Many viruses alter intracellular calcium homeostasis. The rotavirus nonstructural protein 4 (NSP4), an endoplasmic reticulum (ER) transmembrane glycoprotein, increases intracellular levels of cytoplasmic Ca2+ ([Ca2+]cyto) through a phospholipase C-independent pathway, which is required for virus replication and morphogenesis. However, the NSP4 domain and mechanism that increases [Ca2+]cyto are unknown. We identified an NSP4 domain (amino acids [aa] 47 to 90) that inserts into membranes and has structural characteristics of viroporins, a class of small hydrophobic viral proteins that disrupt membrane integrity and ion homeostasis to facilitate virus entry, assembly, or release. Mutational analysis showed that NSP4 viroporin activity was mediated by an amphipathic α-helical domain downstream of a conserved lysine cluster. The lysine cluster directed integral membrane insertion of the viroporin domain and was critical for viroporin activity. In epithelial cells, expression of wild-type NSP4 increased the levels of free cytoplasmic Ca2+ by 3.7-fold, but NSP4 viroporin mutants maintained low levels of [Ca2+]cyto, were retained in the ER, and failed to form cytoplasmic vesicular structures, called puncta, which surround viral replication and assembly sites in rotavirus-infected cells. When [Ca2+]cyto was increased pharmacologically with thapsigargin, viroporin mutants formed puncta, showing that elevation of calcium levels and puncta formation are distinct functions of NSP4 and indicating that NSP4 directly or indirectly responds to elevated cytoplasmic calcium levels. NSP4 viroporin activity establishes the mechanism for NSP4-mediated elevation of [Ca2+]cyto, a critical event that regulates rotavirus replication and virion assembly.

Anti-tumor efficacy of T cells engineered to express chimeric antigen receptors (CARs) is dependent on their specificity, survival, and in vivo expansion following adoptive transfer. Toll-like receptor (TLR) and CD40 signaling in T cells can improve persistence and drive proliferation of antigen-specific CD4 and CD8 T cells following pathogen challenge or in graft-versus-host disease (GvHD) settings, suggesting that these costimulatory pathways may be co-opted to improve CAR-T cell persistence and function. Here, we present a novel strategy to activate TLR and CD40 signaling in human T cells using inducible MyD88/CD40 (iMC), which can be triggered in vivo via the synthetic dimerizing ligand, rimiducid, to provide potent costimulation to CAR-modified T cells. Importantly, the concurrent activation of iMC (with rimiducid) and CAR (by antigen recognition) is required for interleukin (IL)-2 production and robust CAR-T cell expansion and may provide a user-controlled mechanism to amplify CAR-T cell levels in vivo and augment anti-tumor efficacy.

Use of chimeric antigen receptors (CARs) as the basis of targeted adoptive T cell therapies has enabled dramatic efficacy against multiple hematopoietic malignancies, but potency against bulky and solid tumors has lagged, potentially due to insufficient CAR-T cell expansion and persistence. To improve CAR-T cell efficacy, we utilized a potent activation switch based on rimiducid-inducible MyD88 and CD40 (iMC)-signaling elements. To offset potential toxicity risks by this enhanced CAR, an orthogonally regulated, rapamycin-induced, caspase-9-based safety switch (iRC9) was developed to allow in vivo elimination of CAR-T cells. iMC costimulation induced by systemic rimiducid administration enhanced CAR-T cell proliferation, cytokine secretion, and antitumor efficacy in both in vitro assays and xenograft tumor models. Conversely, rapamycin-mediated iRC9 dimerization rapidly induced apoptosis in a dose-dependent fashion as an approach to mitigate therapy-related toxicity. This novel, regulatable dual-switch system may promote greater CAR-T cell expansion and prolonged persistence in a drug-dependent manner while providing a safety switch to mitigate toxicity concerns.

Successful adoptive chimeric antigen receptor (CAR) T-cell therapies against hematological malignancies require CAR-T expansion and durable persistence following infusion. Balancing increased CAR-T potency with safety, including severe cytokine-release syndrome (sCRS) and neurotoxicity, warrants inclusion of safety mechanisms to control in vivo CAR-T activity. Here, we describe a novel CAR-T cell platform that utilizes expression of the toll-like receptor (TLR) adaptor molecule, MyD88, and tumor-necrosis factor family member, CD40 (MC), tethered to the CAR molecule through an intentionally inefficient 2A linker system, providing a constitutive signal that drives CAR-T survival, proliferation, and antitumor activity against CD19+ and CD123+ hematological cancers. Robust activity of MC-enhanced CAR-T cells was associated with cachexia in animal models that corresponded with high levels of human cytokine production. However, toxicity could be successfully resolved by using the inducible caspase-9 (iC9) safety switch to reduce serum cytokines, by administration of a neutralizing antibody against TNF-α, or by selecting “low” cytokine-producing CD8+ T cells, without loss of antitumor activity. Interestingly, high basal activity was essential for in vivo CAR-T expansion. This study shows that co-opting novel signaling elements (i.e., MyD88 and CD40) and development of a unique CAR-T architecture can drive T-cell proliferation in vivo to enhance CAR-T therapies.

