Phase II Randomized Study of CMB305 and Atezolizumab Compared With Atezolizumab Alone in Soft-Tissue Sarcomas Expressing NY-ESO-1.

PURPOSE: CMB305 is a heterologous prime-boost vaccination regimen created to prime NY-ESO-1-specific CD8 T-cell populations and then activate the immune response with a potent TLR-4 agonist. This open-label randomized phase II trial was designed to investigate the efficacy and safety of adding the CMB305 regimen to atezolizumab (anti-programmed death ligand-1 therapy) in comparison with atezolizumab alone in patients with synovial sarcoma or myxoid liposarcoma. PATIENTS AND METHODS: Patients with locally advanced, relapsed, or metastatic synovial sarcoma or myxoid liposarcoma (any grade) were randomly assigned to receive CMB305 with atezolizumab (experimental arm) or atezolizumab alone (control arm). The primary end points were progression-free survival (PFS) and overall survival (OS) analyzed using the Kaplan-Meier method. Safety and immune responses were assessed. RESULTS: A total of 89 patients were enrolled; 55.1% had received ≥ 2 prior lines of chemotherapy. Median PFS was 2.6 months and 1.6 months in the combination and control arms, respectively (hazard ratio, 0.9; 95% CI, 0.6 to 1.3). Median OS was 18 months in both treatment arms. Patients treated with combination therapy had a significantly higher rate of treatment-induced NY-ESO-1-specific T cells (P = .01) and NY-ESO-1-specific antibody responses (P < .0001). In a post hoc analysis of all dosed patients, OS was longer (36 months) in the subset who developed anti-NY-ESO-1 T-cell immune response (hazard ratio, 0.3; P = .02). CONCLUSION: Although the combination of CMB305 and atezolizumab did not result in significant increases in PFS or OS compared with atezolizumab alone, some patients demonstrated evidence of an anti-NY-ESO-1 immune response and appeared to fare better by imaging than those without such an immune response. Combining prime-boost vaccines such as CMB305 with anti-programmed death ligand-1 therapies merits further evaluation in other clinical contexts.

Phase 1/2 study of intratumoral G100 (TLR4 agonist) with or without pembrolizumab in follicular lymphoma.

Intratumoral injection of G100, a toll-like receptor 4 (TLR4) agonist, was shown pre-clinically to stimulate anti-tumor immune responses and tumor regression. This open-label, multicenter, phase 1/2 trial evaluated the safety, tolerability, and preliminary efficacy of intratumoral G100 injections following localized low-dose radiation in patients with follicular lymphoma (ClinicalTrials.gov #NCT02501473). The study was comprised of a G100 dose escalation (5 or 10 µg/dose, or 20 µg/dose for large tumors); a randomized component comparing G100 to G100 plus pembrolizumab; and G100 20 µg/dose expansion. Adverse events grade ≥3 were uncommon in patients treated with G100, and no unexpected toxicities were observed when combined with pembrolizumab. G100 20 µg (n = 18) resulted in an overall response rate of 33.3% and abscopal tumor regression in 72.2% of patients. This early-phase study provides a foundation for combining an intratumoral TLR4 agonist with agents to produce immune-mediated responses in follicular lymphoma with limited added toxicity.

A Phase 1b Study Evaluating the Safety, Tolerability, and Immunogenicity of CMB305, a Lentiviral-Based Prime-Boost Vaccine Regimen, in Patients with Locally Advanced, Relapsed, or Metastatic Cancer Expressing NY-ESO-1.

Preclinical data suggest that a "prime-boost" vaccine regimen using a target-expressing lentiviral vector for priming, followed by a recombinant protein boost, may be effective against cancer; however, this strategy has not been evaluated in a clinical setting. CMB305 is a prime-boost vaccine designed to induce a broad anti-NY-ESO-1 immune response. It is composed of LV305, which is an NY-ESO-1 expressing lentiviral vector, and G305, a recombinant adjuvanted NY-ESO-1 protein. This multicenter phase 1b, first-in-human trial evaluated CMB305 in patients with NY-ESO-1 expressing solid tumors. Safety was examined in a 3 + 3 dose-escalation design, followed by an expansion with CMB305 alone or in a combination with either oral metronomic cyclophosphamide or intratumoral injections of a toll-like receptor agonist (glucopyranosyl lipid A). Of the 79 patients who enrolled, 81.0% had sarcomas, 86.1% had metastatic disease, and 57.0% had progressive disease at study entry. The most common adverse events were fatigue (34.2%), nausea (26.6%), and injection-site pain (24.1%). In patients with soft tissue sarcomas, a disease control rate of 61.9% and an overall survival of 26.2 months (95% CI, 22.1-NA) were observed. CMB305 induced anti-NY-ESO-1 antibody and T-cell responses in 62.9% and 47.4% of patients, respectively. This is the first trial to test a prime-boost vaccine regimen in patients with advanced cancer. This approach is feasible, can be delivered safely, and with evidence of immune response as well as suggestion of clinical benefit.

