Enhanced Antigen Presentation Research Articles

EXTH-15. INHIBITION OF 2-HG LEADS TO UPREGULATION OF A PRO-INFLAMMATORY GENE SIGNATURE IN A NOVEL HLA-A2/HLA-DR1 TRANSGENIC MOUSE MODEL OF IDH1R132H-EXPRESSING GLIOMA

Abstract Recent studies have demonstrated the impacts of 2-HG on the anti-tumor immune response in isocitrate dehydrogenase (IDH)-mutant gliomas. Our laboratory has reported that mutant IDH1 (IDH1R132H) suppresses STAT1, the master regulator of IFN-responses and IFN-inducible chemokines, thereby contributing to the “cold” tumor environment. Our data also indicated that inhibition of 2-HG production, through the use of a small molecular inhibitor of mutant IDH, can restore anti-tumor immunity. As the IDH1R132H mutation has been shown to elicit a CD4+ T-cell response in human leukocyte antigen (HLA)-DR1 hosts, to establish a clinically relevant mouse model of IDH1R132H glioma, we generated a novel IDH1R132H glioma cell line syngeneic to the HLA-A2/HLA-DR1-transgenic mice. The cell line expresses the IDH1R132H protein, produces ~65 µg/ml 2-HG in vitro, and forms 2-HG producing orthotopic glioma in vivo. Furthermore, ex vivo sorted tumor-associated myeloid cells (TAMCs) demonstrate high levels of intracellular 2-HG (60–80ng/1M cells), suggesting 2-HG may be taken up by these TAMCs in vivo. Treatment of tumor-bearing mice with vorasidenib, a pan-mutant IDH inhibitor, resulted in a 10-fold reduction of 2-HG levels in the tumor cells, correlate reduction in intracellular 2-HG levels in TAMCs, and 2-fold smaller tumors at time of sacrifice. Analysis of tumor tissues using the NanoString platform revealed enhanced pro-inflammatory IFN-related responses as a result of IDH1R132H inhibition with vorasidenib. Furthermore, vorasidenib treatment increased CD4+ tumor-infiltrating T-cells coupled with upregulation of HLA-DR on tumor-infiltrating myeloid cells, suggesting the opportunity for enhanced antigen presentation. Currently ongoing studies are evaluating the benefit of combining this enhanced immune response with additional immunomodulatory treatments, such as vaccine therapy and immune checkpoint inhibition. These data represent highly translational findings as our major histocompatibility complex (MHC)-humanized model addresses current challenges in the study of IDH1R132H-specific immunity and utilizes a clinically relevant inhibitor in phase 3 clinical development.

A Pilot Study Evaluating Lenalidomide and CC-486 in Combination with Radiotherapy for Patients with Plasmacytoma (LENAZART study)

A Pilot Study Evaluating Lenalidomide and CC-486 in Combination with Radiotherapy for Patients with Plasmacytoma (LENAZART study)

Immune Suppression in CLL Is Mediated By the L-Amino Acid Oxidase IL4I1, a Reason for the Treatment Failure of IDO1 Inhibitors

