Adoptive Cellular Therapy Research Articles

Expanding Human Breg for Cellular Therapy in Transplantation: Time for Translation.

Regulatory B cells (Breg) are instrumental in protecting allografts in transplantation. Breg signatures are identified in operationally tolerant human kidney transplant recipients and can predict organ survival and acute rejection. Animal models of transplantation and autoimmunity support the use of Breg as an adoptive cellular therapy. Detailed mechanistic studies have identified multiple signaling pathways utilized by Breg in their induction, expansion, and downstream function. These preclinical studies provide the guiding principles, which will inform protocols by which to expand this crucial immunoregulatory population before clinical use. There is an urgent need for novel therapies to improve long-term transplant outcomes and to minimize immunosuppression-related morbidity including life-threatening infection and cancer. Systematic evaluation of the signals, which drive Breg expansion, will be key to transforming the as of yet unharnessed potential of this potent immunoregulatory cell. In this review, we explore the potential avenues of translating Breg subsets from cell culture at the laboratory bench to cell therapy at the patient's bedside. We will discuss the standardization of Breg phenotypes to aid in precursor population selection and quality control of a Breg-cell therapy product. We will evaluate avenues by which to optimize protocols to drive human Breg expansion to levels sufficient for cellular therapy. Finally, we will examine the steps required in process development including scalable culture systems and quality control measures to deliver a viable Breg-cell therapy product for administration to a transplant recipient.

Concomitant Expression of CD39, CD69, and CD103 Identifies Antitumor CD8+ T Cells in Breast Cancer Implications for Adoptive Cell Therapy.

In cancer, an effective immune response involves the action of several different cell types, among which CD8 T cells play a major role as they can specifically recognize and kill cancer cells via the release of cytotoxic molecules and cytokines, being of major importance for adoptive cell transfer (ACT) of ex vivo expanded tumor-infiltrating lymphocytes (TILs). The inflammation resulting from the tumor growth attracts both activated and bystander T cells. For an effective antitumor response, the T cell must express a specific group of chemokine receptors and integrins which include CD103, CD39, CD69, and CD25. These markers had already been analyzed in various cancers, not including breast cancer and their subsequent subtypes, until now. To analyze, the key receptors on ex vivo expanded tumor-infiltrating lymphocytes in luminal A and luminal B breast cancer (BC) subtypes. We were successful in expanding TILs ex vivo using a standard TIL culture condition from a cohort study of 15 primary luminal A and luminal B breast cancer patients. Furthermore, we examined the expression of CD103, CD39, CD69, and CD25 biomarkers after the expansion by flow cytometry. We found that the information about the percentage of TILs obtainable after the ex vivo expansion is not associated to nor it is dependent on the heterogeneity of the TIL population before the expansion and does not differ by the molecular subtype (p>0.05). We also found that there is a major population of memory-resident antitumor CD8+CD103+CD39+ and CD8+CD103+ CD69+ TILs present in the stroma after the expansion when compared to CD4 immunosubtypes (p<0.0001). Only the CD8+CD103+CD39+ subpopulation was related to BC subtype (0.0009). Evidence from our study suggests that CD8 TILs present in the stroma of luminal A and luminal B breast cancer patients can be quantified and phenotyped by flow cytometry and be further expanded ex vivo. The immuno-phenotyping of these markers may be targeted to improve the success of immunotherapeutic approaches, such as adoptive cellular therapy (ACT) in patients with BC.

