WO2002080972A1

WO2002080972A1 - Association of a tumor antigen and a system for delivering a cytokine or a chemokine for cancer immunotherapy purposes

Info

Publication number: WO2002080972A1
Application number: PCT/FR2002/001156
Authority: WO
Inventors: Marina Ostankovitch; Pascal Chaux; Joël Crouzet
Original assignee: Neurotech
Priority date: 2001-04-03
Filing date: 2002-04-03
Publication date: 2002-10-17
Also published as: FR2822707A1

Abstract

The invention concerns an association of two active substances in anti-tumoral immunotherapy to be administered to a patient. The invention is characterised in that the first substance is a tumor antigen or a mixture of tumor antigens and the second substance is a system for delivering an immunostimulatory cytokine or chemokine. The first active substance is advantageously a lysate of tumor cells and the second active substance consists in genetically modified cells expressing in stable manner a gene coding for an immunostimulatory cytokine or chemokine.

Description

COMBINATION OF A TUMOR ANTIGEN AND A SYSTEM FOR DELIVERY OF A CYTOKINE OR CHEMOKINE FOR THE PURPOSES OF CANCER IMMUNOTHERAPY.

The present invention relates to the field of cancer treatment and consists in administering to a cancer patient a combination of components which mutually potentiate their immunotherapeutic effect in vivo. More particularly, the invention relates to the combination of a delivery system of a cytokine or of a chemokine stimulating the immune system and of tumor antigens, delivered for example in the form of cell lysates.

Numerous experimental and clinical arguments have shown that cells of the immune system can control tumor development and growth. The capital role of T lymphocytes as effectors of the anti-tumor immune response is widely confirmed in many animal models, in which the regression and prevention of tumor lesions is most often dependent on this type of cell.

Various mechanisms can generate antigenic epitopes recognized by anti-tumor T lymphocytes. These epitopes can come from normal genes whose expression is limited to cancers and cells of the male germ line (MAGE, BAGE, GAGE, RAGE, NY-ESO-1). Some antigens are the products of genes coding for differentiation antigens (tyrosinase, gpl OO, MelanA, TRP-1 and TRP-2) or come from mutated genes in tumor cells (CDK4 mutation, ca tenin B, caspase 8, Ras, p53). Finally, certain antigens are coded by genes overexpressed in tumors (p53, Her- 2 / neu, PRAME) or are viral antigens (protein E7 of the HPV16 virus) (Curr Opin Immunol 1997 Oct; 9 (5): 684 -93). The induction of an anti-tumor immune response includes the presentation of antigens associated with tumors, the selection and activation of specific T lymphocytes, the migration of these cells to the tumor site and the recognition of tumor antigens leading to elimination. tumor cells.

An antigen, in itself, is not munogenic. It must be presented to T lymphocytes by professional cells presenting the antigen, such as macrophages, dendritic cells, B lymphocytes on the periphery, or such as astrocytes or microglia cells in the central nervous system. T lymphocytes recognize on these antigen-presenting cells peptides derived from endogenous or exogenous proteins, after a biological process called dressing (Immunol. Today 2000 Jul; 21 (7): 317-9) (Immunol. Res. 1999; 20 (3): 195-205). The antigens recognized by CD8 T lymphocytes are generally peptides of 8 to 10 amino acids associated with HLA class I molecules, while the antigens recognized by CD4 T lymphocytes are generally peptides of 14 to 22 amino acids presented by molecules HLA class II. Tumor progression often results from the absence of recognition of tumor antigens by the immune system, or from the absence of an appropriate immune response (Crit Rev Oncog. 2000; 11 (2): 97-133). A first approach in cancer immunotherapy is the identification of antigens associated with tumors recognized by cytotoxic T lymphocytes (CTL). Various techniques have been used to identify tumor peptides presented to CTLs. For each of them, it is important to prove that the CTLs obtained in vi tro and raised against these peptides recognize cells expressing the endogenous parental protein.

Cell death can be achieved by various processes leading to apoptosis or necrosis. The use of tumor material in the form of apoptotic bodies can generate, without restriction related to HLA molecules, the presentation of multiple antigens presented both by HLA class II and class I molecules by antigen presenting cells, to CD4 and CD8 T cells respectively (J. Exp. Med. 2000 Dec 4; 192 (11): 1535-44). Exogenous antigens can indeed access the cytosol and the presentation pathway via HLA class I molecules (Reimann J, Schirmbeck R., Immunol. Rev. 1999 Dec; 172: 131-52). CD4 T lymphocytes restricted by HLA class II molecules can exercise helper-like functions involved in the induction and maintenance of the cytotoxic response. In addition, they can have effects either directly against tumors expressing HLA class II molecules (Qi L, Rojas JM, Ostrand-Rosenberg S. J Immunol 2000 Nov 15; 165 (10) .5451-61), or indirect by activating other effector cells such as macrophages, eosinophils and NK cells. The lysis of tumor cells by necrosis allows their release of "Heat Shock Proteins" (HSP), soluble intracellular proteins, capable of interacting with the antigen presenting cells (Int. Immunol. 2000 Nov; 12 (11): 1539 -46). HSPs are chaperone proteins which bind antigenic peptides (J. Biol. Chem. 2000 Feb 25, -275 (8): 5472-7). They interact with the antigen presenting cells via receptors such as CD91 or CD14, resulting in the synthesis of pro-inflammatory cytokines such as TNF-α, IL-12, GM-CSF and ILl-β and the presentation peptides in association with the molecules of the major histocompatibility class I complex on the antigen presenting cells (Proc. Natl. Acad. Sci. US A. 1997 Nov 25; 94 (24): 13146-51) ( J. Immunol. 1999 Apr 1; 162 (7). 3757-60) (J Immunol. 2000 Jan 1; 164 (1): 13 -7) (J Immunol. 1999 Aug 1; 163 (3): 1398-408 ). Immunization of mice with HSP gp96 induces the maturation and migration of antigen-presenting cells within the lymph nodes. The HSP-peptide complexes isolated from murine cancers have been shown to induce protective immunity and the activation of anti-tumor T cells specific for the cancer from which the HSPs were isolated.

Tumor progression is often associated with a defect in cellular immunity and a decrease in the production of cytokines or chemokines involved in this immunity. The identification and cloning of genes coding for specific cytokines or chemokines has allowed an important advance concerning the understanding of anti-tumor immune responses. Modulation of immune responses by the use of recombinant cytokines or chemokines is one of the strategies used for anti-tumor therapy. In fact, the differentiation of naive T lymphocytes into effector cells is regulated by the cytokines to which these T cells are exposed at the time of antigen stimulation. Pre-clinical studies have shown that cytokines that stimulate immune responses mediated by type I helper T cells, when induced at the tumor site, cause an anti-tumor immune response.

In order to improve the strategies of separately employing a tumor cell lysate and an immunostimulatory cytokine or chemokine in anti-tumor immunotherapy, the Applicant has now developed a technology enabling the local and sustained delivery of an immunostimulatory cytokine or chemokine, in combination with a source of antigen represented by apoptotic and / or necrotic bodies originating from tumor cells.

Since cytokines exert their modulating effects paracrine to the antigenic site, this combination can be placed directly in the vicinity of the tumor or in a peripheral site suitable for the initiation of an immune response. This mode of delivery makes it possible on the one hand to obtain high concentrations of cytokine at the level of the implantation site while minimizing any systemic toxicity, on the other hand a great diversity and high levels of tumor antigens which can be directly treated by the antigen presenting cells present at the implantation site.

The subject of the invention is therefore very particularly a combination of two active components in anti-tumor immunotherapy to be administered to a patient, characterized in that the first component is a tumor antigen or a mixture of tumor antigens and the second component is a delivery system for an immunostimulatory cytokine or chemokine. The combination of active components according to the invention makes it possible to offer a method of immunotherapy for cancers which overcomes the fine determination of tumor antigenic determinants.

The combination according to the invention is remarkable in that it makes it possible to overcome the HLA restriction which encounters the use of synthetic antigenic tumor peptides presented by HLA class I and / or class II molecules. In addition, the present invention offers the advantage of overcoming the absence information on the immunogenicity of synthetic peptides in vivo.

