372
`
`Bioconjugate Chem. 1998, 9, 372-381
`
`Role of Cross-Linking Agents in Determining the Biochemical and
`Pharmacokinetic Properties of Mgr6-Clavin Immunotoxins
`
`Franco Dosio,† Silvia Arpicco,† Elena Adobati,‡ Silvana Canevari,‡ Paola Brusa,† Rita De Santis,§
`Dino Parente,§ Paola Pignanelli,§ Donatella R. M. Negri,‡ Maria I. Colnaghi,‡ and Luigi Cattel*,†
`
`Dipartimento di Scienza e Tecnologia del Farmaco, University of Torino, Torino, Italy, Divisione di Oncologia
`Sperimentale E, Istituto Nazionale per lo Studio e la Cura dei Tumori, Milano, Italy, and Dipartimento di
`Biotecnologie, Menarini Ricerche Sud, Pomezia, Italy. Received October 31, 1997;
`Revised Manuscript Received January 27, 1998
`
`Several immunotoxins (ITs) were synthesized by the attachment of clavin, a recombinant toxic protein
`derived from Aspergillus clavatus, to the monoclonal antibody Mgr6 that recognizes an epitope of the
`gp185HER-2 extracellular domain expressed on breast and ovarian carcinoma cells. Conjugation and
`purification parameters were analyzed in an effort to optimize the antitumor activity and stability of
`the ITs in vivo. To modulate the in vitro and in vivo properties of the immunotoxins, different coupling
`procedures were used and both disulfide and thioether linkages were obtained. Unhindered and
`hindered disulfide with a methyl group linkage ethyl S-acetyl 3-mercaptopropionthioimidate ester
`hydrochloride (AMPT) or ethyl S-acetyl 3-mercaptobutyrothioimidate ester hydrochloride (M-AMPT)
`were obtained by reaction with recombinant clavin, while the monoclonal antibody Mgr6 was
`derivatized with ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate ester hydrochloride (CDPT).
`To achieve higher hindrance (a disulfide bond with a geminal dimethyl group), Mgr6 was derivatized
`with the N-hydroxysuccinimidyl 3-methyl-3-(acetylthio)butanoate (SAMBA) and clavin with CDPT.
`To evaluate the relevance of the disulfide bond in the potency and pharmacokinetic behavior of the
`ITs, a conjugate consisting of a stable thioether bond was also prepared by derivatizing Mgr6 with
`the N-hydroxysuccinimidyl ester of iodoacetic acid (SIA) and clavin with AMPT. The immunotoxins
`were purified and characterized using a single-step chromatographic procedure. Specificity and
`cytotoxicity were assayed on target and unrelated cell lines. The data indicate that the introduction
`of a hindered disulfide linkage into ITs has little or no effect on antitumor activity and suggest that
`disulfide cleavage is essential for activity; indeed, the intracellularly unbreakable thioether linkage
`produced an inactive IT. Analysis of IT stability in vitro showed that the release of mAb by incubation
`with glutathione is proportional to the presence of methyl groups and increases exponentially with
`the increase in steric hindrance. Analysis of the pharmacokinetic behavior of ITs in Balb/c mice given
`intravenous bolus injections indicated that ITs with higher in vitro stability were eliminated more
`slowly; i.e., the disulfide bearing a methyl group doubled the (cid:226)-phase half-life (from 3.5 to 7.1 h)
`compared with that of the unhindered, while a geminal dimethyl protection increased the elimination
`phase to 24 h. The thioether linkage showed its intrinsic stability with a (cid:226)-phase half-life of 46 h.
`The thioether linkage also increased the distribution phase from 17 to 32 min. The in vitro
`characteristics and in vivo stability of Mgr6-clavin conjugates composed of a methyl and dimethyl
`steric hindered disulfide suggest clinical usefulness.
`
`INTRODUCTION
`The potent and highly specific cytocidal activity of
`conjugates between monoclonal antibodies and toxins has
`raised the promise of effective immunotherapy for tumors
`and graft-versus-host disease.
`ITs1 have particular
`potential in the treatment of leukemias and lymphomas
`(1, 2), although some evidence suggests they are effective
`against solid tumors as well (3, 4).
`Extremely active ITs have been constructed by co-
`valently linking mAb to bacterial toxins such as Pseudomo-
`nas exotoxin or to plant toxins such as the ricin A chain,
`saporin, and gelonin (5-8).
`
`* Correspondence should be addressed to Luigi Cattel, Di-
`partimento di Scienza e Tecnologia del Farmaco, v. P. Giuria 9,
`10125 Torino, Italy. Phone: ++39-11-6707697. Fax: ++39-11-
`6707695. E-mail: dosio@pharm.unito.it.
`† University of Torino.
`‡ Istituto Nazionale per lo Studio e la Cura dei Tumori.
