Full Text - Molecular Cancer Therapeutics

Molecular
Cancer
Therapeutics
Therapeutic Discovery
Therapeutic Mechanism and Efficacy of the Antibody–Drug
Conjugate BAY 79-4620 Targeting Human Carbonic
Anhydrase 9
Heike M. Petrul, Christoph A. Schatz, Charlotte C. Kopitz, Lila Adnane, Timothy J. McCabe, Pamela Trail,
Sha Ha, Yong S. Chang, Andrei Voznesensky, Gerald Ranges, and Paul P. Tamburini
Abstract
Carbonic anhydrase IX (CAIX) is a cell surface glycoprotein that is expressed in many different tumors
and yet restricted in normal tissues to the gastrointestinal tract. It is upregulated by hypoxia and
correlates with tumor grade and poor survival in several tumor indications. Monoclonal antibodies
(mAb) with single digit nanomolar binding affinity for CAIX were derived by panning with the
recombinant ectodomain of CAIX against the MorphoSys HUCAL Gold library of human Fabs. Highest
affinity Fabs were converted to full-length IgGs and subjected to further characterization based upon
their avidity and selectivity for CAIX, their capacity to undergo internalization in CAIX-expressing cell
lines, and their selective localization to CAIX-positive human xenografted tumors when administered to
mice as fluorescent conjugates. Through this selection process, the 3ee9 mAb was identified, which upon
conjugation to monomethyl auristatin E through a self-immolative enzyme-cleavable linker yielded
the potent and selective CAIX antibody–drug conjugate CAIX-ADC (BAY 79-4620). In preclinical human
xenograft models in mice representing several tumor indications, BAY 79-4620 showed potent antitumor
efficacy and in some models showed partial and complete tumor shrinkage even following a single
dose. The mechanism of action was shown by histology to involve the sequelae of events typical of
antitubulin agents. Efficacy in murine preclinical models correlated semiquantitatively, with CAIX
expression levels as determined by immunohistochemistry and ELISA. These preclinical data collectively
support the development of BAY 79-4620 for the treatment of cancer patients with CAIX overexpressing
tumors. Mol Cancer Ther; 11(2); 340–9. 2011 AACR.
Introduction
Carbonic anhydrase IX (CAIX) was first identified
by Pastorekova as an endogenous HeLa cell antigen
that was recognized by the antibody M75 raised
against this cervix carcinoma cell line (1). Although
initially referred to as MN antigen, it was shortly
thereafter identified as a new carbonic anhydrase by
the same group (2) and was confirmed to be identical
Authors' Affiliation: Bayer HealthCare AG, Berlin, Germany
Note: Supplementary data for this article are available at Molecular Cancer
Therapeutics Online (http://mct.aacrjournals.org/).
Current address for T.J. McCabe: Johnson & Johnson, PA; current address
for P. Trail: Regeneron Pharmaceuticals, Inc., NY; current address for S. Ha:
Merck&Co., Inc. PA; current address for Y.S. Chang: Aileron Therapeutics,
MA; current address for A. Voznesensky: Novartis Institutes for BioMedical
Research, MA; current address for G. Ranges: Bayer HealthCare AG,
retired; and current address for P.P. Tamburini: Alexion Pharmaceuticals.
Inc, CT.
Corresponding Author: Heike M. Petrul, Bayer HealthCare AG, Berlin
13342, Germany. Fax: 49-30-18079; E-mail: [email protected]
doi: 10.1158/1535-7163.MCT-11-0523
2011 American Association for Cancer Research.
340
Mol Cancer Ther; 11(2) February 2012
to the tumor marker G250 (named after the antibody
with which it was identified) published by Oosterwijk
and colleagues (3).
CAIX is a cell surface glycoprotein that is expressed in
carcinomas of several histologic types, including a strikingly high proportion of renal cell carcinomas (4, 5),
carcinomas of the esophagus (4, 6), cervical carcinomas
(4, 7), malignant colon carcinomas (4, 8), non–small cell
lung carcinomas (NSCLC; refs. 4, 9), and, to a lesser
degree, breast carcinomas (4, 10). By contrast, the expression of CAIX on normal tissues is largely restricted to the
apical surface of cells of the stomach, bile duct mucosa
(4, 11), and small intestine (4, 12). The extracellular
domain of this type I transmembrane protein comprises
both a proteoglycan domain implicated in cell adhesion
through homotypic interaction (13) and the carbonic
anhydrase domain that catalyzes the reversible hydration
of carbon dioxide to bicarbonate and protons (14) and is
involved in the regulation of the pH within the tumor
environment (15).
CAIX gene expression is under the direct control
of the transcription factor hypoxia-inducible factor-1
and is significantly upregulated by tumor hypoxia (16).
