Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Molecular
`Cancer
`Therapeutics
`
`Large Molecule Therapeutics
`
`Construction and Characterization of Novel, Completely
`Human Serine Protease Therapeutics Targeting Her2/neu
`
`Yu Cao, Khalid A. Mohamedali, John W. Marks, Lawrence H. Cheung, Walter N. Hittelman, and
`Michael G. Rosenblum
`
`Abstract
`
`Immunotoxins containing bacterial or plant toxins have shown promise in cancer-targeted therapy, but
`their long-term clinical use may be hampered by vascular leak syndrome and immunogenicity of the
`toxin. We incorporated human granzyme B (GrB) as an effector and generated completely human
`chimeric fusion proteins containing the humanized anti-Her2/neu single-chain antibody 4D5 (designated
`GrB/4D5). Introduction of a pH-sensitive fusogenic peptide (designated GrB/4D5/26) resulted in
`comparatively greater specific cytotoxicity although both constructs showed similar affinity to Her2/
`neu–positive tumor cells. Compared with GrB/4D5, GrB/4D5/26 showed enhanced and long-lasting
`cellular uptake and improved delivery of GrB to the cytosol of target cells. Treatment with nanomolar
`concentrations of GrB/4D5/26 resulted in specific cytotoxicity, induction of apoptosis, and efficient
`downregulation of PI3K/Akt and Ras/ERK pathways. The endogenous presence of the GrB proteinase
`inhibitor 9 did not impact the response of cells to the fusion construct. Surprisingly, tumor cells resistant
`to lapatinib or Herceptin, and cells expressing MDR-1 resistant to chemotherapeutic agents showed no
`cross-resistance to the GrB-based fusion proteins. Administration (intravenous, tail vein) of GrB/4D5/26
`to mice bearing BT474 M1 breast tumors resulted in significant tumor suppression. In addition, tumor
`tissue excised from GrB/4D5/26–treated mice showed excellent delivery of GrB to tumors and a dramatic
`induction of apoptosis compared with saline treatment. This study clearly showed that the completely
`human, functionalized GrB construct can effectively target Her2/neu–expressing cells and displays
`impressive in vitro and in vivo activity. This construct should be evaluated further for clinical use.
`Mol Cancer Ther; 12(6); 979–91. Ó2013 AACR.
`
`Introduction
`Bacterial and plant toxin-based immunotoxins have
`shown remarkable potency and specificity, but a number
`of obstacles limit their clinical application (1, 2). The toxin
`component of these fusion proteins can elicit vascular
`damage leading to loss of vascular integrity (vascular leak
`syndrome, VLS; refs. 3, 4). Immune responses to the toxins
`in patients also result in rapid clearance of subsequent
`courses of therapy (5, 6). Toxin immunogenicity is being
`addressed by engineering B-cell epitopes on the structure
`
`Authors' Affiliation:
`Immunopharmacology and Targeted Therapy Lab-
`oratory, Department of Experimental Therapeutics, MD Anderson Cancer
`Center, Houston, Texas
`
`Note: Supplementary data for this article are available at Molecular Cancer
`Therapeutics Online (http://mct.aacrjournals.org/).
`
`Current address for Y. Cao: Department of Chemistry, The Scripps
`Research Institute, La Jolla, CA 92037.
`
`Corresponding Author: Michael G. Rosenblum, Department of Experi-
`mental Therapeutics, MD Anderson Cancer Center, Houston, TX 77054.
`Phone: 713-792-3554; Fax: 713-794-4261; E-mail:
`mrosenbl@mdanderson.org
`
`doi: 10.1158/1535-7163.MCT-13-0002
`Ó2013 American Association for Cancer Research.
`
`(7, 8), but these molecules may be difficult to humanize
`completely (9).
`A new class of immunotoxins have recently been devel-
`oped containing cytotoxic human proteins (10, 11). Gran-
`zyme B (GrB) is a well-known serine protease generated
`by cytotoxic lymphocytes to induce apoptotic cell death in
`target cells (12, 13). Our group first showed that various
`fusion constructs targeting tumor cells and tumor endo-
`thelium and containing GrB have impressive proapopto-
`tic and cytotoxic activity (14–18). Several other groups
`have since confirmed these findings using other GrB-
`containing constructs (19, 20). Because endogenous GrB
`is present in plasma in both normal and pathologic states,
`it is unlikely that this molecule would engender an
`immune response.
