Engineering hydrophobicity and manufacturability for optimized biparatopic antibody-drug conjugates targeting c-MET.

Optimal combinations of paratopes assembled into a biparatopic antibody have the capacity to mediate high-grade target cross-linking on cell membranes, leading to degradation of the target, as well as antibody and payload delivery in the case of an antibody-drug conjugate (ADC). In the work presented here, molecular docking suggested a suitable paratope combination targeting c-MET, but hydrophobic patches in essential binding regions of one moiety necessitated engineering. In addition to rational design of HCDR2 and HCDR3 mutations, site-specific spiking libraries were generated and screened in yeast and mammalian surface display approaches. Comparative analyses revealed similar positions amendable for hydrophobicity reduction, with a broad combinatorial diversity obtained from library outputs. Optimized variants showed high stability, strongly reduced hydrophobicity, retained affinities supporting the desired functionality and enhanced producibility. The resulting biparatopic anti-c-MET ADCs were comparably active on c-MET expressing tumor cell lines as REGN5093 exatecan DAR6 ADC. Structural molecular modeling of paratope combinations for preferential inter-target binding combined with protein engineering for manufacturability yielded deep insights into the capabilities of rational and library approaches. The methodologies of in silico hydrophobicity identification and sequence optimization could serve as a blueprint for rapid development of optimal biparatopic ADCs targeting further tumor-associated antigens in the future.

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.

Antibody-Secreting Cell Isolation from Different Species for Microfluidic Antibody Hit Discovery.

The recent advent of microfluidic-assisted antibody hit discovery as standard methodology accelerated pharmaceutical research. While work on compatible recombinant antibody library approaches is ongoing, the major source of antibody-secreting cells (ASCs) remains to be primary B cells of mostly rodent origin. As fainting viability and secretion rates can lead to false-negative screening results, careful preparation of these cells is an essential prerequisite for successful hit discovery. We here describe procedures to enrich plasma cells from relevant tissues of mice and rats and plasmablasts from human blood donations. Although freshly prepared ASCs yield the most robust results, suitable freezing and thawing protocols to preserve the viability and antibody secretory function can circumvent extensive process time and allow transferring of samples between laboratories. An optimized procedure is described to yield similar secretion rates after prolonged storage when compared to freshly prepared cells. Finally, the identification of ASC-containing samples can increase the probability of success of droplet-based microfluidics-two methods for pre- or in-droplet staining are described. In summary, the preparative methods described herein can facilitate robust and successful microfluidic antibody hit discovery.

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.

Versatile and rapid microfluidics-assisted antibody discovery.

Recent years have seen unparalleled development of microfluidic applications for antibody discovery in both academic and pharmaceutical research. Microfluidics can support native chain-paired library generation as well as direct screening of antibody secreting cells obtained by rodent immunization or from the human peripheral blood. While broad diversities of neutralizing antibodies against infectious diseases such as HIV, Ebola, or COVID-19 have been identified from convalescent individuals, microfluidics can expedite therapeutic antibody discovery for cancer or immunological disease indications. In this study, a commercially available microfluidic device, Cyto-Mine, was used for the rapid identification of natively paired antibodies from rodents or human donors screened for specific binding to recombinant antigens, for direct screening with cells expressing the target of interest, and, to our knowledge for the first time, for direct broad functional IgG antibody screening in droplets. The process time from cell preparation to confirmed recombinant antibodies was four weeks. Application of this or similar microfluidic devices and methodologies can accelerate and enhance pharmaceutical antibody hit discovery.

A Streamlined Approach for the Construction of Large Yeast Surface Display Fab Antibody Libraries.

Yeast surface display is a versatile platform technology for antibody discovery. Nevertheless, the construction of antibody Fab libraries typically is a tedious multistep process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. Here, we present a focused one-step Golden Gate cloning approach for the generation of yeast surface display Fab libraries that allows for simultaneous introduction of heavy-chain and light-chain variable regions into one single display vector. Thereby, the overall time as well as the materials needed for library generation can be reduced significantly.

A novel one-step approach for the construction of yeast surface display Fab antibody libraries.

BACKGROUND: Yeast surface display (YSD) has proven to be a versatile platform technology for antibody discovery. However, the construction of antibody Fab libraries typically is a tedious three-step process that involves the generation of heavy chain as well as light chain display plasmids in different haploid yeast strains followed by yeast mating. RESULTS: Within this study, we aimed at implementing a focused Golden Gate Cloning approach for the generation of YSD libraries. For this, antibodies heavy and light chains were encoded on one single plasmid. Fab display on yeast cells was either mediated by a two-directional promoter system (2dir) or by ribosomal skipping (bicis). The general applicability of this methodology was proven by the functional display of a therapeutic antibody. Subsequently, we constructed large antibody libraries with heavy chain diversities derived from CEACAM5 immunized animals in combination with a common light chain. Target-specific antibodies from both display systems were readily obtained after three rounds of fluorescence activated cell sorting. Isolated variants exhibited high affinities in the nanomolar and subnanomolar range as well as appropriate biophysical properties. CONCLUSION: We demonstrated that Golden Gate Cloning appears to be a valid tool for the generation of large yeast surface display antibody Fab libraries. This procedure simplifies the hit discovery process of antibodies from immune repertoires.