Peptide vaccine with glucopyranosyl lipid A-stable oil-in-water emulsion for patients with resected melanoma.

Aim: We tested the safety and immunogenicity of a novel vaccine in patients with resected high-risk melanoma. Patients & methods: HLA-A2-positive patients with resected Stage II-IV melanoma were randomized to receive up to three vaccinations of melanoma-associated peptide (MART-1a) combined with a stable oil-in-water emulsion (SE) either with the Toll-like receptor 4 agonist glucopyranosyl lipid A (GLA-SE-Schedule 1) or alone (SE-Schedule 2). Safety and immunogenicity of the vaccines were monitored. Results: A total of 23 patients were registered. No treatment-related grade 3 or higher adverse events were observed. Increases in MART-1a-specific T cells were seen in 70 and 63% of Schedule 1 and Schedule 2 patients, respectively. Conclusion: Both vaccine schedules were well-tolerated and resulted in an increase in MART-1a-specific T cells. Clinical Trial registration: NCT02320305 (ClinicalTrials.gov).

ZAS3 accentuates transforming growth factor ß signaling in epithelial cells.

In mammals, the ZAS family of transcription factors activates or represses transcription depending on the cellular context. In the current study, we explored the interaction between ZAS3 and TGFß1 signaling in epithelial cells using HEK293 cells and the intestinal epithelial cell line, RIE-1. Endogenous ZAS3 expression was detected in each cell line and the small intestine of mice. Additionally, endogenous ZAS3 expression was increased in both whole cell and nuclear lysates by TGFß1 and in vivo in TGFß-overexpressing mice, indicating a potential interaction between ZAS3 and TGFß. ZAS3 transfection enhanced TGFß1 activation of a luciferase reporter in both HEK293 and RIE-1 cells. Analysis of truncated ZAS3 constructs revealed a 155 amino acid, N-terminal sequence between amino acids 106 and 261 that was required for enhancement of TGFß1-mediated transcription. Co-immunoprecipitation experiments with nuclear extracts from TGFß1-stimulated HEK293 cells revealed an association between ZAS3 and the Smad complex. Additionally, transfected ZAS3 decreased the association between the Smad complex and the TGFß transcriptional repressors Ski and SnoN, indicating a possible mechanism for the enhancement of transcription by exogenous ZAS3. These observations were confirmed by site-directed mutagenesis of ZAS domains homologous with Smad-interacting domains in Ski and SnoN. Finally, ZAS3 transfection enhanced the TGFß1-mediated induction of α-smooth muscle actin in HEK293 cells, indicating that ZAS3 plays a functional role in TGFß signaling. In conclusion, we have identified an interaction between ZAS3 and Smad proteins that enhances TGFß signaling. Since TGFß signaling is primarily known as a negatively regulated pathway, the enhancement of signaling by ZAS3 has novel implications for understanding TGFß biology.

Vectorial TGFbeta signaling in polarized intestinal epithelial cells.

Polarized gastrointestinal epithelial cells form tight junctions that spatially separate apical and basolateral cell membrane domains. These domains harbor functionally distinct proteins that contribute to cellular homeostasis and morphogenesis. Transforming growth factor beta (TGFbeta) is a critical regulator of gastrointestinal epithelial cell growth and differentiation. Functional assays of vectorial TGFbeta signaling and immunofluorescence techniques were used to determine the localization of TGFbeta receptors and ligand secretion in polarizing Caco-2 cells, a colon cancer cell line. Results were compared to the nontransformed MDCK cell line. In both Caco-2 and MDCK cells, addition of TGFbeta1 to the basolateral medium resulted in phosphorylation of Smad2. No phosphorylation was observed when TGFbeta1 was added to the apical chamber, indicating that receptor signaling is localized at the basolateral membrane. In support of this, immunofluorescence and biotinylation assays show receptor localization along the basolateral membrane. Secretion of TGFbeta1 from MDCK and Caco-2 cells into the apical or basolateral medium was measured by ELISA. Interestingly, secretion was exclusively apical in the nontransformed MDCK line and basolateral in transformed Caco-2 cells. Collectively, these results show basolateral domain specificity in localization of the TGFbeta receptor signaling apparatus. These observations have important implications for understanding the biology of TGFbeta in polarized epithelia, including elements of communication between epithelial and mesenchymal layers, and will prove useful in the design of therapeutics that target TGFbeta function.

