Programmed death receptor ligand 2 monoclonal antibody 8e3 and uses thereof

Abstract

The present application relates to the technical field of biotechnology, in particular to a programmed death receptor ligand 2 monoclonal antibody and application thereof. The present application provides a PD-L2 monoclonal antibody, amino acid sequences of three CDR regions of a heavy chain thereof; amino acid sequences of three CDR regions of a light chain thereof; or amino acid sequences obtained by substitution, deletion or addition of one or more amino acids of the amino acid sequences, or amino acid sequences functionally identical or similar to the amino acid sequences; or amino acid sequences having at least 80% homology with the sequences. The present application widens the possibility of antibody therapy for blocking PD1 / PD1 ligand, and compared with existing PD-L2 monoclonal antibodies, the 8E3 antibody has unique amino acid sequences and CDR region sequences, high affinity and high specificity. The 8E3 antibody can be used in ELISA, Western blot and flow cytometry detection, and has wide application.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to a programmed death ligand 2 monoclonal antibody and application thereof. BACKGROUND

[0002] Programmed death-1 (PD-1) is a member of the CD28 superfamily, which is an important immunosuppressive molecule and mainly expressed on activated T cells and B cells. The ligands of PD-1, PD-Ls, mainly include PD-L1 (B7-H1 / CD274) and PD-L2 (B7-DC / CD273), and PD-L1 is the main ligand. The currently clinically used antibody drugs for blocking PD-1 / PD-L1 are ineffective for PD-L1 negative patients, and it is urgent to develop a monoclonal antibody for programmed death ligand 2 (PD-L2).

[0003] PD-L2 protein is a type I transmembrane protein containing 274 amino acid residues encoded by PDCD1LG2 gene, which has a homology of 40% with PD-L1, but also has some differences. PD-L2 is only expressed on the membrane surface of macrophages, dendritic cells and some B cell subsets in vivo tissues, but recent studies have shown that under the stimulation of a specific microenvironment, PD-L2 can be induced to express in a variety of immune cells and non-immune cells, such as lung cancer, breast cancer, thymoma and other tumor cells. A large number of studies have shown that PD-L2 can play an important role as an important biomarker in the diagnosis, targeted therapy and prognosis of various tumors. The advantages and disadvantages of PD-L2 monoclonal antibody determine the sensitivity and specificity of various immune detection methods (such as immunohistochemistry, enzyme-linked immunoreaction, flow cytometry detection, etc.), and determine the safety and effectiveness of targeted therapy. At present, the antibodies for PD1 / PD-L1 are vigorously promoted on the market for cancer treatment, and there is a lack of monoclonal antibodies for PD-L2 treatment. The specificity and sensitivity of the PD-L2 antibody for detection are still not high. Therefore, it is of great significance to develop an anti-PD-L2 monoclonal antibody with high affinity and high specificity. SUMMARY

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

[0005] One aspect of the present application provides a monoclonal antibody or antigen binding fragment thereof to Programmed Death Ligand 2, having three heavy chain variable region complementarity determining regions (HCDRs) and three light chain variable region complementarity determining regions (LCDRs) sequences 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 application provides an isolated antibody or antigen binding fragment thereof to Programmed Death Ligand 2, wherein the heavy chain variable region and / or light chain variable region sequences thereof are as follows:

[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 embodiments of the present application, the antibody is an IgGl antibody.

[0018] The present application also provides a nucleotide sequence encoding the monoclonal antibody.

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

[0020] CGGGTCCAGCTGCAGCAGTCTGGACCTGTCCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGTTTCTGGCTACGCATTCAGTAGTTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGACGGAGATACTGACTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTACAACATGGGGAGCCGGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO. 9,

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

[0022] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCGGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTTCGGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAACCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA

[0023] SEQ ID NO. 10.

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

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

[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 or semi-solid medium.

[0027] In another aspect, the present invention provides the use of the above-mentioned antibody or its antigen-binding fragment as a detection reagent, said reagent being used for: enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry (FACS), immunohistochemistry (IHC) detection, or immunoPCR.

