Preclinical development of macrophage-targeting CAR monocytes to improve immunotherapy outcomes in breast cancer
254. Targeting tumor-associated macrophages with CAR-monocytes as a first-in-class approach for cellular therapy in breast cancer
Daniel Michaud (Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, United States) presented a first-in-class platform of using CAR-monocytes to limit tumor-associated macrophage (TAMs) function in breast cancer tumors. TAMs have been well-characterized as inhibitory hurdles for T cell function in solid tumors. TAM-targeting CAR T cells showed some anti-cancer activity in mouse models of ovarian cancer, but trafficking and infiltration of CAR T cells to the tumor and the immunosuppressive tumor microenvironment (TME) of solid tumors are challenges that have not been fully overcome. Monocytes typically thrive in the TME of solid tumors, and prior studies indicate CAR monocytes exhibit significantly superior tumor trafficking activity and persist up to 10 times longer in the TME compared to CAR macrophages. Therefore, this study explored the use of CAR monocytes to infiltrate solid tumors and differentiate to CAR macrophages (CAR macs) that target and destroy immunosuppressive TAMs by phagocytosis. A breast TAM atlas was developed, and SSP1 TAMs (a transcriptomic subtype characterized by high SPP1) that express the TREM2 receptor were identified as the most immunosuppressive. TAMs expressing TREM2 were found to be associated with poor survival outcomes in breast cancer and hypoxic phenotypes. In addition, TREM2 TAMs are present in a multitude of solid tumors, and non-cancerous tissues express TREM2 at limited levels, making TREM2 a strong tumor-specific target. Human TREM2 CAR macs were developed by transducing U-937 AML cells with a TREM2-targeting CAR. TREM2 CAR macs secreted the proinflammatory cytokine in response to TREM2 in vitro and exhibited significant and TREM2-specific phagocytic activity against TREM2-positive macrophages compared to non-TREM2-targeting CAR macs. Syngeneic murine TREM2 CAR monocytes and TREM2 CAR macs were developed. TREM2 CAR macs demonstrated significant TREM2-specific phagocytic activity against TREM2-expressing macrophages. Murine TREM2 CAR monocytes significantly delayed tumor growth of syngeneic orthotopic EO771 tumors (murine triple-negative breast cancer model)and increased CD8 T cell infiltration of tumors in vivo. The combination of TREM2 CAR monocytes and anti-PD-1 antibodies improved survival in this system. Future studies include exploring the role of TREM2 TAMs in metastasis and developing approaches to engineer more effective TREM2-targeting CAR monocytes. These results support additional work in the clinical development of TAM-targeting CAR monocytes as a strategy to improve immunotherapy outcomes for breast cancer and other solid tumors.
Migratory dendritic cells drive response to immunotherapy via sentinel lymph nodes
676. Lymphatic-sparing immunoradiotherapy reprograms migratory dendritic cells to drive tumor rejection via the sentinel lymph node
Robert Saddawi-Konefka (The University of Texas MD Anderson Cancer Center, Houston, TX, United States) presented the development of a novel mouse reporter model that allows for the inducible spatial tracking of immune cells between tumors and lymph nodes. Previous translational studies in mice indicate that tumor draining lymph nodes (TDLNs) are required for a response to immune checkpoint blockade in head and neck cancer. This finding, together with recent studies highlighting the benefits of neoadjuvant immunotherapy, has prompted a paradigm shift in head and neck cancer management. Standard-of-care chemoradiotherapy for head and neck cancers may compromise the response to immunotherapy by ablating or damaging tumor-draining lymph nodes, and rational sequencing of therapies is necessary to preserve lymph nodes and promote anti-tumor immunity. A murine spatiotemporal reporting model was used to develop a tumor-sentinel lymph node immunomigratome. Immune cells were labeled in the tumor, and cells could be tracked as they passed from the tumor to sentinel lymph nodes via the lymphatic channel and to the periphery. Sentinel lymph nodes were found to contain a unique migratory niche of immune cells that passed from the tumor. Sequential therapy strategy involving low-dose, lymph-node sparing, tumor-directed radiotherapy followed by PD-1 inhibition was developed to modulate the tumor immune microenvironment, with the goal to enhance the activity of immune cells in the immunomigratome. Sequenced therapy with tumor targeted radiation and anti-PD-1 antibodies led to durable near CR in mouse models of head and neck cancer. Placing radiotherapy first in the treatment sequence was necessary for immune priming. Radio-immunotherapy increased CCL19 signaling, thus activating CCR7+ dendritic cells in the sentinel lymph nodes and increasing immune activity. Physical or pharmacological disruption of lymphatic channels prevented the entry of dendritic cells to the sentinel lymph nodes and eliminated response to immunotherapy. Results from this study indicate that a population of migratory dendritic cells can be targeted and modulated at the tumor which then prime immune cells and promote anti-tumor immunity at the sentinel lymph nodes. These findings have provided the basis for two phase 2 clinical trials exploring the sequencing of lymph-node sparing radiotherapy with immunotherapy for human papilloma virus-positive and -negative head and neck cancers.
