Bioconjugate Chem. 1990, 7, 325-330
325
Antibody Conjugates with Morpholinodoxorubicin and Acid-Cleavable Linkers Barbara M. Mueller,*y+Wolf A. Wrasidlo,* and Ralph A. Reisfeldt Scripps Clinic and Research Foundation, La Jolla, California 92037, and Biotechnetics, San Diego, California 92121. Received June 7, 1990
Antibody-morpholinodoxorubicin conjugates were prepared for targeted immunotherapy of human melanoma. Spacer molecules that differ in hydrolytic stability were employed between the C-13 of the drug and amino residue of lysine on the monoclonal antibody. Antibody-drug conjugates were made with five structurally different morpholinodoxorubicin derivatives including oxime, phenylhydrazone, (sulfonylphenyl)hydrazone,and acylhydrazone moieties. Hydrolytic stability of the antibody conjugates directly correlated with their in vitro cytotoxicity against melanoma cells. Derivatives or conjugates with the greatest hydrolytic stability showed the least cytotoxicity.
INTRODUCTION Anthracyclines are effective antitumor antibiotics that have been used extensively in the treatment of different cancers (1-5).Although anthracyclines are among the most potent anticancer drugs known, their clinical application is limited by their cardiotoxicity. One possibility to overcome the toxicity of drugs to normal tissues is to attach the drug to a carrier system, such as monoclonal antibodies (Mabl), which are capable of targeting tumor cells. In this way, the drug concentration will be selectively enhanced in tumor tissues, while the systemic concentration is reduced, thereby eliminating some of the toxic side effects of chemotherapy. T o this end, anthracyclines were conjugated to a variety of Mab and these chemoimmunoconjugates were analyzed in several preclinical in vitro and in vivo studies (6-13). Among the extensive list of anthracycline derivatives synthesized, morpholino doxorubicin and its 3-cyano analogue were shown to exhibit cytotoxic potencies against human tumor cell lines that can be from 2- to 10 000fold higher than those of any other anthracyclines (1416). This increase in potency, however, is not accompanied by an increase in cardiotoxicity. An additional advantage of these anthracycline analogues is that, due to different intracellular mechanisms of action than that of their parental drug doxorubicin (DXR), they are able to overcome multiple drug resistance (17-20). Recently, while preparing chemoimmunoconjugates of these compounds and Mab, we found that their potency could be varied over several orders of magnitude, depending on the nature of the spacer molecules employed in the conjugation reactions (21).These findings prompted us to investigate the linker chemistry of these immunoconjugates in greater detail. We describe here conjugates between MRA-HC1 and Mab LM609 directed against the vitronectin receptor a& on human melanoma cells (22,23). The human melanoma cell line used in this study (M21) has 2.5 X lo5 binding
* To whom correspondence should be addressed.
+ Scripps Clinic and
Research Foundation. Biotechnetics. 1 Abbreviations: Mab, monoclonal antibody; DXR, doxorubicin; MRA-HC1,3’-deamino-3’-(4-morpholinyl)doxorubicin, NHS, N-hydroxysuccinimide; EcDi, carbodiimide;FBS, fetal bovine serum; DMA, dimethylacetamide. 1043-1802/90/2901-0325$02.50/0
sites per cell for LM609 and internalizes LM609 with an endocytotic rate constant (k,) of 1X 1W2 (21).This implies that at saturation and 37 “C, 2500 antibody molecules are internalized into each cell per minute. We describe here the synthesis of MRA-HC1-LM609 conjugates with different acid-cleavable linkers and report the results of a study on the hydrolytic stability and cytotoxicity of these conjugates. EXPERIMENTAL PROCEDURES
