Desnoyers 1..10

Tumor-Specific Activation of an EGFR-Targeting Probody Enhances
Therapeutic Index
Luc R. Desnoyers et al.
Sci Transl Med 5, 207ra144 (2013);
DOI: 10.1126/scitranslmed.3006682
Editor's Summary
Seek and Destroy
Cetuximab is a Food and Drug Administration−approved EGFR-targeting antibody used to treat metastatic
colorectal cancer and head and neck cancer, but therapy often results in dose-limiting skin rash. The authors modified
cetuximab to form a Probody (PB1) −−where the antigen-binding sites are masked until the antibody is activated by
proteases commonly found in the tumor microenvironment. The authors found that PB1 was largely inert while in
circulation in mice, but that it had comparable efficacy to cetuximab in the presence of tumor. In nonhuman primates,
PB1 demonstrated safety and decreased toxicity at higher doses than cetuximab. What's more, ex vivo human
primary tumor samples were sufficient to activate PB1. If these data hold true in human trials and for other antibodies,
Probodies could be used to target cancer while minimizing treatment side effects.
A complete electronic version of this article and other services, including high-resolution figures,
can be found at:
http://stm.sciencemag.org/content/5/207/207ra144.full.html
Supplementary Material can be found in the online version of this article at:
http://stm.sciencemag.org/content/suppl/2013/10/11/5.207.207ra144.DC1.html
Information about obtaining reprints of this article or about obtaining permission to reproduce this
article in whole or in part can be found at:
http://www.sciencemag.org/about/permissions.dtl
Science Translational Medicine (print ISSN 1946-6234; online ISSN 1946-6242) is published weekly, except the
last week in December, by the American Association for the Advancement of Science, 1200 New York Avenue
NW, Washington, DC 20005. Copyright 2013 by the American Association for the Advancement of Science; all
rights reserved. The title Science Translational Medicine is a registered trademark of AAAS.
Downloaded from stm.sciencemag.org on November 4, 2013
One of the main problems with current cancer therapies is lack of specificity: Traditional chemotherapeutics
target all dividing cells, and even more restricted drugs, like monoclonal antibodies, may have on-target but off-tumor
side effects. But what if you had a drug that was only turned on in the presence of the tumor? Desnoyers et al. now
report the development of a Probody that targets epidermal growth factor receptor (EGFR) only in the presence of
tumor.
RESEARCH ARTICLE
CANCER
Tumor-Specific Activation of an EGFR-Targeting
Probody Enhances Therapeutic Index
Luc R. Desnoyers,1* Olga Vasiljeva,1* Jennifer H. Richardson,1 Annie Yang,1 Elizabeth E. M. Menendez,1
Tony W. Liang,1 Chihunt Wong,1 Paul H. Bessette,1 Kathy Kamath,1 Stephen J. Moore,1
Jason G. Sagert,1 Daniel R. Hostetter,1 Fei Han,1 Jason Gee,1 Jeanne Flandez,1 Kate Markham,1
Margaret Nguyen,1 Michael Krimm,1 Kenneth R. Wong,1 Shouchun Liu,1 Patrick S. Daugherty,2
James W. West,1 Henry B. Lowman1†
Target-mediated toxicity constitutes a major limitation for the development of therapeutic antibodies. To redirect
the activity of antibodies recognizing widely distributed targets to the site of disease, we have applied a prodrug
strategy to create an epidermal growth factor receptor (EGFR)–directed Probody therapeutic—an antibody that
remains masked against antigen binding until activated locally by proteases commonly active in the tumor microenvironment. In vitro, the masked Probody showed diminished antigen binding and cell-based activities, but when
activated by appropriate proteases, it regained full activity compared to the parental anti-EGFR antibody cetuximab.
In vivo, the Probody was largely inert in the systemic circulation of mice, but was activated within tumor tissue and
showed antitumor efficacy that was similar to that of cetuximab. The Probody demonstrated markedly improved
safety and increased half-life in nonhuman primates, enabling it to be dosed safely at much higher levels than
cetuximab. In addition, we found that both Probody-responsive xenograft tumors and primary tumor samples from
patients were capable of activating the Probody ex vivo. Probodies may therefore improve the safety profile of
therapeutic antibodies without compromising efficacy of the parental antibody and may enable the wider use of
empowered antibody formats such as antibody-drug conjugates and bispecifics.
INTRODUCTION
Monoclonal antibodies have become an important class of targeted
therapeutics for the treatment of a range of human diseases. In particular, more than 100 years after the “magic bullet” concept of attacking cancer cells through the use of antigen-specific antibodies was
formulated by Paul Ehrlich (1), monoclonal antibodies are being approved for the treatment of a growing list of cancers. Antibodies such
as rituximab, brentuximab vedotin, trastuzumab, and cetuximab act in
a highly specific manner through the engagement of specific cell surface
antigens CD20, CD30, HER2/ERBB2, and EGFR (epidermal growth
factor receptor), respectively (2, 3).
Antigens expressed in hematologic malignancies and those expressed in solid tumors represent two distinct classes of targets. In
the former class, CD20 and CD30 represent examples of antigens that
are expressed at similar levels on both malignant and normal cells.
Depletion of these cells by the antibody rituximab (anti-CD20) or the
antibody-drug conjugate (ADC) brentuximab vedotin (anti-CD30) is
followed by repopulation of normal cells from progenitor stem cells in
the hematopoietic lineage, providing a useful therapeutic index. Solid
tumor targets include HER2, the selective up-regulation of which permits the therapeutic use of the antibody trastuzumab (4) or, more recently, the ADC ado-trastuzumab emtansine (T-DM1) (5), to attack tumor
cells while sparing normal tissues that express lower levels of antigen.
On the other hand, in the case of the solid tumor–targeting agent
cetuximab, the presence of relatively high levels of EGFR in healthy
skin leads to dose-limiting skin toxicity (6–10). Although skin reac1
CytomX Therapeutics Inc., 343 Oyster Point Boulevard, Suite 100, South San Francisco, CA
94080, USA. 2Department of Chemical Engineering, University of California, Santa Barbara,
Mail Code 5080, Engineering II, Room 3357, Santa Barbara, CA 93106–5080, USA.
*These authors contributed equally to this work.
†Corresponding author. E-mail: [email protected]
tions can be managed in clinical usage, cetuximab dosing may be decreased or stopped because of severe rash (11), and this side effect
limits the potential benefit of cetuximab therapy to some patients.
Moreover, recent studies have pointed to the potential for combinations of cetuximab with small-molecule tyrosine kinase inhibitors
(TKIs) to overcome the resistance associated with certain EGFR mutations (12–14). However, such combination strategies may be limited
by the overlapping toxicities associated with these classes of therapeutics. The use of antibodies targeting tumor-specific variants may protect against systemic effects of a pan-EGFR inhibitor; however, this
approach is limited to a subset of patients (15). Finally, the potential
development of an ADC based on cetuximab is limited by the widespread expression of EGFR in the skin. Indeed, the administration of
bivatuzumab mertansine, an ADC targeting a similarly widely expressed epithelial antigen, CD44v6, resulted in severe skin toxicities
and fatalities during early clinical trials (16). Unfortunately, it is clear that
magic bullets generally require “magic targets”—targets that are highly
overexpressed in diseased tissue. Despite ongoing research efforts, there
may be relatively few targets that meet these criteria (17, 18). Many
more antigens, although attractive as targets for pathway-blocking or cytotoxic antibodies in diseased tissue, are also expressed in at least one healthy
tissue type that can manifest dose-limiting on-target toxicity.
A solution to the problem of target-mediated toxicity of antibodies
has been proposed, based on the transformation of an antibody into
an antibody-based prodrug that remains inert in healthy tissues and
systemic circulation, but is activated locally at the site of disease (19).
