Distinct FAP-CAR constructs target non-overlapping epitopes on FAP protein

Cellular immunotherapies show remarkable efficacy against hematologic malignancies. However, applying these therapies against solid tumors is challenging. Among the obstacles are the lack of tumor-specific antigens and the immunosuppressive tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) expressing fibroblast activation protein (FAP) are key contributors to shaping this immunosuppressive landscape, yet developing effective strategies for targeting these cells remains an ongoing challenge. In this study, we describe the design, generation, and characterization of MiNK-215, an allogeneic human invariant NK T (iNKT) cell therapy in which iNKT cells were engineered to express an FAP-targeting chimeric antigen receptor (CAR) and to secrete IL15 to remodel the TME and enhance antitumor activity. MiNK-215 modulated multifunctional immune responses by enhancing T-cell responsiveness, dendritic cell activation, M1 macrophage polarization, and tumor killing. In a lung tumor mouse model, MiNK-215 depleted FAP+ CAFs, enhanced antigen-specific T-cell infiltration, and promoted durable antitumor immunity without off-target toxicity. These findings were extended to human organoid models of treatment-refractory microsatellite-stable colorectal cancer liver metastases, establishing FAP-CAR-IL15 iNKT cells as a promising strategy to overcome immunotherapy resistance in solid tumors. See related Spotlight by Albelda, p. 184.

DEGs in antigen activated FAP-CAR iNKT cells with and without the IL-15 transgene​

Both primary and metastatic brain tumors rely on signals from the surrounding environment for their survival and progression. In particular, the most common and lethal brain cancer, glioblastoma (GBM), derived from glial cells (astrocytes or microglia), has been shown to integrate into synaptic networks and to receive paracrine signals from neighbouring tumor microenvironment (TME) cells. There is increasing evidence that metastatic disease in the brain exhibits similar behavior. The TME both maintains malignant cells and is maintained by them, a process that relies on cancer stem cells (CSCs). These stem cells and their signaling mechanisms, including in the case of GBM, “GSCs,” provide possible novel targets for immunotherapy. In this review, we will discuss the integration of primary and malignant brain tumors into normal synaptic networks, the role of tumor stem cells and the TME in this integration, and the potential for immunotherapeutic targeting of these processes.

Despite significant advances in cancer therapies, many malignancies remain resistant to current treatments due to complex immunosuppressive mechanisms, limited neoantigen expression, and dynamic tumor adaptations, underscoring the need for innovative therapeutic strategies. Adoptive cell therapy (ACT), particularly with chimeric antigen receptors (CARs and recombinant TCRs) targeting cancer-associated antigens, has emerged as a transformative strategy. However, conventional CAR-T cell therapies face substantial limitations such as manufacturing challenges, severe toxicities, and limited efficacy against solid tumors. Invariant natural killer T (iNKT) cells, a unique lymphocyte subset bridging innate and adaptive immunity, have emerged as a compelling alternative platform for CAR-based therapies, due to their distinctive ability to persist, penetrate in and remodel the tumor microenvironment (TME). Unlike conventional T cells, iNKT cells exhibit rapid activation without priming, potent cytotoxicity, and extensive immunomodulatory functions. Furthermore, the inherent immunomodulatory properties of iNKT cells through interactions with the monomorphic antigen-presenting molecule CD1d or stress ligands augment endogenous anti-tumor immunity by activating NK cells and cytotoxic T lymphocytes, promoting dendritic cell maturation, and reducing immunosuppressive myeloid cells, unlike other Innate T cells. CAR-engineered iNKT (CAR-iNKT) cells therefore leverage multiple targeting mechanisms through their native semi-invariant T-cell receptor (TCR), NK receptors (NKRs) and engineered CARs, enabling broader and more effective tumor recognition while actively reshaping immunosuppressive TME. Notably, iNKT cells lack alloreactivity, circumventing the risk of graft-versus-host disease (GvHD), positioning CAR-iNKT cells as ideal candidates for “off-the-shelf” allogeneic therapies that can overcome the limitations of existing immunotherapies.

