A Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) for ovarian cancer.

BACKGROUND: Pronectins™ are a new class of fibronectin-3-domain 14th-derived (14Fn3) antibody mimics that can be engineered as bispecific T cell engager (BTCE) to redirect immune effector cells against cancer. We describe here the in vitro and in vivo activity of a Pronectin™ AXL-targeted first-in-class bispecific T cell engager (pAXLxCD3ε) against Epithelial Ovarian Cancer (EOC). METHODS: pAXLxCD3ε T-cell mediated cytotoxicity was evaluated by flow cytometry and bioluminescence. pAXLxCD3ε mediated T-cell infiltration, activation and proliferation were assessed by immunofluorescence microscopy and by flow cytometry. Activity of pAXLxCD3ε was also investigated in combination with poly-ADP ribose polymerase inhibitors (PARPi). In vivo antitumor activity of pAXLxCD3ε was evaluated in immunocompromised (NSG) mice bearing intraperitoneal or subcutaneous EOC xenografts and immunologically reconstituted with human peripheral blood mononuclear cells (PBMC). RESULTS: pAXLxCD3ε induced dose-dependent cytotoxicity by activation of T lymphocytes against EOC cells, regardless of their histologic origin. The addition of PARPi to cell cultures enhanced pAXLxCD3ε cytotoxicity. Importantly, in vivo, pAXLxCD3ε was highly effective against EOC xenografts in two different NSG mouse models, by inhibiting the growth of tumor cells in ascites and subcutaneous xenografts. This effect translated into a significantly prolonged survival of treated animals. CONCLUSION: pAXLxCD3ε is an active therapeutics against EOC cells providing a rational for its development as a novel agent in this still incurable disease. The preclinical validation of a first-in-class agent opens the way to the development of a new 14Fn3-based scaffold platform for the generation of innovative immune therapeutics against cancer.

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

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.

Engineering highly thermostable xylanase variants using an enhanced combinatorial library method.

A new directed evolution method was used to enhance the thermostability of the wild-type GH11 xylanase 2 (known as BD-11) from Hypocrea jecorina (Trichoderma reesei). Both Look-Through Mutagenesis (LTM™), which is a method for rapidly screening selected positions in the protein sequence for amino acids that introduce favorable properties, and Combinatorial Beneficial Mutagenesis (CBM™), which is a method for identifying the best ensemble of individual mutations, were employed to enhance the stability of an enzyme that has been thoroughly engineered by various means during the past 20 years. A diverse set of novel mutations was discovered, including N71D, Y73G, T95G and Y96Q. When these mutations were combined into a single construct (Hjx-81), the purified protein was active even after heating at 100°C for 20 min. This time-effective method should be generally applicable for quickly improving the physico-chemical properties of other industrial and therapeutic enzymes in only several months time.