A better understanding of cancer-induced immune suppression has led to the development of novel immunotherapy approaches, some of which are considered as breakthrough in cancer treatment. One of the promising targets that was followed in clinical trials is indoleamine-2,3-dioxygenase 1 (IDO1) which is a tryptophan (Trp)-catabolizing enzyme that produces metabolites such as kynurenine (Kyn) which promote immune evasion by suppressing T cell activity, and therefore tumor progression. Enhanced expression of IDO1 was described for patients with B cell lymphoma, including chronic lymphocytic leukemia (CLL), and we observed a strong upregulation of IDO1 and a significantly increased Kyn to Trp serum ratio in the Eµ-TCL1 mouse model of CLL. To evaluate the therapeutic potential of this pathway in CLL, we treated Eµ-TCL1 mice with the IDO1 inhibitors 1-Methyl-D-tryptophan or Epacadostat. Both drugs led to initial treatment responses, but failed to improve tumor development over time which is in line with disappointing results of a first phase III clinical trial combining Epacadostat with immune checkpoint blockade for patients with melanoma. In search for an explanation for this failure, we investigated genes that are induced upon resistance to immune checkpoint blockade and identified the phenylalanine-catabolizing enzyme interleukin-4-induced 1 (IL4I1). IL4I1 is strongly expressed in cancer-associated myeloid cells as well as tumor cells of different entities, including CLL and other B cell lymphoma, and was attributed immunosuppressive functions. We thus explored its role in cancer immune escape in bone marrow-chimeric mice that lack IL4I1 in the hematopoietic tumor microenvironment. Adoptive transfer of Eµ-TCL1 leukemia in these mice showed that lack of IL4I1 in hematopoietic cells resulted in a dramatically reduced tumor development compared to respective control mice (Figure 1A). In-depth analysis of the immune compartment of these mice by flow cytometry and transcriptome analyses revealed that IL4I1 deficiency was associated with lower expression of immune checkpoint molecules including PD-1 on CD8 T cells. Moreover, CD8+ effector T cells of IL4I1-deficient mice were highly enriched in a transcriptional signature of genes that was downregulated upon T-cell exhaustion (Figure 1B). We further observed less suppressive regulatory T cells, and enhanced antigen presentation capacity of dendritic cells in the IL4I1-deficient chimeric mice developing CLL. Altogether, these results provide evidence for a central role for IL4I1 in CLL-associated immune suppression and suggest IL4I1 as attractive novel target for immunotherapy of cancer, including tumors that do not respond to IDO1 inhibitors. Figure 1: CLL development and CD8 T cell phenotype in mice with IL4I1-deficient microenvironment A: Bone marrow (BM)-chimeric mice were generated by i.v. transplantation of 5 x 106 BM cells of wildtype (WT) or Il4i1-/-mice in lethally irradiated C57BL/6 mice. After reconstitution of the hematopoietic system, 10 weeks after BM transfer, mice were i.v. transplanted with 1 x 107 malignant B cells of Eµ-TCL1 mice and CLL development was monitored in peripheral blood over time by quantifying absolute number of CD5+ CD19+ CLL cells by flow cytometry. B: 5 weeks after CLL cell transfer, mice were sacrificed and splenic CD8+ effector T cells were sorted for transcriptome analysis by microarrays. Gene set enrichment analysis of ranked-list of genes (fold change) was performed using published signatures which showed an enrichment of genes that are downregulated in exhausted versus memory CD8+ T cells in Il4i1-/- in comparison to WT mice. Figure Disclosures No relevant conflicts of interest to declare.

The anti-B7-H4 checkpoint synergizes trastuzumab treatment to promote phagocytosis and eradicate breast cancer

Trastuzumab is a humanized mAb used to treat HER2-overexpressing breast cancer; however its mechanisms remain to be fully elucidated. Previous studies suggest a role for immunity in mediating trastuzumab-specific antitumor effects. This study evaluated the role(s) of trastuzumab and other antibodies on macrophage activation and Ab-dependent cell-mediated phagocytosis (ADCP) of HER2+ breast cancer cells in vitro and in vivo. We employed orthotopic implantation of HER2+ murine breast cancer (BC) cells in immunocompetent mouse models, a human HER2+ BC xenograft in an immune humanized mouse model, and human PDXs involving adoptive transfer of autologous macrophages to simulate an endogenous mammary tumor-immune microenvironment. Our study demonstrated that trastuzumab greatly and consistently increased macrophage frequency and tumor-cell phagocytosis, and that concurrent knockdown of B7-H4 by a neutralizing antibody increased immune cell infiltration and promoted an antitumor phenotype. Furthermore, neoadjuvant trastuzumab therapy significantly upregulated B7-H4 in the cancer-infiltrating macrophages of HER2+ BC patients, which predicted poor trastuzumab response. We suggest that strategies to specifically enhance ADCP activity might be critical to overcoming resistance to HER2 mAb therapies by inhibiting tumor growth and potentially enhance antigen presentation. Furthermore, these results advance the understanding of macrophage plasticity by uncovering a dual role for ADCP in macrophages involving elimination of tumors by engulfing cancer cells while causing a concomitant undesired effect by upregulating immunosuppressive checkpoints.

Near‐Infrared Triggered Cascade of Antitumor Immune Responses Based on the Integrated Core–Shell Nanoparticle

AbstractThe clinical application of antitumor immunotherapy still faces severe challenges related to efficacy. Here, a light‐triggered core–shell nanosystem is designed to boost antitumor immune response via controlled release of anti‐PD‐L1 (αPD‐L1) antibodies and enhanced antigen presentation. The nanosystem (AZ‐P@P) is constructed via integrating gold nanorods (AuNRs) as a photothermal core and zeolitic imidazolate framework‐8 (ZIF‐8) as a shell for aPD‐L1 delivery, and further PEGylating. In the nanosystem, the ZIF‐8 shell protects αPD‐L1 antibody from the complex physiological environment and hyperthermia. Once accumulated at the tumor site, AZ‐P@P under near‐infrared (NIR) light‐triggered heating induces tumor cell deaths releasing tumor‐derived protein antigens (TDPAs) and adenosine triphosphate (ATP). Thereafter, the released ATP degrades the ZIF‐8 shell to expose the AuNRs, which can promote intratumoral T cell infiltration by capturing TDPAs and transporting them to dendritic cells (DCs). Concurrently, a large amount of αPD‐L1 is released in situ to reinvigorate T cell activity. Mechanistic studies reveal that AZ‐P@P promotes the maturation of DCs and the infiltration of activated T cells, thus eliciting a robust antitumor immunity. It is demonstrated that AZ‐P@P triggered by NIR light can significantly destroy primary tumors and suppress metastasis. This multiple immunoregulatory system provides a promising tool for tumor treatment.

