Pergamon Bioorganic & Medicinal Chemistry, Vol. 3, No. 10, pp. 1305-1312, 1995 Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All fights reserved 0968-0896/95 $9.50 + .00 0968-0896(95)00126-3 Novel Doxorubicin-Monoclonal Anti-carcinoembryonic Antigen Antibody Immunoconjugate Activity in vitro Achilles Lau, a Gervais Btrub~, b* Christopher H. J~ Ford c and Maureen Gallanff aSchool of Pharmacy, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A I B 3V6 bDdpartement de chimie-biologie, Universit~ du Quebec fi Trois-Rivi~res, C. P. 500, Trois-Rivi~res, PQ, Canada, G9A 5H7 C Oncology Research Laboratory, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, AI B 3V6 Abstract--Doxorubicin was modified with five different heterobifunctional reagents to produce drug analogs containing 3'-N- amide or C-13 hydrazone linkage with maleimide. Synthesis and characterization of two new reagents, 4-maleimido- benzohydrazide trifluoroacetate salt (13) and N-(4-maleimidobenzoyl)-6-aminoeaprohydrazide trifluoroacetate salt (14) are described here. All Dox maleimido derivatives were conjugated to thiolated anti-carcinoembryonic antigen monoclonal antibody, 11-285-14, via a Michael addition reaction. Antibody-directed cytotoxicity was demonstrated with the MTr assay using combinations of antigen-positive and antigen-negative cell lines. The immunoconjugates prepared from Dox 3'-N-amide analogs are not active in vitro, however, Dox(hydrazone-linked) immunoconjugates are selectively toxic to the CEA positive cell line. Introduction Recent developments in conjugation chemistry focus on novel methods for producing drug-monoclonal antibody (MAb) immunoeonjugates for site-specific drug deliv- ery with preservation of both drug toxicity and antibody binding. Efforts to investigate the usefulness of antibody mediated targeting (AMT) have led to the development of a variety of cross-linking reagents to produce immunoconjugates in which the linkage can be cleaved by,the target cells. The development and efficacy of AMT have recently been reviewed. L2 Doxorubicin (Dox, 1; Chart 1) plays an important role in cancer treatment and may be the most utilized anti- tumor drug worldwide. 3 However, its efficacy is impeded by toxicity, including myelosuppression, muco- sitis and alopecia. The greatest concern is the unique cumulative cardiac damage, which is the major obstacle to the use of Dox in cancer treatment. 4 Thus, Dox is the logical choice for conjugation with MAbs in targeted chemotherapy. Carcinoembryonic antigen (CEA) is the best known tumor associated antigen which often exists in elevated amounts in various human cancers and in the blood of cancer patients and is associated with the common solid tumors that cause high mortality. 5 Thus, CEA highly specific MAb, 11- 285-14, was selected as a carrier of Dox. Moreover, recent investigations in our laboratory have demon- strated that this particular MAb is internalized in all high CEA-expressing cell lines such as LSI74T. This is an essential requirement to achieve drug targeting and potential in situ release of the drug. 6 Recently, the development of hydrazone linkers has been shown to allow release of the unmodified Dox under mild acidic conditions and to produce complete tumor regression in vivo. 7-9 Deconjugation via a chem- ical process in the intracellular compartment, lyso- some, to release the free drugs will become the major approach to design Dox immunoconjugates for improv- ing therapeutic efficacy. Below, we report the in vitro cytotoxicity results obtain- ed with Dox 3'-N-amide derivatives (2a-4a), Dox C-13 hydrazone derivatives (5a, 6a) and their immuno- conjugates (2b-6b). In vitro testing indicates that the Dox 3'-N-amide linked immunoconjugates are not active. However, preliminary evaluation of Dox C-13 hydrazone linked immunoconjugates suggests selective toxicity for a high CEA-expressing cell line. Results and Discussion This paper reports on our effort to use MAb as a tool in targeted chemotherapy. We also describe the methods for linking Dox to MAbs via an acylhydrazone and arylhydrazone bond at the 13-keto position of Dox. Our newly developed heterobifunctional linkers (9, 10, 13 and 14) are regioselectively reacted with the 3'-N- amine or C-13 keto moiety of Dox. All linkers are easy to prepare and stable during handling. Using these linkers, we obtained immunoconjugates with different linkages (amide or hydrazone linkage) and different linker arms (phenyl, N-benzoylaminopentyl or N- benzoylaminodecyl spacer arm), and then compared the cytotoxic effect from these immunoconjugates in vitro. CEA highly specific MAb, 11-285-14, has been well characterized immunocytochemically and confirmed to 1305
