Potent Antitumor Activity In Mice Research Articles

Augmenting TCR signal strength and ICOS costimulation results in metabolically fit and therapeutically potent human CAR Th17 cells

IL-17-producing antigen-specific human Tcells elicit potent antitumor activity in mice. Yet, refinement of this approach is needed to position it for clinical use. While activation signal strength regulates IL-17 production by CD4+ Tcells, the degree to which Tcell antigen receptor (TCR) and costimulation signal strength influences Th17 immunity remains unknown. We discovered that decreasing TCR/costimulation signal strength by incremental reduction of αCD3/costimulation beads progressively altered Th17 phenotype. Moreover, Th17 cells stimulated with αCD3/inducible costimulator (ICOS) beads produced more IL-17A, IFNγ, IL-2, and IL-22 than those stimulated with αCD3/CD28 beads. Compared with Th17 cells stimulated with the standard, strong signal strength (three beads per Tcell), Th17 cells propagated with 30-fold fewer αCD3/ICOS beads were less reliant on glucose and favored the central carbon pathway for bioenergetics, marked by abundant intracellular phosphoenolpyruvate (PEP). Importantly, Th17 cells stimulated with weak αCD3/ICOS beads and redirected with a chimeric antigen receptor that recognizes mesothelin were more effective at clearing human mesothelioma. Less effective CAR Th17 cells generated with high αCD3/ICOS beads were rescued by overexpressing phosphoenolpyruvate carboxykinase 1 (PCK1), a PEP regulator. Thus, Th17 therapy can be improved by using fewer activation beads during manufacturing, a finding that is cost effective and directly translatable to patients.

Anti-tumor effects and cellular mechanisms of Pistacia atlantica methanolic extract against Ehrlich solid tumor in mice

Objective: To assess the anti-tumor effects of Pistacia atlantica methanolic extract (PAME) compared with cyclophosphamide against Ehrlich solid tumors in mice.Methods: Swiss albino mice (n=40) were divided into five groups: normal control mice, mice with Ehrlich solid tumors treated with normal saline, mice with Ehrlich solid tumors treated with cyclophosphamide intraperitoneally once a day for 14 d, or 50 mg/kg or 100 mg/kg PAME orally once a day for 14 d. Tumor growth inhibition, body weight, tumor markers, liver and kidney enzymes, oxidative stress markers, antioxidant enzymes, tumor necrosis factor-alpha level (TNF-α), and apoptosis-regulatory gene expression were evaluated.Results: Treatment of mice bearing Ehrlich solid tumors with PAME at 50 and 100 mg/kg orally significantly decreased tumor volume, body weight, tumor markers, liver and kidney enzymes, oxidative stress markers and TNF-α level in comparison with mice with Ehrlich solid tumors receiving normal saline. whereas PAME at 50 and 100 mg/kg/day significantly elevated the level of antioxidant enzymes (P<0.05).Conclusions: Pistacia atlantica methanolic extract has potent antitumor activity in mice. Therefore, the extract might be considered as an alternative anticancer agent against tumors, however, additional studies especially in the clinical setting are required to confirm this finding.

Isolation of TCR genes with tumor-killing activity from tumor-infiltrating and circulating lymphocytes in a tumor rejection cynomolgus macaque model

To develop effective adoptive cell transfer therapy using T cell receptor (TCR)-engineered T cells, it is critical to isolate tumor-reactive TCRs that have potent anti-tumor activity. In humans, tumor-infiltrating lymphocytes (TILs) have been reported to contain CD8+PD-1+ T cells that express tumor-reactive TCRs. Characterization of tumor reactivity of TILs from non-human primate tumors could improve anti-tumor activity of TCR-engineered T cells in preclinical research. In this study, we sought to isolate TCR genes from CD8+PD-1+ T cells among TILs in a cynomolgus macaque model of tumor transplantation in which the tumors were infiltrated with CD8+ T cells and were eventually rejected. We analyzed the repertoire of TCRα and β pairs obtained from single CD8+PD-1+ T cells in TILs and circulating lymphocytes and identified multiple TCR pairs with high frequency, suggesting that T cells expressing these recurrent TCRs were clonally expanded in response to tumor cells. We further showed that the recurrent TCRs exhibited cytotoxic activity to tumor cells in vitro and potent anti-tumor activity in mice transplanted with tumor cells. These results imply that this tumor transplantation macaque model recapitulates key features of human TILs and can serve as a platform toward preclinical studies of non-human primate tumor models.

