A Multipronged Unbiased Strategy Guides the Development of an Anti-EGFR/EPHA2-Bispecific Antibody for Combination Cancer Therapy.

PURPOSE: Accumulating analyses of pro-oncogenic molecular mechanisms triggered a rapid development of targeted cancer therapies. Although many of these treatments produce impressive initial responses, eventual resistance onset is practically unavoidable. One of the main approaches for preventing this refractory condition relies on the implementation of combination therapies. This includes dual-specificity reagents that affect both of their targets with a high level of selectivity. Unfortunately, selection of target combinations for these treatments is often confounded by limitations in our understanding of tumor biology. Here, we describe and validate a multipronged unbiased strategy for predicting optimal co-targets for bispecific therapeutics. EXPERIMENTAL DESIGN: Our strategy integrates ex vivo genome-wide loss-of-function screening, BioID interactome profiling, and gene expression analysis of patient data to identify the best fit co-targets. Final validation of selected target combinations is done in tumorsphere cultures and xenograft models. RESULTS: Integration of our experimental approaches unambiguously pointed toward EGFR and EPHA2 tyrosine kinase receptors as molecules of choice for co-targeting in multiple tumor types. Following this lead, we generated a human bispecific anti-EGFR/EPHA2 antibody that, as predicted, very effectively suppresses tumor growth compared with its prototype anti-EGFR therapeutic antibody, cetuximab. CONCLUSIONS: Our work not only presents a new bispecific antibody with a high potential for being developed into clinically relevant biologics, but more importantly, successfully validates a novel unbiased strategy for selecting biologically optimal target combinations. This is of a significant translational relevance, as such multifaceted unbiased approaches are likely to augment the development of effective combination therapies for cancer treatment. See related commentary by Kumar, p. 2570.

Derivative of Extremophilic 50S Ribosomal Protein L35Ae as an Alternative Protein Scaffold.

Small antibody mimetics, or alternative binding proteins (ABPs), extend and complement antibody functionality with numerous applications in research, diagnostics and therapeutics. Given the superiority of ABPs, the last two decades have witnessed development of dozens of alternative protein scaffolds (APSs) for the design of ABPs. Proteins from extremophiles with their high structural stability are especially favorable for APS design. Here, a 10X mutant of the 50S ribosomal protein L35Ae from hyperthermophilic archaea Pyrococcus horikoshii has been probed as an APS. A phage display library of L35Ae 10X was generated by randomization of its three CDR-like loop regions (repertoire size of 2×108). Two L35Ae 10X variants specific to a model target, the hen egg-white lysozyme (HEL), were isolated from the resulting library using phage display. The affinity of these variants (L4 and L7) to HEL ranges from 0.10 µM to 1.6 µM, according to surface plasmon resonance data. While L4 has 1-2 orders of magnitude lower affinity to HEL homologue, bovine α-lactalbumin (BLA), L7 is equally specific to HEL and BLA. The reference L35Ae 10X is non-specific to both HEL and BLA. L4 and L7 are more resistant to denaturation by guanidine hydrochloride compared to the reference L35Ae 10X (mid-transition concentration is higher by 0.1-0.5 M). Chemical crosslinking experiments reveal an increased propensity of L4 and L7 to multimerization. Overall, the CDR-like loop regions of L35Ae 10X represent a proper interface for generation of functional ABPs. Hence, L35Ae is shown to extend the growing family of protein scaffolds dedicated to the design of novel binding proteins.

Interleukin-11 binds specific EF-hand proteins via their conserved structural motifs.

Interleukin-11 (IL-11) is a hematopoietic cytokine engaged in numerous biological processes and validated as a target for treatment of various cancers. IL-11 contains intrinsically disordered regions that might recognize multiple targets. Recently we found that aside from IL-11RA and gp130 receptors, IL-11 interacts with calcium sensor protein S100P. Strict calcium dependence of this interaction suggests a possibility of IL-11 interaction with other calcium sensor proteins. Here we probed specificity of IL-11 to calcium-binding proteins of various types: calcium sensors of the EF-hand family (calmodulin, S100B and neuronal calcium sensors: recoverin, NCS-1, GCAP-1, GCAP-2), calcium buffers of the EF-hand family (S100G, oncomodulin), and a non-EF-hand calcium buffer (α-lactalbumin). A specific subset of the calcium sensor proteins (calmodulin, S100B, NCS-1, GCAP-1/2) exhibits metal-dependent binding of IL-11 with dissociation constants of 1-19 µM. These proteins share several amino acid residues belonging to conservative structural motifs of the EF-hand proteins, 'black' and 'gray' clusters. Replacements of the respective S100P residues by alanine drastically decrease its affinity to IL-11, suggesting their involvement into the association process. Secondary structure and accessibility of the hinge region of the EF-hand proteins studied are predicted to control specificity and selectivity of their binding to IL-11. The IL-11 interaction with the EF-hand proteins is expected to occur under numerous pathological conditions, accompanied by disintegration of plasma membrane and efflux of cellular components into the extracellular milieu.

High-affinity interaction between interleukin-11 and S100P protein.

Interleukin-11 (IL-11) and S100P are oncoproteins co-expressed in numerous cancers, which might favor their interaction during oncogenesis. We have explored the possibility of this interaction by surface plasmon resonance spectroscopy, intrinsic fluorescence, and chemical crosslinking. Recombinant forms of IL-11 and S100P interact with each other under physiological level of calcium ions. IL-11 molecule has at least two S100P-binding sites with dissociation constants of 32 nM and 288 nM, which is 5-13-fold lower than its affinity to extracellular domain of IL-11 receptor subunit α. S100P does not alter IL-11-induced STAT3 activation in HEK293 cells co-expressing IL-11 receptors, but could affect other tumorigenic signaling pathways. The highly specific IL-11 - S100P interaction occurring under physiologically relevant conditions should be taken into consideration upon development of the antineoplastics inhibiting IL-11 signaling.

Extremophilic 50S Ribosomal RNA-Binding Protein L35Ae as a Basis for Engineering of an Alternative Protein Scaffold.

Due to their remarkably high structural stability, proteins from extremophiles are particularly useful in numerous biological applications. Their utility as alternative protein scaffolds could be especially valuable in small antibody mimetic engineering. These artificial binding proteins occupy a specific niche between antibodies and low molecular weight substances, paving the way for development of innovative approaches in therapeutics, diagnostics, and reagent use. Here, the 50S ribosomal RNA-binding protein L35Ae from the extremophilic archaea Pyrococcus horikoshii has been probed for its potential to serve as a backbone in alternative scaffold engineering. The recombinant wild type L35Ae has a native-like secondary structure, extreme thermal stability (mid-transition temperature of 90°C) and a moderate resistance to the denaturation by guanidine hydrochloride (half-transition at 2.6 M). Chemical crosslinking and dynamic light scattering data revealed that the wild type L35Ae protein has a propensity for multimerization and aggregation correlating with its non-specific binding to a model cell surface of HEK293 cells, as evidenced by flow cytometry. To suppress these negative features, a 10-amino acid mutant (called L35Ae 10X) was designed, which lacks the interaction with HEK293 cells, is less susceptible to aggregation, and maintains native-like secondary structure and thermal stability. However, L35Ae 10X also shows lowered resistance to guanidine hydrochloride (half-transition at 2.0M) and is more prone to oligomerization. This investigation of an extremophile protein's scaffolding potential demonstrates that lowered resistance to charged chemical denaturants and increased propensity to multimerization may limit the utility of extremophile proteins as alternative scaffolds.