MXPA96004401A - Immunoterapeutico agent and its - Google Patents

Abstract

The present invention relates to the use of an antigenic and / or immunoregulatory material, derived from vaccum mycobacteria, to retard or prevent the growth or spread of a tumor.

Description

IMMUNOTERAPEUTIC AGENT AND ITS USE FIELD OF THE INVENTION The present invention relates to immunomodulatory agents useful in retarding or preventing the growth of tumors.

BACKGROUND OF THE INVENTION It has been suggested that the immune system is capable of preventing or slowing the growth of tumors, at least in some cases, / some studies have tended to support this point of view. In this way Coley (WB 1894"Treatment of Inoperable Malignant Tumors, with toxins of erysipelas and the prodigious bacillus") described an immunotropic method that appeared to be effective against a restricted range of tumors of mesodermal origin, inducing a necrotizing mechanism similar to induced by Koch's immunotherapy, against tuberculosis. Since that time, most of the attempts in immunotherapy for cancer have had the purpose of inducing rapid tissue necrosis, REF: 23247 similar to that achieved in some cases by Coley. It is known that there are at least 2 maturation patterns of the activating T cells, called TH1 and TH2. The foregoing is associated with the production of interleukin 2 (IL2), interferon gamma (IFN-?), Cytotoxic T cells (CTL), reduced production of antibodies and a down regulation of TH2 cell production. The latter produce IL4, IL5, increase the production of antibodies and regulate the decrease in production of TH1 cells. It is believed that the Koch phenomenon, probably induced by the Coley toxin, is the result of a mixture of mature TH1 + TH2 cells. It has been found that there are two categories of immune reactivity, which can be exploited for the control of tumors, and both can be evoked by the Bacillus Calmet t e-Guerin n (BCG) vaccine. One of these mechanisms is tissue destroyer, as exemplified by Koch and Coley immunotherapy and tumor immunotherapy, with TNFa, and probably involves tissue damage, mediated by cytokines, in inflammatory sites, adequately prepared by the phenomenon of Koch, dependent on T cells. This mechanism seems to be effective against tumors of mesodermal origin. The second mechanism, based on the immune recognition of the tumor cells themselves, could be effective against tumors of any origin. Indirect evidence of the existence of the non-specific, anti-tumor mechanism based on TH1 cells arises from the success of interleukin 12, recombination, a powerful inducer of TH1 cells, in experimental cancers. It is believed that mycobactia, if used in such a way as to avoid induction of the Koch phenomenon, may be capable of primary recognition of cells derived from themselves, subject to fatigue, via heat shock protein epitopes (HSP, by its acronym in English) and epitopes of other proteins with highly conserved sequences, and thus lead to the selective destruction of tumor cells. It is concluded that the variable efficacy of BCG immunotherapy was due, in the past, to a failure to understand that BCG tends to increase the response pattern already loaded or prepared, which has arisen from studies of its effectiveness in the protection against tuberculosis and leprosy. BCG as a vaccine against a mycobacterial disease (both tuberculosis and leprosy) is very variable, giving protection, in different studies, from 80% to 0%. It is therefore significant that BCG delivered at birth appears, in general, to protect against childhood leukemia, only in those geographic locations where it protects against tuberculosis (Grange JM, Stanford JL, BCG vaccination and cancer). Tubercle. (1990); 7_1_: 61-64). That is, it protects against both conditions in those places where it evokes a response to mycobacterial antigens, which is not tissue necrosing, and which is not the Koch phenomenon. Many tumor cells can present on their surfaces, carbohydrate antigens, bacteriomimetics, and protein epitopes subjected to fatigue and other highly conserved proteins, in relation to the complex of primary compatibility (MHC, class 1 and class 2). In the production of antibodies, antibodies to carbohydrates, originally raised against bacterial sugars, and perhaps of the IgA type, could cover tumor cells and block their surface components from cellular immune attack. As described in the International application PC T / GB 93/00463, immunotherapy with M. vaccae induces a predominantly TH1 response pattern, with the activation of macrophages by IFN-α and a small production of antibodies. It is known that TH1 responses drive the generation of CTL cells. The marked reduction in the production of antibodies, combined with increased numbers of CTL, could cause the tumor cells to become exposed to attack, both by activated macrophages after they have been linked to the bacterial sugars on the surface of the cells. the tumor cells, as well as fatigued CTL-recognizing proteins and other highly conserved protein epitopes, presented by the MHC class 1. It is believed that the necrotic mechanism in the tissues, via the Koch phenomenon, is suppressed to a high degree, the use of a preparation derived from M. vaccae.

