Kay Stubenrauch

Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies

We describe a generic approach to assemble correctly two heavy and two light chains, derived from two existing antibodies, to form human bivalent bispecific IgG antibodies without use of artificial linkers. Based on the knobs-into-holes technology that enables heterodimerization of the heavy chains, correct association of the light chains and their cognate heavy chains is achieved by exchange of heavy-chain and light-chain domains within the antigen binding fragment (Fab) of one half of the bispecific antibody. This “crossover” retains the antigen-binding affinity but makes the two arms so different that light-chain mispairing can no longer occur. Applying the three possible “CrossMab” formats, we generated bispecific antibodies against angiopoietin-2 (Ang-2) and vascular endothelial growth factor A (VEGF-A) and show that they can be produced by standard techniques, exhibit stabilities comparable to natural antibodies, and bind both targets simultaneously with unaltered affinity. Because of its superior side-product profile, the CrossMabCH1-CL was selected for in vivo profiling and showed potent antiangiogenic and antitumoral activity.

Progress in overcoming the chain association issue in bispecific heterodimeric IgG antibodies

The development of bispecific antibodies has attracted substantial interest, and many different formats have been described. Those specifically containing an Fc part are mostly tetravalent, such as stabilized IgG-scFv fusions or dual-variable domain (DVD) IgGs. However, although they exhibit IgG-like properties and technical developability, these formats differ in size and geometry from classical IgG antibodies. Thus, considerable efforts focus on bispecific heterodimeric IgG antibodies that more closely mimic natural IgG molecules. The inherent chain association problem encountered when producing bispecific heterodimeric IgG antibodies can be overcome by several methods. While technologies like knobs-into-holes (KiH) combined with a common light chain or the CrossMab technology enforce the correct chain association, other approaches, e.g., the dual-acting Fab (DAF) IgGs, do not rely on a heterodimeric Fc part. This review discusses the state of the art in bispecific heterodimeric IgG antibodies, with an emphasis on recent progress.

Ang-2-VEGF-A CrossMab, a Novel Bispecific Human IgG1 Antibody Blocking VEGF-A and Ang-2 Functions Simultaneously, Mediates Potent Antitumor, Antiangiogenic, and Antimetastatic Efficacy

Purpose: VEGF-A blockade has been clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of tumor angiogenesis and metastasis. Experimental Design: We have applied the recently developed CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on bevacizumab (Avastin), and the other arm recognizing Ang-2 based on LC06, an Ang-2 selective human IgG1 antibody. The potency of Ang-2-VEGF CrossMab was evaluated alone and in combination with chemotherapy using orthotopic and subcutaneous xenotransplantations, along with metastasis analysis by quantitative real-time Alu-PCR and ex vivo evaluation of vessels, hypoxia, proliferation, and apoptosis. The mechanism of action was further elucidated using Western blotting and ELISA assays. Results: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient or cell line-derived human tumor xenografts, especially at later stages of tumor development. Ang-2-VEGF-A CrossMab treatment led to a strong inhibition of angiogenesis and an enhanced vessel maturation phenotype. Neoadjuvant combination with chemotherapy resulted in complete tumor regression in primary tumor-bearing Ang-2-VEGF-A CrossMab-treated mice. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiologic vessels. Moreover, treatment with Ang-2-VEGF-A CrossMab resulted in inhibition of hematogenous spread of tumor cells to other organs and reduced micrometastatic growth in the adjuvant setting. Conclusion: These data establish Ang-2-VEGF-A CrossMab as a promising antitumor, antiangiogenic, and antimetastatic agent for the treatment of cancer. Clin Cancer Res; 19(24); 6730–40. ©2013 AACR.

