表皮生长因子EGF-样域多肽-6（EGFL6）在高级别浆液性卵巢癌中高度表达，并促进内皮细胞增殖/血管生成和癌细胞增殖/转移。因此，它被认为是一个治疗靶点。作为一种分泌因子，EGFL6是抗体治疗的候选人。本研究的目标是创建和验证人源化亲和力成熟的EGFL6中和抗体，以用于临床开发。 选择的小鼠EGFL6抗体通过CDR移植进行了人源化，创建了26个变异的人源化抗体。对它们进行了筛选，选择了领先的候选者并进行了亲和力成熟。筛选了7种人源化亲和力成熟的EGFL6抗体，测试它们在体外阻止EGFL6对癌细胞的活性的能力，其中选择了2种并测试了它们的治疗活性。 人源化亲和力成熟的抗体显示出对EGFL6的高亲和力（150 pM至2.67 nM）。我们发现，几种人源化亲和力成熟的EGFL6抗体特异性地结合到重组和天然人EGFL6。两种领先的抗体能够在体外抑制EGFL6介导的（i）癌细胞迁移，（ii）增殖和（iii）癌细胞ERK磷酸化的增加。两种领先的抗体限制了表达EGFL6的卵巢癌患者源性异种移植物的生长。治疗过的人类肿瘤异种移植物的分析表明，抗EGFL6治疗抑制了血管生成，抑制了肿瘤细胞增殖，并促进了肿瘤细胞凋亡。 我们的研究证实了这些人源化亲和力成熟的抗体中和EGFL6的能力，并作为阻止癌症生长的治疗措施。这项工作支持将这些抗体开发为首个用于人体的临床试验。版权所有©2023 Elsevier Inc.。保留所有权利。

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 proliferation/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.A selected murine EGFL6 antibody was humanized using CDR grafting to create 26 variant humanized 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.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 ERK 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 xenografts indicated that anti-EGFL6 therapy suppressed angiogenesis, inhibited tumor cell proliferation, and promoted tumor cell apoptosis.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.Copyright © 2023 Elsevier Inc. All rights reserved.