Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Construction and Characterization of Novel, Recombinant
` Oncogene Product:
`Her2/neu
`Immunotoxins Targeting the
` Studies
`In vivo
` and
`vitro
` 
`Yu Cao, James D. Marks, John W. Marks, et al.
`Cancer Res  
`
`2009;69:8987-8995. Published OnlineFirst November 24, 2009.
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`IMMUNOGEN2111, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
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`

`

`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
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`Experimental Therapeutics, Molecular Targets, and Chemical Biology
`
`Construction and Characterization of Novel, Recombinant
`Immunotoxins Targeting the Her2/neu Oncogene Product:
`In vitro and In vivo Studies
`
`Yu Cao,1 James D. Marks,2 John W. Marks,1 Lawrence H. Cheung,1 Sehoon Kim,1
`and Michael G. Rosenblum1
`
`1Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics, M.D. Anderson Cancer Center,
`Houston, Texas and 2Department of Anesthesia, University of California, San Francisco, San Francisco, California
`
`Abstract
`The goal of this study was to characterize a series of anti-
`Her2/neu immunotoxin constructs to identify how different
`antibodies and linker choices affect the specificity and cyto-
`toxicity of these proteins. We constructed a series of immuno-
`toxins containing either the human single-chain antibody
`(scFv) C6.5 or the murine scFv e23 fused to the highly toxic
`recombinant gelonin (rGel) molecule. Based on the flexible
`GGGGS linker (L), the fusion construct C6.5-L-rGel was com-
`pared with e23-L-rGel to evaluate the specific cytotoxic effects
`against Her2/neu-positive and Her2/neu-negative tumor cells.
`Both constructs retained the specificity of the original anti-
`body as well as the biological activity of rGel toxin. The two
`constructs displayed similar cytotoxicity against different car-
`cinoma cells. We additionally introduced the modified linkers
`TRHRQPRGWEQL (Fpe) and AGNRVRRSVG (Fdt), which
`contained furin cleavage sites, to determine the effect of these
`design changes on stability and cell killing efficiency. The in-
`troduction of furin cleavage linkers (Fpe or Fdt) into the mo-
`lecules resulted in dissimilar sensitivity to protease cleavage
`compared with the constructs containing the L linker, but
`very similar intracellular rGel release, cytotoxic kinetics,
`and induction of autophagic cell death in vitro. Xenograft
`studies with SKOV3 ovarian tumors were done using various
`C6.5/rGel constructs. C6.5-L-rGel was more efficient in tumor
`inhibition than constructs containing furin linkers, attribut-
`ing to a higher stability in vivo of the L version. Therefore,
`our studies suggest that human C6.5-L-rGel may be an effec-
`tive novel clinical agent for therapy of patients with Her2/neu-
`overexpressing malignancies. [Cancer Res 2009;69(23):8987–95]
`
`Introduction
`The Her2/neu proto-oncogene encodes a 185-kDa transmem-
`brane glycoprotein kinase with extensive homology to the epider-
`mal growth factor receptor (HER1; refs. 1–3). Amplification of
`the gene and overexpression of the Her2/neu protein product in
`tumor cells have been well described in numerous human cancers,
`
`Note: Supplementary data for this article are available at Cancer Research Online
`(http://cancerres.aacrjournals.org/).
`Current address for S. Kim: Merck & Co., Inc., 21 Lafayette, Lebanon, NH 03766.
`Requests for reprints: Michael G. Rosenblum, M.D. Anderson Cancer Center, Box
`0044, 1515 Holcombe Boulevard, Houston, TX 77030. Phone: 713-792-3554; Fax:
`713-794-4261; E-mail: mrosenbl@mdanderson.org.
`©2009 American Association for Cancer Research.
`doi:10.1158/0008-5472.CAN-09-2693
`
`including mammary and ovarian carcinomas and gastric and lung
`tumors (4–6). Because Her2/neu plays a central role in malignant
`transformation and growth, it provides an attractive target for
`focused therapeutic approaches.
