WO2014180288A1 - 具有抑制肿瘤微环境血管再生和激活适应性免疫应答双功能的融合蛋白及其基因和应用 - Google Patents

具有抑制肿瘤微环境血管再生和激活适应性免疫应答双功能的融合蛋白及其基因和应用 Download PDF

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WO2014180288A1
WO2014180288A1 PCT/CN2014/076768 CN2014076768W WO2014180288A1 WO 2014180288 A1 WO2014180288 A1 WO 2014180288A1 CN 2014076768 W CN2014076768 W CN 2014076768W WO 2014180288 A1 WO2014180288 A1 WO 2014180288A1
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seq
cd137l
amino acid
protein
tumstatin
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PCT/CN2014/076768
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English (en)
French (fr)
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王淑珍
陈依军
何东洋
刘楠
马超
高振月
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中国药科大学
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Priority claimed from CN201310163407.1A external-priority patent/CN103232543B/zh
Priority claimed from CN201310162738.3A external-priority patent/CN103214584B/zh
Application filed by 中国药科大学 filed Critical 中国药科大学
Priority to DK14795356.6T priority Critical patent/DK2995626T3/en
Priority to US14/895,959 priority patent/US10875903B2/en
Priority to ES14795356.6T priority patent/ES2686968T3/es
Priority to EP14795356.6A priority patent/EP2995626B1/en
Priority to JP2016512207A priority patent/JP2016520053A/ja
Publication of WO2014180288A1 publication Critical patent/WO2014180288A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • 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
    • 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
    • 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
    • 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
    • C07K2317/00—Immunoglobulins specific features
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • Fusion protein with dual functions of inhibiting tumor microenvironment angiogenesis and activating adaptive immune response, and gene and application thereof
  • the invention belongs to the field of bioengineering technology.
  • the present invention relates to a fusion protein CD137L-Tumstatin having a dual function of inhibiting tumor microenvironment angiogenesis and activating an adaptive immune response, and an expression vector containing the same, and a strain transformed from the vector, and the fusion
  • a method for preparing a protein and relates to the use of a protein having dual functions of CD137L and Tumsstatin for the preparation of a medicament for inhibiting tumor microvascular regeneration and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.) and activating an adaptive immune response drug,
  • the route of administration e.g., oral administration, spray administration, etc.
  • Cancer is a process of multi-gene cooperation and participation by different signaling pathways. By its very nature, cancer is a molecular condition. At present, single-molecule molecular therapy strategies have gradually shown many drawbacks, and multi-target, multi-mechanical combination therapy has shown better therapeutic effects.
  • Tumor growth and metastasis depend on microenvironmental angiogenesis.
  • the theory suggests that by inhibiting tumor microenvironmental angiogenesis, cutting off the nutrient and oxygen supply of the tumor can inhibit the growth and metastasis of tumor cells. Since its publication, the doctrine has been supported by a large number of laboratory and clinical data worldwide, and has become a new strategy in the field of cancer therapy.
  • Human tumor suppressor (Tumstatin) is a tumor angiogenesis inhibitory factor derived from the C-terminal end of the vascular basement membrane type IV collagen ⁇ 3 chain, consisting of 244 amino acids.
  • the three-dimensional network skeleton formed by type IV collagen is the main component of scaffolding, which promotes cell adhesion, migration, differentiation and growth. It is encoded by six unique genes to generate six chains ⁇ 1 ⁇ ⁇ 6 and form a trimer with different or identical ⁇ chains to further form a network structure.
  • Each ⁇ 3 chain of type IV collagen consists of three functional regions (7S domain, triple helix region, non-collagen peptide NC1 domain), distributed in glomeruli, alveolar capillaries, cochlea, lens capsule, ovary And the basement membrane of the testes.
  • Tumstatin specifically inhibits the synthesis of tumor vascular endothelial cell proteins, leading to apoptosis of endothelial cells, inhibiting angiogenesis, thereby inhibiting tumor growth and metastasis.
  • Tumstatin also has a property of directly acting on tumor cells and inhibiting their proliferation.
  • the cells of the immune system of a tumor patient are capable of recognizing antigens expressed by tumor cells, such as tissue differentiation antigens, carcinoembryonic antigens, and products of mutant genes, and the like.
  • tumor antigen recognition and immune response mechanisms studies have shown that providing immune co-stimulatory signals through helper molecules can increase the anti-tumor immune response. Since tumor antigen-specific tau cells require synergistic stimulation to assist the first antigen signal to stimulate the function of effector cells, adjuvant therapy with synergistic stimulation molecules can be used to modulate immune responses against malignant tumors.
  • TCR T cell receptor
  • APC antigen-presenting cells
  • the peptide signal first signal
  • the synergistic stimulus signal ie, the second signal
  • CD137 and its ligand CD137L are another important pair of T cell synergistic stimulation signal molecules newly discovered in addition to CD28/B7.
  • the conjugated molecules can be divided into two categories: Tumor Necrosis Factor Receptor (TNFR) and immunoglobulin superfamily.
  • TNFR Tumor Necrosis Factor Receptor
  • CD137 is a member of the tumor necrosis factor receptor family. It plays an important role in regulating cell proliferation, differentiation, and apoptosis.
  • Its ligand CD137L is also a member of the TNF family, which is a type II transmembrane protein with similar C-terminal amino acids to other T F families.
  • the gene encoding human CD137L is located at 19p3.3, and its product is 254 amino acids, of which 28 amino acids in the cytoplasmic region, 21 amino acids in the transmembrane region, and 205 amino acids in the extracellular region.
  • CD137L was first isolated from mouse thymoma cells by Goodwin et al. using expression screening techniques and subsequently isolated in human CD4+ T cell clones.
  • CD137/CD137L The interaction between CD137 and its ligand CD137L (CD137/CD137L) has been well recognized for its important role in T cell activation. It was observed in both murine and human T cells that CD137 can induce T cell proliferation, synthesize cytokines (such as IFN- ⁇ ), and prolong the survival of activated cells in the presence of CD3 antibodies (first signal). Synergistic stimulation signals enhance effector function by increasing the number of antigen-specific and effector CD8+ T cells; however, in the absence of CD3 antibody signaling, stimulation of CD137 molecules does not alter T cell function, suggesting that CD137 interacts with CD137L. All that is provided is a synergistic stimulus signal.
  • CD137 monoclonal antibody-mediated anti-tumor immunity is dependent on the involvement of CD4+T and CD8+ T cells.
  • CD137 mediates the activation of F-kB, which in turn up-regulates the expression of bcl-xL and bfl-1 molecules, prolongs the survival of CD8+T and CD4+ T cells and promotes their proliferation.
  • Melero et al. used activated murine CD137 monoclonal antibodies for CD137-targeted immunotherapy. The results indicate that CD137 mAb can eliminate P815 solid tumors that have been inoculated in mice.
  • CD137L synergistically stimulates cytotoxicity Cytotoxic lymphocyte (CTL) and anti-tumor effects.
  • CTL Cytotoxic lymphocyte
  • CD137L-/- mice well demonstrate the important role of the CD137/CD137L system in T cell-mediated immune responses to viruses and tumors.
  • Studies in CD137 or CD137L deficient mice indicate that the synergistic stimulation of CD137/CD137L is important for graft versus host disease, antiviral cellular responses of T cells.
  • the stimulation signal provided by CD137/CD137L can synergize with CD28/B7 molecules to further activate T cells and maintain the proliferation and survival of CD8+ T cells.
  • Tumstatin can effectively inhibit the formation of tumor blood vessels and block the survival and metastasis of tumor cells. Therefore, the fusion protein T U m S t a tin-CD137L of the present invention can inhibit tumor growth and metastasis from both inhibition of angiogenesis and enhancement of anti-tumor immune response, thereby avoiding drug tolerance caused by single treatment, for treatment Tumor patients offer new strategies.
  • the present invention synthesizes an anti-angiogenic active fragment of tumstatin and a CD137L extracellular domain protein by a short flexible linker peptide, and has a bifunctional, dual target molecule which enhances T cell immunity and antiangiogenesis; and is selected in the present invention.
  • Tumstatin 45th to 98th amino acid fragment of Tumstatinl, 60th to 132nd amino acid fragment of Tumstatin2, 60th to 98th amino acid fragment of Tumstatin3, 74th to 98th amino acid of Tumstatin7 Fragment
  • CD137L extracellular domain protein amino acid sequence CD137L1 amino acid fragment 46 to 254, CD137L4 50th to 240th amino acid fragment, CD137L5 83th to 254th amino acid fragment, CD137L6 46th to the
  • the ligated fragment of amino acid 85 and amino acid 167 to 254, as shown in Figure 2 is prepared by ligation of a linker peptide, using a prokaryotic or eukaryotic expression system, which is simple to prepare and avoids full-length tumstatin and The side effect of full length CD137L, the present invention will be in the preparation of angiogenesis inhibitors, various Tumor-related diseases (such as melanoma, prostate cancer, lung cancer, colon cancer, kidney
  • Another object of the present invention is to provide a gene encoding the above-described protein having the functions of Tumstatin and CD137L.
  • Another object of the present invention is to provide a process for the preparation of the above-described protein having the functions of Tumstatin and CD137L.
  • Another object of the present invention is to provide the above-mentioned protein having Taustatin and CD137L functions and genes thereof for treating oncology-related diseases (e.g., melanoma, rectal cancer, lung cancer, etc.) and administration routes (e.g., oral administration, spraying Application of the route of administration, etc.).
  • oncology-related diseases e.g., melanoma, rectal cancer, lung cancer, etc.
  • administration routes e.g., oral administration, spraying Application of the route of administration, etc.
  • a bifunctional recombinant protein having tumstatin and CD137L having an amino acid sequence of a Tumstatin active fragment and The CD137L extracellular domain protein fragment amino acid sequence, the CD137L extracellular domain protein fragment amino acid sequence and the Tumstatin active fragment amino acid sequence are fused by a flexible linker peptide, and the Tumstatin active fragment amino acid sequence is selected from the group consisting of SEQ ID N0.65 to SEQ ID One of the amino acid sequences shown in N0.68, wherein the amino acid sequence of the CD137L extracellular domain protein fragment is selected from one of the amino acid sequences shown in SEQ ID N0.77 to SEQ ID NO.
  • the amino acid sequence of the above-described linker peptide can be designed by a conventional technique in the art, preferably one of SEQ ID N0.69 to SEQ ID N0.76.
  • the bifunctional recombinant protein amino acid sequence of the present invention is preferably one of SEQ ID N0.25 to SEQ ID N0.48, and the recombinant protein is schematically shown in Figure 1.
  • the present invention also provides a gene encoding the above bifunctional recombinant protein having Tumsstatin and CD 137L, comprising a gene encoding a CD 137L extracellular domain protein fragment, a gene encoding a linker peptide, and a gene encoding a Tumsstatin active fragment, wherein the encoding CD137L extracellular
  • the gene for the region protein fragment is selected from one of SEQ ID N0.61 - SEQ ID NO. 64, and the gene encoding the active fragment of Tumsstat is selected from one of SEQ ID N0.49 - SEQ ID N0.52.
  • the above gene encoding the linker peptide is preferably one of SEQ ID N0.53-SEQ ID NO.
  • the above recombinant protein gene preferably has the nucleotide sequence of one of SEQ ID NO: 0.1 to SEQ ID NO: 4.
  • the present invention also provides a gene encoding the above recombinant protein T U m S t a tin-CD137L, which has 70% or more homology with the above nucleotide sequence, and can encode the recombinant protein of the present invention. Or a conservative variant polypeptide thereof or an active fragment thereof or a reactive derivative thereof.
  • the present invention also provides the above preparation method of the bifunctional recombinant protein having Tumstatin and CD137L, comprising the following steps:
  • the recombinant protein having the activity of Tusumatin and CD137L of the present invention is isolated and purified.
  • the above expression system may select a prokaryotic expression system or a eukaryotic expression system, and the prokaryotic expression system is preferably an E. coli expression system or a Bacillus subtilis expression system, and the two expression systems are generally adapted to the expression of the recombinant protein of the present invention, wherein the Escherichia coli system
  • the medium expression vector is preferably pET-lla, pET-22b, and the B. subtilis expression system expression vector is preferably pP43;
  • the eukaryotic expression system is preferably a yeast expression system, the expression vector is preferably pPIC9K, pPICZaA, and the yeast host cell is preferably GS115 or SMD1168.
  • a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having TumMatin activity and CD137L activity is digested with Ndel and Nhel, and then ligated to the expression vector pET-lla.
