Immune Responses In Preclinical Models Research Articles

Abstract LB119: The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy

Abstract Immunotherapy, encompassing immune checkpoint blockades (ICBs) and immune stimulators, has become a widespread approach in cancer treatment. However, the effectiveness of these strategies relies significantly on the characteristics of the current tumor microenvironment (TME). This reliance underscores the urgent need for a potent immune modulator capable of inducing a favorable TME, particularly in non-immunogenic cold tumors. Here, we demonstrate that HM16390, a long acting IL-2 analog, has the potential to modify the immunogenicity of the TME by activating, expanding, and recruiting cytotoxic effector cells in cold tumor. In the murine B16F10 melanoma model, following the early upregulated proliferative potential, the proportions of CD8+ T as well as NK cells increased in both blood and tumor. While NK cell frequency initially increased and then recovered, CD8 T cells exhibited a dose-dependent upregulation in the late phase in both blood and tumor. Given the IL-2Rα (CD25) affinity of HM16390, there was an increase in Treg proportions in blood; however, intriguingly, Treg frequency decreased in the tumor. This pattern emphasizes the crucial role of HM16390 in balancing the hyper-activation of peripheral effector cells while simultaneously inducing a profoundly immune-favorable TME. Effector functions were also upregulated in both blood and tumor. Early upregulation of IFN-γ and TNF occurred in both blood and tumor, but the upregulation of granzyme B displayed a sustained expression pattern in the tumor. These immune-modulated features correlated with the exposures of HM16390, particularly noting a negative correlation between Treg down-regulation and Its exposure in blood. The immune modulations of HM16390 were validated in terms of efficacy in the Pancreatic Ductal Adenocarcinoma (PDAC) model. HM16390 was administered subcutaneously to panc02 tumor syngeneic mice via once-a-week administration for 6 weeks, with or without PD-1 combination treatment. HM16390 treatment group was well-tolerated and resulted in a dose-dependent anti-tumor effect. Notably, all mice treated with the highest dose exhibited a complete response. Furthermore, in panc02 syngeneic mice, known for a poor response to ICBs, the combination treatment of HM16390 with mAnti-PD-1 demonstrated synergistic anti-tumor effects. In conclusion, HM16390 demonstrates improved expansion and functions of effector tumor-infiltrating lymphocytes (TILs), correlating with exposure, and exhibits a safe Treg modulation pattern. These modulations culminate in a significant anti-tumor effect and synergies with PD-1 blockade in the B16F10 and PDAC model. Given that both B16F10 melanoma and Panc02 PDAC are recognized as poor immunogenic murine models with low TIL frequency, HM16390 shows promise in modifying the immunogenicity of the TME, thereby activating proper immune responses in preclinical models. Citation Format: Seongju Jeong, Yunjae Kim, Jooyun Byun, Jinyoung Kim, Jaehyuk Choi, Sungmin Bae, Daejin Kim, In Young Choi. The immune-modulation of HM16390, firing up the poor tumor microenvironment to induce a potent anti-tumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB119.

Abstract 170: Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth

Abstract Background: Prostate cancer (PCa) is traditionally considered an immunologically 'cold’ indication, as evidenced in part by significant infiltration of immunosuppressive tumor associated macrophages (TAMs) and few activated effector CD8+ T cells. Attempts to (re)invigorate an anti-prostate cancer immune response with immune checkpoint blockade (ICB) such as anti-CTLA-4 and anti-PD-1 have been largely ineffective clinically, despite promising results in other cancers. This is likely due to a multitude of immunosuppressive cell types and secreted factors within the tumor microenvironment (TME) that diminish T cell activation and trafficking. Reducing the immunosuppressive burden intratumorally by targeting TAMs is a clinically attractive approach and offers opportunity to sensitize PCa to immune checkpoint blockade. While PI3K signaling is ubiquitous in most cell types, specific catalytic isoforms and regulatory elements are differentially expressed depending on cell type, with PI3Kγ being significantly enriched in myeloid cells and therefore offering a unique therapeutic opportunity for specific targeting. Methods: Utilizing novel syngeneic prostate cancer tumors, PI3Kγ deletion and antagonism using Eganelisib (a small molecule inhibitor for PI3Kγ), we tested our hypothesis that targeting PI3Kγ signaling in PCa TAMs would lead to tumor growth inhibition and anti-tumor immune responses. Results: We show here that PCa tumor cells do not express the PI3Kγ isoform and, therapeutic inhibition of PI3Kγ with Eganelisib reduces the TAM suppressive phenotype. We also show PI3Kγ signaling is essential to monocyte anti-apoptotic signaling, and inhibition of PI3Kγ can enhance anti-tumor immune responses in preclinical models of PCa. Specifically, inhibition of PI3Kγ alone is sufficient to enhance anti-tumor immune responses in the less aggressive B6CaP PCa model, but insufficient in the more aggressive RM-1 PCa model. However, treatment of RM-1 tumors with Eganelisib produces improved tumor control when combined with anti-PD-1 therapy, relative to Eganelisib or anti-PD1 as a monotherapy. Conclusions: Altogether, these data highlight the integral role of PI3Kγ signaling in TAMs and the potential for therapeutic targeting with Eganelisib to sensitize PCa to ICB. Citation Format: Kenneth M. Adusei, Thomas R. Nirschl, Alex J. Lee, Fan Shen, Xiaoxu Wang, Monali Praharaj, Jade Alvarez, Iyana Gross, Kara A. Lombardo, Tamara L. Lotan, Judith A. Varner, Jelani C. Zarif. Targeting of macrophage PI3Kγin prostate cancer using Eganelisib (IPI-549) reprograms immune-suppressive infiltrating macrophages to enhance anti-tumor immune responses and promote immunologically mediated tumor growth [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 170.

