Methods of using integrin modulators for treatment of...

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C514S706000

Reexamination Certificate

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06177475

ABSTRACT:

BACKGROUND OF THE INVENTION
Integrins are heterodimeric transmembrane glycoproteins which act, inter alia, as cell receptors for surface molecules of other cells and for extracellular matrix (ECM) proteins, herein termed collectively “integrin ligands.” Both soluble and immobilized integrin ligands are known to be ordinarily bound by integrins. Integrins are found on most types of cells. Ligand binding by integrins may result in a series of additional cellular events. When these additional events occur, they involve one or more cellular functions. These cellular events and functions, some of which are discussed below for illustrative purposes, are integrin-mediated. For a general review of integrins, see,
Guidebook to the Extracellular Matrix and Adhesion Proteins
(Kreis, et al., Eds.), 1993, and Pigot, et al.,
The Adhesion Molecule Facts Book,
Academic Press, 1993.
One such integrin-mediated cellular function is signaling. For instance, certain integrins are known to transfer information from the inside to the outside of the cell (inside-out signaling), and/or from the outside to the inside of the cell (outside-in signaling), although other types of signaling may also occur. An example of inside-out signaling is the process whereby an integrin acquires affinity for ligands in response to cell activation-associated intracellular events (integrin upregulation). Binding of integrin ligands to certain integrins (e.g., in the case of integrin-mediated cell adhesion) may initiate signal transduction events, in a manner similar to that described for other cell surface receptors. The signals thus elicited are termed outside-in signals and regulate various cell responses, such as gene expression, cell differentiation, and cell proliferation.
Signaling may result in integrin clustering, i.e., the association of integrins with each other (and other molecules) by lateral interactions. The formation of such clusters may influence various integrin functions in multiple ways, including, for example, by secondary signaling.
The integrin-mediated function of adhesion is important for a variety of physiological and pathological responses. The extent of adhesion is controlled by integrin signaling. For example, as a result of initial integrin-dependent adhesion to a substratum, certain cells change their shape and start spreading on its surface, using integrins for establishing new contacts with the underlying proteins (e.g., ECN components). In motile cells, the whole array of integrin-mediated events involving adhesion—initial contact, cell shape change, cell spreading, and cell locomotion—is sometimes termed “the adhesion cascade” (Sharar, S.R., et al., The Adhesion Cascade and Anti-Adhesion Therapy: An Overview, 16
Springer Semin. Immunopathol.
359, 1995). Examples of adhesion cascades include tumor cell metastasis, cell migration processes associated with wound healing, and lymphocyte homing, although similar cascade mechanisms are operative in the absence of locomotion (e.g., platelet adhesion and aggregation). Extravasation of neutrophils is described below in greater detail, as a paradigmatic integrin-driven adhesion cascade (Hub, E., et al., Mechanism of Chemokine-Induced Leukocyte Adhesion and Emigration,
Chemoattractant Ligands and Their Receptors
(Horuk, R., Ed.), Boca Raton, CRC Press, 1996, 301).
The onset of extravasation is signaled by the appearance in the circulation of chemotactic factors, or chemoattractants (i.e., specific substances that initiate cell migration along their concentration gradients). Chemoattractants (e.g., chemokines, bacterial peptides, and products of complement activation) activate neutrophils and upregulate their integrin receptors (neutrophil integrins include, e.g., LFA-1 [CD11a/CD18], CR3 [also known as Mac-1, CD11b/CD18], and gpl50,95 [CD11c/CD18].) As a result, the activated neutrophils adhere to endotheliocytes, change shape, and spread on the endothelial surface. Thereafter, the motile apparatus of the neutrophils is stimulated by the chemoattractants, and they start migrating, first across the endothelial layer and further, through the perivascular ECK, towards the source of the chemotactic stimulus, e.g., pathogenic bacteria invading a certain bodily tissue. During the whole process, from the initial firm contact with the endothelium to the cessation of locomotion at the destination site, various integrins serve to attach the neutrophil to the substrata it encounters, enabling its recruitment to the locus of infection.
Another integrin-mediated function is cell-to-cell fusion. Under physiological conditions, fusion is a developmentally regulated stage in the differentiation of certain multinucleate cells (e.g., osteoclasts, myocytes, and syncytiotrophoblasts) and a prerequisite to fertilization (in the case of sperm-egg fusion). Fusion is effected by specialized cellular systems involving integrins (see, e.g., refs. cited in Huovila, A.-P.J., et al., ADAMs and Cell Fusion, 8
Current Olin. Cell. Biol.
692, 1996 and Ohgimoto, S., et al., Molecular Characterization of Fusion Regulatory Protein-1 [FRP-1] that Induces Multinucleate Giant Cell Formation of Monocytes and HIV gp160-Mediated Cell Fusion: FRP-1 and 4F2/CD98 Are Identical Molecules, 155
J. Immunol.
3585, 1995).
The ability to undergo recirculation from intracellular compartments to the cell surface and vice versa is a common property of divers cellular receptors, including integrins (see, e.g., Handagama, P., et al., Kistrin, an Integrin Antagonist, Blocks Endocytosis of Fibrinogen into Guinea-Pig Megakaryocyte and Platelet &agr;-Granules, 91
J. Clin. Invest.
193, 1993). This capability of integrins serves to mediate other cellular functions by transporting into the cell extracellular material (e.g., soluble proteins, particulate matter, and other cells). Integrin-mediated internalization is used by certain microorganisms to invade their targets. For example, CR3 mediates entry of iC3b-opsonized HIV-1 and HIV-2 into CD4-negative lymphocytic and monocytic cells (Boyer, V., et al., Complement Mediates Human Immunodeficiency Virus Type 1 Infection of a Human T cell Line in a CD4- and Antibody-Independent Fashion, 173
J. Exp. Med.
1151, 1991).
The above-delineated functions of integrins are illustrative only, as other characterizations of integrin functions can also be made. Moreover, the integrin-mediated functions as delineated herein are overlapping and interrelated. In the case of neutrophil extravasation, for example, the initial chemotactic signal activating the cells results in integrin upregulation (inside out signalina) and adhesion to the endothelial surface. This adhesion event, in turn, elicits an outside-in signal, enabling the neutrophil to change its shape, which is a prerequisite to spreading and migration. Likewise, when the neutrophil that has arrived to the source of chemoattractants adheres to the bacteria, an outside-in signal transduced via the involved integrins initiates their internalization, together with the attached bacteria (phagocytosis).
Furthermore, regarding outside-in integrin signaling, certain cellular processes are coregulated by several distinct signaling systems acting in a concert. In the case of neutrophils extravasating to the tissues to phagocytose bacteria, the cells receive signals via distinct integrins (first from those that attach it to the substratum and subsequently from those recognizing the bacteria) and the receptors of the chemoattractant (along the concentration gradient of which the movement occurs). This interplay of signals regulates the antibacterial machinery of the neutrophils in such a way that only upon contact with the bacteria, which is established via a particular type of integrin, are the constituents of the intracellular granules released and reactive oxygen species formed. As a result, the formation and release of microbicidal substances take place preferentially at sites of contact with bacteria, enabling their effective killing and preventing the destruction of host

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