University of Kentucky UKnowledge Microbiology, and Molecular Genetics Microbiology, Immunology, and Molecular Faculty Patents Genetics

3-18-1997 Anti-Idiotype Monoclonal 1A7 and Use for the Treatment of Melanoma and Small Cell Carcinoma Malaya Chatterjee University of Kentucky

Kenneth A. Foon University of Kentucky Right click to open a feedback form in a new tab to let us know how this document benefits oy u.

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Recommended Citation Chatterjee, Malaya and Foon, Kenneth A., "Anti-Idiotype 1A7 and Use for the Treatment of Melanoma and Small Cell Carcinoma" (1997). Microbiology, Immunology and Molecular Genetics Faculty Patents. 2. https://uknowledge.uky.edu/microbio_patents/2

This Patent is brought to you for free and open access by the Microbiology, Immunology, and Molecular Genetics at UKnowledge. It has been accepted for inclusion in Microbiology, Immunology and Molecular Genetics Faculty Patents by an authorized administrator of UKnowledge. For more information, please contact [email protected]. llllllllllllll|||llllllllllllllllllllllllllllllllllllllllllllllllllllllllll USOO561203OA United States Patent [19] [11] Patent Number: 5,612,030 Chatterjee et a1. [45] Date of Patent: Mar. 18, 1997

[54] ANTI-IDIOTYPE MONOCLONAL ANTIBODY 5,208,146 5/1993 Irie ...... 435/723 1A7 AND USE 170R THE TREATMENT ()1? 5,240,833 8/1993 Nudelman et 211. .. 435/7021 MELANOMA AND SMALL CELL 5,242,824 9/1993 l-lcllstrom et a1. 435/240.27 5,270,202 12/1993 Raychaudhuri .435/240.27 CARCINOMA 5,305,559 4/1994 Ogawa . . .. 51/323 [75] Inventors: Malaya Chatterjee; Kenneth A. Foon, FOREIGN PATENT DOCUMENTS both of Lexington, Ky. 0280209A2 8/1988 European Pat. O11. . [73] Assignce: University of Kentucky Research WO86/OO909 2/1986 WIPO' FOllIld??Ol'l, Lexington, Ky. OTHER PUBLICATIONS [21] AWL NW 372 676 Chcung, NV. et a1. (1993) Disialogangliosidc GD2 Anti*id ’ iotypic monoclonal . Int. J. Cancer vol. 54 pp. [22] Filed: Jan. 17, 1995 499-505. [51] Int C16 A61K39/395_CO7K16/42_ Scavcr, SS. (1994). Monoclonal Antibodies in Industry: More di?icult than originally thought. Genetic Engineering C12P 21/05; C12N 15/06 News Aug. 1994 pp. 10, 21. [52] U.S. Cl...... 424/1311; 455/70.21; Bhattacharya—Chatterjee et a1. (1993). l. Immunology. vol. 455/172.l; 455/327; 530/3872; 530/3888; 150 (8 part 2) 142A. Abstract 805. 530/3913; 424/ 155-1? 424/1741; 435/3441 Saleh, M.N. et a1 (1993). generation of Human Anti-idio [58] Field Of Search ...... 424/1311, 178.1, typic antibody that Mimics the GD2 ]_ Immuno1_ 424/155.1, 174.1; 436/501, 548, 542, 435/7021, 172'1’ 24021792495’ 7'23; 530/387‘2> Mujoo, K. et a1. (1989). Cancer Research. vol. 49 pp. 388.8, 388.85, 391.3 2857_2861_ [56] References Cited Primary ExaminermChristine M. Nucker Assistant Examiner-Julie E. Reeves U-S- PATENT DOCUMENTS Attorney, Agent, or Firm—Lowe, Price, LeBlanc & Becker 4,675,287 6/1987 Reisfeld et a1...... 435/7

4,849,5094,693,966 7/9/1987 1989 Thuri"HOllghtOl'l e931‘ 81 a1.~ - - - - - ~ - ~ - - - .....~ -- 530/387.. 435/7 The present invention relates isolation of anti-idiotypic 4,904,596 Hakomon ...... raised against anti_GD2 and its 4’918’164 4,1990 Henstmm et a1‘ """ " 530/387 use for the treatment of melanoma and small cell carcinoma. 4,965,498 10/1990 Yokota ...... 318/468 . . . 5,009,995 4/1991 Albino et a1...... 435/723 Th‘? annbody “13y b‘? used as a sllbsmm? f0? ‘5013169 5,053,224 10/1991 Koprowski et a1...... 424/858 P‘m?ed GD2 amlgcn 1“ any aPPmPnale aPPhcallOn 5,091,177 2/1992 Hellstrom et a1...... 424/858 5,134,075 7/1992 Hellstrom et a1...... 530/3873 21 Claims, 6 Drawing Sheets US. Patent Mar. 18, 1997 Sheet 1 0f 6 5,612,030