Switch receptor T3/28 improves long-term persistence and antitumor efficacy of CAR-T cells

BackgroundChimeric antigen receptor (CAR) T cells have been successfully used in tumor immunotherapy due to their strong antitumor responses, especially in hematological malignancies such as B cell acute lymphoid leukemia....

Sophoridine Derivatives Induce Apoptosis and Autophagy to Suppress the Growth of Triple-Negative Breast Cancer through Inhibition of mTOR Signaling.

In order to improve the antitumor potency and therapeutic margins of natural product sophoridine, its novel nitrogen mustard carbamate derivatives were designed and synthesized. In screening their in vitro activity, we found all the tested compounds were more potent against the highly aggressive triple-negative breast cancer cell line MDA-MB-231. Cellular functional assays showed that representative compounds could induce G1-phase arrest and trigger apoptosis, evidenced by the alteration of Bax, Bcl-2, caspase-3 and PARP levels. Furthermore, these compounds significantly enhanced the autophagic flux with increased expression of LC3-II and Beclin-1, as well as decreased level of p62, which may attribute to simultaneously inhibition of the phosphorylation of p70S6K, 4E-BP1 and AKT, the key substrates of the mTOR signaling pathway. In vivo, two compounds revealed potent antitumor activity in mice bearing MDA-MB-231. Altogether, our work describes novel leads to yield more potent chemotherapeutics against triple-negative breast cancers, possibly mesenchymal stem-like subtype.

Abstract 4520: Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice

Abstract Introduction: The STING (STimulator of INterferon Genes) protein has been identified as an attractive target for cancer immunotherapy due to its central role in inducing T-cell mediated tumor control. Here we describe the preclinical characteristics of the novel STING agonist, ALG-031048, that demonstrates potent anti-tumoral activity in in vivo mouse models. Methods: Binding to STING was determined using differential scanning fluorimetry (DSF). Cellular activation and cytokine release were assessed using HEK 293T reporter cell lines as well as THP-1 and Raw264.7 cells. In vitro stability was measured in the presence of snake venom phosphodiesterase (SVPD). In vivo anti-tumoral activity was tested in the syngeneic CT26 and B16F10 mouse model. Results: ALG-031048 demonstrated potent binding to the STING R232 protein in the DSF assay with a mean Kd of 3.12 ± 0.12 μM and a thermal shift of 12.8 °C, similar to the natural ligand 2'3'cGAMP (Kd of 2.75 ± 0.78 μM, shift of 16.7°C) or the clinical stage STING agonist, ADU-S100 (Kd of 4.61 ± 0.42 μM, shift of 10.7°C). In the HEK 293T R232 reporter assay, ALG-031048 was more potent than ADU-S100 with a mean EC50 of 55.9 ± 24.1 and 30.3 ± 17.1 nM for the IFNβ and IRF reporter, respectively, compared to ADU-S100 with EC50 values of 233 ± 179 and 88.7 ± 35.6 nM. Cellular activation was confirmed in THP-1 and Raw264.7 cells where ALG-031048 induced the secretion of IFNβ and CXCL-10. ALG-031048 was highly stable and exhibited no degradation for up to 24 hours in the presence of SVPD while ADU-S100 and 2'3'cGAMP were quickly degraded with less than 10% remaining after 24 hours. In vivo, 3 intra-tumoral (IT) doses of 100 μg ALG-031048 q3d caused complete tumor regression in 90% of animals in the CT26 mouse colon carcinoma model, while treatment with 25 μg ALG-031048 IT 3xq3d resulted in survival of 60 % of animals. In contrast, only 10 and 44% of mice showed complete tumor regression when treated IT 3xq3d with 25 or 100 μg ADU-S100, respectively. To study whether induction of a protective immune response was associated with STING agonist treatment, surviving animals were re-challenged with a second inoculation of CT26 cells on the contra-lateral side. All naïve animals formed tumors within 23-30 days. In contrast, 5 out of 5 and 8 out of 9 mice pre-treated with 25 or 100 μg ALG-031048, respectively, did not exhibit any significant tumor growth over 40 days, while 3 out of 4 animals pretreated with 100 μg ADU-S100 showed complete tumor suppression. Initial results using the B16F10 mouse melanoma model confirmed the potent anti-tumoral activity of ALG-031048 observed in the CT26 model. Summary: ALG-031048 is a novel STING agonist combining high stability and excellent in vitro potency. In preclinical models, ALG-031048 demonstrated potent and long-lasting tumor suppression. ALG-031048 is advancing in toxicology studies for further profiling as a clinical candidate. Citation Format: Andreas Jekle, Santosh Thatikonda, Sarah Stevens, Caroline Williams, April Kinkade, Suping Ren, Ruchika Jaisinghani, Qingling Zhang, Dinah Misner, Antitsa Stoycheva, Jerome Deval, Sucheta Mukherjee, Francois Gonzalvez, Sushmita Chanda, David B. Smith, Julian A. Symons, Lawrence M. Blatt, Leonid Beigelman. Preclinical characterization of ALG-031048, a novel STING agonist with potent anti-tumor activity in mice [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 4520.

