Jörg T. Regula

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.

A Novel Angiopoietin-2 Selective Fully Human Antibody with Potent Anti-Tumoral and Anti-Angiogenic Efficacy and Superior Side Effect Profile Compared to Pan-Angiopoietin-1/-2 Inhibitors

There is increasing experimental evidence for an important role of Angiopoietin-2 (Ang-2) in tumor angiogenesis and progression. In addition, Ang-2 is up-regulated in many cancer types and correlated with poor prognosis. To investigate the functional role of Ang-2 inhibition in tumor development and progression, we generated novel fully human antibodies that neutralize specifically the binding of Ang-2 to its receptor Tie2. The selected antibodies LC06 and LC08 recognize both rodent and human Ang-2 with high affinity, but LC06 shows a higher selectivity for Ang-2 over Ang-1 compared to LC08 which can be considered an Ang-2/Ang-1 cross-reactive antibody. Our data demonstrate that Ang-2 blockade results in potent tumor growth inhibition and pronounced tumor necrosis in subcutaneous and orthotopic tumor models. These effects are attended with a reduction of intratumoral microvessel density and tumor vessels characterized by fewer branches and increased pericyte coverage. Furthermore, anti-Ang-2 treatment strongly inhibits the dissemination of tumor cells to the lungs. Interestingly, in contrast to the Ang-2/Ang-1 cross-reactive antibody LC08 that leads to a regression of physiological vessels in the mouse trachea, the inhibition with the selective anti-Ang-2 antibody LC06 appears to be largely restricted to tumor vasculature without obvious effects on normal vasculature. Taken together, these data provide strong evidence for the selective Ang-2 antibody LC06 as promising new therapeutic agent for the treatment of various cancers.

The use of CrossMAb technology for the generation of bi- and multispecific antibodies

ABSTRACT The major challenge in the generation of bispecific IgG antibodies is enforcement of the correct heavy and light chain association. The correct association of generic light chains can be enabled using immunoglobulin domain crossover, known as CrossMAb technology, which can be combined with approaches enabling correct heavy chain association such as knobs-into-holes (KiH) technology or electrostatic steering. Since its development, this technology has proven to be very versatile, allowing the generation of various bispecific antibody formats, not only heterodimeric/asymmetric bivalent 1+1 CrossMAbs, but also tri- (2+1), tetravalent (2+2) bispecific and multispecific antibodies. Numerous CrossMAbs have been evaluated in preclinical studies, and, so far, 4 different tailor-made bispecific antibodies based on the CrossMAb technology have entered clinical studies. Here, we review the properties and activities of bispecific CrossMAbs and give an overview of the variety of CrossMAb-enabled antibody formats that differ from heterodimeric 1+1 bispecific IgG antibodies.

Low Level Sequence Variant Analysis of Recombinant Proteins: An Optimized Approach

Sequence variants in recombinant biopharmaceuticals may have a relevant and unpredictable impact on clinical safety and efficacy. Hence, their sensitive analysis is important throughout bioprocess development. The two stage analytical approach presented here provides a quick multi clone comparison of candidate production cell lines as a first stage, followed by an in-depth analysis including identification and quantitation of aberrant sequence variants of selected clones as a second stage. We show that the differential analysis is a suitable tool for sensitive and fast batch to batch comparison of recombinant proteins. The optimized approach allows for detection of not only single amino acid substitutions in unmodified peptides, but also substitutions in posttranslational modified peptides such as glycopeptides, for detection of truncated or elongated sequence variants as well as double amino acid substitutions or substitution with amino acid structural isomers within one peptide. In two case studies we were able to detect sequence variants of different origin down to a sub percentage level. One of the sequence variants (Thr → Asn) could be correlated to a cytosine to adenine substitution at DNA( desoxyribonucleic acid) level. In the second case we were able to correlate the sub percentage substitution (Phe → Tyr) to amino acid limitation in the chemically defined fermentation medium.

A Human Neutralizing Antibody Specific to Ang‐2 Inhibits Ocular Angiogenesis

Please cite this paper as: Rennel, Regula, Harper, Thomas, Klein and Bates (2011). A Human Neutralizing Antibody Specific to Ang‐2 Inhibits Ocular Angiogenesis. Microcirculation 18(7), 598–607.

