Bloconjugate Chem. 1993, 4, 521-527
521
(6-Maleimidocaproyl)hydrazone of Doxorubicin—A New Derivative for the Preparation of Immunoconjugates of Doxorubicin David Willner,* Pamela A. Trail/ Sandra J. Hofstead, H. Dalton King, Shirley J. Lasch/ Gary R. Braslawsky/ Robert S. Greenfield,5 Takushi Kaneko,-* and Raymond A. Firestone 1
Bristol-Myers Squibb Company,
5
Research Parkway, Wallingford, Connecticut 06492-7660. Received June 27, 1993®
The (6-maleimidocaproyl)hydrazone of doxorubicin was synthesized and conjugated to several mAbs, including chimeric BR96, via a Michael addition reaction to thiol-containing mAbs. DTT reduction of disulfides present in the mAb was a reliable and general method for generating a consistent number of reactive SH groups. The conjugates, after purification by Bio-Beads, were free of unreacted linker and/ or doxorubicin. All conjugates released doxorubicin under acidic conditions that mimic the lysosomal environment, while they were relatively stable at neutral pH. BR96 conjugates showed antigen-specific cytotoxicity.
Chart I
The antitumor agent doxorubicin (DOX,1 1; Chart I) (and other antitumor anthracyclines) has been linked to monoclonal antibodies (mAb) to enhance its activity against solid tumors, and also to decrease its side effects of cardiac toxicity and myelosuppression (1). Of particular interest are immunoconjugates that release the chemotherapeutic agent due to the acid sensitivity of the linker (2-6). Thus, compound 2, a hydrazone derivative of 1, has been shown to release DOX at pH 4.S-5.5, and furthermore, conjugates of this linker with several mAbs show good in vitro and in vivo activity (3a,b, 5, 6). Following antigen-specific binding, the conjugates are internalized into the acidic environment of the lysosomes where DOX is released from the acid-labile linker (7). When compound 2 is conjugated to a thiol-containing mAb, the linkage formed includes a disulfide bridge (structure 3), which is susceptible to cleavage by thiols (3a, 8). Conjugates of ricin A and other toxins with disulfide linkages are unstable when administered in vivo and this instability could be due to the slow splitting of the disulfide bond by reduced glutathione or to the action of a disulfide reductase (9,10). In some cases (11,12) no decomposition of the conjugate was found, and it was reasoned that the immunotoxins were cleared from the blood so fast that the slower breakdown reaction could not be detected (9). This instability of the disulfide bond was also proposed to explain the need for high therapeutic doses of DOX conjugates in certain carcinoma models (6). A linker containing a thioether group should provide a much more stable conjugate (8). Such a thioether could be formed by reacting a thiol derivative of the therapeutic
1.
o
agent with either a haloacetyl (13) or a maleimide derivative of a mAb (3c, 14,15) or, conversely, by reacting a haloacetyl or maleimide derivative of the drug with a thiol-containing antibody (15). Using the latter approach, we have synthesized the (6-maleimidocaproyl)hydrazone of DOX(MC-DOXHZN), 4, a new hydrazone, and covalently attached it to several mAbs that have free thiols generated by two methods. The resulting conjugates, 5, were tested in vitro and showed antigen-specific cytotoxicity.
EXPERIMENTAL PROCEDURES
General. Melting points were determined on a FisherJohns melting point apparatus and are uncorrected. NMR spectra were obtained on a Bruker AM 300 in various solvents (specified) and are reported as values downfield from tetramethylsilane. Multiplicities of resonances are described as broad (b), singlet (s), doublet (d), triplet (t), quartet (q), or multiplet (m). IR spectra were run as KBr pellets or CHCI3 solutions on a Perkin-Elmer (PE) FTIR Model 1800. MS and HRMS were obtained with a Kratos MS25RFA and MS50TC instrument, respectively. Elemental analyses were performed by Oneida Research
t
Present address: Bristol-Myers Squibb Pharmaceutical Research Institute, P.O. Box 4000, Princeton, NJ 08543. * Present address: 124 Heritage Drive, Glastonbury, CT 06033. * Present address: 10 Sieter Hill Rd, Wallingford, CT 06492. L Present address: Pfizer Central Research, Groton, CT 06430. *
Abstract published in Advance ACS Abstracts, October
1,
Services. Flash chromatography was performed with Woelm silica gel (silica 32-63). TLC was carried out on Analtec silica gel GHLF plates, 250 µ . For HPLC, a PE QC-620 pump equipped with a PE ISS-200 injector and LKB Multitemp II Thermostatic controller was used. The detector was an HP 1046 fluorescent detector and the integrator a PE Nelson Turbochrome. HPLC was performed on a Jones column using a 70:30 mixture of methanol and 0.025 M
1993.