Abstract 6650: Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields)

Abstract Introduction: Tumor Treating Fields (TTFields) are low intensity electric fields that disrupt cellular processes critical for cancer cell viability. TTFields have previously been shown to induce immunogenic cell death, promote dendritic cell maturation, and elicit a systemic anti-cancer immune response in preclinical models. The benefit of applying TTFields together with immune checkpoint inhibitors (ICI) was demonstrated in a phase 3 study in non-small cell lung carcinoma. In the current research, we investigated possible effects of TTFields on regulation of macrophage phenotype. Methods: Bone marrow-derived macrophages (BMDMs) were generated from the femurs and tibias of 5-8-week-old Balb\C mice. LPS+IFN-γ or IL-4 were applied to the BMDMs to induce polarization (M1 and M2, respectively). The BMDMs were then treated with TTFields (150 kHz, 24 h), and then examined by flow cytometry for surface expression of the macrophage biomarker F4/80 and the activation markers CD80, major histocompatibility complex class II (MHC II), inducible nitric oxide synthase (iNOS), CD206, and ARG-1. Cell supernatants were collected and analyzed by multiplexed secretion assay for secretion levels of CXCL1 (KC), IL-18, IL-23, IL-12p70, IL6, TNF-α, IL-12p40, TGF-β1, CCL22 (MDC), IL-10, IL-6, G-CSF, CCL17 (TARC), and IL-1β. Cell lysates were examined using a RhoA activation kit and Western blot to determine GEF-H1, c-Jun, and p65 phosphorylation levels. Results: BMDMs exposed to TTFields demonstrated elevated expression of the pro-inflammatory M1 markers CD80+ and MHC IIhigh, and decreased expression of the M2 markers CD206 and ARG-1. A pro-inflammatory secretion pattern was detected, with increased levels of CXCL1, IL-18, IL-23, IL-12p70, TNF-α, IL-12p40, CCL22, G-CSF, CCL17 and IL-1β. The treated cells further demonstrated RhoA activation and GEF-H1 phosphorylation, with subsequent activation of the transcription factors c-Jun and p65. Conclusions: This study reveals a novel immunomodulatory role for TTFields, promoting in vitro pro-inflammatory polarization of macrophage. Citation Format: Tal Kan, Yiftah Barsheshet, Boris Brant, Tharwat Haj Khalil, Tali Voloshin, Alexandra Volodin, Lilach Koren, Bella Koltun, Anat Klein-Goldberg, Efrat Zemer-Tov, Rom Paz, Adi Haber, Moshe Giladi, Uri Weinberg, Yoram Palti. Macrophage pro-inflammatory phenotype skewing by the application of tumor treating fields (TTFields) [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 6650.

Combination of BET Inhibition with Radiation Induces Anti-Tumor Immune Response in Pre-Clinical Models of ER-Positive Breast Cancer

Despite advances in multidisciplinary care, the risk of late recurrence in Estrogen Receptor-positive (ER+) breast cancer (BC) patients following standard-of-care treatment ranges from 20-52% at 20 years. While immune checkpoint inhibitors (CPI), have shown enormous potential in inducing long term remission in some cases, patients with ER+ BC show poor response to these agents. The objective of this study is to develop effective radiation therapy (RT)-based combination regimens that induce anti-tumor immunity against immunologically cold ER+ breast tumors. We have previously shown that pharmacological inhibition of bromodomain and extraterminal domain (BET) family of epigenetic regulator proteins overcomes both endocrine therapy resistance and radiation resistance in pre-clinical models of ER+ BC by reversing pathological reprogramming of transcriptional and DNA repair pathways, respectively. In this study, we explore the potential of RT+BET inhibition combination to elicit anti-tumor immunity against ER+ BC. A unique ER+ BC model, MXT+, was used to generate tumors in female, BDF1 mice. PD-L1 expression following RT or RT+OTX015 (a BET inhibitor) was assessed by Western blot analysis. Comet assay was used to assess the degree of unrepaired DNA damage following treatment with RT alone, OTX015 alone and RT+OTX015. Multiplex IHC was used to assess changes in tumor microenvironment after various treatments. Tumor growth characteristics were established following treatment with vehicle, RT alone (8 Gy x 3), OTX015 alone (100 mg/kg x 3 days) and RT+OTX015. Using murine ER+ BC model, MXT+, we show that RT is a potent inducer of PD-L1 expression. Combination of RT+OTX015 completely ablated RT-mediated PD-L1 induction at the transcription level. OTX015 also accentuated RT-induced DNA damage through inhibition of NHEJ pathway, resulting in increased micronuclei formation (which activate cGAS/STING pathway, a critical bridge between innate and adaptive immunity). Combination of OTX015 with RT also caused a statistically significant (on unpaired t test) increase tumor infiltrating lymphocytes. In in vivo therapeutic studies, combination of OTX015 with RT (8 Gy x 3) resulted in extraordinary synergy and caused complete inhibition of long-term tumor growth. Treatment with RT alone (8 Gy x 3) or OTX015 alone (on 3 consecutive days) only caused modest inhibition of tumor growth. ANOVA with Dunnett's test was used to adjust for multiple comparisons and showed statistically significant difference in tumor growth between OTX015+RT arm and OTX alone as well as OTX015+RT arm and RT alone. Patients with ER+ BC face high risk of late recurrence after definitive treatment and show poor response to immune CPIs. We show that combination of BET inhibition with RT induces anti-tumor immunity and completely prevents tumor growth. Clinical validation of this regimen is warranted to prevent late recurrences in ER+ BC patients.

