Anti-tl1a nanobodies and uses thereof

Abstract

The application belongs to the technical field of biotechnology, and particularly relates to an anti-TL1A nanobody and application thereof. The amino acid sequences of the complementarity determining regions CDR1, CDR2 and CDR3 of the nanobody are respectively shown as SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:1. The application uses a llama natural nanobody library and combines phage display technology to successfully screen a high-affinity nanobody targeting TL1A. The antibody can specifically recognize human TL1A protein and a TL1A overexpression cell line, and has high affinity, high specificity and good physicochemical stability. Based on the characteristic of targeting TL1A, the nanobody has important application value in the precise diagnosis and targeted treatment of TL1A high-expression related diseases such as inflammatory bowel disease, rheumatoid arthritis, asthma and fibrosis, and is particularly suitable for developing a high-sensitivity in-vitro diagnostic kit and a novel biological therapeutic drug.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an anti-TL1A nanobody and its application. Background Technology

[0002] TL1A (TNF-like ligand 1A, also known as TNFSF15) is a member of the tumor necrosis factor superfamily. It is a type II transmembrane glycoprotein of approximately 25 kDa, with its C-terminus located extracellularly and its N-terminus located intracellularly. Its extracellular region can be cleaved by metalloproteinases to form soluble trimeric cytokines. TL1A specifically binds only to its receptor DR3 (TNFRSF25), a type I transmembrane protein that is primarily highly expressed in activated T cells (especially Th1 and Th17 subsets), regulatory T cells (Tregs), some NK cells, and dendritic cells. TL1A / DR3 signaling recruits the TRADD-TRAF2 / 5 complex, activating pathways such as NF-κB, MAPK, and JNK, significantly enhancing T cell proliferation, cytokine (IFN-γ, IL-13, IL-17) secretion, and the expression of the intestinal homing receptor α4β7, thereby playing a crucial role in mucosal immunity, inflammatory cascade amplification, and tissue repair. Preclinical and clinical studies have shown that TL1A is abnormally highly expressed in inflammatory bowel disease (IBD), rheumatoid arthritis, asthma, and fibrotic diseases, and its serum or tissue levels are positively correlated with disease activity. Gene polymorphisms (such as TNFSF15 rs4263839) are also significantly associated with IBD susceptibility. Based on this, the TL1A / DR3 axis has become a new generation of therapeutic targets for autoimmune and inflammatory diseases. Several blocking monoclonal antibodies (such as PF-06480605 and TEV-48574), fusion proteins, and small molecule inhibitors are currently in Phase I / II clinical evaluation, showing promising safety profiles and potential for improving disease activity.

[0003] Phage display technology can use the extracellular domain of TL1A or its receptor DR3 as immobilized target molecules to perform multiple rounds of "adsorption-elution-amplification" screening on fully human or camelid immune single-domain antibody (VHH) libraries, enriching high-affinity clones. Nanobodies (15kDa), due to their small size, high stability, ease of humanization, and large-scale expression in *E. coli*, can precisely inhibit downstream inflammatory signals by blocking the binding interface between TL1A and DR3. Developing highly specific nanobodies targeting TL1A or DR3 using a phage display platform holds promise for providing next-generation targeted biologics for autoimmune diseases such as IBD, and can also be used in the development of companion diagnostic reagents for inflammatory imaging diagnosis or stratified therapy. Summary of the Invention

[0004] The purpose of this invention is to provide an anti-TL1A nanobody that can specifically recognize Human TL1A and HEK293TL1A overexpressing cell lines.

[0005] The technical solution of this invention to solve its technical problem is as follows:

[0006] In a first aspect of the present invention, an anti-TL1A nanobody is provided, wherein the amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 of the VHH chain of the nanobody are shown in SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5, respectively.

[0007] Preferably, the amino acid sequence of the anti-TL1A nanobody is as shown in SEQ ID NO:1, or has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:1.

[0008] In a second aspect of the invention, an isolated nucleic acid molecule is provided that encodes an anti-TL1A nanobody as described in the first aspect.

[0009] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:2.

[0010] In a third aspect of the invention, an expression vector is provided, the expression vector comprising the nucleic acid molecule described in the second aspect of the invention.

[0011] In a fourth aspect of the invention, a host cell is provided, the host cell containing the expression vector described in the third aspect.

[0012] Preferably, the host cell is selected from mammalian cells.

[0013] In a fifth aspect of the invention, an antibody derivative is provided, the antibody derivative comprising one, two or more nanobodies as described in the first aspect of the invention; preferably, the antibody derivative is selected from: monovalent nanobodies, multivalent nanobodies, bispecific antibodies, multispecific antibodies, heavy chain antibodies, antibody-drug conjugates or antigen-binding fragments thereof.