The abbrevations used are as follows: DOX, doxorubicin; mAb, monoclonal antibody; HRMS, high-resolution mass spectrum; SEC, size-exclusion chromatography; THF, tetrahydrofuran; DMF, N,iV-dimethylformamide; MC-DOXHZN, (6maleimidocaproyl)hydrazone of doxorubicin; SPDP, JV-succinimidyl 3-(2-pyridinyldithio)propionate; DTT, dithiothreitol; PBS, phosphate-buffered saline. MR, molar ratio; mAb-DOX, conjugate of doxorubicin (mAb specified when necessary); cpm, counts per minute. 1
1043-1802/93/2904-0521 $04.00/0
R.O
©
1993 American Chemical Society
522
Bioconjugate Chem., Vol. 4, No. 6, 1993
ammonium phosphate, pH 4.5, as a mobile phase, at a 1.5 mL/min flow rate. The eluted DOX was detected by excitation at ca. 233 nm and emission monitored at ca. 570 nm. Size-exclusion chromatography (SEC) was done on a TSK column, Model G3000SWXL, and respective guard column from Hewlett Packard. The mobile phase was 0.2 KH2PO4 in saline adjusted to pH 6.8 with 4 N KOH, and detection was done by UV and visible light at 280 and 495 nm, respectively. Stirred cells for diafiltration and accessories were from Amicon, Inc. (Beverly, MA). BioBeads (SM-2) were from Bio-Rad Laboratories (Richmond, CA). Antibodies. mAb BR96 is a mouse/human chimeric mAb which identifies a Ley-related tumor-associated antigen expressed on the cell membrane of carcinomas of the lung, breast, colon, and ovary and is internalized following antigen-specific binding (16). The chimeric mAb (humanized IgGl Fc portion) was produced from a murine mAb by a modification of the homologous recombination procedure as described previously (17). mAb BR 64 (6, 16) was obtained from Brunswick BioTechnetics (San Diego, CA). mAb L6, an anticarcinoma mAb which does not internalize following antigen-specific binding (18) was obtained from CellTech Ltd. (Berkshire, U.K.). Conjugates prepared from this antibody were used for synthetic comparisons. Human IgG, obtained from Rockwell Inc. (Gilbertsville, PA), was used to produce control, nonbinding immunoconjugates. Tumor Cell Lines. L2987 is a human lung adenocarcinoma line which expresses the BR96 antigen and was obtained from I. Hellstrom (Bristol-Myers Squibb, Seattle, WA). Chemicals. DOX hydrochloride was obtained from Sanraku, Inc. All other chemicals were obtained from commercial sources.
6-(2,5-Dihydro-2,5-dioxo-l£T-pyrrolo)-l-hexanoic Acid (6-Maleimidocaproic Acid), 8 (20, 21). Maleic
anhydride (29.4 g, 0.3 mol) and 6-aminocaproic acid (39.35 g, 0.3 mol) were refluxed in glacial acetic acid (900 mL) for 16 h. Acetic anhydride (30.6 g, 0.3 mol) was added dropwise over a period of 2 h and reflux was continued for 1 h. The acetic acid was removed under vacuum at 70 °C to yield a yellow syrup which solidified. The material was chromatographed over silica using methylene chloridemethanol-acetic acid (100:5:1) affording a crystalline solid: mp 87-91 °C (27.8 g, 43%); NMR (DMSO) 6.95 (s, 2H), 3.35 (t, 2H), 2.15 (t, 2H), 1.45, 1.18 (2m, 6H).
6-(2,5-Dihydro-2,5-dioxo-l.ff-pyrrolo)-l-hexanoic
Acid Hydrazide (6-Maleimidocaprohydrazide) and Its Trifluoroacetic Acid Salt, 10 (20). 6-Maleimidocaproic acid (2.11 g, 10 mmol) in dry THF (200 mL) was stirred under nitrogen at 4 °C with N-methylmorpholine (1.01 g, 10 mmol) followed by dropwise addition of isobutyl chloroformate (1.36 g, 10 mmol) in THF (10 mL). After 5 min, a solution of tert-butyl carbazate (1.32 g, 10 mmol) in THF (10 mL) was added dropwise. The reaction mixture was kept at 4 °C for 30 min and at room
temperature for 1 h. The solvent was evaporated and the residue was partitioned between ethyl acetate and water. The organic layer was washed with dilute HC1 solution, water, and dilute sodium bicarbonate and dried over anhydrous sodium sulfate, and the solvent was evaporated. The residual foam was chromatographed over silica using a gradient solvent system of methylene chloride-methanol (100:1-2). The protected hydrazide, 9, was obtained in 70% yield (2.24 g). Compound 9 (545 mg, 2.4 mmol) was dissolved in icecold trifluoroacetic acid (10 mL) and stirred in an ice bath
Winner et al.
