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  Pharmaceutical Patents  

 

Title:  Product and method for treatment of conditions associated with receptor-desensitization
United States Patent: 
7,825,224
Issued: 
November 2, 2010

Inventors:
 Vilen; Barbara J. (Chapel Hill, NC), Cambier; John C. (Denver, CO)
Assignee:
  National Jewish Health (Denver, CO)
Appl. No.:
 11/825,147
Filed:
 July 2, 2007


 

Patheon


Abstract

Particular members of the multisubunit immune recognition receptor (MIRR) family of receptors, specifically, the B cell antigen receptor (BCR), the pre-B cell receptor (pre-BCR), the pro-B cell receptor (pro-BCR), Ig Fc receptors (FcR), and NK receptors, can be physically uncoupled from their associated transducers. The invention describes regulatory compounds and methods for mimicking such dissociation/destabilization for the purposes of receptor desensitization and for treatment of conditions in which receptor desensitization or alternatively, enhanced or prolonged receptor sensitization, is desirable. Compounds and methods for enhancing or prolonging receptor sensitization are also disclosed, as are methods for identifying regulatory compounds suitable for use in the present methods.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention generally relates to methods and compounds for desensitizing a receptor selected from the group consisting of a B cell antigen receptor (BCR), a pro-B cell receptor (pro-BCR), a pre-B cell receptor (pre-BCR), immunoglobulin Fc receptor (FcR) and natural killer (NK) cell receptor. Such a method includes the step of contacting a compound with such a receptor that has an extracellular ligand binding component and a transducer component, wherein contact with the compound: (1) causes a dissociation of the extracellular ligand binding component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the extracellular ligand binding component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the receptor.

The present invention also relates to a method and compounds for sensitizing or prolonging/enhancing sensitization of a receptor selected from the group consisting of BCR, pro-BCR, pre-BCR, FcR and NK receptor. Such a method includes the step of contacting a compound with such a receptor that has an extracellular ligand binding component and a transducer component, wherein the compound: (1) causes the extracellular ligand binding component to associate with the transducer component when the two components are not associated with each other prior to contact by the compound; and/or (2) prolongs or enhances the time over which the extracellular ligand binding component is associated with the transducer component when the components are associated prior to contact by the compound, thereby sensitizing the receptor.

More particularly, one embodiment of the present invention relates to a method to desensitize a B cell antigen receptor, and preferably, by selectively desensitizing a B cell antigen receptor which binds to a specific antigen. Such a method is useful for treating any B cell-related disorder in which desensitization of the B cell antigen receptor would provide a therapeutic benefit, alone or in conjunction with another treatment. Such a method is also useful for research and diagnostic assays, and for screening putative regulatory compounds. Such method includes the step of contacting a regulatory compound with a B cell antigen receptor that has an mIg component and a transducer component including Ig.alpha. and/or Ig.beta., wherein contact with the compound: (1) causes a dissociation of the mIg component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the mIg component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the B cell antigen receptor. The mIg component can be either IgD or IgM. A B cell-disorder that can be treated by the present method can include, but is not limited to, autoimmune disease (e.g., rheumatoid arthritis or systemic lupus erythematosus), malignancies and transplantation. A particularly preferred disorder to treat with the method of the present invention is an autoimmune disease. Preferably, the compound selectively targets a BCR having a particular antigen specificity, such as a B cell antigen receptor which specifically binds to an autoantigen. Such a method is advantageous in that functions of normal or desirable B cells can be left intact, while functions of abnormal or undesirable B cells can be inhibited.

Another embodiment of the present invention relates to a method to sensitize or prolong/enhance sensitization of a BCR. Such a method is useful, for example, for increasing or inducing a B cell response to a given antigen or antigens, and can be used in a vaccine or adjuvant system. In one embodiment, the method includes the step of contacting a compound with a B cell antigen receptor that has an mIg component and a transducer component including Ig.alpha. and Ig.beta., wherein the compound: (1) causes the mIg component to associate with the transducer component when the components are not associated with each other prior to contact by the compound; and/or (2) prolongs or enhances the time over which the mIg component is associated with the transducer component when the components are associated prior to contact by the compound, thereby enhancing sensitization of the B cell antigen receptor for treatment of the disorder. In one embodiment of this method, an additional factor can be contacted with the B cell antigen receptor, such as an antigen or other factor which enhances vaccination against a given antigen or enhancement of the B cell antigen response.

One embodiment of the present invention relates to a method to treat autoimmune disease, including the step of contacting a compound with an autoreactive B cell antigen receptor that has an mIg component associated with a transducer component including Ig.alpha. and Ig.beta., wherein contact with the compound: (1) causes a dissociation of the mIg component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the mIg component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the B cell antigen receptor for treatment of an autoimmune disease.

Another embodiment of the present invention relates to a method desensitize an Ig Fc receptor (FcR). Such a method is useful for treating any disorder in which desensitization of an FcR, and particularly, a specific FcR, would provide a therapeutic benefit, alone or in conjunction with another treatment. Such a method is also useful for research and diagnostic assays, and for screening putative regulatory compounds. Such disorders include, but are not limited to, an allergic disorder; and disorders related to inflammatory responses including antibody-dependent cell-mediated cytotoxicity, release of inflammatory mediators and regulation of antibody production, and more particularly include, but are not limited to, thrombocytopenia purpura, rheumatoid arthritis, systemic lupus erythematosus, type II and type III hypersensitivity reactions, and allergic inflammation. The method includes the step of contacting a compound with an Fc receptor that has an .alpha. receptor component and a transducer component, wherein contact with the compound: (1) causes a dissociation of the .alpha. receptor component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the .alpha. receptor component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the Fc receptor.

Another embodiment of the present invention relates to a method to treat allergic disorders, including the step of contacting a compound with an Fc.epsilon.RI receptor that has an .alpha. receptor component and a transducer .beta./.gamma. component, wherein contact with the compound: (1) causes a dissociation of the .alpha. receptor component from the transducer component when the two components are associated prior to contact with the compound, and/or (2) inhibits association of the .alpha. receptor component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the Fc.epsilon.RI receptor for treatment of the allergic disorder.

Another embodiment of the present invention relates to a method to identify compounds useful for desensitizing a receptor, including the steps of: (1) providing an assay system including a receptor selected from a B cell antigen receptor, a pro-B cell receptor, a pre-B cell receptor or an Fc receptor, wherein the receptor includes an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is associated with the transducer component; (2) contacting the receptor with a compound to be evaluated; and, (3) determining whether the compound, when contacted with the receptor, is capable of causing the extracellular ligand binding component to dissociate from the transducer component. In the case of a B cell antigen receptor, the extracellular ligand binding component is mIg and the transducer components are Ig.alpha. and Ig.beta.. In the case of an FcR receptor, the extracellular ligand binding receptor is typically the .alpha. receptor chain, and the transducer component varies depending on the specific FcR, as is known in the art. For the Fc.epsilon.RI, the transducer components are .beta. and .gamma. chains.

Yet another embodiment of the present invention relates to a method to identify compounds useful for desensitizing a receptor, including the steps of: (1) providing an assay system including a receptor selected from a B cell antigen receptor or an Fc receptor, wherein the receptor includes an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is not associated with the transducer component; (2) contacting the receptor with a compound to be evaluated; and, (3) determining whether the compound, when contacted with the receptor, is capable of inhibiting the extracellular ligand binding component from associating with the transducer component.

The above methods can include cell-based assays and non-cell based assays. Compounds identified by the above methods are useful for treating a variety of B cell and FcR-associated disorders, including autoimmune disease and allergic disorders. The methods of desensitizing/sensitizing a receptor and compounds useful in such methods can be performed/used in vivo, in vitro, and/or ex vivo. In addition, the above-described regulatory compounds, including those identified by the present method, can be used in a therapeutic composition and in a method of treatment or desensitization of the present invention.

