WO2024066616A1 - 一种高亲和力pd1蛋白偶联物及其应用 - Google Patents

一种高亲和力pd1蛋白偶联物及其应用 Download PDF

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WO2024066616A1
WO2024066616A1 PCT/CN2023/104977 CN2023104977W WO2024066616A1 WO 2024066616 A1 WO2024066616 A1 WO 2024066616A1 CN 2023104977 W CN2023104977 W CN 2023104977W WO 2024066616 A1 WO2024066616 A1 WO 2024066616A1
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affinity
protein
elp
protein conjugate
polypeptide
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高卫平
高春红
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北京大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
    • 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
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • 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
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedicine technology, and in particular to a high-affinity PD1 protein conjugate and applications thereof.
  • T lymphocytes The activity of T lymphocytes is regulated by a complex signaling system generated by stimulatory and inhibitory receptors. These receptors are expressed on the surface of lymphocytes and mediate intercellular communication to determine their response to different antigens. These stimulatory and inhibitory receptors enable the immune system to respond appropriately to foreign antigens and inhibit responses to self-antigens.
  • Programmed cell death 1 (PD1) is a major inhibitory receptor that is preferentially expressed on activated T cells and B cells. Studies have found that it is also expressed in other subsets, such as natural killer cells, monocytes, and dendritic cells. PD1 is a member of the CD28 superfamily and produces negative signals when interacting with its ligands.
  • PD1 binds to two ligands (PDL1 and PDL2), which are commonly expressed on immune cells and tumor cells.
  • PDL1 and PDL2 Two ligands
  • the interaction between PD1 and its ligands plays a key immunomodulatory role in the activation and tolerance of T lymphocytes.
  • PDL1 plays a role in the second signaling pathway to inhibit T cell proliferation
  • drugs targeting the blockade of PD1/PDL1 binding have attracted much attention in the field of tumor immunotherapy.
  • Antibodies against PD1 and PDL1 block the binding of PDL1 expressed on the surface of tumor cells to PD1.
  • antibodies have inherent defects as a treatment method. For example, due to the large size of antibodies, it is difficult for them to enter solid tumors to antagonize the PD1:PDL1 signaling pathway deep in the tumor, resulting in poor efficacy.
  • Fc-mediated cytotoxic immune responses of antibodies targeting PD1 and PDL1 such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP)
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • the first object of the present invention is to provide a high-affinity PD1 protein conjugate and its related nucleic acid molecules and biomaterials.
  • the second object of the present invention is to provide a preparation method and application of the above-mentioned high-affinity PD1 protein conjugate.
  • the third object of the present invention is to provide the above-mentioned pharmaceutical composition containing the high-affinity PD1 protein conjugate.
  • the present invention provides the following technical solutions:
  • the present invention provides a high-affinity PD1 protein conjugate, wherein the high-affinity PD1 protein conjugate comprises an elastin-like polypeptide and a high-affinity PD1 protein connected to the elastin-like polypeptide.
  • the elastin-like polypeptide is directly connected to the high-affinity PD1 protein or is connected via a connecting peptide.
  • the connecting peptide mentioned above may be a polypeptide rich in glycine and serine.
  • the elastin-like polypeptide is directly linked to the high-affinity PD1 protein.
  • the C-terminus of the elastin-like polypeptide is linked to the N-terminus of the high-affinity PD1 protein.
  • the elastin-like polypeptides are often connected to the C-terminus of the target protein.
  • the present invention unexpectedly found that, compared with other connection methods, the high-affinity PD1 protein conjugate obtained by directly connecting the C-terminus of the elastin-like polypeptide to the N-terminus of the high-affinity PD1 protein has a significantly higher expression level and higher anti-tumor activity.
  • the high-affinity PD1 protein of the present invention has an enhanced affinity for PDL1 compared to the wild-type PD1 protein and can competitively bind PDL1.
  • the high-affinity PD1 protein is a high-affinity variant of the wild-type PD1 protein, which contains a mutation that can improve the affinity of the wild-type PD1 protein for PDL1 compared to the wild-type PD1 protein.
  • the high-affinity PD1 protein lacks the PD1 transmembrane domain and comprises one or more amino acid mutations relative to the extramembrane region of the wild-type PD1 protein.
  • the high-affinity PD1 protein is a soluble fragment of the extracellular domain mutant of the natural PD1 protein and has high affinity for PDL1.
  • the high-affinity PD1 breaks through the intrinsic limitations of antibody drugs and shows the effect of significantly improving the anti-tumor response of anti-PDL1 antibodies.
  • the present invention found that the high-affinity PD1 protein with an amino acid sequence such as SEQ ID NO.3 can better cooperate with the elastin-like polypeptide, and is more conducive to improving the expression and anti-tumor activity of the conjugate.
  • the high-affinity PD1 protein is any one of the following 1) or 2):
  • the amino acid sequence is the sequence obtained by adding one or more selected from protein tags, enzyme cleavage sites, and connecting peptides to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.3.
  • the high-affinity PD1 protein with the amino acid sequence shown in SEQ ID NO.3 comes from the extramembrane domain mutant of the natural PD1 protein, has a molecular weight of 14kD, and has an affinity for PDL1 that is approximately 20,000 times greater than that of the wild-type PD1.
  • the above-mentioned protein tag can be selected from various protein tags known in the art, including but not limited to His-tag, Gst, MBP, Strep, Flag and other tags.
  • the above-mentioned enzyme cleavage site can be selected from various enzymes used to cleave protein tags, including but not limited to TEV protease, transferase Sortase and the like.
  • the connecting peptide is a short peptide rich in glycine and serine, such as GSGGGGS and the like.
  • amino acid sequence of the high-affinity PD1 protein is shown as SEQ ID NO.3 or 4.
  • the elastin-like polypeptide is temperature-responsive, and the high-affinity PD1 protein conjugate is a fusion protein with temperature-responsiveness.
  • the present invention designs and optimizes the sequence of the elastin-like polypeptide so that it can better cooperate with the high-affinity PD1 protein.
  • amino acid sequence of the elastin-like polypeptide described above comprises (X-Gly-X-Pro-Gly)n, wherein 10 ⁇ n ⁇ 200, and X is any natural amino acid except proline.
  • X is one selected from valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine, threonine, serine, leucine, and isoleucine; and the response temperature of the elastin-like polypeptide is 10-60°C.
  • X is valine, 30 ⁇ n ⁇ 150, and the response temperature of the elastin-like polypeptide is 18-40°C.
  • the amino acid sequence of the elastin-like polypeptide is (VGVPG)n, wherein 60 ⁇ n ⁇ 120, preferably 80 ⁇ n ⁇ 100.
  • amino acid sequence of the elastin-like polypeptide is shown as SEQ ID NO.5.
  • the hydration dynamic radius of the high-affinity PD1 protein conjugate is 5-15 nm; more preferably 12.8 nm ⁇ 1.9 nm.
  • High-affinity PD1 is small in size and has a short circulation half-life, requiring daily injections to maintain therapeutically effective blood levels. However, frequent dosing may lead to poor patient compliance and serious side effects, making it difficult to use in clinical practice.
  • the use of the above-mentioned elastin-like polypeptide can better cooperate with the high-affinity PD1 protein of the above-mentioned specific sequence, and the half-life of the obtained high-affinity PD1 conjugate is significantly prolonged, the bioavailability is significantly increased, and it has a sustained release effect. It has a high affinity for PDL1 and thus competitively binds to PDL1, and can significantly reduce the toxicity of the PD1 protein. It has high safety, can effectively penetrate into the tumor tissue, and exert long-term anti-tumor activity, making the high-affinity PD1 protein suitable for clinical application.
  • the high-affinity PD1 protein conjugate provided by the present invention has the following characteristics and functions:
  • the present invention provides a nucleic acid molecule encoding the high-affinity PD1 protein conjugate described above.
  • the nucleic acid molecules include DNA and RNA.
  • nucleotide sequence of the nucleic acid molecule encoding the high-affinity PD1 protein conjugate. Based on the degeneracy of codons, the nucleotide sequence of the above nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the above heavy chain and light chain are within the scope of protection of the present invention.
  • nucleotide sequence of the nucleic acid molecule encoding the high-affinity PD1 protein is shown as SEQ ID NO.1 or 2.
  • the present invention provides a biological material comprising the above-mentioned nucleic acid molecule or expressing the above-mentioned high-affinity PD1 protein conjugate; the biological material is an expression cassette, a vector or a host cell.
  • the expression cassette containing the nucleic acid molecule can be obtained by operably linking a promoter and the nucleic acid molecule.
  • the expression cassette may also contain other transcription and translation regulatory elements such as terminators and enhancers.
  • the vector containing the nucleic acid molecule includes, but is not limited to, a plasmid vector, a phage vector, a viral vector, etc., wherein the plasmid vector includes a replicating vector and a non-replicating vector.
  • the host cells mentioned above include microbial cells or animal cells, wherein the microorganisms include prokaryotic microorganisms (such as Escherichia coli) and eukaryotic microorganisms (such as yeast) and the like.
  • the microorganisms include prokaryotic microorganisms (such as Escherichia coli) and eukaryotic microorganisms (such as yeast) and the like.
  • the present invention provides a method for preparing the high-affinity PD1 protein conjugate described above, the method comprising:
  • step 2) introducing the recombinant expression plasmid of step 1) into a microorganism to obtain a recombinant microorganism;
  • step 2) Cultivating the recombinant microorganism in step 2) to express a high-affinity PD1 protein conjugate, and obtaining the high-affinity PD1 protein conjugate after reversible phase transition (ITC) purification.
  • ITC reversible phase transition
  • the plasmid is selected from the PET series, preferably PET-25b+.
  • the microorganism is Escherichia coli, and the Escherichia coli is selected from the BL21 series, preferably BL21(DE3)PLySs.
  • the reversible phase transition (ITC) purification is 3 times reversible phase transition (ITC) purification.
  • the present invention provides use of the high-affinity PD1 protein conjugate or the nucleic acid molecule or the biomaterial described above in the preparation of a drug.
  • the drug is a drug for preventing or treating tumors.
  • the tumor is preferably a solid tumor, including but not limited to colorectal cancer, melanoma, renal cancer, lung cancer, liver cancer, oral squamous cell carcinoma, glioblastoma, breast cancer, prostate cancer, gastrointestinal cancer, thyroid cancer, lymphoma, uterine cancer, ovarian cancer, head and neck cancer.
  • the drug is a drug for preventing or treating colorectal cancer
  • the drug is a drug for preventing or treating colon cancer.
  • the present invention has been verified through practice that the high-affinity PD1 protein conjugate provided by the present invention has significant anti-tumor activity against colon cancer, can significantly improve the survival rate, prolong the survival period, and exert a long-term anti-tumor effect in vivo.
  • the high-affinity PD1 protein conjugate can be administered by injection.