Inhibitors of tubulin assembly identified through screening a compound library.

Tubulin is the proposed target for drugs against cancer and helminths and is also a validated target in kinetoplastid parasites. With the aim of identifying new lead compounds against Leishmania sp., tubulin isolated from L. tarentolae was used to screen a 10 000 compound library. One compound, Chembridge No. 7992831 (5), displayed an IC(50) of 13 microm against Leishmania tubulin in an in vitro assembly assay and showed a greater than threefold selectivity over mammalian tubulin. Another compound, Chembridge No. 9067250 (8), exhibited good activity against mammalian tubulin (IC(50) = 5.0 microm). This compound was also toxic to several cancer cell lines with IC(50) values in the region of 1 microm. Subsequent testing of analogues of 8 contained within the library identified two compounds with greater potency against mammalian tubulin (IC(50) values of 1.1 and 2.8 microm). The more potent antitubulin agent also showed promising activity against cancer cell lines in vitro, with IC(50) values ranging from 0.18 to 0.73 microm.

Transformation by oncogenic Ras expands the early genomic response to transforming growth factor beta in intestinal epithelial cells.

A substantial body of evidence implicates TGFbeta as a tumor promoter in epithelial cells that have become resistant to its tumor suppressor activity. To better understand early, genome-wide TGFbeta responses in cells resistant to growth inhibition by TGFbeta, we used microarray analysis in a well-defined cell culture system of sensitive and resistant intestinal epithelial cells. TGFbeta-regulated gene expression in TGFbeta-growth-sensitive, nontransformed rat intestinal epithelial cells (RIE-1) was compared to expression in TGFbeta-growth-resistant RIE cells stably transformed by oncogenic Ras(12V). Treatment of RIE-1 cells with 2 ng/ml TGFbeta1 for 1 hour increased the expression of eight gene sequences by 2.6-fold or more, whereas eight were down regulated 2.6-fold. In RIE-Ras(12V) cells, 42 gene sequences were upregulated and only 3 were down-regulated. Comparison of RIE and RIE-Ras(12V) identified 37 gene sequences as unique, Ras-dependent genomic targets of TGFbeta1. TGFbeta-regulation of connective tissue growth factor and vascular endothelial growth factor, two genes up-regulated in RIE-Ras cells and previously implicated in tumor promotion, was independently confirmed and further characterized by Northern analysis. Our data indicate that overexpression of oncogenic Ras in intestinal epithelial cells confers a significantly expanded repertoire of robust, early transcriptional responses to TGFbeta via signaling pathways yet to be fully elucidated but including the canonical Raf-1/MAPK/Erk pathway. Loss of sensitivity to growth inhibition by TGFbeta does not abrogate TGFbeta signaling and actually expands the early transcriptional response to TGFbeta1. Expression of some of these genes may confer to Ras-transformed cells characteristics favorable for tumor promotion.

Synthesis, biological evaluation, and molecular modeling of 3,5-substituted-N1-phenyl-N4,N4-di-n-butylsulfanilamides as antikinetoplastid antimicrotubule agents.

Dinitroanilines are of interest as antiprotozoal lead compounds because of their selective activity against the tubulin of these organisms, but concern has been raised due to the potentially mutagenic nitro groups. Analogues of N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (GB-II-150, compound 2b), a selective antimitotic agent against African trypanosomes and Leishmania, have been prepared where the nitro groups are replaced with amino, chloro, cyano, carboxylate, methyl ester, amide, and methyl ketone moieties. Dicyano compound 5 displays IC(50) values that are comparable to 2b against purified leishmanial tubulin assembly (6.6 vs 7.4 microM), Trypanosoma brucei brucei growth in vitro (0.26 vs 0.18 microM), Leishmania donovani axenic amastigote growth in vitro (4.4 vs 2.3 microM), and in vitro toxicity against Vero cells (16 vs 9.7 microM). Computational studies provide a rationale for the antiparasitic order of activity of these analogues and further insight into the role of the substituents at the 3 and 5 positions of the sulfanilamide ring.