for 8 min. The acid was removed under high vacuum at room temperature. The residue was triturated with ether to yield the crystalline trifluoroacetic acid salt of 6-maleimidocaprohydrazide (10) (384 mg,70%). An analytical sample was prepared by crystallization from methanol-ether: mp 102-5 °C; NMR (CDCI3-DMSO) 7.24 (broad peak superimposed on chloroform peak), 6.55 (s, 2H), 3.34 (t, 2H), 2.06, (t, 2H), 1.46 (m, 4H), 1.15 (m, 2H); MS [MH]+ 226. Anal. Caled for C10H15N3O3· O.8CF3COOH: C, 44.02; H, 4.99; N, 13.28. Found: C, 44.13;
,
5.00; N, 12.75. The salt (220 mg) was converted to the free base by chromatography over silica using a methylene chloridemethanol-ammonium hydroxide (100:5:0.5) solvent system. The material obtained (124 mg, 80 %) was crystallized from methylene chloride-ether: mp 92-93 °C. Anal. Caled for C10H15N3O3: C, 53.33; H, 6.67; N, 18.67. Found: C, 53.12; H, 6.67; N, 18.44. (6-Maleimidocaproyl)hydrazone of DOX, 4. DOX hydrochloride (5.2 g, 8.96 mmol) and 6-maleimidocaprohydrazide trifluoroacetate salt (10) (9.2 g, 27.1 mmol) were dissolved in 1750 mL of methanol. Trifluoroacetic acid (0.5 mL) was added and the solution was stirred at room temperature for 24 h while being protected from light. The methanolic solution was concentrated under reduced pressure at 31 °C to a volume of 250 mL. Acetonitrile (1250 mL) was added and the resulting suspension was allowed to stand at 4 °C for 48 h for crystallization of the product. The red solid hydrazone was isolated by centrifugation, washed with fresh methanol-acetonitrile (1: to yield the (6-maleimi10), and dried under vacuum docaproyl)hydrazone of DOX (5.78 g, 7.7 mmol, 86% yield): mp >200 °C dec; IR (KBr) 3430,2936,1704,1666,
1616, 1582, 1446, 1410, 1378, 1284, 1208, 1014, 986, 824 cm-1; NMR (CD3OD) 7.94 (bd, 1H), 7.82 (t, 1H), 7.55 (d, 1H), 6.78 (s, 2H), 5.48 (s, 1H), 5.07 (t, 1H), 4.59 (d, 1H), 4.21 (m, 1H), 4.02 (s, 3H), 3.63-3.30 (m, 5H), 2.55-2.26 (m, 4H), 2.19-1.88 (m, 3H), 1.69-1.18 (m, 12 H including a doublet, 3H, at 1.26); MS ( + H)+ 751; HRMS caled for C37H43N4O13: 751.2827. Found: 751.2823. Anal. Caled for C37H42N4Oi3-HCl: C, 56.45; H, 5.73; N, 7.12, Cl, 4.50. Found: C, 56.73; H, 5.73; N, 6.89; Cl, 3.76.
Conjugation of mAb with MC-DOXHZN via SPDP Thiolation. The conjugation of hydrazones to mAbs was
described previously (3a,b, 5). The following conjugation of BR96 with MC-DOXHZN is typical for all mAb used in this study. A solution of BR96 in PBS buffer (27.5 mL, 12.53 mg/mL, 7.83 X -5 M, total of 2.15 µ ; the protein concentration was determined by UV absorption at 280 nm, 1 mg/mL =1.4 AU) was treated with a 10 mM ethanolic solution of SPDP (1.7 mL, 5.3 mg, 17 µ ). The solution was incubated at 31 °C for 35 min, chilled in ice, and treated with a 50 mM solution of DTT (1.7 mL, 13.09 mg, 85 µ ) for 15 min at 4 °C. The solution was transferred to a dialysis tube (Spectrum Medical Industries, Los Angeles, CA) and dialyzed 4 X 8 h in PBS-0.1 M histidine buffer, pH 7.4 (4.5 L). The amount of mAb was determined as above (27.5 mL, 9.29 mg/mL, 5.8 X -5 M, total of 1.6 µ ) and the SH concentration was determined by the Ellman method (19) (2.06 X 1(H M, 5.7 µ total); MR SH/mAb 3.6. The thiolated BR96 (17 mL, 0.99 µ ) was treated with an equivalent molar amount of MC-DOXHZN in DMF (5 mg/mL, 6.3 X -3 ; 0.59 mL, 3.7 µ ) and the reaction mixture was incubated overnight at 4 °C. The solution was centrifuged and transferred to a dialysis tube and dialyzed as above. The dialyzed solution was centrifuged and the supernatant shaken gently with Biobeads for a few hours at 4 °C. The solution (19 mL) was freed =