The tumor antigens of the combination according to the invention can come from: - One or more tumor cell lines or primary tumor cells, rendered incapable of proliferation in vivo and representing a source of tumor antigens capable of activating a anti-tumor immune response. - Undefined tumor antigens, purified or not, originating from primary tumor cells or tumor cells originating from the autologous line or from allogenic lines, or from their mixture, rendered incapable of proliferating in vivo and lysed by a biological mechanism, physical, mechanical or other. Antigens can include, for example, cellular debris or purified cellular subfractions, such as hsp.

- Characterized tumor antigens which can be either purified or synthesized and used in the form of peptides and / or recombinant proteins.

According to a preferred embodiment of the invention, the tumor antigen is a lysate of tumor cells. Advantageously, said lysate of tumor cells is constituted by apoptotic bodies and / or by necrotic bodies.

This embodiment of the invention offers the advantage of using antigen presenting cells which are capable of priming the antigens contained in the lysates originating from tumor cells, and of presenting the peptides resulting from a process. physiological to the immune system.

The tumor cells are preferably either cells from one or more lines allogenic, or autologous tumor cells from a patient who will be administered the combination according to the invention.

According to a very preferred embodiment of the invention, the delivery system for an immunostimulatory cytokine or chemokine is a genetically modified cell expressing a gene coding for an immunostimulatory cytokine or chemokine. Advantageously, it is a xenogenic or allogenic cell.

The cells genetically modified to express a cytokine or a chemokine can be primary allogenic or xenogenic cells exhibiting either spontaneously or after immortalization, an extended proliferation capacity. These cells can also come from allogenic or xenogenic lines. These cells can be primary cells, chosen from epithelial cells of the retina, endothelial cells, kidney cells, smooth muscle cells, fibroblasts, cells derived from muscle, cells from the epidermis and dermis.

The immortalization of cells can be obtained by introduction using vectors:

- one or more cellular and viral oncogenes, including SV40T, E1A, E6, E7, v-myc, c-myc, src, ras;

- genes having an anti - senescence effect, such as telomerase;

- genes having an anti - apoptotic effect, such as bcl2.

The term “vector” is intended to mean any nucleotide sequence into which the desired sequence can be inserted by restriction ion and ligat ion and allowing the transfer into said target cells of said inserted sequences. These vectors are for example plasmids or viruses. The viral vectors may be human or non-human viruses, retroviral, adenoviral or associated with adenovirus, lentiviral, poxviral, or viruses derived from Herpes. According to a very preferred embodiment, the cells genetically modified to express a cytokine or a chemokine are encapsulated in microcapsules or macrocapsules of biodegradable polymers or not. These are microcapsules or macrocapsules, compatible with the survival and bioactivity of the encapsulated cells and having no toxicity or immunogenicity in the recipient. The composition of the capsules used according to the present invention allows a controlled and prolonged secretion of the active pharmaceutical agent by diffusion through the pores of said capsule. The pharmaceutical composition of the capsules used in the present invention is highly biocompatible.

The use of genetically modified encapsulated cells to express a cytokine or a chemokine according to the invention has many advantages. First, this encapsulation allows the secretion of a biologically active protein. Secondly, this approach makes it possible to obtain a sustained secretion over time of said cytokine or a chemokine. A phase 1 study was conducted in patients with amyotrophic lateral sclerosis, in which six patients were given capsules containing BHK cells genetically modified to produce CNTF. Nanogram levels of CNTF have been measured in patients' cerebrospinal fluids for at least seventeen weeks post-implantation (P. Aebischer et al., Nat Med 1996, 2 .696-699). Finally, this encapsulation offers protection of said cells against immune rejection by the recipient. This encapsulation also has the advantage of overcoming several limitations linked to the systemic administration of biologically active proteins, such as the need for repeated injections due to the low stability of certain proteins. The rate of release of a protein, biologically active and secreted by genetically modified cells and contained in capsules, is continuous over time, unlike a system based on the passive diffusion of a recombinant protein contained in a polymer. . In addition, the kinetics of diffusion of an encapsulated protein is faster than the secretion then diffusion observed with encapsulated cells producing the same protein and according to the cell encapsulation system described in the present invention.