`§ Menarini Ricerche Sud.
`
`Other interesting ribosome-inactivating proteins (RIPs)
`with potent antitumor activity such as R-sarcin, restric-
`tocin, and mitogillin have been isolated from fungi and
`used to prepare ITs (9-11). RIPs of fungi are extensively
`homologous in their primary structure (>80%) and have
`the same catalytic activity, i.e., hydrolysis of the phos-
`phodiester linkage between the G residue at position
`4325 and the A residue at position 4326 in 28S rRNA
`(12).
`
`1 Abbreviations: ITs, immunotoxins; AMPT, ethyl S-acetyl-
`propionthioimidate; M-AMPT, ethyl S-acetyl-3-mercaptobuty-
`rothioimidate; CDPT, 3-[(4-carboxamidophenyl)dithio]propi-
`onthiomidate; SAMBA, N-hydroxysuccinimidyl 3-methyl-3-(acet-
`ylthio)butanoate; SIA, N-hydroxysuccinimidyl ester of iodoacetic
`acid; RIP, ribosome-inactivating protein; DTNB, Ellman’s re-
`agent; DTT, dithiothreitol; TNB, 5-mercapto-2-nitrobenzoic acid;
`SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel elec-
`trophoresis; GSH, reduced glutathione; SMPT, 4-[(succinimidyl-
`oxy)carbonyl]-R-methyl-R-(2-pyridyldithio)toluene.
`
`© 1998 American Chemical Society
`S1043-1802(97)00192-4 CCC: $15.00
`Published on Web 04/16/1998
`
`IMMUNOGEN 2078, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Properties of Mgr6-Clavin Immunotoxins
`
`Recently, Parente et al. (13) described the cloning and
`expression of a new RIP, clavin, from Aspergillus clava-
`tus. Clavin was highly active on free ribosomes, inducing
`low and transient systemic toxicity and a late, low-level
`antibody response in mice. These characteristics and its
`availability in large amounts identify clavin as a good
`candidate for IT preparation.
`To selectively target clavin to breast and ovarian
`carcinoma cells, we used a mAb (Mgr6) that recognizes
`an epitope of the gp185HER-2 extracellular domain (14).
`The tissue distribution of gp185HER-2 in normal human
`tissues is restricted, and overexpression of the HER-2
`oncogene is associated with poor prognosis in breast and
`ovarian carcinoma patients (15, 16). Existing ITs di-
`rected to gp185HER-2 have already shown immunothera-
`peutic promise (17-19).
`In this study, we analyzed the conjugation and puri-
`fication parameters that might yield efficient and stable
`ITs suitable for preclinical studies.
`We prepared various ITs and characterized them with
`respect to coupling systems (disulfide or thioether link-
`age). In fact, most of the ITs prepared to date have a
`reducible disulfide bond which is required for the intra-
`cellular release of the toxic moiety (20). The major in
`vivo limitations of such ITs rest in the instability of the
`chemical linkage, which restricts the amount of conjugate
`that is able to bind to target cells (21, 22), and in the
`potential competition of the released antibody with the
`intact conjugate since the mAb persists longer in circula-
`tion. Previously released antibody may mask tumor
`antigens, thereby compromising IT potency during mul-
`tiple-dose treatment regimens.
`In an effort to prolong the in vivo half-life of ITs and
`enhance their antitumor potency, we prepared Mgr6-
`clavin conjugates using the thioimidate cross-linking
`system which preserves the positive charge on the
`derivatized proteins and allows modulation of the hin-
`drance on the chemical linkage between clavin and the
`mAb.
`
`EXPERIMENTAL PROCEDURES
`Materials. Anhydrous dichloromethane, Ellman’s
`reagent (DTNB), and all other reagents were from
`Aldrich (Milwaukee, WI).
`Diethyl ether was distilled from lithium aluminum
`hydride, and dry tetrahydrofuran was obtained by distil-
`lation from sodium.
`General Procedures. Melting points were deter-
`mined with a Reichert Kofler apparatus and are uncor-
`rected. 1H NMR spectra were recorded on a JEOL PMX-
`60 spectrometer, operating at 60 MHz, with tetra-
`methylsilane as the internal standard. IR spectra were
`obtained as KBr disks on a Shimadzu FT-IR 8101 M
`spectrophotometer; wavelengths are given in inverse
`centimeters. Mass spectra were obtained with a FINNI-
`GAN-MAT TSQ-700 or with a VG Analytical 70-70 EQ-
`HF spectrometer. Ultraviolet spectra were recorded on
`a Beckman DU-70 spectrophotometer.
`Reactions were checked on F254 silica gel precoated
`sheets (Merck, Milan, Italy). Purification was carried out
`by column flash chromatography on silica gel 60 (Merck,
`230-400 mesh).
