ES2436624T3 - Peptides or peptide analogs for modulation of the binding of a PDZ protein and a PL protein - Google Patents

Abstract

A fusion peptide or peptide analog thereof for modulating the binding of a PDZ protein and a PL protein comprising: (i) a transmembrane transporter amino acid sequence capable of facilitating the entry of a peptide linked to it into a cell through the plasma membrane, (ii) an optional flexible polylinker peptide sequence, and (iii) a carboxyl terminal peptide sequence comprising a PDZ domain binding motif in terminal C, the PDZ domain binding sequence sequence comprising X * - S / TD / EV / I / L, where X * is any non-aromatic amino acid.

Description

Peptides or peptide analogs for modulation of the binding of a PDZ protein and a PL protein

1. FIELD OF THE INVENTION

The present invention relates to peptides and peptide analogs, and methods for using such

5 compositions to regulate the activities of cells of the hematopoietic system. In one aspect, the invention provides methods of modulation of metabolism (eg, activation) of hematopoietic cells (eg, T cells and B cells) by antagonism of an interaction between a protein containing a PDZ domain and a protein that binds. to a PDZ domain. In one aspect, that refers to fusion peptides containing an amino acid sequence corresponding to the carboxyl terminus of a surface receptor expressed by a

10 hematopoietic cell and a transmembrane transport sequence; Such fusion peptides are useful in the regulation of hematopoietic cells by inhibition of cell activation.

2. BACKGROUND OF THE INVENTION

The PDZ domains of the proteins are named according to three prototypical proteins: PSD95 (Drosophila large disk protein and Zonula Occludin 1 protein (Gomperts et al., 1996, Cell 84: 659-662).

15 PDZ domain are involved in synapse formation by transmembrane neurotransmitter receptor organization by intracellular interactions. PDZ domains contain the GLGF signature sequence. In the nervous system, typical proteins containing PDZ domains contain three PDZ domains, an SH3 domain and a guanylate kinase domain. Examples of intracellular proteins that contain PDZ domains include LIN-2, LIN-7 and LIN-10 at pre-synapse, and PSD95 at post-synapse.

The PDZ domains have been shown to bind to the carboxyl terms of transmembrane proteins in neuronal cells. Songyang et al. reported that proteins capable of fixing PDZ domains contain a motif sequence in the carboxyl terminus of E-S / T-X-V / I (Songyang et al., 1997, Science 275: 73). X-ray crystallography studies have revealed the points of contact between the motif sequence and the PDZ domains (Doyle et al., 1996, Cell 88: 1067-1076). While the interaction between PDZ domains and ion channels in neurons is

25 have studied extensively, such interactions have been the subject of limited studies in other biological systems, especially in the hematopoietic system.

The hematopoietic system is composed of different types of cells that perform different functions. Many of its various functions require the coordinated movement of cell surface receptors that include ion channels, surface adhesion molecules to coordinate cell-cell interaction, and 30 cytokine receptors. Despite its various functional activities, it is believed that all hematopoietic cells develop from a multipotent hematopoietic stem cell of the bone marrow. It has been shown that said stem cell expresses a surface marker called CD34. During differentiation, the stem cell gives rise to progenitor cells in each of several specific hematopoietic cell lineages. The progenitor cells then undergo a series of morphological and functional changes to produce hematopoietic cells

35 assigned functionally mature.

Among the functions performed by hematopoietic cells, certain types of cells are involved only in immunity. For example, lymphocytes, which include T cells, B cells and natural aggressive cells (NK), are effectors in immune responses. Monocytes and granulocytes (i.e. neutrophils, basophils and eosinophils) play a role in nonspecific forms of defense. The lymphocytes, monocytes and granulocytes are referred to

40 collectively as white blood cells or leukocytes. On the other hand, other hematopoietic cells perform functions that are not related to the immune system. For example, erythrocytes are involved in gas transport, and thrombocytic series cells are involved in blood clotting.

T cells and B cells recognize antigens and generate an immune response. T cells recognize the

45 antigens by surface heterodimeric receptors called the T-cell receptor (TCR). The TCR is associated with a series of polypeptides referred to collectively as a CD3 complex. B cells recognize the antigens by surface immunoglobulins (Ig), which are also secretory molecules. Additionally, a large number of surface co-stimulatory receptors have been identified in T cells and B cells, which increase cell activation during antigen-induced activation.

In addition to the T / CD3 antigen receptor complex (TCR / CD3), other molecules expressed by T cells that mediate an activation signal include, but are not limited to, CD2, CD4, CD5, CD6, CD8, CD18, CD27, CD28, CD43, CD45, CD152 (CTLA-4), CD154, MHC class I, MHC class II, CDw137 (4-1BB), CDw150, and the like (Barclay et al., The Leucocyte Antigen Facts Book, 1997, second edition, Academic Press; Leucocyte Typing, 1984, Bernard et al. (eds.), Springer-Verlag; Leukocyte Typing II, 1986, Reinherz et

55 al. (eds.), Springer-Verlag; Leukocyte Typing III, 1987, McMichael (ed.), Oxford University Press; Leukocyte Typing IV, 1989, Knapp et al. (eds.), Oxford University Press; CD Antigens, 1996, VI Internat. Workshop and Conference on Human Leukocyte Differentiation Antigens. http://www.nc-bi.nlm.nih.gov/prow). Cell surface antigens operating together with TCR / CD3 are often referred to in the art as coreceptors.

Specific antibodies have been generated against all the surface antigens of the T cells mentioned above. Other molecules that bind to the aforementioned T cell surface receptors include derivatives of antigen binding antibodies such as variable domains, peptides, superantigens, and their natural ligands such as CD58 (LFA-3) for CD2, HIV gp120 for CD4, CD27L for CD27, CD80 or CD86 for CD28 or CD152, ICAM1, ICAM2 and ICAM3 for CD11a / CD18, 4-1BBL for CDw137.

Activation molecules expressed by B cells include, but are not limited to, surface Ig, CD18, CD19, CD20, CD21, CD22, CD23, CD40, CD45, CD80, CD86 and ICAM1. Similarly, natural ligands of these molecules and antibodies directed to them as well as antibody derivatives can be used to deliver an activation signal to B cells.

However, prior to the present invention, it was not known that signal transduction after stimulation of any leukocyte receptor was mediated by receptor interactions with proteins containing PDZ domains. Therefore, it was not even contemplated in the art that an interference of leukocyte surface receptor / PDZ domain interactions could regulate leukocyte activation.

WO 98/23781 A1 discloses peptides that modulate the fixation of a PDZ protein comprising a D-X-V sequence for fixing the C-terminal PDZ domain.

3. SUMMARY OF THE INVENTION

The present invention discloses a fusion peptide or peptide analog thereof for modulation of the binding of a PDZ protein and a PL protein comprising:

(i)

 a transmembrane transporter amino acid sequence capable of facilitating the entry of a peptide bound to it into a cell through the plasma membrane,

(ii)

 an optional flexible polylinker peptide sequence, and

(iii) a peptide sequence of the carboxyl terminal comprising a PDZ domain binding motif in terminal C, the sequence of the PDZ domain binding motif comprising

X * -S / T-D / E-V / I / L, where X * is any non-aromatic amino acid.

Preferred embodiments of the invention are described in the appendix claims.

A method of modulating a biological function of a cell, eg, an endothelial cell or hematopoietic cell (such as a leukocyte, eg T cell or B cell), by introduction into the cell of an antagonist that inhibits is also described herein. the fixation of a PDZ protein and a PL protein in the cell, or an agonist that improves the fixation of a PDZ protein and a PL protein in the cell. In various embodiments, the PL protein is an adhesion protein, an adapter protein, or an intracellular protein. In some embodiments, it is CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, DNAM-1, CD83, CD44, CD4, CD97, CD3n, DOCK2, CD34, FceRIb , or FasLigand. In one embodiment, the PL protein is characterized by a carboxy-terminal amino acid motif that is XSXA, XAD / EV, XV / I / LX * -V, or XS / TXF (where X is any amino acid and X * is any amino acid not aromatic). In embodiments, the PL protein is expressed by T lymphocytes or B lymphocytes. In some embodiments of this method, the PDZ protein is CASK, MPP1, DLG1, PSD95, NeDLG, SYN1a, TAX43, LDP, LIM, LIMK, AF6, PTN- 4, prIL16, 41.8, RGS12, DVL1, TAX 40, TIAM1, MINT1, K303, TAX2, or KIAA561.

In some aspects, the cell is a leukocyte and the biological function is cell activation, cell proliferation, maintenance of cell structure, cellular metabolic activity, or cytokine production. In some embodiments, the method further includes detection of a change in leukocyte activation.

In preferred aspects, the antagonist is an agent that inhibits the binding of a PL peptide to a polypeptide of the PDZ domain in an "A" assay, in an "G" assay, or at the same time in an A test and a G. test. The antagonist may be a polypeptide, such as a polypeptide having at least two residues in the carboxy terminus that are the same as the two carboxy-terminal residues of a PL protein, such that a PL protein is expressed in a hematopoietic cell or cell endothelial which is an adhesion protein, an adapter protein, or an intracellular protein. In a further aspect, at least the four carboxy-terminal residues of the polypeptide are the same as the four carboxy-terminal residues of the PL protein. In a further aspect, the PL protein has a carboxy-terminal amino acid motif selected from XSXA, XAD / EV, XV / I / L, -X * -V, or XS / TXF, where X is any amino acid and X * is Any non-aromatic amino acid. In a further aspect, the PL protein is CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb , or FasLigand.

In a related aspect, the antagonist is a peptidomimetic of a PL inhibitory sequence peptide. In another related aspect, the antagonist is a fusion polypeptide having a PL sequence and transmembrane transporter amino acid sequence (such as HIV tat, Drosophila antenapedia, herpes simplex virus VP22 or anti-DNA CDR 2 and 3).

In another aspect, the description provides a method of determining whether a test compound is a binding inhibitor between a PDZ protein and a PL protein by contact of a PDZ domain polypeptide having a PDZ protein sequence, and a PL peptide under conditions in which they form a complex, in the presence and absence of a test compound, and detection of complex formation in the presence and absence of the test compound, where less complex formation in the presence of the compound test that in the absence of the compound indicates that the test compound is a PDZ protein-PL protein binding inhibitor. The PL peptide has a sequence that includes the C-terminal sequence of a PL protein, such as CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, or FasLigand. In some embodiments, the PDZ domain polypeptide is a fusion polypeptide.

In a related aspect, the description provides a method of determining whether a test compound is a binding agonist between a PDZ protein and a PL protein by contact of a PDZ domain polypeptide, and a PL peptide under conditions in which they form a complex, in the presence and in the absence of a test compound, and detection of the formation of the complex in the presence and absence of the test compound, wherein more complex formation in the presence of the test compound than in the absence of the compound indicates that the test compound is an agonist of the PDZ protein-PL protein binding.

The description additionally provides a binding inhibitor of a PDZ protein and a PL protein. In one aspect, the inhibitor is characterized in that it reduces the fixation of a peptide selected from the group consisting of a PL peptide selected from the group consisting of CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp- 2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand and a PDZ domain polypeptide. In various aspects, the inhibitor is a peptide comprising a sequence that is from 3 to about 20 residues of a C-terminal sequence of a PL protein selected from CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, CAM1 , Clasp-2, CD95, DNAM-1, CD83, CD44, CD97, CD3n, DOCK2, CD34, FceRIb, and FasLigand; a peptide having an X-S-X-A, X-A-D / E-V, X-V / I / L-X * -V, or X-S / T-X-F motif, (where X is any amino acid and X * is any non-aromatic amino acid); a peptidomimetic; or a small organic molecule. The description also provides a pharmaceutical composition containing the inhibitor.

The description also provides a method for the treatment of a disease characterized by activation of leukocytes by administration of a therapeutically effective amount of an inhibitor of a PL-PDZ interaction. In some aspects, the disease is characterized by an inflammatory or humoral immune response or is an autoimmune disease. The description additionally provides a method of reducing the inflammation of an individual, by administration of an agent that inhibits the binding of a PDZ protein and a PDL protein, where the PL protein is an adhesion protein, an adapter protein, or a protein. intracellular

The description also provides the use of a PDZ protein binding inhibitor and a PL protein to inhibit leukocyte activation or to treat a disease mediated by hematopoietic cells, such as a disease characterized by an inflammatory or immune response. humoral The description also provides the use of a binding inhibitor of a PDZ protein and a PL protein in the preparation of a medicament for the treatment of a disease measured by hematopoietic cells.

The description also provides a method of modulating a biological function of a hematopoietic cell, which comprises introducing into the cell an antagonist that inhibits fixation in a PDZ protein and a PL protein in the cell as deduced from Table 2, for example. , where the PL protein is DNAM-1 and the PDZ protein is MPP1, MPP2, DLG1, NeDLG, PSD95, LIM, AF6, 41.8 or RGS12, the PL protein is LPAP and the PDZ protein is DLG1 or MINT1, or the PL protein is DNAM-1 and the PDZ protein is PSD95 or MPP2.

The present description also relates to peptides and peptide analogs that bind to PDZ domains in hematopoietic cells. In particular, it refers to peptides and fusion peptide analogs containing a carboxy-terminal sequence of the hematopoietic cell surface receptor and a transmembrane transporter sequence that facilitates the entry of the peptides into a target cell. The description also refers to methods of using such compositions in inhibiting leukocyte activation as measured by cytokine production, cell proliferation, apoptosis and cytotoxicity.

It is one aspect of the description the administration of a therapeutically effective amount of the above-mentioned peptide or fusion peptide analogs as pharmaceutical compositions to an individual in order to inhibit undesirable events mediated by leukocytes.

