Enhancement Of Antitumor Immunity Research Articles

Potent induction of antitumor immunity by combining cryo-thermal ablation with immune checkpoint inhibitors in hepatocellular carcinoma.

The low response rate of immune checkpoint inhibitors (ICIs) prompts the exploration of novel combination therapies for patients with hepatocellular carcinoma (HCC). Here, we aimed to examine the efficiency and potential mechanism of cryo-thermal ablation (Cryo-A) combined with anti-programmed death protein 1 (αPD1) and/or cytotoxic T-lymphocyte antigen 4 (αCTLA4) inhibitors in a murine hepatoma model. Immunocompetent C57BL/6 mice inoculated with unilateral or bilateral H22 hepatic tumour cells were treated with Cryo-A and/or ICIs (αPD1 and/or αCTLA4). Flow cytometry, immunohistochemistry, ELISpot assay, time-of-flight cytometry, tumour rechallenging, and T-cell depletion assay were used to assess the dynamic changes of immune cell subsets following therapy. We found Cryo-A resulted in immunogenic cell death of tumour cells, activation of dendritic cells, and enhancement of antitumor immunity. Cryo-A alone was insufficient to extend survival, combining Cryo-A with αPD1 and αCTLA4 further modulated the tumour microenvironment, inducing a durable antitumor immune response by tumour-reactive CD8+ T cells and significantly prolonged survival. Time-of-flight cytometry (CyTOF) data revealed that combination therapies reshaped the tumour microenvironment by the increase of intratumoral CD8+ T cells expressed higher levels of cytotoxic markers and immune checkpoint molecules, and by downregulation of intratumoral granulocytes. The combination also resulted in the eradication of remote unablated tumours (abscopal effect). These findings suggested that Cryo-A turned HCC from "cold" tumours to "hot" tumours and the combination of Cryo-A with αPD1 and αCTLA4 may be a promising approach to improve the prognosis of HCC.

Lenvatinib delivery using a Gd/Fe bimetallic MOF: Enhancing antitumor immunity following microwave-based thermal therapy

Microwave (MW) thermal therapy has been developed as an effective clinical strategy that can achieve pronounced antitumor activity and also has the potential to trigger antitumor immunity. However, patients generally face high rates of tumor recurrence following MW treatment, limiting the long-term benefits of such treatment. The combination of MW treatment and immunomodulatory strategies may represent a promising means of reprogramming the immunosuppressive tumor microenvironment (TME) in a manner conducive to lower recurrence rates. In this study, a Lenvatinib-loaded Gd/Fe metal-organic framework (Gd/FeMOF) was designed as a promising approach to enhancing such antitumor immunity. MW-enhanced dynamic Gd/FeMOF sensitization can facilitate high levels of reactive oxygen species production under MW irradiation, resulting in stronger immunogenic tumor cell death. In parallel, the Lenvatinib released from Gd/FeMOF preparations can serve as an immune adjuvant that suppresses programmed death ligand 1 (PD-L1) expression and drives the reprogramming of the immunosuppressive TME. The Gd and Fe present within this MOF preparation also imbue it with magnetic resonance imaging capabilities. Importantly, in vivo animal model experiments confirmed the ability of GdFeMOF treatment to significantly enhance antitumor immunity while protecting against recurrence. Accordingly, this study offers a foundation for promising strategies aimed at the integrated diagnosis and durable treatment of cancer. Statement of SignificanceHigh rates of tumor recurrence following MW thermal therapy limit the long-term benefits of such treatment. We found that the administration of Lenvatinib-loaded Gd/FeMOF nanoparticles significantly reduced tumor recurrence after MW thermal therapy. Under MW irradiation, the Gd/FeMOF nanoparticles were found to augment the immune response due to facilitation of the process of immunogenic cell death. In addition, the released Lenvatinib could act as an immune adjuvant to downregulate the expression of PD-L1 and reprogram the immunosuppressive state of the tumor microenvironment, thus further enhancing the immune response. This is significant because MW-induced immune responses are relatively weak and usually fail to effectively prevent tumor recurrence. The combination of MW treatment with an immunomodulatory strategy may solve this problem.

