Targeting the cancer stroma with a fibroblast activation
`protein-activated promelittin protoxin
`
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`produced significant lysis and growth inhibition of hu-
`man breast and prostate cancer xenografts with mini-
`mal toxicity to the host animal. [Mol Cancer Ther
`2009;8(5):1378–86]
`
`Introduction
`The growth of an epithelial neoplasm requires the formation
`of a supporting tumor stroma to supply nutrients and
`growth factors for tumor cell survival and continued
`growth. This invasive growth is associated with characteris-
`tic changes in the supporting stroma that include the induc-
`tion of tumor blood vessel formation; the recruitment of
`reactive stromal fibroblasts, lymphocytes, and macro-
`phages; the release of peptide-signaling molecules and pro-
`teases; and the production of an altered extracellular matrix
`(1–5). The tumor stroma compartment represents a major
`component of the mass of most carcinomas, with 20% to
`50% commonly seen in breast, lung, and colorectal cancers
`and reaching >90% in carcinomas that have desmoplastic
`reactions (5, 6).
`Reactive tumor stromal fibroblasts differ from fibroblasts
`of normal adult tissues with regard to morphology, gene ex-
`pression profiles, and production of important biological
`mediators such as growth factors and proteases (1, 7, 8).
`A highly consistent trait of tumor stromal fibroblasts is
`the induction of the membrane-bound serine protease fibro-
`blast-activation protein-α (FAP). FAP was originally identi-
`fied as an inducible antigen expressed on reactive stroma
`and given the name Fibroblast Activation Protein. FAP
`was independently identified by a second group as a gela-
`tinase expressed by aggressive melanoma cell lines and was
`given the name “seprase” for surface expressed protease (9).
`Subsequent cloning of FAP and seprase revealed that they
`are the same cell-surface serine protease (10).
`FAP was originally reported to be a cell-surface antigen
`recognized by the F19 monoclonal antibody on human as-
`trocytes and sarcoma cell lines in vitro (11). In one series
`using human tissues, FAP was detected in the stroma of
`over 90% of malignant breast, colorectal, skin, and pancre-
`atic tumors (7, 11). In a small study, FAP was detected in
`the stroma of 7of 7 prostate cancers (12). FAP is also ex-
`pressed by a subset of soft tissue and bone sarcomas (7).
`FAP-positive fibroblasts also accompany newly formed
`tumor blood vessels (10). In nonmalignant tissue, FAP is
`expressed by reactive fibroblasts in wound healing, rheu-
`matoid arthritis, liver cirrhosis, and in some fetal mesen-
`chymal tissues (7). Cheng et al. (13) also showed that,
`such as human FAP, mouse FAP is expressed by reactive
`fibroblasts within human cancer xenografts. In contrast,
`most normal adult tissues show no detectable FAP protein
`expression (7). In a recent study, Ghilardi et al. (14) used
`real-time PCR to quantify gene expression from laser
`
`Mol Cancer Ther 2009;8(5). May 2009
`
`Aaron M. LeBeau,1 W. Nathaniel Brennen,1
`Saurabh Aggarwal,2 and Samuel R. Denmeade1,2,3
`
`Departments of 1Pharmacology and Molecular Sciences and
`2Chemical and Biomolecular Engineering, and 3The Sidney
`Kimmel Comprehensive Cancer Center at Johns Hopkins
`The Johns Hopkins University, Baltimore Maryland
`
`Abstract
`Fibroblast-Activation Protein-α (FAP) is a membrane-
`bound serine protease that is expressed on the surface
`of reactive stromal fibroblasts present within the major-
`ity of human epithelial tumors but is not expressed by
`normal tissues. FAP is a postprolyl peptidase that dif-
`fers from other dipeptidyl prolyl peptidases such as di-
`prolylpeptidase 4 in that it also has gelatinase and
`collagenase endopeptidase activity. Therefore, FAP re-
`presents a potential pan-tumor target whose enzymatic
`activity can be exploited for the intratumoral activation
`of prodrugs and protoxins. To evaluate FAP as a tumor-
`specific target, putative FAP-selective peptide protoxins
`were constructed through modification of the prodo-
`main of melittin, a 26 amino acid amphipathic cytolytic
`peptide that is the main toxic component in the venom
`of the common European honeybee Apis milefera. Me-
`littin is synthesized as promelittin, containing a 22 ami-
`no acid NH2-terminal prodomain rich in the amino acids
`proline and alanine. In this study, peptides containing
`truncated melittin prodomain sequences were tested
`on erythrocytes to determine the optimal prodomain
`length for inhibiting cytolytic activity. Once optimized,
`modified promelittin peptides were generated in which
`previously identified FAP substrate sequences were in-
`troduced into the prodomain. Peptide protoxins were
`identified that were efficiently activated by FAP and se-
`lectively toxic to FAP-expressing cell lines with an IC50
`value in the low micromolar range that is similar to me-
`littin. Intratumoral injection of an FAP-activated protoxin
`
`Received 9/17/08; revised 2/11/09; accepted 2/19/09; published
`OnlineFirst 5/5/09.
