Anti-4-1BB nanobodies, their production and uses

Abstract

The present invention relates to the technical field of biomedicine, in particular to anti-4-1BB nanobodies, their production and use. The complementarity determining regions of the anti-4-1BB nanobodies include CDR1, CDR2 and CDR3 as shown in SEQ ID NOs: 1-7. The anti-4-1BB nanobodies are useful for the prevention and / or treatment of cancer.

Description

[Technical Field]

[0001] The present application relates to the technical field of biomedicine, in particular to anti-4-1BB nanobodies, their production and uses. [Background technology]

[0002] Activation of T cell immune functions 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 persistently persists on T cells. The Pollok KE, Kim SH, and colleagues demonstrated that anti-CD3 antibody or concanavalin A (Con A) stimulates high expression of 4-1BB on splenic T cells. The natural ligand of 4-1BB, 4-1BBL, is present on activated antigen-presenting cells (APCs). When 4-1BB binds to its natural ligand or a stimulatory monoclonal antibody (MoAB), it delivers a unique costimulatory signal, which exerts functions in in vivo and in vitro T cell responses, including stimulating T cell proliferation, upregulating survival-related genes, and producing Th1 cytokines such as IL-2, IFN-γ, and TNF-α. IL-2 is one of the main 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. 4-1BB-mediated signaling also plays an important role in preventing activation-induced cell death, promoting the rejection of cardiac allografts and skin grafts, enhancing the cytolytic activity of CD8+ T cells, and eradicating tumors.

[0003] Nanobodies (Nb) are the variable regions of heavy-chain antibodies (IgG2 and IgG3) from camelids and are known as the smallest antigen-binding fragments found in nature. Compared to conventional full-length monoclonal antibodies (mAbs, approximately 150 kD), Nb have advantages such as a smaller molecular weight (12-15 kD), simpler structure, lower immunogenicity, better tissue penetration, higher stability, higher solubility, less aggregation, and easier cloning. Furthermore, compared to mAb-like products, Nb production costs are significantly lower, making them suitable for most cancer patients.

[0004] Currently, the 4-1BB monoclonal antibody still faces many challenges, including 1) its large molecular size (160-170 kD), which limits its ability to penetrate solid tumors and the blood-brain barrier, and 2) the high production costs due to the extensive work involved in the in vitro eukaryotic expression and cell screening process. Summary of the Invention

[0005] In view of the above, the present invention provides an anti-4-1BB nanobody, its preparation method and use, which has advantages such as small molecular weight, simpler structure, low immunogenicity, high tissue penetration, high stability, high solubility, low aggregation, and ease of cloning, and is useful for enhancing the cytolytic activity of CD8+ T cells and treating or preventing tumors.

[0006] In order to achieve the above object of the invention, the present invention provides the following inventions.

[0007] The present invention provides anti-4-1BB antibodies and variants thereof, or antigen-binding fragments thereof, which comprise three complementarity-determining regions CDR1, CDR2 and CDR3 of VHHs designated 137-1, 137-7, 137-12, 137-16, 137-18, 137-36 and 137-39 (shown in the table below). [Table 1]

[0008] Illustratively, the antibodies and variants thereof, or antigen-binding fragments thereof described herein are humanized variants or identical (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] Furthermore, the antibodies and variants thereof, or antigen-binding fragments thereof described herein are selected from camelid IG, Ig NAR, Fab fragments, Fab' fragments, F(ab)'2 fragments, F(ab)'3 fragments, Fv, scFv, bis-scFv, (scFv)2, microantibodies, two-chain antibodies, three-chain antibodies, four-chain antibodies, disulfide-stabilized Fv proteins and single domain antibodies (sdAb, nanobodies), camelid antibodies, bispecific antibodies or triabodies.

[0010] The present invention also provides fusion proteins comprising the antibodies and variants thereof, or antigen-binding fragments thereof, described herein.

