Anti-Glypican-3 antibody and application thereof

Abstract

The invention relates to the technical field of biomedicine, in particular to an anti-Glypican-3 antibody and application thereof. The antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises at least one of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the light chain variable region comprises at least one of a light chain CDR1, a light chain CDR2 and a light chain CDR3. The invention also discloses a nucleic acid molecule for coding the antibody. The anti-Glypican-3 antibody provided by the invention can efficiently kill human / monkey liver tumor cells in vitro, and has high specificity.

Description

Technical Field

[0001] This application relates to the technical field of biomedicine, and particularly relates to an anti-Glypican-3 antibody and its application. Background Art

[0002] Primary liver cancer (referred to as liver cancer for short) is one of the common malignant tumors globally. Currently, its incidence and mortality rates show an obvious upward trend worldwide. Hepatocellular carcinoma (HCC) is the most common type of liver cancer. Hepatectomy remains the only potentially radical treatment method at present. However, only 5%-10% of newly diagnosed patients meet the indications for radical resection, and the 5-year recurrence-free survival rate after surgery is only 30%-50%. In the field of systemic treatment, although the multi-kinase inhibitor small molecule drug sorafenib can extend the median overall survival of advanced patients by 2.8 months and the median progression-free survival to 5.5 months, its objective response rate is less than 10%, and pathological complete remission cannot be achieved. There are still significant limitations in the overall survival benefit of existing treatment drugs. In view of this, there is an urgent need to develop new treatment strategies to optimize clinical outcomes in multiple dimensions, aiming to achieve early detection of tumors, effective control of the tumor burden, systematic improvement of the quality of life of patients, and significant extension of the survival period.

[0003] Phosphatidylinositol proteoglycan-3 (Glypican-3, GPC3) is a cell membrane surface proteoglycan that is anchored to the cell surface through a glycosylphosphatidylinositol anchor. Its expression pattern has significant spatio-temporal specificity: it shows high functional expression during embryonic development, while it is generally silent or shows low-abundance expression in normal adult tissues. A large number of clinical studies have confirmed that GPC3 is abnormally highly expressed in hepatocellular carcinoma (HCC), and is positively correlated with tumor malignancy. Based on its tumor tissue-specific expression characteristics and molecular accessibility, GPC3 has been confirmed as a highly selective molecular marker for HCC and a reliable target for the diagnosis and treatment of hepatocellular carcinoma, showing important translational value in the fields of early diagnosis, targeted therapy (such as antibody-drug conjugates, CAR-T cell therapy), and efficacy monitoring. Summary of the Invention

[0004] This application provides an anti-Glypican-3 antibody and its application. The anti-Glypican-3 antibody provided by this application can effectively kill human / monkey liver tumor cells and has high specificity.

[0005] The anti-Glypican-3 antibody provided by the present application comprises an antigen-binding domain consisting of heavy-chain complementarity-determining regions CDR-H1 (SEQ ID NO: 1), CDR-H2 (SEQ ID NO: 2), CDR-H3 (SEQ ID NO: 3) and light-chain complementarity-determining regions CDR-L1 (SEQ ID NO: 4), CDR-L2 (SEQ ID NO: 5), CDR-L3 (SEQ ID NO: 6). The variable region sequence thereof is obtained by combining hybridoma fusion technology with a differential binding activity screening strategy and can specifically recognize the GPC3 protein epitope of human and cynomolgus monkey liver tumor cells.

[0006] In a first aspect, the present application provides an anti-Glypican-3 antibody, adopting the following technical solution:

[0007] An anti-Glypican-3 antibody, the antibody comprising a heavy-chain variable region and a light-chain variable region; the heavy-chain variable region comprising at least one of heavy-chain CDR1, heavy-chain CDR2 and heavy-chain CDR3; the light-chain variable region comprising at least one of light-chain CDR1, light-chain CDR2 and light-chain CDR3;

[0008] The amino acid sequence of the heavy-chain CDR1 is as shown in SEQ ID NO: 1; the amino acid sequence of the heavy-chain CDR2 is as shown in SEQ ID NO: 2; the amino acid sequence of the heavy-chain CDR3 is as shown in SEQ ID NO: 3;

[0009] The amino acid sequence of the light-chain CDR1 is as shown in SEQ ID NO: 4; the amino acid sequence of the light-chain CDR2 is as shown in SEQ ID NO: 5; the amino acid sequence of the light-chain CDR3 is as shown in SEQ ID NO: 6.