  • the enzyme cleavage site was transformed into E. coli BL21 (DE3), and the target protein in the form of inclusion bodies was obtained by liquid culture engineering bacteria.
  • the inclusion body protein was subjected to dilution and renaturation, that is, the inclusion body protein was washed several times with a solution containing a low concentration of urea, and then the inclusion body was dissolved at 50 ° C with a denatured solution containing 8 M urea, and finally 0.4 M L-Arg was used.
  • the refolding body is diluted with the refolding inclusion body (the purity of the target product is more than 80%), and the recombinant protein of the present invention is obtained.
  • a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having TumMatin activity and CD137L activity is digested with Pstl and Hindlll, and then ligated to the corresponding restriction site of the expression vector pP43, and then electrotransferred.
  • Bacillus subtilis WB800 a protein of interest secreted into an extracellular soluble form was obtained by liquid culture engineering bacteria. Purification by DEAE anion exchange method, the purity is over 80%, and the recombinant protein of the present invention is obtained.
  • a preferred embodiment of the above preparation method is: the gene encoding the above-mentioned recombinant protein Tumsstatin-CD137L having Tumatatin activity and CD137L activity is double-digested with EcoRI and Notl, and then ligated to the corresponding restriction site of the expression vector pPICZaA, and then electrotransferred.
  • Pichia pastoris GS115 the target protein secreted into the extracellular soluble form was obtained by liquid culture engineering bacteria. Purification by DEAE anion exchange, the purity is over 90%, and the recombinant protein of the present invention is obtained.
  • the present invention also provides the above-mentioned dual-function recombinant protein with tumstatin and CD137L in the preparation of tumor microenvironment-inhibiting angiogenesis and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.), regulation of body immunity, T cell proliferation and body The application of cytokine synthesis and secretion of drugs.
  • the present invention also provides the above-mentioned dual-function recombinant protein gene with tumstatin and CD137L for preparing tumor microvascular angiogenesis and related oncological diseases (eg, melanoma, rectal cancer, lung cancer, etc.), regulating body immunity, T cell proliferation, and The application of the synthesis and secretion of cytokines in the body.
  • tumor microvascular angiogenesis and related oncological diseases eg, melanoma, rectal cancer, lung cancer, etc.
  • body immunity eg, melanoma, rectal cancer, lung cancer, etc.
  • T cell proliferation e.g, T cell proliferation
  • the recombinant protein of the present invention may be used alone or in the form of a pharmaceutical composition.
  • the pharmaceutical composition comprises the recombinant protein of the present invention as an active ingredient and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition has from 0.1 to 99.9% by weight of the recombinant protein of the present invention as an active ingredient.
  • the "pharmaceutically acceptable carrier” does not destroy the pharmaceutical activity of the recombinant protein of the present invention, and its effective amount, i.e., the amount which can be used as a drug carrier, is not toxic to the human body.
  • “Pharmaceutically acceptable carrier” includes, but is not limited to, ion exchange materials, alumina, aluminum stearate, lecithin, self-emulsifying drug delivery systems (SEDDS) such as d-vitamin E polyethylene glycol 1000 succinate, Tween Or other surfactants such as a polymeric medium, serum proteins such as human serum albumin, buffer substances such as phosphate, glycine, sorbic acid, potassium sorbate, saturated glycerides of saturated plant fatty acids, water, salt, Electrolytes such as protamine, dibasic hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, silica gel, magnesium silicate, and the like.
  • SEDDS self-emulsifying drug delivery systems
  • excipients such as fillers (such as anhydrous lactose, starch, lactose beads and glucose), binders (such as microcrystalline cellulose), disintegrants (such as croscarmellose sodium, cross-linked carboxylates) Sodium methylcellulose, low-substituted hydroxypropylcellulose and cross-linked PVP), lubricants (such as magnesium stearate), absorption enhancers, flavoring agents, sweeteners, diluents, excipients, wetting agents Solvents, solubilizers, colorants and the like may also be added to the pharmaceutical compositions of the invention.
  • fillers such as anhydrous lactose, starch, lactose beads and glucose
  • binders such as microcrystalline cellulose
  • disintegrants such as croscarmellose sodium, cross-linked carboxylates
  • lubricants such as magnesium stearate
  • absorption enhancers such as magnesium stearate
  • flavoring agents such as sweeteners,
  • any dosage form that can be optionally used.
  • oral administration forms such as tablets, capsules, granules, powders or liquid preparations, or parenteral administration forms such as injections, topical products or suppositories, which may be formulated in a conventional manner or unconventional.
  • parenteral administration forms such as injections, topical products or suppositories, which may be formulated in a conventional manner or unconventional.
  • Methods such as liposomes and the like.
  • the recombinant protein of the present invention When used as a therapeutic agent, its use amount is in the range of about 0.01 mg to lg per day for an adult, depending on the age, sex, body weight and degree of symptoms of each patient, and the daily dose can be divided into Several doses.
  • the recombinant protein of the present invention further comprises a modified protein which modifies the recombinant protein of the present invention by a conventional method in the prior art.
  • modified peptide drugs can significantly reduce immunogenicity, reduce toxic side effects, increase water solubility, prolong the time of action in vivo, and change Its bio-distribution and so on, significantly improve the efficacy of the drug.
  • the commonly used modification methods of the recombinant protein of the present invention include modification of intermediate residues, amino acid substitution, glycosylation modification and PEG modification, etc.
  • the basic principle is to increase the relative molecular weight and steric hindrance of the polypeptide molecule, and to increase the peptide hydrolase. Stability, reducing glomerular filtration. Replacing a few amino acids in a peptide chain is another way of delaying the degradation of the polypeptide to extend the half-life of the polypeptide drug.
  • the replacement object is usually an easily digestible amino acid in the peptide chain.
  • the intermediate residues of the recombinant protein may be glycosylated, phosphorylated, methylated, acetylated, nitrated, sulfonated or linked to PEG modified or coupled to a protein, wherein:
  • glycosylation-modified peptide is preferably one or more of the oxygen-linked to the sugar at one or more Tyr, Ser or Thr residues in the amino acid sequence of the recombinant protein of the present invention or the amino acid sequence of the recombinant protein of the present invention.
  • the amide nitrogen of the asparagine side chain is attached to the sugar.
  • the phosphorylated modified peptide is preferably phosphorylated at one or more Tyr, Ser or Thr sites in the amino acid sequence of the recombinant protein of the present invention.
  • the methylated modified peptide includes a side chain methylation modified peptide and an N-terminal methylated modified peptide, and the side chain methylation is preferably one or more Lys, Tyr or Arg side chains in the amino acid sequence of the recombinant protein of the present invention.
  • Methylation such as Lys (For), Lys (Me), Lys (Me) 2, Lys (Me) 3, Arg (Me) 2 symmetrical, D-Tyr (Me), D-Tyr (Et);
  • the acetylated modified peptide is preferably subjected to one or more Lys or Ser side chains in the amino acid sequence of the recombinant protein of the present invention. Acylation, such as Ser (Ac) or Lys (Ac).
  • the nitrating or sulfonating modified peptide is preferably nitrolated or sulfonated on one or more Tyr side chains in the amino acid sequence of the recombinant protein of the present invention, Tyr(3-N0 2 ), Tyr(S0 3 H 2 ).
  • the PEG modification of the intermediate residue is preferably PEG-modified with one or more amino groups of the Lys side chain in the amino acid sequence of the recombinant protein of the present invention, and the PEG molecular weight is preferably from 2,000 to 10,000.
  • one or more amino acids in the amino acid sequence of the recombinant protein of the present invention or the above modified protein thereof are replaced with corresponding amino acid derivatives or special amino acids, such as alanine instead of ⁇ -alanine, high Amphetamine or naphthylalanine, replacing proline with hydroxyproline, leucine with norleucine, valine with normal valine, threon with other threonine,
  • the leucine is replaced by a different leucine
  • the asparagine is replaced by 2-acetamido-2-deoxy- ⁇ -D-glucopyranosyl asparagine (Asn (GlcNac(Ac)3-e-D))
  • Lysine is replaced by Lys (palmitoyl).
  • one or more amino acids in the amino acid sequence of the recombinant protein of the present invention or the above-described modified protein thereof are replaced with the corresponding D-type amino acid.
  • the gene encoding the above bifunctional recombinant protein having Tusumatin activity and CD137L activity (GenBank: AAF72632.1 and P_003802.1) in the present invention is obtained by a whole gene synthesis, a PCR method, or a combination thereof, using a conventional strategy, wherein CD137L
  • the full-length sequence vector template can be prepared by the method disclosed in the literature (Wang shuzhen. J Ind Microbiol Biotechnol. 2012 Mar; 39(3): 471-6. doi: 10.1007/sl0295-011-1045-l.).
  • the present invention constructs an expression vector containing the above-described protein gene encoding the dual function of Tumsstatin and CD137L, which is obtained by double-digesting the Numdel and Nhel gene fragments by conventional PCR and restriction enzyme digestion and ligation. , and connected to the corresponding restriction sites of the prokaryotic expression vector pETlla, and verified by sequencing to obtain the correct expression vector.
  • the present invention constructs a genetically engineered bacteria containing the above expression vector, that is, by transforming the target gene into BL21, a positive engineering strain expressing the target protein is screened by a small amount of liquid culture medium.
  • the invention provides a method for obtaining a protein having dual functions of Tumsstatin and CD137L, which is to carry out culture fermentation of a positive engineering strain and frequently induce it to express a protein having dual functions of Tumsstatin and CD137L, and then collect the bacteria, high pressure.
  • the disrupted cells, the pellet obtained by centrifugation were denatured and diluted and renatured to obtain a recombinant protein having a dual function of Tumsstatin and CD137L.
  • the activity of the recombinant protein of the present invention having dual functions of tumstatin and CD 137L was carried out by conducting a human umbilical vein endothelial cell activity assay (HUVEC assay) and a mouse T cell activation assay. Among them, the human umbilical vein endothelial cell activity test results showed that the proliferation of endothelial cells was significantly inhibited.
  • Mouse T cell activation assays have shown that the recombinant protein maintains the biological activity of CD137L and synergizes with anti-CD3 and anti-CD28 monoclonal antibodies to stimulate T cell proliferation. Colonization.
  • the present invention shows that a prokaryotic expression system is used to express a recombinant protein which is active from the extracellular domain of Tumsstatin and CD137L, and a protein which has both the activity of Tumsstatin and CD137L can be produced.
  • the Tumstatin-Linker-CD137L recombinant protein (a protein having both Tumstatin and CD137L functions) produced by the present invention has the advantages of high expression efficiency, large expression amount, short expression period, and easy purification.
  • the fusion protein provided by the present invention has a significant inhibitory effect on the proliferation of human umbilical vein endothelial cells in a dose-dependent manner, and synergistically anti-CD3 and anti-CD28 monoclonal antibodies can stimulate the proliferation of mouse T cells. Therefore, the present invention provides a new and safe way for large-scale production of recombinant proteins of Tumstatin and CD137L, and lays a solid foundation for further research and development as a new generation of anti-tumor drugs, and has broad application prospects in the pharmaceutical industry.
  • Figure 1 is a schematic view showing the structure of a recombinant protein having dual functions of Tumstatin and CD137L according to the present invention.
  • the flexible linker amino acid sequence is selected from one of SEQ ID N0.69 - SEQ ID N0.76.
  • Figure 2 is a schematic representation of the region of CD137L1 (SEQ ID N0.77), CD137L5 (SEQ ID N0.78), CD137L6 (SEQ ID N0.79) and CD137L4 (SEQ ID NO. 80) in the full length sequence of CD137L amino acid.
  • Figure 3 shows the expression of the recombinant protein Tumstatin-linker peptide-CD137L in Escherichia coli with the amino acid sequence as shown in SEQ ID N0.25-SEQ ID N0.48. Lanes 1-24 correspond to SEQ ID N0.25-48, respectively.
  • Figure 4 shows the expression of the representative Tumstatin-linker peptide-CD137L protein amino acid sequence (SEQ ID N0.29-SEQ ID N0.38) in Bacillus subtilis. Lanes 1-8 correspond to SEQ ID NO. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, respectively.
  • Figure 5 is a representation of the representative amino acid sequence of the Tumstatin-linker peptide-CD137L protein (SEQ ID NO. 29 - SEQ ID NO. 36) in yeast. Lanes 1-16 correspond to the protein amino acid sequences SEQ ID NO. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, respectively.
  • Figure 6 is a graph showing the results of SDS-PAGE electrophoresis of a representative Tumtatin-Linker-CD137L protein sample after dilution renaturation.
