Development of systems to produce antibodies to target tumor-stroma crosstalk

Cancer cells can program their tumor microenvironment (TME) in a manner that facilitates their adaptation, thereby influencing cancer survival, progression, and metastasis. In this context, it is crucial to elucidate the stroma components and their roles in various cancer types, as well as to investigate their potential applications in diagnosis, treatment, and follow-up. Previously, we demonstrated that cancer-derived secreted factors, cytokines, alter tumor fate through regulating stromal autophagy, cancer-stroma signaling. This thesis aims to develop systems for producing antibody-based drugs that target cytokines in the tumor microenvironment. To achieve this, we developed systems to produce recombinant cytokines and established a mouse single-chain variable fragment (scFv) library. To obtain these, we first cloned cDNA of cytokines into different IPTG-inducible, lac-promoter-containing vectors, including pGEX4T2 and pGEX4T1-3xFLAG. We validated our positive clones and further optimized the protocols to produce recombinant GST- and GST-FLAG-tagged cytokines using BL21 E. coli cells. In the second part, we focus on optimizing protocols and establishing recombinant scFv antibodies through phage display technology. Hence, we first determined the sequences of the antibody variable light (VL) and variable heavy (VH) chains. To establish a diverse scFv antibody library, we designed at least 36 oligos to produce VL and VH fragments. Fragments were further combined by SOE-PCR (splicing by overlap extension) and cloned into the pCANTAB-5E phage display plasmid for further phage display studies against recombinant cytokines. Overall, we established systems for producing recombinant cytokines and recombinant scFv antibodies. This study could lead to the discovery of new, specific, and drug-potential antibodies that target specific cytokines in the tumor stroma context, potentially serving as a new cancer therapeutic.