An antibody or antigen-binding fragment against human tgf-betal protein and uses thereof

Abstract

The application discloses a recombinant humanized antibody for human TGF-beta 1 protein and application thereof, and belongs to the technical field of biological medicines. The antibody or antigen binding fragment for human TGF-beta 1 protein provided by the application is constructed by variable region complementarity determining regions A, B and C of a TGF-beta 1 protein binding region, and the TGF-beta 1 protein binding region is used for inhibiting tumor cells, thereby providing a new direction for anti-tumor. Meanwhile, the antibody has excellent affinity and specificity, and can be used for preparing a TGF-beta 1 protein inhibitor.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an antibody or antigen-binding fragment targeting human TGF-β1 protein and its application. Background Technology

[0002] TGF-β1 (transforming growth factor-β1) is the most widely used and extensively studied member of the TGF-β superfamily. It is a key multifunctional cytokine regulating cell growth, differentiation, apoptosis, migration, and immune homeostasis. Under physiological conditions, TGF-β1 exerts important regulatory effects through autocrine and paracrine pathways. In the tumor microenvironment, TGF-β1 exhibits a significant "dual role": in the early stages of tumorigenesis, it acts as a tumor suppressor, inhibiting epithelial cell proliferation and promoting apoptosis; while in the middle and late stages of tumor progression, it transforms into a potent tumor promoter, driving tumor invasion, metastasis, and immune escape by inducing epithelial-mesenchymal transition (EMT), promoting angiogenesis, suppressing immune cell function (such as cytotoxic T cells and natural killer cells), and enriching regulatory T cells.

[0003] This complex and contradictory biological function makes direct targeting of the TGF-β1 signaling pathway a highly attractive strategy for cancer treatment. However, existing TGF-β1 inhibitors, such as small molecule kinase inhibitors and antisense oligonucleotides, often face challenges in clinical development due to insufficient target specificity, systemic toxicity (such as valvular heart disease), or limited efficacy.

[0004] Therefore, how to develop an antibody or antigen-binding fragment targeting human TGF-β1 protein has become an important issue that urgently needs to be addressed. Summary of the Invention

[0005] Therefore, the purpose of this invention is to provide an antibody or antigen-binding fragment targeting human TGF-β1 protein and its application thereto, which inhibits tumor cells through the TGF-β1 protein binding region.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] In a first aspect, the present invention provides an antibody or antigen-binding fragment targeting human TGF-β1 protein, comprising a binding site for human TGF-β1 protein composed of a variable region complementarity-determining region A as shown in SEQ ID NO: 11, a variable region complementarity-determining region B as shown in SEQ ID NO: 12, and a variable region complementarity-determining region C as shown in SEQ ID NO: 13.

[0008] Among them, the antibody or antigen-binding fragment is a human single-domain antibody (VHH), which is obtained by screening a fully human synthetic single-chain phage display library.

[0009] Based on the above technical solution, the binding affinity KD with TGF-β1 protein is further between 1E-6 and 1E-9.

[0010] Based on the above technical solution, the sequence of the antibody or antigen-binding fragment is shown in SEQ ID NO: 1.

[0011] Secondly, the present invention provides a nucleic acid molecule encoding the aforementioned antibody or antigen-binding fragment against human TGF-β1 protein.

[0012] Thirdly, the present invention provides the use of the above-mentioned antibody or antigen-binding fragment targeting human TGF-β1 protein in the preparation of TGF-β1 protein inhibitors or in the preparation of antitumor drugs.

[0013] Fourthly, the present invention provides a bispecific antibody, comprising a first antigen-binding unit and a second antigen-binding unit, wherein the first antigen-binding unit is an antibody or antigen-binding fragment targeting human TGF-β1 protein as described above, and the second antigen-binding unit is an antibody or antibody fragment targeting another target.

[0014] Based on the above technical solution, further, the structure is selected from VHH-VHH tandem structure, VHH-IgG fusion structure or multivalent VHH structure.

