Efficacy Of Immune Checkpoint Blockade Research Articles

Overcoming microenvironmental resistance to PD-1 blockade in genetically engineered lung cancer models.

Immune checkpoint blockade (ICB) with PD-1 or PD-L1 antibodies has been approved for the treatment of non-small cell lung cancer (NSCLC). However, only a minority of patients respond, and sustained remissions are rare. Both chemotherapy and antiangiogenic drugs may improve the efficacy of ICB in mouse tumor models and patients with cancer. Here, we used genetically engineered mouse models of Kras G12D/+;p53 -/- NSCLC, including a mismatch repair-deficient variant (Kras G12D/+;p53 -/-;Msh2 -/-) with higher mutational burden, and longitudinal imaging to study tumor response and resistance to combinations of ICB, antiangiogenic therapy, and chemotherapy. Antiangiogenic blockade of vascular endothelial growth factor A and angiopoietin-2 markedly slowed progression of autochthonous lung tumors, but contrary to findings in other cancer types, addition of a PD-1 or PD-L1 antibody was not beneficial and even accelerated progression of a fraction of the tumors. We found that antiangiogenic treatment facilitated tumor infiltration by PD-1+ regulatory T cells (Tregs), which were more efficiently targeted by the PD-1 antibody than CD8+ T cells. Both tumor-associated macrophages (TAMs) of monocyte origin, which are colony-stimulating factor 1 receptor (CSF1R) dependent, and TAMs of alveolar origin, which are sensitive to cisplatin, contributed to establish a transforming growth factor-β-rich tumor microenvironment that supported PD-1+ Tregs Dual TAM targeting with a combination of a CSF1R inhibitor and cisplatin abated Tregs, redirected the PD-1 antibody to CD8+ T cells, and improved the efficacy of antiangiogenic immunotherapy, achieving regression of most tumors.

Fucoidan-Supplemented Diet Potentiates Immune Checkpoint Blockage by Enhancing Antitumor Immunity

Immune checkpoint blockade (ICB) therapies such as PD-1 antibodies have produced significant clinical responses in treating a variety of human malignancies, yet only a subset of cancer patients benefit from such therapy. To improve the ICB efficacy, combinations with additional therapeutics were under intensive investigation. Recently, special dietary compositions that can lower the cancer risk or inhibit cancer progression have drawn significant attention, although few were reported to show synergistic effects with ICB therapies. Interestingly, Fucoidan is naturally derived from edible brown algae and exhibits antitumor and immunomodulatory activities. Here we discover that fucoidan-supplemented diet significantly improves the antitumor activities of PD-1 antibodies in vivo. Specifically, fucoidan as a dietary ingredient strongly inhibits tumor growth when co-administrated with PD-1 antibodies, which effects can be further strengthened when fucoidan is applied before PD-1 treatments. Immune analysis revealed that fucoidan consistently promotes the activation of tumor-infiltrating CD8+ T cells, which support the evident synergies with ICB therapies. RNAseq analysis suggested that the JAK-STAT pathway is critical for fucoidan to enhance the effector function of CD8+ T cells, which could be otherwise attenuated by disruption of the T-cell receptor (TCR)/CD3 complex on the cell surface. Mechanistically, fucoidan interacts with this complex and augments TCR-mediated signaling that cooperate with the JAK-STAT pathway to stimulate T cell activation. Taken together, we demonstrated that fucoidan is a promising dietary supplement combined with ICB therapies to treat malignancies, and dissected an underappreciated mechanism for fucoidan-elicited immunomodulatory effects in cancer.

Combining an Alarmin HMGN1 Peptide with PD-L1 Blockade Results in Robust Antitumor Effects with a Concomitant Increase of Stem-Like/Progenitor Exhausted CD8+ T Cells.

The expansion of intratumoral stem-like/progenitor exhausted CD8+ T (Tstem/Tpex) cells provides a potential approach to improve the therapeutic efficacy of immune checkpoint blockade (ICB). Thus, here we demonstrate a strategy to facilitate Tstem/Tpex cell expansion by combining an alarmin high-mobility group nucleosome binding domain 1 (HMGN1) peptide with programmed death-ligand 1 (PD-L1) blockade. The antitumor effects of HMGN1, anti-PD-L1, and their combined treatment were monitored in the B16F10, LLC, Colon26, or EO771 tumor-bearing mice. The comprehensive immunologic analyses, such as high-dimensional flow cytometry, transcriptome analysis, and single-cell RNA-sequencing (scRNA-seq), were used to investigate the cellular and molecular mechanisms of antitumor immune responses after treatments. We identified the immunostimulatory domain (EPKRR SARLS AKPPA KVEAK PKK) on HMGN1 and synthesized this domain as a therapeutic peptide (minP1). Combined treatment with minP1 and PD-L1 blockade induced durable tumor regression in tumor-bearing mice. minP1 increased the number of intratumoral mature DCs enriched in immunoregulatory molecules (mregDC) and enhanced their MHC class I antigen-presenting program. minP1 also synergized with PD-L1 blockade in augmenting intratumoral Tstem/Tpex cell number. Analysis of our scRNA-seq dataset by CellPhonDB suggested potential interactions between mregDCs and Tstem/Tpex cells in tumors. Our results indicate that HMGN1 peptide (minP1) serves as an immunoadjuvant to promote effective anti-PD-L1 immunotherapy with increased Tstem/Tpex cells in tumors.

