WO2019109954A1 - PD-1-Fc融合蛋白及其制备方法和用途 - Google Patents

PD-1-Fc融合蛋白及其制备方法和用途 Download PDF

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WO2019109954A1
WO2019109954A1 PCT/CN2018/119388 CN2018119388W WO2019109954A1 WO 2019109954 A1 WO2019109954 A1 WO 2019109954A1 CN 2018119388 W CN2018119388 W CN 2018119388W WO 2019109954 A1 WO2019109954 A1 WO 2019109954A1
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fusion protein
molecule
fragment
high affinity
affinity
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French (fr)
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李懿
李艳艳
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广东香雪精准医疗技术有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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
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    • 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
    • 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

Definitions

  • the present invention relates to the field of tumor immunology; more specifically, the present invention relates to a fusion protein formed by a high affinity soluble programmed death receptor (PD-1) molecule and an Fc fragment, and a preparation method thereof and a tumor Application in therapy.
  • PD-1 programmed death receptor
  • tumor suppressor synergistic costimulatory molecules antagonize T cell anti-tumor effect is an important mechanism of tumor immune escape. Blocking such molecules has become a new hot spot in tumor immunotherapy research (Hamanishi J et al., Proceedings of The National Academy of Sciences of the United States of America, 2007, 104, 3360-3365).
  • PD-1 Programmed death 1
  • PD-L1 Programmed death-ligand 1
  • CD28/B7 superfamily protein an important immunosuppressive molecule, which can intercept T cell activation signals and inhibit T cell proliferation.
  • the release of cytokines plays an important role in tumor immune escape, autoimmune diseases and viral infections (Keir ME et al., Annu Rev Immunol, 26, 677-704).
  • a series of preclinical and clinical studies have confirmed that therapeutic monoclonal antibodies against the PD-1/PD-L1 signaling pathway have achieved significant therapeutic effects and have become a hot research direction in the field of cancer therapy.
  • soluble PD-1 (souble PD-1, sPD-1) can block the interaction of membrane-bound PD-1 and PD-L1 and enhance the anti-tumor function of T cells.
  • Highly expressed sPD-1 was found in the serum of arthritic patients, and further studies indicate that sPD-1 can block the inhibition of T cell activation by membrane-bound PD-1 (Liu et al., Arthritis research & therapy, 17, (1) 340), He et al. also found that sPD-1 is capable of enhancing tumor-specific CD8 T cell-mediated killing by binding to PD-L1 (He YF, The Journal of Immunology, 173, (8), 4919-4928).
  • HiPD-1 high-affinity PD-1 molecule
  • the HiPD-1 molecule can effectively recognize the PD-L1 molecule and block the interaction between PD-1 and PD-L1, the natural HiPD-1 has a small molecular weight and a short half-life in vivo, which limits its biological activity. . Therefore, there is an urgent need in the art for a way to improve its half-life in vivo and promote its biological activity.
  • the object of the present invention is to provide a fusion protein of PD-1 and Fc fragment having high affinity for PDL-1 molecule, which can efficiently recognize PD-L1 molecule and block PD-1 and PD-L1 The interaction, and can very effectively promote the killing effect of effector cells on tumor cells; the present invention also provides a preparation method and use of the above fusion protein of PD-1 and Fc fragment.
  • the invention provides a fusion protein comprising a high affinity PD-1 molecule and an Fc fragment of an IgG molecule, wherein:
  • the affinity of the high affinity PD-1 molecule to the PDL-1 molecule is at least 100 times greater than the wild type PD-1 molecule and the PDL-1 molecule;
  • the amino acid sequence of the high affinity PD-1 molecule has at least 90% sequence identity to the wild type PD-1 amino acid sequence (SEQ ID NO. 1).
  • the amino acid sequence of the high affinity PD-1 molecule is 92% identical to the amino acid sequence set forth in SEQ ID NO. 1; preferably, at least 94% (eg, 95%, 96%, 97%) , 98% and 99%) sequence identity.
  • the affinity of the high affinity PD-1 molecule to the PDL-1 molecule is at least 200 times the affinity of the wild type PD-1 molecule to the PDL-1 molecule; preferably at least 500 times; more preferably Ground, at least 1000 times; more preferably, at least 2000 times.
  • the high affinity PD-1 molecule is located at the N-terminus of the Fc fragment of an IgG molecule.
  • the Fc fragment of the IgG is selected from the Fc fragment of human IgGl, IgG2, IgG3 or IgG4; preferably, an Fc fragment selected from IgG4.
  • the Fc fragment comprises a hinge region, a CH2 and a CH3 domain, preferably, the Fc fragment does not comprise a CH1 and CH4 domain, and more preferably, the amino acid sequence of the Fc fragment is SEQ ID NO: 7.
  • amino acid sequence of the high affinity PD-1 molecule is selected from the group consisting of SEQ ID NOs: 2-6.
  • the fusion protein comprises two high affinity PD1 molecules.
  • two high-affinity PD1 molecules in the fusion protein are joined by a disulfide bond to form a dimeric structure; preferably, the disulfide bond is a disulfide bond of a hinge region of an Fc fragment of an IgG molecule .
  • the invention provides a nucleic acid molecule comprising a nucleic acid sequence encoding the fusion protein of the first aspect, or a complement thereof.
  • the present invention provides a vector comprising the nucleic acid molecule of the second aspect.
  • the present invention provides a host cell comprising the nucleic acid molecule of the second aspect, wherein the vector or the chromosome of the second aspect is integrated with an exogenous source.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the fusion protein of the first aspect, and optionally a pharmaceutically acceptable carrier or excipient.
  • the present invention provides a method of treating a disease comprising administering an appropriate amount of the fusion protein of the first aspect or the pharmaceutical composition of the fifth aspect to a subject in need of treatment; preferably, the disease is Tumor; more preferably, the tumor expresses a PD-L1 molecule.
  • the present invention provides the use of the fusion protein of the first aspect for the preparation of a medicament for treating a tumor.
  • the present invention provides a method of preparing the fusion protein of the first aspect, comprising the steps of:
  • Figure 1 is a SDS-PAGE gel of purified wild-type PD-1 protein.
  • M protein molecular weight Mark.
  • Figure 2 is a BIAcore map of the binding of a wild-type PDL-1 molecule to a PD-1 molecule.
  • Figure 3 shows that PD-1 and L5B7 recognize the flow-through detection of PD-L1 on the surface of H1299 cells, indicating that L5B7 has higher ability to recognize PDL-1 on the surface of H1299 cells than PD-1.
  • A anti-PDL-1 antibody (2.5 ul / sample) recognizes PDL-1 on the surface of H1299 cells;
  • B different concentrations of PD-1, L5B7 (concentration of 0.02mg/ml, 0.04mg/ml, 0.08mg) /ml) Flow cytometry to identify PDL-1 on the surface of H1299 cells, wherein the amount of SA-PE is 0.5 ul/sample;
  • C when the concentration is 0.08 mg/ml, the control group, PD-1, L5B7 recognize PDL Flow histogram of -1.
  • Figure 4 is a gel electrophoresis pattern of the expression plasmid.
  • M is a DNA molecular marker
  • Lane 1 is wild-type PD-1-Fc
  • Lane 2 is L2F8-Fc
  • Lane 3 is L2F10-Fc
  • Lane 4 is L45-Fc
  • Lane 5 is L45-123-Fc
  • Lane 6 is L5B7-Fc.
  • Figure 5 is a diagram of SDS-PAGE electrophoresis after purification of the fusion protein. Among them, A is non-reducing; B is reducing. Lane 1 is L2F8-Fc; Lane 2 is L2F10-Fc; Lane 3 is L45-Fc; Lane 4 is L45-123-Fc; Lane 5 is L5B7-Fc.
  • Figure 6 is a graph showing the results of ELISA detection of the Fc fragment of the fusion protein.
  • Figure 7 shows the results of the fusion protein of the present invention promoting killing.
  • A is Mel624; B is H1299; and C is A375.
  • Figure 8 shows the Elispot results of the fusion protein of the present invention promoting PBMC release of IFN- ⁇ .