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L0 98 *t in“ g m a. 3: U) U’) 3% 6 LO. LO. h *- T- 11> O O ‘E co ED 0') [L m . P ‘i ‘n :9 o r : <33 m . : ‘°_ cu m. m a w HC. Lo.. 9-’ 2 5-’ U. 1: <1 1: N h 2 o ‘5. “- 6') 05 < 2 an (5 _.l (\i LU N c: m c5 ,4 Figure4 US. Patent Mar. 18, 1997 Sheet 6 0f 6 5,612,030 5,612,030 1 2 ANTI-IDIOTYPE MONOCLONAL ANTIBODY ential reactivity to melanomas, ncuroblastomas and adeno 1A7 AND USE FOR THE TREATMENT OF carcinomas. They are anti-ganglioside antibodies with MELANOMA AND SMALL CELL speci?c isotopes such as IgG3 and lgG2a. CARCINOMA U.S. Pat. No. 5,242,824 to Hellstrom et al. discloses novel monoclonal antibodies reactive with a glycolipid cell mem TECHNICAL FIELD brane antigen on the surface of human carcinomas. Mono The present invention relates isolation of anti-idiotypic clonal antibodies react with carcinomas of the lung, ovary antibody 1A7 raised against anti-GDZmAb 1462a and its and colon. They show no detectable reactivity with mela use for the treatment and detection of melanoma and small noma cells. cell carcinoma. U.S. Pat. No. 5,270,202 to Rayehaudhuri discloses a novel anti-idiotypic antibody lMelpg2 which is speci?c for BACKGROUND melanoma cells. It can be used for the diagnosis and treatment of melanoma tumors. Monoclonal antibody to the human ganglioside GD2 antigen, and to other melanoma and small cell lung carci U.S. Pat. No. 5,208,146 to lrie discloses murine mono nomas are known. For example, U.S. Pat. No. 4,675,287 to clonal anti-idiotypc antibodies raised against human mono Reisfeld et al. discloses a monoclonal antibody to the human clonal anti-ganglioside antibody known as L612. The mono ganglioside GD2 antigen. This antibody is reactive with clonal antibody is speci?c for melanoma cells. melanoma and oat cell lung carcinoma cells. This mono U.S. Pat. No. 4,904,596 to Hakomori discloses a hybri clonal antibody is tolerated by the human doma cell line and monoclonal antibody to fucoganglioside, and thus human immune system does not remove this 6B, which is present in human colonic adenocarcinoma and antibody by immunoactive mechanisms. lntemational lung carcinoma cells. Patent Publication WO 8600909 to Reisfeld, R. A. ct al is U.S. Pat. No. 5,009,995 to Albino et al. discloses mono directed to a “Monoclonal antibody directed to human clonal antibodies recognized by gpl30 antigen of human ganglioside GDZ.” This patent is the international patent 25 cells. The monoclonal antibodies are useful in the detection publication related to U.S. Pat. No. 4,675,287 to Reisfeld et of the gpl30 antigen and human cells, including melanoma al. described above. More speci?cally, the publication dis cells, which contain the antigen. closes a non-human, mammalian monoclonal receptor pro U.S. Pat. No. 4,918,164 to Hellstrom et al. discloses duced by a hybridoma formed by fusion of cells from a anti-idiotypic antibodies for immunization against tumor, myeloma cell line and that produce antibodies for inhibition of immune suppression mediated by suppres that react with ganglioside GD2 from a mammal immunized sor T cells or suppressor factors expressing an anti-idiotype with a ganglioside GDz-containing is disclosed. against tumors bearing the oncofetal antigen. Monoclonal U.S. Pat. No. 4,693,966 to Houghton et al. discloses antibody recognizes a human melanoma associated GD3 human monoclonal antibodies from lymphocytes of patients ganglioside antigen. with malignant melanoma. The monoclonal antibodies of 35 Journal of Immunology, Volume 150, 142A, 1993 dis Houghton et al. speci?cally bind to found on closes an abstract of Chatterjee et al. entitled “Syngeneic surfaces of renal, lung and breast cancer cells. The antibody Monoclonal Anti-IdiotypeAntibodies Against a Monoclonal also detects the cytoplasmic antigen expressed by cells of Antibody to Human Melanoma-Associated Antigen.” The neuroectodermal origin, such as melanoma cells. abstract generally discloses that the 1A7 antibody was U.S. Pat. No. 4,965,498 to Yamasaki et al. discloses a isolated, but does not disclose a method of obtaining it or monoclonal antibody speci?c to a sugar chain containing an provide any of its particular properties or uses. N-glycolylneuramine acid and has the ability to bind to at Patent No. EP 280209 is directed to “Monoclonal anti least N-glycolyl GM2 ganglioside. Page 1, lines 55—56 bodies against melanoma-associated antigens, hybridoma acknowledges that monoclonal antibodies against human cell lines producing these antibodies, and uses of the mono melanoma which react with glycolipids such as GD2 gan 45 clonal antibodies”. This patent to Thurin et al. discloses glioside are known. hybridomas producing antibodies against ganglioside anti U.S. Pat. No. 5,305,559 to Nicolson et al. is directed to gens GD2 and GD3 