Passariello, Margherita (2018) "Innovative strategies for the generation of novel human therapeutic anti-tumor and immunomodulatory antibodies". [Tesi di dottorato]

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Item Type:	Tesi di dottorato
Resource language:	English
Title:	"Innovative strategies for the generation of novel human therapeutic anti-tumor and immunomodulatory antibodies"
Creators:	Creators Email Passariello, Margherita margherita.passariello@unina.it
Date:	7 December 2018
Number of Pages:	105
Institution:	Università degli Studi di Napoli Federico II
Department:	Medicina Molecolare e Biotecnologie Mediche
Dottorato:	Medicina molecolare e biotecnologie mediche
Ciclo di dottorato:	31
Coordinatore del Corso di dottorato:	nome email Avvedimento, Enrico Vittorio avvedim@unina.it
Tutor:	nome email De Lorenzo, Claudia UNSPECIFIED
Date:	7 December 2018
Number of Pages:	105
Keywords:	Immunotherapy, Phage Display, Tribody.
Settori scientifico-disciplinari del MIUR:	Area 11 - Scienze storiche, filosofiche, pedagogiche e psicologiche > M-EDF/01 - Metodi e didattiche delle attività motorie
Date Deposited:	09 Jan 2019 12:31
Last Modified:	29 Jun 2020 10:58
URI:	http://www.fedoa.unina.it/id/eprint/12501

Collection description

Immunotherapy, based on the use of novel human mAbs endowed with antitumor or immunomodulatory activity, is an increasingly important strategy for cancer therapy. Monoclonal antibodies can be directed against Tumor Associated Antigens (TAAs), to inhibit their oncogenic function, or against Immune Checkpoints (IC), to modulate specific T cell responses against cancer. Furthermore, ongoing clinical trials in oncology are currently testing combinatorial treatments of anti-TAA and immunomodulatory antibodies. In our laboratory novel human antitumor immunoagents have been successfully produced against the TAA ErbB2, which is a Tyrosine Kinase Receptor, overexpressed in breast cancer and several other carcinomas. In particular, a fully human single chain antibody fragment (scFv), named Erbicin, able to bind to an epitope of ErbB2 different from those recognized by the clinically validated mAbs Trastuzumab and Pertuzumab, was isolated by phage display selection on live cells. A human anti-ErbB2 “compact antibody” (100kDa) was also generated by the fusion of Erbicin with a human IgG1 Fc, which was found to efficiently inhibit ErbB2-positive tumor growth both in vitro and in vivo. As a further progress, a trispecific antibody derivative, named Tribody, targeting three noncompeting epitopes on the extracellular domain of ErbB2 was obtained by fusing 3 binding moieties derived from Erb-hcAb, Trastuzumab and Pertuzumab, respectively. The triparatopic Tribody significantly downregulates ErbB2 and inhibits the cell growth of tumor cells, including those resistant to Trastuzumab. We show here that the multiparatope tribody combines and potentiates the therapeutic effects of the 3 different antibodies in 1 single antibody construct, thus allowing for the reduction of costs of antibody production and overcoming the limits related to monotherapy associated drug resistance. On the other hand, we performed a massive parallel screening of phage antibody library to obtain a large repertoire of fully human immunomodulatory antibodies against several-immune regulatory checkpoints to be used in monotherapy or in combinatorial treatments for cancer therapy. We used for the first time an innovative selection strategy on human activated lymphocytes to generate a large collection of scFvs against 10 different IC, called “Immunome Library”, from which scFvs specifically recognizing a given receptor could be pulled out by subsequent affinity selection cycles on recombinant purified proteins used as baits. By Next Generation Sequencing and bioinformatic analysis we ranked individual scFvs in each collection and identified those with the highest level of enrichment. Human IgGs from three of these collections (i.e. PD-1, PD-L1 and LAG-3) were generated and tested for their binding and biological activity. In particular, they were found to specifically bind to their targets with high affinity, to efficiently activate T cell proliferation, induce cytokine secretion and inhibit in vivo tumor growth. Interestingly, the novel isolated mAbs have comparable or even better binding affinity and biological activity than the clinically validated anti-PD-1 mAb Nivolumab. Finally, in order to explore the possibility to enhance the antitumor effects of the anti-TAA and immunomodulatory antibodies we tested some combinatorial treatments. We found that the anti-ErbB2 antibody Erb-hcAb and the novel anti-PD-L1 mAb, generated in our laboratory, showed additive antitumor effects on a panel of ErbB2-positive tumor cells, thus suggesting novel therapeutic approaches for the therapy of breast cancer.

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