CN117777303A - A high-affinity PD1 protein conjugate and its application - Google Patents

Abstract

The invention relates to the technical field of biological medicine, in particular to a high-affinity PD1 protein conjugate and application thereof. The high-affinity PD1 protein conjugate provided by the invention comprises an elastin-like polypeptide and a high-affinity PD1 protein connected with the elastin-like polypeptide. The conjugate remarkably improves the biological half-life and bioavailability of the high-affinity PD1 protein, has tumor targeting property, permeability and tumor microenvironment temperature responsiveness, and can effectively inhibit tumor growth and improve anti-tumor treatment effect by relieving body immunity inhibition and activating body autoimmunity. The high-affinity PD1 protein conjugate has the advantages of simple synthesis, controllable synthesis process, simple and convenient production and the like, is easier to realize large-scale production and commercial application, and has important significance in tumor treatment.

Description

A high-affinity PD1 protein conjugate and its application

Technical Field

The present invention relates to the field of biomedicine technology, and in particular to a high-affinity PD1 protein conjugate and its application.

Background technique

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. The interaction between PD1 and its ligands plays a key immunoregulatory role in the activation and tolerance of T lymphocytes. Because 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. So far, monoclonal antibodies targeting PD1 and PDL1, such as Keytruda (pembrolizumab), Opdivo (Odivo monoclonal antibody), and Atezolizumab (atezolizumab), have made great breakthroughs in clinical practice.

Although the existing antibody technology targeting PD1/PDL1 has made breakthrough progress in clinical practice, antibodies as therapeutic methods have inherent shortcomings. For example, due to their large size, it is difficult to enter solid tumors to antagonize deep tumors. of the PD1:PDL1 signaling pathway, resulting in poor efficacy. Furthermore, Fc-mediated cytotoxic immune responses, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), of antibodies against PD1 and PDL1 may be undesirably depleted It is intended to activate anti-tumor cytotoxic T cells. This is mainly due to the fact that both PD1 and PDL1 are expressed on the surface of anti-tumor cytotoxic T cells. Therefore, the development of antibody replacement therapies with better clinical effects is also a research hotspot in the field of tumor immunity.

Summary of the invention

The first object of the present invention is to provide a high-affinity PD1 protein conjugate and its related nucleic acid molecules and biological materials.

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 a high-affinity PD1 protein conjugate.

Specifically, the present invention provides the following technical solutions:

In a first aspect, the present invention provides a high-affinity PD1 protein conjugate, which contains an elastin-like polypeptide and a high-affinity PD1 protein connected to the elastin-like polypeptide.

In the above-mentioned high-affinity PD1 protein conjugate, the elastin-like polypeptide is directly connected to the high-affinity PD1 protein or connected through a connecting peptide.

The above-mentioned connecting peptide can be a polypeptide rich in glycine and serine.

Preferably, in the high-affinity PD1 protein conjugate, the elastin-like polypeptide is directly linked to the high-affinity PD1 protein.

Further preferably, the C-terminus of the elastin-like polypeptide is linked to the N-terminus of the high-affinity PD1 protein.

In the prior art, when coupling elastin-like polypeptides, 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.

Compared with wild-type PD1 protein, the high-affinity PD1 protein of the present invention has enhanced affinity for PDL1 and can competitively bind to PDL1.

The above-mentioned high-affinity PD1 protein is a high-affinity variant of the wild-type PD1 protein. Compared with the wild-type PD1 protein, it contains mutations that can increase the affinity of the wild-type PD1 protein for PDL1.

Preferably, the high-affinity PD1 protein lacks the PD1 transmembrane domain and contains 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.

Among the currently known high-affinity PD1 proteins, the present invention found that the high-affinity PD1 protein with the amino acid sequence shown in SEQ ID NO. 3 can better cooperate with the elastin-like polypeptide, and is more conducive to improving the conjugate's expression and antitumor activity.

Preferably, the high-affinity PD1 protein is any one of the following 1) or 2):

1) The amino acid sequence is shown in SEQ ID NO.3;

2) The amino acid sequence is a sequence obtained by adding one or more selected from the group consisting of protein tags, enzyme cleavage sites, and linking peptides to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.3.

The high-affinity PD1 protein with an amino acid sequence as shown in SEQ ID NO.3 comes from a mutant of the extracellular region of the natural PD1 protein, has a molecular weight of 14 kD, and has an affinity for PDL1 that is approximately 20,000 times greater than that of the wild-type PD1.

Those skilled in the art know that adding a protein tag, enzyme cleavage site, or connecting peptide to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.3 will usually not affect the structure and function of SEQ ID NO.3.

The above-mentioned protein tags 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, transpeptidase Sortase, etc.

The connecting peptide is a short peptide rich in glycine and serine, such as GSGGGGS and the like.

In some embodiments of the present invention, the amino acid sequence of the high-affinity PD1 protein is shown in 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.

In view of the above-mentioned high-affinity PD1 protein, 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.

The amino acid sequence of the above-mentioned elastin-like polypeptide includes (X-Gly-X-Pro-Gly)n, where 10≤n≤200, and X is any natural amino acid except proline.

Specifically, X is selected from valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine, threonine, serine, leucine, and isoleucine A kind of; the response temperature of the elastin-like polypeptide is 10-60°C.

Preferably, X is valine, 30≤n≤150, and the response temperature of the elastin-like polypeptide is 18-40°C.

In some embodiments of the present invention, the amino acid sequence of the elastin-like polypeptide is (VGVPG)n, where 60≤n≤120, preferably 80≤n≤100.