3’-Deamino-3’-(4-morpholinyl)doxorubicin (MRAHCl). MRA-HC1was prepared by a method similar to that described in the literature (14). DXR was first reacted via 2,2’-oxybisacetaldehyde,followed by a reaction with sodium cyanoborohydride to form the morpholino- and (cyanomorpho1ino)doxorubicinmixtures. These mixtures were separated by silica gel column fractionation and after purification provided morpholino and cyanomorpholino derivatives in 45% and 20% yield, respectively. Homogeneity of these compounds was determined by silica gel thin-layer chromatography (TLC) (19:l chloroform/ methanol) and the purity was found to be 98% by reversephase HPLC analysis (20 mM ammonium acetate, pH 4.5/ methanol). 3’-Deamino-3’-(4-morpholinyl)doxorubicin 13-[ 0(Carboxymethy1)oximel (Compound V). A solution of 6.1 mg (9.7 X 10-5 mol) of MRA-HC1 in 500 pL of anhydrous dimethylacetamide (DMA) and 3 pL of triethylamine (1.1molar excess) was stirred in the dark at 50 “C. A stock solution of 45 pL of carboxymethoxylamine (4.66 mg; 2.13 X mol in 200 pL of dimethylacetamide) was then added with stirring. The course of the reaction was followed by reverse-phase HPLC and the elution time of the MRA-HCl was 20.29 min and that of its acid derivative equaled 15.89 min. The reaction was complete after 90 min and the product was 95 % pure by HPLC analysis. Visible spectrophotometric analysis of the product showed peaks at 530, 488, 474, and 382 nm, characteristic of the doxorubicin chromophores. 13-[ (4-Carboxyphenyl)hydrazono]-3’-deamino-3’(4-morpholinyl)doxorubicin(Compound IV). MRAHCl(4.3 mg, 6.6 X 104 mol) in 200 pL of DMA was stirred in the dark and 10 pL of a 10 mg/100 pL stock solution of 4-hydrazinobenzoic acid was added. The clear reddish 0 1990 American Chemical Society
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326 Bioconjugate Chem., Vol. 1, No. 5, 1990
solution was heated for 60 min at 43 "C. Then another 2 pL of reagent was added and the reaction continued for 60 min. HPLC analysis revealed quantitative conversion to the hydrazone derivative. The visible spectrum of this compound showed peaks at 530, 495, 478, and 389 nm, which are characteristic of doxorubicin, plus an additional absorption peak at 324 nm which is due to the phenylhydrazone moiety. 13-[ [ (4-Carboxyphenyl)sulfonyl]hydrazonol-3'deamino-3'-( 4-morpholinyl)doxorubicin (Compound 111). A solution of 7.16 mg (1.12 X mol) of MRAHC1 in 180 pL of dimethylacetamide was stirred in the dark and 20 pL or 1.14 X 10-5 mol of a stock solution of 3-(hydrazinosulfony1)benzoic acid (25 mg in 200 pL of DMA) was added with stirring for 22 h at ambient temperature. After this period, HPLC analysis revealed the complete disappearance of free drug and showed a single new absorption band of the hydrazone derivative. Analysis of the visible spectrum indicated absorption bands at 528, 489, 474, and 385 nm attributable to doxorubicin and a new band at 338 nm due to the (hydrazinosulfony1)phenyl chromophore. 13-[(Hydrazinoadipyl) hydrazono]-3'-deamino-3'(4-morpholinyl)doxorubicin (Compound 11). A mixture of 6.5 mg (1 X mol) of MRA-HC1 in 300 pL mol) of adipic dihydrazide in of DMA, 15 mg (1.1X 200 p L of dimethylacetamide, and 3 mg of lithium chloride was stirred in the dark. Then, 3 pL of acetic acid was added and the clear red solution was stirred for 18 h. After this period, TLC (19:l chloroform/methanol) revealed the absence of free starting material. The slightly turbid solution was passed through a silica gel plug using methanol as an eluant. The product was purified via preparative silica gel TLC (3:l chloroform/ methanol). Reverse-phase HPLC analysis (20 mM ammonium acetate, pH 4.5/methanol) of this compound showed a purity of 93% and visible spectral analysis revealed bands at 531, 486,474, and 385 nm. 13-[ (3-Carboxy-1-oxopropyl)hydrazonol-3'-deamino3'-(4-morpholinyl)doxorubicin (Compound I ) . A mixture of 4.9 mg (7.5 X 10-6 mol) of MRA-HCl in 250 pL of DMA and 3.2 mg (2.4 x 10-5 mol) of succinic acid monohydrazide was stirred in the dark a t 45 "C for 18 h. The resultant turbid solution was filtered and the product was purified by preparative silica gel