`Dalken and colleagues have described a GrB/FRP5
`fusion construct targeting Her2/neu, which displayed
`selective and rapid tumor cell killing in vitro (21). How-
`ever, studies have shown that
`the fusion construct
`required the presence of the endosome-disrupting agent
`chloroquine for biologic activity and suggested that an
`endosomal release process may be necessary for Her2/
`neu–targeted agents. Studies by Wang and colleagues
`suggested that incorporation of a furin-sensitive linker
`into GrB-based fusion constructs may promote effective
`
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Cao et al.
`
`cytoplasmic delivery of an active GrB fragment into target
`cells (22). However, the recombinant molecule seemed to
`be stable only when generated in situ by protein-expres-
`sing transfected cells.
`We previously examined a series of anti-Her2/neu
`single-chain antibodies (scFv) fused to the recombinant
`gelonin (rGel) toxin, and clearly showed that scFvs with
`11 mol/L) as opposed to high
`intermediate affinity (K
`d 10
`12 mol/L) were optimal carriers of protein
`affinity (K
`d 10
`toxins (23, 24). Therefore, we used an intermediate-affin-
`ity, humanized anti-Her2/neu scFv-designated 4D5 for
`the construction of our GrB-containing fusion constructs.
`In this study, we provided data on the cytotoxicity of
`Her2/neu–targeted GrB fusions against a panel of human
`tumor cell lines and explored the mechanism of in vitro
`activity of these fusion constructs. Finally, we showed the
`in vivo antitumor efficacy of the functionalized GrB chi-
`meric protein against a human breast xenograft model.
`
`Materials and Methods
`Cell lines
`The cell lines BT474 M1, NCI-N87, Calu3, MDA MB435,
`and Me180 were all obtained from American Type Cul-
`ture Collection (ATCC). The human breast cancer cell
`lines MDA MB453 and eB-1 were generously supplied
`by Drs. Zhen Fan and Dihua Yu (MD Anderson Cancer
`Center, Houston, TX). The BT474 M1 Herceptin- and
`lapatinib-resistant cells were derived from BT474 M1 cells
`after a 12-month selection in the continuous presence of 1
`mmol/L Herceptin or 1.5 mmol/L lapatinib. BT474 M1
`MDR-1 cells were generated by the transfection of plas-
`mid pHaMDR1 to parental BT474 M1 cells. The HEK 293T
`cell line was supplied by Dr. Bryant G. Darnay (MD
`Anderson Cancer Center). All cell lines were maintained
`in Dulbecco’s Modified Eagle Medium or RPMI-1640
`medium supplemented with 10% heat-inactivated FBS,
`2 mmol/L L-glutamine, and 1 mmol/L antibiotics.
`Cell lines were validated by short tandem repeat
`(STR) DNA fingerprinting using the AmpF‘STR Identi-
`filer Kit according to the manufacturer’s instructions
`(Applied Biosystems). The STR profiles were compared
`with known ATCC fingerprints (ATCC.org),
`to the
`Cell Line Integrated Molecular Authentication database
`(CLIMA) version 0.1.200808 (http://bioinformatics.
`istge.it/clima/; Nucleic Acids Research 37:D925-D932
`PMCID: PMC2686526) and to the MD Anderson finger-
`print database. The STR profiles matched known DNA
`fingerprints or were unique.
`
`Plasmid construction
`The GrB/4D5/26, GrB/4D5, GrB/26, and GrB DNA
`constructs were generated by an overlapping PCR meth-
`od. Illustrations of the constructs are shown in Fig. 1A. We
`designed a universal 218 linker (GSTSGSGKPGSGEG-
`STKG) incorporated between the individual components
`of GrB, 4D5, or peptide 26. Peptide 26 (AALEALAEA-
`LEALAEALEALAEAAAA) was generated from the 29-
`residue amphipathic peptide without the 3 C-terminal
`
`A
`
`GrB/4D5
`
`GrB
`
`GrB/4D5/26
`
`GrB
`
`26
`
`A.A.
`502
`546
`
`M.W.
`53932.4
`57900.6
`
`P.I.
`9.56
`9.23
`
`GrB/4D5
`GrB/4D5/26
`
`B
`
`kDa
`150
`100
`75
`
`50
`
`35
`
`25
`
`Nonreduced
`
`Reduced
`
`Figure 1. Construction and preparation of GrB-based fusion constructs.
`A, schematic diagram of GrB fusion constructs containing scFv 4D5 and
`GrB without or with fusogenic peptide 26. B, purified GrB-based chimeric
`proteins were analyzed by SDS–PAGE under nonreducing conditions.
`
`amino acids, which are responsible for dimerization (25).
`All construct genes were cloned into the mammalian cell
`expression vector pSecTag (Life Technologies).
