Anti-pd-1 antibodies and biomaterials and products thereof

Abstract

This application relates to the field of biomedical technology, specifically to an anti-PD-1 antibody and its biomaterials and products. The anti-PD-1 antibody of this invention can effectively block the binding of PD-1 to PD-L1, and can be used for the prevention and / or treatment of cancer.

Description

An anti-PD-1 antibody and its biomaterial and product Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to an anti-PD-1 antibody and its biomaterials and products. Background Art

[0002] During tumor development and progression, numerous molecular regulatory mechanisms exist between immune cells and tumor cells. These mechanisms negatively impact the normal function of immune cells, thereby promoting tumor growth and metastasis. Immune checkpoints play a key role in this process. Programmed death-1 (PD-1, also known as CD279) and PD-1 ligand 1 (PDL1) are a pair of key checkpoint molecules that help tumor cells evade clearance by cytotoxic T lymphocytes (CTLs). When T cells interact with cancer cells, PD-1 (expressed on the T cell surface) binds and reacts with PD-L1 (overexpressed on various cancer cells, antigen-presenting cells, and endothelial cells). The formation of the PD-1 / PD-L1 complex negatively regulates T cell activation and effector function and may contribute to tumor immune evasion. Therefore, blocking the PD-1 / PD-L1 signaling pathway can restore the cytotoxic activity of tumor antigen-specific T cells and is widely considered a revolutionary therapy for a variety of malignancies. The FDA has approved several monoclonal antibodies that block PD-1 or PD-L1, including Nivolumab (PD-1, BMS), Atezolizumab (PD-L1, Roche), Pembrolizumab (PD-1, Merck), Durvalumab (PD-L1, AZ), and Avelumab (PD-L1, Merck). These monoclonal antibodies have shown effective anti-tumor activity against a variety of malignancies.

[0003] Although these therapeutic drugs have achieved good therapeutic effects, PD-1 monoclonal antibodies still face many challenges, including 1) large molecular size (160-170kD), which limits their ability to penetrate solid tumors and the blood-brain barrier, and 2) the large workload involved in the in vitro eukaryotic expression and cell screening processes, resulting in high production costs.

[0004] Summary of the Invention

[0005] In view of this, the present invention provides an anti-PD-1 antibody (e.g., nanobody), biomaterial, and product thereof. This anti-PD-1 antibody has one of the following advantages: small molecular weight, simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility, and low aggregation or easy cloning. It can effectively block the binding of PD-1 to PD-L1 and is used to prevent and / or treat PD-1-positive cancers.

[0006] The present invention provides the following technical solutions:

[0007] The present invention provides an anti-PD-1 antibody and a variant thereof, or an antigen-binding fragment thereof, wherein the anti-PD-1 antibody comprises three complementary determining regions CDR1, CDR2 and CDR3 (as indicated by horizontal lines in italics) contained in VHH named PD1-A1, PD1-A2, PD1-A3, PD1-C1, PD1-C2, PD1-C3, PD1-C4, PD1-C5, and PD1-C6.

[0008] Illustratively, for the antibodies and variants thereof described herein, or antigen-binding fragments thereof, the variants are humanized variants or identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) variants.

[0009] Further, the antibodies and variants thereof described herein, or antigen-binding fragments thereof, are selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bi-scFv, (scFv)2, miniantibodies, double-chain antibodies, three-chain antibodies, four-chain antibodies, disulfide-stabilized Fv proteins and single-domain antibodies (sdAb, nanoantibodies), bispecific antibodies or trispecific antibodies.

[0010] The present invention also provides a fusion protein comprising the antibody and variants thereof described herein, or an antigen-binding fragment thereof.

[0011] Exemplarily, the fusion protein described herein further comprises a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an additional epitope (e.g., an epitope directed against or other epitopes different from PD-1) or an additional antibody active fragment (e.g., an antibody or antibody active fragment directed against other epitopes or the same epitope of PD-1, or a ligand capable of binding to PD-1).

[0012] The present invention also provides an antibody-drug conjugate comprising the antibody and variants thereof described herein, or an antigen-binding fragment thereof.

[0013] For the antibody-drug conjugate described herein, the drug is selected from the following: radiolabels, 32 P. 35S, fluorescent dyes, electron-dense reagents, enzymes, biotin, streptavidin, digoxigenin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to a target, or a combination of any of the foregoing; or immunomodulatory compounds, anticancer agents, antiviral agents, antibacterial agents, antifungal agents, and antiparasitic agents, or a combination of any of the foregoing.

[0014] The present invention also provides an isolated polynucleotide expressing an antibody or an antigen-binding fragment thereof, characterized in that the polynucleotide is capable of expressing the antibody and its variants, or its antigen-binding fragment described herein; the polynucleotide is capable of expressing the fusion protein described herein; the polynucleotide is capable of expressing the antibody-drug conjugate described herein.