Five-Day Spacing of Two Fractionated Ablative Radiotherapies Enhances Antitumor Immunity

This study aimed to enhance tumor control and abscopal effects by applying diverse stereotactic ablative radiation therapy (SABR) schedules. FSaII, CT-26, and 4T1 cells were used for tumor growth delay and lung metastases analysis after 1- or 5-day intervals radiation therapy (RT) with 40, 20, and 20 Gy, respectively. Immunodeficient BALB/c-nude, immunocompetent C3H, and BALB/c mouse models were used. For immune monitoring, FSaII tumors were analyzed using flow cytometry, immunofluorescence staining, and real-time quantitative reverse transcription polymerase chain reaction. The spleens were used for the ELISpot assay and flow cytometry to determine effector CD8 T cells. For abscopal effect analysis in CT-26 tumors, the volume of the nonirradiated secondary tumors was measured after primary tumors were irradiated with 1-day or 5-day intervals. Contrary to the high-dose 1-day interval RT, the 5-day interval RT significantly delayed tumor growth in immunocompetent mice, which was not observed in immunodeficient mice. In addition, the 5-day interval RT significantly reduced the number of lung metastases in FSaII and CT-26 tumors. Five-day spacing was more effective than 1-day interval in enhancing the antitumor immunity via increasing the secretion of tumor-specific IFN-γ, activating the CD8 T cells, and suppressing the monocytic myeloid-derived suppressor cells. The 5-day spacing inhibited nonirradiated secondary tumor growth more effectively than did the 1-day interval. Compared with the 1-day interval RT, the 5-day interval RT scheme demonstrated enhanced antitumor immunity of CD8 T cells associated with inhibition of myeloid-derived suppressor cells. Enhancing antitumor immunity leads to significant improvements in both primary tumor control and the abscopal effect.

Sodium Bicarbonate Nanoparticles for Amplified Cancer Immunotherapy by Inducing Pyroptosis and Regulating Lactic Acid Metabolism.

Although immunotherapy has a broad clinical application prospect, it is still hindered by low immune responses and immunosuppressive tumor microenvironment. Herein, a simple and drug-free inorganic nanomaterial, alkalescent sodium bicarbonate nanoparticles (NaHCO3 NPs), is prepared via a fast microemulsion method for amplified cancer immunotherapy. The obtained alkalescent NaHCO3 regulates lactic acid metabolism through acid-base neutralization so as to reverse the mildly acidic immunosuppressive tumor environment. Additionally, it can further release high amounts of Na+ ions inside tumor cells and induce a surge in intracellular osmolarity, and thus activate the pyroptosis pathway and immunogenic cell death (ICD), release damage-associated molecular patterns (DAMPs) and inflammatory factors, and improve immune responses. Collectively, NaHCO3 NPs observably inhibit primary/distal tumor growth and tumor metastasis through acid neutralization remitted immunosuppression and pyroptosis induced immune activation, showing an enhanced antitumor immunity efficiency. This work provides a new paradigm for lactic acid metabolism and pyroptosis mediated tumor treatment, which has a potential for application in clinical tumor immunotherapy.

Sodium Bicarbonate Nanoparticles for Amplified Cancer Immunotherapy by Inducing Pyroptosis and Regulating Lactic Acid Metabolism

AbstractAlthough immunotherapy has a broad clinical application prospect, it is still hindered by low immune responses and immunosuppressive tumor microenvironment. Herein, a simple and drug‐free inorganic nanomaterial, alkalescent sodium bicarbonate nanoparticles (NaHCO3 NPs), is prepared via a fast microemulsion method for amplified cancer immunotherapy. The obtained alkalescent NaHCO3 regulates lactic acid metabolism through acid‐base neutralization so as to reverse the mildly acidic immunosuppressive tumor environment. Additionally, it can further release high amounts of Na+ ions inside tumor cells and induce a surge in intracellular osmolarity, and thus activate the pyroptosis pathway and immunogenic cell death (ICD), release damage‐associated molecular patterns (DAMPs) and inflammatory factors, and improve immune responses. Collectively, NaHCO3 NPs observably inhibit primary/distal tumor growth and tumor metastasis through acid neutralization remitted immunosuppression and pyroptosis induced immune activation, showing an enhanced antitumor immunity efficiency. This work provides a new paradigm for lactic acid metabolism and pyroptosis mediated tumor treatment, which has a potential for application in clinical tumor immunotherapy.

Tertiary lymphoid structures correlate with enhancement of antitumor immunity in esophageal squamous cell carcinoma.

Tertiary lymphoid structures (TLSs) are associated with a favorable prognosis in several cancers. However, the correlation between TLSs and outcomes of esophageal squamous cell carcinoma (ESCC) and the impact of TLSs on the tumor immune microenvironment (TIME) remain unknown. We pathologically evaluated the significance of TLSs in ESCC focusing on TLS maturation using 180 ESCC specimens and performed single-cell RNA sequencing (scRNA-seq) using 14 ESCC tissues to investigate functional differences of immune cells according to TLS presence. TLS+ cases had better recurrence-free-survival (RFS) (p < 0.0001) and overall survival (OS) (p = 0.0016) compared with TLS- cases. Additionally, mature TLS+ cases had better RFS and OS compared with immature TLS+ cases (p = 0.019 and p = 0.015) and TLS- cases (p < 0.0001 and p = 0.0002). The scRNA-seq showed that CD8+ T cells in TLS+ tumors expressed high levels of cytotoxic signatures and antigen-presentation of dendritic cells (DCs) was enhanced in TLS+ tumors. Immunohistochemistry showed that the densities of tumor-infiltrating CD8+ T cells and DCs were significantly higher in TLS+ tumors than those in TLS- tumors. These data suggest the prognostic and functional significance of TLSs in ESCC and provides new insights into TLSs on the TIME.

