Safety and Activity of the First-in-Class Sym004

Transcription

Published OnlineFirst May 11, 2015; DOI: 10.1158/2159-8290.CD-14-1432
Research Article
Safety and Activity of the First-in-Class
Sym004 Anti-EGFR Antibody Mixture in
Patients with Refractory Colorectal Cancer
Rodrigo Dienstmann1,2, Amita Patnaik3, Rocio Garcia-Carbonero4, Andrés Cervantes5, Marta Benavent4,
Susana Roselló5, Bastiaan B.J. Tops6, Rachel S. van der Post6, Guillem Argilés1, Niels J.Ø. Skartved7,
Ulla H. Hansen7, Rikke Hald7, Mikkel W. Pedersen7, Michael Kragh7, Ivan D. Horak7, Stephan Braun7,
Eric Van Cutsem8, Anthony W. Tolcher3, and Josep Tabernero1
Tumor growth in the context of EGFR inhibitor resistance may remain EGFRdependent and is mediated by mechanisms including compensatory ligand upregulation and de novo gene alterations. Sym004 is a two-antibody mixture targeting nonoverlapping EGFR
epitopes. In preclinical models, Sym004 causes significant EGFR internalization and degradation, which
translates into superior growth inhibition in the presence of ligands. In this phase I trial, we observed
grade 3 skin toxicity and hypomagnesemia as mechanism-based dose-limiting events during dose
escalation. In dose-expansion cohorts of 9 and 12 mg/kg of Sym004 weekly, patients with metastatic
colorectal cancer and acquired EGFR inhibitor resistance were enrolled; 17 of 39 patients (44%) had
tumor shrinkage, with 5 patients (13%) achieving partial response. Pharmacodynamic studies confirmed marked Sym004-induced EGFR downmodulation. MET gene amplification emerged in 1 patient
during Sym004 treatment, and a partial response was seen in a patient with EGFRS492R mutation that is
predictive of cetuximab resistance.
Abstract
SIGNIFICANCE: Potent EGFR downmodulation with Sym004 in patients with metastatic colorectal
cancer and acquired resistance to cetuximab/panitumumab translates into significant antitumor activity and validates the preclinical hypothesis that a proportion of tumors remains dependent on EGFR
signaling. Further clinical development and expanded correlative analyses of response patterns with
secondary RAS/EGFR mutations are warranted. Cancer Discov; 5(6); 1–12. ©2015 AACR.
1
Vall d’Hebron University Hospital and Institute of Oncology (VHIO), Universitat Autònoma de Barcelona, Barcelona, Center affiliated to the RTICC
(ISCiii), Spain. 2Sage Bionetworks, Fred Hutchinson Cancer Research
Center, Seattle, Washington. 3START South Texas Accelerated Research
Therapeutics, San Antonio, Texas. 4Hospital Universitario Virgen del Rocío/
Instituto de Biomedicina de Sevilla (HUVR, CSIC, Universidad de Sevilla),
Center affiliated to the RTICC (ISCiii), Sevilla, Spain. 5Biomedical Research
Institute INCLIVA, University of Valencia, Valencia, Spain. 6Department
of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands. 7Symphogen A/S, Ballerup, Denmark. 8Digestive Oncology Department, University Hospitals Leuven and KULeuven, Leuven, Belgium.
OF1 | CANCER DISCOVERY June 2015
Note: Supplementary data for this article are available at Cancer Discovery
Online (http://cancerdiscovery.aacrjournals.org/).
Corresponding Author: Josep Tabernero, Vall d’Hebron University Hospital, Vall d’Hebron Institute of Oncology (VHIO), Universitat Autònoma
de Barcelona, P. Vall d’Hebron 119-129, 08035 Barcelona, Spain. Phone:
34-93-489-4301; Fax: 34-93-274-6059; E-mail: [email protected]
doi: 10.1158/2159-8290.CD-14-1432
©2015 American Association for Cancer Research.
www.aacrjournals.org
Downloaded from cancerdiscovery.aacrjournals.org on October 24, 2016. © 2015 American Association for Cancer
Research.
INTRODUCTION
Metastatic colorectal cancer is expected to cause 50,000
deaths in the United States alone in 2015 (1). Multiple clinical trials have confirmed the benefit from anti-EGFR therapy
in patients with wild-type–KRAS metastatic colorectal cancer
(2–6), recently refined to the wild-type RAS (i.e., wild-type
KRAS and NRAS) population (4). Preclinical findings point to
conservation of EGFR dependency of tumors that progressed
upon cetuximab or panitumumab therapy (7), even in the
case of acquired mutations in RAS genes (8, 9), with EGFR
ligands playing a central role in autocrine and paracrine
resistance networks (10–12). Other mechanisms of acquired
cetuximab resistance include mutations in the extracellular
domain of EGFR and EGFR interactions with the receptor
kinases HER3 (ERBB3) or MET (10, 13–16). These findings
raise the question of whether more effective EGFR targeting,
e.g., more potent receptor downmodulation, could overcome
resistance to available anti-EGFR therapy (17).
Sym004 is a 1:1 mixture of two recombinant, human–
mouse chimeric mAbs directed against nonoverlapping
EGFR epitopes (mAb992 and mAb1024). A unique feature
of Sym004 is its ability to mediate rapid EGFR internalization and subsequent degradation of internalized receptors
via EGFR cross-linking (17, 18). Preclinical studies with
Sym004 showed both superior induction of tumor regression as compared with other EGFR-targeting antibodies
and antitumor activity in models of acquired cetuximab
resistance (7, 17). Toxicology studies in cynomolgus monkeys showed a toxicity profile consistent with other drugs of
the same class, including dermatologic and gastrointestinal
events (19).
In this article, we first present novel preclinical experiments
demonstrating enhanced activity of Sym004 over cetuximab
in the presence of various EGFR ligands, providing a rationale for evaluating the clinical activity of Sym004 in an antiEGFR–refractory metastatic colorectal cancer setting. Next,
we present the safety, tolerability, and pharmacokinetic profile
of escalating doses of Sym004 from the first-in-human phase
I trial in patients with advanced epithelial tumors. Finally, we
describe the pharmacodynamic effects and clinical activity of
Sym004 from two dose-expansion cohorts in patients with
wild-type–KRAS metastatic colorectal cancer and acquired
resistance to approved anti-EGFR antibody treatment, supporting Sym004’s proof of mechanism and proof of concept.