  • HiPD-1 molecule or "high affinity PD-1 molecule” or “high affinity PD-1” or “PD-1 molecule of the invention” or “PD-1 of the invention” as used herein refers to PDL -1 affinity is a soluble high-affinity PD-1 molecule having a affinity of wild-type PD-1 molecule for PDL-1 of at least 100 fold, preferably at least 200 fold, more preferably at least 500 fold, at least 1000 fold, at least 2000 fold, and
  • the amino acid sequence of the PD-1 molecule is at least 90%, preferably at least 92%, more preferably at least 94% (eg, at least 95%, 96%, 97%) of the wild-type PD-1 amino acid sequence (SEQ ID NO. 1). , 98% or 99%) sequence identity; preferably, the amino acid sequence of the PD-1 molecule is selected from the group consisting of SEQ ID NO. 2-6.
  • PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE SEQ ID NO.1, wild type PD-1
  • Fc fragment of IgG refers to the constant region of an immunoglobulin heavy chain, for example, an Fc fragment of IgG includes a combination of two or more domains of the heavy chain CH1, CH2, CH3, CH4 and a hinge region.
  • the Fc fragment of the immunoglobulin used comprises at least one immunoglobulin hinge region, one CH2 domain and one CH3 domain; preferably lacks the CH1 domain, more preferably lacks the CH1, CH4 domain.
  • the amino acid sequence of the Fc fragment is selected from the group consisting of SEQ ID NO.
  • the PD-1-Fc of the present invention refers to a protein formed by fusion of a high affinity PD-1 molecule with a human IgG Fc fragment.
  • the high affinity PD-1 molecule is located at the N-terminus of the Fc fragment of the IgG molecule.
  • the fusion protein comprises two high affinity PD-1 molecules. More preferably, the two high affinity PD-1 molecules are stable dimeric structures formed by disulfide bonds of immunoglobulin Fc fragments.
  • the amino acid sequence of the HiPD-1-Fc fusion protein molecule is selected from the group consisting of SEQ ID NO. 10, 12, 14, 16 or 18; the nucleotide sequence encoding thereof corresponds to SEQ ID NO. 15, 17 or 19.
  • the invention also relates to nucleic acid molecules encoding the HiPD-1-Fc fusion proteins of the invention.
  • the nucleic acid molecule of the invention may be in the form of DNA or in the form of RNA.
  • the DNA can be a coding strand or a non-coding strand.
  • a nucleic acid sequence encoding a fusion protein of the invention may be the same or a degenerate variant of the nucleic acid sequence set forth in the Figures of the invention.
  • "degenerate variant” as used herein denotes a nucleic acid sequence that encodes a protein sequence having wild-type PD-1-Fc (SEQ ID NO: 8) but differs from the sequence of SEQ ID NO: 9.
  • the full length sequence of the nucleic acid molecule of the present invention or a fragment thereof can generally be obtained by, but not limited to, PCR amplification, recombinant methods or synthetic methods. At present, it has been possible to obtain a DNA sequence encoding the Hi-PD-1-Fc fusion protein of the present invention completely by chemical synthesis. The DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art.
  • the expression vector described in the present invention may be a prokaryotic expression vector and a eukaryotic expression vector.
  • the selection and construction of vectors is well within the skill of those in the art. Since the product expressed by the prokaryotic expression system is often in the form of insoluble inclusion bodies, the expressed protein of interest needs to undergo a complex process of denaturation and renaturation to obtain a protein of natural biological activity.
  • the Fc fusion protein molecule is large, the molecule contains large disulfide bonds, and the results and functions are also complex. Generally, it is expressed by eukaryotic expression system, so as to produce natural protein or polypeptide organism through glycosylation modification and natural folding. An active functional glycosylation recombinant fusion protein.
  • mammalian eukaryotic expression systems are preferred in the present invention.
  • the inventors used the eukaryotic secretory expression vector pFUSE-hIgG1e1-Fc2 (InvivoGen). Based on this vector, a pFUSE-HiPD-1-Fc eukaryotic expression vector was constructed.
  • the host cells of the present invention include prokaryotic cells and eukaryotic cells.
  • prokaryotic cells include Escherichia coli and the like, and commonly used eukaryotic host cells include yeast cells, insect cells and mammalian cells.
  • eukaryotic host cells include yeast cells, insect cells and mammalian cells.
  • the inventors used 293T adherent mammalian cells, more preferably expi293FTM expressing host cells, a modified 293 cell line with higher transfection efficiency and protein Yield.
  • the method for preparing a HiPD-1-Fc fusion protein according to the present invention comprises inserting a nucleic acid sequence comprising a HiPD-1-Fc fusion protein into a suitable vector to obtain a corresponding suitable vector, and transfecting a suitable host cell; The transfected cells are cultured under appropriate culture conditions, and the expressed HiPD-1-Fc protein is isolated and purified therefrom.
  • the transformation of the expression vector can be carried out by a conventional method such as a calcium chloride method, an electroporation method, a lipofection method, or the like.
  • the present invention uses the Lipofectamine 2000 transfection reagent, more preferably using ExpiFectamine TM 293 Reagent.
  • the purification process may employ a purification process conventionally used in the art, including but not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting method), centrifugation, osmotic sterilizing, sonication, ultracentrifugation, molecular sieve layer Analysis (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques and combinations of these methods.
  • a purification process specially designed by the inventors can also be employed.
  • the method comprises the steps of: (a) collecting the culture supernatant; (b) separating the nickel column affinity chromatography; and (c) separating the Superdex 200 molecular sieve. Purification was carried out by the process selected by the inventors, and finally a HiPD-1-Fc bivalent fusion protein having a purity greater than 85% was obtained.
  • the HiPD-1-Fc fusion protein of the present invention has not only high affinity for PDL-1 but also excellent stability;
  • the HiPD-1-Fc fusion protein of the present invention is capable of effectively enhancing the function of an effector cell and killing tumor activity.
  • the extracellular amino acid sequence and nucleotide sequence of wild-type PD-1 are SEQ ID NO. 1 and 20, respectively.
  • the ligation product was transformed into E. coli DH5 ⁇ (Vazyme), coated with kanamycin-containing LB plate, inverted culture at 37 ° C overnight, and positive clones were picked for PCR screening, and the positive recombinants were sequenced to determine the correct sequence and then extracted.
  • the recombinant plasmid was transformed into E. coli Rosetta strain (TIANGEN) for expression.
  • the above-mentioned Rosetta colony containing the recombinant plasmid pET28a-PD-1 was inoculated into LB medium containing kanamycin, cultured at 37 ° C until the OD600 was 0.6-0.8, IPTG was added to a final concentration of 0.7 mM, and culture was continued at 37 ° C for 4 h. .
  • the cell pellet was harvested by centrifugation at 6000 g for 15 min, and the cell pellet was lysed with Bugbuster Master Mix (Merck).
  • the inclusion bodies were recovered by centrifugation at 6000 g for 15 min, and then washed with Bugbuster (Merck) to remove cell debris and membrane fractions, centrifuged at 6000 g for 15 min, and collected for inclusion. body.
  • the inclusion bodies were dissolved in a buffer (50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 6 Mguanidine HCl, pH 8.1), and the insolubles were removed by high-speed centrifugation. The supernatant was quantified by BCA and then dispensed at -80 ° C. Save spare.
  • a buffer 50 mM Tris-HCl, 200 mM NaCl, 2 mM EDTA, 6 M guanidine HCl, pH 8.1
  • DTT was further added to a final concentration of 20 mM, and treated at 37 ° C for 1 h.
  • the above treated PD-1 mixture was added dropwise to 100 mL of refolding buffer (50 mM HEPES, pH 7.5, 500 mM L-arginine, 9 mM glutathione, 1 mM glutathione disulfide, 24 mM NaCl, 1 mM KCl), stirred at 4 ° C for 30 min, then The reconstituted solution was placed in a cellulose membrane dialysis bag with a cut-off amount of 3.5 KD, and the dialysis bag was placed in 2 L of pre-cooled water and slowly stirred at 4 ° C overnight.
  • refolding buffer 50 mM HEPES, pH 7.5, 500 mM L-arginine, 9 mM glutathione, 1 mM glutathione disulfide, 24 mM NaCl, 1 mM KCl
  • the dialysate was changed to 2 L of pre-cooled buffer (10 mM Tris-HCl, pH 8.5), and dialysis was continued for 24 h at 4 ° C. The dialysate was then replaced with the same fresh buffer for dialysis for 24 hours.
