400
`
`Bioconjugate Chem. 1990, 7, 400-410
`
`Thiol-Containing Cross-Linking Agent with Enhanced Steric Hindrance
`
`Lawrence Greenfield,'?' Will Bloch,? and Margaret Moreland',$
`PCR Department and Department of Chemistry, Cetus Corporation, 1400 53rd Street, Emeryville, California 94608.
`Received September 17, 1990
`
`Ricin A chain immunotoxins disulfide cross-linked with conventional, sterically unhindered reagents
`have unsatisfactorily short circulating life times in vivo. (Acety1thio)succinic anhydride, a thiolating
`reagent with partial steric hindrance of the sulfur atom, does not remedy this situation. Sulfosuc-
`cinimidyl N- [3-(acetylthio)-3-methylbutyryl]-/3-alaninate, a new cross-linker in which the carbon a to
`the sulfur is doubly methylated, creates disulfide bonds 2 orders of magnitude more resistant to reduction
`than unhindered disulfides. Nevertheless, this deactivated thiolating agent rapidly and reliably cross-
`links ricin A chain and antibodies to create immunotoxins with in vitro cytotoxicities comparable to
`those of 2-iminothiolane-coupled conjugates.
`
`pyridy1dithio)propionate (SPDP) and 2-iminothiolane (2-
`IT), are labile in circulation (23-26,181. Premature cross-
`link cleavage reduces the amount of intact conjugate which
`can bind to target cells. In addition, the released antibody
`remains in the circulation longer than conjugate and can
`compete with the intact conjugate for target-cell binding
`(23, 24, 26-29). Finally, the slow, sustained release of ri-
`cin A chain may contribute to the increased toxicity of di-
`sulfide-linked conjugates compared to that of free ricin A
`chain, which is rapidly cleared by renal filtration (23,30).
`All of these factors probably have reduced immunotoxin
`efficacy in various in vivo models for solid-tumor therapy.
`Thorpe et al. (31) and Worrell et al. (32) synthesized
`cross-linking reagents capable of yielding more stable di-
`sulfide bonds, on the basis of the finding that substitution
`of the a-carbon decreases the reactivity of the adjacent
`sulfur atom (33). One linker, 4-[ (succinimidy1oxy)-
`carbonyl] - a - m e t h y l 4 2-pyridyldithio) toluene (SMPT),
`sterically hindered the disulfide with a methyl group and
`a benzene ring attached to the carbon adjacent to the sulfur
`atom (31). The other linker, N-succinimidyl 3-(2-
`pyridyldithio)butyrate, substituted a methyl group on the
`a-carbon (32). Conjugates made with these new cross-
`linkers were more difficult to reduce chemically and had
`longer circulating half-lives than immunotoxins bearing
`
`INTRODUCTION
`Immunotoxins are protein conjugates in which a toxin
`is covalently attached to a monoclonal antibody (reviewed
`in refs 1-3). These chimeric molecules contain up to four
`functional regions. The antibody targets the drug in vivo
`to the desired cell population. The toxin is the effector
`portion responsible for cell death once bound to the cell
`surface (for surface-acting toxins) or internalized into the
`cytosol (for toxins which act on the protein-synthesis
`machinery). Most toxins are thought to contain a trans-
`location domain which facilitates entry of the catalytic
`portion into the cytosol. Finally, there is a cleavable linking
`region, which must confer stability to the conjugate in vivo
`while in the circulation, but later allow the release of the
`toxin to enable its entry into the cytosol.
`Often the toxin moiety is one of a variety of proteins
`capable of catalytically inactivating the protein-synthesis
`machinery of eukaryotic cells. These include holotoxins
`consisting of a catalytically active A fragment and binding
`B fragment [e.g. diphtheria toxin ( 4 , 5 ) , Pseudomonas ex-
`otoxin A (6, 7), ricin (8, 9), and abrin (10, 11)], hemitox-
`ins consisting of only the enzymatic A fragment [e.g. the
`A chain of diphtheria toxin (12,13), ricin ( I , 141, or abrin
`(15, 1611, and ribosome-inactivating proteins [e.g. gelo-
`nin (17, 181, pokeweed antiviral protein (19,20), and sa-
`porin (2111. In order to be effective in vivo, the toxin must
`remain attached to the antibody in the circulation.
`However, once the immunotoxin is inside the target cell,
`the release of the catalytic portion of the toxin is required
`in order to interact with its cytosolic target; A-chain-
`containing conjugates made with noncleavable thioether
`linkages are less than 1% as active as those containing
`easily reduced disulfide linkages (14, 22). The type of
`linkage required between the toxin and antibody depends
`on the form of the toxin used.
`Conjugates made with the standard heterobifunc-
`tional cross-linking reagents, N-succinimidyl 3-( 2-
`
`* To whom correspondence should be addressed.
`+ PCR Department.
`* Department of Chemistry.
`Current address: Glycomed, 860 Atlantic Ave., Alameda CA,
`94501.
