WO2021115333A1 - 一种融合蛋白及表达此蛋白的工程化免疫细胞及其应用 - Google Patents

一种融合蛋白及表达此蛋白的工程化免疫细胞及其应用 Download PDF

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WO2021115333A1
WO2021115333A1 PCT/CN2020/134961 CN2020134961W WO2021115333A1 WO 2021115333 A1 WO2021115333 A1 WO 2021115333A1 CN 2020134961 W CN2020134961 W CN 2020134961W WO 2021115333 A1 WO2021115333 A1 WO 2021115333A1
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fusion protein
amino acid
group
terminus
amino acids
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PCT/CN2020/134961
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English (en)
French (fr)
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黄倬
林彦妮
赵珣
孔红梅
郑小翠
金锐铭
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苏州克睿基因生物科技有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4611—T-cells, e.g. tumor infiltrating lymphocytes [TIL], lymphokine-activated killer cells [LAK] or regulatory T cells [Treg]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464402—Receptors, cell surface antigens or cell surface determinants
    • A61K39/464411—Immunoglobulin superfamily
    • A61K39/464412—CD19 or B4
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes

Definitions

  • This application relates to the field of biomedicine, in particular to a fusion protein, and in particular to an optimized connection method of the fusion protein that promotes membrane positioning.
  • CAR-T immunotherapy has made remarkable clinical progress in the field of tumor treatment.
  • the protein structure of CAR ie chimeric antigen receptor
  • signal peptide-scFv-hinge region (hinge)-transmembrane region-co-activation signal domain-CD3 ⁇ usually the last C-terminal end of the CAR molecule is the CD3 ⁇ domain.
  • CAR-T cells themselves have achieved remarkable success in the treatment of certain blood cancers. However, so far, it has not been able to destroy solid tumors.
  • researchers are increasingly making T cells express CAR while expressing other functional genes, that is, armored CAR-T (Armored CAR-T) technology.
  • CAR T cell therapy and immune checkpoint inhibitors can enhance the efficacy of CAR T against tumors with poor response and improve the positioning of other functional genes on tumors.
  • furin-2A or 2A can ensure the expression of the two proteins in most cases, when the previous protein is CAR or a membrane-localized protein ending in CD3 ⁇ , furin-2A is connected
  • the method will affect the expression of CAR protein on the cell membrane, significantly reduce the expression of CAR, and greatly affect the function of CAR.
  • This application provides a fusion protein, which includes P1, L1, L2, and P2 in sequence from the N-terminus to the C-terminus, where P1 can be any membrane-localized protein with CD3 ⁇ at the C-terminus, and P2 can be any purpose that can be expressed
  • L1 can be a connecting peptide of any amino acid length
  • L2 is furin-2A.
  • L1 enables the expression of P1 and P2 separately without affecting each other, and overcomes the problem of the decrease in P1 expression caused by the connection mode of the P1 and P2 proteins in the prior art, thereby improving the membrane positioning function of the fusion protein.
  • This application also provides a method for promoting the localization of the fusion protein on the cell membrane.
  • the present application provides a fusion protein comprising P1, L1, L2, and P2 sequentially from the N-terminus to the C-terminus, wherein the P1 is a membrane localization protein and includes a CD3 ⁇ signaling domain at the C-terminus;
  • the L1 is a connecting peptide;
  • the L2 is a self-cleaving linker; and
  • the P2 is any polypeptide of interest.
  • the length of L1 is selected from the group consisting of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, and 5 amino acids.
  • the length of L1 is 1 amino acid, and the amino acid is selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the length of L1 is 1 amino acid, and the amino acid is selected from the following group: T, P, G, D, E, I, V, S, and A.
  • the length of L1 is 2 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GG and GS.
  • the length of L1 is 3 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGG and GGS.
  • the length of L1 is 4 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGGG and GGGS.
  • the length of L1 is 5 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGGGG and GGGGS.
  • the signal transduction domain of CD3 ⁇ comprises the amino acid sequence shown in SEQ ID NO.6.
  • the C terminal of P1 is connected to the N terminal of L1.
  • the P1 comprises a transmembrane domain.
  • the transmembrane domain comprises a transmembrane domain derived from a protein selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TCR ⁇ , TCR ⁇ , TCR ⁇ , TCR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , IL2 receptor, CD5, ICOS, OX40, NKG2D, 2B4, CD244, Fc ⁇ R, Fc ⁇ RI ⁇ , BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L , TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, SLAM, CTLA-4 and LAG-3.
  • the P1 comprises a costimulatory domain.
  • the costimulatory domain comprises a costimulatory domain selected from the following proteins or a combination thereof: CD28, CD137, CD27, CD2, CD7, CD8, CD80, CD86, OX40, CD226, DR3, SLAM, CDS, ICAM, NKG2D, NKG2C, B7-H3, 2B4, Fc ⁇ RI ⁇ , BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, PD-L1, PD-L2, 4- 1BBL, OX40L, ICOS-L, CD30L, CD70, CD83, HLA-G, MICA, MICB, lymphotoxin ⁇ receptor, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, CD83 ligand, CD40 and MyD88 .
  • the P1 includes a hinge region.
  • the hinge region comprises a hinge region derived from a protein selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3.
  • the P1 comprises an antigen binding domain.
  • the hinge region connects the antigen binding domain and the transmembrane domain.
  • the antigen binding domain specifically binds to tumor antigens.
  • the tumor antigen is selected from the group consisting of CD19 and BCMA.
  • the P1 comprises a chimeric antigen receptor CAR.
  • the C terminal of L1 is connected to the N terminal of L2.
  • the L2 includes an endopeptidase cleavage site and a cleavage peptide sequentially from the N-terminus.
  • a peptide linker is included between the endopeptidase cleavage site and the cleaved peptide.
  • the peptide linker comprises an amino acid sequence selected from the group: GSG.
  • the L2 includes a furin enzyme cleavage site.
  • the L2 comprises a cleaved peptide selected from the group consisting of P2A, T2A, F2A, and E2A.
  • the L2 includes a furin cleavage site and P2A sequentially from the N-terminus.
  • the L2 includes the amino acid sequence shown in SEQ ID NO. 2 or SEQ ID NO. 17.
  • the C terminal of the L2 is connected to the N terminal of the P2.
  • the P2 is the same as or different from the P1.
  • the P2 is selected from the following group: chimeric antigen receptor CAR, cytokine, constituent protein of MHC complex, tag protein, and immune checkpoint inhibitor.
  • this application provides an isolated nucleic acid molecule that encodes the fusion protein described in this application.
  • the present application provides a vector comprising the isolated nucleic acid described in the present application.
  • the vector contains the following nucleotide sequences in sequence from the 5'end: a gene encoding the P1, a gene encoding the L1, a gene encoding the L2, and a gene encoding the P2 gene.
  • the present application provides an immune cell that contains or expresses the fusion protein, the nucleic acid molecule, and/or the vector.
  • the immune cells include T cells, B cells, natural killer cells (NK cells), macrophages, NKT cells, monocytes, dendritic cells, granulocytes, lymphocytes, white blood cells And/or peripheral blood mononuclear cells.
  • NK cells natural killer cells
  • the present application provides a pharmaceutical composition, which comprises the immune cell described in the present application and a pharmaceutically acceptable carrier.
  • the present application provides a method for promoting the localization of a fusion protein on the cell membrane, wherein the fusion protein includes P1, L2, and P2, and the P1 is a membrane localization protein and includes a CD3 ⁇ signaling domain at its C-terminus ,
  • the L2 is a self-cleaving linker; and the P2 is any polypeptide of interest, which includes the following steps: connecting the C-terminus of the P1 to the N-terminus of L1, and the N-terminus of the L2 and the L1 The C-terminal is connected, and the L1 is a connecting peptide.
  • the length of L1 is selected from the group consisting of 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, and 5 amino acids.
  • the length of L1 is 1 amino acid, and the amino acid is selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the length of L1 is 1 amino acid, and the amino acid is selected from the following group: T, P, G, D, E, I, V, S, and A.
  • the length of L1 is 2 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GG and GS.
  • the length of L1 is 3 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGG and GGS.
  • the length of L1 is 4 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGGG and GGGS.
  • the length of L1 is 5 amino acids.
  • the L1 comprises an amino acid sequence selected from the group consisting of GGGGG and GGGGS.
  • Figure 1 shows a schematic diagram of the structure of the fusion protein described in the present application
  • FIG. 2 shows a schematic diagram of the CAR structure described in this application
  • Figure 3 shows a schematic structural diagram of the fusion protein CAR19-L1-Furin-GSG-P2A-B2M in the examples of the present application;
  • Figure 4 shows the average fluorescence intensity of CAR expression when the fusion protein CAR19-L1-Furin-GSG-P2A-B2M of this application contains different L1;
  • Figures 5 to 8 show the results of flow cytometry detection of CAR expression when the fusion protein CAR19-L1-Furin-GSG-P2A-B2M contains 1 amino acid length L1;
  • Figure 9 shows the results of flow cytometry detection of CAR expression when the fusion protein CAR19-L1-Furin-GSG-P2A-B2M of this application contains L1 of different lengths;
  • FIG. 10A shows the average fluorescence intensity of CAR expression when the fusion protein CAR19-L1-Furin-GSG-P2A-EGFRt of the present application contains different L1;
  • Figure 10B shows the average fluorescence intensity of CAR expression when the fusion protein CAR19-L1-Furin-GSG-P2A-GFP of the present application contains different L1;
  • Figure 11 shows the average fluorescence intensity of CAR expression when the fusion protein CAR19-L1-Furin-GSG-T2A-B2M of the present application contains different L1.
  • polypeptide polypeptide
  • peptide protein (if single chain)
  • the terms “polypeptide”, “peptide” and “protein (if single chain)” are used interchangeably herein and generally refer to amino acid polymers of any length.
  • the polymer can be linear or branched, it can contain modified amino acids, or it can be interrupted by non-amino acids.
  • the term also includes amino acid polymers that have been modified (e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with labeled components).
  • Polypeptides can be isolated from natural sources, can also be produced from eukaryotic or prokaryotic hosts through recombinant technology, or can be artificially synthesized products, as long as they can be expressed.
  • connecting peptide generally refers to any amino acid sequence that can connect two polypeptides.
  • the connecting peptide can be a connecting peptide consisting of one or more amino acids, such as one, two, four. 1, 3, 5, 6, 7, 8, 9, 10, 15, 20, 30 amino acid or more connecting peptides.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • TAA tumor-associated antigen
  • the CAR can be combined with the T cell receptor activation intracellular domain based on the antigen (eg CD19) specificity of the antibody.
  • Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens.
  • the term "antigen-binding domain” generally refers to a domain capable of binding to a target antigen.
  • the antigen-binding domain may include money and antigen receptors and fragments thereof, antibodies or antigen-binding fragments thereof that can specifically bind to an antigen.
  • the antigen-binding domain may be a domain capable of binding to tumor-associated antigens, and the tumor-associated antigens may include, but are not limited to: CD19, CD20, CD22, CD123, CD33/IL3Ra, CD138, CD33, BCMA, CS1, C-Met, EGFRvIII, CEA, Her2, GD2, MAG3, GPC3, NY-ESO-1.
  • Binding domain provides a domain or fragment of a CAR that has the ability to specifically bind to a target antigen of interest.
  • the antigen binding domain can be of natural origin, synthetic origin, semi-synthetic origin, or recombinant origin.
  • an antibody generally refers to a polypeptide molecule that can specifically recognize and/or neutralize a specific antigen.
  • an antibody may comprise an immunoglobulin consisting of at least two heavy (H) chains and two light (L) chains connected to each other by disulfide bonds, and includes any molecule comprising an antigen binding portion thereof.
  • the term “antibody” includes monoclonal antibodies, antibody fragments or antibody derivatives, including but not limited to human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies (e.g., dAb), single chain antibodies (e.g., scFv), And antibody fragments that bind to the antigen (e.g., Fab, Fab' and (Fab) 2 fragments).
  • antibody also includes all recombinant forms of antibodies, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and any antigen-binding antibody fragments and derivatives thereof described in this application.
  • Each heavy chain can be composed of a heavy chain variable region (VH) and a heavy chain constant region.
  • Each light chain can be composed of a light chain variable region (VL) and a light chain constant region.
  • the VH and VL regions can be further divided into hypervariable regions called complementarity determining regions (CDR), which are interspersed in more conserved regions called framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL can be composed of three CDRs and four FR regions, which can be arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain binding domains that interact with antigens.
  • the constant region of the antibody can mediate the binding of the immunoglobulin to host tissues or factors including various cells of the immune system (for example, effector cells) and the first component (Clq) of the classical complement system.
  • single-chain antibody may be an antibody formed by the heavy chain variable region and the light chain variable region or comprising a linker.
  • transmembrane domain Transmembrane Domain
  • Transmembrane Domain generally refers to the domain in the CAR that passes through the cell membrane, which is connected to the intracellular signal transduction domain and plays a role in transmitting signals.
  • costimulatory domain generally refers to an intracellular domain that can provide immune costimulatory molecules, which are cell surface molecules required for effective response of lymphocytes to antigens.
