Anti-claudin 6 Antibody and Antibody-drug Conjugate

Abstract

The present disclosure discloses an anti-Claudin 6 antibody and an antibody-drug conjugate. The present disclosure relates to an antigen sequence for preparing the Claudin 6 (CLDN6) antibody, a corresponding nucleotide sequence thereof, and a gene engineering vector, engineering bacterium or cell line for expressing the nucleotide sequence. A highly specific CLDN6 antibody may be produced by immunizing an animal with the antigenic polypeptide. Further, a corresponding monoclonal antibody may be obtained by preparing a hybridoma cell with a cell of the immunized animal. The anti-Claudin 6 antibody may be combined with a linking structure and a drug to form an antibody-drug conjugate. The antibody-drug conjugate may deliver the drug to a tumor cell having a positive expression of CLDN6 depending on the targeting effect of the CLDN6, thereby achieving the efficacy of specifically killing the tumor cell, which is of great significance for the treatment of the tumor.

Claims

1. An antigenic polypeptide which has an amino acid sequence as set forth in SEQ ID NO: 1.

2. An encoding gene for the antigenic polypeptide according to claim 1.

3. An expression vector, engineering bacterium, or cell line containing the encoding gene for the antigenic polypeptide according to claim 2.

4. A method for preparing an anti-Claudin 6 antibody, comprising preparing the anti-Claudin 6 antibody with the antigenic polypeptide according to claim 1.

5. A method for preparing an anti-Claudin 6 antibody, comprising preparing the anti-Claudin 6 antibody with the encoding gene according to claim 2.

6. A method for preparing an anti-Claudin 6 antibody, comprising preparing the anti-Claudin 6 antibody with the expression vector, engineering bacterium, or cell line containing the encoding gene according to claim 3.

7. A mouse hybridoma monoclonal cell C6-e11 which is prepared through the antigenic polypeptide according to claim 1, and is deposited on Nov. 12, 2019 with a deposit number of CCTCC NO. C2019282.

8. An anti-Claudin 6 monoclonal antibody which is produced by the mouse hybridoma monoclonal cell C6-e11 according to claim 7.

9. An antibody-drug conjugate comprising the anti-Claudin 6 monoclonal antibody according to claim 8, a linking structure, and a drug, wherein the linking structure is connected to the anti-Claudin 6 monoclonal antibody and the drug respectively.

10. The antibody-drug conjugate according to claim 9, wherein the linking structure is SMCC, and the drug is maytansine.

11. A method of preparing a medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6, comprising preparing the medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6 with the antibody according to claim 8.

12. A method of preparing a medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6, comprising preparing the medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6 with the antibody-drug conjugate according to claim 9.

13. A method of preparing a medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6, comprising preparing the medicant or pharmaceutical composition for treating a tumor having a positive expression of Claudin 6 with the antibody-drug conjugate according to claim 10.

14. The method according to claim 11, wherein the tumor having a positive expression of Claudin 6 is a liver tumor.

15. The method according to claim 12, wherein the tumor having a positive expression of Claudin 6 is a liver tumor.

16. The method according to claim 13, wherein the tumor having a positive expression of Claudin 6 is a liver tumor.

17. A pharmaceutical composition, comprising the antibody-drug conjugate according to claim 9.

18. The pharmaceutical composition according to claim 17, further comprising sorafenib.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0033] FIG. 1 is a UV absorption spectrum for DM1, Ab, and Ab-DM1.

[0034] FIG. 2 is an IR absorption spectrum for DM1, Ab, and Ab-DM1.

[0035] FIG. 3 shows the antigen epitope binding effects of the antibody-drug conjugate in different liver cancer cell lines.

[0036] FIG. 4 shows the endocytosis result of the monoclonal antibody and the antibody-drug conjugate prepared therewith in the liver cancer cell lines.

[0037] FIG. 5 shows the killing effect of the antibody-drug conjugate on the liver cancer cell line HepG2.