Over the past 30 years, work of immunologists worldwide has phenotypically and functionally defined “Natural Killer T cells” (NKT) and their subsets, including “invariant Natural Killer T cells” (iNKT). NKT cells make up a substantial fraction of T cells that express NK cell markers and have TCRs restricted to either conventional MHC molecules or the monomorphic CD1d molecule. Among these, iNKT cells are CD1d-restricted and more common within NKT cells than T cells without NK markers. While the definition of NKT cells, whether based on phenotype, function, or both, remains a topic of debate, iNKT cells represent a distinct T cell population characterized by a recurrent, conserved TCR rearrangement (TRAV10–TRAJ18 in humans) paired with a limited Vβ repertoire (mostly encoded by TRBV25-1 in humans). iNKT cells are restricted by CD1d, which, unlike CD1a-c molecules, is expressed not only on professional antigen-presenting cells and thymocytes but also on certain non-hematopoietic somatic tissues, both normal and neoplastic. Like all CD1 family members, CD1d presents various lipid antigens by accommodating their long hydrophobic tails in deep binding pockets, in contrast to the shallow peptide grooves of conventional MHC molecules. However, the ligand repertoire of CD1d is distinct from that of CD1a-c. This review focuses on CD1d-restricted iNKT cells. Activation of iNKT cells via their semi-invariant TCR, often in synergy with NK receptors and other co-stimulatory molecules, triggers a rapid, polyfunctional response. Unlike conventional MHC-restricted T cells, individual iNKT cells can simultaneously produce both Th1- and Th2-type cytokines and exert cytotoxic activity in an immune synapse-directed fashion. Through this combination of direct cytotoxicity and cytokine-mediated immunomodulation, iNKTs can eliminate target cells while activating myeloid and other lymphoid populations to amplify immune responses. Their versatility has fueled growing interest in harnessing iNKT cells across inflammatory, infectious, and oncological diseases, where early-phase studies have demonstrated their safety and preliminary efficacy. Moreover, because they are restricted by the non-polymorphic CD1d molecule and possess immune-regulatory properties, iNKT cells lack graft-versus-host potential, making them ideal candidates for allogeneic, off-the-shelf therapies. This review summarizes how iNKT cells are being reimagined as innovative tools for immune intervention across a range of clinical settings.

Abstract Imvax has developed the Goldspire™ platform to create IGV-001, an autologous biologic-device combination product for the treatment of newly diagnosed glioblastoma (ndGBM). IGV-001 consists of autologous GBM tumor cells and an antisense oligonucleotide against IGF-1R mRNA (IMV-001), irradiated and administered via biodiffusion chambers (BDCs) implanted in the abdomen. Together, these components stimulate immunogenic cell death and antigen release. IGV-001 was well tolerated and multiple efficacy signals were observed in a Phase 1b study in patients with ndGBM (Andrews et al., 2021), including significant improvements in progression-free survival (PFS), radiographic evidence of tumor response, and changes in immune response biomarkers. The Phase 1b study enrolled 33 patients in four different cohorts implanted with 10 or 20 BDCs for 24 or 48 hours. Here we report on 6 subjects with survival beyond 4 years, including 5 subjects who survived 5 years or more (15.2%). There were 4 male and 2 female subjects, with a median age of 52 years (range 32-75). At diagnosis the MGMT promoter was methylated in 4 of 5 subjects that survived 5 years or more. We also report on T cell receptor Vβ CDR3 region sequencing of peripheral blood mononuclear cells and tissue infiltrated lymphocytes that was performed in a subset of 9 patients, including 3 subjects who survived beyond 4 years. Immune correlates of IGV-001 suggest an association between peripheral T cell clonal expansion and PFS/OS outcomes. A Phase 2b randomized, multicenter, double-blind, placebo-controlled study to assess the safety and efficacy of IGV-001 in patients with ndGBM (NCT04485949) has completed enrollment and results are expected to be available in 2025.

Purpose To date, immunotherapeutic approaches in glioblastoma (GBM) have had limited clinical efficacy as compared to other solid tumors. Here we explore autologous cell treatments that have the potential to circumvent treatment resistance to immunotherapy for GBM. Methods We performed literature review and assessed clinical outcomes in phase 1 safety trials as well as phase 2 and 3 autologously-derived vaccines for the treatment of newly-diagnosed GBM. In one recent review of over 3,000 neuro-oncology phase 2 and phase 3 clinical trials, most trials were nonblinded (92%), single group (65%), nonrandomized (51%) and almost half were GBM trials. Only 10% involved a biologic and only 2.2% involved a double-blind randomized trial design. Results With this comparative literature review we conclude that our autologous cell product is uniquely antigen-inclusive and antigen-agnostic with a promising safety profile as well as unexpected clinical efficacy in our published phase 1b trial. We have since designed a rigorous double-blinded add-on placebo-controlled trial involving our implantable biologic drug device. We conclude that IGV-001 provides a novel immunotherapy platform for historically intransigent ndGBM in this ongoing phase 2b trial (NCT04485949).