This approach was demonstrated for a potential diagnostic agent,
termed a “proantibody,” directed to the widely expressed endothelial
antigen VCAM (vascular cell adhesion molecule). This proantibody
consists of a masked scFv antibody fragment linked via a substrate peptide that is activated by matrix metalloproteinases (MMPs) to an antibody
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
1
RESEARCH ARTICLE
Fc region. The resulting proantibody was shown to be locally activated
in the atherosclerotic plaques of diseased mice, yet remained inactive in
healthy tissues, providing an opportunity to detect VCAM expression
in combination with up-regulated MMP activity as a diagnostic marker
in vivo or in vitro. This success raises the question of whether therapeutic
antibodies might be modified into a prodrug form that could spare healthy
tissues while directing antigen-dependent activity to the site of disease.
Here, we report the efficacy and enhanced safety of a Probody
therapeutic in preclinical in vivo models. As a prototype of an antibody targeting a tumor antigen that is also widely expressed in healthy
tissues, we chose cetuximab. We based our molecular design of the
Probody on a conventional immunoglobulin G1 (IgG1), with a small
peptide extension at the N terminus of the light chain containing a
masking peptide and a linker that is cleavable by a selective set of proteases known to be up-regulated in activity in the tumor microenvironment. Such a construct allows direct in vitro and in vivo
comparisons with an approved therapeutic to evaluate the Probody’s
potential to improve therapeutic index—the ratio of maximal tolerated
dose to therapeutic dose. We provide evidence that such a proteaseactivated EGFR Probody, PB1, is relatively inert in healthy nonhuman
primates, yet locally activated and efficacious in mouse xenograft
models. Furthermore, by using the Probody itself as a probe of proteolytic activity, we were able to screen xenograft samples and patient
tumors for the presence of both target antigen and proteolytic activity
sufficient to achieve functional activation. Hence, the results presented
here suggest that an EGFR Probody can provide patient benefit
through expanded therapeutic index compared to cetuximab, and provide a path to selection of patients likely to benefit from the therapy.
well as inhibitors outside the tumor microenvironment in the case
of legumain, which is not normally secreted (26). On the other hand,
protease activity is generally up-regulated within the tumor environment
through up-regulation of protease expression, activation of zymogen,
down-modulation of inhibitor expression, or a combination of these
effects (21).
The fully recombinant EGFR Probody (PB1) consists of an authentic IgG heavy chain and a modified light chain (Fig. 1A). The 21–amino
acid masking peptide is biosynthetically fused to the N terminus of the
light chain through a 26–amino acid linker carrying the 8-residue proteasecleavable substrate linker, flanked by flexible Gly-Ser–rich peptide linkers.
We also produced a control construct, PB-NSUB, consisting of the same
masking peptide and antibody structure but without a cleavable substrate linker.
The activated form of Probody PB1 has in vitro activity
equivalent to cetuximab
To determine whether the activity of the activated PB1 was comparable to that of cetuximab, we purified a sample of PB1 that had been
RESULTS
The EGFR Probody PB1 is based on cetuximab
We engineered an EGFR Probody (PB1) by first identifying a masking
peptide derived from a diverse bacterial peptide display library using
previously described techniques (20) for binding to cetuximab (11).
The masking peptide was selected for specific binding to cetuximab
and its ability, when linked to the antibody, to block binding to EGFR.
In selecting a cleavable substrate linker sequence, we reasoned that sensitivity to multiple proteases in the tumor microenvironment would be
desirable because the activity of any one protease might be up-regulated
to various degrees in different patients or stages of tumor development
(21). The mask linker sequence is as follows (mask sequence is underlined; substrate region is double-underlined): QGQSGQCISPRGCPDGPYVMYGSSGGSGGSGGSGLSGRSDNHGSSGT. Our substrate selection
process included counterselection against active proteases found in healthy
tissues to reduce the potential for systemic activation of the substrate
and Probody. With a CLiPS library (22), the cleavable linker was selected for sensitivity to proteases known to be up-regulated in a variety
of human carcinomas: urokinase-type plasminogen activator (uPA)
(23), membrane-type serine protease 1 (MT-SP1/matriptase) (24, 25),
and legumain, a lysosomal protease found to be released and active in
the acidic extracellular tumor microenvironment (26). Although these
proteases are active in tumor tissue, the data presented here support previous findings that proteolytic activity because of these proteases in
healthy tissues is minimal. This control of proteolytic activity in healthy
tissues is likely due to the presence of endogenous inhibitors in the case
of uPA (23) and matriptase (24), and unfavorable pH conditions as
Fig. 1. Design and in vitro activation of Probody PB1. (A) Cartoon
model of a Probody showing the masking peptide (red), substrate linker
(green), flexible peptide linkers (gray lines), and IgG (gray ellipses). The left
Fab arm represents the intact Probody form with the masking peptide
tethered and bound in the antigen-combining site, whereas the right
Fab arm represents an activated Probody from which the masking peptide
has dissociated. (B) Capillary electrophoretic analysis of PB1 before (blue)
and after proteolytic cleavage with uPA (red) or MT-SP1 (black). LC, light
chain. (C) Intact PB1 shows decreased binding to immobilized EGFR by
ELISA, whereas digestion of PB1 with uPA protease (Activated PB1) restores
binding comparable to cetuximab. This plot is representative of three
independent experiments. Mean EC50 (median effective concentration) ±
SEM values for cetuximab, PB1, and activated PB1 were 11 ± 2 pM, 520 ±
240 pM, and 12 ± 4 pM, respectively. (D) Inhibitory activity of cetuximab,
PB1, or activated PB1 on the proliferation of H292 cells. Equivalent results
were obtained from four experiments; representative results are presented.
Mean IC50 (median inhibitory concentration) ± SEM values for cetuximab,
PB1, and activated PB1 were 0.012 ± 0.003 mg/ml, 4.8 ± 1.0 mg/ml, and 0.02 ±
0.007 mg/ml, respectively.
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
2
RESEARCH ARTICLE
completely activated by incubation with purified protease. PB1 was
coincubated with matriptase (MT-SP1) or uPA and analyzed by capillary electrophoresis (Fig. 1B). The molecular weight of the heavy
chain (74.9 ± 0.3 kD) of PB1 was not affected by addition of protease,
whereas hydrolysis with either of these proteases yielded a light chain
product of 32.0 kD compared to 36.1 kD for the uncleaved light
chain; this difference reflects the expected loss of the masking peptide.
Furthermore, N-terminal sequencing of the light chains after SDS–
polyacrylamide gel electrophoresis (PAGE) of the digestion products
confirmed cleavage at the predicted P1 Arg site. The kinetic parameters of substrate hydrolysis were kcat/Km = 2200 ± 424 M−1 s−1 for
matriptase and kcat/Km = 500 ± 79 M−1 s−1 for uPA. We also evaluated
the sensitivity of PB1 to cleavage by a broader panel of proteases including proteases not restricted to the tumor microenvironment. In
these experiments, PB1 was not cleaved by tissue plasminogen activator (tPA), ADAM10, ADAM17, plasmin, or KLK5 (fig. S1).
To evaluate the antigen binding of the masked and unmasked Probody, we used a direct EGFR enzyme-linked immunosorbent assay
(ELISA). Although this assay format is prone to avidity effects resulting in higher apparent affinities than in solution or on cells, it serves as
a rapid and useful readout of intact and activated Probody concentrations. In this case, the binding of the activated PB1 to EGFR by ELISA
was equivalent to that of cetuximab (Fig. 1C and table S1). However,
the binding of intact (nonactivated) PB1 appeared 48-fold weaker than
that of cetuximab, reflecting a masking
effect in the intact Probody. Fluorescenceactivated cell sorting (FACS) binding analysis also demonstrated effective masking
of PB1 with respect to binding EGFR on
H292 cells (fig. S2).
To evaluate the functional activity of
the Probody, we compared the inhibitory
activity of intact PB, activated PB1, and
cetuximab on H292 cell proliferation
in vitro (Fig. 1D and table S2). Activated
PB1 activity was comparable to that of
cetuximab. However, the activity of intact PB1 was reduced by 400-fold relative
to that of cetuximab, reflecting a functional masking effect in the intact Probody.
