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Probody therapeutics (Pb-Txs) are conditionally activated antibody–drug conjugates (ADCs) designed to remain inactive until proteolytically activated in the tumor microenvironment, enabling safer targeting of antigens expressed in both tumor and normal tissue. Previous attempts to target CD71, a highly expressed tumor antigen, have failed to establish an acceptable therapeutic window due to widespread normal tissue expression. This study evaluated whether a probody–drug conjugate targeting CD71 can demonstrate a favorable efficacy and tolerability profile in preclinical studies for the treatment of cancer. CX-2029, a Pb-Tx conjugated to maleimido-caproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E, was developed as a novel cancer therapeutic targeting CD71. Preclinical studies were performed to evaluate the efficacy and safety of this anti-CD71 PDC in patient-derived xenograft (PDX) mouse models and cynomolgus monkeys, respectively. CD71 expression was detected at high levels by IHC across a broad range of tumor and normal tissues. In vitro, the masked Pb-Tx form of the anti-CD71 PDC displayed a >50-fold reduced affinity for binding to CD71 on cells compared with protease-activated, unmasked anti-CD71 PDC. Potent in vivo tumor growth inhibition (stasis or regression) was observed in >80% of PDX models (28/34) at 3 or 6 mg/kg. Anti-CD71 PDC remained mostly masked (>80%) in circulation throughout dosing in cynomolgus monkeys at 2, 6, and 12 mg/kg and displayed a 10-fold improvement in tolerability compared with an anti-CD71 ADC, which was lethal. Preclinically, anti-CD71 PDC exhibits a highly efficacious and acceptable safety profile that demonstrates the utility of the Pb-Tx platform to target CD71, an otherwise undruggable target. These data support further clinical development of the anti-CD71 PDC CX-2029 as a novel cancer therapeutic.

The RAS/RAF/MEK pathway is activated in more than 30% of human cancers, most commonly via mutation in the K-ras oncogene and also via mutations in BRAF. Several allosteric mitogen-activated protein/extracellular signal–regulated kinase (MEK) inhibitors, aimed at treating tumors with RAS/RAF pathway alterations, are in clinical development. However, acquired resistance to these inhibitors has been documented both in preclinical and clinical samples. To identify strategies to overcome this resistance, we have derived three independent MEK inhibitor–resistant cell lines. Resistance to allosteric MEK inhibitors in these cell lines was consistently linked to acquired mutations in the allosteric binding pocket of MEK. In one cell line, concurrent amplification of mutant K-ras was observed in conjunction with MEK allosteric pocket mutations. Clonal analysis showed that both resistance mechanisms occur in the same cell and contribute to enhanced resistance. Importantly, in all cases the MEK-resistant cell lines retained their addiction to the mitogen-activated protein kinase (MAPK) pathway, as evidenced by their sensitivity to a selective inhibitor of the ERK1/2 kinases. These data suggest that tumors with acquired MEK inhibitor resistance remain dependent on the MAPK pathway and are therefore sensitive to inhibitors that act downstream of the mutated MEK target. Importantly, we show that dual inhibition of MEK and ERK by small molecule inhibitors was synergistic and acted to both inhibit the emergence of resistance, as well as to overcome acquired resistance to MEK inhibitors. Therefore, our data provide a rationale for cotargeting multiple nodes within the MAPK signaling cascade in K-ras mutant tumors to maximize therapeutic benefit for patients. Mol Cancer Ther; 11(5); 1143–54. ©2012 AACR.

Alterations of the phosphoinositide-3 kinase (PI3K)/Akt signaling pathway occur broadly in cancer via multiple mechanisms including mutation of the PIK3CA gene, loss or mutation of phosphatase and tensin homolog (PTEN), and deregulation of mammalian target of rapamycin (mTOR) complexes. The dysregulation of this pathway has been implicated in tumor initiation, cell growth and survival, invasion and angiogenesis, thus, PI3K and mTOR are promising therapeutic targets for cancer. We discovered GDC-0980, a selective, potent, orally bioavailable inhibitor of Class I PI3 kinase and mTOR kinase (TORC1/2) with excellent pharmacokinetic and pharmaceutical properties. GDC-0980 potently inhibits signal transduction downstream of both PI3K and mTOR, as measured by pharmacodynamic (PD) biomarkers, thereby acting upon two key pathway nodes to produce the strongest attainable inhibition of signaling in the pathway. Correspondingly, GDC-0980 was potent across a broad panel of cancer cell lines, with the greatest potency in breast, prostate, and lung cancers and less activity in melanoma and pancreatic cancers, consistent with KRAS and BRAF acting as resistance markers. Treatment of cancer cell lines with GDC-0980 resulted in G1 cell-cycle arrest, and in contrast to mTOR inhibitors, GDC-0980 induced apoptosis in certain cancer cell lines, including those with direct pathway activation via PI3K and PTEN. Low doses of GDC-0980 potently inhibited tumor growth in xenograft models including those with activated PI3K, loss of LKB1 or PTEN, and elicited an exposure-related decrease in PD biomarkers. These preclinical data show that GDC-0980 is a potent and effective dual PI3K/mTOR inhibitor with promise for the clinic. Mol Cancer Ther; 10(12); 2426–36. ©2011 AACR.