`
`The abbreviations used are as follows: BSA, bovine serum
`albumin; DTDP, 4,4'-dithiodipyridine; DTNB, 5,5'-dithiobis(2-
`nitrobenzoic acid); DTT, dithiothreitol; EDTA, ethylenediam-
`inetetraacetic acid; GSH, reduced glutatione; GSSTNB, 2-nitro-
`5-mercaptobenzoic acid disulfide of glutathione; GSSTP, 4-mer-
`captopyridine disulfide of glutathione; HEPES, 4-(2-hydroxy-
`ethyl)-1-piperazineethanesulfonic acid; HNSA, 4-hydroxy-3-
`nitrobenzenesulfonic acid; IAM, iodoacetamide; 2-IT, 2-imino-
`thiolane; MEA, (3-mercaptoethylamine; MTT, 3-(4,5-dimeth-
`ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NaPi, sodium
`phosphate buffer; NHS, N-hydroxysuccinimide; PSH, penicil-
`lamine; PSSTNB, 2-nitro-5-mercaptobenzoic acid disulfide of
`PSH; PSSTP, 4-mercaptopyridine disulfide of PSH; rRA,
`recombinant ricin A chain; rRA-TNB, 2-nitro-5-mercaptoben-
`zoic acid disulfide of rRA; rRA-TP, 4-mercaptopyridine disul-
`fide of rRA; SAMSA, (acety1thio)succinic anhydride; SMPT, 4-
`[ (succinimidyloxy)carbonyl]-r~-methyl-c~-(2-pyridyldithio)-
`toluene; sNHS, N-hydroxysulfosuccinimide; sNHS-ATMBA, sul-
`fosuccinimidyl N- [3-(acetylthio)-3-methylbutyryl]-~-alaninate;
`SPDP, succinimidyl3-(2-pyridyldithio)propionate; TCA, trichlo-
`roacetic acid; TFA, trifluoroacetic acid; TMBA, N-(3-mercapto-
`3-methylbutyryl)-(3-alanine; TNB, 2-nitro-5-mercaptobenzoic acid;
`TP, 4-mercaptopyridine.
`1043-1802/90/2901-0400$02.50/0 0 1990 American Chemical Society
`
`IMMUNOGEN 2087, pg. 1
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Cross-Linker with Enhanced Steric Hindrance
`the standard disulfide cross-links (31, 32). Conjugates
`made with SMPT were as cytotoxic in vitro as conjugates
`synthesized with SPDP or 2-IT and had improved efficacy
`in vivo (34). Attempts to make an active-ester cross-
`linking reagent from 3-(2-pyridyldithio)isovaleric acid, a
`compound containing two methyl substituents on the
`carbon a to the disulfide, failed (32).
`During an effort to make immunotoxins which would
`resist undesired disulfide cleavage in circulation, we found
`that conjugates cross-linked with (acety1thio)succinic
`anhydride (SAMSA), in which the a-carbon is substituted
`with either a carboxylate or a methyl carboxylate, are
`unstable in vivo. Therefore, we developed a cross-
`linking reagent, sulfosuccinimidyl N-[3-(acetylthio)-3-
`methylbutyryll-@-alaninate (sNHS-ATMBA), in which the
`a-carbon is substituted with two methyl groups. The dis-
`ulfide bond involving the sulfur adjacent to the tertiary
`carbon is 2 orders of magnitude more difficult to reduce
`than the analogous glutathione disulfide bond. Never-
`theless, most conjugates made with this cross-linker have
`in vitro cytotoxic activity similar to that of analogous
`conjugates made with 2-IT.
`EXPERIMENTAL PROCEDURES
`Reagents and Chemicals. (35S)methionine (1086 Ci/
`mmol, cat. no. NEG-OOSA) and [ l-14C]iodoacetamide (24.1
`mCi/mmol) were purchased from New England Nuclear
`(Boston, MA); 2-IT, SAMSA, N-hydroxysulfosuccini-
`mide (sNHS), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB),
`and 4,4'-dithiodipyridine (DTDP) were from Pierce
`(Rockford, IL); @-mercaptoethylamine (MEA), iodoace-
`tamide (IAM), tert-butyl-@-alanine hydrochloride, N-hy-
`droxysuccinimide (NHS), dicyclohexyl carbodiimide, and
`carboxymethylated BSA were from Sigma (St. Louis, MO);
`chloroform (CHCL), methanol (MeOH), and methylene
`chloride (CH2C12) were from Burdick and Jackson
`(Muskegon, MI); triethylamine, dimethylacryloyl chloride,
`thiolacetic acid, trifluoroacetic acid (TFA), and D,L-
`penicillamine (a-amino-P-methyl-P-mercaptobutyric acid,
`PSH) were from Aldrich Chemical Co. (Milwaukee, WI);
`N,N-Dimethylformamide was from Fisher Scientific Co.