[0038] FIG. 6 shows the killing effect of the antibody-drug conjugate on the liver cancer cell line PLC-8024.

[0039] FIG. 7 shows the body weight comparison between the group of the isotype control-drug conjugate and the group of the antibody-drug conjugate in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0040] FIG. 8 shows the body weight comparison between the group of the combination of the isotype control-drug conjugate and sorafenib and the group of the combination of the antibody-drug conjugate and sorafenib in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0041] FIG. 9 shows the tumor growth comparison between the group of the isotype control-drug conjugate and the group of the antibody-drug conjugate in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0042] FIG. 10 shows the tumor growth comparison between the group of the combination of the isotype control-drug conjugate and sorafenib and the group of the combination of the antibody-drug conjugate and sorafenib in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0043] FIG. 11 shows the tumor size comparison between the group of the isotype control-drug conjugate and the group of the antibody-drug conjugate in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0044] FIG. 12 shows the tumor size comparison between the group of the combination of the isotype control-drug conjugate and sorafenib and the group of the combination of the antibody-drug conjugate and sorafenib in the in vivo experiment of subcutaneous tumorigenesis in nude mice.

[0045] FIG. 13 shows the results of the drug sensitive test for primary organoids derived from five patients with liver cancer.

[0046] FIG. 14 shows the correlation between the increasing factor of the CLDN6 expression in the cancer tissues of five patients with liver cancer and the sensitivity thereof to the ADC drug (IC50).

DETAILED DESCRIPTION

[0047] For the purpose of better describing the object, the technical solutions, and the advantages of the present disclosure, the present disclosure will be further described below with reference to the drawings and particular examples.

Example 1 The Antigen Sequence and its Preparation

[0048] A fragment of polypeptide sequence having the highest score and the smallest crossover with other tight junction structure related proteins was screened through bioinformatic analysis and comparison:

[0049] LPMWKVTAFIGN (SEQ ID NO: 1).

[0050] The polypeptide sequence corresponds to amino acids 27 to 38 in the protein sequence of Claudin 6.

[0051] For the convenience of connecting to a corresponding vector KIH, a linking structure C (cysteine) was added to the end of the above sequence to synthesize the following polypeptide fragment with a purity up to 90%:

[0052] LPMWKVTAFIGNC (SEQ ID NO: 2)

Example 2 Preparation of the Antibody

[0053] The synthesized polypeptide sequence (SEQ ID NO: 2) was coupled with a vector protein (such as KLH), and 3 BALB/c mice were boosted through a standard procedure. The mouse having the best immune response was selected. Spleen cells and myeloma cells were taken from the mouse for cell fusion to prepare hybridoma cells.

[0054] The hybridoma cells obtained were subjected to proliferation and ELISA screening to obtain clones with a high titer. Then, 2-8 positive parent cells were subcloned by a limiting dilution method. Finally, a strain of positive clones were determined for subtype identification. The positive clone cells were deposited in China Center for Type Culture Collection (address: Wuhan University, Luojia Hill, Bayi Road, Wuchang District, Wuhan, Hubei, China) on Nov. 12, 2019 with a deposit number of CCTCC NO.C2019282.

[0055] The positive clone cells were amplified and cultured for ascites production in 5 mice respectively, and subjected to protein G purification o obtain corresponding monoclonal antibodies.

