Joerg Thomas Regula

Structure-based prediction of asparagine and aspartate degradation sites in antibody variable regions.

Monoclonal antibodies (mAbs) and proteins containing antibody domains are the most prevalent class of biotherapeutics in diverse indication areas. Today, established techniques such as immunization or phage display allow for an efficient generation of new mAbs. Besides functional properties, the stability of future therapeutic mAbs is a key selection criterion which is essential for the development of a drug candidate into a marketed product. Therapeutic proteins may degrade via asparagine (Asn) deamidation and aspartate (Asp) isomerization, but the factors responsible for such degradation remain poorly understood. We studied the structural properties of a large, uniform dataset of Asn and Asp residues in the variable domains of antibodies. Their structural parameters were correlated with the degradation propensities measured by mass spectrometry. We show that degradation hotspots can be characterized by their conformational flexibility, the size of the C-terminally flanking amino acid residue, and secondary structural parameters. From these results we derive an accurate in silico prediction method for the degradation propensity of both Asn and Asp residues in the complementarity-determining regions (CDRs) of mAbs.

Impact of molecular processing in the hinge region of therapeutic IgG4 antibodies on disposition profiles in cynomolgus monkeys

The IgG4 isotype antibody is a potential candidate for immunotherapy when reduced effector functions are desirable. However, antigen binding fragment (Fab) arm exchange leads to functional monovalency with potentially reduced therapeutic efficacy. Mutagenesis studies suggested that the CH3 domain and not the core hinge is dominantly involved in in vivo molecular processing. This work investigated whether stabilization of the core hinge of a therapeutic IgG4 antibody by mutation of Ser228 to Pro (S228P) would be sufficient to prevent in vivo Fab arm exchange. In vitro experiments evaluated the influence of different levels of oxidation-reduction conditions in buffer and serum on Fab arm exchange (swapping) of wild-type (WT) IgG4 and IgG1 and of IgG4 S228P, which included a sterically neutral second mutation (Leu235 replaced by Glu). The objective of single-dose pharmacokinetic experiments in cynomolgus monkeys was to determine whether the mutation reduced IgG4 swapping in vivo. The results indicated that S228P mutation did not completely prevent Fab arm exchange in vitro in buffer under reducing conditions relative to IgG4 WT. The immunoassay findings were confirmed by mass spectrometry measurements. Results of the in vivo studies suggested that the therapeutic IgG4 WT antibody exchanged Fab arms with endogenous cynomolgus monkey IgG4, resulting in bispecific IgG4 antibodies with monovalency for the therapeutic target. In contrast, serum from cynomolgus monkeys dosed with the IgG4 mutant was virtually free of swapped IgG4. In conclusion, the results indicated that IgG4 swapping in vivo was markedly attenuated by S228P mutation.

Crystal structure of an anti-Ang2 CrossFab demonstrates complete structural and functional integrity of the variable domain.

Bispecific antibodies are considered as a promising class of future biotherapeutic molecules. They comprise binding specificities for two different antigens, which may provide additive or synergistic modes of action. There is a wide variety of design alternatives for such bispecific antibodies, including the “CrossMab” format. CrossMabs contain a domain crossover in one of the antigen-binding (Fab) parts, together with the “knobs-and-holes” approach, to enforce the correct assembly of four different polypeptide chains into an IgG-like bispecific antibody. We determined the crystal structure of a hAng-2-binding Fab in its crossed and uncrossed form and show that CH1-CL-domain crossover does not induce significant perturbations of the structure and has no detectable influence on target binding.

Development and validation of a novel SPR-based assay principle for bispecific molecules

Surface plasmon resonance (SPR) is increasingly applied in drug discovery, early development and production. However, there are remarkably few reports describing the application of SPR in a regulated environment. Here, we describe a novel SPR-based assay, which enables us to assess the binding activity of a bivalent-bispecific anti-Ang-2/anti-VEGF antibody to both targets in a single setup. Validation of the assay revealed a high level of precision, accuracy, linearity and specificity. Upon analysis of temperature stressed samples it could be shown that firstly, the assay is able to indicate function-loss and secondly, it allows the parallel analysis of an additional interaction. Therefore, the described assay is highly suitable for quality assessment of the Ang-2/VEGF CrossMab. Additionally, the use of SPR in the context of assay development and routine use in a GMP environment is discussed.

Variable heavy–variable light domain and Fab-arm CrossMabs with charged residue exchanges to enforce correct light chain assembly

Abstract Technologies for the production of bispecific antibodies need to overcome two major challenges. The first one is correct heavy chain assembly, which was solved by knobs-into-holes technology or charge interactions in the CH3 domains. The second challenge is correct light chain assembly. This can be solved by engineering the Fab-arm interfaces or applying the immunoglobulin domain crossover approach. There are three different crossovers possible, namely Fab-arm, constant domain and variable domain crossovers. The CrossMabCH1–CL exchange does not lead to the formation of unexpected side products, whereas the CrossMabFab and the CrossMabVH–VL formats result in the formation of typical side products. Thus, CrossMabCH1–CL was initially favored for therapeutic antibody development. Here, we report a novel improved CrossMab design principle making use of site-specific positional exchanges of charged amino acid pairs in the constant domain of these CrossMabs to enable the correct light chain assembly in the CrossMabVH–VL and improvements for the CrossMabFab design.

