Designed protein logic to target cells with precise combinations of surface antigens

Abstract

Precise cell targeting is challenging because most mammalian cell types lack a single surface marker that distinguishes them from other cells. A solution would be to target cells using specific combinations of proteins present on their surfaces. In this study, we design colocalization-dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations. These switches activate through a conformational change only when all conditions are met, generating rapid, transcription-independent responses at single-cell resolution within complex cell populations. We implement AND gates to redirect T cell specificity against tumor cells expressing two surface antigens while avoiding off-target recognition of single-antigen cells, and three-input switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo designed proteins can perform computations on the surface of cells, integrating multiple distinct binding interactions into a single output.

Figure 1.. A de novo designed protein switch performs AND logic on the cell surface.

a. The ability to compute logic operations on the surface of cells could increase targeting selectivity, provide flexibility for heterogeneous tissue, and avoid healthy tissue. b. Structure of Cage design used to create Co-LOCKR; the x-ray crystal structure (white, PDB ID: 7JH5) matches the computational design model (green) with 1.1 Å RMSD across all backbone atoms. Cross-sections illustrate asymmetric packing of hydrophobic residues (red square) and an asymmetric hydrogen bond network (blue square). c. Colocalization-dependent protein switches are tuned so that Cage and Key do not interact in solution but strongly interact when colocalized on a surface via targeting domains. d. Flow cytometry discriminates Her2⁺/EGFR⁺ cells in a mixed population of K562 cells expressing Her2-eGFP, EGFR-iRFP, both, or neither. e. An Effector protein is recruited only when Cage and Key are colocalized on the surface of the same cell (‘AND’ logic). f. The mixed population of cells from Fig 1d was incubated with 111 nM Her2-targeted Cage, 111 nM EGFR-targeted Key, and 50 nM Bcl2-AF594. Bcl2 binding was only observed for the K562/Her2/EGFR cells. g. The mixed population of cells from Fig 1d was incubated with a dilution series of Her2-targeted Cage and EGFR-targeted Key, washed, and then incubated with 50 nM Bcl2-AF594 Bcl2 binding is reported relative to K562 cells incubated with 3000 nM Her2-targeted Cage, 3000 nM EGFR-targeted Key, and 50 nM Bcl2-AF594.

Figure 2.. Tuning Co-LOCKR sensitivity.

a. Design model of Co-LOCKR with the Bim functional peptide in yellow. Three buried hydrophobic amino acids were mutated to Ala or Ser to weaken the Cage–Latch affinity, thereby favoring Cage–Key binding. b. Tuned Co-LOCKR variants exhibit greater colocalization-dependent activation than the unmutated parental variant. CL_C_HK_E variants recruiting Bcl2-AF594 were evaluated by flow cytometry using the mixed population of cells from Fig 1d. The data shown represent 12.3 nM CL_C_HK_E (n = 1), and Fig S5c shows the complete dilution series for each variant. c. Confocal microscopy of HEK293T cell lines shows that Co-LOCKR switches recruit Bcl2-AF680 Effector proteins only where Her2 and EGFR are colocalized. Each cell line was incubated with CL_C_HK_E (I269S Cage) and Bcl2-AF680 before imaging. NucBlue is a nuclear stain, eGFP indicates Her2 localization, mCherry indicates EGFR localization, AF680 indicates Bcl2 binding in response to Co-LOCKR activation, and white indicates the intersection of Her2-eGFP and EGFR-mCherry signal Scale bars are 10 μm. Uncropped versions of these images are included in Fig S16a–c. d. Heat map showing the intensity of AF680 signal (Co-LOCKR activation) versus eGFP (Her2) and mCherry (EGFR) pixel intensity. Calculations were based on the uncropped 293T/Her2/EGFR image in Fig S16a.

Figure 3.. Co-LOCKR performs 2- and 3-input logic operations in mixed cell populations.

a. Co-LOCKR was used to recruit Bcl2-AF594 for two populations of K562 cells expressing different combinations of Her2, EGFR, and EpCAM. Marker expression for each cell line and identity of the Cage and Key targeting domains are indicated below each bar plot. Red highlighting indicates the expected magnitude of Bcl2-AF594 signal based on relative antigen expression. b. Schematic of [Her2 AND either EGFR OR EpCAM] logic mechanism. c. [Ag₁ AND either Ag₂ OR Ag₃] logic combinations were used to recruit Bcl2-AF594. d. Schematic of [Her2 AND EpCAM NOT EGFR] logic mechanism. The Decoy acts as a sponge to sequester the Key, thereby preventing Cage activation. e. CL_C_HK_EpD_E was used to recruit Bcl2-AF594. Compared to the simple CL_C_HK_Ep ‘AND’ gate (left), recruitment of Decoy to EGFR-expressing cells reduced activation to near background levels. For all panels, population 1 was [K562/EpCAM^lo, K562/EGFR/EpCAM^lo, K562/EpCAM^lo/Her2, and K562/EGFR/EpCAM^lo/Her2], and population 2 was [K562/EpCAM^lo, K562/EGFR/EpCAM^lo, K562/EpCAM^hi/Her2, and K562/EGFR/EpCAM^hi/Her2]. Error bars represent SEM of 6 independent replicates for K562 and K562/EGFR and 3 independent replicates for all others. Statistics are reported in Table S4.

Figure 4.. Co-LOCKR directs CAR T cell specificity using 2- and 3-input logic operations.

a,d,g. Mean IFN-γ concentration in cell supernatants 24 hours after co-culture of Cage, Key, and K562 cells with CAR T cells. Marker expression for each cell line and identity of the Cage and Key targeting domains are indicated below each bar plot. Red highlighting indicates the expected magnitude of signal based on the target cell’s relative antigen expression. Error bars represent SEM of n = 4 (a) or 3 (d,g) healthy T cell donors. AND/NOT logic is demonstrated with EpCAM^lo target K562 cells because T cell effector function was leaky for EpCAM^hi target cells (see Fig S15a). b,e,h. CAR T cell proliferation in response to [Her2 AND EpCAM] (c), [Her2 AND EGFR OR EpCAM] (e), or [Her2 AND EpCAM NOT EGFR] (h) logic. Bar plots are the percent of T cells that have undergone at least one cell division by 72 hours after co-culture of CAR T cells, Cage, Key, and target K562 cells. Histograms show flow cytometric analysis of CFSE dye dilution gated on CD8⁺ lymphocytes. The data are representative of n = 3 biological replicates with healthy T cell donors. c,f,i. CAR T cell cytotoxicity against mixed populations of target Raji cells expressing combinations of Her2, EpCAM, and EGFR. Line graphs show mean frequency of Raji target cells after 0 or 48 hours of co-culture with CAR T cells. n = 4 (c,f) or 3 (i) healthy donors. Arrows indicate cell lines targeted by Co-LOCKR.