Intra-Tumoral Delivery of IL-27 Using Adeno-Associated Virus Stimulates Anti-tumor Immunity and Enhances the Efficacy of Immunotherapy.

IL-27 is an anti-inflammatory cytokine that has been shown to have potent anti-tumor activity. We recently reported that systemic delivery of IL-27 using recombinant adeno-associated virus (rAAV) induced depletion of Tregs and significantly enhanced the efficacy of cancer immunotherapy in a variety of mouse tumor models. A potential caveat of systemic delivery of IL-27 using rAAV is that there is no practical method to terminate IL-27 production when its biological activity is no longer needed. Therefore, in this work, we tested if directly injecting AAV-IL-27 into tumors could lead to similar anti-tumor effect yet avoiding uncontrolled IL-27 production. We found that high levels of IL-27 was produced in tumors and released to peripheral blood after AAV-IL-27 intra-tumoral injection. AAV-IL-27 local therapy showed potent anti-tumor activity in mice bearing plasmacytoma J558 tumors and modest anti-tumor activity in mice bearing B16.F10 tumors. Intra-tumoral injection of AAV-IL-27 induced infiltration of immune effectors including CD8+ T cells and NK cells into tumors, caused systemic reduction of Tregs and stimulated protective immunity. Mechanistically, we found that IL-27 induced T cell expression of CXCR3 in an IL-27R-dependent manner. Additionally, we found that AAV-IL-27 local therapy had significant synergy with anti-PD-1 or T cell adoptive transfer therapy. Importantly, in mice whose tumors were completely rejected, IL-27 serum levels were significantly reduced or diminished. Thus, intra-tumoral injection of AAV-IL-27 is a feasible approach that can be used alone and in combination with anti-PD-1 antibody or T cell adoptive transfer for the treatment of cancer.

Abstract 3854: Preclinical characterization of a novel non-cyclic dinucleotide small molecule STING agonist with potent antitumor activity in mice