Chemical Strategies to Boost Cancer Vaccines.

Personalized cancer vaccines (PCVs) are reinvigorating vaccine strategies in cancer immunotherapy. In contrast to adoptive T-cell therapy and checkpoint blockade, the PCV strategy modulates the innate and adaptive immune systems with broader activation to redeploy antitumor immunity with individualized tumor-specific antigens (neoantigens). Following a sequential scheme of tumor biopsy, mutation analysis, and epitope prediction, the administration of neoantigens with synthetic long peptide (SLP) or mRNA formulations dramatically improves the population and activity of antigen-specific CD4+ and CD8+ T cells. Despite the promising prospect of PCVs, there is still great potential for optimizing prevaccination procedures and vaccine potency. In particular, the arduous development of tumor-associated antigen (TAA)-based vaccines provides valuable experience and rational principles for augmenting vaccine potency which is expected to advance PCV through the design of adjuvants, delivery systems, and immunosuppressive tumor microenvironment (TME) reversion since current personalized vaccination simply admixes antigens with adjuvants. Considering the broader application of TAA-based vaccine design, these two strategies complement each other and can lead to both personalized and universal therapeutic methods. Chemical strategies provide vast opportunities for (1) exploring novel adjuvants, including synthetic molecules and materials with optimizable activity, (2) constructing efficient and precise delivery systems to avoid systemic diffusion, improve biosafety, target secondary lymphoid organs, and enhance antigen presentation, and (3) combining bioengineering methods to innovate improvements in conventional vaccination, "smartly" re-educate the TME, and modulate antitumor immunity. As chemical strategies have proven versatility, reliability, and universality in the design of T cell- and B cell-based antitumor vaccines, the union of such numerous chemical methods in vaccine construction is expected to provide new vigor and vitality in cancer treatment.

Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells.

Fibroblastic reticular cells (FRCs) are stromal cells that actively promote the induction of immune responses by coordinating the interaction of innate and adaptive immune cells. However, whether and to which extent immune cell activation is determined by lymph node FRC reprogramming during acute viral infection has remained unexplored. Here, we genetically ablated expression of the type I interferon-α receptor (Ifnar) in Ccl19-Cre+ cells and found that sensing of type I interferon imprints an antiviral state in FRCs and thereby preserves myeloid cell composition in lymph nodes of naive mice. During localized lymphocytic choriomeningitis virus infection, IFNAR signaling precipitated profound phenotypic adaptation of all FRC subsets enhancing antigen presentation, chemokine-driven immune cell recruitment, and immune regulation. The IFNAR-dependent shift of all FRC subsets toward an immunostimulatory state reduced exhaustive CD8+ T cell activation. In sum, these results unveil intricate circuits underlying type I IFN sensing in lymph node FRCs that enable protective antiviral immunity.

Self-Assembled Nanoparticles: Exciting Platforms for Vaccination.

Vaccination is successfully advanced to control several fatal diseases and improve human life expectancy. However, additional innovations are required in this field because there are no effective vaccines to prevent some infectious diseases. The shift from the attenuated or inactivated pathogens to safer but less immunogenic protein or peptide antigens has led to a search for effective antigen delivery carriers that can function as both antigen vehicles and intrinsic adjuvants. Among these carriers, self-assembled nanoparticles (SANPs) have shown great potential to be the best representative. For the nanoscale and multiple presentation of antigens, with accurate control over size, geometry, and functionality, these nanoparticles are assembled spontaneously and mimic pathogens, resulting in enhanced antigen presentation and increased cellular and humoral immunity responses. In addition, they may be applied through needle-free routes due to their adhesive ability, which gives them a great future in vaccination applications. This review provides an overview of various SANPs and their applications in prophylactic vaccines.

