Search

We report here the discovery and optimization of a novel T cell retargeting anti-GUCY2C x anti-CD3ε bispecific antibody for the treatment of solid tumors. Using a combination of hybridoma, phage display and rational design protein engineering, we have developed a fully humanized and manufacturable CD3 bispecific antibody that demonstrates favorable pharmacokinetic properties and potent in vivo efficacy. Anti-GUCY2C and anti-CD3ε antibodies derived from mouse hybridomas were first humanized into well-behaved human variable region frameworks with full retention of binding and T-cell mediated cytotoxic activity. To address potential manufacturability concerns, multiple approaches were taken in parallel to optimize and de-risk the two antibody variable regions. These approaches included structure-guided rational mutagenesis and phage display-based optimization, focusing on improving stability, reducing polyreactivity and self-association potential, removing chemical liabilities and proteolytic cleavage sites, and de-risking immunogenicity. Employing rapid library construction methods as well as automated phage display and high-throughput protein production workflows enabled efficient generation of an optimized bispecific antibody with desirable manufacturability properties, high stability, and low nonspecific binding. Proteolytic cleavage and deamidation in complementarity-determining regions were also successfully addressed. Collectively, these improvements translated to a molecule with potent single-agent in vivo efficacy in a tumor cell line adoptive transfer model and a cynomolgus monkey pharmacokinetic profile (half-life>4.5 days) suitable for clinical development. Clinical evaluation of PF-07062119 is ongoing.

Rab GTPases recruit effector proteins, via their GTP-dependent switch regions, to distinct subcellular compartments. Rab11 and Rab25 are closely related small GTPases that bind to common effectors termed the Rab11 family of interacting proteins (FIPs). The FIPs are organized into two subclasses (class I and class II) based on sequence and domain organization, and both subclasses contain a highly conserved Rab-binding domain at their C termini. Yeast two-hybrid and biochemical studies have revealed that the more distantly related Rab14 also interacts with class I FIPs. Here, we perform detailed structural, thermodynamic, and cellular analyses of the interactions between Rab14 and one of the class I FIPs, the Rab-coupling protein (RCP), to clarify the molecular aspects of the interaction. We find that Rab14 indeed binds to RCP, albeit with reduced affinity relative to conventional Rab11-FIP and Rab25-FIP complexes. However, in vivo, Rab11 recruits RCP onto biological membranes. Furthermore, biophysical analyses reveal a noncanonical 1:2 stoichiometry between Rab14-RCP in dilute solutions, in contrast to Rab11/25 complexes. The structure of Rab14-RCP reveals that Rab14 interacts with the canonical Rab-binding domain and also provides insight into the unusual properties of the complex. Finally, we show that both the Rab coupling protein and Rab14 function in neuritogenesis., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objective: To compare tooth loss between patients who received surgical therapy for chronic periodontitis and those who received nonsurgical therapy alone., Methods: A retrospective chart study was conducted at Dalhousie University. All patients who had periodontal treatment and were active cases for at least 10 years were included (n = 335). The sample consisted of 120 males (35.8%) and 215 females (64.2%). Ages ranged from 16 to 77 (mean = 46.1 +/- 12.0 years). All patients received nonsurgical therapy; 44.8% received periodontal surgery as well. Variables recorded were demographics, initial attachment loss, treatment type, recall frequency, patient compliance and history of extracted teeth. Independent t-tests or chi-squared tests were used to compare these for surgical and nonsurgical patients. ANOVA was used to test for interactions between initial attachment loss, age, gender, compliance and type of therapy a patient received as reasons for tooth loss., Results: 521 teeth were lost in 69 patients (20.6% of sample). Of teeth lost, 61.8% were due to periodontal disease; 24.8% to caries; 13.2% to other reasons. Patients initially diagnosed with early attachment loss lost an average of 0.37 (+/- 1.33) teeth. Patients diagnosed with moderate attachment loss lost an average of 1.50 (+/- 2.54) teeth and those diagnosed with advanced attachment loss lost an average of 3.11 (+/- 3.01) teeth. Those who received surgical therapy lost more teeth (mean = 1.31 +/- 2.36) than those who received nonsurgical treatment (mean = 0.68 +/- 1.87; p = 0.001). However, initial attachment loss was the only factor that could predict tooth loss. The type of therapy (surgical or nonsurgical) was not statistically significant., Conclusions: Most periodontal patients (79.4%) who received treatment at this dental school clinic did not lose any teeth due to periodontal disease over at least 10 years. Although patients who had surgical therapy lost more teeth than those who had nonsurgical therapy alone, this was not an important predictor of tooth loss.