Investigating therapeutic targeting to intercellular adhesion molecule 1 through epitope, isoform, and cross-reactivity selection

Abstract

[eng] AIM AND HYPOTHESIS: Based on the previous literature and indicated gaps of knowledge, the hypothesis for this study was that by generating cell lines expressing only specific forms of ICAM-1, it may be possible to examine the cellular targeting of new and existing ICAM-1 targeted therapies, investigating also parameters such as epitope specificity, animal cross-species reactivity and isoform expression, which have been thus far overlooked in the design of ICAM-1 targeted therapies. To validate this hypothesis, this study focused on the following goals or aims: 1. Development of controlled cellular models expressing full-length ICAM-1 from several species relevant for therapeutic translation. To minimize ICAM-1 expression variability, new recombinant cells models were generated and characterized using an anti-ICAM-1 Ab as a control, either alone or coated on polymer NCs. Results verified the utility of these new models for mechanistic tests on targeting and subcellular transport, providing a valuable tool for in vivo-in vitro as well as human-animal comparisons. 2. Examination of the epitope role by comparing different commercial anti-ICAM-1 antibodies. To discern the role of epitope selectivity on the targeting, sub-cellular transport, and species cross-reactivity of ICAM-1 targeting systems, four different commercial anti-ICAM-1 Abs (alone or coated on NCs) recognizing different ICAM-1 extracellular domains were compared in cells naturally or recombinantly expressing ICAM- 1 from human and animal sources. This study identified different targeting, sub-cellular transport, and species cross-reactivity behaviors, providing valuable information for future therapeutic designs. 3. Characterization of newly generated anti-ICAM-1 antibodies. In parallel, five new anti- ICAM-1 Abs were developed and mechanistic studies regarding their targeting, uptake, and sub-cellular trafficking were performed applying them (alone or coated on NCs) to cell models naturally or recombinantly expressing ICAM-1. This allowed the identification of new Ab candidates with particular transport and multi-species cross-reactivity patterns to help with future pre-clinical studies. 4. Study the role of ICAM-1 variants on the targeting and transport behavior of anti-ICAM- 1 systems. ICAM-1 expression at the mRNA and protein levels was assessed in different cell lines under control or pathological conditions, suggesting the presence of ICAM-1 variants. Two different ICAM-1 isoforms were recombinantly expressed and their behavior

regarding targeting and transport of anti-ICAM-1 Abs (alone or coated on NCs) were tested and compared to a full-length ICAM-1 cellular system. This study revealed interesting isoform-dependent differences, useful for future translation.