`Grant support: NIH grant 5RO1CA124764 to SRD and a DOD prostate
`cancer predoctoral mentorship grant W81XWH-07 (W.N. Brennen).
`The costs of publication of this article were defrayed in part by the
`payment of page charges. This article must therefore be hereby marked
`advertisement in accordance with 18 U.S.C. Section 1734 solely to
`indicate this fact.
`Requests for reprints: Samuel R. Denmeade, Department of Oncology, The
`Johns Hopkins University School of Medicine, Cancer Research Building I,
`Rm 1M43, 1650 Orleans Street, Baltimore, MD 21231. Phone: 410-955-
`8875; Fax: 410-614-8397. E-mail: denmesa@jhmi.edu
`Copyright © 2009 American Association for Cancer Research.
`doi:10.1158/1535-7163.MCT-08-1170
`
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`capture dissected tumor endothelium and found a signifi-
`cant increase in FAP expression compared with normal en-
`dothelium. This suggests that FAP expression may also be
`induced in both reactive tumor stroma and endothelium.
`FAP is a member of the enzyme class known as postprolyl
`peptidases that are uniquely capable of cleaving the Pro-Xxx
`amino acid bond (15). This group of proteases includes the
`well-characterized dipeptidyl peptidase 4 (DPP4) as well as
`DPP2, DPP6, DPP7, DPP8, DPP9, prolyl carboxypeptidase,
`and prolyl endopeptidase. The substrate preferences for
`many of these prolyl peptidases are not entirely known
`but, such as DPP4, they all have dipeptidase activity. Like
`DPP4, FAP is a type II integral membrane protein able to
`cleave peptides containing proline as the penultimate amino
`acid. FAP differs from DPP4 in that it also has gelatinase
`and collagenase activity (16). This additional gelatinase/col-
`lagenase activity may be unique to FAP among the family of
`prolyl proteases.
`The selective tumor expression and unique enzymatic
`activity of FAP make it a potentially attractive therapeutic
`target. Recently, our laboratory mapped all of the FAP
`cleavage sites in recombinant human gelatin and identified
`a series of peptide substrates that are efficiently cleaved by
`FAP (17). These peptide substrates can be coupled to cyto-
`toxic small molecules to make FAP-activated prodrugs. Al-
`ternatively, the peptides could be incorporated into the
`activation domain of cytolytic proteins and peptides to
`produce FAP-activated protoxins. In this regard, we have
`generated an FAP-activated peptide toxin by incorporating
`an FAP-selective peptide sequence into the prodomain of
`the cytolytic peptide melittin.
`Melittin, a 26 amino acid amphipathic peptide, is the
`main toxic component in the venom of the common Euro-
`pean honeybee Apis milefera (18). The ability of melittin to
`induce the lysis of prokaryotic and eukaryotic cells has been
`well-documented (19–21). The exact mechanism by which
`melittin disrupts both natural and synthetic phospholipid
`bilayers is still largely unknown. In an aqueous milieu, me-
`littin has a net + 6 charge and exists as a random coiled
`monomer. It has been suggested that melittin can produce
`its toxic effects either by forming a transmembrane pore
`structure made up of melittin aggregates or by binding to
`the membrane surface and acting in a detergent-like manner
`leading to an increase in membrane permeability (18, 21).
