Immune cell-based therapies for solid tumors, current challenges and therapeutic advances

BackgroundIt is incompletely understood which populations of tumor-infiltrating lymphocytes (TIL) respond to checkpoint blockade (CB) and when. Recent studies in murine MC-38 colon carcinoma demonstrate CD4+ T cells are among...

Advancing together and moving forward: Combination gene and cellular immunotherapies

Research Highlights: Immunotherapy

5th Annual Immuno-Oncology 360° Conference: Spanning Science and Business to Bring New Therapies to Patients

Background: Success of Chimeric Antigen Receptor (CAR) T cells therapy for solid tumors are limited by the suppressive solid tumor microenvironment (TME), T cell exhaustion, lack of persistence, and poor trafficking to tumors. Strategies to improve this include therapeutic combinations, such as checkpoint inhibition (CPI) or cytokine support. However, CPI therapy has largely failed because of refractory and resistant tumors and cytokine administration can lead to lethal toxicities. To this end, we propose that enabling CAR T cells to secrete bi-functional fusion proteins consisting of cytokine modifiers (ie: IL12, IL15, or TGFβ-trap) combined with checkpoint inhibition (αPDL1 scFv) can provide tumor PDL1 sequestered cytokine activity and local tumor-immune modulation to boost solid tumor CAR T cell efficacy, enhance CPI impacts, and safely improve durable outcomes. Methods: Mouse T cells dual transduced to express surface CAR targeting prostate or ovarian solid tumors in addition to secretable cytokine fused to an αPDL1 scFv were generated. In vitro, the PDL1 blockade capacity and tumor-surface cytokine presentation of supernatants from dual transduced CAR T cells was assessed on PDL1 induced tumor cells. Further, secreted fusion protein transduced CAR T cells were assessed for function, phenotype, and cytokine release in a repetitive tumor rechallenge assay. In vivo, mouse CAR T cells dual transduced with fusion proteins and appropriate controls were assessed for anti-tumor efficacy, survival kinetics, and toxicity in immunocompetent solid tumor mouse models of prostate cancer and intraperitoneal disseminated ovarian cancer. Results: CAR T cells were shown to be successfully dual transduced, and secreted αPDL1-cytokine fusion proteins exhibiting functional PDL1 binding characteristics in vitro. CAR T cells engineered with αPDL1-IL12 fusion protein had greater anti-tumor activity and CAR specific expansion compared to other tested fusion proteins in in vitro co-culture assays. In a syngeneic prostate tumor model, mice receiving CAR T cells with αPDL1-IL12 fusion T cells out competed other cytokine fusion combinations. In a syngeneic ovarian tumor model we safely achieved 100% curative response rate with CAR secreting αPDL1-IL12 fusion in contrast to CAR with αPDL1mutIL12 control along with tumor regional PDL1 blockade on myeloid subsets within the TME. Conclusions: Our findings suggest that CAR T cells engineered to secrete αPD-L1-IL12 fusion protein show improved tumor control, less systemic toxicity, and enhanced expansion which promotes eradication of disease in two independent in vivo tumor models with two unique solid tumor CAR targets. We believe this strategy has the potential to improve solid tumor CAR T-cell efficacy and enhance durable innate immune responses. Citation Format: Lea Christian, John P. Murad, Lupita Lopez, Anthony Park, Jason Yang, Eric Lee, Candi Trac, Stephen Forman, Saul J. Priceman. Secreted cytokine-αPDL1 fusion proteins improve solid tumor chimeric antigen receptor (CAR) T-cell therapy [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 3996.

Thoracic malignancies (lung cancers and malignant pleural mesothelioma) are prevalent worldwide and are associated with high morbidity and mortality. Effective treatments are needed for patients with advanced disease. Cell therapies are a promising approach to the treatment of advanced cancers that make use of immune effector cells that have the ability to mediate antitumor immune responses. In this review, we discuss the prospect of chimeric antigen receptor-T (CAR-T) cells, natural killer (NK) cells, T cell receptor-engineered (TCR-T) cells, and tumor-infiltrating lymphocytes (TILs) as treatments for thoracic malignancies. CAR-T cells and TILs have proven successful in several hematologic cancers and advanced melanoma, respectively, but outside of melanoma, results have thus far been unsuccessful in most other solid tumors. NK cells and TCR-T cells are additional cell therapy platforms with their own unique advantages and challenges. Obstacles that must be overcome to develop effective cell therapy for these malignancies include selecting an appropriate target antigen, combating immunosuppressive cells and signaling molecules present in the tumor microenvironment, persistence, and delivering a sufficient quantity of antitumor immune cells to the tumor. Induced pluripotent stem cells (iPSCs) offer great promise as a source for both NK and T cell-based therapies due to their unlimited expansion potential. Here, we review clinical trial data, as well as recent basic scientific advances that offer insight into how we may overcome these obstacles, and provide an overview of ongoing trials testing novel strategies to overcome these obstacles.

