Emmanuel Adebowale Fajemisin

Conference 2024 Live Talk

Talk Title

SNAP-tag antibody conjugates targeting CD64 overexpressing cells in chronic diseases

Authors and Affiliations

Emmanuel Adebowale Fajemisin 1, Olusiji Alex Akinrinmade 1, 2, Stefan Barth 1, 3.
1. Medical Biotechnology and Immunotherapy Research Unit, Institute of Infectious Disease and Molecular Medicine, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa
2. Department of Molecular Pharmacology, Albert Einstein College of Medicine, Bronx, New York 10461, United States
3. South African Research Chair in Cancer Biotechnology, Faculty of Health Sciences, Department of Integrative Biomedical Sciences , University of Cape Town, Cape Town 7700, South Africa

Abstract

Background

Dysfunctional macrophages are associated with the initiation and maintenance of many chronic inflammatory diseases and cancers. Unfortunately, there is an urgent need for the generation of a curative therapeutic that selectively eliminates disease-causing macrophages without harming healthy cells. Interestingly, the Fc gamma receptor I (FcγRI) or CD64 is a surface receptor expressed on normal and dysfunctional macrophages. Hence, protein engineering leveraging SNAP-tag technology was used to develop anti-CD64 antibody conjugates that could selectively target and deliver cytotoxic agents into disease-causing macrophages. This allows the chemical coupling of a CD64-specific antibody fragment (H22(scFv)) to the cytostatic drug monomethyl auristatin F (ADC) or the light-sensitive dye IR700 (PIC). Both ADC and PIC explored in this study offer superior benefits over conventional drugs or protein therapeutics like immunotoxins used in previous studies.

Methods

Anti-CD64 SNAP-tag proteins were expressed and structurally validated. Afterwards, they are conjugated to auristatin F and IR700 to generate immunoconjugates, H22(scFv)-SNAP-AURIF and H22(scFv)-SNAP-IR700. Next, functional binding and cytotoxicity studies were performed on CD64+ expressing cells (U937) and polarized ex vivo macrophages prepared from human blood monocytes (hMDMs). 500 nM maximal concentrations of ADC and PIC was used for treatment of 5×105 cells seeded in 96-well plates, and dose-dependent cytotoxicity (with IC50 values and p-values) was determined using statistical analysis

Results

Full-length H22 (scFv)-SNAP fusion protein was successfully expressed and structurally validated (at 53.2 kDa) with functional binding (85%) to U937. With ethical approval, hMDMs were successfully prepared, polarized and characterized ex vivo. H22(scFv)-SNAP-AURIF (ADC) showed a dose-dependent killing of U937 cells at nanomolar (nM) concentrations. ADC offers superior benefits over immunotoxins because its drug moiety (AURIF) is unaffected by endosomal proteases. Similarly, H22(scFv)-SNAP-IR700 compromised cell viability in all polarized ex vivo macrophage subtypes. Hence, this potentially allows for selective elimination of the dominant disease-causing macrophages. Because IR700 is a theranostic agent, it allows for dual clinical benefits of diagnosis and therapy in a minimally invasive manner in most chronic inflammatory disease and solid cancers

Conclusions

Anti-CD64 SNAP-tag antibody conjugates demonstrate preliminary therapeutic potential and could serve as targeted therapy for CD64 overexpressing cells in chronic diseases.