Melanoma Cancer Models Research Articles

TNP-470 skews DC differentiation to Th1-stimulatory phenotypes and can serve as a novel adjuvant in a cancer vaccine

AbstractFumagillin is an antiangiogenic and antineoplastic fungal natural product, and TNP-470 is one of its most potent analogs. Decades of studies revealed that TNP-470 has potent anticancer activities via destruction of neovasculature. In stark contrast, TNP-470 has been reported to suppress lymphocyte proliferation, thereby limiting its clinical potentials. In an attempt to investigate whether the similar or opposite immunomodulatory effect of TNP-470 could act on myeloid cells, we found that TNP-470 potentiates the immunogenicity of dendritic cells (DCs) toward a phenotype with T helper cell type 1 (Th1)–stimulatory features. Using DC vaccine on a murine melanoma cancer model, the TNP-470–treated DC vaccine could significantly induce tumor-specific immunogenicity and substantially enhance tumor eradication when compared with vehicle-treated DC vaccine in a prophylactic setting. Enhanced tumor-specific immunogenicity and delayed tumor progression were observed in a therapeutic setting upon the TNP-470–treated DC vaccine. Our data showed that TNP-470 potentiates Toll-like receptor signaling, including NF-κB activation, in DCs to transcriptionally activate interleukin-12 production, thus inducing a Th1-immune response. Our current study uncovers a novel immune function of TNP-470 in DCs and redefines its role as a novel class of small molecule immune adjuvant in DC-based cancer vaccine given potentiation of DC immunogenicity is a major roadblock in DC vaccine development. Our study not only provides a novel adjuvant for ex vivo–cultured patient-specific DC vaccines for cancer treatment but also discovers the distinct immunostimulatory function of TNP-470 in DCs of myeloid lineage that differs from its immunosuppressive function in lymphoid cells.

A designer self-assembled supramolecule amplifies macrophage immune responses against aggressive cancer.

Effectively activating macrophages that can ‘eat’ cancer cells is challenging. In particular, cancer cells secrete macrophage colony stimulating factor (MCSF), which polarizes tumour-associated macrophages from an antitumour M1 phenotype to a pro-tumourigenic M2 phenotype. Also, cancer cells can express CD47, an ‘eat me not’ signal that ligates with the signal regulatory protein alpha (SIRPα) receptor on macrophages to prevent phagocytosis. Here, we show that a supramolecular assembly consisting of amphiphiles inhibiting the colony stimulating factor 1 receptor (CSF-1R) and displaying SIRPα-blocking antibodies with a drug-to-antibody ratio of 17,000 can disable both mechanisms. The supramolecule homes onto SIRPα on macrophages, blocking the CD47-SIRPα signalling axis while sustainedly inhibiting CSF-1R. The supramolecule enhances the M2-to-M1 repolarization within the tumour microenvironment, and significantly improves antitumour and antimetastatic efficacies in two aggressive animal models of melanoma and breast cancer, with respect to clinically available small-molecule and biologic inhibitors of CSF-1R signalling. Simultaneously blocking the CD47-SIRPα and MCSF-CSF-1R signalling axes may constitute a promising immunotherapy.

Abstract 3565: T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors

Abstract Adoptive cell therapy (ACT) using patient-derived tumor-specific T cells is a promising approach for cancer treatment especially for the treatment of hematological malignancies. However, therapeutic approaches using ACT in patients with solid tumors, such as hepatocellular carcinoma (HCC), has not shown substantially improved clinical outcomes in particular due to immunosuppressive tumor microenvironments. Therefore, strategies to enhance ACT T cell functionality in vivo and to overcome the immunosuppressive microenvironments in solid tumors are strongly needed. We developed a strategy combining nanomedicine with ACT, based on the chemical conjugation of cytokine-loaded nanoparticles (NPs) as synthetic “backpacks” to the surfaces of live lymphocytes for adoptive therapy. Novel protein nanoparticles were synthesized by crosslinking of single chain IL-12 (scIL-12)- or IL-15 superagonist through disulfide-containing crosslinkers, forming nanogels that when bound to the surface of T cells, release the supporting cytokines in response to antigen receptor signaling-mediated changes in cell surface redox state. This TCR-mediated drug release allowed drug delivery to the T cells to be focused in tumors and tumor-draining lymph nodes. Coupling of cytokine nanogels to T cell membranes induced significant cell activation in terms of interferon-γ production or proliferation in a concentration-dependent manner. Antigen-specific CD8+ T cells “backpacked” with scIL-12 or IL-15SA nanogels were capable of substantial tumor cell killing in a B16F10 melanoma cancer model in vivo, while avoiding lethal systemic toxicities triggered by systemically-administered immunomodulatory cytokines. Ongoing studies are seeking to apply this approach in HCC, and this approach bears the potential for the development of successful immunotherapeutic strategies against this and other solid tumors where effective treatment options are still lacking. Citation Format: Michael Fichter. T cell receptor signaling-responsive single chain IL-12 and IL-15 superagonist nanogel “backpacks” to enhance adoptive cell therapy in solid tumors [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 3565.

