A group of PD-1 molecules and mutants thereof that block the binding of anti-PD-1 antibodies to cell surface PD-1 molecules and uses thereof

Abstract

The present invention belongs to the field of biomedicine, and specifically relates to a group of PD-1 molecules and their mutants that block the binding of anti-PD-1 antibodies to cell surface PD-1 molecules and their uses. The present invention provides a protein molecule, which has 80%-100% sequence identity compared with the wild-type protein shown in SEQ ID NO:1. The recombinant protein H126 and H134 molecules provided by the present invention can competitively bind anti-PD-1 antibody drugs with the endogenous PD-1 molecules expressed by T cells, without affecting the binding of PD-1 to endogenous PD-L1 molecules, thereby restoring the functional inhibition of activated T cells and alleviating the symptoms of immune therapy-related adverse reactions. The protein molecule provides a new approach for the clinical treatment of immune therapy-related adverse reactions.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and specifically relates to a group of PD-1 molecules and mutants thereof that block the binding of anti-PD-1 antibodies to cell surface PD-1 molecules, and their uses. Background Art

[0002] PD-1 is an immunosuppressive molecule expressed on T cells. When anti-PD-1 antibodies bind to PD-1, they block its binding to its ligand, PD-L1, thereby releasing PD-1's inhibitory signal and restoring T cell tumor-killing activity. Anti-PD-1 antibodies are currently the mainstay of immunotherapy for malignant tumors, but more than half of patients experience immune-related adverse events (irAEs) following treatment. Over 20% of patients require discontinuation of treatment, and nearly 10% require treatment with glucocorticoids (GCs). Managing irAEs caused by anti-PD-1 therapy is one of the most pressing challenges in cancer immunotherapy.

[0003] Although GCs are a major and effective treatment for irAEs, some patients are still resistant to treatment, and long-term GCs treatment has significant side effects for patients with malignant tumors. Currently, there are some drugs in the field that treat irAEs. For example, Chinese patent application 202310565962.0 discloses a CD80 and CD86 binding protein composition and its use. This invention relates to a cytotoxic T-lymphocyte-associated antigen-4 (CTLA-4) protein composition and its use in alleviating autoimmune adverse events associated with cancer immunotherapy. U.S. patent application 202017625226 discloses a method for detecting follicular regulatory T cells (TFR), the method comprising: obtaining a biological sample from a subject, and contacting the biological sample with an antibody for detecting CD3+CD4+FOXP3+BCL6+T cells, CD3+CD4+CXCR5+GITR+T cells, or both, and detecting an increase in TFR in the tumor sample. The invention also includes a combination therapy for depleting follicular regulatory T lymphocytes (TFR) in a manner that minimizes the effect on regulatory T cells (TREGS) to reduce immune-related adverse reactions (irAEs).

[0004] Therefore, more drugs for treating irAEs are needed in this field. Summary of the Invention

[0005] The cause of irAE is anti-PD-1 drug treatment. When the therapeutic drug cannot bind to the endogenous PD-1 expressed by T cells, this inducement will disappear. To this end, the present invention designs molecules that can block the binding of anti-PD-1 antibody drugs to the endogenous PD-1 expressed by T cells, in order to treat patients with irAE.

[0006] In order to overcome the defects of the prior art, the present invention proposes the following technical solutions:

[0007] In one aspect, the present invention provides a protein molecule having 80%-100% sequence identity with the wild-type protein shown in SEQ ID NO: 1.

[0008] In some embodiments, the protein molecule is a functional variant having at least one amino acid position difference compared to the wild-type protein shown in SEQ ID NO: 1;

[0009] The difference in the amino acid sites is achieved by at least one of addition, deletion, modification and / or substitution of amino acids;

[0010] The functional variant has the same, similar or better function as the wild-type protein shown in SEQ ID NO: 1 in blocking the binding of anti-PD-1 antibodies to PD-1 molecules.

[0011] In some preferred embodiments, the difference in the amino acid positions is achieved by amino acid substitution, preferably conservative amino acid substitution.

[0012] In some embodiments, the protein molecule comprises an amino acid substitution at position 126 or 134 compared to the wild-type protein shown in SEQ ID NO: 1.

[0013] In some preferred embodiments, the protein molecule comprises an amino acid substitution of I126A compared to the wild-type protein shown in SEQ ID NO: 1.