Example 3 The Preparation and Identification of the Antibody-Drug Conjugate

[0056] 1. Main materials [0057] Antibody: the monoclonal antibody obtained in Example 2 (hereinafter abbreviated as Ab); [0058] Linking structure: SMCC, purchased from AAT Bioquest Company (USA); [0059] Drug: maytansine (hereinafter abbreviated as DM1). [0060] 2. Preparation method [0061] 1) SMCC was dissolved in DMSO, and stored at 4° C. [0062] 2) Ab and SMCC were mixed in a conjugated buffer (50 mm potassium phosphate, 50 mm sodium chloride, and 2 mm EDTA, at pH 7.2) in a molar ratio of 1:10, and stirred at 4° C. for 4-6 h. [0063] 3) The mixed solution was placed in a dialysis bag (M =5000) after reaction, and dialyzed at 4° C. for three days. [0064] 4) The dialyzate was collected, and freeze dried in a vacuum freeze dryer. [0065] 5) The protein sample was quantitatively analyzed with BCA. [0066] 6) The modified antibody and the DM1 molecule were mixed in a PBS buffer (pH=7.0) in a molar ratio of 1:10, and stirred at 4° C. for 4-6 h. [0067] 7) The mixed solution was added to a dialysis bag (M=5000), and dialyzed at 4° C. for three days. [0068] 8) The dialyzate was collected, and freeze dried in a vacuum freeze dryer. [0069] 9) The absorbance of the Ab-DM1 conjugate was determined at 252 nm, and the antibody was quantified with BCA. [0070] 3. Complex determination [0071] 1) UV-vis absorption spectrum

[0072] The UV absorption spectra of DM1, Ab, and Ab-DM1 were determined respectively. The determination results were shown in FIG. 1.

[0073] As can be seen from FIG. 1, DM1 has the highest absorption peak at 252 nm, Ab has the highest absorption peak at 280 nm, and Ab-DM1 has the highest absorption peak at 268 nm. [0074] 2) Analysis of drug-to-antibody ratio

[0075] A DM1 concentration-absorbance value standard curve was created. The mass of DM1 in Ab-DM1 was calculated from the standard curve. The mass of the antibody was obtained through BCA quantification. The mole numbers of the molecules were calculated according to the molar law, n=m/M. The experiment was repeated three times, and the average value was calculated.

[0076] It was calculated from the antibody-to-drug molar ratio that about 3.6 DM1s were covalently bound to each antibody. [0077] 3) IR absorption spectrum

[0078] The IR absorption spectra for SMCC, Ab, Ab-SMCC, DM1, and Ab-DM1 were determined respectively, and the results were shown in FIG. 2.

[0079] The results in FIG. 2 show that SMCC has specific —C═O bonds at 1660 nm and 1676 nm, and a —C═C— bond at 1485 nm; Ab has a —CNH.sub.2— bond at 1006 nm; Ab-SMCC has a —C═O bond at 1632 nm, and a specific —NH.sub.2 bond at 3375 nm; and DM1 has a —NH— bond at 3113 nm. The specific chemical bonds of various molecular groups can be seen from the absorption spectrum for Ab-DM1, indicating that the construction is successful. [0080] 4) Particle size and potentiometric analysis

[0081] The particle sizes and Zeta potentials of Ab, Ab-SMCC, and Ab-DM1 were determined respectively, and the results were shown in FIG. 1. As can be seen from Table 1, the particle size of Ab-SMCC significantly changed, which was probably because the strong polarity of SMCC allowed it to be polymerized with the antibody, and the Zeta potential of Ab-SMCC also changed, indicating that the antibody was successfully modified.

TABLE-US-00001 TABLE 1 Particle size and Zeta potential Ab Ab-SMCC Ab-DM1 Particle size (±SD nm)  33.12 ± 7.61  338.4 ± 9.33 29.89 ± 7.55 Zeta potential (±SD mV) −14.51 ± 1.32 −18.60 ± 1.46 −5.48 ± 1.42

