| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Cancer Therapy: Preclinical |
Pfizer Global Research and Development, Inc., Groton, Connecticut
Requests for reprints: Bruce D. Cohen, Pfizer Global Research and Development, Inc., Eastern Point Road, Groton, CT 06340. Phone: 860-715-0655; E-mail: bruce_d_cohen{at}groton.pfizer.com.
 |
ABSTRACT |
|---|
 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Experimental Design: By use of XenoMouse technology, we generated CP-751,871, a fully human IgG2 antibody with high affinity (Kd = 1.5 nmol/L) for human IGF-1R and evaluated its biological, pharmacologic, and antitumor properties.
Results: This antibody blocks binding of IGF-1 to its receptor (IC50 1.8 nmol/L), IGF-1-induced receptor autophosphorylation (IC50 0.42 nmol/L) and induced the down-regulation of IGF-1R in vitro and in tumor xenografts. The extent of IGF-1R down-regulation in vivo was proportional to CP-751,871 concentrations in the serum of tumor-bearing mice. Pharmacokinetic profiles in cynomolgus monkeys indicated a close to linear increase of exposure following i.v. dosing of antibody in the range of 3 to 100 mg/kg. CP-751,871 showed significant antitumor activity both as a single agent and in combination with Adriamycin, 5-fluorouracil, or tamoxifen in multiple tumor models. A biomarker assay was developed to establish the relationship between circulating antibody concentrations and down-regulation of IGF-1R in peripheral blood cells. The concentration of CP-751,871 required to down-regulate 50% of IGF-1R on peripheral blood cells was 0.3 nmol/L.
Conclusion: These data suggest that inhibition of the IGF cascade by use of this monoclonal antibody may be of clinical benefit in the treatment of human cancers.
Key Words: IGF-1R • Antibody • Therapy • Biomarker
|
INTRODUCTION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
IGF-1R is also crucial in tumor biology and malignant transformation. Fibroblasts lacking IGF-1R because of targeted disruption cannot be transformed by most oncogenes, but susceptibility to transformation is restored if IGF-1R expression is provided by transfection (3). Numerous studies have linked higher plasma concentrations of IGF-I with increased cancer risk (1), especially increased risk of prostate and colorectal cancer (4, 5). Also, many tumors and tumor cell lines have increased expression of IGF-I or IGF-1R (2). In addition to a role in transformation of cells, continued signals from IGF-1R are important for tumor cell proliferation and survival. Interruption of those signals by various means produce antiproliferative effects in cell culture and antitumor activity in tumor xenograft models. Down-regulation of IGF-1R by antisense inhibits growth of melanoma cells in soft agar and growth of these cells as xenografts in athymic mice (6). Similar results were found for glioblastoma cells (7) and breast cells (8). Blocking IGF-1R signals with antagonistic IGF-I peptide analogues inhibit the growth of prostate cancer cell lines (9). Expression of a dominant-negative form of IGF-1R, introduced by transfection into HT29 human colon cancer cells, virtually eliminated growth of these cells in soft agar and greatly slowed growth of the cognate tumors in athymic mice (10). A secreted truncated (extracellular region) IGF-1R was shown to block IGF-1R autophosphorylation, induce tumor cell apoptosis, and inhibit the formation of tumors from C6 rat glioblastoma cells (11). Thus, multiple independent approaches indicate that inhibition of IGF-1R signals may be an effective approach to cancer therapy.
Monoclonal antibodies that prevent IGF-1 binding to IGF-1R have also been used to inhibit IGF-1R function. One such antibody, the murine monoclonal antibody 
IR-3, identified almost 20 years ago, inhibits binding of IGF-I to MDA-231 human breast cancer cells and inhibits the growth of these cells in soft agar and as tumor xenografts (12–14). IR-3 also had antitumor activity against multiple tumor xenografts (15–18). These experiments support the applicability of anti-IGF-1R antibodies in chemotherapy, but the expected human anti-mouse antibody response to IR-3 would limit the use of this agent in the clinic.
