Regression Of Established Tumors Research Articles

Recombinant ferritin-based nanoparticles as neoantigen carriers significantly inhibit tumor growth and metastasis

BackgroundTumor neoantigen peptide-based vaccines, systemic immunotherapies that enhance antitumor immunity by activating and expanding antigen-specific T cells, have achieved remarkable results in the treatment of a variety of solid tumors. However, how to effectively deliver neoantigens to induce robust antitumor immune responses remains a major obstacle.ResultsHere, we developed a safe and effective neoantigen peptide delivery system (neoantigen-ferritin nanoparticles, neoantigen-FNs) that successfully achieved effective lymph node targeting and induced robust antitumor immune responses. The genetically engineered self-assembled particles neoantigen-FNs with a size of 12 nm were obtained by fusing a neoantigen with optimized ferritin, which rapidly drainage to and continuously accumulate in lymph nodes. The neoantigen-FNs vaccine induced a greater quantity and quality of antigen-specific CD8+ T cells and resulted in significant growth control of multiple tumors, dramatic inhibition of melanoma metastasis and regression of established tumors. In addition, no obvious toxic side effects were detected in the various models, indicating the high safety of optimized ferritin as a vaccine carrier.ConclusionsHomogeneous and safe neoantigen-FNs could be a very promising system for neoantigen peptide delivery because of their ability to efficiently drainage to lymph nodes and induce efficient antitumor immune responses.Graphical

Abstract 4127: Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models

Abstract Despite promising results from recent FDA-approved therapies, many advanced melanoma patients develop resistance to both immunotherapy and targeted therapy. A common resistance mechanism to targeted therapy is upregulation of the PI3K/AKT signaling pathway, which has also been shown to promote the development of melanoma brain metastases. Historically, AKT inhibitors have failed in the clinic due to their limited efficacy or intolerable toxicity. Proteomic analysis comparing non-metastatic vs brain metastatic primary tumors in mice revealed focal adhesion kinase (FAK) as an AKT1 specific effector and a potential alternative therapeutic target. FAK is a non-receptor tyrosine kinase that localizes primarily to focal adhesions to regulate cell migration. To determine whether targeting FAK alone or in combination with the RAF/MEK inhibitor avutometinib reduces brain metastases and prolongs survival, we utilized both autochthonous and syngeneic melanoma mouse models. Mice with either subcutaneous tumors or established brain metastases were treated with FAK inhibitor, RAF/MEK inhibitor, or the combination of FAK and RAF/MEK inhibitors. Each cohort was assessed for tumor onset, growth, metastasis, and survival. Our results show that combined RAF/MEK/FAK inhibition significantly delays tumor onset, causes regression of established tumors, prevents the development of brain metastases, promotes the regression of established brain metastases, and prolongs survival. In addition, patient-derived BRAF V600E melanoma xenograft mouse models resistant to the BRAF inhibitor dabrafenib and the MEK inhibitor trametinib, were sensitive to combined RAF/MEK/FAK blockade. The addition of the BRAF inhibitor encorafenib to these models further enhanced the effect on tumor growth. These results support the initiation of a clinical trial evaluating the efficacy of the RAF/MEK inhibitor avutometinib in combination with the FAK inhibitor defactinib in patients with brain metastases from cutaneous melanoma. Additionally, we are assessing non-canonical roles of FAK in modulating the tumor microenvironment to determine whether avutometinib and defactinib also enhance the efficacy of immune checkpoint inhibition in this disease. Citation Format: Karly A. Stanley, Jared D. Almazan, Tursun Turapov, David A. Kircher, Gennie L. Parkman, MiKaela N. Field, Katie M. Culver, Christopher M. Stehn, Silvia Coma, Jonathan A. Pachter, Howard Colman, Sheri L. Holmen. Combined inhibition of RAF, MEK, and FAK attenuates melanoma brain metastases and prolongs survival in preclinical models [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 4127.

Combination IFNβ and Membrane-Stable CD40L Maximize Tumor Dendritic Cell Activation and Lymph Node Trafficking to Elicit Systemic T-cell Immunity.

Oncolytic virus therapies induce the direct killing of tumor cells and activation of conventional dendritic cells (cDC); however, cDC activation has not been optimized with current therapies. We evaluated the adenoviral delivery of engineered membrane-stable CD40L (MEM40) and IFNβto locally activate cDCs in mouse tumor models. Combined tumor MEM40 and IFNβ expression induced the highest cDC activation coupled with increased lymph node migration, increased systemic antitumor CD8+ T-cell responses, and regression of established tumors in a cDC1-dependent manner. MEM40 + IFNβ combined with checkpoint inhibitors led to effective control of distant tumors and lung metastases. An oncolytic adenovirus (MEM-288) expressing MEM40 + IFNβ in phase I clinical testing induced cancer cell loss concomitant with enhanced T-cell infiltration and increased systemic presence of tumor T-cell clonotypes in non-small cell lung cancer (NSCLC) patients. This approach to simultaneously target two major DC-activating pathways has the potential to significantly affect the solid tumor immunotherapy landscape.

