Antitumor Memory Response Research Articles

Abstract 5293: Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade

Abstract NI-3201 is a PD-L1xCD28 bispecific antibody generated on the κλ body platform that blocks the PD-L1/PD-1 immune checkpoint pathway and conditionally provides T cell activation “signal 2” when engaging PD-L1+ tumor or immune cells (e.g., antigen-presenting cells). By combining two distinct mechanisms of action into a single molecule, NI-3201 overcomes two major immune suppression mechanisms commonly observed in a variety of solid tumors, namely: [1] suppression of T cell response (induced by PD-L1+ cells engaging PD-1 on T cells) and [2] restricted/limited T cell activation (due to the lack of costimulatory signal associated to the scarcity of CD28 ligands in the TME). Importantly, NI-3201 was designed to require primary T cell stimulation (“signal 1”) to afford its activity: in the absence of TCR engagement, NI-3201 is unable to activate T cells, thus preventing unintended systemic inflammation. The single agent pharmacological activity of NI-3201 was assessed in vitro through a Cytomegalovirus (CMV) recall assay in which CMV+ PBMCs were cocultured with PD-L1+ cancer cell lines ectopically loaded with CMV-specific MHC-I peptides, thus providing “signal 1”. NI-3201 induced the activation and proliferation of CMV-specific T cells which resulted in killing of the target tumor cells. In vivo, the single agent activity of NI-3201 was shown in immunocompetent huCD28-transgenic mice engrafted with huPD-L1-expressing murine colon adenocarcinoma MC38 cells, a model in which NI-3201 induced durable anti-tumor immunological memory response. In humanized mouse models where T cell engagers (TCEs) were ineffective, NI-3201 was able to synergize with TCEs to control tumor growth, even inducing tumor regression in one model. The pharmacokinetics (PK) and tolerability of NI-3201 were investigated in non-human primates (NHP) after single and repeated-dosing. NI-3201 demonstrated favorable PK with close to dose-proportional concentrations being achieved. Upon repeated NI-3201 administration, no hypercytokinemia was observed; only a mild and transient IL-6 release and associated increase in C-reactive protein (CRP) levels were seen, with no clinical signs reported. The favorable profile seen in NHP was supported by data generated in vitro further de-risking the cytokine release syndrome (CRS) potential. Quantitative systems pharmacology (QSP) modelling integrating both in vitro and NHP data predicted a drug concentration required for optimal NI-3201 activity in the TME, and preliminary data suggests optimal patient activity at Q2W-Q3W dosing schedule. The anti-tumor activity as a single agent or as universal combination partner for TCEs, shown in vitro and in vivo, together with the favorable safety profile of NI-3201, support its clinical development planned to start in Q1 2025. Citation Format: Sara Majocchi, Nadia Anceriz, Alizée Viandier, Adeline Lesnier, Laura Cons, Lise Nouveau, Emily A. Pace, Bjorn L. Millard, Bruno Daubeuf, Valéry Moine, Susana Salgado-Pires, José Saro, Limin Shang, Krzysztof Masternak, Walter G. Ferlin. Preclinical development of NI-3201, a PD-L1xCD28 bispecific antibody mediating CD28 costimulation upon PD-L1 blockade [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 5293.

Abstract 3979: PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses

Abstract Cytotoxic CD8+ T cells play a key role in response to anti-PD-1/PD-L1 immune checkpoint inhibitors (ICI) therapy. However, the exact effector molecules that are regulated by PD-1 signals in cytotoxic CD8+ T cells have not been completely defined. Lack of this knowledge is a crucial problem in addressing how to overcome the low clinical response rate to ICI therapy. When exploring why certain patients not responding to ICI therapy, we found non-responders did not have an elevated NKG7 expression in CD8+ T cells as responders to ICI therapy. The difference in NKG7 expression in CD8+ T cells between non-responders and responders to anti-PD-1 therapy prompted us to investigate the potential role of PD-1 signaling in regulating NKG7 expression. To that end, we generated a CD8+ T cell-specific Pdcd1 conditional knockout mice (CD8 E8ICre-Pdcd1fl/fl, also known as CD8-PD-1 cKO) where Pdcd1 is deleted after thymic selection of single positive CD8+ T cells. We observed a delayed tumor growth and prolonged survival in CD8-PD-1 cKO mice compared to control mice after B16-OVA melanoma tumor challenge. On Day 12, when the CD8-PD-1 cKO mice demonstrated their ability to control tumor growth, we conducted single cell RNA sequencing on isolated tumor infiltrating CD8+ T cells, revealing a significant increase of Nkg7 expression in effector CD8+ T cells in the Pdcd1 deficient CD8+ T cells compared to the control wild type T cells. Pdcd1-deficient effector CD8+ T cells also had increased expression of genes linked to NFAT/Calcineurin signaling pathway including Nfatc1, S100a4, Tbet and Tox, suggesting Nkg7 could be regulated by Pdcd1 via the NFAT pathway. To determine the impact of increased NKG7 expression in CD8+ T cells, we used OVA protein as a surrogate tumor antigen in vaccination. We found a complete B16-OVA tumor rejection in CD8-PD-1 cKO mice compared to control mice when tumor cells were injected on day 7 after vaccination with OVA protein and poly (I:C) (as an adjuvant), but only a partial rejection on day 21. The CD8-PD-1 cKO mice that were tumor-free for 30-40 days rejected 2nd tumor challenge with B16-OVA tumors. This long-term protection is tumor antigen (OVA) specific because these mice did not reject B16F10 tumor cells that do not express OVA antigen. We also found effector memory CD8+ T cells with high cytotoxicity increased in the spleen of CD8-PD-1 cKO mice after OVA antigen immunization compared to control. In conclusion, we found NKG7 is one of the effector molecules in cytotoxic CD8+ T cells, which can be regulated by PD-1 signals through NFAT signaling. Targeting the PD-1-NFAT pathway may be a new option to overcome low responses of T cells to ICI therapy in treatment of refractory cancer. Citation Format: Zhiming Mao, Joanina Gicobi, Jacob Hirdler, Xin Liu, Michelle Hsu, Emilia Dellacecca, Wenjing Zhang, Fabrice Lucien-Matteoni, Hai dong. PD-1 signals regulate NKG7 that is required for CD8+ T cells to sustain cytotoxicity and durable antitumor memory responses [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 3979.

