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Bispecific antibodies have recently gained major interest as they allow novel mechanisms-of-action and/or therapeutic applications that cannot be achieved using conventional IgG-based antibodies. A major issue in engineering IgG-based bispecific antibodies has been to enable the correct association of heavy and light chains resulting in correct assembly of the desired bispecific antibody in sufficient yield. Various approaches have been described during recent years to tackle this challenge. We have developed the so-called CrossMab technology that enforces correct light chain association based on the domain crossover of immunoglobulin domains in the Fab region of the bispecific antibody. This versatile technology allows the generation of different bispecific antibody formats including asymmetric heterodimeric monovalent 1 + 1 bispecific antibodies and asymmetric heterodimeric bispecific antibodies with 2 + 1 valency in combination with approaches enabling Fc-hetermodimerization like knob-into-hole technology as well as the generation of tetravalent symmetric bispecific antibodies with 2 + 2 valency, also known as Tandem-Fab based IgG antibodies, using processes suitable for the large scale production of therapeutic bispecific antibodies. Notably, as of now, at least eight different bispecific antibodies using CrossMab technology entered clinical development, and additional CrossMabs are in late preclinical development. This review provides a summary of the status and progress with the engineering and generation of CrossMab technology based bispecific antibodies as well as their therapeutic application.

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.

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.

Therapeutic antibodies and antibody derivatives comprise the majority of today's biotherapeutics. Routine methods to generate novel antibodies, such as immunization and phage-display, often give rise to several candidates with desired functional properties. On the contrary, resource-intense steps such as the development of a cell line, a manufacturing process, or a formulation, are typically carried out for only one candidate. Therefore, "developability," that is, the likelihood for the successful development of a lead candidate into a stable, manufacturable, safe, and efficacious drug, may be used as an additional selection criterion. Employing a set of small-scale, fast, and predictive tests addressing biochemical and biophysical features, as well as in vivo fate can help to identify a clinical candidate molecule with promising properties at an early stage of drug development. This article gives an overview of existing methods for developability testing and shows how these assays can be interlaced in the lead selection process.

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.

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.

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

Abstract 4304 Background: VEGF 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 blood vessel remodeling and tumor angiogenesis. Several publications described that VEGF and Ang-2 are up-regulated in several hematological tumors like B-CLL, AML or multiple myeloma. In particular, Ang-2 may qualify as a novel prognostic marker in AML and CLL. We have generated TAvi6, a novel bispecific antibody targeting VEGF-A and Ang-2 and tested its anti-tumor and anti-angiogenic activity. TAvi6 is a tetravalent IgG-like bispecific antibody based on bevacizumab and targets Ang-2 with 2 disulfide-stabilized scFvs (LC06) fused to the C-terminus of the heavy chain. Material and Methods: TAvi 6 was profiled in biochemical and cellular (angiogenesis) assays. Antitumoral and anti-angiogenic efficacy was assessed in established s.c. Colo205, s.c. Calu-3 and orthotopic i.m.f.p. KPL-4 xenografts in SCID beige mice. Mice were treated with bevacizumab or antibody LC06 (10 mg/kg), the respective combination (each 10 mg/kg) and TAvi6 (13.3 mg/kg). In addition, TAvi6 was evaluated for inhibition of metastasis to the lung quantified by Alu-PCR. Tumors were explanted for histological analysis of angiogenesis and inhibition of angiogenesis was assessed in the cornea angiogenesis assay. Results: In biochemical assays (affinity, Tie2-Ang-2 interaction) and cellular assays (Tie2 phosphorylation, HUVEC proliferation, tube formation) TAvi6 shows properties identical to the parental antibodies bevacizumab and LC06. In the orthotopic KPL-4-003 xenograft tumor growth inhibition was 79% for bevacizumab; 39% for LC06; 90% for the combination and 91% for TAvi6. In the s.c. Colo205-009 xenograft TGI was 66% for bevacizumab; 50% for LC06; 78% for the combination and 87% for TAvi6. TAvi6 was able to suppress growth of tumors refractory to 1st-line treatment with bevacizumab (Colo205 xenograft) and suppressed metastasis to the lung significantly (KPL-4 xenograft). In the s.c. Calu-3 xenograft we observed a strong inhibition of angiogenesis by in vivo and ex vivo imaging and an advantage of TAvi6 compared to the antibody combination. Furthermore, TAvi6 potently inhibited VEGF-induced angiogenesis in the mouse corneal angiogenesis assay. Conclusions: We have generated a novel tetravalent IgG-like bispecific antibody targeting VEGF-A and Ang-2 simultaneously. TAvi6 shows identical properties compared to the respective parental antibodies. In particular, TAvi6 blocks angiogenesis in vivo efficacously and mediates strong tumor growth inhibition in various xenograft models with a slight advantage of TAvi6 over the combination of the respective single agents bevacizumab and LC06 in several models. Further studies are foreseen to assess the application of bispecific antibodies targeting Ang-2 and VEGF as novel therapeutic approach for the treatment of hematological cancers. Disclosures: Weidner: Roche: Employment, Equity Ownership. Scheuer:Roche: Employment, Patents & Royalties. Thomas:Roche: Employment, Patents & Royalties. Baehner:Roche: Employment, Patents & Royalties. Seeber:Roche: Employment, Patents & Royalties. Kettenberger:Roche: Employment, Patents & Royalties. Schanzer:Roche: Employment, Patents & Royalties. Brinkmann:Roche: Employment, Patents & Royalties. Regula:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.