[0015] The present invention also provides a vector comprising the polynucleotide described herein, preferably a plasmid vector.

[0016] The present invention also provides a host cell comprising the polynucleotide described herein or the vector described herein. Preferably, the host cell is a eukaryotic cell.

[0017] The present invention also provides a pharmaceutical composition comprising the antibody and variants thereof described herein, or antigen-binding fragments thereof, the fusion protein described herein, the antibody-drug conjugate described herein, and optionally, a pharmaceutically acceptable carrier.

[0018] The present invention also provides use of the antibody and variants thereof described herein, or antigen-binding fragments thereof, the fusion protein described herein, and the antibody-drug conjugate described herein in the preparation of drugs for treating and / or preventing PD-1-related diseases.

[0019] The present invention also provides a method for treating and / or preventing PD-1-related diseases and related symptoms, comprising administering an effective amount of the antibody and variants thereof described herein, or an antigen-binding fragment thereof, the fusion protein described herein, the antibody-drug conjugate described herein, or the pharmaceutical composition described herein to a subject.

[0020] The present invention also provides a method for detecting whether a sample contains T cells that highly express PD-1, comprising the step of contacting the antibody and variants thereof described herein, or antigen-binding fragments thereof, or the fusion protein described herein with the sample. Optionally, the detection can be for diagnostic purposes or non-diagnostic purposes.

[0021] The present invention also provides a detection product, which comprises the antibody and variants thereof described herein, or an antigen-binding fragment thereof.

[0022] Illustratively, for the detection product described herein, the product is selected from one or more of a detection reagent, a kit, a chip, or a test paper.

[0023] In order to achieve the above-mentioned object of the invention, the present invention further provides the following technical solutions:

[0024] In a first aspect, the present invention provides an anti-PD-1 antibody (e.g., a nanobody), wherein the heavy chain variable region of the anti-PD-1 antibody (e.g., a nanobody) consists of a framework region FR and a complementarity determining region CDR, wherein the complementarity determining region CDR comprises any one or at least one of the following groups:

[0025] (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3;

[0026] (2) CDR1 shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10;

[0027] (3) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17;

[0028] (4) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24;

[0029] (5) CDR1 shown in SEQ ID NO: 29, CDR2 shown in SEQ ID NO: 30, and CDR3 shown in SEQ ID NO: 31;

[0030] (6) CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 37, and CDR3 shown in SEQ ID NO: 38;

[0031] (7) CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 44, and CDR3 shown in SEQ ID NO: 45;

[0032] (8) CDR1 shown in SEQ ID NO: 50, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 52;

[0033] (9) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 58, and CDR3 shown in SEQ ID NO: 59.

[0034] Nanobodies (Nb) are the variable regions of heavy-chain antibodies (IgG2 and IgG3) found in camelids and are known as the smallest antigen-binding fragments found in nature. Compared to traditional full-length monoclonal antibodies (mAbs, approximately 150 kD), Nb have advantages such as a smaller molecular weight (12-15 kD), a simpler structure, lower immunogenicity, higher tissue permeability, higher stability, higher solubility, lower aggregation, and ease of cloning. Furthermore, compared to mAb counterparts, Nb production costs are significantly lower, making them accessible to most cancer patients. In September 2018, the European Medicines Agency approved the first nanobody drug, caplacizumab (trade name Cablivi), for the treatment of acquired thrombotic thrombocytopenic purpura (aTTP) in adults. Therefore, Nb are expected to have a wide range of applications in cancer treatment and diagnosis.

[0035] In this study, camels were immunized with recombinant PD-1 antigens to construct a VHH phage library. After biopanning, nine functional anti-PD-1 Nbs were obtained. For example, by comparing the blocking effects of Nbs on PD-1 and PD-L1, Nbs designated PD1-A2, PD1-C1, and PD1-C6, respectively, were shown to be effective in blocking the PD-1 / PD-L1 signaling pathway.

[0036] In the embodiment provided by the present invention, the framework region FR includes any one or at least one of the following groups:

[0037] (1) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7;

[0038] (2) FR1 shown in SEQ ID NO: 11, FR2 shown in SEQ ID NO: 12, FR3 shown in SEQ ID NO: 13, and FR4 shown in SEQ ID NO: 14;

[0039] (3) FR1 shown in SEQ ID NO: 18, FR2 shown in SEQ ID NO: 19, FR3 shown in SEQ ID NO: 20, and FR4 shown in SEQ ID NO: 21;

[0040] (4) FR1 set forth in SEQ ID NO: 25, FR2 set forth in SEQ ID NO: 26, FR3 set forth in SEQ ID NO: 27, and FR4 set forth in SEQ ID NO: 28;

[0041] (5) FR1 set forth in SEQ ID NO: 32, FR2 set forth in SEQ ID NO: 33, FR3 set forth in SEQ ID NO: 34, and FR4 set forth in SEQ ID NO: 35;