[0011] Illustratively, the fusion proteins described herein further comprise a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.) or an IgG1-Fc protein sequence, or an epitope tag (e.g., against 4-1BB or another epitope different therefrom) or an additional antibody active fragment (e.g., another epitope against 4-1BB or an antibody or antibody active fragment of the same epitope, or a ligand capable of binding to 4-1BB).

[0012] The present invention also provides antibody-drug conjugates comprising the antibodies and variants thereof, or antigen-binding fragments thereof, described herein.

[0013] In the antibody-drug conjugates described herein, the drug may be a radiolabel, 32 P, 35S, fluorescent dyes, electron-sensitive reagents, enzymes, biotin, streptavidin, digitoxin, haptens, immunogenic proteins, nucleic acid molecules having a sequence complementary to a target, or a combination of any of the above, or immunomodulatory compounds, anti-cancer drugs, anti-viral drugs, anti-bacterial drugs, anti-fungal drugs and anti-parasitic drugs, or a combination of any of the above.

[0014] The present invention also provides isolated expressed antibody or antigen-binding fragment thereof polynucleotides, wherein the polynucleotides are capable of expressing the antibodies and variants thereof, or antigen-binding fragments thereof, described herein; the polynucleotides are capable of expressing the fusion proteins described herein; and the polynucleotides are capable of expressing the antibody-drug conjugates described herein.

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

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

[0017] The present invention also provides pharmaceutical compositions comprising the antibodies and variants thereof, or antigen-binding fragments thereof, as described herein ...

[0018] The present invention also provides the use of the antibodies and variants thereof, or antigen-binding fragments thereof described herein, the fusion proteins described herein, and the antibody-drug conjugates described herein in the manufacture of a medicament for treating and / or preventing a 4-1BB-associated disease.

[0019] The present invention also provides methods for treating and / or preventing 4-1BB-related diseases and associated symptoms, comprising administering to a subject an effective amount of an antibody and variant thereof, or antigen-binding fragment thereof, a fusion protein described herein, an antibody-drug conjugate described herein, or a pharmaceutical composition described herein.

[0020] The present invention also provides a method for detecting whether a sample contains T cells that highly express 4-1BB, the method comprising the step of contacting the sample with an antibody and variant thereof, or an antigen-binding fragment thereof, or a fusion protein described herein, wherein the detection may be for diagnostic or non-diagnostic purposes.

[0021] The present invention also provides detection products comprising the antibodies and variants thereof, or antigen-binding fragments thereof, described herein.

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

[0023] In order to achieve the above object of the invention, the present invention also provides the following inventions.

[0024] In a first aspect of the present invention, the heavy chain variable region comprises a framework region FR and a complementarity determining region CDR, and the complementarity determining region CDR is (1) CDR1, CDR2 and CDR3 as shown in SEQ ID NO: 1; (2) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 2; (3) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 3; (4) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 4; (5) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 5; (6) CDR1, CDR2 and CDR3 shown in SEQ ID NO: 6; (7) CDR1, CDR2 and CDR3 shown in SEQ ID NO:7. The present invention provides an anti-4-1BB nanobody comprising at least one selected from the group consisting of:

[0025] Nanobodies (Nb) are the variable regions of heavy-chain antibodies (IgG2 and IgG3) in camelids and are known as the smallest antigen-binding fragments found in nature. Compared to conventional full-length monoclonal antibodies (mAbs, approximately 150 kD), Nb have advantages such as a smaller molecular weight (12–15 kD), simpler structure, lower immunogenicity, better tissue penetration, higher stability, higher solubility, less aggregation, and easier cloning. Furthermore, compared to mAb-like products, Nb production costs are significantly lower, making them suitable for most cancer patients. In September 2018, the European Medicines Agency approved the first nanobody drug, caplacizumab (trade name Cablivi), primarily for the treatment of congenital thrombotic thrombocytopenic purpura (aTTP) in adult patients. Therefore, Nb are expected to be widely used in cancer therapy and diagnosis.