Human monoclonal antibodies can be generated by use of the "XenoMouse," in which the human immunoglobulin loci was transplanted into the genome of mice with an inactivated endogenous immunoglobulin locus (19). This approach has been successfully used to generate human therapeutic antibodies (e.g., an antibody directed against the epidermal growth factor receptor; ref. 20). We report here the discovery and characterization of a human monoclonal antibody that binds the human IGF-1R with high affinity and specificity, blocks IGF-I binding, and prevents activation of IGF-1R. This antibody induces a depletion of cellular IGF-1R protein, inhibits tumor growth as a single agent and enhances the efficacy of other cancer agents in human tumor xenograft models.
|
MATERIALS AND METHODS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Cell Lines. The Colo-205 human colorectal carcinoma cell line, MCF7 breast carcinoma, and RPMI multiple myeloma cell lines were obtained from the American Type Culture Collection (Manassas, VA) repository. The 3T3-IGF-1R cells are 3T3 cells transfected with full length human IGF-1R (kindly provided by Dr. Renato Baserga, Kimmel Cancer Center at Thomas Jefferson University, Philadelphia, PA). Colo-205 cells were maintained using DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% heat inactivated FSC (Invitrogen), 2 mmol/L L-glutamine (Invitrogen), and penicillin-streptomycin (Invitrogen). 3T3-IGF-1R cells were maintained using the same medium as Colo-205, supplemented with 0.5 mg/mL Geneticin (Invitrogen). MCF7 cells were maintained using DMEM F12 (Invitrogen) supplemented with 10% heat inactivated FCS, 2 mmol/L L-glutamine, and penicillin-streptomycin.
[125I]-IGF-1 Cell Binding Assay. 3T3-IGF-1R cells were plated in 96-well plates, in full medium. Following overnight incubation, the cells were changed to serum-free DMEM and incubated for 1 hour. CP-751,871 was serially diluted in serum-free DMEM. Medium was decanted from the cell plates, and 50 µL of each antibody dilution added to the cell plate. The plate was incubated at 37°C for 10 minutes. [125I]-IGF-I (NEN, Wilmington, DE) was diluted in serum-free DMEM to 1 µCi/mL. Fifty microliters were added per well of the cell plate. Addition of 100 ng/mL cold IGF-1 (R&D Systems, Minneapolis, MN) was used as control for background. The cell plate was incubated at 37°C for 10 minutes. Medium was decanted and the plates washed twice with serum-free DMEM. The cells were lysed in 0.1 N NaOH + 0.1% SDS, then transferred to a scintillation plate, mixed with OptiPhase Supermix liquid scintillant (Wallac, Shelton, CT), and cell-associated [125I]-IGF-1 quantitated on a Wallac Micro-Beta counter.
IGF-1-R Autophosphorylation. Capture ELISA plates were prepared by incubating Reacti-Bind Goat anti-rabbit plates (Pierce, Rockford, IL) with anti-IGF-1R antibody (Santa Cruz Biotech, Santa Cruz, CA), diluted in 5% bovine serum albumin (Sigma, St. Louis, MO)/TBST [50 mmol/L Tris (pH 8.0), 138 mmol/L NaCl, 2.7 mmol/L KCl, and 0.1% Tween 20]. Plates were incubated while shaking at room temperature for 1 hour. Plates were washed with TBST before addition of the cell lysates.
3T3-IGF-1R cells were plated in 96-well plates in growth medium (see above) and incubated overnight at 37°C, 5% CO2. Media was decanted from the cell plates and 100 µL fresh growth medium added. CP-751,871 to the indicated final concentration was added to the cell plate (all samples were in triplicate). The plates were incubated at 37°C for 1 hour. Cells were stimulated by addition of 100 ng/mL IGF-1 (final concentration in growth medium) and incubated at room temperature for 10 minutes. Media was decanted from the plates. Cells were lysed in 50 µL lysis buffer [50 mmol/L HEPES (pH 7.4), 10 mmol/L EDTA, 150 mmol/L NaCl, 1.5 mmol/L MgCl2, 1% Triton X-100, 1.6 mmol/L NaVO4, and 1% glycerol], and lysates transferred to dilution plates; 100 µL dilution buffer [50 mmol/L HEPES (pH 7.4) and 1.6 mmol/L NaVO4] were added to each lysate, mixed, and 100 µL of each diluted lysate were transferred to the ELISA capture plate.
The ELISA plates were incubated shaking gently at room temperature for 2 hours, washed with wash buffer, then treated with HRP-PY20 (BD Transduction Labs, Lexington, KY) diluted in bovine serum albumin/TBST and incubated at room temperature for 30 minutes with shaking. The ELISA plates were washed, color developed by addition of 3,3',5,5' tetramethylbenzidine substrate (Calbiochem, San Diego, CA), and stopped with 0.9 mol/LH2SO4. IGF-1R phosphotyrosine signal was quantitated by measurement at absorbance 450 nm.
