Monoclonal antibody 8E3 of programmed death receptor ligand 2 and application of monoclonal antibody 8E3

Abstract

The invention relates to the technical field of biology, in particular to a programmed death receptor ligand 2 monoclonal antibody and application thereof. The invention provides a PD-L2 monoclonal antibody. The PD-L2 monoclonal antibody comprises amino acid sequences of three CDR regions of a heavy chain; amino acid sequences of three CDR regions of a light chain; or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence, or an amino acid sequence with the same or similar function as the shown amino acid sequence; or an amino acid sequence having at least 80% homology with the sequence. According to the present invention, the possibility of the antibody therapy for blocking PD1 / PD1 ligand is broadened, and compared with the existing PD-L2 monoclonal antibody, the 8E3 antibody has characteristics of unique amino acid sequence, unique CDR region sequence, high affinity and high specificity. The kit can be used for ELISA (enzyme-linked immunosorbent assay), Western blot and flow cytometry detection, and is wide in application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to programmed death receptor ligand 2 monoclonal antibodies and applications thereof. Background Art

[0002] Programmed death receptor (PD-1), a member of the CD28 superfamily, is an important immunosuppressive molecule primarily expressed on activated T and B cells. PD-1's ligands, PD-Ls (programmed death-1 ligands), primarily include PD-L1 (B7-H1 / CD274) and PD-L2 (B7-DC / CD273), with PD-L1 being the primary ligand. Currently available clinically used antibody drugs that block PD-1 / PD-L1 are ineffective in PD-L1-negative patients, necessitating the development of monoclonal antibodies targeting programmed death receptor ligand 2 (PD-L2).

[0003] The PD-L2 protein is a type I transmembrane protein consisting of 274 amino acid residues, encoded by the PDCD1LG2 gene. It shares 40% homology with PD-L1, but also exhibits certain differences. In vivo, PD-L2 is exclusively expressed on the membranes of macrophages, dendritic cells, and some B cell subsets. However, recent studies have shown that, under specific microenvironmental stimuli, PD-L2 expression can be induced in a variety of other immune and non-immune cells. For example, it is highly expressed in tumor cells such as lung cancer, breast cancer, and thymoma. Numerous studies have demonstrated that PD-L2 can serve as an important biomarker in the diagnosis, targeted therapy, and prognosis of various tumors. The quality of PD-L2 monoclonal antibodies determines the sensitivity and specificity of various immunoassays (such as immunohistochemistry, enzyme-linked immunosorbent assay, and flow cytometry), and thus the safety and efficacy of targeted therapies. While antibodies targeting PD1 / PD-L1 are being marketed for cancer treatment, there is a lack of therapeutic monoclonal antibodies targeting PD-L2, and the specificity and sensitivity of PD-L2 antibodies used for detection remain low. Therefore, it is of great significance to develop anti-PD-L2 monoclonal antibodies with high affinity and specificity. Summary of the Invention

[0004] To solve the above problems, the present invention provides a programmed death receptor ligand 2 monoclonal antibody or an antigen-binding fragment thereof, which specifically binds to programmed death receptor ligand 2. The present invention also confirms that the PD-L2 antibody provided by the present invention has a good anti-tumor effect.

[0005] In one aspect, the present invention provides a monoclonal antibody or an antigen-binding fragment thereof against programmed death receptor ligand 2, wherein the sequences of the complementarity determining regions (HCDRs) of the three heavy chain variable regions and the complementarity determining regions (LCDRs) of the three light chain variable regions are as follows:

[0006] 8E3-HCDR1: SSWMN SEQ ID No: 1;

[0007] 8E3-HCDR2:RIYPGDGDTDYNGKFKG SEQ ID No: 2;

[0008] 8E3-HCDR3: WGAG SEQ ID No: 3;

[0009] 8E3-LCDR1: KSSQSLLDSDGKTYLN SEQ ID No: 4;

[0010] 8E3-LCDR:2: LVSNLDS SEQ ID No: 5; and

[0011] 8E3-LCDR3: WQGTHYPYT SEQ ID No: 6.

[0012] Another aspect of the present invention provides an isolated programmed death receptor ligand 2 antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region and / or light chain variable region sequence are respectively:

[0013] 8E3 heavy chain variable region:

[0014] RVQLQQSGPVLVKPGASVKISCKVSGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTDYNGKFKGKATLTADKSSTTAYMQLSSLTSVDSAVYFCTTWGAGWGQGTTLTVSS SEQ ID No: 7

[0015] 8E3 light chain variable region:

[0016] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFRRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHYPYTFGGGTKLEIK SEQ ID No: 8.

[0017] In some specific embodiments of the present invention, the antibody is an IgG1 antibody.

[0018] Another aspect of the present invention also provides a nucleotide sequence encoding the monoclonal antibody.