  • the costimulatory domain may include the costimulatory domain of CD28, and may also include the costimulatory domain of the TNF receptor family, such as the costimulatory domain of CD28, OX40, 4-1BB or ICOS.
  • hinge region generally refers to the connecting region between the antigen binding domain and the transmembrane region.
  • the term "signal transduction domain” generally refers to a domain located inside a cell capable of transducing signals.
  • the intracellular signal transduction domain can transmit signals into the cell.
  • a signal transduction domain is any continuous amino acid sequence used to direct protein targeting.
  • the intracellular signaling domain is the intracellular signaling domain of the chimeric antigen receptor.
  • the intracellular signaling domain may be selected from the group consisting of CD3 ⁇ intracellular domain, CD28 intracellular domain, 4-1BB intracellular domain, and OX40 intracellular domain.
  • membrane localization protein generally refers to any protein, peptide and/or polypeptide that can be localized on the cell membrane.
  • Membrane localization proteins may be localized near the membrane by binding to other proteins.
  • the co-localization of the membrane localization protein and the cell membrane can be observed under the microscope by techniques such as labeling and immunofluorescence staining.
  • the membrane localization protein comprises a signaling domain, for example, a CD3 ⁇ signaling domain.
  • self-cleaving linker generally refers to a peptide that can be cleaved that contains a recognition site for restriction enzyme cleavage.
  • endopeptidases generally refers to those proteolytic peptidases that cleave peptide bonds in the inner region of the peptide chain away from the end. Endopeptidases can be divided into trypsin, chymotrypsin, elastase, thermolysin, pepsin and glutamyl endopeptidase . In this application, the endopeptidase and its cleavage site can be selected from Table 1 below:
  • Furin can recognize specific amino acid sequences. Normally, Arg-X-X-Arg ⁇ (where X represents any amino acid and ⁇ represents the cleavage site) is the shortest sequence that furin can recognize when cleaving the substrate. In individual cases, furin can cleave proteins that do not completely conform to the sequence.
  • the term "cleaved peptide” generally refers to a type of polypeptide that can realize the function of cleaving a protein.
  • the cleaved peptide can be cleavaged by ribosome instead of protease hydrolysis to achieve protein cleavage.
  • the cleaved peptide may be a cleaved 2A peptide, which may include, but is not limited to, P2A, T2A, F2A, and E2A.
  • the "constituent protein of the MHC complex” generally refers to a type of protein closely related to the immune system.
  • the MHC complexes can be divided into three types: type I, type II, and type III.
  • the MHC complex may be a class I MHC molecule.
  • the constituent proteins of MHC class I molecules may include an alpha chain that spans the cell membrane.
  • the constituent proteins of MHC class I molecules may include ⁇ -2 microglobulin.
  • the MHC complex may be a class II MHC molecule.
  • the constituent proteins of MHC class II molecules may include an alpha chain that spans the cell membrane.
  • the constituent proteins of MHC class II molecules may include ⁇ chains that span cell membranes.
  • the MHC complex may be a class III MHC molecule.
  • the constituent proteins of class III MHC molecules may include secreted proteins with immune functions (for example, components of the complement system or inflammatory molecules).
  • B2M usually refers to a modified ⁇ -2 microglobulin, which is the light chain of a class I MHC molecule.
  • a genetically engineered immune lymphocyte and its preparation Method the Chinese patent application with the application number "201910746355.8”.
  • EGFRt usually refers to a truncated EGFR, which is obtained by truncating domain III and domain IV from wild-type epidermal growth factor receptor (EGFR).
  • the term "tag protein” generally refers to a polypeptide or protein expressed as a fusion with the target protein.
  • the tag protein can be used for the expression, detection, tracing and purification of the target protein.
  • the tag protein can be used to track cells expressing the tag protein.
  • other biomolecules can target the tag protein to eliminate, activate, and inhibit cells expressing the tag protein.
  • the tag protein may include a fluorescent protein.
  • the tag protein may be green fluorescent protein GFP.
  • the tag protein may be red fluorescent protein.
  • the term "isolated” generally refers to an antibody that has been separated from a component in its natural environment.
  • the antibody is purified to greater than 95% or 99% purity by, for example, electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis) or chromatography (e.g., ion exchange or reverse Phase HPLC) confirmed.
  • electrophoresis e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis
  • chromatography e.g., ion exchange or reverse Phase HPLC
  • nucleic acid generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides or their analogs of any length isolated from their natural environment or artificially synthesized.
  • the nucleic acid molecules described in this application may be isolated. For example, it can be produced or synthesized by the following methods: (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) produced by clonal recombination, (iii) purified , For example, fractionation by restriction enzyme digestion and gel electrophoresis, or (iv) synthesized, for example, by chemical synthesis.
  • PCR polymerase chain reaction
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the nucleic acid encoding the antibody or its antigen-binding fragment can be prepared by a variety of methods known in the art. These methods include, but are not limited to, the use of restriction fragment operations or the use of synthetic oligonucleotides.
  • overlapping extension PCR please refer to Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Ausube et al. Current Protocols in Molecular Biology, Greene Publishing, and Wiley-Interscience New York NY, 1993.
  • the "vector” generally refers to a nucleic acid molecule capable of self-replication in a suitable host, and is used to transfer the inserted nucleic acid molecule into and/or between host cells.
  • the vector may include a vector mainly used for inserting DNA or RNA into cells, a vector mainly used for replicating DNA or RNA, and a vector mainly used for expression of DNA or RNA transcription and/or translation.
  • the carrier also includes a carrier having a variety of the above-mentioned functions.
  • the vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell.
  • the vector can produce the desired expression product.
  • the vector may contain one or more of the nucleic acid molecules.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host.
  • control elements are well known to those skilled in the art. For example, they may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the vectors described in this application can be selected from plasmids, retroviral vectors and lentiviral vectors.
  • the plasmids, retroviral vectors and lentiviral vectors described in this application may contain CAR.
  • immune cell generally refers to a cell that participates in an immune response, such as promoting an immune effector response.
  • immune cells include, but are not limited to, T cells, B cells, natural killer (NK) cells, mast cells, granulocytes, and phagocytes derived from bone marrow.
  • NK natural killer
  • the term also includes engineered immune cells, such as immune cells that have been genetically modified by adding exogenous genetic material in the form of DNA or RNA to the total genetic material of the cell.
  • Plasmid usually refers to DNA molecules other than chromosomes or nucleoids in bacteria, yeasts and other organisms. They exist in the cytoplasm and have the ability to replicate autonomously, enabling them to maintain a constant copy in the progeny cells. Count and express the genetic information carried. Plasmids are used as gene carriers in genetic engineering research.
  • retroviral vector generally refers to a virus particle that can control and express foreign genes, but cannot self-package into a virus particle that has the ability to proliferate. Most of these viruses have reverse transcriptase. Retroviruses contain at least three genes: gag, which contains the genes that make up the virus's center and structure; pol, which contains the genes for reverse transcriptase, and env, which contains the genes that make up the virus coat. Through retroviral transfection, the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome. For example, the CAR molecule can be integrated into the host cell.
  • the term "lentiviral vector” generally refers to a diploid RNA viral vector belonging to retrovirus.
  • the lentiviral vector is based on the genome of the lentivirus. Many of the sequence structures related to the viral activity are removed to make it biologically safe, and then the sequence of the target gene required for the experiment is introduced into the genome skeleton And express the structure, and prepare it into a vector.
  • the retroviral vector can randomly and stably integrate its own genome and the foreign genes it carries into the host cell genome.
  • the CAR molecule can be integrated into the host cell.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.
  • the present application provides a fusion protein, which may include a membrane localization protein P1, a linker peptide L1, a self-cleaving linker L2, and any target polypeptide P2.
  • the present application provides a method for promoting the localization of a fusion protein on the cell membrane, wherein the fusion protein includes P1, L2, and P2, and the P1 is a membrane localization protein and includes a CD3 ⁇ signaling domain at its C-terminus , Said L2 is a self-cleaving linker; and said P2 is any desired polypeptide, which includes the following steps: connecting the C-terminus of P1 to the N-terminus of L1, and connecting the N-terminus of L2 to the N-terminus of L1 The C-terminal is connected, and the L1 is connected to the peptide.
  • the inventor of the present application found that when there is a connecting peptide L1 between P1 and L2, the P1 protein can be efficiently expressed, and its localization on the cell membrane can be promoted, so that P1 and P2 can be expressed mutually under the self-cleavage function of L2. Does not affect, and effectively realizes the biological functions of P1 and P2.
  • the L1 may be a connecting peptide with a length of 1 or more than 1 amino acid.
  • the fusion protein of the present application may include membrane localization protein P1.
  • the P1 described in the present application can be any membrane localization protein, as long as its C-terminus contains the CD3 ⁇ signaling domain.
  • the CD3 ⁇ signaling domain may include the amino acid sequence shown in SEQ ID NO.6.
  • P1 may include a transmembrane domain.
  • the transmembrane domain may comprise a transmembrane domain derived from a protein selected from: CD8, CD28, CD27, CD7, TRAC, TRBC, CD3 ⁇ , CD4, 4-1BB, OX40, ICOS, CTLA-4 , PD-1, LAG-3, 2B4 and BTLA.
  • the P1 may include a costimulatory domain.
  • the costimulatory domain may comprise a costimulatory domain selected from the following proteins or a combination thereof: CD137, CD28, OX40, ICOS, DAP10, 2B4, CD27, CD30, CD40, CD40L, TIM1, CD226, DR3, SLAM, NKG2D, CD244, FceRI ⁇ , BTLA, GITR, HVEM, CD2, NKG2C, LIGHT and DAP12.
  • the P1 may include a hinge region.
  • the hinge region may comprise a hinge region derived from a protein selected from the group consisting of CD8, CD28, IgG, 4-1BB, CD4, CD27, CD7, PD-1, and CH2CH3.
  • the P1 described in this application may also include an antigen binding domain.
  • the antigen binding domain specifically binds to tumor antigens.
  • the tumor antigen may be selected from the following group: B-cell maturation antigen (BCMA), mesothelin (MSLN), promise specific membrane antigen (PSMA), promise stem cell antigen (PCSA), carbonic anhydrase IX( CAIX),carcinoembryonic antigen(CEA),CD5,CD7,CD10,CD19,CD20,CD22,CD30,CD33,CD34,CD38,CD41,CD44,CD49f,CD56,CD74,CD123,CD133,CD138,CD33/IL3Ra,CS1 ,C-Met,epithelial glycoprotein2(EGP2),epithelial glycoprotein-40(EGP-40),epithelial cell adhesion molecule(EpCAM),folate-binding protein(FBP),fetal acetylcholine receptor(AChR),folate
  • the P1 may comprise a chimeric antigen receptor CAR.
  • the CAR may include an extracellular antigen binding domain, a hinge region, a transmembrane domain, a costimulatory domain, and a signal transduction domain in sequence from the N-terminus to the C-terminus.
  • the antigen binding domain may be a tumor antigen scFv, such as the tumor antigen CD19
  • the costimulatory domain may include a costimulatory domain selected from the following proteins: 4 -1BB (CD137)
  • the signaling domain may include a CD3 ⁇ signaling domain.
  • the CAR in this application may include an amino acid sequence selected from SEQ ID NOs. 5, 7, and 8.
  • the tumor antigen in the CAR described in the present application may be BCMA, for example, the CAR may include an amino acid sequence selected from SEQ ID NO. 9-12.
  • the fusion protein may include the linking peptide L1, and the L1 may be any amino acid length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid length) connecting peptides.
  • the L1 may be a connecting peptide composed of 1-5 (such as 1, 2, 3, 4, or 5) amino acids.
  • the length of L1 may be selected from the following group: 1 amino acid, 2 amino acids, 3 amino acids, 4 amino acids, and 5 amino acids.
  • the L1 may be a connecting peptide composed of 1 amino acid, for example, the 1 amino acid may be selected from the following group: A, R, N, D, C, Q, E, G, H , I, L, K, M, F, P, S, T, W, Y, and V.
  • the length of the L1 may be a connecting peptide consisting of 1 amino acid, and the amino acid is selected from the following group: T, P, G, D, E, I, V, S, and A.
  • the length of the L1 may be a connecting peptide consisting of 2 amino acids, and the 2 amino acids may be the same or different 2 amino acids consisting of any amino acid selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the L1 composed of 2 amino acids may include GG; or, may include GS.
  • the length of the L1 may be a connecting peptide consisting of 3 amino acids, and the 3 amino acids may be the same or different 3 amino acids consisting of any amino acid selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • L1 composed of the three amino acids may include GGG; or, may include GGS.
  • the length of the L1 may be a connecting peptide consisting of 4 amino acids, and the 4 amino acids may be the same or different 4 amino acids consisting of any amino acid selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the L1 composed of 4 amino acids may include GGGG; alternatively, it may include GGGS.
  • the length of L1 may be 5 amino acids, and the 5 amino acids may be the same or different 5 amino acids composed of any amino acid selected from the group consisting of: A, R, N, D , C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the 5-amino acid L1 may include GGGGG; alternatively, it may include GGGGS.