Bioconjugate Chem., VoI. 4, No. 6, 1993
BR96 Conjugates of Doxorubicin
from the Bio-Beads by centrifugation and the amount of DOX was determined by UV absorption at 495 nm (e495 = 8030 cm-1 M-1; 117 µ , 67.9 Mg/mL). The amount of mAb was determined by absorbance at 280 nm according to the formula (3a) mAb (mg/mL)
=
A gap
Scheme I ,o Ac^O H2N{CH2)5COOH
N-Methyl Morpholine I so butyl chloroformate
N(CH2)5C02H
CK3COOK
7
t-Butyl carbazate
o 8
(0.724 X A495)
L4 (A is the observed absorbance at noted wavelength) which includes a correction for the absorbance of DOX at the same wavelength (6.53 mg/mL, 4.08 X 10~5 M; yield 72%). The MR DOX/mAb was 2.9. Conjugation of mAbs with MC-DOXHZN via DTT Thiolation. The following procedure for mAb BR96 was found applicable to other mAbs in this study. mAb BR96 in PBS buffer (500 mL, 10.49 mg/mL, 6.56 X 10~5 M determined as above, total of 32.8 Mmol) was heated under nitrogen to 37 °C, stirred gently, and treated with DTT (10 mM solution in PBS, 23 mL, 230 Mmol). The solution was kept under these conditions for 3 h and then transferred into 2 400-mL Amicon stirred ultrafiltration cells (Model #8400) each fitted with a YM 30 ultrafilter (molecular weight cutoff 30 000) and connected to a reservoir (Model #RC800) containing PBS solution. The mAb solution was diafiltered until no more thiol groups could be detected in the effluent by the Ellman method (19). The mAb solution (526 mL) was transferred from the cells to a sterile container and kept in ice under nitrogen. The concentration of protein and thiol was determined as above (9.91 mg/mL, 6.19 X 10~5 M, 32.6 Mmol and 5.05 X 10-4 M, 265.6 Mmol, respectively; MR of SH/protein was 8.16) and 1 molar equiv of MC-DOXHZN (50.6 mL of a 5 mg/mL, 6.3 X 1(H M distilled water solution, 319.1 Mmol) was added with stirring. The solution was kept at 4 °C for 30 min after which it was filtered through a 0.22-mm cellulose acetate membrane. A 2.5 X 50 cm column was packed with a slurry of 100 g of Bio-Beads in PBS buffer. The beads had been prepared by soaking in methanol and washing in succession with several volumes of distilled water and buffer. The filtered conjugate solution was percolated through this column at a rate of 2-2.5 mL/min. After that, the solution (530 mL) was filtered again through a cellulose acetate membrane and the concentration of mAb and DOX determined (9.45 mg/ mL, 5.91 X 1(H M; 232 mg/mL, 0.400 X 10™3 M, respectively). The mAb yield was 96% and the MR of DOX to mAb was 6.78. As shown in Table I the mAb yields for BR96 conjugates were 90-100%. The conjugates were frozen in liquid nitrogen and stored at-80 °C. Other mAbs were conjugated in a similar manner, and the data are given in Table I. Relationship between DTT/mAb Ratio and the Number of Thiol Groups Generated. mAb BR96 or BR64 in PBS buffer, at a concentration of approximately 10 mg/mL, was treated for 3 h at 37 °C under an atmosphere of argon with 10-150 mM DTT in PBS to achieve the desired ratio of DTT to mAb (see Figure 1). The mAb sample was then passed through a Pharmacia PD-10 column with deoxygenated PBS buffer at pH 7.4. The protein fraction was collected in a sealed test tube under argon. SH groups were determined using Ellman’s reagent (19). For large samples (>100 mg mAb) the passage over a PD-10 column was substituted by ultrafiltration against PBS buffer, pH 7.4, in an Amicon stirred cell fitted with an Amicon YM-30 membrane and pressurized with argon at 40 psi. Dialysis was continued until the stirred cell eluant tested negative with Ellman’s reagent (19), and the number of SH groups was determined in the same manner.
