Microenvironment Of Solid Tumors Research Articles

Advances in 2,3-Dimethylmaleic Anhydride (DMMA)-Modified Nanocarriers in Drug Delivery Systems.

Cancer represents a significant threat to human health. The cells and tissues within the microenvironment of solid tumors exhibit complex and abnormal properties in comparison to healthy tissues. The efficacy of nanomedicines is inhibited by the presence of substantial and complex physical barriers in the tumor tissue. The latest generation of intelligent drug delivery systems, particularly nanomedicines capable of charge reversal, have shown promise in addressing this issue. These systems can transform their charge from negative to positive upon reaching the tumor site, thereby enhancing tumor penetration via transcytosis and promoting cell internalization by interacting with the negatively charged cell membranes. The modification of nanocarriers with 2,3-dimethylmaleic anhydride (DMMA) and its derivatives, which are responsive to weak acid stimulation, represents a significant advance in the field of charge-reversal nanomedicines. This review provides a comprehensive examination of the recent insights into DMMA-modified nanocarriers in drug delivery systems, with a particular focus on their potential in targeted therapeutics. It also discusses the synthesis of DMMA derivatives and their role in charge reversal, shell detachment, size shift, and ligand reactivation mechanisms, offering the prospect of a tailored, next-generation therapeutic approach to overcome the diverse challenges associated with cancer therapy.

Mechanoimmunology in the solid tumor microenvironment.

The tumor microenvironment (TME) is a complex and dynamic ecosystem that adjoins the cancer cells within solid tumors and comprises distinct components such as extracellular matrix, stromal and immune cells, blood vessels, and an abundance of signaling molecules. In recent years, the mechanical properties of the TME have emerged as critical determinants of tumor progression and therapeutic response. Aberrant mechanical cues, including altered tissue architecture and stiffness, contribute to tumor progression, metastasis, and resistance to treatment. Moreover, burgeoning immunotherapies hold great promise for harnessing the immune system to target and eliminate solid malignancies; however, their success is hindered by the hostile mechanical landscape of the TME, which can impede immune cell infiltration, function, and persistence. Consequently, understanding TME mechanoimmunology - the interplay between mechanical forces and immune cell behavior - is essential for developing effective solid cancer therapies. Here, we review the role of TME mechanics in tumor immunology, focusing on recent therapeutic interventions aimed at modulating the mechanical properties of the TME to potentiate T cell immunotherapies, and innovative assays tailored to evaluate their clinical efficacy.

Immunomodulatory alpha neutrophils: The first-in-class neutrophil progenitor-based allogeneic immuno-cell therapy with cytotoxic and immunomodulatory functionality for the treatment of solid tumours.

e14523 Background: Adoptive cell therapy for the treatment of cancer has largely focused on the administration of autologous engineered αβ T cells. Although successful in some haematological malignancies, the solid tumour microenvironment (TME) poses significant challenges to the efficacy of CAR-T cells, including physical barriers, heterogeneous antigen expression or antigen escape and immunosuppressive mediators. Remodeling the immunosuppressive solid TME is pivotal to the success of immunotherapies. Recent studies have highlighted key roles for neutrophils in tumour control and in facilitating successful responses to cancer immunotherapy, with neutrophil functional heterogeneity within the TME becoming increasingly appreciated. Methods: At LIfT Biosciences we have developed the first-in-class, stem cell-derived immuno-cell therapy with anti-cancer neutrophil properties called Immunomodulatory Alpha Neutrophils (IMANs). IMANs are derived from CD34+ stem cells expanded and differentiated in proprietary media compositions in a simple and scalable GMP-compliant manufacturing process. IMANs are designed to recapitulate those neutrophil states that are both cytotoxic and capable of immunomodulating the solid TME for long-lasting tumour control. Off-the-shelf, allogeneic, innate IMANs have the potential to provide major clinical benefits in the treatment of multiple solid tumours. Results: IMANs multimodal mechanisms of action (MoA) have been demonstrated in different state-of-the-art solid tumour models, including co-culture with patient-derived xenograft organoids (PDX-O) and with 3D ex vivo patient tissues (tumour-on-a-chip). Direct antigen-independent cytotoxicity towards PDX-O from a range of solid tumour indications has been demonstrated. Additionally, and fundamental to IMANs MoA is their ability to migrate to the solid TME and recruit and activate recipients’ anti-tumour immune responses. We have demonstrated IMANs ability to rapidly migrate into the solid TME and promote recruitment of patients’ immune cells leading to enhanced tumour cell death using tumour-on-a-chip technology accompanied by daily live cell imaging. Mechanistically, co-culture with IMANs enhanced expression of co-stimulatory receptors on T and NK cells, such as OX40 and 4-1BB, as well as promoting secretion of TME-modulating factors, such as CXCL10. Furthermore, co-culture with IMANs enhanced secretion of IFN-γ by activated tumour-infiltrating lymphocytes. Conclusions: These cytotoxic and immunomodulatory functions enable IMANs to remodel the TME, leaving the solid tumour more susceptible to host immune responses.