`(Fair Lawn, NJ); and activated Type 4A, 8-12 mesh
`molecular sieve was from J. T. Baker Chemical Co. (Phill-
`ipsburg, NJ). The 4-hydroxy-3-nitrobenzenesulfonic acid
`(HNSA) sodium salt was synthesized as previously
`described ( 3 5 ) . Preparative chromatography was
`performed on a Chromatotron (Harrison Instruments, Palo
`Alto, CA).
`Strains and Medium. Hybridoma cells were grown in
`HL-1 medium (Ventrex, Portland, ME) supplemented with
`Fungibact (Irvine Scientific, Santa Ana, CA) and 8 mM
`glutamine. When labeling the antibody metabolically by
`incorporation of (35S)methionine, HL-1 medium deficient
`in methionine was used. For the in vitro kinetic studies,
`cells were grown in RPMI 1640 medium with and without
`methionine (Flow Laboratories, McLean, VA).
`Mice. Female Balb/C nude (nu/nu) mice were obtained
`from Charles River Breeding Labs (Kingston, NY).
`Hybridomas and Antibodies. The following mouse
`monoclonal antibodies and hybridomas were used in the
`study (14, 36-38).
`antibody
`isotype
`113F1
`IgGi, K
`2G3
`IgGi, K
`260F9
`W i ,
`31705
`IgGi, K
`454A12
`520C9 Wit K
`MOPC2l
`IgG1,
`
`antigen
`37/60/93/200 kDa
`HMW protein
`55 kDa
`44-kDa glycoprotein
`transferrin receptor
`210 kDa
`none known
`
`K
`
`Bioconjugate Chem., Vol. 1, No. 6, 1990 401
`Cell Lines. The human breast cancer cell line SK-
`Br-3 was a generous gift from Dr. J. Fogh (Sloan Ketter-
`ing, New York, NY), and MCF-7 was obtained from E. G.
`& G. Mason Research Institute, Worcester, MA. The in
`vitro human breast cancer cell line MX-1 was a line
`adapted from the in vivo MX-1 tumor (39,40) by C. Vitt
`and A. Creasey (Department of Cell Biology, Cetus Corp.).
`The newborn human foreskin cell line HS27F (ATCC
`CRL1634) was used as a negative control because it bound
`none of the antibodies.
`Chemical Analysis. 1H NMR spectra were taken on
`a Varian FT-80A spectrometer. Chemical shifts of
`compounds in CDC13 are reported in ppm downfield from
`internal tetramethylsilane.
`Synthesis of ATMBA. The tert-butyl ester of @-ala-
`nine hydrochloride (structure 11, Figure 1) (1.8 g, 10 mmol)
`in 10 mL of CH2Cl2 was neutralized with 1 equiv of tri-
`ethylamine (1.4 mL, 10 mmol). The precipitated trieth-
`ylamine hydrochloride was filtered. The solution of @-ala-
`nine tert-butyl ester and 1.4 mL of triethylamine was
`placed in a dropping funnel and added dropwise to a
`nitrogen-flushed 100-mL three-neck round-bottom flask
`containing dimethylacryloyl chloride (structure I, Figure
`1) (1.1 mL, 10 mmol) dissolved in 10 mL of CH2C12. The
`mixture was stirred at room temperature for 2 h. The
`reaction mixture was diluted to about 50 mL with CH2-
`Cl2, washed with water (15 mL X 2) and brine (saturated
`aqueous sodium chloride), and dried over MgS04. The
`crude product was purified by Chromatotron chromato-
`graphy on a 4-mm silica gel plate. The plate was eluted
`first with 0.5 % MeOH in CHC13 to remove an impurity,
`then with 2 % MeOH in CHC13 to elute the product tert-
`butyl ester of dimethylacryloyl-P-alanine (structure 111,
`Figure 1). 'H NMR: 6 6.25 (br s, 1 H), 5.5 (s, 1 H), 3.45
`(q, 2 H), 2.4 (t, 2 H), 2.2 (s, 3 H), 1.8 (s, 3 H), 1.45 (s, 9
`H). Yield = 1.5 g (66%).
`To 1.2 g (5.4 mmol) of the tert-butyl ester of dimeth-
`ylacryloyl-&alanine in a 25-mL round-bottom flask was
`added 5 mL of freshly distilled thioloacetic acid. The
`reaction mixture was refluxed under nitrogen for 4 h. The
`solution was cooled and diluted with about 50 mL of ethyl
`ether. The ether solution was washed with 5% acetic acid,
`water, and brine and dried over MgS04. Evaporation of
`the ether gave a colorless oil which was not further purified.
`The crude product (structure IV, Figure 1) was dissolved
`in 10 mL of TFA and stirred at room temperature for 1
`h. The TFA was evaporated and the crude product was
`purified by Chromatotron chromatography on a 4 mm silica
`gel plate. Chromatography was started in CHCl3 and the
`product eluted with 5% MeOH in CHC13. The product
`N- [ 3- (acetylthio)-3-methylbutyryl] +-alanine (structure V,
`Figure 1) crystallized on evaporation of the solvent and
`'H NMR: 6 8.3
`was recrystallized from CHCl3-hexane.
