JPH08504172A - Anti-erbB-2 monoclonal antibody combination and method of use - Google Patents

Abstract

(57) Summary The present invention relates to a combination of at least two monoclonal antibodies capable of preventing and treating human malignancies in which malignant cells or gp185 ^erbB-2 are overexpressed. The monoclonal antibodies of this combination recognize different epitopes of the gp185 expression product of erbB-2 and therefore the antibodies do not cross-react with each other. Preferably, the combination reduces the expression product of the erbB-2 gene. In other embodiments, the combination does not essentially increase tyrosine phosphorylation of the gp185 expression product.

Description

Detailed Description of the Invention Anti-erbB-2 monoclonal antibody combination and method of use The present application describes a combination of monoclonal antibodies capable of preventing and treating tumors. Regarding combination. More particularly, the monoclonal antibody is Is a single chain monoclonal antibody capable of preventing and treating   Amplification and / or overexpression of this erbB-2 gene (also referred to as HER-2 or neu) results in overexpression of bB mRNA and protein, and chest (1-5), ovary (3), lung Proven within 20-30% of adenocarcinoma of (6) and stomach (7) ing. Two lines of evidence indicate that erbB-2 is involved in the pathogenesis of human neoplasia. Involvement of overexpression, firstly, overexpression, chest (8-11), ovary (12), Associated with poor prognosis in stomach (13) and lung cancer (14) , Indicating that overexpression modifies the cancer cells. Second, er Artificial overexpression of bB-2 results in NIH / 3T3 fibroblasts (15,16) and mammary epithelial cells (17 ) Induces a transformed phenotype, and overexpression directly leads to the development of a deleterious phenotype. Suggest that it can contribute to.   gp185^erbB-2Due to the extensive homology between EGFR and epidermal growth factor receptor (EGFR) It is widely inferred that their activation may proceed through similar mechanisms. It is fixed. One such mechanism is receptor dimerization / oligomerization. (Dlmerization / oligomerization) and the above-mentioned EGFR endogenous tyrosine kiner Including what is believed to be an important step in the activation of the ze machinery (18,19 ). Interference with receptor-receptor interactions is associated with erbB-2 overexpression in cancer. Has been evaluated as a potential therapeutic approach to the treatment of Advanced research Research has shown that erbB-2 (20) and related agents as potential therapeutic agents for the treatment of cancer. A single monoclonal directed against the skin growth factor receptor (EGFR) protein (21) Is evaluating the use of antibody.   A single module directed against erbB-2, which is being evaluated as a potential therapeutic agent. The use of noclonal antibodies has been shown to increase the activity of tyrosine kinase (tyrosine klnase). Increased gp185 due to increase in^erbB-2Leading to the autophosphorylation of There is. Single antibody agents have shown promise as a potential anti-cancer treatment (20,27).Summary of the Invention   One purpose of Applicants' invention is that malignant cells are gp185^erbB-2Overexpressing At least 2 that can cure or prevent malignancies A combination of two monoclonal antibodies. At least this combination Is also the first antibody and the second antibody, each of which recognizes the gp185 extracellular domain of erbB-2. Comprises what you perceive. The activity demonstrated by treatment of the combination antibody is Than predicted by the sum of its individual antibodies at the same overall antibody concentration It shows great activity.   Another object of the invention is for a monoclonal antibody which is a single chain monoclonal antibody. Provide use. This single antibody is used to make a bispecific antibody Can be done.Detailed description of the drawings   Figure 1A． Specificity of monoclonal antibodies # 21 and # 23. Subconfluent SK -Br-3 monolayer was metabolically labeled with 35S-Cys (specific activity (spec.act.) 1000 Ci / mm ol). Over 10 μg of all cellular proteins The antibody was immunoprecipitated. This immune complex was labeled with protein G agarose (G enex, Galthersburg, MD) and on 8-16% Tris-glycine gel. Was analyzed by SDS-PAGE. This gel was intensified at -70 ° C Exposed to film overnight.   