AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics: Discovery, Biology, and Clinical Applications: Abstracts
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A125
Identification of EGFr and K-RAS Somatic Gene Mutations from a
Panitumumab Phase 2 NSCLC Clinical Trial: Association of Mutations, Preclinical
Characterization, and Clinical Outcome. Todd Juan*,1 Dan Freeman*,1
Ildiko Sarosi,1 Jeffrey Crawford,2 Alan Sandler,3 Joan Schiller,4 Diane Prager,5 David
Johnson,3 Michael Wolf,1 Robert Radinsky.1 Amgen Inc.,1 Thousand Oaks, CA,
Duke University Medical Center,2 Durham, NC, Vanderbilt-Ingram Cancer Center,3
Nashville, TN, University of Wisconsin,4 Madison, WI, UCLA Medical Center,5 Los
Angeles, CA.
Background:
Mutant epidermal growth factor receptor (EGFr) and mutant K-RAS
have been associated with non-small cell lung cancer (NSCLC) responsiveness, or lack
there of, to small molecule tyrosine kinase inhibitors. Unlike clinical associations that
have been made with gefitinib and erlotinib, little is known about how patients with
EGFr and K-RAS mutations respond to antibodies against EGFr. Panitumumab, a fully
human monoclonal antibody directed against the EGFr extracellular domain, is currently
being studied in a NSCLC trial comparing chemotherapy (carboplatin/paclitaxel)
vs chemotherapy plus panitumumab (n=175; primary endpoint = time to progression).
Study objectives were to associate EGFr and K-RAS mutations with patient clinical outcome
and to determine in vitro if panitumumab has differential activity versus gefitinib
against mutant EGFr.
Methods:
Genomic DNA was isolated from dissected FFPE tumor
sections (pretreatment); PCR was performed on EGFr gene exons 18, 19, 20, 21,
and 23 and K-RAS exon 2. PCR products were subcloned, and >30 colonies per exon
were sequenced. After mutations were found in tumors, genomic DNA was sequenced
to confirm the existence of somatic mutations. These data were linked to clinical outcome
data (investigator assessed per RECIST every 6 weeks). To determine inhibition of
EGF-induced EGFr autophosphorylation in vitro, WT and mutant EGFr-overexpressing
CHO cells were treated with 0-2 µM of either panitumumab or gefitinib prior to EGF
stimulation. Results: Of 59 samples tested, 5 different somatic mutations in EGFr and
K-RAS were identified in 17 patients (table). The respective IC50 for EGF-induced autophosphorylation
of EGFr WT, (750-759), L858R and T790M cells were 14.6, 1.4,
3.2 and >2000nM with gefitinib and 0.23, 0.17, 0.18 and 0.23nM with panitumumab.
Conclusion:
Eight patients harbored EGFr somatic gene mutations, whereas 10 patients
harbored K-RAS mutations. In EGFr-overexpressing CHO cells, panitumumab inhibited
EGF-induced EGFr autophosphorylation regardless of mutational status. A de
novo EGFr exon 20 gate-keeper mutation was found in this study. This exon 20 EGFr
mutation, previously shown to be resistant to gefitinib in vitro and in vivo, was sensitive
to panitumumab in vitro. The patient carrying this exon 20 mutation experienced stable
disease for 12 weeks (2 assessments) in response to treatment with panitumumab
plus chemotherapy.
B43
Panitumumab Induces Internalization of the Epidermal Growth Factor
Receptor (EGFr). Ian N. Foltz,1 Chadwick T. King,1 Meina Liang,2 Kenneth A. Schooley,
3 Douglas P. Cerretti,3 Dan Freeman,4 Robert Radinsky,4 Xiao-Dong Yang.2 Abgenix
Biopharma Inc.,1 Burnaby, BC, Canada, Abgenix Inc.,2 Fremont, CA, Amgen,3 Seattle,
WA, Amgen,4 Thousand Oaks, CA.
PURPOSE:
To determine if panitumumab, a fully human monoclonal antibody to
EGFr, is internalized upon binding to EGFr.