The Prognosis and Immune Checkpoint Blockade Efficacy Prediction of Tumor-Infiltrating Immune Cells in Lung Cancer.

BackgroundsThe high morbidity and mortality of lung cancer are serious public health problems. The prognosis of lung cancer and whether to apply immune checkpoint blockade (ICB) are currently urgent problems to be solved.MethodsUsing R software, we performed Kaplan–Meier (K-M) analysis, Cox regression analysis, functional enrichment analysis, Spearman correlation analysis, and the single-sample gene set enrichment analysis.ResultsOn the Tumor IMmune Estimation Resource (TIMER2.0) website, we calculated the abundance of tumor-infiltrating immune cells (TIICs) of lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) patients. B cell and myeloid dendritic cell (DC1) were independent prognostic factors for LUAD and LUSC patients, respectively. Enrichment analysis confirmed that genes highly related to B cell or DC1 were closely related to the immune activation of lung cancer patients. In terms of adaptive immune resistance markers, CD8A, CD8B, immunomodulators (immunostimulants, major histocompatibility complex, receptors, and chemokines), immune-related pathways, tumor microenvironment score, and TIICs, high B cell/DC1 infiltration tissue was inflamed and immune-activated and might benefit more from the ICB. Genes most related to B cell [CD19, toll-like receptor 10 (TLR10), and Fc receptor-like A (FCRLA)] and DC1 (ITGB2, LAPTM5, and SLC7A7) partially clarified the roles of B cell/DC1 in predicting ICB efficacy. Among the 186 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, there were three and four KEGG pathways, which partially explained the molecular mechanisms by which B cell and DC1 simultaneously predicted the prognosis and efficacy of immunotherapy, respectively. Among five immune subtypes, the abundance of B cell/DC1 and expression of six hub genes were higher in immune C2, C3, and C6.ConclusionB cell and DC1 could predict the prognosis and ICB efficacy of LUAD and LUSC patients, respectively. The six hub genes and seven KEGG pathways might be novel immunotherapy targets. Immune C2, C3, and C6 subtypes of lung cancer patients might benefit more from ICB therapy.

Expanding the immune armoury against myelodysplastic syndrome.

Expanding the immune armoury against myelodysplastic syndrome.

Tumor microenvironment evaluation promotes precise checkpoint immunotherapy of advanced gastric cancer

BackgroundDurable efficacy of immune checkpoint blockade (ICB) occurred in a small number of patients with metastatic gastric cancer (mGC) and the determinant biomarker of response to ICB remains unclear.MethodsWe developed...

Evaluating the impact of age on immune checkpoint therapy biomarkers.

SUMMARYBoth tumors and aging alter the immune landscape of tissues. These interactions may play an important role in tumor progression among elderly patients and may suggest considerations for patient care. We leverage large-scale genomic and clinical databases to perform comprehensive comparative analysis of molecular and cellular markers of immune checkpoint blockade (ICB) response with patient age. These analyses demonstrate that aging is associated with increased tumor mutational burden, increased expression and decreased promoter methylation of immune checkpoint genes, and increased interferon gamma signaling in older patients in many cancer types studied, all of which are expected to promote ICB efficacy. Concurrently, we observe age-related alterations that might be expected to reduce ICB efficacy, such as decreases in T cell receptor diversity. Altogether, these changes suggest the capacity for robust ICB response in many older patients, which may warrant large-scale prospective study on ICB therapies among patients of advanced age.

Prognostic value and immunological characteristics of a novel autophagy-related signature in pancreatic cancer

Autophagy affects the development, progression, and prognosis of various cancers including pancreatic cancer. To develop an autophagy-related prognostic model of pancreatic cancer, we systematically analyzed gene expression profile from The Cancer Genome Atlas and Genotype-Tissue Expression. Ten autophagy-relevant genes with potential prognostic values were identified, based on which a prognostic model was constructed. We divided patients into a high- and a low-risk group with this model. Time-dependent receiver operating characteristic and Kaplan-Meier curves were conducted to evaluate the accuracy of the model. The Area Under Curvevalues of this model at 12, 18, and 24 months were 0.76, 0.73, and 0.78, respectively. The model was further validated in two Gene Expression Omnibus datasets. Gene set enrichment analysis and Cibersort were applied to analyze immune infiltration patterns and immune checkpoint blockade (ICB) molecules. The expression of ICB molecules, such as PD-L1 and PD1, presented significant correlation with the risk score. In conclusion, the risk score model established herein has been proved to be robust for evaluating the prognosis of pancreatic cancer and facilitate to improve the efficacy of ICB.