  • the amino acid sequence of the first lane sample is SEQ ID N0.25; the amino acid sequence of the second lane sample is SEQ ID N0.26; the amino acid sequence of the third lane sample is SEQ ID N0.27; the amino acid sequence of the fourth lane sample is SEQ ID N0.37; the amino acid sequence of the fifth lane sample is SEQ ID N0.38; the amino acid sequence of the sixth lane sample is SEQ ID N0.41; the amino acid sequence of the seventh and tenth lane samples is SEQ ID N0.43;
  • the amino acid sequence of the 11 lane sample is SEQ ID N0.44; the amino acid sequence of the 9th and 12th lane samples is SEQ ID N0.48; the amino acid sequence of the 13th and 15th lane samples is SEQ ID N0.45; lanes 14 and 16
  • Figure 7 is a graph showing the effect of a representative Tumstatin-linker peptide-CD137L protein sample on the proliferation of human umbilical vein endothelial cells.
  • Figure 8 is a graph showing the effect of a representative Tumstatin-linker peptide-CD137L protein sample on the proliferation of mouse T lymphocytes.
  • the invention has designed 24 bifunctional recombinant proteins having Tumsstatin activity and CD137L activity, and the nucleotide sequence thereof is the sequence shown in SEQ ID NO. 1 to SEQ ID N0.24, and the translation encoding amino acid sequence is SEQ ID N0.25 to The sequence shown in SEQ ID N0.48.
  • the histone has amino acid fragments 45 to 98 of Taustantin, amino acid fragments 60 to 132 of Tumstatin 2, amino acid fragments 60 to 98 of Tumstatin 3 or amino acid fragments 74 to 98 of Tumatatin7 and CD137L cells.
  • amino acid sequence of the outer region protein (amino acid fragment 46 to 254 of CD137L1, amino acid fragment 83 to 254 of CD137L5, and the framing fragment of amino acids 46 to 85 of CD137L6 and amino acids 167 to 254
  • One of the two is fused by a flexible linker peptide, wherein the amino acid sequence of the linker peptide is as shown in SEQ ID N0.69 to SEQ ID N0.76.
  • the present invention is linked to the above-described Tumstatin active fragment gene and the linker peptide gene.
  • the 5' end and 3' of the nucleotide sequence (shown as SEQ ID N0.81-SEQ ID N0.94) were designed with EcoRI and BamHI restriction sites, and submitted to Shanghai Jierui Bioengineering Co., Ltd. for total synthesis.
  • the pBluescriptll SK(+) vector with the corresponding restriction site was inserted, and the recombinant vector was provided by Shanghai Jierui Bioengineering Co., Ltd.
  • Various expression vectors were provided by Novagen ToplO and BL21 (DE3) strains were purchased from Invitrogen.
  • pMD18-T vector, solution I (Cat. No.
  • Example 1 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in Escherichia coli expression system
  • CD137L1, CD137L4, CD137L5, and CD137L6 are derived from amino acids 46-254, 50-240 amino acids, 83-254 amino acids, and amino acids 46-85 and 167-254, respectively, of the full-length amino acid sequence of CD137L. Amino acid ( Figure 2).
  • the nucleotide sequence of the protein is a sequence of 8£010 ⁇ «).1 to 8£010 ⁇ «).24, and the amino acid sequence of the translation encodes the sequence of SEQ ID N0.25 to SEQ ID N0.48, and the linker peptide One selected from the sequences shown in SEQ ID N0.69 to SEQ ID N0.76.
  • CD137L Encoding the extracellular domain of CD137L (amino acid fragment 46 to 254 of CD137L1, amino acid fragment 50 to 240 of CD137L4, amino acid fragment 83 to 254 of CD137L5, amino acid 46 and 85 of CD137L6 and
  • the nucleotide sequence of the amino acid sequence from position 167 to position 254 is shown in SEQ ID N0.61-SEQ ID N0.64, and amplified by PCR, wherein the CD137L full-length sequence template reference (Wang shuzhen. J The method disclosed in Ind Microbiol Biotechnol. 2012 Mar; 39(3): 471-6. doi: 10.1007/sl0295-011-1045-l.) was prepared.
  • the CD137L template used in this patent was prepared by the method mentioned in the above literature. Using the upstream primer of SEQ ID NO. 61 (SEQ IDN0.95) and the downstream primer of PSEQ ID NO. 61 (SEQ IDN0.96) as a primer, rTaq DNA polymerase catalyzed amplification to obtain a CD137L1 (SEQ ID NO. 61) gene fragment (this time The restriction sites at both ends of the gene were 5' BamHI and 3' Notl), respectively, and the product was ligated to the pMD18-T vector.
  • the CD137L1 gene fragment was ligated from the pMD18-T vector by restriction enzyme ligation and other conventional molecular biological methods, and the Tumstatin and the linker peptide (SEQ ID NO. 87 - SEQ ID N0. 94) on the pBluescriptll SK(+) vector.
  • the entire fusion protein gene (SEQ ID N0.5 - SEQ ID N0.12) has been integrated into the pBluescriptll SK(+) vector.
  • the upstream primer SEQ ID N0.97 and the downstream primer SEQ ID N0.98 were used to amplify the fusion protein gene by PCR.
  • the 5' cleavage site was Nhel, and the 3' cleavage site was Ndel.
  • Linked to the expression vector pETlla The CD137L template used in this patent was prepared by the method mentioned in the above literature. Using the upstream primer (SEQ ID NO. 100) of SEQ ID NO. 62 and the downstream primer (SEQ ID NO. 100) of SEQ ID NO. 62 as primers, rTaq DNA polymerase catalyzes the CD137L5 (SEQ ID NO. 62) gene. The fragment (in this case, the restriction sites of the two ends of the gene are 5' BamHI and 3' Notl, respectively), and the product was ligated to the pMD18-T vector.
  • the CD137L5 gene fragment was ligated from the pMD18-T vector by restriction enzyme ligation and other conventional molecular biological methods, and the Tumstatin and the linker peptide (SEQ ID N0.81-SEQ ID NO. 86) on the pBluescriptll SK(+) vector.
  • the entire fusion protein gene (SEQ ID NO. 1, SEQ ID NO, SEQ ID NO. 13 - SEQ ID NO. 16) has been integrated into the pBluescriptll SK (+) vector.
  • SEQ ID NO. 15 - SEQ ID NO. 16 upstream primer 104 SEQ ID NO. 1 SEQ ID NO. 3, SEQ ID NO. 13 - SEQ ID NO. 16 downstream primer SEQ ID NO. 105, amplification of the fusion protein gene by PCR
  • the product 5' cleavage site is Nhel
  • the product 3' cleavage site is Ndel
  • ligated to the expression vector pETlla The CD137L template used in this patent was prepared by the method mentioned in the above literature.
  • the primer 1 (SEQ ID NO. 107) of the upstream primer 1 (SEQ ID NO. 106) SEQ ID NO. 63 of SEQ ID NO. 63 is used as the primer
  • 63 SEQ ID NO. .108
  • the downstream primer 2 SEQ ID NO. 109 of SEQ ID N0.63 are primers
  • rTaq DNA polymerase catalyzes the amplification of two gene fragments of CD137L6 (SEQ IDN0.63)
  • the agarose electrophoresis gel recovers the two Segment gene.
  • the recombinant two-stage gene was used as a template, and the upstream primer 1 (SEQ ID NO. 107) of CD137L6 and the downstream primer 2 (SEQ ID NO. 109) of CD137L6 were used as primers.
  • the final CD137L6 (SEQ ID N0.63) fragment (both restriction sites 5' BamHI and 3' Notl) was ligated and ligated into the pMD18-T vector.
  • the CD137L6 (SEQ IDN0.63) gene fragment was ligated from the pMD18-T vector and ligated to the already synthesized Tusstatin and the linker peptide (SEQ ID N0.8U SEQ ID NO. 82, SEQ ID NO. 84) synthesized by Shanghai Jierui Company. , SEQ ID NO. 86) on pBluescriptll SK (+) vector.
  • the entire fusion protein gene (SEQ ID NO. 2, SEQ ID NO. 4, SEQ ID NO.
  • the primers involved in the above experimental procedures are:
  • the correctly sequenced fragment was ligated to the prokaryotic expression vector pET1 la.
  • the reaction system was T4 ligase 10 X buffer 1 ⁇ , ⁇ ⁇ , target gene 7 ⁇ , ⁇ 4 ligase 1 ⁇ , reaction condition 16 ° C, 16 h. After double enzyme digestion, it was transformed into E. coli expression strain BL21 (DE3).
  • the wet weight of the cells was added to 10 mL of PBS and suspended, and the cells were lysed by a high-pressure cell disrupter. The effluent was collected, centrifuged at 4 ° C, 12000 rpm, 30 min, the supernatant was discarded, and the precipitate was left for SDS-PAGE analysis.
  • the SDS-PAGE experiment operation flow is as follows:
  • the bacterial body precipitate obtained after high-pressure crushing was collected by the above method, and washed repeatedly by washing liquids A and B twice, at 12,000 rpm for 10 minutes, and the supernatant was centrifuged and collected for use.
  • the preparation of the washing solution A was 20 mM Tris-HCL, pH 8.5, 2 M urea, 2% Triton X-100 ;
  • the washing liquid B formulation was 20 mM Tris-HCL, pH 8.5, 2 M urea, 5 mM EDTA.
  • the solution was denatured overnight at 50 ° C with a solution (20 mM Tris, pH 8.5, 8 M urea).
  • the denaturant was added to the refolding solution through a constant flow device in a 4 °C chromatography cabinet, and the whole process was continued for 12 h with slow agitation.
  • the renaturation solution was formulated as 20 mM Tris-HCL pH 8.5, 2 M urea, 0.4-0.6 M L-Arg, 1 mM EDTA.
  • the obtained reconstituted solution containing the sample was concentrated and replaced.
  • the apparatus used for concentration was an Amicon Ultra-15 concentrating tube from Millpore, 4500 rpm, 30 min, 4 °C.
  • the concentrate was replaced with pH 7.4 in 100 mM PBS buffer to a final volume of 1 mL. Reserve at 4 ° C ( Figure 6). It has been estimated that the purity of the target product after renaturation is over 80%. It can be seen from the figure that the renaturation of the denatured inclusion body protein is good.
  • Example 2 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in Bacillus subtilis expression system
  • pBluescriptll SK(+) vector containing the complete recombinant protein gene sequence (SEQ ID N0.1-24) constructed in Example 1 as a template, by designing different primers (5 'Pstl, 3 ' Hindlll) and performing PCR, The different fusion protein genes ligated to the pBluescriptll SK(+) vector were amplified and ligated into the expression vector pP43 (Pstl, Hindlll double-cleaved the vector before ligation). The steps of PCR and restriction enzyme ligation are the same as those of the fusion protein gene of Example 1. After the expression vector is constructed, the electroporation operation is carried out (for details, see Bio-Rad electroscopy instructions), and the recombinant strain of B. subtilis expression system WB800 is constructed.
  • Seed culture was carried out in a clean bench, and 10 ⁇ L of the above-preserved Bacillus subtilis WB800 strain containing the target gene plasmid was added to 5 mL of test tube medium in LB medium containing 50 ⁇ g/mL kanamycin. , placed on a shaker, incubated at 37 ° C, 250 rpm for 12 h.
  • Recombinant WB800 Fermentation Transfer the activated seed broth to a 50 ⁇ g/mL kanamycin sterile 2 X YT medium at a pH of 7.0 at 37 ° C for 96 h after transfer. The supernatant was collected by centrifugation at °C and sampled for SDS-PAGE analysis.
  • the protein of interest was purified by GE's DEAE anion exchange method.
  • the purification buffer was PBS phosphate buffer, pH 8.5.
  • Example 3 Expression of recombinant protein Tumstatin-CD137L with Tumstatin activity and CD137L activity in yeast expression system
  • the different fusion protein genes (SEQ ID NOS. 1-24) integrated on the pBluescriptll SK(+) vector were digested with EcoRI and Notl and ligated into pPIC9K to complete the vector construction work. Then carry out the electric operation (for details, see Bio-Rad The electrorotator instructions were used to complete the construction of the recombinant strain of Pichia pastoris GS115.
  • a BMGY-enriched cell, BMMY-induced expression strategy was used to ferment to obtain the desired product.
  • the expression strain constructed as described above was inoculated to YPD seed medium and cultured overnight at 37 °C.
  • the appropriate amount of seed culture solution was transferred to BMGY medium for 48 hours.
