Volume 15, Number 23, 2009
`
`ISSN: 1381-6128
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`
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`figment pharmaceutical design.
`V4.5, no. 23 (2009)
`Géneral Collection
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`1009-08-17 11:03:40
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`LIBRARY OF
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`%Apoptosis
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`The number 1
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`IMMUNOGEN 2292, pg. 1
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`2676
`Current Pharmaceutical Design, 2009, 15, 2676-2692
`Development of Novel, Highly Cytotoxic Fusion Constructs Containing
`Granzyme B: Unique Mechanisms and Functions
`
`M.G. Rosenblum1,* and S. Barth2,3
`
`1Immunopharmacology and Targeted Therapy Laboratory, Dept. of Experimental Therapeutics, M. D. Anderson Cancer
`Center, Houston, TX 77030, USA; 2Fraunhofer Institute for Molecular Biology and Applied Ecology, Dept. of Pharma-
`ceutical Product Development, 52074 Aachen, Germany and 3Helmholtz Institute for Biomedical Engineering at the
`RWTH Aachen, Dept. of Experimental Medicine and Immunotherapy, 52074 Aachen, Germany
`
`Abstract: Recombinant fusion proteins are an expanding, important class of novel therapeutic agents. The designs of
`these constructs typically involve a cell-targeting motif genetically fused to a highly toxic class of enzymes capable of
`ruthlessly attacking critical cellular machinery once delivered successfully to the cytoplasm of the target cell. Initial de-
`velopment of this class of constructs typically contained recombinant growth factors or single-chain antibodies as the cell-
`targeting motif fused to highly cytotoxic plant or bacterial toxins. This review describes second-generation molecules
`composed of cell-targeting molecules fused to highly cytotoxic human enzymes capable of generating intense apoptotic
`response once delivered to the cytoplasm. The human serine protease granzyme B has been shown to be extremely effec-
`tive as a cytotoxic molecule when incorporated into numerous cell-targeting constructs. The biological activity of GrB-
`containing constructs rivals that of plant or bacterial toxins and appears to represent a new generation and class of com-
`pletely human proteins with unique biological activities.
`Key Words: Fusion proteins, granzyme B, immunotoxins, serine protease, gp240, VEGF, serpins, H22.
`
`INTRODUCTION
` The successful development of targeted therapeutics for
`cancer applications depends on the identification of ligands
`and antigens specific for tumor cells (or their micro-
`environment), generation of molecules capable of targeting
`those components specifically after systemic administration
`and, finally, delivery of highly toxic molecules to the tumor
`(or its surroundings). Immunoconjugates composed of anti-
`bodies and small, toxic drugs or radioisotopes have been
`successfully tested in vitro, in animal models and have dem-
`onstrated activity in the clinical setting. This field has been
`the subject of numerous excellent reviews [1-7].
`
`In addition to the use of small molecules for the toxin
`component, a number of groups have utilized highly cyto-
`toxic protein toxins such as diphtheria toxin, ricin A-chain,
`Pseudomonas exotoxin, gelonin (rGel) in addition to others
`[8-16]. However, problems such as capillary leak syndrome,
`immunogenicity and toxicity continue to limit enthusiasm
`for long-term or chronic applications of these agents in the
`cancer setting. Studies by Pastan et al. [17] have demon-
`strated engineered toxin analogs of Pseudomonas exotoxin
`with reduced antigenicity compared to the original molecule.
`Studies in our laboratory have also demonstrated rGel ana-
`logs with reduced antigenicity and size [18] although immu-
`notoxins containing rGel have demonstrated a low degree of
`immunogenicity in the clinical setting [19] even after
`repeated administration.
`
`*Address correspondence to this author at the Immunopharmacology and
`Targeted Therapy Laboratory, Dept. of Experimental Therapeutics, M. D.
`Anderson Cancer Center, Houston, TX 77030, USA; Tel: 713-792-3554;
`Fax: 713-745-6339; E-mail: mrosenbl@mdanderson.org
`
` Although there are a number of exquisitely cytotoxic
`payloads as mentioned above which are available for the
`construction of targeted therapeutic agents, there are a num-
`ber of considerations which are relevant to the identification
`of molecules which constitute a class of “perfect” protein
`payloads. One of the first characteristics we considered was
`that the payload should be a relatively small human protein
`in the size range of the current toxins (approximately 25
`kDa) or smaller if possible. In addition, this payload should
`not have a nominal cell-binding and internalization route or
`at least should have a cell-binding component, which can be
`engineered out of the molecule. Another characteristic would
`be that the molecule should be an enzyme, which acts, in a
`multi-component cellular cascade to reduce the possibility of
`developing cellular resistance to the delivered therapeutic
`agent. Finally, the cellular pathways necessary for cellular
`cytotoxic effects would have to be present in all cells and, in
`particular, all cancer cells.