Senescent cancer cell vaccines induce cytotoxic T cell responses targeting primary tumors and disseminated tumor cells

BackgroundImmune tolerance contributes to resistance to conventional cancer therapies such as radiation. Radiotherapy induces immunogenic cell death, releasing a burst of tumor antigens, but this appears insufficient to stimulate an...

Abstract 4214: LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation

Abstract Although increased TIGIT expression on TILs is associated with poor survival in patients with cancer, monotherapy TIGIT antibody blockade has not yet demonstrated significant clinical activity. PD-1 is commonly co-expressed on TILs, and PD-1 mediated SHP-2 signaling inhibits DNAM-1 (CD226), preventing PVR co-stimulation in the setting of TIGIT blockade. The interaction between PD-1 and CD226 explains why TIGIT blockade only translates to clinical benefit in the setting of co-blockade of PD-1/L1, however does not explain why co-blockade of TIGIT and PD-1/L1 does not provide benefit in advanced and PD-1/L1 resistant tumors. In advanced and PD-1/L1 resistant tumors, progressive downregulation of CD226 has been reported. Thus, we hypothesized that the lack of CD226 may underlie the lack of clinical responses to combined TIGIT/PD-1/L1 blockade in PD-1/L1 experienced tumors and we sought to identify alternative costimulatory receptors with high-expression. Analysis of TCGA, as well as single-cell RNASeq from TILs, identified HVEM and LTβR as two costimulatory receptors with higher expression in advanced cancers as compared to CD226. HVEM and LTβR directly activate CD8+ T, natural killer (NK), and myeloid cells when bound by their TNF superfamily ligand, known as LIGHT. LIGHT potentiates effector lymphocyte function through signaling that obviates the co-stimulatory role of DNAM-1. We demonstrated that when the extracellular domain (ECD) of LIGHT is combined with the ECD of TIGIT on a TIGIT-Fc-LIGHT bi-functional fusion protein, the simultaneous blockade of PVR, PVRL2, PVRL3, and Nectin-4 and immune co-stimulation by LIGHT increased CD8+ T and NK infiltration into tumors. This translated into tumor cell killing, regression of established tumors, and improved survival in preclinical models of checkpoint primary and acquired resistance. The translation of TIGIT-Fc-LIGHT activity was evaluated in cynomolgus macaques and was well tolerated at doses up to 40 mg/kg. A series of adaptive immune and proinflammatory cytokines, including IL-2, CCL2, CCL4, IL-10, CXCL10, and CCL17, were induced within two hours of TIGIT-Fc-LIGHT infusion. Additionally, receptor engagement led to the rapid margination of HVEM+CD8+ T cells from the periphery into secondary immune tissues. This network of cytokines and post-dose immune cell margination identified in the monkey, was identical to findings in murine models that ultimately translated into significant anti-tumor responses. Lastly, the combination of TIGIT-Fc-LIGHT with anti-PD(L)1 broadened anti-tumor activity of the checkpoint antibodies in aggressive CPI-resistant tumors. These results suggest that LIGHT could be the differentiator that extends the clinical activity of conventional CPIs into PD-L1 low or CPI acquired resistance tumors. Citation Format: Kyung Jin Yoo, Louis Gonzalez, Kellsey Johannes, Jayalakshmi Miriyala, Karen Lenz, Kristen Campbell, Suresh de Silva, Taylor H. Schreiber, George J. Fromm. LIGHT (TNFSF14) costimulation with TIGIT blockade broadens the activity of checkpoint inhibitors (CPIs) into CPI refractory and resistant tumors through targeted myeloid cell and effector lymphocyte activation [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 4214.

Abstract 5550: QLS31904: An anti-DLL3/CD3 bispecific antibody for T cell immunotherapy of small cell lung cancer