Adaptable antigen matrix platforms for peptide vaccination strategies and T cell-mediated anti-tumor immunity

Injection of antigenic peptides has been widely used as a vaccine strategy to boost T cell immunity. However, the poor immunogenicity of single peptides can potentially be strengthened through modification of the tertiary structure and the selection of the accompanying adjuvant. Here, we generated antigenic peptides into non-linear trimers by solid phase peptide synthesis, thereby enhancing antigen presentation by dendritic cells to CD8+ T cells in vitro and in vivo. CD8+ T cells from mice vaccinated with trimers showed an KLRG1+ effector phenotype and were able to recognize and kill antigen-expressing tumor cells ex vivo. Importantly, trimers outperformed synthetic long peptide in terms of T cell response even when equal number of epitopes were used for immunization. To improve the synthesis of trimers containing difficult peptide sequences, we developed a novel small molecule that functions as conjugation platform for synthetic long peptides. This platform , termed Antigen MAtriX (AMAX) improved yield, purity and solubility of trimers over conventional solid phase synthesis strategies. AMAX outperformed synthetic long peptides in terms of both CD8+ and CD4+ T cell responses and allowed functionalization with DC-SIGN-binding carbohydrates for in vivo dendritic cell targeting strategies, boosting T cell responses even further. Moreover, we show that agonistic CD40 antibody combined with MF59 (AddaVax) emulsion synergistically improves the antigen-specific T cell response of the AMAX in vivo. Also, tumor-associated antigens and neo-antigens could be incorporated in AMAX for tumor-specific CD8+ T cell responses. Importantly, immunization with a mix of neoantigen AMAX could reduce tumor growth in a pre-clinical syngeneic mouse model. Hence, we provide pre-clinical support for the induction of effector CD8+ T cells through the adaptable AMAX platform as easy implementable peptidic vaccination strategy against any antigen of choice, including neoantigens for anti-tumor immunity.

Abstract 5215: Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma

Abstract The PIM kinases are highly expressed in activated B-cell (ABC) diffuse large B-cell lymphoma (DLBCL). Oncogenic cooperation between PIMs and MYC has been demonstrated. Transgenic mice co-expressing Eμ-PIM and Eμ-MYC showed accelerated lymphomagenesis. Conversely, knockdown of PIMs dramatically decreased cMYC levels and lowered tumor incidence. Based on these preclinical data, a treatment strategy aiming at disrupting the oncogenic cooperation between PIMs and MYC may improve the outcome of DLBCL. Therefore, we treated a panel of DLBCL cell lines with increasing dose of the clinically relevant pan-PIM inhibitor (PIMi) AZD1208 (from 0.1 to 10μM) for 48 hours (Hrs), which resulted in a dose-dependent growth inhibition with a stronger efficacy in ABC DLBCL cell lines. The analysis of a CRISPR loss-of-function screening in three ABC (LY3, TMD8, HBL1) and three GCB (SUDHL-4, Pfeiffer, BJAB) DLBCL cell lines (Reddy et al, 2017) showed that PIM2 silencing led to significantly decreased viability irrespective of cell-of-origin, suggesting that this oncogene is essential for cell proliferation in DLBCLs. To identify the genes through which PIMs drive the lymphoma phenotype we performed gene expression profiling using 4 ABC DLBCL cell lines (RIVA, TMD8, SUDHL-2, U2932) treated with either DMSO or AZD1208 at 1μM for 4, 8 and 12 Hrs. We observed induction of 3,439 genes whereas 2,473 genes were downregulated. Gene pathway analysis showed that AZD1208 led to downregulation of genes regulated by MYC, including its known downstream p53 and NFKB target genes. On the other hand, AZD1208 treatment broadly induced MHC class II and antigen presentation genes as well as PI3K/AKT, cell cycle and glutaminase genes. Using a high-throughput screening approach, we found that the inhibitors of cell cycle (such as the BCL2 inhibitor venetoclax/ABT199 and the PLK4 inhibitor CFI-400945) and of glutaminase (CB839) enhanced the antiproliferative effect of AZD1208, whereas combinations with the PI3K/AKT/mTOR inhibitors had negligible synergistic effect. In conclusion, our study revealed previously unknown mechanisms of action of PIM inhibitors and provides a framework for future combination strategies. Citation Format: Patrizia Mondello, Chiara Tarantelli, Luciano Cascione, Alberto J. Arribas, Andrea Rinaldi, Anas Younes, Francesco Bertoni. Inhibition of PIM kinases targets synthetic vulnerabilities and enhances antigen presentation in B cell lymphoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5215.

