Preclinical and Clinical Observations Implying Combination Therapy to Enhance the Efficacy of the Her-2/neu B-Cell Peptide-Based Vaccine HER-Vaxx and to Prevent Immune Evasion.

Her-2/neu-targeting therapy by passive application with trastuzumab is associated with acquired resistance and subsequent metastasis development, which is attributed to the upregulation of tumoral PD-L1 expression and the downregulation of Her-2/neu. We aimed to investigate this association, following active immunization with our recently constructed B-cell peptide-based Her-2/neu vaccines in both preclinical and clinical settings. Immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), and combined positive score (CPS) were applied to evaluate Her-2/neu and PD-L1 expression using a murine syngeneic tumor model for Her-2/neu lung metastases and tumor biopsies from a gastric cancer patient with disease progression. A significant and concomitant reduction in Her-2/neu and the upregulation of PD-L1 expression was observed in vaccinated mice after 45 days, but not after 30 days, of metastases development. A significant increase in tumor-infiltrating B lymphocytes was observed at both time points. The downregulation of Her-2/neu and the upregulation of PD-L1 were observed in a patient's primary tumor at the disease progression time point but not prior to vaccination (Her-2/neu IHC: 3 to 0, FISH: 4.98 to 1.63; PD-L1 CPS: 0% to 5%). Our results further underline the need for combination therapy by targeting PD-L1 to prevent metastasis formation and immune evasion of Her-2/neu-positive and PD-L1-negative tumor cells.

An oncolytic poxvirus encoding hNIS, shows anti-tumor efficacy and allows tumor imaging in a liver cancer model.

Oncolytic viruses (OVs) are live viruses that can selectively replicate in cancer cells. We have engineered an OV (CF33) to make it cancer-selective through the deletion of its J2R (thymidine kinase) gene. Additionally, this virus has been armed with a reporter gene, human sodium iodide symporter (hNIS), to facilitate non-invasive imaging of tumors using positron emission tomography (PET). In this study we evaluated the oncolytic properties of the virus (CF33-hNIS) in liver cancer model, and its usefulness in tumor imaging. The virus was found to efficiently kill liver cancer cells and the virus-mediated cell death exhibited characteristics of immunogenic death based on the analysis of 3 damage associate molecular patterns (DAMPs): calreticulin, ATP and HMGB1. Furthermore, local or systemic administration of a single dose of the virus showed anti-tumor efficacy against a liver cancer xenograft model in mice and significantly increased survival of treated mice. Lastly, PET scanning was performed following injection of the radioisotope I-124, for imaging of tumors, and a single dose of virus as low as 1E03 pfu, administered intratumorally (I.T.) or intravenously (I.V.), allowed for PET imaging of tumors. In conclusion, CF33-hNIS is safe and effective in controlling human tumor xenografts in nude mice, and it also facilitates non-invasive imaging of tumors.

An Oncolytic Poxvirus Encoding hNIS, Shows Antitumor Efficacy and Allows Tumor Imaging in a Liver Cancer Model.

Oncolytic viruses (OV) are live viruses that can selectively replicate in cancer cells. We have engineered an OV (CF33) to make it cancer-selective through the deletion of its J2R (thymidine kinase) gene. In addition, this virus has been armed with a reporter gene, human sodium iodide symporter (hNIS), to facilitate noninvasive imaging of tumors using PET. In this study, we evaluated the oncolytic properties of the virus (CF33-hNIS) in liver cancer model, and its usefulness in tumor imaging. The virus was found to efficiently kill liver cancer cells and the virus-mediated cell death exhibited characteristics of immunogenic death based on the analysis of 3 damage-associated molecular patterns: calreticulin, ATP, and high mobility group box-1. Furthermore, local or systemic administration of a single dose of the virus showed antitumor efficacy against a liver cancer xenograft model in mice and significantly increased survival of treated mice. Finally, PET scanning was performed following injection of the radioisotope I-124, for imaging of tumors, and a single dose of virus as low as 1E03 pfu, administered intra-tumorally or intravenously, allowed for PET imaging of tumors. In conclusion, CF33-hNIS is safe and effective in controlling human tumor xenografts in nude mice, and it also facilitates noninvasive imaging of tumors.

Solid-phase synthesis and screening of a library of C-terminal arginine peptide aldehydes against Murray Valley encephalitis virus protease.

