Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
  • A61K2039/507—Comprising a combination of two or more separate antibodies
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a combination o monoclonal antibodies capable of preventing and treatin tumors. More specifically, the monoclonal antibodies ar single chain monoclonal antibodies which are capable o treating and preventing tumors.
• erbB-2 gen also called HER-2 or neu
• One object of Applicant's invention relates to combination of at least two monoclonal antibodies capable treating or preventing human malignancies wherein t malignant cells overexpress gpl85 ⁇ .
• the combinati comprises at least a first and second antibody each of whi recognizes the gpl85 extracellular domain of erbB-2.
• T activity demonstrated by the combination antibody treatme has shown greater activity than expected by the sum of t individual antibodies at the same overall antibo concentration.
• Another object of the present invention provides f the use of monoclonal antibodies which are single cha monoclonal antibodies.
• the single chain antibodies can used to form a bispecific antibody.
• FIG. 1A Specificity of monoclonal antibodies # and #23.
• Subconfluent SK-Br-3 monolayers were metabolical labeled with 35S-Cys (spec. act. 1000 Ci/m ol).
• Total cel proteins were immunoprecipitated with 10 ⁇ g of the indicat antibodies.
• the immune complexes were recovered by Protei G Agarose (Genex, Gaithersburg, MD) and analyzed by SDS-PA on an 8- 16% Tri s-Glycine gel . The gel was exposed to fi l at -70°C overnight with an intensifying screen .
• Figure IB gpl85 overexpression in the gastri cell line N87 and a tumor from N87 mouse xenografts compare to high and low gpl85erbB—2 overexpressers. Cells or tumo were lysed in sample buffer which contained 0.125
• Detection of gpl85 was performed with a monoclona antibody to the c-terminal portion of the protein.
• FIG. lC Southern blot analysis of the erbB-2 gen in N87 (gastric), SK-Br-3 (breast), and SK-OV-3 (ovarian cell lines and human placenta.
• DNA was extracted from cel lines and human placenta tissue using guanidine thiocyanat and cesium gradient centrifugation.
• DNA (15 ⁇ g) was cleave with restriction enzyme HinDIII, separated b electrophoresis on a 1% agarose gel, transferred t nitrocellulose, and probed with radioactive erbB-2 cDN probe as previously described (26).
• the cDNA prob corresponds to the entire erbB-2 protein coding region.
• Figure 2 Effects of Ab#21 and Ab#23 on the growth o human N87 gastric tumor cells in a monolayer MTT growt assay.
• PBS, Ab#21, Ab#23 or combination of Ab#21 and Ab#23 at the indicate concentration were then added.
• the plates were grown a 37°C in a 5% CO. humidified atmosphere.
• FIG. 3A Effects of treatment with Ab#21 ( ) Ab#23( ), a combination of Ab#21 and Ab#23 ( ) , or PB ( ) on the growth of N87 tumor xenografts in BNX mice
• Tumor cells (5 X 10 /mouse) were subcutaneously injecte into the flanks of BNX (beige, nude, xid) mice.
• Treatmen begun on day 1 consisted of four trial groups (3 mice pe group) each given 0.2 ml intraperitoneal injections twice week of either PBS ( ) , 200 ⁇ g purified Ab#21 (0), 200 ⁇ purified Ab#23 ( ) , or a mixture of 100 ⁇ g purified Ab#2 and 100 ⁇ g of purified Ab#23 ( ) for three weeks. Tumo growth is reported as an average relative tumor volume ⁇ .e.m. ⁇ 15%. Two repeats of the experiment gave the sam results.
• Figure 3B Effect of treatment after the formation o small tumors.
• Cells were injected using the same treatmen protocol as above except for the fact the treatment wa begun 4 days after cell injection instead of 1 day after Animal care was in accordance with institutional guidelines
• Figure A Effect of antibody binding on erbB- protein turnover.
• Subconfluent N87 cell monolayers were pulse-labeled 1 h with 20 ⁇ Ci 35S-Cysteine and then chase with 5 mM Cys in the presence of Ab#21 alone, Ab#23 alone or a 1:1 combination of Ab#21 and Ab#23 (10 ⁇ g/ml)for 24 h
• Total cellular protein was immunoprecipitated as describe in Figure 1 using a monoclonal antibody directed against th c-terminus of gpl85 coupled to Sepharose and analyzed by SDS-PAGE. The gel was exposed to film at -70°C overnight with an intensifying screen.