Abstract 4585: LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth

Abstract The majority of Merkel cell carcinomas (MCC), a rare and highly-aggressive type of neuroendocrine skin cancer, are associated with Merkel cell polyomavirus (MCPyV) infection. MCPyV integrates into the host genome, resulting in expression of a truncated form of the viral large T antigen (LT) in infected cells and making LT an attractive target for therapeutic cancer vaccines. While induction of tumor-reactive CD8+ T cells is a major goal of cancer therapy, CD4+ T cells provide essential support to CD8+ T cells by promoting their expression of cytotoxic effector molecules and increasing their migratory capacity. Cytokines secreted by CD4+ T cells, such as IFNγ, can also exert desirable effects on the tumor microenvironment. Therefore, we set out to design a cancer vaccine that promotes potent, antigen-specific CD4+ T cell responses to MCPyV-LT. To activate antigen-specific CD4+ T cells in vivo, we utilized our nucleic acid platform, UNITE (UNiversal Intracellular Targeted Expression), which fuses a tumor-associated antigen with lysosomal-associated membrane protein 1 (LAMP1). This lysosomal targeting technology results in enhanced antigen presentation and a balanced T cell response. LTS220A, encoding a mutated form of MCPyV-LT that abrogates its pro-oncogenic properties, was introduced into the UNITE platform. Intradermal vaccination with LTS220A-UNITE promoted a potent, antigen-specific CD4+ T cell response to MCPyV-LT. Additionally, prophylactic vaccination with LTS220A-UNITE prevented growth of B16F10 tumors expressing LTS220A in 100% of mice. Therefore, we find that DNA vaccination using the UNITE platform enhances CD4+ T cell responses to MCPyV-LT and prevents tumor growth when given prophylactically. Future studies will test the efficacy of LTS220A-UNITE for use as a therapeutic cancer vaccine. Citation Format: Claire Buchta Rosean, Pratima Sinha, David M. Koelle, Paul Nghiem, Teri Heiland. LAMP1 targeting of the large T antigen of Merkel cell polyomavirus elicits potent CD4+ T cell responses and prevents tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4585.

Abstract 955: Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. As most patients present with disseminated disease, the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term Gemcitabine on the tumor microenvironment in order to identify potential therapeutic options in chemo-refractory PDAC. Using a combination of mouse models, patient-derived xenografts, primary and established cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, Gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring Gemcitabine-resistance to cancer-associated fibroblasts in vitroand further enhancingTGFβ1 biosynthesis. We therefore combined Gemcitabine with anti-PD-1 in transgenic models of murine PDAC, which failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signal deficiency, Gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that Gemcitabine may prime PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation, but requires the inhibition of an immunosuppressive cytokine barrier. Given the current lack of third line treatment options, this approach warrants consideration in the clinical management of Gemcitabine-refractory PDAC. Citation Format: Daniel R. Principe, Matthew Narbutis, Sandeep Kumar, Alex Park, Navin Viswakarma, Matthew J. Dorman, Suneel D. Kamath, Paul Grippo, Melissa L. Fishel, Rosa F. Hwang, Dinesh Thummuri, Patrick W. Underwood, Hidayatullah G. Munshi, Jose G. Trevino, Ajay Rana. Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 955.

Abstract 4449: Plinabulin, a microtubule destabilizing agent, improves tumor control by enhancing dendritic cell maturation and CD8 T cell infiltration in combination with immunoradiotherapy

Abstract There are theoretical benefits of combining immune checkpoint blockade with radiotherapy, however, there are few indications that such combinations have translated to significant clinical benefit to patients. There is an unmet need to further enhance the effectiveness of these “immunoradiotherapy” combinations. We hypothesize that enhancing antigen presentation through augmenting dendritic cell (DC) activation could potentially improve the effectiveness of immunoradiotherapy. Towards this end, we have investigated the potential for combining the microtubule destabilizing first-in-class agent Plinabulin as the candidate DC activator, with radiotherapy and anti-PD1 immunotherapy (αPD1). First, we investigated how DCs could be affected by Plinabulin and irradiation in in vitro assays. We irradiated SP37A3 and XS106 DC lines with 10Gy and added Plinabulin to those cells. With the addition of Plinabulin, the phosphorylation of JNK and NFκB was upregulated and DC maturation was promoted with high MHC-II, CD86, CD80 and CD40 surface expressions (p<0.05, Dunnett's test). Combining Plinabulin and radiotherapy further augmented the response, particularly in sequence with plinabulin but not in combination. In in vivo models. TSA syngeneic breast cancer cells were implanted to the flank of BALB/c mice, irradiated with 24Gy/3fr, and treated with αPD1 and Plinabulin. There were no anti-tumoral effects with αPD1 alone, Plinabulin alone, and αPD1+Plinabulin groups. However, triple therapy showed much stronger tumor regression than the double therapy of irradiation and αPD1. Tumors were collected from the mice at 2.5-3 weeks after the irradiation and dissociated tumor cells were analyzed for DC activation and immune repertoire using flow cytometry. The percentage of CD8 T cells and CD86+ DCs in the tumor were significantly increased in the triple therapy group that were not seen for the monotherapy or bimodal therapy groups (p<0.05, Dunnett's test). Our findings may serve as a rationale for establishing clinical trial designs of synergistic combinational approaches to enhance immunoradiotherapy. Citation Format: Shinya Neri. Plinabulin, a microtubule destabilizing agent, improves tumor control by enhancing dendritic cell maturation and CD8 T cell infiltration in combination with immunoradiotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4449.

