The method of combination immunotherapy of malignant brain tumors

 

(57) Abstract:

The invention relates to medicine, namely to Oncology, and can be used for the treatment of malignant brain tumors. Prompted to enter LAC-cells in the tumor bed only after surgical removal of tumor of the substrate, combined with the introduction of lymphocytes stimulated with tumor antigen in the presence of IL-2, and then conduct a course of subcutaneous injections of autologous immunostimulatory factors, obtained by culturing lymphocytes of a patient with tumor antigen in the presence of IL-2. The method allows to increase the efficiency of treatment of patients with malignant brain tumors. 3 Il.

The invention relates to medicine, namely to methods of immunotherapy using autologous lymphocytes and produced by these factors in the treatment of patients with malignant brain tumors.

There are two approaches to immunotherapy of malignant tumor - antigen-specific and nonspecific immunotherapy.

In the first approach, the known method (PCT/US97/18718, WO 98/16238, a 61 K 35/14, 1997), according to which the immunotherapy of cancer is carried out with the method of antigen specific immunotherapy of malignant tumors using an intravenous infusion of autologous lymphocytes, activated in vitro culture of tumor cells and a mixture of native cytokines resulting from prior stimulation of lymphocytes (1, 2). In this way intravenous autologous lymphocytes is carried out in mode 1 times per month for 6 months. However, due to the presence of the blood-brain barrier intravenous autologous lymphocytes may not be effective against malignant tumors "barrier tissues, particularly intracerebral localization.

There is also known a method of antigen specific immunotherapy of malignant neoplasms in the postoperative period, based on the use of autologous immunostimulatory factors, obtained by stimulation of lymphocytes with tumor antigen. In this way the course immunostimulating therapy was fractional subcutaneous received autologous factors (3, 4). This method was tested in the treatment of patients with tumors of the maxillofacial area, gastrointestinal tract, and melanoma. However, the effectiveness of this approach in the treatment of malignant brain tumors has not been studied.

It is important to note that a significant increase PR as a rule, in patients with initial stages of cancer development, provided a radical surgical resection of the tumor. At the same time malignant brain tumors are diagnosed, usually in the late stages (III-IV stage). In addition, due to the lack of capsule problematic is the radical removal of the tumour substrate. Therefore, high efficiency isolated using antigen-specific immunotherapy in the treatment of these patients is questionable.

Under the second approach is the most widely currently used cytokines in the treatment of tumors of different localization (EP 832653 A1, 96250206, a 61 K 38/20, 1996), including brain tumors (PCT/US97/18455, WO 98/16246, a 61 K 39/00, 1997). Also known is a method of treatment involving intravenous administration of autologous lymphocytes activated by interleukin-2 (IL-2), - the so-called lymphokine-activated killer cells (LAK-therapy) (5, 6). The disadvantage LACQUER therapy is the limited scope of use (efficiency at 25% clinical response is detected only when melanoma and kidney cancer), the toxicity of high doses of IL-2 and high cost of the method.

To reduce toxicity and Polychaeta Loco-regional introduction of LAC-cells in combination with IL-2 directly into the tumor (7). This approach is characterized by good clinical tolerability and efficacy in 33% of cases. However, the survival rate for 18 months. was not different from that obtained with traditional treatment.

The objective of the invention is to increase the efficiency of treatment of malignant brain tumors.

The problem is solved in the invention by the fact that in the proposed method of Loco-regional non-specific immunotherapy autologous IL-2-activated lymphocytes in combination with IL-2 is carried out only after surgical removal of tumor of the substrate and is combined with the antigen-specific immunotherapy, namely with the introduction of lymphocytes stimulated with tumor antigen in the presence of IL-2, in the bed of the removed tumor, and thereafter immunotherapy autologous immunostimulating factors obtained by culturing lymphocytes of a patient with tumor antigen in the presence of IL-2.

The treatment is preceded by a step for obtaining suspensions of tumor cells from a slice of the removed tumor. The obtained cell suspension is used as a source of tumor antigens, inactivated and preserved until use. Immunotherapy begins roznoy the patient's blood, add 60 ml of Gelatines and incubated for 45 min at 37oC. the Washed RBC mass return to the patient on the day of geoexpat. Collected lykousis collected in a separate vial, centrifuged at 1000 rpm for 20 min and remove adosados. Cells lykousis once washed phosphate-buffered saline (SFR), layered on a density gradient ficoll-urografin (1,078) and centrifuged for 20 min at 3000 rpm/min Collected from the interphase mononuclear cells (MNCs) twice washed SFR and resuspended in culture medium RPMI-1640, supplemented with 5% serum donors AB (IV) group, 5 mm HEPES-buffer and gentamicin at a dose of 80 U/ml of Cells adjusted to a concentration 1,5106/ml and cultured for 48 h in two modes: 1) with recombinant IL-2 at a dose of 100 IU/ml and 2) with recombinant IL-2 in the same dose and tumor antigen in the optimum ratio. The treatment by the proposed method consists of three procedures for obtaining the IL-2-activated cells and three procedures for obtaining cells stimulated with tumor antigen in the presence of IL-2. The obtained cells are introduced through four stereotactic puncture in the bed of the removed tumor in combination with IL-2 in an optimal dose. Sevastopolya factors obtained by culturing lymphocytes of a patient with tumor antigen in the presence of IL-2, in the form of a course of 10-15 subcutaneous injection.