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`1306 A. LAU et al. o OH 0 6o.2o'6." '? CH3~~~ j I NH HO \ RI R 1 R 1 1. H 2a. O ~ N--ArCO o 2b. O MAb--S~N_ArC O O 3=t. O ~ N--ArCONH(CHz)sCO O 3b. o MAb--S~N--ArCONH(CHa)sCO O 4a. 0 ~ N--ArCONH(CHz)I 0CO o 4b. Chgl. O MAb--S~N--ArCONH(C Hz)loC O o be reactive only with CEA and non-reactive with norm- al cross-reacting antigens (NCA). 10-12 The potential of targeted chemotherapy using vindesine 11-285-14 conjugates had previously been demonstrated in vitro; the efficacy and specificity have been correlated with CEA density in different cancer cell lines. ~3 These conjugates have shown their efficacy in vivo with xenografts in a nude mouse model, t4 Preliminary results with Dox-ll-285-14 conjugates have also shown efficacy in vitro, ns Therefore, we further investigated the efficacy of Dox-ll-285-14 conjugates using novel heterobifunctional linkers. Dox has been coupled to an antibody via the amino group of the sugar moiety (daunosamine) via either the carbonyl group at C-13 or the C-14 aglycon side chain. Modifications of the amino sugar of anthracyclines had been shown to decrease the cytotoxic activity of the drug. m Our experimental results indicated that all Dox 3'-N-amide derivatives were less toxic than the uncon- jugated drug against the CEA positive cell line, LS174T. The binding of Dox to DNA by intercalation is one of the most accepted mechanisms postulated for its cytotoxicity. Structure-activity studies with anthra- cyclines have shown that the sugar residue is an important contributor to cytotoxicity. +'~6 It has been suggested that anthracycline immuno- conjugates show no significant cytotoxicity if the drugs are not deconjugated from the antibody carder at the target site. 17 The attachment of Dox to the antibody with acid-sensitive linkage and the release of Dox from immunoconjugates after internalization inside the lyso- some compartment enable the drug to retain pharma- cological activity. The very poor cytotoxicity of Dox 3'- N-amide immunoconjugates is probably due to a reduced intracellular release of the active drug from the conjugates. The amide linkage has been shown to be too stable to produce an effective conjugate, ts However, the bioconjugates prepared from Dox or Daunorubicin
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`Doxorubicin-MAb immunoeonjugate activity 1307 3'-N-amide analogs that contain a phenyl isothio- cyanate group have shown efficacy in vitro) 9 Moreover, there have been a few reports in the literature where drug derivatives were inactive in vitro but after being linked to an antibody, showed increased cytotoxicity of the immunoconjugates. 2°~1 In vitro testing results from Dox 3'-N-amide derivatives and immunoconjugates were matched with each other; no activity was obtained from any drug derivative nor its conjugate. If the drug derivatives do not exhibit any cytotoxicity, one might expect that the immunoconjugates should not show any activity when tested in vitro. Synthesis of Dox immunoconjugates Synthesis of the immunoconjugates was achieved by thiolating the MAbs with 2-iminothiolane (2-IT) and reacting the thiolated MAbs with Dox derivatives which contained maleimide. The Dox derivatives and their corresponding immunoconjugates are listed in Charts 1 and 2. Dox:MAb ratios of 1.75-2.63 were achieved when 10 equivalents of Dox derivatives were mixed with MAb containing 6.26--7.14 thiol groups. Final protein yields following conjugation of drug to MAb are 32--60%. Cytotoxicity of Dox derivatives and immunoconjugates on human colon adenocarcinoma cell lines Dox derivatives and immunoconjugates were tested in vitro for cytotoxicity using the MTT assay with antigen positive (LS174T) and antigen negative (COLO- 320DM) cell lines. All Dox 3'-N-amide derivatives did not show significant cytotoxic effects on the LS174T cell line when compared to Dox (Fig.l). Derivative 2a showed the highest cytotoxicity and caused 57% cell mortality at 40 ttM while 3a caused 50% cell death at 40 IxM. Compound 4a was much less cytotoxic and showed 30% inhibition of cell growth at 40 gM. However, ioo ~ -~ 80~ T ._> ~" 6o~ it) 40 L • 3a = • 4a 20:- OL 2 5 10 50 Log [Dox or Derivative] (ttM) Rgure 1. Dose-response curves for Dox and Dox 3'N-amide derivatives on LS174T cells (mean + SD). 0 OH II ~C--CI'I20H ~ '"'OH I NN HO B 2 5a. O ~ N--ArCONHN o 5b. O MAb--S~N__ArCONHN O 6a. O ~ N-- ArC ONH(CI-.12) 5CONHN O 6b. O MAb-- ,,~N _.q. II ArCONH(CH2) sC ONHN II O Chart 2.