It is also an aspect of the description to administer a therapeutically effective amount of the above-mentioned peptide or fusion peptide analogs as pharmaceutical compositions to an individual in order to treat an autoimmune disorder or to prevent transplant rejection of a solid organ transplant.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURES 1A-1D show the results of illustrative tests in which the binding of biotinylated peptides having a sequence of the carboxyl term ("term C") of various leukocyte proteins to PDZ domains (ie, GST-fusion proteins) was determined. PDZ domain) using the "G" assay described below. The PDZ domains are: PSD95 (Fig. 1A); NeDLG (Fig. 1B); DLG1 (Fig. 1C), and 41.8 (Fig. 1D). These and other PDZ domain fusion proteins are described below (e.g., TABLE 2). In FIGURE, peptides 1-31 refer to the biotinylated PL peptide used in the assay, and are identified in the key, below). The "Identifications (IDs) of the peptides" are defined in TABLE 3. Key:

10 FIGURES 2A and 2B show the Determination of Apparent Affinity for PDZ binding interactions. Variable concentrations of biotinylated CLASP-2 (Fig. 2A; TABLE 4) or Fas (Fig. 2B; TABLE 4) were reacted with the immobilized GST polypeptide (fixed to the plate) or GST-PDZ (GST-DLG1) fusion proteins. , GST-NeDLG, and GST-PSC95). GST binding alone (<0.2 OD units) was subtracted from fusion protein binding to obtain the signal for each peptide concentration. This signal is

15 then normalized by dividing the signal for each peptide concentration by the maximum signal observed for each peptide-PDZ pair (ie, the signal obtained at 30 μM from the CLASP2 peptide or the 100 μM Fas peptide; 0.41.0 OD units for CLASP2 and 1.2-2.0 OD units for Fas). The normalized signals were then plotted and adjusted to a saturation fixation curve for CLASP2 and 1.2-2.0 OD units for Fas). The normalized signals were then plotted and adjusted to a curve of

20 saturation fixation, producing an apparent affinity of 21 μM for the DLG1-CLASP2 interaction, 7.5 μM for the NeDLG-CLASP2 interaction, 45 μM for the PSD95-CLASP2 interaction, 54 μM for the DLG1-Fas interaction, 54 μM for the NeDLG-Fas interaction, and 85 μM for the PSD95-Fas interaction. Data are average values of duplicate data points, with standard errors between duplicate data points <20%.

FIGURES 3A-3F show the inhibition of PDZ-PL peptide interactions. A fixed concentration of biotinylated C-terminal peptide having a sequence based on the C-terminal sequence of a cell surface receptor protein (Clasp2, CD46, Fas and KV1.3; see TABLE 4) was fixed to immobilized GST polypeptide or the GST fusion protein indicated in the upper left of each frame, in the presence or absence of the competing peptides indicated in the legend of each frame and the

30 level of inhibition. Fig. 3A-BLG1; Fig. 3B-TSP95; Fig. 3C NeDLG; Fig. 3D-DLG1; Fig. 3E, PSD95; Fig. 3F

41.8. In Fig. 3A-B the competing peptides are present at 100 μM; in Figs. 3C-F the competitor is present at the indicated concentration.

FIGURES 4A and 4B show the results of the introduction of a Tat-CD3 fusion peptide on the activation of T cells. The activation of antigen-specific T cells was measured by production.

35 cytokines Fusion peptides containing tat and a carboxyl term of the T cell surface molecule inhibited the production of γ interferon (IFN) by a T cell line in response to stimulation of the myelin basic protein (MBP). The level of inhibition was determined by first subtracting the binding of the GST-labeled peptide only from the binding to the fusion protein and dividing by the signal in the absence of the competing peptide.

5.1 A "fusion protein" or "fusion polypeptide" as used herein refers to a composite protein, that is, a single contiguous amino acid sequence, consisting of two (or more) different heterologous polypeptides that are not normally fused to each other in a single amino acid sequence. Thus, a fusion protein may include a single amino acid sequence containing two totally different amino acid sequences or two similar or identical polypeptide sequences, provided that these sequences are not normally found in the same configuration in a single amino acid sequence. Found in nature. Fusion proteins can generally be prepared using recombinant nucleic acid methods, that is, as a result of transcription and translation of a recombinant gene fusion product, fusion comprising a segment encoding a polypeptide of the invention and a segment encoding a heterologous protein, or by chemical synthesis methods well known in the art.

Boards

Table 1

Amino Acid Classification

Table 2

Protein-Ligand Pairs

Table 3

PDZ domains

Table 3A

Note about Table 3

Table 4

PL peptides

Table 5A & B

Illustrative PL reasons

5. DEFINITIONS

5.2 A "fusion protein construct" as used herein is a polynucleotide that encodes a fusion protein.

5.3 As used herein, the term "PDZ domain" refers to the protein sequence (ie, the modular protein domain) of approximately 90 amino acids, characterized by homology to the PSD-95 cerebral synaptic protein, the protein Discs-Large of the septate junction of Drosophila (DLG) and the strong epithelial junction protein ZO1 (ZO1). PDZ domains are also known as Discs-Large homology repeats ("DHRs"), and GLGF repeats. PDZ domains generally seem to maintain a central consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-1076).

PDZ domains are found in various membrane-associated proteins that include members of the MAGUK family of guanylate kinase homologs, several phosphatases and kinase proteins, neuronal nitric oxide synthase, several dystrophin-associated proteins, collectively known as syndrophins.

Illustrative proteins containing PDZ domain and PDZ domain sequences are shown in TABLE 3. The term "PDZ domain" also encompasses variants (eg naturally occurring variants) of the sequences of TABLE 3 (eg, polymorphic variants, variants with conservative substitutions). , and the like). Typically, PDZ domains are substantially identical to those shown in TABLE 3, eg, at least about 70%, at least about 80%, or at least about 90% amino acid residue identity when compared and aligned for maximum correspondence

5.4 As used herein, the term "PDZ protein" refers to a naturally occurring protein that contains a PDZ domain, e.g., a human protein. Illustrative PDZ proteins include CASK, MPP1, DLG1, PSD95, NeDLG, TAX33, SYN1a, TAX43, LDP, LIM, LIMK1, LIMK2, MPP2, NOS1, AF6, PTN-4, prIL16, 41.8kD, KIAA0559, RGS12, KIAA0316 , TAX40, TIAM1, MINT1, KIAA0303, CBP, MINT3, TAX2, KIAA0561. Illustrative PDZ proteins are listed in TABLE 2 and TABLE 3.

5.5 As used herein, the term "PDZ domain polypeptide" refers to a polypeptide containing a PDZ domain, such as a fusion protein that includes a PDZ domain sequence, a naturally occurring PDZ protein, or a peptide. PDZ domain isolated.

5.6 As used herein, the term "PL protein" or "PDZ ligand protein" refers to a naturally occurring protein that forms a molecular complex with a PDZ domain, or a protein whose carboxy term, when expressed separately from The total length protein (eg, as a peptide fragment of 4-25 residues, eg, 16 residues), forms such a molecular complex. The molecular complex can be observed in vitro using "test A" or "test G" described below, or in vivo. The illustrative PL proteins shown in TABLE 2 have been shown to bind specific PDZ proteins. This definition is not intended to include anti-PDZ antibodies and the like.

5.7 As used herein, a "PL sequence" refers to the amino acid sequence of the C-terminus of a PL protein (eg C-terminal residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 20 or 25) ("Cterminal PL sequence") or an internal sequence known to be fixed to a PDZ domain ("internal PL sequence").

5.8 As used herein, a "PL peptide" is a peptide that has a sequence of, or based on, the sequence of the C-terminus of a PL protein. Illustrative (biotinylated) PL peptides are listed in TABLE 4.

5.9 As used herein, a "PL fusion protein" is a fusion protein that has a PL sequence as a domain, typically as the C-terminal domain of the fusion protein. An illustrative PL fusion protein is a fusion of tat-PL sequences.

5.10 As used herein, the term "PL inhibitor peptide sequence" refers to a PL peptide or an amino acid sequence that (in the form of a PL fusion peptide or protein) inhibits the interaction between a domain peptide PDZ and a PL peptide (eg, in a test A or a test G).

5.11 As used herein, a "PDZ domain coding sequence" means a segment of a polynucleotide encoding a PDZ domain. In various embodiments, the polynucleotide is DNA, RNA, single stranded or double stranded.

 5.12 As used herein, the terms "antagonist" and "inhibitor", when used in the context of modulation of a binding interaction (such as fixing a PDZ domain sequence to a PL sequence) are used interchangeably and refer to an agent that reduces the fixation of the, eg PL sequence (e.g. PL peptide) and the, e.g., PDZ domain sequence (e.g., PDZ protein, PDZ domain peptide).

5.13 As used herein, the terms "agonist" and "enhancer", when used in the context of modulation of a binding interaction (such as fixing a PDZ domain sequence to a PL sequence), are used interchangeably and they refer to an agent that increases the fixation of the, eg, PL sequence (eg, PL peptide), and the, eg, PDZ domain sequence (eg, PDZ protein, PDZ domain peptide).

5.14 As used herein, the terms "peptide mimetic," "peptidomimetic," and "peptide analog" are used interchangeably and refer to a synthetic chemical compound that has substantially the same structural and / or functional characteristics of an inhibitor peptide. of PL or PL binding peptide of the invention. The mimetic can be composed entirely of synthetic, unnatural amino acid analogs, or is a chimeric molecule of partially natural peptide amino acids and partially unnatural amino acid analogs. The mimetic may also incorporate any amount of conservative substitutions of natural amino acids as long as such substitutions do not substantially alter the structure of the mimetic and / or the inhibitory activity or binding activity. As in the case of polypeptides of the invention that are conservative variants, routine experimentation will determine if a mimetic is within the scope of the invention, that is, that its structure and / or function are not substantially altered. Thus, a mimetic composition is within the scope of the invention if it is capable of binding to a PDZ domain and / or inhibiting a PL-PDZ interaction.

Mimetic polypeptide compositions may contain any combination of unnatural structural components, which are typically of 3 structural groups: a) residue binding groups other than natural amide binding bonds ("peptide bond"); b) non-natural residues instead of naturally occurring amino acid residues; or c) residues that induce secondary structural mimicry, that is, that induce or stabilize a secondary structure, e.g. a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.

A polypeptide can be characterized as a mimetic when all or some of its residues are bound by chemical means other than natural peptide bonds. Individual peptidomimetic residues may be linked by peptide bonds, other chemical bonds or coupling means, such as, eg, glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N, N-dicyclohexylcarbodiimide (DCC) or N, N = diisopropylcarbodimide ( DEC). Linker groups that may be an alternative to the traditional amide linkage ("peptide bond") include, eg, ketomethylene (eg, -C (= O) -CH2-for-C (= O) -NH-), aminomethylene (CH2- NH), ethylene, olefin (CH = CH), ether (CH2-O), thioether (CH2-S), tetrazol (CN4-), thiazole, retroamide, thioamide, or ester (see, eg, Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267357, A Peptide Backbone Modifications, Marcel Dekker, NY).

A polypeptide can also be characterized as a mimetic for containing all or some unnatural residues instead of naturally occurring amino acid residues. Unnatural waste is described in detail in the scientific and patent literature; A few illustrative non-natural compositions useful as mimetics of natural amino acid residues and guidance lines are described below.

Aromatic amino acid mimetics can be generated by replacing, e.g., D-or L-naphthylalanine; D-o Lphenylglycine; D-o L-2-thienylalanine; D-o L-1, 2, 3 or 4-pirenylalanine; D-o L-3-thienylalanine; D- or L- (2-pyridinyl) -alanine; D- or L- (3-pyridinyl) -alanine; D-o L- (2-pyrazinyl) -alanine; D-o L- (4-isopropyl) -phenylglycine; D- (trifluoromethyl) -phenylglycine; D (trifluoromethyl) -phenylalanine; D-p-fluorophenylalanine; D-o L-p-biphenyl-phenylalanine; K-o L-p-methoxyphenylphenylalanine; D-o L-2-indole (alkyl) alanines; and D-o L-alkylamines, where alkyl can be methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, substituted or unsubstituted or a non-acidic amino acid.

Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Acidic amino acid mimetics can be generated by substitution by, e.g., amino acids other than carboxylate while maintaining a negative charge; (phosphono) alanine; sulfated threonine. Side carboxyl groups (eg, aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R = -NCNR =) such as, eg, 1-cyclohexyl-3 (2-morpholinyl- (4-ethyl) carbodimide or 1-ethyl -3 (4-azonia-4,4-dimethylpentyl) -carbodimide Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or acid (guanidino) -acetic acid, or (guanidino) -alkyl-acetic acid, where alkyl has been defined above. A nitrile derivative (e.g., containing the CN-moiety instead of COOH) can be substituted instead of asparagine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to give aspartyl residues

or corresponding glutamil.

Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, or ninhydrin, preferably under alkaline conditions.

Mimetics of tyrosine residues can be generated by reacting tyrosyl with e.g., aromatic compounds of diazonium or tetranitromethane, N-acetylimidazole and tetranitromethane can be used to form O-acetyltyrosyl and 3-nitroderivative species, respectively.

Mimetics of cysteine residues can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Mimetics of cysteine residues can also be generated by reacting cysteinyl residues with e.g., bromotrifluoroacetone, alphabromo-beta- (5-imidazoyl) -propionic acid; chloroacetyl phosphate, Nalkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl-2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole.

Lysine mimetics (and some amino-terminal residues) can be generated by lysinyl reaction with, e.g., succinic anhydride or other carboxylic acid anhydrides. Lysine and other residue mimetics containing alpha-amino can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4pentanedione, and glyoxylate reactions catalyzed by transamidases

Methionine mimetics can be generated by reaction with, e.g., methionine sulfoxide. Proline mimetics include, e.g., pipecolic acid, thiazolidine-carboxylic acid, 3-or 4-hydroxyproline, dehydroproline, 3-or 4-methylproline,

or 3,3-dimethylproline. Mimetics of histidine residues can be generated by reaction of histidyl with e.g., diethyl procarbonate or parabromophenacyl bromide.

Other mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of amine residues of the main chain or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.

A component of a natural polypeptide (e.g., a PL polypeptide or PDZ polypeptide) can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid that exists naturally in the L configuration (which can also be referred to as R or S, depending on the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, generally referred to as the D-amino acid, but which can also be designated as the R or S form.

The mimetics of the description may also include compositions containing a structural mimetic residue, particularly a residue that induces or mimics secondary structures, such as a beta turn, beta sheet, alpha helix structures, gamma turns, and the like. For example, the substitution of natural amino acid residues with D-amino acids; N-alpha-methyl amino acids; C-alpha-methyl amino acids; or dehydroamino acids comprised within a peptide may induce or stabilize beta turns, gamma turns, beta sheets or alpha helix conformations. Mimetic structures of beta back have been described, e.g., by Nagai (1985) Tet. Lett. 26: 647-650; Feigl (1986) J. Amer. Chem. Soc. 108: 181-182; Kahn (1988) J. Amer. Chem. Soc. 110: 1638-1639; Kemp (1988) Tet. Lett. 29: 5057-5060; Kahn (1988) J. Molec. Recognition 1: 75-79. Mimetic beta sheet structures have been described, e.g., by Smith (1992) J. Amer. Chem. Coc. 114: 10672-10674. For example, a type VI beta tour induced by a cis-amide substitute, 1,5-disubstituted tetrazole, has been described by Beusen (1995) Biopolymers 36: 181-200. The incorporation of aquatic omega-amino acid residues to generate polymethylene units as a replacement for amide bonds has been described by Banerjee (1996) Biopolymers

39: 769-777. Secondary polypeptide structures can be analyzed by, e.g., 1H NMR high-field spectroscopy or 2D NMR spectroscopy, see, e.g., Higgins (1997) J. Pept. Res. 50: 421-435. See also Hruby (1997) Biopolymers 43: 219-266, Balaji, et al., U.S. Pat. No. 5,612,895.

