HIGH-AFFINITY MONOCLONAL T-CELL RECEPTOR (mTCR) FUSIONS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 483

FIGURE 32.6 ImmTAC-MAGE displays high potency despite low ligand densities. Top: The
expression of MHC class I on A375 cells. Flow cytometry shows only a low level of HLA-A1
expression (red), against an isotype control (blue). Bottom: Visualization of cell killing by CD8 þ T
cells in the presence of ImmTAC-MAGE. Time-lapse fluorescence microscopy over a period of 10 h
shows progressive lysis of the MAGE-A3 positive A375 tumor cells (large red-stained cells) by the
CD8 þ T cells (small unstained cells, indicated by the black arrow) in the presence of ImmTAC-
MAGE at 50 pM concentration. While the population of the A375 cells disappears entirely by the end
of the observation period, the control MAGE-A3 negative cells (Colo205, large green-cells) remain
intact. (No effect was registered in the presence of an irrelevant ImmTAC—data not shown.)

32.6 , top). Yet in the presence of 50pM ImmTAC-MAGE,
the cancer cells were targeted and killed by purified CD8

molecule (peptide-MHC). This path of antigen presentation
allows TCRs to detect the presence of intracellular proteins,
which are largely inaccessible to antibodies.
The mTCR and ImmTAC technologies, which have
their roots in research carried out at Oxford University
and have been developed by Immunocore Ltd, have dem-
onstrated that it is possible to target cancer cells with
precious few antigens per cell. These types of cells will
normally escape immune detection. However, ImmTACs
redirect T cells with good efficacy when presented with as
little as 10 targets per cell.
While naturally selected TCRs exhibit only micromolar
affinities toward their peptide-MHC ligands, mTCRs have

þ
CTLs over the course of 10 h (Figure 32.6, bottom), while
the HLA-A1 þ MAGE-A3 negative cells

(Colo205),

remained unaffected.

32.5 CONCLUSIONS AND FUTURE
PERSPECTIVES

T-cell receptors (TCRs) recognize cell surface-presented anti-
gens; each consisting of an endogenously processed peptide
carried in the context of a major histocompatibility complex

3

FIGURE 32.5 (continued) specific for the HIV gag epitope was used as a negative control.
(B) Biological activity of ImmTAC-NY-ESO-1. Purified CD8 þ T cells were incubated overnight
with IM9 cells and increasing concentrations of ImmTAC-NY-ESO-1. Dose-dependent IFN- g release
(demonstrating T-cell activation, left) and IM9 specific lysis (right) was observed. While significant
levels of T-cell activation were observed in both assays at NY-ESO-ImmTAC concentrations as low
as 10pM (top line), the addition of free (non-CD3-fused) high-affinity NY-ESO mTCR abrogated the
effect (middle line, left), demonstrating the specificity of the interaction. A control cell line
expressing HLA-A2 and lacking NY-ESO-1 expression was unaffected by the addition of the
full titration range of ImmTAC (bottom line, left) or the top concentration of 10nM protein (closed
circle, right). Further controls include CD8 þ T cells with 10nM ImmTAC-NY-ESO-1 (open square,
left), CD8 þ T cells with IM9 cells (open diamond, left) and IM9 cells with 10nM ImmTAC-NY-
ESO-1 (open circle, left). (C) Affinity-driven potency of ImmTAC-NY-ESO. ImmTACs were made
with several NY-ESO mTCRs, ranging in their peptide: MHC-binding affinities from 1.1 to 215 nM
(top). The IFN- g release assays (bottom), shows that enhancing mTCR affinity enhances cellular
potency as demonstrated by increased T-cell activation.