`
`Expression, purification, and activation of GrB-based
`proteins
`The GrB-based proteins were expressed in HEK
`293T host cells and purified by immobilized metal
`affinity chromatography as described in Supplementary
`Methods.
`
`Determination of K
`d by ELISA
`The K
`d value and specificity of GrB-based protein
`samples were evaluated by ELISA on Her2/neu extracel-
`lular domain (ECD), Her2/neu-positive BT474 M1 cells,
`and Her2/neu–negative Me180 cells. Rabbit anti-c-myc
`antibody and horseradish peroxidase–conjugated goat
`anti-rabbit immunoglobulin G were used as tracers in
`this assay, as described previously (24).
`
`GrB activity assays
`The enzymatic activity of the GrB component was
`determined in a continuous colorimetric assay using
`N-a-t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thio-
`benzylester (BAADT) as a specific substrate (18). Assays
`consisted of commercial human GrB (Enzyme Systems
`
`980
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`Mol Cancer Ther; 12(6) June 2013
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Products) or GrB-based fusion proteins in BAADT at
`
`C. The change in absorbance at 405 nm was measured
`25
`on a Thermomax plate reader. Increases in sample absor-
`bance were converted to enzymatic rates by using an
`1 (mol/L)
`1 at 405 nm.
`extinction coefficient of 13,100 cm
`The specific activity of GrB-based fusion proteins was
`calculated using native GrB as the standard.
`
`Internalization analysis
`Immunofluorescence-based internalization studies
`were conducted using BT474 M1 and Me180 cells. Cells
`were treated with 25 nmol/L GrB/4D5/26 for 4 hours
`and subjected to immunofluorescent staining with anti-
`GrB antibody [fluorescein isothiocyanate (FITC)-conju-
`gated secondary antibody]. Nuclei were counterstained
`with propidium iodide (PI). Visualization of immuno-
`fluorescence was conducted with a Zeiss LSM510 con-
`focal laser scanning microscope Zeiss LSM510 (Carl
`Zeiss).
`
`In vitro cytotoxicity assays
`Log-phase cells were seeded (5  103/well) in 96-well
`plates and allowed to attach overnight. Cells were further
`incubated with various concentrations of GrB-based
`
`fusion proteins, GrB, or medium at 37
`C for 72 hours.
`Cell viability was determined using the crystal violet
`staining method as described previously (23).
`
`Annexin V/PI staining
`The Annexin V/PI staining assay was used to quanti-
`tatively determine the percentage of cells undergoing
`apoptosis after exposure to GrB/4D5/26. Cells were seed-
`ed onto 6-well plates (5  105 cells/ well) and incubated
`
`with 100 nmol/L GrB/4D5/26 at 37
`C for 24 or 48 hours.
`Aliquots of cells were washed with PBS and then incu-
`bated with Annexin V–FITC antibody. PI solution was
`added at the end of the incubation, and the cells were
`analyzed immediately by flow cytometry.
`
`Cytochrome c release assay and Bax translocation
`After treatment with GrB/4D5 or GrB/4D5/26, cells
`were collected and resuspended with 0.5 mL of 1
`cytosol extraction buffer mix (BioVision) and then
`homogenized in an ice-cold glass homogenizer. The
`homogenate was centrifuged, and the supernatant was
`collected and labeled as the cytosolic fraction. The
`pellet was resuspended in 0.1 mL of mitochondrial
`extraction buffer and saved as the mitochondrial
`fraction. Aliquots of each cytosolic and mitochondrial
`fraction were analyzed by Western blotting with anti-
`bodies recognizing cytochrome c and Bax (Santa Cruz
`Biotechnology).
`
`Assays for caspase activation and apoptosis
`Western blot analysis was used to identify activation of
`caspases-3 and -9 as well as PARP cleavage. In addition,
`apoptosis was analyzed using antibodies recognizing Bcl-
`2 and BID (Santa Cruz Biotechnology).
`
`Novel Functionalized GrB-Targeting Her2/neu
`
`Impact on cell signaling pathways
`After treatment, cell lysates were analyzed by Western
`blotting with antibodies recognizing Her2/neu and phos-
`phorylated (p)-mTOR (S2448; Cell Signaling Technology)
`as well as p-Her2/neu (Tyr877), p-Her2/neu (Tyr 1221/
`1222), EGF receptor, p-EGF receptor (Thr845), Her3, p-
`Her3 (Tyr1328), IGF1 receptor, p-IGF1 receptor (Tyr 1165/
`1166), estrogen receptor (ER), progesterone receptor (PR),
`Akt, p-Akt, extracellular signal–regulated kinase (ERK),
`p-ERK (Thr 177/Thr 160), PTEN, proteinase inhibitor 9
`(PI-9), and b-actin (all from Santa Cruz Biotechonology).