  • the drug can be an injection preparation.
  • the present invention provides a pharmaceutical composition comprising the high-affinity PD1 protein conjugate described above.
  • the pharmaceutical composition further comprises a drug selected from chemotherapeutic drugs, targeted therapeutic drugs, endocrine therapeutic drugs and immunotherapeutic drugs.
  • a drug selected from chemotherapeutic drugs, targeted therapeutic drugs, endocrine therapeutic drugs and immunotherapeutic drugs.
  • chemotherapeutic drugs selected from chemotherapeutic drugs, targeted therapeutic drugs, endocrine therapeutic drugs and immunotherapeutic drugs.
  • the active ingredient of the pharmaceutical composition described above may be only the high-affinity PD1 protein conjugate, or may further include active ingredients such as chemotherapeutic drugs, targeted therapeutic drugs, endocrine therapeutic drugs, and immunotherapeutic drugs.
  • chemotherapeutic drugs include paclitaxel, docetaxel, gemcitabine, vinorelbine, cisplatin, carboplatin, oxaliplatin, etoposide, etoposide, doxorubicin, and oxaliplatin; targeted therapy drugs include small molecule tyrosine kinase inhibitors for the treatment of lung cancer, such as gefitinib, erlotinib, afatinib, osimertinib, etc., and monoclonal antibody targeted drugs for anti-tumor angiogenesis, such as bevacizumab, etc.; endocrine therapy drugs include letrozole, anastrozole, exemestane, CDK-46 inhibitors, etc. for the treatment of breast cancer; immunotherapy drugs include interferon, interleukin-2, etc.
  • the pharmaceutical composition comprises the high-affinity PD1 protein conjugate described above and oxaliplatin.
  • the present invention finds that the high-affinity PD1 protein conjugate and oxaliplatin described above can play a synergistic role in the treatment of colon cancer, significantly improve the therapeutic effect of colon cancer, and have high safety.
  • the pharmaceutical composition described above can be administered by injection, etc.
  • the pharmaceutical composition described above can contain excipients permitted in the pharmaceutical field.
  • the present invention provides a high-affinity PD1 protein conjugate ELP-PD1, which significantly improves the biological half-life and bioavailability of the high-affinity PD1 protein by connecting the high-affinity PD1 with ELP to form a fusion protein, increases its drug-making possibility, has tumor targeting and permeability, and tumor microenvironment temperature responsiveness, and can effectively inhibit tumor growth and improve anti-tumor therapeutic effects by relieving the body's immunosuppression and activating the body's own immunity.
  • Drugs developed from high-affinity PD1 protein conjugates have the advantages of simple prescription, easy operation, stable quality, strong controllability and good reproducibility. Compared with PD1 antibody drugs or other chemically modified drugs of immune checkpoint inhibitors, they have simple synthesis, controllable synthesis process, simple prokaryotic expression and production, and are easier to mass produce and commercialize.
  • the present invention also provides a pharmaceutical composition for use in combination with a high-affinity PD1 protein conjugate and other chemotherapeutic drugs, which effectively improves the efficiency of existing anti-tumor immunotherapy and is of great significance in tumor treatment.
  • FIG. 1 shows the SDS-PAGE electrophoresis diagram of protein characterization in Example 4 of the present invention.
  • FIG. 2 shows the exact molecular weights of the prepared proteins ELP(V)90-PD1 and PD1 determined by ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) in Example 4 of the present invention.
  • FIG3 shows the circular dichroism spectrum of the protein prepared in Example 4 of the present invention.
  • FIG. 4 shows the phase transition temperature of the protein ELP(V)90-PD1 prepared in Example 4 of the present invention.
  • FIG. 5 shows the particle sizes of proteins ELP(V)90-PD1 and PD1 prepared in Example 4 of the present invention.
  • FIG6 shows the qualitative results of laser confocal microscopy imaging of the competitive binding of ELP(V)90-PD1 and PD1 to the PDL1 receptor by the PDL1 antibody in Example 5 of the present invention, wherein the scale bars are all 10 ⁇ m.
  • FIG. 7 shows the flow cytometry quantitative results of competitive binding of ELP(V)90-PD1 and PD1 with PDL1 antibody to PDL1 receptor in Example 5 of the present invention.
  • FIG8 shows the maximum tolerated dose determination of ELP(V)90-PD1 and PD1 in Example 6 of the present invention in wild BALB/c mice.
  • FIG. 9 shows the in vivo pharmacokinetic changes of ELP(V)90-PD1 and PD1 proteins in Example 7 of the present invention in wild BALB/c mice.
  • FIG. 10 shows the sustained release results of ELP(V)90-PD1 in Example 8 of the present invention in wild BALB/c mice.
  • Figures 11 and 12 show the qualitative and quantitative results of the drug distribution of ELP(V)90-PD1 and PD1 proteins in major tissues and organs and tumor tissues in BALB/c mice bearing colon cancer tumors in Example 9 of the present invention.
  • FIG. 13 shows the permeability study of ELP(V)90-PD1 and PD1 proteins in tumors of BALB/c mice bearing colon cancer tumors in Example 10 of the present invention.
  • FIG. 14 shows the efficacy results of ELP(V)90-PD1 and PD1 proteins in Example 11 of the present invention in BALB/c mice bearing colon cancer tumors.
  • FIG. 15 shows the results of investigating the survival rate of BALB/c mice bearing colon cancer tumors after administration of ELP(V)90-PD1 and PD1 proteins in Example 11 of the present invention.
  • FIG. 16 shows the results of the combined administration of OX in Example 12 of the present invention on the inhibition of tumors in BALB/c mice bearing colon cancer tumors.
  • FIG. 17 shows the HE staining results of tumor sections of BALB/c mice bearing colon cancer tumors, showing the inhibition of combined OX administration in Example 12 of the present invention.
  • FIG. 18 shows the statistical comparison results of tumor sizes of various groups in the tumor inhibition of BALB/c mice bearing colon cancer tumors by combined OX administration in Example 12 of the present invention.
  • FIG. 19 shows the HE staining results of the damage of major tissues and organs of BALB/c mice bearing colon cancer tumors by combined OX administration in Example 12 of the present invention.
  • FIG. 20 shows the results of blood cell analysis in the blood of each group of mice after the combined administration of OX in Example 12 of the present invention on BALB/c mice bearing colon cancer tumors.
  • FIG. 21 shows the results of blood biochemical analysis in the blood of each group of mice after the combined administration of OX in Example 12 of the present invention on the efficacy of BALB/c mice bearing colon cancer tumors.
  • the gene sequence of the high-affinity PD1 mimetic polypeptide is a mutant of the wild-type PD1 protein gene sequence, which is used to block the interaction between the natural protein and its ligand PDL1 in in vivo and in vitro methods. Although the high-affinity PD1 mimetic polypeptide lacks the transmembrane domain of the natural protein, its affinity for the receptor PDL1 is greatly increased.
  • the gene sequence of the high-affinity PD1 protein used in the fusion protein is as follows:
  • the gene encoded by the GSGGGGSLPETGGHHHHHH protein sequence was inserted into the 3' end of the target gene PD1 through screening. After codon modification, the gene sequence containing the target gene and the tag protein is:
  • the high-affinity PD1 protein sequence encoded by the above gene is:
  • the protein sequence containing the purification tag and the cutting enzyme Sortase is:
  • the gene company (Suzhou Jinweizhi Biotechnology Co., Ltd.) was commissioned to insert the gene sequence shown in SEQ ID NO.2 into the E. coli plasmid PET-25b+ plasmid (100-300ng plasmid was mixed with 50 ⁇ l E. coli competent cells, ice-bathed for 30 minutes, and then heat-shocked at 42°C for 90 seconds).
  • the positive recombinants were screened on the ampicillin resistance plate and verified by DNA sequencing.
  • the plasmid was stored at -80°C for a long time, and the strain with the correct sequencing was stored at -80°C with 20% glycerol in a 1:1 ratio.
  • the amplified plasmid was transformed into BL21(DE3)PLysS expression competent cells, positive recombinants were screened on ampicillin resistance plates, 4-8 monoclonal bacteria were selected and inoculated into 150 mL LB medium, cultured at 37°C for 12-16 hours, then the cultured bacterial liquid was transferred to TB Escherichia coli medium and cultured for 6-8 hours, induced by IPTG at 25°C for 16 hours, and the bacterial liquid was collected.
  • This example uses a denaturation method to purify the PD1 protein that forms inclusion bodies.
  • the specific method is as follows:
  • Example 1 The bacterial precipitate collected in Example 1 was ultrasonically disrupted and then centrifuged to obtain the precipitate.
  • the suspension after the bacterial cell disruption was collected from the precipitate and centrifuged at 10,000 rpm for 10 minutes at 4°C to separate the inclusion bodies from the soluble proteins, and the precipitate and supernatant were collected separately.
  • Inclusion body washing Add the crude inclusion body to 20mL of cold washing solution (20mmol/L phosphate buffer solution, pH 8.0, containing 0.5mol/L NaCl, 2mol/L urea, 1% Triton X-100) and stir for 15-30min. Centrifuge at 4°C, 12000rpm for 30min, discard the supernatant and take the precipitate. Repeat the washing once (wash for 2-4h to remove membrane fragments and membrane proteins). Wash the obtained precipitate once with 50mmol/L phosphate buffer solution under the same centrifugation conditions, and the precipitate is the washed inclusion body.
  • cold washing solution 20mmol/L phosphate buffer solution, pH 8.0, containing 0.5mol/L NaCl, 2mol/L urea, 1% Triton X-100
  • Dissolution of inclusion bodies resuspend the washed precipitate in 20 mL of inclusion body denaturing solution (20 mmol/L phosphate buffer solution pH 8.0, 0.5 mol/L NaCl, 6 mol/L urea, 1 mmol/L ⁇ -mercaptoethanol, 1% TritonX-100), stir at room temperature for 60-90 min to fully dissolve, centrifuge at 12000 rpm for 20 min at 4°C, discard the supernatant, and filter the supernatant with a 0.45 ⁇ m filter membrane.
  • inclusion body denaturing solution 20 mmol/L phosphate buffer solution pH 8.0, 0.5 mol/L NaCl, 6 mol/L urea, 1 mmol/L ⁇ -mercaptoethanol, 1% TritonX-100
  • the column was washed with refolding buffer (20mmol ⁇ L -1 phosphate buffer, 500mmol ⁇ L -1 NaCl, pH 8.0) containing 5, 4, 3, 2, 1, and 0mol ⁇ L -1 urea, 10mL for each gradient, and the flow rate was 0.2mL ⁇ min -1 .