[0014] In some preferred embodiments, the protein molecule has an amino acid sequence as shown in SEQ ID NO: 2.

[0015] In some embodiments, the protein molecule comprises an amino acid substitution of I134A compared to the wild-type protein shown in SEQ ID NO: 1.

[0016] In some preferred embodiments, the protein molecule has an amino acid sequence as shown in SEQ ID NO: 3.

[0017] SEQ ID NO: 1:

[0018] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。

[0019] SEQ ID NO:2:

[0020] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAASLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。

[0021] The nucleotide sequence encoding SEQ ID NO:2 is shown in SEQ ID NO:4:

[0022] ATGCAGATCCCCCAGGCTCCTTGGCCTGTGGTGTGGGCTGTGCTGCAGCTGGGATGGAGGCCTGGATGGTTCCTGGACTCCCCTGACCGTCCTTGGAACCCTCCTACCTTCTCCCCCGCTCTCCTCGTTGTCACCGAGGGAGATAACGCCACCTTCACCTGCTCCTTCTCCAACACCTCCGAGTCCTTCGTCCTCAACTGGTATCGCATGTCTCCCTCCAACCAAACCGACAAGCTGGCCGCTTTCCCCGAAGATAGAAGCCAACCCGGCCAGGACTGCAGATTCCGTGTGACACAGCTGCCCAACGGGCGAGATTTCCACATGAGCGTCGTGAGGGCCAGAAGAAACGACTCCGGGACCTACCTGTGTGGCGCAGCTTCTCTGGCTCCCAAGGCTCAAATCAAAGAAAGCCTCCGGGCCGAGCTGAGAGTGACTGAAAGAAGAGCTGAGGTGCCCACCGCCCACCCTTCTCCTTCTCCTAGACCCGCTGGACAGTTCCAAACCCTCGTGTGA。

[0023] SEQ ID NO:3:

[0024] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQAKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLV。

[0025] The nucleotide sequence encoding SEQ ID NO:3 is shown in SEQ ID NO:5:

[0026] ATGCAGATCCCCCAGGCTCCTTGGCCTGTGGTGTGGGCTGTGCTGCAGCTGGGATGGAGGCCTGGATGGTTCCTGGACTCCCCTGACCGTCCTTGGAACCCTCCTACCTTCTCCCCCGCTCTCCTCGT TGTCACCGAGGGAGATAACGCCACCTTCACCTGCTCCTTCTCCAACACCTCCGAGTCCTTCGTCCTCAACTGGTATCGCATGTCTCCCTCCAACCAAACCGACAAGCTGGCCGCTTTCCCCGAAGATAG AAGCCAACCCGGCCAGGACTGCAGATTCCGGTTGACACAGCTGCCCAACGGGCGAGATTTCCACATGAGCGTCGTGAGGGCCAGAAGAAACGACTCCGGGACCTACCTGTGTGGCGCAATCTCCCTGG CTCCAAAAGCCCAAGCCAAAGAAAGCCTCCGGGCCGAACTGAGAGTGACCGAAAGAAGAGCCGAGGTGCCCACCGCACACCCTTCTCCTTCACCTCGACCTGCTGGACAGTTCCAAAACCCTCGTGTGA.

[0027] In another aspect, the present invention provides a recombinant protein comprising any of the aforementioned protein molecules.

[0028] In some embodiments, the recombinant protein further comprises a biologically active protein or a functional fragment thereof that assists its expression and / or secretion, or prolongs its half-life in vivo.

[0029] In some embodiments, the biologically active polypeptide or functional fragment thereof is selected from at least one of an immunoglobulin Fc domain, serum albumin, an albumin-binding polypeptide, prealbumin, a carboxyl-terminal peptide, an elastin-like polypeptide, a His tag, a GST tag, an MBP tag, a FLAG tag, and a SUMO tag.

[0030] In another aspect, the present invention provides a blocker for an anti-PD-1 antibody, wherein the blocker competitively binds to the anti-PD-1 antibody with endogenous PD-1, does not bind to endogenous PD-L1, and does not block the binding of endogenous PD-L1 to endogenous PD-1, and the blocker comprises any of the aforementioned protein molecules or any of the aforementioned recombinant proteins.

[0031] In some embodiments, the blocking agent further comprises a buffer.

