Tumour necrosis factor-alpha (TNF-alpha) is a potent pro-inflammatory agent produced primarily by activated monocytes and macrophages. TNF-alpha is synthesized as a precursor protein of M(r) 26,000 (26K) which is processed to a secreted 17K mature form by cleavage of an Ala-Val bond between residues 76-77. The enzyme(s) responsible for processing pro-TNF-alpha has yet to be identified. Here, we describe the capacity of a metalloproteinase inhibitor, GI 129471, to block TNF-alpha secretion both in vitro and in vivo. The inhibition is specific to TNF-alpha; the production of other secreted cytokines, such as the interleukins IL-1 beta, IL-2, or IL-6, is not inhibited. The mechanism of inhibition occurs at a post-translational step in TNF-alpha production. Our data suggest that TNF-alpha processing is mediated by a unique Zn2+ endopeptidase which is inhibited by GI 129471 and would represent a novel target for therapeutic intervention in TNF-alpha associated pathologies.

BackgroundEnadenotucirev (formerly ColoAd1) is a tumor-selective chimeric adenovirus with demonstrated preclinical activity. This phase 1 Mechanism of Action study assessed intravenous (IV) delivery of enadenotucirev in patients with resectable colorectal cancer (CRC), non-small-cell lung cancer (NSCLC), urothelial cell cancer (UCC), and renal cell cancer (RCC) with a comparator intratumoral (IT) dosed CRC patient cohort.MethodsSeventeen patients scheduled for primary tumor resection were enrolled. IT injection of enadenotucirev (CRC only) was administered as a single dose (≤ 3 × 1011 viral particles [vp]) on day 1, followed by resection during days 8–15. IV infusion of enadenotucirev was administered by three separate doses (1 × 1012 vp) on days 1, 3, and 5, followed by resection during days 8–15 (CRC) or days 10–25 (NSCLC, UCC, and RCC). Enadenotucirev activity was measured using immunohistochemical staining of nuclear viral hexon and quantitative polymerase chain reaction for viral genomic DNA.ResultsDelivery of enadenotucirev was observed in most tumor samples following IV infusion, with little or no demonstrable activity in normal tissue. This virus delivery (by both IV and IT dosing) was accompanied by high local CD8+ cell infiltration in 80% of tested tumor samples, suggesting a potential enadenotucirev-driven immune response. Both methods of enadenotucirev delivery were well tolerated, with no treatment-associated serious adverse events.ConclusionsThis study provides key delivery and feasibility data to support the use of IV infusion of enadenotucirev, or therapeutic transgene-bearing derivatives of it, in clinical trials across a range of epithelial tumors, including the ongoing combination study of enadenotucirev with the checkpoint inhibitor nivolumab. It also provides insights into the potential immune-stimulating properties of enadenotucirev.Trial registrationThis MOA study was a phase 1, multicenter, non-randomized, open-label study to investigate the administration of enadenotucirev in a preoperative setting (ClinicalTrials.gov: NCT02053220).Electronic supplementary materialThe online version of this article (doi:10.1186/s40425-017-0277-7) contains supplementary material, which is available to authorized users.

Effective immunotherapy of stromal-rich tumors requires simultaneous targeting of cancer cells and immunosuppressive elements of the microenvironment. Here, we modified the oncolytic group B adenovirus enadenotucirev to express a stroma-targeted bispecific T-cell engager (BiTE). This BiTE bound fibroblast activation protein on cancer-associated fibroblasts (CAF) and CD3e on T cells, leading to potent T-cell activation and fibroblast death. Treatment of fresh clinical biopsies, including malignant ascites and solid prostate cancer tissue, with FAP-BiTE-encoding virus induced activation of tumor-infiltrating PD1 þ T cells to kill CAFs. In ascites, this led to depletion of CAF-associated immunosuppressive factors, upregulation of proinflammatory cytokines, and increased gene expression of markers of antigen presentation, T-cell function, and trafficking. M2-like ascites macrophages exhibited a proinflammatory repolarization, indicating spectrum-wide alteration of the tumor microenvironment. With this approach, we have actively killed both cancer cells and tumor fibroblasts, reversing CAF-mediated immunosuppression and yielding a potent single-agent therapeutic that is ready for clinical assessment. Significance: An engineered oncolytic adenovirus that encodes a bispecific antibody combines direct virolysis with endogenous T-cell activation to attack stromal fibroblasts, providing a multimodal treatment strategy within a single therapeutic agent. Cancer Res; 78(24); 6852-65. Ó2018 AACR.

