synNotch Receptor Research Articles

Aβ-targeting synNotch Receptor for Alzheimer's Disease: Expanding Applications to Extracellular Protein Aggregates.

The synthetic Notch receptor (synNotch) system is a versatile platform that induces gene transcription in response to extracellular signals. However, its application has been largely confined to membrane-bound targets due to specific activation requirements. Whether synNotch can also target extracellular protein aggregates, such as amyloid beta (Aβ) in Alzheimer's disease (AD), is unclear. To address this, we engineered an Aβ-targeting synNotch receptor controlling the production of chimeric human-mouse versions of Lecanemab (Leqembi®) or Aducanumab (Aduhelm®), both FDA-approved antibodies for AD. We demonstrate that NIH 3T3 cells expressing this synNotch system detect and respond to extracellular Aβ aggregates by synthesizing and secreting Aducanumab or Lecanemab. These findings broaden the potential applications of synNotch, extending its targets beyond membrane-bound proteins to extracellular protein aggregates, providing obvious benefits to research in this scientific arena.

Engineering programmable material-to-cell pathways via synthetic notch receptors to spatially control differentiation in multicellular constructs

Synthetic Notch (synNotch) receptors are genetically encoded, modular synthetic receptors that enable mammalian cells to detect environmental signals and respond by activating user-prescribed transcriptional programs. Although some materials have been modified to present synNotch ligands with coarse spatial control, applications in tissue engineering generally require extracellular matrix (ECM)-derived scaffolds and/or finer spatial positioning of multiple ligands. Thus, we develop here a suite of materials that activate synNotch receptors for generalizable engineering of material-to-cell signaling. We genetically and chemically fuse functional synNotch ligands to ECM proteins and ECM-derived materials. We also generate tissues with microscale precision over four distinct reporter phenotypes by culturing cells with two orthogonal synNotch programs on surfaces microcontact-printed with two synNotch ligands. Finally, we showcase applications in tissue engineering by co-transdifferentiating fibroblasts into skeletal muscle or endothelial cell precursors in user-defined micropatterns. These technologies provide avenues for spatially controlling cellular phenotypes in mammalian tissues.

DIPG-15. NOVEL CNS SENSING SYNNOTCH-CAR T CELLS FOR TARGETING DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG), including Diffuse intrinsic pontine glioma (DIPG), is an aggressive brain tumor in children with limited treatment options. Recent developments of phase 1 clinical trials have shown early promise for chimeric antigen receptor (CAR) T cells in patients with DMG/DIPG. However, several barriers such as the absence of tumor-specific antigens, restricted trafficking to the tumor site, and poor persistence hinder the full therapeutic potential of CAR T cell therapy in DMG/DIPG. METHODS To safely target the glioma-associated antigens (GAAs) without attacking nomal tissues expressing the same antigens, we adopted a novel synthetic Notch (synNotch) receptor system and engineered T cell circuits employing a “prime-and-kill” strategy. In this system, a synNotch receptor recognizing the priming antigen, Brevican (BCAN), exclusively expressed on cells in the central nervous system (CNS) but not on non-CNS cells, locally induces the expression of a tandem CAR against GAAs, ephrin type A receptor (EphA2) and interleukin-13 receptor a2 (IL13Ra2), there by homogenously eliminating DMG/DIPG cells. RESULTS The α-BCAN synNotch⋄α-EphA2/IL-13Rα2 CAR (B-SYNC) T cells efficiently killed DMG/DIPG (BT-245, SF8628, SU-DIPGXIII, and SU-DIPGXVII) cells in vitro in the presence of priming cells (K562 cells expressing BCAN). Moreover, a single intravenous infusion of B-SYNC T cells significantly (P< 0.001) prolonged the survival of immunodeficient mice bearing aggressive, orthotopic SF8628 DIPG xenografts and completely eradicated the tumor in more than 50% (4/7) of mice. Strikingly, we observed excellent homing, priming, activation, and persistence of B-SYNC T cells in the brain stem of mice bearing SF8628 xenografts. In contrast, constitutively active α-EphA2/IL-13Rα2 CAR T cells or untransduced T cells demonstrated a failure to migrate to the brain stem and improve the survival of mice. CONCLUSIONS Collectively, our findings strongly support the development of clinical trials evaluating the efficacy of B-SYNC T cells in DMG/DIPG patients.