[0032] In some embodiments, the anti-PD-1 antibodies include, but are not limited to, pembrolizumab, nivolumab, sintilimab, tislelizumab, and toripalimab.

[0033] In another aspect, the present invention provides the use of any of the aforementioned protein molecules, any of the aforementioned recombinant proteins, or any of the aforementioned blockers in the preparation of a medicament for treating immunotherapy-related adverse events (irAEs).

[0034] In some embodiments, the immunotherapy-related adverse reaction is caused by an anti-PD-1 antibody.

[0035] In another aspect, the present invention provides a use of a protein molecule in the preparation of a drug for treating adverse reactions associated with immunotherapy, wherein the protein molecule has an amino acid sequence as shown in SEQ ID NO: 1.

[0036] The H126 and H134 recombinant proteins provided by this invention can competitively bind to anti-PD-1 antibody drugs with endogenous PD-1 molecules expressed by T cells without affecting the binding of PD-1 to endogenous PD-L1 molecules, thereby restoring the functional inhibition of activated T cells and alleviating the symptoms of immunotherapy-related adverse reactions. This provides a new approach for the clinical treatment of immunotherapy-related adverse reactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The SDS-PAGE results of H126 recombinant protein are shown, wherein M is protein marker, R is reduced H126 recombinant protein, NR is non-reduced H126 recombinant protein, and the unit of marker is KD.

[0038] Figure 2 The SDS PAGE results of H134 recombinant protein are shown, wherein M is protein marker, R is reduced H134 recombinant protein, NR is non-reduced H134 recombinant protein, and the unit of marker is KD.

[0039] Figure 3 Shown are the results of SEC-HPLC analysis of H126 recombinant protein.

[0040] Figure 4 Shown are the results of SEC-HPLC analysis of the H134 recombinant protein.

[0041] Figure 5 PD-1 WT , H126 recombinant protein and H134 recombinant protein competitively bind to pembrolizumab. Where A is different concentrations of PD-1 WT, H126 recombinant protein and H134 recombinant protein competed with PD-1 expressed on T cells for binding to pembrolizumab in three repeated experiments; B is the statistical analysis result of A.

[0042] Figure 6 The results show that H126 recombinant protein and H134 recombinant protein are comparable to PD-1. WT The release of free PD-1 (FPR) on T cells. A represents the difference in the expression of H126 recombinant protein and H134 recombinant protein at different concentrations compared to PD-1. WT The blocking efficiency was determined by three replicate experiments; B is the statistical analysis result of A.

[0043] Figure 7 PD-1 WT , H126 recombinant protein and H134 recombinant protein competitively bind to nivolumab. Where A is different concentrations of PD-1 WT The results of three repeated experiments were as follows: H126 recombinant protein and H134 recombinant protein competed with PD-1 expressed on T cells and bound to nivolumab; B is the statistical analysis result of A.

[0044] Figure 8 The results show that H126 recombinant protein and H134 recombinant protein are comparable to PD-1. WT The release of free PD-1 (FPR) on T cells. A represents the difference in the expression of H126 recombinant protein and H134 recombinant protein at different concentrations compared to PD-1. WT The blocking efficiency was determined by three replicate experiments; B is the statistical analysis result of A.

[0045] Figure 9 PD-1 WT , H126 recombinant protein and H134 recombinant protein competitively bind to sintilimab. Where A is different concentrations of PD-1 WT The competitive binding of A, H126 recombinant protein and H134 recombinant protein to PD-1 expressed on T cells and sintilimab was repeated three times; B is the statistical analysis result of A.

[0046] Figure 10 The results show that H126 recombinant protein and H134 recombinant protein are comparable to PD-1. WT The release of free PD-1 (FPR) on T cells. A represents the difference in the expression of H126 recombinant protein and H134 recombinant protein at different concentrations compared to PD-1. WT The blocking efficiency was determined by three replicate experiments; B is the statistical analysis result of A.

[0047] Figure 11 PD-1 WT, H126 recombinant protein and H134 recombinant protein competitively bind to tislelizumab. Where A is different concentrations of PD-1 WT The competitive binding of A, H126 recombinant protein and H134 recombinant protein to PD-1 expressed on T cells and tislelizumab were repeated three times; B is the statistical analysis result of A.

