Bioconjugate Chem. 1990, 1, 245-250
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Preparation of Antigastric Cancer Monoclonal Antibody MGbz-Mitomycin C Conjugate with Improved Antitumor Activity Song Li,'?? Xue-Yong Zhang,+ Su-Yin Zhang,i Xi-Tao Chen,? Ling-Ji Chen,g Yu-Hua Shu,x Jia-Liu Zhang,t and Dai-Ming Fan? Laboratory of Gastroenterology, Xijing Hospital, Xi'an, Shaanxi Province 710032, China, and Shanghai Institute of Materia Medica, Chinese Academy of Sciences, Shanghai 200031, China. Received February 28,1990
In the present study, an antigastric cancer monoclonal antibody, MGb2, was chosen to prepare antibodymitomycin C conjugate with dextran T-40 as intermediary. Up to 20 molecules of mitomycin C were specifically bound per molecule of antibody, without significantly impairing the antigen-binding capacity of the antibody and the pharmacological activity of mitomycin C. The conjugate showed selective cytotoxicity upon human gastric cancer cell line SGC-7901 in vitro. Radioimmunoimaging and biodistribution studies indicated that, after conjugation with mitomycin C via dextran T-40 as intermediary, the tumor localization capacity of the antibody was well-retained. When tested in nude mice inoculated with human gastric carcinoma GAII in bilateral subrenal capsules, intraperitoneal injection of the conjugate twice a week for 3 weeks at the dose of 1 mg/kg of drug gave a tumor inhibitory rate of 152.29%, the result being far better than that of free mitomycin C or an irrelevant conjugate. A similar result was found in another nude mouse model of human gastric carcinoma SGC-7901. Meanwhile, after conjugation with antibody, the toxicity of mitomycin C on tested animals was significantly reduced.
INTRODUCTION Although it has been years since anticancer therapy developed, there are still many problems in the treatment of gastric cancer. Most cases are inoperable in the late stage. Conventional chemotherapy also has a drawback, that is, agents effective in killing neoplastic cells also have detrimental effects on normal cells, thus limiting the amount of drugs given ( I ) . One possible approach to overcome these limitations is to attach anticancer agents to monoclonal antibodies against gastric cancer so that the toxicity could be selectively restricted to tumor cells. It could improve therapeutic effect, minimize toxic effect, and overcome the resistance of tumors against free drugs (2). Recently, a hybridoma cell line which produces an antigastric cancer IgGl monoclonal antibody (MGb2) was established in our laboratory (3). Previous studies showed that it was highly specific against tumor cells and could be well-localized in the tumor tissues in nude mice bearing human gastric carcinoma xenografts and even in the patients with gastric cancer ( 4 ) . In the present study, MGb2 was chosen to prepare antibody-MMCl conjugate via dextran T-40 as intermediary; the in vitro and in vivo cytotoxic effect of the conjugate upon human gastric cancer cells was investigated. EXPERIMENTAL PROCEDURES
Cells and Medium. Human gastric cancer cell line SGC-7901 was obtained from Dr. X. W. Feng, Academy of Military Medical Sciences, Beijing, China. Human normal embryonic lung cell line SL, was kindly provided by Dr. L. S. Tan, Shanghai First Pulmonary Disease Hospital, Shanghai, China. They were grown as mono+ Xijing f
Hospital.
Chinese Academy of Sciences.
1 Abbreviations: MMC, mitomycin C; PAD, polyaldehyde dextran; PBS, phosphate-buffered saline; SPECT, single photon emission computed tomography; SPF, specific pathogen free.