Mathematical modeling insights into improving CAR T cell therapy for solid tumors with bystander effects

As an adoptive cellular therapy, Chimeric Antigen Receptor T cell (CAR T cell) therapy has shown remarkable success in hematological malignancies but only limited efficacy against solid tumors. Compared with blood cancers, solid tumors present a series of challenges that ultimately combine to neutralize the function of CAR T cells. These challenges include, but are not limited to, antigen heterogeneity - variability in the expression of the antigen on tumor cells, as well as trafficking and infiltration into the solid tumor tissue. A critical question for solving the heterogeneity problem is whether CAR T therapy induces bystander effects, such as antigen spreading. Antigen spreading occurs when CAR T cells activate other endogenous antitumor CD8 T cells against antigens that were not originally targeted. In this work, we develop a mathematical model of CAR T cell therapy for solid tumors that considers both antigen heterogeneity and bystander effects. Our model is based on in vivo treatment data that includes a mixture of target antigen-positive and target antigen-negative tumor cells. We use our model to simulate large cohorts of virtual patients to better understand the relationship involving bystander killing. We also investigate several strategies for enhancing bystander effects, thus increasing CAR T cell therapy’s overall efficacy for solid tumors.

DNA Nanostructures: Advancing Cancer Immunotherapy

AbstractCancer immunotherapy is a groundbreaking medical revolution and a paradigm shift from traditional cancer treatments, harnessing the power of the immune system to target and destroy cancer cells. In recent years, DNA nanostructures have emerged as prominent players in cancer immunotherapy, exhibiting immense potential due to their controllable structure, surface addressability, and biocompatibility. This review provides an overview of the various applications of DNA nanostructures, including scaffolded DNA, DNA hydrogels, tetrahedral DNA nanostructures, DNA origami, spherical nucleic acids, and other DNA‐based nanostructures in cancer immunotherapy. These applications explore their roles in vaccine development, immune checkpoint blockade therapies, adoptive cellular therapies, and immune‐combination therapies. Through rational design and optimization, DNA nanostructures significantly bolster the immunogenicity of the tumor microenvironment by facilitating antigen presentation, T‐cell activation, tumor infiltration, and precise immune‐mediated tumor killing. The integration of DNA nanostructures with cancer therapies ushers in a new era of cancer immunotherapy, offering renewed hope and strength in the battle against this formidable foe of human health.

Accelerating accessibility of CAR-T/NK therapies – Are AlloCARs and rapid manufacturing platforms the road ahead in improving access in multiple myeloma?

While the advent of CAR-T therapies has heralded a new era of efficacious therapies in relapsed/refractory Multiple Myeloma, access continues to be a major limiting factor due to prolonged manufacturing times of autologous products and apheresis and/or manufacturing failures. Allogeneic adoptive cellular therapy products (CAR-T, CAR-NK), currently investigational, are “off-the-shelf” products that may address availability and manufacturing bottlenecks. Novel rapid manufacturing platforms that decrease adoptive cell therapy product development time by weeks are currently being tested in clinical trials and may additionally help bridge the demand-supply chasm. This review provides a comprehensive overview of allogeneic adoptive cellular therapies and rapid manufacturing platforms in development.

The winding road: Infectious disease considerations for CAR-T and other novel adoptive cellular therapies in the era of COVID-19

Adoptive cellular therapies (ACT) are novel, promising treatments for life-threatening malignancies. In addition to the better known chimeric antigen receptor (CAR) T cells, ACTs include tumor infiltrating lymphocytes (TIL), cancer antigen-specific T cell receptors (TCRs), and CAR-NK (natural killer) cells. In key historic milestones, several adoptive therapies recently received FDA approvals, including 6 CAR-T products for the treatment of hematologic malignancies and the first TIL therapy for the treatment for metastatic melanoma. The rapid pace of clinical trials in the field and the discoveries they provide are ushering in a new era of cancer immunotherapy. However, the potential complications of these therapies are still not fully understood. In particular, patients receiving ACT may be at increased risk for severe infections due to immunocompromise resulting from their underlying malignancies, which are further compounded by the immune derangements that develop in the setting of cellular immunotherapy and/or the preconditioning treatment needed to enhance ACT efficacy. Moreover, these treatments are being readily implemented at a time following the height of the COVID-19 pandemic, and it remains unclear what additional risks these patients may face from SARS-CoV-2 and similar infections. Here, we examine the evidence for infectious complications with emerging adoptive therapies, and provide a focused review of the epidemiology, complications, and clinical management for COVID-19 in CAR-T recipients to understand the risk this disease may pose to recipients of other forms of ACT.