Murray Valley encephalitis virus is a member of the flavivirus group, a large family of single-stranded RNA viruses, which cause serious disease in all regions of the world. Unfortunately, no suitable antivirals are available, and there are commercial vaccines for only three flaviviruses. The solid-phase synthesis of a library of 400 C-terminal arginine peptide aldehydes and their screening against Murray Valley encephalitis virus protease are demonstrated. The library was utilised to elucidate several tripeptide sequences that can be used as inhibitors in further SAR studies.

Preclinical Studies of a Novel Human PD-1 B-Cell Peptide Cancer Vaccine PD1-Vaxx From BALB/c Mice to Beagle Dogs and to Non-Human Primates (Cynomolgus Monkeys).

Immunotherapy with monoclonal antibodies to checkpoint inhibitors against the PD-1/PD-L1 signaling pathway is a landmark achievement in cancer therapy. Some anti-PD-1 inhibitors such as nivolumab and pembrolizumab have shown clinical success, in a percentage of patients with prolonged survival rates. However, adverse effects accompany these benefits. In this case, strategies with lower toxicity and increased specificity are urgently required. Cancer vaccines have the ability to stimulate the native immune system and in particular, an engineered B-cell epitope can elicit high-affinity polyclonal antibodies with similar efficacy to PD-1 monoclonal antibodies in murine animal models. We have previously designed and synthesized a unique B-cell vaccine, PD1-Vaxx [MVF-PD-1(92-110)], and we have tested the immunogenicity and antitumor properties in CT26 colon cancer BALB/c syngeneic mice model. This manuscript provides results from comprehensive preclinical pharmacology studies encompassing primary and secondary pharmacodynamics, biodistribution, and safety studies. The results from these preclinical studies support the use of PD1-Vaxx in a first-in-human clinical trial in patients with non-small cell lung cancer (NSCLC). A phase I trial in patients with NSCLC has commenced.

A newly discovered PD-L1 B-cell epitope peptide vaccine (PDL1-Vaxx) exhibits potent immune responses and effective anti-tumor immunity in multiple syngeneic mice models and (synergizes) in combination with a dual HER-2 B-cell vaccine (B-Vaxx).

Blockade of checkpoint receptors with monoclonal antibodies against CTLA-4, PD-1 and PD-L1 has shown great clinical success in several cancer subtypes, yielding unprecedented responses albeit a significant number of patients develop resistance and remain refractory. Both PD-1/PD-L1 and HER-2 signaling pathway inhibitors have limited efficacy and exhibits significant toxicities that limit their use. Ongoing clinical studies support the need for rationale combination of immuno-oncology agents to make a significant impact in the lives of cancer patients. We introduce the development of a novel chimeric PD-L1 B-cell peptide epitope vaccine (amino acid 130-147) linked to a "promiscuous" T cell measles virus fusion (MVF) peptide (MVF-PD-L1(130); PDL1-Vaxx) or linked to tetanus toxoid (TT3) TT3-PD-L1 (130) via a linker (GPSL). These vaccine constructs are highly immunogenic and antigenic in several syngeneic animal models. The PD-L1 vaccines elicited high titers of polyclonal antibodies that inhibit tumor growth in multiple syngeneic cancer models, eliciting antibodies of different subtypes IgG1, IgG2a, IgG2b and IgG3, induced PD-1/PD-L1 blockade, decreased proliferation, induced apoptosis and caused ADCC of tumor cells. The PDL1-Vaxx induces similar inhibition of tumor growth versus the standard anti-mouse PD-L1 antibody in both syngeneic BALB/c and C57BL/6J mouse models. The combination of PDL1-Vaxx with HER-2 vaccine B-Vaxx demonstrated synergistic tumor inhibition in D2F2/E2 carcinoma cell line. The anti-PDL1-Vaxx block PD-1/PD-L1 interaction and significantly prolonged anti-tumor responses in multiple syngeneic tumor models. The combination of HER-2 vaccine (B-Vaxx) with either PDL1-Vaxx or PD1-Vaxx demonstrated synergistic tumor inhibition. PDL1-Vaxx is a promising novel safe checkpoint inhibitor vaccine.

Development of the B cell cancer vaccine HER-vaxx for the treatment of her-2 expressing cancers.