Thus, although the antigen binding and
biological activity of protease-activated PB1
were equivalent to those of cetuximab, PB1
(in the intact state) appeared greatly reduced in its biological activity. This suggested that the intact Probody would prove
safer than cetuximab in vivo while retaining its therapeutic activity in a local,
proteolytically active environment.
PB1 is activated ex vivo by
xenograft tumors
To evaluate the ability of tumor tissue to
activate the Probody, we developed a technique, termed Probody IHZ, whereby activation and binding of Probody can be
evaluated on frozen tumor sections (see
Materials and Methods). This technique
is based on the use of fluorescently labeled Probody that is incubated
on frozen tissue sections, leading to activation in situ by tissue-derived
protease and subsequent binding of the activated Probody to cell surface antigen. We tested this approach on xenograft tumors derived from
the human non–small cell lung cancer (NSCLC)–derived cell line
H292, known to express EGFR. Labeled PB1, PB-NSUB, or cetuximab
was incubated under the same conditions for normalization of fluorescence. A similar level of fluorescence signal was detected at the membranous surface of tumor cells for PB1 and cetuximab (Fig. 2A, middle
and left panels, respectively), indicating the presence of active proteases
capable of activating the Probody. As predicted, a negative signal was
observed on the tumor tissue incubated with the noncleavable PBNSUB construct (Fig. 2A, right panels), supporting the conclusion that
proteolytic processing was required to activate the Probody. To provide
additional evidence that binding of PB1 on the tumor tissue is a result
of proteolysis, we pretreated the tissue section with broad-spectrum
protease inhibitors. Compared to the positive staining for PB1 in the
absence of inhibitor (Fig. 2B, left panel), no binding of PB1 was observed on the tissue pretreated with inhibitor (Fig. 2B, middle panel).
Furthermore, pretreatment of the tissue with nonlabeled cetuximab at
high concentration abolished the PB1 signal (Fig. 2B, right panel), indicating that specific EGFR binding was also necessary for the Probody signal. In a time-course analysis, ex vivo PB1 activation was
detectable within 30 min and was maximal after 1 hour (fig. S3).
Fig. 2. In vitro Probody activation by frozen H292 tumor xenograft sections using IHZ analysis. (A)
Fluorescently labeled cetuximab, PB1, and PB-NSUB were incubated with frozen tissue sections. After
1 hour of incubation, positive staining was detected for cetuximab and PB1 (red). No fluorescent signal
was detected for PB-NSUB Probody at identical conditions. (B) The fluorescent signal of PB1 (left panel)
was inhibited by pretreatment of the tissue by preincubation with a broad-spectrum inhibitor cocktail
(middle panel) or by preincubation with unlabeled cetuximab. 4′,6-Diamidino-2-phenylindole (DAPI) nuclear staining appears in blue. Original magnification, ×40. Scale bars, 100 mm.
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
3
RESEARCH ARTICLE
PB1 is activated in vivo in xenograft tumors
To determine whether the Probody could be activated in an intact tumor environment, we evaluated the distribution of PB1 in mice bearing H292 tumor xenografts. Lung-derived tumor cells express active
matriptase (27) and secrete active uPA (28). Optical imaging was
carried out 48 hours after injection of PB1, PB-NSUB, or cetuximab,
each conjugated to the fluorophore Alexa750. The results showed accumulation of PB1 or cetuximab at the tumor site (Fig. 3A and fig. S4)
compared to PB-NSUB.
Furthermore, we have performed ex vivo analysis of tumor-bound
PB1 with an anti-human IgG detection antibody. Immunohistochemistry (IHC) analysis of tumors excised from PB1-treated animals
showed a staining pattern consistent with that observed in tumors from
cetuximab-treated animals (Fig. 3B); in contrast, tumor sections from
PB-NSUB–treated mice showed no specific staining. These results
suggested that, as in the in vitro and ex vivo experiments described
above, the Probody remains masked until activated by proteases in
the tumor environment.
croenvironment and the antiproliferative activity of PB1, we found
that the IHC staining for pERK was reduced in tumors of PB1-treated
mice compared to that in tumors from vehicle control–treated mice
(fig. S5). As expected, the pERK staining in tumors of cetuximab-treated
mice was also reduced, reflecting effective down-modulation of the
signaling pathway similar to that of the Probody. In contrast, the pERK
staining was not substantially reduced in tumors from PB-NSUB–
treated mice, again reflecting the requirement of proteolytic activity
for activation of the Probody.
PB1 suppresses tumor growth in vivo in mouse
xenograft models
We next investigated whether local activation of PB1 would translate
into antitumor efficacy in vivo using the H292 xenograft model in
nude mice. Weekly injections of PB1 and cetuximab demonstrated
significant efficacy [78% tumor reduction (P = 0.009) and 87% tumor
reduction (P = 0.002), respectively, versus vehicle control] after 20 days
(Fig. 4A and table S3). No statistically significant efficacy was observed
in the PB-NSUB group (P = 0.14 at day 20).
Because the intracellular signaling cascade initiated by the activation of many cell surface receptors, including EGFR, triggers the phosphorylation of extracellular signal–regulated kinase (ERK), we examined
tumor samples from treated animals using a phosphorylated ERK
(pERK) IHC assay to evaluate the degree to which EGFR signaling
was inhibited (29). Consistent with activation of PB1 in the tumor mi-
Fig. 3. In vivo activation of Probody PB1 in H292 mouse xenograft
tumors. (A) Optical imaging of H292 xenograft tumor–bearing mice
48 hours after intraperitoneal administration of Alexa750-conjugated PB1,
cetuximab, and noncleavable Probody (PB-NSUB). n = 3 mice per group
were injected with fluorescent conjugate. A high-intensity fluorescent signal was detected only in the tumors of mice dosed with PB1 or cetuximab,
suggesting that PB1 was activated and accumulated in the tumor through
EGFR binding. (B) IHC staining for human IgG in H292 xenograft tumors
72 hours after the mice (n = 3 per group) were injected with vehicle, or
with Alexa750-conjugated PB-NSUB, cetuximab, or PB1. Original magnification, ×40. Scale bars, 100 mm.
Fig. 4. PB1 inhibits tumor growth in xenograft models. (A) H292 xenograft tumor–bearing mice (n = 8 per treatment group) were treated weekly
with cetuximab (25 mg/kg, green), PB1 (25 mg/kg, red), PB-NSUB (25 mg/kg,
blue), or vehicle (10 ml/kg; black). Data are presented as mean tumor volume ± SEM. Efficacy was evaluated by measuring the reduction of the
mean tumor volume at day 20 relative to vehicle control (PB1: 78% tumor
growth inhibition, P = 0.009; cetuximab: 87% tumor growth inhibition, P =
0.002). No statistically significant efficacy was observed in the PB-NSUB
group (25% tumor growth inhibition, P = 0.14 at day 20). (B) Antiproliferative activity of a single dose of cetuximab (25 mg/kg, green), PB1 (25 mg/kg,
red), or vehicle (black) on LXFA677 xenograft tumors. Data are presented as
mean tumor volume ± SEM. Relative to a vehicle control, tumor growth
was inhibited by 96% (P = 0.0003) and 100% (P = 0.0003) on day 21 in
the cetuximab- and PB1-treated groups, respectively. Statistical analyses
used an unpaired Student’s t test.
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
4
RESEARCH ARTICLE
To further compare the antitumor activity of PB1 and cetuximab,
we used a patient-derived human NSCLC transplant model, LXFA677.
Mice with established tumors were treated once with PB1, cetuximab,
or a vehicle control. At a dosing of 25 mg/kg, PB1 and cetuximab were
equally efficacious at suppressing tumor growth in this model (Fig. 4B
and table S4). At a time point 21 days after treatment, cetuximab had
inhibited tumor growth by 96% (P = 0.0003) and PB1 by 100% (P = 0.0003)
relative to a vehicle-treated control group. Thus, the two molecules appeared equally efficacious.
