Antibody to CD137 Activated by Extracellular Adenosine Triphosphate Is Tumor Selective and Broadly Effective In Vivo without Systemic Immune Activation.

Agonistic antibodies targeting CD137 have been clinically unsuccessful due to systemic toxicity. Because conferring tumor selectivity through tumor-associated antigen limits its clinical use to cancers that highly express such antigens, we exploited extracellular adenosine triphosphate (exATP), which is a hallmark of the tumor microenvironment and highly elevated in solid tumors, as a broadly tumor-selective switch. We generated a novel anti-CD137 switch antibody, STA551, which exerts agonistic activity only in the presence of exATP. STA551 demonstrated potent and broad antitumor efficacy against all mouse and human tumors tested and a wide therapeutic window without systemic immune activation in mice. STA551 was well tolerated even at 150 mg/kg/week in cynomolgus monkeys. These results provide a strong rationale for the clinical testing of STA551 against a broad variety of cancers regardless of antigen expression, and for the further application of this novel platform to other targets in cancer therapy. SIGNIFICANCE: Reported CD137 agonists suffer from either systemic toxicity or limited efficacy against antigen-specific cancers. STA551, an antibody designed to agonize CD137 only in the presence of extracellular ATP, inhibited tumor growth in a broad variety of cancer models without any systemic toxicity or dependence on antigen expression.See related commentary by Keenan and Fong, p. 20.This article is highlighted in the In This Issue feature, p. 1.

Exploitation of Elevated Extracellular ATP to Specifically Direct Antibody to Tumor Microenvironment.

The extracellular adenosine triphosphate (ATP) concentration is highly elevated in the tumor microenvironment (TME) and remains tightly regulated in normal tissues. Using phage display technology, we establish a method to identify an antibody that can bind to an antigen only in the presence of ATP. Crystallography analysis reveals that ATP bound in between the antibody-antigen interface serves as a switch for antigen binding. In a transgenic mouse model overexpressing the antigen systemically, the ATP switch antibody binds to the antigen in tumors with minimal binding in normal tissues and plasma and inhibits tumor growth. Thus, we demonstrate that elevated extracellular ATP concentration can be exploited to specifically target the TME, giving therapeutic antibodies the ability to overcome on-target off-tumor toxicity.

Optimizing the Physicochemical Properties of Raf/MEK Inhibitors by Nitrogen Scanning.

Substituting a carbon atom with a nitrogen atom (nitrogen substitution) on an aromatic ring in our leads 11a and 13g by applying nitrogen scanning afforded a set of compounds that improved not only the solubility but also the metabolic stability. The impact after nitrogen substitution on interactions between a derivative and its on- and off-target proteins (Raf/MEK, CYPs, and hERG channel) was also detected, most of them contributing to weaker interactions. After identifying the positions that kept inhibitory activity on HCT116 cell growth and Raf/MEK, compound 1 (CH5126766/RO5126766) was selected as a clinical compound. A phase I clinical trial is ongoing for solid cancers.

The sulfamide moiety affords higher inhibitory activity and oral bioavailability to a series of coumarin dual selective RAF/MEK inhibitors.

Introducing a sulfamide moiety to our coumarin derivatives afforded enhanced Raf/MEK inhibitory activity concomitantly with an acceptable PK profile. Novel sulfamide 17 showed potent HCT116 cell growth inhibition (IC50=8 nM) and good PK profile (bioavailability of 51% in mouse), resulting in high in vivo antitumor efficacy in the HCT116 xenograft (ED50=4.8 mg/kg). We confirmed the sulfamide moiety showed no negative impact on tests run on the compound to evaluate DMPK (PK profiles in three animal species, CYP inhibition and CYP induction) and the safety profile (hERG and AMES tests). Sulfamide 17 had favorable properties that warranted further preclinical assessment.

Enhanced inhibition of ERK signaling by a novel allosteric MEK inhibitor, CH5126766, that suppresses feedback reactivation of RAF activity.