`(br s, 1 H), 6.4 (br t, 1 H), 3.5 (9, 2 H), 2.75 (s, 2 H), 2.55
`(t, 2 H), 2.25 (s, 3 H), 1.5 (s, 6 H). Yield = 0.7 g (52%).
`HNSA or sNHS Esters. N-[3-(Acetylthio)-3-meth-
`ylbutyryll-0-alanine (617 mg, 2.5 mmol) was weighed into
`a 10-mL round-bottom flask. Sodium HNSA (602 mg, 2.5
`mmol) or sNHS (542 mg, 2.5 mmol) was dissolved in about
`3 mL of dimethylformamide and added to the flask,
`followed by 515 mg of dicyclohexylcarbodiimide, and the
`mixture was stirred at room temperature for 18 h. The
`mixture was filtered to remove dicyclohexylurea and added
`dropwise to 50 mL of ethyl ether with rapid stirring. The
`ether was stirred for about 0.5 h, then the precipitate was
`allowed to settle. The ether was decanted and the
`precipitate was washed with fresh ether four times. The
`solid product (structure VI, Figure 1) was collected by
`filtration and dried. Yield (HNSA linker) = 535 mg (46%).
`
`Accession No.
`HB 8490
`HB 8491
`HB 8488
`HB 8484
`IVI 10075
`HB 8696
`
`IMMUNOGEN 2087, pg. 2
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`402 Bioconjugate Chem., Vol. 1, No. 6, 1990
`
`I
`
`I1
`
`111
`
`V
`
`DCC 1 H 0 - L 3 s 0 3 N a
`
`0
`
`)-N/"
`0
`
`Vi
`Figure 1. Synthesis of sNHS-ATMBA. The structures are (I)
`dimethylacryloyl chloride; (11) tert-butyl ester of &alanine; (111)
`tert-butyl ester of (dimethylacryloyl)-j3-alanine; (IV) tert-butyl
`ester of N- [3-(acetylthio)-3-methylbutyryl]-P-alanine; (V) N - [3-
`(acetylthio)-3-methylbutyryl]-@-alanine; and (VI) sulfosuccin-
`imidyl ester of N - [3-(acetylthio)-3-methylbutyryl]-@-alanine.
`NHS Ester. N- [ 3-(Acetylthio)-3-methylbutyryl] -0-
`alanine (494 mg, 2 mmol) was weighed into a 25-mL round-
`bottom flask. NHS (230 mg, 2 mmol), in 10 mL of CH2-
`Clz, was added followed by 412 mg of dicyclohexylcarbo-
`diimide. The reaction was stirred at room temperature
`for 18 h. The precipitated dicyclohexylurea was filtered
`off and the solvent evaporated to give a white powder. The
`product was recrystallized from ethanol. 'H NMR 6 6.3
`(br s, 1 H), 3.6 (9, 2 H), 2.85 (s, 4 H), 2.8 (t, 2 H), 2.75 (s,
`2 HI, 2.25 (s, 3 H), 1.5 (s, 6 H). Yield = 350 mg (50%).
`Characterization of Stock Concentrations of
`Linker. Stock 10 mM solutions of the HNSA ester of the
`linker were freshly prepared prior to use by dissolving ca.
`2 mg in 500 pL of water. The concentration of active ester
`was determined in buffer (either 100 mM NaPi, 1 mM
`EDTA, pH 7.6, or 100 mM HEPES, 0.2 M NaC1,l mM
`EDTA, pH 7.6) as described by Aldwin and Nitecki (35).
`Briefly, the free HNSA dianion concentration was
`measured at 406 nm with a molar extinction coefficient
`of 4.6 X 103 M-' cm-'. The initial concentration of the free
`HNSA dianion was determined. Following the addition
`of 5 N NaOH to a final concentration of 240 mM the total
`concentration of HNSA was determined. The concen-
`tration of ester was calculated from the difference in the
`initial and final values.
`A stock (25 mM) solution of the NHS ester of the linker
`was prepared by dissolving 3 mg in acetonitrile and storing
`at -20 "C. The amount of the anion of NHS was measured
`at 259 nm with an extinction coefficient of 8600 M-l cm-l
`(41,42). To determine the concentration of ester, the stock
`solution was diluted into 0.25 M Tris-C1, pH 8.0, and the
`absorbance at 259 nm was monitored with time. The ester
`concentration was determined from the difference between
`the final and initial (extrapolated) absorbances.
`Stock (10 mM) solutions of the sNHS ester of the linker
`were made in dry dimethylformamide and stored at -70
`"C. Following dilution of the stock in 100 mM NaPi, pH
`6.0, the initial absorbance at 269 nm was measured. Hy-
`droxylamine was added to a final concentration of 5 mM,
`and the measurement was repeated 1 min later. The ami-
`nolysis of the ester was complete in 10 s. Ester concen-
`tration was determined from the difference in the two
`
`Greenfield et al.