Abstract Background: More than 150,000 patients are diagnosed with small cell lung cancer (SCLC) each year in the world. Standard of care for patients at different stages of SCLC includes radiation, etoposide, cisplatin/carboplatin, and atezolizumab. However, relapse is quite common. Therefore, there is a strong need to develop a more effective treatment. Delta-like ligand 3 (DLL3) is a target selectively expressed in SCLC rather than normal tissue. We have developed an anti-DLL3/CD3 bispecific antibody QLS31904 that is engineered to direct and activate T cells through CD3 to kill SCLC cells expressing DLL3. Methods: Tumor cell killing, T cell activation, and cytokine production of QLS31904 in a concentration range were assessed in the presence of SCLC cells and human T cells in vitro. Antitumor activity of QLS31904 was evaluated in SCLC xenograft models, in which NCG mice were co-implanted with SHP-77 and human T cells first, then treated with intraperitoneal QLS31904 or control IgG1AA isotype. The in vivo tumor growth inhibition was assessed. Pharmacokinetic analyses were performed in non-human primates (NHP). The cross-reactivity of QLS31904 with NHP DLL3 and CD3 were established, and the tolerability was also evaluated in NHP model. Results: QLS31904 displayed the anticipated bispecific binding properties and mediated potent and selective lysis of DLL3-positive cancer cell lines through recruitment and activation of human T cells. No cytokine activation was observed with human peripheral blood mononuclear cell alone. In xenograft models, QLS31904 induced significant tumor growth inhibition at doses as low as 20 µg/kg, including complete regression of established tumors. QLS31904 exhibits about 100 hours of T1/2 in NHP, supporting feasibility of weekly dosing. QLS31904 was well tolerated with no related adverse findings up to the highest tested dose (five weekly doses of 10mg/kg) in NHP. Conclusions: QLS31904 has an impressive efficacy and safety profile in the preclinical studies, making it a potentially viable option for DLL3-expressing SCLC in the clinical setting. Citation Format: Liuqing Yang, Ruimei Li, Jiahua Jiang, Hongliang Qian, Hongxiu Ge, Ting Lu, Yan Fang, Jun Liu, Shuyong Zhao. QLS31904: An anti-DLL3/CD3 bispecific antibody for T cell immunotherapy of small cell lung cancer [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 5550.

Cancer therapy in mice using a pure population of CD8+\u2009T cell specific to the AH1 tumor rejection antigen

There is a growing interest in the use of patient-derived T cells for the treatment of various types of malignancies. The expansion of a polyclonal and polyspecific population of tumor-reactive T cells, with a subsequent infusion into the same donor patient, has been implemented, sometimes with positive results. It is not known, however, whether a set of T cells with a single antigen specificity may be sufficient for an effective therapy. To gain more insights in this matter, we used naturally occurring T cells recognizing a retroviral peptide (AH1), which is endogenous in many tumor cell lines of BALB/c origin and which serves as potent tumor rejection antigen. We were able to isolate and expand this rare population of T cells to numbers suitable for therapy experiments in mice (i.e., up to 30 × 106 cells/mouse). After the expansion process, T cells efficiently killed antigen-positive tumor cells in vitro and demonstrated tumor growth inhibition in two syngeneic murine models of cancer. However, AH1-specific T cells failed to induce complete regressions of established tumors. The incomplete activity was associated with a failure of injected T cells to survive in vivo, as only a very limited amount of T cells was found in tumor or secondary lymphoid organs 72 h after injection. These data suggest that future therapeutic strategies based on autologous T cells may require the potentiation of tumor-homing and survival properties of cancer-specific T cells.

Abstract PR13: PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas

Abstract Melanoma is one of the most highly mutated cancer types, harboring numerous alterations with unknown significance. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase (PTP) that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten and Cdkn2a null mice, tet-inducible and melanocyte-specific PTPN11E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRASQ61K-mutant melanoma. Using a quantitative tyrosine phospho-proteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3α/β signaling pathways. This study also identifies PTPN11 as a novel and actionable therapeutic target for BRAF wild-type melanoma. This abstract is also being presented as Poster A14. Citation Format: Kristen S. Hill, Evan R. Roberts, Xue Wang, John M. Koomen, Jane L. Messina, Jamie K. Teer, Youngchul Kim, Jie Wu, Charles E. Chalfant, Minjung Kim. PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR13.

Abstract 4858: Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma

Abstract Yes associated protein YAP1 (YAP1) is a cofactor of gene transcription and exerts its action through association with transcription factors from the TEAD family. Activity of YAP1-TEAD is tightly regulated by the HIPPO pathway and upon activation the expression of pro-survival and anti-apoptotic genes is induced. The HIPPO pathway consists of a series of Ser/Thr kinases, which lead to the phosphorylation of YAP1, and hence to its inactivation by either sequestration in the cytoplasm, or degradation. HIPPO pathway deletions and YAP1 activation are particularly prevalent (> 60 %) in patients with malignant pleural mesothelioma. We use a malignant mesothelioma tumor cell line with a HIPPO deleted genetic background (MSTO-211H, LATS1 loss) to demonstrate, that downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression, the induction of apoptosis and the inhibition of cell growth in vitro. When these cell lines are grown as an in vivo subcutaneous xenograft in mice, downregulation of YAP1 leads to the inhibition of YAP1-dependent gene expression and eventually results in the regression of established tumors. This effect is specific to YAP1 activated mesothelioma models and is not observed in the HIPPO pathway independent HCT116 colon cancer model. We show here, that YAP1 activity alone is required for tumor maintenance in vivo in the context of HIPPO pathway genetic alterations, and thus confirm the relevance of this target for a drug discovery approach. Citation Format: Loreley Calvet, Odette Dos Santos, Véronique Jean-Baptiste, Emmanuel Spanakis, Yvette Ruffin, Isabelle Sanchez, Jessica Mestadier, Stéphane Soubigou, Sylvie Feteanu, Pascale Picard, Chagri Boumaya, Jack Pollard, Colette Dib, Sukhvinder Sidhu, Laurent Debussche, Iris Valtingojer. Oncogenic HIPPO-YAP1: in vivo target validation of YAP1 in malignant mesothelioma [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 4858.

