Anti-4-1BB nano antibody as well as preparation and application thereof

Abstract

Relates to the technical field of biological medicines, in particular to an anti-4BB nano antibody as well as preparation and application thereof. The complementarity determining region of the anti-4BB-1BB nano antibody comprises CDR1, CDR2, CDR3 and the like shown in SEQ ID NOs: 1-7. The invention further discloses a preparation method of the anti-4BB-1BB nano antibody. The anti-4-1BB nano antibody can be used for preventing and / or treating cancers.

Description

An anti-4-1BB nanobody and its preparation and application Technical Field

[0001] The present application relates to the field of biomedicine technology, and specifically to an anti-4-1BB nanobody and its preparation and application. Background Art

[0002] Effective stimulation of T cell immune function requires a costimulatory process and cytokine environment to promote cell cycle progression, survival, and proliferation. 4-1BB (CD137), a costimulatory member of the tumor necrosis factor receptor superfamily (TNFRSF), is an inducible T cell surface receptor that is not only highly expressed on activated T cells but also remains on them. Pollok KE, Kim SH's team demonstrated that anti-CD3 antibodies or concanavalin A (Con A) stimulate high expression of 4-1BB on splenic T cells. 4-1BB's natural ligand, 4-1BBL, is present on activated antigen-presenting cells (APCs). When 4-1BB binds to its natural ligand or an agonistic monoclonal antibody (MoAb), it transmits a unique costimulatory signal that plays a role in T cell responses in vivo and in vitro, including stimulating T cell proliferation, upregulating survival-related genes, and producing Th1 cytokines such as IL-2, IFN-γ, and TNF-α. Among them, IL-2 is one of the major positive growth factors for T cells. High levels of IL-2 secreted by CD8+ T cells play an important role in inducing cell cycle progression, producing cytokines such as IFN-γ, and inducing memory CD8+ T cells. In addition, 4-1BB-mediated signaling plays a key role in preventing activation-induced cell death, promoting rejection of cardiac allografts and skin grafts, increasing the cytolytic capacity of CD8+ T cells, and eradicating tumors.

[0003] Nanobodies (Nb) are the variable regions of heavy-chain antibodies (IgG2 and IgG3) in camelids and are considered 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 penetration, greater stability, higher solubility, lower aggregation, and easier cloning. Furthermore, compared to their mAb counterparts, Nb production costs are significantly lower, making them suitable for most cancer patients.

[0004] Currently, 4-1BB 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 process, resulting in high production costs.

[0005] Summary of the Invention

[0006] In view of this, the present invention provides an anti-4-1BB nanobody and its preparation method and use. The anti-4-1BB nanobody has the advantages of small molecular weight, simpler structure, low immunogenicity, high tissue permeability, high stability, high solubility and low aggregation, and easy cloning, and can be used to increase the cytolytic ability of CD8+ T cells and treat or prevent tumors.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0008] The present invention provides an anti-4-1BB antibody and a variant thereof, or an antigen-binding fragment thereof, wherein the anti-4-1BB antibody comprises three complementary determining regions CDR1, CDR2 and CDR3 of VHH named 137-1, 137-7, 137-12, 137-16, 137-18, 137-36 and 137-39 (as shown in the following table).

[0009] 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.

[0010] 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), camel antibodies, bispecific antibodies or trispecific antibodies.

[0011] The present invention also provides a fusion protein comprising the antibody and variants thereof described herein, or an antigen-binding fragment thereof.

[0012] 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 4-1BB) or an additional antibody active fragment (e.g., an antibody or antibody active fragment directed against other epitopes or the same epitope of 4-1BB, or a ligand capable of binding to 4-1BB).

[0013] The present invention also provides an antibody-drug conjugate comprising the antibody and variants thereof described herein, or an antigen-binding fragment thereof.

[0014] For the antibody-drug conjugate described herein, the drug is selected from the following: radiolabels, 32 P. 35 S, 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.

[0015] 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.

[0016] The present invention also provides a vector comprising the polynucleotide described herein, preferably a plasmid vector.

[0017] 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.

[0018] 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.

[0019] 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 a medicament for treating and / or preventing diseases associated with 4-1BB.

[0020] The present invention also provides a method for treating and / or preventing 4-1BB-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.

[0021] The present invention also provides a method for detecting whether a sample contains T cells that highly express 4-1BB, which comprises 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.