Abstract 962: The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer

Abstract The oral DPP8/9 inhibitor BXCL701 in combination with an anti-PD-1 antibody (aPD-1) [AACR 2017] or in triple combination with aPD-1 and pegylated IL-2 (NKTR-214) [ASCO 2018] has demonstrated inhibition of tumor growth or complete regression respectively in animal models of pancreatic cancer. In the present study, the mechanism of action of BXCL701 has been elucidated in a sub-chronic pharmacokinetic / pharmacodynamic study at molecular and cellular level, by administering BXCL701 as a single agent. BXCL701 (20 μg) was administered orally every day for 14 days in tumor (Pan02) bearing mice. Tumor and serum samples were harvested after 0, 1, 8 and 16 hrs after BXCL701 administration on day 1, 7 and 14. Serum samples were analysed for cytokines and tumor tissues were analysed for infiltrating immune cells and gene expression. BXCL701 is known to inhibit regulatory proteases DPP8/9, consequently activating Nlrp1b inflammasome, which in turn activates pro-caspase-1 to mediate pyroptosis in mouse macrophages [Okondo et al, 2018]. Caspase-1 is involved in cleavage of pro-IL-1β and pro-IL-18 to their active forms, IL-1β and IL-18 respectively [Walle et al, 2016,]. IL-18 was observed to be significantly (p<0.05) upregulated (>50 fold) at 8 hrs on day 1 and achieved steady state levels by day 7 in BXCL701-treated animals. Other cytokines like IL-1β, IFN-γ, G-CSF, IL-5, IL-6, CXCL9, MCP-1, KC and Eotaxin were also observed to be upregulated in BXCL701-treated animals at the 8 hr timepoint on day 1, 7 and 14 in comparison to day 1, 0 hr. BXCL701 significantly upregulates T cells (total T cells, CD4+ T helper cells and CD8+ T cells) infiltration along with NK cells within the tumor microenvironment. It also appears to enhance antigen presentation by upregulating MHC class I genes and MHC class I expressing cells within the tumor. The mechanism of action of BXCL701 was evaluated at the molecular level as well. The comparison of genes in tumor tissues from BXCL701-treated animals vs respective vehicle-treated animals on Day 7 and 14 demonstrated that the upregulated gene clusters were innate and adaptive immune response genes, T-cell receptor genes and MHC genes. Also, genes associated with T cell and NK cell mediated apoptosis and cytolysis e.g. FasL, GzmA, were upregulated and indicates enhanced cell death (e.g. upregulation of pdcd1) within tumor. On the other hand, downregulated gene clusters belonged to functional categories like cell cycle, DNA repair, several genes associated with cancer progression (GPCRs and Olfactory receptors) and extra cellular matrix (ECM) modification (collagen and metalloproteases). In conclusion, BXCL701-treatment induces innate and adaptive immune responses that leads to tumor growth inhibition probably via inducing cell death and reducing ECM modification. Citation Format: Veena Agarwal, John MacDougall, shubhendu Trivedi, Dimple Bhatia, Zeenia Jagga, Hemant Banga, Diane Healy, Sreenivas Adurthi, Vince O'Neill. The dipeptidyl peptidase inhibitor BXCL701 activates innate immunity followed by adaptive immunity on a molecular and cellular level in a mouse model of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 962.