Figure 1B． Gp185 in gastric cell line N87^erbB-2Overexpression and high and low gp185^{erb B-2} Tumors from N87 mouse xenografts compared to overexpressers. Cells or tumors were treated with 0.125M Tris-HCl, 4% SDS, 0.00 Lysis in 2% bromophenol blue and 15% glycerol It was After measuring the protein concentration, 5% β-mercaptoethanol was added. Service Samples (10 μg of total protein) are boiled for 3 minutes and S-loaded on 8-16% Tris-glycine gel. Fractionated by DS-PAGE and transferred to nitrocellulose. gp185^erbB-2The detection of , A monoclonal antibody against the c-terminal part of the protein.   Figure 1C． N87 (stomach), SK-Br-3 (chest), and SK-OV-3 (ovary) cell lines and human embryos Southern blot analysis of the erbB-2 gene within the placenta. DNA, cell lines and From human placental tissue, guanidine thiocyanate and Extracted using cesium gradient centrifugation. Restriction fermentation of DNA (15 μg) Cleavage with HindIII and separation by electrophoresis on a 1% agarose gel. Transferred to trocellulose and previously described by radioactive erbB-2 cDNA probe (26) probed as described. This cDNA probe is a complete erbB-2 protein Match the code region.   Figure 2.Ab # 21 and A for proliferation of human N87 gastric tumor cells in a monolayer MTT proliferation assay The effect of b # 23. Insulin (10,000 cells / well) 5 μg / ml), human transferrin (tra nsferrin) (10 μg / ml), 17-β-estradiol (estradiol) (10 nM), sub R supplemented with sodium selenite (5 nM) and 10 mM Hepes It was placed in a chemically defined medium consisting of PMI-1640. PBS at a given concentration , Ab # 21, Ab # 23 or a combination of Ab # 21 and Ab # 23 was then added. This play 5% CO₂Incubated at 37 ° C in a humidified atmosphere. After 7 days, 50 μl of 3- (4,5- Dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide ( 0.1 mg) was added and incubated at 37 ° C. for 4 hours. This culture 90% of the ground was then removed and the crystals solubilized in 0.175 ml DMSO. Absorbance at 540 nm in a Molecular Devices Vmax kinetic microplate reader. I measured it. Results are the average of 8 wells with standard deviation. Article used Under conditions, the cell number is directly proportional to the decrease in MTT.   Figure 3A． Ab # 21 (), A against growth of N87 tumor xenografts in BNX mice Effect of b # 23 (), treatment with combination of Ab # 21 and Ab # 23 (), or PBS. Tumor cells (5x10⁶/ Mouse, BNX (beige, nude, x) id) injected subcutaneously into the flanks of mice. The treatment started on the first day It consisted of 4 test groups (3 mice per group), each for 3 weeks. PBS (), 200 μg purified Ab # 21 (0), 200 μg purified Ab # twice each week 23 (0) or a mixture of 100 μg purified Ab # 21 and 100 μg purified Ab # 23 () in the peritoneum Was injected into. Tumor growth is reported as the mean relative tumor volume, s.e.m. ± 15%. Two replicates of this experiment gave the same results.   Figure 3B． The effect of treatment after the formation of small tumors. Cells using the same treatment procedure as above I made an injection. However, the treatment was started on the 4th day after the cell injection instead of on the 1st day. Excluding the facts. Institutionalize the care of animals It was done according to the combined guidelines.   Figure 4A． Effect of antibody binding on erbB-2 protein turnover. Sub Confluence N87 Cell monolayer, 20 μCi³⁵Pulse labeled with S-cysteine for 1 hour and then Ab Existence of # 21 alone, Ab # 23 alone, or a 1: 1 combination of Ab # 21 and Ab # 23 (10 μg / ml) It was chased with 5 mM Cys for 24 hours in the air. Whole-cell protein, Sephar gp185 bound to ose^erbB-2Use a monoclonal antibody directed against the C-terminus of Was immunoprecipitated as described in Figure 1 and analyzed by SDS-PAGE. The gel was exposed to film overnight at -70 ° C with an intensifying screen.   Figure 4B． Gp185 after incubation with antibody combination^erbB-2-2Tiroshi Measurement of tyrosine phosphorylation. The cells should be plated as in Figure 4A. I gave it. After 1 hour, cells were treated as in Figure IB. This protein, nitro Electroblot onto cellulose paper and anti-phosphotyrosine IgG (polynucleotide Ronal, Upstate Biotechnology, Inc.) and ECL Immunodetection was performed using a Western blotting detector (Amersham). This Film was exposed for 5 minutes at room temperature.   Figure 5.Human Calu-3 lung adenocarcinoma adenocarcinoma tumor cell line in monolayer MTT proliferation assay Effect of Ab # 21 and Ab # 23 on cell proliferation. 