Chondrosarcoma is a rare and usually slow growing sarcoma. This tumor is notoriously resistant to chemotherapy and radiation treatment. To improve survival rates in patients with chondrosarcoma exploring targeted therapies approaches seems promising. Thus we performed a preclinical study evaluating the efficacy of mTOR inhibitor everlimus in an orthotopic rat chondrosarcoma model and showed the potential of this mTORC 1 inhibitor to significantly delay tumor progression and relapse. Based on these data, we initiated preclinical studies in the same chondrosarcoma model to investigate how mTOR inhibition can be optimized. We first evaluated the antitumor effect of a PI3K (GDC-0941), an Akt (G-594) and a dual PI3K/mTOR inhibitor (GDC-0980). In a second step and in order to overcome forms of resistance we combined inhibition of PI3K to Akt or PI3K to the one of mTOR. GDC-0941 based drug combinations (GDC-0941+G-594; GDC-0941+RAD001) potency to inhibit tumor progression was compared to the one of each agent alone. For each setting, the effect of each treatment on mTOR and ERK pathways, apoptosis and cell proliferation was also tested. Results: As monotherapy, PI3K inhibitor (GDC-0941) was found to be the most efficient inducing an inhibition of tumor progression of 58 % whereas RAD001 induced a 49 % inhibition. The comparison of therapeutic efficiency of GDC-0941 based combinations showed an additive effect of G-594 or RAD001 to GDC-0941. Each of these combination caused an inhibition >60% in chondrosarcoma progression, whereas each agent alone induced a 22% to 45 % inhibition of tumor progression. GDC-0941 exposure inhibited chondrosarcoma proliferation as evaluated by Ki67 staining and induced Caspase 3/7 mediated apoptosis when used as single agent or in combined therapies. Our data reveal that all combination treatments blocked Akt activation, and inhibited phosphorylation of both 4EBP1 and S6K1 which resulted in. The tested treatments induced a similar inhibition of mTOR mediated signaling. Chondrosarcoma exposure to GDC-0941 or G-594 caused a 20% inhibition of p70S6K and 4EBP activation similar to the one induced by the rapalog RAD001. The PI3K inhibitor GDC-0941 caused an activation of Erk Pathway as noted by the strong increase in bRaf phosphorylation. Taken together these data confirm that targeting the PI3K/mTOR pathway is an efficient treatment for chondrosarcoma and combined therapy best way to cause inhibition of tumor progression. The effect noted on Erk pathway suggests that new combination approaches targeting the mTOR and Erk pathway could be of interest. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C260. Citation Format: Delphine Jury, Anne Valerie Decouvelaere, Amandine Pasquier, Marcia Belvin, Lorie Friedman, Jean Baptiste Langlois, Jean Philippe Michot, Jean Yves Blay, Aurelie Dutour. Dual targeting of the PI3K/Akt/mTOR pathway significantly delays chondrosarcoma progression and relapse. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C260.

Introduction: The phosphoinositide-3 kinase (PI3K)/Akt signaling pathway is deregulated in a wide variety of cancers. Somatic activating mutations and amplifications in PI3K are common in multiple cancers including breast, colon, and lung cancer. PTEN, a phosphatase that converts PIP3 to PIP2 and thus has an opposing function to PI3K, is a commonly mutated tumor suppressor in multiple cancers including prostate and glioblastoma. Alterations of this pathway have been implicated in tumor initiation, progression, survival, angiogenesis, and metastasis. PI3K/Akt pathway activation has also been implicated in therapeutic resistance. Thus PI3K is considered to be a promising therapeutic target for cancer. Summary of Results: Medicinal chemistry efforts resulted in the discovery of GDC-0980, a selective, orally bioavailable inhibitor of PI3 Kinase and mTOR kinase with promising pharmacokinetic and pharmaceutical properties. Here we report the first pre-clinical profile of GDC-0980. This compound is a potent ATP-competitive Class I PI3 kinase inhibitor with an IC50 of 4.8 nM against the PI3K p110apha subunit, 26.8 nM p110beta, 6.7 nM p110delta, 13.8 nM p110gamma, and an mTOR Kiapp of 17.3 nM. GDC-0980 demonstrates selectivity against a large panel of protein kinases as well as selectivity over PIK family kinases including Class II, Class III, and DNA-PK. GDC-0980 inhibits the PI3K signaling pathway in vitro causing a reversible G1 cell cycle arrest, and apoptosis induction in a subset of tumor cell lines. Levels of signaling pathway markers such as phosphorylated AKT (pAKT), PRAS40 (pPRAS40), and S6 (pS6) are rapidly and dramatically reduced following exposure of cells to GDC-0980. Oral dosing of GDC-0980 potently inhibits tumor growth in xenograft models including those mutated in PI3K and PTEN, and elicits an exposure-related concomitant decrease in PD biomarkers. Conclusion: These preclinical data provide compelling evidence in support of GDC-0980 as a clinical candidate, and Phase I studies are ongoing. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C201.

The PTEN/PI3K pathway is commonly mutated in cancer and therefore represents a therapeutic target. In order to investigate the primary phenotypes mediated by mutant PIK3CA in a controlled and patient-relevant context, we utilized human non-tumorigenic MCF10A parental and isogenic knock-in cell lines that harbor a common activating PIK3CA mutation (H1047R). We found that endogenous introduction of a mutated PIK3CA allele results in a marked epithelial-mesenchymal transition (EMT) and invasive phenotype compared to isogenic wild-type cells. Moreover, a potent and selective inhibitor of PIK3CA (GDC-0941) was highly effective in reversing this phenotype in 3D cell culture. Our results suggest that invasion assays and biomarkers should be used to reveal PIK3CA ‘oncogeneaddiction’ responses of pathway-targeted agents. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C68.