Vetrei, Cinzia (2022) COMBINATORIAL APPROACHES BASED ON NOVEL HUMAN IMMUNOMODULATORY ANTIBODIES AND APTAMERS FOR SAFE CANCER THERAPY. [Tesi di dottorato]

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Item Type: Tesi di dottorato
Resource language: English
Title: COMBINATORIAL APPROACHES BASED ON NOVEL HUMAN IMMUNOMODULATORY ANTIBODIES AND APTAMERS FOR SAFE CANCER THERAPY
Creators:
CreatorsEmail
Vetrei, Cinziacinzia.vetrei@unina.it
Date: 7 March 2022
Number of Pages: 92
Institution: Università degli Studi di Napoli Federico II
Department: Medicina Molecolare e Biotecnologie Mediche
Dottorato: Medicina molecolare e biotecnologie mediche
Ciclo di dottorato: 34
Coordinatore del Corso di dottorato:
nomeemail
Santoro, Massimomassimo.santoro@unina.it
Tutor:
nomeemail
De Lorenzo, ClaudiaUNSPECIFIED
Date: 7 March 2022
Number of Pages: 92
Keywords: Cancer Immunotherapy, Monoclonal antibodies, Phage display
Settori scientifico-disciplinari del MIUR: Area 05 - Scienze biologiche > BIO/10 - Biochimica
Date Deposited: 17 Mar 2022 15:01
Last Modified: 28 Feb 2024 14:16
URI: http://www.fedoa.unina.it/id/eprint/14573

Collection description

Cancer Immunotherapy consists of multiple approaches aimed at improving immune responses against cancer cells. Among them, we focused on the use of monoclonal antibodies (mAbs) specifically targeting either tumor associated antigens (TAA), overexpressed on tumor cells, or immunecheckpoints (ICs), key regulators of the immune system, for activating T lymphocytes against cancer cells. In our laboratory, novel fully human monoclonal antibodies targeting the ICs Programmed death-ligand 1 (PD-L1) or Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) were successfully generated by massive parallel screening of phage antibody libraries, by using for the first time a selection strategy on human activated lymphocytes to generate large collections of single-chain variable fragments (scFvs) recognizing multiple ICs. In order to identify human-mouse cross-reactive mAbs to be used in future in vivo studies, parallel panning rounds were performed in this thesis project on purified mouse and human CTLA-4 proteins. The selection for cross-reactive mAbs was guaranteed by a high throughput sequencing to identify the clones commonly enriched by the two parallel pannings on human and mouse CTLA-4. We identified two clones, named ID-1 and ID-8, able to bind with high affinity and specificity to both human and mouse CTLA-4 antigen, either expressed on lymphocytes (in its native conformation) or as purified recombinant proteins, showing nanomolar or sub-nanomolar Kd values. They were found able to activate both human and mouse PBMCs, as confirmed by the high levels of IL-2 and IFN-γ cytokines secretion induced by their treatments. Interestingly, ID-1 and ID-8 activated also Natural Killer (NK) cells and efficiently inhibited cancer cell growth. Since recent studies showed the expression of ICs also on tumor cells, we treated them with the clinically validated Ipilimumab and Nivolumab and the novel human antibodies targeting CTLA-4 (ID-1 and ID-8) and PD-L1 (PD-L1_1 and 10_12) mAbs, previously produced in our laboratory, to further investigate on their effects on intracellular pathways. Unexpectedly, we found that they are able to inhibit ERK phosphorylation and to activate NF-kB transcription factor, with following upregulation of IC expression. On the contrary, treatments with agonistic PD-L1 and Programmed Cell Death 1 (PD-1) recombinant proteins showed opposite effects, suggesting also the probable existence of a crosstalk in tumor cells among multiple ICs. Since anti-ICs mAbs show their highest anti-tumor efficacy by activating lymphocytes against cancer cells, we also set up in vitro systems based on co-cultures of lymphocytes with tumor cells or human fetal cardiomyocytes, in order to identify the most efficient combinations of immunomodulatory mAbs associated with the lowest cardiac adverse events. We found that novel combinations involving the immunomodulatory mAbs, PD-L1_1 and ID-1, have a more potent anti-cancer activity than the clinically validated Ipilimumab and Nivolumab combination, while lacking their cardiotoxic side effects. We think that these co-cultures-based assays represent useful tools to test also other combinatorial treatments of emerging immunomodulatory mAbs against different ICs to early screen combinatorial therapeutic regimens for both anti-tumor potency and safety. Finally, we also combined the novel antibodies with other drugs, such as aptamers, since these oligonucleotides have been recently proposed as promising drugs due to their low size, and lack of immunogenicity. In particular, we decided to combine an anti- epidermal growth factor receptor (EGFR) aptamer (CL4) with anti-tumor mAbs, in order to create bi-specific molecules overcoming aptamers’ limits, such as short half-life and lack of effector functions. Here, we constructed, for the first time, three novel bispecific conjugates, made up of CL4 aptamer linked either with an immunomodulatory (anti-PD-L1 or anti-CTLA-4) antibody or with an anti-epidermal growth factor receptor 2 (ErbB2) compact antibody. These three novel immuno-conjugates retain the targeting ability of both the parental moieties and acquire a more potent cancer cell killing activity by combining the anti-tumor properties of the two parental moietes. Furthermore, the conjugation of the anti-EGFR aptamer with the anti-PD-L1 (10_12) or anti-CTLA-4 (Ipilimumab) antibody allowed for efficient redirection and activation of T cells against cancer cells, leading to a dramatic enhancement of the cytotoxicity of the two conjugated partners.

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