[0022] The present invention also provides a detection product, which comprises the antibody and variants thereof, or antigen-binding fragments thereof described herein.

[0023] 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.

[0024] In order to achieve the above-mentioned object of the invention, the present invention further provides the following technical solutions:

[0025] The first aspect of the present invention provides an anti-4-1BB nanobody, wherein the heavy chain variable region of the anti-4-1BB nanobody consists of a framework region FR and a complementarity determining region CDR, and the complementarity determining region CDR comprises at least one of the following groups:

[0026] (1) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 1;

[0027] (2) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 2;

[0028] (3) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 3;

[0029] (4) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 4;

[0030] (5) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 5;

[0031] (6) CDR1, CDR2, and CDR3 shown in SEQ ID NO: 6;

[0032] (7) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 7.

[0033] 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.

[0034] In this study, camels were immunized with recombinant 4-1BB antigen to construct a VHH phage library. After biopanning, seven functional anti-4-1BB Nbs (e.g., antibodies described in SEQ ID Nos: 1-7) were obtained.

[0035] In an embodiment provided by the present invention, the framework region FR includes at least one of the following groups:

[0036] (1) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 1;

[0037] (2) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 2;

[0038] (3) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 3;

[0039] (4) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 4;

[0040] (5) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 5;

[0041] (6) FR1, FR2, FR3, and FR4 shown in SEQ ID NO: 6;

[0042] (7) FR1, FR2, FR3 and FR4 shown in SEQ ID NO: 7.

[0043] 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.

[0044] The second aspect of the present invention provides a polynucleotide encoding the above-mentioned anti-4-1BB nanobody.

[0045] The third aspect of the present invention provides a recombinant expression vector comprising the above-mentioned polynucleotide.

[0046] In the embodiments provided by the present invention, the expression vector includes a prokaryotic expression vector or a eukaryotic expression vector.

[0047] 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.

[0048] In embodiments provided herein, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0049] In specific embodiments provided by the present invention, the host cell is selected from Escherichia coli or yeast cells.

[0050] In specific embodiments provided by the present invention, the host cell is selected from HEK293T cells, HEK293F cells, Expi293F cells or CHO cells.

[0051] The fifth aspect of the present invention provides a method for preparing the above-mentioned anti-4-1BB nanobody, comprising the following steps:

[0052] Inserting the polynucleotide encoding the anti-4-1BB nanobody into an expression vector to obtain a recombinant expression vector;

[0053] Transforming the recombinant expression vector into a host cell to obtain a recombinant host cell;

[0054] culturing the recombinant host cell to obtain a culture;

[0055] The culture was purified to obtain anti-4-1BB nanoantibodies.

[0056] In the embodiments provided by the present invention, purification is performed using Protein A agarose purification resin.

[0057] The sixth aspect of the present invention provides a bispecific antibody, which comprises the above-mentioned anti-4-1BB nanobody and a second antibody.

[0058] 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.

[0059] In a seventh aspect, the present invention provides the use of the above-mentioned anti-4-1BB nanobody or bispecific antibody in the preparation of drugs for preventing and / or treating cancer and the detection of 4-1BB protein.

[0060] 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.

[0061] An eighth aspect of the present invention provides a pharmaceutical composition, comprising: the above-mentioned anti-4-1BB nanobody or bispecific antibody; and pharmaceutically acceptable excipients.

[0062] In the embodiments provided herein, the dosage forms of the pharmaceutical composition include but are not limited to injection, powder injection, tablet or capsule.

[0063] In a ninth aspect, the present invention provides a kit for detecting 4-1BB protein, comprising: the above-mentioned anti-4-1BB nanobody or bispecific antibody; and a reagent acceptable in detection science. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 shows the FACS binding affinity of the VHH-hFc recombinant antibody to human 4-1BB-overexpressing 293F cells;

[0065] FIG2 shows the IL-2 release of PBMC stimulated by VHH-hFc recombinant antibody (in the presence of OKT3);

[0066] FIG3 shows the IL-2 release of PBMC stimulated by VHH-hFc recombinant antibody (in the absence of OKT3). DETAILED DESCRIPTION

[0067] The present invention discloses an anti-4-1BB nano antibody and its biomaterial and product. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described by preferred embodiments, and relevant personnel can obviously change or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0068] Explanation of terms:

[0069] Nanobodies (Nb) are naturally light-chain-deficient antibodies found in the peripheral blood of alpacas. These antibodies contain only a single heavy-chain variable region (VHH) and two conventional CH2 and CH3 regions. However, unlike artificially engineered single-chain antibody fragments (scFv), they are not prone to adhesion or even aggregation. More importantly, the individually cloned and expressed VHH structure possesses comparable structural stability and antigen-binding activity to the original heavy-chain antibody, representing the smallest known unit capable of binding to the target antigen.