METHODS:
Five human cancer cell lines
were used: SKMES, A431, A549, H1975 and HeLa. Confocal microscopy was used to
directly visualize the internalization of EGFr in cells treated with either panitumumab (0,
0.5, 6.5, 30.5 or 54.5 hrs) or EGF (0.5 hr). The cells were then fixed, permeabilized, and
stained with a non-competitive murine antibody against EGFr conjugated to FITC to
detect total EGFr and an anti-human IgG conjugated with phycoerythrin to detect panitumumab.
As an alternative approach to quantitate internalization of panitumumab,
EGFr-expressing cells were sequentially treated with a saturating concentration of panitumumab
and an anti-human IgG-Fc Fab conjugated to a disulfide-linked fluorochrome
at 4C. The cells were then incubated at either 4C or 37C for various times followed by
treatment with or without a non-cell permeable reducing reagent to remove any extracellular
disulfide-linked fluorochrome from the cell. The geometric mean fluorescence
of each sample was determined using FACS and used to quantitate internalization. RESULTS:
Panitumumab and EGF were found to be internalized on SKMES, A431, and
A549 cells using confocal microscopy. Treatment with EGF led to a rapid internalization
of EGFr with nearly complete removal of cell surface EGFr by 0.5 hr. While the
internalization of EGFr in response to panitumumab treatment was visually less than
that induced by EGF, EGFr and panitumumab were clearly co-localized inside the cell
from 0.5 hr to 54.5 hrs. Of note, the internalization induced by EGF was observed
as punctate perinuclear staining, while internalization induced by panitumumab was
diffuse throughout the inside of the cell. By FACS, it was observed that panitumumab
was internalized at a constant rate over the first hour in A431 cells. The percent of panitumumab
internalized after 1 hour was 20%, 36%, 74% and 28% for A431, A549,
H1975 and HeLa cells, respectively. This internalization was shown to be an energy dependent
process as it was completely inhibited by sodium azide pre-treatment of cells.
To correlate panitumumab-induced internalization with EGFr expression in vivo, H1975
tumor xenografts were treated with panitumumab or an isotype control antibody for
three weeks. Western analysis indicated that panitumumab significantly reduced total
EGFr expression, but not total AKT expression, as compared to the control antibody.
CONCLUSIONS:
These results using two distinct approaches demonstrate that panitumumab
is internalized upon binding to EGFr expressed on tumor cells. However, the
internalization of EGFr induced by panitumumab varied among the different cell lines
tested, displayed slower kinetics than EGF, and may be mechanistically distinct from
that induced by EGF. Interestingly, the strong internalization observed in H1975 cells
correlates with the down-regulation of EGFr in H1975 tumor xenografts, and suggests
that the internalization of EGFr may represent a mechanism of action of panitumumab
in certain tumors.
B44 Effects of an Antibody to VEGFR-3, mF4-31C1,
B64
Activity of Panitumumab Alone and in combination with Chemotherapy
Against Mutant Epidermal Growth Factor Receptor (EGFr)- Expressing Non-
small Cell Lung Carcinoma (NSCLC) Cell Lines and Xenografts. Tammy Bush*,1
Dan Freeman*,1 Selam Ogbagabriel,1 Brian Belmontes,1 Carl Kozlosky,2 Angelo Baher,
1 Carol Johnson,1 Gwyneth Van,1 Doug Cerretti,2 Robert Radinsky.1 Amgen Inc.,1
Thousand Oaks, CA, Amgen Washington,2 Seattle, WA.
Background:
EGFr kinase domain mutations render the receptor hyperresponsive
to ligand and hypersensitive to small molecule tyrosine kinsase inhibitors. However,
little is know about how these mutations respond to antibodies against EGFr. Panitumumab,
a fully human monoclonal antibody, binds to EGFr with high affinity (5x10-
11M), prevents ligand-induced activation, and results in arrest of tumor cell proliferation.
The primary study objectives were to measure inhibition of ligand-dependent
EGFr phosphorylation by panitumumab in vitro in mutant EGFr-expressing NSCLC cells
and to assess the anti-tumor activity of panitumumab alone and in combination with
chemotherapy in xenograft models in vivo. The secondary objective was to address
the ability of panitumumab to inhibit phosphorylation of the mutant EGFr in liganddependent
and -independent cell lines.