`In the honeybee, melittin is secreted into the venom
`glands as promelittin possessing an NH2-terminal prodo-
`main made up of 22 amino acids. The prodomain is highly
`negatively charged containing nine acidic amino acid resi-
`dues (22). The presence of the prodomain confers an overall
`negative charge to the molecule and decreases the ability of
`melittin to interact with the surface of the cell membrane. In
`the prodomain amino acid sequence, every second amino
`acid is either proline or alanine. Promelittin activation
`in vivo is the result of the stepwise cleavage of the prodo-
`main into 11 dipeptide fragments by a DPP4-like protease
`present in honeybee venom gland extracts (22). By acetylat-
`ing the promelittin peptide or adding an extra amino acid
`residue at the NH2 terminus, the stepwise activation of pro-
`
`Mol Cancer Ther 2009;8(5). May 2009
`
`melittin by DPP4 dipeptidase activity is prevented. This ob-
`servation suggested that the promelittin prodomain could
`be readily reengineered to produce a prodomain that can
`be removed by a non–DPP4-like endopeptidase such as
`FAP. In this study, we report studies done to determine
`the minimal prodomain length required to inactivate the
`cytolytic activity of melittin. Subsequently, we substituted
`putative FAP peptide substrates into this truncated prodo-
`main to identify an FAP-melittin peptide that is selectively
`toxic to FAP-producing cells. Finally, we evaluated the anti-
`tumor effect of an FAP-melittin protoxin after intratumoral
`injection of peptide into human prostate and breast cancer
`xenografts.
`
`Materials and Methods
`All reagents for Fmoc solid-phase peptide synthesis were
`purchased from Anaspec. Unless stated otherwise, all other
`reagents were purchased from Sigma. His-tagged FAP lack-
`ing the transmembrane domain was produced and purified
`in our laboratory as previously described (17). FAP activity
`was confirmed through activation of the dipeptide substrate
`Ala-Pro-AFC.
`Cell Lines
`The human prostate cancer cell line LNCaP and the hu-
`man breast cancer cell line MCF-7 were purchased from
`American Type Culture Collection. LNCaP was maintained
`in RPMI 1640 and MCF-7 in DMEM media supplemented
`with 10% serum, 1% pen/strep, and 2 mmol/L L-glutamine
`(Invitrogen) in a 37°C incubator with 5% CO2 and 98% hu-
`midity as previously described (23).
`Generation of FAP-Transfected Cells
`The full-length human FAP cDNA was generated as pre-
`viously described (17) and cloned into the multiple cloning
`site of a pIRESneo3 vector (Clontech). Neomycin-selected
`colonies were obtained and evaluated for FAP expression
`through fluorescence-activated cell sorting analysis using
`supernatant from an anti-FAP F19 monoclonal antibody
`producing hybridoma line obtained from American Type
`Culture Collection as the primary antibody. Colonies ex-
`pressing the highest levels of FAP were expanded and main-
`tained under neomycin selection for use in in vitro studies.
`Peptide Synthesis
`Promelittin peptides were synthesized on Fmoc-Gln(Trt)
`Rink amide 4-methyl benzhydrylamine resin and were elon-
`gated using standard Fmoc solid-phase peptide conditions
`on an AAPPTEC Apex 396 peptide synthesizer as previous-
`ly described (24). The prodomain for each peptide was of
`variable length, but the mature melittin peptide sequence,
`NH2-GIGAVLKVLTTGLPALISWIKRKRQQ-NH2, was the
`same for each peptide. The cleavage and deprotection of
`the peptides from the resin were carried out using a cleav-
`age cocktail of trifluoroacetic acid/thioanisole/water/phe-
`nol/EDT (82.5:5:5:5:2.5, v/v) for 4 h. The peptides were
`precipitated from the cleavage cocktail using cold ether
`and dissolved in water for reversed-phase high-perfor-
`mance liquid chromatography purification. Reversed-phase
`high-performance liquid chromatography purification was
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`Table 1. Prodomain amino acid sequence of promelittin peptides
`
`Toxin
`
`HD50 (μmol/L) Net charge
`
`>100
`PM11 APEPEPAPEPEAEADAEADPEA
`>100
`PM11a PEPEPAPEPEAEADAEADPEA
`>100
`PM10
`EPEPAPEPEAEADAEADPEA
`>100
`PM10a
`PEPAPEPEAEADAEADPEA
`PM9
`EPAPEPEAEADAEADPEA 95.5 ± 3.4
`PM9a
`PAPEPEAEADAEADPEA 73.0 ± 4.7
`PM8
`APEPEAEADAEADPEA 64.0 ± 4.2
`PM8a
`PEPEAEADAEADPEA 59.3 ± 2.7
`PM7
`EPEAEADAEADPEA 66.6 ± 2.9
`PM7a
`PEAEADAEADPEA 52.0 ± 2.3
`PM6
`EAEADAEADPEA 55.9 ± 3.5
`PM6a
`AEADAEADPEA 48.4 ± 1.9
`PM5
`EADAEADPEA 37.6 ± 2.5
`PM5a
`ADAEADPEA 29.2 ± 1.8
`PM4
`DAEADPEA 22.3 ± 1.1
`PM4a
`AEADPEA 11.8 ± 0.6
`PM3
`EADPEA
`8.6 ± 0.3
`PM3a
`ADPEA
`6.2 ± 0.2
`PM2
`DPEA
`4.7 ± 0.3
`PM2a
`PEA
`1.8 ± 0.1
`PM1
`EA
`1.7 ± 0.1
`PM1a
`A
`1.5 ± 0.1
`PM0
`1.3 ± 0.1
`
`−3
`−3
`−3
`−2
`−2
`−1
`−1
`−1
`−1
`0
`0
`1
`1
`2
`2
`3
`3
`4
`4
`5
`5
`6
`6
`
`NOTE: HD50, concentration required to lyse 50% of RBC in a 2% RBC solu-
`tion. Charge, net charge on the full length peptide.
