Drug – bio-affecting and body treating compositions – In vivo diagnosis or in vivo testing
Reexamination Certificate
2000-09-11
2003-04-01
Nolan, Patrick J. (Department: 1644)
Drug, bio-affecting and body treating compositions
In vivo diagnosis or in vivo testing
C435S029000
Reexamination Certificate
active
06540977
ABSTRACT:
BACKGROUND OF THE INVENTION
Immunoglobulin G (IgG) Receptors (Fc&ggr;R)
An IgG antibody molecule has two binding determinants (known as Fab) for its specific cognate antigen, and one binding determinant (Fc) for its cellular receptor (Fc&ggr;R; reviewed in Ravetch, J. V. and J-P. Kinet, 1991,
Ann. Rev. Imm.
9:457). There are three classes of Fc&ggr;R, designated Fc&ggr;RI, Fc&ggr;RII and Fc&ggr;RIII. Fc&ggr;RI has the highest affinity for IgG Fc, and is present on monocytes and macrophages, and on human neutrophils when induced by gamma-interferon (IFN-&ggr;), which also enhances Fc&ggr;RI expression on monocytes and macrophages (Guyre, P. M. et al., 1983,
J.Clin.Investig.
72:393). Interleukin-10 (IL-10) is also a potent up-regulator of Fc&ggr;RI expression on monocytes (Capsoni, F. et al., 1995,
J. Leukoc. Biol.
58:351). Neutrophil expression of Fc&ggr;RI is increased almost four-fold by granulocyte-colony stimulating factor (G-CSF; Repp, R. et al., 1991,
Blood
78:885), and about three-fold during streptococcal pharyngitis (Guyre, P. M. et al., 1990,
J. Clin. Investig.
86:1892). G-CSF stimulation is associated with enhanced neutrophil-mediated cytotoxicity of tumor cells (Gosselin, E. et al., 1992,
J. Immunol.
149:3477).
Cellular distribution of the low affinity receptor Fc&ggr;RII is broader, extending to neutrophils, platelets, B cells, T cell, and possibly mast cells and basophils, in addition to monocytes and macrophages. Human Fc&ggr;RIII receptors are restricted to macrophages and macrophage cell lines, natural killer (NK) cells, myeloid precursor cells, and a neutrophil cell line (Ravetch and Kinet, supra).
The interaction of antibody-antigen complexes with cells via Fc&ggr;R triggers a range of responses including antibody-dependent cytotoxicity, degranulation, phagocytosis, and immunomodulatory signals that regulate lymphocyte proliferation and antibody secretion. Fc&ggr;RI, for example, is active in enhancing antigen presentation (Valerius, T. et al., 1993,
Blood
82:931) and the Fc&ggr;RI complementarity designation CD64 has been used to distinguish granulomonocytic progenitor cell populations within a human progenitor cell mixture (CD34
+
/CD38
+
; European Patent Application EP O 708 336 A2).
Further, a monoclonal antibody (mAb) that binds Fc&ggr;RI has been shown to cause down-regulation of expression of this receptor, and administration of this mAb to a chronic immune thrombocytopenia purpura patient resulted in clinical improvement (Ericson, S. G., 1996,
Brit. J. Haematol.
92:718). The analysis of in vivo function of Fc&ggr;RI receptors is complicated by the finding that four healthy members of one family lack Fc&ggr;RI (Ceuppens, J. L., 1988,
J. Clin. Invest.
82
:
571
).
Cell Staining with PE-Cy5
The tandem dye PE-Cy5 comprises the combination of two fluorescent dyes, R-phycoerythrin (PE) and Cy5.18.OSu (Cy5; Mujumdar, R. B. et al., 1993,
Bioconj. Chem.
4:105). PE-Cy5 is commonly used conjugated to a monoclonal antibody (mAb), as described in for example, the Jan. 1, 1996, issue of Blood that reviews the structure and biology of CD34, accompanied by a cover photograph of a flow cytometric experiment with PE-Cy5 conjugated to an anti-CD34 mAb (Krause D S, et al., 1996,
Blood
87:1). PE-Cy5 tandem dye is known as “tricolor” since it is frequently used with other dye-mAb conjugates for three-color flow cytometric analyses (Waggoner A S, et al., In: Clinical Flow Cytometry, p.185 (Eds) A. Landay et al. The New York Academy of Sciences, New York, N.Y., 1993).