523
CF3COOH
N(CH2)5C0NHNHC02tBu
[I
O 9
N(CH2)5CONHNH2
0
'
cf3co2h
10
Relationship between SPDP/mAb and Number of Thiols Generated. This experiment was carried out similarly to the previous one, except that the thiolating agent was SPDP. BR64 and BR96 were treated with an ethanolic solution of SPDP for 30 min at 30 °C under argon, cooled to 0 °C, and treated with a 5-fold molar
amount of DTT for 15 min. The solution was then purified by passing it through a PD-10 column with deoxygenated PBS, pH 7.4, and collected in a sealed test tube under argon. The SH titer was determined by the Ellman method (19). The results are plotted in Figure 2. Air Reoxidation of DTT Reduced BR64. BR64 in PBS (10.72 mg/mL, 6.7 X 10~® , 1 mL) was treated with DTT solution in PBS (50 mL of 134 mM solution) and the reaction mixture was incubated at 37 °C for 1 h. The mAb was purified on a PD-10 column and 1.5 mL solution was collected in a 12 X 75 mm glass tube which was left open to atmosphere for 21 h. Aliquots were removed at the beginning (t = 0) and subsequent intervals and the SH concentration determined as above. The results are shown in Figure 3. In Vitro Cytotoxicity Assays. The activity and specificity of BR96-DOX conjugates was evaluated as described previously (6). Briefly, monolayer cultures were harvested using trypsin-EDTA (GIBCO, Grand Island, NY), and the cells counted and resuspended to 1 X 10s/ mL in RPMI-1640 containing 10% heat-inactivated fetal calf serum (RPMI-10% FCS). Cells (0.1 mL/well) were added to each well of 96-well microtiter plates and incubated overnight at 37 °C in a humidified atmosphere of 5% CO2. Media were removed from the plates and serial dilutions of DOX or mAb-DOX conjugates added to the wells. All dilutions were performed in quadruplicate. Cells were exposed to DOX or mAb-DOX conjugates for 2 h at 37 °C in a humidified atmosphere of 5% CO2. The mAb-DOX conjugate or DOX was removed and the cells washed three times with RPMI-10% FCS and cultured in RPMI-10% FCS (37 °C, 5% CO2) for an additional 48 h. At this time the cells were pulsed for 2 h with 1.0 mCi/ well of [3H] thymidine (New England Nuclear, Boston, MA). The cells were harvested onto glass-fiber mats (Skatron Instruments, Inc., Sterling, VA) and dried, and filter-bound [3H] thymidine radioactivity was determined
(0-Plate scintillation counter, Pharmacia LKB Biotechnology, Piscataway, NJ). Inhibition of [3H]thymidine uptake was determined by comparing the mean cpm for treated samples with that of the untreated control.
RESULTS AND DISCUSSION
Preparation of Linker 4. 6-Maleimidocaproic acid (8) (Scheme I) was previously prepared from 6-aminoca-
proic acid and N- (methyoxycarbonyl)maleimide which in
524
Winner et al.
Bioconjugate Chem., Vol. 4, No. 6, 1993
Table I. Conjugates Prepared via DTT and SPDP Thiolation DTT mAb
SH
MR DOX
BR96
8.6 6.4 8.7 8.2 8.2 8.6 7.9 7.8
7.9 5.8 8.4 7.6 6.8 7.2 7.0 5.3
MR
BR64
L6 IgG
9.8 5.3 8.8 8.9
7.4 4.9 7.2 8.1
SPDP
yield of
MR
mAb(%)
SH
100 100 95 100 95 93 94 97
3.1 3.6
100 84 96 100
MR DOX
yield of mAb(%)
6.5 7.8 6.9
3.5 2.9 4.9 5.6 5.3
73 72 46 30 23
10.6 7.3 8.5 5.7 6.6 8.9
7.1
95
6.0 7.2 4.8 6.0 7.3
77 77
99 88 54
7.9
6.23
82
turn was prepared from maleimide and methyl chloroformate (20). An alternate synthesis (22) started from the amino acid and maleic anhydride to form 6-[(3carboxy-l-oxo-2-propenyl)amino]hexanoic acid, which was cyclized in toluene in the presence of triethylamine. Acid 8 (as well as its homologues) could be synthesized in a single step by heating maleic anhydride and 6-aminocaproic acid (or its homologue) in acetic acid followed by treatment with acetic anhydride.2 It was converted to its hydrazide, 10, via a mixed anhydride and ieri-butyl carbazate followed by removing the tert-butoxycarbonyl group with trifluoroacetic acid. The hydrazone 4 was prepared by treating DOX hydrochloride and 10 in methanol overnight with catalytic trifluoroacetic acid (5). The final product still contained 3-5 % of unreacted 1 but this did not interfere with the conjugation and purification of the conjugate. Generation of Reactive Thiol Groups on the mAh. Previous conjugations of DOX hydrazones to mAbs were done through thiolation of the mAb and reacting it with linker 2 to form a disulfide bridge between the mAb and the drug, e.g. 3 (3,5,6; however also see 4). The thiolation of the mAb was achieved by reacting the lysine moieties of the mAb with SPDP and reducing the S-S bond with DTT. Initially, we used the same thiolation methodology for our four mAbs (see Table I). Although as the SPDP ratio to BR64 and BR96 rose to 50 and the MR of SH groups to these mAb increased smoothly up to 12 (see Figure 2), thiolated BR96 with MR above 8-9 suffered from reduced binding to the antigen,3 presumably because of reaction on active-site lysines. An additional problem arose when we switched from mAb BR64 to chimeric BR96. Conjugates that were prepared via SPDP thiolation and had a MR of 8 were soluble in PBS buffer when BR64 was used, but not with BR96. Owing to precipitation, yields of conjugates plummeted when final MRs were raised above 3.5, which is too low for good antitumor activity (6). Adding L-histidine or mannitol to the buffer gave little benefit. The tendency to precipitate resided in the antibody, not the linker, because it persisted with either mono or dithio links, and with thiolation by either SPDP or iminothiolane (data not shown). The use of the latter reagent shows also that loss of positive charge, which occurs when lysine amino groups react with SPDP but not with We thank Dr. I. Monkovic for these procedures. Binding determinations were done according to ref 3a,b. We thank Ms. K. Kadow for performing these measurements. 2
3
Figure 1. Number of thiol groups generated from mAb by treatment with DTT at 37 °C as a function of MR of DTT to mAb (BR64 ; BR96 A). 16-i
U1-1-1-1-1-1-1-1 0
10
20
30
40
50
60
70
SPDP/Ab
2. Number of thiol groups generated from mAb by treatment with SPDP at 30 °C as a function of MR of SPDP to mAb (BR64 ; BR96 A).