which are non-reactive with other methods and compositions for the identi?cation of meta ganglioside antigens. static human tumors. Monoclonal antibodies of this patent 50 None of the patents nor literature recognize an anti- _ react with human tumor cells and are prepared against a 580 idiotypic monoclonal antibody speci?c for melanoma and kilodalton glycoprotein antigen gp580. Antibodies are spe small cell carcinoma cells which is not tolerated by the ci?c for lung metastasis from breast tissue and are not human immune system. reactive with melanoma tumors. Neuroblastomas are highly malignant tumors occurring U.S. Pat. No. 5,091,177 to Hellstrom et al. issued is 55 during infancy and early childhood. Except for Wilms’ directed to monoclonal antibodies which de?ne a glycolipid tumor, they are the most common retroperitoneal tumors in antigen associated with human non-small cell lung carcino children. Neuroblastomas arise most commonly in the adre mas. Activity with melanoma cells is not disclosed. The nal medulla, but they may also develop in other sympathetic monoclonal antibody has an IgG2 isotope. ganglia within the thorax or abdomen. These tumors metas U.S. Pat. No. 5,134,075 to Hellstrom et al. discloses a 60 tasize early with wide spread involvement of lymph nodes, monoclonal antibody which binds strongly to a protein liver, bone, lung and marrow. The prognosis is often good antigen associated with human tumors, including lung when the tumor is diagnosed prior to obvious metastasis, but tumors as well as melanomas and sarcomas. The monoclonal with metastasis, prognosis is poor despite the extensive use antibody is of the subclass IgG2a. of radical surgery, deep X-ray therapy, and chemotherapeu U.S. Pat. No. 5,240,833 to Nudelman et al. discloses 65 tie agents. monoclonal antibodies that bind to tumor-associated gan Several antigenic determinants have recently been gliosides. The monoclonal antibodies have selected prefer detected on neuroblastoma cells with monoclonal antibodies 5,612,030 3 4 (Mabs). See Seeger, Ann. Intern. Med, 97, 873 (1982); determinant on fetal tissues, especially fetal brain, as well as Wikstrand et al., Cancer Res., 42, 267(1982); Wikstrand et on adult brain and other neural tissues. In addition, cross al., J. Neuroimmunlogy, 3, 43 (1982); Eisenbarth et al., Proc. reactions of such antibodies have also been reported with Nat’l Acad. Sci. (USA), 76, 4913 (1979); Liao et al., Eur. J. normal kidney, fibroblasts, rnyoblasts, and , See Immunol, 11, 450 (1981); Seeger et al., Cancer Res., 4, ger et al., Cancer Res., supra, and Seeger et al., J. Immunol, 2714 (1981); Kennett et al., Advances in Neuroblastoma supra, with islet cells, Eisenbarth et al., Proc. Nat’l Acad. Research, p. 209, Raven Press, New York (Evans ed.) Sci. (USA), supra, and with spleen cells, Wikstrand et al., (1980); Seeger et al., J. Immunol., 128, 983 (1982); Kems Cancer Res., supra. head et al., Pediatr. Res., 15, 1282 (1981). Furthermore, some of the monoclonal antibodies reported A panel of such antibodies has been reported to be helpful in the literature are not only restricted in their reactivity to in the differential diagnosis of neuroblastoma and lympho neuroectoderrnal tumors, such as neuroblastoma, melanoma blastic disorders, Kemshead et al., Pediatr. Res., supra; and glioma, but also show binding to some forms of leuke Kemshead et al., Lancet, I2 (1983). In these same studies, mia, osteogenic sarcoma, rhabdomyosarcoma, leiomyosar antibodies were used either in immunoperoxidase assays coma and even to carcinomas of the lung and breast, Seeger, with tumor tissue sections or in direct immuno?uorescence Ann. Intern. Med, supra. assays to detect tumor cells in bone marrow aspirates. A monospeci?c human monoclonal antibody, (anti-OFA The effective use of Mabs directed to any tumor-associ— I-2), produced in vitro by a lymphoblast cell line that ated antigens as diagnostic reagents depends on the quantity, originated from a melanoma patient was reported to detect expression and chemical nature of the corresponding anti a GD2 ganglioside on human melanoma, glioma and neu gen. In this regard, Mabs directed to tumor-associated gan 20 roblastoma cells, while reportedly not reacting with a variety