Induce Tumor Rejection Research Articles

Abstract PR-11: Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity - From the bench to the bedside and back

Abstract PR-11: Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity - From the bench to the bedside and back

CD200 depletion in glioma enhances antitumor immunity and induces tumor rejection.

We demonstrate mechanisms of the druggable glioma-derived CD200 checkpoint on tumor growth and immune suppression.

Intratumoral Fc-optimized agonistic CD40 antibody induces tumor rejection and systemic antitumor immunity in patients with metastatic cancer.

While CD40 agonism is an attractive approach for activating antigen-presenting cells and initiating antitumor responses, previous attempts have encountered limited clinical efficacy coupled with toxicity. We previously demonstrated that interactions between the antibody Fc domain and the inhibitory receptor FcγRIIB are critical for enhanced antitumor activity. Here, we present the results of a phase 1 study on intratumoral administration of an anti-CD40 agonistic antibody (2141-V11) Fc-engineered to enhance FcγRIIB binding. Primary endpoints included safety, maximum tolerated dose (MTD), and recommended phase 2 dose. Secondary objectives included preliminary clinical activity and correlative studies from biospecimens. 2141-V11 was well-tolerated without dose-limiting toxicities and MTD was not reached. In ten evaluable patients with metastatic cancer, the overall response rate was 20%, with complete responses in two patients (melanoma and breast carcinoma) and stable disease in six patients. 2141-V11 induced tumor regression in injected and non-injected lesions, with increased leukocyte infiltration and tertiary lymphoid structures (TLS) formation in post-treatment biopsies. In a humanized mouse model for CD40 and FcγRs, 2141-V11 induced TLS formation in mice bearing orthotopic breast carcinoma, correlating with local and abscopal antitumor effects, systemic immune activation, and immune memory. These findings support the safety and efficacy of 2141-V11, warranting phase 2 studies and suggesting a unique mechanism of action for this Fc-enhanced immunotherapy (NCT04059588).

Abstract 6597: CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors

Abstract Cytotoxic CD8 T cells are the canonical mediators of tumor clearance by the adaptive immune system. As such, current immunotherapies, including immune checkpoint blockade (ICB), target exhausted CD8 T cells with the hope of reinvigorating the CD8 T cells to drive tumor clearance. However, many tumors, including pancreatic ductal adenocarcinoma (PDAC), can evade CD8 T cell detection via downregulation of the antigen presentation molecule, major histocompatibility complex I (MHC I) as either primary or acquired resistance to immunotherapy. We have previously reported that agonistic CD40 antibody (αCD40), which promotes the maturation and licensing of dendritic cells to drive T cell priming, synergizes with ICB to induce tumor rejection dependent on both CD4 and CD8 T cells in mouse models of PDAC. Given the potential mechanisms of resistance to CD8 T cell immunity, we hypothesized that interrogation of the mechanisms underlying αCD40-induced CD4 T cell rejection of PDAC will reveal targets for improving CD4 T cell-directed immunotherapies. Here, we show that implantation of an MHC I deficient PDAC cell line derived from the genetically engineered KrasG12DTrp53R172HPdx1-Cre (KPC) mouse model results in normal tumor establishment and progression. Upon treatment with combined αCD40 and ICB, tumor growth was significantly decreased in a CD4, but not CD8, T cell-dependent manner. Using genetic knockout mice, we determined that tumor regression was perforin-independent and identified host expression of IFNγ as requisite for therapeutic efficacy. To identify the cellular subset mediating CD4 T cell-rejection of the tumor, we assessed the major histocompatibility complex II (MHC II) positive cells in the tumor microenvironment (TME), which possess the ability to directly synapse with CD4 T cells to drive anti-tumorigenic T cell programs. We determined that macrophage and myeloid cells were dispensable for treatment efficacy, however non-immune cells in the TME can also interact with CD4 T cells through expression of MHC II. Interrogation of stromal cells reveals an increase in the proportion of MHC II+ subsets after combined αCD40/ICB treatment, with a concomitant decrease in the proportion of MHC II+ tumor cells. These data support the notion that CD4 T cell control of tumors may be mediated by both direct and indirect mechanisms of tumor killing. Findings from this work will elucidate non-canonical mechanisms of CD4 T cell-mediated rejection of tumors and may be leveraged to discover novel translational targets for immunotherapy. Citation Format: Margaret E. Haerr, Samuel I. Kim, Charu Arora, Kyle P. Gribbin, Robert H. Vonderheide, Katelyn T. Byrne. CD40 agonism induces CD4 T cell mediated rejection of major histocompatibility complex class I deficient pancreatic tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6597.

Tumor Burden Dictates the Neoantigen Features Required to Generate an Effective Cancer Vaccine.

