Synthetic biology approaches for improving the specificity and efficacy of cancer immunotherapy

Abstract

Immunotherapy has shown robust efficacy in treating a broad spectrum of hematological and solid cancers. Despite the transformative impact of immunotherapy on cancer treatment, several outstanding challenges remain. These challenges include on-target off-tumor toxicity, systemic toxicity, and the complexity of achieving potent and sustainable therapeutic efficacy. Synthetic biology has emerged as a promising approach to overcome these obstacles, offering innovative tools for engineering living cells with customized functions. This review provides an overview of the current landscape and future prospects of cancer immunotherapy, particularly emphasizing the role of synthetic biology in augmenting its specificity, controllability, and efficacy. We delineate and discuss two principal synthetic biology strategies: those targeting tumor surface antigens with engineered immune cells and those detecting intratumoral disease signatures with engineered gene circuits. This review concludes with a forward-looking perspective on the enduring challenges in cancer immunotherapy and the potential breakthroughs that synthetic biology may contribute to the field.

Keywords: Adoptive cell therapy.; Cancer immunotherapy; Gene circuit; Gene therapy; Synthetic biology.

Fig. 1

CAR designs that enhance tumor-targeting specificity. A An AND gate design based on splitting the T-cell activation motif (signal 1) and the costimulation motif (signal 2) into two tumor-targeting receptors. Only when the target cell expresses both antigens 1 and 2 to trigger both receptors, will the integrated signal fully activate T cells. B The design of the AND-gated LINK CAR. This design links the LAT protein to a scFv recognizing antigen 1 and the SLP-76 protein to another scFv recognizing antigen 2. Upon T cells encounter both antigens, LAT and SLP-76 interact and trigger T-cell activation. Several mutant variants have been designed to reduce on-target off-tumor toxicity, including cysteine residue mutations in the CD28 TM domain (2CA mutation) and GADS-binding site deletions in both LAT and SLP-76. C A synNotch receptor-based CAR design. The synNotch receptor is used to recognize antigen 1, which in turn activates the expression of CAR-targeting antigen 2. Therefore, only when the target cell expresses both antigens will the T-cell kill the target cell. D AND-NOT gate design. The activation CAR exhibits a standard CAR design and recognizes a tumor antigen (antigen 1). The inhibitory CAR (iCAR) recognizes a normal cell antigen (antigen 2) and delivers an inhibitory signal to T cells. Only when the target cell expresses antigen 1 but not antigen 2 will the AND-NOT gate trigger the T-cell to kill the target cell

Fig. 2

Tunable switches for controlling CAR-T-cell activity. A The design of an “ON-switch”. This type of design splits a conventional CAR into two individual components. Upon the administration of a small molecule drug, both components will assemble into a functional CAR and enable T cells to be activated by the target antigen. B An “ON-switch” CAR based on lenalidomide-induced receptor dimerization. Lenalidomide induces dimerization of an IKZF3 variant and a CRBN variant, leading to functional CAR formation that can be activated by the target antigen. C The design of the LiCAR. This design utilizes light-inducible dimerization of LOV2-ssrA and sspB to control the assembly of two separate CAR chains into a functional chain that can be activated by the target antigen. D The design of an “OFF” switch. This design consists of two separate CAR components and can naturally form a functional CAR. When a small molecule drug is administered, the functional CAR will disassemble and lose its function. E The design of an “OFF” switch based on small molecule-induced protein degradation. An IKZF3-based degron is tagged at the CAR. When lenalidomide is administered, it recruits the CRL4^CRBN E3 ubiquitin ligase to trigger the degradation of CAR

Fig. 3

CAR designs that can mitigate tumor antigen loss. A The design of the biotin-binding immune receptor (BBIR) CAR. BBIR can bind to the administered scFv-biotin molecule and therefore enable T cells to target a specific tumor antigen. By administering different scFv-biotin molecules, BBIR CARs can target a variety of antigens. B The design of an anti-PNE CAR that works with the administration of a PNE-fused tumor-targeting domain. By utilizing different PNE-fused tumor-targeting molecules, the anti-PNE CAR can target various tumor antigens. C The design of the SUPRA CAR. This system requires the expression of a universal receptor (zipCAR) expressed on T cells and the administration of a tumor-targeting adapter molecule (zipFv). The two leucine zippers will allow a functional CAR to assemble, which can then be activated by tumor antigens. By administering different zipFv molecules, the SUPRA CAR can target different antigens. D The design of an anti-FITC CAR for targeting multiple tumor antigens. This system requires the expression of an anti-FITC CAR on T cells and the administration of FITC-conjugated tumor-targeting moieties such as FITC-folate, FITC-DUPA and FITC-AZA adapter, which enable T cells to target antigens such as FRα, PSMA and CA IX on tumor cells, respectively. E The design of a bispecific CAR. This design links two tumor-targeting scFvs in tandem for targeting two tumor antigens. F The design of a multispecific CAR. This design links three DARP proteins in tandem to target three different tumor antigens

