Escape tag

In the unlikely case that our therapeutic cells escape from the capsules and to prevent their dissemination through the body we designed a safety mechanism that would enhance the recognition and destruction of the escaped therapeutic cells by the innate immune system.

We introduced an escape tag that labels the cells with a surface protein that alerts natural killer cells of the host organism to recognize and destroy cells.

HEK293 cells expressing MICA protein were efficiently killed by human NK cells.

We considered several variants to ensure destruction of escaped cells such as:

1. quorum sensing which would provide the survival signal only for cell clusters within microcapsules,
2. conjugation of microcapsule material with a ligand for a receptor that provides a survival signal,
3. activation of apoptosis for escaped cells when they encounter the extracellular matrix and
4. tagging cells for the recognition and destruction by the cells of the immune system.

After considering many factors we selected the immuno-tagging variant because of its elegant simplicity and since it seems less sensitive to the spontaneous loss of the constructs and leaky apoptosis than other options.

The MICA/NKG2D system

Though our microencapsulation system is designed in such a way that the capsules are permeable only to nutrients, signalling molecules and produced protein therapeutics, we cannot completely exclude the possibility that some cells could escape from the microcapsules, e.g. due to mechanical damage. We designed a safety mechanism that would ensure that the escaped therapeutic cells could not survive outside microcapsules because of enhanced recognition by the innate immune system.

Innate immune cells, such as natural killer cells (NK cells) and the γδT cells, patrol the tissues of the organism and recognize foreign, infected or damaged cells and destroy them without inflicting dammage to the surrounding tissue. One of the proteins that marks cells for destruction is MICA, a distant homolog of the major histocompatibility complex class I. It has been demonstrated that some cancer cells downregulate MICA to escape recognition (Salih et al., 2002) and that the overexpression of this surface protein marks them for destruction (Groh et al.,1999). MICA is recognized by the activating receptor NKG2D, which is expressed on most natural killer cells, CD8+ T cells, and γδT cells (Bauer et al., 1999), and recognition activates the cytolytic response of NK cells (Steinle et al., 2001) (Figure 1).

Our cells should therefore express an abundant amount of MICA protein (equivalent role could also be fufilled by MICB), which has strong affinity for the NKG2D receptor expressed by NK cells that would recognize and induce apoptosis of the escaped therapeutic cells.

Figure 1. The principle of our "Escape-tag" safety mechanism.

Results

Cells have to express MICA on their surface constitutively since we don't know when a cell might escape. We therefore put MICA under the control of the constitutive promoter. We transfected HEK293T cells with DNA encoding MICA and BFP and incubated them with different ratios (1:1, 1:5, 1:10) of NK-92 cells. Flow cytometry analysis allowed us to quantify the efficiency of recognition and killing of HEK293T expressing MICA escape tag by NK-92 cells.

Figure 2. The efficiency of NK-92 mediated cell killing. HEK293T cells were transfected with MICA tag. HEK293T cells and NK target cells K562, which were used as a positive control, were stained with CFSE. Target cells (HEK293T and K562) were mixed with NK-92 cells and incubated for 4 h. Cells were then stained with propidium iodide and flow cytometry was used to determine the percentage of specific lysis.

Results demonstrate that HEK293T cells tagged with MICA are destroyed as efficiently as the standard target cells, which are used as a control for the killing assay (Figure 2). This demonstrates that the escape tag is appropriate as an additional safety mechanism to prevent the spreading of the therapeutic cells through the body. The removal of foregin cells would probably be even more efficient in vivo, since the immune system is comprised of several different immune cell types, apart from NK cells that are capable of recognizing and destructing foreign cells. However for the real therapeutic application, in vivo tests for the detection of any remaining cells will be neccessary.

References

Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., Spies, T. (1999) Activation of NK Cells and T Cells by NKG2D, a receptor for Stress-Inducible MICA. Science 285, 727-729.

Borrego, F., Kabat, J., Kim, D.K., Lieto, L., Maasho, K., Peña, J., Solana, R., Coligan J.E. (2001) Structure and function of major histocompatibility complex (MHC) class I specific receptors expressed on human natural killer (NK) cells. Mol. Immunol. 38, 637-660.

Groh, V., Rhinehart, R., Secrist, H., Bauer., S., Grabstein, K.H., Spies, T. (1999) Broad tumor-associated expression and recognition by tumor-derived gd T cells of MICA and MICB. Proc. Natl. Acad. Sci. 96, 6879–6884.

Salih, H.R., Rammensee, H.G., Steinle, A. (2002) Cutting Edge: Down-Regulation of MICA on Human Tumors by Proteolytic Shedding. J Immunol. 169, 4098-4102.

Stenile, A., Li, P., Morris, D.L., Groh, V., Lanier, L.L., Strong, R.K., Spies, T. (2001) Interactions of human NKG2D with its ligands MICA, MICB, and homologs of the mouse RAE-1 protein family. Immunogenet. 53, 279-287.