RTN4RL2 monoclonal antibody and use thereof

Abstract

Provided are an RTN4RL2 monoclonal antibody and the use thereof. Specifically, the provided monoclonal antibody can specifically target human RTN4RL2. The amino acid sequences of complementarity determining regions CDR1, CDR2 and CDR3 of a heavy chain variable region of the monoclonal antibody are as shown in SEQ ID NOs: 1, 2 and 3, respectively; the amino acid sequences of complementarity determining regions CDR1 and CDR3 of a light chain variable region thereof are as shown in SEQ ID NOs: 4 and 5, respectively; and the amino acid sequence of a complementarity determining region CDR2 is GAT. The antibody can activate an anti-tumor immune response by blocking the inhibitory effect of RTN4RL2 on T cells.

Description

An RTN4RL2 monoclonal antibody and its application Technical Field

[0001] This invention relates to an RTN4RL2 monoclonal antibody and its application, belonging to the field of biomedical technology. Background Technology

[0002] Immune checkpoints are a collective term for receptors and their corresponding ligands on T cells. The binding of these receptors and ligands activates or inhibits downstream signaling pathways on T cells, thus playing a role in immune activation or suppression. In recent years, many co-inhibitory and co-stimulatory immune checkpoints have been discovered on different cells in the tumor microenvironment (TME). Immune checkpoint inhibitors are a class of immunotherapy drugs used in cancer treatment. Their main mechanism of action is to restore the immune system's ability to kill cancer cells by blocking proteins called immune checkpoints. This approach can effectively treat tumors.

[0003] Global cancer incidence and mortality rates continue to rise, with cancer now the third leading cause of death worldwide. Primary liver cancer is one of the most serious malignant tumors threatening human health; according to WHO statistics, there are 1 to 1.5 million new cases of liver cancer globally each year. In recent years, immunotherapy, represented by PD-1 / PD-L1 and other immune checkpoint monoclonal antibodies, has changed the landscape of liver cancer treatment. Immunotherapy combined with targeted therapy has become the preferred first-line treatment for advanced liver cancer, and has brought unlimited possibilities for neoadjuvant therapy, surgical conversion therapy, and downstaging therapy before liver transplantation. However, in the field of liver cancer treatment, the overall response rate of immune checkpoint monotherapy is only about 15%, and the performance of existing CAR-T and ADC drugs targeting specific targets (such as GPC3, AFP, and HBV-related antigens) in clinical trials has been less than ideal.

[0004] Therefore, effective treatments for solid tumors, especially hepatocellular carcinoma, are currently lacking. RTN4RL2 is an innovative tumor therapeutic target, highly expressed in hepatocellular carcinoma and also significantly highly expressed in many other solid tumors, including breast cancer, thyroid cancer, gastric cancer, and kidney cancer. Furthermore, this molecule promotes tumor growth by inhibiting T cell activation. Developing a high-affinity blocking antibody targeting RTN4RL2, which can activate an anti-tumor immune response by blocking RTN4RL2's inhibitory effect on T cells, would be a highly promising anti-tumor therapeutic drug and treatment method. Summary of the Invention

[0005] The purpose of this invention is to provide an antibody that specifically targets human RTN4RL2 and its application.

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

[0007] A first aspect of the present invention provides an antibody or antigen-binding fragment of RTN4RL2, said antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, wherein:

[0008] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4 and 5, respectively, and the amino acid sequence of the complementarity-determining region CDR2 is GAT.

[0009] In some embodiments of the present invention, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7.

[0010] In some embodiments of the present invention, the antibody or its antigen-binding fragment is selected from monoclonal antibodies, Fab, Fab″, F(ab″)2, Fv, single-chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.

[0011] In a second aspect, the present invention provides a nucleotide that encodes an antibody or antigen-binding fragment thereof of the aforementioned RTN4RL2.

[0012] Based on the amino acid sequence of the antibody or its antigen-binding fragment described above, those skilled in the art can obtain the nucleotide sequence of the nucleic acid molecule encoding the antibody or its antigen-binding fragment. Due to the degeneracy of codons, the nucleotide sequence encoding the nucleic acid molecule of the antibody or its antigen-binding fragment is not unique, and all nucleic acid molecules capable of encoding the antibody or its antigen-binding fragment are within the scope of protection of this invention.

[0013] In some embodiments of the present invention, the nucleotides include nucleotides with sequences as shown in SEQ ID NO: 8-14 and nucleotides with the sequence GGTGCAACA.

[0014] In a third aspect, the present invention provides a biomaterial containing the above-mentioned nucleotides, wherein the biomaterial is an expression cassette, a vector, or a host cell.

[0015] The aforementioned expression cassette can be obtained by linking a transcriptional or translational regulatory element such as a promoter upstream of the nucleotide and / or a transcriptional or translational regulatory element such as a terminator downstream of it.

