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WO2017117890A1 - Il-12/cd107a融合蛋白及其制法和用途 - Google Patents

Il-12/cd107a融合蛋白及其制法和用途 Download PDF

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WO2017117890A1
WO2017117890A1 PCT/CN2016/080417 CN2016080417W WO2017117890A1 WO 2017117890 A1 WO2017117890 A1 WO 2017117890A1 CN 2016080417 W CN2016080417 W CN 2016080417W WO 2017117890 A1 WO2017117890 A1 WO 2017117890A1
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protein
cd107a
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cells
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杨世成
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杨世成
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    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/20—Interleukins [IL]
    • 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
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • 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
    • 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
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of medicine, in particular to a high-efficiency and low-toxic tumor therapeutic agent based on transient expression of IL-12 membrane migration, a preparation method thereof and use thereof.
  • IL-12 is a very important immune stimulating factor.
  • IL-12 is a heterodimeric cytokine linked by a covalent bond, consisting of the p35 and p40 subunits, secreted by activated immune cells in vivo.
  • IL-12 is an important cellular immune regulatory factor that acts through NK cells and CTLs in immunity against infection and malignancy.
  • IL-12 has a significant antitumor effect, it is extremely limited due to systemic systemic administration which causes systemic toxic side effects.
  • Prior to the present invention the clinical application of IL-12 was greatly limited in clinical application due to the systemic toxic side effects induced by it and the accidental death of two patients caused by systemic administration in early clinical trials.
  • the present inventors have attempted to maximize the antitumor effect by genetically modifying anti-tumor T cells to continuously secrete IL-12, but for unknown reasons (a possible cause is the side effects of IL-12) T cells that have been genetically modified by sustained secretion of the IL-12 gene are not efficiently amplified in vitro and cause a large number of T cell apoptosis.
  • Tumors have become the first killer of human health beyond cardiovascular disease, and there is a lack of IL-12-based antitumor drugs that are highly effective and have low side effects. Therefore, there is an urgent need in the art to develop a tumor therapeutic drug that is highly effective and has low toxic side effects.
  • the object of the present invention is to provide a tumor therapeutic drug with high efficiency and low toxic side effects, and a preparation method and application thereof.
  • the present invention constructs a series of fusion proteins of IL-12/CD107a, including the entire sequence of IL-12 (including the secretory fragment of IL-12), and a link to the N-terminus of CD107a at the C-terminus of IL-12 Molecular strategies for the attachment of peptides (SGSG) to the full length sequence of CD107a and CD107a with the secreted peptide removed.
  • the experimental data show that the above two fusion protein genes can enhance the anti-tumor response in vitro and in vivo after anti-tumor CTL, and the CD107a fusion protein without secretory peptide can not only prolong the survival of tumor-bearing mice, but also anti-tumor for gene modification.
  • the in vitro expansion of T cells has no obvious toxic and side effects, and it is an anti-tumor synergistic solution with very clinical application prospects.
  • FIG. 1 Schematic diagram of the synergistic molecular mechanism of the anti-tumor CTL expression fusion protein hscIL-12/CD107a.
  • CD107a also known as LAMP1
  • LAMP1 LAMP1
  • N-terminus is located on the side of the lysosomal vesicle, and the C-terminus is a small stretch of peptide, facing the cytoplasmic side.
  • T cells When anti-tumor T cells recognize tumor antigens, lysosomes fuse with T cell membranes to produce degranulation effects of T cells or NK cells, ie T cell or NK cells release effector granzymes (Granzyme B) and perforin (perforin) And to achieve the process of killing tumor cells.
  • effector granzymes Gramzyme B
  • perforin perforin
  • the present invention constructs a series of fusion proteins of IL-12/CD107a, including the entire sequence of IL-12 (including the secretory fragment of IL-12). And at the C-terminus of IL-12 and the N-terminus of CD107a, a linker peptide is fused to the full-length CD107a molecule and the CD107a molecule that removes the secreted peptide, and when the T cell recognizes the tumor cell, the IL-12 molecule The anti-tumor synergistic response is achieved by migration of CD107a to the surface of the T cell membrane.
  • FIG. 1 Construction of the hscIL-12/CD107a fusion protein series and transduction of anti-tumor T cell mediated expression.
  • the genes to be expressed namely MART-1 TCR and the IL-12/CD107a fusion protein series.
  • the 5' and 3' LTR (long terminal repeat) of this vector was transformed into SIN-LTR (self-inactivating-LTR), aiming at reducing the probability of lentiviral recombination, enhancing safety performance, and combining WPRE to terminate RNA transcription.
  • SIN-LTR self-inactivating-LTR
  • C Expression of IL-12 molecules on the surface of anti-tumor T cell membranes. After co-culture for 4 hours, the expression of hscIL-12 on the surface of T cell membrane was detected by flow cytometry.
  • the experimental data showed that the IL-12 on the surface of the cell membrane was detected only in the fusion protein group expressing IL-12/CD107a and the MHC A2+ reaction group expressing MART-1, and the surface of the secreted IL-12 membrane was not expressed.
  • FIG. 3 Synergistic response of lentiviral vectors expressing hscIL-12/CD107a transduced T cells. As shown in the figure, T cells were sequentially transduced with anti-tumor TCR and hscIL-12/CD107a fusion protein series for 14 days, and co-cultured with tumor cells 526 and 938. After 16 hours, the expression levels of IFN ⁇ and IL-12 in the supernatant were passed. ELISA kit detection.
  • the sequential transfer to the fusion protein group showed a significant anti-tumor synergistic response, that is, secretion of high levels of IFN ⁇ ; however, in the experimental supernatant, only the C1 group (sustained secretion of IL-12 group) can be detected.
  • C1 group sustained secretion of IL-12 group
  • * indicates that p ⁇ 0.001 compared with the control group.
  • the C2 group transduced with IL-12/CD107a was significantly reduced compared with the C3 group transfected with IL-12/(no signal peptide) without secretion peptide CD107a, **, P ⁇ 0.01.
  • the C3 group was close to the amplification factor, and no significant difference was observed.
  • Activation was carried out in the presence of IL-2 (5 IU/ml); on day 2, T cells were transduced with the lentiviral vector shown in the figure, and then cultured for 6 days, cells were collected, and injected into the tail vein of the mice (IV) 5X 106 T cells.
  • the asterisk (*) indicates that the experimental group was compared with the other groups, p ⁇ 0.001; the C3 group was compared with the C2 group (**), and the survival days of the C3 group were significantly prolonged, statistically significant, p ⁇ 0.05.
  • IL-12 is present in the lysosome of the cytoplasm, and no expression can be detected on the cell membrane surface.
  • IL-12 is expressed in T cells, it is transiently and effectively displayed on the surface of T cells only when the anti-tumor CTL recognizes the tumor.
  • the IL-12/deletion secreting peptide CD107a fusion protein series has little effect on the viability of T cells, and the anti-tumor T cells carrying the fusion protein of the invention are close to tumor cells, IL-12 It is transiently expressed on the surface of the cell membrane, resulting in IL-12 located on the surface of the cell membrane to act more effectively and safely on tumor cells.
  • IL-12 by attacking the local part of the tumor by T cells and changing the immune microenvironment of the T cell-tumor tissue by IL-12 which transiently migrates on the cell surface, synergistically and effectively achieve the maximization of the anti-tumor immunity effect, and is extremely remarkable. Reduce the toxic side effects of IL-12.
  • the present invention has been completed on this basis.
  • the present inventors developed a novel fusion protein of IL12/deletion secreting CD107a by expressing IL-12 by CD107a, and modified anti-tumor T cells by a lentiviral gene.
  • the experimental data showed that the IL12/deletion secreting peptide CD107a lentiviral gene modified anti-tumor T cells transiently expressed on the surface of T cells when the anti-tumor CTL recognized the tumor, and the response to the tumor antigen showed an effect of enhancing the immune response.
  • the preclinical tumor-bearing mouse model showed that the modified T cells could significantly prolong the survival of tumor-bearing mice, and no obvious cytotoxic effects were found.
  • T cells expressing the fusion protein by the transient membrane are effective in killing tumor cells on the one hand, and have been confirmed to have safety in vitro and in vivo on the other hand.
  • the modified T cells themselves appear to be only slightly adversely affected or substantially unaffected by the expressed IL-12 (in vitro, the fusion protein of the invention is directed against T cells compared to T cells that continue to secrete IL-12) Amplification has no significant effect).
  • head refers to the N-terminus of a polypeptide or fragment thereof, particularly the N-terminus of a wild-type polypeptide or fragment thereof.
  • tail refers to the C-terminus of a polypeptide or fragment thereof, particularly the C-terminus of a wild-type polypeptide or fragment thereof.
  • containing includes “including”, “consisting essentially of”, “consisting essentially of”, and “consisting of”;
  • the subordinate concepts of “consisting of”, “consisting essentially of” and “consisting of” are “contained,” “having,” or “including.”
  • CD107a also known as LAMP1
  • LAMP1 is a transmembrane protein expressed on lysosomes. Its N-terminus is located on the side of the lysosomal vesicle. (Eskelinen 2006) is a small stretch of peptide at the C-terminus, facing the cytosol. Side, when anti-tumor T cells recognize tumor antigens, lysosomes fuse with T cell membranes to produce degranulation effects of T cells or NK cells, ie T cell or NK cell release effector granzyme (Granzyme B) and perforin (perforin) to achieve the process of killing tumor cells.
  • the property of CD107a to migrate to the T cell membrane has been widely used as an indicator of the T cell killing function when T cells react with tumor cells.
  • Secreted peptide-deficient CD107a The secreted peptide-deficient CD107a of the present invention is mature CD107a (Seq ID NO. 10), ie, the secreted peptide (signal peptide) of wild-type CD107a (Seq ID NO.: 8) was removed by gene editing. Amino acid sequence --MAAPGSARRPLLLLLLLLLLGLMHCASAA, linked to IL-12 by gene synthesis into a novel fusion protein.
  • the CD107a may be from a mammal (human or non-human mammal) or may be derived from other eukaryotic species.
  • CD107a is wild-type CD107a from humans.
  • IL-12 is a very important immune stimulating factor.
  • IL-12 is a heterodimeric cytokine linked by a covalent bond, consisting of the p35 and p40 subunits, secreted by activated immune cells in vivo.
  • the IL-12 may be derived from a human or non-human mammal, and may be a full-length, mature form of IL-12, or an active fragment thereof.
  • the IL-12 (or IL-12 protein element) can be a single subunit or multiple subunits.
  • the first protein element may comprise one or more (e.g., two) subunits of an IL-12 protein.
  • the first protein element comprises the IL-12 protein P40 and P35 subunits joined together.
  • the manner of connecting the P40 and P35 subunits is not particularly limited, and includes "head-to-head”, “head-to-tail”, “tail-head”, and “tail-tail”, wherein “ “Head” refers to the N-terminus of a polypeptide, and “tail” refers to the C-terminus of a polypeptide.