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SummaryAlthough thyroglobulin (Tg), the thyroid prohormone, is well known as a T cell dependent autoantigen in human and experimental autoimmune thyroid disease, very little is known about the molecular basis of Tg recognition by T cells. In this paper, we have characterized the epitopes recognized by .two clonotypically distinct, murine Tg autoreactive T cell hybridomas, CH9 and ADA2 . In vitro iodination of a Tg preparation which was deficient in in vivo organified iodine was first used to confirm our previous observation that these T cells recognize iodination-related epitopes in the Tg molecule . Affinity chromatography of tryptic peptides derived from normally iodinated human Tg revealed that these epitopes were exclusively located in thyroxine (T4) containing peptides. Through the use of synthetic T4-containing peptides, representing the four major hormonogenic sites in Tg, we demonstrated that both CH9 and ADA2 recognize an epitope containing the T4 at position 2553 in human Tg. Sets of overlapping 5mer to 12mer peptides around this T4 showed that the most potent peptide was a 9mer beginning at Asp 2551. The T4 was shown to be a critical residue, since its replacement with any of the 20 naturally occurring amino acids produced only nonstimulatory peptides. Since the T cell hybridomas could also be stimulated by major histocompatibility complex class II positive (interferon-, y-treated) thyroid epithelial cells in vitro, and their parent T cell lines can induce thyroiditis on adoptive transfer, the T4-containing Tg sequence described here is implicated as a pathogenic epitope in murine thyroid autoimmunity. T he production of the thyroid hormones thyroxine (T4),t tri-iodothyronine (T3) and reverse T3 (rT3) is dependent on the organification of iodine into thyroglobulin (Tg), the major protein product of the thyroid (1, 2) . This involves thyroid peroxidase catalyzed iodination of tyrosine residues in Tg to form mono-and di-iodotyrosines and their subsequent crosslinking to form the iodothyronines T3 and T4. These mature Tg molecules are stored in a colloidal form in the lumen of thyroid follicles. Secretion of T4 and T3 involves the endocytosis and subsequent proteolysis of colloidal Tg, which releases the hormone residues for diffusion into the circulation . The recent cloning of the genes coding for Tg from several species has allowed the precise localization 1 Abbreviations used in this paper.

BackgroundEnadenotucirev is a chimeric adenovirus with demonstrated preclinical tumor-selective cytotoxicity and a short half-life. Further clinical mechanism of action data showed that enadenotucirev can gain access to and replicate within different types of epithelial tumors. This phase 1 dose escalation study assessed intravenous (IV) dose escalation with enadenotucirev to establish the maximum tolerated dose (MTD) and subsequently identify a suitable schedule for repeated cycles.MethodsSixty-one patients with advanced epithelial tumors unresponsive to conventional therapy were enrolled and received enadenotucirev monotherapy as part of this study. During the phase 1a dose escalation (n = 22) and expansion (n = 9), delivery of enadenotucirev between 1 × 1010 and 1 × 1013 viral particles (vp) on days 1, 3, and 5 (single cycle) was used to determine an appropriate MTD. Subsequent treatment cohorts (phase 1a, n = 6 and phase 1b, n = 24) examined the feasibility of repeated dosing cycles in either 3-weekly or weekly dosing regimens.ResultsEnadenotucirev displayed a predictable and manageable safety profile at doses up to the MTD of 3 × 1012 vp, irrespective of infusion time or dosing schedule. The most commonly reported treatment-emergent adverse events (TEAEs) of grade 3 or higher were hypoxia, lymphopenia, and neutropenia. The frequency of all TEAEs (notably pyrexia and chills) was highest within 24 h of the first enadenotucirev infusion and decreased upon subsequent dosing. Additionally, delivery of three doses of enadenotucirev over 5 days optimized pharmacokinetic and chemokine profiles in the circulation over time.ConclusionsThis study provides key clinical data in patients with solid epithelial tumors following treatment with IV enadenotucirev monotherapy and supports further investigation of enadenotucirev in combination with other therapeutic agents at doses up to the MTD of 3 × 1012 vp.Trial registration(ClinicalTrials.gov Identifier: NCT02028442). Trial registration date: 07 January 2014 – Retrospectively registered.Electronic supplementary materialThe online version of this article (10.1186/s40425-019-0510-7) contains supplementary material, which is available to authorized users.

Summal"y A synthetic peptide based on a sequence containing thyroxine at position 2553 in thyroglobulin (Tg), and already shown to be recognized by two clonotypically distinct murine Tg autoreactive T cell hybridomas, can trigger primed lymph node cells to transfer thyroiditis to naive recipients. Donor lymph node cells could be prepared from mice immunized either with intact mouse Tg or with this peptide itself. After a second exposure to the priming antigen in vitro, both these populations induced 100% thyroiditis in recipient animals. The importance of the T4 residue in the development of disease was demonstrated by the failure of Tg tryptic peptides depleted of T4 to stimulate pathogenic effectors in vitro, even when the lymph node cells had been taken from mice primed with whole Tg. We conclude that this T4-containing 12mer sequence is a major thyroiditogenic epitope in CBA/J mice although we cannot exclude the possibility that there are other pathogenic epitopes present in the whole Tg molecule.

Affinity matured murine monoclonal antibody producing cell lines can now be rapidly generated using a novel repetitive, multiple site immunization strategy designated RIMMS. RIMMS capitalizes on rapid hypermutation and affinity maturation events which occur in B cell populations localized within secondary lymphatic tissue early in response to antigenic challenges. A murine myeloma cell line, P3XBcl-2-13, stably transfected with Bcl-2, enhances the outgrowth of hybridomas following somatic fusion with immune lymphocytes isolated from pooled peripheral lymph nodes (PLN) 8-14 days after the initial immunization. Immunizations somatic fusion, screening and isolation of affinity matured IgG secreting monoclonal antibody cell lines occur within a one month time period. By using RIMMS, we have been able to expedite the isolation of affinity matured monoclonal antibodies to numerous antigens, including a drug hapten.