`
`done on a Waters Δ 600 semiprep system using a Phe-
`nomenex Luna 10u C18 250 × 10 mm semiprep column.
`The high-performance liquid chromatography gradient
`profile was linear starting at 100% solvent A (0.1% tri-
`fluoroacetic acid in H2O) and changing to 100% solvent
`B (0.1% trifluoroacetic acid in acetonitrile) over 25 min
`with a flow rate of 8 mL/min. Fractions of the desired
`purity (>95% as determined using an analytic reversed-
`phase high-performance liquid chromatography) were
`pooled and lyophilized. The purified promelittin peptides
`were mass analyzed on an Applied Biosystems Voyager
`DE-STR MALDI-TOF mass spectrometer at the Johns
`Hopkins School of Medicine Mass Spectrometry and Pro-
`teomics Facility using a matrix of 10 mg/mL 2,5-dihy-
`droxy benzoic acid in 50% ethanol/water. The mass
`spectrometer was calibrated using the ProteoMass Peptide
`MALDI Calbration kit (Sigma). All spectra were acquired
`in the positive ion mode.
`Hemolysis Assays
`Hemolysis assays were done as previously described
`(23). Briefly, peptides were dissolved in DMSO and seri-
`ally titrated by 2-fold dilution using 1× PBS buffer. The
`peptides were incubated over a range of concentrations
`with washed human RBC at a concentration of 2% v/v
`for 1 h at 37°C. The control for zero hemolysis was RBCs
`suspended in PBS buffer alone, and the 100% hemolysis
`control consisted of RBCs in the presence of 1% Triton
`X-100. After incubation with the peptides, the RBCs were
`pelleted and 50 μL of each sample were transferred in
`triplicate to a clear flat-bottomed 96-well polystyrene
`
`plate. Hemolysis was assessed by measuring the absor-
`bance of the samples at 540 nm with a Molecular Devices
`Spectra Max Plus automatic plate reader.
`Promelittin FAP Digestion
`One hundred micrograms of each promelittin peptide
`were incubated with 2 μg of purified FAP in 200 μL of
`FAP assay buffer containing 100 mmol/L Tris, 100 mmol/L
`NaCl (pH 7.8) at 37°C. Aliquots of the digests were taken
`every hour for 8 h, desalted using P10-C18 ZipTips (Milli-
`pore), and spotted (0.5 μL) on a MALDI-TOF plate using
`the 2,5-dihydroxy benzoic acid matrix. Spectra were collected
`on an Applied Biosystems Voyager DE-STR MALDI-TOF
`mass spectrometer in positive ion mode.
`Cytotoxicity Assays
`Assays were done using MCF-7 breast cancer cells trans-
`fected with a full-length FAP expression vector. Vector only–
`transfected MCF-7 cells served as a control. Cells were
`exposed to peptides over a range of concentrations for
`72 h prior and then cell viability was determined using an
`3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
`mide cell proliferation assay (Promega) as previously de-
`scribed and according to manufacturer's instructions (23).