  • the temperature-responsive fusion protein (ELP(V)90-PD1) prepared in this example is obtained by fusing the temperature-responsive elastin-like polypeptide and the high-affinity PD1 protein, and the C-terminus of the elastin-like polypeptide is connected to the N-terminus of the high-affinity PD1 protein, wherein the amino acid sequence of the high-affinity PD1 protein is shown in SEQ ID NO.3, and the encoding gene sequence is shown in SEQ ID NO.1.
  • the temperature-responsive elastin-like polypeptide is composed of 90 VGVPG repeating pentapeptides connected in sequence, and its amino acid sequence SEQ ID NO.5 and gene sequence SEQ ID NO.6 are as follows:
  • the above ELP(V)90 gene fragment was inserted into the PET-24b(+) plasmid by Gene Company. After designing primers and polymerase chain reaction (PCR), the PCR fragment product containing restriction sites was double-digested (BseR I and Acu I) and inserted into the PET-24b(+) plasmid.
  • the primers involved are as follows:
  • the PD1 and ELP(V)90 plasmids were double-digested with two restriction endonucleases (AcuI and BglI) and (BseRI and BglI), respectively, and then the digested fragments were ligated overnight at 4°C using T4 ligase.
  • the ligated recombinants were transformed into the Top10 strain for amplification, and the positive recombinants were screened on kanamycin resistance plates and verified by DNA sequencing.
  • the plasmids were stored at -80°C for long-term storage, and the strains with correct sequencing were stored at -80°C with 20% glycerol in a 1:1 ratio.
  • the amplified plasmid was transformed into BL21(DE3)PLysS expression competent cells, and positive recombinants were screened on kanamycin resistance plates. Select 4-8 monoclonal bacteria and inoculate them into 150mL LB medium and culture them at 37°C for 12-16 hours. Then transfer the cultured bacterial liquid to TB Escherichia coli medium and culture it for 6-8 hours. After induction with IPTG at 25°C for 16 hours, collect the bacterial liquid. Resuspend the Escherichia coli cells collected by centrifugation in 40mL 10mM phosphate buffer solution and lyse the cells with an ultrasonic cell disruptor.
  • the ultrasonic power is 300w
  • the time is 45min
  • the ultrasonic mode is selected to run for 5s and pause for 5s.
  • the precipitate is discarded; then 2mL 10% (w/w) polyethyleneimine (PEI) is added and centrifuged again at 4°C and 14000rpm for 15 minutes to remove nucleic acids. Collect the supernatant and obtain the target protein by reversible phase change ITC technology.
  • PEI polyethyleneimine
  • the high-affinity PD1 protein and ELP(V)90-PD1 fusion protein prepared in Examples 2 and 3 were analyzed by polyacrylamide gel electrophoresis (SDS-PAGE) to analyze the expression and purity of the target protein ( FIG. 1 ), and the exact molecular weight of the prepared protein was determined by ultra-high performance liquid chromatography-mass spectrometry ( FIG. 2 ).
  • the absorbance value at OD350nm was measured by an ELISA instrument to determine the phase transition temperature of ELP(V)90-PD1 at different concentrations, and to explore the existence form of ELP(V)90-PD1 in the physiological environment in vivo. The results are shown in Figure 4.
  • the dynamic hydration radius of the protein PD1 and the fusion protein ELP(V)90-PD1 in solution at different time points was analyzed by a light dynamic scattering particle size analyzer (DLS) to preliminarily determine whether the fused protein can be filtered through the glomerulus and prolong the half-life.
  • LDS light dynamic scattering particle size analyzer
  • Figure 5 shows that the hydration dynamic radius of the fusion protein is about 4 times higher than that of the PD1 protein, which is higher than the minimum diameter of 10nm for kidney clearance, but much lower than the maximum cutoff size of 200nm of the reticuloendothelial system, indicating that the fusion protein can reduce the renal clearance rate and penetrate into tissue cells, thereby prolonging the half-life and increasing the uptake of tumor cells.
  • high-affinity PD1 protein has binding activity not only to human PDL1 receptors, but also to mouse PDL1 receptors.
  • Mouse colon cancer CT26 cells in the logarithmic growth phase were taken, the cell density was adjusted to 1 ⁇ 10 5 cells/mL, 2 mL/well was spread on a laser confocal dish, and cultured in a 37°C, 5% CO 2 incubator for 12 hours.
  • Mouse IFN ⁇ was added to induce upregulation of PDL1 receptors on the cell surface.
  • Cy5-labeled ELP(V)90-PD1 or high-affinity PD1 protein was added and cultured at 37°C, 5% CO After incubation in the incubator for 6 hours, the unbound labeled proteins were washed away with 10 mM phosphate buffer, and then the cells were fixed with 4% paraformaldehyde for 15 minutes, washed three times with 10 mM phosphate buffer, blocked with 5% bovine serum albumin (BSA) for 1 hour, washed three times with 10 mM phosphate buffer, added with goat anti-rabbit PDL1 primary antibody, incubated the cells at room temperature for 1 hour, washed the cells three times with 10 mM phosphate buffer, then added with FITC-labeled secondary antibody, incubated the cells at room temperature for 2 hours, washed the cells three times with 10 mM phosphate buffer, and added with 1 mL 10 mM phosphate buffer, and imaged and analyzed using a laser
  • the results are shown in Figure 6.
  • the competitive binding activity results in Figure 6 show that compared with the PD1 group, although the activity of the fusion protein ELP(V)90-PD1 in binding to the PDL1 receptor decreased by about 45% due to the fusion of the ELP(V)90 protein, the ELP(V)90-PD1 protein still has a strong activity in binding to the PDL1 receptor.
  • Flow cytometry was used to quantitatively analyze the competitive binding of ELP(V)90-PD1 or high-affinity PD1 protein to PDL1 on the surface of CT26 cells.
  • Mouse colon cancer CT26 cells in the logarithmic growth phase were taken, the cell density was adjusted to 2 ⁇ 10 5 cells/mL, and 4 mL/well was spread on a 6-well plate at 37°C and 5% CO 2 The cells were cultured in an incubator for 12 hours, and mouse IFN ⁇ was added to induce upregulation of PDL1 receptors on the cell surface. After 12 hours of induction, Cy5-labeled ELP(V)90-PD1 or PD1 protein was added.
  • the proteins were then characterized at the individual level in vivo.
  • the ELP(V)90-PD1 fusion protein prepared in Example 3 was tested for maximum tolerated dose in wild BALB/c mice.
  • 6-8 week old female BALB/c mice were purchased from the Animal Experimental Science Center of Peking University Health Science Center, and all operations on animals were performed in accordance with the guidelines of the Animal Ethics Committee of Peking University Health Science Center.
  • Female BALB/c mice were intraperitoneally injected with high-affinity PD1 protein at doses of 4.6, 8.5 and 12.6 mg/kg body weight and ELP(V)90-PD1 fusion protein at doses of 28.0, 32.0 and 48.0 mg/kg (three mice per group), and the weight changes of each group and the living conditions of each group of mice were monitored and recorded.
  • the maximum tolerated dose was the dose with a weight loss of no more than 10% at the highest dose.
  • the weight changes of each mouse per day in each dose group are shown in Figure 8.
  • Example 3 the pharmacokinetics of the ELP(V)90-PD1 fusion protein prepared in Example 3 was tested.
  • the heparin blood sample was centrifuged at 4°C and 3500 rpm for 5 minutes, and the pharmacokinetic change was determined by measuring the fluorescence intensity of the upper plasma.
  • the concentration of each group of drugs in the blood sample was calculated based on the fluorescence intensity curve of the mixture of Cy5-labeled PD1 protein and ELP(V)90-PD1 with the blank group plasma, and the pharmacokinetic parameters of each group were calculated using the drug analysis system 3.0 software.
  • the drug-drug curve and pharmacokinetic parameters of ELP(V)90-PD1 are shown in Figure 9 and Table 1.
  • the pharmacokinetic parameters of PD1 and ELP(V)90-PD1 were analyzed using the compartment model in the DAS software.
  • the half-life of PD1 was 20.9 ⁇ 0.9 hours, which was extended to 551.5 ⁇ 12.8 hours of ELP(V)90-PD1.
  • the area under the drug-drug curve of ELP(V)90-PD1 was 74275.5 ⁇ 113.2 mg/L ⁇ h, which was 21.1 times that of PD1 (3515.8 ⁇ 8.1 mg/L ⁇ h).
  • the peak drug concentration of ELP(V)90-PD1 (227.7 ⁇ 2.7 mg/L ⁇ h) was significantly higher than that of PD1 (3515.8 ⁇ 8.1 mg/L ⁇ h).
  • /L) is 2.7 times that of PD1 (88.6 ⁇ 2.7mg/L)
  • the plasma clearance rate of ELP(V)90-PD1 (0.04 ⁇ 0.09mL/h) is 25 times slower than that of PD1 (0.5 ⁇ 0.1mL/h)
  • the average residence time of ELP(V)90-PD1 (203.3 ⁇ 2.5h) is 6.68 times that of PD1 (30.4 ⁇ 1.4h).
  • the sustained release of the drug allows the drug to be released continuously for a longer period of time, thereby improving the local therapeutic effect of the drug.
  • This example evaluates the sustained release of the ELP(V)90-PD1 fusion protein in mice.
  • the Cy7-labeled high-affinity PD1 protein and ELP(V)90-PD1 fusion protein were intraperitoneally injected into mice (three mice in each group) at the maximum tolerated dose (4.6 mg/kg and 28.0 mg/kg, respectively).
  • the mice were anesthetized with isoflurane at 2h, 1d, 3d, 7d, 10d, 15d, and 18d after injection.
  • the results of the imaging analysis by the Spectrum imaging system are shown in Figure 10.
  • the results in Figure 10 show that the PD1 protein without ELP fusion metabolized all the drugs within 3 days, while the ELP(V)90-PD1 fusion protein still had some drugs not metabolized at 18 days.
  • the ELP(V)90-PD1 fusion protein formed a drug reservoir in the peritoneal mucosa and slowly released the drug, which was consistent with the results of drug metabolism kinetics.
  • Example 3 the tissue distribution of the ELP(V)90-PD1 fusion protein obtained in Example 3 was investigated.
  • BALB/c mice inoculated with colon cancer cells were intraperitoneally injected with the maximum tolerated dose of fluorescently labeled high-affinity PD1 protein and ELP(V)90-PD1 fusion protein, and the relative fluorescence intensity of the remaining PD1 in each major tissue organ was measured 2h, 1d, 3d, 7d, and 15d after administration, respectively, to reflect the drug distribution in each tissue organ.
  • the results are shown in Figures 11 and 12.
  • ELP(V)90-PD1 By detecting the relative fluorescence intensity of the drugs in various organs, it was found that at 2 hours, PD1 and ELP(V)90-PD1 were widely distributed in the liver, kidney, spleen, lung and tumor, respectively, but the distribution in the heart was lower than that in other tissues and organs. At two hours, the content of ELP(V)90-PD1 in tumor tissue was lower than that of PD1, which shows that the sustained-release drug has a slow onset. However, after 24 hours, the distribution of ELP(V)90-PD1 in tumor tissue reached its maximum, which was similar to PD1.