Example 4 Cell Experiment

[0082] (I) Cell membrane staining experiment by flow cytometry

[0083] The binding effects of the antibody-drug conjugate prepared in Example 3 with the HepG2 cells and the PLC-8024 cells respectively were determined through a cell membrane staining experiment by flow cytometry, which comprised the following steps. [0084] 1. The cells were resuspended in 0.5-1 mL 1×PBS, distributed into flow tubes, and centrifuged. [0085] 2. The cells were resuspended in 1×PBS, and centrifuged, and the supernatant was discarded. This step was repeated twice. [0086] 3. The cells were resuspended in 100 μL diluted antibody or antibody-drug conjugate. [0087] 4. The suspension was incubated on ice for 15-30 minutes, and then centrifuged, and the supernatant was discarded. [0088] 5. The cells were resuspended in 1×PBS, and centrifuged, and the supernatant was discarded. This step was repeated twice. [0089] 6. The cells were resuspended in 100 μL diluted secondary antibody labeled with a fluorescent dye. [0090] 7. The suspension was incubated on ice for 30 minutes, and then centrifuged, and the supernatant was discarded. [0091] 8. The cells were resuspended in 1×PBS, and centrifuged, and the supernatant was discarded. This step was repeated. [0092] 9. The cells were resuspended in 1×PBS, and analyzed by a flow cytometer.

[0093] The results of the flow cytometry were shown in FIG. 3. The antibody-drug conjugate prepared in the present disclosure had significantly different antigen epitope binding effects in different liver cancer cell lines. The antigen binding capacity was strong on the membrane surface of the HepG2 cell line having a positive expression of Claudin 6, and was weak on the membrane surface of the PLC-8024 cell line having a negative expression of Claudin 6.

[0094] (II) The endocytosis of the antibody-drug conjugate in the liver cancer cell lines

[0095] The endocytosis of the antibody-drug conjugate prepared in Example 3 in the HepG2 cells was further determined. The cells were divided into two groups: the CLDN6-Ab group and the CLDN6-DM1 group, wherein the monoclonal antibody prepared in Example 2 was used in the CLDN6-Ab group, and the antibody-drug conjugate prepared in Example 3 was used in the CLDN6-DM1 group. The experiment procedure was as follows. [0096] 1. The HepG2 cells were cultured to a cell density of about 3×10.sup.4/well. [0097] 2. The monoclonal antibody or antibody-drug conjugate was added to the cell culture medium at a final concentration of 1 μM. [0098] 3. The cells were cultured in an incubator at 37° C. for 1-8 hours. [0099] 4. The incubation was ended with the culture medium absorbed, and the cells were washed with PBS twice. [0100] 5. A methanol solution was added to fix the cells at room temperature for 20 min. [0101] 6. The methanol solution was removed, and the cells were washed with PBS three times at room temperature, 5 min for each time. [0102] 7. The secondary antibody was added dropwise, and the cells were incubated at room temperature away from light for 1 h. [0103] 8. The cells were washed with PBS 3 times, 5 min for each time. [0104] 9. The cells were observed and photographed with a confocal fluorescent microscope.

[0105] The results were shown in FIG. 4. Both the monoclonal antibody (CLDN6-Ab) and its corresponding antibody-drug conjugate (CLDN6-DM1) could be bound to the antigen on the membrane surface of the liver cancer cell lines to be endocytosed into the cells, and then specifically kill the cells.

[0106] (III) The efficacy of the antibody-drug conjugate:

[0107] The liver cancer cell line HepG2 having a positive expression of Claudin 6 and the liver cancer cell line PLC-8024 having a negative expression of Claudin 6 were evaluated for efficacy respectively. There were 3 groups, the group of single drug in which DM1 was directly used, the group of the isotype control-drug conjugate in which IgG-DM1 was used, and the group of the antibody-drug conjugate in which the antibody-drug conjugate prepared in Example 3, CLDN6-DM1, was used.

[0108] The experiment procedure was as follows.

[0109] The cells were seeded into a 96-well plate with 1000 cells in each well. After 24 hours of cell culture, different concentrations (0-172 μM) of drugs (DM1, CLDN6-DM1, and IgG-DM1) were added into each well respectively. After 72 hours of drug treatment, the inhibition rate of the cell growth was determined.