Her-2/neu is a tumor-associated protein that is overexpressed in a number of malignancies, including advanced cancer of the stomach, and has been proposed as a human cancer vaccine target. Overexpression of Her-2/neu in human breast and gastric carcinomas correlates with a more aggressive course of disease that results in poorer overall survival rates and shorter times to disease progression than in patients with tumors without overexpression of Her-2/neu. Cancer vaccines have the ability to stimulate the native immune system and in particular engineered B cell epitopes can elicit high affinity polyclonal antibodies with similar efficacy to Her-2 monoclonal antibodies such as trastuzumab (Roche). HER-Vaxx is under development as a therapeutic B cell vaccine for the treatment of gastric cancer in patients with Her-2/neu overexpressing metastatic or advanced adenocarcinoma of the stomach or gastroesophageal junction, referred to as advanced cancer of the stomach. P467-CRM197, the vaccine's immunogenic component, contains a single peptide antigen composed of 3 individual linear B cell epitope peptide sequences selected from the oncoprotein Her-2/neu that induce the patient's own B cells to produce endogenous anti-Her-2/neu antibodies. This review provides results from comprehensive preclinical studies encompassing primary and secondary pharmacodynamics, biodistribution and safety studies. These studies were performed to support clinical development of HER-Vaxx. Results from the GLP toxicology study in rodents showed that the vaccine did not produce any observable adverse effects suggesting that the doses proposed for the clinical trial should be well tolerated in patients.

Crystal structures of novel allosteric peptide inhibitors of HIV integrase identify new interactions at the LEDGF binding site.

An optimised method of solution cyclisation gave us access to a series of peptides including SLKIDNLD (2). We investigated the crystallographic complexes of the HIV integrase (HIV-IN) catalytic core domain with 13 of the peptides and identified multiple interactions at the binding site, including hydrogen bonds with residues Thr125 and Gln95, that have not previously been described as being accessible within the binding site. We show that the peptides inhibit the interaction of lens epithelium-derived growth factor (LEDGF) with HIV-IN in a proximity AlphaScreen assay and in an assay for the LEDGF enhancement of HIV-IN strand transfer. The interactions identified represent a potential framework for the development of new HIV-IN inhibitors.

Clinical and Immunologic Responses to a B-Cell Epitope Vaccine in Patients with HER2/neu-Overexpressing Advanced Gastric Cancer-Results from Phase Ib Trial IMU.ACS.001.

PURPOSE: HER2/neu is overexpressed in up to 30% of gastroesophageal adenocarcinomas (GEA) and linked to poor prognosis. Recombinant mAbs to treat HER2/neu-overexpressing cancers are effective with limitations, including resistance and toxicity. Therefore, we developed a therapeutic B-cell epitope vaccine (IMU-131/HER-Vaxx) consisting of three fused B-cell epitopes from the HER2/neu extracellular domain coupled to CRM197 and adjuvanted with Montanide. This phase Ib study aimed to evaluate the optimal/safe dose leading to immunogenicity and clinical responses (https//clinicaltrials.gov/ct2/show/NCT02795988). PATIENTS AND METHODS: A total of 14 patients with HER2/neu-overexpressing GEA were enrolled, and dose escalation (10, 30, 50 µg) was performed in three cohorts (C). Immunogenicity was evaluated by HER2-specific Abs and cellular responses, clinical responses by CT scans according to RECIST version 1.1. RESULTS: IMU-131 was safe without vaccine-related significant local/systemic reactions or serious adverse events. A total of 11 of 14 patients were evaluable for changes in tumor size and vaccine-specific immune responses. One patient showed complete, 5 partial responses, and 4 stable diseases as their best response. HER2-specific IgG levels were dose dependent. In contrast to patients in C1 and C2, all patients in C3 mounted substantial HER2-specific Ab levels. In addition, cellular vaccine responses, such as Th1-biased cytokine ratios and reduced regulatory T cell numbers, were generated. Progression-free survival was prolonged in C3, correlating with the vaccine-specific humoral and cellular responses. CONCLUSIONS: IMU-131 was well tolerated and safe. The induced HER2-specific Abs and cellular responses were dose dependent and correlated with clinical responses. The highest dose (50 µg) was recommended for further evaluation in a phase II trial, with chemotherapy + IMU-131 or chemotherapy alone, which is currently ongoing.

Beyond Rf tagging.