Ang-2/VEGF bispecific antibody reprograms macrophages and resident microglia to anti-tumor phenotype and prolongs glioblastoma survival

Significance Improving survival of patients with glioblastoma (GBM) using antiangiogenic therapy remains a challenge. In this study we show that dual blockade of angiopoietin-2 and vascular endothelial growth factor delays tumor growth and enhances survival benefits through reprogramming of tumor-associated macrophages toward an antitumor phenotype as well as by pruning immature tumor vessels. The antitumor immunomodulatory potential of this dual blockade supports clinical testing of this approach for GBM with other immunotherapeutic approaches such as checkpoint blockers. Inhibition of the vascular endothelial growth factor (VEGF) pathway has failed to improve overall survival of patients with glioblastoma (GBM). We previously showed that angiopoietin-2 (Ang-2) overexpression compromised the benefit from anti-VEGF therapy in a preclinical GBM model. Here we investigated whether dual Ang-2/VEGF inhibition could overcome resistance to anti-VEGF treatment. We treated mice bearing orthotopic syngeneic (Gl261) GBMs or human (MGG8) GBM xenografts with antibodies inhibiting VEGF (B20), or Ang-2/VEGF (CrossMab, A2V). We examined the effects of treatment on the tumor vasculature, immune cell populations, tumor growth, and survival in both the Gl261 and MGG8 tumor models. We found that in the Gl261 model, which displays a highly abnormal tumor vasculature, A2V decreased vessel density, delayed tumor growth, and prolonged survival compared with B20. In the MGG8 model, which displays a low degree of vessel abnormality, A2V induced no significant changes in the tumor vasculature but still prolonged survival. In both the Gl261 and MGG8 models A2V reprogrammed protumor M2 macrophages toward the antitumor M1 phenotype. Our findings indicate that A2V may prolong survival in mice with GBM by reprogramming the tumor immune microenvironment and delaying tumor growth.

Subset analysis of patients experiencing clinical events of a potentially immunogenic nature in the pivotal clinical trials of tocilizumab for rheumatoid arthritis: Evaluation of an antidrug antibody ELISA using clinical adverse event-driven immunogenicity testing.

BACKGROUND Tocilizumab (TCZ) is a recombinant humanized monoclonal antibody directed against the interleukin-6 receptor. In Europe, TCZ is approved for use in combination with methotrexate in the treatment of adult patients with moderate to severe rheumatoid arthritis (RA) who have failed to respond to or were unable to tolerate previous therapy with one or more disease-modifying antirheumatic drugs or tumor necrosis factor (TNF) antagonists; in the United States, it is approved for the treatment of adult patients with moderate to severe active RA who have failed to respond to one or more TNF antagonists. As part of the Phase III clinical development program, the immunogenicity of TCZ was evaluated using a bridging ELISA; however, this assay is considered limited in detecting low-affinity or immunoglobulin G4 subisotype antidrug antibodies (ADAs). OBJECTIVE This study assessed the validity of the ELISA for detecting anti-TCZ ADAs by using complementary bioanalytic assays to test samples from a subgroup of patients with clinical adverse events (AEs) of a potentially immunogenic nature, who were considered highly likely to have ADAs. The goal was to determine whether use of these additional assays led to detection of ADAs not found on the ELISA, thus minimizing the risk of false-negative results. METHODS The Phase III program for TCZ consisted of 5 core studies in which adult patients with RA received either TCZ 4 or 8 mg/kg IV or control every 4 weeks, with or without concomitant antirheumatic therapy. Blood samples obtained at baseline and at regular intervals thereafter were tested using the ELISA for ADA screening and confirmation. Regardless of the results on ADA screening, samples from patients who developed clinical AEs of a potentially immunogenic nature (ie, falling within predefined system organ classes, occurring during or within 24 hours of TCZ infusion, considered related to TCZ therapy, or leading to study withdrawal) were subjected to additional testing with a surface plasmon resonance (SPR) assay for isotyping and epitope localization and a standard ImmunoCAP immunoglobulin E (IgE) assay made specific for TCZ. RESULTS The 5 core studies and their open-label, longterm extension studies enrolled a total of 4199 adult patients with RA (82.1% female; 74.0% white; mean age, 52.0 years [range, 18-89 years]; mean weight, 73.4 kg [range, 35-150 kg]); 2928 patients received TCZ and 1271 received control. Of the 2816 samples from TCZ-treated patients tested, 64 (2.3%) had samples that tested positive at least once on the ELISA screening and confirmation assay, 48 (75.0%) of them at baseline. A clinical AE of a potentially immunogenic nature occurred during TCZ treatment in 21 patients, 8 of whom had an anaphylactic reaction. Eleven of the samples from these 21 patients had tested negative for AD As on the screening ELISA. Only 1 of these 11 patients tested positive for ADAs on both additional assays; all others tested negative. The results of the ELISA, SPR, and IgE assays were consistent for 16 of 18 tested patients (88.9%) who provided data on at least 2 of the 3 assays. CONCLUSIONS Based on the findings of this analysis in a relevant patient population, the bridging-type screening and confirmation ELISA was a valid method of detecting anti-TCZ ADAs. Immunogenicity testing of samples from patients with clinical AEs of a potentially immunogenic nature using assays complementary to the ELISA added valuable information about the incidence and character of ADAs.