Endogenous CCL21-Ser deficiency reduces B16–F10 melanoma growth by enhanced antitumor immunity

The chemokine CCL21 regulates immune and cancer cell migration through its receptor CCR7. The Ccl21a gene encodes the isoform CCL21-Ser, predominantly expressed in the thymic medulla and the secondary lymphoid tissues. This study examined the roles of CCL21-Ser in the antitumor immune response in Ccl21a-knockout (KO) mice. The Ccl21a-KO mice showed significantly decreased growth of B16–F10 and YUMM1.7 melanomas and increased growth of MC38 colon cancer, despite no significant difference in LLC lung cancer and EO771 breast cancer. The B16–F10 tumor in Ccl21a-KO mice showed melanoma-specific activated CD8+ T cell and NK cell infiltration and higher Treg counts than wild-type mice. B16–F10 tumors in Ccl21a-KO mice showed a reduction in the positive correlation between the ratio of regulatory T cells (Tregs) to activated CD8+ T cells and tumor weight. In Ccl21a-KO tumor, the intratumoral Tregs showed lower co-inhibitory receptors TIM-3 and TIGIT. Taken together, these results suggest that endogenous CCL21-Ser supports melanoma growth in vivo by maintaining Treg function and suppressing antitumor immunity by CD8+ T cells.

Killing three birds with one stone: Tumor-membrane-decorated Prussian blue nanovaccines for synergistic management of skin tumors, radiation dermatitis and wounds

Unhealed wounds following the resection of malignant skin tumors, featuring incomplete tumor tissue resection, full-thickness cutaneous defects, wound infection and radiation dermatitis, represent the main causes of prolonged recovery time, poor prognosis, and elevated tumor recurrence. Herein, a tumor cell membrane-decorated Prussian blue (PB) nanovaccine, referred to as PBVac, was fabricated via camouflage of PB nanozymes with the tumor cell membrane as tumor antigens and CpG oligodeoxynucleotides (ODNs) as adjuvants. Reinforcement by the tumor cell membrane substantially enhanced the tumor targeting potential of PBVac, achieving tumor-selective targeting and therapeutic efficacy. The bioactivity of PB nanozymes conferred PBVac with remarkable photothermal activity, antibacterial and antioxidant effects, curing wounds and radiation dermatitis by stimulating angiogenesis, antioxidation and anti-infection. The enhanced antitumor immunity of PBVac conferred by improved antigenicity was also assessed in a tumor challenge assay. PBVac could provide tumor antigens and molecular adjuvants for the priming of an efficient adaptive immune response, suppressing tumor growth. The combined merits of PBVac (photothermal, antibacterial and antioxidant capacities, and tumor membrane antigen) make it a novel nanoplatform that could be utilized as a safe and efficient therapeutic for synergistic management of skin tumors, radiation dermatitis and wounds.

A Dual-Mechanism Based Nutrient Partitioning Nanoregulator for Enhanced Immunotherapy against Anti-PD-1 Resistant Tumors.

Competitive consumption of nutrients between rapidly proliferating cancer cells and T cells results in an immunosuppressive tumor microenvironment (TME) and nutrient deprivation of T cells, which can cause low response rate and resistance to immunotherapies. In this study, we proposed a dual-mechanism based nutrient partitioning nanoregulator (designated as DMNPN), which can simultaneously regulate the immunosuppressive TME and enhance T cell nutrient availability. DMNPN consists of a charge-reversal biodegradable mesoporous silica, encapsulating glycolysis inhibitor lonidamine, and small interfering RNA against glutaminase. Through inhibiting glycolysis to decrease the lactic acid production and downregulating glutaminase expression to reduce the uptake of glutamine by tumor cells, DMNPN enables effective remodeling of metabolism and nutrient partitioning, which alleviates the immunosuppressive TME and boosts nutrient availability for T cells with enhanced antitumor immunity. Such a nutrient partitioning nanoregulator can effectively inhibit the growth of anti-programmed death receptor 1 (anti-PD-1) resistant tumors and prevent tumor metastasis and recurrence. Overall, this dual-mechanism based nutrient reallocation strategy provides a promising approach for cancer therapy.