10,000 cells / well single cell suspension , Insulin (5 μg / ml), human transferrin (10 μg / ml), 17-β-E Supplemented with stradiol (10 nM), sodium selenide (5 nM), and 10 mM Hepes Cultures were placed in a chemically defined medium consisting of RPMI-1640. To a predetermined concentration PBS, Ab # 21, Ab # 23 or a combination of Ab # 21 and Ab # 23 was then added. This Plate of 5% CO₂Incubated at 37 ° C in a humidified atmosphere. After 7 days, 50 μl of 3 -(4,5-Dimethylthiazol-2-yl)- Add 2,5-diphenyl tetrazolium bromide (0.1 mg) and 37 ° C Incubation was carried out for 4 hours. 90% of this medium is then removed and The crystals were then solubilized in 0.175 ml DMSO. Absorbance, Molecular Devices It was measured at 54 Onm in a Vmax kinetic microplate reader. The result is a standard deviation Average of 8 wells with differences. Under the conditions used, its cell number is MTT It is directly proportional to the decrease.   Figure 6.Ab # for proliferation of human Calu-3 lung adenocarcinoma tumor cells in a monolayer MTT proliferation assay The effect of 23 and Ab # 94. Insulin (5μ) g / ml), human transferrin (10 μg / ml), 17-β-estradiol (10 nM ), Sodium selenide (5 nM), and RPMI-1640 supplemented with 10 mM Hepes It was placed in a chemically defined medium consisting of PBS, Ab # 23, Ab # 9 at a given concentration 4 or a combination of Ab # 21 and Ab # 23 was then added. This plate is 5% CO₂ Incubated at 37 ° C in a humidified atmosphere. After 7 days, 50 μl of 3- (4,5-dimethylthia Zol-2-yl) -2,5-diphenyl tetrazolium bromide (0.1 mg) And incubated at 37 ° C. for 4 hours. 90% of this medium It was then removed and the crystals solubilized in 0.175 ml DMSO. Absorbance, Mo Measured at 540 nm in lecular Devices Vmax kinetic microplate reader . Results are the average of 8 wells with standard deviation. Under the conditions used, Cell number is directly proportional to MTT decrease.   Figure 7.* CDNA sequence for single chain anti-erbB2 antibody, Ab # 23.   Figure 8.* CDNA sequence for single chain anti-erbB2 antibody, Ab # 21 (e22).Detailed Description of the Invention   One object of the present invention is the ability to prevent or treat human malignancies. A combination of at least two monoclonal antibodies whose malignant cells are gp 185^erbB-2Of at least two different antibodies Each recognizes a different epitope of the gp185 expression product of erbB-2, and therefore , A combination in which the antibodies do not cross-react with each other. Aspects of the invention include , The combination has a first to a second obtained ratio of the expression of the erbB-2 gene. The first is preferably combined to be effective in reducing the current product. It is provided that it comprises one antibody and a second antibody. erbB-2 gene expression product A convenient method for measurement can be found in Figure 4A. This erbB-2 gene A decrease in product expression is a set that reduces the intracellular half-life of erbB-2 protein. It is the result of the combination. In another aspect of the invention, the antibody combination Characteristically does not essentially increase tyrosine phosphorylation of gp185 expression products. It has characteristics.   Pairs with a second antibody that has the activity required to reduce the expression product of the erbB-2 gene. An example of a ratio of primary antibodies comprising comprises a ratio of about 1: 2 to about 2: 1. Preferably, Such a ratio is 1: 1. However, the present invention is discussed herein. It is not intended to be limited to the antibody ratio. Other ratios are effective, and with examples The fact that it can produce higher activity than the 1: 1 ratio used for It is recognized and known by the inventor to be within the scope of the invention.   The activity of this combination is illustrated in FIGS. 1A-C, 2 and 3A, B as follows. Figures 1A-C show that N87 cells overexpress the gp185 erbB-2 protein as a result of erbB-2 gene amplification. Prove that it shows. Figure 2 shows the combination of Ab # 21 and Ab # 23 Of N87 cells in vitro It is shown to suppress proliferation. Similar results have been shown to correlate with the growth of human Calu-3 lung adenocarcinoma. Then, use the combination of Ab # 23 and Ab # 94 and the combination of Ab # 23 and Ab # 21. Have been demonstrated (see FIGS. 5 and 6). 3A and B show Ab # 21 and Ab Combination with # 23 suppresses N87 cell proliferation as a tumor in immunodeficient mice It shows the activity of controlling and reversing. These results are in the book The general properties of the antibody combinations of the specification are shown.   