  • the length of L1 may be 6 or more selected from the following group (such as 7, 8, 9, 10, 12, 15 or more amino acids in length)
  • L1 can be designed as a sequence rich in glycine, glutamate, and/or serine residues, or can refer to the design ideas of protein linking peptides in the existing literature, such as Chen et al., Adv Drug Deliv Rev.
  • the C terminal of the P1 may be connected to the N terminal of the L1.
  • the CD3 ⁇ signaling domain at the C-terminus of P1 may be connected to the N-terminus of L1.
  • the fusion protein may include a self-cleaving linker L2, and the L2 may sequentially include an endopeptidase cleavage site and a cleavage peptide from the N-terminus.
  • the endopeptidase cleavage site may include a cleavage site of the shortest sequence that can be recognized by any endopeptidase.
  • the endopeptidase cleavage site may be a furin cleavage site.
  • the furin protease cleavage site can contain the following sequence: Arg-X-X-Arg ⁇ , where X represents any amino acid, and ⁇ represents the cleavage site.
  • furin can cleave proteins that do not completely conform to the above sequence.
  • the cleaved peptide may be a 2A peptide.
  • the cleaved peptide may be a 2A peptide selected from the group consisting of P2A, T2A, F2A, and E2A.
  • the amino acid sequences of various 2A peptides are shown in Table 2 below:
  • the cleavage peptide can be P2A.
  • P2A is a self-cleaving polypeptide linking sequence, which is a common linking sequence for expressing two independent proteins at the same time in one transcript in a vector. The protein is broken at the end of the P2A sequence during translation. Separate, make the two proteins before and after connected by P2A separate and function separately.
  • the amino acid sequence of P2A is ATNFSLLKQAGDVEENPGP.
  • a linker such as a peptide linker
  • the peptide linker is not a necessary condition for the function of L2, but it can usually improve the efficiency of cleavage peptide-induced cleavage.
  • Peptide linkers are usually artificially synthesized or naturally occurring, and consist of linear amino acid chains.
  • the peptide linker usually has a length of 1-50 amino acids (e.g., 1-28 amino acids, 1-25 amino acids, 3-25 amino acids).
  • the linker may include a repetitive amino acid sequence, or a naturally-occurring polypeptide sequence, such as a polypeptide having a hinge function.
  • Peptide linkers can promote the correct folding and proper presentation of peptides, thereby implementing their biological activities.
  • the linker may be a synthetic peptide linker that is designed to be rich in glycine, glutamic acid, and/or serine residues.
  • Examples of peptide linkers may include, but are not limited to, GSG, GSGS, (GGS)n, (GGGS)n, and (EAAAK)n, where n can be any positive integer.
  • the L2 of the fusion protein described in the present application may sequentially include a furin cleavage site and P2A from the N-terminus.
  • the peptide linker GSG may also be included between the furin cleavage site and P2A.
  • the L2 may include the amino acid sequence shown in SEQ ID NO.2.
  • the C terminal of the L1 may be connected to the N terminal of the L2.
  • the fusion protein may include any desired polypeptide P2.
  • the any polypeptide of interest P2 can be any polypeptide that can be expressed.
  • P2 may include, but is not limited to, a polypeptide selected from the group consisting of fluorescent protein, membrane-bound polypeptide, glycoprotein, globular protein, immune polypeptide (such as antibody), toxin, antibiotic, peptide hormone, growth factor, cytokine, transcription factor, coagulation Factors, RNA binding proteins, cytoskeletal proteins, ion channels, G protein coupled receptors, enzymes (such as proteases, kinases, phosphatases), vaccines, receptors, ligands, bactericidal and/or endotoxin binding proteins, structural polypeptides , Transport proteins and transmembrane proteins.
  • the P2 and the P1 may be the same or different.
  • the P2 may be selected from the following group: chimeric antigen receptor CAR, cytokine, constituent protein of MHC complex, tag protein, and immune checkpoint inhibitor.
  • the P2 may include an immune checkpoint inhibitor, such as an antibody or an antigen-binding fragment thereof selected from the following group of immune checkpoints: PD-1, CTLA-4, LAG-3, and TIM-3.
  • the P2 may include cytokines or functional fragments thereof, for example, interleukins (such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5, IL -6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12), interferons (such as IFN- ⁇ , IFN- ⁇ , IFN- ⁇ ), chemokines (such as CC chemokine, CXCL chemokine, XCL1, XCL2, CX3CL1), epidermal growth factor, hepatocyte growth factor, fibroblast growth factor, tumor necrosis factor (such as TNF- ⁇ , TNF- ⁇ ), colony stimulating factor (such as M-CSF, GM-CSF, G-CSF) and transforming growth factor, and other polypeptide factors, including LIF and kit ligand (KL).
  • interleukins such as IL-1, IL-1 ⁇ , IL-2, IL-3, IL-4, IL-5
  • the P2 may include a chimeric antigen receptor CAR.
  • a CAR that targets a binding portion of a tumor antigen selected from the following group: B-cell maturation antigen (BCMA), mesothelin (MSLN), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PCSA), carbonic anhydrase IX (CAIX), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41 , CD44, CD49f, CD56, CD74, CD123, CD133, CD138, CD33/IL3Ra, CS1, C-Met, epithelial glycoprotein2 (EGP 2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion (EpCAM), folate-binding protein(FBP),fetal acetylcholinereceptor(AChR),folatereceptor-
  • the P2 described in this application may include B2M, and the B2M may include the amino acid sequence shown in SEQ ID NO.1.
  • the P2 of the present application may include EGFRt, and the EGFRt may include the amino acid sequence shown in SEQ ID NO.13.
  • the P2 described in this application may include GFP, and the GFP may include the amino acid sequence shown in SEQ ID NO.16.
  • the C terminal of the L2 may be connected to the N terminal of the P2.
  • the fusion protein may include the P1, the L1, the L2, and the P2 in sequence from the N-terminus to the C-terminus.
  • the C-terminus of the P1 can be connected to the N-terminus of the L1
  • the C-terminus of the L1 can be connected to the N-terminus of the L2
  • the C-terminus of the L2 The terminal can be connected to the N terminal of the P2.
  • the P1 may include CAR19, and the L2 may include furin, the peptide linker GSG, and the P2A cleavage peptide in sequence from the N-terminus to the C-terminus.
  • P2 can contain B2M.
  • the fusion protein may include the amino acid sequence shown in SEQ ID NO. 4, where X represents any amino acid.
  • the P1 may include CAR19
  • the L2 may include furin
  • the peptide linker GSG may include furin
  • the P2 may include EGFRt.
  • the fusion protein may include the amino acid sequence shown in SEQ ID NO.14.
  • this application provides an isolated nucleic acid molecule, which can encode the fusion protein described in this application.
  • the isolated nucleic acid molecule encoding the fusion protein described in this application may include the nucleic acid sequence shown in SEQ ID NO. 3 and 15 (wherein "nnn” represents any nucleotide that can encode L1) or its functionality Variants.
  • the nucleic acid molecules described in this application may be isolated.
  • the isolated nucleic acid is a nucleic acid molecule prepared by recombinant DNA technology.
  • the present application provides a vector, which can contain the isolated nucleic acid molecule.
  • the vector may be selected from one or more of plasmids, retroviral vectors and lentiviral vectors.
  • the vector of the present application includes the following nucleotide sequences in sequence from the 5'end: the gene encoding the P1, the gene encoding the L1, the gene encoding the L2, and the gene encoding the P2.
  • the vector may also contain other genes, such as a marker gene that allows the vector to be selected in a suitable host cell and under suitable conditions.
  • the vector may also contain expression control elements that allow the coding region to be correctly expressed in a suitable host. Such control elements are well known to those skilled in the art.
  • the expression control sequence may include promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation.
  • the expression control sequence is a tunable element.
  • the specific structure of the expression control sequence may vary according to the function of the species or cell type, but usually includes 5'non-transcribed sequences and 5'and 3'non-translated sequences involved in transcription and translation initiation, such as TATA box, plus Cap sequence, CAAT sequence, etc.
  • the 5' non-transcriptional expression control sequence may include a promoter region, and the promoter region may include a promoter sequence for transcriptional control functionally linked to the nucleic acid.
  • the vector may include, for example, a plasmid, a cosmid, a virus, a phage, or other vectors commonly used in, for example, genetic engineering.
  • the vector may include an expression vector, such as a lentiviral purpose expression plasmid, and a helper vector, such as a packaging helper plasmid.
  • the vector may be a lentiviral vector.
  • the lentiviral vector contains a long terminal repeat sequence 5'LTR and a truncated 3'LTR, RRE, rev response element (cPPT), central termination sequence (CTS) and post-translational regulatory element (WPRE).
  • the molecule can be constructed on the lentiviral vector by digestion with BamHI and SalI.
  • the present application provides an immune cell, which can contain or express the fusion protein, the nucleic acid molecule, and/or the vector described in the present application.
  • the immune cells include T lymphocytes, B cells, natural killer cells, macrophages, NKT cells, monocytes, dendritic cells, and granulocytes.
  • the immune cells may include T lymphocytes.
  • the T lymphocytes may include thymocytes, natural T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes or activated T lymphocytes.
  • the T cell may be a helper T cell (Th), such as a helper T cell 1 (Th1) or a helper T cell 2 (Th2) cell.
  • the T lymphocytes may be CD4+ helper T cells (HTL; CD4+ T cells), cytotoxic T cells (CTL; CD8+ T cells), tumor infiltrating cytotoxic T cells (TIL; CD8+ T cells), CD4+/ CD8+ T cells, CD4-/CD8-T cells or any other T lymphocyte subtype.
  • the T lymphocytes may be derived from peripheral blood cells, cord blood cells and/or white blood cells.
  • the T cell may be a Jurkat cell.
  • the immune cells may include B cells.
  • the B cells may include effector B cells (plasma cells) and memory B cells.
  • the B cells may include B2 cells, B1 cells, marginal zone B cells, follicular B cells, and regulatory B cells.
  • the immune cells may include macrophages.
  • the B cells may include type I macrophages (M1) and type II macrophages (such as M2a, M2B, M2c).
  • the immune cells may include NK cells.
  • the NK cells may include CD56bright and CD56dim.
  • the NK cells may include NK1 and NK2.
  • the NK cells may include A-NK and NA-NK.
  • the present application provides a method for preparing immune effector cells, which may include introducing the vector described in the present application into the immune effector cells.
  • the vector described in this application can be introduced into the immune effector cells, such as T lymphocytes, B cells, macrophages, or natural killer (NK) cells.
  • each or each cell may contain one or one of the vectors described in this application.
  • each or each cell may contain multiple (e.g., 2 or more) or multiple (e.g., 2 or more) vectors described in the present application.
  • the vector described in this application can be introduced into the cell.
  • a retroviral vector can be used to transfect immune effector cells, and the viral genome with the nucleic acid encoding the fusion protein can be integrated into the host genome to ensure long-term and stable expression of the target gene.
  • a transposon is used to introduce a plasmid carrying the nucleic acid encoding the fusion protein and a plasmid carrying a transposase into the target cell.
  • nucleic acid molecules can be added to the genome by means of gene editing (such as CRISPR/Cas9).
  • the vector with the nucleic acid encoding the fusion protein described in this application can be introduced into the cell by methods known in the art, such as electroporation (Neon electroporator), liposome transfer Dyeing and so on.
  • the present application provides a pharmaceutical composition, which may include the immune cell described in the present application and a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable adjuvant may include buffers, antioxidants, preservatives, low molecular weight polypeptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, counter ions, metal complexes and/or non-ionic surfaces Active agent, etc.
  • the pharmaceutical composition can be formulated for oral administration, intravenous administration (e.g., intravenous injection, IV), intramuscular administration (e.g., intramuscular injection, IM), and the original Local administration, inhalation, rectal administration, vaginal administration, transdermal administration or administration via subcutaneous depot.
  • the pharmaceutical composition described in the present application may include a therapeutically effective amount of the fusion protein.
  • the therapeutically effective amount is a dose required to prevent and/or treat (at least partially treat) a disorder or disorder (such as cancer) and/or any complications thereof in a subject suffering from or at risk of development.
  • a lentiviral vector containing the P1-L1-L2-P2 gene of the fusion protein of the present application As a negative control, a P1-L2-P2 lentiviral vector was constructed. The vector is a sequence without L1, and P1 and L2 are directly connected.
  • L2 is Furin-GSG-P2A (SEQ ID NO. 2).
  • L1 is a single amino acid selected from the following group: A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V.
  • the nucleotide sequence of the formed whole gene sequence is shown in SEQ ID NO. 3 (nnn represents any codon that encodes L1), and the amino acid sequence is shown in SEQ ID NO. 4 (X represents any amino acid), and the schematic diagram is as shown in image 3.
  • Example sequence 2 "CAR19-L1-Furin-GSG-P2A-EGFRt” P1 is CAR19 (SEQ ID NO. 5), and P2 is EGFRt (SEQ ID NO. 13).
  • L2 is Furin-GSG-P2A (SEQ ID NO. 2).
  • L1 is any single amino acid.
