Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction

<p>Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of pro-amyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (<b>18a</b>) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, <b>18a</b> significantly lowers murine serum retinol-binding protein 4 (RBP4) levels despite a lack of binding at that protein’s all-<i>trans</i>-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via <b>18a</b> is allosterically hindering all-<i>trans</i>-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.</p><br>

Identification of Transthyretin Tetramer Kinetic Stabilizers That Are Capable of Inhibiting the Retinol-Dependent Retinol Binding Protein 4-Transthyretin Interaction

<p>Dissociation of transthyretin (TTR) tetramers may lead to misfolding and aggregation of pro-amyloidogenic monomers, which underlies TTR amyloidosis (ATTR) pathophysiology. ATTR is a progressive disease resulting from the deposition of toxic fibrils in tissues that predominantly presents clinically as amyloid cardiomyopathy and peripheral polyneuropathy. Ligands that bind to and kinetically stabilize TTR tetramers prohibit their dissociation and may prevent ATTR onset. Drawing from clinically investigated AG10, we designed a constrained congener (<b>18a</b>) that exhibits excellent TTR tetramer binding potency, prevents TTR aggregation in a gel-based assay, and possesses desirable pharmacokinetics in mice. Additionally, <b>18a</b> significantly lowers murine serum retinol-binding protein 4 (RBP4) levels despite a lack of binding at that protein’s all-<i>trans</i>-retinol site. We hypothesize that kinetic stabilization of TTR tetramers via <b>18a</b> is allosterically hindering all-<i>trans</i>-retinol-dependent RBP4-TTR tertiary complex formation and that the compound could present ancillary therapeutic utility for indications treated with RBP4 antagonists, such as macular degeneration.</p><br>

Identification of Functional HLA-A*01:01–Restricted Epstein-Barr Latent Membrane Protein 2–Specific T-Cell Receptors

Background Adoptive transfer of genetically engineered T cells expressing antigen-specific T-cell receptors (TCRs) is an appealing therapeutic approach for Epstein-Barr virus (EBV)–associated malignancies of latency type II/III that express EBV antigens (LMP1/2). Patients who are HLA-A*01:01 positive could benefit from such products, since no T cells recognizing any EBV-derived peptide in this common HLA allele have been found thus far. Methods HLA-A*01:01–restricted EBV-LMP2–specific T cells were isolated using peptide major histocompatibility complex (pMHC) tetramers. Functionality was assessed by production of interferon gamma (IFN-γ) and cytotoxicity when stimulated with EBV-LMP2–expressing cell lines. Functionality of primary T cells transduced with HLA-A*01:01–restricted EBV-LMP2–specific TCRs was optimized by knocking out the endogenous TCRs of primary T cells (∆TCR) using CRISPR-Cas9 technology. Results EBV-LMP2–specific T cells were successfully isolated and their TCRs were characterized. TCR gene transfer in primary T cells resulted in specific pMHC tetramer binding and reactivity against EBV-LMP2–expressing cell lines. The mean fluorescence intensity of pMHC-tetramer binding was increased 1.5–2 fold when the endogenous TCRs of CD8+ T cells was knocked out. CD8+/∆TCR T cells modified to express EBV-LMP2–specific TCRs showed IFN-γ secretion and cytotoxicity toward EBV-LMP2–expressing malignant cell lines. Conclusions We isolated the first functional HLA-A*01:01–restricted EBV-LMP2–specific T-cell populations and TCRs, which can potentially be used in future TCR gene therapy to treat EBV-associated latency type II/III malignancies.

HLA-DQ:gluten tetramer test in blood gives better detection of coeliac patients than biopsy after 14-day gluten challenge

ObjectiveInitiation of a gluten-free diet without proper diagnostic work-up of coeliac disease is a frequent and demanding problem. Recent diagnostic guidelines suggest a gluten challenge of at least 14 days followed by duodenal biopsy in such patients. The rate of false-negative outcome of this approach remains unclear. We studied responses to 14-day gluten challenge in subjects with treated coeliac disease.DesignWe challenged 20 subjects with biopsy-verified coeliac disease, all in confirmed mucosal remission, for 14 days with 5.7 grams per oral gluten daily. Duodenal biopsies were collected. Blood was analysed by multiplex assay for cytokine detection, and by flow cytometry using HLA-DQ:gluten tetramers.ResultsNineteen participants completed the challenge. Villous blunting appeared at end of challenge in 5 of 19 subjects. Villous height to crypt depth ratio reduced with at least 0.4 concomitantly with an increase in intraepithelial lymphocyte count of at least 50% in 9 of 19 subjects. Interleukin-8 plasma concentration increased by more than 100% after 4 hours in 7 of 19 subjects. Frequency of blood CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells increased by more than 100% on day 6 in 12 of 15 evaluated participants.ConclusionA 14-day gluten challenge was not enough to establish significant mucosal architectural changes in majority of patients with coeliac disease (sensitivity ≈25%–50%). Increase in CD4+ effector-memory gut-homing HLA-DQ:gluten tetramer-binding T cells in blood 6 days after gluten challenge is a more sensitive and less invasive biomarker that should be validated in a larger study.Trial registration numberNCT02464150

