We generated panels of recombinant human IgG Abs and tested their reactivity towards different cofactor molecules. These analyses demonstrated that the immune repertoire of healthy humans have a high frequency (>10 %) of heme-binding Abs. During these studies we discovered that human immune repertoires contain also a fraction of antibodies that bind to another cofactor molecule – folic acid. We found that cofactor-binding antibodies are present in all types of B cells, naïve, memory, and plasma cells and they are spontaneously generated by the immune system but not derived by specific immune responses. Further our 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. Indeed, heme binding antibodies contain higher frequency of specific amino acid residues in their antigen-binding site. 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. It was found that heme binds to regions overlapping with the most diverse part of the V region, CDR3 loops and the binding of heme causes conformational changes in the antibody. Furthermore, we provide evidence that antibodies use heme as an interfacial cofactor for recognition of novel antigens, thus expending the antigen recognition specificity of the immune system. By addressing another objective in the project, 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. Moreover, heme-binding antibodies were able to accelerate the clearance of pro-inflammatory and pro-oxidative products of intravascular hemolysis in vivo. This finding has significant translational potentilla.
Finally, we observed that a significant fraction of clinical-stage therapeutic antibodies are able to bind heme. Importantly, we demonstrated that heme binding can be used as a predictive marker for some liabilities in the physicochemical properties of antibodies. In addition we demonstrated that theinteraction of heme with a therapeutic anti-cancer antibody, Trastuzumab causes formation of well-organized oligomers that have a higher capacity to kill cancer cells. Notably, we succeeded to underscore the underlining mechanism and to design an engineering strategy for transfer of this phenomenon to other monoclonal antibodies, with goal of potentiating their cytotoxic activities. This finding provided a proof of concept that cofactor binding can be exploited for optimization of the therapeutic properties Abs.
The results of this project were presented at 5 scientific forums, published in 12 peer-reviewed articles (three additional manuscripts are currently under preparation) and part of them protected by a patent.