Abstract 3745: Exogenous and endogenous sources of serine contribute to colon cancer metabolism and growth

Abstract Serine is a non-essential amino acid that is generated by the sequential actions of phosphoglycerate dehydrogenase (PHGDH), phosphoserine aminotransferase (PSAT1) and phosphoserine phosphatase (PSPH). Increased serine biosynthesis occurs in several cancers and supports tumor growth. In addition to serine synthesis, exogenous serine is taken up by cells and can also fuel tumor growth. Interestingly, colon cancer cells increase expression of serine biosynthesis enzymes in the absence of exogenous serine, suggesting a compensatory adaptive response to reduced availability of serine. This study explored the relative contributions of exogenous and synthesized serine to colon cancer cell growth, metabolism and response to anti-cancer therapy. We found that PSAT1 expression was markedly increased in human colonic adenomas and colorectal cancer (CRC) compared to normal adjacent tissue. Additionally, high levels of PSAT1 in CRC were associated with reduced patient survival. Deletion of PSAT1 only modestly reduced colon cancer cell proliferation in vitro. Removal of serine from the medium strongly suppressed cell proliferation whereas the combination of removing exogenous serine and deleting PSAT1 caused even greater inhibition of cell proliferation. Metabolite profiling revealed altered nucleotide levels upon exogenous serine depletion, an effect that was enhanced when PSAT1 was deleted. Global gene expression profiling showed approximately 3,000 differentially expressed genes when only exogenous serine was removed, with an additional ~800 changes in PSAT1 knockout (KO) cells grown in serine deficient medium. A marked DNA damage response was induced selectively in PSAT1KO cells in serine deficient medium, which was prevented by the addition of formate. Xenografts derived from PSAT1 sufficient and PSAT1KO cells showed that neither PSAT1 deletion alone nor removal of dietary serine alone affected tumor growth, but the combination resulted in profound growth suppression. Additionally, treatment with 5-fluorouracil (5-FU) in the xenograft model induced a strong regression of established tumors derived from mice carrying PSAT1KO cells fed a serine deficient diet. Complementary in vitro studies showed enhanced sensitivity of PSAT1KO cells grown in serine deficient medium to 5-FU induced death, which was preceded by specific metabolite changes including markedly reduced levels of TMP and TTP. Taken together, our results suggest that both exogenous and endogenous sources of serine are important contributors to colon cancer cell proliferation and metabolism. Limiting exogenously available and synthesized serine may be an effective strategy to limit colon cancer growth and enhance the efficacy of treatment. Citation Format: David C. Montrose, Miguel Foronda, Suchandrima Saha, Erin M. McNally, Xi Kathy Zhou, Jan Krumsiek, Akanksha Verma, Olivier Elemento, Rhonda K. Yantiss, Qiuying Chen, Steven S. Gross, Lorenzo Galluzzi, Lukas E. Dow, Andrew J. Dannenberg. Exogenous and endogenous sources of serine contribute to colon cancer metabolism and 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 3745.

A paradoxical role for classical dendritic cells and Flt3 ligand in tumor immune response

Abstract Flt3 ligand (FL) induces differentiation and proliferation of classical dendritic cells (cDCs) and is a promising molecule in therapies of cancer and vaccination, but has mixed effects in clinical trials. The role of cDCs in antitumor response is ambiguous as they may induce priming of anti-tumor lymphocytes, but also may favor recruitment of tumor-protecting regulatory T cells (Tregs). To better understand the role of FL/DCs axis in cancer, we monitored tumor growth in a mouse model of melanoma in the context of low, normal or high levels of FL. Paradoxically, both genetic FL depletion and overexpression stalled tumor growth and increased animal survival as compared to control. We show that FL-deficient mice tumor growth is predominantly controlled by the adaptive immune system unleashed by a diminution of Tregs, while high FL levels favor the innate anti-tumor response through the recruitment of NK cells. Analysis of human gene expression data confirmed the paradoxical effect of FL as well as cDCs on survival in several types of cancer. Tumor samples from patients in these groups expressed Treg and NK cells signatures in correlation with the levels of cDCs, FL, IL15 and IL15RA expression. As a proof-of-principle, we showed that the combination of FL overexpression and Tregs depletion resulted in regression of established tumors in mice. We propose that FL is a master regulator of immune control of tumor growth owing to its key role in DCs function.