[0070] The framework region (FR) is the backbone region within the variable region of an antibody. The approximately 110 amino acids near the N-termini of the immunoglobulin H and L chains vary significantly, while the remaining amino acid sequences are relatively constant. This is how the light and heavy chains are divided into the variable (V) and constant (C) regions. The variable region contains the hypervariable regions (HVRs) or complementarity-determining regions (CDRs) and the FR framework regions. FRs are less variable than CDRs. There are four FR molecules: FR1, FR2, FR3, and FR4. During antibody recognition, the four FR molecules curl together to bring the CDR molecules closer together.

[0071] Complementarity Determining Region (CDR): The entire antibody molecule can be divided into two parts: the constant region and the variable region. Within the variable region, a small portion of amino acid residues varies significantly. These regions, where the composition and order of amino acid residues are more susceptible to variation, are called hypervariable regions. There are three hypervariable regions (HVRs) in the V region of the L and H chains. These regions are also called complementarity determining regions because their spatial structure allows for precise complementarity with antigenic determinants.

[0072] Bispecific antibodies: They are artificial antibodies containing two specific antigen-binding sites. They can build a bridge between target cells and functional molecules (cells) to stimulate a directed immune response. They are a type of genetically engineered antibody and have become a hot topic in the field of antibody engineering. They have broad application prospects in tumor immunotherapy.

[0073] The names and abbreviations of the 20 amino acids:

[0074] The reagents, instruments, strains or biological materials used in the present invention can all be obtained commercially.

[0075] The present invention will be further described below in conjunction with the embodiments:

[0076] Example 1: Antigen preparation and animal immunization

[0077] 1. Animal immunization:

[0078] The human 4-1BB extracellular domain recombinant protein (hFc tag) was injected into multiple masses at the subcutaneous and intramuscular sites of the neck of two alpacas, and the absorption of the subcutaneous injection masses was tracked to confirm correct immunization.

[0079] 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 in a volume of 1 ml per alpaca.

[0080] Second immunization: 3 weeks after the first 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;

[0081] 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;

[0082] Fourth immunization: 3 weeks after the third 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.

[0083] 2. Serum processing and titer detection:

[0084] One week after the third and fourth immunizations, 2 mL of peripheral blood was collected and serum was isolated. Recombinant human 4-1BB 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 titers of the four immunization sera from both alpacas were >1:32,000, meeting the library construction criteria.

[0085] Example 2: Construction of phage display immune antibody library

[0086] Since the serum titer of the alpaca after the fourth immunization was >1:32000, indicating the presence of high-affinity antibodies against human 4-1BB in the serum, we constructed a phage display immune antibody library according to the following steps:

[0087] ① Collect 50 mL of peripheral blood from each of the two alpacas after the fourth immunization, separate the PBMCs, and combine the PBMCs of the two alpacas; take 2×10 7PBMCs were collected 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. ② The IgG2 and IgG3 heavy chain variable region sequences (VHH of the nanobody heavy chain variable region) were obtained step by step by nested PCR. The experimental steps are as follows: 1) A pair of specific nested outer primers were designed and amplified in the first round of PCR using cDNA as a template. The amplified region was the Leader-CH2 region of the alpaca heavy chain antibody gene, and the product sizes were 700 bp and 900 bp respectively. The 700 bp PCR product was recovered by DNA gel electrophoresis and gel excision. 2) Three pairs of nested inner primers were designed and amplified in the second round of PCR using the 700 bp first-round PCR product as a template. The amplified region was the VHH fragment of the alpaca heavy chain antibody variable region, and the product size was 400 bp. The second-round PCR product was purified and recovered using a PCR product purification kit. 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). 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 4-1BB Alpaca is 4.60×10 9 20 monoclonal clones were randomly picked from each plate for sequencing, and the results showed that the VHH insertion efficiency of the alpaca antibody library Lib 4-1BB Alpaca was 90%.

