Minibinders adapt readily to next-generation synthetic proteolytic receptors.

A) Schematic representation of a synthetic intramembrane proteolysis receptor (SNIPR) that enables customized genetic responses upon binding a target. LCB1 binder structure cartoon loosely based on PDB 7JZU. B) Assay for testing SNIPR function, whereby SNIPRs with various minibinder antigen sensors are expressed in Jurkat cells and cells are mixed with SARS- CoV-2 Spike-expressing K562 cells. SNIPR activation is readout as BFP expression. C) Histograms of BFP expression in SNIPR-expressing Jurkats after 72-hour incubation with K562s or K562s expressing Spike.

Minibinders perform similarly across different synthetic proteolytic receptors.

A) Expression of surface-displayed Spike protein (as assessed by anti-Myc labeling of an N-terminal extracellular Myc tag) for untransduced K562s and K562 lines sorted for moderate (M) and high (H) expression of Spike (median fluorescent counts for populations across 3 replicates). B) Surface expression levels of multiple minibinder-coupled synthetic receptors on Jurkat cells as assessed by anti-Myc labeling of an N-terminal extracellular Myc tag on each receptor (median fluorescent counts for populations across 3 replicates). C) Histograms describing BFP expression in minibinder receptor-expressing Jurkats after 72 hours of incubation with K562s with 3 different levels of Spike expression (pooled populations from 3 replicates). D) Mean log BFP expression in the activated populations of Jurkats after 72 hour incubation with Spike-K562 (H) cells as calculated by the estimated mean of the high BFP expression population after fitting a two component Gaussian mixture model on each of 3 replicates per condition. Data points are estimated mean of ‘on’ population in each replicate. E) The fraction of BFP-expressing Jurkats in the mixture model-defined activated population for each receptor as a function of Spike expression. All error bars (including E) are standard deviation.

Novel synthetic proteolytic receptors enable minibinder-dependent detection of live SARS-CoV-2 virus.

A) Schematic of live virus sensing experiment. Minibinder receptor-expressing Jurkats were incubated with live SARS-CoV-2 virus for 72 hours, and then fixed with paraformaldehyde and assessed for BFP expression as a sign of receptor activation. B) Median BFP expression in Jurkat cells exposed to no virus or WA1 or Delta isolates of SARS-CoV-2 at a multiplicity of infection of 1. C) Fraction of Jurkat population expressing BFP as a function of MOI of SARS-CoV-2 that cells were exposed to. All error bars are standard deviation.

Minibinders enable chimeric antigen receptor-mediated killing of SARS-CoV-2 Spike-expressing cells.

A) Schematic of a chimeric antigen receptor with 4-1BB costimulatory domain using the minibinder LCB1 as its antigen sensor. B) Assay schematic indicating that primary CD8+ human T cells expressing the LCB1-CAR were mixed with Spike-expressing K562s for 72 hours and then were assessed for proliferation, T cell activation, and killing of the target cells. C) Proliferation of T cells as a function of which target cells they were incubated with. Histogram showing Cell Trace dye fluorescence across a population of T cells, with homogenous high staining indicating no proliferation and multiple populations indicating proliferation. D) T cell activation as assessed by CD25 antibody-labeling as a function of which targets T cells were exposed to (median fluorescent for population in each of 3 replicates). E) The fraction of target cell populations that were lysed after T cell incubation as a function of the ratio of effector T cells to K562 targets that were incubated together. All error bars are standard deviation.

Short linker sequences optimize minibinder-coupled receptor function.

A) The fraction of K562s expressing no ligand, CD19, or Spike that were lysed after 72 hour incubation with LCB1-CAR, LCB3-CAR, or the benchmark CD19-CAR at a 1:1 effector:target ratio. B) Schematic describing 4 different linkers that were evaluated to explore the effect of linker length and rigidity on minibinder receptor function. Fibcon linker structure cartoon loosely based on PDB 3TEU. C) The fraction of K562 of Spike-K562 targets lysed after 24 hour incubation with CD8+ T cells expressing 4 variants of the LCB1-CAR at a 1:1 effector:target ratio. D) The percent of proteolytic receptor-expressing cells that were expressing BFP after 72 hour incubation with K562 or Spike-K562 (M) target cells for 4 different linker variations. All error bars are standard deviation.