In some embodiments of the present invention, the amino acid sequence of the elastin-like polypeptide is shown as SEQ ID NO.5.

Preferably, 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 has a smaller volume and shorter circulation half-life and requires daily injections to maintain therapeutically effective blood levels. However, frequent administration may lead to poor patient compliance and severe 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 invention has the following characteristics and functions:

1) Temperature responsiveness;

2) Sustained release;

3) Tumor targeting;

4) Tumor tissue permeability;

5) Activate and enhance the body’s own immunity;

6) Anti-tumor.

In a second aspect, the present invention provides nucleic acid molecules encoding the high-affinity PD1 protein conjugates described above.

The nucleic acid molecules include DNA and RNA.

Based on the amino acid sequence of the high-affinity PD1 protein conjugate described above, those skilled in the art can obtain the 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-mentioned nucleic acid molecule is not unique, and all nucleic acid molecules capable of encoding the above-mentioned heavy chain and light chain are within the protection scope of the present invention.

In some embodiments of the present invention, the nucleotide sequence of the nucleic acid molecule encoding the high-affinity PD1 protein is shown in SEQ ID NO. 1 or 2.

In a third aspect, the present invention provides biological materials comprising the nucleic acid molecules described above or expressing the high-affinity PD1 protein conjugates described above; the biological materials are expression cassettes, vectors or host cells.

Wherein, the expression cassette containing the nucleic acid molecule can be obtained by operably connecting a promoter and the nucleic acid molecule.

Depending on the expression needs and the upstream and downstream sequences of the expression cassette, the expression cassette may also contain other transcription and translation regulatory elements such as terminators and enhancers.

Vectors containing the nucleic acid molecules include but are not limited to plasmid vectors, phage vectors, viral vectors, etc., where plasmid vectors include replicating vectors and non-replicating vectors.

The above-mentioned host cells include microbial cells or animal cells, wherein the microorganisms include prokaryotic microorganisms (such as Escherichia coli) and eukaryotic microorganisms (such as yeast).

In a fourth aspect, the present invention provides a method for preparing the high-affinity PD1 protein conjugate described above, the method comprising:

1) Insert the nucleic acid molecule encoding the high-affinity PD1 protein conjugate into the expression plasmid to obtain a recombinant expression plasmid;

2) introducing the recombinant expression plasmid of step 1) into a microorganism to obtain a recombinant microorganism;

3) 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.

In some embodiments of the invention, the plasmid is selected from the PET family, preferably PET-25b+.

In some embodiments of the invention, the microorganism is Escherichia coli, and the Escherichia coli is selected from the BL21 series, preferably BL21(DE3)PLySs.

In some embodiments of the present invention, the reversible phase transition (ITC) purification is 3 times reversible phase transition (ITC) purification.

In a fifth aspect, 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.

Preferably, 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.

Preferably, the drug is a drug for preventing or treating colorectal cancer

Further preferably, 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, extend the survival period, and exert long-lasting anti-tumor effects in the body.

The above-mentioned high-affinity PD1 protein conjugate can be administered by injection. The drug may be an injectable preparation.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising the high-affinity PD1 protein conjugate described above.

Preferably, the pharmaceutical composition further comprises one or more selected from chemotherapeutic drugs, targeted therapeutic drugs, endocrine therapeutic drugs and immunotherapeutic drugs.

The active ingredient of the above-mentioned pharmaceutical composition may only be the high-affinity PD1 protein conjugate, or may further include active ingredients such as chemotherapy drugs, targeted therapy drugs, endocrine therapy drugs, and immunotherapy drugs.

Among them, chemotherapy drugs include paclitaxel, docetaxel, gemcitabine, vinorelbine, cisplatin, carboplatin, oxaliplatin, etoposide, etoposide, doxorubicin, and platinum oxalate; 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 against tumor angiogenesis, such as bevacizumab Tizumab, 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.

In some embodiments of the invention, the pharmaceutical composition includes the high-affinity PD1 protein conjugate described above and oxaliplatin.

The present invention finds that the above-mentioned high-affinity PD1 protein conjugate and oxaliplatin can play a synergistic effect in the treatment of colon cancer, significantly improve the therapeutic effect of colon cancer, and have high safety.

The pharmaceutical compositions described above can be administered by injection or other methods. Depending on the mode of administration, the above pharmaceutical composition may contain excipients permitted in the pharmaceutical field.

The beneficial effects of the present invention include at least the following points:

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 in which a high-affinity PD1 protein conjugate is used in combination with other chemotherapeutic drugs. The pharmaceutical composition effectively improves the efficiency of existing anti-tumor immunotherapy and is of great significance in tumor treatment.

Description of the drawings

In order to explain the present invention or the technical solutions in the prior art more clearly, the drawings needed to be used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are of the present invention. For some embodiments of the invention, those of ordinary skill in the art can also obtain other drawings based on these drawings without exerting creative efforts.

Figure 1 shows the SDS-PAGE electrophoresis pattern 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.

Figure 3 shows the circular dichroism spectrum of the protein prepared in Example 4 of the present invention.

Figure 4 shows the phase transition temperature of the protein ELP(V)90-PD1 prepared in Example 4 of the present invention.

Figure 5 shows the particle size of the proteins ELP(V)90-PD1 and PD1 prepared in Example 4 of the present invention.

Figure 6 shows the qualitative results of laser confocal microscopy imaging of ELP(V)90-PD1 and PD1 competing with PDL1 antibodies for binding to PDL1 receptors in Example 5 of the present invention, in which the scale bars are all 10 μm.

