Manna, Lorenzo (2025) Generation and characterization of novel anti-tumor immunomodulatory bi-specific tribodies and novel human mAbs for other therapeutic approaches. [Tesi di dottorato]

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Abstract

Cancer immunotherapy, based on monoclonal antibodies (mAbs) targeting immune checkpoints (ICs), is an efficient approach to restore the activity of the immune system against tumor cells. We previously isolated novel monoclonal antibodies targeting different ICs and we characterized their biological properties. In particular, two novel mAbs named PD-L1_1 and ID-1, targeting Programmed Death-Ligand-1 (PD-L1) and Cytotoxic T-Lymphocyte Antigen-4 (CTLA-4), respectively, were further characterized in this thesis project. We investigated their differential effects on immune cell subpopulations of Natural Killer (NK) cells and Pan T cells, in comparison with the effects of clinically validated Atezolizumab and Ipilimumab, targeting the same ICs. The results showed that the novel PD-L1_1 and ID-1 mAbs were able to activate more efficiently the NK cells subpopulation, leading to stronger tumor cell lysis when NK cells were co-cultured with tumor cells, either when used as single agents or in combinatorial treatments. On the other hand, the clinically validated Atezolizumab and Ipilimumab induced stronger anti-tumor effects when used in co-cultures of Pan T cells with tumor cells. These results suggest that the novel antibodies might be suitable for the treatment of tumor cells characterized by the loss of Major Histocompatibility Complex (MHC) expression, as they efficiently involve NK cells in the anti-tumor response. Given these promising results, we decided to combine the binding moiety derived from PD-L1_1 mAb with those from two different mAbs, also previously generated in our laboratory against Programmed cell Death protein-1 (PD-1) and Lymphocyte Activation Gene-3 (LAG-3), to generate four novel bi-specific immunomodulatory constructs, called tribodies. These new constructs contain two different immune checkpoint inhibitors in one single molecule, with the goal of inducing a more potent T cell activation. All the tribodies were able to efficiently bind to their targets, and to induce T cell activation. In particular, the tribodies TR0304 (αPD-L1/LAG-3) and TR0506 (αPD-1/LAG-3) activated T cells more efficiently and led to a stronger tumor cell lysis than the combination of their respective parental mAbs, proving the validity of the strategy of combining two different IC binding moieties into one single molecule. Moreover, when tested by BLI analyses, the novel tribodies showed similar binding affinities to those observed with the clinically validated mAbs, and recognized distinct or only partially overlapping epitopes. On the basis of the data obtained with bi-specific tribodies, we also generated new tri-specific T-cell engager tribodies with the aim of recruiting T cells against cancer cells, overcoming immunosuppressive environment of tumors. These tribodies are endowed with three different binding moieties, two of them (shared by all the constructs) recognizing CD3 T cells co-receptor and 5T4 tumor-associated antigen, and a third additional binding site recognizing either PD-L1, PD-1 or LAG-3, from the same mAbs mentioned above. The novel tribodies retained the ability to efficiently bind to their 3 targets, and they also showed more potent anti-tumor activity in co-cultures of human Peripheral Blood Mononuclear Cells (hPBMCs) and tumor cells than the combination of the parental T-cell engager bi-specific tribody (a bi-specific tribody targeting only CD3 and 5T4) with each of parental anti-ICs mAbs. In parallel, we also identified, by using an innovative phage display strategy, new binders for two different targets: the Factor V of the coagulation cascade and the Spike protein of the novel SARS-CoV-2 virus. The novel anti-Factor V monoclonal antibody, called D9, was found able to efficiently bind to both the Factor V and its active form, Factor Va, and is currently under evaluation for its biological properties. On the other hand, D3, the newly-generated mAb against the Spike protein of SARS-CoV-2 virus was tested in this thesis project for its binding to the Omicron variant of the virus. D3 showed to retain its binding ability against the Omicron variant of the Spike protein, either when used as purified recombinant protein or when expressed on the surface of pseudoviral particles, suggesting that it could be potentially used both as a new therapeutic agent to treat the infections caused by the Omicron variant of the virus, or as a diagnostic tool to detect viral particles in biological samples.

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