Overcoming resistance to ERBB pathway inhibitors

[0120] Example 1: MM-121 blocks ligand-induced ErbB3 activation

[0121] In this example, the anti-ErbB3 monoclonal antibody MM-121 (Ab#6, as disclosed in U.S. Patent No. 7,846,440) was examined in a series of in vitro experiments for its inhibition of ligand-induced activation of ErbB3 phosphorylation and Ability to transduce signals.

[0122] In vitro signaling studies measured by ELISA

[0123] In the first set of experiments, 3 cancer cell lines (ACHN (renal cell adenocarcinoma, ATCC #CRL-1611 TM ), Du145 (prostate cancer, ATCC #HTB-81 TM ) and OvCAR8 (ovarian adenocarcinoma, ATCC #HTB-161 TM) cell line; obtained from the Developmental Therapeutics Program (Developmental Therapeutics Program) of the National Cancer Institute (National Cancer Institute)) were seeded into 96-well plates and cultured overnight (maintenance medium was supplemented with 10% fetal calf serum , 2mM L-glutamine, and Pen-Strep in RPMI-1640 medium; in a humidity-controlled atmosphere at 5%...

[0126] Example 2: MM-121 overcomes resistance to erlotinib in an in vitro model of EGFR-wild-type non-small cell lung cancer

[0127] method

[0128] Nine non-small cell lung cancer (NSCLC) cell lines were obtained from the American Type Culture Collection: A549 (ATCC #CCL-185 TM ), EKVX (NCI vial0502454), NCI-H2170 (ATCC #CRL-5928 TM ), NCI-H2347 (ATCC #CRL-5942 TM ), NCI-H322M (ATCC #CRL-5806 TM ), NCI-H358 (ATCC #CRL-5806 TM ), NCI-H441 (ATCC #HTB-174 TM ), NCI-H661 (ATCC #HTB-183 TM ) and SW-900 (ATCC #HTB-59 TM ). The cell lines carry no mutations in their EGFR gene and represent 3 distinct histological subtypes (see Table X below). Cells carrying Ras mutations are indicated.

[0129] At 5,000 cells / well, cells were seeded in a 96-well 3D-culture system (low-binding NanoCulture plate, ScivaX Corporation), and cultured at 37°C in RPMI-1640 medium supplemented with 10% fetal bovine serum and Pen-Strep. After 48 h (after which time spheroids had f...

[0139] Example 3: Inhibition of pAKT production in human ovarian cancer cells in vitro

[0140] Materials and methods:

[0141] The A2780 human ovarian carcinoma cell line was initially established from tumor tissue obtained from untreated patients. The A2780cis cell line is cisplatin resistant (Catalog #93112517, Sigma). It is formed by chronic exposure of the cisplatin-sensitive parental A2780 cell line (cat. no. 93112519, Sigma) to increasing concentrations of cisplatin. A2780cis is cross-resistant to melphalan, doxorubicin and irradiation. To maintain resistance, cisplatin was added to the medium every 2-3 passages after attachment.

[0142]Resistance to cisplatin was demonstrated by treating A2780 and A2780cis cells with serial dilutions of cisplatin (0.01-10 [mu]M) for 72 hours. Cell viability was measured using the Cell Titer Glo Assay (Cat# G7570, Promega) according to the manufacturer's instructions.

[0143] The effect of cisplatin on the AKT pathway was determi...