[0014] In a sixth aspect of the invention, a pharmaceutical composition is provided comprising the anti-TL1A nanobody of the first aspect or the antibody derivative of the seventh aspect, and a pharmaceutically acceptable carrier.

[0015] In a seventh aspect of the invention, a kit for detecting human TL1A protein is provided, the kit comprising the anti-TL1A nanobody described in the first aspect.

[0016] In an eighth aspect of the invention, the use of the anti-TL1A nanobody as described in the first aspect is provided in the preparation of a diagnostic reagent for in vitro detection of cells expressing human TL1A.

[0017] In a ninth aspect of the invention, the use of the anti-TL1A nanobody as described in the first aspect is provided in the preparation of a diagnostic reagent for detecting human TL1A protein levels in biological samples to aid in the diagnosis of TL1A-related diseases, wherein the TL1A-related diseases are selected from inflammatory bowel disease, rheumatoid arthritis, asthma, and fibrotic diseases.

[0018] Compared with the prior art, the present invention has the following technical effects:

[0019] This invention utilizes phage display technology to obtain nanobodies targeting TL1A. These antibodies specifically recognize Human TL1A and HEK293 TL1A-overexpressing cell lines. The anti-TL1A nanobodies provided by this invention exhibit high specificity, high affinity, and good physicochemical stability. Based on their TL1A-targeting properties, these nanobodies demonstrate significant advantages in the precise diagnosis and targeted therapy of TL1A-overexpressing diseases such as inflammatory bowel disease, rheumatoid arthritis, asthma, and fibrosis, and are particularly suitable for developing highly sensitive in vitro diagnostic kits and novel biotherapeutic drugs.

[0020] This invention develops nanobodies based on a natural alpaca nanobody library. Compared to immune libraries constructed through immunization, this method offers the following advantages: a. It saves time, eliminating the need for animal immunization; b. It circumvents antigen immunogenicity limitations, allowing screening against weakly immunogenic or toxic antigens; c. It directly enriches specific nanobodies from a diverse natural library, shortening the development cycle. This technical approach provides a stable and universal platform for the efficient discovery of nanobodies, contributing to the advancement of novel diagnostic and therapeutic tools in the biomedical field. Attached Figure Description

[0021] Figure 1 This is a graph showing the initial ELISA screening results of 94 monoclonal antibodies in Example 1.

[0022] Figure 2 The image shows the FACS validation results of 94 monoclonal antibodies in Example 1.

[0023] Figure 3 This is a map of the antibody-mammal system expression vector in Example 2.

[0024] Figure 4 The image shows the SDS-PAGE analysis of the purified antibody in Example 2 (M: protein marker; R: reduction conditions; NR: non-reduction conditions).

[0025] Figure 5 The SEC-HPLC chromatogram of the purified antibody in Example 2 is shown (A: 214 nm; B: 280 nm).

[0026] Figure 6 The image shows the binding curve of the purified antibody in Example 3 to the Human TL1A / His protein.

[0027] Figure 7 The image shows the binding curve of the purified antibody in Example 4 to HEK293 Human TL1A overexpressing cells.

[0028] Figure 8 The image shows the binding curve of the purified antibody in Example 4 to HEK293 blank cells. Detailed Implementation

[0029] The present invention will be further explained below with reference to specific embodiments. However, it should be noted that the following embodiments are only used to explain the present invention and cannot be used to limit the present invention. All technical solutions that are the same as or similar to the present invention are within the protection scope of the present invention.

[0030] For any techniques or conditions not specified in this embodiment, the operation shall be carried out in accordance with conventional technical methods and instrument manuals in this field; for reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained commercially.

[0031] Example 1: Screening anti-TL1A nanobodies from a natural library of alpaca nanobodies

[0032] Phage display technology was used to screen anti-TL1A nanobodies from a natural library of alpaca nanobodies.

[0033] S1. Construction of phage nanobody library: PBMCs were isolated from peripheral blood of alpacas that had never been immunized. Total RNA was extracted from these PBMCs and cDNA was obtained by reverse transcription PCR. The gene fragment of alpaca nanobody VHH was amplified using cDNA as a template. The target gene was then cloned into a phage vector and transformed into competent cells to construct a natural library of alpaca nanobody.

[0034] S2. Panning: The amplified bacterial culture was plated and cultured using liquid-phase panning. The next day, 94 clones from one plate were selected for specific screening. Using TL1A / His protein as the target antigen, the above phage library was subjected to three rounds of liquid-phase panning: in each round, the phage library was incubated with the antigen, unbound phages were washed away, and specifically bound phages were washed away. The library was then infected with TG1 bacteria for amplification. After the third round, an enriched anti-TL1A phage population was obtained.

