RU2345788C2 - Laser vaccination method for patients suffering from metastatic cancer type - Google Patents

Abstract

FIELD: medicine; oncology.

SUBSTANCE: postoperative 4-6 skin areas of diameter 5-15 mm are exposed to laser radiation on copper vapour, once a week, within 10-15 procedures of treatment course, combined with area introduction of produced infiltration containing increased amount of Langerhans' cells derived from patient's biopsy material.

EFFECT: decrease in metastases.

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Description

Technical field

The invention relates to the field of biotechnology, clinical immunology and oncology.

State of the art

A known method of immunotherapy with bone marrow dendritic cells (DC) of patients with solid tumors, which consists in the intradermal administration after surgical removal of the tumor focus of bone marrow DC in a dose of 3 × 10 ⁶ cells / kg of patient’s body weight in 3.0 ml of official 0.9% - physiological saline for injection and 0.6 ml of a solution of human albumin into the tumor area and the region of regional lymph nodes in 12 points of 0.3 ml. Moreover, the first 3 vaccinations are carried out every 7th day, and the subsequent ones from 3 to 12 every 21st day [RF patent No. 2203683, A61K 39/395, A61P 35/00, stated 09/20/2001, publ. 05/10/2003].

The method allows you to activate a specific immune response to ongoing immunotherapy, however, its effectiveness is insufficient, in addition, the technology of obtaining antigen presenting cells (APC) from bone marrow precursors is expensive, technologically challenging and very time consuming.

Known methods of vaccinating patients using genetically modified vaccines obtained by introducing genes into autologous or allogeneic tumor cells that cause the synthesis of proteins that activate the antitumor immune response, and in particular heat shock proteins (HSP). In this case, HSP is isolated from the removed tumor and injected intradermally.

Known methods for vaccinating patients with vaccines based on DC and antigens from autologous tumor cells in disseminated tumors after radical surgery have low efficiency and so far are only an experimental treatment method [V.M. Moiseenko et al. Vaccine therapy for skin melanoma. - Practical Oncology, No. 4 (8), December, 2001, p. 58-64].

A known method of treating malignant tumors using photodynamic therapy in the treatment of cancer [RF patent No. 2196623, A61N / 067, the application. July 21, 2000, publ. January 20, 2003].

The method consists in heating a zone of a malignant tumor, introducing a photosensitizer and conducting a session of photodynamic therapy and thermotherapy. In this case, before the introduction of the photosensitizer or during its introduction in the area of the malignant tumor, a laser-induced thermotherapy session is performed. The area of the malignant tumor is heated to a temperature of 40-45 ° C, while the heating is carried out by laser radiation with a radiation spectrum in the near infrared region of the optical range other than the absorption spectrum of the photosensitizer. Then conduct combined sessions of photodynamic therapy and laser-induced therapy. Treatment in this way is repeated with an interval of 4-5 days, at least twice.

The method allows to increase the effectiveness of the treatment of malignant neoplasms while reducing its time. However, the lack of additional activation of antigen-presenting Langerhans cells of the skin reduces the effectiveness of the antitumor immune response.

A known method of immunotherapy and immunoprophylaxis of metastasis, which consists in the postoperative exposure of a patient to vaccination with an encapsulated antitumor antigenic material using a polyacrylamide capsule [RF patent No. 2236868, A61K 39/39, A61K 47/30, A61K 48/00, decl. 01/15/2003, publ. 09/27/2004]. Cancer cells and, in particular, xenogenic cancer cells can be used as antigenic material.

The disadvantage of the proposed solutions is the low efficiency of the antitumor immune response.

The closest in technical essence to the claimed solution is a method of laser vaccination of patients with metastatic forms of cancer, including as a postoperative exposure to the skin surface of the radiation of a CO ₂ laser using copper vapor and introducing into the zones of the formed infiltrate cultures of tumor cells obtained from the biopsy material of the patient [G .A.Baranov, S.B. Onikienko, A.V. Zemlyanoy, et al. In situ antitumor vaccines based on laser technologies. Report at the I All-Russian Conference on Cancer Biotherapy, M., 2002].

The disadvantage of this method is the lack of effectiveness of the antitumor immune response.

Technical result

The technical result of the invention is to create a new immunotherapy technology, in particular cancer biotherapy with vaccines with increased immunogenicity.

Disclosure of invention

To achieve the technical result indicated in the method of laser vaccination of patients with metastatic forms of cancer, including as a postoperative exposure to the skin surface of the radiation of a CO ₂ laser using copper vapor and introducing into the zones of the formed infiltrate the cultures of tumor cells obtained from the biopsy material of the patient, according to the proposal, exposure to radiation CO ₂ laser is produced on 4-6 sections of the skin of the back with a diameter of 5-15 mm, 1 time per week, 10-15 procedures are performed per treatment course to obtain cellular inf iltrate containing an increased number of Langerhans cells, followed by intradermal administration after 24-48 hours of tumor cell cultures.

The authors obtained therapeutic antitumor vaccines modified based on laser technology. The concept of the vaccine includes the pre-activation of antigen-presenting LA skin and the introduction of targeted antigens with increased immunogenicity in situ, obtained from the patient’s biopsy specimens (auto-vaccine), other patients with the same disease (hetero-vaccine), tumor cell cultures or embryonic tissues of birds and echinoderms (xenovaccine).

The immunogenicity of the target antigens was increased by modifying them with pulsed laser radiation of a CO ₂ laser with a wavelength (λ) of 10 μm and a radiation power (P) of 0.5-1.0 kW / cm ² , by exposing a droplet stream to the cell suspension, and LC activated by exposure to the skin surface of high-intensity (P> 3 W / cm ² ) radiation from a copper vapor laser (λ = 512 nm and λ = 578 nm) or infrared laser (λ = 830 nm). The introduction of modified target antigens in situ into the APC migration zones led to the activation of LA and a specific antitumor immune response.

The first laser vaccine therapy studies in the surgical treatment of patients with colorectal cancer, gastric cancer, pancreas and prostate cancer showed the promise of using laser technology to increase neoadjuvant cancer therapy.

However, subsequent studies received sufficient development for an application for an invention by 2005 and were published as abstracts at the I Russian-American Conference “Biotechnology and Oncology” [Abstract, ISTC, St. Petersburg, Russia, May 29-31, 2005, p.51 -52].

In the claimed solution to create a vaccine using antigenic material with increased immunogenicity obtained by modification with a CO ₂ laser beam or an electron beam of tumor cells (auto vaccine), antigens are introduced in situ into the patient’s back skin in the area of cellular infiltrate containing an increased amount of LC that migrated to zones of increase in the concentration of heat shock proteins (HSP) in the skin in vivo, the formation and secretion of which were caused by exposure to the surface of the skin with a copper vapor laser or an infrared laser.

The radiation wavelength of the CO ₂ laser is 10 μm, the radiation power is in the range of 0.5-1.0 kW / cm ² . In another embodiment, the modification of tumor cells is carried out by a wide-aperture linear electron accelerator, which operates in a pulsed mode, the radiation field size is 270 × 270 mm, the electron energy is 0.1-0.8 MeV, preferably 0.2 MeV, the absorbed dose is 2- 20 kGy, preferably 5 kGy.

The activation of in vivo HSP formation and the subsequent in situ activation of LCs is carried out by a pulsed-periodic copper vapor laser with simultaneous radiation at two wavelengths of 510.6 nm and 578 nm with a radiation power exceeding 3 W / cm ² and short pulses 10-12 ns with peak values of power up to 10-12 kW / cm ² and a pulse repetition rate of 5-15 kHz. This activation can also be carried out by infrared laser radiation with a wavelength of 830 nm and a power density of 1.5 to 15 W / cm ² , preferably 5 W / cm ² .

An antitumor vaccine based on skin LK, preactivated HSP and antigens obtained from a patient’s biopsy (autologous vaccine), or other patients with the same disease (heterovaccine), or tumor cell cultures, or embryonic tissues of birds or echinoderms (xenovaccine), whose immunogenicity modification increased CO ₂ laser or electron beam radiation is applied after removal of the primary tumor mass, antigens after their modifications are introduced intradermally into the formation zone of cellular infiltrates, containing LC yshennoe amount, which were obtained by subjecting the back portions of the patient's skin surface rays copper vapor laser or an infrared laser.

Laser vaccination of patients with metastatic forms of cancer is carried out after conditionally radical (cytoreductive) surgery by exposure to 4-6 sections of the patient’s back skin with a diameter of 5-15 mm using copper vapor laser beams to obtain a cell infiltrate containing an increased number of LCs, the procedures are carried out once a week, 10-15 procedures per treatment course, followed by 24-48 hours after laser irradiation of the skin by intradermal injection in the area of formation of a modified tumor cell infiltrate out cells.

The use of a laser auto vaccine with antigens from stage IV surgical material showed no signs of disease progression within one year in 62% of patients with colorectal cancer (10 out of 18), gastric cancer (3 out of 7), non-small cell lung cancer (10 out of 14), hypernephroma ( 4 out of 5). The use of a dendritic cell vaccine obtained ex vivo in patients of the comparison group showed the absence of signs of disease progression within one year in 71% of patients (17 out of 24 people).

However, the financial and time costs required to obtain the ex vivo vaccine exceed 5-7 times the amount of funds required for the implementation of therapeutic vaccination using in situ laser technologies. A 1.6-fold increase in average life expectancy was noted in the main group compared with the control group. This was accompanied by the development of a specific T-cell immune response (according to skin tests, inhibition of leukocyte migration (RTML), associated with the activation of the functional activity of the APC.

The rationale for obtaining a positive clinical effect is the pulsed effect of laser radiation on a drop of liquid that causes explosive boiling of its surface layer (0.5-1.0 μm) and the formation of shock (shear) waves in it — a light-hydraulic effect.

The shock wave causes the deployment of globular structures of proteins and biomembranes and the release of hydrophobic sites on their outer surface. This leads to a change in the antigenic composition, "xenogenization" of the cells. The redistribution of peptides of the outer surface of cells into immunogenic clusters promotes the presentation of target antigens to the body’s immune surveillance system using APC, stimulates the secretion of cytokines by lymphocytes, and develops an antitumor immune response.

Conformational changes are recorded as a result of interaction with the "active" cluster structures of water, which are formed from its macromolecule as a result of breaking of "hydrogen" bonds under the influence of a shock wave. Cluster structures of water interact with hydrophobic sections of cell membranes and proteins, which contributes to the emergence of their functionally active intermolecular structures. Stabilization of conformational rearrangements of macromolecules using the cluster structures of water molecules promotes the activation of “binding sites” and the interaction of antibodies and antigens, increasing their immunogenicity.

Another mechanism for laser modification of the immunogenicity of the antigens of the membranes of target cells is an increase in their surface due to the fusion of cells, their aggregation into "giant cells". “Molecular shaking” of the cell’s membrane structures when interacting with the shock wave field during laser irradiation helps to free them from screening blocking factors (antibodies) and expose the target antigenic repertoire of target cells, followed by the emergence of a population of cytotoxic T lymphocytes sensitized to target antigens. Blocking factors interfere with the recognition of target antigens by the immune surveillance system.

The experiments showed that activation of the formation of heat shock proteins in vivo, followed by in situ activation of skin LK, allows increasing the content of LK in the irradiation zone up to 10 times due to photodamage of skin cells and the release of HSPs, endogenous adjuvants and chemokines from them. And the introduction of antigens with increased immunogenicity in situ in the migration zones of LA to the region of cell infiltrate causes activation of LA and a specific antitumor immune response.