High-Risk Neuroblastoma Challenges and Opportunities for Antibody-Based Cellular Immunotherapy.

Immunotherapy has emerged as an attractive option for patients with relapsed or refractory high-risk neuroblastoma (HRNB). Neuroblastoma (NB), a sympathetic nervous system cancer arising from an embryonic neural crest cell, is heterogeneous clinically, with outcomes ranging from an isolated abdominal mass that spontaneously regresses to a widely metastatic disease with cure rates of about 50% despite intensive multimodal treatment. Risk group stratification and stage-adapted therapy to achieve cure with minimal toxicities have accomplished major milestones. Targeted immunotherapeutic approaches including monoclonal antibodies, vaccines, adoptive cellular therapies, their combinations, and their integration into standard of care are attractive therapeutic options, although curative challenges and toxicity concerns remain. In this review, we provide an overview of immune approaches to NB and the tumor microenvironment (TME) within the clinical translational framework. We propose a novel T cell-based therapeutic approach that leverages the unique properties of tumor surface antigens such as ganglioside GD2, incorporating specific monoclonal antibodies and recent advancements in adoptive cell therapy.

A “Prime and Expand” strategy using the multifunctional fusion proteins to generate memory-like NK cells for cell therapy

Adoptive cellular therapy (ACT) using memory-like (ML) natural killer (NK) cells, generated through overnight ex vivo activation with IL-12, IL-15, and IL-18, has shown promise for treating hematologic malignancies. We recently reported that a multifunctional fusion molecule, HCW9201, comprising IL-12, IL-15, and IL-18 domains could replace individual cytokines for priming human ML NK cell programming (“Prime” step). However, this approach does not include ex vivo expansion, thereby limiting the ability to test different doses and schedules. Here, we report the design and generation of a multifunctional fusion molecule, HCW9206, consisting of human IL-7, IL-15, and IL-21 cytokines. We observed > 300-fold expansion for HCW9201-primed human NK cells cultured for 14 days with HCW9206 and HCW9101, an IgG1 antibody, recognizing the scaffold domain of HCW9206 (“Expand” step). This expansion was dependent on both HCW9206 cytokines and interactions of the IgG1 mAb with CD16 receptors on NK cells. The resulting “Prime and Expand” ML NK cells exhibited elevated metabolic capacity, stable epigenetic IFNG promoter demethylation, enhanced antitumor activity in vitro and in vivo, and superior persistence in NSG mice. Thus, the “Prime and Expand” strategy represents a simple feeder cell-free approach to streamline manufacturing of clinical-grade ML NK cells to support multidose and off-the-shelf ACT.

An aptamer-mediated base editing platform for simultaneous knockin and multiple gene knockout for allogeneic CAR-T cells generation

Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene editing platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction of genetic modifications; however, these modifications occur via the generation of DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations and genotoxicity. Here, we apply a novel modular RNA aptamer-mediated Pin-point™ base editing platform to simultaneously introduce multiple gene knockouts and site-specific integration of a transgene in human primary T cells. We demonstrate high editing efficiency and purity at all target sites and significantly reduced frequency of chromosomal translocations compared to the conventional CRISPR-Cas9 system. Site-specific knock-in of a chimeric antigen receptor (CAR) and multiplex gene knockout are achieved within a single intervention and without the requirement for additional sequence-targeting components. The ability to perform complex genome editing efficiently and precisely highlights the potential of the Pin-point platform for application in a range of advanced cell therapies.