A non-toxic molecule that binds specifically to Fc&ggr;RI in vivo would be useful in a variety of diagnostic and therapeutic applications.
SUMMARY OF THE INVENTION
In general the invention relates to agents that specifically bind IgG receptors of the class Fc&ggr;RI on white blood cells (leukocytes). Because of the functional binding of these compounds, the class of compositions is referred to herein as “GRIL” to indicate an IgG receptor I ligand. The agents are members of the cyanidin family of naturally-occurring plant pigments, exemplified by cyanin, idaein, keracyanin and asterin.
In one aspect, the invention features a class of cyanin compositions of which the fluorescent dye Cy5.18.OSu (referred to as Cy5), and conjugates and derivatives, are examples. The general features of these compositions are firstly functional, which is that they bind with high affinity and specificity to Fc&ggr;RI IgG receptors found on monocytes and macrophages, and which can also be induced to appear on neutrophils. The second feature is the chemical structure, which may be comprised of at least two moieties, one of which being the cyanin succinimidyl ester composition class. In one embodiment, the second moiety is derived from any of the phycobilisome proteins of which PE is an exemplary but not limiting member.
In another aspect, the present invention features therapeutic compositions comprising a GRIL, for example, a cyanin compound exemplified by a Cy5 reagent, and a therapeutic agent, which therapeutic agent in a preferred embodiment comprises at least one epitope. In one embodiment, the GRIL directs delivery of the useful epitope to Fc&ggr;RI, and thus comprises a composition which is both vaccine and delivery system. The epitope can be selected from the broad group consisting of a pathogenic organism, a cancer marker, and an allergenic substance. The compounds can thus be used to vaccinate a subject against a pathogenic organism, such as a bacterium, a protozoan, a virus or a fungus. In a preferred embodiment, the epitope for a vaccine is from a Gram positive pathogen, for example, from a cellular or secreted epitope of
Staphyloccoccus aureus
or
Clostridium tetani.
The invention also features a vaccine against one or more types of cancer, comprising a GRIL and an epitope from a cancer marker which, in a preferred embodiment, is selected from the group consisting of the human EGF receptor family, the mucine TAG72 family, and the carcinoembryonic family.
In another aspect, the present invention features therapeutic compositions comprising a GRIL and a chemotherapeutic cytotoxic anticancer agent, for example, actinomycin D, mitomycin C, adriamycin, taxol, notirimine, or a therapeutic composition comprising a Cy5 reagent and a radionuclide. The therapeutic agent is thus targetted to Fc&ggr;RI by virtue of the presence of the cyanin, for example a Cy5 or similar reagent, and is useful for treatment of, for example, a myelocytic leukemia, a promyelocytic leukemia or a leukocyte adhesion deficiency.
Methods for targetting Fc&ggr;RI receptors with a chemotherapeutic or radiotherapeutic GRIL is another aspect of this invention, for the purpose of treating for example, a patient with an autoimmune disease, such as idiopathic thrombocytopenic purpura. Since the agents of the instant invention target IgG receptors, which are located on white blood cells, the methods of the invention include delivery of these compositions ex vivo to cells obtained from a blood sample or a bone marrow sample removed from the patient for such treatment, which blood or bone marrow cell sample or its cellular progeny is subsequently returned to that patient. Further, an interferon, an interleukin or a colony stimulating factor may also be administered to the patient or to the ex vivo blood or bone marrow sample or to cells derived from such samples. In preferred embodiments, the patient is administered IFN-&ggr;, IL-10, or G-CSF.
Another aspect of the invention features compositions and diagnostic methods for detecting and quantifying Fc&ggr;RI (CD64) in a blood sample or a bone marrow sample from a subject, comprising the steps of contacting the sample with a GRIL stain, removing nonspecifically-bound reagent, illuminating the sample with light of an appropriate excitation wavelength, and detecting the fluorescent cells as an indication of Fc&ggr;RI-bearing cells in the blood sample. Preferably, at least one additional dye reagent, for example, a fluorescein reagent or a phycoerythrin reagent is used in conjunction wi
DeConti, Jr. Esq. Giulio A.
Lahive & Cockfield LLP
Medarex Inc.
Nolan Patrick J.
Remillard, Esq. Jane E.
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