  • a linker may or may not be present between the P40 and P35 subunits.
  • the linker is a flexible 4-20 amino acid linker, more preferably the linker is GGGGGGS (G6S) (SEQ ID NO.: 14).
  • the bifunctional fusion proteins of the invention may optionally contain a peptide linker.
  • the size and complexity of the peptide linker may affect the activity of the protein.
  • the peptide linker should be of sufficient length and flexibility to ensure that the two proteins attached have sufficient freedom in space to perform their function. At the same time, the effect of the formation of an alpha helix or a beta sheet in the peptide linker on the stability of the fusion protein is avoided.
  • the length of the linker peptide is generally from 0 to 15 amino acids, preferably from 1 to 15 amino acids.
  • linker peptides examples include, but are not limited to, the ligated fragments set forth in SEQ ID NO.: 11-14.
  • Signal peptides and leader peptides The fusion proteins of the invention may also contain other elements, including but not limited to: signal peptides, leader peptides and the like.
  • the fusion protein contains a signal peptide.
  • Representative examples include, but are not limited to, a signal peptide of the human IL-12P40 subunit.
  • the fusion protein is an isolated protein that is not associated with other proteins, polypeptides or molecules, is expressed by a recombinant host cell, or is isolated or Purified product.
  • fusion protein in the present invention, "recombinant bifunctional fusion protein", "protein of the present invention”, “fusion protein of the present invention”, “bifunctional fusion protein”, “IL-12-deficient secretion peptide CD107a fusion protein”, “IL-12” “Non-secretory peptide CD107a fusion protein” is used interchangeably and refers to a structure having the structure of Formula Ia, or the structure described in IIa, ie, a fusion protein comprising a protein element comprising a IL-12 protein element, a deletion secretory peptide CD107a, and a linker element. .
  • a representative example is the IL12-deficient secretory peptide CD107a.
  • the protein of the invention may be a monomer or a multimer (e.g., a dimer) formed from a monomer. Furthermore, it is to be understood that the term also encompasses active fragments and derivatives of fusion proteins.
  • a preferred fusion protein has the sequence set forth in SEQ ID NO.: 4, wherein positions 1-328 are the P40 subunit of IL-12; positions 329-335 are G6S linker peptides; and positions 336-532 are IL. P35 subunit of -12; SGSG linker peptide (SEQ ID NO.: 12) at positions 533-537; and signal-free peptide CD107a amino acid sequence at positions 537-924.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is isolated and purified, as separated from other substances present in the natural state.
  • isolated recombinant fusion protein means that the recombinant fusion protein is substantially free of other proteins, lipids, carbohydrates or other materials with which it is naturally associated.
  • One skilled in the art can purify recombinant fusion proteins using standard protein purification techniques. Substantially pure proteins produce a single major band on a non-reducing polyacrylamide gel.
  • the polynucleotide of the present invention may be in the form of DNA or RNA.
  • DNA forms include cDNA, genomic DNA or synthetic DNA.
  • DNA can be single-stranded or double-stranded.
  • the DNA can be a coding strand or a non-coding strand.
  • the present invention also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present invention.
  • Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants, and insertion variants.
  • an allelic variant is an alternative form of a polynucleotide that may be a substitution, deletion or insertion of one or more nucleotides, but does not substantially alter the function of the polypeptide encoded thereby.
  • the term "primer” refers to a generic term for a oligodeoxynucleotide that, in pairing with a template, can be used to synthesize a DNA strand complementary to a template under the action of a DNA polymerase.
  • the primer may be native RNA, DNA, or any form of natural nucleotide.
  • the primer may even be a non-natural nucleotide such as LNA or ZNA.
  • the primer is “substantially” (or “substantially") complementary to a particular sequence on a strand on the template.
  • the primer must be sufficiently complementary to a strand on the template to initiate extension, but the sequence of the primer need not be fully complementary to the sequence of the template.
  • a sequence that is not complementary to the template is added to the 5' end of a primer complementary to the template at the 3' end, such primers are still substantially complementary to the template.
  • the non-fully complementary primers can also form a primer-template complex with the template for amplification.
  • the fusion protein of the present invention can conveniently prepare by various known methods. These methods are, for example but not limited to, recombinant DNA methods, artificial synthesis, and the like.
  • the full length nucleotide sequence of the element of the fusion protein of the present invention (e.g., IL12 or no signal peptide CD107a) or a fragment thereof can be generally obtained by a PCR amplification method, a recombinant method or a synthetic method.
  • primers can be designed according to published nucleotide sequences, particularly open reading frame sequences, and used as commercially available cDNA libraries or cDNA libraries prepared by conventional methods known to those skilled in the art.
  • the template is amplified to obtain the relevant sequence. When the sequence is long, it is often necessary to perform two or more PCR amplifications, and then the amplified fragments are spliced together in the correct order.
  • the recombinant sequence can be used to obtain the relevant sequences in large quantities. This is usually done by cloning it into a vector, transferring it to a cell, and then isolating the relevant sequence from the proliferated host cell by conventional methods.
  • synthetic sequences can be used to synthesize related sequences, especially when the fragment length is short.
  • a long sequence of fragments can be obtained by first synthesizing a plurality of small fragments and then performing the ligation.
  • a method of amplifying DNA/RNA using PCR technology is preferably used to obtain the gene of the present invention.
  • the primers for PCR can be appropriately selected according to the sequence information of the present invention disclosed herein, and can be synthesized by a conventional method.
  • the amplified DNA/RNA fragment can be isolated and purified by conventional methods such as by gel electrophoresis.
  • the invention also relates to vectors comprising the polynucleotides of the invention, as well as host cells genetically engineered using the vector or fusion protein coding sequences of the invention, and methods of producing the proteins of the invention by recombinant techniques.
  • polynucleotide sequences of the present invention can be utilized to express or produce recombinant proteins by conventional recombinant DNA techniques. Generally there are the following steps:
  • Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the proteins of the invention and suitable transcription/translation control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like.
  • the DNA sequence can be operably linked to an appropriate promoter in an expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase for eukaryotic cell culture, neomycin resistance, and green Fluorescent protein (GFP), or tetracycline or ampicillin resistance for E. coli.
  • Vectors comprising the appropriate DNA sequences described above, as well as appropriate promoters or control sequences, can be used to transform appropriate host cells to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a mammalian cell.
  • Representative examples are: Escherichia coli, bacterial cells of the genus Streptomyces; fungal cells such as yeast; plant cells; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS, or 293 cells, and the like.
  • a particularly preferred cell is a cell of a human and a non-human mammal, especially an immune cell, including T cells, NK cells.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention.
  • the medium used in the culture may be selected from various conventional media depending on the host cell used.
  • the cultivation is carried out under conditions suitable for the growth of the host cell.
  • the selected promoter is induced by a suitable method (such as temperature conversion or chemical induction) and the cells are cultured for a further period of time.
  • the protein in the above method can be expressed intracellularly, or on the cell membrane, or secreted outside the cell. If desired, the protein can be isolated and purified by various separation methods using its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • conventional renaturation treatment treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, super treatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer Analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and
  • the present invention also provides an immune cell (abbreviated as "immune cells of the present invention") expressing the fusion protein of the present invention, which carries the fusion protein on the cell surface.
  • immune cells of the present invention an immune cell (abbreviated as "immune cells of the present invention") expressing the fusion protein of the present invention, which carries the fusion protein on the cell surface.
  • the fusion protein is located on the cell membrane of the immune cell, and the first protein element, i.e., the IL-12 protein element, is located extracellularly.
  • a preferred class of immune cells includes T cells, particularly human T cells.
  • said T cell surface carries a MART-1 TCR.
  • a lentiviral expression system gene-modified T cell which expresses an anti-tumor TCR (T-cell receptor) and simultaneously expresses an hscIL-12/signal-free peptide CD107a fusion protein.
  • T-cell receptor anti-tumor TCR
  • the T cells may optionally also express wild-type CD107a or hscIL-12 (human single-stranded IL-12) or a combination thereof.
  • CD107a is present in the cytoplasmic lysosome in the inactive state of T cells.
  • the fusion protein IL-12 can migrate to the surface of the cell membrane by transient fusion of CD107a membrane, thereby exerting biological effects.
  • the fusion protein retains both the biological activity of Il-12 and the function of T cells.
  • compositions and Methods of Administration also provides a composition comprising (a) an effective amount of a fusion protein of the invention and/or an effective amount of an immune cell of the invention, and a pharmaceutically acceptable carrier.
  • the fusion proteins of the invention may be formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium wherein the pH is usually from about 5 to about 8, preferably from about 6 to about 8.
  • the term "effective amount” or “effective amount” refers to an amount that is functional or active to a human and/or animal and that is acceptable to humans and/or animals, such as from 0.001 to 99% by weight; preferably 0.01-95 wt%; more preferably, 0.1-90 wt%.
  • an effective amount or “effective amount” means 1 x 10 3 - 1 x 10 7 of said immune cells/mL.
  • a "pharmaceutically acceptable” ingredient is one that is suitable for use in humans and/or mammals without excessive adverse side effects (eg, toxicity, irritation, and allergies), ie, having a reasonable benefit/risk ratio.
  • pharmaceutically acceptable carrier refers to a carrier for the administration of a therapeutic agent, including various excipients and diluents.
  • compositions of the present invention comprise a safe and effective amount of a fusion protein of the invention and a pharmaceutically acceptable carrier.
  • Such carriers include, but are not limited to, saline, buffer, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the pharmaceutical preparation should be matched to the mode of administration, and the pharmaceutical composition of the present invention can be prepared into an injection form, for example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants.
  • the pharmaceutical composition is preferably manufactured under sterile conditions.
  • the amount of active ingredient administered is a therapeutically effective amount.
  • the pharmaceutical preparation of the present invention can also be formulated into a sustained release preparation.
  • the effective amount of the fusion protein of the present invention may vary depending on the mode of administration and the severity of the disease to be treated and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on various factors (e.g., by clinical trials). The factors include, but are not limited to, pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.; severity of the disease to be treated by the patient, body weight of the patient, immune status of the patient, administration Ways, etc.
  • pharmacokinetic parameters of the fusion protein of the invention such as bioavailability, metabolism, half-life, etc.
  • severity of the disease to be treated by the patient body weight of the patient, immune status of the patient, administration Ways, etc.
  • a satisfactory effect can be obtained.
  • several separate doses may be administered per day, or the dose may be proportionally reduced, as is critical to the condition of the treatment.
  • the fusion proteins of the invention are particularly suitable for use in the treatment of diseases such as tumors.
  • Representative tumors include, but are not limited to, brain tumors, colorectal cancer tumors, lung cancer tumors, liver cancer tumors, breast cancer tumors, gastric cancer tumors, and pancreatic cancer tumors.
