US005382428A United States Patent (19) 11) Patent Number: 5,382,428 Livingston-Wheeler et al. 45 Date of Patent: Jan. 17, 1995 54 METHOD FOR PREPARNG A PURIFIED 56) References Cited EXTRACTION RESIDUE FRACTION AND TSUSE IN STMULATING THE IMMUNE U.S. PATENT DOCUMENTS RESPONSE 3,958,025 5/1976 Livingston .......................... 424/317 OTHER PUBLICATIONS 75 Inventors: Virginia Livingston-Wheeler, San Livingston, et al., Trans. N.Y. Acad. Sci., vol. 36, p. 569 Diego County, Calif.; John J. (1974). Majnarich, King County, Wash. Chen et al., Proc. Soc. Exp. Biol. and Med., vol. 152, pp. 408-410 (1976). (73 Assignees: John J. Mainarich; Virginia Maruo et al., Proc. Natl. Acad. Sci., vol. 76, pp. Livingston-Wheeler, both of 6622-6626, 1979. Redmond, Wash. Acevedo et al., Cancer, vol. 41, pp. 1217-1229, 1978. Acevedo et al., Injection and Immunity, vol. 24, pp. 920-924, 1979. 21 Appl. No.: 336,567 Primary Examiner-Douglas W. Robinson Assistant Examiner-Deborah K. Ware 22 Fled: Apr. 11, 1989 Attorney, Agent, or Firm-Seed and Berry 57 ABSTRACT Related U.S. Application Data A method is disclosed for preparing a purified extrac 63 Continuation of Ser. No. 150,512, Feb. 8, 1988, aban tion residue fraction (antigen) of a microorganism iso doned, which is a continuation of Ser. No. 935,123, lated from the blood or urine of a warm-blooded animal Nov. 26, 1986, abandoned, which is a continuation of or tumor carried by a warm-blooded animal, the micro Ser. No. 712,468, Mar. 15, 1985, abandoned, which is a organism having the capacity to synthesize the polypep continuation of Ser. No. 523,679, Aug. 16, 1983, aban tide hormone known as "chorionic gonadotropin' in its doned, which is a continuation-in-part of Ser. No. total form or in its alpha and beta subunits. The purified 255,678, Apr. 20, 1981, Pat. No. 4,410,510, which is a extraction residue fraction is an activator and modula continuation-in-part of Ser. No. 128,919, Mar. 10, 1980, tor of immunological response and is capable of evoking abandoned, which is a continuation of Ser. No. pronounced prophylactic and therapeutic effects 957,206, Nov. 3, 1978, abandoned. against a variety of tumors in laboratory animals and man. The immune response is stimulated by administer (51) Int. Cl. ..................... A61K 37/00; A61K 39/02; ing an effective immuno-stimulating amount of the resi C12N 1/20; A01N 37/18 due fraction and/or co-administering the residue frac (52) U.S. C. .............................. 424/234.1; 424/282.1; tion together with an adjuvant such as the complete 435/68. 1; 435/71.1; 435/252.1; 514/2 Freund's adjuvant. 58) Field of Search ................. 424/92, 93 D; 435/68, 435/252.1, 253, 71.7; 514/2 8 Claims, No Drawings 5,382,428 1. 2 Livingston and E. Alexander-Jackson, “A Specific METHOD FOR PRE PARING A PURIFED Type of Organism Cultivated from Malignancy: Bacte EXTRACTION RESOUE FRACTION AND ITS USE riology and Proposed Classification,” Ann. N. Y. Acad. NST MULATING THE MMUNE RESPONSE Sci., 174: 636-654 (1970); and V. W-C. Livingston and A. M. Livingston, "Some Cultural, Immunological and CROSS-REFERENCE TO RELATED Bio-Chemical Properties of Progenitor Cryptocides,” APPLICATION Trans. N. Y. Acad. Sci, 36:569-582 (1974). This application is a continuation of U.S. application The avian leukosis complex comprises the neoplastic Ser. No. 07/150,512, filed Feb. 8, 1988, now abandoned, diseases of the hematopoietic system of the domestic which is a continuation of U.S. patent application Ser. 10 chicken, together with several other neoplastic and No. 935,123, filed Nov. 26, 1986, now abandoned under non-neoplastic conditions which are related either etio C.F.R. 8 1.62, which is a continuation of U.S. patent logically or pathologically. The following diseases are application Ser. No. 712,468, filed Mar. 15, 1985, now included in the complex leukosis: lymphoid leukosis, abandoned, which was a continuation of U.S. patent erythroid leukosis, and myeloid leukosis. Diseases etio application Ser. No. 523,679, filed Aug. 16, 1983, now 15 logically related to leukosis include sarcoma, neophrob abandoned, which was a continuation-in-part (CIP) of lastoma, endothelioma and osteopletrosis. Marek's dis U.S. patent application Ser. No. 255,678, filed Apr. 20, ease includes the neural form, visceral form and ocular 1981, now U.S. Pat. No. 4410,510, which was a con form. See The Merck Veterinary Manual, 3rd edition, tinuation-in-part of U.S. Ser. No. 128,919, filed Mar. 10, Merck & Co., Inc., New Jersey, pp. 1081-1091 (1976). 