Abstract PR20: A novel combination immunotherapy with a vaccine targeting tumor neoepitopes that mediates immune cascade in murine tumor models

Abstract Clinical successes utilizing checkpoint inhibitors demonstrate the potential of immune-based therapies for the treatment of cancer. Research suggests that these successes are a result of T cells recognizing nonsynonymous mutations uniquely expressed by a patient’s tumor. The advent of comparative tumor-normal DNA sequencing and tumor RNA sequencing for accurate prediction of presented somatic mutations has made it possible to rapidly identify these neoepitopes, allowing for the development of personalized cancer vaccines. However, such vaccines are often ineffective due to the immunosuppressive nature of the tumor microenvironment, and their clinical efficacies would be improved when used in an orchestrated combination approach with additional immune-oncology agents. In the present study, we investigated the combination of three immune-oncology agents, each targeting a unique mechanism of immune therapy, with a vaccine targeting tumor neoepitopes. We combined a neoepitope vaccine, delivered as a peptide or via a novel E2b-deleted adenovirus vehicle, with N-IL15 (previously called ALT-803), an IL-15 superagonist, to enhance the development of antigen-specific immunity; NHS-IL12, a tumor-targeting IL-12 molecule, to promote the maintenance and expansion of T cells within the tumor microenvironment; and PD-L1 blockade to decrease immune cell exhaustion within the tumor microenvironment. This combination therapy resulted in the regression of established tumors, which associated with the generation of robust immunity against neoepitopes contained in the vaccine. It also showed evidence of inducing a neoepitope cascade by causing development of T-cell responses to neoepitopes expressed by the tumor but not contained within the vaccine. This report is the first demonstration of an E2b-deleted adenovirus neoantigen vaccine generating robust immunity. Together, this study demonstrates the importance of a multifaceted, orchestrated treatment regimen to promote the generation of effective antitumor immunity. This abstract is also being presented as Poster A18. Citation Format: Karin L. Lee, Claudia Palena, John Lee, Kayvan Niazi, Andrew Nguyen, Frank Jones, Shahrooz Rabizadeh, Patrick Soon-Shiong, Jeffrey Schlom, Duane H. Hamilton. A novel combination immunotherapy with a vaccine targeting tumor neoepitopes that mediates immune cascade in murine tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR20.

Abstract PR18: A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer

Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of immune evasion in cancer and explore novel therapeutic modalities. Recently, glyco-immune checkpoints have been identified as an important modulator in cancer immune escape. Upon ligation of cancer-specific sialylated glycans, glyco-immune checkpoints suppress multiple facets of cancer immunity, including cancer antigen release, cancer antigen presentation, and priming and activation anticancer T-cell immunity. However, due to the complexity and heterogeneity of cancer sialylated glycans and the profound species differences of the glyco-immune checkpoints between human and mouse, this immunomodulatory pathway remains unexplored for therapeutic interventions. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), to target this glyco-immune checkpoint axis. EAGLE overcomes the complexity of cancer glycans and specifically cleaves the immunosuppression-mediating key sugar moieties, the terminal sialic acids of various sialylated glycans on tumor cells. We hypothesized that EAGLE could block the immunosuppressive glyco-immune checkpoints in the tumor microenvironment and potentiate innate and adaptive immune responses to treat cancer. Indeed, systematic delivery of an EAGLE molecule decreased the levels of immunosuppressive sialic acids on tumor cells, and increased T-cell infiltration and activation in syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy, and 100% cures in combination with anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced antitumor immunologic memory. In summary, we demonstrated that this novel therapeutic modality, EAGLE, can release glyco-immune checkpoint-mediated immunosuppression in the tumor microenvironment and potentiate innate and adaptive antitumor immunity, which offers a novel and promising immunomodulatory strategy to treat cancer. This abstract is also being presented as Poster A91. Citation Format: Li Peng, Adam Petrone, Lizhi Cao, James Broderick. A novel immunomodulatory strategy of targeting glyco-immune checkpoints using EAGLE technology to treat cancer [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr PR18.

Abstract LB-085: PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas

Abstract Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase (PTP) that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten and Cdkn2a null mice, tet-inducible and melanocyte-specific PTPN11 E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11 E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRAS Q61K-mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma. Citation Format: Kristen Hill, Jane Messina, Jie Wu, Charles E. Chalfant, Minjung Kim. PTPN11 plays oncogenic roles and is a therapeutic target for BRAF wild-type melanomas [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 LB-085.