Abstract The cGAS-STING pathway plays a pivotal role in sensing aberrant cytoplasmic DNA fragments derived from pathogens or host tumor cells and initiating an innate immune response. STING activation induces type I IFNs and a cascade of other pro-inflammatory cytokines that activate the innate immune system and subsequently recruit adaptive immune cells. STING functions as a key mediator bridging the innate and adaptive immune responses by stimulating anti-tumor antigen presentation and promoting effective T cell priming, thereby making STING activation an attractive target for cancer immunotherapy. Currently, all known STING agonists in clinical trials are based on a cyclic dinucleotide (CDN) scaffold. These molecules mimic the endogenous STING ligand cGAMP which displays limited potency and cell permeability. Here we present the preclinical characterization of a novel non-CDN small molecule STING agonist, TTI-10001. TTI-10001 exhibited potent and broad activity in a panel of HEK293T-STING reporter cell lines expressing each of the five human STING (hSTING) alleles or murine STING (mSTING). Activation of the STING pathway was confirmed by an increase of both phospho-STING and phospho-IRF3 after TTI-10001 treatment, and TTI-10001-dependent induction of reporter activity was ablated in the presence of an inhibitor of the downstream STING signaling kinase TBK1. TTI-10001 showed a favorable safety profile with no activity on hERG or major CYP450 isoforms and was well tolerated in mice with no weight loss or overt morbidity after repeat intratumoral administration. Mice dosed with TTI-10001 displayed increased levels of phospho-STING and phospho-IRF3 in tumors as well as elevated expression of various pro-inflammatory cytokines including IFNβ, TNFα, and IL-6. This in vivo activation of STING signaling correlated with significant anti-tumor activity in multiple syngeneic mouse tumor models. In summary, we have generated a novel non-CDN small molecule STING agonist with potent activity on the five hSTING alleles and mSTING. Our compound exhibits strong in vitro and in vivo induction of the STING pathway as well as potent anti-tumor activity. Taken together, these results highlight the potential of TTI-10001 and support further evaluation and development of this molecule as a novel cancer immunotherapy agent. Citation Format: Zezhou Wang, Peter Dove, David Rosa, Bolette Bossen, Simone Helke, Marilyse Charbonneau, Laura Brinen, Karen Dodge, Gloria H. Lin, Carole Galligan, Natasja N. Viller, Mark Wong, Vivian Lee, Tina Catalano, Robert A. Uger, Malik Slassi, Jeff Winston. Preclinical characterization of a novel non-cyclic dinucleotide small molecule STING agonist with potent antitumor activity in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3854.

Optimized Expression and Characterization of a Novel Fully Human Bispecific Single-Chain Diabody Targeting Vascular Endothelial Growth Factor165 and Programmed Death-1 in Pichia pastoris and Evaluation of Antitumor Activity In Vivo.

Bispecific antibodies, which can bind to two different epitopes on the same or different antigens simultaneously, have recently emerged as attractive candidates for study in various diseases. Our present study successfully constructs and expresses a fully human, bispecific, single-chain diabody (BsDb) that can bind to vascular endothelial growth factor 165 (VEGF165) and programmed death-1 (PD-1) in Pichia pastoris. Under the optimal expression conditions (methanol concentration, 1%; pH, 4.0; inoculum density, OD600 = 4, and the induction time, 96 h), the maximum production level of this BsDb is achieved at approximately 20 mg/L. The recombinant BsDb is purified in one step using nickel-nitrilotriacetic acid (Ni-NTA) column chromatography with a purity of more than 95%. Indirect enzyme-linked immune sorbent assay (ELISA) and sandwich ELISA analyses show that purified BsDb can bind specifically to VEGF165 and PD-1 simultaneously with affinities of 124.78 nM and 25.07 nM, respectively. Additionally, the BsDb not only effectively inhibits VEGF165-stimulated proliferation, migration, and tube formation in primary human umbilical vein endothelial cells (HUVECs), but also significantly improves proliferation and INF-γ production of activated T cells by blocking PD-1/PD-L1 co-stimulation. Furthermore, the BsDb displays potent antitumor activity in mice bearing HT29 xenograft tumors by inhibiting tumor angiogenesis and activating immune responses in the tumor microenvironment. Based on these results, we have prepared a potential bispecific antibody drug that can co-target both VEGF165 and PD-1 for the first time. This work provides a stable foundation for the development of new strategies by the combination of an angiogenesis inhibition and immune checkpoint blockade for cancer therapy.

Abstract 748: Non-internalizing site-specific antibody-drug conjugates based on maytansinoids display curative properties

Abstract Antibody-Drug Conjugates (ADCs) are highly potent biopharmaceuticals that use the targeting ability of monoclonal antibodies to selectively bind to tumor cells where the conjugated cancer drug is released. Traditionally, the anticancer efficacy of ADCs relies on their selective internalization by cancer cells. However, it has recently been shown that non-internalizing, linkerless ADCs targeting the tumor extracellular environment can display a potent therapeutic activity. LGALS3BP (aka Mac-2 BP or 90K) represents an appealing target for non-internalizing ADC development, as the protein is largely secreted by the majority of human tumors, while being virtually undetectable in normal adult tissues except for the prostate and the colorectum. Additionally, once secreted the protein stops from spreading and clusters on the plasma membrane where it binds to some endogenous ligands. Here, we show that an engineered variant of the humanized 1959 antibody to human LGALS3BP selectively localizes around the tumor cells and once site-specifically coupled to the maytansinoid drugs DM3 or DM4 by means of disulfide linker can display a potent therapeutic activity. Both the 1959sss-DM3 and -DM4 conjugates mediated potent antitumor activity in mice bearing human tumor xenografts. Quantitative biodistribution studies confirmed a preferential accumulation of 1959sss-DM3 and -DM4 conjugates around the tumor cells. Notably, we found that both conjugates were well tolerated and cured 100% of tumor-bearing mice. Our findings offer a preclinical proof-of-concept for curative non-internalizing ADC endowed with an innovative mechanism of action. Citation Format: Gianluca Sala, Emily Capone, Enza Piccolo, Sara Ponziani, Roberta Gentile, Rodolfo Ippoliti, Francesco Giansanti, Stefano Iacobelli. Non-internalizing site-specific antibody-drug conjugates based on maytansinoids display curative properties [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 748.

