Anti-Human IGF1 Receptor Recombinant Antibody (Figitumumab) (CAT#: TAB-232)

Figure 1 Two gastrointestinal cancer cell lines, pancreatic adenocarcinoma, BxPC3 (A), and colorectal adenocarcinoma, HT29 (B) were cultured with different doses of figitumumab 30 minutes, then were stimulated with 20 mg/mL IGF-I 5 minutes.

Western blotting shows that CP-751,871 blocks IGF-I–induced autophosphorylation of IGF-IR completely in both cells. Figitumumab terminated IGF-I–induced activation of and Akt-1, but not ERKs completely. C, in hepatocellular carcinoma, PLC/PRF/5, ligand induced both phospholyration of Akt and ERKs were blocked by 3 hours treatment with this mAb. With incubation with 1 μg/mL figitumumab from 1 to 48 hours, this agent effectively blocked IGF-I–stimulated autophosphorylation of IGF-IR and both activation of Akt and ERKs in PLC/PRF/5.

Ii, M., Li, H., Adachi, Y., Yamamoto, H., Ohashi, H., Taniguchi, H.,... & Shinomura, Y. (2011). The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status. Clinical Cancer Research, 17(15), 5048-5059.

Figure 2 The effect of figitumumab on established tumor on mice.

A, 125 μg CP-751,871 (twice a week, i.p.) tended to reduce relative size of s.c. tumors of HT29 on nude mice. The combination of CP-751,871 and 5-FU (once a week, i.p.) suppressed tumor growth (#, P = 0.0268 compared to control, each group n = 8). There are no significant differences between each single therapy and the combination. There are not significant differences in both body weight and blood glucose level on sacrifice (B) CP-751,871 alone reduced relative size of BxPC3 tumors on nude mice (§, P = 0.0133 compared to control, each group n = 8). The combination with CP-751,871 and gemcitabine blocked tumor growth most (#, P < 0.0001 compared to control). The effects of combination were more than the monotherapies (¶, P = 0.0274 compared to mAb; fl, P = 0.0036 to gemcitabine). Both body weight and blood glucose level on sacrifice were not different among 4 groups. C, ELISA showed that CP-751,871 did not affect blood concentrations of IGF-I (mean ± SE in mice treated with CP-751,871 was 415 ng/mL ± 122 and that in mice treated with control was 420 ng/mL ± 137, P = 0.9775), growth hormone (1.58 ng/mL ± 0.42 and 1.74 ng/mL ± 0.27, respectively, P = 0.7470), insulin (0.92 ng/mL ± 0.19 and 0.73 ng/mL ± 0.16, respectively, P = 0.4446), and IGF binding protein-3 (92. 07 ng/mL ± 24.92 and 66.45 ng/mL ± 20.97, respectively, P = 0.4446) on sacrifice. D, receptors expressions and apoptosis induction in SC tumors on sacrifice were assessed by immunostaining. CP-751,871 alone reduced the expression of IGF-IR in BxPC3 tumors but not in HT29. Combination treatments reduce both expression of IGF-IR in both cell types and InsR in HT29, but up-regulated InsR in BxPC3. TUNEL assay shows that CP-751, 871 induced apoptosis in HT29 and enhanced both gemcitabine and 5-FU induced apoptosis. Post hoc t test was by Fischer's PLSD. Cont, control; CP, CP-751,871 (figitumumab); Gem, gemcitabine.

Ii, M., Li, H., Adachi, Y., Yamamoto, H., Ohashi, H., Taniguchi, H.,... & Shinomura, Y. (2011). The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status. Clinical Cancer Research, 17(15), 5048-5059.

Figure 3 The effect of signal transduction on gastrointestinal cancer cells with k-ras mutation.

A, with incubation with 1 μg/mL figitumumab from 1 to 48 hours, this mAb effectively blocked IGF-I–stimulated autophosphorylation of IGF-IR and both activation of Akt and ERKs in 3 cell lines; colorectal adenocarcinoma, DLD-1; pancreatic adenocarcinoma, MIAPaca2; and esophageal squamous cell carcinoma, TE1. B, ligand induced phospholyration of down-stream, Akt and ERKs, was blocked by 3 hours treatment with figitumumab, the former tend to be seen in lower dose of figitumumab than the latter. C, in DLD-1, this mAb blocked IGF-II stimulated both phosphorylation of Akt and ERKs. D, to block 10 nmol/L insulin-induced signal transduction, more than 10 μg/mL figitumumab (3 hours incubation) are needed in DLD-1.

Ii, M., Li, H., Adachi, Y., Yamamoto, H., Ohashi, H., Taniguchi, H.,... & Shinomura, Y. (2011). The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status. Clinical Cancer Research, 17(15), 5048-5059.

Figure 4 The effect of figitumumab on colony formation and survival in k-ras mutated GI cancer cells.

