C H A P T E R E I G H T
`
`Cell-Targeting Fusion Constructs
`Containing Recombinant Gelonin
`
`Mi-Ae Lyu, Yu (Joshua) Cao, Khalid A. Mohamedali, and
`Michael G. Rosenblum
`
`Contents
`1. Introduction
`2. Engineered Proteins Targeting Her2/neu
`2.1. Background
`2.2. Results
`2.3. Translational relevance
`3. rGel/BLyS Targeting Malignant B-Cells
`3.1. Background
`3.2. Results
`3.3. Translational relevance
`4. VEGF121/rGel Targeting Tumor Vasculature and Skeletal Metastases
`4.1. Receptor specificity of VEGF121/rGel by ELISA
`4.2. Cytotoxicity of VEGF121/rGel fusion toxin
`4.3. Internalization of VEGF121/rGel
`4.4. Angiogenesis assessment in chicken chorioallantoic
`membranes
`4.5. Targeting of VEGF121/rGel to blood vessels of orthotopic
`tumors in mice
`4.6. In vivo efficacy of VEGF121/rGel against solid tumors in nude
`mice
`4.7. In vivo efficacy of VEGF121/rGel against pulmonary
`metastases of MDA-MB-231 breast tumors
`4.8. In vivo efficacy of VEGF121/rGel against PC-3 prostate tumor
`growth in bone
`4.9. VEGF121/rGel inhibits the osteoblastic growth of MDA PCa
`118b cells in bone and normalizes the bone volume of 118b-
`tumor-containing bone
`4.10. Translational relevance
`References
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`168
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`Immunopharmacology and Targeted Therapy Laboratory, Department of Experimental Therapeutics,
`M.D. Anderson Cancer Center, Houston, Texas, USA
`
`Methods in Enzymology, Volume 502
`ISSN 0076-6879, DOI: 10.1016/B978-0-12-416039-2.00008-2
`
`# 2012 Elsevier Inc.
`All rights reserved.
`
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`Abstract
`Therapeutic agents capable of targeting tumor cells present as established
`tumors and micrometastases have already demonstrated their potential in
`clinical trials. Immunotoxins targeting hematological malignancies and solid
`tumors have additionally demonstrated excellent clinical activity. This review
`focuses on our design and characterization studies of constructs composed of
`recombinant gelonin toxin fused to either growth factors or single-chain anti-
`bodies targeting solid tumor cells, tumor vasculature or hematological malig-
`nancies. These agents demonstrate cytotoxicity at nanomolar or sub-nanomolar
`levels. All of these constructs display impressive selectivity and specificity for
`antigen-bearing target cells in vitro and in vivo and are excellent clinical trial
`candidates.
`
`1. Introduction
`
`Although they are capable of selectively binding to cancer cells, most
`antibodies and other cell-targeting therapeutic agents alone have only
`modest antitumor properties primarily through ADCC activation, direct
`tumor cell signaling generating apoptosis or interference with essential
`growth factor homeostatic mechanisms. The majority of these agents have
`defined clinical utility primarily when used in combination with chemo-
`therapy or radiation therapy or as a part of a regimen using all three
`modalities. In order to improve the effectiveness of targeted therapeutic
`molecules, numerous groups have developed highly cytotoxic payloads
`conjugated or fused to these cell-targeting molecules. The concept is that
`the cell-targeting component serves to direct the agent to tumor cells in the
`body and to internalize into the cells themselves once they arrive. This
`specialized entry route also internalizes the attached cytotoxic payload
`directly into the tumor cells sparing normal tissues.
`There is now significant scientific and commercial interest in the devel-
`opment and application of antibody-directed cytotoxic molecules such as
`antibody-drug conjugates (ADCs) (Alley et al., 2010; Hughes, 2010; Mayes
`et al., 2011; Teicher, 2009; Webb, 2011).These constructs have gained
`considerable attention recently based on several promising clinical trial
`results demonstrating efficacy against a number of tumor targets (Kovtun
`and Goldmacher, 2007; Morrow et al., 2009; Murphy and Modi, 2009;
`Polson and Sliwkowski, 2009; Polson et al., 2009; Senter, 2009). It is
`important to note that the recent positive results with ADCs come as a
`second “Golden Age” well after notable disappointing clinical results of
`conjugates produced in the 1980s.