RESULTS
Preclinical Studies
A high-throughput cell line screen was performed to determine the activity of Sym004 in the presence of the EGFR ligands
EGF, TGFα, and epiregulin (EREG) in a panel of 22 colorectal
cancer cell lines (Fig. 1A and B). Approximately 60% of the tested
cell lines (13/22) were sensitive to antibody targeting (defined by
an activity area above 2). No clear differentiation of Sym004 and
cetuximab was seen in the absence of ligands. In addition, no correlation between KRAS or BRAF mutational status and response
to antibody targeting was observed, although the two cell lines
harboring the BRAF V600E mutation (HT29 and COLO205) were
completely resistant. We did observe that the cells stimulated by
EGFR ligands were those that also responded to EGFR inhibition, supporting the notion that EGFR dependency in colorectal
cancer is largely driven by the various EGFR ligands.
June 2015 CANCER DISCOVERY | OF2
Research.
Dienstmann et al.
RESEARCH ARTICLE
DEL
MUT
–10
200
100
Cetuximab
Sym004
0
0.0001 0.001 0.01 0.1
1
10
Antibody concentration (µg/mL)
GEO+TGFα
50
150
100
50
Cetuximab
Sym004
0
0.0001 0.001 0.01 0.1
1
10
100
100
200
150
50
and PIK3CAE542k)
EGF
TGFα
EREG
Cetuximab
Sym004
150
100
50
0
0.0001 0.001 0.01 0.1
1
10
Concentration (nmol/L)
200
150
100 1,000
T84 (KRASG12D)
EGF
TGFα
EREG
Sym004
Cetuximab
100
50
0
1
10
0.0001 0.001 0.01 0.1
100 1,000
100 1,000
E
150
100
+
Cetuximab
Sym004
0
0.0001 0.001 0.01 0.1
1
10
500
250
100
GEO+EREG
50
SW948 (KRAS
1,000
100 1,000
G12A
)
GEO (KRAS
EGF
TGFα
EREG
Cetuximab
Sym004
0
0.0001 0.001 0.01 0.1
1
10
10
200
Number of viable cells (%)
GEO+EGF
0
Activity area
Inhibition
Stimulation
150
50
100
250
200
200
150
0
1
10
0.0001 0.001 0.01 0.1
N/A
EREG
EGF
TGFα
Sym004
Cetuximab
C
400
Genetic annotation
600
Q61L
1,500
EGF
TGFα
EREG
Cetuximab
Sym004
SMAD4
APC
PTEN
PIK3CA
RAF1
BRAF
HRAS
NRAS
KRAS
TP53
ERBB4
ERBB3
ERBB2
EGFR
800
Cetuximab 24 h
Sym004 24 h
Cetuximab 24 h
Sym004 24 h
Cetuximab 24 h
Sym004 24 h
Cetuximab 24 h
Sym004 24 h
Anti-EGFR antibody resistant
H508 (BRAFG596R)
1,000
Anti-EGFR antibody sensitive
LOVO
HCT116
DLD1
H716
SW620
LS1034
HT29
COLO205
HCT15
B
GEO
H508
SW403
LS174T
T84
SW1463
SW837
SW948
SNUC2A
CACO2
COLO678
GP5D
SW480
A
+
+ +
+
100
+ +
1 nmol/L EGF 15 minutes
1 nmol/L EREG 15 minutes
+ + 1 nmol/L Heregulin 15 minutes
EGFR
pEGFR (Tyr1068)
SW948
500
Untreated
***P < 0.0001
***P < 0.00001
300
200
100
0
5 µg/mL Cetuximab
pHER2 (Tyr1221)
HER3
100
pHER3 (Tyr1289)
pERK1/2
50
0
HER2
**P < 0.005
150
ERK1/2
Untreated
1 nmol/L EGF
1 nmol/L EREG
1 nmol/L TGFα
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
400
***P < 0.00005
100
0
GEO
200
Untreated
1 nmol/L EGF
1 nmol/L EREG
1 nmol/L TGFα
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
***P < 0.00001
300
200
H508
***P < 0.00001
***P < 0.00001
400
Untreated
1 nmol/L EGF
1 nmol/L EREG
1 nmol/L TGFα
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
Untreated
1 nmol/L EGF
1 nmol/L TGFα
1 nmol/L EREG
1 nmol/L Heregulin
500
D
pAKT (Ser473)
AKT
pS6R
Actin
5 µg/mL Sym004
Figure 1. Functional comparison of Sym004 and cetuximab in the presence and absence of HER family ligands. A, heat map showing activity area
calculated from dose–response curves of EGF, TGFα, EREG, Sym004, and cetuximab. A positive activity area (red) indicates stimulation of cell growth,
whereas a negative activity area (blue) indicates inhibition of cell growth. B, examples of dose–response curves for ligands, cetuximab, and Sym004 in
selected colorectal cancer cell lines. C, dose–response curves for Sym004 and cetuximab in the presence of 1 nmol/L EGF, 1 nmol/L TGFα, or 1 nmol/L
EREG in the GEO cell line. D, inhibitory activity of 5 μg/mL of cetuximab and Sym004 in the presence of 1 nmol/L of the high-affinity ligands EGF or
TGFα, or the low-affinity ligand EREG in the cell lines SW948, H508, and GEO. E, immunoblot analyses of the effect of 20 μg/mL of cetuximab and
Sym004 on the HER family receptors and selected downstream signaling nodes in the presence and absence of EGF, EREG, and heregulin. DEL, deletion;
MUT, mutation; N/A, not available.
Knowing that high-affinity EGFR ligands confer resistance to cetuximab in preclinical models (11, 12), we assessed
whether Sym004, with its distinguished mechanism of action,
had superior activity as compared with cetuximab in the presence of various EGFR ligands. Proliferation experiments of
colorectal cancer cell lines in the presence of the high-affinity
ligands EGF and TGFα as well as the low-affinity ligand EREG
(Fig. 1C and D) clearly demonstrated that Sym004 outperformed cetuximab in the presence of the high-affinity ligands,
whereas the difference was less pronounced in the presence of
the low-affinity ligand. Mechanistically, Sym004 led to more
pronounced EGFR degradation than cetuximab following
24 hours of treatment, with increased inhibition of ERK1/2 and
S6R phosphorylation following ligand stimulation (Fig. 1E).