  • the OD600 was 10
  • the medium was changed to BMMY
  • the expression was induced for 96 hours, and the supernatant was collected by centrifugation at 12,000 rpm for 30 minutes while sampling for SDS-PAGE analysis.
  • Example 4 Effect of recombinant protein with dual functions of Tumstatin and CD137L on proliferation of human umbilical vein endothelial cells.
  • MTT colorimetric assay Take logarithmic growth phase cells, inoculate 5000 cells per well in 96-well culture plates, add 100 L of cell suspension to each well, and incubate in a cell culture incubator for 12 h. After adding 100 L of different concentrations of protein samples, the positive control was T P-470, the negative control group was added with the same volume of PBS, and each group was set with 3 duplicate wells for 48 h. Add 4 ⁇ g of 5 mg/mL MTT solution to each well in a 96-well culture plate 4 h before the end of the culture, and continue to incubate for 4 h in a cell culture incubator, then gently aspirate the medium, then add 150 ⁇ L of DMSO to each well and shake for 10 min. The blue-violet precipitate is fully soluble. The absorbance (A490) was measured with a microplate reader.
  • T P-470 is a positive control
  • "115” is Tumstatin1-linked peptide 1-CD137L5 (SEQ ID N0.25)
  • 155 is Tumstatin1-linked peptide 5-CD137L5 (SEQ ID N0.27)
  • “116” is the Tumstatin1-linker peptide 1-CD137L6 (SEQ ID N0.26)
  • "215" is the Tumstatin2-linker peptide 1-CD137L5 (SEQ ID N0.37)
  • “255” is the Tumstatin2-linker Peptide 5-CD137L5 (SEQ ID N0.38)
  • “256” is Tumstatin2-linker peptide 5-CD137L6 (SEQ IDN0.41)
  • “711” is Tumstatin7-linker peptide 1-CD137L1 (SEQ ID N0.29)
  • “721 Tumstatin7-linked peptide 2-CD137L1 (SEQ ID NO.
  • Example 5 Effect of recombinant protein with dual function of tumtatin and CD137L on proliferation of mouse spleen T lymphocytes.
  • the human CD3 monoclonal antibody was coated in 96-well plates at 7.5 g/mL, overnight at 4 °C, and the isolated mouse spleen T cells were added to 100 ⁇ l/well (10 5 cells) on the second day, and divided into 4 experimental groups: 1) T cell blank group (Medium + T cell) without any antibody stimulation; 2) Anti-CD3 (7.5 g/mL) and anti-CD28 monoclonal antibody (2.5 g/mL) combined with stimulator group (CD3/CD28); 3) anti-CD137 monoclonal antibody combined with anti-CD3 and anti-CD28 monoclonal antibody stimulator group (CD3/CD28/anti-CD137mAb); 4) fusion protein samples combined with anti-CD3 and anti-CD28 monoclonal antibody stimulator group (CD3/CD28) /Tumstatinl-CD137L), 3 replicate wells per group.
  • Tumstatin2-linker peptide 1-CD137L5 SEQ ID N0.37
  • 255 is the Tumstatin2-linker peptide 5-CD137L5
  • 256 is the Tumstatin2-linker peptide 5-CD137L6 (SEQ. IDNO.41)
  • 711 is Tumstatin7-linked peptide 1-CD137L1 (SEQ ID N0.29)
  • 721 is Tumstatin7-linked peptide 2-CD137L1
  • 114 is the Tumstatin1-linker peptide 1-CD137L4 (SEQ ID N0.43)
  • 214 is the Tumstatin2-linker peptide 1-CD137L4 (SEQ IDN0.44)
  • 314" is the Tumstatin3-linker peptide 1-CD137L4 (SEQ IDN0) .45)
  • 154 is Tumstatin1-linking peptide 5-CD137L4 (SEQ ID N0.46)
  • 254" is Tumstatin2-linked peptide 5-CD137L4

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Abstract

公开了具有Tumstatin活性片段和CD137L胞外区的双功能融合蛋白。该蛋白兼有抑制人脐静脉内皮细胞增殖和协同刺激T细胞增殖的活性，可用于制备血管生成抑制剂、各种肿瘤相关疾病和机体免疫调节药物等。

Description

具有抑制肿瘤微环境血管再生和激活适应性免疫应答双功能 的融合蛋白及其基因和应用 技术领域
本发明属于生物工程技术领域。 本发明涉及一种具有抑制肿瘤微环境血管再生和激活适应 性免疫应答双功能的融合蛋白 CD137L-Tumstatin及其基因， 以及含有该基因的表达载体与由 该载体转化得到的菌株， 还涉及该融合蛋白的制备方法， 同时涉及具有 CD137L和 Tumstatin 双功能的蛋白在制备抑制肿瘤微血管再生以及相关肿瘤学疾病 （如， 黑色素瘤、 直肠癌、 肺 癌等） 和激活适应性免疫应答功效药物方面的应用， 另外涉及给药途径方面的应用 （如， 口 服给药、 喷雾给药途径等）。
背景技术
癌症发生是一个多基因合作、 不同信号途径参与的过程。 就其本质而言， 癌症是一种分子 病症。 当前， 靶点单一的分子治疗策略逐渐显示出诸多弊端， 而多靶点、 多机制联合用药的 治疗方法显示出更好的治疗效果。
1971年哈佛大学 Folkman教授提出 "肿瘤的生长和转移依赖于微环境血管再生"学说。 该 理论认为， 通过抑制肿瘤微环境血管再生， 切断肿瘤的营养和氧气供应能够抑制肿瘤细胞的 生长和转移。 自发表至今， 该学说在世界范围内已得到了大量实验室和临床上的数据支持， 因而成为了近来肿瘤治疗领域内的新策略。 人肿瘤抑制素 （Tumstatin) 是一个源于血管基底 膜 IV型胶原 α 3链羧基 C末端的肿瘤血管再生抑制因子， 由 244个氨基酸组成。
在所有血管基底膜中， IV型胶原蛋白形成的三维网状骨架结构是起支架作用的主要成分， 可促进细胞的黏附、 迁移、 分化和生长。 它由 6个独特的基因分别编码产生 6条链 α 1~ α 6， 并以不同或相同的 α链形成三聚体， 进一步形成网状结构。 IV型胶原蛋白的每条 α 3链都由 3部分功能区组成 （7S结构域、 三螺旋区、 非胶原肽 NC1结构域）， 分布于肾小球、 肺泡毛 细血管、 耳蜗、 晶状体囊、 卵巢和睾丸的基底膜。
新生血管的形成是肿瘤生长和转移的关键性步骤， 包含了一系列复杂的过程。 肿瘤组织内 由于低氧， 使血管形成剌激因子合成增加， 从而剌激肿瘤组织微环境新生血管的生成。 近来 研究表明， Tumstatin可特异性地抑制肿瘤血管内皮细胞蛋白的合成， 导致内皮细胞凋亡， 使 血管生成受到抑制， 从而抑制肿瘤生长和转移。 另外， Tumstatin还具有直接作用于肿瘤细胞 并抑制其增殖的特性。 其作用机制为通过与整合素受体 α ν β 3结合， 特异地抑制肿瘤微环境 血管内皮细胞蛋白的合成， 进而阻断血管再生， 抑制肿瘤的生长和转移。 目前学术界就抗肿瘤免疫已达成共识，即在人和动物体内对恶性肿瘤存在着某种程度的免 疫反应。 肿瘤患者免疫系统的细胞能够识别肿瘤细胞表达的抗原， 如组织分化抗原、 癌胚抗 原以及突变基因的产物等等。 随着对肿瘤抗原识别及免疫应答机制的了解， 研究已经表明通 过辅助分子提供免疫共剌激信号可以提高抗肿瘤免疫应答。 由于肿瘤抗原特异性 τ细胞需要 协同剌激来辅助第一抗原信号激发效应细胞的功能， 因而， 协同剌激分子的辅助治疗可用于 调节针对恶性肿瘤的免疫反应。
共剌激分子在免疫应答中起了极其重要的作用。 一般而言， 激活 τ淋巴细胞需要两个信号 的参与，分别是 T细胞受体（T cell receptor, TCR)接受抗原递呈细胞（Antigen Presenting Cells, APC) 传导的主要组织相容复合体 -抗原肽信号 （第一信号）， 和细胞膜表面黏附分子提供的 协同剌激信号 （亦即第二信号）。 增强 τ 细胞介导的抗肿瘤免疫的重要方法之一即是提供 T 淋巴细胞活化所需要的协同剌激信号。 CD137与其配体 CD137L是继 CD28/B7之外新发现的 另一对重要的 T细胞协同剌激信号分子。
根据结构可将共剌激分子分为两类： 肿瘤坏死因子受体超家族 （Tumor Necrosis Factor Receptor, TNFR) 和免疫球蛋白超家族。 CD137是肿瘤坏死因子受体家族的成员。 它在调节 细胞增殖、 分化、 凋亡中发挥着重要作用。 其配体 CD137L也是 TNF家族的成员， 系细胞表 面 II型跨膜蛋白，与其他 T F家族有相似的 C-端氨基酸。编码人 CD137L的基因位于 19p3.3， 其产物为 254个氨基酸， 其中胞浆区 28个氨基酸， 跨膜区 21个氨基酸， 胞外区 205个氨基 酸。 CD137L首先由 Goodwin等人利用表达筛选技术在小鼠胸腺瘤细胞中分离提出， 随后又 在人 CD4+T细胞克隆中分离得到。
CD137与其配体 CD137L之间的相互作用 （CD137/CD137L) 对 T细胞活化的重要作用已 得到充分认可。 在鼠和人 T细胞中均可观察到， 在 CD3 抗体 （第一信号） 存在下， CD137 便可诱导 T细胞增殖、 合成细胞因子 （如 IFN- α ), 以及延长活化细胞的生存期。 协同剌激 信号可通过提高抗原特异性和效应 CD8+T细胞的数量来增强效应功能； 但在缺乏 CD3抗体 信号时， CD137分子的剌激并不能改变 T细胞的功能，表明 CD137与 CD137L相互作用所提 供的只是一种协同剌激信号。