`The use of protein toxins as payloads linked to cell-targeting molecules is
`an interesting analogous approach to ADC technology. There have been a
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`number of highly cytotoxic protein payloads developed including ricin
`toxin A-chain (RTA) (Kreitman, 2001; Lord et al., 2003; Wawrzynczak
`and Derbyshire, 1992), saporin (Flavell, 1998), pseudomonas exotoxin A
`(PE) (Kreitman, 2009; Pastan, 2003; Wolf and Elsasser-Beile, 2009), diph-
`theria toxin (DT) (Coll-Fresno et al., 1997; Hertler and Frankel, 1989; Negro
`and Skaper, 1997), and gelonin (Hertler and Frankel, 1989; Huang et al.,
`2010; Kwon et al., 2008; Sandvig and van Deurs, 2005). More recently,
`several groups (Hursey et al., 2002; Newton et al., 2001) including ours
`(Kurschus and Jenne, 2010; Kurschus et al., 2004; Rosenblum and Barth,
`2009; Stahnke et al., 2008) have developed completely human cytotoxic
`proteins for use as payloads in an attempt to circumvent concerns regarding
`the development of immunogenicity during long-term use with these agents.
`The highly cytotoxic plant toxin gelonin was first identified by Stirpe
`et al. (1980) as an excellent payload for the development of immunotoxins.
`This protein was identified as a new member of a class of ribosome-
`inactivating proteins (RIPs) with n-glycosidase activity similar to that of
`RTA. The crystal structure of gelonin has been reported (Hosur et al., 1995;
`Satyamurthy et al., 1994). A number of groups have utilized this molecule
`for generation of chemical conjugates (Chu et al., 2006; Fishwild et al.,
`1994; Harris et al., 1991; McGraw et al., 1994; McIntyre et al., 1994; Mehta
`et al., 2004; Mujoo et al., 1991; Pagliaro et al., 1998; Rosenblum et al., 1991,
`1992, 1999; Schwartz et al., 1987). In 1995, two separate groups reported
`the cloning and sequencing of a gene encoding the gelonin protein (Nolan
`et al., 1993; Rosenblum et al., 1995) and described a recombinant version
`(rGel) with enzymatic activity virtually identical to the native material. The
`recombinant version of the toxin has been employed by a number of groups
`as an excellent, flexible fusion partner (Cao et al., 2009; Lyu et al., 2007;
`Nimmanapalli et al., 2007; Veenendaal et al., 2002) allowing the construc-
`tion of fusion proteins at either the N-terminus or C-terminus of the rGel
`molecule.
`In this chapter, we will review some of the unique features—including
`the molecular events, unique signaling events, specific cytotoxicity, and
`in vivo studies of three fusion constructs we have generated which contain
`the toxin rGel.
`
`2. Engineered Proteins Targeting Her2/neu
`
`2.1. Background
`
`The Her2/neu proto-oncogene encodes a 185kDa transmembrane glyco-
`protein kinase with extensive homology to the epidermal growth factor
`receptor (EGFR, HER1). Amplification of the gene and overexpression of
`the Her2/neu protein product on tumor cells have been well described in
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`numerous human cancers, including mammary and ovarian carcinomas, and
`gastric and lung tumors. Because Her2/neu plays a central role in malignant
`transformation and growth, it provides an attractive target for focused
`therapeutic approaches (Abramson and Arteaga, 2011; Cai et al., 2010;
`Mannocci et al., 2010).
`A number of approved immunotherapeutic agents directed at tumors
`which express high levels of Her2/neu, such as the monoclonal antibody
`trastuzumab (Herceptin) and small molecule inhibitors such as gefitinib
`(Iressa), have shown promising results, but the development of resistance
`to treatment remains a well-known problem (Garrett and Arteaga, 2011).
`To enhance its clinical potential, cell-surface Her2/neu has been targeted
`using ADCs or immunotoxins, composed of plant or bacterial toxins linked
`with a targeting molecule composed of monoclonal antibodies or antibody
`fragments (Govindan and Goldenberg, 2010; Isakoff and Baselga, 2011;
`Zielinski et al., 2009). Previously, a recombinant, murine anti-Her2/neu
`single-chain antibody (scFv) designated e23 has been fused to catalytic
`toxins such as Pseudomonas exotoxin A (PE) (Liu et al., 2009; Shinohara
`et al., 2002), to specifically target Her2/neu expressing cells. A major
`drawback of such proteins is their potential for immune response after
`repeated administration. Further complications could result from nonspe-
`cific binding of foreign proteins to vascular endothelial cells leading to
`vascular leak syndrome and ultimately interstitial edema and organ failure.