PD-1-CD28-enhanced receptor and CD19 CAR-modified tumor-infiltrating T lymphocytes produce potential anti-tumor ability in solid tumors

The limited expansion ability and functional inactivation of T cells within the solid tumor microenvironment are major problems faced during in the application of using tumor-infiltrating lymphocytes (TILs) in vivo. We sought to determine whether TILs carrying a PD-1-CD28-enhanced receptor and CD19 CAR could overcome this limitation and mediate tumor regression. First, anti-tumor effects of PD-1-CD28-enhanced receptor or CD19 CAR modified NY-ESO-1-TCR-T cells to mimic the TILs function (hereafter “PD-1-CD28-TCR-T” or “CD19 CAR-TCR-T” cells, respectively) were tested using the NY-ESO-1 over-expressed tumor cell line in vitro and in a tumor-bearing model. Furthermore, the safety and anti-tumor ability of S-TILs (TILs modified through transduction with a plasmid encoding the PD-1-CD28-T2A-CD19 CAR) were evaluated in vivo. PD-1-CD28-TCR-T cells showed a formidable anti-tumor ability that was not subject to PD-1/PD-L1 signaling in vivo. CD19 CAR-TCR-T cells stimulated with CD19+ B cells exhibited powerful expansion and anti-tumor abilities both in vitro and in vivo. Three patients with refractory solid tumors received S-TILs infusion. No treatment-related mortality was observed, and none of the patients experienced serious side effects. One patient with melanoma achieved a partial response, and two patients with colon or kidney cancer achieved long-term stable disease following S-TILs therapy. To the best of our knowledge, this is the first study describing the safety and efficacy of the adoptive transfer of autologous S-TILs to control disease in patients with advanced cancers, suggesting that S-TILs may be a promising alternative therapy for cancer.

Abstract PR010: Exploratory analysis of tumor tertiary lymphoid structures using a novel artificial intelligence–based approach in patients with muscle-invasive urothelial carcinoma from the CheckMate 274 trial

Abstract Introduction: The phase 3 randomized CheckMate 274 trial in patients (pts) with high-risk muscle-invasive urothelial carcinoma (MIUC) demonstrated improved disease-free survival (DFS) with nivolumab (NIVO) vs placebo (PBO) in intent-to-treat (ITT) and tumor programmed death ligand 1 expression ≥1% pts (primary endpoints). With extended follow-up, continued efficacy was observed, and immature overall survival favored NIVO in both populations. Extending from previous exploratory tumor biomarker analyses (Necchi et al. ESMO 2022), we conducted a post hoc analysis of tertiary lymphoid structures (TLS), which are ectopic lymphoid structures often found within the solid tumor microenvironment. A subset of TLS has been associated with improved outcomes to neoadjuvant immunotherapy in UC. Using artificial intelligence (AI)–based algorithms, we explored the biomarker utility of TLS from CheckMate 274. Methods: We used a reproducible AI-based algorithm to identify and classify TLS in situ using hematoxylin and eosin (H&E) images from pretreatment tumor tissue specimens (n=272/699 treated pts). Images were scored for the presence or absence of lymphoid aggregate (LA), immature TLS (iTLS), and mature TLS (mTLS) and scored as either positive (+) or negative (−) for the respective TLS category. Relationships between TLS categories and DFS were explored (minimum follow-up, 5.9 months). Results: Most tumors exhibited some form of LA/TLS (245/272; 90.1%), with a higher incidence of LA (237/272; 87.1%) compared with mTLS (89/272; 32.7%). There was heterogeneity of TLS within the tumors, with most tumors demonstrating more than 1 form of TLS (198/272; 72.8%). In the PBO arm, median DFS was modest for most TLS categories (5.7–8.3 months) but was substantially improved for mTLS+ tumors (22.1 months; 95% CI, 5.4–not estimable [NE]); n=41), representing a 2.7- to 3.9-fold increase relative to any other category. In contrast, treatment with NIVO resulted in 2.1- to 4.0-fold improvements in median DFS relative to PBO across all but the mTLS+ category (22.9 months; 95% CI,11.9–NE; n=48), which was similar to the PBO arm. The largest increases in median DFS (95% CI) for NIVO vs PBO were observed for LA+ (27.6 [16.1–NE; n=114] vs 8.3 [5.6–11.9; n=123] months; HR, 0.56; 95% CI, 0.39–0.80; P=0.0015), iTLS+ (27.0 [16.1–NE; n=91] vs 8.2 [5.4–21.2; n=105] months; HR, 0.61; 95% CI, 0.41–0.91; P=0.0143), and mTLS− tumors (24.6 [11.4–NE; n=81] vs 6.1 [5.2–8.5; n=102] months; HR, 0.53; 95% CI, 0.36–0.78; P=0.0012]). Conclusion: These results suggest that a novel AI-based TLS maturity classification using H&E images is feasible to investigate the tumor microenvironment in MIUC, with potential broad application in other studies. Citation Format: Johannes Alfred Witjes, Matthew D. Galsky, Jürgen E. Gschwend, Dean F. Bajorin, Matthew I. Milowsky, Gautam Jha, Roger Li, Jun Li, Vanessa Matos-Cruz, Scott Ely, George Lee, Vipul Baxi, Joshua Zhang, Saurabh Gupta, Justin M. David. Exploratory analysis of tumor tertiary lymphoid structures using a novel artificial intelligence–based approach in patients with muscle-invasive urothelial carcinoma from the CheckMate 274 trial [abstract]. In: Proceedings of the AACR Special Conference on Bladder Cancer: Transforming the Field; 2024 May 17-20; Charlotte, NC. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(10_Suppl):Abstract nr PR010.