Our preclinical results thus provide evidence that Sym004
could overcome acquired resistance to cetuximab mediated
by EGFR ligands and a rationale for clinical evaluation of
Sym004 in this setting.
Clinical Trial Patients
Between March 2010 and April 2012, a total of 62 patients
were enrolled in the trial. Of these, 20 patients with solid
Research.
Sym004 in Advanced Solid Tumors
RESEARCH ARTICLE
Table 1. Demographic baseline characteristics of patients
Solid epithelial tumorsa
Dose escalation
Metastatic colorectal cancer
12 mg/kg
9 mg/kg
20 (100)
29 (100)
13 (100)
61
35–81
63
35–84
68
52–79
Sex (N, %)
Female
Male
13 (65)
7 (35)
14 (48)
15 (52)
6 (46)
7 (54)
Previous treatment for mCRC (N, %)
Bevacizumab
Irinotecan
Oxaliplatin
NA
NA
NA
18 (62)
25 (86)
24 (83)
5 (39)
12 (92)
10 (77)
Previous anti-EGFR treatment
Any
Any cetuximab treatment
Any panitumumab treatment
More than one line of mAb treatment
NA
NA
NA
NA
29 (100)
22 (76)
13 (45)
13 (45)
13 (100)
11 (85)
3 (23)
1 (8)
Previous treatment lines (N, %)b
1–3
>3
NA
NA
15 (52)
14 (48)
9 (69)
4 (31)
Metastatic sitesc
Liver
Lung
Lymph nodes
Other
NA
NA
NA
NA
23 (79)
12 (41)
6 (21)
4 (14)
8 (62)
7 (54)
5 (39)
2 (15)
Full analysis set
Age (years)
Median
Min–max
Abbreviations: NA, not assessed; mCRC, metastatic colorectal cancer.
Of 20 patients with solid epithelial tumors, 14 had colorectal cancer, 3 pancreatic cancer, 2 non–small cell lung cancer,
and 1 spinal chordoma.
a
Including antitumor compounds aflibercept, sorafenib, ramucirumab, carlumab, deforolimus, ganitumab, trebananib,
and cediranib.
b
Multiple nominations of metastatic sites identified as target lesions per patient possible.
c
epithelial tumors received escalating doses of Sym004, ranging from 0.4 to 12 mg/kg administered weekly. The remaining 42 patients had metastatic colorectal cancer and received
either 9 or 12 mg/kg weekly in the dose-expansion phase and
were evaluated for pharmacodynamic effects and response
(Table 1). All 62 patients were assessed for toxicity.
Prior treatment with anti-EGFR antibodies was required
for eligibility only in the dose-expansion cohorts. Fourteen
patients had more than 1 line of anti-EGFR mAb treatment
(Table 1), and the majority (90%) had at least 1 line of antiEGFR mAb treatment in combination with chemotherapy.
All patients had a documented response to previous antiEGFR mAb treatment followed by disease progression,
with a median time until administration of the first dose
of Sym004 of 54 and 33 days for patients treated at 9 and
12 mg/kg, respectively. Of note, the median time to disease progression after termination of the latest anti-EGFR
treatment was 8 days, and the median time from the last
anti-EGFR mAb treatment until the first dose of Sym004
was 81 days.
Safety and Exposure
A formal MTD was not reached, and the maximum administered dose, declared in a joint decision of the investigators,
sponsor, and the independent data-monitoring committee,
was set at 12 mg/kg. The decision to stop dose escalation was
based on the observation of accumulating skin and serum
electrolyte (hypomagnesemia) toxicities observed beyond the
4-week observation period for dose-limiting toxicities (DLT).
Adverse events of any grade were reported in 61 patients
(98%; Table 2). Serious adverse events that investigators considered to be related to treatment occurred in 11 of 36
patients (31%; Supplementary Table S1). The most common drug-related adverse events of any grade were skin rash
(69%), dry skin (45%), hypomagnesemia (53%), pruritus (39%),
mucosal inflammation (31%), and diarrhea (27%; Supplementary Table S1).
Skin toxicity and hypomagnesemia events of grade 3
or higher were reported for 50% and 21% of the patients,
respectively, whereas only 3% of the patients experienced
diarrhea of grade 3 or higher (Table 2). Skin-related adverse
Research.
Dienstmann et al.
RESEARCH ARTICLE
Table 2. Summary of treatment-emergent adverse events and relative dose intensity
Dose expansion
Dose escalation (N = 20)
12 mg/kg (N = 29)
9 mg/kg (N = 13)
Number of patients (%)
Any adverse event
Any grade ≥3 adverse event
Any serious adverse event
Drug-related fatal adverse
events
Discontinuation due to
adverse event
19 (95)
11 (55)
10 (50)
0 (0)
29 (100)
28 (97)
16 (55)
1 (3)
13 (100)
13 (100)
10 (77)
0 (0)
4 (20)
0 (0)
0 (0)
Skin toxicity adverse eventsa
15 (75)
29 (100)
12 (92)
Grade ≥3
4 (20)
18 (62)
9 (69)
Diarrhea
6 (30)
10 (34)
7 (54)
Grade ≥3
0 (0)
1 (3)
1 (8)
Infusion-related reactions
4 (20)
1 (3)
0 (0)
Grade ≥3
1 (5)
0 (0)
0 (0)
Hypomagnesemia
6 (30)
21 (72)
6 (46)
Grade ≥3
2 (10)
11 (38)
0 (0)
Sym004 exposure
Dose reduced (N, %)
Treatment interrupted (N, %)
NA
NA
14 (48)
26 (90)
4 (31)
10 (77)
NA
72 (52–82)
Median% (q25–q75)
Relative dose intensity of
Sym004
67 (55–79)
No grade 4 events observed; reported preferred terms were acne, cellulitis, dermatitis acneiform, dry skin, erysipelas,
erythema, folliculitis, hypertrichosis, paronychia, pruritus, rash, rash maculopapular, rash vesicular, skin exfoliation,
skin hyperpigmentation, and xerosis.
a
events and hypomagnesemia were mainly controlled by supportive care, dose delays, and reductions as per protocol.
