IRX-2, a Novel Immunotherapeutic, Enhances Functions of Human Dendritic Cells

Background: In a recent phase II clinical trial for HNSCC patients, IRX-2, a cell-derived biologic, promoted T-cell infiltration into the tumor and prolonged overall survival. Mechanisms responsible for these IRX-2-mediated effects are unknown. We hypothesized that IRX-2 enhanced tumor antigen-(TA)-specific immunity by up-regulating functions of dendritic cells (DC). Methodology/Principal Findings: Monocyte-derived DC obtained from 18 HNSCC patients and 12 healthy donors were matured using IRX-2 or a mix of TNF-α, IL-1β and IL-6 ("conv. mix"). Multicolor flow cytometry was used to study the DC phenotype and antigen processing machinery (APM) component expression. ELISPOT and cytotoxicity assays were used to evaluate tumor-reactive cytotoxic T lymphocytes (CTL). IL-12p70 and IL-10 production by DC was measured by Luminex® and DC migration toward CCL21 was tested in transwell migration assays. IRX-2-matured DC functions were compared with those of conv. mix-matured DC. IRX-2-matured DC expressed higher levels (p<0.05) of CD11c, CD40, CCR7 as well as LMP2, TAP1, TAP2 and tapasin than conv. mix-matured DC. IRX-2-matured DC migrated significantly better towards CCL21, produced more IL-12p70 and had a higher IL12p70/IL-10 ratio than conv. mix-matured DC (p<0.05 for all). IRX-2-matured DC carried a higher density of tumor antigen-derived peptides, and CTL primed with these DC mediated higher cytotoxicity against tumor targets (p<0.05) compared to the conv. mix-matured DC. Conclusion: Excellent ability of IRX-2 to induce ex vivo DC maturation in HNSCC patients explains, in part, its clinical benefits and emphasizes its utility in ex vivo maturation of DC generated for therapy. © 2013 Schilling et al

A general process for the development of peptide-based immunoassays for monoclonal antibodies

Monoclonal antibodies (mAb) are an important and growing class of cancer therapeutics, but pharmacokinetic analyses have in many cases been constrained by the lack of standard and robust pharmacologic assays. The goal of this project was to develop a general method for the production of immunoassays that can measure the levels of therapeutic monoclonal antibodies in biologic samples at relevant concentrations. Alemtuzumab and rituximab are monoclonal approved for the treatment of B-cell malignancies and were used as a model system. Phage-displayed peptide libraries were screened for peptide sequences recognized by alemtuzumab (anti-CD52) or rituximab (anti-CD20). Synthetic biotinylated peptides were used in enzyme-linked immunosorbent assays (ELISA). Peptides directly synthesized on polymer resin beads were used in an immunofluorescent-based assay. Peptide mimetope sequences were recovered for both mAb and confirmed by competitive staining and kinetic measurements. A peptide-based ELISA method was developed for each. The assay for rituximab had a limit of detection of 4 μg/ml, and the assay for alemtuzumab had a limit of detection of 1 μg/ml. Antibody-specific staining of peptide conjugated beads could be seen in a dose-dependent manner. Phage-displayed peptide libraries can be a source of highly specific mimetopes for therapeutic mAb. The biotinylated forms of those peptides are compatible with conventional ELISA methods with sensitivities comparable to other assay methods and sufficient for pharmacological studies of those mAb given at high dose. The process outlined here can be applied to any mAb to enable improved pharmacokinetic analysis during the development and clinical use of this class of therapies

Rituximab in B-Cell Hematologic Malignancies: A Review of 20 Years of Clinical Experience

