Neutralizing antibody with AQP3 channel blocking function as well as preparation method and application thereof

Abstract

The invention discloses a neutralizing antibody with an AQP3 channel blocking function as well as a preparation method and application thereof, and belongs to the technical field of biological medicines. The neutralizing antibody can be specifically combined with an extracellular domain of AQP3, after combination, a water channel and a small molecule transport function of the AQP3 are blocked through steric hindrance and conformation change, and meanwhile, antigen combination specificity is reserved. The preparation method is realized through'recombinant antigen-immunization-B cell screening culture-recombinant antibody expression and affinity purification-ELISA detection ', and the functions of the antibody are ensured. The antibody can be efficiently combined with AQP3 positive cells, is strong in targeting property and high in specificity (the serum titer reaches 1: 1024 * 10, and the purified antibody titer is greater than or equal to 1: 128 * 10), and can be independently used or combined with a nano-carrier for treating related diseases, so that the technical bottleneck that a common AQP3 antibody can only be combined and cannot efficiently play a blocking role is solved.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a neutralizing antibody with AQP3 channel blocking function, its preparation method, and its application. Background Technology

[0002] Lung adenocarcinoma is the most common pathological subtype of lung cancer, accounting for approximately 40% of all lung cancer cases. The bottleneck in its treatment lies in the immunosuppressive properties of the tumor microenvironment (TME)—among which M2 tumor-associated macrophages (M2 TAMs) are a core pro-cancer factor. M2 TAMs suppress anti-tumor immune responses by secreting immunosuppressive factors such as IL-10 and TGF-β, while simultaneously promoting tumor angiogenesis, directly leading to lung adenocarcinoma progression, drug resistance, and worsened prognosis. Therefore, they have become a key target for precision treatment of lung adenocarcinoma.

[0003] Aquaporin 3 (AQP3) is a transmembrane transport protein widely expressed on the cell membrane, possessing both aquaporin and small molecule (such as glycerol and urea) transport functions. It acts as a "molecular switch" in the polarization of M2-type TAMs: AQP3 mediates the entry of glycerol into TAMs, activating the PPAR-γ / NF-κB signaling pathway, thereby upregulating the expression of pro-M2 polarization factors such as IL-6 and IL-10, ultimately enhancing the immunosuppressive function of TAMs. Therefore, targeting AQP3 is an important strategy for regulating M2-type TAMs and improving the immune microenvironment of lung adenocarcinoma.

[0004] In existing technologies, interventions targeting AQP3 mainly include small molecule inhibitors and conventional antibodies. Small molecule inhibitors suffer from poor targeting, easy off-target effects, and significant toxic side effects. Conventional AQP3 antibodies can only bind to the extracellular domain of AQP3 through the antigen-binding domain to achieve the "recognition and localization" function, but they cannot interfere with the channel transport activity of AQP3—that is, they cannot block AQP3-mediated glycerol / water transport at the molecular level. This results in insufficient regulatory efficiency on M2 type TAMs polarization (only reducing the CD206 positivity rate by ≤15%), which is insufficient to meet clinical treatment needs.

[0005] In summary, there is an urgent need to develop a novel antibody that combines the dual characteristics of "AQP3 specific binding" and "channel function blocking" to overcome the functional limitations of ordinary antibodies, achieve efficient regulation of M2 type TAMs, and provide a safe and precise new treatment option for lung adenocarcinoma. Summary of the Invention

[0006] The purpose of this invention is to provide a neutralizing antibody with AQP3 channel blocking function, its preparation method and application, so as to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: an AQP3 neutralizing antibody that specifically binds to the extracellular domain of AQP3, blocking its water channel and small molecule transport functions; comprising three positive clones: 3G2, 4D7, and 4G6, wherein the amino acid sequence of the heavy chain variable region of the 3G2 antibody is QSVEESGGRLVTPGTPLTLTCTLSGFSLSNYAIIWVRQAPGEGLEYIGFINSRGSTYYATWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARYAADTSTAIWGPGTLVTVSS, and the amino acid sequence of the light chain variable region is QVLTQTPSSVSAAVGGTVTINCQASQSLYNNKNSAWYQQKPGQSPKLLIYKASTLASGVPSRFKGSGSTQFTLTISDLECDDAATYFCQGEFSCSSADCFAFGGGTEVVVKG; this antibody avoids non-specific interaction with normal tissue cells through specific epitope binding, and has a stable titer (≥1:128×10³ after purification).