IGF-1R Confocal Analysis. 3T3IGF-1R cells were plated onto 8-well no. 1.5 cover glass chamber slides in a phenol red–free cell culture medium and grown to 80% confluence. LysoTracker Red (1 mmol/L stock solution, Molecular Probes, Eugene, OR) was diluted to 50 nmol/L in each chamber for a period of 2 hours. CP-751,871 (1 µg/mL final culture chamber concentration) was added in a time course fashion to track IGF-1R internalization. At the conclusion of the time course incubation, culture medium was aspirated and cells were quickly washed with fresh warm culture medium. The washed cells were again aspirated and fixed with ice-cold methanol for 20 minutes. Fixed cells were washed with PBS at room temperature, and nonspecific binding was blocked using 1% normal goat serum for 20 minutes at 37°C. CP-751,871 was detected in these cells using a goat anti-human secondary antibody conjugated to Alexa488 (1:100 dilution from 2 mg/mL stock, Molecular Probes) for 60 minutes at 37°C. Cells were again washed thrice in PBS. Coverslips were mounted with VectaShield anti-fade medium containing the DNA dye 4',6-diamidino-2-phenylindole and sealed with nail polish. Digital images were collected sequentially using the Leica SP laser-scanning confocal microscope using appropriate lasers and spectrophotometer setting matched to the fluorochromes.
Western Blotting. Cells were lysed in lysis buffer (as above) cleared by centrifugation, boiled in Laemmli buffer and the proteins separated on 4-12% Tris/Bis gels (Invitrogen). Western blots were done with anti-IGF1R antibody sc-713 (Santa Cruz). PY54 (OSIP), phospho-AKT (Cell Signaling, Santa Cruz, CA), or actin monoclonal (Sigma) as a loading control, with appropriate secondary antibodies.
Hybrid IGF-1R/IR Detection. IGF-1R and IR were immunprecipitated from MCF7 lysates with either CP-751,871 or a polyclonal rabbit anti-IR antibody PP5 (21). Immune complexes were washed and boiled in SDS dissociation buffer. Denatured complexes were divided and analyzed by Western blot probing for IGF-1R (Santa Cruz) or IR (PP5). In some cases, MCF7 cells were pretreated with CP-751,871 (1 µg/mL) for 15 hours in complete medium under normal growth conditions.
CP-751,871 Quantitation. Serum concentrations of CP-751,871 were determined by an ELISA method. Serum samples (15-20 µL) were diluted in lysis buffer (described above), to be within the linear range of the assay (3-30 ng/mL in assay plate). CP-751,871 was diluted similarly for the assay standards. The immunosorbent assay plates (Costar, Cambridge, MA) were coated with recombinant human IGF-1R extracellular domain (ECD, R&D Systems), and then blocked with 5% bovine serum albumin/TBST. The diluted samples (50 µL) were added to the plates with an equal volume (50 µL) of 10% milk/TBST and incubated with shaking at room temperature for 1 to 2 hours, washed in TBST, and probed with horseradish peroxidase–conjugated anti-human IgG antibody (Jackson ImmunoResearch, West Grove, PA) for 30 minutes. The plates were developed by color reaction with TMB substrate (see above) and stopped with 0.9 mol/L H2SO4. Absorbance was determined at a wavelength of 450 nm. The CP-751,871 standard dilutions were used to construct a standard curve. Serum concentrations of CP-751,871 were calculated using linear least square regression with uniform weighting.
Animal Care and Use. Three to 4-week-old female athymic mice (CD-1 nu/nu) were used for tumor xenografts. Mice were obtained from Charles River Laboratories (Wilmington, MA) and were housed in specific pathogen-free conditions according to the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care. All studies were carried out under approved institutional experimental animal care and use protocols. During these studies, animals were provided pelleted food and water ad libitum. Sentinel mice were monitored routinely at 4-week intervals by serologic assays and were found to be free of exposure to the following agents: murine hepatitis virus, Senai virus, pneumonia virus of mice, minute virus of mice, mouse poliovirus, type 3 Reovirus, Mycoplasma pulmonis, mouse parvovirus, and epizootic diarrhea of infant mice. In addition, the sentinels were monitored on a quarterly basis for lymphocytic choriomeningitis virus, mouse adenovirus, ectromelia, mouse pneumonitis, and polyomavirus. For all studies, the mice were allowed to acclimate for 3 days after receipt of shipment; test animals were randomized before commencement of treatments.