[0035] S3. Single-clone screening:

[0036] After panning and plating, single colonies were picked and the third round of panning products were used to infect TG1 cells. After plating, single clones were picked. Helper phage M13K07 was added, and the culture supernatant was collected. Positive clones binding to human TL1A were initially screened by ELISA. The binding activity of positive clones to human TL1A was further tested in parallel, and their specific binding to HEK293-hTL1A cells was verified by FACS. Based on the comprehensive screening results, the target clones were sequenced to obtain anti-TL1A nanobodies.

[0037] The ELISA screening process for monoclonal antibodies was as follows: Human TL1A / His protein and blocking buffer (3% non-fat powdered milk in PBS) were coated separately onto microplates (Corning, 3590). After incubation, the plates were washed thoroughly, and then incubated with blocking buffer (3% non-fat powdered milk in PBS) for 1 h. After washing thoroughly, the phage display nanobody samples expressed by each monoclonal antibody were added. The positive control was anti-Human TL1A (Tulisokibart), and the negative control was Anti-HELIgG1 hFc (Biointron, B117901). The plates were incubated for 1 h. After washing thoroughly again, Mouse anti-M13 mAb HRP (Sino Biolo, 11973-MM05T-H) was added as the secondary antibody. The control antibody secondary antibody was Goat Anti-Human IgG-Fc, HRP (Sigma, A0170). The plates were incubated for 1 h and then washed thoroughly again. Add 100 μL of ABTS chromogenic solution (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) to each well, and incubate at room temperature in the dark for 10-15 min. Then, read the values ​​at 415 nm using a microplate reader.

[0038] Criteria for determining a positive clone: ​​Human TL1A / Milk > 3 and Milk < 0.3, as shown in the test results. Figure 1 As shown, the positive rate of monoclonal and Human TL1A / His protein binding was 86%.

[0039] The FACS screening procedure for monoclonal phages is as follows: HEK293 Human TL1A Cell Line and HEK293GFP negative control cells were mixed at a 1:1 ratio and seeded at 2E+5 cells per well in a 96-well V-plate. The cells were centrifuged and the supernatant was discarded. 100 μL of monoclonal phage supernatant was added to each well of the 96-well V-plate to resuspend the cells, and a positive control well was included. 10 μg / mL of anti-Human TL1A antibody (Tulisokibart) was used as a positive control. The cells were incubated at 4°C for 0.5 h, centrifuged, and the supernatant was discarded. The cells were washed once with PBS. Anti-M13 Bacteriophage and AlpHcAbs were added to the sample wells. ® Rabbit antibody (iFlour647), 1:2000, 100 μL / well, incubated at 4℃ for 0.5 h. For the positive control wells, add Goat Anti-hIgG (FcγSpecific) pAb [Alexa Fluor 647] (Jackson, 109-605-190), 1:800, 100 μL / well, incubate at 4℃ for 0.5 h, centrifuge and discard the supernatant, wash twice with PBS, and finally resuspend the cells in 100 μL / well PBS buffer for flow cytometry analysis.

[0040] Positive clone determination: Sample / negative control Median APC-H (≥3). Detection results are as follows: Figure 2 The results showed that the positive rate of binding to monoclonal antibodies and HEK293 Human TL1A Cell Line cells was 91%.

[0041] Based on the screening results of ELISA and FACS, and after sequencing analysis and comparative screening, the high-affinity anti-TL1A nanobody TL1A-C2R3-D4-P2 was finally obtained. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:1, and its encoding DNA sequence is shown in SEQ ID NO:2. The amino acid sequence of the complementarity-determining region (CDR1) of nanobody TL1A-C2R3-D4-P2 is shown in SEQ ID NO:3, the amino acid sequence of the complementarity-determining region (CDR2) is shown in SEQ ID NO:4, and the amino acid sequence of the complementarity-determining region (CDR3) is shown in SEQ ID NO:5.

[0042] Example 2: Expression and purification of anti-TL1A nanobodies in a lactational system

[0043] The mammalian system expression vector pcDNA3.4 was used to construct the TL1A-C2R3-D4-P2 nanobody. (See diagram below.) Figure 3Then, plasmids were prepared using this plasmid. CHO cells were selected as the host cells for antibody expression, with an expression volume of 20 mL. The supernatant after expression was purified using a Protein A affinity chromatography column to obtain high-purity expressed antibodies. The purity was determined by SDS-PAGE and SEC-HPLC, and the results are shown below. Figure 4 and Figure 5 As shown, the antibody purity all reached over 95%, indicating high purity.