Tumors with mutant RAS are often dependent on extracellular signal-regulated kinase (ERK) signaling for growth; however, MEK inhibitors have only marginal antitumor activity in these tumors. MEK inhibitors relieve ERK-dependent feedback inhibition of RAF and cause induction of MEK phosphorylation. We have now identified a MEK inhibitor, CH5126766 (RO5126766), that has the unique property of inhibiting RAF kinase as well. CH5126766 binding causes MEK to adopt a conformation in which it cannot be phosphorylated by and released from RAF. This results in formation of a stable MEK/RAF complex and inhibition of RAF kinase. Consistent with this mechanism, this drug does not induce MEK phosphorylation. CH5126766 inhibits ERK signaling output more effectively than a standard MEK inhibitor that induces MEK phosphorylation and has potent antitumor activity as well. These results suggest that relief of RAF feedback limits pathway inhibition by standard MEK inhibitors. CH5126766 represents a new type of MEK inhibitor that causes MEK to become a dominant-negative inhibitor of RAF and that, in doing so, may have enhanced therapeutic activity in ERK-dependent tumors with mutant RAS.

Fluorine Scanning by Nonselective Fluorination: Enhancing Raf/MEK Inhibition while Keeping Physicochemical Properties.

A facile methodology effective in obtaining a set of compounds monofluorinated at various positions (fluorine scan) by chemical synthesis is reported. Direct and nonselective fluorination reactions of our lead compound 1a and key intermediate 2a worked efficiently to afford a total of six monofluorinated derivatives. All of the derivatives kept their physicochemical properties compared with the lead 1a and one of them had enhanced Raf/MEK inhibitory activity. Keeping physicochemical properties could be considered a benefit of monofluorinated derivatives compared with chlorinated derivatives, iodinated derivatives, methylated derivatives, etc. This key finding led to the identification of compound 14d, which had potent tumor growth inhibition in a xenograft model, excellent PK profiles in three animal species, and no critical toxicity.

Cell binding isoforms of vascular endothelial growth factor-A (VEGF189) contribute to blood flow-distant metastasis of pulmonary adenocarcinoma.

Vascular endothelial growth factor A (VEGF-A) has two kinds of isoforms depending on cellular binding domains. VEGF189 is the largest molecule with the strongest cellular binding ability, and is thought to be most potent for vascularization in various cancers. This study aims to clear the clinicopathological characteristics of VEGF189 in the pulmonary adenocarcinoma. We finely and quantitatively examined the expression of VEGF-A isoforms (VEGF121, VEGF165 and VEGF189) by real-time polymerase chain reaction in a total of 100 pulmonary adenocarcinomas resected by surgical operation. The VEGF isoform expression status was analyzed on clinicopathological features including stromal vascularization, vascular involvement, distant metastasis, lymph nodal metastasis, postoperative relapse time and prognosis of long-term observation periods. All the pulmonary adenocarcinomas showed significant expression of VEGF-A. Twenty-two cases with the adenocarcinomas overexpressing VEGF-A significantly showed earlier postoperative relapse and poorer prognosis between 5- to 15-year periods (p = 0.0093 and p = 0.0240, Kaplan Meier, log-rank test). The expression levels of VEGF189 increased in 13% of the pulmonary adenocarcinoma. These 13 cases with increased VEGF189 expression significantly showed higher distant metastases, earlier postoperative relapse, and poorer prognosis (p = 0.0006, Fisher's test; p = 0.0016 and p = 0.0084, Kaplan Meier, log-rank test) than the other 87 cases. The 13 lung cancers with VEGF189 overexpression also showed increased vessel counts, areas (p = 0.0091 and p < 0.0001, Mann-Whitney U test) and enhanced venous involvement (p = 0.0056, Fisher's test). The cellular binding isoform VEGF189 confers pulmonary adenocarcinoma patients with poorer prognosis with distant metastasis via blood flow.

The modifier subunit of glutamate cysteine ligase (GCLM) is a molecular target for amelioration of cisplatin resistance in lung cancer.

It is unclear whether a subunit of glutamate cysteine ligase [a modifier subunit (GCLM) and a catalytic subunit (GCLC)] is an effective target for ameliorating cisplatin (CDDP)-resistance. We inhibited each subunit of GCL mRNA using a specific ribozyme (M-Rz and C-Rz) in the pulmonary adenocarcinoma cell line A549. GCL activity was suppressed by the ribozyme. CDDP-resistance was more effectively ameliorated when GCLM rather than GCLC was inhibited. GCLM is a potentially more effective pharmacologic target for ameliorating CDDP-resistance in non-small cell lung cancer than GCLC.