  • ELP( The content of ELP(V)90-PD1 protein in tumor tissue is higher than that of PD1, and its content in other tissues and organs is lower than that in tumor tissue. This suggests that ELP(V)90-PD1 can target tumor tissue and penetrate into tumor tissue. As time goes by, no signal of PD1 protein can be detected in various tissues and organs on the 7th and 15th days, while ELP(V)90-PD1 still has a high signal intensity in tumor tissue, which once again proves the sustained release effect and tumor targeting enrichment effect of ELP(V)90-PD1.
  • This example analyzes the penetration ability of the ELP(V)90-PD1 fusion protein prepared in Example 3 in tumor tissue, as follows:
  • ELP(V)90-PD1 fusion protein The penetration ability of ELP(V)90-PD1 fusion protein in tumor tissue was determined using BALB/c mice inoculated with CT26 colon cancer cells. High affinity PD1 protein and ELP(V)90-PD1 fusion protein were labeled with Cy5.
  • Example 11 In vivo anti-tumor biological activity detection of ELP(V)90-PD1 fusion protein
  • a subcutaneous tumor model of BALB/c mice bearing colon cancer cells was used to evaluate the in vivo antitumor activity of the ELP(V)90-PD1 fusion protein.
  • mice 100 ⁇ l of 1 ⁇ 10 6 /mL CT26 cells were subcutaneously inoculated on the right hind leg of the back of BALB/c mice.
  • high-affinity PD1 protein and ELP(V)90-PD1 fusion protein were intraperitoneally injected into the tumor-bearing mice at the maximum tolerated dose.
  • the survival status of the mice and the tumor growth status were observed every day, and the weight and tumor size of the mice were dynamically detected and recorded. After the treatment, the survival rate was calculated.
  • the tumor volume of each mouse in each group grew to 1500 mm 3 or the weight decreased by more than 10%, the mouse was considered dead and recorded as "1".
  • the mice whose tumors did not reach the standard were considered alive and recorded as "0".
  • the survival curves and survival rates of mice in each group are shown in Figures 14 and 15.
  • Figures 14 and 15 show that after a single maximum tolerated dose, although PD1 protein has a certain effect on the treatment of colon cancer tumors, the therapeutic effect of the ELP(V)90-PD1 group on the mouse colon cancer model is significantly better than that of the PD1 group. This is mainly due to the fact that after a single maximum tolerated dose, ELP(V)90-PD1 The group can be given a higher dose of the drug, and the sustained-release effect of ELP ensures that the release of PD1 is always at a relatively stable blood concentration, which fully stimulates the body's immunity and effectively inhibits the tumor.
  • the survival curve after drug efficacy also shows that after ELP(V)90-PD1 protein treatment, the overall survival time of mice in the ELP(V)90-PD1 group was significantly prolonged, with a median survival of 28 days, which is 1.5 times (19 days) and 1.9 times (15 days) of that in the PD1 and PBS treatment groups, respectively. This once again proves the long-term effect of ELP(V)90-PD1.
  • Example 12 In vivo antitumor activity of ELP(V)90-PD1 fusion protein combined with first-line chemotherapy drug oxaliplatin
  • mice 100 ⁇ l of 1 ⁇ 10 6 /mL CT26 cells were subcutaneously inoculated on the right hind leg of BALB/c mice.
  • 90 mice with tumors of the same size were evenly divided into 6 groups, with 15 mice in each group, namely PBS group, oxaliplatin (OX) group, PD1 group, ELP(V)90-PD1 group, PD1+OX group, and ELP(V)90-PD1+OX group.
  • PD1 protein and ELP(V)90-PD1 protein were intraperitoneally injected into the tumor-bearing mice in the PD1 group, ELP(V)90-PD1 group, PD1+OX group, and ELP(V)90-PD1+OX group at the maximum tolerated dose.
  • OX was given to the tumor-bearing mice in the OX group at a dose of 2 mg/kg.
  • OX was given to the PD1+OX group and the ELP(V)90-PD1+OX group at a dose of 2 mg/kg.
  • the survival status of the mice and the growth status of the tumor were observed every day, and the body weight and tumor size of each mouse in each group were dynamically detected and recorded.
  • the efficacy diagram is shown in Figure 16, the HE staining of tumor tissue is shown in Figure 17, the comparison of tumor size of each group is shown in Figure 18, the HE staining diagram of the main tissue organs of each group is shown in Figure 19, and the blood cell and blood biochemical analysis of some mice in each group are shown in Figures 20 and 21, respectively.
  • the results of Figure 17 show that after drug treatment, the number of tumor cells in the ELP(V)90-PD1+OX group ⁇ ELP(V)90-PD1 group ⁇ PD1+OX group ⁇ PD1 ⁇ OX group ⁇ PBS group, which is consistent with the results of the drug efficacy, indicating that after the combined administration of the fusion protein, a synergistic effect is produced to jointly exert an anti-tumor therapeutic effect.
  • the efficacy of each group can also be clearly seen in the tumor size comparison chart extracted in Figure 18.
  • the results of Figure 19 show that no obvious damage occurred to the tissues and organs after the protein drugs used and the combined administration, indicating the safety of drug administration.