`In vivo Assays: Tumor Xenograft Studies
`Mouse care and treatment was approved by and done in
`accordance with the guidelines of the Animal Care and Use
`Committee of the Johns Hopkins University School of Med-
`icine. Cells maintained under standard conditions were de-
`tached by treatment with 0.25% trypsin-EDTA solution and
`washed in HBSS. They were then suspended in a 60% mix-
`ture of Matrigel Matrix (BD Biosciences) in HBSS at a con-
`centration of 2.0 × 106 cells per 100 μL of solution. LNCaP
`cells were then injected into the subcutis overlying the rear
`flanks of 6-week-old male nude mice (Harlan). MCF-7 cells
`were injected s.c. into 6-wk-old female nude mice previous-
`ly implanted s.c. with a slow release estrogen pellet (0.72 mg
`of 17β-estradiol; Innovative Research of America) in the
`contralateral flank. Weekly tumor measurements were
`made with calipers and the tumor volume (in cm3) was cal-
`culated by the formula 0.5236 × L × W × H. The mice were
`euthanized by CO2 overdose, and the tumors were weighed
`and processed for histochemical analysis as previously de-
`scribed (23). Balb-c mice (Harlan) were used for i.v. toxicity
`studies as previously described (23).
`Statistical Analysis
`For the in vitro proliferation studies, P values were de-
`rived from the Student's t test. All statistical tests were
`two-sided, and P value of <0.05 was considered to be statis-
`tically significant. For the in vivo studies, data, presented as
`mean ± SE, were evaluated using ANOVA analysis. P value
`of <0.05 was considered statistically significant.
`
`Results
`Promelittin Prodomain Truncation
`A total of 22 promelittin peptides, representing every
`possible prodomain length, were synthesized (Table 1). Us-
`ing the truncated promelittin peptides, we investigated
`how much of the prodomain was necessary to inhibit the
`
`Mol Cancer Ther 2009;8(5). May 2009
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`cytolytic ability of melittin. The goal was to find the min-
`imal length melittin prodomain that could be subsequently
`modified to produce the minimal length FAP-activated me-
`littin peptide toxin. Whereas PM11 represents the full-
`length prodomain, peptides PM0-PM10 represent products
`of the stepwise two amino acid cleavage of promelittin by
`DPP4. Peptides PM1a-PM11a are non-DPP4 substrates be-
`cause they do not contain dipeptide units at the NH2 ter-
`minus ending with either proline or alanine. To assess the
`relative degree of inhibition of the lytic ability of each pro-
`melittin peptide toward eukaryotic cells, human erythro-
`cytes were used as a model membrane. The hemolytic
`dose necessary to lyse 50% of the erythrocytes (i.e., HD50)
`was determined for each promelittin peptide (Table 1).
`These studies revealed that the promelittin peptides
`containing the longest prodomains were the least hemolytic
`toward human erythrocytes. The full-length promelittin
`peptide (PM11), PM11a, PM10, and PM10a, all had HD50
`values above 100 μmol/L. Appreciable hemolysis was not
`
`observed until approximately half of the pro-domain had
`been removed. PM6, with a 12 amino acid prodomain se-
`quence and a net charge of 0, had an HD50 of 55.9 μmol/L.
`As the pro-domain sequence decreased one amino acid at a
`time and the net negative charge of the peptide increased,
`the HD50 for each peptide steadily decreased (Table 1). PM0
`(melittin) was found to have an HD50 of 1.3 μmol/L. Like-
`wise, the 7 shortest promelittin peptides were hemolytic with
`HD50 values at or below 10 μmol/L.
`Based on these results, the 14 amino acid pro-domain
`length of PM7, which had an HD50 of 66.6 μmol/L,
`was selected for further studies aimed at developing an
`FAP-activated toxin. PM7 was found to be ∼50-fold less
`hemolytic than the fully processed melittin. Although lon-
`ger length prodomains had higher HD50 values in the he-
`molysis assay, the 40 amino acid PM7 was selected
`because this starting peptide length allowed for the intro-
`duction of modifications and additions to the prodomain
`that would produce peptides that were <50 amino acids
`
`Figure 1.
`FAP cleavage of the modified promelittin peptides. The prodomain sequence of modified protoxins (FAP 1-5) with cleavage site and mass of
`cleavage fragment delineated. MALDI-TOF analysis was used to evaluate the extent of cleavage. The relative intensity of each mass fragment is based on a
`comparison of the relative peak height for each individual trace, with the largest peak for each experiment arbitrarily set to 100. Bottom, representative
`MALDI trace for FAP1 and FAP2 (100 μg) at time 0 and 8 h after exposure to active FAP (2 μg total).
`
`Mol Cancer Ther 2009;8(5). May 2009
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`in length. Peptides longer than 50 amino acids were tech-
`nically difficult to synthesize and this precluded the use
`of longer length promelittins (e.g., PM11) as the starting
`sequence.