`The development of immunotoxins containing human or humanized
`components may circumvent these problems. Such immunotoxins may
`display reduced immunogenicity although antibodies to the toxin compo-
`nents may still
`limit prolonged therapy. We previously reported
`(Rosenblum et al., 1999) in vitro characterization and in vivo antitumor
`efficacy studies of an immunotoxin composed of the human chimeric
`anti-Her2/neu antibody (BACH-250) chemically conjugated to recombi-
`nant gelonin (rGel). rGel is a 29kDa ribosome-inactivating plant toxin with
`a potency and mechanism of action similar to RTA but with improved
`stability and reduced toxicity. The BACH-250/rGel conjugate demon-
`strated potent and specific cytotoxicity against Her2/neu overexpressing
`human tumor cells in culture and against SKOV3 tumor xenografts. How-
`ever, the treatment of solid tumors presents a potential problem since full-
`length antibodies must diffuse into the tumor against a hydrostatic pressure
`gradient and into disordered vasculature.
`Schier et al. (1995) and Adams et al. (1998) 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. Utilizing scFv C6.5,
`McCall et al. (1999) constructed and characterized a bispecific scFv com-
`posed of C6.5 and anti-CD16 scFv, displaying a high level of in vitro tumor
`cell cytotoxicity and in vivo tumor targeting. Studies by Park et al. (2001,
`2002) generated immunoliposomes containing doxorubicin which were
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`Cell-Targeting Fusion Constructs Containing Recombinant Gelonin
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`targeted to tumor cells using antibody C6.5. These constructs demonstrated
`selective enhancement of the therapeutic index of doxorubicin chemother-
`apy. Most recently, Robinson et al. (Abramson and Arteaga, 2011; Adams
`et al., 2000; Cai et al., 2010; Mannocci et al., 2010; Robinson et al., 2008)
`successfully utilized a C6.5 diabody construct as a radioimmunotherapeutic
`agent containing (211At) for the treatment of Her2/neu positive solid
`tumors in xenograft models, demonstrating that scFv C6.5 could be utilized
`effectively in vehicles for targeted radioimmunotherapy by using powerful,
`short-lived a-emitting radioisotopes.
`In the present study, we describe the construction and characterization of
`several rGel-based chimaeric toxins composed of the scFv e23 or C6.5 and
`employing various linker configurations to examine how different antibodies
`and linker choices impact the in vitro and in vivo efficacy of fusion constructs.
`
`2.2. Results
`2.2.1. Construction, expression, and purification of rGel-based
`fusions
`The initial
`rGel-based immunotoxins consisted of a flexible linker
`(GGGGS, “L”) tethering the C-terminus of the e23 or C6.5 to the native
`rGel N-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 incorporating two different
`enzymatically sensitive furin cleavage linkers between the scFv and rGel
`toxin components. The two furin sensitive sequences designated
`“Fpe” (TRHRQPRGWEQL) and “Fdt” (AGNRVRRSVG), respectively
`(Fig. 8.1A). Several biochemical studies have demonstrated that the serine
`protease furin efficiently cleaves proteins containing these recognition
`sequences.
`Following purification, all the rGel-based immunotoxins migrated on
`SDS-PAGE at the expected molecular weight of 55kDa (Fig. 8.1B). How-
`ever, 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 occurring precisely at the predicted furin
`cleavage site producing the 27–28kDa fragments of
`scFv and rGel.
`Further analysis indicated the yields for each protein (per liter of bacterial
`culture) were 1.55mg for e23-L-rGel, 1.05mg for C6.5-L-rGel, 1.08mg for
`C6.5-Fpe-rGel, and 0.70mg for C6.5-Fdt-rGel.
`
`2.2.2. Characterization of e23-L-rGel and C6.5-L-rGel immunotoxins
`2.2.2.1. Binding activity To ensure that immunotoxins retained antigen
`binding ability, the fusion proteins were compared in an ELISA-based
`binding assay (Fig. 8.2A) using Her2/neu positive SKOV3 and Her2/neu
`negative MCF7 cells. The equilibrium dissociation constant Kd was further
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`Mi-Ae Lyu et al.