Preclinical efficacy of a HER2 synNotch/CEA-CAR combinatorial immunotherapy against colorectal cancer with HER2 amplification

HER2 amplification occurs in about 5% of colorectal cancer (CRC) cases and is associated only partially with clinical response to combined HER2/EGFR targeted treatment. An alternative approach based on adoptive cell therapy (ACT) using T-cells engineered with anti-HER2 chimeric antigen receptor (CAR) proved to be toxic due to "on-target off-tumor" activity. Here we describe a combinatorial strategy to safely target HER2 amplification and CEA expression in CRC using a synNotch-CAR based artificial regulatory network. The natural killer cell line NK-92 was engineered with an anti-HER2 synNotch receptor driving the expression of a CAR against CEA only when engaged. After being transduced and sorted for HER2-driven CAR expression, cells were cloned. The clone with optimal performances in terms of specificity and amplitude of CAR induction demonstrated significant activity in vitro and in vivo specifically against HER2amp/CEA+ CRC models, with no effects on cells with physiological HER2 levels. The HER2-synNotch/CEA-CAR-NK system provides an innovative, scalable and safe off-the shelf cell therapy approach with potential against HER2amp CRC resistant or partially responsive to HER2/EGFR blockade.

Fluorescein-Based SynNotch Adaptors for Regulating Gene Expression Responses to Diverse Extracellular Cues.

We introduce an adaptor-based strategy for regulating fluorescein-binding synthetic Notch (SynNotch) receptors using ligands based on conjugates of fluorescein isomers and analogs. To develop a versatile system, we evaluated the surface expression and activities of multiple constructs containing distinct extracellular fluorescein-binding domains. Using an optimized receptor, we devised ways to regulate signaling via fluorescein-based chemical transformations, including an approach based on a bio-orthogonal chemical ligation and a spatially controllable strategy via the photo-patterned uncaging of an o -nitrobenzyl-caged fluorescein conjugate. We further demonstrate that fluorescein-conjugated extracellular matrix (ECM)-binding peptides can regulate SynNotch activity depending on the folding state of collagen-based ECM networks. Treatment with these conjugates enabled cells to distinguish between folded versus denatured collagen proteins and enact dose-dependent gene expression responses depending on the nature of the signaling adaptors presented. To demonstrate the utility of these tools, we applied them to control the myogenic conversion of fibroblasts into myocytes with spatial and temporal precision and in response to denatured collagen-I, a biomarker of multiple pathological states. Overall, we introduce an optimized fluorescein-binding SynNotch as a versatile tool for regulating transcriptional responses to extracellular ligands based on the widely used and clinically-approved fluorescein dye.

Augmenting the Antitumor Efficacy of Natural Killer Cells via SynNotch Receptor Engineering for Targeted IL-12 Secretion.

Natural killer (NK) cells are crucial components of innate immunity, known for their potent tumor surveillance abilities. Chimeric antigen receptors (CARs) have shown promise in cancer targeting, but optimizing CAR designs for NK cell functionality remains challenging. CAR-NK cells have gained attention for their potential to reduce side effects and enable scalable production in cancer immunotherapy. This study aimed to enhance NK cell anti-tumor activity by incorporating PD1-synthetic Notch (synNotch) receptors. A chimeric receptor was designed using UniProt database sequences, and 3D structure models were generated for optimization. Lentiviral transduction was used to introduce PD1-Syn receptors into NK cells. The expression of PD1-Syn receptors on NK cell surfaces was assessed. Engineered NK cells were co-cultured with PDL1+ breast cancer cells to evaluate their cytotoxic activity and ability to produce interleukin-12 (IL-12) and interferon-gamma (IFNγ) upon interaction with the target cells. This study successfully expressed the PD1-Syn receptors on NK cells. CAR-NK cells secreted IL-12 and exhibited target-dependent IFNγ production when engaging PDL1+ cells. Their cytotoxic activity was significantly enhanced in a target-dependent manner. This study demonstrates the potential of synNotch receptor-engineered NK cells in enhancing anti-tumor responses, especially in breast cancer cases with high PDL1 expression.