  • the fusion protein of the present invention has no significant toxicity to T cells. This may be due to the fact that IL-12 is normally present in the lysosomal enzyme in the cytoplasm and cannot be effectively contacted or acted on T cells;
  • Fig. 2 is an engineering vector pLenti-MSCV containing a gene of interest, which is a promoter which is optimized for transduction of T cells by MSCV as a promoter;
  • the envelope protein particle pMD2.G contains VSV-G; gag/pol helper plasmid; and pRev plasmid system.
  • the plasmids pRRLSIN.cPPT.MSCV/GFP and 293FT cells used were commercially available, and the reagents used were also commercially available.
  • CD107a without a signal peptide refers to a mature CD107a protein lacking a secreted peptide.
  • hscIL-12/CD107a refers to hscIL-12/wild type CD107a or is expressed as CD107a.
  • Tumor cells 938 and 526 are conventional melanoma cell lines (supplied by Dr. Rosenberg of the National Cancer Institute), and subcultured in vitro with 10% FCS RPMI medium, 2 per -3 days was subcultured by 0.25% trypsin. Both tumors express MART-1 antigen, of which 938 is MHC I A2-(negative) and 526 is MHC I A2+ (positive), and the genetically modified anti-tumor MART-1 T cells recognize only A2+ cell line, ie 526 cells.
  • PBMC peripheral blood cells were derived from healthy human peripheral blood, and T cells were stimulated for growth with CD3/CD28 magnetic beads or CD3 antibody for 1 day, and cultured in IL-2 (100 IU/ml) X-VIVO medium. After recombinant lentivirus transduction of T cells, the fluorescence intensity of MART-1 in T cells was detected by flow cytometry through the MART-1 tetramer peptide. At the same time, the expression of cell surface CD62L, membrane surface CD107a, hscIL-12/CD107a and hscIL-12/no signal peptide CD107a was detected by flow cytometry.
  • a lentiviral vector a recombinant lentiviral vector carrying a human TCR ⁇ and ⁇ chain gene that specifically recognizes the human melanoma-associated antigen MART-1 is transfected into autologous peripheral blood lymphocytes using a molecular biological technique to make a recombinant TCR Expressed in T lymphocytes for the purpose of effectively killing tumors.
  • a TCR expression vector containing a self-cleaving 2A peptide, furin (Furin) and a spacer sequence was constructed using a lentiviral vector with an optimized promoter.
  • CD107a wild-type CD107a
  • no signal CD107a no signal CD107a
  • the fusion protein of hscIL-12/wild type CD107a and hscIL-12/no signal peptide was ligated with the peptide G6S and SGSG peptides, respectively.
  • Preparation of fusion protein expression in lentiviral expression system 293T cells were cultured, and the cell density was adjusted in DMEM medium containing 10% fetal bovine serum one day before transfection, and then 25 ⁇ 10 6 293T cells were inoculated per 15 cm cell culture dish. The cells were cultured in a 5% CO 2 incubator at 37 ° C, and were used for transfection after the cells were grown to 80% to 90% after 16 h to 24 h. On the day of transfection, the medium was changed to complete medium without antibiotics (P/S) (DMEM + 10% FBS).
  • P/S DMEM + 10% FBS
  • the lentiviral backbone of LVV-MSCV-MART-1TCR, hscIL-12/CD107a, and hscIL-12/signal-free peptide CD107a fusion protein was co-transfected into 293T cells with three other packaging plasmids, using commercially available calcium phosphate. For the medium. After 6 hours of culture, the medium was discarded, washed 3 times with PBS and replaced with 20 ml of fresh complete medium (DMEM + 10% FBS + P / S). The culture supernatant of 30-72 h after transfection was collected, centrifuged at 6000 rpm for 10 min, and the cell debris was discarded.
  • the supernatant was filtered through a 0.45 ⁇ m PVDF filter into a 50 ml round bottom centrifuge tube, centrifuged at 50,000 g for 2 h at 4 ° C, and carefully discarded. Clear, DMEM (free of serum, double antibody) resuspended virus precipitate, according to the amount of virus used each time into a clean 15ml centrifuge tube, stored in a -80 ° C refrigerator, used to infect T cells.
  • the titer of the virus detected by the Lentivirus-Associated p24 ELISA Kit was 5 ⁇ 10 7 -1.5 ⁇ 10 8 IFU. For details, see the instructions of the Lentivirus-Associated p24 ELISA Kit. (Yang, Cohen et al. 2008).
  • T cells modified by anti-MART-1 TCR gene were co-cultured with 526, 938 cell lines, and placed at a ratio of 1:1, that is, 1 ⁇ 106 of each cell was placed in a 14 ml round bottom.
  • the total volume of 1 ml was transferred to a 37 ° C CO2 incubator for 4 h.
  • the cells were centrifuged at 800 x g for 10 minutes, the supernatant was collected, and the cells were lysed by RIPA lysate.
  • the content of IFN ⁇ and IL-12 in the supernatant was detected by an ELISA reagent.
  • IL-12/CD107a on the surface of the membrane or IL-12 in the IL-12/no signal peptide CD107a was manipulated by live cell staining of a conventional flow cytometer.
  • the wild type or no signal peptide CD107a and the intracellular fusion protein in the supernatant were detected by an ELISA kit (R&D Systems, Minneapolis, MN).
  • CD3, CD8, CD62L, CD107a, IL-12 and CD45RO on the cell surface were detected by fluorescently labeled corresponding antibodies, including isothiocyanate (FITC), allophycocyanin (APC), Phycoerythrin (PE), PE-Cy7, and APC-Cy7 (BD Biosciences, San Jose, CA).
  • FITC isothiocyanate
  • APC allophycocyanin
  • PE Phycoerythrin
  • PE-Cy7 BD Biosciences, San Jose, CA
  • APC-Cy7 APC-Cy7
  • the cells were washed twice with FACS staining solution (PBS containing 2% FBS), then 0.2 ml (106/ml) was added to the flow tube, incubated at 4 ° C for 30 minutes, and then washed twice.
  • the dead cells were separated by adding 20 ⁇ l PI (l5 ⁇ g/ml propidium iodide) (Sigma-Aldrich, Saint Louis, MO) and cell subpopulation before the sample was taken to achieve the purpose of separation.
  • the streaming data is analyzed by FlowJo8.1.1 for post-processing.
  • mice Female pmel mice (6-8 weeks, 7 mice per group) were selected for intracranial tumor inoculation (IC). B16F10-MART-1 tumor cells were stopped by 0.25% trypsinization with 0.02% EDTA and washed once with serum-containing medium to wash the trypsin reaction, followed by washing twice with PBS. Tumor cells were finally mixed with methylcellulose in zinc option medium in a volume of 1:1, and 5000 cells were diluted in a 5 ⁇ l liquid and loaded onto a 250- ⁇ l syringe (Hamilton, Reno, NV) using a 25-gauge needle.
  • IC intracranial tumor inoculation
  • the Quinesential Stereotaxic Injector System (Stoelting Co. Wood Dale, IL) was injected into the right brain caudate nucleus of the mice. Five days after inoculation of the tumor cells, the mice received whole body 5 Gy radiation. On day 2, the mice received a 0.5-1X 106DC vaccine subcutaneously, or a 1X 107 reinfusion via the tail vein IV. The anti-MART-1 TCR, IL-12/wild-type CD107a or IL-12/signal-free peptide CD107a was sequentially transduced. Lentiviral gene-modified T cells.
  • Mouse T cells were obtained from mouse spleen cells, activated with 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); day 2, transduced with Lentiviral vector T The cells were then cultured for 6 days, cells were collected, and T cells were injected through the tail vein of the mice.
  • DC cells of DC group mice were obtained from mouse bone marrow cells, induced differentiation and maturation in vitro for 8 days, and inoculated intraperitoneally. The deaths of the mice were then recorded daily, growth curves were recorded and plotted by Prism mapping software. Asterisks indicate that the experimental group was compared with the other groups, p ⁇ 0.001.
  • the fusion gene was synthesized by Invitrogen, and the length and sequence of the fusion gene were confirmed by 1% agarose electrophoresis and sequencing.
  • the structure of the obtained IL-12/CD107a fusion gene series was constructed as shown in SEQ ID NOS.: 1, 3, and the amino acid sequence of the encoded fusion protein is shown in SEQ ID NO.: 2, 4.
  • mixture The composition is as follows, take 2ml Optimum I and add pLenti-MSCV (22.5ug), pMD2.G (7.5ug), gag/pol (15ug), pRev (10ug), mix; take 2ml Optimum I and add Lipofectamine 160ul (Invitrogen), mix. The two suspensions were mixed, incubated at room temperature for 5 minutes, and then uniformly added dropwise to the Petri dish.
  • the supernatant containing the genetically engineered vector was harvested, centrifuged to remove cell debris at 2000 g, and the supernatant was collected and filtered through a 0.45 uM filter to remove possible contamination, dispensed and stored in a negative 80 refrigerator. According to different needs, the collected virus supernatant can be subjected to ultracentrifugation at 50,000 g to obtain a higher concentration of the viral vector.
  • the obtained lentiviral expression vectors were designated as LV-hscIL-12/CD107a, LV-hscIL-12/signal-free peptide CD107a, LV-hscIL-12, respectively.
  • the method is as follows: CD3/CD28 magnetic beads or anti-CD3 antibody activates PBMC, and on day 2, the T cells are modified by lentiviral gene.
  • the brief method is as follows: Wash T cells three times in PBS buffer, according to virus titer and T cell The appropriate amount of lentivirus was added in a ratio of 3:1, centrifuged at 2000 ⁇ g for 2 h, and after 6 h, the culture was continued by adding 100 IU/ml IL-2; the second transduction was performed on the 5th day, or combined co-transduction. The flask was divided according to the growth of the cells, and two weeks later, the cells modified by the T cells were examined by flow cytometry.
  • Example 4 IL-12/CD107a series lentiviral gene modified human anti-tumor T cells achieve IL-12 transient membrane expression
  • a lentiviral vector that continuously secretes LVV-hscIL-12, hscIL-12/CD107, and hscIL-12/secretory peptide IL-12 is constructed, wherein IL- The 12 gene and the CD107a gene are linked by an amino acid peptide SGSG, which is a third generation lentiviral vector and the promoter is MSCV.
  • the 5' and 3' LTR (long terminal repeat) of the vector was engineered into SIN-LTR (self-inactivating-LTR) to reduce the probability of lentiviral recombination and enhance safety performance.
  • T-cells were transduced with anti-tumor TCR and IL-12/CD107a fusion protein series, and anti-tumor T cells were cultured for 14 days in vitro with tumor cells. 526, 938 co-culture.
  • the phenotype of transient membrane expression of tumor antigen reactivity in the IL-12/CD107a series was observed, and it was further confirmed that IL-12/CD107a can achieve IL-12 membrane expression by antigen-reactive membrane migration of CD107a.
  • the signal-free peptide CD107a showed high expression level, which may be related to partial secretion fragmentation caused by the processing of IL-12/CD107a in the cytoplasm.