`Immunoreactive proteins were visualized by enhanced
`chemiluminescence.
`
`In vivo efficacy studies
`We used Balb/c nude mice to evaluate the in vivo effect
`of GrB/4D5/26 against aggressive breast cancer. Each
`mouse received a weekly subcutaneous injection of 3
`mg/kg estradiol cypoinate (26) starting 2 weeks before
`the injection of 1  107 BT474 M1 cells into the right flank.
`On the third day after cell inoculation, mice were injected
`intravenously (tail vein) either with saline or GrB/4D5/26
`(44 mg/kg) 5 times per week for 2 weeks. Animals were
`monitored and tumors were measured (calipers) for an
`additional 50 days.
`
`Immunofluorescence analysis
`Twenty-four hours after the final injection of saline or
`GrB/4D5/26, mice were sacrificed and tumor samples
`were frozen immediately in preparation for section
`slides. The sample slides were incubated with either
`anti-GrB antibody (FITC-conjugated secondary anti-
`body) or a terminal deoxynucleotidyl transferase–medi-
`ated dUTP nick end labeling (TUNEL) reaction mixture,
`as well as with an anti-mouse CD31 antibody (phyco-
`erythrin-conjugated secondary antibody), and were fur-
`ther subjected to nuclear counterstaining with Hoechst
`33342. Immunofluorescence observation was conducted
`under a Zeiss Axioplan 2 imaging microscope (Carl
`Zeiss).
`
`Results
`Construction, expression, and purification of GrB-
`based fusions
`The sequence of the humanized anti-Her2/neu scFv
`4D5 was derived from the published Herceptin light-
`and heavy-chain variable domain sequences (27). Pre-
`vious observations suggested that use of fusogenic
`peptides facilitates endosomal escape and delivery of
`large molecules into the cytotol (28, 29). We therefore
`incorporated the fusogenic peptide 26 (Fig. 1A). GrB-
`based fusions were generated by fusing GrB to 4D5
`with (designated GrB/4D5/26) or without (designated
`GrB/4D5) the addition of pH-sensitive fusogenic pep-
`tide 26 (AALEALAEALEALAEALEALAEAAAA)
`to
`the C-terminal of the construct. Furthermore, GrB and
`GrB/26 were used as controls. All fusion proteins were
`expressed in human embryonic kidney cells (HEK
`
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Cao et al.
`
`293T). Following purification, the final products migrat-
`ed at the expected molecular weights, with a purity of
`more than 95% (Fig. 1B).
`
`Analysis of binding affinity
`The binding affinities (K
`d values) of GrB/4D5/26 and
`GrB/4D5 were assessed by ELISA using purified Her2/
`neu ECD, Her2/neu–positive BT474 M1 human breast
`cancer cells, and Her2/neu–negative Me180 human cer-
`vical cancer cells. Both fusions specifically bound to Her2/
`neu ECD and BT474 M1 cells but not to Me180 cells (Fig.
`2A). The apparent K
`d values were determined by calcu-
`lating the concentration of fusion constructs that pro-
`duced half-maximal specific binding. GrB/4D5 and
`GrB/4D5/26 showed apparent K
`d values of 0.329 and
`0.469 nmol/L, respectively, to Her2/neu ECD and 0.383
`and 0.655 nmol/L, respectively, to BT474 M1 cells. These
`results are in general agreement with the published K
`d
`value for native Herceptin to the Her2/neu receptor (0.15
`nmol/L; ref. 27).
`
`Enzymatic assay of GrB-based fusions
`To assess the biologic activity of the GrB component of
`the fusions, we compared the ability of the constructs to
`cleave the substrate BAADT with that of native, authentic
`GrB (Fig. 2B). GrB/4D5 and GrB/4D5/26 had intact GrB
`enzymatic activity (1.54  105 and 1.57  105 U/mmoL,
`respectively). These activities were comparable with that
`of the native GrB standard (1.19  105 U/mmoL). Because
`the pro-GrB fusion constructs contain purification tags on
`the N-terminal end of GrB and render the molecule
`enzymatically inactive, these proteins were unable to
`cause hydrolysis of BAADT.