5.15 As used herein, "peptide variants" and "conservative amino acid substitutions" refer to peptides that differ from a reference peptide (eg, a peptide having the carboxy terminus sequence of a specified PL protein) by substitution of an amino acid residue having similar properties (based on size, polarity, hydrophobicity, and the like). Thus, to the extent that the compounds that are encompassed within the scope of the invention are partially defined in terms of amino acid residues of designated classes, the amino acids can generally be classified into three main classes: hydrophilic amino acids, hydrophobic amino acids and related amino acids. cysteine, depending mainly on the characteristics of the amino acid side chain. These main classes can be further divided into subclasses. Hydrophilic amino acids include amino acids that have acidic, basic or polar side chains, and hydrophobic amino acids include amino acids that have aromatic or apolar side chains. Apolar amino acids can be further subdivided to include, among others, aliphatic amino acids. The definitions of the amino acid classes as used herein are as follows:

"Hydrophobic Amino Acid" refers to an amino acid that has a side chain that is devoid of charge at physiological pH and that is repelled by aqueous solutions. Examples of genetically encoded hydrophobic amino acids include Ile, Leu and Val. Examples of hydrophobic amino acids not genetically encoded include t-BuA.

"Aromatic Amino Acid" refers to a hydrophobic amino acid that has a side chain that contains at least one ring that has a conjugated π electron system (aromatic group). The aromatic group may be further substituted with groups such as alkyl, alkenyl, alkynyl, hydroxyl, sulfanyl, nitro and amino groups, and others. Examples of genetically encoded aromatic amino acids include Phe, Tyr and Trp. Commonly found and not commonly found aromatic amino acids include phenylglycine, 2-naphthylalanine, β-2-thienylalanine, 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, 4-chloro-phenylalanine, 2-fluorophenylalanine, 3-fluorophenylalanine and 4-fluorophenylalanine.

"Apolar amino acid" refers to a hydrophobic amino acid that has a side chain that is generally devoid of charge at physiological pH and that is apolar. Examples of genetically encoded apolar amino acids include Gly, Pro and Met. Examples of non-encoded apolar amino acids include Cha.

"Aliphatic Amino Acid" refers to an apolar amino acid that has a saturated or unsaturated, linear, branched or cyclic hydrocarbon side chain. Examples of genetically encoded aliphatic amino acids include Ala, Leu, Val and Ile. Examples of uncoded aliphatic amino acids include Nle.

"Hydrophilic Amino Acid" refers to an amino acid that has a side chain that is attracted to aqueous solutions. Examples of genetically encoded hydrophilic amino acids include Ser and Lys. Examples of uncoded hydrophilic amino acids include Cit and hCys.

"Acidic Amino Acid" refers to a hydrophilic amino acid that has a side chain pK value of less than 7. Acidic amino acids typically have side chains negatively charged to physiological pH due to the loss of a hydrogen ion. Examples of genetically encoded acidic amino acids include Asp and Glu.

"Basic Amino Acid" refers to a hydrophilic amino acid that has a pK value of the side chain greater than

7. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Examples of genetically encoded basic amino acids include Arg, Lys and His. Examples of basic non-genetically encoded amino acids include the noncyclic amino acids ornithine, 2,3-diaminopropionic acid, 2,4-diaminobutyric acid and homoarginine.

"Polar amino acid" refers to a hydrophilic amino acid that has a side chain that is not charged at physiological pH, but that has a bond in which the pair of electrons shared in common by two atoms is more closely retained by one of the atoms . Examples of genetically encoded polar amino acids include Asx and Glx. Examples of non-genetically encoded polar amino acids include citrulline, N-acetyl-lysine and methionine-sulfoxide.

"Cysteine-like Amino Acid" refers to an amino acid having a side chain capable of forming a covalent bond with a side chain of another amino acid residue, such as a disulfide bond. Typically, cysteine-related amino acids generally have a side chain that contains at least one thiol group (SH). Examples of genetically encoded cysteine-related amino acids include Cys. Examples of cysteine-like or genetically encoded amino acids include homocysteine and penicillamine.

As will be appreciated by those skilled in the art, the above classification is not absolute, exhibiting several more amino acids than one characteristic property, and can therefore be included in more than one category. For example, tyrosine has both an aromatic ring and a polar hydroxyl group. Thus, tyrosine has dual properties and can be included in both categories, aromatic and polar. Similarly, in addition to being able to form disulfide bonds, cysteine also has a nonpolar character. Thus, although it is not strictly classified as hydrophobic or apolar amino acid, in many cases cysteine can be used to confer hydrophobicity to a peptide.

Certain commonly found amino acids that are not genetically encoded of which they may be

5 constituted peptides and peptide analogs of the invention include, but are not limited to, β-alanine (b-Ala) and other omega-amino acids such as 3-aminopropionic acid (Dap), 2,3-diaminopropionic acid (Dpr) , 4-aminobutyric acid, and so on; α-aminoisobutyric acid (Aib); ε-aminohexanoic acid (Aha); δ-aminovaleric acid (Ava), Ava); N-methylglycine or sarcosine (MeGly); Ornithine (Orn); citrulline (Cit); t-butylalanine (t-BuA); t-butylglycine (t-BuG); N-methylisoleucine (Me-lle); phenylglycine (Phg); cyclohexylalanine (Cha); norleucine (Nle); 2-naphthylalanine (2

10 Nal); 4-chlorophenylalanine (Phe (4-Cl)); 2-fluorophenylalanine (Phe (2-F)); 3-fluorophenylalanine (Phe (3-F)); 4fluorophenylalanine (Phe (4-F)); penicillamine (Pen); 1, 2, 3, 4-tetrahydroisoquinoline-3-carboxylic acid (Tic); β 2-thienylalanine (Thi); methionine sulfoxide (MSO); homoarginine (hArg); N-acetyl lysine (AcLys); 2, 3diaminobutyric acid (Dab); 2,3-diaminobutyric acid (Dbu); p-aminophenylalanine (Phe (pNH2)); N-methyl valine (MeVal);

homocysteine (hCys) and homoserine (hSer). These amino acids also conveniently fall into the categories defined above.

The above-described genetically encoded and uncoded amino acid classifications are summarized below in TABLE 1. It should be understood that TABLE 1 is given for illustrative purposes only and is not intended to be an exhaustive list of amino acid residues that may comprise peptides and peptide analogs described herein. Other amino acid residues that are useful for producing peptides and

20 peptide analogs described herein can be found, e.g., in Fasman, 1989, CRC Practical Handbook of Biochemistry and Molecular Biology, CRC Press, Inc., and references cited therein. Amino acids not specifically mentioned herein can be conveniently classified in the categories described above based on their known behavior and / or their chemical and / or physical characteristic properties compared to specifically identified amino acids.

25 TABLE 1

Classification

Genetically Coded Not Genetically Coded

Hydrophobe

Aromatic

F, Y, W Phg, Nal, Thi, Tic, Phe (4-Cl), Phe (2-F), Phe (3-F), Phe (4-F), Pyridyl Ala, Benzothienil Ala

Apolar

M, G, P

Aliphatic

A, V, L, I t-BuA, t-BuG, Me-lle, Nle, MeVal, Cha, bAla, MeGly, Aib

Hydrophilic

Acidic

FROM

Basic

H, K, R Dpr, Om, hArg, Phe (p-NH2), DBU, A2BU

Polar

Q, N, S, T, Y Cit, AcLys, MSO, hSer

Similar to cysteine

C Pen, hCys, p-methyl Cys

5.16 As used herein, a "detectable label" has ordinary meaning in the art and refers to an atom (eg radionuclide), molecule (eg, fluorescein), or complex, which is used or can be used to detect (eg. due to a physical or chemical property), indicate the presence of a molecule or allow the fixation of another molecule to which it is covalently bound or otherwise associated. The term "marker" also refers to covalently linked or otherwise associated molecules (e.g., a biomolecule such as an enzyme), which act on a substrate to produce a detectable atom, molecule or complex. Detectable markers suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful markers in

The present invention includes biotin for staining with labeled streptavidin conjugate, magnetic beads

(eg, Dynabeads TM), fluorescent dyes (eg fluorescein, Texas red, rhodamine, green fluorescent protein, intensified green fluorescent protein, and the like), radiolabels (eg, 3H, 125I, 35S, 14C, or 32P), enzymes (eg , hydrolases, particularly phosphatases such as alkaline phosphatase, esterases and glycosidases, or oxidoreductases, particularly peroxidases such as horseradish peroxidase, and others commonly used in ELISAs), substrates, cofactors, inhibitors, chemiluminescent groups, chromogens, and colorimetric markers such as colloidal gold or colored glass or plastic beads (eg, polystyrene, polypropylene, latex, etc.). Patents that teach the use of such markers include U.S. Pat. No. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149, and 4,366,241. Means for detecting such markers are well known to those skilled in the art. Thus, for example, radiolabels and chemiluminescent markers can be detected using photographic film or scintillation counters; fluorescent markers can be detected using a photodetector to detect the emitted light (e.g., as the classification of cells activated by fluorescence). Enzymatic markers are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric markers are detected by simple visualization of the colored marker. Thus, a marker is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. The label can be directly or indirectly coupled to the desired test component according to methods well known in the art. Non-radioactive markers are often fixed by indirect means. Generally, a ligand molecule (e.g., biotin) binds covalently to the molecule. The ligand is then fixed to an anti-ligand molecule (e.g. streptavidin) that is either inherently detectable or covalently binds to a signal generating system such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in association with the naturally occurring labeled anti-ligands. Alternatively, any haptic or antigenic compound can be used in combination with an antibody. The molecules can also be conjugated directly to signal generators, e.g., by conjugation with an enzyme or fluorophore. Marker detection means are well known to those skilled in the art. Thus, for example, where the marker is a radioactive marker, the means for detection include a scintillation counter, a photographic film as in autoradiography, or luminescence storage imaging. Where the marker is a fluorescent marker, it can be detected by fluorochrome excitation with the light wavelength coupled and detection of the resulting fluorescence. Fluorescence can be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupling devices (CCDs) or photomultipliers and the like. Similarly, enzyme markers can be detected by providing appropriate substrates for the enzyme and detecting the resulting reaction product. Also, simple colorimetric markers can be detected by observing the color associated with the marker. It will be appreciated that when pairs of fluorophores are used in a test, it is often preferred that they have different emission patterns (wavelengths) such that they can be easily differentiated.

5.17 As used herein, the term "substantially identical" in the context of amino acid sequence comparison means that the sequences have at least about 70%, at least about 80%, or at least about 90% identity. of amino acid residues when compared and aligned for maximum correspondence. An algorithm that is suitable for determining sequence identity and percentage sequence similarity is the FASTA algorithm, which is described in Pearson, W.R. & Lipman, D.J., 1988, Proc. Natl. Acad. Sci. U.S.A. 85: 2444. See also W. R. Pearson, 1996, Methods Enzymol.

266: 227-258. Preferred parameters used in an FASTA alignment of DNA sequences to calculate percent identity are optimized, BL50 Matrix 15: -5, k-tuple = 2; union penalty = 40, optimization = 28, lagoon penalty -12, lagoon length penalty = -2; and width: 16.

5.18

As used herein, "hematopoietic cells" include leukocytes, comprising lymphocytes (T cells, B cells and NK cells), monocytes, and granulocytes (ie, neutrophils, basophils and eosinophils), macrophages, dendritic cells, megakaryocytes, reticulocytes, erythrocytes, and CD34 + stem cells.

6.

 DETAILED DESCRIPTION OF THE INVENTION

The authors of the present invention have discovered that interactions between PDZ proteins and PL proteins play an important and extensive role in the biological function of hematopoietic cells and other cells involved in the immune response. Although PDZ-PL interactions were known in the nervous system (i.e. in neurons), their universal importance in the function of hematopoietic cells, especially in the function of T cells and B cells, and their fundamental role in Immune response modulation has not been recognized so far. In particular, the authors of the present invention have surprisingly discovered that cell adhesion molecules that mediate cell-cell interaction in the hematopoietic system are PDZ binding proteins (PL proteins) and are fixed to PDZ proteins. The inventors have identified numerous interactions between PDZ proteins and PL proteins present in the cells of the immune system, and the invention provides reagents and methods that affect biological function in the immune system by inhibiting these interactions. As used herein, the term "biological function" in the context of a cell, refers to a detectable biological activity normally carried out by a cell, eg, a phenotypic change such as proliferation, cell activation (eg, activation). of T cells, activation of B cells, formation of TB cell conjugates), cytokine release, degranulation, tyrosine phosphorylation, ion flow (eg, calcium), metabolic activity, apoptosis, changes in gene expression, maintenance of cell structure, cell migration, adherence to a substrate, signal transduction, cell-cell interactions, and others described herein or known in the art.

In one aspect, the present invention relates to peptides or peptide analogs as defined in the

5 claims Mimetics, pharmaceutical compositions, and methods of using such compositions to regulate the biological activities of hematopoietic cells, eg, T cells and B cells, or other cells (eg, endothelial cells) that are necessary for immune function are also described. in this memory. The description further relates to methods of using the compositions to modulate the activation and immune function of hematopoietic cells, as well as assays for such inhibitors.

10 TABLE 2 summarizes an extensive analysis of protein interactions in T cells and B cells. PDZ proteins, the vast majority of which were not previously known to be expressed in immune system cells, are listed in the row. TABLE 2. The first column of the table lists PL proteins. The positions in the matrix designated with the letter "A" or "G" indicate that an interaction between the PDZ protein and the PL has been detected in new binding assays (described in detail in, eg, Section 6.2, below) .

A blank cell indicates that no interaction has been detected using the assays of the invention. An asterisk (*) denotes a PL-PDZ interaction previously recorded in the scientific literature.

