Dual anti-idiotypic purification of a novel, native-format biparatopic anti-MET antibody with improved in vitro and in vivo efficacy

Bispecific antibodies are of great interest due to their ability to simultaneously bind and engage different antigens or epitopes. Nevertheless, it remains a challenge to assemble, produce and/or purify them. Here we present an innovative dual anti-idiotypic purification process, which provides pure bispecific antibodies with native immunoglobulin format. Using this approach, a biparatopic IgG1 antibody targeting two distinct, HGF-competing, non-overlapping epitopes on the extracellular region of the MET receptor, was purified with camelid single-domain antibody fragments that bind specifically to the correct heavy chain/light chain pairings of each arm. The purity and functionality of the anti-MET biparatopic antibody was then confirmed by mass spectrometry and binding experiments, demonstrating its ability to simultaneously target the two epitopes recognized by the parental monoclonal antibodies. The improved MET-inhibitory activity of the biparatopic antibody compared to the parental monoclonal antibodies, was finally corroborated in cell-based assays and more importantly in a tumor xenograft mouse model. In conclusion, this approach is fast and specific, broadly applicable and results in the isolation of a pure, novel and native-format anti-MET biparatopic antibody that shows superior biological activity over the parental monospecific antibodies both in vitro and in vivo

GDF15/MIC1 and MMP9 Cerebrospinal Fluid Levels in Parkinson's Disease and Lewy Body Dementia.

Based on animal and ex-vivo experiments, Growth/Differentiation Factor-15 (GDF15, also called Macrophage Inhibitory Cytokine-1, MIC1), a member of the transforming growth factor-beta family, and Matrix Metalloproteinase-9 (MMP9), a member of the matrix metalloprotease family may be potential markers for Lewy body disorders, i.e. Parkinson's disease with (PDD) and without dementia (PDND) and Lewy body dementia (DLB). GDF15 has a prominent role in development, cell proliferation, differentiation, and repair, whereas MMP9 degrades, as a proteolytic enzyme, components of the extracellular matrix. In this study, cerebrospinal fluid GDF15 and MMP9 levels of 59 PDND, 17 PDD and 23 DLB patients, as well as of 95 controls were determined, and associated with demographic, clinical and biochemical parameters. Our analysis confirmed the already described association of GDF15 levels with age and gender. Corrected GDF15 levels were significantly higher in PDD than in PDND patients, and intermediate in DLB patients. Within Lewy body disorders, GDF15 levels correlated positively with age at onset of Parkinsonism and dementia, Hoehn & Yahr stage and cerebrospinal fluid t-Tau and p-Tau levels, and negatively with the Mini Mental State Examination. Remarkably, it does not relevantly correlate with disease duration. MMP9 was not relevantly associated with any of these parameters. Cerebrospinal GDF15, but not MMP9, may be a potential marker of and in Lewy body disorders

Acceptance, reliability and confidence of diagnosis of fetal and neonatal virtuopsy compared with conventional autopsy: A prospective study

Objectives To compare prospectively maternal acceptance of fetal and neonatal virtuopsy with that of conventional autopsy and to determine the confidence with which magnetic resonance (MR) virtuopsy can be used to diagnose normality/abnormality of various fetal anatomical structures. Methods MR and/or computed tomography virtuopsy and conventional autopsy were offered to 96 women (102 fetuses/neonates) following termination of pregnancy (TOP), intrauterine fetal death (IUFD) or neonatal death. Multivariable logistic regression analysis was used to investigate the effect on maternal acceptance of virtuopsy and/or conventional autopsy of the age of the mother, gestational age at TOP or delivery after IUFD, order of pregnancy, parity, religion, type of caregiver obtaining consent and reason for death. When parents consented to both MR virtuopsy and conventional autopsy of fetuses ≥ 20 weeks of gestation or neonates, the confidence with which MR virtuopsy could be used to diagnose normality/abnormality of various anatomical structures was determined on a scale in which conventional autopsy was considered gold standard. On autopsy we classified fetuses/neonates as having either 'normal' or 'abnormal' anatomical structures; these groups were analyzed separately. At virtuopsy, we indicated confidence of diagnosis of normality/abnormality of every anatomical structure in each of these two groups defined at autopsy, using a scale from 0 (definitely abnormal) to 100 (definitely normal). Results Of the 96 women, 99% (n = 95) consented to virtuopsy and 61.5% (n = 59) to both conventional autopsy and virtuopsy; i.e. 36 (37.5%) consented to virtuopsy alone. Maternal acceptance of conventional autopsy was independently positively related to singleton pregnancy, non-Moslem mother, earlier gestation at TOP or delivery afer IUFD and a maternal-fetal medicine specialist obtaining consent. Thirty-three fetuses ≥ 20 weeks of gestation had both conventional autopsy and MR virtuopsy, of which 19 had a full autopsy including the brain. In fetuses with normal anatomical structures at conventional autopsy, MR virtuopsy was associated with high diagnostic confidence (scores > 80) for the brain, skeleton, thoracic organs except the heart, abdominal organs except the pancreas, ureters, bladder and genitals. In fetuses with abnormal anatomical structures at autopsy, MR virtuopsy detected the anomalies with high confidence (scores < 20) for these same anatomical structures. However, in three cases, virtuopsy diagnosed brain anomalies additional to those observed at conventional autopsy. Conclusion MR virtuopsy is accepted by nearly all mothers while conventional autopsy is accepted by about two-thirds of mothers, in whom refusal depends mainly on factors over which we have no control. Although conventional autopsy remains the gold standard, the high acceptance of virtuopsy makes it an acceptable alternative when the former is declined. Copyright © 2012 ISUOG. Published by John Wiley & Sons, Ltd.SCOPUS: ar.jFLWINinfo:eu-repo/semantics/publishe