Drug-related adverse events (rash, acneiform dermatitis, and
infusion-related reaction) led to discontinuation in 3 patients
in the dose-escalation part. In the dose-expansion cohorts, the
predominant reason to discontinue was disease progression
(88%), the median number of Sym004 infusions was 10 (range,
2–47), and the median relative dose intensity was 71% (Table
2). Treatment was delayed at least once in 86% of the patients,
and the assigned dose was reduced at least once in 43% of the
patients, predominantly due to grade 3 skin adverse events
and in accordance with the protocol. Both interventions were
observed more often in patients treated at 12 mg/kg (Table 2).
Infusion-related reactions were observed in 5 of 62 patients
(8%). We observed only a single grade 3 infusion-related reaction in a patient from the 9 mg/kg dose-escalation cohort.
This event led to the expansion of an already-implemented
premedication scheme with antihistamines by adding anti
pyretics (for the first 4 infusions) and glucocorticoids (for the
first 2 infusions) for the remaining patients.
Pharmacokinetics and Pharmacodynamics
Serum mAb levels increased in a dose-dependent manner from 0.4 to 12 mg/kg in patients of the dose-escalation
cohort. Doses of 3 mg/kg or less were rapidly cleared and
had a terminal serum half-life of 2 days or less (Supplementary Table S2 and Supplementary Fig. S1A). The relationship
between doses and geometric mean area under the curve
appeared to be linear for doses of 6 mg/kg and higher (Supplementary Table S2 and Supplementary Fig. S1B). The serum
half-life of the doses chosen for cohort expansion, estimated
by compilation of serum elimination curves, was 3 days after
the first infusion and 4 to 5 days after the fourth infusion
(Supplementary Table S2). In patients of the dose-expansion
cohort who received uninterrupted weekly treatment, trough
levels revealed a continuous drug exposure (Supplementary
Fig. S1C). A single patient treated at 9 mg/kg showed a transient anti-Sym004 antibody response of low magnitude.
The pharmacodynamic response in tumor biopsies of patients
with metastatic colorectal cancer, obtained before the first and
the planned fifth infusion, showed a statistically significant
durable downmodulation of EGFR and decrease in Ki67 expression (Fig. 2A), providing clinical proof of mechanism of Sym004.
Internal validation for this observation was obtained from skin
biopsies obtained on the identical schedule (Fig. 2B). To confirm the molecular status of the metastatic colorectal cancer of
patients enrolled to the study, we conducted next-generation
sequencing (NGS) of baseline biopsies from metastatic lesions.
Research.
decreased Ki67 expression in skin and tumor biopsies of patients with metastatic colorectal cancer
treated with weekly Sym004. A, membrane EGFR
and nuclear Ki67 histoscores for tumor biopsies
that were obtained before the first and the
planned fifth infusion (in week 4) with Sym004,
respectively. In 22 paired samples, EGFR downregulation was seen in 16 patients (P = 0.0004);
in 21 paired samples, decreased nuclear Ki67
histoscores were seen in 15 patients (P = 0.005).
B, membrane EGFR and nuclear Ki67 histoscores
for skin biopsies that were obtained before the
first and the planned fifth infusion (in week 4)
with Sym004, respectively. In 33 paired samples,
EGFR downregulation was seen in 32 patients
(P < 0.0001; Wilcoxon signed rank test, twotailed); the corresponding decrease in Ki67
expression was not significantly different. The
span of interquartile range (q1–q3) is represented by the vertical line. The horizontal bar
represents the median value.
A 250
200
Histoscore
Figure 2. EGFR downmodulation and
RESEARCH ARTICLE
150
100
50
0
5th infusion
Baseline
5th infusion
Baseline
Ki67
EGFR
B 250
Histoscore
200
150
100
50
0
5th infusion
Baseline
EGFR
In 20 samples with enough neoplastic tissue available for analysis, we found 3 mutations of KRAS, 2 of PIK3CA, and 1 of EGFR
(Table 3). MET gene FISH analysis revealed 6 cases of polysomy
among 20 baseline samples, but no MET amplification (Table 3).
Antitumor Activity
Of 42 patients with metastatic colorectal cancer resistant
to EGFR treatment enrolled in the dose-expansion cohorts,
39 had imaging data available for central radiological evaluation. Five patients (13%) achieved a partial response of target
lesions (Fig. 3). Overall, 17 patients (44%) had some degree
of tumor shrinkage during Sym004 therapy, of which 4
(33%) of 12 and 13 (48%) of 27 patients had received 9 and
12 mg/kg, respectively (Fig. 3). The overall disease control
rate (partial response and stable disease) was 67% (58% and
5th infusion
Baseline
Ki67
70% for patients on 9 and 12 mg/kg, respectively). In addition, Fig. 3 shows treatment duration and time points of
first onset of grade >2 skin rash, confirming that this toxicity
occurred early, did not lead to significant treatment discontinuation, and was not indicative of predicting response.
Overall, median progression-free survival (PFS) was 3.3 (95%
confidence interval, 2.6–4.9) months, and individual PFS
intervals are shown in Fig. 3. At the time of data cutoff, 41
of 42 patients (98%) had died due to progression of cancer.
In an exploratory analysis of potential associations between
molecular profile and antitumor response, we found that all
patients experiencing partial response to Sym004 therapy
with individual PFS intervals of 5.0 to 6.7 months had
tumors with wild-type status of KRAS, NRAS, and BRAF and
lacked MET amplification (Table 3). One patient experiencing
Research.
Dienstmann et al.
