少一 种shRNA或其编码序列或包含其编码序列的载体的组合时，重组细胞和至少一种shRNA或其编码序列或包含其编码序列的载体可顺序施用或同时施用。
  67. 如权利要求65或66中所述的治疗方法，所述方法进一步包括向受试者施用其他的抗HIV药物，包括但不限于其他的核苷类抑制剂、HIV疫苗、广谱中和抗体和/或CAR-T细胞。
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