[0016] The aforementioned vectors include, but are not limited to, plasmid vectors, bacteriophage vectors, viral vectors, and artificial chromosome vectors.

[0017] The host cells mentioned above include microbial cells, insect cells, or other animal cells.

[0018] In a fourth aspect, the present invention provides an antibody conjugate obtained by conjugating an antibody or antigen-binding fragment of the aforementioned RTN4RL2 with a label and / or a cytotoxic drug; wherein the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

[0019] The cytotoxic drugs mentioned above are mainly used to treat tumor diseases, and can inhibit cell growth and replication, and reduce the number of tumor cells.

[0020] Common cytotoxic drugs include alkaloids, metabolites, and antibiotics, such as paclitaxel, gemcitabine, and epirubicin.

[0021] A fifth aspect of the present invention provides an antibody composition comprising an antibody against RTN4RL2, the antibody comprising a heavy chain variable region and a light chain variable region, wherein:

[0022] The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4 and 5, respectively, and the amino acid sequence of the complementarity-determining region CDR2 is GAT.

[0023] The antibody composition may also include other pharmaceutically active ingredients and / or pharmaceutically acceptable carriers and excipients, wherein the pharmaceutically active ingredients are small molecule chemical drugs, biological agents, antibodies, immune checkpoint inhibitors, etc., with antitumor activity.

[0024] In a sixth aspect, the present invention provides uses for the antibody or antigen-binding fragment of the above-described RTN4RL2, nucleotides thereof, the above-described biological materials, the above-described antibody conjugates, and the above-described antibody compositions, wherein the uses are selected from the group consisting of:

[0025] a) Used in the preparation of antitumor drugs;

[0026] b) Reagents or kits used to prepare for the detection (tracing) of tumors.

[0027] Preferably, the tumor is a tumor that highly expresses RTN4RL2, including but not limited to solid tumors such as liver cancer, breast cancer, thyroid cancer, gastric cancer, and kidney cancer. More preferably, the tumor is liver cancer.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides a high-affinity blocking antibody targeting RTN4RL2, which has high affinity and specific binding ability to RTN4RL2-positive tumor cells, thereby relieving T cell suppression in tumors. It can activate anti-tumor immune responses by blocking the inhibitory effect of RTN4RL2 on T cells, and has no obvious toxic side effects on normal tissues and organs, showing broad prospects for anti-tumor applications. Attached Figure Description

[0030] Figure 1: ELISA detection of the specific binding affinity of monoclonal antibody 23B5 targeting RTN4RL2 to the antigen.

[0031] Figure 2: BLI assay for the affinity of monoclonal antibody 23B5 targeting RTN4RL2 with the antigen.

[0032] Figure 3: T cell proliferation assay to detect the blocking effect of monoclonal antibody 23B5 targeting RTN4RL2 on RTN4RL2-mediated T cell inhibition. Detailed Implementation

[0033] To make the present invention more apparent and understandable, preferred embodiments are described in detail below with reference to the accompanying drawings.

[0034] Unless otherwise specified, the experimental methods in the following examples are performed according to conventional methods and conditions or according to the product instructions; the materials and reagents used are all commercially available products unless otherwise specified.

[0035] Example 1: Screening and preparation of monoclonal antibodies that specifically bind to human RTN4RL2

[0036] 1. Mouse Immunization: Six- to eight-week-old female BALB / c mice were selected and immunized with human RTN4RL2 protein (expressed and purified in the laboratory) as the immunogen. After immunization, the serum of the immunized animals was detected by ELISA to determine the level of immune response. If the immunized animals achieved an immune response against the immunogen after routine immunization, cell fusion could be performed.

[0037] 2. Screening: The supernatant of fused cells was screened using the ELISA method, and cells that specifically bind to human RTN4RL2 protein were selected.

[0038] 3. Clonal expansion culture and cryopreservation: Positive maternal clones were transferred to 24-well plates for expansion culture. The supernatant from each expanded culture clone was collected for detection using the indirect ELISA method.

[0039] 4. Subcloning: Positive maternal clones were subcloned using the limiting dilution method, and subclones were screened using ELISA. The supernatant of the subcloned cells was then screened using Biolayer Interferometry (BLI) to select cells that specifically bind to and have high affinity for human RTN4RL2 protein; this clone was designated 23B5.

[0040] 5. Hybridoma Cell Antibody Gene Sequencing: Total RNA was extracted from hybridoma cells and reverse transcribed into cDNA using RT-PCR. The antibody light and heavy chain sequences were cloned and constructed into a T vector. DNA sequencing analysis was then performed to obtain the antibody gene sequence. The sequencing results are shown in Tables 1 and 2 below:

[0041] Table 1 Antibody amino acid sequence

[0042] Table 2 Antibody nucleotide sequences

[0043] 6. Antibody production and purification: The antibody gene sequence obtained in step 5 was transfected into HEK293 cells and cultured in a large scale. The antibody was purified by protein A / G affinity chromatography and the purified antibody was stored in phosphate-buffered saline (PBS) by dialysis.