CAR-macrophage versus CAR-T for solid tumors: The race between a rising star and a superstar.

Adoptive cell therapy (ACT) has been demonstrated to be one of the most promising cancer immunotherapy strategies due to its active antitumor capabilities in vivo. Engineering T cells to overexpress chimeric antigen receptors (CARs), for example, has shown potent efficacy in the therapy of some hematologic malignancies. However, the efficacy of chimeric antigen receptor T cell (CAR-T) therapy against solid tumors is still limited due to the immunosuppressive tumor microenvironment (TME) of solid tumors, difficulty in infiltrating tumor sites, lack of tumor-specific antigens, antigen escape, and severe side effects. In contrast, macrophages expressing CARs (CAR-macrophages) have emerged as another promising candidate in immunotherapy, particularly for solid tumors. Now at its nascent stage (with only one clinical trial progressing), CAR-macrophage still shows inspiring potential advantages over CAR-T in treating solid tumors, including more abundant antitumor mechanisms and better infiltration into tumors. In this review, we discuss the relationships and differences between CAR-T and CAR-macrophage therapies in terms of their CAR structures, antitumor mechanisms, challenges faced in treating solid tumors, and insights gleaned from clinical trials and practice for solid tumors. We especially highlight the potential advantages of CAR-macrophage therapy over CAR-T for solid tumors. Understanding these relationships and differences provides new insight into possible optimization strategies of both these two therapies in solid tumor treatment.

Key players of immunosuppression in epithelial malignancies: Tumor-infiltrating myeloid cells and γδ T cells.

The tumor microenvironment of solid tumors governs the differentiation of otherwise non-immunosuppressive macrophages and gamma delta (γδ) T cells into strong immunosuppressors while promoting suppressive abilities of known immunosuppressors such as myeloid-derived suppressor cells (MDSCs) upon infiltration into the tumor beds. In epithelial malignancies, tumor-associated macrophages (TAMs), precursor monocytic MDSCs (M-MDSCs), and gamma delta (γδ) T cells often acquire strong immunosuppressive abilities that dampen spontaneous immune responses by tumor-infiltrating T cells and B lymphocytes against cancer. Both M-MDSCs and γδ T cells have been associated with worse prognosis for multiple epithelial cancers. Here we discuss recent discoveries on how tumor-associated macrophages and precursor M-MDSCs as well as tumor associated-γδ T cells acquire immunosuppressive abilities in the tumor beds, promote cancer metastasis, and perspectives on how possible novel interventions could restore the effective adaptive immune responses in epithelial cancers.

Signaling crosstalk between tumor endothelial cells and immune cells in the microenvironment of solid tumors.

