Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues – Blood proteins or globulins – e.g. – proteoglycans – platelet...
Reexamination Certificate
1997-11-17
2002-08-13
Scheiner, Laurie (Department: 1648)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Blood proteins or globulins, e.g., proteoglycans, platelet...
C530S387100, C530S387200, C530S388150, C530S387700, C436S548000
Reexamination Certificate
active
06433148
ABSTRACT:
FIELD OF THE INVENTION
The present invention is related to the field of Immunology and in particular to the selection and use of antidiotypic monoclonal antibodies (AB2) IgG type with the main characteristic of being highly connected to the idiotipic network and recognition of B and T human lymphocytes, which are major participants in the immune response.
Accordingly the objective of this invention is to provide antidiotypic monoclonal antibodies IgG type connected to the immune network, able to interact with T and B lymphocytes and able to exert an immunoregulatory effect.
DESCRIPTION OF THE PRIOR ART
The immune response is mainly based on the antigen stimulation of some determined B lymphocyte clones bearing in the surface specific receptors for this antigen. T cells have the ability of producing positive and negative signals as a way of regulation of this process. However the immune system regulation mechanisms are very complex as was postulated by Jerne (Jerne N K (1974) Ann Immunol. Inst. Pasteur 125, C:373:389): the immune system can be “seen” as an organized structure of antibody molecules and interconnected lymphocytes by idiotipic directed interactions.
Idiotypes in antibodies are defined as epitopes in the variable regions in such a way each antibody presents particular idiotipical determinants and among them the antigen- antibody recognition region is included. The theory of the idiotipic network emerges from the demonstrated fact (Urbain .J (1976) Ann Immunol. 1231 C; 357) that the idiotipic determinants in the immunoglobulin molecule (AB1) are recognized by antidiotypic antibodies (AB2) obtained against it.
The binding of an idiotypic determinant with its corresponding antidiotypic occurs in a dynamic state that could be perturbed during the course of immune response.
The theory of the idiotypic network proposed by Jerne in the seventies has implicit that the immune repertoire contains and recognize all the possible antigens. Ignoring the dicotomy between self and non sell antigens, thus the existence of autoreactive clones of lymphocytes occurs in a physiological mode.
Studies performed in the last ten years have demonstrated the presence of natural autoreactive antibodies in normal subjects (Avrameas.S., (1991) Immunol. Today, 132C: 231:236, Antin et al (1986), J Immunol. 136,505:510, Lundkvist (1989), PNAS USA 86:5074-5078, Longtenberg .T (1989)J Exp Med, 170:1347.1335)).
These experimental facts have prompted the concept that considers the immune system as a highly connected network of specific receptors ontogenically organized through the internal references (self recognition) philogenetically selected for the maintenance of the homeostatic physiological functions, and more than the normal defense against infections. In this sense recognition of the self is normal and tolerance to the self and memory are not any more considered clone properties as postulates by Burnet in his clone selection theory (Burnet F. M. (1956) London Cambridge University Press) but they are emerging properties of the immune network with an integral point of view (Coutinho, A (1984) Immunol. Rev. 79:151-168, Coutinho. A (1989) Immunol. Rev 110:63-87).
This new concept in relation to the immune system have had its impact in understanding the basic mechanisms of autoimmune diseases and also in the new therapy approach Zanetti. M (1985) Immunol. Today 6: 299-302; Kaveri et al (1991) Clin Exp. Immunol. 96:192-198; Kazatchine. D. M (1994) Immunol. Rev 139:79-107.
An amazing therapeutic effect with different immunoglobulin compounds have observed in the wide variety of systemic and organ specific autoimmune diseases that is useful for the clinical validation of this new concept. (Kazatchine et al (1993) Autoimmunity: physiology and disease Wiley-Liss, Inc., New York).