`Generation of an FAP-Cleavable Promelittin Protoxins
`Previous studies in our laboratory and others have
`documented that the most preferred FAP-cleavable pep-
`tide sequences contain Pro in the P1 position and Gly in
`the P2 position with a suggestion that Ala in the P′1
`position is also favored (17, 25). Based on our previous
`studies characterizing FAP cleavage substrates from a
`map of cleavage sites within human gelatin, five candidate
`protoxins were synthesized using the prodomain of PM7
`(i.e., FAP1-5; Fig. 1A; ref. 16). In FAP1, the Asp-4 of the
`PM7 prodomain was changed to a Gly to reproduce the
`Gly-Pro preference in the P1 and P2 positions ascribed
`to FAP (17). Because the effect on the ability of FAP to hy-
`drolyze a peptide containing an acidic Glu residue in the
`P1 position was not known, FAP2 was designed such that
`the prodomain sequence was kept the same as that for
`FAP1 with the exception that Glu-2 of the prodomain of
`FAP1 was removed to create the FAP preferred P2-P1-P′1
`sequence of Gly-Pro-Ala. For FAP3, the P2-P1-P′1 sequence
`of Gly-Pro-Ala was inserted between the NH2 terminus of
`melittin and the full-length native PM7 prodomain se-
`quence. FAP4 had a seven amino acid FAP cleavable peptide
`substrate (SGEAGPA) inserted between the NH2 terminus
`and the PM7 prodomain, whereas FAP5 had a repetitive
`(Pro-Gly-Pro)2 motif inserted between the NH2 terminus of
`melittin and the prodomain of PM7. FAP4 and FAP5 were
`the two largest peptides synthesized, 46 and 47 amino acids,
`respectively. FAP2 was the shortest, consisting of only 39
`amino acids. The hemolytic activity of these FAP candidate
`protoxins was assayed and all were found to have HD50
`values between 50 and 70 μmol/L (Table 2).
`FAP Cleavage Assays
`To assess FAP cleavage, the FAP candidate protoxins
`were assayed in vitro with purified recombinant FAP to
`characterize the extent of FAP-mediated cleavage. The pep-
`tides (100 μg) were digested with FAP (2 μg) for a total of
`8 hours at 37°C. Every 2 hours, aliquots were taken and the
`progress of the digest was monitored using matrix-assisted
`laser desorption/ionization time-of-flight (MALDI-TOF)
`mass spectrometry (Fig. 1). After 8 hours, the only protoxin
`that was completely digested by FAP was FAP2 (Fig. 1). The
`digested FAP2 yielded only 1 cleavage product with a mass
`of 2,918.32 m/z, corresponding to the hydrolysis of the Gly-
`Pro↓Ala bond. FAP1, which differed from FAP2 by only one
`Glu residue, did show some of the desired cleavage prod-
`uct at 3,046.89 m/z (Fig. 1). However, the FAP1 digest was
`incomplete, leaving uncut starting material and other
`cleavage by-products. FAP3, FAP4, and FAP5 were
`cleaved to varying degrees, but none were cleaved as well
`as FAP2 (Fig. 1). Finally, although mature melittin also
`contains an internal proline residue, MALDI-TOF anal-
`ysis showed that it was not cleaved by FAP (data not
`shown).
`
`FAP Promelittin Protoxins Selectively Kill FAP-
`Expressing Human Breast Cancer Cell Lines
`To evaluate the selectivity of each FAP-activated protoxin
`for the ability to kill FAP-positive versus FAP-negative can-
`cer cells, we transfected the human breast cancer cell line
`MCF-7 with either FAP or vector only controls. These cells
`were then used to assess the effect of each protoxin on
`growth as assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
`phenyltetrazolium bromide assay. In this assay, mature me-
`littin showed no selectivity and was able to kill both cell
`lines at approximately equally low micromolar concentra-
`tions (Table 2). Compared with melittin, the modified pro-
`melittin peptides were ∼30- to 40-fold less toxic against the
`vector only–transfected FAP-negative MCF-7 cells. In con-
`trast, against the transfected FAP-producing cell line,
`FAP2 was the most toxic peptide surveyed with an IC50 of
`5.2 μmol/L. This peptide was also the most selective and
`was ∼7-fold more active against the FAP-positive versus
`FAP-negative MCF-7 cells. All of the other promelittin pep-
`tides had fold differences in cytotoxicity of less than two
`(Table 2). FAP2 was the only protoxin in this series that
`showed a significant therapeutic index in vitro.
