A computational method for predicting the aggregation propensity of IgG1 and IgG4(P) mAbs in common storage buffers.

The propensity for some monoclonal antibodies (mAbs) to aggregate at physiological and manufacturing pH values can prevent their use as therapeutic molecules or delay time to market. Consequently, developability assessments are essential to select optimum candidates, or inform on mitigation strategies to avoid potential late-stage failures. These studies are typically performed in a range of buffer solutions because factors such as pH can dramatically alter the aggregation propensity of the test mAbs (up to 100-fold in extreme cases). A computational method capable of robustly predicting the aggregation propensity at the pH values of common storage buffers would have substantial value. Here, we describe a mAb aggregation prediction tool (MAPT) that builds on our previously published isotype-dependent, charge-based model of aggregation. We show that the addition of a homology model-derived hydrophobicity descriptor to our electrostatic aggregation model enabled the generation of a robust mAb developability indicator. To contextualize our aggregation scoring system, we analyzed 97 clinical-stage therapeutic mAbs. To further validate our approach, we focused on six mAbs (infliximab, tocilizumab, rituximab, CNTO607, MEDI1912 and MEDI1912_STT) which have been reported to cover a large range of aggregation propensities. The different aggregation propensities of the case study molecules at neutral and slightly acidic pH were correctly predicted, verifying the utility of our computational method.

Engineering the Fab fragment of the anti-IgE omalizumab to prevent Fab crystallization and permit IgE-Fc complex crystallization.

Immunoglobulin E (IgE) plays a central role in the allergic response, in which cross-linking of allergen by FcεRI-bound IgE triggers mast cell and basophil degranulation and the release of inflammatory mediators. The high-affinity interaction between IgE and FcεRI is a long-standing target for therapeutic intervention in allergic disease. Omalizumab is a clinically approved anti-IgE monoclonal antibody that binds to free IgE, also with high affinity, preventing its interaction with FcεRI. All attempts to crystallize the pre-formed complex between the omalizumab Fab and the Fc region of IgE (IgE-Fc), to understand the structural basis for its mechanism of action, surprisingly failed. Instead, the Fab alone selectively crystallized in different crystal forms, but their structures revealed intermolecular Fab/Fab interactions that were clearly strong enough to disrupt the Fab/IgE-Fc complexes. Some of these interactions were common to other Fab crystal structures. Mutations were therefore designed to disrupt two recurring packing interactions observed in the omalizumab Fab crystal structures without interfering with the ability of the omalizumab Fab to recognize IgE-Fc; this led to the successful crystallization and subsequent structure determination of the Fab/IgE-Fc complex. The mutagenesis strategy adopted to achieve this result is applicable to other intractable Fab/antigen complexes or systems in which Fabs are used as crystallization chaperones.

Electrostatic interactions modulate the differential aggregation propensities of IgG1 and IgG4P antibodies and inform charged residue substitutions for improved developability.

Native state aggregation is an important concern in the development of therapeutic antibodies. Enhanced knowledge of mAb native state aggregation mechanisms would permit sequence-based selection and design of therapeutic mAbs with improved developability. We investigated how electrostatic interactions affect the native state aggregation of seven human IgG1 and IgG4P mAb isotype pairs, each pair having identical variable domains that are different for each set of IgG1 and IgG4P constructs. Relative aggregation propensities were determined at pH 7.4, representing physiological conditions, and pH 5.0, representing commonly used storage conditions. Our work indicates that the net charge state of variable domains relative to the net charge state of the constant domains is predominantly responsible for the different native state aggregation behavior of IgG1 and IgG4P mAbs. This observation suggests that the global net charge of a multi domain protein is not a reliable predictor of aggregation propensity. Furthermore, we demonstrate a design strategy in the frameworks of variable domains to reduce the native state aggregation propensity of mAbs identified as being aggregation-prone. Importantly, substitution of specifically identified residues with alternative, human germline residues, to optimize Fv charge, resulted in decreased aggregation potential at pH 5.0 and 7.4, thus increasing developability.

