Exploration of immune-modulatory effects of amivantamab in combination with pembrolizumab in lung and head and neck squamous cell carcinoma.

Immune checkpoint inhibitors are effective first-line therapy for solid cancers. However, low response rate and acquired resistance over time has led to the need for additional therapeutic options. Here, we evaluated synergistic anti-tumor efficacy of EGFR x MET targeting bispecific antibody, amivantamab with PD-L1 immunotherapy, pembrolizumab in head and neck squamous cell carcinoma (HNSCC) and lung squamous cell carcinoma (LUSC) tumor bearing humanized PDX models. We demonstrated that pembrolizumab or amivantamab alone was ineffective and that combination treatment induced a significant reduction of tumor growth in both models (p<0.0001 and p<0.01, respectively). It appeared that combination of amivantamab and pembrolizumab significantly enhanced infiltration of granzyme B-producing CD8 T cells was in the TME of HNSCC PDX (p<0.01), and enhanced neoantigen-associated central memory CD8 T cells in circulating immune cells. Analysis of single cell RNA transcriptomics suggested that the tumor cells dramatically upregulated EGFR and MET in response to PD-L1 immunotherapy, potentially creating a metabolic state fit for tumor persistence in the tumor microenvironment (TME) and rendered pembrolizumab ineffective. We demonstrated that EGFRHIGHMETHIGH subcluster displayed an increased expression of genes implicated in production of lactate (SLC16A3 and LDHA) compared to the EGFRLOWMETLOW cluster. Accumulation of lactate in the TME has been associated with immunosuppression by hindering the infiltration of tumor killing CD8 T and NK cells. This study proved that amivantamab reduced glycolytic markers in the EGFRHIGHMETHIGH subcluster including SLC16A3 and LDHA and highlighted remodeling of the TME by combination treatment, providing rationale for additional therapy of amivantamab with PD-1 immunotherapy.

CD81 and CD82 expressing tumor-infiltrating lymphocytes in the NSCLC tumor microenvironment play a crucial role in T-cell activation and cytokine production.

Introduction: To understand the immune system within the tumor microenvironment (TME) of non-small cell lung cancer (NSCLC), it is crucial to elucidate the characteristics of molecules associated with T cell activation. Methods: We conducted an in-depth analysis using single-cell RNA sequencing data obtained from tissue samples of 19 NSCLC patients. T cells were classified based on the Tumor Proportion Score (TPS) within the tumor region, and molecular markers associated with activation and exhaustion were analyzed in T cells from high TPS areas. Results: Notably, tetraspanins CD81 and CD82, belonging to the tetraspanin protein family, were found to be expressed in activated T cells, particularly in cytotoxic T cells. These tetraspanins showed strong correlations with activation and exhaustion markers. In vitro experiments confirmed increased expression of CD81 and CD82 in IL-2-stimulated T cells. T cells were categorized into CD81highCD82high and CD81lowCD82low groups based on their expression levels, with CD81highCD82high T cells exhibiting elevated activation markers such as CD25 and CD69 compared to CD81lowCD82low T cells. This trend was consistent across CD3+, CD8+, and CD4+ T cell subsets. Moreover, CD81highCD82high T cells, when stimulated with anti-CD3, demonstrated enhanced secretion of cytokines such as IFN-γ, TNF-α, and IL-2, along with an increase in the proportion of memory T cells. Bulk RNA sequencing results after sorting CD81highCD82high and CD81lowCD82low T cells consistently supported the roles of CD81 and CD82. Experiments with overexpressed CD81 and CD82 showed increased cytotoxicity against target cells. Discussion: These findings highlight the multifaceted roles of CD81 and CD82 in T cell activation, cytokine production, memory subset accumulation, and target cell cytolysis. Therefore, these findings suggest the potential of CD81 and CD82 as promising candidates for co-stimulatory molecules in immune therapeutic strategies for cancer treatment within the intricate TME.

Successful expansion and cryopreservation of human natural killer cell line NK-92 for clinical manufacturing.

Natural killer (NK) cells have recently shown renewed promise as therapeutic cells for use in treating hematologic cancer indications. Despite this promise, NK cell manufacturing workflows remain largely manual, open, and disconnected, and depend on feeders, as well as outdated unit operations or processes, often utilizing research-grade reagents. Successful scale-up of NK cells critically depends on the availability and performance of nutrient-rich expansion media and cryopreservation conditions that are conducive to high cell viability and recovery post-thaw. In this paper we used Cytiva hardware and media to expand the NK92 cell line in a model process that is suitable for GMP and clinical manufacturing of NK cells. We tested a range of cryopreservation factors including cooling rate, a range of DMSO-containing and DMSO-free cryoprotectants, ice nucleation, and cell density. Higher post-thaw recovery was seen in cryobags over cryovials cooled in identical conditions, and cooling rates of 1°C/min or 2°C/min optimal for cryopreservation in DMSO-containing and DMSO-free cryoprotectants respectively. Higher cell densities of 5x107 cells/ml gave higher post-thaw viability than those cryopreserved at either 1x106 or 5x106 cells/ml. This enabled us to automate, close and connect unit operations within the workflow while demonstrating superior expansion and cryopreservation of NK92 cells. Cellular outputs and performance were conducive to clinical dosing regimens, serving as a proof-of-concept for future clinical and commercial manufacturing.