  • the sample was passed through 0.45 ⁇ m. The filter was filtered, vacuum degassed and injected onto an anion exchange column (HiTrap Q HP, GE Healthcare). The protein was purified by linear gradient elution with 0-1 M NaCl in 10 mM Tris-HCl pH 8.5 and the collected fractions were subjected to SDS-PAGE analysis.
  • the target PD-1 fraction was collected and further purified by a gel filtration column (Superdex 75 10/300, GE Healthcare), and the target component was also subjected to SDS-PAGE analysis, and the results are shown in Fig. 1.
  • the binding activity of the wild-type PD-1 molecule to PDL-1 was detected using a BIAcore T200 real-time analysis system.
  • the anti-streptavidin antibody (GenScript) was added to a coupling buffer (10 mM sodium acetate buffer, pH 4.77), and then the antibody was passed through a CM5 chip previously activated with EDC and NHS to immobilize the antibody on the surface of the chip. Finally, the unreacted activated surface was blocked with a solution of ethanolamine in hydrochloric acid to complete the coupling process at a coupling level of about 15,000 RU.
  • a low concentration of streptavidin is passed over the surface of the coated antibody chip, then biotinylated PD-1 is passed through the detection channel, and the other channel is used as a reference channel, and 0.05 mM biotin is then 10 ⁇ L.
  • the flow rate of /min flows through the chip for 2 min, blocking the remaining binding sites of streptavidin.
  • the affinity was determined by single-cycle kinetic analysis.
  • PD-1 was diluted to several different concentrations with HEPES-EP buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% P20, pH 7.4) at 30 ⁇ L/min.
  • the flow rate which flows through the surface of the chip in turn, is 120 s for each injection and is dissociated for 600 s after the last injection.
  • the chip was regenerated with 10 mMGly-HCl, pH 1.75, after each round of assay. Kinetic parameters were calculated using BIAcore Evaluation software.
  • amino acid sequence and nucleotide sequence of PDL-1 used in this example are respectively SEQ ID NO. 21
  • the purified PDL-1 molecule was concentrated using a Millipore ultrafiltration tube while the buffer was replaced with 10 mM Tris pH 8.0, followed by biotinylation reagent 0.05 MBicine pH 8.3, 10 mM ATP, 10 mM MgOAc, 50 ⁇ M D-Biotin, 100 ⁇ g/ml BirA enzyme. (GST-BirA), the mixture was incubated overnight at room temperature, and biotinylation was detected by SDS-PAGE.
  • Biotinylated labeled PDL-1 molecules were concentrated to 500 ⁇ l using a Millipore ultrafiltration tube. Biotinylated PDL-1 was purified by gel filtration chromatography, and Superdex 75 10/300 gel was pre-equilibrated with filtered PBS. Filter column (GE General Electric Company), further load 500 ⁇ l of concentrated biotinylated PDL-1 molecule, then elute with PBS at a flow rate of 1 ml/min, and collect the collected components for SDS-PAGE analysis. The components of the target protein were concentrated using a Millipore ultrafiltration tube, the protein concentration was determined by BCA method (Thermo), and the biotinylated PDL-1 molecule was dispensed at -80 °C.
  • the KD value of the binding affinity of the wild-type PD-1 molecule to the PDL-1 molecule detected by the above procedure of the present example was 2.815E-06M, and the BIAcore binding map is shown in FIG.
  • the extracellular sequence of wild-type PD-1 described in Example 1 was used as a template strand, and high affinity was performed according to the phage display and screening method described by Li et al. (2005) Nature Biotech 23(3): 349-354). Screening of PD-1. After several rounds of screening, the phage library has a strong binding signal to PD-1, picking up the monoclonal and performing sequence analysis.
  • the high affinity PD-1 molecule of the present invention was expressed, renatured and purified as described in Example 1, and its affinity for the PDL-1 molecule was determined as described in Example 2.
  • the affinity of the high-affinity PD-1 molecule obtained in the present invention to the PDL-1 molecule is at least 100 times that of the wild-type PD-1 molecule and the affinity of the PDL-1 molecule, and the amino acid sequence thereof and its affinity value with the PDL-1 molecule are as follows Table 1 shows.
  • Example 4 recognizes the ability of PD-L1 on the surface of H1299 cells to be higher than PD-1
  • Biacore results showed that PD-1 mutants with improved affinity were obtained after screening, but whether this affinity change affects the binding of PDL-1 on the cell surface under physiological conditions still needs to be confirmed experimentally. Therefore, we selected H1299 cells with positive expression of PDL-1, added different concentrations of biotinylated PD-1 and L5B7 proteins, and analyzed the ability of PD-1 and L5B7 to recognize PDL-1 on the cell surface by flow cytometry.
  • the HiPD-1-Fc fusion protein is a dimeric soluble protein formed by two single-stranded peptides via an interchain disulfide bond. Its main structure comprises the head of two high-affinity PD-1 molecules (HiPD-1) and an IgG4. The Fc tail.
  • the single-stranded peptide comprising the fusion protein comprises 315 amino acids, and from the N-terminus to the C-terminus: HiPD-1 molecule, IgG4Fc fragment, His tag, wherein 1-116 is HiPD-1 molecule; 117-345 is IgG4 Fc fragment; 118-128, hinge region of Fc fragment; 129-236, CH2 domain of Fc fragment; 237-345, CH3 domain of Fc fragment; 346-351, 6 histidine composition for His tag For protein purification.
  • the two peptides form two interchain disulfide bonds at positions 124 and 127 (hinge region), respectively, to ensure HiPD-1-Fc dimerization.
  • the fusion amino acid sequence of wild-type PD-1 molecule and IgG4Fc fragment was optimized by the nucleotide sequence of the eukaryotic expression system, and the nucleotide fragment was synthesized in vitro by Nanjing Kingsray.
  • the cells were inverted and cultured overnight, and the positive recombinants were sequenced to identify the correct wild-type PD-1-Fc fusion protein particles.
  • the HiPD-1-Fc recombinant clone and His tag were constructed by PCR mutation technology, and transformed into Top10 competent state. The successful recombinants were sequenced and identified, and the bacterial liquid was stored at -20 °C for use.
  • the correct bacterial solution containing the HiPD-1-Fc plasmid was inoculated in 200 ml of LB medium (containing 100 ⁇ g/ml ampicillin) at 1:100, and cultured at 37 ° C overnight, and collected by centrifugation at 4500 g for 30 min. After the plasmid was extracted in accordance with standard process large plasmid was extracted (see PureLink TM HiPure Plasmid Filter Maxiprep Kit standard techniques procedures, Life), as shown in FIG, OD 260/280 plasmid concentration was measured 4, plasmid concentration was adjusted 1mg / ml, dispensing Store at -20 °C.
  • the density of overnight cultured cells was first adjusted to 2.5*10 6 /ml in 25.5 ml (required cell viability greater than 95%); secondly, 30 ⁇ g plasmid and 81 ⁇ l ExpiFectamine TM 293 Reagent (purchased from Life) was diluted into opti-MEM medium (1.5 ml each), and after standing for 5 min, the transfection reagent mixture was slowly added dropwise to the plasmid mixture, and allowed to stand for 20 min.
  • a mixed droplet of the plasmid and the transfection reagent was added to the cell culture, cultured at 37 ° C, 125 rpm, 5% CO 2 for 16-18 h, and 150 ⁇ l of enhancer 1 (kit, purchased from life) and 1.5 were added.
  • Ml enhancer 2 (kit, purchased from life), continue to culture for 3-4 days (exploration conditions, found that after 4-5 days of culture, cell viability is 40-50%), 4 ° C, 12000g centrifugation to collect the supernatant, 0.45 Mm filtration.
  • the protein was purified by a linear gradient eluate of 500 mM imidazolium phosphate buffer (pH 7.2) through a nickel column (GE Healthcare) at a flow rate of 2 ml/min, and the collected fractions were subjected to SDS-PAGE analysis. According to the analysis results, the target HiPD-1-Fc fraction was collected and further purified by molecular sieve Superdex 200 (GE Healthcare), and the target fraction was collected and subjected to SDS-PAGE analysis to identify the protein purity. The result is shown in Figure 5.
  • the fusion protein can recognize both the HiPD-1 molecule-specific ligand PD-L1 and the Fc fragment activity.
  • the ability of the HiPD-1-Fc fusion protein to recognize PD-L1 was determined by Octet system based on biofilm interferometry.
