Cancer immunotherapy has revolutionized the field of oncology by giving the possibility to ligands (e.g., antibodies) to selectively target tumor antigens and accumulate at the site of the disease while sparing normal tissues. During the past years, the number of patient eligible for immune-based cancer treatments has seen an exponential increase as these therapies are becoming first line treatment for many cancer indications. A promising anticancer strategy consists in the targeted delivery of bioactive compounds (e.g., cytokines) to the tumor microenvironment with high-affinity ligands specific for tumor-associated antigens. This approach improves the efficacy of the drug by reducing related side effects and increasing the therapeutic index of the payload. Currently, antibodies represent one of the most successful class of ligands used for this purpose as they can be generated against virtually any antigen. Many methodologies have been described for the generation and isolation of antibodies with high antigen-binding specificity. Among these, phage display technology has emerged as a powerful and versatile tool for the in vitro discovery of antibodies and peptides. Since it was invented in mid 1980s, phage-display has paved the way to the generation of more than 70 phage–derived monoclonal antibodies (mAbs) that entered clinical studies, and 14 of which have been approved in the market. Cytokines are proteins capable of modulating the activity of the immune system and some cytokine-products have gained marketing authorization for the treatment of cancer. In order to increase the therapeutic index of cytokine payloads, the generation of fusion proteins with tumor-homing antibodies has been proposed. These so-called “immunocytokine” products constitute a class of “armed” antibody products, in which a tumor-targeting immunoglobulin is fused with a cytokine. In this thesis, we present the generation and characterization of antibodies specific for two tumor microenvironment-associated antigens: Tenascin C and Fibroblast Activation Protein. Both antigens are undetectable in healthy tissues but abundantly expressed in the tumor stroma. In the first part of the thesis, we have isolated antibodies specific for the spliced domain D of Tenascin C from the synthetic phage library “ETH2Gold”. Antibodies were affinity matured randomizing key residues of CDR1 of heavy and light chains. The highest affinity clone, R6N, was characterized in vitro and in vivo showing selective accumulation at the tumor site in mouse models of cancer. An immunocytokine featuring IL12 as payload has been generated and its therapeutic activity evaluated in tumor bearing mice. R6N-IL12 exhibited potent antitumor activity in immunodeficient mice bearing SKRC52 renal cell carcinoma, as well as in immunocompetent mice bearing SMA-497 glioma. In the second part of this thesis, a monoclonal antibody has been isolated against Fibroblast Activation Protein. After affinity maturation of the CDR2 of heavy and light chains of the parental antibody C5, the selected 7NP2 antibody showed improved affinity and excellent tumor targeting properties in SKRC52-hFAP tumor bearing mice. When fused to IL12, 7NP2 was able to induce tumor growth retardation and tumor remission in mouse models of cancer. Collectively, in this thesis we have isolated and validated two monoclonal antibodies selective for tumor microenvironment-associated antigens. Both antibodies when fused to IL12 induced tumor growth retardation and remission in immunodeficient and immunocompetent mouse models providing a rationale for possible future applications of R6N and 7NP2 antibodies for the treatment of cancer patients.

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Le ciblage de l’activité de l’IFNα est une stratégie développée afin d’augmenter l’index thérapeutique de cette cytokine, dont l’efficacité requiert de fortes doses au site d’action, responsables d’une toxicité systémique. Du fait de l’expression ubiquitaire de son récepteur, le ciblage de l’IFNα par immunocytokine est limité. En se basant sur le concept d'immunocytokine et utilisant un IFNα muté, peu actif, une efficacité de ciblage de 3 log a pu être obtenue, dans le système humain et murin, grâce au rétablissement de l'activité de l'IFNα sur les cellules ciblées. Ces IFNα ciblés sont doués d'une activité biologique, notamment antitumorale, dont la cible cellulaire reste à déterminer. Une stratégie inverse, en cours d'optimisation, permet d'inhiber l’activité des IFN-I spécifiquement sur les cellules ciblées. Cette double stratégie de ciblage de l’activité et de l’inhibition de l’IFN devrait permettre de déterminer les cibles des effets bénéfiques et néfastes des IFN-I, avec des applications thérapeutiques éventuelles. / Targeting IFN activity is a strategy developed to increase the therapeutic index of thiscytokine, whose efficiency requires high doses to site of action, responsible for systemictoxicity. Due to the ubiquitous expression of its receptor, the targeting efficiency of IFNbasedimmunocytokine is limited. Using a mutated IFNα, poorly active, a 3 log targetingefficiency was achieved, in the human and mouse system, by restoring the activity of IFNα ontargeted cells. The biological activity of these targeted IFNα include an antitumoral effect,the cellular target remains to be determined. A reverse strategy being optimized, can inhibitIFN-I activity specifically on targeted cells. This dual targeting strategy of the activity andinhibition of IFN should identify the targets of beneficial and deleterious effects of IFN-I,with possible therapeutic applications.