Bispecific T cell engager (BiTE®) antibody constructs can mediate bystander tumor cell killing.

For targets that are homogenously expressed, such as CD19 on cells of the B lymphocyte lineage, immunotherapies can be highly effective. Targeting CD19 with blinatumomab, a CD19/CD3 bispecific antibody construct (BiTE®), or with chimeric antigen receptor T cells (CAR-T) has shown great promise for treating certain CD19-positive hematological malignancies. In contrast, solid tumors with heterogeneous expression of the tumor-associated antigen (TAA) may present a challenge for targeted therapies. To prevent escape of TAA-negative cancer cells, immunotherapies with a local bystander effect would be beneficial. As a model to investigate BiTE®-mediated bystander killing in the solid tumor setting, we used epidermal growth factor receptor (EGFR) as a target. We measured lysis of EGFR-negative populations in vitro and in vivo when co-cultured with EGFR-positive cells, human T cells and an EGFR/CD3 BiTE® antibody construct. Bystander EGFR-negative cells were efficiently lysed by BiTE®-activated T cells only when proximal to EGFR-positive cells. Our mechanistic analysis suggests that cytokines released by BiTE®-activated T-cells induced upregulation of ICAM-1 and FAS on EGFR-negative bystander cells, contributing to T cell-induced bystander cell lysis.

MDM2 antagonists synergize broadly and robustly with compounds targeting fundamental oncogenic signaling pathways.

While MDM2 inhibitors hold great promise as cancer therapeutics, drug resistance will likely limit their efficacy as single agents. To identify drug combinations that might circumvent resistance, we screened for agents that could synergize with MDM2 inhibition in the suppression of cell viability. We observed broad and robust synergy when combining MDM2 antagonists with either MEK or PI3K inhibitors. Synergy was not limited to cell lines harboring MAPK or PI3K pathway mutations, nor did it depend on which node of the PI3K axis was targeted. MDM2 inhibitors also synergized strongly with BH3 mimetics, BCR-ABL antagonists, and HDAC inhibitors. MDM2 inhibitor-mediated synergy with agents targeting these mechanisms was much more prevalent than previously appreciated, implying that clinical translation of these combinations could have far-reaching implications for public health. These findings highlight the importance of combinatorial drug targeting and provide a framework for the rational design of MDM2 inhibitor clinical trials.

Design and synthesis of novel amide AKT1 inhibitors with selectivity over CDK2.

Through the analysis of X-ray crystallographic information and previous SAR studies, a novel series of protein kinase B (PKB/AKT) inhibitors was developed. The compounds showed nanomolar inhibition of AKT1 and were selective against cyclin-dependent kinase 2 (CDK2).

Azole-based inhibitors of AKT/PKB for the treatment of cancer.

Through a combination of screening and structure-based rational design, we have discovered a series of N(1)-(5-(heterocyclyl)-thiazol-2-yl)-3-(4-trifluoromethylphenyl)-1,2-propanediamines that were developed into potent ATP competitive inhibitors of AKT. Studies of linker strand-binding adenine isosteres identified SAR trends in potency and selectivity that were consistent with binding interactions observed in structures of the inhibitors bound to AKT1 and to the counter-screening target PKA. One compound was shown to have acceptable pharmacokinetic properties and to be a potent inhibitor of AKT signaling and of in vivo xenograft tumor growth in a preclinical model of glioblastoma.

Kinetic mechanism of AKT/PKB enzyme family.

AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.

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.