Phenylbutyl isoselenocyanate induces reactive oxygen species to inhibit androgen receptor and to initiate p53-mediated apoptosis in LNCaP prostate cancer cells.

Previous studies have established the in vivo bioavailability and efficacious dosages of phenylbutyl isoselenocyanate (ISC-4), a selenium-substituted isothiocyanate, against mouse xenograft models of human melanoma and colorectal cancer. To explore its potential attributes against prostate cancer, we treated human LNCaP prostate cancer cells with ISC-4 and examined their apoptosis responses, and interrogated the signaling mechanisms through pharmacological and siRNA knockdown approaches. Our results show that ISC-4 was more potent at inducing apoptosis than its sulfur analog phenylbutyl isothiocyanate (PBITC) without suppressing protein kinase AKT Ser473 phosphorylation. ISC-4 induced apoptosis in concentration- and time-dependent manners, and the apoptosis execution was attenuated by pre-incubation with a pan caspase inhibitor. ISC-4 decreased the abundance of androgen receptor (AR) and its best known target prostate specific antigen (PSA) without decreasing their steady state mRNA. ISC-4 upregulated the abundance of p53 protein and its Ser15 -phosphorylative activation, and that of DNA double strand break marker Ser139 -p-H2A.X coincident with apoptotic exposure. Similar to the rapid induction of reactive oxygen species (ROS) by isothiocyanates, ISC-4 increased dihydroethidium-detectable signals in LNCaP cells. Pre-incubation with ROS scavenger N-acetyl-l-cysteine preserved AR and PSA abundance, markedly reduced ISC-4-induced apoptosis and attenuated p53 Ser phosphorylation, p21Cip1, and p-H2A.X. Furthermore, siRNA knockdown of p53 did not suppress ROS production, but decreased ISC-4-induced apoptosis. Knocking down p53-targets PUMA and Bax exerted greater protective effect on ISC-4-induced apoptosis than depleting p21Cip1. In summary, ISC-4 inhibited LNCaP cell growth and survival with ROS-mediated suppression of AR axis signaling and induction of p53-PUMA-Bax mitochondrial apoptosis.

Literature Highlights: Impactful Papers Published Elsewhere

Engineering safer immunolocks and keys (by Wapcaplet via Wikimedia Commons)IL2 therapy is used with caution due to adverse events at IL2 concentrations that are clinically useful. A modified IL2 receptor, which only binds to a modified form of IL2, was inserted into T cells destined for adoptive antitumor therapy. The modified IL2 was designed to only bind the modified receptor, and, as administration of this modified IL2 activates only the transferred T cells, the IL2 provides not only therapeutic benefit but also is not associated with identifiable toxicity.Sockolosky JT, …, Garcia KC. Science 2018 Mar 2;359:1037–42.Fibroblast engulfing debris (From Lakins et al.)Tumor fibroblasts were found to engulf and process tumor cell debris, then cross-present it to T cells. However, instead of activating T cells, this presentation is suppressive and reduces T-cell viability. The T-cell death results from increased expression of FasL and PD-L2 on the fibroblasts that, in turn, activates Fas and PD-1 pathways in the T cells. Blockade of these interactions restored antigen-specific T-cell killing of tumor cells.Lakins MA, …, Shields JD. Nat Commun 2018 Mar 5. DOI: 10.1038/s41467-018-03347-0.Tumor-associated macrophage clusters (from Perry et al., Fig. 2)Immunotherapeutics are hampered by suppressive elements in the tumor microenvironment, including M2 (suppressive) macrophages. Perry et al., using an authochthonous mouse model (mutant Braf/Pten) of melanoma, and Hoves et al., primarily with the MC38 model, show that activating CD40 while inhibiting CSF-1R signaling changes transcriptional modules, reduces M2s, increases inflammatory macrophages, and improves tumor control. The effects are strong, but not permanent, and both CD8+ T cells and macrophages are necessary for tumor eradication.Perry CJ, …, Kaech SM. J Exp Med 2018 Feb 7;215:877–93.Hoves S, …, Ries CH. J Exp Med 2018 Feb 7;215:859–76.Fc affinity affects checkpoint blockade effectiveness (from Arce Vargas et al.)Optimization of the interaction between human FcγRs and anti– CTLA-4 in humanized mice expressing human FcγRs efficiently depletes Tregs and controls tumor growth. However, removal of Tregs is only important in tumors that are highly inflamed and infiltrated by CD8+ T cells. Patients carrying FcγR polymorphisms with higher binding affinities for IgGs, and that have higher neoantigen burdens, show greater responses to CTLA-4 blockade.Arce Vargas F, …, Quezada SA. Cancer Cell 2018 Mar 22. DOI: 10.1016/j.ccell.2018.02.010.4-1BB–activated mitochondria (from Teijeira et al., Fig. 6)Removing checks on T-cell activation is often insufficient for reactivating CD8+ T cells. Inducing 4-1BB signaling while blocking PD-1 (Menk et al.) or during adoptive T-cell therapy (Teijeira et al.) induces robust antitumor immunity in mouse models of melanoma or colon cancer. 4-1BB appears to regulate mitochondrial mass and function, reprogramming the metabolic state of antitumor T cells to counter exhaustion.Menk AV, …, Delgoffe GM. J Exp Med 2018 Mar 6. DOI: 10.1084/jem.20171068.Teijeira A, …, Melero I. Cancer Immunol Res 2018 Apr. DOI: 10.1158/2326-6066.CIR-17-0767.Core of hydrogel (by Edgar181 via Wikimedia Commons)Surgery removes primary tumors, but the wound healing process produces an immunosuppressive environment that may be conducive to regrowth of residual tumor cells and distal metastasis. Placing a hydrogel laced with agonists for innate immune responses into post-surgical wounds from which tumors had been extracted resulted in activation of innate and specific antitumor responses and a decrease in metastasis.Park CG, …, Goldberg MS. Sci Transl Med 2018 Mar 21;10:eaar1916.