[0008] The method for preparing AQP3 neutralizing antibody includes the following steps: (1) Preparation of recombinant AQP3 antigen: - Vector construction: Using the AQP3 extracellular domain sequence as a template (>SIP-hFc-AQP3) SIP-EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLYITREPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTK PREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALKFHYTQKSLSLSP (GA- YDAIWHFADNQLFVSGPNGTAGIFATYPSGHLDM-GGGGSGGGGS-LAGWGSAVFTTGQHWWW), primers containing EcoRI / NotI restriction sites were designed, and the target fragment was amplified by PCR; after double digestion of the fragment with the SIP-hFc vector, it was ligated with T4 DNA ligase at 16℃ for 0.5-1h to construct the recombinant expression vector SIP-hFc-AQP3; - Transformation and screening: Transform the recombinant vector into TOP10 competent cells, plate them on LB plates containing the resistance, and incubate at 37°C for 12-16 h; pick single colonies for colony PCR verification, and send positive clones for sequencing (sequencing results are ≥99% consistent with the AQP3 extracellular domain sequence). - Expression and purification: The correctly sequenced recombinant plasmid was transfected into HEK293 cells and cultured at 37℃ and 130 rpm until Day 6. The supernatant was collected. After centrifugation at 8000 rpm for 10 min and filtration at 0.22 μm, the supernatant was loaded onto a Protein A resin column. The column was equilibrated with PBS (pH 7.4), eluted with 0.1 M glycine (pH 3.0), and the elution buffer was neutralized with 1 M Tris-Cl (pH 8.5). The purified product was detected by SDS-PAGE. Coomassie brilliant blue staining showed a single target band with a purity ≥ 90%. The final concentration of recombinant AQP3 protein was 0.733 mg / ml and the yield was 3665 μg. The protein was stored at -80℃ for later use.

[0009] (2) Animal immunization: - Immunization subjects: 4-month-old, 2.1kg SPF-grade female New Zealand white rabbits (without pre-stored AQP3 antibodies); - Immunization regimen: Multiple injections on the back, first immunization on day 1 (French complete adjuvant + recombinant AQP3 protein, 1:1 emulsion), second immunization on day 14, third immunization on day 28, fourth immunization on day 42, and fifth immunization on day 56 (all French incomplete adjuvant + recombinant AQP3 protein, 1:1 emulsion). - Potency assay: 1 ml of blood was collected from the ear vein on day 49, incubated overnight at 4°C, and then centrifuged to obtain serum. ELISA was performed (coating antigen 6 μg / mL, incubated overnight at 4°C; blocking with 5% skim milk powder PBST at 37°C for 1 h; serum serially diluted 1:1000, incubated at 37°C for 1 h; HRP-labeled goat anti-rabbit secondary antibody diluted 1:8000, incubated at 37°C for 45 min; TMB color development for 5-10 min, terminated with 2M H2SO4). The results showed that rabbit A serum at a concentration of 1:1024×10... 3 The OD value at dilution was 0.373, significantly higher than the negative control (OD value 0.038), indicating successful immunization. - Cell collection: Whole blood was collected from the carotid artery on day 63. At the same time, the rabbit was sacrificed and the spleen was removed. Peripheral blood and spleen B cells were separated and cryopreserved in liquid nitrogen.

[0010] (3) B cell selection culture: - Sorting of positive B cells: Flow cytometry was used to sort out antigen-specifically bound positive B cells using fluorescently labeled recombinant AQP3 protein as a probe. - Limiting dilution culture: After counting positive B cells, dilute with special culture medium to 1 cell / well, plate in a 96-well plate, and culture at 37°C and 5% CO2 for 15 days; - Supernatant detection: Collect the culture supernatant from each well and screen it by indirect ELISA (operation is the same as serum titer detection). Candidate clones with OD value ≥ 0.451 (3G2, 4D7, 4G6) were screened out. Among them, the 3G2 clone had the highest OD value in the supernatant (1.867) and was identified as the core positive clone.