Human natural killer T (NKT) cells have been proposed as a promising cell platform for chimeric antigen receptor (CAR) therapy in solid tumors. Here we generated murine CAR-NKT cells and compared them with CAR-T cells in immune-competent mice. Both CAR-NKT cells and CAR-T cells showed similar antitumor effects in vitro, but CAR-NKT cells showed superior antitumor activity in vivo via CD1d-dependent immune responses in the tumor microenvironment. Specifically, we show that CAR-NKT cells eliminate CD1d-expressing M2-like macrophages. In addition, CAR-NKT cells promote epitope spreading and activation of endogenous T cell responses against tumor-associated neoantigens. Finally, we observed that CAR-NKT cells can co-express PD1 and TIM3 and show an exhaustion phenotype in a model of high tumor burden. PD1 blockade as well as vaccination augmented the antitumor activity of CAR-NKT cells. In summary, our results demonstrate the multimodal function of CAR-NKT cells in solid tumors, further supporting the rationale for developing CAR-NKT therapies in the clinic.

Driving the Chimeric Antigen Receptor: The Road Ahead for Cell Therapy

Enhancing CAR T Cell Anti-Tumor Efficacy through Secreted Single Chain Variable Fragment (scFv) Immune Checkpoint Blockade

Adoptive cell therapy with chimeric antigen receptor (CAR) immunotherapy has made tremendous progress with five CAR T therapies approved by the US Food and Drug Administration for hematological malignancies. However, CAR immunotherapy in solid tumors lags significantly behind. Some of the major hurdles for CAR immunotherapy in solid tumors include CAR T cell manufacturing, lack of tumor-specific antigens, inefficient CAR T cell trafficking and infiltration into tumor sites, immunosuppressive tumor microenvironment (TME), therapy-associated toxicity, and antigen escape. CAR Natural Killer (NK) cells have several advantages over CAR T cells as the NK cells can be manufactured from pre-existing cell lines or allogeneic NK cells with unmatched major histocompatibility complex (MHC); can kill cancer cells through both CAR-dependent and CAR-independent pathways; and have less toxicity, especially cytokine-release syndrome and neurotoxicity. At least one clinical trial showed the efficacy and tolerability of CAR NK cell therapy. Macrophages can efficiently infiltrate into tumors, are major immune regulators and abundantly present in TME. The immunosuppressive M2 macrophages are at least as efficient as the proinflammatory M1 macrophages in phagocytosis of target cells; and M2 macrophages can be induced to differentiate to the M1 phenotype. Consequently, there is significant interest in developing CAR macrophages for cancer immunotherapy to overcome some major hurdles associated with CAR T/NK therapy, especially in solid tumors. Nevertheless, both CAR NK and CAR macrophages have their own limitations. This comprehensive review article will discuss the current status and the major hurdles associated with CAR T and CAR NK therapy, followed by the structure and cutting-edge research of developing CAR macrophages as cancer-specific phagocytes, antigen presenters, immunostimulators, and TME modifiers.