• Figure 4B Measurement of tyrosine phosphorylation of gp!85 ⁇ after incubation with antibody combination.
• Cells were plated as in Figure 4A. After 1 h cells were processed as in Figure IB.
• the proteins were electroblotted onto nitrocellulose paper and incubated with anti-phosphotyrosine IgG (polyclonal, Upstate Biotechnology, Inc.) and immunodetected using an ECL western blotting detection system (Amersham) . The film was exposed for 5 min at room temperature.
• FIG. 5 Effects of Ab#21 and Ab#23 on the growth of human Calu-3 lung adenocarcinoma tumor cells in a monolayer MTT growth assay.
• a single cell suspension of 10,000 cells/well was plated in a chemically defined medium consisting of RPMI-1640 supplemented with Insulin (5 ⁇ g/ml), human transferrin (10 ⁇ g/ml) , 17- ⁇ -estradiol (10 nM), sodium selenite (5 nM), and 10 mM Hepe ⁇ .
• PBS, Ab#21, Ab#23 or a combination of Ab#21 and Ab#23 at the indicated concentration were then added.
• the plates were grown at 37°C in a 5% CO- humidified atmosphere.
• FIG. 6 Effects of Ab#23 and Ab#94 on the growth of human Calu-3 lung adenocarcinoma tumor cells in a monolayer MTT growth assay.
• a single cell suspension of 10,000 cells/well was plated in a chemically defined medi consisting of RPMI-1640 supplemented with Insulin (5 ⁇ g/ml human transferrin (10 ⁇ g/ml), 17- ⁇ -estradiol (10 nM), sodi selenite (5 nM), and 10 mM Hepes.
• PBS, Ab#23, Ab#94 or combination of Ab#21 and Ab#23 at the indicat concentration were then added.
• the plates were grown 37°C in a 5% C0 2 humidified atmosphere.
• Figure 7 The cDNA sequence for the *single cha anti-erbB2 antibody, Ab#23.
• Figure 8 The cDNA sequence for the *single cha anti-erbB2 antibody, Ab#21 (e22).
• One object of the present invention is a combination at least two monoclonal antibodies, which is capable preventing and treating human malignancies, wherein t malignant cells overexpress gpl85 ⁇ and wherein said least two different antibodies each recognize a differe epitope of the gpl85 expression product of erbB-2, therefo the antibodies do not cross react with each other.
• embodiment of the present invention provides for t combination to comprise first and second antibodies whi are preferably combined such that the resulting ratio of t first to second is effective for decreasing the expressi product of the erbB-2 gene.
• a convenient method f measuring the expression product of erbB-2 gene may be found in Figure 4A.
• the decrease in the expression of the erbB-2 gene product is the result of the combination decreasing the half life of erbB-2 protein in the cell.
• the combination of the antibodies has the characteristic trait of essentially not increasing the tyrosine phosphorylation of gpl85 expression product.
• An example of a first to second antibodies ratio having the activity necessary to decrease the expression product of the erbB-2 gene comprises a ratio of from about 1:2 to about 2:1. Preferably, such a ratio is 1:1.
• the present invention is not intended to be limited to the antibody ratios discussed herein. The fact that other ratios are effective and may yield higher activity than the 1:1 ratio used as an example is recognized and acknowledged by the inventors as being within the scope of this invention.
• Figures 1A-C, 2 and 3A, B The activity of this combination is exemplified in Figures 1A-C, 2 and 3A, B as follows.
• Figure 1A-C demonstrate that the N87 cells overexpress the gpl85 erbB-2 protein as a result of erbB-2 gene amplification.
• Figure 2 shows that a combination of Ab#21 and Ab#23 inhibits the growth of N87 cells dji vitro. Similar results have been demonstrated using the combination of Ab#23 and Ab#94 as well as Ab#23 and Ab#21, on the growth of human Calu-3 lung adenocarcinoma (See Figures 5 and 6).
• Figure 3A and B show the activity of combinations of Ab#21 and Ab#23 inhibiting and reversing the growth of N87 cells growing as tumors in immunodeficient mice. These results indicate the general nature of the application of combinations of antibodies.
• the antibodies against the erbB2 gene encoded product used in this invention can be designed as chimeric antibodies.