Novel Combination of Silver Nanoparticles and Carbon Nanotubes for Plasmonic Photo Thermal Therapy in Melanoma Cancer Model.

Purpose: Plasmonic photo thermal therapy (PPTT) is a therapeutic method in which the photon energy is rapidly transformed into heat via a series of radiative and non-radiative phenomena to ablate cancer. Plasmonic NPs, such as silver NPs (Ag NPs), have considerable properties in optical absorbance. Furthermore, good thermal conductivity and cell penetration ability of carbon nanotubes (CNTs) could improve the efficacy of Ag NPs for PPTT. Decoration of the multi-walled carbon nanotubes (MWCNTs) with silver has been developed to enhance thermal conductivity of the MWCNT particles.Methods: The Ag NPs were decorated on the CNTs and the ability of these particles (CNT/Ag NPs) in reduction of melanoma tumor size after PTT was evaluated experimentally. For comparison, the PTT of silver nanorods (Ag NRs) and CNTs were investigated. The melanoma tumor was induced by injection of B16/F10 cell line to the inbred mice. Different NPs were injected into the tumors and then irradiated via laser diode (λ=670 nm, P=500 mW, and I= 3.5 W/cm2) at scheduled time.Results: Monitoring of tumor sizes showed that integration of CNTs with silver could enhance the optical absorption of CNTs and improve tumor destruction in PPTT technique.Conclusion: The CNT/Ag NPs could act as a potent agent in PPTT method in curing solid tumors.

RANKL blockade improves efficacy of PD1-PD-L1 blockade or dual PD1-PD-L1 and CTLA4 blockade in mouse models of cancer

ABSTRACT Receptor activator of NF-κB ligand (RANKL) and its receptor RANK, are members of the tumor necrosis factor and receptor superfamilies, respectively. Antibodies targeting RANKL have recently been evaluated in combination with anti-CTLA4 in case reports of human melanoma and mouse models of cancer. However, the efficacy of anti-RANKL in combination with antibodies targeting other immune checkpoint receptors such as PD1 has not been reported. In this study, we demonstrated that blockade of RANKL improves anti-metastatic activity of antibodies targeting PD1/PD-L1 and improves subcutaneous growth suppression in mouse models of melanoma, prostate and colon cancer. Suppression of experimental lung metastasis following combination anti-RANKL with anti-PD1 requires NK cells and IFN-γ, whereas subcutaneous tumor growth suppression with this combination therapy is attenuated in the absence of T cells and IFN-γ. Furthermore, addition of anti-RANKL to anti-PD1 and anti-CTLA4 resulted in superior anti-tumor responses, irrespective of the ability of anti-CTLA4 isotype to engage activating FcR, and concurrent or delayed RANKL blockade was most effective. Early-during-treatment assessment reveals this triple combination therapy compared to dual anti-PD1 and anti-CTLA4 combination therapy further increased the proportion of tumor-infiltrating CD4+ and CD8+ T cells that can produce both IFN-γ and TNF. Finally, RANKL expression appears to identify tumor-specific CD8+ T cells expressing higher levels of PD1 which can be modulated by anti-PD1. These data set the scene for clinical evaluation of denosumab use in patients receiving contemporary immune checkpoint blockade.

Arthrospira Platensis - Potential in Dermatology and Beyond.

The search for natural products with benefits for health in general and of potential for treating human disease has gained wider interest world-wide. Here, we analyse current data on the microalga Arthrospira platensis (AP), that has been used in nutrition since ancient times in Fare East and African communities, for medical purposes with a focus on dermatology. Extracts of AP have been investigated in vitro and in vivo. The alga is rich in proteins, lipopolysaccharides and gamma-linolenic acid. AP extracts, phycocyanin compounds and polysaccharide calcium spirulan (Ca-SP) have been evaluated in various models. It could be demonstrated, that AP has significant antioxidant activity, prevents viruses from entry into target cells and inhibits the colonisation of wounds by multi-resistant bacteria. Furthermore, anti-cancer activity was documented in models of oral cancer, melanoma, and UV-induced non-melanoma skin cancer.

