Bovine lactation associated immunotropic protein (CD14),...

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Reexamination Certificate

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C426S657000, C426S801000, C514S008100, C514S021800, C530S351000

Reexamination Certificate

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06676985

ABSTRACT:

FIELD OF INVENTION
This invention, in the fields of immunology, biochemistry and cell and molecular biology, relates to proteins or proteins that are co- and/or post-translationally modified, termed LAIT proteins, that activate B cells. This invention is also directed to the use of such protein in pharmaceutical preparations, and pharmaceutical compositions comprising LAIT protein or functional derivatives thereof. This invention is also directed to nucleic acid molecules encoding the bovine LAIT protein or functional derivatives thereof and methods for the purification of native and recombinant forms of said proteins that activate B cells.
BACKGROUND OF THE INVENTION
Bone marrow-derived “B” lymphocytes, commonly called B cells, are a type of white blood cell present in the lymph, the blood, and in secondary lymphoid organs of the immune system. B cells are the precursors of antibody secreting cells, plasma cells, and as such are central to the induction of humoral immune responses.
The induction of most humoral immune responses in the adult involves a number of cellular interactions among thymus-derived T lymphocytes, commonly called T cells, antigen presenting cells (APC), and B cells [J. Exp. Med 147:1159, 1978; PNAS 77:1612, 1982; PNAS 79:1989, 1982; Immunol. Rev. 95:914, 1987].
As currently understood, T cell-dependent B cell activation involves activation of T cells upon their recognition of antigen, as presented by APC in conjunction with proteins encoded within the major histocompatibility complex (MHC), which are expressed on the cell surface of the APC. This antigen specific and MHC restricted T cell-APC interaction results in reciprocal activation of the two cell types, and the alteration of T cell physiology such that “helper function” becomes manifest.
Helper T cells can activate antigen specific B cells. Antigen specificity of the T cell-B cell interaction is maintained as a consequence of the ultimate capacity of the B cell to function as an APC. Thus, while resting, quiescent B cells are not efficient APC (PNAS 79: 1989, 1982), they specifically interact with antigen through membrane associated immunoglobulin, the specificity of which reflects that of the immunoglobulin their daughter cells will secrete (J. Exp. Med. 140:904, 1974).
Immunoglobulin mediated internalization of antigen by the specific B cell, which may involve presentation by yet another sort of APC, the follicular dendritic cell, results in the initiation of antigen processing by the B cell, the up-regulation of MHC Class II and B7 expressior., and the presentation of antigen derived peptides in the context of MHC (J. Exp. Med. 178: 2055, 1993). The B cell activated by this route is a target for the activated helper T cell.
T cell helper function includes signals delivered through both T cell-B cell contact, and the interaction of T cell derived soluble mediators, referred to as cytokines, with their cognate ligands expressed on the B cell plasma membrane. T cell-B cell contact is also MHC restricted, analogous to the T cell-APC interaction (Eur. J. Immunol. 12:627, 1982; Eur. J. Immunol. 12:634, 1982). However, the specific interaction of the molecules which mediate the MHC restricted interaction between the two lymphocyte lineages, specifically, the T cell receptor for antigen (TcR), and the MHC/antigen complex expressed by the B cells, do not predicate the induction of B cell growth and differentiation (Eur. J. Immunol. 18:375, 1988).
The essential molecular interaction, reflected by the requirement for T cell-B cell contact, is mediated by CD40 expressed on the plasma membrane of the B cell, and its cognate ligand, gp39 (or CD40L), expressed on the plasma membrane of the T cell (PNAS 89:6550, 1992; Nature 357:80, 1992). Consistent with this paradigm is the observation that membrane expression of the latter increases upon T cell-APC interaction, as well as subsequent to T cell-B cell interaction (PNAS 89:6550, 1992). Further, membrane immunoglobulin mediated B cell interaction with antigen results in the increased membrane expression of CD40 (Sem. in Immunol 6:303, 1994). The interaction between CD40 and CD40L predicates the induction of B cell growth, B cell differentiation into immunoglobulin secreting cells, and immunoglobulin isotype switching (J. Exp. Med. 178:1567, 1993).
Consistent with this model is the observation that soluble CD40L, or monoclonal antibody (mAb) specific for CD40 can induce B cell growth and differentiation to immunoglobulin secretion (Sem. in Immunol. 6:267, 1994; PNAS 83:4494, 1986; J. Immunol. 140:1425, 1988;).
In addition to the obligate requirement for T cell-B cell contact, a number of T cell derived cytokines, IL-2, IL-4 and IL-5 are central to B cell growth and differentiation. B cell susceptibility to these cytokines is for the most part limited by prior contact with a T cell. Thus, subsequent to T cell contact, the B cells increase expression of cytokine specific membrane receptors (PNAS 80:6628, 1983; J. Immunol. 145:2025, 1990; J. Immunol. 146:1118, 1991). IL-2 and IL-5 have been demonstrated to support the growth of activated B cells (PNAS 77:1612, 1980; Immunol. Rev. 52;115, 1980). Further, IL-4 and anti-immunoglobulin have been shown to synergize in supporting B cell growth (J. Exp. Med. 155:914, 1982).
Notable exceptions in this context are the quiescent B cell responses to IL-4 and IL-5. IL-4 induces the de novo transcription and translation of MHC Class II proteins (J. Exp. Med. 155:914, 1982; PNAS 81:6149, 1984; J. Exp. Med. 160;679, 1984), and IL-5 is able to support the differentiation of quiescent B cells into high rate immunoglobulin secreting cells in the absence of cell growth (Eur. J. Immunol. 22:2323, 1992).
In any event, signals derived from molecular interactions amongst membrane molecules on T cells and B cells, and from those of T cell derived cytokines interacting with their cognate receptors on B cells are parts of a complex signaling system. Each signal drives the B cell to another stage of activation, rendering it susceptible to subsequent progression signals. These signals complement one another, rather than having the capacity, individually, to drive the complete process of B cell growth and differentiation (Immunol. Rev. 95:177, 1987).
In 1988, a unique activity in ovine colostrum was discovered (J. Immunol. 140:1366, 1988). Proline Rich Protein (PRP) had been partially purified using classical techniques of protein purification. This material was shown to support the induction of quiescent B cells into the cell cycle, and to support their differentiation into high rate immunoglobulin secreting cells. This was apparently the first report of a protein of mammalian origin that mediates these functions.
A monoclonal antibody specific for ovine PRP was subsequently prepared. When PRP preparations were passed over an affinity column prepared using the antibody, all of the PRP was retained by the column, as assessed by Western blotting analysis of eluate and effluent. However, all of the B cell stimulatory activity was found in the effluent. Thus, the published characterization of the B cell tropic bioactivity present in ovine colostrum was not attributable to PRP (unpublished information).
SUMMARY OF THE INVENTION
This invention features a novel bovine protein and isolated nucleotide sequences encoding the protein, the said protein being capable of activating B-cells of mammalian origin. A substantially pure LAIT protein or co- and/or post-translationally modifiel form of the protein may be produced by biochemical purification, or by recombinant means in a prokaryotic or eukaryotic host substantially free of other proteins with which it is natively associated. Also included in this invention is a process for purifying LAIT protein or a co- and/or post-translationally modified form of LAIT protein of this invention from bovine colostral whey comprising:
(i) salting out of proteins contained within said samples
(ii) enrichment and ultimate purification of LAIT protein from proteins salted out in step (i) utilizing classical pr

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