Figure
2-iminothiolane, was not responsible for the precipitation. Only when thiolation of lysines was abandoned altogether was the precipitation problem overcome (vide infra). Another way to generate free SH groups in the mAb is by reduction of the mAb with DTT. The light and heavy chains of antibodies are held together through noncovalent interactions, with interchain disulfide bridges providing additional stability to the structure (22). These interchain disulfide bonds can be reduced in preference to the interchain disulfide in the absence of a denaturing reagent (Figure 4) (23-25). Under mild reduction conditions, the mAb remains intact, retaining its binding, while providing free SH groups for conjugation (26). Further, it was shown that when immune serum globulins (a mixture of isotypes with predominating) were treated similarly with DTT only four disulfide linkages were reduced corresponding to the number of interchain linkages present in an IgGl type (27). Very recently (28) mAb BR96 was subjected to
Bioconjugate Chem., Vol. 4, No. 6, 1993
BR96 Conjugates of Doxorubicin
525
Figure 5. SEC-HPLC of BR96-DOX conjugate in saline-0.2M KH2PO4 buffer. Absorbance (mAU)
was
measured at 280
nm.
Time (hrs.)
Figure
3. Reoxidation
of DTT-reduced BR64 in open atmo-
sphere. interchain disulfides
6. Cytotoxicity of BR96-DOX conjugates against the L2987 lung carcinoma line. Cells were exposed for 2 h at 37 °C to DOX or DOX conjugates, washed and cultured for an additional 48 h prior to the addition of [3H]thymidine. Data are presented as percent inhibition of [3H] thymidine uptake relative to control cells. Cells were treated with DOX (A), BR96-DOX, MR = 7.59 (O); or nonbinding IgG-DOX, MR = 8.11 (·).
Figure
Figure 4. Schematic drawing of BR96 mAb. The heavy (H) and light (L) chain are held together through noncovalent interactions and the interchain disulfides provide additional stability to the molecule.
DTT reduction but with the generation of only four SH
groups rather than the expected eight. We investigated the DTT reduction of mAb BR96 by carrying out the reaction at 37 °C under nitrogen and using varied molar ratios of DTT to mAb. The data, given in Figure 1, clearly indicate that the number of SH groups levels off at eight no matter how much excess DTT is used. Thus, we were able to improve on the earlier procedure (28) and apply this thiolation method to the preparation of conjugates. In our best procedure, BR96 (and other mAbs; see Table I) is treated with 7 molar equiv of DTT at 37 °C for 3 h. This reaction and all subsequent steps through the final conjugation must be done with air excluded because reoxidation of thiols is facile (see Figure 3). Excess DTT is removed by diafiltration (which takes only hours, as opposed to dialysis, which takes days) and the thiol molar concentration is determined. This value is used to determine the amount of hydrazone needed for conjugation. The advantage of this method is that it yields consistently the same number of SH groups and hence a consistent MR of drug to mAb. Immunoconjugates made from BR96, in which thiols were generated in this way, do not precipitate, regardless of whether a mono or dithio linker is used, and high yields are always obtained. This method of producing thiols was then adopted for all subsequent conjugation, working equally well for mAb BR64, L6, and IgG (see Table I).
Most importantly, these immunoconjugates maintain their molecular integrity despite the loss of bridging disulfide units, presumably owing to noncovalent forces between the chains. This was shown in four ways. (1) Size-exclusion chromatography finds a single entity of mw
150 000 (Figure 5). (2) Gel electrophoresis under nondenaturing conditions gives the same result (data not shown). (3) Binding is not significantly ( 33 µ based on DOX content, respectively) and approximately 4-fold less potent than unconjugated DOX (IC50 = 0.2 µ ). A detailed description of both in vitro and in vivo experiments will be published elsewhere (30). In summary, the (6-maleimodocaproyl)hydrazone of DOX was synthesized and conjugated to several mAbs, 5
Unpublished results by L. Padilla and G. Dubowchik.
Willner et al.
including chimeric BR96. The conjugation was done via a Michael addition reaction to thiol containing mAbs. Generation of thiols on the mAb was done in three ways but best by DTT, and the procedure was developed into a reliable general method providing a consistent number of SH groups. All conjugates released DOX under conditions that mimic the lysosomal environment, but were relatively stable at neutral pH. The BR96 conjugates showed good specificity in in vitro studies, being much more active than a conjugate made from an irrelevant antibody. These findings suggest further studies in vivo. ACKNOWLEDGMENT We wish to thank the Analytical Research Group for spectral and analytical data, in particular Mr. Ed Pack for the HPLC data. We thank also Dr. Kirk Leister and his group at Bristol-Myers Squibb AR&D, Syracuse, NY, for their help. Finally, we thank Drs. Terrence W. Doyle and Dinesh M. Vyas for their encouragement and support.