An antibody against the erbB2 gene-encoded product used in the present invention is a chimeric anti-antibody. Can be designed as a body. Chimeric antibody is of non-human (eg, murine) origin Has a variable region (antigen-binding region) and a constant region of human origin. They are excellent Being predominantly human, chimeric antibodies are less immunogenic in humans. And this is to overcome the problems associated with the administration of foreign proteins to humans. I can help.   Furthermore, the antibody of the present invention may be used as a Kohler-Milst antibody for gene recombination or production of the antibody. It can be made through the ein hybridoma method. A fragment of the antibody itself, a homolog The body or derivative can be used in the present invention instead of its whole antibody It is also recognized.   Another object of the invention is the erbB-2 gene coding product designed as a single chain antibody. Antibodies to. Single-chain antibodies are now allowed to overlap with the light variable region of the antibody. The variable region is bound to each other to form a single chain antibody. Of these antibodies The combination includes the combined antibodies as a mixture as discussed above. It is used herein to include antibodies that have been combined to produce bispecific antibodies. Consider.   Bispecific antibodies are usually two single-chain antibodies, each of which is different. It is an artificially made antibody consisting of one that recognizes the antigen-binding site.   The following: Adenocarcinoma of the breast, ovary, lung and stomach are treated or prevented using the present invention. Here are some examples of human malignancies that can:   Other aspects of Applicants' invention prevent human malignancies as described above and Provide a method for eradicating. This method produces an effective amount of anti-erbB -2 antibody combination is administered to a patient and the antibody combination is Effective concentration; including achieving a concentration of at least 1 μg / ml. Preferred Preferably, the concentration at the tumor site does not exceed about 10 μg / ml. Generally, the tumor site In order to achieve the desired concentration of this combination in Is administered at a dose of about 0.1 mg / kg to about 10 mg / kg body weight.   Another aspect of Applicant's invention is that the antibody combination is useful in the passive treatment of tumors. Is used as an effective dose of the antibody combination. Malignant tumor overexpression gp185^erbB-2With a pharmaceutically acceptable carrier for patients suffering from Or in it. Examples of vehicles are non-toxic buffers, physiological Physiological saline.   Moreover, the applicant's invention is that at least one of the antibodies in this antibody combination is It is provided to be used as a component of immunotoxin. Im For the treatment of notoxin, at least one antibody in the combination is It can be made into an immunotoxin by binding to a cancer drug or cytotoxin. Wear. Various pharmaceutical or cytotoxic agents have been used to combine this drug, whether chemically or genetically engineered. Let Can be attached to an object. Examples of some useful therapeutic agents are radioactive compounds ( For example, isotopes of boron and rhenium); agents that bind to DNA, such as Rukylating agents or various antibodies (eg daunomysin, adria) Adriamycin, chlorambucil, anti-metabolites (eg. For example, methotrexate; and inhibitors of protein synthesis (eg, Includes diphteria toxin and toxic plant proteins. Book combination A combination in which at least one of the antibodies in the product is bound to the immunotoxin The use of the combination will increase the therapeutic effects mentioned above.   Administration of an effective amount of the antibody combination described herein to a patient is treated. Sufficient time to cause regression or eradication of existing human malignancies Can be achieved through chronic intravenous administration over time. Also, this combination The administration of the quasi-administration involves direct injection or delivery of the combination to the tumor site. Can be achieved within the patient by Such administration will It will have sufficient duration and concentration to effect eradication or reduction.   The scope of the invention is not intended to be limited to any rationale. But the theory below is that gp185^erbB-2Malignant tumors that overexpress Down-regulation and protection from cells is achieved by the combination of the above two antibodies The mechanism that can be achieved can be explained.   One hypothesized mechanism is that the combination of the two antibodies is gp185.