`Synthesis of Cross-Linkers. Preparation of Thio-
`imidate Ester Hydrochlorides (CDPT, AMPT, and
`M-AMPT). Hydrogen chloride gas was bubbled through
`ice-cold ethanethiol (3.25 mL, 0.0435 mol) for 1 h. The
`nitriles 4-[(2-cyanoethyl)dithio]benzamide, 3-(acetylthio)-
`propionitrile, and 3-(acetylthio)butyronitrile, synthesized
`
`Bioconjugate Chem., Vol. 9, No. 3, 1998 373
`
`as described by Arpicco (23), were diluted in anhydrous
`diethyl ether or dry tetrahydrofuran and quickly added
`to the cold solution with stirring, and the reaction
`mixture was left overnight at 0 °C. Anhydrous cold
`diethyl ether was then added, and the reaction mixture
`was left at -20 °C until a crystalline solid formed. The
`supernatant was decanted, and the precipitate was
`washed with anhydrous diethyl ether under argon and
`dried under reduced pressure at room temperature.
`Ethyl 3-[(4-carboxamidophenyl)dithio]propionthioimidate
`ester hydrochloride (CDPT): yield 0.64 g (65%); mp 110-
`115 °C; 1H NMR (CD3OD) (cid:228) 7.85 (q, 4H, Ar-H), 3.4-3.15
`(m, 6H, SCH2CH3 and CH2CH2), 1.47 (t, 3H, SCH2CH3);
`IR (KBr) 3500-2200 (NH2 and NH2
`+), 1650 (CdO), 1620
`(CdN); MS (FAB+) 301 (M+ + 1).
`Ethyl S-acetyl 3-mercaptopropionthioimidate ester hy-
`drochloride (AMPT): yield 0.87 g (88%); mp 64-66 °C;
`1H NMR (CDCl3) (cid:228) 3.48 (q, 2H, SCH2CH3), 3.26 (m, 4H,
`CH2CH2), 2.38 (s, 3H, SAc), 1.45 (t, 3H, SCH2CH3); IR
`(KBr) 3300-2400 (NH), 1690 (CdO), 1620 (CdN), 1360
`(SAc).
`Ethyl S-acetyl 3-mercaptobutyrothioimidate ester hy-
`drochloride (M-AMPT): yield 0.79 g (75%); mp 112 °C;
`1H NMR (CDCl3) (cid:228) 3.4-3.2 (m, 5H, SCH2CH3, CH, and
`CH2), 2.43 (s, 3H, SAc), 1.6 (d, 3H, CH3), 1.43 (t, 3H,
`SCH2CH3); IR (KBr) 3300-2400 (NH), 1690 (CdO), 1620
`(CdN), 1360 (SAc); MS-EI m/z (relative intensity) 206
`(M+, 7), 162 (100), 145 (53), 130 (20), 102 (65), 89 (20),
`75 (33), 61 (52), 43 (100).
`Preparation of N-Hydroxysuccinimidyl 3-Methyl-3-
`(acetylthio)butanoate (SAMBA). The method of Carrol
`(24) was used with minor modifications. Briefly, 3-meth-
`yl-3-(acetylthio)butanoic acid (1.5 g, 0.0085 mol) dissolved
`in 12 mL of dry dichloromethane was mixed with 1.3 g
`of NHS (0.011 mol) in 5 mL of dichloromethane, and
`dicyclohexylcarbodiimide (2.3 g, 0.011 mol) dissolved in
`1 mL of dry dichloromethane was added dropwise. The
`reaction mixture was stirred at room temperature for 22
`h. After filtration and evaporation, the mixture was
`purified by flash chromatography with elution in hexane/
`EtOAc (80/20). The ester was obtained as a pale yellow
`oil that crystallized rapidly at room temperature: yield
`1.6 g (70%); mp 63 °C; TLC (80/20 hexane/EtOAc) Rf )
`0.15; 1H NMR (CDCl3) (cid:228) 3.23 (s, 2H, CH2), 2.80 (s, 4H,
`NHS ester), 2.25 (s, 3H, SAc), 1.6 (s, 6H, CH3); MS-EI
`m/z (relative intensity) 273 (10), 159 (90), 117 (30), 75
`(15), 43 (55).
`The N-hydroxysuccinimidyl ester of iodoacetic acid
`(SIA) was prepared as described (25).