Figure 7 shows the flow cytometry quantitative results of competitive binding of ELP(V)90-PD1 and PD1 with PDL1 antibodies to the PDL1 receptor in Example 5 of the present invention.

Figure 8 shows the maximum tolerated dose determination of ELP(V)90-PD1 and PD1 in wild BALB/c mice in Example 6 of the present invention.

Figure 9 shows the in vivo pharmacokinetic changes of ELP(V)90-PD1 and PD1 protein in wild BALB/c mice in Example 7 of the present invention.

Figure 10 shows the sustained release results of ELP(V)90-PD1 in wild BALB/c mice in Example 8 of the present invention.

Figures 11 and 12 show the qualitative and quantitative results of drug distribution of ELP(V)90-PD1 and PD1 protein in the main tissues, organs and tumor tissues of BALB/c mice bearing colon cancer tumors in Example 9 of the present invention. .

Figure 13 shows the investigation of the permeability of ELP(V)90-PD1 and PD1 protein in tumors of BALB/c mice bearing colon cancer tumors in Example 10 of the present invention.

Figure 14 shows the efficacy results of ELP(V)90-PD1 and PD1 protein in BALB/c mice bearing colon cancer tumors in Example 11 of the present invention.

Figure 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 protein in Example 11 of the present invention.

Figure 16 shows the tumor inhibition results of combined OX administration on BALB/c mice bearing colon cancer tumors in Example 12 of the present invention.

Figure 17 shows the HE staining results of tumor sections of BALB/c mice bearing colon cancer tumors that were inhibited by combined administration of OX in Example 12 of the present invention.

Figure 18 shows the statistical comparison results of tumor size in each group in the tumor inhibition of BALB/c mice bearing colon cancer tumors by combined administration of OX in Example 12 of the present invention.

Figure 19 shows the HE staining results of damage to major tissues and organs in BALB/c mice bearing colon cancer tumors when combined with OX administration in Example 12 of the present invention.

Figure 20 shows the analysis results of blood cells in the blood of each group of mice after combined administration of OX in Example 12 of the present invention on BALB/c mice bearing colon cancer tumors.

Figure 21 shows the blood biochemical analysis results of the blood of mice in each group after combined administration of OX in Example 12 of the present invention on BALB/c mice bearing colon cancer tumors.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present invention more clear, the technical solutions in the present invention will be clearly and completely described below in conjunction with the accompanying drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention. , not all examples. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without making creative efforts fall within the scope of protection of the present invention.

Example 1 Prokaryotic preparation of high-affinity PD1 mimetic polypeptide

The gene sequence of the high-affinity PD1 mimetic polypeptide (high-affinity PD1 protein) is a mutant of the wild-type PD1 protein gene sequence and is used to block the interaction between the native protein and its ligand PDL1 in in vivo and in vitro methods. Although the high-affinity PD1 mimetic peptide lacks the transmembrane domain of the native protein, its affinity with the receptor PDL1 is greatly increased. In this example, the gene sequence of the high-affinity PD1 protein used in the fusion protein is as follows:

SEQ ID NO.1:

ATGGATTCCCCAGATAGACCATGGAACCCACCAACTTTCTCCCCAGCTTTGTTGGTCGTCACTGAAGGTGATAACGCTACTTTCACTTGTTCCTTCTCCAACACTTCCGAATCCTTCCATGTTGTTTGGCATCGTGAATCCCCATCCGGTCAAACTGATACATTGGCTGCTTTCCAGAAGATAGATCCCAACCAGGTCAAGATGCTAGATTCAGAGTTACTCAATTGCCAAACGGTAGAGATTTCCACATGTCCGTCGTCA GAGCTAGAAGAAACGATTCCGGTACTTATGTTTGGTGGTGTTATTTCCCTTGCTCCAAAGATTCAAATTAAGGAATCCTTGAGAGCTGAATTGAGAGTCACTGAAAGAGGATCC.

In order to facilitate later purification, the gene encoded by the GGGGGGSLPETGGHHHHHH 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 tag protein is:

SEQ ID NO.2:

ATGGACAGCCCGGATCGTCCGTGGAATCCGCCTACGTTCTCCGGCACTGCTGGTCGTGACCGAGGGTGATAACGCAACTTTTACCTGTTCTTTTTCTAACACCAGCGAGAGCTTCCATGTTGTATGGCACCGTGAATCTCCATCCGGTCAAACCGACACTCTGGCGGCCTTCCCGGAAGATCGTTCTCAGCCAGGCCAGGACGCGTTTCCGTGTGACCCAGCTGCCGAACGGCCGCGATTTCCACATGAGCG TAGTTCGTGCTCGTCGCAACGACTCCGGTACCTACGTTTGCGGCGTTATCCCTGGCGCCGAAGATTCAGATCAAAGAATCCCTGCGCGCTGAACTGCGCGTGACTGAACGTGGATCCGGTGGCGGTGGCTCTCTGCCGGAAACCGGTGGCCACCATCATCATCATCAT.

The sequence of the high-affinity PD1 protein encoded by the above genes is:

SEQ ID NO.3:

MDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTER.

The protein sequence containing the purification tag and cutting enzyme Sortase is:

SEQ ID NO.4：

MDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFHVVWHRESPSGQTDTLAAFPEDRSQPGQDARFRVTQLPNGRDFHMSVVRARRNDSGTYVCGVISLAPKIQIKESLRAELRVTERGSGGGGSLPETGGHHHHHH.