PB1 has reduced cutaneous toxicity in nonhuman primates
Because our findings in mouse models suggested that the Probody is
locally activated, we investigated the potential safety benefits conferred
by this selectivity. Cetuximab is known to induce cutaneous toxicity in
nonhuman primates (11). We therefore compared the relative safety
of PB1 and cetuximab administered by intravenous infusion in female
cynomolgus monkeys (n = 3 per group) at 25 mg/kg per week for 4 weeks,
after a loading dose of 40 mg/kg. Consistent with previous findings,
skin toxicity was observed in all three cetuximab-treated monkeys after 5 weeks of treatment (Table 1). In contrast, none of the animals
treated with PB1 showed any signs of dermal toxicity. On the basis of
the lack of toxicity at this dose, PB1 was administered to three additional monkeys at 75 mg/kg per week for 7 weeks after a loading dose
of 120 mg/kg. Mild dermal toxicity was observed in two of three
monkeys at this dose, and this effect was less severe than the toxicity
observed in cetuximab-treated animals at the lower dosing of 25 mg/kg.
In both groups, the skin manifestations were fully reversible.
Plasma concentrations of PB1 and cetuximab were measured
throughout the study and confirmed sustained, high-level exposure
to the Probody and antibody during the dosing phase (Fig. 5A). Pharmacokinetic analysis demonstrated that exposure to PB1 as measured
by Cmax and the area under the plasma concentration–time curve over
a 4-week dosing period (AUC1–29) was 1.3-fold higher than exposure
to cetuximab (Table 2). Exposure to PB1 increased proportionally with
the increase in loading/weekly dose from 40/25 to 120/75 mg/kg. The
elimination half-life of PB1 averaged 6.7 and 7.3 days in the low- and
high-dose groups, and was about twice that of cetuximab at 3.2 days
(Table 2). The increase in dose-normalized exposure and extended
half-life of PB1 relative to cetuximab is consistent with a lack of binding to EGFR and a corresponding reduction in target-mediated clearance. These pharmacokinetic findings and the reduced toxicity of PB1
Table 1. Preclinical toxicology results in cynomolgus monkeys. NO,
not observed.
Dermatologic findings
Test
article*
Dose (loading/
weekly), mg/kg
Cetuximab
Time to onset
(study day)†
Extent and
severity
40/25
22, 23, 25
Mild to moderate
PB1
40/25
NO, NO, NO
Not applicable
PB1
120/75
9, 19, NO
Mild
*Plasma exposure to human IgG was 1.7-fold higher in Probody-treated monkeys when compared to the cetuximab-treated group (dose groups of 40/25 mg/kg).
†Time to onset of
dermatologic lesions, where observed, is shown for each animal (n = 3 animals per group) and
is comparable to that previously reported for cetuximab (data available at http://www.accessdata.
fda.gov/drugsatfda_docs/bla/2004/125084_ERBITUX_PHARMR_P3.PDF).
relative to cetuximab in cynomolgus monkeys are apparently the result of effective masking of the circulating Probody. Only trace levels
of EGFR-binding activity were observed (Fig. 5A, right panel) in plasma samples from PB1-treated monkeys despite the presence of high
concentrations of total Probody. Consistent with this observation, PB1
was not activated by ex vivo cyno skin samples by IHZ analysis (fig. S6).
Over the course of the study, the amount of activated PB1, expressed
as a percentage of total PB1 in plasma, averaged 5.9 and 7.7% at the
low and high dose, respectively. IHC staining with an anti-human IgG
antibody showed that, unlike cetuximab, there is no appreciable accumulation of PB1 in the skin of monkeys treated at the same dose level
of 40/25 mg/kg (Fig. 5B). Treatment-emergent anti-drug antibodies
were detected in two study animals: one in the cetuximab-treated
group and one in the 75 mg/kg PB1–treated group (table S5). Thus,
the PB1 Probody did not demonstrate increased immunogenicity
compared to cetuximab in this small pilot study. Together, these data
demonstrate that the Probody remains stably masked and inactive in
circulation as well as in healthy tissues, thus providing a basis for improved safety over cetuximab.
PB1 is activated ex vivo by primary human tumor samples
To test the ability to translate our preclinical findings on the Probody
to the clinical setting, we obtained primary frozen tissue samples from
human NSCLC and colorectal cancer (CRC) patients for analysis of in
situ protease activity. Probody IHZ analysis was carried out using fluorescently labeled PB1 or cetuximab (see above and Materials and Methods). As in the case of the H292 xenograft tumors described above, we
were able to detect a positive PB1 binding signal in both NSCLC and
CRC tumor sections, often comparable in intensity to that of sections
incubated with fluorescently labeled cetuximab (Fig. 6).
For quantitative assessment of a panel of tumor samples, EGFR
expression in the tissue was first evaluated by cetuximab staining using
a 3+ scale. Next, PB1 incubations were carried out and PB1 fluorescence was scored on the basis of comparison with cetuximab antibody
staining and defined as follows: −, no staining; 1+, weak staining compared to parental antibody; 2+, moderate staining compared to parental
antibody; and 3+, analogous staining to parental antibody. We scored
20 NSCLC samples, of which 12 were cetuximab- and PB1-positive;
none of the cetuximab-positive samples were negative for PB1 (table S6).
Among 30 CRC samples, 27 were cetuximab- and PB1-positive, and
only one cetuximab-positive sample was negative for PB1 activation
and binding (table S7), suggesting that a minority of CRC tumors
may lack proteolytic activity toward the PB1 linker.
Although these results currently represent limited sample sizes, the
positive signal observed in these sections suggests that, as in the case of
the PB1-responsive H292 xenograft model, most EGFR-positive human tumors have the potential to activate and respond to PB1 treatment. The Probody IHZ methodology described here, applied to
frozen biopsy samples, may be useful for the identification of patients
likely to respond to Probody therapy.
DISCUSSION
Our results demonstrate that a therapeutic antibody with known ontarget toxicity can be reengineered as a Probody retaining potent in
vivo efficacy, but with greatly reduced side effects. In particular, the
clinically validated anti-EGFR antibody cetuximab was converted into
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
5
RESEARCH ARTICLE
a prodrug form, PB1, using a masking peptide that was linked recombinantly to the light chain of the antibody through a proteasecleavable linker peptide. In this case, our choice of protease selectivity
was based on literature reports of uPA, matriptase, and legumain as
proteases that are widely expressed in solid tumors of epithelial origin
(23–28). We confirmed that some combinations of these proteases
were active in tumors by using the Probody itself as a probe of proteolytic activity, both via analysis of frozen tissue sections and by
in vivo imaging in mice. The activation of Probody PB1 was sufficient
in mouse xenograft models to yield antitumor efficacy similar to that
of the parental antibody cetuximab, whereas a noncleavable version of
the Probody, PB-NSUB, showed little activity. In the LXFA677 xenograft study in mice, a nonbinding species for cetuximab and PB1, total
pharmacologic exposure of Probody was similar to that of cetuximab,
and this exposure is similar to that achieved in humans under the
dosing schedule prescribed for cetuximab (11). Like cetuximab, PB1
accumulated in the tumors of treated animals and attenuated EGFRmediated signaling as measured by a decrease in ERK phosphorylation.
Together, these data suggest that local activation of PB1 gives rise to
sufficient levels of activated Probody to produce therapeutic benefit.
The studies presented here provide a starting point for further investigation of the mechanisms of activation and the applications of the
Probody platform. Our conclusions are limited by the fact that cetuximab
and PB1 do not cross-react with rodent EGFR. However, we have observed robust activity for PB1 in two mouse models at clinically accessible drug exposures. Although we have selected a substrate linker for
PB1 based on defined protease activities and have characterized some
of the proteases that can activate the PB1 Probody, it seems likely that
additional proteases will contribute to PB1 activation. Our study of
PB1 efficacy in a limited number of preclinical models and safety in
nonhuman primates has been encouraging; however, ultimately, its
safety, including immunogenicity, and efficacy in humans will need
to be determined in clinical trials.