Combination of AAV-delivered tumor suppressor PTEN with anti-PD-1 loaded depot gel for enhanced antitumor immunity

Recent clinical studies have shown that mutation of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene in cancer cells may be associated with immunosuppressive tumor microenvironment (TME) and poor response to immune checkpoint blockade (ICB) therapy. Therefore, efficiently restoring PTEN gene expression in cancer cells is critical to improving the responding rate to ICB therapy. Here, we screened an adeno-associated virus (AAV) capsid for efficient PTEN gene delivery into B16F10 tumor cells. We demonstrated that intratumorally injected AAV6-PTEN successfully restored the tumor cell PTEN gene expression and effectively inhibited tumor progression by inducing tumor cell immunogenic cell death (ICD) and increasing immune cell infiltration. Moreover, we developed an anti-PD-1 loaded phospholipid-based phase separation gel (PPSG), which formed an in situ depot and sustainably release anti-PD-1 drugs within 42 days in vivo. In order to effectively inhibit the recurrence of melanoma, we further applied a triple therapy based on AAV6-PTEN, PPSG@anti-PD-1 and CpG, and showed that this triple therapy strategy enhanced the synergistic antitumor immune effect and also induced robust immune memory, which completely rejected tumor recurrence. We anticipate that this triple therapy could be used as a new tumor combination therapy with stronger immune activation capacity and tumor inhibition efficacy.

Combination therapy with PD-1 inhibition plus rapamycin and metformin enhances anti-tumor efficacy in triple negative breast cancer

Immunotherapy using PD-1/PD-L1 inhibitors has been proved to be effective in triple negative breast cancer (TNBC), albeit only in a fraction of patients. Emerging evidences indicate mTOR blockade and metformin may re-orchestrate the immune system in tumors. Herein, in this study we aimed to evaluate the anti-tumor efficacy of PD-1 monoclonal antibody with mTOR inhibitor rapamycin or with the anti-diabetic drug metformin. The status of PD-1/PD-L1 and mTOR pathway was determined through analyzing the TCGA and CCLE data in TNBCs as well as by detection at mRNA and protein level. The inhibition of tumor growth and metastasis by anti-PD-1 combined with rapamycin or with metformin was evaluated in allograft mouse model of TNBC. The effects of combination therapy on the AMPK, mTOR and PD-1/PD-L1 pathways were also evaluated. The combination treatment with PD-1 McAb and rapamycin/metformin had additive effects on suppression of tumor growth and distant metastasis in mice. Compared with the control group and the monotherapy, combined PD-1 McAb with either rapamycin or metformin exhibited more obvious effects on induction of necrosis, CD8+ T lymphocytes infiltrating and inhibition of PD-L1 expression in TNBC homograft. In vitro study showed either rapamycin or metformin not only decreased PD-L1 expression, but increased p-AMPK expression and therefore led to down-regulation of p-S6. In summary, combination of PD-1 antagonist with either rapamycin or metformin led to more infiltrating TILs and decreased PD-L1 resulting in enhanced antitumor immunity and blockade of PD-1/PD-L1 pathway. Our results suggested such combination therapy may be a potential therapeutic strategy for TNBC patients.

Microfluidics-Enabled Nanovesicle Delivers CD47/PD-L1 Antibodies to Enhance Antitumor Immunity and Reduce Immunotoxicity in Lung Adenocarcinoma.

The CD47/PD-L1 antibodies combination exhibits durable antitumor immunity but also elicits excessive immune-related adverse events (IRAEs) caused by the on-target off-tumor immunotoxicity, hindering their clinical benefits greatly. Here, a microfluidics-enabled nanovesicle using ultra-pH-sensitive polymer mannose-poly(carboxybetaine methacrylate)-poly(hydroxyethyl piperidine methacrylate) (Man-PCB-PHEP) is developed to deliver CD47/PD-L1 antibodies (NCPA) for tumor-acidity-activated immunotherapy. The NCPA can specifically release antibodies in acidic environment, thereby stimulating the phagocytosis of bone marrow-derived macrophages. In mice bearing Lewis lung carcinoma, NCPA shows significantly improved intratumoral CD47/PD-L1 antibodies accumulation, promoted tumor-associated macrophages remodeling to antitumoral status, and increased infiltration of dendritic cells and cytotoxic T lymphocytes, resulting in more favorable treatment effect compared to those of free antibodies. Additionally, NCPA also shows less IRAEs, including anemia, pneumonia, hepatitis, and small intestinal inflammation in vivo. Altogether, a potent dual checkpoint blockade immunotherapy utilizing NCPA with enhanced antitumor immunity and reduced IRAEs is demonstrated.

