Cembrola, Biancamaria (2017) Affinity maturation of novel human antibodies for cancer immunotherapy, by yeast surface display technology. [Tesi di dottorato]

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The affinity engineering is a key step to increase the therapeutic efficacy of monoclonal antibodies (mAbs). Yeast surface display (YSD) is the most widely used and powerful affinity maturation approach, allowing for the achievement of low picomolar antibody binding affinities. A great number of mAbs approved for clinics are used in cancer immunotherapy, for the targeting of either tumor neoantigens or immune checkpoint components; this second approach aims to re-activate the T cell-mediated anti-tumor immunity, which is often impaired by cancer cells through several immune escape mechanisms. In this study, we describe an optimization of the YSD methodology, applied to the generation of potentially therapeutic high affinity single chain antibody fragments (scFvs) targeting PD-L1, an immune checkpoint component which is often upregulated on cancer cell surface. We generated two different yeast libraries with high mutant frequency and diversity, by multi-step random mutagenesis of the heavy chain variable region CDR3 of an anti PD-L1 scFv. By panning the libraries against soluble PD-L1 antigen and through few sequential rounds of fluorescence-activated cell sorting (FACS), we quickly isolated mutated yeast clones with conserved mutation hotspots. Among these scFv-yeast clones, 6 of them were enriched and showed a 6,3- to 9,8-fold affinity improvement compared with the parental one. These scFvs maintained some binding improvement also when converted into IgGs and tested on PD-L1 protein showed on the plasma membrane of human activated lymphocytes. For this reason, these novel antibodies could be good candidates for an antibody-based, PD-L1-targeted cancer immunotherapy.

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