TABLE 2 SUMMARY OF PDZ-LIGANDO / PDZ INTERACTIONS

CODE

* Interactions described in the scientific literature

TABLE 2 (continued) SUMMARY OF PDZ-LIGANDO / PDZ INTERACTIONS

* Interactions described in the scientific literature

As set forth in detail herein, the PDZ proteins listed in TABLE 2 are naturally existing proteins that contain a PDZ domain. The present invention is particularly directed to the detection and modulation of interactions between PDZ proteins and PL protein in hematopoietic cells. Illustrative PL proteins are listed in TABLE 2. Notably, as discussed below, many of these PL proteins have not previously been recognized as such in any cellular system. A variety of classes of PL proteins are known, and the PL proteins described herein can be characterized as (1) "adhesion PL proteins" (2) "ion channel PL proteins" (3) "adaptive PL proteins" (4 ) "intracellular PL proteins" and (5) "cytokine receptor PL proteins".

As used herein, an adhesion protein is a cell surface protein involved in cell-cell interactions by direct contact with cell surface molecules (e.g., transmembrane proteins or surface proteins) in a different cell. Thus, when a cell expressing an adhesion PL protein comes into contact with another appropriate cell, the adhesion PL protein is located at the interface of the two cells and comes into contact directly with a cell surface molecule of the second cell. . A cell-cell interface is a region in which the plasma membranes of two different cells are in a close position (generally <10 nm, often approximately 1 nm). Typically, direct molecular contact means interaction of molecules at distances where Van der Waals forces are important, as a rule less than about 1 nm. Illustrative adhesion PL proteins include CD6; CD49E (alpha-4); CD49F (one form, alpha6); CD138 (syndecan); CLASP-1; CLASP-4; VCAM1; CLASP-2; DNAM-1; CD83; CD44 (long form); CD97; (CD55L); CD3n; DOCK2; CD34; and FceRlb. Thus, in one embodiment, the PL proteins of the invention are adhesion PL proteins. In one embodiment, the invention provides methods and reagents, as detailed herein, to inhibit interactions between adhesion PL proteins and PDZ proteins in order to modulate an immune response. In one embodiment, inhibition or modulation occurs in a hematopoietic cell. In a related embodiment, inhibition or modulation occurs in an endothelial cell. In a related embodiment, inhibition or modulation occurs in an endothelial cell. In a related embodiment, inhibition or modulation occurs in epithelial cells, keratinocytes, hepatocytes and cardiac myocytes.

As used herein, an ionic channel protein means a transmembrane protein that catalyzes on its own the passage of an ion to an aqueous solution on one side of a membrane of the lipid bilayer to an aqueous solution on the other side (eg, formation of a small pore in the membrane). An illustrative ion channel PL protein is Kv1.3. Thus, in one embodiment, the PL proteins of the invention are PL proteins of ion channels. In one embodiment, the invention provides methods and reagents, as detailed herein, for inhibition of interactions between PL proteins of ion channels and PDZ proteins to modulate an immune response. In one embodiment, inhibition or modulation occurs in a hematopoietic cell. In a related embodiment, the invention or modulation occurs in an endothelial cell.

As used herein, an intercellular protein (ie, cytosolic) has the normal meaning in the art and refers to a protein that is not fixed to the membrane, e.g., has no transmembrane domain. Thus, in one embodiment, the PL proteins of the invention are intercellular PL proteins. Illustrative intercellular PL proteins include Glycoforin C and LPAP. In one embodiment, the invention provides methods and reagents, as detailed herein, for inhibition of interactions between cytoplasmic PL proteins and PDZ proteins to modulate an immune response. In one embodiment, inhibition or modulation occurs in a hematopoietic cell. In a related embodiment, inhibition or modulation occurs in an endothelial cell.

As used herein, a cytokine receptor has the normal meaning in the art and refers to a membrane protein with an extracellular domain that specifically binds a cytokine. Illustrative cytokine receptor PL proteins include CDW125 (IL5R), CDW128A (IL8RA), and BRL-1. Thus, in one embodiment, the PL proteins of the invention are cytokine PL proteins. In one embodiment, the invention provides methods and reagents, as detailed herein, to inhibit interactions between cytokine PL proteins and PDZ proteins that modulate an immune response. In one embodiment, inhibition or modulation occurs in a hematopoietic cell. In a related embodiment, inhibition or modulation occurs in an endothelial cell.

As used herein, an adapter protein means a molecule (e.g., protein) that contributes to the formation of a multimolecular complex by binding two or more different biomolecules. The fixation of the two or more different molecules by the adapter molecule / protein generally implies a direct molecular contact between the adapter protein and each of the two or more different molecules. An illustrative PL adapter protein is LPAP. Thus, in one embodiment, the PL proteins of the invention are adaptive PL proteins. In one embodiment, the invention provides methods and reagents, as detailed herein, to inhibit interactions between adapter PL proteins and PDZ proteins to modulate an immune response. In one embodiment, inhibition or modulation occurs in a hematopoietic cell. In a related embodiment, inhibition or modulation occurs in an endothelial cell.

In various embodiments, the PL proteins of the invention are characterized by specific amino acid sequences or C-terminal amino acid motifs (i.e., from the PL domain), as described elsewhere in this description.

In various embodiments of the invention, the PL proteins of the invention are fixed to a PDZ protein expressed in T lymphocytes, B lymphocytes, or at the same time in T lymphocytes and B lymphocytes. In one embodiment, the PL protein is fixed to a PDZ protein. expressed in endothelial cells. In various embodiments, the PL protein and / or the PDZ protein to which it is bound are not expressed in the nervous system (e.g., neurons).

In various embodiments of the invention, the PL protein of the invention is fixed only to a PDZ protein listed in TABLE 2. In another embodiment, the PL protein is set to 1 to 3, 3 to 5, or more than 5 PDZ proteins. different listed in TABLE 2.

In various embodiments of the invention, the PL protein is expressed or regulated incrementally after cell activation (eg in activated B lymphocytes, T lymphocytes), or after entry into mitosis (eg, increased regulation in proliferating cell populations quickly).

In various embodiments of the invention, the PL protein is (i) a protein that mediates the activation or migration of an immune cell (eg, hematopoietic cell), (ii) a protein that does not mediate apoptosis in one type of cell, (iii ) a protein that is distinct from a G-protein coupled receptor of seven transmembrane helices, (iv) a protein that is G protein coupled to a seven transmembrane helices receptor but not a cytokine receptor, or (v) a protein that does not It is a G protein coupled to a seven transmembrane helices receptor and is a cytokine receptor.

6.1 Detection of Proteins Containing PDZ Domain Expressed in Hematopoietic Cells

As indicated above, the authors of the present invention surprisingly discovered that numerous PDZ proteins are expressed in cells of the immune system, and play a fundamental biological role in modulating the immune response. It has been previously described that PDZ DLG1 and TIAM-1 proteins are found in T cells. The authors of the present invention discovered, using a BLAST search of the human EST database and the experiments described below, that several additional PDZ proteins They are present in hematopoietic cells, including MPP1, P-DLG, VELI-1, PSD95, T-cell and CASK, DLG1, DLG2, KINASE ZIP, Sintrophin 2, P-dlg, PSD95, and synthenin in cells B.

To determine the full extent of the involvement of PDZ proteins in hematopoietic function, the inventors undertook a systematic investigation of PDZ proteins in T and B cells. An exhaustive list of proteins containing PDZ domain was recovered from the database. from the Sanger Center (Pfam) searching for the keyword, PDZ. The corresponding cDNA sequences were recovered from GenBank using the NCBI "entrez" database (hereinafter, "GenBank PDZ protein cDNA sequences"). The portion of DNA encoding PDZ domains was identified by cDNA alignment and protein sequence using CLUSTALW. Based on the DNA / protein alignment information, primers were designed that encompassed the PDZ domains. The expression of certain PDZ-containing proteins in immune cells was detected by amplification of the polymerase chain region ("PCR") of cDNAs obtained by reverse transcription ("RT") of RNA derived from immune cells (ie, "RT -PCR "). PCR, RT-PCR and other methods for nucleic acid analysis and manipulation are well known and are generally described in Sambrook et al., (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2ND ED., VOLS. 1-3, Cold Spring Harbor Laboratory, hereinafter "Sambrook"); and Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing and Wiley-Interscience, New York (1997), as it has been supplemented until January 1999 (hereinafter "Ausubel").

In the experiments summarized in TABLE 2, T cells (Jurkat E6 cell line) and B cells (MV 4-11 cell line) were tested for the expression of genes containing the specific PDZ domain by RT-PCR. RNA was prepared using the "trizol" RNA preparation kit (GIBCO-BRL; Cat. # 15596-018) according to the manufacturer's recommendations. In summary, 1-5 x 107 lymphoblasts were harvested by centrifugation at 200 x g for 10 minutes at 20 ° C. The cells were resuspended in 100 µl of PBS buffer and 1 ml of TRIZOL reagent was added for every 5 x 106 cells. The resuspension of cells was mixed and after 5 minutes of incubation at room temperature (RT), 0.2 mg chloroform per ml of TRIZOL was added. The resuspension was shaken vigorously and incubated for another 3 minutes at RT. The samples were then centrifuged at 12,000 x g for 15 minutes at 4 ° C, the aqueous phase was recovered and the RNA was precipitated with 2-propanol. The precipitate was collected by centrifugation at 12,000 xg for 15 minutes at 4 ° C, washed with 55% ethanol, finally collected by another centrifugation at 12,000 xg for 15 minutes at 4 ° C, air dried and resuspended in an appropriate volume of DEPC treated water.

The concentration and purity of the RNA were determined by the measurement of light absorption at 270/280 nm by the nucleic acid. The SUPERCRIPT II reverse transcriptase cDNA kit (GIBCO-BRL; Cat. # 18064-014) was used for cDNA synthesis. The RNA contribution per 200 μl of the cDNA reaction sample was 10 μg. Prior to cDNA synthesis, RNA was treated with 1 unit / μl of DNase in 110 μl of water at 37 ° C for 20 minutes. DNase I was then deactivated by incubation for 10 minutes at 70 ° C. The random primer was used for cDNA priming; 10 µl of random hexamer primer (100 ng / µl) was added, samples were heated at 70 ° C for 5 minutes and cooled on ice. Subsequently, 40 μl of the "first chain" SUPERSCRIPT II buffer, 20 μl of 0.1 M DDT, 10 μl of a 10 mM mixture of deoxynucleotide triphosphates (dATP, dCTP, dGTP, dUTP) and 10 μl of SUPERSCRIPT II reverse transcriptase and cDNA synthesis was carried out for 45 minutes at 42 ° C. The reactions were stopped by incubation for 5 minutes at 95 ° C and 2-4 µl of such cDNA samples were typically used for PCR.

A portion of the cDNA (typically, 1/5 of a 20 µl reaction) was used for the PCR. PCR was conducted using primers designed to specifically amplify regions containing PDZ domain of 5 PDZ proteins of interest. Oligonucleotide primers were designed to amplify one or more PDZ coding domains. The DNA sequences encoding the various PDZ domains of interest were identified by inspection (ie, conceptual translation of the PDZ protein cDNA sequences obtained from GenBank, followed by alignment with the amino acid sequence of the PDZ domain). TABLE 3 shows the PCR primers, the amplified PDZ-encoded domains, and the GenBank accession number of the proteins they contained.

10 PDZ domain. To facilitate subsequent cloning of the PDZ domains, the PCR primers included restriction endonuclease sequences at their ends in order to allow ligation with the cloning vector pGEX-3X (Pharmacia, GenBank XXI 13852) under glutathione- S-transferase (GST).

TABLE 3 lists the proteins detected in the aforementioned assays. The results showed that PDZ proteins are widely used in T and B cells both in a specific way of lineage and independently of lineage. INADL 2/3 (PDZ domain), KIAA0316, and p27 of the 26s subunit were detected in T cells but not in B cells. MCASK, KIAA0559, PTN-4 and X11beta were detected in B cells, but not in T cells. AF6 , BAII-associated protein, Cytohexine-bound protein, DLG1, DLG5 (pdlg), DVL1, DVL3, GTPase, 41.8 kd hipot., KIAA147, KIAA0300, KIAA0303, KIAA0380, KIAA0440, KIAA0545, KIAA0561 LIM, KIAA0561 LIMI . from the LIM domain, LIM protein, MINT1, MINT3, MPP1, MPP2, NE-DLG, NOS1, serine

20 new protease, PTN-3, prIL 16, PSD95, RGS12, serine protease, SYNTENIN, SYNTR 1 alpha, TAX1, TAX2, TAX33, TAX40, Tax43 (SYN, Beta1), TIAM wwp3, and prot. X11 were detected in both T cells and B cells.

TABLE 3 PDZ DOMAINS (continued) (continued) (continued) (continued) (continued) (continued) (continued) (continued)

-Key: The names of genes and the corresponding gene products are provided. In some cases, cDNA sequences that represent the same gene have several entries in the databases under different access numbers and names. The access numbers represented correspond to the gene name used in this description, and the nucleotide and amino acid numbering is correlated with those entries in GenBank. The amino acid sequences represented correspond to the cloned DNA portions of genes that contain the PDZ domain. The linker amino acid sequences (e.g., DNA-encoded amino acids flanking the cloning site of the cloning vector pGEX-3X) are shown in italics.

