Diabody-Ig: a novel platform for the generation of multivalent and multispecific antibody molecules

ABSTRACT

Multivalent mono- or bispecific antibodies are of increasing interest for therapeutic applications, such as efficient receptor clustering and activation, or dual targeting approaches. Here, we present a novel platform for the generation of Ig-like molecules, designated diabody-Ig (Db-Ig). The antigen-binding site of Db-Ig is composed of a diabody in the V_H-V_L orientation stabilized by fusion to antibody-derived homo- or heterodimerization domains, e.g., C_H1/C_L or the heavy chain domain 2 of IgE (EHD2) or IgM (MHD2), further fused to an Fc region. In this study, we applied the Db-Ig format for the generation of tetravalent bispecific antibodies (2 + 2) directed against EGFR and HER3 and utilizing different dimerization domains. These Db-Ig antibodies retained the binding properties of the parental antibodies and demonstrated unhindered simultaneous binding of both antigens. The Db-Ig antibodies could be purified by a single affinity chromatography resulting in a homogenous preparation. Furthermore, the Db-Igs were highly stable in human plasma. Importantly, only one short peptide linker (5 aa) per chain is required to generate a Db-Ig molecule, reducing the potential risk of immunogenicity. The presence of a fully functional Fc resulted in IgG-like pharmacokinetic profiles of the Db-Ig molecules. Besides tetravalent bispecific molecules, this modular platform technology further allows for the generation of other multivalent molecules of varying specificity and valency, including mono-, bi-, tri- and tetra-specific molecules, and thus should be suitable for numerous applications.     

Specific and high-resolution identification of monoclonal antibody fragments detected by capillary electrophoresis–sodium dodecyl sulfate using reversed-phase HPLC with top-down mass spectrometry analysis

ABSTRACT

In recent years, capillary electrophoresis–sodium dodecyl sulfate (cSDS) has been widely used for high resolution separation and quantification of the fragments and aggregates of monoclonal antibodies (mAbs) to ensure the quality of mAb therapeutics. However, identification of the low-molecular-weight (LMW) and high-molecular-weight (HMW) species detected in cSDS electropherograms has been based primarily on the approximate MWs calculated from standard curves using known MW standards and correlations with fragments and aggregates identified by other methods. It is not easy to collect sufficient amounts of H/LMW species from cSDS for analysis by orthogonal methods and the direct coupling of cSDS with mass spectrometry (MS) is very difficult due to interference from SDS. In this study, we describe the precise identification of H/LMW species detected by cSDS using reversed-phase high performance liquid chromatography (RP-HPLC) coupled with top-down tandem MS analysis. The H/LMW species were first identified by on-line RP-HPLC MS analysis and the RP-HPLC fractions were then analyzed by cSDS to connect the identified H/LMW species with the peaks in the cSDS electropherogram. With this method, 58 unique H/LMW species were identified from an immunoglobulin G1 (IgG1) mAb. The identified fragments ranged from 10 kDa single chain fragments to 130 kDa triple chain fragments, including some with post-translational modifications. This is the first study to clearly identify the antibody fragments, including the exact clipping sites, observed in cSDS electropherograms. The methodology and results presented here should be applicable to most other IgG1 mAbs.     

Proteome Variations in Pancreatic Stellate Cells upon Stimulation with Proinflammatory Factors

Pancreatic stellate cells are key mediators in chronic pancreatitis and play a central role in the development of pancreatic fibrosis, stromal formation, and progression of pancreatic cancer. This study was aimed at investigating molecular changes at the level of the proteome that are associated with the activation of pancreatic stellate cells by proinflammatory factors, namely TNF-α, FGF2, IL6, and chemokine (C-C motif) ligand 4 (CCL4). They were added individually to cells growing in serum-free medium next to controls in medium supplemented with serum, thus containing a mixture of them all, or in serum-free medium alone. Variations were detected by means of a microarray of 810 antibodies targeting relevant proteins. All tested factors triggered increased proliferation and migration. Further analysis showed that TNF-α is the prime factor responsible for the activation of pancreatic stellate cells. CCL4 is associated with cellular neovascularization, whereas FGF2 and IL6 induction led to better cellular survival and decreased apoptotic activity of the stellate cells. The identified direct effects of individual cytokines on human pancreatic stellate cells provide new insights about their contribution to pancreatic cancer promotion.     

