Background Despite demonstrated efficiency in antibody generation, classical immunization strategies and subsequent hybridoma generation often face strong limitations when it comes to complex targets like GPCRs or tetraspanins. Using WT, KO or Alloy therapeutics ATX-Gx™ humanized transgenic mice, we have developed innovative approaches combining mRNA immunization and Bruker Beacon® single cell screening platform to provide unique opportunities to dramatically speed up antibody discovery against such challenging targets.

Methods WT BALB/c or humanized Alloy BALB/c mice (5 mice per immunization group) were immunized with either standard protocol using recombinant proteins and adjuvant or formulated mRNA encoding same extracellular domain protein. In a separate experiment, BALB/c mice were immunized with full length formulated mRNA encoding a 7 transmembrane domain GPCR.

Splenocytes and Bone marrow cells were recovered after a final immunizing boost, plasma cells were purified based on CD138 expression , and single cell analysis was performed either on recombinant antigen coated beads or on transfected cell expressing immunogen on the Beacon platform.

Individual plasma cells demonstrating antigen recognition were exported, and VH/VL sequences were recovered by single cell RT-PCR. VH/VL pairs were cloned, produced and purified as recombinant mAbs and antigen recognition was characterized on transfected cells.

Results For a model antigen where recombinant protein was available, mRNA immunization led to greater percentage of positive plasma cells (number of positive wells divided by total number of single cell wells) detected on the machine (1,86% versus 0,6%) coming from both spleen and bone marrow. Parallel immunization of Alloy mice with recombinant protein led to similar numbers of positive plasma cells, with high diversity of human VH and VL domain, compared to WT mice.

For a GPCR antigen, where multiple hybridoma campaigns failed to provide collection of antigen-specific mAbs, mRNA immunization followed by Beacon process provided >26 specific recombinant mAbs with EC₅₀ in the nanomolar range or lower, and high sequence and functional diversity (crossreactivity on cynomolgus antigen, and 2 ligand blocking mAbs).

Conclusions Combining mRNA immunization, Beacon single cell technology, and the possibility to use ATX-Gx™ humanized mice, time to therapeutic candidate antibody delivery can now be significantly shortened for regular and difficult antigens, where no recombinant well folded immunogen is available.

Ethics Approval This study involving mice experiments has obtained ethics approval in France (N° APAFIS#28553-2020112314486902 v2).