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

CASK

CASK

Y17138 AA495-584; PDZ domain 1 (of 1) Bam HI / Eco RI 6CAFN1471-1494 7CARN1761-1738

MPP1

55 Kd erythrocyte membrane protein M64925 AA101-186; PDZ domain 1 (of 1) Bam HI / Bam HI 62MPFN296-320 63MPRN568-543

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

DLG1

human counterpart of Drosophila discs-large protein U13897 AA275-477; PDZ domains 1-2 (of 3) Bam HI / Eco RI 1DFN815-841 2DRN1442-1421

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

PSD95

human protein postsynaptic density95 U83192 AA387-724; PDZ domains 1-3 (of 3) Bam HI / Eco RI 8PSFN1150-1173 11PSRN2191-2168

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

NeDLG

presynaptic protein102 (neuroendocrine-dlg) U49089 AA205-1171; PDZ domains 1-2 (of 3) Bam HI / Eco RI 71NEDFN608-635 72NEDRN1186-1161

TAX33

interaction protein Tax 33 AF028826 AA73-162; PDZ domain 1 (of 1) Bam HI / Eco RI 92TAFN208-234 93TARN497-468

SYN 1 α

alpha1-syntrophin U40571 AA96-189 PDZ domain 1 (of 1) Bam HI / Eco RI 124SYFN279-301 125SYRN576-551

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

TAX43

human interaction protein tax 43 AF028828 AA15-85 domain PDZ 1 (of 1) Bam HI / Eco RI 97TAFN37-63 98TARN267-231

LDP

lim clp-36 domain protein U90878 AA46-88 PDZ domain 1 (of 1) Bam HI / Eco RI 146LIFN129-155 147LIRN276-239

LIM

Human LIM protein AF061258 AA29-112; PDZ domain 1 (of 1) Bam HI / Eco RI 182LFN86-115 183LRN350-320

LIMK1

kinase 1 of human LIM domain NM_ 002314 AA194-291; PDZ domain 1 (of 1) SMA I 52LIFP 53LIRP

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

N570-597 N874-851

LIMK2

kinase 2 of human LIM domain D45906 AA185-275; PDZ domain 1 (of 1) Bam HI / Eco RI 185LFN545-573 186LRN834-805

MPP2

member 2 of the maguk p55 family (DLG2) X82895 AA185-273; PDZ domain 1 (of 1) Bam HI / Eco RI 142MFN542-569 143MRN828-801

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

US1

Human Syntasaneuronal Nitric Oxide U17327 AA239-988; PDZ domain 1 (of 1) Bam HI / Eco RI 155NOFN711-733 156NORN994-970

AF6

af-6 protein U02478 AA985-1077; PDZ domain 1 (of 1) Bam HI / Eco RI 66AFFN2946-2970 67AFRN3239-3214

PTN-4

Proteytyrosine phosphatase meg1 M68941 AA774-862; PDZ domain 1 (of 1) Bam HI / Eco RI 247PTFN2312-2338 248PTRN2595-2569

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

prIL16

putative precursor of interleukin 16 S81601 AA170-383; PDZ1-2 domain (of 2) Bam HI / EcoRI 75PRFN503-528 76PRR

41.8 kD

41.8 k protein AF007156 AA4-85; PDZ domain 1 (of 1) Bam HI / EcoRI 145HFN4-30 146HRN267-240

K559

KIAA0559 AB011131 AA766-870; PDZ1 (of 1) Bam HI / EcoRI 130KIFN2290-2312 131KIRN2623-2595

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

RGS12

human regulator of the G12 proteinization AF035152 AA35-103; PDZ domain 1 (of 1) Bam HI / Eco RI 64RGFN93-119 65RGRN316-291

K316

KIAA0316 AB002314 AA197-284; PDZ domain 1 (of 1) Bam HI / Eco RI 158KIFN586-611 159KIRN866-839

DVL1

human homologue of the protein depolarity of the disordered segment AF006011 AA248-340; PDZ domain 1 (of 1) Bam HI / Eco RI First PCR: 55DVISF N652-673 Second PCR, nested: 37DVF First PCR: 56DVISR N1195-1174Second PCR, nested: 38DVR

(continued) (continued) (continued)

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

TAX40

human interaction protein tax 40 AF028827 AA35-137; PDZ domain 1 (of 1) Bam HI / Eco RI 136TFN97-123 137TRN421-393

TIAM1

protein 1 invasion and metastasis of T lymphoma NM_ 003253 AA1001-1088; PDZ 1 (of 1) Bam HI / Eco RI 39TFN2995-3019 40TRN3275-3253

MINT1

human protein X11 L04953 AA717-894; PDZ domains 1-2 (of 2) Eco RI / Eco RI 34MIFN2149-2167 20MRN2690-2666

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

K3.03

KIAA0303 Ab002301 AA652-742; PDZ domain 1 (of 1) Bam HI / Eco RI 152KIF 153KIR

N1948-1976 N2237-2209

CBP

Protein HE cytohesin defixation AF68836 AA85-176; PDZ domain 1 (of 1) Bam HI / EcoRI 235CYFN246-274 236CYRN535-510

MINT3

Human MINT3 AF029110 AA11-52; PDZ domain 1 (of 1) Bam HI / EcoRI 188MFN23-51 189MRN165-138

GEN SYMBOL

PROTEIN ACC. # SEQUENCE OF AMINO ACIDS * DECLONATION SITES DIRECT PRIMER REVERSE PRIMER

TAX2

Human tax interaction protein 2 AF028824 AA54-140; PDZ domain 1 (of 1) Bam HI / Eco RI 197 TF 198 TRN429-401

N154-182

K561

KIAA0561 AB011133 AA948-1038; PDZ domain 1 (of 1) Bam HI / Eco RI 161KIFN2836-2863 162KIRN3120-3095

* Note concerning TABLE 3

In several cases, the sequence analysis of the PDZ clones revealed differences with respect to the DNA and / or protein sequence with respect to that published in the databases, summarized in TABLE 3A.

TABLE 3A

GEN

ENTRY IN GENBANK *** REAL CONSTRUCTION

AF6

N 3060: C N 3060: T *

DLG1

N 1021: A, = AA 340: Gln N 1021: G, = AA 340: Arg

Lim domain

N202: G N 203: C, = AA 68: Arg N202: C * N 203: G, = AA 68: Gly

LIMK1

N 855: C, = AA 285: Leu N 855: A, = AA 285: lle

MINT1

N 2386: G, = AA 796: Glu ** N 2386: A, = AA 796: Lys **

NE-DLG

N713: T N 766: G, = AA 255: Gly N803: G, = AA 267: Glu N861: G, = AA 287: Val N 713: C * N 766: A, = AA 255: Glu N 803: C, = AA 267: Asp N 861: A, = AA 287: Met

TIAM1

N 3224: A N 3224: G *

(*) = Silent mutation, does not affect the sequence of AA; (**) = MINT1 is the same as X11a. The database entry for X11a shows the same sequence as the current construct of the inventors in relation to N2386 of the MINT1 entry in GenBank. (***) = The nucleotide ("N") and amino acid ("AA") annotations correspond to the numbering as found in the GenBank files.

5 6.2 Tests for Detection of Interactions Between PDZ Domain Polypeptides and PDZ Candidate Ligand Proteins (PL Proteins)

Two complementary assays, called "A" and "G", were developed to detect binding between a PDZ domain polypeptide and the candidate PDZ ligand. In each of the two different assays, binding is detected between a peptide having a sequence corresponding to the C-terminus of a protein that is expected to be fixed to one or more 10 PDZ domains (ie a candidate PL peptide) and a polypeptide of PDZ domain (typically a fusion protein that contains a PDZ domain). In the "A" assay, the candidate PL peptide is immobilized and the binding of a soluble PDZ domain polypeptide to the immobilized peptide is detected (the "A" assay is named for the fact that in one embodiment a surface of avidin to immobilize the peptide). In the "G" assay, the PDZ domain polypeptide is immobilized and the fixation of a soluble PL peptide is detected (the "G" assay is

15 so called by the fact that in one embodiment a GST binding surface is used to immobilize the PDZ domain polypeptide). Preferred embodiments of these tests are described in detail below. However, it will be appreciated by technicians with ordinary experience that these tests can be modified in numerous ways while still being useful for the purposes of the present invention.

6.2.1 Production of Fusion Proteins Containing PDZ Domains

20 GST-PDZ domain fusion proteins were prepared for use in the assays of the invention. PCR products containing PDZ coding domains (as described in § 6.1, above) were subcloned into an expression vector that allowed the expression of fusion proteins containing a PDZ domain and a heterologous domain (i.e., a sequence of glutathione-S-transferase, "GST"). PCR products (i.e., DNA fragments) representing DNA encoding the PDZ domain were extracted from agarose gels using the system of

25 gel extraction "Sephaglas" (Pharmacia) according to the manufacturer's recommendations.

As indicated above, PCR primers were designed to include restriction endonuclease sites to facilitate ligation of the PCR fragments to a GST gene fusion vector (PGEX-3X; Pharmacia, GenBank accession number No. XXU13852 ) in frame with the glutathione-S-transferase coding sequence. This vector contains a lacZ promoter inducible by IPTG. The pGEX-3X vector was linearized using BamHI and EcoRI or, in some cases, EcoRI or Sma1, as shown in TABLE 3, and dephosphorylated. For most cloning operations, double digestion was performed with BamHI and EcoRI), such that the ends of the PCR fragments to be cloned were BamHI and EcoRI. In some cases, the restriction endonuclease combinations used were Bgl II and EcoRI, BamHI and MfeI or RcoRI only, SmaI only, or BamHI only (see TABLE 3). When more than one PDZ domain was cloned, the cloned DNA portion represents the PDZ domains and the portion of

cDNA located between individual domains. The precise locations of the cloned fragments used in the assays are indicated in TABLE 3. The DNA binding sequences between the GST portion and the DNA portion containing the PDZ domain vary slightly, depending on which of the cloning sites and methods. described above were used. As a consequence, the amino acid sequence of the GST-PDZ fusion protein varies in the linker region between GST and the PDZ domain. The protein linker sequences corresponding to different cloning sites / approaches are shown below. The linker sequences (encoded vector DNA) are shown in bold, and sequences derived from the gene containing the PDZ domain are shown in italics.

1) GST-BamHI / BamHI-PDZ domain insertion

Gly - Ile-PDZ Domain Insertion

2) GST-BamHI / Bglll-PDZ domain insertion

Gly-Ile-PDZ Domain Insertion

3) GST-EcoRI / EcoI-PDZ domain insertion

Gly-Ile-Pro-Gly - Asn-PDZ Domain Insertion

4) GST - SmaIlSmaI-PDZ domain insertion

Gly-Ile-Pro-PDZ Domain Insertion

The PDZ-encoding PCR fragment and the linearized vector pGEX-3X were precipitated with ethanol and resuspended in 10 µl of standard ligation buffer. Ligation was performed for 4-10 hours at 37 ° C using T4 DNA ligase. It will be understood that some of the resulting constructs include very short linker sequences and that, when multiple PDZ domains were cloned, the constructs included some DNA located between the individual PDZ domains.

The ligation products were transformed into bacterial strains of E. coli DH5α or BL-21. The colonies were screened for the presence and identity of the DNA containing the cloned PDZ domain, as well as for the correct fusion with the portion of the glutathione-S-transferase coding DNA by PCR and by sequence analysis. Positive clones were tested in a small-scale assay for the expression of the GST-PDZ domain fusion protein and, if expressed, these clones were subsequently grown for large-scale preparations of the GST-fusion protein. PDZ

The GST-PDZ domain fusion protein was overexpressed after the addition of IPTG to the culture medium and purified. The detailed procedure for the expression and purification of small-scale and large-scale fusion protein is described in "GST Gene Fusion System" (2nd edition, revision 2; published by Pharmacia). In summary, a small culture (3-5 ml) containing a bacterial strain (DH5α, BL21 or JM9) with the fusion protein construct was allowed to grow overnight in LB medium at 37 ° C with the appropriate antibiotic selection (100 μg / ml ampicillin; also known as LB-amp). The night culture was poured into a recent application of LB-amp (typically 250-500 ml) and allowed to grow until the optical density (OD) of the culture was between 0.5 and 0.9 (approximately 2.5 hours). ). IPTG (isopropyl- (β-D-thiogalactopyranoside) was added at a final concentration of 1.0 mM to induce the production of GST fusion protein, and the culture was allowed to grow for an additional 1.5-2.5 hours. bacteria by centrifugation (4500 g) and were resuspended in Buffer A- (50 mM Tris, pH 8.0, 50 mM dextrose, 1 mM EDTA, 200 μM phenylmethylsulfonyl fluoride) An equal volume of Buffer A + (Buffer A was added -, 4 mg / ml lysozyme) and incubated on ice for 3 minutes to lyse the bacteria An equal volume of Buffer B (10 mM Tris, pH 8.0, 50 mM KCl, 1 mM EDTA, 0, was added) 5% Tween-20, 0.5% NP40 (also known as IGEPAL CA-630), 200 µM phenylmethylsulfonyl fluoride) and incubated for an additional 20 minutes.The bactrian cell lysate was centrifuged (20,000 g), and added supernatant to glutathione-sepharose 4B (Pharmacia, Cat. No. 17-0765-01) previously swollen (rehydrated) in 1X phosphate buffered saline (PBS). or supernatant-sepharose was poured into a column and washed with at least 20 bed volumes of 1 X PBS. The GST fusion protein was eluted from glutathione sepharose by application of 0.5-1.0 ml aliquots of 5 mM glutathione and collected as separate fractions. The concentrations of the fractions were determined using BioRadProtein Assay (Cat. No. 500-0006) according to the manufacturer's specifications. Those fractions containing the maximum concentration of the fusion protein were pooled and dialyzed against 1X PBS / 35% glycerol. Fusion proteins were tested for size and quality by SDS gel electrophoresis (PAGE) as described in "Sambrook." Aliquots of the fusion protein were stored at -80 ° C and -20 ° C.

6.2.2 Identification of PL Candidate Proteins and Peptide Synthesis.

In some non-hematopoietic cells (e.g., neurons, epithelial cells), it is known that certain PDZ domains are linked by the C-terminal residues of PDZ binding proteins. To identify PL proteins that function in hematopoietic and endothelial cells, cell surface receptor proteins were identified and peptides were synthesized that had the sequence corresponding to the C-terminus of each protein. TABLE 4 lists these proteins, and provides corresponding C-terminal sequences and GenBank access numbers. "Clasp1" is described in WO 00/20434 (published April 13, 2000). "CLASP2" and "CLASP4" are described in applications also under processing USSN 09/547276 (agent file No. 20054-000200) and 60/196527 (agent file No. 20054-000400), both filed on April 11, 2000 .

Synthetic peptides of defined sequence (eg, corresponding to the carboxyl terms of the indicated proteins) can be synthesized by any standard resin-based method (see, eg, US Patent No. 4,108,846; see also Caruthers et al., 1980, Nucleic Acids Res. Symp. Ser., 215-223; Horn et al., 1980, Nucleic Acids Res. Symp. Ser., 225-232; Roberge, et al., 1995, Science 269: 202). The peptides used in the assays described herein were prepared by the FMOC (see, eg, Guy and Fields, 1997, Met. Enz. 289: 67-83; Wellings and Atherton, 1997, Met. Enz. 289: 44- 67. In some cases (eg, for use in tests A and G of the invention), the peptides were labeled with biotin at the amino terminus by reaction with a 4-fold excess of biotin methyl ester in dimethylsulfoxide with a catalytic amount The peptides were cleaved from the resin using an acid containing halide (eg trifluoroacetic acid) in the presence of appropriate antioxidants (eg ethanedithiol) and excess lyophilized solvent.

