Surface Charge-Modification Prevents Sequestration and Enhances

Article pubs.acs.org/bc

Surface Charge-Modification Prevents Sequestration and Enhances Tumor-Cell Specificity of a Recombinant Granzyme B−TGFα Fusion Protein Robert A. Jabulowsky, Pranav Oberoi, Hayat Baḧ r-Mahmud, Benjamin Dal̈ ken, and Winfried S. Wels* Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Str. 42-44, 60596 Frankfurt am Main, Germany S Supporting Information *

ABSTRACT: The serine protease granzyme B (GrB) plays an important role in the immune defense mediated by cytotoxic lymphocytes. Recombinant derivatives of this pro-apoptotic protein fused to tumor-targeting ligands hold promise for cancer therapy, but their applicability may be limited by promiscuous binding to nontarget tissues via electrostatic interactions. Here, we investigated cell binding and specific cytotoxicity of chimeric molecules consisting of wild-type or surface-charge-modified human GrB and the natural EGFR ligand TGFα for tumor targeting. We mutated two cationic heparin-binding motifs responsible for electrostatic interactions of GrB with cell surface structures, and genetically fused the resulting GrBcs derivative to TGFα for expression in the yeast Pichia pastoris. Purified GrBcs-TGFα (GrBcs-T) and a corresponding fusion protein employing wild-type GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFRoverexpressing breast carcinoma cells in the presence of an endosomolytic reagent. However, unspecific binding of the modified GrBcs-T variant to EGFR-negative cells was dramatically reduced, preventing the sequestration by nontarget cells in mixed cell cultures and increasing tumor-cell specificity. Likewise, modification of the GrB domain alleviated unspecific extracellular effects such as cell detachment indicative of extracellular matrix degradation. Our data demonstrate improved selectivity and functionality of surface-charge-modified GrBcs, suggesting this strategy as a general approach for the development of optimized GrB fusion proteins for therapeutic applications.

■

INTRODUCTION Cancer cells are frequently insensitive to apoptotic stimuli due to deregulation of apoptotic cell death pathways. Targeted delivery of a potent apoptosis inducer into such cells that can concurrently activate multiple targets within the apoptotic cascade may effectively overcome apoptosis resistance and selectively induce cancer cell death. One such strategy with therapeutic potential is based on recombinant granzyme B (GrB) fused to tumor-targeting ligands.1,2 These chimeric molecules structurally and functionally reflect immunotoxins,3,4 but in contrast to the latter, they employ an effector domain of human origin. This can be expected to result in lower immunogenicity and circumvent complications frequently observed with classical immunotoxins due to their toxin domain of plant or bacterial origin.5 The serine protease GrB plays an important role in the immune defense against virus-infected and transformed cells.6 It is naturally produced by cytotoxic T lymphocytes and NK cells, where it resides together with other granzymes in cytotoxic granules. Upon target cell recognition and induction of degranulation, GrB is released in a directed fashion into the immunological synapse formed between effector and target cells. Following cellular uptake aided by the pore-forming protein perforin, GrB rapidly induces target cell apoptosis via caspase-dependent and caspase-independent mechanisms.7 © 2012 American Chemical Society

Recombinant human GrB and GrB−ligand fusion proteins have been successfully produced in bacterial, yeast, and mammalian expression systems,8−10 and have been shown to retain potent cell-death inducing activity upon targeted delivery into tumor cells.11−14 Nevertheless, while natural GrB is released complexed with the negatively charged chondroitin sulfate proteoglycan serglycin that shields its positively charged surface,15,16 the applicability of recombinant GrB derivatives may be limited by promiscuous binding of uncomplexed GrB to negatively charged cell surface proteoglycans via electrostatic interactions. 17−19 Two major heparin-binding motifs, RKAKRTR (residues 116 to 122; GrB numbering) and KKTMKR (residues 241 to 246) were identified on GrB surface loops, both electrostatically interacting with glucosaminoglycans on the cell surface. Exchange of basic amino acid residues within these sites with neutral alanines diminished cell binding and endocytosis, but also disturbed perforin-assisted cytotoxicity.17 Here, we investigated intrinsic and ligand-mediated cell binding, and in vitro antitumoral activity of recombinant proteins employing wild-type or surface-charge-modified Received: February 9, 2012 Revised: June 11, 2012 Published: July 3, 2012 1567

dx.doi.org/10.1021/bc3000657 | Bioconjugate Chem. 2012, 23, 1567−1576

Bioconjugate Chemistry

Article

column (Qiagen, Hilden, Germany). Specifically bound proteins were eluted with 250 mM imidazole, 1 M NaCl in PBS (pH 8). Protein fractions containing GrB derivatives were identified by SDS-PAGE and immunoblotting with GrB-specific monoclonal antibody (mAb) 2C5 (Santa Cruz Biotechnology, Heidelberg, Germany), pooled, dialyzed against PBS (pH 7.4), concentrated, and stored until use at −80 °C. Native human GrB purified from NK cells was purchased from Enzo Life Sciences (Lörrach, Germany). GrB Activity Assays. Enzymatic activity of recombinant GrB proteins was analyzed using the synthetic GrB substrate Ac-IETD-pNA (Alexis, Grünberg, Germany) as described.9 Purified proteins were incubated with reaction buffer (10 mM Hepes pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and 200 μM AcIETD-pNA in a total volume of 100 μL per sample in 96-well plates. Triplicate samples were incubated for 3 h at 37 °C, and substrate cleavage was quantified by measuring the absorbance (A) at 405 nm (corrected for background by subtracting A490) with a microplate reader (Molecular Devices, Ismaning, Germany). Buffered substrate solution without recombinant proteins served as blank. GrB dependence of cleavage was confirmed by preincubation of recombinant proteins with 400 μM of GrB-specific peptide aldehyde inhibitor Ac-IETD-CHO (Alexis) for 15 min at room temperature prior to the addition of peptide substrate. Binding Analysis. Expression of EGFR on the surface of tumor cells was investigated by flow cytometry. Trypsinized MDA-MB468 and MDA-MB453 cells (2.5 to 5 × 105) were incubated with antihuman EGFR antibody R-1 (Santa Cruz Biotechnology) for 30 min at 4 °C, followed by phycoerythrin (PE)-conjugated goat antimouse IgG secondary antibody (Jackson ImmunoResearch, West Grove, PA, USA). For flow cytometric analysis of binding of recombinant GrB proteins to the surface of tumor cells, trypsinized MDA-MB468 and MDAMB453 cells (2.5 to 5 × 105) were incubated with up to 0.5 μg of purified GrB proteins for 30 min at 4 °C. Unbound proteins were removed, cells were washed, and bound proteins were detected with 20 μg/mL of Alexa Fluor 647-conjugated antihuman GrB antibody (BD Biosciences, Heidelberg, Germany). Fluorescence of cells was analyzed using FACSCalibur and FACSCanto II flow cytometers, and CELLQuest Pro and FACSDiva software, respectively (BD Biosciences). To investigate the contribution of the positively charged GrB domain to intrinsic cell binding, GrB proteins were incubated with an 18-fold molar excess of heparin (Ratiopharm, Ulm, Germany) for up to 60 min at room temperature before cellbinding analysis. For analysis of binding of GrB-T and GrBcs-T to mixed cell populations, first 4 × 106 washed EGFR-positive MDA-MB468 cells were labeled with 4 μL calcein violet AM stock solution (Molecular Probes, Invitrogen, Karlsruhe, Germany) for 1 h at 4 °C and washed twice with RPMI medium. Then, the labeled cells were mixed with unlabeled EGFR-negative MDA-MB453 cells prior to incubation with the GrB fusion proteins and analysis of binding by flow cytometry as described above. Confocal Laser Scanning Microscopy. Cells were grown overnight on coverslips coated with poly(L-lysine) (SigmaAldrich, Taufkirchen, Germany), and then treated with 2.5 μg/ mL (62.5 nM) of recombinant GrB proteins for 60 min at 4 °C. Cells were washed, fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, and incubated with 1 μg/mL of GrB-specific mAb 2C5 in PBS, 3% BSA, followed by Alexa Fluor 488-conjugated donkey antimouse IgG (Molecular

