Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-11-12
2002-01-22
Chan, Christina Y. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S029000, C435S325000, C435S326000, C435S372200, C530S388700, C530S388730
Reexamination Certificate
active
06340569
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates generally to hybridomas capable of producing antibodies, even more particularly to antibodies specific for cell surface antigens, and even more particularly to the methods for utilizing the antibodies, the cells they identify, and the antigens with which they interact.
BACKGROUND OF THE INVENTION
The immune system is crucial for survival, as it initiates the destruction and elimination of both invading organisms (e.g., a virus) and toxic materials produced by such organisms. There are two broad types of immune system responses: (1) “antibody” responses; and (2) “cell mediated” responses. Antibodies circulate in the bloodstream and permeate other body fluids and tissues. Antibodies generally bind with specificity to the antigens which induced the antibody response. This binding works to both inactivate the antigen as well as aid in the destruction of the antigen. The cell mediated response involves the production of specialized cells which react with antigens on the surface of virus-infected host cells. These specialized cells work to destroy the virus-infected host cell before the virus is able to replicate and further infect other cells.
The cells involved in the immune system response are a class of white blood cells referred to as “lymphocytes.” These lymphocytes are generally classified into two types: “B cells” and “T cells.” B cells are generally described as having “receptors” which can “recognize” an antigen alone, leading to production of antibodies specific for the recognized antibody. T cells, on the other hand, are known to have receptors which cannot recognize an antigen unless it is associated with cell-membrane proteins referred to as “major histocompatability complex” (MHC) molecules. There are generally two classes of MHC molecules, referred to as “class I” and “class II”. In the cell mediated response, the antigen is degraded by the cell, forming small peptide fragments that form physical complexes with a “class I” or “class II” MHC molecule. The peptide-class I or peptide-class II MHC complex is then exported to the surface of the cell, and this complex is then capable of being recognized by T cells.
Dendritic cells (DCs) are a diverse population of morphologically similar cell types found in a variety of lymphoid and non-lymphoid tissue. Most dendritic cells are potent antigen-presenting cells specialized in initiating primary T cell immune responses. Dendritic cells generally have long, tentacle-like projections which express high levels of class II MHC molecules. Dendritic cells represent less than 0.1% of white blood cells (these are referred to as “blood dendritic cells”). Dendritic cells are also found in lymphoid organs (such as the tonsil) and are referred to as “interdigitating dendritic cells” when associated with T cell areas (e.g., lymphoid organs), and “follicular dendritic cells” when associated with B cell areas. They are also known as veiled cells present in afferent lymph, as Langerhans cells in the epidermis and as dermal DCs in the dermis of the skin. One function for dendritic cells is capturing antigens and stimulating T cell response. In essence, dendritic cells “pick-up” antigens and migrate the captured antigens to T cells. Because dendritic cells can induce a T cell response to an antigen without other adjuvants, DCs are often referred to as “nature's adjuvant.” As “nature's adjuvant,” dendritic cells have attracted attention for therapeutic purposes.
PCT Publication WO 94/02156 purportedly describes a method for isolating human DCs to present antigens to induce antigen specific T cell mediated responses. Uses mentioned include cellular immunotherapy and cancer treatment. U.S. Pat. No. 5,788,963 purportedly teaches methods and compositions for use of human dendritic cells to activate T cells for immunotherapeutic response against primary and metastatic prostrate cancer cells.
SUMMARY OF THE INVENTION
An embodiment of the present invention relates to kits, e.g., diagnostic assay kits, utilizing the antibody to tumor-associated antigens and carrying out the method disclosed here.
An embodiment of the present invention may be described as a hybridoma formed by fusion of a tumor cell and a non-tumor cell, more specifically, lymphocytes and tumor cells. The hybridoma provides continuously replicating (hybrid) cells which exhibit some or all the characteristics of the non-tumor cell. The hybridomas of the present invention are used to produce or secrete antibodies which interact with cells having a dendritic morphology. Preferably, the monoclonal antibodies of the present invention produced by the hybridomas recognize cells having both a dendritic morphology and a B cell phenotype. Even more preferably the hybridoma is specific for cells which are defined herein as B-DC cells. A hybridoma in accordance with the present invention has been deposited with the American Type Culture Collection (A.T.C.C.) and has an A.T.C.C. Accession Number HB-12430 (reported with the A.T.C.C. 10801 University Boulevard, Manassas, Va. 20110-2209; U.S.A., U.S.A., Nov. 14, 1997, determined viable by the A.T.C.C. on Nov. 18, 1997).
Another embodiment of the present invention is a hybridoma comprised of lymphocytes and tumor cells wherein at least a portion of the lymphocytes and tumor cells are fused to form a hybridoma capable of producing antibodies. The antibodies preferably recognize a 220 kDa protein on the surface of cells. The 220 kDa protein located on the cells is recognized by the antibodies produced by the hybridoma and is further capable of being reduced to four subunits which consist of about 55 kDa, about 65 kDa, about 80 kDa, and about 85 kDa subunits. It is preferable that the 220 kDa protein be an antigen, and that the hybridoma be designated hybridoma 5G9 with A.T.C.C. Accession Number HB-12430. The monoclonal antibody expressed by hybridoma 5G9 (mAb 5G9) is a preferred embodiment capable of reacting with cells expressing the 220 kDa protein. It is to be understood that other monoclonal antibodies having substantially the same function (i.e., able to recognize 220 kDa proteins) are encompassed within the scope of the present invention.
Another embodiment of the present invention is a monoclonal antibody or fragment thereof which is capable of reacting with cells expressing a protein on the surface of the cell which is indicative of the cells' morphology, preferably a dendritic like morphology. As discussed above, it is preferable that the antibodies are produced by a hybridoma cell line, most preferably hybridoma cell line 5G9, having A.T.C.C. Accession N HB-12430, or subclones thereof. It is preferable that the monoclonal antibody of this embodiment react with specific lymphoma cells, even more preferably lymphoma cells known as chronic lymphocitic leukemia cells.
Another embodiment of the present invention is a cell which is isolated from a blood cell population wherein the cell expresses the 220 kDa protein discussed above. It is more preferable that the cell expresses the protein on the surface of the cell. It is also preferred that the cell which is isolated have a dendritic morphology and even more preferably also a B-cell phenotype. Additionally, it is preferable that the cell have a function which is similar to that of classic dendritic cells. More preferably the cells react with the monoclonal antibody 5G9 or a monoclonal antibody specific for the 220 kDa cell surface protein. It is even more preferable that the cell be isolated from peripheral blood mononuclear cells and even more preferable that the cell express a phenotype which is selected from the group CD19+, CD20+, CD40+, CD83+ (after positive selection), HLA-D- hi positive, immunoglobulin &mgr; chain positive, Kappa light chain positive, lamda-light chain positive, CD3−, CD4−, CD5−, CD10−, CD13−, CD14−, CD15−, CD16−, CD33−, CD56−, CD64−, and any combination of one or more of the above phenotypes. Even more preferably the phenot
Ball Edward D.
Zhong Rui-Kun
Chan Christina Y.
Reed Smith LLP
University of Pittsburgh
VanderVegt F. Pierre
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