[0019] In some specific embodiments of the present invention, the sequence encoding the heavy chain variable region in the nucleotide sequence is shown in SEQ ID NO.9

[0020] CGGGTCCAGCTGCAGCAGTCTGGACCTGTCCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGTTTCTGGCTACGCATTCAGTAGTTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGACGGAG ATACTGACTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTACAACATGGGGAGCCGGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO.9,

[0021] The sequence encoding the light chain variable region in the nucleotide sequence is shown in SEQ ID NO.10

[0022] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCGGCCTCCATCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTTCGGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTG TCTAACCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA

[0023] SEQ ID NO.10.

[0024] Another aspect of the present invention provides an expression vector comprising a nucleotide encoding the monoclonal antibody.

[0025] Another aspect of the present invention provides a host cell comprising the aforementioned vector or vector group. Preferably, the host cell is prokaryotic or eukaryotic, more preferably selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or antigen-binding fragments thereof.

[0026] Based on the above research, the present invention also provides a conjugate prepared by coupling the monoclonal antibody or the conjugate with a solid medium or a semi-solid medium.

[0027] Another aspect of the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, wherein the reagent is used for the following purposes: enzyme-linked immunosorbent assay (ELISA), immunoblotting (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immuno-PCR.

[0028] In the above-mentioned immunological tests, antibodies or antigen-binding fragments thereof can be coupled alone or with conjugates such as horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads and agarose through chemical bonding, electrostatic adsorption or hydrophilic adsorption.

[0029] In the technical solution of the present invention, the detection reagent can be used for non-diagnostic or therapeutic detection purposes.

[0030] Another aspect of the present invention provides use of the above-mentioned antibody or antigen-binding fragment thereof as an anti-tumor drug.

[0031] In another aspect, the present invention provides a medicine comprising the monoclonal antibody and pharmaceutically acceptable excipients.

[0032] In another aspect, the present invention provides a kit comprising the monoclonal antibody and acceptable adjuvants.

[0033] Beneficial effects

[0034] This invention broadens the potential for antibody therapies that block PD1 / PD1 ligands. Compared to existing PD-L2 monoclonal antibodies, the 8E3 antibody has a unique amino acid sequence and CDR region sequence, resulting in high affinity and specificity. In particular, the monoclonal antibody of the invention can be used to treat tumors and as a detection reagent. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0036] Figure 1 Shows the results of ELISA detection of PD-L2 monoclonal antibody;

[0037] Figure 2 Figure 3 shows the PD-L2 8E3 antibody detection vector introduced into 293T cells expressing PD-L2. DETAILED DESCRIPTION

[0038] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0039] As used herein, the term "antibody" refers to a molecule comprising at least one antigen binding site that immunospecifically binds to a specific target antigen. Thus, the term "antibody" includes, but is not limited to, full-length antibodies and / or variants thereof, fragments thereof, peptibodies and variants thereof, monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed by at least two complete antibodies, human antibodies, humanized antibodies, and antibody mimics or designated fragments or portions thereof, including single-chain antibodies and fragments thereof, that mimic the structure and / or function of an antibody. The binding of an antibody to a target can cause a variety of effects, such as, but not limited to, regulating, reducing, increasing, antagonizing, exciting, alleviating, slowing down, blocking, inhibiting, eliminating, and / or interfering with at least one target activity or binding, or receptor activity or binding, in vitro, in situ, and / or in vivo. Thus, the antibodies of the present disclosure encompass antibody fragments capable of binding to a biomolecule (e.g., an antigen or receptor) or a portion thereof, including but not limited to Fab, Fab' and F(ab')2, pFc', Fd, single domain antibodies (sdAb), variable fragments (Fv), single chain variable fragments (scFv) or disulfide-linked Fv (sdFv); bifunctional antibodies or bivalent bifunctional antibodies; linear antibodies; single chain antibody molecules; multispecific antibodies formed from antibody fragments. The antibody can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass.

[0040] As used herein, the term "monoclonal antibody" refers to an antibody derived from a group of substantially homologous antibodies, i.e., the individual antibodies constituting the group are identical except for the possible presence of a small amount of naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations comprising different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to its specificity, the advantage of monoclonal antibodies is that they can be synthesized without contaminating other antibodies. The modifier "monoclonal" should not be interpreted as requiring the production of antibodies by any particular method.

[0041] As used herein, the term HCDR has the same meaning as the heavy chain complementarity determining region, and LCDR has the same meaning as the light chain complementarity determining region.

[0042] As used herein, monoclonal antibodies include "chimeric" antibodies in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to the corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass, and fragments of these antibodies exhibit the desired biological activity.