A mixture of functionally oligoclonal humanized monoclonal antibodies that neutralize Clostridium difficile TcdA and TcdB with high levels of in vitro potency shows in vivo protection in a hamster infection model.

Clostridium difficile infections are a major cause of antibiotic-associated diarrhea in hospital and care facility patients. In spite of the availability of effective antibiotic treatments, C. difficile infection (CDI) is still a major cause of patient suffering, death, and substantial health care costs. Clostridium difficile exerts its major pathological effects through the actions of two protein exotoxins, TcdA and TcdB, which bind to and disrupt gut tissue. Antibiotics target the infecting bacteria but not the exotoxins. Administering neutralizing antibodies against TcdA and TcdB to patients receiving antibiotic treatment might modulate the effects of the exotoxins directly. We have developed a mixture of three humanized IgG1 monoclonal antibodies (MAbs) which neutralize TcdA and TcdB to address three clinical needs: reduction of the severity and duration of diarrhea, reduction of death rates, and reduction of the rate of recurrence. The UCB MAb mixture showed higher potency in a variety of in vitro binding and neutralization assays (∼10-fold improvements), higher levels of protection in a hamster model of CDI (82% versus 18% at 28 days), and higher valencies of toxin binding (12 versus 2 for TcdA and 3 versus 2 for TcdB) than other agents in clinical development. Comparisons of the MAb properties also offered some insight into the potential relative importance of TcdA and TcdB in the disease process.

Relative stabilities of IgG1 and IgG4 Fab domains: influence of the light-heavy interchain disulfide bond architecture.

The stability of therapeutic antibodies is a prime pharmaceutical concern. In this work we examined thermal stability differences between human IgG1 and IgG4 Fab domains containing the same variable regions using the thermofluor assay. It was found that the IgG1 Fab domain is up to 11°C more stable than the IgG4 Fab domain containing the same variable region. We investigated the cause of this difference with the aim of developing a molecule with the enhanced stability of the IgG1 Fab and the biological properties of an IgG4 Fc. We found that replacing the seven residues, which differ between IgG1 C(H) 1 and IgG4 C(H) 1 domains, while retaining the native IgG1 light-heavy interchain disulfide (L-H) bond, did not affect thermal stability. Introducing the IgG1 type L-H interchain disulfide bond (DSB) into the IgG4 Fab resulted in an increase in thermal stability to levels observed in the IgG1 Fab with the same variable region. Conversely, replacement of the IgG1 L-H interchain DSB with the IgG4 type L-H interchain DSB reduced the thermal stability. We utilized the increased stability of the IgG1 Fab and designed a hybrid antibody with an IgG1 C(H) 1 linked to an IgG4 Fc via an IgG1 hinge. This construct has the expected biophysical properties of both the IgG4 Fc and IgG1 Fab domains and may therefore be a pharmaceutically relevant format.

Towards a universal disulphide stabilised single chain Fv format: importance of interchain disulphide bond location and vL-vH orientation.

Engineered introduction of interface interchain disulphide bonds is perceived to be a simple method to increase the stability of single chain Fv (scFv). Six disulphide bond locations have been cited within the literature but the potential for the broad use of each has not been examined. Five of these disulphide bond locations were introduced into one scFv in order to compare their relative effects on expression, thermal stability, percent monomer formation and retention of antigen binding. The disulphide bond position vH44-vL100 was observed to enable the most favourable balance of biophysical properties. The vH44-vL100 disulphide bond was introduced into five additional scFv in both vL-vH and vH-vL orientations in order to investigate its general applicability. Data are presented to show the relative influence of scFv sequence, v-region organisation and interchain disulphide bond on expression yield, thermal stability and percent monomer. Introduction of the vH44-vL100 disulphide bond typically resulted in no or little increase in thermal stability and no change in percent monomer but did confer the benefit of permanently fixing monomer:dimer ratios during purification and analysis.