[0010] Optionally, the heavy-chain sequence of the antibody comprises the amino acid sequence as shown in SEQ ID NO: 7.

[0011] Optionally, the light-chain sequence of the antibody comprises the amino acid sequence as shown in SEQ ID NO: 8.

[0012] Optionally, the full-length amino acid sequence of the antibody is as shown in SEQ ID NO: 9.

[0013] Optionally, the molecular hook type of the antibody is scFv-hIgG1Fc tag.

[0014] Optionally, the gene sequence of the antibody is the nucleotide sequence as shown in SEQ ID NO: 13.

[0015] In a second aspect, the present application provides a nucleic acid molecule. This nucleic acid molecule can encode the above-mentioned antibody.

[0016] In a third aspect, the present application provides a kit. The kit includes the above-mentioned antibody.

[0017] In a fourth aspect, the present application provides the use of the above-mentioned antibody and the above-mentioned kit in the preparation of a composition for the treatment and diagnosis of liver cancer.

[0018] In summary, the present application has the following beneficial effects:

[0019] The anti-Glypican-3 antibody provided by the present application can efficiently kill human / monkey liver tumor cells and has high specificity. Description of the Drawings

[0020] Figure 1 Results of SDS-PAGE analysis ((a) Native, i.e., control antibody; (b) Reduced, i.e., the antibody of the present application).

[0021] Figure 2 Results of the detection of the affinity of the anti-Glypican-3 antibody of the present application for the full-length human GPC3ECD protein.

[0022] Figure 3 Results of the detection of the affinity of the anti-Glypican-3 antibody of the present application for the GPC3 polypeptide.

[0023] Figure 4 Results of flow cytometry ((a) GPC3-scfv; (b) GC33-scfv; (c) Human IgG).

[0024] Among them, black is Negative cell line (CHO-K1) + Samples + secondary antibody; red is Positive cell line-1 (CHO-K1 human GPC3) + Samples + secondary antibody; yellow is Positive cell line-2 (CHO-K1 Cyno GPC3) + Samples + secondary antibody; blue is Positive cell line-3 (HepG2) + Samples + secondary antibody. Detailed Embodiments

[0025] Before describing the embodiments of the present application in detail, it should be understood that the terms used herein are for the purpose of describing specific embodiments only. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this term belongs.

[0026] It should be noted that the terms "first" and "second" are only used for descriptive purposes and should not be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, features defined with "first" and "second" may explicitly or implicitly include one or more of such features. Further, in the description of the present application, unless otherwise specified, the meaning of "a plurality" is two or more.

[0027] In the scope disclosed in the present application, the endpoints and any values of the scope are not limited to the exact scope or value. These scopes or values should be understood to include values close to these scopes or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values, they can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0028] In the present application, the term "comprising" or "including" is an open expression, that is, it includes the content specified in the present application, but does not exclude other aspects of the content.

[0029] The present application adopts a cross-species immune strategy combined with a high-throughput differential screening platform to obtain high-affinity epitopes that can simultaneously recognize human / monkey GPC3.

[0030] To make the objectives, technical solutions, and advantages of the present application clearer, the technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the accompanying drawings. Based on the embodiments of the present application, all other embodiments obtained by those skilled in the art without creative efforts shall fall within the scope of protection of the present application. The embodiments described below are exemplary and are only used to explain the present application and should not be construed as a limitation of the present application.

[0031] For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified as to the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0032] In the following embodiments, the Human Glypican 3 antigen / GPC3 Protein sequence is the amino acid sequence shown in SEQ ID NO 10, with a content of 2 mg (purity > 45%), and is used for immunization and screening.

[0033] The content of GPC3 peptide (511D→560S) (GPC3 polypeptide) is at least 1 mg (concentration > 85%), and it is used for screening. The amino acid sequence is as shown in SEQ ID NO 11, and a biotinylation label (Biotin) is added at the N-terminus (the immunogenicity of the N-terminus is weaker than that of the C-terminus) for detection.

[0034] The GPC3 target benchmark is Codrituzumab (GC33), with a content of 3 mg. As a positive control antibody, its sequence is the amino acid sequence shown in SEQ ID NO 12.

[0035] The following further elaborates on this application in conjunction with examples and test results.

[0036] Examples

[0037] Example 1

[0038] This example provides a preparation process for an anti-Glypican-3 antibody.