  • the nucleotide sequence of the formed whole gene sequence is shown in SEQ ID NO. 15 (nnn represents any codon encoding L1), and the amino acid sequence is shown in SEQ ID NO. 14 (X represents any amino acid).
  • Example sequence 3 "CAR19-L1-Furin-GSG-P2A-GFP”: P1 is CAR19 (SEQ ID NO. 5), and P2 is GFP (SEQ ID NO. 16).
  • L2 is Furin-GSG-P2A (SEQ ID NO. 2).
  • L1 is any single amino acid.
  • the nucleotide sequence of the formed whole gene sequence is shown in SEQ ID NO.19 (nnn represents any codon encoding L1), and the amino acid sequence is shown in SEQ ID NO.18 (X represents any amino acid).
  • the virus expression plasmid (Addgene ID: #12252) is digested with BamHI/SalI as the backbone. After the above-mentioned various P1-L1-L2-P2 sequences are fully synthesized, they are respectively connected to the digested virus expression backbone. The CAR19 sequence of the control group and the sequence P1-L2-P2 without L1 were also connected to the viral expression backbone with BamHI/SalI.
  • a three-plasmid system is used: the lentiviral target expression plasmid constructed as described above, and the packaging auxiliary plasmids psPAX2 (Addgene ID: #12260) and pMD2.G (Addgene ID: #12259).
  • the virus packaging was carried out in HEK293T cells (purchased from Shanghai Institute of Cell Research, Chinese Academy of Sciences). The preparation process is as follows: the HEK293T cells in the frozen working cells are resuscitated, and they are cultured in DMEM medium (+10%FBS+1%P/S) (Cellgro 10-013-CMR) in a 10cm petri dish, and the second is resuscitated. Change the liquid after days.
  • the mixture of plasmid and PEI was added to Opti-MEM medium (Gibco, cat#31985-070), and then the mixed solution was added to HEK293T cells passaged to the 4th generation. After 6 hours of transfection, the medium was changed with 2% FBS fresh medium, and then the culture was continued to 72 hours, and the supernatant of HEK293T cells was collected.
  • the collected virus supernatant was concentrated by ultra-isolation (82200g, 4-8°C centrifugation for 2 hours), and the concentrated virus was filtered and sterilized with a 0.22 ⁇ m filter membrane and resuspended for later use.
  • the medium used is a complete medium, ImmunoCult TM -XFT Cell Expansion Medium (Stem Cell Technology, cat#10981).
  • Example sequence 1 of P1-L1-L2-P2 described in Example 1 is used, where L1 is any one of 20 amino acids, or 1-5 Gs.
  • the fluorescence intensity (MFI) expressed by CAR19 after virus transfection is detected.
  • Three days after virus transfection remove a small amount of cells (about 1 ⁇ 10 5 cells), add 1ml PBS (Gibco, cat#C10010500BT) to wash the cells once, resuspend with 100 ⁇ L PBS, and add 3 ⁇ L anti-FMC63 antibody primary antibody (detection CAR19 )
  • After incubating for 30 minutes at 4°C add 1ml PBS and mix well, centrifuge at 350g for 3 minutes to collect the cells to remove the supernatant, and add 0.5 ⁇ L of secondary antibodies (primary and secondary antibodies from anti-Mouse FMC63 kit, Shanghai Xingwan, R19PB-100 The product) into the cells, mix well, incubate at 4°C for 30min, add 1ml PBS to mix, and centrifuge at 350g for 3min to collect the cells to remove the supernatant, add 200 ⁇ L PBS to
  • Figures 5 to 8 are flow cytometry detection results of different 1 amino acid L1.
  • Figure 4 shows the statistics of the influence of 20 different amino acids as L1 on the fluorescence intensity of CAR expression in the model T cell line Jurkat, reflecting the results in Figures 5 to 8.
  • the CAR19 control group is a group where cells only express CAR.
  • the average fluorescence intensity (MFI) of CAR expression in this group is set to 1, and the other groups are normalized to CAR19.
  • the normalized calculation is performed using the MFI expressed by CAR alone.
  • the ordinate shows the multiple of the MFI of each CAR measured by different constructions relative to the MFI when CAR is expressed alone, that is, (CAR MFI when L1 is a certain amino acid)/( CAR MFI of the CAR19 control group).
  • the L1 group was used as a negative control.
  • the CD3 ⁇ at the C-terminus of the CAR structure of this group was directly connected to the linking sequence without L1 (ie, without any amino acid interval).
  • the CAR expression intensity of this group was significantly reduced, which was about 20% of that of the CAR19 control group.
  • Figure 9 shows that after replacing L1 in the example sequence 1 of P1-L1-L2-P2 described in Example 1 with 1 G, GG(2G), GGG(3G), GGGG(4G), GGGGG(5G) , The result of using flow cytometry to detect the fluorescence intensity of CAR. Combined with the statistical results of Figure 4, it is shown that increasing the number of amino acids (more than one) can also restore or increase the expression intensity of CAR.
  • L2 is a self-cleaving linker.
  • L1 is also suitable for other linkers such as T2A, E2A and F2A.
  • T2A as an example for testing.
  • the structure is shown in Table 4.
  • Table 4 The structure of CAR19-L1-Furin-GSG-T2A-P2 with L2 including T2A

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Abstract

一种融合蛋白，以及编码其的核酸分子、包含其的载体和免疫细胞、药物组合物，以及一种促进融合蛋白在细胞膜上定位的方法。该融合蛋白能提高CAR分子的表达和细胞膜定位，从而提高CAR的功能。

Description

一种融合蛋白及表达此蛋白的工程化免疫细胞及其应用 技术领域
本申请涉及生物医药领域，具体的涉及一种融合蛋白，尤其涉及促进膜定位的融合蛋白的优化连接方式。
背景技术
目前，CAR-T免疫疗法在肿瘤治疗领域取得了令人瞩目的临床进展。CAR(即嵌合抗原受体)的蛋白结构通常为：信号肽-scFv-铰链区(hinge)-跨膜区-共激活信号域-CD3ζ。通常CAR分子的最后C端结尾是CD3ζ结构域。
虽然CAR-T细胞本身在治疗某些血液癌症方面取得了显著的成功。然而，到目前为止，其并没有足够的能力消灭实体肿瘤。为增强CAR-T的功能和疗效，研究人员越来越多的使T细胞在表达CAR的同时再表达其他的功能基因，即装甲CAR-T(Armored CAR-T)技术。研究表明，CAR T细胞疗法与免疫检查点抑制剂联合使用能增强CAR T针对原来反应较差的肿瘤的功效，提高其他功能性基因对肿瘤的定位。研究人员通常采用自切割连接序列，如furin-2A或2A，连接CAR基因和其他功能基因，使两者分开表达，形成独立蛋白(参见Rafiq,S.,Yeku,O.,Jackson,H.等人.Targeted delivery of a PD-1-blocking scFv by CAR-T cells enhances anti-tumor efficacy in vivo.Nat Biotechnol 36,847–856(2018)doi:10.1038/nbt.4195)。
然而，虽然furin-2A或2A这种连接方式在大多数情况下可以保证前后两个蛋白表达，但当前面的蛋白是CAR或是以CD3ζ结尾的膜定位的蛋白时，furin-2A这种连接方式会影响CAR蛋白在细胞膜上的表达，使CAR表达量显著下降，且很大程度的影响CAR的功能。
因此，亟需开发新的技术，以提高CAR分子的高效表达和细胞膜定位功能，从而提高CAR与其他基因联合蛋白的功能。
发明内容
本申请提供了一种融合蛋白，其以N端至C端的方向依次包含P1、L1、L2及P2，其中，P1可以为C端为CD3ζ的任意膜定位蛋白，P2可以为能够表达的任意目的多肽，L1可以为任意氨基酸长度的连接肽，L2为furin-2A。L1的存在能使P1和P2分别表达，互不影响，克服了现有技术中P1和P2蛋白的连接方式使P1表达量下降的问题，从而提高融合蛋白的膜定位功能。本申请还提供了促进融合蛋白在细胞膜上定位的方法。
一方面，本申请提供了一种融合蛋白，其以N端至C端的方向依次包含P1、L1、L2及P2，其中所述P1为膜定位蛋白且在其C端包含CD3ζ信号传导结构域；所述L1为连接肽；所述L2为自切割连接子；且所述P2为任意目的多肽。
在某些实施方式中，所述L1的长度选自以下组：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸和5个氨基酸。
在某些实施方式中，所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。
在某些实施方式中，所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：T、P、G、D、E、I、V、S和A。