[0015] Fifthly, the present invention provides a fusion protein comprising an antibody or antigen-binding fragment against human TGF-β1 protein as described above and a functional domain connected thereto.

[0016] Based on the above technical solution, the functional domain is further defined as the immunoglobulin Fc region.

[0017] Based on the above technical solution, the structural units are further connected by flexible linker peptides, wherein the linker peptides are selected from (GGGGS)n.

[0018] Compared with the prior art, the present invention has the following beneficial effects: The antibody or antigen-binding fragment against human TGF-β1 protein provided by this invention has a TGF-β1 protein binding region constructed from variable complementarity-determining regions A, B, and C. It inhibits tumor cells through the TGF-β1 protein binding region, providing a new direction for anti-tumor therapy. At the same time, the antibody has excellent affinity and specificity and can be used to prepare TGF-β1 protein inhibitors. Attached Figure Description

[0019] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0020] Figure 1 This is a liquid chromatography analysis chromatogram of the unpurified antibody sample from Example 3 of the present invention; Figure 2 This is a liquid chromatography analysis chromatogram of the purified antibody sample from Example 3 of the present invention; Figure 3 This is a diagram illustrating the biomolecular interaction dynamics analysis of Example 4 of the present invention; Figure 4 This is a diagram showing the cell migration results in Example 5 of the present invention; Figure 5 This is a comparison of scratch healing rates among different treatment groups in Example 5 of the present invention. Detailed Implementation

[0021] The present invention will now be described in detail with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention. In the embodiments, when nM is used as a concentration unit, it refers to mmol / L; when M is used as a concentration unit, it refers to mol / L.

[0022] Example 1 A single-chain VH phage library was synthesized, and three rounds of solid-phase panning were performed using TGF-β1 as the screening antigen. Two screening schemes were implemented: one with a constant antigen concentration and the other with a decreasing antigen concentration. After panning, the output increased to 8 × 10^9, and single-clone ELISA screening was performed on two 96-well plates. The initial screening yielded 98 positive clones, and the retest yielded 64 positive clones (OD450 > 0.5). Sequencing yielded 23 unique antibody sequences. Further SPR analysis of the candidate clones in the supernatant showed that most clones exhibited significant binding signals to TGF-β. Eight clones were selected to construct VH-huFc recombinant antibodies, which were then expressed and purified in CHO cells. The results showed that seven recombinant antibodies specifically bound to TGF-β, with 51-huFc exhibiting the best binding activity (Biacore affinity of 5.49 × 10^9). -8 M. The above results demonstrate that high-affinity anti-TGF-β antibodies can be effectively screened using a fully human single-chain VH synthetic library. The final antibody sequence targeting human TGF-β1 protein is as follows.

[0023] Sequence of the complementary determinant region A of the variable region: SEQ ID NO: 11 GFTFSDYAVS Sequence of the complementary determinant region B of the variable region SEQ ID NO: 12 VISGDGGSTYYADSVKG Sequence of the complementary determinant region C of the variable region: SEQ ID NO: 13 AKENYYIHDYFDY Sequence of antibody or antigen-binding fragment targeting human TGF-β1 protein SEQ ID NO: 1 EVQLLESGGGLVQPGGSLRLSCAASGFTFSDYAVSWVRQAPGKGLEWVSVISGDGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKENYYIHDYFDYWGQGTLVTVSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEV KFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0024] Example 2 To adapt to the ExpiCHO system, the plasmid vector used should contain strong mammalian cell promoters such as the CMV promoter, as well as necessary selection markers (such as ampicillin resistance genes for amplification in bacteria). The antibody heavy chain gene obtained in the previous step is then cloned into this type of expression vector.