TLC (3:l chloroform/ methanol). The purity of this material by HPLC was 97%. The compound showed the characteristic visible spectrum with maxima at 534,488,474, and 380 nm. Coupling Reactions with Mabs. Mab LM609 used in this study is directed against the vitronectin receptor expressed on human melanoma cells. LM609 is of IgGl isotype and was purified from tissue culture supernatants by protein A Sepharose affinity chromatography. The doxorubicin derivatives were coupled to the NH3 of the lysine side chains of this antibody via the carboxyl terminals by N-hydroxysuccinimide (NHS) activation. In a typical coupling procedure, the compound was dissolved in DMA and reacted with equal molar quantities of carbodiimide (EcDi) and NHS in the dark at 4 "C with stirring for 18 h. Conversion rates to the NHS esters were nearly quantitative under these reactions conditions, as determined by HPLC analysis. Solutions of the active esters were then added to Mab LM609 dissolved at approximately 10 mg/mL of solution in 100 nM phosphate-buffered saline (PBS) in a 30-fold molar excess at pH 7.8-8.0. After stirring for 3 h, the drugantibody conjugates were purified by Sephadex G-50 column chromatography, filtered, and stored at 4 "C until
200
280
360
440
520
al.
600
Nanometers
Figure 1. UV absorption spectrum of MRA-NNHC&CONHMab conjugate showing three distinct maxima corresponding to Mab (at 280 nm), linker (at 324 nm), and drug (at 488 nm) chro-
mophores.
further use. The drug to antibody ratios were determined by spectroscopy (see Figure 1) at 280 and 480 nm using extinction coefficients of 9.9 and 13 mM-l cm-l, respectively. Stability Experiments. The stability of various preparations was tested by incubating samples in PBS under controlled conditions of temperature and pH by determining the extent of degradation by HPLC analysis of aliquots that were periodically removed from the preparation. Typically, 10 pL of such aliquots were diluted with 30 pL of methanol prior to injection on a Bondapak C18 column (Waters Assoc.). The elution was carried out with a gradient of 10 mM potassium phosphate, pH 5.6, and 60% methanol. The extent of degradation was determined by the ratios of areas under the peaks for the derivatized versus the free drug. Degradation studies of the antibody-drug conjugates were done by incubating samples at 37 "C, removing 100-pLaliquots, and separating free drug from the conjugate by Sephadex G-50 gel chromatography. The residually bound drug was then determined by fluorescence spectroscopy (Perkin-Elmer LS-SR spectrophotometer) at an excitation frequency of 480 nm and drug concentration was measured a t an emission frequency of 585 nm. I n Vitro Cytotoxicity. The human melanoma cell line UCLA-SO-M21 was kindly provided by Dr. D. L. Morton (UCLA, Loti Angeles, CA) and a subclone (M21) established from these cells in our laboratory was used for our studies. M21 has 2.5 X lo5 binding sites for Mab LM609 and internalizes LM609 with an endocytotic rate constant (k,) of 1X (22). The M21 cells were grown in RPMl tissueculture media supplemented with 10% fetal bovine serum (FBS). The adherent growing cell line was detached with 0.5 mM EDTA, 0.15 M NaC1,0.02 M HEPES and plated at lo4cells in 100pL of RPMI containing 10%FBS in each well of a 96-well tissue-culture plate. These cells were allowed to adhere overnight and then dilutions of drug, antibody, or conjugate were added in 10-pL volumes and incubated with the cells for 2 h. Thereafter, the plates were washed three times in tissue-culture media and incubated overnight. Each well received 1 pCi of [sH]thymidine and after a 16-h incubation, the cells were harvested onto glass-fiber filters with a Skatron cell harvester. The filters were placed in Ecolume scintillation solution (ICN, Irvine, CA) and were counted in a @-scintillationcounter. [3H]Thymidine incorporation, as percentage of untreated control cells, is used to express cytotoxicity.