`values, using a molar extinction coefficient at 269 nm of
`6100 M-l cm-' at pH 6.0.
`Kinetic Studies. Hydrolysis rates were studied spec-
`trophotometrically in the indicated buffers as a function
`of time at 25 "C. Reactions were carried out for 15 min
`and monitored at 15-s intervals. Hydrolysis was followed
`at 406 nm for the HNSA ester and at 269 nm for the sNHS
`ester. The total ester was determined as described above.
`Second-order rate constants for thiol-disulfide exchange
`reactions with DTNB or DTDP, releasing 2-nitro-5-
`mercaptobenzoic acid (TNB) [molar extinction coefficient
`at 412 nm of 13 600 M-' cm-' (43)] or 4-mercaptopyri-
`dine (TP) [molar extinction coefficient at 324 nm of 19 800
`M-' cm-l(44)], were determined by monitoring the reaction
`spectrophotometrically at 5-9 intervals for 10 min at 23
`"C in 100 mM NaPi, pH 7.0 or 8.0. When one reagent was
`in great excess, the reaction exhibited first-order kinetics
`with respect to the limiting reagent, and the pseudo-first-
`order rate constant was calculated from the initial linear
`part of the graph of In [unreacted reagent] vs time. The
`second-order rate constant was calculated by dividing the
`pseudo-first-order constant by the concentration of the
`excess species. For fast reactions, equal concentration of
`the two reagents were added, and the second-order rate
`constant was calculated from a plot of (l/[A]) - (1/[&])
`vs time. Finally, when the concentrations of the two
`reagents were not equal but neither was in great excess,
`a plot of (l/(IAol- [BO])) X In (([AI X [Bol)/([Aol X [Bl))
`vs time yielded the second-order rate constant.
`The reactivity of the TMBA thiol was examined
`following derivatization of antibody, deacetylation with
`hydroxylamine, and desalting.
`Synthesis of Immunoconjugates with t-lminothi-
`olane. Recombinant ricin A (rRA) chain produced in Es-
`cherichia coli (45) and 2-IT conjugates were prepared by
`the Cetus Process and Product Development group using
`previously published methods (46, 47).
`Synthesis of Radioactive Immunoconjugates with
`SAMSA. For the preparation of metabolically 35s-
`labeled 260F9 antibody [ (F3)-260F9], hybridoma cells were
`grown in HL-1 medium at 37 "C under 10% COz for 2 days.
`Cells were harvested, washed, and resuspended in HL-1
`medium deficient in methionine to a final cell density of
`1 X lo6 viable cells per mL. (35S)methionine was added
`to a final specific activity of 50 pCi/mL (50 nM), and the
`culture was incubated an additional 24 h at 37 "C under
`10 5% Con. The radiolabeled antibody was concentrated
`from the supernatant by chromatography over a Bio-Gel
`HPHT hydroxyapatite HPLC column (100 mm X 7.8 mm,
`Bio-Rad Laboratories, Richmond, CA) using a sodium
`phosphate gradient. The antibody was further purified
`by chromatography over a BioGel TSK-phenyl-5-PW
`HPLC column (75 mm X 7.5 mm, Bio-Rad, Richmond, CA)
`with a simultaneously descending gradient of ammonium
`sulfate (1.0-0 M) and ascending gradient of propylene
`glycol (0-3074 ) in phosphate buffer (100 mM NaPi, pH
`8.0). The antibody was at least 95% pure based on SDS-
`PAGE and autoradiography.
`A stock solution (5 mM) of SAMSA was prepared in ac-
`etonitrile. (=S)-260F9 (640 pg/mL, 1.1 X 105dpmlpg) was
`derivatized with a 15-fold excess of SAMSA in 10 mM
`HEPES, 0.2 M NaC1, 1 mM EDTA, pH 7.6, for 16 h to
`generate approximately 1.8 linkers per antibody, and the
`acetyl group was removed by treatment with 50 mM hy-
`droxylamine at 23 "C for 1 h. Following activation of the
`thiol by reaction with 1 mM DTNB at 23 "C for 1 h, the
`preparation was dialyzed against 100 mM NaPi, pH 8.0.
`rRA was freshly reduced in 100 mM NaPi, pH 8.0, with
`
`IMMUNOGEN 2087, pg. 3
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`Cross-Linker with Enhanced Steric Hindrance
`1 mM dithiothreitol (DTT) and desalted over a PD-10
`column (Pharmacia, Piscataway, NJ) in 100 mM NaPi, pH
`8.0. For conjugation, a 2.5 molar excess of rRA thiols over
`antibody thiols was reacted at 23 "C for 72 h under
`nitrogen. Conjugate containing one rRA per antibody (1-
`mer) was purified as described below.
`Synthesis of Immunoconjugates with TMBA.