Tumor-associated endothelial cells (TECs) are crucial mediators of immune surveillance and immune escape in the tumor microenvironment (TME). TECs driven by angiogenic growth factors form an abnormal vasculature which deploys molecular machinery to selectively promote the function and recruitment of immunosuppressive cells while simultaneously blocking the entry and function of anti-tumor immune cells. TECs also utilize a similar set of signaling regulators to promote the metastasis of tumor cells. Meanwhile, the tumor-infiltrating immune cells further induce the TEC anergy by secreting pro-angiogenic factors and prevents further immune cell penetration into the TME. Understanding the complex interactions between TECs and immune cells will be needed to successfully treat cancer patients with combined therapy to achieve vasculature normalization while augmenting antitumor immunity. In this review, we will discuss what is known about the signaling crosstalk between TECs and tumor-infiltrating immune cells to reveal insights and strategies for therapeutic targeting.

Hypoxia-associated markers in the prognosis of oral canine melanoma.

Canine oral malignant melanoma (COMM) is the most common neoplasm in the oral cavity characterized by local invasiveness and high metastatic potential. Hypoxia represents a crucial feature of the solid tumor microenvironment promoting cancer progression and drug resistance. Hypoxia-inducible factor-1α (HIF-1α) and its downstream effectors, vascular endothelial growth factor A (VEGF-A), glucose transporter isoform 1 (GLUT1), C-X-C chemokine receptor type 4 (CXCR4), and carbonic anhydrase IX (CAIX), are the main regulators of the adaptive response to low oxygen availability. The prognostic value of these markers was evaluated in 36 COMMs using immunohistochemistry. In addition, the effects of cobalt chloride-mediated hypoxia were evaluated in 1 primary COMM cell line. HIF-1α expression was observed in the nucleus, and this localization correlated with the presence or enhanced expression of HIF-1α-regulated genes at the protein level. Multivariate analysis revealed that in dogs given chondroitin sulfate proteoglycan-4 (CSPG4) DNA vaccine, COMMs expressing HIF-1α, VEGF-A, and CXCR4 were associated with shorter disease-free intervals (DFI) compared with tumors that were negative for these markers (P = .03), suggesting hypoxia can influence immunotherapy response. Western blotting showed that, under chemically induced hypoxia, COMM cells accumulate HIF-1α and smaller amounts of CAIX. HIF-1α induction and stabilization triggered by hypoxia was corroborated by immunofluorescence, showing its nuclear translocation. These findings reinforce the role of an hypoxic microenvironment in tumor progression and patient outcome in COMM, as previously established in several human and canine cancers. In addition, hypoxic markers may represent promising prognostic markers, highlighting opportunities for their use in therapeutic strategies for COMMs.

An Electron Donor-Acceptor Structured Rhenium(I) Complex Photo-Sensitizer Evokes Mutually Reinforcing "Closed-Loop" Ferroptosis and Immunotherapy.

The hypoxic microenvironment of solid tumors severely lowers the efficacy of oxygen-dependent photodynamic therapy (PDT). The development of hypoxia-tolerant photosensitizers for PDT is an urgent requirement. In this study, a novel rhenium complex (Re-TTPY) to develop a "closed-loop" therapy based on PDT-induced ferroptosis and immune therapy is reported. Due to its electron donor-acceptor (D-A) structure, Re-TTPY undergoes energy transfer and electron transfer processes under 550nm light irradiation and displays hypoxia-tolerant type I/II combined PDT capability, which can generate 1O2, O2 -, and ·OH simultaneously. Further, the reactive oxygen species (ROSs) leads to the depletion of 1,4-dihydronicotinamide adenine dinucleotide (NADH), glutathione peroxidase 4 (GPX4), and glutathione (GSH). As a result, ferroptosis occurs in cells, simultaneously triggers immunogenic cell death (ICD), and promotes the maturation of dendritic cells (DCs) and infiltration of T cells. The release of interferon-γ (IFN-γ) by CD8+ T cells downregulates the expression of GPX4, further enhancing the occurrence of ferroptosis, and thereby, forming a mutually reinforcing "closed-loop" therapeutic approach.

ScRNAseq and High-Throughput Spatial Analysis of Tumor and Normal Microenvironment in Solid Tumors Reveal a Possible Origin of Circulating Tumor Hybrid Cells.

Metastatic cancer is a leading cause of death in cancer patients worldwide. While circulating hybrid cells (CHCs) are implicated in metastatic spread, studies documenting their tissue origin remain sparse, with limited candidate approaches using one-two markers. Utilizing high-throughput single-cell and spatial transcriptomics, we identified tumor hybrid cells (THCs) co-expressing epithelial and macrophage markers and expressing a distinct transcriptome. Rarely, normal tissue showed these cells (NHCs), but their transcriptome was easily distinguishable from THCs. THCs with unique transcriptomes were observed in breast and colon cancers, suggesting this to be a generalizable phenomenon across cancer types. This study establishes a framework for HC identification in large datasets, providing compelling evidence for their tissue residence and offering comprehensive transcriptomic characterization. Furthermore, it sheds light on their differential function and identifies pathways that could explain their newly acquired invasive capabilities. THCs should be considered as potential therapeutic targets.