Despite the success of HER2 targeted therapies in HER2+ breast and gastric cancer, additional therapies are needed to address treatment-resistant metastatic disease. Adoptive immune cell therapy is a promising therapeutic modality given the remarkable clinical responses seen with autologous chimeric antigen receptor (CAR) T cells in hematological malignancies. However, success of cell therapy in solid tumors has been more limited. Three major impediments to the success of adoptive cell therapies in solid tumors are the heterogeneity of antigen expression, the immunosuppressive tumor microenvironment (TME), and the inherent challenges of manufacturing autologous cells and consequent variability of these cell products. Engineered, off-the-shelf, allogeneic Natural Killer (NK) cells provide a solution to these challenges. We describe here CAT-179, a novel engineered CAR-NK cell therapeutic for HER2+ solid tumors. CAT-179 cells express three transgenes: a HER2-directed CAR to effectively eliminate tumor cells, a Transforming Growth Factor (TGF) β dominant negative receptor (DNR) for resistance to TGFβ -mediated immunosuppression in the TME, and Interleukin 15 (IL15) cytokine to enhance NK cell persistence and activity for durable response. High efficiency engineering of the large (~3.7Kb) cargo containing CAR, IL15, and DNR in CAT-179 is enabled by the non-viral TC Buster™ Transposon System. Transposon engineering of CAT-179 results in high and stable expression of CAR (45% CAR at day 7 post gene delivery) without the need for post-engineering selection. CAT-179 demonstrates both CAR-dependent and innate NK receptor-dependent tumor cell killing in vitro, reducing the likelihood of tumor escape through antigen loss. CAT-179 effectively kills in vitro both high HER2-expressing SKOV3 cells as well as lower HER2-expressing HT-29 cells. CAT-179 also demonstrates resistance to TGFβ mediated immunosuppression, as evidenced by 75% reduction in TGFβ -induced phosphorylation of SMAD2 as well as prevention of TGFβ induced downregulation of NK cell activating receptors and restoration of NK cell cytotoxic activity. These data suggest CAT-179 cells will be protected from TGFβ -mediated immune suppression in the TME. Finally, the addition of IL15 in CAT-179 significantly enhances persistence for at least fourteen days in vitro without the need for exogenous cytokines. Moreover, CAT-179 administration to NSG mice showed expansion and persistence of the transferred cell product. CAT-179 addresses key hurdles to allogeneic cell therapy for solid tumors and is a promising new therapeutic approach for HER2 expressing breast, gastric and other tumors. Citation Format: Celeste Richardson, Finola Moore, Andres Alvarez, Alexia Barandiaran, Luke Barron, Eugene Choi, Tucker Ezell, Charlotte Franco, Bashar Hamza, Jennifer Johnson, Annie Khamhoung, Taeyoon Kyung, Marilyn Marques, Dominic Picarella, Jared Sewell, Alex Storer, Meghan Walsh, Vipin Suri. Allogeneic Natural Killer cells engineered to express HER2 CAR, Interleukin 15 and TGF beta dominant negative receptor effectively control HER2+ tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 555.

Tumor-infiltrating lymphocyte (TIL) therapy has evolved from a pioneering experimental approach to a clinically validated treatment strategy, underscored by the recent approval of lifileucel (Amtagvi) by the Food and Drug Administration (FDA) for advanced melanoma refractory to existing therapies. Initially successful in melanoma due to its high tumor mutational burden (TMB) and immune-reactivity, contemporary efforts extend TIL applications to other solid tumors, including lung, cervical, and colorectal cancers. However, these lower-TMB malignancies typically require the selective enrichment of tumor-specific T cells to achieve significant clinical efficacy. The therapeutic potential of TILs is influenced by critical factors, including cell dose, T-cell phenotype and differentiation state, tumor-specific reactivity, and the ability to persist and expand within patients post-infusion. Emerging techniques, including single-cell transcriptomics and biomarker-guided TIL selection (e.g., CD137, CD103 markers), have provided deeper insights into the characteristics correlating with successful outcomes. Ongoing clinical trials highlight future directions, including genetically engineered TILs with chimeric antigen receptor (CAR) or immune checkpoint knockout, improved cytokine support strategies to enhance T-cell expansion and reduce toxicity, and optimized lymphodepletion regimens. Establishing clear quality attributes for TIL manufacturing will be essential for consistent clinical success, paving the way toward personalized and robust immunotherapeutic approaches across diverse cancer types.