`Clavin and Mgr6 Production. For clavin produc-
`tion, a 100 mL culture of Escherichia coli HB101 cells
`carrying the pMRS38 plasmid was used to start the final
`cultivation step in a Chemap bioreactor with a working
`volume of 1.5 L. The cultivation was performed in
`complex medium [20 g/L glucose, 25 g/L yeast extract,
`40 g/L casamino acid (Difco), 0.5 g/L NaCl, 5 g/L KCl,
`2.6 g/L K2SO4, 0.86 g/L MgCl2(cid:226)6H2O, 6.6 mg/L CaCl2(cid:226)
`6H2O, and trace amounts of oligo elements], supple-
`mented with 100 mg/mL ampicillin. Cultivation param-
`eters such as temperature, pH, and oxygen dissolved in
`tension (DOT) were computer-controlled. The culture
`supernatant was recovered by centrifugation and filtered
`on a Millipore 0.22 (cid:237)m filter. Clavin was purified as
`described (13).
`Mgr6 hybridoma, directed against the extracellular
`domain of gp185HER-2, was obtained by immunization of
`Balb/c mice with the adenocarcinoma cell line CaLu3, and
`its characterization is described elsewhere (14). mAb
`Mgr6 was obtained by cultivating hybridoma cells in a
`
`IMMUNOGEN 2078, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`374 Bioconjugate Chem., Vol. 9, No. 3, 1998
`
`hollow fiber bioreactor (Acusyst R, Endotronics) using a
`serum-free medium. Medium, prepared in our facilities,
`consisted of a basal mixture (1/1) of RPMI 1640 and
`DMEM supplemented with transferrin, insulin, albumin,
`and Ex-Cite (Miles Inc.) as a lipid source. Additional
`ingredients were 2-mercaptoethanol, ethanolamine, and
`sodium selenite.
`Cell cultivation was performed as continuous perfusion
`for approximately 1 month, during which parameters
`such as glucose and lactic acid concentrations, pH, oxygen
`and carbon dioxide levels, and antibody production were
`monitored along with operator responses.
`Conditioned media harvested during cultivation were
`pooled after thawing and adjusted to pH 5.5. Material
`was diluted and filtered on 4.5 (cid:237)m filters (Millipore Co.,
`Bedford, MA) before being loaded on a Bakerbond Abx
`column (Mallinckrodt Baker B. V., Deventer, The Neth-
`erlands). The eluted protein peak was desalted on a
`Sephadex G-25 (Pharmacia-Biotech, Uppsala, Sweden)
`column and loaded onto a Q-Sepharose FF column
`(Pharmacia-Biotech). The eluted peak was desalted as
`indicated above and subjected to the final purification
`step on a S-Sepharose FF column (Pharmacia-Biotech).
`Elution was obtained by applying a linear gradient from
`20 to 200 mM NaCl in 20 mM phosphate buffer.
`Preparation of Immunotoxins. Disulfide Bridge.
`In a standard preparation, a recombinant clavin solution
`(357 (cid:237)M, 1 mL) was stirred with AMPT (122 mM, 30 (cid:237)L
`dissolved in ethanol), M-AMPT (98 mM, 30 (cid:237)L dissolved
`in ethanol), or CDPT (139 mM, 40 (cid:237)L in dry dimethyl-
`formamide) for 30 min at 25 °C to incorporate an average
`of 1-1.3 linkers per mole of clavin. The mixture was
`purified by gel filtration on a 1 (cid:2) 20 cm Bio-Gel P6-DG
`column (Bio-Rad, Hercules, CA) eluted with HBS (0.1 M,
`0.2 M NaCl and 1 mM EDTA disodium salt at pH 7.4) at
`20 °C. The protein solution was concentrated to 1 mL
`with an Amicon concentrator (Amicon, Beverly, MA)
`equipped with a Y10 membrane.
`mAb Mgr6, dissolved in HBS, was separately reacted
`with two different cross-linkers, CDPT and SAMBA. The
`reaction of Mgr6 with CDPT proceeded as follows: CDPT
`in dry dimethylformamide (13 mM, 40 (cid:237)L) was added to
`the mAb in solution (33 (cid:237)M, 1 mL), and the mixture was
`stirred for 30 min at 25 °C. The Mgr6/CDPT molar ratio
`was 1/1.1. Derivatization of Mgr6 with SAMBA (11 mM,
`30 (cid:237)L) dissolved in ethanol was carried out for 30 min at
`25 °C to introduce two acetylthio groups. In both cases,
`the mixture was purified by gel centrifugation on a 15 (cid:2)
`55 mm Bio-Gel P6-DG column preequilibrated in HBS
`at 20 °C.
`The number of thioacetylated groups linked to the
`protein was calculated spectrophotometrically by reaction
`of the sample with the deacetylating reagent hydroxyl-
`amine hydrochloride (0.5 M, 12.5 mM EDTA, pH 7.4)
`followed by thiol disulfide exchange with DTNB as
`described (26).
