A feasibility study of combined epigenetic and vaccine therapy in advanced colorectal cancer with pharmacodynamic endpoint.

Epigenetic therapies may modulate the tumor microenvironment. We evaluated the safety and optimal sequence of combination DNA methyltransferase inhibitor guadecitabine with a granulocyte macrophage-colony-stimulating-factor (GM-CSF) secreting colon cancer (CRC) vaccine (GVAX) using a primary endpoint of change in CD45RO + T cells. 18 patients with advanced CRC enrolled, 11 underwent paired biopsies and were evaluable for the primary endpoint. No significant increase in CD45RO + cells was noted. Grade 3-4 toxicities were expected and manageable. Guadecitabine + GVAX was tolerable but demonstrated no significant immunologic activity in CRC. We report a novel trial design to efficiently evaluate investigational therapies with a primary pharmacodynamic endpoint.Trial registry Clinicaltrials.gov: NCT01966289. Registered 21 October, 2013.

Evaluation of Cyclophosphamide/GVAX Pancreas Followed by Listeria-Mesothelin (CRS-207) with or without Nivolumab in Patients with Pancreatic Cancer.

PURPOSE: Two studies in previously treated metastatic pancreatic cancer have been completed combining GVAX pancreas vaccine (GM-CSF-secreting allogeneic pancreatic tumor cells) with cyclophosphamide (Cy) and CRS-207 (live, attenuated Listeria monocytogenes-expressing mesothelin). In the current study, we compared Cy/GVAX followed by CRS-207 with (Arm A) or without nivolumab (Arm B). PATIENTS AND METHODS: Patients with pancreatic adenocarcinoma who received one prior therapy for metastatic disease and RECIST measurable disease were randomized 1:1 to receive treatment on Arm A or Arm B. The primary objective was to compare overall survival (OS) between the arms. Additional objectives included assessment of progression-free survival, safety, tumor responses, CA19-9 responses, and immunologic correlates. RESULTS: Ninety-three patients were treated (Arm A, 51; Arm B, 42). The median OS in Arms A and B were 5.9 [95% confidence interval (CI), 4.7-8.6] and 6.1 (95% CI, 3.5-7.0) months, respectively, with an HR of 0.86 (95% CI, 0.55-1.34). Objective responses were seen in 3 patients using immune-related response criteria (4%, 2/51, Arm A; 2%, 1/42, Arm B). The grade ≥3 related adverse event rate, whereas higher in Arm A (35.3% vs. 11.9%) was manageable. Changes in the microenvironment, including increase in CD8+ T cells and a decrease in CD68+ myeloid cells, were observed in long-term survivors in Arm A only. CONCLUSIONS: Although the study did not meet its primary endpoint of improvement in OS of Arm A over Arm B, the OS was comparable with standard therapy. Objective responses and immunologic changes in the tumor microenvironment were evident.

A phase 2 study of GVAX colon vaccine with cyclophosphamide and pembrolizumab in patients with mismatch repair proficient advanced colorectal cancer.

BACKGROUND: Mismatch repair proficient (MMRp) colorectal cancer (CRC) has been refractory to single-agent programmed cell death protein 1 (PD1) inhibitor therapy. Colon GVAX is an allogeneic, whole-cell, granulocyte-macrophage colony-stimulating factor -secreting cellular immunotherapy that induces T-cell immunity against tumor-associated antigens and has previously been studied in combination with low-dose cyclophosphamide (Cy) to inhibit regulatory T cells. METHODS: We conducted a single-arm study of GVAX/Cy in combination with the PD1 inhibitor pembrolizumab in patients with advanced MMRp CRC. Patients received pembrolizumab plus Cy on day 1, GVAX on day 2, of a 21-day cycle. The primary endpoint was the objective response rate by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1. Secondary objectives included safety, overall survival, progression-free survival, changes in carcinoembryonic antigen (CEA) levels, and immune-related correlates. RESULTS: Seventeen patients were enrolled. There were no objective responses, and the disease control rate was 18% by RECIST 1.1. The median progression-free survival was 82 days (95% confidence interval [CI], 48-97 days) and the median overall survival was 213 days (95% CI 179-441 days). Biochemical responses (≥30% decline in CEA) were observed in 7/17 (41%) of patients. Grade ≥ 3 treatment-related adverse events were observed in two patients (hemolytic anemia and corneal transplant rejection). Paired pre- and on-treatment biopsy specimens showed increases in programmed death-ligand 1 expression and tumor necrosis in a subset of patients. CONCLUSIONS: GVAX/Cy plus pembrolizumab failed to meet its primary objective in MMRp CRC. Biochemical responses were observed in a subset of patients and have not previously been observed with pembrolizumab monotherapy in MMRp CRC, indicating that GVAX may modulate the antitumor immune response.