Quality requirements for critical assay reagents used in bioanalysis of therapeutic proteins: what bioanalysts should know about their reagents

Ligand-binding assays are the standard technology used for bioanalysis of therapeutic proteins, for example, for drug quantification (pharmacokinetics assays) and immunogenicity testing (antidrug antibody assays). Besides the selection of the most suitable technology platform (e.g., ELISA, electrochemiluminescence assays and surface plasmon resonance assays) and assay procedure, a pivotal prerequisite for good assay performance on any technology platform is the design, production and characterization of high quality reagents. To enable bioanalytical project support over the complete product life cycle, an appropriate long-term reagent supply is needed. This perspective describes our opinion on the requirements for generation and QC of critical reagents used in ligand-binding assays for drug quantification and antidrug antibody detection to enable high-quality assays and long-term supply, including reagent batch switches. The critical parameters during reagent design, production and long-term supply, along with the appropriate analytical methods for QC testing and appropriate certification, are discussed.

Conjugation of an antibody Fv fragment to a virus coat protein: cell-specific targeting of recombinant polyoma-virus-like particles

The development of cell-type-specific delivery systems is highly desirable for gene-therapeutic applications. Current virus-based vector systems show broad cell specificity, which results in the need to restrict the natural tropism of these viral systems. Here we demonstrate that tumour-cell-specific virus-like particles can be functionally assembled in vitro from recombinant viral coat protein expressed in Escherichia coli. The insertion of a negatively charged peptide in the HI loop of polyoma VP1 interferes with the binding of VP1 to the natural recognition site on mammalian cells and also serves as an adapter for the coupling of antibody fragments that contain complementary charged fusion peptides. A recombinant antibody fragment of the tumour-specific anti-(Lewis Y) antibody B3 could be coupled to the mutant VP1 by engineered polyionic peptides and an additional disulphide bond. With this system an entirely recombinant cell-specific delivery system assembled in vitro could be generated that transfers genes preferentially to cells presenting the tumour-specific antigen on the cell surface.

Evaluation of an immunoassay for human-specific quantitation of therapeutic antibodies in serum samples from non-human primates