  • no abnormalities were found in various blood cells of mice in each group, and the results of blood biochemical indicators also showed no obvious abnormalities, which once again demonstrated the safety of drug administration.
  • the present invention innovatively proposes a temperature-responsive high-affinity PD1 fusion protein, combined with oxaliplatin, a first-line anti-colon cancer drug, which greatly improves the anti-tumor efficacy.
  • the biological half-life of high-affinity PD1 is significantly extended.
  • the drug can be released continuously for 20 days, which is the longest circulation time currently achieved by non-ELP fusion protein delivery systems.
  • a single administration can achieve the best therapeutic effect, and the number of administrations is reduced, which improves patient compliance.
  • the fusion protein can also effectively penetrate into the tumor tissue, bind to the PDL1 receptor on the surface of tumor cells, and exert an anti-tumor immune effect.
  • the present invention provides a high-affinity PD1 protein conjugate and its application.
  • the high-affinity PD1 protein conjugate provided by the present invention comprises an elastin-like polypeptide and a high-affinity PD1 protein connected to the elastin-like polypeptide.
  • the conjugate significantly improves the biological half-life and bioavailability of the high-affinity PD1 protein, has tumor targeting and permeability, and tumor microenvironment temperature responsiveness, and can effectively inhibit tumor growth and improve the anti-tumor treatment effect by relieving the body's immunosuppression and activating the body's autoimmunity.
  • the high-affinity PD1 protein conjugate has the advantages of simple synthesis, controllable synthesis process, simple production, etc., is easier to mass produce and commercially apply, is of great significance in tumor treatment, and has good economic value and application prospects.

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Abstract

提供的是一种高亲和力PD1蛋白偶联物，其包含类弹性蛋白多肽以及与类弹性蛋白多肽连接的高亲和力PD1蛋白。该偶联物显著提高了高亲和力PD1蛋白的生物半衰期和生物利用度，具有肿瘤靶向性和渗透性以及肿瘤微环境温度响应性。

Description

一种高亲和力PD1蛋白偶联物及其应用
交叉引用
本申请要求2022年9月27日提交的专利名称为“一种高亲和力PD1蛋白偶联物及其应用”的第202211185337.5号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明涉及生物医药技术领域，尤其涉及一种高亲和力PD1蛋白偶联物及其应用。
背景技术
T淋巴细胞的活性受到刺激性和抑制性受体产生的复杂信号系统的调节。这些受体在淋巴细胞表面表达，并介导细胞间的通信，以确定它们对不同抗原的反应。这些刺激性和抑制性受体使免疫系统能够对外源抗原做出适当的反应，并抑制对自身抗原的反应。程序性细胞死亡1(PD1)是一种主要的抑制性受体，优先表达在活化的T细胞和B细胞上。研究发现，它也在其他亚群中表达，如自然杀伤细胞、单核细胞和树突状细胞。PD1是CD28超家族中的一员，在与其配体相互作用时产生负信号。PD1与两种配体(PDL1和PDL2)结合，这两种配体通常表达于免疫细胞和肿瘤细胞上。PD1与其配体之间的相互作用在T淋巴细胞的活化和耐受性中起着关键的免疫调节作用。由于PDL1起着第二信号通路抑制T细胞增殖的作用，因此针对阻断PD1/PDL1结合的靶点药物在肿瘤免疫治疗领域备受关注。抗PD1和PDL1的抗体阻断了肿瘤细胞表面表达PDL1与PD1的结合，到目前为止，针对PD1和PDL1的单克隆抗体如Keytruda(帕博利珠单抗)、Opdivo(欧狄沃单抗)、Atezolizumab(阿特珠单抗)等在临床实践中取得了巨大的突破。
虽然现有针对于PD1/PDL1的抗体技术已在临床实践中获得了突破性的进展，然而，抗体作为治疗方法存在固有的缺陷，例如，由于抗体体积大，很难进入实体肿瘤以拮抗肿瘤深部的PD1：PDL1信号通路，从而导致疗效不佳。此外，针对PD1和PDL1的抗体的Fc介导的细胞毒性免疫反应，如抗体依赖的细胞介导的细胞毒性作用(ADCC)和抗体依赖性细胞吞噬(ADCP)作用，可能会不可取地耗尽其打算激活的抗肿瘤细胞毒性T细胞。这主要是由于PD1和PDL1都在抗肿瘤细胞毒性T细胞的表面表达。因此，开发临床效果更优的抗体替代疗法也是肿瘤免疫领域的研究热点。
发明内容
本发明的第一目的是提供一种高亲和力PD1蛋白偶联物及其相关核酸分子和生物材料。
本发明的第二目的是提供上述高亲和力PD1蛋白偶联物的制备方法和应用。
本发明的第三目的是提供上述含有高亲和力PD1蛋白偶联物的药物组合物。
具体地，本发明提供以下技术方案：
第一方面，本发明提供一种高亲和力PD1蛋白偶联物，所述高亲和力PD1蛋白偶联物包含类弹性蛋白多肽以及与类弹性蛋白多肽连接的高亲和力PD1蛋白。
以上所述的高亲和力PD1蛋白偶联物中，类弹性蛋白多肽与高亲和力PD1蛋白直接连接或通过连接肽连接。
以上所述的连接肽可为富含甘氨酸和丝氨酸的多肽。
优选地，所述高亲和力PD1蛋白偶联物中，类弹性蛋白多肽与高亲和力PD1蛋白直接连接。
进一步优选地，类弹性蛋白多肽的C末端与高亲和力PD1蛋白的N末端连接。
在进行类弹性蛋白多肽偶联时，现有技术多将类弹性蛋白多肽连接于目标蛋白的C末端。本发明意外地发现，与其他连接方式相比，将类弹性蛋白多肽的C末端直接与高亲和力PD1蛋白的N末端连接得到的高亲和力PD1蛋白偶联物具有明显更高的表达水平以及更高的抗肿瘤活性。
本发明所述的高亲和力PD1蛋白相较于野生型PD1蛋白，对PDL1的亲和力增强，且能够竞争性结合 PDL1。
上述高亲和力PD1蛋白为野生型PD1蛋白的高亲和力变体，其与野生型PD1蛋白相比，含有能够使得野生型PD1蛋白对PDL1的亲和力提高的突变。
优选地，所述高亲和力PD1蛋白缺乏PD1跨膜域，并且相对于野生型PD1蛋白的膜外区包含一或多个氨基酸突变。
上述高亲和力PD1蛋白是天然PD1蛋白的胞外结构域突变体的一个可溶性片段，与PDL1具有高亲和力，高亲和力PD1突破了抗体药的内在限制，显示出显著改善抗PDL1抗体的抗肿瘤反应的作用。
在目前已知的高亲和力PD1蛋白中，本发明发现采用氨基酸序列如SEQ ID NO.3所示的高亲和力PD1蛋白能够更好地与类弹性蛋白多肽配合作用，更有利于提高偶联物的表达和抗肿瘤活性。
优选地，所述高亲和力PD1蛋白为以下1)或2)中的任一种：
1)氨基酸序列如SEQ ID NO.3所示；
2)氨基酸序列为在SEQ ID NO.3所示序列的N端或C端添加选自蛋白标签、酶切位点、连接肽中的一种或多种后得到的序列。
氨基酸序列如SEQ ID NO.3所示的高亲和力PD1蛋白来自于天然PD1蛋白的膜外区突变体，分子量为14kD，与野生型PD1相比其与PDL1的亲和力提高约2万倍。
本领域技术人员知晓，在SEQ ID NO.3所示序列的N端或C端添加选自蛋白标签、酶切位点、连接肽通常不会影响SEQ ID NO.3的结构和功能。
上述蛋白标签可选自本领域知晓的各种蛋白标签，包括但不限于His-tag、Gst、MBP、Strep、Flag等标签。
上述酶切位点可选自各种用于切割蛋白标签的酶，包括但不限于TEV蛋白酶、转肽酶Sortase等。
上述连接肽为富含甘氨酸和丝氨酸的短肽，例如GSGGGGS等。
在本发明的一些实施方式中，所述高亲和力PD1蛋白的氨基酸序列如SEQ ID NO.3或4所示。
类弹性蛋白多肽具有温度响应性，上述高亲和力PD1蛋白偶联物为具有温度响应性的融合蛋白。
针对上述高亲和力PD1蛋白，本发明对类弹性蛋白多肽的序列进行了设计和优化，以使得其与高亲和力PD1蛋白能够更好地配合作用。
以上所述的类弹性蛋白多肽的氨基酸序列包含(X-Gly-X-Pro-Gly)n，其中，10≤n≤200，X是除脯氨酸以外的任一天然氨基酸。
具体地，X为选自缬氨酸、苯丙氨酸、色氨酸、酪氨酸、丙氨酸、甘氨酸、甲硫氨酸、苏氨酸、丝氨酸、亮氨酸、异亮氨酸中的一种；所述类弹性蛋白多肽的响应温度为10-60℃。
优选地，X为缬氨酸，30≤n≤150，所述类弹性蛋白多肽的响应温度为18-40℃。
在本发明的一些实施方式中，类弹性蛋白多肽的氨基酸序列为(VGVPG)n，其中，60≤n≤120，优选为80≤n≤100。
在本发明的一些实施方式中，所述类弹性蛋白多肽的氨基酸序列如SEQ ID NO.5所示。
优选地，所述高亲和力PD1蛋白偶联物的水合动力学半径为5-15nm；更优选为12.8nm±1.9nm。
高亲和力PD1的体积较小，循环半衰期较短，需要每天注射，以维持治疗有效的血液水平，但频繁给药可能会导致患者依从性差和严重的副作用，较难用于临床实践。
采用上述类弹性蛋白多肽能够与上述特定序列的高亲和力PD1蛋白更好地配合作用，得到的高亲和力PD1偶联物的半衰期显著延长，生物利用度显著增加，具有缓释效果，对PDL1具有高亲和力进而竞争性结合PDL1，且能够明显降低PD1蛋白的毒性，具有较高的安全性，能够有效渗透至肿瘤组织内部，长效发挥抗肿瘤活性，使得高亲和力PD1蛋白能够适用于临床应用。
