Journal: PLOS ONE

Article Title: A simplified function-first method for the discovery and optimization of bispecific immune engaging antibodies

doi: 10.1371/journal.pone.0273884

Figure Lengend Snippet: Novel monoclonal antibodies were generated via mouse immunization and hybridoma screening. Four monoclonal antibodies are shown which have high reactivity to (A) Jurkat T cells and (B) primary human T cells. Following this, antibody heavy and light chains were sequenced. (C) scFv DNA was then synthesized and assembled into CD19 or EGFRvIII BiTE molecules using single-pot restriction ligation with Esp3I restriction enzyme. (D) Ten unique CD19 or EGFRvIII BiTE plasmids were then transfected into HEK293T cells and supernatants were tested using Jurkat cells in 1:1 co-cultures with EGFR-vIII+ U87vIII cells ( left ) or CD19+ Raji cells ( right ) using varying doses of BiTE supernatant as shown. (E) Four BiTE plasmids found to produce active BiTE molecules were tested again against EGFRvIII+ U87-vIII cells, EGFRvIII- U87-WT cells, or CD19+ Raji cells. (F) BiTE supernatants for CD19 or EGFRvIII targeted molecules incorporating the 1E2 CD3-scFv were added to co-cultures with 10 000 primary human T cells and 2000 target cells. Graphs show the relative fluorescent signal of red-fluorescent target cells over 5 days in co-culture. Results are derived from a single experiment, but are representative of at least 3 repeated observations.

Article Snippet: Swapping CD3-targeting domains was performed similarly as described above using Esp3I enzyme to integrate synthetic CD3-scFv fragments (Twist Biosciences, USA), derived from in-house generated CD3-specific murine monoclonal antibodies, into the pBiTE construct.

Techniques: Bioprocessing, Generated, Synthesized, Ligation, Transfection, Co-Culture Assay, Derivative Assay