在某些实施方式中，所述L1的长度为2个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GG和GS。
在某些实施方式中，所述L1的长度为3个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGG和GGS。
在某些实施方式中，所述L1的长度为4个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGGG和GGGS。
在某些实施方式中，所述L1的长度为5个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGGGG和GGGGS。
在某些实施方式中，所述CD3ζ的信号传导结构域包含SEQ ID NO.6所示的氨基酸序列。
在某些实施方式中，所述P1的C端与L1的N端连接。
在某些实施方式中，所述P1包含跨膜结构域。
在某些实施方式中，所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ、CD3γ、CD3ζ、IL2受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154、SLAM、CTLA-4和LAG-3。在某些实施方式中，所述P1包含共刺激结构域。
在某些实施方式中，所述共刺激结构域包含选自下述蛋白的共刺激结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
在某些实施方式中，所述P1包含铰链区。
在某些实施方式中，所述铰链区包含源自选自下述蛋白的铰链区：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。
在某些实施方式中，所述P1包含抗原结合结构域。
在某些实施方式中，所述铰链区连接所述抗原结合结构域和所述跨膜结构域。
在某些实施方式中，所述抗原结合结构域与肿瘤抗原特异性结合。
在某些实施方式中，所述肿瘤抗原选自以下组：CD19和BCMA。
在某些实施方式中，所述P1包含嵌合抗原受体CAR。
在某些实施方式中，所述L1的C端与所述L2的N端连接。
在某些实施方式中，所述L2自N端起依次包含内肽酶切割位点和剪切肽。
在某些实施方式中，所述内肽酶切割位点和剪切肽之间包含肽接头。
在某些实施方式中，所述肽接头包含选自以下组的氨基酸序列：GSG。
在某些实施方式中，所述L2包含弗林蛋白(furin)酶切割位点。
在某些实施方式中，所述L2包含剪切肽，所述剪切肽选自以下组：P2A、T2A、F2A和E2A。
在某些实施方式中，所述L2自N端起依次包含弗林蛋白酶切割位点和P2A。
在某些实施方式中，所述L2包含SEQ ID NO.2或SEQ ID NO.17所示的氨基酸序列。
在某些实施方式中，所述L2的C端与所述P2的N端连接。
在某些实施方式中，所述P2与所述P1相同或不同。
在某些实施方式中，所述P2选自以下组：嵌合抗原受体CAR、细胞因子、MHC复合物的组成蛋白、标签蛋白和免疫检查点抑制剂。
另一方面，本申请提供了一种分离的核酸分子，其编码本申请所述的融合蛋白。
另一方面，本申请提供了一种载体，其包含本申请所述的分离的核酸。
在某些实施方式中，所述的载体自5’端依次包含以下的核苷酸序列：编码所述P1的基因、编码所述L1的基因、编码所述L2的基因和编码所述P2的基因。
另一方面，本申请提供了一种免疫细胞，其包含或表达所述的融合蛋白，所述的核酸分子，和/或所述的载体。
在某些实施方式中，所述的免疫细胞包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。另一方面，本申请提供了一种药物组合物，其包含本申请所述的免疫细胞和药学上可接受的载体。
另一方面，本申请提供了一种促进融合蛋白在细胞膜上定位的方法，其中所述融合蛋白包括P1、L2和P2，所述P1为膜定位蛋白且在其C端包含CD3ζ信号传导结构域，所述L2为自切割连接子；且所述P2为任意目的多肽，其包括以下的步骤：使所述P1的C端与L1的N端连接，所述L2的N端与所述L1的C端连接，所述L1为连接肽。
在某些实施方式中，所述L1的长度选自以下组：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸和5个氨基酸。
在某些实施方式中，所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。
在某些实施方式中，所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：T、P、G、D、E、I、V、S和A。
在某些实施方式中，所述L1的长度为2个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GG和GS。
在某些实施方式中，所述L1的长度为3个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGG和GGS。
在某些实施方式中，所述L1的长度为4个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGGG和GGGS。
在某些实施方式中，所述L1的长度为5个氨基酸。
在某些实施方式中，所述L1包含选自以下组的氨基酸序列：GGGGG和GGGGS。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1显示的是本申请所述融合蛋白的结构示意图；
图2显示的是本申请所述CAR的结构示意图；
图3显示的是本申请实施例中融合蛋白CAR19-L1-Furin-GSG-P2A-B2M的结构示意图；
图4显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-P2A-B2M包含不同L1时CAR表达的平均荧光强度；
图5-图8显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-P2A-B2M包含1个氨基酸长度的L1时CAR表达的流式细胞术检测结果；
图9显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-P2A-B2M包含不同长度的L1时CAR表达的流式细胞术检测结果；
图10A显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-P2A-EGFRt包含不同L1时CAR表达的平均荧光强度；
图10B显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-P2A-GFP包含不同L1时的CAR表达的平均荧光强度；
图11显示的是本申请所述融合蛋白CAR19-L1-Furin-GSG-T2A-B2M包含不同L1时的CAR表达的平均荧光强度。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
在本申请中，术语“多肽”、“肽”和“蛋白质(如果为单链)”在本文中可互换地使用，通常是指任意长度的氨基酸聚合物。该聚合物可以是线形的或分支的，它可以包含修饰的氨基酸，也可以由非氨基酸隔断。该术语也包括已经被修饰(例如，二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作，如以标记组分缀合)的氨基酸聚合物。多肽可以从天然来源分离，也可以通过重组技术从真核或原核宿主产生，还可以是人工合成的产物，只要其能被表达即可。
在本申请中，术语“连接肽”通常是指可连接两个多肽的任意氨基酸序列，所述连接肽可以为由1个或多个氨基酸组成的连接肽，如，1个、2个、4个、3个、5个、6个、7个、8个、9个、10个、15个、20个、30个氨基酸或更多个组成的连接肽。
在本申请中，术语“嵌合抗原受体(Chimeric Antigen Receptor，CAR)”通常是指包含能够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括抗原(例如，肿瘤相关抗原(tumor-associated antigen，TAA))结合结构域、跨膜结构域、共刺激结构域和胞内信号结构域。在本申请中，所述CAR可以基于抗体的抗原(例如CD19)特异性与T细胞受体活化胞内结构域组合在一起。经遗传修饰表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。关于CAR和CAR-T细胞的描述，可参见例如Sadelain M,Brentjens R,Rivi`ere I.The basicprinciples of chimeric antigen receptor design.Cancer Discov.2013；3(4):388-398；Turtle CJ,Hudecek M,Jensen MC,Riddell SR.Engineered T cells for anti-cancer therapy.Curr Opin Immunol.2012；24(5):633-639；Dotti G,Gottschalk S,Savoldo B,Brenner MK.Design and development of therapies using chimeric antigen receptor-expressing T cells.Immunol Rev.2014；257(1):107-126；以及WO2013154760、WO2016014789。
在本申请中，术语“抗原结合结构域”通常是指能够与靶抗原结合的结构域。抗原结合结构域可包含能特异性结合抗原的钱和抗原受体及其片段，抗体或其抗原结合片段。抗原结合结构域可以能够与肿瘤相关抗原结合的结构域，所述肿瘤相关抗原可包括但不限于：CD19、CD20、CD22、CD123、CD33/IL3Ra、CD138、CD33、BCMA、CS1、C-Met、EGFRvIII、CEA、Her2、GD2、MAG3、GPC3、NY-ESO-1。
在本申请中使用的术语“抗原结合结构域”、“结合结构域”、“胞外结构域”、“胞外结合结构域”、“抗原特异性结合结构域”和“胞外抗原特异性结合结构域”可互换使用，并且提供了具有特异性结合目标靶抗原的能力的CAR的结构域或片段。抗原结合结构域可以为天然来源、合成来源、半合成来源或重组来源。
在本申请中，术语“抗体”通常是指一种能够特异性识别和/或中和特定抗原的多肽分子。例如，抗体可包含通过二硫键相互连接的至少两条重(H)链和两条轻(L)链组成的免疫球蛋白，并且包括任何包含其抗原结合部分的分子。术语“抗体”包括单克隆抗体、抗体片段或抗体衍生物，包括但不限于人抗体、人源化抗体、嵌合抗体、单域抗体(例如，dAb)，单链抗体(例如，scFv)，以及与抗原结合的抗体片段(例如，Fab、Fab’和(Fab) 2片段)。术语“抗体”还包括抗体的所有重组体形式，例如在原核细胞中表达的抗体、未糖基化的抗体以及本申请所述的任何与抗原结合的抗体片段及其衍生物。每条重链可由重链可变区(VH) 和重链恒定区构成。每条轻链可由轻链可变区(VL)和轻链恒定区构成。VH和VL区可进一步被区分为称为互补决定区(CDR)的高变区，它们散布在称为构架区(FR)的更保守的区域中。每个VH和VL可由三个CDR和四个FR区构成，它们从氨基端至羧基端可按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3和FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(例如，效应细胞)和经典补体系统的第一成分(Clq)。
在本申请中，术语“单链抗体(scFv)”可以是由所述重链可变区和所述轻链可变区或包含通过连接子(linker)连接而成的抗体。
在本申请中，术语“跨膜结构域(Transmembrane Domain)”通常是指CAR中穿过细胞膜的结构域，其与细胞内信号转导结构域相连接，起着传递信号的作用。
在本申请中，术语“共刺激结构域”通常是指可以提供免疫共刺激分子的胞内结构域，所述共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子。所述共刺激结构域可包括CD28的共刺激结构域，还可包括TNF受体家族的共刺激结构域，例如CD28、OX40、4-1BB或ICOS的共刺激结构域。
在本申请中，术语“铰链区”通常是指抗原结合结构域和跨膜区之间的连接区。
在本申请中，术语“信号传导结构域”通常是指位于细胞内部能够转导信号的结构域。在本申请中，所述胞内信号传导结构域可以将信号传导至细胞内。通常，信号传导结构域为用于指导蛋白质寻靶的任何一段连续的氨基酸序列。例如，所述胞内信号传导结构域是所述嵌合抗原受体的胞内信号传导结构域。例如，在某些实施方式中，所述胞内信号传导结构域可选自CD3ζ胞内域、CD28胞内域、4-1BB胞内域和OX40胞内域。
在本申请中，术语“膜定位蛋白”通常是指能够在细胞膜上定位的任意蛋白、肽和/或多肽。膜定位蛋白有可能通过结合其他蛋白定位于膜附近。通常，可以通过标签、免疫荧光染色等技术在显微镜下观察到膜定位蛋白与细胞膜共定位。在某些实施方式中，膜定位蛋白包含信号传导结构域，例如，CD3ζ信号传导结构域。
在本申请中，术语“自切割连接子”通常是指含有酶切识别位点的能够被切割的肽。
在本申请中，术语“内肽酶”通常是指那些在远离末端的肽链内部区域切割肽键的蛋白 水解肽酶。内肽酶可分为胰蛋白酶(trypsin)、胰凝乳蛋白酶(chymotrypsin)、弹性蛋白酶(elastase)、嗜热菌素(thermolysin)、胃蛋白酶(pepsin)和谷胱酰内肽酶(glutamyl endopeptidase)。本申请中，所述内肽酶及其切割位点可以选自下表1：
表1
内肽酶 主要切割识别位点
Xa因子蛋白酶 Ile-(Glu/Asp)-Gly-Arg的Arg之后
肠激酶 Asp-Asp-Asp-Asp-Lys的Lys之后
弗林蛋白酶 Arg-X-X-Arg
弗林蛋白酶能识别特定的氨基酸序列。通常情况下，Arg-X-X-Arg↓(其中，X代表任意氨基酸，↓代表切割位点)是弗林蛋白酶切割底物时所能识别的最短序列。在个别情况下，弗林蛋白酶可以对不完全符合该序列的蛋白进行切割。
在本申请中，术语“剪切肽”通常是指能够实现剪切蛋白的功能的一类多肽。例如，所述剪切肽可以经核糖体跳跃而非蛋白酶水解来实现蛋白质剪切。例如，所述剪切肽可为剪切2A肽，其可包括但不限于P2A、T2A、F2A和E2A。
在本申请中，所述“MHC复合物的组成蛋白”通常是指与免疫系统密切相关的一类蛋白。所述MHC复合物可以分为三类：I类、II类和III类。在某些情形中，所述MHC复合物可以是I类MHC分子。例如，I类MHC分子的组成蛋白可以包括跨越细胞膜的α链。例如，I类MHC分子的组成蛋白可以包括β-2微球蛋白。在某些情形中，所述MHC复合物可以是II类MHC分子。例如，II类MHC分子的组成蛋白可以包括跨越细胞膜的α链。例如，II类MHC分子的组成蛋白可以包括跨越细胞膜的β链。在某些情形中，所述MHC复合物可以是III类MHC分子。例如，III类MHC分子的组成蛋白可以包括具有免疫功能的分泌蛋白(例如，补体系统成分或炎性分子)。
在本申请中，术语“B2M”通常是指一种改造的β-2微球蛋白，是I类MHC分子的轻链，可参见发明名称为“一种基因工程化的免疫淋巴细胞及其制备方法”、申请号为“201910746355.8”的中国专利申请。