Tumor neoantigens (nAg) represent a promising target for cancer immunotherapy. The identification of nAgs that can generate T-cell responses and have therapeutic activity has been challenging. Here, we sought to unravel the features of nAgs required to induce tumor rejection. We selected clinically validated Great Ape-derived adenoviral vectors (GAd) as a nAg delivery system for differing numbers and combinations of nAgs. We assessed their immunogenicity and efficacy in murine models of low to high disease burden, comparing multi-epitope versus mono-epitope vaccines. We demonstrated that the breadth of immune response is critical for vaccine efficacy and having multiple immunogenic nAgs encoded in a single vaccine improves efficacy. The contribution of each single neoantigen was examined, leading to the identification of 2 nAgs able to induce CD8+ T cell-mediated tumor rejection. They were both active as individual nAgs in a setting of prophylactic vaccination, although to different extents. However, the efficacy of these single nAgs was lost in a setting of therapeutic vaccination in tumor-bearing mice. The presence of CD4+ T-cell help restored the efficacy for only the most expressed of the two nAgs, demonstrating a key role for CD4+ T cells in sustaining CD8+ T-cell responses and the necessity of an efficient recognition of the targeted epitopes on cancer cells by CD8+ T cells for an effective antitumor response. This study provides insight into understanding the determinants of nAgs relevant for effective treatment and highlights features that could contribute to more effective antitumor vaccines. See related Spotlight by Slingluff Jr, p. 382.

PD-1 limits differentiation and plasticity of Tc17 cells.

Blockade of surface co-inhibitory receptor programmed cell death-1 (PD-1; CD279) has been established as an important immunotherapeutic approach to treat malignancies. On a cellular level, PD-1 is demonstrated to be of particular importance in inhibiting differentiation and effector function of cytotoxic Tc1 cells (CTLs). Nevertheless, the role of PD-1 in modulating interleukin (IL)-17-producing CD8+ T-cells (Tc17 cells), which generally display suppressed cytotoxic nature, is not well understood. To evaluate the impact of PD-1 in Tc17 responses, we examined its functioning using different in vitro and in vivo models. Upon activation of CD8+ T-cells in Tc17 environment, we found that PD-1 was rapidly expressed on the surface of CD8+ T-cells and triggered a T-cell-internal mechanism that inhibited the expression of IL-17 and Tc17-supporting transcription factors pSTAT3 and RORγt. Expression of type17-polarising cytokine IL-21 and the receptor for IL-23 were also suppressed. Intriguingly, adoptively transferred, PD-1-/- Tc17 cells were highly efficient in rejection of established B16 melanoma in vivo and displayed Tc1 like characteristics ex vivo. When using IL-17A-eGFP reporter mice for in vitro fate tracking, IL-17A-eGFP expressing cells lacking PD-1 signaling upon re-stimulation with IL-12 quickly acquired Tc1 characteristics such as IFN-γ, and granzyme B expression, implicating lineage independent upregulation of CTL-characteristics that are needed for tumor control. In line with plasticity characteristics, absence of PD-1 signaling in Tc17 cells increased the expression of the stemness and persistence-associated molecules TCF1 and BCL6. Thus, PD-1 plays a central role in the specific suppression of Tc17 differentiation and its plasticity in relation to CTL-driven tumor rejection, which provides further explanation as to why the blockade of PD-1 is such an efficient therapeutic target for inducing tumor rejection.

Role of NKT cells in cancer immunotherapy-from bench to bed.

Natural killer T (NKT) cells are a specific T cell subset known to express the αβ-T cell receptor (TCR) for antigens identification and express typical NK cell specifications, such as surface expression of CD56 and CD16 markers as well as production of granzyme. Human NKT cells are divided into two subgroups based on their cytokine receptor and TCR repertoire. Both of them are CD1-restricted and recognize lipid antigens presented by CD1d molecules. Studies have demonstrated that these cells are essential in defense against malignancies. These cells secret proinflammatory and regulatory cytokines that stimulate or suppress immune system responses. In several murine tumor models, activation of type I NKT cells induces tumor rejection and inhibits metastasis's spread. However, type II NKT cells are associated with an inhibitory and regulatory function during tumor immune responses. Variant NKT cells may suppress tumor immunity via different mechanisms that require cross-talk with other immune-regulatory cells. NKT-like cells display high tumor-killing abilities against many tumor cells. In the recent decade, different studies have been performed based on the application of NKT-based immunotherapy for cancer therapy. Moreover, manipulation of NKT cells through administering autologous dendritic cell (DC) loaded with α-galactosylceramide (α-GalCer) and direct α-GalCer injection has also been tested. In this review, we described different subtypes of NKT cells, their function in the anti-tumor immune responses, and the application of NKT cells in cancer immunotherapy from bench to bed.

Comparing Intraperitoneal and Intravenous Personalized ErbB2CAR-T for the Treatment of Epithelial Ovarian Cancer.