`The number of aryldithio groups linked to Mgr6 or
`clavin was determined following the release of the
`thiolated anion at 313 nm, after incubation of the protein
`sample (1 mL) with 2-mercaptoethanol in PBS/EDTA (11
`mM sodium phosphate and 3 mM EDTA, 50 (cid:237)L) and
`NaOH (1 M, 40 (cid:237)L), to a final pH of 8.8-9.4. The molar
`absorptivity value of the 4-carboxamidophenylthiolate
`anion under these conditions at 313 nm was 15 200 (
`300 M-1 cm-1.
`All conjugations were performed by mixing the deriva-
`tized mAb and RIP in the presence of a solution of
`hydroxylamine (1/10, v/v). Reactions were allowed to
`proceed for 5 h at 25 °C followed by 18-35 h at 4 °C; at
`
`Dosio et al.
`
`the end of the reaction, a solution of N-ethylmaleimide
`(20 mM, 20 (cid:237)L) was added to block free thiol groups.
`Thioether Bridge. Clavin (357 (cid:237)M, 1 mL) was deriva-
`tized with AMPT as described above; Mgr6 (33 (cid:237)M, 1 mL)
`was reacted with SIA (5.7 mM, 30 (cid:237)L) dissolved in
`ethanol, for 30 min at 25 °C. The iodoacetyl groups
`inserted on Mgr6 were identified by reaction with the
`TNB (5-mercapto-2-nitrobenzoic acid) reagent previously
`prepared; briefly, 25 mg of dithiothreitol (DTT) was added
`to 50 mg of DTNB dissolved in 300 (cid:237)L of 1 M aqueous
`NaOH under an inert atmosphere. After 30 min, the
`reaction mixture was acidified with 1 M HCl and the
`mixture was extracted with diethyl ether under an inert
`atmosphere. The organic layers were then anhydrifi-
`cated and evaporated under reduced pressure. The
`residue was washed several times with ethanol to obtain
`TNB as a dark yellow product.
`The Mgr6 derivatization degree was determined by
`reaction of mAb (3.3 (cid:237)M, 500 (cid:237)L) with TNB dissolved in
`ethanol (3.4 mM, 10 (cid:237)L) for 90 min at 25 °C. After
`purification by gel centrifugation, the number of io-
`doacetyl groups was determined spectrophotometrically
`(TNB molar absorption coefficient was 8800 M-1 cm-1 at
`339 nm). The mean derivatization degree was 1.7.
`The derivatized mAb and RIP were mixed in the
`presence of a solution of hydroxylamine (1/10, v/v). The
`conjugation reactions proceeded for 18 h at 4 °C; at the
`end of the reaction, a solution of N-ethylmaleimide (20
`mM, 20 (cid:237)L) was added to block free thiol groups.
`Immunotoxin Purification. Conjugates were puri-
`fied from unconjugated clavin and Mgr6 in a one-step
`procedure using CM MemSep cartridges (1000 or 1010)
`(Millipore) for analytical or preparative application and
`a Merck-Hitachi 655A-12 HPLC gradient system equipped
`with an L-5000 LC controller. The eluting fractions were
`monitored at 280 nm using a L4000 UV detector. Peak
`heights were recorded and processed on a CBM-10A
`Shimadzu interface. The mobile phase was sodium
`acetate buffer (20 mM at pH 5.5) flushed at 1 mL/min.
`Fractions containing immunoconjugates were dialyzed
`and concentrated. The purity of the immunotoxins was
`monitored by sodium dodecyl sulfate-polyacrylamide gel
`electrophoresis (SDS-PAGE) using 4-15% precast gels
`(Bio-Rad) under nonreducing conditions and Coomassie
`blue staining.
`In Vitro Evaluation of Disulfide Bond Stability.
`The in vitro stability of the bond in the various conjugates
`was evaluated as follows. Samples (1 mg/mL, 3 (cid:237)L) were
`incubated for 1 h at 37 °C with solutions of reduced
`glutathione (GSH, 3 (cid:237)L) in increasing excesses (from 3
`to 10000-fold), and the reaction was stopped by addition
`of excess iodoacetamide. Following SDS-PAGE under
`nonreducing conditions, the 7.5% precast gels (Bio-Rad)
`were stained with Coomassie blue, dried, and scanned
`on a Compact 4800 flatbed scanner using Twain compat-
`ible software. Band densities were analyzed using Im-
`agePC (Scion Co., Frederick, MD) to calculate the amount
`of Mgr6 released.
`Cell Lines. Human breast carcinoma cell line SKBr3
`(ErbB2+) was purchased from ATCC (Rockville, MD),
`and human melanoma cell line MeWo (ErbB2-) was
`kindly provided by the late J. Fogh (Memorial Sloan-
`Kettering Cancer Center, New York). Both cell lines
`were grown in RPMI 1640 containing 10% fetal calf
`serum and gentamicin (100 (cid:237)g/mL).
`Binding Inhibition Assay. mAb activity after con-
`jugation was assayed as the ability to inhibit binding of
`[125I]Mgr6 to adherent glutaraldehyde-fixed SKBr3 cells.