CAIX expression was found to correlate with (i) a high
Efficacy of Anti-CA9 Antibody–Drug Conjugate BAY 79-4620
mean vessel density, cancer stage, and degree of necrosis
in head and neck carcinoma (17); (ii) poor survival in
nasopharyngeal carcinoma (18); (iii) tumor grade, negative estrogen receptor status, higher relapse rate, and poor
survival for invasive breast carcinoma (10). This association with tumor grade and overall survival, together with
its cell surface distribution and restricted expression in
normal tissues, implicates CAIX as an important therapeutic target for monoclonal antibody (mAb)-based therapy. In this article, we report the identification of a potent
and selective internalizing human antibody directed
against CAIX that, when conjugated to monomethylauristatin E (MMAE), yielded a highly efficacious antibody–
drug conjugate BAY 79-4620, with activity against a variety of solid tumor types.
Materials and Methods
Reference antibodies
The hybridoma producing CAIX mAb M75 (1, 13) was
obtained from American Type Culture Collection (ATCC)
and used to express and purify mAb M75 using standard
protocols.
Antibody discovery using the HuCAL Gold
Fab-phage library
The HuCAL Gold Fab-phage library was obtained from
MorphoSys AG and was prepared as described elsewhere
(19), comprising a highly diverse library of 1010 different
monovalent phage encoded within phagemid vector
pMORPH23 and allowing for monovalent CysDisplay of
Fab fragments. Solid phase panning was carried out as
described in the Supplementary Data.
Fab expression
Soluble Fab fragments were produced from the isolated
phage clones as described in the Supplementary Data.
Identification of CAIX-binding Fabs
Isolated Fabs were tested for binding to the purified
ectodomain of CAIX in an ELISA, positive Fabs were
recloned in the IgG format and expressed in Chinese
hamster ovary cells (see Supplementary Data). AntiCAIX IgG antibodies were purified using protein A
sepharose.
Antibody binding kinetics using surface plasmon
resonance
Surface plasmon resonance (SPR) was carried out on a
BIAcore 3000 instrument (BIAcore; see Supplementary
Data).
Immunoprecipitation
The complexes formed between the antibodies and
biotin-labeled cell proteins were immunoprecipitated and
visualized by immunoblots developed with enzymelinked streptavidin using an adaptation of methods
described elsewhere (20).
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Cell binding by flow cytometry
Adherent CAIX-expressing PC-3 mm2 cells, non-CAIX
expressing MiaPaCa-2 cells, and MiaPaCa-2 cells transfected with full-length CAIX were detached from their
flasks with 1:10 trypsin/Versene in PBS solution for 5 to 10
minutes. Cells were spun down (1,000 rpm, 5 minutes),
washed once with ice-cold RPMI 10% FBS, and resuspended in ice-cold staining buffer [Caþ Mgþ-free PBS, 2%
bovine serum albumin (BSA), and 0.05% sodium azide] to
achieve 6 106 cells/mL. Anti-CAIX IgG1 or control
human IgG1 antibodies at 25 mg/mL were incubated with
6 105 cells on ice for 1 hour. Unbound antibody was
removed by washing the cells with the ice-cold staining
buffer. The cells were fixed with 2% formaldehyde in PBS
for 10 minutes, then washed twice with staining buffer.
The cell pellet was resuspended in 100 mL ice-cold staining
buffer containing a 1:200 dilution of Alexa Fluor 488–
labeled secondary antibody (Molecular Probes/Invitrogen) and incubated on ice for 1 hour. The unbound
antibody was washed from the cells 2 times with flow
buffer (PBS containing 2% BSA), and the cells were resuspended in 1 mL flow buffer. Fluorescence-activated cell
sorting (FACS) analysis of the resuspended cells was done
on a Beckman FACS Calibur instrument. All cell lines
were obtained from ATCC; MiaPaCa-2-CAIX was generated using standard transfection methods. Cell lines are
regularly checked for authenticity by DNA fingerprinting
at the DSMZ (German Collection of Microorganisms and
Cell Cultures), Braunschweig, Germany.
Assessment of cellular internalization of mAbs
Antibody internalization was assessed using a Cellomics Array Scan automated confocal microscope system
(see Supplementary Data).
Generation of immunoconjugates with
monomethylauristatin E
mAb conjugation with MMAE was done essentially as
described elsewhere for conjugation with an anti-CD30
mAb (21).
Cell cytotoxicity assays
Antigen-positive and antigen-negative cells were plated at 5,000 cells per well in 96-well plates in 100 mL media
per well overnight at 37 C, in 5% CO2 to adhere. The
media was replaced with fresh media containing antibody
or antibody-vcMMAE conjugate and the plates further
incubated at 37 C, in 5% CO2 for 72 hours. Cytotoxicity
was assessed using the Alamar Blue cell viability assay.
Alamar Blue was added at a final concentration of 10% for
the last 4 hours of the incubation, and its transformation to
fluorescent product by viable cells determined spectrofluorometrically by plate reader (544 nm excitation–590
nm emission).