Recent Advances in Glioma Therapy: Combining Vascular Normalization and Immune Checkpoint Blockade.

Simple SummaryGlioblastoma is the most malignant tumor of the brain. Over the years, prognosis for patients with glioblastoma has remained dismal despite advances in medical sciences. Glioblastoma is a highly vascularized tumor; however, antiangiogenic therapy has not achieved the expected outcome. Recent promising results from immunotherapies for other cancer types such as melanoma have prompted the further investigation of combining antiangiogenic therapy with immune checkpoint blockade. This article aims to provide an overview concerning the development of a potential intervention that may enhance the efficacy of immune checkpoint blockade as glioblastoma therapy.Glioblastoma (GBM) accounts for more than 50% of all primary malignancies of the brain. Current standard treatment regimen for GBM includes maximal surgical resection followed by radiation and adjuvant chemotherapy. However, due to the heterogeneity of the tumor cells, tumor recurrence is often inevitable. The prognosis of patients with glioma is, thus, dismal. Glioma is a highly angiogenic tumor yet immunologically cold. As such, evolving studies have focused on designing strategies that specifically target the tyrosine kinase receptors of angiokines and encourage immune infiltration. Recent promising results from immunotherapies on other cancer types have prompted further investigations of this therapy in GBM. In this article, we reviewed the pathological angiogenesis and immune reactivity in glioma, as well as its target for drug development, and we discussed future directions in glioma therapy.

Enhanced Antitumor Response to Immune Checkpoint Blockade Exerted by Cisplatin-Induced Mutagenesis in a Murine Melanoma Model.

Sequencing data from different types of cancers including melanomas demonstrate that tumors with high mutational loads are more likely to respond to immune checkpoint blockade (ICB) therapies. We have previously shown that low-dose intratumoral injection of the chemotherapeutic DNA damaging drug cisplatin activates intrinsic mutagenic DNA damage tolerance pathway, and when combined with ICB regimen leads to tumor regression in the mouse YUMM1.7 melanoma model. We now report that tumors generated with an in vitro cisplatin-mutagenized YUMM1.7 clone (YUMM1.7-CM) regress in response to ICB, while an identical ICB regimen alone fails to suppress growth of tumors generated with the parental YUMM1.7 cells. Regressing YUMM1.7-CM tumors show greater infiltration of CD8 T lymphocytes, higher granzyme B expression, and higher tumoral cell death. Similarly, ex-vivo, immune cells isolated from YUMM1.7-CM tumors-draining lymph nodes (TDLNs) co-incubated with cultured YUMM1.7-CM cells, eliminate the tumor cells more efficiently than immune cells isolated from TDLNs of YUMM1.7 tumor-bearing mice. Collectively, our findings show that in vitro induced cisplatin mutations potentiate the antitumor immune response and ICB efficacy, akin to tumor regression achieved in the parental YUMM1.7 model by ICB administered in conjunction with intratumoral cisplatin injection. Hence, our data uphold the role of tumoral mutation burden in improving immune surveillance and response to ICB, suggesting a path for expanding the range of patients benefiting from ICB therapy.

Abstract 2964: Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy

Abstract Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy. These differences in sensitivity can be either innate or acquired. Yet, there has been limited 3D in vitro models to assess tumor immune-reactivity. These systems are ideal for isolating specific molecular mechanisms that dictate cell behavior and interactions. Our goal was to create an organoid model containing cancer cells paired with cytotoxic T-cells to model immune checkpoint blockade (ICB) efficacy. This model could then be used to examine novel microbiome-ICB interactions shown by recent research to alter therapeutic response levels in patients. Methods: We created tumor organoids consisting of tumor and immune cells, embedded in extracellular matrix (ECM)-like hydrogels. Organoids were treated with therapeutic equivalent doses of anti-PD-1 and anti-CTLA-4 or single dose of anti-CD-47 and cell viability assays, flow cytometry, RT-qPCR, and immunohistochemistry staining were performed to determine the effects of ICB. Physiologic concentrations of metabolites derived from likely effector bacterial species were then evaluated for their effects on genetic expression and immunotherapy efficacy. Results and Discussion: We showed that ICB therapy stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing. ICB treated samples resulted significant loss of viability with corroborating readings from the other methods of characterization. RT-qPCR resulted in some elucidation of potential cellular changes due to bacterial metabolites with further quantification ongoing. Conclusion: We have created an ex-vivo tumor immune-reactive organoid model for studying immunotherapy. We can now continue to change individual microenvironment factors, such as microbiome metabolites, and observe their impacts on immunotherapy efficacy to better understand what conditions are conducive or detrimental to successful ICB treatment. Citation Format: Ethan Shelkey, David Soto-Pantoja, Yong Lu, Shay Soker. Immuno-reactive cancer organoid models to examine microbiome metabolite effects on immune checkpoint blockade efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2964.