Bloconjug8te Chem., Vol. 1, NO. 5, 1990 327
Antibody Conjugates with Morphoiinodoxorubicin
woH
Scheme I
I
Me0
0
I
0
X
=
OH
R-X-NH,
''.OH
o
W
OH O
N/'\RH
L
OH 0
"OH
Me0
NH and R = -CO-CH,CH,-COOH
= -CO(CH,),-CONHNH,
0
I II
-SO,-@OOH
111
O C O O H
IV
X = 0 and R = -CH,-COOH
OH 0
4
o i 40
20
0
V
60
80
Time, hours
Scheme I1 0
OH
N-NH-R-COOH OH
@#k?9
Me0
0
Figure 2. Hydrolytic stability of MRA-HCl derivatives expressed as percent of nonhydrolyzed derivative after incubation for various times at pH 4.5 and 37 "C. 0
OH
-
1w
Bo80-
-
70
H
€050-
Me0
0
OH 0
ri
401
IV,V
ll
10
RESULTS
The (2-13 substituted morpholinoanthracyclines were prepared according to Scheme I. Conversions of the parent MRA-HC1 to the hydrazones took place in near quantitative yields (>go% ) under mild reaction conditions and the derivatives were readily purified by preparative TLC. With the exception of compound 11, all other derivatives were conjugated to Mabs via their N-hydroxysuccinimide esters as shown in Scheme 11. The intermediate NHS esters were determined by HPLC by first converting small aliquots to stable derivatives with ethanolamine. Then the intermediate NHS esters were linked directly to Mabs without any further purification. Immunoconjugates were prepared by the addition of about 30 molar excess of NHS ester to antibody and the Sephadex G-50 purified preparations yielded drug to Mab ratios of 3 to 6. In these conjugates, the specific binding characteristics of the antibody were not significantly affected as measured by testing serial dilutions of Mab and conjugate in a n ELISA assay with goat antimouse horseradish peroxidase as secondary reagent (data not shown). Compounds I-V were tested for hydrolytic stability at pH 4.5 and 7 a t 37 "C. HPLC analysis of samples taken periodically showed retention times corresponding to free MRA-HC1 without any secondary hydrolysis products being present. The results of these experiments are shown in Figures 2 and 3. Except for the oxime derivative (Compound V), which was completely stable under these conditions, all other derivatives exhibited hydrolysis a t rates that depended on the structure of the R group incorporated into these compounds. At pH 7, only the aliphatic acylhydrazone showed significant hydrolytic instability, while the other hydrazone derivatives exhibited relatively good resistance to hydrolytic degradation. At pH 4.5 both acyl (I and 11)and sulfonylhydrazones (111)
I
,
.
I
.
20
0
I
.
I
40
.
1
60
I
,
80
.
100
I
120
Time Hrs
Figure 3. Hydrolytic stability of MRA-HC1derivatives expreased as percent of nonhydrolyzed derivative after incubation for various times at pH 7.0 and 37 O C .
90 50 30 -
110
70
10
-101
.9
.
I
-8
.
, -7
.
, -6
.
, -5
.
, .4
. 3
Concentration (10E.)Molar
Figure 4. Cytotoxicity of MRA-HCl and its hydrazone derivatives on the human melanoma cell line M21. Cells were incubated with drugs for 2 h and their ability to incorporate [SHIthymidine was measmec! ?4 h later.
exhibited relative rapid hydrolytic reversal rates to free MRA-HC1and only the oxime derivative remained stable. Figure 4 depicts the cytotoxicity of MRA-HC1 and its hydrazone and oxime derivatives on the human melanoma cell line M21. These substitutions of the C-13 position of the original drug result in a loss of cytotoxicity of 1 and 2 log units, respectively. Hydrolysis data for antibody-drug conjugates of I, 111,
328
Mueller et 81.