`A number of approved immunotherapeutic agents are directed
`at tumors that express high levels of Her2/neu, such as the mono-
`clonal antibody trastuzumab (Herceptin), and small-molecule inhi-
`bitors such as gefitinib (Iressa) have shown promising results, but
`the development of resistance to treatment remains a well-known
`problem (7, 8). To enhance its clinical potential, cell-surface Her2/
`neu has been targeted using antibody-drug conjugates (9) or im-
`munotoxins, composed of plant or bacterial toxins linked with a
`targeting molecule composed of monoclonal antibodies or anti-
`body fragments (10, 11). Previously, a recombinant, murine anti-
`Her2/neu single-chain antibody (scFv), designated e23, has been
`fused to catalytic toxins such as Pseudomonas exotoxin A and diph-
`theria toxin (DT) to specifically target Her2/neu-expressing cells
`(12, 13). A major drawback of such proteins is their potential for
`immune response after repeated administration. Further complica-
`tions could result from nonspecific binding of foreign proteins to
`vascular endothelial cells, leading to vascular leak syndrome and,
`ultimately, interstitial edema and organ failure (14, 15).
`The development of immunotoxins containing human or hu-
`manized components may circumvent these problems. Such im-
`munotoxins may display reduced immunogenicity although
`antibodies to the toxin components may still limit prolonged ther-
`apy (16). We previously reported in vitro characterization and
`in vivo antitumor efficacy studies of an immunotoxin composed
`of the human chimeric anti-Her2/neu antibody (BACH-250) chem-
`ically conjugated to recombinant gelonin (rGel; ref. 17). rGel is a
`29-kDa ribosome-inactivating plant toxin with a potency and
`mechanism of action similar to that of ricin toxin A-chain, but with
`improved stability and reduced toxicity (18, 19). The BACH-250/
`rGel conjugate showed potent and specific cytotoxicity against
`Her2/neu-overexpressing human tumor cells in culture and against
`SKOV3 tumor xenografts. However, the treatment of solid tumors
`presents a potential problem because full-length antibodies must
`diffuse into the tumor against a hydrostatic pressure gradient and
`into disordered vasculature (20, 21).
`Marks and colleagues previously described an anti-Her2/neu
`scFv, designated C6.5, which was selected from a human scFv
`phage display library and affinity-matured in vitro (22). Using scFv
`C6.5, McCall and colleagues constructed and characterized a bis-
`pecific scFv composed of C6.5 and anti-CD16 scFv, displaying a
`high level of in vitro tumor cell cytotoxicity and in vivo tumor tar-
`geting (23). Studies by Park and colleagues generated immunolipo-
`somes containing doxorubicin, which were targeted to tumor cells
`
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`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Cancer Research
`
`using antibody C6.5. These constructs showed selective enhance-
`ment of the therapeutic index of doxorubicin chemotherapy (24).
`Most recently, Adams and colleagues successfully used a C6.5 dia-
`body construct as a radioimmunotherapeutic agent containing
`(211At) for the treatment of Her2/neu-positive solid tumors in xe-
`nograft models, showing that scFv C6.5 could be used effectively in
`vehicles for targeted radioimmunotherapy by using powerful,
`short-lived α-emitting radioisotopes (25).
`In the present study, we describe the construction and charac-
`terization of several rGel-based chimeric toxins composed of the
`scFv e23 or C6.5 and using various linker configurations to exam-
`ine how different antibodies and linker choices affect the in vitro
`and in vivo efficacy of fusion constructs.
`
`Materials and Methods
`Plasmid construction. The gene encoding murine scFv e23 was ob-
`tained from Oncologix, Inc., and human scFv C6.5 was kindly supplied by
`Prof. James D. Marks (University of California, San Francisco, San Francisco,
`CA). Illustrations of the immunotoxin constructs are shown in Fig. 1A.
`Recombinant immunotoxins containing rGel and either e23 or C6.5 and
`the flexible linker (GGGGS, designated “L”) were constructed by overlapping
`PCR. These proteins were designated e23-L-rGel and C6.5-L-rGel, respec-
`tively. In addition, we generated two different linkers (TRHRQPRGWEQL,
`designated “Fpe,” and AGNRVRRSVG, designated “Fdt”) containing furin
`cleavage sites from Pseudomonas exotoxin A and DT, incorporated between
`the C6.5 and rGel components. The intent was that furin cleavage would
`allow more facile release of the toxin from the complex once internalized
`by the endosomal compartment. The resulting proteins were designated
`C6.5-Fpe-rGel and C6.5-Fdt-rGel, respectively. All the fusion genes were
`cloned into a T7 promoter–based E. coli expression vector, pET-32a(+).