Methods:
Mutant EGFr expressing NSCLC
(NCI-H1975 [L858R+T790M], NCI-H1650 [ 746-752]), ligand-independent 293T, and
ligand-dependent CHO cells were treated with panitumumab prior to EGF stimulation
to determine the inhibition of EGFr autophosphorylation. The inhibition of specific
EGFr phosphorylation sites (Y845, Y1045, Y1068, Y1086, and Y1173) was assayed by
western blot or immunoassay. Established tumors (200 mm3) were treated with panitumumab
(25, 100 or 500 µg/mouse 2Xweek) alone or in combination with docetaxel
(10 or 20 mg/kg 1Xweek) or cisplatin (7.5 mg/kg 1Xweek). FACS and immunohistochemistry
were performed to evaluate EGFr levels, panitumumab penetration, and
proliferation levels. Results: Treatment of mutant EGFr-expressing NSCLC cells with
panitumumab resulted in inhibition of ligand-dependent autophosphorylation at all 5
tyrosine phosphorylation sites. In 293T (ligand independent) cells, no significant inhibition
of autophoshorylation was observed. Treatment of established NCI-H1975 and
NCI-H1650 xenografts with panitumumab resulted in statistically significant antitumor
effect compared to IgG2 treatment (50% inhibition, p<0.0001). Combination treatment
of panitumumab plus docetaxel enhanced antitumor efficacy against NCI-H1975
and NCI-H1650 xenografts compared to either agent alone (70% inhibition, p=0.1390
and 0.0534, respectively). In contrast, combination treatment of panitumumab plus
cisplatin did not enhance antitumor efficacy of panitumumab. FACS analysis revealed
that EGFr levels were 6-fold higher in NCI-H1650 vs NCI-H1975 while immunohistochemistry
demonstrated tumor penetration by panitumumab in both tumors. With
combination therapy of panitumumab plus docetaxel, Ki67 and pMAPK staining decreased
> 60% compared to either agent alone in the NCI-H1650 xenografts, but
not in the NCI-H1975 xenografts, on day 12.
Conclusions:
Panitumumab effectively
inhibited ligand-dependent phosphorylation of mutant EGFr. Treatment with panitumumab
as monotherapy or in combination with docetaxel resulted in a significant
growth inhibition of established tumors expressing mutant EGFr. Panitumumab was
detected in tumor tissues, which correlated with a reduction in tumor cell proliferation.
These data support the potential clinical application of panitumumab alone and
in combination with chemotherapy for the treatment of NSCLC patients with EGFr kinase
domain mutations.
B72
Antitumor Efficacy of Panitumumab Alone or in Combination with
Chemotherapy against Human Pancreatic Carcinoma Xenografts. Tammy
Bush*, Dan Freeman*, Selam Ogbagabriel, Beth Ziegler, Brian Belmontes, Gwyneth
Van, Carol Johnson, Robert Radinsky. Amgen Inc., Thousand Oaks, CA.
Background:
The prognosis for pancreatic cancer patients is poor and emphasizes
the need for new, effective pancreatic cancer therapies. Panitumumab, a fully human
monoclonal antibody, binds to the epidermal growth factor receptor (EGFr) with
high affinity (Kd=5x10-11 M), prevents ligand-induced activation, and results in arrest
of tumor cell proliferation. Because many primary pancreatic carcinomas express EGFr,
targeting this receptor has the potential to effectively treat pancreatic cancer. The objective
of this study was to assess the antitumor activity of panitumumab alone or in
combination with chemotherapy against 2 established xenograft models of pancreatic
cancer.