Kim 等对 T 细胞及细胞因子的研究显示， CD137 单克隆抗体介导的抗肿瘤免疫依赖于 CD4+T、 CD8+T细胞的共同参与。 CD137介导 F-kB的活化， 进而上调 bcl-xL和 bfl-1分子 的表达，延长 CD8+T、 CD4+T细胞的生存并促进其增殖。 Melero等人利用激活型鼠源 CD137 单克隆抗体来开展 CD137靶向免疫治疗。 结果表明 CD137单抗能够消除小鼠体内已接种的 P815实体瘤。 与激活型 CD137单克隆抗体的抗肿瘤效果一致， CD137L可协同激发细胞毒性 T 淋巴细胞效应 （Cytotoxic lymphocyte,CTL) 和抗肿瘤效应。 CD137L-/-小鼠很好地说明了 CD137/CD137L系统在 T细胞介导的对病毒和肿瘤免疫应答中所起的重要作用。对 CD137或 CD137L缺陷型小鼠的研究表明 CD137/CD137L的协同剌激作用对移植物抗宿主病、 T细胞 的抗病毒细胞性应答很重要。
综上所述， CD137/CD137L提供的剌激信号能协同 CD28/B7分子对进一步活化 T细胞， 维 持 CD8+T细胞的增殖和生存。 而 Tumstatin可以有效抑制肿瘤血管的生成， 对肿瘤细胞的存 活、 转移起阻滞作用。 所以本发明中的融合蛋白 TUmStatin-CD137L可以从抑制血管生成与增 强抗肿瘤免疫应答两个方面共同抑制肿瘤的生长和转移，从而避免单一治疗引起的药物耐受， 为治疗肿瘤患者提供新的策略。
发明内容
本发明通过短的柔性连接肽将 Tumstatin抗血管生成活性片段与 CD137L胞外区蛋白合成一 个同时具有增强 T细胞免疫与抗血管生成的双功能、 双靶点的分子； 并且在本发明中选取了 Tumstatin不同的相关活性位点 （Tumstatinl第 45位至第 98位氨基酸片段， Tumstatin2第 60 位至第 132位氨基酸片段， Tumstatin3第 60位至第 98位氨基酸片段， Tumstatin7第 74位至 第 98位氨基酸片段） 与 CD137L胞外区蛋白氨基酸序列 （CD137L1第 46位至第 254位氨基 酸片段， CD137L4第 50位至第 240位氨基酸片段， CD137L5第 83位至第 254位氨基酸片段， CD137L6第 46位至第 85位氨基酸和第 167位至第 254位氨基酸的接合片段， 示意图如图 2 所示） 通过连接肽相连接， 利用原核或真核表达系统制备， 此方法具有制备简单及避免了全 长 Tumstatin和全长 CD137L的副作用问题， 本发明将在制备血管生成抑制剂、各种肿瘤相关 疾病 （如黑色素瘤、 前列腺癌、 肺癌、 大肠癌、 肾癌、 膀胱癌等）、 视网膜病变、 细胞增殖、 机体细胞因子合成与分泌、 调节机体免疫力等药物， 以及在制备以口服或注射等相关剂型药 物中有很好的应用前景与市场价值。
本发明的目的是提供一个兼有 Tumstatin和 CD137L功能的蛋白。
本发明的另一个目的是提供编码上述具有 Tumstatin和 CD137L功能的蛋白的基因。
本发明的另一个目的是提供上述具有 Tumstatin和 CD137L功能的蛋白的制备方法。
本发明的另一个目的是提供上述具有 Tumstatin和 CD137L功能的蛋白及其基因在治疗肿瘤 学相关疾病 （如， 黑色素瘤、 直肠癌、 肺癌等） 和给药途径方面 （如， 口服给药、 喷雾给药 途径等） 的应用。
本发明技术方案具体如下：
一种具有 Tumstatin和 CD137L 双功能重组蛋白， 具有 Tumstatin 活性片段氨基酸序列和 CD137L胞外区蛋白片段氨基酸序列， 所述 CD137L胞外区蛋白片段氨基酸序列和 Tumstatin 活性片段氨基酸序列通过柔性连接肽融合而成，所述 Tumstatin活性片段氨基酸序列选自 SEQ ID N0.65至 SEQ ID N0.68所示氨基酸序列中的一种， 所述 CD137L胞外区蛋白片段氨基酸 序列选自 SEQ ID N0.77至 SEQ ID NO.80所示氨基酸序列中的一种。
上述连接肽氨基酸序列可采用本领域常规技术手段进行设计， 优选 SEQ ID N0.69至 SEQ ID N0.76中的一种。
本发明所述双功能重组蛋白氨基酸序列优选 SEQ ID N0.25至 SEQ ID N0.48所述中的一 种， 所述重组蛋白结构示意图如图 1所示。
本发明还提供了编码上述具有 Tumstatin和 CD 137L双功能重组蛋白基因，包括编码 CD 137L 胞外区蛋白片段的基因、编码连接肽的基因和编码 Tumstatin活性片段的基因， 其中所述编码 CD137L胞外区蛋白片段的基因选自 SEQ ID N0.61- SEQ ID NO.64 中的一种， 所述编码 Tumstatin活性片段的基因选自 SEQ ID N0.49- SEQ ID N0.52中的一种。
上述编码连接肽的基因优选 SEQ ID N0.53- SEQ ID NO.60中的一种。
上述重组蛋白基因， 其优选具有 SEQ ID N0.1至 SEQ ID N0.24之一的核苷酸序列。
本发明还提供了一种编码上述重组蛋白 TUmStatin-CD137L的基因， 与上述的核苷酸序列相 比具有 70%及以上的同源性， 能编码本发明所述重组蛋白或其保守性变异多肽或其活性片段 或其活性衍生物。
本发明还提供了上述具有 Tumstatin和 CD137L双功能重组蛋白的制备方法，包括如下步骤：
( 1 ) 设计得到本发明所述编码具有 Tumstatin活性与 CD137L活性的重组蛋白基因序列；
(2)构建含上述基因序列表达系统， 包括构建表达载体再将表达载体转化入宿主细胞， 形成 可表达本发明所述具有 Tumstatin活性与 CD137L活性的重组蛋白的重组细胞；
(3 ) 培养步骤 （2) 重组细胞；
(4) 分离纯化得到本发明所述具有 Tumstatin活性与 CD137L活性的重组蛋白。
上述表达系统可选用原核表达系统或真核表达系统， 原核表达系统优选大肠杆菌表达系统 或枯草芽孢杆菌表达系统， 这两个表达系统普遍适应于本发明所述重组蛋白的表达， 其中大 肠杆菌系统中表达载体优选 pET-lla、 pET-22b, 枯草芽孢杆菌表达系统表达载体优选 pP43 ; 真核表达系统优选酵母表达系统，表达载体优选 pPIC9K、pPICZaA，酵母宿主细胞优选 GS115 或 SMD1168。
上述制备方法的一种优选方案为：将编码上述具有 Tumstatin活性与 CD137L活性的重组蛋 白 Tumstatin-CD137L的基因通过 Ndel及 Nhel双酶切， 然后连接至表达载体 pET-lla的相应 酶切位点， 再转化大肠杆菌 BL21(DE3)， 经液体培养工程菌获得包涵体形式的目的蛋白。 通 过将包涵体进行稀释复性的方法， 即用含低浓度尿素的溶液多次洗涤包涵体蛋白， 然后用含 8M尿素的变性液在 50°C溶解包涵体，最后用含 0.4M L-Arg的复性液稀释复性包涵体（目的产 物纯度达 80%以上)， 得到本发明所述重组蛋白。
上述制备方法的一种优选方案为：将编码上述具有 Tumstatin活性与 CD137L活性的重组蛋 白 Tumstatin-CD137L的基因通过 Pstl及 Hindlll双酶切，然后连接至表达载体 pP43的相应酶 切位点， 再电转枯草杆菌 WB800, 经液体培养工程菌获得分泌至胞外可溶形式的目的蛋白。 利用 DEAE阴离子交换的方法进行纯化， 纯度达 80%以上， 得到本发明所述重组蛋白。
上述制备方法的一种优选方案为：将编码上述具有 Tumstatin活性与 CD137L活性的重组蛋 白 Tumstatin-CD137L的基因通过 EcoRI及 Notl双酶切， 然后连接至表达载体 pPICZaA的相 应酶切位点， 再电转毕赤酵母 GS115, 经液体培养工程菌获得分泌至胞外可溶形式的目的蛋 白。 通过 DEAE阴离子交换的方法进行纯化， 纯度达 90%以上， 得到本发明所述重组蛋白。 本发明还提供了上述具有 Tumstatin和 CD137L双功能重组蛋白在制备抑制肿瘤微环境血管 再生以及相关肿瘤学疾病 （如， 黑色素瘤、 直肠癌、 肺癌等）、 调节机体免疫力、 T细胞增殖 和机体细胞因子的合成与分泌的药物中的应用。
本发明还提供了上述具有 Tumstatin和 CD137L双功能重组蛋白基因在制备抑制肿瘤微环境 血管再生以及相关肿瘤学疾病 （如， 黑色素瘤、 直肠癌、 肺癌等）、 调节机体免疫力、 T细胞 增殖和机体细胞因子的合成与分泌的药物中的应用。
上述应用中， 本发明所述的重组蛋白可以单独使用或以药物组合物的形式使用。 药物组合 物包括作为活性成分的本发明所述的重组蛋白和可药用载体。 较佳的， 药物组合物有 0.1-99.9%重量百分比的作为活性成分本发明所述的重组蛋白。 "可药用载体"不会破坏本发 明重组蛋白的药学活性， 同时其有效用量， 即能够起药物载体作用时的用量对人体无毒。
"可药用载体"包括但不限于： 离子交换材料、 氧化铝、 硬脂酸铝、 卵磷脂、 自乳化药物 传递系统 （SEDDS) 如 d-维生素 E聚乙二醇 1000琥珀酸酯、 吐温或其他类似聚合介质等药 物制剂用的表面活性剂、 血清蛋白如人血清白蛋白、 缓冲物质如磷酸盐、 氨基乙酸、 山梨酸、 山梨酸钾、 饱和植物脂肪酸部分甘油酯混合、 水、 盐、 电解质如硫酸盐精蛋白、 磷酸氢二钠、 磷酸氢钾、 氯化钠、 锌盐、 硅胶、 硅酸镁等。 聚乙烯吡咯酮、 纤维素物质、 聚乙烯醇、 羧甲 基纤维素钠、 聚丙烯酸酯、 乙烯-聚氧乙烯-嵌段聚合物和羊毛脂、 环糊精如 α -、 β -、 Υ -环 糊精或其经化学修饰的衍生物如 2-和 3-羟丙基 - β -环糊精等羟烷基环糊精或其他可溶性衍生 物等均可用于促进本发明所述重组蛋白的药物传递。 其他可药用辅料如填充剂 （如无水乳糖、 淀粉、 乳糖珠粒和葡萄糖）、 粘合剂 （如微晶纤维 素）、崩解剂（如交联羧甲基淀粉钠、交联羧甲基纤维素钠、低取代羟丙基纤维素和交联 PVP)、 润滑剂 （如硬脂酸镁）、 吸收促进剂、 香味剂、 甜味剂、 稀释剂、 赋形剂、 润湿剂、 溶剂、 增 溶剂和着色剂等也可加入本发明的药物组合物中。
在上述的药物组合物中， 没有限制可以任选使用的任何剂型。 例如， 可举例说明的有口服 给药形式如片剂、 胶囊剂、 颗粒剂、 粉剂或液体制剂， 或胃肠外给药形式如注射、 局部产品 或栓剂， 他们可以以常规方法配制或非常规方法如脂质体等。
当使用本发明所述重组蛋白作为治疗剂时， 其使用量对于成人大致每天 O.Olmg至 lg的范 围内， 这取决于各患者的年龄、 性别、 体重和症状程度， 并且日剂量可分为几个剂量。
本发明所述的重组蛋白还包括采用现有技术领域常规方法对本发明所述重组蛋白进行修饰 的修饰蛋白。
对于蛋白质和肽类药物， 在多数情况下， 机体内的氨肽酶及羧肽酶很容易从常见的直链肽 的两端进行逐步的切割分解， 使直链肽被降解。多肽修饰是改变肽链主链结构和侧链基团的 重要手段， 已有大量文献表明经过修饰后的多肽药物可以显著降低免疫原性、减少毒副作用、 增加水溶性、 延长体内作用时间、 改变其生物分布状况等等， 明显改善药物的疗效。
本发明所述重组蛋白常用修饰方法包括中间残基的修饰、 氨基酸替换、 糖基化修饰及 PEG 修饰等， 基本原理都是增加多肽分子的相对分子量和空间位阻， 提高其对多肽水解酶的稳定 性， 减少肾小球的滤过作用。 替换肽链中的某几个氨基酸是另一种推迟酶降解使多肽药物的 半衰期延长的方式， 替换对象通常为肽链中的易酶解的氨基酸。 具体的说， 可对重组蛋白中 间残基进行糖基化、 磷酸化、 甲基化、 乙酰化、 硝基化、 磺酸化或者连接 PEG修饰或者偶联 蛋白质， 其中：
糖基化修饰最常用的为 N-糖基化和 0-糖基化。糖基化修饰肽优选在本发明所述重组蛋白氨基 酸序列中的一个或多个 Tyr、 Ser或 Thr残基上的氧与糖相连或本发明所述重组蛋白的氨基酸 序列中的一个或多个天冬酰胺侧链的酰胺氮与糖相连。
磷酸化修饰肽优选在本发明所述重组蛋白氨基酸序列中的一个或多个 Tyr、 Ser或 Thr位点 进行磷酸化。
甲基化修饰肽包括侧链甲基化修饰肽和 N端甲基化修饰肽， 侧链甲基化优选在本发明所述 重组蛋白氨基酸序列中的一个或多个 Lys、 Tyr或 Arg侧链上进行甲基化，如 Lys(For),Lys(Me), Lys(Me)2, Lys(Me)3, Arg(Me)2 symmetrical, D-Tyr(Me),D-Tyr(Et)；
乙酰化修饰肽优选在本发明所述重组蛋白氨基酸序列中的一个或多个 Lys或 Ser侧链进行乙 酰化， 如 Ser(Ac)或 Lys(Ac)。