本发明提供的高亲和力PD1蛋白偶联物具有以下特性和功能：
1)温度响应性；
2)缓释；
3)肿瘤靶向性；
4)肿瘤组织渗透性；
5)激活、增强机体自身免疫；
6)抗肿瘤。
第二方面，本发明提供编码以上所述的高亲和力PD1蛋白偶联物的核酸分子。
所述核酸分子包括DNA、RNA。
根据以上所述的高亲和力PD1蛋白偶联物的氨基酸序列，本领域技术人员可以获得编码高亲和力PD1蛋白偶联物的核酸分子的核苷酸序列。基于密码子的简并性，上述核酸分子的核苷酸序列并不唯一，所有能够编码上述重链和轻链的核酸分子均在本发明的保护范围内。
在本发明的一些实施方式中，编码高亲和力PD1蛋白的核酸分子的核苷酸序列如SEQ ID NO.1或2所示。
第三方面，本发明提供包含以上所述的核酸分子或表达以上所述的高亲和力PD1蛋白偶联物的生物材料；所述生物材料为表达盒、载体或宿主细胞。
其中，含有所述核酸分子的表达盒可由启动子和所述核酸分子可操作性地连接得到。
根据表达需要以及表达盒上下游序列的不同，表达盒中还可包含终止子、增强子等其他转录、翻译调控元件。
含有所述核酸分子的载体包括但不限于质粒载体、噬菌体载体、病毒载体等，其中质粒载体包括复制型载体和非复制型载体。
以上所述的宿主细胞包括微生物细胞或动物细胞，其中微生物包括原核微生物(例如大肠杆菌)和真核微生物(例如酵母)等。
第四方面，本发明提供以上所述的高亲和力PD1蛋白偶联物的制备方法，所述方法包括：
1)将编码高亲和力PD1蛋白偶联物的核酸分子插入在表达质粒中，得到重组表达质粒；
2)将步骤1)的重组表达质粒导入微生物中，得到重组微生物；
3)培养步骤2)的重组微生物使其表达高亲和力PD1蛋白偶联物，经可逆相变(ITC)纯化后得到高亲和力PD1蛋白偶联物。
在本发明的一些实施方式中，所述质粒选自PET系类，优选为PET-25b+。
在本发明的一些实施方式中，所述微生物为大肠杆菌，所述大肠杆菌选自BL21系列，优选为BL21(DE3)PLySs。
在本发明的一些实施方式中，可逆相变(ITC)纯化为3次可逆相变(ITC)纯化。
第五方面，本发明提供以上所述的高亲和力PD1蛋白偶联物或所述核酸分子或所述生物材料在制备药物中的应用。
优选地，所述药物为用于预防或治疗肿瘤的药物。
所述肿瘤优选为实体肿瘤，包括但不限于结直肠癌、黑色素瘤、肾癌、肺癌、肝癌、口腔鳞癌、脑胶质母细胞瘤、乳腺癌、前列腺癌、胃肠癌、甲状腺癌、淋巴瘤、子宫癌、卵巢癌、头颈癌。
优选地，所述药物为用于预防或治疗结直肠癌的药物
进一步优选地，所述药物为用于预防或治疗结肠癌的药物。
本发明通过实践验证，本发明提供的高亲和力PD1蛋白偶联物对于结肠癌具有显著的抗肿瘤活性，能够显著提高生存率、延长生存期，并且在体内长效发挥抗肿瘤作用。
上述高亲和力PD1蛋白偶联物可采用注射方式进行给药。所述药物可为注射制剂。
第六方面，本发明提供一种药物组合物，所述药物组合物包含以上所述的高亲和力PD1蛋白偶联物。
优选地，所述药物组合物还包含选自化学治疗药物、靶向治疗药物、内分泌治疗药物及免疫治疗药物中的 一种或多种。
以上所述的药物组合物的活性成分可仅为所述高亲和力PD1蛋白偶联物，也可进一步包含化学治疗药物、靶向治疗药物、内分泌治疗药物、免疫治疗药物等活性成分。
其中，化学治疗药物包括紫杉醇、多西紫杉醇、吉西他滨、长春瑞滨、顺铂、卡铂、奥沙利铂、依托泊苷、足叶乙苷、阿霉素、草酸铂；靶向治疗药物包括治疗肺癌的小分子酪氨酸激酶抑制剂，比如吉非替尼、厄洛替尼、阿法替尼、奥希替尼等，抗肿瘤血管生成的单克隆抗体类靶向药物，比如贝伐珠单抗等；内分泌治疗药物包括治疗乳腺癌的来曲唑、阿那曲唑、依西美坦、CDK-46抑制剂等；免疫治疗药物包括干扰素、白介素-2等。
在本发明的一些实施方式中，所述药物组合物包含以上所述的高亲和力PD1蛋白偶联物以及奥沙利铂。
本发明发现，以上所述的高亲和力PD1蛋白偶联物和奥沙利铂在结肠癌的治疗中能够发挥协同作用，显著提高结肠癌的治疗效果，且具有较高的安全性。
以上所述的药物组合物可采用注射等方式给药。根据给药方式不同，上述药物组合物可包含药学领域允许的赋形剂。
本发明的有益效果至少包括以下几点：
本发明提供一种高亲和力PD1蛋白偶联物ELP-PD1，该偶联物通过将高亲和力PD1与ELP连接形成融合蛋白，显著提高了高亲和力PD1蛋白的生物半衰期和生物利用度，增加其成药的可能性，具有肿瘤靶向性和渗透性以及肿瘤微环境温度响应性，通过解除机体免疫抑制，激活机体自身免疫，可以有效抑制肿瘤的生长，提高抗肿瘤治疗效果。由高亲和力PD1蛋白偶联物开发药物具有处方简单、易操作、质量稳定、可控性强及重现性好等优点，相较于PD1抗体药物或其他的免疫检查点抑制剂化学修饰药物，具有合成简单，合成过程可控，原核表达生产简便，更易于大规模生产和商业化应用。
本发明还提供高亲和力PD1蛋白偶联物与其他化学治疗药物联合使用的药物组合物，该药物组合物有效提升了现有抗肿瘤免疫疗法的效率，在肿瘤治疗中具有重要意义。
附图说明
为了更清楚地说明本发明或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作一简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1显示了本发明实施例4中蛋白表征的SDS-PAGE电泳图。
图2显示了本发明实施例4中通过超高液相飞行时间质谱(UHPLC-MS)测定的所制备的蛋白ELP(V)90-PD1和PD1的确切分子量。
图3显示了本发明实施例4中所制备蛋白的圆二色图谱。
图4显示了本发明实施例4中所制备蛋白ELP(V)90-PD1的相转变温度。
图5显示了本发明实施例4中所制备蛋白ELP(V)90-PD1和PD1的粒径大小。
图6显示了本发明实施例5中ELP(V)90-PD1和PD1与PDL1抗体竞争性结合PDL1受体的激光共聚焦显微成像定性结果，其中标尺均为10μm。
图7显示了本发明实施例5中ELP(V)90-PD1和PD1与PDL1抗体竞争性结合PDL1受体的流式细胞术定量结果。
图8显示了本发明实施例中6中ELP(V)90-PD1和PD1在野生BALB/c小鼠身上的最大耐受剂量测定。
图9显示了本发明实施例7中ELP(V)90-PD1和PD1蛋白在野生BALB/c小鼠的体内药物代谢动力学变化。
图10显示了本发明实施例8中ELP(V)90-PD1在野生BALB/c小鼠身上的缓释结果。
图11、图12显示了本发明实施例9中ELP(V)90-PD1和PD1蛋白在荷结肠癌肿瘤BALB/c小鼠体内的主要组织脏器及肿瘤组织内的药物分布定性及定量结果。
图13显示了本发明实施例10中ELP(V)90-PD1和PD1蛋白在荷结肠癌肿瘤BALB/c小鼠肿瘤中的渗透性考察。
图14显示了本发明实施例11中ELP(V)90-PD1和PD1蛋白在荷结肠癌肿瘤BALB/c小鼠身上的药效结果。
图15显示了本发明实施例11中给药ELP(V)90-PD1和PD1蛋白后，荷结肠癌肿瘤BALB/c小鼠的生存率的考察结果。
图16显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠肿瘤的抑制结果。
图17显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠肿瘤抑制的肿瘤切片的HE染色结果。
图18显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠肿瘤抑制的各组肿瘤大小统计比较结果。
图19显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠各主要组织脏器损伤情况HE染色结果。
图20显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠药效后各组小鼠血液中血细胞分析结果。
图21显示了本发明实施例12中联合OX给药对荷结肠癌肿瘤BALB/c小鼠药效后各组小鼠血液中血生化分析结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将结合本发明中的附图，对本发明中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1高亲和力PD1模拟多肽的原核制备
高亲和力PD1模拟多肽(高亲和力PD1蛋白)的基因序列是野生型PD1蛋白基因序列的突变体，以用于体内及体外方法中阻断天然蛋白与其配体PDL1之间的相互作用。高亲和力PD1模拟多肽虽然缺少天然蛋白的跨膜结构域，但与受体PDL1的亲和力却大大增加。在本实施例中，融合蛋白中使用的高亲和力PD1蛋白的基因序列如下：
SEQ ID NO.1：
为了便于后期纯化，通过筛选将GSGGGGSLPETGGHHHHHH蛋白序列所编码的基因插入在目的基因PD1的3’端，经密码子修饰后含目的基因及标签蛋白的基因序列为：
SEQ ID NO.2：

上述基因编码的高亲和力PD1蛋白序列为：
SEQ ID NO.3：
含纯化标签及切割酶Sortase的蛋白序列为：
SEQ ID NO.4：
委托基因公司(苏州金唯智生物科技有限公司)将SEQ ID NO.2所示的基因序列插入到大肠杆菌质粒PET-25b+质粒中(100-300ng质粒与50μl大肠杆菌感受态混合后，冰浴30分钟后，42℃热击90秒)，经氨苄抗性平板筛选出阳性重组子，对其进行DNA测序验证。质粒置于-80℃长期保存，将测序正确的菌株与20％甘油1：1保存于-80℃。
将经扩增后的质粒转化至BL21(DE3)PLysS表达感受态细胞中，经氨苄抗性平板筛选出阳性重组子，挑选4-8个单克隆菌接种至150mL LB培养基中，于37℃培养12-16小时，然后将培养后的菌液转至TB大肠杆菌培养基中培养6-8小时后，在25℃下经IPTG诱导16小时，收集菌液。
实施例2纯化PD1蛋白混合物制备高亲和力PD1蛋白
本实施例采用变复性的方法对形成包涵体的PD1蛋白进行纯化，具体方法如下所示：
将实施例1收集的菌液沉淀经超声破碎后离心取沉淀，将沉淀收集菌体破碎后的混悬液，于4℃下10000rpm离心10分钟，使包涵体与可溶性蛋白分离，分别收集沉淀与上清。
包涵体洗涤：包涵体粗品加入到20mL冷的洗涤液(20mmol/L磷酸盐缓冲溶液中，pH 8.0，含0.5mol/L NaCl，2mol/L尿素，1％Triton X-100)中，搅拌15-30min。4℃，12000rpm离心30min，弃上清，取沉淀。重复洗涤一次(洗涤2～4h去除膜碎片和膜蛋白)。所得沉淀再用50mmol/L磷酸盐缓冲溶液洗1次，相同离心条件，取沉淀即为洗涤后的包涵体。
包涵体的溶解：将洗涤后的沉淀加入20mL包涵体变性溶解液重悬(20mmol/L磷酸盐缓冲溶液pH 8.0，0.5mol/L NaCl，6mol/L尿素，1mmol/Lβ-巯基乙醇，1％TritonX-100)，室温搅拌60-90min以充分溶解，4℃下12000rpm离心20min，弃取上清，将上清用0.45μm滤膜过滤。
柱上复性及纯化：取5-10mL变性上样缓冲液(20mmol·L-1磷酸盐缓冲溶液，300mmol·L-1NaCl，6mol·L-1尿素，5mM·L-1咪唑，1mM·L-1巯基乙醇，pH＝8.0)平衡Ni柱，流速为0.5mL·min-1。将经0.45μm滤膜过滤后的包涵体溶解液上样，流速为0.5mL·min-1。取10mL洗涤缓冲液(20mM·L-1磷酸盐缓冲液，300mmol·L-1NaCl，6mol·L-1尿素，5mM·L-1咪唑，1mM·L-1巯基乙醇，pH＝8.0)洗柱，直至OD280不再变化，流速为0.5mL·min-1。依次用含有5、4、3、2、1、0mol·L-1尿素的复性缓冲液(20mmol·L-1磷酸盐缓冲液，500mmol·L-1NaCl，pH 8.0)洗柱，每个梯度10mL，流速为0.2mL·min-1。再以含有300mmol·L-1咪唑的复性缓冲液洗脱，流速为0.5mL·min-1，复性过程中OD值会随时间的变化，并收集各浓度阶段的洗脱液，4℃、10000r/min下离心10min，取上清液进行SDS-PAGE电泳分析。