在本申请中，术语“EGFRt”通常是指EGFR截短体，由野生型表皮生长因子受体(EGFR) 截取domain III及domain IV得到。
在本申请中，术语“标签蛋白”通常是指与目的蛋白一起融合表达的一种多肽或者蛋白。在本申请中，所述标签蛋白可以用于目的蛋白的表达、检测、示踪和纯化等。在某些情形中，所述标签蛋白可以用来追踪表达标签蛋白的细胞。在某些情形中，其它生物分子可以靶向标签蛋白，以达到清除、激活、抑制表达该标签蛋白的细胞。在某些情形中，所述标签蛋白可以包括荧光蛋白。例如，所述标签蛋白可以是绿色荧光蛋白GFP。例如，所述标签蛋白可以是红色荧光蛋白。
在本申请中，术语“分离的”通常是指抗体是已经与它的天然环境中的组分分离的抗体。在一些实施方案中，将抗体纯化至大于95％或99％纯度，所述纯度通过例如电泳(例如，SDS-PAGE、等电聚焦(IEF)、毛细管电泳)或色谱(例如，离子交换或反相HPLC)确定。关于评价抗体纯度的方法的综述可参见Flatman，S.等，J.Chrom.B 848(2007)79-87。
在本申请中，术语“核酸”通常是指从其天然环境中分离的或人工合成的任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸或其类似物。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。在本申请中，可以通过本领域已知的多种方法来制备编码所述抗体或其抗原结合片段的核酸，这些方法包括但不限于，采用限制性片段操作或采用合成性寡核苷酸的重叠延伸PCR，具体操作可参见Sambrook等人，Molecular Cloning,A Laboratory Manual,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,N.Y,1989；和Ausube等人Current Protocols in Molecular Biology,Greene Publishing and Wiley-Interscience,New York N.Y.,1993。
在本申请中，所述“载体”通常是指能够在合适的宿主中自我复制的核酸分子，用以将插入的核酸分子转移到宿主细胞中和/或宿主细胞之间。所述载体可包括主要用于将DNA或RNA插入细胞中的载体、主要用于复制DNA或RNA的载体，以及主要用于DNA或RNA的转录和/或翻译的表达的载体。所述载体还包括具有多种上述功能的载体。所述载体可以是当引入合适的宿主细胞时能够转录并翻译成多肽的多核苷酸。通常，通过培养包含所述载体的合适的宿主细胞，所述载体可以产生期望的表达产物。在本申请中，所述载体中可包含一 种或多种所述核酸分子。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。本申请所述载体可选自质粒、逆转录病毒载体和慢病毒载体。本申请所述质粒、逆转录病毒载体和慢病毒载体可包含CAR。
在本申请中，术语“免疫细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。免疫细胞的示例包括但不限于T细胞、B细胞、天然杀伤(NK)细胞、肥大细胞、粒细胞以及来源于骨髓的吞噬细胞。该术语还包括工程化的免疫细胞，如通过将DNA或RNA形式的外源遗传物质加入细胞的总遗传物质而被基因修饰的免疫细胞。
在本申请中，术语“质粒”通常是指细菌、酵母菌等生物中染色体或拟核以外的DNA分子，存在于细胞质中，具有自主复制能力，使其能够在子代细胞中保持恒定的拷贝数，并表达所携带的遗传信息。质粒在遗传工程研究中被用作基因的载体。
在本申请中，术语“逆转录病毒载体”通常是指可以可控并表达外源基因，但不能自我包装成有增殖能力的病毒颗粒。此类病毒多具有反转录酶。反转录病毒至少含有三种基因：gag，包含组成病毒中心和结构的蛋白质的基因；pol，包含反转录酶的基因和env，包含组成病毒外壳的基因。通过逆转录病毒转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“慢病毒载体”通常是指属于逆转录病毒的一种二倍体RNA病毒载体。慢病毒载体是以慢病毒的基因组为基础，将其中多个和病毒活性相关的序列结构去除，使其具有生物学的安全性，然后再在这个基因组骨架中引入实验所需要的目标基因的序列和表达结构，并将之制备成载体。通过慢病毒载体转染，逆转录病毒载体可将自身基因组及其携带的外源基因随机、稳定地整合入宿主细胞基因组中，例如，可将CAR分子整合进宿主细胞中。
在本申请中，术语“和/或”应理解为意指可选项中的任一项或可选项的两项。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％、或10％的范围内变动。
融合蛋白
一方面，本申请提供一种融合蛋白，其可包含膜定位蛋白P1、连接肽L1、自切割连接子L2和任意目的多肽P2。
另一方面，本申请提供了一种促进融合蛋白在细胞膜上定位的方法，其中所述融合蛋白包括P1、L2和P2，所述P1为膜定位蛋白且在其C端包含CD3ζ信号传导结构域，所述L2为自切割连接子；且所述P2为任意目的多肽，其包括以下的步骤：使所述P1的C端与L1的N端连接，所述L2的N端与所述L1的C端连接，所述L1连接肽。
本申请的发明人发现，当P1和L2之间存在连接肽L1时，能使P1蛋白高效表达，促进其在细胞膜上的定位，进而使P1和P2在L2的自切割功能下分别表达切互不影响，有效实现P1和P2的生物学功能。所述L1可以为1个或多于1个氨基酸长度的连接肽。
P1
本申请的融合蛋白可包含膜定位蛋白P1。本申请所述P1可以是任意膜定位蛋白，只要其C端包含CD3ζ信号传导结构域即可。例如，所述CD3ζ信号传导结构域可包含SEQ ID NO.6所示的氨基酸序列。
在某些情形中，P1可包含跨膜结构域。例如，所述跨膜结构域可包含源自选自下述蛋白的跨膜结构域：CD8、CD28、CD27、CD7、TRAC、TRBC、CD3ζ、CD4、4-1BB、OX40、ICOS、CTLA-4、PD-1、LAG-3、2B4和BTLA。在某些情形中，所述P1可包含共刺激结构域。例如，所述共刺激结构域可包含选自下述蛋白的共刺激结构域或其组合：CD137、CD28、OX40、ICOS、DAP10、2B4、CD27、CD30、CD40、CD40L、TIM1、CD226、DR3、SLAM、NKG2D、CD244、FceRIγ、BTLA、GITR、HVEM、CD2、NKG2C、LIGHT和DAP12。在某些情形中，所述P1可包含铰链区。例如，所述铰链区可包含源自选自下述蛋白的铰链区：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。在某些情形中，本申请所述P1还可包含抗原结合结构域。所述抗原结合结构域与肿瘤抗原特异性结合。在某些情形 中，所述肿瘤抗原可以选自下组：B-cell maturation antigen(BCMA),mesothelin(MSLN),prostate specific membrane antigen(PSMA),prostate stem cell antigen(PCSA),carbonic anhydrase IX(CAIX),carcinoembryonic antigen(CEA),CD5,CD7,CD10,CD19,CD20,CD22,CD30,CD33,CD34,CD38,CD41,CD44,CD49f,CD56,CD74,CD123,CD133,CD138,CD33/IL3Ra,CS1,C-Met,epithelial glycoprotein2(EGP 2),epithelial glycoprotein-40(EGP-40),epithelial cell adhesion molecule(EpCAM),folate-binding protein(FBP),fetal acetylcholine receptor(AChR),folate receptor-αandβ(FRαandβ),Ganglioside G2(GD2),Ganglioside G3(GD3),human Epidermal Growth Factor Receptor 2(HER-2/ERB2),Epidermal Growth Factor Receptor vIII(EGFRvIII),ERB3,ERB4,human telomerase reverse transcriptase(hTERT),Interleukin-13 receptor subunit alpha-2(IL-13Rα2),κ-light chain,kinase insert domain receptor(KDR),Lewis A(CA19.9),Lewis Y(LeY),L1 cell adhesion molecule(LlCAM),melanoma-associated antigen 1(melanoma antigen family A1,MAGE-A1),Mucin 16(Muc-16),Mucin 1(Muc-1),NKG2D ligands,cancer-testis antigen NY-ESO-1,oncofetal antigen(h5T4),tumor-associated glycoprotein 72(TAG-72),vascular endothelial growth factor R2(VEGF-R2),Wilms tumor protein(WT-1),type 1tyrosine-protein kinase transmembrane receptor(ROR1),B7-H3(CD276),B7-H6(Nkp30),Chondroitin sulfate proteoglycan-4(CSPG4),DNAX Accessory Molecule(DNAM-1),Ephrin type A Receptor 2(EpHA2),Fibroblast Associated Protein(FAP),Gp100/HLA-A2,Glypican 3(GPC3),HA-1H,HERK-V,IL-11Rα,Latent Membrane Protein 1(LMP1),MAG3,Neural cell-adhesion molecule(N-CAM/CD56),NY-ESO-1,and Trail Receptor(TRAIL R)。例如，所述肿瘤抗原可选自以下组：CD19和BCMA。在一些情况下，所述P1的抗原结合结构域可通过所述铰链区与所述跨膜结构域连接。
在某些情形中，所述P1可包含嵌合抗原受体CAR。所述CAR从N端至C端可依次包含胞外的抗原结合结构域、铰链区、跨膜结构域、共刺激结构域、信号传导结构域。
例如，如图2所示，在所述CAR中，抗原结合结构域可以为肿瘤抗原scFv，如，肿瘤抗原CD19，所述共刺激结构域可包含选自下述蛋白的共刺激结构域：4-1BB(CD137)，所述信号传导结构域可包含CD3ζ信号传导结构域，例如，本申请中所述CAR可以包含选自SEQ ID NO.5、7、8所示的氨基酸序列。
在另一些具体的情形中，本申请中所述CAR中的肿瘤抗原可以是BCMA，例如，所述 CAR可以包含选自SEQ ID NO.9-12所示的氨基酸序列。
L1
在本申请中，所述融合蛋白可包含连接肽L1，所述L1可以为任意氨基酸长度(如，1个、2个、3个、4个、5个、6个、7个、8个、9个、10个或更多个氨基酸长度)的连接肽。
在某些情况下，所述L1可以为由1-5个(如1个、2个、3个、4个或5个)氨基酸组成的连接肽。在某些情形中，所述L1的长度可选自以下组：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸和5个氨基酸。
在某些情形中，所述L1可以为由1个氨基酸组成的连接肽，例如，所述1个氨基酸可以选自以下组：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。在某些情形中，所述L1的长度可以为由1个氨基酸组成的连接肽，且所述氨基酸选自以下组：T、P、G、D、E、I、V、S和A。
在某些情形中，所述L1的长度可以为由2个氨基酸组成的连接肽，所述2个氨基酸可以为由选自下组的任意氨基酸组成的相同的或不同的2个氨基酸：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。例如，所述2个氨基酸组成的L1可包含GG；或者，可包含GS。
在另一些情形中，所述L1的长度可以为由3个氨基酸组成的连接肽，所述3个氨基酸可以为由选自下组的任意氨基酸组成的相同的或不同的3个氨基酸：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。例如，所述3个氨基酸组成的L1可包含GGG；或者，可包含GGS。在另一些情形中，所述L1的长度可以为由4个氨基酸组成的连接肽，所述4个氨基酸可以为由选自下组的任意氨基酸组成的相同的或不同的4个氨基酸：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。例如，所述4个氨基酸组成的L1可包含GGGG；或者，可包含GGGS。在另一些情形中，所述L1的长度可以为5个氨基酸，所述5个氨基酸可以为由选自下组的任意氨基酸组成的相同的或不同的5个氨基酸：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。例如，所述5个氨基酸组成的L1可包含GGGGG；或者，可包含GGGGS。
在其他情况下，所述L1的长度可以为由选自下组的6个或更多个(如7个、8个、9个、10个、12个、15个或更多个氨基酸长度)相同或不同的任意氨基酸组成的连接肽：A、R、 N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。在某些情况下，L1可被设计为富含甘氨酸、谷氨酸和/或丝氨酸残基的序列，或可参考现有文献中对于蛋白连接肽的设计思路，如Chen et al.,Adv Drug Deliv Rev.2013 Oct 15；65(10):1357–1369；Klein et al.,Protein Eng Des Sel.2014 Oct；27(10):325–330.；Liu et al.,Bioinformatics,2015,31(22):3700–3702
在某些情形中，所述P1的C端可与所述L1的N端连接。在某些情形中，所述P1的C端的CD3ζ信号传导结构域可与所述L1的N端连接。
L2
在本申请中，所述融合蛋白可包含自切割连接子L2，所述L2自N端起可依次包含内肽酶切割位点和剪切肽。所述内肽酶切割位点可以包括任意内肽酶能够识别的最短序列的切割位点。
在某些情形中，所述内肽酶切割位点可以是弗林蛋白(furin)酶切割位点。通常情况下，furin蛋白酶切割位点可包含以下序列：Arg-X-X-Arg↓，其中，X代表任意氨基酸，↓代表切割位点。在个别情况下，弗林蛋白酶可以对不完全符合上述序列的蛋白进行切割。
在某些情形中，所述剪切肽可以是2A肽。例如，剪切肽可以是选自下组的2A肽：P2A、T2A、F2A和E2A。各种2A肽的氨基酸序列如下表2所示：
表2
2A肽 氨基酸序列
T2A EGRGSLLTCGDVEENPGP