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`1308 A. LAU et aL the Dox hydrazone derivatives 5a and 6a showed simi- lar cytotoxic effects when compared to Dox on both the LS174T and COLO320DM cell lines (Figs 2 and 3). The LS174T cell line showed higher sensitivity to Dox and Dox hydrazone derivatives than the COLO320DM cell line. 100 "~ T ~" • Dox 80 ~","~ • 5a ~ 411 2O 0 J i 0.1 0.5 1 5 10 50 Log [Oox or Derivative] (~.M) Figure 2. Dose-response curves for Dox and Dox C-13 hydrazone derivatives on LS174T cells (mean + SD). T 100 | • Doz -- 80 ~ • 5a ,,q W) 20 I 0 ; 10 so Log [Dox or Dorivative] (O.M) Figure 3. Dose-response curves for Dox and DOx C-13 hydrazone derivatives on COLO320DM cells (mean + SD). Immunoconjugates synthesized from Dox 3'-N-amide derivatives were not sufficiently cytotoxic to produce 50% inhibition of growth of the LS174T cell line (Fig. 4). These immunoconjugates exhibited only very low cytotoxicity even at 10 ~tM. However, the immuno- conjugates prepared from Dox C-13 hydrazone deriva- tives were sufficiently cytotoxic to achieve 50% inhibition of LS174T cell growth at -8 laM (Fig. 5) and had no appreciable cytotoxic effects on the COLO- 320DM cell line (Fig. 6). _ 80 1- 40 20 0 0.5 I .. 1 s Log [Dox or Immunoconlugate] (pM) Figure 4. Dose-response curves for Dox and Dox 3'-N-amide immunoconjugates on LS174T cells (mean + SD). 100 8O cn 40 o< 2O I ...... I ; 1o Log [Dox or Immunoconjugate] (j~l) Figure 5. Dose-response curves for Dox and Dox C- 13 hydrazone immunoconjugates on LSI74T cells (mean + SD). 140 120 100 2O I 015 1 5 Log [Dox or ImmunoconJugate] ~MI 0 Figure 6. Dose-response curves for Dox and Dox C-13 hydrazone immunoconjugates on COLO320DM cells (mean + SD). The Dox(hydrazone-linked) immunoconjugates were more potent than Dox 3'-N-amide immunoconjugates, but not as potent as their starting material, Dox C-13 hydrazone derivatives. A gradual reduction in the percentage of surviving cells as the immunoconjugate concentration increased was observed when the high CEA-expressing cell line, LS174T, was evaluated. Selectivity was confirmed by the absence of conjugate effect on the low CEA-expressing cell line, COLO- 320DM, with the same concentration range. The Dox(hydrazone-linked) immunoconjugates showed ef- ficacy in in vitro testing; they were sufficiently cytotoxic to achieve 50% inhibition of LS174T cell growth at -8 ~tM. The efficacy of immunoconjugates depends on the density of antigen expressed on the target cells and the quantity of drug delivered per antibody molecule. Thus, immunoconjugates prepared with a higher drug substitution should be more potent than low drug substitution, because more drug is delivered per antibody molecule bound to the target cell. Higher Dox:MAb ratio can be simply achieved by increasing the thiol groups on the MAb and/or Dox derivative for the conjugation reaction. However, we observed that significant losses in protein yield occurred following conjugation. Thus, around seven thiol groups per MAb and a 10-fold molar excess of Dox derivative to MAb were adopted for the final con- jugation reaction. Dox:MAb ratios of 1.75-2.63 obtained in our immunoconjugates are probably too low for good antitumor activity. The present report describes the synthesis of two new linkers, 13 and 14. The preliminary testing results show
