Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1998-04-15
2001-03-06
Schwadron, Ronald B. (Department: 1644)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007200, C435S007240, C436S501000, C436S506000, C436S536000, C436S811000
Reexamination Certificate
active
06197524
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to methods for detecting, identifying, isolating, and selectively labelling and targeting TH1 lymphocytes and, more particularly, to such methods which use the presence of LAG-3 protein on the surface thereof as markers for the identity of TH1 lymphocytes. The present invention also related to methods of treating infectious diseases, cancer, Th1-mediated diseases and disorders associated with an imbalance of Th1 and Th2 cells.
2. Description of the Background Art
The lymphocyte activation gene (LAG-3) is a member of the immunoglobulin superfamily, that is selectively transcribed in human activated T (both CD4
+
and CD8
+
) and NK cells (Triebel et al, 1990; see also WO91-110682). The sequence data, the compared exon/intron organization, and the chromosomal localization revealed that LAG-3 is closely related to CD4 (Baixeras et al, 1992). The close relationship between LAG-3 and CD4 was further strengthened by the demonstration that both share the same ligand, i.e., MHC class II molecules (Baixeras et al, 1992). However, in contrast to CD4, LAG-3 does not bind the human immunodeficiency virus gp120 (Baixeras et al, 1992). In vivo, LAG-3 expression was neither found in primary lymphoid organs, such as spleen, mucosa-associated lymphoid tissue or normal lymph nodes. However, it was readily detected in inflamed tonsils, or lymph nodes with follicular hyperplasia, supporting the view that even in vivo LAG-3 is expressed following activation (Huard et al, 1994A) The physiological role of encoded LAG-3 protein is still unclear. Antigen-specific stimulation of T-cell clones in the presence of anti-LAG-3 monoclonal antibody (mAb) led to increased thymidine incorporation, higher expression of activation marker CD25 and enhanced cytokine production (Huard et al, 1994B). Accordingly, addition of a soluble recombinant form of LAG-3 inhibited antigen-specific T-cell proliferation, suggesting a regulatory role of LAG-3 in CD4
+
T-lymphocyte activation (Huard, 1995).
Studies of both murine and human CD4
+
T-cell clones have shown that CD4
+
T helper (Th) cells comprise functionally heterogenous populations based on their profile or cytokine production (Mosmann et al, 1986; Del Prete et al, 1991). Th1 cells produce interleukin (IL)-2, interferon (IFN)-&ggr; and tumor necrosis factor (TNF)-&bgr;, whereas Th2 cells produce IL-4 and IL-5. In the absence of a prominent differentiation of Th1 or Th2 cells, the large majority of CD4
+
T-cells produce both Th1- and Th2-type cytokines (i.e., Th0 cells) (Mosmann et al, 1986; Del Prete et al, 1991; Sher et al, 1992; Romagnani, S., 1994). Recently, we have shown that human Th1 and Th2 clones not only exhibit different functional properties but also show differential expression of CD30 (Del Prete et al, 1995A), an activation marker belonging to the TNF receptor family (Smith et al, 1990).
It has been suggested that Th1 cells contribute to the pathogenesis of organ-specific autoimmune diseases while Th2 cells prevents them (Liblau et al, 1995). Thus, it would be useful to have a simple way to identify and isolate Th1 cells to the exclusion of Th2 cells.
Citation of any document herein is not intended as an admission that such document is pertinent prior art, or considered material to the patentability of any claim of the present application. Any statement as to content or a date of any document is based on the information available to applicant at the time of filing and does not constitute an admission as to the correctness of such a statement.
SUMMARY OF THE INVENTION
It has now been discovered that the LAG-3 expression is preferentially associated with CD4
+
T-cells addressed to the production of Th1-type cytokines. Thus, LAG-3 expression can be used as a marker to detect and identify Th1 lymphocytes and differentiate them from Th2 lymphocytes. Monoclonal antibodies against LAG-3 can be used to detect and identify Th1 lymphocytes which express the LAG-3 protein.
