An anti-HER2 biparatopic antibody that induces unique HER2 clustering and complement-dependent cytotoxicity.

Human epidermal growth factor receptor 2 (HER2) is a receptor tyrosine kinase that plays an oncogenic role in breast, gastric and other solid tumors. However, anti-HER2 therapies are only currently approved for the treatment of breast and gastric/gastric esophageal junction cancers and treatment resistance remains a problem. Here, we engineer an anti-HER2 IgG1 bispecific, biparatopic antibody (Ab), zanidatamab, with unique and enhanced functionalities compared to both trastuzumab and the combination of trastuzumab plus pertuzumab (tras + pert). Zanidatamab binds adjacent HER2 molecules in trans and initiates distinct HER2 reorganization, as shown by polarized cell surface HER2 caps and large HER2 clusters, not observed with trastuzumab or tras + pert. Moreover, zanidatamab, but not trastuzumab nor tras + pert, elicit potent complement-dependent cytotoxicity (CDC) against high HER2-expressing tumor cells in vitro. Zanidatamab also mediates HER2 internalization and downregulation, inhibition of both cell signaling and tumor growth, antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP), and also shows superior in vivo antitumor activity compared to tras + pert in a HER2-expressing xenograft model. Collectively, we show that zanidatamab has multiple and distinct mechanisms of action derived from the structural effects of biparatopic HER2 engagement.

Defective apoptotic cell contractility provokes sterile inflammation, leading to liver damage and tumour suppression.

Apoptosis is characterized by profound morphological changes, but their physiological purpose is unknown. To characterize the role of apoptotic cell contraction, ROCK1 was rendered caspase non-cleavable (ROCK1nc) by mutating aspartate 1113, which revealed that ROCK1 cleavage was necessary for forceful contraction and membrane blebbing. When homozygous ROCK1nc mice were treated with the liver-selective apoptotic stimulus of diethylnitrosamine, ROCK1nc mice had more profound liver damage with greater neutrophil infiltration than wild-type mice. Inhibition of the damage-associated molecular pattern protein HMGB1 or signalling by its cognate receptor TLR4 lowered neutrophil infiltration and reduced liver damage. ROCK1nc mice also developed fewer diethylnitrosamine-induced hepatocellular carcinoma (HCC) tumours, while HMGB1 inhibition increased HCC tumour numbers. Thus, ROCK1 activation and consequent cell contraction are required to limit sterile inflammation and damage amplification following tissue-scale cell death. Additionally, these findings reveal a previously unappreciated role for acute sterile inflammation as an efficient tumour-suppressive mechanism.

AlbuCORE: an albumin-based molecular scaffold for multivalent biologics design.

As biologics have become a mainstay in the development of novel therapies, protein engineering tools to expand on their structural advantages, namely specificity, affinity, and valency are of interest. Antibodies have dominated this field as the preferred scaffold for biologics development while there has been limited exploration into the use of albumin with its unique physiological characteristics as a platform for biologics design. There has been a great deal of interest to create bispecific and more complex multivalent molecules to build on the advantages offered by protein-based therapeutics relative to small molecules. Here, we explore the use of human serum albumin (HSA) as a scaffold for the design of multispecific biologics. In particular, we describe a structure-guided approach to the design of split HSA molecules we refer to as AlbuCORE, that effectively and spontaneously forms a native albumin-like molecule, but in a heterodimeric state upon co-expression. We show that the split AlbuCORE designs allow the creation of novel fusion entities with unique alternate geometries. We also show that, apart from these AlbuCORE fusion entities, there is an opportunity to explore their albumin-like small hydrophobic molecule carrying capacity as a drug conjugate in these designs.

Nonclinical antiangiogenesis and antitumor activities of axitinib (AG-013736), an oral, potent, and selective inhibitor of vascular endothelial growth factor receptor tyrosine kinases 1, 2, 3.