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`Doxorubicin-MAb immunoconjugate activity 1309 that Dox 3'-N-amide immunoconjugates 2b, 3b and 4b are not active in vitro while Dox(hydrazone-linked) immunoconjugates 5b and 6b are active in vitro and appear to provide a more promising approach. Further investigation of the correct spacer groups for effective release of the drug appears to be essential for de- velopment of doxorubicin immunoconjugates. The im- provement of conjugation conditions is also essential to prevent protein aggregation and precipitation. These will hopefully contribute to the development of better conjugates for antibody mediated targeting. Experimental Monoclonal antibody Thiolation of MAbs Thiolation of MAbs with 2-iminothiolane (2-IT) was carded out as described before. 25 MAbs (1.48 or 1.70 mg mL -~ in 0.1 M phosphate buffer, 1 mM EDTA, pH 8.0) were treated with 0.1 M 2-IT to make the molar ratio of MAb to 2-1T 1:100. The reaction mixture was incubated for 2 h at rt and thiolated MAbs were separated from excess 2-1T on a Sephadex G-25 column equilibrated with PBS (0.9% NaCI:I mM EDTA, pH 7.0). The thiolated MAbs were concentrated down using an Amicon ultrafiltration cell fitted with an Amicon PM10 ultrafilter (molecular weight cutoff 10,000). The aver- age number of thiol groups per protein molecule was determined by reaction with 5,5'-dithiobis-(2-nitro- benzoic acid) (DTNB). 26 MAb (11-285-14) was purified from ascitic fluid on a protein-A affinity column according to the procedure of Ford et al. 22 Cell lines LS174T and COLO320DM (both are human colon adenocarcinoma cell lines) were obtained from the American Type Culture Collection (Maryland). LS174T cells were propagated in RPMI-1640 containing 8.8% fetal calf serum, glutamine and penicillin-streptomycin. COLO320DM were maintained in minimum essential medium with 8.8% fetal calf serum, glutamine, non- essential amino acids and penicillin-streptomycin. Cells were grown at 37 *C in a humid atmosphere with 5% CO2. Materials Anhydrous reactions were performed under an inert atmosphere of nitrogen. Unless otherwise noted, starting material, reactant and solvents were obtained com- mercially from Aldrich and were used as such or purified and/or dried by standard means. 23 Dox-HCI was a gift from Adria Laboratories Inc. (Ohio). Organic solvents were dried over MgSO4, evaporated on a rotatory evaporator and under reduced pressure. All reactions were monitored by TLC. The plates were visualized by UV fluorescence. Commercial TLC plates were Sigma T6145 (polyester silica gel 60 A 0.25 mm). Flash chromatography was performed according to the method of Still et al. on Merck grade 60 silica gel, 230- 400 mesh. 24 Melting points were recorded on an Electrothermal 9100 apparatus and are uncorrected. The IR spectra were taken on a Nicolet model 205 FF-IR spectrophotometer. MS assays, (m/z) were obtained using a VG