Furthermore, the LAG-3 marker can be used to isolate Th1 cells from Th2 cells. It is known, for example, that Th1 cells contribute to the pathogenesis of organ-specific autoimmune diseases while Th2 cells prevent them. Thus, autologous T-cells from autoimmune disease patients can be subjected to separation into Th1 and Th2 rich fractions ex vivo and the Th2 cells reinfused to help fight the autoimmune disease. The same is true for any disease or condition which is preferentially mediated by Th1 cells, such as contact dermatitis.
Another therapeutic method using the discovery that LAG-3 is a selective marker for Th1 lymphocytes is by means of immunotoxins using monoclonal antibodies specific for the extracellular portion of the LAG-3 protein. If a toxic moiety is attached to such antibodies by means well known in the art, Th1 lymphocytes can be selectively targeted for destruction. By thus effectively shifting the balance of CD4
+
helper T-cells from the Th1 type to the Th2 type, Th1 mediated diseases can be mitigated.
Alternately, the anti-LAG-3 monoclonal antibodies is used to modulate the balance of Th1/Th2 cell population and polarize the differentiation of Th0 cells to Th1 cells.
Labelled monoclonal antibodies against LAG-3 can be used to selectively label Th1 cells. Thus, if a radioactive label is used, the location of Th1 cells can be followed by appropriate viewing means.
Another aspect of the present invention is that anti-LAG-3 antibodies, either immobilized on a solid support or labeled with a fluorescent compound, can bind Th1 cells to separate Th1 cells from Th2 cells.
A further aspect of the present invention is to expand the isolated or enriched Th1 or Th2 cells for reinfusion back into a patient from whom they were obtained in order to increase phagocyte dependent or phagocyte independent host defenses, respectively.
In addition, the present invention also provides a method for diagnosing Th1-mediated diseases or disorders by measuring in a fluid sample from a patient the amount of soluble LAG-3 that is bound to soluble LAG-3 specific antibody.
Accordingly, the present invention is directed to all of the above methods for using the discovery that the LAG-3 protein is preferentially associated with Th1 lymphocytes.
REFERENCES:
patent: 5874250 (1999-02-01), Hercend et al.
patent: 9110682 (1991-07-01), None
patent: 9530750 (1995-11-01), None
Huard et al., “Lymphocyte-activation gene 3/major histocompatibility complex class II interaction modulates the antigenic response of CD4 T lymphocytes”,Eur. J. Immunol., 24:3216-3221, (1994).
Del Prete et al., “Preferential expression of CD30 by human CD4 T cells producing Th2-type cytokines”The FASEB Journal, 9:81-86, (1995).
Maggi et al., “Reciprocal Regulatory Effects of IFN-y and IL-4 on the in vitro Development of Human Th1 and Th2 Clones”,The Journal of Immunology, vol. 148, No. 7, pp. 2142-2147, (1992).
Romagnani et al., “Human Th1 and Th2 cells”,Allergologie, vol. 19, No. 4, pp. 175-179, (1996).
Annuziato, F. et al., “Expression and release of LAG-3-encoded protein by human CD4+T cells are associated with IFN-gamma production.”, The FASEB Journal, vol. 10, pp. 769-776 (1996).
Waldmann, T., “Monoclonal antibodies in diagnosis and therapy.”, Science, vol. 253, pp. 1657-1662 (1991).
Harris, W. et al., “Therapeutic antibodies—the coming of age.”, Forum-Tibtech, vol. 11, pp. 42-44 (1993).
Foon, K. et al., “Biological response modifiers: the new immunotherapy.”, Cancer Research, vol. 49, pp. 1621-1639 (1989).
Riddell, S. et al., “Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones.”, Science, vol. 257, pp. 238-241 (1992).
Bruijin, M. et al., “Peptide loading of empty major histocompatibility complex molecules on RMA-A cells allows the induction of primary cytotoxic T lymphocyte responses.”, Eur. J. Immunol., vol. 21, pp. 2963-2970 (1991).
Macatonia, S. et al., “Primary stimulation by dendritic cells induces antiviral proliferative and cytotoxic T cell responses i
Browdy and Neimark
Institute National de la Sante et de la Recherche Medicale
Schwadron Ronald B.
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