[0110] The results show that the antibody-drug conjugate has more sensitive killing effect than single drug in the liver cancer cell line HepG2 having a positive expression of Claudin 6 (as shown in FIG. 5), whereas the antibody-drug conjugate substantially has no effect in the liver cancer cell line PLC-8024 having a negative expression of Claudin 6 (as shown in FIG. 6). Thus, it can be indicated that this antibody-drug conjugate has specific killing effect in vitro.

Example 5 Animal Experiment

[0111] The liver cancer cell line HepG2 having a positive expression of Claudin 6 was selected for subcutaneously injecting 10.sup.6 cells on the dorsal side of a nude mouse. The tumor mass was removed after it was observed that the tumor volume reached 1000 mm.sup.3. The tumor mass was divided into small tumor masses with the same volume (around 1-5 mm.sup.3), which were subcutaneously inoculated on the dorsal sides of a plurality of nude mice. The administration was performed after it was observed that the tumor volume reached 50-150 mm.sup.3.

[0112] The tumor-bearing mice were randomly divided into four groups (with 5 mice in each group): the group of the isotype control-drug conjugate (in which IgG-DM1 was used), the group of the complex of the Claudin 6 antibody-drug conjugate (in which CLDN6-DM1 was used), the group of the combination of the isotype control-drug conjugate and sorafenib (in which sorafenib and IgG-DM1 were used), and the group of the combination of the antibody-drug conjugate and sorafenib (in which sorafenib and CLDN6-DM1 were used).

[0113] Here, sorafenib was administrated by intraperitoneal injection at a concentration of 20 mg/kg. The isotype control-drug conjugate and the antibody-drug conjugate were administrated by tail intravenous injection at a concentration of 4 mg/kg. In the administration experiments of the latter two groups (the group of the combination of the isotype control-drug conjugate and sorafenib, and the group of the combination of the antibody-drug conjugate and sorafenib), single drug of sorafenib was firstly administrated, and the tumor was observed and measured; on the 18th day of tumor growth, the isotype control-drug conjugate or the antibody-drug conjugate was further added for the combination; after administration, the subcutaneous tumor growth and the body weight change of the mice were observed, and measured twice per week. The results of the body weight measurements were shown in FIGS. 7 and 8, and the tumor growth curves were shown in FIGS. 9 and 10. The result of body weight change showed that there was no obvious difference in body weight between the groups, indicating that the medicant of each group had no obvious toxicity in vivo.

[0114] After the experiment, subcutaneous tumors of the nude mice in each group were removed, and the results were shown in FIGS. 11 and 12.

[0115] As shown in FIGS. 9-12, each group inhibited the growth of the tumor, but the inhibition effects were as follows: the group of the combination of the antibody-drug conjugate and sorafenib>the group of the Claudin 6 antibody-drug conjugate>the group of the combination of the isotype control-drug conjugate and sorafenib>the group of the isotype control-drug conjugate.

Example 6 In Vitro Efficacy Evaluation of Primary Drug

[0116] The cancer tissues were collected from five patients with liver cancer for organoid culture. After about 2 weeks of culture, the cancer tissues were subcultured and seeded in a 96-well culture plate. 24 hours after seeding, different concentrations of medicants (the isotype control-drug conjugate or the antibody-drug conjugate) were added respectively. The cell activities were determined respectively after 72 hours, and efficacy curves were drawn. The results were shown in FIG. 13.

[0117] The difference in Claudin 6 expression between the cancer tissues and corresponding normal liver tissues of five patients with liver cancer were determined and analyzed respectively. The result was shown in FIG. 14. It can be found from FIG. 14 that the increasing factor of the CLDN6 expression in the cancer tissues relative to corresponding normal liver tissues is negatively correlated to the drug IC50.

[0118] Finally, it should be noted that the above examples are only intended to describe the technical solutions of the present disclosure, but not to limit the protection scope of the present disclosure. Although the present disclosure is described in detail with reference to preferred examples, it should be appreciated by those skilled in the art that modification or equivalent substitution can be made on the technical solutions of the present disclosure without departing from the spirit and scope of the technical solutions of the present disclosure.