Drug – bio-affecting and body treating compositions – Immunoglobulin – antiserum – antibody – or antibody fragment,...
Reexamination Certificate
2001-11-09
2003-12-16
Chan, Christina (Department: 1644)
Drug, bio-affecting and body treating compositions
Immunoglobulin, antiserum, antibody, or antibody fragment,...
C424S179100, C424S193100, C424S194100
Reexamination Certificate
active
06663861
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to antibodies against molecules, particularly haptens and other B cell antigens, and methods for their preparation. In the methods of the present invention, the molecule is conjugated to a carrier comprising a mixture of charge-modified proteins prior to raising of the antibodies in a host animal. The host animal is pre-immunized with unmodified proteins of the carrier. The host animal is then immunized with the molecule complexed with the carrier wherein proteins in the mixture making up the carrier have been charge-modified. Antibodies or B cells producing antibodies can then be routinely isolated from the host animal. The present invention also relates to methods for use of these antibodies in immunoassays as well as in therapeutic applications. In addition, the present invention relates to vaccine therapies and methods of invoking and/or enhancing an immune response in animals via molecules coupled to carriers comprising a mixture of charge-modified proteins.
BACKGROUND OF THE INVENTION
The general humoral immune response is based on the cooperation of antigen presenting cells, antigen specific T lymphocytes and antigen specific B lymphocytes.
T lymphocytes are selected and stimulated by T cell epitopes, which associate with Major Histocompatibility Complex (MHC) and are presented on the surface of antigen presenting cells. A T cell epitope is a short, linear amino acid sequence most frequently localized to the interior portion of a protein and thus inaccessible from the protein surface. A T cell epitope reacts with the antigen specific receptor of the T cell only when bound to an MHC molecule thus forming part of a ternary complex. Helper T cells participate in determining what kinds of immunoglobulins are produced by B cells and stimulate the proliferation of specified lymphocytes. Peptides arising from the digestion of internalized and endocytosed proteins by a B cell are bound to MHC proteins and stimulate and activate helper T cells.
Accordingly, these T cell epitopes react with the antigen specific receptor of the helper T cell only when the helper T cell epitope is bound to an MHC Class II molecule.
B lymphocytes are selected by antigens which bind to the antigen specific receptor (a membrane bound antibody) of the B cell. Almost any molecular configuration can act as a B cell antigen or B cell epitope. With respect to proteins, the epitopes are most frequently positioned on the outer accessible face of the protein as direct binding to the antigen specific receptor is required. The T cell epitope of the antigen is then presented on the surface of the B cell, associated with MHC class II molecules. MHC class II proteins, which comprise the T cell epitopes, arise from the degradation of proteins that have been internalized. This presentation entails the selected and stimulated T accessory cells to be associated with the antigen specific B lymphocyte, which is now stimulated and matured via hormone like factors from the T cell. The final stage of the B cell after stimulation and maturing is the antibody producing plasma cell, which secretes antigen specific antibody (Biochemistry ed. Lubert Stryer 4th Edition, Freeman and Co., New York pp. 379).
Thus, antibody formation against an antigen is conditional upon the presence of both B cell epitopes and T cell epitopes. Further, it has been demonstrated that the two types of epitopes must be physically associated to generate an antibody immune response (Rajewsky et al.
J. Exp. Med.
1969 129:1131; Mitchison, N. A. Eur. J. Immunol. 1971 1:10; and Mitchison, N. A. Eur. J. Immunol. 1971 1:18).
Accordingly, the antibody response to low molecular weight molecules such as haptens is often quite poor and can be induced in a mammal only by presentation of the low molecular weight molecule coupled to an immunogenic carrier. Coupling to an immunogenic carrier is also required for relatively large molecular weight molecules with only limited immunogenicity such as larger peptides, low immunogenic proteins (such as self-proteins), carbohydrates, lipids, viruses and nucleic acids. Coupling to an immunogenic carrier also provides a means for enhancing the immunogenicity of immunogenic proteins wherein a more powerful immunogenicity is required. Some examples of carrier molecules which have been disclosed for inducing or enhancing an immune response to such molecules include serum albumin such as bovine serum albumin (BSA), human serum albumin (HSA) and others, keyhole limpet hemocyanin (KLH), ovalbumin (OA), chicken immunoglobulin (IgY) and diphtheria toxoid.
Conjugation methods have also been described for increasing the immunogenicity of low molecular weight molecules. Pioneering work to enhance the immunochemistry of low molecular weight molecules began as early as 1917 when antigens were covalently conjugated by azo reaction on histidine, tyrosine and tryptophan residues (Landsteiner, K. The Specificity of Serological Reactions” Harvard Univ. Press, Cambridge, Mass., 1945). More recently, methods have been described for preparing hapten-carrier conjugates via formation of covalent bonds between the low molecular weight hapten and a carrier protein selected from a wide range of globulin fractions (Erlanger, B. F. The Preparation of Antigenic Hapten-Carrier Conjugates: A Survey” in Methods of Enzymology, 70, Academic Press, Inc. 1980). In these methods, the functional groups of the hapten determine which synthesis is selected for conjugation of the functional groups of the carrier to the antigen. A wide range of chemical syntheses have been described for conjugating hapten on the basis of hapten functionality.
In addition to invoking an antibody response, it is also desirable to avoid antigen-specific suppression wherein prior immunity against a carrier protein reduces the response to re-inoculation with the same carrier protein coupled to an antigen. This effect can be avoided or reduced, enabling expansion of native B cell populations, if the carrier molecules, for example secretory proteins from BCG, are sufficiently modified at the surface. Reduction of B cell response to the carrier molecule can be achieved by chemical or physical modification of the carrier molecule. Examples of chemical and/or physical processes, which have been described for modification of the carrier molecule include heat denaturation and chemical methylation or formaldehyde treatment of the carrier molecule prior to the coupling to the hapten molecule.
Modification of the carrier to avoid epitope-specific suppression has several additional advantages. Specifically, since T cell immunity can be induced as a general principal in animals, the T cell immunity can be retrained against the modified carrier while considerably reducing the degree of B cell immunity against the carrier molecule.
Accordingly, subsequent immunization with a B cell antigen coupled to a modified form of the carrier ensures a rapid development of antibodies, a high level of antibodies after only one or two immunizations, a sustained immune response, and the possibility of rapid development of antibodies having a high binding strength.
WO 94/12213 describes a process for modifying an immunogenic protein to be exclusively or predominantly a T cell antigen by blocking charged groups, either —NH
3
+
or —COO
−
. —NH
3
+
groups are blocked by means of formaldehyde, while disulfide bridges are reduced by means of dithiothreitol or &bgr;-mercaptoethanol. Denaturation of the protein antigen is then performed by heat treatment.
The present invention relates to a new method for raising antibodies against molecules with low antigenicity by conjugating the molecule to a carrier comprising a balanced charge mixture of proteins chemically modified to carry a net positive or negative charge.
SUMMARY OF THE INVENTION
An object of the present invention is to provide a method for raising antibodies against molecules including, but not limited to, those with low immunogenicity. The method comprises coupling the molecule to a carrier com
Hansen Trine Overgaard
Recke Camilla
AntibodyShop A/S
Belyavskyi Michail
Chan Christina
Licata & Tyrrell P.C.
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