T Cell Stimulation and Expansion

As T cell stimulation in vitro requires the mimicking of both natural signals, most of the contemporary activating reagents rely on multivalent anti-CD3 and anti-CD28 antibodies. However, the activation still poses the need for a precise modulation of the T cell surface interactions as well as the signal duration and intensities to meet a sufficient signal threshold, otherwise the cells can become anergic or even degraded. This modulation is frequently realized by immobilization of at least one of the antibodies on either plate- or bead-based solid supports. In doing so, a favorable microenvironment for the clustering of TCRs and the corresponding co-receptors can be achieved. Nonetheless, approaches based on culture plates rather result in an inefficient use of space by formation of antibody monolayers. Another inconvenience arises by the fact that prolonged activation can cause down-regulation or even auto-inhibition, leading to suboptimal yields. This is especially a disadvantage in widely used magnetic bead-based approaches, where it is often neither possible to remove the particles completely, nor at a distinct point of time. These circumstances are further complicated by tedious assessment of other modulation factors, such as culture density, medium composition, bead-to-cell ratio and debeading time. 

Our CD3/CD28 Streptamer® principle is based on our Strep-tag® technology and offers an alternative T cell activation and expansion approach. In this method, anti-CD3 and anti-CD28 Fab fragments fused to a Twin-Strep-tag® (Fab-Streps) are immobilized on Strep-Tactin® multimer backbones to generate soluble polymeric protein complexes (CD3/CD28 Streptamers). Consequently, the regular drawbacks of solid supports are overcome, and a defined and consistent activation signal is induced. In addition, the 3D structure provides an increased surface area for interactions. Moreover, simply adding excessive innocuous biotin leads to quick dissociation of the stimulatory reagents from the activated cells, which does not only result in an instantaneous signal termination, but also delivers viable and unmanipulated targets. It is flexibly usable from small scale experiments to high-throughput screens and further provides an unparalleled level of modulation options and reproducibility as well as intensity- and time-resolution.