[0048] Figure 12 The results show that H126 recombinant protein and H134 recombinant protein are comparable to PD-1. WT The release of free PD-1 (FPR) on T cells. A represents the difference in the expression of H126 recombinant protein and H134 recombinant protein at different concentrations compared to PD-1. WT The blocking efficiency was determined by three replicate experiments; B is the statistical analysis result of A.

[0049] Figure 13 PD-1 WT , H126 recombinant protein and H134 recombinant protein competitively bind to Teplizumab. Where A is different concentrations of PD-1 WT The results of three repeated experiments were as follows: A, H126 recombinant protein and H134 recombinant protein competed with PD-1 expressed on T cells and bound to toripalimab; B is the statistical analysis result of A.

[0050] Figure 14 The results show that H126 recombinant protein and H134 recombinant protein are comparable to PD-1. WT The release of free PD-1 (FPR) on T cells. A represents the difference in the expression of H126 recombinant protein and H134 recombinant protein at different concentrations compared to PD-1. WT The blocking efficiency was determined by three replicate experiments; B is the statistical analysis result of A.

[0051] Figure 15 Surface plasmon resonance technology is used to detect PD-1. WT Binding to PD-L1.

[0052] Figure 16 The figure shows the binding of H126 recombinant protein to PD-L1 detected by surface plasmon resonance technology.

[0053] Figure 17 The figure shows the binding of H134 recombinant protein to PD-L1 detected by surface plasmon resonance technology. DETAILED DESCRIPTION

[0054] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0055] Unless the context clearly dictates otherwise, as used herein, the expressions "a" and "an" include plural references. For example, reference to "a cell" includes a plurality of such cells and equivalents thereof known to those skilled in the art, and so forth.

[0056] As used herein, numerical ranges are to be understood as including all numbers within the range. For example, a range of 1 to 20 is to be understood as including any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0057] As used herein, the term "comprises" or "comprising" means "including but not limited to". The term is intended to be open-ended to specify the presence of any stated features, elements, integers, steps, or components, but does not preclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" encompasses the more restrictive terms "consisting of" and "consisting essentially of". In one embodiment, the term "comprising" as used throughout the application, particularly in the claims, may be replaced by the term "consisting of".

[0058] As used herein, the terms "optional," "either," "any," or "any" mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur. As used herein, "a" and "an" are used to refer to one or more than one of the grammatical objects of the present invention.

[0059] The term "and / or" used herein should be understood to mean any one of the options or a combination of any two or more of the options.

[0060] The experimental method involved in the present invention is:

[0061] Flow cytometry:

[0062] Dilute the blood with PBS at a ratio of 1:1, then slowly add the diluent and human lymphocyte separation medium at a ratio of 2:1 to the upper layer of the 15 mL centrifuge tube containing lymphocyte separation medium;

[0063] Centrifuge for 22 minutes at room temperature, 2000 RPM, acceleration 1, and deceleration 1.

[0064] After centrifugation, the mixture consists of PBS and unabsorbed plasma, buffy coat layer, lymphocyte separation fluid layer and blood cell layer from top to bottom. PBMC is in the buffy coat layer in the middle. Pipette the buffy coat layer into another sterile centrifuge tube.

[0065] Add sterile PBS three times the volume of the buffy coat aspirate, centrifuge at 1500 RPM for 10 minutes at room temperature to wash away the residual lymphocyte separation solution, and discard the supernatant.

[0066] Resuspend the pellet in a small amount of PBS, add an appropriate amount of PBS, centrifuge again at 1200 RPM for 8 minutes to wash off platelets and some cell debris, and resuspend in PBS to obtain a PBMC suspension.

[0067] ·Prepare antigen (PD-1 WT , H126, H134), and antibody (pembrolizumab, nivolumab, sintilimab, tislelizumab, and toripalimab) systems, with final antigen concentrations of 2.5 μg / mL, 5 μg / mL, and 10 μg / mL, respectively, and final antibody concentrations of 5 μg / mL, with antigen:antibody ratios of 0.5:1, 1:1, and 2:1;

[0068] Add different concentrations of antigen and commercial PD-1 antibody into EP tubes, pipette and mix thoroughly, and incubate at 4°C for 30 minutes;

[0069] Take 50 μL of PBMC suspension, add the above reaction system to the corresponding cell suspension, mix well, and incubate at 4°C for 30 minutes;

[0070] Wash cells twice with 1 mL PBS / tube and centrifuge at 300 g for 5 min.