[0039] The above process specifically includes the following steps:

[0040] (I) Animal immunization

[0041] Commercially available Human Glypican 3 antigen is used to immunize SPF-grade C57BL / 6 mice (or Balb / c, or LOU / c rats).

[0042] The immunization process specifically includes the following steps:

[0043] (1) Four days before immunization (T = -4 days), blood is collected from the mice.

[0044] (2) Primary immunization (T = 0 days): s.c. injection (subcutaneous injection), with an injection volume of 50 μg of Human Glypican 3 antigen per animal.

[0045] (3) First boost (T = 14 days): i.p. injection (intraperitoneal injection), with an injection volume of 25 μg of Human Glypican 3 antigen per animal.

[0046] (4) Test serum (T = 21 days): ELISA is used to detect the serum against GPC3 protein.

[0047] (5) Second boost (T = 28 days): s.c. injection, with an injection volume of 25 μg of Human Glypican 3 antigen per animal.

[0048] (6) Test serum (T = 35 days): ELISA is used to detect the serum against GPC3 protein.

[0049] (7) Final immunization (T = 56 ± 7 days): i.p. injection, with an injection volume of 25 μg of Human Glypican 3 antigen per animal.

[0050] (8) Fusion (T = final immunization + 4 days).

[0051] (2) Cell fusion

[0052] Cell fusion was started on the fourth day after the final immunization of C57BL / 6 mice.

[0053] The process of cell fusion specifically includes the following steps:

[0054] (1) Preparation of splenocyte suspension: On the fourth day after the final immunization of C57BL / 6 mice, blood was collected by enucleating the eyeballs of C57BL / 6 mice, and the serum of C57BL / 6 mice was separated as a positive control for antibody detection; at the same time, C57BL / 6 mice were sacrificed by cervical dislocation, and their spleens were taken. The spleens were removed under sterile conditions to prepare a splenocyte suspension;

[0055] Preparation of myeloma cell suspension: Myeloma cells (ATCC, catalog number BNCC100908) were resuscitated two weeks in advance (to ensure that the myeloma cells were in the logarithmic growth phase when used) to prepare a myeloma cell suspension;

[0056] Preparation of feeder cells: One day before cell fusion, peritoneal macrophages and splenocytes of blank C57BL / 6 mice were taken and added to a 96-well plate for culture to obtain a cell plate containing feeder cells (cell concentration: 1×10 4 / well), and feeder cells were prepared.

[0057] (2) Cell fusion process:

[0058] The myeloma cell suspension and the splenocyte suspension were fused at a ratio of 5:1 by electrofusion. The fused cells were resuspended with fusion medium and mixed evenly to prepare a cell suspension. The cells were inoculated into a sterile 96-well plate at 2 - 8 clones / well and cultured in a carbon dioxide cell incubator. After 6 - 7 days of culture, the fusion rate was counted.

[0059] (3) ELISA screening of positive hybridoma cells

[0060] The cell plate containing fused cells - feeder cells was cultured for 4 days for semi-medium change, and then continued to be cultured for 7 days for full-medium change. When the fused cells covered 10 - 50% of the well bottom, positive wells were screened by the conventional indirect ELISA method.

[0061] The indirect ELISA method specifically includes the following steps:

[0062] (1) Coating plate: Using Human Glypican 3 antigen as the coating antigen, it is diluted to 2 μg / mL with 0.05 mol / L CB buffer (31.8 g of Na2CO3, 58.8 g of NaHCO3, and made up to 2 L with ultrapure water) with a pH value of 9.6. Add 100 μL per well to the ELISA plate, coat overnight at 4 °C, then pat dry. Block with 1% gelatin-PBS buffer at a volume of 300 μL per well, block at 37 °C for 2 h, and then pat dry for standby.

[0063] (2) Detection: Add the cell culture supernatant in the cell plate containing fused cells - feeder cells to the ELISA plate at 100 μL per well. Incubate at 37 °C for 60 min, then wash 3 times with 0.01 mol / L PBST buffer containing Tween-20 and pat dry. Add 100 μL per well of HRP-labeled goat anti-mouse secondary antibody (product number TJ-211229CN, Xiamen Taijing), incubate at 37 °C for 60 min, then wash 3 times and pat dry. Add 100 μL per well of TMB chromogenic solution, incubate at 37 °C in the dark for 10 min, and add 50 μL per well of 1 mol / L HCl to terminate the reaction.