`To eliminate the potential for nonspecific cleavage of the
`FAP2 sequence by DPP4, we subsequently generated a
`DPP4-“resistant” version of FAP2 by adding an acetylated
`NH2-terminal glycine to the FAP2 peptide to generate Ac-
`FAP6. Ac-FAP6 was cleaved by FAP to the same extent as
`FAP2 (data not shown) and had the highest HD50 (72
`μmol/L) of all of the FAP-activated protoxins (Table 2). This
`acetylated peptide showed increased specificity with an
`IC50 of 47.9 versus 35.1 μmol/L for FAP2 against FAP-neg-
`ative cells. However, Ac-FAP6 was nearly as potent as FAP2
`with an IC50 value of 6.1 μmol/L against FAP-positive cells
`for an overall higher ∼8-fold difference in toxicity against
`FAP-positive and FAP-negative cells.
`In vivo Antitumor Activity of FAP Promelittin Protoxins
`Before performing in vivo efficacy studies, we did toxicity
`studies in vivo with the administration of the protoxins i.v.
`and intratumorally. Melittin is a nonspecific cytolytic
`toxin. Therefore, as expected, melittin was highly toxic to
`mice with an i.v. LD100 (i.e., single dose that kills 100% of
`
`Table 2. HD50 values and cytotoxicity of FAP Melittin protoxins
`against FAP-negative and FAP-positive MCF-7 human breast
`cancer cells
`
`Toxin
`
`HD50 (μmol/L)
`
`FAP1
`FAP2
`FAP3
`FAP4
`FAP5
`Ac-FAP6
`Melittin
`
`56.9 ± 3.1
`54.2 ± 2.2
`60.0 ± 3.7
`70.5 ± 5.1
`67.5 ± 3.5
`72.2 ± 3.6
`1.3 ± 0.1
`
`IC50 (μmol/L)
`FAP pos
`
`Fold diff
`
`26.8 ± 1.1
`5.2 ± 0.4
`18.9 ± 1.7
`28.1 ± 2.2
`18.3 ± 2.0
`6.1 ± 0.3
`1.3 ± 0.2
`
`1.7
`6.7
`1.8
`1.8
`1.5
`7.9
`1.1
`
`FAP neg
`
`45.6 ± 5.6
`35.1 ± 2.0
`33.1 ± 2.3
`50.1 ± 4.9
`27.6 ± 1.2
`47.9 ± 2.9
`1.4 ± 0.1
`
`Abbreviations: FAP neg, FAP negtive; FAP pos, FAP positive; fold diff, fold
`difference.
`
`Mol Cancer Ther 2009;8(5). May 2009
`
`Petitioner GE Healthcare – Ex. 1038, p. 1382
`
`

`

`Molecular Cancer Therapeutics
`
`1383
`
`Figure 2. Nude mice bearing LNCaP human
`prostate cancer xenografts on both flanks were
`treated with single intratumoral dose of FAP2 at
`the indicated concentration of FAP2 into the tu-
`mor in one flank, with saline injected into the tu-
`mor on the contralateral flank. Representative
`tumor response is shown for 1 animal at each dose
`level over a 34-d period.
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`
`animals) of ∼3 mg/kg. i.v., a dose of 1 mg/kg of melittin
`was the maximally tolerated dose. In contrast, for PM11,
`FAP2, and Ac-FAP6, a single dose of 40 mg/kg i.v. was tol-
`erated in Balb-c mice, whereas a dose of 100 mg/kg was
`100% lethal. The LD100 for FAP2 was subsequently found
`to be lower in tumor-bearing nude mice used in efficacy ex-
`periments as a single 40 mg/kg i.v. dose proved lethal to all
`mice within 1 week posttreatment. For the intratumoral in-
`jection studies, the maximum tolerated dose of intratumoral
`melittin was determined to be 5.7 mg/kg (50 nmoles). In
`contrast, an intratumoral dose of 40 mg/kg (250 nmoles)
`of FAP2 was well-tolerated, whereas a dose of 200 mg/kg
`(1,250 nmoles) was lethal to ∼33% of treated animals by 24
`hours posttreatment.