[0042] (6) FR1 set forth in SEQ ID NO: 39, FR2 set forth in SEQ ID NO: 40, FR3 set forth in SEQ ID NO: 41, and FR4 set forth in SEQ ID NO: 42;

[0043] (7) FR1 set forth in SEQ ID NO: 46, FR2 set forth in SEQ ID NO: 47, FR3 set forth in SEQ ID NO: 48, and FR4 set forth in SEQ ID NO: 49;

[0044] (8) FR1 set forth in SEQ ID NO: 53, FR2 set forth in SEQ ID NO: 54, FR3 set forth in SEQ ID NO: 55, and FR4 set forth in SEQ ID NO: 56;

[0045] (9) FR1 shown in SEQ ID NO: 60, FR2 shown in SEQ ID NO: 61, FR3 shown in SEQ ID NO: 62, and FR4 shown in SEQ ID NO: 63.

[0046] However, the framework region FR of the present invention is not limited to the above sequences, and any sequence that can achieve its function is within the scope of protection of the present invention.

[0047] In the embodiments provided by the present invention, the amino acid sequence of the anti-PD-1 nanobody is shown in SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 or SEQ ID NO: 72.

[0048] The CDR and FR sequences of PD1-A2, PD1-C1, and PD1-C6 are as follows:

[0049] The CDR and FR sequences of PD1-A1, PD1-A3, and PD1-C2 to C5 are as follows:

[0050] SEQ ID NO: 64 (PD1-A1):

[0051] SEQ ID NO: 65 (PD1-A2):

[0052] SEQ ID NO: 66 (PD1-A3):

[0053] SEQ ID NO: 67 (PD1-C1):

[0054] SEQ ID NO: 68 (PD1-C2):

[0055] SEQ ID NO: 69 (PD1-C3):

[0056] SEQ ID NO: 70 (PD1-C4):

[0057] SEQ ID NO: 71 (PD1-C5):

[0058] SEQ ID NO: 72 (PD1-C6):

[0059] The second aspect of the present invention provides a polynucleotide encoding the above-mentioned anti-PD-1 antibody (eg, nanobody).

[0060] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned polynucleotide.

[0061] In the embodiments provided by the present invention, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.

[0062] The fourth aspect of the present invention provides a recombinant host cell, which comprises the above-mentioned polynucleotide or the above-mentioned recombinant expression vector.

[0063] In embodiments provided herein, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0064] In specific embodiments provided by the present invention, the host cell is selected from Escherichia coli or yeast cells.

[0065] In specific embodiments provided by the present invention, the host cell is selected from HEK293T cells, HEK293F cells, Expi293F cells or CHO cells.

[0066] A fifth aspect of the present invention provides a method for preparing the above-mentioned anti-PD-1 antibody (e.g., nanobody), comprising the following steps:

[0067] Inserting a polynucleotide encoding the above-mentioned anti-PD-1 antibody (e.g., Nanobody) into an expression vector to obtain a recombinant expression vector;

[0068] Transforming the recombinant expression vector into a host cell to obtain a recombinant host cell;

[0069] culturing the recombinant host cell to obtain a culture;

[0070] The culture medium is purified to obtain anti-PD-1 antibodies (eg, nanobodies).

[0071] In the embodiments provided by the present invention, purification is performed using Protein A agarose purification resin.

[0072] In a sixth aspect, the present invention provides a bispecific antibody comprising the above-mentioned anti-PD-1 antibody (eg, nanobody) and a second antibody.

[0073] In the embodiments provided herein, the second antibody includes but is not limited to 4-1BB nanobody, CD47 nanobody, VEGF nanobody, HER2 nanobody, EGFR nanobody, HER3 nanobody, B7H3 nanobody, TIGIT nanobody, OX-40 nanobody, CD40 nanobody or PD-L1 nanobody.

[0074] In a seventh aspect, the present invention provides the use of the above-mentioned anti-PD-1 antibodies (such as nanobodies) or bispecific antibodies in the preparation of drugs for preventing and / or treating cancer, and in the detection of PD-1 protein.

[0075] In the embodiments provided herein, cancer includes but is not limited to gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colorectal cancer, breast cancer, large intestine cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor or bladder tumor.

[0076] An eighth aspect of the present invention provides a pharmaceutical composition, comprising: the above-mentioned anti-PD-1 antibody (such as a nanobody) or bispecific antibody; and pharmaceutically acceptable excipients.

[0077] In the embodiments provided herein, the dosage forms of the pharmaceutical composition include but are not limited to injection, powder injection, tablet or capsule.

[0078] A ninth aspect of the present invention provides a kit for detecting PD-1 protein, comprising: the above-mentioned anti-PD-1 antibody (eg, nanobody) or bispecific antibody; and a reagent acceptable in detection science.