^erbB-2Live Forced to be in the sexual conformation, which causes the agonist ligand (agon It works by imitating ist ligand). This two antibody combination is When imitating a ligand, Subsequent treatment using this combination will increase gp185^erbB-2Automatic phosphorus It should lead to autophosphorylation. Anti-phosphotyrosine im Anti-Phosphotyrosine immu noblots test this hypothesis Used for. As shown in Figure 4B, gp185 from N87 cells.^erbB-2Of tyrosine The increase in phosphorylation is also apparent 1 or 2 hours after addition of the antibody combination. Was not observed by 24 hours of treatment. This means that this antibody combination Gp185^erbB-2Does not increase the autophosphorylation of and therefore its It suggests that it does not act to inhibit the activity of the kinase.   The result is that the combination of anti-receptor antibodies differed from the single antibody and It has been shown to lead to potent anti-tumor activity. More particularly, the result is the book Combination antibody treatment is gp185^erbBFor the treatment of human malignant tumors that overexpress It is a good approach. This approach results in gastric cancer, namely Especially important in the treatment of illnesses that rarely respond to recent systemic chemotherapy There can be.   The invention is further described in the following examples which are not intended to limit the scope of the invention. This will be described below.Example 1 Antibody preparation   As a source of human erbB-2 protein, we express human erbB-2 protein on its surface NIH / 3T3 cells (N / erbB-2) engineered as described above were used. Membrane preparation of these cells Of the nuclei by hypertonic lysis in 2 mM Hepes pH 7.4, centrifugation at 5,000 x g. Prepared by removal and membrane isolation by centrifugation at 100,000 x g. Mau 200μ Immunization with 100 μg N / erb B-2 membrane preparation in a 50:50 mixture of adjuvants in It was Adjuvant is Freund's complete for the first injection, then Freund's incomplete It was all adjuvant. Mice received 4 intraperitoneal injections over 4 weeks. One week after the last boost, serum was obtained and the anti-erbB-2 immune response was metabolized. Presence of gp185 erbB-2 protein from immunolabeled cells was determined by immunoprecipitation analysis. Decided. Immunized mice were then selected and treated with 100 μg N / erbB-2 membrane preparation. Boost and fuse with Ag8.653 myeloma cells according to standard methods. went. Hybrid clone selection was again followed by standard methods, Hypoxanthine, aminopterin, and thymine mine) (HAT) -containing medium. Anti-erb B-2 Monoclonal Anti Identification of hybridomas secreting the body was performed by identifying the antigen N / erbB-2 membrane protein attached to a 96-well dish. Was performed by ELISA using. ELISA reaction was performed using goat anti-matrix conjugated with peroxidase. Color was developed using Us antibody and standard methods. Ha secreting anti-erbB-2 antibody The ibridoma colonies were subjected to single cell cloning and ELISA as described above. Subjected to two rounds of identification of positive subclones.   The above procedure was named as Ab # 21; Ab # 23; and Ab # 94 for three monoclonals. Used to make antibodies.   gp185^erbB-2Monoclonal antibody directed against the extracellular domain of ELI The SA assay was tested for specific reaction against N / erbB-2 cell membrane. Proliferation test Of these named Ab # 21 and Ab # 23 after screening in Two showed the highest bioactivity and were used in this study. Antibody, large Isolated from ascites fluid in volume and loaded with Gammabind Ultra columns (genex, Gaithersburg, Purified by HPLC according to MD). Standard SDS-PAGE gel electrophoresis Were run under non-reducing conditions using coomassie blue staining and a single van at 130 kd Was observed to represent a> 98% purified preparation (data not shown).   Both antibodies were tested for SK-Br-3 cells (gp185) as shown in Figure 1A.^erbB-2Excessive protein emission Revealing breast cell line) (22) MW 185,000 single³⁵S-labeled protein specifically Immunoprecipitated. This gp185^erbB-2Cells that do not overexpress the protein (eg MDA-MB- 468, data not shown), no immunoprecipitation was detected.   