• Chimeric antibodies have variable region (antigen binding regions) of nonhuman (e.g., murine) origi and constant regions of human origin. Because they ar predominantly human, chimeric antibodies are les immunogenic in humans, which can help overcome problem associated with administering foreign proteins to humans.
• the antibodies of the present inventio may be produced through genetic recombination or th Kohler-Milstein hybridoma method for production o antibodies. It is also recognized that fragments, analogue or derivatives of the antibodies themselves can be utilize in this invention in place of the entire antibody.
• Another object of the present invention provides fo antibodies against erbB-2 gene encoded product which ar designed as single chain antibodies.
• a single chai antibody is one in which the light and heavy variabl regions of the antibody are linked together to form a singl chain antibody. It is contemplated in this application tha a combination of these antibodies include antibodies whic are combined as an admixture as discussed above an antibodies which are combined to form a bispecific antibody
• a bispecific antibody is an artificially produce antibody usually comprised of two single chain antibodie each of which is recognizes a different antigen bindin site.
• huma malignancies which may be treated or prevented using th present invention
• adenocarcinonas of the breast, ovary lung and stomach are examples of some of the huma malignancies which may be treated or prevented using th present invention.
• Another embodiment of applicants' invention provides method for preventing and eradicating the human malignancie described above.
• the method involves administering to patient an effective dose of a combination of anti-erbB- antibodies to achieve an effective concentration of the antibody combination at the tumor site; for example, a concentration of at least l ⁇ g/ml.
• concentration at the tumor site does not exceed about lO ⁇ g/ml.
• the combination is administered in a dose from about .1 mg/kg to about 10 mg/kg of body weight.
• Another embodiment of Applicants' invention provides for the antibody combination to be used in passive tumor therapy, wherein an effective dose of the antibody combination is administered in or with a pharmaceutically acceptable vehicle to a patient afflicted with a human malignancy overexpressing gpl85erbB-2.
• a pharmaceutically acceptable vehicle examples include non-toxic buffers, physiological saline, etc.
• Applicants' invention also provides for at least one of the antibodies of the antibody combination to be used as a component of an immunotoxin.
• at least one antibody of the combination can be linked to an anti-cancer pharmaceutical or a cytotoxin to form an immunotoxin.
• Various pharmaceutical or cytotoxic agents can be chemically or genetically coupled to the combination.
• radioactive compounds e.g., isotopes of Boron and Rhenium
• agents which bind DNA such as alkylating agents or various antibodies (e.g., daunomysin, adriamycin, chlorambucil)
• anti-metabolites e.g., methotrexate
• inhibitors of protein synthesis e.g., diphtheria toxin and toxic plant proteins.
• Administration to a patient of an effective dose of the combination of antibodies described herein may be accomplished via chronic intraveneous administration for period of time sufficient to result in the regression o eradication of the human malignancy being treated
• Administration of the combination may also be accomplishe in a patient by direct injection or delivery of th combination to the tumor site. Such administration would b of sufficient duration and concentration to result i eradication or reduction of the tumor.
• tw antibody combination acts by constraining gpl85 er " int an activated conformation thus mimicking an agonist ligand If the two antibody combination mimics the ligand, the treatment using the combination should result in increase gpl85 ⁇ autophosphorylation.
• Anti-phosphotyrosin immunoblots were used to test this hypothesis. As shown i Figure 4B, no increase in tyrosine phosphorylation o gpl85 er ⁇ from N87 cells was observed 1 or 2 hours afte the addition of the antibody combination or up to 24 h o treatment. This suggests that the antibody combination doe not increase the autophosphorylation of gpl85 an therefore does not act to inhibit the activity of th tyrosine kinase.
• results demonstrate that a combination o anti-receptor antibodies leads to different and more poten anti-tumor activities than single antibodies.
• results indicate that the combination antibod therapy is a useful approach to treatment of huma ___ *hR malignancies overexpressing gpl85 This approach may b particularly important in the treatment of gastric cancer, disease which responds poorly to current systemi chemotherapies.
• a source of human erbB-2 protein we used a NIH/3T cell engineered to express the human erbB-2 protein on it surface (N/erbB-2).
• Membrane preparations of these cell were prepared by hypotonic lysis in 2mM Hepes pH 7.4, removal of nuclei by centrifugation at 5,000 x g an isolation of membranes by centrifugation at 100,000 x g.