[0043] The sequences of the three heavy chain variable regions' complementarity determining regions (HCDRs) and the three light chain variable regions' complementarity determining regions (LCDRs) of a monoclonal antibody or antigen-binding fragment thereof of the present invention against programmed death receptor ligand 2 are as follows:

[0044] 8E3-HCDR1: SSWMN SEQ ID No: 1;

[0045] 8E3-HCDR2:RIYPGDGDTDYNGKFKG SEQ ID No: 2;

[0046] 8E3-HCDR3: WGAG SEQ ID No: 3;

[0047] 8E3-LCDR1: KSSQSLLDSDGKTYLN SEQ ID No: 4;

[0048] 8E3-LCDR:2: LVSNLDS SEQ ID No: 5; and

[0049] 8E3-LCDR3: WQGTHYPYT SEQ ID No: 6.

[0050] Among them, the heavy chain variable region and / or light chain variable region of antibody 8E3 is:

[0051] 8E3 heavy chain variable region:

[0052] RVQLQQSGPVLVKPGASVKISCKVSGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTDYNGKFKGKATLTADKSSTTAYMQLSSLTSVDSAVYFCTTWGAGWGQGTTLTVSS SEQ ID No: 7

[0053] 8E3 light chain variable region:

[0054] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFRRPGQSP KRLIYLVSNLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHYP YTFGGGTKLEIK SEQ ID No: 8;

[0055] In a specific embodiment, the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO.9

[0056] CGGGTCCAGCTGCAGCAGTCTGGACCTGTCCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGTTTCTGGCTACGCATTCAGTAGTTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGACGGAG ATACTGACTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTACAACATGGGGAGCCGGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO.9,

[0057] The sequence encoding the light chain variable region in the nucleotide sequence is shown in SEQ ID NO.10

[0058] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCGGCCTCCATCTCTTGCAAGTCAAGTCAGAGCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTTCGGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTG TCTAACCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA

[0059] SEQ ID NO.10.

[0060] The present invention will be further described below in conjunction with the embodiments:

[0061] Example 1 Production of PD-L2 Monoclonal Antibody

[0062] The hybridoma method (first proposed by Kohler et al., Nature, 256:495 (1975)) was used to immunize female BALB / c mice (6 weeks old) with PD-L2 protein antigen (purchased from Sino Biological, catalog number 10292-h08h-1mg). The antigen was emulsified with Freund's complete adjuvant for the first immunization. Starting from the second immunization, the antigen was emulsified with Freund's incomplete adjuvant and injected subcutaneously at 5-6 sites. The amount of antigen injected per mouse was ~100 μg. Ten days after the third immunization, a small amount of blood was collected by tail clipping for serum titer ELISA testing. Mice with high antibody titers (>1:100,000) were selected for a fourth booster immunization, with 100 μg of antigen protein injected intraperitoneally per mouse. 3-5 days after the fourth immunization, the mice were sacrificed and their spleen cells were fused with SP2 / 0 cells. Stable hybridoma cells were cultured in HAT medium to obtain stable hybridoma cells. Hybridoma cells that can secrete PD-L2 antibodies were screened by ELISA, and subcloned by limiting dilution. Monoclonal hybridoma cell line 8E3 ( Figure 1 ), through step-by-step expansion of culture, and cryopreservation in liquid nitrogen.

[0063] Example 2 Sequencing of the variable regions of the antibody gene of PD-L2 monoclonal antibody hybridoma cells

[0064] Monoclonal antibody 8E3 hybridoma cells in the logarithmic growth phase were harvested and lysed with TRIZOL for RNA extraction. cDNA was obtained after reverse transcription, and the heavy and light chain variable regions were amplified and obtained. The non-functional VK gene was removed and cloned into the pMD18-T vector for sequencing. The sequencing results were compared with the IMGT / V-QUEST database for further analysis.

[0065] 8E3 heavy chain variable region:

[0066] RVQLQQSGPVLVKPGASVKISCKVSGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTDYNGKFKGKATLTADKSSTTAYMQLSSLTSVDSAVYFCTTWGAGWGQGTTLTVSS SEQ ID No: 7

[0067] 8E3 light chain variable region:

[0068] DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFRRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHYPYTFGGGTKLEIK SEQ ID No: 8;

[0069] The sequences of the complementarity determining regions (HCDRs) of the three heavy chain variable regions and the complementarity determining regions (LCDRs) of the three light chain variable regions are shown below:

[0070] 8E3-HCDR1: SSWMN SEQ ID No: 1;

[0071] 8E3-HCDR2:RIYPGDGDTDYNGKFKG SEQ ID No: 2;

[0072] 8E3-HCDR3: WGAG SEQ ID No: 3;

[0073] 8E3-LCDR1: KSSQSLLDSDGKTYLN SEQ ID No: 4;

[0074] 8E3-LCDR:2: LVSNLDS SEQ ID No: 5; and

[0075] 8E3-LCDR3: WQGTHYPYT SEQ ID No: 6.