[0044] Example 3: Detection of binding between anti-TL1A nanobody and Human TL1A antigen

[0045] The ELISA detection procedure for the binding of TL1A-C2R3-D4-P2 nanobody to TL1A / His protein is as follows: Human TL1A / His recombinant protein is coated onto an ELISA plate (Corning, 3590), incubated, and then thoroughly washed. Blocking buffer (3% non-fat powdered milk in PBS) is added, and the plate is incubated for 1 hour, followed by thorough washing. The purified TL1A-C2R3-D4-P2 antibody and control antibody from Example 2 are diluted to 100 nM as the initial well concentration, and serially diluted 4-fold, with the last well being a blank. 100 μL / well is used, and the plate is incubated for 1 hour, followed by thorough washing. Secondary antibody Goat Anti-Human IgG-Fc, HRP (Sigma, A0170) is used, and the plate is incubated for 1 hour, followed by thorough washing again. Add 100 μL of ABTS chromogenic solution (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) to each well, and incubate at room temperature in the dark for 10-15 min. Read the results using a microplate reader at 415 nm. The positive control was anti-Human TL1A (Tulisokibart), and the negative control was Anti-HEL VHH-Human IgG1 Fc (Biointron, B117901). The detection results are as follows: Figure 6 As shown, TL1A-C2R3-D4-P2 binds to the Human TL1A / His protein with an EC50 value of 0.6018 nM, demonstrating high affinity at the sub-nanomolar level.

[0046] Example 4: Detection of binding of anti-TL1A nanobody to HEK293 Human TL1A cells

[0047] The FACS assay for the binding of TL1A-C2R3-D4-P2 nanobody to HEK293 Human TL1A cells was performed as follows: HEK293 Human TL1A cells and HEK293 blank cells were seeded separately (2E+5 cells per well in a 96-well V plate), centrifuged at 800g for 3 min, and the supernatant was discarded. The purified TL1A-C2R3-D4-P2 antibody and control antibody were diluted to 400 nM as the initial concentration, serially diluted 4-fold, with the last well being a blank, 100 μL / well, incubated at 4℃ for 0.5 h, centrifuged at 800g for 3 min, and the supernatant was discarded. GoatAnti-hIgG (Fcγ Specific) pAb [Alexa Fluor 647] was added at a 1:800 ratio, 100 μL / well, incubated at 4℃ for 0.5 h, centrifuged at 800g for 3 min, and the supernatant was discarded. Add 200 μL / well PBS buffer, mix well, centrifuge at 800g for 3 min, and discard the supernatant. Resuspend cells in 100 μL / well PBS buffer and analyze by flow cytometry. The positive control was anti-Human TL1A (Tulisokibart), and the negative control was Anti-HEL IgG1 hFc (Biointron, B117901). The binding results of TL1A-C2R3-D4-P2 to HEK293 Human TL1A cells are shown below. Figure 7 As shown: TL1A-C2R3-D4-P2 can bind to HEK293 Human TL1A with an EC50 of 2.634 nM, but does not bind to control cells (HEK293 blank cells). Figure 8 As shown, this nanobody can specifically recognize the natural conformation of Human TL1A and has the ability to maintain high affinity binding in physiologically relevant environments.

[0048] Finally, it should be noted that the above embodiments are merely illustrative of the principles, performance, and effects of the present invention, and are not intended to limit the present invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. An anti-TL1A nanobody, characterized in that, The amino acid sequence of the complementarity-determining region (CDR1) of the nanobody is shown in SEQ ID NO:3, the amino acid sequence of the complementarity-determining region (CDR2) is shown in SEQ ID NO:4, and the amino acid sequence of the complementarity-determining region (CDR3) is shown in SEQ ID NO:

5.

2. The anti-TL1A nanobody as described in claim 1, characterized in that, The amino acid sequence of the anti-TL1A nanobody is as shown in SEQ ID NO:1, or has at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with SEQ ID NO:

1.

3. An isolated nucleic acid molecule encoding an anti-TL1A nanobody as described in claim 1 or 2.

4. The nucleic acid molecule as described in claim 3, characterized in that, The nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO:

2.

5. An expression carrier, characterized in that, The expression vector comprises the nucleic acid molecule as described in claim 3 or 4.

6. A host cell, characterized in that, The host cell contains the expression vector as described in claim 5.

7. An antibody derivative comprising the anti-TL1A nanobody according to claim 1 or 2.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) the anti-TL1A nanobody as described in claim 1 or 2, or the antibody derivative as described in claim 7; and (ii) Pharmaceutically acceptable carriers.

9. A kit for detecting human TL1A protein, characterized in that, The kit contains the anti-TL1A nanobody as described in claim 1 or 2.

10. The use of the anti-TL1A nanobody as described in claim 1 or 2 in the preparation of a diagnostic reagent for detecting human TL1A protein levels in biological samples to aid in the diagnosis of TL1A-related diseases, wherein the TL1A-related diseases are selected from inflammatory bowel disease, rheumatoid arthritis, asthma, and fibrotic diseases.

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