Phase I/II Trial Of Immucin a Pan HLA, Anti-MUC1 Signal Peptide Vaccine, In Multiple Myeloma Patients With Residual Disease Or Progression Following Autologous Stem Cell Transplantation

Background Anti-multiple myeloma (MM) vaccinations have recently emerged as a strategy to eradicate MM cells in patients with residual tumor mass. However, most vaccination studies reported transient immune responses, involving CD8+ T-cells or antibodies responses in patients with predefined MHC repertoire. Signal peptide (SP) domains, a 13-50 amino acid long peptides, have been demonstrated to present an exceptionally high number of antigen specific MHC class I, class II and B-cell epitopes per sequence length. Therefore, they are suitable to induce a robust immune response combining CD8+, CD4+ T-cells and antibodies across HLA barrier. Following this rationale we developed ImMucin, A 21-mer therapeutic vaccine encoding the entire SP domain of the MUC1 tumor-associated antigen (TAA), which is over expressed by most carcinomas and hematological tumors including MM. Methods A phase I/II study to assess the safety (primary endpoint) and efficacy (secondary endpoint) of ImMucin was conducted in MM subjects with stable or progressive residual asymptomatic MUC1+ disease who have already had upfront auto-SCT. Patients recruited into the study initially received 6 bi-weekly vaccination of intra-dermal 100ug ImMucin plus 250ug hGM-CSF. Based on immune and clinical response eligible patient could receive additional 6 bi-weekly vaccination of 100ug or 250ug ImMucin plus 250ug hGM-CSF. Immunomonitoring analysis included; ImMucin-specific IFN-g production in CD4+ and CD8+ T-cells, HLA-A2.1 tetramer binding in CD8+ T-cells, proliferation and antibodies production. Positive response in these assays vs. pre vaccination levels were; x2 increase in IFN-g and >0.5%+ T-cells, any increase in tetramer binding levels, x2 increase and stimulation index (SI)>2 and any increase in anti-ImMucin titer respectively. Results 15 patients, median age 58 years participated in the study. The median number of prior therapies was 2. Median time from diagnosis and from auto-SCT to vaccination were 25 months (12-143) and 15 months (3-134) respectively. 9 patients had post auto-SCT residual MM and 6 had an evidence for biochemical progression. 11 patients completed the vaccination program while 4 discontinued the program due to PD. ImMucin was shown to be safe with no vaccine related grade ≥3 adverse events (AEs). Patients had temporary, grade 1,2 AEs, including injection related rash (n=8), fatigue (n=6), weakness, self-resolving fever and muscle pain (n=5). IFN-g production response to ImMucin was highly positive in all patients with 4-80 folds increase for CD4+ and 18-80 folds increase for CD8+. Mean baseline and peak post vaccination levels for ImMucin-specific CD4+ T-cells were 0.21% and 4.07%, respectively (P<0.001, t-test) and of CD8+ T-cells 0.21% and 11.76%, respectively (P<0.0001, t-test). All 4 HLA-A2.1 positive patients demonstrated < x2 increase in tetramer-positive cells after vaccination, with mean levels of 0.33% and 2.11% at baseline and peak post vaccination, respectively. Proliferative response to ImMucin increased signifcantly in all patients with mean baseline and peak of 3.24 and 15.92, respectively (P<0.024, t-test). An increased ImMucin humoral response of up to 40 folds from baseline levels was detected in 10 of 15 patients (66.6%). The vaccination efficacy was further confirmed by CD8+ T-cells and antibody mediated cytotoxicity against autologous BM cells. In these assays MUC1 control epitope didn’t show positive activation. Since ImMucin target MUC1’s SP domain which is not part of soluble MUC1 (sMUC1), a reduction in sMUC1 following vaccination can reflect a specific tumor destruction. A reduction of up to 17 folds, 5129.44 pg/ml as a mean levels at baseline vs. 792.33 pg/ml at maximal response (P<0.002, t-test) was detected in 9 of 10 patients, initially presenting abnormally elevated sMUC1 levels. Response assessment revealed disease stabilization in the majority of the patients, lasting for up to 29 months post study completion (ongoing follow up), including in patients entering study with PD. Conclusions The ImMucin therapeutic vaccine is directed to the less studied SP domain of MUC1 TAA. The current phase I/II results confirmed its high safety profile. ImMucin induced a robust CD8+ and CD4+ specific T-cell response in all patients and a marked anti-tumor humoral response. Further studies exploring ImMucin’s efficacy in patients with residual myeloma are under development. Disclosures: Carmon: Vaxil BioTherapeutics Ltd. : Employment, Equity Ownership, Patents & Royalties. Avivi:Vaxil BioTherapeutics Ltd. : Consultancy. Riva:Vaxil BioTherapeutics Ltd. : Employment. Shapira:Vaxil BioTherapeutics Ltd. : Consultancy.