[0044] In this invention, the preparation of the above-mentioned monoclonal antibody involved multiple rounds of repeated screening of clones and subclones before the ideal monoclonal antibody that specifically binds to human RTN4RL2 was obtained.

[0045] Example 2: Specificity detection of monoclonal antibody 23B5

[0046] The specific binding affinity of the monoclonal antibody 23B5 targeting RTN4RL2 to the antigen was detected by enzyme-linked immunosorbent assay (ELISA). The method and results are as follows:

[0047] 1. Dilute RTN4RL2 protein to 2 μg / mL with PBS and add 100 μL to each well of an ELISA plate. Seal the plate with sealing film and incubate overnight at 4°C.

[0048] 2. Discard the liquid in the wells, blot dry the microplate, wash the plate with PBST washing buffer (300 μL / well for 30 seconds), blot dry the microplate, and perform the next washing. Repeat the washing process 3 times in total.

[0049] 3. Add 100 μL of blocking agent (PBST washing buffer containing 5% BSA) to each well, seal the plate with sealing film, incubate at 37°C, and then wash.

[0050] 4. Add 100 μL of cell culture supernatant from the different clones to each well of an ELISA plate. Seal the plate with sealing film, incubate at 37°C, and then wash.

[0051] 5. Dilute HRP-Anti-Mouse IgG to 0.05 μg / mL with sample dilution buffer, add 100 μL to each well, seal with sealing film, incubate at 37°C, and then wash.

[0052] 6. Add 100 μL of colorimetric solution to each well, seal the plate with sealing film, and incubate at 37°C in the dark.

[0053] 7. Add 50 μL of stop solution to each well and gently shake the microplate until the color development is uniform.

[0054] 8. Use an ELISA reader to read the absorbance values ​​at 450nm and 630nm. Subtract the OD630 value from the OD450 value to obtain the absorbance value (OD value). The detection results are shown in Figure 1.

[0055] 9. Select a clone that strongly binds to the human RTN4RL2 protein. The selected clone was 23B5. The antibody of clone 23B5 strongly binds to the human RTN4RL2 protein. The results showed that the EC50 value of the monoclonal antibody 23B5 targeting human RTN4RL2 was 0.05091 mM (Figure 1).

[0056] Example 3: Affinity detection of monoclonal antibody 23B5 with antigen

[0057] The affinity of the monoclonal antibody 23B5 targeting RTN4RL2 for the antigen was detected by BLI. The method and results are as follows:

[0058] 1. Run the AMC biosensor in the first column of 1×PBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 60 seconds. This step is the baseline step.

[0059] 2. Run the sensor in the cell supernatant of the different clones for a period of time until it solidifies on the sensor for 200 seconds.

[0060] 3. Run the biosensor in 1×PBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 60 seconds. This step is the baseline step.

[0061] 4. Dilute the RTN4RL2 protein to 200 nM with 1×PBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA. Run the sensor for 60 s. During this process, antibodies from different clones on the sensor will be able to bind to the RTN4RL2 protein.

[0062] 5. Run the sensor in 1×PBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 90 s. During this process, the RTN4RL2 protein dissociates from the sensor.

[0063] 6. Regenerate the sensor in 10mM Glycine-HCl solution (pH 1.5) for 5 seconds, then neutralize it in 1×PBS solution (pH 7.4) containing 0.02% Tween-20 and 0.1% BSA for 5 seconds. Repeat this regeneration process three times.

[0064] The test results are shown in Figure 2. The results show that the monoclonal antibody 23B5 targeting human RTN4RL2 has good affinity for the antigen. d The value is 0.001nM.

[0065] Example 4: Detection of the blocking effect of monoclonal antibody 23B5 on RTN4RL2-mediated T cell suppression.

[0066] The blocking effect of the monoclonal antibody 23B5 targeting RTN4RL2 on RTN4RL2-mediated T cell inhibition was detected by T cell proliferation assay. The methods and results are as follows:

[0067] 1. Reagent Preparation: The fluorescent dye CFSE (5,6-carboxyfluorescein diacetate, succinimidyl ester) is used as the label. CFSE needs to be dissolved in DMSO to prepare a 5 mmol / L stock solution and stored at -20℃ protected from light. When using, dilute with serum-free DMEM culture medium to prepare a 5 μmol / L working solution.

[0068] 2. Cell markers:

[0069] 1) Preparation of cell suspension: Prepare a cell suspension from the T cells to be tested.