`
`DRUG TARGETING SYSTEMS
` There are a large number of molecules which potentially
`fit the considerations mentioned above including several
`kinases, phosphatases, nucleases [20-23] and proteases [24-
`26]. One candidate molecule we identified involves the
`granule-associated serine proteases called granzymes. The
`serine protease granzyme B (GrB) is integrally involved in
`apoptotic cell death induced in target cells upon their expo-
`sure to cytotoxic T-lymphocytes (CTL) and natural killer
`(NK) cells (Fig. (1)).
` The granule secretion pathway appears to require the
`direct intracellular delivery of this family of proteases (gran-
`zyme A and GrB), that activate both caspase-independent
`
`
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`1381-6128/09 $55.00+.00
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`© 2009 Bentham Science Publishers Ltd.
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`IMMUNOGEN 2292, pg. 2
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`IPR2014-00676
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`Development of Novel, Highly Cytotoxic Fusion Constructs
`
`Current Pharmaceutical Design, 2009, Vol. 15, No. 23 2677
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`Fig. (1). Intracellular mechanism of action of GrB.
`
`and -dependent death programs to ensure that the targeted
`cell dies [27-29]. Perforin, well known for its pore-forming
`capacity, has long been considered the vehicle that provides
`the gateway for entry of granzymes through the plasma
`membrane [30-32]. In CTL-mediated cytolysis, perforin is
`initially inserted into the target cell membranes and polymer-
`izes to form transmembrane pores which facilitates access of
`NK or CTL-released GrB to the target cell cytoplasm. GrB
`appears to have the most potent apoptotic activity of all
`granzymes, as a result of its caspase-like ability to cleave
`substrates at key aspartic acid residues. The cell death-
`inducing properties of GrB have recently been studied in
`detail [33-37]. GrB can cleave and directly activate several
`procaspases, and it can also directly cleave downstream
`caspase substrates such as the inhibitor of caspase-activated
`DNase [38]. Although many procaspases are efficiently
`cleaved in vitro, GrB-induced caspase activation occurs in a
`hierarchical manner in intact cells, commencing at the level
`of “executioner caspases” such as caspase-3, followed by
`caspase-7 [39]. Overexpression of the anti-apoptotic Bcl-2
`protein in mitochondria inhibits GrB completely, indicating
`that mitochondrial disruption is an indispensable feature of
`granzyme-mediated cell death [40]. In addition to caspase-
`dependent mechanisms, there are also caspase-independent
`pathways: cells in which caspase activity is blocked can also
`be killed by granzymes, although the caspase-independent
`mechanisms are poorly understood [41]. In addition to the
`caspase-mediated cytotoxic events, GrB can also rapidly
`translocate to the nucleus and cleave poly (ADP-ribose) po-
`lymerase and nuclear matrix antigen, utilizing different
`cleavage sites than those preferred by caspases [42,43]. In
`
`addition, some studies have shown that GrB can direct dam-
`age to non-nuclear structures such as mitochondria, subse-
`quently induce cell death through caspase-independent
`pathways [44-46].
`
`Since almost all cells contain mechanisms responsible for
`mediating cell death (apoptosis) we propose that targeted
`delivery of GrB to the interior of cells will result in cell
`death through apoptotic mechanisms assuming that sufficient
`quantities of active enzyme can be successfully delivered to
`the appropriate subcellular compartment (Fig. (2)).
`
`In addition to providing a cytotoxic insult directly to
`target cells, an additional aspect of delivering pro-apoptotic
`agents is the potential for impacting radio-sensitivity, metas-
`tatic spread and sensitivity to chemotherapeutic agents.
`Numerous studies have suggested that the apoptotic status of
`cells impacts all three phenomenon and an additional ration-
`ale for targeting pro-apoptotic agents is the potential for
`impacting these cellular events in a unique fashion.