human GrB fused to the natural epidermal growth factor receptor (EGFR) ligand transforming growth factor α (TGFα). EGFR overexpression has been found in many tumors of epithelial origin, and has been shown to contribute to cellular transformation.20 Due to its accessibility from the extracellular space, this tumor-associated surface antigen is an attractive therapeutic target for monoclonal antibodies and other targeted reagents.3,21 We mutated the two cationic heparin-binding motifs responsible for nonselective electrostatic interactions of GrB with cell surface structures, and fused the resulting GrBcs derivative to TGFα for expression in the yeast Pichia pastoris. Purified GrBcs-TGFα (GrBcs-T) and a corresponding TGFα fusion protein employing wild-type GrB (GrB-T) displayed similar enzymatic activity and targeted cytotoxicity against EGFR-overexpressing breast carcinoma cells. However, unspecific binding of the modified GrBcs-T variant to EGFRnegative cells was dramatically reduced, preventing the sequestration by nontarget cells in mixed cell cultures seen for GrB-T, and increasing tumor-cell specificity.

■

EXPERIMENTAL PROCEDURES Cells and Culture Conditions. Human MDA-MB468 and MDA-MB453 breast carcinoma, HeLa cervix carcinoma, and LN-18 glioblastoma cells (all ATCC, Manassas, VA) were cultured in DMEM (Lonza, Köln, Germany) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/mL penicillin, 100 μg/mL streptomycin. Construction of Granzyme B Expression Vectors. Plasmids pPIC9-GrB and pPIC9-GrB-T for expression of mature human wild-type GrB and the GrB−TGFα fusion protein GrB-T in the yeast Pichia pastoris were described previously.8,13 The surface-charge-modified GrB variant GrBcs was generated by stepwise replacement of codons encoding amino acid residues R116, R120, R122, K241, K242, K245, and R246 (GrB numbering) within cationic sites of GrB17 with codons encoding alanine residues by PCR-based site-directed mutagenesis using human GrB cDNA as a template. The resulting GrBcs PCR product was fused to a sequence encoding a Myc- and a hexa-histidine-tag, and subcloned as an XhoI-NotI fragment into the yeast vector pPIC9 (Invitrogen, Karlsruhe, Germany), resulting in plasmid pPIC9-GrBcs. A fusion gene encoding the GrBcs domain followed by a (Gly4Ser)4 linker, an internal hexa-histidine-tag, the EGFR ligand TGFα,22 and Cterminal Myc- and hexa-histidine-tags was assembled in silico, synthesized in codon-optimized form for expression in Pichia pastoris (GeneArt, Regensburg, Germany), and subcloned as an XhoI-NotI fragment into the vector pPIC9, resulting in plasmid pPIC9-GrBcs-T. Expression and Purification of Recombinant Proteins. Recombinant human GrB, GrBcs, GrB-T, and GrBcs-T were expressed in Pichia pastoris and purified from culture supernatants basically as described.13 Briefly, Pichia pastoris GS115 cells (Invitrogen) were transformed with pPIC9 expression plasmids by electroporation, and positive clones were selected following the manufacturer’s recommendations. For large-scale protein expression, single colonies were grown in 80 mL YPD medium (Invitrogen) for 2 days at 28 °C. For induction of expression, cultures were diluted to an OD600 of 1 to 2 in buffered methanol complex medium (pH 6) containing 2% methanol (v/v), and incubated for another 3 to 4 days at 28 °C with addition of methanol every 24 h. Supernatants containing recombinant proteins were harvested by centrifugation, adjusted to pH 8, filtered, and applied to a Ni-NTA superflow 1568