Blood exosomes regulate the tissue distribution of grapefruit-derived nanovector via CD36 and IGFR1 pathways.

Tumor-specific delivery of therapeutics is challenging. One of the major hurdles for successfully delivering targeted agents by nanovectors is the filtering role of the liver in rapidly sequestering nanovectors from the circulation. Exosomes, a type of endogenous nanoparticle, circulate continuously in the peripheral blood and play a role in intercellular communication. The aim of this study was to determine whether the level of endogenous exosomes has an effect on nanovector delivery efficiency of targeted agents.Methods: Exosomes were isolated from peripheral blood and intravenously (I.V.) injected into tumor-bearing mice. Subsequently, 1,1-dioctadecyl-3,3,3'3'-tetramethylindotricarbocyanine-iodide (DiR) fluorescent dye-labeled nanoparticles, including grapefruit nanovectors (GNV) and standard liposomes, were I.V. injected in the mice. The efficiency of redirecting GNVs from liver to other organs of injected mice was further analyzed with in vivo imaging. The concentration of chemo drugs delivered by GNV was measured by HPLC and the anti-lung metastasis therapeutic effects of chemo drugs delivered by GNVs in mouse breast cancer and melanoma cancer models were evaluated.Results: We show that tail vein-injected exosomes isolated from mouse peripheral blood were predominately taken up by liver Kupffer cells. Injection of peripheral blood-derived exosomes before I.V. injection of grapefruit-derived nanovector (GNV) decreased the deposition of GNV in the liver and redirected the GNV to the lung and to the tumor in breast and melanoma tumor-bearing mouse models. Enhanced therapeutic efficiency of doxorubicin (Dox) or paclitaxel (PTX) carried by GNVs for lung metastases was demonstrated when there was an I.V. injection of exosomes before therapeutic treatment. Furthermore, we found that CD36 and IGFR1 receptor-mediated pathways played a critical role in the exosome-mediated inhibitory effect of GNV entry into liver macrophages.Conclusions: Collectively, our findings provide a foundation for using autologous exosomes to enhance therapeutic vector targeted delivery to the lung.

Pharmacokinetics and efficacy of doxorubicin-loaded plant virus nanoparticles in preclinical models of cancer.

To compare the pharmacokinetics and efficacy of doxorubicin containing plant virus nanoparticles (PVNs) with PEGylated liposomal doxorubicin (PLD) and small molecule doxorubicin in two mouse models of cancer. Studies were performed in A375 melanoma and intraperitoneal SKOV3ip1 ovarian cancer xenografts. The PVNs were administered in lower and more frequent doses in the ovarian model. The PVNs were more efficacious than PLD and small molecule doxorubicin in the ovarian cancer model, but not in the melanoma cancer model. The pharmacokinetics profiles of the PVNs showed fast plasma clearance, but more efficient tumor delivery as compared with other carrier-mediated agents. PVNs administered at lower repeated doses provide both pharmacologic and efficacy advantages compared with PLD.

1662P - Synergistic antitumor effects of OT-101 (trabedersen), a transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) and chemotherapy in preclinical tumor models

Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combination strategies. Methods: OT-101 was administered as single agent (1-64 mg/kg, qdx3/wk or qdx21) and in combination with Gemcitabine (GEM, 15 mg/kg, qdx2/wk), Dacarbazine (DTIC, 1-10 mg/kg, qdx4/wk) or Paclitaxel (PTX, 10 mg/kg, qdx5) to nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) SC glioblastoma (U87) or (iv) SC ovarian (SKOV-3) tumors. Mice were monitored for adverse effects, body weight loss, tumor size and survival outcome. Lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-BrdUrd and CD31 antibodies to determine tumor cell proliferation and vascularization, respectively. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p < 0.0001) versus untreated control in the PAC model. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. DTIC alone), glioblastoma (U87, p = 0.001 vs. PTX) and ovarian (SKOV-3, p < 0.05 vs. PTX) cancer models when combined with either DTIC (C8161) or PTX (U87 and SKOV-3). No synergy was observed with GEM (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at HED of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day. Conclusions: The preclinical data laid the groundwork for establishing combination therapies in the clinic. Of interest is the preferential synergy between OT-101 and PTX or DTIC, but not with GEM. Legal entity responsible for the study: Autotelic Inc Funding: None Disclosure: All authors have declared no conflicts of interest.