Yet another embodiment of the present invention relates to a compound useful for treating a condition selected from the group consisting of a B cell-associated disorder and an FcR-associated disorder. Such a compound is characterized by its ability to: (1) cause a dissociation of an extracellular ligand binding component from a transducer component in a B cell antigen receptor or an Fc receptor when the two components are associated prior to contact with the compound, and/or (2) inhibit association of the extracellular ligand binding component with the transducer component when the two components are dissociated prior to contact with the compound, thereby desensitizing the receptor. In a preferred embodiment, the compound is selected from an antibody, a peptide, or a mimetope thereof. In one embodiment, the compound is an antibody. Such an antibody can be a monovalent antibody, a divalent antibody, or a bi-specific antibody.

Another embodiment of the present invention relates to an isolated antibody which selectively binds to a BCR, wherein the antibody, upon binding to such a BCR having an mIg component associated with Ig.alpha. and Ig.beta. components, is capable of inducing the mIg component to dissociate from the Ig.alpha. and Ig.beta. components, thereby desensitizing the BCR. In one embodiment, the BCR is expressed by a B cell. Such an antibody can be used in a method of the present invention to desensitize a B cell antigen receptor.

Another embodiment of the present invention relates to an isolated antibody which selectively binds to an FcR, wherein the antibody, upon binding to such an FcR that has an .alpha. receptor component associated with at least one transducer component, is capable of inducing the .alpha. receptor component to dissociate from the transducer component, thereby desensitizing the FcR. In one embodiment, the FcR is Fc.epsilon.RI, and the transducer components are .beta. and .gamma. chains. In another embodiment, the Fc.epsilon.RI is expressed by a mast cell, a basophil or an eosinophil. Such an antibody can be used in a method of the present invention to desensitize an Fc receptor.

DETAILED DESCRIPTION OF THE INVENTION

B cell antigen receptor (BCR) ligation leads to receptor desensitization wherein BCR remain competent to bind antigen yet fail to transduce signals. Desensitized BCR exhibit a defect at the most proximal level of signal transduction, consistent with failed transmission of signals through the receptor complex. The present inventors have discovered that antigen stimulation leads to dissociation or destabilization of the BCR reflected by inability to coimmunoprecipitate Ig-.alpha./Ig-.beta. with mIg. This destabilization is temporally correlated with desensitization and occurs in BCR containing mIgM and mIgD. Induction of BCR destabilization requires tyrosine kinase activation, but is not induced by phosphatase inhibitors. BCR destabilization occurs at the cell surface and "dissociated" Ig-.alpha./Ig-.beta. complexes remain responsive to anti-IG-.beta. stimulation, indicating that mIg-transducer uncoupling mediates receptor desensitization. More particularly, the present inventors have shown that upon binding of moderate to low affinity antigen, the Ig-.alpha./Ig-.beta. subunits of the BCR become destabilized or physically dissociated from mIg. This event requires specific activation of the BCR signaling cascade. Most interestingly, although desensitized receptors fail to respond to receptor ligation, the Ig-.alpha./Ig-.beta. complex retains signaling function if aggregated, indicating that transducer dissociation from mIg mediates the unresponsive state.

The present invention generally relates to the discovery that particular members of the multisubunit immune recognition receptor (MIRR) family of receptors, specifically, the B cell antigen receptor (BCR), the pre-B cell receptor (pre-BCR), the pro-B cell receptor (pro-BCR), Ig Fc receptors (FcR), and NK receptors, can be physically uncoupled from their associated transducers. One embodiment of the present invention relates to a method to desensitize a receptor selected from the group of a B cell antigen receptor, a pro-B cell receptor, a pre-B cell receptor and an Ig Fc receptor. Another embodiment of the present invention relates to a method to desensitize a receptor selected from the group of a B cell antigen receptor, a pro-B cell receptor, a pre-B cell receptor, an Ig Fc receptor and an NK receptor. The method includes the steps of contacting such a receptor with a regulatory compound, wherein the receptor has a transducer component and an extracellular ligand binding component. The regulatory compound: (1) causes a dissociation of the extracellular ligand binding component from the transducer component when the components are associated with each other prior to contact with the compound; or (2) inhibits association of the extracellular ligand binding component with the transducer component when the components are dissociated from each other prior to contact with the compound.

In the case of the B cell antigen receptor (BCR), the present inventors have discovered that desensitization of the receptor involves an uncoupling of Ig-.alpha./.beta. from mIg. The present inventors provide evidence herein that uncoupling of the BCR transducer (Ig-.alpha./.beta.) from the antigen binding receptor (mIg heavy and light chains) is responsible for the desensitized phenotype exhibited by B cells following antigen stimulation. The present inventors' discovery regarding the BCR has led to the additional conclusion that the FcR, and particularly, Fc.epsilon.RI will, upon desensitization of this receptor in mast cells, exhibit uncoupling of the transducer component (e.g., .beta./.gamma. subunit for Fc.epsilon.RI) from the receptor. This discovery has tremendous therapeutic potential in that one could intervene and prevent signaling through these receptors. In the case of mast cell degranulation, this mechanism of desensitization can be manipulated to treat allergic disorders, for example. In the case of the BCR, such a mechanism can be manipulated for the treatment of autoimmune disease, for example. Additionally, using the method of the present invention, the extracellular ligand binding component of the natural killer (NK) cell receptor, referred to as KIRDL, can be desensitized by affecting the association of the KIRDL with its transducer, referred to as DAP12.

According to the present invention, a transducer, or transducer component, is a component (i.e., portion, constituent, element) of a receptor, and typically one or more chains of a receptor, which transduces a signal from the receptor to other signal transduction molecules inside a cell when the receptor is ligated by its ligand. More specifically, by associating with an extracellular ligand binding component, a transducer component allows the extracellular ligand binding component(s) of a receptor to associate indirectly with cytosolic enzymes that provide the means of intracellular signaling when the extracellular ligand binding component is bound by its ligand, or an equivalent stimulus. In B cells, including pro-B, pre-B, immature and mature B cells, the transducer component is typically an Ig.alpha. and an Ig.beta. chain, which form a dimer. For Fc receptors, a transducer component is typically one or more chains associated with an .alpha. chain of the receptor, which are sufficient to associate with cytosolic enzymes that provide the means of intracellular signaling when the extracellular ligand binding component is bound by its ligand, or an equivalent stimulus. For example, tranducer components of an Fc.epsilon.RI include a .beta. chain and a dimer of .gamma. chain. An Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIc, Fc.gamma.RIII and Fc.alpha.R's typically have a .gamma. chain dimer transducer component. An NK cell transducer component is DAP12. As used herein, a transducer component can include naturally occurring transducer components or any portion thereof that interacts with an extracellular ligand binding component as described herein and that is sufficient to transduce a signal from the receptor upon appropriate stimulation.

According to the present invention, an extracellular ligand binding component is a component (i.e., portion, constituent, element) of a receptor, and typically one or more chains of a receptor, at least a portion of which extends out of the cell surface (i.e., is extracellular) and which is the portion of a receptor which binds to a ligand. The extracellular ligand binding component may also have a transmembrane and a cytoplasmic portion. Typically, the extracellular ligand binding component is not a component of the receptor which is responsible for intracellular signaling by the receptor. By way of example, the extracellular ligand binding component of an immature B cell is a mIgM, which includes the antigen binding portion of the B cell antigen receptor; the extracellular ligand binding component of receptors on a mature, naive B cell includes both mIgM and mIgD. For Fc receptors, the extracellular ligand binding component is typically an .alpha. chain of the receptor, and is the portion of the receptor which binds the Fc region of an immunoglobulin. For NK receptors, the extracellular ligand binding component is referred to as KIRDL. As used herein, an extracellular ligand binding component can include a naturally occurring extracellular ligand binding component or any portion of thereof that interacts with a transducer component as described herein and/or that is sufficient to bind to a ligand for the receptor or to an equivalent stimulus.