Reprogramming of tumor-induced emergency granulopoiesis overcomes immune checkpoint therapy resistance

Abstract In established cancer in mice and humans, “emergency” myelopoiesis usually occurs to facilitate malignancy progression. Neutrophils and immature myeloid cells accumulated in the bone marrow, periphery and tumor are potent mediators of immunosuppression and tumor promotion. Here, we demonstrated that Cysteinyl leukotriene receptor 1 (CysLTR1), a key player for asthma, and other inflammatory conditons, sustained tumor-induced emergency granulopoiesis. Genetic ablation and pharmacological inhibition of CysLTR1 resulted in diminished tumor growth with enhanced antitumor immunity. The anti-tumor effect of CysLTR1 inhibition was linked with transcriptomic rewiring of granulopoiesis and neutrophil reprogramming toward an anti-tumor immune phenotype; this process required the distinct transcriptional factors to specifically dictate neutrophil progenitor commitment and differentiation in association with controlled degranulation. Furthermore, single-cell RNA sequencing analysis provided a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in tumor bearers. CysLTR1-mediated changes in the differentiation and degranulation also occurred in human neutrophils and correlated with the survival and ICT responsiveness in cancer patients. Consistently, targeting CysLTR1 with clinically available antagonists overcame resistance to immune checkpoint PD-1 inhibitors in several mouse tumor models. These findings thus have important implications for understanding the abnormal myelopoiesis during cancer progression and suggest new therapeutic approaches to improve ICT. Supported by grants from NCI (R01CA250101) and DoD (W81XWH-20-1-0024).

In Situ STING-Activating Nanovaccination with TIGIT Blockade for Enhanced Immunotherapy of Anti-PD-1-Resistant Tumors.

Immunotherapies comprising programmed cell death protein 1/PD ligand 1 (PD-1/PD-L1) immune checkpoint inhibitors are effective cancer treatments. However, the low response rate and immunoresistance resulting from alternative immune checkpoint upregulation and inefficient immune stimulation by T cells are problematic. The present report describes a biomimetic nanoplatform that simultaneously blocks the alternative T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory motif domain (TIGIT) checkpoint and activates the stimulator of interferon genes (STING) signaling pathway in situ for enhanced antitumor immunity. The nanoplatform is engineered by fusing a red blood cell membrane with glutathione-responsive liposome-encapsulated cascade-activating chemoagents (β-lapachone and tirapazamine), and anchoring them with a detachable TIGIT block peptide (named as RTLT). In the tumor environment, the peptide is spatiotemporally released to reverse T-cell exhaustion and restore antitumor immunity. The cascade activation of chemotherapeutic agents causes DNA damage and inhibits the repair of double-stranded DNA, which induces robust in situ STING activation for an efficient immune response. The RTLT inhibits anti-PD-1-resistant tumor growth, and prevents tumor metastasis and recurrence in vivo by inducing antigen-specific immune memory. This biomimetic nanoplatform thus provides a promising strategy for in situ cancer vaccination.

Enhancing anti-tumor immunity through liposomal oxaliplatin and localized immunotherapy via STING activation

The cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) pathway is a promising approach for anti-cancer immunotherapy by bridging innate and adaptive immunity. Recent evidence suggests that chemotherapy-induced DNA damage can directly induce dendritic cell (DC) maturation and recruitment, which synergizes with STING activation to enhance anti-tumor effects. As an immunogenic cell death (ICD) inducer, oxaliplatin generates massive double-stranded DNA (dsDNA) crosslinks, release of tumor-associated antigens and promoting the “eat me” signal. STING activation improves antigen immunogenicity, which can promote T cell activation and infiltration. In this study, we developed liposomes encapsulating oxaliplatin and combine this formulation with a STING agonist (ADU-S100) for treating colorectal cancer. The liposomes efficiently inhibited the proliferation of tumor cells while induced ICD in CT26 colorectal cancer cells, which enhanced dendritic cell maturation and phagocytosis in vitro. The liposome-based immunochemotherapy exhibited the strongest efficacy, resulting in complete remission upon tumor inoculation. Mechanistic studies showed this potent anti-cancer effect was related to the significant recruitment of infiltrating CD8 and CD4 T cells, reduction of suppressive Treg cells, and a shift in the phenotype of tumor-associated suppressive macrophages that promote cancer to immune stimulating macrophages. Thus, our study demonstrated the potential of combining oxaliplatin-loaded liposomes with a STING agonist to reduce tumor growth by regulating the immunosuppressive state in the tumor.