[0079] The tenth aspect of the present invention provides a chimeric antigen receptor (CAR), the extracellular domain of which comprises the anti-PD-1 antibody described herein.

[0080] In an eleventh aspect, the present invention provides a cell comprising or expressing the CAR described herein, such as a T cell or NK cell.

[0081] A twelfth aspect of the present invention provides a method for treating cancer, which comprises administering to a subject the antibody described herein or cells comprising or expressing the CAR described herein, such as T cells or NK cells.

[0082] Compared with the prior art, the present invention has one of the following beneficial effects:

[0083] The present invention has learned through the affinity experiment of antibodies (such as nanobodies) and antigen recombinant proteins that the affinity EC of PD1-A2, PD1-C1, PD1-C6 VHH-his recombinant antibodies and human PD-1 extracellular region recombinant protein is 50 =0.13~42.17μg / mL;

[0084] The present invention has learned through affinity experiments between antibodies (such as nanobodies) and antigen recombinant proteins that the VHH-his recombinant antibodies of PD1-A2, PD1-C1, and PD1-C6 can bind to human PD-1 overexpressing 293F cells;

[0085] The present invention learned through PD-1 / PD-L1 blocking experiments that compared with the positive drug Keytruda (IgG full-length antibody), PD1-A2, PD1-C1 and PD1-C6 (VHH-His recombinant antibodies) can block the binding of PD-1 and PD-L1 and restore the TCR signaling pathway; further, the PD1-C1 recombinant antibody can completely block the binding of PD-1 and PD-L1, IC 50 =0.68μg / mL; IC of Keytruda blocking the binding of PD-1 and PD-L1 50 =0.57μg / mL. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is the ELISA binding curve of VHH-His recombinant antibody to human PD-1 (hFc tag);

[0087] Figure 2 shows the dose-effect curve of PD-1 antibody blocking PD-1 / PD-L1 binding;

[0088] FIG3 is an ELISA binding curve of VHH-His recombinant antibody to monkey PD-1 (his tag). DETAILED DESCRIPTION

[0089] The present invention discloses an anti-PD-1 antibody (e.g., nanobody) and its biomaterials and products. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0090] The present invention will be further described below in conjunction with the embodiments:

[0091] Example 1: Antigen preparation and animal immunization

[0092] 1. Animal immunization:

[0093] The human PD-1 extracellular domain recombinant protein (hFc tag) was injected into multiple masses at multiple points in the subcutaneous and intramuscular areas of the neck and back of an alpaca and a Bactrian camel, respectively. The absorption of the subcutaneous injection masses was tracked to confirm correct immunization.

[0094] For the first immunization, 0.5 mg of antigen was mixed with Freund's complete adjuvant in a ratio of 1:1 and injected after emulsification. The volume was 1 mL per alpaca or camel.

[0095] Second immunization: 3 weeks after the first immunization, use 0.25 mg of antigen mixed with Freund's incomplete adjuvant in a ratio of 1:1, emulsify and inject, with an injection volume of 1 mL per alpaca or camel;

[0096] The third immunization: 3 weeks after the second immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant in a ratio of 1:1, emulsified and injected with an injection volume of 1 mL per alpaca or camel;

[0097] Fourth immunization: 3 weeks after the third immunization, use 0.25 mg of antigen mixed with Freund's incomplete adjuvant in a ratio of 1:1, emulsify and inject, with an injection volume of 1 mL per alpaca or camel.

[0098] 2. Serum processing and titer detection:

[0099] One week after the third and fourth immunizations, 2 mL of peripheral blood was collected and serum was isolated. Recombinant human PD-1 extracellular domain protein (his tag) was coated onto a 96-well ELISA plate, and serum antibody titers were determined using ELISA. ELISA results showed that the titer of serum from three alpaca immunizations was >1:64,000, meeting the library construction criteria; and the titer of serum from four camel immunizations was >1:64,000, also meeting the library construction criteria.

[0100] Example 2: Construction of phage display immune antibody library

[0101] Since the serum titer of alpacas after the third immunization was >1:32000 and that of camels after the fourth immunization was >1:32000, it indicated that high-affinity antibodies against human PD-1 were present in the serum. Therefore, a phage display immune antibody library was constructed according to the following steps:

[0102] ① Collect 50 mL of peripheral blood from alpacas after the third immunization and camels after the fourth immunization, and separate PBMCs; take 2×10 7 PBMCs were cultured and total RNA was extracted using an RNA extraction kit; an appropriate amount of RNA (e.g., 3-5 μg) was taken and cDNA was obtained using an RT-PCR reverse transcription kit.