After lyophilization, the peptides can be redissolved and purified by reverse phase high performance liquid chromatography (HPLC). An appropriate HPLC solvent system involves a Vydac C18 semi-preparative column that operates at 5 ml per minute with increasing amounts of acetonitrile plus 0.1% trifluoroacetic acid in a water based solvent plus 0.1% trifluoroacetic acid. After HPLC purification, the identities of the peptides are confirmed by MALDI mass spectrometry in cation mode. As indicated, illustrative biotinylated peptides are provided in TABLE 4.

6.2.3 Detection of PDZ-PL Interactions

Based on the determination that the immune system cells contain both PDZ proteins and similarly many candidate PL proteins at the same time, it seemed clear to the inventors that the characterization of specific PDZ-PL interactions between these proteins would require reliable and rapid assays for such interactions A variety of assay formats known in the art can be used to select ligands that are specifically reactive as a particular protein. For example, solid phase ELISA immunoassays, immunoprecipitation, Biacore, and Western blotting assays can be used to identify peptides that specifically bind to PDZ domain polypeptides. As stated above, two different complementary assays were developed to detect PDZ-PL interactions. In each, a fixing pair of a PDZ-PL pair is immobilized, and the ability of the second member of the fixing pair to be fixed is determined. These assays, which are described below, can easily be used to screen hundreds to thousands of potential PDZ-ligand interactions in a few hours. Thus, these assays can be used to identify newer PDZ-PL interactions still in hematopoietic cells. Additionally, they can be used to identify antagonists of PDZ-PL interactions (see below).

6.2.3.1 Detection in "One assay" of the PDZ-Ligand Fixation Using Immobilized PL Peptide

In one aspect, the invention provides an assay in which biotinylated candidate PL peptides are immobilized on an avidin coated surface. The binding of the PDZ domain fusion protein to this surface is then measured. In a preferred embodiment, the PDZ domain fusion protein is then measured by attaching a PDZ fusion domain protein to this surface. In a preferred embodiment, the PDZ domain fusion protein is a GST / PDZ fusion protein and the assay is carried out as follows:

(one)

 Avidin is fixed to a surface, e.g. a protein binding surface. In one embodiment, avidin is fixed to a 96-well plate of polystyrene (eg, Nunc Polisorb (Cat. # 475094) by adding 100 μl per well of 20 μg / ml of avidin (Pierce) in phosphate buffered saline without calcium and magnesium, pH 7.4 ("PBS", GibcoBRL) at 4 ° C. for 12 hours The plate is then treated to block nonspecific interactions by adding 200 μl per well of PBS containing 2 g per 100 ml of bovine serum albumin Protease-free ("PBS / BSA") for 2 hours at 4 ° C. The plate is then washed 3 times with PBS by repeated addition of 200 μl per well of PBS to each well of the plate followed by overturning the plate contents into a waste container and tap the plate on a dry surface.

 (2)

 Biotinylated PL peptides (or candidate PL peptides, see TABLE 4) are immobilized on the surface of the plate wells by adding 50 μl per well of 0.4 μM peptide in PBS / BSA for 30 minutes at 4 ° C. Usually, each different peptide is added to at least 8 different wells so that multiple measurements can be made (eg duplicates as well as measures that use different (3ST / PDZ fusion domains and a negative GST control alone), and they are also prepared Additional negative control wells in which no peptide has been immobilized After immobilization of the PL peptide on the surface, the plate is washed 3 times with PBS.

(3)

 The GST / PDZ domain fusion protein (prepared as described above) is allowed to react with the surface by adding 50 µl per well of a solution containing 5 µg / ml of GST / PDZ domain fusion protein in PBS / BSA for 2 hours at 4 ° C. As a negative control, GST is added alone (i.e. not

a fusion protein) to specific wells, generally at least two wells (ie duplicate measurements) for each immobilized peptide. After the 2 hour reaction, the plates are washed 3 times with PBS to remove the unbound fusion protein.

(4)

 The binding of the GST / PDZ domain fusion protein to the avidin-biotinylated peptide surface can be detected using a variety of methods, and detectors known in the art. In one embodiment, 50 µl per well of an anti-GST antibody in PBS / BSA (e.g. 2.5 µg / ml goat polyclonal goat anti-GST antibody, Pierce) is added and allowed to react for 20 minutes at 4 ° C. The plate is washed 3 times with PBS and a second detectably labeled antibody is added. In one embodiment, 50 µl per well of 2.5 µg / ml of rabbit goat anti-immunoglobulin polyclonal antibody conjugated to horseradish peroxidase (HRP) is added to the plate and allowed to react for 20 minutes at 4 ° C. The plate is washed 5 times with 50 mM Tris pH 8.0 containing 0.2% Tween 20, and is revealed by adding 100 µl per well of HRP substrate solution (TMB, Dako) for 20 minutes at room temperature (RT). The reaction of the HRP and its substrate is terminated by the addition of 100 μl per well of 1 M sulfuric acid and the optical density (D.O.) of each well of the plate is read at 450 nm.

(5)

Specific binding of a PL peptide and a PDZ domain. The polypeptide is detected by comparing the signal from the well or wells in which the PL peptide and the PDZ domain polypeptide are combined with the background noise signal (s). The background noise signal is the signal found in the negative controls. Typically, a specific or selective reaction will be at least twice the background noise signal, more generally more than 5 times the background noise, and very generally 10 or more times the background noise signal. Additionally, a statistically significant reaction will involve multiple reaction measurements in which the signal and background noise will differ by at least two standard errors, more typically 4 standard errors, and very typically 6 or more standard errors. Correspondingly, a statistical test (eg a T test) comparing repeated measurements of the signal with repeated measurements of background noise will result in a p-value <0.05, more typically a p-value <0.01, and very typically a p value <0.001 or less.

As indicated above, in one embodiment of the "A" assay, the signal of binding of a GST / PDZ domain fusion protein to an avidin surface not exposed to (i.e. not covered with) the PL peptide is a control adequate negative (sometimes referred to as "B"). The signal from the fixation of the GST polypeptide alone (i.e. not a fusion protein) to an avidin coated surface that has been exposed (i.e. covered with) the PL peptide is a second suitable negative control (referred to sometimes as "B2"). Because all measurements are made in multiples (ie at least in duplicate) the arithmetic mean (or, equivalently, the average value) of several measurements is used to determine the fixation, using the standard error of the mean in the determination of the probable error in the measurement of the fixation. The standard error of the mean of N measures is equal to the square root of the following: the sum of the squares of the difference between each measure and the mean, divided by the product of (N) and (N-1). Thus, in one embodiment, the specific binding of the PDZ protein to the PL peptide attached to the plate is determined by comparison of the average signal ("S average") and the standard signal error ("SE") for a PL combination -PDZ particular with the average B1 and / or the average B2. In TABLE 2, it was detected that the fixation was specific (denoted by an "A" in the matrix) when (1) the mean S was at least twice the average B1 and at least twice the average B2, and (2 ) the mean S was at least 6 standard errors (six SE) greater than the average B1 and the average B2. Additionally, in the experiments summarized in TABLE 2, an additional criterion was used to ensure that none of the interactions defined as specific arose from a combined trend of both the particular PDZ fusion protein and the PL peptide tested to each give a noise background greater than usual. This criterion was that (3) the mean S was at least 20 times the product of the average B1 and the average B2. The factor 20 times reflects that at least one of B1 and B2 is generally less than 0.1 OD units, and therefore 20 times the product of the average B1 and the average B2 is as a rule less than twice the average B1 and twice the average B2, which makes the criterion (3) less severe than the criterion (1). Only in a small number of cases in which the average B1 and the average B2 are both greater than 0.1 D.O. (ie both the particular PDZ fusion protein and the PL peptide tested tend to give a background noise greater than usual) criterion (3) is more severe than criterion (1).

Table 4: PL Peptides

CODE

PROTEIN NAME GENBANK ACCESS SEQUENCE

AA1L

Clasp-1 ISKATPALPTVSISSSAEV

AA2L

Clasp-2 ISGTPTSTMVHGMTSSSSVV

AA3L

Clasp-4 CAISGTSSDRGYGSPRYAEV

AA4L

CD3n M33158 SVFSIPTLWSPWPPSSSSQL

AASL-M *

CD4 M12807 SEKKTSQSPHRFQKTCSPI

AA6L

CD6 X60992 SPQPDSTDNDDYDDISAA

AA7L

CD34 M81104 QATSRNGHSARQHVVADTEL

AA9L

CD44 M69215 QFMTADETRNLQNVDMKIGV

AA10L

CD46 (Form 1) M58050 KKGTYLTDETHREVKFTSL

AA11L

CD49E (4) X06256 PYGTAMEKAQLKPPATSDA

AA12L

CD49F X53586 HKAEIHAQPSDKERLTSDA

AA13L

CD95 M67454 KDITSDSENSNFRNEIQSLV

AA14L

CD97 X84700 TSGTGHNQTRALRASESGI

AA15L

CD98 J02939 ERLKLEPHEGLLLRFPYAA

AA16L

CD105 X72012 STNHSIGSTQSTPCSTSSMA

AA17L

VCAM1 M73255 ARKANMKGSYSLVEAQKSKV

AA18L

CD138 J05392 PKQANGGAYQKPTKQEEFYA

AA19L

CD148 D37781 ENLAPVTTFGKTNGYIA

AA20L

CD166 L38608 DLGNMEENKKLEENNHKTEA

AA22L

DNAM-1 U56102 TREDIYVNYPTFSRRPKTRV

AA23L-M *

FasL U11821 SSKSKSSEESQTFFGLYKL

AA25L

FceRIb D10583 YSATYSELEDPGEMSPPIDL

AA28L

CDW125 (IL5R) X62156 EVICYIEKPGVETLEDSVF

AA29.1L

CDW128A (ILBRA) M68932 ARHRVTSYTSSSVNVSSNL

AA29.2L

CDW128B (IL8RB) M73969 KDSRPSFVGSSSGHTSTTL

AA30L

LPAP X81422 AWDDSARAAGGQGLHVTAL

AA33L

KV1.3 AAC31761 TTNNNPNSAVNIKKIFTDV

AA34.2L

NMDA NP000824 LNSCSNRRVYKKMPSIESDV

AA37L

Glycoforin C AAA52574 QGDPALQDAGDSSRKEYFI

AA38L

Neurexin AB011150 SSAKSSNKNKKNKDKEYYV

AA39L

Syndecan-2 A33880 GERKPSSAAYQKAPTKEFYA

AA40L

DOCK2 BAA13200 LASKSAEEGKQIPDSLSTDL

AA41L

CC CKR-1R L09230 LERVSSTSPSTGEHELSAGF

AA42L

CC CKR-2 U03882 GKGKSIGRAPEASLQDKEGA

AA43L

CC CKR-3 HSU28694 LERTSSVSPSTAEPELSIVF

AA44L

CC CKR-4 X85740 DTPSSSYTQSTMDHDLHDAL

AA45L

BLR-1 S56162 PSW RRSSLSESENATSLTTF

AA47L

CD83 Z11697 VTSPNKHLGLVTPHKTELV

AA48L

CD62E M30640 SSSQSLESDGSYQKPSYIL

AA49L

CD5 X04391 SMQPDNSSDSDYDLHGAQRL

AA55L

CD148 D37781 TIYENLAPVTTFGKTIA

* The sequence studied is mutated at positions> 10 amino acids from term C to increase water solubility and / or eliminate intramolecular disulfides.

6.2.3.2 "G Test" - Detection of PDZ Ligand Fixation Using Immobilized PDZ Domain Fusion Polypeptide

In one aspect, the invention provides an assay in which a GST / PDZ fusion protein is immobilized on the surface ("G" assay). The binding of the labeled PL peptide (as listed in TABLE 4) to this surface is then measured. In a preferred embodiment, the test is carried out as follows:

(1) A polypeptide of the PDZ domain is fixed to a surface, e.g. a protein binding surface. In a preferred embodiment, a GST / PDZ fusion protein containing one or more PDZ domains is attached to a 96-well polystyrene plate. The GST / PDZ fusion protein can be fixed to the plate by anyone

10 of a variety of standard methods known to one skilled in the art, although some care must be taken so that the plate fusion protein binding process does not alter the ligand binding properties of the PDZ domain. In one embodiment, the GST / PDZ fusion protein is fixed by an anti-GST antibody that is coated on the 96-well plate. Proper fixing to the plate can be achieved when:

15 a. 100 μl per well of 5 μg / ml of Goce Pierce goat anti-GST polyclonal antibody in PBS is added to a 96-well polystyrene plate (e.g., Nunc. Polisorb) at 4 ° for 12 hours.

b.

 The plate is blocked by adding 200 µl per well of PBS / BSA for 2 hours at 4 ° C.

C.

 The plate is washed 3 times with PBS.

d. 50 μl per well of a 5 μg / ml GST / PDZ fusion protein or, as a negative control, GST polypeptide alone (i.e. not a fusion protein) in PBS / BSA for 2 hours at 4 ° C is added to the plate .

and. The plate is washed again 3 times with PBS.

(2) Biotinylated PL peptides (or candidate PL peptides, eg as shown in TABLE 4) are allowed to react with the surface by adding 50 µl per well of 20 µM solution of the biotinylated peptide in PBS / BSA for 10 minutes at 4 ° C , followed by an additional incubation for 20 minutes at 25 ° C. The

25 plate is washed 3 times with ice cold PBS.

(3) Biotinylated peptide binding to the surface of the GST / PDZ fusion protein can be detected using a variety of methods and detectors known to one skilled in the art. In one embodiment, 100 µl per well of streptavidin-horseradish peroxidase (HRP) conjugate of 0.5 µg / ml dissolved in BSA / PBS is added and allowed to react for 20 minutes at 4 ° C. The plate is then washed 5 times with

30 mM Tris pH 8.0 containing 0.2% Tween 20, and is revealed by adding 100 µl per well of HRP substrate solution (TMB, Dako) for 20 minutes at room temperature (RT). The reaction of the HRP and its substrate is terminated by the addition of 100 μl per well of 1 M sulfuric acid, and the optical density (D.O.) of each well of the plate is read at 450 μM.