  • Example 5 lentiviral vector IL-12/CD107a series transduced T cells co-culture with tumor can enhance the expression of IFN ⁇
  • IL-12/CD107a fusion protein vectors in order to reduce systemic cytotoxicity induced by IL-12 release, a series of transient membrane-expressed IL-12/CD107a fusion protein vectors was constructed.
  • human (species) T cells were sequentially transduced with anti-tumor TCR and IL-12/CD107a series fusion proteins for 14 days, and co-cultured with tumor cells 526 and 938. After 24 hours, the supernatant was IFN ⁇ . And IL-12 expression levels were detected by ELISA kit.
  • the surface of the IL-12/CD107a membrane was consistently detected, and IL-12/CD107a could not be cleaved from the cell membrane, so IL-12 secretion could not be detected in the supernatant of the mixed cultured cells.
  • Expression of IL-12 was only detected in the group that continued to secrete IL-12.
  • the other groups were able to detect a significant increase in the level of secreted IL-12 compared with the persistent IL-12 group, P ⁇ 0.001.
  • the IL-12/CD107a series can induce transient IL-12 membrane expression, and the expression is limited to the surface of the T cell membrane and cannot be secreted into the culture supernatant, minimizing the secretion of IL-12. Systemic cytotoxicity.
  • Example 6 Lentiviral Vector IL-12/No Signal Peptide CD107a Transduced T Cells Can Effectively Avoid the Side Effects of In Vitro T Cell Expansion Caused by Persistent Secretion of IL-12
  • this example further observes the transduction of the IL-12/CD107a fusion protein series. The effect of T cell expansion in vitro.
  • T-cell As shown in Figure 4, after T cells were sequentially transduced with anti-tumor TCR and IL-12/CD107a fusion protein series for 14 days, the amplification ratios of each group of cells and control transduction group (T-cell) were compared. The amplification factor of the fixed T-cell group was 100%, and the values of the other groups were compared with the T-cell group.
  • the results showed that the number of amplifications in the transduced IL-12 group was significantly lower than that in the other groups, p ⁇ 0.001.
  • the fusion hscIL-12/no signal peptide fusion protein group was the same as the TCR group, and no significant difference was observed.
  • the amplification fold of the hscIL-12/no signal peptide CD107a fusion protein group was significantly higher than that of the hscIL-12/CD107a fusion protein group.
  • the possible cause is due to the partial breakdown of the secreted peptide caused by the signal peptide of CD107a during the intracytoplasmic processing of the IL-12/CD107a fusion protein, resulting in the cleavage of IL-12 from the fusion protein and secretion. Sex cytotoxicity.
  • Example 7 lentiviral vector hscIL-12/CD107a series transduced murine T cell mediated cell transfusion therapy significantly prolonged survival of tumor-bearing mice
  • mice Female pmel mice (7 mice per group) were selected to be intracranial (5000 cells/only) (IC) for 5 days by B16F10 cells, and 5Gy whole body radiotherapy was received 1 day before cell return.
  • Pmel mice are transgenic mice whose T cells stably express anti-tumor TCR by transgene modification and can recognize the tumor antigen gp100 on B16F10 cells.
  • Rat T cells were obtained from mouse spleen cells and activated by 10 ug/ml concanavalin (Con A) in the presence of IL-2 (5 IU/ml); One day, T cells were transduced with a lentiviral vector, and then cultured for 6 days, cells were collected, and (X) 5 ⁇ 106 T cells were injected through the tail vein of the mice.
  • the DC cells of the DC group were obtained from bone marrow cells, and induced to differentiate and mature for 8 days in vitro, and 1X 106 cells were inoculated intraperitoneally. As shown in Figure 5, the groups of DC and T cells are described on the right.
  • the asterisk (*) indicates transduction of IL-12 compared to the other groups, p ⁇ 0.001; asterisk (**) indicates that the transduction of IL-12 / no signal peptide CD107a group has significantly better survival than IL-12 /CD107a group. Among them, the continuous secretion of IL-12 group showed significant systemic cytotoxicity, and the mice all died 4-7 days after cell reinfusion.
  • CD107a is a transmembrane protein expressed on lysosomes. Its N-terminus is located on the side of the lysosomal vesicle. (Eskelinen 2006) is a small peptide at the C-terminus, facing the cytoplasmic side, when anti-tumor When T cells recognize tumor antigens, lysosomes fuse with T cell membranes to produce degranulation effects of T cells or NK cells, that is, T cells or NK cells release effector granzymes (Granzyme B) and perforin. The process of killing tumor cells. When T cells react with tumor cells, the property of CD107a to migrate to the T cell membrane has been It is widely used as an indicator of the T cell killing function.
  • IL-12 has a significant antitumor effect, it is extremely limited due to systemic systemic administration which causes systemic toxic side effects.
  • Prior to the present invention the clinical application of IL-12 was greatly limited in clinical application due to the systemic toxic side effects induced by it and the accidental death of two patients caused by systemic administration in early clinical trials.
  • the transient membrane migration of tumor antigen reactivity of IL-12 can be achieved by migrating CD107a membrane by constructing a strategy of IL-12/CD107a fusion protein series.