It can then be expected that immunotherapy with M. vaccae is effective against tumors of mesodermal, endodermal and ectodermal origin, including breast and bronchial tumors, preventing or delaying the growth or spread of these tumors. Accordingly, the present invention provides the use of antigenic and / or immunoregulatory materials derived from Mycobacteria vaccae for the manufacture of a medicament useful in retarding or preventing the growth and spread of tumors. That material can be administered to a subject, in a sufficient amount, to at least retard or prevent the growth or spread of a tumor.

DESCRIPTION OF THE INVENTION The therapeutic agent of the invention comprises, conveniently and therefore preferentially, dead cells of M. vaccae, and most preferably, cells that have been killed by autoclaving. The infectious agent usually comprises more than 108 microorganisms per ml of diluent, and preferably from 10β to 1111 killed or killed M. vaccae microorganisms by my diluent. The invention includes within its material, antigenic and / or immunoregulatory scope, of M. vaccae, for use in therapy, to retard or prevent the growth or spread of a tumor. The diluent can be pyrogen-free saline solution, for injection only, or a borate buffer of pH 8.0. The diluent must be sterile. A suitable borate buffer is: N a 2 B 07. 10 H20 3.63 g H 3 B O 3 5.25 g N a C l 6.19 g Twe e n 0.0005% Distilled water up to 1 liter The strain of M. vaccae is one denoted by R877R, isolated from mud samples from the Lango district of Central Uganda (J.L. Stanford and R.C. Paul, Ann. Soc. Belge Med, Trop. 1973, 5_3_, 141-389). The strain is a rough, stable variant, and can be identified as belonging to M. vaccae, by biochemical and antigenic criteria (R. Bonic e, S. E. Juhasz., Zentr albl Bakteriol, Parasitenkd, Infection skr. Hgy. Abt. 1, Orig., 1964, 192, 133). The strain denoted by R877R has been deposited in the National Type Culture Collection (NCTC) Central Public Health Laboratory, Colindale Avenue, London NW9 5a., United Kingdom, on February 13, 1984, under the NCTC No. 11659. For the preparation of the immunoreactive agent, the M. vaccae microorganism can be cultured or grown on a suitable solid medium. A modified sauton liquid medium (S. V. Boyden and E. Sorkin., J. Immunol, 1955, 7_5, 15) solidified with agar is preferred. Preferably the solid medium contains 1.3% agar. The medium inoculated with the microorganisms is incubated aerobically to allow the growth of the microorganisms to take place, generally at 32 ° C for 10 days. The organisms are collected, then weighed and suspended in a diluent. The diluent may be a non-buffered saline but is preferably buffered with borates and contains a surfactant such as Tween 80, as described above. The suspension is diluted to give 100 mg of micorga no smo / mi. For further dilution, saline buffered with borates is preferably used, so that the suspension contains 10 mg by wet weight, of microorganisms / ml of diluent. Then the suspension can be poured into small bottles of 5 ml, multiple doses. Although the microorganisms, in the flasks, can be exterminated using irradiation, for example of Cobalt at a dose of 2.5 megarads, or through other means, for example chemically, it is preferred to exterminate the microorganisms by the use of autoclave, for example 0.7 kg / cm2 (10 psi) for 10 minutes (115-125 ° C). It has been found that using the autoclave produces a more effective preparation than by irradiation. The immunotherapeutic agent is generally administered by injection, in a volume that is in the range of 0.1 to 0.2 ml, preferably 0.1 ml, supplied in t thermally. A single dosage will generally contain from 107 to 1010 M. vaccae organisms, killed or exterminated. It is preferred to administer to patients a single dose containing 10'10: M. vaccae exterminated or killed. However, the dose can be repeated depending on the patient's condition. Although the i nmunot agent was generally administered by i n t -radio-injection, other routes, such as for example oral administration, can also be used. The invention includes within its scope, a method for retarding or preventing the growth or spread of a tumor, which comprises administering to a subject suffering from a tumor, antigenic and / or immunoregulatory material, derived from the Mycobacteria vaccae. , in an amount sufficient to elicit an effective immune response to retard or prevent the growth or spread of a tumor. It may be advantageous, and it is within the scope of the invention, to use more than one strain of M. vaccae and / or to include in the therapeutic agent other antigens of my cobac terium. You can also include tuberculin. The therapeutic agent may also contain BCG (Bacillus Calmete-Guerin) vaccine, in particular the freeze-dried form, of the vaccine, to promote its effect. The therapeutic agent may also contain additional ingredients such as adjuvants, preservatives, stabilizers, etc. It can be supplied in the form of injectable, sterile liquid, or in the sterile, freeze-dried form, which is reconstituted before use. M. vaccae can be used as such or as an extract or fractional portion of the organism, to prepare therapeutic agents according to the invention. The invention is further illustrated by the following examples.