Abstract 439: BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer

Abstract BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer Background: Immune checkpoint inhibitors (ICIs) has received approval due to its encouraging clinical efficacy with improving the outcome of cancer patients. However, current immunotherapeutic regimens benefit merely a minority of populations. Thus, combining ICIs with other potential modalities is of great significance to enhance the response of single drug alone. Modifying tumor-associated macrophages (TAMs) strategies have attracted attention for ameliorating tumor immunosuppression and improving anti-tumor immunity. Here we exhibit APG2575, a proven BCL-2 inhibitor, can act as an antitumor immune modulator that resets TAMs from M2 to pro-tumoral M1 phenotype and further augment immunotherapeutic efficacy. Methods: By applying non-small cell lung cancer cell lines and murine syngeneic tumor xenograft, we investigated the effects of combined APG-2575 and PD-1 antibody on tumor growth. Furthermore, we explored the role of APG-2575 in regulating TAMs polarization through utilizing human and murine macrophages, involving immortalized and primary models. Besides, IL-10 secretion in cell culture supernatants was detected via ELISA assay. In addition, we also assessed the tumor immune microenvironment regulation of APG-2575 using flow cytometry. Results: In vitro, APG-2575, a BCL-2 inhibitor, augmented the tumor cell lysis induced by T cells when combined with ICIs. Moreover, mice treated with APG-2575 and ICIs showed remarkable tumor regression and prolonging survival which were associated with the increase of granzyme-B, IFN-γ and TNF-α release of CD8+ cytotoxic lymphocytes. Importantly, we found that APG-2575-mediated antitumor T cell immunity is dependent on macrophages. Furthermore, we discovered APG-2575 could induce M2 macrophage switching to M1 phenotype and suppress IL-10 secretion in vitro. Meanwhile, reduced M2 macrophages as well as increased M1 polarization were also observed in the tumor-infiltrating immune cells from mice treated with APG-2575. Additionally, APG-2575-mediated macrophage transition could improve tumor immunosuppression, with decreasing number of myeloid-derived suppressor cells (MDSCs) and regulatory T (Treg) cells in tumor microenvironment. Conclusions: Our data demonstrate that APG-2575 can synergizes with ICIs in vitro and in vivo, which was involved in switching TAMs from M2 to M1 polarization and further improving tumor immunosuppression, finally enhancing T cell anti-tumor activity. These results warranted future clinical evaluation of combination therapy of APG-2575 and ICIs. Key words: BCL-2, APG-2575, ICIs, macrophages, non-small cell lung cancer Citation Format: Fan Luo, Fei Teng Lu, Miao Zhen Qiu, Wen Tao Pan, Lin Zhang, Hong Yun Zhao, Li Zhang, Da Jun yang. BCL-2 inhibitor APG-2575 promotes anti-tumor efficacy of immune checkpoint blockade through converting tumor-associated macrophages into M1 phenotype in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 439.

Abstract 2977: Organoid-T-cell co-cultures for preclinical testing of adoptive cell and cancer immunotherapy

Abstract Recent advances in cancer immunotherapy had a positive impact on the life expectancy of patients with a large range of clinical indications. With new treatment strategies and druggable targets being identified at an increasing pace, the number of patients eligible for cancer immunotherapy is expected to expand steadily. However, promising therapeutic developments face hurdles in translating preclinical findings into therapy. Challenges range from lack of confidence in animal cancer models, inter-tumor heterogeneity and the absence of cellular microenvironment. Hubrecht Organoid Technology (HUB) offers an innovative alternative, build on the discovery that adult stem cells proliferate and organize into three-dimensional organotypic structures when they are embedded into Matrigel. HUBOrganoids™ are generated from healthy and disease tissues and stored as “living” biobanks with high quality and reproducibility. HUB Organoids recapitulate complex characteristics of the original parental tissue, including molecular heterogeneity, and morphological and functional traits. HUB has developed organoid-T-cell co-culture systems that allow a physiologic representation of the complexity of interactions in the tumor microenvironment, which is important for evaluating the effects of novel therapies. HUB offers an innovative platform combining image-based analysis with flow cytometry to visualize and quantify T-cell-tumor interactions as well as measurements of T-cell cytotoxicity against tumor (and normal-matched) organoids for colorectal and several other cancers. Our platform offers a powerful strategy for the development and validation of cancer immunotherapy and may help to screen the efficacy of immune checkpoint blockade or bispecific antibodies. In addition, cancer organoid-T-cell co-culture may also predict the functionality of tumor-infiltrating lymphocytes (TIL) and chimeric antigen receptor (CAR)-engineered T cells. As such, our platform offers opportunities in preclinical development and could become a diagnostic tool to predict individual patient response to immunotherapy. Citation Format: Pleun Hombrink, Soura Mardpour, Ewout Spaan, Jarmil Hanrath, Farzin Pourfarzad, Jasper Mullenders, Rene Overmeer, G.J Robert, Vries, F Sylvia, Boj. Organoid-T-cell co-cultures for preclinical testing of adoptive cell and cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2977.