As discussed above, B cell receptors which are suitable targets for the methods of the present invention include B cell antigen receptors (e.g., on immature or mature B cells), pro-B cell receptors, and pre-B cell receptors. A pro-B cell appears during B cell differentiation before immunoglobulin gene rearrangement has begun, and is identified by surface markers characteristic of B cells, including extracellular components (e.g., calnexin) that can associate with an Ig.alpha.-Ig.beta. transducer component. In late pro-B cell development, a V.sub.H segment becomes joined to the DJ.sub.H segment (discussed in detail below), producing a pre-B cell that expresses both low levels of surface and high levels of cytoplasmic .mu. heavy chain. An immature B cell expresses both light chains and .mu. heavy chains as surface IgM molecules (i.e., mIgM). Finally, mature, naive B cells express both mIgM and mIgD. The cytoplasmic tail of these transmembrane B cell receptor components, such components generally referred to herein as an extracellular ligand binding component, consists of only a few amino acids and is too short to interact with the proteins required for intracellular signaling. Transmission of signals instead depends on two other chains associated with the extracellular ligand binding component. These transducer components are called Ig.alpha. and Ig.beta.. The transducer components allow the extracellular ligand binding components of the receptor to associate with cytosolic enzymes that provide the means of intracellular signaling when B cells come into contact with antigen.

According to the present invention, reference to "B cells" or "B lymphocytes" includes splenic B cells, lymph node B cells, myeloma cells, peripheral blood B cells, bone marrow B cells and hybridoma cells. Hybridoma cells refer to hybrid cell lines comprising myeloma cells (tumor cells capable of being maintained in tissue culture but do not produce immunoglobulin) fused with, for example, a spleen cell capable of producing an immunoglobulin molecule. Reference to a "B cell antigen receptor" or "BCR" is intended to reference the B cell antigen receptor, which includes a membrane immunoglobulin (mIg) antigen binding component (i.e., an extracellular ligand binding component), or a biologically active portion thereof (i.e., a portion capable of binding a ligand and/or capable of associating with a transducer component), and transducer Ig-.alpha. and Ig-.beta. components, or biologically active portions thereof (i.e., a portion capable of transducing an intracellular signal and/or capable of associating with an extracellular ligand binding portion).

According to the present invention, Fc receptors include Fc gamma receptors (Fc.gamma.R) which bind gamma immunoglobulin (IgG), Fc epsilon receptors (Fc.epsilon.R) which bind epsilon immunoglobulin (IgE), Fc alpha receptors (Fc.alpha.R) which bind alpha immunoglobulin (IgA). Fc.gamma.R include, Fc.gamma.RI, which is a high a affinity receptor for IgG; Fc.gamma.RII, which are low affinity receptors for IgG that avidly bind to aggregates immune complexes; and Fc.gamma.RIII, which are low affinity receptors that bind to immune complexes.

According to the present invention, NK receptors include an extracellular ligand binding component, KIRDL, and a transducer component, DAP12.

According to the present invention, reference to an "Fc receptor" or "FcR" refers to one or more members of a family of highly related receptors that specifically bind to the Fc portion of immunoglobulin (Ig). FcR can also be referred to herein as immunoglobulin Fc receptors or Ig Fc receptors (Ig FcR). These receptors have major roles in normal immunity and resistance to infection and provide the humoral immune system with a cellular effector arm. Receptors have been defined for each of the immunoglobulin classes and as such are defined by the class of Ig of which they bind (i.e. Fc gamma receptor (Fc.gamma.R) bind gamma immunoglobulin (IgG), Fc epsilon receptor (Fc.epsilon.R) bind epsilon immunoglobulin (IgE), Fc alpha receptor (Fc.alpha.R) bind alpha immunoglobulin (IgA)). Among the Fc.gamma.R receptors, three subfamily members have been defined; Fc.gamma.RI, which is a high a affinity receptor for IgG; Fc.gamma.RII, which are low affinity receptors for IgG that avidly bind to aggregates immune complexes; and Fc.gamma.RIII, which are low affinity receptors that bind to immune complexes. These receptors are highly related structurally but perform different functions.

Fc.gamma.R are expressed on most hematopoietic cells, and through the binding of IgG play a key role in homeostasis of the immune system and host protection against infection. Fc.gamma.RII is a low affinity receptor for IgG that essentially binds only to IgG immune complexes and is expressed on a variety of cell types including, for example monocytes, macrophages, neutrophils, eosinophils, platelets and B lymphocytes. Fc.gamma.RII is involved in various immune and inflammatory responses including antibody-dependent cell-mediated cytotoxicity, clearance of immune complexes, release of inflammatory mediators and regulation of antibody production. The binding of IgG to an Fc.gamma.R can lead to disease indications that involve regulation by Fc.gamma.R. For example, the autoimmune disease thrombocytopenia purpura involves tissue (platelet) damage resulting from Fc.gamma.R-dependent IgG immune complex activation of platelets or their destruction by Fc.gamma.R+ phagocytes. In addition, various inflammatory disease are known to involve IgG immune complexes (e.g. rheumatoid arthritis, systemic lupus erythematosus), including type II and type III hypersensitivity reactions. Type II and type III hypersensitivity reactions are mediated by IgG, which can activate either complement-mediated or phagocytic effector mechanisms, leading to tissue damage.

Fc.epsilon.R are expressed on mast cells, basophils and eosinophils, and through the binding of IgE, trigger an inflammatory immune response which is primarily due to the release of inflammatory mediators upon degranulation of the mast cell (e.g., histamine and serotonin). Release of these mediators causes localized vascular permeability and increase in fluids in the local tissues, including an influx of polymorphonuclear cells into the site. Thus, binding of IgE to an Fc.epsilon.RI can lead to disease indications that involve discharge of fluids from the gut and increased mucus secretion and bronchial contraction, such indications typically being associated with diseases involving allergic inflammation.

Natural killer (NK) cells are identified by their ability to kill certain lymphoid tumor cell lines in vitro without the need for prior immunization or activation. In vivo, NK cells play a role in innate immunity by providing early protection from a range of pathogens. Such mechanisms can hold infection in check during its early phases while the adaptive immune response is developing.

According to the present invention, "receptor desensitization" refers to a state of "unresponsiveness" of a receptor, or in other words, a state wherein the receptor remains competent to bind its ligand, yet fails, or has a reduced ability, to transduce signals. Although a desensitized receptor fails to respond to receptor ligation by transducing an intracellular signal, the present inventors have discovered that the transducer component retains signaling function if aggregated, indicating that transducer dissociation from the extracellular ligand binding component mediates the unresponsive state. Receptor desensitization is characterized, for example, by the inability of ligand to elicit tyrosine phosphorylation or renewed Ca.sup.2+ mobilization in a cell expressing the receptor, despite the continued expression of ligand binding receptors. More particularly, a receptor desensitization occurs according to the present invention by a physical uncoupling, dissociation or destabilization, of the extracellular ligand binding component of the receptor from the transducer component of the receptor.