  • the HiPD-1-Fc fusion protein was diluted to 0.5 ⁇ M and loaded onto a NTA probe (PALL) needle.
  • the PD-L1 antigen was diluted to a different concentration in a 2-fold gradient, and HBS dilution buffer (70 mM NaCl, 750 mM Na 2 HPO 4 ⁇ 2H 2 O, 25 mM HEPS) was used as a baseline to bind to the HiPD-1-Fc fusion protein.
  • HBS dilution buffer 70 mM NaCl, 750 mM Na 2 HPO 4 ⁇ 2H 2 O, 25 mM HEPS
  • Data was collected using Data Acquisition software, and Data Analysis software analyzed the data and calculated the kinetic parameters.
  • the chip was regenerated with 10 mM Gly-HCl, pH 1.75, after each round of assay.
  • the affinity of the HiPD-1-Fc fusion protein for antigen binding to PD-L1 is shown in Table 1.
  • the HiPD-1-Fc molecule is present in a soluble, dimeric form, which requires the Fc to be active.
  • HiPD-1-Fc fusion protein promotes specific killing of PD-L1 + tumor cells by PBMC
  • the downstream signaling pathway of T cell activation thereby targeting T cells specifically killing tumor cells.
  • the present inventors firstly incubate 10 ⁇ g/ml of HiPD-1-Fc fusion protein with tumor cells (2*10 4 /well) for 30-60 min, and then add PBMC cells according to PBMC: tumor cells at a ratio of 5:1.
  • the concentration of ImmTAC-IG4 was adjusted to 2*10 -10 M for a total of 150 ⁇ l.
  • the supernatant was taken to detect the release of LDH (Promega).
  • the HiPD-1-Fc fusion protein significantly promoted the specific killing of tumor cells by PBMC.
  • the killing of the tumor was increased by at least 30%.
  • the killing of the tumor was increased by 245.2% after the addition of L45-123-Fc.
  • HiPD-1-Fc fusion protein promotes release of IFN- ⁇ from PBMC cells
  • HiPD-1-Fc fusion protein was incubated with tumor cells (1*10 4 /well 150 ⁇ l system) for 30-60 min, and PBMC was added according to PBMC: tumor cells at a ratio of 2:1. The cells were adjusted to a concentration of 4*10 -9 M of ImmTAC-IG4, and after being placed in an incubator for 36 hours, IFN- ⁇ release was measured according to a standard procedure.
  • the HiPD-1-Fc fusion protein shown in Fig. 5 can promote the specific release of IFN- ⁇ by PBMC.
  • the HiPD-1-Fc fusion protein can promote the specific release of IFN- ⁇ by PBMC, wherein L2F8-Fc promotes the release of IFN- ⁇ of ImmTAC to 77.42%, and L2F10-Fc promotes the release of IFN- ⁇ from ImmTAC.

Abstract

本发明提供了含高亲和力PD-1和人免疫球蛋白IgG的Fc片段元件的融合蛋白HiPD-1-Fc，编码该融合蛋白的核酸序列，制备该融合蛋白的方法以及所述融合蛋白在提高T细胞效应功能和杀伤肿瘤活性中的应用。

Description

PD-1-Fc融合蛋白及其制备方法和用途 技术领域
本发明涉及肿瘤免疫学领域；更具体地说，本发明涉及高亲和力的可溶性程序性死亡受体(Programmed Death-1,PD-1)分子与Fc片段形成的融合蛋白及其制备方法和在肿瘤治疗中的应用。
背景技术
正常情况下，机体的免疫系统能够监视、清除病变的细胞。但是，肿瘤仍然能够继续生长进而影响到生活质量及生命安全，说明肿瘤本身存在一定的机制来逃逸机体的免疫监视。近年来研究发现：肿瘤擅用抑制性协同共刺激分子拮抗T细胞抗肿瘤效应是肿瘤免疫逃逸的重要机制，阻断这类分子成为肿瘤免疫治疗研究新的热点(Hamanishi J et al.,Proceedings of the National Academy of Sciences of the United States of America,2007,104,3360-3365)。
PD-1(Programmed death 1)及其配体PD-L1(Programmed death-ligand 1)属于CD28/B7超家族蛋白，是一种重要的免疫抑制分子，可截断T细胞活化信号，抑制T细胞增殖及细胞因子的释放，在肿瘤免疫逃逸、自身免疫性疾病及病毒感染过程中发挥重要作用(Keir ME et al.,Annu Rev Immunol,26,677-704)。一系列临床前和临床研究证实针对PD-1/PD-L1信号通路的治疗性单克隆抗体取得了显著疗效，成为肿瘤治疗领域的热点研究方向。这些抗体作用的本质是其针对抗原的高亲和力，及占据了野生型PD-1和PD-L1相互作用的结合位点，从而中断了PD-1与PD-L1结合引起的负性信号的转导，解除了肿瘤微环境中T细胞受抑制的状态(Liu K et al.,Cell Res,27,151-153；Na Z et al.,Cell Res,27,147-150；Tan S et al.,Nat Commun,8,14369)。
研究发现：可溶性PD-1(souble PD-1,sPD-1)能够封闭膜结合性PD-1和PD-L1的相互作用，增强T细胞的抗肿瘤功能。在关节炎患者的血清中发现了高表达的sPD-1，进一步研究表明sPD-1能够封闭膜结合性PD-1对T细胞活化的抑制(Liu et al.,Arthritis research&therapy,17,(1),340)，He等也发现sPD-1能够通过结合PD-L1，增强肿瘤特异性的CD8T细胞介导的杀伤(He YF,The Journal of Immunology,173,(8),4919-4928)。通过分子定向进化和噬菌体高通量筛选技术，发现高亲和力PD-1分子(HiPD-1)能够以较野生型PD-1分子更高的亲和力识别肿瘤细胞表面的PD-L1。
尽管HiPD-1分子能够有效的识别PD-L1分子并能封闭PD-1和PD-L1的相互作用，但是由于天然的HiPD-1分子量小，体内应用后半衰期短，限制其体内生物活性的发挥。因此，本领域迫切需要一种改善其体内半衰期，促进其生物学活性发挥的途径。
发明内容