`A number of studies have documented that FAP expres-
`sion in mouse stromal cells occurs in a wide variety of hu-
`man cancer xenografts (13, 26, 27). On the basis of these
`dose finding studies, an initial cohort of animals (n = 6
`per group) bearing LNCaP human prostate cancer xeno-
`grafts received a single intratumoral injection of either 40
`or 200 mg/kg of FAP2 (Fig. 2). Tumors were then imaged
`serially over a 34-day period. Representative results are
`shown in Fig. 2. Treated tumors developed a necrotic center
`and overlying eschar, which eventually healed as the under-
`lying tumor regressed over the observation period. Com-
`plete regressions were observed in select animals in the
`200 mg/kg group (Fig. 2), but this dose level also resulted
`in the death of 1 of 3 of the treated animals. No complete
`regressions were observed in the 40 mg/kg dosing group.
`The next experiment was designed to compare the extent
`of FAP specific versus nonspecific killing after the injection
`of a series of promelittin toxins into human MCF-7 breast
`cancer xenografts. This line was selected based on previous
`studies demonstrating that MCF-7 possesses a moderate
`amount of stroma and can induce expression of human
`FAP by human fibroblasts coinoculated with MCF-7 cells
`(28). For these studies, we compared the single dose
`efficacy of Ac-FAP6 (FAP-activated, DPP4-resistant, HD50
`
`Mol Cancer Ther 2009;8(5). May 2009
`
`of 72 μmol/L) to PM11 (FAP-resistant, DPP4-activated,
`HD50 of >100 μmol/L). In addition, to generate a toxin that
`would not be cleaved by FAP or DPP4, we evaluated the ef-
`fects of acetylating the amino terminus of the PM toxins. In
`this analysis, we determined that acetylation can lower the
`HD50 compared with the unacetylated protoxin in some
`instances. From this analysis, we selected the acetylated ver-
`sion of sequence PM9 for in vivo studies because this acetylat-
`ed protoxin had the highest HD50 of all of the acetylated
`peptides tested. Although PM9 had an HD50 of 95 μmol/L,
`Ac-PM9 had an HD50 of 76 μmol/L, which was similar to
`the HD50 for Ac-FAP-6. Like Ac-FAP6, Ac-PM 9 is not a sub-
`strate for dipeptidyl peptidase IV due to acetylation of the
`amino terminus and is not cleaved by FAP due to lack of
`the FAP-preferred Gly-Pro dipeptide in the prodomain.
`Therefore Ac-PM9 can be considered FAP resistant and
`DPP4 resistant.
`Based on previous toxicity studies, tumor-bearing ani-
`mals were treated with a single intratumoral dose of 250
`nmoles (∼40-50 mg/kg) of each of these promelittin toxins.
`Tumors (n = 3 per group) were then harvested 96 hours
`postinjection, fixed, and stained. Areas of necrotic tissue
`were easily seen under low-power magnification with high-
`er power magnification demonstrating areas with pyknotic
`nuclei in a field of cellular debris (Fig. 3A). Under low-
`power magnification using image analysis, the total area
`of the tumor slice was determined as previously described
`(23). Subsequently, the total area of nonviable tumor tissue
`was determined and the % area of necrosis was determined
`from the ratio of these two areas (Fig. 3B). Using this meth-
`odology, injection of PM11 resulted in tumors with ∼25%
`necrosis of total tumor cross-sectional area, which was not
`significantly different than the 16% necrosis seen in control
`tumors injected with saline (Fig. 3C). Ac-PM9 induced ne-
`crosis that was not significantly different than that seen
`for PM11 (Fig. 3C). In contrast, Ac-FAP6 injection resulted
`in significant increase in the area of necrosis with ∼60% ne-
`crosis of tumors at 96 hours postinjection, consistent with
`
`Petitioner GE Healthcare – Ex. 1038, p. 1383
`
`

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`
`1384
`
`FAP-Activated Promelittin Protoxin
`
`the enhanced distribution and activation of the Ac-FAP6
`toxin compared with the non-FAP activated PM11 and Ac-
`PM9 toxins (Fig. 3C).
`In the final experiment, the effect of Ac-FAP6 on the
`growth rate of tumors after intratumoral injection was com-
`pared with the growth rate of saline injected controls
`(Fig. 4A). Studies were done using an intratumoral treat-
`ment approach to evaluate the full extent of activation of
`the FAP-activated toxin within tumor tissue. After random-
`ization to afford groups of relatively equal starting average
`tumor size, animals (n = 8 per group) received a single in-
`tratumoral injection of eit