Systematic pharmacological analysis of agonistic and antagonistic fibroblast growth factor receptor 1 MAbs reveals a similar unique mode of action.

Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase that plays a major role in developmental processes and metabolism. The dysregulation of FGFR1 through genetic aberrations leads to skeletal and metabolic diseases as well as cancer. For this reason, FGFR1 is a promising therapeutic target, yet a very challenging one due to potential on-target toxicity. More puzzling is that both agonistic and antagonistic FGFR1 antibodies are reported to exhibit similar toxicity profiles in vivo, namely weight loss. In this study, we aimed to assess and compare the mechanism of action of these molecules to better understand this apparent contradiction. By systematically comparing the binding of these antibodies and the activation or the inhibition of the major FGFR1 signaling events, we demonstrated that the molecules displayed similar properties and can behave either as an agonist or antagonist depending on the presence or the absence of the endogenous ligand. We further demonstrated that these findings translated in xenografts mice models. In addition, using time-resolved FRET and mass spectrometry analysis, we showed a functionally distinct FGFR1 active conformation in the presence of an antibody that preferentially activates the FGFR substrate 2 (FRS2)-dependent signaling pathway, demonstrating that modulating the geometry of a FGFR1 dimer can effectively change the signaling outputs and ultimately the activity of the molecule in preclinical studies. Altogether, our results highlighted how bivalent antibodies can exhibit both agonistic and antagonistic activities and have implications for targeting other receptor tyrosine kinases with antibodies.

Correction: Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

Combined MEK and ERK inhibition overcomes therapy-mediated pathway reactivation in RAS mutant tumors.

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

High cell-surface density of HER2 deforms cell membranes.

Breast cancers (BC) with HER2 overexpression (referred to as HER2 positive) progress more aggressively than those with normal expression. Targeted therapies against HER2 can successfully delay the progression of HER2-positive BC, but details of how this overexpression drives the disease are not fully understood. Using single-molecule biophysical approaches, we discovered a new effect of HER2 overexpression on disease-relevant cell biological changes in these BC. We found HER2 overexpression causes deformation of the cell membranes, and this in turn disrupts epithelial features by perturbing cell-substrate and cell-cell contacts. This membrane deformation does not require receptor signalling activities, but results from the high levels of HER2 on the cell surface. Our finding suggests that early-stage morphological alterations of HER2-positive BC cells during cancer progression can occur in a physical and signalling-independent manner.

Discovery of a Noncovalent, Mutant-Selective Epidermal Growth Factor Receptor Inhibitor.

Inhibitors targeting the activating mutants of the epidermal growth factor receptor (EGFR) have found success in the treatment of EGFR mutant positive non-small-cell lung cancer. A secondary point mutation (T790M) in the inhibitor binding site has been linked to the acquired resistance against those first generation therapeutics. Herein, we describe the lead optimization of a series of reversible, pan-mutant (L858R, del746-750, T790M/L858R, and T790M/del746-750) EGFR inhibitors. By use of a noncovalent double mutant (T790M/L858R and T790M/del746-750) selective EGFR inhibitor (2) as a starting point, activities against the single mutants (L858R and del746-750) were introduced through a series of structure-guided modifications. The in vitro ADME-PK properties of the lead molecules were further optimized through a number of rational structural changes. The resulting inhibitor (21) exhibited excellent cellular activity against both the single and double mutants of EGFR, demonstrating target engagement in vivo and ADME-PK properties that are suitable for further evaluation. The reversible, noncovalent inhibitors described complement the covalent pan-mutant EGFR inhibitors that have shown encouraging results in recent clinical trials.

Noncovalent Mutant Selective Epidermal Growth Factor Receptor Inhibitors: A Lead Optimization Case Study.

Because of their increased activity against activating mutants, first-generation epidermal growth factor receptor (EGFR) kinase inhibitors have had remarkable success in treating non-small-cell lung cancer (NSCLC) patients, but acquired resistance, through a secondary mutation of the gatekeeper residue, means that clinical responses only last for 8-14 months. Addressing this unmet medical need requires agents that can target both of the most common double mutants: T790M/L858R (TMLR) and T790M/del(746-750) (TMdel). Herein we describe how a noncovalent double mutant selective lead compound was optimized using a strategy focused on the structure-guided increase in potency without added lipophilicity or reduction of three-dimensional character. Following successive rounds of design and synthesis it was discovered that cis-fluoro substitution on 4-hydroxy- and 4-methoxypiperidinyl groups provided synergistic, substantial, and specific potency gain through direct interaction with the enzyme and/or effects on the proximal ligand oxygen atom. Further development of the fluorohydroxypiperidine series resulted in the identification of a pair of diastereomers that showed 50-fold enzyme and cell based selectivity for T790M mutants over wild-type EGFR (wtEGFR) in vitro and pathway knock-down in an in vivo xenograft model.

Blocking NRG1 and other ligand-mediated Her4 signaling enhances the magnitude and duration of the chemotherapeutic response of non-small cell lung cancer.