High-grade serous ovarian carcinoma (HGSOC) is the most common type of epithelial ovarian cancer. The majority of cases are diagnosed at advanced stages, when intraperitoneal (IP) spread has already occurred. Despite significant surgical and chemotherapeutic advances in HGSOC treatment over the past decades, survival rates with HGSOC have only modestly improved. Chimeric antigen receptor (CAR)-T cells enable T cells to directly bind to tumor-associated antigens in a major histocompatibility complex-independent manner, thereby inducing tumor rejection. While CAR-T cell therapy shows great promise in hematological malignancies, its use in solid tumors is limited. Therefore, innovative approaches are needed to increase the specificity of CAR-modified T cells against solid tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal (IP) versus intravenous (IV) CAR-T cell therapy in the treatment of HGSOC. We constructed a CAR that targets the ErbB2/HER2 protein (ErbB2CAR), which is overexpressed in HGSOC, and evaluated the functionality of ErbB2CAR on ovarian cancer cell lines (OVCAR8, SKOV3, and NAR). Our findings show that an IP injection of ErbB2CAR-T cells to tumor-bearing mice led to disease remission and increased survival compared to the IV route. Moreover, we found that IP-injected ErbB2CART cells circulate to a lesser extent, making them safer for non-tumor tissues than IV-injected cells. Further supporting our findings, we show that the effect of ErbB2CAR-T cells on primary HGSOC tumors is correlated with ErbB2 expression. Together, these data demonstrate the advantages of an IP administration of CAR-T cells over IV administration, offering not only a safer strategy but also the potential for counteracting the effect of ErbB2CAR in HGSOC. Significance: IP-injected ErbB2CAR-T cells led to disease remission and increased survival compared to the IV route. These findings demonstrate the advantages of IP administration, offering a safe treatment strategy with the potential for counteracting the effect of ErbB2CAR in HGSOC.

Intra-peritoneal CAR-T cell therapy shows promising results in a murine model of epithelial ovarian cancer (313)

<b>Objectives:</b> Epithelial ovarian cancer is often diagnosed at an advanced stage due to intraperitoneal spread. Modern treatment strategies have only modestly improved the survival rates of this disease. Chimeric antigen receptor (CAR) T cells enable T cells to directly bind tumor-associated antigens in an MHC-independent manner inducing tumor rejection. A major barrier is the lack of true tumor-specific antigens followed by risk of "on-target off-tumor" toxicity. Innovative approaches are therefore needed to increase the specificity of CAR-modified T cells exclusively against tumors. The aim of this study was to assess the efficacy and safety of intraperitoneal versus intravenous CAR T cell therapy. Our working hypothesis was that intraperitoneal CAR T cell therapy will demonstrate higher cytotoxic activity towards a tumor tissue while sparing non-tumor tissues. <b>Methods:</b> We have constructed CARs targeting <i>HER2</i> (ErbB2) that are overexpressed in ovarian cancers. CARs were expressed in human lymphocytes, and their functionality toward human ovarian cancer cell lines was evaluated <i>in-vitro</i> and in a murine model. <b>Results:</b> We have found that the anti-ErbB2 CARs secreted IFN-g (600-1800pg/ml) after stimulation with OVCAR8, SKOV3, or NAR cells compared to untransduced lymphocytes CAR T cells (50pg/ml). Treatment with an intraperitoneal injection of anti-ErbB2 CAR T cells leads to remission of the tumor and increased survival compared to the intravenous route. Intraperitoneal treatment of anti-ErbB2 CARs was less toxic to non-tumor tissues compared to the intravenous treatment. <b>Conclusions:</b> Our results demonstrate that the intra-peritoneal approach of CAR T cells offers a safe strategy with a clinical potential for the treatment of ovarian cancer.

Abstract 4188: CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells

Abstract Identifying and interrogating novel immune-mediated mechanisms of tumor rejection is a major clinical opportunity. Immune checkpoint blockade (ICB) induces tumor rejection primarily via cytolytic CD8+ T cells, but many patients have primary or acquired resistance to CD8+ T cell immunity. Reduced major histocompatibility complex I (MHC I) expression is a contributing factor to resistance, and pancreatic ductal adenocarcinoma (PDAC) is historically treatment-refractory due to, among other features, low MHC expression and a dearth of T cells in the tumor. Interrogating interventions in the context of the PDAC tumor microenvironment will reveal novel mechanisms regulating sensitivity to immune-mediated destruction. Using a genetically engineered mouse model of PDAC, we previously reported that combination immunotherapy (agonistic anti-CD40 and dual ICB, anti-PD-1/CTLA-4) induced both a CD8+ and a CD4+ T cell response sufficient to mediate tumor rejection and long-lived protection. Here, we utilized CRISPR-Cas9 to disrupt the dominant MHC I (H-2Kb allele) expression in PDAC cell lines, precluding direct antigen presentation to CD8+ T cells via this molecule. Subcutaneous injection of MHC I-deficient tumor clones into wild-type mice revealed normal tumor growth at baseline, and significantly delayed tumor growth kinetics in response to combination immunotherapy (CD40/ICB) in a CD4+ T cell-dependent manner (p &amp;lt; 0.0001 vs. vehicle-treated mice). These findings were observed in parental PDAC clones lacking H-2Kb expression, as well as in an ovalbumin (OVA)-expressing PDAC clone. CD4+ T cells mediated tumor rejection including in OVA-tolerized mice, excluding the role of tumor-neoantigens as a major determinant of response. There was no direct contribution of tumor rejection by CD8+ T cells, despite the generation of an activated, tumor-specific CD8+ T cell response in the tumor site (24.5% vs. 1.98% in vehicle-treated mice, p &amp;lt; 0.003). CD4+ T cells upregulated IFN-γ production (52.3% vs. 11.9% in vehicle-treated mice, p &amp;lt; 0.0001), and host IFN-γ and IFN-γ receptor expression were required for response. No changes were observed in the expression of cytotoxic molecules (Granzyme A or B) by CD4+ T cells after CD40/ICB, and perforin was not required for tumor rejection. Furthermore, depletion of macrophage and myeloid cells or genetic knockout of inducible nitric oxide synthase did not dampen CD4+ T cell immunity against established PDAC tumors. Thus, CD4+ T cells mediated PDAC tumor rejection via non-canonical mechanisms, excluding cytotoxic molecules and re-direction of anti-tumor macrophage effector functions after treatment with anti-CD40/ICB. This study provides critical insight into novel mechanisms of tumor clearance beyond direct CD8+ T cell:tumor cell interactions, and opportunities to exploit CD4 T+ cells as regulators of tumor immunity. Citation Format: Samuel I. Kim, Charu Arora, Ioannis I. Verginadis, Christopher R. Cassella, Nune Markosyan, Constantinos Koumenis, Robert H. Vonderheide, Katelyn T. Byrne. CD4+ T cells mediate non-canonical rejection of major histocompatibility class-I deficient pancreatic tumors independently of CD8+ T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4188.