[0025] Heavy chain gene SEQ ID NO: 2 The vector used for transient transfection expression in this invention is the pcDNA3.2 series of eukaryotic expression plasmids (derived from Thermofisher). The pcDNA3.2 plasmid is a high-copy-number mammalian cell expression vector containing strong promoter elements (such as the CMV promoter), enabling efficient transient expression of exogenous genes in mammalian host cells. This plasmid also includes a multiple cloning site (MCS) to facilitate the cloning and insertion of the target gene, as well as transcription termination and polyadenylation signal sequences (such as BGHpolyA) to ensure transcript stability and integrity. Furthermore, the pcDNA3.2 plasmid contains a replication origin suitable for prokaryotic cell amplification and antibiotic resistance selection markers, which can be used for plasmid amplification and purification in *E. coli*.

[0026] In this embodiment, the nucleic acid sequence encoding the target protein is cloned downstream of the multiple cloning site of the pcDNA3.2 plasmid to construct a recombinant expression vector, which is then introduced into mammalian cells for expression via transient transfection to obtain the target protein for subsequent functional analysis and characterization studies.

[0027] 2. Host cell: The host cell used in this system is ExpiCHO-S cell, a modified Chinese hamster ovary cell line that can grow in suspension in serum-free medium. It exhibits high-density growth and efficient protein expression. These ExpiCHO-S cells were purchased directly from Thermo Fisher Scientific.

[0028] Cell culture: Before use, cells need to be routinely passaged in ExpiCHO expression medium at 37°C, 8% CO2, and with shaking on a fixed-track shaker, maintaining a viability of over 95%. When the cell density reaches approximately 4 × 10⁻⁶ cells / year... 6 -6×10 6 When the cell / mL ratio is reached, it can be used for transfection.

[0029] 3. Transfection The specific steps for introducing the gene into the host (ExpiCHO transient transfection) are as follows.

[0030] Preparation before transfection (Day 1): Centrifuge ExpiCHO-S cells in the logarithmic growth phase and resuspend them in fresh culture medium, adjusting the density to 3 × 10⁻⁶. 6 -4×10 6 cells / mL, incubated overnight.

[0031] Day 0 (on the day of transfection) The cells were further diluted to 6 × 10⁶. 6cells / mL. For example, add an appropriate amount of cell suspension to a shake flask or deep-well plate.

[0032] Preparation of the transfection complex: Dilute the purified plasmid DNA in OptiPRO SFM serum-free medium. A typical final concentration target is approximately 0.8 µg / mL culture volume.

[0033] Add ExpiFectamine CHO transfection reagent to the diluted DNA and mix gently.

[0034] Incubate at room temperature for about 10-30 minutes to form a DNA-transfection reagent complex.

[0035] Add the complex dropwise to the cell culture and mix gently.

[0036] Post-transfection treatment: Feeding (approximately 18-22 hours after transfection): Add ExpiFectamine CHO Enhancer and ExpiCHO Feed to the culture system.

[0037] Culture conditions optimization: After transfection, the culture temperature can be reduced from 37°C to 32°C and the CO2 concentration can be reduced to 5% to improve the expression level and stability of the antibody against human TGF-β1 protein (high expression protocol).

[0038] Continue cultivating for 7 to 10 days until harvest.

[0039] Example 3 This embodiment uses the cell culture medium from Example 2 as the starting material to purify the antibody against human TGF-β1 protein. The specific steps are as follows.

[0040] Initial harvest: Centrifuge (e.g., 30 minutes, 3200 rpm) or filter the culture to remove cells and cell debris to obtain a cell supernatant containing antibodies against human TGF-β1 protein.

[0041] The steps for purifying antibodies from the harvested supernatant include: Affinity chromatography: This is the most crucial step. It utilizes the high affinity between recombinant protein A packing material and the antibody Fc fragment for capture. The clarified supernatant is loaded onto a Protein A column, thoroughly washed, and then eluted with a low pH buffer (e.g., 0.1 M glycine, pH 3.0). The elution is immediately followed by neutralization with a neutralization buffer (e.g., 1 M Tris-HCl, pH 9.0) to preserve antibody activity.