Micromass 7070 HS instrument with an ionization energy of 70 eV. Elemental analysis was conducted by Microanalysis Laboratories Limited, Markham, Ontario. NMR spectra were obtained in deuterated Me2SO4, MeOH and CHCI3 on a General Electric GE 300-NB (300 MHz) instrument: chemical shifts were measured relative to internal standards: tetramethylsilane (TMS, ~ 0.0 ppm) for ~H and =3C NMR spectra. Synthesis of heterobifunctional linkers and Dox deriv- atives The synthesis and characterization of the linkers, 4- maleimido-benzoic acid (8), N-(4-maleimidobenzoyl)- 6-aminocaproic acid (9) and N-(4-maleimidobenzoyl)- l l-aminoundecanoic acid (10) (Scheme 1), and their Dox derivatives (2a--4a) were reported in the preceding paper. 25 HzN'-~CO2H 7 I 1. (CH3CO)20 2. Ac20 AcO" Na + o t° -\OH o 9 1. Et3N CICO2iBu 2. (Me3Si)zNH H2N(CH2)nCO2H o ~N .__~ ~0 - \ NH(CH2)nCO2 H O 9,10 9:n=5 10: n-- 10 Scheme 1. Derivatives 13 and 14 were prepared directly from acids 8 and 9, respectively by converting the carboxylic acid group to protected hydrazide followed by treatment with
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`1310 A. LAU etaL trifluoroacetatic acid (TFA) (Scheme 2), using a procedure modified from Willner et al. 8 Compound 8 (2.68 g, 1.3 mmol) was suspended in 60 mL CH2CI 2 and cooled to 0 "12 before the addition of triethylamine (1.89 mL, 13.6 mmol) and isobutyl chloroformate (1.76 mL, 13.6 mmol). The mixture was stirred for 1 h at 0 *C and then tert-butyl carbazate (1.62 g, 12.3 mmol) in 10 mL CH2C12 was added dropwise. Stirring was continued for 2 h at rt. The reaction mixture was diluted with EtOAc (150 mL) and washed with saturated NaHCO3 (2 x 50 mL), 0.1 N HCI (2 x 50 mL), saturated NaCI (2 x 50 mL), H20 (50 mL), then dried (MgSO4) and evaporated to give crude protected hydrazide 11. The product was purified by flash chromatography, using two mixtures of solvent: initially 1:2 and then 2:3 Me2CO:hexane, to yield 2.44 g (60%) of 11: mp 182-184 *(2; IR (KBr) 3545, 3329, 3093, 2991 and 1710 cm-I; IH NMR (Me2CO-d6): 8 9.60 (s, 1H), 8.02 and 7.53 (two d, 4H, J = 8.6 Hz), 7.07 (s, 2H), 2.99 (s, 1H), 1.45 (s, 9H); 13C NMR (Me2CO-d6): ~i 169.06 (2), 165.63, 155.47, 134.79, 134.34 (2), 131.38, 127.68 (2), 125.59 (2), 79.50, 27.30 (3); Anal. Calcd for CI6I-II7N305: C, 57.97; H, 5.17; N, 12.69. Found: C, 58.06; H, 5.14; N, 12.73. so N---R--C\o H O 8, 9 1. Et3N CICO2iBu 2. H2NNHCO2tBu O ° NHNHCO2tBu o 11,12 I TFA N.~R.~C. JO "NHNH2 " CF3CO2H O 13, 14 11, 13 : R='~ 12, 14 : R" ~--NH(CH2)s--- Scheme 2. Compound 11 (500 mg, 1.51 mmol) was dissolved in ice-cold TFA (5 mL) and stirred for 30 min in an ice- bath. The TFA was removed under vacuum at rt to give compound 13 quantitively: mp 142-144 °C; IR (KBr) 3500-2500, 3277, 3111, 2901, 2720 and 1710 cm-', 'H NMR (DMSO-d6): 8 11.62 (bs, 1H), 8.63 (bs, 3H), 8.01 and 7.56 (two d, 4H, J = 8.5 Hz), 7.24 (s, 2H); '3C NMR (DMSO-d6): ~ 169.60 (2), 165.43, 135.47, 134.97 (2), 129.38, 128.38 (2), 126.46 (2); Anal. Calcd for CI3H18N3OsF3: C, 44.17; H, 5.14; N, 11.89. Found: C, 44.16; H, 5.06; N, ll.93. Compound 9 (1.05 g, 3.18 mmol) was treated with the same procedure