Depleting MET-expressing tumor cells by ADCC provides a therapeutic advantage over inhibiting HGF/MET signaling

International audienceHepatocyte growth factor (HGF) and its receptor MET represent validated targets for cancer therapy. However, HGF/MET inhibitors being explored as cancer therapeutics exhibit cytostatic activity rather than cytotoxic activity, which would be more desired. In this study, we engineered an antagonistic anti-MET antibody that, in addition to blocking HGF/MET signaling, also kills MET-overexpressing cancer cells by antibody-dependent cellular cytotoxicity (ADCC). As a control reagent, we engineered the same antibody in an ADCC-inactive form that is similarly capable of blocking HGF/MET activity, but in the absence of any effector function. In comparing these two antibodies in multiple mouse models of cancer, including HGF-dependent and -independent tumor xenografts, we determined that the ADCC-enhanced antibody was more efficacious than the ADCC-inactive antibody. In orthotopic mammary carcinoma models, ADCC enhancement was crucial to deplete circulating tumor cells and to suppress metastases. Prompted by these results, we optimized the ADCC-enhanced molecule for clinical development, generating an antibody (ARGX-111) with improved pharmacologic properties. ARGX-111 competed with HGF for MET binding, inhibiting ligand-dependent MET activity, downregulated cell surface expression of MET, curbing HGF-independent MET activity, and engaged natural killer cells to kill MET-expressing cancer cells, displaying MET-specific cytotoxic activity. ADCC assays confirmed the cytotoxic effects of ARGX-111 in multiple human cancer cell lines and patient-derived primary tumor specimens, including MET-expressing cancer stem-like cells. Together, our results show how ADCC provides a therapeutic advantage over conventional HGF/MET signaling blockade and generates proof-of-concept for ARGX-111 clinical testing in MET-positive oncologic malignancies. (C) 2015 AACR

Neonatal Fc receptor antagonist efgartigimod safely and sustainably reduces IgGs in humans

BACKGROUND: Intravenous Ig (IVIg), plasma exchange, and immunoadsorption are frequently used in the management of severe autoimmune diseases mediated by pathogenic IgG autoantibodies. These approaches modulating IgG levels can, however, be associated with some severe adverse reactions and a substantial burden to patients. Targeting the neonatal Fc receptor (FcRn) presents an innovative and potentially more effective, safer, and more convenient alternative for clearing pathogenic IgGs.METHODS: A randomized, double-blind, placebo-controlled first-in-human study was conducted in 62 healthy volunteers to explore single and multiple ascending intravenous doses of the FcRn antagonist efgartigimod. The study objectives were to assess safety, tolerability, pharmacokinetics, pharmacodynamics, and immunogenicity. The findings of this study were compared with the pharmacodynamics profile elicited by efgartigimod in cynomolgus monkeys.RESULTS: Efgartigimod treatment resulted in a rapid and specific clearance of serum IgG levels in both cynomolgus monkeys and healthy volunteers. In humans, single administration of efgartigimod reduced IgG levels up to 50%, while multiple dosing further lowered IgGs on average by 75% of baseline levels. Approximately 8 weeks following the last administration, IgG levels returned to baseline. Efgartigimod did not alter the homeostasis of albumin or Igs other than IgG, and no serious adverse events related to efgartigimod infusion were observed.CONCLUSION: Antagonizing FcRn using efgartigimod is safe and results in a specific, profound, and sustained reduction of serum IgG levels. These results warrant further evaluation of this therapeutic approach in IgG-driven autoimmune diseases.TRIAL REGISTRATION: Clinicaltrials.gov NCT03457649.FUNDING: argenx BVBA.</p

Correlation coefficients (r²) of CSF GDF15 and MMP9 and demographic, clinical and neurodegenerative parameters.

<p>Correlation coefficients (r²) of CSF GDF15 and MMP9 and demographic, clinical and neurodegenerative parameters.</p

Significant correlations of cerebrospinal fluid GDF15 levels with clinical and routine neurodegenerative markers in Lewy body disorders.

<p>(A) GDF15 and age of onset of Parkinsonism. (B) GDF15 and age of onset of dementia. (C) GDF15 and Hoehn and Yahr score. (D) GDF15 and t-Tau. (E) GDF15 and p-Tau. (F) GDF15 and Mini Mental State Examination. Aao, age at onset; CSF, cerebrospinal fluid; GDF15, growth differentiation factor 15; H&Y, Hoehn and Yahr score; MMSE, Mini Mental State Examination; p-Tau, phospho-Tau; r², correlation coefficient; t-Tau, total-Tau.</p

Demographical, clinical and biochemical data of the included cohorts.

<p>Demographical, clinical and biochemical data of the included cohorts.</p