Carl Dolman

“Cytokine Storm” in the Phase I Trial of Monoclonal Antibody TGN1412: Better Understanding the Causes to Improve PreClinical Testing of Immunotherapeutics

The CD28-specific mAb TGN1412 rapidly caused a life-threatening “cytokine storm” in all six healthy volunteers in the Phase I clinical trial of this superagonist, signaling a failure of preclinical safety testing. We report novel in vitro procedures in which TGN1412, immobilized in various ways, is presented to human white blood cells in a manner that stimulates the striking release of cytokines and profound lymphocyte proliferation that occurred in vivo in humans. The novel procedures would have predicted the toxicity of this superagonist and are now being applied to emerging immunotherapeutics and to other therapeutics that have the potential to act upon the immune system. Data from these novel procedures, along with data from in vitro and in vivo studies in nonhuman primates, suggest that the dose of TGN1412 given to human volunteers was close to the maximum immunostimulatory dose and that TGN1412 is not a superagonist in nonhuman primates.

Batches of intravenous immunoglobulin associated with adverse reactions in recipients contain atypically high anti‐Rh D activity

Background and Objectives The presence of anti‐Rh D in intravenous immunoglobulin (IVIG) products has been claimed to be associated with adverse reactions in recipients. There is currently no regulatory specification to control the level of anti‐D in IVIG products and it is unclear what this should be. Two reports of haemolysis occurring in recipients of IVIG manufactured from US plasma provided a rare opportunity to investigate whether high anti‐D levels could have induced the haemolysis.

Validation of an SEC‐HPLC Method for the Analysis of rhG‐CSF in Pharmaceutical Formulations

Abstract Granulocyte colony‐stimulating factor (G‐CSF) is a hematopoietic cytokine produced by recombinant DNA technology and used clinically to treat neutropenia. An isocratic high performance liquid chromatography procedure was developed for the assay of filgrastim in pharmaceutical formulations. HPLC separation was carried out by size‐exclusion chromatography on a TSK gel G2000 SW column (60 cm × 7.5 mm I.D.). The mobile phase was composed of phosphoric acid (pH 2.5; 0.1 M), run at a flow rate of 1.0 mL min−1 and with UV detection at 214 nm. Method validation investigated parameters such as the range, linearity (r 2 = 0.9998), precision, accuracy, and robustness; the method yielded good results with a quantitation limit of 45 µg mL−1 and a detection limit of 12 µg mL−1. The results demonstrate the validity of the SEC‐HPLC method for the analysis of filgrastim.

International collaborative study to evaluate a candidate reference preparation to define an appropriate specified limit of anti-D in intravenous immunoglobulin products

Background and Objectives  The aim of the study was to evaluate a lyophilized intravenous immunoglobulin (IVIG) preparation containing anti‐D (02/228; nominal reciprocal titre of 8) for its suitability to define the maximum limit of anti‐D in IVIG products when used in a proposed reference method of direct haemagglutination of papain‐treated erythrocytes, in an international collaborative study.

Characterization of Complete Particles (VSV-G/SIN-GFP) and Empty Particles (VSV-G/EMPTY) in Human Immunodeficiency Virus Type 1-Based Lentiviral Products for Gene Therapy: Potential Applications for Improvement of Product Quality and Safety

Lentiviral vectors persist in the host and are therefore ideally suited for long-term gene therapy. To advance the use of lentiviral vectors in humans, improvement of their production, purification, and characterization has become increasingly important and challenging. In addition to cellular contaminants derived from packaging cells, empty particles without therapeutic function are the major impurities that compromise product safety and efficacy. Removal of empty particles is difficult because of their innate similarity in particle size and protein composition to the complete particles. We propose that comparison of the properties of lentiviral products with those of purposely expressed empty particles may reveal potential differences between empty and complete particles. For this, three forms of recombinant lentiviral samples, that is, recombinant vesicular stomatitis virus glycoprotein (VSV-G) proteins, empty particles (VSV-G/Empty), and complete particles (VSV-G/SIN-GFP) carrying viral RNA, were purified by size-exclusion chromatography (SEC). The SEC-purified samples were further analyzed by immunoblotting with six antibodies to examine viral and cellular proteins associated with the particles. This study has demonstrated, for the first time, important differences between VSV-G/Empty particles and complete VSV-G/SIN-GFP particles. Differences include the processing of Gag protein and the inclusion of cellular proteins in the particles. Our findings support the development of improved production, purification, and characterization methods for lentiviral products.

05.09 The role of the european consensus finding study group (ecfsg) in characterising new tentative reference standards for autoantibody measurement

Background Since 1988, the European Consensus Finding Study Group on autoantibodies in rheumatic diseases (ECFSG), also known as the EULAR (European League Against Rheumatism) autoantibody study group, has been distributing sera with unspecified antibodies to European laboratories (presently n=43) for evaluation of different autoantibody measurement techniques in a clinical context. Use of international reference standards helps align test results by adopting internationally used measurement units, but there are no standards for many autoantibody specificities. Recently, the scope of ECFSG was expanded to include unbiased autoantibody characterisation of serum/plasma specimens with potential for serving as future autoantibody reference standards. Materials and methods Four samples were evaluated as tentative international reference standards for four different autoantibody specificities: double stranded/native DNA (dsDNA: 2013/14), IgG anti-beta2 glycoprotein 1 (b2GP1), proteinase 3 (PR3) and myeloperoxidase (MPO; 2015/16). The samples were evaluated ‘blind’ for multiple autoantibody specificities by participating laboratories. Results All or almost all participating laboratories detected the target specificities, and all samples showed restricted autoantibody specificities related to the target specificity. Anti-dsDNA was detected in the tentative anti-dsDNA standard by all laboratories using Crithidia luciliae, ELISA/EIA, FARR assay or ALBIA, together with a homogenous ANA pattern. Other specificities were restricted to histones, nucleosomes and anti-Ku. All laboratories but one detected IgG anti-b2GP1 and IgG anti-cardiolipin, mostly in high levels, in the tentative IgG anti-b2GP1 reference standard, whereas corresponding IgA and IgM antibodies were absent. All laboratories detected anti-MPO, mostly monospecific and in high levels together with P-ANCA pattern in the anti-MPO reagent. Anti-proteinase 3 and C-ANCA pattern, mostly in high levels/titers were detected by all laboratories in the tentative anti-PR3 reagent, Conclusions The expanded scope of ECFSG has enabled broad characterisation of new tentative autoantibody reference standards. The anti-dsDNA specimen has been processed by the National Institute for Biological Standards and Control (NIBSC) for consideration as the 2nd WHO anti-dsDNA reference standard. The other materials are basis for certified reference material for IgG anti-myeloperoxidase (ERM-DA476/IFCC), and the candidate reference materials for IgG anti-proteinase 3 (in certification) and for IgG anti-β2PG1 (in evaluation) from the Joint Research Centre of the EU Science Hub.