The radiation of a copper vapor laser activates the secretion of HSP, in particular HSP-70, the receptors for which are present on the surface of LCs. An increase in HSP causes their migration to the skin irradiation zone and activation of T-cell immunity. A copper vapor laser emits simultaneously in the green (510.6 nm) and yellow ranges (578 nm), the pulse-periodic nature of its action is the formation of short (10-20 ns) pulses with high peak values (10-12 kW / cm ² ) and a high frequency (5-15 kHz) makes it possible to selectively excite metabolic structures, for example, riboflavin, which is a two-photon acceptor of radiation from a copper vapor laser. The energy of electronic excitation of its molecule is transformed into the work of the tissue respiration chain with the formation of macroergic compounds for trophic support of cell functions.

During 1998 - 2005, laser vaccine therapy was carried out in 42 patients with metastatic forms of cancer after carrying out conditionally radical surgery. The main group included patients with colorectal cancer - 18 people, stomach cancer - 7 people, non-small cell lung cancer - 14 people, hypernephroma - 5 people. The comparison group consisted of 24 patients with metastatic colorectal cancer.

The treatment was carried out as follows:

1) Exposure to 5 areas of the skin surface of the back with a laser using a copper vapor laser in order to create a cell infiltrate containing an increased amount of LC, 1 time per week - 12 injections per course.

2) Intradermal injection into the cell infiltrate zone (5 regions) after 24 hours of a tumor cell lysate (gamma-ray inactivated cell culture (200 kGy)), modified with CO ₂ laser radiation, once a week, 12 injections per course.

3) As adjuvants used interleukin-2 at a dose of 250-500 thousand units; or a 0.05-1% solution of oligosaccharides obtained from galactose and / or manose containing polysaccharides by exposure to radiation from a CO ₂ laser with an energy of more than 1.0 kW / cm ² or an electron beam in a dose of more than 100 kGy; or a suspension of perfluorodecalin ("Perftoran") 0.3-1.0 ml in each zone of formation of cellular infiltrate, 2 times a week, 15-30 injections per treatment course.

In the comparison group, vaccination was carried out after the in vitro (by electroporation) introduction of the target antigens into DCs obtained from autologous blood monocytes by ex vivo incubation in the presence of granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). DCs were cultured for 5 days from monocytes attached to plastic, isolated from 80-100 ml of autologous blood in RPMI-1640 medium supplemented with 10% serum IV gr.

For induction of DC maturation, GM-CSF, 3000 U / ml, (Leukomax) and IL-4, 500 U / ml were added to the medium. On day 5, the target AH was introduced into DC using electroporation. True DCs have a characteristic morphology; their markers are CD80, CD86, and CD83. The number of DCs required for a single injection is 5 × 10 ⁶ .

Treatment of patients of the comparison group was carried out as follows.

A DC-based vaccine was administered intradermally once a week, for a total of 12 injections. Adjuvants were used similarly to their use in the main group. The treatment was performed in 24 patients with a vaccine based on DC and autoantigens from tumor cell lysate. To determine the effectiveness of the immune response, we used the results of RTML with tumor antigens, investigated the cytotoxic activity of natural killers, the functional activity of monocytes, an “oxygen explosion” in peripheral blood neutrophils, and performed scintigraphy with labeled lymphocytes (Kasi LP, Lamki LM, Saranti S. et al. - Indium-111 labeled leukocytes in evaluation of active specific immunotherapy responses- Int. J. Gynecol. Cancer. 1995; vol. 5, No. 3, p. 226-232).

The use of autologous laser-modified tumor antigens in RTML makes it possible to detect lymphocytes sensitized to tumor antigens and determine the effectiveness of vaccine therapy. Modified autologous antigens had the greatest antigenicity. Adjuvants enhanced the specific immune response, which developed 3-5 weeks after initiation of vaccine therapy in situ. The key role in determining the effectiveness of immunotherapy was played by metabolic and functional indicators of the activity of APC and lymphocytes - the activity of succinate dehydrogenase (LDH), lactate dehydrogenase (LDH), glutathione reductase (GR), 6-phosphogluconate dehydrogenase (6-FGDH), tetrazom Nitrosim test, lysotrosin cationic test (LKT), secretory activity of monocytes.