  • Technical problems can not only enhance the anti-tumor response, but also have no effect on the in vitro expansion of tumor T cells. It is an anti-tumor strategy with broad application prospects and will play an important role in the treatment of tumor immune cells.

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Abstract

本发明提供了一种IL-12/CD107a融合蛋白及其制法和用途。利用含IL-12/CD107a融合基因的慢病毒修饰抗肿瘤T细胞，从而实现肿瘤细胞膜表面IL-12的释放。

Description

[根据细则37.2由ISA制定的发明名称]　IL-12/CD107A融合蛋白及其制法和用途 技术领域
本发明涉及医药领域，具体地涉及一种基于IL-12膜迁移瞬时表达的高效低毒的肿瘤治疗剂及其制法和用途。
背景技术
IL-12是一种非常重要的免疫刺激因子。IL-12是由共价键链接的异质性二聚体细胞因子，由p35和p40亚基组成，在体内由激活的免疫细胞分泌。IL-12是重要的细胞免疫调控因子，在抗感染免疫及恶性肿瘤的免疫中通过NK细胞以及CTL发挥作用。
虽然IL-12具有显著的抑瘤效果，但是由于其系统性全身给药会引起全身性毒副反应，因而受到极大的限制。在本发明之前，IL-12的临床应用由于其所诱导的全身性毒副作用，以及早期临床试验通过全身系统性给药引起的两例患者的意外死亡，使其临床应用受到大大的限制。
为了避免全身性给药的毒副作用从而实现临床应用的目的，临床科研工作者们通过肿瘤局部注射、瞬时表达及多点注射等途径应用于肿瘤的临床试验，但是由于IL-12的治疗效果同回输的IL-12的剂量直接相关，上述局部的临床方案并未有取得显著的抑瘤效果。
为了解决这一问题，本发明人曾经试图通过基因修饰抗肿瘤T细胞持续分泌IL-12来实现抗肿瘤效果的最大化，但是由于未知的原因(一种可能的原因是IL-12的毒副作用)通过持续分泌IL-12方案基因修饰的T细胞在体外不能有效扩增，并引起大量的T细胞凋亡。
肿瘤已经成为超越心血管疾病危害人类健康的第一杀手，本领域尚缺乏高效且低毒副作用的基于IL-12的抗肿瘤药物。因此，本领域迫切需要开发高效且低毒副作用的肿瘤治疗药物。
发明内容
本发明的目的就是提供了一种高效且毒副作用低的肿瘤治疗药物及其制法和应用。
本发明构建了一系列IL-12/CD107a的融合蛋白，包括IL-12的全序列(包括IL-12的分泌片段)，及在IL-12的C-端与CD107a的N－端加入一个链接肽段(SGSG)分别同CD107a的全长序列及去掉分泌肽的CD107a连接的分子策略。实验数据显示，上述两种融合蛋白基因修饰抗肿瘤CTL后，可以增强体内外的抗肿瘤反应，其中无分泌肽的CD107a融合蛋白不但可以显著延长荷瘤小鼠生存，同时对基因修饰的抗肿瘤T细胞的体外扩增没有明显的毒副作用，是一种非常有临床应用前景的抗肿瘤增效方案。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.抗肿瘤CTL表达融合蛋白hscIL-12/CD107a的增效分子机制示意图。CD107a(又称为LAMP1)是一种表达于溶酶体上的跨膜蛋白，它的N-端位于溶酶体腔囊一侧，C-端是一小段肽段，朝向胞浆一侧，当抗肿瘤T细胞在识别肿瘤抗原时，溶酶体与T细胞膜融合而发生T细胞或NK细胞的脱颗粒效应，即T细胞或NK细胞释放效应分子颗粒酶(Granzyme B)及穿孔素(perforin)而达到杀伤肿瘤细胞的过程。在T细胞与肿瘤T细胞发生反应的时，CD107a迁移到T细胞膜上的特性已经被广泛地应用于反映T细胞杀伤功能的一个指标。如图左侧所示，当抗肿瘤T细胞识别肿瘤的同时，T细胞内的溶酶体向胞膜一侧迁移并融合，CD107a分子暴露于细胞膜外，并可以检测，标示为CD107a阳性细胞。如图右侧所示，为了实现IL-12的溶酶体囊内表达，本发明构建了一系列IL-12/CD107a的融合蛋白，包括IL-12的全序列(包括IL-12的分泌片段)及在IL-12的C-端与CD107a的N－端加入一个链接肽段分别与全长的CD107a分子及去除分泌肽段的CD107a分子融合，当T细胞识别肿瘤细胞时，IL-12分子通过CD107a迁移到T细胞膜表面，实现抗肿瘤的增效反应。
图2.hscIL-12/CD107a融合蛋白系列的构建及转导抗肿瘤T细胞介导的表达。A.慢病毒表达IL-12/CD107a融合蛋白系列载体的构建。通过第三代慢病毒载体表达融合蛋白，所选用的启动子为MSCV及EF-1α，已经被广泛地应用于人体的肿瘤免疫细胞治疗，具有表达的稳定性及安全性，表达基因构件可以替代为需要表达的基因，即MART-1TCR以及IL-12/CD107a融合蛋白系列等。该载体的5’及3’LTR(long terminal repeat)被改造成SIN-LTR(self-inactivating-LTR),目的是降低慢病毒重组的机率，增强安全性能，联合WPRE终止RNA转录的功能，是目前广泛应用于临床的最新一代的基因工程载体结构。B.T细胞的基因修饰策略。T细胞活化后，通过两次转导即MART-1TCR(第一天)及图示中三个载体的序贯转导(第4天)，于第14天，与表达MART-1识别靶点的肿瘤细胞526、938共培养。C.抗肿瘤T细胞膜表面IL-12分子的表达。共培养4小时后，通过流式细胞仪检测T细胞膜表面hscIL-12的表达。
实验数据显示，细胞膜表面的IL-12只有在表达IL-12/CD107a的融合蛋白组同表达MART-1的MHC A2+反应组中可以检测出来，分泌型的IL-12膜表面没有表达。
图3.慢病毒载体表达hscIL-12/CD107a转导的T细胞的增效反应。如图所示，T细胞序贯转导抗肿瘤TCR及hscIL-12/CD107a融合蛋白系列14天后，与肿瘤细胞526及938共培养，16小时后，上清中IFNγ及IL-12表达水平通过ELISA试剂盒检测。同TCR基因修饰组比较，序贯转到融合蛋白组表现出明显的抗肿瘤增效反应，即分泌高水平的IFNγ；但是实验上清中，只有C1组(持续分泌IL-12组)可以检测到分泌的IL-12。*表示与对照组比较，p<0.001。
图4.慢病毒载体表达IL-12/CD107a系列转导的T细胞可以有效避免持续性分泌IL-12造成的体外T细胞扩增的毒副作用。T细胞序贯转导抗肿瘤TCR及IL-12/CD107a融合蛋白系列14天后，各组细胞与对照转导组(T-cell)的扩增倍数比较，转导持续分泌IL-12组较其它各组的扩增倍数显著降低，*,p<0.001。其中，转导IL-12/CD107a的C2组与转到IL-12/(no signal peptide)无分泌肽CD107a的C3组比较，扩增倍数显著降低，**，P<0.01。C3组与TCR-T组比较，扩增倍数接近，未见显著差异。
图5.慢病毒载体hscIL-12/CD107a系列转导鼠T细胞介导的细胞回输治疗显著延长荷瘤小鼠的生存。雌性pmel小鼠(每组7只小鼠)通过B16F10细胞(5000细胞/鼠)植入颅内(IC)5天，细胞回输前1天小鼠接受5Gy全身放疗。鼠的T细胞取自小鼠的脾脏细胞，通过10ug/ml的刀豆蛋白(Con A)在 IL-2(5IU/ml)存在的条件下活化；第2天，用图示中慢病毒载体转导T细胞，然后继续培养6天，收集细胞，通过小鼠尾静脉注射(IV)5X 106个T细胞。星号(*)表示该实验组与其它组比较，p<0.001；其中C3组与C2组比较(**)，C3组的存活天数显著延长，有统计学意义，p<0.05。
具体实施方式
本发明人经过广泛而深入的研究，首次开发了一种结构新颖的、具有高效杀伤肿瘤细胞活性且毒副作用很小的IL-12融合蛋白。该IL-12/CD107a或IL-12/缺失分泌肽的CD107a融合蛋白通过基因修饰抗肿瘤T细胞后，IL-12存在于细胞浆的溶酶体内，细胞膜表面不能检测到表达。实验表明，当本发明融合蛋白在T细胞中表达后，只有在抗肿瘤CTL识别杀伤肿瘤时会瞬时有效展示在T细胞的表面。出乎意料的是，该IL-12/缺失分泌肽CD107a融合蛋白系列对T细胞的活力(viability)基本无影响，并且携带本发明融合蛋白的抗肿瘤T细胞在接近肿瘤细胞时，IL-12才会瞬时表达在细胞膜表面，导致位于细胞膜表面IL-12更有效而安全地作用于肿瘤细胞。在本发明中，通过T细胞攻击肿瘤的局部并通过细胞表面瞬时迁移的IL-12改变T细胞-肿瘤组织的免疫微环境，从而协同而有效地实现抗肿瘤免疫效果的最大化，同时极其显著地降低IL-12的毒副作用。在此基础上完成了本发明。
具体地，本发明人利用通过CD107a表达IL-12，开发了一种IL12/缺失分泌CD107a的全新的融合蛋白，并通过慢病毒基因修饰抗肿瘤T细胞。实验数据，显示IL12/缺失分泌肽CD107a慢病毒基因修饰抗肿瘤T细胞后，在抗肿瘤CTL识别肿瘤时瞬时表达于T细胞表面，对肿瘤抗原的反应表现出增强免疫反应的效应。临床前荷瘤鼠模型显示，该方案修饰的T细胞可以显著延长荷瘤小鼠的生存期，没有发现明显的细胞毒效应。换言之，瞬时膜表达所述融合蛋白的T细胞一方面可有效地杀灭肿瘤细胞，另一方面经证实具有体内外的安全性。此外,所述经修饰的T细胞自身似乎仅受到所表达IL-12轻微的不利影响或基本上不受其影响(与持续分泌IL-12的T细胞比较，本发明融合蛋白对T细胞的体外扩增无明显影响)。
术语
如本文所用，术语“头部”指多肽或其片段的N端，尤其是野生型多肽或其片段的N端。
如本文所用，术语“尾部”指多肽或其片段的C端，尤其是野生型多肽或其片段的C端。
如本文所用，所述的“含有”，“具有”或“包括”包括了“包含”、“主要由……构成”、“基本上由……构成”、和“由……构成”；“主要由……构成”、“基本上由……构成”和“由……构成”属于“含有”、“具有”或“包括”的下位概念。
CD107a又称为LAMP1，是一种表达于溶酶体上的跨膜蛋白，它的N-端位于溶酶体腔囊一侧，(Eskelinen 2006)C-端是一小段肽段，朝向胞浆一侧，当抗肿瘤T细胞在识别肿瘤抗原时，溶酶体与T细胞膜融合而发生T细胞或NK细胞的脱颗粒效应，即T细胞或NK细胞释放效应分子颗粒酶(Granzyme B)及穿孔素(perforin)而达到杀伤肿瘤细胞的过程。在T细胞与肿瘤细胞发生反应的时，CD107a迁移到T细胞膜上的特性已经被广泛地应用于反应T细胞杀伤功能的一个指标。
分泌肽缺失型CD107a：本发明中的分泌肽缺失型CD107a是成熟的CD107a(Seq ID NO.10),即通过基因编辑去掉了野生型CD107a(Seq ID NO.:8)的分泌肽(信号肽)氨基酸序列 --MAAPGSARRPLLLLLLLLLLGLMHCASAA，通过基因合成与IL-12连接成全新的融合蛋白。
应理解，在本发明中，所述的CD107a可以是来自哺乳动物(人或非人哺乳动物)，也可以来自其他真核物种。优选地，CD107a是来自人的野生型CD107a。
IL-12是一种非常重要的免疫刺激因子。IL-12是由共价键链接的异质性二聚体细胞因子，由p35和p40亚基组成，在体内由激活的免疫细胞分泌。
应理解，在本发明中，所述的IL-12可以来自人或非人哺乳动物，可以是全长的、成熟形式的IL-12，或其活性片段。此外，所述的IL-12(或IL-12蛋白元件)可以是单个亚基或多个亚基。例如，在本发明中，所述的第一蛋白元件可包括IL-12蛋白的一个或多个(如两个)亚基。
在另一优选例中，所述的第一蛋白元件包括连接在一起的IL-12蛋白P40和P35亚基。
在另一优选例中，所述的P40和P35亚基的连接方式没有特别限制，包括“头-头”、“头-尾”、“尾-头”、“尾-尾”相连，其中“头”指多肽的N端，“尾”指多肽的C端。
此外，在P40和P35亚基之间可以存在或不存在接头(linker)。较佳地，所述的接头为柔性的4-20个氨基酸的接头，更佳地，所述的接头为GGGGGGS(G6S)(SEQ ID NO.:14)。
肽接头：本发明的双功能融合蛋白可任选地含有肽接头。肽接头大小和复杂性可能会影响蛋白的活性。通常，肽接头应当具有足够的长度和柔韧性，以保证连接的两个蛋白在空间上有足够的自由度以发挥其功能。同时避免肽接头中形成α螺旋或β折叠等对融合蛋白的稳定性的影响。
连接肽的长度一般为0-15个氨基酸，较佳地1-15个氨基酸。
优选的连接肽例子包括(但并不限于)：SEQ ID NO.:11-14所示的连接片断。
信号肽和前导肽：本发明中融合蛋白还可含有其他元件，代表性的元件包括(但并不限于)：信号肽、前导肽等。
在本发明的一个实例中，融合蛋白含有信号肽。代表性的例子包括(但并不限于)：人源的IL-12P40亚基的信号肽。
双功能融合蛋白及其制备：如本文所用，除非另外说明，所述的融合蛋白是一种分离的蛋白，与其它蛋白、多肽或分子无联系，是重组宿主细胞所表达的，或经分离或纯化的产物。