实施例3温度响应性融合蛋白的制备
本实施例制备的温度响应性融合蛋白(ELP(V)90-PD1)由温度响应性类弹性蛋白多肽和高亲和力PD1蛋白融合得到，类弹性蛋白多肽的C末端与高亲和力PD1蛋白的N末端连接，其中，高亲和力PD1蛋白的氨基酸序列如SEQ ID NO.3所示，编码基因序列如SEQ ID NO.1所示。温度响应性类弹性蛋白多肽是由90个VGVPG重复五肽顺次连接而成，其氨基酸序列SEQ ID NO.5和基因序列SEQ ID NO.6如下：
SEQ ID NO.5：
SEQ ID NO.6：
将上述ELP(V)90基因片段由基因公司插入在PET-24b(+)质粒上，设计引物并通过聚合酶链式反应(PCR)后，将含酶切位点的PCR片段产物双酶切(BseR I和Acu I)后插入至PET-24b(+)质粒中。上述涉及的引物如下：
上游引物：
5'-3'GAGGAGTACATATGGGCGACAGCCCGGATCGT；
下游引物：
5'-3'CTGAAGATCATTATTATCAGCCACCGGATCCACG。
使用两种限制性内切酶(AcuI和BglI)以及(BseRI和BglI)分别双酶切PD1和ELP(V)90质粒，然后将酶切后的片段通过T4连接酶4℃过夜连接。将连接后的重组子转化至Top10菌株中进行扩增，经卡那霉素抗性平板筛选出阳性重组子，对其进行DNA测序验证。质粒置于-80℃长期保存，将测序正确的菌株与20％甘油1:1保存于-80℃。
经扩增后的质粒转化至BL21(DE3)PLysS表达感受态细胞中，经卡那霉素抗性平板筛选出阳性重组子， 挑选4-8个单克隆菌接种至150mL LB培养基37℃中培养12-16小时，然后将培养后的菌液转至TB大肠杆菌培养基中培养6-8小时后，在25℃下经IPTG诱导16小时，收集菌液。将离心收集大肠杆菌菌体使用40mL10mM磷酸盐缓冲溶液重悬后用超声细胞破碎仪裂解菌体，超声功率为300w，时间为45min，超声模式选择超声运行5s、暂停5s。超声后破碎的菌体在4℃、14000rpm离心15分钟后，弃去沉淀；之后加入2mL10％(w/w)聚乙烯亚胺(Polyethyleneimine,PEI)，并在4℃、14000rpm下再次离心15分钟，以去除核酸。收集上清液，通过可逆相变ITC技术获得目标蛋白。具体方法如下：
在蛋白上清液中加入终浓度为3M的NaCl，并在37℃水浴中孵育使之充分溶解，之后用16000rpm离心20分钟(37℃)，收集沉淀；沉淀加入12mL预冷的10mM PBS充分溶解沉淀，之后4℃、16000rpm离心20分钟以收集上清液。以上步骤即为1次ITC循环。通过3次ITC循环获得目标蛋白，其中，NaCl的浓度分别是3M、2M、1M，加入预冷10mM PBS的体积分别为12mL、8mL、6mL。将制备好的蛋白储存在-80℃冰箱中备用。
实施例4测定PD1和ELP(V)90-PD1蛋白物理化学表征参数
将实施例2、3中制备的高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白用聚丙烯酰胺凝胶电泳(SDS-PAGE)分析目的蛋白的表达情况和纯度(图1)，结合超高液相质谱联用仪确定所制备蛋白的确切分子量(图2)。
在初步确定成功制备出蛋白后，用圆二色谱仪对两种蛋白的二级结构进行分析，以验证融合了ELP(V)90的蛋白是否对PD1蛋白的二级结构产生影响。结果如图3所示，结果表明融合了ELP(V)90蛋白的PD1蛋白的二级结构未发生改变，提示PD1蛋白的功能可能不会受ELP(V)90蛋白的影响。
用酶标仪测定OD350nm处的吸光度值来测定不同浓度下ELP(V)90-PD1的相转变温度，探索ELP(V)90-PD1在体内生理环境下的存在形态。结果如图4所示，结果表明随着温度的增加，ELP(V)90-PD1蛋白的OD350值在达到临界温度后有一个突跃，随后进入平台期，且随着浓度的增加，ELP(V)90-PD1的相转变温度逐渐降低，这提示生理温度下，一定浓度的ELP(V)90-PD蛋白可能在体内发生相转变而成凝聚态，从而缓释PD1药物。
通过光动态散射粒度仪(DLS)来分析不同时间点蛋白PD1及融合蛋白ELP(V)90-PD1在溶液状态下的水合动力学半径大小，初步确定融合后的蛋白是否能够经过肾小球的滤过而延长半衰期。结果如图5所示，结果表明，融合蛋白的水合动力学半径与PD1蛋白相比，增加约4倍，高于肾脏清除的最小直径10nm，但远低于网状内皮系统的最大截止尺寸200nm，这说明融合蛋白可降低肾清除率，且能渗透进组织细胞中，从而延长半衰期，增加肿瘤细胞的摄取。
实施例5 ELP(V)90-PD1融合蛋白的体外竞争性结合活性分析
根据已有专利文献显示，高亲和力PD1蛋白不仅对人的PDL1受体有结合活性，而且对小鼠的PDL1受体也有结合活性，取对数生长期的小鼠结肠癌CT26细胞，调整细胞密度为1×105个/mL，2mL/孔铺激光共聚焦小皿，37℃，5％CO2培养箱中培养，孵育12小时，加入鼠源IFNγ以诱导细胞表面的PDL1受体上调，诱导12小时后，加入Cy5标记的ELP(V)90-PD1或高亲和力PD1蛋白，37℃，5％CO2培养箱中孵育6小时后，用10mM磷酸盐缓冲液洗去未结合的标记蛋白，之后用4％多聚甲醛固定细胞15分钟后，用10mM磷酸盐缓冲液洗细胞三遍，将细胞用5％牛血清白蛋白(BSA)封闭1小时后，用10mM磷酸盐缓冲液洗细胞三遍，加入羊抗兔PDL1一抗，室温下孵育细胞1小时，用10mM磷酸盐缓冲液洗细胞三遍，随后加入FITC标记的二抗，室温下孵育细胞2小时，用10mM磷酸盐缓冲液洗细胞三遍后加入1mL 10mM磷酸盐缓冲液，用激光共聚焦显微成像仪LSM900进行成像分析，结果见图6。图6的竞争性结合活性结果显示，与PD1组相比，虽然融合蛋白ELP(V)90-PD1因融合了ELP(V)90蛋白，结合PDL1受体的活性下降约45％，但ELP(V)90-PD1蛋白仍有很强的结合PDL1受体的活性。
用流式细胞术定量分析ELP(V)90-PD1或高亲和力PD1蛋白与CT26细胞表面的PDL1竞争性结合受体情况，取对数生长期的小鼠结肠癌CT26细胞，调整细胞密度为2×105个/mL，4mL/孔铺6孔板，37℃，5％CO2 培养箱中培养，孵育12小时，加入鼠源IFNγ以诱导细胞表面的PDL1受体上调，诱导12小时后，加入Cy5标记的ELP(V)90-PD1或PD1蛋白，37℃，5％CO2培养箱中孵育6小时后，用10mM磷酸盐缓冲液洗去未结合的标记蛋白，细胞用PE标记的PDL1一抗室温下孵育20分钟，洗去未结合的抗体后，用流式细胞仪进行分析。结果见图7，结果表明与PD1+anti-PDL1组相比，虽然ELP(V)90-PD1蛋白与PDL1受体结合活性降低约22％，但仍然具有与PDL1竞争结合的活性。
在初步验证通过原核体系制备的高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白具有一定的生物活性的基础上，接下来对蛋白在体内个体水平进行表征。
实施例6 ELP(V)90-PD1融合蛋白小鼠体内最大耐受剂量测定
本实施例对实施例3中制备得到的ELP(V)90-PD1融合蛋白在野生BALB/c小鼠中进行最大耐受剂量测试。6-8周龄的BALB/c雌性小鼠购自北京大学医学部动物实验科学中心，对动物的所有操作均按照北京大学医学部动物伦理委员会的指导原则进行操作。雌性的BALB/c小鼠经腹腔注射剂量分别为4.6、8.5和12.6mg/kg体重的高亲和力PD1蛋白和28.0、32.0和48.0mg/kg的ELP(V)90-PD1融合蛋白(每组三只小鼠)，监测每组每只体重变化及各组小鼠的生活状态并记录。以最高剂量下体重下降不超过10％的剂量为最大耐受剂量。每个剂量组每只小鼠每天的体重变化情况如图8所示。
结果显示，未融合的高亲和力PD1蛋白的最大耐受剂量测得为4.6mg/kg，而ELP(V)90-PD1融合蛋白的最大耐受剂量为28mg/kg，这说明融合后的蛋白在体内的毒性显著降低，充分说明了融合蛋白可以降低PD1蛋白的毒性。
实施例7 ELP(V)90-PD1融合蛋白体内药代动力学测试
本实施例对实施例3中制备得到的ELP(V)90-PD1融合蛋白的药物代谢动力学进行测试。以体重为200±10g的Sprague-Dawley(SD，购自北京维通利华实验动物技术有限公司)大鼠为实验对象，经腹腔注射最大耐受剂量下的Cy5标记的高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白，分别于注射后的0,0.5,1,2,8,24,72,120,168,240,288,336,408和480小时通过眼眶内眦静脉丛取血。肝素血样经4℃，3500转离心5分钟，通过测定上层血浆的荧光强度进行药代变化测定，根据将Cy5标记PD1蛋白和ELP(V)90-PD1与空白组血浆混合测定的荧光强度曲线计算出血样中各组药物的浓度，用药物分析系统3.0软件计算各组的药代参数。
ELP(V)90-PD1药时曲线及药代参数见图9和表1。用DAS软件中的房室模型分析PD1和ELP(V)90-PD1的药代动力学参数，PD1的半衰期为20.9±0.9小时，延长到了ELP(V)90-PD1的551.5±12.8小时，ELP(V)90-PD1的药时曲线下面积为74275.5±113.2mg/L·h，是PD1(3515.8±8.1mg/L·h)的21.1倍，并且ELP(V)90-PD1的药物达峰浓度(227.7±2.7mg/L)是PD1(88.6±2.7mg/L)的2.7倍，ELP(V)90-PD1的血浆清除率(0.04±0.09mL/h)比PD1(0.5±0.1mL/h)慢25倍，ELP(V)90-PD1的平均驻留时间(203.3±2.5h)是PD1(30.4±1.4h)的6.68倍，这说明由于融合后的蛋白的尺寸与未融合前相比变大，使得肾脏的清除速率减慢，药物半衰期显著延长，生物利用度显著增加。
表1 ELP(V)90-PD1和PD1蛋白在野生BALB/c小鼠体内的药物代谢动力学参数
实施例8 ELP(V)90-PD1融合蛋白体内缓释评估
药物的缓释使得药物在较长时间持续释放药物，进而提高药物的局部治疗效果，本实施例对ELP(V)90-PD1融合蛋白在小鼠体内的缓释情况进行评估。
将Cy7标记的高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白以最大耐受剂量(分别为4.6mg/kg和28.0mg/kg)腹腔注射进小鼠体内(每组三只)，分别于注射后的2h,1d,3d,7d,10d,15d,18d用异氟烷进行麻醉后，用Spectrum成像系统成像分析，结果见图10。图10结果显示，未融合ELP的PD1蛋白在3天内药物全部代谢完，而ELP(V)90-PD1融合蛋白在18天时仍有部分药物未被代谢，ELP(V)90-PD1融合蛋白在腹腔粘膜处形成药物贮库而缓慢释放药物，这与药物代谢动力学的结果一致。
实施例9 ELP(V)90-PD1融合蛋白的在体内各主要组织脏器的分布情况
本实施例对实施例3中得到的ELP(V)90-PD1融合蛋白进行组织分布的考察。利用接种了结肠癌细胞的BALB/c小鼠一次腹腔注射最大耐受剂量荧光标记的高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白，分别于给药2h，1d,3d，7d,15d后测定各主要组织脏器中剩余的PD1的相对荧光强度，以此来反映各组织脏器中的药物分布情况，结果见图11，图12。通过检测各脏器中的药物的相对荧光强度发现，在2h时，PD1和ELP(V)90-PD1分别在肝脏、肾脏、脾脏、肺及肿瘤中广泛分布，但在心脏中的分布较其他组织脏器中含量低，且两小时时，ELP(V)90-PD1在肿瘤组织中的含量相比PD1较低，这说明了药物缓释起效慢的特点，但24小时后，ELP(V)90-PD1在肿瘤组织中的分布达到最大，与PD1近似，在第3天检测时，ELP(V)90-PD1蛋白在肿瘤组织中的含量相比PD1更高，且在其它组织脏器中的含量低于肿瘤组织中的含量，这提示ELP(V)90-PD1能够靶向肿瘤组织，且能够渗透进肿瘤组织内，并且随着时间的延长，PD1蛋白在第7天，15天时各组织脏器中均检测不到信号，而ELP(V)90-PD1在肿瘤组织中仍然有较高的信号强度，再次证明了ELP(V)90-PD1的缓释作用及肿瘤靶向富集作用。