In vivo antibody distribution in mice
Noninvasive in vivo imaging studies were conducted
using the CRI Maestro in vivo imaging system with
Mol Cancer Ther; 11(2) February 2012
341
Petrul et al.
multispectral acquisition and analysis (CRI; see Supplementary Data).
In vivo tumor growth inhibition and
pharmacodynamic studies
All animal experiments were conducted in accordance
with either the United Kingdom Coordinating Committee
on Cancer Research regulations for the welfare of animals,
the German animal welfare law, or the Institutional Animal Care and Use Committee, in addition to approval by
local authorities. For xenograft experiments based on
human tumor cell lines, immunocompromised mice (8–
12 weeks of age) were subcutaneously implanted on day 0
with the respective tumor cells in 0.1 mL volume as
detailed in Supplementary Table ST2. For xenograft
experiments based on patient-derived NSCLC tumors,
tumor fragments were subcutaneously passaged on naive
NMRI nu/nu mice (Lu7506, Lu7298, Lu7913, Lu7406,
Lu7747; refs. 22, 23). Treatment was initiated when tumors
reached an average size of 60 to 160 mm3 depending on the
model used (Supplementary Table ST2). Dosing of test
compounds was carried out according to the dosing
levels, schedules, and routes of administration described
in Supplementary Table ST2. Carboplatin was obtained
from Hexal, taxol (Paclitaxel) from Bristol Myers Squibb,
cisplatin from Sigma Aldrich, and gemcitabine from Eli
Lilly. A dose volume of 0.1 mL/10 g body weight was
used throughout and intravenous administration was by
tail vein injection. The health status of animals was monitored daily. The length and width of each tumor was
measured by electronic calipers 2 to 3 times per week and
tumor volumes (mm3) were calculated as [length (mm) width (mm)2]/2. Percentage increases in tumor size during the study were calculated by the formula [100 tumor
volume at treatment end/tumor volume at treatment
start] 100 for every single animal. Final tumor volume
was defined by the last time point in which the vehicle
treated control remained within the experiment. Antitumor efficacy was also assessed from the incidence of
regression in which a tumor regression is defined as a
reduction in tumor size of more than 30% at study end
relative to the initial size.
In pharmacodynamic experiments, tumors were collected at 4 hours, 1, 3, and 5 days following single administration of BAY 79-4620, formalin-fixed, paraffin-embedded (FFPE), sectioned at 5 mm, deparaffinized, and stained
using standard protocol for fluorescent immunohistochemistry (IHC) of human tissues using mouse antibodies against -a/b-tubulin, phospho-Histone H3, and
DNA. Slides were observed by fluorescent microscopy.
Immunohistochemistry
In parallel with efficacy studies, a group of animals
carrying each tumor type was sacrificed, the tumors were
preserved as FFPE and cryo (frozen in Tissue-Tek) tissues.
Tumor tissue was cut in 3-mm slices and stained with the
anti-CAIX mouse mAb M75 at 0.5 mg/mL (cryo sections)
and 1 mg/mL (FFPE sections) final concentrations in
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Mol Cancer Ther; 11(2) February 2012
conjunction with Animal Research Kit Peroxidase (Dako),
mouse IgG block, peroxidase block and biotin block.
Epitope demasking for FFPE tissue was done for 17
minutes in a vegetable steamer in Tris-buffered solution
(pH 9).
IHC slides were analyzed by a pathologist with an
intensity scale of 0, 1þ, 2þ, or 3þ.
Correlation of CAIX level with antitumor efficacy of
CAIX-ADC across a variety of human tumor xenografts. IHC slides were quantified using Histoscore
(H-score). H-score considers staining intensity per cell
as well as the distribution of the staining in the tumor
tissue and is determined as percentage of tumor cells
showing 3þ staining intensity 3 þ (% cells staining
2þ) 2 þ 1 (% cells staining 1þ); range 0–300. Percent
tumor growth inhibition (TGI) at 1 mg/kg BAY 79-4620
was plotted against the average H-Score for each model.
CAIX content of several tumors was also determined
by Western blot (Supplementary Data).
Statistical analysis
To compare TGI between treatment groups and their
respective vehicle group, 2-sided Dunnett tests (24) were
carried out. The Dunnett test is a one-step test procedure
that is very powerful in the given situation of comparing
several groups in a common control. Dunnett’s procedure
keeps the multiple significance level. In each study, a
significance level of 0.05 was chosen. The log-transformed
ratios of tumor volume at the end of experiment to
baseline tumor volume at day 0 were defined as the
response variable of interest.
All calculations were carried out using SAS 9.1.3.
