Engineering receptor-based decoys for the treatment of cancer using a yeast surface display platform

Decoy receptors form a distinct class of targeted therapeutics that offer unique advantages compared to traditional antibody therapy. Since they are derived from preexisting signaling processes, they possess inherent ligand specificity and immune tolerance, which can significantly reduce off-target effects and enhance clinical efficacy. The use of receptor-based biologics to target aberrant signalling pathways that promote cancer cell proliferation and metastasis has emerged as a new frontier in cancer therapy. The major drawback of using “wild-type” receptor-based biologics is their limited competitive advantage over cellular receptors. Therefore, in this thesis, I employed directed evolution approaches to engineer high affinity TACI and RAGE receptor-based therapeutics that target tumour-promoting components in diffused large B cell lymphoma (DLBCL) and triple-negative breast cancer (TNBC), respectively.
DLBCL is characterised by the expansion of malignant B cells in the lymphocytes and the overexpression of autoantibodies. APRIL and BAFF are essential ligands for B cell development, which have been shown to promote DLBCL growth. In this study, I used yeast surface display-based affinity maturation processes to develop TACI variants with enhanced APRIL and BAFF binding affinity. Structural and biochemical analyses were carried out to investigate the basis of the enhanced ligand binding affinity. The TACI variants were reformatted into Fc fusion proteins to examine their in vitro and in vivo antitumour activity. The affinity-enhanced TACI variant, namely BS7-10, was shown to have an 8-fold increase in binding affinity to both APRIL and BAFF compared to atacicept, a commercially available WT-TACI decoy receptor. Also, BS7-10-Fc showed higher efficacy in B cell proliferation assay but demonstrated modest anti-tumour activity in a DLBCL mouse model. Collectively, our findings indicated that BS7-10 is an affinity-enhanced decoy receptor that binds APRIL and BAFF with higher affinity that should perform better than atacicept in clinical indications for both malignant diseases as well as not malignant indications such as lupus erythematosus.
TNBC is an aggressive type of breast cancer that fails to express three hormonal regulatory receptors at targetable levels: estrogen, progesterone and HER2. Due to the lack of these three receptors, there is limited approaches for the treatment of TNBC. Receptor for advanced glycation end productions (RAGE) is a multi-ligand receptor that has been associated with TNBC metastasis. RAGE ligands pleiotropically interact with a range of receptors that can trigger TNBC progression and inflammation. In this study, I used yeast surface display and directed evolution approaches to engineer a high affinity RAGE V domain, the ligand binding domain of the RAGE receptor, that has an enhanced binding capacity to multiple RAGE ligands. Structural analysis was performed to investigate the basis of the improved binding ability. IgG1 Fc fusion proteins of RAGE V decoy receptors were produced to evaluate their anti-tumour activity in both in vitro and in vivo studies. IP-MS and RNA sequencing were used to investigate the underlying tumour-suppressive mechanism of the RAGE V variants. Overall, RAGE V13 was the most robust variant which had elevated binding capacity to multiple RAGE ligands compared to WT-RAGE V and demonstrated superior anti-proliferative and antimigratory activity in both in vitro and in vivo studies. I found through IP-MS analysis that GRP78 was a novel interactor with both WT-RAGE V-Fc and RAGE V13-Fc. In addition, RNA-seq data revealed that RAGV13-Fc treatment resulted in CXCL3 downregulation in MDA-MB-231. I hypothesize that RAGE V13-Fc suppresses TLR4 signalling by sequestering GRP78, which in turn downregulates CXCL3 expression by reducing phosphor-IRF3 level. Given the complexity of RAGE-ligands interactions, further studies are needed to fully understand the underlying anti-tumour mechanism of RAGE V13-Fc.

Advisor: Giaccia, A
Moon, E
Sibson, N