Risk of Second Tumors and T-Cell Lymphoma after CAR T-Cell Therapy

BackgroundThe risk of second tumors after chimeric antigen receptor (CAR) T-cell therapy, especially the risk of T-cell neoplasms related to viral vector integration, is an emerging concern.MethodsWe reviewed our clinical experience with adoptive cellular CAR T-cell therapy at our institution since 2016 and ascertained the occurrence of second tumors. In one case of secondary T-cell lymphoma, a broad array of molecular, genetic, and cellular techniques were used to interrogate the tumor, the CAR T cells, and the normal hematopoietic cells in the patient.ResultsA total of 724 patients who had received T-cell therapies at our center were included in the study. A lethal T-cell lymphoma was identified in a patient who had received axicabtagene ciloleucel therapy for diffuse large B-cell lymphoma, and both lymphomas were deeply profiled. Each lymphoma had molecularly distinct immunophenotypes and genomic profiles, but both were positive for Epstein–Barr virus and were associated with DNMT3A and TET2 mutant clonal hematopoiesis. No evidence of oncogenic retroviral integration was found with the use of multiple techniques.ConclusionsOur results highlight the rarity of second tumors and provide a framework for defining clonal relationships and viral vector monitoring. (Funded by the National Cancer Institute and others.)

Targeting Cancers with oHSV-Based Oncolytic Viral Immunotherapy.

The recent success of cancer immunotherapies, such as immune checkpoint inhibitor (ICIs), monoclonal antibodies (mAbs), cancer vaccines, and adoptive cellular therapies (ACTs), has revolutionized traditional cancer treatment. However, these immunotherapeutic modalities have variable efficacies, and many of them exhibit adverse effects. Oncolytic viral Immunotherapy (OViT), whereby viruses are used to directly or indirectly induce anti-cancer immune responses, is emerging as a novel immunotherapy for treating patients with different types of cancer. The herpes simplex virus type-1 (HSV-1) possesses many characteristics that inform its use as an effective OViT agents and remains a leading candidate. Its recent clinical success resulted in the Food and Drug Administration (FDA) approval of Talimogene laherparevec (T-VEC or Imlygic) in 2015 for the treatment of advanced melanoma. In this review, we discuss recent advances in the development of oncolytic HSV-1-based OViTs, their anti-tumor mechanism of action, and efficacy data from recent clinical trials. We envision this knowledge may be used to inform the rational design and application of future oHSV in cancer treatment.

The role of obesity on outcomes of adoptive cellular therapy in solid tumors.

2544 Background: Anti-tumor response and toxicity outcomes with adoptive cell therapy (ACT) have been heterogeneous in solid tumors, and it is still unclear how host factors such as obesity may contribute to efficacy and immune-related safety events. Methods: We conducted a retrospective study including 95 consecutive advanced solid tumor patients who received ACT on protocol in the Department of Investigational Cancer Therapeutics between August 2017 and June 2023. We analyzed baseline clinical characteristics, immune-related safety events, response to ACT, and survival outcomes between obese and non-obese patients. Overweight was defined as body mass index (BMI) 25-29.99 kg/m2 and obesity was defined as BMI ≥ 30 kg/m2 at the time of treatment. Fisher’s exact test was used to compare the proportions of categorical variables between two groups. Pairwise comparison with Bonferroni correction was performed if necessary. T-test was used to compare the mean of continuous variables between two groups. The distributions of overall survival (OS) and progression-free survival (PFS) were estimated by Kaplan-Meier method. Log-rank test was used to compare the distributions of OS and PFS by baseline characteristics. Hazard ratio was estimated in Cox proportional hazard regression model. A two-sided p-value &lt; 0.05 was considered statistically significant. Results: The median age at treatment was 60.9 (range 20.5 – 84). Thirty-nine patients (41.1%) had a BMI (kg/m2) defined as normal weight, 26 (27.4%) as overweight, 27 (28.4%) as obese, and 3 (3.2%) as underweight. Eighty-four (88.4%) were Caucasian; 54 (56.8%) were female. The most prevalent cancers were gastrointestinal (21.1%), gynecologic (15.8%), sarcoma (11.6%), mesothelioma (10.5%), breast (8.4%). The median follow-up was 39.9 months, with a median PFS of 2.8 months (95% CI: 2.3, 3.5) and a median OS of 8.4 months (95% CI: 6.4, 11.0). The risk of death among the obese patients was 0.56 (95% CI: 0.31, 0.99) times the risk of death among normal weight patients (p = 0.045). No weight difference was found between responder and non-responder. While BMI was not associated with the development of cytokine release syndrome (CRS), the patients with normal weight had a higher proportion of immune effector cell-associated neurotoxicity syndrome (ICANS) than the obese patients (23.1% vs 0%, p = 0.049). Conclusions: Obesity was associated with improved survival and decreased ICANS rates following ACT. Our findings may suggest a differential interaction between obesity, different immunotherapeutic modalities, and hematologic vs nonhematologic cancers.