According to the method of the present invention, a receptor is contacted with a regulatory compound. As used herein, a regulatory compound can be any compound which, when contacted with a receptor according to the present invention: (1) causes a dissociation of the extracellular ligand binding component from the transducer component when the components are associated with each other (i.e., prior to contact with the compound); or (2) inhibits association of the extracellular ligand binding component with the transducer component when the components are dissociated from each other (i.e., prior to contact with the compound). A regulatory compound is a compound that mimics the desensitized state through inducing uncoupling of the transducers of these receptors. A regulatory compound for desensitization of a receptor according to the present invention therefore preferably does not stimulate the receptor upon contact with the receptor, or has substantially reduced stimulatory ability as compared to a natural ligand or its equivalent stimulus, for example. Such compounds are useful in the method of desensitizing a receptor of the present invention. Without being bound by theory, the present inventors believe that the uncoupling of a receptor from its transducer components may be due to a mechanism that includes phosphorylation of serine/threonine/tyrosine residues in TM (transmembrane) domain of the extracellular ligand binding component, such as .mu. (i.e., mIg) in the BCR. In the BCR, this phosphorylation-mediated mechanism is supported by both the rapidity of uncoupling/dissociation and the requirement for kinase activation in BCR destabilization. This may seem somewhat counterintuitive given the .mu.TM would be "shielded" from kinase by its location in lipid bilayer. However, the definition of "TM" residues versus "cytoplasmic" residues is based on protein algorithms, and although Kyle/Doolittle define this as lipid embedded, the present inventors believe that these residues can be cytoplasmic under certain conditions (i.e., antigen binding). The present inventors believe that the region may be kinase accessible only following antigen binding since: 1) the receptors are initially coupled prior to antigen binding; and 2) only antigen or anti-idiotype, but not anti-Ig-.beta., anti-Ig-.alpha., anti-.lamda. or pervanadate can induce dissociation. This suggests an antigen induced conformational change that renders the target residue accessible to kinase located in the cytoplasm. Additionally, and without being bound by theory, the present inventors believe that a portion of the extracellular portion of a BCR may be associating with the Ig-.alpha. or Ig-.beta. chain prior to antigen binding, such region moving to become accessible to phosphorylation subsequent to antigen binding. An equivalent scenario applies to Fc receptors.

Therefore, in one embodiment of the present invention, a regulatory compound useful in the present invention preferably binds to a site on a receptor which includes the antigen binding site or a site that is extracellular prior to antigen/ligand binding, and which is associated with a transducer component of the receptor and/or is phosphorylated in the cytoplasmic region subsequent to antigen/ligand binding. Suitable sites include a site on the transducer component or on the extracellular ligand binding component. Preferably, the site is on the transducer component. When the site is on the extracellular ligand binding component, it is preferably to target a site that is unique to the membrane form of the extracellular ligand binding site, or to target this component in receptors which have low levels of circulating (soluble) receptor. Alternate sites to target include transmembrane and/or cytoplasmic sites which are involved in association of the receptor components and or is phosphorylated upon antigen/ligand binding. Such transmembrane or cytoplasmic sites may be made available using delivery vehicles which are known in the art, and include liposomes and viral delivery systems. When the method of the present invention is directed to desensitization of the receptor, the target site is preferably a site other than the antigen/ligand binding site, to minimize signal induction through the receptor prior to dissociation of the components, although the antigen binding site is not excluded as a target site. When the method of the present invention is directed to sensitization of the receptor, the target site preferably includes the antigen/ligand binding site, to optimize, enhance and/or prolong signal induction through the receptor.

In a preferred embodiment, the regulatory compound binds to a site which prevents or inhibits association of the extracellular ligand binding component with the transducer component when the components are dissociated from each other. In one embodiment, the regulatory compound selectively binds to a portion of the transducer component that contacts a portion of the extracellular ligand binding component when the receptor is bound by its natural ligand. Therefore, the regulatory compound prevents or inhibits contact of the transducer component with the extracellular ligand binding component. In another embodiment, the regulatory compound selectively binds to a portion of the transducer component which contacts a portion of the extracellular ligand binding component that is phosphorylated when the receptor is bound by its natural ligand. Therefore, the regulatory compound prevents or inhibits phosphorylation of the extracellular ligand binding component. In one embodiment, when the receptor is a B cell antigen receptor, the regulatory compound binds to a portion of an Ig.alpha.-Ig.beta. dimer which prevents or inhibits association of the dimer with a mIg.

It is to be noted that while the above discussion pertains primarily to compounds for desensitizing a receptor, with regard to compounds that prolong or enhance sensitization of the receptor, the target sites for binding can be similar or the same, however, the compounds are selected on the basis of being capable of preventing or inhibiting dissociation of the receptor components, causing the receptor components to associate, and/or prolonging the time during which the receptor components are associated subsequent to stimulation, such as by the receptor ligand.

Regulatory compounds as referred to herein include, for example, compounds that are products of rational drug design, natural products and compounds having partially defined signal transduction regulatory properties. A putative compound can be a protein-based compound, a carbohydrate-based compound, a lipid-based compound, a nucleic acid-based compound, a natural organic compound, a synthetically derived organic compound, an anti-idiotypic antibody and/or catalytic antibody, or fragments thereof. In one embodiment, a regulatory compound suitable for use in the present invention can include an antibody (i.e., an immunoglobulin), a peptide, or a mimetope thereof. In one embodiment, the regulatory compound is an antibody.

A naturally-occurring immunoglobulin molecule is a plasma protein comprising immunoglobulin domains. An immunoglobulin molecule comprises two types of chains. One type of chain is referred to as the heavy or H chain and the other is referred to as the light or L chain. The two chains are present in an equimolar ratio, and each immunoglobulin molecule has at least two H chains and at least two L chains. The two H chains are linked together by disulfide bonds and each H chain is linked to a L chain by a disulfide bond. There are only two types of L chains referred to as lambda (.lamda.) and kappa (.kappa.) chains. In contrast, there are five major H chain classes referred to as isotypes. The five classes include immunoglobulin M (IgM or .mu.), immunoglobulin D (IgD or .delta.), immunoglobulin G (IgG or .lamda.), immunoglobulin A (IgA or .alpha.), and immunoglobulin E (IgE or .epsilon.). The distinctive characteristics between such isotypes are defined by the constant domain of the immunoglobulin and are discussed in detail below. Human immunoglobulin molecules comprise nine isotypes, IgM, IgD, IgE, four subclasses of IgG including IgG1 (.gamma.1), IgG2 (.gamma.2), IgG3 (.gamma.3) and IgG4 (.gamma.4), and two subclasses of IgA including IgA1 (.alpha.1) and IgA2 (.alpha.2).

Each H or L chain of an immunoglobulin molecule comprises two domains referred to as L chain variable regions (V.sub.L regions) and L chain C regions (C.sub.L), and H chain V regions (V.sub.H regions) and H chain C regions (C.sub.L regions). A complete C.sub.H region comprises a CH1, CH2, CH3 domain and a hinge region. Together, one H chain and one L chain form an arm of an immunoglobulin molecule having an immunoglobulin V region. A complete immunoglobulin molecule comprises two arms, di-sulfide bonded by the H chain C regions of each arm. Thus, each arm of an immunoglobulin comprises two domains referred to as a V.sub.H+L region, and a C.sub.H+L region. As used herein, the term "variable region" or "V region" refers to a V.sub.H+L region, a V.sub.L region or a V.sub.H region. Also as used herein, the term "constant region" or "C region" refers to a C.sub.H+L region, a C.sub.L region or a C.sub.H region.

Limited digestion of an immunoglobulin with a protease produces two fragments. An antigen binding protease fragment is referred to as an Fab or an F(ab').sub.2 fragment. A protease fragment lacking the ability to bind to antigen is referred to as an Fc fragment. An Fab fragment comprises one arm of an immunoglobulin molecule containing a L chain (V.sub.L region+C.sub.L region) paired with the V.sub.H region and a portion of the C.sub.H region (CH1 domain). An F(ab').sub.2 fragment corresponds to two di-sulfide bonded arms of an immunoglobulin molecule, each arm containing a L chain (V.sub.L region+C.sub.L region) paired with a V.sub.H region and a CH1 domain.

A C.sub.H region defines the isotype of an immunoglobulin and confers different functional characteristics depending upon the isotype. For example, .mu. C regions enable the formation of pentameric aggregates of IgM molecules and .alpha. C regions enable the formation of dimers.