[0103] ② The IgG2 and IgG3 heavy chain variable region sequences (the heavy chain variable region VHH of the nanobody) were obtained step by step by nested PCR. The experimental steps are as follows: 1) Design a pair of specific nested outer primers and use cDNA as a template for the first round of PCR amplification. The amplified region is the Leader-CH2 region of the heavy chain antibody gene of alpaca and camel, and the product size is 700bp and 900bp; use DNA gel electrophoresis and gel cutting to recover the 700bp PCR product; 2) Design nested inner primers (3 pairs for alpaca and 5 pairs for camel) and use the 700bp first round PCR product as a template for the second round of PCR amplification. The amplified region is the VHH fragment of the heavy chain antibody variable region of alpaca and camel, and the product size is 400bp; use a PCR product purification kit to purify and recover the second round PCR product.

[0104] ③ Insert the heavy chain variable region sequence into the linearized phagemid vector VHH-libTemplate that has been treated with enzymes through homologous recombination or enzyme ligation to obtain a recombinant vector; after purification and recovery, transform supercompetent SS320 cells (containing helper phage M13K07); the transformed bacterial liquid is resuspended in SOC medium and activated for 1 hour; a small amount of bacterial liquid is diluted 10-fold in a serial dilution, and the appropriate dilution titer is selected. Plate the plate on LB / tet10 and LB / Carb50 culture plates and place them in a 37°C biochemical incubator overnight. The next day, the library capacity is calculated; the remaining bacterial liquid is transferred to a large volume of 2YT / Carb50 / Kan25 liquid medium and placed in a shaker at 37°C for overnight incubation. The supernatant is harvested the next day, and 1 / 4 volume of PEG / NaCl solution is added to precipitate the phage. After that, an appropriate amount of PBT solution is used to resuspend and dilute to the required concentration to obtain the phage display immune antibody library (stored at -80°C for future use).

[0105] ④ Count the number of clones on the LB / Carb50 plate and calculate the library capacity: the library capacity of the alpaca antibody library Lib PD-1 Alpaca is 1.78×10 9The library capacity of camel antibody library Lib PD-1 Camel is 4.6×10 9 . 20 monoclonal clones were randomly picked from each plate for sequencing. The results showed that the VHH insertion efficiency of the alpaca antibody library Lib PD-1 Alpaca was 75%, and the VHH insertion efficiency of the camel antibody library Lib PD-1 Camel was 80%.

[0106] Example 3: Screening of antibody library

[0107] 5 μg / mL of human PD-1 extracellular domain recombinant protein (his tag) was added to a 96-well plate (100 μL / well) and coated overnight at 4°C. NEB5αF' Escherichia coli was streaked onto a 2YT / Tet10 plate and incubated overnight at 37°C. The next day, NEB5αF' monoclonal colonies were picked from the overnight 2YT / Tet10 plate and added to 3 mL of 2YT / Tet10 liquid culture medium. The cells were shaken and grown at 37°C until the OD 600 =0.8; at the same time, remove the antigen supernatant from the 96-well plate, add 200 μL of 1% BSA to each well for blocking, and add 200 μL of 1% BSA to the blank well as a negative control well, and place it on a 3D rotary shaker at room temperature for 2 hours; then, remove the supernatant from the protein wells and control wells, wash with 200 μL of PT, add 100 μL of phage antibody library to each well, and place it on a 3D rotary shaker at room temperature for 2 hours; remove the supernatant from the protein wells and control wells, wash with 200 μL of PT; add 100 μL of 100 mM HCl to the wells and place them at room temperature for 5 minutes; aspirate the supernatant, add it to a 1.5 mL centrifuge tube, and neutralize it with 1 M Tris-HCl. The above mixture was added to a centrifuge tube containing 1 mL of NEB5αF' bacteria and cultured at 37°C for 1 hour. 20 μL of the culture medium in the centrifuge tube was diluted to an appropriate multiple and plated on an LB / Carb50 culture plate. The plate was placed in a 37°C biochemical incubator overnight and used for titer and enrichment calculation the next day. 1 μL of helper phage M13K07 (final concentration of 10 10 The above culture medium was transferred into 35 mL of 2YT / Carb50 / Kan25 culture medium, placed in a shaker, and cultured at 37°C overnight to collect phages to form the antibody library of each round.

[0108] Repeat the above steps for 2-3 rounds until phage enrichment occurs. Enrichment is considered successful if the number of colonies in the antigen-binding wells on the LB / Carb50 plate is at least 10 times that of the negative control wells. In this experiment, after the third round of screening for Lib PD-1 Alpaca, the number of colonies in the antigen-binding wells was 100 times that of the negative control wells, and after the second round of screening for Lib PD-1 Camel, the number of colonies in the antigen-binding wells was 10 times that of the negative control wells, indicating successful enrichment.

[0109] Randomly select clones from the enrichment round and expand them in 96-well plates. After centrifugation, use the supernatant for Phage ELISA screening. Select clones with an OD value > 2 for binding to the human PD-1 extracellular domain recombinant protein (his tag) and blocking buffer. These clones are defined as positive clones and sequenced and aligned to obtain unique sequences.