A, the mAb reduced colony formation with dose dependency in both DLD-1 and MIAPaca2. One μg/mL figitumumab effectively blocked colony formation in TE1. B, caspase-3 assay shows that CP-781871 enhanced chemotherapy induced apoptosis; synergistically with 5-FU in DLD-1, and additively with gemcitabine in MIAPaca2 and with 5-FU in MIAPaca2 and TE1.

Ii, M., Li, H., Adachi, Y., Yamamoto, H., Ohashi, H., Taniguchi, H.,... & Shinomura, Y. (2011). The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status. Clinical Cancer Research, 17(15), 5048-5059.

Figure 5 The effects of figitumumab on k-ras mutated GI cancer on mice.

A, figitumumab suppressed tumor growth rate of DLD-1 on mice (n = 8, P = 0.0053). Tumor volume of mice treated with combination of this mAb and 5-FU showed least of all groups (P = 0.0151, the combination vs. control, each group n = 8), however there are no significant differences between each monotherapy and the combination. Body weight of mice on sacrifice was not influenced by treatments. B, figitumumab inhibited tumor growth rate of MIAPaca2 on mice (n = 8, P = 0.0288). The combination of this antibody and gemcitabine suppressed tumor volume most effectively of all (P < 0.0001, the combination vs. control, each group n = 8), however there are no significant differences between each monotherapy and the combination. Gemcitabine decreased murine weight on sacrifice and figitumumab single therapy up-regulated blood glucose on sacrifice. C, the combination treatment reduced Ki-67 label in DLD1. Gemcitabine alone reduce tumor cell growth and figitumumab enhanced this effect. In both tumors, 5-FU induced TUNEL positive area and figitumumab strengthened this effect. Post hoc t test was by Fischer's PLSD. Cont, control; CP, CP-751,871 (figitumumab); Gem, gemcitabine.

Ii, M., Li, H., Adachi, Y., Yamamoto, H., Ohashi, H., Taniguchi, H.,... & Shinomura, Y. (2011). The efficacy of IGF-I receptor monoclonal antibody against human gastrointestinal carcinomas is independent of k-ras mutation status. Clinical Cancer Research, 17(15), 5048-5059.

Figure 6 Anti-proliferative effect of figitumumab.

A) Analysis of the anti-proliferative effect of figitumumab on gastric and hepatocellular carcinoma cells. Two groups of cancer cells, including nine gastric cancer cell lines and eight hepatocellular carcinoma cell lines, were treated with increasing concentrations of figitumumab (0, 0.1, 1, and 10 µg/mL) for 120 h to inhibit the growth of the control cells by 30%. Cell proliferation was assessed by an MTT assay. Six replicate wells were used for each analysis, and at least three independent experiments were conducted. Data from replicate wells are presented as the mean of the remaining cells. Bars = ±SE. B) Effect of figitumumab on the IGF1R signaling pathway. Immunoblotting analysis was performed to observe the dose-response effect of figitumumab (0.1–10 µg/mL) on IGF1R signaling. SNU638, SNU719, SNU354, HepG2, and SNU368 cells were exposed to increasing concentrations of figitumumab for 72 h. The levels of proteins associated with the IGF1R pathway and their activated forms were analyzed. Differences relative to the control are shown. In each panel, representative blots from three independent experiments are shown. C) Effect of figitumumab on the cell cycle distribution. Figitumumab-sensitive cells (SNU719, HepG2, and SNU368) were treated with increasing concentrations of the drug [0 µg/mL (black solid bar), 0.1 µg/mL (gray solid bar), 1 µg/mL (white bar), and 10 µg/mL (dark gray hatched bar)] for 48 h and then stained with propidium iodide, and analyzed by flow cytometry. The percentage of cells in the G0/G1, S, and G2/M phases are shown. Columns represent the mean of three independent experiments; Bars = ±SE. *P-values <0.05, **P-values <0.01. D) Effect of figitumumab on tumor growth in mice bearing HepG2 xenografts. HepG2 cells (1×107) were injected into the right flank of nude mice (n = 5). Treatment with figitumumab (125 µg/mL [6.3 mg/kg body weight], once per week for 3 wk) was initiated once the tumor volume had reached 200 mm3. No significant body weight loss was observed during the course of the study. The tumors were measured with calipers at regular intervals. Solid circles = treatment with vehicle control alone (control), Open triangles = treatment with figitumumab. Differences between the two groups (tumor sizes of the control mice and those of mice treated with figitumumab) were compared from day 17 until the end of the treatment period (day 21) using a two-sided Student's t test. *P-values <0.05; **P-values <0.01 versus control.

Kim, J. G., Kang, M. J., Yoon, Y. K., Kim, H. P., Park, J., Song, S. H.,... & Im, S. A. (2012). Heterodimerization of glycosylated insulin-like growth factor-1 receptors and insulin receptors in cancer cells sensitive to anti-IGF1R antibody. PLoS One, 7(3), e33322.