[0026] In this study, a VHH phage library was constructed using recombinant 4-1BB antigen-immunized camels, and seven functional anti-4-1BB Nbs (eg, antibodies shown in SEQ ID Nos: 1-7) were obtained through biopanning.

[0027] In the examples provided herein, the framework region FR is (1) FR1, FR2, FR3 and FR4 as shown in SEQ ID NO: 1; (2) FR1, FR2, FR3 and FR4 as set forth in SEQ ID NO: 2; (3) FR1, FR2, FR3 and FR4 as set forth in SEQ ID NO: 3; (4) FR1, FR2, FR3 and FR4 shown in SEQ ID NO: 4; (5) FR1, FR2, FR3 and FR4 as set forth in SEQ ID NO: 5; (6) FR1, FR2, FR3 and FR4 shown in SEQ ID NO: 6; (7) FR1, FR2, FR3 and FR4 shown in SEQ ID NO:7. The compound contains at least one selected from the group consisting of:

[0028] However, the framework region FR of the present invention is not limited to the above sequences, and all sequences that can achieve the function are included within the scope of the present invention.

[0029] A second aspect of the present invention provides a polynucleotide encoding the anti-4-1BB Nanobody described above.

[0030] A third aspect of the present invention provides a recombinant expression vector comprising the above polynucleotide.

[0031] In the embodiments provided herein, the expression vector comprises a prokaryotic expression vector or a eukaryotic expression vector.

[0032] A fourth aspect of the present invention provides a recombinant host cell comprising the polynucleotide described above or comprising the recombinant expression vector described above.

[0033] In the embodiments provided herein, the host cell comprises a prokaryotic or eukaryotic cell.

[0034] In specific embodiments provided herein, the host cell is selected from E. coli or yeast cells.

[0035] In specific embodiments provided herein, the host cells are selected from HEK293T cells, HEK293F cells, Expi293F cells or CHO cells.

[0036] A fifth aspect of the present invention relates to a method for producing a recombinant 4-1BB nanobody, comprising the steps of: inserting a polynucleotide encoding the anti-4-1BB nanobody into an expression vector to obtain a recombinant expression vector; introducing the recombinant expression vector into a host cell to obtain a recombinant host cell; Culturing the recombinant host cells to obtain a culture; and purifying the culture to obtain the anti-4-1BB Nanobody.

[0037] In the example provided herein, the purification is performed using a Protein A agarose purification resin.

[0038] A sixth aspect of the invention provides a bispecific antibody comprising an anti-4-1BB Nanobody as described above and a second antibody.

[0039] In embodiments provided herein, the second antibody includes, but is not limited to, a 4-1BB nanobody, a CD47 nanobody, a VEGF nanobody, a HER2 nanobody, an EGFR nanobody, a HER3 nanobody, a B7H3 nanobody, a TIGIT nanobody, an OX-40 nanobody, a CD40 nanobody, or a PD-L1 nanobody.

[0040] A seventh aspect of the present invention provides the use of the above-mentioned anti-4-1BB nanobody or bispecific antibody in the manufacture of a medicament for the prevention and / or treatment of cancer, and in the detection of 4-1BB protein.

[0041] In embodiments provided herein, the cancer includes, but is not limited to, gastric cancer, liver cancer, leukemia, kidney tumor, lung cancer, small intestine cancer, bone cancer, prostate cancer, colon / rectal cancer, breast cancer, colorectal cancer, prostate cancer, cervical cancer, lymphoma, adrenal tumor, or bladder tumor.

[0042] An eighth aspect of the present invention provides a pharmaceutical composition comprising an anti-4-1BB nanobody or bispecific antibody as described above, and a pharmaceutically acceptable adjuvant.

[0043] In the embodiments provided herein, the dosage forms of the pharmaceutical compositions include, but are not limited to, injections, powder injections, tablets, or capsules.

