This year, team SYSU-CHINA aims to provide a safer yet affordable CAR T therapy. Taken the shortcomings of the current methods into
consideration, we reasoned that the following criteria is essential in order to develop an effective safe switch:
1.The inhibition is preferred to be confined to transferred T cells population alone.
2.The inhibition is reversible, meaning that the inhibition is achieved without killing the cells and the cells can be
reactivated when drug is removed.
3.The safe switch is universal and is compatible with most of the mainstream, if not all, CAR receptors. It is also preferable that
this safe switch can be used in TCR T cells.
4.The cost for the controlling method is low, without adding additional financial burden on the already expensive therapy.
With the criteria above, we developed a reversible safe switch based on the tet-inducible CMV promoter and U24 protein of Human
Herpesvirus 6. By expressing U24 protein of under the control of tet-ON promoter, we can downregulate CAR molecules on the cell
surface through endosomal recycling inhibition in the presence of doxycycline, an inexpensive drugs already approved by the FDA. This
could potentially be used as a universal add-on for all CAR-Ts and TCR-Ts to ensure safety.
Human herpesvirus 6 (HHV-6) of betaherpesvirus family is ubiquitous among the human population. Although both of its subtypes, HHV-6A
and HHV-6B, may infect and persist in different peripheral blood mononuclear cells population, CD4+ T cells are believed to be their
primary target (Lusso et al., 1988)
. Similar to other viruses, HHV-6 encodes a variety of proteins for immune evasion, one of them is
U24 protein.
U24 is a small (87aa) tail-anchored protein (Sullivan and Coscoy, 2010)
that can downregulate TCR/CD3 complex from the cell surface by
exclusion of CD3 from Rab11-containing recycling endosomes and thus inhibiting TCR complex recycling back to the surface(Sullivan and
Coscoy, 2008). While it was demonstrated later that U24 also downregulate transferrin(Sullivan and Coscoy, 2010), its action is
relatively specific, without affecting the surface level of ICAM-1, MHC class I, ULBP1, ULBP2, CD4 and CD8 (Sullivan and Coscoy,
2008). Since U24 does not colocalized with CD3, it is believed that the downregulation does not rely on the interaction of U24 with
CD3 but instead results from interaction of U24 and the endosomal recycling machinery (Sullivan and Coscoy, 2010). In addition, unlike
proteins from other herpesviruses that downregulate TCRs or B cell receptors (BCRs), U24 does not activate lymphocyte signaling
pathways(Sullivan and Coscoy, 2008). Furthermore, it was demonstrated that U24 can impair T cell activation by antigen presenting
cells(Sullivan and Coscoy, 2008).
In summary, U24 is able to relatively specifically downregulate TCRs from cell surface by interfering with endosomal recycling, a
common pathway, and subsequently impair T cell activation. Considering the similarity of CAR receptors and the endogenous TCR/CD3
complex, we reasoned that U24 may also downregulate CARs from cell surface, and subsequently inhibit CAR T cells activation. In
support with our hypothesis, it has been observed that anti-CD19 CAR also undergoes internalization and rapid recycling (Kalos et al.,
2011). The ITAM motifs in TCR complex, which is believed to mediate TCR recycling (Liu et al., 2000), also presents in CARs.
Furthermore, different surface CAR level can lead to activation signaling with different intensity (Walker et al., 2017).
In addition, the unique mechanism of U24 opens the possibility to construct a universal safe switch, for it targets a common pathway
that affects potentially all CARs and TCRs, and thus effective in all CAR T cells and modified TCR T cells.
With the molecule for inhibition in mind, we next sought for a mechanism to regulate its function.
As a proof of principle, we first considered the Tet-inducible transcription system (Gossen et al., 1995) for it is well-
characterized and the molecule for its induction is tetracycline (tet) or doxycycline (dox), two inexpensive antibiotics already
approved by the FDA. The tet-ON system utilizes the sequence-specific DNA binding property of tet repressor protein (tetR) from
Escherichia coli in the presence of tet or dox, and consists of two parts: The Tet-inducible CMV promoter (tet-ON promoter) and
reverse tetracycline-controlled transactivator (rtTA). The tet-ON promoter consists of tandem tetracycline-responsive elements (TRE)
followed by a minimal CMV promoter. The rtTA protein comprises of reverse-tetR (rtetR, mutant of tetR) and activation domains from
herpes simplex virus VP16. When tet or dox is added, the rtTA binds to TRE, and VP16 domain will recruit factors of RNA polymerase II
to initiate transcription. In the absence of tet or dox, the rtTA detaches from tet-ON promoter, and thus no transcription.
Taken together, our device works as follows:
In a CAR T cell with our device, the rtTA protein is constutively expressed, the U24 protein is expressed under the control of tet-ON
promoter. In the absence of tet or dox, no U24 is expressed and CARs are internalized and recycled without disruption, ensuring
enough CARs on the cell surface to recognize cancer cells. In the presence of dox, however, the rtTA binds to tet-on promoter,
facilitating the expression of U24. U24 in turns inhibiting CAR recycling, resulting in a lower number of CARs on cell surface and
thus inhibit CAR T cells function.