Abstract 1387: High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma

Abstract Effective cancer immunotherapy, such as immune checkpoint therapy (ICT) [e.g., anti-CTLA-4 and anti-PD-1/PD-L1], is dependent on T cell recognition of tumor antigens presented on major histocompatibility complex (MHC) and leads to durable responses in certain cancer patients. Recent advances have facilitated identification of tumor mutant neoantigens and led to efforts to develop effective personalized neoantigen cancer vaccines. However, little is known about how expression of multiple neoantigens with variable peptide-MHC (pMHC) binding or TCR affinity/avidity affects T cell responses to both discrete neoantigens and shared non-mutant antigens, and if the localization of T cells with distinctive pMHC specificity differs within the tumor. To address this, we genetically introduced multiple combinations of MHC-I and MHC-II neoantigens in the poorly immunogenic Brafv600e Pten-/- Cdkn2a-/- YUMM1.7 (Y1.7) mouse melanoma line that contains a paucity of neoantigens. We expressed neoantigens previously found in a mouse sarcoma line of the same MHC haplotype as Y1.7. The MHC-I neoantigens used were formed by G1254V mutation in Laminin alpha subunit-4 (Lama4) and A506T mutation in the Asparagine-linked glycosylation 8 (Alg8) and the MHC-II neoantigen was formed by A710T mutation in Integrin beta-1 (Itgb1). We generated Y1.7 lines expressing minigenes for mLama4, mAlg8, or mLama4 + mAlg8 MHC-I neoantigens. All three lines also expressed the mItgb1 MHC-II neoantigen. Whereas ICT-treated mice bearing the parental Y1.7 melanoma line showed rapid tumor growth, modified Y1.7 lines expressing mLama4 and/or mAlg8 plus mItgb1 were rendered sensitive to ICT. Tumors expressing mLama4 and/or mAlg8 contained mLama4- and/or mAlg8-specific CD8+ T cells, respectively. Preliminary analysis revealed expression of either MHC-I neoantigen alters the frequency of CD8+ T cells specific for non-mutant endogenous antigens in Y1.7. Interestingly, therapeutic synthetic long-peptide (SLP) vaccines incorporating mLama4 and/or mAlg8 neoantigens plus poly I:C induced tumor rejection comparable to ICT. Moreover, SLP vaccines amplified intratumoral mLama4- and mAlg8-specific CD8+ T cells, as well as in tumor-draining lymph nodes, where these neoantigen-specific CD8+ T cells were found to express IFN-γ. During either vaccination or ICT, CD8+ T cell response to mLama4 was higher than that to mAlg8 when both neoantigens were co-expressed. Altogether, these melanoma models will enable us to determine whether expression of one or more MHC-I neoantigens influences CD8+ T cell responses to both distinct neoantigens and non-mutant antigens (e.g., gp100, Trp2) during ICT and SLP vaccination. Furthermore, high-dimensional analyses approaches including scRNA-seq. and CODEX imaging are ongoing and will facilitate development of improved immunotherapies and may help uncover novel biomarkers of response. Citation Format: Sunita Keshari, Charmelle D. Williams, Elizabeth D. Sanchez, Alexander S. Shavkunov, Matthew M. Gubin. High-dimensional analysis of T cell responses to mutant neoantigens and nonmutant antigens in mouse models of melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1387.

Glutamine is essential for overcoming the immunosuppressive microenvironment in malignant salivary gland tumors.