A 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 μl E. coli competent cells, and then incubated on ice for 30 Minutes later, heat shock at 42°C for 90 seconds), the positive recombinants were screened out by ampicillin-resistant plates, and DNA sequencing was performed to verify them. Plasmids were stored at -80°C for long-term storage, and correctly sequenced strains were stored at -80°C with 20% glycerol at a ratio of 1:1.

Transform the amplified plasmid into BL21(DE3)PLysS expression competent cells, screen out positive recombinants on ampicillin-resistant plates, select 4-8 single clones and inoculate them into 150 mL LB medium, and incubate at 37°C Cultivate for 12-16 hours, then transfer the cultured bacterial liquid to TB E. coli culture medium, culture for 6-8 hours, induce with IPTG at 25°C for 16 hours, and collect the bacterial liquid.

Example 2 Purification of PD1 protein mixture to prepare high affinity PD1 protein

In this example, the PD1 protein that forms inclusion bodies is purified by denaturation and renaturation. The specific method is as follows:

The bacterial liquid precipitate collected in Example 1 was broken by ultrasonic and then centrifuged to obtain the precipitate. The precipitate was collected into a suspension after the bacterial cells were broken, and centrifuged at 10,000 rpm for 10 minutes at 4°C to separate inclusion bodies from soluble proteins. Collect the precipitate and soluble protein respectively. Supernatant.

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℃, 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, and centrifuge under the same conditions. The precipitate is the washed inclusion body.

Dissolution of inclusion bodies: The washed precipitate was resuspended 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 β-mercaptoethanol, 1% TritonX-100), stirred at room temperature for 60-90 min to fully dissolve, centrifuged at 12000 rpm for 20 min at 4°C, discarded the supernatant, and filtered with a 0.45 μm filter membrane.

On-column renaturation and purification: Take 5-10mL of denaturing loading buffer (20mmol〃L ^-1 phosphate buffer solution, 300mmol〃L ^-1 NaCl, 6mol〃L ^-1 urea, 5mM〃L ^-1 imidazole, 1mM〃 L ^-1 mercaptoethanol, pH = 8.0) to balance the Ni column, with a flow rate of 0.5 mL "min ^-1 . Load the dissolved inclusion body solution filtered through a 0.45 μm filter membrane at a flow rate of 0.5 mL"min ^-1 . Take 10mL washing buffer (20mM〃L ^-1 phosphate buffer, 300mmol〃L ^-1 NaCl, 6mol〃L ^-1 urea, 5mM〃L ^-1 imidazole, 1mM〃L ^-1 mercaptoethanol, pH=8.0) and wash column until OD ₂₈₀ no longer changes, and the flow rate is 0.5mL"min ^-1 . Wash the column sequentially with renaturation buffer (20mmol" ^{L -1 phosphate} buffer, 500mmol ^{"L -1} NaCl, pH 8.0) containing 5, 4, 3, 2, 1, and 0mol"L -1 urea. The gradient is 10 mL, and the flow rate is 0.2 mL"min ^-1 . Then use the refolding buffer containing 300mmol"L ^-1 imidazole to elute, the flow rate is 0.5mL"min ^-1 , the OD value will change with time during the refolding process, and collect the eluate at each concentration stage, 4℃ , centrifuge at 10000r/min for 10min, and take the supernatant for SDS-PAGE electrophoresis analysis.

Example 3 Preparation of temperature-responsive fusion protein

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:

SEQ ID NO.5:

VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG VGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGV GVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPGVGVPG.

SEQ ID NO.6:

GGCGTGGGTGTTCCGGGCGTAGGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTG TCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGT GTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGG GTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTTCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTA GGTGTCCCAGGTGTGGGCGTACCGGTGTCGGCGTGCCGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTTGGTGTTCCTGGTGTCGGCGTGCCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTGGGCGTACCGGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGCGTGGGTGT TCCGGGCGTAGGTGTCCCAGGCGTGGGTGTTCCGGGCGTAGGTGTCCCAGGTGTTCCGGGCGTAGGTGTCCCAGGTGTTCCG.

The above-mentioned ELP(V)90 gene fragment was inserted into the PET-24b(+) plasmid by a gene company. After designing primers and passing the polymerase chain reaction (PCR), the PCR fragment product containing the enzyme cutting site was double-digested. (BseR I and Acu I) and then inserted into the PET-24b(+) plasmid. The primers involved above are as follows:

Upstream primer:

5'-3'GAGGAGTACATATGGGCGACAGCCCGGATCGT;

Downstream primers:

5'-3'CTGAAGATCATTATTATCAGCCACCGGATCCACG.

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, positive recombinants were screened out by kanamycin resistance plate, and 4-8 monoclonal bacteria were selected and inoculated into 150 mL LB medium at 37°C. Cultivate for 12-16 hours, then transfer the cultured bacterial liquid to TB E. coli culture medium, culture for 6-8 hours, induce with IPTG at 25°C for 16 hours, and collect the bacterial liquid. The E. coli cells collected by centrifugation were resuspended in 40 mL of 10mM phosphate buffer solution and then lysed with an ultrasonic cell disrupter. The ultrasonic power was 300w and the time was 45 minutes. In the ultrasonic mode, select ultrasonic running for 5 seconds and pause for 5 seconds. The broken bacterial cells after sonication were centrifuged at 4°C and 14000rpm for 15 minutes, and the precipitate was discarded; then 2 mL of 10% (w/w) polyethyleneimine (PEI) was added and centrifuged again at 4°C and 14000rpm for 15 minutes. , to remove nucleic acids. The supernatant is collected and the target protein is obtained through reversible phase change ITC technology. The specific methods are as follows:

Add NaCl with a final concentration of 3M to the protein supernatant, incubate it in a 37°C water bath to fully dissolve it, then centrifuge it at 16000rpm for 20 minutes (37°C) to collect the precipitate; add 12 mL of pre-cooled 10mM PBS to the precipitate to fully dissolve it. After precipitation, the supernatant was collected by centrifugation at 4°C and 16,000 rpm for 20 minutes. The above steps are one ITC cycle. The target protein was obtained through three ITC cycles, in which the concentrations of NaCl were 3M, 2M, and 1M respectively, and the volumes of pre-cooled 10mM PBS were added to be 12mL, 8mL, and 6mL respectively. Store the prepared protein in a -80°C refrigerator for later use.

Example 4 Determination of physical and chemical characterization parameters of PD1 and ELP(V)90-PD1 proteins

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) for expression and purity of the target protein (Figure 1). High-performance liquid chromatography mass spectrometry was used to determine the exact molecular weight of the prepared protein (Figure 2).

After preliminary confirmation that the proteins were successfully prepared, the secondary structures of the two proteins were analyzed using a circular dichroism spectrometer to verify whether the protein fused with ELP(V)90 had an impact on the secondary structure of the PD1 protein. The results are shown in Figure 3. The results show that the secondary structure of the PD1 protein fused with the ELP(V)90 protein has not changed, suggesting that the function of the PD1 protein may not be affected by the ELP(V)90 protein.

Use a microplate reader to measure the absorbance value at OD350nm to determine the phase transition temperature of ELP(V)90-PD1 at different concentrations and explore the existence form of ELP(V)90-PD1 in the physiological environment in vivo. The results are shown in Figure 4. The results show that with the increase of temperature, the OD350 value of ELP(V)90-PD1 protein has a sudden jump after reaching the critical temperature, and then enters the plateau phase, and as the concentration increases, ELP ( The phase transition temperature of V)90-PD1 gradually decreases, which suggests that at physiological temperature, a certain concentration of ELP(V)90-PD protein may undergo phase transition in the body to form a condensed state, thereby sustaining the release of PD1 drugs.

The dynamic light scattering particle size analyzer (DLS) was used to analyze the hydration kinetic radius of the protein PD1 and the fusion protein ELP(V)90-PD1 in the solution state at different time points to initially determine whether the fused protein can pass through the glomerulus. Filtration increases half-life. The results are shown in Figure 5. The results show that the hydration kinetic radius of the fusion protein is increased by approximately 4 times compared with the PD1 protein, which is higher than the minimum diameter of 10 nm for renal clearance, but much lower than the maximum cutoff size of the reticuloendothelial system of 200 nm. , which shows that the fusion protein can reduce the renal clearance rate and can penetrate into tissue cells, thereby extending the half-life and increasing the uptake of tumor cells.

Example 5 In vitro competitive binding activity analysis of ELP(V)90-PD1 fusion protein

According to existing patent documents, the high-affinity PD1 protein not only has binding activity to human PDL1 receptors, but also has binding activity to mouse PDL1 receptors. Mouse colon cancer CT26 cells in the logarithmic growth phase were taken and the cell density was adjusted. Spread laser confocal small dishes at 1 × 10 ⁵ cells/mL, 2 mL/well, and incubate them in a 37°C, 5% CO ₂ incubator for 12 hours. Add mouse IFNγ to induce the upregulation of PDL1 receptors on the cell surface, and induce 12 hours. After 6 hours, add Cy5-labeled ELP(V)90-PD1 or high-affinity PD1 protein, incubate for 6 hours in a 37°C, 5% _CO2 incubator, and then wash away the unbound labeled protein with 10mM phosphate buffer. After fixing the cells with 4% paraformaldehyde for 15 minutes, wash the cells three times with 10mM phosphate buffer. After blocking the cells with 5% bovine serum albumin (BSA) for 1 hour, wash the cells three times with 10mM phosphate buffer. , add goat anti-rabbit PDL1 primary antibody, incubate the cells at room temperature for 1 hour, wash the cells three times with 10mM phosphate buffer, then add FITC-labeled secondary antibody, incubate the cells at room temperature for 2 hours, wash the cells with 10mM phosphate buffer After three times, 1 mL of 10 mM phosphate buffer was added, and imaging analysis was performed using a laser confocal microscope LSM900. The results are shown in Figure 6. The competitive binding activity results in Figure 6 show that compared with the PD1 group, although the fusion protein ELP(V)90-PD1 is fused with the ELP(V)90 protein, the activity of binding to the PDL1 receptor is reduced by approximately 45%, but ELP( V) 90-PD1 protein still has strong activity in binding to PDL1 receptor.

Use flow cytometry to quantitatively analyze the competitive binding of ELP(V)90-PD1 or high-affinity PD1 protein to PDL1 on the surface of CT26 cells. Take mouse colon cancer CT26 cells in the logarithmic growth phase and adjust the cell density to 2. ×10 ⁵ cells/mL, 4 mL/well was spread on a 6-well plate, cultured in a 37°C, 5% CO ₂ incubator for 12 hours, and mouse IFNγ was added to induce the upregulation of PDL1 receptors on the cell surface. After induction for 12 hours, Add Cy5-labeled ELP(V)90-PD1 or PD1 protein, incubate for 6 hours at 37°C in a 5% _CO2 incubator, then wash away the unbound labeled protein with 10mM phosphate buffer, and use PE-labeled PDL1 in the cells. The primary antibody was incubated at room temperature for 20 minutes, and unbound antibodies were washed away before analysis by flow cytometry. The results are shown in Figure 7. The results show that compared with the PD1+anti-PDL1 group, although the binding activity of the ELP(V)90-PD1 protein to the PDL1 receptor is reduced by approximately 22%, it still has the activity to compete with PDL1 for binding.