Invariant natural killer T (iNKT) cells, a unique T cell population, lend themselves for use as adoptive therapy due to diverse roles in orchestrating immune responses. Originally developed for use in cancer, agenT-797 is a donor-unrestricted allogeneic ex vivo expanded iNKT cell therapy. We conducted an open-label study in virally induced acute respiratory distress syndrome (ARDS) caused by the severe acute respiratory syndrome-2 virus (trial registration NCT04582201). Here we show that agenT-797 rescues exhausted T cells and rapidly activates both innate and adaptive immunity. In 21 ventilated patients including 5 individuals receiving veno-venous extracorporeal membrane oxygenation (VV-ECMO), there are no dose-limiting toxicities. We observe an anti-inflammatory systemic cytokine response and infused iNKT cells are persistent during follow-up, inducing only transient donor-specific antibodies. Clinical signals of associated survival and prevention of secondary infections are evident. Cellular therapy using off-the-shelf iNKT cells is safe, can be rapidly scaled and is associated with an anti-inflammatory response. The safety and therapeutic potential of iNKT cells across diseases including infections and cancer, warrants randomized-controlled trials.

Goldspire (TM) is a personalized immunotherapy platform that combines whole tumor-derived cells with antisense oligonucleotide (IMV-001) against Insulin-Like Growth Factor-1 Receptor (IGF-1R) in biodiffusion chambers (BDCs; 0.1 mu m pore). BDCs are exposed to 5-6 Gy and implanted at abdominal sites for similar to 48 h to deliver an antigenic payload and immunostimulatory factors to train the immune system. Lead product IGV-001 was evaluated in newly diagnosed glioblastoma (ndGBM) patients in Phase 1a and 1b trials (NCT02507583). A Phase 2b study (NCT04485949) recently completed enrollment. Preventative treatment with tumor-specific products manufactured with Goldspire limited tumor progression and extended overall survival in mice challenged with bladder, pancreatic, ovarian, colorectal, or renal carcinomas. The benefit of this immunotherapy was enhanced with anti-PD-1; combination treatment was superior to either monotherapy in orthotopic GBM and melanoma models. Lastly, Goldspire elicited immune T cell activation and memory phenotypes against patient-derived endometrial tumor-derived products in co-cultures with matching immune cells.

Abstract INTRODUCTION Imvax is developing GoldspireTM, a personalized immunotherapy that combines whole tumor-derived cells with an antisense oligonucleotide against insulin-like growth factor 1 receptor (IGF-1R; IMV-001) in biodiffusion chambers (BDCs; 0.1 µm pore). BDCs are irradiated and implanted at abdominal sites for ~48h to deliver an antigenic payload and immunostimulatory factors that together train the immune system to attack tumor cells. The lead product, IGV-001, was evaluated in newly diagnosed glioblastoma (ndGBM) patients in a phase 1b study. Median overall survival (OS) of highest exposure IGV-001-treated Stupp-eligible patients (n=10) was 38.2 mos compared with 16.2 mos in standard-of-care-treated patients (p=0.044; ClinicalTrials.gov NCT02507583). A Phase 2b study in ndGBM is currently underway (ClinicalTrials.gov NCT04485949), where a primary endpoint of progression-free survival and key secondary endpoints of overall survival and safety will be evaluated. METHODS The orthotopically implanted GBM (luciferase-expressing GL261-luc2) model was utilized to evaluate the efficacy of mIGV-001 (the murine version of IGV-001) alone or in combination with anti-PD-1 immunotherapy. The following four groups were compared: mIGV-001 or BDC placebo + isotype or anti-PD-1 antibody (4 doses total every 3-4 days). GL261-luc2 cells were treated with IGF-1R antisense IMV-001. The treated cell suspension was loaded into clinical-grade BDCs (0.1-µm pore size, polyvinylidene difluoride membrane) at a density of 1 × 106 cells per BDC and then irradiated with 5–6 Gy. mIGV-001 or BDCs filled with saline were administered 28 days prior to tumor challenges and tumor progression and survival were monitored for an additional 100 days. RESULTS Preventative treatment with mIGV-001 limited tumor progression and extended OS in mice orthotopically (i.e., intracranially) challenged with GL261-luc2 cells. The therapeutic benefit of mIGV-001 was enhanced with anti-PD-1 treatment and the combination therapy was superior to either monotherapy.

