Novel antibodies specific for CTHRC1 and use thereof

Abstract

The present invention relates to antibodies binding to CTHRC1 and uses thereof.

Description

Example 2-4: Efficacy Test of Humanized Antibodies In order to test the efficacy of the candidate clones of the 4H5 and 9E6 humanized antibodies, a migration assay was performed using the pancreatic cancer cell line Panc-1. 5 pg / mL of each antibody and 5X104 cells were diluted with a serum-free medium and dispensed into the upper chamber of the Transwell. Then, a medium containing serum was placed in the lower chamber to create an environment in which cells can migrate. The number of migrated cells was measured by culturing the antibodies for 24 hours, and the efficacy of the candidate antibodies was evaluated. As a result, it was confirmed that the degree of migration of cancer cells was 28 2020293734   26 Aug 2026 significantly reduced in the groups treated with cCMAb45 and cCMAb96 compared to the control IgG (Figs. 4 to 7) As a result, the candidate clones of the 4H5 humanized antibody reduced the cell migration ability by 30% to 50% compared to the control IgG, and the candidate clones of the 9E6 humanized antibody reduced the cell migration ability by 30% to 40%. In conclusion, it was confirmed that all of the candidates for the 4H5 and 9E6 humanized antibodies had efficacy similar to or higher than cCMAb45 and cCMAb96 (Fig. 15). Example 2-5: Selection of Humanized Antibodies and Re-evaluation of Affinity and Efficacy Antibodies were selected by evaluating the candidates clones of the 4H5 and 9E6 humanized antibodies based on three criteria: affinity, efficacy, and yield. SA2759, SA2761, SA2762 and SA2765 were selected for the 4H5 humanized antibody, and SA2768, SA2769, and SA2770 were selected for the 9E6 humanized antibody. In order to re-evaluate the affinity and efficacy of the antibodies selected based on the above criteria, indirect ELISA was used for the evaluation of affinity, and migration assay was used for the evaluation of efficacy. The experimental method is the same as described in Examples 2-3 and 2-4. As a result, as for the affinity, it was confirmed that the Kd values were measured to be similar or lower than those of cCMAb45 and cCMAb96, similar to the first measurement, and the cell migration ability was also reduced by 30% to 50%. Specifically, it was confirmed that the selected 4H5 humanized antibody had an affinity of several pM and reduced cell migration ability by 40%, and the 9E6 humanized antibody had an affinity of several tens of pM and reduced cell migration ability by 50% (Figs. 16 and 17). Finally, SA2765 was named hCMAb45, and SA2770 was named hCMAb96. Example 2-6: Analysis of Specificity of hCMAb45 and hCMAb96 Indirect ELISA was performed to determine whether hCMAb45 and hCMAb96 specifically bind to CTHRC1. An immuno-plate was coated with CTHRC1-His and a 2020293734   26 Aug 2026 number of non-specific antigens. On the next day, hCMAb45 and hCMAb96 were reacted with antigens at concentrations of 0 pg / mL, 1 pg / mL, and 5 pg / mL, and then OD values were measured using the anti-human kappa light chain-HRP as a secondary antibody. As a result, it was confirmed that hCMAb45 and hCMAb96 specifically bind to CTHRC1 (Fig. 18). Example 2-7: Confirmation of Inhibitory Effect on Migration of Cancer Cells In order to verify the therapeutic efficacy of hCMAb45 and hCMAb96, the inhibitory effect on the migration of cancer cells was confirmed. Pancreatic cancer, breast cancer, ovarian cancer, bladder cancer, lung cancer, and malignant melanoma were used, and the experimental method is the same as that described in Example 17. As a result, it was confirmed that the migration ability of cancer cells was significantly reduced in the groups treated with hCMAb45 and hCMAb96 compared to the control IgG (Figs. 19 to 22). Example 2-8: Confirmation of Inhibitory Effect on Invasion of Cancer Cells In the same manner as in the experimental method described in Example 1-8, the inhibitory effect on the invasion of cancer cells of hCMAb45 and hCMAb96 was confirmed. As a result, the invasion ability of cancer cells was significantly reduced in the groups treated with hCMAb45 and hCMAb96 compared to the control IgG (Figs. 23 to 26). Example 2-9: Confirmation of in vivo Anticancer Effect in Pancreatic Cancer Animal Model The inhibitory ability of the hCMAb45 and hCMAb96 humanized antibodies that specifically bind to CTHRC1 protein on cancer cell migration and invasion was confirmed in vitro through Examples 2-7 and 2-8. Accordingly, it was confirmed 2020293734   26 Aug 2026 whether the anticancer effect was identically observed in vivo. Specifically, a subcutaneous xenograft mouse model was prepared using the pancreatic cancer cell line BxPC-3 overexpressing CTHRC1. 1x106 cells were transplanted subcutaneously in the right leg of the mice, and after 6 days, mice developed with tumors with a size of 73 mm3 to 77 mm3 were selected and divided into groups. The drug was injected into the tail vein, and the control IgG and the hCMAb45 and hCMAb96 experimental groups were administered at a dose of 10 mg / kg twice a week for a total of 4 weeks, and the size of the tumor was measured twice a week using a caliper. After treatment of the antibodies, and the experiment was terminated on day 28 (treatment 8), and the tumor size was compared relative to the tumor size of the control IgG. As a result, it was confirmed that the size of the tumor in the groups administered with hCMAb45 and hCMAb96 was reduced compared to the group administered with the control IgG (Fig. 27). In particular, it was confirmed that the group treated with hCMAb96 had superior anticancer effect than the group treated with hCMAb45. Through the above Examples, the in vivo anticancer effects of the hCMAb45 and hCMAb96 humanized antibodies that specifically bind to CTHRC1 were confirmed, and their potential as substances delaying or inhibiting tumor growth was verified. From the foregoing, a skilled person in the art to which the present invention pertains will be able to understand that the present invention may be embodied in other specific forms without modifying the technical concepts or essential characteristics of the present invention. In this regard, the exemplary embodiments disclosed herein are only for illustrative purposes and should not be construed as limiting the scope of the present invention. The scope of the present invention is therefore indicated by the appended claims rather than by the foregoing description. All changes which come within the meaning and range of equivalency of the claims are to be embraced within the scope of the present invention.