Abstract 2924: In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes

Abstract Microsatellite instability is a hallmark of DNA mismatch repair (MMR)-deficient colorectal cancer (CRC), and is accompanied by genome-wide somatic hypermutation and enhanced immunogenicity. Clinical trials have demonstrated remarkable efficacy of immune checkpoint blockade (i.e. anti-PD-1) in treating MMR-deficient cancers, which is thought to be driven by an enhanced burden of mutation-derived tumor specific antigens (neoantigens). Despite these successes, tumor mutation burden is an imperfect clinical predictor of immunotherapy response, and major questions remain regarding the mechanistic role of neoantigens and other features of MMR-deficiency in the anti-tumor immune response. Notably, most genetically engineered mouse models (GEMMs) fail to recapitulate the mutation burden of their associated human cancer and lack prominent immune involvement, precluding careful study of these questions. To model microsatellite instability in colon cancer, we developed novel autochthonous GEMMs utilizing CRISPR/Cas9-based knockout of DNA MMR genes in the distal colon. Colonoscopy-guided submucosal injection was used to deliver lentivirus containing two single-guide RNAs (sgRNAs) against Apc (the initiating event in the majority of colon cancers) and MMR pathway genes (Msh2, Mlh1, Msh3, Msh6). Whole-exome sequencing of MMR-targeted colon tumors revealed a high mutation burden with signatures consistent with those found in MMR-deficient human cancer. In addition, sequencing revealed a number of recurrently mutated microsatellites, including those within putative tumor suppressor genes Asxl1, Acvr2a, and Acvr1b. To interrogate the functional relevance of these genes in MMR-deficient colon cancer, we developed a novel ex vivo organoid competition assay. Specifically, Apc null organoids expressing Cas9 were infected with lentivirus expressing the fluorophore mScarlet and sgRNAs targeting the putative tumor suppressor genes. Flow cytometry was used to longitudinally assess kinetics of the mScarlet positive fraction. Preliminary results suggest a selective advantage following deletion of Asxl1 and Acvr2a. Further assessment of tumorigenesis in vivo will be performed to confirm the role of these putative tumor suppressors. Altogether, these genetically manipulable systems can be used for rapid functional interrogation of tumor suppressor genes, facilitating downstream mechanistic studies and preclinical testing of therapies in these important subsets of CRC. Citation Format: Daniel D. Zhang, Peter M. Westcott, Olivia Smith, Nate Sacks, Haley Hauck, Mary C. Beytagh, Abbey Jin, Tyler Jacks. In vivo CRISPR/Cas9 models of microsatellite instable colon cancer reveal recurrent mutations in putative tumor supressor genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2924.

Abstract 1914: Orally available oncolytic reovirus, RC402, effectively promotes anti-cancer immunity and synergizes with immune checkpoint blockade in colon cancer

Abstract Introduction: Reoviruses are replication-competent and selectively infect and destroy cancer cells. Although the most common route of oncolytic virotherapy is intratumoral or intravenous administration, these routes are often not clinically feasible because of tumor inaccessibility, tumor multiplicity, and systemic toxicities. Here, we developed an orally available oncolytic reovirus, RC402, to elicit potent anti-tumor immune responses without the above mentioned limitations and thereby maximize the efficacy of PD-1 immune checkpoint blockade in colon cancer. Method: MC38 or CT26 colon cancer-bearing immunocompetent mice were treated with RC402 and/or anti-PD-1 antibody. Tumor growth was monitored after treatment and comprehensively analyzed by flow cytometry and/or multiplex tissue imaging. Results: RC402 treatment effectively delayed tumor growth regardless of the route of administration (per os as well as intratumorally). In particular, orally administered RC402 was well tolerated without gross toxicity and interacted with intestinal immune cells, eliciting adaptive T cell responses within intestinal lymphoid organs such as Peyer's patch. Most of the RC402 was cleared in the gastrointestinal tract within 10 days of the first treatment, and it was not detectable in the blood stream or major organs after oral administration. However, RC402 selectively induced strong and durable anti-tumor immunity in distant tumor tissues. Oral RC402 monotherapy markedly increased CD8+ cytotoxic T cells and decreased CD4+CD25+Foxp3+ regulatory T cells in distant colon cancers, while there were no significant changes in tumor-associated myeloid cells. Finally, oral RC402 treatment cooperated with anti-PD-1 blockade to further suppress colon cancer growth and augment anti-tumor immunity within the tumor microenvironment, leading to complete regression of tumors and survival benefit of tumor-bearing mice. Conclusion: Our study demonstrates that orally available oncolytic reovirus, RC402, could induce potent anti-cancer immune responses and effectively suppress colon cancer progression in combination with anti-PD-1 blockade. Citation Format: Chan Kim, Hong Jae Chon, Hye Jin Lee, Won Suk Lee, Hannah Yang, Jeong Hun Kim, So Jung Kong, Yu Seong Lee, Seung Joon Lee, Enkhtaivan Gansukh, Ki-Hoon Song, Yeon-Sook Lee, Beodeul Kang. Orally available oncolytic reovirus, RC402, effectively promotes anti-cancer immunity and synergizes with immune checkpoint blockade in colon cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1914.