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

Turning up the heat: Oncolytic virus MEM-288 induces anti-tumoral immune response in pre-clinical models of high grade serous ovarian cancer (190)

CLDN18.2 BiTE Engages Effector and Regulatory T Cells for Antitumor Immune Response in Preclinical Models of Pancreatic Cancer

BiTE (bispecific T-cell engager) immune therapy has demonstrated clinical activity in multiple tumor indications, but its influence in the tumor microenvironment remains unclear. CLDN18.2 is overexpressed in solid tumors including gastric cancer (GC) and pancreatic ductal adenocarcinoma (PDAC), both of which are characterized by the presence of immunosuppressive cells, including regulatory T cells (Tregs) and few effector T cells (Teffs). We evaluated the activity of AMG 910, a CLDN18.2-targeted half-life extended (HLE) BiTE molecule, in GC and PDAC preclinical models and cocultured Tregs and Teffs in the presence of CLDN18.2-HLE-BiTE. AMG 910 induced potent, specific cytotoxicity in GC and PDAC cell lines. In GSU and SNU-620 GC xenograft models, AMG 910 engaged human CD3+ T cells with tumor cells, resulting in significant antitumor activity. AMG 910 monotherapy, in combination with a programmed death-1 (PD-1) inhibitor, suppressed tumor growth and enhanced survival in an orthotopic Panc4.14 PDAC model. Moreover, Treg infusion enhanced the antitumor efficacy of AMG 910 in the Panc4.14 model. In syngeneic KPC models of PDAC, treatment with a mouse surrogate CLDN18.2-HLE-BiTE (muCLDN18.2-HLE-BiTE) or the combination with an anti-PD-1 antibody significantly inhibited tumor growth. Tregs isolated from mice bearing KPC tumors that were treated with muCLDN18.2-HLE-BiTE showed decreased T cell suppressive activity and enhanced Teff cytotoxic activity, associated with increased production of type I cytokines and expression of Teff gene signatures. Our data suggest that BiTE molecule treatment converts Treg function from immunosuppressive to immune enhancing, leading to antitumor activity in immunologically "cold" tumors.

Targeting the Siglec-sialic acid axis promotes antitumor immune responses in preclinical models of glioblastoma.

Glioblastoma (GBM) is the most aggressive form of primary brain tumor, for which effective therapies are urgently needed. Cancer cells are capable of evading clearance by phagocytes such as microglia- and monocyte-derived cells through engaging tolerogenic programs. Here, we found that high expression of sialic acid-binding immunoglobulin-like lectin 9 (Siglec-9) correlates with reduced survival in patients with GBM. Using microglia- and monocyte-derived cell-specific knockouts of Siglec-E, the murine functional homolog of Siglec-9, together with single-cell RNA sequencing, we demonstrated that Siglec-E inhibits phagocytosis by these cells, thereby promoting immune evasion. Loss of Siglec-E on monocyte-derived cells further enhanced antigen cross-presentation and production of pro-inflammatory cytokines, which resulted in more efficient T cell priming. This bridging of innate and adaptive responses delayed tumor growth and resulted in prolonged survival in murine models of GBM. Furthermore, we showed the combinatorial activity of Siglec-E blockade and other immunotherapies demonstrating the potential for targeting Siglec-9 as a treatment for patients with GBM.

