Figure 1: Compact geometry improves TCE activity against targets with low antigen density in vitro.
(A) TRP2/H2-Kb antigen expression on different tumor cell lines: B16F10 were either left untreated or incubated with 100 ng/mL IFNγ and/or 1 μM TRP2 peptide (SVYDFFVWL) for 24 hours and stained with 100 nM TRP2 TCE or no antibody followed by secondary staining with an anti-mouse IgG2a antibody. Shown is the difference between TRP2 TCE-stained and secondary antibody only for each cell line and condition as indicated. The same staining was performed for MC38 cells transduced to stably express different levels TRP2.
(B) Representation of the designed TCE variants: a full-length IgG2a-based TCE (left) with the retained flexible hinge domain, and a compact diabody TCE (right), also including the CH2 and CH3 domains of IgG2a. The approximate distances between the CD3 binding paratope and target binding paratope are indicated. To prevent Fc-mediated effector functions, all TCEs carried the D265A mutation.
(C) Polyacrylamide gel-electrophoresis of TRP2 Db and full-length IgG2a TCE variants under non-reducing (left) and reducing conditions (right).
(D) Effector T cells from C57BL/6 mice were co-cultured with the indicated target cells (Effector-to-target ratio, 1:1) for 36 hours in the presence of TRP2 diabody TCE at the indicated concentration. B16F10 cells were pretreated with IFNγ and/or TRP2 peptide as described in (A) prior to T cell addition. T cell activation was determined by the fraction of CD8+ T cells expressing CD137.
(E) T cell activation as measured by CD137 upregulation in the presence of TRP2 TCE (full length IgG2a). Co-culture conditions are identical to those described in (D).
(F) Cytotoxicity (specific lysis) of OT-I effector T cells (Effector-to-target ratio, 1:1) against the indicated cell lines was measured after 36 h of coculture. B16F10 cells were pretreated with IFNγ and/or TRP2 peptide as described in (A) prior to T cell addition.
(G) Cytotoxicity (specific lysis) after 36 h induced by DLL3 TCE against murine SCLC cell lines with different antigen expression levels.
All values represent mean ± s.e.m. of intra-assay duplicates unless otherwise specified. In D and F one outlier was removed for cell lines MC38low and MC38hi at a 16 nM concentration due to pipetting error. See also Supplemental Figure S1.