&bgr;2 microglobulin fusion proteins and high affinity variants

Details &bgr;2 microglobulin fusion proteins and high affinity variants &bgr;2 microglobulin fusion proteins and high affinity variants

2001-03-19

2004-01-27

Gambel, Phillip (Department: 1644)

Drug, bio-affecting and body treating compositions

Antigen, epitope, or other immunospecific immunoeffector

Fusion protein or fusion polypeptide

C424S184100, C424S185100, C424S093100, C424S093200, C424S093210, C424S093700, C530S350000, C536S023100, C536S023400, C435S325000, C435S320100

Reexamination Certificate

active

06682741

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to compositions based on &bgr;
2
microglobulin, and the use of such compositions in immunological methods pertaining to the targeting of proteins to cell surfaces. The disclosed compositions and methods have particular application to vaccination and tumor therapy.
BACKGROUND OF THE INVENTION
MHC I and Activation of Cytotoxic T-cells
The beta-2 microglobulin (&bgr;
2
m) protein is a component of the class I major histocompatibility complex (MHC I). MHC I is formed by the association of &bgr;
2
m and an alpha protein (also known as the “heavy” chain), which comprises three domains, a1, a2 and a3. MHC I is found on the surface of most types of nucleated cells, where it presents antigens derived from proteins synthesized in the cytosol to CD8
+
T-cells. Two signals are required for activation of naive CD8
+
T-cells. The first signal is provided by the interaction of the T-cell receptor (TCR) with the MHC I-antigen complex on the antigen-presenting cell surface. The second signal is generated by the interaction of a ligand on the antigen-presenting cell (APC) with a second receptor present on the T-cell surface. This second signal is termed co-stimulation, and the APC ligand is often referred to as a co-stimulatory molecule. The best characterized co-stimulatory molecules on APCs are the structurally related glycoproteins B7.1 (CD80) and B7.2 (CD86) which interact with the CD28 receptor on the T-cell surface. Activation of CD8
+
T-cells by these two signals leads to the proliferation of antigen-specific cytotoxic T-cells, which recognize and destroy cells presenting the signaling antigen. These cytotoxic T-cells play an important role in the immunological defense against intracellular pathogens such as viruses, as well as tumors. A detailed presentation of the immunological basis of the cytotoxic T-cell response can be found in Janeway and Travers (
Immunobiology: the immune system in health and disease,
Current Biology Ltd./Garland Publishing, Inc. New York, 1997).
The failure of an exogenous (non-self) antigen to stimulate a cytotoxic T-cell response can result from a block in the above-described cytotoxic T-cell activation pathway at one of many points (see Ploegh, 1998,
Science
280:248-53). Failure of the cytotoxic T-cell activation pathway is of great significance in two particular areas of medicine: vaccination and tumor immunology.
Cytotoxic T-cells and Vaccination
Vaccine technology has focused in recent years on sub-unit vaccines. Sub-unit vaccines comprise isolated pathogen components, such as viral capsid or envelopes, or synthetic peptides that mimic an antigenic determinant of a pathogen-related protein. For example, U.S. Pat. No. 4,974,168 describes leukemia associated immunogens that are peptides based on envelope proteins of a leukemia-associated virus. However, while sub-unit vaccines can stimulate CD4
+
helper T-cells (which play a key role in humoral immunity), attempts to stimulate CD8
+
cytotoxic T-cells in vivo with such vaccines have been largely unsuccessful. It has been postulated that the reason for this is the inability of the exogenously administered vaccine peptide to associate with the MHC I molecules on the cell surface (Liu, 1997,
Proc. Natl. Acad Sci. USA
94:10496-8). In other words, the block in the cytotoxic T-cell activation pathway occurs at the stage where the antigen is loaded into the MHC I molecule.
One proposed solution to this problem is to combine the antigenic peptide with a molecule that is readily taken up into cells (reviewed by Liu, 1997,
Proc. Natl. Acad Sci. USA
94:10496-8). Thus, this strategy is based on getting the antigen into the cytosol so that it can join the normal pathway by which antigens are processed for presentation by MHC I. In contrast, Rock et al. (