RESEARCH ARTICLE
Table 3. KRAS, NRAS, BRAF, PIK3CA, and EGFR point mutations and MET amplification status in tumor biopsies before
and during treatment with Sym004 in patients with metastatic colorectal cancer
Patient
Dose
(mg/kg)
KRAS, NRAS, BRAF, PIK3CA, EGFR point
mutationsa
Baseline
Prior to 5th infusion
MET gene copy-number alterationsa
Baseline
Prior to 5th infusion
Response
PFS
(mo)
1
12
Wild-type
Wild-type
Diploid
—
PR
6.7
2
9
Wild-type
Wild-type
Polysomy (G:CN 0.9)
Diploid
PR
5.3
3
12
Wild-type
—
Polysomy (G:CN 0.7)
—
PRc
5.0
5
12
EGFR c.1474A>C
—
Diploid
—
PR
5.0
7
12
—
—
—
Loss of METb
SD
3.2
10
12
—
NRAS c.35G>T
—
Polysomy (G:CN 0.8)
SD
3.3
11
12
PIK3CA c.1624G>A
PIK3CA c.1624G>A
—
Diploid
SD
3.2
14
12
Wild-type
—
Polysomy (G:CN 0.8)
—
SD
3.1
16
9
—
—
Diploid
—
PDd
0.9
17
12
KRAS c.183A>C
—
Polysomy (G:CN 0.9)
—
SD
7.1
18
12
PIK3CA c.1633G>A
—
—
—
SD
2.6
19
12
Wild-type
KRAS c.35G>C;
c.183A>C
Polysomy (G:CN 0.8)
Diploid
PDd
1.2
22
12
KRAS c.35G>A
—
Diploid
—
SD
4.8
24
12
Wild-type
Wild-type
Diploid
Diploid
SD
3.2
25
12
KRAS c.35G>A
—
Loss of METb
Amplified (G:CN 15.0)
SD
2.6
26
12
Wild-type
Wild-type
Diploid
Diploid
SD
1.3
28
9
Wild-type
Wild-type
Diploid
Loss of METb
SD
3.0
1.3
29
12
Wild-type
—
Polysomy (G:CN 0.9)
—
PD
30
9
Wild-type
—
Diploid
—
SD
2.6
32
12
—
Wild-type
Diploid
—
PD
1.4
35
9
—
BRAF c.1799T>A
—
—
PD
1.5
36
12
—
Wild-type
—
Diploid
PD
0.7
37
12
Wild-type
NRAS c.183A>T
Diploid
Polysomy (G:CN 0.9)
PD
1.2
38
12
Wild-type
Wild-type
Diploid
Polysomy (G:CN 1.7)
PD
1.4
41
9
Wild-type
—
Diploid
—
PDc,d
1.0
42
12
Wild-type
—
Diploid
—
PDc,d
0.8
Abbreviations: G:CN, MET gene to chromosome 7 copy-number ratio; wild-type, no point mutations detected in analyzed KRAS, NRAS, BRAF, PIK3CA,
and EGFR exons; —, biopsy not evaluable; PD, progressive disease; PR, partial response; SD, stable disease.
Analyses based on a minimum of 20% neoplastic cells in a corresponding hematoxylin and eosin–stained slide.
Diploid with a focal heterozygous gene loss.
c
Unconfirmed PR or PD, as per RECIST criteria.
d
Clinical assessment of PD.
a
b
a partial response to Sym004 had a baseline EGFRS492R mutation, predictive of resistance to cetuximab (14, 15). The 3
patients with KRAS mutations at baseline had stable disease
with PFS figures of 2.6, 4.8, and 7.1 months (Table 3).
DISCUSSION
In this trial, the anti-EGFR mAb mixture Sym004 induced
clinically meaningful rates of partial response (13%) or minor
tumor regression (44%) of target lesions in patients with metastatic colorectal cancer with acquired resistance to anti-EGFR
mAb treatment. To the best of our knowledge, this is the first trial
that demonstrates antitumor activity of a mixture of two mAbs
targeting nonoverlapping eptitopes of the same receptor, with
encouraging results that support targeted therapy alone in a
very refractory patient population. Acknowledging the limited
number of patients treated with Sym004, it remains noteworthy that the response rate seen in this trial was similar
to that assessed in previous phase III studies with cetuximab
and panitumumab monotherapy in EGFR treatment–naïve
patients with metastatic colorectal cancer (5, 6). In addition,
clinical trials with cetuximab and panitumumab monotherapy
have shown no clinically relevant activity in patients resistant
to previous anti-EGFR mAb treatment (20, 21).
Research.
75
RESEARCH ARTICLE
75
PFS (months):
50
25
0
Stable disease
25
0
–25
–50
Partial response
–25
Percentage change from baseline
Percentage change from baseline
50
Progression
1.3 1.0 0.8 1.2 1.4 1.2 0.7 1.5 1.2 1.3 1.4 1.6 2.6 1.3 3.0 3.2 1.3 2.6 3.2 1.6 4.8 2.1 3.5 1.2 7.1 2.6 0.9 6.4 3.1 4.3 1.8 3.2 3.3 5.1 5.1 3.2 3.3 5.0 4.2 5.0 5.3 6.7
–50
SLD of TL (cm):
NE NE NE 11 17 15 18 23 16 4 13 15 7 15 8 13 23 18 13 9 14 20 8 19 15 17 14 5 21 4 14 18 5
5
7 16 2
5 15 7 12 2
–75
–75
15
15
12 mg/kg Sym004
9 mg/kg Sym004
Partial response
Progressive disease
10
Not evaluable
10
Onset of skin rash:
Grade 2
Grade 3
5
5
0
0
Treatment duration (months)
Treatment duration (months)
Stable disease
Figure 3. Treatment duration, occurrence of skin toxicity, and antitumor activity of weekly Sym004. The waterfall plot (top) shows the change in sum
of the longest diameter of tumor target lesions (SLD of TL) after patients received Sym004 at weekly doses of either 9 or 12 mg/kg. The bars indicate
the largest percentage change in target lesions from baseline; the baseline SLD of TL (cm) and individual PFS intervals are shown at the bottom and top
of the waterfall plot, respectively. The lower green horizontal line indicates a 30% reduction from baseline; a >30% reduction from baseline is defined
as partial response as per RECIST 1.1. Progressive disease, defined as a >20% increase from baseline, is indicated by the upper black horizontal line. The
plot shows response data from 39 patients evaluable for response, including 2 patients with tumor shrinkage that was nonevaluable as per RECIST and
excluding 3 patients without post-baseline imaging. Corresponding to the sequence of patients illustrated in the waterfall plot, the treatment duration
plot (bottom) also illustrates occurrence of the first maximum grade 2 or grade 3 skin rash, and emergence of best overall response. The plot shows
exposure data from all 42 patients treated with Sym004, including 2 patients with tumor shrinkage that was nonevaluable as per RECIST and 3 patients
without post-baseline imaging indicated by black diamonds. NE, not evaluable for response.
Research.
Dienstmann et al.