A Chemically Defined TLR3 Agonist with Anticancer Activity

ABSTRACT Toll-like receptor 3 (TLR3) agonists such as polyinosinic:polycytidylic acid (poly(I:C)) have immunostimulatory effects that can be taken advantage of to induce anticancer immune responses in preclinical models. In addition, poly(I:C) has been introduced into clinical trials to demonstrate its efficacy as an adjuvant and to enhance the immunogenicity of locally injected tumors, thus reverting resistance to PD-L1 blockade in melanoma patients. Here, we report the pharmacokinetic, pharmacodynamic, mechanistic and toxicological profile of a novel TLR3 agonist, TL-532, a chemically synthesized double-stranded RNA that is composed by blocks of poly(I:C) and poly(A:U) (polyadenylic – polyuridylic acid). In preclinical models, we show that TL-532 is bioavailable after parenteral injection, has an acceptable toxicological profile, and stimulates the production of multiple chemokines and interleukins that constitute pharmacodynamic markers of its immunostimulatory action. When given at a high dose, TL-532 monotherapy reduced the growth of bladder cancers growing on mice. In addition, in immunodeficient mice lacking formylpeptide receptor-1 (FPR1), TL-532 was able to restore the response of orthotopic subcutaneous fibrosarcoma to immunogenic chemotherapy. Altogether, these findings may encourage further development of TL-532 as an immunotherapeutic anticancer agent.

Inhibition of SUMOylation modulates the immunosuppressive microenvironment of pancreatic cancer

Abstract Objective Pancreatic cancer (PC) is a highly aggressive disease that is poorly responsive to available immunotherapy approaches, such as checkpoint inhibitors. Post-translational protein modification (PTM) by small ubiquitin-like modifier (SUMO) is upregulated in cancer, and high expression of SUMOylation-related factors correlates with poor survival in PC. TAK-981 is a novel inhibitor of SUMOylation that has demonstrated induction of anti-tumor immune responses in preclinical models. Our hypothesis is that TAK-981 will decrease SUMOylated proteins and subsequently modulate the tumor microenvironment to increase anti-tumor immunity. Methods In order to recapitulate the microenvironment of human PC, a 3-dimensional organoid cell line derived from a genetically-engineered “KPC” mouse was used. Organoids were injected orthotopically into the head of the pancreas via laparotomy. Once tumors reached 5–7 mm in diameter on ultrasound imaging, mice were randomized to one of two treatment groups: Control/Vehicle versus TAK-981 (15 mg/kg) delivered daily via intraperitoneal injection. Tumors were harvested on day 14 for gene expression analysis by quantitative real time PCR (RT-qPCR). Results Tumor growth (Fig. 1) in the TAK-981 15 mg/KG daily group (mean + SD volume = 207 + 109 mm3) was significantly inhibited compared to the vehicle group (595 + 141 mm3, p < 0.01). Immunohistochemical staining for CD 31 (Fig. 2) and Caspase 3 indicated a reduction of angiogenesis and increased apoptosis of tumor cells, consistent with the well-established roles of SUMOylation in angiogenesis and tumorigenesis. Gene expression analysis of tumor lysates showed a 26-fold increase in expression of Interferon beta (p<0.05), and an almost 2-fold increase in expression of genes related to dendritic cell activation, including CD80 (p<0.05) and CD86 (p=0.14) in the TAK-981 group, suggesting a modulation of the immune microenvironment. Conclusion Our results suggest that the inhibition of SUMOylation with TAK-981 is associated with improved local tumor control and changes in the immunosuppressive tumor microenvironment. We expect that TAK-981 will improve PC responsiveness to immunotherapy, such as with checkpoint inhibitors.

Abstract P013: Germinal center hypoxia in tumor-draining lymph nodes negatively regulates humoral immune responses and affects the activation of tumor-infiltrating T cells

Abstract Efforts to understand intrinsic and acquired resistance to anti-tumor immunotherapies have largely focused on characterizing factors within the primary tumor. However, lymph nodes are specialized hubs of immune response development, and an improved understanding of the microenvironmental conditions impacting immune cell activation in tumor-draining lymph nodes (TDLNs) may yield novel approaches for enhancing responses to anti-tumor immunotherapies. Using mouse models of breast cancer, we identified hypoxic regions amongst the B cell germinal center reactions of TDLNs. Injection of lethally irradiated tumor cells was sufficient to induce GC hypoxia, and hypoxia in TDLNs associated with the frequency of antibody- secreting cells. In vitro, hypoxia impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel- Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in TDLNs, and reduced class-switched and tumor-specific antibodies in the circulation, indicating that the hypoxic response negatively-regulates tumor-induced humoral immune responses. pVHL deletion in class-switched B cells also reduced the expression of activation markers amongst tumor-infiltrating T cells and increased the presence of M2-like macrophages within the primary tumor. We detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing the first line of evidence that hypoxia develops within human GCs and validating our observations of hypoxia in TDLNs of pre-clinical mammary tumor models. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in pre-clinical models, with consequent impacts on the phenotype tumor-infiltrating T cells and macrophages. Citation Format: Natalie Firmino, Kevin Bennewith. Germinal center hypoxia in tumor-draining lymph nodes negatively regulates humoral immune responses and affects the activation of tumor-infiltrating T cells [abstract]. In: Abstracts: AACR Virtual Special Conference: Tumor Immunology and Immunotherapy; 2021 Oct 5-6. Philadelphia (PA): AACR; Cancer Immunol Res 2022;10(1 Suppl):Abstract nr P013.