Example 1 M. vaccae is grown or grown on a solid medium comprising modified Sauton's medium, solidified with 1.3% agar. The medium is inoculated with the microorganisms and incubated for 10 days at 32 ° C to allow the growth of the microorganism to take place. Then the microorganisms are collected and weighed and suspended in diluent, to give 100 mg of microorganisms / ml of diluent. The suspension is then further diluted with buffered saline to give a suspension containing 10 mg by wet weight of microorganism / ml of diluent and poured into or placed in 5 ml vials for multiple doses. The vials containing live microorganisms are then autoclaved for 10 minutes at 0.7 kg / cm 2 (10 psi) to kill or kill the microorganisms and produce the immunotherapeutic agent of the invention, which can be diluted (if desired). you want) in additional form for your use. This agent can not be administered by intradermal injection, in the manner already described.

E j e plo 2 Injections of M. vaccae were administered to several patients with tumors. Some of the results obtained are reported as follows: Breast carcinoma 1) At the National Institute of Hygiene and Epidemiology, in Hanoi, Vietnam, where a number of studies are in progress, using vaccinal mycobacteria, as immunotherapy, a team member developed breast carcinoma with axillary secondaries. After the removal or extraction by operation, the patient was recommended to have BCG immunotherapy. After observing these treatment regimens in other patients, the patient did not want to accept BCG treatment, and requested the treatment of M. vaccae. She was given a normal injection of 109 M. vaccae, exterminated or killed, and for at least 3 years, the patient has been well and without evidence of additional tumor. 2) After a few weeks of receiving an injection of M. vaccae, for the treatment of persistent pityriasis, it was found, by routine mammography, that a 50-year-old woman had a lump in her breast or breast. The biopsy showed that the tumor was malignant, and was removed by extraction with surgery. The hipathopathy showed a stenosis of the breast, marked, around the axillary lymph nodes that were free of tumor. Approximately 9 months later, pityriasis returned and a second dose of M. vaccae was given, almost with immediate relief of pityriasis. There were no secondary signs, 21 months after tumor removal. 3) A woman, a little over 70 years old, had a breast tumor removed or removed several years ago. He had a history of back pain, due to arthritis, for several years and received an injection of M. vaccae in an attempt to alleviate this. A short time later, the X-rays of the spine or back showed a number of lesions considered secondary deposits of tumor, and he was referred to radiotherapy. After a delay of 8 weeks, he presented for radiotherapy and on a repeated radiograph it was found that he no longer had signs of secondary deposits; Radiotherapy stopped. Two and a half years later the woman is well without recurrence of tumors. 4) A general practitioner, 55 years of age, presented with a 2-day history that was thought to be an acute chest abscess. The incision showed that the mass was a very rapidly growing carcinoma, with secondary deposits already present in the axilla. Immediate removal was performed, and immediately after leaving the hospital an injection of M. vaccae was given. Two additional injections followed at 3-month intervals. The wound of the operation healed well and after a year there is no evidence of secondary deposits in a patient in whom the rapid progression of the disease was feared.

) A 50-year-old nurse, who had complained of hip pain for 1 year, was diagnosed as having secondary bone, from a small breast tumor. After an initial injection of M. vaccae, he made good progress, endured chemotherapy very well and was given a second injection of M. vaccae. Everything is going well with the patient.

Lung Tumors 1) A 55-year-old woman developed shortness of breath in October 1993, and it was found to be due to several inoperable tumors in the lung. It was thought that these were secondary deposits of an uncertain primary tumor. With the consent of the oncologist, an injection of M. vaccae was given and a chemotherapy and radiotherapy procedure was initiated. The patient endured this remarkably with few side effects and did not want to be given a second injection of M. vaccae when this was suggested after 6 months. Ten months after the presentation, her illness worsened, the chemotherapy was changed, but the patient was given a poor or pessimistic prognosis. At the beginning of October 1994, she was given a second injection of M. vaccae, followed by which the condition of the patient improved and she was given two additional injections, one in December 1994, and the other one. March 1995. This patient, not only progressed much better than expected, but endured 20 chemotherapy procedures without inconvenience.