On the basis of preliminary verification that the high-affinity PD1 protein and ELP(V)90-PD1 fusion protein prepared through the prokaryotic system have certain biological activities, the proteins will be characterized at the individual level in vivo.

Example 6 Determination of the maximum tolerated dose of ELP(V)90-PD1 fusion protein in mice

In this example, the maximum tolerated dose of the ELP(V)90-PD1 fusion protein prepared in Example 3 was tested in wild BALB/c mice. BALB/c female mice aged 6-8 weeks were purchased from the Experimental Animal Science Center of Peking University Health Science Center. 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 injected intraperitoneally with doses of 4.6, 8.5 and 12.6 mg/kg of body weight of high-affinity PD1 protein and 28.0, 32.0 and 48.0 mg/kg of ELP(V)90-PD1 fusion protein (three in each group). mice), monitor and record changes in the weight of each mouse in each group and the living conditions of each group of mice. The maximum tolerated dose is the dose that results in a weight loss of no more than 10% at the highest dose. The daily weight changes of each mouse in each dose group are shown in Figure 8.

The results showed that the maximum tolerated dose of the unfused high-affinity PD1 protein was 4.6 mg/kg, while the maximum tolerated dose of the ELP(V)90-PD1 fusion protein was 28 mg/kg. This indicates that the toxicity of the fused protein in vivo is significantly reduced, which fully demonstrates that the fusion protein can reduce the toxicity of the PD1 protein.

Example 7 In vivo pharmacokinetic test of ELP(V)90-PD1 fusion protein

This example tests the pharmacokinetics of the ELP(V)90-PD1 fusion protein prepared in Example 3. Sprague-Dawley (SD, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.) rats weighing 200±10g were used as experimental subjects. Cy5-labeled high-affinity PD1 protein and ELP were injected intraperitoneally at the maximum tolerated dose. (V) 90-PD1 fusion protein, blood was collected from the orbital canthal venous plexus at 0, 0.5, 1, 2, 8, 24, 72, 120, 168, 240, 288, 336, 408 and 480 hours after injection. The heparin blood sample was centrifuged at 4°C and 3500 rpm for 5 minutes, and the pharmacokinetic changes were determined by measuring the fluorescence intensity of the upper plasma. The calculation was based on the fluorescence intensity curve measured by mixing Cy5-labeled PD1 protein and ELP(V)90-PD1 with the plasma of the blank group. The concentration of each group of drugs in the blood samples was calculated using Drug Analysis System 3.0 software to calculate the pharmacokinetic parameters of each group.

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). In addition, 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), and the average residence time of ELP(V)90-PD1 (203.3±2.5h) is 6.68 times that of PD1 (30.4±1.4h). This indicates that the size of the fused protein is larger than that before fusion, which slows down the renal clearance rate, significantly prolongs the drug half-life, and significantly increases the bioavailability.

Table 1 Pharmacokinetic parameters of ELP(V)90-PD1 and PD1 protein in wild BALB/c mice

Example 8 Evaluation of sustained release of ELP(V)90-PD1 fusion protein in vivo

The sustained release of the drug allows the drug to be released continuously over 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.

Cy7-labeled high-affinity PD1 protein and ELP(V)90-PD1 fusion protein were intraperitoneally injected into mice (three in each group) at the maximum tolerated dose (4.6 mg/kg and 28.0 mg/kg, respectively), respectively. After anesthesia with isoflurane at 2h, 1d, 3d, 7d, 10d, 15d and 18d after injection, use Spectrum imaging system imaging analysis, the results are shown in Figure 10. The results in Figure 10 show that the PD1 protein without ELP was completely metabolized within 3 days, while the ELP(V)90-PD1 fusion protein still had some drugs that were not metabolized at 18 days. The ELP(V)90-PD1 fusion protein was The drug reservoir is formed in the peritoneal mucosa and the drug is slowly released, which is consistent with the results of pharmacokinetics.

Example 9 Distribution of ELP(V)90-PD1 fusion protein in major tissues and organs in the body

In this example, 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 at 2h, 1d, 3d, 7d, and 15d respectively. Finally, the relative fluorescence intensity of the remaining PD1 in each major tissue and organ was measured to reflect the drug distribution in each tissue and organ. The results are shown in Figure 11 and Figure 12. By detecting the relative fluorescence intensity of 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 tumors respectively, but the distribution in the heart was lower than that in other organs. The content in tissues and organs is low, and at two hours, the content of ELP(V)90-PD1 in tumor tissues is lower than that of PD1, which illustrates the slow onset of sustained release of the drug. However, after 24 hours, ELP(V)( The distribution of V)90-PD1 in tumor tissue reaches the maximum, which is similar to PD1. When detected on the third day, the content of ELP(V)90-PD1 protein in tumor tissue is higher than that of PD1, and it is more abundant in other tissues. The content in the organ is lower than that in the tumor tissue, which suggests that ELP(V)90-PD1 can target the tumor tissue and penetrate into the tumor tissue. As time goes by, the PD1 protein increases in the tumor tissue at day 7 and 15 No signal was detected in various tissues and organs, but ELP(V)90-PD1 still had high signal intensity in tumor tissues, once again proving the sustained release effect and tumor targeting of ELP(V)90-PD1 enrichment effect.