STING activator 2'3'-cGAMP enhanced HSV-1-based oncolytic viral therapy.

Oncolytic viruses (OVs) can selectively replicate in tumor cells and remodel the microenvironment of immunologically cold tumors, making them a promising strategy to evoke antitumor immunity. Similarly, agonists of the stimulator of interferon genes (STING)-interferon (IFN) pathway, the main cellular antiviral system, provide antitumor benefits by inducing the activation of dendritic cells (DC). Considering how the activation of the STING-IFN pathway could potentially inhibit OV replication, the use of STING agonists alongside OV therapy remains largely unexplored. Here, we explored the antitumor efficacy of combining an HSV-1-based OV, C-REV, with a membrane-impermeable STING agonist, 2'3'-GAMP. Our results demonstrated that tumor cells harbor a largely defective STING-IFN pathway, thereby preventing significant antiviral IFN induction regardless of the permeability of the STING agonist. In vivo, the combination therapy induced more proliferative KLRG1-high PD1-low CD8+ T-cells and activated CD103+ DC in the tumor site and increased tumor-specific CD44+ CD8+ T-cells in the lymph node. Overall, the combination therapy of C-REV with 2'3'-cGAMP elicited antitumor immune memory responses and significantly enhanced systemic antitumor immunity in both treated and non-treated distal tumors.

Differential tumor immune microenvironment coupled with tumor progression or tumor eradication in HPV-antigen expressing squamous cell carcinoma (SCC) models.

Human papilloma virus (HPV) is an etiological factor of head and neck squamous cell carcinoma (HNSCC). To investigate the role of HPV antigen in anti-tumor immunity, we established mouse models by expressing HPV16 E6 and E7 in a SCC tumor cell line. We obtained two HPV antigen-expressing clones (C-225 and C-100) transplantable into C57BL/6 recipients. We found that C-225 elicited complete eradication in C57BL/6 mice (eradicated), whereas C-100 grew progressively (growing). We examined immune tumor microenvironment (TME) using flow cytometry and found that eradicated or growing tumors exhibited differential immune profiles that may influence the outcome of anti-tumor immunity. Surprisingly, the percentage of CD8 and CD4 tumor-infiltrating lymphocytes (TILs) was much higher in growing (C-100) than eradicated (C-225) tumor. However, the TILs upregulated PD-1 and LAG-3 more potently and exhibited impaired effector functions in growing tumor compared to their counterparts in eradicated tumor. C-225 TME is highly enriched with myeloid cells, especially polymorphonuclear (PMN) myeloid-derived suppressor cells (MDSC), whereas the percentage of M-MDSC and tumor-associated macrophages (TAMs) was much higher in C-100 TME, especially M2-TAMs (CD206+). The complete eradication of C-225 depended on CD8 T cells and elicited anti-tumor memory responses upon secondary tumor challenge. We employed DNA sequencing to identify differences in the T cell receptor of peripheral blood lymphocytes pre- and post-secondary tumor challenge. Lastly, C-225 and C-100 tumor lines harbored different somatic mutations. Overall, we uncovered differential immune TME that may underlie the divergent outcomes of anti-tumor immunity by establishing two SCC tumor lines, both of which express HPV16 E6 and E7 antigens. Our experimental models may provide a platform for pinpointing tumor-intrinsic versus host-intrinsic differences in orchestrating an immunosuppressive TME in HNSCCs and for identifying new targets that render tumor cells vulnerable to immune attack.

Dosimetric and biologic intercomparison between electron and proton FLASH beams

Background and purposeThe FLASH effect has been validated in different preclinical experiments with electrons (eFLASH) and protons (pFLASH) operating at an average dose rate above 40 Gy/s. However, no systematic intercomparison of the FLASH effect produced by eFLASHvs. pFLASH has yet been performed and constitutes the aim of the present study. Materials and methodsThe electron eRT6/Oriatron/CHUV/5.5 MeV and proton Gantry1/PSI/170 MeV were used to deliver conventional (0.1 Gy/s eCONV and pCONV) and FLASH (≥110 Gy/s eFLASH and pFLASH) dose rates. Protons were delivered in transmission. Dosimetric and biologic intercomparisons were performed using previously validated dosimetric approaches and experimental murine models. ResultsThe difference between the average absorbed dose measured at Gantry 1 with PSI reference dosimeters and with CHUV/IRA dosimeters was −1.9 % (0.1 Gy/s) and + 2.5 % (110 Gy/s). The neurocognitive capacity of eFLASH and pFLASH irradiated mice was indistinguishable from the control, while both eCONV and pCONV irradiated cohorts showed cognitive decrements. Complete tumor response was obtained after an ablative dose of 20 Gy delivered with the two beams at CONV and FLASH dose rates. Tumor rejection upon rechallenge indicates that anti-tumor immunity was activated independently of the beam-type and the dose-rate. ConclusionDespite major differences in the temporal microstructure of proton and electron beams, this study shows that dosimetric standards can be established. Normal brain protection and tumor control were produced by the two beams. More specifically, normal brain protection was achieved when a single dose of 10 Gy was delivered in 90 ms or less, suggesting that the most important physical parameter driving the FLASH sparing effect might be the mean dose rate. In addition, a systemic anti-tumor immunological memory response was observed in mice exposed to high ablative dose of electron and proton delivered at CONV and FLASH dose rate.

MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER

You have accessJournal of UrologyCME1 Apr 2023MP04-13 IMMUNOPHENOTYPE ANALYSIS OF TUMOR-INFILTRATING IMMUNE CELLS TO ELUCIDATE THE MECHANISM OF ANTITUMOR EFFECT OF IL-7 AND CCL19 PRODUCING CAR-T CELLS AGAINST SOLID CANCER Shunsuke Goto, Koji Tamada, and Masatoshi Eto Shunsuke GotoShunsuke Goto More articles by this author , Koji TamadaKoji Tamada More articles by this author , and Masatoshi EtoMasatoshi Eto More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003215.13AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Chimeric antigen receptor (CAR)-T cell therapy against solid cancer is challenging. We reported that mouse CAR-T cells modified to produce interleukin- (IL)-7 and CCL19 (7×19 CAR-T) enhance the therapeutic efficacy against solid tumors by recruiting not only administered CAR-T cells but also endogenous T cells and DCs into tumors and inducing long-term antitumor memory responses. Furthermore, we reported that human 7×19 CAR-T cells show enhanced antitumor efficacy against patient-derived xenograft (PDX) tumor expressing mesothelin, for which 10% of renal cell carcinoma are positive. In this research, we explore the mechanism of antitumor activity of 7×19 CAR-T cells and object to prevail the difference between conventional CAR-T cells (Conv.) and 7×19, especially focusing on the immunophenotype of tumor-infiltrating lymphocytes (TILs). METHODS: We produced a novel anti-human mesothelin 7×19 CAR-T cells, then assessed the antitumor activity against solid tumor in vivo using a malignant mesothelioma orthotopic tumor and a pancreatic cancer PDX models. Tumor was resected after the administration of Conv. or 7×19 CAR-T cells and analyzed TILs by flowcytometry focusing on the number, exhaustion marker, phenotype and cytokine producing ability. RESULTS: 7×19 CAR-T cells showed superior antitumor efficacy compared to Conv. CAR-T cells in the orthotopic and PDX tumor models. In the PDX treatment model, TIL analysis revealed that not only CAR-T cells but also non-CAR-T cells were infiltrated effectively into tumors by 7×19 treatment compared to Conv. Exhaustion marker expressions such as PD-1 and TIGIT were significantly downregulated in 7×19 than Conv. The examination of the ability of TILs to express cytokines and effector molecules showed that significant increases of TNF-α and Granzyme B, as well as a trend of increased IFN-γ in 7×19 CAR-T cells. The analysis of splenocyte after the treatment revealed that 7×19 CAR-T cells maintained their effector memory phenotype. CONCLUSIONS: It was found that 7×19 CAR-T cells showed enhanced antitumor potential by the maintenance of their effector functions. These data support the foresight of this therapy as a treatment option for patients with mesothelin-positive renal cell carcinoma. Source of Funding: Practical Research for Innovative Cancer Control, and Project for Cancer Research and Therapeutic Evolution (P-CREATE) 16770206 by Japan Agency for Medical Research and Development (AMED) © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e38 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Shunsuke Goto More articles by this author Koji Tamada More articles by this author Masatoshi Eto More articles by this author Expand All Advertisement PDF downloadLoading ...

Abstract 1385: The influence of MHC class I and II on T cell responses in mouse melanoma

Abstract Introduction: Using an in situ vaccine (ISV) regimen consisting of radiation combined with immunocytokine (tumor-targeting mAb linked to IL2), we can cure mice of well established B78 melanoma tumors (B78s). Mice cured of their B78s with ISV demonstrate long-term immune memory, evidenced by rejection of engraftment of a tumor rechallenge >180 days post initial cure of tumor. Traditionally, immaune memory is thought to be mediated via CD8+ T cells, which require antigen presentation via MHC Class I (MHCI). However, B78s express little to no MHCI but do express MHCII when stimulated with IFNγ. While not commonly expressed on solid tumors, MHCII is expressed on 50-60% of melanomas in humans. Here we explored implications of MHCI and MHCII expression in both the primary and long-term memory anti-tumor responses generated with ISV. Methods: CD4+ or CD8+ T cells were depleted in mice during the primary anti-tumor response (i.e. B78 tumor bearing mice receiving the ISV regimen) or during tumor rechallenge experiments (i.e. B78-cured mice rechallenged with B78s). Tumor growth was monitored. In separate studies, tumors and tumor draining lymphnodes (TDLNs) were harvested during the primary antitumor response and analyzed via flow cytometry to assess T cell activation and immune infiltrate. Finally, TDLNs were harvested from B78-cured mice, 7 days after tumor rechallenge, to define memory T cell subsets. Results: Depletion studies revealed CD4+ T cells are required for both the antitumor response to ISV and the long-term memory response in B78-cured mice, but CD8+ T cells are not required for either of these responses. Increased CD8+ and CD4+ T cell infiltrates are observed in the tumor microenvironment during the primary anti-tumor response. In B78-cured mice, though not required for memory responses, CD8+ central memory T cells are significantly increased in the TDLNs compared to naïve or primary tumor bearing mice. The amount of CD4+ effector memory T cells are significantly increased in the TDLN of B78-cured mice compared to CD8+ effector memory T cells. Conclusion: Often not expressed on solid tumors, MHCII is expressed on some melanoma tumors, and its expression has been correlated with a positive response to immunotherapies. CD4+ cytotoxic T cells can directly engage MHCII on tumors, suggesting this interaction has an important role in the response to immunotherapy for MHCII expressing tumors. Our data suggest that CD4+ T cells drive both the primary anti-tumor and long-term immune memory responses in the B78 model when treating with this effective ISV. We are continuing our efforts to understand the characteristics of the B78 cell line that may be relevant to its response to immunotherapy and its resistance to single agent checkpoint blockade. Understanding the cellular and molecular mechanisms involved in ISV-induced immune recognition and destruction of B78s may guide future improvements of this clinically-relevant immunotherapy regimen. Citation Format: Amy K. Erbe, Arika Feils, Mackenzie Heck, Sabrina VandenHeuvel, Julianna Castillo, Alina Hampton, Lizzie Frankel, Anna Hoefges, Peter Carlson, Alex A. Pieper, Taylor Aiken, Lauren Zebertavage, David Komjathy, Dan Spiegelman, Noah Tsarovsky, Zachary S. Morris, Ravi Patel, Alexander Rakhmilevich, Paul M. Sondel. The influence of MHC class I and II on T cell responses in mouse 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 1385.