Example 1.

Patient N. , 43 years old, IB 2055, was in the clinic of neurosurgery with a diagnosis of Intracerebral malignant tumor of the right parieto-occipital region (polymorphonucleocytes glioblastoma).

Was admitted to the hospital 22.09.98 with complaints of a headache mainly in the frontal and occipital areas, reduced vision, insecurity when walking, podtashnivaet, hiccups. The history of the disease: the disease progredient course. Considers himself ill since may 1998, when it began to be troubled with headaches, soon noticed limiting the field of view.

In neurological status: tendon reflexes D>S, coordinatorsee sample performs with ataxia rougher on the right, in the Romberg staggers in both directions. Continuous hiccups. Assessment on a scale Karnofsky - 60 points. Radiographically: review kronograf in 2 projections is determined by the moderate strengthening of figure diplococci vessels, marked osteoporosis of the back of the Turkish saddle. The ophthalmologist: visus OD/OS=1,0/0,8. Tubular narrowing of visual fields in both eyes. The fundus of the eye: expressed stagnant oluseyi on the background of the expressed cerebral changes involving midline structures. Echoes: offset M-echo from right to left in the rear and middle sections 13 mm in anterior sections 7 mm. Signs of intracranial hypertension. MRI with contrast enhancement "OmniScan": intracerebral tumor with cystic inclusions in the right occipital lobe with marked mass effect, causing compression of the right lateral ventricle and the shift of median structures.

28.09.98 operation: osteoplastic craniotomy in the right parieto-occipital region, total removal of intracerebral tumors.

After receiving the results of histological studies patients received a course of combination immunotherapy on the proposed method. 02.10.98, 04.10.98, 09.10.98 and 11.10.98 by stereotactic puncture under aseptic conditions in the bed of the removed tumor introduced autologous activated lymphocytes in the total dose 1,4109in combination with interleukin-2. Then spent 15 subcutaneous injection of autologous immunostimulatory factors.

The postoperative period was uneventful, healing by primary intention. The patient was discharged in satisfactory condition with a rating on a scale Karnofsky score of 90.

After 17 months. after surgery sostoyaniye to work (works watchmaker). Score on the Karnofsky scale is 100 points.

Cerebral changes according to MRI in the dynamics of treatment is shown in Fig. 1 and 2.

Example 2.

Patient D. , aged 32, IB 96, was in the clinic of neurosurgery with a diagnosis of Intracerebral malignant tumor of the right parieto-occipital region (polymorphonucleocytes glioblastoma). Spasms with rare generalized seizures.

Was admitted to the hospital 14.01.99 complaining of constant diffuse headache, shakiness, lack of confidence when walking, short-term (few seconds) episodes of loss of consciousness. Medical history: In September and December 1998, after exhaustion and alcohol abuse was mentioned briefly (30 seconds) episodes of loss of consciousness. Since the beginning of January 1999 headaches have become permanent.

In the neurological status of focal pathology it is not revealed. Assessment on a scale Karnofsky 80 points. Radiographically: review kronograf in 2 projections is determined by the moderate strengthening of figure diplococci vessels. The shape and size of the Turkish saddle usual. The ophthalmologist: visus OD/OS= 0,7/0,9. The fundus of the eye: the disks of optic nerves pale pink, clear boundaries. Artery in normal veins on the of rector. Echoes: offset M-the echo is not defined. MRI with contrast enhancement "OmniScan": the brain tumor in the parietal-occipital region on the right, consisting of a solid minor and mainly of cystic components.

26.01.99 operation performed: bone-plastic decompressive craniotomy in the right parieto-occipital region, total removal of intracerebral tumors.

After receiving the results of histological studies patients received a course of combination immunotherapy on the proposed method. 17.02.99, 19.02.99, 24.02.99 and 26.02.99 by stereotactic puncture under aseptic conditions in the bed of the removed tumor introduced autologous activated lymphocytes in the total dose 3,43109in combination with interleukin-2. Then spent 10 subcutaneous injections of autologous immunostimulatory factors.

