AXL-Receptor Targeted 14FN3 Based Single Domain Proteins (Pronectins™) from 3 Synthetic Human Libraries as Components for Exploring Novel Bispecific Constructs against Solid Tumors

Abstract

A highly specific AXL-receptor targeted family of non-immunoglobulin, single domain protein binders (Pronectins™) have been isolated from three (3) synthetic libraries that employ the human scaffold of the 14th domain of Fibronectin III (14FN3) and evolutionary CDRs diversity of over 25 billion loop sequences. The three libraries, each containing diversity in two loops, were designed to expand upon a human database of more than 6000 natural scaffold sequences and approximately 3000 human loop sequences. We used a bioinformatic-based approach to maximize “human” amino acid loop diversity and minimize or prevent altogether CDR immunogenicity created by the use of mutagenesis processes to generate diversity. A combination of phage display and yeast display was used to isolate 59 AXL receptor targeted Pronectins with KD ranging between 2 and 100 nM. FACS analysis with tumor cells over-expressing AXL and the use of an AXL knock-out cell line allowed us to identify Pronectin candidates with exquisite specificity for AXL receptor. Based upon several in vitro cell-based tests, we selected the best candidate, AXL54, to further characterize its in vitro cancer cells killing activity. Finally, AXL54 was used to produce the first bi-specific T cell engager protein (AXL54 [Pronectin]-linker-scFV CD3), a “new in class” protein for further testing of its anti-tumor activity in vitro and in vivo.

The First-In-Class Anti-AXL×CD3ε Pronectin™-Based Bispecific T-Cell Engager Is Active in Preclinical Models of Human Soft Tissue and Bone Sarcomas

“…Consistently, here we demonstrated that pAXL×CD3ε indeed redirects T cells toward AXL-expressing sarcoma cells, leading to a dose-dependent T-cell activation, with a consequent release of inflammatory cytokines and cytolytic molecules. Our results are in accordance with data recently reported, showing that pAXL×CD3ε exhibits cytotoxic effects on AXL-positive MDA-MB-231 cells and minimal cytotoxicity on AXL-negative CHO cells [ 38 ]. Moreover, we found enhanced cytotoxic effects as a function of increased concentrations of pAXL×CD3ε, which was highly promising taking into account the presence of immune cells in the TME of sarcomas.…”

“…A highly specific anti-AXL Pronectin™, a non-immunoglobulin and single-domain protein, has been isolated from synthetic libraries based on the human scaffold of the 14th domain of fibronectin III (14FN3), as previously described [ 38 ]. By bioinformatic analysis aimed to select the best candidate within the amino acid loop diversity and minimize or prevent immunogenicity, 6 Pronectins™ with a KD < 10 nM were identified and AXL54 was chosen for targeting purposes (KD = 8 nM).…”

“…This Pronectin™ was used to develop a first-in-class BTCE (AXL54 (Pronectin™)-linker-scFV CD3, pAXL×CD3ε), for investigation as an anti-tumor novel agent. The linker is made of a single unit of Gly4–Ser (GGGGS) [ 38 ].…”