`A fixed amount of [125I]Mgr6 (1 nM) was mixed with serial
`
`IMMUNOGEN 2078, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`Properties of Mgr6-Clavin Immunotoxins
`
`dilutions of cold Mgr6 or IT, starting from a 100-fold
`molar excess. The mixture was added to SKBr3 fixed
`cells (adherent in 96-well plates) and incubated for 3 h
`at 37 °C. Cells were washed 10 times with PBS and
`incubated with 2 N NaOH (100 (cid:237)L/well) for 20 min at
`room temperature. The supernatant was collected and
`radioactivity determined in a (cid:231)-counter (Beckman In-
`struments, Fullerton, CA).
`Percent inhibition was calculated as follows:
`
`% inhibition ) (1 - Ci/Cni) x 100
`
`where Ci is the average counts per minute in the presence
`of cold inhibitor and Cni is the average counts per minute
`without inhibitor.
`Inhibition of Protein Synthesis. The assay was
`carried out essentially as described (13). Briefly, SKBr3
`and MeWo cells were suspended in culture medium
`containing the appropriate concentration of IT, toxin, or
`mAb alone and incubated for 3 h at 4 °C. Control cells
`were incubated with medium alone. Cells were centri-
`fuged, resuspended in fresh culture medium, and seeded
`in 96-well plates (3 (cid:2) 105 cells/well). After incubation
`at 37 °C for 48 h, [3H]proline (1 (cid:237)Ci/well) was added. After
`48 h, cells were washed and [3H]proline incorporation was
`determined by liquid scintillation in a (cid:226)-counter. Results
`are expressed as a percentage of [3H]proline incorporation
`in control cells.
`Radioiodination Procedure. ITs, clavin, and Mgr6
`were 125I-labeled using the Iodogen method according to
`the manufacturer’s instructions, to a mean specific activ-
`ity of 1.5 mCi/mg for ITs, 8.5 mCi/mg for Mgr6, and 4.5
`mCi/mg for clavin. The integrity of the radiolabeled
`protein was tested by ascending paper chromatography
`in 10% trichloroacetic acid and by SDS-PAGE analysis.
`Pharmacokinetic Evaluation. Experiments were
`performed on 6-week-old female Balb/c mice (Charles
`River, Como, Italy) maintained according to the provi-
`sions of the European Economic Community Council
`directive 86/209 recognized and adopted by the Italian
`Government.
`Mice received a Lugol solution (0.02% I2) and 0.6 mg/
`mL KClO4 in their drinking water 3 days before admin-
`istration of radioiodinated ITs and throughout the ex-
`periments to block free iodine uptake by the thyroid gland
`and the stomach mucosa. Animals were injected intra-
`venously with a single dose of 125I-labeled IT, ranging
`from 1.7 to 2.5 (cid:237)g/mouse. Blood samples were drawn
`from the retro-orbital sinus at fixed times after injection.
`Pharmacokinetic analysis was carried out on serum
`samples. Each serum sample was diluted 1/20 and run
`in SDS-PAGE under nonreducing conditions. Gels were
`fixed, dried, and exposed in a PhosphorImager screen
`(Molecular Dynamics, Sunnyvale, CA). Densitometric
`analysis, performed using Image Quant software
`(Molecular Dynamics), was used to determine the per-
`centage of intact immunoconjugates in the serum at each
`time point. Pharmacokinetic parameters were calculated
`using PCNONLIN software (SCI Software, Lexington,
`KY).
`
`RESULTS
`Chemistry. The cross-linkers, prepared as described
`(23-25), were characterized by NMR, IR, and MS.
`Before conjugation, the reactivity of the linkers toward
`the lysyl (cid:15)-amino groups of IgG as a model protein was
`evaluated to insert 1-2 groups. Scheme 1 shows the
`structures of the cross-linkers.
`
`Bioconjugate Chem., Vol. 9, No. 3, 1998 375
`
`Scheme 1. Structures of Cross-Linkers Used to
`Produce Mgr6-Clavin ITs
`
`Preparation of Immunotoxins. Four different cou-
`pling procedures (Schemes 2 and 3) were followed to
`compare the relative activity, stability, and in vivo fate
`of the immunotoxins.
`Disulfide Bridge Immunotoxins. Linkers AMPT and
`M-AMPT were reacted with recombinant clavin to intro-
`duce 1-1.3 S-acetylthio groups per protein molecule,
`whereas a mean value of 1.1 aryldithio residues was
`introduced into Mgr6 with CDPT; the derivatized pro-
`teins were coupled in the presence of hydroxylamine to
`deprotect the acetylthio groups (IT-1 and IT-2 in Scheme
`2). Clavin modified with CDPT was reacted with the
`Mgr6 previously reacted with the hindered N-hydroxy-
`succinimidyl linker SAMBA (IT-4) (Scheme 3).