PURPOSE: Axitinib (AG-013736) is a potent and selective inhibitor of vascular endothelial growth factor (VEGF) receptor tyrosine kinases 1 to 3 that is in clinical development for the treatment of solid tumors. We provide a comprehensive description of its in vitro characteristics and activities, in vivo antiangiogenesis, and antitumor efficacy and translational pharmacology data. EXPERIMENTAL DESIGN: The potency, kinase selectivity, pharmacologic activity, and antitumor efficacy of axitinib were assessed in various nonclinical models. RESULTS: Axitinib inhibits cellular autophosphorylation of VEGF receptors (VEGFR) with picomolar IC(50) values. Counterscreening across multiple kinase and protein panels shows it is selective for VEGFRs. Axitinib blocks VEGF-mediated endothelial cell survival, tube formation, and downstream signaling through endothelial nitric oxide synthase, Akt and extracellular signal-regulated kinase. Following twice daily oral administration, axitinib produces consistent and dose-dependent antitumor efficacy that is associated with blocking VEGFR-2 phosphorylation, vascular permeability, angiogenesis, and concomitant induction of tumor cell apoptosis. Axitinib in combination with chemotherapeutic or targeted agents enhances antitumor efficacy in many tumor models compared with single agent alone. Dose scheduling studies in a human pancreatic tumor xenograft model show that simultaneous administration of axitinib and gemcitabine without prolonged dose interruption or truncation of axitinib produces the greatest antitumor efficacy. The efficacious drug concentrations predicted in nonclinical studies are consistent with the range achieved in the clinic. Although axitinib inhibits platelet-derived growth factor receptors and KIT with nanomolar in vitro potencies, based on pharmacokinetic/pharmacodynamic analysis, axitinib acts primarily as a VEGFR tyrosine kinase inhibitor at the current clinical exposure. CONCLUSIONS: The selectivity, potency for VEGFRs, and robust nonclinical activity may afford broad opportunities for axitinib to improve cancer therapy.

Functional roles of the rho/rho kinase pathway and protein kinase C in the regulation of cerebrovascular constriction mediated by hemoglobin: relevance to subarachnoid hemorrhage and vasospasm.

Although there is evidence that the Rho/Rho kinase pathway and protein kinase C (PKC) are involved in the development of cerebral vasospasm, the mechanism by which subarachnoid hemorrhage (SAH) activates these pathways is unclear. A large body of evidence points to oxyhemoglobin (OxyHb) as a major causative component of blood clot responsible for vasospasm. Therefore, the present studies were conducted to explore whether the Rho/Rho kinase and PKC may be involved in a sustained vasoconstriction induced by OxyHb in cerebral arteries. OxyHb evoked sustained vasoconstriction in the endothelium-denuded rabbit basilar arteries, which was reversed by the selective inhibitors of Rho kinase, Y-27632, and HA-1077, with the IC50 values of 0.26+/-0.02 and 0.74+/-0.1 micromol/L, respectively. In quiescent cerebrovascular smooth muscle (CVSM) cells, OxyHb induced Rho translocation, as assessed by immunoblotting, with a time course, which paralleled the contractile action of OxyHb. Rho translocation was also observed in intact arteries stimulated with OxyHb for 24 hours (219%) and 48 hours (160%). The increase in Rho translocation was fully inhibited by GGTI-297, an inhibitor of Rho prenylation. OxyHb also caused significant translocation of both PKCalpha and PKCepsilon (P<0.01), which was maximal at the time corresponding to maximal tension developed in response to OxyHb. Ro-32-0432, an inhibitor of PKC, attenuated vasoconstriction mediated by OxyHb in basilar artery. These results show, for the first time, that OxyHb-mediated signaling in CVSM utilizes the Rho/Rho kinase and PKC-based mechanisms.

Differing contributions of LIMK and ROCK to TGFß-induced transcription, motility and invasion.

The ability of transforming growth factor ß (TGFß) to induce epithelial-mesenchymal transition (EMT) is mediated by SMAD-dependent and SMAD-independent pathways such as the activation of Rho GTPase signalling. Upon activation, GTP-bound Rho stimulates the ROCK kinases, which in turn phosphorylate numerous substrates including the LIM kinases (LIMK). The net result of ROCK activation is increased actin-myosin contractile force generation, with a contribution from LIMK-induced actin filament stabilisation. In this study, we made use of siRNA-mediated knockdown and selective inhibitors to determine the contributions of ROCK and LIMK to TGFß-induced responses. We find that both ROCK and LIMK are required for TGFß stimulation of serum-response factor (SRF) transcriptional activity and actin stress fibre formation during EMT. In contrast, although LIMK inhibition had little effect on cell motility in scratch wound and Transwell migration assays, ROCK inhibition actually promoted TGFß-induced cell motility by helping individual cells to break free from the epithelial sheet. Furthermore, we demonstrate that selective inhibition of LIMK, but not ROCK, effectively blocked TGFß driven invasion through a layer of matrigel extracellular matrix protein. These results indicate that the roles of LIMK and ROCK in the Rho signalling pathway downstream of TGFß are not identical and suggest that LIMK represents an attractive therapeutic target in TGFß driven organ fibrosis and metastatic cancer spread.

Targeting activin receptor-like kinase 1 inhibits angiogenesis and tumorigenesis through a mechanism of action complementary to anti-VEGF therapies.

