Stefan Loverix

Species-Specific Determinants in the IgG CH3 Domain Enable Fab-Arm Exchange by Affecting the Noncovalent CH3–CH3 Interaction Strength

A distinctive feature of human IgG4 is its ability to recombine half molecules (H chain and attached L chain) through a dynamic process termed Fab-arm exchange, which results in bispecific Abs. It is becoming evident that the process of Fab-arm exchange is conserved in several mammalian species, and thereby represents a mechanism that impacts humoral immunity more generally than previously thought. In humans, Fab-arm exchange has been attributed to the IgG4 core-hinge sequence (226-CPSCP-230) in combination with unknown determinants in the third constant H chain domain (CH3). In this study, we investigated the role of the CH3 domain in the mechanism of Fab-arm exchange, and thus identified amino acid position 409 as the critical CH3 determinant in human IgG, with R409 resulting in exchange and K409 resulting in stable IgG. Interestingly, studies with IgG from various species showed that Fab-arm exchange could not be assigned to a common CH3 domain amino acid motif. Accordingly, in rhesus monkeys (Macaca mulatta), aa 405 was identified as the CH3 determinant responsible (in combination with 226-CPACP-230). Using native mass spectrometry, we demonstrated that the ability to exchange Fab-arms correlated with the CH3–CH3 dissociation constant. Species-specific adaptations in the CH3 domain thus enable Fab-arm exchange by affecting the inter-CH3 domain interaction strength. The redistribution of Ag-binding domains between molecules may constitute a general immunological and evolutionary advantage. The current insights impact our view of humoral immunity and should furthermore be considered in the design and evaluation of Ab-based studies and therapeutics.

Quantitative Analysis of the Interaction Strength and Dynamics of Human IgG4 Half Molecules by Native Mass Spectrometry

Native mass spectrometry (MS) is a powerful technique for studying noncovalent protein-protein interactions. Here, native MS was employed to examine the noncovalent interactions involved in homodimerization of antibody half molecules (HL) in hinge-deleted human IgG4 (IgG4Δhinge). By analyzing the concentration dependence of the relative distribution of monomer HL and dimer (HL)(2) species, the apparent dissociation constant (K(D)) for this interaction was determined. In combination with site-directed mutagenesis, the relative contributions of residues at the CH3-CH3 interface to this interaction could be characterized and corresponding K(D) values quantified over a range of 10(-10)-10(-4) M. The critical importance of this noncovalent interaction in maintaining the intact dimeric structure was also proven for the full-length IgG4 backbone. Using time-resolved MS, the kinetics of the interaction could be measured, reflecting the dynamics of IgG4 HL exchange. Hence, native MS has provided a quantitative view of local structural features that define biological properties of IgG4.

Structural basis of IL-23 antagonism by an Alphabody protein scaffold

Protein scaffolds can provide a promising alternative to antibodies for various biomedical and biotechnological applications, including therapeutics. Here we describe the design and development of the Alphabody, a protein scaffold featuring a single-chain antiparallel triple-helix coiled-coil fold. We report affinity-matured Alphabodies with favourable physicochemical properties that can specifically neutralize human interleukin (IL)-23, a pivotal therapeutic target in autoimmune inflammatory diseases such as psoriasis and multiple sclerosis. The crystal structure of human IL-23 in complex with an affinity-matured Alphabody reveals how the variable interhelical groove of the scaffold uniquely targets a large epitope on the p19 subunit of IL-23 to harness fully the hydrophobic and hydrogen-bonding potential of tryptophan and tyrosine residues contributed by p19 and the Alphabody, respectively. Thus, Alphabodies are suitable for targeting protein–protein interfaces of therapeutic importance and can be tailored to interrogate desired design and binding-mode principles via efficient selection and affinity-maturation strategies.

Abstract 3850: First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth in vivo

We have developed Cell Penetrating Alphabodies (CPABs), a novel and unique therapeutic class of proteins engineered to efficiently enter cells. In vitro, uptake in a range of tumor and non-tumor cell types occurs rapidly with cytosol levels of up to 1 μM concentration after 2 hours of CPAB exposure. Early forms of these CPABs suffered from rapid serum clearance, thereby limiting their efficacy in vivo and amenability to drug development. The incorporation of an albumin binding region in the body of the protein has allowed extension of serum half-life in mice from a few minutes to more than one hour. These CPABs have been shown to be efficiently delivered to xenograft tumors in mice after IV bolus injection by tissue ELISA and immunohistochemistry. CPABs can be used to target and interfere with intracellular protein-protein interactions involved in tumor survival in a highly specific way. The anti-apoptotic protein Myeloid Cell Leukaemia-1 (Mcl-1) promotes through its interaction with Bak, the survival of a range of different tumor types including myeloid leukemia, breast cancer and non-small cell lung cancer. Moreover, Mcl-1 overexpression is often associated with chemotherapeutic resistance and disease relapse. Mcl-1, however, has proven difficult to target using the conventional small molecule approach. Alphabodies which bind to Mcl-1 were engineered by a combination of rational design and phage display library screening. The affinities for Mcl-1 ranged between 18 pM and 750 pM with binding to the closely related proteins Bcl-2 and Bcl-XL being below the limit of detection for the assay. In a Mammalian Two Hybrid assay, these Alphabodies inhibited Bak-Mcl-1 but not Bak-Bcl-XL interactions. Anti-Mcl-1 CPABs were shown to efficiently kill the Mcl-1 dependent multiple myeloma cell line NCI-H929 with IC50s ranging from 0.5 μM to 2 μM as monitored in cell viability assays. The dose responsive cell killing correlated with caspase-3/7 activation in NCI-H929 cells. Other Mcl-1 dependent tumor cell types including non-small cell lung cancer (NCI-H23) and Burkitt9s lymphoma (Raji) or tumor cell types with high Mcl-1 expression such as ovarian cancer (A2780) and colorectal adenocarcinoma (COLO-320DM) were also killed efficiently using anti-Mcl-1 CPABs. Despite its short half-life, daily intraperitoneal administration of a prototype Mcl-1 targeting CPAB (without half-life extension) at 30 mg/kg for 14 days resulted in tumor inhibition of 33% as compared to vehicle control. Experiments are underway in mouse models using the more optimal CPABs with extended serum half-life and tumor exposure. CPABs represent the best-in-class cell penetrating protein therapeutics both in terms of efficiency of uptake and amenability to conversion to viable drugs opening unprecedented opportunities to tackle intracellular protein-protein interactions critical to diseases with unmet medical need. Citation Format: Sabrina Deroo, Sophie Thiolloy, Johan Desmet, Franky Baatz, Stefan Loverix, Karen Vandenbroucke, Eric Lorent, Paula Henderikx, Irma Lemmens, Philippe Alard, Ignace Lasters, Yvonne McGrath. First-in-class cell-penetrating proteins targeting Mcl-1 induce tumor cell apoptosis and inhibition of tumor growth in vivo. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3850.