• Mice were immunized with lOO ⁇ g of N/erbB-2 membran preparation in a 50:50 mix of adjuvant in 200 ⁇ l.
• Adjuvan was Freund's complete for the first injection followed b Freund' ⁇ incomplete adjuvant. Mice were given intraperitoneal injections over 4 weeks.
• ELISA reaction was develope using peroxidase coupled goat anti-mouse antibody an standard methods. Hybridoma cultures secreting a anti-erbB-2 antibody were subjected to two rounds of singl cell cloning and identification of positive subclones b ELISA as described above.
• Monoclonal antibodies directed against th erbB-2 extracellular domain of gpl85 were tested for specifi reaction to N/erbB-2 cell membranes in an ELISA assay. Tw of these designated Ab#21 and Ab#23 after screening i growth assays exhibited the highest biological activity an were used in this study. Antibodies were isolated in larg amounts from ascites fluid and purified by HPLC with
• N87 tumor xenografts The efficacy of combination antibody therapy was teste on the growth of N87 tumor xenografts.
• One inoculation o five million N87 cells were injected subcutaneou ⁇ ly int nude mice produce rapidly growing tumors, with a shor latency. Tumor growth at the injection site was easil quantitated.
• the N87 cells did no form tumors in the animals treated twice a week for thre weeks with a total of 200 ⁇ g of antibodies per injectio with the combination of Ab#21 and Ab#23. In sharp contras they were potently tumorigenic in animals treated with th
• each monoclonal antibody alone may hav limited activity to partially restrict the rate of tumo growth.
• the activity exhibited by the combinatio far exceeded the cumulative effect expected from th combination.
• Th activation of the murine neu oncogene is accomplished b point mutation as evidenced by qualitative interference i the structure and function of the neu gene, whereas th human erbB-2 oncogene is activated by overexpres ⁇ ion o erbB-2, a quantitative interference of the apparently norma protein which results in tumor formation.
• Antibodies #21; Ab#23; and Ab#94 have been deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852, USA. Ab#21 was deposited on and given ATCC # . Ab#23 was deposited on and given ATCC # . Ab#94 was deposited on and given ATCC # .
• cDNA was prepared using random primer (N & ) (Boerhinger Mannheim).
• the immunoglobulin light and heavy chain clones were isolated using PCR and the primers: light chain, 5' CAC GTC GAC ATT CAG CTG ACC CAC TCT CCA and GAT GGA TCC AGT TGG TGC AGC ATC3*; heavy chain 5'C GGA ATT TCA GGT TCT GCA GIA GTC WGG3' and 5' AGC GGA TCC AGG GGC CAG TGG ATA GAC3' [G,A,C, stand for standard nucleotides; I for inosine, W for A
• the light and heavy chain coding regions were joined by synthetic linker GSTSGSGKSSEGKG specified by overlappin oligonucleotides as described.
• the intact scFv codin region was inserted in frame with an E.coli OMPA leade sequence under direction of the lambda P_ promoter
• cDNA wa prepared using random primer (N ⁇ ) (Boerhinger Mannheim) The immunoglobulin light and heavy chain clones were isolated using PCR and the primers: light chain, 5' CAC GT GAC ATT CAG CTG ACC CAC TCT CCA and GAT GGA TCC AGT TGG TG AGC ATC3'; heavy chain 5'C GGA ATT TCA GGT TCT GCA GIA GT WGG3' and 5' AGC GGA TCC AGG GGC CAG TGG ATA GAC3* [G,A,C, stand for standard nucleotides; I for inosine, W for A o T] .
• the products of the PCR reaction wre cloned into PUC18 Linkage into a scFv was by PCR giving the individual ligh and heavy cDNA clones and 4 oligonucleotides 5' - cgagatgagtccagctgacccagtctc 5' - gaagatttaccagaaccagaggtagaaccttttatttccagcttgga 5' - ctggttctggtaaatcttctgaaggtaaggtgtgcagctgcaggag 5' - cgagtgcaagcttaggagacggtgaccgt.
• the light and heavy chain coding regions were joined by synthetic linker GSTSGSGKSSEGKG specified by overlappi oligonucleotides as described.
• the intact scFv codi region was inserted in frame with an E.coli OMPA lead sequence under direction of the lambda P. L_ promote

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