The antigen specificity of an immunoglobulin molecule is conferred by the amino acid sequence of a variable, or V, region. As such, V regions of different immunoglobulin molecules can vary significantly depending upon their antigen specificity. Certain portions of a V region are invariant and are referred to as framework regions (FW regions). In contrast, certain portions of a V region are highly variable and are designated hypervariable regions. When the V.sub.L and V.sub.H domains pair in an immunoglobulin molecule, the hypervariable regions from each domain associate and create hypervariable loops that form antigen binding sites. Thus, the hypervariable loops determine the specificity of an immunoglobulin and are termed complementarity-determining regions (CDRs) because their surfaces are complementary to antigens.

Further variability of V regions is conferred by combinatorial variability of gene segments that encode an immunoglobulin V region. Immunoglobulin genes comprise multiple germline gene segments which somatically rearrange to form a rearranged immunoglobulin gene that encodes an immunoglobulin molecule. V.sub.L regions are encoded by a L chain V gene segment and J gene segment (joining segment). V.sub.H regions are encoded by a H chain V gene segment, D gene segment (diversity segment) and J gene segment (joining segment).

Both a L chain and H chain V gene segment contains three regions of substantial amino acid sequence variability. Such regions are referred to as L chain CDR1, CDR2 and CDR3, and H chain CDR1, CDR2 and CDR3, respectively. The length of an L chain CDR1 can vary substantially between different V.sub.L regions. For example, the length of CDR1 can vary from about 7 amino acids to about 17 amino acids. In contrast, the lengths of L chain CDR2 and CDR3 typically do not vary between different V.sub.L regions. The length of a H chain CDR3 can vary substantially between different V.sub.H regions. For example, the length of CDR3 can vary from about 1 amino acid to about 20 amino acids. In contrast, the lengths of H chain CDR1 and CDR2 typically do not vary between different V.sub.H regions. Each H and L chain CDR region is flanked by FW regions.

Another portion of an immunoglobulin molecule important in the targeting aspect of the present invention is the spacer or transmembrane spanning region.

Other functional aspects of an immunoglobulin molecule include the valency of an immunoglobulin molecule, the affinity of an immunoglobulin molecule, and the avidity of an immunoglobulin molecule. As used herein, affinity refers to the strength with which an immunoglobulin molecule binds to an antigen at a single site on an immunoglobulin molecule (i.e., a monovalent Fab fragment binding to a monovalent antigen). Affinity differs from avidity which refers to the sum total of the strength with which an immunoglobulin binds to an antigen. Immunoglobulin binding affinity can be measured using techniques standard in the art, such as competitive binding techniques, equilibrium dialysis or BIAcore methods. As used herein, valency refers to the number of different molecules an immunoglobulin molecule can combine with at one time. For example, a monovalent immunoglobulin molecule can only bind to one antigen at one time, whereas a bivalent immunoglobulin molecule can bind to two or more antigens at one time, and so forth.

In one embodiment of the present invention, a regulatory compound is a monovalent antibody. Such an antibody is not capable of aggregating receptors, which, without being bound by theory, is believed to enhance the ability of such an antibody to desensitize receptors expressed by a cell. Divalent antibodies can also be used in the present invention. Examples of divalent antibodies suitable as regulatory compounds of the present invention are described in detail in Example 9.

In one embodiment, the antibody is a bi-specific antibody, such as chimeric antibody. A bi-specific antibody is capable of binding two or more antigens, as with a divalent antibody, but in this case, the antigens are different antigens (i.e., the antibody exhibits dual specificity). A bi-specific antibody suitable for use in the present method includes an antibody having: (a) a first portion (e.g., a first antigen binding portion) which binds to the receptor and: (1) causes a dissociation of the extracellular ligand binding component from the transducer component when the components are associated with each other prior to contact with the compound; or (2) inhibits association of the extracellular ligand binding component with the transducer component when the components are dissociated from each other prior to contact with the compound; and (b) a second portion which binds to a cell surface molecule expressed by a cell which expresses the receptor. In this embodiment, the second portion can bind to any cell surface molecule, including to the receptor to be desensitized (i.e., the second portion of the bi-specific antibody binds to a different region of the receptor than the first portion of the bi-specific antibody). In a preferred embodiment, the second portion is capable of targeting the regulatory antibody to a specific receptor (i.e., the regulatory antibody binds to a target receptor, because it specifically recognizes that receptor, and it does not bind to other, different receptors). For example, the second portion of the bi-specific antibody can be an anti-idiotype antibody such that it binds to the antigen-binding region of a B cell antigen receptor. In the case of an Fc receptor, the second portion of the bi-specific antibody can be an antibody that specifically (i.e., selectively) recognizes a particular Fc receptor type (e.g., Fc.epsilon.RI), and therefore does not substantially bind to other Fc receptor types (e.g., Fc.gamma.R, Fc.alpha.R). Preferably, however, such antibodies do not substantially stimulate the receptor.

In one embodiment, the second portion binds to a cell surface molecule which is expressed by an autoreactive B cell. Such a cell surface molecule preferably distinguishes the autoreactive B cell from other B cells. In another embodiment, the second portion binds to a cell surface molecule which is expressed by a B cell involved in graft rejection.

Isolated antibodies of the present invention can include serum containing such antibodies, or antibodies that have been purified to varying degrees. Antibodies of the present invention can be polyclonal or monoclonal, functional equivalents such as antibody fragments (e.g., Fab fragments or Fab.sub.2 fragments) and genetically-engineered antibodies, including single chain antibodies or chimeric antibodies that can bind to more than one epitope (i.e., bi-specific antibodies).

Generally, in the production of an antibody, a suitable experimental animal, such as a rabbit, hamster, guinea pig or mouse, is exposed to an antigen against which an antibody is desired. Typically, an animal is immunized with an effective amount of antigen that is injected into the animal. An effective amount of antigen refers to an amount needed to induce antibody production by the animal. The animal's immune system is then allowed to respond over a pre-determined period of time. The immunization process can be repeated until the immune system is found to be producing antibodies to the antigen. In order to obtain polyclonal antibodies specific for the antigen, serum is collected from the animal that contains the desired antibodies. Such serum is useful as a reagent. Polyclonal antibodies can be further purified from the serum by, for example, treating the serum with ammonium sulfate. In order to obtain monoclonal antibodies, the immunized animal is sacrificed and B lymphocytes are recovered from the spleen. The differentiating and proliferating daughter cells of the B lymphocytes are then fused with myeloma cells to obtain a population of hybridoma cells capable of continual growth in suitable culture medium. Hybridomas producing a desired antibody are selected by testing the ability of an antibody produced by a hybridoma to bind to the antigen.

A preferred method to produce antibodies of the present invention includes (a) administering to an animal an effective amount of a protein, peptide or mimetope thereof of the present invention to produce the antibodies and (b) recovering the antibodies. In another method, antibodies of the present invention are produced recombinantly. Antibodies raised against defined proteins or mimetopes can be advantageous because such antibodies are not substantially contaminated with antibodies against other substances that might otherwise cause interference in a diagnostic assay or side effects if used in a therapeutic composition. As used herein, the term "selectively binds to" refers to the ability of antibodies of the present invention to preferentially bind to specified proteins and mimetopes thereof. Binding can be measured using a variety of methods standard in the art including enzyme immunoassays (e.g., ELISA), immunoblot assays, etc.

As used herein, an isolated protein according to the present invention can be a full-length protein or any homologue of such a protein, such as a protein in which amino acids have been deleted (e.g., a truncated version of the protein, such as a peptide), inserted, inverted, substituted and/or derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristoylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol). A homologue of a protein is a protein having an amino acid sequence that is sufficiently similar to the natural protein amino acid sequence that a nucleic acid sequence encoding the homologue is capable of hybridizing under stringent conditions to (i.e., with) a nucleic acid molecule encoding the natural protein (i.e., to the complement of the nucleic acid strand encoding the natural protein amino acid sequence).