[0064] Meanwhile, use the serum of BALB / c female mice obtained by eyeball blood collection and separation in "Cell Fusion" as a positive control to screen out the fused cells with higher antibody titers, which are the positive hybridoma cells.

[0065] (4) Cloning of positive hybridoma cells

[0066] In order to obtain completely homogeneous monoclonal antibodies, it is necessary to clone the positive hybridoma cells.

[0067] One day before cloning, prepare feeder cells and plate them according to the method of step (1) in "Cell Fusion" to obtain a cell plate containing feeder cells; suspend the positive hybridoma cells obtained by screening with HT medium and pipette to mix evenly, inoculate them into the cell plate containing feeder cells, and dilute the cells in the wells of the cell plate to 1 cell per well with HT medium. Place at 37 °C, 5% CO2 humidified culture for 7 - 10 d. When visible cloned cells appear, the antibody can be detected.

[0068] Among them, the following reagents are required to prepare 500 mL of HT medium: 100 mL of fetal bovine serum (product number 11011-8611, Sijiqing), 5 mL of penicillin-streptomycin double antibody for cell culture (100×, product number E607011-0100, Shanghai Sangon), 10 mL of HT medium additive (product number H0137, sigma company), and 385 mL of 1640 medium (product number C3010-0500, VivaCell).

[0069] Observed under an inverted microscope, the wells with only single clone growth and positive ELISA detection were marked, and the cell lines were expanded and established. Finally, a hybridoma cell line was screened and named. The antibody produced by this hybridoma cell is the anti-Glypican-3 antibody provided in this application. It was used for the production of 30 ml of recombinant antibody based on the Expi-CHOS system. The molecular configuration is scFv-hIgG1Fc tag.

[0070] The hybridoma cells in the logarithmic growth phase were resuspended, centrifuged, and then cell cryopreservation solution was added. After programmed cooling, they were stored in liquid nitrogen.

[0071] Example 2

[0072] This example provides the sequencing results of the anti-Glypican-3 antibody screened in Example 1.

[0073] The hybridoma cell line screened in Example 1 was expanded in culture, and 5×10 6 cells / ml were collected in a centrifuge tube. The supernatant was blotted dry, stored in dry ice, and sent to a third-party company for sequencing of the hybridoma cells. The sequencing results are as follows:

[0074] (1) Heavy chain sequence information

[0075] The amino acid sequence of the heavy chain: It includes the amino acid sequence shown in SEQ ID NO: 7.

[0076] Among them, the heavy chain variable region includes heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3. Heavy chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 1. Heavy chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 2. Heavy chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 3.

[0077] (2) Light chain sequence information

[0078] The amino acid sequence of the light chain: It includes the amino acid sequence shown in SEQ ID NO: 8.

[0079] Among them, the light chain variable region includes light chain CDR1, light chain CDR2, and light chain CDR3. Light chain CDR1 includes the amino acid sequence shown in SEQ ID NO: 4. Light chain CDR2 includes the amino acid sequence shown in SEQ ID NO: 5. Light chain CDR3 includes the amino acid sequence shown in SEQ ID NO: 6.

[0080] (4) Full length

[0081] The full-length amino acid sequence of the above anti-Glypican-3 antibody: including the amino acid sequence shown in SEQ ID NO: 9. The gene sequence of the above anti-Glypican-3 antibody is the nucleotide sequence shown in SEQ ID NO: 13.

[0082] Detection test

[0083] (I) SDS-PAGE analysis

[0084] The anti-Glypican-3 antibody (GPC3-scfv) obtained in Example 1 was subjected to SDS-PAGE analysis to verify the molecular weight of the Glypican-3 antibody and detect its purity.

[0085] The detection results are as Figure 1 shown. It can be seen from Figure 1 that the antibody provided in this application is 53.61 Dka.

[0086] (II) Affinity detection

[0087] ELISA was used to detect the affinity of the anti-Glypican-3 antibody for the full-length human GPC3ECD protein (amino acid sequence shown in SEQ ID NO 14) and GPC3 polypeptide (EC50; starting concentration 10 nM, 2-fold dilution, 10 concentration points; positive control antibody (GC33scFv) and Human IgG (brand: Equitech-Bio, catalog number: SLH56) were used as controls).

[0088] The detection results are shown in Table 1 and Figure 2-3 shown.