IgG Immunoglobulin preparations are obtained from blood pools of healthy donors representing the autoreactive physiological network of antibodies. The reactivity analysis in these preparations have demonstrated the existence of antibodies interacting with idiotypes expressed in the autoantibodies and autoantibodies per se (Rossi,et al (1988) Clin Exp. Immunol. 74: 311-316; Rossi et al (1989) J Immunol. 143:4104-4109. Both types of reactivity are in high concentration in the total number of IgG molecules that are more connected than other molecules inside the pool (Dietrich et al (1992) Eur J Immunol. 22:1701-1706). In this way the immunoregulatory effect of these preparations justify the design of new therapeutic strategies addressed to restores the connectivity and diversity in the autoimmune patient. Through this clinical value the concept of emergency of autoimmune disease has been validated, through the fail in the regulatory network in the variable regions of the connected antibodies.
In previous research a panel of monoclonal antibodies against tumor associated gangliosides was obtained, the antigen, classified as T cell independent self antigen EPO: No:0 657 471 A1) These Mabs are IgM type bearing idiotypes able to activate the whole idiotypic network and thus generating an antibody response in absence of the antigen, i.e. they are recurrent idiotypes. These antibodies have been previously described as recognizing specifically the NeucAc GM2 ganglioside (Alfonso. M et al (1995) Hibridoma 14(3): 209-216).
The present invention provides novelty in the generation and selection of a panel of antidiotypic monoclonal antibodies (AB2) against the original IgM antibody recognizing ganglioside.
From this panel a further characterization was performed to one of the antibodies, named B7 showing it was an IgG2a with medium affinity for the antigen (Kd 10 E-8)and alpha type even though this type, it was able to induce an antibody (AB1) independent response in a singeneic model (Balb/C).
Surprising properties have been shown in the antidiotypic antibody B7 studies: High idiotypic connectivity with immunoglobulins of different species human B and T lymphocyte recognition and the immunoregulatory effect on previously activated B and T lymphocytes.
Up to know there isn't any therapy proposing this property in antiidioptype antibodies for the treatment of autoimmune, and infectious diseases and cancer.
The novelty of the present invention consists in the selection of the antidiotypic monoclonal antibodies IgG type highly connected to the immune network through their variable regions able to interact with human B and T lymphocytes and their immunoregulatory effect.
DETAILED DESCRIPTION OF THE INVENTION
GENERATION AND PRODUCTION OF MONOCLONAL ANTIDIOTYPIC ANTIBODIES HIGHLY CONNECTED TO THE IMMUNE NETWORK
For the generation and production of monoclonal antidiotypic antibodies, the Mab against ganglioside is used as immunogen with an immunization procedure is performed as described elsewhere (European Patent Application 657 471 A1).
Mice with high titers of antidiotypic antibodies in serum are reimmunized and three days afterwards the spleen is removed and splenocytes are obtained. Antibody producing cells can be obtained from any other organ if preferred.
These cells are fused with myeloma calls, cell fusion may be performed according to any of the known methods and thus hybridomas are obtained. This method is described in detail in any of the procedures for this purpose.
The antibodies produced are assayed through any immunoassay method preferably an immunoenzimatic method, in which the supernatant are assessed for the presence of specific recognition of the proper antigen. The antibody- antibody binding is detected through a second antibody labeled with an enzyme.
The supernatants are selected for their capacity of blocking the antiganglioside Mab (E1) binding to the antigen (Neu Ac GM2) the supernatants in adequate concentrations are incubated and then the E1 Mab with the antigen.
Once the desired hybridoma has been selected and cloned at least twice by limiting dilution, the resulting Mab can be produced by several culture techniques.
One method can be inoculating the proper amount of cells in the peritoneal cavity of mice producin
Abrahan Amparo Emilia Macias
Lopez Gumersinda Bombino
Marichal Orlando Pena
Obaya Teresita Rodriguez
Rodriguez Rolando Perez
Centro de Immunologia Molecular (CIM)
Lackenbach Siegel
Scheiner Laurie
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