as above for the preparation of 11 to yield 12 (1.01 g, 72%): mp 137-139 °C; IR (KBr) 3310, 3082, 2978, 2934 and 1707 cm-I; 1H NMR (Me2CO-d6): 6 8.80 (s, 1H), 8.00 and 7.48 (d, 2H, J = 8.7 Hz), 7.73 (t, IH, J = 4.7 Hz), 7.48 (d, 2H, J = 8.7 Hz), 7.07 (s, 2H), 3.41 (q apparent, 2H, J = 6.9 Hz), 2.88 (s, 1H), 2.21 (t, 2H, J = 7.3 Hz), 1.64 (m, 4H), 1.44 (m, 2H), 1.41 (s, 9H); 13C NMR (Me2CO-d6): 8 172.14, 170.22 (2), 166.47, 156.45, 135.39 (2), 135.19, 134.94, 128.45 (2), 126.65 (2), 80.17, 40.24, 34.15, 29.91 (super- imposed on acetone peak), 28.36 (3), 27.08, 25.64; Anal. Calcd for C22H2gN406: C, 59.43; H, 6.35; N, 12.61. Found: C, 59.48; H, 6.31; N, 12.58. Compound 12 (500 mg, 1.12 mmol) was deprotected with the same procedure as above for the preparation of 13 to yield 14 quantitatively as an oily yellowish gum: IR (KBr) 3500-2500, 3107, 2944 and 1717 cm-'; IH NMR (DMSO-d6): 8 10.91 (bs, 1H), 9.40 (bs, 3H), 8.57 (t, 1H, J = 5.5 Hz), 7.95 and 7.45 (two d, 4H, J = 8.5 Hz), 7.22 (s, 2H), 3.28 (q apparent, 2H, J = 6.6 Hz), 2.25 (t, 2H, J = 7.3 Hz), 1.57 (m, 4H), 1.34 (m, 2H); ~3C NMR (DMSO-d6): ~ 171.79, 169.75 (2), 165.51, 134.86 (2), 133.95, 133.78, 127.82 (2), 126.22 (2), 40.22 (super- imposed on DMSO peak), 32.73, 28.84, 26.01, 24.49. Anal. Calcd for CI9H2,NaO6F3: C, 49.77; H, 4.62; N, 12.23. Found: C, 49.80; H, 4.57; N, 12.27. Synthesis of derivative 5a. Dox-HCI (20.58 mg, 0.036 mmol) and 13 (41.28 mg, 0.120 mmol) were dissolved in 5 mL MeOH with the addition of 3 ttL TFA. The mixture, protected from light, was stirred at rt for 1 day. The methanolic solution was concentrated to 1 mL under nitrogen at rt. Acetonitrile (4 mL) was added for precipitation and the suspension was allowed to stand at 4 °C for 1 day. The red precipitate was isolated by centrifugation and then dried under vacuum to give compound 5a (20.2 mg, 72%). The product can be reprecipitated from the remaining solution and further purified by TLC. 5a: IH NMR (DMSO-d6): 8 14.07 (s, 1H), 13.35 (bs, 1H), 11.86 (s, 1H), 7.93-7.64 (m, 9H), 7.50 (d, 2H, J = 8.4 Hz), 7.22 (s, 2H), 5.49 (d, 1H, J = 5.6 Hz), 5.31 (bs, 1H), 5.12 (bs, 1H), 4.63 (s, 2H), 4.56 (d, 1H, J = 5.7 Hz), 3.98 (s, 3H), 3.55 (bs, 1H), 1.18 (d, 3H). Synthesis of derivative 6a. Dox-HC1 (20.34 mg, 0.035 mmol) and 14 (53.65 mg, 0.117 mmol, assuming 100% yield obtained from 52 mg of 12) were dissolved in 5 mL MeOH with the addition of 3 lxL TFA. The reaction condition and the precipitation procedure were similar to the preparation of 5a. The red precipitate was dried
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`Doxorubicin-MAb immunoconjugate activity 131 i under vacuum to give 28.2 mg (90%) of 6a; IH NMR (DMSO-d6): 8 14.10 (s, 1H), 13.32 (s, 1H), 10.34 (s, 1H), 8.46 (t, IH, J = 5.3 Hz), 7.90 (m, 7H), 7.60 (dd, 1H, J = 4.3 and 2.6 Hz), 7.42 (d, 2H, J = 7.4 Hz), 7.21 (s, 2H), 5.49 (bs, 1H), 5.31 (bs, 1H), 4.95 (t, 1H, J = 6.4 Hz), 4.42 (s, 2H), 4.04 (q, 1H), 3.95 (s, 3H), 1.18 (d, 3H). Conjugation of thiolated MAbs with Dox derivatives Derivative 2a, 3a, 4a, 5a or 6a was dissolved in DMF and added to 2-IT thiolated MAbs in PBS (0.9% NaCI:I mM EDTA, pH 