Results
Human antibody fragments (Fabs) directed at the
ectodomain of CAIX were selected by in vitro "panning"
of the MorphoSys HuCal Gold nonimmune biased Fabphage display library with a purified preparation of the
extracellular domain of human CAIX. More than 50
CAIX-binding Fabs with unique VH and/or VL
sequences were identified that bound CAIX by ELISA
with signal to noise ratios of more than 10. The top 10
Fabs disclosed in Tamburini and colleagues (25) with
respect to selective binding and internalization by
CAIX-expressing cancer cell lines were converted to
full-length human IgG1 antibodies, expressed in Chinese hamster ovary cells, and purified by affinity chromatography. Seven of these antibodies with acceptable
expression and solubility were further characterized
and found to exhibit high affinity binding to the CAIX
ectodomain in vitro, with several mAbs exhibiting single-digit nmol/L Kd values by SPR (Supplementary
Table ST1). Next, the capacity of 6 of these antibodies
to selectively immunoprecipitate antigen from wholecell lysates derived from biotin-labeled PC-3mm2 whole
cells that express CAIX (CAIXþ) or DLD-1 cells that do
not (CAIX) was examined by immunoblots developed
Molecular Cancer Therapeutics
Efficacy of Anti-CA9 Antibody–Drug Conjugate BAY 79-4620
None;
biotinylated
ctrl
5A6
3a4
5 aa 3
3 ef 2
1 aa 1
3 ee 9
Control IgG
5A6
3a4
5 aa 3
3 ef 2
1 aa 1
3 ee 9
Control IgG
A
191
97
64
51
PC3mm2
DLD1
B
PC-3MM2
MiaPaCa-2
MiaPaCa-2-CAIX
Untreated
Sec. antibody
Anti-CAIX 3ee9
Figure 1. In vitro selectivity of fully human mAbs directed against human carbonic anhydrase IX. A, immunoprecipitation of biotinylated cell surface proteins
þ
from CAIX PC-3mm2 cells (left) and CAIX DLD-1 cells (right). Lanes are labeled according to the particular anti-CAIX mAb used. Far right lane contains
biotinylated CAIX ectodomain directly applied to the lane. Arrows locate positions of migration of Mr markers of the indicated sizes (kDa). B, binding of mAb
3ee9 to CAIX-positive PC-3mm2 and CAIX-transfected MiaPaca-2-CAIX cells (left and right) but not to CAIX-negative parental MiaPaCa-2 cell line (middle) by
flow cytometry.
with streptavidin–horseradish peroxidase (Fig. 1A).
mAbs 3ee9 and 1aa1 selectively coimmunoprecipitated
from biotin-labeled CAIXþ cells—but not from the
CAIX cells—a single protein that comigrated with
recombinant biotinylated CAIX (Fig. 1A). mAbs 3ef2,
5aa3, and 5a6 were not selective and coprecipitated
many additional cell surface proteins in addition to
CAIX. Importantly, the conjugate of mAb 3ee9 with
MMAE (BAY 79-4620; CAIX-ADC) retained the affinity
of the parent antibody for CAIX (Supplementary Table
ST1), exhibiting a kDa of 3.6 nmol/L by SPR. The
conjugate did not bind to 13 other carbonic anhydrases
tested (data not shown). Binding to mitochondria-associated CA5 was observed, but this isozyme would be inaccessible to the antibody in vivo. BAY 79-4620 selectively
bound (Fig. 1B) and underwent internalization by (Supplementary Fig. S1) CAIXþ PC-3mm2 cells in culture but
did not bind CAIX MiaPaca2 cells (Fig. 1B) or undergo
internalization by CAIX DLD-1 cells (Supplementary
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Fig. S1). A similar selectivity was observed with the ADC
comprising mAb1aa1 (data not shown), and both CAIXspecific mAb conjugates selectively killed CAIXþ cells in
culture. For example, BAY 79-4620 killed CAIX-transfected MiaPaCa-2-CAIX cells, with an IC90 of 10
nmol/L, whereas CAIX-negative MiaPaca2 wild-type
cells were quite resistant (Fig. 2), with at least a 10-fold
higher IC90. The mechanism of cell killing by BAY 79-4620
involved targeted tubulin disruption. Thus although
treatment of CAIXþ PC-3mm2 cells in culture resulted in
fragmented fibers and prevention of normal spindle formation, as visualized by tubulin staining, CAIX H460
cells retained normal spindle formation in the presence of
the conjugate (Supplementary Fig. S2).
The high selectivity of the 3ee9 targeting antibody
component of BAY 79-4620 was further shown using
established tumors derived from the CAIX-negative MIAPaCa-2 pancreatic carcinoma cell line, and its CAIX-positive transfectant. Administration of an 3ee9-Alexa Fluor
Mol Cancer Ther; 11(2) February 2012
343
Petrul et al.
100
% Inhibition
80
60
MiaPaCa-2-CAIX
40
MiaPaCa-2
20
0
0
20
40
60
80
100
120
–20
c (nmol/L)
Figure 2. Cytotoxicity of BAY 79-4620 toward CAIX-transfected
MiaPaCa-2 (diamonds) but not CAIX-negative MiaPaCa-2 cells (circles).
