Drug – bio-affecting and body treating compositions – Radionuclide or intended radionuclide containing; adjuvant... – Molecular bilayer structure
Reexamination Certificate
1999-03-15
2004-02-03
Paras, Peter (Department: 1632)
Drug, bio-affecting and body treating compositions
Radionuclide or intended radionuclide containing; adjuvant...
Molecular bilayer structure
C424S184100, C435S070100, C435S377000
Reexamination Certificate
active
06685911
ABSTRACT:
The object of the invention is a novel process for sensitizing antigen-presenting cells, novel means for the implementation of the process and novel membrane vesicles having an immunogenic potency.
Since the demonstration of the existence of CD8+ cytotoxic T lymphocytes specific for tumor antigens presented in the context of class I molecules (Rosenberg et al., 1996; Boon, 1992), several laboratories have been able to show that anti-tumor immuno-therapy is an efficacious therapeutic strategy in animal models (Pardoll, 1995). The principle of immunotherapy is to induce an effective immune response against specific tumor antigens. Up to the present, it has been possible to carry this out in different ways. First, tumor cells expressing recombinant co-stimulatory molecules and/or immunomodulatory cytokines are capable of stimulating anti-tumor responses capable of eradicating solid tumors in vivo (Zitvogel et al., 1996 {a}). Similarly, peptide derivatives of tumor antigens (or exogenous antigens expressed in tumor cells) injected in different chemical forms including the use of liposomes or viruses (adenovirus or poxvirus, for example) as vectors are capable of causing tumors to regress. Finally, professional antigen-presenting cells, such as dendritic cells sensitized with peptides derived from tumor antigens, reinjected in vivo induce potent anti-tumor responses as well as the regression of solid tumors established in mice (Mayordomo et al., 1995).
Immunotherapy based on the use of dendritic cells has been able to show its efficacy in the studies conducted in the mouse. As a result, this therapy has recently been transposed to the clinic. In the Unites States, trials are presently underway to demonstrate that dendritic cells loaded with tumor peptides significantly increase the frequency of specific cytotoxic T lymphocytes (CTL).
A first limitation to this approach is the sensitization of the dendritic cells with peptides derived from tumor antigens. In fact, in the majority of tumors specific antigens have not been identified. Antigens specific to the tumors are only known in cases of tumors induced by viruses (carcinoma of the uterine cervix), in cases of melanoma (self antigens, mutated antigens, differentiation antigens) or in a small percentage of breast tumors (oncogenes or products of tumor suppressor genes having undergone mutations). However, the direct implication of these peptides or tumor antigens in the elimination of the tumors in man remains to be demonstrated. Novel sensitization methods of the antigen-presenting cells such as dendritic cells thus prove to be necessary. The object of these methods is to induce specific anti-tumor responses in the context of class I and class II molecules of the MHC.
Most of the sensitization methods of dendritic cells at present use peptides corresponding to epitopes presented in combination with the class I molecules and identified in tumor cells by means of CTL clones specific for the tumor. However, these methods are probably not optimal since they do not take into account epitopes recognized in the context of the class II molecules which are critical for the proliferation of the helper T lymphocytes necessary for obtaining optimal cytotoxic responses. Furthermore, epitopes presented by the tumor cells and those presented by antigen-presenting cells (as for example the dendritic cells) are probably not the same. Finally, tumor peptides recognized by the CTL are only available in the case of a small percentage of patients having molecules of the appropriate class I haplotype.
The ideal sensitization method, one which would be applicable to any tumor with a minimal risk of immunoselection, must not be limited to a small number of identified tumor antigens. Similarly, such a method ought to make use of intact protein antigens rather than peptides in order to enable the dendritic cell to prepare them and to present the adequate combination of peptides in combination with the class I and class II molecules, and do so for any individual.
Recently, Gilboa and collaborators (Boczkowsky et al., 1996) have been able to show that messenger RNAs prepared from tumors biopsies loaded in the dendritic cells may have an in vivo anti-tumor effect. However, the RNAs are very unstable and the quantity of potentially interesting RNA compared with the total RNA is probably very low. Zitvogel et al. (Zitvogel et al., 1996 {b}) have shown that tumor peptides prepared from an acidic tumor eluate (acidic peptide eluate: APE) may be used to load dendritic cells. These cells thus loaded, once injected, have the capacity to cause tumors to regress. However, in the case of tumors which do not express tumors of class I (which represent the majority of metastatic human tumors) or in the case of tumors which may not be dissociated in a cellular suspension, the approach using the acidic eluates is not very efficacious and not reproducible.
A second limitation to immunotherapy based on the use of dendritic cells is linked to the phenotypic changes which may occur when these cells are maintained in culture or subjected to different treatments. This may in fact lead to cell populations which are not very homogeneous and inadequately characterized for therapeutic use.
Hence there exists a real need to improve the methods for sensitizing antigen-presenting cells in order to enhance the efficacy of these approaches and to broaden their applications as well as to develop novel means for the vectorization of antigens and other molecules.
The present invention provides solutions to these questions. The object of the present invention is in fact to provide novel methods for sensitizing antigen-presenting cells, in particular dendritic cell, as well as for the identification, isolation and characterization of the novel membrane vesicles having remarkable immunogenic properties.
One of the features of the invention is more particularly to provide a novel reproducible process for sensitizing antigen-presenting cells by tumor antigens.
Another feature of the invention is to provide a novel reproducible process for sensitizing antigen-presenting cells by tumor antigens, in which it is not necessary that the tumor antigens are known.
Another feature of the invention is to provide the means which make it possible to set up a library of tumor antigens.
Another feature of the invention resides in lipid membrane vesicles produced by the tumor cells or by the dendritic cells and endowed with immunogenic properties as well as their use for the production of antigen libraries, the sensitization of antigen-presenting cells or the vectorization of antigens, in particular in the context of immunotherapeutic approaches.
In this respect a first object of the invention relates to a vesicle derived from tumor cells having the following characteristics:
it is freed from its natural environment,
it comprises a lipid bilayer (designated by “surface”) which surrounds a cytosolic fraction,
and optionally,
it exhibits on its surface molecules of class I of the major histocompatibility complex (MHC) and/or of class II of the major histocompatibility complex (MHC), optionally loaded with antigenic peptides and/or adhesion molecules and/or lymphocytic costimulatory molecules, and/or,
it contains in its cytosolic fraction tumor antigen molecules and/or immunomodulators and/or chemo-attractors and/or hormones and/or nudeic acids.
The secretion of the vesicles by cells is a phenomenon described in the prior art (reticulocytes, B lymphocytes, macrophages). These vesicles are usually designated by the generic term “exosome” which reflects their mechanism of production by exocytosis of internal vesicles. However, the physiological role of these vesicles has not been really established. Furthermore, the structural characteristics, properties and functions of these vesicles vary depending on the cell type from which they are derived.
Unexpectedly, the inventors have now demonstrated that tumor cells are capable of secreting vesicles exhibiting particularly interesting immunogeni
Amigorena Sebastian
Raposo Graça
Regnault Armelle
Zitvogel Laurence
Institut National de la Sante et de la Recherche Medicale
Paras Peter
Preston Gates Ellis & Rouvelas Meeds LLP
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