`
`Cellular uptake and GrB delivery of fusion constructs
`Immunofluorescence staining was conducted with
`BT474 M1 and Me180 cells. The GrB moiety of both fusions
`was observed primarily in the cytosol after treatment with
`a fusion protein in BT474 M1 cells but not in Me180 cells
`(Fig. 2C), showing that both constructs were efficient in
`cell binding and internalization after exposure to Her2/
`neu–positive cells. The internalization efficiency of the
`
`ELISA of GrB-based fusions on Me180 cells
`0.8
`
`GrB/4D5
`GrB/4D5/26
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`100
`10–3 10–2 10–1
`Conc. (nmol/L)
`
`101
`
`102
`
`Absorbance at 405 nm
`
`GrB/4D5
`GrB/4D5/26
`Control
`
`ELISA of GrB-based fusions on Her2/neu ECD
`1.8
`1.5
`1.2
`0.9
`0.6
`0.3
`0.0
`100
`10–3 10–2 10–1
`Conc. (nmol/L)
`
`101
`
`102
`
`Absorbance at 405 nm
`
`A
`
`Absorbance at 405 nm
`
`ELISA of GrB-based fusions on Her2/neu ECD
`1.8
`1.5
`1.2
`0.9
`0.6
`0.3
`0.0
`100
`10–3 10–2 10–1
`Conc. (nmol/L)
`
`GrB/4D5
`GrB/4D5/26
`Control
`
`101
`
`102
`
`C
`
`PBS
`
`GrB/4D5
`
`GrB/4D5/26
`
`GrB standard
`Pro-GrB/4D5
`GrB/4D5
`Pro-GrB/4D5/26
`GrB/4D5/26
`
`BT474 M1
`
`Me 180
`
`0
`
`10
`
`20
`
`40
`30
`Time (min)
`
`50
`
`60
`
`70
`
`D
`
`GrB/4D5
`
`GrB/4D5/26
`
`0 0.5 1 2 4 8 12 24 36 48 h
`
`0 0.5 1 2 4 8 12 24 36 48 h
`
`Full-length GrB fusion
`
`Free GrB
`β-Actin
`
`0.20
`
`0.15
`
`0.10
`
`0.05
`
`0.00
`
`B
`
`Absorbance at 405 nm
`
`Figure 2. Characterization and comparison of GrB-based fusion proteins. A, Kd of the fusion constructs to Her2/neu ECD, Her2/neu–positive BT474 M1 cells,
`and Her2/neu–negative Me180 cells by ELISA. B, enzymatic activity of GrB moiety of fusion proteins compared with native GrB. C, internalization
`analysis of BT474 M1 cells and Me180 cells after 4 hours of treatment with 25 nmol/L functionalized GrB fusions. Cells were subjected to immunofluorescent
`staining with anti-GrB antibody (FITC-conjugated secondary), with PI nuclear counterstaining. D, Western blot analysis of intracellular behavior of
`25 nmol/L GrB fusion constructs in BT474 M1 cells.
`
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Novel Functionalized GrB-Targeting Her2/neu
`
`Table 1. Comparative IC50 values of GrB-based fusion constructs against various types of tumor cell lines
`IC50 (nmol/L)
`
`PI-9
`level
`þ
`þþþþþ
`þ
`þ
`
`Her2/neu
`GrB/4D5/26
`level
`Cell line
`Type
`þþþþ
`29.3
`BT474 M1
`Breast
`þþþþ
`40.5
`Calu3
`Lung
`þþþþ
`90.4
`NCI-N87
`Gastric
`þþþ
`56.8
`MDA MB453
`Breast
`þþ
`93.1
`eB-1
`Breast
`þ
`>500.0
`MDA MB435
`Breast
`þ
`þ
`>500.0
`Me180
`Cervical
`NOTE: þ, indicates the Her2/neu expression level in different cancer cells.
`
`—
`
`—
`
`GrB/4D5
`253.3
`242.4
`629.0
`436.0
`551.3
`>750.0
`>750.0
`
`GrB/26
`905.5
`863.0
`1,106.0
`694.2
`1,134.5
`1,031
`>1,500.0
`
`GrB
`>1,500.0
`>1,500.0
`>1,500.0
`>1,500.0
`>1,500.0
`>1,500.0
`>1,500.0
`
`fusions was further examined by time-dependent West-
`ern blot analysis of the GrB signal (full-length GrB fusion
`þ free GrB; Fig. 2D). Both constructs internalized rapidly
`into BT474 M1 cells within 30 minutes. Compared with
`GrB/4D5, GrB/4D5/26 displayed enhanced and long-
`lasting cell internalization. The intracellular delivery of
`GrB after endocytosis of GrB/4D5 or GrB/4D5/26 also
`was assessed by time-dependent Western blotting (free
`GrB). We observed no GrB delivery by GrB/4D5 up to 48
`hours of treatment, whereas GrB delivery by GrB/4D5/26
`was observed starting at approximately 4 hours of treat-
`ment and presented a tremendously high level of free GrB
`up to 48 hours (Fig. 2D).