Ex vivo Studies. For tumor implantation, cells (3T3-IGF-1R) were grown in vitro, harvested with Trypsin-EDTA, washed 1X with PBS and resuspended in Matrigel (BD Biosciences, Bedford, MA) to a final concentration of 2.5 x 107 cells/mL. Athymic mice were injected with 0.2 mL of this suspension s.c. in the flank region. Once tumor sizes reached 400 to 500 mm3, the animals were dosed with either CP-751,871 or vehicle (i.p.). CP-751,871 was prepared in Dulbecco's phosphate-buffer saline (Invitrogen). At 24 hours post-dose, animals were euthanized according to Pfizer IACUC guidelines. Tumors were removed and immediately flash frozen in liquid nitrogen. Blood was collected into serum separation tubes and kept on ice. Tumors were stored at –80°C until time of analysis. Serum samples were stored at 4°C until time of analysis.
Tumor extract preparation. 3T3-IGF-1R tumors were homogenized (1 mL lysis buffer supplemented with protease inhibitor cocktail tablets; Roche, Indianapolis, IN; per 100 mg of tumor weight) on ice. The homogenates were centrifuged for clarification at 4°. Each lysate was analyzed for total protein content using the bicinchoninic acid Protein Assay kit (Pierce).
Ex vivo ELISA. Reacti-Bind goat anti-Rabbit coated plates were blocked for 30 minutes by shaking at room temperature with SuperBlock (Pierce). Blocker was aspirated and plates incubated with rabbit polyclonal anti-IGF-1R (Santa Cruz Biotechnology) diluted in SuperBlock. The plates were washed with TBST. Tumor extracts were added to plates and incubated for 1 hour at room temperature. Plates were washed with TBST and incubated with biotinylated goat anti-IGF-1R (R&D Systems) for 1 hour. Plates were washed and probed with streptavidin horseradish peroxidase (Zymed, South San Francisco, CA) for 30 minutes. Plates were washed with TBST and developed by addition of TMB. Signal was stopped by addition of 0.9 mol/L H2SO4. Quantitation of signal was measured by absorbance 450 nm. All readings were normalized to the total protein content of each sample.
Tumor Growth Inhibitory Experiments. Treatment was initiated when all mice in the experiment had established tumors of 200 to 250 mm3. CP-751,871 was prepared in PBS. Vehicle control groups were dosed with PBS alone. The general health of the mice was monitored and body weight recorded at the time of tumor measurement. Tumor dimensions (volume = length x width2 x 0.5) were recorded thrice/wk, starting with the first day of treatment. Statistical analysis by Student's t test was used to compare treatments. Animals were euthanized according to IACUC guidelines. Antitumor efficacy was measured as the percent tumor growth inhibition.
Monkey Pharmacokinetic Studies. CP-751,871, prepared in acetate formulation buffer [20 mmol/L Na acetate, 140 mmol/L NaCl, and 2 mg/mL polysorbate 80 (pH 5.5)], was given i.v. via cephalic vein to cynomolgus monkeys (1 per sex per dose) naive to CP-751,871 and other monoclonal antibodies at a bolus dose of 3, 30, and 100 mg/kg. Blood (1 mL) was collected from the femoral vein into microtainer serum separator tubes at 0.08, 2, 6, 24, 48, 72, and 168 hours post-dose and then weekly thereafter until the serum concentration of CP-751,871 reached the lower limit of quantitation (0.3 µg/mL) or day 70. Serum was prepared from whole blood by centrifugation, transferred to clean microtubes, and stored at 4°C before analysis. Serum concentrations of CP-751,871 were determined by the ELISA method as described for analysis of mouse serum samples.
Pharmacokinetic Analysis. The pharmacokinetic variables of CP-751,871 were calculated by fitting the data into a two-compartmental model using WinNonlin 3.2 (Pharsight, Corporation, Mountain View, CA): distribution and elimination coefficients and rate constants (A, B, , and ß, respectively) were directly calculated from the program, the distribution and elimination half-lives were calculated as 0.693 / and 0.693 / ß, respectively, and the other pharmacokinetic variables (AUC, CL, and Vdss) were obtained from standard formulas.