` The rationale described above was the impetus for our
`original studies focusing on developing targeted therapeutic
`agents targeting tumor vasculature by using vascular endo-
`thelial growth factor-A (VEGF) and melanoma-associated
`antigen gp240 by using the single chain Fv antibody
`scFvMEL.
`
`GrB/scFvMEL FUSION CONSTRUCT
` To target melanoma cells, we chose the recombinant
`single-chain antibody scFvMEL, which recognizes the high-
`molecular-weight glycoprotein gp240, found on a majority
`
`IMMUNOGEN 2292, pg. 3
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`IPR2014-00676
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`2678 Current Pharmaceutical Design, 2009, Vol. 15, No. 23
`
`Rosenblum and Barth
`
`Fig. (2). The impact of apoptosis on various growth and anti-growth signals.
`
`Fig. (3). Schematic Representation of the gene encoding GrB/scFvMEL.
`
`of melanoma cell lines and fresh tumor samples [47,48].
`Others and we have demonstrated that this antibody pos-
`sesses high specificity for melanoma and is minimally reac-
`tive with a variety of normal tissues, making it a promising
`candidate for further study [49-52]. In the present study, we
`used scFvMEL as a tumor cell-targeting carrier and designed
`a novel recombinant fusion construct designated GrB/
`scFvMEL, containing human pro-apoptotic enzyme GrB
`(Fig. (3)). The purpose of these studies was to determine
`whether an antibody delivery vehicle would be sufficient to
`deliver active GrB enzyme to drive cellular apoptotic events
`specifically in melanoma target cells.
` The fusion protein was generated by PCR, sequenced and
`cloned into a bacterial expression system (pET-32, Novagen)
`containing a
`thioredoxin
`tag upstream of
`the coding
`sequence for the final protein. The material was purified
`from bacterial paste using immobilized metal affinity chro-
`matography and the final product was generated by en-
`terokinase cleavage to uncover the N-terminal Ile of the GrB
`molecule, which is essential for enzymatic activity[53]. An
`ELISA was performed to determine the binding specificity
`of the GrB/scFvMEL fusion construct to antigen-positive
`A375-M and to antigen negative SKBR3 cells. As shown in
`Fig. (4), GrB/scFvMEL specifically bound to antigen-
`positive A375-M cells but we were able to detect little bind-
`ing to antigen-negative SKBR3 cells.
`
`Fig. (4). ELISA binding of the GrB/scFvMEL fusion construct
`to antigen-positive A375-M and antigen-negative SKBR3 cells.
`ELISA of GrB/scFvMEL on gp240 Ag-positive A375-M versus
`gp240 Ag-negative SKBR3 cells detected using an anti-GrB mAb.
`Ninety-six-well plates containing adherent A375-M or SKBR3 cells
`(5 x 104 cells/well) were blocked by addition of 5% BSA and then
`treated with purified GrB/scFvMEL at various concentrations. After
`washing, the cells were incubated first with anti-GrB mAb, and
`then with HRP-GAM. Then, substrate solution (ABTS plus 1 (cid:1)l/ml
`30% H2O2) was added. A405 nm was measured after 30 min.
`
`IMMUNOGEN 2292, pg. 4
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`Development of Novel, Highly Cytotoxic Fusion Constructs
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`Current Pharmaceutical Design, 2009, Vol. 15, No. 23 2679
`
` To assess the functionality of the GrB component of the
`fusion construct, the ability of the enzyme to cleave a
`BAADT substrate was assessed and compared to a known
`GrB standard (Table 1). The fusion construct GrB/scFvMEL
`was shown to have intact GrB enzymatic activity with a spe-
`cific activity comparable to that of the unmodified enzyme,
`(SA = 2.6 (cid:4) 105 units/(cid:1)mole for the GrB/scFvMEL com-
`pared to 4.8 (cid:4) 105 units/(cid:1)mole for native GrB). As expected,
`the fusion construct which had the thioredoxin tag on the
`molecule (non-rEK cut) had no activity since the N-terminal
`Ile of the GrB was hindered.