Enhancement of antitumor immunotherapy using mitochondria-targeted cancer cell membrane-biomimetic MOF-mediated sonodynamic therapy and checkpoint blockade immunotherapy

Immunotherapeutic interventions represent a promising approach to treating cancer, with strategies such as immune checkpoint blockade (ICB), immunogenic sonodynamic therapy (SDT), and immune adjuvant T cell delivery having exhibited clinical promise. In this report, we describe the use of cancer cell membrane-coated triphenylphosphonium (TPP) decorated nano-metal–organic framework (nMOF) constructs [Zr-TCPP(TPP)/R837@M] that were used to generate homologous, mitochondria-targeted platforms with a high rate of sonosensitizer loading. This construct was utilized to simultaneously promote tumor antigen presentation via enhancing SDT while synergistically promoting dendritic cell (DC) maturation through the delivery of the Toll-like receptor agonist R837. In vitro, these functionalized nMOFs were readily internalized by homologous tumor cells in which they were efficiently targeted to the mitochondria, promoting DC activation through the induction of immunogenic cell death (ICD) following ultrasound exposure. Moreover, this nanoplatform was able to achieve in vivo synergy with anti-CTLA-4 ICB to reverse immunosuppression tumor microenvironment (TME), thus achieving more robust antitumor efficacy capable of suppressing metastatic disease progression and facilitating the development of durable antitumor memory responses. Together, these results highlight a promising approach to achieving enhanced SDT activity while overcoming an immunosuppressive TME, thereby achieving more robust antitumor immunity.

Intratumoral delivery of a novel oncolytic adenovirus encoding human antibody against PD-1 elicits enhanced antitumor efficacy

To date, diverse combination therapies with immune checkpoint inhibitors (ICIs), particularly oncolytic virotherapy, have demonstrated enhanced therapeutic outcomes in cancer treatment. However, high pre-existing immunity against the widely used adenovirus human serotype 5 (AdHu5) limits its extensive clinical application. In this study, we constructed an innovative oncolytic virus (OV) based on a chimpanzee adenoviral vector with low seropositivity in the human population, named AdC68-spE1A-αPD-1, which endows the parental OV (AdC68-spE1A-ΔE3) with the ability to express full-length anti-human programmed cell death-1 monoclonal antibody (αPD-1). In vitro studies indicated that the AdC68-spE1A-αPD-1 retained parental oncolytic capacity, and αPD-1 was efficiently secreted from the infected tumor cells and bound exclusively to human PD-1 (hPD-1) protein. In vivo, intratumoral treatment with AdC68-spE1A-αPD-1 resulted in significant tumor suppression, prolonged overall survival, and enhanced systemic antitumor memory response in an hPD-1 knockin mouse tumor model. This strategy outperformed the unarmed OV and was comparable with combination therapy with intratumoral injection of AdC68-spE1A-ΔE3 and systemic administration of commercial αPD-1. In summary, AdC68-spE1A-αPD-1 is a cost-effective approach with potential clinical applications. ‬‬‬‬

IM-4 Impact of oHSV activated NOTCH signaling in tumor microenvironment and its impact on anti-tumor immunity

Oncolytic herpes simplex virus-1 (oHSV) is novel FDA-approved immunotherapy for advanced melanoma patients in US. Also, oHSV is recently approved for the treatment of recurrent GBM in Japan. We have shown that oHSV treatment of GBM cells induces NICD cleavage and NOTCH activation in adjacent uninfected glioma cells via HSV-1 microRNA-H16 (Otani Y and Yoo JY, Clin Cancer Res, 2020), however, the consequences of NOTCH on immunotherapy in GBM is unknow. Here we have investigated the impact of oHSV-induced NOTCH signaling on the tumor microenvironment (TME). Analysis of TCGA GBM data and experimental murine models revealed NOTCH induced immunosuppressive myeloid cell recruitment and limited anti-tumor immunity. In oHSV treated tissue, viral infection educated tumor associated macrophages to secrete CCL2 which recruited monocytic myeloid derived suppressor cell (MDSC) that attenuated anti-tumor immunity. Consistent with this, CCL2 induction was also observed in serum of recurrent GBM patients treated with oHSV (NCT03152318). Importantly, blockade of NOTCH signaling reduced the oHSV induced immunosuppressive environment and activated a CD8 dependent anti-tumor memory response. These findings present the opportunities for combination therapies that can help improve therapeutic benefit and anti-tumor immunity in GBM.