gliosides have been useful in de?ning antigens associated of cell lines derived from carcinomas and from diiferent with melanoma, neuroblastoma, colon carcinoma, and lymphoid tumors, Cahan et al., Proc. Nat’l Acad. Sci. adenocarcinoma, Hakomori et al., J. Natl. Cancer Inst., (USA), supra, and Irie et al., Proc. Nat’l Acad. Sci. (USA), 71,231 (1983). One of these antibodies was reported to 79, 5666 (1982). However, problems have arisen when such detect a ganglioside antigen shed into the serum of patients 25 a human monoclonal antibody is used for immunoperoxi with colon carcinomas, Koprowski et al., Science, 212, 53 dase assays of human tissues in that the anti-human second (1981). Some of the above neuroblastoma—associated anti ary antibody required for such assays causes a large amount gens are present in fetal neural tissues whereas others are of non-speci?c background reactivity. expressed by both fetal and adult neural tissues. Seeger, Ann. 30 Heterogeneity of neuroblastomas with regard to cell sur Intern. Med., supra. face antigenic expression has been reported in Seeger, Ann. Most of the monoclonal antibodies utilized to detect the Intern. Med, supra; Kemshead et al., Pediatr. Res., supra; neuroblastoma-associated antigens are not restricted in their Kemshead et al., Int. J. Cancer, 27, 447(1981); and, Kems reactivity to neuroectoderrnal tumors like melanoma and head et al., Proc. Am. Assoc. Cancer Res., 2, 399 (1981). As glioma but also recognize common antigens on other malig 35 discussed in these publications, Mab A2 B5 failed to react nancies such as a variety of sarcomas and leukemias, Seeger, with some human neuroblastoma lines tested, and quantita Ann. Intern. Med., supra. In addition, only some of the tive differences in antigenic expression were observed antigenic structures on neuroblastoma cells recognized by between di?‘erent cell cultures. Analysis of tumor cells in monoclonal antibodies have been partially characterized by heavily in?ltrated bone marrow aspirates indicated that only immunochemical means. Thus, a monoclonal antibody des 40 70 percent of the samples reacted with A2 B5, suggesting ignated Mab 390 was reported to react with an antigenic that the heterogeneity seen in the expression of antigen on determinant of human Thy-l that had a molecular weight of cell lines is paralleled in fresh tumor material, Kemshead et 25,000 daltons. Seeger et al., J. Immunol., supra. al., Int. J. Cancer, supra. Another Mab, designated A2 B5, was reported to recog Thus there is a need in the art for new methods of nize a GD2 ganglioside on neurons, Eisenbarth et al., Proc. detecting and treating melanoma and small cell carcinoma. Nat’l Acad. Sci. (USA), supra. A human monoclonal anti The present invention overcomes the de?ciencies of the body produced in vitro by a lymphoblast cell line from a prior art by providing an anti-idiotypic antibody 1A7 raised melanoma patient was also reported to react with a GD2 against anti-GD2mAb l4G2a, which is not tolerated by the ganglioside present on neuroectoderm-derived tumors, human immune system, and thus may be used as a vaccine Cahan et al., Proc. Nat’l Acad. Sci. (USA), 79, 7629 (1982). 50 to stimulate the immune system. This property of the present From a biological point of view, gangliosides are of monoclonal antibody makes it ideal for a new immuno considerable interest since they have been implicated in a therapeutic approach to cancer. variety of cellular functions, including cell-cell adhesion and communication, as well as cell-substrate interactions, Hako Disclosure of the Invention mori et al., J. Nat’l Cancer Inst., supra. Recent studies have 55 emphasized the importance of gangliosides for tumor It is an object of the present invention to provide an growth regulation by demonstrating di?erences in ganglio anti-idiotype monoclonal antibody 1A7, which is the inter side composition among cells expressing various degrees of nal image of the GD2 ganglioside antigen which is highly tumorigenicity, Itaya et al., Proc. Nat’l Acad. Sci. (USA), 73, expressed on malignant melanoma cell and small cell car 1568 (1976). Consequently, the use of monoclonal antibod cinoma cells. ies directed to ganglioside determinants aids in further