Immunotherapeutic Strategies Targeting Breast Cancer Stem Cells.

Breast cancer is the most commonly diagnosed cancer in women and is a leading cause of cancer death in women worldwide. Despite the implementation of multiple treatment options, including immunotherapy, breast cancer treatment remains a challenge. In this review, we aim to summarize present challenges in breast cancer immunotherapy and recent advancements in overcoming treatment resistance. We elaborate on the inhibition of signaling cascades, such as the Notch, Hedgehog, Hippo, and WNT signaling pathways, which regulate the self-renewal and differentiation of breast cancer stem cells and, consequently, disease progression and survival. Cancer stem cells represent a rare population of cancer cells, likely originating from non-malignant stem or progenitor cells, with the ability to evade immune surveillance and develop resistance to immunotherapeutic treatments. We also discuss the interactions between breast cancer stem cells and the immune system, including potential agents targeting breast cancer stem cell-associated signaling pathways, and provide an overview of the emerging approaches to breast cancer stem cell-targeted immunotherapy. Finally, we consider the development of breast cancer vaccines and adoptive cellular therapies, which train the immune system to recognize tumor-associated antigens, for eliciting T cell-mediated responses to target breast cancer stem cells.

Research advances on TIL therapy for colorectal cancer.

Colorectal cancer (CRC) is a prevalent gastrointestinal malignancy. Tumor-infiltrating lymphocyte (TIL) therapy, a form of adoptive cellular therapy (ACT), involves isolating T lymphocytes from tumor tissues, in vitro expansion, and reintroduction into the body to target and eliminate tumor cells. This article presents an overview of the development and application of TIL therapy in CRC, as well as the associated challenges.

Deleting the mitochondrial respiration negative regulator MCJ enhances the efficacy of CD8+ T cell adoptive therapies in pre-clinical studies

Mitochondrial respiration is essential for the survival and function of T cells used in adoptive cellular therapies. However, strategies that specifically enhance mitochondrial respiration to promote T cell function remain limited. Here, we investigate methylation-controlled J protein (MCJ), an endogenous negative regulator of mitochondrial complex I expressed in CD8 cells, as a target for improving the efficacy of adoptive T cell therapies. We demonstrate that MCJ inhibits mitochondrial respiration in murine CD8+ CAR-T cells and that deletion of MCJ increases their in vitro and in vivo efficacy against murine B cell leukaemia. Similarly, MCJ deletion in ovalbumin (OVA)-specific CD8+ T cells also increases their efficacy against established OVA-expressing melanoma tumors in vivo. Furthermore, we show for the first time that MCJ is expressed in human CD8 cells and that the level of MCJ expression correlates with the functional activity of CD8+ CAR-T cells. Silencing MCJ expression in human CD8 CAR-T cells increases their mitochondrial metabolism and enhances their anti-tumor activity. Thus, targeting MCJ may represent a potential therapeutic strategy to increase mitochondrial metabolism and improve the efficacy of adoptive T cell therapies.

The role and novel use of natural killer cells in graft-versus-leukemia reactions after allogeneic transplantation.