Bioconjugafe Chem., Vol. 1, No. 5, 1990 120
Scheme I11
0
N-X-
N-X-
I
MRA-C=N-X-
I
MRA-C-
_.)
I
OH,
I O
CH,OH
_*
MRA-C-OH
I
CH,OH
CH,OH
0
It
MRA-C-
CH,OH
-H
1
+
CH,OH
1 0
20
40
60
80
100
120
140
Time his
Figure 5. Hydrolytic stability of LM609-MRA conjugates expressed as percent of MRA-HC1bound to Mab after incubation for various times at pH 4.5 and 37 O C . 110-
9070
-
50-
3010
-10
! 0
I 10
20
Concentration 11OE-7 MI
Figure 6. Effect of linker stability on the cytotoxicity of LM609MRA conjugates. M21 cells were incubated with LM609MRA conjugates for 2 h and their ability to incorporate [3H]thymidine was measured 24 h later.
and V with Mab LM609 are summarized in Figure 5. The results obtained are similar to those shown in Figures 2 and 3. The acylhydrazone I conjugate dissociated about 90% within the first 8 h of incubation at pH 4.5 and 37 "C. However, under these same conditions, the acylsulfonyl-MRA-HC1-antibody (111) conjugate hydrolyzed at a rate of 1 order of magnitude slower, while the oximeMRA-HC1-antibody conjugate (V) remained essentially unchanged. The effect of linker structure on the cytotoxicity of these conjugates was also investigated. Plots of [3H]thymidine incorporation into M21 melanoma cell as a function of molar concentration of the free drug MRA-HC1 or molar equivalents of drug linked to Mab LM609 are shown in Figure 6. The hydrolytically stable oxime V conjugate did not exhibit any significant cytotoxicity over the time period investigated, while both the hydrazones I11 and I were cytotoxic, with the acylhydrazone being significantly more so, approaching the cytotoxicity of free MRA-HC1. DISCUSSION
The most significant result from this study has been the direct correlation between hydrolytic stability of the intermediate drug derivatives and their antibody conjugate with the cytotoxicity against human tumor cells. In every case, derivatives or conjugates with the greatest hydrolytic stability showed the least cytotoxicity. Since the amino group of daunosamine, which is commonly used when modifying anthracyclines with a linkable group, is unavailable in the morpholino derivatives of anthracy-
clines, we choose to react these through their C-13 carbonyl group. It has been shown previously (24) that the carbonyl group of daunomycin can be reacted with hydrazines and semicarbazide, yielding the corresponding hydrazones and semicarbazone, respectively. We found that morpholinoanthracyclines could be readily modified for antibody conjugation through their (2-13 carbonyl group. Furthermore, condensation reactions with hydrazines or oximes yielded the corresponding hydrazono and hydroxyimino derivatives under mild reaction conditions in near quantitative yields. The use of heterobifunctional linkers of the general formula HzNNHRCOOH or HzNORCOOH resulted in derivatives with terminal carboxy functionalitiea as shown in Scheme I. These reactive derivatives could readily be activated via carbodiimidel N-hydroxysuccimide intermediates and conjugated to lysine residues of monoclonal antibodies via the reactions of Scheme 11. Our initial effort involved the reaction of both morpholino- and (cyanomorpho1ino)doxorubicin with carboxymethoxylamine and subsequent conjugationto several Mabs directed against different tumor-associated antigens expressed on human melanoma cells, including the vitronectin receptor, chondroitin sulfate proteoglycan, gangliosides, and epidermal growth factor receptor. In vitro assays with these chemoimmunoconjugates indicated good immunoreactivityand specifcity. However, a substantial loss of 2 orders of magnitude in cytotoxicity was observed with both intermediate oxime derivatives,as well as their final antibody conjugates. These findings are similar to those of earlier studies (25) which indicated that the cytotoxic properties of daunorubicin are lost whenever the drug is attached to carrier proteins in an irreversible manner. Thus, we were prompted to investigate other hydrazino derivatives