`Protein expression in E. coli. To express the recombinant fusion pro-
`teins, bacterial cultures were incubated at 37°C in LB growth medium with
`strong antibiotic selection (400 μg/mL ampicillin, 70 μg/mL chloramphen-
`icol, and 15 μg/mL kanamycin) and grown to log phase (A600 nm = 0.8). The
`target protein expression was induced at 18°C with 0.1 mmol/L isopropyl-L-
`thio-β-D-galactopyranoside for 16 h. Induced bacterial cultures were then
`centrifuged and stored frozen at −80°C.
`Isolation and purification of immunotoxins. Frozen bacterial pellets
`containing different immunotoxins were purified as follows: The pellets
`were allowed to thaw with the addition of 50 mmol/L Na-phosphate (pH
`7.6), 300 mmol/L NaCl, 5 mmol/L imidazole. The bacterial suspension was
`further lysed by passing the material through a microfludizer (Microflui-
`dics). The solution was then clarified by centrifugation (40,000 rpm), and
`the supernatants were loaded onto columns (2.5-cm internal diameter × 13
`cm) containing cobalt-charged IMAC resins. After washing with 50 mmol/L
`Na-phosphate (pH 7.6), 300 mmol/L NaCl, 15 mmol/L imidazole, the bound
`protein was eluted with 50 mmol/L Na-phosphate (pH 7.6), 300 mmol/L
`NaCl, 300 mmol/L imidazole. Fractions containing immunotoxin were
`dialyzed in 20 mmol/L Tris-HCl (pH 7.4) and 150 mmol/L NaCl, followed
`by digestion with recombinant enterokinase. The purified immunotoxins
`were dialyzed in PBS, filter sterilized, and stored at 4°C.
`Binding affinity and internalization analyses. The binding affinity
`and specificity of rGel-based immunotoxins containing either scFv e23
`or C6.5 were evaluated by ELISA on Her2/neu-positive SKOV3 cells and
`Her2/neu-negative MCF7 cells. Rabbit anti-rGel antibody and horseradish
`peroxidase–conjugated goat anti-rabbit IgG was used as a tracer in this
`assay as described previously (26).
`Immunofluorescence-based internalization studies were also done on
`SKOV3 and MCF7 cells (27). Cells treated with immunotoxins or rGel
`were subjected to immunofluorescent staining with anti-rGel antibody
`(FITC-conjugated secondary antibody). Nuclei were counterstained with
`propidium iodine. Visualization of immunofluorescence was done with a
`confocal laser scanning microscope (Zeiss LSM510, Carl Zeiss).
`Reticulocyte lysate in vitro translation assay. The rGel-induced
`inhibition of [3H]leucine incorporation into protein in a cell-free protein
`
`Figure 1. Preparation of e23/rGel and C6.5/rGel series immunotoxins. A,
`schematic diagram of immunotoxin constructs containing scFv (e23 or C6.5),
`peptide linker (L, Fpe, or Fdt), and rGel toxin. B, SDS-PAGE analysis of purified
`immunotoxins.
`
`synthesizing system after the administration of various doses of immuno-
`toxins was done as described previously (28).
`Furin cleavage assay. Various C6.5/rGel fusions containing different
`linkers were treated with recombinant human furin (NEB) at various pH.
`For pH 5.4, 50 mmol/L sodium acetate buffer was used, and for pH 7.2, 10
`mmol/L HEPES buffer was used. A dose of 2 units of purified furin was
`applied to 25 μg of fusion protein in each reaction. After incubation for
`16 h at room temperature, the proteins were analyzed by Western blot with
`anti-rGel antibody. The rate of cleavage by furin was calculated using
`AlphaEaseFC software (version 4.0.1) from the following formula: cleavage
`rate = value of gray scale of cleaved protein / value of gray scale of total
`protein.