Tumor and Peritoneum-Associated Macrophage Gene Signature as a Novel Molecular Biomarker in Gastric Cancer.

A spectrum of immune states resulting from tumor resident macrophages and T-lymphocytes in the solid tumor microenvironment correlates with patient outcomes. We hypothesized that in gastric cancer (GC), macrophages in a polarized immunosuppressive transcriptional state would be prognostic of poor survival. We derived transcriptomic signatures for M2 (M2TS, MRC1; MS4A4A; CD36; CCL13; CCL18; CCL23; SLC38A6; FGL2; FN1; MAF) and M1 (M1TS, CCR7; IL2RA; CXCL11; CCL19; CXCL10; PLA1A; PTX3) macrophages, and cytolytic T-lymphocytes (CTLTS, GZMA; GZMB; GZMH; GZMM; PRF1). Primary GC in a TCGA stomach cancer dataset was evaluated for signature expressions, and a log-rank test determined overall survival (OS) and the disease-free interval (DFI). In 341 TCGA GC entries, high M2TS expression was associated with histological types and later stages. Low M2TS expression was associated with significantly better 5-year OS and DFI. We validated M2TS in prospectively collected peritoneal fluid of a GC patient cohort (n = 28). Single-cell RNA sequencing was used for signature expression in CD68+CD163+ cells and the log-rank test compared OS. GC patients with high M2TS in CD68+CD163+ cells in their peritoneal fluid had significantly worse OS than those with low expression. Multivariate analyses confirmed M2TS was significantly and independently associated with survival. As an independent predictor of poor survival, M2TS may be prognostic in primary tumors and peritoneal fluid of GC patients.

Abstract LB446: Decoding the mechanism of metastasis: A novel CAF-therapy that blocks the tumor microenvironment and reverses metastatic traits in all solid tumors

Abstract Progression and metastatic spreading of cancer cells are modulated by the carcinoma-associated fibroblasts (CAFs) within the primary tumor microenvironment in solid tumors. However, the mechanisms through which CAFs exert their pro-metastatic effects on cancer cells remain unknown. In this study, we have developed a small molecule that blocks Neuropilin-1 signaling in metastatic CAFs (mCAFs), reversing the mechanisms that promote metastasis in prostate, breast, colorectal and pancreatic cancer. To address these critical needs, in this study, we sought to assess the potential role of proteoglican neuropilin 1 (PG-NRP1) signaling in the maintenance of the activated CAF phenotype. Our results by gene expression profile, qPCR, WB, and ELISA indicate that CAFs isolated from primary tumors of metastatic patients (mCAFs) are functionally different from CAFs isolated from primary tumors of non-metastatic patients, promoting metastasis traits of cancer cells. Among the most prominent differences is an increased PG-NRP1 expression. siRNA studies demonstrated that the knockdown of NRP-1 in mCAFs showed the reversion of the activated phenotype, evaluated by the activation markers a-SMA, Tenascin C, and Fibronectin. Furthermore, conditioned media from PG-NRP1-siRNA-mCAFs failed to increase cancer cells' metastasis traits (migration and invasion) in vitro and tumor progression in vivo. Furthermore, we engineered a small molecule designed to bind PG-NRP1 in mCAFs. Our findings, demonstrate in vitro and in vivo models that the inhibition of PG-NRP1 impedes tumor growth and metastatic dissemination of cancer cells in spontaneous metastasis assay. We designed and validated, using in vivo models, an inhibitor that blocks NRP1-mCAF. Our research demonstrates that blocking PG-NRP1 in mCAF, with a small molecule, is a transversal therapeutic strategy to prevent progression and metastatic spread in solid cancer tumors. Citation Format: Benjamin Prieto, Muriel Núñez, Valentina I. Cerdfa, Javier Cerda-Infante. Decoding the mechanism of metastasis: A novel CAF-therapy that blocks the tumor microenvironment and reverses metastatic traits in all solid 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 LB446.