Article

Probes). Nucleic acids were counterstained with TO-PRO-3 (Invitrogen). Samples were then analyzed with a Leica TCS SL laser scanning microscope (Leica Mikrosysteme, Bensheim, Germany). Cell Viability Assays. Cytotoxicity of GrB and GrB fusion proteins was analyzed in WST-1 cell viability assays (Roche Diagnostics, Mannheim, Germany) following the manufacturer’s recommendations. Cells were seeded in 96-well plates at a density of 1.5 × 104 cells/well in triplicates, and incubated for 14 to 48 h at 37 °C with varying concentrations of purified GrB proteins in the presence or absence of 50 μM chloroquine. After addition of WST-1 reagent for 4 h, the relative number of viable cells in comparison to cells grown without recombinant GrB proteins was determined by measuring the absorbance (A) at 450 nm (corrected for background by subtracting A690). To compete binding of GrBcs-T to EGFR, cells were preincubated with a 23-fold molar excess of anti-EGFR antibody cetuximab23 for 30 min at 4 °C prior to the addition of recombinant protein and determination of cytotoxicity. To evaluate sequestration of EGFR-specific GrB-T and GrBcs-T fusion proteins by nontarget cells, EGFR-negative MDAMB453 cells were seeded in 96-well plates at a density of 1 × 105 cells/well and incubated in triplicate with 2.5 nM (100 ng/mL) of purified GrB fusion proteins for 4 h at 37 °C. Supernatants containing unbound proteins were collected, and analyzed for specific cytotoxicity toward EGFR-positive MDAMB468 cells in the presence of 50 μM chloroquine as described above. Analysis of Granzyme B-Induced Cell Detachment. HeLa cells were seeded in 96-well plates at a density of 1 × 104 cells/well and allowed to adhere for 24 h. Cells were then treated for 24 h at 37 °C with different concentrations of purified GrB proteins, or GrB proteins preincubated for 30 min at room temperature with an 18-fold molar excess of heparin. Control cells were treated with PBS or 1 μM staurosporine. Morphological changes of cells were analyzed after 24 h by light microscopy using a Nikon ECLIPSE TE3000 inverted microscope (Nikon, Düsseldorf, Germany) at 200× magnification. To quantify cell detachment, nonadherent cells were removed by washing once with PBS, and the relative number of remaining attached cells was determined in WST-1 cell viability assays as described above. Statistical Analysis. Differences between values were evaluated using the two-tailed unpaired Student’s t test. P values 0.05.

■

RESULTS Cell Binding and Cytotoxic Activity of the Granzyme B Fusion Protein GrB-T. The chimeric fusion protein GrB-T consists of human granzyme B (GrB) (amino acid residues 21− 247) genetically fused via a flexible linker to transforming growth factor-α (TGFα), a peptide ligand specific for epidermal growth factor receptor (EGFR).13 Recombinant GrB-T was produced as a secreted protein in Pichia pastoris transformed with expression plasmid pPIC9-GrB-T, which encodes GrB-T under the control of the methanol-inducible alcohol oxidase AOX1 promoter (Figure 1A). Release of GrB-T into the culture supernatant is mediated by the α-factor signal peptide from Saccharomyces cerevisiae, which is cleaved off during secretion, thereby generating the N-terminus of mature GrB.9,13

Cell binding of GrB-T purified from yeast culture supernatants was investigated by flow cytometry using human MDAMB468 and MDA-MB453 breast cancer cells. While MDAMB468 cells express high levels of EGFR, MDA-MB453 cells are EGFR-negative (Figure 1B). As expected, we observed strong binding of GrB-T to EGFR-expressing MDA-MB468 cells utilizing fluorochrome-labeled GrB-specific antibody for direct detection of surface-bound protein (Figure 1C, left 1569



Article

panel). However, considerable binding of GrB-T to EGFRnegative MDA-MB453 cells was also found (Figure 1C, right panel). Similar binding to both cell lines was also observed for recombinant wild-type GrB and native GrB purified from NK cells (see Supporting Information Figure S1). Preincubation of untargeted GrB with heparin markedly reduced cell binding, suggesting the previously reported interaction of cationic residues within the GrB domain with negatively charged structures on the cell surface as the underlying mechanism.17,19 Even at high concentrations, intrinsic cell binding of recombinant and purified native GrB did not result in cell killing (see Supporting Information Figure S2). To investigate cytotoxicity of EGFR-targeted GrB-T, MDA-MB468, and MDA-MB453 cells were treated with 2.5 or 12.5 nM of purified fusion protein for 24 or 48 h. While prolonged incubation of the EGFR-positive cells with high concentrations of GrB-T resulted in significant cell killing in comparison to PBS-treated controls (Figure 1D, left panel), no reduction in cell viability was observed upon treatment of the EGFRnegative cells (Figure 1D, middle panel). Addition of chloroquine as an endosomolytic activity strongly enhanced target cell-specific cytotoxicity of GrB-T,13 facilitating marked and concentration-dependent killing of MDA-MB468, but not MDA-MB453 cells already upon short exposure to the fusion protein (Figure 1D, right panel). These data demonstrate an ability of GrB-T to bind to cells irrespective of the presence of the TGFα receptor EGFR. However, cell killing activity was restricted to EGFR-expressing cells, indicating that cell binding via electrostatic interactions is not sufficient to mediate routing of the protein to intracellular GrB substrates. Expression and Functional Characterization of a Surface-Charge-Modified Granzyme B Derivative. To reduce intrinsic cell binding of the GrB domain, we replaced seven positively charged amino acid residues within two cationic clusters on the surface of GrB by alanine residues following the strategy reported by Bird et al.17 (Figure 2A). Upon expression in Pichia pastoris and purification from culture supernatants, the identity of this mutant GrBcs derivative was confirmed by immunoblot analysis with GrB-specific antibody. Recombinant wild-type GrB was included for comparison (Figure 2B). GrB expressed in Pichia pastoris in secreted form is glycosylated.8 This resulted in a higher apparent molecular mass for GrB and GrBcs than calculated from the amino acid sequence (38 versus 29 kDa). Modification of residues on the surface of GrB had no effect on enzymatic activity of the protein. GrBcs and wild-type GrB cleaved the GrB-specific peptide substrate Ac-IETD-pNA equally well, and enzymatic activity of both proteins was abolished upon preincubation with the peptide aldehyde inhibitor Ac-IETD-CHO (Figure 2C). Flow cytometric analysis with fluorochrome-labeled GrBspecific antibody revealed a marked reduction in the binding of untargeted GrBcs to MDA-MB468 and MDA-MB453 cells when compared to wild-type GrB (Figure 2D). Quantification of flow cytometric data demonstrated reduction of binding of GrBcs to 1.2% of that of GrB in the case of MDA-MB468 cells, and to 2.8% of that of GrB in the case of MDA-MB453 cells (see Supporting Information Table S1). Generation and Functional Characterization of EGFRSpecific Fusion Protein GrBcs-T. Next, we generated an EGFR-targeted GrBcs fusion protein by replacing wild-type GrB cDNA in the pPIC9-GrB-T vector (see Figure 1A) with the mutant GrBcs sequence. The modified GrBcs-T protein