Claims

1. An antibody that specifically binds to collagen triple helix repeat containing-1 (CTHRC1) protein, which comprises: (a) a heavy chain variable region comprising a heavy chain CDR1 of SEQ ID NO: 11, a heavy chain CDR2 of SEQ ID NO: 12, and a heavy chain CDR3 of SEQ ID NO: 13; and(b) a light chain variable region comprising a light chain CDR1 of SEQ ID NO: 14, a light chain CDR2 of SEQ ID NO: 15, and a light chain CDR3 of SEQ ID NO: 16.

2. The antibody of claim 1, wherein the heavy chain variable region consists of an amino acid sequence of SEQ ID NO: 17 or 25, and the light chain variable region consists of an amino acid sequence of SEQ ID NO: 19, or 27.

3. A polynucleotide encoding the antibody of claim 1 or claim 2.

4. An expression vector comprising the polynucleotide of claim 3.

5. A host cell comprising the expression vector of claim 4.

6. A pharmaceutical composition for preventing or treating cancer, comprising the antibody of claim 1 or claim 2.

7. The composition of claim 6, wherein the cancer is selected from the group consisting of pancreatic cancer, ovarian cancer, breast cancer, melanoma, liver cancer, stomach cancer, lung cancer, colorectal cancer, oral cancer, cervical cancer, and bladder cancer.

8. A method of providing information for diagnosing cancer, comprising detecting CTHRC1 protein in a biological sample isolated from a subject suspected of having cancer through an antigen-antibody reaction, using the antibody of claim 1 or claim 2.

9. A method of providing information for predicting prognosis of cancer, comprising detecting CTHRC1 protein in a biological sample isolated from a subject suffering from cancer through an antigen-antibody reaction, using the antibody of claim 1 or claim 2.2020293734   26 Aug 2026

10. A composition for diagnosing cancer or predicting prognosis of cancer, comprising the antibody of claim 1 or claim 2.

11. A kit, when used in diagnosing cancer or predicting prognosis of cancer, comprising the composition of claim 10.

12. A kit, when used in detecting CTHRC1, comprising the antibody of claim 1 or claim 2.

13. The kit of claim 12, wherein the kit is in the form of ELISA.

14. The kit of claim 13, wherein the antibody is a capture antibody.

15. The kit of claim 13, wherein the ELISA is an indirect ELISA comprising:(i) a polyclonal antibody that binds to CTHRC1;(ii) a capture antibody in the form of the antibody of claim 1 or claim 2; and(iii) an antibody bound to a detection-labeled IgG Fc.

16. A method for detecting CTHRC1 protein in a sample, comprising detecting anCTHRC1 antigen-antibody complex using the antibody of claim 1 or claim 2

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