硝基化或磺酸化修饰肽优选在本发明所述重组蛋白氨基酸序列中的一个或多个 Tyr侧链上 进行硝基化或磺酸化， Tyr(3-N02)， Tyr(S03H2)。
中间残基的 PEG修饰优选在本发明所述重组蛋白氨基酸序列中的一个或多个 Lys侧链的氨 基进行 PEG修饰， PEG分子量优选为 2000-10000。
或者， 将本发明所述重组蛋白或其上述修饰蛋白的氨基酸序列中的一种或多种氨基酸替换 成相应的氨基酸衍生物或特殊氨基酸，如将丙氨酸替换成 β -丙氨酸、高苯丙氨或萘基丙氨酸， 将脯氨酸替换成羟脯氨酸， 亮氨酸替换成正亮氨酸， 缬氨酸替换成正缬氨酸， 苏氨酸替换成 别苏氨酸， 异亮氨酸替换成别异亮氨酸， 天冬酰胺替换成 2-乙酰氨基 -2-脱氧 - β -D-吡喃葡萄 糖基天冬酰胺 （Asn ( GlcNac(Ac)3- e -D))， 赖氨酸替换成 Lys (palmitoyl)。
或者， 将本发明所述重组蛋白或其上述修饰蛋白的氨基酸序列中的一种或多种氨基酸替换 成相应的 D型氨基酸。
本发明中编码上述具有 Tumstatin活性与 CD137L活性的双功能重组蛋白的基因（GenBank: AAF72632.1与 P_003802.1 )使用常规策略通过全基因合成、 PCR方法或其两者结合的方法 获得， 其中 CD137L 全长序列载体模板可参考文献 （Wang shuzhen. J Ind Microbiol Biotechnol. 2012 Mar;39(3):471-6. doi: 10.1007/sl0295-011-1045-l. ) 中公开的方法制备得到。 本发明构建了含有上述编码具有 Tumstatin和 CD137L双功能的蛋白基因的表达载体，即通 过常规 PCR技术及酶切、 连接将 Tumstatin-连接肽 -CD137L胞外区基因片段经 Ndel和 Nhel 双酶切后，连接入原核表达载体 pETlla的相应酶切位点之间，经过测序验证得到正确的表达 载体。
本发明构建了含上述表达载体的基因工程菌， 即通过将目的基因转化 BL21 , 通过液体培养 基小量培养筛选表达目的蛋白的阳性工程菌。
本发明提供了获得具有 Tumstatin和 CD137L双功能的蛋白的方法，该方法是将阳性工程菌 种进行培养发酵并经常温诱导使其高效表达具有 Tumstatin和 CD137L双功能的蛋白，再收集 菌体， 高压破碎细胞、 离心得到的菌体沉淀经变性溶解和稀释复性获得具有 Tumstatin 和 CD137L双功能的重组蛋白。
本发明对具有 Tumstatin和 CD 137L双功能的重组蛋白的活性测定是通过开展人脐静脉内皮 细胞活性试验 （HUVEC assay) 和小鼠 T细胞激活试验来进行的。 其中， 人脐静脉内皮细胞 活性试验结果显示， 对内皮细胞的增殖有显著的抑制作用。 小鼠 T细胞激活试验表明， 所述 重组蛋白能保持 CD137L的生物活性，可协同抗 CD3和抗 CD28单克隆抗体剌激 T细胞的增 殖。
本发明的有益效果：
本发明表明， 采用原核表达系统表达由 Tumstatin和 CD137L胞外区活性片段重组蛋白， 可以产生兼具 Tumstatin和 CD137L 双活性的蛋白。 利用本发明生产的 Tumstatin-连接肽 -CD137L重组蛋白 （兼具 Tumstatin和 CD137L功能的蛋白）， 具有表达效率高、 表达量大、 表达周期短， 易于纯化等优点。 同时， 本发明提供的融合蛋白对人脐静脉内皮细胞的增殖有 显著的抑制作用， 并且具有剂量依赖性， 同时也可协同抗 CD3和抗 CD28单克隆抗体剌激小 鼠 T细胞的增殖。 因此， 本发明为大规模生产 Tumstatin和 CD137L重组蛋白提供了新的、安 全的途径， 为进一步研究和开发成为新一代抗肿瘤药物奠定了扎实的基础， 在制药行业具有 广阔的应用前景。
附图说明
图 1为本发明所述具有 Tumstatin和 CD137L双功能的重组蛋白结构示意图。柔性连接肽氨 基酸序列选自 SEQ ID N0.69- SEQ ID N0.76中的一种。
图 2为 CD137L1 ( SEQ ID N0.77)、 CD137L5 ( SEQ ID N0.78)、 CD137L6 ( SEQ ID N0.79 ) 禾口 CD137L4 ( SEQ ID NO.80 ) 在 CD137L氨基酸全长序列中的区域示意图。
图 3 为氨基酸序列如 SEQ ID N0.25- SEQ ID N0.48所示的重组蛋白 Tumstatin-连接肽 -CD137L在大肠杆菌中的表达。 泳道 1-24分别对应 SEQ ID N0.25-48。
图 4为代表性 Tumstatin-连接肽 -CD137L蛋白氨基酸序列 （SEQ ID N0.29-SEQ ID N0.38 ) 在枯草芽孢杆菌中的表达。 泳道 1-8分别对应 SEQ ID NO. 29、 30、 31、 32、 33、 34、 35、 36、 37、 38。
图 5为代表性 Tumstatin-连接肽 -CD137L蛋白氨基酸序列（SEQ ID NO. 29-SEQ ID NO. 36 ) 在酵母菌中的表达。 泳道 1-16分别对应蛋白氨基酸序列 SEQ ID NO. 29、 30、 31、 32、 33、 34、 35、 36、 37、 38、 39、 40、 41、 42。
图 6为代表性 Tumstatin-连接肽 -CD137L蛋白样品经稀释复性后进行的 SDS-PAGE电泳结 果图。其中，第 1泳道样品氨基酸序列为 SEQ ID N0.25 ;第 2泳道样品氨基酸序列为 SEQ ID N0.26; 第 3泳道样品氨基酸序列为 SEQ ID N0.27; 第 4泳道样品氨基酸序列为 SEQ ID N0.37; 第 5泳道样品氨基酸序列为 SEQ ID N0.38 ; 第 6泳道样品氨基酸序列为 SEQ ID N0.41 ; 第 7与第 10泳道样品氨基酸序列为 SEQ ID N0.43 ; 第 8与第 11泳道样品氨基酸序 列为 SEQ ID N0.44; 第 9与第 12泳道样品氨基酸序列为 SEQ ID N0.48 ; 第 13与第 15泳道 样品氨基酸序列为 SEQ ID N0.45 ; 第 14与第 16泳道样品氨基酸序列为 SEQ ID N0.46; 第 17与第 18泳道样品氨基酸序列为 SEQ ID N0.47。
图 7为代表性 Tumstatin-连接肽 -CD137L蛋白样品对人脐静脉内皮细胞增殖的影响。
图 8为代表性 Tumstatin-连接肽 -CD137L蛋白样品对小鼠 T淋巴细胞增殖的影响。
具体实施方式
下面结合附图及实施例对本发明作进一步描述：
本发明共设计 24种具有 Tumstatin活性与 CD137L活性的双功能重组蛋白， 其核苷酸序 列为 SEQ ID NO. l至 SEQ ID N0.24所示序列，翻译编码氨基酸序列为 SEQ ID N0.25至 SEQ ID N0.48所示序列。 该组蛋白具有 Tumstatinl第 45位至第 98位氨基酸片段、 Tumstatin2第 60位至第 132位氨基酸片段、 Tumstatin3第 60位至第 98位氨基酸片段或 Tumstatin7第 74 位至第 98位氨基酸片段与 CD137L胞外区蛋白氨基酸序列 （CD137L1第 46位至第 254位氨 基酸片段， CD137L5第 83位至第 254位氨基酸片段， CD137L6第 46位至第 85位氨基酸和 第 167位至第 254位氨基酸的接合片段中的一种， 两者通过柔性连接肽连接融合而成， 其中 连接肽氨基酸序列如 SEQ ID N0.69至 SEQ ID N0.76所示。 本发明在连接有上述 Tumstatin 活性片段基因和连接肽基因的核苷酸序列的 （如 SEQ ID N0.81-SEQ ID N0.94所示） 的 5 ' 端和 3 ' 设计 EcoRI和 BamHI酶切位点， 交由上海捷瑞生物工程有限公司进行全合成， 并连 入具有相应酶切位点 pBluescriptll SK(+)载体， 重组载体由上海捷瑞生物工程有限公司提供。 各类表达载体由 Novagen公司购得， ToplO和 BL21 (DE3 ) 菌株由 Invitrogen公司购得。 pMD18-T载体、 溶液 I (货号： D103A)、 逆转录酶、 T4DNA连接酶和 Ndel、 Nhel等限制性 内切酶均购自 TAKARA公司。引物的合成和核苷酸序列测序由上海英骏生物技术有限公司完 成。 变性溶解和稀释复性所采用的尿素 （分析纯） 购自南京试剂化学品有限公司。 人脐静脉 内皮细胞株购自南京凯基生物科技发展有限公司。 小鼠 T细胞增殖试验所使用的 EasySep™ Negative Selection Kit购自 Stem Cell公司。 抗 CD3和抗 CD28单克隆抗体购自 Santa Cruz公 司。 其他试剂均为国产分析纯。
实施例 1:具有 Tumstatin活性与 CD137L活性的重组蛋白 Tumstatin-CD137L在大肠杆菌 表达系统中的表达
1. 表达系统的构建
共设计了 24种具有 Tumstatin活性与 CD137L活性的双功能重组蛋白， 该蛋白为 Tumstatinl/ 成 （图 1 )。 其中， CD137L1、 CD137L4, CD137L5, CD137L6分别来自 CD137L全长氨基酸 序列的 46-254位氨基酸、 50-240位氨基酸、 83-254位氨基酸，以及 46-85位氨基酸 和 167-254 位氨基酸 （图 2)。 该蛋白核苷酸序列为 8£010^«).1至8£010^«).24所示序列， 翻译编码 氨基酸序列为 SEQ ID N0.25至 SEQ ID N0.48所示序列，连接肽选自 SEQ ID N0.69至 SEQ ID N0.76所示序列中的一种。
编码 CD137L胞外区蛋白（CD137L1第 46位至 254位氨基酸片段， CD137L4第 50位至第 240位氨基酸片段， CD137L5第 83位至第 254位氨基酸片段， CD137L6第 46位至第 85位 氨基酸和第 167位至第 254位）氨基酸序列的核苷酸序列如 SEQ ID N0.61- SEQ ID N0.64所 示， 采用 PCR方法扩增得到， 其中， CD137L全长序列模板参考文献 （Wang shuzhen. J Ind Microbiol Biotechnol.2012 Mar;39(3):471-6. doi: 10.1007/sl0295-011-1045-l. )中公开的方法制 备得到。
以上述文献中提及方法制备本专利所用的 CD137L模板。 以 SEQ ID NO. 61 的上游引物 (SEQIDN0.95)禾 PSEQIDNO.61的下游引物 （SEQIDN0.96) 为引物， rTaqDNA聚合酶 催化扩增得到 CD137L1 (SEQ ID NO. 61) 基因片段 （此时该基因两端酶切位点分别为 5' BamHI和 3' Notl), 并将该产物连至 pMD18-T载体。 随后通过酶切连接等常规分子生物学 手段， 将 CD137L1 基因片段从 pMD18-T 载体上切下连接至由捷瑞公司全合成的已含有 Tumstatin和连接肽 (SEQ ID NO.87- SEQ ID N0.94)的 pBluescriptll SK(+)载体上。到这一步， 已将完整的融合蛋白基因 （SEQ ID N0.5- SEQ ID N0.12) 整合在 pBluescriptll SK(+)载体上。 之后选用上游引物 SEQ ID N0.97, 下游引物 SEQ ID N0.98, 通过 PCR的方法扩增该融合蛋 白基因， 产物 5' 酶切位点为 Nhel， 产物 3' 酶切位点为 Ndel， 最终连接至表达载体 pETlla。 以上述文献中提及方法制备本专利所用的 CD137L模板。 以 SEQ ID NO. 62的上游引物 (SEQ ID N0.99) 禾 P SEQ ID NO.62的下游引物 （SEQ ID NO.100) 为引物， rTaqDNA聚合 酶催化扩增得到 CD137L5 (SEQIDNO.62) 基因片段 （此时该基因两端酶切位点分别为 5' BamHI和 3' Notl), 并将该产物连至 pMD18-T载体。 随后通过酶切连接等常规分子生物学 手段， 将 CD137L5 基因片段从 pMD18-T 载体上切下连接至由捷瑞公司全合成的已含有 Tumstatin和连接肽 (SEQ ID N0.81- SEQ ID NO.86)的 pBluescriptll SK(+)载体上。到这一步， 已将完整的融合蛋白基因 （SEQIDN0.1、 SEQIDN0.3, SEQ ID NO.13- SEQ ID NO.16) 整 合在 pBluescriptll SK(+)载体上。 