[0088] Example 3: Screening of antibody library

[0089] 5 μg / mL of human 4-1BB 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 on a 2YT / Tet10 plate and cultured overnight in an incubator at 37°C; the next day, NEB5αF' monoclonal clones were picked from the overnight 2YT / Tet10 plate and added to 3 mL of 2YT / Tet10 liquid culture medium, and the bacteria were grown at 37°C to OD600 = 0.8; at the same time, the antigen supernatant of the 96-well plate was removed, 200 μL of 1% BSA was added to each well for blocking, and 200 μL of 1% BSA was added to the blank well. 1% BSA was used as a negative control well and placed on a 3D rotating shaker at room temperature for 2 hours. Afterwards, the supernatant from the protein well and control well was removed, washed with 200 μL of PT, and 100 μL of phage antibody library was added to each well and placed on a 3D rotating shaker at room temperature for 2 hours. The supernatant from the protein well and control well was removed and washed with 200 μL of PT. 100 μL of 100 mM HCl was added to the well and placed at room temperature for 5 minutes. The supernatant was aspirated and added to a 1.5 mL centrifuge tube and neutralized with 1 M Tris-HCl. The above mixture was added to a centrifuge tube containing 1 mL of NEB5αF' bacteria and incubated at 37°C for 1 hour. 20 μL of the culture medium in the centrifuge tube was diluted appropriately 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.

[0090] 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 second round of screening, the number of colonies in the antigen-binding wells of Lib 4-1BB Alpaca was 100 times that of the negative control wells, indicating successful enrichment.

[0091] 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 4-1BB extracellular domain recombinant protein (his tag). These clones are defined as positive clones and sequenced and aligned to obtain unique sequences.

[0092] Example 4: NGS sequencing

[0093] 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.

[0094] Example 5: Comparison and statistics of positive sequences and NGS high-frequency sequences

[0095] The phage ELISA-positive sequences and NGS high-frequency sequences (Top 50) were aligned and grouped according to the differences in CDR H3. The results are shown in Table 1.

[0096] Table 1. 4-1BB nanobodies obtained by immunization of alpacas

[0097] Note: The underlined region is CDR

[0098] Example 6: Eukaryotic expression of nanobodies

[0099] Seven sequences in Table 1 of Example 5 were eukaryotically expressed using the following experimental steps: 1) VHH fragments from these sequences were amplified by PCR and inserted into the eukaryotic expression vector pFcIG containing a human IgG1 Fc tag using homologous recombination or enzyme ligation. The fragments were then electroporated into the Escherichia coli trans5α host strain, screened with bleomycin, and the correct recombinant plasmids were obtained by sequencing. The host strain containing the recombinant plasmids was then expanded and sterile, endotoxin-free plasmids were obtained using an endotoxin removal kit. 2) HEK293F cells were cultured in serum-free medium and transfected with the recombinant expression plasmids using Polyplus suspension cell transfection reagent for expression. Feeds were added 24 and 72 hours after transfection, and the supernatant was collected on day 5. The antibodies were purified using Protein A agarose purification resin and then exchanged for storage in PBS. The experimental results (Table 2) showed that the yields of the seven VHH-hFc recombinant antibodies transiently expressed in 293F cells ranged from 53 to 231.4 mg / L. SDS-PAGE electrophoresis analysis showed that the VHH-hFc bands for sequences 137-1, 137-7, 137-12, 137-18, and 137-36 were normal in size and >95% pure. The recombinantly expressed antibodies for sequences 137-16 and 137-39 exhibited nonspecific bands and were <95% pure.

[0100] Table 2. Eukaryotic transient expression results of VHH-His recombinant antibodies