Over the past 20 years, dendritic cells (DCs) have been utilized to activate immune responses capable of eliminating cancer cells. Currently, ex vivo DC priming has been the mainstay of DC cancer immunotherapies. However, cell-based treatment modalities are inherently flawed due to a lack of standardization, specialized facilities and personnel, and cost. Therefore, direct modes of DC manipulation, circumventing the need for ex vivo culture, must be investigated. To facilitate the development of next-generation, in vivo targeted DC vaccines, we characterized the DC interaction and activation potential of the Tobacco Mosaic virus (TMV), a plant virus that enjoys a relative ease of production and the ability to deliver protein payloads via surface conjugation. In this study we show that TMV is readily taken up by mouse bone marrow-derived DCs, in vitro. Footpad injection of fluorophore-labeled TMV reveals preferential uptake by draining lymph node resident DCs in vivo. Uptake leads to activation, as measured by the upregulation of key DC surface markers. When peptide antigen-conjugated TMV is injected into the footpad of mice, DC-mediated uptake and activation leads to robust antigen-specific CD8+ T cell responses, as measured by antigen-specific tetramer analysis. Remarkably, TMV priming induced a greater magnitude T cell response than Adenovirus (Ad) priming. Finally, TMV is capable of boosting either Ad-induced or TMV-induced antigen-specific T cell responses, demonstrating that TMV, uniquely, does not induce neutralizing self-immunity. Overall, this study elucidates the in vivo DC delivery and activation properties of TMV, and indicates its potential as a vaccine vector in stand alone or prime-boost strategies.

TPS2679 Background: In 2022 approximately 1.7 million Americans will die from solid cancers. Recently there have been significant advances in the genetic engineering of T lymphocytes to recognize neoantigens on tumors in vivo, resulting in remarkable cases of tumor regression and remission. Cancer cells frequently harbor KRAS, TP53, and EGFR somatic hotspot mutations that can be processed and presented by tumor HLA as neoantigens to T cells through their T-cell receptor (TCR). These neoantigens are not present in the normal tissues; thus, they are attractive targets for adoptive T cell therapy. Given the number and complexity of different neoantigen/HLA combinations on solid tumors, a TCR library approach is warranted. Therefore, we have developed a library of TCR-T cell therapies including those targeting shared KRAS, TP53 and EGFR mutations. Methods: Patients for whom a TCR matching the subject’s somatic mutation(s) and HLA type is available in our TCR library, and have progressive or recurrent disease following standard therapy are eligible for enrollment on this protocol. Patients with the following tumor types will be enrolled: ovarian, endometrial, colorectal, pancreatic, cholangiocarcinoma, and non-small cell lung cancer. This first-in-human study includes Screening, Pre-Treatment, Treatment and Follow-up Periods. During the Pre-Treatment Period, subjects will undergo apheresis for PBMCs isolation. The PBMCs will be transposed using the Sleeping Beauty system to express the subject’s mutation specific TCR. Bridging therapy after apheresis is allowed once the apheresis product has been accepted. During the Treatment Period, patients will undergo lymphodepletion with cyclophosphamide and fludarabine. After which, the TCR-T cell drug product will be administered to the subject by infusion at the assigned dose level. The starting dose level of Arm A (monotherapy) will be DL1 (5 x10 9 TCR+ Cells) administered on Day 0. Dose escalation will continue utilizing the accelerated BOIN design (planned escalation dose levels: 5 x10 9, 4 x10 10 and 1 x10 11 TCR+ Cells). In Arm B, if initiated by protocol, subjects will also receive aldesleukin (interleukin-2) infusion starting on Day 0 (within 24 hours of TCR-T cell product infusion) at 720K IU/kg, every eight hours for up to 4 days. The Follow-up Period will begin after the subject completes their Day 28 visit. Clinical and radiologic response will be assessed at 6 and 12 weeks after TCR-T cell drug product infusion and every 12 weeks thereafter until up to 2 years or study discontinuation (e.g., disease progression, initiation of new anti-cancer therapy, consent withdrawn), whichever occurs first. All subjects will continue to be followed in the Long-Term Follow-up Protocol for up to 15 years post-TCR-T cell drug product infusion. Clinical trial information: NCT05194735.