Abstract CT170: Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid tumors

Abstract Checkpoint immunotherapies have revolutionized solid tumor treatment yet durably benefit a minority of patients, as they rely on endogenous anti-tumor T cells. A potential solution for patients lacking functional endogenous anti-tumor T cells is engineering their T cells with exogenous T cell receptors (TCRs) to target and kill tumor cells. Initial clinical trials with TCR engineered T cell therapies (TCR-Ts) targeted single tumor antigens on single HLA and produced partial, short-lasting responses. Solid tumors are notoriously heterogenous with highly variable antigen expression. Recent discoveries also identified HLA loss of heterozygosity in up to 40% of solid tumors, allowing tumor cells to evade T cell attack. To overcome this heterogeneity, TScan has developed T-Plex, a multiplexed cell therapy comprising 2-3 different TCR-Ts, chosen from a collection of TCR-Ts called the ImmunoBank, to target different tumor antigens on different HLA types with confirmed tumor expression. To deepen clinical responses, TCR-T cells are engineered to express CD8α/β co-receptors that, in preclinical experiments, enable CD4+ helper T cells to have >100-fold improved cytotoxicity and cytokine secretion over CD4+ cells expressing the TCR alone. Finally, to allow T cell persistence despite immunosuppressive TGF-β in the solid tumor microenvironment, TCR-T cells also express the dominant negative TGF-βreceptor, enabling ~10-fold improved proliferation in the presence of TGF- β compared to T cells expressing the TCR and CD8α/β co-receptors alone. A proprietary transposon vector with larger cargo limit enables the inclusion of these additional genes. The Phase 1 study utilizes a separate screening protocol to pre-identify patients with head and neck, cervical, anogenital cancers, NSCLC, and melanoma, any time during standard clinical care, enabling rapid enrollment into the treatment protocol upon disease progression. Screening comprises germline HLA testing, and archival tumor testing for antigen expression and exclusion of HLA loss. Treatment includes standard non-myeloablative lymphodepletion followed by one or 2 doses of T-Plex infused 28 days apart. Dose escalation under the interval 3+3 design starts with testing single TCR-Ts in dose levels 1 and 2. Thereafter, TCR-Ts are combined and escalated in dose levels 3 and 4. TCR-Ts currently in the master protocol target PRAME, MAGE-A1 or HPV16 on HLA-A*02:01 or MAGE-A1 on HLA-C*07:02. Additional TCR-Ts added to the ImmunoBank and master protocol go through dose levels 1 and 2 as single therapies before becoming available for multiplexed dose levels 3 and 4. Primary endpoints include safety, feasibility and identifying the recommended Phase 2 dose. Secondary endpoints are rates and durations of response and exploratory endpoints measure T cell activation and persistence. Two additional TCR-Ts are on track to be added to the ImmunoBank, which could allow 50-80% of common solid tumor patients to qualify for multiplexed TCR-T therapy. Citation Format: Justin Moser, Brian Pico, Brian Henick, Rom Leidner, Jared Weiss, James Isaacs, Jaspreet Grewal, Michael Hurwitz, Jim Murray, Marlyane Motta, Yun Wang, Shrikanta Chattopadhyay, Debora Barton, Gavin MacBeath, Sajeve Thomas. Trial in progress: A phase 1, first in human clinical trial for T-Plex, a multiplexed, enhanced T cell receptor-engineered T cell therapy (TCR-T) for solid 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 CT170.

Abstract LB068: Repurposing chimeric antigen receptor regulatory T cells for treating cancer