7.0). In a typical experiment, 10 equiv- alents of 5a were added to MAbs containing 6.55 thiol groups. Conjugation reaction was incubated at rt for 1.5 h. Then, the immunoconjugates was separated from unreacted $a by passage through a Sephadex G-25 column equilibrated with PBS (pH 7.2). The conjugate solution was concentrated down by ultrafiltration and then subjected to dialysis (2 x 8 h in PBS, pH 7.2). The absorbances of conjugates were determined at 280 nm and 495 nm to estimate protein and drug concentrations respectively, using the molar extinction coefficients summarized in Table 1. To correct for the overlap of Dox absorbance at 280 nm, the amount of MAb was determined according to the following formula MAb (gM)= A280 -- (E280 X A495/E495) 214,600 where A is the observed absorbance at noted wavelength and e is the molar extinction coefficient of the Dox derivative at noted wavelength. The Dox:MAb molar ratios ot the conjugates are presented in Table 2. Microcytostasis assay Chemosensitivity of cell lines to Dox, Dox derivatives and the immunoconjugates were determined using the MTT assay. 27 The cells (1 x 104 cells well -I) were grown in sterile 96-well culture plates at 37 °(2 in a humidified incubator with 5% CO2 for 24 h, then incubated for 24 h with Dox, Dox derivatives or Dox immunoeonjugates diluted in culture medium. After incubation, the cells were washed with PBS and incubated for 24 h, and then treated with MTT for 4 Ix The precipitate was solubilized in DMSO and the plate was read spectrophotometrieally at 570 and 630 nm. Absorbance was recorded and percentage cell survival was determined by comparing drug-, drug derivative-, or immunoconjugate-treated cells with the untreated control. Acknowledgements Financial support from the School of Pharmacy is acknowledged. We also thank Dr Gregory, Ms Baggs and Ms Brunet, Department of Chemistry, Memorial University of Newfoundland, for the spectra. References 1. Ford, C. I-L J.; Richardson, V. J.; Reddy, V. S. Ind/an J Pediatr. 1990, 57, 29. 2. Pietersz, G. A.; MeKenzie, I.F.C. Immunol. Rev. 1992, 129, 57. 3. Weiss, R. B.; Sarosy, G.; Clagett-Carr, K.; Russo, IVL; Leyland-Jones, B. Cancer Chemother. Pharmacol. 1986, 18, 185. 4. Wiemik, P. H. In Anthracyclines. Current Status and New Developments; Crooke S. T.; Reich S. D., Eds; Academic: New York, 1980; pp. 273-294. 5. Bates, S. E.; Longo, D. L Semin. Oncol. 1987, 14,102. Table 1. Molar extinction coefficient (E) of Dox derivatives 2a-6a Compound ~m~ (nm) c en0 c,n Solvent 2a 267 16853 12508 12380 DMF 3a 267 16248 11413 13236 DMF 4a 267 16017 11754 13061 DMF 5a 268 26320 18510 12531 DMF 6a 268 16028 12315 13051 DMF IgG 280 214600 PBS Dox T-N-amine derivatives (2a-4a). Dox C-13 hydrazone derivatives ($a and 6a). Table 2. Spectrophotometric evaluation of the Dox-I 1-285-14 conjugates C C C Dox MAb Dox MAb Dox/ A 280nm A 495nm A 280rim A 280rim (p.M) (lxM) MAb 2b 1.82 0.24 0.24 1.58 19.38 7.36 2.63 3b 2.10 0.24 0.21 1.89 18.13 8.81 2.06 4b 1.13 0.11 0.I0 1.03 8.42 4.80 1.75 511 1.41 0.15 0.23 1.18 11.97 5.50 2.18 6b 1.16 0.14 O. 13 1.03 10.72 4.80 2.23 C = Conjugate, A = absorbancc.