The application of anti-EGFR antibodies as cancer therapy has the
potential to be expanded, in terms of clinical outcome, quality of life,
and scope of patient populations who may ultimately benefit. Dysregulation of the expression or activity of
EGFR is a component in the etiology of
multiple human carcinomas including head
and neck squamous cell cancer, NSCLC,
CRC, breast cancer, pancreatic cancer, and
brain cancer. Although both the antiEGFR antibodies and the small-molecule
TKIs approved or being tested in these indications (6, 7) are relatively specific with
respect to EGFR, this target, like many
being considered for targeted immunotherapy, is widely expressed in both healthy
and diseased tissues. The therapeutic benefit
of EGFR inhibition in cancer cells is accompanied in the case of pan-EGFR inhibitors
by inhibition in normal tissues, producing
undesirable adverse events. The most serious manifestation of these side effects is
in tissues where EGFR activity is crucial
for tissue growth and maintenance such
as skin. Indeed, papulopustular rash, dry
Fig. 5. Evaluation of PB1 and cetuximab concentration and activity in cynomolgus monkeys. (A) and itching skin, and hair and periungual
Plasma concentration–time curves for total human IgG (blue) and EGFR-binding human IgG (red) in PB1alterations are among the most common
and cetuximab-treated monkeys. Plasma from cynomolgus monkeys treated with cetuximab or PB1 (n = 3
per group for both groups through day 39; n = 2 per group from day 40 to day 61) was analyzed by ELISA side effects found in patients treated with
for total human IgG and for IgG capable of binding to EGFR. (B) IHC staining for anti-human IgG in skin EGFR-targeting agents. These effects retissue from monkeys treated with cetuximab or PB1. IHC staining was scored on a 3+ scale: −, no staining; sult in decreased quality of life as well as a
1+, weak staining; 2+, moderate staining; and 3+, strong staining. In the cetuximab section, the IHC signal decrease, interruption, or discontinuation
was scored as “+++”; in the PB1 section, the IHC signal was scored as “−.” Scale bars, 100 mm.
of treatment, limiting the efficacy of the
Table 2. Pharmacokinetic parameters in cynomolgus monkeys. Results are means ± SD from n = 3 animals (n = 2 for cetuximab half-life
determination).
Test article (loading/weekly
dose, mg/kg)
Cetuximab (40/25)
Cmax
(mg/ml)
Tmax
(days)
AUC1–29
(day mg/ml)
Dose-normalized
AUC1–29 (day mg/ml per mg/kg)
Half-life
(days)
779 ± 217
11.9 ± 10.8
13,024 ± 5,711
113.25 ± 49.66
3.24 ± 0.62
PB1 (40/25)
1,042 ± 98
25.4 ± 9.8
17,162 ± 1,940
149.23 ± 16.87
6.68 ± 0.51
PB1 (120/75)
3,894 ± 1,019
21.1 ± 18.5
52,010 ± 16,731
150.75 ± 48.50
7.32 ± 2.86
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
6
RESEARCH ARTICLE
Fig. 6. IHZ analysis of PB1 activation in human primary lung and colorectal tumor samples. Frozen tissue sections of human primary NSCLC
or CRC were incubated with fluorescently labeled PB1 or cetuximab. Red
staining indicates the binding of active antibody/Probody in these sections.
DAPI nuclear staining appears in blue. Original magnification, ×40. Scale
bars, 100 mm. For the results of additional NSCLC and CRC tissue screening,
see tables S6 and S7.
therapy (10). In addition, although the use of combinations of EGFR
antibodies and TKIs has appeared promising preclinically with antiEGFR antibodies that do not cross-react with mouse EGFR (and therefore do not produce systemic toxicity in rodent models), the use of such
combinations in humans is likely to be limited by severe skin toxicity
arising from both agents (12–14).
The pilot toxicity study in nonhuman primates (cynomolgus monkeys) shows that the PB1 Probody alleviates the dose-limiting cutaneous toxicity previously reported in this species. In particular, at a
weekly dose of 25 mg/kg after a loading dose of 40 mg/kg, we observed no skin rash or redness in the Probody treatment group,
whereas all three animals treated with cetuximab showed some evidence of cutaneous toxicity at this dose level, consistent with previous
studies of cetuximab in cynomolgus monkeys (11). The improvement
in safety for PB1 is even larger when considered in terms of exposure.
Although PB1 and cetuximab were dosed at the same level and schedule, the Probody achieved higher exposure because of its prolonged
pharmacokinetic half-life. This effect is consistent with the observation
that little Probody is activated in the circulation of these animals, protecting it from the effects of antigen-dependent clearance. Even at the
weekly dose of 75 mg/kg (with a loading dose of 120 mg/kg)—a nominal dose about 10-fold higher than the clinical dose of cetuximab—
the Probody produced only mild skin effects, with two of three animals
showing some signs of skin redness or rash. In contrast, previous studies
of cetuximab at this dose level produced severe rash in all animals,
with some animals succumbing to sepsis; 50% mortality was reported
in a group dosed at 75 mg/kg weekly (11). When considering the prolonged half-life and higher exposure of PB1 compared to cetuximab,
we estimate that the safety factor may be 3- to 15-fold greater for the
Probody—well beyond the range of typical clinical dosing—compared
to the parental antibody.
The efficacy and safety findings for PB1 suggest that PB1 may provide clinical benefit in patients by reducing rash associated with anti-
EGFR therapy, enabling higher doses of drug compared to the parental
antibody, with higher exposure of locally active antibody in the tumor
environment and potentially enhanced efficacy. Indeed, a previous
study with dose-escalating anti-EGFR therapy suggested that higher
antibody doses may provide a clinical benefit, although no improvement in overall survival was found in that study (30). Moreover, by
reducing skin rash effects for anti-EGFR monotherapy, the door may
be opened to allow combination therapies with TKIs and other therapeutics that suffer from cutaneous safety liabilities.
To examine the ability of PB1 to be activated in patients, we analyzed a collection of human tumor samples from lung and colon cancer patients for their ability to activate the Probody ex vivo. The results
of these experiments suggest that a large fraction of tumors across different stages of disease can activate the Probody. Indeed, across a combination of stage I, II, and III colon cancer samples, 90% (n = 30) of
the samples showed evidence of ex vivo Probody activation. This high
fraction of protease-positive samples compares favorably with a recent
study (25), which found that 68% (n = 152) of colon tumors expressed
active matriptase. We speculate that the higher fraction of positive
results in our experiment may reflect the ability of PB1 to be cleaved
by uPA and legumain, in addition to matriptase. The use of the Probody itself as a probe of protease activity, coupled with binding to target antigen, therefore offers a direct measure of tumor-associated
protease activity and antibody binding capacity that may prove to be
of utility in selecting patients for Probody therapy.
We believe that the enhanced targeting of antibodies to diseased
tissue made possible by Probodies will improve the therapeutic index
for many antibody therapeutics by reducing on-target toxicity in normal tissues. This approach has the potential not only to improve the
safety profile and clinical outcome of antibodies directed against validated
targets but also to expand the landscape for targets not previously druggable with antibodies because of on-target toxicities. More generally,
our findings suggest that a wide range of tumor antigens may be accessible to a Probody approach in various antibody-derived formats,
including Probody-drug conjugates and bispecific Probodies. Although
the findings described in this paper relate to translational clinical opportunities for Probodies in solid tumors, a Probody approach could
also be applied to targeting a host of diseases outside of oncology
where monoclonal antibodies have become an important modality
in the treatment armamentarium but have also been limited by ontarget toxicities in healthy tissue.
MATERIALS AND METHODS
Study design
The objectives of the in vivo efficacy studies were to evaluate the activity of PB1 on the growth of human xenograft tumors in nude mice
and compare it to the activity of the parental anti-EGFR antibody
cetuximab. The effect of PB1 was evaluated in a cell line tumor model
and in a patient-derived tumor model. Sample sizes (n = 8 to 10 per
group) were determined on the basis of homogeneity and consistency
of tumor growth characteristics in the selected models and were sufficient to detect statistically significant differences in tumor size between
groups. Tumor volumes were calculated with the formula (ab2)/2,
where a is the longer and b is the smaller of two perpendicular diameters. Animals were randomly assigned to groups based on tumor size
and were sacrificed once the tumor volume reached 2000 mm3. All
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
7
RESEARCH ARTICLE
tumor measurements were included in the analysis. Mean tumor volume with SEM was plotted. Technicians treating animals and measuring endpoints were blinded to the identity of the test articles.