Human Lymphocyte Treatment. The expression of IGF-1R on human lymphocytes was tested using samples of five subjects and a flow cytometry assay that employs 1H7 (mouse IgG1, 
; BD-PharMingen, San Diego, CA), an anti-IGF-1R monoclonal antibody conjugated with R-Phycoerythrin, as an analytic reagent. 1H7 effectively detected the expression of IGF-1R in human lymphocytes. Immunophenotyping of T helper and cytotoxic cells, B cells, monocytes and granulocytes using CD3, CD4, CD8, CD19, CD15 markers and forward scatter channel and side scatter channel characteristics was also done to determine cell type specific levels of anti-IGF-1R signal. Range of expression was found to be 
300 to 500 molecules of equivalent soluble fluorochrome (MESF) for T-helper cells, 250 to 450 MESFs for T cytotoxic cells, 200 to 500 MESFs for B cells, 500 to 600 MESFs for monocytes, and 1,500 to 2,300 MESFs for granulocytes. The ratio of positive cells (cells with detectable IGF-1R expression) was 5% to 10% for lymphocytes, 30% for monocytes, and 90% for granulocytes. Incubation of the blood samples for 24 hours in the presence of CP-751,871 resulted in a concentration-dependent down-regulation of IGF-1R expression; 106 leucocytes were incubated with 10 µL of each analytic antibody per test for 20 minutes at 4°C, washed twice, and acquired using a Becton Dickinson FACSort with CELL Quest v3.1 software. A minimum of 50,000 total cells were analyzed. Cells were identified using a sequential gating strategy, assessing CD8, CD4 or CD19 and IGF1R (Becton Dickinson, Sunnyvale, CA) fluorescence, as well as forward and side light-scatter characteristics). MESF standards were spectrally matched with the fluorochrome used for the analysis.
CP-751,871 was tested for analytic interference with the monoclonal antibody 1H7 used in the determination of surface IGF-1R expression. Following RBC lysis with fluorescence-activated cell sorter lysis buffer (Becton Dickinson, Franklin Lakes, NJ), triplicate aliquots of 20 µL per 106 nucleated cells were added 5 µg/mL of CP-751,871 or PBS and flow cytometry analysis of IGF1R with 1H7 antibody done using a Becton Dickinson FACSCalibur. The percentage of granulocytes stained for IGF-1R in the control (PBS) and CP-751,871 samples were 94.7 ± 2.5% and 91.3 ± 3.2%, respectively. Thus, no significant analytic interference of CP-751,871 on 1H7 was detected.
|
RESULTS |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
|
|
The time course of IGF-1R loss on CP-751,871 treatment was examined in more detail. Treatment of tumor cells in culture with CP-751,871 at 1 µg/mL resulted in a time-dependent decrease in total cellular IGF-1R, which was maximal between 3 and 4 hours as determined by Western blot analysis (Fig. 2B). CP-751,871-induced internalization of IGF-1R could be monitored indirectly by following the localization of CP-751,871 over time by confocal microscopy (Fig. 2C). NIH 3T3/IGF-1R cells were exposed to CP-751,871 for different time periods, fixed, and stained with an anti-human Alexa488 conjugated antibody. Binding of CP-751,871 to the cell surface was detected within 15 minutes, and the complex translocated to the cytoplasmic compartment within 1 hour after addition of antibody. CP-751,871 also induced the down-regulation of IGF-1R protein on MCF7 breast carcinoma cells and multiple myeloma cells (RPMI 8226) in vitro.
CP-751,871 Recognizes the IGF-1R/IR Heterodimer Complex. IGF-1R and insulin receptors have been shown to associate in both homodimer and heterodimer complexes in multiple cell and tissue types (22, 23). The ability of CP-751,871 to recognize the heterodimer complex was investigated using MCF7 cells that express both IR and IGF-1R in homodimer and heterodimer complexes (24). IGF-1R complexes from cell lysates were immunoprecipitated by CP-751,781 (CP-751,871 does not cross react with IR; data not shown). Immunoblot analysis of these IGF-1R immune complexes with an anti-insulin receptor antibody showed the presence of the IR (data not shown). Conversely, immunoprecipitation with an insulin receptor antibody and immunoblotting with an IGF-1R antibody also revealed the IGF-1R/IR complex (data not shown). These data show that CP-751,871 recognizes an IR/IGF-1R heterodimer complex, and treatment of the MCF7 cells with CP-751,871 results in the down-regulation of both IGF-1R homodimers and heterodimers of IGF1-R with insulin receptor.