It is another object of the invention to provide an antibody delineating the role of gangliosides in these processes. which generates an active to GD2 antigen which Most of the monoclonal antibodies directed against neu is highly expressed on malignant melanoma cell and small roblastoma-associated antigens that have been reported thus cell carcinoma cells. far, Wikstrand et al., Cancer Res., supra; Wikstrand et al., J. 65 A further object of the invention is to provide a pharma Neuro-immunology, supra; Eisenbarth et al., Proc Nat’l ceutical composition comprising anti—idiotype monoclonal Acad. Sci. (USA), supra, recognize a common antigenic antibody 1A7, and a pharmaceutically acceptably carrier. 5,612,030 5 6 A still further object of the invention provides a method During the pendency of this application, access to the of treatment of metastatic melanoma and small cell lung deposit will (a) be forwarded to one determined by the cancer comprising administering a pharmaceutically effec Commissioner to be entitled thereto; tive amount of a pharmaceutical composition of the inven (b) all restrictions imposed by the depositor on the avail tion to a patient in need of treatment. ability to the public of the deposited material will be In a preferred embodiment the method may be adminis— irrevocably removed upon the granting of the patent, tered to a patient who has had disease removed by surgery, (c) the deposit will be maintained for a period of at least radiation or chemotherapy and remains at high risk for thirty years or at least ?ve years after the most recent request recurrence of metastatic melanoma and small cell lung for the furnishings of a sample of the deposited material; and cancer. (d) the deposit will be replaced should it become neces Another object of the invention is to provide a lA7 sary due to inviability, contamination or loss of capability to monoclonal antibody which can be used as a substitute for function in the manner described in the speci?cation. GD2 antigen in all biochemical and serological assays, such as a monoclonal antibody probe. The 1A7 monoclonal DESCRIPTION OF THE INVENTION 15 antibody probe may be incorporated into a test kit in Anti-idiotype monoclonal antibody 1A7, is the internal accordance with the present invention, such as a diagnostic image of a GD2 ganglioside antigen which is expressed on test kit. malignant melanoma cells and on small cell carcinoma cells. The above and other objects of the invention will become The anti-idiotype antibody 1A7, raised against a known readily apparent to those of skill in the relevant an from the anti-GD2 antibody (1462a), mimics GD2 antigen. However, following detailed description and ?gures, wherein only the it is not tolerated by the human immune system. This preferred embodiments of the invention are shown and property of the present monoclonal anti-idiotype antibody described, simply by way of illustration of the best mode of makes it ideal for a new immuno-therapeutic approach to carrying out the invention. As is readily recognized the cancer. invention is capable of modi?cations within the skill of the 25 Discussion below represents the results of immunization relevant art without departing from the spirit and scope of and treatment of monkeys with anti-Id 1A7 ( IgGl-k) the invention. which mimics GD2. Murine monoclonal anti—Id 1A7 was raised against anti-GD2 mAb l4G2a (isotype IgG2a—k) BRIEF DESCRIPTION OF DRAWINGS obtained from Scripps Research Institute, La lolla. In pre vious studies of the inventors, small animals, such as mice FIG. 1 shows the binding of puri?ed Ab3 to Ab2 1A7 on and rabbits, were immunized three to four times bi-weekly the plate by sandwich radioimmunoassay. with anti-Id 1A7 coupled to KLH and mixed with Freund’s FIG. 2 demonstrates the inhibition of Abl-Ab2 binding Adjuvant. The production of anti-GD2 antibodies were by monkey Ab3 or vice-versa (Ab2—Ab3 binding inhibition) induced in the animals. which shows that Abl and Ab2 share idiotopes and Ab3 is 35 A murine monoclonal antibody mAB (IgGZak) which true anti-anti-idiotypic in nature. binds to the ganglioside GD2 in human melanoma, neuro FIG. 3 shows the inhibition of binding of 1251 labelled blastoma, glioma and sarcoma has been