Rationale: Immunosuppression in the tumor microenvironment (TME) is key to the pathogenesis of solid tumors. Tumor cell-intrinsic autophagy is critical for sustaining both tumor cell metabolism and survival. However, the role of autophagy in the host immune system that allows cancer cells to escape immune destruction remains poorly understood. Here, we determined if attenuated host autophagy is sufficient to induce tumor rejection through reinforced adaptive immunity. Furthermore, we determined whether dietary glutamine supplementation, mimicking attenuated host autophagy, is capable of promoting antitumor immunity.Methods: A syngeneic orthotopic tumor model in Atg5+/+ and Atg5flox/flox mice was established to determine the impact of host autophagy on the antitumor effects against mouse malignant salivary gland tumors (MSTs). Multiple cohorts of immunocompetent mice were used for oncoimmunology studies, including inflammatory cytokine levels, macrophage, CD4+, and CD8+ cells tumor infiltration at 14 days and 28 days after MST inoculation. In vitro differentiation and in vivo dietary glutamine supplementation were used to assess the effects of glutamine on Treg differentiation and tumor expansion.Results: We showed that mice deficient in the essential autophagy gene, Atg5, rejected orthotopic allografts of isogenic MST cells. An enhanced antitumor immune response evidenced by reduction of both M1 and M2 macrophages, increased infiltration of CD8+ T cells, elevated IFN-γ production, as well as decreased inhibitory Tregs within TME and spleens of tumor-bearing Atg5flox/flox mice. Mechanistically, ATG5 deficiency increased glutamine level in tumors. We further demonstrated that dietary glutamine supplementation partially increased glutamine levels and restored potent antitumor responses in Atg5+/+ mice.Conclusions: Dietary glutamine supplementation exposes a previously undefined difference in plasticity between cancer cells, cytotoxic CD8+ T cells and Tregs.

Abstract LBA022: IMM20324, a first-in-class, anti-interleukin-38 monoclonal antibody, rescues myeloid cell activation in vitro and induces robust anti-tumor responses in vivo

Abstract Immune checkpoint inhibitors that overcome T cell suppressive mechanisms in tumors have revolutionized the treatment of cancer. However, therapies targeting innate immune cell subsets that orchestrate adaptive immune responses could significantly improve the incidence of clinical response by more effectively activating a multi-lineage response to the tumor. Here, we show that IMM20324, an antibody that binds and neutralizes IL-38, enhances innate immune cell function in vitro and inhibits tumor growth in vivo. IMM20324 binds human, mouse and cynomolgus IL-38 and inhibits the binding of IL-38 to its putative receptors, IL1RAPL1 and IL-36R. In vivo, IMM20324 administration leads to inhibition of tumor growth and improved survival in two different syngeneic tumor models. Importantly, while IMM20324 targets innate immune cells, antibody treatment leads to the induction of immunological memory responses that induce tumor rejection following tumor rechallenge. Together, our data support the hypothesis that IL-38 plays an important role in tumor immunity and that therapies targeting IL-38 could reverse suppressive mechanisms in tumor microenvironment. IMM20324 demonstrated favorable in vitro potency and in vivo efficacy and therefore represents a promising development candidate with the potential to treat multiple cancers. Citation Format: John P Dowling, Pavel A Nikitin, Fang Shen, James P Finn, Nirja Patel, Cezary Swider, Jamie Steele, Halley Shukla, Matthew K Robinson, Karen Lundgren, Benjamin C Harman. IMM20324, a first-in-class, anti-interleukin-38 monoclonal antibody, rescues myeloid cell activation in vitro and induces robust anti-tumor responses in vivo [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr LBA022.

Targeting Hypoxia-A2A Adenosinergic Immunosuppression of Antitumor T Cells During Cancer Immunotherapy.

The blockade of immunological negative regulators offered a novel therapeutic approach that revolutionized the immunotherapy of cancer. Still, a significant portion of patients fail to respond to anti-PD-1/PD-L1 and/or anti-CTLA-4 therapy or experience significant adverse effects. We propose that one of the major reasons that many patients do not respond to this form of therapy is due to the powerful physiological suppression mediated by hypoxia-adenosinergic signaling. Indeed, both inflamed and cancerous tissues are hypoxic and rich in extracellular adenosine, in part due to stabilization of the transcription factor hypoxia-inducible factor 1 alpha (HIF-1α). Adenosine signals through adenosine A2A receptors (A2AR) to suppress anti-tumor and anti-pathogen immune responses. Several classes of anti-hypoxia-A2AR therapeutics have been offered to refractory cancer patients, with A2AR blockers, inhibitors of adenosine-generating enzymes such as CD39 and CD73, and hypoxia-targeting drugs now reaching the clinical stage. Clinical results have confirmed preclinical observations that blockade of the hypoxia-adenosine-A2AR axis synergizes with inhibitors of immune checkpoints to induce tumor rejection. Thus, A2AR blockers provide a new hope for the majority of patients who are nonresponsive to current immunotherapeutic approaches including checkpoint blockade. Here, we discuss the discoveries that firmly implicate the A2AR as a critical and non-redundant biochemical negative regulator of the immune response and highlight the importance of targeting the hypoxia-adenosine-A2AR axis to manipulate anti-pathogen and anti-tumor immune responses.