LITERATURE CITED (1) Hermentin, P., and Seiler, E. R. (1988) Investigations with monoclonal antibody drug (anthracycline) conjugates. Bhering Inst. Mitt. 82, 197-215. (2) (a) Shen, W. C., and Reiser, J. P. (1981) cis-Aconityl spacer
between daunomycin and macromolecular carriers: a model of pH-sensitive linkage releasing drug from a lysosomotropic conjugate. Biochem. Biophys. Res. Commun. 102,1048-1054. (b) Yang, . M., and Reisfeld, R. A. (1988) Doxorubicin conjugates with a monoclonal antibody directed to a human melanoma-associated proteoglycan suppresses the growth of established tumor xenographs in nude mice. Proc.Natl. Acad. Sci. U.S.A. 85, 1189-1193. (3) (a) Greenfield, R. S., Kaneko, T., Daues, A., Edson, . A., Fitzgerald, K. A., Olech, L., Grattan, J. A., and Braslawsky, G. R. (1990) Evaluation in vitro of adriamycin-immunoconjugates synthesized using an acid sensitive hydrazone linker. Cancer Res. 50,6600-6607. (b) Braslawsky, G. R., Edson, M. A., Pearce, W., Kaneko, T., and Greenfield, R. S., (1990) Antitumor activity of adriamycin (hydrazone linked) immunoconjugates compared to free adriamycin and specificity of tumor cell killing. Cancer Res. 50,6608-6614. (c) Greenfield, R. S., Braslawsky, G. R., Olech, L. and Kaneko, T. (1989) Anthracycline protein conjugates having a novel linker and methods for their production. EP # 0 328 147. (d) Kato, Y., Endo, N., and Kara, K. (1988) Hydrazides and method for their preparation. J. P. Kokai No. 63-57569. (4) Mueller, B. M., Wrasidlo, W. A., and Reisfeld, R. A. (1990) Antibody conjugates with morpholinodoxorubicin and acid cleavable linkers. Bioconjugate Chem. 1, 325-330. (5) Kaneko, T., Willner, D., Monkovic, I., Knipe, J. O., Braslawsky, G. R., Greenfield, R. S., and Vyas, D. M. (1991) New hydrazone derivatives of adriamycin and their immunoconjugates—A correlation between acid stability and cytotoxicity. Bioconjugate Chem. 2, 133-141. (6) Trail, P. A., Willner, D., Lasch, S. H., Henderson, A. J., Greenfield, R. S., King, D., Zoeckler, . E., and Braslawsky, G. R. (1992) Antigen specific activity of carcinoma reactive BR64-adriamycin conjugates evaluated in vitro and in human tumor xenograft models. Cancer Res. 52, 5693-5700. (7) Braslawsky, G. R., Kadow, K., Knipe, J., McGoff, K., Edson, M., Kaneko, T., and Greenfield, R. (1991) Adriamycin (hydrazone)-antibody conjugates require internalization, intracellular hydrolysis for antitumor activity. Cancer Immunol. Immunother. 33, 367-374. (8) (a) Means, G. E., and Feeney, R. E. (1990) Chemical modification of protein: history and application. Bioconjugate Chem. 1, 2-12 (b) Koppel, G. A. (1990) Recent advances with monoclonal antibody drug targeting for the treatment of human cancer. Bioconjugate Chem. 1,13-23. (9) Thorpe, P. E., Wallace, P. M., Knowless, P. P., Relf, M. G., Brown, A. N. F., Watson, G. J., Knyba, R. E., Wawrzynczak, E. J., and Blakey, D. C. (1987) New coupling agents for the
BR96 Conjugates of Doxorubicin
synthesis of immunotoxins containing a hindered disulfide bond with improved stability in vivo. Cancer Res. 47, 59245931. (10) Candiani, C., Franceschi, A., Chignola, R, Fasti, M., Anselmi, C. , Benoni, G., Tridente, G., and Colombatti, M. (1992)
Blocking effect of human serum but not of cerebrospinal fluid on ricin A chain immunotoxin potentiation of carrier proteinmonensin conjugates. Cancer Res. 52, 623-630. (11) Raso, V., and Basóla, M. (1984) Monoclonal antibodies as cell targeted carriers of covalently and non-coyalently attached toxins. NATO Adv. Study Inst. Ser. A Life Sci. 82,119-138. (12) Bourrie, B. J. P., Casellas, P., Blythman, . E. and Jansen, F. K. (1986) Study of the plasma clearance of antibody-ricinA-chain immunotoxins. Evidence for specific recognition site on the A chain that mediate rapid clearance of the immunotoxin. Eur. J. Biochem. 155, 1-10. -
(13) Li, P., Medon, P. P., Skingle, D. C., Lanser, J. A., and Symons, R. H. (1987) Enzyme linked synthetic oligonucleotides
radioactive detection of enterotoxigenic Escherichia coll in faecal specimens. Nucleic Acids Res. 15, 5275probes:
non
5287. (14) Senter, P. D., Saunier, M. G., Schreiber, G. J., Hirschberg, D. L., Brown, J. P., Hellstrom, I., and Hellstrom, K. E. (1988) Antitumor effects of antibody-alkaline phosphatase conjugates
in combination with etoposide phosphate. Proc. Natl. Acad.