A synthetic notch (synNotch) system linking intratumoral immune-cancer cell communication to a synthetic blood biomarker assay.

Introduction: Cellular immunotherapy has greatly improved cancer treatment in recent years. For instance, chimeric antigen receptor (CAR) T cell therapy has been proven highly effective in treating hematological malignancies, and many CAR cell designs are being explored for solid tumors. However, many questions remain why responses differ across patients and some tumor types are resistant. Improved and relatively inexpensive ways to monitor these cells could provide some answers. Clinically, blood tests are regularly used to monitor these therapies, but blood signals often do not reflect the activity of immune cells within the tumor(s). Here, using the synthetic Notch (synNotch) receptor that tethers antigen binding to customized transgene expression, we linked intratumoral immune-cancer cell communication to a simple secreted reporter blood test. Specifically, we engineered immune cells with a CD19-targeted synNotch receptor and demonstrated that binding to CD19 on cancer cells in vivo resulted in the production of secreted embryonic alkaline phosphatase (SEAP) at levels that are readily detected in the blood. Methods and Results: Jurkat T cells were engineered via sequential lentiviral transduction of two components: an anti-CD19 synNotch receptor and a synNotch response element encoding SEAP. Co-culture of engineered cells with CD19+, but not CD19-, Nalm6 cells, resulted in significantly elevated SEAP in media. Nod-scid-gamma (NSG) mice were subcutaneously injected with either CD19+ or CD19- Nalm6 cells. Intratumoral injection of engineered T cells (1x107) resulted in significantly elevated blood SEAP activity in mice bearing CD19+ tumors (n = 7), but not CD19- tumors (n = 5). Discussion: Our synNotch reporter system allows for the monitoring of antigen-dependent intratumoral immune-cancer cell interactions through a simple and convenient blood test. Continued development of this system for different target antigens of interest should provide a broadly applicable platform for improved monitoring of many cell-based immunotherapies during their initial development and clinical translation, ultimately improving our understanding of design considerations and patient-specific responses.

Post-translational covalent assembly of CAR and synNotch receptors for programmable antigen targeting

Chimeric antigen receptors (CARs) and synthetic Notch (synNotch) receptors are engineered cell-surface receptors that sense a target antigen and respond by activating T cell receptor signaling or a customized gene program, respectively. Here, to expand the targeting capabilities of these receptors, we develop “universal” receptor systems for which receptor specificity can be directed post-translationally via covalent attachment of a co-administered antibody bearing a benzylguanine (BG) motif. A SNAPtag self-labeling enzyme is genetically fused to the receptor and reacts with BG-conjugated antibodies for covalent assembly, programming antigen recognition. We demonstrate that activation of SNAP-CAR and SNAP-synNotch receptors can be successfully targeted by clinically relevant BG-conjugated antibodies, including anti-tumor activity of SNAP-CAR T cells in vivo in a human tumor xenograft mouse model. Finally, we develop a mathematical model to better define the parameters affecting universal receptor signaling. SNAP receptors provide a powerful strategy to post-translationally reprogram the targeting specificity of engineered cells.

A recombinant adenovirus vector containing the synNotch receptor gene for the treatment of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is known as the most difficult molecular subtype of breast cancer to treat. Recent studies revealed that cancer stem cells (CSCs) play a critical role in TNBC recurrence and metastasis. In this study, we developed a recombinant replication-deficient adenoviral vector (Ad-CD44-N-HIF-3α4), which contains a gene encoding a synthetic Notch (synNotch) receptor composed of the extracellular domain of CD44 (CD44-ECD) and the hypoxia-inducible factor (HIF)-3α4 connected by the Notch core regulatory region. CD44 is a transmembrane glycoprotein and known as a CSC marker in breast cancer and other malignancies. HIF-3α4 is a dominant-negative regulator of HIF-1α and HIF-2α and inhibits hypoxia-inducing effect. Both CD44 and HIF signals contribute cancer stemness and maintaining CSCs in breast cancer. The CD44-ECD in the synNotch receptor acts as the CD44 decoy receptor, and after a ligand such as a hyaluronic acid binds to the CD44-ECD, HIF-3α4 is released from the Notch core domain. We performed an in vivo study using a mouse xenograft model of MDA-MB-231, a highly invasive TNBC cell, and confirmed the significant antitumor activity of the intratumoral injections of Ad-CD44-N-HIF3α4. Our findings in this study warrant the further development of Ad-CD44-N-HIF3α4 for the treatment of patients with TNBC.