Abstract B86: 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 efficiency

Purpose: VEGF-A blockade has been validated clinically 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 recently described a novel generic method for the production of bivalent bispecific human IgG1 antibodies. Subsequently, we have applied the CrossMab technology for the generation of a bispecific antibody recognizing VEGF-A with one arm based on Bevacizumab, and the other arm recognizing Ang-2 based on LC06, a highly Ang-2 selective human IgG1 antibody. Ang-2-VEGF CrossMab was evaluated in vitro and in vivo using molecular, biochemical, cellular and histological analyses to determine its pharmacologic properties, along with its mechanism of action. Results: Ang-2-VEGF-A CrossMab showed potent tumor growth inhibition in a panel of orthotopic and subcutaneous syngeneic mouse tumors and patient and cell line-derived human tumor xenografts at both early and late stages of tumor development. Furthermore, a strong inhibition of angiogenesis and an enhanced vessel normalization phenotype were observed. In contrast to Ang-1 inhibition, anti-Ang-2-VEGF-A treatment did not aggravate the adverse effect of anti-VEGF treatment on physiological 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: Our data indicate that Ang-2-VEGF-A CrossMab mediates potent anti-tumor, anti-angiogenic, and anti-metastatic efficacy and represents a promising therapeutic agent for the therapy of cancer. Citation Format: Erica Lorenzon, Yvonne Kienast, Romi Raemsch, Werner Scheuer, Frank Herting, Dirk Bernicke, Wolfgang Schaefer, Joerg Regula, Kay Stubenrauch, Sidney Yu, Huynh Hung The, Laurent Martarello, Christian Klein, Markus Thomas. 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 efficiency. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr B86.

Abstract 2319: Dual inhibition of Ang-2 and VEGF via a novel human bispecific bivalent IgG1 CrossMAb shows potent anti-angiogenic, antitumoral, and antimetastatic efficacy and leads to a reduced side effect profile compared to single therapies

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL VEGF-A blockade has been extensively clinically validated as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling, tumor angiogenesis, and metastasis. In tumors, Ang-2 is up-regulated and associated with poor prognosis. Recent data demonstrated that Ang-2 inhibitors, both as single agents or in combination with chemo- or anti-VEGF therapy, mediate anti-tumor effects. Additionally, it has been shown that the Ang-2/-1/Tie and the VEGF/VEGFR systems act in complementary ways suggesting that dual targeting may be more effective than targeting each pathway alone. Based on bevacizumab and the Ang-2 selective antibody LC06 we have generated a novel human bispecific bivalent IgG1 CrossMab antibody blocking VEGF-A and Ang-2 function simultaneously. Here we show in multiple subcutaneous and orthotopic in vivo models including models (semi-) resistant to anti-VEGF treatment that the systemic application of the Ang-2-VEGF CrossMAb effectively reduces angiogenesis, tumor growth and metastasis. Furthermore, we demonstrate that a highly selective anti-Ang-2 approach has safety related advantages in this model over an unselective treatment with an antibody targeting Ang-1 and Ang-2 simultaneously. Whereas anti-Ang-1/Ang-2 long-term treatment resulted in regression of healthy vessels in the mouse trachea, an anti-Ang-2 selective treatment did not affect the physiological vessels in the trachea of the mice at all. These results imply a clear differentiation between selective Ang-2 and unselective Ang-1/Ang-2 inhibition. Although anti-tumoral efficacy is retained selective Ang-2 inhibition did not lead to a further impairment of healthy vessels compared to anti-VEGF-A treatment only. Finally, we demonstrate a clear disadvantage of Ang-2 monotherapy compared to Ang-2-VEGF dual inhibition due to strong up-regulation of VEGF resulting not only in revascularization and tumor growth, but also in toxicity with macroscopic evidence of histopathological subcapsular peliosis-like changes in the liver. These pathological effects were inhibited by dual inhibition of Ang-2 and VEGF-A. Taken together, our data indicate that Ang-2 and VEGF-A exhibit angiogenic synergy in a mutually compensatory fashion and that their inhibition via the novel Ang-2-VEGF CrossMab mediates potent anti-tumoral, anti-metastatic and anti-angiogenic efficacy. Additionally the CrossMAb is expected to exhibtit a better side effect profile compared to the respective monotherapies and thereby represents a promising therapeutic agent for the therapy of cancer. These data support the investigation of the Ang-2-VEGF CrossMAb in PhI clinical trials scheduled for 2012. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2319. doi:1538-7445.AM2012-2319