[0042] Fine purification: In some embodiments of the present invention, the antibody is further finely purified by size exclusion chromatography (SEC) after initial purification by affinity chromatography and / or ion exchange chromatography.

[0043] The following materials were used: Cytiva Superdex 200 packing material; equilibration / elution buffer: 20 mM PB, 11% trehalose, pH 7.3 ± 0.1; the column was isocratically equilibrated with 20 mM PB, 11% trehalose, pH 7.3 ± 0.1 buffer. Clarified antibody samples filtered through a 0.22 μm filter were loaded at a volume not exceeding 3% of the column volume and eluted isocratically at a low linear velocity (approximately 10–15 cm / h) to improve resolution. Elution peaks were monitored using UV 280 nm; the leading and shoulder peaks of enriched aggregates were discarded, and only the central portion of the main peak was collected as the product pool.

[0044] The molecular sieve chromatography separates proteins based on differences in molecular size, effectively removing antibody aggregates, fragments, and other impurities of different molecular weights, thereby improving the purity and uniformity of antibodies against human TGF-β1 protein.

[0045] Specifically, the antibody sample against human TGF-β1 protein obtained through pretreatment steps is loaded into a pre-equilibrated molecular sieve column after buffer replacement or dilution. The equilibration buffer is a physiologically compatible buffer system. The antibody sample against human TGF-β1 protein is separated under isocratic elution conditions, and the elution peaks are collected according to the elution volume. The protein elution is monitored by UV absorption or other conventional methods. The target elution fraction containing the antibody against human TGF-β1 protein is collected, combined, concentrated, and buffer-replaced to obtain a purified antibody formulation against human TGF-β1 protein.

[0046] like Figure 1 and 2 As shown, after purification by molecular sieve chromatography, the antibody against human TGF-β1 protein achieved a SEC purity of over 90% and an SDS-PAGE purity of over 95%. Aggregates and low molecular weight impurities were effectively removed, making the resulting antibody against human TGF-β1 protein suitable for subsequent structural characterization, functional studies, or formulation development. Antibody aggregates were removed, and buffer replacement was performed. Finally, the antibody against human TGF-β1 protein was placed in a formulation buffer such as PBS. The purified antibody solution against human TGF-β1 protein was sterile filtered and stored at -80°C.

[0047] Example 4 This experiment employed surface plasmon resonance (SPR) technology to determine the binding affinity between antibodies against human TGF-β1 protein and TGFβ antigen using a "capture method." The specific procedure is as follows: 1. Chip surface immobilization: Anti-Fab antibodies were covalently immobilized on the surface of a CM5 chip using an "amino-coupled chemical method".

[0048] 2. Target antibody capture: An expression supernatant sample containing an antibody against human TGF-β1 protein is injected into a flow cell and flows through a chip surface immobilized with anti-Fab antibody. The anti-Fab antibody specifically captures the antibody against human TGF-β1 protein in the expression supernatant, thereby effectively immobilizing the antibody against human TGF-β1 protein on the chip sensing surface to form a detection complex.

[0049] 3. Antigen binding and dissociation kinetics analysis: A 100 nM GFβ antigen solution was injected into the flow cell and passed over the surface of a chip containing an antibody targeting human TGF-β1 protein. The binding process between the TGF-β1 antigen and the immobilized antibody against human TGF-β1 protein was monitored in real time for 150 seconds, and the binding rate (kon) was recorded. Subsequently, the flow was switched to buffer, and the dissociation process of the antigen-antibody complex was monitored in real time for 150 seconds, and the dissociation rate (koff) was recorded. Results are as follows: Figure 3 As shown.

