Drug – bio-affecting and body treating compositions – Antigen – epitope – or other immunospecific immunoeffector – Fusion protein or fusion polypeptide
Reexamination Certificate
1997-10-21
2001-05-29
Saunders, David (Department: 1644)
Drug, bio-affecting and body treating compositions
Antigen, epitope, or other immunospecific immunoeffector
Fusion protein or fusion polypeptide
C424S278100, C514S019300, C530S350000, C530S380000
Reexamination Certificate
active
06238670
ABSTRACT:
The present invention relates to administration of immunogens and modulation, either inhibition or augmentation, of the immune response.
The initial encounter with antigen is the most difficult phase of an immune response. The threshold for activating unprimed, as opposed to primed, T cells is higher and B cells express unmutated, generally low affinity antigen receptors. This can be problematic when there is a need or desire for raising an immune response to an immunogen of interest, for example for therapeutic purposes or in order to obtain antibodies for subsequent manipulation and use.
The Complement system plays a major role in enhancing the immune response. Almost twenty years ago Pepys showed that depleting mice of C3 decreased their immune response to sheep erythrocytes, as measured by their antibody response to this antigen (1). Subsequent studies of humans, guinea pigs and dogs with genetically determined deficiencies of C3 or of C4 and C2, the proteins that form the enzymes that activate C3, confirmed the conclusion that activation of C3 augmented the primary immune response (2).
C3 is a plasma protein that contains a thioester that mediates covalent attachment to other biological molecules (3). Activation of C3 to C3b by a “C3 convertase” of the classical or alternative pathway makes the thioester accessible to attack by weak nucleophiles such as the —OH or carbohydrates, causing attachment of C3b, via an ester linkage, to target—OH—bearing molecule in the immediate vicinity of the activation reaction. C3b (or proteolytically processed fragments iC3b and C3dg) complexes with antigen then binds receptors CR1, CR2 and CR3. Binding of the complexes to C3 receptors on follicular dendritic cells in the primary follicles and germinal centres of secondary lymphoid organs may promote development and maintenance of memory B lymphocytes. Binding of the complexes to antigen-specific B lymphocytes causes cross-linking of CR2 to the antigen receptor, membrane immunoglobulin (mIg). CR2 is associated with the B lymphocyte membrane protein CD19 which amplifies signalling through mIg by approximately 100-fold when coligated to it.
A limiting step in this overall reaction is the attachment of C3b. It requires the antigen be capable of activating the Complement system, through either the classical or the alternative pathway, and that it have a site to which C3b can covalently attach. Problems then arise when the immunogen of interest is, for instance, a soluble peptide and protein, since these tend not to be activators of the alternative pathway nor effectively to bind C3b. Furthermore, low affinity, cross-reactive IgM antibodies do not bind monovalent protein antigens with sufficient avidity to activate the classical pathway.
These difficulties may be especially relevant when immunising with small peptides, for example that have been defined as epitopes for B or T cells, and with DNA immunisation (4). Although adjuvants may be used to enhance immunogenicity of peptides and non-aggregated proteins when immunising experimental animals such as mice, in general their administration to humans is not possible or at least not preferred.
The present invention now provides a way of enhancing the immune response to an immunogen.
Additionally, there are circumstances when it is desirable to inhibit, ie at least reduce, the immune response to an immunogenic substance. For instance, there is much interest in the administration of therapeutic antibodies. Historically, however, the antibodies available have been murine monoclonal antibodies. Because the human body recognises such murine antibodies as foreign an immune response is mounted against them, resulting in their rapid clearance from the bloodstream. Numerous techniques of “humanising” non-human antibodies and other techniques for reducing their foreign character are available but often require highly-skilled workers to perform them. Furthermore, even following “humanisation”, the immune system may mount an anti-idiotypic response, unless the antibody is compatible with the host idiotype.
Additionally, there are many other foreign, ie non-human, substances administered to individuals for a variety of purposes, including therapy, and for which “humanising” techniques are not available. One of numerous examples well known to those skilled in the art is the “clot-busting” drug streptokinase administered to heart-attack patients. This enzyme is bacterial in origin and an immune response is mounted against it by recipient individuals.
The present invention additionally provides a way of inhibiting the immune response to an immunogen.
The present invention in its various aspects is founded on the surprising discovery that coupling of the Complement C3 fragment C3d (a ligand for CD21 (CR2)) to immunogen of interest enables modulation of the immune response to the immunogen. The modulation may be an increase or a decrease in the level of antibody response to immunogen administration. Completely unexpectedly, it has been found that the immune response may be enhanced by coupling a plurality of C3d molecules, to the immunogen and reduced by coupling of one C3d molecule to the immunogen. In view of this, the present invention in various aspects relates to modulation of an immune response by associating an antigen with a ligand for CD21 or, since CD21 associates with CD19 on the surface of B cells, a ligand for CD19.
Thus, according to the present invention there is provided a method of altering the immunogenicity of an immunogen, the method comprising forming a composition comprising the immunogen in association with (or “coupled to”) a ligand for CD21 (CR2) or CD19. In a preferred embodiment the method comprises coupling one or more C3d molecules to the immunogen. If the C3d coupled is monomeric, the immunogenicity of the immunogen is reduced. If the C3d coupled is multimeric, eg dimeric, the immunogenicity of the immunogen is increased. With increased or decreased immunogenicity, the immune response of an individual who receives the immunogen will be enhanced or reduced, respectively. The level of immune response may be measured, for example, with reference to antibody levels.
The association of antigen and ligand may be such that there is modulation of a response in a B cell through two receptors on the B cell; a membrane immunoglobulin (mIg) specific for the antigen and a CD21/CD19 complex. Preferably, a response in a B cell is stimulated, but in certain circumstances, e.g. as discussed, a response in a B cell may be inhibited. Experimental assays for assessing ability to modulate B cell responses through mIg and the CD21/CD19 complex are described herein. For example, an increase in intracellular calcium concentration upon stimulation of antigen-specific B cells using the invention (as compared with stimulation with antigen alone) may be used as an indicator that the composition stimulates B cells through mIg and CD21/CD19.
The experimental work described herein includes evidence of T-cell involvement in the immune response. B-cell responses to protein antigens are T-cell-dependent. Furthermore, T-cell-independent responses do not develop memory: memory is demonstrated herein. Also, isotype switching is characteristic of T-cell-dependent B-cell responses.
In a second aspect, the invention provides a composition comprising an antigen/immunogen in association with (or “coupled to”) a ligand for CD21(CR2) or CD19. Such a composition may comprise a molecule (conjugate) comprising an antigen or immunogen coupled to one or more C3d molecules. If a plurality of C3d molecules are linked to the antigen it may be preferred to use 2 or 3, as exemplified herein. Since the experimental evidence described below indicates an increase in effect with increasing number of C3d molecules, it may be preferred, depending on circumstances, to use a greater number, e.g. 4 or 5 or more.
Since a preferred way of coupling to C3d is by a peptide bond, the conjugate may be a contiguous polypeptide, ie a fusion protein. In a fusion protein according to the present invention,
Dempsey Paul W.
Fearon Douglas T.
Cambridge University Technical Services Limited
DeCloux Amy
Flehr Hohbach Test Albritton & Herbert LLP
Saunders David
Trecartin Richard F.
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