Anti-IGF1R Recombinant Antibody (Dalotuzumab) (CAT#: TAB-736)

Figure 1 Cotreatment with dalotuzumab and ridaforolimus enhances antitumor activity in IGF1Rexpressing tumors.

A, Western blot analysis of PI3K pathway components in cancer cells treated with dalotuzumab, ridaforolimus, or the combination. In IGF1R-expressing cancer cell lines (H2122), combination treatment suppresses the activation of AKT demonstrated by decreased phosphorylation of AKT and downstream targets FOXO3A and PRAS40. B, combination therapy with ridaforolimus and dalotuzumab results in increased cell death in anchorage-independent growth inhibition assays in IGF1R-expressing cells (H2122). No such combination benefit was observed in cancer cells expressing low levels of IGF1R (H1703). C, In vivo, combination therapy with ridaforolimus and dalotuzumab significantly inhibits growth over monotherapies. Tumor growth of primary human tumor-derived lung adenocarcinoma xenograft model LXFA629 in control (vehicle) or experimental agenttreated mice (n =10/group) were plotted. Combination therapy with ridaforolimus and dalotuzumab significantly enhanced tumor growth inhibition compared to vehicle-treated (P < 0.0001) or single-agent treated groups (P < 0.05).

Di Cosimo, S., Sathyanarayanan, S., Bendell, J. C., Cervantes, A., Stein, M. N., Braña, I.,... & Jha, S. (2015). Combination of the mTOR inhibitor ridaforolimus and the anti-IGF1R monoclonal antibody dalotuzumab: preclinical characterization and phase I clinical trial. Clinical Cancer Research, 21(1), 49-59.

Figure 2 Combination therapy with mTOR and IGF1R inhibitors block feedback activation of AKT.

A, diversity in feedback activation of AKT by ridaforolimus was observed in a panel of breast cancer cell lines. B, cell lines exhibiting feedback activation of AKT following ridaforolimus treatment showed sensitivity to anti-IGF1R therapy. C, knockdown of IGF1R and its signaling pathway components blocked feedback activation of AKT. A lentiviral shRNA screen identified mTOR as an enhancer of the activity of dalotuzumab in tumor cell lines, and AKT activation by mTOR was confirmed to be mediated via the IGF1R signaling pathway. D, representative results from a shRNA screen targeting various PI3K and MAPK kinases in HT-29 CRC cells. mTOR, mammalian target of rapamycin; IGF1R, insulin-like growth factor 1 receptor; shRNA, short hairpin RNA; PI3K, phosphatidylinositol 3-kinase; CRC, colorectal cancer; PTEN, phosphatase and tensin homolog.

Di Cosimo, S., Sathyanarayanan, S., Bendell, J. C., Cervantes, A., Stein, M. N., Braña, I.,... & Jha, S. (2015). Combination of the mTOR inhibitor ridaforolimus and the anti-IGF1R monoclonal antibody dalotuzumab: preclinical characterization and phase I clinical trial. Clinical Cancer Research, 21(1), 49-59.

Figure 3 IGF-1R inhibition (MK-0646) does not enhance hormonal therapy (letrozole or tamoxifen) in MCF-7/AC-1 xenografts.

Ovariectomized female nu/nu mice between the ages of 7–8 weeks old were inoculated with MCF-7/AC-1 tumor cells in each flank and immediately supplemented with androstenedione (100 μg/day). Once bilateral flank tumors (both left and right) reached the appropriate size (250–300 mm3), mice were randomized (n ≥ 8 mice/cohort) and the appropriate treatments initiated (control, MK-0646, letrozole (LET), LET + MK-0646, tamoxifen (TAM), TAM + MK-0646) for a total of 28 days. Tumor volumes were measured twice weekly and depicted as percent change relative to day 0. Error bars represent SEM and results are representative of three independent experiments.