Abstract 1568: A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer

Abstract Extracellular adenosine impairs immune function in tumors and limits the efficacy of anti-CTAL4 immune checkpoint blockade. We previously showed that adenosine 2A receptor (A2aR) blockade with AZD4635 moderately inhibited tumor growth, in part by improving antigen presentation and cytotoxic T cell (CTL) activity in mouse Pten-deficient prostate cancer. Here, we continue to investigate the antitumor activity of AZD4635 and its effects when combined with CTLA4 blockade (aCTLA4) in preclinical models of Pten-deficient prostate cancer. Prostate tumors from Pten conditional knockout (KO) mice treated with aCTLA4 for five days had increased expression levels of adenosinergic genes. Furthermore, gene expression, flow cytometric and IHC analyses showed increased T regulatory cell differentiation and accumulation that was offset with the addition of AZD4635. Moreover, expression levels of genes associated with Th 1 cell differentiation and T cell-mediated cytotoxicity were increased in AZD4635/aCTLA4 treated tumors. Adding AZD4635 to a four-week regimen of aCTLA4 therapy improved the antitumor response by two-fold in an early-stage intervention model of Pten-deficient prostate cancer. Androgen deprivation (AD) has the potential to promote T cell infiltration in prostate tumors and using the AZD4635/aCTLA4 treatment combination as neoadjuvant therapy to AD via surgical castration led to a greater antitumor response. The antitumor activity of this treatment combination was further examined and confirmed in a subcutaneous syngeneic genome-derived allograft model using tumor-bearing conditional Pten-deficient knockout mice grafted with Pten-deficient CRPC tumor fragments. In a Pten/Trp53 conditional double knockout (DKO) mouse model of advanced prostate cancer, the AZD4635/aCTLA4 treatment combination did not improve overall survival rates. However, when AZD4635/aCTLA4 was used as neoadjuvant therapy to AR inhibition with the anti-androgen apalutamide, it improved median survival time from 21 days in monotherapy treated mice to 34 days in AZD4635/aCTLA4 treated mice. These results provide preclinical evidence to support the rational combination of A2AR blockade with AZD4635 and aCTLA4 immune checkpoint inhibition for PTEN-deficient prostate cancer. Citation Format: Marco A. De Velasco, Yurie Kura, Noriko Sako, Naomi Ando, Kazuko Sakai, Alwin Schuller, Kazutoshi Fujita, Masahiro Nozawa, Kazuhiro Yoshimura, Kazuto Nishio, Hirotsugu Uemura. A2aR inhibition enhances the antitumor activity of CTLA4 blockade in mouse Pten-deficient prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1568.

Abstract 1554: PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model

Abstract Purpose: Breast cancer is generally viewed as immunologically ‘cold', imposing an immune-suppressive tumor microenvironment (TME) and responding poorly to lone immune checkpoint blockade (ICB). As an adjunct to ICB, radiation therapy (RT) holds promise in terms of in situ tumor vaccination effect, although it is known to promote immune suppression, increasing regulatory T cells (Treg), myeloid-derived suppressor cells (MDSCs), and M2 tumor-associated macrophages (TAMs). It was our contention that combined use of RT and a PI3Kγδ inhibitor to combat immune suppression might enhance the efficacy of ICB. Methods: Murine breast cancer cells (4T1) were grown in both immune-competent and -deficient BALB/c mice, and tumors were irradiated by 3 fractions of 24 Gy. A PD-1 blockade (10 mg/kg) and a PI3Kγδ inhibitor (IPI145; 15 mg/kg) were then administered every other day for 2 weeks. We analyzed mRNA-sequencing data from humanized patient-derived xenograft (PDX) model to identify differentially activated immune-related pathways. Transcriptomic and clinical data were acquired from The Cancer Genome Atlas (TCGA) pan-cancer cohort, and the ‘xCell' deconvolution algorithm was used to profile immune cellular distributions at certain levels of PIK3C and PIK3CD expression. Results: In the immune-competent syngenic 4T1 murine tumor model, PD-1 blockade alone led to tumor hyperprogression, whereas a three-pronged strategy of PI3Kγδ inhibitor, RT, and PD-1 blockade significantly delayed primary tumor growth, boosted abscopal effect, and improved animal survival by comparison. The immune-deficient syngeneic 4T1 murine tumor model failed to show this synergism in delaying tumor growth and the abscopal effect. RT significantly increased not only CD8+cytotoxic T-cell fractions but also immune-suppressive Tregcells, MDSCs, and M2 TAMs. However, PI3Kγδ inhibitor significantly lowered proportions of Treg, MDSCs, and M2 TAMs, achieving dramatic gains in splenic, nodal, and tumor CD8+ T-cell populations after triple combination therapy. Triple combination therapy significantly delayed tumor growth in humanized PDX model as well. Analyses of RNA sequencing data of humanized PDX samples showed triple combination increased immune response pathways and increased CD8+ T-cell and decreased Treg and M2 TAM. In the TCGA pan-cancer cohort, higher tumor purity-adjusted Treg/CD8+T-cell and M2/M1 TAM ratios and worse overall patient survival were associated with high PIK3CG (PI3Kγ) or PIK3CD (PI3Kδ) gene expression. Conclusion: These findings collectively indicate that PI3Kγ and PI3Kδ are clinically relevant targets in an immunosuppressive TME. Combining PI3Kγδ inhibitor, RT, and PD-1 blockade may thus be a viable approach, helping to overcome the therapeutic resistance of immunologically cold tumors such as breast cancer. Citation Format: Min Guk Han, Bum-Sup Jang, Mi Hyun Kang, In Ah Kim. PI3Kγδ inhibitor combined with radiation enhances the antitumor immune effect of PD-1 blockade in syngenic murine breast cancer model and humanized patient-derived xenograft model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1554.