Abstract 2800: Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models

Abstract Background: Overexpression of TGF-β2 has been implicated in the malignant progression of tumors by inducing immunosuppression, proliferation, angiogenesis and metastasis. Clinical failures of TGF-β inhibitors in targeting the tumor promoting arm of TGF-β signaling is attributed to multiple isoforms and receptor functions. OT-101 (Trabedersen) is a phosphorothioate ASO designed to specifically target human TGF-β2 mRNA with superior clinical activity in patients with TGF-β2-overexpressing tumors. Herein, we report the synergizing effect of OT-101 with chemotherapy in multiple human tumor xenograft models for further exploration of clinical combinations. Methods: The in vivo efficacy studies of intraperitoneal (IP, 16 or 50 mg/kg) or subcutanous (SC, 1-64 mg/kg) repeated administration (qdx3/wk or qdx21) of OT-101 as single agent and in combination with Gemcitabine (15 mg/kg, qdx2/wk, IP), Dacarbazine (1-10 mg/kg, qdx4/wk, IP) or Paclitaxel (10 mg/kg, qdx5, IV) were evaluated in nude mice (10/subgroup) bearing either (i) orthotopic human L3.6pl pancreatic cancer (PAC), (ii) human metastatic C8161 melanoma, (iii) subcutaneous glioblastoma (U87-MG) or (iv) subcutaneous ovarian (SK-OV-3) tumors. Mice were monitored thrice a week for adverse effects, body weight loss, tumor size and survival outcome. The incidence of lymph node and liver surface and micro-metastases as well as size and weight of the pancreatic tumors were determined. Tumor sections were stained with anti-5-bromo-2'-deoxy-uridine antibody to determine tumor cell proliferation and with anti-CD31/PECAM-1 antibody to determine vascularization. Results: OT-101 significantly reduced tumor growth (p = 0.0084), lymph node metastasis (p = 0.023), and tumor angiogenesis (p &amp;lt;0.0001) in the orthotopic PAC model. Mean tumor vessel density was significantly reduced (p &amp;lt;0.0001) in all groups in comparison to untreated control. OT-101 demonstrated synergy in tumor growth inhibition and increased survival in human malignant melanoma (C8161, p = 0.038, vs. Dacarbazine alone), glioblastoma (U87-MG, p = 0.001 vs. Paclitaxel) and ovarian (SKOV-3, p &amp;lt;0.05 vs. Paclitaxel) cancer models when combined with either Dacarbazine (C8161) or Paclitaxel (U87-MG and SK-OV-3). No syngergy was observed with Gemcitabine (PAC). The combination regimen tested was effective and tolerable. Significant antitumor activity was achieved at human dose equivalent of 80 mg/m2/day which is well below the optimized clinical dose used for IV infusion of patients at 140 mg/m2/day. Conclusions: The preclinical data lay the groundwork for further exploration of combination therapy in the clinic as well as demonstrating that TGF-β2 is a druggable target. Of interest is the preferential synergy between OT-101 and Paclitaxel or Dacarbazine, but not with Gemcitabine. Citation Format: Osmond D'Cruz, Cynthia Lee, Vuong Trieu, Larn Hwang. Transforming growth factor-beta 2 (TGF-β2) antisense oligonucleotide (ASO) OT-101 synergizes with chemotherapy in preclinical tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2800. doi:10.1158/1538-7445.AM2017-2800

Abstract 5932: Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model

Abstract Prior work by us and others has demonstrated that the extracellular pH (pHe) of solid tumors is acidic, due to a combination of increased fermentative metabolism, resulting in lactic acid production and poor perfusion. This acidity promotes tumor progression and metastasis formation. Recently we have shown in melanoma and pancreatic cancer models that acidity inhibits antitumor immunity by preventing T-cell activation. Reversal of acidity with buffer therapy (200mM NaHCO3) synergized with checkpoint blockade (anti-CTLA4 and anti-PD1) and adoptive T-cell therapy has resulted in cures. While this is promising, concerns are high regarding the ingestion of such large amounts of sodium bicarbonate, which makes clinical translation a challenge. Hence, we hypothesize that alternative pharmacological interventions can neutralize the pHe of tumors and remove this immunosuppressive effect. To study this we first investigated a series of agents for their ability to inhibit metastasis in the PC3 prostate cancer model, which is exquisitely sensitive to inhibition with buffer therapy. In this study, male SCID mice were grouped in to 6 groups (n=5) and treated with tap water, 200 mM bicarbonate ad lib, 30 mg/kg daily (q.d.) intraperitoneal (i.p.) Acetazolamide (CA inhibitor), 1.2 mg/kg q.d.i.p. Furosemide (diuretic), 10 mg/kg q.d.i.p. DH348 (selective CAIX inhibitor) or 2.1 mg/kg q.d.i.p. FX-11 (LDHA inhibitor). Mice were intravenously injected with 5*10^6 PC3M-luc cells and ventral bioluminescence images were acquired at time 0 and weekly thereafter. Our results showed that FX-11, acetazolamide, DH348 and bicarbonate were able to effectively (p&amp;lt;0.004) suppress metastasis formation in this system, whereas furosemide was not. Based on these results, a subsequent study investigated the combination of bicarbonate, FX-11 or DH348 with immune checkpoint blockade in a Panc02 mouse model of pancreatic cancer. Animals were inoculated orthotopically with Panc02 cells and randomized into 8 groups (n=10). Once tumors reached 0.3 cc in size, mice were treated with either 15 mg/kg anti-PD-1 antibody or normal rat IgG controls twice weekly alone or in combination with bicarbonate; FX-11 or DH348 and tumor response was evaluated using US 3D-Mode imaging and volume analysis (FUJIFILM VisualSonics) and histopathology. Anti-PD-1 antibody was ineffective as a monotherapy in this system. Both bicarbonate and DH348 neutralized tumor acidity and reduced tumor growth as monotherapies. Notably, despite the fact that FX-11 was shown to inhibit LDH-A in pancreatic cancer models, it did not affect pH and had no effect on tumor growth as monotherapy. However, FX-11 significantly reduced tumor growth (p &amp;lt;0.01) and metastasis formation in combination with anti-PD-1 antibody, suggesting that inhibition of LDH-A might be effective in combination with checkpoint blockade. Citation Format: Arig A. Ibrahim Hashim, Dominique Abrahams, Liping Xu, Barbra Centeno, Enakshi Sunassee, Rasha Abddelgader, Ludwig Dubois, Philippe Lambin, Robert A. Gatenby, Robert J. Gillies. Targeting tumor acidity with the LDHA inhibitor (FX11) and CAIX inhibitor (DH348) overcomes resistance to PD-1 blockade and inhibits metastasis in a pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5932. doi:10.1158/1538-7445.AM2017-5932