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`1312 A.L.~a.I etal. 6. Tsaltas, G.; Ford, C. H. J.; Gallant M. Anticancer Res. 1992, 12,2133. 7. Greenfield, R. S.; Kaneko, T.; Danes, A.; Edson, M. A.; Fitzgerald, K. A.; Olech, L. J.; Grattan, J. A.; Spitalny, G. L.; Braslawsky, G. R. Cancer Res. 1990, 50, 6600. 8. Willner, D.; Trail, P. A.; Hofstead, S. J.; King, H. D.; Lasch, S. J.; Braslawsky, G. R.; Greenfield, R. S.; Kaneko, T.; Firestone, R. A. Bioconj. Chem. 1993, 4, 521. 9. Trail, P. A.; Willner, D.; Lasch, S. J.; Henderson, A. J.; Hofstead, S.; Casazza, A. M.; Firestone, R. A.; Hellstr6m, L; Hellstr0m, K. E. Science 1993, 261,212. 10. Hockey, M. S.; Stokes, H. J.; Thompson, H.; Woodhouse, C. S.; MacDonnald, F.; Fielding, J. W. L.; Ford, C. H. J. Br. I Cancer 1984, 49,129. 11. Ford, C. H. J.; Gallant, M. E.; Ali, S. K. Pediatr. ICes. 1985, 19,385. 12. Price, M. R. Br. J. Cancer 1988, 57, 165. 13. Ford, C. H. J.; Bartlett, S. E.; Casson, A. G., Marsden, C. H.; Gallant, M. E. NCIMonogr. 1987, 3, 107. 14. Casson, A. G.; Ford, C. H. J.; Marsden, C. H.; Gallant, M E.; Bartlett, S. E. NCIMonogr. 1987,3, 117. 15. Richardson, V. J.; Ford, C. H. J.; Tsaltas, G.; Gallant, IV[ K Eur. J. Cancer Clin. Oncol. 1969, 25,633. 16. Yamamoto, K.; Acton, E. M.; Henry, P. W. J. Med. Chem. 1972, 15,872. 17. Hermentin, P.; Seiler, F. R. Behring InsL Mitt. 19~i, 82, 197. 18. Hermentin, P.; Doenges, R.; Gronski, P.; Bosslet, K.; Kraemer, H. P.; Hoffmann, D.; Zilg, H.; Steinstraesser, A.; Schwarz, A.; Kuhlmann, L.; Luben, G.; Seiler, F. R. Bioconj. Chent 1990, 1, 100. 19. Sweet, F.; Rosik, L O.; Sommers, G. M.; Collins, J. L Gynecol. Oncol. 1989, 34,305. 20. Garnett, M. C.; Baldwin, R. W. Cancer Res. 1986, 46, 2407. 21. Smyth, M. J.; Pietersz, G. A.; Classon, B. J.; McKenzie, F. C. JNCI 1986, 76,503. 22. Ford, C. I-L J.; Bartlett, S. E.; Casson, A. G.; Marsden, C. H.; Gallant, M. E. TumorBiol. 1987, 8, 241. 23. Perdn, D. D.; Armarego, L F. Purification of Laboratory Chemicals, 3rd ed.; Pergamon Press: Oxford, 1988. 24. Still, W. C.; Kalm, M.; Mitra, A. J. Org. Chem~ 1978, 43, 2923. 25. Lau, A.; B6rub6, G.; Ford, C. I-I. J. Bioorg. Med. Chem. 1995,3, 1299. 26. Riddles, P. W.; Blakeley, R. L.; Zemer, B. Anal Biochem. 1979, 94, 75. 27. Ford, C. FL J.; Richardson, V. J.; Tsaltas, G. Cancer Chemother. Pharmacol. 1989, 24,295. (Received in U.S.A. 8 November 1994; accepted 28 June 1995)
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