NOVEL MONO-, BI- AND TRI-SPECIFIC IMMUNOMODULATORY ANTIBODIES FOR CANCER THERAPY AND TOOLS FOR OTHER THERAPEUTIC AND DIAGNOSTIC APPLICATIONS

Cancer represents one of the leading causes of death worldwide, and the development of novel immunotherapeutic strategies, such as that of immune checkpoint inhibitors (ICIs), has generated long-lasting responses in multiple malignancies. Even though safer than chemotherapies, some adverse events, including rare cases of cardiotoxicity, have also been reported for ICIs, especially when used in combination. In this context, the aim of my research project was to identify combinations of novel human immunomodulatory monoclonal antibodies targeting different ICs, with improved efficacy and safety profiles. Some novel ICIs were previously isolated in our laboratory and characterized for their biological properties. Here, they were tested in combinations to identify those with stronger anti-tumor effects and reduced cardiotoxic side effects on cardiomyocytes, compared to combinatorial treatments with clinically validated monoclonal antibodies (mAbs). Interestingly, the combination of novel human PD-L1_1 (anti-PD-L1 mAb) and ID-1 (anti-CTLA-4 mAb) induced efficient lysis of triple-negative breast cancer cells and lower cytotoxicity on cardiomyocytes, when co-cultured with lymphocytes, compared to the treatments with Ipilimumab, Atezolizumab or their combination. We further evaluated the pro-inflammatory effects of FDA-approved combinatorial treatments of ICIs, and we found that these combinations, especially regimens including the anti-LAG-3 antibody Relatlimab, promoted an important increase of pro-inflammatory cytokines secretion, such as IL-6, IL-1β, TNF-α, IFNγ and granzyme B. Since combinations of novel mAbs showed a better profile, we decided to fuse the binding moieties derived from LAG-3_1 with those of PD-L1_1 and PD-1_1, generating four novel bi-specific immunomodulatory tribodies. The novel compounds showed binding affinities for their targets comparable to those of validated mAbs (Atezolizumab, Pembrolizumab or Relatlimab) and, more interestingly, an increased ability to induce lymphocyte activation and stronger in vitro tumor cytotoxicity compared to the combination of clinically validated mAbs. In parallel, with the aim of recruiting T cells against cancer cells, we also generated novel tri-specific T-cell engager (TCE) tribodies, able to simultaneously recognize the oncofetal antigen 5T4 on tumor cells, CD3 on T cells, and an IC (PD-L1, PD-1 or LAG-3) by including the scFvs derived from the mentioned novel mAbs. We found that the novel TCEs enhanced cytokine release from hPBMCs, with a strong secretion of IL-2 and IFNγ and showed more potent antitumor effects than the combination of the parental bispecific compound and clinically validated mAbs targeting the same ICs. Notably, the TCE including the anti-PD-L1 moiety induced complete tumor regression in a mouse model. To increase the collection of mAbs specific for ICs, human anti-OX-40 scFvs were identified by phage display selections on hPBMCs and purified receptor and used to generate four novel fully human mAbs targeting this immunostimulatory receptor expressed on immune cells. The novel mAbs were tested for their biological effects on human lymphocytes alone or in co-cultures with tumor cells and enhanced both T cell activation and tumor cell killing, displaying additive effects when combined with Atezolizumab. Finally, a novel human antibody targeting the coagulation cascade, and in particular Factor V, was also generated by using phage display. The novel mAb, called D9, was found able to efficiently bind to Factor V in both its native and activated forms, with nanomolar KD values and to slow down the coagulation by prolonging the activated partial thromboplastin time (aPTT). These features could make this mAb a potential tool for both diagnostic and therapeutic applications. Overall, these findings highlight the potential of the newly generated bi- and tri-specific antibodies to overcome the limitations of conventional IC inhibitors, paving the way for next-generation anti-tumor immunotherapeutics with improved safety and efficacy and show the possibility to enlarge the combinatorial strategies including novel immunostimulatory mAbs. Furthermore, novel antibody-based approach has been used also for facing cardiovascular disorders.