Cathepsin B Is Dispensable for Cellular Processing of Cathepsin B-Cleavable Antibody-Drug Conjugates.

Antibody-drug conjugates (ADC) are designed to selectively bind to tumor antigens via the antibody and release their cytotoxic payload upon internalization. Controllable payload release through judicious design of the linker has been an early technological milestone. Here, we examine the effect of the protease-cleavable valine-citrulline [VC(S)] linker on ADC efficacy. The VC(S) linker was designed to be cleaved by cathepsin B, a lysosomal cysteine protease. Surprisingly, suppression of cathepsin B expression via CRISPR-Cas9 gene deletion or shRNA knockdown had no effect on the efficacy of ADCs with VC(S) linkers armed with a monomethyl auristatin E (MMAE) payload. Mass spectrometry studies of payload release suggested that other cysteine cathepsins can cleave the VC(S) linker. Also, ADCs with a nonprotease-cleavable enantiomer, the VC(R) isomer, mediated effective cell killing with a cysteine-VC(R)-MMAE catabolite generated by lysosomal catabolism. Based on these observations, we altered the payload to a pyrrolo[2,1-c][1,4]benzodiazepine dimer (PBD) conjugate that requires linker cleavage in order to bind its DNA target. Unlike the VC-MMAE ADCs, the VC(S)-PBD ADC is at least 20-fold more cytotoxic than the VC(R)-PBD ADC. Our findings reveal that the VC(S) linker has multiple paths to produce active catabolites and that antibody and intracellular targets are more critical to ADC efficacy. These results suggest that protease-cleavable linkers are unlikely to increase the therapeutic index of ADCs and that resistance based on linker processing is improbable. Cancer Res; 77(24); 7027-37. ©2017 AACR.

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.

Ligand-independent HER2/HER3/PI3K complex is disrupted by trastuzumab and is effectively inhibited by the PI3K inhibitor GDC-0941.

Herceptin (trastuzumab) is the backbone of HER2-directed breast cancer therapy and benefits patients in both the adjuvant and metastatic settings. Here, we describe a mechanism of action for trastuzumab whereby antibody treatment disrupts ligand-independent HER2/HER3 interactions in HER2-amplified cells. The kinetics of dissociation parallels HER3 dephosphorylation and uncoupling from PI3K activity, leading to downregulation of proximal and distal AKT signaling, and correlates with the antiproliferative effects of trastuzumab. A selective and potent PI3K inhibitor, GDC-0941, is highly efficacious both in combination with trastuzumab and in the treatment of trastuzumab-resistant cells and tumors.

Erlotinib directly inhibits HER2 kinase activation and downstream signaling events in intact cells lacking epidermal growth factor receptor expression.

Erlotinib (Tarceva), is an orally available, reversible inhibitor of epidermal growth factor receptor (EGFR; HER1) that exhibits inhibitory activity on purified HER2 kinase at much higher concentrations. Despite the minimal activity on purified protein in vitro, in vivo studies show that erlotinib inhibits the growth of HER2-driven systems effectively. Several hypotheses have been put forward to explain this discrepancy. In particular, it has been suggested that erlotinib might indirectly suppress the activity of HER2 by blocking the ability of EGFR to transactivate it when the two receptors are part of a heterodimer complex. However, an alternative possibility that has not been adequately addressed is whether the direct inhibitory action of erlotinib on the HER2 kinase might account for the observed biological responses. To distinguish between a direct effect of erlotinib on HER2 kinase in intact cells or an indirect effect of erlotinib on HER2 activity that is mediated through EGFR, we generated cell lines that express either EGFR-H2 chimeric receptor or HER2 and HER3 receptors in an EGFR-negative background. We show that dose-dependent inhibition of HER2 was achieved at the receptor level, on downstream signaling molecules, and more importantly was also translated into inhibition of cell growth. Our findings imply that the inhibitory effect of erlotinib in HER2-expressing cells may in part be mediated through direct interaction with HER2 rather than indirectly through a process that requires the presence of EGFR.

Preclinical studies with Erlotinib (Tarceva).

Erlotinib HCl (Tarceva; Genentech, Inc, South San Francisco, CA) is an orally available, highly selective, reversible inhibitor of epidermal growth factor receptor (HER1/EGFR) tyrosine kinase. Inhibition of tyrosine kinase activity prevents HER1/EGFR phosphorylation, the associated downstream signaling events, and may block tumorigenesis mediated by inappropriate HER1/EGFR signaling. In vitro and in vivo studies show that erlotinib has activity against human colorectal, head and neck, non-small cell lung, and pancreatic tumor cells. Recent preclinical studies suggest that erlotinib may also have activity against tumors that are dependent on HER2 activation for growth and/or survival. Preclinical studies have addressed the feasibility of using erlotinib in combination with various chemotherapeutic agents, radiotherapy, and targeted agents. Combining agents that have different mechanisms of action has the potential to improve efficacy and inhibit the development of resistance. For example, in preclinical studies, combining erlotinib with cisplatin, doxorubicin, gemcitabine, or low-dose paclitaxel has an additive effect on antitumor activity with no increase in toxicity. Preclinical data provide a strong rationale for investigating erlotinib in the clinical setting. However, additional studies are required to gain further insights into the processes that regulate or influence the antitumor activity of erlotinib.

Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth.

ErbB2 is a ligand-less member of the ErbB receptor family that functions as a coreceptor with EGFR, ErbB3, and ErbB4. Here, we describe an approach to target ErbB2's role as a coreceptor using a monoclonal antibody, 2C4, which sterically hinders ErbB2's recruitment into ErbB ligand complexes. Inhibition of ligand-dependent ErbB2 signaling by 2C4 occurs in both low- and high-ErbB2-expressing systems. Since the ErbB3 receptor contains an inactive tyrosine kinase domain, 2C4 is very effective in blocking heregulin-mediated ErbB3-ErbB2 signaling. We demonstrate that the in vitro and in vivo growth of several breast and prostate tumor models is inhibited by 2C4 treatment.

Identification of a region within the ErbB2/HER2 intracellular domain that is necessary for ligand-independent association.

Ligand-independent ErbB2 activation occurs principally by two distinct mechanisms: overexpression and mutation. Overexpression of ErbB2 at the plasma membrane drives receptor self-association in a concentration-dependent manner, which in turn leads to constitutive receptor activation. Subsets of human breast cancers contain a molecular alteration that leads to erbB2 gene amplification and subsequent protein overexpression. Although not recognized to occur in human cancers, mutation can also lead to increased ErbB2 association. A well characterized mutant of the rodent ortholog neu involves substitution of glutamate for valine within the transmembrane domain. In each case, a number of explanations have been proposed to explain the resulting ErbB2 activation. These include stabilization of receptor oligomers, release of negative constraints, and altered receptor conformations. Here we define a short amino acid segment comprising amino acids 966-968 in the intracellular domain that seemingly disrupts receptor-receptor association that is driven either by overexpression or mutation in the transmembrane region. Because of the hydrophobic nature of these amino acids (VVI), we propose that alteration of this segment likely results in a global conformational change in an area that has been proposed previously to be a dimerization motif for ErbB homomeric association.