[0071] Resuspend with appropriate amount of PBS, add flow cytometry antibodies PE-Cy7 anti-human CD3 and APC anti-human PD-1 to the ISO tube, and add flow cytometry antibodies PE-Cy7 anti-human CD3 and APC anti-human PD-1 to the experimental tube respectively, vortex to mix, and incubate at 4°C in the dark for 30 minutes;

[0072] Add 2 mL of PBS, centrifuge for 5 minutes, discard the supernatant, and repeat once;

[0073] Add 2 mL of PBS, centrifuge for 5 minutes, discard the supernatant, and repeat once.

[0074] Surface Plasmon Resonance Technology:

[0075] Biacore X optical biosensors were used at 25°C. Human PD-L1 fusion protein was immobilized on the surface of a CM5 sensor chip via free amine coupling and then deactivated with ethanolamine. To determine the equilibrium dissociation constant (Kd), PD-1 or its mutants were sequentially injected into the flow cell at a rate of 20 L / min over a concentration range (0.35–20 M). The background response of the reference flow cell was subtracted from the total response of the experimental flow cell to obtain the final response level. The data were fitted to a nonlinear 1:1 Langmuir binding model to obtain the Kd using the relationship: R / Rmax / (C / Kd), where R is the response unit (RU) of bound analyte, Rmax is the maximum response level, C is the micromolar concentration of free analyte, and Kd is the micromolar equilibrium dissociation constant. For rapid qualitative analysis of PD-1 mutants, an equal amount of protein was injected into the flow cell at a concentration of 1.7 μM (below saturation).

[0076] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the examples. If the specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for all reagents or instruments, they are all conventional products that can be purchased commercially. In order to better illustrate the present invention, numerous specific details are provided in the following specific embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concept of the present invention. Such structures and technologies are also described in many publications, such as "Molecular Cloning Laboratory Manual (4th Edition)" (Cold Spring Harbor Laboratory Science Press), Ausubel, FM et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience.

[0077] Example 1 Expression and purification of recombinant protein

[0078] 1.1 Expression vector construction

[0079] H126 recombinant protein: The nucleotide sequence shown in SEQ ID NO: 4 was cloned into the expression vector pcDNA3.4 (with restriction enzyme cutting sites NotI / XbaI). After screening positive clones, sequencing was performed using universal sequencing primers to obtain an expression vector encoding the H126 recombinant protein.

[0080] H134 recombinant protein: The nucleotide sequence shown in SEQ ID NO: 5 was cloned into the expression vector pcDNA3.4 (with restriction enzyme cutting sites NotI / XbaI). After screening positive clones, sequencing was performed using universal sequencing primers to obtain an expression vector encoding the H134 recombinant protein.

[0081] 1.2 Recombinant protein expression, purification and quality testing

[0082] (1) Cell transfection:

[0083] Take HEK293 cells of appropriate confluency, centrifuge and take the supernatant;

[0084] Add about 0.5 mL of electroporation solution to the cells, mix well, and then add an appropriate amount of plasmid (the expression vector constructed above) (concentration 500 ng / μL);

[0085] After thoroughly mixing the above cell plasmid suspension, take 1 mL and add it to a 1 mL electroporation tube. Place the electroporation tube into the electroporator for electroporation.

[0086] After the electroporation is completed, the cells in the electroporated tube were divided into shake flasks containing 20 mL of culture medium prepared in advance and incubated statically for 40 minutes;

[0087] After incubation, place the shake flask in 37°C, 270 rpm, 8% CO2 for incubation. After 24 hours, add feed / sodium butyrate / double antibody and continue incubation for 3-7 days.

[0088] (2) Protein A affinity chromatography column purification

[0089] The recombinant protein expressed by the cultured cells is purified by affinity chromatography.

[0090] Balance chromatography column: 1×PBS, flow rate 1mL / min, 20mL

[0091] Sample loading: flow rate 1 mL / min

[0092] Wash: 1×PBS, flow rate 1mL / min, 20mL

[0093] Elution: Citrate buffer (pH 3.4), 1 mL / min, collect in 10 tubes, approximately 500 μL per tube. Read absorbance at 280 nm using a NanoDrop instrument.