Other Malignant Formations 1) A 30-year-old dentist, with terminal illness due to a malignant teratoma, was given an injection of M. vaccae just before he had been sent home to die. The patient showed signs of improvement in 1 or 2 days, and 3 weeks later showed a reduction in the size of the visible tumors and returned to eating for the first time after a few weeks. 2) A 36-year-old woman developed adenocarcinoma of the stomach, around an ulcer resulting from infection with helicobactor pylori. Despite the chemotherapy, the patient had a large number of secondary disseminations. The patient received a first injection of M. vaccae and is intended to give continuous injections at intervals of 2 months Tumors associated with HIV 1) A patient in the initial phase of AIDS, with multiple Kaposi sarcomas, received an injection of M. vaccae in November 1993. The patient has not developed more tumors, and his existing tumors show signs of resolution. In December 1994 he was given a second injection of M. vaccae, after he developed secondary infections related to HIV. The patient will receive repeated injections of M. vaccae every 2 months. 2) One patient, almost without CD4 * cells, but without obvious signs of AIDS, received an injection of M. vaccae in June 1994, and a few weeks later he was found to have a small KS lesion on one thigh, approximately 0.5 cm in diameter. Additional injections of M. vaccae were given at 2-month intervals and in March 1995 the patient showed no additional lesions and the initial size of his lesion did not increase. 3) A - a 48-year-old man with recurrent, multiple oropharyngeal carcinomas was given an injection of M. vaccae in June 1994 and given additional injections at 2-month intervals. By March of 1995 there was no recurrence of tumors. The anecdotes discussed above provide support for the treatment of tumors with M. vaccae. The use of M. vaccae has not been associated with any adverse side effects, and on the contrary it may have helped to reduce the toxic effects of chemotherapy in at least two of the patients. These incipient practical observations support the theoretical expectations and provide a first indication of the valuable antitumor activity.

E j us 3 A preliminary experiment was carried out using Balb / c mice challenged with MM3 strain of mouse mammary carcinoma. Three groups of 10 mice were challenged, with 107 MM3 cells. One week later, a first group was injected with * 107 M. vaccae, a second group - it received 109 M. vaccae and a third group acted as control. The animals were sacrificed 40 days after the challenge and the development of tumors was examined.

Group 1 Group 2 Group 3 107 M. 109 Controls vaccae M. vaccae Developed Tumors p = 0.056 No tumors 5 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, the content of the following is claimed as property:

Claims (13)

1. The use of an antigenic and / or immunoregulatory material, derived from Mycobacteria vaccae, for the manufacture of a drug useful in retarding or preventing the growth or spread of tumors.

2. The use according to claim 1, wherein the antigenic and / or immunoregulatory material, derived from M. vaccae, comprises dead cells of M. vaccae.

3. The use according to claim 2, wherein the M. vaccae cells have been killed by the use of an autoclave.

4. The use according to any of the preceding claims, wherein the material derived from M. vaccae is derived from the strain that is deposited in the National Collection of Type Crops (NCTC) of the Central Public Health Laboratory, Colindale Avenue, London NW9 5th., United Kingdom, on February 13, 1984, under number NCTC 11659.

5. The use according to any of the preceding indications, wherein the material derived from M. vaccae is contained in a medicament comprising from 107 to 10 microorganisms per dose.

6. Use in accordance with any of the above indications, wherein the tumor is a bronchial tumor or a breast breast tumor.

7. A method for retarding or preventing the growth or spread of a tumor characterized in that it comprises administering to a subject, antigen and / or immunoregulatory material, derived from mycobacterium vaccae, in an amount sufficient to at least retard or prevent the growth or spread of the tumor.

8. A method according to claim 7, characterized in that the tumor is a breast or breast tumor.

9. A method according to claim 7 or 8, characterized in that the material derived from M. vaccae is as defined in any of claims 2 to 5.

10. The products that comprise the material derived from the Mycobacteria vaccae, characterized in that they are used to delay or prevent the growth or spread of a tumor.

The products according to claim 10, characterized in that the material derived from M. vaccae is such as that defined in any of the claims from i.a. 2 to 5.

12. A specific immune agent, characterized in that it comprises an antigenic and / or immunoregulatory material, derived from Mycobacteria vaccae, for use in therapy to delay or prevent the growth or spread of a tumor.

13. An agent according to claim 12, characterized in that the material derived from M. vaccae is as defined in any of claims 2 to 5.