used to generate l4G2a antibody to GD2 positive melanoma cell line M2l/P6 monoclonal antibodies (Ab2) in BALB/c mice. The culture in presence of different concentrations of Abl and Ab3. supernatants from primary fusion cells were initially Parallel inhibition curves were obtained using either puri?ed screened by a sandwich radioimmunoassay using Id as Abl or Ab3 form monkey sera. This suggests that Abland antigen. Ab3 bind to the same on GD2. Several Ab2 hybridomas were obtained that reacted with FIG. 3A shows the inhibition of binding of I25 I-labelled the immunizing Id of l4G2a (Abl) and did not react with 14G2a antibody to puri?ed GD2 on the plate by Abl and any isotype or matched control immunoglobulins. Ab3. 45 Three of the mAb2s reacted with the antigen binding site FIG. 4 shows the binding of Abl and Ab3 to different (), since they inhibited the binding between 1251 gangliosides on the plate by ELISA assay. 250 ng of GD2 labelled 14G2a and the target melanoma cell line M21/P6. and other gangliosides were coated per well of 96-well One of these clones lAI-lA7 is used to raise anti-anti microtiter plates, blocked and incubated with different con idiotype antibodies (Ab3) in rabbits. Polyclonal rabbit A3 centrations of puri?ed Abl and Ab3. The bound antibody sera competed with Abl for binding to M2l/P6 cell lines was detected using anti-Human-Ig-alkaline phosphatase available from Scripps Institute, La Jolla, Calif, and inhib labelled reagent and substrate. The CD. value obtained after ited the binding of radiolabelled Abl to Ab2. 2 hr. using 1.5 micrograms of different antibodies per well The anti-idiotype monoclonal antibody to the anti-GD2 were plotted. At this concentration, there was only reactivity 55 antibody (designated 1462a) is designated 1A7. This anti with GD2. Idiotype antibody is, therefore, the internal image of the FIG. 5 shows the ELISA results con?rmed by dot blot GD2 ganglioside antigen which is highly expressed on analysis. malignant melanoma cell and small cell carcinoma cells. The anti-idiotype antibody mimics the GD2 ganglioside antigen. The present inventors have determined in mice, STATEMENT OF DEPOSIT rabbits and monkeys, all acceptable experimental animal A deposit of the hybridoma producing the 1A7 mono models, that when the anti-idiotype antibody is injected clonal antibody was made prior to the ?ling date of the intraeutaneously into these animals that they develop an above-identi?ed patent application under the terms of the anti-anti-idiotype antibody that is like anti-GD2 and mimics Budapest Treaty with the American Type Culture Collection, 65 the original anti-GD2 (l4G2a). Parklawn Drive, Rockville, Md., USA, Accession No. A pair of male and female cynomolgus monkeys were HB-ll786. immunized with 2 mg of 1A7 (intact IgGl) mixed with 100 5,612,030 7 8 pg QS-2l (Cambridge Biotech), 3 to 4 times. Sera obtained Radioimmuno assay (RIA) from monkeys 2 weeks after 3rd and 4th immunizations were analyzed. Additional characterization of Ab2 was done by RIA. For the direct binding assay between Abl and Ab2, puri?ed Abl Anti-anti-Id(Ab3) antibody from monkey sera was puri ?ed ?rst by adsorption and then elution from a?inity column was used to coat plate (500 ng/well) and the binding of lA7-sepharose 4B and then by passing through a negative radiolabeled Ab2 to Abl was tested in the presence of a?inity column of mouse IgG-sepharose 4B. The flow different Abl, Ab2 or several control mouse myeloma pro terns. through material has been used as “puri?ed” Ab3 and compared with the reactivity of Abl (l4G2a) in different assays. 2.6 mg of puri?ed Ab3 was recovered from 10 ml of Anti-Idiotype assay sera (i.e. about 260 micrograms Ab3 per ml of serum) from To determine whether Ab2 recognizes the paratope of monkey #PRO 685 and #PRO 778. Abl, the following inhibition assays were done. Target This anti-idiotype antibody can be used to treat patients tumor cells (M2l/P6) which contain GD2 antigen as a cell with malignant melanoma and small cell lung cancer to surface constituent, were grown as con?uent monolayer in generate an active immunity to the GD2 antigen which is 96-well tissue culture plates. The binding of radiolabeled highly expressed on their tumor cells. These patients are not Abl to cultured cells was tested for inhibition in the presence capable in vivo of generating the active immunity to GD2, of different Ab2 culture supernatant and also puri?ed Ab2 but by using the anti-idiotype as a surrogate antigen, they are preparation. able to overcome tolerance to this antigen and generate an active immunity to GD2 antigen. 