Figure 2. Expression and functional characterization of wild-type and surface-charge-modified Granzyme B. (A) Constructs pPIC9-GrB and pPIC9-GrBcs for expression of human wild-type GrB and the GrBcs mutant protein in the yeast Pichia pastoris. In GrBcs, amino acid residues R116, R120, R122, K241, K242, K245, and R246 within cationic sites are replaced by alanine residues (lower panel). AOX1, methanol-inducible alcohol oxidase I promoter; SP, α-factor signal peptide; GrB21−247, cDNA encoding mature human GrB or mutant GrBcs (amino acids 21−247); M, Myc tag; H, polyhistidine tag. (B) GrB and GrBcs proteins were purified from yeast culture supernatants by Ni2+-affinity chromatography. Eluate fractions were analyzed by SDS-PAGE and immunoblotting with GrB-specific antibody followed by horseradish-peroxidase-coupled secondary antibody and chemiluminescent detection. (C) To determine enzymatic activity, GrBspecific peptide substrate Ac-IETD-pNA was incubated with the indicated concentrations of purified GrB (open circle) or GrBcs proteins (filled circle) for 3 h. Cleavage of substrate was determined by measuring the absorbance at 405 nm. GrB dependence of cleavage was confirmed by preincubation of recombinant proteins with 400 μM of GrB-specific peptide aldehyde inhibitor Ac-IETD-CHO (open and filled diamonds). Mean values ± SEM are shown (n = 3). (D) Binding of purified GrB (regular line) and GrBcs (bold line) proteins to MDAMB468 and MDA-MB453 breast carcinoma cells was determined by flow cytometry with Alexa Fluor 647 conjugated GrB-specific antibody (open areas). Control cells were treated with GrB-specific antibody in the absence of GrB proteins (shaded areas).

was expressed in Pichia pastoris, purified from culture supernatants, and analyzed in comparison to GrB-T by immunoblot analysis with GrB-specific antibody (Figure 3A). Both proteins exhibited an apparent molecular mass of approximately 40 kDa. Subsequently, enzymatic activity of GrBcs-T was investigated in colorimetric cleavage assays using Ac-IETD-pNA as a GrB-specific peptide substrate. Like in the case of untargeted GrBcs, the activity of the targeted GrBcs-T variant was indistinguishable from that of the corresponding 1570



Article

stronger reduction of GrBcs-T binding to cells that do not express EGFR (Figure 3C, lower panel; see also Supporting Information Table S2). Interestingly, in comparison to GrB-T we observed weaker binding of GrBcs-T to EGFR-expressing cells (Figure 3C, upper panel; see also Supporting Information Table S2). This suggests that, in addition to TGFα−EGFR interactions, also in the case of cognate target cells, electrostatic interactions via the GrB domain contribute to cell binding. In an independent experiment, binding of GrB-T and GrBcs-T to the surface of MDA-MB468 and MDA-MB453 cells was analyzed by confocal laser scanning microscopy. Thereby, strong binding of both proteins to EGFR-positive cells was observed (Figure 3D, left panels). However, while unmodified GrB-T also displayed significant binding to the surface of the EGFR-negative cells, this was not the case for GrBcs-T (Figure 3D, right panels). To investigate the potential influence of the mutations introduced into the effector domain on cytotoxic activity of GrBcs-T, MDA-MB468 and MDA-MB453 cells were treated for 14 h with increasing concentrations of the fusion protein in the presence of chloroquine. Cells treated with unmodified GrB-T were included for comparison. Incubation of the EGFRpositive cells with GrBcs-T or GrB-T resulted in effective and concentration-dependent cell killing in comparison to PBStreated controls (Figure 4A, left panel). While GrBcs-T appeared slightly more active than GrB-T in these experiments (IC50 values of 125 versus 225 pM), this difference was statistically not significant (P > 0.05). No reduction in cell viability was observed upon treatment of the EGFR-negative cells with increasing concentrations of GrBcs-T or GrB-T (Figure 4A, right panel). Also, human LN-18 glioblastoma and HeLa cervix carcinoma cells that express more moderate EGFR levels than MDA-MB468 cells were killed equally well by GrBcs-T and GrB-T (see Supporting Information Figure S3). However, due to the lower amount of target receptor available on the cell surface this required higher concentrations of GrB fusion proteins and prolonged exposure of the cells. Specificity of target cell killing was confirmed by competition of GrBcs-T binding to EGFR with anti-EGFR antibody cetuximab, which blocks ligand binding to the receptor.23 While in this experiment in the absence of competitor treatment of MDAMB468 cells with 2.5 nM of GrBcs-T resulted in 66% of cell killing, cytotoxicity was reduced to 26% in the presence of a 23fold molar excess of cetuximab (Figure 4B). In contrast, antiErbB2 antibody trastuzumab included as a control had no significant effect on GrBcs-T activity (62% of cell killing). In addition to specific target cell recognition via the TGFα domain, cytotoxic activity of GrBcs-T was also dependent on enzymatic activity of the GrB domain. Preincubation of GrBcsT with the peptide aldehyde inhibitor Ac-IETD-CHO abolished cytotoxic activity of the fusion protein against MDA-MB468 cells (Figure 4C). These results demonstrate that surface-charge-modified GrBcs can functionally replace wild-type GrB in the context of a tumor-specific fusion protein. While intrinsic cell binding of GrBcs-T via electrostatic interactions was markedly reduced in comparison to GrB-T, this did not affect specific binding to EGFR-expressing cells via the TGFα domain, resulting in potent targeted cytotoxicity. Sequestration of GrB-T and GrBcs-T by Binding to Non-Target Cells. Fusion of GrB to a target-cell-specific ligand such as TGFα should enable selective recognition and elimination of tumor cells by the chimeric molecule while not

Figure 3. Expression, enzymatic activity, and cell binding of surfacecharge-modified Granzyme B - TGFα fusion protein GrBcs-T. (A) GrB-T and GrBcs-T proteins were purified by Ni 2+ -affinity chromatography from culture supernatants of Pichia pastoris cells carrying pPIC9-GrB-T or pPIC9-GrBcs-T vectors. Eluate fractions were analyzed by SDS-PAGE and immunoblotting with GrB-specific antibody followed by horseradish-peroxidase-coupled secondary antibody and chemiluminescent detection. (B) Enzymatic activity of purified GrB-T (open circle) or GrBcs-T proteins (filled circle) was determined with synthetic GrB substrate Ac-IETD-pNA as described in the legend of Figure 2. GrB dependence of cleavage was confirmed by preincubation of recombinant proteins with 400 μM of GrB-specific inhibitor Ac-IETD-CHO (open and filled diamonds). Mean values ± SEM are shown (n = 3). (C) Binding of purified GrB-T (regular line), GrB-T preincubated with heparin (dotted line) and GrBcs-T (bold line) proteins to MDA-MB468 and MDA-MB453 cells was determined by flow cytometry with Alexa Fluor 647 conjugated GrB-specific antibody (open areas). Control cells were treated with GrB-specific antibody in the absence of GrB proteins (shaded areas). Data for GrB-T are the same as in Figure 1C and shown here for comparison. (D) Binding of GrB-T and GrBcs-T to the surface of MDA-MB468 and MDA-MB453 cells was also analyzed by confocal laser scanning microscopy with GrBspecific antibody followed by Alexa Fluor 488 conjugated secondary antibody (green). Nucleic acids were counterstained with TO-PRO-3 (red). Merged images are shown. Size bar: 10 μm.