[0110] Example 4: NGS sequencing

[0111] Primers were designed to flank the constant regions of the VHHs to amplify the variable regions of the phage library. PCR products were analyzed on an E-gel and purified using a kit. The PCR library fragments were then recovered for NGS sequencing. The resulting data were statistically analyzed using SPSS 2.0 and Microsoft Excel 2019, with the data sorted by DNA number and frequency.

[0112] Example 5: Comparison and statistics of positive sequences and NGS high-frequency sequences

[0113] The phage ELISA-positive sequences and NGS high-frequency sequences (Top 100-500) were aligned and grouped according to the differences in CDR H3. The results are shown in Tables 1 and 2.

[0114] Table 1. PD-1 nanoantibodies obtained by alpaca immunization Note: The underlined part is the CDR region.

[0115] Table 2. PD-1 Nanobodies Obtained from Camel Immunization Note: The underlined part is the CDR region.

[0116] Example 6: Eukaryotic expression of nanobodies

[0117] The sequences in Table 1 and Table 2 of Example 5, a total of 9 sequences, were eukaryotically expressed. The experimental steps were as follows:

[0118] 1) Amplify the VHH fragments of these sequences by PCR and insert the fragments into the eukaryotic expression vector pFcIG-His containing a His tag using homologous recombination or enzyme ligation. Electrophoretically transform the fragments into the Escherichia coli trans5α host bacteria, select with bleomycin, and sequence the single clones to obtain the correct recombinant plasmids. The host bacteria containing the recombinant plasmids are then expanded and sterile and endotoxin-free plasmids are obtained using an endotoxin removal kit.

[0119] 2) HEK293F cells were cultured in serum-free medium and transfected with the recombinant expression plasmid using Polyplus suspension cell transfection reagent for expression. Feed was added 24 and 72 hours after transfection. Supernatant was collected on day 5 and the antibody was purified using Protein A agarose resin and then stored in PBS.

[0120] The experimental results (Table 3) showed that the yields of the nine VHH-his recombinant antibodies transiently expressed in 293F cells ranged from 5.4 to 36.4 mg / L. SDS-PAGE electrophoresis analysis showed that the VHH-his bands for the PD1-A1, PD1-A2, PD1-A3, PD1-C1, PD1-C2, and PD1-C3 sequences were of normal size and had a purity >95%. The recombinantly expressed antibodies for the PD1-C4, PD1-C5, and PD1-C6 sequences exhibited nonspecific bands with a purity <95%.

[0121] Table 3. Eukaryotic transient expression results of VHH-His recombinant antibodies

[0122] Example 7: Affinity EC of Nanobodies and Antigen Recombinant Proteins 50

[0123] The affinity of nine recombinant VHH-his antibodies for the human PD-1 extracellular domain recombinant protein was determined using ELISA. Human PD-1 extracellular domain recombinant protein (hFc tag) was added to a 96-well ELISA plate at 100 ng / well and coated overnight at 4°C. The VHH-his recombinant antibody was diluted to various concentrations (0.01-10 μg / mL) and reacted with the antigen in an ELISA. HRP-labeled anti-His secondary antibody was used for color development, and the absorbance at 450 nm was measured using a microplate reader.

[0124] The experimental results (Table 4 and Figure 1) show that the affinity EC values ​​of the VHH-his recombinant antibodies of PD1-A1, PD1-A2, PD1-A3, PD1-C1, PD1-C2, PD1-C3, and PD1-C5 to the recombinant protein of the extracellular region of human PD-1 are 50 =0.13~9.86μg / mL.

[0125] Table 4. Binding affinity of VHH-His recombinant antibodies to human PD-1 (hFc tag)

[0126] Example 8: Binding of Nanobodies to Human PD-1 Overexpressing Cells

[0127] Flow cytometry was used to detect the affinity of nanoantibodies to human PD-1 overexpressing 293F cells:

[0128] 1) Transfect 293F cells with a eukaryotic expression plasmid containing the full-length PD-1 gene and continue culturing for 24 hours;

[0129] 2) Take 0.3×10 6 293F cells transiently overexpressing human PD-1 were washed twice with PBS and then resuspended in 100 μL PBS;

[0130] 3) Incubate with 10 μg / mL or 50 μg / mL VHH-his recombinant antibody or PD-L1 (his tag) recombinant protein for 1 hour;

[0131] 4) Wash the cells three times with PBS, resuspend the cells in 100 μL PBS, add 0.2 μg / mL PE-labeled Anti-his antibody, and incubate for 1 hour;

[0132] 5) After washing the cells three times with PBS, the cells were resuspended in 300 μL PBS and fluorescence was detected using a flow cytometer. The results are shown in Table 4.

