Andreas Nechansky

HAHA ― nothing to laugh about. Measuring the immunogenicity (human anti-human antibody response) induced by humanized monoclonal antibodies applying ELISA and SPR technology

Immunogenicity induced by passively applied proteins is a serious issue because it is directly related to the patients safety. The out-come of an immune reaction to a therapeutic protein can range from transient appearance of antibodies without any clinical significance to severe life threatening conditions. Within this article, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) methodology to measure immunogenicity are compared and the pros and cons are discussed.

Correlation of ADCC activity with cytokine release induced by the stably expressed, glyco-engineered humanized Lewis Y-specific monoclonal antibody MB314

A major limitation to the application of therapeutic monoclonal antibodies (mAbs) is their reduced in vivo efficacy compared with the high efficacy measured in vitro. Effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) are dramatically reduced in vivo by the presence of high amounts of endogenous IgG in the serum. Recent studies have shown that modification of the glycosylation moieties attached to the Fc part of the mAb can enhance binding affinity to FcγRIIIα receptors on natural killer cells and thus may counteract the reduced in vivo efficacy. In the present study, a humanized IgG1/κ monoclonal antibody recognizing the tumor-associated carbohydrate antigen Lewis Y was stably produced in a moss expression system that allows glyco-engineering. The glyco-modified mAb (designated MB314) showed a highly homogeneous N-glycosylation pattern lacking core-fucose. A side-by-side comparison to its parental counterpart produced in conventional mammalian cell-culture (MB311, formerly known as IGN311) by fluorescence-activated cell sorting analysis confirmed that the target specificity of MB314 is similar to that of MB311. In contrast, ADCC effector function of MB314 was increased up to 40-fold whereas complement dependent cytotoxicity activity was decreased 5-fold. Notably, a release of immunostimulatory cytokines, including interferon γ, monocyte chemotactic protein-1 (MCP-1), interleukin-6 and tumor necrosis factor (TNF) was particularly induced with the glyco-modified antibody. TNF release was associated with CD14+ cells, indicating activation of monocytes.

Comparison of the Calibration Standards of Three Commercially Available Multiplex Kits for Human Cytokine Measurement to WHO Standards Reveals Striking Differences

Serum parameters as indicators for the efficacy of therapeutic drugs are currently in the focus of intensive research. The induction of certain cytokines (or cytokine patterns) is known to be related to the status of the immune response e.g. in regulating the TH1/TH2 balance. Regarding their potential value as surrogate parameters in clinical trials and subsequently for the assignment of treatment efficacy, the accurate and reliable determination of cytokines in patient serum is mandatory. Because serum samples are precious and limited, test methods–-like the xMAP multiplex technology–-that allow for the simultaneous determination of a variety of cytokines from only a small sample aliquot, can offer great advantages. We here have compared multiplex kits from three different manufactures and found striking differences upon standardizing using WHO standards for selected cytokines. We therefore extended our xMAP multiplex measurements investigations to an ex-vivo situation by testing serum samples and found that the cytokine amounts measured was critically influenced by the actual kit used. The presented data indicate that statements regarding the quantitive determination of cytokines–-and therefore their use as biomarkers–-in serum samples have to be interpreted with caution.

Immunogenicity of therapeutics: a matter of efficacy and safety

Importance of the field: The unwanted immunogenicity of therapeutic proteins is a major concern regarding patient safety. Furthermore, pharmacokinetic, pharmacodynamic and clinical efficacy can be seriously affected by the immunogenicity of therapeutic proteins. Authorities have fully recognized this issue and demand appropriate and well-characterized assays to detect anti-drug antibodies (ADAs). Areas covered in this review: We provide an overview of the immunogenicity topic in general, the regulatory background and insight into underlying immunological mechanisms and the limited ability to predict clinical immunogenicity a priori. Furthermore, we comment on the analytical testing approach and the status-quo of appropriate method validation. What the reader will gain: The review provides insight regarding the analytical approach that is expected by regulatory authorities overseeing immunogenicity testing requirements. Additionally, the factors influencing immunogenicity are summarized and key references regarding immunogenicity testing approaches and method validation are discussed. Take home message: The unwanted immunogenicity of protein therapeutics is of major concern because of its potential to affect patient safety and drug efficacy. Analytical testing is sophisticated and requires more than one assay. Because immunogenicity in humans is hardly predictable, assay development has to start in a timely fashion and for clinical studies immunogenicity assay validation is mandatory prior to analyzing patient serum samples. Regarding ADAs, the question remains as to when such antibodies are regarded of clinical relevance and what levels are, if at all, acceptable. In summary, the detection of ADAs should raise the awareness of the physician concerning patient safety and of the sponsor/manufacture concerning the immunogenic potential of the drug product.

