Abstract 5764: Discovery of therapeutic human monoclonal antibody combinations using microfluidics and molecular genomics
Authors: Johnson David S. , Adler Adam S. , Mizrahi Rena , Keller Carter 
Published: August 2018

Monoclonal antibodies (mAbs) have proven to be extremely effective immuno-oncology therapeutics that lead to previously unseen durable responses in melanoma, bladder cancer, RCC, lung cancer, and other malignancies. Most mAb therapeutics, including current anti-PD-1 drugs, were discovered by mouse immunization followed by hybridoma isolation. Unfortunately, mouse hybridomas are highly inefficient, capturing only about 0.1% of the total B-cell diversity in immunized mice. As a result, discovery programs that use mouse hybridomas often struggle to find high-affinity, developable antibodies against certain challenging targets, even when using humanized mice. We have developed a novel method for mouse mAb discovery that leverages microfluidics, multiplex PCR, yeast single chain variable fragment (scFv) display, and fluorescence-activated cell sorting (FACS). Our technology is significantly faster and more comprehensive than conventional hybridoma screening. We recently embarked on a discovery campaign for 17 immuno-oncology targets, including PD-1, CTLA-4, GITR, and OX40. We injected chimeric humanized mice (Trianni) with antigen twice weekly for three weeks. Lymph nodes, splenocytes, and bone marrow were removed from the mice and made into single-cell suspensions. We then isolated B cells using negative selection magnetic beads, and used microfluidics to process &gt;4 million cells per mouse, generating DNA libraries that keep single cell heavy and light chain immunoglobulin (Ig) pairing intact. We built yeast scFv libraries for each of the 17 antigen-specific B cell repertoires with a C-terminal c-myc tag. To select scFv-expressing cells that bind to antigen, biotinylated antigen was added to the yeast cells at 7-250nM and then stained with fluorescent streptavidin. We then flow sorted for the top double-positive cells (c-myc+/antigen+). Finally, we deep sequenced the libraries of enriched high-affinity scFvs, which yielded the sequences for &gt;2,000 candidate scFv binders. Hundreds of these candidate scFvs were engineered into full-length mAb constructs and expressed transiently in Chinese hamster ovary (CHO) cells. To develop candidates for clinical use, we performed assays for binding kinetics, cell surface antigen binding, in vitro T-cell activation, and epitope binning. Candidates that are high affinity, bind cell surface antigen, and induce immune cell activation are now being tested in combinations for in vitro and in vivo efficacy. Combinations that have significantly better in vitro and in vivo efficacy than benchmark checkpoint inhibitors currently on the market will be moved forward into clinical studies.

Citation Format: David S. Johnson, Adam S. Adler, Rena Mizrahi, Carter Keller. Discovery of therapeutic human monoclonal antibody combinations using microfluidics and molecular genomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5764.