`In vitro cytotoxicity assays. Log-phase cells were seeded (∼5 × 103 per
`well) in 96-well plates and allowed to attach overnight. Cells were further
`incubated with various concentrations of immunotoxins, rGel, or medium
`at 37°C for 72 h, and cell viability was determined by using the crystal violet
`staining method followed by solubilization of the dye in Sorensor's buffer as
`described previously (26).
`Intracellular release of rGel. The intracellular rGel release from C6.5/
`rGel fusion constructs was analyzed in SKOV3 cells. After incubation for
`various times, the cells were treated with acidic glycine buffer [500
`mmol/L NaCl and 0.1 mol/L glycine (pH 2.5)] to strip cell-surface–bound
`proteins and lysed in lysis buffer [10 mmol/L Tris-HCl (pH 8.0), 60 mmol/L
`KCl, 1 mmol/L EDTA, 1 mmol/L DTT, 0.2% NP40]. Cytosolic fractions were
`further quantified and analyzed by Western blot using anti-rGel antibody.
`Western blot analysis of apoptosis and autophagy. SKOV3 cells
`treated with 200 nmol/L immunotoxins were pelleted and lysed. Proteins
`
`Cancer Res 2009; 69: (23). December 1, 2009
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`IPR2014-00676
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`

`

`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Novel Immunotoxins Targeting Her2/neu
`
`from each cell lysates were analyzed by Western blot with antibodies
`that recognized poly(ADP-ribose) polymerase (PARP), β-actin, high mo-
`bility group box 1 (HMGB1; Santa Cruz Biotechnology), and microtu-
`bule-associated protein 1 light chain 3 (MAP LC3; Novus Biological).
`For HMGB1 release assay, the medium was harvested, concentrated,
`and further analyzed by Western blot.
`In vivo efficacy studies. BALB/c nude mice were injected s.c. with
`SKOV3 cells (5 × 106 per mouse). Once tumors were measurable (∼40–50
`mm3), animals were treated (i.v. via tail vein) with PBS or rGel as control or
`with different C6.5/rGel fusion proteins, every other day for 10 d. Animals
`were monitored and tumors were measured for an additional 40 d.
`Immunofluorescence analysis. Twenty-four hours after i.v. injection of
`C6.5-L-rGel or rGel, the mice were sacrificed and tumor samples were col-
`lected and frozen immediately for sectioning. The sample slides were fixed
`in 3.7% paraformaldehyde, permeabilized with PBS containing 0.2% Triton
`X-100, and blocked in 5% nonfat milk in PBS. After incubation with anti-
`rGel antibody, the samples were subjected to immunofluorescent staining
`with FITC-conjugated secondary antibody and nuclear counterstaining
`with propidium iodine. The slides were mounted and delivered for the im-
`munofluorescence observation under a Nikon Eclipse TS-100 fluorescence
`microscope (Nikon).
`
`the native rGel NH2 terminus. VH/VL orientations determined the
`best binding activity of VL-VH for e23 and VH-VL for C6.5 (data not
`shown). The C6.5/rGel construct was further engineered by incor-
`porating two different enzymatically sensitive furin cleavage lin-
`kers between the scFv and rGel toxin components. The two furin
`sensitive sequences are designated Fpe (TRHRQPRGWEQL) and
`Fdt (AGNRVRRSVG; Fig. 1A). Several biochemical studies have
`shown that the serine protease furin efficiently cleaves proteins
`containing these recognition sequences (29, 30).
`Following purification, all the rGel-based immunotoxins migrat-
`ed on SDS-PAGE at the expected molecular weight of 55 kDa
`(Fig. 1B). However, with the introduction of sensitive furin linker,
`C6.5-Fdt-rGel, but not C6.5-Fpe-rGel, displayed cleavage bands to
`some extent after rEK digestion. The cleavage was found to be oc-
`curring precisely at the predicted furin cleavage site producing the
`27- to 28-kDa fragments of scFv and rGel. Further analysis indicat-
`ed that the yields for each protein (per liter of bacterial culture)
`were 1.55 mg for e23-L-rGel, 1.05 mg for C6.5-L-rGel, 1.08 mg for
`C6.5-Fpe-rGel, and 0.70 mg for C6.5-Fdt-rGel.