P2A ATNFSLLKQAGDVEENPGP
E2A QCTNYALLKLAGDVESNPGP
F2A VKQTLNFDLLKLAGDVESNPGP
例如，剪切肽可以是以P2A，P2A是一种自切割多肽连接序列，是在一个载体中一个转录本同时表达两个独立蛋白常用的连接序列，蛋白在翻译过程中在P2A序列的末端断裂分开，使得由P2A所连接的前后两个蛋白分开，分别发挥功能，P2A的氨基酸序列为 ATNFSLLKQAGDVEENPGP。
在另一些情形中，所述内肽酶切割位点和剪切肽之间还可包含接头，如肽接头。肽接头对L2的功能不是必要条件，但是通常能提高剪切肽诱导剪切的效率。肽接头通常是人工合成的或天然存在的，由线性氨基酸链组成。肽接头通常具有1-50个氨基酸的长度(如1-28个氨基酸、1-25个氨基酸、3-25个氨基酸)。接头可以包含重复的氨基酸序列，或天然存在的多肽序列，例如具有绞合部功能的多肽。肽接头可以促进肽正确地折叠和适当地呈递，从而实施其生物学活性。在某些实施方案中，接头可以是合成的肽接头，其被设计为富含甘氨酸、谷氨酸和/或丝氨酸残基。肽接头的实例可以包括但不限于GSG、GSGS、(GGS)n、(GGGS)n和(EAAAK)n，其中，n可以为任意正整数。
在某些情形中，本申请所述融合蛋白的L2自N端起可依次包含弗林蛋白酶切割位点和P2A。在另一些情形中，弗林蛋白酶切割位点和P2A之间还可包括肽接头GSG。例如，所述L2可包含SEQ ID NO.2所示的氨基酸序列。
在某些情形中，所述L1的C端可与所述L2的N端连接。
P2
在本申请中，所述融合蛋白可包含任意目的多肽P2。所述任意目的多肽P2可以是任何能被表达的多肽。P2可包括但不限于选自下组的多肽：荧光蛋白、膜结合多肽、糖蛋白、球状蛋白质、免疫多肽(如抗体)、毒素、抗生素、肽激素、生长因子、细胞因子、转录因子、凝血因子、RNA结合蛋白、细胞骨架蛋白、离子通道、G蛋白偶联受体、酶(如蛋白酶、激酶、磷酸酶)、疫苗、受体、配体、杀菌和/或内毒素结合蛋白、结构多肽、转运蛋白和跨膜蛋白。所述P2与所述P1可以相同或不同。
在某些情形中，所述P2可选自以下组：嵌合抗原受体CAR、细胞因子、MHC复合物的组成蛋白、标签蛋白和免疫检查点抑制剂。在某些情形中，所述P2可以包含免疫检查点抑制剂，如选自下组免疫检查点的抗体或其抗原结合片段：PD-1、CTLA-4、LAG-3和TIM-3。
在另一些情形中，所述P2可以包含细胞因子或其功能片段，例如，白细胞介素(如IL-1、IL-1α、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-9、IL-10、IL-11、IL-12)、干扰素(如IFN-α、IFN-β、IFN-γ)、趋化因子(如CC趋化因子、CXCL趋化因子、XCL1、XCL2、CX3CL1)、表皮生长因子、肝细胞生长因子、成纤维细胞生长因子、肿瘤坏死因子(如TNF- α、TNF-β)、集落刺激因子(如M-CSF、GM-CSF、G-CSF)和转化生长因子，和其它多肽因子，包括LIF和kit配体(KL)。
在另一些情形中，所述P2可以包含嵌合抗原受体CAR，例如，可以含有针对选自下组的肿瘤抗原的结合部分的CAR：B-cell maturation antigen(BCMA),mesothelin(MSLN),prostate specific membrane antigen(PSMA),prostate stem cell antigen(PCSA),carbonic anhydrase IX(CAIX),carcinoembryonic antigen(CEA),CD5,CD7,CD10,CD19,CD20,CD22,CD30,CD33,CD34,CD38,CD41,CD44,CD49f,CD56,CD74,CD123,CD133,CD138,CD33/IL3Ra,CS1,C-Met,epithelial glycoprotein2(EGP 2),epithelial glycoprotein-40(EGP-40),epithelial cell adhesion molecule(EpCAM),folate-binding protein(FBP),fetal acetylcholine receptor(AChR),folate receptor-αandβ(FRαandβ),Ganglioside G2(GD2),Ganglioside G3(GD3),human Epidermal Growth Factor Receptor 2(HER-2/ERB2),Epidermal Growth Factor Receptor vIII(EGFRvIII),ERB3,ERB4,human telomerase reverse transcriptase(hTERT),Interleukin-13 receptor subunit alpha-2(IL-13Rα2),κ-light chain,kinase insert domain receptor(KDR),Lewis A(CA19.9),Lewis Y(LeY),L1 cell adhesion molecule(LlCAM),melanoma-associated antigen 1(melanoma antigen family A1,MAGE-A1),Mucin 16(Muc-16),Mucin 1(Muc-1),NKG2D ligands,cancer-testis antigen NY-ESO-1,oncofetal antigen(h5T4),tumor-associated glycoprotein 72(TAG-72),vascular endothelial growth factor R2(VEGF-R2),Wilms tumor protein(WT-1),type 1tyrosine-protein kinase transmembrane receptor(ROR1),B7-H3(CD276),B7-H6(Nkp30),Chondroitin sulfate proteoglycan-4(CSPG4),DNAX Accessory Molecule(DNAM-1),Ephrin type A Receptor 2(EpHA2),Fibroblast Associated Protein(FAP),Gp100/HLA-A2,Glypican 3(GPC3),HA-1H,HERK-V,IL-11Rα,Latent Membrane Protein 1(LMP1),MAG3,Neural cell-adhesion molecule(N-CAM/CD56),NY-ESO-1,and Trail Receptor(TRAIL R)。
例如，本申请所述P2可包含B2M，所述B2M可包含SEQ ID NO.1所示的氨基酸序列。
例如，本申请所述P2可包含EGFRt，所述EGFRt可包含SEQ ID NO.13所示的氨基酸序列。
例如，本申请所述P2可包含GFP，所述GFP可包括SEQ ID NO.16所示的氨基酸序列。
在某些情形中，所述L2的C端可与所述P2的N端连接。
在本申请中，所述融合蛋白以N端至C端的方向依次可包含所述P1、所述L1、所述L2和所述P2。如图1所示，在所述融合蛋白中，所述P1的C端可与所述L1的N端连接，所述L1的C端可与所述L2的N端连接，所述L2的C端可与所述P2的N端连接。
例如，如图3所示，在所述融合蛋白中，所述P1可以包含CAR19，所述L2自N端到C端可以依次包含弗林蛋白furin、肽接头GSG和P2A剪切肽，所述P2可以包含B2M。如，所述融合蛋白可包含SEQ ID NO.4所示的氨基酸序列，其中，X表示任意氨基酸。
例如，在所述融合蛋白中，所述P1可以包含CAR19，所述L2自N端到C端可以依次包含弗林蛋白furin、肽接头GSG和P2A剪切肽，所述P2可以包含EGFRt。如，所述融合蛋白可包含SEQ ID NO.14所示的氨基酸序列。
核酸、载体、细胞和制备方法
另一方面，本申请提供了一种分离的核酸分子，其可编码本申请所述的融合蛋白。本申请所述编码所述融合蛋白的分离的核酸分子，其可包含SEQ ID NO.3、15(其中，“nnn”表示任意可编码L1的核苷酸)所示的核酸序列或其功能性变体。本申请所述的核酸分子可以为分离的。例如，其可以是通过以下方法产生或合成的：(i)在体外扩增的，例如通过聚合酶链式反应(PCR)扩增产生的，(ii)通过克隆重组产生的，(iii)纯化的，例如通过酶切和凝胶电泳分级分离，或者(iv)合成的，例如通过化学合成。在某些实施方式中，所述分离的核酸是通过重组DNA技术制备的核酸分子。
另一方面，本申请提供了一种载体，其可包含所述的分离的核酸分子。在本申请中，所述载体可选自质粒、逆转录病毒载体和慢病毒载体中的一种或多种。例如，本申请所述载体自5’端依次包含以下的核苷酸序列：编码所述P1的基因、编码所述L1的基因、编码所述L2的基因和编码所述P2的基因。此外，所述载体中还可包含其他基因，例如允许在适当的宿主细胞中和在适当的条件下选择该载体的标记基因。此外，所述载体还可包含允许编码区在适当宿主中正确表达的表达控制元件。这样的控制元件为本领域技术人员所熟知的，例如，可包括启动子、核糖体结合位点、增强子和调节基因转录或mRNA翻译的其他控制元件等。在某些实施方式中，所述表达控制序列为可调的元件。所述表达控制序列的具体结构可根据物种或细胞类型的功能而变化，但通常包含分别参与转录和翻译起始的5’非转录序列和5’ 及3’非翻译序列，例如TATA盒、加帽序列、CAAT序列等。例如，5’非转录表达控制序列可包含启动子区，启动子区可包含用于转录控制功能性连接核酸的启动子序列。本申请所述的一种或多种核酸分子可以与所述表达控制元件可操作地连接。所述载体可以包括，例如质粒、粘粒、病毒、噬菌体或者在例如遗传工程中通常使用的其他载体。例如，所述载体可以包括表达载体，如慢病毒目的表达质粒，和辅助载体，如包装辅助质粒。例如，所述载体可以为慢病毒载体。慢病毒载体包含长末端重复序列5’LTR和截短的3’LTR，RRE，rev应答元件(cPPT)，中央终止序列(CTS)和翻译后调控元件(WPRE)。所述分子可以通过BamHI和SalI酶切构建到慢病毒载体上。
另一方面，本申请提供了一种免疫细胞，其可包含或表达本申请所述的融合蛋白，所述的核酸分子，和/或所述的载体。在本申请中，所述的免疫细胞包括T淋巴细胞、B细胞、天然杀伤细胞、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞。在某些情形中，所述免疫细胞可包括T淋巴细胞。所述T淋巴细胞可包括胸腺细胞、天然T淋巴细胞、未成熟T淋巴细胞、成熟T淋巴细胞、静息T淋巴细胞或活化的T淋巴细胞。所述T细胞可以是辅助T细胞(Th)，例如辅助T细胞1(Th1)或辅助T细胞2(Th2)细胞。所述T淋巴细胞可以是CD4+辅助T细胞(HTL；CD4+T细胞)、细胞毒性T细胞(CTL；CD8+T细胞)、肿瘤浸润细胞毒性T细胞(TIL；CD8+T细胞)、CD4+/CD8+T细胞、CD4-/CD8-T细胞或任何其他T淋巴细胞亚型。在某些实施方式中，所述T淋巴细胞可来自外周血细胞、脐带血细胞和/或白细胞。在某些实施方式中，所述T细胞可以是Jurkat细胞。
在本申请中，所述免疫细胞可包括B细胞。在某些情形中，所述B细胞可包括效应B细胞(浆细胞)、记忆B细胞。所述B细胞可包括B2细胞、B1细胞、边缘区B细胞、滤泡B细胞、调节性B细胞。
在本申请中，所述免疫细胞可包括巨噬细胞。所述B细胞可包括I型巨噬细胞(M1)、II型巨噬细胞(如M2a、M2B、M2c)。
在本申请中，所述免疫细胞可包括NK细胞。在某些情形中，所述NK细胞可包括CD56bright和CD56dim。在某些情形中，所述NK细胞可包括NK1和NK2。在某些情形中，所述NK细胞可包括A-NK和NA-NK。
另一方面，本申请提供了一种制备免疫效应细胞的方法，其可包括向免疫效应细胞中引入本申请所述的载体。例如，可将本申请所述的载体引入所述免疫效应细胞中，例如T淋巴 细胞、B细胞、巨噬细胞或天然杀伤(NK)细胞。在某些实施方式中，每种或每个细胞可包含一个或一种本申请所述的载体。在某些实施方式中，每种或每个细胞可包含多个(例如，2个或以上)或多种(例如，2种或以上)本申请所述的载体。例如，可将本申请所述的载体引入所述细胞中。例如，可以通过逆转录病毒载体进行转染免疫效应细胞，将带有编码所述融合蛋白核酸的病毒基因组能整合到宿主基因组，保证目的基因长期、稳定地表达。又例如，利用转座子，通过携带编码所述融合蛋白的核酸的质粒和携带转座酶的质粒导入到靶细胞中。又例如，可以通过基因编辑的方式(例如CRISPR/Cas9)将核酸分子添加进基因组中。在本申请中，可通过本领域已知的方法将本申请所述的带有编码所述融合蛋白的核酸的载体引入所述细胞中，例如电穿孔(Neon电转仪)、脂质体法转染等。
另一方面，本申请提供了一种药物组合物，其可包含本申请所述的免疫细胞和药学上可接受的载体。所述药学上可接受的佐剂可以包括缓冲剂、抗氧化剂、防腐剂、低分子量多肽、蛋白质、亲水聚合物、氨基酸、糖、螯合剂、反离子、金属复合物和/或非离子表面活性剂等。在本申请中，所述药物组合物可被配制用于口服给药，静脉内给药(例如，静脉注射，I.V.)，肌肉内给药(例如，肌肉注射，I.M.)，在肿瘤部位的原位给药，吸入，直肠给药，阴道给药，经皮给药或通过皮下储存库给药。
本申请所述的药物组合物可以包含治疗有效量的所述融合蛋白。所述治疗有效量是能够预防和/或治疗(至少部分治疗)患有或具有发展风险的受试者中的病症或病症(例如癌症)和/或其任何并发症而所需的剂量。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的融合蛋白、制备方法和用途等，而不用于限制本申请发明的范围。
实施例
实施例1.慢病毒载体的构建
构建包含本申请融合蛋白P1-L1-L2-P2基因的慢病毒载体。作为阴性对照，构建了P1-L2-P2慢病毒载体，该载体为不含L1的序列，P1与L2直接相连。P1-L1-L2-P2的示例序列1“CAR19-L1-Furin-GSG-P2A-B2M”：P1为CAR19(SEQ ID NO.5)，P2为改造的B2M蛋白(SEQ ID NO.1)。L2为Furin-GSG-P2A(SEQ ID NO.2)，在L2中，弗林蛋白(Furin) 剪切位点的氨基酸序列为RRKR，肽接头为GSG，剪切肽为P2A。其中，不同融合蛋白的慢病毒载体中，L1分别为选自下组的单个氨基酸：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。形成的全基因序列的核苷酸序列如SEQ ID NO.3所示(nnn代表编码L1的任意的密码子)，氨基酸序列如SEQ ID NO.4所示(X代表任意一个氨基酸)，示意图如图3。
在“CAR19-L1-Furin-GSG-P2A-P2”的连接方式中，除B2M外，设计了不同的P2，分别是代表细胞膜定位的蛋白EGFRt和细胞质具备游离属性的绿色荧光蛋白GFP的序列。L1选择了具有代表性的氨基酸，如表3所示。
表3连接不同P2的CAR19-L1-Furin-GSG-P2A-P2结构
Figure PCTCN2020134961-appb-000001