`Antibody (10 mg/mL) was derivatized in 100 mM HEPES,
`200 mM NaCl, 0.1 mM EDTA, pH 7.6, with 3.3-fold excess
`of sNHS-ATMBA (or 8.5-fold excess of HNSA-
`ATMBA) for 16 h at 23 "C, resulting in 1.8-2 thiols per
`antibody. The linker thiol was deprotected by treatment
`of the modified antibody with 50 mM hydroxylamine at
`pH 8.0 for 1 h and the preparation desalted over a PD-
`10 column in 100 mM NaPi, pH 8.0. rRA was freshly
`reduced with 1 mM DTT and desalted over a PD-10
`column, the thiol was activated by treatment with 1 mM
`DTNB, and the unreacted products were removed by
`dialysis. Conjugation proceeded by mixing 1.5 mol of
`activated rRA-TNB disulfides per titratable antibody thiol
`at 4 "C for 16 h. Unreacted thiols were blocked by addition
`of 40 mM IAM for 1 h at 23 "C, and the conjugate was
`purified as described by Ferris et al. (47).
`Synthesis of Radioactive Immunoconjugate with
`TMBA. (35S)-260F9 (850 pg, 1 mg/mL) in 10 mM
`HEPES, 200 mM NaC1, 1 mM EDTA, pH 7.6 was de-
`rivatized with 15-fold excess of HNSA-ATMBA at 23 "C
`for 18 h. The preparation was deacetylated and conjugated
`as described above. The conjugate mixture was chro-
`matographed over a Bio-Gel TSK-phenyl-5-PW HPLC
`column (75 mm X 7.5 mm) and eluted at 1 mL/min with
`a simultaneously descending gradient of sodium chloride
`(1.5-0 M) and ascending gradient of propylene glycol (0-
`30%) in phosphate buffer (100 mM, pH 6.8). Fractions
`containing predominantly intact 1-mer, as determined by
`estimated molecular weight from SDS nonreducing PAGE
`and autoradiography, were pooled. Contaminating free
`rRA was removed by chromatography at 1 mL/min over
`a Zorbax Bio Series GF-250 HPLC column (25 cm X 9.4
`cm, Du Pont, Wilmington, DE) equilibrated with 100 mM
`sodium phosphate, pH 6.8, 250 pg/mL human serum
`albumin (Travenol, Laboratores, Inc, Glendale, CA). The
`final conjugate had a specific activity of 7.5 X lo5 dpm.
`Detection of Cysteine Thiols following Deacet-
`ylation of 260F9-ATMBA. Monoclonal antibody 260F9
`(33 mg/mL) was derivatized in 100 mM HEPES, 0.2 M
`NaCl, 1 mM EDTA, pH 7.6, with 6.7 molar excess of sNHS-
`ATMBA, deacetylated with 50 mM hydroxylamine, pH
`8, and desalted over a PDlO column. DTNB analysis
`indicated 1.9 thiols per antibody. To 1 mg of underiva-
`tized antibody (260F9 in 397 pL), 1 mg of derivatized
`antibody prior to deacetylation (260F9-ATMBA, in 344
`pl), or 1 mg of derivatized, deacetylated antibody (260F9-
`TMBA, in 166 pL) was added a 5 molar excess of [1-14C]-
`IAM (24.1 mCi/mmol, 16 pL) at 23 "C for 16 h. Unin-
`corporated label was removed by desalting over a PDlO
`column equilibrated with 20 mM N-ethylmorpholine, pH
`8.0. In addition, [1J4C]IAM was reacted with a 12-fold
`molar excess of cysteine and deacetylated TMBA. For acid
`hydrolysis, each sample was dried under vacuum, resus-
`pended in 300 pL of 6 N HC1, sealed in glass capillary tubes
`under vacuum, and incubated at 100 "C for 16 h. Samples
`were chromatographed on a microcrystalline cellulose
`Baker-flex plate (J.T. Baker, Phillipsburg, NJ) in n-bu-
`tanol-pyridine-glacial acetic acid-water (90:6018:72), dried,
`and autoradiographed at -70 "C.
`In Vitro MTT Assay. The in vitro activity of con-
`jugates was measured by using a colorimetric assay based
`
`Bioconjugate Chem., Vol. 1, No. 6, 1990 403
`
`on the ability of mitochondrial dehydrogenase enzymes
`to cleave the tetrazolium ring of the salt 3-(4,5-dimeth-
`ylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to
`the violet crystal formazan (48). Assays were performed
`by the Cetus Assay Development group, using a procedure
`developed by C. Vitt of Cetus. Briefly, wells of a 96-well
`tissue culture plate were filled with 50 pL of MEM Eagles
`medium (Irvine Scientific, Santa Ana, CA) containing 10%
`fetal calf serum (Flow Laboratories, McLean, VA) and 1 %
`penicillin-streptomycin (Irvine Scientific, Santa Ana, CA).