Abstract 741: Targeting trastuzumab and T-DM-1 resistant breast cancer cells with EV20/MMAF, an antibody drug-conjugate against HER3

Abstract Trastuzumab and its derivative Trastuzumab-emtansine (T-DM1) are drugs approved to treat HER-2+ tumors. Despite the impressive clinical efficacy observed in many patients, intrinsic and acquired resistance to both drugs has emerged as a challenge. The HER-3 receptor is emerging as an attractive molecule in therapeutics because of its overexpression across many human cancers and because of its role in resistance to certain anticancer drugs. We recently showed that the EV20 humanized anti-HER-3 antibody has the ability of selectively and efficiently deliver the plant toxin Saporin to tumor cells. Here, we describe the antitumor activity of a novel ADC derivative of EV20 coupled to the cytotoxic drug monomethyl auristatin F (MMAF). This HER-3/ADC (EV20/MMAF) demonstrated a powerful and target-dependent killing activity in a panel of HER2+ breast cancer cells, including those resistant to trastuzumab and T-DM1 with an IC50 in the pico/nanomolar range. EV20/MMAF mediated potent antitumor activity in mice bearing trastuzumab resistant xenograft models. Notably, we found that the novel conjugate was well tolerated and caused tumor shrinkage in 100% of tumor-bearing mice. In summary, these findings open the possibility to use EV20/MMAF as a novel ADC targeting HER-3 and support its clinical development for malignancies, such as HER-2+ breast cancer, in which HER-3 is frequently overexpressed. Citation Format: Gianluca Sala, Lucia Gandullo, Emily Capone, Maria Elena Diaz-Rodriguez, Daniela D'Agostino, Vincenzo De Laurenzi, Jean-Frederic Sauniere, Stefano Iacobelli, Atanasio Pandiella. Targeting trastuzumab and T-DM-1 resistant breast cancer cells with EV20/MMAF, an antibody drug-conjugate against HER3 [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 741.

Targeting Attenuated Interferon-α to Myeloma Cells with a CD38 Antibody Induces Potent Tumor Regression with Reduced Off-Target Activity.

Interferon-α (IFNα) has been prescribed to effectively treat multiple myeloma (MM) and other malignancies for decades. Its use has waned in recent years, however, due to significant toxicity and a narrow therapeutic index (TI). We sought to improve IFNα’s TI by, first, attaching it to an anti-CD38 antibody, thereby directly targeting it to MM cells, and, second, by introducing an attenuating mutation into the IFNα portion of the fusion protein rendering it relatively inactive on normal, CD38 negative cells. This anti-CD38-IFNα(attenuated) immunocytokine, or CD38-Attenukine™, exhibits 10,000-fold increased specificity for CD38 positive cells in vitro compared to native IFNα and, significantly, is ~6,000-fold less toxic to normal bone marrow cells in vitro than native IFNα. Moreover, the attenuating mutation significantly decreases IFNα biomarker activity in cynomolgus macaques indicating that this approach may yield a better safety profile in humans than native IFNα or a non-attenuated IFNα immunocytokine. In human xenograft MM tumor models, anti-CD38-IFNα(attenuated) exerts potent anti-tumor activity in mice, inducing complete tumor regression in most cases. Furthermore, anti-CD38-IFNα(attenuated) is more efficacious than standard MM treatments (lenalidomide, bortezomib, dexamethasone) and exhibits strong synergy with lenalidomide and with bortezomib in xenograft models. Our findings suggest that tumor-targeted attenuated cytokines such as IFNα can promote robust tumor killing while minimizing systemic toxicity.