Abstract 1901: Diglyceride acyltransferase 1 reprograms lipid metabolism in myeloid-derived suppressor cells and augments immune checkpoints cancer immunotherapy

Abstract A better understanding of the tumor immune microenvironment and current challenges facing immunotherapy will advance research in pursuit of effective cancer therapy. Although huge clinical successes have been recorded with the blockade of programmed death-1 receptor/programmed death-ligand 1 (PD-1/PD-L1) signaling pathway, studies have shown that immunosuppressive cells such as myeloid-derived suppressor cells (MDSCs) contribute to its resistance in a fraction of cancer patients. Our group and others previously showed that lipid metabolism drives the immunosuppressive attribute of MDSCs thus leading to the inability of T-cells activation for anti-tumor activity. Lipidomics analysis showed that triglyceride species are increased in tumor-bearing mice compared to their control counterparts; however, it remains unreported if triglyceride synthesis could contribute to MDSCs suppressive function in cancer. Here we found an increased expression of diglyceride acyltransferase 1(DGAT1) in MDSCs, a key enzyme regulating exogenous fatty acid uptake for triglyceride synthesis. MDSCs generated in the presence of unsaturated fatty acids upregulated DGAT1 expression. Therapeutic targeting of DGAT1 in MDSCs cultured in the presence of tumor explanted supernatant (TES) and unsaturated fatty acids reduced lipid accumulation and their suppressive function on T-cells. Also, inhibiting DGAT1 expression in TES-induced MDSCs downregulated mRNA for ARG1, IL-6, and IL-10. Treatment with DGAT1 inhibitor compromised tumor growth in melanoma-B16F10 and Lewis lung carcinoma-LLC bearing C57/BL6 mice. Immune cell profile data showed a decreased population of MDSCs, polymorphonuclear-MDSCs, and macrophages, but an increased proportion of CD3, CD4, CD8, and dendritic cells while there was no significant change in monocytic-MDSCs from the tumor of mice treated with DGAT1 inhibitor. On the contrary, DGAT1 blockade in immunocompromised mice failed to reduce B16F10-tumor progression, this suggests that the effect of the DGAT1 inhibitor may be immune dependent. Remarkably, DGAT1 inhibition provided additional therapeutic benefits for immune checkpoint blockade (ICB) and delayed B16F10 and LLC growth in immunocompetent mice. Combination treatment of DGAT1 and anti-PD-L1 antibody blocked MDSCs accumulation and their immunosuppressive function leading to reactivation of T cells anti-tumor response through enhanced production of TNFα and IFN-γ. Altogether, our data demonstrate that DGAT1 promotes the immunosuppressive role of MDSCs and suggest a new target to inhibit MDSCs function; hence, improve the efficacy of ICB in cancer therapy. Citation Format: Adeleye Oluwatosin Adeshakin, Dehong Yan, Funmilayo O. Adeshakin, Lukman O. Afolabi, Menqgi Zhang, Xiaochun Wan. Diglyceride acyltransferase 1 reprograms lipid metabolism in myeloid-derived suppressor cells and augments immune checkpoints cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1901.

Resolution of tissue signatures of therapy response in patients with recurrent GBM treated with neoadjuvant anti-PD1

The response of patients with recurrent glioblastoma multiforme to neoadjuvant immune checkpoint blockade has been challenging to interpret due to the inter-patient and intra-tumor heterogeneity. We report on a comparative analysis of tumor tissues collected from patients with recurrent glioblastoma and high-risk melanoma, both treated with neoadjuvant checkpoint blockade. We develop a framework that uses multiplex spatial protein profiling, machine learning-based image analysis, and data-driven computational models to investigate the pathophysiological and molecular factors within the tumor microenvironment that influence treatment response. Using melanoma to guide the interpretation of glioblastoma analyses, we interrogate the protein expression in microscopic compartments of tumors, and determine the correlates of cytotoxic CD8+ T cells, tumor growth, treatment response, and immune cell-cell interaction. This work reveals similarities shared between glioblastoma and melanoma, immunosuppressive factors that are unique to the glioblastoma microenvironment, and potential co-targets for enhancing the efficacy of neoadjuvant immune checkpoint blockade.