Abstract 532: A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 activity

Abstract Background Adaptive immunity to cancer cells has been shown to form a crucial part of cancer immunotherapy. Recently, the importance of tumor B-cell signatures were shown to correlate with melanoma survival. We investigated whether anti-tumor antibodies could be isolated from a patient with metastatic melanoma that was tumor-free for 6 years following adoptive transfer of ex vivo expanded autologous T cells (Verdegaal 2011). Methods Peripheral blood memory B cells were immortalized using AIMM's immmortalisation technology (ectopic Bcl-6 and Bcl-xL; Kwakkenbos 2010) expression and analyzed for the presence of tumor-reactive B cells. Results AT1412 antibody was identified by virtue of its differential binding to melanoma cells as compared to healthy melanocytes. AT1412 was found to bind the tetraspanin CD9, a broadly expressed protein involved in multiple cellular activities and disease progression. The crystal structure of the CD9 large extracellular loop in complex with an AT1412 Fab fragment revealed that AT1412 binds the CD9 epitope in an extended and unfolded conformation. In addition to melanoma, AT1412 binds other tumor types including gastric, colon- and pancreatic cancer. AT1412 was shown to induce antibody dependent cellular cytotoxicity (ADCC) and antibody dependent cellular phagocytosis (ADCP) of cancer cells. In addition, AT1412 demonstrated activation of monocytes, most likely via activation of CD9 on platelets-bound monocytes. In mice carrying a human immune system (HIS-mice; van Lent 2010) AT1412 strongly enhanced tumor rejection of A375 and SKMEL-5 tumor cells. AT1412 treatment was shown to enhance tumor infiltration of CD8 T cells and macrophages. AT1412 efficacy was further synergistically enhanced when combined with an anti-PD1 antibody (nivolumab). Other groups have shown that anti-CD9 antibodies can induce tumor rejection in mice. However, these antibodies could not be advanced due to induction of platelet aggregation. We show that AT1412 does not induce aggregation of both human and cynomolgus platelets. Also affinity matured versions of AT1412 do not induce platelet aggregation, supporting that AT1412 binds a unique epitope on CD9. In addition, we studied the safety of AT1412 during a one month exposure in a weekly administration in cynomolgus monkeys. Besides a transient thrombocytopenia AT1412 was well tolerated up to 10 mg/kg antibody (highest dose tested) and did not lead to other adverse events nor were any coagulation factors affected Conclusions Taken together, applying AIMM's proprietary B-cell immortalization technology we isolated antibody AT1412 that targets a unique epitope on CD9. AT1412 was shown to induce tumor rejection as a single agent and enhances the activity of anti-PD-1 antibodies, while not inducing platelet aggregation. One month exposure of AT1412 in monkeys indicated that AT1412 can be safely administered. Preclinical development of AT1412 is ongoing to initiate clinical evaluation in 2020. Funding Dutch Cancer Society, grant UVA 2010-4822 Citation Format: Remko Schotte, Julien Villaudy, Martijn Kedde, Wouter Pos, Koen Wagner, Viviana Neviani, Daniel Go, Etsuko Yasuda, Christien Fatmawati, Els Verdegaal, Susan van Hal, Yvonne Claasen, Hans van Eenennaam, Pauline van Helden, Piet Gros, Sjoerd van der Burg, Hergen Spits. A patient-derived anti-CD9 antibody induces tumor rejection and synergistically enhances anti-PD1 activity [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 532.

Abstract 6615: Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ

Abstract Despite improved ability to generate strong tumor-specific T cell responses, clinical benefits remain relatively moderate. Only a small proportion of predicted neoepitopes was shown to induce specific cytotoxic T cells (CTLs) or serve as an efficient target for T cell-based therapy. We suggest here that low efficacy of CTLs could be at least partially explained by the fact that tumor microenvironment (TME) modulates antigen presentation and epitope profile expressed by tumor. As a result, tumor escapes recognition by CTLs. TME is enriched in peroxynitrite (PNT), a potent oxidant. It is mostly produced by infiltrating myeloid cells, but also by some tumors (as melanoma). We hypothesized that PNT is able to affect antigen presentation on tumor cells and facilitates tumor immune escape. To check this hypothesis, we treated murine EG7 tumor cells with PNT, isolated MHC I peptides and compared their expression on treated versus non-treated cells using SILAC mass spectrometry. We found that PNT treatment significantly decreased expression of a portion of MHC I peptides presented by tumor cells, whereas other peptides remained relatively intact. We found that PNT-sensitive and resistant peptides demonstrated similar binding capacity to MHC I. However, the off-rate of PNT-sensitive peptides was significantly higher. PNT strongly affected proteasomal activity of tumor cells suggesting the mechanism for under-representation of MHC I peptides with high off-rate level. To investigate the biological role of this effect, we generated CTLs specific to a pool of PNT-sensitive or resistant peptides. In vitro we observed that both types of CTLs effectively killed tumor cells. Pre-treatment of tumor cells with PNT significantly reduced killing only by CTLs specific to PNT-sensitive peptides. By ELISPOT we showed that most of antitumor-specific CTL responses in mice with different tumors were induced against PNT-resistant, but not PNT-sensitive species. Moreover, the adoptive transfer of CTLs specific to PNT-resistant peptides combined with immune checkpoint inhibition (ICI) was able to induce tumor rejection in 70% of mice, whereas similar setting for CTLs specific to PNT-sensitive peptides didn't affect tumor growth. PNT inhibition combined with ICI significantly delayed the tumor growth in mice. In addition, we showed that PNT had similar effect on MHC I peptide profile for human melanoma cells. Finally, we measured nitrotyrosine (NT) level (marker of PNT presence) in tumor tissues resected from melanoma patients before the start of ICI therapy. We retrospectively correlated NT levels with patient ability to respond to ICI therapy. We found that high NT level was a predictor of poor prognosis and patient un-responsiveness to ICI. Overall, our study demonstrates PNT ability to affect antigen presentation in tumor site. In addition, we suggest PNT as a promising target for ICI-combined therapy as well as a biomarker for resistance to ICI. Citation Format: Evgenii N. Tcyganov, Dmitry I. Gabrilovich. Peroxynitrite mediates immune escape of tumor cells from cytotoxic T cells in situ [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 6615.