[0076] Example 3 Identification of subtypes of PD-L2 monoclonal antibodies

[0077] A commercial mouse monoclonal antibody isotyping kit (Sino Biological, SEK003) was used to identify the monoclonal antibody subtype, and 8E3 was an IgG1 antibody.

[0078] Example 4 Anti-PD-L2 Monoclonal Antibody Detection Plasmid Vector Introduced into PD-L2 293T Cells

[0079] 1. Transfect 293T cells with PD-L2 expression vector (Sino Biological HG 10292

[0080] 2. Collect 293T cells, blow off and count, and divide into 5 centrifuge tubes, 3×10 5 cells, centrifuged at 500 g for 5 min, removed the supernatant, added 1 mL PBS, centrifuged at 500 g for 5 min, and removed the supernatant.

[0081] 3. Add reagents according to Table 2:

[0082] Table 1

[0083]

[0084] Place on ice and incubate for 30 min.

[0085] 4. Add 1 mL of PBS + 5% FBS to each tube, centrifuge at 500 g for 5 min, remove the supernatant, add 100 μL of DMEM culture medium + 1 μL of PE goat anti-mouse IgG (minimal x-reactivity) antibody (biolegend, 405307) to each tube, and incubate on ice for 20 min.

[0086] 5. Add 1 mL of PBS + 5% FBS to each tube, centrifuge at 500 g for 5 min, and remove the supernatant.

[0087] 6. Add 300 μL PBS + 5% FBS to each tube, resuspend, and detect using Beckman CytoFLEXCM.

[0088] like Figure 2 As shown, the 8E3 antibody can bind to the PD-L2 protein overexpressed on the surface of 293T cells with better affinity and sensitivity than BD's commercial anti-PD-L2 antibody and can be used for flow cytometry detection.

[0089] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. An isolated antibody against programmed death ligand 2 or an antigen-binding fragment thereof, characterized in that, the sequences of the complementarity-determining regions (HCDR) of its three heavy-chain variable regions and the complementarity-determining regions (LCDR) of its three light-chain variable regions are as follows: 8E3-HCDR1: SSWMN SEQ ID No: 1; 8E3-HCDR2: RIYPGDGDTDYNGKFKG SEQ ID No: 2; 8E3-HCDR3: WGAG SEQ ID No: 3; 8E3-LCDR1: KSSQSLLDSDGKTYLN SEQ ID No: 4; 8E3-LCDR:2: LVSNLDS SEQ ID No: 5; and 8E3-LCDR3: WQGTHYPYT SEQ ID No:

6.

2. An isolated antibody against programmed death ligand 2 or an antigen-binding fragment thereof, characterized in that, the sequence of the heavy-chain variable region and / or the light-chain variable region of the monoclonal antibody against programmed death ligand 2 is: 8E3 heavy-chain variable region: RVQLQQSGPVLVKPGASVKISCKVSGYAFSSSWMNWVKQRPGQGLEWIGRIYPGDGDTDYNGKFKGKATLTADKSSTTAYMQLSSLTSVDSAVYFCTTWGAGWGQGTTLTVSS SEQ ID No: 7 8E3 light-chain variable region: DVVMTQTPLTLSVTIGQPASISCKSSQSLLDSDGKTYLNWLFRRPGQSPKRLIYLVSNLDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYYCWQGTHYPYTFGGGTKLEIK SEQ ID No:

8.

3. The antibody against programmed death ligand 2 according to claim 1 or claim 2, characterized in that, the antibody is an IgG1 antibody.

4. A nucleotide sequence, characterized in that, the nucleotide sequence encodes the antibody according to any one of claims 1-3.

5. An expression vector, characterized in that, it comprises a nucleotide encoding the antibody according to any one of claims 1-3.

6. A host cell, characterized in that, it contains the expression vector according to claim 5, preferably, the host cell is prokaryotic or eukaryotic, and more preferably selected from yeast cells, mammalian cells or other cells suitable for preparing antibodies or their antigen-binding fragments.

7. Use of the antibody or its antigen-binding fragment according to any one of claims 1-3 as a detection reagent for the following uses: enzyme-linked immunosorbent assay (ELISA), immunoblot (Western Blot), flow cytometry (FACS), immunohistochemistry (IHC) detection or immunological PCR.

8. Use of the antibody or its antigen-binding fragment according to any one of claims 1-3 in the preparation of an anti-tumor drug.

9. A drug or detection reagent, characterized in that, the drug or detection reagent comprises the antibody according to any one of claims 1-3 and a pharmaceutically acceptable excipient.

10. A kit, characterized in that, it comprises the antibody according to any one of claims 1-3 and an acceptable adjuvant.

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