The objectives of the nonhuman primate study were to compare
the toxicity and toxicokinetics of cetuximab and PB1 administered by
intravenous infusion to cynomolgus monkeys. Animals were randomly
assigned to experimental groups to achieve a similar group mean body
weight. The sample size chosen (n = 3 per group) is standard for preliminary safety evaluation and exposure assessment in nonhuman primates to minimize animal use. Test articles were coded to maintain
blinding of the study staff. Full experimental details are provided below.
Probody expression and purification
The complementary DNAs (cDNAs) coding for the heavy chain and
the light chain of each Probody were separately cloned into a modified
pcDNA3.1 mammalian expression vector (Life Technologies). CHO-S
cells (Life Technologies) were transiently transfected with the plasmids
encoding each Probody for 5 to 7 days using FreeStyle MAX transfection
reagent (Life Technologies) according to the manufacturer’s instructions.
Probodies were affinity-purified with MabSelect SuRe protein A columns (GE Healthcare) coupled to an AKTA FPLC (GE Healthcare). The
purity of purified Probodies was analyzed by SDS-PAGE in reducing
and nonreducing conditions, and its homogeneity was analyzed by
size exclusion chromatography with a Superdex 200, 10/300 GL column (GE Healthcare).
Proteolytic activation of Probody and kcat/Km determination
Recombinant human uPA and MT-SP1 (R&D Systems) were active
site–titrated and diluted in 50 mM tris-HCl and 0.01% Tween 20
(pH 8.5). PB1 stock solutions were added to protease samples at a final
concentration of 500 nM. All samples were incubated at 37°C for
24 hours. To stop the reaction, we incubated 5 ml of samples with 7 ml
of HT Protein Express Sample Buffer (Caliper Life Sciences) containing 20 mM 2-mercaptoethanol for 10 min at 95°C. Samples were applied to an HT Protein Express LabChip (Caliper Life Sciences) on the
GX-II Capillary Electrophoresis instrument (Caliper Life Sciences),
and concentrations of cleaved and uncleaved light chain were determined with LabChip GX software (Caliper Life Sciences); concentrations were converted to molarity with molecular weights of 24,318
daltons (cleaved) and 27,849 daltons (uncleaved). kcat/Km values were
determined with the following equation:
kcat
¼ −lnð1 − CÞ=ðt*pÞ
Km
where C is product conversion [cleaved light chain/(cleaved + uncleaved
light chain)], t is time (s), and p is protease concentration (M), which assumes that the substrate concentration is below the Km and in excess of the
protease. Protease-mediated activation of PB1 for in vitro studies was
achieved by incubating PB1 (100 mg) with uPA [50 nM in phosphatebuffered saline (PBS), pH 7.2] or MT-SP1 (10 nM in PBS, pH 7.2) at room
temperature. The uPA-cleaved PB1 was purified by protein A–agarose affinity chromatography. The cleavage of PB1 was analyzed by SDS-PAGE
and capillary electrophoresis (LabChip GXII; Caliper Life Sciences).
Cell culture and proliferation assays
H292 human lung cancer cells [American Type Culture Collection
(ATCC)] were maintained at 37°C (5% CO2) in RPMI medium sup-
plemented with 10% fetal bovine serum (FBS). For proliferation assays, the cells were seeded at 3000 cells per well in a 96-well plate
under low serum conditions (RPMI containing 1% FBS). The following day, antibodies or Probodies were added at the indicated concentrations, and cells were incubated for an additional 4 days at 37°C.
Cell viability was measured with CellTiter-Glo (Promega) according to
the manufacturer’s instructions.
EGFR-binding assay
Ninety-six–well plates (Nunc) were coated with EGFR-Fc (50 ng per
well; R&D Systems) in Hanks’ balanced salt solution (HBSS) (pH 7.4,
10 mM Hepes) and blocked with HBSS containing 1% bovine serum
albumin (BSA). The plates were incubated with the indicated concentrations of antibody or Probody in HBSS/1% BSA for 1 hour at room
temperature. The plates were then incubated with horseradish peroxidase
(HRP)–conjugated anti-human F(ab′)2 (Jackson ImmunoResearch Laboratories) in HBSS for 30 min, and detection was performed by the addition of 3,3′,5,5′-tetramethylbenzidine substrate (1-Step Ultra-TMB,
Pierce) followed by an equal volume of 1 M hydrochloric acid. Absorbance at 450 nm was then measured and reported as optical density (OD 450 nm).
In some cases, the relative EGFR binding was converted to an IgG
concentration with a cetuximab standard curve (1 nM starting concentration, 10-point serial dilutions). The standard curve was fitted
on a four-parameter curve, and the results were interpolated with
the ELx800 software.
Evaluation of Probody distribution in vivo by optical imaging
H292 xenograft tumor–bearing mice were injected intraperitoneally
with Alexa750-conjugated Probodies (12.5 mg/kg). The mice were imaged 48 hours after Probody injection with an IVIS Spectrum/CT imaging system (Caliper Life Sciences, PE) at excitation and emission
wavelengths of 745 and 800 nm, respectively. During the procedure,
the mice were kept under gaseous anesthesia (5% isoflurane) at 37°C.
In vivo efficacy studies
In vivo studies, conducted at The Jackson Laboratory, were reviewed
and approved by the Institutional Animal Care and Use Committee
(IACUC). In vivo studies conducted at Oncotest GmbH were reviewed and approved by the Regierungspräsidium Freiburg, Germany,
and conducted according to the guidelines of the German Animal
Welfare Act.
For H292 xenograft studies conducted at The Jackson Laboratory,
6- to 8-week-old female NU/J (JAX #2019) mice were inoculated subcutaneously in the right hind flank with 5 × 106 NCI-H292 cells
(ATCC) suspended 1:1 with Matrigel in serum-free medium. Clinical
observations, body weights, and digital caliper tumor volume measurements were made three times weekly once tumors become measureable.
Animals were tumor size rank–matched in cohorts (n = 12 mice per
group) with average tumor volumes of ~150 to 200 mm3, and treatments were started. Animals were treated intravenously once weekly
(25 mg/kg) for 4 weeks. Tumors were measured with calipers twice a
week for the duration of the study. Four mice per group were euthanized on day 3, and tumors were collected and snap-frozen for analysis.
The LXFA677 xenograft model was established at Oncotest GmbH
from primary patient material after informed consent. Xenografts
were subcutaneously grown in athymic NMRI nu/nu mice. Animals
were randomized after tumors reached volumes of 100 to 300 mm3.
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
8
RESEARCH ARTICLE
Mice (n = 10 per group) were injected intraperitoneally with a single
dose of antibody or Probody, and tumors were measured with calipers
twice weekly.
In situ zymography (Probody IHZ analysis)
H292 xenograft tumor samples were flash-frozen in liquid nitrogen
and embedded in OCT (optimal cutting temperature) compound.
Human tissue samples were provided by the Cooperative Human
Tissue Network, which is funded by the National Cancer Institute.
Other investigators may have received specimens from the same subjects. The samples were frozen with either liquid nitrogen or dry ice
within 1 hour of surgical removal. Frozen tissue sections (5 mm) were
laid over glass slides. A solution containing labeled PB1 or cetuximab
(1 mg/ml) was applied on the tissue and incubated 1 hour at room temperature in 50 mM tris-HCl buffer (pH 7.4), containing 150 mM
NaCl, 100 mM ZnCl2, 5 mM CaCl2, and 0.05% Tween 20. The tissue
was then washed to remove nonbound material, and detectable label
was visualized with a fluorescence microscope (Olympus IX 81) and
an Imaging Software for Life Science Microscopy Cell.