Highlights of This Issue

Metronomic chemotherapy, or the frequent administration of low, minimally toxic doses, inhibits the proliferation of endothelial cells. Previs, Armaiz-Pena, Lin, and colleagues tested the efficacy of nab-paclitaxel and topotecan (increases TSP-1 and suppresses carbonic anhydrase IX) at metronomic doses on multiple orthotopic ovarian cancer models and demonstrated decreased angiogenesis and increased apoptosis. Additionally, metronomic treatment suppresses hypoxia in the tumor microenvironment. Treatment with both agents led to a significantly prolonged overall survival compared to vehicle and other monotherapy groups (P &lt; 0.001). These findings support further clinical development of this combination, which has potent antiangiogenic effects.SS1P is a recombinant immunotoxin (RIT) that targets mesothelin. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. To overcome this impediment, a RIT with low immunogenicity was developed by removal of eight T-cell epitopes. LMB-T20 is more active than SS1P in vitro. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. LMB-T20 is a favorable candidate for evaluation in clinical trials.The murine double minute 2 (Mdm2) protein is elevated in many therapy-refractive breast cancers, making it an attractive therapeutic target. In triple-negative breast cancer (TNBC) cells, Nutlin-3a modulation of Mdm2 protein–protein interactions sensitized mutant p53 TNBC cells to carboplatin. Combination Nutlin-3a/carboplatin was synergistic in promoting p73a-dependent cell death. This correlated with persistence of DNA damage as assessed by increased γH2AX and association of Mdm2 with chromatin. Moreover, in a human breast-to-lung metastatic TNBC model, combination Nutlin-3a/carboplatin resulted in decreased tumor growth at primary and metastatic sites. This investigation provides rationale for developing novel combination therapies that target Mdm2 in TNBC.Continued refinements in the delivery of ionizing radiation to solid tumors and development of novel radiosensitizing agents have great potential to improve patient treatment. However, identifying reliable, predictive markers of genotoxic stress and radiation response remains elusive. Sandulache, Chen, and colleagues demonstrate that irradiation triggers quantifiable, transient, and reversible perturbations in tumor lactate levels, which correlate with intrinsic and acquired radioresistance. These findings provide a critical biochemical foundation for using noninvasive imaging techniques to assess tumor lactate levels. Incorporation of lactate as a real-time biomarker of radiation effectiveness in the treatment of solid tumors could revolutionize current clinical paradigms.

Recombinant Immunotoxin with T-cell Epitope Mutations That Greatly Reduce Immunogenicity for Treatment of Mesothelin-Expressing Tumors.

SS1P is a recombinant immunotoxin (RIT) that targets mesothelin. It consists of an antimesothelin Fv fused to a portion of Pseudomonas exotoxin A. In clinical studies, it has produced dramatic responses in patients with advanced mesothelioma, when combined with immunosuppressive therapy so that several treatment cycles could be given. Otherwise its activity is limited by its immunogenicity. In this work, we describe the development and characterization of LMB-T20, a highly potent RIT targeted at mesothelin-expressing cancers with low immunogenicity due to removal of its eight T-cell epitopes. LMB-T20 was more active than SS1P when tested on four different mesothelin-expressing cell lines as well as on cells obtained from patients with mesothelioma. It also has potent antitumor activity in mice, and has reduced immunogenicity as measured by cytokine secretion assays. In conclusion, LMB-T20 is a favorable candidate for evaluation in clinical trials due to its reduced immunogenicity and excellent activity.

Curative Properties of Noninternalizing Antibody–Drug Conjugates Based on Maytansinoids