1043-1802/90/2901-0245$02.50/0
layers in RPMI 1640 (GIBCO) supplemented with 10% fetal calf serum (Lan Zhou Institute of Biological Products, Lan Zhou, China). The cells were routinely passaged after detachment with trypsin/0.2% EDTA. Animals and Tumor Models. Nude mice of SwissDF-nu/nu and BALB/c-nu/nu genetic background (Shanghai Institute of Materia Medica, Shanghai, China) were used for the passage, tumor inoculation, and the in vivo studies. They were maintained under SPF conditions. SGC-7901 cloned human gastric carcinoma previously established in ascitic form from cultured cell line and maintained in Swiss-DF nude mice was used as a source of solid tumor. The solid tumor was induced by inoculating the tumor cell suspension (in ascitic fluid) subcutaneously. It was revealed to possess fast growth and a short latent period of 3-4 days. Ten days after the inoculation, the tumor could reach 1 cm in diameter. The human gastric carcinoma GAII was established in the Department of Pathology of Beijing Medical University and maintained in B A L B / c nude mice by serial transplantation with a duration of 4-5 weeks. Monoclonal Antibody MGb2. T h e monoclonal antibody (IgG1) was prepared in BALB/c mice by hybridoma clone established in our laboratory and purified from ascitic fluid by (NH4)2S04precipitation and ion-exchange chromatography on DEAE-cellulose (DE-52). Preparation of Immunoconjugates. Dextran T-40 was oxidized by the following Malaprade reaction (5).Dextran T-40 (Pharmacia) (1.0 g) was mixed with sodium periodate (0.33 g) in 200 mL of distilled water and incubated overnight a t room temperature in the dark. The oxidized dextran was then dialyzed extensively against distilled water and lyophilized. Sixty milligrams of PAD was incubated with 20 mg of MGb2 in 2 mL of 0.1 M PBS, pH 7.2, for 24 h a t 4 "C, following which 1 2 mg of MMC (Kyowa Hakko Co., Tokyo, Japan) in 4 mL of PBS was added. The solution was stirred for another 24 h. The Schiff bases thus formed were then reduced by the addition of 0.1 mL of sodium borohydride PBS solution (1 mg/mL). The 0 1990 American Chemical Society
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reaction mixture was incubated with the reducing agent for 2 h a t 4 OC and then applied to a Sephadex G-200 column (1.8 X 65 cm) equilibrated with PBS. The antibody peak was collected and analyzed for the MMC/IgG molar ratio. The concentration of MMC was determined by measuring t h e a b s o r b a n c e a t 363 nm. D e x t r a n concentration was determined by the modified anthronesulfuric acid method (6). The IgG concentration was measured by the Bio-Rad protein assay (7). Similarly, normal mouse IgG-PAD-MMC (irrelevant conjugate) was prepared. D e t e r m i n a t i o n of A n t i b o d y A c t i v i t y i n t h e Conjugate. Indirect enzyme linked immunosorbent assay was employed to evaluate the antigen-binding capacity of the conjugate. Human gastric cancer cells SGC-7901 were plated in a tissue-culture plate with 40 flat-bottomed wells and fixed with 0.25 % glutaraldehyde. Different dilutions of conjugate and free antibody in 0.1 mL was added. Following incubation a t 37 OC for 1 h, rabbit antimouse immunoglobulin G antibody labeled with horseradish peroxidase (Tianjing Institute of Hematology, Tianjing, China) was added. The peroxidase reaction was initiated by the addition of o-phenylenediamine and the optical density a t 495 nm of each well was recorded. Cytotoxicity Assay. The tetrazolium dye colorimetric assay was used in the cytotoxicity test with human gastric cancer cell line SGC-7901 as target and human normal embryonic lung cell line SL, as nontarget as described previously (8). The cytotoxic effect of MGb2PAD-MMC was compared with that of free MMC and irrelevant conjugate. Stability Study. To assess the release of MMC from t h e antibody-PAD-MMC conjugate, MGb2-PADMMC conjugate was incubated under the physiological conditions of pH 7.4 and 37 "C. The MMC remaining in the conjugate was determined after gel filtration on Sephadex G-25. MGb2 IgG content in the conjugate after gel filtration was also determined. Iodination of MGb2-PAD-MMC Conjugate. MGbzPAD-MMC was labeled with 1251 (Institute of Atomic Energy, Beijing, China) by the chloramine-T method (9) to a specific activity of 3.4 gCi/gg of protein. Imaging and Biodistribution Study. For the imaging and biodistribution study, six Swiss-DF nude mice bearing human gastric carcinoma SGC-7901 about 1.5 cm X 1.5 cm in size at 2 weeks posttransplantation of 1-2 mm3 tissues into their right flank were used. The mice were divided into two groups and were then given intraperitoneal injections of radiolabeled MGb2-PAD-MMC and irrelevant conjugate respectively at a dose of 10 pCi per mouse. Ninty-six hours following the administration, the mice had SPECT taken and were then sacrificed. Blood, tumor, and tissue samples were taken, weighed, and assayed for radioactivity. The result of analysis was expressed as the ratio of radioactivity per milligram of tumor to that per milligram of blood or tissue (T/NT). I n h i b i t o r y E f f e c t of MGb2-PAD-MMC on t h e Growth of Human Gastric Carcinoma SGC-7901. The subrenal capsule assay was used to investigate the in vivo therapeutic effect of the conjugate (10, 11). Treatment was initiated soon after fragments (about 1mm3 in size) of tumor tissue were inoculated under bilateral renal capsules. MGb2-PAD-MMC, free MMC, free MGb2, a mixture of MMC and MGb2, irrelevant conjugate, and saline were given intraperitoneally daily for 6 days at the dose of 1 mg/kg of drug. Eight days after the tumor inoculation, the mice were sacrificed by cervical dislocation. Kidneys were removed and the final in situ tumor size
Table 1. Enzyme Linked Immunosorbent Assay of Binding of MGbz-PAD-MMC and Free MGbz to Human Gastric Cancer Cells SGC-7901
concn, nmol/L 300 30 3 0.3
absorbance at 495 nma MG bz-PAD-MMC MGbz 1.24 1.53 0.87 1.04 0.43 0.73 0.14 0.27
(Algs,,)control = 0.043.
measurements were taken. The in vivo antitumor activity of the tested agents were evaluated by analyzing the change in tumor size pre- and posttreatment. Similarly, the inhibitory effect of MGbTPAD-MMC and MMC in different dose level (1,2,3 mg/kg) on the tumor growth was also studied. I n h i b i t o r y E f f e c t of MGb2-PAD-MMC on the Growth of Human Gastric Carcinoma GAII. The subrenal capsule assay was also employed to assess the in vivo cytotoxic effect of MGb2-PAD-MMC conjugate on a slow-growinghuman gastric carcinoma GAII. Treatment was initiated the day after tumor inoculation. MGbzPAD-MMC, MGb2, MMC, irrelevant conjugate, and saline were given twice a week for 3 weeks a t the dose of 1 mg/ kg of drug. Eighteen days after tumor inoculation, the mice were sacrificed. The next steps were performed as described above. RESULTS
Preparation of Conjugate a n d Its Immunoreactivity. According to the methods described above, MGB-
PAD-MMC was successfully prepared. It was calculated that the MGbZ/PAD/MMC molar ratio in the conjugate was about 1:1.4:20 with the protein-recovery rate being 72 5%. On this basis, it was estimated that the average molecular weight of the MGbz-PAD-MMC conjugate waa around 220 kDa. This was confirmed by the gel-filtration profile of MGb2-PAD-MMC on Sephadex G-200 (data not shown). Table I shows the antibody activity of the conjugate, as measured by indirect enzyme linked immunosorbent assay. The MGb2-PAD-MMC conjugate retained up to 50% of the antibody activity, compared with that of equimolar amount of unmodified antibody. At the concentration of 3 nmol/L, the conjugate could still be bound to tumor cells. Cytotoxic Effect of MGb2-PAD-MMC Conjugate. The 48-h cytotoxic effect of MGb2-PAD-MMC, free MMC, and irrelevant conjugate on target cells and nontarget cells is shown in Figure 1. The cytotoxicity of MGbz-PADMMC on human gastric cancer cell line SGC-7901 was quite similar to that of free MMC, but greater than that of irrelevant conjugate 03 < 0.01). Compared with free MMC, the MGb2-PAD-MMC conjugate had only a little effect on nontarget cells, similar to that of irrelevant conjugate ( p > 0.05). Figure 2 shows the cytotoxic activity of MGb2-PADMMC on target cells and nontarget cells after a 30-min exposure to the conjugate. The cytotoxic effect of MGbzPAD-MMC on target cells was greater than that of free MMC ( p < 0.01). In contrast, MGbz-PAD-MMC showed statistically less cytotoxic effect than MMC against nontarget cells 03 < 0.01). MMC exhibited comparable cytotoxicity to target cells and nontarget cells. Stability of the Conjugate. Figure 3 shows the time course for MMC remaining in the conjugate. The release of MMC from the conjugate was less than 10% of the original content in the conjugate during incubation in PBS
Bioconjugate Chem., Vol. 1, No. 4, 1990 247
MGb,-Mitomycin C Conjugate
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Time (days) Figure 3. Time course of MMC in MGb2-PAD-MMC conjugate. MGb2-PAD-MMC in 0.1 M PBS, pH 7.4, was incubated a t 37 "C. The MMC remaining in the conjugate was determined after gel filtration on Sephadex G-25.
h
5
3
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Table 11. Biodistribution of 12sI-MGb2-PAD-MMC and 1251-NormalMouse IgGPAD-MMC Conjugate in Swiss-DF Nude Mice Bearing Human Gastric Carcinoma SGC-7901
R 0 ,
T/NTa 10 1
34
0.38
I 1
tissues
mol/L)
Concentration (x
Figure 1. Cytotoxic effects of antigastric cancer monoclonal antibody MGb2-PAD-MMC (e),free MMC ( O ) , and normal mouse IgG-PAD-MMC (w) on (A) human gastric cancer cell line SGC-7901 and (B) human normal embryonic lung cell line SL7. Cells (2 X lO5/mL) were cultured with the test drugs for 48 h. A
blood heart lung liver spleen kidney stomach
MGb,-PAD-MMC 0.79 f 0.03 2.62 f 0.72 1.65 f 0.14 3.46 f 0.56 3.46 f 0.71 4.11 f 0.88 5.50 f 1.38
normal mouse IgGPAD-MMC 0.19 f 0.013 0.83 f 0.240 0.34 f 0.100 1.03 f 0.270 0.93 f 0.210 0.86 f 0.190 1.31 f 0.320
a Ratio of radioactivity per milligram of tumor to that per milligram of nontumor tissues.
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Figure 2. Cytotoxic effects of antigastric cancer monoclonal antibody MGb2-PAD-MMC (e),free MMC (O), and normal mouse IgGPAD-MMC (m)on (A) human gastric cancer cell line SGC-7901 and (B) human normal embryonic lung cell line SL7. Cells (2 X lO5/mL) were cultured for 48 h after a 30-min drug exposure.
a t 37 "C for 3 days. MGb2 IgG content in the conjugate was not changed, suggesting that MGb2-MMC coupled by a dextran bridge was stable. Tumor L o c a l i z a t i o n o f MGb2-PAD-MMC Conjugate in Nude Mice with Tumor Xenografts. Table I1 shows the result of biodistribution study of 12YMGb2-PAD-MMC and 1251-normal mouse IgG-PADMMC conjugate. It could be seen t h a t MGb2-PADMMC conjugate was taken up by the tumor preferentially. SPECT imaging confirmed the result of biodistribution
Figure 4. Immunoscintigraphy performed by emission computed tomography (SPECT) a t 96 h after injection of 10 pCi of 1251MGb2-PAD-MMC conjugate.
study. 1251-MGb2-PAD-MMC could visualize tumors clearly while 1251-normalmouse IgGPAD-MMC showed negative results (Figure 4). In Vivo Inhibitory Effect of MGb2-PAD-MMC on the Growth of Human Gastric Carcinoma SGC7901. Table I11 shows the response of SGC-7901 gastric carcinoma growing under the renal capsules of nude mice to MGb2-PAD-MMC conjugate, irrelevant conjugate, free MGb2, free MMC, MMC mixed with but not conjugated to MGb2, and saline. Significant responses were found in those groups treated with MGb2-PAD-MMC conjugate, free MMC, and a mixture of MMC with MGb2, while MGb2-PAD-MMC conjugate showed greater inhibitory effect on tumor growth than MMC or the mixture of MMC with MGb2 did. No synergetic effect was found with regard to the mixture of MMC with MGb2.
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Table 111. Inhibitory Effects of MGb2-PAD-MMC, MGb2, MMC, a Mixture of MGbz with MMC, and Normal Mouse IgG-PAD-MMC on the Growth of Human Gastric Carcinoma SGC-7901 Implanted under Renal Capsules in Swiss-DF Nude Mice. agents tested MGb2-PAD-MMC MGbz MMC MGbz + MMC normal mouse IgG-PAD-MMC saline control
dose, mg/kg 1
10 1 1 1
tumor size (omub) initial final 11.13 k 0.75 20.88 f 1.10 39.88 f 2.93 12.63 f 0.63 12.63 f 0.63 30.38 f 3.45 13.00 f 1.08 29.88 f 1.80 11.00 f 0.91 11.50 f 0.95
37.88 f 3.82 42.50 f 3.85
change in tumor size (day 8-day 0) 9.75 f 1.26 27.25 f 2.40 17.75 f 2.40 16.88 f 1.84 26.88 f 4.26 31.00 f 3.92
YO inhibition rate 68.55c*d 12.00 42.74c 45.50' 13.29
Two mice (four capsules) per test group; four mice (eight capsules) in control group. Omu, ocular micrometer units; 1 mm = 10 omu.
p
< 0.001 (vs control). p < 0.001 (conjugate vs mixture of MMC and MGbz).
Table IV. Inhibitory Effects of MGb2-PAD-MMC and Free MMC of Different Dose Levels on Human Gastric Carcinoma SGC-7901 Implanted under Renal Capsules in Swiss-DF Nude Mice' tumor size (omu) change in tumor agents tested dose, mg/ kg initial final size (day 8-day 0) 55 inhibition rate MG bZ-PAD-MMC 1 13.25 f 1.85 25.25 f 8.00 12.00 f 6.86 68.00' 1 11.88 f 3.00 31.88 f 4.00 20.00 f 3.24 46.67 MMC 2 12.38 f 1.25 11.88 f 1.49 -0.50 f 2.16 101.33 MGbz-PAD-MMC MMC 2 12.50 f 0.82 19.88 f 7.16 7.38 f 6.80 80.32 MGbz-PAD-MMC 3 12.50 f 1.00 12.00 f 1.78 -0.50 f 1.47 101.33b MMC 3 12.75 f 1.85 13.00 f 0.00 0.25 99.33d 49.56 f 9.66 37.50 f 9.44 saline control 12.06 f 1.01 d
a Two mice (four capsules) per test group; four mice (eight capsules) in control group. p < 0.01. p < 0.001 (MGbz-PAD-MMC vs MMC). One of the two mice tested died of toxicity.
Table V. Inhibitory Effects of MGb2-PAD-MMC, MGb2, MMC, Normal Mouse IgG, and Normal Mouse IgG-PAD-MMC on the Growth of Human Gastric Carcinoma GAII Implanted under Renal Capsules in BALB/c Nude Mice' tumor size (omu) change in tumor agents tested dose, mg/kg initial final size (day 18-day 0) 55 inhibition rate MGb2-PAD-MMC 1 12.13 f 2.46 8.25 f 4.19 -3.88 f 2.49 152.2gbve 21.38 f 4.23 10.00 f 5.79 MGbz 10 11.38 f 1.65 1 12.00 f 1.41 16.00 f 2.48 4.00 f 2.97 46.19 MMC normal mouse IgG 10 12.63 f 2.17 24.63 f 2.32 12.00 f 3.94 normal mouse 1 11.63 f 1.18 17.00 f 2.08 5.38 f 3.20 27.49 IgG-PAD-MMC 20.00 f 2.99 7.42 f 3.10 saline control 12.58 f 1.35 a Two mice (four capsules) per test group; six mice (12 capsules) in control group. p < 0.001 (vs control). p < 0.001 (vs MMC).
Table IV shows the effect of various doses of MGb2PAD-MMC c o n j u g a t e i n c o m p a r i s o n w i t h t h e chemotherapeutic ability of free MMC. With increasing dose, the in vivo cytotoxic effect of both conjugate and free MMC was augmented, but compared to free drug, the conjugate exhibited higher activity. Besides, a t the dose above 3 mg/kg, one of the two mice in the free MMCtreated group died of toxicity. No lethal toxicity was found in the conjugate-treated group. In Vivo Inhibitory Effect of MGb2-PAD-MMC on the Growth of Human Gastric Carcinoma GAII. Table V shows the suppressive effect of MGb2-PADMMC on the growth of human gastric carcinoma GAII. It could be seen that MMC alone had some effect on GAII. However, when coupled to antigastric cancer monoclonal antibody MGb2, its activity was significantly increased. At the dose of 1mg/ kg of drug, the conjugate could completely inhibit the tumor growth. MGb2 alone and irrelevant conjugate demonstrated no inhibition on GAII. DISCUSSION
MMC, an antibiotic, has potent antitumor activity and has been used in the treatment of various solid tumors including gastric cancer and colorectal carcinoma. However, its clinical use is limited by its detrimental effect on normal cells especially rapidly proliferating cells such
as those in bone marrow (12). One possible approach to overcome this defect is to link MMC with antibodies against tumors so that the toxicity could be selectively restricted to tumor cells. Suzuki et al. (13) first prepared an antibody-MMC conjugate with cyanogen bromide as the coupling agent. But, only one molecule of MMC could be introduced into each molecule of antibody. Kat0 et al. (14) presented an active ester method by which the molar ratio of antibody/MMC in the conjugate reached 1:8 with the protein-recovery rate being as high as 85-90%. However, with direct linkage, the number of drug molecules that could be attached to antibody molecule without significantly damaging its activity is limited. Attempts to increase t h e druglantibody molar ratio not only decreased t h e yield of immunoconjugate b u t also dramatically damaged antibody activity (15, 16). In order to get active conjugate with increased drug content, an indirect linkage method was adopted. We chose dextran T-40 in the present study as intermediary to prepare antibody-MMC conjugate because it is highly soluble and it may be able to decrease the recipient's immune response to foreign proteins (17). Dextrans of different molecular size have been used as a carrier system in the past. However, the conjugates obtained varied greatly in their in vitro and in vivo antitumor potency (18-20). This may be due to, among other factors, the degree of oxidation of dextran and the extent
MGb*-Mitomycin C Conjugate
of reduction of Schiff bases by sodium borohydride. We adopted 25% oxidation of dextran and partial reduction by sodium borohydride to prepare the MGb2-PADMMC conjugate. In this manner, no severe loss of antigenbinding capacity of the antibody and the pharmacological activity of MMC occurred. T h e MGb2-PAD-MMC conjugate was also proved to be quite stable. The MGb2-PAD-MMC conjugate exhibited selective cytotoxicity upon human gastric cancer cells SGC-7901 in vitro. This cytotoxicity was apparent in the 30-min pretreatment cytotoxicity assay in which conjugate was only briefly exposed to cells, a situation closely mimicking the in vivo action of a conjugate where there may be only a brief encounter with tumor or normal tissue followed by prolonged association with antigen-positive cells. We once chose another antigastric cancer monoclonal antibody, MGll, to prepare the antibody-MMC conjugate. The cytotoxicity of MG11-PAD-MMC conjugate on human gastric cancer cell line SGC-7901 was weaker than that of MGb2-PAD-MMC (Li et al., unpublished observation). This may be due to the quantitative difference of the corresponding antigens on tumor cells, suggesting that the selective cytotoxicity of the immunoconjugates on target cells be mediated by the antibodies chosen. Retention of tumor localization capacity is an important component of the therapeutic approaches with monoclonal antibodies and their anticancer agent conjugates. Different methods of conjugation may affect greatly the in vivo distribution behavior of the conjugates obtained (21). O u r imaging a n d b i o d i s t r i b u t i o n s t u d i e s demonstrated clearly t h a t t h e MGb2-PAD-MMC conjugate could be satisfactorily localized in the tumor tissue in nude mice bearing human gastric carcinoma xenografts. Compared with unmodified antibody, no obvious increase in the uptake of conjugate by liver was observed, as noted in the conjugate of antibody with ricin toxin A chain (22)or the conjugate of antibody with methotrexate utilizing serum albumin as intermediary (23). Having firmly established the specific in vitro cytotoxic effect of MGb2-PAD-MMC conjugate and its in vivo tumor localization capacity, studies were carried out to appraise its in vivo antitumor activity. The subrenal capsule (SRC) assay method was used. Because of the rich blood flow in the renal capsules and thus the relative rapid growth of tumor xenografts and the easy access of tested agents to the tumor tissues, SRC required much less time as compared to the tumor models of subcutaneous or ascitic form which were widely used to test the therapeutic effect of immunoconjugates (24,25).By inoculating the tumor tissues in bilateral capsules instead of in the left one, as introduced in the literature (IO),fewer nude mice were used. Furthermore, because of the graft size and site of implantation, it mimics the residual metastases more closely, it is the metastases against which immunochemotherapy is directed after removal of primary tumor loci (26). The result of SRC assay showed that MMC alone had some inhibitory effect on human gastric carcinoma xenografts in nude mice. However, when coupled to monoclonal antibody MGb2, its antitimor activity was significantly augmented, which was more obviously manifested in GAII, a slow-growing human gastric carcinoma model. Meanwhile, by conjugation with antibody, the toxicity of MMC on nude mice was also reduced. This should allow a greater amount of immunoconjugates to be given to achieve better therapeutic effect. Of course, one should bear in mind the high sensitivity of SRC assay when appraising the in vivo therapeutic efficacy of the immunoconjugate.
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We are aware of the fact that the oxidized dextrans are multifunctional with many reactive aldehyde groups per molecule, just as immunoglobulin is with many reactive amine groups. Therefore, the coupling between those two types of molecules may yield a complex mixture of products differing in t h e i r molecular size. However, t h e chromatographic analyses of the products showed that they were not grossly heterogenous and that products as those with one molecule of dextran and two molecules of immunoglobulins or more were not formed. The thing which should be more taken into account is perhaps t h e heterogeneity in their coupling sites. If the sites necessary for antigen-binding were involved, great reduction to the immunological activity of antibody may occur. The site specific linkage method presented by Shih et al. (27)may provide a solution to this problem. In summary, an antigastric cancer monoclonal antibodyMMC conjugate was prepared with dextran T-40 as intermediary. In vitro studies demonstrated that the immunoconjugate exhibited selective cytotoxicity upon human gastric cancer cells SGC-7901. The conjugate was proved to be quite stable and could be well-localized in the tumor tissue in nude mice xenografted with human gastric carcinoma. SRC assay indicated that MGb2PAD-MMC conjugate could inhibit t h e growth of transplanted tumor much more effectively than unconjugated MMC or irrelevant conjugate. Recent studies showed that the MGb2-PAD-MMC conjugate could be satisfactorily localized in the tumor tissue in the patients with gastric cancer. Even the metastatic lymph nodes could be recognized by the immunoconjugate, the tumor to nontumor uptake ratio ranging from 2.6 to 5.7 (Li et al., manuscript in preparation). The above results are encouraging us to make continuous effect to further improve this conjugate and perform a clinical trial. ACKNOWLEDGMENT
We thank Mrs. Xiao-Yan Cao for her assistance in preparing this manuscript. LITERATURE CITED (1) Zhang, X. Y. (1984) Further study on the etiology, mechanism and early diagnosis of gastric cancer. Chin. J. Dig. 4 , 29. (2) Baldwin, R. W. (1985) Monoclonal antibody targeting of anticancer agents. Eur. J. Cancer Clin. Oncol. 21, 1231-1235. ( 3 ) Fan, D. M., Zhang, X. Y., Chen, X. T., Mu, Z. X., Hu, J. L., Qiao, T. D., and Chen, B. J. (1988) Mouse and human monoclonal antibodies against gastric cancer: Preparation and clinical application. Chin. Med. J. 101, 488-489. (4) Deng, J. L., Jiang, M. D., Tan, X. Y., Gao, R. Z., Chen, M., Fan, D. M., Hu, J. L., Zhang, X. Y., Chen, X. T., and Mu, Z. X. (1990) Tumor localizationand radioimmunoimaging by monoclonal antibodies in nude mice and patients with gastric cancer. Chin. J . Dig., in press. (5) Bernstein, A., Hurwitz, E., Maron, R., Arnon, R., Sela, M., and Wilchek, M. (1978) Higher antitumor efficacy of daunomycin when linked to dextran: In vivo and in vitro studies. JNCI, J. Natl. Cancer Znst. 60, 379-384. (6) Bradford, N. M. (1976) A rapid and sensitive method of quantitation of microgram quantities of protein utilising the principle of protein-dye binding. Anal. Biochem. 72, 248-253. (7) Termyn, M. A. (1975) Increasing the sensitivity of the anthrone method for carbohydrate. Anal. Biochem. 68, 332335. (8) Li, S., Zhang, X. Y., Chen, X. T., Fan, D. M., Tan, L. S., Pan, H. Z., and Huang, L. Q. (1989) Preparation and characterization of immunotoxin with blocked galactose-binding sites of ricin. Chin. J . Pharmacol. Toxicol. 3, 81-84.
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