[0044] A ninth aspect of the present invention provides a kit for detecting 4-1BB protein, comprising the above-mentioned anti-4-1BB nanobody or bispecific antibody and a detectably acceptable reagent. [Brief explanation of the drawings]

[0045] [Figure 1] 1 shows the FACS binding affinity between VHH-hFc recombinant antibodies and 293F cells overexpressing human 4-1BB. [Figure 2] This shows IL-2 release from PBMCs stimulated with VHH-hFc recombinant antibodies (in the presence of OKT3). [Figure 3] IL-2 release from PBMCs upon stimulation with VHH-hFc recombinant antibodies (in the absence of OKT3) is shown. DETAILED DESCRIPTION OF THE INVENTION

[0046] The present invention discloses anti-4-1BB Nanobody and its biological materials and products, but those skilled in the art can realize the invention by referring to the contents of this specification and appropriately modifying the process parameters. In particular, all similar substitutions and modifications are obvious to those skilled in the art and are intended to be encompassed by the present invention. Although the method and use of the present invention have been described in terms of preferred embodiments, it is clear that those skilled in the art can modify or combine the method and use described herein with appropriate changes and modifications in order to realize and utilize the technology of the present invention without departing from the content, spirit, and scope of the present invention.

[0047] Technical terms explained

[0048] Nanobodies (Nb) are antibodies found in alpaca peripheral blood that naturally lack light chains and contain only one heavy chain variable region (VHH) and two constant regions, CH2 and CH3. However, they do not clump together or cluster like artificially modified single-chain antibody fragments (scFv). More importantly, individually cloned and expressed VHH structures have the same structural stability and antigen-binding activity as the original heavy chain antibodies, and are the smallest known units capable of binding to a target antigen.

[0049] The framework region (FR) is the framework region within the variable region of an antibody. The sequences of approximately 110 amino acids at the N-terminus of the H and L chains of immunoglobulins are highly variable, while the remaining amino acid sequences are relatively stable. Therefore, the light and heavy chains can be divided into variable regions (V) and constant regions (C). The variable region contains regions known as hypervariable regions (HVRs) or complementarity-determining regions (CDRs) and FR framework regions. The variability of FRs is lower than that of CDRs. There are four FR molecules in total: FR1, FR2, FR3, and FR4. When labeling an antibody, the four FR molecules curl to bring the CDR molecules closer together.

[0050] Complementarity determining region (CDR): The entire antibody molecule is divided into two regions: the constant region and the variable region. In the variable region, some amino acid residues are highly variable, and the region where the residue composition and sequence of these amino acids are more likely to change is called the hypervariable region. There are three hypervariable regions (HVR) in the V regions of the L chain and H chain, and because these regions can form exact complements with antigen determinants in terms of spatial structure, the hypervariable regions are also called complementarity determining regions.

[0051] Bispecific antibodies: These are artificial antibodies with two different antigen-binding sites that can bridge target cells and functional molecules (cells) and stimulate induced immune responses. They are a type of genetically engineered antibody that has attracted attention in the field of antibody engineering and are expected to be widely used in tumor immunotherapy.

[0052] [Table 2]

[0053] All of the reagents, instruments, bacterial strains, biological materials, etc. used in the present invention can be purchased commercially.

[0054] The present invention will be further described below with reference to examples.

[0055] Example 1: Antigen preparation and animal immunization

[0056] 1.Animal immunity Human 4-1BB extracellular domain recombinant protein (hFc tag) was injected subcutaneously and intramuscularly at multiple sites on the back of the neck of two alpacas, forming multiple clots. The absorption of the clots was monitored and confirmed to be successful. For the initial immunization, 0.5 mg of antigen was mixed with Freund's complete adjuvant at a 1:1 ratio, emulsified, and then injected in an injection volume of 1 ml per alpaca. Second immunization: Three weeks after the first immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified, and then injected in an injection volume of 1 ml per alpaca. Third immunization: Three weeks after the second immunization, 0.25 mg of antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified, and then injected in an injection volume of 1 ml per alpaca. Fourth immunization: Three weeks after the third immunization, 0.25 mg of the antigen was mixed with Freund's incomplete adjuvant at a ratio of 1:1, emulsified, and then injected in an injection volume of 1 ml per alpaca.