YH29407 with anti-PD-1 ameliorates anti-tumor effects via increased T cell functionality and antigen presenting machinery in the tumor microenvironment.

Among cancer cells, indoleamine 2, 3-dioxygenase1 (IDO1) activity has been implicated in improving the proliferation and growth of cancer cells and suppressing immune cell activity. IDO1 is also responsible for the catabolism of tryptophan to kynurenine. Depletion of tryptophan and an increase in kynurenine exert important immunosuppressive functions by activating regulatory T cells and suppressing CD8+ T and natural killer (NK) cells. In this study, we compared the anti-tumor effects of YH29407, the best-in-class IDO1 inhibitor with improved pharmacodynamics and pharmacokinetics, with first and second-generation IDO1 inhibitors (epacadostat and BMS-986205, respectively). YH29407 treatment alone and anti-PD-1 (aPD-1) combination treatment induced significant tumor suppression compared with competing drugs. In particular, combination treatment showed the best anti-tumor effects, with most tumors reduced and complete responses. Our observations suggest that improved anti-tumor effects were caused by an increase in T cell infiltration and activity after YH29407 treatment. Notably, an immune depletion assay confirmed that YH29407 is closely related to CD8+ T cells. RNA-seq results showed that treatment with YH29407 increased the expression of genes involved in T cell function and antigen presentation in tumors expressing ZAP70, LCK, NFATC2, B2M, and MYD88 genes. Our results suggest that an IDO1 inhibitor, YH29407, has enhanced PK/PD compared to previous IDO1 inhibitors by causing a change in the population of CD8+ T cells including infiltrating T cells into the tumor. Ultimately, YH29407 overcame the limitations of the competing drugs and displayed potential as an immunotherapy strategy in combination with aPD-1.

Immune checkpoint silencing using RNAi-incorporated nanoparticles enhances antitumor immunity and therapeutic efficacy compared with antibody-based approaches.

BACKGROUND: Cytotoxic CD8+ T cell-based cancer immunotherapy has been extensively studied and applied, however, tumor cells are known to evade immune responses through the expression of immune checkpoints, such as programmed death ligand 1 (PD-L1). To overcome these issues, antibody-based immune checkpoint blockades (eg, antiprogrammed cell death 1 (anti-PD-1) and anti-PD-L1) have been revolutionized to improve immune responses. However, their therapeutic efficacy is limited to 15%-20% of the overall objective response rate. Moreover, PD-L1 is secreted from tumor cells, which can interrupt antibody-mediated immune reactions in the tumor microenvironment. METHODS: We developed poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) encapsulating PD-L1 small interfering RNA (siRNA) and PD-1 siRNA, as a delivery platform to silence immune checkpoints. This study used the TC-1 and EG7 tumor models to determine the potential therapeutic efficacy of the PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs, on administration twice per week for 4 weeks. Moreover, we observed combination effect of PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs using TC-1 and EG7 tumor-bearing mouse models. RESULTS: PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs boosted the host immune reaction by restoring CD8+ T cell function and promoting cytotoxic CD8+ T cell responses. We demonstrated that the combination of NP-based therapeutic vaccine and PLGA (siRNA)-NPs resulted in significant inhibition of tumor growth compared with the control and antibody-based treatments (p<0.001). The proposed system significantly inhibited tumor growth compared with the antibody-based approaches. CONCLUSION: Our findings suggest a potential combination approach for cancer immunotherapy using PLGA (PD-L1 siRNA+PD-1 siRNA)-NPs and PLGA (antigen+adjuvant)-NPs as novel immune checkpoint silencing agents.

Incorporation of SKI-G-801, a Novel AXL Inhibitor, With Anti-PD-1 Plus Chemotherapy Improves Anti-Tumor Activity and Survival by Enhancing T Cell Immunity.