Becker, M. A., Hou, X., Tienchaianada, P., Haines, B. B., Harrington, S. C., Weroha, S. J.,... & Haluska, P. (2016). Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer. BMC cancer, 16(1), 814.

Figure 4 MK-0646 increases Insulin Receptor A Isoform expression in MCF-7/AC-1 xenografts.MCF-7/AC-1 xenograft tumors were harvested and immediately flash frozen following 28 days of treatment.

RNA and protein were extracted for Insulin Receptor and IGF-1R quantification by qPCR (a) and western blot analysis (b) as described in the methods. a Absolute IGF1R (Top) and IRA/B isoform (Bottom) copy number normalized to RPL19 housekeeper. b Protein from pooled group replicates isolated at indicated time points were subject to western blotting with indicated antibodies, as described in Materials and Methods section. Error bars represent SEM. *, P < 0.05; **, P < 0.01; ***, P <0.001; **** P < 0.0001.

Becker, M. A., Hou, X., Tienchaianada, P., Haines, B. B., Harrington, S. C., Weroha, S. J.,... & Haluska, P. (2016). Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer. BMC cancer, 16(1), 814.

Figure 5 Persistent mTOR signaling in response to IGF-1R inhibition is overcome by MK-8669.

Western Blot analysis of MCF-7/AC-1 (a) and MCF-7/AC-1/IGF-2 (b) cells treated with one or more of the following agents for 24 h at the indicated dose(s): LET (1uM), MK-0646 (1.25, 2.5, 5 ng/uL) and/or MK-8669 (100, 500, 1000 nM). IGF-1 was added for 10 min prior to harvest for the indicated samples.

Becker, M. A., Hou, X., Tienchaianada, P., Haines, B. B., Harrington, S. C., Weroha, S. J.,... & Haluska, P. (2016). Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer. BMC cancer, 16(1), 814.

Figure 6 In vivo activity of LET +/− MK-8669 +/− MK-0646 in MCF-7/AC-1 xenografts.

a As previously described, ovariectomized female nu/nu mice between the ages of 7–8 weeks old were inoculated with MCF-7/AC-1 tumor cells in each flank and immediately supplemented with androstenedione (100 μg/day). Once bilateral flank tumors (both left and right) reached the appropriate size (250–300 mm3), mice were randomized (n ≥ 9 mice/cohort) and the appropriate treatments initiated (Control, LET, MK-8669, MK-0646, MK-8669 + MK-0646, LET + MK-0646, LET + MK-8669 or LET + MK-0646 + MK-8669) for a total of 28 days. Tumor volumes were measured weekly and depicted as percent change relative to day 0. Error bars represent SEM and results are representative of two independent experiments. b Tumors from treatment groups were collected 28 days post treatment initiation, immediately snap frozen and lysates pooled (n ≥ 3 samples/treatment cohort) for Western Blot analysis.

Becker, M. A., Hou, X., Tienchaianada, P., Haines, B. B., Harrington, S. C., Weroha, S. J.,... & Haluska, P. (2016). Ridaforolimus (MK-8669) synergizes with Dalotuzumab (MK-0646) in hormone-sensitive breast cancer. BMC cancer, 16(1), 814.

Figure 1 IGF1R Recombinant Antibody (TAB-736) in SDS-PAGE

SDS-PAGE analysis of TAB-736 in non-reduced (lane 1) and β-mercaptoethanol-reduced (lane 2) conditions. Gel stained for 30 minutes with Coomassie Blue. As a result of different β-mercaptoethanol-reduced proteins (Heavy chain and Light chain) migrate as about 50 kDa and 25 kDa, respectively. And non-reduced protein migrates as 130-180 kDa.

Figure 2 IGF1R Recombinant Antibody (TAB-736) in HPLC

The purity of TAB-736 was greater than 95% as determined by SEC-HPLC.

Column: 3 µm, 7.8 x 300 nm
Mobile phase: 150 mM Sodium Phosphate Buffer, pH 7.0
Detection: UV 280 nm
Injection: 10 µl