`
`rGel
`
`rGel
`
`rGel
`
`rGel
`
`L
`
`L F
`
`pe
`
`Fdt
`
`VL–VH
`
`VH–VL
`
`VH–VL
`
`VH–VL
`
`M arker 1
`
`M arker 2
`
`C 6.5-F dt-r G el
`C 6.5-F pe-r G el
`C 6.5-L-r G el
`e23-L-r G el
`
`A
`e23-L-rGel
`
`C6.5-L-rGel
`
`C6.5-Fpe-rGel
`
`C6.5-Fdt-rGel
`
`B
`
`kDa
`
`150
`100
`75
`
`50
`
`35
`
`25
`
`Figure 8.1 Preparation of e23/rGel and C6.5/rGel series immunotoxins. (A) Sche-
`matic 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.
`
`¼8.5
`calculated (Graphpad Prism, V4.03). The affinity of e23-L-rGel (Kd
`¼12.6 nM).
`nM) for SKOV3 cells was similar to that for C6.5-L-rGel (Kd
`The Kd values were consistent with those previously measured in an in vitro
`live cell assay using scFv itself. In addition, both immunotoxins demon-
`strated significant specificity based on the background of binding to MCF7
`cells. ELISA assay suggested that the human scFv C6.5 displayed similar
`binding specificity compared to the murine e23.
`
`2.2.2.2. 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 incorporation by
`isolated
`rabbit
`reticulocytes.
`Inhibition
`curves
`for
`the
`fusion
`constructs e23-L-rGel, C6.5-L-rGel, and native rGel were compared
`(Fig. 8.2B), and IC50 values for the three molecules were found to be
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`173
`
`SKOV3
`
`MCF7
`
`e23-L-rGel
`C6.5-L-rGel
`rGelonin
`
`0
`
`10
`20
`30
`40
`Concentration (nM)
`
`50
`
`60
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Absobance at 405 nm
`
`e23-L-rGel
`C6.5-L-rGel
`rGelonin
`
`0
`
`30
`20
`40
`10
`Concentration (nM)
`
`50
`
`60
`
`A
`
`0.8
`
`0.6
`
`0.4
`
`0.2
`
`0.0
`
`Absobance at 405 nm
`
`e23-L-rGel
`
`C6.5-L-rGel
`
`rGelonin
`
`C
`
`SKOV3
`
`MCF7
`
`e23-L-rGel, IC50= 15.4 pM
`C6.5-L-rGel, IC50= 15.5 pM
`rGelonin, IC50= 10.6 pM
`
`10-13
`
`10-12
`10-11
`10-10
`Concentration (M)
`
`10-9
`
`10-8
`
`B
`
`120
`
`100
`
`80
`
`60
`
`40
`
`20
`
`Percent control
`
`0
`10-14
`
`Figure 8.2 Characterization and comparison of e23-L-rGel and C6.5-L-rGel immu-
`notoxins. (A) Evaluation 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 (RRLA).
`(C) Internalization of e23-L-rGel and C6.5-L-rGel on SKOV3 and MCF7 cells.
`Cells were subjected to immunofluorescent staining with anti-rGel antibody (FITC-
`conjugated secondary), with propidium iodine nuclear counterstaining.
`
`virtually identical (15.41, 15.52 vs. 10.6 pM, respectively), suggesting that
`no loss of toxin activity occurred in the fusion molecules.
`
`2.2.2.3. Cellular uptake of immunotoxins We next examined whether
`the e23-L-rGel and C6.5-L-rGel fusions could specifically internalize into
`target cells. Immunofluorescence staining was performed on SKOV3
`and MCF7 cells after exposure to the constructs. As shown in Fig. 8.2C,
`the rGel moiety of both fusions was observed primarily in the cytosol
`after treatment of SKOV3, but not in MCF7 cells, demonstrating that
`both constructs were comparable in efficient cell binding and rapid
`internalization after exposure to Her2/neu positive cells.