`All coupling reactions were allowed to proceed for 5 h
`at 25 °C and for 18-35 h at 4 °C; aliquots of the reaction
`mixtures were analyzed by SDS-PAGE.
`Thioether Bridge Immunotoxin. To obtain a thioether
`linkage, Mgr6 was derivatized with SIA (1.7 iodoacetyl
`groups) and clavin with AMPT as described above. After
`addition of hydroxylamine, the reaction proceeded for 18
`h at 4 °C (IT-3 in Scheme 2).
`The yield of conjugation for the thioether-linked IT was
`12%, while for the disulfide-ITs, the yield was ap-
`proximately 10% for IT-1 and IT-2 and 5% for IT-4. This
`suggested an inverse correlation between disulfide bond
`stability and conjugation efficiency. The low yield can
`be attributed in part to the low (range of 1-1.7) number
`of reactive groups per protein.
`Purification of Immunotoxins. All ITs were puri-
`fied from the reaction mixture in a single step by HPLC-
`ion exchange chromatography using a 1010 CM MemSep
`cartridge.
`Figure 1A shows an example of the elution profile of
`the crude mixture eluted using a discontinuous gradient
`of sodium chloride ranging from 0 to 250 mM. Fractions
`from each of the four regions of the elution peaks were
`separately pooled and subjected to nonreducing SDS-
`
`IMMUNOGEN 2078, pg. 4
`Phigenix v. Immunogen
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`376 Bioconjugate Chem., Vol. 9, No. 3, 1998
`
`Dosio et al.
`
`Scheme 2. Preparation of Mgr6-(CDPT)SS(AMPT)-Clavin (IT-1), Mgr6-(CDPT)SS(M-AMPT)-Clavin (IT-2), and
`Mgr6-(SIA)CS(AMPT)-Clavin (IT-3)
`
`Scheme 3. Preparation of Mgr6-(SAMBA)SS(CDPT)-Clavin (IT-4)
`
`PAGE analysis (Figure 1B). By comparison with purified
`Mgr6 and clavin electrophoresed in parallel, peak 1 was
`composed only of unreacted Mgr6, peak 2 was 80% IT
`with a molecular mass of 167 kDa, peak 3 was >99% IT
`(167 kDa), and peak 4 contained only unreacted clavin
`(17 kDa).
`Disulfide Bond Stability of Conjugates in Vitro.
`To test the in vitro stability of the immunotoxins, samples
`were incubated with reduced GSH in different excesses,
`subjected to SDS-PAGE, and assessed densitometrically
`(Figure 2). The profile of the disulfide rupture as a
`function of GSH excess used is shown in Figure 3. An
`increase in disulfide stability in relation to the steric
`hindrance around the disulfide of the conjugates was
`observed. In comparison, the presence of a thioether
`bond precluded the breakage by GSH. The increase in
`stability was exponential, i.e., 10-fold for one methyl
`group and 100-fold for a geminal dimethyl group. When
`GSH was used in an excess higher than 7000-fold,
`
`cleavage of the mAb disulfide bond was measurable (data
`not shown).
`In Vitro Activity of ITs. To determine whether Mgr6
`retained its binding capacity after derivatization and
`conjugation, competitive inhibition experiments were
`conducted with the four ITs. A small, reproducible drop
`in binding capacity was observed after derivatization that
`was independent of the cross-linker used (see Figure 4,
`Mgr6-CDPT curve). All ITs competed with the native
`[125I]mAb, with a titration similar to that of cold deriva-
`tized Mgr6, and no significant differences due to the
`linkage method adopted were evident (Figure 4).
`Each IT was tested for its ability to inhibit protein
`synthesis in ErbB2-positive SKBr3 cells compared with
`the inhibition by unconjugated clavin. All ITs specifically
`inhibited protein synthesis in the SKBr3 cells, whereas
`none did so in ErbB2-negative MeWo cell. As shown in
`Figure 5, the IC50s of the conjugates containing a disulfide
`bridge were similar and ranged between 1 and 4 nM; the
`
`IMMUNOGEN 2078, pg. 5
`Phigenix v. Immunogen
`IPR2014-00676
`
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`

`Properties of Mgr6-Clavin Immunotoxins
`
`Bioconjugate Chem., Vol. 9, No. 3, 1998 377
`
`Figure 1. Elution profile of the IT reaction mixture from the CM-MemSep column (A) and SDS-PAGE analysis of eluted fractions
`(B). For preparative purposes, a MemSep 1010 cartridge eluted in sodium acetate buffer (20 mM at pH 5.5) was loaded with 400 (cid:237)L
`of conjugation mixture, and a step gradient of the same buffer and 1 M NaCl was applied, at a 0.5 mL/min flow rate for the first 12
`min and at 2 mL/min thereafter. After 20 min, the gradient was raised over the course of 10 min to 100 mM NaCl and kept constant
`for another 10 min. To remove unconjugated clavin, the gradient was increased to 250 mM over the course of 10 min and held there
`for 20 min. The collected fractions (about 3.5 mL) were immediately brought to pH 7.4 by addition of a solution of 1 M PBS (pH 8).