ErbB- and MUC1-targetted CAR-T cell immunotherapy of oral squamous cell carcinoma

Chimeric antigen receptor T (CAR-T) cell therapy has shown great success in treating B cell malignancies however, there are many challenges which limit their therapeutic efficacy in solid tumours. Immunotherapy of head and neck squamous cell carcinoma (HNSCC), and in particular, oral squamous cell carcinoma (OSCC), presents a unique set of challenges including lack of consistently expressed tumour associated antigens (TAAs) and the immunosuppressive tumour microenvironment (TME). Currently, there are few clinical trials investigating the use of CAR-T cells in HNSCC/OSCC however results from trials investigating similar solid tumours, such as breast cancer, can be adopted to help evaluate the use of CAR-T in this cancer. In this review, the process of CAR-T cell engineering, and different generations of these cells will be summarised, highlighting their potential use in treating HNSCC through targeting ErbB and MUC1; TAAs highly expressed by this solid tumour. Potential strategies including combination therapy, utilising both TAA-targeting CAR-Ts and immune checkpoint inhibitors, such as PD-L1, has been discussed, in an attempt to develop synergistic anti-tumour responses. In addition to this, the use of dual-targeting CAR-T cells, synthetic NOTCH (synNOTCH) receptors and alternative non-tumour targets of the TME have been reviewed. Such combination therapies have been shown to help limit solid tumour progression and enhance both the safety and efficacy of CAR-T cell immunotherapy, which may be adopted for the treatment and management of OSCC.

Tension-tuned receptors for synthetic mechanotransduction and intercellular force detection.

Cells interpret mechanical stimuli from their environments and neighbors, but the ability to engineer customized mechanosensing capabilities has remained a synthetic and mechanobiology challenge. Here we introduce tension-tuned synthetic Notch (SynNotch) receptors to convert extracellular and intercellular forces into specifiable gene expression changes. By elevating the tension requirements of SynNotch activation, in combination with structure-guided mutagenesis, we designed a set of receptors with mechanical sensitivities spanning the physiologically relevant picoNewton range. Cells expressing these receptors can distinguish between varying tensile forces and respond by enacting customizable transcriptional programs. We applied these tools to design a decision-making circuit, through which fibroblasts differentiate into myoblasts upon stimulation with distinct tension magnitudes. We also characterize cell-generated forces transmitted between cells during Notch signaling. Overall, this work provides insight into how mechanically induced changes in protein structure can be used to transduce physical forces into biochemical signals. The system should facilitate the further programming and dissection of force-related phenomena in biological systems.

Synthetic cytokine circuits that drive T cells into immune-excluded tumors.

Chimeric antigen receptor (CAR) T cells are ineffective against solid tumors with immunosuppressive microenvironments. To overcome suppression, we engineered circuits in which tumor-specific synNotch receptors locally induce production of the cytokine IL-2. These circuits potently enhance CAR T cell infiltration and clearance of immune-excluded tumors, without systemic toxicity. The most effective IL-2 induction circuit acts in an autocrine and T cell receptor (TCR)- or CAR-independent manner, bypassing suppression mechanisms including consumption of IL-2 or inhibition of TCR signaling. These engineered cells establish a foothold in the target tumors, with synthetic Notch-induced IL-2 production enabling initiation of CAR-mediated T cell expansion and cell killing. Thus, it is possible to reconstitute synthetic T cell circuits that activate the outputs ultimately required for an antitumor response, but in a manner that evades key points of tumor suppression.