[0094] (3) Recombinant protein purity detection

[0095] 1) SDS PAGE detection

[0096] Preparation: Install the electrophoresis tank: Remove the glass holder containing the gel and insert it into the electrode holder, with the lower glass facing the red strip. Place the electrode holder into the motor base and turn on the switch. Remove the glass holder containing the gel and insert it into the electrode holder, with the lower glass facing the red strip. Place the electrode holder into the motor base and turn on the switch. Place the base into the electrophoresis tank and add electrophoresis buffer to the center of the electrode holder until it reaches 1 cm above the stacking gel. Then, add electrophoresis buffer to the tank until the liquid level reaches the top of the stacking gel.

[0097] Sample heating treatment: reducing heating at 99°C for 6 min; non-reducing heating at 99°C for 3 min.

[0098] Load the sample, run the gel, stain and destain, and photograph. Load 10 μL per well. Run at 180V for 45 minutes, stain and destain for 15 minutes. Set the gel camera to white light and photograph.

[0099] The results of SDS PAGE detection were as follows Figure 1 and Figure 2 shown.

[0100] 2)SEC-HPLC detection

[0101] SEC experiments were performed using a high performance liquid chromatograph LC-20AT and a gel chromatography column. The experimental conditions are shown in Table 1:

[0102] Table 1

[0103] parameter Numerical flow rate 1mL / min Injection volume 20 μL Column temperature 35℃ Detection wavelength 214nm, 280nm Collection time 15min

[0104] Replace the mobile phase with water and slowly increase the flow rate to 1.000 mL / min until the baseline stabilizes. Transfer 50 μL of antibody to the corresponding injection vial and place the vial in the corresponding position on the instrument. Inject for 15 minutes. Analyze and save the data. Replace the mobile phase with deionized water and rinse for 1.5 hours.

[0105] SEC-HPLC test results are as follows Figure 3-Figure 4 shown.

[0106] (4) Endotoxin detection

[0107] Preparation of sample positive control solution: 2 times the concentration of test solution + endotoxin standard (0.24EU / mL), 1:1 mixture

[0108] Preparation of test solution:

[0109] Sample dilution factor: MVD = C·L / λ, where MVD is the maximum effective dilution factor of the test sample, L is the bacterial endotoxin limit of the test sample (1 EU / mg), C is the test sample concentration, and λ is the labeled sensitivity of the limulus amebocyte lysate. The dosage of each test solution is shown in Table 2.

[0110] Table 2

[0111] Limulus amebocyte lysate (0.06 EU / mL) Endotoxin test water sample Negative control tube 200 μL none Positive control tube 100 μL 100 μL endotoxin standard (0.12 EU / mL) Sample positive control tube 100 μL 100 μL sample positive control solution Test tube 100 μL 100 μL test solution

[0112] Sample testing: Seal the tube, shake gently, and place vertically in a 37°C constant temperature incubator for 60 minutes.

[0113] The test results showed that the horseshoe crab reagent was clear and transparent and did not coagulate, indicating that the purified protein had a low endotoxin content.

[0114] Example 2 Determination of the blocking effect of recombinant protein on pembrolizumab (Keytruda)

[0115] The inventors defined PD-1 on T cells that is not bound to anti-PD-1 antibodies as free PD-1 (Free PD-1). After incubating the anti-PD-1 antibody drug with T cells, flow cytometry was used to detect the expression of CD3, CD4, CD8, and PD-1 on peripheral blood T cells of tumor patients. If the PD-1 molecules on T cells are bound by anti-PD-1 antibody drugs, they will not be detected by fluorescently labeled anti-PD-1 flow cytometry antibodies. The expression level of PD-1 molecules on peripheral blood T cells before incubation with anti-PD-1 antibodies is used as the baseline. When the free PD-1 recovery reaches more than 50% of the baseline, it is defined as free PD-1 recovery (Free PD-1Recover, FPR).

[0116] Pembrolizumab is a monoclonal antibody that binds to the PD-1 receptor, blocking the interaction between PD-1 and PD-L1 and PD-L2, relieving the PD-1 pathway-mediated immune response inhibition and restoring tumor-specific T cell immunity.

[0117] In this example, flow cytometry was used to detect different concentrations of wild-type PD-1 recombinant protein (PD-1 WT , amino acid sequence as shown in SEQ ID NO: 1), H126 recombinant protein, H134 recombinant protein and endogenous PD-1 molecules competitively bind to pembrolizumab.