As used herein, a mimetope or mimic of a protein or antibody according to the present invention refers to any compound that is able to mimic the activity of a given protein or antibody, often because the mimetope has a structure that mimics the protein or antibody. Mimetopes can be, but are not limited to: peptides that have been modified to decrease their susceptibility to degradation; anti-idiotypic and/or catalytic antibodies, or fragments thereof; non-proteinaceous immunogenic portions of an isolated protein (e.g., carbohydrate structures); and synthetic or natural organic molecules, including nucleic acids. Such mimetopes can be designed using computer-generated structures of proteins or antibodies. Mimetopes can also be obtained by generating random samples of molecules, such as oligonucleotides, peptides or other organic or inorganic molecules, and screening such samples by affinity chromatography techniques using the corresponding binding partner.

In accordance with the present invention, an isolated nucleic acid molecule is a nucleic acid molecule that has been removed from its natural milieu (i.e., that has been subject to human manipulation) and can include DNA, RNA, or derivatives of either DNA or RNA. As such, "isolated" does not reflect the extent to which the nucleic acid molecule has been purified. An isolated nucleic acid molecule of the present invention can be isolated from its natural source or produced using recombinant DNA technology (e.g., polymerase chain reaction (PCR) amplification, cloning) or chemical synthesis. Isolated nucleic acid molecules can include, for example, natural allelic variants and nucleic acid molecules modified by nucleotide insertions, deletions, substitutions, and/or inversions in a manner such that the modifications do not substantially interfere with the nucleic acid molecule's ability to encode a protein or to form stable hybrids under stringent conditions with natural gene isolates. An isolated nucleic acid molecule can include degeneracies. As used herein, nucleotide degeneracies refers to the phenomenon that one amino acid can be encoded by different nucleotide codons. Thus, the nucleic acid sequence of a nucleic acid molecule that encodes a protein can vary due to degeneracies.

A nucleic acid molecule homologue can be produced using a number of methods known to those skilled in the art (see, for example, Sambrook et al.). For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, by classic mutagenesis and recombinant DNA techniques (e.g., site-directed mutagenesis, chemical treatment, restriction enzyme cleavage, ligation of nucleic acid fragments and/or PCR amplification), or synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecule homologues can be selected by hybridization with a naturally occurring gene or by screening the function of a protein encoded by a nucleic acid molecule.

As used herein, stringent hybridization conditions refer to standard hybridization conditions under which nucleic acid molecules are used to identify similar nucleic acid molecules. Such standard conditions are disclosed, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press, 1989. Sambrook et al., ibid., is incorporated by reference herein in its entirety (see specifically, pages 9.31-9.62, 11.7 and 11.45-11.61). In addition, formulae to calculate the appropriate hybridization and wash conditions to achieve hybridization permitting varying degrees of mismatch of nucleotides are disclosed, for example, in Meinkoth et al., 1984, Anal. Biochem. 138, 267-284; Meinkoth et al., ibid., is incorporated by reference herein in its entirety.

More particularly, stringent hybridization conditions, as referred to herein, refer to conditions which permit isolation of nucleic acid molecules having at least about 70% nucleic acid sequence identity with the nucleic acid molecule being used to probe in the hybridization reaction, more particularly at least about 75%, and most particularly at least about 80%. Such conditions will vary, depending on whether DNA:RNA or DNA:DNA hybrids are being formed. Calculated melting temperatures for DNA:DNA hybrids are 10.degree. C. less than for DNA:RNA hybrids. In particular embodiments, stringent hybridization conditions for DNA:DNA hybrids include hybridization at an ionic strength of 0.1.times.SSC (0.157 M Na.sup.+) at a temperature of between about 20.degree. C. and about 35.degree. C., more preferably, between about 28.degree. C. and about 40.degree. C., and even more preferably, between about 35.degree. C. and about 45.degree. C. In particular embodiments, stringent hybridization conditions for DNA:RNA hybrids include hybridization at an ionic strength of 0.1.times.SSC (0.157 M Na.sup.+) at a temperature of between about 30.degree. C. and about 45.degree. C., more preferably, between about 38.degree. C. and about 50.degree. C., and even more preferably, between about 45.degree. C. and about 55.degree. C. These values are based on calculations of a melting temperature for molecules larger than about 100 nucleotides, 0% formamide and a G+C content of about 50%. Alternatively, T.sub.m can be calculated empirically as set forth in Sambrook et al., supra, pages 11.55 to 11.57.

As used herein, reference to a percent (%) identity refers to a BLAST homology search with the default parameters identified by the manufacturer.

Regulatory and therapeutic compounds of the present invention, including antibodies, proteins, peptides and mimetopes thereof, can be designed using structure based drug design. Structure based drug design refers to the use of computer simulation to predict a conformation of a peptide, polypeptide, protein, or conformational interaction between a peptide or polypeptide, and a therapeutic compound. For example, generally, for a protein to effectively interact with a therapeutic compound, it is necessary that the three dimensional structure of the therapeutic compound assume a compatible conformation that allows the compound to bind to the protein in such a manner that a desired result is obtained upon binding. According to the present invention, the step of designing can include creating a new chemical compound or searching databases of libraries of known compounds (e.g., a compound listed in a computational screening database containing three dimensional structures of known compounds). Designing can also be performed by simulating chemical compounds having substitute moieties at certain structural features. The step of designing can include selecting a chemical compound based on a known function of the compound. A preferred step of designing comprises computational screening of one or more databases of compounds in which the three dimensional structure of the compound is known and is interacted (e.g., docked, aligned, matched, interfaced) with the three dimensional structure of a receptor of the present invention by computer (e.g. as described by Humblet and Dunbar, Animal Reports in Medicinal Chemistry, vol. 28, pp. 275-283, 1993, M Venuti, ed., Academic Press). Methods to synthesize suitable chemical compounds are known to those of skill in the art and depend upon the structure of the chemical being synthesized. Methods to evaluate the bioactivity of the synthesized compound depend upon the bioactivity of the compound (e.g., inhibitory or stimulatory) and are disclosed herein.

Various other methods of structure-based drug design are disclosed in Maulik et al., 1997, Molecular Biotechnology: Therapeutic Applications and Strategies, Wiley-Liss, Inc., which is incorporated herein by reference in its entirety. Maulik et al. disclose, for example, methods of directed design, in which the user directs the process of creating novel molecules from a fragment library of appropriately selected fragments; random design, in which the user uses a genetic or other algorithm to randomly mutate fragments and their combinations while simultaneously applying a selection criterion to evaluate the fitness of candidate ligands; and a grid-based approach in which the user calculates the interaction energy between three dimensional receptor structures and small fragment probes, followed by linking together of favorable probe sites.

As discussed above, in one embodiment of the present invention, the receptor is selected from the group of a B cell antigen receptor, a pro-B cell receptor, and a pre-B cell receptor. In this embodiment, the transducer component is selected from the group of Ig.alpha. and Ig.beta.. In one aspect of the invention, the regulatory compound selectively binds to the transducer component and prevents or inhibits association of the extracellular ligand binding component with the transducer component. In one embodiment, the extracellular binding component comprises an mIg selected from the group consisting of mIgD and mIgM, although the extracellular ligand binding component can be different, for example, when the B cell expressing the receptor is a pro-B cell or a pre-B cell. In one embodiment, the B cell antigen receptor selectively binds to an antigen associated with an autoimmune disease; in another embodiment, the B cell antigen receptor selectively binds to an antigen associated with a graft cell. B cells which express receptors that are suitable for desensitizing according to the present invention include, but are not limited to, an autoreactive B cell, a B cell comprising a B cell antigen receptor that selectively binds to an antigen on a graft, a B cell lymphoma and a chronic lymphocytic leukemia cell. B cells that are suitable for sensitizing according to the present invention include any normal B cell. In one embodiment of the present invention, the regulatory compound is contacted with the B cell receptor by administering the regulatory compound to a patient that has an autoimmune disease selected from the group of rheumatoid arthritis, systemic lupus erythematosus, insulin dependent diabetes mellitis, multiple sclerosis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, or polyarteritis nodosa.