`
`Figure 2. SDS-PAGE analysis of IT-2 incubated with GSH
`at different concentrations for 1 h at 37 °C.
`
`Figure 3. Release of mAb from the immunoconjugates. The
`conjugates were incubated with GSH at different concentrations
`for 1 h at 37 °C. The amounts released were measured by
`densitometry after SDS-PAGE. Results are the average of three
`independent experiments (SDs < 10% of the mean).
`conjugation to the mAb resulted in a 150-650-fold
`increase in cytotoxic activity compared to that of clavin
`alone. No toxicity was observed after treatment with the
`conjugate containing the thioether bond (IT-3), which
`cannot be cleaved inside the cell. The specificity of the
`
`Figure 4. Competition of [125I]Mgr6 binding to SKBr3 (HER2+)
`glutaraldehyde-fixed cells with increasing molar concentrations
`of unlabeled mAb, derivatized mAb, and ITs (SD < 5% of the
`means).
`
`cytotoxicity was confirmed by the absence of an effect on
`MeWo cells (IC50s for all ITs are >1 (cid:237)M vs an IC50 of 0.8
`(cid:237)M for clavin).
`Pharmacokinetics. To evaluate the in vivo stability
`of ITs with a hindered (IT-2 and IT-4) or an unhindered
`(IT-1) disulfide bond or a thioether bond (IT-3), the
`pharmacokinetic behavior of each species was evaluated
`after intravenous bolus administration of 125I-labeled IT
`in groups of three Balb/c mice. Blood samples were
`collected at various times, and radioactivity was deter-
`mined. Plasma samples were analyzed by SDS-PAGE,
`autoradiography, and densitometry. The unhindered
`disulfide conjugate (IT-1) broke down to release the free
`
`IMMUNOGEN 2078, pg. 6
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`

`378 Bioconjugate Chem., Vol. 9, No. 3, 1998
`
`Dosio et al.
`
`Figure 5. Inhibition of [3H]proline incorporation in SKBr3
`target cells. Values represent the arithmetic means of three
`determinations (SDs < 10% of the means).
`
`antibody in vivo at the fastest rate, and after 6 h, only
`12% of the intact IT was present as compared with 35%
`of the IT-2 at the same time point. Plasma samples
`collected from IT-4 injected mice contained approximately
`equal amounts of intact immunotoxin and released mAb
`47 h after injection. IT-3 appeared to be stable, and by
`densitometric analysis, the conjugate represented 86-
`90% of the total radioactivity 72 h after injection.
`Plasma concentrations of IT at each time point were
`used to construct pharmacokinetic curves for each con-
`jugate (Figure 6). Table 1 lists the parameters obtained
`from computerized analysis of the clearance data using
`an open two-compartment pharmacokinetic model. Data
`for the single components of the ITs are included for
`comparison. Mgr6 showed a long serum elimination
`phase, whereas clavin, due to its low molecular mass and
`relevant charge, was cleared rapidly with only 3% of the
`initial radioactivity present after 1 h. The clearance
`values for clavin are shorter than those we reported
`previously (13) due possibly to the different method used
`for evaluation.
`The elimination curves of the ITs were biphasic, with
`a similar initial rapid R phase (about 17 min) followed
`by a slower (cid:226) phase. The immunoconjugates with higher
`in vitro stability were eliminated more slowly. Indeed,
`IT-1 was cleared rapidly; IT-2 doubled the (cid:226)-phase half-
`life (from 3.5 to 7.1 h), and IT-4 increased the elimination
`phase to 24 h. The thioether linkage showed its intrinsic
`stability, with a (cid:226)-phase half-life of 46 h. The difference
`in the elimination rate (144 h for the mAb and 46 h for
`IT-3) is due only to the presence of the clavin molecule.
`The thioether linkage stability also increased the distri-
`bution phase (from 17 to 32 min).
`
`DISCUSSION
`Immunotoxins have shown promise in therapeutic
`applications for hematological malignancies (1, 2, 27, 28);
`however, the therapy of disseminated solid tumors has
`proven to be a greater challenge, and only few trials have
`provided encouraging results (3, 29). Several investiga-
`tors used a reduced size IT Fab linked to the toxic domain
`
`Figure 6. Pharmacokinetic serum curves for ITs, clavin, and
`Mgr6. Mice were injected intravenously with the