` The GrB moiety of GrB/scFvMEL was delivered into the
`cytosol of A375-M cells after treatment with GrB/scFvMEL
`
`for 1 h or 6 h assessed by analysis of confocal microscope
`imaging as detected by anti-GrB antibody (Fig. (5)). GrB
`was found in the cytosol after treatment for 1 h, and the sig-
`nals were stronger after treatment for 6 h than that after 1 h
`demonstrating localization and concentration of the construct
`over time. Antibody ZME-018 is the parental murine anti-
`body for the scFvMEL recombinant fragment. Both agents
`recognize the same antigenic domain on the gp240 target
`antigen present on the cell surface of human melanoma cells.
`When cells were pre-treated with ZME-018, GrB fluorescent
`signal could not be detected in the cytosol after treatment
`with the construct, demonstrating that the uptake of the con-
`struct is dependant on specific interaction with gp240 on the
`cell surface.
`
`Table 1. Enzymatic Activity of GrB and GrB Fusion Constructs
`
`Samples
`
`Native GrB
`
`GrB/scFvMEL (Un-rEK cut)
`
`GrB/scFvMEL (rEK-cut)
`
`(cid:1)mOD/min
`
`Units (U)
`
`48.2
`
`2.0**
`
`68.6
`
`1.0
`
`-
`
`1.42
`
`U/(cid:1)g
`
`19.2
`
`-
`
`4.7
`
`MW (kDa)
`
`Specific Activity (U/(cid:1)M)
`
`25
`
`70
`
`53
`
`4.8 x 105
`
`-
`
`2.6 x 105
`
`* BAADT: N-(cid:2) t-butoxycarbonyl-L-alanyl-L-alanyl-L-aspartyl-thiobenzyl ester.
`** The rate of non-enzymatic hydrolysis of BAADT at 0.2 nM, in 0.3 nM Ellman’s Buffer at 25 °C is (cid:3) 5 (cid:1) mOD/min.
`
`Fig. (5). Rapid internalization of GrB/scFvMEL fusion construct into target cells is blocked by pre-treatment with an anti-gp240
`antibody.
`Internalization of GrB/scFvMEL into A375-M cells assessed by confocal microscopy. A375-M cells were pretreated with ZME-018 (3 (cid:1)M)
`for 2 h, and the cells were then treated with 40 nM GrB/scFvMEL for 1 or 6 h. Molecules bound to the cell surface were removed by brief
`treatment with glycine buffer (pH 2.5). Cells were fixed in 3.7% formaldehyde and permeabilized in 0.2% Triton X-100. Samples were
`blocked with 3% BSA, incubated with goat anti-GrB mAb, and then incubated with FITC-coupled anti-goat IgG and PI. The slides were
`mounted with DABCO containing 1 (cid:1)g/ml of PI and analyzed by Zeiss LSM 510 confocal laser scanning microscopy. A, no GrB/scFvMEL
`treatment control. B, pretreatment with ZME-018 (3 (cid:1)M), then GrB/scFvMEL treatment for 1 h. C, pretreatment with ZME-018, then
`GrB/scFvMEL treatment for 6 h. D, GrB/scFvMEL treatment for 1 h. E, GrB/scFvMEL treatment for 6 h.
`
`IMMUNOGEN 2292, pg. 5
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`2680 Current Pharmaceutical Design, 2009, Vol. 15, No. 23
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`Rosenblum and Barth
`
`4 h. Moreover, cytochrome c was released from mitochon-
`dria into the cytosol on A375-M but not on SKBR3 cells
`after treatment with GrB/scFvMEL at 50 nM for 16 h (Fig.
`(9)).
`
`Fig. (6). Cytotoxic effects of GrB/scFvMEL fusion construct on
`log phase target and non target cells.
`Cytotoxicity of the GrB/scFvMEL fusion toxin on A375-M and
`SKBR3. Log-phase cells were plated into 96-well plates at a density
`of 2.5 x 103 cells per well and allowed to attach for 24 h. The
`medium was replaced with medium containing different concentra-
`tions of GrB/scFvMEL. After 72 h, the effect of fusion toxin on the
`growth of cells in culture was determined using crystal violet stain-
`ing. The IC50 of GrB/scFvMEL was 20 nM on A375-M cells. In
`contrast, no cytotoxicity was observed on SKBR3 cells.
` The cytotoxicity of GrB/scFvMEL was next assessed
`against log-phase A375-M and SKBR3 cells in culture. A
`50% growth inhibitory effect was found at a concentration of
`~20 nM on A375-M cells. However, no cytotoxic effects
`were found on SKBR3 cells at doses of up to 1 (cid:1)M (Fig.
