Modular design of synthetic receptors for programmed gene regulation in cell therapies

Abstract

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.

Keywords: CAR-T cells; cancer immunotherapy; cell therapy; synNotch; synthetic biology.

Figure 1.. Design of synthetic RIP receptors for customized antigen-dependent gene regulation in therapeutic cells.

(A) Design of Synthetic Intramembrane Proteolysis Receptors. Receptors are comprised of a ligand binding domain (LBD), an extracellular domain (ECD), a transmembrane domain (TMD), a juxtamembrane domain (JMD), and a transcription factor (TF). Receptor circuits are designed to maximize clinical translation potential (B). A synRobo receptor replaces the Notch1 core with one from human Robo1. Compared to synNotch, a synRobo receptor fails to induce BFP. By replacing the TMD and JMD of Robo1 with those of Notch1, control of BFP production is lost. Deletion of a known ADAM10 cleavage site in the Robo1 ECD rescues ligand-dependent receptor behavior. (C) Same as B, but with minimal SNIPRs constructed using simple (GGS)_n ECDs, and the TMD/JMD from Notch1. Statistics were calculated using unpaired T-tests, ***P≤0.001.

Figure 2.. The ECD module defines activation triggers and diversifies sensor functions.

(A) SNIPR ECDs with exposed cleavage sites display ligand-independent signaling. Deleting the NRR from synNotch produces a receptor that is sensitive to both ligand and TCR stimulation. A variety of hinge domains utilized in CARs also demonstrate ligand-dependent signaling. (B) Same as A, but with two methods of T cell stimulation. A SNIPR with the Notch1 NRR core domain displays enhanced activation with a Bi-specific T cell Engager (BiTE) targeting a K562 antigen, and a co-expressed second-generation CAR targeting a separate antigen. A SNIPR with a truncated Notch1 NRR activates with these stimuli independent of the presence of ligand. (C) Same as A, but with variations of the CD8α Hinge. The CD8α Hinge can be optimized to enhance SNIPR expression and activation. Statistics were calculated using unpaired T-tests, ***P≤0.001.

Figure 3.. Transmembrane and juxtamembrane domain libraries enable modular assembly of SNIPR architectures.

(A) To identify functional receptor TMDs and JMDs for modular assembly, 88 TMDs and 76 JMDs were cloned into a human synNotch scaffold, replacing either the Notch1 TMD or JMD, respectively. Jurkat T cells expressing an inducible BFP reporter were transduced with these SNIPR libraries in an arrayed format. (B) Normalized results of TMD screening in Jurkat T cells. An alignment of the best performing TMDs shows a common Gly-Val motif (Dark blue = >80% agreement with consensus sequence, blue = >60% agreement, light blue = >40% agreement). An alanine scan of the human Notch1 TMD in primary T cells supports the importance of this motif. (C) Same as B, but with the JMD library. High-performing JMDs are strongly basic at their N-termini and may include polar residues but not acidic or hydrophobic residues. (D) Compared to a reference SNIPR containing the Notch1 TMD/JMD, a SNIPR containing the CLSTN2 TMD/JMD is inactive, but receptor function is restored when the CLSTN2 JMD is replaced with the Notch1, AGER or PTPRF JMD.

Figure 4.. Enhanced sensitivity and tunable gene regulation through SNIPR engineering.

(A) From analyzing activity of high-performing SNIPR-BFP circuits, the Notch1 TMD was selected for further testing. Three JMDs and two TMD alanine mutants were selected to produce a wide output range. (B) K562 cells transduced with a doxycycline-inducible FLAG-tagged ALPPL2 cassette express ALPPL2 in a dose-dependent manner. (C) CD4+ T cells expressing anti-ALPPL2 SNIPR-MCAM CAR circuits were co-incubated with sender cells for 48 hours and CAR output was measured using a t2a GFP system. (D) Graphical representation of C. (E) Supernatant IL-2 concentration was assayed using ELISA. (F) T cells stained with Cell Trace Violet were co-incubated with irradiated sender cells in media without IL-2 for 9 days. T cell proliferation was measured using flow cytometry.

Figure 5.. Humanization of SNIPRs to reduce immunogenicity potential for cell-based therapies.

(A) Humanized TF and RE construction. (B) Activity of fully humanized SNIPRs. (C) SNIPR receptor scaffold compatibility with humanized TFs. (D) Assessing SNIPR immunogenicity. 9mer peptide sequences for SNIPRs with Gal4-VP64, Pax6, and HNF1A transcription factors were assessed for MHC I immunogenicity. Relative immunogenic potential across receptors was examined by comparing immunogenicity scores in regions derived from non-contiguous human protein sources (highlighted in red dashed boxes). Average scores: Pax6 0.039, HNF1A 0.156, BBz 0.102. (E) HNF1A SNIPR RNA-sequencing analysis. HNF1A SNIPR T cells were induced with target cells for 48 hours and sorted to remove targets for RNA-sequencing analysis. Correlation of transcriptomes against non-SNIPR T cells in two donors show few differences apart from SNIPR circuit components. Pearson correlation coefficients (left panel) were calculated for native transcripts (gray). Differential gene analysis shows few upregulated or downregulated genes compared to control cells following circuit induction (right panel).

Figure 6.. Humanized SNIPR – CAR circuits exhibit precise dual antigen targeting in preclinical in vivo models of solid tumors.

(A) Incucyte live cell imaging showing killing kinetics and specificity of humanized anti-ALPPL2 SNIPR –> anti-HER2 CAR circuits against SK-OV-3 ovarian tumor cells. (B) Quantitation of incucyte assay killing in (A). (C) Humanized SNIPR → CAR circuits clear dual positive ALPPL2+/HER2+ SK-OV-3 tumors in vivo. Statistics calculated using one-way analysis of variance (ANOVA) with Dunnet’s test post hoc comparing anti-HER2 CAR-T cells to circuit T cells (top) and untransduced T cells to anti-HER2 CAR and Circuit T cells (bottom). ***P ≤ 0.001. (D) In vivo assessment of fully human SNIPR circuit activation and trafficking. (E) Quantitation of T cells in the spleen and tumors. (F) Circuit activation of humanized SNIPR circuits in the spleen and tumors. (G) Quantitation of CAR surface expression in (F). Statistics were calculated using Mann-Whitney U-test (E and G), **P ≤ 0.01.

Figure 7.. Design framework for next generation synthetic receptors for custom transcriptional regulation in therapeutic cells.

SNIPRs can be built through design of the receptor ECD, TMD, and JMD. The receptor ECD represents the first regulatory site and affects receptor activation parameters, expression, and stringency for ligand. Several known C-terminal motifs in the receptor TMD, commonly found in the Notch and Calsyntenin families, appear to be important for receptor signaling. Highly basic residues in the receptor JMD are required for signaling, and the choice of JMD can strongly affect receptor expression and output levels. By combining these elements, clinically relevant SNIPRs can be built that utilize fully human proteins and are compact, highly expressed, and regulatable. Our SNIPR design framework opens the possibility to build customized precision cellular therapeutics.