Pharmacokinetic characterization of therapeutic antibodies plays an important role during preclinical and clinical development. However, accurate pharmacokinetic evaluation of therapeutic antibodies in serum samples from non-human primates is often complicated by insufficient specificity of the assays to measure drug levels. The present paper describes the use of a murine monoclonal antibody in an immunoassay format to specifically and quantitatively measure human therapeutic antibodies in serum from non-human primates. This murine antibody is directed against a unique epitope on the constant region CH2 domain of all isotypes of human immunoglobulin G (IgG). The antibody, designated anti-human Fcgamma-pan: R10Z8E9, does not cross-react with serum from mouse, rat, and the non-human primates marmoset, rhesus macaque, cynomolgus monkey and baboon when using an enzyme-linked immunosorbent assay (ELISA) or surface plasmon resonance technology (Biacore) format for measurement of the therapeutic antibody. Use of the antibody anti-human Fcgamma-pan: R10Z8E9 as capturing and detection reagent allowed human-specific quantitation of total therapeutic antibody anti-IGF-1R in spiked cynomolgus monkey serum via a Sandwich ELISA format. In contrast, a commercially available polyclonal antibody (PAB) directed to the Fcgamma fragment of human IgG only specifically measured the therapeutic antibody in buffer samples, but not in serum from cynomolgus monkeys. This generic human IgG assay was already applied in several pharmacokinetic studies in cynomolgus monkeys to determine serum levels of different therapeutic antibodies, including the anti-IGF-1R. Validation of the assay for a humanized IgG1 therapeutic antibody against a membrane protein revealed a lower limit of quantitation of 8 ng/mL in undiluted serum. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 15%. Dilutional linearity was evidenced by a recovery of 98.7-114% of expected concentrations. In conclusion, the monoclonal antibody anti-human Fcgamma-pan: R10Z8E9 provides a standard means for human-specific quantitation of therapeutic antibodies with high sensitivity in serum samples from non-human primates in a generic human IgG assay.

Evaluation of a generic immunoassay with drug tolerance to detect immune complexes in serum samples from cynomolgus monkeys after administration of human antibodies.

Current state of the art bridging ELISA technologies for detection of anti-drug antibodies (ADAs) against therapeutic antibodies bear the risk of false-negative results due to interference by circulating drug. Methods to remove the drug in the sample or sample pre-treatment techniques such as acid dissociation of the immune complexes are limited, laborious and may destroy ADAs resulting again in false-negative results. The immune complex ELISA described in this publication provides a simple solution. It is designed to analyze samples from cynomolgus monkeys dosed with human antibodies; it can be used for all human antibodies since it is independent of the specific antibody and its target. The generic applicability of the ADA assay is enabled by the use of (1) a murine anti-human Fc monoclonal antibody (MAb) as capture reagent; (2) a murine anti-cynomolgus monkey IgG MAb as detection reagent; and (3) an ADA positive control conjugate consisting of cynomolgus IgG complexed with human IgG. In its basic version, the generic ADA ELISA specifically detects only immune complexes formed in vivo. Validation of the ADA assay revealed a lower limit of quantitation of 15.6 ng/mL in serum samples. Intra-assay and inter-assay precision was characterized by a coefficient of variation of less than 10% and accuracy was within 8%. Matrix effects were low as evidenced by a mean recovery of 95%. In vitro pre-incubation of the serum samples with drug makes also the free ADA in the sample amenable to measurement by the immune complex ELISA as demonstrated by analysis of ADAs from two cynomolgus monkey studies with two different antibodies. The generic and versatile nature of this ADA assay favors its use in pilot pharmacokinetic and safety studies in cynomolgus monkeys during candidate selection of antibodies. The assay can help to explain unexpected drug clearance profiles, loss of efficacy or safety events caused by immune complexes and guide further development.

Advanced glycation end products and receptor for advanced glycation end products in AA amyloidosis

Advanced glycation end products (AGEs) may be involved in either amyloidogenesis or complications related to amyloid. We hypothesized that AGEs may influence the pathogenesis of AA amyloidosis, and investigated the spatial and temporal relationship between AGEs, carboxy methyl lysine (CML), the AGE receptor (RAGE), and AA amyloid in humans and mice. Specimens from patients with AL and ATTR amyloidosis served as a control. Using immunohistochemistry, AGEs, CML, and RAGE were found within amyloid deposits, more commonly in AA amyloid than in AL amyloid and not in ATTR amyloid. Western blotting showed that multiple proteins (between 12 and >60 kd) are modified, but not the AA amyloid fibril protein itself. In the murine model of AA amyloidosis, we found a marked interindividual variability with respect to local and systemic CML levels, as well as to splenic RAGE transcription. Serum levels of CML correlated with the duration of the inflammatory response but not with amounts of splenic RAGE mRNA. Other as yet unidentified variables, especially of the heterogeneous group of AGEs, probably modulate transcription of RAGE and influence amyloidogenesis. CML serum levels, in turn, may prove useful in predicting patients at risk.