Combination of PD-1 Inhibitor and OX40 Agonist Induces Tumor Rejection and Immune Memory in Mouse Models of Pancreatic Cancer

Advanced pancreatic ductal adenocarcinoma (PDAC) is resistant to therapy, including immune checkpoint inhibitors. We evaluated the effects of a neutralizing antibody against programmed cell death 1 (PD-1) and an agonist of OX40 (provides a survival signal to activated T cells) in mice with pancreatic tumors. We performed studies in C57BL/6 mice (controls), KrasG12D/+;Trp53R172H/+;Pdx-1-Cre (KPC) mice, and mice with orthotopic tumors grown from Panc02 cells, KrasG12D;P53flox/flox;PDX-1-Cre;Luciferase (KPC-Luc) cells, or mT4 cells. After tumors developed, mice were given injections of control antibody or anti-OX40 and/or anti-PD-1 antibody. Some mice were then given injections of antibodies against CD8, CD4, or NK1.1 to deplete immune cells, and IL4 or IL7RA to block cytokine signaling. Bioluminescence imaging was used to monitor tumor growth. Tumor tissues collected and single-cell suspensions were analyzed by time of flight mass spectrometry analysis. Mice that were tumor-free 100 days after implantation of orthotopic tumors were rechallenged with PDAC cells (KPC-Luc or mT4) and survival was measured. Median levels of PD-1 and OX40 mRNAs in PDACs were determined from The Cancer Genome Atlas and compared with patient survival times. In mice with orthotopic tumors, all those given control antibody or anti-PD-1 died within 50 days, whereas 43% of mice given anti-OX40 survived for 225 days; almost 100% of mice given the combination of anti-PD-1 and anti-OX40 survived for 225 days, and tumors were no longer detected. KPC mice given control antibody, anti-PD-1, or anti-OX40 had median survival times of 50 days or less, whereas mice given the combination of anti-PD-1 and anti-OX40 survived for a median 88 days. Mice with orthotopic tumors that were given the combination of anti-PD-1 and anti-OX40 and survived 100 days were rechallenged with a second tumor; those rechallenged with mT4 cells survived an additional median 70 days and those rechallenged with KPC-Luc cells survived long term, tumor free. The combination of anti-PD-1 and anti-OX40 did not slow tumor growth in mice with antibody-mediated depletion of CD4+ T cells. Mice with orthotopic tumors given the combination of anti-PD-1 and anti-OX40 that survived after complete tumor rejection were rechallenged with KPC-Luc cells; those with depletion of CD4+ T cells before the rechallenge had uncontrolled tumor growth. Furthermore, KPC orthotopic tumors from mice given the combination contained an increased number of CD4+ T cells that expressed CD127 compared with mice given control antibody. The combination of agents reduced the proportion of T-regulatory and exhausted T cells and decreased T-cell expression of GATA3; tumor size was negatively associated with numbers of infiltrating CD4+ T cells, CD4+CD127+ T cells, and CD8+CD127+ T cells, and positively associated with numbers of CD4+PD-1+ T cells, CD4+CD25+ T cells, and CD8+PD-1+ T cells. PDACs with high levels of OX40 and low levels of PD-1 were associated with longer survival times of patients. Pancreatic tumors appear to evade the immune response by inducing development of immune-suppressive T cells. In mice, the combination of anti-PD-1 inhibitory and anti-OX40 agonist antibodies reduces the proportion of T-regulatory and exhausted T cells in pancreatic tumors and increases numbers of memory CD4+ and CD8+ T cells, eradicating all detectable tumor. This information can be used in development of immune-based combination therapies for PDAC.

Immunotherapy for Lymphangioleiomyomatosis and Tuberous Sclerosis: Progress and Future Directions

Pulmonary lymphangioleiomyomatosis (LAM) is a rare genetic multisystem disease characterized by the nodular proliferation of smooth muscle-like LAM cells, progressive cystic changes of the lung, lymphatic abnormalities, and renal angiomyolipomas (AMLs). LAM can arise sporadically or in women with the autosomal dominant disorder, tuberous sclerosis complex (TSC), in which hamartomatous tumors of brain, heart, skin, kidney, and lung are found. LAM and TSC are caused by mutations in the TSC1 or TSC2 tumor suppressor genes leading to elevated mechanistic/mammalian target of rapamycin complex activity. Recent data indicate that Tcells within LAM nodules and renal AMLs exhibit features of T-cell exhaustion, with coinhibitory receptor programmed cell death protein 1 (PD-1) expression on tumor-infiltrating Tcells. Treatment of animal models of TSC and LAM with anti-PD-1 antibodies or with the combination of anti-PD-1 and anti-CTLA4 antibodies has led to remarkable results, suppressing TSC2-null tumor growth and inducing tumor rejection. Here we review our current knowledge about the potential for immunotherapy for the treatment of LAM and TSC and highlight critical unknowns and key next steps.