Methods:
Inhibition of ligand-induced autophosphorylation by panitumumab
was determined both in vitro and in vivo. In vitro, BxPC-3 and MiaPaCa-2 cells
were treated with 0.5, 2, and 10 g/ml of panitumumab for 1 hour prior to 100 ng/ml
EGF stimulation. In vivo, mice bearing established tumors were treated with 500 µg
panitumumab or control 96 hours prior to stimulation with 100 µg of EGF. To determine
in vivo efficacy, mice bearing established BxPC-3 and MiaPaCa-2 tumors (200
mm3) were treated with panitumumab (100, 200, or 500 g) alone or in combination
with gemcitabine (80 mg/kg once weekly). Immunohistochemistry was performed to
evaluate EGFr levels, panitumumab tumor penetration, and proliferation levels at day
18 post-treatment. Results: Treatment of BxPC-3 and MiaPaCa-2 cells in vitro and
of xenograft tumors in vivo with panitumumab resulted in greater than 90% inhibition
of ligand-induced autophosphorylation of EGFr. Monotherapy treatment with
either panitumumab or gemcitabine resulted in significant growth inhibition of Mia-
PaCa-2 (36% inhibition, p = 0.0009 and 38% inhibition, p = 0.0012, respectively)
and BxPC-3 (44% inhibition, p < 0.0001 and 25% inhibition, p = 0.0007, respectively)
xenograft tumors when compared to the control group. Combination therapy
with panitumumab plus gemcitabine resulted in enhanced antitumor efficacy against
MiaPaCa-2 (58% inhibition, p = 0.04 vs either monotherapy) and against BxPC-3 (61%
inhibition, p = 0.1575 vs panitumumab monotherapy and p < 0.0001 vs gemcitabine
monotherapy) xenograft tumors compared with either agent alone. EGFr levels and
tumor penetration by panitumumab were detected by immunohistochemistry in both
tumor models. Combination treatment with panitumumab plus gemcitabine resulted
in a greater than 60% reduction in Ki67 and BrdU staining at day 18 post-treatment
compared to either agent alone.
Conclusions:
Panitumumab inhibited ligand-induced
EGFr autophosphorylation in vitro and in vivo in both BxPC-3 and MiaPaCa-2 pancreatic
models. Panitumumab is present in the tumor tissues and correlates with a significant
reduction in Ki67 levels and cell proliferation when combined with gemcitabine, resulting
in growth inhibition of established models of pancreatic cancer. These data provide
preclinical evidence for the potential clinical application of panitumumab alone or in
combination for the treatment of pancreatic cancer.
B73
Antitumor Activity of AMG 706, A Novel Tyrosine Kinase Inhibitor, in
Combination with Panitumumab, A Fully Human Antibody Targeting the EGF
Receptor, Against Multiple Established Human Tumor Xenograft Models in
Nude Mice. C Starnes, D Freeman, T Bush, J Leal, K McDorman, A Coxon, V Patel,
T Polverino, R Kendall, R Radinsky. Amgen Inc., Thousand Oaks, CA.
Background:
AMG 706 is a potent, oral, small molecule, multi-kinase inhibitor
with both antiangiogenic and direct antitumor activity achieved by selectively targeting
VEGF, PDGF, and Kit receptors.
AMG 706 demonstrated potent antitumor activity
against multiple established human tumor xenograft models in nude mice. Panitumumab
is a fully human IgG2 antibody that specifically targets the human EGF
receptor with high affinity and inhibits ligand-induced receptor tyrosine phosphorylation,
resulting in antitumor activity against various xenograft models. The present
studies examined 3 xenograft models (with varying degrees of responsiveness to either
monotherapy) for their response to combination treatment with both targeted
agents.
Methods:
For each model, female CD1 nu/nu mice were challenged subcutaneously
with 1x107 tumor cells on day 0. Treatment (range 10-75 mg/kg AMG
706/dose; 20-500 µg/mouse 2x/wk panitumumab) was initiated after the tumors became
established (average tumor volume range ~200-800 mm3) and continued for
approximately 4-5 weeks. Tumor size was measured with a caliper and reported as a
function of time. Bodyweights were recorded as an index of toxicity. Individual groups
are compared by p values calculated from Repeated Measures Analysis of Variance
(RMANOVA) followed by Scheffe post hoc testing for multiple comparisons. Results:
In each of the 3 models, the combination of panitumumab + AMG 706 resulted in a
therapeutic effect that was statistically superior to that seen with either single agent
alone at the given dose levels employed (A431 epidermoid carcinoma, combination vs.
AMG 706 alone, p < 0.0001; vs. panitumumab alone, p = 0.0003; HT29 colorectal
carcinoma, p = 0.0013 and p < 0.0001, respectively; Calu-6 non-small cell lung cancer,
p = 0.0204 and p < 0.0001, respectively). No adverse drug-drug interactions were
observed.
Conclusions:
A beneficial therapeutic effect of the combination of AMG
706 and panitumumab was observed in 3 different xenograft models with no apparent
adverse effects. These results support the potential of combining targeted therapies
that interfere with different pathways to provide additive clinical benefit. Specifically,
the combined use of AMG 706 and panitumumab in clinical trials is warranted. |