Abstract 4983: Discovery and characterization of next-generation small molecule direct STING agonists

Abstract Accumulating evidence highlights an important role of type I interferon response in the immune surveillance mechanisms. IFNβ release by antigen-presenting cells promotes spontaneous anti-tumor CD8+ T cell priming being largely dependent on activation of Stimulator of Interferon Genes (STING). STING agonists promote regression of established tumors and generation of long-term immunologic memory in preclinical animal models. Herein we report the discovery of potent and selective, first-in-class non-nucleotide, non-macrocyclic, small molecule direct STING agonists with molecular weight below 500, structurally unrelated to known cyclic dinucleotide chemotypes with potential for systemic administration. Activation of STING pathway was monitored in THP-1 Dual reporter monocytic cell line as well as peripheral blood mononuclear cells (PBMC) or antigen presenting cells from human and mouse origin. Surface expression of the antigen-presenting cell maturation markers i.e. CD80, CD86, CD83 and HLA-DR was assessed by flow cytometry. Binding affinity was confirmed by three independent assays. RNA sequencing was performed on total RNA isolated from THP-1 cells and PBMC isolated from 2 healthy human donors. Direct binding to both mouse and human STING protein of Selvita agonists have been confirmed in biophysical binding assays (FTS, MST and FP) and by crystallography studies. The compounds have fine-tunable ADME properties with good solubility, permeability and human plasma stability. They selectively activates STING-dependent signaling in both THP-1 reporter assays and in primary cells of human and mouse origin. In addition, RNA sequencing data confirmed selectivity of the Selvita compounds. In vitro functional assays demonstrated their ability to induce cytokine responses (IFNβ, TNFα) in a panel of human peripheral blood mononuclear cell (PBMC), human monocyte derived macrophage (HMDM) and human dendritic cells samples with various STING haplotypes including refractory alleles. Additionally, the compounds efficiently induced cytokine release in mouse bone marrow-derived macrophages and dendritic cells. Pro-inflammatory cytokine profile was accompanied by up-regulation of the maturation markers, i.e. CD80, CD86, CD83 and HLA-DR, on the surface of human antigen presenting cells. These data demonstrate potent, novel, next-generation small molecule STING agonists activating STING-dependent signaling in both mouse and human immune cells to promote potential antitumor immunity. The compounds show good selectivity and in vitro ADME properties enabling further development for systemic administration as a single agent or in combinatory immunotherapies for cancer treatment. Citation Format: Monika Dobrzańska, Stefan Chmielewski, Magdalena Zawadzka, Jolanta Mazurek, Karolina Gluza, Katarzyna Wójcik-Jaszczyńska, Maciej Kujawa, Grzegorz Topolnicki, Grzegorz Ćwiertnia, Aleksandra Poczkaj, Izabela Dolata, Magdalena Mroczkowska, Agnieszka Gibas, Marcin Leś, Sylwia Sudoł, Adam Radzimierski, Kinga Michalik, Magdalena Sieprawska-Lupa, Katarzyna Banaszak, Katarzyna Wiklik, Federico Malusa, Michał Combik, Karolina Wiatrowska, Agnieszka Adamus, Lukasz Dudek, Jose Alvarez, Charles Fabritius, Anna Rajda, Maciej Rogacki, Faustyna Gajdosz, Peter Littlewood, Luigi Stasi, Krzysztof Brzózka. Discovery and characterization of next-generation small molecule direct STING agonists [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 4983.