Example 10 Penetration ability of ELP(V)90-PD1 fusion protein in tumor tissue

This example analyzes the penetration ability of the ELP(V)90-PD1 fusion protein prepared in Example 3 in tumor tissue, as follows:

BALB/c mice inoculated with CT26 colon cancer cells were used to determine the penetration ability of ELP(V)90-PD1 fusion protein in tumor tissue. High-affinity PD1 protein and ELP(V)90-PD1 fusion protein were labeled with Cy5.

100 μl of CT26 cells at 1×10 ⁶ /mL were inoculated subcutaneously on the back of the right hind leg of BALB/c mice. When the tumor grew to a size of 50 mm ³ , Cy5-labeled high-affinity PD1 protein and ELP (V )90-PD1 protein was injected intraperitoneally into tumor-bearing mice, and tumor tissues were collected at 24h, 3d, and 7d after administration, and frozen sections were prepared. CD31 primary antibody was used to label tumor blood vessels, FITC-labeled IgG secondary antibody was used to detect the primary antibody, and finally the nuclei were stained with DAPI and mounted. The fluorescence intensity of tumor tissues in each group was observed using an LSM880 laser confocal microscope. The imaging results are shown in Figure 13. The results showed that both PD1 and ELP(V)90-PD1 had a certain degree of penetration at 24 hours, and compared with the PD1 group, ELP(V)90-PD1 still had drug accumulation farther away from the tumor blood vessels, which shows that ELP(V)90-PD1 can penetrate into the tumor tissue, and on the third day, ELP(V)90-PD1 penetrates further into the tumor blood vessels, and on the seventh day, it penetrates further than the third day, which is sufficient. It shows that after fusion with ELP protein, although the molecular weight increases, it does not affect the penetration of PD1 into tumor tissue.

Example 11 In vivo anti-tumor biological activity detection of ELP(V)90-PD1 fusion protein

In this example, 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.

Inoculate 100 μl of CT26 cells at 1×10 ⁶ /mL subcutaneously on the back of the right hind leg of BALB/c mice. When the tumor grows to an average size of 50 mm ³ , high-affinity PD1 protein and ELP(V) are administered at the maximum tolerated dose. The 90-PD1 fusion protein was injected intraperitoneally into tumor-bearing mice. The survival status and tumor growth of the mice were observed every day. The weight and tumor size of the mice were dynamically detected and recorded. After the treatment, the survival rate was calculated. When the tumor volume of each mouse in each group grew to ^1500mm3 or the body weight decreased by more than 10%, the mouse was considered dead and recorded as "1". The mice whose tumors did not reach the target were considered alive. , recorded as "0". The survival curves and survival rates of mice in each group are shown in Figure 14 and Figure 15.

Figures 14 and 15 show that after a maximum tolerated dose administration, 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 PD1 group, this is mainly due to the maximum tolerated dose administration at one time, the ELP(V)90-PD1 group can be given a higher dose of the drug, and the sustained release effect of ELP keeps the release of PD1 at a relatively stable blood concentration. Fully stimulating immunity in the body, so that the tumor is well suppressed. The survival curve after drug effect can also be seen. After treatment with ELP(V)90-PD1 protein, the overall survival of mice in the ELP(V)90-PD1 group The time was significantly prolonged, with the median survival days being 28 days, which was 1.5 times (19 days) and 1.9 times (15 days) that of the PD1 and PBS treatment groups respectively, once again proving the long-term effect of ELP(V)90-PD1.

Example 12 In vivo anti-tumor activity of ELP(V)90-PD1 fusion protein combined with first-line chemotherapy drug oxaliplatin

100 μl of CT26 cells at 1×10 ⁶ /mL were subcutaneously inoculated on the back of the right hind leg of BALB/c mice. When the tumors grew to an average size of 50 mm ³ , 90 mice with the same tumor size were divided into 6 groups. There are 15 mice in each group, which are PBS group, oxaliplatin (OX) group, PD1 group, ELP(V)90-PD1 group, PD1+OX group, ELP(V)90-PD1+OX group, and PD1 protein and ELP(V)90-PD1 protein were intraperitoneally injected into 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. In vivo, tumor-bearing mice in the OX group were given OX at a dose of 2 mg/kg. On the fifth day, the PD1+OX group and ELP(V)90-PD1+OX group were given OX at a dose of 2 mg/kg. The survival status of the mice was observed every day. and tumor growth status. The weight and tumor size of each mouse in each group were dynamically detected and recorded.

After the drug efficacy experiment, the blood of each mouse in each group was taken by removing the eyeballs, 100 μl of fresh blood was taken for blood cell analysis, 500 μl of blood was centrifuged for blood biochemical analysis, and then the tumor tissue and heart of the mice in each group were taken. , liver, spleen, kidney, lung and other major tissues, some were fixed in tissue fixative for HE staining, and some were immersed in sterile 10mM PBS to detect immune-related indicators. The drug efficacy diagram is shown in Figure 16, the HE staining of tumor tissue is shown in Figure 17, the comparison of tumor size in each group is shown in Figure 18, the HE staining diagram of the main tissues and organs in each group is shown in Figure 19, each group The blood cells and blood biochemical analysis of some mice are shown in Figure 20 and Figure 21 respectively.