Allogeneic hematopoietic cell transplantation (HCT) has transformed over the past several decades through enhanced supportive care, reduced intensity conditioning (RIC), improved human leukocyte antigen (HLA) typing, and novel graft-versus-host disease (GVHD)-prevention and treatment strategies. Most notably, the implementation of post-transplantation cyclophosphamide (PTCy) has dramatically increased the safety and availability of this life-saving therapy. Given reductions in nonrelapse mortality (NRM) with these advances, the HCT community has placed even greater emphasis on developing ways to reduce relapse - the leading cause of death after HCT. When using RIC HCT, protection from relapse relies predominantly on graft-versus-leukemia (GVL) reactions. Donor lymphocyte infusion (DLI), adoptive cellular therapy, checkpoint inhibition, and post-HCT maintenance strategies represent approaches under study that aim to augment or synergize with the GVL effects of HCT. Optimizing donor selection algorithms to leverage GVL represents another active area of research. Many of these strategies seek to harness the effects of T cells, which for decades were felt to be the primary mediators of GVL and the focus of investigation in relapse reduction. However, there is growing interest in capitalizing on the ability of natural killer (NK) cells to yield potent anti-tumor effects. A potential advantage of NK cell-based approaches over T cell-mediated is the potential to reduce NRM in addition to relapse. By decreasing infection, without increasing the risk of GVHD, NK cells may mitigate NRM, while still yielding relapse reduction through identification and clearance of cancer cells. Most T cell-focused relapse-prevention strategies must weigh the benefits of relapse reduction against the increased risk of NRM from GVHD. In contrast, NK cells have the potential to reduce both, potentially tipping the scales significantly in favor of survival. Here, we will review the role of NK cells in GVL, optimization of NK cell match or mismatch, and burgeoning areas of research in NK cell therapy such as adoptive transfer and chimeric antigen receptor (CAR) NK cells.

Unpaired cysteine insertions favor transmembrane dimerization and induce ligand-independent constitutive cytokine receptor signaling.

Naturally occurring gain-of-function (GOF) mutants have been identified in patients for a variety of cytokine receptors. Although this constitutive activation ofcytokine receptors is strongly associated with malignant disorders, ligand-independent receptor activation is also a useful tool in synthetic biology e.g. to improve adoptive cellular therapies with genetically modified T-cells. Balanced Interleukin (IL-)7 signaling via a heterodimer of IL-7 receptor (IL-7Rα) and the common γ-chain (γc) controls T- and B-cell development and expansion, whereas uncontrolled IL-7 signaling can drive acute lymphoid leukemia (ALL) development. The ALL-driver mutation PPCL in the transmembrane domain of IL-7Rα is a mutational insertion of the four amino acids proline-proline-cysteine-leucine and leads to ligand-independent receptor dimerization and constitutive activation. We showed here in the cytokine-dependent pre-B-cell line Ba/F3 that the PPCL-insertion in a synthetic version of the IL-7Rα induced γc-independent STAT5 and ERKphosphorylation and also proliferation of the cells and that booster-stimulation by arteficial ligands additionally generated non-canonical STAT3 phosphorylation via the synthetic IL-7Rα-PPCL-receptors. Transfer of theIL-7Rα transmembrane domain with the PPCL insertion intonatural and synthetic cytokine receptor chains of the IL-6, IL-12 and Interferon families also resulted in constitutive receptor signaling. In conclusion, our data suggested that the insertion of the mutated PPCL IL-7Rα transmembrane domain is an universal approach to generate ligand-independent, constitutively active cytokine receptors.