Abstract Solid tumors are the deadliest form of cancer. Over the past decade, chimeric antigen receptor (CAR) T-cell therapy has led to remarkable regression rates in leukemia and lymphoma. However, solid tumors have remained refractory to CAR T-cell therapy, with CAR T cells either failing to penetrate the solid tumor microenvironment (TME) or becoming exhausted once in it. Mechanisms behind these properties of the TME include nutrient scarcity, aberrant vascularization, stromal cell deposition, expression of inhibitory immune receptors by cancer cells, and the presence of immunosuppressive cells. Interestingly, there is a subset of T cells found in the TME - regulatory T cells (Tregs). Dedicated to suppressing immune responses, Tregs constitute a barrier to anticancer immunity by migrating to and heavily accumulating in solid tumors, inhibiting local immunity. Tregs can be redirected towards any cell surface antigen using a CAR and have been shown to be cytotoxic towards target antigen presenting cells. Can Tregs be converted from tumor protectors to anti-tumor effector cells? To test the anti-tumor potential of CAR Tregs, we modified human Tregs with an anti-CD19 CD28-CD3z CAR. Tregs activated via the CAR or endogenous TCR/CD28 were evaluated at the functional and gene expression levels. CAR and endogenous TCR/CD28 activation resulted in similar cell expansion and Treg stability (FOXP3 and HELIOS expression). However, CAR-mediated activation markedly increased Treg proinflammatory cytokine production. Moreover, compared to their TCR/CD28-activated counterparts, CAR Tregs displayed increased cytotoxic activity towards tumor cells and a diminished capacity to modulate antigen-presenting cells and suppress T cell proliferation, suggesting a global shift from suppression to inflammation. Strikingly, CAR Tregs suppressed tumor growth in immunodeficient mice inoculated with antigen-expressing B-cell leukemia, myeloid leukemia, and lung epithelial carcinoma. Single-cell RNA sequencing identified a cell cluster enriched in CAR Tregs characterized by high expression of effector T cell genes, such as IFNG, GZMB, and CD40LG, revealing that CAR activation creates a highly active population of proinflammatory Tregs. Multiplex cytokine analysis, whole-transcriptome RNA-seq, and intracellular cytokine staining confirmed that IFNG production by FOXP3+ Tregs is induced specifically by CAR activation. Of note, murine CAR Tregs are also cytotoxic towards tumor cells. Current experiments are focused on pinpointing and validating transcription factors and surface markers characteristic of proinflammatory CAR Tregs and further assessing CAR Treg antitumor efficacy in vivo. In conclusion, CAR-mediated activation induces a novel proinflammatory Treg phenotype that can be exploited in the next generation of engineered immune cell therapies against solid tumors. Citation Format: Leonardo M. Ferreira, Russell W. Cochrane, Rob A. Robino, Bryan Granger, Eva Allen, Vishnu Gudivada, Aguirre A. de Cubas, Stefano Berto. Repurposing chimeric antigen receptor regulatory T cells for treating cancer [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 LB068.

Nanomedicine Remodels Tumor Microenvironment for Solid Tumor Immunotherapy.

Although immunotherapy is relatively effective in treating hematological malignancies, their efficacy against solid tumors is still suboptimal or even noneffective presently. Compared to hematological cancers, solid tumors exhibit strikingly different immunosuppressive microenvironment, severely deteriorating the efficacy of immunotherapy: (1) chemical features such as hypoxia and mild acidity suppress the activity of immune cells, (2) the pro-tumorigenic domestication of immune cells in the microenvironment within the solid tumors further undermines the effectiveness of immunotherapy, and (3) the dense physical barrier of solid tumor tissues prevents the effective intratumoral infiltration and contact killing of active immune cells. Therefore, we believe that reversing the immunosuppressive microenvironment are of critical priority for the immunotherapy against solid tumors. Due to their unique morphologies, structures, and compositions, nanomedicines have become powerful tools for achieving this goal. In this Perspective, we will first briefly introduce the immunosuppressive microenvironment of solid tumors and then summarize the most recent progresses in nanomedicine-based immunotherapy for solid tumors by remodeling tumor immune-microenvironment in a comprehensive manner. It is highly expected that this Perspective will aid in advancing immunotherapy against solid tumors, and we are highly optimistic on the future development in this burgeoning field.

Armored TGFβRIIDN ROR1-CAR T cells reject solid tumors and resist suppression by constitutively-expressed and treatment-induced TGFβ1

BackgroundChimeric antigen receptor (CAR) T-cell therapy target receptor tyrosine kinase-like orphan receptor 1 (ROR1) is broadly expressed in hematologic and solid tumors, however clinically-characterized ROR1-CAR T cells with single chain...

RAD23B mediated proteasomal degradation occurs through p38 MAPK/ATF-2/RAD23B axis under nutrient-deprived conditions in breast cancer.

Metabolic reprogramming in cancer occurs due to interaction of cells with the surrounding tumor microenvironment. In the microenvironment of solid tumors, nutrient deprivation is induced by high consumption of nutrients and insufficient vasculature. Tumor cells alter their metabolic strategies to adapt to the microenvironment. To understand the role of these metabolic changes, in the current study, we have mimicked nutrient deprivation condition in vitro to evaluate the associated signaling pathways in breast cancer cells. In our study, we have shown that nutritional deprivation activated p38 MAPK and activating transcription factor-2 (ATF-2) by increased phosphorylation of Thr180/Tyr182 and Thr71, respectively, in breast cancer cells. Pharmacological inhibition of p38 MAPK showed increased cell viability and reduced expression of ATF-2 and RAD23B under nutrient starvation conditions. Further, silencing of ATF-2 showed increased cell viability and decreased expression of RAD23B under nutrient starvation conditions. This suggests the involvement of p38 MAPK/ATF-2/RAD23B axis as a signaling pathway under nutrition starvation in breast cancer cells. The RAD23B mediated proteasome activity was shown to be much higher under stress conditions indicating a crucial role of RAD23B as a target for breast cancer.

