Figure 2
Validation of the anti-CD2 mAb GGTA1 targeting construct. (A) Top, GGTA1 genomic structure. Middle, knock-in backbone showing 5′ and 3′ homology arms (5′-HA and 3′-HA), neomycin resistance cassette (NeoR), multiple cloning site (MCS), and polyadenylation signal (pA). Into this was cloned (bottom) the CMV immediate early enhancer (CMV IE), mouse H-2Kb promoter, intron, and coding regions for the heavy and light chains of anti-CD2 mAb diliximab (αCD2-HC and αCD2-LC) linked by a furin cleavage site-F2A ribosome skip signal (f2A). The isotype of diliximab is human IgG3. (B) Detection of anti-CD2 mAb diliximab secreted by stably transfected COS-7 cells. Human leukocytes were incubated with culture supernatant, and mAb binding to CD3+ T cells was detected with anti-human IgG3. Red line, supernatant (S/N) from COS-7 cells transfected with the anti-CD2 knock-in construct; blue line, positive control (62.5 ng/ml purified diliximab); black line, supernatant from vector-transfected cells.