`
`In vitro cytotoxic effects of GrB-based fusions
`GrB-based fusions were then tested against a number of
`tumor cell lines. After 72 hours exposure, GrB/4D5/26
`showed specific cytotoxicity to Her2/neu–positive cells,
`with IC50 values of less than 100 nmol/L (Table 1), and
`GrB/4D5 showed cytotoxic effects at somewhat higher
`doses (>200 nmol/L). In addition, GrB/26 showed min-
`imal cytotoxicity at doses more than 600 nmol/L, but no
`significant activity of GrB itself was observed at doses up
`to 1.5 mmol/L. When Her2/neu–positive MDA MB453
`cells were pretreated with Herceptin (5 mmol/L) for 6
`hours and then treated with GrB/4D5/26 for 72 hours, the
`cytotoxicity of GrB/4D5/26 was reduced (Supplementary
`
`Fig. S1), thereby showing a requirement for antigen bind-
`ing of the GrB/4D5/26 construct.
`We further investigated the expression levels of the
`endogenous PI-9 in different tumor cells (Supplementary
`Fig. S2 and Table 1). These studies failed to find an
`association between the response of cells to the cytotox-
`icity of the GrB constructs and the endogenous expression
`of PI-9. This may suggest that factors other than PI-9 may
`account for the observed differences in GrB/4D5/26 cyto-
`toxicity to Her2/neu–expressing target cells.
`
`Cytotoxic effects of GrB/4D5/26 against cells
`resistant to Herceptin or lapatinib
`Acquired resistance to Herceptin or lapatinib can be
`mediated by concomitant upregulation of Her2/neu
`downstream signaling pathways or activation of signaling
`through the ER pathway (30). In this study, we developed
`a model of Herceptin- and lapatinib-resistant variants of
`BT474 M1 cells. Parental BT474 M1 cells were readily
`sensitive to both Herceptin (IC50, 52.5 nmol/L) and lapa-
`tinib (IC50, 34.7 nmol/L; Table 2). Herceptin-resistant cells
`showed resistance to Herceptin [IC50, 10.1 mmol/L; fold
`resistance (FR), 192] but remained sensitive to lapatinib
`(IC50, 32.4 nmol/L). Lapatinib-resistant cells showed
`resistance to high micromolar concentrations of both
`Herceptin (IC50, 74.1 mmol/L; FR, 1411) and lapatinib
`(IC50, 8.2 mmol/L; FR, 237). As shown in Table 2, cells
`
`Table 2. Cytotoxic effects of Her2/neu–targeted therapeutic agents on IC50 values in BT474 M1 cells and
`resistant variants
`
`IC50 (nmol/L) with (FR)a
`
`Agent
`Herceptin
`Lapatinib
`GrB/4D5/26
`
`BT474 M1
`52.5 (1)
`34.7 (1)
`32.9 (1)
`
`BT474 M1
`Herceptin-
`resistant
`10,100.5 (192)
`32.4 (1)
`26.8 (1)
`
`BT474 M1
`lapatinib-
`resistant
`74,100.0 (1,411)
`8,225.0 (237)
`66.1 (2)
`
`BT474 M1 þ
`EGFb
`26,305.0 (501)
`543.0 (16)
`21.7 (1)
`
`BT474 M1 þ
`NRG-1c
`23,033.0 (439)
`547.1 (16)
`18.1 (1)
`
`BT474 M1 þ
`b-estradiold
`74.1 (1)
`33.9 (1)
`31.3 (1)
`
`aFR represents IC50 of agent on BT474 M1–resistant variants/that on BT474 M1 parental cells.
`b,c,dCells were pretreated with b20 ng/mL EGF, c50 ng/mL NRG-1, or d10 ng/mL b-estradiol for 72 hours before drug treatment.
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`Cao et al.
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`resistant to Herceptin showed equivalent sensitivity to the
`
`GrB/4D5/26 construct (IC50, 30 nmol/L for both Her-
`
`ceptin-resistant and parental BT474 M1 cells, respective-
`ly). For lapatinib-resistant cells, the IC50 was marginally
`increased (2-fold) compared with parental cells (66.1 vs.
`32.9 nmol/L).