Abstract 3206: Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors

Abstract Near infrared photoimmunotherapy (NIR-PIT) is a newly developed cancer treatment that employs a targeted monoclonal antibody-photo-absorber conjugate (APC). Following antibody localization of the APC to a tumor cell surface antigen, NIR light is used to induce highly selective cytolysis. Extensive pre-clinical evidence demonstrates that NIR-PIT is effective at inducing tumor cell lysis using a number of different antibody-APC conjugates and NIR-PIT targeting EGFR with a cetuximab-APC conjugate has shown promising results in phase 2 clinical evaluation for the treatment of recurrent head and neck squamous cell carcinoma. NIR-PIT induces rapid, necrotic cell death that yields innate immune ligands that activate dendritic cells, consistent with immunogenic cell death. Yet, NIR-PIT treatment of syngeneic tumors in wild-type mice has mostly failed to induce durable regression of established tumors, suggesting the presence of one or more mechanisms of resistance to formation of meaningful anti-tumor immunity. In this study, we hypothesized that NIR-PIT could induce anti-tumor immunity that was being restricted by the PD-1/PD-L1 signaling axis, and that PD-1 could reverse innate immune resistance to induce durable, effective anti-tumor immune responses. Using a CD44-targeting APC, we demonstrated the ability of PD-1 immune checkpoint blockade to significantly enhance antigen-specific anti-tumor immunity induced by NIR-PIT in multiple syngeneic tumor models (MC38, LLC, and MOC1 cancer cell line). In two of three models, NIR-PIT monotherapy halted tumor growth, enhanced dendritic cell tumor infiltration, and induced de novo tumor antigen-specific T-cell responses absent at baseline. The addition of PD-1 blockade appeared to reverse adaptive immune resistance, resulting in both enhanced pre-existing tumor antigen-specific T-cell responses and enhanced de novo T-cell responses induced by NIR-PIT. Enhanced immune responses appeared to correlate with shared tumor antigen expression, suggesting that antigenicity is a major determinant of response to combination NIR-PIT and PD-1 blockade. Combination treatment induced complete rejection of MC38 tumors treated with NIR-PIT as well as untreated, distant tumors. Accordingly, tumor antigen-specific T-cell responses were measured in both treated and untreated tumors, validating the development of systemic anti-tumor immunity. Cured mice resisted tumor challenge, indicating the presence of systemic immune memory. Cumulatively these results demonstrate reversal of adaptive immune resistance following induction of innate and adaptive immunity by NIR-PIT, resulting in high rates of tumor rejection and/or significant tumor growth control in highly antigenic syngeneic models of cancer. Citation Format: Tadanobu Nagaya, Jay Friedman, Yasuhiro Maruoka, Fusa Ogata, Shuhei Okuyama, Paul E. Clavijo, Peter L. Choyke, Clint Allen, Hisataka Kobayashi. Host immunity following near infrared photoimmunotherapy is enhanced with PD-1 checkpoint blockade to eradicate established highly antigenic tumors [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 3206.

Unbiased functional identification and therapeutic targeting of T cell neoantigens in a spontaneous murine squamous cell carcinoma

Abstract The comparative resistance of some cancers including head and neck squamous cell carcinoma (HNSCC) to checkpoint blockade is speculated to derive from the low frequency of expressed somatic mutations targeted by T cells as neoantigens (NeoAg). SCC VII, a spontaneously arising murine squamous carcinoma resembling human HNSCC in several key features, is likewise poorly immunogenic as irradiated tumor cells alone fail to induce protective immunity in syngeneic hosts. Justifying use of this model to identify NeoAgs, we confirm activated CD4+ and CD8+ T cells are detectable and essential for vaccine efficacy of SCC VII and polyI:C co-administration. Whole-exome sequencing tumor versus normal genome identified 39 nonsynonymous missense mutations that were synthesized into 81 representative 20-mers. NeoAg-specific CD4+ T cell IFN-γ responses were found against mutations of Pik3ca, Ctnnd1, and Otud5 while both CD4+ and CD8+ T cells produced IFN-γ when stimulated by a single Cltc mutation containing overlapping minimal epitopes during in vitro recall assays. NeoAgs eliciting CD4+ T cell responses alone were not protective in vivo. However, the more immunogenic Cltc NeoAg prophylactically protected hosts from tumor challenge and caused regression of established tumors as an immunotherapy. Anti-PD-1 combinatorial blockade resulted in synergistic tumor rejection by boosting Cltc-specific responses and increasing response diversification via epitope spreading. These data show that a functional NeoAg identification platform can be used to select immunotherapeutically relevant targets and filtration of neoepitopes that co-prime both CD4+ and CD8+ T cell responses is superior for practical intervention of poorly immunogenic tumors.

PTPN11 Plays Oncogenic Roles and Is a Therapeutic Target for BRAF Wild-Type Melanomas

Melanoma is one of the most highly mutated cancer types. To identify functional drivers of melanoma, we searched for cross-species conserved mutations utilizing a mouse melanoma model driven by loss of PTEN and CDKN2A, and identified mutations in Kras, Erbb3, and Ptpn11. PTPN11 encodes the SHP2 protein tyrosine phosphatase that activates the RAS/RAF/MAPK pathway. Although PTPN11 is an oncogene in leukemia, lung, and breast cancers, its roles in melanoma are not clear. In this study, we found that PTPN11 is frequently activated in human melanoma specimens and cell lines and is required for full RAS/RAF/MAPK signaling activation in BRAF wild-type (either NRAS mutant or wild-type) melanoma cells. PTPN11 played oncogenic roles in melanoma by driving anchorage-independent colony formation and tumor growth. In Pten- and Cdkn2a-null mice, tet-inducible and melanocyte-specific PTPN11E76K expression significantly enhanced melanoma tumorigenesis. Melanoma cells derived from this mouse model showed doxycycline-dependent tumor growth in nude mice. Silencing PTPN11E76K expression by doxycycline withdrawal caused regression of established tumors by induction of apoptosis and senescence, and suppression of proliferation. Moreover, the PTPN11 inhibitor (SHP099) also caused regression of NRASQ61K -mutant melanoma. Using a quantitative tyrosine phosphoproteomics approach, we identified GSK3α/β as one of the key substrates that were differentially tyrosine-phosphorylated in these experiments modulating PTPN11. This study demonstrates that PTPN11 plays oncogenic roles in melanoma and regulates RAS and GSK3β signaling pathways. IMPLICATIONS: This study identifies PTPN11 as an oncogenic driver and a novel and actionable therapeutic target for BRAF wild-type melanoma.