Heterologous Prime-Boost Vaccination with a Peptide-Based Vaccine and Viral Vector Reshapes Dendritic Cell, CD4+ and CD8+ T Cell Phenotypes to Improve the Antitumor Therapeutic Effect.

Simple SummaryDeveloping new therapeutic cancer vaccines is of paramount importance to counteract tumor escape observed after conventional therapies in certain types of cancer. We have previously shown that the combination of two different vaccine platforms, targeting tumor-specific antigens, resulted in potent immune responses in preclinical models. Here, we show that the heterologous prime-boost combination with a protein vaccine and a viral vector vesicular stomatitis virus immunologically reshapes the immune-excluded TC-1 tumor model as well as the inflamed MC-38 tumor model, leading to beneficial therapeutic efficacy. Furthermore, the treatment with a multi-epitope vaccine allowed us to appreciate the various repartition among three antigen-specific cytotoxic T-cell responses combined with the viral boost. The combination leads to improved efficacy in all animals and highlights the importance of combining tumor epitopes. Our vaccine strategy could be further extended to prophylactic cancer vaccines and beyond, for infectious diseases.Heterologous prime-boost settings with a protein vaccine and the viral vector vesicular stomatitis virus, both expressing tumor-associated antigens (KISIMA-TAA and VSV-GP-TAA), have been previously shown to generate potent antitumor immunity. In the cold TC-1 model (HPV antigen) and the immune-infiltrate MC-38 model (Adpgk, Reps1 and Rpl18 neo-antigens), we further investigated pivotal immune cells that educate CD8+ T cells. Heterologous prime-boost vaccination induced a superior antitumor response characterized by the increase in number and functionality of antigen-specific CD8+ T cells, recruitment of cross-presenting dendritic cells, and polarization of CD4+ T cells towards an antitumor Th1 phenotype within the tumor and tumor-draining lymph nodes, turning the cold TC-1 tumor into a hot, inflamed tumor. In the inflamed MC-38 tumor model, treatment combination markedly prolonged the overall survival of mice. Treatment with multi-epitope vaccines also induced high frequencies of multiple antigen specificities in the periphery and in the tumor. Prime-boost treatment reduced tumor-infiltrating regulatory CD4+ T cells whilst increasing cross-presenting dendritic cells in tumor-draining lymph nodes. In conclusion, heterologous prime-boost vaccination possesses the ability to induce a potent anti-tumor response in both immune-excluded and immune-infiltrated mouse tumor models. Additionally, this study highlights the design of a multi-epitope vaccine for cancer immunotherapy.

Targeting cholesterol homeostasis in hematopoietic malignancies

AbstractCholesterol is a vital lipid for cellular functions. It is necessary for membrane biogenesis, cell proliferation, and differentiation. In addition to maintaining cell integrity and permeability, increasing evidence indicates a strict link between cholesterol homeostasis, inflammation, and hematological tumors. This makes cholesterol homeostasis an optimal therapeutic target for hematopoietic malignancies. Manipulating cholesterol homeostasis by either interfering with its synthesis or activating the reverse cholesterol transport via the engagement of liver X receptors affects the integrity of tumor cells both in vitro and in vivo. Cholesterol homeostasis has also been manipulated to restore antitumor immune responses in preclinical models. These observations have prompted clinical trials involving acute myeloid leukemia to test the combination of chemotherapy with drugs interfering with cholesterol synthesis (ie, statins). We review the role of cholesterol homeostasis in hematopoietic malignancies as well as in cells of the tumor microenvironment and discuss the potential use of lipid modulators for therapeutic purposes.

Inhibition of SUMOylation enhances responses to irreversible electroporation in pancreatic cancer

Abstract Objective The revolutionary results of immunotherapy have not yet translated to pancreatic cancer (PC). Irreversible electroporation (IRE) is a non-thermal ablative therapy that can generate tumor-specific immune responses, yet not sufficient to eradicate distant metastatic disease. Post-translational protein modification (PTM) by small ubiquitin-like modifier (SUMO) is involved in carcinogenesis and PD-L1 mediated immunosuppression. TAK-981 is a novel inhibitor of SUMOylation that has demonstrated induction anti-tumor immune responses in preclinical models. Our hypothesis is that TAK-981 will augment the effects of IRE in an immunocompetent orthotopic mouse model of PC. Methods The PC cell line (KPC4580P) was derived from an autochthonous tumor arising in a genetically engineered mouse model (KPC). The tumors were implanted orthotopically into the pancreas via laparotomy. Once tumors reached 5-7 mm in diameter, mice were randomized to one of four treatment groups: IRE alone, TAK-981 alone, IRE + TAK-981, or no treatment. IRE versus sham laparotomy (150 x 90 microsec pulses at 1500 V/cm) was performed once through second laparotomy. TAK-981 (7.5mg/kg) versus vehicle was delivered via subcutaneous injection twice weekly x four doses, starting on the day of IRE. Tumors were harvested on day 14 for flow cytometric analysis. Results Ultrasound was performed on days 8, 14 and 20 after initiation of treatment. Tumor growth in the IRE + TAK-981 group was significantly inhibited compared to sham-treated tumors (Fig. 1, *p=0.0002), and this effect persisted to day 20, even after treatment with TAK-981 was stopped on day 11 (p < 0.01). Flow cytometry revealed a greater than 2-fold increase in CD8+ T-cells (Fig. 2a, *p <0.05) and 4-fold increase in IFN-gamma+ CD8+ T-cells (Fig. 2b, *p=0.01) in tumors treated with the combination relative to sham-treated tumors. Conclusion Our results suggest that the combination of IRE with TAK-981 is associated with better local tumor control and a significant increase of CD8+ T-cells and IFN-gamma+ CD8+ T-cells. We expect that this combination might contribute systemic immune responses that would prevent or even eradicate distant metastasis. Given that immunosuppression mediated by PD-L1 is regulated by ubiquitination and is seen in 50% of PC tumors, targeting PTM with TAK-981 might also render PC responsive to immunotherapy with checkpoint inhibitors.