A persistent decrease in cytotoxicity and the number of natural killers, functional and metabolic parameters of APC and lymphocytes, secretory activity of monocytes, a significant decrease in the activity of GR, 6-FGDG indicate a lack of effectiveness of the therapy and an increase in secondary immunosuppression.

The data obtained indicate the promise of using in situ therapeutic vaccines based on laser technologies and the use of electron beams in the immunoprophylaxis of cancer relapse after conditionally radical (cytoreductive) surgical treatment, as well as the fact that cancer immunotherapy can only be effective when combined with surgical removal of the bulk of the tumor. In patients with common forms of oncopathology, this allows in some cases to increase life expectancy by 1.6-3 times and improve its quality, inhibit the progression of the disease and the occurrence of relapse of tumors.

Clinical example. Patient T., born in 1945 In September 2003, cancer of the left kidney with metastatic foci in the lungs was diagnosed.

Computed tomography (November, 2003) is a neoplasm of the left kidney, the size of the formation is 10.8 × 7.9 × 15 cm. The lymph nodes of the paraaortic group are determined at the level of the portal of the kidneys with a diameter of 6-8 mm; 15 mm, lymph nodes of the bronchopulmonary group on the left with a diameter of up to 15 mm and tracheobronchial group with a diameter of 12 mm are detected.

In December 2003, an operation was performed - left-sided nephrectomy. Histology - clear cell kidney cancer.

From the biopsy material, a culture of tumor cells was obtained, which were treated with an electron beam of 0.2 MeV energy, with an absorbed dose of 2 kGy, the tumor cell suspension (3 million / ml) was exposed to a CO ₂ laser with a power of 1 kW / cm ² , exposure time 5 ms .

5 areas of the back skin with a diameter of 10 mm were irradiated with a laser with active media on copper vapor with simultaneous radiation at two wavelengths of 510.6 nm and 578.0 nm with a radiation power of 3.5 W / cm ² , pulses of 10 ns duration with peak power values up to 12 kW / cm ² and a pulse frequency of 10 kHz.

24 hours after irradiation, a suspension of tumor cells inactivated by an electron beam and modified by CO ₂ laser radiation was injected intracutaneously into the cell infiltrate in the irradiation zone in 1 ml of physiological saline in an amount of 3 million cells. Laser vaccination procedures were carried out once a week, namely 12 procedures per treatment course.

Criteria for vaccination effectiveness are the formation of a delayed-type cellular hypersensitivity reaction (similar to the Mantoux reaction).

In patient T., the formation of a targeted immune response developed after 4 procedures - at the fifth week of vaccination. Laboratory criteria for vaccination effectiveness is the formation of a positive inhibition of leukocyte migration (RTML) with an antigen from an auto vaccine. Positive RTML with a modified tumor antigen was recorded in patient T. 6 weeks after the start of the course of vaccine therapy. Within 3.5 years, the patient showed no signs of relapse and disease progression. There is no lymphadenopathy in the lungs.

During control CT of the lungs, one metastasis with a diameter of up to 1.5 cm was detected (in 2003 there were more than 5 metastases). In a laboratory study 11/21/2006: an increase in the content of activated T-lymphocytes (CD25), circulating T-helpers (CD4), cytotoxic T-cells (CD8), natural killer cells (CD 16), the functional activity of lymphocytes and neutrophils characterizes the presence of an active immune response .

Claims (1)

A method of laser vaccination of patients with metastatic forms of cancer, including as a postoperative exposure to the surface of the skin of copper laser radiation and introducing into the zones of the resulting infiltrate the cultures of tumor cells obtained from the biopsy material of the patient modified by the radiation of a CO ₂ laser, characterized in that the exposure a copper vapor laser is produced on 4-6 skin areas with a diameter of 5-15 mm, 1 time per week, by conducting 10-15 procedures per treatment course to obtain cellular infiltrate, containing an increased number of Langersans cells, followed by intradermal administration after 24-48 hours of culture of tumor cells inactivated by gamma rays, modified by the radiation of a CO ₂ laser.