734 Background: PSCA is a cell surface protein overexpressed in approximately 60% of pancreatic cancers. BPX-601 is an autologous GOCAR-T cell therapy engineered to express a PSCA-CD3ζ CAR and the MyD88/CD40 (iMC) costimulatory domain activated by rimiducid (Rim), designed to boost CAR-T performance in solid tumors. The safety and activity of BPX-601 activated with Rim in PSCA+ metastatic pancreatic cancer is being assessed in a Phase 1/2 clinical trial, BP-012 (NCT02744287). Methods: Phase 1 of BP-012 is a 3+3 dose escalation of BPX-601 (1.25-5 x106/kg) administered on Day 0 with a single, fixed-dose of Rim (0.4 mg/kg) on Day 7 in subjects with previously treated PSCA+ metastatic pancreatic cancer. All 5 subjects in cohort 5B received Flu/Cy lymphodepletion followed by BPX-601 (5 x106/kg) and Rim. BPX-601 kinetics, PBMC phenotype, and serum cytokines were assayed by qPCR, flow cytometry, and cytokine multiplex, respectively. Baseline and on-treatment biopsies were evaluated by RNAscope in situ hybridization. Results: BPX-601 cells expanded in all subjects and persisted up to 9 months (median 42 days). Transient reduction in BPX-601 vector copy number and total T cell count concurrent with Rim infusion, supports margination of activated BPX-601 cells. Increased serum cytokines, such as IFN-γ and GM-CSF, were observed following BPX-601 infusion with further elevation after Rim activation. All subjects with evaluable on-treatment biopsies had infiltration of BPX-601 cells (n = 3) proximal to tumor cells 7-15 days after Rim, but not in an end of treatment biopsy > 200 days after Rim (n = 1). Stratification by best response (RECIST 1.1) revealed stable disease in 3 subjects and progressive disease in 2 subjects was potentially associated with distinct cytokine signatures. Conclusions: BPX-601 GOCAR-T cells expand and persist in patients with PSCA+ metastatic pancreatic cancer and infiltrate metastatic lesions. A peripheral cytokine signature was observed following BPX-601 infusion. Select cytokines were enhanced after GOCAR-T cell activation and may correlate with clinical response. A cohort of subjects exploring serial administration of Rim is open for enrollment. Clinical trial information: NCT02744287.

Background: While chimeric antigen receptor (CAR)-T immunotherapies are remarkably effective against a subset of leukemias and lymphomas, three current hurdles for broad deployment include lack of regulation once administered to the patient, modest efficacy against solid tumors, and the necessity to make separate GMP vectors for each tumor target. Methods: We developed two methods that utilize chemical induction of protein dimerization (CID) to regulate the activity of engineered T cells containing a CAR of broad utility. “Uni-iC9CAR” combines Bellicum's caspase-9-based, rimiducid-inducible safety switch, CaspaCIDe, with a first generation CD16/FCGR3A–CAR. Antigen receptor specificity relies on the interaction of the Fc-binding domain of CD16 with various tumor-targeted antibodies. In the “Uni-GoCAR” strategy, signaling domains from MyD88 and CD40 are fused to two copies of FKBPv36 to generate iMC, which is co-expressed with the CD16-based CAR. In each strategy, the same dimerizer, rimiducid, binds to FKBPv36 with sub-nanomolar affinity to cause the activation of signaling molecules with distinct functions and outcomes. Lastly, we generated the “Uni-CIDeCAR” vector that combines the iCaspase-9 and CD16-CAR activities from Uni-iC9CAR with augmented ligand-independent MyD88/CD40 costimulation to generate a potent universal CAR with a rapid and effective suicide gene, activated by the normally bio-inert ligand, rimiducid. Results: Both Uni-GoCAR and Uni-iC9CAR constructs demonstrated rapid, effective and durable, rituximab-dependent antitumor activity when expressed in human T cells and mixed with Raji B cells as early as 7 days after T cell co-culture at a 1:1 ratio. Additionally, rimiducid induced robust cytokine production, including IL-2 and IL-6, and proliferation in T cells transduced with the Uni-GoCAR vector, which expresses the iMC activation switch. In contrast, co-expression of iC9 in the Uni-iC9CAR vector demonstrated robust rimiducid-dependent T cell apoptosis, thus providing a valuable safety mechanism for clinical applications. Finally, in the enhanced, but regulated, Uni-CIDeCAR vector, iC9 maintains safety in a CD16-CAR that is functionally enhanced by rimiducid-independent, basal MC activity. Conclusion: We report an improved “universal” CAR-T technology that employs a CD16-based CAR (described by Kudo et al (14) Cancer Res) coupled with Bellicum's costimulatory and safety switches to effectively target tumor cells while providing a broad clinical safety net. Citation Format: MyLinh T. Duong, Matthew R. Collinson-Pautz, Aaron E. Foster, J. Henri Bayle, David M. Spencer. Uni-CIDeCAR-T cells: MyD88/CD40-enhanced, Ab-directed CAR incorporating the CaspaCIDe® safety switch. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr A057.