Optimized polyepitope neoantigen DNA vaccines elicit neoantigen-specific immune responses in preclinical models and in clinical translation

BackgroundPreclinical studies and early clinical trials have shown that targeting cancer neoantigens is a promising approach towards the development of personalized cancer immunotherapies. DNA vaccines can be rapidly and efficiently manufactured and can integrate multiple neoantigens simultaneously. We therefore sought to optimize the design of polyepitope DNA vaccines and test optimized polyepitope neoantigen DNA vaccines in preclinical models and in clinical translation.MethodsWe developed and optimized a DNA vaccine platform to target multiple neoantigens. The polyepitope DNA vaccine platform was first optimized using model antigens in vitro and in vivo. We then identified neoantigens in preclinical breast cancer models through genome sequencing and in silico neoantigen prediction pipelines. Optimized polyepitope neoantigen DNA vaccines specific for the murine breast tumor E0771 and 4T1 were designed and their immunogenicity was tested in vivo. We also tested an optimized polyepitope neoantigen DNA vaccine in a patient with metastatic pancreatic neuroendocrine tumor.ResultsOur data support an optimized polyepitope neoantigen DNA vaccine design encoding long (≥20-mer) epitopes with a mutant form of ubiquitin (Ubmut) fused to the N-terminus for antigen processing and presentation. Optimized polyepitope neoantigen DNA vaccines were immunogenic and generated robust neoantigen-specific immune responses in mice. The magnitude of immune responses generated by optimized polyepitope neoantigen DNA vaccines was similar to that of synthetic long peptide vaccines specific for the same neoantigens. When combined with immune checkpoint blockade therapy, optimized polyepitope neoantigen DNA vaccines were capable of inducing antitumor immunity in preclinical models. Immune monitoring data suggest that optimized polyepitope neoantigen DNA vaccines are capable of inducing neoantigen-specific T cell responses in a patient with metastatic pancreatic neuroendocrine tumor.ConclusionsWe have developed and optimized a novel polyepitope neoantigen DNA vaccine platform that can target multiple neoantigens and induce antitumor immune responses in preclinical models and neoantigen-specific responses in clinical translation.

MRNA-encoded, constitutively active STINGV155M is a potent genetic adjuvant of antigen-specific CD8+ T cell response

mRNA vaccines induce potent immune responses in preclinical models and clinical studies. Adjuvants are used to stimulate specific components of the immune system to increase immunogenicity of vaccines. We utilized a constitutively active mutation (V155M) of the stimulator of interferon (IFN) genes (STING), which had been described in a patient with STING-associated vasculopathy with onset in infancy (SAVI), to act as a genetic adjuvant for use with our lipid nanoparticle (LNP)-encapsulated mRNA vaccines. mRNA-encoded constitutively active STINGV155M was most effective at maximizing CD8+ T cell responses at an antigen/adjuvant mass ratio of 5:1. STINGV155M appears to enhance development of antigen-specific T cells by activating type I IFN responses via the nuclear factor κB (NF-κB) and IFN-stimulated response element (ISRE) pathways. mRNA-encoded STINGV155M increased the efficacy of mRNA vaccines encoding the E6 and E7 oncoproteins of human papillomavirus (HPV), leading to reduced HPV+ TC-1 tumor growth and prolonged survival in vaccinated mice. This proof-of-concept study demonstrated the utility of an mRNA-encoded genetic adjuvant.

Germinal center hypoxia in tumor-draining lymph nodes negatively regulates tumor-induced humoral immune responses in mouse models of breast cancer

ABSTRACT Hypoxia develops in germinal centers (GCs) induced by model antigens; however, it is unknown whether tumor-reactive GCs are also hypoxic. We identified GC hypoxia in lymph nodes (LNs) draining murine mammary tumors and lethally irradiated tumor cells, and found that hypoxia is associated with the levels of antibody-secreting B cells. Hypoxic culture conditions impaired the proliferation of activated B cells, and inhibited class-switching to IgG1 and IgA immunoglobulin isotypes in vitro. To assess the role of the hypoxic response in tumor-reactive GCs in vivo, we deleted von Hippel-Lindau factor (VHL) in class-switched B cells and found decreased GC B cells in tumor-draining LNs, reduced class-switched and tumor-specific antibodies in the circulation, and modified phenotypes of tumor-infiltrating T cells and macrophages. We also detected the hypoxia marker carbonic anhydrase IX in the GCs of LNs from breast cancer patients, providing evidence that GC hypoxia develops in humans. We conclude that GC hypoxia develops in TDLNs, and that the hypoxic response negatively regulates tumor-induced humoral immune responses in preclinical models.