CAR T therapy from haematological malignancies to aging-related diseases: An ever-expanding universe.

Short but impactful, the two-decade story of gene editing allowed a significant breakthrough in the treatment of haematological malignancies. However, despite different generations of chimeric antigen receptor T (CAR T), such a successful therapy has not yet been replicated in solid tumours and non-oncological diseases. This narrative review discusses how CAR T therapy still faces challenges in overcoming the complexity of the solid tumour microenvironment and the concerns that its long-term activity raises about potential unknown and unpredictable consequences in non-oncological diseases. In the most recent studies, the senolytic potential of CAR T is becoming an exciting field of research. Still, experimental but promising results indeed indicate the clearance of senescent cells as an effective strategy to improve exercise capacity and metabolic dysfunction in physiological ageing, with long-term therapeutic and preventive effects. However, an effective expansion of a CAR T population requires a lympho-depleting chemotherapy prior to infusion. While this procedure sounds reasonable for rescue therapy of oncological diseases, it poses genotoxic risks that may not be justified for non-malignant diseases. Those represent the leading gaps for applying CAR T therapy in non-oncological diseases. More is expected from current studies on the other classes of CAR cells now under investigation. Engineering NK cells and macrophages are candidates to improve cytotoxic and immunomodulating properties, potentially able to broaden application in solid tumours and non-oncological diseases. Finally, engineering autologous T cells in old individuals may generate biologically deteriorated CAR T clones with impaired function and unpredictable effects on cytokine release.

Abstract 1241: Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE)

Abstract With an annual incidence rate exceeding 660,000 cases, and a death toll surpassing 325,000 per year, head and neck cancer (HNC) ranks as the seventh most common cancer in the world. Surgery, radiation and chemotherapy are used to treat HNC patients with modest and variable clinical success. However, these treatments prove less effective for human papillomavirus negative (HPV-) HNC patients, a subset of HNC patients with markedly worse prognosis. While significant advancements have been made in cancer immunotherapy over the past decade, its success also remains elusive for HNC due to factors such as the hypoxic solid tumor microenvironment (TME). To address the critical need for an improved therapeutic intervention, we leveraged the ability of natural killer (NK) cells in killing cancer cells without prior sensitization by developing a novel tri-specific killer engager (TriKE) that can direct NK cell killing of tumor within the hypoxic solid TME. The TriKE is composed of three domains: a humanized nanobody arm binding the activating receptor CD16 on NK cells, an interleukin (IL)-15 moiety that can drive expansion of NK cells, and a nanobody arm binding B7H3, a protein which high expression can be negatively correlated with overall survival of HPV- HNC patients. B7H3 is a prime target candidate because it is highly expressed on HPV- HNC cells, but minimally expressed on healthy tissues. In vitro testing using HPV- HNC patient blood samples revealed that B7H3 TriKE enhances activation (measured by NK cell degranulation and interferon-gamma production) and expansion of NK cells from these patients at levels equivalent to those observed in healthy controls. Furthermore, B7H3 TriKE is efficacious in its ability to drive high NK cell cytotoxicity in both acute (<48-hours) and prolonged (7-days) hypoxic (1% oxygen) models of HNC. Under acute hypoxia, B7H3 TriKE induces significantly more killing of targets by NK cells compared to IL-15 treatment, where NK cell cytotoxicity is impaired. In addition, B7H3 TriKE can boost the killing efficacy of NK cells exposed to prolonged hypoxia, surpassing limitations seen with IL-15 treatment. These findings strongly suggest that the B7H3 TriKE can bypass hypoxic suppression of NK effector functions in the solid TME. In vivo studies using immunocompromised mice engrafted with HPV- HNC cells revealed that B7H3 TriKE treatment significantly extends the survival of mice, compared to IL-15 treatment. Moreover, NK cells persisted in the blood of B7H3 TriKE-treated mice 28 days post-NK cell injection, highlighting the promising clinical translation of this immunotherapy. More in-depth characterization of NK cells from HPV- HNC patients are underway but altogether, these robust pre-clinical data present a novel avenue for the management of HNC for these patients. We plan to translate results from these studies to clinical trials in fall 2024. Citation Format: Melissa Khaw, Nicholas A. Zorko, Carly Selleck, Laura Bendzick, Zachary Davis, Peter Hinderlie, Madison Shackelford, Ann Lu, James Lim, Naomi Fujioka, Margaret MacMillan, John Wagner, Martin Felices, Jeffrey S. Miller. Enhancing NK cell therapy for head and neck cancer within the solid tumor microenvironment using a B7H3-targeting tri-specific killer engager (TriKE) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1241.