Immunohistochemistry
Xenograft tumors and cynomolgus monkey skin were fixed in 10%
neutral-buffered formalin overnight and then processed for paraffin
embedding. Formalin-fixed, paraffin-embedded samples of xenograft
tumors or cynomolgus monkey skin were sectioned (5 mm) and affixed to glass slides. Slides were deparaffinized and rehydrated through
graded alcohol to distilled water followed by retrieval with citrate
buffer (pH 6.0) (Thermo Scientific). Endogenous peroxidase was
quenched with 0.3% hydrogen peroxide. Sections were blocked with
the avidin/biotin blocking kit (Vector Laboratories) followed by 3%
BSA. The sections were stained with biotin-conjugated goat anti-human
IgG antibodies (American Qualex) at 20 mg/ml, followed by biotinconjugated donkey anti-goat IgG antibody (Jackson) at 5 mg/ml to
amplify the signal. Detection used the ABC Elite Detection kit (Vector
Laboratories), and staining was visualized with DAB (Pierce Scientific).
The slides were counterstained with hematoxylin, dehydrated, cleared, and
coverslipped. The stained sections were imaged with a bright-field microscope (Leica DM750) and LAS EZ software (Leica Application Suite).
Toxicity study in cynomolgus monkeys
All animals were housed, maintained, and treated in accordance to
standard ethical animal handling guidelines. The study was reviewed
and approved by the IACUC at Xenometrics. Nine experimentally
naïve female cynomolgus monkeys (Macaca fascicularis) were randomly assigned to dosing groups (n = 3 per group) at Xenometrics.
PB1 and cetuximab supplied at 2 mg/ml in PBS were administered to
three monkeys each by intravenous infusion for 5 weeks followed by a
4-week recovery period. A loading dose of 40 mg/kg was administered
over 3 days (study days 1, 3, and 5; 13.3 mg/kg per day) followed by a
weekly maintenance dose of 25 mg/kg administered over 2 days per
week (study days 8/11, 15/18, 22/25, and 29/32; 12.5 mg/kg per day).
A third group of monkeys received PB1 (a loading dose of 120 mg/kg)
as a single infusion followed by once weekly infusions of 75 mg/kg for
seven consecutive weeks. Animals were observed twice daily throughout the study for overall health, including evidence of dermatologic
toxicity. Plasma samples were collected at intervals for evaluation of
total and EGFR-binding Probody or cetuximab concentration. One
animal in each 40/25 mg/kg group and all three animals in the PB1
high-dose group were euthanized at the end of the dosing phase (dosing
for one high-dose animal was terminated early on day 29 because of a
possible infusion reaction). Areas of skin were collected and preserved
in neutral-buffered formalin for microscopic examination and IHC.
Human IgG ELISA for concentration measurements in
cynomolgus monkey plasma samples
Ninety-six–well MaxiSorp plates (Nunc) were coated with goat antihuman IgG antibody (preadsorbed against cynomolgus monkey IgG;
American Qualex Antibodies) in PBS and blocked with PBS containing 1% BSA. Samples were added to the wells and incubated for 1 to
2 hours at room temperature. The plates were then incubated with
HRP-conjugated goat anti-human IgG (preadsorbed as above) in PBS
for 1 hour, and detection was performed by the addition of 3,3′,5,5′tetramethylbenzidine substrate (1-Step Ultra-TMB, Pierce) followed
by an equal volume of 1 M hydrochloric acid. Absorbance at 450 nm
was then measured and reported as optical density (OD 450 nm). A
standard curve was generated for each plate using the corresponding
analyte (cetuximab or PB1; starting concentration of 10 mg/ml, 11-point
serial dilutions). The standard curve was fitted on a four-parameter
curve, and the results were interpolated with the ELx800 software. Noncompartmental pharmacokinetic analysis of plasma concentration–time
data was performed with Phoenix WinNonlin version 6.3 (Pharsight).
Statistical analysis
In vitro assays were repeated three to four times. Plotted values represent
means ± SEM. Descriptive statistics were generated with GraphPad
Prism where n ≥ 3. Linear and nonlinear regression analyses were
used to fit curves.
For in vivo studies, a one-sided Student’s t test was performed with
Microsoft Excel to assess the statistical significance of tumor size differences between treated and control groups. P values of ≤0.05 were
considered statistically significant.
SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/5/207/207ra144/DC1
Fig. S1. Selectivity of PB1 Probody linker cleavage.
Fig. S2. FACS binding of Probody PB1 and cetuximab to H292 cells.
Fig. S3. Time course of ex vivo Probody activation.
Fig. S4. Quantification of tumor/normal Probody and cetuximab accumulation in mice.
Fig. S5. EGFR pathway inhibition in tumors from mice treated with cetuximab or Probody PB1.
Fig. S6. EGFR expression and lack of Probody activation in skin from normal cynomolgus monkey.
Table S1. EGFR ELISA binding data from individual experiments.
Table S2. H292 cell–based activity inhibition data from individual experiments.
Table S3. Tumor volumes from H292 efficacy study.
Table S4. Tumor volumes from LXFA677 efficacy study.
Table S5. Anti-drug antibody observations in nonhuman primates.
Table S6. Probody IHZ screening of NSCLC patient tumor samples.
Table S7. Probody IHZ screening of CRC patient tumor samples.
REFERENCES AND NOTES
1. K. Strebhardt, A. Ullrich, Paul Ehrlich’s magic bullet concept: 100 Years of progress.
Nat. Rev. Cancer 8, 473–480 (2008).
2. J. M. Reichert, Marketed therapeutic antibodies compendium. MAbs 4, 413–415 (2012).
3. B. A. Teicher, Antibody-drug conjugate targets. Curr. Cancer Drug Targets 9, 982–1004 (2009).
4. C. A. Hudis, Trastuzumab—Mechanism of action and use in clinical practice. N. Engl. J. Med.
357, 39–51 (2007).
5. S. Verma, D. Miles, L. Gianni, I. E. Krop, M. Welslau, J. Baselga, M. Pegram, D. Y. Oh, V. Diéras,
E. Guardino, L. Fang, M. W. Lu, S. Olsen, K. Blackwell; EMILIA Study Group, Trastuzumab emtansine for HER2-positive advanced breast cancer. N. Eng. J. Med. 367, 1783–1791 (2012).
6. J. Baselga, C. L. Arteaga, Critical update and emerging trends in epidermal growth factor
receptor targeting in cancer. J. Clin. Oncol. 23, 2445–2459 (2005).
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
9
RESEARCH ARTICLE
7. N. E. Hynes, H. A. Lane, ERBB receptors and cancer: The complexity of targeted inhibitors.
Nat. Rev. Cancer 5, 341–354 (2005).
8. N. E. Hynes, G. MacDonald, ErbB receptors and signaling pathways in cancer. Curr. Opin.
Cell Biol. 21, 177–184 (2009).
9. H. Modjtahedi, S. Essapen, Epidermal growth factor receptor inhibitors in cancer treatment:
Advances, challenges and opportunities. Anticancer Drugs 20, 851–855 (2009).
10. R. Peréz-Soler, L. Saltz, Cutaneous adverse effects with HER1/EGFR-targeted agents: Is
there a silver lining? J. Clin. Oncol. 23, 5235–5246 (2005).
11. Erbitux (cetuximab) prescribing information, ImClone LLC, a wholly owned subsidiary of Eli
Lilly and Company, and Bristol-Myers Squibb Company, 693US13PBS01901 (2013).
12. L. Regales, Y. Gong, R. Shen, E. de Stanchina, I. Vivanco, A. Goel, J. A. Koutcher, M. Spassova,
O. Ouerfelli, I. K. Mellinghoff, M. F. Zakowski, K. A. Politi, W. Pao, Dual targeting of EGFR can
overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer.
