RU2192884C2 - Vaccine for antitumor immunity stimulation - Google Patents

Abstract

FIELD: medicine, oncology. SUBSTANCE: invention relates to vaccine used for stimulation of antitumor immunity. Invention involves vaccine composed of xenogenic tumor cells (murine carcinoma and melanoma cells) and xenogenic nontumor cells (porcine testicle cells) that are able to stimulate human antitumor immune responses efficiently. After penetration in body xenogenic cells are subjected for acute rejection mediated by natural antibodies and this process is accompanying with active implication of professional antigen-presenting (dendritic and macrophagial) cells in antitumor immune processes. EFFECT: enhanced antitumor immunity, valuable medicinal properties. 2 cl, 1 dwg, 1 tbl

Description

 The invention relates to medicine, namely to the treatment and prevention of cancer. To stimulate antitumor immunity, it is proposed to immunize the body with a vaccine made from xenogenic tumor and non-tumor cells expressing a wide range of tumor-associated antigens (OAS).

It is known that tumor cells carry immunogenic determinants on their surface. The fact of tumor immunogenicity implies the possibility of using immunotherapy both in the treatment and in the prevention of cancer. According to modern concepts [1-8], tumor-associated antigens (OAS) are divided as follows:
1. Products encoded by genes belonging to the families MAGE, BAGE, GAGE and RAGE. Normally, these genes are expressed only in organs that are barrier to the immune system (MAGE, BAGE, GAGE in the testes, RAGE in the retina).

 2. Differentiated antigens (tirosinase, Melan-A / Mart-1, gp100, gp75, p15 and some others), as well as heat shock proteins (gp96, hsp70). Despite the fact that these antigens are expressed on normal cells, they are able to induce in some cases effective antitumor immunity.

 3. Products of mutant genes (p53, Ras, beta-catenin).

 4. Oncoproteins expressed in excess on tumor cells (HER-2 / neu).

 5. Viral antigens. Epstein-Barra virus can induce immunoblastic lymphoma. Papilloma virus (HPV) 16 gene expression has been detected in some carcinoma cells.

 6. Oncofetal antigens (CEA, alfa-fetoprotein).

 7. Mucin and related hydrocarbon structures.

 8. Ganglisides.

 It is important that the majority (OAS) belong to the so-called common antigens that can be expressed on tumors of different histological types. Such antigenic similarity of different tumors, apparently, is a consequence of the involvement of similar intracellular mechanisms in the processes underlying the malignancy of different types of cells. An illustration of the above can be a table with published data [1] on the distribution of the expression of products of the MAGE, BAGE, GAGE and RAGE genes among tumors of different histogenesis.

 It is also important to note that the OAS in the vast majority are conservative molecules. The consequence of this is a high degree of homology between OAS in humans and animals, which, in turn, implies the possibility of inducing tumor-specific immune responses in the body through immunization with xenogenic OAS. In particular, it was shown that immunization of C57BL6 mice with human glycoproteins gp75 and gp100 can prevent the development of B 16 melanoma in their body, the cells of which express the corresponding murine analogues [9-12].

However, the presence of OAS on the tumor cells, in itself, in most cases, is not enough for the development of a stable effective antitumor immunity. According to published data [13-15], low tumor immunogenicity may be due to:
1) the lack of membrane expression of the products of the main histocompatibility complex (MHC), capable of complexing with OaAG;
2) the lack of membrane expression of co-stimulatory molecules (LFA3, CD40, CD54, CD80 / 86) necessary for the full-scale activation of T-lymphocytes that recognize OaAG in combination with MHC products;
3) deficiency in the tumor microenvironment of immunostimulating cytokines (primarily interferon (IF) -gamma and interleukin (IL) -2);
4) production of tumor cells of immunosuppressive cytokines such as, for example, transforming growth factor (TRF) -beta and IL-10;
5) the development of a tumor of immunosuppressive gangliosides.

 The purpose of specific immunotherapy is to overcome the barriers to the development of an effective antitumor immune process by increasing the number of sensitized T-lymphocytes in the body that can respond to OAS in a secondary manner. As is known, in comparison with native T cells, these lymphocytes have a sharply reduced threshold of sensitivity to an antigenic stimulus and in their functional activities are significantly less dependent on membrane and cytokine co-stimulation. An increase in the number of such lymphocytes in the body creates all the prerequisites for full-scale T-cell activation by relatively low immunogenic intact tumor cells [13, 16, 17].

 According to available data, vaccination with tumor-associated peptides, in principle, can lead to the formation of stable antitumor immunity [17]. The presentation of peptides to T-lymphocytes in this case is carried out by dendritic cells and macrophages that have a complete set of tools for triggering T-cell activation. It is important, however, to note that the development of an immune response to one or more tumor-associated determinants often does not slow down the development of the tumor process, but only gives selective growth benefits to those tumor cells that do not express these determinants [17]. Immunization of the body with autologous or allogeneic tumor cells in this regard seems more preferable, as it allows the induction of immune responses to a wide range of OAS. However, as already noted above, the immunogenicity of these cells in most cases is insufficient to induce effective antitumor immunity. Therefore, approaches aimed at the genetic modification of immunizing cells have become widespread in order to increase their immunogenicity (introducing genes encoding immunostimulatory cytokines or membrane co-stimulatory molecules into the cellular genome). These approaches, however, are difficult to implement in clinical practice, since the possibility of modifying tumor cells has not only technical, but also time limitations.

The aim of the invention is the development of a new vaccine that can effectively stimulate immune responses directed against a wide range of OAS, and ensure the formation of an effective long-term antitumor immunity. The developed vaccine includes three components, namely:
1) LLC mouse carcinoma cells (C57BL / 6, H-2 ^b );
2) cells of murine melanoma B16 (C57BL / 6, H-2 ^b );
3) pork testicle cells.

 The cells used in the vaccine were either irradiated (2000 R) or destroyed by a freeze-thaw procedure. Particular attention should be paid to the presence of non-tumor testicular cells in the vaccine. As noted above, these cells express the genes of the families MAGE, BAGE, GAGE, encoding a range of testicular antigens, widely represented on tumor cells of different histogenesis (see table). It was previously shown that the development of an immune response directed to these antigens can lead to the elimination of tumor cells from the body [18].

 Man and mouse (pig) are discordant species in relation to each other. This means that naturally occurring antibodies are naturally present in human serum, which can cause acute rejection of transplanted xenogenic cells. Most of these antibodies (approximately 1% of serum immunoglobulins) are recognized on xenogenic cells by the alpha-galactosyl epitope (human cells do not express this epitope due to the absence of the alpha-1,3-galactosyltransferase enzyme). Based on the available data [19-21], it can be assumed that the ingress of xenogenic cells into the human body includes the following immune mechanism.

 1. Serum anti-galactosyl antibodies and complement cover the surface of xenogenic cells and induce the process of their destruction.

 2. Through interactions with Fc receptors and receptors for complement components, antigenic material enters macrophages and dendritic cells.

3. Macrophages and dendritic cells present antigenic determinants in combination with MHC products of class II and I to T-helper cells (CD4 ⁺ ) and precursors of cytolytic T-lymphocytes (CD8 ⁺ ), respectively. The interaction of co-stimulatory molecules expressed on antigen-presenting cells with the corresponding T-cell membrane ligands, as well as immunostimulatory cytokines produced by these cells (IL-12, tumor necrosis factor (TNF) -alpha, IL-4, etc.) carry out effective co-stimulation of antigen-specific CD4 ⁺ T lymphocytes.

 4. T-helpers activated by OAS and xenoantigens, through the production of cytokines (IF-gamma, IL-2 and IL-15, etc.) stimulate the functional activity of cells with non-specific antitumor activity (macrophages, bone marrow cytostatic effectors, natural cytotoxic and killer cells). Activation of these cells leads to inhibition of tumor growth and is associated with increased expression of MHC products on tumor cells.

5. Tumor-specific CD8 ⁺ cytolytic T-lymphocytes, as well as their precursors, recognize complexes expressed on the surface of tumor cells consisting of OAG and class I MH products, and after contact and cytokine co-stimulation by activated T-helpers and professional antigen-presenting cells enter the phase of active growth.

 6. High activity of T-cell cytolytic clones directed against a wide range of OAS leads to the elimination of the tumor from the body.

As part of a clinical trial conducted at the Institute of Clinical Immunology SB RAMS (protocol 6 dated October 6, 1998), specific immune reactivity directed against melanoma OAS was studied in 28 patients with stage III and IV disease (melanoma - 12; colorectal cancer - 4 ; esophageal cancer - 1; breast cancer - 2; cancer of the uterus - 1; lung cancer - 3; ovarian cancer - 1; saliva cancer - 1; glioma - 3), who received inducing and consolidating courses of vaccine therapy. The initial level of functional activity of patients according to Karnovsky was at least 70%. They have not received any systemic treatment for at least 2 months from the start of immunotherapy. One vaccination dose included 5-8 • 10 ⁷ destroyed cells; the induction course consisted of 4-5 intradermal / subcutaneous immunizations performed at intervals of 7-14 days; subsequent consolidation course of 4-5 immunizations at intervals of 15-30 days. Studies have shown that, as a result of the treatment, T-cell reactivity to melanoma OAS (as measured by skin tests, as well as from HRT and in vitro lymphoblast transformation reactions) significantly increased in the vast majority of 24 (85%) of 28 patients studied. In this regard, it should be noted that, according to existing notions [1], increased T-cell sensitization to general OAS is a favorable prognostic sign for the cancer patient.

No serious systemic complications of vaccine therapy have been reported. Short-term local reactions in the form of redness of the injection site were noted in most patients. Within 24-48 hours after vaccination, 50% of patients had a moderate increase in temperature (up to 38 ^o C) and the development of a flu-like state. Toxic manifestations of the II-IV degrees were not recorded.

 The drawing shows the data on the 1.5-year survival rate of multiple vaccinated melanoma patients (15 people, 11 women and 4 men aged 24 to 69 years) with stage IV disease (9 with visceral and 14 with soft-tissue metastases) and control patients, not receiving any systemic treatment, or undergoing chemotherapy (dacarbazine). Control patients were comparable with experienced patients in the localization and prevalence of the tumor process, gender, age and time of observation.

 According to the data presented in figure 1, the number of surviving patients at the end of 1.5 years of observation in the experimental group was 9 people (60%). Similar indicators in the control groups did not exceed 14% (2 and 1 people). The differences between the experimental and control values were statistically significant (P = 0.033 according to the Gehan criterion).

The advantages of xenogenic cell vaccines over autologous or allogeneic cell vaccines are as follows:
1) it does not require the use of bacterial or cytokine adjuvants;
2) involves natural (pre-existing) immunity in the formation of antitumor cellular reactions
3) solves the problem of effective (immunogenic) presentation of OAS to T-lymphocytes.

Sources of information
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Claims (2)

 1. A vaccine for stimulating antitumor immunity, characterized in that it is represented by xenogenic (with respect to humans) cells capable of inducing immune responses directed against a wide range of tumor-associated antigens.  2. The vaccine according to claim 1, characterized in that it includes murine melanoma and carcinoma cells, as well as cells of the swine testicle.

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