The effect of these cells on cell proliferation was demonstrated by the gastric cell line, namely N87 and SK- Gp185 at a level balanced with Br-3^erbB-2For those that overexpress did. Immunoblot of this N87 cell line and nude mouse tumor xenografts from N87 Immunoblot of one of the breast cell lines SK-Br-3 (high levels of gp185^erbB-2Overexpression) and And MDA-MB-231 (low level gp185^erbB-2Overexpression) and is shown in FIG. 1B. Figure 1 The level of erbB-2 gene amplification in N87 as shown in C is shown in SK-Br-3 and SK-OV-3 cells. Surpasses the levels in its well that characterize the system (22).Example 2 Effect of antibodies on tumor growth in vitro   The effect of these antibodies on proliferation was assessed using a semi-automatic colorimetric MIT assay. First studied in Russia. Single cell suspension at 10,000 cells / well , Human transferrin, 17-estradiol, sodium selenide and And a platen in a chemically defined medium consisting of RPMI-1640 supplemented with Hepes I'm sorry PBS, Ab # 21, Ab # 23 or a combination of Ab # 21 and Ab # 23 was then added . 5% CO₂Incubate at 37 ° C in a humidified atmosphere It was After 7 days, 50 μl of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl. Add tetrazolium bromide (MTT reagent) and at 37 ° C for 4 hours It was subjected to incubation. 90% of this medium is then removed and the crystals removed , Solubilized in DMSO. Absorbance of molecular devices Vmax kinetic microplat Measured at 540 nm in the e reader. Administration of antibody effects on N87 cell proliferation A dose response analysis is shown in Figure 2. Antibody Ab # 21 or Ab # 23, administered separately, was 10 μg / It had no effect on cell monolayer growth up to a concentration of ml (6 μM) . However, administration of the 1: 1 combination of Ab # 21 and Ab # 23 was the same as 1 μg / ml. It had a marked effect on cell proliferation at very low doses. Also both antibodies Fabe fragments prepared from Escherichia coli, alone or in combination, were used for total growth of cells. And had no effect (data not shown). Slightly overdue by immunoblot analysis In similar experiments with three other gastric cell lines showing overexpression or no overexpression Growth inhibition, even at the highest doses, is dependent on the antibody combination or It was not observed at all by the body alone.Example 3 Treatment with prophylactic combination antibodies   The effect of combination antibody therapy was tested on the growth of N87 tumor xenografts. It was Subcutaneous injection of one inoculum of 5 million N87 cells into nude mice , Created a fast growing tumor with a short latency. This injection site Tumor growth in the was easily quantified. This N87, as shown in Figure 3A Cells containing Ab # 21 and Ab # 23 in combination twice a week for 3 weeks Tumors in animals treated with 200 μg total antibody per shot Did not make Quite the contrary, they are not treated with single antibody or PBS. Is potentially tumorigenic in the body, and the tumor is Within 1cm³Has grown to over. Contrary to in vitro experiments, it Each of the monoclonal antibodies alone was limited to partially limit the rate of tumor growth. It can have a defined activity. However, this is shown by this combination. The activity exerted far exceeded the cumulative effect expected from the combination.   Ab # 21 and Ab # 23 combination therapy can eradicate established tumors. Tumor to a measurable size prior to antibody treatment to determine if Experiments were carried out to grow the. The results are illustrated in Figure 3B. Outbreak of the tumor The same size (100mm)³) Smell in a group of animals disaggregated to be near The animals were treated with Ab # 21 or Ab # 23 single antibody (200 μg / injection, 2 injections / week, 3 weeks). , 6 mice) were allowed to grow. On the contrary, between Ab # 21 and Ab # 23 In animals given a combination treatment of the two antibodies, the results shown were: Tumors were completely remissioned after 11 days on average for 6 animals (200 μg total 4 treatments with antibody). This is a combination of anti-erbB-2 monoclonal antibodies This is the first reported observation of more derivatized tumor xenograft regression. Advanced research The research showed that two anti-neu antibodies were activated by mutation in the neu oncogene (oncogen tumor growth by murine cells transformed by e) can be suppressed (23). Activation of this murine neu oncogene Point mutations (as evidenced by qualitative interference in the structure and function of genes point mutation), whereas the human erbB-2 oncogene has an excess of erbB-2. Expression, ie the quantity of apparently normal proteins leading to tumorigenesis It is activated by a simple interference.   