[0133] The experimental results showed that the VHH-his recombinant antibodies of PD1-A1, PD1-A2, PD1-C1, PD1-C2, PD1-C3, and PD1-C5 can bind to human PD-1 overexpressing 293F cells.

[0134] Table 5. Binding affinity of VHH-His recombinant antibodies to human PD-1 overexpressing 293F cells

[0135] Example 9: PD-1 / PD-L1 blocking experiment

[0136] The PD-1 / PD-L1 Blockade Bioassay was used to detect the blocking effect of the Nanobody on PD-1 binding to PD-L1:

[0137] 1) Add aAPC / CHO-K1 cells stably expressing human PD-L1 into 96-well plates, 5×10 4 / well, culture overnight.

[0138] 2) The recombinant antibody was incubated with Jurcat effector cells (1×10 5 The cells were added to a 96-well plate and co-cultured with aAPC / CHO-K1 cells expressing human PD-L1 for 6 hours.

[0139] 3) Add luciferase substrate, incubate at room temperature for 10 minutes, read the plate using a plate reader, and analyze the data using GraphPad Prism.

[0140] First, a single-concentration blocking experiment was performed using 10 μg / mL of recombinant antibodies. The experimental results (Table 6) showed that compared with the positive drug Keytruda (full-length IgG antibody), PD1-A2, PD1-C1, and PD1-C6 (VHH-His recombinant antibodies) could block the binding of PD-1 to PD-L1 and restore the TCR signaling pathway.

[0141] A concentration gradient of PD1-A2 and PD1-C1 recombinant antibodies was further set (the starting concentration was 30 μg / mL of recombinant antibody, 3-fold dilution, a total of 9 concentrations), and PD-1 / PD-L1 blocking experiments were performed using recombinant antibodies of different concentrations. The experimental results (Figure 2) showed that the PD1-C1 recombinant antibody could completely block the binding of PD-1 and PD-L1, and the IC 50 =0.68μg / mL; IC of Keytruda blocking the binding of PD-1 and PD-L1 50 =0.57μg / mL.

[0142] Table 6. PD-1 / PD-L1 blocking assay of VHH-His recombinant antibodies (10 μg / mL)

[0143] Example 10: ELISA binding of PD1-A2 and PD1-C1 to monkey and mouse PD-1 extracellular domain antigen recombinant proteins

[0144] The affinity of PD1-A2 and PD1-C1 VHH-his recombinant antibodies to monkey or mouse PD-1 extracellular domain recombinant proteins was determined using ELISA: 100 ng / well of monkey or mouse PD-1 extracellular domain recombinant protein (his tag) was added to a 96-well ELISA plate and coated overnight at 4°C. The VHH-his recombinant antibody was diluted to various concentrations (0.01-10 μg / mL) and reacted with the antigen in an ELISA. HRP-labeled anti-VHH secondary antibody was used for color development, and the absorbance at 450 nm was measured using a microplate reader.

[0145] The experimental results (Table 7 and Figure 3) show that the affinity EC of PD1-A2 and PD1-C1 VHH-his recombinant antibodies to monkey PD-1 extracellular domain recombinant protein is 50 The concentrations of PD1-A2 and PD1-C1 were 1.69 μg / mL and 0.93 μg / mL, respectively; however, PD1-A2 and PD1-C1 did not bind to the mouse PD-1 extracellular domain antigen recombinant protein.

[0146] Table 7. Binding affinity of VHH-His recombinant antibodies to monkey PD-1 (hFc tag)

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An anti-PD-1 antibody (e.g., a nanobody), It is characterized in that The anti-PD-1 antibody comprises the CDR in any one of SEQ ID NOs: 64-72. Exemplarily, the antibody comprises a complementary determining region CDR, and the complementary determining region CDR comprises any one of the following groups: (1) CDR1 shown in SEQ ID NO: 1, CDR2 shown in SEQ ID NO: 2, and CDR3 shown in SEQ ID NO: 3; (2) CDR1 shown in SEQ ID NO: 8, CDR2 shown in SEQ ID NO: 9, and CDR3 shown in SEQ ID NO: 10; (3) CDR1 shown in SEQ ID NO: 15, CDR2 shown in SEQ ID NO: 16, and CDR3 shown in SEQ ID NO: 17; (4) CDR1 shown in SEQ ID NO: 22, CDR2 shown in SEQ ID NO: 23, and CDR3 shown in SEQ ID NO: 24; (5) CDR1 shown in SEQ ID NO: 29, CDR2 shown in SEQ ID NO: 30, and CDR3 shown in SEQ ID NO: 31; (6) CDR1 shown in SEQ ID NO: 36, CDR2 shown in SEQ ID NO: 37, and CDR3 shown in SEQ ID NO: 38; (7) CDR1 shown in SEQ ID NO: 43, CDR2 shown in SEQ ID NO: 44, and CDR3 shown in SEQ ID NO: 45; (8) CDR1 shown in SEQ ID NO: 50, CDR2 shown in SEQ ID NO: 51, and CDR3 shown in SEQ ID NO: 52; (9) CDR1 shown in SEQ ID NO: 57, CDR2 shown in SEQ ID NO: 58 and CDR3 shown in SEQ ID NO:

59.