GrB-T protein, and specifically inhibited by preincubation with Ac-IETD-CHO (Figure 3B). In flow cytometric analysis with fluorochrome-labeled GrBspecific antibody, GrBcs-T displayed strong binding to EGFRpositive MDA-MB468 cells, but only minimal remaining binding to EGFR-negative MDA-MB453 cells (Figure 3C). While preincubation of unmodified GrB-T with heparin also decreased intrinsic GrB-mediated binding to MDA-MB453 cells, replacement of positively charged amino acid residues within the GrB domain was more effective, resulting in a 1571



Article

free protein and selective cytotoxicity against EGFR-positive cells (Figure 5B, right panel). This was not the case for GrBcsT, which upon prior contact with MDA-MB453 cells remained in solution, and subsequently killed MDA-MB468 cells to a comparable degree as purified GrBcs-T not preadsorbed to MDA-MB453 cells. Extracellular Effects of GrBcs-T. In addition to its apoptosis-inducing activity upon cleavage of intracellular substrates, granzyme B can also process components of the extracellular matrix.24 To investigate potential differences in the extracellular activities of wild-type GrB and GrBcs derivatives, we employed human HeLa cervix carcinoma cells as a model. These cells undergo morphological changes upon degradation of their extracellular matrix by GrB,8 but were not sensitive to the apoptosis-inducing effects of GrB-T or GrBcs-T in the absence of chloroquine (data not shown). Cultures of adherent HeLa cells were treated for 24 h with increasing concentrations of GrB, GrBcs, GrB-T, or GrBcs-T without addition of an endosomolytic activity, before cellular morphology was analyzed by light microscopy (Figure 6A). While low concentrations of GrB proteins had no visible effects (Figure 6B and data not shown), at higher GrB or GrB-T concentrations HeLa cells rounded up and partially lost contact to the culture dish (Figure 6A, middle panels). Nevertheless, we did not observe membrane blebbing, cellular fragmentation or other characteristics of apoptotic morphology, which were seen in staurosporine-treated cells. In contrast to GrB and GrB-T, treatment of HeLa cells with GrBcs or GrBcs-T had no visible effects (Figure 6A, right panels), resulting in cellular morphology very similar to that of PBS-treated controls. Similar results were obtained upon treatment of MDA-MB453 cells with GrBcs and GrBcs-T (data not shown). The marked reduction in the extracellular activity of the GrBcs derivatives was confirmed by quantification of cells still attached to the culture dish after prolonged exposure to GrB proteins (Figure 6B). While exposure to GrB and GrB-T resulted in a concentration-dependent loss of adherent cells, GrBcs and GrBcs-T induced only minimal cell detachment. This was also the case upon pretreatment of GrB and GrB-T with heparin, indicating that electrostatic interactions of the GrB domain also play a role for the extracellular proteolytic activity of GrB and GrB fusion proteins.

Figure 4. Cytotoxic activity of surface-charge-modified Granzyme B− TGFα fusion protein GrBcs-T. (A) EGFR-positive MDA-MB468 (left panel) and EGFR-negative MDA-MB453 cells (right panel) were treated with the indicated concentrations of purified GrB-T (open circles) or GrBcs-T protein (filled circles) in the presence of 50 μM of the endosomolytic reagent chloroquine. Control cells were treated with PBS. After 14 h, the relative number of viable cells was determined in WST-1 assays. (B) To compete binding of GrBcs-T to EGFR, MDA-MB468 cells were preincubated with a 23-fold molar excess of anti-EGFR antibody cetuximab prior to the addition of 2.5 nM of purified protein and determination of cytotoxicity as described above (filled bar). Control cells were treated with GrBcs-T in the absence of competitor (open bar), or preincubated with isotypematched anti-ErbB2 antibody trastuzumab before addition of GrBcs-T (shaded bar). (C) Dependence of cell killing on GrB activity was investigated by preincubation of GrBcs-T with 400 μM of GrB-specific inhibitor Ac-IETD-CHO before addition to MDA-MB468 cells (filled bar). Control cells were treated with GrBcs-T in the absence of inhibitor (open bar). For all experiments, mean values ± SEM are shown (n = 3). ***, P < 0.001; ns, P > 0.05.

■

affecting target antigen-negative bystander cells. As a model system to investigate the selectivity of GrB-T and GrBcs-T in mixed cell populations, we performed flow cytometric cell binding analyses with GrB-specific antibody using mixtures of MDA-MB468 and MDA-MB453 cells. To distinguish between the two cell lines, prior to the experiment MDA-MB468 cells were fluorescently labeled with calcein violet AM. While in 1:1 mixed cell populations GrB-T displayed similar binding to EGFR-positive and EGFR-negative cells, this was not the case for GrBcs-T, which selectively bound only to EGFR-positive MDA-MB468 cells (Figure 5A). Similar results were also obtained with 5:1 and 10:1 mixtures, where EGFR-negative cells were in excess (data not shown). Next, we investigated the effect of sequestration by nontarget cells on selective cytotoxicity of GrB-T and GrBcs-T. MDAMB453 cells were incubated with equal amounts of the fusion proteins for 4 h, before culture supernatants containing unbound GrB-T and GrBcs-T proteins were collected, and added for 14 h to MDA-MB468 cells in the presence of chloroquine (Figure 5B, left panel). Preincubation of GrB-T with EGFR-negative cells significantly reduced the amount of