[0101] Example 7: Affinity EC50 of Nanobodies and Antigen Recombinant Proteins

[0102] The affinity of seven recombinant VHH-hFc antibodies for the human and monkey 4-1BB extracellular domain recombinant proteins was determined using ELISA. Human and monkey 4-1BB extracellular domain recombinant proteins (his-tagged) were added to a 96-well ELISA plate at 200 ng / well and coated overnight at 4°C. The VHH-hFc recombinant antibodies were diluted to varying concentrations (0.014-10 μg / mL) and reacted with the antigen in an ELISA. HRP-labeled anti-VHH secondary antibodies were used for color development, and absorbance at 450 nm was measured using a microplate reader. The results (Tables 3 and 4) show that all seven recombinant VHH-hFc antibodies were able to bind to the human or monkey 4-1BB extracellular domain recombinant proteins, with CD137-1, CD137-7, CD137-16, and CD137-18 ranking in the top four in terms of affinity for the human or monkey 4-1BB extracellular domain recombinant proteins. The same experimental method was used to detect the affinity of the recombinant antibodies to the mouse 4-1BB extracellular region recombinant protein. The results (Table 5) showed that the VHH-hFc recombinant antibodies of CD137-1, CD137-7, CD137-16, and CD137-18 did not bind or weakly bound to the mouse 4-1BB extracellular region recombinant protein.

[0103] Table 3. ELISA binding of VHH-hFc recombinant antibodies to human 4-1BB (his tag)

[0104] Table 4. ELISA binding of VHH-hFc recombinant antibodies to monkey 4-1BB (his tag)

[0105] Table 5. ELISA binding of VHH-hFc recombinant antibodies to mouse 4-1BB (his tag)

[0106] Example 8: Affinity KD value of nanobody and antigen recombinant protein

[0107] Biacore was used to test the affinity KD values ​​of VHH-hFc recombinant antibodies with four sequences (CD137-1, CD137-7, CD137-16, and CD137-18) for the human 4-1BB extracellular domain recombinant protein. The Biacore 8k was set at 25°C for the sample chamber and flow chamber. The VHH-hFc sample was captured on a Protein A chip as the ligand, and the corresponding target protein was diluted in HBS-EP+pH 7.4 buffer as the analyzer. The analyzer flow rate was 30 μL / min, with an association time of 120 s and a dissociation time of 600 s. Data were analyzed using a 1:1 binding and a local model fit. The experimental results (Table 6) show that all four VHH-hFc recombinant antibodies bound to the human 4-1BB extracellular domain recombinant protein with KD values ​​ranging from 3.3 to 38 nM.

[0108] Table 6. SPR binding affinity of VHH-hFc recombinant antibodies to human 4-1BB (his tag)

[0109] Example 9: Binding of Nanobodies to Antigen Recombinant Protein Overexpressing Cells

[0110] Flow cytometry was used to detect the affinity of the nanobody to human 4-1BB-overexpressing 293F cells: 1) 293F cells were transfected with a eukaryotic expression plasmid containing the full-length 4-1BB gene and cultured for 24 hours; 2) 0.3×10 6 293F cells transiently overexpressing human 4-1BB were washed twice with PBS and then resuspended in 100 μL PBS; 3) incubated with 10 μg / mL VHH-hFc recombinant antibody for 1 hour; 4) after washing the cells 3 times with PBS, the cells were resuspended in 100 μL PBS, 0.2 μg / mL FITC-labeled anti-human Fc antibody was added, and incubated for 1 hour; 4) after washing the cells 3 times with PBS, the cells were resuspended in 300 μL PBS, and fluorescence was detected by flow cytometry. The experimental results (Figure 1) show that the VHH-hFc recombinant antibodies of CD137-1, CD137-7, CD137-16, and CD137-18 can bind to 293F cells overexpressing human 4-1BB.

[0111] Example 10: PBMC activation experiment

[0112] An ELISA kit was used to detect the level of IL-2 released after human PBMCs were stimulated with VHH-hFc recombinant antibodies of four sequences (CD137-1, CD137-7, CD137-16, and CD137-18): 96-well cell culture plates were coated or not coated with CD3 antibody OKT3 and incubated overnight at 4°C; the next day, PBMCs (1X10e5 / well) and different concentrations of 4-1BB recombinant antibodies were added, and culture was continued for 72 hours before the supernatant was collected for ELISA detection of IL-2 levels. The experimental results (Figures 2 and 3) show that VHH-hFc recombinant antibodies of CD137-1, CD137-7, CD137-16, and CD137-18, when coated with OKT3, can stimulate human PBMCs to release IL-2. However, in the absence of OKT3 coating, the ability of CD137-1, CD137-7, CD137-16, and CD137-18 to activate PBMCs for IL-2 release was significantly reduced, and the antibody EC50 values ​​were significantly increased (Table 7). Furthermore, the experimental results (Figure 3) show that the maximum IL-2 release (Max value) of CD137-1 and CD137-18 after PBMC stimulation was similar in the presence or absence of OKT3, indicating that both CD137-1 and CD137-18 VHH-hFc recombinant antibodies activated PBMCs 100% in both the presence and absence of OKT3. However, in the absence of OKT3, the CD137-7 and CD137-16 VHH-hFc recombinant antibodies failed to activate PBMCs 100%. The maximum IL-2 release (Max value) of these two antibodies in the absence of OKT3 was significantly lower than in the presence of OKT3. These data indicate that 100% activation of PBMCs by CD137-1 and CD137-18 is independent of CD3 antibody, whereas 100% activation of PBMCs by CD137-7 and CD137-16 is dependent on CD3 antibody.