实施例10 ELP(V)90-PD1融合蛋白的在肿瘤组织内的渗透性能力
本实施例分析实施例3中制备的ELP(V)90-PD1融合蛋白在肿瘤组织中的渗透能力，具体如下：
用接种了CT26结肠癌细胞的BALB/c小鼠测定ELP(V)90-PD1融合蛋白在肿瘤组织中的渗透能力。高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白用Cy5标记。
将1×106/mL的CT26细胞100μl皮下接种在BALB/c小鼠背部右后腿侧，待肿瘤长至50mm3大小时，以最大耐受剂量将Cy5标记高亲和力PD1蛋白和ELP(V)90-PD1蛋白腹腔注射进荷瘤小鼠体内，分别于给药后的24h,3d,7d取肿瘤组织，制备冷冻切片。用CD31一抗标记肿瘤血管，用FITC标记的IgG二抗检测一抗，最后用DAPI染细胞核后封片。用LSM880激光共聚焦显微镜呈现观察各组肿瘤组织的荧光强度。成像结果如图13所示。结果发现，PD1和ELP(V)90-PD1在24h均有一定程度的渗透，且与PD1组相比，在距离肿瘤血管较远处，ELP(V)90-PD1仍有药物聚集，这说明ELP(V)90-PD1可以渗透进肿瘤组织内部，且第三天时，ELP(V)90-PD1渗透至肿瘤血管更远处，第七天时渗透至比第三天更远处，这充分说明了融合ELP蛋白后，虽然分子量有所增加，但并不影响PD1渗透至肿瘤组织内部。
实施例11 ELP(V)90-PD1融合蛋白的体内抗肿瘤生物活性检测
本实施例中采用荷瘤结肠癌细胞的BALB/c小鼠皮下肿瘤模型来评价ELP(V)90-PD1融合蛋白的体内抗肿瘤活性。
将1×106/mL的CT26细胞100μl皮下接种在BALB/c小鼠背部右后腿侧，待肿瘤长至平均50mm3大小时，以最大耐受剂量将高亲和力PD1蛋白和ELP(V)90-PD1融合蛋白腹腔注射进荷瘤小鼠体内，每天观察小鼠生存状态及肿瘤生长状况，动态检测小鼠体重及肿瘤大小，并作记录。治疗结束后，统计生存率，每组每只小鼠肿瘤体积长至1500mm3或体重下降超过10％时，视为小鼠死亡，记录为“1”，肿瘤未达标小鼠视为小鼠存活，记录为“0”。各组小鼠的生存曲线及生存率如图14，图15所示。
图14，15显示，一次最大耐受剂量给药后，虽然PD1蛋白对于治疗结肠癌肿瘤有一定的作用，但ELP(V)90-PD1组对小鼠结肠癌模型的治疗效果显著优于PD1组，这主要是由于一次最大耐受剂量给药，ELP(V)90-PD1 组可以给予更高剂量的药物，且ELP的缓释效应使得PD1的释放始终处于一个较稳定的血药浓度，充分激起体内免疫，使得肿瘤被很好地抑制，药效后的生存曲线也可以看出，经ELP(V)90-PD1蛋白治疗后，ELP(V)90-PD1组小鼠的整体生存时间显著延长，中位生存天数为28天，分别是PD1和PBS治疗组的1.5倍(19天)和1.9倍(15天)，再次证明了ELP(V)90-PD1的长效作用。
实施例12 ELP(V)90-PD1融合蛋白联合一线化疗药物奥沙利铂后的体内抗肿瘤活性
将1×106/mL的CT26细胞100μl皮下接种在BALB/c小鼠背部右后腿侧，待肿瘤长至平均50mm3大小时，将90只肿瘤大小一致的小鼠平均分为6组，每组15只小鼠，分别为PBS组，奥沙利铂(OX)组，PD1组，ELP(V)90-PD1组，PD1+OX组，ELP(V)90-PD1+OX组，以最大耐受剂量将PD1蛋白和ELP(V)90-PD1蛋白腹腔注射进PD1组，ELP(V)90-PD1组，PD1+OX组，ELP(V)90-PD1+OX组荷瘤小鼠体内，以2mg/kg剂量的OX给予OX组荷瘤小鼠，在第五天时给予PD1+OX组，ELP(V)90-PD1+OX组2mg/kg剂量的OX，每天观察小鼠生存状态及肿瘤生长状况，动态检测每组每只小鼠体重及肿瘤大小，并作记录。
药效实验结束后，通过摘眼球取各组每只小鼠的血液，取100μl新鲜血液用于血细胞分析，取500μl血液离心后用于血生化分析，然后取各组小鼠的肿瘤组织，心脏，肝脏，脾脏，肾脏以及肺等主要组织，部分用组织固定液固定用以HE染色，部分浸入无菌10mM PBS中用以检测免疫相关指标。药效图如图16所示，肿瘤组织的HE染色如图17所示，各组肿瘤大小比对如图18所示，各组主要组织脏器的HE染色图如图19所示，各组部分小鼠的血细胞及血生化分析分别如图20和图21所示。
图16结果显示，对于CT26结肠癌肿瘤模型，ELP(V)90-PD1+OX组的治疗效果显著优于其他给药组，且有85％的小鼠肿瘤消失，被治愈，均未见复发。ELP(V)90-PD1组有14％的小鼠肿瘤消失，被治愈。与PBS组相比，OX组，PD1组及PD1+OX组小鼠的肿瘤显著被抑制，未有小鼠肿瘤消失。并且，ELP(V)90-PD1组的治疗效果明显优于PD1+OX联合给药组，再次证明了融合蛋白缓释长效的优势。
图17结果显示，经过给药治疗，肿瘤细胞的数量ELP(V)90-PD1+OX组<ELP(V)90-PD1组<PD1+OX组<PD1<OX组<PBS组，这与药效的结果一致，说明了融合蛋白联合给药后，产生协同作用，共同发挥抗肿瘤治疗作用。在图18所摘取的肿瘤大小比较图中也可以很明显看出各组的药效。图19的结果显示，所用蛋白药物及联合给药后各组织脏器均未发生明显的损伤，说明了给药的安全性，同时结合图20和图21结果，各组给药小鼠各种血细胞均未发现有异常，血生化指标结果也显示出没有明显的异常，这再次说明了给药的安全性。
综上所述，本发明创新性地提出了一种温度响应性的高亲和力PD1融合蛋白，结合抗结肠癌一线治疗药物奥沙利铂，大大提高了抗肿瘤药效，同时高亲和力PD1的生物半衰期显著延长，一次最大耐受剂量下给药，可持续20天释药，这是目前非ELP融合蛋白递送系统所达到的最长循环时间，同时一次给药即可达到最佳治疗效果，且减少给药次数，提高了病人顺应性。融合蛋白也能有效渗透进肿瘤组织内部，与肿瘤细胞表面的PDL1受体结合，发挥抗肿瘤免疫效应。
最后应说明的是：以上实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
工业实用性
本发明提供一种高亲和力PD1蛋白偶联物及其应用。本发明提供的高亲和力PD1蛋白偶联物包含类弹性蛋白多肽以及与类弹性蛋白多肽连接的高亲和力PD1蛋白。该偶联物显著提高了高亲和力PD1蛋白的生物半衰期和生物利用度，具有肿瘤靶向性和渗透性以及肿瘤微环境温度响应性，通过解除机体免疫抑制，激活机体自身免疫，可以有效抑制肿瘤生长，提高抗肿瘤治疗效果。高亲和力PD1蛋白偶联物具有合成简单，合成过程可控，生产简便等优势，更易于大规模生产和商业化应用，在肿瘤治疗中具有重要意义，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种高亲和力PD1蛋白偶联物，其特征在于，所述高亲和力PD1蛋白偶联物包含类弹性蛋白多肽以及与类弹性蛋白多肽连接的高亲和力PD1蛋白。
  2. 根据权利要求1所述的高亲和力PD1蛋白偶联物，其特征在于，所述类弹性蛋白多肽与所述高亲和力PD1蛋白直接连接或通过连接肽连接；
    优选地，所述类弹性蛋白多肽的C末端与所述高亲和力PD1蛋白的N末端连接。
  3. 根据权利要求1或2所述的高亲和力PD1蛋白偶联物，其特征在于，所述高亲和力PD1蛋白相较于野生型PD1蛋白，对PDL1的亲和力增强，且能够竞争性结合PDL1；
    优选地，所述高亲和力PD1蛋白缺乏PD1跨膜域，并且相对于野生型PD1蛋白的膜外区包含一或多个氨基酸突变。
  4. 根据权利要求3所述的高亲和力PD1蛋白偶联物，其特征在于，所述高亲和力PD1蛋白为以下1)或2)中的任一种：
    1)氨基酸序列如SEQ ID NO.3所示；
    2)氨基酸序列为在SEQ ID NO.3所示序列的N端或C端添加选自蛋白标签、酶切位点、连接肽中的一种或多种后得到的序列；
    优选地，所述高亲和力PD1蛋白的氨基酸序列如SEQ ID NO.3或4所示。
  5. 根据权利要求1～4任一项所述的高亲和力PD1蛋白偶联物，其特征在于，所述类弹性蛋白多肽的氨基酸序列包含(X-Gly-X-Pro-Gly)n，其中，10≤n≤200，X是除脯氨酸以外的任一天然氨基酸。
  6. 根据权利要求5所述的高亲和力PD1蛋白偶联物，其特征在于，X为选自缬氨酸、苯丙氨酸、色氨酸、酪氨酸、丙氨酸、甘氨酸、甲硫氨酸、苏氨酸、丝氨酸、亮氨酸、异亮氨酸中的一种；所述类弹性蛋白多肽的响应温度为10-60℃；
    优选地，X为缬氨酸，30≤n≤150，所述类弹性蛋白多肽的响应温度为18-40℃；
    更优选地，所述高亲和力PD1蛋白偶联物的水合动力学半径为5-15nm。
  7. 核酸分子，其特征在于，所述核酸分子编码权利要求1～6任一项所述的高亲和力PD1蛋白偶联物。
  8. 生物材料，其特征在于，所述生物材料包含权利要求7所述的核酸分子或表达权利要求1～6任一项所述的高亲和力PD1蛋白偶联物；所述生物材料为表达盒、载体或宿主细胞。
  9. 权利要求1～6任一项所述的高亲和力PD1蛋白偶联物或权利要求7所述的核酸分子或权利要求8所述的生物材料在制备药物中的应用；
    优选地，所述药物为用于预防或治疗肿瘤的药物；
    更优选地，所述药物为用于预防或治疗结肠癌的药物。
  10. 一种药物组合物，其特征在于，所述药物组合物包含权利要求1～6任一项所述的高亲和力PD1蛋白偶联物；
    优选地，所述药物组合物还包含选自化学治疗药物、靶向治疗药物、内分泌治疗药物及免疫治疗药物中的一种或多种；
    更优选地，所述药物组合物包含权利要求1～6任一项所述的高亲和力PD1蛋白偶联物以及奥沙利铂。
PCT/CN2023/104977 2022-09-27 2023-06-30 一种高亲和力pd1蛋白偶联物及其应用 WO2024066616A1 (zh)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101500606A (zh) * 2005-06-24 2009-08-05 杜克大学 基于热反应生物聚合物的直接药物送递系统
CN103230598A (zh) * 2006-09-06 2013-08-07 费斯生物制药公司 融合肽治疗组合物
CN107108707A (zh) * 2014-08-08 2017-08-29 小利兰斯坦福大学理事会 高亲和力pd‑1药剂以及使用方法
CN110101868A (zh) * 2019-05-24 2019-08-09 北京大学 一种环境刺激响应性蛋白质高分子偶联物自组装体及其制备方法与应用
WO2022187488A2 (en) * 2021-03-03 2022-09-09 Shattuck Labs, Inc. Mutant pd-1 extracellular domains

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101500606A (zh) * 2005-06-24 2009-08-05 杜克大学 基于热反应生物聚合物的直接药物送递系统
CN103230598A (zh) * 2006-09-06 2013-08-07 费斯生物制药公司 融合肽治疗组合物
CN107108707A (zh) * 2014-08-08 2017-08-29 小利兰斯坦福大学理事会 高亲和力pd‑1药剂以及使用方法
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