Preferential Identification of Agonistic OX40 Antibodies by Using Cell Lysate to Pan Natively Paired, Humanized Mouse-Derived Yeast Surface Display Libraries

To discover therapeutically relevant antibody candidates, many groups use mouse immunization followed by hybridoma generation or B cell screening. One modern approach is to screen B cells by generating natively paired single chain variable fragment (scFv) display libraries in yeast. Such methods typically rely on soluble antigens for scFv library screening. However, many therapeutically relevant cell-surface targets are difficult to express in a soluble protein format, complicating discovery. In this study, we developed methods to screen humanized mouse-derived yeast scFv libraries using recombinant OX40 protein in cell lysate. We used deep sequencing to compare screening with cell lysate to screening with soluble OX40 protein, in the context of mouse immunizations using either soluble OX40 or OX40-expressing cells and OX40-encoding DNA vector. We found that all tested methods produce a unique diversity of scFv binders. However, when we reformatted forty-one of these scFv as full-length monoclonal antibodies (mAbs), we observed that mAbs identified using soluble antigen immunization with cell lysate sorting always bound cell surface OX40, whereas other methods had significant false positive rates. Antibodies identified using soluble antigen immunization and cell lysate sorting were also significantly more likely to activate OX40 in a cellular assay. Our data suggest that sorting with OX40 protein in cell lysate is more likely than other methods to retain the epitopes required for antibody-mediated OX40 agonism

GMP Manufacturing and IND-Enabling Studies of a Recombinant Hyperimmune Globulin Targeting SARS-CoV-2

Conventionally, hyperimmune globulin drugs manufactured from pooled immunoglobulins from vaccinated or convalescent donors have been used in treating infections where no treatment is available. This is especially important where multi-epitope neutralization is required to prevent the development of immune-evading viral mutants that can emerge upon treatment with monoclonal antibodies. Using microfluidics, flow sorting, and a targeted integration cell line, a first-in-class recombinant hyperimmune globulin therapeutic against SARS-CoV-2 (GIGA-2050) was generated. Using processes similar to conventional monoclonal antibody manufacturing, GIGA-2050, comprising 12,500 antibodies, was scaled-up for clinical manufacturing and multiple development/tox lots were assessed for consistency. Antibody sequence diversity, cell growth, productivity, and product quality were assessed across different manufacturing sites and production scales. GIGA-2050 was purified and tested for good laboratory procedures (GLP) toxicology, pharmacokinetics, and in vivo efficacy against natural SARS-CoV-2 infection in mice. The GIGA-2050 master cell bank was highly stable, producing material at consistent yield and product quality up to >70 generations. Good manufacturing practices (GMP) and development batches of GIGA-2050 showed consistent product quality, impurity clearance, potency, and protection in an in vivo efficacy model. Nonhuman primate toxicology and pharmacokinetics studies suggest that GIGA-2050 is safe and has a half-life similar to other recombinant human IgG1 antibodies. These results supported a successful investigational new drug application for GIGA-2050. This study demonstrates that a new class of drugs, recombinant hyperimmune globulins, can be manufactured consistently at the clinical scale and presents a new approach to treating infectious diseases that targets multiple epitopes of a virus