[0118] The results showed that PD-1 WT , H126 recombinant protein, and H134 recombinant protein can compete with endogenous PD-1 molecules to bind to pembrolizumab ( Figure 5). When the PD-1 molecules on T cells are bound by anti-PD-1 antibody drugs, they cannot be detected by fluorescently labeled anti-PD-1 flow cytometry antibodies. Therefore, the more PD-1 molecules that can be detected (that is, the higher the PD-1 expression level), the better the blocking effect (competitive binding with endogenous PD-1 molecules). According to the statistical analysis of the test results, when the concentration of H134 recombinant protein with pembrolizumab is 1:1-2, the blocking effect of pembrolizumab on H134 recombinant protein is the best, followed by PD-1 WT , and finally the H126 recombinant protein ( Figure 5 ).

[0119] The determination of free PD-1 recovery (FPR) was also consistent with the above results. H134 recombinant protein had the best blocking effect on pembrolizumab, which could restore free PD-1 to more than 50% of the baseline, which was better than H126 recombinant protein ( Figure 6 ).

[0120] Example 3 Determination of the Blocking Effect of Recombinant Protein on Nivolumab (Opdivo)

[0121] Nivolumab can bind to programmed death receptor 1 (PD-1), blocking its interaction with PD-L1 and PD-L2, and blocking the immunosuppressive response mediated by the PD-1 pathway, including anti-tumor immune response.

[0122] In this example, flow cytometry was used to detect different concentrations of wild-type PD-1 recombinant protein (PD-1 WT ), H126 recombinant protein, H134 recombinant protein and endogenous PD-1 molecules compete for binding to nivolumab.

[0123] The results showed that PD-1 WT , H126 recombinant protein, and H134 recombinant protein can all compete with endogenous PD-1 molecules to bind to nivolumab ( Figure 7 ). According to the statistical analysis of the test results, when the concentration of H126 to nivolumab was 2:1, the blocking effect of H126 recombinant protein on nivolumab was the best, followed by H134 recombinant protein, and finally PD-1 WT ( Figure 7 ).

[0124] The determination of free PD-1 recovery (FPR) was also consistent with the above results. At a concentration of 2:1 with nivolumab, H126 recombinant protein had the best blocking effect on nivolumab, followed by H134 recombinant protein. Both groups of recombinant proteins could restore free PD-1 to above the baseline ( Figure 8 ).

[0125] Example 4 Determination of the Blocking Effect of Recombinant Protein on Sintilimab

[0126] Sintilimab can bind to the programmed death receptor 1 (PD-1) receptor expressed on T cells, blocking its interaction with PD-L1 and PD-L2, and blocking the immunosuppressive response mediated by the PD-1 pathway, including anti-tumor immune response.

[0127] In this example, flow cytometry was used to detect different concentrations of wild-type PD-1 recombinant protein (PD-1 WT ), H126 recombinant protein, H134 recombinant protein and endogenous PD-1 molecules compete for binding to sintilimab.

[0128] The results showed that PD-1 WT , H126 recombinant protein, and H134 recombinant protein can compete with endogenous PD-1 molecules to bind to sintilimab ( Figure 9 According to the statistical analysis of the test results, at a concentration of 2:1 with sintilimab, the blocking effects of H126 recombinant protein and H134 recombinant protein on sintilimab were comparable, and both were superior to PD-1 WT ( Figure 9 ).

[0129] The determination of free PD-1 recovery (FPR) was also consistent with the above results. When the concentration of H126 recombinant protein and H134 recombinant protein was 0.5-2:1 with sintilimab, both H126 recombinant protein and H134 recombinant protein could restore free PD-1 to more than 50% of the baseline ( Figure 10 At a 2:1 concentration of sintilimab, H126 and H134 recombinant proteins were equally effective, both restoring free PD-1 to above baseline.

[0130] Example 5 Determination of the blocking effect of recombinant protein on Tislelizumab

[0131] Tislelizumab can bind to the programmed death receptor 1 (PD-1) receptor expressed on T cells, blocking its interaction with PD-L1 and PD-L2, and blocking the immunosuppressive response mediated by the PD-1 pathway, including anti-tumor immune response.