Although standard chemotherapies are commonly used to treat most types of solid tumors, such treatment often results in inadequate response to, or relapse after, therapy. This is particularly relevant for lung cancer because most patients are diagnosed with advanced-stage disease and are treated with frontline chemotherapy. By studying the residual tumor cells that remain after chemotherapy in several in vivo non-small cell lung cancer models, we found that these cells have increased levels of human epidermal growth factor receptor (HER) signaling due, in part, to the enrichment of a preexisting NRG1(HI) subpopulation. Neuregulin 1 (NRG1) signaling in these models can be mediated by either the HER3 or HER4 receptor, resulting in the differential activation of downstream effectors. Inhibition of NRG1 signaling inhibits primary tumor growth and enhances the magnitude and duration of the response to chemotherapy. Moreover, we show that inhibition of ligand-mediated Her4 signaling impedes disease relapse in cases where NRG1 inhibition is insufficient. These findings demonstrate that ligand-dependent Her4 signaling plays an important role in disease relapse.

Noncovalent wild-type-sparing inhibitors of EGFR T790M.

UNLABELLED: Approximately half of EGFR-mutant non-small cell lung cancer (NSCLC) patients treated with small-molecule EGFR kinase inhibitors develop drug resistance associated with the EGF receptor (EGFR) T790M "gatekeeper" substitution, prompting efforts to develop covalent EGFR inhibitors, which can effectively suppress EGFR T790M in preclinical models. However, these inhibitors have yet to prove clinically efficacious, and their toxicity in skin, reflecting activity against wild-type EGFR, may limit dosing required to effectively suppress EGFR T790M in vivo. While profiling sensitivity to various kinase inhibitors across a large cancer cell line panel, we identified indolocarbazole compounds, including a clinically well-tolerated FLT3 inhibitor, as potent and reversible inhibitors of EGFR T790M that spare wild-type EGFR. These findings show the use of broad cancer cell profiling of kinase inhibitor efficacy to identify unanticipated novel applications, and they identify indolocarbazole compounds as potentially effective EGFR inhibitors in the context of T790M-mediated drug resistance in NSCLC. SIGNIFICANCE: EGFR-mutant lung cancer patients who respond to currently used EGFR kinase inhibitors invariably develop drug resistance, which is associated with the EGFR T790M resistance mutation in about half these cases. We unexpectedly identified a class of reversible potent inhibitors of EGFR T790M that do not inhibit wild-type EGFR, revealing a promising therapeutic strategy to overcome T790M-associated drug-resistant lung cancers.

Reorienting the Fab domains of trastuzumab results in potent HER2 activators.

The structure of the Fab region of antibodies is critical to their function. By introducing single cysteine substitutions into various positions of the heavy and light chains of the Fab region of trastuzumab, a potent antagonist of HER2, and using thiol chemistry to link the different Fabs together, we produced a variety of monospecific F(ab')(2)-like molecules with activities spanning from activation to inhibition of breast tumor cell growth. These isomers (or bis-Fabs) of trastuzumab, with varying relative spatial arrangements between the Fv-regions, were able to either promote or inhibit cell-signaling activities through the PI3K/AKT and MAPK pathways. A quantitative phosphorylation mapping of HER2 indicated that the agonistic isomers produced a distinct phosphorylation pattern associated with activation. This study suggests that antibody geometric isomers, found both in nature and during synthetic antibody development, can have profoundly different biological activities independent of their affinities for their target molecules.

A two-in-one antibody against HER3 and EGFR has superior inhibitory activity compared with monospecific antibodies.

Extensive crosstalk among ErbB/HER receptors suggests that blocking signaling from more than one family member may be essential to effectively treat cancer and limit drug resistance. We generated a conventional IgG molecule MEHD7945A with dual HER3/EGFR specificity by phage display engineering and used structural and mutational studies to understand how a single antigen recognition surface binds two epitopes with high affinity. As a human IgG1, MEHD7945A exhibited dual action by inhibiting EGFR- and HER3-mediated signaling in vitro and in vivo and the ability to engage immune effector functions. Compared with monospecific anti-HER antibodies, MEHD7945A was more broadly efficacious in multiple tumor models, showing that combined inhibition of EGFR and HER3 with a single antibody is beneficial.

Structure of the complex between HER2 and an antibody paratope formed by side chains from tryptophan and serine.

Engineered antibody paratopes with limited sequence diversity permit assessment of the roles played by different amino acid side chains in creating the high-affinity, high-specificity interactions characteristic of antibodies. We describe a paratope raised against the human ErbB family member HER2, using a binary diversity tryptophan/serine library displayed on phage. Fab37 binds to the extracellular domain of HER2 with sub-nanomolar affinity. An X-ray structure at 3.2 A resolution reveals a contact paratope composed almost entirely of tryptophan and serine residues. Mutagenesis experiments reveal which of these side chains are more important for direct antigen interactions and which are more important for conformational flexibility. The crystal lattice contains an unprecedented trimeric arrangement of HER2 closely related to previously observed homodimers of the related epidermal growth factor receptor.