Sci. U.S.A. 85, 4842-4846. (15) Ghosh, S. S., Kao, P. M., McCue, A. W., and Chappelle, H. L. (1990) Use of maleimidothiol chemistry for efficient
synthesis of oligonucleotide-enzyme conjugates hybridization probes. Bioconjugate Chem. 1, 71-76. (16) Hellstrom, I., Garrigues, H. J., Garrigues, U., and Hellstrom, K. E. (1990) Highly tumor-reactive, internalizing, mouse monoclonal antibodies to Ley-related cell surface antigen. Cancer Res. 50, 2183-2190. (17) Fell, . P., Yarnold, S., Hellstrom, I., Hellstrom, K. E., and Folger, K. R. (1989) Homologous recombination in hybridoma cells: heavy chain chimeric antibody produced by gene targeting. Proc. Natl. Acad. Sci. U.S.A. 86, 8507-8511. (18) Hellstróm, I., Beaumier, P. L., and Hellstrom, K. E. (1986) Antitumor effects of L6, an IgG2a antibody reacting with most human carcinomas. Proc. Natl. Acad. Sci. U.S.A. 83, 70597063. (19) Riddles, P. W., Blakeley, R. L., and Zerner, B. (1979)
Ellmans’s reagent: 5,5'-Dithiobis(2-nitrobenzoic acid)—A reexamination. Anal. Biochem. 94, 75-81.
Bioconjugate Chem., Vol. 4, No. 6, 1993
527
(20) Keller, O., and Rudinger, J. (1975) Preparation and some
properties of maleimido acids and maleoyl derivatives of peptides. Helv. Chim. Acta 58, 531-541. (21) Rich, D. H., Gesellchen, P. D., Tong, A., Cheung, A., and Buckner, C. K. (1975) Alkylating derivatives of amino acids and peptides. Synthesis of maleoylamino acids [1-(Nmaleoylglycyl)cysteinyl]oxytocin and [l-(N-maleoyl-ll-aminoundecanoyl)cysteinyl] oxytocin. Effect of vassopresinstimulated water loss from isolated toad bladder. J. Med. Chem. 15,1004-1010. (22) Marquart, M., and Deisenhofer, J. (1982) The three dimensional structure of antibodies. Immunol. Today 3,160166. (23) Nissonof, A. (1984) Introduction to molecular immunology, p 20. Sinauer Associates Inc., Sunderland, MA. (24) Gunewardena, P., and Cook, K. B. (1966) 1,4-dithiothreitol
as a reagent for reductive degradation of human J-G globulin. Biochem. J. 99, 8 p. (25) Gorevic, P. D., Prelli, F. C., and Frangione, B. (1985) Immunoglobulin G. Methods Enzymol. 116, 3-25. (26) Goldberg, M., Knudsen, K. L., Platt, D., Kohen, A., Bayer, E. A., and Wilchek, M. (1991) Specific interchain cross linking of antibodies using bis maleimide. Repression of ligand leakage in immunoaffinity chromatography. Bioconjugate Chem. 2, 275-280. (27) Schroeder, D. D., Tankersly, D. L., and Lundblad, J. L. (1981) A new preparation of modified immune serum globulin (human) suitable for intravenous administration. Vox Sang. 40, 373-382. (28) Siegall, C. B., Gawlak, S. L., Chin, J. L., Zoeckler, . E., Kadow, K. F., Brown, J. P. and Braslawsky, G. R. (1992) Cytotoxicity of chimeric (human-murine) monoclonal antibody BR96, IgG, F(ab')2. and Fab' conjugated to Pseudomonas exotoxin. Bioconjugate Chem. 3, 302-307. (29) Zhao, H., Willner, D., Cleveland, J. S., Braslawsky, G. R., and Brown, J. P. (1992) Determination of immunoreactivity of doxorubicin antibody immunoconjugates by a Ley competitive RIA. Bioconjugate Chem. 3, 549-553. (30) Trail, P. A., Willner, D., Lasch, S. J., Henderson, A. J., Hofstead, S. J., Casazza, A. M., Firestone, R. A., Hellstrom, I. and Hellstrom, K. E. (1993) Cure of xenografted human carcinomas by BR96-doxorubicin Immunoconjugates. Science 261, 212-215.