`
`Results
`
`Construction, expression, and purification of rGel-based fu-
`sions. The initial rGel-based immunotoxins consisted of a flexible
`L linker (GGGGS) tethering the COOH terminus of e23 or C6.5 to
`
`Characterization of e23-L-rGel and C6.5-L-rGel
`Immunotoxins
`Binding activity. To ensure that immunotoxins retained anti-
`gen binding ability, the fusion proteins were compared in an
`ELISA-based binding assay (Fig. 2A) using Her2/neu-positive
`
`Figure 2. Characterization and comparison of e23-L-rGel and C6.5-L-rGel immunotoxins. A, evaluation of the binding activity of the fusion constructs to SKOV3
`and MCF7 cells by whole-cell ELISA. B, the enzymatic (N-glycosidase) activity of the rGel component of the fusion was assessed using rabbit reticulocyte lysate assay.
`C, internalization of e23-L-rGel and C6.5-L-rGel into SKOV3 and MCF7 cells. Cells were subjected to immunofluorescent staining with anti-rGel antibody
`(FITC-conjugated secondary) with propidium iodine nuclear counterstaining.
`
`www.aacrjournals.org
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`8989
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`Cancer Res 2009; 69: (23). December 1, 2009
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`cancerres.aacrjournals.org Downloaded from
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`on October 7, 2014. © 2009 American Association for Cancer
`Research.
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`IMMUNOGEN2111, pg. 4
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`IPR2014-00676
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`

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`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Cancer Research
`
`SKOV3 and Her2/neu-negative MCF7 cells. The equilibrium disso-
`ciation constant, Kd, was further calculated (GraphPad Prism,
`v4.03). The affinity of e23-L-rGel (Kd = 8.5 nmol/L) for SKOV3 cells
`was similar to that of C6.5-L-rGel (Kd = 12.6 nmol/L). The Kd values
`were consistent with those previously measured in an in vitro live
`cell assay using scFv itself (22). In addition, both immunotoxins
`showed significant specificity based on the background of binding
`to MCF7 cells. ELISA assay suggested that the human scFv C6.5 dis-
`played similar binding specificity compared with the murine e23.
`Cell-free protein synthesis inhibitory activity. The biological
`activity of toxins can be severely compromised when incorporated
`into fusion constructs. To examine the N-glycosidic activity of the
`rGel component of the immunotoxins, these materials were added
`to an in vitro protein translation assay using [3H]leucine incorpo-
`ration by isolated rabbit reticulocytes. Inhibition curves for the fu-
`sion constructs e23-L-rGel and C6.5-L-rGel and native rGel were
`compared (Fig. 2B), and IC50 values for the three molecules were
`found to be virtually identical (15.41, 15.52, and 10.6 pmol/L, re-
`spectively), suggesting that no loss of toxin activity occurred in
`the fusion molecules.
`Cellular uptake of immunotoxins. We next examined whether
`the e23-L-rGel and C6.5-L-rGel fusions could specifically internal-
`ize into target cells. Immunofluorescence staining was done on
`SKOV3 and MCF7 cells after exposure to the constructs. As shown
`in Fig. 2C, the rGel moiety of both fusions was observed primarily
`in the cytosol of SKOV3 after treatment, but not in MCF7 cells,
`showing that both constructs were comparable in efficient cell
`binding and rapid internalization and delivery of rGel toxin to
`the cytoplasm after exposure to Her2/neu-positive cells.
`In vitro cytotoxicity. The e23-L-rGel and C6.5-L-rGel constructs
`and rGel were tested against a number of different tumor cell lines
`(Table 1). The SKBR3 cells with the highest level of Her2/neu ex-
`pression were killed most efficiently by both antibody fusion con-
`structs, with IC50 values of 6.0 and 9.1 nmol/L for e23-L-rGel and
`C6.5-L-rGel, respectively. IC50 values for rGel toxin were ∼200-fold
`higher (1,671 nmol/L). For the other Her2/neu-positive cells, both
`immunotoxins also showed similar IC50 values, showing that the
`two fusion proteins possess very similar cell killing activity and
`specificity. Furthermore, MCF7 and 4T1 cells, which express rela-
`tively low levels of Her2/neu, showed little to no specific cytotoxi-
`city of the fusion constructs compared with rGel itself, clearly
`showing that the presence of higher levels of cell-surface Her2/
`neu is required for specific cytotoxicity of the constructs.