[0011] (4) Recombinant antibody expression and affinity purification: - Gene amplification: Total RNA was extracted from 3G2, 4D7, and 4G6 clones, reverse transcribed into cDNA, and the variable regions of the antibody heavy chain and light chain were amplified using specific primers. Sequencing was used to verify the correctness of the sequences. - Vector construction: The heavy chain and light chain variable region genes were subcloned into a eukaryotic expression vector, transformed into the TOP10 strain, and a large number of recombinant plasmids were extracted (purity A260 / A280=1.8-2.0). - Cell transfection: HEK293 cells were passaged to a density of 1.5-2.0 × 10⁶ cells / year. 6 For each cell / ml sample, 80 μg of recombinant plasmid was mixed with 8 ml of PBS, and 0.32 ml of PEI solution (0.5 mg / ml) was added. The mixture was incubated at room temperature for 20 min. The mixture was then added to the cell culture system and cultured at 37°C, 120 rpm, and 5% CO2 for 6 days. - Antibody purification: Centrifuge at 3000g, 4℃ for 5 min to collect the supernatant, filter at 0.22μm and load onto a Protein A / G resin column; wash the column with PBS (pH 7.4) until the effluent does not change color in G250 detection, elute with PBS (pH 3.0), neutralize and concentrate; SDS-PAGE verification showed clear heavy chain (approximately 55kDa) and light chain (approximately 25kDa) bands, with a purity ≥95%; finally obtained 2.5mg (2.5mg / ml) of 3G2 antibody, 1.36mg (1.36mg / ml) of 4D7 antibody, and 1.54mg (1.54mg / ml) of 4G6 antibody, dialyzed with PBS (pH 7.4) and aliquoted for storage.

[0012] (5) Antibody identification: - Specificity verification: Western blot analysis showed that the antibody only bound to the lysate of AQP3 positive cells (such as A549) and had no cross-reactivity with AQP3 negative cells (such as HUVEC); immunofluorescence showed that the antibody specifically bound to A549 cells.

[0013] - Potency assay: The purified antibodies were tested by ELISA. The OD value of the 3G2 antibody was 0.691 when diluted at 1:128×10³, and the OD value of the 4G6 antibody was 0.123 when diluted at 1:128×10³, both of which meet the functional application requirements. Attached Figure Description

[0014] Figure 1 This is a flowchart of the monoclonal antibody development process of the present invention; Figure 2 This is a schematic diagram of the recombinant antibody expression and purification test of the present invention; Figure 3 This is a schematic diagram of the cell binding test of the present invention; Figure 4 This is a schematic diagram of the polarization structure of M2-type TAMs interfered with by the present invention. Detailed Implementation

[0015] Example 1: Preparation of recombinant AQP3 protein 1. Primer design: The upstream primer contains an EcoRI restriction site (5'-GAATTCATGGCCTGGCTGCTGCTG-3'), and the downstream primer contains a NotI restriction site (5'-GCGGCCGCTTAGTGGTGGTGGTGGTG-3'); 2. PCR amplification: Using human AQP3 cDNA as a template, the reaction system (50 μl) consisted of: F 1 μl, R 1 μl, template 1 μl (20 ng), 10*pfu buffer 5 μl, pfu 1 μl, dNTP 2 μl, and deionized water 39 μl. The reaction conditions were: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 55℃ annealing at 2 Kb / min, 72℃ extension for 1 min (32 cycles), and 72℃ final extension for 10 min. 3. Vector construction: PCR products and SIP-hFc vector were digested with EcoRI / NotI (37℃ for 1.5h), and after gel recovery, they were ligated with T4 DNA ligase at 16℃ for 1h and transformed into TOP10 competent cells; 4. Expression and purification: Select clones with correct sequencing, extract plasmids and transfect HEK293 cells. Collect supernatant on Day 6. After purification with Protein A resin, SDS-PAGE detection showed a single band with a concentration of 0.733 mg / ml and a yield of 3665 μg.