EXTH-33. PRIMING OF SYNNOTCH CAR T CELLS VIA CNS-SPECIFIC ANTIGEN ALLOWS SPATIAL AND TEMPORAL REGULATION OF CAR EXPRESSION, EFFECTIVE HOMING AND PERSISTENCE OF T CELLS IN THE CNS

Abstract One of the key challenges in the development of CAR-T therapy is the absence of target antigens that are tumor-specific and have homogenous expression. To safely target glioblastoma-associated antigens (GAAs) in the tumor without attacking normal tissue expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system and established a “prime and kill” sequential two-receptor CAR circuit. We used glioblastoma- (GBM) specific neoantigen EGFRvIII as a priming signal for synNotch receptor and have reported robust antitumor response in the mice bearing intracerebral PDX tumor with heterogenous EGFRvIII expression. However, less than 20% of adult GBM cases express EGFRvIII. Furthermore, EGFRvIII expression can diminish over time even after its detection in the primary GBM, and the EGFRvIII-synNotch primed CAR T cells may deplete EGFRvIII-expressing GBM cells via their cytotoxic effects, thereby losing the priming signal. To overcome these inherent challenges of the EGFRvIII-priming strategy, we used CNS tissue-specific antigens as priming signal to drive localized expression of the CAR against EphA2 and IL-13Rα2 and bypassed the need for tumor-specific antigen. We have found BCAN (also known as Brevican) as the most promising priming antigen among several CNS-specific cell surface proteins that we evaluated. When mice bearing intracerebral GBM6 PDX received a single IV infusion of T cells engineered with the α-BCANsynNotch- > α-EphA2/IL-13Rα2 CAR (B-SYNC) circuit, all mice (10/10) demonstrated complete regression of the tumor. Furthermore, these B-SYNC T cells were significantly more efficacious than constitutively expressed EphA2/IL-13Rα2 CAR T cells, associated with superior persistence and less exhausted phenotype. Thus, the B-SYNC approach represents a conceptual novelty of CNS tissue-specific CAR activation. This also enhances the translational significance of synNotch-CAR T cells by widely extending the eligibility to EGFRvIII-negative glioma patients. We will develop and conduct a phase I study to evaluate our hypothesis.

Tuning the ignition of CAR: optimizing the affinity of scFv to improve CAR-T therapy.

How single-chain variable fragments (scFvs) affect the functions of chimeric antigen receptors (CARs) has not been well studied. Here, the components of CAR with an emphasis on scFv were described, and then several methods to measure scFv affinity were discussed. Next, scFv optimization studies for CD19, CD38, HER2, GD2 or EGFR were overviewed, showing that tuning the affinity of scFv could alleviate the on-target/off-tumor toxicity. The affinities of scFvs for different antigens were also summarized to designate a relatively optimal working range for CAR design. Last, a synthetic biology approach utilizing a low-affinity synthetic Notch (synNotch) receptor to achieve ultrasensitivity of antigen-density discrimination and murine models to assay the on-target/off-tumor toxicity of CARs were highlighted. Thus, this review provides preliminary guidelines of choosing the right scFvs for CARs.

The Molecular Landscape and Essential Pathways Involved in Bone Marrow-Mediated Carfilzomib Resistance of Multiple Myeloma