`
`In vitro cleavage of C6.5/rGel fusions by furin. From the
`in vitro study, it was evident that no significant differences were
`observed between murine e23– and human C6.5–based fusion con-
`structs. Therefore, we focused on C6.5/rGel for further studies by
`incorporation of proteolytically cleavable linkers (Fpe and Fdt) to
`examine whether this change would improve killing efficiency. To
`investigate the susceptibility of various chimeric toxins to proteo-
`lytic cleavage, purified fusions were subjected to proteolysis with
`recombinant furin (Fig. 3A). At pH 7.2, cleavage of Fpe (18.5% of
`total) and Fdt (100%) was observed. At pH 5.4, Fpe was cleaved less
`efficiently (4.5% of total), but Fdt still displayed high cleavage effi-
`ciency (100%). In contrast, fusion with L linker was found to be
`highly stable and could not be cleaved at either pH. As indicated,
`the Fdt linker was the most sensitive to cleavage among all the con-
`structs. In contrast, cleavage of the molecule containing the Fpe
`linker was highly dependent on pH. The L linker was found to be
`comparatively resistant to protease action without regard to the pH.
`Kinetics of cytotoxicity by C6.5/rGel fusions. To investigate
`the kinetics of cytotoxicity by different C6.5/rGel fusions, their cell
`killing activities were assessed against SKBR3, SKOV3, Calu3, and
`MDA MB435S cells at various time points (Supplementary
`Table S1). Interestingly, the cell lines showed no differences in
`overall sensitivity to the fusion constructs with furin cleavage lin-
`kers compared to those with flexible L linker. All the fusions
`showed potent cytotoxicity after 48 hours and exerted highly po-
`tent cell killing at 72 hours. This suggests that the cleavage efficien-
`cy of different linkers for these chimeric toxins was not a major
`determinant of the overall cytotoxic effects observed with different
`linkers. Surprisingly, the cytotoxic kinetics of the constructs there-
`fore seemed to be independent of the sensitivity of the constructs
`to proteolytic cleavage.
`Intracellular release of rGel from various constructs. The in-
`tracellular release of rGel after endocytosis of various C6.5/rGel fu-
`sion constructs was assessed by Western blot with an anti-rGel
`antibody (Fig. 3B). During the treatment of SKOV3 cells, rGel re-
`lease was found to be maximal at 2 hours after treatment with
`C6.5-L-rGel and 4 hours after exposure to C6.5-Fpe-rGel. For
`C6.5-Fdt-rGel, the rGel component was released within 1 to 2
`hours and degraded simultaneously, corresponding to the status
`of full-length protein. The decreasing intracellular level of full-
`length C6.5-Fdt-rGel could be ascribed to rapid instability of the
`construct after internalization. Although the maximal rGel release
`of different fusions was achieved at different time points, the
`
`Table 1. Comparative IC50 values of the e23-L-rGel and C6.5-L-rGel fusion constructs against various types of tumor cell lines
`
`Cell line
`
`Origin
`
`Her2/neu level
`
`IC50 (nmol/L)
`
`Targeting index*
`
`e23-L-rGel
`
`C6.5-L-rGel
`
`rGel
`
`e23-L-rGel
`
`C6.5-L-rGel
`
`SKBR3
`NCI-N87
`Calu3
`SKOV3
`BT474
`MDA MB435S
`MCF7
`4T1
`
`Human, breast
`Human, gastric
`Human, lung
`Human, ovarian
`Human, breast
`Human, breast
`Human, breast
`Mouse, breast
`
`High
`High
`High
`High
`High
`Medium
`Low
`No
`
`6.0
`59.2
`41.1
`16.3
`27.1
`24.6
`266.3
`>1,000
`
`9.1
`45.0
`31.3
`18.0
`25.2
`28.8
`200.9
`>1,000
`
`1,671.0
`1,334.0
`879.7
`378.9
`325.2
`359.0
`260.4
`>1,000
`
`279
`23
`21
`23
`12
`15
`1
`1
`
`184
`30
`28
`21
`13
`12
`1
`1
`
`*Targeting index represents IC50 of rGel/ IC50 of immunotoxin.
