"Stapling" scFv for multispecific biotherapeutics of superior properties.

Single-chain fragment variable (scFv) domains play an important role in antibody-based therapeutic modalities, such as bispecifics, multispecifics and chimeric antigen receptor T cells or natural killer cells. However, scFv domains exhibit lower stability and increased risk of aggregation due to transient dissociation ("breathing") and inter-molecular reassociation of the two domains (VL and VH). We designed a novel strategy, referred to as stapling, that introduces two disulfide bonds between the scFv linker and the two variable domains to minimize scFv breathing. We named the resulting molecules stapled scFv (spFv). Stapling increased thermal stability (Tm) by an average of 10°C. In multiple scFv/spFv multispecifics, the spFv molecules display significantly improved stability, minimal aggregation and superior product quality. These spFv multispecifics retain binding affinity and functionality. Our stapling design was compatible with all antibody variable regions we evaluated and may be widely applicable to stabilize scFv molecules for designing biotherapeutics with superior biophysical properties.

Functional, Biophysical, and Structural Characterization of Human IgG1 and IgG4 Fc Variants with Ablated Immune Functionality.

Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically "silent".

Discovering Molecules That Regulate Efferocytosis Using Primary Human Macrophages and High Content Imaging.

Defective clearance of apoptotic cells can result in sustained inflammation and subsequent autoimmunity. Macrophages, the "professional phagocyte" of the body, are responsible for efficient, non-phlogistic, apoptotic cell clearance. Controlling phagocytosis of apoptotic cells by macrophages is an attractive therapeutic opportunity to ameliorate inflammation. Using high content imaging, we have developed a system for evaluating the effects of antibody treatment on apoptotic cell uptake in primary human macrophages by comparing the Phagocytic Index (PI) for each antibody. Herein we demonstrate the feasibility of evaluating a panel of antibodies of unknown specificities obtained by immunization of mice with primary human macrophages and show that they can be distinguished based on individual PI measurements. In this study ~50% of antibodies obtained enhance phagocytosis of apoptotic cells while approximately 5% of the antibodies in the panel exhibit some inhibition. Though the specificities of the majority of antibodies are unknown, two of the antibodies that improved apoptotic cell uptake recognize recombinant MerTK; a receptor known to function in this capacity in vivo. The agonistic impact of these antibodies on efferocytosis could be demonstrated without addition of either of the MerTK ligands, Gas6 or ProS. These results validate applying the mechanism of this fundamental biological process as a means for identification of modulators that could potentially serve as therapeutics. This strategy for interrogating macrophages to discover molecules regulating apoptotic cell uptake is not limited by access to purified protein thereby increasing the possibility of finding novel apoptotic cell uptake pathways.

EGF potentiation of VEGF production is cell density dependent in H292 EGFR wild type NSCLC cell line.

Non-small cell lung cancer (NSCLC) affects millions of patients each year worldwide. Existing therapies include epidermal growth factor receptor (EGFR) inhibition using small molecules or antibodies with good efficacy. Unfortunately, intrinsic and acquired resistance to EGFR therapy remains a persistent complication for disease treatment. A greater understanding of the role of EGFR in NSCLC etiology is crucial to improving patient outcomes. In this study, the role of EGFR in tumor angiogenesis was examined in H292 NSCLC cells under the pretense that confluent cells would exhibit a more angiogenic and growth-centered phenotype. Indeed, confluent H292 cells potentiated endothelial cell angiogenesis in co-culture models in an EGFR-dependent manner. While confluent H292 cells did not exhibit any change in EGFR protein expression, EGFR localization to the extracellular membrane was increased. EGFR membrane localization coincided with a comparable potentiation of maximal EGFR phosphorylation and was followed by a 3-fold increase in vascular endothelial growth factor A (VEGF-A) production as compared to subconfluent cells. EGFR-mediated VEGF-A production was determined to be dependent on signal transducer and activator of transcription 3 (STAT3) activation and not phosphoinositide 3-kinase (PI3K) signaling. These results identify unique cell density dependent phenotypes within a monoclonal NSCLC cell line and provide a potential mechanism of resistance to anti-EGFR therapy in metastatic NSCLC.