Method for treatment of oncology diseases

FIELD: medicine, in particular treatment of malignant neoplasm.

SUBSTANCE: claimed method includes administration of vaccine representing complex of heat shock protein and tumor peptide, isolated from subject tumor tissue, and radioactive preparation Oncofer. Oncofer is administered both during vaccination course and after vaccination.

EFFECT: stable antitumor immunity, enhanced tumor cell apoptosis and blocked blood supply due to alteration of erythrocyte properties in tumor caused by radioactive preparation and activation of clot formation in said vessels.

2 cl, 4 ex, 2 dwg

 

The technical field to which the invention relates.

The invention relates to medicine and can be used for the treatment of malignant tumors by the combination of immunotherapy and radiotherapy techniques, in particular using autologous vaccines.

The level of technology

Immunological approaches to the regulation of tumor growth known for a long time. Attempts to use tumor antigens as the basis for vaccines was a success, but with great reservations. Unlike viral nature, which often T-cell antigens known, in the vast majority of tumors, the structure of tumor antigens is unknown. In addition, they often mutate and not always represented by tumor cells because the tumor may have weak immunogenicity and to have abnormalities in the mechanisms related to the expression of subunits major histocompatibility complex type I (MHC I), as one of the most important structural formations that are necessary for the presentation of antigen CD8+T-lymphocytes.

Modern immunotherapy adopted as bases antitumor protection heat shock proteins (HSP) in complex with tumor peptides (OP). These complexes are contained in large amounts in the tumor tissue of the patient to be removed during surgery. They can be the ü isolated from tumor and returned to the same patient for the induction of specific antitumor response (so-called autologous vaccination).

Immunostimulirutuyu activity of complexes of HSP-OP has a molecular-biological basis. HSP are polyfunctional objects cellular metabolism. On the one hand, they perform the function of chaperones, ensuring the correct folding of denaturirovannykh in the cellular stress proteins, and on the other, are nonspecific stimulators of the immune system, which is very important in a weakened body. In addition, HSP very seldom or never mutate in tumors: identity HSP sequences at the nucleotide level from normal and tumor tissues of the same origin is 100%. So the tumor is seeking to save the most important for its livelihood options based on the solution of the structural fate of other proteins. The founder of directions associated with the immunotherapy of tumors with autologous heat shock proteins, is .Srivastava. The method proposed by scientists, protected by many patents: US Patent 5837251, US Patent 5935576, US Patent 5750119, US Patent 5948646, US Patent 5961979, US Patent 5985270, US Patent 6475490, US Patent 6468540, US Patent 6461615, US Patent 6455503, US Patent 6447781, US Patent 6410028, US Patent 6403095, US Patent 6399070, US Patent 6322790, US Patent 6162436, US Patent 5830464.

Along with topical immunotherapy radiotherapy, which is an additional tool and is used in therapeutic and diagnostic purposes (see, the example EN 2053776, 10.02.1996). However, the application of only one of the isotopes are not able to protect the patient from relapse and to generate antitumor immunity: they can, in case of recurrence of the tumor, to be used again, which may exceed the permissible dose. In this regard, there is a real need for a combined approach, in combination immunostimulating and radiological components that can successfully complement each other in terms of enhancing apoptotic, antiproliferative activities.

Widely used at present, radiotherapy of tumors also has many shortcomings. First of all, under uncontrolled exposure beam fall and healthy cells, including cells of the immune system. Furthermore, in tumor cells that survive after such exposure is the induction of proliferation of the endothelium, which provides a molecular basis for neoangiogenesis. On a background of spent radiation, thus, creates the conditions for further tumor growth.

It is impossible not to take into account the fact that surgical removal of the tumor from which further highlighted the complexes of HSP-OP, strongly stimulates the growth and invasiveness of metastatic possibly existing in the body by the time of surgical intervention. Cells of the parent tumor former is rezerwat angiostatin - protein, "inhibiting the growth of new blood vessels at the periphery. With removal of the tumor, the source of angiostatin, the growth of the metastases become uncontrollable. In this regard, the use of a component that blocks the blood supply of metastases, is a priority on the background applied immunotherapy.

There is a method of treatment involving the administration to a patient of heat shock proteins, covalently linked with radioisotopes (US Patent 6455493). An alternative method of using HSP described in another patent, US Patent 6455493. According to the method, HSP, or its fragment, which is used in the complex (covalent, non-covalent, as a fused protein with toxins, and radioactive isotopes, i.e. apply HSP-immunotoxins. The authors proposed a method of reducing the number of immune cells through induction of their apoptosis induced cytotoxic T-lymphocytes. This biocidal activity is mediated due to the fact that the target cells have their own HSP, which are antigens. On the one hand, the classical immunological scenario, according to which endogenous HSP are proteosomal the path of degradation and present their antigens, against which produces an immune response. However, a disadvantage of this approach is the fact that HSP-immunotoxins, communicating with their recipe is Rami on the surface of antigen-presenting cells (APC), can cause apoptosis of APC, which weakens the body's ability to present antigens. It is obvious that for anticancer therapy, this approach is not applicable, and the use of toxins can be used in combination with other protein factors that do not have receptors on the surface of the APC.

