Characterization of scFv against S100A7 and Calprotectin

Abstract

This work aimed to characterize single chain fragment variables (scFvs) against S100A7 and Calprotectin, proteins part of the Ca2+ binding S100 family that have been associated with inflammatory diseases and cancer. The mechanisms governing the physiological functions of S100 proteins are not fully understood, but it is known that many of them contribute to maintaining homeostasis in the intracellular space, while functioning as alarmins which promote inflammatory responses (mainly via interactions with pattern recognition receptors TLR-4 and RAGE) once excreted. Reliable probes targeting these proteins could facilitate further research, and could potentially be used therapeutically in the clinic. A scFv constitutes the antigen-binding site of an antibody, a naturally derived probe which can be evolved to bind almost any molecule with high specificity. The scFvs studied here were developed by phage display technology and were provided by SciLifeLab Drug Discovery and Development, Human Therapeutic Antibody Facility as part of the EUbOPEN consortium, an IMI financed project run in an open science context. As part of the process to identify interesting scFv candidates for further development, these scFvs were investigated not only with respect to specificity but also with respect to several other characteristics known to affect their developability. These included expression levels, homogeneity, reaction kinetics and cross-reactivity. In line with previous research about phage display derived scFvs, it was found that many of the scFvs studied in this work were aggregated to some extent and that some of them were poly-specific. Others showed good binding strength in enzyme linked immunosorbent assays (ELISAs) and were highly specific even in the presence of other S100 family members which have high structural similarity. However, many scFvs that performed well in ELISAs were not able to bind their target antigen in other analytical assays. This sensitivity towards the assay format might call for a reconsideration of the screening process used to obtain the studied scFvs, alternatively affinity maturation with a selected subset of the scFvs could be performed to improve affinity and stability of the scFvs. Nevertheless, the results presented in this study should serve as guidance for the continued efforts to identify future research and therapeutic probes against Calprotectin and S100A7.

Popular Abstract

Characterization of molecules which specifically recognize S100A7 and Calprotectin

S100A7 and Calprotecin are proteins part of the calcium binding S100 family of proteins which have been shown to stimulate inflammatory responses and have been associated with several diseases including cancer. Molecules which can specifically recognize S100A7 and Calprotectin could potentially be used therapeutically in the clinic or facilitate basic research which is needed to expand our knowledge of how these proteins work. In this study, molecules specific for S100A7 and Calprotectin have been investigated with respect to several characteristics known to affect the molecules’ functions in different situations. By determining these characteristics, patient safety can be predicted and the cost of production processes can be reduced.
The molecules studied in this work were developed by a technology called phage display, and were provided by SciLifeLab Drug Discovery and Development, Human Therapeutic Antibody Facility. In phage display, a big population of molecules is screened to identify molecules that can recognize a specific target. This screening process can be manipulated to identify molecules with desired properties. It was found in this work that many of the molecules provided by SciLifeLab had a tendency to self-associate (form aggregates or clusters) and that some of them were poly-specific (recognized several proteins including Calprotectin/S100A7). Others could bind S100A7 and Calprotectin strongly, and were highly specific even in the presence of other (very similar) S100 family members. High specificity is important for both therapeutic and research applications, to decrease potential medicational side-effects and to ensure reliable experimental results. Additionally, a few pairs of molecules which bound different parts of the proteins were identified. Such pairs could potentially be used together to increase the sensitivity and specificity of tests used to determine the presence of Calprotectin and S100A7 in e.g biological samples.
Surprisingly, a majority of the molecules were only functional in one type of experiment (out of three tested types). This sensitivity towards the assay format might call for a reconsideration of the screening process used to identify the molecules, alternatively efforts could be made to improve a selected subset of the molecules studied in this work. Nevertheless the results presented in this study should serve as guidance for the continued efforts to identify future research and therapeutic probes against Calprotectin and S100A7.

• author: Tallgren, Emma
• supervisor: Mats Ohlin
• organization: Department of Immunotechnology
• course: KIMM05 20221
• year: 2022
• type: H2 - Master's Degree (Two Years)
• subject: Biology and Life Sciences
• language: English
• id: 9094131
• date added to LUP: 2022-06-30 11:43:13
• date last changed: 2022-06-30 11:43:13