Periodic Reporting for period 4 - CoBABATI (Cofactor Binding Antibodies – Basic Aspects and Therapeutic Innovations)
Periodo di rendicontazione: 2021-03-01 al 2023-02-28
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 identified the following objective: 1) to understand the origin and prevalence of cofactor-binding Abs in immune repertoires; 2) to characterize the molecular mechanisms of interaction of cofactors with Abs; 3) to understand the physiopathological roles of cofactor-binding Abs, and 4) to explore the therapeutic potential of cofactor-binding 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.
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.
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.
The results obtained in this project demonstrate that cofactor-binding Abs are important components of the immune repertoires of healthy human. These Abs possess untypical functional characteristics. We demonstrate that cofactor-binding antibodies play a role in maintenance of homeostasis and in immune defense. Particularly, heme binding antibodies can contribute to protection of organism from the consequences of intravascular hemolysis. This finding represents a novel unprecedented role of antibodies. Our results also disclose that cofactor-binding antibodies might be exploited for development of new generation of therapeutics. These finding extend the fundamental understanding about antibodies and their role in maintenance of immune homeostasis.
As a consequence of this project, we anticipate there will be following fundamental studies of cofactor-binding antibodies. The role of diversification of immune specificity of antibodies by binding of heme to antibodies should be better clarified. We also expect that the project results will ignite interest in translational aspects as for example to explore the potential of cofactor-binding antibodies to be used as therapeutics in various pathological situations.
As a consequence of this project, we anticipate there will be following fundamental studies of cofactor-binding antibodies. The role of diversification of immune specificity of antibodies by binding of heme to antibodies should be better clarified. We also expect that the project results will ignite interest in translational aspects as for example to explore the potential of cofactor-binding antibodies to be used as therapeutics in various pathological situations.