A prototype of the proposed invention is adopted a method of treating cancer, involving the administration to a patient of a vaccine containing the composition of the anti-apoptotic heat shock proteins, consisting of HSP70, BTS, BTS (US Patent 6468540). According to the data, the range of concentrations required for immunization of the patient, is from 7.5 to 18.75 mg per person average weight of 75 kg At the same time, according to clinical trials, adequate antitumor immunity is achieved in 75% of patients. This suggests that the total final concentration of HSP is a factor that depends on the feedstock, which is not always, for obvious reasons, is sufficient. Therefore the injected concentration it is difficult to clearly define or standardize: the immune response is always personal, and at the same concentration of different patients react differently. The remaining 25% of patients have growth of metastases, which were immunotherapy failed to neutralize. By the way in case of insufficient concentration of HSP complexes required an additional factor that enhances the apoptosis of tumor cells. Considering the specifics of surgical stimulation of tumor metastases, a necessary factor blocking their blood supply.

The invention

The present invention is to develop an integrated method of treatment of tumors of various kinds, which provides, on the one hand, immunotherapy of the tumor, and on the other, the blockage of blood supply to tumor metastasis stimulated after removal of the parent tumor.

The problem is solved by the described method of treatment of malignant tumors, including the introduction of the vaccine, representing a complex of heat shock proteins-tumor peptide", isolated from the tumor tissue of the patient, in combination with the introduction of radioisotopes in effective amounts, as during the course of vaccination and after vaccination recommended by a doctor period.

The effectiveness of the proposed method of treatment is based on the following. Depending on the stress, which is experiencing the tumor (hypoxia, oxygen or glucose starvation, radiation, hyperthermia, and so on) increases the fraction of the inducible forms of anti-apoptotic HSP, and immunogenicity of the tumor may rise significantly. On the e surface of the formed complexes MNS OP", and in this moment it becomes available for attack CD8+ T - lymphocytes. In fact, traditional anticancer treatment is to increase the immunogenicity of tumor formation stressful conditions, the use of non-specific cytotoxic drugs and irradiation, which kills, including necessary in this situation, normal cells T-cell protection. The use of complexes of HSP-OP" as immunotherapy has advantages over traditional treatment. First, in this situation don't need to know the structure of the antigen, which forms a complex with HSP. Isolation and purification of the complexes is entirely dependent on HSP as to the media.

Secondly, immunization with autologous HSP stimulates cytotoxic and macrophage activity - the most valuable achievement of nonspecific antitumor protection, which is not in the traditional approach. Third, immunization complexes "HSP-OP" builds lasting antitumor immunity against the tumor, where the complexes were selected. Activated CD4+ and CD8+T-lymphocytes, NK-cells, maturation of dendritic cells.

As part of the combined anticancer treatment was selected "Uncover", - radioactive iron Fe59with the induced radioactivity of 0.8 MCI. The choice in favor of this drug is based is by following its properties. First, Fe59forms a complex with the protein transport system iron - transferrin. As part of this complex, due to transferring receptors, which are abundant in cancer cells by receptor-mediated endocytosis Fe59enters the tumor cell and selectively causing her to necrosis. Secondly, radioactive iron displaces normal iron of hemoglobin, which changes the properties of erythrocytes in the blood vessels that feed the tumor. When this is active stimulation of blood clots that disrupts the blood supply to the tumor and subsequent death. The use of this approach assumes a kind of therapeutic and diagnostic value.

Radiotherapy, in combination with vaccination, allows to achieve more effective results, especially in the case of reduced immunity in patients.

Information confirming the possibility of carrying out the invention

The following are specific examples of the activation of anti-tumour response, not limiting the scope of invention:

Example 1. Evaluation of protective immunity induced by complexes of HSP-OP, and cell necrosis caused by radioactive isotope conducted on animals. For immunization were taken mouse BALB/C Tumor cells taken from the animal carriers of tumour with carcinoma of the breast. From puhalovich cells was prepared vaccine enriched fractions of HSP. The first group of mice received the vaccine "Btsm", the second is a combination vaccine with the drug, "Uncover" (HSP+Fe59). Animals from the control group were injected with phosphate-saline buffer. Animals of experimental groups the vaccine was administered subcutaneously in a volume of 100-500 μl in the neck twice with one week interval -14 day -7 day), while in group (Btsm+Fe59) vaccination was carried out on the background of the introduction of the isotope. The drug is crushed and injected into mice with 15 ml of water for injection. The resulting mixture was administered orally to each mouse in a volume of 0.5 ml one week after the second vaccination all animals were injected with live tumor cells, within 1 month conducted monitoring of tumor growth. After decapitate estimated the size of the grafted tumors, texture, signs of inflammation, necrosis. Visual assessment showed that all mice in the control group, which received live tumor cells were observed growth and the rapid development of tumor tissue. In the group Btsh was observed weak signs of tumor formation, with a confident tendency to necrosis, and in the group Btsm+Fe59areas of tumor growth was practically absent. It shows a strong influence of the radiological component of the modulation of tumor growth and acceleration of apoptosis of tumor cells by enhancing the immunogenicity of tumor tissue in vivo.