Fig. 4

Synthetic gene circuits for detecting intracellular signaling. A Design of the RASER system. This system contains two components. One component is a fusion protein consisting of a membrane-tethered SH2 domain, an NS3 cleavage site, and a therapeutic payload [e.g., OFP-Bid (left panel) or dCas9-VP64 (right panel)]. The other component is a fusion protein consisting of a PTB domain, a HIF1α degron, and an NS3 protease domain. In ErbB-hyperactive tumor cells, the two components colocalize, and the NS3 protease in one component cleaves its target site on the other component, releasing the therapeutic payload to induce apoptosis (left panel) or to activate the transcription of endogenous genes such as GM-CSF (right panel). B The design of the CHOMP system. This system utilizes interacting synthetic proteases for computations. For example, TEVP can be split into two halves (cTEVP and nTEVP) and can function as a protease by interacting with a pair of leucine zippers. A synthetic TVMVP can be designed to abrogate the reconstitution of the cTEVP and the nTEVP, inhibiting the function of TEVP. These interacting proteases can be used to accomplish various logic computations. CHOMP requires two components to detect the upstream signals that activate the RAS. One component consists of the RAS protein fused with the nTEVP, and the other component consists of a RAS-binding domain (RBD), which binds to the active form of RAS fused with the cTEVP. The two components are reconstituted together in cells with high RAS-activating signals and form a functional TEVP. This TEVP further cleaves its substrate sequence and releases active Caspase 3 (Cas p3) from a membrane-anchored complex to trigger apoptosis

Fig. 5

Synthetic gene circuits for detecting transcription factor activities or microRNA expression profiles. A The design of the inducible RheoSwitch® Therapeutic System (RTS). The Gal4-EcR and VP16-RXR fusion proteins are expressed constitutively under the Ubiquitin C promoter. The activating ligand will enable both fusion proteins to form functional heterodimers and therefore activate the transcription of IL-12. B The design of the Dual Promoter Integrator. To accomplish AND gate computation, two native tumor-specific promoters are used as tumor sensors to drive the expression of two protein components. One component is a fusion protein consisting of the GAL4 DNA binding domain fused with Coh2 (GAL4-DBD-Coh2). The other component is a fusion protein consisting of the VP16 transcription activation domain fused with DocS (DocS-VP16 TAD). When both components are expressed, the interaction of Coh2 and DocS will form a functional GAL4-VP16 complex to activate the output expression. C The design of a circuit that targets cells with low p53 activity. This design contains two modules. One module utilizes p53-repressed elements to suppress the expression of the output (i.e., HSV-TK). The other module utilizes p53-activated elements to trigger the production of shRNA that depletes the output. Therefore, this circuit will express high levels of HSV-TK when p53 activity is low. D The design of synthetic promoters. A synthetic promoter is usually built by fusing one type of TFBS in tandem upstream of a minimal promoter. Various synthetic promoter libraries have been built according to this design principle. High-throughput promoter activity analysis can be achieved by leveraging FACS sorting and next-generation sequencing analysis. E The design of an RNA-based immunomodulatory gene circuit for cancer immunotherapy. This circuit senses the activities of c-Myc and E2F1, two cancer-associated TFs, and triggers therapeutic output production only when both activities are high. Specifically, module 1 senses the activity of c-Myc and utilizes this activity to trigger the transcription of GAD, a synthetic TF, along with a microRNA (miRNA) that inhibits GAD transcript accumulation. Module 2 senses the activity of E2F1 and utilizes this activity to trigger the expression of a miRNA “sponge” that titrates the inhibitory miRNA. Hence, only when both c-Myc and E2F1 are highly active (AND gate) will  GAD accumulate and drive the production of the therapeutic outputs encoded in module 3. This circuit enables tumor-localized combinatorial immunotherapy. GAD: a fusion protein consisting of the GAL4 DNA binding domain and the VP16 transcription activation domain. STE: surface T-cell engager (a potent synthetic universal tumor antigen). F The design of a mini gene circuit based on CRISPReader. The c-Myc- and Get1-responsive elements are used to regulate the expression of the Cas9-VP64 protein and two sgRNAs (sgRNA1 and sgRNA2). Cas9-VP64 complexed with a 14 nt long sgRNA exhibited transcriptional activation only. When the activities of both c-Myc and Get1 are high, Cas9-VP64 and the two sgRNAs are produced. The produced Cas9-VP64 and the two sgRNAs can further form transcription-activating complexes to activate transcription. As there are sgRNA binding sites immediately after the c-Myc and Get1 responsive elements, the transcription-activating complexes further enhance the production of Cas9-VP64 and the two sgRNAs, forming a positive feedback loop. In addition, the expressed Cas9-VP64 and sgRNA2 can form a gene knockout complex to knock out the LacI gene. Knocking out the LacI gene will then enhance the final output production. As a result, only when the activities of both c-Myc and Get1 are high will the circuit trigger high-output production. G The design of the HeLa cell classifier. This classifier circuit contains sensors for both HeLa-high (miR-21 and miR-17) and HeLa-low (miR-141, miR-142(3p), and miR-146a) miRNAs. In HeLa cells where HeLa-high miRNAs are abundant, the production of rtTA and LacI will be low, and therefore, the circuit will trigger high therapeutic output. Furthermore, in HeLa cells where HeLa-low miRNAs are scarce, the therapeutic yield will remain high. H The design of a “SOX9/10 AND HNF1A/B AND (NOT let-7c)” gate for targeting HCC. This design consists of two transcription components. The first component can sense SOX9 or SOX10 and trigger the expression of a transactivator (PIT-VP16). The second component needs to sense both PIT-VP16 and HNF1A/B to trigger the expression of HSV-TK. Therefore, only when both SOX9/10 and HNF1A/B are expressed will HSK-TK be produced. The binding sites for highly expressed miRNAs in normal tissue (e.g., let-7c) were also incorporated into the 3’-UTRs of both the PIT-VP16 and HSV-TK genes to decrease off-target effects on normal cells. Abbreviations: ITR, inverted terminal repeat; RE_SOX, binding site for SOX9/10; RE_HNF1, binding site for HNF1A/B; RE_PIT, binding site for PIT-VP16 or PIT-RelA; Tlet7c, four repeats of a fully complementary let-7c target