Activity of and initial mechanistic studies on a novel antileishmanial agent identified through in silico pharmacophore development and database searching.

A 3D pharmacophore was generated to describe the antileishmanial activity of dinitroaniline sulfonamides by CATALYST 3D-QSAR methodology, and this pharmacophore was used to search the Maybridge database. Two compounds identified in this search, BTB 06237 and BTB 06256, were highly active with IC(50) values against L. donovani amastigotes of 0.5 +/- 0.2 and 2.3 +/- 0.8 microM, respectively. BTB 06237 also reduced parasite burdens in L. mexicana-infected J774 macrophages at low micromolar concentrations. Unlike the dinitroaniline sulfonamides, the active compounds did not display antimitotic effects against Leishmania. Transmission electron microscopy showed that the single parasite mitochondrion becomes dilated following incubation with BTB 06237, and fluorescence microscopy demonstrated that this organelle fragments into intensely staining spheres when treated with a mitochondrion-specific dye. The mitochondrial membrane potential was also dissipated in BTB 06237-treated parasites. These results indicate that BTB 06237 is an intriguing antileishmanial lead compound that likely interferes with mitochondrial function.

Leishmania tarentolae: purification and characterization of tubulin and its suitability for antileishmanial drug screening.

Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, L. amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of alpha- and beta-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly disassembly cycle in 1% overall recovery based on total cellular protein. Leishmania tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae alpha- and beta-tubulin genes were 99.6 and 99.4% identical to the corresponding amino acid sequences from the Leishmania major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.

Antikinetoplastid antimitotic activity and metabolic stability of dinitroaniline sulfonamides and benzamides.

N(1)-Phenyl-3,5-dinitro-N(4),N(4)-di-n-propylsulfanilamide (1) and N(1)-phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (2) show potent in vitro antimitotic activity against kinetoplastid parasites but display poor in vivo activity. Seventeen new dinitroaniline sulfonamide and eleven new benzamide analogs of these leads are reported here. Nine of the sulfonamides display in vitro IC(50) values under 500 nM against African trypanosomes, and the most active antikinetoplastid compounds also inhibit the in vitro assembly of purified leishmanial tubulin with potencies similar to that of 2. While several of the potent compounds are rapidly degraded by rat liver S9 fractions in vitro, N(1)-(3-hydroxy)phenyl-3,5-dinitro-N(4),N(4)-di-n-butylsulfanilamide (21) displays an IC(50) value of 260 nM against African trypanosomes in vitro and is more stable than 2 in the in vitro metabolism assay.

Cytotoxic 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienes mounted on alicyclic scaffolds.

The 5-aryl-1-(4-nitrophenyl)-3-oxo-1,4-pentadienyl pharmacophore was incorporated into four series of compounds 1-4. Compounds 1a-g comprised a cluster of 3-arylidene-1-(4-nitrophenylmethylene)-2-oxo-3,4-dihydro-1H-naphthalenes while the analogues 2a-g consisted of a group of 6-arylidene-2-(4-nitrophenylmethylene)cyclohexanones. Three other compounds prepared in this study were 1-(4-nitrophenylmethylene)-3-(3,4,5-trimethoxyphenylmethylene)-2-oxo-2,3-dihydro-1H-indene 3a as well as two 5-arylidene-2-(4-nitrophenylmethylene)cyclopentanones 4a,b. The compounds were evaluated against human Molt 4/C8 and CEM T-lymphocytes as well as murine L1210 cells. In general, the compounds in series 1 displayed marked cytotoxicity having IC50 values in the 1-5 microM range while the related cyclohexyl analogues in series 2 were slightly less potent (IC50 figures were mainly 5-10 microM). The relative locations of two aryl rings present in all four series were considered to contribute significantly to bioactivity and may have accounted for the virtual absence of cytotoxic properties in series 3 and 4. Most of the compounds were administered intraperitoneally to mice using doses up to and including 300 mg/kg. No mortalities were noted. The inhibiting effect of most of the compounds towards Helicobacter pylori is noteworthy. The modes of action of representative compounds include the induction of apoptosis while some compounds weakly inhibited tubulin polymerisation and human N-myristoyltransferase.