CP-751,871 Induces the Down-Regulation of IGF-1R In vivo. Athymic mice bearing NIH3T3/IGF-1R tumors were used to investigate the biochemical activities of CP-751,871 in vivo; i.p. injection of CP-751,871 resulted in a serum Cmax between 12 and 24 hours. At 24 hours, there was a dose-dependent reduction of IGF-1R protein in tumors, with 50% reduction observed at a serum concentration of 15 µg/mL (Fig. 3A). A time course analysis relating CP-751,871 serum concentrations with IGF-1R tumor levels showed strong relationship between antibody levels with IGF-1R down-regulation (Fig. 3B). An injected dose of 125 µg of CP-751,871 resulted in serum levels of approximately 40 µg/mL CP-751,871 and >90% down-regulation of IGF-1R from the tumor at 24 hours. The serum antibody concentrations drop to 20 µg/mL at 48 to 72 hours and IGF-1R levels rebound to 70% of control levels. CP-751,871 serum concentrations and IGF-1R reduction are maintained at this level throughout the remainder of the experiment (7 days). The half-life of CP-751,871 in an athymic mouse was determined to be 4 to 6 days by longer-term studies (data not shown).
|
6 days in the monkeys. The observed maximal serum concentration of CP-751,871 following i.v. injections of 3, 30, and 100 mg/kg doses reached 0.1 to 2 mg/mL. The elimination of CP-751,871 in cynomolgus monkeys may have been influenced by a PAHA (primate anti-human antibody) response. Direct measurement of primate anti-CP-751,871 antibodies in these monkeys were not conducted for this study, but when evaluated in subsequent studies total and neutralizing anti-CP-751,871 antibodies were detected in the monkeys treated with CP-751,871 (data not shown).
|
90% IGF-1R down-regulation in tumors at Cmax of 24 hours (Fig. 3A), and 90% tumor growth inhibition by day 10 (Fig. 5A). Tumor growth inhibition studies were extended to human tumor xenograft models. As a single agent, CP-751,871 inhibited the growth of s.c. xenografts derived from colon (Colo-205, Fig. 6A), breast (MCF7, Fig. 7), and lung (H460) cancer cell lines (data not shown).
|
|
|
Combinations of CP-751,871 were also extended to other tumors and other cytotoxic agents. Combining a single dose of CP-751,871 with 5-fluorouracil (5-FU, 100 mg/kg i.v., 50% maximal tolerated dose) in animals bearing Colo-205 tumors resulted in enhanced efficacy relative to either agent alone (Fig. 6B). Extension of these dosing combinations to a weekly schedule resulted in initial minor regression and 100% inhibition of tumor growth when both drugs were used in combination, which was superior to the single agent activities of either agent alone (Fig. 6C).
Combination of CP-751,871 with a more targeted therapeutic agent was also investigated. Tamoxifen is an anti-estrogen and is a front line therapy for estrogen receptor–positive tumors. MCF7 breast carcinoma cells are estrogen receptor positive, dependent upon estrogen for growth in athymic mice, and responsive to anti-estrogens such as tamoxifen (24). Tamoxifen was combined with CP-751,871 in the treatment of MCF7 tumors and showed an enhanced antitumor growth activity when used in combination versus either agent alone (Fig. 7). These data show that CP-751,871 can inhibit tumor growth as a single agent, and enhances the efficacy of both cytotoxic and targeted cancer therapies.
CP-751-871 Biomarker Activity. Measurements of IGF-1R depletion in patients would be a valuable biomarker to monitor the biochemical activity of an antibody directed against the IGF-1R. Ex vivo treatment of blood with CP-751,871 resulted in the down-regulation of IGF-1R on human peripheral blood mononuclear cells as monitored by flow cytometry (Fig. 8). A rapid dose-dependent depletion was observed with an IC50 of 40 to 50 ng/mL. This concentration is similar to the IC50 of CP-751,871 for in vitro inhibition of IGF-1 binding (180 ng/mL) and IGF-1-induced receptor autophosphorylation (80 ng/mL) but is far lower than the plasma concentrations needed to down-regulate IGF-1R in tumors (EC50 15 µg/mL) presumably due to poor tumor penetration.
|
|
DISCUSSION |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CP-751,871 is of the IgG2 subtype and was specifically chosen because that subtype is a poor activator of cellular immune modulators. Therefore, any tumor growth inhibition seen in vivo following antibody treatment is likely attributed to the blockade of IGF-1R function and not to an antibody-dependent cell cytotoxic (ADCC) or complement activity. Subsequent to the preparation of this article, Burtrum et al. (25) and Maloney et al. (26) have reported the creation of distinct anti-IGF-1R antibodies of the IgG1 isotype. Both of these antibodies show similar activities such as IGF-1 antagonism, IGF-1R internalization and single agent antitumor activity as compared with CP-751,871. The major distinguishing feature between these antibodies and CP-751,871 is the use of an IgG1 Fc domain versus an IgG2 for CP-751,871. These two isotypes differ in that IgG2s have a longer half-life in humans, and the IgG1 isotype in humans is capable of activating immune cell effector functions (antibody-dependent cell cytotoxic) but the IgG2 isotype is inefficient in this process. Whereas it remains to be determined whether efficacy in humans is linked to this immune effector activity, the isotype differences may also impact the safety profile of these candidates. Furthermore, given the antibody-induced IGF-1R down-regulation, antigen density may be insufficient to trigger an antibody-dependent cell cytotoxic response.