230 Preclinical efficacy of CLEC-1 antagonist as novel myeloid immune checkpoint therapy for oncology

BackgroundC-type lectin receptors (CLRs) are powerful pattern recognition receptors shaping immune cell-mediated tissue damage by positively or negatively regulating myeloid cell functions and hence tumor elimination or evasion. We previously reported that the orphan CLR CLEC-1 expressed by dendritic cells (DCs) tempers T cell’s responses in vivo by limiting antigen cross-presentation by cDC1. Furthermore, we observed that CLEC-1 is highly expressed by myeloid cells purified from human tumor microenvironment, in particular tumor-associated macrophages.MethodsMacrophages were generated from monocytes of healthy volunteers for phagocytosis assays. MC38 and Hepa 1.6 murine tumor cells were implanted in Clec1a KO or KI mice for immunotherapeutic treatment evaluation.ResultsUsing newly developed anti-human CLEC-1 monoclonal antibodies (mAbs), we found that antagonist anti-CLEC-1 mAbs with the capacity to block CLEC-1/CLEC-1Ligand interaction, as opposed to non-antagonist CLEC-1 mAbs, increase the phagocytosis of CLEC-1Ligand-positive human tumor cells by human macrophages, in particular when opsonized by tumor-associated antigen mAbs (Rituximab, Cetuximab, Trastuzumab) or with anti-CD47 mAb (Magrolimab). In-vivo, CLEC-1 knock-out (KO) mice (n=19) display significant prolonged survival in monotherapy as compared to wild-type littermates (n=12) in an orthotopic hepatocellular carcinoma (HCC) model and anti-tumor memory responses was demonstrated by tumor rechallenge in cured mice. CLEC1 KO mice also illustrate significant eradication of MC38 colorectal tumors in combination with chemotherapy promoting CLEC-1Ligand expression by tumor cells (n=16 with Gemcitabine or n=11 with Cyclophosphamide). HCC tumor microenvironment analysis after 2 weeks of tumor implantation shows significantly higher number of CD8+ and memory CD8+ T cells with reduced PD1 expression in CLEC1 KO animals (n=16 versus n=12 for KO vs WT mice respectively). Finally, we recently generated human CLEC-1 knock-in mice expressing the extracellular human CLEC1 domain fused to the intracellular mouse CLEC1 tail and confirmed preclinical efficacy in vivo with anti-human CLEC1 antagonist mAb in monotherapy in the orthotopic HCC model.ConclusionsThese data illustrate that CLEC-1 inhibition represents a novel therapeutic target for immuno-oncology modifying T cell immune responses and tumor cell phagocytosis by macrophages.

An engineered IL-21 with half-life extension enhances anti-tumor immunity as a monotherapy or in combination with PD-1 or TIGIT blockade

Interleukin-21 (IL-21) has exhibited anti-tumor activity in preclinical and clinical studies; however, its modest efficacy and short half-time has limited its therapeutic utility as a monotherapy. Therefore, we engineered a fusion protein (IL-21-αHSA) in which a nanobody targeting human serum albumin (HSA) was fused to the C-terminus of rhIL-21. The αHSA nanobody displayed broad species cross-reactivity and bound to a HSA epitope that does not overlap with the FcRn binding site, thus providing a strategic design for half-life extension. The IL-21-αHSA fusion protein showed increased stability compared to rhIL-21, while retaining its bioactivity in a liquid solution for at least 6 months. Moreover, IL-21-αHSA showed a dramatically extended half-life and prolonged exposure in cynomolgus monkeys, with the t1/2 and AUC nearly 10 and 50 times greater than that of rhIL-21, respectively. Furthermore, IL-21-αHSA displayed enhanced anti-tumor efficacy in two syngeneic mouse models. Notably, IL-21-αHSA increased the anti-tumor effect of programmed cell death protein 1 (PD-1) and T cell immunoglobulin and ITIM domain (TIGIT) blockades when used in combination, with a protection against tumor rechallenge, suggesting the formation of long-term anti-tumor memory response. KEGG analysis identified significantly enriched pathways associated with anti-tumor immune response, with increased expression of genes associated with CD8+ T and NK cell cytotoxicity. Overall, these data support further clinical evaluation of IL-21-αHSA as a monotherapy or in combination with immune checkpoint blockades.

Radiation-induced neoantigens broaden the immunotherapeutic window of cancers with low mutational loads

Immunotherapies are a promising advance in cancer treatment. However, because only a subset of cancer patients benefits from these treatments it is important to find mechanisms that will broaden the responding patient population. Generally, tumors with high mutational burdens have the potential to express greater numbers of mutant neoantigens. As neoantigens can be targets of protective adaptive immunity, highly mutated tumors are more responsive to immunotherapy. Given that external beam radiation 1) is a standard-of-care cancer therapy, 2) induces expression of mutant proteins and potentially mutant neoantigens in treated cells, and 3) has been shown to synergize clinically with immune checkpoint therapy (ICT), we hypothesized that at least one mechanism of this synergy was the generation of de novo mutant neoantigen targets in irradiated cells. Herein, we use KrasG12D x p53-/- sarcoma cell lines (KP sarcomas) that we and others have shown to be nearly devoid of mutations, are poorly antigenic, are not controlled by ICT, and do not induce a protective antitumor memory response. However, following one in vitro dose of 4- or 9-Gy irradiation, KP sarcoma cells acquire mutational neoantigens and become sensitive to ICT in vivo in a T cell-dependent manner. We further demonstrate that some of the radiation-induced mutations generate cytotoxic CD8+ T cell responses, are protective in a vaccine model, and are sufficient to make the parental KP sarcoma line susceptible to ICT. These results provide a proof of concept that induction of new antigenic targets in irradiated tumor cells represents an additional mechanism explaining the clinical findings of the synergy between radiation and immunotherapy.

Fc-Optimized Anti-CCR8 Antibody Depletes Regulatory T Cells in Human Tumor Models.

FOXP3+ regulatory T cells (Treg) play a critical role in mediating tolerance to self-antigens and can repress antitumor immunity through multiple mechanisms. Therefore, targeted depletion of tumor-resident Tregs is warranted to promote effective antitumor immunity while preserving peripheral homeostasis. Here, we propose the chemokine receptor CCR8 as one such optimal tumor Treg target. CCR8 was expressed by Tregs in both murine and human tumors, and unlike CCR4, a Treg depletion target in the clinic, CCR8 was selectively expressed on suppressive tumor Tregs and minimally expressed on proinflammatory effector T cells (Teff). Preclinical mouse tumor modeling showed that depletion of CCR8+ Tregs through an FcyR-engaging anti-CCR8 antibody, but not blockade, enabled dose-dependent, effective, and long-lasting antitumor immunity that synergized with PD-1 blockade. This depletion was tumor Treg-restricted, sparing CCR8+ T cells in the spleen, thymus, and skin of mice. Importantly, Fc-optimized, nonfucosylated (nf) anti-human CCR8 antibodies specifically depleted Tregs and not Teffs in ex vivo tumor cultures from primary human specimens. These findings suggest that anti-CCR8-nf antibodies may deliver optimal tumor-targeted Treg depletion in the clinic, providing long-term antitumor memory responses while limiting peripheral toxicities. SIGNIFICANCE: These findings show that selective depletion of regulatory T cells with an anti-CCR8 antibody can improve antitumor immune responses as a monotherapy or in combination with other immunotherapies. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/81/11/2983/F1.large.jpg.

Oncolytic reovirus-mediated recruitment of early innate immune responses reverses immunotherapy resistance in prostate tumors

Prostate cancers are considered “cold” tumors characterized by minimal T cell infiltrates, absence of a type I interferon (IFN) signature, and the presence of immunosuppressive cells. This non-inflamed phenotype is likely responsible for the lack of sensitivity of prostate cancer patients to immune checkpoint blockade (ICB) therapy. Oncolytic virus therapy can potentially overcome this resistance to immunotherapy in prostate cancers by transforming cold tumors into “hot,” immune cell-infiltrated tumors. We investigated whether the combination of intratumoral oncolytic reovirus, followed by targeted blockade of Programmed cell death protein 1 (PD-1) checkpoint inhibition and/or the immunomodulatory CD73/Adenosine system can enhance anti-tumor immunity. Treatment of subcutaneous TRAMP-C2 prostate tumors with combined intratumoral reovirus and anti-PD-1 or anti-CD73 antibody significantly enhanced survival of mice compared with reovirus or either antibody therapy alone. Only combination therapy led to rejection of pre-established tumors and protection from tumor re-challenge. This therapeutic effect was dependent on CD4+ T cells and natural killer (NK) cells. NanoString immune profiling of tumors confirmed that reovirus increased tumor immune cell infiltration and revealed an upregulation of the immune-regulatory receptor, B- and T-lymphocyte attenuator (BTLA). This expression of BTLA on innate antigen-presenting cells (APCs) and its ligand, Herpesvirus entry mediator (HVEM), on T cells from reovirus-infected tumors was in keeping with a role for the HVEM-BTLA pathway in promoting the potent anti-tumor memory response observed.

Abstract 5203: MGC018, a duocarmycin-based antibody-drug conjugate targeting B7-H3, exhibits immunomodulatory activity and enhanced antitumor activity in combination with checkpoint inhibitors

Abstract Introduction: B7-H3, a member of the B7 family of immunomodulatory molecules, is overexpressed in a wide range of solid tumors. B7-H3 tumor overexpression has been correlated with disease severity and poor outcome. MGC018 is a duocarmycin-based antibody-drug conjugate (ADC) targeting B7-H3. MGC018 exhibits a favorable preclinical profile, with strong reactivity toward tumor cells and tumor-associated vasculature, limited normal tissue reactivity, and potent antitumor activity toward B7-H3-expressing tumor xenografts. With the emergence of immune-checkpoint blockade as a promising treatment for cancer, interest has grown in understanding the potential of cytotoxic agents to promote immune surveillance or stimulate immune responses to dying cancer cells, leading to immunological memory. ADCs bearing tubulin and DNA modifying cytotoxic payloads have been reported to induce immunological cell death (ICD), mediate antitumor immunity in immunocompetent mouse models, and synergistically combine with checkpoint inhibitors to deliver enhanced antitumor responses. Based on those results, we investigated the immunomodulatory potential of MGC018 and the prospect to combine with checkpoint blockade to enhance antitumor responses. Methods: Syngeneic mouse models expressing human B7-H3 were employed to investigate the antitumor activity of MGC018 in an immune competent setting. Studies were conducted to assess the role of the immune system in the MGC018-mediated antitumor responses, whether MGC018 could impart antitumor memory responses in vivo, and the potential to enhance antitumor responses by combining MGC018 with PD-1 blockade. Results: MGC018 demonstrated specific, dose-dependent in vivo antitumor activity toward human B7-H3-bearing tumors in immunocompetent syngeneic mouse models. Depletion of CD8+ T cells led to reduced antitumor responses, indicating that CD8+ T cells contributed to MGC018-mediated antitumor activity. Antitumor activity in these models was enhanced when MGC018 was combined with anti-PD-1. Treatment with MGC018 alone, or in combination with anti-PD-1, led to complete antitumor responses, and the majority of mice rejected subsequent tumor rechallenge. Conclusion: MGC018, a clinical-stage therapeutic comprised of a humanized antibody targeting B7-H3, conjugated to a duocarmycin-based DNA alkylating payload, exhibits a favorable preclinical profile. Results from these syngeneic model studies support the hypothesis that the antitumor activity of the duocarmycin-based MGC018 ADC (1) mediates immunomodulatory activity, (2) is enhanced by combination with checkpoint blockade, and (3) induces immunological memory. Our findings support a clinical strategy that combines MGC018 with checkpoint blockade for the treatment of B7-H3-expressing solid cancers. Citation Format: Juniper A. Scribner, Michael Chiechi, Pam Li, Thomas Son, Jeff Hooley, Ying Li, Anushka De Costa, Peter Lung, Nicholas Yee-Toy, Francine Chen, Bhaswati Barat, Christina Wolff, Valentina Ciccarone, James Tamura, Scott Koenig, Chet Bohac, Jon Wigginton, Paul A. Moore, Ezio Bonvini, Deryk Loo. MGC018, a duocarmycin-based antibody-drug conjugate targeting B7-H3, exhibits immunomodulatory activity and enhanced antitumor activity in combination with checkpoint inhibitors [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 5203.

Abstract 4577: ONM-500: A STING-activating therapeutic nanovaccine platform for cancer immunotherapy

Abstract Background: The efficacy of cancer vaccines requires induction of cancer-specific cytotoxic T-lymphocytes (CTL) to effectively clear established tumors. Orchestration of antigen presentation, co-stimulatory signaling, and innate cytokine signals are necessary steps for tumor-specific T cell activation. The ONM-500 nanovaccine platform utilizes a novel pH-sensitive polymer that forms an antigen-encapsulating nanoparticle and functions both as a carrier for antigen delivery to dendritic cells and as an adjuvant, activating the stimulator of interferon genes (STING) pathway and generating a CD8+ CTL response. Peptide antigens have translational challenges due to limited stability, HLA-type-specific antigen sequence recognition and presentation. Full-length protein antigens alleviate HLA subtype limitation, allowing coverage of multi-immunogenic T cell epitopes in patients. Using the ONM-500 platform with the full-length recombinant E6/E7 oncoproteins from human papillomavirus (HPV) cancers, we have demonstrated great antitumor efficacy in an HPV positive cancer model in animals. Methods: Based on the previously demonstrated STING-dependent T cell activation by ONM-500, the nanovaccine was formulated with full-length HPV16 E6/E7 proteins, and the nanoparticle properties and antigen loading were characterized. In vivo lymph node accumulation following subcutaneous administration was evaluated using fluorophore-labeled ONM-500. Direct binding of ONM-500 polymer to human STING was evaluated using a pull-down assay. Antitumor efficacy of ONM-500 nanovaccine with full length E6/E7 was evaluated using a TC-1 model which overexpresses HPV16 E6/E7 and compared with ONM-500 formulated with peptide antigen. Long-term anti-tumor memory was evaluated in a rechallenge study after 60 days in tumor-free animals. Results: Characterization of ONM-500 nanovaccine shows reproducible particle chemical and physical properties and antigen loading. The nanoparticle size substantiates the effective lymph node accumulation for antigen cross-presentation in dendritic cells following subcutaneous administration. The pull-down assay showed effective binding of the ONM-500 polymer to STING. In TC-1 tumors, ONM-500 nanovaccine containing full-length E6/E7 protein showed 100% overall survival at 55 days. Tumor growth inhibition was also improved over peptide antigen formulated ONM-500. A rechallenge study demonstrated long-term antigen-specific anti-tumor memory response. Conclusions: ONM-500 STING-activating nanovaccines effectively deliver antigens in vivo to lymph nodes to elicit an antigen-specific CTL response. The anti-tumor efficacy in the animal tumor model demonstrates the potential of ONM-500 as a general STING-activating cancer vaccine platform, and full-length E6/E7 incorporated ONM-500 is being developed for HPV-associated cancers. Citation Format: Jason Miller, Min Luo, Hua Wang, Zhaohui Wang, Xinliang Ding, Ashley Campbell, Jonathan Almazan, Stephen Gutowski, Zhijian Chen, Jinming Gao, Tian Zhao. ONM-500: A STING-activating therapeutic nanovaccine platform for cancer immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4577.

Abstract 4548: SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor activity

Abstract CD112R (PVRIG) is a recently identified inhibitory receptor of the PVR gene family, which includes TIGIT, CD96, and CD226, that is expressed on NK cells and T cells. CD112R suppresses immune cell activation through its association with the cell adhesion molecule CD112 (PVRL2), a ligand that it competes for with the activating receptor CD226. CD112 binding to CD112R induces downstream signaling via recruitment of SHP-1, SHP-2, and SHIP-1 phosphatases to the ITIM in the cytoplasmic tail, resulting in dampened effector cell activation. As CD112 is commonly upregulated on cancer cells and is also expressed on tumor infiltrating myeloid cells, it was hypothesized that disruption of the CD112:CD112R interaction would promote anti-tumor immune responses. Phenotypic screening of a panel of antibodies generated against CD112R led to the identification of SRF813, a high affinity, fully human CD112R antibody that blocks the interaction of CD112R with CD112. To explore the therapeutic potential of CD112R blockade, the ability of SRF813 to increase immune cell activation was evaluated in human peripheral blood mononuclear cells. In vitro assays showed that SRF813 treatment enhanced NK cell activation in response to multiple tumor cell lines. This activation was characterized by upregulation of several cell surface markers, including 4-1BB and ICAM-1, in addition to increased cytokine production and degranulation. The activity of SRF813 was highly dependent on the antibody isotype and Fc receptor engagement. Mechanistically, the enhanced immunologic responses observed in NK cells treated with SRF813 could be reversed in the presence of a CD226 blocking antibody, confirming the role of CD112R in regulating CD226 mediated signaling. Blockade of CD112R in mouse syngeneic tumor models using a murine surrogate of SRF813 demonstrated reduced tumor growth with an accompanying increase in TIL activation following treatment. In the CT-26 tumor model, anti-CD112R treatment resulted in complete tumor regression in a subset of treated mice. These mice rapidly rejected tumors upon re-challenge, signifying the development of an immunologic anti-tumor memory response in these animals. Additional depletion studies highlighted that the anti-tumor efficacy was dependent on both CD8 T cells and NK cells. Moreover, the combination of anti-CD112R with PD-1 blockade led to greater inhibition of tumor growth than either treatment alone. Collectively, these preclinical data demonstrated that CD112R is a negative regulator of immune responses and that CD112R blockade can potentiate anti-tumor responses in cancers that express CD112. Citation Format: Marisella Panduro, Roy M. Dornbrook, Kshama A. Doshi, Jing Hua, Jamie Strand, Vito J. Palombella, Pamela M. Holland, Jonathan A. Hill, James F. Mohan. SRF813, a fully human monoclonal antibody targeting the inhibitory receptor CD112R, enhances immune cell activation and demonstrates preclinical in vivo anti-tumor 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 4548.

In situ Vaccine Plus Checkpoint Blockade Induces Memory Humoral Response.

In a syngeneic murine melanoma (MEL) model, we recently reported an in situ vaccination response to combined radiation (RT) and intra-tumoral (IT) injection of anti-GD2 hu14. 18-IL2 immunocytokine (IC). This combined treatment resulted in 71% complete and durable regression of 5-week tumors, a tumor-specific memory T cell response, and augmented response to systemic anti-CTLA-4 antibody checkpoint blockade. While the ability of radiation to diversify anti-tumor T cell response has been reported, we hypothesize that mice rendered disease-free (DF) by a RT-based ISV might also exhibit a heightened B cell response. C57BL/6 mice were engrafted with 2 × 106 GD2+ B78 MEL and treated at a target tumor size of ~200 mm3 with 12 Gy RT, IT-IC on day (D)6-D10, and anti-CTLA-4 on D3, 6, and 9. Serum was collected via facial vein before tumor injection, before treatment, during treatment, after becoming DF, and following rejection of subcutaneous 2 × 106 B78 MEL re-challenge on D90. Flow cytometry demonstrated the presence of tumor-specific IgG in sera from mice rendered DF and rejecting re-challenge with B78 MEL at D90 after starting treatment. Consistent with an adaptive endogenous anti-tumor humoral memory response, these anti-tumor antibodies bound to B78 cells and parental B16 cells (GD2-), but not to the unrelated syngeneic Panc02 or Panc02 GD2+ cell lines. We evaluated the kinetics of this response and observed that tumor-specific IgG was consistently detected by D22 after initiation of treatment, corresponding to a time of rapid tumor regression. The amount of tumor-specific antibody binding to tumor cells (as measured by flow MFI) did not correlate with host animal prognosis. Incubation of B16 MEL cells in DF serum, vs. naïve serum, prior to IV injection, did not delay engraftment of B16 metastases and showed similar overall survival rates. B cell depletion using anti-CD20 or anti-CD19 and anti-B220 did not impact the efficacy of ISV treatment. Thus, treatment with RT + IC + anti-CTLA-4 results in adaptive anti-tumor humoral memory response. This endogenous tumor-specific antibody response does not appear to have therapeutic efficacy but may serve as a biomarker for an anti-tumor T cell response.

Abstract IA15: Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition

Abstract Radiation is commonly used in the definitive management of for head and neck cancer. Yet, many patients still fail treatment and develop resistance, especially non-virally driven tumors. Head and neck tumors are highly enriched in regulatory T cells that dampen the response to radiotherapy by creating an immune-inhibitory microenvironment. A key way by which CD4 T cells convert to FOXP3 positive regulatory T cells (Tregs) is through activation of STAT3. We explored mechanisms of Treg signaling and infiltration and assessed their modulation by RT in murine models of head and neck squamous cell carcinoma (HNSCC). Mechanisms of Treg infiltration were investigated in orthotopic murine HNSCC tumors using flow cytometry, multiplex cytokine arrays and targeted therapeutics. Mice were treated with STAT3 ASO, anti-CTLA-4, anti-CD25, and/or anti-PD-L1 alone and in combination with RT. Tumor growth and survival were assessed. Flow cytometry was used to assess phenotypic and functional changes in intratumoral T-cell populations. Multiplex ELISA was performed for assessment of cytokines. Treatment with anti-CD25 concurrently with RT led to tumor eradication, enhanced T-cell cytotoxicity, induced an antitumor antigen-specific memory response, decreased Tregs, and improved survival in orthotopic HNSCC tumor-bearing mice. Treg depletion induced an influx of CD8 and CD4 T cells when combined with RT. In addition, Treg depletion in combination with RT transformed myeloid populations, decreasing M2 macrophages and myeloid-derived suppressor cells (MDSCs) and increasing M1 macrophages. In contrast, RT alone or when combined with anti-CTLA4 and/or anti-PD-L1 did not lead to durable tumor control. To assess whether STAT3 inhibition can target Tregs we employed the STAT3 anti-sense oligonucleotide, AZD5149. STAT3 inhibition in combination with RT, but not as single agent, led to significant tumor growth delay and resulted in significant downregulation of immunosuppressive cytokines including TGF-β1 and IL10 and upregulation of IL2. Analysis of immune populations showed that STAT3 inhibition when combined with RT significantly decreases intratumoral Tregs and enhanced effector T-cell function. Experiments in nude mice demonstrated complete abolishment of the benefit of STAT3 ASO and RT, demonstrating the dependency on T cells for achieving tumor control. These data reveal a critical role for Tregs in mediating resistance to RT. Targeted depletion of Tregs represents an important mechanism of sensitizing tumors to RT. We propose that STAT3 inhibition is a viable and potent therapeutic target against Tregs. Our data support the design of clinical trials integrating STAT3 ASO in the standard of care for cancer patients receiving RT. Citation Format: Sana Karam. Suppression of intratumoral regulatory T cells restores radiosensitivity in head and neck cancer: Role for Stat3 inhibition [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; 2019 Apr 29-30; Austin, TX. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(12_Suppl_2):Abstract nr IA15.