Background: Imvax is developing a novel personalized immunotherapy platform that combines whole-tumor derived cells with an antisense oligonucleotide against insulin-like growth factor 1-receptor in implantable biodiffusion chambers (BDCs; 0.1 μm pore-size). The lead product, IGV-001, was evaluated in newly diagnosed glioblastoma (GBM) patients in a phase 1b clinical trial. Median overall survival of highest exposure IGV-001-treated ‘Stupp-eligible patients (n=10) was 38.2 months compared with 16.2 months in current standard-of-care-treated patients (p=0.044) (Andrews 2021). Imvax also reported anti-tumor activity of the murine variant of this product, mIGV-001, in the GL261 GBM mouse model and detected mIGV-001-induced immune responses in BDC-draining lymph nodes. Since reactive oxygen species (ROS) overproduction can result in immunogenic cell death, we investigated the role of ROS formation as a mediator of cell death in mIGV-001 as well as the generation of subcellular particles that, when released from the BDCs, may provide a tumor antigen payload with potential anti-tumor activity. Methods: Mouse (m) or human (h) variants of IGV-001 were prepared using mouse GL261 or human T98G GBM cells, respectively, and BDCs were incubated for 24-48 h. ROS levels were detected by flow cytometry and fluorescence microscopy via oxidation of H2DCF (a fluorescein analog used to quantify ROS in cells) in the presence or absence of the antioxidant N-acetyl-cysteine (NAC). m/hIGV-001 viability was assessed by flow cytometry using Annexin V/7-AAD staining. Particle size distribution in hIGV-001 BDC contents was analyzed using a Nanosight NS300. The transport of subcellular particles (25-90 nm) across the BDC membrane was modeled under dynamic and static conditions using MATLAB® 2022a and confirmed by in vitro studies. Results: After 24 h in vitro, ROS levels in mIGV-001 were significantly increased along with overt cell death. ROS scavenging with NAC resulted in a cell viability rescue of approximately 50% (from ~40% to ~60% viable cells). Similar trends, although of reduced magnitude, were observed in hIGV-001. Particle analysis within hIGV-001 BDCs showed that after 48 h in culture, dead cells produced particles small enough to diffuse through the BDC membrane. Computational models corroborated that under static conditions, particle equilibrium inside and outside BDCs was achieved after 40 h, 80 h, and 260 h for 25 nm, 50 nm, and 90 nm particles, respectively. Under dynamic conditions, equilibrium was reached in less than 5 h irrespective of particle size. Conclusions: The use of a surgically implantable drug-device combination product induces ROS-associated immunogenic cell death and generation of a tumor-derived immunogenic payload that can be taken up by local DCs, which travel to the proximal draining lymph nodes to activate anti-tumor T cells. Citation Format: Christopher Cultrara, Kenneth Kirby, Essam Elrazaq, Christopher Uhl, Amelia Zellander, Lorenzo Galluzzi, Mark Exley, Jenny Zilberberg. ROS-dependent activation of immunogenic glioblastoma cell death & release of immunogenic particles by an autologous cell-based immunotherapeutic platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1812.

Determination of severity for adverse events

Pan-Cancer Immunity Panel for advanced analysis of T cell activation scores

Background IGV-001 is a personalized, autologous cancer cell-based immunotherapy conceived to deliver a tumor-derived antigenic payload in the context of immunostimulatory signals to patients with glioblastoma (GBM). IGV-001 consists of patient-derived GBM cells treated with an antisense oligodeoxynucleotide against insulin-like growth factor 1 receptor (IGF1R) and placed in proprietary biodiffusion chambers (BDCs). The BDCs are then exposed to 5–6 Gy radiation and implanted at abdominal sites for ~48 hours. IGV-001 has previously been shown to be generally safe with promising clinical activity in newly diagnosed GBM patients. Methods Mouse (m) or human (h) variants of IGV-001 were prepared using GL261 mouse GBM cells or human GBM cells, respectively. BDCs containing vehicle or mIGV-001 were implanted in the flanks of C57BL/6 albino female mice in preventative and therapeutic experiments, optionally in combination with a programmed cell death 1 (PD-1) blocker. Bioactivity of the general approach was also measured against hepatocellular carcinoma Hepa 1–6 cells. Mice were followed for the growth of subsequently implanted or pre-existing tumors and survival. Draining lymph nodes from mice receiving mIGV-001 were immunophenotyped. mIGV-001 and hIGV-001 were analyzed for extracellular ATP and high mobility group box 1 (HMGB1) as indicators of immunogenic cell death (ICD), along with flow cytometric analysis of viability, surface calreticulin, and reactive oxygen species. Stress and cell death-related pathways were analyzed by immunoblotting. Results IGV-001 causes oxidative and endoplasmic reticulum stress in GL261 cells, resulting in a cytotoxic response that enables the release of antigenic material and immunostimulatory, ICD-associated molecules including ATP and HMGB1 from BDCs. Immunophenotyping confirmed that IGV-001 increases the percentage of dendritic cells, as well as effector, and effector memory T cells in BDC-draining lymph nodes. Consistent with these observations, preventative IGV-001 limited tumor progression and extended overall survival in mice intracranially challenged with GL261 cells, a benefit that was associated with an increase in tumor-specific T cells with effector features. Similar findings were obtained in the Hepa 1–6 model. Moreover, therapeutically administered IGV-001 combined with PD-1 delayed progression in GBM-bearing mice. Conclusions These results support treatment with IGV-001 to induce clinically relevant ICD-driven anticancer immune responses in patients with GBM.