p> Example 2. Evaluation of the cytotoxicity of the complexes of HSP-tumor peptide" in combination with "Oncopera" was carried out in vitro splenocytes, T-lymphocytes. Immunization of animals were conducted in accordance with the scheme described in the previous example. At 7-10 days mice were deceptional. 8 ml splenocytes were cultured with 40000 tumor cells treated with gamma radiation (10000 rad) in 3 ml of medium RRM containing 10% fetal bovine serum. After 6 days evaluated the cytotoxicity of T-lymphocytes (CTL). Differences in CTL activity in groups Btsm and Btsm+Fe59in comparison with the control were statistically significant (P<0.05), although differences between them were insignificant. These results suggest that background the use of Fe59does not affect the immune response (i.e. does not affect the activity of T-cells), and in combination with in vivo experiments, allows to make a conclusion about the need for more stimulation of apoptosis of tumor cells due to induced radioactivity.

Example 3. Conducted comparative evaluation of antitumor activity between autologous HSP-complexes, taken from tumorous and non-tumorous tissue, in combination with the drug, "Uncover". Participated in the experiment, 3 groups of animals C57BL/6 mice. On the background of Oncopera" the first group immunized with lysate of tumor cells of melanoma VR, the second group is isatom, enriched HSP from the liver, the third group received only "Uncover". Induction of anti-tumor activity was evaluated according to the size of the tumor (Figure 1). Differences in tumor size is set using the T-criterion Student (significance level P<0.05). It is shown that the antitumor activity have complexes "HSP-peptide in combination with a radioactive isotope, while the HSP complexes not from the tumor, even in combination with a radioactive isotope, did not possess sufficient antitumor activity.

Example 4. We also studied the effect of Oncopera" on the blockade of the blood supply of some types of tumor tissues in vivo. The drug was administered in accordance with the standard plans as a diagnostic tool for monitoring proliferative activity. It is shown that, regardless of the type and size of the tumor, was selective accumulation of Fe59in the vasculature, causing thrombosis and necrosis of tumor cells (Figure 2).

Thus, the advantage of the proposed method anticancer therapy is expressed as follows.

First, the use of only one vaccination causes, depending on the type of tumor, the stimulation of antitumor activity in 75% of patients. On the one hand, these figures are quite high, however, and they require a certain level actually is about, nonspecific immunity of the patient. In some cases the patient's condition does not allow to achieve the desired effect. Radiological component enables you to make an additional contribution to the selective death of tumor cells, disrupting the blood supply of tumor metastases, whose growth and invasive activity immediately stimulated after surgical removal of the parent tumor.

Secondly, to highlight the required number HSP-proteins for vaccination requires a certain amount of tumor tissue: according to clinical trials, the minimum number is 1-3 g of the tumor. It was not always possible to obtain the desired amount of tissue, which is a significant disadvantage of mono-immunotherapy: if you have a smaller amount of tissue is likely that the antitumor immunity is insufficient. Background radiotherapy can increase the antitumor activity by increasing the immunogenicity of the tumor and/or metastases when induced isotopes reactivity.

Thirdly, the use of Oncopera is also a diagnostic method that allows you to monitor the vaccination status of the patient, visualizing the accumulation of Fe59in the remaining tumor tissue.

The proposed method of treatment, representing particularly the successful combination immunotherapy, diagnostic and radiotherapy treatment of tumors and metastases, recommended for use in medicine, veterinary medicine and/or research purposes.

Brief description of drawings

Figure 1. Induction of antitumor activity of complexes of HSP-OP"taken their tumor tissue from non-neoplastic tissue, in combination with the "Oncopera" (HSP+Fe, HSP+Fe Control).

Figure 2. Historiarum uveal melanoma (a) and mammary gland tumors (). Both drugs are clearly visible areas of necrosis caused by Oncotherm", as well as the surviving parts of the tumor with inclusions of iron granules. The drug selectively accumulate in the tumor (dotted arrow) depending on its size, causes in the place of accumulation of thrombus formation in the microvasculature, which induces the disruption of the blood supply of the tumor with subsequent necrosis and fatty arrow).

1. The method of treatment of malignant tumors, including the introduction of the vaccine, representing complexes of heat shock protein - tumor peptide", isolated from the tumor tissue of the patient, and radioactive drug "Uncover".

2. The method according to claim 1, characterized in that the "Uncover" enter as during the course of vaccination and after vaccination.



 

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