Abstract 4246: SENTI-101, a novel genetically modified allogeneic cell product expressing IL12 and IL21, elicits a tumor-localized, robust, and multimodal immune response in preclinical models of solid tumors

Abstract While immunotherapies based on single recombinant cytokines such as IL12 and IL21 have shown great promise in preclinical models of solid tumors, clinical translation has proven challenging due to limited mechanisms of action, narrow therapeutic windows upon systemic administration, and short half-lives resulting in poor pharmacokinetics and distribution. Thus, there is a need for tumor-localized cytokine therapies capable of driving sustained efficacy with a wide therapeutic window. SENTI-101 is a cell-based immunotherapy comprising allogeneic bone marrow-derived mesenchymal stromal cells (BM-MSCs) genetically modified to express IL12 and IL21. Consistent with prior studies, we demonstrated that SENTI-101 innately homes to disseminated tumors in the peritoneal cavity and induces durable anti-tumor responses and immune memory in various preclinical models of peritoneal tumors. In this study, we investigated the mechanisms of action of SENTI-101. Our results demonstrate that the IL12 and IL21 combination elicits pleiotropic and complementary effects that drive a multi-modal immune response across various steps of the cancer immunity cycle. Treatment of preclinical murine models of peritoneal tumors (e.g., CT26 and B16F10) with SENTI-101 significantly increased the local production of IFNg by more than 40-fold (p<0.02). Concurrently, mice treated with SENTI-101 had significantly increased levels of cytokines and chemokines such as CXCL9 (p<0.02), which have previously been associated with better prognosis and response to immunotherapy in multiple cancer types. We used flow cytometry and multiplexed IHC to characterize the immune landscape in response to SENTI-101. The number of antigen-presenting cells (F4/80negCD11c+MHC-II+CD103+) more than doubled in peritoneal lymph nodes 72 h after treatment with SENTI-101 (p=0.016). We also observed an increase in pSTAT1 positivity in the myeloid compartment, indicating a favorable immune phenotype. This change was accompanied by an increase in T-cell infiltrates into tumors (p=0.0003) that were in close proximity with B-cells and that were organized in tertiary lymphoid structures, which are known to correlate with improved prognosis in cancer patients. T-cell activation markers (CD38, CD25, IFNg, GranzymeB) also increased by more than 6 times (p=0.015) in the tumor microenvironment (TME) and peritoneal fluid after treatment with SENTI-101. In accordance with increased T-cell infiltration and activation in the TME, SENTI-101 showed a synergistic anti-tumor effect when combined with checkpoint inhibitor anti-PD1. Overall, our preclinical studies show that SENTI-101 modulates the tumor immune landscape via multiple complementary modes of action, resulting in long-term anti-tumor immunity. Furthermore, this work demonstrates the therapeutic potential of tumor-localized cell therapies armed with gene circuits expressing combinatorial immune effectors to trigger a multi-factorial anti-tumor response. Citation Format: Alba Gonzalez, Frances D. Liu, Archana Nagaraja, Alyssa Mullenix, Russell M. Gordley, Daniel O. Frimannsson, Anissa Benabbas, Chen-Ting Lee, Tiffany A. Truong, Allison Quach, Mengxi Tian, Rowena Martinez, Rishi Savur, Alyssa Perry-McNamara, Don-Hong Wang, Ori Maller, Dharini Iyer, Ashita Magal, Sravani Mangalampalli, Christina J. Huynh, Carmina C. Blanco, Jack T. Lin, Brian S. Garrison, Philip Lee, Timothy K. Lu, Gary Lee. SENTI-101, a novel genetically modified allogeneic cell product expressing IL12 and IL21, elicits a tumor-localized, robust, and multimodal immune response in preclinical models of solid tumors [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 4246.

