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.

Introduction of the hybcell-based compact sequencing technology and comparison to state-of-the-art methodologies for KRAS mutation detection.

The detection of KRAS mutations in codons 12 and 13 is critical for anti-EGFR therapy strategies; however, only those methodologies with high sensitivity, specificity, and accuracy as well as the best cost and turnaround balance are suitable for routine daily testing. Here we compared the performance of compact sequencing using the novel hybcell technology with 454 next-generation sequencing (454-NGS), Sanger sequencing, and pyrosequencing, using an evaluation panel of 35 specimens. A total of 32 mutations and 10 wild-type cases were reported using 454-NGS as the reference method. Specificity ranged from 100% for Sanger sequencing to 80% for pyrosequencing. Sanger sequencing and hybcell-based compact sequencing achieved a sensitivity of 96%, whereas pyrosequencing had a sensitivity of 88%. Accuracy was 97% for Sanger sequencing, 85% for pyrosequencing, and 94% for hybcell-based compact sequencing. Quantitative results were obtained for 454-NGS and hybcell-based compact sequencing data, resulting in a significant correlation (r = 0.914). Whereas pyrosequencing and Sanger sequencing were not able to detect multiple mutated cell clones within one tumor specimen, 454-NGS and the hybcell-based compact sequencing detected multiple mutations in two specimens. Our comparison shows that the hybcell-based compact sequencing is a valuable alternative to state-of-the-art methodologies used for detection of clinically relevant point mutations.

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.

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 patient's 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.

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.

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.

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.

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 T(H)1/T(H)2 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.

Analysis of lysine clipping of a humanized Lewis-Y specific IgG antibody and its relation to Fc-mediated effector function.

During the analytical characterization of the humanized Lewis-Y specific monoclonal antibody IGN311 (IgG1/kappa) used for passive anti-cancer therapy in humans, isoelectric focusing (IEF) experiments revealed that IGN311 batches produced in serum-containing and serum-free medium, respectively, displayed different banding patterns. The additional bands in the IEF pattern correlated with additional peaks observed by subsequent cation exchange (CEX)-HPLC analysis. Since the IEF pattern is one of the specification criteria in the quality control of monoclonal antibodies and a non-matching pattern may be indicative for lot-to-lot inconsistency, this phenomenon was investigated in detail. First, we investigated whether a difference in antibody glycosylation was the cause for the observed charge heterogeneity. De-N-glycosylation experiments demonstrated that charge heterogeneity observed in the IEF pattern is not a consequence of glycosylation. In contrast, sample treatment by carboxypeptidase B, removing the carboxy-terminal lysine residues from the two heavy chains of the antibody, resulted in reduced charge heterogeneity eliminating the two most basic bands observed in IEF. These data were supported by reversed phase HPLC-MALDI-TOF-MS analysis of enzymatically cleaved peptides of the antibody as well as by carboxy-terminal sequencing of the heavy chains. It was demonstrated that the differences in the IEF banding pattern were due to lysine clipping occurring during the production of the antibody. The antibody batch produced under serum-free conditions was less affected by lysine clipping. Both antibody variants--clipped and unclipped--elicited the same potency in a complement dependent cytotoxicity (CDC) assay demonstrating that lysine clipping of IGN311 does not impair Fc-mediated effector functions.

Compensation of endogenous IgG mediated inhibition of antibody-dependent cellular cytotoxicity by glyco-engineering of therapeutic antibodies.

A major limitation to the application of therapeutic IgG antibodies (Abs) is their reduced in vivo efficacy compared to their high efficacy as measured in vitro. Recently, Preithner et al. showed that the high amount of endogenous serum IgG impairs the antibody-dependent cellular cytotoxicity effector function (ADCC) of therapeutic Abs in vivo by competing for binding to Fcgamma-RIII on the effector cells. Modification of the glycosylation moieties attached to the Fc part of the Ab, e.g. de-fucosylation, has been shown to increase ADCC activity. We here show that the ADCC activity of a fucose-deficient, moss-produced therapeutic IgG is not impaired by normal human serum. The increased ADCC activity of the fucose-deficient Ab variant even in the presence of high endogenous IgG indicates that glyco-engineering of Abs may translate into improved clinical efficacy. Noteworthy, moss production of glyco-modified Abs should be applicable to a broad variety of therapeutic Abs currently in use indicative for the potential of this technology platform.