In another embodiment of the present invention, the receptor to be targeted by the method of the present invention is a human Ig Fc receptor selected from the group consisting of Fc.alpha.RI, Fc.epsilon.RI, Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIc, Fc.gamma.IIIb. In this embodiment, the regulatory compound selectively binds to an extracellular ligand binding domain of the Fc receptor. Typically, the extracellular binding component comprises an .alpha. receptor. In one embodiment, the receptor is an Fc.epsilon.RI receptor comprising an .alpha. receptor extracellular ligand binding component and a .beta./.gamma. transducer component. In another embodiment, the receptor is expressed by a cell selected from the group consisting of a mast cell and a basophil. In one embodiment, the regulatory compound is contacted with the receptor by administration of the regulatory compound to a patient that has a condition associated with inflammation. In one embodiment, the condition is associated with allergic inflammation. Various inflammatory conditions suitable for treatment using the method of the present invention are discussed below.

The method of the present invention includes a step of contacting the regulatory compound with the receptor to be desensitized. According to the present invention, the step of contacting can be conducted in vivo, in vitro and/or ex vivo. When the step of contacting is conducted in vitro, such as in an assay, the step of contacting can include contacting the compound and receptor such as by mixing or combining the compound and receptor in culture. When the method is used in an assay, the receptor can be free receptor or a expressed by a cell (i.e., a cell based assay). As used herein, in vivo delivery refers to the administration of a regulatory compound directly to a subject. Suitable methods of administration are discussed below. Ex vivo delivery of a regulatory compound refers to a method that includes the steps of contacting a population of cells removed from an subject with a regulatory compound of the present invention under conditions such that the regulatory compound interacts with targeted cell types (i.e., T cells) and returning the contacted cells to the subject. Methods to achieve such interaction include, but are not limited to, transfection, retroviral infection, electroporation, lipofection, bacterial transfer, spheroplast fusion, and adsorption.

Acceptable protocols to administer compositions in an effective manner include individual dose size, number of doses, frequency of dose administration, and mode of administration. Determination of such protocols can be accomplished by those skilled in the art. A suitable single dose is a dose that is capable of desensitizing at least about 10% of the target receptors on at least one cell, and in increasing order of preference, more preferably about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% and most preferably, about 100%, of the target receptors on at least one cell when administered one or more times over a suitable time period. For example, a preferred single dose of a regulatory compound can range from about 1 ng (nanogram) to 1 microgram (.mu.g, also denoted .mu.g) to about 10 milligrams (mg) of the composition per kilogram body weight of the animal. Additional doses preferably are administered when the response of the animal becomes insufficient to achieve the desired desensitization effect.

The manner of administration of a regulatory compound and/or therapeutic composition of the present invention can depend upon the particular purpose for the delivery (e.g., treatment of disease, use as a diagnostic reagent), the overall health and condition of the recipient and the judgement of the physician or technician administering the target vehicle. A therapeutic composition of the present invention can be administered to an animal using a variety of methods. Such delivery methods can include parenteral, topical, oral or local administration, such as intradermally or by aerosol. A therapeutic reagent can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration to the intestinal region of an animal include powder, tablets, pills and capsules. Preferred delivery methods for a therapeutic composition of the present invention include intravenous administration, local administration by, for example, injection, intradermal injection, intramuscular injection and inhalation. For particular modes of delivery, a therapeutic composition of the present invention can be formulated in a carrier or an excipient of the present invention.

As used herein, a pharmaceutically acceptable carrier refers to any substance suitable as a vehicle for delivering an compound or therapeutic composition to a suitable in vitro or in vivo site of action. As such, carriers can act as an excipient for formulation of a therapeutic or experimental reagent. Preferred carriers are capable of maintaining the compound or therapeutic composition in a form that is capable of interacting with a receptor according to the present invention. Examples of such carriers include, but are not limited to water, phosphate buffered saline, saline, Ringer's solution, dextrose solution, serum-containing solutions, Hank's solution and other aqueous physiologically balanced solutions. Aqueous carriers can also contain suitable auxiliary substances required to approximate the physiological conditions of the recipient, for example, enhancement of chemical stability and isotonicity. Suitable auxiliary substances include, for example, sodium acetate, sodium chloride, sodium lactate, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and other substances used to produce phosphate buffer, Tris buffer, and bicarbonate buffer. Auxiliary substances can also include preservatives, such as thimerosal, m or o-cresol, formalin and benzyl alcohol. Preferred auxiliary substances for aerosol delivery include surfactant substances nontoxic to a recipient, for example, esters or partial esters of fatty acids containing from about 6 to about 22 carbon atoms. Examples of esters include, caproic, octanoic, lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleic acids. Reagents of the present invention can be sterilized by conventional methods and/or lyophilized. Standard formulations can either be liquid injectables or solids which can be taken up in a suitable liquid as a suspension or solution for injection. Thus, in a non-liquid formulation, the excipient can comprise dextrose, human serum albumin, preservatives, etc., to which sterile water or saline can be added prior to administration. Carriers also include compounds that increase the half-life of a therapeutic composition in the treated animal. Such suitable carriers include, but are not limited to, polymeric controlled release vehicles, biodegradable implants, liposomes, bacteria, viruses, other cells, oils, esters, and glycols.

According to the present invention, the method of desensitizing a receptor can be used to treat a condition associated with inflammation, such as allergic inflammation. Allergic inflammation is a condition in which the elicitation of one type of immune response (e.g., a Th2-type immune response) against a sensitizing agent, such as an allergen, can result in the release of inflammatory mediators that recruit cells involved in inflammation in a mammal, the presence of which can lead to tissue damage and sometimes death. Preferred diseases associated with allergic inflammation which are preferable to treat using the method and composition of the present invention include, allergic airway diseases, allergic rhinitis, allergic conjunctivitis and food allergy. Other conditions which are associated with inflammation, are well known in the art and include, but are not limited to, autoimmune diseases and infectious diseases.

An autoimmune disease to treat can be any autoimmune disease, including, but not limited to, rheumatoid arthritis, systemic lupus erythematosus, insulin dependent diabetes mellitis, multiple sclerosis, myasthenia gravis, Grave's disease, autoimmune hemolytic anemia, autoimmune thrombocytopenia purpura, Goodpasture's syndrome, pemphigus vulgaris, acute rheumatic fever, post-streptococcal glomerulonephritis, and polyarteritis nodosa. Preferred BCR autoantigen specificities to target include, but are not limited to, at least a portion of a thyroid-stimulating hormone receptor, pancreatic .beta. cell antigens, epidermal cadherin, acetyl choline receptor, platelet antigens, nucleic acids, nucleic acid:protein complexes, myelin protein, thyroid antigens, joint antigens, antigens of the nervous system, salivary gland proteins, skin antigens, kidney antigens, heart antigens, lung antigens, eye antigens, erythrocyte antigens, liver antigens and stomach antigens.

The methods of the present invention can be used to treat any animal, and preferably, those of the mammalian class Mammalia, and more preferably, include domestic animals, commercially valuable animals, laboratory animals and humans.