750 conjugate, but not a control hIgG Alexa Fluor 750
conjugate, to mice with bilaterally implanted CAIX-negative and CAIX-positive MiaPaca tumors resulted in
exclusive retention of the Alexa-750 conjugate in the
CAIX-positive tumor following elimination of the bulk
of the unbound fluorescence via the liver and bladder on
or around day 4 (Supplementary Fig. S3). Furthermore,
BAY 79-4620 exhibited minimal TGI (50%) when administered to mice implanted with established CAIX-negative
MiaPaca2 tumors, even at the higher dose of 10 mg/kg,
but exhibited significant antitumor activity when administered to mice implanted with established CAIX-positive
tumors derived with MiaPaca2 cells transfected with
CAIX (data not shown), in which doses of 2.5, 5, and 10
mg/kg yielded TGI of 63%, 82%, and 94%, respectively,
and 100% shrinkage at the latter dose.
BAY 79-4620 was highly active against colorectal cancer
HT-29 tumors using a range of dosing schedules, including, quite remarkably, single intravenous doses (Fig. 3A,
left panel) in the range 0.625 to 10 mg/kg. To explore the in
vivo mechanism of action, tumors were harvested following single BAY 79-4620 doses of 1.25 and 5 mg/kg and
evaluated by IHC (Fig. 3B). Whereas little effect was
observed at 4 hours, increased numbers of cells in
G2/M arrest, the appearance of multipolar spindles, and
a decreased level of tubulin was clearly seen by day 1. This
pattern intensified through to 5 days postdosing when
almost all cells in the 5 mg/kg dose group were severely
affected as judged by the presence of defective spindles or
apoptosis. These data indicate a mechanism of tumor
killing by BAY 79-4620 that is both consistent with the
effects of the compound on CAIX-expressing cells (Supplementary Fig. S2) and in line with the known properties
of MMAE as a tubulin polymerization inhibitor that
induces G2/M arrest through prevention of normal spindle formation (26).
Despite the impressive results obtained in this model,
the tumor response to single dose (Fig. 3A, left panel) and
the 1 dose every 7 days (not shown) schedules, was, unlike
the effects of the 3 doses every 4 days schedule (Fig. 3A,
right panel), transient in nature. Therefore, for all follow-
344
Mol Cancer Ther; 11(2) February 2012
ing animal experiments the dosing schedule of 3 doses
every 4 days was used. At this dose scheduling, BAY 794620 was highly active against established tumors
derived from the HT-29 cell line (Fig. 3A, right panel).
Whereas tumors from untreated, PBS vehicle treated, and
10 mg/kg unconjugated CAIX mAb-treated control
groups grew progressively, with mean doubling times of
around 6.4 days, treatment with BAY 79-4620 on a 3 doses
every 4 days schedule, intravenous schedule produced
robust antitumor efficacy at all doses examined, with
0.625, 1.25, 2.5, 5, and 10 mg/kg doses yielding 54, 72,
97, 100, and 100% TGI, respectively. The percentages of
the tumors that exhibited regression were 20, 90, 100, 100,
respectively, at the 1.25, 2.5, 5, and 10 mg/kg doses, and
BAY 79-4620 was well tolerated with no weight loss at all
doses. Paclitaxel (Taxol) at a dose of 15 mg/kg showed
efficacy very similar to 2.5 mg/kg of BAY 79-4620. Free
MMAE toxophore administered at a dose of 0.2 mg/kg,
equivalent to the amount of MMAE administered in
10 mg/kg of BAY 79-4620 was much less active than the
antibody conjugate, producing 60% TGI and no tumor
regression. Although this dose of free MMAE was well
tolerated, a 1 mg/kg dose produced 50% lethality with
severe weight loss across all survivors (not shown).
BAY 79-4620 was highly active against a variety of other
established tumor types (Fig. 4), including those established using the following CAIXþ human cancer cell lines:
cervix carcinoma HeLa-MaTu, colorectal Colo205, prostate PC3mm2, gastric NCI-N87, lung A549, gastric SNU16,
and gastric MKN45. Tumors derived using HeLa-MaTu
were the most sensitive wherein a minimal effective dose
of 0.625 mg/kg was observed. The 60 mg/kg dose of
BAY 79-4620 produced 10% lethality and 20% body
weight loss establishing this dose of BAY 79-4620 as the
MTD, whereas all other treatment levels were well tolerated. Eighty percent of the HeLa-MaTu tumors exhibited
tumor shrinkage at the 1.25 mg/kg dose, whereas at the
2.5 mg/kg dose and higher, 100% of the tumors showed
regression or complete regression. BAY 79-4620 was also
active against established tumor xenografts derived from
the human prostate PC-3mm2 cell line (Fig. 4). Thus,
whereas PBS vehicle or unconjugated mAb (not shown)
at 30 mg/kg exhibited no antitumor efficacy, BAY 79-4620
produced a dose-dependent TGI ranging from 45% at
1 mg/kg to 100% at doses of 10 mg/kg and higher and
was well tolerated at all doses.