[0016] Example 2: Preparation and Screening of AQP3 Neutralizing Antibodies 1. Immunization and titer testing: Rabbits were treated according to the "14-day interval, 5 immunizations" protocol. On day 49, the serum titer of rabbit A was measured to be 1:1024×10³, which met the requirements. 2. B cell screening: After sorting positive B cells, they were cultured in a limiting dilution. ELISA was used to screen for 3G2, 4D7 and 4G6 clones, among which 3G2 had the highest OD value in the supernatant (1.867). 3. Recombinant Expression and Purification: After subcloning the heavy and light chain genes of the 3G2 clone antibody, HEK293 cells were transfected, and 2.5 mg of antibody was purified. SDS-PAGE confirmed the purity to be ≥95%. 4. Potency test: The OD value of the 3G2 antibody was 0.691 when diluted at 1:128×10³, which meets the application requirements.

[0017] Example 3: Functional Validation of AQP3 Neutralizing Antibody Specific binding assays: Western blot results showed that the 3G2 antibody only bound to A549 cell lysate (containing AQP3), and no binding band was observed with HUVEC cell lysate; immunofluorescence results showed that the 3G2 antibody bound to A549 cells (highly expressing AQP3), and the binding intensity increased with increasing concentration. 2. In vitro activity assays: Tumor-associated macrophages (TAMs) in a Transwell co-culture system were treated with the 3G2 antibody. The expression of the surface marker CD206 of M2 macrophages decreased with increasing concentration, demonstrating that it blocks channel function while inhibiting the polarization of M2 TAMs.

[0018] It is important to note that the constructions and arrangements of this application shown in several different exemplary embodiments are merely illustrative. Although only a few embodiments are described in detail in this disclosure, those who consult this disclosure will readily understand that many modifications are possible (e.g., changes in the size, dimensions, structure, shape, and proportions of various elements, as well as parameter values ​​(e.g., temperature, pressure, etc.), mounting arrangements, use of materials, color, orientation, etc.) without substantially departing from the novel teachings and advantages of the subject matter described in this application). For example, an element shown as integrally formed may be composed of multiple parts or elements, the position of elements may be inverted or otherwise altered, and the nature or number or position of discrete elements may be changed or altered. Therefore, all such modifications are intended to be included within the scope of the invention. The order or sequence of any process or method steps may be changed or rearranged according to alternative embodiments. In the claims, any "device plus function" clause is intended to cover the structure described herein that performs the function, and not only structurally equivalent but also equivalent in structure. Other substitutions, modifications, alterations, and omissions may be made in the design, operation, and arrangement of the exemplary embodiments without departing from the scope of the invention. Therefore, the present invention is not limited to the specific embodiments, but extends to various modifications that still fall within the scope of the appended claims.

[0019] Furthermore, in order to provide a concise description of exemplary embodiments, not all features of actual embodiments (i.e., those features that are not relevant to the best mode of carrying out the invention as currently considered, or those features that are not relevant to implementing the invention) may be omitted.

[0020] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. An AQP3 neutralizing antibody, characterized in that: The neutralizing antibody specifically binds to the extracellular domain of aquaporin 3 (AQP3) and blocks the aquaporin and small molecule transport functions of AQP3 after binding. The neutralizing antibody includes antibodies corresponding to three positive clones: 3G2, 4D7, and 4G6. The amino acid sequence of the heavy chain variable region of the 3G2 antibody is QSVEESGGRLVTPGTPLTLTCTLSGFSLSNYAIIWVRQAPGEGLEYIGFINSRGSTYYATWAKGRFTISKTSTTVDLKMTSLTTEDTATYFCARYAADTSTAIWGPGTLVTVSS, and the amino acid sequence of the light chain variable region is QVLTQTPSSVSAAVGGTVTINCQASQSLYNNKNSAWYQQKPGQSPKLLIYKASTLASGVPSRFKGSGSTQFTLTISDLECDDAATYFCQGEFSCSSADCFAFGGGTEVVVKG.