Abstract LB337: Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells

Abstract The E3 ubiquitin ligase CBL-B is a master regulator of effector T cells through its downstream regulation of T-cell receptor and co-stimulatory stimulation, but it has also been shown to play a negative feedback role in NK cells. CBL-B depletion enhances the production of IFNγ and the cytotoxicity of activated NK cells. Similar to the effect of depleting CD8 T cells in mice, depletion of NK cells abolishes the tumor growth delay observed in Cbl-b-/- mice. With emerging data supporting the potential role of exogenous NK cells in anti-tumor immunity, inhibition of CBL-B represents a promising approach to enhance both adaptive and innate immunity for cancer immunotherapy. Using one of our small molecules, allosteric CBL-B inhibitors, HOT-A, we demonstrated that CBL-B inhibition enhanced NK activation, IFNγ production and granzyme B secretion in response to the various stimulation conditions, including cytokines (such as IL-15 and IL12 + IL-18) and stimulatory receptors (such as NKp30 and NKG2D + CD244). NK cell proliferation was also enhanced in the presence of HOT-A. Single cell analysis further suggested that HOT-A increased NK polyfunctional cytokine expression during IL-12 + IL-18 activation. In a coculture system of NK cells and K562 target cells, HOT-A dose-dependently promoted primary human NK cell activation, cytokine production and cytotoxicity in the K562 cells. In the in vivo CT-26 tumor model, granzyme B+ NK cells in the TIL were significantly increased in the HOT-A treated tumor bearing mice and NK function gene signature was also increased by Nanostring analysis, suggesting an enhanced NK function in the tumor microenvironment. Taken together, the pre-clinical data presented here demonstrate that our CBL-B inhibitor enhances NK cell function in vitro and in vivo, suggesting that its enhancement of anti-tumor immunity may be driven through both effector T cell and NK cell-driven biology. Citation Format: Yilin Qi, Jun Kuai, Yingzhi Bi, David Greco, Samira Jaeger, Ken Carson, Hadi Danaee, Timothy Reilly, Geraldine Harriman, Fang Wang. Inhibition of the E3 ligase CBL-B enhances the effector function and proliferation of natural killer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB337.

Abstract 6657: Phase I trial of first-in-class anti-PVR mAb NTX1088: Restoration of DNAM1 expression as MOA for enhanced antitumor immunity

Abstract NTX1088, a first-in-class anti-PVR (CD155) monoclonal antibody (mAb), is currently evaluated in a Phase 1, open-label, multi-center study (NCT05378425), initiated at MD Anderson Cancer center. Study objectives are safety, dose-finding, and efficacy with focus on pharmacokinetics, pharmacodynamics, and biomarker discovery. NTX1088 will be investigated as a single agent and combined with the anti-PD1 mAb, pembrolizumab (Keytruda) in patients with locally advanced and metastatic solid malignancies. NTX1088 is a humanized, IgG4-S228P mAb that binds PVR with sub-nM affinity and blocks all known interacting receptors with a single nM EC50. PVR, is a membranal protein, highly upregulated on tumor cells, across multiple cancer types. PVR expression has been associated with worse patient outcomes, due to its role in immune suppression. PVR’s impact on immune cells is mediated through interaction with the key stimulatory receptor, DNAM1 (CD226), on T and NK cells, leading to internalization and degradation of DNAM1. Additionally, PVR is the ligand for the inhibitory immune checkpoint receptors, TIGIT, CD96 and KIR2DL5A. Blocking PVR by NTX1088, therefore, has a multi-faceted immune-stimulating role, through restoration of DNAM1 expression and its immune activation function, while simultaneously neutralizing TIGIT, CD96 and KIR2DL5A inhibitory signals in immune cells. Importantly, DNAM1 downmodulation was recently identified as a key resistance mechanism to approved immune checkpoint inhibitors (ICIs), and its restoration by NTX1088 is a novel MoA, not demonstrated by other therapies. In vitro, as a monotherapy, NTX1088 significantly increased immune cell activation, and was superior to TIGIT, CD112R, and PD1 antibody blockade, leading to greater immune-mediated tumor cell killing, IFNγ secretion, and CD137 induction. Importantly, only NTX1088 was able to restore DNAM1 to the surface of immune cells in all experimental settings. Synergy was observed when NTX1088 was combined with PD1 blockers, or with the anti-CD112R mAb, NTX2R13, in line with the restoration of DNAM1 expression. Numerous humanized murine xenograft models were investigated. NTX1088 exhibited robust tumor growth inhibition as a standalone and in combination with PD1 blockade. Syngeneic models of PVRK.O resulted in complete immune-mediated tumor regression, including hard to treat colorectal tumors. In conclusion, PVR blockade by NTX1088 has a remarkable pre-clinical efficacy, suggesting a potential clinical breakthrough, based on the ability of simultaneously overcome multiple tumor escape mechanisms. First-in-human (FIH) trial is currently ongoing and biomarker data is analyzed to assess clinical impact of the drug and prepare for patient stratification. Citation Format: Anas Atieh, Akram Obiedat, Alon Vitenshtein, Guy Cinamon, Keren Paz, Tihana lenac, Paola Kucan, Marija Mazor, Ofer Mandelboim, Stipan Jonji, Pini Tsukerman. Phase I trial of first-in-class anti-PVR mAb NTX1088: Restoration of DNAM1 expression as MOA for enhanced antitumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6657.