在本发明中，“重组双功能融合蛋白”、“本发明蛋白”、“本发明融合蛋白”、“双功能融合蛋白”、“IL-12-缺失分泌肽CD107a融合蛋白”、“IL-12/无分泌肽CD107a融合蛋白”可互换使用，指具有式Ia所述结构，或者IIa所述结构，即含有包括IL-12蛋白元件，缺失分泌肽CD107a的蛋白元件和连接肽元件的融合蛋白。一个代表性的例子是IL12-缺失分泌肽CD107a。本发明蛋白可以是单体或由单体形成的多聚体(如二聚体)。此外，应理解，所述术语还包括融合蛋白的活性片段和衍生物。
一种优选的融合蛋白的序列如SEQ ID NO.:4所示，其中第1-328位为IL-12的P40亚基；第329-335位为G6S连接肽；第336-532位为IL-12的P35亚基；第533-537位为SGSG连接肽(SEQ ID NO.:12)；第537-924位为无信号肽CD107a氨基酸序列。
如本文所用，“分离的”是指物质从其原始环境中分离出来(如果是天然的物质，原始环境即是天然环境)。如活体细胞内的天然状态下的多核苷酸和多肽是没有分离纯化的，但同样的多核苷酸或多肽如从天然状态中同存在的其他物质中分开，则为分离纯化的。
如本文所用，“分离的重组融合蛋白”是指重组融合蛋白基本上不含天然与其相关的其它蛋白、脂类、糖类或其它物质。本领域的技术人员能用标准的蛋白质纯化技术纯化重组融合蛋白。基本上纯的蛋白在非还原聚丙烯酰胺凝胶上能产生单一的主带。
本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。
本发明还涉及上述多核苷酸的变异体，其编码与本发明有相同的氨基酸序列的蛋白质片段、类似物和衍生物。此多核苷酸的变异体可以是天然发生的等位变异体或非天然发生的变异体。这些核苷酸变异体包括取代变异体、缺失变异体和插入变异体。如本领域所知的，等位变异体是一个多核苷酸的替换形式，它可能是一个或多个核苷酸的取代、缺失或插入，但不会从实质上改变其编码多肽的功能。
如本文所用，术语“引物”指的是在与模板配对，在DNA聚合酶的作用下能以其为起点进行合成与模板互补的DNA链的寡居核苷酸的总称。引物可以是天然的RNA、DNA，也可以是任何形式的天然核苷酸。引物甚至可以是非天然的核苷酸如LNA或ZNA等。引物“大致上”(或“基本上”)与模板上一条链上的一个特殊的序列互补。引物必须与模板上的一条链充分互补才能开始延伸，但引物的序列不必与模板的序列完全互补。比如，在一个3’端与模板互补的引物的5’端加上一段与模板不互补的序列，这样的引物仍大致上与模板互补。只要有足够长的引物能与模板充分的结合，非完全互补的引物也可以与模板形成引物-模板复合物，从而进行扩增。
根据本发明提供的氨基酸序列，本技术领域人员可方便地用各种已知方法制得本发明的融合蛋白。这些方法例如但不限于：重组DNA法，人工合成等。
本发明融合蛋白的元件(如IL12或无信号肽CD107a)的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据已公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。
此外，还可用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。
应用PCR技术扩增DNA/RNA的方法被优选用于获得本发明的基因。用于PCR的引物可根据本文所公开的本发明的序列信息适当地选择，并可用常规方法合成。可用常规方法如通过凝胶电泳分离和纯化扩增的DNA/RNA片段。
本发明也涉及包含本发明的多核苷酸的载体，以及用本发明的载体或融合蛋白编码序列经基因工程产生的宿主细胞，以及经重组技术产生本发明所述蛋白质的方法。
通过常规的重组DNA技术，可利用本发明的多核苷酸序列可用来表达或生产重组蛋白。一般来说有以下步骤：
(1).用本发明的编码本发明蛋白的多核苷酸(或变异体)，或用含有该多核苷酸的重组表达载体转化或 转导合适的宿主细胞；
(2).在合适的培养基中培养的宿主细胞；
(3).从培养基或细胞中分离、纯化蛋白质。
本领域的技术人员熟知的方法能用于构建含本发明蛋白的编码DNA序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外，表达载体优选地包含一个或多个选择性标记基因，以提供用于选择转化的宿主细胞的表型性状，如真核细胞培养用的二氢叶酸还原酶、新霉素抗性以及绿色荧光蛋白(GFP)，或用于大肠杆菌的四环素或氨苄青霉素抗性。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：大肠杆菌，链霉菌属的细菌细胞；真菌细胞如酵母；植物细胞；果蝇S2或Sf9的昆虫细胞；CHO、COS、或293细胞的动物细胞等。
一种特别优选的细胞为人和非人哺乳动物的细胞，尤其是免疫细胞，包括T细胞、NK细胞。
用重组DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。另一种方法是使用MgCl2。如果需要，转化也可用电穿孔的方法进行。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养，表达本发明的基因所编码的多肽。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。
在上面的方法中的蛋白质可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相层析技术及这些方法的结合。
经修饰的免疫细胞：本发明还提供一种表达本发明所述融合蛋白的免疫细胞(简称为“本发明免疫细胞”)，所述免疫细胞在细胞表面携带所述的融合蛋白。
在本发明中，至少一部分或全部所述的融合蛋白位于所述免疫细胞的细胞膜上，并且所述的第一蛋白元件即IL-12蛋白元件位于胞外。
一类优选的免疫细胞包括T细胞，尤其是人的T细胞。优选地，所述的T细胞表面携带MART-1TCR。
例如，在一个优选例中，提供了一种慢病毒表达体系基因修饰的T细胞，该T细胞表达抗肿瘤TCR(T-cell receptor)同时表达hscIL-12/无信号肽CD107a融合蛋白。
此外，所述的T细胞还可任选地同时表达野生型CD107a或hscIL-12(人源单链IL-12)或其组合。
膜表达IL-12/无信号肽CD107a的机理。为了便于理解，本发明人提供以下机理供参考。应理解，本发明的保护范围并不受所述机理的限制。
膜表达IL-12/CD107a或IL-12/无信号肽CD107a成熟蛋白的作用机制如图1所示。CD107a在T细胞非激活状态下存在于细胞胞浆的溶酶体内，在T细胞活化的情况下，融合蛋白IL-12可以通过CD107a的膜瞬时融合迁移到细胞膜表面，进而发挥生物学效应。最为关键的是该融合蛋白既保留了Il-12的生物学活性又不影响T细胞的功能。
药物组合物及施用方法：本发明还提供了一种组合物，它含有(a)有效量的本发明融合蛋白和/或有效量的本发明的免疫细胞，以及药学上可接受的载体。
通常，可将本发明的融合蛋白配制于无毒的、惰性的和药学上可接受的水性载体介质中，其中pH通常约为5-8，较佳地，pH约为6-8。
如本文所用，术语“有效量”或“有效剂量”是指可对人和/或动物产生功能或活性的且可被人和/或动物所接受的量，如0.001-99wt％；较佳的0.01-95wt％；更佳的，0.1-90wt％。
当本发明的药物组合物含有免疫细胞时，“有效量”或“有效剂量”是指1×103-1×107个所述的免疫细胞/mL。
如本文所用，“药学上可接受的”的成分是适用于人和/或哺乳动物而无过度不良副反应(如毒性、刺激和变态反应)的，即具有合理的效益/风险比的物质。术语“药学上可接受的载体”指用于治疗剂给药的载体，包括各种赋形剂和稀释剂。
本发明的药物组合物含有安全有效量的本发明的融合蛋白以及药学上可接受的载体。这类载体包括(但并不限于)：盐水、缓冲液、葡萄糖、水、甘油、乙醇、及其组合。通常药物制剂应与给药方式相匹配，本发明的药物组合物可以被制成针剂形式，例如用生理盐水或含有葡萄糖和其他辅剂的水溶液通过常规方法进行制备。所述的药物组合物宜在无菌条件下制造。活性成分的给药量是治疗有效量。本发明的药物制剂还可制成缓释制剂。
本发明融合蛋白的有效量可随给药的模式和待治疗的疾病的严重程度等而变化。优选的有效量的选择可以由本领域普通技术人员根据各种因素来确定(例如通过临床试验)。所述的因素包括但不限于：本发明融合蛋白的药代动力学参数例如生物利用率、代谢、半衰期等；患者所要治疗的疾病的严重程度、患者的体重、患者的免疫状况、给药的途径等。通常，当本发明的融合蛋白每天以约5mg-20mg/kg动物体重(较佳的5mg-10mg/kg动物体重)的剂量给予，能得到令人满意的效果。例如，由治疗状况的迫切要求，可每天给予若干次分开的剂量，或将剂量按比例地减少。
本发明融合蛋白特别适合用于治疗肿瘤等疾病。代表性的肿瘤包括(但并不限于)：脑肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、乳腺癌肿瘤、胃癌肿瘤、胰腺癌肿瘤。
本发明的主要优点包括：
(a)本发明的融合蛋白对于T细胞无明显毒性。这可能是由于IL-12正常情况下存在于胞浆内的溶酶体酶内，无法有效接触或作用于T细胞；
(b)当携带本发明融合蛋白的抗肿瘤T细胞在接近肿瘤细胞时，融合蛋白IL-12会通过CD107a的瞬 时膜迁移转移到抗肿瘤T细胞表面，从而不会释放出游离的IL-12(即IL-12瞬时地保留在细胞膜表面)。这使得膜表面的IL-12能够更有效且只能作用于近距离的肿瘤细胞，从而显著提高了安全性；
(c)通过T细胞攻击肿瘤的局部并通过细胞表面的IL-12改变T细胞-肿瘤组织的免疫微环境，从而协同而有效地实现抗肿瘤免疫效果的最大化。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
材料和通用方法：所用引物及DNA序列均由Invitrogen公司合成。
本发明实施例所使用的质粒LVV的图谱如图2所示，为含有目的基因的工程载体pLenti-MSCV,该载体由MSCV作为启动子，是最优化的可以有效转导T细胞的启动子；包膜蛋白质粒pMD2.G含有VSV-G；gag/pol辅助质粒；及pRev质粒系统。所使用的质粒pRRLSIN.cPPT.MSCV/GFP、293FT细胞为市售商品，所使用的试剂亦为市售商品。
实施例中，无信号肽的CD107a指缺失分泌肽的成熟CD107a蛋白。
实施例中，hscIL-12/CD107a指hscIL-12/野生型CD107a或表述为CD107a。
肿瘤细胞及T细胞的培养：肿瘤细胞938和526为常规的黑色素瘤细胞株(由美国国立癌症研究院的Dr.Rosenberg所赠)，体外传代培养条件为含10％FCS RPMI培养基，每2-3天通过0.25％的胰酶传代培养。两种肿瘤均表达MART-1抗原，其中938为MHC I A2-(阴性)，526为MHC I A2+(阳性)，基因修饰的抗肿瘤MART-1T细胞只识别A2+细胞株，即526细胞。
PBMC来自健康人外周血，用CD3/CD28磁珠或CD3抗体刺激T细胞生长1天，并在IL-2(100IU/ml)的X-VIVO培养基中维持培养。重组慢病毒转导T细胞后利用流式细胞仪通过MART-1四聚体肽段检测MART-1在T细胞的荧光强度。同时应用流式检测细胞表面CD62L、膜表面CD107a、hscIL-12/CD107a及hscIL-12/无信号肽CD107a的表达。
慢病毒载体的构建：采用分子生物学的技术方法将携带特异性识别人黑色素瘤相关抗原MART-1的人源TCRα和β链基因的重组慢病毒载体转染外周血自体淋巴细胞，使重组TCR表达在T淋巴细胞中以达到高效杀伤肿瘤的目的。利用已优化启动子的慢病毒载体构建含自我剪切2A肽、弗林蛋白酶(Furin)及间隔序列的TCR表达载体。
表达野生型CD107a(简称为“CD107a”)和无信号肽CD107a(简称为“no signal CD107a”)的慢病毒表达载体的构建参照发表的文献(Yang,Cohen et al.2008)，hscIL-12的构建参考发表的文献(Zhang,Kerkar et al.2011)。其中hscIL-12/野生型CD107a和hscIL-12/无信号肽的融合蛋白均分别用肽段G6S及SGSG肽段进行连接。
慢病毒表达体系表达融合蛋白的制备：培养293T细胞，转染前一天，以含10％胎牛血清的DMEM培养基调整细胞密度后，按照每15cm细胞培养皿接种25×106个293T细胞，置于37℃，5％CO2培养箱中培养，16h～24h后待细胞密度生长到80％～90％时即可用于转染。转染当天更换培养基为不含抗生素(P/S)的完全培养基(DMEM+10％FBS)。分别将LVV-MSCV-MART-1TCR、hscIL-12/CD107a以及hscIL-12/无信 号肽CD107a融合蛋白的慢病毒骨架与另外三种包装质粒一起共转染293T细胞，利用可以商业化的磷酸钙为媒介。培养6h后弃去培养基，用PBS洗3次后更换为20ml新鲜的完全培养基(DMEM+10％FBS+P/S)。收集转染后30-72h的培养上清，6000rpm离心10min，弃去细胞碎片，上清液以0.45μm PVDF滤器过滤至50ml圆底离心管中，4℃，50000g高速离心2h，小心弃去上清，DMEM(不含血清、双抗)重悬病毒沉淀，按每次使用的病毒量分装到洁净的15ml离心管中，-80℃冰箱保存，用于感染T细胞。Lentivirus-Associated p24ELISA Kit检测病毒的滴度为5×107-1.5×108IFU，具体步骤参见Lentivirus-Associated p24ELISA Kit的说明书。(Yang,Cohen et al.2008)。