`(6)). By comparison, the cytotoxic effects of GrB/scFvMEL
`were approximately the same as that of another fusion toxin,
`scFvMEL/rGel on A375-M (Fig. (7)). When A375-M cells
`were pre-treated with ZME-018 (40 mg/mL) for 6 h and then
`treated with GrB/scFvMEL for 72 h, the cytotoxicity of
`GrB/scFvMEL was abolished (Fig. (7)) thereby demonstrat-
`ing a requirement for antigen recognition in the cytotoxic
`effect of the GrB/scFvMEL fusion construct. In addition, we
`examined the cytotoxic effects of GrB/scFvMEL construct
`still containing the thioredoxin tag and therefore containing
`GrB, which was enzymatically inactive. As shown in Fig.
`(7), this molecule demonstrated no cytotoxic effects at the
`highest doses tested. This demonstrates that the enzymatic
`activity of the GrB molecule is essential for generating the
`cytotoxic effect.
` Both antigen-positive and antigen-negative cells were
`treated with an IC50 concentration of the GrB/scFvMEL
`fusion construct. At various times (0, 8 and 16 h) after
`administration, the cells were stained for apoptosis using the
`TdT-mediated dUTP-biotin nick end labeling (TUNEL)
`assay. Apoptotic cells were shown up at 8 h treatment.
`Within 16 h after administration, virtually all antigen-
`positive cells were positive for apoptosis (Fig. (8)). In con-
`trast, there was no apoptosis found in non-target cells treated
`with identical doses of the fusion construct.
` As demonstrated in Fig. (9), treatment with GrB/scFvMEL
`induced caspase 3 cleavage on antigen-positive A375-M cells
`but not on antigen-negative SKBR3 cells after treatment for
`
`Fig. (7). Influence of the purification tag on the cytotoxicity of
`the GrB/scFvMEL.
`Comparative cytotoxicity of GrB/scFvMEL and MEL sFv/rGel and
`effect of addition of ZME-018 on cytotoxicity of GrB/scFvMEL
`against A375-M cells. Log-phase A375-M cells were plated into
`96-well plates (2.5 x 103 cells per well) and allowed to attach for
`24 h. The medium was replaced with medium containing different
`concentrations of GrB/scFvMEL or MEL sFv/rGel. Cells were also
`pretreated with ZME-018 (40 mg/ ml) for 6 h and then co-treated
`with various concentrations of GrB/scFvMEL. After 72 h, the cells
`were stained with crystal violet. Plates were read on a microplate
`ELISA reader at 595 nm. The IC50 of GrB/scFvMEL was
`approximately identical to that of MEL sFv/rGel on A375-M.
`ZME-018 pretreatment inhibited the cytotoxicity of GrB/scFvMEL
`on A375-M cells.
`
`Finally, we generated large amounts of purified, endo-
`
`toxin free fusion protein and administered the construct (or
`saline) to groups of 8 mice (by iv administration) bearing
`well-developed A-375 tumors growing subcutaneously. We
`used a QOD X5 schedule and as shown in Fig. (10), tumors
`from control mice increased from 50–1200 mm3 while the
`tumors in the treated group increased to 200 mm3. There was
`a long-term inhibitory effect noted since tumors from the
`treated group remained static.
`
`Bearing A375 Xenografts
` These preliminary mouse experiments clearly demon-
`strated the in vivo potential of constructs targeting tumor
`cells and containing GrB. The doses used showed no obvious
`toxicity and were likely well below the maximum tolerated
`dose. In vivo efficacy, pharmacokinetics and tissue disposi-
`tion studies are continuing.
`
`GrB FUSED TO VEGF121 (GrB/VEGF121)
` Various soluble cytokines have been shown to mediate
`angiogenesis in vitro and in vivo [54,55]. VEGF plays a cen-
`tral role in both normal vascular tissue development and in
`
`IMMUNOGEN 2292, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
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`

`Development of Novel, Highly Cytotoxic Fusion Constructs
`
`Current Pharmaceutical Design, 2009, Vol. 15, No. 23 2681
`
`Fig. (8). Time-course of apoptosis generated by GrB/scFvMEL.
`
`A.
`
`B.
`
`Fig. (9). Effects of GrB/scFvMEL on various pro-apoptotic signals.