A recently developed treatment strategy for lung cancer that combines immune checkpoint inhibitors with chemotherapy has been applied as a standard treatment for lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), and it has improved the outcomes of chemotherapy. Maintenance treatment with anti-PD-1 antibody (aPD-1) enhances the effect of immunochemical combination therapy and improves therapeutic efficacy, which contributes toward a significant improvement in patient survival rates. The AXL receptor tyrosine kinase (AXL), which is expressed in tumor cells, plays an essential role in the resistance of cancers to chemotherapy and immunotherapy, and stimulates signaling associated with epithelial-mesenchymal transition (EMT) in metastatic cancer. AXL is thus an attractive target for controlling resistance to anti-tumor therapies. In this study, we examined the effect of AXL inhibitors on immune activation and tumor growth in TC1 and C3PQ mouse tumor models, in the context of clinical immunotherapy/chemotherapy and maintenance treatment, using an aPD-1 with/without pemetrexed. To determine the optimal timing for administration of SKI-G-801, an AXL inhibitor, we investigated its anti-tumor effects based on inclusion at the immunochemotherapy and maintenance therapy stages. We also performed flow cytometry-based immune profiling of myeloid cells and lymphoid cells at different points in the treatment schedule, to investigate the immune activation and anti-tumor effects of the AXL inhibitor. The addition of SKI-G-801 to the immune checkpoint inhibitor and chemotherapy stage, as well as the maintenance therapy stage, produced the best anti-tumor results, and significant tumor growth inhibition was observed in both the TC1 and C3PQ models. Both models also exhibited increased proportion of effector memory helper T cells and increased expression of CD86+ macrophages. Especially, regulatory T cells were significantly reduced in the TC1 tumor model and there was an increase in central memory cytotoxic T cell infiltration and an increased proportion of macrophages with high CD80 expression in the C3PQ tumor model. These results suggest increased infiltration of T cells, consistent with previous studies using AXL inhibitors. It is expected that the results from this study will serve as a stepping stone for clinical research to improve the existing standard of care.

SKI-G-801, an AXL kinase inhibitor, blocks metastasis through inducing anti-tumor immune responses and potentiates anti-PD-1 therapy in mouse cancer models.

OBJECTIVES: AXL-mediated activation of aberrant tyrosine kinase drives various oncogenic processes and facilitates an immunosuppressive microenvironment. We evaluated the anti-tumor and anti-metastatic activities of SKI-G-801, a small-molecule inhibitor of AXL, alone and in combination with anti-PD-1 therapy. METHODS: In vitro pAXL inhibition by SKI-G-801 was performed in both human and mouse cancer cell lines. Immunocompetent mouse models of tumor were established to measure anti-metastatic potential of SKI-G-801. Furthermore, SKI-G-801, anti-PD-1 or their combination was administered as an adjuvant or neoadjuvant in the 4T1 tumor model to assess their potential for clinical application. RESULTS: SKI-G-801 robustly inhibited pAXL expression in various cell lines. SKI-G-801 alone or in combination with anti-PD-1 potently inhibited metastasis in B16F10 melanoma, CT26 colon and 4T1 breast models. SKI-G-801 inhibited the growth of B16F10 and 4T1 tumor-bearing mice but not immune-deficient mice. An antibody depletion assay revealed that CD8+ T cells significantly contributed to SKI-G-801-mediated survival. Anti-PD-1 and combination group were observed the increased CD8+Ki67+ and effector T cells and M1 macrophage and decreased M2 macrophage, and granulocytic myeloid-derived suppressor cell (G-MDSC) compared to the control group. The neoadjuvant combination of SKI-G-801 and anti-PD-1 therapy achieved superior survival benefits by inducing more profound T-cell responses in the 4T1 syngeneic mouse model. CONCLUSION: SKI-G-801 significantly suppressed tumor metastasis and growth by enhancing anti-tumor immune responses. Our results suggest that SKI-G-801 has the potential to overcome anti-PD-1 therapy resistance and allow more patients to benefit from anti-PD-1 therapy.

Primary Tumor Suppression and Systemic Immune Activation of Macrophages through the Sting Pathway in Metastatic Skin Tumor.

PURPOSE: Agonists of the stimulator of interferon genes (STING) play a key role in activating the STING pathway by promoting the production of cytokines. In this study, we investigated the antitumor effects and activation of the systemic immune response of treatment with DMXAA (5,6-dimethylxanthenone-4-acetic acid), a STING agonist, in EML4-ALK lung cancer and CT26 colon cancer. MATERIALS AND METHODS: The abscopal effects of DMXAA in the treatment of metastatic skin nodules were assessed. EML4-ALK lung cancer and CT26 colon cancer models were used to evaluate these effects after DMXAA treatment. To evaluate the expression of macrophages and T cells, we sacrificed the tumor-bearing mice after DMXAA treatment and obtained the formalin-fixed paraffin-embedded (FFPE) tissue and tumor cells. Immunohistochemistry and flow cytometry were performed to analyze the expression of each FFPE and tumor cell. RESULTS: We observed that highly infiltrating immune cells downstream of the STING pathway had increased levels of chemokines after DMXAA treatment. In addition, the levels of CD80 and CD86 in antigen-presenting cells were significantly increased after STING activation. Furthermore, innate immune activation altered the systemic T cell-mediated immune responses, induced proliferation of macrophages, inhibited tumor growth, and increased numbers of cytotoxic memory T cells. Tumor-specific lymphocytes also increased in number after treatment with DMXAA. CONCLUSION: The abscopal effect of DMXAA treatment on the skin strongly reduced the spread of EML4-ALK lung cancer and CT26 colon cancer through the STING pathway and induced the presentation of antigens.