Abstract 5168: KO-947, a potent ERK inhibitor with robust preclinical single agent activity in MAPK pathway dysregulated tumors

Abstract The RAS/RAF/MEK pathway is a major driver of malignant progression, particularly in cancers arising from mutations in RAS, BRAF and NF1. Although both BRAF and MEK inhibitors have been approved for treatment of melanoma, their clinical activity is commonly limited by acquired resistance due to reactivation of the pathway downstream of their targets. Since the ERK1/2 kinases are the final node in the MAPK signaling pathway, they are not subject to the feedback reactivation mechanisms that can undermine RAF or MEK blockade, offering the possibility of clinical benefit in settings where earlier drugs are ineffective. Here we describe the characterization of KO-947, a potent and selective inhibitor of ERK1/2 kinases, in biochemical, cellular and in vivo antitumor activity assays. KO-947 was profiled in vitro in biochemical activity assays, competition binding assays, and a probe-based competition binding assay in cell lysates, and subsequently screened in a large panel of human tumor cell lines focused on MAPK-dysregulated tumor types. In vivo studies were carried out in well-characterized xenograft models of BRAF-, KRAS- and NRAS-mutant disease and subsequently extended to a large panel of early-passage patient-derived xenograft (PDX) models representative of a wide range of tumor types and molecular profiles. Biochemical assays reveal that KO-947 is a 10nM inhibitor of ERK with at least 50-fold selectivity against a panel of 450 kinases. KO-947 blocks ERK signaling and proliferation of tumor cells exhibiting dysregulation of MAPK pathway signaling, including mutations in BRAF, NRAS or KRAS, at low nanomolar concentrations. KO-947 is differentiated from other published ERK inhibitors by an extended residence time and high potency in cell engagement that translate into prolonged pathway inhibition in vitro and in vivo. In cell-line derived xenograft studies, the drug profoundly suppresses ERK signaling for up to five days after a single dose and induces regressions in RAS- and RAF-mutant melanoma, NSCLC and pancreatic cancer models on administration schedules ranging from daily to weekly. Intermittent dosing enables comparable antitumor activity at reduced dose-intensity. PDX screening confirms and extends these findings to include RAS and BRAF mutant colorectal, gastric and cervical carcinoma models, and robust activity is also seen on both weekly and Q2D schedules in tumor models lacking BRAF/RAS mutations but with other dysregulation of the MAPK pathway. Thus, the favorable ADME properties of KO-947 enable the achievement of optimal antitumor activity with intermittent dosing, which may provide an opportunity to maximize the therapeutic window with flexible administration routes and schedules. These results demonstrate the potential clinical utility of KO-947 in MAPK pathway dysregulated tumors. Citation Format: Francis Burrows, Linda Kessler, Jeffrey Chen, Xin Gao, Rasmus Hansen, Shuangwei Li, Carol Thach, Levan Darjania, Yvonne Yao, Yi Wang, Ata Zarieh, Ke Yu, Tao Wu, Jingchuan Zhang, Dana Hu-Lowe, Liansheng Li, Pingda Ren, Yi Liu. KO-947, a potent ERK inhibitor with robust preclinical single agent activity in MAPK pathway dysregulated tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5168. doi:10.1158/1538-7445.AM2017-5168

Abstract 4610: Towards understanding the cellular uptake patterns of nano-particles among different immune cell lines

