Generation and characterization of humanized affinity-matured EGFL6 antibodies for ovarian cancer therapy

Objectives. Epidermal growth factor EGF-like domain multiple-6 (EGFL6) is highly expressed in high grade serous ovarian cancer and promotes both endothelial cell proliferation/angiogenesis and cancer cell prolifera-tion/metastasis. As such it has been implicated as a therapeutic target. As a secreted factor, EGFL6 is a candidate for antibody therapy. The objectives of this study were to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. Methods. A selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant human-ized antibodies. These were screened and the lead candidate was affinity matured. Seven humanized affinity -matured EGFL6 antibodies were screened for their ability to block EGFL6 activity on cancer cells in vitro, two of which were selected and tested their therapeutic activity in vivo. Results. Humanized affinity matured antibodies demonstrated high affinity for EGFL6 (150 pM to 2.67 nM). We found that several humanized affinity-matured EGFL6 antibodies specifically bound to recombinant, and native human EGFL6. Two lead antibodies were able to inhibit EGFL6-mediated (i) cancer cell migration, (ii) proliferation, and (iii) increase in ERIC phosphorylation in cancer cells in vitro. Both lead antibodies restricted growth of an EGFL6 expressing ovarian cancer patient derived xenograft. Analysis of treated human tumor xeno-grafts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis. Conclusions. Our studies confirm the ability of these humanized affinity-matured antibodies to neutralize EGFL6 and acting as a therapeutic to restrict cancer growth. This work supports the development of these antibody for first-in-human clinical trials. (c) 2023 Elsevier Inc. All rights reserved.

Automated summary

The study aims to create and validate humanized affinity-matured EGFL6 neutralizing antibodies for clinical development. The results demonstrate that these antibodies have high affinity for EGFL6 and can effectively neutralize it, inhibiting cancer cell proliferation and metastasis, suppressing angiogenesis, and promoting tumor cell apoptosis.