`A. GrB/scFvMEL induced caspase-3 cleavage on antigen-positive A375-M. A375-M and SKBR3 cells (2 x 105) were treated with
`GrB/scFvMEL at 50 nM for various times (2, 4, 8, and 16 h). Whole cell lysate (30 (cid:1)g) was analyzed by 12% SDS-PAGE and followed by
`immunoblotting to detect caspase-3 or cleaved caspase-3. Pro-caspase-3 was cleaved into one fragment at 4 h and further cleaved into smaller
`fragments after treatment for 8 h by GrB/scFvMEL on A375-M cells. We found no caspase-3 cleavage on SKBR3 cells treated with
`GrB/scFvMEL.
`B. Cytochrome c released from mitochondria to cytosol by GrB/scFvMEL on A375-M. Cells (5 x 107) were treated with GrB/scFvMEL at 50
`nM for various times (2, 4, 8, and 16 h). Cells were collected, and the cytosolic and mitochondrial fractions were isolated. Fractions (30 (cid:1)g)
`from nontreated and treated cells were analyzed by 15% SDS-PAGE and immunoblotting, detected with an anti-cytochrome c antibody.
`Cytochrome c was found to be released on A375-M cells but not on SKBR3 cells after 4 h treatment by GrB/scFvMEL.
`
`IMMUNOGEN 2292, pg. 7
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`2682 Current Pharmaceutical Design, 2009, Vol. 15, No. 23
`
`Rosenblum and Barth
`
`Fig. (10). Effect of iv administration of GrB/scFvMEL to nude mice bearing A375 xenografts.
`
`tumor neovascularization [56-58]. The lowest molecular
`weight isoform of VEGF, VEGF121, is a soluble, non-
`heparin-binding variant that exists in solution as a disulfide-
`linked homodimer. VEGF121 has been demonstrated in pre-
`vious studies to contain the full biological activity of the
`larger variants [59,60]. The angiogenic actions of VEGF are
`mediated via two closely related endothelium-specific recep-
`tor tyrosine kinases, the human VEGF receptor 1 (fms-
`related tyrosin kinase-1 (FLT-1)) and VEGF receptor 2
`(kinase insert domain receptor (KDR) or fetal liver kinase-1
`(FLK-1)). Both are largely restricted to vascular endothelial
`cells [61-64].
` We previously selected VEGF121 for our studies because
`we consider it an appropriate carrier to deliver toxic agents
`to tumor endothelial cells that overexpress the KDR/FLK-1
`receptors. Recently, we described a fusion toxin composed
`of VEGF121 and the recombinant plant toxin rGel [65]. This
`construct was shown to be selectively cytotoxic to vascular
`endothelial cells overexpressing the KDR/FLK-1 receptor
`for VEGF in both in vitro and xenograft models. We demon-
`strated that VEGF121 ligand is an excellent delivery platform
`with which to target tumor vascular endothelium cells in vivo
`in PC-3 tumor xenografts. In addition, studies against nu-
`merous tumor xenograft models demonstrated impressive
`activity as a single agent [66,67]. Finally, we found that both
`VEGF121 itself and fusion constructs containing VEGF121
`were capable of providing excellent imaging information for
`solid tumors [68] as a potential predictor of response to
`targeted therapeutics using VEGF121. In addition, we found
`these fusion constructs demonstrated exceptional activity as
`therapeutics against non-tumor applications such as ocular
`neovascularization [69].
` The GrB/VEGF121 construct (Fig. (11)) was one of the
`first we described in a paper originally published in Molecu-
`lar Cancer Therapeutics [70].
` The fusion construct GrB-VEGF121 was composed of
`GrB engineered to contain an enterokinase cleavage site
`upstream of the GrB protein so that enterokinase digestion
`would leave an isoleucine residue as the N-terminal amino
`
`acid of the GrB protein. In its natural activation process in-
`side cytotoxic cells, active GrB is generated from a zymogen
`by the action of dipeptidyl peptidase I-mediated proteolysis
`[71], which removes the two residue (Gly Glu) propeptide
`and exposes Ile-21. The N-terminal Ile-Ile-Gly-Gly sequence
`of GrB is necessary for the mature, active GrB. The GrB was
`fused to the VEGF121 coding sequence tethered by a short,
`flexible G4S tether to relieve steric stress on both molecules.