Abstract P2-20-09: Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases

Abstract Breast cancer is one of the most commonly diagnosed cancer types and a leading cause of cancer death in women in the United States. However, it is difficult to treat with immunotherapy because of its immunological “cold” status. This underscores the importance of a novel clinical approach to increase efficacy of immunotherapy including acute upregulation of Type I Interferon (IFN) response. One strategy to enhance Type I IFN response is by release of damage-associated molecular patterns (DAMPs) from mitochondria, including mitochondrial DNA, RNA and oxidized proteins that are recognized by pattern recognition receptors (PRRs) in cytoplasm and activate downstream IFN signaling. Because many cancers including breast cancer experience increased and dysregulated mitochondrial biogenesis at baseline, they contain significantly higher levels of DAMPs compared to normal cells, but these substrates are sequestered in mitochondria and not accessible to PRRs. We set out to develop a strategy to exploit increased levels of DAMPs in cancer cell mitochondria to enhance Type I IFN response. While DAMPs are released from mitochondria upon apoptosis caused by mitochondrial outer membrane permeabilization (MOMP), Type I IFN response is still silenced due to cleavage of PRRs by activated caspases. One way to induce MOMP is by inhibition of Myeloid Cell Leukemia-1 (Mcl-1), an anti-apoptotic family protein that is frequently upregulated in breast cancer. Interestingly, breast cancer cell lines showed the highest dependency on Mcl-1 among solid cancers and inhibitors of Mcl-1 have shown efficacy in breast cancer early clinical trials. Based on our preliminary observations showing that Mcl-1 inhibition activates a robust Type I IFN response in MCF7 breast cancer cells deficient for caspase 3, we hypothesized that combining Mcl-1 inhibitors with caspase inhibitors would greatly enhance Type I IFN response in a robust and specific manner by facilitating release on DAMPs into the cytoplasm while preventing inactivation of PRRs. Strikingly, in contrast with Mcl-1 inhibitor treatment alone, combination treatment led to dramatic increase in expression levels of IFN-β and interferon-stimulated genes (ISGs) in MCF7 and ZR-75-1 ER+ breast cancer cell lines with minimal effects on cytotoxicity. In addition, T47d, a breast cancer line highly resistant to Mcl-1 inhibition due to compensation by other Bcl-2 family proteins, showed greatly enhanced Type I IFN response upon combined Mcl-1, Bcl-2 and caspase inhibition. Because reduced expression of MHC class I molecules on cell surface is one of the most important immune escape mechanisms in tumors including breast cancer, we examined whether the proposed combinatorial treatment would overcome this by enhancing expression of MHC class I. Immunofluorescence (IF) staining confirmed that the combination treatment dramatically increased total HLA-ABC protein expression on plasma membrane of ZR-75-1. Furthermore, combinatorial treatment upregulated transcription of most genes responsible for antigen presentation, a multistep process consisting of antigen peptide generation and loading of MHC class I molecules. Lastly, we sought to examine how elevated chemokine expression may impact patients with breast cancer. We examined association of cytokines highly upregulated by the combination treatment with immune cell infiltration using a TCGA dataset that included over a thousand breast cancer samples. This analysis was performed by xCell algorithm and demonstrated strong association of that cytokine expression with CD8+ T, B and DC infiltrate. Our results demonstrate that the combination of Mcl-1 and caspase inhibitors greatly enhances Type I IFN response and may improve anti-tumor immunity. We plan to further test this drug combination in immunocompetent mouse models, laying the foundation for a clinical trial assessing the combination treatment in patients with metastatic ER+ breast cancer. Citation Format: Jae Kyo Yi, Alexander Spektor. Enhancement of anti-tumor immunity in ER+ breast cancer through dual inhibition of MCL1 and caspases. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-20-09.