The split-pool diversity orientated synthesis method, which requires some form of encoding to track the synthesis of discrete compounds, has been the lynchpin of most combinatorial synthesis efforts. The use of encoding methods in combinatorial chemistry has matured, and depending on their level of resources, chemists now have a diverse choice of encoding methods available. New methods of encoding have been developed that are inexpensive, simple to incorporate into any laboratory, and utilize analytical equipment such as MS, FTIR and NMR that are readily available to most organic chemists.

Multiple parallel synthesis of peptides on SynPhase grafted supports.

The Multipin peptide synthesis approach originated as an immunological tool for epitope mapping. However, continuing evolution of the basic technology has permitted the synthesis of peptides at scales up to 25 micromol per modular grafted surface. At this loading, the methodology can no longer be considered just a screening tool and is now used for the synthesis of micromoles of peptide and other small drug-like molecules for high throughput compound screening. Recent developments such as the introduction of novel grafted polymeric surfaces, new linkers, as well as novel cleavage formats has extended the scope of applications for modular grafted surfaces. This review summarizes the important achievements over the last 15 years of applications of the Multipin synthesis approach and also introduces a new modular grafted surface for peptide and small molecule synthesis called the SynPhase Lantern.

Novel solid-phase synthesis of 1,2-dialkoxyindoles.

[reaction: see text] A novel solid-phase synthesis of 1,2-dialkoxyindoles on SynPhase lanterns is described. A unique C-C bond formation involving a nucleophilic displacement of a solid-bound aryl fluorine by dimethyl malonate afforded the arylnitro methyl ester, which upon treatment with tin(II) chloride dihydrate gave the N-hydroxyindolone. Alkylation of the N-hydroxyindolones afforded the corresponding N-hydroxy-2-alkoxyindoles, which were further alkylated to give the 1,2-dialkoxyindole. A library of 64 (8R(1) x 8R(2)) discrete 1,2-dialkoxyindoles was prepared using a color encoding technique on SynPhase A-series lanterns.

Examination of methodology for the synthesis of cyclic thioether peptide libraries derived from linear tripeptides.

Work was undertaken to examine methodology for the cyclization of linear tripeptides on the solid phase via intramolecular S-alkylation using the Multipin(trade mark) Solid-Phase Peptide Synthesis platform. While previous work had shown that this chemistry could be used to efficiently cyclize linear tetrapeptide libraries, application of this synthetic strategy to the model linear tripeptide sequence Leu-Ser-Lys resulted in significant cyclic dimer formation. Ultimately, it was found that the addition of a large excess of lithium in the form of LiCl to the cyclization solution, significantly reduced cyclic dimer formation affording highly pure crude cyclic monomer. The application of this modified cyclization protocol to the preparation of cyclic peptide libraries was successfully demonstrated with the synthesis of a 20-membered library 4{1-20} based on the linear tripeptide sequence Leu-Xxx-Lys in which the position Xxx was varied with the standard 20 proteogenic residues.

Efficient methodology for the cyclization of linear peptide libraries via intramolecular S-alkylation using Multipin solid phase peptide synthesis.

Methodology is described here for the efficient parallel synthesis and cyclization of linear peptide libraries using intramolecular S-alkylation chemistry in combination with Multipin solid phase peptide synthesis (Multipin SPPS). The effective use of this methodology was demonstrated with the synthesis of a 72-member combinatorial library of cyclic thioether peptide derivatives of the conserved four-residue structural motif DD/EXK found in the active sites of the five crystallographically defined orthodox type II restriction endonucleases, EcoRV, EcoRI, PvuII, BamHI and BglI.

A broad-spectrum fluorescence-based peptide library for the rapid identification of protease substrates.

Identification of peptide substrates for proteases can be a major undertaking. To overcome issues such as feasibility and deconvolution, associated with large peptide libraries, a 'small but smart' generic fluorescence resonance energy transfer rapid endopeptidase profiling library (REPLi) was synthesised as a tool for rapidly identifying protease substrates. Within a tripeptide core, flanked by Gly residues, similar amino acids were paired giving rise to a relatively small library of 3375 peptides divided into 512 distinct pools each containing only 8 peptides. The REPLi was validated with trypsin, pepsin, the matrix metalloprotease (MMP)-12 and MMP-13 and calpains-1 and -2. In the case of calpain-2, a single iteration step involving LC-MS, provided the definitive residue specificity from which a highly sensitive fluorogenic substrate, (FAM)-Gly-Gly-Gly-Gln-Leu-Tyr-Gly-Gly-DPA-Arg-Arg-Lys-(TAMRA), was then designed. The thorough validation of this 'small but smart' peptide library with representatives from each of the four mechanistic protease classes indicates that the REPLi will be useful for the rapid identification of substrates for multiple proteases.