RESEARCH ARTICLE
It is noteworthy that some metastatic colorectal cancer
tumors appear to remain “EGFR-addicted” despite progression
on anti-EGFR treatment, which is supported by our preclinical
and emerging clinical data (7). The clinical antitumor activity
seen with Sym004 is in line with the superior antitumor activity of Sym004 over cetuximab in preclinical animal models of
acquired cetuximab resistance (7, 17). In addition, we showed
here that Sym004 is highly efficient at blocking colorectal cancer
cell line growth in the presence of the high-affinity ligands EGF
and TGFα, factors known to be both upregulated in response
to anti-EGFR antibody treatment and determinants of EGFR
inhibitor resistance (13). We further hypothesize that the pharmacodynamic effects of Sym004, with sustained decrease in
EGFR expression in tumor biopsies, relate to the ability of
the mAbs to cross-link the EGFR, causing internalization and
subsequent degradation of the antibody–receptor complexes
(17). Thus, effective and broad blockade of ligand–receptor
interaction together with receptor downmodulation may be
responsible for the promising antitumor activity observed in
this trial, a hypothesis that might also be translated to the
setting of upfront anti-EGFR mAb treatment.
KRAS mutations are well-established predictors of clinical
resistance to anti-EGFR treatment in metastatic colorectal
cancer (4, 22), although this association is not strong in preclinical models (23–25). Our initial clinical development program focused on metastatic colorectal cancer with wild-type
KRAS (determined on archived tumor samples), anticipating
that Sym004 could overcome an acquired nongenetic mechanism of resistance to anti-EGFR treatment, even though we
did see some activity of Sym004 in colorectal cancer cell lines
with mutated KRAS in the presence of ligands. In an exploratory analysis, we investigated molecular events potentially associated with response and resistance to Sym004. The results
must be viewed with caution, considering that baseline tumor
biopsies and paired sets of biopsies with sufficient tissue for
NGS were available from only 43% and 17% of the 42 enrolled
patients, respectively. Although partial responses were enriched
in patients without genetic mechanisms of EGFR inhibitor
resistance (KRAS, NRAS, BRAF, MET), we did see antitumor
activity in a patient with acquired EGFRS492R mutation. The
activity of Sym004 mAbs in preclinical models of acquired EGFR
extracellular domain mutations will be further explored. Finally,
with regard to the potential clinical benefit of Sym004 in the
setting of acquired RAS mutations, it is important to emphasize
that the clinical significance of these genomic events in the antiEGFR refractory setting remains poorly understood. Nongenetic
resistance mechanisms, such as upregulation of EGFR ligands in
the tumor microenvironment and cross-talk between EGFR and
other receptors, most likely coexist with RAS mutations (11).
The effect of Sym004 on different disease subclones selected
under treatment pressure needs additional investigation.
This trial has not expanded the spectrum of known adverse
events of anti-EGFR drugs. Furthermore, consistent with the
experience from approved anti-EGFR antibodies, Sym004related grade 3 skin toxicity and hypomagnesemia could be
managed without treatment discontinuation in both doseexpansion cohorts. The observed grade ≥3 event rate may
be related to pronounced EGFR downmodulation and/or
the re-exposure to anti-EGFR mAb treatment. In addition,
a propensity to develop high-grade skin toxicities might be
explained by the inclusion of patients who had previously
had a clinical benefit from anti-EGFR mAbs, which is typically linked to an experience of high-grade skin toxicity.
In this trial, we validated the EGFR pathway as an important
target for therapeutic intervention in wild-type–KRAS refractory metastatic colorectal cancer even beyond cetuximab/panitumumab. The disease control achieved with Sym004 in 26 of
39 patients in the acquired resistance setting forms a robust
clinical basis to be tested in future trials. Such trials are currently under way to further investigate an optimal dose and
dosing schedule in patients with metastatic colorectal cancer
(NCT02083653 and NCT01117428).
METHODS
Preclinical Studies
Anti-EGFR mAb-sensitive (GEO, H508, SW403, LS174T, T84,
SW1463, SW837, SW948, SNUC2A, CACO2, COLO678, GP5D,
SW480) and mAb-resistant (LOVO, HCT116, DLD1, H716, SW620,
LS1034, HT29, COLO205, HCT15) colorectal cancer cell lines were
used for the experiments. The cell lines, with the exception of GEO,
were obtained from the ATCC. The colon cancer cell line GEO has been
described previously (26) and was a generous gift from Professor Douglas Boyd (MD Anderson Cancer Center, Houston, TX). All cell lines were
Mycoplasma free, cultured according to the suppliers’ recommendations,
and used within 6 months of resuscitation. The cell lines were obtained
in the period from 2010 to 2014. Cell lines from the ATCC are routinely
tested for authenticity using short tandem repeat profiling.
A standard 4-day WST-1 viability assay (Roche Diagnostics) was
used to measure growth and growth inhibition following treatment
with mAbs and mAb mixtures and was performed as previously
described (17). The number of viable cells was calculated as percentage of untreated control. All antibodies were stored individually at
−80°C or at 4°C for shorter periods. Antibody mixtures were generated before performing experiments, mixed in ratios of 1:1 (w/w), and
immediately added to experimental wells.
Cells were seeded in media supplemented with 2% FBS and allowed
to adhere overnight. Cells were treated with antibodies for 24 hours
and then with ligands for 15 minutes. Following ligand stimulation,
cells were lysed in RIPA buffer, and immunoblot analyses were performed using 10 μg of total protein. The ligands EGF, TGFα, EREG,
and heregulin were all obtained from R&D Systems. Immunoblotting
was conducted according to the antibody manufacturers’ recommendations. Anti-HER2 (1:1,000), anti-pHER2 (Tyr1221; 1:1,000), antiEGFR (1:5,000), anti-pEGFR (Tyr1068; 1:1,000), anti-HER3 (1:1,000),
anti-pHER3 (Tyr1289; 1:200), anti-ERK1/2 (1:1,000), anti-pERK1/2
(Thr202/Tyr204; 1:1,000), anti-pAKT (Ser473; 1:1,000), anti-AKT
(1:1,000), anti-pS6R (Ser235/236; 1:1,000), and anti–β-actin (1:2,000)
antibodies were from Cell Signaling Technology. Bands were visualized using IRDye 800CW Secondary Antibodies (LI-COR Biosciences).
Clinical Trial Design and Conduct
This trial was conducted as a multicenter phase I trial with dose
escalation in patients with recurrent advanced solid tumors and dose
expansion in patients with metastatic colorectal cancer and acquired
EGFR inhibitor resistance (Trial registration ID: NCT01117428). The
trial protocol was approved by the Institutional Review Boards/ethics
committees at the participating centers, and the trial was conducted
in accordance with the Declaration of Helsinki. All patients provided
written informed consent. The trial was designed by senior academic
authors and the sponsor, Symphogen A/S. Trial medications were
provided by the sponsor. R. Dienstmann, S. Braun, and J. Tabernero
wrote all drafts of the article, with editorial support provided by a
medical writer and funded by the sponsor.