[0113] Table 7. EC50 values ​​of VHH-hFc recombinant antibodies stimulating PBMC to produce IL-2

[0114] 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-4-1BB antibody and its variant, or an antigen-binding fragment thereof, wherein the anti-4-1BB antibody comprises three complementary determining regions (CDR1, CDR2, and CDR3) of VHHs named 137-1, 137-7, 137-12, 137-16, 137-18, 137-36, and 137-39, For example, for the antibodies and variants thereof, or antigen-binding fragments thereof, the variants are humanized variants or have an 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%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 96%, 97%, 98%, 99%, 99.5%, 99.9%) variants; Or, exemplarily, the antibody and its variant, or its antigen-binding fragment, is selected from camel Ig, Ig NAR, Fab fragment, Fab' fragment, F(ab)'2 fragment, F(ab)'3 fragment, Fv, scFv, bi-scFv, (scFv)2, miniantibody, double-chain antibody, three-chain antibody, four-chain antibody, disulfide bond-stabilized Fv protein and single domain antibody (sdAb, nanoantibody), camel antibody, bispecific antibody or trispecific antibody, and further, the antibody is named 137-1, 137-7, 137-12, 137-16, 137-18, 137-36, 137-39.

2. A fusion protein comprising the antibody and variants thereof, or antigen-binding fragments thereof according to claim 1, Exemplarily, the fusion protein 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 4-1BB) or an additional antibody active fragment (e.g., an antibody or antibody active fragment directed against other epitopes of 4-1BB or the same epitope, or a ligand capable of binding to 4-1BB).

3. An antibody-drug conjugate comprising the antibody and variants thereof according to claim 1, or an antigen-binding fragment thereof.

4. An isolated polynucleotide expressing an antibody or antigen-binding fragment thereof, characterized in that: The polynucleotide can express the antibody and variant thereof, or antigen-binding fragment thereof, described in the claims; the polynucleotide can express the fusion protein described herein; the polynucleotide can express the antibody-drug conjugate described herein. A vector comprising the polynucleotide according to claim 4 , preferably a plasmid vector.

6. A host cell comprising the polynucleotide according to claim 4 or the vector according to claim 5, preferably, the host cell is a eukaryotic cell.

7. A pharmaceutical composition comprising the antibody and variants thereof, or antigen-binding fragments thereof, according to claim 1, the fusion protein according to claim 2, the antibody-drug conjugate according to claim 3, and optionally, a pharmaceutically acceptable carrier.

8. Use of the antibody and variant thereof according to claim 1, or an antigen-binding fragment thereof, the fusion protein according to claim 2, or the antibody-drug conjugate according to claim 3 in the preparation of a medicament for treating and / or preventing diseases associated with 4-1BB.

9. A method for treating and / or preventing 4-1BB-related diseases and related symptoms, comprising administering an effective amount of the fusion protein of claim 2, the antibody-drug conjugate of claim 3, or the pharmaceutical composition of claim 7 to a subject.

10. A method for detecting whether a sample contains T cells that highly express 4-1BB, comprising the step of contacting the sample with the antibody and variant thereof, or an antigen-binding fragment thereof, the fusion protein of claim 2, or the antibody-drug conjugate of claim 3, wherein the detection can be for diagnostic or non-diagnostic purposes.

11. A kit for detecting 4-1BB protein, characterized in that: The kit comprises: the antibody and variants thereof, or antigen-binding fragments thereof according to claim 1, the fusion protein according to claim 2, or the antibody-drug conjugate according to claim 3; and a reagent acceptable for detection.

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