INTRODUCTIONOver the past decade, immunotherapy has redefined the landscape for cancer treatment, providing unprecedented survival benefits across a broad swath of tumors. The ability to harness and modulate the immune system has transformed outcomes for patients, from immune checkpoint inhibitors (ICIs) to advanced cellular therapies, such as chimeric antigen receptor (CAR) T cells and CAR macrophages (CAR-MΦ). However, these advancements have come with new challenges, such as variability in efficacy, toxicities, and lack of efficacy against the immunosuppressive tumor microenvironment (TME), especially in solid tumors[1]. In this editorial, we explore the major advances in immunotherapy, the potential of combination therapies with CAR-MΦ, and the need for these combination approaches to overcome evolving challenges. Immune Checkpoint Inhibitors: In cancers such as melanoma, NSCLC, and RCC, ICIs have become the cornerstone of immunotherapy. ICIs block inhibitory receptors such as PD-1, PD-L1, and CTLA-4, and thereby enable the immune system to overcome the suppression and unleash the immune T cells for effective anti-tumor response. In many patients, these therapies have provided durable responses and some patients have survived more than five years. We have landmark trials that show significant improvements in overall survival (OS) and progression-free survival (PFS) vs. chemotherapy in metastatic and refractory cancers[2]. Nevertheless, despite all these advances, not all patients respond to ICIs. Resistance is due to tumor antigen heterogeneity, immune evasion mechanisms, and the immunosuppressive TME. In addition, immune-mediated adverse events (images), including gastrointestinal, dermatologic, and endocrine toxicities, continue to be significant barriers. Biomarker discovery including PD-L1 expression and tumor mutational burden will become increasingly important as the field evolves for identifying patients most likely to benefit from ICIs for personalized therapy with minimal risk and cost[3]. Cellular Therapies: CAR-T Cells and the Emerging Role of CAR-MacrophagesHowever, ICIs are transformative; cellular therapies are now the new frontier in immunotherapy. Remarkable success with CAR-T cells, which involve engineering T cells to express tumor-specific receptors, has been shown in hematologic malignancies, particularly leukemia and lymphoma. Despite these barriers, however, the efficacy of these approaches in solid tumors is still limited[4]. New CAR-MΦ therapies emerging as a novel solution to meet these challenges. Chimeric antigen receptors engineered into macrophages are capable of targeting tumor cells while modifying the hostile TME. After CAR-MΦ binds to tumor cells through phagocytosis, they actively engulf tumor cells while secreting pro-inflammatory cytokines to reprogram the TME to be immunostimulatory. In addition to stimulating other immune cells like T cells and natural killer (NK) cells, these engineered macrophages also amplify anti-tumor responses[5]. Promising safety and efficacy have been demonstrated by early clinical trials, including those in HER2-expressing solid tumors. CAR-MΦ therapies can persist within the tumor, can overcome physical barriers, and can synergize with other immunotherapies. Safety issues, however, remain, most notably the possibility of cytokine release syndrome (CRS) and macrophage activation syndrome (MAS)[6]. Macrophages' intrinsic role in inflammation regulation, however, may provide a more controlled cytokine response than CAR T cells. These risks are being mitigated by tailored engineering strategies, such as IL-10 expression, to ensure safe clinical application[7]. Combination Therapies: The future of immunotherapy will be in combination with strategies to overcome its limitations and improve efficacy. In preclinical models, CAR-MΦ has synergistic activity with immune checkpoint inhibitors like anti-PD-L1 and anti-CTLA-4. Checkpoint blockade reinvigorates exhausted T cells, and CAR-MΦ remodels the TME, making it hospitable to sustained immune attack[8]. Efforts to overcome the physical and biochemical barriers within the TME are equally required. Since engineering CAR cells to secrete pro-inflammatory cytokines or enzymes that digest the extracellular matrix enhances immune cell infiltration and persistence in tumors, we hypothesized that CAR cells could be engineered to secrete diphtheria toxin, which elicits an immune response within the tumor site. Moreover, immunotherapies alone, when combined with conventional therapies like chemotherapy and radiotherapy, may increase antigen presentation, increase T cell infiltration, and increase overall immune response[9]. CONCLUSION Immunotherapy has fundamentally changed how cancer is treated and has given many patients who were once untreatable hope. However, immunosuppressive TME and durable response in solid tumors remain challenges. CAR-MΦ therapy is a promising innovation with unique advantages because they are capable of phagocytosing tumor cells, presenting antigens, and reprogramming the TME.A rational combination approach with ICIs, cellular therapies, and conventional treatments would be the future. Additionally, safety concerns need to be addressed through rigorous clinical trials and long-term follow-up to optimize treatment strategies. As we stand on the brink of this new frontier, the challenge for the scientific community is clear: Immunotherapy needs to be refined to expand its reach, to ensure it becomes a mainstay of cancer care, and to offer cures where none existed before.

β-Glycoglycosphingolipid-Induced Alterations of the STAT Signaling Pathways Are Dependent on CD1d and the Lipid Raft Protein Flotillin-2

Chimeric antigen receptor (CAR) T-cell therapy has ushered in substantial advancements in the management of various B-cell malignancies. However, its integration into chronic lymphocytic leukemia (CLL) treatment has been challenging, attributed largely to the development of very effective chemo-free alternatives. Additionally, CAR T-cell responses in CLL have not been as high as in other B-cell lymphomas or leukemias. However, a critical void exists in therapeutic options for patients with high-risk diseases who are resistant to the current CLL therapies, underscoring the urgency for adoptive immunotherapies in these patients. The diminished CAR T-cell efficacy within CLL can be traced to factors such as compromised T-cell fitness due to persistent antigenic stimulation inherent to CLL. Resistance mechanisms encompass tumor-related factors like antigen escape, CAR T-cell-intrinsic factors like T-cell exhaustion, and a suppressive tumor microenvironment (TME). New strategies to combat CAR T-cell resistance include the concurrent administration of therapies that augment CAR T-cell endurance and function, as well as the engineering of novel CAR T-cells targeting different antigens. Moreover, the concept of "armored" CAR T-cells, armed with transgenic modulators to modify both CAR T-cell function and the tumor milieu, is gaining traction. Beyond this, the development of readily available, allogeneic CAR T-cells and natural killer (NK) cells presents a promising countermeasure to innate T-cell defects in CLL patients. In this review, we explore the role of CAR T-cell therapy in CLL, the intricate tapestry of resistance mechanisms, and the pioneering methods studied to overcome resistance.