High throughput screen for the improvement of inducible promoters for tumor microenvironment cues

Abstract

Cancer immunotherapies are highly potent and are gaining wide clinical usage. However, severe side effects require focusing effector immune cell activities on the tumor microenvironment (TME). We recently developed a chimeric antigen receptor tumor-induced vector (CARTIV), a synthetic promoter activated by TME factors. To improve CARTIV functions including background, activation levels, and synergism, we screened a library of promoters with variations in key positions. Here, we present a screening method involving turning ON/OFF stimulating TNFα and IFNγ cytokines, followed by sequential cell sorting. Sequencing of enriched promoters identified seventeen candidates, which were cloned and whose activities were then validated, leading to the identification of two CARTIVs with lower background and higher induction. We further combined a third hypoxia element with the two-factor CARTIV, demonstrating additional modular improvement. Our study presents a method of fine-tuning synthetic promoters for desired immunotherapy needs.

The Contribution of the Minimal Promoter Element to the Activity of Synthetic Promoters Mediating CAR Expression in the Tumor Microenvironment

“…Being short sequences (60-25 bp), these are very easy to work with: simply order an oligo with the sequence of interest and flanking restriction-sites, or any other mode of cloning, such as gateway [32] or Gibson Assembly ® [33]. Moreover, the ability to work with a relatively short oligo opens an intriguing option to further refine such minimal promoters, just as we recently did with our CARTIV promoters [14]. Considering the known core transcription-initiating complex, and the structure of the protein-DNA complex, one may pick a few key nucleotides and change them to gain variations over the basic minimal promoter.…”

“…The minimal promoter, or core promoter, is commonly a short sequence that allows the formation of a transcription initiation complex right at the transcription start site. We had previously developed CARTIV promoters [14,15] focusing on the PRE portion to gain response to common TME factors. We sought to further adjust expression levels by switching the minimal promoter portion.…”

“…We cloned either YB_TATA, miniTK, or miniCMV right after the G1K0.6H1 part of our recently published CARTIV [14,15], getting each minimal promoter with the very same PRE (Figure 1A,B). Our constructs drive either a reporter florescent RFP670 or a third generation Herceptin-based CAR [28].…”

“…A different safety approach is limiting the CAR expression to the tumor site. The CARTIV 2 of 12 method showed that it is possible to restrict CAR expression to the tumor by inducible promoters specific to the tumor microenvironment (TME) [14,15].…”

“…The PREs can be customized to respond to specific TME signals, such as inflammatory signals or the tumor's hypoxic environment. The second main component is the minimal promoter [14]. The minimal promoter, or core promoter, is a short sequence that allows for the formation of the initiation complex.…”