Genetic and molecular studies suggest that activin receptor-like kinase 1 (ALK1) plays an important role in vascular development, remodeling, and pathologic angiogenesis. Here we investigated the role of ALK1 in angiogenesis in the context of common proangiogenic factors [PAF; VEGF-A and basic fibroblast growth factor (bFGF)]. We observed that PAFs stimulated ALK1-mediated signaling, including Smad1/5/8 phosphorylation, nuclear translocation and Id-1 expression, cell spreading, and tubulogenesis of endothelial cells (EC). An antibody specifically targeting ALK1 (anti-ALK1) markedly inhibited these events. In mice, anti-ALK1 suppressed Matrigel angiogenesis stimulated by PAFs and inhibited xenograft tumor growth by attenuating both blood and lymphatic vessel angiogenesis. In a human melanoma model with acquired resistance to a VEGF receptor kinase inhibitor, anti-ALK1 also delayed tumor growth and disturbed vascular normalization associated with VEGF receptor inhibition. In a human/mouse chimera tumor model, targeting human ALK1 decreased human vessel density and improved antitumor efficacy when combined with bevacizumab (anti-VEGF). Antiangiogenesis and antitumor efficacy were associated with disrupted co-localization of ECs with desmin(+) perivascular cells, and reduction of blood flow primarily in large/mature vessels as assessed by contrast-enhanced ultrasonography. Thus, ALK1 may play a role in stabilizing angiogenic vessels and contribute to resistance to anti-VEGF therapies. Given our observation of its expression in the vasculature of many human tumor types and in circulating ECs from patients with advanced cancers, ALK1 blockade may represent an effective therapeutic opportunity complementary to the current antiangiogenic modalities in the clinic.

Dual functional monoclonal antibody PF-04605412 targets integrin alpha5beta1 and elicits potent antibody-dependent cellular cytotoxicity.

Integrin α5ß1 is overexpressed in tumor-associated stroma and cancer cells, and has been implicated in angiogenesis, tumor survival, and metastasis. Antibody-dependent cellular cytotoxicity (ADCC) by immune effector cells has been shown to contribute to clinical efficacy for several IgG1 monoclonal antibody (mAb) therapeutics. Taking advantage of these two mechanisms, we generated a fully human, fragment crystalizable (Fc)-engineered IgG1 mAb, PF-04605412 (PF-5412), which specifically neutralizes α5 and binds the Fcγ receptors (FcγR) with enhanced affinity. In vitro, PF-5412 potently inhibited α5ß1-mediated intracellular signaling, cell adhesion, migration, and endothelial cell (EC) tubulogenesis. PF-5412 induced significantly greater ADCC in α5-expressing tumor cells and ECs compared with a wild-type IgG1 (IgG1/wt) or IgG2 of identical antigen specificity. The degree of ADCC correlated with the abundance of natural killer (NK) cells in the peripheral blood mononuclear cells but was independent of donor FcγRIIIa polymorphism. In animal studies, PF-5412 displayed robust and dose-dependent antitumor efficacy superior to that observed with IgG1/wt, IgG2, or IgG4 of identical antigen specificity. The degree of efficacy correlated with α5 expression, macrophage and NK cell infiltration, and NK activity in the tumor. Depletion of host macrophages abrogated antitumor activity, suggesting a critical contribution of macrophage-mediated antitumor activity of PF-5412. Combination of PF-5412 with sunitinib significantly improved antitumor efficacy compared with either agent alone. The dual mechanism of action and robust antitumor efficacy of PF-5412 support its clinical development for the treatment of a broad spectrum of human malignancies.

LIM kinases are required for invasive path generation by tumor and tumor-associated stromal cells.

LIM kinases 1 and 2 (LIMK1/2) are centrally positioned regulators of actin cytoskeleton dynamics. Using siRNA-mediated knockdown or a novel small molecule inhibitor, we show LIMK is required for path generation by leading tumor cells and nontumor stromal cells during collective tumor cell invasion. LIMK inhibition lowers cofilin phosphorylation, F-actin levels, serum response factor transcriptional activity and collagen contraction, and reduces invasion in three-dimensional invasion assays. Although motility was unaffected, LIMK inhibition impairs matrix protein degradation and invadopodia formation associated with significantly faster recovery times in FRAP assays indicative of reduced F-actin stability. When LIMK is knocked down in MDA-MB-231 cells, they lose the ability to lead strands of collectively invading cells. Similarly, when LIMK activity is blocked in cancer-associated fibroblasts, they are unable to lead the collective invasion of squamous carcinoma cells in an organotypic skin model. These results show that LIMK is required for matrix remodeling activities for path generation by leading cells in collective invasion.