Rare, high-affinity anti-pathogen antibodies from human repertoires, discovered using microfluidics and molecular genomics

Abstract

Affinity-matured, functional anti-pathogen antibodies are present at low frequencies in natural human repertoires. These antibodies are often excellent candidates for therapeutic monoclonal antibodies. However, mining natural human antibody repertoires is a challenge. In this study, we demonstrate a new method that uses microfluidics, yeast display, and deep sequencing to identify 247 natively paired anti-pathogen single-chain variable fragments (scFvs), which were initially as rare as 1 in 100,000 in the human repertoires. Influenza A vaccination increased the frequency of influenza A antigen-binding scFv within the peripheral B cell repertoire from <0.1% in non-vaccinated donors to 0.3-0.4% in vaccinated donors, whereas pneumococcus vaccination did not increase the frequency of antigen-binding scFv. However, the pneumococcus scFv binders from the vaccinated library had higher heavy and light chain Replacement/Silent mutation (R/S) ratios, a measure of affinity maturation, than the pneumococcus binders from the corresponding non-vaccinated library. Thus, pneumococcus vaccination may increase the frequency of affinity-matured antibodies in human repertoires. We synthesized 10 anti-influenza A and nine anti-pneumococcus full-length antibodies that were highly abundant among antigen-binding scFv. All 10 anti-influenza A antibodies bound the appropriate antigen at KD<10 nM and neutralized virus in cellular assays. All nine anti-pneumococcus full-length antibodies bound at least one polysaccharide serotype, and 71% of the anti-pneumococcus antibodies that we tested were functional in cell killing assays. Our approach has future application in a variety of fields, including the development of therapeutic antibodies for emerging viral diseases, autoimmune disorders, and cancer.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

“…43 In recent years, novel single-cell methodologies based on droplet microfluidics have been adapted to screen naturally paired V L and V H genes by yeast display. [44][45][46] A similar approach of cloning natural V H :V L pairs into our synthetic antibody HDR scaffold would offer an attractive way to functionally screen antibody repertoires in mammalian cells.…”

Antibody repertoire analysis of mouse immunization protocols using microfluidics and molecular genomics

“…Using our previously described high-throughput microfluidic antibody discovery platform, 12,18,19 B cells isolated from each tissue (>2 million total cells per immunization method; Supplementary Table S1) were encapsulated into droplets such that each droplet contained a single B cell ( Figure 1b). The heavy and light (kappa only) chain sequences from each cell were amplified within droplets to generate an scFv sequence that retains the proper heavy:light Ig pairing of the original B cell.…”

“…Clonal cluster analysis 18,19 reveals clonal lineages of similar antibody sequences, generated by processes such as affinity maturation and selection in vivo. Though our libraries were derived from different mice, and it is therefore impossible to distinguish clonal clusters from similar clones arising in different mice, clonal cluster analysis is still useful for visualizing similar sequences across different immunization methods and tissues.…”

“…Paired heavy and light chain libraries were generated as previously reported. 12,18 During the development of this method, several spike-in experiments with known antibody sequences were performed that indicated proper antibody pairing was maintained; however, there is always the possibility that a minority of sequences could still have improper pairing.…”

Ion channels as therapeutic antibody targets

“…Of course, the latter approach will be dependent on the viability of the knock-out animal. With regards to mining the immune repertoire, a number of platform technologies, e.g., direct B-cell cloning and/or deep sequencing, [97][98][99] have been developed recently that increase the probability of identifying rare antibodies and avoid standard hybridoma-based technologies where valuable antibodies may be missed owing to inefficient fusion events or the loss of rare B cell clones. 100 Following the identification of antibodies that specifically recognize the ion channel target, it is important to select extracellular binders by some means to advance these clones into functional characterization.…”