Abstract 3242: CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment

Abstract Despite recent advances in T cell immunotherapy for the treatment of human cancer, metastatic solid tumors remain an intractable challenge. Macrophages are often the most abundant immune cell in the tumor microenvironment (TME) where, as immunosuppressive tumor associated macrophages (TAMs), they participate in disease progression. Currently, most macrophage based immunotherapeutic approaches are focused on the depletion, repolarization, or phagocytic disinhibition of TAMs. We have developed a new paradigm based on the adoptive transfer of genetically engineered CAR macrophages (CAR-M) for the treatment of human cancer. CAR-M can be efficiently produced using the chimeric adenoviral vector Ad5f35. We have previously shown that the primary mechanism of action of CAR-M is antigen dependent phagocytosis, and that a single dose of primary human anti-HER2 CAR-M leads to significantly improved overall survival in multiple solid tumor xenograft models. Given that Ad5f35-transduced anti-HER2 CAR-M (CT-0508) adopt a unique pro-inflammatory M1-like phenotype, we hypothesized that CT-0508 may have the capacity to reprogram the TME toward an activated state. Functional evaluation and transcriptome-wide characterization revealed that CT-0508 maintain a pro-inflammatory phenotype despite challenge with immunosuppressive environments in vitro. By engrafting immunodeficient mice with human hematopoietic cells and human cancer cells we established a novel xenografted human TME model. We demonstrate with single cell resolution that CT-0508 maintain their M1 phenotype within the human TME. Additionally, CT-0508 augmented the human TME by inducing a pro-inflammatory signature in surrounding immune cells, characterized by induction of MHC-II and TNFα. To further investigate the potential of CT-0508 for TME activation, we modeled the interaction of CT-0508 with primary human M2 macrophages, dendritic cells, and T cells in vitro. CT-0508 repolarized bystander M2 macrophages toward a pro-inflammatory phenotype, induced activation and maturation markers on immature dendritic cells, and recruited resting as well as activated T cells in chemotaxis assays. CT-0508 demonstrated enhanced antigen presentation when compared to control human macrophages and cross-presented tumor derived intracellular antigens to CD8 T cells after tumor phagocytosis. Our results show that in addition to direct anti-tumor activity, the anti-HER2 CAR macrophage cell product CT-0508 is capable of promoting a pro-inflammatory tumor microenvironment and has the potential to induce epitope spreading via T cell recruitment and antigen presentation. Citation Format: Michael Klichinsky, Konrad Gabrusiewicz, Nicholas Anderson, Maggie Schmierer, Andrew Best, Martha Zeeman, Sotheavy Chhum, Yumi Ohtani, Olga Shestova, Xueqing Lu, Nicholas Petty, Xinhe Shan, Feng Shen, Saar Gill. CT-0508 is an anti-HER2 chimeric antigen receptor (CAR) macrophage with targeted anti-tumor activity that promotes a pro-inflammatory solid tumor microenvironment [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3242.

Abstract 6647: Sensitizing microsatellite stable colorectal cancer to immune checkpoint therapy utilizing Wnt pathway inhibition

Abstract Immunotherapies that target immune regulatory checkpoints such as CTLA-4 and PD-1 are widely used among many cancer types and have shown positive results in CRC with high microsatellite instability. However, in microsatellite stable (MSS) CRC there is a dismal response rate of 0%. The limited efficacy has shown to be partially due to the lack of T-cells in the tumor microenvironment and/or no activation/regulation of paramount cells in the immune system. The Wnt pathway is the most commonly altered pathway in CRC and is highly involved in driving tumor initiation and progression. Recent evidence also demonstrates the Wnt pathway is involved in T-lymphocyte development, maturation/activation of CD8+ effector T cells and recruitment of dendritic cells. Therefore, targeting the Wnt pathway utilizing a Porcupine (PORCN) inhibitor (ETC-159) in MSS CRC may be a promising strategy to sensitize tumors to immune checkpoint inhibition. Human Immune System BRGS (BALB/c, Rag2−/−, IL2RγC−/−, NODSIRPα) mice were engrafted with MSS CRC PDX (hPDX). The hPDX were randomized according to human chimerism into the following drug treatments groups: Vehicle, ETC-159, nivolumab, and the combination. Treatments began when tumors reached 100-300mm3 and tumors were measured twice weekly. At the end of study, sera, lymph nodes, spleen, and tumor tissue were collected for immunohistochemistry, single cell suspensions, and flow cytometry analysis. Combination therapy resulted in a significant decrease in tumor volume compared to both single agents and vehicle. Flow cytometric analysis demonstrated an increase in human immune cells, in particular human CD4 and CD8 cells in the combination compared to the vehicle and nivolumab treated groups. Additionally, these T-cells showed increased signs of activation and effector function, as indicated by increased CD69+ expression, effector memory subsets, and granzyme B+ cells in the TILs, with a further reduction in Treg populations, suggesting an overall increase in inflammation. An increase in MHC II expression on tumor cells was observed in the ETC-159 single agent with a statistically significant increase in the combination treated tumors demonstrating enhanced antigen presentation. Furthermore, PD-1 expression was upregulated on CD4+ T-cells in the ETC-159 single agent. Lastly, VECTRA analysis corroborates the flow cytometry data showing a changing tumor immune landscape through an increase in CD4+ and CD8+ T cells in the tumor and surrounding stroma. Our data demonstrates the combination treatment of ETC-159 + nivolumab in MSS CRC hPDX show increased tumor infiltration of human immune cells. Further preclinical data is compulsory but these results support further development of this combination in clinical trials. Citation Format: Stacey M. Bagby, Sarah J. Hartman, Natalie M. Navarro, Betelehem W. Yacob, Jeremy Shulman, Jessica Barkow, Christopher H. Lieu, S. Lindsey Davis, Alexis D. Leal, Wells A. Messersmith, Angela Minic, Kimberly R. Jordan, Julie Lang, Todd M. Pitts. Sensitizing microsatellite stable colorectal cancer to immune checkpoint therapy utilizing Wnt pathway inhibition [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6647.

A secreted pore-forming protein modulates cellular endolysosomes to augment antigen presentation.

