Ramakrishnan Melarkode

Nimotuzumab with chemoradiation confers a survival advantage in treatment-naïve head and neck tumors over expressing EGFR

Head and neck cancer associated with the chewing of betel preparations, including tobacco, is common to South East Asia. We report a Phase IIB study in which ninety-two treatment naïve patients with advanced squamous cell carcinoma received standard therapy with or without an anti-Epidermal Growth Factor Receptor (EGFR) monoclonal antibody (Nimotuzumab). In pretreatment samples, the tissue expression of ErbB family proteins and downstream molecules, including their association with clinical response and survival. Marker expression in tumor adjacent sections was evaluated by immunohistochemistry. The clinical benefit of Nimotuzumab (200 mg/dose, once a week for six weeks) in combination with radiotherapy or chemoradiation was assessed with respect to EGFR expression and intensity. Two antibodies, which recognized independent epitopes, were used to assess EGFR expression levels by immunohistochemistry. EGFR detection using mR3, an antibody with similar specificity to Nimotuzumab, correlated significantly with the expression of ErbB3 (p < 0.05), PCNA and pMAPK (p < 0.001). Although EGFR expression showed a significant relationship to patient survival in patients treated with Nimotuzumab and chemoradiation (p=0.02), pMAPK expression did not (p=0.07). Interestingly, EGFR overexpression (as defined by mR3) correlated directly with overall survival in this group (p=0.01). This data supports a model of basal activation of the EGFR signal transduction pathway in these oropharyngeal tumors. Detection of EGFR by immunohistochemistry could define a subset of treatment naïve Head and Neck cancer patients who may benefit from receiving EGFR targeted therapies in combination with chemoradiation.

CD6 synergistic co-stimulation promoting proinflammatory response is modulated without interfering with the activated leucocyte cell adhesion molecule interaction.

The CD6 membrane‐proximal scavenger receptor cysteine‐rich domain (SRCR3) includes the activated leucocyte cell adhesion molecule (ALCAM) binding site. CD6‐ALCAM mediates a low‐affinity interaction and their long‐term engagement contributes to the immunological synapse. Their ligation may play a dual function, facilitating stable adhesion between the antigen‐presenting cells and T cells during the early activation phase and later in the proliferative phase of the immune response. This study explored the strength of the CD6 co‐stimulatory effect and whether CD6 co‐stimulation with its natural ligand ALCAM also contributes to the lymphocyte effector differentiation. It was found that CD6–ALCAM interaction in vitro induced a synergistic co‐stimulation of normal human peripheral blood mononuclear cells, defined by Bliss analysis. CD6 co‐stimulation enhanced the CD3 proliferative efficacy by 23–34%. Moreover, a fivefold increment in the CD25 molecules number with a distinct gene transcription profile associated with cell activation, differentiation, survival and adhesion molecules was observed over CD3 single activation. Additionally, CD6 co‐stimulation in excess interleukin (IL)‐2 promotes a preferentially proinflammatory response. Besides, a CD6 membrane‐distal domain (SRCR1)‐specific non‐depleting monoclonal antibody (mAb) inhibited the induced proliferation in the presence of ALCAM, reducing interferon‐γ, IL‐6 and tumour necrosis factor‐α production. These results suggest that CD6 co‐stimulation enhances the intrinsic activity of the CD3 activation pathway and contributes to the T helper type 1 subset commitment, enhancing the IL‐2 sensitivity of recent activated human lymphocytes. It supports the role of CD6 as a susceptibility gene for pathological autoimmunity leading to tissue inflammation, and its relevance for targeted therapy.

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His-Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.

T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

CD6 is associated with T-cell modulation and is implicated in several autoimmune diseases. We previously demonstrated that Itolizumab, a CD6 domain 1 (CD6D1) specific humanized monoclonal antibody, inhibited the proliferation and cytokine production by T lymphocytes stimulated with anti-CD3 antibody or when co-stimulated with ALCAM. Aberrant IL-17 producing CD4+ helper T-cells (Th17) have been identified as pivotal for the pathogenesis of certain inflammatory autoimmune disorders, including psoriasis. Itolizumab has demonstrated efficacy in human diseases known to have an IL-17 driven pathogenesis. Here, in in vitro experiments we show that by day 3 of human PBMC activation using anti-CD3 and anti-CD28 co-stimulation in a Th17 polarizing milieu, 15–35% of CD4+ T-cells overexpress CD6 and there is an establishment of differentiated Th17 cells. Addition of Itolizumab reduces the activation and differentiation of T cells to Th17 cells and decreases production of IL-17. These effects are associated with the reduction of key transcription factors pSTAT3 and RORγT. Further, transcription analysis studies in these conditions indicate that Itolizumab suppressed T cell activation by primarily reducing cell cycle, DNA transcription and translation associated genes. To understand the mechanism of this inhibition, we evaluated the effect of this anti-human CD6D1 mAb on ALCAM-CD6 as well as TCR-mediated T cell activation. We show that Itolizumab but not its F(ab’)2 fragment directly inhibits CD6 receptor hyper-phosphorylation and leads to subsequent decrease in associated ZAP70 kinase and docking protein SLP76. Since Itolizumab binds to CD6 expressed only on human and chimpanzee, we developed an antibody binding specifically to mouse CD6D1. This antibody successfully ameliorated the incidence of experimental autoimmune encephalitis in the mice model. These results position CD6 as a key molecule in sustaining the activation and differentiation of T cells and an important target for modulating autoimmune diseases.

EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs

Nimotuzumab, an anti‐epidermal growth factor receptor (anti‐EGFR) monoclonal antibody, has been used extensively in many solid tumors and confers significant survival advantage. The antibody has limited skin toxicity and is generally well tolerated. Similar to other anti‐EGFR therapies, patients may relapse a few months after treatment. In this study we show for the first time, the use of Nimotuzumab along with Sirolimus has synergistic effect on tumor inhibition as compared with the drugs used individually, in Nimotuzumab responsive and nonresponsive cell lines. In vitro studies prove that while Sirolimus (25 nmol/L) affects the signal downstream to mammalian target of rapamycin (mTOR), Nimotuzumab (83 nmol/L) downregulates pTYR, pMAPK and pSTAT3 by 40%, 20% and 30%, respectively. The combination, targeting these two different signaling hubs, may be associated with the synergistic inhibition observed. In vivo, the use of half human therapeutic equivalent doses for both the drugs substantially reduces tumors established in nude as well as severe combined immunodeficiency (SCID) mice by EGFR overexpressing A‐431 cells. The drug combination reduces cell proliferation and the expression of signal transduction molecules. Treated tumors are better differentiated as compared with those established in the control mice. Tumor microarray demonstrates that Nimotuzumab and the combination groups segregate independently to the Sirolimus and the control treatment. The combination uniquely downregulated 55% of the altered tumor genes, extending beyond the typical pathways associated with Nimotuzumab and Sirolimus downstream pathways inhibition. These results would suggest that this nontoxic drug combination improves therapeutic benefit even in patients with low‐EGFR expression and severely immunocompromised because of their current medication.

Development and validation of a cell based assay for the detection of neutralizing antibodies against recombinant insulins

Recombinant biopharmaceuticals can induce generation of anti-drug antibodies, which could potentially neutralize therapeutic drug activity. In this report, we describe development and validation of a cell-based assay for detection of neutralizing antibodies (Nab) against insulin and insulin analogues. In order to achieve clinically meaningful sensitivity the method used an early signalling event, insulin induced insulin receptor phosphorylation as the endpoint. Percentage insulin receptor phosphorylation in cell lysates was measured using ECL based ELISA. Presence of neutralizing antibodies (Nab) in samples will inhibit insulin induced receptor phosphorylation and consequently lead to a reduction in the percentage of phosphorylated insulin receptor. Additionally, usage of human insulin receptor overexpressing recombinant CHO cell line further improved the assay sensitivity by reducing the fixed drug (EC50) concentration used for induction of receptor phosphorylation. To ensure adequate free drug tolerance a pre-treatment step was introduced, where serum samples underwent acid dissociation and charcoal extraction before drug incubation. In order to distinguish ADA positive samples containing true Nab from samples containing non-antibody phosphorylation inhibitory serum factors, a confirmatory tier was integrated based on immunodepletion using protein AGL mix. Assay parameters including determination of screening and confirmatory cut-points, intra and inter assay precision, selectivity, specificity and stability were assessed during validation in accordance with recent regulatory guidelines and white papers. The advantage of selecting insulin receptor phosphorylation as assay endpoint made the assay capable of detecting Nab against insulin and insulin analogues.

Development and validation of an electrochemiluminescent ELISA for quantitation of oral insulin tregopil in diabetes mellitus serum

AIM Tregopil, a novel PEGylated human insulin is in clinical development for oral delivery in diabetes treatment. The aim of the study was to develop and validate a sensitive and specific ELISA method for quantitating Tregopil in diabetes subjects on basal Glargine, since most commercially available insulin kits either do not detect Tregopil or show significant reactivity to Glargine. METHODS An electrochemiluminescent ELISA was developed and validated for Tregopil quantitation in diabetes serum. RESULTS The method has a LLOQ of 0.25 ng/ml, shows minimum cross-reactivity to Glargine and was successfully tested using a subset of samples from Tregopil-dosed Type 1 diabetes mellitus patients. CONCLUSION The ELISA method is sensitive and can be used to support accurate measurement of Tregopil with no cross-reactivity to Glargine and its metabolites in clinical studies.

Correction: T cell activation and differentiation is modulated by a CD6 domain 1 antibody Itolizumab

[This corrects the article DOI: 10.1371/journal.pone.0180088.].