2. The anti-PD-1 antibody according to claim 1, It is characterized in that The antibody further comprises a framework region FR, wherein the framework region FR comprises any one of the following groups: (1) FR1 shown in SEQ ID NO: 4, FR2 shown in SEQ ID NO: 5, FR3 shown in SEQ ID NO: 6, and FR4 shown in SEQ ID NO: 7; (2) FR1 shown in SEQ ID NO: 11, FR2 shown in SEQ ID NO: 12, FR3 shown in SEQ ID NO: 13, and FR4 shown in SEQ ID NO: 14; (3) FR1 shown in SEQ ID NO: 18, FR2 shown in SEQ ID NO: 19, FR3 shown in SEQ ID NO: 20, and FR4 shown in SEQ ID NO: 21; (4) FR1 shown in SEQ ID NO: 25, FR2 shown in SEQ ID NO: 26, FR3 shown in SEQ ID NO: 27, and FR4 shown in SEQ ID NO: 28; (5) FR1 shown in SEQ ID NO:32, FR2 shown in SEQ ID NO:33, FR3 shown in SEQ ID NO:34, and FR4 shown in SEQ ID NO:35; (6) FR1 shown in SEQ ID NO:39, FR2 shown in SEQ ID NO:40, FR3 shown in SEQ ID NO:41, and FR4 shown in SEQ ID NO:42; (7) FR1 set forth in SEQ ID NO:46, FR2 set forth in SEQ ID NO:47, FR3 set forth in SEQ ID NO:48, and FR4 set forth in SEQ ID NO:49; (8) FR1 set forth in SEQ ID NO:53, FR2 set forth in SEQ ID NO:54, FR3 set forth in SEQ ID NO:55, and FR4 set forth in SEQ ID NO:56; (9) FR1 shown in SEQ ID NO:60, FR2 shown in SEQ ID NO:61, FR3 shown in SEQ ID NO:62, and FR4 shown in SEQ ID NO:

63.

3. Anti-PD-1 antibodies, It is characterized in that The anti-PD-1 antibody comprises any of the following amino acid sequences or its humanized sequence or a sequence with more than 90% identity: SEQ ID NO: 64, SEQ ID NO: 65, SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68, SEQ ID NO: 69, SEQ ID NO: 70, SEQ ID NO: 71 or SEQ ID NO:

72.

4. A polynucleotide, It is characterized in that The polynucleotide is a polynucleotide encoding the anti-PD-1 antibody according to any one of claims 1 to 3.

5. A recombinant expression vector, It is characterized in that The recombinant expression vector comprises the polynucleotide according to claim 4.

6. A recombinant host cell, It is characterized in that The recombinant host cell comprises the polynucleotide according to claim 4, or comprises the expression vector according to claim 5.

7. A method for preparing the anti-PD-1 antibody according to any one of claims 1 to 3, It is characterized in that The steps include: Inserting a polynucleotide encoding the anti-PD-1 antibody according to any one of claims 1 to 3 into an expression vector to obtain a recombinant expression vector; Transforming the recombinant expression vector into a host cell to obtain a recombinant host cell; Cultivating the recombinant host cell to obtain a culture; The culture medium was purified to obtain anti-PD-1 antibody.

8. A bispecific antibody, It is characterized in that The bispecific antibody comprises the anti-PD-1 antibody according to any one of claims 1 to 3 and a second antibody.

9. Use of the anti-PD-1 antibody according to any one of claims 1 to 3 or the bispecific antibody according to claim 8 in the preparation of a drug for preventing and / or treating cancer or detecting PD-1 protein.

10. A pharmaceutical composition, It is characterized in that The pharmaceutical composition comprises: the anti-PD-1 antibody according to any one of claims 1 to 3 or the bispecific antibody according to claim 8; and a pharmaceutically acceptable excipient.

11. A kit for detecting PD-1 protein, It is characterized in that The kit comprises: the anti-PD-1 antibody according to any one of claims 1 to 3 or the bispecific antibody according to claim 8; and a reagent acceptable in detection.

12. A chimeric antigen receptor (CAR), the extracellular domain of which comprises the anti-PD-1 antibody according to any one of claims 1 to 3.

13. A cell comprising or expressing the CAR of claim 12, such as a T cell or a NK cell.

14. A method for treating cancer, comprising administering to a subject the antibody of any one of claims 1 to 3 or a cell comprising or expressing a CAR thereof, such as a T cell or a NK cell.

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