之后选用 SEQ ID NO.l上游引物 SEQ ID NO.101、 SEQ ID N0.3上游弓 I物 SEQ ID NO.102、 SEQ ID NO.13- SEQ ID NO.14上游弓 |物 SEQ ID NO.103、 SEQ IDNO.15-SEQIDNO.16上游引物 104， SEQ ID NO.1 SEQIDNO.3, SEQ ID NO.13- SEQ ID NO.16下游引物 SEQ IDNO.105, 通过 PCR的方法扩增该融合蛋白基因， 产物 5' 酶切位点 为 Nhel， 产物 3' 酶切位点为 Ndel， 最终连接至表达载体 pETlla。 以上述文献中提及方法制备本专利所用的 CD137L模板。 以 SEQ ID N0.63的上游引物 1 (SEQIDNO.106)^P SEQ ID NO.63的下游引物 1(SEQ ID NO.107)为引物，以 SEQ ID NO.63 的上游引物 2 (SEQ ID NO.108) 和 SEQ ID N0.63的下游引物 2 (SEQ ID NO.109) 为引物， rTaqDNA聚合酶催化扩增得到 CD137L6 (SEQIDN0.63)的两个基因片段， 琼脂糖电泳切胶 回收该两段基因。 随即使用分子生物学 OverlapPCR技术， 将回收得到的两段基因用做模板， 以 CD137L6的上游引物 1 (SEQ ID NO.107) 和 CD137L6的下游引物 2 (SEQ ID NO.109) 为引物， 扩增出最终的 CD137L6 (SEQ ID N0.63) 片段 （两端酶切位点 5' BamHI禾口 3' Notl) 并连接至 pMD18-T载体。 随后将 CD137L6 (SEQIDN0.63)基因片段从 pMD18-T载 体上切下连接至由上海捷瑞公司合成的已含有 Tumstatin和连接肽 （SEQ ID N0.8U SEQ ID NO.82, SEQ ID NO.84, SEQ ID NO.86) 的 pBluescriptll SK(+)载体上。 到这一步， 已把完整 的融合蛋白基因（SEQIDN0.2、 SEQ ID NO.4, SEQ ID NO.17-18)整合在 pBluescriptll SK (+) 载体上。之后分别选用 SEQIDN0.2、 8£010^3.4上游引物8£010^3.110、 SEQ ID NO.17 上游引物 SEQIDN0.111、 SEQ ID NO.18上游引物 SEQ ID NO.112, SEQIDNO.2, SEQ ID N0.4、 SEQ ID N0.17、 SEQ ID NO.18下游引物 SEQ ID NO.113, 通过 PCR的方法扩增该融 合蛋白基因 （SEQIDN0.2、 SEQIDNO.4, SEQ ID NO.17、 SEQ ID NO.18), 并连接至最终 表达载体 pETlla。
上述实验操作过程中涉及到的引物有：
SEQ ID NO.61的上游引物
5' -GGATCCGCCGTCTTCCTCGCCTGCC-3， (下划线部分为 BamHI酶切位点）
SEQIDNO.61的下游引物
5' -GCGGCCGCTTCCGACCTCGGTGAAGGGAGT-3 ' (下划线部分为 Notl酶切位点） 融合蛋白基因 （SEQ ID N0.5- SEQIDNO.12) 的上游引物
5' -GCTAGCACAATGCCATTCTTATTCTGCAATG-3 ' (下划线部分为 Nhel酶切位点） 融合蛋白基因 （SEQIDN0.5- SEQIDNO.12) 的下游引物
5' -CATATGTTCCGACCTCGGTGAAGGGAGTCCG-3 ' (下划线部分为 Ndel酶切位点） SEQ ID NO.62的上游引物
5' -CGGGATCCGCCTCTTGGACCTGCGGCAG-3 ' (下划线部分为 BamHI酶切位点） SEQIDNO.62的下游引物
5， -GCGGCCGCTTCCGACCTCGGTGAAGGGAG-3 ' (下戈 'J线部分为 Notl酶切位点） SEQIDN0.1上游引物 5' -GCTAGCGGTTTTTCTTTCTTATTTGTTCAAG-3 ' (下戈 ij线部分为 Nhel酶切位点） SEQIDN0.3上游引物
5' -GCTAGCGGTTTTTCTTTCTTATTTGTTCAAG-3 ' (下戈 'J线部分为 Nhel酶切位点） SEQ ID NO.13- SEQ ID NO.14上游引物
5' -GCTAGCC AAGATTTAGGTACTTTGGGCTCTT-3 ' (下划线部分为 Nhel酶切位点） SEQ ID NO.15- SEQ ID NO.16上游弓 |物
5' -GCTAGCAAGAGCCCAAAGTACCTAAATCTTG-3 ' (下划线部分为 Nhel酶切位点) SEQ ID NO.1、 SEQIDN0.3, SEQ ID NO.13- SEQ ID NO.16下游引物
5， - CATATGTTCCGACCTCGGTGAAGGGAG-3， (下划线部分为 Ndel酶切位点） SEQIDN0.63的上游引物 1
5' -CGGGATCCGCCGTCTTCCTCGCCTGC-3， (下戈 'J线部分为 BamHI酶切位点）
SEQIDN0.63的下游引物 1
5， -CGCAAACATGCCCTGCCCTG-3 '
SEQ ID N0.63的上游引物 2
5， -CAGGGCAGGGCATGTTTGCGGGTTTCCAGGGCCGCTTGC -V
SEQ ID N0.63的下游引物 2
5， -GCGGCCGCTTCCGACCTCGGTGAAGGGAG -3， (下戈 'J线部分为 Notl酶切位点） SEQIDN0.2, SEQIDN0.4上游引物
5' -GCTAGCGGTTTTTCTTTCTTATTTGTTCAAG-3 ' (下戈 'J线部分为 Nhel酶切位点） SEQ ID NO.17上游引物
5' -GCTAGCCAAGATTTAGGTACTTTG-3， (下划线部分为 Nhel酶切位点）
SEQIDN0.18上游引物
5， -GCTAGCCAAGATTTAGGTACTTTGGGCTCTT-3， (下划线部分为 Nhel酶切位点）
SEQIDN0.2, SEQIDN0.4, SEQIDN0.17, SEQ ID NO.18下游引物
5' -CATATGTTCCGACCTCGGTGAAGGGAG-3， (下划线部分为 Ndel酶切位点） 上述 PCR扩增条件均为：
94 V 5min
94 V lmin, 60 °C 30s, 72 °C 30s, 30 cycles
72 °C 5min,
4°C∞, Hold 将 PCR产物回收后，与 pMD-18T载体连接，体系为 ρΜϋ-18Τ1μί， 目的片段 4μί，溶液 Ι5μί， 连接反应条件为 16°C， 反应时间为 16h。 化学氯化钙法转化大肠杆菌 ToplO菌株。 在含有氨 苄青霉素的 LB 固体培养基上培养 12h后挑取单克隆， 提取质粒后用限制性内切酶 Ndel和 Nhel进行双酶切鉴定。 经酶切验证正确的菌落送交上海英骏生物技术有限公司测序， 以确定 基因序列的正确性。 将测序正确的目的片段与原核表达载体 pETl la连接， 反应体系为 T4连 接酶 10 X缓冲液 1μί， ρΕΤΙ ΙαΙμί, 目的基因 7μί， Τ4连接酶 1μί， 反应条件为 16°C， 16h。 双酶切验证后， 转化入大肠杆菌表达菌株 BL21 (DE3 )。
重组蛋白的表达
在超净台中， 向含 lOO g/mL氨苄青霉素的 LB培养基中分别加入 lmL上述保存的含有 目的基因质粒的大肠杆菌 BL21 (DE3 ) 菌种至 lOOmL菌液中， 置于摇床上， 37°C， 250rpm 条件下过夜培养。 将活化后的种子培养基转接到无菌 LB培养基中， 于 OD6,值为 0.9时加 入 IPTG, 诱导剂浓度为 ΙΟΟμΜ, 37°C培养 8h后室温离心收菌， 转速 12000rpm， 30min。 菌 体湿重 lg加入 lOmLPBS吹悬，加入高压破细胞仪中裂解细胞，收集流出液， 4°C， 12000rpm, 30min离心， 弃上清， 留沉淀做 SDS-PAGE分析。
SDS-PAGE实验操作流程如下所示：
1 ) 每管各取适量裂解菌体沉淀于 EP管中， 用 lmLddH20洗涤， 离心 12000rpm, lmin,弃上 清， 再用 100 μ !χ!(1Η2Ο重悬。
2) 取 20 μ L重悬菌液加 5 μ L5 X SPS-PAGE上样缓冲液， 沸水浴 5min。
3 )参照《分子克隆手册》灌制 SDS-PAGE胶， 5%浓缩胶， 12%分离胶， l X Tri-Gly蛋白电 泳缓冲液。
4) 各取 20 L上清液上样， 进行电泳， 参数为恒压 90V,30min而后 130V， lh。 电泳结束 后取下 SDS-PAGE胶， 考马斯亮蓝染色 l-2h。 取出后用去离子水冲洗三次， 置脱色液中浸泡 过夜。
5 ) 蛋白电泳结果详见附图。 与阴性对照相比， 经过诱导之后， 阳性菌种得到了显著表达。 具体表达情况见图 3所示。 由图可见以上所述蛋白均已在大肠杆菌 BL21(DE3)中表达。
3. 重组蛋白的纯化
包涵体复性研究
采用上述方法收集高压破碎后所得的菌体沉淀， 经过洗涤液 A和 B 反复洗涤 2 次， 12000rpm, 10min,离心弃上清， 收集备用。 洗涤液 A的配方为 20mMTris-HCL,pH8.5,2M尿素， 2%TritonX-100; 洗涤液 B配方为 20mMTris-HCL,pH8.5,2M尿素， 5mMEDTA。 洗涤完毕后， 用溶解液（20mMTris,pH8.5， 8M尿素）于 50°C过夜变性。 经过完全变性后， 在 4°C层析柜中 将变性液通过恒流装置加入复性溶液中， 整个过程在缓慢搅动中持续 12h。 复性溶液配方为， 20mMTris-HCL pH8.5 , 2M尿素， 0.4-0.6M L-Arg， lmM EDTA。 将所得含有样品的复性溶液 进行浓缩、 置换。 浓缩使用的装置为 Millpore 公司的 Amicon Ultra- 15 浓缩管， 4500rpm,30min,4°C。同时将浓缩液置换至 pH7.4, 100mM PBS缓冲液中，最终体积为 lmL。4°C 保藏备用 （图 6)。 经测算， 复性后目的产物的纯度达 80%以上， 由图可见以上所述变性包涵 体蛋白复性情况良好。
实施例 2:具有 Tumstatin活性与 CD137L活性的重组蛋白 Tumstatin-CD137L在枯草芽孢 杆菌表达系统中的表达
1. 表达系统的构建
使用实施例 1中构建好的包含完整重组蛋白基因序列（SEQ ID N0.1-24)的 pBluescriptll SK(+)载体为模板，通过设计不同引物（5 ' Pstl、 3 ' Hindlll)并进行 PCR，扩增连在 pBluescriptll SK(+)载体上的不同融合蛋白基因并将该完整片段连接至表达载体 pP43 (连接实验前需 Pstl、 Hindlll双酶切该载体）。 PCR以及酶切连接等操作步骤同实施例 1中融合蛋白基因的构建部 分。 待表达载体构建完毕， 即进行电转操作 （具体方法详见 Bio-Rad电转仪使用说明）， 完成 枯草芽孢杆菌表达系统 WB800重组菌株的构建。
2. 重组蛋白的表达
种子液的培养 在超净台中， 向含 50 μ g/mL卡那霉素的 LB培养基中分别加入 10 μ L上 述保存的含有目的基因质粒的枯草芽孢杆菌 WB800菌种至 5mL试管培养基中，置于摇床上， 37°C， 250rpm条件下培养 12h。 重组 WB800发酵 将活化后的种子菌液按 10%的转接量转 接到含有 50 μ g/mL卡那霉素无菌 2 X YT培养基中， pH7.0， 37°C培养 96h后 4°C低温离心收 集上清同时取样做 SDS-PAGE分析。
3. 重组蛋白的纯化
重组蛋白的纯化采用 GE公司的 DEAE阴离子交换法纯化该目的蛋白，纯化缓冲液为 PBS 磷酸盐缓冲液， pH8.5。
实施例 3:具有 Tumstatin活性与 CD137L活性的重组蛋白 Tumstatin-CD137L在酵母表达 系统中的表达
1.表达系统的构建
将整合在 pBluescriptll SK(+)载体上的不同融合蛋白基因 （SEQ ID NO.1-24)用 EcoRI和 Notl双酶切并连接至 pPIC9K，完成载体构建工作。随后进行电转操作（具体方法详见 Bio-Rad 电转仪使用说明）， 完成毕赤酵母 GS115重组菌株的构建。
2. 重组蛋白的表达