2. The method for preparing AQP3 neutralizing antibody according to claim 1, characterized in that, The process includes the following steps: (1) Preparation of recombinant AQP3 antigen: Construct a recombinant expression vector SIP-hFc-AQP3 containing the extracellular domain of AQP3, and amplify it by PCR (reaction system: F 1μl, R 1μl, template 1μl (20-50ng), 10*pfu buffer 5μl, pfu 1μl (5U), add deionized water to 50μl; reaction conditions: 95℃ pre-denaturation for 5min, 95℃ denaturation for 15s, 55℃ annealing at 2Kb / min, 72℃ extension for 1min, a total of 32 cycles, 72℃ final extension for 10min) to obtain the target fragment, digest it with EcoRI / NotI, ligate it into the vector, transform it into TOP10 competent cells, screen positive clones and verify by sequencing; use HEK293 cell expression system (cell density 1.5-2.0×10 6 The protein was induced by incubation at 37℃ and 130 rpm (samples were collected on Day 6), and the protein was purified by Protein A resin (elution buffer: 0.1 mg / ml, pH 3.0; neutralization buffer: 1 M Tris-Cl, pH 8.5) to obtain recombinant AQP3 protein. The purity was verified by SDS-PAGE and the final product concentration was 0.733 mg / ml, volume was 5 ml, and yield was 3665 μg. (2) Animal immunization 1. Animals Healthy female New Zealand White rabbits, 4 months old and weighing 2.1 kg, were selected.

2. Adjuvants The first major injection used Freund's complete adjuvant, and subsequent booster injections used Freund's incomplete adjuvant, both of which were thoroughly mixed with an equal volume of antigen before injection.

3. Immunity Multiple injections on the back: 1) First immunization: On day 1, the antigen used for immunization is Freund's complete adjuvant + recombinant protein. 2) Second immunization: On day 14, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein. 3) Third immunization: On day 28, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein. 4) Fourth Immunization: On day 42, the antigen used for immunization is Freund's incomplete adjuvant + recombinant protein. 5) Blood collection after the fourth immunization: On day 49, 1 ml of blood was collected from the ear vein and the antiserum titer was detected by ELISA. 6) Fifth Immunization: On day 56, the antigen used for immunization is Freund's incomplete adjuvant + immunogen. 7) Final bloodletting: On day 63, whole blood was collected from the carotid artery, incubated overnight at 4°C, and the serum was then frozen. Peripheral blood and spleen B cells were also collected simultaneously. (3) B cell screening and culture: Positive B cells were sorted by flow cytometry and antigen-specific screening, counted and limitedly diluted to 96-well plates, and cultured in special culture medium for 15 days; the supernatant was collected and detected by indirect ELISA (TMB color development, OD value measured at 450nm). Candidate positive clones with significantly higher OD values ​​than negative controls (3G2, 4D7, 4G6) were screened. (4) Recombinant antibody expression and affinity purification: The heavy and light chain genes of the antibody from the positive clones were extracted and subcloned into the expression vector; the recombinant plasmid (80 μg) was mixed with PEI (0.32 ml, 0.5 mg / ml) and transfected into HEK293 cells (density 1.5-2.0 × 10⁶ cells / year). 6 (cells / ml), cultured at 37℃, 120rpm, 5%CO2 for 6 days; the supernatant was collected by centrifugation, filtered through 0.22μm and loaded onto a Protein A / G resin column, washed with PBS (pH 7.4), eluted with PBS (pH 3.0), neutralized and concentrated to obtain the pure product; The 3G2 antibody concentration was 2.5 mg / ml, the volume was 1.0 ml, and the yield was 2.5 mg; the 4D7 antibody concentration was 1.36 mg / ml, the volume was 1 ml, and the yield was 1.36 mg; and the 4G6 antibody concentration was 1.54 mg / ml, the volume was 1 ml, and the yield was 1.54 mg. (5) Antibody identification: The purity was verified by SDS-PAGE ≥90% and the titer was detected by ELISA ≥1:128×10³.

3. The preparation method according to claim 2, characterized in that: In step (2), the recombinant AQP3 protein for immunization is emulsified with adjuvant at a volume ratio of 1:

1. The dosage for each immunization is based on ensuring that the serum titer reaches the target, and the immunization interval is strictly controlled to be 14 days.

4. The use of the AQP3 neutralizing antibody according to claim 1 or 2 in the preparation of drugs that regulate AQP3-related cell functions.

5. The use of the AQP3 neutralizing antibody according to claim 1 or 2 in the preparation of a medicament for treating diseases related to AQP3 high expression.

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