Design, Development, and Production of Therapeutic Immunoglobulins for Inhibition of Carboxyethylpyrrole-Induced Angiogenesis
Year: 2014
Type: article
Abstract: YALUN CUICarboxyethylpyrrole (CEP) protein derivatives are uniquely generated from oxidative fragmentation of docosahexaenoate (DHA)-containing lipids that are especially abundant in the retina and brain.High levels of CEP protein modifications accumulate on Bruch's membrane and in the photoreceptor cell rod outer segments and retinal pigmented epithelial (RPE) cells of individuals with wet age-related macular degeneration (AMD).Previous investigations by the Salomon group revealed the angiogenicity of CEPs that promote the choroidal neovascularization (CNV), which is the primary cause of severe vision loss in the wet AMD, and also facilitates tumor growth in a mouse melanoma allograft.Compared to vascular endothelial growth factor (VEGF) angiogenesis, the independent biological pathway of CEP-induced angiogenesis suggests that the neutralization of CEP-modified proteins is a potential therapy for inhibiting the CNV of wet AMD and for causing the death of tumor cells by blocking tumor vascularization.In line with the above suggestion, a therapeutic immunoglobulin was developed by humanizing an anti-CEP murine monoclonal antibody (mumAb).Using assembly PCR, six complementarity-determining regions (CDRs) from murine variable regions were grafted into human frameworks homologous to the original murine ones.The xxi recombinant plasmids pFUSE2-huL and pFUSE-huH encoding the humanized light and heavy chains were introduced into a Chinese hamster ovary suspension (CHO-S) cell line for expression of anti-CEP humanized antibody (huAb).The recombinant plasmid pFabCEP was also created for transient expression of anti-CEP humanized Fab (huFab).A stable cell line clone A1 for full length huAb expression was isolated by subcloning.The affinity (KD) of anti-CEP huAb was determined to be 1.58 nM that is ten times lower than that of its mouse counterpart (0.16 nM).The cross-reactivities of huAb and mumAb to other carboxyalkylpyrroles were found to be similar.To improve the affinity of anti-CEP huAb, a phage display library of anti-CEP huFab variants was constructed using the combination of random mutagenesis by error-prone PCR and phage display technology.The clone 2F1, which exhibited the most improved affinity (KD = 0.74 nM), was one of six mutated candidates that were identified.Only two mutations (F29L and D101G) were observed in the clone 2F1, in CDRH1 and CDRH2, respectively.Preclinical studies are now underway to determine the efficacy of clone A1 for inhibiting angiogenesis and the growth of human cancer xenografts on mice.
Author: Yalun Cui
Institution: Case Western Reserve University
Language: English
Subfield: Immunology
Field: Immunology and Microbiology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2344304009