本发明的目的在于提供一种对PDL-1分子具有较高亲和力的PD-1与Fc片段的融合蛋 白，所述融合蛋白既能够有效的识别PD-L1分子并封闭PD-1和PD-L1的相互作用，并且能够非常有效地促进效应细胞对肿瘤细胞的杀伤作用；本发明还提供了上述PD-1与Fc片段的融合蛋白的制备方法及用途。
在第一方面，本发明提供一种融合蛋白，所述融合蛋白包含高亲和力PD-1分子和IgG分子的Fc片段，其中：
(i)所述高亲和力PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的至少100倍；和
(ii)所述高亲和力PD-1分子的氨基酸序列与野生型PD-1氨基酸序列(SEQ ID NO.1)有至少90％的序列相同性。
在优选的实施方式中，所述高亲和力PD-1分子的氨基酸序列与SEQ ID NO.1所示的氨基酸序列有92％；优选地，至少94％(例如，95％、96％、97％、98％和99％)的序列相同性。
在优选的实施方式中，所述高亲和力PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力的至少200倍；优选地，至少500倍；更优选地，至少1000倍；更优选地，至少2000倍。
在具体的实施方式中，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。
在具体的实施方式中，所述的IgG的Fc片段选自人IgG1、IgG2、IgG3或IgG4的Fc片段；优选地，选自IgG4的Fc片段。
在具体的实施方式中，所述Fc片段包括铰链区、CH2和CH3结构域，优选地，所述Fc片段不包含CH1和CH4结构域，更有选地，所述Fc片段的氨基酸序列为SEQ ID NO:7。
在具体的实施方式中，所述高亲和力PD-1分子的氨基酸序列选自SEQ ID NO:2-6。
在具体的实施方式中，所述融合蛋白中包含两个高亲和力的PD1分子。
在具体的实施方式中，所述融合蛋白中两个高亲和力的PD1分子通过二硫键连接形成二聚体结构；优选地，所述二硫键是IgG分子的Fc片段铰链区的二硫键。
在第二方面，本发明提供一种核酸分子，所述核酸分子包含编码第一方面所述融合蛋白的核酸序列或其互补序列。
在第三方面，本发明提供一种载体，所述载体含有第二方面所述的核酸分子。
在第四方面，本发明提供一种宿主细胞，所述宿主细胞中含有第三方面所述载体或染色体中整合有外源的第二方面所述的核酸分子。
在第五方面，本发明提供一种药物组合物，所述组合物中含有第一方面所述的融合蛋白，和任选的药学上可接受的载体或赋形剂。
在第六方面，本发明提供一种治疗疾病的方法，包括给需要治疗的对象施用适量的第一方面所述的融合蛋白或第五方面所述的药物组合物；优选地，所述疾病为肿瘤；更优选地，所述肿瘤表达PD-L1分子。
在第七方面，本发明提供第一方面所述融合蛋白的用途，用于制备治疗肿瘤的药物。
在第八方面，本发明提供一种制备第一方面所述融合蛋白的方法，包括步骤：
(a)培养第四方面所述的宿主细胞，从而表达第一方面所述的融合蛋白；
(b)任选分离或纯化所述的融合蛋白。
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为野生型PD-1蛋白纯化后的SDS-PAGE胶图。M，蛋白分子量Mark。
图2为野生型PDL-1分子与PD-1分子结合的BIAcore图谱。
图3为PD-1、L5B7识别H1299细胞表面PD-L1的流式检测，显示L5B7识别H1299细胞表面PDL-1的能力高于PD-1。注：A，anti-PDL-1抗体(2.5ul/样品)识别H1299细胞表面的PDL-1；B，不同浓度的PD-1、L5B7(浓度为0.02mg/ml、0.04mg/ml、0.08mg/ml)识别H1299细胞表面的PDL-1的流式检测图，其中SA-PE的用量为0.5ul/样品；C，当浓度为0.08mg/ml时，对照组、PD-1、L5B7识别PDL-1的流式柱状图。
图4为表达质粒的凝胶电泳图。其中，M为DNA分子标记；泳道1为野生型PD-1-Fc；泳道2为L2F8-Fc；泳道3为L2F10-Fc；泳道4为L45-Fc；泳道5为L45-123-Fc；泳道6为L5B7-Fc。
图5为融合蛋白纯化后SDS-PAGE电泳图。其中，A为非还原性的；B为还原性的。泳道1为L2F8-Fc；泳道2为L2F10-Fc；泳道3为L45-Fc；泳道4为L45-123-Fc；泳道5为L5B7-Fc。
图6为融合蛋白的Fc片段ELISA检测结果图。
图7显示了本发明融合蛋白促进杀伤的结果。其中A为Mel624；B为H1299；C为A375。
图8显示了本发明融合蛋白促进PBMC释放IFN-γ的Elispot结果。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现将高亲和力的可溶性PD-1与IgG Fc片段融合后得到高亲和力PD-1-Fc融合蛋白，所述融合蛋白不仅能够高效识别PD-L1配体，而且能够非常有效地促进效应细胞对肿瘤细胞的杀伤作用。
术语定义
除非另外定义，否则本文中所用的全部技术与科学术语均具有如本发明所属领域的普通技术人员通常理解的相同含义。
本文所用的术语“HiPD-1分子”或“高亲和力PD-1分子”或“高亲和力PD-1”或“本发明的PD-1分子”或“本发明的PD-1”是指对PDL-1的亲和力是野生型PD-1分子对PDL-1的亲和力至少100倍、优选至少200倍、更优选至少500倍、至少1000倍、至少2000倍的可溶性高亲和力PD-1分子，并且所述PD-1分子的氨基酸序列与野生型PD-1氨基酸序列(SEQ ID NO.1)有至少90％、优选至少92％、更优选至少94％(例如，至少95％、96％、97％、98％或99％)的序列相同性；优选地，所述PD-1分子的氨基酸序列选自SEQ ID NO.2-6。
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAE(SEQ ID NO.1，野生型PD-1)
Figure PCTCN2018119388-appb-000001
Figure PCTCN2018119388-appb-000002
Figure PCTCN2018119388-appb-000003
Figure PCTCN2018119388-appb-000004
Figure PCTCN2018119388-appb-000005
本文所用的术语“IgG的Fc片段”指免疫球蛋白重链的恒定区，例如IgG的Fc片段包括重链CH1、CH2、CH3、CH4的两个或更多结构域与铰链区的组合。在一个优选的实施例中，所用的免疫球蛋白的Fc片段包括至少一个免疫球蛋白铰链区，一个CH2结构域和一个CH3结构域；优选缺少CH1结构域，更优选缺少CH1、CH4结构域。最优选地，所述Fc片段的氨基酸序列选自SEQ ID NO.7。
ESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.7，IgG4Fc片段)
本文所用的术语“HiPD-1-Fc融合蛋白”或“高亲和力PD-1-Fc融合蛋白”或“高亲和力PD-1-Fc”或“本发明的PD-1-Fc融合蛋白”或“本发明的PD-1-Fc”是指由高亲和力PD-1分子与人的IgG Fc片段融合形成的蛋白。优选地，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。优选地，所述融合蛋白中包含两个高亲和力PD-1分子。更优选地，所述两个高亲和力PD-1分子是通过免疫球蛋白Fc片段二硫键形成的稳定的二聚体结构。最优选地，所述HiPD-1-Fc融合蛋白分子的氨基酸序列选自SEQ ID NO.10、12、14、16或18；其编码核苷酸序列分别对应于SEQ ID NO.11、13、15、17或19。
PPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDSRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK(SEQ ID NO.8，野生型PD-1-Fc)