Preferred examples of association according to the invention are the following:

A first formulation comprises: i) Tumor antigens in the form of cell lysates obtained for example by physical, chemical or biological processes, combined or not, inducing apoptosis and / or necrosis in tumor cells. The tumor cells or their derivatives used are either cells originating from one or more allogenic lines possibly characterized for the expression of certain tumor genes by RT-PCR, or autologous tumor cells. ii) Genetically modified xenogenic or allogenic cells stably expressing a gene coding for an immunostimulatory cytokine or chemokine. This paracrine delivery method has the advantage of being closely related to the natural biology of cytokines or chemokines. They may be interleukins known to activate T cells, such as IL-2, IL-4, IL-7, IL-12, IL-18, or chemokines having a direct or indirect effect on the antigen presenting cells, in particular on their migration and activation capacity, such as GM-CSF, M-CSF, MlP-lalpha, MlP-lbeta, MIP-5, MCP -3, MCP-4, RANTES, TECK, SDF-1 or MIP-3beta / ELC, 6Ckine / SLC.

A second formulation comprises: i) Tumor antigens in the form of cell lysates obtained by physical, chemical or biological methods, combined or not, inducing apoptosis and / or necrosis in tumor cells. The tumor cells or their derivatives used are either cells originating from one or more allogenic lines possibly characterized for the expression of certain tumor genes by RT-PCR, or autologous tumor cells. ii) Genetically modified xenogenic or allogeneic cells which stably express a gene coding for an immunostimulatory cytokine or chemokine, also known as “immunostimulatory cells”, protected by encapsulation, before their implantation in the patient, from mediated rejection by the the patient's immune system.

An adequate amount of the two components of the combination according to the invention can be administered to a cancer patient simultaneously. Administrations can be single or multiple in time and space. The administration can be carried out either directly in the vicinity of the tumor, or in a peripheral site suitable for the initiation of an anti-tumor immune response. In the case where the cells are encapsulated, the implantation of capsule (s) and of tumor antigens may be carried out sequentially in the vicinity of the tumor or in a site device suitable for initiating an anti-tumor immune response.

It can be an intramuscular, intracranial, subcutaneous, intra-dermal administration, or within a cavity, such as the peritoneal cavity, the renal capsule or the prostate.

Thus, in a first embodiment of the invention consisting in administering the two components jointly, the invention relates to a pharmaceutical composition comprising as active components a mixture of an effective amount of the two components defined above. In a second embodiment of the invention consisting in administering the two components sequentially, the invention relates to a pharmaceutical pack comprising two pharmaceutical compositions, each comprising an effective amount of one of the two components defined above.

More particularly, the invention relates to the use of a combination defined above for the preparation of a medicament intended for anti-tumor immunotherapy in a patient. This medicament can be in the form of a composition or a pharmaceutical pack above according to whether the two components are administered in mixture or separately. A first medicament according to the invention is intended to be administered directly to the tumor site or vicinity or at the level of a lymphoid organ suitable for the initiation of an immune response, for example at the level of the lymph nodes draining the tumor. A second medicament according to the invention is intended to be administered at the periphery of the tumor, for example by the subcutaneous route. A third medicament according to the invention is intended to be administered in the cavity housing the tumor, such as the peritoneal cavity in the case of colon cancers, the pleural cavity in the case of mesotheliomas or the cranial cavity after possible removal of the tumor.

A preferred example using this new approach is immunotherapy of gliomas.

Long considered an immuno-privileged site, the brain nevertheless has antigenic presentation mechanisms, distinct from those of the periphery. The presence of tumor specific antigens, the presence of HLA molecules on gliomas and the increase in their expression under appropriate conditions (Int J Cancer. 1988 Nov 15; 42 (5): 780-6) (J Neuroimmunol. 1991 Dec; 35 (1-3): 139-52) suggest that gliomas are sensitive to a cellular immune response. Lymphocytes infiltrate, sometimes in large numbers, most inflammatory diseases of the central nervous system. In particular, gliomas are infiltrated mainly by CD4 and CD8 T lymphocytes, while B lymphocytes and NK cells are more rare. These tumor infiltrating lymphocytes correspond to polyclonal expansions against antigens of gliomas not yet identified (Int Immunol. 1999 Aug; 11 (8): 1337-50). Although in some circumstances tumor cells are able to present their antigens directly to T lymphocytes, it is unlikely that the initiation of an anti-tumor immune response is possible without the intervention of cells specialized in antigen presentation, in the presence of 'tumor antigens. The presentation of antigens from cell lysates delivered in the brain could be ensured, among other things, by microglial cells. These cells, which make up 5 to 15% of the brain's cell population, have most of the characteristics of dendritic cells, such as adhesion and co-stimulation molecules. They are abundant in tumors and in inflammatory lesions. The strictly cerebral localization of gliomas and the usual absence of metastases would suggest that it is preferable to inject the immunogenic material directly at the tumor site. However, it is likely that this site is not favorable to the establishment of an anti-tumor immune response, due to locoregional immunosuppression due for example to TGF-β2, to prostaglandins