DISCUSSION Targeted delivery of pro-apoptotic proteins to tumor cells represents a promising strategy to induce selective tumor cell death.25 We and others have described prototypic granzyme B fusion proteins that carry natural peptide ligands or antibody fragments specific for tumor-associated surface antigens for cell binding.11−14,26 After uptake, such molecules cleaved natural GrB substrates within the target cells, and rapidly induced programmed cell death with kinetics similar to that of GrB/ perforin-mediated cell killing by cytotoxic lymphocytes.13 Targeted GrB derivatives so far have employed an active fragment of the wild-type enzyme. Hence, in addition to specific and high-affinity binding mediated by their heterologous targeting ligands, such molecules likely retained the GrBintrinsic ability to also interact with natural GrB binding sites present on the surface of cells17−19 and components of extracellular matrix.24,27 Here, we have functionally characterized recombinant GrB-TGFα fusion proteins either harboring the wild-type enzyme (GrB-T) or a variant where the two cationic heparin-binding motifs of GrB were mutated (GrBcs1572



Article

Figure 5. Differential binding of GrB-T and GrBcs-T to EGFR-negative cells. (A) EGFR-positive MDA-MB468 cells (black) were fluorescently labeled with calcein violet AM and mixed at a 1:1 ratio with unlabeled EGFR-negative MDA-MB453 cells (gray) prior to incubation with GrB-T (middle panel) or GrBcs-T fusion proteins (right panel) and analysis of binding with Alexa Fluor 647 conjugated GrB-specific antibody by flow cytometry. Control cells were treated with GrB-specific antibody in the absence of GrB fusion proteins (left panel). (B) Sequestration of GrB fusion proteins by binding to nontarget cells was analyzed by incubation of EGFR-negative MDA-MB453 cells with 2.5 nM of purified fusion proteins for 4 h. Then, EGFR-positive MDA-MB468 cells were treated for 14 h with MDA-MB453 culture supernatants containing unbound GrB-T (open bars) or GrBcs-T (filled bars) proteins in the presence of 50 μM chloroquine before determination of the relative number of viable MDA-MB468 cells in WST-1 assays. Control cells were treated with PBS (shaded bar) or purified proteins without prior adsorption to MDA-MB453 cells as indicated. The relative amounts of GrB fusion proteins in the samples was analyzed by immunoblotting with GrB-specific antibody followed by horseradishperoxidase-coupled secondary antibody and chemiluminescent detection. Mean values ± SEM are shown (n = 3). **, P < 0.01; *, P < 0.05; ns, P > 0.05.

enzymatic activity when expressed in yeast on its own or fused to the natural EGFR ligand TGFα. To determine the consequences of GrB-mediated intrinsic cell binding for selectivity of a targeted GrB fusion protein, binding of GrBcs-T and GrB-T to EGFR-overexpressing human MDA-MB468 and EGFR-negative MDA-MB453 breast carcinoma cells was investigated utilizing directly fluorochromelabeled anti-GrB antibody for detection of cell-bound fusion proteins. This detection system proved much more sensitive than indirect detection with an anti-Myc tag antibody that we employed previously.13 We observed strong binding of GrB-T to MDA-MB468 cells, but also considerable binding to EGFRnegative control cells. Preincubation of GrB-T with heparin markedly reduced cell binding, suggesting interaction of cationic residues within the GrB domain with negatively charged structures on the cell surface as the underlying mechanism.17,19,28 In contrast, modified GrBcs-T displayed strong binding only to EGFR-positive MDA-MB468 cells. Interestingly, this binding of GrBcs-T to EGFR-expressing cells was less pronounced than observed for GrB-T. Masking of the positive surface charge of GrB-T by heparin also reduced binding to MDA-MB468 cells, suggesting a general contribu-

T). In the presence of an endosomolytic activity, GrBcs-T retained the high and specific cytotoxicity of GrB-T toward EGFR-expressing targets. However, sequestration by promiscuous binding to EGFR-negative cells as observed for GrB-T was greatly diminished, resulting in increased availability of the modified GrBcs-T molecule for specific cell killing. With a calculated pI of ∼10 GrB is a highly basic protein, which enables binding to glucosaminoglycans and other negatively charged structures such as gangliosides, sulfated lipids, and phospholipid head groups on the surface of different cell types.17,19,28 Bird and colleagues identified two cationic sequence loops RKAKRTR (residues 116 to 122; GrB numbering) and KKTMKR (residues 241 to 246) within GrB that electrostatically interact with heparan-sulfate-containing molecules. 17 Mutation of these sequences resulted in diminished binding to the plasma membrane and suppression of subsequent endocytosis. More recently, residues K133 and K137 were also implicated in nonselective cell binding of GrB.28 Following the strategy described by Bird et al., we generated a surface-charge-modified GrB derivative.17 This recombinant GrBcs molecule displayed markedly reduced binding to cells derived from epithelial tissues, but retained 1573



Article

reduced the amount of free protein and selective cytotoxicity against EGFR-positive cells. This was not the case for GrBcs-T, which upon prior contact with MDA-MB453 control cells remained in solution, and subsequently killed MDA-MB468 tumor cells to a comparable degree as purified GrBcs-T not preadsorbed to cells. Hence, modification of the GrB domain in GrBcs-T prevented the sequestration by nontarget cells seen for GrB-T, and increased tumor-cell specificity. This will likely be important for in vivo applications, where unmodified GrB fusion proteins could be trapped to a large extent by nontarget tissues before reaching the site of the tumor. Experiments are underway to scale up protein production for the investigation of differential effects of GrB-T and GrBcs-T in in vivo models. In addition to its apoptosis-inducing activity within cells, GrB can also process components of the extracellular matrix,24,27 which may be important for tissue remodeling in the course of an ongoing immune reaction. However, excessive extracellular activity of GrB has been linked to pathophysiological conditions such as rheumatoid arthritis30,31 and atherosclerosis,32,33 and may complicate application of large doses of recombinant GrB proteins for therapeutic purposes. We analyzed morphological changes and cell detachment in the presence of GrB-T and GrBcs-T to assess potential differences in the extracellular activities of the fusion proteins. While exposure to GrB-T or untargeted GrB resulted in a concentration-dependent loss of adherent cells, GrBcs-T and GrBcs induced only minimal cell detachment. This confirms that electrostatic interactions also play a role for the extracellular proteolytic activity of GrB and GrB fusion proteins, which can be controlled by surface charge modification as in the case of unspecific cell binding. Taken together, our data demonstrate that surface charge modification of the GrB domain reduces promiscuous binding of a targeted GrB fusion protein and alleviates unwanted extracellular effects without interfering with enzymatic activity and ligand-mediated tumor cell recognition. Reduced sequestration by nontarget cells enhanced availability of the GrBcs-T molecule for tumor cell binding, resulting in increased specific cytotoxicity. Hence, this strategy may serve as a general approach for the development of optimized immunotoxin-like GrB fusion proteins for therapeutic applications.