使用 BMGY富集菌体， BMMY诱导表达的策略来发酵获得目的产物。 第一天将按上述 方法构建完成的表达菌株接菌至 YPD种子培养基， 37°C过夜培养。第二天转移适量种子培养 液至 BMGY培养基中， 生长 48h， 待 OD600为 10时， 将培养基更换为 BMMY, 诱导表达 96h, 12,000rpm30min离心收集上清同时取样做 SDS-PAGE分析。
3. 重组蛋白的纯化
重组蛋白的纯化采用 GE公司的 DEAE阴离子柱进行离子交换纯化分析。 磷酸盐缓冲液 体系下 pH为 8.5， 产物纯度可达 80%以上。
实施例 4: 具有 Tumstatin和 CD137L双功能的重组蛋白对人脐静脉内皮细胞增殖的影响。
1) 细胞培养： 人脐静脉内皮细胞系 （HUVEC) 用含 10%胎牛血清、 100U/mL青霉素及 100U/mL链霉素的 RPMI-1640培养基， 在 37°C， 5%C02、 饱和湿度的培养箱中培养， 2~3天 待细胞贴壁长满后传代， 取对数生长期细胞用于实验。
2) MTT比色法测定： 取对数生长期细胞， 以每孔 5000个细胞接种于 96孔培养板中， 每 孔加入细胞悬液 100 L， 在细胞培养箱中培养 12h， 待细胞贴壁后加入 lOO L不同浓度蛋 白样品， 阳性对照为 T P-470,阴性对照组加入相同体积的 PBS， 每组设置 3个复孔， 分别处 理 48h。培养结束前 4h于 96孔培养板中每孔加入 5mg/mL的 MTT液 20 μ L,置细胞培养箱中 继续培养 4h后轻轻吸去培养基， 然后每孔加入 DMSO150 μ L,震荡 lOmin使蓝紫色沉淀充分 溶解。 用酶标仪测定吸光值 （A490)。
实验结果如图 7所示，其中， T P-470为阳性对照， "115"为 Tumstatinl-连接肽 1-CD137L5 (SEQ ID N0.25), "155" 为 Tumstatinl-连接肽 5-CD137L5 (SEQ ID N0.27), "116" 为 Tumstatinl-连接肽 1-CD137L6(SEQ ID N0.26), "215"为 Tumstatin2-连接肽 1-CD137L5 ( SEQ IDN0.37), "255"为 Tumstatin2-连接肽 5-CD137L5 ( SEQ ID N0.38), "256"为 Tumstatin2- 连接肽 5-CD137L6(SEQIDN0.41)，"711"为 Tumstatin7-连接肽 1-CD137L1 ( SEQ ID N0.29), "721" 为 Tumstatin7-连接肽 2-CD137L1 (SEQ ID NO.30), "731" 为 Tumstatin7-连接肽 3-CD137L1 (SEQIDN0.31), "741"为 Tumstatin7-连接肽 4-CD137L1 ( SEQ ID N0.32), "751" 为 Tumstatin7-连接肽 5-CD137L1 (SEQ ID N0.33), "761"为 Tumstatin7-连接肽 6-CD137L1 (SEQ ID N0.34), "771" 为 Tumstatin7-连接肽 7-CD137L1 (SEQ ID N0.35), "781" 为 Tumstatin7-连接肽 8-CD137L1 ( SEQ IDN0.36), "114"为 Tumstatinl-连接肽 1-CD137L4 ( SEQ IDN0.43), "214"为 Tumstatin2-连接肽 1-CD137L4 ( SEQ ID N0.44), "314"为 Tumstatin3- 连接肽 1-CD137L4(SEQ ID N0.45)，" 154"为 Tumstatinl-连接肽 5-CD137L4( SEQ IDN0.46),
"254" 为 Tumstatin2-连接肽 5-CD137L4 (SEQ ID N0.47), "354" 为 Tumstatin3-连接肽 5-CD137L4 (SEQ ID N0.48)。 上述结果表明所选的代表性具有 Tumstatin和 CD137L功能的 重组蛋白对人脐静脉内皮细胞显示出抑制效应， 其中 SEQIDN0.26、 SEQIDN0.37, SEQ ID N0.4U SEQIDNO.30, SEQ ID N0.31显示出的抑制效应高于阳性对照。
实施例 5:具有 Tumstatin和 CD137L双功能的重组蛋白对小鼠脾脏 T淋巴细胞增殖的影响。 按照 Stemcell公司的 EasySep Negative Selection Mouse T cell Enrichment Kit试剂盒说明书 来操作。 从小鼠脾脏中分离获得纯化的 T细胞群体。 人 CD3单克隆抗体按 7.5 g/mL包被 96孔板， 4°C过夜， 第 2天加入分离的小鼠脾脏 T细胞 lOOul/孔 （105个）， 共分成 4个实验 组： 1) 未加任何抗体剌激的 T细胞空白组 （Medium +T cell); 2) 抗 CD3 (7.5 g/mL) 和 抗 CD28单克隆抗体 （2.5 g/mL) 联合剌激组 (CD3/CD28); 3) 抗 CD137单抗联合抗 CD3 和抗 CD28单克隆抗体剌激组 (CD3/CD28/anti-CD137mAb); 4)融合蛋白样品分别联合抗 CD3 和抗 CD28单克隆抗体剌激组 (CD3/CD28/Tumstatinl-CD137L)，每组置 3个复孔。培养 92h后， 每孔加 lOyLalamarblue, 12h后测吸光度 A570值， 以 A600为参照。 实验结果如图 8所示， 其中， "115"为 Tumstatinl-连接肽 1-CD137L5 (SEQ ID N0.25), "155"为 Tumstatinl-连接 肽 5-CD137L5 (SEQ IDN0.27), "116"为 Tumstatinl-连接肽 1 -CD 137L6 (SEQ IDN0.26),
"215" 为 Tumstatin2-连接肽 1-CD137L5 (SEQ ID N0.37), "255" 为 Tumstatin2-连接肽 5-CD137L5 (SEQ IDN0.38), "256"为 Tumstatin2-连接肽 5-CD137L6 ( SEQ IDNO.41), "711" 为 Tumstatin7-连接肽 1-CD137L1 (SEQ ID N0.29), "721"为 Tumstatin7-连接肽 2-CD137L1
(SEQ ID NO.30), "731" 为 Tumstatin7-连接肽 3-CD137L1 (SEQ ID NO.31), "741" 为 Tumstatin7-连接肽 4-CD137L1 ( SEQ ID N0.32), "751"为 Tumstatin7-连接肽 5-CD137L1 ( SEQ IDN0.33), "761"为 Tumstatin7-连接肽 6-CD137L1 ( SEQ ID N0.34), "771"为 Tumstatin7- 连接肽 7-CD137LKSEQ ID N0.35)，"781"为 Tumstatin7-连接肽 8-CD137LKSEQ IDN0.36),
"114" 为 Tumstatinl-连接肽 1-CD137L4 (SEQ ID N0.43), "214" 为 Tumstatin2-连接肽 1-CD137L4(SEQIDN0.44), "314"为 Tumstatin3-连接肽 1-CD137L4 (SEQ IDN0.45), "154" 为 Tumstatinl-连接肽 5-CD137L4 (SEQ ID N0.46), "254"为 Tumstatin2-连接肽 5-CD137L4
(SEQIDN0.47), "354"为 Tumstatin3-连接肽 5-CD137L4 ( SEQ ID N0.48)。 结果表明所选 的代表性具有 Tumstatin和 CD137L功能的重组蛋白在终浓度为 2 μ g/mL时对 T细胞的增殖 作用显著高于 "CD3+CD28"协同剌激组， 证明 CD137L和 CD28具有协同效应。 由此表明， 本发明所获得的代表性具有 Tumstatin和 CD137L功能的重组蛋白具有良好的协同剌激 T细胞 增殖的生物学活性。
以上所述实施例仅表达了本发明的实施方法， 是结合具体的优选实施方法对本发明所作的 进一步详细说明， 不能因此理解为本发明的具体实施只局限于这些说明。 除此之外， 实施例 仅选取了不同活性位点的 Tumstatin基因与 CD137L胞外区相连接， 不能理解为本发明中的 Tumstatin基因只与 CD137L胞外区相连接，其也能够与其他蛋白或多肽相连接,或者不与其他 蛋白基因相连接。 对于本领域的普通技术人员来说， 在不脱离本发明构思的前提下， 对实施 例做出的若干简单推演或替换， 都应视为属于本发明的保护范围。

Claims

WO 2014/180288 权 利 要 求 书 PCT/CN2014/076768
1. 一种具有 Tumstatin和 CD137L活性的双功能重组蛋白， 其特征在于该蛋白具有 Tumstatin 活性片段氨基酸序列和 CD137L胞外区蛋白片段氨基酸序列，所述 Tumstatin活性片段氨基酸 序列和 CD137L胞外区蛋白片段氨基酸序列通过柔性连接肽融合而成，所述 Tumstatin活性片 段氨基酸序列选自 SEQ ID N0.65至 SEQ ID N0.68中的一种， 所述连接肽氨基酸序列选自 SEQ ID NO.69至 SEQ ID NO.76中的一种，所述 CD137L蛋白胞外区片段氨基酸序列选自 SEQ ID N0.77至 SEQ ID NO.80所示的氨基酸序列中的一种。
2. 如权利要求 1所述的双功能重组蛋白，其特征在于具有 SEQ ID N0.25至 SEQ ID N0.48之 一的氨基酸序列。
3. 编码如权利要求 1所述的双功能重组蛋白基因， 其特征在于由编码 Tumstatin活性片段的 基因、编码连接肽的基因和编码 CD137L蛋白胞外区片段的基因组成， 所述编码 Tumstatin活 性片段的基因选自 SEQ ID N0.49至 SEQ ID N0.52中的一种、 所述编码连接肽的基因选自 SEQ ID N0.53至 SEQ ID NO.60中的一种、所述编码 CD137L蛋白胞外区片段的基因选自 SEQ ID N0.61至 SEQ ID N0.64中的一种。
4. 编码如权利要求 1所述的双功能重组蛋白基因， 其特征在于具有 SEQ ID N0.1至 SEQ ID N0.24之一的核苷酸序列。
5. —种具有 Tumstatin和 CD137L活性的双功能重组蛋白的基因，其特征在于与权利要求 3-4 所述任一项的核苷酸序列相比具有 70 %及以上的同源性。
6. 一种如权利要求 1所述的具有 Tumstatin和 CD137L双功能重组蛋白的制备方法， 其特征 在于包括如下步骤：
( 1 ) 设计得到如权利要求 3-5任一项所述的核苷酸序列；
(2)构建含如权利要求 3-5任一项所述的核苷酸序列表达系统， 包括构建表达载体并将表达 载体转化入宿主细胞，形成可表达如权利要求 1所述的具有 Tumstatin活性与 CD137L活性的 重组蛋白的重组细胞；
( 3 ) 培养步骤 （2) 重组细胞；
(4) 分离纯化得到如权利要求 1所述的具有 Tumstatin和 CD137L双功能重组蛋白。
7. 如权利要求 6所述的制备方法，其特征在于所述表达系统为原核表达系统或真核表达系统， 所述原核表达系统选自大肠杆菌表达系统或芽孢杆菌表达系统； 所述真核表达系统选自酵母 表达系统。
8. 如权利要求 1所述的具有 Tumstatin和 CD137L双功能重组蛋白在制备抑制肿瘤微环境血 管再生、 各种肿瘤相关疾病、 调节机体免疫力、 T 细胞增殖和机体细胞因子的合成与分泌药 物中的应用。
PCT/CN2014/076768 2013-05-06 2014-05-05 具有抑制肿瘤微环境血管再生和激活适应性免疫应答双功能的融合蛋白及其基因和应用 WO2014180288A1 (zh)

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WO2020012486A1 (en) 2018-07-11 2020-01-16 Kahr Medical Ltd. SIRPalpha-4-1BBL VARIANT FUSION PROTEIN AND METHODS OF USE THEREOF
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CN116640226B (zh) * 2022-04-02 2024-04-26 广东东阳光药业股份有限公司 一种抑制宿主抗外源免疫细胞hvg反应的嵌合受体

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