`
`2.2.2.4. In vitro cytotoxicity The e23-L-rGel, C6.5-L-rGel constructs
`and rGel were tested against a number of different tumor cell
`lines
`(Table 8.1). The SKBR3 cells with the highest level of Her2/neu expres-
`sion were killed most efficiently by both antibody-fusion constructs, with
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`Table 8.1 Comparative IC50 values of e23-L-rGel and C6.5-L-rGel fusion constructs against various types of tumor cell lines
`
`Cell line
`
`Origin
`
`Her2/neu level
`
`IC50 (nM)
`
`Targeting index
`
`a
`
`SKBR3
`NCI-N87
`Calu3
`SKOV3
`BT474
`MDA MB435S
`MCF7
`4T1
`
`High
`Human, breast
`High
`Human, gastric
`High
`Human, lung
`Human, ovarian High
`Human, breast
`High
`Human, breast
`Medium
`Human, breast
`Low
`Mouse, breast
`No
`
`a Targeting index represents IC50 of rGel/IC50 of immunotoxin.
`
`e23-L-rGel
`
`C6.5-L-rGel
`
`rGel
`
`e23-L-rGel
`
`C6.5-L-rGel
`
`6.0
`59.2
`41.1
`16.3
`27.1
`24.6
`266.3
`>1000
`
`9.1
`45.0
`31.3
`18.0
`25.2
`28.8
`200.9
`>1000
`
`1671.0
`1334.0
`879.7
`378.9
`325.2
`359.0
`260.4
`>1000
`
`279
`23
`21
`23
`12
`15
`1
`1
`
`184
`30
`28
`21
`13
`12
`1
`1
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`IC50 values of 6.0 and 9.1 nM for e23-L-rGel and C6.5-L-rGel, respec-
`
`tively. IC50 values for rGel toxin were 200-fold higher (1671nM). For the
`
`other Her2/neu positive cells, both immunotoxins also showed similar IC50
`values demonstrating that the two fusion proteins possess very similar cell-
`killing activity and specificity. Further, the MCF7 and 4T1 cells which
`express relatively low levels of Her2/neu demonstrated little to no specific
`cytotoxicity of the fusion constructs compared to rGel itself, clearly demon-
`strating that the presence of higher levels of cell-surface Her2/neu is
`required for specific cytotoxicity of the constructs.
`
`2.2.2.5. 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 constructs. 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 chi-
`maeric toxins to proteolytic cleavage, purified fusions were subjected to
`proteolysis with recombinant furin (Fig. 8.3A). 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 efficiency
`(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 constructs. 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.
`
`2.2.2.6. 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 (Table 8.2). Interestingly, the cell lines
`showed no differences in overall sensitivity to the fusion constructs with the
`incorporation of furin cleavage linkers compared with flexible L linker. All
`the fusions showed potent cytotoxicity after 48h and exerted highly potent
`cell-killing at 72h. This suggests that the cleavage efficiency of different
`linkers for these chimaeric toxins was not a major determinant to the overall
`cytotoxic effects significant observed on different linkers. Surprisingly, the
`cytotoxic kinetics of the constructs therefore appeared to be independent of
`the sensitivity of the constructs to proteolytic cleavage.
`
`2.2.2.7. Intracellular
`from various constructs The
`rGel
`release of
`intracellular release of rGel after endocytosis of various C6.5/rGel fusion
`constructs was assessed by Western blot with an anti-rGel antibody
`(Fig. 8.3B). During the treatment of SKOV3 cells, rGel release was found
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`Mi-Ae Lyu et al.
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`B
`
`C6.5-L-rGel
`
`C6.5-Fpe-rGel
`
`C6.5-Fdt-rGel
`
`kDa
`
`1 2 4 8 12 24 h
`
`1 2 4 8 12 24 h 1 2 4 8 12 24 h
`
`C6.5-L-rGel
`
`C6.5-Fpe-rGel C6.5-Fdt-rGel
`
`pH 5.4
`pH 7.2
`uncut
`
`pH 5.4
`pH 7.2
`uncut
`
`pH 5.4
`pH 7.2
`uncut
`
`0 h
`24 h
`48 h
`72 h
`
`C6.5-L-rGel
`C6.5-Fpe-rGel C6.5-Fdt-rGel
`rGel-based fusions
`
`58
`46
`
`30
`25
`
`b-actin
`
`60
`50
`40
`30
`20
`10
`0
`
`IC50 (nM)
`
`18.5 4.5 5.8 100 100
`
`C6.5-L-rGel
`C6.5-Fpe-rGel
`C6.5-Fdt-rGel
`
`0
`
`10
`
`20
`30
`40
`50
`60
`Incubation time (h)
`
`70
`
`80
`
`176
`
`A
`
`kDa
`
`58
`46
`
`30
`25
`
`Cleavage
`rate (%)
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`C
`
`% Maximal binding
`
`Figure 8.3 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) Func-
`tional stability analysis of the fusions by whole-cell ELISA and cytotoxicity on SKOV3
`
`C for up to 72h before test.