BAY 79-4620 was also active in the gastric carcinoma
model NCI-N87 (Fig. 4). This aggressive tumor model
responded well to the higher dose of 60 mg/kg on a
3 doses every 4 days schedule, although neither cisplatin
nor paclitaxel treatment reached statistical significance
compared with the vehicle group on day 37, when the
vehicle group was sacrificed.
Efficacy of BAY 79-4620 in heterogeneous tumors
derived from patient tumor explants was shown in several
lung carcinoma models (Lu series). As these tumors
consist of both CAIX-positive and CAIX-negative cells
(Fig. 5), the observed efficacy in these models suggested
Molecular Cancer Therapeutics
Efficacy of Anti-CA9 Antibody–Drug Conjugate BAY 79-4620
A
Vehicle
10 mg/kg 3ee9
10 mg/kg 3ee9 + 0.2 mg/kg MMAE
0.625 mg/kg BAY 794620
1.25 mg/kg BAY 794620
PBS
0.625 mg/kg BAY 794620
1.25 mg/kg BAY 794620
2.5 mg/kg BAY 794620
5 mg/kg BAY 794620
10 mg/kg BAY 794620
2.5 mg/kg BAY 794620
5 mg/kg BAY 794620
10 mg/kg BAY 794620
15 mg/kg Taxol
0.2 mg/kg MMAE
2,500
Tumor volume [mm3]
Tumor volume [mm3]
2,000
Mean ± SD
1,500
1,000
500
Mean ± SD
2,000
1,500
1,000
500
0
0
0
10
20
30
40
50
60
0
Time after tumor cell inoculation [d]
10
20
30
40
50
Time after tumor cell inoculation [d]
60
B
Day 1
Day 5
0 mg/kg
1.25 mg/kg
5.0 mg/kg
Figure 3. Antitumor efficacy of CAIX-ADC against HT-29 tumors. A, dose-dependent TGI resulting from single dose (left) and 3 doses every 4 days
(right) treatments. Mean values SD are plotted. Red arrows indicate time points of treatment. B, immunohistochemistry of HT-29 tumors at 1 or 5 days
after a single dose of 0, 1.25, or 5 mg/kg BAY 79-4620. Sections were stained for a/b tubulin (green fluorescence), phospho-histone-H3 (red
fluorescence), and DNA (blue fluorescence). Highlighted features are normal M-phase cells with chromosomes and spindles (arrows); M-phase cells
with defective or absent spindles (arrowheads); normal tubulin in the cytosol (asterisks); apoptotic cells with condensed or fragmented DNA,
aggregation or absence of tubulin (stars).
that the release of toxophore from the CAIX-positive cells
kills the CAIX-negative cells through a bystander effect.
In contrast to the effects observed in the above tumor
models, BAY 79-4620 was inactive against the MDR line
HCT-15 that overexpresses P-glycoprotein (P-gp). The Pgp substrate paclitaxel was similarly inactive, whereas
gemcitabine, a non-Pgp substrate, exhibited TGI in this
tumor model (Fig. 4).
Anti-CAIX–antibody drug conjugates are expected to
release the attached toxophore specifically in antigenexpressing tumors. To test whether the activity of BAY
79-4620 correlated with CAIX expression levels in
tumors, an immunohistochemical staining protocol for
CAIX was developed. In parallel to the above studies of
the antitumor activity of BAY 79-4620 in the various
xenograft models, the levels of CAIX expression within
these tumors was determined. Groups of animals were
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sacrificed at the time when treatment was started and
tested for CAIX expression by IHC. CAIX expression
was semiquantified from the average pixel intensities
per cell, the percentage of the tumor cells staining
positive for CAIX and the location of CAIX within the
tumor tissue (Fig. 5). Most of the tumors, including
A431, A549, Lu7913, Lu7298, and Lu7506, expressed
CAIX in the area around necrotic cores. In most cases,
CAIX staining was absent from the immediate vicinity of blood vessels consistent with its upregulation
by hypoxia. The intensity of CAIX expression across
the various tumor types varied strongly. HeLa-MaTu
showed a strong homogenous CAIX expression pattern, whereas Hs746T showed almost no staining. The
efficacy of BAY 79-4620 in tumor models showed a
strong positive association with tumor CAIX expression
level and/or H-score (Fig. 6). HeLa-MaTu, HT29, and
Mol Cancer Ther; 11(2) February 2012
345
Increase of tumor size during study
[% of size at treatment start]
Increase of tumor size during study
[% of size at treatment start]
Petrul et al.