J. Clin. Invest. 119, 3000–3010 (2009).
13. Y. Y. Janjigian, C. G. Azzoli, L. M. Krug, L. K. Pereira, N. A. Rizvi, M. C. Pietanza, M. G. Kris, M. S. Ginsberg,
W. Pao, V. A. Miller, G. J. Riely, Phase I/II trial of cetuximab and erlotinib in patients with lung
adenocarcinoma and acquired resistance to erlotinib. Clin. Cancer Res. 17, 2521–2527 (2011).
14. A. Cavazzoni, R. R. Alfieri, D. Cretella, F. Saccani, L. Ampollini, M. Galetti, F. Quaini, G. Graiani,
D. Madeddu, P. Mozzoni, E. Galvani, S. La Monica, M. Bonelli, C. Fumarola, A. Mutti, P. Carbognani,
M. Tiseo, E. Barocelli, P. G. Petronini, A. Ardizzoni, Combined use of anti-ErbB monoclonal
antibodies and erlotinib enhances antibody-dependent cellular cytotoxicity of wild-type
erlotinib-sensitive NSCLC cell lines. Mol. Cancer 11, 91 (2012).
15. D. Patel, A. Lahiji, S. Patel, M. Franklin, X. Jimenez, D. J. Hicklin, X. Kang, Monoclonal antibody cetuximab binds to and down-regulates constitutively activated epidermal growth
factor receptor vIII on the cell surface. Anticancer Res. 27, 3355–3366 (2007).
16. H. Riechelmann, A. Sauter, W. Golze, G. Hanft, C. Schroen, K. Hoermann, T. Erhardt, S. Gronau,
Phase I trial with the CD44v6-targeting immunoconjugate bivatuzumab mertansine in head
and neck squamous cell carcinoma. Oral Oncol. 44, 823–829 (2008).
17. T. T. Hansel, H. Kropshofer, T. Singer, J. A. Mitchell, A. J. George, The safety and side effects
of monoclonal antibodies. Nat. Rev. Drug Discov. 9, 325–338 (2010).
18. K. Even-Desrumeaux, D. Baty, P. Chames, State of the art in tumor antigen and biomarker
discovery. Cancers 3, 2554–2596 (2011).
19. O. Erster, J. M. Thomas, J. Hamzah, A. M. Jabaiah, J. A. Getz, T. D. Schoep, S. S. Hall, E. Ruoslahti,
P. S. Daugherty, Site-specific targeting of antibody activity in vivo mediated by disease-associated
proteases. J. Control. Release 161, 804–812 (2012).
20. J. J. Rice, A. Schohn, P. H. Bessette, K. T. Boulware, P. S. Daugherty, Bacterial display using
circularly permuted outer membrane protein OmpX yields high affinity peptide ligands.
Protein Sci. 15, 825–836 (2006).
21. L. C. van Kempen, K. E. de Visser, L. M. Coussens, Inflammation, proteases and cancer.
Eur. J. Cancer 42, 728–734 (2006).
22. K. T. Boulware, P. S. Daugherty, Protease specificity determination by using cellular libraries of
peptide substrates (CLiPS). Proc. Natl. Acad. Sci. U.S.A. 103, 7583–7588 (2006).
23. S. Ulisse, E. Baldini, S. Sorrenti, M. D’Armiento, The urokinase plasminogen activator system:
A target for anti-cancer therapy. Curr. Cancer Drug Targets 9, 32–71 (2009).
24. K. Uhland, Matriptase and its putative role in cancer. Cell. Mol. Life Sci. 63, 2968–2978 (2006).
25. A. M. LeBeau, M. Lee, S. T. Murphy, B. C. Hann, R. S. Warren, R. D. Santos, J. Kurhanewicz,
S. M. Hanash, H. F. VanBrocklin, C. S. Craik, Imaging a functional tumorigenic biomarker
in the transformed epithelium. Proc. Natl. Acad. Sci. U.S.A. 110, 93–98 (2013).
26. C. Liu, C. Sun, H. Huang, K. Janda, T. Edgington, Overexpression of legumain in tumors is
significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy.
Cancer Res. 63, 2957–2964 (2003).
27. X. Jin, T. Hirosaki, C. Y. Lin, R. B. Dickson, S. Higashi, H. Kitamura, K. Miyazaki, Production of
soluble matriptase by human cancer cell lines and cell surface activation of its zymogen by
trypsin. J. Cell. Biochem. 95, 632–647 (2005).
28. G. Liu, M. A. Shuman, R. L. Cohen, Co-expression of urokinase, urokinase receptor and
PAI-1 is necessary for optimum invasiveness of cultured lung cancer cells. Int. J. Cancer
60, 501–506 (1995).
29. D. Raben, B. Helfrich, D. C. Chan, F. Ciardiello, L. Zhao, W. Franklin, A. E. Barón, C. Zeng,
T. K. Johnson, P. A. Bunn Jr., The effects of cetuximab alone and in combination with radiation
and/or chemotherapy in lung cancer. Clin. Cancer Res. 11, 795–805 (2005).
30. E. Van Cutsem, S. Tejpar, D. Vanbeckevoort, M. Peeters, Y. Humblet, H. Gelderblom, J. B. Vermorken,
F. Viret, B. Glimelius, E. Gallerani, A. Hendlisz, A. Cats, M. Moehler, X. Sagaert, S. Vlassak,
M. Schlichting, F. Ciardiello, Intrapatient cetuximab dose escalation in metastatic colorectal
cancer according to the grade of early skin reactions: The randomized EVEREST study. J. Clin.
Oncol. 10, 2861–2868 (2012).
Acknowledgments: We thank our CytomX colleagues D. Ray, K. Polu, and C. T. Verser for
insightful discussions. Funding: CytomX Therapeutics Inc. and the NIH National Cancer Institute Small Business Innovation Research grant 1R43CA139790. Author contributions: L.R.D.,
O.V., J.H.R., A.Y., J.G.S., J.W.W., H.B.L., and P.S.D. (as consultant to CytomX Therapeutics Inc.)
designed the studies; A.Y., D.R.H., J.G.S., J.F., J.W.W., K.M., M.N., S.J.M., T.W.L., C.W., P.H.B., and
K.K. performed the experiments; A.Y., D.R.H., E.E.M.M., J.G.S., J.H.R., J.W.W., L.R.D., M.N., O.V., S.L.,
S.J.M., M.K., K.R.W., T.W.L., P.H.B., and K.K. collected and analyzed the data; K.R.W. and O.V.
performed in vivo imaging studies; E.E.M.M. performed IHC staining; F.H., J.G., and S.L.
expressed and purified Probodies; C.W. prepared the final figures; L.R.D. and H.B.L. wrote
the manuscript, with essential input from J.G.S., S.J.M., O.V., D.R.H., A.Y., and P.S.D. Competing
interests: All authors, with the exception of P.S.D., are current or former paid employees of
CytomX. P.S.D. is a consultant, scientific advisory board member, and stockholder of CytomX.
CytomX has filed or licensed patent applications related to the work described herein: “Activatable
binding polypeptides and methods of identification and use thereof” (WO 2009/025846),
“Modified antibody compositions, methods of making and using thereof” (WO 2010/081173),
and “Activatable antibodies that bind epidermal growth factor receptor and methods of
use thereof” (PCT US2013/038540). Data and materials availability: Data and materials are
available from CytomX under a material transfer agreement.
Submitted 31 May 2013
Accepted 28 August 2013
Published 16 October 2013
10.1126/scitranslmed.3006682
Citation: L. R. Desnoyers, O. Vasiljeva, J. H. Richardson, A. Yang, E. E. M. Menendez, T. W. Liang,
C. Wong, P. H. Bessette, K. Kamath, S. J. Moore, J. G. Sagert, D. R. Hostetter, F. Han, J. Gee,
J. Flandez, K. Markham, M. Nguyen, M. Krimm, K. R. Wong, S. Liu, P. S. Daugherty, J. W. West,
H. B. Lowman, Tumor-specific activation of an EGFR-targeting probody enhances therapeutic
index. Sci. Transl. Med. 5, 207ra144 (2013).
www.ScienceTranslationalMedicine.org
16 October 2013
Vol 5 Issue 207 207ra144
10