Rituximab is a human/murine, chimeric anti-CD20 monoclonal antibody with established efficacy, and a favorable and well-defined safety profile in patients with various CD20-expressing lymphoid malignancies, including indolent and aggressive forms of B-cell non-Hodgkin lymphoma. Since its first approval 20 years ago, intravenously administered rituximab has revolutionized the treatment of B-cell malignancies and has become a standard component of care for follicular lymphoma, diffuse large B-cell lymphoma, chronic lymphocytic leukemia, and mantle cell lymphoma. For all of these diseases, clinical trials have demonstrated that rituximab not only prolongs the time to disease progression but also extends overall survival. Efficacy benefits have also been shown in patients with marginal zone lymphoma and in more aggressive diseases such as Burkitt lymphoma. Although the proven clinical efficacy and success of rituximab has led to the development of other anti-CD20 monoclonal antibodies in recent years (e.g., obinutuzumab, ofatumumab, veltuzumab, and ocrelizumab), rituximab is likely to maintain a position within the therapeutic armamentarium because it is well established with a long history of successful clinical use. Furthermore, a subcutaneous formulation of the drug has been approved both in the EU and in the USA for the treatment of B-cell malignancies. Using the wealth of data published on rituximab during the last two decades, we review the preclinical development of rituximab and the clinical experience gained in the treatment of hematologic B-cell malignancies, with a focus on the well-established intravenous route of administration. This article is a companion paper to A. Davies, et al., which is also published in this issue

Two mechanisms of the enhanced antibody-dependent cellular cytotoxicity (ADCC) efficacy of non-fucosylated therapeutic antibodies in human blood

<p>Abstract</p> <p>Background</p> <p>Antibody-dependent cellular cytotoxicity (ADCC) has recently been identified as one of the critical mechanisms underlying the clinical efficacy of therapeutic antibodies, especially anticancer antibodies. Therapeutic antibodies fully lacking the core fucose of the Fc oligosaccharides have been found to exhibit much higher ADCC in humans than their fucosylated counterparts. However, data which show how fully non-fucosylated antibodies achieve such a high ADCC in human whole blood have not yet been disclosed. The precise mechanisms responsible for the high ADCC mediated by fully non-fucosylated therapeutic antibodies, even in the presence of human plasma, should be explained based on direct evidence of non-fucosylated antibody action in human blood.</p> <p>Methods</p> <p>Using a human <it>ex vivo </it>B-cell depletion assay with non-fucosylated and fucosylated anti-CD20 IgG1s rituximab, we monitored the binding of the therapeutic agents both to antigens on target cells (target side interaction) and to leukocyte receptors (FcγR) on effector cells (effector side interaction), comparing the intensities of ADCC in human blood.</p> <p>Results</p> <p>In the target side interaction, down-modulation of CD20 on B cells mediated by anti-CD20 was not observed. Simple competition for binding to the antigens on target B cells between fucosylated and non-fucosylated anti-CD20s was detected in human blood to cause inhibition of the enhanced ADCC of non-fucosylated anti-CD20 by fucosylated anti-CD20. In the effector side interaction, non-fucosylated anti-CD20 showed sufficiently high FcγRIIIa binding activity to overcome competition from plasma IgG for binding to FcγRIIIa on natural killer (NK) cells, whereas the binding of fucosylated anti-CD20 to FcγRIIIa was almost abolished in the presence of human plasma and failed to recruit NK cells effectively. The core fucosylation levels of individual serum IgG1 from healthy donors was found to be so slightly different that it did not affect the inhibitory effect on the ADCC of fucosylated anti-CD20.</p> <p>Conclusion</p> <p>Our results demonstrate that removal of fucosylated antibody ingredients from antibody therapeutics elicits high ADCC in human blood by two mechanisms: namely, by evading the inhibitory effects both of plasma IgG on FcγRIIIa binding (effector side interaction) and of fucosylated antibodies on antigen binding (target side interaction).</p