The data presented here show that CP-751,871 is able to bind to IGF-1R with high affinity and inhibit its function both by inhibiting IGF-I binding and by inducing IGF-1R down-regulation in vitro and in tumor xenografts in vivo. Down-regulation of surface receptors upon antibody binding has been frequently observed, for example with antibodies to HER2 (27) as well as antibodies to IGF1R (25, 26). IGF-1R down-regulation by CP-751,871 was proportional to the serum concentrations and was maintained for >7 days after a single dose in athymic mice. The concentration of CP-751,871 in the serum required for maximal inhibition of tumor growth as a single agent (40 µg/mL) corresponded to 90% down-regulation of IGF-1R at day 1. Serum concentrations decreased to 20 µg/mL at day 7, which corresponded to 30% reduction of IGF-1R. These data provided a basis for estimating the efficacious concentrations of CP-751,871 required for the treatment of solid tumors as a single agent.
Survival signals from IGF-1R can reduce the effectiveness of chemotherapy and various approaches for inhibition of IGF-1R have been shown to increase the activity of cytotoxic drugs (28–30). Maloney et al. have reported that a mouse monoclonal anti-IGF-1R antibody inhibited tumor growth as a single agent and enhanced the activity of gemcitabine in a pancreatic tumor model (26). We have extended these findings by use of a fully human anti-IGF-1R antibody in combination with Adriamycin, 5-fluorouracil, and tamoxifen. In all cases, submaximum tolerated doses of chemotherapy in combination with CP-751,871 enhanced efficacy and produced a more durable response. Similarly, dose escalation studies in the NIH3T3/IGF-1R tumor model showed that doses of CP-751,871 that were not efficacious as a single agent, could substantially enhance the antitumor activity of Adriamycin. These data suggest that usage of sub–maximal tolerated dose of both agents in combination may reduce high-dose associated toxicities without sacrificing clinical efficacy.
IGF-1R has been shown to be expressed on many tissues, and that widespread expression could present an obstacle to generating a therapeutic antibody, in that saturation of the target is either not possible (nonlinear PK) or requires large amounts of antibody. However, our studies in the cynomolgus monkeys, indicated a nearly linear increase in antibody concentrations with doses of 3, 30, and 100 mg/kg. Thus, depletion of anti-IGF1R by binding to normal tissues does not preclude reasonably low projected human doses.
A trend in drug development today is to shorten the time required to make critical decisions regarding the continued development of clinical candidates. The use of biomarkers can prove that the drug is inhibiting its intended target at clinically obtainable concentrations and allow comparison with those required for biochemical effects in preclinical models. One such model for CP-751,871 is to measure the down-regulation of IGF-1R on peripheral blood lymphocytes, which can be readily obtained from both human volunteers and cancer patients. Ex vivo treatment of human blood with CP-751,871 resulted in a dose-dependent down-regulation of IGF-1R at concentrations which are in line with the in vitro inhibition of IGF-1R autophosphorylation and ligand binding. Whereas the measurement of IGF-1R receptor down-regulation in circulating cells could provide evidence in clinical trials for functional biochemical modulation, it may not reflect the concentrations required to impact the growth of solid tumors due to inefficient penetration of macromolecules into solid tumors. Nonetheless, our results with this assay suggest that it may be useful for establishing proof of biochemical mechanism in conjunction with clinical trials of this agent.
CP-751,871 is a human monoclonal anti-IGF-1R antibody that can block the function of IGF-1R through multiple mechanisms. The activity showed in both in vitro and in vivo assays suggests IGF-1R provides a critical survival signal in multiple tumor types. These data together with the observations that IGF-1R is an indicator of poor prognosis, is up-regulated in human tumors, and promotes tumor cell survival, suggest that blockade of IGF-1R will successfully inhibit the progression of multiple tumor types in cancer patients. This antibody is currently in phase I clinical trials.
|
FOOTNOTES |
|---|
Received 6/ 2/04; revised 9/24/04; accepted 12/13/04.