Atypical Antigen Recognition Mode of a Shark Immunoglobulin New Antigen Receptor (IgNAR) Variable Domain Characterized by Humanization and Structural Analysis

The immunoglobulin new antigen receptors (IgNARs) are a class of Ig-like molecules of the shark immune system that exist as heavy chain-only homodimers and bind antigens by their single domain variable regions (V-NARs). Following shark immunization and/or in vitro selection, V-NARs can be generated as soluble, stable, and specific high affinity monomeric binding proteins of ∼12 kDa. We have previously isolated a V-NAR from an immunized spiny dogfish shark, named E06, that binds specifically and with high affinity to human, mouse, and rat serum albumins. Humanization of E06 was carried out by converting over 60% of non-complementarity-determining region residues to those of a human germ line Vκ1 sequence, DPK9. The resulting huE06 molecules have largely retained the specificity and affinity of antigen binding of the parental V-NAR. Crystal structures of the shark E06 and its humanized variant (huE06 v1.1) in complex with human serum albumin (HSA) were determined at 3- and 2.3-Å resolution, respectively. The huE06 v1.1 molecule retained all but one amino acid residues involved in the binding site for HSA. Structural analysis of these V-NARs has revealed an unusual variable domain-antigen interaction. E06 interacts with HSA in an atypical mode that utilizes extensive framework contacts in addition to complementarity-determining regions that has not been seen previously in V-NARs. On the basis of the structure, the roles of various elements of the molecule are described with respect to antigen binding and V-NAR stability. This information broadens the general understanding of antigen recognition and provides a framework for further design and humanization of shark IgNARs.     

Simultaneous Surface Display and Secretion of Proteins from Mammalian Cells Facilitate Efficient in Vitro Selection and Maturation of Antibodies

A mammalian expression system has been developed that permits simultaneous cell surface display and secretion of the same protein through alternate splicing of pre-mRNA. This enables a flexible system for in vitro protein evolution in mammalian cells where the displayed protein phenotype remains linked to genotype, but with the advantage of soluble protein also being produced without the requirement for any further recloning to allow a wide range of assays, including biophysical and cell-based functional assays, to be used during the selection process. This system has been used for the simultaneous surface presentation and secretion of IgG during antibody discovery and maturation. Presentation and secretion of monomeric Fab can also be achieved to minimize avidity effects. Manipulation of the splice donor site sequence enables control of the relative amounts of cell surface and secreted antibody. Multi-domain proteins may be presented and secreted in different formats to enable flexibility in experimental design, and secreted proteins may be produced with epitope tags to facilitate high-throughput testing. This system is particularly useful in the context of in situ mutagenesis, as in the case of in vitro somatic hypermutation.     

Antibody engineering & therapeutics,the annual meeting of the antibody society December 7–10, 2015, San Diego,CA, USA

The 26th Antibody Engineering & Therapeutics meeting, the annual meeting of The Antibody Society united over 800 participants from all over the world in San Diego from 6–10 December 2015. The latest innovations and advances in antibody research and development were discussed, covering a myriad of antibody-related topics by more than 100 speakers, who were carefully selected by The Antibody Society. As a prelude, attendees could join the pre-conference training course focusing, among others, on the engineering and enhancement of antibodies and antibody-like scaffolds, bispecific antibody engineering and adaptation to generate chimeric antigen receptor constructs. The main event covered 4 d of scientific sessions that included antibody effector functions, reproducibility of research and diagnostic antibodies, new developments in antibody-drug conjugates (ADCs), preclinical and clinical ADC data, new technologies and applications for bispecific antibodies, antibody therapeutics for non-cancer and orphan indications, antibodies to harness the cellular immune system, building comprehensive IgVH-gene repertoires through discovering, confirming and cataloging new germline IgVH genes, and overcoming resistance to clinical immunotherapy. The Antibody Society's special session focused on “Antibodies to watch” in 2016. Another special session put the spotlight on the limitations of the new definitions for the assignment of antibody international nonproprietary names introduced by the World Health Organization. The convention concluded with workshops on computational antibody design and on the promise and challenges of using next-generation sequencing for antibody discovery and engineering from synthetic and in vivo libraries.     

Aberrant bispecific antibody pharmacokinetics linked to liver sinusoidal endothelium clearance mechanism in cynomolgus monkeys

Bispecific antibodies (BsAbs) can affect multiple disease pathways, thus these types of constructs potentially provide promising approaches to improve efficacy in complex disease indications. The specific and non-specific clearance mechanisms/biology that affect monoclonal antibody (mAb) pharmacokinetics are likely involved in the disposition of BsAbs. Despite these similarities, there are a paucity of studies on the in vivo biology that influences the biodistribution and pharmacokinetics of BsAbs. The present case study evaluated the in vivo disposition of 2 IgG-fusion BsAb formats deemed IgG-ECD (extracellular domain) and IgG-scFv (single-chain Fv) in cynomolgus monkeys. These BsAb molecules displayed inferior in vivo pharmacokinetic properties, including a rapid clearance (> 0.5 mL/hr/kg) and short half-life relative to their mAb counterparts. The current work evaluated factors in vivo that result in the aberrant clearance of these BsAb constructs. Results showed the rapid clearance of the BsAbs that was not attributable to target binding, reduced neonatal Fc receptor (FcRn) interactions or poor molecular/biochemical properties. Evaluation of the cellular distribution of the constructs suggested that the major clearance mechanism was linked to binding/association with liver sinusoidal endothelial cells (LSECs) versus liver macrophages. The role of LSECs in facilitating the clearance of the IgG-ECD and IgG-scFv BsAb constructs described in these studies was consistent with the minimal influence of clodronate-mediated macrophage depletion on the pharmacokinetics of the constructs in cynomolgus monkeys The findings in this report are an important demonstration that the elucidation of clearance mechanisms for some IgG-ECD and IgG-scFv BsAb molecules can be unique and complicated, and may require increased attention due to the proliferation of these more complex mAb-like structures.     