1980, now abandoned, which was a continuation of U.S. Marek's disease, for example, is seen in birds three patent application Ser. No. 957,206, filed Nov. 3, 1978 weeks of age onward to adults. The commonest is two now abandoned. to four months. Young chicks are more susceptible than TECHNICAL FIELD older birds. Genetic factors strongly influence the re sponse of birds to agents of the leukosis complex. See A. This invention relates to a method of preparing a 25 E. Churchill and P. M. Briggs, “Agent of Marek's Dis water-soluble, purified extraction residue fraction (anti ease in Culture,” Nature, London 215: 28–530 (1967), gen) of a microorganism, the purified residue fraction and J. J. Solomon, R. L. Witter, K. Nazerian and B. B. itself, and its use alone or in combination with an adju Burmester, “Studies on the Etiology of Marek's Dis vant for stimulating the immunological response in ease: Ch. I. Propagation of the Agent in Cell Culture,” warm-blooded animals against a variety of tumors. 30 Proc. Soc. Exp. Biol. Med., 127:173-177 (1968). Avian BACKGROUND ART leukosis is presumably caused by a group B herpes vi The purified extraction residue fraction (antigen) of rus. See J. J. Solomon, R. L. Witter, K. Nazerian and B. microorganisms described in detail hereafter has been B. Burmester, “Studies on the Etiology of Marek's Dis found to be useful in the treatment of avian leukosis as 35 ease: Ch. II. Finding of Herpes Virus in Cell Culture,” well as in the treatment of warm-blooded animals, in Proc. Soc. Exp. Biol. Med., 127:177-182 (1968). A live cluding man, by stimulation of their immune response but attenuated form of turkey herpes virus vaccine has against a variety of tumors. The microorganism from been effective until recently in preventing the incidence which the purified extraction residue fraction is ob of Marek's disease tumors; however, its effectiveness tained is a pleomorphic, refractile and filterable form of 40 has markedly declined. The mechanism of protection is bacteria which has been repeatedly isolated from both not clear. Birds vaccinated develop a viremia with the human and animal malignant tissue and blood of tumor vaccine. See Kermani-ARAB, V. T. Moll, B. R. Cho, bearing hosts. These bacteria have been described by W. D. Davis and Y-S. Lu, “Effect of Cyclophospha many investigators, but not many agree on their taxon mide on the Response of Chickens to a Virulent Strain omy because of their pleomorphism. Using standard 45 of Marek's Disease Virus,' Infection & Immunity, culture and fermentation techniques, they resemble 12:1058–1064 (1975). A group of investigators have just common saphophytes. Their various growth phases recently reported protection of chickens against Rous have been described as viruses, depetheroids, micro sarcoma virus with the use of methanol extracts of BCG cocci, bacilli, and fungi. See V. W-C. Livingston and E. challenged with Rous sarcoma. They also reported that Alexander-Jackson, "A Specific Type of Organism 50 chickens with palpable tumors, and then treated, devel Cultivated from Malignancy: Bacteriology and Pro oped necrosis of the tumor. See Y. Markson, F. Dol posed Classification, Ann. N. Y. Acad. Sci, jansky and D. W. Weiss, “Effects of Prophylactic 174:636-654 (1970); G. J. Dominque and J.U. Schlegel, Treatment with the Methanol Extraction Residue Frac "Novel Bacterial Structures in Human Blood: Cultural tion of Tubercle Bacili (MER) on the Development of Isolation,” Infection & Immunity, 15:621-627 (1977); 55 Rous Sarcomas of Chickens Following Challenge with and V. W-C. Livingston and A. M. Livingston, "Some the Rous Sarcoma Virus,” Immunological Parameters of Cultural, Immunological and Biochemical Properties of Host-Tumor Relationships, Vol. 5, D. W. Weiss (Ed.), Progenitor Cryptocides,” Trans. N. Y. Acad. Sci, Academic Press, New York, pp. 51-59 (1978). 36:569-582 (1974). Treatment of warm-blooded animals by subcutaneous In 1970, Virginia Livingston-Wheeler and Eleanor 60 injection of the purified extraction residue fraction is Alexander-Jackson proposed a new taxon for this or based on stimulation of the immune response of the host ganism within the order Actinomycetale. They named to the injected substance. Immunotherapy is a therapeu the organism "Progenitor cryptocides.' Progenitor crypto tic approach to the treatment of cancer which is based cides has been repeatedly isolated from human and ani on the concept that there are distinctive antigens in or mal malignant tumors and has many interesting features, 65 on most tumor cells that distinguish them from normal such as pleomorphism, intermittent acid-fastness, a host cells. Most tumor immunologists favor the view unique ability to pass through bacterial filters, and the that potentially malignant cells constantly arise in the ability to produce chorionic gonadotropin. See V. W-C. body; but because of their foreign nature, they are nor 5,382,428 3 4 mally eliminated by the body's immune system. On chickens an effective immunostimulating amount of a occasion, however, tumor cells escape this immune purified extraction residue fraction of the microorgan surveillance and continue to reproduce, resulting in ism Progenitor cryptocides and an adjuvant.