`We also showed that addition of EGF or neuregulin-1
`(NRG-1) growth factor, but not b-estradiol, to BT474
`M1 parental cells can circumvent the cellular cytotoxic
`responses to Herceptin and lapatinib. Seventy-two hours
`of pretreatment of BT474 M1 cells with 20 ng/mL EGF or
`50 ng/mL NRG-1 resulted in a 400- to 500-fold increase in
`resistance to Herceptin and a 16-fold increase in resistance
`to lapatinib (Table 2). However, treatment of these resis-
`tant cells resulted in no cross-resistance to GrB/4D5/26
`fusions compared with parental BT474 M1 cells.
`A significant observation was that incubation of cells
`with GrB/4D5/26 in the presence of chloroquine did
`not improve cytotoxicity toward these cells (Supple-
`mentary Fig. S3). This finding showed that the fuso-
`
`genic peptide 26 efficiently releases GrB fusion pro-
`teins from intracellular vesicles,
`thereby allowing
`access to cytosolic GrB substrates and induction of
`apoptosis.
`
`Mechanistic studies of GrB/4D5/26 cytotoxicity
`We conducted a panel of experiments to assess the
`potential of GrB-based fusions to initiate the proteolytic
`cascade culminating in apoptosis of BT474 M1 parental,
`Herceptin-, and lapatinib-resistant cells.
`Annexin V/PI staining. Cells were incubated for 24 or
`48 hours with 100 nmol/L GrB/4D5/26 and apoptosis
`was detected via Annexin V/PI staining. GrB/4D5/26
`induced apoptosis in BT474 M1 parental, Herceptin- and
`lapatinib-resistant cells, as indicated by the reduced viable
`population combined with greater populations of early
`apoptosis (Fig. 3A). No apoptosis was induced by 100
`nmol/L GrB/4D5 in any of these cells (Supplementary
`Fig. S4). Her2/neu–negative Me180 cells were not affected
`by either construct.
`
`Figure 3. Effects of GrB-based fusions on apoptotic pathways of BT474 M1 parental, Herceptin-, and lapatinib-resistant cells. A, detection of apoptosis of
`GrB/4D5/26 by Annexin V/PI staining assay. Me180 cells served as a Her2/neu–negative control group. B, Western blot analysis of cleavage and
`activation of caspases-3 and -9 as well as PARP by GrB-based fusion constructs. C, Western blot analysis of apoptosis kinetics and specificity of GrB/4D5/26.
`Cells were treated with GrB/4D5/26 for up to 24 hours with or without 100 mmol/L zVAD-fmk for 24 hours in parental or Herceptin-resistant cells and for up to
`48 hours in lapatinib-resistant cells.
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
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`Novel Functionalized GrB-Targeting Her2/neu
`
`Activation of caspases. Caspase activation in BT474
`M1 cells was detected by Western blot analysis. Treatment
`with GrB/4D5/26 resulted to the cleavage of caspase-3,
`9, and PARP in all cells, but no activation occurred when
`cells were treated with GrB/4D5 (Fig. 3B). Compared with
`BT474 M1 parental and Herceptin-resistant cells, the acti-
`vations of caspase-9, -3, and PARP were delayed in lapa-
`tinib-resistant cells, which coincided with the observed
`decreased cytotoxic effects.
`We further assessed the kinetics of PARP cleavage
`induced by GrB/4D5/26 on BT474 M1 parental, Her-
`ceptin- and lapatinib-resistant cells, and found that
`cleavage occurred after 2 hours of drug exposure for
`parental and Herceptin-resistant cells but at 24 hours for
`lapatinib-resistant cells (Fig. 3C). In addition, in the
`presence of the pan-caspase inhibitor zVAD-fmk, PARP
`
`cleavage of GrB/4D5/26 was partially inhibited in all
`cells. This finding is in agreement with a mechanism
`relying on GrB activity for caspase-3 cleavage followed
`by PARP cleavage.
`Impact on mitochondrial pathways. We detected cell
`death induced by GrB/4D5/26 via several mitochondrial-
`related pathways. In BT474 M1 parental, Herceptin- and
`lapatinib-resistant cells, GrB/4D5/26 treatment activated
`BID and downregulated the antiapoptotic Bcl-2 protein
`(Fig. 4A), and it triggered the release of cytochrome c from
`the mitochondria into the cytosol (Fig. 4B). Bax was
`normally present in both the cytosol and mitochondria
`of untreated cells. However, when the cells were treated
`with GrB/4D5/26, Bax was decreased in cytosol and
`increased in mitochondria (Fig. 4B). As previously
`described, treatment for 24 hours with GrB/4D5/26 was
`
`Figure 4. Effects of GrB fusion
`constructs on mitochondrial
`pathway in BT474 M1 parental,
`Herceptin-, and lapatinib-resistant
`cells. A, effects of GrB-based fusion
`proteins on the upstream
`components Bcl-2 and BID in
`mitochondrial pathway. B, effects of
`the fusion constructs on cytochrome
`c release and Bax translocation.