CCTCCTACATTTTCTCCTGCTCTGCTGGTCGTGACAGAGGGCGACAACGCCACCTTTACCTGCTCTTTCTCCAACACCTCTGAGTCTTTCGTGCTGAACTGGTACAGGATGTCTCCTTCTAACCAGACCGATAAGCTGGCTGCTTTCCCTGAAGATAGATCTCAGCCTGGACAGGATTGCAGGTTTAGAGTCACACAGCTGCCTAACGGTCGCGATTTTCATATGTCCGTCGTCAGAGCTAGGAGAAACGATTCTGGAACCTACCTGTGCGGTGCTATTTCTCTGGCTCCTAAAGCTCAGATCAAAGAGTCCCTGAGAGCTGAACTGAGAGTCACAGAAAGAAGGGCTGAATCAAAGTATGGACCACCTTGCCCATCCTGTCCAGCACCAGAGTTTCTGGGCGGACCCTCCGTGTTCCTGTTTCCACCCAAGCCTAAAGATACACTGATGATTTCCCGCACACCTGAAGTCACTTGCGTGGTCGTGGACGTGAGCCAGGAGGATCCAGAAGTCCAGTTCAACTGGTACGTGGACGGAGTCGAGGTGCACAATGCCAAGACCAAACCCCGGGAGGAACAGTTTAACAGTACATACAGAGTCGTGTCAGTCCTGACTGTGCTGCATCAGGACTGGCTGAACGGCAAGGAGTATAAGTGCAAAGTGTCTAATAAGGGACTGCCTTCATCCATCGAGAAAACAATTAGTAAGGCAAAAGGCCAGCCTAGAGAACCACAGGTGTACACTCTGCCTCCAAGTCAGGAGGAAATGACTAAGAACCAGGTCTCACTGACCTGTCTGGTGAAAGGGTTCTATCCAAGCGATATCGCTGTGGAGTGGGAATCTAATGGTCAGCCCGAGAACAATTACAAGACAACTCCCCCTGTGCTGGACAGCGATGGCTCTTTCTTTCTGTATTCCCGTCTGACTGTGGACAAGAGCAGGTGGCAGGAGGGAAACGTCTTTAGCTGTTCTGTGATGCACGAAGCTCTGCACAATCATTACACCCAGAAGAGTCTGTCACTGTCCCTGGGCAAA(SEQ ID NO.9，野生型PD-1-Fc)
Figure PCTCN2018119388-appb-000006
Figure PCTCN2018119388-appb-000007
Figure PCTCN2018119388-appb-000008
Figure PCTCN2018119388-appb-000009
Figure PCTCN2018119388-appb-000010
Figure PCTCN2018119388-appb-000011
Figure PCTCN2018119388-appb-000012
Figure PCTCN2018119388-appb-000013
Figure PCTCN2018119388-appb-000014
Figure PCTCN2018119388-appb-000015
Figure PCTCN2018119388-appb-000016
Figure PCTCN2018119388-appb-000017
Figure PCTCN2018119388-appb-000018
本发明还涉及编码本发明HiPD-1-Fc融合蛋白的核酸分子。本发明的核酸分子可以是DNA形式或RNA形式。DNA可以是编码链或非编码链。例如，编码本发明融合蛋白的核酸序列可以与本发明附图中所示的核酸序列相同或是简并的变异体。例如，本文所用的“简并的变异体”表示编码具有野生型PD-1-Fc(SEQ ID NO:8)的蛋白序列，但与SEQ ID NO:9的序列有差别的核酸序列。
本发明的核酸分子全长序列或其片段通常可以用但不限于PCR扩增法、重组法或人工合成的方法获得。目前，已经可以完全通过化学合成来得到编码本发明Hi-PD-1-Fc融合蛋白的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。
本发明中所述的表达载体可以是原核表达载体及真核表达载体。载体的选择及构建是本领域技术人员通常所掌握的。由于原核表达体系所表达的产物常以不溶性的包涵体形式存在，表达的目的蛋白需经过复杂的变性及复性过程后，才能获得具有天然生物学活性的目的蛋白。而Fc融合蛋白分子较大，分子中含有较大二硫键，结果及功能也较复杂，一般均采用真核表达系统表达，以便通过糖基化修饰和自然折叠，产生具有天然蛋白或多肽生物学活 性功能的糖基化重组融合蛋白。因此本发明优选了哺乳动物真核表达系统。在优选的一个方案中，本发明人使用了真核分泌型表达载体pFUSE-hIgG1e1-Fc2(InvivoGen)。在此载体基础上，构建了pFUSE-HiPD-1-Fc真核表达载体。
在本发明所述的宿主细胞包括原核细胞和真核细胞，常用的原核细胞包括大肠杆菌等，常用的真核宿主细胞包括酵母细胞，昆虫细胞和哺乳动物细胞。在一个优选的实施例中，本发明人使用了293T贴壁哺乳动物细胞，更优选的使用expi293FTM表达宿主细胞(life)，一种改良型的293细胞系，具有更高的转染效率和蛋白产量。
在本发明所述的HiPD-1-Fc融合蛋白制备方法包括将含有编码HiPD-1-Fc融合蛋白的核酸序列，插入到合适的载体中，得到相应的合适载体，转染适宜的宿主细胞；并在在适宜的培养条件下，培养转染细胞，并从中分离纯化出表达的HiPD-1-Fc蛋白。
表达载体的转化可采用常规的方法，如氯化钙法、电穿孔法、脂质体转染法等。优选地，本发明人使用了Lipofectamine 2000转染试剂，更优选地使用了ExpiFectamine TM 293 Reagent。
纯化工艺可采用本领域中常规使用的纯化工艺，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、超声处理、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。也可采用本发明人专门设计的纯化工艺。优选地，包括以下步骤：(a)收集培养上清；(b)镍柱亲和层析分离；(c)Superdex 200分子筛分离。用本发明人选择的工艺进行纯化，最终可获得纯度大于85％的HiPD-1-Fc双价融合蛋白。
本发明的主要优点在于：
(1)本发明的HiPD-1-Fc融合蛋白不仅对PDL-1具有高亲和力，还具备优异的稳定性；和
(2)本发明的HiPD-1-Fc融合蛋白能够有效提高效应细胞的功能和杀伤肿瘤活性。
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，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。
实施例中的实验方法，如无特别说明，均采用本领域常规技术，实验试剂均为市售产品。虽然在本发明的实施或测试中可以使用与本发明中所述相似或等价的任何方法和材料，本文在此处例举优选的方法和材料。
实施例1.野生型PD-1的表达、复性和纯化
野生型PD-1的胞外氨基酸序列及核苷酸序列分别为SEQ ID NO.1和20
(CCTCCTACATTCTCCCCGGCACTGCTGGTTGTTACCGAAGGCGATAATGCGACCTTTACCTGTAGTTTCTCCAATACGAGCGAATCGTTTGTCCTGAACTGGTATCGTATGAGCCCG TCTAATCAGACCGATAAACTGGCGGCCTTCCCGGAAGATCGCTCTCAGCCGGGCCAAGACAGCCGTTTTCGCGTTACGCAACTGCCGAACGGTCGTGATTTCCATATGAGTGTGGTTCGCGCCCGTCGCAATGACTCCGGCACCTACCTGTGTGGTGCAATTTCACTGGCTCCGAAAGCCCAAATCAAAGAATCGCTGCGTGCGGAACTGCGTGTTACCGAACGTCGTGCCGAA)，将携带野生型PD-1的胞外序列的目的基因经Nco Ⅰ和Not Ⅰ双酶切，与经过Nco Ⅰ和Not Ⅰ双酶切的pET28a载体(Novagen)该载体经过优化后带有biotin tag标签)连接。连接产物转化至E.coli DH5α(Vazyme)，涂布含卡那霉素的LB平板，37℃倒置培养过夜，挑取阳性克隆进行PCR筛选，对阳性重组子进行测序，确定序列正确后抽提重组质粒转化至E.coli Rosetta菌株(TIANGEN)中用于表达。
将上述含有重组质粒pET28a-PD-1的Rosetta菌落接种于含有卡那霉素的LB培养基中，37℃培养至OD600为0.6-0.8，加入IPTG至终浓度为0.7mM，37℃继续培养4h。6000g离心15min收获细胞沉淀物，用BugbusterMaster Mix(Merck)裂解细胞沉淀物，6000g离心15min回收包涵体，再用Bugbuster(Merck)进行洗涤以除去细胞碎片和膜组分，6000 g离心15min,收集包涵体。将包涵体溶解在缓冲液(50mM Tris-HCl，200 mM NaCl,2mM EDTA，6Mguanidine HCl，pH 8.1)中，高速离心去除不溶物，上清液用BCA法定量后进行分装，于-80℃保存备用。