Immunization of Rhesus monkeys with a SialylTn–mAb17-1A conjugate vaccine co-formulated with QS-21 induces a temporary systemic cytokine release and NK cytotoxicity against tumor cells

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are frequently over-expressed on cancer cells and provide potential targets for cancer vaccination. Immunization of Rhesus monkeys with SialylTn coupled to a highly immunogenic carrier molecule and formulated on aluminum hydroxide induced a strong immune response against the carrier protein but only a moderate IgM immune response against the SialylTn carbohydrate antigen. Co-formulation with QS-21 adjuvant dramatically enhanced the anti-SialylTn immune response and resulted in a SialylTn-specific IgG switch. The kinetics of the carbohydrate-specific IgG response correlated with a temporary release of cytokines such as IFNγ, IL-2, IL-1β, TNFα and GM-CSF which was measurable in the immune serum by xMAP Multiplex technology. Furthermore, tumor cell killing by activated natural killer cells was induced. These data demonstrate that immunization with a tumor-associated carbohydrate antigen in a highly immunogenic formulation results in a temporary release of type 1 cytokines which may be required for the induction of a specific IgG immune response against the carbohydrate antigen as well as for activation of effector cells against tumor cells.

Interaction of human IgE with Fc epsilon RI alpha exposes hidden epitopes on IgE.

Background: Binding of human IgE via the heavy–chain constant region domain 3 (Cε3) to the α–chain of its high affinity receptor (FcεRIα) is a key event in mediating allergic reactions. We wanted to identify epitopes within Cε3 that are stable to denaturation and to evaluate whether such structures are involved in receptor binding. The existence of stable epitopes would facilitate the generation of compounds that inhibit the IgE–FcεRIα interaction. Methods: Monoclonal anti–human IgE–antibodies against recombinant bacterially synthesized Cε3, which is known to be partly misfolded, were raised in mice. These antibodies were probed for binding to native, immobilized and receptor–bound IgE, respectively, providing tools for the identification of the indicated stable epitopes. Results: Two of the generated antibodies (8E7, 3G9) discriminate between IgE in solution and IgE attached to FcεRIα, pointing towards a steric rearrangement within Cε3 induced upon receptor binding. The described antibodies represent tools for studying the mechanism of the Fcε–FcεRIα interaction and may be of diagnostic value since serum IgE from various human donors was differently recognized by 8E7, which is indicative for naturally occurring IgE molecules with different steric conformation. Conclusion: The presented data support the hypothesis of a conformational change within IgE Cε3 upon receptor binding by showing that monoclonal antibodies raised against recombinant Cε3 differently recognize soluble and receptor–bound IgE. The presence of an IgE portion in sera of human donors that is recognized by 8E7 indicates the existence of IgE molecules in different steric conformations in human blood, which may be related to pathologic parameters.

Qualification of a microfluidics-based electrophoretic method for impurity testing of monoclonal antibodies.

In this work, we present a comprehensive evaluation of the Agilent Bioanalyzer, a microfluidics-based electrophoretic device that was used for impurity testing of a monoclonal antibody (mAb). We compared the system to SDS-PAGE, both operated under non-reducing conditions and found a significant improvement of accuracy for the Bioanalyzer. In addition, the latter exhibited a larger assay range and lower limit of quantitation (LOQ) based on a predefined total error limit of +/-30%. However, during method qualification applying a three-factor nested design with two operators performing duplicate measurements per day, each on 4 different days, we observed unpredictable recurring quantitative outliers using the chip-based system. In-depth analysis on multiple runs with various chip lots confirmed the above finding and indicated that most likely on-chip dye labeling and/or post-column background fluorescence elimination are not compatible with the large size of the intact antibody as similar findings were observed for myosin used as upper marker for time correction. Interestingly, after reducing the intact antibody into light and heavy chain, we resolved the outlier issue. Eventually, requalification of the micro-fabricated analytical device under reducing conditions revealed only 1 out of 32 quality control samples (QCs) exceeding the +/-30% total error limits.

Application of Lectin Array Technology for Biobetter Characterization: Its Correlation with FcγRIII Binding and ADCC

Lectin microarray technology was applied to compare the glycosylation pattern of the monoclonal antibody MB311 expressed in SP2.0 cells to an antibody-dependent cellular cytotoxic effector function (ADCC)-optimized variant (MB314). MB314 was generated by a plant expression system that uses genetically modified moss protoplasts (Physcomitrella patens) to generate a de-fucosylated version of MB311. In contrast to MB311, no or very low interactions of MB314 with lectins Aspergillus oryzae l-fucose (AOL), Pisum sativum agglutinin (PSA), Lens culinaris agglutinin (LCA), and Aleuria aurantia lectin (AAL) were observed. These lectins are specific for mono-/biantennary N-glycans containing a core fucose residue. Importantly, this fucose indicative lectin-binding pattern correlated with increased MB314 binding to CD16 (FcγRIII; receptor for the constant region of an antibody)—whose affinity is mediated through core fucosylation—and stronger ADCC. In summary, these results demonstrate that lectin microarrays are useful orthogonal methods during antibody development and for characterization.

In Response to: ‘Impact of Glycosylation on Effector Functions of Therapeutic IgG’ (Pharmaceuticals 2010, 3, 146–157)

To complete the review article by Abes and colleagues (Pharmaceuticals 2010, 3, 146–157) which provides a good overview on recently developed approaches for generation of glyco-modified antibodies and the impact of glyco-modification of antibodies on their effector functions, important information should be added, namely that — besides the Glycart and the Biowa approach to generate de-fucosylated antibodies — innovative, moss derived methods have been shown to generate glyco-modified antibodies with improved effector function profile.