Docosahexaenoic acid reduces inflammation and joint destruction in mice with collagen-induced arthritis.

OBJECTIVE: This study was designed to determine the anti-inflammatory activity of docosahexaenoic acid (DHA), alone and in combination with eicosapentaenoic acid (EPA), in a murine model of rheumatoid arthritis, collagen induced arthritis (CIA). METHODS: The CIA was induced in DBA/1OlaHsd mice by the injection of bovine type II collagen in Freunds's complete adjuvant on days 0 and 21. Mice were fed modified diets containing DHA and/or EPA for 4 weeks prior to the initial collagen injection until study termination at day 45. The severity of CIA was assessed by measuring erythema, edema and mobility of the digits on the fore and hind paws, as well as histology. The level of serum anti-collagen antibodies was determined by ELISA. The ex vivo effects of DHA and/or EPA on splenocyte proliferation and cytokine production were evaluated by BrdU method and ELISA. RESULTS: Prophylactic treatment with DHA, and not DHA/EPA, significantly reduced arthritis severity and joint damage. Treatment with DHA also decreased anti-collagen (CII) antibodies in vivo, downregulated interleukin-1ß, interferonγ and upregulated protective interleukin-10 ex vivo. CONCLUSION: Prophylactic treatment with DHA was efficacious in a mouse model of rheumatoid arthritis and may be a useful intervention strategy against inflammatory arthritis.

Growth, clinical chemistry and immune function in domestic piglets fed varying ratios of arachidonic acid and DHA.

In the USA, infant formulas contain long-chain PUFA arachidonic acid (ARA) and DHA in a ratio of 2:1 and comprise roughly 0·66 g/100 g and 0·33 g/100 g total fatty acids (FA). Higher levels of dietary DHA appear to provide some advantages in visual or cognitive performance. The present study evaluated the effect of physiologically high dietary ARA on growth, clinical chemistry, haematology and immune function when DHA is 1·0 g/100 g total FA. On day 3 of age, formula-reared (FR) piglets were matched for weight and assigned to one of six milk replacer formulas. Diets varied in the ratio of ARA:DHA as follows (g/100 g FA/FA): A1, 0·1/1·0; A2, 0·53/1·0; A3-D3, 0·69/1·0; A4, 1·1/1·0; D2, 0·67/0·62; D1, 0·66/0·33. A seventh group was maternal-reared (MR) and remained with the dam during the study. Blood collection and body weight measurements were performed weekly, and piglets were killed on day 28 of age. No significant differences were found among any of the FR groups for formula intake, growth, clinical chemistry, haematology or immune status measurements. A few differences in clinical chemistry, haematology and immune function parameters between the MR pigs and the FR groups probably reflected a difference in growth rate. We conclude that the dietary ARA level up to 1·0 g/100 g total FA is safe and has no adverse effect on any of the safety outcomes measured, and confirm that DHA has no adverse effect when ARA is at 0·66 g/100 g FA.

Docosahexaenoic acid (DHA) and docosapentaenoic acid (DPAn-6) algal oils reduce inflammatory mediators in human peripheral mononuclear cells in vitro and paw edema in vivo.

The anti-inflammatory activity associated with fish oil has been ascribed to the long-chain polyunsaturated fatty acids (LC-PUFA), predominantly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Here we examined the anti-inflammatory effects of two DHA-rich algal oils, which contain little EPA, and determined the contribution of the constituent fatty acids, particularly DHA and docosapentaenoic acid (DPAn-6). In vitro, lipopolysaccharide (LPS)-stimulated Interleukin-1 beta (IL-1beta) and Tumor Necrosis Factor-alpha (TNF-alpha) secretion in human peripheral blood mononuclear cells (PBMC) was inhibited with apparent relative potencies of DPAn-6 (most potent) > DHA > EPA. In addition, DPAn-6 decreased intracellular levels of cyclooxygenase-2 (COX-2) and was a potent inhibitor of pro-inflammatory prostaglandin E2 (PGE2) production. DHA/DPAn-6-rich DHA-S (DHA-S) algal oil was more effective at reducing edema in rats than DHA-rich DHA-T (DHA-T), suggesting that DPAn-6 has anti-inflammatory properties. Further in vivo analyses demonstrated that feeding DPAn-6 alone, provided as an ethyl ester, reduced paw edema to an extent approaching that of indomethacin and enhanced the anti-inflammatory activity of DHA when given in combination. Together, these results demonstrate that DPAn-6 has anti-inflammatory activity and enhances the effect of DHA in vitro and in vivo. Thus, DHA-S algal oil may have potential for use in anti-inflammatory applications.