E2, to IL-10 or the IL-1 receptor antagonist secreted by tumor cells. Another combined injection site could be the periphery. It has been clearly established that activated T cells outside the brain can re-circulate and reach their tumor target inside the central nervous system.

Several preferred examples of implementation of the invention will be indicated below. These examples are given by way of illustration and should not limit the scope of the request.

Example 1 Encasulation of rat endothelial cells producing human IL-2.

The IL-2 producing cells were encapsulated in macrocapsules having a PES 14 membrane, a PET matrix network coated with laminin and type IV collagen. Three loading densities were used: 4xl0 ^s , 8xl0 ⁵ or 1.6xl0 ⁶ cells per capsule. Human IL-2 production was measured over time by ELISA (R&D).

The results obtained on day 1, day 3 and on day 7 after loading are shown in Figures 1, 2 and 3 respectively. Example 2; Encapsulation of rat endothelial cells producing human IL-2.

The production of IL-2 presented as an average by type of capsule and monitored over time is presented in Figure 4.

The capsules were loaded on day 0 with different quantities of CNT-121 cells which are rat endothelial cells producing human IL-2, then kept in culture.

The amount of IL-2 secreted into the culture medium was measured by ELISA on days 1, 3, 7, 10, 14 and 21 following the loading of the capsules.

Example 3; Release of IL-2 in vivo using encapsulated cells.

This example shows that capsules containing the NTC-121 cells release IL-2 in vivo. The NTC-121 cells were encapsulated as described in Examples 2, in 21 capsules. The capsules were then kept in 1 ml of Endo-SFM supplemented with 0.25 ng / of human bFGF, in an incubator at 37 ° C under 5% CO ₂ for 7 days. 18 capsules were implanted in the peritoneal cavity of BD-IX rats; the implantations of capsules were carried out simultaneously with an injection of 2 ml of PBS.

The IL-2 concentrations present in the intraperitoneal cavity were determined as follows. Peritoneal washes were performed with 5 ml of PBS. An ELISA test made it possible to determine the IL-2 concentration.

Figure 5 in appendix represents the concentration of IL-2 in the rat peritoneal cavity BD-IX implanted with devices loaded with NTC-121 cells. 18 devices were loaded with 4x10 ⁵ NTC-121 cells and were maintained for 7 days in vitro before implantation. The peritoneal cavities were washed with 5 ml of PBS on days 1, 2, 3, 7, 14 and 20. The measurement of IL-2 in the peritoneal cavity was measured by ELISA.

The following concentrations are observed from 1 day to 20 days post implantation: J = l 1000-2300 pg / ml

J = 2 1300-2700 pg / ml

J = 3700-3700 pg / ml

J = 7,300-1100 pg / ml

J = 8 0-300 pg / ml J = 20 100-4200 pg / ml

This shows that the devices can continuously release doses of IL-2 in vivo and can therefore be used for anti-tumor immunotherapy applications comprising a system for delivering one or more cytokines and tumor antigens.

Claims

1) A combination of two active components in anti-tumor immunotherapy to be administered to a patient, characterized in that the first component is a tumor antigen or a mixture of tumor antigens and the second a system for delivering a cytokine or an immunostimulatory chemokine.

2) A combination of two active components in anti-tumor immunotherapy according to claim

1, characterized in that the tumor antigen is a lysate of tumor cells.

3) A combination of two active components in anti-tumor immunotherapy according to claim

2, characterized in that the lysate of tumor cells consists of apoptotic bodies and / or by necrotic bodies.