` The construct was cloned, sequenced and expressed in
`bacterial cells using the pET bacterial expression system
`(Novagen). Bacterial paste from a small-scale (5 L) fermen-
`tor was lysed and the soluble fraction was applied to immo-
`bilized metal affinity columns followed by imidazole elution,
`enterokinase digestion and final purification. Purity was
`assessed by SDS-PAGE and Western blot analysis. The
`dimeric nature of the complex was established by SDS-
`PAGE under reducing and non-reducing conditions. The
`purified GrB/VEGF121 construct was then assessed against
`log-phase transfected porcine aortic endothelial (PAE) cells
`transfected with either KDR/FLK-1 or FLT-1 human recep-
`tors for VEGF. As shown, the construct was highly cytotoxic
`to cells expressing the KDR/FLK-1 receptor but were not
`cytotoxic to cells expressing the FLT-1 receptor. IC50 values
`were found to be ~ 10 nM and were similar to the values
`obtained with the VEGF121/rGel fusion toxin against the
`same cell line (Fig. (12)).
` Clonogenic studies (Fig. (13)) were performed against
`these same transfected PAE cells and IC50 values obtained
`
`Fig. (11). Schematic Representation of GrB/VEGF121.
`
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`Development of Novel, Highly Cytotoxic Fusion Constructs
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`Current Pharmaceutical Design, 2009, Vol. 15, No. 23 2683
`
`Fig. (12). Cytotoxic effects of GrB/VEGF121 on log-phase endothelial cells.
`Cytotoxicity of the GrB/VEGF121 fusion toxin on transfected endothelial cells. XTT cytotoxicity assay. Log-phase PAE cells were plated
`into 96-well plates at a density of 2.5 x 103 cells/well and allowed to attach for 24 h. The medium was replaced with medium containing
`different concentrations of GrB/VEGF121. After 72 h, the effect of fusion toxin on the growth of cells in culture was determined using XTT.
`Plates were read on a microplate ELISA reader at 540 nm. IC50 of GrB/VEGF121 was 10 nM on PAE/FLK-1 cells; it was not cytotoxic on
`PAE/FLT-1 cells.
`
`Fig. (13). Effects of various doses of GrB/VEGF121 on clonality of endothelial cells.
`Cologenic assay. 5 x 105 cells/ml were incubated at 37°C and 5% CO2 for 72 h with different concentrations of GrB/VEGF121 and 100 nM
`of irrelevant fusion protein GrB/scFvMEL. Cells were then washed with PBS, trypsinized, counted, and diluted serially. The serial cell sus-
`pensions were then plated in triplicate and cultured in six-well plates for 5–7 days. Cells were stained with crystal violet and colonies consist-
`ing of >20 cells were counted. Columns, percentage of colonies in relation to the number of colonies formed by untreated cells.
`
`for the GrB/VEGF121 construct were similar to that found for
`log-phase culture (20 nM vs 10 nM respectively). Internali-
`zation studies were also performed. Transfected endothelial
`cells were treated for 4 h with the fusion construct and the
`cells were treated with a low pH wash to remove cell-surface
`
`bound material, fixed and immunostained for GrB. As shown
`in Fig. (14), the construct rapidly internalized into cells ex-
`pressing the FLK-1 receptor, but not in cells expressing the
`FLT-1 receptor. The observed specificity of VEGF121-related
`fusion constructs for targeting the FLK-1 receptor compared
`
`IMMUNOGEN 2292, pg. 9
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`2684 Current Pharmaceutical Design, 2009, Vol. 15, No. 23
`
`Rosenblum and Barth
`
`Fig. (14). Rapid internalization of GrB/VEGF121 on endothelial cells.
`PAE cells were plated onto 16-well chamber slides (1 x 104 cells/well). Cells were treated with 100 nM of GrB/VEGF121 for 4 h and then
`washed briefly with PBS. The cell surface was stripped with glycine buffer (pH 2.5) and the cells were fixed in 3.7% formaldehyde and per-
`meabilized in PBS containing 0.2% Triton X-100. After blocking, samples were incubated with anti-GrB antibody and treated with FITC-
`coupled anti-mouse IgG. The slides were analyzed under a fluorescence microscope. The GrB moiety of GrB/VEGF121 is delivered into the
`cytosol of PAE/FLK-1 but not into that of PAE/FLT-1 after 4-h treatment.