1,400
1,200
1,000
800
600
400
200
0
–50
–100
–150
*
****
HT-29
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0
–50
–100
–150
*
**
Lu7913
**
**
*
**
*
*
** *
*
*
* *
**
**
Lu7298
*
*
Lu7406
**
Lu7506
Lu7747
Discussion
Although some recently published small molecule
inhibitors (27, 28) successfully target the enzymatic
activity of CAIX, the work described here uses a different therapeutic approach in exploiting CAIX as an
anchor for the selective delivery of a toxic payload to
tumors via an antibody-based therapeutic. A rigorous
discovery cascade was implemented to generate and
PC3mm2
Lu7298
Lu7506
HCT116
select fully human antibodies that are highly selective
for binding to, and internalization by, CAIX-expressing
cancer cell lines. These antibodies exhibited undetectable binding to other proteins within complex target
cell proteomes by immunoprecipitation, did not bind
to other extracellular carbonic anhydrases, and in the
case of the CAIX-targeting antibody 3ee9 (BAY 794682), showed specific retention in CAIX-positive tumors in vivo by whole animal imaging using a fluorescently labeled version. The binding selectivity of 3ee9
was fully retained in the corresponding antibody–drug
conjugate BAY 79-4620, which proved to be both potent
and highly selective in the killing of cultured CAIXexpressing cell lines through a mechanism that involved
tubulin disruption. Consistent with the in vitro selectivity of the conjugates, conjugate BAY 79-4620 exhibited
A549
A431
Hs746T
Overview
HT29
PC3mm2 HeLaMaTu
Vehicle
0.625 mg/kg BAY 794620
1 mg/kg BAY 794620
1.25 mg/kg BAY 794620
2.5 mg/kg BAY 794620
3 mg/kg BAY 794620
5 mg/kg BAY 794620
10 mg/kg BAY 794620
30 mg/kg BAY 794620
60 mg/kg BAY 794620
Gemcitabine
Taxol
Cisplatin
75 mg/kg Carboplatin
PC3mm2 models characterized by the highest expression of CAIX showed 56% to 92% TGI, when treated
with 1 mg/kg BAY 79-4620. This dose did not show TGI
in the Hs746T, A549, and A431 models—characterized
by the lowest CAIX levels observed–-but these models
did respond to doses higher than 1 mg/kg.
HeLa-MaTu Lu7913
******
Colo205 HCT-15 NCI-N87 MKN45 SNU16 A549
*
**
60x
20x
Figure 5. Distribution of CAIX
expression in xenograft models.
Staining intensity of CAIX-positive
tumor cells was done as 0, 1þ, 2þ,
or 3þ. Representative images of
the tumor models are shown.
The 3 panels show an overview.
Magnifications, 20 and 60.
Cervix cancer
346
Figure 4. CAIX-ADC exhibits potent
antitumor activity against a variety
of human tumor types. Waterfall
plots for change in tumor size are
shown for tumors derived from
various human cancer cell lines
(top row) and patient-derived
tumor fragments (bottom row).
Colored bars identify the particular
treatment depicted in the inset.
Mean þ SD is plotted. Asterisks
indicate statistical significance
versus respective vehicle controls
(detailed statistical information are
presented in Supplementary Table
ST3).
Lung cancer Colon cancer Prostate cancer Lung cancer
Mol Cancer Ther; 11(2) February 2012
Lung cancer
Colon cancer
Lung cancer
Skin cancer
Gastric cancer
Molecular Cancer Therapeutics
Efficacy of Anti-CA9 Antibody–Drug Conjugate BAY 79-4620
%Tumor growth inhibition (TGI)
at 1 mg/kg CA9-ADC
100
80
HeLa-MaTu
Hs746T
HT29
A549
HCT116
A431
PC3mm2
Lu 7506
Lu 7913
Lu7298
60
50% TGI
40
20
0
0
100
200
300
H-Score [0-300]
3 x (% cells staining 3+) + 2 x (% cells staining 2+) + 1 x (% cells staining 1+)
Figure 6. Correlation of CAIX level with antitumor efficacy of CAIX-ADC
across a variety of human tumor xenografts. Tumor-bearing animals
were treated with vehicle or 1 mg/kg BAY 79-4620 3 doses every 4 days,
intravenously. Tumor size was determined using caliper. Percent TGI was
determined at the end of the experiment. IHC slides were quantified using
Histoscore (H-score), which was determined by 3 (% of tumor cells
staining 3þ) þ 2 (% of tumor cells staining 2þ) þ 1 (% of tumor cells
staining 1þ); range 0 to 300. Percent TGI at 1 mg/kg BAY 79-4620 is
plotted against the average H-Score in that model.
little or no antitumor activity against a human tumor
xenograft with low or no CAIX expression but was
highly potent against xenografts comprising the same
cell line that had been stably transfected with CAIX. The
selectivity of the conjugate BAY 79-4620 was further
confirmed in human and primate normal tissue binding
studies, wherein the binding pattern mimicked exactly
the expected tissue distribution of CAIX with significant
staining only in the gastrointestinal tract and where no
off-target binding was observed (data not shown).