The results in Figure 16 show that for the CT26 colon cancer tumor model, the therapeutic effect of the ELP(V)90-PD1+OX group was significantly better than that of other administration groups, and 85% of the mice's tumors disappeared and were cured, with no recurrence seen. . 14% of the mice in the ELP(V)90-PD1 group had tumors disappearing and were cured. Compared with the PBS group, the tumors of mice in the OX group, PD1 group and PD1+OX group were significantly inhibited, and no mouse tumors disappeared. Moreover, the therapeutic effect of the ELP(V)90-PD1 group was significantly better than that of the PD1+OX combined administration group, once again proving the advantage of the fusion protein's sustained release and long-acting effect.

The results in Figure 17 show that after drug treatment, the number of tumor cells in ELP(V)90-PD1+OX group <ELP(V)90-PD1 group<PD1+OX group<PD1<OX group<PBS group, which is consistent with the drug The results of the efficacy are consistent, indicating that the fusion proteins produce a synergistic effect after combined administration and jointly exert an anti-tumor therapeutic effect. The drug efficacy of each group can also be clearly seen in the tumor size comparison chart extracted in Figure 18. The results in Figure 19 show that no obvious damage occurred to the tissues and organs after the protein drugs and combined administration, which illustrates the safety of the administration. At the same time, combined with the results in Figure 20 and Figure 21, mice in each group were administered various kinds of No abnormalities were found in the blood cells, and the blood biochemical index results also showed no obvious abnormalities, which once again illustrates the safety of the administration.

In summary, the present invention innovatively proposes a temperature-responsive high-affinity PD1 fusion protein, which is combined with oxaliplatin, a first-line anti-colon cancer treatment drug, to greatly improve the anti-tumor efficacy, and at the same time, the biological activity of high-affinity PD1 The half-life is significantly extended. Once administered at the maximum tolerated dose, the drug can be released continuously for 20 days. This is the longest circulation time currently achieved by non-ELP fusion protein delivery systems. At the same time, the best therapeutic effect can be achieved with one administration, and Reduce the frequency of administration and improve patient compliance. The fusion protein can also effectively penetrate into tumor tissue and bind to the PDL1 receptor on the surface of tumor cells to exert anti-tumor immune effects.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that it can still be used Modifications are made to the technical solutions described in the foregoing embodiments, or equivalent substitutions are made to some of the technical features; however, these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A high-affinity PD1 protein conjugate, characterized in that the high-affinity PD1 protein conjugate comprises an elastin-like polypeptide and a high-affinity PD1 protein connected to the elastin-like polypeptide. 2. The high-affinity PD1 protein conjugate according to claim 1, characterized in that the elastin-like polypeptide is directly connected to the high-affinity PD1 protein or connected through a connecting peptide; Preferably, the C-terminus of the elastin-like polypeptide is connected to the N-terminus of the high-affinity PD1 protein. 3. The high-affinity PD1 protein conjugate according to claim 1 or 2, characterized in that, compared with wild-type PD1 protein, the high-affinity PD1 protein has enhanced affinity for PDL1 and can competitively bind to PDL1; Preferably, 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. 4. The high-affinity PD1 protein conjugate according to claim 3, wherein the high-affinity PD1 protein is any one of the following 1) or 2): 1) The amino acid sequence is shown in SEQ ID NO.3; 2) The amino acid sequence is a sequence obtained by adding one or more selected from the group consisting of protein tags, enzyme cleavage sites, and connecting peptides to the N-terminus or C-terminus of the sequence shown in SEQ ID NO.3; Preferably, the amino acid sequence of the high-affinity PD1 protein is as shown in SEQ ID NO. 3 or 4. 5. The high-affinity PD1 protein conjugate according to any one of claims 1 to 4, wherein the amino acid sequence of the elastin-like polypeptide includes (X-Gly-X-Pro-Gly)n, wherein , 10≤n≤200, X is any natural amino acid except proline. 6. The high affinity PD1 protein conjugate according to claim 5, characterized in that, X is selected from valine, phenylalanine, tryptophan, tyrosine, alanine, glycine, methionine One of amino acid, threonine, serine, leucine and isoleucine; the response temperature of the elastin-like polypeptide is 10-60°C; Preferably, X is valine, 30≤n≤150, and the response temperature of the elastin-like polypeptide is 18-40°C; More preferably, the hydration dynamic radius of the high-affinity PD1 protein conjugate is 5-15 nm. 7. Nucleic acid molecule, characterized in that the nucleic acid molecule encodes the high-affinity PD1 protein conjugate according to any one of claims 1 to 6. 8. Biological material, characterized in that the biological material contains the nucleic acid molecule of claim 7 or expresses the high-affinity PD1 protein conjugate of any one of claims 1 to 6; the biological material is an expression cassette , vector or host cell. 9. Application of the high-affinity PD1 protein conjugate of any one of claims 1 to 6 or the nucleic acid molecule of claim 7 or the biological material of claim 8 in the preparation of medicines; Preferably, the drug is a drug for preventing or treating tumors; More preferably, the drug is a drug for preventing or treating colon cancer. 10. A pharmaceutical composition, characterized in that the pharmaceutical composition contains the high-affinity PD1 protein conjugate according to any one of claims 1 to 6; Preferably, the pharmaceutical composition further includes one or more selected from the group consisting of chemotherapeutic drugs, targeted therapy drugs, endocrine therapy drugs and immunotherapy drugs; More preferably, the pharmaceutical composition comprises the high-affinity PD1 protein conjugate according to any one of claims 1 to 6 and oxaliplatin.