Research.
Eligibility Criteria
Inclusion criteria included: age of 18 years or older; Eastern
Cooperative Oncology Group performance status of 2 or better; and
adequate hematologic, hepatic, and renal function. Patients in the
dose-escalation cohort had to have advanced solid tumors refractory to available standard treatment. Patients in the dose-expansion
cohorts had to have KRAS–wild-type metastatic colorectal cancer
with documented radiological response to an anti-EGFR mAbcontaining regimen. Response was defined as complete response,
partial response, or stable disease for at least 16 weeks. Patients also
had to have documented disease progression (verified by computerized tomography or MRI) during or within 6 months after cessation
of primary anti-EGFR mAb treatment, with the last anti-EGFR dose
no more than 6 months before first Sym004 infusion. Exclusion
criteria for all cohorts included symptomatic central nervous system
metastases and antitumor treatment within 4 weeks of first planned
Sym004 infusion, or within 12 weeks for experimental vaccines.
Treatments
Sym004 was administered as an intravenous infusion at a maximum rate of 10 mg/min once every week. A premedication schedule
included a glucocorticoid (before the first 2 infusions), an antihistamine, and an antipyretic (before the first 4 infusions).
The starting dose of the dose-escalation cohort was derived based
on a recently published toxicology study in cynomolgus monkeys
(19). Patients were enrolled sequentially, and cohorts received weekly
doses of 0.4, 0.75, 1.5, 3, 6, 9, or 12 mg/kg; 1 patient in the 2 first
cohorts followed by 3 to 6 patients in subsequent cohorts according to the traditional 3+3 design. Dose escalation proceeded if no
DLTs were observed during the first 4 weeks. DLTs were defined as
treatment-related grade ≥3 hematologic and nonhematologic toxicities (adapted from NCI CTCAE, version 4.02) observed during the
first 4 weeks of treatment (corresponding to a minimum of 3 and a
maximum of 4 infusions) and within 48 hours of the most recent
infusion (allowing at most 30 days between first and most recent
infusions). Exceptions were grade 3 fatigue improving in less than 2
weeks, grade 3 EGFR inhibition–associated skin toxicity (if improved
to grade ≤2 within 2 weeks from onset), and grade 3 diarrhea (of less
than 2 days’ duration). All adverse events were coded with the use of
the Medical Dictionary for Regulatory Activities.
Following dose escalation, dose-expansion cohorts with weekly doses
of 9 or 12 mg/kg were conducted to obtain additional safety, pharmacokinetic, and pharmacodynamic data and to explore antitumor activity in patients with metastatic colorectal cancer. Doses were adjusted
(paused, decreased, or discontinued) in response to treatment-emergent
toxicities. All patients remained on treatment until disease progression, unacceptable toxicities, or withdrawal of consent. Toxicities were
assessed through clinical examination and laboratory assessments and
were graded by NCI CTCAE version 4.02. Safety evaluations were
conducted for all treated patients at baseline and at regular intervals,
including assessment of the onset and degree of skin toxicities.
Immunogenicity, Pharmacokinetics, and
Pharmacodynamics
For pharmacokinetic analyses, we collected serial blood samples
to measure serum levels of the individual Sym004 antibodies using
ELISAs. Blood samples were taken before and after all Sym004 infusions in the dose-escalation and dose-expansion cohorts. In addition,
postinfusion pharmacokinetic profile samples were taken 1, 2, 4, 8, 24,
and 48 hours after the first infusion and 1, 2, 4, 8, and 24 hours after
the fourth infusion. For immunogenicity, a validated double antigen
ELISA with immobilized Sym004 and horseradish peroxidase–labeled
Sym004 in solution was used for detection of anti-Sym004 antibodies before the first and planned fifth infusions (27). The lower limit
of detection was 150 ng/mL using rabbit anti-Sym004 IgG from
RESEARCH ARTICLE
hyperimmunized animals as a positive control for assay performance.
Serum concentrations of Sym004 were calculated as the sum of the
concentration of individual antibodies mAb992 and mAb1024 following infusion of Sym004, using two validated competitive ELISAlabeled anti-idiotypic antibody fragments specific for mAb992 or
mAb1024 (AbD Serotec). The lower limit of quantification for the
individual antibodies mAb992 and mAb1024 was 0.25 μg/mL.
For the pharmacodynamic analyses, serial skin and tumor biopsies
[before the first (baseline) and planned fifth Sym004 infusion (week
4)] were obtained from patients enrolled in the dose-expansion cohorts
after the patients had provided signed informed consent and investigators had assessed the risk before the procedure. Formalin-fixed and
paraffin-embedded skin and tumor biopsy samples were sent to Covance Laboratories Ltd. and Pathology Diagnostics Ltd. for immunohistochemistry analysis and evaluation of EGFR expression (0.86 µg/mL
anti-EGFR mAb; Dako M7239) and proliferation (0.92 µg/mL antiKi67 mAb; Dako M7240). A histopathologic score (H-score) with a
range of 0 to 300 was calculated according to the following formula:
(3 × percentage of cells with strong staining) + (2 × percentage of cells
with moderate staining) + (1 × percentage of cells with weak staining).
Tumor samples were assessed for tissue content and content of
neoplastic cells by a pathologist, and then analyzed for targeted mutation and gene copy-number profile (Department of Pathology, Radboud University Medical Center, Nijmegen, the Netherlands). Using
NGS (IonTorrent platform), frequently mutated positions in AKT1
(reference sequence NM_005163.2; exon 3), BRAF (reference sequence
NM_004333.4; exon 15), EGFR (reference sequence NM_005228.3;
exons 12, 18–21), ERBB2 (reference sequence NM_004448.2; exon
20), KRAS (reference sequence NM_004985.3; exons 2–4), NRAS
(reference sequence NM_002524.3; exons 2–4), and PIK3CA (reference sequence NM_006218.2; exons 10, 21) were assessed. MET copynumber changes were analyzed with FISH. Amplification was defined
as MET gene(G)-to-copy number (CN) control probe (centromere
chromosome 7) ratio (G:CN) of greater than 2.2 scored in >20 tumor
nuclei. Equivocal G:CN ratios of <2.2 were considered negative for
amplification, as was polysomy, i.e., increasing numbers of chromosomes carrying a single copy of the MET gene.