[0089] Table 1 Affinity of anti-Glypican-3 antibody for full-length human GPC3ECD protein and GPC3 polypeptide

[0090] EC50 GPC3-scfv GC33scFv Human IgG Full-length human GPC3 ECD protein 0.06658 0.1008 ～1.904e+008 GPC3 polypeptide 0.6740 0.1873 17.66

[0091] It can be seen from Table 1 that, in comparison, the anti-Glypican-3 antibody provided in this application has better affinity.

[0092] (III) Flow cytometry detection

[0093] The specific details of the materials are as follows:

[0094] 1) Cell line

[0095] Negative cell line: C2864501P0-1G: CHO-K1(-) (Probio, P12, 97.2%), 1×10 5 cells / reaction, 50 μL;

[0096] Positive cell line 1: C2864501P0-1D: CHO-K1 human GPC3(+)(Probio, P1, 98.7%), 1×10 5 cells per reaction, 50 μL;

[0097] Positive cell line 2: C2864501P0-1F: CHO-K1 Cyno GPC3(+)(Probio, P7, 93.5%), 1×10 5 cells per reaction, 50 μL;

[0098] Positive cell line 3: C2864501P0-1E: HepG2(Probio, P8, 72.8%), 1×10 5 cells per reaction, 50 μL;

[0099] 2) Main antibodies

[0100] Positive control: C2864501P0-1H: Codrituzumab(50 nM);

[0101] Isotype control: human IgG(Equitech-Bio, SLH56, 210112-0356)(50 nM);

[0102] Negative control: PBS;

[0103] Sample: purified antibody(50 nM);

[0104] 3) Secondary antibody

[0105] alexa 647 AffiniPure goat anti-human IgG, Fcγ fragment specific(min X Bov, Hrs, MsSr Prot)(Jackson, 109-605-098, 144613)(1 μg / mL).

[0106] The detection results are shown in Table 2 and Figure 4 as follows.

[0107] Table 2 Flow cytometry detection of the affinity of purified anti-GPC3 for cells

[0108]

[0109]

[0110] As can be seen from Table 2, at the cellular level, the affinity of GPC 3-scFv is stronger than that of GC33-scfv.

[0111] In the description of this specification, the descriptions referring to terms such as "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of this application. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials, or characteristics described can be combined in a suitable manner in any one or more embodiments or examples. In addition, without contradiction, those skilled in the art can combine and combine the different embodiments or examples described in this specification and the features of different embodiments or examples.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, not to limit them; although this application has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements for some of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of this application.

Claims

1. An anti-Glypican-3 antibody, characterized in that: The antibody comprises a heavy chain variable region and a light chain variable region; the heavy chain variable region comprises at least one of a heavy chain CDR1, a heavy chain CDR2 and a heavy chain CDR3; the light chain variable region comprises at least one of a light chain CDR1, a light chain CDR2 and a light chain CDR3; The amino acid sequence of the heavy chain CDR1 is shown in SEQ ID NO: 1; the amino acid sequence of the heavy chain CDR2 is shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain CDR3 is shown in SEQ ID NO: 3; The amino acid sequence of the light chain CDR1 is shown in SEQ ID NO: 4; the amino acid sequence of the light chain CDR2 is shown in SEQ ID NO: 5; and the amino acid sequence of the light chain CDR3 is shown in SEQ ID NO:

6.

2. The anti-Glypican-3 antibody according to claim 1, characterized in that The heavy chain sequence of the antibody includes the amino acid sequence shown in SEQ ID NO:

7.

3. The anti-Glypican-3 antibody according to claim 1, characterized in that The light chain sequence of the antibody includes the amino acid sequence shown in SEQ ID NO:

8.

4. The anti-Glypican-3 antibody according to claim 1, characterized in that The full-length amino acid sequence of the antibody is shown in SEQ ID NO:

9.

5. The anti-Glypican-3 antibody according to claim 1, characterized in that The molecular hook type of the antibody is scFv-hIgG1 Fc tag.

6. The anti-Glypican-3 antibody according to claim 1, characterized in that The gene sequence of the antibody is the nucleotide sequence shown in SEQ ID NO:

13.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody of any one of claims 1-5.

8. A kit, characterized in that: The kit comprises the antibody according to any one of claims 1 to 5.

9. Use of the antibody according to any one of claims 1 to 5 or the kit according to claim 7 in the preparation of a composition for diagnosing and treating hepatocellular carcinoma.

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