向7mg溶解的PD-1包涵体蛋白中，加入2mL缓冲液(50mM Tris-HCl，200mM NaCl，2mM EDTA，6M guanidine HCl，pH 8.1)，再加入DTT至终浓度为20mM，37℃处理1h。向100mL复性缓冲液(50mM HEPES，pH 7.5，500mM L-arginine，9mM glutathione，1mM glutathione disulfide，24mM NaCl，1mM KCl)中滴加上述处理后的PD-1混合液，4℃搅拌30min，然后将复性液装入截留量为3.5KD的纤维素膜透析袋,透析袋置于2L预冷的水中，4℃缓慢搅拌过夜。24小时后，将透析液换成2L预冷的缓冲液(10mMTris-HCl，pH 8.5)，4℃继续透析24h，然后将透析液换成相同的新鲜缓冲液继续透析24小时，样品经0.45μm滤膜过滤，真空脱气后进样至阴离子交换柱(HiTrap Q HP，GE Healthcare)。用10mMTris-HCl pH 8.5配制的0-1MNaCl线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析。根据分析结果，收集目标PD-1组分浓缩后进一步用凝胶过滤柱(Superdex 75 10/300，GEHealthcare)纯化，目标组分也进行SDS-PAGE分析，结果如图1所示。
实施例2.结合表征
BIAcore分析
使用BIAcore T200实时分析系统检测野生型PD-1分子与PDL-1的结合活性。将抗链霉亲和素的抗体(GenScript)加入偶联缓冲液(10mM醋酸钠缓冲液，pH 4.77)，然后将抗体流过预先用EDC和NHS活化过的CM5芯片，使抗体固定在芯片表面，最后用乙醇胺的盐酸溶液封闭未反应的活化表面，完成偶联过程，偶联水平约为15，000 RU。
使低浓度的链霉亲和素流过已包被抗体的芯片表面，然后将生物素化的PD-1流过检测通道，另一通道作为参比通道，再将0.05mM的生物素以10μL/min的流速流过芯片2min， 封闭链霉亲和素剩余的结合位点。采用单循环动力学分析方法测定其亲和力，将PD-1用HEPES-EP缓冲液(10mM HEPES，150mMNaCl，3mM EDTA,0.005％P20，pH 7.4)稀释成几个不同的浓度，以30μL/min的流速，依次流过芯片表面，每次进样的结合时间为120s，最后一次进样结束后让其解离600s。每一轮测定结束后用pH 1.75的10mMGly-HCl再生芯片。利用BIAcore Evaluation软件计算动力学参数。
本实施例中所用的PDL-1的氨基酸序列和核苷酸序列分别如SEQ ID NO.21
Figure PCTCN2018119388-appb-000019
(TTTACGGTTACGGTTCCGAAAGACCTGTATGTGGTTGAATACGGC
TCTAATATGACGATTGAATGCAAATTCCCGGTTGAAAAACAACTGGATCTGGCGGCCCTGATTGTGTATTGGGAAATGGAAGACAAAAACATCATCCAATTCGTGCATGGCGAAGAAGATCTGAAAGTTCAGCACAGCTCTTACCGTCAACGCGCACGTCTGCTGAAAGACCAGCTGAGCCTGGGCAATGCAGCTCTGCAGATCACGGATGTTAAACTGCAAGACGCCGGTGTCTATCGCTGCATGATTTCTTATGGCGGTGCAGACTACAAACGTATCACCGTCAAAGTGAACGCTCCGTACAACAAAATTAATCAGCGCATCCTGGTGGTTGATCCGGTTACGTCCGAACATGAACTGACCTGTCAAGCGGAAGGCTATCCGAAAGCCGAAGTCATTTGGACCAGTTCCGATCACCAGGTGCTGTCAGGTAAAACCACGACCACGAACTCGAAACGCGAAGAAAAACTGTTTAATGTCACGAGCACCCTGCGTATTAACACCACGACCAATGAAATCTTCTACTGCACCTTTCGTCGTCTGGACCCGGAAGAAAATCATACGGCGGAACTGGTTATCCCGGAACTG)所示，其表达、复性和纯化过程与实施例1中野生型PDL-1的表达、复性和纯化过程相同。其生物素化的过程如下：
a.生物素化
用Millipore超滤管将纯化的PDL-1分子浓缩，同时将缓冲液置换为10mMTris pH 8.0，然后加入生物素化试剂0.05MBicine pH 8.3、10mM ATP、10mMMgOAc、50μM D-Biotin、100μg/ml BirA酶(GST-BirA)，室温孵育混合物过夜，SDS-PAGE检测生物素化是否完全。
b.纯化生物素化后的复合物
用Millipore超滤管将生物素化标记后的PDL-1分子浓缩至500μl，采用凝胶过滤层析纯化生物素化的PDL-1，先用过滤过的PBS预平衡Superdex 75 10/300凝胶过滤柱(GE通用电气公司)，再加载500μl浓缩过的生物素化PDL-1分子，然后用PBS以1ml/min流速洗脱,将收集到的组分进行SDS-PAGE分析，根据结果合并含有目标蛋白质的组分，用Millipore超滤管浓缩，BCA法(Thermo)测定蛋白质浓度，将生物素化的PDL-1分子分装保存在-80℃。
通过本实施例上述过程检测到野生型PD-1分子与PDL-1分子的结合亲和力的KD值为2.815E-06M，其BIAcore结合图谱如图2所示。
实施例3.高亲和力PD-1分子的产生
将实施例1中所述的野生型PD-1的胞外序列作为模板链，根据Li等，(2005)Nature Biotech 23(3):349-354)描述的噬菌体展示和筛选方法，进行高亲和力PD-1的筛选。经过几轮筛选后的噬菌体文库均和PD-1有较强的结合信号，从中挑取单克隆，并进行序列分析。
按照实施例1中所述方法表达、复性和纯化本发明高亲和性PD-1分子，并按实施例2中所述方法测定其与PDL-1分子的亲和力。本发明中获得的高亲和力PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的亲和力至少100倍，其氨基酸序列及其与PDL-1分子的亲和力数值如下表1所示。
表1.高亲和力克隆对PDL-1分子的BIAcore结果
Figure PCTCN2018119388-appb-000020
实施例4.L5B7识别H1299细胞表面PD-L1的能力高于PD-1
Biacore结果显示，经过筛选后确实得到了亲和力提高的PD-1突变体，但这种亲和力的改变是否会影响其与生理条件下细胞表面PDL-1的结合仍需实验确认。因此，我们选用PDL-1表达阳性的H1299细胞，加入不同浓度的生物素化PD-1、L5B7蛋白，流式细胞术分析PD-1、L5B7识别细胞表面PDL-1的能力。
图3结果显示，随着加入的PD-1、L5B7蛋白浓度的升高，其对PDL-1的识别能力逐渐增强；在同一浓度条件下，L5B7蛋白识别PDL-1的能力高于PD-1，这种识别能力的改变可能是由于L5B7的亲和力较高所引起的，与生化结果一致。
实施例5.HiPD-1-Fc融合蛋白的设计及表达载体构建
HiPD-1-Fc融合蛋白是由两条单链肽段经链间二硫键形成的二聚体可溶性蛋白，其主要结构包含两个高亲和力PD-1分子(HiPD-1)的头部和一个IgG4的Fc尾部。组成该融合蛋白的单链肽段包含315个氨基酸，从N端到C端依次为：HiPD-1分子、IgG4Fc片段、His tag，其中，1-116为HiPD-1分子；117-345为IgG4的Fc片段；118-128，Fc片段的铰链区；129-236，Fc片段的CH2的结构域；237-345，Fc片段的CH3结构域；346-351，6个组氨酸组成His tag用于蛋白纯化。两条肽段在124位和127位(铰链区)各自形成两个链间二硫键，保证HiPD-1-Fc二聚体化。
基于此设计，野生型PD-1分子和IgG4Fc片段的融合氨基酸序列(SEQ ID NO:8)经真核表达系统核苷酸序列优化后，由南京金斯瑞公司体外合成核苷酸片段(SEQ ID NO:9)，采用同源重组技术(参照ClonExpress II One Step Cloning Kit标准技术流程，南京诺唯赞生物科技有限公 司)由EcoRⅠ和NheⅠ酶切位点整合到pFUSE-hIgG1e1-Fc2(InvivoGen)表达载体(该载体采用IL-2信号肽，融合蛋白分泌表达在上清)，并化转到Top10大肠杆菌(广州真知生物科技有限公司)中，涂布含博莱霉素的LB平板，37℃倒置培养过夜，对阳性重组子进行测序，鉴定正确的野生型PD-1-Fc融合蛋白质粒。在此基础上，通过PCR突变技术构建HiPD-1-Fc重组克隆子及His tag，并转化到Top10感受态，测序鉴定构建成功的重组子，菌液冻存-20℃备用。
将鉴定正确的含有HiPD-1-Fc质粒的菌液按照1:100接种于200ml LB培养基(含100μg/ml氨苄青霉素)中，37℃过夜培养后，4500g，30min离心收集沉淀。按照大提质粒标准流程(参照PureLink TM HiPure Plasmid Filter Maxiprep Kit标准技术流程，life)抽提质粒，如图4所示，OD 260/280测定质粒浓度，调整质粒浓度为1mg/ml，分装后于-20℃保存。
实施例6.HiPD-1-Fc融合蛋白的表达和纯化