One embodiment of the present invention relates to an isolated regulatory compound that desensitizes a receptor selected from the group consisting of a B cell antigen receptor and an Ig Fc receptor, wherein the receptor has an extracellular ligand binding component and a transducer component, the regulatory compound being identified by its ability to selectively bind to the receptor and upon the binding, is capable of: (1) inducing the extracellular ligand binding component to dissociate from the transducer component; and/or (2) inhibiting the extracellular ligand binding component from associating with said transducer component. Another embodiment of the present invention relates to an isolated regulatory compound that enhances or prolongs sensitization of a receptor selected from the group of BCR, pro-BCR, pre-BCR, FcR and NK receptor. Such a compound is identified as being capable of: (1) causing the extracellular ligand binding component to associate with the transducer component when the two components are not associated with each other prior to contact by the compound; or (2) prolonging or enhancing the time over which the extracellular ligand binding component is associated with the transducer component when the components are associated prior to contact by the compound, thereby sensitizing the receptor. Various aspects of such regulatory compounds have been described in detail above.

One embodiment of the present invention relates to a method for sensitizing or enhancing sensitization of a receptor selected from the group of BCR, pro-BCR, pre-BCR, FcR and NK receptor. Such a method includes the step of contacting a compound with a receptor that has an extracellular ligand binding component and a transducer component, wherein the compound: (1) causes the extracellular ligand binding component to associate with the transducer component when the two components are not associated with each other prior to contact by the compound; or (2) prolongs or enhances the time over which the extracellular ligand binding component is associated with the transducer component when the components are associated prior to contact by the compound, thereby sensitizing the receptor. Such a method is particularly useful when a receptor has become refractory to sensitization, and sensitization is desired, or when an enhanced receptor response is desired (i.e., the method increases sensitization of the receptor, typically by prolonging sensitization). For example, prolonged or enhanced immune response are desirable in vaccination protocols or treatments against particular diseases, such as cancer or HIV infection. In general, the methods described herein with regard to receptor desensitization, can be applied appropriately to receptor sensitization and to methods of identifying regulatory compounds that enhance or prolong sensitization of receptors. In one embodiment of this method, an additional factor can be contacted with the B cell antigen receptor, such as an antigen or other factor which enhances vaccination against a given antigen or enhancement of the B cell antigen response.

Another embodiment of the present invention relates to a method to identify compounds useful for desensitizing a receptor. One such method includes the steps of: (a) contacting with a putative regulatory compound a receptor selected from the group of a B cell antigen receptor, a pro-B cell receptor, a pre-B cell receptor, an Fc receptor, and an NK receptor, wherein the receptor comprises an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is associated with the transducer component prior to contact of the receptor with the putative regulatory compound; and, (b) detecting whether the putative regulatory compound, when contacted with the receptor, causes the extracellular ligand binding component to dissociate from the transducer component. A putative regulatory compound that causes the dissociation is identified as a regulatory compound.

Another such method includes the steps of: (a) contacting with a putative regulatory compound, a receptor selected from the group of a B cell antigen receptor, an Fc receptor, and an NK receptor, wherein the receptor includes an extracellular ligand binding component and at least one transducer component, and wherein the extracellular ligand binding component is not associated with the transducer component prior to contact with the putative regulatory compound; and, (b) detecting whether the putative regulatory compound, when contacted with the receptor, inhibits the extracellular ligand binding component from associating with the transducer component. A putative regulatory compound that inhibits the association is identified as a regulatory compound. As used herein, the term "putative" refers to compounds having an unknown regulatory activity, at least with respect to the ability of such compounds to desensitize receptors as described herein.

In both embodiments of the method of identifying a regulatory compound according to the present invention, the method can be a cell-based assay, or non-cell-based assay. In a preferred embodiment, the receptor is expressed by a cell (i.e., a cell-based assay). In accordance with the present invention, such a method is conducted under conditions which are effective to screen for regulatory compounds useful in the method of the present invention. Effective conditions include, but are not limited to, appropriate media, temperature, pH and oxygen conditions that permit cell growth. An appropriate, or effective, medium refers to any medium in which a cell of the present invention, when cultured, is capable of cell growth and expression of BCR, FcR or NK receptor. Such a medium is typically a solid or liquid medium comprising growth factors and assimilable carbon, nitrogen and phosphate sources, as well as appropriate salts, minerals, metals and other nutrients, such as vitamins. Culturing is carried out at a temperature, pH and oxygen content appropriate for the cell. Such culturing conditions are within the expertise of one of ordinary skill in the art.

In the first embodiment, the conditions under which a receptor according to the present invention is contacted with a putative regulatory compound, such as by mixing, are conditions in which the receptor components are associated if essentially no regulatory compound is present. For example, such conditions include normal culture conditions in the absence of a stimulatory compound, such as the natural ligand for the receptor, an anti-idiotype antibody, or other equivalent stimulus. In this embodiment, when the assay is a cell-based assay, the putative regulatory compound is then contacted with the receptor. Without being bound by theory, given the experimental data provided herein, the present inventors believe that receptor complexes may normally exist in an equilibrium between stable and unstable configurations. Therefore, in the absence of an appropriate stimulator, such as the receptor's natural ligand, an anti-idiotype antibody, or an equivalent stimulus, both associated and dissociated receptors are likely to be present on the cell surface. Therefore, the step of detecting in such an assay is designed to indicate whether a physical association between components can be disrupted by the putative regulatory compound.

In the second embodiment, the conditions under which a receptor according to the present invention is contacted with a putative regulatory compound, such as by mixing, are conditions in which the receptor components are not normally associated if essentially no regulatory compound is present. Such conditions can include, for example, contact of said receptor with a stimulator molecule which causes the receptor components to dissociate. Alternatively, as discussed above, such conditions include normal culture conditions in the absence of a stimulus, since the present inventors believe that complexes may normally exist in an equilibrium between stable and unstable configurations. Preferably, however, such conditions include contacting the receptor with its natural ligand or an equivalent stimulus, such that the components are induced to dissociate.

The assay of the present invention can also be a non-cell based assay. In this embodiment, the putative regulatory compound can be directly contacted with an isolated receptor, or a receptor component (i.e., an isolated transducer component or an isolated extracellular ligand binding component), and the ability of the putative regulatory compound to bind to the receptor or receptor component can be evaluated, such as by an immunoassay or other binding assay. The assay can then include the step of further analyzing whether putative regulatory compounds which bind to a portion of the receptor (e.g., a portion of a transducer component or extracellular ligand binding component), is capable of inhibiting association of the receptor components or is capable of causing dissociation of the receptor components. Such further steps can be performed by cell-based assay, as described above, or by non-cell-based assay.

In either embodiment of the present invention, the step of detecting includes any method that measures: (1) the ability of a receptor to transduce a signal as a result of stimulation of the receptor, wherein a reduced ability of the receptor to transduce a signal as a result of stimulation when contacted with the putative regulatory compound, as compared to in the absence of contact with the compound, indicates that the compound desensitizes the receptor; and/or (2) the physical association or dissociation state of the transducer and extracellular ligand binding component. Such assays include bioassays and molecular assays, including, but not limited to, calcium mobilization assays, phosphorylation assays, kinase assays, immunofluorescence microscopy, flow cytometry, immunoprecipitation assays, immunoblots, enzyme-linked immunosorbant assays, radioimmunoassays, and other binding assays, biological assays and/or combinations thereof. Several of such assays are described in the Examples section.
 

Claim 1 of 7 Claims

1. An isolated regulatory compound that desensitizes a B cell antigen receptor, receptor, wherein the isolated regulatory compound is an antibody or antigen-binding fragment thereof, wherein said receptor has an extracellular ligand binding component and a transducer component, wherein said antibody or fragment thereof is identified by its ability to selectively bind to said transducer component consisting of Ig.alpha. and Ig.beta. of said receptor and upon said binding: (1) induces said extracellular ligand binding component to dissociate from said transducer component; and/or (2) inhibits said extracellular ligand binding component from associating with said transducer component and wherein said antibody or fragment thereof does not substantially stimulate said receptor.
 

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