`cells. The proteins were incubated in human plasma at 37
`
`Table 8.2 Kinetics of cytotoxicity of rGel-based immunotoxins
`
`IC50 (nM)
`
`SKBR3
`
`SKOV3
`
`Calu3
`
`MDA MB435S
`
`24
`─a
`─
`─
`
`48
`
`30.1
`36.4
`44.3
`
`72
`24
`48
`9.4 ─ 102.3
`9.3 ─ 175.1
`10.4 ─ 203.2
`
`72
`24
`48
`26.6 ─ 250.5
`34.2 ─ 185.8
`30.9 ─ 468.8
`
`72
`24
`48
`31.9 ─ 251.7
`27.5 ─ 260.1
`88.1 ─ 277.4
`
`72
`
`25.3
`25.9
`30.5
`
`Toxins duration (h)
`
`C6.5-L-rGel
`C6.5-Fpe-rGel
`C6.5-Fdt-rGel
`
`a –, not detected.
`
`to be maximal at 2h after treatment with C6.5-L-rGel and 4h after exposure
`to C6.5-Fpe-rGel. For C6.5-Fdt-rGel, the rGel component was released
`within 1–2h 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 inter-
`nalization. Although the maximal rGel release of different fusions was
`achieved at different time points, the absolute amounts of delivered rGel
`
`IMMUNOGEN 2113, pg. 10
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`
`

`

`Cell-Targeting Fusion Constructs Containing Recombinant Gelonin
`
`177
`
`were virtually identical. Therefore, this data confirms the observation that
`introduction of an unstable furin cleavage linker does not improve the
`intracellular rGel release of the constructs.
`
`2.2.2.8. Functional stability analysis of C6.5/rGel fusions The linkers
`between C6.5 and rGel demonstrated a differential sensitivity to protease
`action which may result in different clearance and metabolic kinetics in vivo
`(Clemente and de la Torre, 2007; Zhang et al., 2008). 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. 8.3C). Our results showed
`that in the presence of human plasma, the C6.5-Fdt-rGel construct dis-
`played a reduction in binding activity within 6h of incubation and a 20%
`loss of binding activity after 72h incubation. In contrast, C6.5-L-rGel
`and C6.5-Fpe-rGel fusion constructs demonstrated only a 9% and 12%
`reduction, respectively, after 72h incubation.
`In addition, the immunotoxins were evaluated for cytotoxic activity fol-
`lowing incubation in human plasma for 0, 24, 48, and 72h (Fig. 8.3C). For the
`C6.5-Fdt-rGel construct, the cell-killing activity was reduced over twofold
`after 48h as indicated by increasing IC50 values of 20 nM versus 48 nM.
`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 48h, and displayed a little
`influence on IC50 after 72h incubation in plasma (16 nM vs. 22 nM and 17 nM
`vs. 25 nM for each construct). This functional stability analysis indicated that
`compared with L and Fpe linker, the Fdt linker was much more unstable in
`human plasma and this may reduce the in vivo potency of potential therapeutic
`applications using constructs containing this linker design.
`
`2.2.2.9. Mechanistic studies 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. 8.4A,
`C6.5/rGel fusions did not demonstrate activation of caspase-dependent
`apoptosis in SKOV3 cells and showed no cleavage of caspase substrate
`PARP. The TUNEL results (Fig. 8.4B) confirmed that the cytotoxic effects
`of the rGel-based fusions were not mediated by apoptosis and DNA
`fragmentation.
`To assess whether necrotic cell death was induced, we examined LDH
`release which is a marker of abrupt membrane lysis. In this case, treatment of
`SKOV3 cells with Triton X-100 serves as a positive control causing LDH
`release (Fig. 8.5). In contrast, treatment with the fusion constructs failed to
`demonstrate LDH release indicating that the observed cytotoxicity did not
`appear to be the result of necrosis.
`
`IMMUNOGEN 2113, pg. 11
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`IPR2014-00676
`
`

`

`178
`
`A
`
`24 h
`
`PARP
`
`b-actin
`
`B
`
`C 6.5-F pe-rG el
`C 6.5-F dt-rG el
`C 6.5-L-rG el
`
`-
`
`rG el
`
`48 h
`
`PARP
`
`b-actin
`
`Mi-Ae Lyu et al.