A combination of TLR-4 agonist and saponin adjuvants increases antibody diversity and protective efficacy of a recombinant West Nile Virus antigen

Members of the Flaviviridae family are the leading causes of mosquito-borne viral disease worldwide. While dengue virus is the most prevalent, the recent Zika virus outbreak in the Americas triggered a WHO public health emergency, and yellow fever and West Nile viruses (WNV) continue to cause regional epidemics. Given the sporadic nature of flaviviral epidemics both temporally and geographically, there is an urgent need for vaccines that can rapidly provide effective immunity. Protection from flaviviral infection is correlated with antibodies to the viral envelope (E) protein, which encodes receptor binding and fusion functions. TLR agonist adjuvants represent a promising tool to enhance the protective capacity of flavivirus vaccines through dose and dosage reduction and broadening of antiviral antibody responses. This study investigates the ability to improve the immunogenicity and protective capacity of a promising clinical-stage WNV recombinant E-protein vaccine (WN-80E) using a novel combination adjuvant, which contains a potent TLR-4 agonist and the saponin QS21 in a liposomal formulation (SLA-LSQ). Here, we show that, in combination with WN-80E, optimized SLA-LSQ is capable of inducing long-lasting immune responses in preclinical models that provide sterilizing protection from WNV challenge, reducing viral titers following WNV challenge to undetectable levels in Syrian hamsters. We have investigated potential mechanisms of action by examining the antibody repertoire generated post-immunization. SLA-LSQ induced a more diverse antibody response to WNV recombinant E-protein antigen than less protective adjuvants. Collectively, these studies identify an adjuvant formulation that enhances the protective capacity of recombinant flavivirus vaccines.

Adenoviral vector type 26 encoding Zika virus (ZIKV) M-Env antigen induces humoral and cellular immune responses and protects mice and nonhuman primates against ZIKV challenge.

In 2015, there was a large outbreak of Zika virus (ZIKV) in Brazil. Despite its relatively mild impact on healthy adults, ZIKV infection during pregnancy has been associated with severe birth defects. Currently, there is no ZIKV vaccine available, but several vaccine candidates based on the ZIKV membrane (M) and envelope (Env) structural proteins showed promising results in preclinical and clinical studies. Here, the immunogenicity and protective efficacy of a non-replicating adenoviral vector type 26 (Ad26) that encodes the ZIKV M-Env antigens (Ad26.ZIKV.M-Env) was evaluated in mice and non-human primates (NHP). Ad26.ZIKV.M-Env induced strong and durable cellular and humoral immune responses in preclinical models. Humoral responses were characterized by Env-binding and ZIKV neutralizing antibody responses while cellular responses were characterized by ZIKV reactive CD4+ and CD8+ T cells. Importantly, a single immunization with a very low dose of 4x107 vp of Ad26.ZIKV.M-Env protected mice from ZIKV challenge. In NHP, a single immunization with a typical human dose of 1x1011 vp of Ad26.ZIKV.M-Env also induced Env-binding and ZIKV neutralizing antibodies and Env and M specific cellular immune responses that associated with complete protection against viremia from ZIKV challenge as measured in plasma and other body fluids. Together these data provide the rationale to progress the Ad26.ZIKV.M-Env candidate vaccine to clinical testing.

Abstract 3764: Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of cancer

Abstract Background: RXC004, a potent and selective porcupine (PORCN) inhibitor, is being investigated in a safety and tolerability study in cancer patients with solid tumours (CT 2017-000720-98). In addition to the tumour targeting role of RXC004 and other Wnt pathway inhibitors, we present pre-clinical data which suggests further potential for RXC004 in modulating the immune system of the tumour microenvironment. Materials and Methods: To evaluate the potential of a novel porcupine inhibitor, RXC004, as an immunomodulatory anti-cancer agent, sub cutaneous B16F10 melanoma (C57BL/6 mice) and CT26 colorectal (BALB/c mice) murine tumour models were utilised. Mice in both models were treated with RXC004 alone or in combination with mouse anti-PD-1 antibody. Flow cytometry analysis was utilised to measure key immune cell populations in the tumour microenvironment. To probe the underlying mechanism of immune modulation in these models and to provide a link to the emerging clinical data suggesting a role for Wnt pathway activation in immune escape, human monocytic cells were isolated from PBMCs and human dendritic cells were derived in vitro. The Wnt pathway was induced in derived Dendritic cells and expression of IDO was measured. Results: In the murine CT26 model, RXC004 treatment reduced tumour size when dosed in combination with anti-PD-1 antibody, causing regression and cures in some animals. Furthermore, flow cytometry showed RXC004 in combination with anti-PD-1 antibody increased the proportion of CD8+ cytotoxic T cells as well as decreasing FoxP3+ regulatory T cells when compared to the monotherapy anti-PD-1 arm. In a syngeneic murine melanoma B16F10 model, RXC004 monotherapy at a dose of 5mg/kg QD orally significantly inhibited tumour growth, as did RXC004 combined with anti-PD-1. RXC004 had no effect on the proliferation of B16F10 cells in vitro, suggesting this was not caused by the compound directly affecting B16 cell proliferation. Flow cytometry analysis of the B16 tumours showed significant immune modulatory effects in the tumour microenvironment. In addition to mouse model data, Wnt pathway activation in human dendritic cells was shown to increase IDO expression. Conclusion: Taken together, data from murine syngeneic mouse models corroborate literature data suggesting that inhibiting the Wnt pathway may promote the immune response against human cancers. Citation Format: Inder Bhamra, Richard Armer, Matilda Bingham, Catherine Eagle, Alicia Edmenson Cook, Caroline Phillips, Simon Woodcock. Porcupine inhibitor RXC004 enhances immune response in pre-clinical models of cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3764.