Complement C1q Interacts With LRP1 Clusters II and IV Through a Site Close but Different From the Binding Site of Its C1r and C1s-Associated Proteases.

LRP1 is a large endocytic modular receptor that plays a crucial role in the scavenging of apoptotic material through binding to pattern-recognition molecules. It is a membrane anchored receptor of the LDL receptor family with 4 extracellular clusters of ligand binding modules called cysteine rich complement-type repeats that are involved in the interaction of LRP1 with its numerous ligands. Complement C1q was shown to interact with LRP1 and to be implicated in the phagocytosis of apoptotic cells. The present work aimed at exploring how these two large molecules interact at the molecular level using a dissection strategy. For that purpose, recombinant LRP1 clusters II, III and IV were produced in mammalian HEK293F cells and their binding properties were investigated. Clusters II and IV were found to interact specifically and efficiently with C1q with K Ds in the nanomolar range. The use of truncated C1q fragments and recombinant mutated C1q allowed to localize more precisely the binding site for LRP1 on the collagen-like regions of C1q (CLRs), nearby the site that is implicated in the interaction with the cognate protease tetramer C1r2s2. This site could be a common anchorage for other ligands of C1q CLRs such as sulfated proteoglycans and Complement receptor type 1. The use of a cellular model, consisting in CHO LRP1-null cells transfected with full-length LRP1 or a cluster IV minireceptor (mini IV) confirmed that mini IV interacts with C1q at the cell membrane as well as full-length LRP1. Further cellular interaction studies finally highlighted that mini IV can endorse the full-length LRP1 binding efficiency for apoptotic cells and that C1q has no impact on this interaction.

Unbiased Combinatorial Screening Identifies a Bispecific IgG1 that Potently Inhibits HER3 Signaling via HER2-Guided Ligand Blockade.

HER2-driven cancers require phosphatidylinositide-3 kinase (PI3K)/Akt signaling through HER3 to promote tumor growth and survival. The therapeutic benefit of HER2-targeting agents, which depend on PI3K/Akt inhibition, can be overcome by hyperactivation of the heregulin (HRG)/HER3 pathway. Here we describe an unbiased phenotypic combinatorial screening approach to identify a bispecific immunoglobulin G1 (IgG1) antibody against HER2 and HER3. In tumor models resistant to HER2-targeting agents, the bispecific IgG1 potently inhibits the HRG/HER3 pathway and downstream PI3K/Akt signaling via a "dock & block" mechanism. This bispecific IgG1 is a potentially effective therapy for breast cancer and other tumors with hyperactivated HRG/HER3 signaling.

A new approach for generating bispecific antibodies based on a common light chain format and the stable architecture of human immunoglobulin G1.

Bispecific antibodies combine two different antigen-binding sites in a single molecule, enabling more specific targeting, novel mechanisms of action, and higher clinical efficacies. Although they have the potential to outperform conventional monoclonal antibodies, many bispecific antibodies have issues regarding production, stability, and pharmacokinetic properties. Here, we describe a new approach for generating bispecific antibodies using a common light chain format and exploiting the stable architecture of human immunoglobulin G1 We used iterative experimental validation and computational modeling to identify multiple Fc variant pairs that drive efficient heterodimerization of the antibody heavy chains. Accelerated stability studies enabled selection of one Fc variant pair dubbed "DEKK" consisting of substitutions L351D and L368E in one heavy chain combined with L351K and T366K in the other. Solving the crystal structure of the DEKK Fc region at a resolution of 2.3 Å enabled detailed analysis of the interactions inducing CH3 interface heterodimerization. Local shifts in the IgG backbone accommodate the introduction of lysine side chains that form stabilizing salt-bridge interactions with substituted and native residues in the opposite chain. Overall, the CH3 domain adapted to these shifts at the interface, yielding a stable Fc conformation very similar to that in wild-type IgG. Using the DEKK format, we generated the bispecific antibody MCLA-128, targeting human EGF receptors 2 and 3. MCLA-128 could be readily produced and purified at industrial scale with a standard mammalian cell culture platform and a routine purification protocol. Long-term accelerated stability assays confirmed that MCLA-128 is highly stable and has excellent biophysical characteristics.

Recombinant Expression of the Full-length Ectodomain of LDL Receptor-related Protein 1 (LRP1) Unravels pH-dependent Conformational Changes and the Stoichiometry of Binding with Receptor-associated Protein (RAP).

LDL receptor-related protein 1 (LRP1) is a highly modular protein and the largest known mammalian endocytic receptor. LRP1 binds and internalizes many plasma components, playing multiple crucial roles as a scavenger and signaling molecule. One major challenge to studying LRP1 has been that it is difficult to express such a large, highly glycosylated, and cysteine-rich protein, limiting structural studies to LRP1 fragments. Here, we report the first recombinant expression of the complete 61 domains of the full-length LRP1 ectodomain. This advance was achieved with a multistep cloning approach and by using DNA dilutions to improve protein yields. We investigated the binding properties of LRP1 using receptor-associated protein (RAP) as a model ligand due to its tight binding interaction. The LRP1 conformation was studied in its bound and unbound state using mass spectrometry, small-angle X-ray scattering, and negative-stain electron microscopy at neutral and acidic pH. Our findings revealed a pH-dependent release of the ligand associated with a conformational change of the receptor. In summary, this investigation of the complete LRP1 ectodomain significantly advances our understanding of this important receptor and provides the basis for further elucidating the mechanism of action of LRP1 in a whole and integrated system.