Bacterial pore-forming toxin aerolysin-like proteins are widely distributed in animals and plants. Emerging evidence supports their roles in host innate immunity, but their direct actions in adaptive immunity remain elusive. In this study, we found that βγ-CAT, an aerolysin-like protein and trefoil factor complex identified in the frog Bombina maxima, modulated several steps of endocytic pathways during dendritic cell antigen presentation. The protein augmented the antigen uptake of dendritic cells and actively neutralized the acidification of cellular endocytic organelles to favor antigen presentation. In addition, the release of functional exosome-like extracellular vesicles was largely enhanced in the presence of βγ-CAT. The cellular action of βγ-CAT increased the number of major histocompatibility complex (MHC) I-ovalbumin and MHC II molecules on dendritic cell surfaces and the released exosome-like extracellular vesicles. An enhanced antigen presentation capacity of dendritic cell for priming of naive T cells was detected in the presence of βγ-CAT. Collectively, these effects led to strong cytotoxic T lymphocyte responses and antigen-specific antibody responses. Our findings provide evidence that a vertebrate-secreted pore-forming protein can augment antigen presentation by directly modulating cellular endocytic and exocytic pathways, leading to robust activation of adaptive immunity.

Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line-derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC. SIGNIFICANCE: These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3101/F1.large.jpg.

Small molecule AZD4635 inhibitor of A2AR signaling rescues immune cell function including CD103+ dendritic cells enhancing anti-tumor immunity.

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated.MethodsWe report here the development of a small molecule A2AR inhibitor including characterization of binding and inhibition of A2AR function with varying amounts of a stable version of adenosine. Functional activity was tested in both mouse and human T cells and dendritic cells (DCs) in in vitro assays to understand the intrinsic role on each cell type. The role of adenosine and A2AR inhibition was tested in DC differentiation assays as well as co-culture assays to access the cross-priming function of DCs. Syngeneic models were used to assess tumor growth alone and in combination with alphaprogrammed death-ligand 1 (αPD-L1). Immunophenotyping by flow cytometry was performed to examine global immune cell changes upon A2AR inhibition.ResultsWe provide the first report of AZD4635, a novel small molecule A2AR antagonist which inhibits downstream signaling and increases T cell function as well as a novel mechanism of enhancing antigen presentation by CD103+ DCs. The role of antigen presentation by DCs, particularly CD103+ DCs, is critical to drive antitumor immunity providing rational to combine a priming agent AZD4635 with check point blockade. We find adenosine impairs the maturation and antigen presentation function of CD103+ DCs. We show in multiple syngeneic mouse tumor models that treatment of AZD4635 alone and in combination with αPD-L1 led to decreased tumor volume correlating with enhanced CD103+ function and T cell response. We extend these studies into human DCs to show that adenosine promotes a tolerogenic phenotype that can be reversed with AZD4635 restoring antigen-specific T cell activation. Our results support the novel role of adenosine signaling as an intrinsic negative regulator of CD103+ DCs maturation and priming. We show that potent inhibition of A2AR with AZD4635 reduces tumor burden and enhances antitumor immunity. This unique mechanism of action in CD103+ DCs may contribute to clinical responses as AZD4635 is being evaluated in clinical trials with IMFINZI (durvalumab, αPD-L1) in patients with solid malignancies.ConclusionWe provide evidence implicating suppression of adaptive and innate immunity by adenosine as a mechanism for immune evasion by tumors. Inhibition of adenosine signaling through selective small molecule inhibition of A2AR using AZD4635 restores T cell function via an internal mechanism as well as tumor antigen cross-presentation by CD103+ DCs resulting in antitumor immunity.

Recent Advances in Nanotechnology for Dendritic Cell-Based Immunotherapy.

Dendritic cells (DCs) are the most important antigen-presenting cells that determine cancer immune responses by regulating immune activation and tolerance, especially in the initiation stage of specific responses. Manipulation of DCs to enhance specific antitumor immune response is considered to be a powerful tool for tumor eradication. Nanotechnology, which can incorporate multifunction components and show spatiotemporal control properties, is of great interest and is widely investigated for its ability to improve immune response activity against cancer and even for prevention and avoiding recurrence. In this mini-review, we aim to provide a general view of DC-based immunotherapy, including that involving the promising nanotechnology. Particularly we discuss: (1) manipulation or engineering of DCs for adoptive vaccination, (2) employing DCs as a combination to more existing therapeutics in tumor treatment, and (3) direct modulation of DCs in vivo to enhance antigen presentation efficacy and priming T cells subsequently. We comprehensively discuss the updates on the application of nanotechnology in DC-based immunotherapy and provide some insights on the challenges and opportunities of DC-based immunotherapeutics, including the potential of nanotechnology, against cancers.