Mechanisms for tumor growth differ significantly between murine and human Therefore, a similar mechanism of neutralization of related genes would be effective. In addition, this effect is anti- Suppressing leukemia tumor cells using transferrin monoclonal antibody Seen (24).Example 4 Antiproliferative effect of antibody combination   To investigate the molecular basis for the antiproliferative effects of Ab # 21 and Ab # 23, we Gp185 in the presence or absence of antibody^erbB-2The rate of turnover was measured. N87 cells ,³⁵Pulse-labeled with S-Cys, and then for varying times a single antibody or Ab Chase in the presence of the # 21 / Ab # 23 combination. 24-hour chase results, Shown in Figure 4A. This antibody gp185^erbB-2The combination is gp185^erbB-2Fast disassembly of This individual antibody treatment, on the other hand, has no or little effect. I was Thus, the antiproliferative effect of Ab # 21 / Ab # 23 treatment was^erbB-2Turnover of May be explained by their ability to increase (turnover).Example 5 Combination antibody therapy with effects on Calu-3 cell proliferation   The effect of the combination of anti-erbB-2 antibodies Ab # 21 and Ab # 23 on N87 gastric cancer cell line To prove that the effect is not limited, we have established that the human lung adenocarcinoma cell line Ca I researched lu-3. The cells were determined as determined by immunoblot assay. Overexpresses gp185 erbB-2 protein (data not shown). Very similar to those listed above In an experiment similar to, the combination of Ab # 21 and Ab # 23 was tested in the MTT assay. Demonstrating a dramatic inhibition of cell proliferation as measured by (FIG. 5). In this experiment In addition, a single cell suspension of 10,000 cells / well was added to insulin and human trans Ferrin, 17-estradiol, sodium selenide and Hepes buffer The plating was performed in a chemically defined medium consisting of supplemented RPMI-1640. P BS, Ab # 21, Ab # 23 or a combination of Ab # 21 and Ab # 23 was then added. This cell 5% CO₂Incubated at 37 ° C in a humidified atmosphere. After 7 days, add MTT reagent, Then, the plate was incubated at 37 ° C for 4 hours. 90% of this medium is then And the crystals were solubilized in DMSO. Absorbance, Molecular Device It was measured at 540 nm in a s Vmax kinetic microplate reader. Effect of antibody treatment The fruit dose response analysis is shown in FIG. This result indicates that combination antibody treatment Showing that it is not limited in its effect on N87 cells or gastric cancer cells ing. In addition, combination antibody treatment may be effective in treating lung adenocarcinoma. It shows that and can be done.Example 6 Effect of other combinations of antibodies on the inhibition of cell proliferation   Ab # 21 and Ab # 23 implicate the ability of their combination to cause growth inhibition. In order to determine whether the The combination of Ab # 94 and Ab # 23 was investigated for growth of lu-3 cells. cell MTT assay of proliferation was performed as described in Example 5. As shown in FIG. The antibody combination suppresses cell proliferation and individual antibodies do not. This Has the ability to combine antibodies to create deeper growth suppression. It shows that it is not limited to a particular antibody combination.   Antibodies # 21; Ab # 23; and Ab # 94 are available from American Type Culture Collection, 12301 Par. klawn Drive, Rockville, Md. 20852, deposited in USA. Ab # 21 is Deposited with, and ATCC#Was given. Ab # 23 is Deposited with Then ATCC#Was given. Ab # 94 is Deposited with, and ATCC#   Was given.   Although the present invention has been described in connection with those specific aspects, changes, modifications and variations are It will be apparent that the conversion will be apparent to those skilled in the art in view of the above description. Shi Accordingly, the invention resides in the broadest scope of the following claims and any subsequent claims within the core. It is intended to include all such changes, modifications and variations.Example 7 Preparation of single chain (Fv) from mAb e23   Poly A RNA, oligo dT affinity chromatography (In vitrogen) Were used to extract from hybridoma cells. cDNA to random primer ( N₆) (Boerhinger Mannheim). Immunoglobulin light chain and A heavy chain clone was isolated using PCR and primers: light chain, 5'CAC GTC G. AC ATT CAG CTG ACC CAC TCT CCA and GAT GGA TCC AGT TGG TGC AGC ATC 3 '; Heavy Chain 5'C GGA ATT TCA GGT TCT GCA GIA GTC WGG 3'and 5'AGC GGA TCC AGG GGC CAG TGG ATA GAC 3 '[G, A, C, T represent standard nucleotides; I represents Syn represents, and W represents A or T. ]. The product of the PCR reaction was cloned into PUC18. Made a loan. Binding into the SC (Fv) is dependent on individual light and heavy chain cDNA clones and 4 It was performed by PCR giving the oligonucleotide. 