`
`C 6.5-F pe-rG el
`C 6.5-F dt-rG el
`C 6.5-L-rG el
`
`-
`
`rG el
`
`No treatment
`
`DNase I
`
`rGel
`
`C6.5-L-rGel
`
`C6.5-Fpe-rGel
`
`C6.5-Fdt-rGel
`
`Figure 8.4 Apoptosis analysis of C6.5/rGel immunotoxins on SKOV3 cells. (A) Anal-
`ysis of PARP cleavage after 24 and 48h of C6.5/rGel fusions treatment. (B) Assessment
`of apoptosis in SKOV3 cells treated with rGel-based immunotoxins by TUNEL assay.
`SKOV3 cells were treated with 25 nM of C6.5/rGel fusions or rGel for 72h, then
`stained for DNA fragmentation. As shown, neither the C6.5/rGel fusions nor the rGel
`itself affected DNA fragmentation.
`
`48 h
`
`100
`
`75
`
`50
`
`25
`
`LDH release (%)
`
`0
`
`Triton X-100
`
`-
`
`rGel
`
`C6.5-Fdt-rGel
`C6.5-Fpe-rGel
`C6.5-L-rGel
`
`24 h
`
`100
`
`75
`
`50
`
`25
`
`0
`
`LDH release (%)
`
`Triton X-100
`
`-
`
`rGel
`
`C6.5-L-rGel
`C6.5-Fdt-rGel
`C6.5-Fpe-rGel
`
`Figure 8.5 Evaluation of necrosis by LDH release in the SKOV3 cells after treatment
`with C6.5/rGel fusions or Triton X-100; no LDH is released in the fusion-treated cells
`(meanS.D. from three replicates).
`
`IMMUNOGEN 2113, pg. 12
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Cell-Targeting Fusion Constructs Containing Recombinant Gelonin
`
`179
`
`C 6.5-L-r G el
`
`C 6.5-F pe-r G el
`
`C 6.5-F dt-r G el
`
`r G el
`
`-
`
`r G el
`
`C 6.5-F dt-r G el
`C 6.5-F pe-r G el
`C 6.5-L -r G el
`
`48 h
`
`LC3-I
`LC3-II
`
`b-actin
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`LC3-II/b-actin ratio
`
`C 6.5-L-r G el
`
`C 6.5-F pe-r G el
`
`C 6.5-F dt-r G el
`
`r G el
`
`-
`
`r G el
`
`C 6.5-F dt-r G el
`C 6.5-F pe-r G el
`C 6.5-L -r G el
`
`Cells
`
`C 6.5-L-r G el
`
`C 6.5-F pe-r G el
`
`C 6.5-F dt-r G el
`
`r G el
`
`Medium
`
`C 6.5-L-r G el
`
`C 6.5-F pe-r G el
`
`C 6.5-F dt-r G el
`
`r G el
`
`A
`
`24 h
`
`LC3-I
`LC3-II
`
`b-actin
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`LC3-II/b-actin ratio
`
`B
`
`HMGB1
`b-actin
`
`Figure 8.6 Western blot analysis of cell-killing mechanism of C6.5/rGel immunotox-
`ins on SKOV3 cells. (A) Analysis of PARP cleavage after 24 and 48h of C6.5/rGel
`fusions treatment. (B) Analysis of LC3 after treated with C6.5/rGel fusions. The
`the ratio of LC3-II compared with b-actin.
`histogram shows quantitation of
`(C) Analysis of cell extract and medium for HMGB1 protein after C6.5/rGel treatment
`for 48h.
`
`the immunotoxins activate autophagic signaling
`We next asked if
`in SKOV3 cells. MAP LC3-I, known to be usually present in the cytosol, is
`palmitoylated during autophagy to form membrane-bound LC3-II and
`is associated with autophagosomes. As shown in Fig 8.6A, the ratio of
`LC3-II formation to the b-actin control was increased after treatment with
`the fusion constructs demonstrating 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. 8.6B). Tumor cells that are dying with autophagy selectively
`release the nuclear HMGB1 protein, without displaying characteristics of
`necrosis. These data indicated that the observed cytotoxic effects of C6.5/rGel
`fusions in SKOV3 cells appeared to be mediated not through an apoptotic
`or necrotic mechanisms but by the efficient induction of autophagic cell death.