20 Inhibition assay These new anti-idiotype antibodies represent a new Percent inhibition of the assay was calculated according immunotherapeutic approach to cancer. The antibodies may to the formula: be used for the treatment and therapy of metastatic mela noma and small cell lung cancer. The antibodies may also be 25 used as a prevention for recurrent disease in patients who %1nhrb1tion —l [—————RmaX_RC ] X 100 have had disease removed by surgery, radiation or chemo therapy and who remain at high risk for recurrence. in which R, is the average cpm of the experimental well with inhibitors, RC is the average background cpm and RMAX is 30 the average maximum binding without any inhibitors. Production of Ab2 (1A7) Puri?cation of Ab2 The murine mAb l4G2a is an anti-GD2 antibody that mediates antibody-dependent cytotoxicity and complement To get enough puri?ed Ab2, ascites of Ab2 (1A7) hybri mediated lysis of neuroblastoma and melanoma cell lines in domas was prepared by injecting individual pristane primed vitro. Murine monoclonal antibody l4G2a (Abl) was used 35 BALB/c mice intraperitoneally with 2>

Dot Blot Further, the 1A7 monoclonal antibody of the present invention is useful in pharmaceutical compositions for sys Reactivity of immunized sera and puri?ed Ab3 for anti temic administration to humans and animals in unit dosage GD2 antibodies against various gangliosides was also mea forms, sterile parenteral solutions or suspensions, sterile sured by immunoblotting. Puri?ed gangliosides (2 pg each non-parenteral solutions or suspensions oral solutions or of GM3, GM2, GMl, GD3, GD2 and GTlb) were spotted on suspensions, oil in water or water in oil emulsions and the strips of PVDF cellulose membrane at 1 cm intervals. After like, containing suitable quantities of an active ingredient. blocking with 3% BSA in PBS, the strips were incubated Formulations for parenteral and nonparenteral drug delivery with puri?ed Ab3 or Abl (l 0 pg ml) overnight at room temp. 65 are known in the art as set forth in Reminglon’s Pharma After washing, the strips were incubated with alkaline ceutical Sciences, 18th Ed, Mack Publishing (1989) incor phosphatase conjugated second antibody (1:1000 dilution) porated herein by reference in its entirety. 5,612,030 11 12 The compounds are useful in pharmaceutical composi We claim: tions (wt %) of the active ingredient with a carrier or vehicle 1. An antibody having all the identifying characteristics of in the composition in about 1 to 20% and preferably about monoclonal antibody 1A7 produced by the hybridoma 5 to 15%. deposited under ATCC Accession No. HB—11786. The above 1A7 monoclonal antibody can be present alone 2. Hybridoma designated 1 A7 having ATCC Accession or in combination form with pharmaceutical carriers. The No. HB-11786, or progeny thereof producing a monoclonal antibody having all the identifying characteristics of the pharmaceutical carriers acceptable for the purpose of this antibody produced by said hybricloma 1A7. invention are the art known carriers that do not adversely 3. Monoclonal antibody produced by the hybridoma or a?'ect the drug, the host, or the material comprising the drug the progeny thereof according to claim 2. delivery device. Suitable pharmaceutical carriers include 4. Monoclonal antibody puri?ed from the hyb?doma or sterile water; saline, dextrose; dextrose in water or saline; the progeny thereof according to claim 2. and the like. 5. A puri?ed antibody having all the identifying charac The effective dosage for mammals may vary due to such teristics of an antibody produced by the hybridoma accord factors as age, weight, activity level or condition of the ing to claim 2. subject being treated. Typically, an effective dosage of a 6. An antibody according to claim 1 wherein said antibody compound according to the present invention is about 2mg further comprises a detectable label. per injection in humans. A preferred dosage is 100 pg of 7. An antibody according to claim 6, wherein said detect Ab2-KLH (KLH=keyhole limpet hemocyanin) when able label is selected from the group consisting of radiola injected i.p. with Freund’s complete adjuvant in small bels, fluorescent labels and chemiluminescent labels. animals. A more preferred dosage range is 0.001 mg to 10 20 8. A diagnostic test kit for detecting an anti-GD2 antibody mg of 1A7 (intact IgGl) mixed with QS-2l (Cambridge m a biological sample, comprising an antibody according to Biotech) in monkeys. claim 1 in a suitable container. 