5'-cgagatgagtccagctgacccagtctc 5'-gaagatttaccagaaccagaggtagaaccttttatttccagcttgga 5'-ctggttctggtaaatcttctgaaggtaaaggtgtgcagctgcaggag 5'-cgagtgcaagcttaggagacggtgaccgt The light and heavy chain coding regions are overlapped with the oligonucleotides as described. They were linked by a synthetic linker GSTSGSGKSSEGKG which was specialized. Intact Lambda P in the scFv code area_LE. coli in frame under the direction of the promoter(E .coli) The OMPA leader sequence was inserted. Protein induction and bacterial lysis and refolding The refolding was the same as previously described (28). CM for scFv Purified as a single peak from chromatography and by SDS gel electrophoresis. >> 70%.Example 8 Preparation of scFv from mAb e21   Poly A RNA, oligo dT affinity chromatography (In vitrogen) Were used to extract from hybridoma cells. cDNA to random primer ( N₆) (Boerhinger Mannheim). Immunoglobulin light chain and A heavy chain clone was isolated using PCR and primers: light chain, 5'CAC GTC. GAC ATT CAG CTG ACC CAC TCT CCA and GAT GGA TCC AGT TGG TGC AGC ATC 3 '; Heavy chain 5'C GGA ATT TCA GGT TCT GCA GIA GTC WGG 3'and 5'AGC GGA TCC AGG GG C CAG TGG ATA GAC 3 '[G, A, C, T represent standard nucleotides; I represents Syn represents, and W represents A or T. ]. The product of the PCR reaction was cloned into PUC18. Made a loan. Binding into the scFv is dependent on individual light and heavy chain cDNA clones and 4 It was performed by PCR giving gonucleotides. 5'-cgagatgagtccagctgacccagtctc 5'-gaagatttaccagaaccagaggtagaaccttttatttccagcttgga 5'-ctggttctggtaaatcttctgaaggtaaaggtgtgcagctgcaggag 5'-cgagtgcaagcttaggagacggtgaccgt The light and heavy chain coding regions are overlapped with the oligonucleotides as described. They were linked by a synthetic linker GSTSGSGKSSEGKG which was specialized. Intact Lambda P in the scFv code region_LE. coli in frame under the direction of the promoter(E. coli) The OMPA leader sequence was inserted. Protein induction and bacterial lysis and refolding The folds were the same as previously described (28). CM chromatography of scFv Purified as a single peak from the gel and determined to be> 70% by SDS gel electrophoresis. I refused.Literature 1. C.R. King, M.H. Kraus, S.A. Aaronson, Science 229, 974       (1985). 2. D.J. Slamon, et al., Science 235, 177 (1987). 3. D.J. 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Claims (1)

[Claims] 1. Treatment or prevention of human malignant tumors in which malignant cells overexpress erbB-2 An antibody combination for:   A few recognizing different epitopes of the erbB-2 gp185 expression product At least two different monoclonal antibodies, An antibody combination such as. 2. The combination is:   Comprising first and second different monoclonal antibodies, and the erbB-2 gene 2. The antibody combination according to claim 1, which reduces the expression products of. 3. The combination essentially increases tyrosine phosphorylation of gp185 expression products The antibody combination according to claim 2, which is absent. 4. At least one of the antibodies in the antibody combination is an immunotoxin (im The antibody combination according to claim 2, which is bound to a munotoxin) molecule. 5. Patients with human malignancies where malignant cells overexpress erbB-2 How to treat:   Different erbB-2 gp185 expression products were detected in patients with the human malignancy. Combination of at least two different monoclonal antibodies that recognize a pitope Of an effective amount of. 6. The combination is:   Comprising first and second different monoclonal antibodies, and the erbB-2 gene 6. The method of claim 5, wherein the expression product of L. 7. The combination essentially increases tyrosine phosphorylation of gp185 expression products 7. The method of claim 6, which is not. 8. 7. The effective amount provides a concentration of at least 1 μg / ml at the tumor site. The method described in. 9. 9. The effective amount provides a concentration of 10 μg / ml or less at the tumor site. the method of. 10. 7. An effective amount or a dose of about 0.1 mg / kg to about 10 mg / kg of patient body weight, in claim 6. The method described. 11. At least one of the antibodies of the combination binds to the immunotoxin molecule 7. The method of claim 6, wherein 12. Prevent human malignancies where malignant cells overexpress erbB-2 or A composition that can be treated:   (A) at least two recognizing different epitopes of erbB-2 gp185 expression product Effective amount of a combination of different monoclonal antibodies of   (B) a pharmaceutically acceptable carrier, A composition comprising: