Overexpressed tumor associated antigens (e.g., HER2 and epidermal growth factor receptor) are attractive targets for therapeutic T cells, but toxic “off-tumor” cross-reaction with normal tissues expressing low levels of target antigen can occur with Chimeric Antigen Receptor (CAR) T cells. Inspired by natural ultrasensitive response circuits, we engineered a two-step positive feedback circuit that allows T cells to discriminate targets based on a sigmoidal antigen density threshold. In this circuit, a low affinity synthetic Notch receptor for HER2 controls the expression of a high affinity CAR for HER2. Increasing HER2 density thus has cooperative effects on T cells—it both increases CAR expression and activation—leading to a sigmoidal response. T cells with this circuit show sharp discrimination between target cells expressing normal amounts of HER2 and cancer cells expressing 100-fold more HER2, both in vitro and in vivo.

Transcription from bacteriophage T4 middle promoters uses Escherichia coli RNA polymerase together with the T4 transcriptional activator MotA and the T4 coactivator AsiA. AsiA binds tightly within the C-terminal portion of the 70 subunit of RNA polymerase, while MotA binds to the 9-bp MotA box motif, which is centered at ؊30, and also interacts with 70 A programmed cascade of transcriptional events is initiated when bacteriophage T4 infects its host Escherichia coli (reviewed in reference 57). T4 early genes are transcribed immediately after infection by using the existing host RNA polymerase holoenzyme comprising the core (␣ 2 ␤␤Ј) and the 70 subunit. Early T4 promoters do not require T4-encoded transcription factors, since they contain excellent matches to the ideal 70 sequences in their Ϫ10 and Ϫ35 regions (61; reviewed in reference 60). In contrast, transcription from T4 middle promoters uses two T4 early gene products, the transcriptional activator MotA and the coactivator AsiA (23,39,43,44; reviewed in reference 57). Late promoter utilization requires the replacement of 70 by the T4 sigma factor, gp55, as well as other phage-encoded activators and coactivators (reviewed in reference 62).The MotA protein binds as a monomer (6, 33) to a 9-bp element (MotA box) centered at position Ϫ30 of middle promoter DNA (3,19,23). In addition, MotA forms a complex with 70 (18). Nuclear magnetic resonance and crystallographic studies indicate that the 211 amino acids of MotA are organized into an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a small flexible linker (15,33,34 (44,55,56). Binding sites for AsiA have been mapped within C-terminal amino acids (regions 4.1 and 4.2) of 70 (8,49,50,53,59). Residues within region 4.2 normally contact the Ϫ35 element of host promoter DNA (5,9,17,29,54). In the absence of MotA, AsiA binding to 70 inhibits transcription by polymerase from promoters that require recognition of the Ϫ35 canonical sequences (8,45,51), suggesting that the presence of AsiA inhibits the 70 region 4.2-DNA interaction. In this paper we show that the interaction of a MotA Nterminal peptide (amino acids 1 to 97) with 70 , like the interaction of AsiA with 70 , involves the C-terminal region of 70 . In addition, deletions of the amino acids within the far-Cterminal region of 70 (amino acids 604 to 613) impair the ability of RNA polymerase to perform MotA-dependent activation in vitro. We also show that a MotA C-terminal peptide, beginning at amino acid 102, binds DNA with an apparent dissociation constant like that of wild-type MotA. Our results support a model for MotA-dependent activation in which the interaction between the DNA-bound MotA and the C-terminal region of 70 helps to substitute functionally for an interaction between 70 and a promoter Ϫ35 element. MATERIALS AND METHODSStrains. E. coli KS1 (12) contains a chromosomal lacZ reporter gene under the control of a derivative of the lac promoter P lac that carries a lambda operator (O R 2) centered at position Ϫ62 in place of the ...

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