Figure 6. Morphological changes and cell detachment induced by recombinant GrB proteins. Human HeLa cervix carcinoma cells were treated for 24 h with 25, 50, or 125 nM of GrB proteins, or 125 nM of proteins preincubated with heparin. Control cells were treated with PBS or 1 μM staurosporine. (A) Morphology of cells treated with 125 nM of GrB, GrBcs, GrB-T or GrBcs-T, PBS, or staurosporine as indicated. Representative microscopic fields at 200× magnification are shown. (B) To quantify cell detachment, nonadherent cells were removed by washing with PBS, and the relative number of remaining attached cells was determined in WST-1 cell viability assays as indicated. Mean values ± SEM are shown (n = 9).

tion of electrostatic interactions via the GrB domain to cell binding of GrB-T that is independent from TGFα−EGFR interactions. Importantly, GrB-mediated cell binding did not affect cytotoxic activity, which was very similar for GrB-T and GrBcs-T and restricted to EGFR-expressing target cells, thereby correlating with the amount of target receptor on the cell surface. Hence, promiscuous charge-mediated cell binding of the GrB-T fusion protein alone did not mediate access to GrB substrates in intracellular compartments and induction of apoptosis, even when the endosomolytic reagent chloroquine was present.8,13 Chloroquine accumulates in acidic compartments such as late endosomes and lysosomes, where it interferes with the pH equilibrium, leading to osmotic rupture of the vesicles.29 While GrB fusion proteins taken up via binding to growth factor receptors such as EGFR are routed to chloroquine-sensitive endosomes,13 this is not the case for GrB bound via electrostatic interactions, which is transported to neutral vesicles insensitive to the endosomolytic reagent.8 Promiscuous binding of targeted GrB to the surface of cells did not result in nonspecific cytotoxicity, but could limit the amount of protein available for specific tumor cell killing. In binding experiments with mixed populations of EGFR-positive and EGFR-negative cells, GrB-T interacted with both cell types to a similar extent, while GrBcs-T exclusively bound to cells expressing the tumor-associated antigen. Consequently, preincubation of GrB-T with EGFR-negative cells significantly

■

ASSOCIATED CONTENT

S Supporting Information *

Supplementary Figure S1 presents data on the cell binding of recombinant and native GrB, Supplementary Figure S2 presents data on the cytotoxicity of untargeted GrB, Supplementary Table S1 compares cell binding of GrB and GrBcs, Supplementary Table S2 compares cell binding of GrBT and GrBcs-T fusion proteins, and Supplementary Figure S3 presents data on targeted cytotoxicity of GrB-T and GrBcs-T toward cells with moderate EGFR expression. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*Telephone: +49-69-63395-188. Telefax: +49-69-63395-189. E-mail: [email protected]. Notes

The authors declare no competing financial interest. 1574



■

Article

Cytotoxic cell granule-mediated apoptosis: perforin delivers granzyme B-serglycin complexes into target cells without plasma membrane pore formation. Immunity 16, 417−428. (16) Raja, S. M., Metkar, S. S., Höning, S., Wang, B., Russin, W. A., Pipalia, N. H., Menaa, C., Belting, M., Cao, X., Dressel, R., and Froelich, C. J. (2005) A novel mechanism for protein delivery: granzyme B undergoes electrostatic exchange from serglycin to target cells. J. Biol. Chem. 280, 20752−20761. (17) Bird, C. H., Sun, J., Ung, K., Karambalis, D., Whisstock, J. C., Trapani, J. A., and Bird, P. I. (2005) Cationic sites on granzyme B contribute to cytotoxicity by promoting its uptake into target cells. Mol. Cell. Biol. 25, 7854−7867. (18) Kurschus, F. C., Bruno, R., Fellows, E., Falk, C. S., and Jenne, D. E. (2005) Membrane receptors are not required to deliver granzyme B during killer cell attack. Blood 105, 2049−2058. (19) Shi, L., Keefe, D., Durand, E., Feng, H., Zhang, D., and Lieberman, J. (2005) Granzyme B binds to target cells mostly by charge and must be added at the same time as perforin to trigger apoptosis. J. Immunol. 174, 5456−5461. (20) Hynes, N. E., and MacDonald, G. (2009) ErbB receptors and signaling pathways in cancer. Curr. Opin. Cell Biol. 21, 177−184. (21) Groner, B., Hartmann, C., and Wels, W. (2004) Therapeutic antibodies. Curr. Mol. Med. 4, 539−547. (22) Schmidt, M., Vakalopoulou, E., Schneider, D. W., and Wels, W. (1997) Construction and functional characterization of scFv(14E1)ETA - a novel, highly potent antibody-toxin specific for the EGF receptor. Br. J. Cancer 75, 1575−1584. (23) Mendelsohn, J., and Baselga, J. (2000) The EGF receptor family as targets for cancer therapy. Oncogene 19, 6550−6565. (24) Boivin, W. A., Cooper, D. M., Hiebert, P. R., and Granville, D. J. (2009) Intracellular versus extracellular granzyme B in immunity and disease: challenging the dogma. Lab. Invest. 89, 1195−1220. (25) Mahmud, H., Dälken, B., and Wels, W. S. (2009) Induction of programmed cell death in ErbB2/HER2-expressing cancer cells by targeted delivery of apoptosis-inducing factor. Mol. Cancer Ther. 8, 1526−1535. (26) Liu, Y., Zhang, W., Niu, T., Cheung, L. H., Munshi, A., Meyn, R. E., and Rosenblum, M. G. (2006) Targeted apoptosis activation with GrB/scFvMEL modulates melanoma growth, metastatic spread, chemosensitivity, and radiosensitivity. Neoplasia 8, 125−135. (27) Buzza, M. S., Zamurs, L., Sun, J., Bird, C. H., Smith, A. I., Trapani, J. A., Froelich, C. J., Nice, E. C., and Bird, P. I. (2005) Extracellular matrix remodeling by human granzyme B via cleavage of vitronectin, fibronectin, and laminin. J. Biol. Chem. 280, 23549−23558. (28) Kurschus, F. C., Fellows, E., Stegmann, E., and Jenne, D. E. (2008) Granzyme B delivery via perforin is restricted by size, but not by heparan sulfate-dependent endocytosis. Proc. Natl. Acad. Sci. U. S. A. 105, 13799−13804. (29) Zenke, M., Steinlein, P., Wagner, E., Cotten, M., Beug, H., and Birnstiel, M. L. (1990) Receptor-mediated endocytosis of transferrinpolycation conjugates: an efficient way to introduce DNA into hematopoietic cells. Proc. Natl. Acad. Sci. U. S. A. 87, 3655−3659. (30) Tak, P. P., Spaeny-Dekking, L., Kraan, M. C., Breedveld, F. C., Froelich, C. J., and Hack, C. E. (1999) The levels of soluble granzyme A and B are elevated in plasma and synovial fluid of patients with rheumatoid arthritis (RA). Clin. Exp. Immunol. 116, 366−370. (31) Ronday, H. K., van der Laan, W. H., Tak, P. P., de Roos, J. A., Bank, R. A., TeKoppele, J. M., Froelich, C. J., Hack, C. E., Hogendoorn, P. C., Breedveld, F. C., and Verheijen, J. H. (2001) Human granzyme B mediates cartilage proteoglycan degradation and is expressed at the invasive front of the synovium in rheumatoid arthritis. Rheumatology (Oxford) 40, 55−61. (32) Skjelland, M., Michelsen, A. E., Krohg-Sorensen, K., Tennoe, B., Dahl, A., Bakke, S., Brosstad, F., Damas, J. K., Russell, D., Halvorsen, B., and Aukrust, P. (2007) Plasma levels of granzyme B are increased in patients with lipid-rich carotid plaques as determined by echogenicity. Atherosclerosis 195, e142−146.