[0070] 2) Add CFSE working solution: Mix CFSE working solution with an equal volume of cell suspension and incubate at 37°C for 10 minutes.

[0071] 3) Termination labeling: Immediately terminate the labeling 10 minutes with 40% volume of cold fetal serum.

[0072] 4) Washing cells: After washing twice by centrifugation, resuspend the cells in an appropriate amount of complete culture medium.

[0073] 3. Cell culture:

[0074] Labeled T cells were cultured in a 37°C, 5% CO2 incubator for 48 hours using a T cell-specific culture medium (containing the stimulant concanavalin A, or ConA, at a concentration of 1 μg / mL) to allow the cells to proliferate. RTN4RL2 protein (20 μg / mL) and monoclonal antibody 23B5 (20 μg / mL) were added to the corresponding experimental groups.

[0075] 4. Detection and Analysis:

[0076] Flow cytometry: Flow cytometry is used to detect the fluorescence intensity of cells. Because CFSE is evenly distributed among daughter cells during cell division, the fluorescence intensity gradually decreases. By detecting changes in fluorescence intensity, cell division and proliferation can be analyzed, reflecting the activation and proliferative capacity of T cells under stimulation conditions.

[0077] As shown in Figure 3, in the T cell proliferation system, the exogenously added RTN4RL2 protein significantly inhibited T cell proliferation, while the monoclonal antibody had a significant blocking effect on RTN4RL2-mediated T cell inhibition.

[0078] Example 5: Safety and in vitro / in vivo activity assay of monoclonal antibody 23B5

[0079] Cellular experiments:

[0080] The activity and cytotoxicity of the monoclonal antibody were evaluated on a variety of RTN4RL2 positive and negative cell lines.

[0081] Animal experiments:

[0082] The in vivo antitumor activity of monoclonal antibodies 23G12 and 22F7 was evaluated in a BALB / c nude mouse subcutaneous hepatocellular carcinoma xenograft model.

[0083] In vitro and in vivo experiments have shown that the monoclonal antibody of the present invention has good anti-tumor activity and safety.

[0084] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make several improvements and additions without departing from the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof of RTN4RL2, characterized in that, The antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region, wherein: The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4 and 5, respectively, and the amino acid sequence of the complementarity-determining region CDR2 is GAT.

2. The antibody or antigen-binding fragment of RTN4RL2 according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

7.

3. The antibody or antigen-binding fragment of RTN4RL2 according to claim 1, characterized in that, The antibody or its antigen-binding fragment is selected from monoclonal antibodies, Fab, Fab″, F(ab″)2, Fv, single-chain antibodies, animal-derived antibodies, chimeric antibodies, humanized antibodies, bispecific antibodies, or multispecific antibodies.

4. A nucleotide, characterized in that, It encodes the antibody or antigen-binding fragment of RTN4RL2 as described in any one of claims 1 to 3.

5. The nucleotide according to claim 4, characterized in that, The nucleotides include nucleotides with sequences as shown in SEQ ID NO: 8-14 and nucleotides with the sequence GGTGCAACA.

6. A biomaterial, characterized in that, It contains the nucleotides as described in claim 4 or 5, and the biological material is an expression cassette, vector, or host cell.

7. An antibody conjugate, characterized in that, It is obtained by conjugating the antibody or antigen-binding fragment of RTN4RL2 as described in any one of claims 1 to 3 with a label or cytotoxic drug; the label is selected from one or more of enzyme labeling, biotin labeling, fluorescent dye labeling, chemiluminescent dye labeling, and radioactive labeling.

8. An antibody composition, characterized in that, The antibody composition comprises an antibody against RTN4RL2, the antibody including a heavy chain variable region and a light chain variable region, wherein: The amino acid sequences of the complementarity-determining regions CDR1, CDR2, and CDR3 of the heavy chain variable region are shown in SEQ ID NO: 1, 2, and 3, respectively; the amino acid sequences of the complementarity-determining regions CDR1 and CDR3 of the light chain variable region are shown in SEQ ID NO: 4 and 5, respectively, and the amino acid sequence of the complementarity-determining region CDR2 is GAT.

9. Use of the antibody or antigen-binding fragment of RTN4RL2 according to any one of claims 1 to 3, the nucleotide according to claim 4, the biomaterial according to claim 6, the antibody conjugate according to claim 7, or the antibody composition according to claim 8, wherein the use is selected from the group consisting of: a) Used in the preparation of antitumor drugs; b) Reagents or kits used to prepare for the detection of tumors.

10. The application according to claim 9, characterized in that, The tumors mentioned are tumors that highly express RTN4RL2, including solid tumors such as liver cancer, breast cancer, thyroid cancer, gastric cancer, and kidney cancer.

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