[0028] In the aforementioned immunological assays, antibodies or their antigen-binding fragments can be used alone or linked by chemical bonds, electrostatic adsorption, or hydrophilic / hydrophobic adsorption. The conjugates include horseradish peroxidase (HRP), alkaline phosphatase (AP), biotin, fluorescein isothiocyanate (FITC), Cy3, Cy5, magnetic beads, and agarose, among other conjugates.

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

[0030] In another aspect, the present invention provides the use of the above-described antibody or its antigen-binding fragment as an antitumor medicament.

[0031] In another aspect, the present invention provides a medicament 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 expands the possibilities of antibody therapies that block PD1 / PD1 ligands. Furthermore, compared to existing PD-L2 monoclonal antibodies, the 8E3 antibody possesses a unique amino acid sequence and CDR region sequence, exhibiting high affinity and high specificity. In particular, the monoclonal antibody of this invention can be used to treat tumors and as a diagnostic reagent. Attached Figure Description

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced as follows.

[0036] Figure 1 Results of using the anti-PD-L2 monoclonal antibody for ELISA detection;

[0037] Figure 2 Anti-PD-L2 8E3 antibody detects the carrier-introduced PD-L2-expressing 293T cells. DETAILED DESCRIPTION

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

[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 target. 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 from at least two intact antibodies, human antibodies, humanized antibodies, and antibody mimetics or specified fragments or portions thereof that mimic the structure and / or function of antibodies, including single-chain antibodies and fragments thereof. Binding of an antibody to a target can elicit a variety of effects, such as, but not limited to, modulating, decreasing, increasing, antagonizing, agonizing, mitigating, slowing, blocking, inhibiting, abrogating, 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, 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 (sdAbs), 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. Antibodies 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] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that can be present. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The modifier "monoclonal" is not to be construed as requiring production of the antibody by any particular method.

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

[0042] As used herein, a monoclonal antibody includes "chimeric" antibodies in which a portion of the heavy and / or light chain is identical with or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, that exhibit the desired biological activity.

[0043] A monoclonal antibody or antigen-binding fragment thereof of Programmed Death Receptor Ligand 2 of the present application, the complementarity determining regions (HCDRs) of three heavy chain variable regions and the complementarity determining regions (LCDRs) of three light chain variable regions 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] Wherein the heavy chain variable region and / or the 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 one specific embodiment, the nucleotide sequence encoding the heavy chain variable region is set forth in SEQ ID NO. 9

[0056] CGGGTCCAGCTGCAGCAGTCTGGACCTGTCCTGGTGAAGCCTGGGGCCTCAGTGAAGATTTCCTGCAAAGTTTCTGGCTACGCATTCAGTAGTTCTTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACGGATTTATCCTGGAGACGGAGATACTGACTACAATGGGAAGTTCAAGGGCAAGGCCACACTGACTGCAGACAAATCCTCCACCACAGCCTACATGCAGCTCAGCAGCCTGACCTCTGTGGACTCTGCGGTCTATTTCTGTACAACATGGGGAGCCGGCTGGGGCCAAGGCACCACTCTCACAGTCTCCTCA SEQ ID NO. 9,

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

[0058] GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTTACCATTGGACAACCGGCCTCCATCTCTTGCAAGTCAAGTCAGAGCCTCTTAGATAGTGATGGAAAGACATATTTGAATTGGTTGTTTCGGAGGCCAGGCCAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAACCTGGACTCTGGAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTATTGCTGGCAAGGTACACATTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAGATAAAA

[0059] SEQ ID NO. 10.