示例序列2“CAR19-L1-Furin-GSG-P2A-EGFRt”：P1为CAR19(SEQ ID NO.5)，P2为EGFRt(SEQ ID NO.13)。L2为Furin-GSG-P2A(SEQ ID NO.2)。L1为任意单个氨基酸。形成的全基因序列的核苷酸序列如SEQ ID NO.15所示(nnn代表编码L1的任意的密码子)，氨基酸序列如SEQ ID NO.14所示(X代表任意一个氨基酸)。
示例序列3“CAR19-L1-Furin-GSG-P2A-GFP”：P1为CAR19(SEQ ID NO.5)，P2为GFP(SEQ ID NO.16)。L2为Furin-GSG-P2A(SEQ ID NO.2)。L1为任意单个氨基酸。形成的全基因序列的核苷酸序列如SEQ ID NO.19所示(nnn代表编码L1的任意的密码子)，氨基酸序列如SEQ ID NO.18所示(X代表任意一个氨基酸)。
病毒表达质粒(Addgene ID：#12252)用BamHI/SalI酶切作为骨架，上述各种P1-L1-L2-P2序列分别全基因合成后，分别连入已酶切的病毒表达骨架中。对照组CAR19序列、不含L1的序列P1-L2-P2同样用BamHI/SalI连入病毒表达骨架中。
实施例2.慢病毒制备方法
采用三质粒系统：如上述方法构建的慢病毒目的表达质粒，包装辅助质粒psPAX2(Addgene ID：#12260)和pMD2.G(Addgene ID：#12259)。在HEK293T细胞(购自中科院上海细胞研究所)中进行病毒包装。制备流程如下：将冻存的工作细胞中的HEK293T细胞复苏，用DMEM培养基(+10％FBS+1％P/S)(Cellgro 10-013-CMR)置于10cm培养皿培养，复苏第2天后换液。待细胞长满后开始传代(通常1个培养皿长满之后可传至5个培养皿)，将细胞传代4代后可进行质粒转染。转染采用PEI作为转染试剂，PEI：质粒(质量比)＝2:1的条件转染。将质粒与PEI的混合物加入到Opti-MEM培养基(Gibco,cat#31985-070)中，再将此混合溶液加入到传代至第4代的HEK293T细胞中。转染6小时后用2％FBS的新鲜培养基换液，之后继续培养至72小时，收集HEK293T细胞上清。收集的病毒上清采用超离的方式(82200g，4-8℃离心2小时)进行浓缩，浓缩好的病毒用0.22μm滤膜过滤除菌后重悬待用。
实施例3.慢病毒基因转导方法
T细胞选择Jurkat模式细胞株(Jurkat细胞购自中科院上海细胞研究所)。取3×10 5个细胞于24孔培养板，以细胞数的3倍量加入制备好的慢病毒，即MOI＝3(Multiplicity of Infection，感染复数，病毒量与细胞数的比值)，补充培养基至500μL。24h后补充培养基至1mL，以利于细胞生长。
所用培养基为完全培养基，ImmunoCult TM-XF T Cell Expansion Medium(Stem Cell  Technology，cat#10981)。
实施例4.病毒转染效率检测
利用实施例1所述P1-L1-L2-P2的示例序列1，其中L1为20种氨基酸中的任意一个，或为1-5个G。
当L1为1个氨基酸长度时，检测病毒转染后，CAR19表达的荧光强度(MFI)。病毒转染3天后，取出少量细胞(约1×10 5个)，加入1ml PBS(Gibco，cat#C10010500BT)清洗细胞1遍，用100μL PBS重悬，加入3μL抗FMC63的抗体一抗(检测CAR19)4℃孵育30min后，加入1ml PBS混匀，350g离心3min收集细胞去上清，顺次加入0.5μL二抗(一抗、二抗来自抗-Mouse FMC63试剂盒，上海星湾，R19PB-100的产品)至细胞中，混合均匀，4℃孵育30min，加入1ml PBS混匀，350g离心3min收集细胞去上清，加入200μL PBS重悬细胞，流式细胞仪上机检测。流式结果如图5-图8所示，图4为统计结果。
图5-图8为不同的1个氨基酸的L1的流式细胞术检测结果。图4为在模式T细胞株Jurkat中，20种不同氨基酸作为L1对CAR表达荧光强度的影响的统计，反映了图5-图8中的结果。CAR19对照组为细胞只表达CAR的组别，该组CAR表达的平均荧光强度(MFI)设置为1，其他组相对于CAR19进行归一化。使用CAR单独表达的MFI进行归一化计算，纵坐标显示每个不同构建测出的CAR的MFI相对于CAR单独表达时的MFI的倍数，即(L1为某个氨基酸时的CAR MFI)/(CAR19对照组的CAR MFI)。不含L1组作为阴性对照，该组CAR结构C端的CD3ζ直接与连接序列连接，没有L1(即，没有任何氨基酸间隔)，该组CAR表达强度明显降低，约是CAR19对照组的20％左右。
其他组别显示，引入一个不同的氨基酸可以明显改变CAR的表达强度。其中，T、P、G、D、E、I、V、S、A能够有效恢复CAR的表达。
图9显示将实施例1所述P1-L1-L2-P2的示例序列1中的L1分别替换为1个G，GG(2G)，GGG(3G)，GGGG(4G)，GGGGG(5G)后，利用流式细胞术检测CAR的荧光强度的结果。结合图4的统计结果显示，增加氨基酸的数量(大于1个)也可以恢复或提高CAR的表达强度。
将实施例1所述P1-L1-L2-P2的示例序列2中的L1分别替换为1个G，T，P，R，GG (2G)，GGG(3G)后，利用流式细胞术检测CAR的荧光强度的结果。结合图10A的统计结果显示，不含L1的组限制影响CAR的表达，筛选得到的较优的L1，如G，T，P，R或多个氨基酸如2G，3G等，可以有效恢复CAR的表达强度。
将实施例1所述P1-L1-L2-P2的示例序列3中的L1分别替换为1个T，P，R，GGG(3G)后，利用流式细胞术检测CAR的荧光强度的结果。结合图10B的统计结果显示，不含L1的组限制影响CAR的表达，筛选得到的较优的L1，如T，P，R或多个氨基酸3G等，可以有效恢复CAR的表达强度。
结果说明在不同种类的P2的情况下，L1均能恢复或提高CAR的表达强度。
L2是自切割连接子，除P2A外，L1也适用于其他的连接子如T2A,E2A和F2A等，下图以T2A为例进行检测，结构如表4所示。
表4 L2包含T2A的CAR19-L1-Furin-GSG-T2A-P2结构
Figure PCTCN2020134961-appb-000002
结果如图11所示，当L2是T2A时，不含L1的组限制影响CAR的表达，而筛选得到的较优的L1，如T，P，R或是多个氨基酸如3G等，可以有效恢复CAR的表达强度。
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (54)

  1. 融合蛋白，其以N端至C端的方向依次包含P1、L1、L2及P2，
    其中所述P1为膜定位蛋白且在其C端包含CD3ζ信号传导结构域；
    所述L1为连接肽；
    所述L2为自切割连接子；且
    所述P2为任意目的多肽。
  2. 根据权利要求1所述的融合蛋白，其中所述L1的长度选自以下组：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸和5个氨基酸。
  3. 根据权利要求1-2中任一项所述的融合蛋白，其中所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其中所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：T、P、G、D、E、I、V、S和A。
  5. 根据权利要求1-4中任一项所述的融合蛋白，其中所述L1的长度为2个氨基酸。
  6. 根据权利要求5所述的融合蛋白，其中所述L1包含选自以下组的氨基酸序列：GG和GS。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中所述L1的长度为3个氨基酸。
  8. 根据权利要求7所述的融合蛋白，其中所述L1包含选自以下组的氨基酸序列：GGG和GGS。
  9. 根据权利要求1-8中任一项所述的融合蛋白，其中所述L1的长度为4个氨基酸。
  10. 根据权利要求9所述的融合蛋白，其中所述L1包含选自以下组的氨基酸序列：GGGG和GGGS。
  11. 根据权利要求1-10中任一项所述的融合蛋白，其中所述L1的长度为5个氨基酸。
  12. 根据权利要求11所述的融合蛋白，其中所述L1包含选自以下组的氨基酸序列：GGGGG和GGGGS。
  13. 根据权利要求1-12中任一项所述的融合蛋白，其中所述CD3ζ的信号传导结构域包含SEQ ID NO.6所示的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的融合蛋白，其中所述P1的C端与L1的N端连接。
  15. 根据权利要求1-14中任一项所述的融合蛋白，其中所述P1包含跨膜结构域。
  16. 根据权利要求15所述的融合蛋白，其中所述跨膜结构域包含源自选自下述蛋白的跨膜结构域：CD8、CD28、4-1BB、CD4、CD27、CD7、PD-1、TCRα、TCRβ、TCRγ、TCRδ、CD3ε、CD3δ、CD3γ、CD3ζ、IL2受体、CD5、ICOS、OX40、NKG2D、2B4、CD244、FcεR、FcεRIγ、BTLA、CD30、GITR、HVEM、DAP10、CD2、NKG2C、LIGHT、DAP12，CD40L、TIM1、CD226、DR3、CD45、CD80、CD86、CD9、CD16、CD22、CD33、CD37、CD64、CD134、CD137、CD154、SLAM、CTLA-4和LAG-3。
  17. 根据权利要求1-16中任一项所述的融合蛋白，其中所述P1包含共刺激结构域。
  18. 根据权利要求17所述的融合蛋白，其中所述共刺激结构域包含选自下述蛋白的共刺激结构域或其组合：CD28、CD137、CD27、CD2、CD7、CD8、CD80、CD86、OX40、CD226、DR3、SLAM、CDS、ICAM、NKG2D、NKG2C、B7-H3、2B4、FcεRIγ、BTLA、GITR、HVEM、DAP10、DAP12、CD30、CD40、CD40L、TIM1、PD-1、PD-L1、PD-L2、4-1BBL、OX40L、ICOS-L、CD30L、CD70、CD83、HLA-G、MICA、MICB、淋巴毒素β受体、LFA-1、LIGHT、JAML、CD244、CD100、ICOS、CD83的配体、CD40和MyD88。
  19. 根据权利要求1-18中任一项所述的融合蛋白，其中所述P1包含铰链区。
  20. 根据权利要求19所述的融合蛋白，其中所述铰链区包含源自选自下述蛋白的铰链区：CD8、CD28、IgG、4-1BB、CD4、CD27、CD7、PD-1和CH2CH3。
  21. 根据权利要求1-20中任一项所述的融合蛋白，其中所述P1包含抗原结合结构域。
  22. 根据权利要求21所述的融合蛋白，其中所述铰链区连接所述抗原结合结构域和所述跨膜结构域。
  23. 根据权利要求21-22中任一项所述的融合蛋白，其中所述抗原结合结构域与肿瘤抗原特异性结合。
  24. 根据权利要求23所述的融合蛋白，其中所述肿瘤抗原选自以下组：CD19和BCMA。
  25. 根据权利要求1-24中任一项所述的融合蛋白，其中所述P1包含嵌合抗原受体CAR。
  26. 根据权利要求1-25中任一项所述的融合蛋白，其中所述L1的C端与所述L2的N端连接。
  27. 根据权利要求1-26中任一项所述的融合蛋白，其中所述L2自N端起依次包含内肽酶切割位点和剪切肽。
  28. 根据权利要求1-27中任一项所述的融合蛋白，其中所述内肽酶切割位点和剪切肽之间包含肽接头。
  29. 根据权利要求28所述的融合蛋白，其中所述肽接头包含选自以下组的氨基酸序列：GSG。
  30. 根据权利要求1-29中任一项所述的融合蛋白，其中所述L2包含弗林蛋白(furin)酶切割位点。
  31. 根据权利要求1-30中任一项所述的融合蛋白，其中所述L2包含剪切肽，所述剪切肽选自以下组：P2A、T2A、F2A和E2A。
  32. 根据权利要求1-31中任一项所述的融合蛋白，其中所述L2自N端起依次包含弗林蛋白酶切割位点和P2A。
  33. 根据权利要求1-32中任一项所述的融合蛋白，其中所述L2包含SEQ ID NO.2或SEQ ID NO.17所示的氨基酸序列。
  34. 根据权利要求1-33中任一项所述的融合蛋白，其中所述L2的C端与所述P2的N端连接。
  35. 根据权利要求1-34中任一项所述的融合蛋白，其中所述P2与所述P1相同或不同。
  36. 根据权利要求1-35中任一项所述的融合蛋白，其中所述P2选自以下组：嵌合抗原受体CAR、细胞因子、MHC复合物的组成蛋白、标签蛋白和免疫检查点抑制剂。
  37. 分离的核酸分子，其编码权利要求1-36中任一项所述的融合蛋白。
  38. 载体，其包含权利要求37所述的分离的核酸。
  39. 根据权利要求38所述的载体，其自5’端依次包含以下的核苷酸序列：编码所述P1的基因、编码所述L1的基因、编码所述L2的基因和编码所述P2的基因。
  40. 免疫细胞，其包含或表达权利要求1-36中任一项所述的融合蛋白，权利要求37所述的核酸分子，和/或权利要求38所述的载体。
  41. 根据权利要求40所述的免疫细胞，其包括T细胞、B细胞、天然杀伤细胞(NK细胞)、巨噬细胞、NKT细胞、单核细胞、树突状细胞、粒细胞、淋巴细胞、白细胞和/或外周血单个核细胞。
  42. 药物组合物，其包含权利要求40-41中任一项所述的免疫细胞和药学上可接受的载体。
  43. 促进融合蛋白在细胞膜上定位的方法，其中所述融合蛋白包括P1、L2和P2，所述P1为膜定位蛋白且在其C端包含CD3ζ信号传导结构域，所述L2为自切割连接子；且所述P2为任意目的多肽，
    其包括以下的步骤：使所述P1的C端与L1的N端连接，所述L2的N端与所述L1的C端连接，所述L1为连接肽。
  44. 根据权利要求43所述的方法，其中所述L1的长度选自以下组：1个氨基酸、2个氨基酸、3个氨基酸、4个氨基酸和5个氨基酸。
  45. 根据权利要求43-44中任一项所述的方法，其中所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：A、R、N、D、C、Q、E、G、H、I、L、K、M、F、P、S、T、W、Y和V。
  46. 根据权利要求43-45中任一项所述的方法，其中所述L1的长度为1个氨基酸，且所述氨基酸选自以下组：T、P、G、D、E、I、V、S和A。
  47. 根据权利要求43-46中任一项所述的方法，其中所述L1的长度为2个氨基酸。
  48. 根据权利要求47所述的方法，其中所述L1包含选自以下组的氨基酸序列：GG和 GS。
  49. 根据权利要求43-48中任一项所述的方法，其中所述L1的长度为3个氨基酸。
  50. 根据权利要求49所述的方法，其中所述L1包含选自以下组的氨基酸序列：GGG和GGS。
  51. 根据权利要求43-50中任一项所述的方法，其中所述L1的长度为4个氨基酸。
  52. 根据权利要求51所述的方法，其中所述L1包含选自以下组的氨基酸序列：GGGG和GGGS。
  53. 根据权利要求43-52中任一项所述的方法，其中所述L1的长度为5个氨基酸。
  54. 根据权利要求53所述的方法，其中所述L1包含选自以下组的氨基酸序列：GGGGG和GGGGS。
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