The postoperative period was uneventful, the wound healed by first intention. The patient was discharged in satisfactory condition with a rating on a scale Karnofsky score of 90.

After 13 months. after surgery the patient was in good condition. An epileptic seizure is not resumed. Headaches do not bother. In the area of bone defect webwhen what about the data NMRI in the dynamics of treatment is shown in Fig. 3.

According to the literature about 90% of patients with malignant glioblastomas brain die within 10-11 months. after surgical treatment, combined with radiotherapy (8). The inclusion in the comprehensive treatment of patients with malignant brain tumors, the method of combination immunotherapy on the proposed method reduced the mortality in the first year after surgery to 28%. Of the 7 patients died 2 people. The remaining 5 patients currently alive and under observation. In 2 of the surviving patients, the duration of recurrence-free period exceeded 12 months. (13 and 17 months). It should be noted that in the group of patients in the treatment which used the method of combination immunotherapy, the quality of life in the postoperative period was higher score on the Karnofsky scale was 77 points, whereas in patients treated by traditional schemes, only 61 points. After 6 months. since the operation level of quality of life in patients receiving combination therapy, 75.5 points, whereas in patients treated by traditional schemes, the score on the Karnofsky scale was reduced to 45 points.

Thus, using golovnogo brain.

Literature

1. Krane, R. J., Carpinito G. A., Ross, S. D., Lavin, P. T., M. E. Osband Treatment of metastatic renal cell carcinoma with autolymphocyte therapy. Low toxicity outpatient approach to adoptive immunotherapy without the use of in vivo interleukin-2.//Of Urology. - 1990. - Vol. 35. - 5. - P. 417-422.

2. Osband M. E. , Ross, S. D. Problems in the investigational study and clinical use of cancer immunotherapy.//Immunol. Today. - 1990. - Vol. 11. - 6. - R. 193-195.

3. Vlasov, A. A., Novikov, C. I., content Century. And., Sidorovich, I. Postoperative immunotherapy of patients with malignant neoplasms. //Russian honey. log. - 1996. - 6. - S. 17-19.

4. Novikov, C. I., Vlasov, A. A., Content C. I., N. Ryazanov.To. Immunotherapy of malignant tumors of the maxillofacial area of human autologous drugs in the postoperative period.//Immunology. - 1995. - 2. - S. 54-56.

5. Rosenberg S. A., Lotze, M. T., L. M. Muul et al. A progress report on the treatment of 157 patients with advanced cancer using lymphokine activated killer cells and interleukin-2 or high dose interleukin-2 alone.//N. Engl. J. Med. - 1987. - Vol. 316. - 5. - P. 889-897.

6. Rosenberg S. A. Immunotherapy of cancer using interleukin-2: current status and future prospects.//Immunol. Today. - 1988. - Vol. 9. - 1. - P. 58-62.

7. Boiardi, A., Silvani A., Ruffini, P. A., et al. Loco-regional immunotherapy with recombinant interleukin-2 and adherent lymphokine-activated killer-cells (A-LAK) in recurrent glioblastoma patients.//Cancer Immunology Immunotherapy. - 1994. - Vol. 39. - 3. - R. 193-197.

8. Korshunov, A. G., Golan Century A., Sycheva, R. C., Proshin I. N. Prognostic value is rgii. - 1999. - 1. - N-3-7.

The method of combination immunotherapy of malignant brain tumors, which consists in obtaining from the patients blood mononuclear cells, culturing them with interleukin-2 and their subsequent Loco-regional introduction in combination with IL-2, characterized in that the introduction of LAK cells in the tumor bed is carried out only after surgical removal of tumor of the substrate, combined with the introduction of lymphocytes stimulated with tumor antigen in the presence of IL-2, followed by a course of subcutaneous injections of autologous immunostimulatory factors, obtained by culturing lymphocytes of a patient with tumor antigen in the presence of IL-2.

 

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The invention relates to medicine

FIELD: medicine, phthisiology.

SUBSTANCE: method involves administration of roncoleukin by lymphotropic route: subcutaneously, in region of pretracheal cellular tissue in the equal dose 1/4-1/5 of the average daily therapeutic dose, once in one or two days, 3 injections for a course. Invention provides the local delivery of roncoleukin to the injure zone directly that promotes to making depot of the latter in the injure focus and to the rapid elimination of pathogen from body of patients. Invention can be used for correction of immune insufficiency in patients with pulmonary tuberculosis.

EFFECT: improved method for correction.

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