`Dilutions of the test conjugate were added in 25 pL followed
`by the addition of 100 pL of cell suspension (at 1 X 105
`cells/mL). Following incubation at 37 "C (6% COz for 72
`h) 75 pg of MTT was added, and the plates were incubated
`for an additional 4-6 h at 37 "C under 5% COz. The liquid
`was removed by aspiration, 150 pL of 3% SDS, 0.04 N
`HC1-2-propanol was added, and the plates were incubated
`for 30-60 min to allow color development. The plates were
`read at 570 nm in a Titertek Multiscan plate reader.
`Kinetics of in Vitro Cytotoxicity. OVCAR3 cells were
`trypsinized, counted, and seeded in 96-well plates at a cell
`density of 6.7 X lo4 viable cells/mL followed by the
`addition of 10 nM conjugate. At the indicated times, the
`cells were washed, incubated for 45 min with methionine-
`deficient RPMI 1640 medium supplemented with 8 pCi/
`mL (35S)methionine, washed, and harvested onto filter
`paper. Incorporated radioactivity was precipitated with
`TCA and counted.
`Analysis of Radioactive Conjugates in Vitro. Six
`female Balb/C nude (nu/nu) mice were injected with 0.1
`mL of (35S)-260F9-SAMSA-rRA (6 pg, 5.4 X 105 dpm) in
`100 mM NaPi, pH 7,200 pg/mL carboxymethylated BSA.
`At 0.25, 1.25,2.25,4.25,8.25, and 12.33 h, mice were bled
`retroorbitally with heparinized capillaries and sacrificed.
`Alternatively, eight mice were injected with 0.1 mL of (3%)-
`260F9-TMBA-rRA (3.6 pg); four mice were bled and
`sacrificed at both 5 and 24 h. Samples of blood were
`counted, and plasma was electrophoresed on a 5-10%
`gradient polyacrylamide SDS gel, dried, and autoradio-
`graphed at -70 "C using an intensifying screen.
`RESULTS
`Analysis of (35S)-260F9-SAMSA-rRA in Vitro.
`Thorpe et al. (31) and Worrell et al. (32) have reported
`that cross-linkers with a methyl substituent on the carbon
`a to the thiol stabilize conjugates in vivo. We investigated
`the commercially available reagent SAMSA, which is
`branched at the carbon a to the sulfur (Figure 2). Due
`to reagent asymmetry, the substituent following deriva-
`tization can either be a carboxymethyl or a carboxylate
`group (Figure 2). Despite the substitution, SAMSA
`conjugates were unstable in vivo (Figure 3), as has been
`observed with 2-IT (26, 23-25) and SPDP (11, 16, 26)
`conjugates. Because this substitution at the a-carbon failed
`to stabilize the conjugate in vivo, we investigated a new
`cross-linking reagent in which the a-carbon is substituted
`with two methyl groups.
`Synthesis of ATMBA. Initially, penicillamine was
`chosen for the linker backbone (Figure 2). The 4-nitro-
`phenyl ester of N-carbobenzoxy-S-benzylpenicillamine has
`been used in peptide synthesis (49). Preparation of several
`esters (NHS, HNSA, 2,4-dinitrophenol, and 4-nitrophe-
`nol) of N,S-diacetylpenicillamine was attempted. Only
`4-nitrophenol yielded an ester product (data not shown).
`Unfortunately, this reagent, 4-nitrophenyl N,S-diacetyl-
`penicillaminate, was not sufficiently water soluble to be
`useful as a protein cross-linker.
`The inability to esterify the penicillaminecarboxylate
`efficiently was ascribed to steric hindrance by the adjacent
`
`IMMUNOGEN 2087, pg. 4
`Phigenix v. Immunogen
`IPR2014-00676
`
`

`
`404 Bioconjugate Chem., Vol. 1, No. 6, 1990
`
`Greenfield et at.
`
`Table I. Reactivity of Various Nucleophiles with
`Activated Thiols.
`
`GMalhone
`
`ACwH
`
`HS
`
`NHZ
`
`Penicillamine
`
`\Np
`
`NH-Antibody
`
`rRA-s - s H,C
`
`(x.
`
`2-lminothmlane
`
`0
`
`SAMSA
`
`S u b NHS-ATMBA
`Figure 2. Chemical structures of thiol-containing compounds
`and cross-linkers. The thiol-containing compounds glu-
`tathione and penicillamine were used to compare the reactivity
`of sulfur atoms adjacent to a primary and tertiary carbon,
`respectively (Table I). The structures of the cross-linkers used
`in this study (2-IT, SAMSA, and TMBA) both before (left) and
`after (right) reacting with proteins are shown.