Abstract Today with the privilege of being able to synthesize extremely small particles in the nano-range and make use of nanoscience and nanotechnology, we can reach superior properties than the bulk scale. Nanoparticles have their own physicochemical properties that make them promising in the field of cancer immunotherapy in form of drug delivery, diagnostic and theranostic modalities. Manipulation of the immune response by therapeutic intervention is becoming of great interest owing to the significant role of immunity in the general health and disease control. APCs like Dendritic cells and macrophages are important targets for the particulate delivery system due to their ability to trigger cascade of events on both levels cellular and humoral immune response specially DCs. Successful targeting of DCs and macrophages will have a great impact on T-cell activation and priming. The design of successful particulate system that can elicit preferential targeting towards specific immune cell in the tumor microenvironment is becoming crucial. This might represent a novel avenue for enhancing the antitumor immunity. Liposomes are made of phospholipid bilayer with a composite nature. Their properties are highly linked to their physicochemical characteristics such as size, surface charge and composition. These physicochemical properties can trigger certain immune response.The hypothesis here whether passive targeting via particulate system can offer a platform for preferential targeting for critical immune cells residing in spleen and tumor. Moreover, test whether there will be differences in uptake according to certain preference towards surface charge in specific time frame. In the current study, three sets of fluorescently labelled nano-liposomes were engineered as a model for different surface charges, the cationic DOTAP NP, anionic DOPG NP and near neutral DOPC NP with mean diameter of ~ 200 nm. Physical stability of the NPs was evaluated by monitoring the changes in size and zeta potential. B16 melanoma cancer model was induced subcutaneously in C57BL/6 black mice, divided into four groups each of five mice. CD11c Dendritic Cells (DCs), CD11b macrophages, CD90.2 T-cells and CD49b Natural Killer (NK) cells were isolated from the tumors and spleens of each group. The three sets of NPs were tested against the isolated cell lines. The cellular uptake (internalization) was assessed by normalizing the fluorescence of the cells against their protein concentration, then all samples were acquired to flow cytometry, and shifts in fluorescence histograms on horizontal axis were monitored against PE channel on the vertical axis. Results reveal the presence of preferential internalization of specific surface charge over others in some cell lines in different time frames. For the first time differences in the internalization pattern are reported in the same immune cell line isolated from two different contexts tumor and spleen. Note: This abstract was not presented at the meeting. Citation Format: Noha Ismail, Ashish Kulkarni, Siva Kumar, Vineeth krishna, Shiladitya Sengupta. Towards understanding the cellular uptake patterns of nano-particles among different immune cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4610. doi:10.1158/1538-7445.AM2017-4610

Dual Targeting Nanoparticle Stimulates the Immune System To Inhibit Tumor Growth.

We describe the development of a nanoparticle platform that overcomes the immunosuppressive tumor microenvironment. These nanoparticles are coated with two different antibodies that simultaneously block the inhibitory checkpoint PD-L1 signal and stimulate T cells via the 4-1BB co-stimulatory pathway. These "immunoswitch" particles significantly delay tumor growth and extend survival in multiple in vivo models of murine melanoma and colon cancer in comparison to the use of soluble antibodies or nanoparticles separately conjugated with the inhibitory and stimulating antibodies. Immunoswitch particles enhance effector-target cell conjugation and bypass the requirement for a priori knowledge of tumor antigens. The use of the immunoswitch nanoparticles resulted in an increased density, specificity, and in vivo functionality of tumor-infiltrating CD8+ T cells. Changes in the T cell receptor repertoire against a single tumor antigen indicate immunoswitch particles expand an effective set of T cell clones. Our data show the potential of a signal-switching approach to cancer immunotherapy that simultaneously targets two stages of the cancer immunity cycle resulting in robust antitumor activity.

Intratumoral delivery of inactivated modified vaccinia virus Ankara (iMVA) induces systemic antitumor immunity via STING and Batf3-dependent dendritic cells.

Advanced cancers remain a therapeutic challenge despite recent progress in targeted therapy and immunotherapy. Novel approaches are needed to alter the tumor immunosuppressive microenvironment and to facilitate the recognition of tumor antigens that leads to antitumor immunity. Poxviruses, such as modified vaccinia virus Ankara (MVA), have potential as immunotherapeutic agents. We show that infection of conventional dendritic cells (DCs) with heat- or ultraviolet-inactivated MVA leads to higher levels of interferon induction than MVA alone through the cGAS (cyclic guanosine monophosphate-adenosine monophosphate synthase)-STING cytosolic DNA-sensing pathway. Intratumoral injection of inactivated MVA (iMVA) was effective and generated adaptive antitumor immunity in murine melanoma and colon cancer models. iMVA-induced antitumor therapy was less effective in STING- or Batf3-deficient mice than in wild-type mice, indicating that both cytosolic DNA sensing and Batf3-dependent CD103+/CD8α+ DCs are essential for iMVA immunotherapy. The combination of intratumoral delivery of iMVA and systemic delivery of immune checkpoint blockade generated synergistic antitumor effects in bilateral tumor implantation models as well as in a unilateral large established tumor model. Our results suggest that inactivated vaccinia virus could be used as an immunotherapeutic agent for human cancers.