Molecular Pathways Responsible for Adaptive Resistance to Single-Checkpoint Inhibition May Reveal Biomarkers and Immunotherapy Targets in Multiple Myeloma Patients

Immune checkpoints blocking antibodies represent a promising strategy to harness anti-tumor immune responses and improve the clinical outcome of patients with hematological malignancies. Early studies based on single-agent PD-1 blockade have fallen short of clinical expectations in multiple myeloma patients. Thus, several strategies are under consideration to implement and optimize the clinical efficacy of immune checkpoint blockade in multiple myeloma patients. We have focused on Vγ9Vδ2 T cells because we believe that these cells are very sensitive tools to assess the immune competence of multiple myeloma (MM) patients in the tumor microenvironment. By interrogating the bone marrow (BM) Vγ9Vδ2 T-cell immune reactivity to phosphoantigens, we have revealed a very early and unexpected tumor microenvironment commitment to immune suppression which is already detectable in monoclonal gammopathy of undetermined significance (MGUS) and not fully reverted in clinical remission after autologous stem cell transplantation. Multiple cell subsets (myeloma cells, myeloid-derived suppressor cells, regulatory T cells, bone marrow stromal cells) are involved in Vγ9Vδ2 T-cell inhibition via several immune suppressive mechanisms including the immune checkpoint circuity.We have shown that there is a multifaceted immune checkpoint expression in the tumor microenvironment which is dependent on the clinical stage and cannot be neutralized with single-agent PD-1 blockade. To test our hypothesis that adaptive resistance to PD-1 blockade in MM patients may be due to the compensatory up-regulation of other immune checkpoints, we analyzed the expression of other inhibitory receptors (TIM-3, LAG-3, TIGIT and CTLA-4) in PB and BM Vg9Vd2 T cells from healthy donors and MM patients and observed that also TIM-3 beyond PD-1 is significantly upregulated in BM Vγ9Vδ2 T cells from MM patients at diagnosis. Unlike healthy immune cells, the attempt to activate functionally exhausted immune cells can further compromise their anti-tumor functions. pAg stimulation of PD-1+ and TIM-3+ BM Vγ9Vδ2 T cells further increases their inhibitory receptor expression. Interestingly, TIM-3 up-regulation is even more pronounced than PD-1 up-regulation in BM Vγ9Vδ2 T cells and it is specular to Vγ9Vδ2 T -cell proliferation, confirming that any inappropriate attempt to activate these cells leads further worsen their immune competence status. Our observations about the compensatory up-regulation of multiple inhibitory receptors (PD-1 and TIM-3) on BM Vγ9Vδ2 T cells and the further up-regulation of TIM-3 after anti-PD1 treatment suggested us to assess the combination of both immune checkpoint inhibitors (anti-PD1 and anti-TIM3) to overcome the PD-1 single blocking resistance and recover anti-myeloma reactivity. TIM-3 blockade alone was able to partially recover pAg-induced Vγ9Vδ2 T-cell proliferation but the best recovery was obtained when pAg stimulation is carried out in the presence of concurrent PD-1 and TIM-3 blockade. To investigate the molecular mechanisms behind the up-regulation of multiple immune checkpoints responsible for PD-1-mediated hyporesponsiveness and the effector cells exhaustion, we analyzed the main PD-1-mediated (PI3K/AKT) and TIM-3-mediated (JAK/STAT) signal transduction pathways. After pAg stimulation, purified Vγ9Vδ2 T cells from peripheral blood (PB) CTRL, PB MM and BM MM has been tested by western blot analysis for the expression of several intracellular proteins: Ras, phospho-ERK1/2, ERK1/2, phospho-Akt, Akt, phospho- JAK1, JAK1, phospho-STAT1, STAT1, phospho-STAT3, STAT3. Vγ9Vδ2 T cells isolated from BM MM and PB MM showed a remarkable down-modulation of pAKT, a key player of PD-1-mediated signal transduction, and pJAK-1 and pSTAT1, both involved in TIM-3-mediated signaling pathway. Morever, we observed a significantly reduced expression of the transcription factor T-bet, the downstream target gene of pSTAT1, selectively in Vγ9Vδ2 T cells isolated from the tumor site, suggesting that low T-bet expression is a further hallmark of BM Vγ9Vδ2 T cells exhaustion.Altogether, our findings allow to identify potential molecular mechanisms responsible for the poor efficacy of single immune checkpoint blockade in the tumor microenvironment and to design rational combination therapeutic strategy to be applied to the different clinical settings. DisclosuresBoccadoro:Celgene: Honoraria, Research Funding; Sanofi: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; AbbVie: Honoraria; Mundipharma: Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Massaia:Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Research Funding; Novartis: Research Funding.