Preparation of cyclic peptide libraries using intramolecular oxime formation.

A new method for the synthesis of cyclic head-to-side chain peptide libraries has been developed in which the key cyclization step involves reaction between a C-terminal ketone and an N-terminal hydroxylamine to form a macrocyclic oxime. This methodology efficiently delivers cyclic products that consist of mixtures of syn and anti isomers.

Induction of long-term memory CD8(+) T cells for recall of viral clearing responses against influenza virus.

Induction of cytotoxic T-cell-mediated virus-clearing responses by influenza virus T cell determinant-containing peptide immunogens was examined. The most potent synthetic immunogens for eliciting pulmonary viral-clearing responses contained peptides representing determinants for CD4 and CD8 T cells (TH and CTL peptides, respectively) together with two or four palmitic acid (Pal) groups. Inoculated in adjuvant, these Pal2- or Pal4-CTL-TH lipopeptides and the nonlipidated CTL peptide induced equivalent levels of cytolytic activity in the primary effector phase of the response. The ability to recall lytic responses, however, diminished much more rapidly in CTL peptide-primed than in lipopeptide-primed mice. By 15 months postpriming, the recalled lytic activity in lipopeptide-inoculated mice remained potent, but the response induced by the CTL peptide was weak. Enumeration of specific gamma interferon-secreting CD8 T cells revealed that a greater number of these T cells had entered or remained in the memory pool in lipopeptide-primed mice, arguing for a quantitative rather than qualitative enhancement of the response on recall. Addition of either the lipid or the TH peptide to the CTL peptide was not sufficient to provide these long-lived antiviral responses, but inclusion of both components augmented the response. CD4 T cells elicited by the lipopeptides did not influence the rate of viral clearance upon challenge and most likely had a role in induction or maintenance of the memory response. It therefore appears that the lipopeptide immunogens, although not significantly superior at inducing primary effector CD8 T cells, elicit a much more effective memory population, the recall of which may account for their superiority in inducing pulmonary protection after viral challenge.

Synthesis of tetrahydro-1,4-benzodiazepine-2-ones on hydrophilic polyamide SynPhase lanterns.

Solid-phase synthesis is greatly dependent on the solid support. Here, we report the use of a new hydrophilic grafted surface on SynPhase lanterns in solid-phase organic chemistry. A convenient and facile solid-phase synthesis of disubstituted 1,4-benzodiazepine-2-ones on polyamide SynPhase lanterns is described. The key step of the synthesis involved a reduction-cyclization of a nitroaryl methyl ester with a mixture of tin(II) chloride dihydrate and ammonium acetate in water and ethanol at elevated temperature to give the desired target compounds. A library of 21 disubstituted 1,4-benzodiazepine-2-ones was prepared.

Efficient syntheses of benzothiazepines as antagonists for the mitochondrial sodium-calcium exchanger: potential therapeutics for type II diabetes.

Type II diabetes mellitus is a chronic metabolic disorder that can lead to serious cardiovascular, renal, neurologic, and retinal complications. While several drugs are currently prescribed to treat type II diabetes, their efficacy is limited by mechanism-related side effects (weight gain, hypoglycemia, gastrointestinal distress), inadequate efficacy for use as monotherapy, and the development of tolerance to the agents. Consequently, combination therapies are frequently employed to effectively regulate blood glucose levels. We have focused on the mitochondrial sodium-calcium exchanger (mNCE) as a novel target for diabetes drug discovery. We have proposed that inhibition of the mNCE can be used to regulate calcium flux across the mitochondrial membrane, thereby enhancing mitochondrial oxidative metabolism, which in turn enhances glucose-stimulated insulin secretion (GSIS) in the pancreatic beta-cell. In this paper, we report the facile synthesis of benzothiazepines and derivatives by S-alkylation using 2-aminobenzhydrols. The syntheses of other bicyclic analogues based on benzothiazepine, benzothiazecine, benzodiazecine, and benzodiazepine templates are also described. These compounds have been evaluated for their inhibition of mNCE activity, and the results from the structure-activity relationship (SAR) studies are discussed.