and, in particular, to study the stability of these new derivatives and their respective immunoconjugates. Of particular interest to us was the stability observed a t 37 "C under acidic conditions, since previous studies of acid-labilelinkers (2628) suggested that cleavage in the acidic milieu of the endosomes of cancer cells could release the toxic agent from the conjugate which then could transverse the endosome membrane and cause cell death. The results of hydrolysis studies of five structurally different MRA-HC1derivatives, summarized in Figure 2, indicated that oxime or phenylhydrazone derivatives were quite stable while those with either sulfonyl or acyl groups in the linker were readily hydrolyzed to regenerate the carbonyl moiety. The general mechanism of hydrolysis of carbon-nitrogen double bonds involves initial addition of water, followed by elimination of the nitrogen moiety (29, 30). Thus, the hydrolytic regeneration of MRA-HCl from the derivatized conjugates can be viewed as shown in Scheme 111. For the acid-catalyzed reaction, the specific mechanism shown in Scheme IV would apply. Thus, the nature of X should have a significant influence on the above mechanism. The relatively good hydrolytic stability of the oxime and phenylhydrazone can be attributed to the
Antibody Conjugates with Morphollnodoxorubicin
Scheme IV
CH,OH Scheme V H
04
presence of the neighboring oxygen and NH groups in the linker. On the other hand, the relative ease of hydrolysis of the acyl and sulfonyl derivatives can be attributed to the delocalization of the lone electron pair of the neighboring carbonyl or sulfonyl groups (Scheme V). Of the five compounds investigated, the acylhydrazone derivative (structure I) was the least stable. In fact, slow hydrolysis was evident with this compound even at neutral pH, which could pose a storage problem with immunoconjugates, as well as result in premature drug release of these antibody conjugates in in vivo experiments. Ideally, the optimal drug-linker-antibody system is one which exhibits the greatest stability under physiological conditions, followed by rapid cleavage after endocytosis. From the data presented here, it appears that the best candidate is intermediate HI, which is quite stable at pH 7 and 37 "C (Figure 3) and when conjugated with Mab LM609 is reasonably cytotoxic against the M21 human melanoma cell line. ACKNOWLEDGMENT
We wish to thank George L. Tong for his help in synthesizing morpholinodoxorubicin and Lynne Kottel for preparing this manuscript. This work was supported in part by a grant from the National Cancer Institute, Outstanding Investigator Award lR35CA42508. This is the Research Institute of Scripps Clinic Manuscript Number 6425-IMM. LITERATURE CITED (1) DiMarco, A., Gaetani, M., and Scarpinato, B. (1969) Adriamycin (NCS-123,127): A new antibiotic with antitumor activity. Cancer Chemother. Rep. 53, 133-137. (2) Bonadonna, G., De Lena, M., and Beretta, G. (1971) Preliminary clinical screening with adriamycin in lung cancer. Eur. J . Cancer 7, 365-367. (3) Davis, H. L., and Davis, T. E. (1979) Daunorubicin and adriamycin in cancer treatment: An analysis of their roles and limitations. Cancer Treat. Rep. 63, 809-815. (4) Oki, T. (1984) Structure-activity relationship of antitumor anthracyline antibiotics and drug development. Stud. Biophys. 104, 169-200. (5) Weiss, R. B., Sarosy, G., Clagett-Carr, K., Russo, M., and Leyland-Jones, B. (1986) Anthracycline analogues: The past, present, and future. Cancer Chemother. Pharmacol. 18,185197. (6) Hurwitz, E., Maron, R., Arnon, R., Wilchek, M., and Sela, M. (1978) Daunomycin-immunoglobulin conjugates, uptake and activity in vitro. Eur. J . Cancer 14, 1213-1220. (7) Hurwitz, E., Maron, R., Bemstein, A., Wilchek, M., Sela, M., and Arnon, R. (1978) The effect in vivo of chemotherapeutic drug-antibody conjugates in two murine experimental tumor systems. Int. J . Cancer 21, 747-755.
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