Mitigation of reversible self-association and viscosity in a human IgG1 monoclonal antibody by rational,structure-guided Fv engineering

Undesired solution behaviors such as reversible self-association (RSA), high viscosity, and liquid-liquid phase separation can introduce substantial challenges during development of monoclonal antibody formulations. Although a global mechanistic understanding of RSA (i.e., native and reversible protein-protein interactions) is sufficient to develop robust formulation controls, its mitigation via protein engineering requires knowledge of the sites of protein-protein interactions. In the study reported here, we coupled our previous hydrogen-deuterium exchange mass spectrometry findings with structural modeling and in vitro screening to identify the residues responsible for RSA of a model IgG1 monoclonal antibody (mAb-C), and rationally engineered variants with improved solution properties (i.e., reduced RSA and viscosity). Our data show that mutation of either solvent-exposed aromatic residues within the heavy and light chain variable regions or buried residues within the heavy chain/light chain interface can significantly mitigate RSA and viscosity by reducing the IgG's surface hydrophobicity. The engineering strategy described here highlights the utility of integrating complementary experimental and in silico methods to identify mutations that can improve developability, in particular, high concentration solution properties, of candidate therapeutic antibodies.     

Estimating Neospora caninum prevalence in wildlife populations using Bayesian inference

Prevalence of disease in wildlife populations, which is necessary for developing disease models and conducting epidemiologic analyses, is often understudied. Laboratory tests used to screen for diseases in wildlife populations often are validated only for domestic animals. Consequently, the use of these tests for wildlife populations may lead to inaccurate estimates of disease prevalence. We demonstrate the use of Bayesian latent class analysis (LCA) in determining the specificity and sensitivity of a competitive enzyme‐linked immunosorbent assay (cELISA; VMRD^®, Inc.) serologic test used to identify exposure to Neospora caninum (hereafter N. caninum) in three wildlife populations in southeastern Ohio, USA. True prevalence of N. caninum exposure in these populations was estimated to range from 0.1% to 3.1% in American bison (Bison bison), 51.0% to 53.8% in Père David's deer (Elaphurus davidianus), and 40.0% to 45.9% in white‐tailed deer (Odocoileus virginianus). The accuracy of the cELISA in American bison and Père David's deer was estimated to be close to the 96% sensitivity and 99% specificity reported by the manufacturer. Sensitivity in white‐tailed deer, however, ranged from 78.9% to 99.9%. Apparent prevalence of N. caninum from the test results is not equal to the true prevalence in white‐tailed deer and Père David's deer populations. Even when these species inhabit the same community, the true prevalence in the two deer populations differed from the true prevalence in the American bison population. Variances in prevalence for some species suggest differences in the epidemiology of N. caninum for these colocated populations. Bayesian LCA methods could be used as in this example to overcome some of the constraints on validating tests in wildlife species. The ability to accurately evaluate disease status and prevalence in a population improves our understanding of the epidemiology of multihost pathogen systems at the community level.     

Characterization of mAb dimers reveals predominant dimer forms common in therapeutic mAbs

The formation of undesired high molecular weight species such as dimers is an important quality attribute for therapeutic monoclonal antibody formulations. Therefore, the thorough understanding of mAb dimerization and the detailed characterization mAb dimers is of great interest for future pharmaceutical development of therapeutic antibodies. In this work, we focused on the analyses of different mAb dimers regarding size, surface properties, chemical identity, overall structure and localization of possible dimerization sites. Dimer fractions of different mAbs were isolated to a satisfactory purity from bulk material and revealed 2 predominant overall structures, namely elongated and compact dimer forms. The elongated dimers displayed one dimerization site involving the tip of the Fab domain. Depending on the stress applied, these elongated dimers are connected either covalently or non-covalently. In contrast, the compact dimers exhibited non-covalent association. Several interaction points were detected for the compact dimers involving the hinge region or the base of the Fab domain. These results indicate that mAb dimer fractions are rather complex and may contain more than one kind of dimer. Nevertheless, the overall appearance of mAb dimers suggests the existence of 2 predominant dimeric structures, elongated and compact, which are commonly present in preparations of therapeutic mAbs.