BackgroundMultiple myeloma (MM) remains an incurable malignancy, with most patients relapsing and dying from the disease. Anti-myeloma drugs, such as proteasome inhibitors (PIs) bortezomib and carfilzomib (CFZ), have considerably improved prognosis in myeloma. Despite these advances, disease heterogeneity, early relapse and treatment resistance still pose major challenges in MM treatment. Understanding the mechanisms that mediate PI resistance provide a key to targeting both, PI-resistant minimal residual disease that drives relapsed MM after prolonged PI-containing frontline therapy, as well as PI-refractory, aggressive advanced MM. While key mechanisms of the in vitro-generated PI resistance in MM have been revealed in cell line models, we lack understanding of PI resistance in vivo, where in particular clonal heterogeneity and the tumor microenvironment (TME) within the bone marrow (BM) add additional levels of complexity. Therefore, the aim of our study was to analyze the molecular landscape and changes occurring during MM progression under CFZ treatment in vivo and to identify key molecular processes contributing to CFZ-resistance of MM cells in the presence of stromal cells in vitro, to ultimately identify new molecular pathways and develop innovative treatment strategies in PI-resistant MM.MethodsThe NSG mice intrafemorally engrafted with human RPMI-8226 cells were either untreated or treated long-term with 4 mg/kg CFZ (intravenously) until they became drug resistant. At this point, CFZ naïve and CFZ-resistant cells were isolated and processed for single-cell RNA sequencing (scRNA-seq, 10x Genomics) with the aim to characterize a transcriptional CFZ-resistance signature in refractory cells. To investigate the role of the TME as well as the importance of cell-cell interactions in CFZ-resistance in vitro, we performed two independent genome-wide CRISPR/Cas9 library screenings. In the first one, Brunello library transduced RPMI-8226 cells were co-cultured with human stromal cells (HS5) and treated with CFZ to identify CFZ sensitivity/resistance candidate genes. In the second experiment, Brunello library and synthetic Notch (synNotch) receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells to identify genes that are essential for establishing cell-cell contacts between stromal and MM cells. Subsequent functional analysis of the highest-ranking CFZ sensitivity/resistance candidates in the RPMI-8226+HS5 co-culture included shRNA-silencing, single-gene knockouts, viability assays, cell cycle analysis and protein synthesis analysis using the SUnSET assay.ResultsScRNA-seq analysis of CFZ-refractory RPMI-8226 cells growing in the BM of NSG mice showed a different transcriptional landscape, compared to CFZ-naïve cells isolated from the BM of untreated mice. The unsupervised clustering analysis, using UMAP, revealed that cells exposed to CFZ show distinct populations with a strong increase in the OXPHOS and protein folding capacity as well as down-regulation of several genes involved in proliferation and apoptosis, when compared to naïve cells. The CRISPR/Cas9 library screening where RPMI-8226 cells were co-cultured with HS5 cells and exposed to CFZ revealed several CFZ sensitivity candidates at the cut-off of false discovery rate (FDR) < 0.01 and fold change above 1.5-fold. Those genes are involved in cytokine signaling, cell growth, invasion, metastasis and quality control of translational elongation. At the same time, the CRISPR/Cas9 library screening, where synNotch receptor transduced HS5 cells were co-cultured with synNotch ligand transduced RPMI-8226 cells revealed gene candidates at the cut-off of FDR < 0.01 and fold change greater than 1.5-fold, which mediate stronger or weaker cell-cell interaction. Those genes are particularly involved in cytokine signaling and mitochondrial metabolism.ConclusionIn conclusion, MM cells that acquired CFZ-resistance upon cell-cell contact with certain cell types within the TME, such as stromal cells, differ significantly from CFZ-naïve cells. CFZ-resistance, caused by cell-cell contact with stromal cells, is presumably mediated via decreased proliferative as well as protein synthesis capacity of MM cells. Therefore, stimulation of MM cells to proliferate and synthesize more proteins may be a key to targeting CFZ-resistance in vivo. DisclosuresNo relevant conflicts of interest to declare.

Getting better mileage with logically primed CARs

Although remarkably successful against liquid tumors, chimeric antigen receptor (CAR)-T cell therapy has been stymied by solid tumors, limited by inadequate specificity and poor efficacy. Pairing synthetic Notch (synNotch) receptors with CARs, Choe etal. and Hyrenius-Wittsten etal. engineer Tcells that more precisely and potently combat solid tumors.

A logic to cell-cell recognition

Synthetic Biology There has been exciting progress in the field of cancer immunotherapy, which harnesses a patient's own immune system to kill cancer cells. However, achieving precise recognition of cancer cells remains challenging. Cells engineered with synthetic Notch (synNotch) receptors bind to specific antigens, and binding induces the expression of defined genes. Williams et al. used synNotch modules as transcriptional connectors that daisy-chain together multiple receptors. They engineered T cells that can recognize up to three target antigens expressed on or inside cancer cells and integrated these inputs to achieve NOT, AND, and OR logic. The engineered cells achieved precise recognition of targeted cancer cells. Science , this issue p. [1099][1] [1]: /lookup/doi/10.1126/science.abc6270

Precise T cell recognition programs designed by transcriptionally linking multiple receptors.