[0057] 2. Serum Processing and Titer Measurement One week after the third and fourth immunizations, 2 mL of peripheral blood was collected and serum was separated. Human 4-1BB extracellular domain recombinant protein (His tag) was loaded onto a 96-well ELISA plate, and antibody titers in the serum were measured by ELISA. ELISA results showed that serum titers after the fourth immunization in two alpacas were >1:32,000, meeting the requirements for library construction.

[0058] Example 2: Generation of a phage-display immune antibody library

[0059] 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. Therefore, a phage-display immune antibody library was constructed using the following procedure.

[0060] (1) After the fourth immunization, 50 mL of peripheral blood was collected from each of the two alpacas, and lymphocyte PBMCs were separated. The PBMCs from the two alpacas were combined and used to generate 2 × 10 7PBMCs were collected, total RNA was extracted using an RNA extraction kit, and an appropriate amount of RNA (e.g., 3-5 μg) was collected and used to obtain cDNA using an RT-PCR reverse transcription kit. (2) 1) A pair of specific nested outer primers was designed, and a first-round PCR amplification was performed using the cDNA as a template. The amplified region was the leader-CH2 region of the alpaca heavy chain antibody gene, with product sizes of 700 bp and 900 bp. The 700 bp PCR product was isolated by DNA gel electrophoresis and excised from the gel. 2) Three pairs of nested inner primers (alpaca) were designed, and a second-round PCR amplification was performed using the 700 bp first-round PCR product as a template. The amplified region was the alpaca heavy chain antibody VHH fragment, with product size of 400 bp. The second-round PCR product was purified and isolated using a PCR product purification kit. The IgG2 and IgG3 heavy chain variable region sequences (VHH of nanobody heavy chain variable regions) were obtained stepwise by nested PCR. (3) The heavy chain variable region sequence was inserted into the enzymatically digested linearized phage vector VHH-libTemplate by homologous recombination or enzymatic digestion and ligation to obtain the recombinant vector. After purification and recovery, it was transformed into hypersensitive SS320 cells (containing the auxiliary phage M13K07). The transformed bacterial solution was resuspended in SOC medium and activated for 1 hour. A small amount of the bacterial solution was collected and diluted 10-fold. An appropriate dilution titer was selected and transformed into LB / tet10 and LB / Carb5. The resulting phage was coated onto a LB / Carb50 plate and placed in a biochemical incubator at 37°C overnight. The next day, the volume of the library was calculated. The remaining bacterial suspension was transferred to a large volume of 2YT / Carb50 / Kan25 liquid medium, placed in a shaking incubator at 37°C, and cultured overnight. The next day, the supernatant was removed, and 1 / 4 the volume of PEG / NaCl solution was added to precipitate the phage. The phage was then resuspended in an appropriate amount of PBT solution and diluted to the required concentration to obtain a phage-displayed immune antibody library (stored at -80°C). (4) The number of clones on the LB / Carb50 plate was counted, and the library volume was calculated. The library volume of the alpaca antibody library, Lib 4-1BB Alpaca, was found to be 4.60 x 10 9Twenty monoclonal antibodies were randomly selected from the plate and sequenced, revealing that the VHH insertion efficiency of the alpaca antibody library Lib 4-1BB Alpaca was 90%.

[0061] Example 3: Screening of antibody libraries

[0062] 5 μg / mL of human 4-1BB extracellular domain recombinant protein (His tag) was added to a 96-well plate (100 μL / well) and incubated overnight at 4°C. NEB5αF' E. coli cells were streaked onto a 2YT / Tet10 plate and grown in a 37°C incubator. The next day, NEB5αF' monoclonal cells were selected from the overnight-cultured 2YT / Tet10 plate, added to 3 mL of 2YT / Tet10 liquid medium, and grown at 37°C with shaking until an OD600 of 0.8 was reached. Simultaneously, the antigen supernatant was removed from the 96-well plate, and 200 μL of 1% BSA was added to each well to close them. 200 μL of 1% BSA was added to blank wells to serve as negative controls. The plates were then placed in a 3D rotary shaker at room temperature for 2 hours. The supernatant from the protein wells and control wells was then removed and washed with 200 μL of PT. 100 μL of the phage antibody library was added to each well and placed in a 3D rotary shaker at room temperature for 2 hours. The supernatant from the protein wells and control wells was then removed and washed with 200 μL of PT. 100 μL of 100 mM HCl was added to the wells and placed in a 3D rotary shaker at room temperature for 5 minutes. The supernatant was then aspirated, transferred 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 from the centrifuge tube was diluted appropriately, coated onto an LB / Carb50 culture plate, and placed in a biochemical incubator at 37°C overnight. The next day, the titer and concentration were calculated. The remaining culture medium was added with 1 μL of helper phage M13K07 (final concentration: 10 10The culture medium was then transferred to 35 mL of 2YT / Carb50 / Kan25 culture medium, placed on a shaker, and cultured overnight at 37°C. Phages were collected to form antibody libraries for each round.

[0063] The above procedure was repeated two or three times until phage enrichment was observed. Enrichment was considered successful if the number of colonies in the antigen-binding wells on the LB / Carb50 culture plate was 10 times or more that in the negative control wells. In this experiment, Lib 4-1BB Alpaca was found to have been successfully enriched after the second round of screening, as the number of colonies in the antigen-binding wells was 100 times that of the negative control wells.

[0064] Clones from the enrichment round were randomly selected and expanded in a 96-deep-well plate. After centrifugation, the supernatant was used for Phage ELISA screening. Clones with an OD value >2 that bound to the human 4-1BB extracellular domain recombinant protein (His tag) and the blocking solution were selected as positive clones. They were sequenced and compared to obtain unique sequences.

[0065] Example 4: NGS sequencing

[0066] Primers were designed to amplify the constant regions of the VHHs on either side of the phage library, and the PCR products were subjected to E-gel detection. The PCR library fragments were then purified and collected using a kit and subjected to NGS sequencing. Statistical analysis of the data was performed using SPSS 2.0 and Microsoft Excel 2019 to rank the DNA by number and frequency.

[0067] Example 5: Comparison and statistics of positive sequences and NGS high frequency sequences

[0068] The phage ELISA-positive sequences were compared with the NGS high-frequency sequences (Top 50) and grouped according to differences in CDR H3. The results are shown in Table 1.

[0069] [Table 3]

[0070] Example 6: Eukaryotic expression of nanobodies

[0071] For the seven sequences listed in Table 1 in Example 5, 1) the VHH fragments of these sequences were amplified by PCR, and then inserted into the human IgG1 Fc-tagged eukaryotic expression vector pFcIG by homologous recombination or enzymatic cleavage and ligation. The fragments were then electroporated into E. coli trans5α host cells, screened with bleomycin, and the monoclonal clones were sequenced to obtain the correct recombinant plasmids. Next, the recombinant plasmid-containing host cells were cultured and an endotoxin removal kit was used to obtain sterile, endotoxin-free plasmids. 2) HEK293F cells were cultured in serum-free medium, and the recombinant expression plasmids were introduced into HEK293F cells using Polyplus suspension cell transfection reagent for expression. Replenishment solution was added after 24 and 72 hours of transfection. The supernatant was collected on the fifth day, and the antibodies were isolated and purified using Protein A agarose purification resin and stored in PBS. The experimental results (Table 2) showed that the yields of the seven VHH-hFc recombinant antibodies transiently expressed in 293F cells were 53-231.4 mg / L. SDS-PAGE electrophoresis showed that the VHH-hFc bands of the 137-1, 137-7, 137-12, 137-18, and 137-36 sequences were normal in size and had a purity of >95%, whereas the recombinantly expressed antibodies of the 137-16 and 137-39 sequences had nonspecific bands and a purity of <95%.

[0072] [Table 4]

[0073] Example 7: Affinity EC50 of Nanobodies to Antigen Recombinant Proteins

[0074] The affinity of seven VHH-hFc recombinant antibodies for human and monkey 4-1BB extracellular domain recombinant proteins was measured using ELISA. Human and monkey 4-1BB extracellular domain recombinant proteins (His-tagged) were added at 200 ng / well to a 96-well ELISA plate and incubated overnight at 4°C. The VHH-hFc recombinant antibodies were diluted to different concentrations (0.014-10 μg / mL) and reacted with the antigen in ELISA. Color development was performed using an HRP-conjugated anti-VHH secondary antibody, and the absorbance at 450 nm was measured using a microplate reader. The experimental results (Tables 3 and 4) showed that all seven VHH-hFc recombinant antibodies were able to bind to human or monkey 4-1BB extracellular domain recombinant proteins. Among these, CD137-1, CD137-7, CD137-16, and CD137-18 were ranked in the top four for affinity to human or monkey 4-1BB extracellular domain recombinant proteins. The affinity of the recombinant antibodies for the mouse 4-1BB extracellular region recombinant protein was measured using the same experimental method. The results (Table 5) show that the VHH-hFc recombinant antibodies of CD137-1, CD137-7, CD137-16, and CD137-18 did not bind or only weakly bound to the mouse 4-1BB extracellular region recombinant protein.

[0075] [Table 5]

[0076] [Table 6]

[0077] [Table 7]

[0078] Example 8: Affinity KD values ​​of nanobodies to antigenic recombinant proteins

[0079] The affinity KD values ​​of four VHH-hFc recombinant antibodies (CD137-1, CD137-7, CD137-16, and CD137-18) for human 4-1BB extracellular domain recombinant protein were measured using Biacore. The temperature of the Biacore 8k sample compartment and flow cell was set to 25°C. VHH-hFc samples were captured as ligands on a Protein A chip. The corresponding target proteins were diluted with HBS-EP pH 7.4 buffer to prepare analytes. The analyte flow rate was set to 30 μL / min, and binding was allowed for 120 s and dissociation for 600 s. Data were subjected to fitting analysis using a 1:1 binding and Fit local model. The experimental results (Table 6) showed that all four VHH-hFc recombinant antibodies were able to bind to human 4-1BB extracellular domain recombinant protein, with KD values ​​ranging from 3.3 to 38 nM.

[0080] [Table 8]

[0081] Example 9: Binding of nanobodies to cells overexpressing antigenic recombinant proteins

[0082] The affinity of the nanobody to human 4-1BB-overexpressing 293F cells was measured using a flow cytometer. 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 293F cells transiently overexpressing human 4-1BB were cultured. 6Cells were harvested, washed twice with PBS, resuspended in 100 μL of PBS, and incubated for 1 hour with 10 μg / mL VHH-hFc recombinant antibodies. Cells were washed three times with PBS, resuspended in 100 μL of PBS, and incubated for 1 hour with 0.2 μg / mL FITC-labeled anti-human Fc antibodies. Cells were washed three times with PBS, resuspended in 300 μL of PBS, and fluorescence was detected using a flow cytometer. Experimental results (Figure 1) showed that the VHH-hFc recombinant antibodies CD137-1, CD137-7, CD137-16, and CD137-18 bound to human 4-1BB-overexpressing 293F cells.