`
`C6.5/rGel
`2.2.2.10. Antitumor
`xenograft
`in
`fusions
`of
`activity
`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.
`
`IMMUNOGEN 2113, pg. 13
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Mi-Ae Lyu et al.
`
`PBS
`rGelonin (20 mg/kg)
`
`C6.5-L-rGel (20 mg/kg)
`C6.5-Fpe-rGel (20 mg/kg)
`
`C6.5-Fdt-rGel (20 mg/kg)
`
`1500
`
`1250
`
`1000
`
`750
`
`500
`
`250
`
`B
`
`Tumor volume (mm3)
`
`PBS
`
`rGelonin (20 mg/kg)
`
`C6.5-L-rGel (40 mg/kg)
`
`C6.5-Fpe-rGel (40 mg/kg)
`
`C6.5-Fdt-rGel (40 mg/kg)
`
`180
`
`1500
`
`1250
`
`1000
`
`750
`
`500
`
`250
`
`A
`
`Tumor volume (mm3)
`
`0
`
`0
`
`10
`
`20
`
`30
`
`40
`50
`Time (days)
`
`0
`
`0
`
`10
`
`20
`
`30
`
`40
`50
`Time (days)
`
`FITC/rGel
`
`PI/nucleus
`
`C
`
`C6.5-L-rGel
`
`rGelonin
`
`Figure 8.7 The antitumor activity of C6.5/rGel immunotoxins against SKOV3 tumor
`xenografts in nude mice. (A, B) Treatments of SKOV3 flank tumors with C6.5/rGel
`fusions at the dose of 40 and 20mg/kg. Antitumor effects of i.v. injections of PBS, rGel,
`C6.5-L-rGel, C6.5-Fpe-rGel, or C6.5-Fdt-rGel on SKOV3 tumors. Mean tumor vol-
`
`ume was calculated by WLH as measured by digital calipers. (C) Immunofluores-
`
`cence staining of tumor samples after i.v. injection of C6.5-L-rGel and rGel. 24h after
`injection, animal was sacrificed and frozen tumor sections were prepared and detected
`by anti-rGel antibody. Propidium iodide was used for DNA staining.
`
`injection of SKOV3 cells on day 0, and treatment was initiated on day 9
`postinjection when the tumors were well-established. Treatment consisted
`of five i.v. injections every other day. Groups of mice were treated at doses
`of 40 and 20mg/kg for each fusion construct. Control mice were treated
`with PBS or 20mg/kg rGel only. As shown in Fig. 8.7A and B, treatment
`with C6.5-L-rGel exhibited a significant antitumor effect. Mice treated at
`the 40mg/kg dose of C6.5-L-rGel demonstrated a long-lasting antitumor
`effect which lasted more than 1month until the animals were sacrificed.
`With mice treated at the 20mg/kg dose level, 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 40mg/kg of
`
`IMMUNOGEN 2113, pg. 14
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`IPR2014-00676
`
`

`

`Cell-Targeting Fusion Constructs Containing Recombinant Gelonin
`
`181
`
`C6.5-Fpe-rGel, resulted in a significant delay in tumor 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 (20mg/kg) dose level. In
`contrast, mice treated with either dose (40 or 20mg/kg) of C6.5-Fdt-rGel
`showed no specific antitumor effect above that observed with rGel alone.
`We next examined the localization of C6.5-L-rGel and rGel after
`administration to mice bearing SKOV3 tumors. Immunofluorescence
`staining confirmed that the C6.5-L-rGel localized specifically in tumor
`tissue, but no staining was observed in tumors after administration of rGel
`itself (Fig. 8.7C). This suggests that the fusion construct C6.5-L-rGel can
`effectively target tumor cells overexpressing Her2/neu in vivo and can
`demonstrate significant tumor growth suppressive effects in the absence of
`observable toxicity.
`
`2.3. Translational relevance
`
`In conclusion, we have designed and developed several novel immunotox-
`ins containing the human scFv C6.5 and toxin rGel. These agents exhibit
`efficient cytotoxicity for Her2/neu overexpressing tumor cells, and the
`human antibody appears to be virtually identical as an effective carrier of
`rGel toxin compares to the murine e23. The introduction of a furin
`cleavable linker between C6.5 and rGel did not result in improved intrac