Evolution of the clinical-stage hyperactive TcBuster transposase as a platform for robust non-viral production of adoptive cellular therapies

Cellular therapies for the treatment of human diseases, such as chimeric antigen receptor (CAR) T and NK cells have shown remarkable clinical efficacy in treating hematological malignancies, however current methods mainly utilize viral vectors which are limited by their cargo size capacities, high cost, and long timelines for production of clinical reagent. Delivery of genetic cargo via DNA transposon engineering is a more timely and cost-effective approach, yet has been held back by less efficient integration rates. Here, we report the development of a novel hyperactive TcBuster (TcB-M) transposase engineered through structure guided and in vitro evolution approaches that achieves high-efficiency integration of large, multicistronic CAR-expression cassettes in primary human cells. Our proof of principle TcB-M engineering of CAR-NK and CAR-T cells show low integrated vector copy number, a safe insertion site profile, robust in vitro function, and improves survival in a Burkitt lymphoma xenograft model in vivo. Overall, TcB-M is a versatile, safe, efficient and open-source option for the rapid manufacture and preclinical testing of primary human immune cell therapies through delivery of multicistronic large cargo via transposition.

Abstract CT160: A phase I trial to evaluate allogeneic NKG2DL-targeting chimeric antigen receptor-grafted γδ T cells in subjects with advanced solid tumors or hematological malignancies (the ANGELICA Trial)

Abstract Background: Adoptive cellular therapy (ACT) has been transformative in the treatment of hematological malignancies. Limitations of ACTs include difficulty identifying suitable tumor antigens for solid tumors, the ability to target only one antigen per construct which commonly results in secondary resistance due to antigen escape, and largely autologous approaches which are challenging in heavily pre-treated patients with poor marrow function and those with rapidly progressive disease who may not receive their personalised cellular products in time. This is a phase I study evaluating a novel off-the-shelf allogenic mRNA-electroporated NKG2D ligand-targeting CAR-grafted γδ T cell therapy. NKG2DLs are a group of 8 types of stress-induced cancer antigens which are preferentially and widely expressed on tumor cells from diverse tissue origins but are not typically present on normal tissue. NKG2DL-targeting γδ T cells will be manufactured from peripheral blood mononuclear cells isolated from healthy donors enrolled on the donor protocol of the ANGELICA trial. ANGELICA is a phase I study evaluating the safety, tolerability and recommended phase 2 dose (RP2D) of NKG2DL-targeting CAR-grafted γδ T cells in patients with treatment refractory tumors. Methods: Patients will be enrolled in a 3+3 design. Dose escalation will be performed at 3 dose levels: 1x108, 3x108 and 1x109 per infusion (adjusted for body weight). Lymphodepletion with fludarabine 25mg/m2/day and cyclophosphamide 250mg/m2/day will given for 3 days and completed at least 2 days prior to the first cycle of treatment. Patients will receive 4 doses of weekly infusions for the first cycle, with up to 5 subsequent infusions every 2 months as maintenance. Patients will receive intravenous zoledronic acid 1mg prior to each cellular infusion and subcutaneous IL-2 1x106 IU/m2 within 2 hours of each cellular infusion as pre-clinical data demonstrated cancer cell sensitisation and prolonged γδ T cell survival with these adjuncts. Dose-limiting toxicities will be assessed over the first 8 weeks. Adverse events, response rates (RECIST v1.1), survival outcomes and immunomonitoring (immune cell phenotyping and serum cytokine analysis) will be assessed. The trial is currently enrolling healthy donors; enrolment of patients at the first dose level will begin in February 2024. NCT05302037 TABLE 1: NAND Dose Level and Schedule Dose Level Dose per infusion Infusion schedule Number of patients 1 Weight 65kg and above: 1x108.Weight less than 65kg: 1.5x106. Cycle 1: weekly x 4 infusions. Maintenance phase: 2-monthly x 5 infusions. 3-6 2 Weight 65kg and above: 3x108.Weight less than 65kg: 4.6x106. 3-6 3 Weight 65kg and above: 1x109.Weight less than 65kg: 1.5x107. 3-6 Citation Format: Joan Choo, Wee Kiat Tan, Lucas Luk, Jieming Zeng, Teck Guan Soh, Sou Yen Soon, Jedidah Lieow, Calista Wong, Mei Yan Pang, Sudipto Bari, Michelle Poon, Liang Piu Koh, Wee Joo Chng, Anand Jeyasekharan, Lip Kun Tan, Esther Chan, Raghav Sundar. A phase I trial to evaluate allogeneic NKG2DL-targeting chimeric antigen receptor-grafted γδ T cells in subjects with advanced solid tumors or hematological malignancies (the ANGELICA Trial) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT160.