[0060] The application will be further described below in conjunction with examples:

[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. Female BALB / c mice (6 weeks old) were immunized with PD-L2 protein antigen (purchased from sino biological, item number 10292-h08h-1mg). The first immunization used Freund's complete adjuvant to emulsify the antigen, and the second immunization used Freund's incomplete adjuvant to emulsify the antigen, and was injected subcutaneously at 5-6 points, with each mouse injected with ~100 μg of antigen. Ten days after the third immunization, the mice were tail-clipped to collect a small amount of blood for serum titer ELISA detection, and the mouse with high antibody titer (>1:100000) was selected for a fourth booster immunization by intraperitoneal injection of antigen protein, with each mouse injected with 100 μg. Three to five days after the fourth immunization, the mice were sacrificed and their spleen cells were fused with SP2 / 0 cells, and stable hybridoma cells were obtained by HAT medium culture. Hybridoma cells that could secrete PD-L2 antibody were screened by ELISA, and monoclonal hybridoma cell strain 8E3 ( Figure 1 ) that could secrete PD-L2 antibody was obtained by subcloning by the limited dilution method and ELISA screening, and was expanded by stepwise culture and cryopreserved in liquid nitrogen.

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

[0064] ​Harvest monoclonal antibody 8E3 hybridoma cells in logarithmic growth phase, TRIZOL lysis for RNA extraction, obtain cDNA after reverse transcription, amplify and obtain heavy chain and light chain variable regions, remove non-functional VK gene, clone into pMD18-T vector, sequence, use IMGT / V-QUEST database for sequence alignment, and further analyze.

[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 three heavy chain variable region complementarity determining regions (HCDRs) and the three light chain variable region complementarity determining regions (LCDRs) are as follows:

[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 Subtype identification of PD-L2 monoclonal antibody

[0077] The monoclonal antibody isotyping kit (sino biological, SEK003) was used for monoclonal antibody subtype identification, and 8E3 was an IgG1 type antibody.

[0078] Example 4 Anti-PD-L2 monoclonal antibody detection plasmid vector introduction PD-L2 293T cells

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

[0080] 2. Collect 293T cells, blow and count, divide into 5 centrifuge tubes, 3x10 5 cells per tube, 500g centrifuge for 5min, remove supernatant, add 1mL PBS respectively, 500g centrifuge for 5min, remove supernatant.

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

[0082] Table 1

[0083]

[0084] Put on ice, incubate, 30min.

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

[0086] 5. Add 1mL PBS+5%FBS respectively, 500g centrifuge for 5min, remove supernatant.

[0087] 6. Add 300μL PBS+5%FBS to each tube, resuspend, and use Beckman flow cytometer (Beckman CytoFLEX CM) for detection.

[0088] As shown in Figure 2 , the 8E3 antibody can bind to the PD-L2 protein overexpressed on the surface of 293T cells, and the affinity and sensitivity are better than the commercial anti-PD-L2 antibody of BD company, which can be applied to flow cytometry detection.

[0089] The above only describes the preferred embodiments of the present application, and it should be noted that for ordinary skilled persons in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should be considered as the protection scope of the present application.

Claims

1. An antibody against or an antigen-binding fragment of isolated programmed death receptor ligand 2, characterized in that, 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: 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 antibody against or an antigen-binding fragment of isolated programmed death receptor ligand 2, characterized in that, The heavy chain variable region and / or light chain variable region sequences of the monoclonal antibody against programmed death receptor ligand 2 are as follows: 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 receptor ligand 2 according to claim 1 or claim 2, characterized in that, The antibody mentioned is an IgG1 antibody.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the antibody according to any one of claims 1-3.

5. An expression carrier, characterized in that, Includes nucleic acid molecules encoding the antibodies of any one of claims 1-3.

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

7. The host cell according to claim 6, characterized in that, The host cell is either prokaryotic or eukaryotic.

8. The host cell according to claim 6, characterized in that, The host cell is selected from yeast cells, mammalian cells, or other cells suitable for preparing antibodies or their antigen-binding fragments.

9. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1-3 in the preparation of a detection reagent, wherein the detection reagent is used as a reagent for: enzyme-linked immunosorbent assay (ELISA), Western blotting, flow cytometry (FACS), immunohistochemistry (IHC) detection, or immunoPCR.

10. A drug or diagnostic reagent, characterized in that, The drug or diagnostic reagent includes the antibody as described in any one of claims 1-3 and pharmaceutically acceptable excipients.

11. A reagent kit, characterized in that, Includes the antibody as described in any one of claims 1-3 and an acceptable adjuvant.

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