Engineering B cells that evade CD19 CAR T cells to prevent B cell aplasia
1033. Empowering CD19 CART therapy precision editing on CD19 in primary B cells to prevent prolonged B cell aplasia in B-ALL patients
Jayadev Mavuluri (St. Jude Children’s Research Hospital, Memphis, TN, United States) CD19-directed CAR T therapies, specifically those expressing the FMC63-based CAR construct, are highly effective against hematologic malignancies. B-cell aplasia is a common adverse event that is associated with CD19-targeting CAR T cells (CD19 CAR T cells), because non-cancerous B cells can also express CD19. B cell aplasia cannot be prevented and patients experiencing this toxicity are vulnerable to infection, increasing patient mortality. The goal of this study was to engineer a version of CD19 that could evade CD19 CAR T cells while preserving B cell function. This strategy could enable reconstitution of functional B cells following CAR T therapy, reducing infection risk and improving long-term safety. These engineered B cells could be introduced to patients after CAR T cell infusion, restoring immunity and preventing infection. Potential FMC63 binding sites of CD19 were identified, and it was found that a H218R/serine insertion mutation on human CD19 (hCD19 HAS) prevented FMC63 binding. Ramos tumor cell lines expressing hCD19 HAS escaped CD19 CAR T cell killing in vitro and in vivo. Human B cell lines expressing hCD19 HAS also evaded CD19 CAR T cell killing in vitro and in mouse models, and B cell clones expressing hCD19 HAS exhibited wild type levels of IgM expression, calcium flux signaling, maturation, and immunoglobulin production. hCD19 HAS was introduced in induced pluripotent stem cells and hematopoietic stem cells by CRISPR/Cas9 homology-directed repair, and cells were implanted in immunodeficient mice. hCD19 HAS cells exhibited similar levels of B cell engraftment and differentiation compared to cells expressing wild type CD19 and were able to evade CD19 CAR T cell therapy. These results provide new opportunities to explore adoptive transfer of B cells or hematopoietic stem cells expressing CD19 HAS after CAR T cell infusion, making cellular therapy safer and reducing patient mortality. BASE and PRIME gene editing technologies are currently being explored to streamline the generation of CD19 CAR T cell resistant B cells.
Preclinical studies of the effects of aging on gamma delta T cells and anti-tumor immunity in mouse models of melanoma
1289. Role of the aging on the gamma delta-T-cells in metastatic cutaneous melanoma progression
Kelly Coutant (Fox Chase Cancer Center, Philadelphia, PA, United States) reported a study investigating melanoma progression in mouse models of aging. In humans, cutaneous melanoma is a disease of aging, where incidence peaks at ages 65-79 and then decreases. To explore this phenomenon and melanoma metastasis in mouse models of aging, melanoma cells were injected intravenously into the tails of mice of various ages. Liver metastases were significantly increased in middle aged mice compared to young and geriatric mice, and liver metastases from middle aged mice contained decreased levels of gamma delta T cells, compared to liver metastases from young and from geriatric mice. To explore whether age-related decreases in gamma delta T cells promoted melanoma progression, gamma delta T cell knockouts (gamma delta knockouts) were generated in young murine melanoma models. Interestingly, after injection with melanoma cells, gamma delta knockout mice developed smaller tumors compared to wild type mice, but gamma delta knockouts exhibited significantly higher levels of lung and liver metastases compared to wild type mice. Lung and liver metastases from middle aged mice contained higher levels of gamma delta T cells than metastases from young mice, and geriatric mice exhibited more gamma delta T cells and fewer metastases than middle aged mice, suggesting that aging dysregulated gamma delta T cell expression and promoted melanoma metastasis. Geriatric mice that were depleted of gamma delta T cells and injected with melanoma cells produced significantly more lung and liver metastases than geriatric mice that were not depleted of gamma delta T cells, suggesting age-related depletion of gamma delta T cells leads to increased melanoma metastases. Melanoma cells overexpressing PROS1 were associated with growth of lung and liver metastases and decreased levels of gamma delta T cells in tumors, indicating PROS1 played a major role in age-related gamma delta T cell dysfunction. Overexpression of PROS1 also increased levels of polymorphonuclear myeloid-derived suppressor cells (PMN MDSCs), building upon previous findings that PMN MDSCs suppressed the anti-tumor activity of gamma delta T cells. Finally, MDSCs were found to be increased in liver metastases from middle aged mice and not in metastases from young and geriatric mice. These results have led to a model in which reactivated melanoma cells in the aging tumor microenvironment increase expression of PROS1, activating PMN MDSCs, which depletes gamma delta T cell and promotes progression of liver metastases. Future directions for this work include carrying out these studies in humanized mouse models to further elucidate the mechanisms behind gamma delta T cell dysregulation and depletion in the aged melanoma tumor microenvironment.