Translation Levels Control Multi-Spanning Membrane Protein Expression

Attempts to express eukaryotic multi-spanning membrane proteins at high-levels have been generally unsuccessful. In order to investigate the cause of this limitation and gain insight into the rate limiting processes involved, we have analyzed the effect of translation levels on the expression of several human membrane proteins in Escherichia coli (E. coli). These results demonstrate that excessive translation initiation rates of membrane proteins cause a block in protein synthesis and ultimately prevent the high-level accumulation of these proteins. Moderate translation rates allow coupling of peptide synthesis and membrane targeting, resulting in a significant increase in protein expression and accumulation over time. The current study evaluates four membrane proteins, CD20 (4-transmembrane (TM) helixes), the G-protein coupled receptors (GPCRs, 7-TMs) RA1c and EG-VEGFR1, and Patched 1 (12-TMs), and demonstrates the critical role of translation initiation rates in the targeting, insertion and folding of integral membrane proteins in the E. coli membrane

Engineering T cells for cancer therapy

It is generally accepted that the immune system plays an important role in controlling tumour development. However, the interplay between tumour and immune system is complex, as demonstrated by the fact that tumours can successfully establish and develop despite the presence of T cells in tumour. An improved understanding of how tumours evade T-cell surveillance, coupled with technical developments allowing the culture and manipulation of T cells, has driven the exploration of therapeutic strategies based on the adoptive transfer of tumour-specific T cells. The isolation, expansion and re-infusion of large numbers of tumour-specific T cells generated from tumour biopsies has been shown to be feasible. Indeed, impressive clinical responses have been documented in melanoma patients treated with these T cells. These studies and others demonstrate the potential of T cells for the adoptive therapy of cancer. However, the significant technical issues relating to the production of natural tumour-specific T cells suggest that the application of this approach is likely to be limited at the moment. With the advent of retroviral gene transfer technology, it has become possible to efficiently endow T cells with antigen-specific receptors. Using this strategy, it is potentially possible to generate large numbers of tumour reactive T cells rapidly. This review summarises the current gene therapy approaches in relation to the development of adoptive T-cell-based cancer treatments, as these methods now head towards testing in the clinical trial setting

Therapeutic vaccines and cancer: focus on DPX-0907

Mohan Karkada,1,2 Neil L Berinstein,3 Marc Mansour1 1ImmunoVaccine Inc, 2Department of Microbiology/Immunology, Dalhousie University, Halifax, NS, Canada; 3Ontario Institute for Cancer Research, Toronto, ON, Canada Abstract: In an attempt to significantly enhance immunogenicity of peptide cancer vaccines, we developed a novel non-emulsion depot-forming vaccine platform called DepoVax&trade; (DPX). Human leukocyte antigen (HLA)-A2 restricted peptides naturally presented by cancer cells were used as antigens to create a therapeutic cancer vaccine, DPX-0907. In a phase I clinical study, the safety and immune-activating potential of DPX-0907 in advanced-stage breast, ovarian, and prostate cancer patients were examined, following encouraging results in HLA-A2 transgenic mice. The DPX-0907 vaccine was shown to be safe and well tolerated, with injection-site reactions being the most commonly reported adverse event. Vaccinated cancer patients exhibited a 61% immune response rate, with higher response rates in the breast and ovarian cancer patient cohorts. In keeping with the higher immune efficacy of this vaccine platform, antigen-specific responses were detected in 73% of immune responders after just one vaccination. In 83% of responders, peptide-specific T-cells were detected at two or more time points post-vaccination, with 64% of these patients showing evidence of immune persistence. Immune monitoring also demonstrated the generation of antigen-specific T-cell memory, with the ability to secrete multiple type 1 cytokines. The novel DPX formulation promotes multifunctional effector/memory responses to peptide-based tumor-associated antigens. The data support the capacity of DPX-0907 to elicit type-1 biased immune responses, warranting further clinical development of the vaccine. In this review, we discuss the rationale for developing DPX-based therapeutic cancer vaccine(s), with a focus on DPX-0907, aimed at inducing efficient anti-tumor immunity that may eventually be shown to prolong patient survival. Keywords: immunotherapy, DepoVax&trade;, cancer vaccine, DPX-090