There remains a high unmet need for safe and effective antitumor agents targeting different tumor
indications, yet despite the large amount of information about the association of CAIX with various solid
tumors, there is no approved therapeutic drug targeting this antigen. Our approach uses an anti-CAIX
antibody with a linker toxophore system based on the
tubulin inhibitor MMAE that offers potency at the
site of action and a high level of stability in the periphery. Accordingly, the conjugate BAY 79-4620 proved
to be highly effective and well tolerated when used
against CAIX-expressing human xenografts representing cervical, prostate, colorectal, gastric, and lung
tumors. In each case, a 100% complete response rate
could be shown at the higher doses within a 3 doses
every 4 days schedule. At lower doses of BAY 79-4620
on or around 1 mg/kg, differences in the antitumor
activity across the various models was observed with
higher activity associated with tumor types exhibiting
high CAIX expression, such as HeLa-MaTu, and lower
activity against xenograft models with less CAIX
expression such as PC-3mm2. The data suggests that
the antitumor activity of BAY 79-4620 at 1 mg/kg is
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driven mainly by targeted delivery of the MMAE
toxophore to CAIX-expressing tumor cells. Targeting
of a CAIX-expressing tumor by the anti-CAIX antibody
was shown in in vivo imaging studies, in which the
antibody was selectively retained in the antigen-positive, but not the antigen-negative, tumor implanted on
the same animal. The therapeutic efficacy of targeted
delivery of the toxophore was also shown in the
patient-derived lung tumor model Lu7406, in which
both carboplatin and paclitaxel show little efficacy,
whereas BAY 79-4620—which delivers a much lower
total dose of cytotoxic compound to the tumor—
showed high efficacy. The efficacy against this and
other patient-derived models with heterogeneous
expression of CAIX is consistent with BAY 79-4620–
dependent tumor cell killing both by direct and
bystander mechanisms. The mechanistic IHC studies
of HT-29 tumors showed that the response to a single
dose of CAIX-ADC was rapid, being detectable at
day 1, and seemed to affect the majority of tumor cells
by day 5. The efficacy of BAY 79-4620 seems to be
limited to tumors that are susceptible to tubulin inhibition, as was shown by the lack of response to the
ADC in the highly chemoresistant HCT-15 colon carcinoma model.
The safety and toxicologic evaluation of the ADCs
described presently has been facilitated by cross-reactivity with primate CAIX and BAY 79-4620 is currently in
phase I of clinical development.
We anticipate that the high potency and selectivity of
the ADCs described presently will translate into the clinic
and that these compounds may possess single-agent
efficacy, possibly rising to become first-line therapeutics.
We note that these therapeutics are combinable preclinically with standard of care therapeutics such as capecitabine (Xeloda; data not shown), and it is conceivable
that therapeutic use could be further augmented in
combination.
The mechanism of action of BAY 79-4620 suggests
clinical development in tumor indications characterized
by a high expression of CAIX and a reported sensitivity to
spindle poisons. NSCLC and gastric cancer are 2 of several
tumor indications showing CAIX expression as well as
sensitivity to taxanes (29–31). The expression of CAIX
varies between tumor indications as well as between
individual patients with a tumor of the same histologic
origin. Clinical development of BAY 79-4620 should,
therefore, be supported by biomarker measurements of
CAIX expression. Clinical studies should explore cut-offs
for CAIX expression associated with response to BAY 794620.
Noninvasive assays to identify patients likely to
respond to BAY 79-4620 treatment and assays monitoring
early response to treatment would aid clinical development of this drug. Potential approaches currently under
evaluation include the quantification of circulating levels
of CAIX ectodomain and imaging-based methods such as
positron emission tomography.
Mol Cancer Ther; 11(2) February 2012
347
Petrul et al.
Disclosure of Potential Conflicts of Interest
All authors are current or former employees of Bayer Healthcare.
Acknowledgments
BAY 79-4620 uses the Seattle Genetics linker-toxophore chemistry;
the antibody 3ee9 is derived from the HuCAL phage display library
(Morphosys AG). The authors thank Silvia Pastorekova and Jaromir
Pastorek, Institute of Virology, for scientific consultancy and also the
following individuals for their important contributions to this work:
Dana Wirak, Richard Altman, Tom Donaldson, Haren Vasavada
(molecular biology); Dave Wunderlich, Jennifer Pendleton, Karla D’
Agostino (protein expression); Carla Pellegrino, Robert Dreyer, Steven
Fisk (protein chemistry), Elizabeth Bourret, Peggy Bourguillon, Marina
Ichetovkin, Susan Gawlak, Marina Reinelt, Katrin Weidner (cellular
assays), Elizabeth Bortolon, Arris Henderson, Dahai Xue, Bianka Timpner, Karola Henschel, Katrin J€ansch (preclinical animal studies), Sabine
Jabusch (IHC), Anna Behnke (biochemical assays) and Tina M€
uller
(statistics).
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 July 20, 2011; revised November 2, 2011; accepted November
11, 2011; published OnlineFirst December 6, 2011.
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