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`Cancer Res 2009; 69: (23). December 1, 2009
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`IPR2014-00676
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`

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`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Novel Immunotoxins Targeting Her2/neu
`
`Figure 3. Functional analysis of C6.5/rGel series immunotoxins in vitro. A, Western blot analysis of furin cleavage of purified C6.5/rGel fusion constructs. B, Western
`blot analysis of intracellular rGel release of C6.5/rGel fusions in SKOV3 cells. C, functional stability analysis of the fusions by whole-cell ELISA and cytotoxicity on
`SKOV3 cells. The proteins were incubated in human plasma at 37°C for up to 72 h before analysis.
`
`absolute amounts of delivered rGel found in the cytosol were vir-
`tually identical. Therefore, these data confirm the observation
`that introduction of an unstable furin cleavage linker does not
`improve the intracellular rGel release of the constructs.
`Functional stability analysis of C6.5/rGel fusions. The linkers
`between C6.5 and rGel showed a differential sensitivity to protease
`action, which may result in different clearance and metabolic ki-
`netics in vivo (31, 32). To estimate the stability of various C6.5/rGel
`fusions, we incubated the purified proteins at 37°C for varying
`times in the presence of human plasma before testing cellular
`Her2/neu binding to SKOV3 cells (Fig. 3C). Our results showed that
`in the presence of human plasma, the C6.5-Fdt-rGel construct dis-
`played a reduction in binding activity within 6 hours of incubation
`and a 20% loss of binding activity after 72 hours of incubation. In
`contrast, the C6.5-L-rGel and C6.5-Fpe-rGel fusion constructs
`showed only 9% and 12% reductions, respectively, after 72 hours
`of incubation.
`In addition, the immunotoxins were evaluated for cytotoxic ac-
`tivity following incubation in human plasma for 0, 24, 48, and 72
`hours (Fig. 3C). For the C6.5-Fdt-rGel construct, the cell killing ac-
`tivity was reduced more than 2-fold after 48 hours, as indicated by
`increasing IC50 values of 20 versus 48 nmol/L. However, this was
`not the case for C6.5-L-rGel and C6.5-Fpe-rGel, which retained
`most of its cytotoxic activity even after 48 hours and displayed a
`little influence on IC50 after 72 hours of incubation in plasma (16
`versus 22 nmol/L and 17 versus 25 nmol/L for each construct).
`This functional stability analysis indicated that compared with
`
`the L and Fpe linkers, the Fdt linker was much more unstable in
`human plasma, and this may reduce the in vivo potency of poten-
`tial therapeutic applications using constructs containing this linker
`design.
`Mechanisms of cytotoxic effects. The cytotoxic effects
`mediated by C6.5/rGel fusions were analyzed to evaluate whether
`the cytotoxic mechanisms of the constructs observed included
`elements of apoptosis, necrosis, or autophagy in SKOV3 cells. As
`shown in Fig. 4A, C6.5/rGel fusions did not show activation of
`caspase-dependent apoptosis in SKOV3 cells and showed no cleav-
`age of caspase substrate PARP. The terminal deoxyribonucleotidyl
`transferase–mediated dUTP nick end labeling (TUNEL) results
`(Supplementary Fig. S1) confirmed that the cytotoxic effects of
`the rGel-based fusions were not mediated by apoptosis and DNA
`fragmentation.
`To assess whether the necrotic cell death was induced, we ex-
`amined lactate dehydrogenase (LDH) release, which is a marker of
`abrupt membrane lysis (33). In this case, treatment of SKOV3 cells
`with Triton X-100 serves as a positive control causing LDH release
`(Supplementary Fig. S2). In contrast, treatment with the fusion
`constructs failed to show LDH release, indicating that the observed
`cytotoxicity did not seem to be a result of necrosis.