Evaluation of Tumor Response
Tumors were evaluated by computerized tomography or MRI per
RECIST version 1.1 (28) against a baseline assessment performed
within 3 weeks before the first dose. Reassessments were performed
after the first 6 weeks of treatment and thereafter every 8 weeks.
Tumor responses were evaluated both locally (by investigators to
enable treatment allocation) and centrally (for independent review
and primary analysis).
Statistical Analysis
The primary endpoint of this trial was safety and tolerability;
secondary endpoints included antitumor activity, and pharmacokinetic and pharmacodynamic effects (cutoff date, October 29, 2014).
We evaluated baseline characteristics and adverse events in all 62
patients (full analysis set) who were treated with Sym004. For the
assessments of pharmacokinetic and pharmacodynamic data, only
the patients with available samples were considered. Descriptive statistics were provided for demographic, safety, exposure, antitumor,
pharmacokinetic, and pharmacodynamic data. Categorical data were
summarized by frequency and percentages; continuous data were
summarized by median and range, or geometric mean and coefficient
of variation for pharmacokinetics. H-scores of skin and tumor biopsies were presented using box plots. Mean pharmacokinetic profiles
and geometric mean plots of AUC0–168 h by dose (after first dose) were
presented for the dose-escalation cohort, and dose-normalized Ctrough
values over time until dose reduction or interruption were presented
for the dose-expansion cohorts.
Research.
Dienstmann et al.
RESEARCH ARTICLE
Disclosure of Potential Conflicts of Interest
R. Dienstmann has received speakers’ bureau honoraria from
Symphogen. A. Patnaik is a consultant/advisory board member for
Symphogen. E. Van Cutsem has received research funding from
Amgen, Bayer, Boehringer, Lilly, Merck Serono, Novartis, Roche,
and Sanofi. A.W. Tolcher is a consultant/advisory board member for
AbbVie, Adnexus, Ambit, AP Pharma, Aragon, Ariad, ArQule, Asana,
Astex, Astellas, Avid, Bayer, Bind, BioMed Valley Discoveries, Blend
Therapeutics, Bristol-Myers-Squibb, Celator, Clovis, Curis, Daiichi
Sankyo, Dicerna, Eisai, Emergent Product Development, Endo, Five
Prime, Galapagos, Heron, Janssen, Lilly, MedImmune, Merck, Sharp
& Dohme, Mersana, Merus, Micromet, Nanobiotix, Nektar, Neumedicines, Novartis, OncoGenex, Onyx, Otsuka, Pfizer, Pharmacyclics,
Pierre Fabre, ProNai, Proximagen, Sanofi-Aventis, Santaris, Symphogen, Vaccinex, Valent Technologies, and Zyngenia. J. Tabernero
is a consultant/advisory board member for Amgen, Celgene, Chugai,
Imclone, Lilly, Merck KGaA, Millennium, Novartis, Roche, Sanofi,
Symphogen, and Taiho. No potential conflicts of interest were
disclosed by the other authors.
Disclaimer
The authors were fully responsible for all content and editorial
decisions, were involved at all stages of manuscript development, and
have approved the final version.
Authors’ Contributions
Conception and design: R. Dienstmann, A. Patnaik, M.W. Pedersen,
I.D. Horak, E. Van Cutsem, A.W. Tolcher, J. Tabernero
Development of methodology: R. Dienstmann, M. Benavent, N.J.Ø.
Skartved, R. Hald, M.W. Pedersen, M. Kragh, E. Van Cutsem, A.W.
Tolcher, J. Tabernero
Acquisition of data (provided animals, acquired and managed
patients, provided facilities, etc.): R. Dienstmann, A. Patnaik,
R. Garcia-Carbonero, A. Cervantes, M. Benavent, S. Roselló, B.B.J. Tops,
R.S. van der Post, G. Argilés, M.W. Pedersen, M. Kragh, E. Van Cutsem, A.W. Tolcher, J. Tabernero
Analysis and interpretation of data (e.g., statistical analysis,
biostatistics, computational analysis): R. Dienstmann, A. Patnaik,
R. Garcia-Carbonero, R.S. van der Post, U.H. Hansen, M.W. Pedersen,
S. Braun, E. Van Cutsem, A.W. Tolcher, J. Tabernero
Writing, review, and/or revision of the manuscript: R. Dienstmann,
A. Patnaik, R. Garcia-Carbonero, A. Cervantes, S. Roselló, R.S. van der
Post, G. Argilés, N.J.Ø. Skartved, U.H. Hansen, R. Hald, M.W. Pedersen,
M. Kragh, I.D. Horak, S. Braun, E. Van Cutsem, A.W. Tolcher, J. Tabernero
Administrative, technical, or material support (i.e., reporting or
organizing data, constructing databases): B.B.J. Tops, G. Argilés,
S. Braun, J. Tabernero
Study supervision: R. Dienstmann, U.H. Hansen, S. Braun, A.W.
Tolcher, J. Tabernero
Acknowledgments
The authors thank P. Pamperin, Symphogen A/S, and H. Liedman,
TFS, for assistance in the preparation and editing of this article,
and M. Düring, BioStata ApS, for statistical assistance. European
sites thank the European Union Seventh Framework Programme,
grant 259015 (COLTHERES) for supporting translational research
in colorectal cancer.
Grant Support
This study was funded by Symphogen A/S and Merck KGaA
(ClinicalTrials.gov trial registration ID: NCT01117428).
The costs of publication of this article were defrayed in part by
the payment of page charges. This article must therefore be hereby
marked advertisement in accordance with 18 U.S.C. Section 1734
solely to indicate this fact.
Received December 1, 2014; revised March 14, 2015; accepted
March 25, 2015; published OnlineFirst May 11, 2015.
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Safety and Activity of the First-in-Class Sym004 Anti-EGFR
Antibody Mixture in Patients with Refractory Colorectal Cancer
Rodrigo Dienstmann, Amita Patnaik, Rocio Garcia-Carbonero, et al.
Cancer Discovery Published OnlineFirst May 11, 2015.
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http://cancerdiscovery.aacrjournals.org/content/suppl/2015/05/08/2159-8290.CD-14-1432.DC1.ht
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Research.

Safety and Activity of the First-in-Class Sym004

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