Abstract 4454: Identification of BAY-218, a potent and selective small-molecule AhR inhibitor, as a new modality to counteract tumor immunosuppression

Abstract Re-constitution of anti-tumor T-cell responses by clinically-approved immune checkpoint inhibitors (ICIs) targeting CTLA4 or PD-1/PD-L1 represents a breakthrough cancer therapy. Nevertheless, a substantial number of patients do not benefit from these new therapeutic modalities chiefly due to local immunosuppression in the tumor microenvironment. In addition, the long circulation time of ICIs restricts options to modify dosing regimens for management of adverse effects. Oral small molecule inhibitors as next generation immune-oncology agents may - in contrast to antibodies - allow targeting of intracellular targets for a defined duration of time. This will permit a fine-tuning of efficacy versus tolerability in single-agent treatment as well as in combination with approved ICIs. The overexpression of indole dioxygenase (IDO1) and tryptophan dioxygenase (TDO2) by many tumors results in increased metabolism of tryptophan (TRP) into kynurenine (KYN), which induces immunosuppression via activation of the aryl hydrocarbon receptor (AhR). Inhibition of AhR was proposed to restore T-cell function and induce tumor rejection. However, it was expected that identification of selective lead candidates for AhR inhibition would be challenging due to the known affinity of the AhR ligand binding site for polyaromatic ligands such as 2,3,7,8-tetrachlorodibenzodioxine (TCDD). A library of 4 million compounds was screened in a cell-based HTS campaign. A thorough hit reduction process was performed based on stringent filter parameters for lead-likeness. This process delivered a hit set of significant chemical diversity. Out of several compound classes with drug-like properties, 1,3-diaryl-pyrazin-6-one-5-carboxylic amides were selected as a preferred lead series. A comprehensive SAR exploration, including in vitro mechanistic and functional validation, was performed. Lead optimization was strongly emphasized on improving lipophilicity efficiency (LLE) to balance potency with a viable PK and CYP450 interaction profile. Several candidates suitable for in vivo profiling were identified and BAY-218 was advanced to in-depth pharmacodynamic and pharmacokinetic in vivo assessments. BAY-218 showed mono-therapeutic efficacy that was comparable to ICI treatment and further therapeutic improvement was achieved by combination with an anti-PD-L1 antibody. We were able to characterize 1,3-diaryl-pyrazin-6-one-5-carboxylic amides as a new and unprecedented class of AhR inhibitors as well as identify the key substitutions that contribute to the overall compound profile. Citation Format: Norbert Schmees, Ilona Gutcher, Ulrike Roehn, Horst Irlbacher, Detlef Stoeckigt, Benjamin Bader, Christina Kober, Lars Roese, Rafael Carretero, Iris Oezen, Ludiwg Zorn, Michael Platten, Ingo Volker Hartung, Bertolt Kreft, Hilmar Weinmann. Identification of BAY-218, a potent and selective small-molecule AhR inhibitor, as a new modality to counteract tumor immunosuppression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4454.

PC-3 A reappraisal of CTLA-4 checkpoint blockade hypothesis in cancer immunotherapy

The classic checkpoint blockade hypothesis states that cancer immunity is restrained by 2 distinct checkpoints: the first is the CTLA-4:B7 interaction that limits priming of naive T cells in lymphoid organs, while the second is the PD-1/B7-H1(PD-L1) interaction that results in exhaustion of effector T cells within the tumor microenvironment. Ipilimumab, a clinically-used so-called immune checkpoint inhibitor (ICI) has been FDA approved for the treatment of metastatic melanoma and might get clinical approval for additional oncological indications, most likely in combination with other drugs, for example anti-PD-1/PD-L1. It is assumed that anti-CTLA-4 antibodies cause tumor rejection by blocking negative signaling from the B7-CTLA-4 interactions. Surprisingly, at concentrations considerably higher than plasma levels achieved by Ipilimumab blocks neither B7 transendocytosis by CTLA-4 nor CTLA-4 binding to immobilized or cell-associated B7. In Ctla4 h/m mice expressing both human and mouse CTLA4 genes, anti-CTLA-4 antibodies that bind to human but not mouse CTLA-4 efficiently induce Treg-depletion and Fc receptor-dependent tumor rejection. The blocking antibody L3D10 is comparable to the non-blocking Ipilimumab in causing tumor rejection. Remarkably, L3D10 progenies (HL12/HL32) that lost blocking activity during humanization remain fully competent in Treg depletion and tumor rejection. We also set out to test 3 commercially available anti-mouse Ctla-4 mAbs(4F10/9H10/9D9) that had been shown to induce tumor rejection for their ability to block the B7-Ctla-4 interaction under physiologically relevant conditions, these data also argue against a role for blocking the B7-Ctla-4 interaction in the induction of antitumor immunity by anti-mouse Ctla-4 mAbs. Since these mAbs showed comparable immunotherapeutic effect and comparable depletion of Treg cells in the tumor microenvironment. Thus, both anti-human CTLA-4 mAbs and anti-mouse CTLA-4 mAbs cause tumor rejection by mechanisms that are independent of checkpoint blockade but dependent on host Fc receptor. These data have important implications for the development of the next generation of immunotherapeutic anti-CTLA-4 mAbs and call for a reappraisal of the checkpoint blockade hypothesis.