It is generally thought that the anticancer efficacy of antibody-drug conjugates (ADC) relies on their internalization by cancer cells. However, recent work on an ADC that targets fibronectin in the tumor microenvironment suggests this may not be necessary. The alternatively spliced extra domains A and B (EDA and EDB) of fibronectin offer appealing targets for ADC development, because the antigen is strongly expressed in many solid human tumors and nearly undetectable in normal tissues except for the female reproductive system. In this study, we describe the properties of a set of ADCs based on an antibody targeting the alternatively spliced EDA of fibronectin coupled to one of a set of potent cytotoxic drugs (DM1 or one of two duocarmycin derivatives). The DM1 conjugate SIP(F8)-SS-DM1 mediated potent antitumor activity in mice bearing DM1-sensitive F9 tumors but not DM1-insensitive CT26 tumors. Quantitative biodistribution studies and microscopic analyses confirmed a preferential accumulation of SIP(F8)-SS-DM1 in the subendothelial extracellular matrix of tumors, similar to the pattern observed for unmodified antibody. Notably, we found that treatments were well tolerated at efficacious doses that were fully curative and compatible with pharmaceutical development. Our findings offer a preclinical proof-of-concept for curative ADC targeting the tumor microenvironment that do not rely upon antigen internalization.

Mouse, but not Human STING, Binds and Signals in Response to the Vascular Disrupting Agent 5,6-Dimethylxanthenone-4-Acetic Acid

Vascular disrupting agents such as 5,6-dimethylxanthenone-4-acetic acid (DMXAA) represent a novel approach for cancer treatment. DMXAA has potent antitumor activity in mice and, despite significant preclinical promise, failed human clinical trials. The antitumor activity of DMXAA has been linked to its ability to induce type I IFNs in macrophages, although the molecular mechanisms involved are poorly understood. In this study, we identify stimulator of IFN gene (STING) as a direct receptor for DMXAA leading to TANK-binding kinase 1 and IFN regulatory factor 3 signaling. Remarkably, the ability to sense DMXAA was restricted to murine STING. Human STING failed to bind to or signal in response to DMXAA. Human STING also failed to signal in response to cyclic dinucleotides, conserved bacterial second messengers known to bind and activate murine STING signaling. Collectively, these findings detail an unexpected species-specific role for STING as a receptor for an anticancer drug and uncover important insights that may explain the failure of DMXAA in clinical trials for human cancer.

Induction of Potent Antigen-specific Cytotoxic T Cell Response by PLGA-nanoparticles Containing Antigen and TLR Agonist

Previously we showed that biodegradable nanoparticles containing poly-IC or CpG oligodeoxynucleotide (ODN) together with ovalbumin (OVA) were efficient at inducing MHC-restricted presentation of OVA peptides in dendritic cells. The CTL-inducing activities of the nanoparticles were examined in the present study. Nanoparticles containing poly-IC or CpG ODN together with OVA were prepared using biodegradable polymer poly(D,L-lactic acid-co-glycolic acid), and then were opsonized with mouse IgG. The nanoparticles were injected into the tail vein of mice, and 7 days later the OVA-specific CTL activities were measured using an in vivo CTL assay. Immunization of mice with the nanoparticles containing poly-IC or CpG ODN together with OVA elicited potent OVA-specific CTL activity compared to those containing OVA only. In accordance with these results, nanoparticles containing poly-IC or CpG ODN together with OVA exerted potent antitumor activity in mice that were subcutaneously implanted with EG7.OVA tumor cells. These results show that encapsulation of poly-IC or CpG ODN together with antigen in biodegradable nanoparticles is an effective approach for the induction of potent antigen-specific CTL responses in vivo.

Tamoxifen inhibits malignant peripheral nerve sheath tumor growth in an estrogen receptor-independent manner

Few therapeutic options are available for malignant peripheral nerve sheath tumors (MPNSTs), the most common malignancy associated with neurofibromatosis type 1 (NF1). Guided by clinical observations suggesting that some NF1-associated nerve sheath tumors are hormonally responsive, we hypothesized that the selective estrogen receptor (ER) modulator tamoxifen would inhibit MPNST tumorigenesis in vitro and in vivo. To test this hypothesis, we examined tamoxifen effects on MPNST cell proliferation and survival, MPNST xenograft growth, and the mechanism by which tamoxifen impeded these processes. We found that 1-5 μM 4-hydroxy-tamoxifen induced MPNST cell death, whereas 0.01-0.1 μM 4-hydroxy-tamoxifen inhibited mitogenesis. Dermal and plexiform neurofibromas, MPNSTs, and MPNST cell lines expressed ERβ and G-protein-coupled ER-1 (GPER); MPNSTs also expressed estrogen biosynthetic enzymes. However, MPNST cells did not secrete 17β-estradiol, exogenous 17β-estradiol did not stimulate mitogenesis or rescue 4-hydroxy-tamoxifen effects on MPNST cells, and the steroidal antiestrogen ICI-182,780 did not mimic tamoxifen effects on MPNST cells. Further, ablation of ERβ and GPER had no effect on MPNST proliferation, survival, or tamoxifen sensitivity, indicating that tamoxifen acts via an ER-independent mechanism. Consistent with this hypothesis, inhibitors of calmodulin (trifluoperazine, W-7), another known tamoxifen target, recapitulated 4-hydroxy-tamoxifen effects on MPNST cells. Tamoxifen was also effective in vivo, demonstrating potent antitumor activity in mice orthotopically xenografted with human MPNST cells. We conclude that 4-hydroxy-tamoxifen inhibits MPNST cell proliferation and survival via an ER-independent mechanism. The in vivo effectiveness of tamoxifen provides a rationale for clinical trials in cases of MPNSTs.