肿瘤细胞/T细胞的共培养体系的建立：通过anti-MART-1TCR基因修饰的T细胞与526、938细胞系共培养，按1：1比例，即每种细胞1×106放置在14ml圆底的polypropylene培养管中，总体积1ml,转移至37℃的CO2培养箱中温浴4h。4h后，800×g离心细胞10分钟，收集上清，细胞通过RIPA裂解液裂解。上清中的IFNγ、IL-12的含量通过ELISA试剂检测。膜表面的IL-12/CD107a或IL-12/无信号肽CD107a中的IL-12通过常规的流式细胞仪的活细胞染色进行操作。上清中的野生型或无信号肽CD107a及细胞内的融合蛋白通过ELISA试剂盒检测(R&D Systems,Minneapolis,MN)。
流式细胞仪检测的分析：细胞表面的CD3、CD8、CD62L、CD107a、IL-12和CD45RO通过荧光标记的对应的抗体进行检测，所应用的荧光抗体包括isothiocyanate(FITC),allophycocyanin(APC),phycoerythrin(PE),PE-Cy7,and APC-Cy7(BD Biosciences,San Jose,CA)。MART-1:27–35四聚体通过设计由公司合成(iTAg MHC Tetramer,Beckman Coulter,Fullerton,CA),用来检测基因修饰的TCR表达水平。具体流程如下，首先细胞经过FACS染色液(PBS containing 2％FBS)洗涤两次,然后加入0.2ml(106/ml)于流式细胞管中，于4℃孵育30分钟,然后洗涤两次。死细胞通过样本上机前加入20μl PI(l5μg/ml propidium iodide)(Sigma-Aldrich,Saint Louis,MO)以及细胞亚群的划分来实现分离的目的。流式数据通过FlowJo8.1.1进行上机后处理分析。
荷瘤鼠模型的建立，本实验所涉及的方法如下：选择雌性pmel小鼠(6-8周，每组7只小鼠)进行颅内肿瘤接种(IC)。B16F10-MART-1肿瘤细胞通过含有0.02％EDTA的0.25％胰酶消化，并用含有血清的培养液洗涤一次来终止胰酶的反应，然后用PBS洗涤两次。肿瘤细胞最终以1：1的体积与methylcellulose in zinc option medium混合，5000个细胞稀释在5μl液体中上样到250-μl syringe(Hamilton,Reno,NV)，选用25-gauge针头。应用Quitessential Stereotaxic Injector System(Stoelting Co.Wood Dale,IL)注射到小鼠的右侧脑caudate核中。接种肿瘤细胞5天后，小鼠接受全身5Gy的射线照射。第2天，小鼠皮下接受0.5-1X 106DC疫苗，或者通过尾静脉IV回输1X 107经过序贯转导anti-MART-1TCR、IL-12/野生型CD107a或IL-12/无信号肽CD107a慢病毒基因修饰的T细胞。鼠的T细胞取自小鼠的脾脏细胞，利用10ug/ml的刀豆蛋白(Con A)在IL-2(5IU/ml)存在的条件下活化；第2天，用慢病毒载体转导T细胞，然后继续培养6天，收集细胞，通过小鼠尾静脉注射T细胞。DC组小鼠的DC细胞取自小鼠的骨髓细胞，经过体外诱导分化成熟8天，通过腹腔接种。然后每天记录小鼠的死亡情况，记录生长曲线并通过Prism绘图软件制图。星号表示该实验组与其它组比较，p<0.001。
实施例1融合基因的构建
融合基因由Invitrogen公司合成,融合基因的长度及序列通过1％的琼脂糖电泳及测序证实。
构建获得的IL-12/CD107a融合基因系列的结构如SEQ ID NO.:1、3所示，所编码的融合蛋白的氨基酸序列如SEQ ID NO.:2，4所示。
实施例2慢病毒表达载体的构建
采用通用方法中“慢病毒载体的构建及T细胞的基因修饰”所述的方法：体外低代DMEM(含10％的FBS)培养基培养的人源化293T细胞经计数后，传到15CM的培养皿中，培养皿的底部经过poly-D-Lysine处理，每个培养皿中铺20X 106细胞，第二天，每个转染培养皿中加入由DNA混合液和Lipofectamine的混合液:混合液的组成如下，取2ml Optimum I并加入pLenti-MSCV(22.5ug)，pMD2.G(7.5ug)，gag/pol(15ug)，pRev(10ug),混匀；同时取2ml Optimum I并加入Lipofectamine 160ul(Invitrogen)，混匀。把两种混悬液混合，放置室温孵育5分钟，然后均匀地滴加到培养皿中。48-72小时后，收获含有基因工程载体的上清，2000g离心去除细胞碎片，收集上清，并用0.45uM的滤膜过滤去除可能的污染，分装并存放于负80冰箱。根据不同的需要，收集的病毒上清可以进行50000g超速离心，得到更高浓度的病毒载体。
获得的慢病毒表达载体分别命名为LV-hscIL-12/CD107a、LV-hscIL-12/无信号肽CD107a、LV-hscIL-12。
实施例3表达IL-12/无信号肽CD107a融合蛋白的T细胞的制备
方法如下：CD3/CD28磁珠或抗-CD3抗体激活PBMC，第2天，利用慢病毒基因修饰T细胞，简要的方法如下：PBS缓冲液洗涤T细胞3次，按病毒滴度与T细胞的比例3：1加入适量的慢病毒，2000X g离心2h，6h后，加入100IU/ml IL-2继续培养；于第5天进行第2次转导，或联合共转导。根据细胞生长情况进行分瓶，二周后，根据T细胞所修饰的基因通过流式细胞仪进行检查。
实施例4IL-12/CD107a系列慢病毒基因修饰的人抗肿瘤T细胞实现IL-12瞬时膜表达
在本实施例中，构建了持续性分泌LVV-hscIL-12、hscIL-12/CD107和hscIL-12/无分泌肽IL-12的慢病毒载体(结构如图2.所示)，其中IL-12基因与CD107a基因通过氨基酸肽段SGSG链接，所述慢病毒为第三代慢病毒载体，启动子为MSCV。该载体的5’及3’LTR(long terminal repeat)被改造成SIN-LTR(self-inactivating-LTR),以降低慢病毒重组的机率，增强安全性能。
为了明确IL-12/CD107a融合蛋白系列在抗肿瘤T细胞的表达情况，依次对T细胞转导了抗肿瘤TCR及IL-12/CD107a融合蛋白系列，抗肿瘤T细胞体外培养14天后与肿瘤细胞526、938共培养。
结果观察到了IL-12/CD107a系列的肿瘤抗原反应性的瞬时膜表达的表型，同时也进一步证实了IL-12/CD107a可以通过CD107a的抗原反应性的膜迁移从而实现IL-12膜表达的理论推测。其中无信号肽CD107a显示出高表达水平，可能与IL-12/CD107a在胞浆内的加工过程而导致的部分分泌片切割有关。
实施例5慢病毒载体IL-12/CD107a系列转导的T细胞与肿瘤共培养可以增强IFNγ的表达
在本发明中，为了降低IL-12释放诱导产生的系统性细胞毒性，构建了瞬时膜表达的IL-12/CD107a融合蛋白载体系列。
如图3所示，对人的(物种)的T细胞依次转导抗肿瘤TCR及IL-12/CD107a系列融合蛋白14天后，与肿瘤细胞526及938共培养，24小时后，上清中IFNγ及IL-12表达水平通过ELISA试剂盒检测。
结果显示，持续性IL-12分泌组、IL-12/CD107a系列组较TCR基因修饰T细胞组比较，可以显著增强抗肿瘤T细胞与肿瘤的反应性，表现为显著增强IFNγ分泌的水平，P<0.001。
同图2中IL-12/CD107a膜表面可以检测到一致，IL-12/CD107a不能从细胞膜上切割，因此混合培养的细胞上清中不能检到IL-12的分泌。IL-12的表达只能在持续分泌IL-12的组中检测到。其它组同持续性分泌IL-12组比较，可以检测到分泌的IL-12的水平显著升高，P<0.001。
因此，这证实了IL-12/CD107a系列可以诱导瞬时的IL-12膜表达，并且表达仅限于T细胞膜表面，不能分泌到培养的上清中，最大程度地降低了IL-12的分泌而导致的系统性细胞毒性。
实施例6慢病毒载体IL-12/无信号肽CD107a转导的T细胞可以有效避免持续性分泌IL-12造成的体外T细胞扩增的毒副作用
在解决了肿瘤抗原反应性的IL-12膜瞬时表达的技术上和理论上的可行性及增强抗肿瘤反应性的问题后，本实施例进一步观察转导该IL-12/CD107a融合蛋白系列对T细胞体外扩增的影响。
如图4所示，对T细胞依次转导抗肿瘤TCR及IL-12/CD107a融合蛋白系列14天后，各组细胞与对照转导组(T-cell)的扩增倍数比较，数据分析采用设定T-cell组的扩增倍数为100％,其它各组的值系与T-cell组的比值。
结果表明，转导持续分泌IL-12组较其它各组的扩增倍数显著降低，p<0.001。其中，转导hscIL-12/无信号肽融合蛋白组同TCR组比较扩增倍数相同，未见显著差异。但是，转导hscIL-12/无信号肽CD107a融合蛋白组的扩增倍数显著高于转导了hscIL-12/CD107a融合蛋白组。推测可能的原因是由于CD107a的信号肽在IL-12/CD107a融合蛋白在胞浆内加工过程中导致的分泌肽部分切割，从而导致IL-12从融合蛋白上的断裂，进而分泌而导致的系统性细胞毒性。
实施例7慢病毒载体hscIL-12/CD107a系列转导鼠T细胞介导的细胞回输治疗显著延长荷瘤小鼠的生存
在清楚了IL-12/CD107a的肿瘤抗原反应性膜瞬时表达、肿瘤抗原反应的增效性及体外扩增的基础上，在本实施例中，进一步采用了临床前荷瘤小鼠模型，观测体内抑瘤效应。方法如下：。
选择雌性pmel小鼠(每组7只小鼠)通过B16F10细胞植入颅内(5000细胞/只)(IC)5天，细胞回输前1天小鼠接受5Gy全身放疗。pmel小鼠是转基因小鼠，其T细胞通过转基因修饰稳定表达抗肿瘤TCR，可以识别B16F10细胞上的肿瘤抗原gp100。(Overwijk,Tsung et al.1998)鼠的T细胞取自小鼠的脾脏细胞，通过10ug/ml的刀豆蛋白(Con A)在IL-2(5IU/ml)存在的条件下活化；第2天，用慢病毒载体转导T细胞，然后继续培养6天，收集细胞，通过小鼠尾静脉注射(IV)5X 106个T细胞。DC组小鼠的DC细胞取自骨髓细胞，经过体外诱导分化成熟8天，通过腹腔接种1X 106细胞。如图5所示，DC和T细胞的各组说明见右边标识。星号(*)表示转导IL-12该实验组与其它组比较，p<0.001；星号(**)表示转导IL-12/无信号肽CD107a组的生存期显著优于IL-12/CD107a组。其中持续分泌IL-12组表现为显著的系统性细胞毒性，小鼠于细胞回输后4-7天全部死亡。
讨论
CD107a是一种表达于溶酶体上的跨膜蛋白，它的N-端位于溶酶体腔囊一侧，(Eskelinen 2006)C-端是一小段肽段，朝向胞浆一侧，当抗肿瘤T细胞在识别肿瘤抗原时，溶酶体与T细胞膜融合而发生T细胞或NK细胞的脱颗粒效应，即T细胞或NK细胞释放效应分子颗粒酶(Granzyme B)及穿孔素(perforin)而达到杀伤肿瘤细胞的过程。在T细胞与肿瘤细胞发生反应的时，CD107a迁移到T细胞膜上的特性已经被 广泛地应用于反应T细胞杀伤功能的一个指标。
虽然IL-12具有显著的抑瘤效果，但是由于其系统性全身给药会引起全身性毒副反应，因而受到极大的限制。在本发明之前，IL-12的临床应用由于其所诱导的全身性毒副作用，以及早期临床试验通过全身系统性给药引起的两例患者的意外死亡，使其临床应用受到大大的限制。
为了避免全身性给药的毒副作用从而实现临床应用的目的，临床科研工作者们通过肿瘤局部注射、瞬时表达及多点注射等途径应用于肿瘤的临床试验，但是由于IL-12的治疗效果同回输的IL-12的剂量直接相关，上述局部的临床方案并未有取得显著的抑瘤效果。
本发明为了解决IL-12系统性分泌而导致的系统性细胞毒性，通过构建IL-12/CD107a融合蛋白系列的策略，通过CD107a膜迁移从而实现IL-12的肿瘤抗原反应性的瞬时膜迁移的技术难题，不但可以增强抗肿瘤反应，对抗肿瘤T细胞的体外扩增亦无影响，是一种有广泛应用前景的抗肿瘤策略，在肿瘤的免疫细胞的治疗中将发挥重要的作用。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
参考文献
Cohen,J.(1995)."IL-12deaths:explanation and a puzzle."Science 270(5238):908.