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 IFNgamma, IL-2, IL-1beta, TNFalpha 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.

Cancer immunotherapy.

Cancer is the second leading cause of death in the industrialized world. Most cancer patients are treated by a combination of surgery, radiation and/or chemotherapy. Whereas the primary tumor can, in most cases, be efficiently treated by a combination of these standard therapies, preventing the metastatic spread of the disease through disseminated tumor cells is often not effective. The eradication of disseminated tumor cells present in the blood circulation and micro-metastases in distant organs therefore represents another promising approach in cancer immunotherapy. Main strategies of cancer immunotherapy aim at exploiting the therapeutic potential of tumor-specific antibodies and cellular immune effector mechanisms. Whereas passive antibody therapy relies on the repeated application of large quantities of tumor antigen-specific antibodies, active immunotherapy aims at the generation of a tumor-specific immune response combining both humoral and cytotoxic T cell effector mechanisms by the host's immune system following vaccination. In the first part of this review, concurrent developments in active and passive cancer immunotherapy are discussed. In the second part, the various approaches for the production of optimized monoclonal antibodies used for anti-cancer vaccination are summarized.

Immunization of Rhesus monkeys with the conjugate vaccine IGN402 induces an IgG immune response against carbohydrate and protein antigens, and cancer cells.

Tumor-associated antigens resulting from aberrant glycosylation, such as the SialylTn carbohydrate antigen, are over-expressed on cancer cells and provide potential targets for cancer vaccination. However, as T-cell-independent antigens carbohydrates are poorly immunogenic, and fail to induce memory. In order to increase the immunogenicity we have coupled the SialylTn carbohydrate antigen to a highly immunogenic carrier molecule, the murine monoclonal antibody mAb17-1A. An immunogenic formulation of the SialylTn-mAb17-1A conjugate on alhydrogel, IGN402, with or without additional adjuvants was tested in Rhesus monkeys for tolerability and immunogenicity. A significant antibody response against mAb17-1A antibody was found. Importantly, also a specific immune response against SialylTn carbohydrate and binding to tumor cells was induced. Immunization in the presence of additional adjuvants, such as QS-21, strongly enhanced the immune response against the carbohydrate antigen, and resulted in induction of SialylTn-specific IgG antibodies. Noteworthy, also an induced temporary release of cytokines including IFNgamma and IL-2, indicative for T-cell activation, was measured. The data indicate that carrier-induced T-cell help together with strong adjuvant is sufficient for carbohydrate specific class switch induction.

SialylTn-mAb17-1A carbohydrate-protein conjugate vaccine: effect of coupling density and presentation of SialylTn.

Carbohydrate antigens resulting from aberrant glycosylation of tumor cells, such as SialylTn, represent attractive targets for cancer vaccination. However, T-cell-independent carbohydrate antigens are poorly immunogenic and fail to induce memory and IgG class switch. Clustered expression patterns of some carbohydrates on the cell surface add further complexity to the design of carbohydrate-based vaccines. We describe here a vaccine consisting of SialylTn carbohydrate epitopes coupled to a highly immunogenic carrier molecule, mAb17-1A, adsorbed on alhydrogel and coformulated with a strong adjuvant, QS-21. The SialylTn-mAb17-1A conjugate vaccine was administered in Rhesus monkeys, and the immune responses against mAb17-1A, SialylTn, ovine submaxillary mucin, and tumor cells were analyzed. The data demonstrate that the density of carbohydrate epitopes on the carrier is an essential parameter for induction of anti-carbohydrate specific memory IgG immune responses. Furthermore, the influence of different types of presentation of SialylTn (monomeric vs trimers vs clustered via a branched polyethylenimine linker) on antibody titers and specificity was studied. High-density coupling of SialylTn epitopes to mAb17-1A induced the strongest immune response against synthetic SialylTn and showed also the highest reactivity against natural targets, such as OSM and tumor cells.