[0050] 4. Surface regeneration: After each binding-dissociation cycle, a glycine solution at pH 1.5 is injected as a regeneration reagent. This low pH condition effectively disrupts the binding between antigen and antibody, as well as between the capture antibody and the target antibody, restoring the chip surface to its initial state (with only anti-Fab antibody immobilized), ensuring the repeatability of subsequent detection cycles.

[0051] The final antibody targeting human TGF-β1 protein had a binding affinity KD of 5.49 × 10⁻⁶. -8 M.

[0052] Example 5 This embodiment verifies the inhibitory effect of the antibody against human TGF-β1 protein purified in Example 2 on tumor cells.

[0053] First, cervical squamous cell carcinoma cell line SiHa (1×10⁵ cells / well) was seeded into 6-well plates. When the adherent cells reached confluence of over 95%, a scratch was made in each well using a pipette tip. After scratching, the cells were washed twice with sterile PBS to remove the suspended cells, and then cultured in serum-free medium. Simultaneously, an antibody against human TGF-β1 protein (code 51) and a negative control IgG1 were added to different wells, with a final concentration of 6.25 nM.

[0054] After placing the cell culture plate in a 37°C incubator for 72 hours, the cell migration was photographed using an inverted microscope. Figure 4 As shown; simultaneously, ImageJ software was used to measure and statistically analyze the cell migration distances in the captured images, and the results are as follows. Figure 5 As shown, the cell migration rate was 58.5% in the blank control group without any antibodies, 58.5% in the negative control group with IgG1, and 58.5% in the group with antibody against human TGF-β1 protein (code 51).

[0055] Based on the characteristics that faster scratch healing leads to stronger cell migration and a higher risk of tumor invasion and distant metastasis, it can be concluded that the antibody (code 51) targeting human TGF-β1 protein can significantly inhibit the migration of tumor cells (cervical cancer) at an effective concentration of 6.25 nM.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. An antibody or antigen binding fragment against human TGF-β1 protein, characterized in that, It includes a binding site for human TGF-β1 protein composed of the variable region complementarity-determining region A as shown in SEQ ID NO: 11, the variable region complementarity-determining region B as shown in SEQ ID NO: 12, and the variable region complementarity-determining region C as shown in SEQ ID NO:

13. Among them, the antibody or antigen-binding fragment is a human single-domain antibody (VHH), which is obtained by screening a fully human synthetic single-chain phage display library.

2. The recombinant humanized antibody against human TGF-β1 protein according to claim 1, characterized in that, The binding affinity (KD) to TGF-β1 protein is between 1E-6 and 1E-9.

3. The recombinant humanized antibody against human TGF-β1 protein according to claim 1, characterized in that, The sequence of the antibody or antigen-binding fragment is shown in SEQ ID NO:

1.

4. A nucleic acid molecule, characterized in that, Encoding an antibody or antigen-binding fragment against human TGF-β1 protein as described in any one of claims 1 to 3.

5. The use of the antibody or antigen-binding fragment against human TGF-β1 protein as described in any one of claims 1 to 3 in the preparation of TGF-β1 protein inhibitors or antitumor drugs.

6. A bispecific antibody, characterized in that, It includes a first antigen-binding unit and a second antigen-binding unit, wherein the first antigen-binding unit is an antibody or antigen-binding fragment against human TGF-β1 protein as described in any one of claims 1 to 3, and the second antigen-binding unit is an antibody or antibody fragment against another target.

7. The bispecific antibody according to claim 6, characterized in that, Selected from VHH-VHH tandem structure, VHH-IgG fusion structure or multivalent VHH structure.

8. A fusion protein, characterized in that, It includes the antibody or antigen-binding fragment against human TGF-β1 protein as described in any one of claims 1 to 3 and the functional domain linked thereto.

9. The fusion protein according to claim 7, characterized in that, The functional domain is the Fc region of immunoglobulin.

10. The bispecific antibody according to claim 6 or 7 or the fusion protein according to claim 8 or 9, characterized in that, The structural units are linked by flexible linker peptides selected from (GGGGS)n.

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