Abstract A42: Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen

Abstract While cancer immunotherapies like checkpoint inhibitors have resulted in unprecedented clinical success, they only benefit a subset of patients. To improve therapeutic outcomes for greater numbers of patients, one strategy is to rationally combine different immunotherapy modalities. We recently demonstrated that attaching albumin-binding lipophilic tails to peptide antigens or molecular adjuvants (creating amphiphile vaccines) results in enhanced T-cell responses. Additionally, we observed significant tumor regression upon combining tumor-antigen targeting antibodies with extended half-life IL-2 (exPK-IL-2) in mouse models of melanoma and prostate cancer. In the present work, we combined both approaches to treat a spontaneous model of lung adenocarcinoma expressing carcinoembryonic antigen (CEA), an oncofetal protein expressed in some human lung cancers. KrasLSL-G12D/+;;p53fl/fl mice were crossed with transgenic mice expressing human-CEA to generate a CEA-tolerant background. Lung tumors were induced by infection with a lentivirus expressing Cre recombinase and human CEA. Due to the extended latency of tumor initiation in this model, we also generated a CEA-expressing cell line from these mice to test the efficacy of different combination immunotherapy regimens. We discovered that weekly treatments combining a CEA-targeting amphiphile-vaccine, exPK-IL-2, and an anti-CEA antibody with checkpoint inhibitors (anti-PD-1 and -CTLA4) resulted in sustained tumor regression in 50% of mice bearing established tumors. We are currently testing this combination immunotherapy on autochthonous lung tumors. Our results suggest that breaking tolerance to a tumor-associated self-antigen (CEA) and combining immunotherapies to recruit both innate and adaptive immune effectors can have a potent therapeutic effect in intractable tumors like lung cancer. Citation Format: Kavya Rakhra, Eric F. Zhu, Wuhbet Abraham, Kelly D. Moynihan, Naveen Mehta, Karl D. Wittrup, Darrell J. Irvine. Combination immunotherapy of an autochthonous murine lung cancer model expressing human CEA as a tumor-associated self-antigen. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A42.

Validation of syngeneic mouse models of melanoma and non-small cell lung cancer for investigating the anticancer effects of the soy-derived peptide Lunasin

Background: Lunasin is a naturally occurring peptide present in soybean that has both chemopreventive and therapeutic activities that can prevent cellular transformation and inhibit the growth of several human cancer types. Recent studies indicate that Lunasin has several distinct potential modes of action including suppressing integrin signaling and epigenetic effects driven by modulation of histone acetylation. In addition to direct effects on cancer cells, Lunasin also has effects on innate immunity that may contribute to its ability to inhibit tumor growth in vivo. Methods: Standard assays for cell proliferation and colony formation were used to assess Lunasin’s in vitro activity against murine Lewis lung carcinoma (LLC) and B16-F0 melanoma cells. Lunasin’s in vivo activity was assessed by comparing the growth of tumors initiated by subcutaneous implantation of LLC or B16-F0 cells in Lunasin-treated and untreated C57BL/6 mice. Results: Lunasin was found to inhibit growth of murine LLC cells and murine B16-F0 melanoma cells in vitro and in wild-type C57BL/6 mice. The effects of Lunasin in these two mouse models were very similar to those previously observed in studies of human non-small cell lung cancer and melanoma cell lines. Conclusions: We have now validated two established syngeneic mouse models as being responsive to Lunasin treatment. The validation of these two in vivo syngeneic models will allow detailed studies on the combined therapeutic and immune effects of Lunasin in a fully immunocompetent mouse model.

Validation of syngeneic mouse models of melanoma and non-small cell lung cancer for investigating the anticancer effects of the soy-derived peptide Lunasin

Background: Lunasin is a naturally occurring peptide present in soybean that has both chemopreventive and therapeutic activities that can prevent cellular transformation and inhibit the growth of several human cancer types. Recent studies indicate that Lunasin has several distinct potential modes of action including suppressing integrin signaling and epigenetic effects driven by modulation of histone acetylation. In addition to direct effects on cancer cells, Lunasin also has effects on innate immunity that may contribute to its ability to inhibit tumor growth in vivo. Methods : Standard assays for cell proliferation and colony formation were used to assess Lunasin’s in vitro activity against murine Lewis lung carcinoma (LLC) and B16-F0 melanoma cells. Lunasin’s in vivo activity was assessed by comparing the growth of tumors initiated by subcutaneous implantation of LLC or B16-F0 cells in Lunasin-treated and untreated C57BL/6 mice. Results : Lunasin was found to inhibit growth of murine LLC cells and murine B16-F0 melanoma cells in vitro and in wild-type C57BL/6 mice. The effects of Lunasin in these two mouse models were very similar to those previously observed in studies of human non-small cell lung cancer and melanoma cell lines. Conclusions : We have now validated two established syngeneic mouse models as being responsive to Lunasin treatment. The validation of these two in vivo syngeneic models will allow detailed studies on the combined therapeutic and immune effects of Lunasin in a fully immunocompetent mouse model.