4) An association of two active components in anti-tumor immunotherapy according to one of claims 2 or 3, characterized in that the tumor cell lysate is a lysate of one or more lines of tumor cells or cells primary tumors.

5) A combination of two active components in anti-tumor immunotherapy according to claim 1, characterized in that the tumor antigen (s) are antigens, purified or not, originating from the lysis of primary tumor cells or of tumor cells derived of the autologous line or of allogenic lines, or of their mixture. 6) A combination of two active components in anti-tumor immunotherapy according to claim 1, characterized in that the tumor antigen (s) are recombinant peptides or proteins or a mixture of these.

7) A combination of two active components in anti-tumor immunotherapy according to any one of the preceding claims, characterized in that the delivery system of an immunostimulatory cytokine or chemokine consists of genetically modified cells expressing a coding gene for an immunostimulatory cytokine or chemokine.

8) A combination of two active components in anti-tumor immunotherapy according to claim 7, characterized in that the genetically modified cells expressing a gene coding for an immunostimulatory cytokine or chemokine are xenogenic or allogenic cells.

9) A combination of two active components in anti-tumor immunotherapy according to one of claims 7 or 8, characterized in that the cells genetically modified to express a cytokine or a chemokine are primary cells or cells derived from lines allogenic or xenogenic.

10) A combination of two active components in anti-tumor immunotherapy according to claim

9, characterized in that the cells are chosen from the group comprising retinal epithelial cells, endothelial cells, kidney cells, smooth muscle cells, fibroblasts, cells derived from muscle, cells of the epidermis and dermis.

11) An association according to any one of claims 7 to 10, characterized in that the cells genetically modified to express a cytokine or a chemokine are encapsulated in microcapsules or macrocapsules.

12) An association according to any one of the preceding claims, characterized in that the cytokine is an interleukin activating T lymphocytes.

13) An association according to claim 12, characterized in that the interleukin is chosen from the group comprising: IL-2, IL-4, IL-7, IL-12, IL - 18.

14) An association according to any one of claims 1 to 11, characterized in that the chemokine is chosen from those having a direct or indirect effect on the cells presenting the antigen, in particular on their migration and activation capacity .

15) An association according to claim 14, characterized in that the chemokine is chosen from the group comprising: GM-CSF, M-CSF, MlP-lalpha, MlP-lbeta, MIP-5, MCP-3, MCP-4, RANTES, TECK, SDF-1 or MIP-3beta / ELC, 6Ckine / SLC.

16) A pharmaceutical composition for anti-tumor immunotherapy, characterized in that it comprises, as active components, a mixture of an effective amount of the two components defined in any one of claims 1 to 15. 17) A pharmaceutical pack for anti-tumor immunotherapy, characterized in that it comprises two pharmaceutical compositions, each comprising an effective amount of one of the two components defined in any one of claims 1 to 15.

18) Use of a combination according to any one of claims 1 to 15, for the preparation of a pharmaceutical composition or of a pharmaceutical pack intended for anti-tumor immunotherapy in a patient.

19) Use of a combination according to any one of claims 1 to 15, for the preparation of a medicament intended for anti-tumor immunotherapy in a patient administered directly to the tumor site or neighborhood.

20) Use of a combination according to any one of claims 1 to 15, for the preparation of a medicament intended for anti-tumor immunotherapy in a patient administered in the vicinity of lymphoid organs suitable for the initiation of an immune response, such as the lymph nodes draining the tumor.

21) Use of a combination according to any one of claims 1 to 15, for the preparation of a medicament intended for anti-tumor immunotherapy in a patient administered on the periphery of the tumor, for example subcutaneously . 22) Use of a combination according to any one of claims 1 to 15, for the preparation of a medicament intended for anti-tumor immunotherapy in a patient administered in the cavity sheltering the tumor, such as the peritoneal cavity, the pleural cavity, the cranial cavity, after possible excision of the tumor.

23) Use of a combination according to any one of claims 1 to 15, for the preparation of a medicament intended for the immunotherapy of gliomas.

24) Use according to claim 22, characterized in that the cytokine is chosen from the group comprising IL-2, IL-4, IL-7, IL-12, IL-18.

25) Use according to claim 22, characterized in that the chemokine is GM-CSF.