Abstract PD4-03: PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial

Abstract Background The status of the tumor microenvironment (TME) can profoundly affect the response to immune-based therapies for the treatment of cancer. Results previously reported from the AWARE-1 study in early breast cancer patients demonstrated that pelareorep (pela, an oncolytic reovirus), alone or in combination with checkpoint inhibitor (CPI) therapy, modified the inflammatory state of the TME. We also showed that many of pela’s effects on the TME were enhanced by the addition of checkpoint blockade. Here, we report the effect of treatment with pela on selected molecular markers associated with enhanced anti-tumor immunity. Methods Newly diagnosed HR+/HER2- early BC patients were enrolled into two cohorts: Cohort 1 (C1): pela + letrozole (n=10); and Cohort 2 (C2): pela + letrozole + atezolizumab (n=10). Pela was intravenously administered on days 1, 2 and 8, 9, and atezolizumab was given on day 3. For this analysis, tumor biopsies (FFPE samples) collected pre-treatment (D1) and on days 3 (D3, prior to the atezolizumab administration) were examined by GeoMx digital spatial profiling (DSP, using Nanostring’s Cancer Transcriptome Atlas [CTA]). Moreover, the expression of 770 immune-related genes was analyzed using a specific immune panel (n=20). Gene Set Enrichment Analysis (GSEA) (version 4.1.0) was used to assess pela-induced activation pathways. Results GeoMx DSP showed that pela therapy significantly activated IFN-gamma signaling and associated interferon response genes from D1 to D3 (Normalized Enrichment Score [NES] = 3.3, p-values &amp;lt; 0.02) in the cytokeratin-positive subset of the tumor samples. GSEA of the immune dataset (730 immune genes + 30 housekeeping genes) from the whole tissue also showed a significant upregulation of IFN-gamma signaling pathway genes (FDR &amp;lt; 25%, p-value&amp;lt; 0.001). Increases were also observed in genes reported to be associated with an enhanced tumor inflammatory signal (TIS) including PD-L1, IDO1, HLA-E and STAT1 (p-values &amp;lt; 0.005). Conclusions These results demonstrate that treatment with pela alters the TME to induce and enhance anti-tumor immunity. This enhancement of anti-tumor immunity may potentiate the TME for CPI therapy. Citation Format: Houra Loghmani, Joaquín Gavilá, Luis Manso, Matt Coffey, Richard Trauger, Fernando Salvador, Tomás Pascual, Aleix Prat, Thomas Heineman. PD4-03 Pelareorep primes the tumor for checkpoint inhibition therapy by activating the interferon-gamma signaling pathway and tumor inflammation signature in early breast cancer patients - results of the AWARE-1 trial [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr PD4-03.

Gastric cancer (GC) cohort of a phase 2 trial of E7389-LF (liposomal formulation of eribulin) in combination with nivolumab.

339 Background: Due to a lack of efficacy and long-term survival seen in previously-studied therapies, new therapies for pretreated advanced GC are warranted. E7389-LF is a new formulation that uses liposomes to encapsulate eribulin; this is anticipated to improve eribulin concentration in tumor tissues. E7389-LF and nivolumab have both shown efficacy as monotherapy in pretreated GC. E7389-LF may also synergize well with PD-1 inhibitors by acting as a cytotoxic therapy and by modulating the tumor microenvironment. Methods: Patients (pts) with unresectable and measurable GC, esophageal cancer, or small cell lung cancer who were previously treated with 1 (2 for GC) chemotherapy regimens were enrolled in the phase 2 part of Study 120 and treated with E7389-LF 2.1 mg/m2 plus nivolumab 360 mg every 3 weeks (Q3W). In the GC cohort, pts had to show disease progression from combination therapy including a platinum drug + fluoropyrimidine (1st-line therapy) and a taxane-containing regimen (2nd-line therapy). The primary objective of the phase 2 part was to evaluate the objective response rate (ORR); secondary objectives included safety, progression-free survival (PFS), and pharmacokinetics. Other efficacy (including overall survival [OS]) and biomarker objectives were exploratory. Tumor responses were assessed by the investigators per RECIST v1.1. Results: 31 GC pts were included; median age was 63 years; 18 pts were male, 13 were female. By the data cutoff date (May 31, 2022), 29 pts discontinued (26 due to disease progression, 3 due to adverse events). 8 Pts had a partial response (PR); the ORR was 25.8% (95% CI 11.9–44.6). The median PFS was 2.69 months (95% CI 1.91–2.99). The median OS was 7.85 months (95% CI 4.47–not estimable). The 6-month OS rate was 61.3%; the 9-month OS rate was 44.7%. 30 Pts had ≥1 treatment-related TEAE, most commonly neutropenia (n = 24); 25 pts had at least 1 grade ≥3 treatment-related TEAE, most commonly neutropenia (n = 22). 16 Pts had ≥1 TEAE resulting in dose reduction of E7389-LF. 4 Pts had ≥1 TEAE resulting in withdrawal of E7389-LF or nivolumab, including asthma, cerebral hemorrhage, decreased appetite, pulmonary edema, and upper gastrointestinal hemorrhage (each n = 1). 29 Pts had an evaluable PD-L1 combined positive score (CPS); 5 of 20 pts (25.0%) with a CPS of &lt; 5 and 2 of 9 (22.2%) with a CPS of ≥5 had a PR. Increases in pharmacodynamic markers including vasculature-related markers were observed; antitumor immunity was observed per changes in interferon gamma (IFNγ) and IFNγ-related markers. Conclusions: E7389-LF 2.1 mg/m2 in combination with nivolumab 360 mg Q3W had promising efficacy for pretreated GC, as evidenced by the notable ORR of 25.8% as well as by PFS and OS. No new safety signals were identified for this combination. Biomarker changes suggested vascular remodeling activity and enhancement of antitumor immunity via IFNγ signaling. Clinical trial information: NCT04078295 .