`We next asked if the immunotoxins activate autophagic sig-
`naling in SKOV3 cells. MAP LC3-I, known to be usually pres-
`ent in the cytosol,
`is palmitoylated during autophagy to form
`membrane-bound LC3-II and is associated with autophagosomes
`(34). As shown in Fig. 4B, the ratio of LC3-II formation to the
`
`www.aacrjournals.org
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`8991
`
`Cancer Res 2009; 69: (23). December 1, 2009
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`Downloaded from
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`cancerres.aacrjournals.org
`
`on October 7, 2014. © 2009 American Association for Cancer
`Research.
`
`IMMUNOGEN2111, pg. 6
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`IPR2014-00676
`
`

`

`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Cancer Research
`
`β-actin control was increased after treatment with the fusion con-
`structs, showing that autophagic flux was induced by C6.5/rGel
`fusions in SKOV3 cells. In addition, autophagy induction by C6.5/
`rGel fusions was further validated by the selective release of
`HMGB1 (Fig. 4C). Tumor cells that are dying by autophagy selec-
`tively release the nuclear HMGB1 protein without displaying char-
`acteristics of necrosis (35). These data indicated that the observed
`cytotoxic effects of C6.5/rGel fusions on SKOV3 cells seemed to be
`mediated not through an apoptotic or necrotic mechanism but by
`the efficient induction of autophagic cell death.
`Antitumor activity of C6.5/rGel fusions in xenograft models.
`We evaluated the ability of various C6.5/rGel fusion constructs to
`inhibit the growth of established SKOV3 tumor xenografts in nude
`mice after systemic administration. Tumors were induced in nude
`mice by s.c. injection of SKOV3 cells on day 0, and treatment was
`initiated on day 9 post-injection when the tumors were well estab-
`
`lished. Treatment consisted of five i.v. injections every other day.
`Groups of mice were treated at doses of 40 and 20 mg/kg for each
`fusion construct. Control mice were treated with PBS or 20 mg/kg
`rGel only. As shown in Fig. 5A and B, treatment with C6.5-L-rGel
`exhibited a significant antitumor effect. Mice treated with the 40
`mg/kg dose of C6.5-L-rGel showed a long-lasting antitumor effect
`that lasted more than 1 month until the animals were sacrificed. In
`mice treated with the 20 mg/kg dose, tumor growth was, in most
`cases, arrested for the duration of the treatment and resumed a
`couple of weeks after its completion. Otherwise, treatment of mice
`with 40 mg/kg C6.5-Fpe-rGel resulted in a significant delay in tu-
`mor growth. This was similar to the effect observed with the same
`dose of C6.5-L-rGel, but no significant effect could be observed at
`the lower (20 mg/kg) dose level. In contrast, mice treated with
`C6.5-Fdt-rGel at either dose (40 or 20 mg/kg) showed no specific
`antitumor effect above that observed with rGel alone.
`
`Figure 4. Western blot analysis of the
`cell killing mechanism of C6.5/rGel
`immunotoxins on SKOV3 cells. A, analysis
`of PARP cleavage after 24 and 48 h of
`C6.5/rGel fusion treatment. B, analysis of
`LC3 after treatment with C6.5/rGel fusions.
`The histogram shows quantitation of the
`ratio of LC3-II to β-actin. C, analysis of cell
`extract and medium for HMGB1 protein
`after C6.5/rGel treatment for 48 h.
`
`Cancer Res 2009; 69: (23). December 1, 2009
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`8992
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`www.aacrjournals.org
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`Downloaded from
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`on October 7, 2014. © 2009 American Association for Cancercancerres.aacrjournals.org
`
`
`Research.
`
`IMMUNOGEN2111, pg. 7
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Published OnlineFirst November 24, 2009; DOI: 10.1158/0008-5472.CAN-09-2693
`
`Novel Immunotoxins Targeting Her2/neu
`
`with reduced immunogenicity without affecting efficacy, toxicity,
`or specificity have not generally focused on anti-Her2/neu agents
`(26, 38, 39). To the best of our knowledge