Heavy and light chain pairing of bivalent quadroma and knobs-into-holes antibodies analyzed by UHR-ESI-QTOF mass spectrometry

The quadroma antibody represents the first attempt to produce a bispecific heterodimeric IgG antibody by somatic fusion of 2 hybridoma cells each expressing monoclonal antibodies with distinctive specificities. However, because of random heavy and light chain pairing, the desired functional bispecific antibody represents only a small fraction of the protein produced. Subsequently, the knobs-into-holes (KiH) approach was developed to enforce correct heavy chain heterodimerization. Assuming equimolar expression of 4 unmodified chains comprising 2 heavy and 2 light chains, the statistical distribution of all paired combinations can be calculated. With equimolar expression as the goal, we transfected HEK cells with 1:1:1:1 plasmid ratios and analyzed the protein A affinity-purified antibodies from the quadroma and KiH approaches qualitatively and quantitatively with regard to the estimated relative amounts of the products using electrospray quadrupole time-of-flight mass spectrometry. Our results show that all expected species are formed, and that, within the methodological limits, the species distribution in the mixtures corresponds approximately to the statistical distribution.

Anti-tumoral, anti-angiogenic and anti-metastatic efficacy of a tetravalent bispecific antibody (TAvi6) targeting VEGF-A and angiopoietin-2

ABSTRACT Vascular endothelial growth factor (VEGF)-A blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) is a key regulator of blood vessel remodeling and maturation. In tumors, Ang-2 is up-regulated and an unfavorable prognostic factor. Recent data demonstrated that Ang-2 inhibition mediates anti-tumoral effects. We generated a tetravalent bispecific antibody (Ang-2-VEGF-TAvi6) targeting VEGF-A with 2 arms based on bevacizumab (Avastin®), and targeting Ang-2 with 2 arms based on a novel anti-Ang-2 antibody (LC06). The two Ang-2-targeting single-chain variable fragments are disulfide-stabilized and fused to the C-terminus of the heavy chain of bevacizumab. Treatment with Ang-2-VEGF-A-TAvi6 led to a complete abrogation of angiogenesis in the cornea micropocket assay. Metastatic spread and tumor growth of subcutaneous, orthotopic and anti-VEGF-A resistant tumors were also efficiently inhibited. These data further establish Ang-2-VEGF bispecific antibodies as a promising anti-angiogenic, anti-metastatic and anti-tumor agent for the treatment of cancer.

Determination of antibody glycosylation by mass spectrometry.

Immunoglobulin (Ig) G is formed by two antigen-binding moieties termed Fabs and a conserved Fc -portion, which interacts with components of the immune systems. Within the Fc, N-linked carbohydrates are attached to each conserved asparagine residue at position 297 within the CH2 domain. These oligosaccharide moieties introduce a higher degree of heterogeneity within the molecule, by influencing stability of the antibody and its mediated effector functions, such as antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). The carbohydrate moieties can vary strongly depending on the production host and can be manipulated by different fermentation conditions, thereby influencing the function of the antibody. Therefore it is necessary to carefully monitor changes in the carbohydrate composition during cell line development and production processes. This chapter describes two different mass spectrometry based methods used for analyses of the carbohydrate moieties attached to the Fc-part of human IgG1. In the first approach, the glycans are released from the antibody by endoglycosidase (Peptide N Glycosidase F) digestion and monitored by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MS), whereas in the second method the carbohydrate structures, still attached to an enzymatically produced Fc-fragment, are analyzed by electrospray ionization mass spectrometry.

SPR-based assays enable the full functional analysis of bispecific molecules.

Graphical abstract The comparison of results from the assay described here to a recently described assay (Gassner et al., J. Pharm. Biom. Anal. 102, 144–149 (2014)) reveals the contribution of individual binding events to the overall binding of the bispecific drug, and also shows that both assay principles can be applied equally. The introduced SPR‐based assay principles represent generic approaches enabling the “full” functional analysis of bispecific molecules in only one assay. Figure. No Caption available. HighlightsA novel SPR‐based dual‐binding assay for bispecifics measures both interactions.Simultaneous binding can be calculated based on both measured individual readouts.Comparison to a SPR‐based bridging assay displayed good correlation.Assay principle selection should be guided by the molecular properties of a given bispecific molecule.Both assays allow a “full functional analysis” of bispecifics. ABSTRACT The increasing complexity of novel biotherapeutics such as bispecific antibodies or fusion proteins raises new challenges for functional characterization. When compared to standard antibodies, two individual interactions and the inter‐dependency of binding events need to be considered for bispecific antibodies. We have previously described an SPR‐based assay setup, which enables us to assess the binding activity of a bivalent‐bispecific molecule to both targets simultaneously and − in addition to one individual target − in a single setup. However, there might be some pitfalls when applying the bridging assay, e.g. change of antigen activity upon immobilization. Therefore, we have developed an alternative SPR‐based assay principle, which allows the individual assessment of both targets in solution. Comparison of data between the assays showed that simultaneous binding can be calculated based on both individual readouts, and revealed a good correlation. Hence, both SPR‐based assay principles allow a “full” functional analysis of a bispecific CrossMab in only one assay. The assay principles can be qualified and enable an efficient drug development.