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`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
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`Cao et al.
`
`Figure 5. Western blot analyses of
`the effects of GrB/4D5 and GrB/
`4D5/26 in BT474 M1 parental,
`Herceptin-, and lapatinib-resistant
`cells on Her2/neu and ER signaling
`pathways. Cells were treated with
`100 nmol/L functionalized GrB
`constructs for 24 or 48 hours, and
`total cell lysates were quantified
`and further measured by Western
`blot analysis of pHer2/neu, pAkt,
`pmTOR, pERK, ER, PR, and PI-9
`level.
`
`shown to activate the mitochondrial pathway in both
`BT474 M1 parental and Herceptin-resistant cells, but this
`activation was delayed in lapatinib-resistant cells.
`
`Effects of GrB fusions on Her- and ER-associated
`signaling pathways
`We next examined the mechanistic effects of the con-
`structs on Her- and ER-related signaling events in BT474
`M1 parental cells and the resistant variants. As shown in
`Supplementary Fig. S5, cells resistant to Herceptin had
`enhanced Her family receptor activity but reduced levels
`of PR and PI-9. In contrast, in lapatinib-resistant cells there
`was total downregulation of Her family receptor activity
`but higher levels of ER, PR, and PI-9.
`Cells treated with GrB/4D5 or GrB/4D5/26 showed the
`effects on these signaling pathways, corresponding to the
`cytotoxic results we observed (Fig. 5). Treatment with
`GrB/4D5/26 remarkedly inhibited phosphorylation of
`Her2/neu and its downstream molecules Akt, mTOR,
`and ERK, which are critical events in Her2/neu signaling
`cascade. In contrast, GrB/4D5 showed a comparatively
`reduced effect on these pathways. We observed a reduced
`ER level among all cells. Evidence from other researchers
`
`has shown that upregulation of the ER pathway in ER-
`and Her2/neu–positive cell lines with lapatinib creates
`an escape/survival pathway (30, 31), but GrB/4D5/26
`seem to be able to inactivate all the signaling pathways
`in these cells. We also observed the delaying signaling
`effects of GrB/4D5/26 on lapatinib-resistant cells com-
`pared with parental or Herceptin-resistant cells, which
`was in agreement with the apoptotic cell death results
`observed for the lapatinib-resistant cells. Notably, there
`was an increased mRNA and protein level of PI-9 in this
`resistant line but not in the parental or Herceptin-resis-
`tant cells (Supplementary Figs. S5 and S6). Taken
`together, these results suggest that activation of the ER
`pathway upregulates the expression of PI-9 and this
`results in a slight inhibition of GrB/4D5/26 activity and
`a delay in apoptotic cell death compared with parental
`cells.
`Our investigation suggests that the GrB/4D5/26 fusion
`is more cytotoxic than GrB/4D5 construct to Her2/
`neu–positive cells, even those that have acquired resis-
`tance to the traditional Her2/neu therapeutic agents Her-
`ceptin and lapatinib. The cytotoxicity results coincide
`with the observed effects on signal transduction and
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`

`

`Published OnlineFirst March 14, 2013; DOI: 10.1158/1535-7163.MCT-13-0002
`
`Table 3. Cytotoxicity of chemical agents and
`GrB-based fusions on MDR-1–expressing cells
`
`IC50 (nmol/L)
`
`BT474
`M1
`5.2
`1.3
`311.8
`34.1
`
`BT474 M1
`MDR-1
`1,047.3
`105.1
`318.9
`35.5
`
`FRa
`209
`89
`1
`1
`
`Taxol
`Vinblastin
`GrB/4D5
`GrB/4D5/26
`
`aFR represents IC50 of agent on BT474 M1 MDR-1 cells/that
`on BT474 M1 parental cells.
`
`monitoring these pathways may be useful as a monitor of
`drug efficacy.
`
`Effects of GrB/4D5/26 on the MDR-1–expressing
`cells
`Multidrug resistance (MDR) is a phenomenon, which
`results from various reasons. The most-characterized
`cause of MDR is the overexpression of a 170-kDa mem-
`brane glycoprotein known as P-glycoprotein (Pgp). To
`verify the effects of GrB-based fusi