(4) The specific binding of a PL peptide and a PDZ domain polypeptide is determined by comparison of

The signal of the well (s) in the one or which the PL peptide and the PDZ domain polypeptide are combined with the background noise signal (s). The background noise signal is the signal found in the negative control (s). Typically, a specific or selective reaction will be equal to at least twice the background noise signal, more typically more than 5 times the background noise, and very typically 10 or more times the background noise signal. Additionally, a statistically significant reaction will involve multiple measurements of the reaction, the signal and background noise differing by at least two standard errors, more typically four or more standard errors, and very typically six or more standard errors. Correspondingly, a statistical test (eg a T-test) comparing repeated measurements of the signal with repeated measurements of background noise will result in a p-value <0.05, more typically a p-value <0.01, and very typically a p value <0.001 or less. As indicated, in one embodiment of the "G" test, the binding signal of a PL peptide given to an immobilized GST polypeptide (fixed to the surface) is only a suitable negative control (sometimes referred to as " B1 "). Since all measurements are made in multiples (ie at least in duplicate) the arithmetic mean (or, equivalently, the average value) of several measurements is used in determining the fixation, and the standard error of the mean is used in determining the probable error in the measurement of the fixation. The standard error of the mean of N measures is equal to the square root of the following: the sum of the squares of the difference between each measure and the average value, divided by the product of (N) and (N) -1. Thus, in one embodiment, the specific binding of the PDZ protein to the plate-bound peptide is determined by comparison of the average signal ("S average") and the standard signal error ("SE") for a PL combination -PDZ particular with the average B1. In experiments summarized in TABLE 2, it was determined that the fixation was specific (denoted by a "G" in the matrix) when (1) the mean S was at least twice the average of B1 and (2) the mean S was at least six standard errors (six SE) greater than the mean B1. The results of illustrative "G" tests are shown in FIGURES 1A-1D.

6.2.4 Test Variations

As stated above, it will be appreciated that many of the steps in the tests described above can be modified; for example, various substrates can be used to fix the PL and PDZ-containing proteins; different types of fusion proteins containing PDZ can be used; different markers can be used to detect PDZ / PL interactions, and different detection pathways can be used.

PDZ-PL detection assays can employ a variety of surfaces to fix proteins containing PL and PDZ. For example, a surface can be a "test plate" that is formed of a material

(e.g. polystyrene) that optimizes the adhesion of the PL protein or the PDZ-containing protein to it. Generally, the individual wells of the test plate will have a high surface-to-volume ratio and therefore a suitable shape is a flat bottomed well (where the test proteins are adherent). Other surfaces include, but are not limited to, polystyrene or glass beads, polystyrene or glass slides, and the like.

For example, the test plate may be a "microtiter" plate. The term "microtiter plate", when used herein, refers to a multi-well test plate, e.g. which has between about 30 and 200 individual wells, usually 96 wells. Alternatively, high density networks can be used. Often, the individual wells of the microtiter plate will contain a maximum volume of approximately 250 μl. Conveniently, the test plate is a 96-well polystyrene plate (such as that sold by Becton Dickinson Labware, Lincoln Park, L.J.), which allows high capacity automation and screening. Other surfaces include polystyrene microtiter ELISA plates such as that sold by Nunc Maxisorp, Inter Med., Denmark. Often, approximately 50 μl to 300 μl, more preferably 100 μl to 200 μl, of an aqueous sample comprising buffers suspended therein will be added to each well of the test plate.

The detectable markers can be any detectable compound or composition that is directly conjugated

or indirectly with a molecule (such as the one described above). The label may itself be detectable (e.g., radioisotope markers or fluorescent labels) or, in the case of an enzymatic label, it may catalyze a chemical alteration of a substrate compound or composition that is detectable. The preferred label is an enzymatic label that catalyzes a color change of a non-radioactive colored reagent.

Sometimes, the label is indirectly conjugated with the antibody. An experienced person is knowledgeable about various techniques for indirect conjugation. For example, the antibody can be conjugated with biotin and any of the categories of markers mentioned above can be conjugated with avidin, or vice versa (see also assay "A" and "G" above). Biotin is selectively fixed to avidin and therefore, the label can be conjugated with the antibody in this indirect way. See, Ausubel, supra, for a review of techniques involving biotin-avidin conjugation and similar assays. Alternatively, to achieve indirect conjugation of the label with the antibody, the antibody is conjugated with a small hapten (e.g., digoxin) and one of the different types of markers mentioned above is conjugated with an anti-hapten antibody (e.g., anti-digoxin antibody). In this way, indirect conjugation of the label with the antibody can be achieved.

Test variations may include different washing steps. By "washing" is meant to expose the solid phase to an aqueous solution (usually a buffer or cell culture medium) such that the unbound material

(e.g., non-adherent cells, non-adherent capture agent, unbound ligand, receptor, receptor construct, cell lysate, or HRP antibody) is separated from it. To reduce background noise, it is convenient to include a detergent (e.g., Triton X) in the wash solution. Usually, the aqueous wash solution is decanted from the wells of the test plate after washing. Conveniently, the washing can be performed using an automatic washing device. Sometimes several washing steps may be necessary (e.g., between about 1 and 10 washing steps).

Various buffers can also be used in PD-PL detection assays. For example, various blocking buffers can be used to reduce the background noise of the test. The term "blocking buffer" refers to an aqueous, pH buffered solution that contains at least one blocking compound that is capable of binding to exposed surfaces of the substrate that are not coated with a PL protein or that contains PDZ. The blocking compound is normally a protein such as bovine serum albumin (BSA), gelatin, casein or milk powder and does not cross-react with any of the test reagents. The blocking buffer is generally provided at a pH between about 7 and 7.5, and suitable buffer agents include phosphate and TRIS.

Various enzyme-substrate combinations can also be used in the detection of PDZ-PL interactions. Examples of enzyme-substrate combinations include, for example:

(i)

Horseradish peroxidase (HRPO) with hydrogen peroxidase as a substrate, where hydrogen peroxidase oxidizes a dye precursor (eg orthophenylene diamine [OPD] or 3,3 ', 5,5'-tetramethylbenzidine [TMB] hydrochloride) (as has been described above);

(ii)

 Alkaline phosphatase (AP) with para-nitrophenyl phosphate as a chromogenic substrate.

(iii) β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g., p-nitrophenyl-β-D-galactosidase) or 4-methylumbelliferyl-β-D-galactosidase fluorogenic substrate.

Numerous other enzyme-substrate combinations are available to those skilled in the art. For a general review of these, see U.S. Pat. No. 4,275,149 and 980.

Additionally, it will be appreciated that, although for convenience the present disclosure relates mainly to antagonists of PDZ-PL interactions, agonists of PDZ-PL interactions can be identified using the methods described herein or readily apparent variations thereof.

6.2.5 Results of PDZ-PL Interaction Tests

TABLE 2, supra, shows the results of tests in which specific binding was detected using the "A" and "G" assays described herein. The top row of the table specifies the source of the PDZ domain used in the GST-PDZ fusion proteins (see TABLE 3). The first column lists the cell surface proteins from which C-terminal peptide sequences were derived and the second column ("code") identifies the peptide used in the assay (see TABLE 4). The third column, "Seq" provides sequence of the four (4) C-terminal residues of the protein and the cell surface peptide. In the matrix, "A" indicates specific binding as detected in test "A". "G" indicates specific binding as shown in the "G" test. A blank indicates that no specific binding was detected using the "A" or "G" assays. An asterisk (*) indicates that a paired interaction between the PDZ protein and the cell surface protein (or subdomains of any of them) has been described by others.

6.2.5.1 New PL Reasons

As indicated above, TABLE 2 shows the test results (referred to as "PRISM MATRIX") to detect binding between PDZ proteins and candidate PL peptides. A number of specific PDZ-PL interactions are identified by the MATRIX and from these results key amino acids and important positions in PDZ fixation ("PL motifs") are deduced. The MATRIX is not only useful for comprehensively cataloging PDZ-PL binding combinations, but the assay can further assist in the rapid discovery and characterization of new PL proteins and PL motifs to aid rational drug design and synthesis of inhibitors of PL-PDZ interaction.

Other researchers have recorded certain important PL motifs in the fixation of PDZ, eg, the C / S / TXV / I / L (for DLG1) and Y / FY / FY / L / F motives for MPP1 (see Doyle et al., 1996 , Cell 85, 1067; Songyang et al., 1997, Science 275, 73). However, the reasons given are not specific enough (that is, a large number of proteins meet these criteria but are not necessarily real PDZ ligands) and cover only a small number of PDZ proteins (approximately 10). The PRISM MATRIX can be used to determine ligand specificity and deduce ligand binding motifs for any PDZ protein, since it can accurately determine amino acid sequences that give or not result in specific PDZ binding. Additionally, the assay has revealed the importance of new PDZ domain binding motifs (ie PL motifs): CD6 C-terminal sequence, ISAA; C-terminal sequence of CD49E, TSDA; C-terminal sequence of CD49F, TSDA; C-terminal sequence of Clap-1, SAEV; C-terminal sequence of CLASP-4, YAEV; Cterminal sequence of CD44, KIGV; C-terminal sequence of IL5R, DSVF; C-terminal sequence of BLR-1, LTTF. The identification of these new PL sequences makes it possible to define new PL motifs (see TABLE 5A, below). The specificity with which these new motifs are defined is enhanced by the fact that the MATRIX records both positive results (i.e. PDZ-PL combinations that result in specific binding interactions) and negative results (i.e. PDZ combinations- PLO that do not result in specific fixation). For example, the C-terminal sequence of CD6, SAA and the C-terminal sequence of CD49E, SDA are fixed to the PDZ domain polypeptide 41.8, while the related C-terminal sequence of CD166, TEA and the Cterminal sequence of CD148, YIA do not. This identifies the new PL motif (Reason 1, below) of alanine-terminating polypeptides with serine at position -2 and excludes threonine and tyrosine polypeptides at position -2. This reason is therefore more specific than most of the reasons identified above. Other new reasons are described in TABLE 5A.

TABLE 5A

Position: -3 -1 C-terminal

-

2 Reason 1 X X A

S Reason 2 X D / E V

A Reason 3 X X * V

V / I / L S / T

4 X X F reason

X * is any non-aromatic amino acid (any residue other than T, F or W)

10 6.2.5.2 Fixing Groups

TABLE 2 is arranged in such a way that both PDZ / GST fusion proteins and PL peptide ligands are arranged with structurally similar molecules close to each other. In the preparation of TABLE 2 and the conduct of the experiments used to create this table, the PDZ domains of each of the GST-PDZ fusion proteins were classified in terms of amino acid sequence similarity using the software package of CLUSTAL multiple sequence alignment. Proteins with greater sequence similarity are closer to each other in the matrix. PL peptide ligands were also ordered on the basis of amino acid similarity, but assigning weight to residues that are considered important in PDZ binding (Doyle et al., 1996, Cell 85, 1067). In particular, the peptides were first ordered on the basis of the residue closest to the C terminal (zero position) in the following amino acid order: G, A, C, S, T, N,

20 Q, D, E, H, K, R, V, I, L, M, P, F, Y, W. Among the peptides with identical C terms, the same classification scheme was then applied to the following most important residue for peptide fixation, position -2, followed by position -1 and position -3. (In an alternative approach, GST-PDZ functions can also be arranged so that they give additional weight to residues known to be important for ligand fixation when proteins are aligned).

25 The regions of the TABLE 2 matrix that are densely filled with important binding interactions indicate that a particular ligand structure tends to bind to a particular family of PDZ domains. The results indicate that PDZ domains with similar structures fix ligands with similar structures, as indicated by the presence in the matrix of some clusters of many important binding interactions and other areas of few important binding interactions. These results reveal a certain number of relationships

30 structural between different PDZ proteins and their ligands. The compression of these structural interrelationships can be used to scan databases for probable ligands of specific PDZ proteins, facilitating the design of inhibitors of such new interactions and the prediction of the side effect profile of such inhibitors.

The most notable example of a grouping occurs in columns 1-5 of the matrix (PDZ domains of CASK, MPP1,

35 DLG1, PSD95, and NeDLG). In the tests performed by the authors of the invention, CASK was significantly fixed only to one of the ligands tested, neurexin (this interaction had previously been identified in neuronal cells). MPP1, the closest relative to CASK in the sorting system used, fixed a set of 5 different ligands, including neurexin. All identified MPP1 ligands are structurally similar in that they terminate in valine (V) and have an amino acid

40 loaded (E or K) in position -3 from the term C ((C-terminal motif: E / K-X-X-V).

Of the five MPP1 ligands identified, four of them are also fixed to DLG1, the next protein most similar to CASK and MPP1. DLG is closely related to two other proteins, PSD95 and NeDLG. Each of these three fixed protein ligands ending in the S / T / Y-X-V / I / L motif, as previously described (Songyang et al., 1997, Science 275, 73). Interestingly, if the terminal ligand residue is valine, other residues not previously identified at position -2 are compatible with binding, resulting in the AEV and ELV Cterminales sequences important binding events. While this reflects a flexibility not previously recognized in the binding of PDZ ligands, the specificity of the interactions detected in the assays

made by the authors of the invention is reflected in the large areas at the top and bottom of columns 1-5 that do not contain any significant fixing event. These areas reflect that none of the potency ligands ending in residues other than V / I / L were significantly set to CASK, MPP1, DLG1, PSD95, or NeDLG.

5 (With respect to MPP1, it is noteworthy that glycoforin C, a known ligand of MPP1, Marfatia et al., 1997,

J. Biol. Chem. 272, 24691, was not significantly fixed to MPP1 in the tests carried out by the authors of the present invention, but terminates in isoleucine, an amino acid very similar to valine, and in fact has a residue charged in the position -3 of the term C.)

Other smaller groupings in the matrix also provide valuable information about specificity.

10 ligand of certain PDZ domains. For example, five ligands are significantly fixed in both "G" and "A" assays to the 41.8 kD protein, a protein containing PDZ domain not previously studied.

All these ligands terminate at A, V, I, or L, and four of the five have serine (S) at position -2, defining the C-terminal ligand motif S-X-A / V / I / L for this PDZ domain. Similarly, two of three ligands that specifically bind to KIAA0561 also conform to the C-terminal motif X * -S / T-D / E-V / I / L where X * is any non-aromatic amino acid. Interestingly, one of these ligands is also fixed to the closest relative to KIAA0561's, TAX2. Similarly, the two ligands that bind prIL16 both have an amino acid charged at the -3 position, a hydrophobic amino acid at the -2 position and valine at the terminal position. An interesting final example of defining the ligand specificity of a PDZ domain involves PTN-4. Although the two ligands that are specifically bound to PTN-4 are not closely related in their 2 C-terminal residues, both conform to motif C-terminal D / E-S-X-V / I / L / F / Y. These motifs that characterize the ligand specificity of particular PDZ proteins are summarized in TABLE 5B. Such motifs allow database investigations for new ligands (PL proteins) that bind to these particular PDZ proteins. The knowledge of the PL proteins that bind to these PDZ proteins allows the design of inhibitors of these interactions, using the methods described herein below. Additionally, knowledge of the set of PL proteins that are attached to a

Particular PDZ protein can be used to predict the utility and side effects of compounds that are targeted to this PDZ protein.

