Mammalian display to secretion switchable libraries for antibody preselection and high throughput functional screening.

Recently, there has been a co-evolution of mammalian libraries and diverse microfluidic approaches for therapeutic antibody hit discovery. Mammalian libraries enable the preservation of full immune repertoires, produce hit candidates in final format and facilitate broad combinatorial bispecific antibody screening, while several available microfluidic methodologies offer opportunities for rapid high-content screens. Here, we report proof-of-concept studies exploring the potential of combining microfluidic technologies with mammalian libraries for antibody discovery. First, antibody secretion, target co-expression and integration of appropriate reporter cell lines enabled the selection of in-trans acting agonistic bispecific antibodies. Second, a functional screen for internalization was established and comparison of autocrine versus co-encapsulation setups highlighted the advantages of an autocrine one cell approach. Third, synchronization of antibody-secreting cells prior to microfluidic screens reduced assay variability. Furthermore, a display to secretion switchable system was developed and applied for pre-enrichment of antibody clones with high manufacturability in conjunction with subsequent screening for functional properties. These case studies demonstrate the system's feasibility and may serve as basis for further development of integrated workflows combining manufacturability sorting and functional screens for the identification of optimal therapeutic antibody candidates.

Efficient Microfluidic Downstream Processes for Rapid Antibody Hit Confirmation.

Microfluidics has been recently applied to better understand the spatial and temporal progression of the immune response in several species, for tool and biotherapeutic production cell line development and rapid antibody hit discovery. Several technologies have emerged that allow interrogation of large diversities of antibody-secreting cells in defined compartments such as picoliter droplets or nanopens. Mostly primary cells of immunized rodents but also recombinant mammalian libraries are screened for specific binding or directly for the desired function. While post-microfluidic downstream processes appear as standard steps, they represent considerable and interdependent challenges that can lead to high attrition rates even if original selections had been successful. In addition to next-generation sequencing recently described in depth elsewhere, this report aims at in detail explanations of exemplary droplet-based sorting followed by single-cell antibody gene PCR recovery and reproduction or single-cell sub-cultivation for crude supernatant confirmatory studies.

An integrated mammalian library approach for optimization and enhanced microfluidics-assisted antibody hit discovery.

Recent years have seen the development of a variety of mammalian library approaches for display and secretion mode. Advantages include library approaches for engineering, preservation of precious immune repertoires and their repeated interrogation, as well as screening in final therapeutic format and host. Mammalian display approaches for antibody optimization exploit these advantages, necessitating the generation of large libraries but in turn enabling early screening for both manufacturability and target specificity. For suitable libraries, high antibody integration rates and resulting monoclonality need to be balanced - we present a solution for sufficient transmutability and acceptable monoclonality by applying an optimized ratio of coding to non-coding lentivirus. The recent advent of microfluidic-assisted hit discovery represents a perfect match to mammalian libraries in secretion mode, as the lower throughput fits well with the facile generation of libraries comprising a few million functional clones. In the presented work, Chinese Hamster Ovary cells were engineered to both express the target of interest and secrete antibodies in relevant formats, and specific clones were strongly enriched by high throughput screening for autocrine cellular binding. The powerful combination of mammalian secretion libraries and microfluidics-assisted hit discovery could reduce attrition rates and increase the probability to identify the best possible therapeutic antibody hits faster.