ACKNOWLEDGMENTS This work was supported in part by Deutsche Forschungsgemeinschaft (DFG) grant WE 2589/2-1 and DFG Graduiertenkolleg GRK1172, and institutional funds of the Georg-SpeyerHaus. The Georg-Speyer-Haus is funded jointly by the German Federal Ministry of Health (BMG) and the Ministry of Higher Education, Research and the Arts of the State of Hessen (HMWK). We thank Dr. Christian Brendel, Georg-SpeyerHaus, for help with CLSM analysis, and Drs. Kurt Schönfeld and Martin Zörnig, Georg-Speyer-Haus, for helpful suggestions.

■

REFERENCES

(1) Rosenblum, M. G., and Barth, S. (2009) Development of novel, highly cytotoxic fusion constructs containing granzyme B: unique mechanisms and functions. Curr. Pharm. Des. 15, 2676−2692. (2) Kurschus, F. C., and Jenne, D. E. (2010) Delivery and therapeutic potential of human granzyme B. Immunol. Rev. 235, 159−171. (3) Wels, W., Biburger, M., Müller, T., Dälken, B., Giesübel, U., Tonn, T., and Uherek, C. (2004) Recombinant immunotoxins and retargeted killer cells: employing engineered antibody fragments for tumor-specific targeting of cytotoxic effectors. Cancer Immunol. Immunother. 53, 217−226. (4) FitzGerald, D. J., Wayne, A. S., Kreitman, R. J., and Pastan, I. (2011) Treatment of hematologic malignancies with immunotoxins and antibody-drug conjugates. Cancer Res. 71, 6300−6309. (5) von Minckwitz, G., Harder, S., Hövelmann, S., Jäger, E., AlBatran, S. E., Loibl, S., Atmaca, A., Cimpoiasu, C., Neumann, A., Abera, A., Knuth, A., Kaufmann, M., Jäger, D., and Wels, W. S. (2005) Phase I clinical study of the recombinant antibody-toxin scFv(FRP5)-ETA specific for the ErbB2/HER2 receptor in patients with advanced solid malignomas. Breast Cancer Res. 7, R617−R626. (6) Cullen, S. P., Brunet, M., and Martin, S. J. (2010) Granzymes in cancer and immunity. Cell Death Differ. 17, 616−623. (7) Afonina, I. S., Cullen, S. P., and Martin, S. J. (2010) Cytotoxic and non-cytotoxic roles of the CTL/NK protease granzyme B. Immunol. Rev. 235, 105−116. (8) Giesübel, U., Dälken, B., Mahmud, H., and Wels, W. S. (2006) Cell binding, internalization and cytotoxic activity of human granzyme B expressed in the yeast Pichia pastoris. Biochem. J. 394, 563−573. (9) Dälken, B., Jabulowsky, R. A., Oberoi, P., Benhar, I., and Wels, W. S. (2010) Maltose-binding protein enhances secretion of recombinant human granzyme B accompanied by in vivo processing of a precursor MBP fusion protein. PLoS One 5, e14404. (10) Gehrmann, M., Doss, B. T., Wagner, M., Zettlitz, K. A., Kontermann, R. E., Foulds, G., Pockley, A. G., and Multhoff, G. (2011) A novel expression and purification system for the production of enzymatic and biologically active human granzyme B. J. Immunol. Methods 371, 8−17. (11) Liu, Y., Cheung, L. H., Thorpe, P., and Rosenblum, M. G. (2003) Mechanistic studies of a novel human fusion toxin composed of vascular endothelial growth factor (VEGF)121 and the serine protease granzyme B: directed apoptotic events in vascular endothelial cells. Mol. Cancer Ther. 2, 949−959. (12) Kurschus, F. C., Kleinschmidt, M., Fellows, E., Dornmair, K., Rudolph, R., Lilie, H., and Jenne, D. E. (2004) Killing of target cells by redirected granzyme B in the absence of perforin. FEBS Lett. 562, 87− 92. (13) Dälken, B., Giesübel, U., Knauer, S. K., and Wels, W. S. (2006) Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition. Cell Death Differ. 13, 576−585. (14) Stahnke, B., Thepen, T., Stöcker, M., Rosinke, R., Jost, E., Fischer, R., Tur, M. K., and Barth, S. (2008) Granzyme B-H22(scFv), a human immunotoxin targeting CD64 in acute myeloid leukemia of monocytic subtypes. Mol. Cancer Ther. 7, 2924−2932. (15) Metkar, S. S., Wang, B., Aguilar-Santelises, M., Raja, S. M., Uhlin-Hansen, L., Podack, E., Trapani, J. A., and Froelich, C. J. (2002) 1575



Article

(33) Saito, Y., Kondo, H., and Hojo, Y. (2011) Granzyme B as a novel factor involved in cardiovascular diseases. J. Cardiol. 57, 141− 147.

1576


Surface Charge-Modification Prevents Sequestration and Enhances

Recommend Documents