|
REFERENCES |
|---|
|
TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
|
|
 |
|
  M. Q. Lacy, M. Alsina, R. Fonseca, M. L. Paccagnella, C. L. Melvin, D. Yin, A. Sharma, M. Enriquez Sarano, M. Pollak, S. Jagannath, et al. Phase I, Pharmacokinetic and Pharmacodynamic Study of the Anti-Insulinlike Growth Factor Type 1 Receptor Monoclonal Antibody CP-751,871 in Patients With Multiple Myeloma J. Clin. Oncol., July 1, 2008; 26(19): 3196 - 3203. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. Piao, Y. Wang, Y. Adachi, H. Yamamoto, R. Li, A. Imsumran, H. Li, T. Maehata, M. Ii, Y. Arimura, et al. Insulin-like growth factor-I receptor blockade by a specific tyrosine kinase inhibitor for human gastrointestinal carcinomas Mol. Cancer Ther., June 1, 2008; 7(6): 1483 - 1493. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. R. Hecht Current and emerging therapies for metastatic colorectal cancer: Applying research findings to clinical practice Am. J. Health Syst. Pharm., June 1, 2008; 65(11_Supplement_4): S15 - S21. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Sachdev and D. Yee Disrupting Insulin-Like Growth Factor Signaling as a Potential Cancer Therapy Am. Assoc. Cancer Res. Educ. Book, April 12, 2008; 2008(1): 39 - 58. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Kusada, T. Morizono, A. Matsumoto-Takasaki, K. Sakai, S. Sato, H. Asanuma, A. Takayanagi, and Y. Fujita-Yamaguchi Construction and Characterization of Single-chain Antibodies Against Human Insulin-like Growth Factor-I Receptor from Hybridomas Producing 1H7 or 3B7 Monoclonal Antibody J. Biochem., January 1, 2008; 143(1): 9 - 19. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. T. Kurmasheva, J. B. Easton, and P. J. Houghton Combined Targeting of mTOR and the Insulin-like Growth Factor Pathway ASCO Educational Book, January 1, 2008; 2008(1): 460 - 464. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. D. Ryan and P. E. Goss The Emerging Role of the Insulin-Like Growth Factor Pathway as a Therapeutic Target in Cancer Oncologist, January 1, 2008; 13(1): 16 - 24. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P. Haluska, H. M. Shaw, G. N. Batzel, D. Yin, J. R. Molina, L. R. Molife, T. A. Yap, M. L. Roberts, A. Sharma, A. Gualberto, et al. Phase I Dose Escalation Study of the Anti Insulin-Like Growth Factor-I Receptor Monoclonal Antibody CP-751,871 in Patients with Refractory Solid Tumors Clin. Cancer Res., October 1, 2007; 13(19): 5834 - 5840. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Chi-Hang Li and R. Li RAV12 Accelerates the Desensitization of Akt/PKB Pathway of Insulin-like Growth Factor I Receptor Signaling in COLO205 Cancer Res., September 15, 2007; 67(18): 8856 - 8864. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Q.-s. Ji, M. J. Mulvihill, M. Rosenfeld-Franklin, A. Cooke, L. Feng, G. Mak, M. O'Connor, Y. Yao, C. Pirritt, E. Buck, et al. A novel, potent, and selective insulin-like growth factor-I receptor kinase inhibitor blocks insulin-like growth factor-I receptor signaling in vitro and inhibits insulin-like growth factor-I receptor dependent tumor growth in vivo Mol. Cancer Ther., August 1, 2007; 6(8): 2158 - 2167. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. S. de Bono, G. Attard, A. Adjei, M. N. Pollak, P. C. Fong, P. Haluska, L. Roberts, C. Melvin, M. Repollet, D. Chianese, et al. Potential Applications for Circulating Tumor Cells Expressing the Insulin-Like Growth Factor-I Receptor Clin. Cancer Res., June 15, 2007; 13(12): 3611 - 3616. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Imsumran, Y. Adachi, H. Yamamoto, R. Li, Y. Wang, Y. Min, W. Piao, K. Nosho, Y. Arimura, Y. Shinomura, et al. Insulin-like growth factor-I receptor as a marker for prognosis and a therapeutic target in human esophageal squamous cell carcinoma Carcinogenesis, May 1, 2007; 28(5): 947 - 956. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Fonseca and A. K. Stewart Targeted therapeutics for multiple myeloma: The arrival of a risk-stratified approach Mol. Cancer Ther., March 1, 2007; 6(3): 802 - 810. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. A. Samani, S. Yakar, D. LeRoith, and P. Brodt The Role of the IGF System in Cancer Growth and Metastasis: Overview and Recent Insights Endocr. Rev., February 1, 2007; 28(1): 20 - 47. [Abstract] [Full Text] [PDF]
|
|