Abstract B034: A novel immunomodulatory strategy of targeting glyco-immune checkpoints with EAGLE technology

Abstract Cancer therapy has been revolutionized by inhibiting immune checkpoints to harness the power of the immune system in fighting cancer. However, the majority of patients are resistant to the current immuno-oncology drugs. There is a strong need to identify novel mechanisms of cancer immune evasion and explore novel therapeutic modalities. Glyco-immune checkpoint axis plays a critical role in modulating innate and adaptive immune responses against cancer. However, this pathway is underexplored for therapeutic interventions of cancer, because the complexity and heterogeneity of glycan-ligands on tumor cells pose grand challenges for conventional therapeutic modalities. Here we described a novel multifunctional antibody-like therapeutic modality, named EAGLE (Enzyme-Antibody Glyco-Ligand Editing), which can overcome the heterogeneity and complexity problems and specifically edit tumor-specific glycans. We evaluated the efficacy of an EAGLE molecule and studied its mechanism of actions in a breast cancer EMT6 syngeneic tumor model. Systematic delivery of the EAGLE molecule decreased the expression levels of immunosuppressive glycan-ligands on tumor cell surfaces, and increased T-cell infiltration and activation in the syngeneic tumor models. EAGLE treatment led to 50% complete regressions of established tumors as a monotherapy and 100% cures in combination with an anti-PD1 mAb. Furthermore, cured mice from EAGLE treatment completely rejected the rechallenge of tumor cells, suggesting that EAGLE induced anti-tumor immunologic memory. In summary, the novel therapeutic modality, EAGLE, blocked the glyco-immune checkpoint pathway in the tumor microenvironment and potentiated innate and adaptive antitumor immunity, which offers a novel immunomodulatory strategy to treat cancer. Citation Format: Li Peng. A novel immunomodulatory strategy of targeting glyco-immune checkpoints with EAGLE technology [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr B034.

Abstract B82: Identification of tumor neoantigens for combination therapy in murine tumor models

Abstract The adoption of anti-CTLA-4 and anti-PD-1/PD-L1 blockade has dramatically changed the treatment approaches in multiple cancer types, and has demonstrated the power of the immune system to control the outgrowth of neoplastic lesions. Accumulating evidence suggests these successes are mediated by T cells targeting nonsynonymous mutations unique to the patient’s tumor. When used in combination with checkpoint inhibition, or other standard of care treatment modalities, vaccines targeting tumor neoantigens have the potential to greatly enhance patient survival. Recent advances in DNA/RNA sequencing and bioinformatic analysis have made it possible to rapidly identify nonsynonymous mutations being expressed in patient tumors. As the number of nonsynonymous mutations detected in patient samples can range from less than 10 to several thousand, it is necessary to develop strategies to identify and rank neoantigens with a greater probability of being immunogenic and mediating regression of established tumors. In the present study, we harvested tumors from mice bearing syngeneic tumors, and performed DNA/RNA sequencing to identify nonsynonymous mutations to use as tumor specific neoantigens. These potential neoantigens were ranked based upon their proposed in-silico MHC binding affinity, and the relative abundance of each transcript as determined by RNA-seq. Top ranking 9-mer neoantigens peptides were synthesized and evaluated. We assessed neoantigens for their ability to bind MHC in vitro, and expand antigen-specific T cells in vivo. To further augment the immunogenicity of our neoantigen 9-mer peptides, we used them in combination with an IL-15 super-agonist (ALT-803) and an anti-PD-L1 antibody. When used in combination with these immune-modulators, we observed a dramatic increase in the magnitude of the immune response generated against our neoantigens, which associated with increased tumor therapy. This study provides the rational for the combination of neoantigen vaccines with immune modulators to slow tumor growth and extend survival. Citation Format: Karin L. Lee, Andrew T. Nguyen, Steven C. Benz, Shahrooz Rabizadeh, James W. Hodge, Claudia Palena, Jeffrey Schlom, Duane H. Hamilton. Identification of tumor neoantigens for combination therapy in murine tumor models [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B82.