The immune repertoire of healthy individuals contains a fraction of antibodies (Abs) that bind with high affinity various endogenous or exogenous low molecular weight compounds, including some cofactor molecules essential for the aerobic life, such as riboflavin, heme and...
The immune repertoire of healthy individuals contains a fraction of antibodies (Abs) that bind with high affinity various endogenous or exogenous low molecular weight compounds, including some cofactor molecules essential for the aerobic life, such as riboflavin, heme and adenosine triphosphate (ATP). Despite identification of cofactor-binding Abs as a constituent of normal immune repertoires, their fundamental characteristics have never been systematically investigated. Thus, we do not know the origin, prevalence and physiopathological significance of cofactor-binding Abs. Moreover, the molecular mechanisms of interaction of cofactors with Abs are ill defined. Different proteins use cofactors to extend the chemistry intrinsic to the amino acid sequence of their polypeptide chain. Thus, one can speculate that the association of Abs with cofactors would results in the emergence of untypical properties for Abs. Indeed the binding of heme to certain Abs result in acquisition of new antigen-binding specificities i.e. this interaction can contribute to diversification of the immune repertoires. The principal goal of the present proposal is to gain a basic understanding about the fraction of cofactor-binding Abs in immune repertoires of healthy individuals and to use this knowledge for rational design of novel classes of therapeutic Abs. In this project, we have been addressing the following questions: 1) understanding of the origin and prevalence of cofactor-binding Abs in immune repertoires; 2) characterization of the molecular mechanisms of interaction of cofactors with Abs; 3) understanding of the physiopathological roles of cofactor-binding Abs, and 4) using the cofactor binding for the development of novel types of therapeutic Abs. A comprehensive understanding of various aspects of cofactor-binding Abs should lead to advances in the fundamental knowledge and in the development of innovative therapeutic strategies and diagnostic tools.
During the first phase of the project we generated panels of recombinant human IgG Abs and tested their reactivity towards different cofactor molecules. These analyses showed that the immune repertoire of healthy humans have a high frequency of heme binding Abs. In addition, these studies helped to uncover the molecular characteristics of cofactor-binding Abs and delineate the features that distinguish them from Abs that are specific for conventional antigens. Further, by using various biophysical and biochemical approaches as well as molecular modelling and site-directed mutagenesis we provided understanding about the intimate details of the interaction of heme with the Ab molecule and the molecular mechanism of the heme-mediated diversification of the immune specificity. In a parallel line of research, it has been shown that cofactor-binding Abs exert potent anti-inflammatory activity. Free extracellular heme is noxious molecule able to trigger inflammation and oxidative stress. Our data illustrated that a fraction of human Abs has a capacity to inhibit pro-inflammatory activity of heme, thus reducing the cellular damage elicited by activation of the innate immune system. In addition these lines of study have revealed a novel pathophysiological mechanism linking free extracellular heme, innate immune receptors (TLR4) and activation of the complement system. Finally, we observed that the interaction of heme with a therapeutic anti-cancer Ab improved its cytotoxic activity. This finding provided a proof of concept that cofactor binding can be exploited for optimization of the therapeutic properties Abs.
The results obtained in the first half of the project demonstrate that cofactor-binding Abs are important components of the immune repertoires. These Abs possess untypical functional characteristics. Our results also disclose that cofactor-binding Abs might be exploited for development of new generation of therapeutics. These finding extend the fundamental understanding about Abs and their role in maintenance of immune homeostasis.
In the next phase of the project we anticipate to provide more details about immune modulatory and immune defence functions of Abs that interact with cofactors. The pathophysiological role of diversification of antigen-binding specificity by binding of heme to Abs should be better clarified. We also expect to gain insights about potential of cofactor-binding Abs to be utilized as therapeutics in actual pathological situations. As continuation of the molecular studies we envisage to obtain direct structural characterization of the interaction of Ab with heme.