Antitumor Activity and Immune Response Induction of a Dual Agonist of Toll-Like Receptors 7 and 8

Viral and synthetic single-stranded RNAs are the ligands for Toll-like receptors 7 and 8 (TLR7 and TLR8). We have reported a novel class of synthetic oligoribonucleotides, referred to as stabilized immune-modulatory RNA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8 depending on the sequence composition and the presence of specific chemical modifications. In the present study, we evaluated the antitumor activity of a dual TLR7/8 agonist in tumor-bearing mice with peritoneal disseminated CT26.CL25 colon and 3LL-C75 lung carcinomas. Peritoneal administration of dual TLR7/8 agonist in mice bearing CT26.CL25 colon carcinomas had potent dose-dependent antitumor activity, which was associated with a marked decrease in CD4(+)CD25(+)Foxp3(+) T regulatory cells and a significant increase in tumor antigen-specific IFN-gamma-secreting effector cell responses in splenocytes and local tumor-infiltrating cells. In 3LL-C75 lung carcinoma, dual TLR7/8 agonist induced strong immune responses and antitumor effects in C57BL/6 and TLR9(-/-) mice, but not in TLR7(-/-) and MyD88(-/-) mice, indicating that the agonist induces immune responses via TLR7 and through the MyD88-dependent signaling pathway. TLR8 is not functional in mice. Additionally, s.c. administration of TLR7/8 agonist effectively prevented lung metastasis of tumors in the CT26.CL25 pulmonary metastasis model. These studies show that the dual TLR7/8 agonist induced Th1-type immune responses and potent antitumor activity in mice via TLR7 and through the MyD88-dependent pathway.

Development of novel 5‐fluorouracil carrier erythrocyte with pharmacokinetics and potent antitumor activity in mice bearing malignant ascites

To investigate pharmacokinetics and potency of antitumor activity of a novel 5-fluorouracil carrier erythrocyte (RBC-FU) in mice bearing malignant ascites. RBC-FU was synthesized with a hyperosmotic technique. The entrapment efficiency of targeted carrier erythrocytes was determined by reverse dialysis method with high-performance liquid chromatography (HPLC) for analyzing the quantity of 5-fluorouracil (5-FU). After a H22 hepatocarcinoma malignant ascites model was established in Kunming mice, 5-FU encapsulated by carrier erythrocytes (for Group A) and 5-FU solution (for Group B) at 20 mg per kg were injected into the peritoneal cavity of the mice, respectively. Blood and ascites samples were collected at different times to detect 5-FU quantity by HPLC. Body weight and survival time of mice were recorded in Group A, B and the Control Group in which mice were injected with normal saline only. 5-FU was effectively encapsulated into erythrocytes, with an encapsulating effect as 55 +/- 0.50%. In Group A, the maximum concentration (Cmax) and the area under curve (AUC) in peritoneal exudates were significantly higher than those of Group B (P < 0.05). On the other hand, 5-FU level in serum was significantly lower than that in peritoneal exudates of Group A and B (P < 0.05). High drug levels in the abdominal cavity in Group A were maintained longer than those in Group B. Compared with that in Group B and the control, the quantity of malignant ascites in Group A had significant regression and the survival time was prolonged. The hyperosmotic method described here could be suitable for producing this novel RBC-FU as a liposomal drug of potential value for treating malignant ascites by intraperitoneal administration.