Overexpression of the thrombospondin 2 (TSP2) gene modulated by the matrix metalloproteinase family expression and production in human colon carcinoma cell line.

Thrombospondin 2 (TSP2) is an extracellular matrix glycoprotein involved in tumor progression and angiogenesis. We evaluated whether overexpression of the TSP2 gene show an alteration of various genes by cDNA arrays in the colon carcinoma cell line SW480. The transformants with the human TSP2 gene overexpression showed a down-regulation of matrix metalloproteinase 2 (MMP2) and MMP9 in comparison to those with vector-control. Protein production of MMP2 and MMP9 decreased in the transformants overexpressing the TSP2 gene. Conversely, the SW480 transformants showed up-regulation of MMP12 and MMP17. These results suggested that the TSP2 gene is a multifunctional modulator of remodeling tissue in which matrix degradation is required.

Unique properties of 189 amino acid isoform of vascular endothelial growth factor in tumorigenesis.

The 189 amino acid isoform of vascular endothelial growth factor (VEGF189) has been shown to be more strongly associated with the cell membrane than other isoforms of human VEGF (VEGF121, VEGF165). To analyze the biological activities of these VEGF isoforms on tumor growth, we transfected human VEGF121, VEGF165 or VEGF189 cDNA into the human colon cancer cell line SW-480, and established several clones overexpressing these VEGF isoforms. The total amounts of VEGF protein in the culture supernatants of the VEGF189-transfectants were less than those of VEGF121 and VEGF165-transfectants. These transfectants showed no significant differences in growth in culture. Nevertheless, the rate of in vivo tumor growth of VEGF189-transfectants was faster than or equivalent to that of VEGF121-transfectants, while the VEGF165-transfectant showed the greatest enhancement of tumor growth. The protein levels of VEGF were markedly increased only in the VEGF189-transfectants cultured in the presence of heparin. The enhanced in vivo tumor growth of VEGF189-transfectants can be partly explained by the cell-associated features of VEGF189 molecules. The VEGF189 molecule, which is strongly bound to the cell surface, has unique properties and high potential in local angiogenesis and tumor growth in the cancer inductive microenvironment.

Ribozyme mediated suppression of vascular endothelial growth factor gene expression enhances matrix metalloproteinase 1 expression in a human hepatocellular carcinoma cell line.

The levels of expression of various genes were altered in cellular transformants with manipulation of expression of single genes. Vascular endothelial growth factor A (VEGF-A) is a key molecule for tumor progression, although it is unclear how VEGF-A expression regulates various genes. Multiple gene expression levels were evaluated using cDNA arrays in a human hepatocellular carcinoma cell line (HLF) with suppression of the VEGF-A gene by anti-VEGF-A ribozyme (alphaVRz). The ribozyme-mediated suppression of VEGF-A gene solely up-regulated matrix metalloproteinase 1 (MMP1) gene level in HLF/alphaVRz. Levels of expression of other members of MMP family or tissue inhibitors of MMPs did not show any alteration. These results suggested that intracellular suppression of VEGF-A gene was specifically linked to up-regulation of MMP1 in human hepatocellular carcinoma cells.

Human thrombospondin 2 inhibits proliferation of microvascular endothelial cells.

This study was performed to characterize human thrombospondin 2 (TSP2). TSP2 has recently attracted attention as an endogenous negative regulator of angiogenesis in tumorigenesis. We cloned and transfected human TSP2 cDNA into the human colon cancer cell line SW-480. Stable transfectants (TSP2-1, TSP2-6) overexpressing TSP2 were established. Growth characteristics of TSP2-transfectants were investigated in vitro and in vivo. TSP2-transfectants showed similar growth properties to vector-transfectants and wild-type SW-480 cells. The overexpression of transfected human TSP2 cDNA did not affect proliferation of SW-480 cells. When the conditioned media of TSP2-transfectants were added to cultures of bovine pulmonary microvascular endothelial cells (BPMEC), the BPMEC proliferation was significantly inhibited. These results suggested that human TSP2 is a potential inhibitor of angiogenesis.