[0132] In this example, flow cytometry was used to detect different concentrations of wild-type PD-1 recombinant protein (PD-1 WT ), H126 recombinant protein, H134 recombinant protein and endogenous PD-1 molecules compete for binding to tislelizumab.

[0133] The results showed that PD-1 WT, H126 recombinant protein, and H134 recombinant protein can all compete with endogenous PD-1 molecules to bind to tislelizumab ( Figure 11 ). According to the statistical analysis of the test results, when the concentration of PD-1 was 0.5-2:1 with tislelizumab, WT The blocking effect was the best, followed by H134 recombinant protein, and finally H126 recombinant protein ( Figure 11 ).

[0134] The determination of free PD-1 recovery (FPR) was also consistent with the above results. When the concentration of H134 recombinant protein with tislelizumab was 1-2:1, the free PD-1 recovery reached more than 50% of the baseline, which was better than that of H126 recombinant protein ( Figure 12 ).

[0135] Example 6 Determination of the Blocking Effect of Recombinant Protein on Treprinumab

[0136] Tepliizumab can bind to the programmed death receptor 1 (PD-1) receptor expressed on T cells, blocking its interaction with PD-L1 and PD-L2, and blocking the immunosuppressive response mediated by the PD-1 pathway, including anti-tumor immune response.

[0137] In this example, flow cytometry was used to detect different concentrations of wild-type PD-1 recombinant protein (PD-1 WT ), H126 recombinant protein, H134 recombinant protein and endogenous PD-1 molecules compete for binding to toripalimab.

[0138] The results showed that PD-1 WT , H126 recombinant protein, and H134 recombinant protein can all compete with endogenous PD-1 molecules to bind to Teplizumab ( Figure 13 When the concentration of H126 recombinant protein was 1-2:1 with that of Teplizumab, the blocking effect of H126 recombinant protein was better, followed by H134 recombinant protein. The blocking effects of both groups of recombinant proteins were better than those of PD-1. WT .

[0139] The determination of free PD-1 recovery (FPR) was also consistent with the above results. Both H126 and H134 recombinant proteins could restore free PD-1 to above the baseline ( Figure 14 ).

[0140] Example 7 Recombinant protein does not bind to endogenous PD-L1

[0141] PD-L1 expressed on tumor cells binds to PD-1 expressed on T cells, transmitting inhibitory signals to T cells and suppressing their anti-tumor effects. PD-1 antibody drugs block the binding between PD-1 expressed on T cells and PD-L1 expressed on tumor cells, activating T cells. Because PD-1 antibody drugs are non-selective in vivo, they can also activate T cells targeting self-antigens, leading to immunotherapy-related adverse reactions.

[0142] This embodiment uses surface plasmon resonance technology to detect PD-1 WT , the binding of H126 recombinant protein, H134 recombinant protein and PD-L1.

[0143] The results showed that PD-1 WT Can bind to endogenous PD-L1, while H126 recombinant protein and H134 protein do not bind to endogenous PD-L1 ( Figure 15-17 ).

[0144] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A protein molecule, characterized in that, the protein molecule is: a functional variant having at least 1 amino acid site difference compared with the wild-type protein shown in SEQ ID NO:1; the amino acid sequence of the protein molecule is shown in SEQ ID NO:2 or SEQ ID NO:3; the functional variant has the same, similar or better function of blocking the binding of anti-PD-1 antibody to PD-1 molecule as the wild-type protein shown in SEQ ID NO:

1.

2. A recombinant protein, characterized in that, the recombinant protein comprises the protein molecule according to claim 1.

3. The recombinant protein according to claim 2, characterized in that, the recombinant protein further comprises a bioactive protein or its functional fragment that assists its expression and / or secretion, or prolongs its half-life in vivo.

4. A blocking agent for anti-PD-1 antibody, characterized in that, the blocking agent competitively binds to the anti-PD-1 antibody with endogenous PD-1, does not bind to endogenous PD-L1, and does not block the binding of endogenous PD-L1 to endogenous PD-1, and the blocking agent comprises the protein molecule according to claim 1 or the recombinant protein according to any one of claims 2-3.

5. The blocking agent according to claim 4, characterized in that, the blocking agent further comprises a buffer.

6. Use of the protein molecule according to claim 1, the recombinant protein according to any one of claims 2-3, or the blocking agent according to claim 4 in the preparation of a drug for treating immune therapy-related adverse reactions.

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