Supplementary Figure 3C-D from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer
Molecularly targeted therapeutics continue to offer great promise in the treatment of human cancers. The receptor tyrosine kinase, HER2/ErbB2, is a validated clinical target for HER2-amplified breast cancer, as evidenced by the U.S.F.D.A approval of the humanized HER2 antibody trastuzumab (Herceptin®) and the dual HER2/EGFR small molecule tyrosine kinase inhibitor lapatinib (Tykerb®). Although many patients respond to trastuzumab, innate resistance or acquired resistance upon prolonged treatment also occur. Mechanisms of resistance to trastuzumab include upregulation of compensatory signaling pathways through activation of receptor tyrosine kinases such as EGFR, IGF-1R and met, or through mutational activation of the PI3 kinase (PI3K) pathway. An alternative approach for targeting HER2 is the direct covalent coupling of a cytotoxic drug to trastuzumab. We have previously reported the potent in vitro and in vivo efficacy of trastuzumab linked to the microtubule polymerization inhibitor mertansine (trastuzumab-DM1) in trastuzumab-sensitive and -refractory breast tumor models. Inhibition of signaling through PI3K, which is hyperactivated in HER2-amplified breast cancer due to constitutive activity of overexpressed HER2 and/or through mutation of the p110-\#945; subunit of PI3K, offers an additional therapeutic approach. We describe here the enhanced anti-tumor activity of trastuzumab-DM1 combined with the PI3K inhibitor GDC-0941, in HER2-amplified breast cancer lines with mutated PI3K: BT474 (K111N), MDA-361.1 (E545K), and KPL4 (H1047R). Combination treatment in vitro resulted in additive or synergistic inhibition of cell proliferation, as well as increased apoptosis. Similarly, in vivo efficacy was augmented with combined drug treatment compared to single agent activityin the MDA-MB-361.1 and Fo5 HER2-amplified xenograft models. Biochemical analyses of biomarkers for each agent showed inhibition of phospho-Akt and phospho-ERK by both trastuzumab-DM1 and GDC-0941, decreased phosphorylation of Rb and PRAS40 by GDC-0941, and increased levels of the mitotic markers phospho-histone H3 and cyclin B1 after treatment with trastuzumab-DM1. In addition, trastuzumab-DM1 treatment resulted in apoptosis in these breast cancer models as determined by appearance of the 23 kDa PARP-cleavage fragment, decreased levels of Bcl-XL, as well as activation of capases 3 and 7. Addition of GDC-0941 to trastuzumab-DM1 further enhanced apoptosis induction. These studies provide evidence for the use of rational drug combinations in HER2-amplified breast cancer and offer additional therapeutic approaches for patients whose disease progresses on trastuzumab or lapatinib-based therapy. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3239.
Supplementary Figure 3A-B from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer
AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA
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Antibody drug conjugates (ADCs) are a promising technology for specifically targeting cytotoxic agents to tumor cells. The current method for preparation of ADCs involves the usage of either lysine residues or cysteine residues involved in inter-chain disulfide bonds. Either method results in a heterogeneous population and inconsistent preparation, thereby posing a manufacturing challenge to produce ADCs for therapeutic utility. We have developed a novel robust biochemical assay (PHESELECTOR) to screen reactive thiols on antibody surface with an objective of utilizing these thiol groups for site-specific conjugation of cytotoxic drugs. We have screened and identified several potential ThioMab variants to conjugate cytotoxic drugs to either light or heavy chain (Fab or Fc region). These ThioMabs can then be derivatized through the reactive thiol group of engineered cysteine to obtain ADCs with defined number of drugs (~2 drugs/ab). A number of ThioMab-ADCs were produced and tested in both efficacy and toxicity models. The efficacy of the ThioMab-ADCs is equal if not superior to ADCs produced by the conventional method. Surprisingly, the safety of these conjugates is considerably better than that observed with the conventional ADCs. Therefore, ThioMab technology provides a unique advantage over conventional ADCs to overcome unresolved manufacturing challenges and offers a solution to potential ADC toxicity issues.
Supplementary Figure 3C-D from Engineered Thio-Trastuzumab-DM1 Conjugate with an Improved Therapeutic Index to Target Human Epidermal Growth Factor Receptor 2–Positive Breast Cancer