[0083] Example 10: PBMC activation experiments

[0084] After human PBMCs were stimulated with VHH-hFc recombinant antibodies of four sequences (CD137-1, CD137-7, CD137-16, and CD137-18), the level of IL-2 release was measured using an ELISA kit. A 96-well cell culture plate was incubated overnight at 4°C with or without the CD3 antibody OKT3. The next day, PBMCs (1x10e5 / well) and different concentrations of 4-1BB recombinant antibody were added. After 72 hours of incubation, the supernatants were collected and used for ELISA detection of IL-2 levels. The experimental results (Figures 2 and 3) showed that the CD137-1, CD137-7, CD137-16, and CD137-18 VHH-hFc recombinant antibodies were all able to stimulate human PBMCs to release IL-2 when loaded with OKT3, but without OKT3, the ability of CD137-1, CD137-7, CD137-16, and CD137-18 to stimulate PBMCs to release IL-2 was significantly reduced, and the antibody EC50 values ​​were significantly increased (Table 7). Furthermore, the experimental results (Figure 3) showed that, regardless of the presence or absence of OKT3, CD137-1 and CD137-18 had similar maximum IL-2 release values ​​after PBMC stimulation, indicating that the CD137-1 and CD137-18 VHH-hFc recombinant antibodies were able to fully activate PBMCs regardless of the presence or absence of OKT3. However, in the absence of OKT3, the VHH-hFc recombinant antibodies CD137-7 and CD137-16 failed to fully activate PBMCs, and the maximum IL-2 release values ​​for these two antibodies in the absence of OKT3 were significantly lower than those in the presence of OKT3. These data indicate that full PBMC activation by CD137-1 and CD137-18 is independent of CD3 antibodies, whereas full PBMC activation by CD137-7 and CD137-16 is dependent on CD3 antibodies.

[0085] [Table 9]

[0086] The above is merely a preferred embodiment of the present invention, and those skilled in the art can make some improvements and modifications without departing from the principles of the present invention, and it should be understood that these improvements and modifications also fall within the scope of the present invention.

Claims

1. An anti-4-1BB antibody or variant thereof, or an antigen-binding fragment thereof, comprising three complementarity determining regions CDR1, CDR2, and CDR3 of VHHs designated 137-1, 137-7, 137-12, 137-16, 137-18, 137-36, and 137-39, 【Table 1】 Illustratively, the variant is a humanized variant or an identical (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%) variant; Alternatively, illustratively, camel IG, Ig NAR, Fab fragment, Fab' fragment, F(ab)' ２ Fragment, F(ab)' ３ Fragment, Fv, scFv, bis-scFv, (scFv) ２ , microantibodies, double-chain antibodies, triple-chain antibodies, quadruple-chain antibodies, disulfide-stabilized Fv proteins and single domain antibodies (sdAbs, nanobodies), camelid antibodies, bispecific antibodies or trispecific antibodies, and further, the antibodies are designated as 137-1, 137-7, 137-12, 137-16, 137-18, 137-36, 137-39, or variants thereof, or antigen-binding fragments thereof.

2. A fusion protein comprising the antibody of claim 1, a variant thereof, or an antigen-binding fragment thereof, Illustratively, the fusion protein further comprises a tag sequence (e.g., Poly-His, Hemagglutinin, c-Myc, GST, Flag-tag, etc.), an IgG1-Fc protein sequence, or an epitope tag (e.g., for 4-1BB or another epitope different therefrom), or an additional antibody active fragment (e.g., another epitope for 4-1BB or an antibody or antibody active fragment for the same epitope, or a ligand capable of binding to 4-1BB).

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

4. 1. An isolated expressed antibody or antigen-binding fragment thereof polynucleotide, wherein the polynucleotide is capable of expressing the claimed antibody and variants thereof, or antigen-binding fragments thereof; wherein the polynucleotide is capable of expressing a fusion protein as described herein; and wherein the polynucleotide is capable of expressing an antibody-drug conjugate as described herein.

5. A vector comprising the polynucleotide of claim 4, which is preferably a plasmid vector.

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

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

8. Use of the antibody and its variant, or its antigen-binding fragment according to claim 1, the fusion protein according to claim 2, or the antibody-drug conjugate according to claim 3 in the manufacture of a pharmaceutical for treating and / or preventing a 4-1BB-associated disease.

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

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

11. A kit for detecting 4-1BB protein, comprising the antibody and its variant, or its antigen-binding fragment, of claim 1, the fusion protein of claim 2, or the antibody-drug conjugate of claim 3, and a detectably acceptable reagent.

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