Eskelinen,E.L.(2006)."Roles of LAMP-1and LAMP-2in lysosome biogenesis and autophagy."Mol Aspects Med 27(5-6):495-502.
Overwijk,W.W.,A.Tsung,et al.(1998)."gp100/pmel 17is a murine tumor rejection antigen:induction of"self"-reactive,tumoricidal T cells using high-affinity,altered peptide ligand."J Exp Med 188(2):277-286.
Yang,S.,C.J.Cohen,et al.(2008)."Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition."Gene Ther 15(21):1411-1423.
Zhang,L.,S.P.Kerkar,et al.(2011)."Improving adoptive T cell therapy by targeting and controlling IL-12expression to the tumor environment."Mol Ther 19(4):751-759.

Claims (10)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包括融合在一起的以下元件：
    (i)任选的位于N端的信号肽和/或前导肽；
    (ii)第一蛋白元件；
    (iii)第二蛋白元件；以及
    (iv)任选的位于第一蛋白元件和第二蛋白元件之间的连接肽元件；
    其中，所述信号肽可操作地连于由(ii)、(iii)和(iv)所构成的融合元件；
    并且第一蛋白元件为IL-12蛋白元件；第二蛋白元件为CD107a蛋白元件；
    在另一优选例中，所述的CD107a是去掉CD107a分泌肽的CD107a成熟的蛋白元件；
    在另一优选例中，所述的“可操作地连于”指所述信号肽可引导所述融合元件的表达或跨膜转移(定位)；
    在另一优选例中，所述的融合蛋白具有选自下组的结构：
    (1)式Ia所述结构：
    D-A-B   (Ia)，或
    (2)式Ⅱa所述结构：
    D-A-C-B   (Ⅱa)，
    其中，
    A为IL-12蛋白元件；
    B为CD107a或CD107a成熟蛋白元件；
    C为任选的连接肽元件；
    D为任选的信号肽信号肽和/或前导肽序列；
    “-”表示连接上述元件的肽键或肽接头；
    在另一优选例中，所述的连接肽元件包括序列如SEQ ID NO.:12所示的连接肽；
    在另一优选例中，所述的IL-12蛋白来源于人或非人哺乳动物；
    在另一优选例中，所述的IL-12蛋白包括野生型和突变型；
    在另一优选例中，所述的IL-12蛋白包括全长的、成熟形式的IL-12，或其活性片段；
    在另一优选例中，所述的第一蛋白元件包括IL-12蛋白的一个或两个亚基；
    在另一优选例中，所述的IL-12蛋白的亚基选自下组：P40和P35亚基；
    在另一优选例中，所述的第一蛋白元件包括连接在一起的IL-12蛋白P40和P35亚基；
    在另一优选例中，所述的P40和P35亚基为“头-头”、“头-尾”、“尾-尾”相连；
    在另一优选例中，所述的P40和P35亚基之间存在或不存在接头(linker)；
    较佳地，所述的接头为柔性的4-20个氨基酸的接头，更佳地，所述的接头为GGGGGGS(G6S)(SEQ ID NO.:14)；
    在另一优选例中，所述的IL12蛋白元件的序列如SEQ ID NO.:6所示；
    在另一优选例中，所述的成熟CD107a蛋白元件中缺少了分泌肽；
    在另一优选例中，所述的CD107a蛋白来源于人或非人哺乳动物；
    在另一优选例中，所述的CD107a蛋白包括全长的、成熟形式的CD107a，或其活性片段；
    在另一优选例中，所述的CD107a蛋白元件的序列如SEQ ID NO.:8所示；
    在另一优选例中，所述的肽接头的长度为0-15个氨基酸，较佳地1-10个氨基酸；
    在另一优选例中，所述融合蛋白还包括信号肽元件D；
    在另一优选例中，所述的融合蛋白中，在单链IL-12(第一蛋白元件)和CD107a(第二蛋白元件)之间设有连接肽，优选SGSG序列(SEQ ID NO.:12)；
    在另一优选例中，所述的融合蛋白中，第一蛋白元件为单链IL-12，在所述单链IL-12中，在P40亚基及P35亚基设有连接肽G6S(SEQ ID NO.:14)；
    在另一优选例中，所述融合蛋白的氨基酸序列如SEQ ID NO.:2所示；
    在另一优选例中，所述融合蛋白具有以下多种特征：
    a)所述融合蛋白CD107a去掉了分泌肽；
    b)所述融合蛋白包含IL-12的两条亚基,即P40和P35亚基,并由GGGGGGS(G6S)连接；
    在另一优选例中，所述的融合蛋白为单体、或二聚体。
  2. 一种分离的多核苷酸，其特征在于，所述的多核苷酸编码权利要求1所述的融合蛋白；
    在另一优选例中，所述的多核苷酸的序列如SEQ ID NO.:1所示。
  3. 一种载体，其特征在于，它含有权利要求2所述的多核苷酸；
    在另一优选例中，所述的载体包括质粒、病毒载体；
    在另一优选例中，所述的病毒载体包括：慢病毒载体、腺病毒载体、黄热病毒载体；
    在另一优选例中，所述的载体包括表达载体。
  4. 一种宿主细胞，其特征在于，它含有权利要求3所述的载体或基因组中整合有权利要求2所述的多核苷酸；
    在另一优选例中，所述的宿主细胞包括原核细胞和真核细胞；
    在另一优选例中，所述的宿主细胞包括哺乳动物细胞；
    在另一优选例中，所述的宿主细胞包括免疫细胞，较佳地T细胞、NK细胞。
  5. 一种产生权利要求1所述的蛋白的方法，它包括步骤：
    (1)在适合表达的条件下，培养本发明权利要求4所述的宿主细胞，从而表达出权利要求1所述的融合蛋白；和
    (2)任选地分离所述融合蛋白。
  6. 一种免疫细胞，其特征在于，所述的免疫细胞在膜表面上携带权利要求1所述的融合蛋白；
    在另一优选例中，所述的免疫细胞为至少103个(较佳地103-109个，更佳地较佳地104-108个)所述免疫细胞的细胞群；
    在另一优选例中，至少一部分或全部所述的融合蛋白位于所述免疫细胞的细胞膜上，并且所述的第一蛋白元件即IL-12蛋白元件位于胞外；
    在另一优选例中，所述的免疫细胞包括T细胞；
    在另一优选例中，所述的T细胞表面携带MART-1 TCR。
  7. 一种药物组合物，其特征在于，所述的组合物包含：
    权利要求6所述的免疫细胞，以及
    药学上可接受的载体；
    在另一优选例中，所述的药物组合物为液态；
    在另一优选例中，所述的药物组合物含有1×103-1×107个所述的免疫细胞/mL。
  8. 一种如权利要求1所述的融合蛋白和/或权利要求6所述的免疫细胞的用途，其特征在于，用于制备治疗肿瘤的药物；
    在另一优选例中，所述肿瘤包括：脑肿瘤、大肠癌肿瘤、肺癌肿瘤、肝癌肿瘤、乳腺癌肿瘤、胃癌肿瘤、胰腺癌肿瘤。
  9. 一种治疗肿瘤的方法，其特征在于，包括步骤：给需要的对象施用权利要求1所述的融合蛋白和/或权利要求6所述的免疫细胞；
    在另一优选例中，所述的融合蛋白以单体和/或二聚体形式施用；
    在另一优选例中，所述的对象是人。
  10. 一种治疗性的体外杀灭肿瘤细胞的方法，其特征在于，包括步骤：将权利要求6所述的免疫细胞与肿瘤细胞进行接触，从而杀灭所述肿瘤细胞。
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092737A1 (en) * 2002-04-30 2003-11-13 Molmed Spa Fusions of cytokines and tumor targeting proteins
CN1665933A (zh) * 2002-04-30 2005-09-07 莫尔梅德股份有限公司 用于治疗肿瘤的免疫缀合物
CN101638657A (zh) * 2008-12-17 2010-02-03 张文卿 重组人白细胞介素12真核表达载体的构建及其应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003092737A1 (en) * 2002-04-30 2003-11-13 Molmed Spa Fusions of cytokines and tumor targeting proteins
CN1665933A (zh) * 2002-04-30 2005-09-07 莫尔梅德股份有限公司 用于治疗肿瘤的免疫缀合物
CN101638657A (zh) * 2008-12-17 2010-02-03 张文卿 重组人白细胞介素12真核表达载体的构建及其应用

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