9. The diagnostic kit of claim 8, wherein the antibody is Probe capable of binding anti-GD2. 25 10. The diagnostic kit of claim 8, wherein the antibody is The 1A7 anti-idiotype monoclonal antibody according to labeled with a detectable label. invention may be labeled and used as a probe for the 11. The diagnostic kit of claim 8, wherein the detectable detection of melanoma or small cell carcinoma. The probes label is selected from the group consisting of radiolabels, ?uorescent labels, and chemiluminescent labels. may be incorporated into a diagnostic test kit including a 12. The diagnostic kit of claim 8, also comprising an detectable label or marker for the probe. anti-immunoglobulin reagent labeled with a detectable label. 13. The diagnostic kit of claim 8, wherein the biological Diagnostic Kit sample is obtained from an individual suspected of having a GD2 antigen associated cancer. The diagnostic kit may further comprise, where necessary, 14. The diagnostic kit of claim 8, wherein the sample is other components of the signal-producing system, including 35 obtained from an individual suspected of having a cancer agents for reducing background interference, control selected from the group consisting of melanoma, neuroblas reagents or an apparatus or container for conducting the test. toma, glioma, sarcoma, and small cell carcinoma. Examples of imaging reagents that can be used include, 15. The diagnostic kit of claim 8, wherein the biological but are not limited to, radiolabels such as 1311, 1111n, 1231, sample is obtained from an individual treated with an 99mTc, 32P, 1251, 31-1, and 14C, ?uorescent labels such as antibody according to claim 1. ?uorescein and rhodarnine, and cherniluminescers, such as 16. A method of preparing monoclonal antibody 1A7, luciferin. Other labels known to those of skill in the art are comprising purifying antibody from the hybridoma or the set forth in US. Pat. No. 4,366,241 and are incorporated progeny thereof of claim 2. herein by reference. The monoclonal antibody can be 17. A method of preparing monoclonal antibody 1A7, labeled with such reagents using techniques known in the 45 comprising growing the hybridoma or the progeny thereof of art. For example, see Wensel and Meares, Radioimrnunoim claim 2. aging and Radioimmunotherapy, Esevier, New York (1983), 18. A method for detecting an anti-GD2 antibody in a for techniques relating to the radiolabeling of proteins. (See biological sample, comprising the steps of providing the diagnostic kit of claim 8, and contacting the antibody also, Sambrook, M. J., Fritsch, E. F. & Marriatis, T. (1989) provided in the container with any anti-GD2 antibody in the Molecular Cloning: A Laboratory Manual (Cold Spring biological sample. Harbor Lab., Cold Spring Harbor, N.Y., section 10, incor 19. A method for eliciting active immunity to ganglioside porated herein by reference in its entirety). GD2 in an individual, comprising administering to the The purpose of the above description and examples is to individual the antibody of claim 1. illustrate some embodiments of the present invention with 20. The method of claim 19, wherein the active immunity out implying any limitation. It will be apparent to those of comprises production of anti-GD2 antibody by the indi skill in the art that various modi?cations and variations may vidual. be made to the composition and method of the present 21. The method of claim 19, further comprising mixing invention without departing from the spirit or scope of the the antibody with QS-21. invention. All patents and publications cited herein are incorporated by reference in their entireties.