J. Immunol.
150:1244-52, 1993) adopted a strategy of enhancing the binding of the vaccine peptide to MHC I already present on the cell surface. Rock et al. (
J. Immunol.
150:1244-52, 1993) report that the administration of exogenous purified &bgr;
2
m along with the vaccine peptide produces enhanced loading of the peptide onto MHC I in vivo and thereby stimulates a cytotoxic T-cell response against the peptide. The use of exogenous &bgr;
2
m as a vaccine adjuvant is also described in U.S. Pat. No. 5,733,550 (to Rock et al.), which is incorporated herein by reference.
Tumor Cells and Immune System Evasion
Tumor cell immunity is primarily cell-mediated, involving both CD8
+
cytotoxic T-cells and CD4
+
helper T-cells. However, despite the fact that tumor cells express tumor-specific proteins that are not recognized as self-antigens by the immune system, they often escape recognition by the immune system. A number of factors may contribute to the ability of tumor cells to evade immune recognition, including the down-regulation of expression of co-stimulatory proteins. TCR stimulation in the absence of co-stimulatory molecules can result in failure to activate the T-cell and the induction of clonal anergy. Thus, down-regulation of co-stimulatory proteins in tumor cells prevents normal activation of T-cells that do bind to tumor antigens on the cell surface, permitting the tumor cell to escape recognition.
Several research groups have attempted to address this issue by removing tumor cells from a patient, providing exogenous co-stimulatory molecules on the surface of the removed tumor cells and then reintroducing the tumor cells to the patient so that immune recognition can occur. For example, European patent application number 96302009.4 describes a method by which tumor cells are removed from a patient, transfected to express both B7 and CD2 (a co-receptor involved in T-cell adhesion and activation) on the tumor cell surface, and then reintroduced to the patient. The reintroduced cells are reported to stimulate a broad immunological response against both the reintroduced transfected tumor cells and the non-transfected tumor cells within the patient's body, resulting in tumor regression.
Adopting an alternative approach to this problem, Gerstmayer et al. (
J. Immunol.
158:4584-90, 1997) describe a chimeric B7-antibody protein, in which the antibody is specific for the erbB2 proto-oncogene product. This chimeric molecule localizes specifically on the surface of erbB2 expressing tumor cells, and presents the B7 co-stimulatory molecule to cytotoxic T-cells, resulting in enhanced proliferation of cytotoxic T-cells. Gerstmayer et al. (
J. Immunol.
158:4584-90, 1997) thus propose that fusion proteins comprising an anti-tumor antibody and a co-stimulatory molecule could be useful as tumor immunotherapeutics. However, this approach would require prior knowledge and characterization of tumor-specific antigens expressed on the tumor cells of each individual patient, and the use of an antibody specific for that particular type of tumor cell.
SUMMARY OF THE INVENTION
The present invention employs various forms of beta-2 microglobulin to address the problems associated with failure of the cytotoxic T-cell activation pathway in both vaccination and tumor therapy. The invention also provides compositions and methods based on &bgr;
2
m that are broadly applicable to achieve expression of any desired target protein on the surface of any mammalian cell.
In one embodiment, the invention provides fusion proteins comprising a first amino acid sequence and a second amino acid sequence, wherein the second amino acid sequence is a &bgr;
2
-microglobulin. In particular applications, the first amino acid sequence may be a co-stimulatory protein, such as B7.1 or B7.2, or another protein having immunological activity, such as a cytokine, an integrin or a cellular adhesion molecule. Examples of such proteins include interleukins (e.g., IL-2, IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), lymphocyte function-associated proteins (e.g., LFA-1, LFA-3) and intercellular adhesion molecules (e,g., ICAM-1, ICAM-2). In other embodiments, the first amino acid sequence of the fusion protein

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