`
`to the FLK-1 receptor had been observed originally with
`VEGF121/rGel [72]. We attributed this finding to the possibil-
`ity that the FLK-1 receptor but not the FLT-1 receptor had a
`strong kinase activity, which may contribute to its internaliz-
`ing characteristics. Subsequent studies in our group have
`demonstrated that FLT-1 receptors expressed on osteoclast
`progenitor cells appear to internalize well into cells and are
`capable of mediating selective sensitivity to VEGF-related
`fusion constructs.
`
` We next examined the cytotoxic mechanism of GrB/
`VEGF121 action against target cells and related the effects to
`those known to be related to GrB activity. PAE cells were
`treated for various times with IC50 doses of GrB/VEGF121
`construct and were stained for apoptosis via TUNEL stain-
`ing. As shown in Fig. (15) (left), within 24 h after admini-
`stration of the agent, FLK-1-positive cells demonstrated in-
`tense apoptotic staining. Quantitative assessment (Fig. (15),
`
`Fig. (15). Rapid development of apoptosis in endothelial cells caused by GrB/VEGF121.
`GrB/VEGF121 induces apoptosis on PAE/FLK-1 cells. Cells (1 x 104 cells/well) were treated with GrB/VEGF121 at an IC50 concentration
`for different times (0, 24, and 48 h) and washed with PBS. Cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-
`100 and 0.1% sodium citrate. Cells were incubated with TUNEL reaction mixture, incubated with Converter-AP, and finally treated with
`Fast Red substrate solution. The slides were analyzed under a light microscope. A, microscopic appearance of PAE cells after various treat-
`ments. Apoptosis cells are stained red (400x). B, apoptotic cells. Columns, percentage of the total counted cells (>200 cells) in randomly
`selected fields (200x); bars, SD.
`
`IMMUNOGEN 2292, pg. 10
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Development of Novel, Highly Cytotoxic Fusion Constructs
`
`Current Pharmaceutical Design, 2009, Vol. 15, No. 23 2685
`
`right graph) demonstrates that 75–90% of cells were apop-
`totic (TUNEL positive) within 48 h after treatment.
` The pro-apoptotic effects of GrB/VEGF121 were further
`confirmed by examining DNA degradation in transfected
`endothelial cells. As shown in Fig. (16), the GrB fusion con-
`struct caused an impressive degradation of DNA in FLK-1-
`transfected cells but not in the FLT-1-transfected cells thus
`confirming our observation of specificity and activity of the
`fusion protein. Again, this is in sharp contrast to studies with
`VEGF121/rGel, which found specific cytotoxic effects of the
`construct but the activity appeared to be a necrotic damage
`rather than an apoptotic effect since we could find no
`evidence of DNA laddering, TUNEL positivity or caspase
`activation in treated cells (M.G. Rosenblum, et al. manu-
`script in preparation).
`
`of Hodgkin’s lymphoma [73]. Initially we developed our
`own bacterial vectors for periplasmic expression in E. coli
`[74] and a novel expression technology [75]. We subse-
`quently used this for the generation of the first Pseudomonas
`exotoxin-based immunotoxins targeting cluster of differen-
`tiation (CD)30 overexpressed on Hodgkin-Reed/Sternberg
`cells [76-79]. During this time we also used this expression
`system to develop the first human immunotoxins also target-
`ing CD30 [80].
`
`Fig. (16). DNA degradation specifically on FLK-1-transfected
`cells treated with GrB/VEGF121
`GrB/VEGF121 induces DNA laddering in PAE/FLK-1 cells. Cells
`were plated into six-well plates at a density of 2 x 105 cells/well
`and exposed to 20-nM GrB/VEGF121 for 24 h. DNA was isolated
`from cell lysates and fractionated on 1.5% agarose gel.
`
`Further mechanistic studies were performed to evaluate
`
`the activation profile for intracellular caspase cascade, which
`is a well-described mechanism of GrB cytotoxic effect. PAE
`cells were treated with GrB/VEGF121, and total cell lysates
`were loaded onto 12% SDS-PAGE and standard Western
`blotting was performed. The results showed that treatment
`with GrB/VEGF121 cleaved caspase-8, caspase-3, and PARP
`on PAE/FLK-1-transfected cells but not on PAE/FLT-1-
`transfected cells (Fig. (17)). These data c