Living cells often identify their correct partner or target cells by integrating information from multiple receptors, achieving levels of recognition that are difficult to obtain with individual molecular interactions. In this study, we engineered a diverse library of multireceptor cell-cell recognition circuits by using synthetic Notch receptors to transcriptionally interconnect multiple molecular recognition events. These synthetic circuits allow engineered T cells to integrate extra- and intracellular antigen recognition, are robust to heterogeneity, and achieve precise recognition by integrating up to three different antigens with positive or negative logic. A three-antigen AND gate composed of three sequentially linked receptors shows selectivity in vivo, clearing three-antigen tumors while ignoring related two-antigen tumors. Daisy-chaining multiple molecular recognition events together in synthetic circuits provides a powerful way to engineer cellular-level recognition.

IMMU-33. MULTI-ANTIGEN RECOGNITION CIRCUITS OVERCOME CHALLENGES OF SPECIFICITY, HETEROGENEITY, AND DURABILITY IN T-CELL THERAPY FOR GLIOBLASTOMA

Abstract Treatment of solid cancers with chimeric antigen receptor (CAR) T-cells is challenging because of a lack of target antigens that are both tumor-specific and homogenously expressed. While epidermal growth factor receptor (EGFR)vIII represents a glioblastoma (GBM)-specific antigen, its expression is heterogeneous within the tumor resulting in tumor escape. In contrast, more homogenously expressed GBM-associated antigens (GAA), such as EphA2, are non-ideal because of expression in other normal organs, yielding potential cross-reactive toxicity. As a way to safely target GAAs in the tumor without attacking normal cells expressing the same GAAs outside of the brain, we adapted a novel synthetic Notch (synNotch) receptor system and established a “prime and kill” sequential two-receptor CAR circuit. A synNotch receptor recognizes a specific priming antigen; the heterogeneous GBM neoantigen EGFRvIII or a brain tissue-specific antigen to prime the local expression of a CAR that mediates cytotoxicity against a GAA (e.g. EphA2). In orthotopic GBM6 glioma model, a patient-derived xenograft (PDX) with heterogeneous expression of EGFRvIII, intravenous infusion of T-cells transduced with EGFRvIII synNotch→anti-IL-13Rα2/EphA2 tandem CAR circuit resulted in long-term (over 100 days) survival and eradication of the heterogeneous tumor in all of 12 mice in two independent experiments. In contrast, constitutive CARs targeting EGFRvIII or IL-13Rα2/EphA2 (as a tandem CAR) failed to exhibit long-term anti-tumor response. Moreover, T-cells transduced with synNotch-regulated CAR maintain a less differentiated state which is associated with higher durability compared with ones with constitutive CAR in vivo. T-cells transduced with a synNotch→CAR circuit primed by a brain-specific antigen, myelin oligodendrocyte glycoprotein (MOG), exhibited a precise and potent local control of intracranial PDX without evidence of priming in extracranial organs. These data support the utility of synNotch→CAR circuits in EGFRvIII-negative GBM cases. By integrating multiple imperfect but complementary antigens, we improve both the specificity and persistence of T-cells directed against GBM.

Synthetic Notch-Receptor-Mediated Transmission of a Transient Signal into Permanent Information via CRISPR/Cas9-Based Genome Editing

Synthetic receptor biology and genome editing are emerging techniques, both of which are currently beginning to be used in preclinical and clinical applications. We were interested in whether a combination of these techniques approaches would allow for the generation of a novel type of reporter cell that would recognize transient cellular events through specifically designed synthetic receptors and would permanently store information about these events via associated gene editing. Reporting cells could be used in the future to detect alterations in the cellular microenvironment, including degenerative processes or malignant transformation into cancer cells. Here, we explored synthetic Notch (synNotch) receptors expressed in human embryonic kidney cells to investigate the efficacy of antigen recognition events in a time- and dose-dependent manner. First, we evaluated the most suitable conditions for synNotch expression based on dsRed-Express fluorophore expression. Then, we used a synNotch receptor coupled to transcriptional activators to induce the expression of a Cas9 nuclease targeted to a specific genomic DNA site. Our data demonstrate that recognition of various specific antigens via synNotch receptors robustly induced Cas9 expression and resulted in an indel formation frequency of 34.5%–45.5% at the targeted CXCR4 locus. These results provide proof of concept that reporter cells can be designed to recognize a given event and to store transient information permanently in their genomes.