`
`1 2 3 4 5 6 7 0
`
`%
`
`P
`
`Figure 3. In vivo lability of 260F9-SAMSA-rRA conjugate. Six
`mice were injected intravenously with 6 pg of (35S)-260F9-
`SAMSA-rRA and sacrificed at 0.25 (lane 3), 1.25 (lane 4), 2.25
`(lane 5), 4.25 (lane 6), 8.25 (lane 7) and 12.3 h (lane 8). Plasma
`was isolated and electrophoresed on a 5-10% gradient SDS poly-
`acrylamide gel. The gel was dried and autoradiographed. Lane
`1 is (35S)-260F9 and lane 2 is (35S)-260F9-SAMSA-rRA injec-
`tate.
`
`doubly branched a-carbon. To avoid the problem, the
`linker backbone was elongated before the introduction of
`the sulfur, as outlined in Figure 1. On the basis of the
`approach of Worrell et al. (32), dimethylacryloyl chloride
`was used as the starting material but the linker backbone
`was extended by reacting with 0-alanine tert-butyl ester.
`Thioloacetic acid was added to the double bond, and the
`tert-butyl blocking group was removed. The carboxy-
`late group, now five atoms removed from the sterically
`hindered site, was readily esterified with three different
`
`activated thiol
`DTNB
`DTDP
`GSSTNB
`GSSTP
`PSSTNB
`PSSTP
`rRA-TNB
`
`GSH
`2200 (5)
`1900 (3)
`200 (2)
`360 (1)
`1 (1)
`1.6 (2)
`nd
`
`DTT
`nd
`nd
`nd
`nd
`1.7 (1)
`1.4 (1)
`nd
`
`PSH
`MEA
`2100 (6)
`nd*
`1700 (4)
`nd
`140 (3)
`770 (1)
`210 (1)
`890 (1)
`0.73 (2)
`3.2 (2)
`nd
`1.8 (2)
`160 (1)
`nd
`k2, M-1 S-1
`activated thiol hydroxylamine 260F9-TMBA 520C9-TMBA
`nd
`nd
`diacetyl-PSH
`0.34 (1)
`DTNB
`nd
`nd
`88 (8)
`nd
`nd
`0.018 (6)
`sNHS-ATMBA
`rRA-TNB
`nd
`nd
`7 (1)
`nd
`3.6 (1)
`rRA-TP
`nd
`a The indicated two reagents were mixed and the release of the
`chromophore, TNB or TP, was followed spectrophotometrically at
`23 "C as described in the Experimental Procedures. The concen-
`tration of the nucleophiles and the activated thiols ranged between
`M. The rate of deacetylation of diacetyl-
`and 2 X
`1 X
`PSH (7 X 10-5 M) and sNHS-ATMBA (5 X
`M) with hydroxy-
`lamine (50 mM) was monitored by measuring the exposed thiols by
`the inclusion of DTNB (1 X 10-4 M). The second-order rate constants
`(122) were determined during the initial part of the reaction. The
`number of determinations is indicated in parentheses. (rRA-
`TNB, rRA-2-nitro-5-mercaptobenzoate, rRA-TP, rRA-4-mercap-
`topyridine; 260F9-TMBA and 520C9-TMBA, derivatized antibody
`in which the thiol of the linker has been deblocked.) * Nd, not
`determined.
`
`alcohols (HNSA, NHS, and sNHS). All three esters were
`evaluated for coupling efficiency.
`Reactivity of Blocked Thiol. Penicillamine (Figure
`2) was used to model the thiol reactivity of TMBA (Table
`I). The reactivity of the penicillamine (PSH) thiol with
`DTNB, DTDP, GSSTNB (2-nitro-5-mercaptobenzoic acid
`disulfide of glutathione), and GSSTP (4-mercaptopyri-
`dine disulfide of glutathione) was similar to those of
`reduced glutathione (GSH) and MEA (Table I). The
`reactivity of the penicillamine thiol with GSSTNB (I32 =
`140 M-l s-l) was similar to that with activated rRA, in
`which the thiol of rRA was activated with TNB (122 = 160
`M-l s-l). In contrast, the 2-nitro-5-mercaptobenzoic acid
`(PSSTNB) and 4-mercaptopyridine (PSSTP) disulfides
`of penicillamine were at least 2 orders of magnitude less
`reactive than the corresponding GSH derivatives toward
`reduction by GSH or MEA (Table I). Rabenstein and The-
`riault (50) have reported similar findings in which the pen-
`icillamine thiol acts as an efficient nucleophile while di-
`sulfide bonds involving the penicillamine sulfur resist
`cleavage.
`Forms of Ester. Efficiency of protein modification with
`ATMBA is dependent upon competing aminolysis and hy-
`drolysis reactions. As the hydrolysis rate is buffer
`dependent (Table 11), more efficient derivatization could
`be achieved by selecting buffers which minimize this rate.
`It was found that HEPES buffer resulted in both a lower
`hydrolysis rate and a higher derivatization efficiency when
`compared to phosphate buffer (Tables I1 and 111). This
`buffer effect on both hydrolysis rate and derivatization
`efficiency was greater for the HNSA ester than the sNHS
`ester (Tables I1 and 111).
`HNSA is a water-soluble and spectrophotometrically
`monitorable active-ester leaving grou