Abstract LB065: Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors

Abstract Introduction: Adoptive cellular therapies (ACT) have encountered challenges in solid tumors due in part to the immunosuppressive tumor microenvironment (TME). We have developed OBX-115, TIL engineered to express mbIL15 regulatable using the cytoDRiVE® platform, which allows for TIL expansion, persistence, and anti-tumor efficacy under control of the FDA-approved small-molecule ligand, acetazolamide (ACZ), eliminating the need for co-administration of IL2 (NCT05470283). LIGHT, a tumor necrosis factor family member, interacts with lymphotoxin beta receptor (LTbR) and herpes virus entry mediator (HVEM) found on various TME cell types, including stromal cells such as cancer associated fibroblasts (CAF). In preclinical studies, LIGHT expression within a tumor has been linked to the formation of tertiary lymphoid structures and vascular normalization (Ramachandran Cancer Cell 2023), both associated with better clinical outcomes (Sautès-Fridman Nat Rev Cancer 2019). We hypothesized that engineering TIL with regulatable mbIL15 and LIGHT expression could enhance their efficacy by modifying the TME. Methods: TIL from colorectal (CRC) and head and neck squamous cell carcinoma (HNSCC) were transduced with retroviral vectors to express regulatable mbIL15 and LIGHT. ACZ-induced surface expression of mbIL15 and LIGHT in expanded TIL was examined using flow cytometry. Functional signaling of LIGHT was assessed through co-culture with Jurkat-HVEM-NF-kappaB reporter cells and LTbR+ HUVEC cells. In vitro, engineered TIL were tested in stromal-rich tumor models (CRC and HNSCC) by co-culturing with autologous patient-derived tumor/CAF hybrid spheroids. In vivo, antigen-independent TIL persistence was assessed in NSG mice without exogenous IL2. Syngeneic studies were performed to assess the efficacy of adoptively transferred mbIL15 and LIGHT-engineered Pmel cells (CD8+ T cells transgenic for a gp100-specific T cell receptor) in a subcutaneous cold tumor model (B16-F10). Results: Engineered TIL were successfully expanded without exogenous IL2. ACZ-dependent mbIL15 and LIGHT expression were confirmed, validating co-regulation and functionality in vitro. TIL engineered with mbIL15 and LIGHT displayed significantly increased cytotoxicity against autologous tumor/CAF spheroids compared with TIL expressing mbIL15 alone (p&amp;lt;0.005) in CRC and HNSCC tumor/CAF hybrid models. TIL with mbIL15 and LIGHT expanded in vivo and persisted for ≥42 days without exogenous IL2 support. Moreover, Pmel cells engineered with mbIL15 and LIGHT demonstrated durable anti-tumor efficacy in B16-F10 tumor-bearing mice, which was greater than Pmel cells engineered with mbIL15 alone (p&amp;lt;0.01). Conclusions: These preclinical results suggest that TIL engineered with regulatable mbIL15 and LIGHT using the cytoDRiVE platform have the potential to address the high unmet clinical need in cold tumors with suppressive TME, which are currently not amenable to ACT. Citation Format: Balazs Koscso, Zheng Ao, Carmela Passaro, Nirzari Shah, Ngoc Ly, Patricia Timpug, Bulent A. Aksoy, Dexue Sun, Dan Jun Li, Kerri-Lynn Sheahan, Violet Young, Theresa Ross, Benjamin Primack, Meghan Langley, Jeremy Tchaicha, Dhruv K. Sethi, Jan ter Meulen, Michelle Ols. Tumor-infiltrating lymphocytes (TIL) engineered with regulatable membrane-bound IL15 (mbIL15) and LIGHT (TNFSF14) show enhanced efficacy in fibroblast-containing cold tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB065.