复苏expi293F TM细胞株(购自Life)，并于37℃,125rpm,5％CO 2稳定培养2-3代。于转染前一天，调整细胞密度为2*10 6个/ml，过夜培养。转染当天(以30ml转染体系为例)，首先调整过夜培养细胞的密度为2.5*10 6个/ml于25.5ml(要求细胞活率大于95％)中；其次，将30μg质粒和81μl ExpiFectamine TM 293 Reagent(购自Life)分别稀释到opti-MEM培养基(各共1.5ml)中，静置5min后，将转染试剂混合液缓慢滴加到质粒混合液中，静置放置20min。最后，将质粒和转染试剂的混合液滴加到细胞培养物中，于37℃,125rpm,5％CO 2条件下培养16-18h，加入150μl enhancer 1(试剂盒，购自life)和1.5ml enhancer 2(试剂盒，购自life)，继续培养3-4天(摸索条件发现，在培养4-5天后，细胞活率在40-50％)，4℃，12000g离心收集上清，0.45μm过滤。
以2ml/min的进样流速经过镍柱(GE Healthcare)，用500mM咪唑磷酸盐缓冲液(pH 7.2)线性梯度洗脱液纯化蛋白，收集的洗脱组分进行SDS-PAGE分析。根据分析结果，收集目标HiPD-1-Fc组分浓缩后进一步用分子筛Superdex 200(GE Healthcare)纯化，收集目标组分并进行SDS-PAGE分析，鉴定蛋白纯度。结果如图5所示。
实施例7.HiPD-1-Fc融合蛋白的结合活性
为了评估实施例2中制备和纯化的HiPD-1-Fc融合蛋白的活性，确定该融合蛋白是否既可以识别HiPD-1分子特异性配体PD-L1，又具有Fc片段活性。首先，通过基于生物膜干涉技术的Octet system测定HiPD-1-Fc融合蛋白对PD-L1的识别能力。将HiPD-1-Fc融合蛋白稀释成0.5μM，加载到NTA探(PALL)针上。将PD-L1抗原按照2倍梯度稀释成不同浓度，HBS稀释缓冲液(70mM NaCl，750mM Na 2HPO 4·2H 2O，25mM HEPS)作为基线，结合到HiPD-1-Fc融合蛋白上。利用Data Acquisition软件收集数据，Data Analysis软件分析数据，并计算动力学参数。每一轮测定结束后用pH 1.75的10mM Gly-HCl再生芯片。HiPD-1-Fc融合蛋白与PD-L1的抗原结合的亲和力如表1。结果显示，融合蛋白的高亲和力PD-1段仍然能够以相当高的亲和力结合PD-L1，与Fc片段融合后并没有改变高亲和力PD-1结合PD-L1的能力。其次，HiPD-1-Fc分子是以可溶性、二聚体形式存在，这要求Fc需要具有活 性。本发明人通过酶联免疫吸附测定(ELISA)证明融合蛋白的Fc片段是完整有活性的。前一天，将HiPD-1-Fc融合蛋白用PBS稀释到20μg/ml，每孔100μl加入到ELISA板中，4℃过夜。第二天，PBS洗3次后，用5％milk-PBS室温封闭ELISA板中非特异性结合位点2h。0.05％PBST洗板三次，加入1:750稀释的HRP偶联的抗人IgG抗体并室温孵育1h。0.05％PBST洗三次，加入100μl/孔(TMB BioPanda Diagnostics)显色10min，1M H 2SO 4 50μl/孔终止显色，OD 450读数。结果如图6，HiPD-1-Fc融合蛋白结合anti-IgG抗体的信号明显高于阴性对照组，说明HiPD-1-Fc融合蛋白的Fc片段是有活性的。
表1HiPD-1-Fc融合对PD-L1分子的亲和力测定(Octet system)
Figure PCTCN2018119388-appb-000021
实施例8.HiPD-1-Fc融合蛋白促进了PBMC对PD-L1 +肿瘤细胞的特异性杀伤
为了进一步检测HiPD-1-Fc融合蛋白对PBMC特异性杀伤的效应，我们使用乳酸酶脱氢法(LDH)来检测PBMC对PD-L1 +的Mel624、A375、H1299肿瘤细胞系的特异性杀伤。ImmTACs分子能够重定向T细胞特异性杀伤肿瘤细胞已在多篇研究中报道(Jakobsen,2013；Oates et al.,2015)。其基本原理是ImmTACs可以模拟T细胞活化发挥效应功能的关键信号，一方面通过其高亲和的特异性TCR识别肿瘤细胞表面的MHC-肽复合物，另一方面通过其anti-CD3抗体端激活T细胞活化的下游信号通路，从而定向T细胞特异性杀伤肿瘤细胞。本发明人首先将10μg/ml的HiPD-1-Fc融合蛋白与肿瘤细胞(2*10 4个/孔)提前孵育30-60min，再按照PBMC:肿瘤细胞为5:1的细胞比例加入PBMC细胞，同时调整ImmTAC-IG4的浓度为2*10 -10M，共150μl体系，放置培养箱48h后，取上清检测LDH(Promega)的释放。结果如图7所示，HiPD-1-Fc融合蛋白能显著促进PBMC对肿瘤细胞的特异性杀伤。并且，对于不同的肿瘤细胞系都有明显的促进作用。相对于PBMC+肿瘤细胞系+ImmTAC组，对肿瘤的杀伤至少提高了30％，在Mel624肿瘤细胞中，加入L45-123-Fc后对肿瘤的杀伤提高了245.2％。
实施例9.HiPD-1-Fc融合蛋白促进了PBMC细胞IFN-γ的释放
为了进一步检测HiPD-1-Fc融合蛋白对PBMC活化时细胞因子释放的影响，我们使用酶联免疫斑点测定法(Elispot，BD)来检测PBMC特异性的IFN-γ释放。首先将15μg/ml的Hi-PD-1-Fc融合蛋白与肿瘤细胞(1*10 4个/孔150μl体系)提前孵育30-60min，再按照PBMC:肿瘤细胞为2:1的细胞比例加入PBMC细胞，同时调整ImmTAC-IG4的浓度为4*10 -9M，放置培养箱36h后，按照标准流程检测IFN-γ释放。结果如图5所示HiPD-1-Fc融合蛋白能促进PBMC特异性释放IFN-γ。结果如图8所示，HiPD-1-Fc融合蛋白能促进PBMC特异性释 放IFN-γ,其中L2F8-Fc促进ImmTAC的IFN-γ释放为77.42％,L2F10-Fc促进ImmTAC的IFN-γ释放为96.1％，L45-Fc促进ImmTAC的IFN-γ释放为82.9％，L45-123-Fc促进ImmTAC的IFN-γ释放为100.3％，L5B7-Fc促进ImmTAC的IFN-γ释放为67.4％。
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。

Claims (14)

  1. 一种融合蛋白，其特征在于，所述融合蛋白包含高亲和力PD-1分子和IgG分子的Fc片段，其中：
    (i)所述高亲和力PD-1分子与PDL-1分子的亲和力是野生型PD-1分子与PDL-1分子的至少100倍；和
    (ii)所述高亲和力PD-1分子的氨基酸序列与野生型PD-1氨基酸序列(SEQ ID NO.1)有至少90％的序列相同性。
  2. 如权利要求1所述的融合蛋白，其特征在于，所述高亲和力PD-1分子位于IgG分子的Fc片段的N端。
  3. 如权利要求1所述的融合蛋白，其特征在于，所述的IgG的Fc片段选自人IgG1、IgG2、IgG3或IgG4的Fc片段，优选地，选自IgG4的Fc片段。
  4. 如权利要求1所述的融合蛋白，其特征在于，所述Fc片段包括铰链区、CH2和CH3结构域，优选地，所述Fc片段不包含CH1和CH4结构域，更有选地，所述Fc片段的氨基酸序列为SEQ ID NO:7。
  5. 如权利要求1所述的融合蛋白，其特征在于，所述高亲和力PD-1分子的氨基酸序列选自SEQ ID NO:2-6。
  6. 如以上任一权利要求所述的融合蛋白，其特征在于，所述融合蛋白中包含两个高亲和力的PD1分子。
  7. 如权利要求6中所述的融合蛋白，其特征在于，所述融合蛋白中两个高亲和力的PD1分子通过二硫键连接形成二聚体结构，优选地，所述二硫键是IgG分子的Fc片段铰链区的二硫键。
  8. 一种核酸分子，其特征在于，所述核酸分子包含编码权利要求1-7中任一项所述融合蛋白的核酸序列或其互补序列。
  9. 一种载体，其特征在于，所述载体含有权利要求8所述的核酸分子。
  10. 一种宿主细胞，其特征在于，所述宿主细胞中含有权利要求9中所述载体或染色体中整合有外源的权利要求8中所述的核酸分子。
  11. 一种药物组合物，其特征在于，所述组合物中含有权利要求1-7中任一项所述的融合蛋白，和任选的药学上可接受的载体或赋形剂。
  12. 一种治疗疾病的方法，其特征在于，包括给需要治疗的对象施用适量的权利要求1-7中任一项所述的融合蛋白或权利要求11中所述的药物组合物；优选地，所述疾病为肿瘤；更优选地，所述肿瘤表达PD-L1分子。
  13. 权利要求1-7中任一所述融合蛋白的用途，用于制备治疗肿瘤的药物。
  14. 一种制备权利要求1-7中任一所述融合蛋白的方法，包括步骤：
    (a)培养权利要求10中所述宿主细胞，从而表达权利要求1-7中任一所述融合蛋白；
    (b)任选分离或纯化所述的融合蛋白。
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