Metabolism and biological production of resolvins derived from docosapentaenoic acid (DPAn-6).

17S-HDPAn-6 (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid) and 10S,17S-HDPAn-6 (10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E-pentaenoic acid) are potent anti-inflammatory resolvins derived from DPAn-6 (docosapentaenoic acid n-6) and are analogous in structure and action to DHA (docosahexaenoic acid)-derived resolvins. These resolvins have proven to be potential drug candidates, albeit with therapeutic profiles that need optimization. The main objectives of this study were to evaluate key features of DPAn-6 derived resolvins that are important for therapeutic efficacy, demonstrate that these DPAn-6 resolvins could be produced naturally, and could therefore have physiological significance. Here we demonstrate biological production, examine pharmacokinetic profiles and identify key routes of metabolic inactivation of DPAn-6 derived resolvins. We compare their metabolic stability to a known resolvin, 17S-HDHA (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid) and show that order of their stabilities is 10S,17S-HDPAn-6>17S-HDPAn-6>17S-HDHA. We show that both these compounds are not strong inhibitors of cytochrome-P450 enzymes. We evaluate activity of compounds in the delayed-type hypersensitivity model, results of which show that compounds need optimization for enhanced duration and magnitude of action. Analysis of the metabolic stability and identification of metabolites of these compounds could play an important role in the design of better analogs with longer durations of action and hence better efficacy.

Biogenic synthesis, purification, and chemical characterization of anti-inflammatory resolvins derived from docosapentaenoic acid (DPAn-6).

Enzymatically oxygenated derivatives of the omega-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197-1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677-14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.

In vivo genetic mutations define predominant functions of the human T-cell leukemia/lymphoma virus p12I protein.

The human T-cell leukemia/lymphoma virus type 1 (HTLV-1) ORF-I encodes a 99-amino acid hydrophobic membrane protein, p12(I), that affects receptors in different cellular compartments. We report here that proteolytic cleavage dictates different cellular localization and functions of p12(I). The removal of a noncanonical endoplasmic reticulum (ER) retention/retrieval signal within the amino terminus of p12(I) is necessary for trafficking to the Golgi apparatus and generation of a completely cleaved 8-kDa protein. The 8-kDa protein in turn traffics to the cell surface, is recruited to the immunologic synapse following T-cell receptor (TCR) ligation, and down-regulates TCR proximal signaling. The uncleaved 12-kDa form of p12(I) resides in the ER and interacts with the beta and gamma(c) chains of the interleukin-2 receptor (IL-2R), the heavy chain of the major histocompatibility complex (MHC) class I, as well as calreticulin and calnexin. Genetic analysis of ORF-I from ex vivo samples of HTLV-1-infected patients reveals predominant amino acid substitutions within ORF-I that affect proteolytic cleavage, suggesting that ER-associated functions of p12(I) may contribute to the survival and proliferation of the infected T cells in the host.

Memory lineage relationships in HTLV-1-specific CD8+ cytotoxic T cells.

Cytotoxic memory T cells play a critical role in combating viral infections; however, in some diseases they may contribute to tissue damage. In HAM/TSP, HTLV-1 Tax 11-19+ cells proliferate spontaneously in vitro and can be tracked using the Tax 11-19 MHC Class I tetramer. Immediately ex vivo, these cells were a mix of CD45RA-/CCR7- TEM and CD45RA+/CCR7- TDiff memory CTL. The subsequent proliferating Tax 11-19 tetramer+ population expressed low levels of IL-7Ralpha, failed to respond to IL-7 and IL-15, and did not develop a TCM phenotype. Thus, chronic exposure to viral antigen may result in a sustained pool of TEM cells that home to the CNS and mediate the spinal cord pathology seen in this disease.