TABLE 5B

PDZ -3 -2 -10

41.8 kD X X X A / V / I / L

KIAA 0561 X * S / T D / E V / I / L

TAX 2 X S D / EV

PRIL 16 D / E / K / R V / I / L / F / Y X V

PTN4 D / E S X V / I / L / F / Y

X * is any non-aromatic amino acid

6.3 Measuring Affinity of Ligand PDZ

The "A" and "G" assays of the invention can be used to determine the "apparent affinity" of binding a PDZ ligand peptide to a PDZ domain polypeptide. Apparent affinity is determined based on the concentration of a molecule required to saturate the binding of a second molecule (e.g., the binding of a ligand to a receptor). Two particularly useful approaches for quantifying the apparent affinity for binding the PDZ ligand are provided below.

35 (1) A GST / PDZ fusion protein, as well as GST only as a negative control, are fixed to a surface

(e.g. a 96-well plate) and the surface is blocked and washed as described above for the "G" test.

(2) 50 μl per well of a biotinylated PL peptide solution (eg as shown in TABLE 4) is added to the surface in increasing concentrations in PBS / BSA (eg 0.1 μM, 0.33 μM, 1 μM, 3.3 μM, 10

40 μM, 33 μM, and 100 μM). In one embodiment, the PL peptide is allowed to react with the fixed GST / PDZ fusion protein (as well as the negative GST control alone) for 10 minutes at 4 ° C followed by 20 minutes at 25 ° C. The plate is washed 3 times with ice-cold PBS to remove the unbound labeled peptide.

(3)

 The binding of the PL peptide to the immobilized PDZ domain polypeptide is detected as described above for the "G" assay.

(4)

For each peptide concentration, the net binding signal is determined by subtraction of the peptide binding to GST only from the peptide binding to the GST / PDZ fusion protein. The net fixation signal is then plotted as a function of ligand concentration and the graph is adjusted (eg by using the curve fitting algorithm of the Kaleidagraph software package) to the following equation, where "Signal [ligand]" is the net fixation signal at the concentration of the PL peptide "[ligand]", "Kd" is the apparent affinity of the fixation event, and "Saturation Fixation" is a constant determined by the curve fitting algorithm to optimize the fit to the experimental data:

Signal [ligand] = Saturation Setting x ([ligand] / ([ligand] + Kd))

For reliable application of the above equation it is necessary that the maximum concentration of experimentally tested peptide ligand be greater than, or at least similar to, the calculated Kd (equivalently, the maximum observed fixation should be similar to the calculated saturation fixation) . In cases where the satisfaction of the above criteria is difficult, an alternative approach can be used (below).

The results obtained when using approach 1 are shown in FIGURES 2A AND 2B. FIGURE 2 shows varying concentrations of CLASP-2 (FIGURE 2A) or biotinylated Fas (FIGURE 2B). The Cterminales peptides reacted with the immobilized GST polypeptide (plaque bound) or the GST / PDZ fusion proteins (GST / DLG1, GST / NeDLG, and GST / PSD95) in duplicate. The signals were normalized, plotted and adjusted to a saturation fixation curve, producing an apparent affinity of 21 μM for the DLG1-CLASP-2 interaction, 7.5 μM for the NeDLG-CLASP-2 interaction, 45 μM for the PSD95-CLASP-2 interaction, and 54 μM for the DLG1-Fas interaction, 54 μM for the NeDLG-Fas interaction, and 85 μM for the PSD95-Fas interaction.

Approach 2:

(one)

A fixed concentration of a PDZ domain polypeptide and increasing concentrations of labeled PL peptide (labeled with, for example, biotin or fluorescein, see TABLE 4 for representative peptide amino acid sequences) are mixed in solution and allowed to react. In one embodiment, preferred concentrations of peptide are 0.1 μM, 1 μM, 10 μM, 100 μM, 1 mM. In various embodiments, appropriate reaction times may range from 10 minutes to 2 days at temperatures between 4 ° C and 37 ° C. In some embodiments, the identical reaction can also be carried out using a protein that does not contain a PDZ domain as a control (e.g., if the PDZ domain polypeptide is a fusion protein, the fusion partner can be used).

(2)

PDZ-ligand complexes can be separated from the unbound labeled peptide using a variety of methods known in the art. For example, complexes can be separated using high resolution size exclusion chromatography (HPSEC, gel filtration) (Rabinowitz et al., 1998, Immunity 9: 699), affinity chromatography (eg using glutathione-Sepharose beads, and affinity absorption (eg, by attachment to a plate coated with anti-GST as described above).

(3)

 The PDZ-ligand complex is detected based on the presence of the marker in the peptide ligand using a variety of methods and detectors known to a person skilled in the art. For example, if the marker is fluorescein and separation is achieved using HPSEC, an online fluorescence detector can be used. Fixation can also be detected as described above for test G.

(4) The PDZ-ligand binding signal is plotted as a function of ligand concentration and the graph is adjusted (eg, using the curve fitting algorithm of the Kaleidagraph software package) to the following equation, where "Signal [ ligand] "is the net binding signal at the concentration of the PL peptide" [ligand] "," Kd "is the apparent affinity of the binding event, and" Saturation Fixing "is a constant determined by the curve fitting algorithm to optimize the fit to the experimental data:

Signal [ligand] = Saturation Setting x ([ligand] / ([ligand + Kd])

The measurement of the affinity of a labeled peptide ligand that binds to a polypeptide n of the PDZ domain is useful because knowledge of the affinity (or apparent affinity) of this interaction allows the design of concentration inhibitors with known potency ( see EXAMPLE 2). The potency of inhibitors in inhibition should be similar to (ie within an order of magnitude of) the apparent affinity of the labeled peptide ligand that binds to the PDZ domain.

6.4 Tests to Identify New PDZ Domain Fixation Remains and to Identify PDZ Protein-PL Protein Fixation Inhibitors

Although described above primarily in terms of identification of interactions between PDZ domain polypeptides and PL proteins, the assays described above and other assays can be used to identify the binding of other molecules (eg peptide mimetics, small molecules and the like) to the PDZ domain sequences. For example, using the assays described herein, they can be screened

combinatorial libraries and other compound libraries. Library screening can be performed by any of a variety of commonly known methods. See, e.g., the following references, which disclose the screening of peptide libraries: Parmley and Smith, 1989, Adv. Exp. Med. Biol. 251: 215-218; Scott and Smith, 1990, Science 249: 386-390; Fowlkes et al., 1992; BioTechniques 13: 422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393-5397; Yu et al., 1994, Cell 76: 933-945; Staudt et al., 1988, Science 241: 577-580; Bock et al., 1992, Nature 355: 564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6988-6992; Ellington et al., 1992, Nature 355: 850-852; U.S. Patent No. 5,096,815, U.S. Patent No. 5,223,409, and U.S. Pat. No. 5,198,346, all of them granted to Ladner et al .; Rebar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO 94/18318.

In a specific aspect, screening can be performed by contacting members of the library with a PDZ domain polypeptide of hematopoietic cells immobilized on a solid support (eg as described above in the "G" assay) and collecting those members of the library that bind to protein. Examples of such screening methods, called "panning techniques" are described by way of example in Parmley and Smith, 1988, Gene 73: 305-318; Fowlkes et al., 1992, BioTechniques 13: 422-427; PCT Publication No. WO 94/18318; and in the references cited above in this report.

In another aspect, the two hybrid system can be used to select yeast interacting proteins (Fields and Song, 1989, Nature 340: 245-246; Chien et al., 1991, Proc. Natl. Acad. Sci. USA 88: 9578 -9582) to identify molecules that specifically bind to a protein that contains PDZ domain. Additionally, the identified molecules are further tested for their ability to inhibit transmembrane receptor interactions with a PDZ domain.

In one aspect, antagonists of an interaction between a PDZ protein and a PL protein are identified. In one embodiment, a modification of the "A" assay described above is used to identify antagonists. In one embodiment, a modification of the "G" assay described above is used to identify antagonists.

In one aspect, screening assays are used to detect molecules that specifically bind to PDZ domains in hematopoietic cells. Such molecules are useful as agonists or antagonists of cellular function mediated by PDZ proteins (eg, cell activation, eg, T cell activation, vesicle transport, cytokine release, growth factors, transcription changes, rearrangement of cytoskeletin, cell movement, chemotaxis, and the like). In one aspect, such assays are performed for screening with respect to leukocyte activation inhibitors for drug development. The description thus provides assays to detect molecules that specifically bind to proteins that contain PDZ domain in hematopoietic cells. For example, recombinant cells expressing nucleic acids encoding the PDZ domain can be used to produce PDZ domains in these assays and screen for molecules that bind to the domains. The molecules are contacted with the PDZ domain (or fragment thereof) under conditions conducive to fixation, and molecules that specifically bind to such domains are then identified. Methods that can be used to perform the above are commonly known in the art.

By way of example, various libraries, such as random or combinatorial peptide libraries or non-peptide libraries can be screened for molecules that specifically bind PDZ domains in hematopoietic cells. Many libraries that can be used are known in the art, e.g., libraries synthesized by chemical means, recombinant libraries (e.g. phage display libraries), and libraries based on in vitro translation.

Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251: 767-773; Houghten et al., 1991, Nature 354: 84-86; Lam et al., 1991, Nature 354: 82-84; Medynski, 1994, Bio / Technology 12: 709-710; Gallop et al., 1994, J. Medicinal Chemistry 37 (9): 1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. USA 90: 10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11422-11426; Houghten et al., 1992, Biotechniques 13: 412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90: 11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89: 5381-5383.

Examples of phage display libraries are described in Scott and Smith, 1990, Science 249: 386-390; Devlin et al., 1990, Science, 249: 404-406; Christian, R.B., et al., 1992, J. Mol. Biol. 227: 711-718); Lenstra, 1992, J. Immunol. Meth. 152: 149-157; Kay et al., 1993, Gene 128: 59-65; and PCT Publication No. WO 94/18318 dated August 18, 1994.

Libraries based on in vitro translation include, but are not limited to, those described in PCT Publication No. WO91 / 05058 dated April 18, 1991; and Mattheakis etal., 1994, Proc.Natl.Acad.Sci.USA 91: 9022- 9026

As examples of non-peptide libraries, a benzodiazepine library can be adapted for use (see, e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91: 4708-4712). Peptoid libraries can also be used (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9367-9371). Another example of a library that can be used, in which the amide functionalities in the peptides have been subjected to permethylation to generate a chemically transformed combinatorial library, is described by Osthres et al. (1994) Proc. Natl. Acad. Sci. USA

91: 11138-11142).

It will be appreciated by a technician with ordinary experience that, in one aspect, the antagonists are identified by performing the A or G tests in the presence and absence of a known antagonist or candidate. When a reduced fixation is observed in the presence of a compound, said compound is identified as an antagonist. Increased fixation in the presence of a compound means that the compound is an agonist.

For example, in a test a test compound can be identified as a binding inhibitor (antagonist) between a PDZ protein and a PL protein by contact of a PDZ domain polypeptide and a PL peptide in the presence or absence of the test compound, under conditions in which they would form a complex (except for the presence of the test compound), and detection of the formation of the complex in the presence and absence of the test compound. It will be appreciated that a lower formation of the complex in the presence of the test compound which in the absence of the compound indicates that the test compound is a PDZ protein-PL protein binding inhibitor. In various embodiments, the PL peptide comprises an amino acid sequence substantially identical to the C-terminal sequence of a PL protein (eg, CD6, CD49E, CD49F, CD138, Clasp-1, Clasp-4, VCAM1, Clasp-2, CD95 , DNAM-1, CD83, CD44, CD4, CD97, Neurexin, CD3n, DOCK2, CD34, FceRIb, or FasLigand).

In one aspect, the "G" assay is used in the presence or absence of a candidate inhibitor. In one embodiment, the "A" assay is used in the presence or absence of a candidate inhibitor.

In one aspect (in which a G-test is used), one or more GST fusion proteins containing PDZ domain are fixed to the surface of the wells of a 96-well plate as described above (with appropriate controls including protein GSP not fusion). All fusion proteins are fixed in multiple wells, so that appropriate controls and statistical analysis can be performed. A test compound in BSA / PBS (typically at different multiple concentrations) is added to the wells. Immediately thereafter, 30 µl of a detectably labeled peptide (e.g., biotinylated) known to bind the relevant PDZ domain (see, e.g., TABLE 2) is added to each well at a final concentration of, e.g. between about 2 μM and about 40 μM, typically 5 μM, 15 μM, or 25 μM. This mixture is then allowed to react with the PDZ fusion protein fixed to the surface for 10 minutes at 4 ° C, followed by 20 minutes at 25 ° C. The surface is washed 3 times to leave it free of unbound peptide with ice-cold PBS and the amount of peptide binding is determined in the presence and absence of the test compound. Usually, the level of fixation for each set of replicate wells (e.g. duplicates) is measured by subtraction of the average value of the GST exclusive background noise from the mean value of the crude measurement of peptide fixation in these wells.

In an alternative aspect, test A is carried out in the presence or absence of a test candidate to identify inhibitors of PL-PDZ interactions.

In one aspect, a test compound is determined that is a specific inhibitor of PDZ domain binding.

(P) and a PL (L) sequence when, at a concentration of the test compound less than or equal to 1 μM (eg, less than or equal to 500 μM, 100 μM, 10 μM, 1 μM, 100 nM or 1 nM ) the fixation of P to L in the presence of the test compound is less than about 50% of the fixation in the absence of the test compound (in various embodiments, less than about 25%, less than about 10%, or less than about 1 %). Preferably, the net fixation signal from P to L in the presence of the test compound plus six (6) times the standard error of the signal in the presence of the test compound is less than the fixation signal in the absence of the test compound.

In one aspect, assays are performed for an inhibitor using a single PDZ protein-PL protein pair (e.g., a fusion protein of the PDZ domain and a PL peptide). In a related embodiment, the tests are carried out using a plurality of pairs, such as a plurality of different pairs listed in TABLE 2. In some aspects, it is desirable to identify compounds that, at a given concentration, inhibit the fixation of a PL-PDZ pair, but do not inhibit (or inhibit to a lesser extent) the fixation of a second specified PL-PDZ pair. These antagonists can be identified by performing a series of assays using a candidate inhibitor and different PL-PDZ pairs (e.g., as shown in the matrix of TABLE 2) and comparing the test results. All such combinations are contemplated in pairs (e.g., the test compound inhibits the fixation of PL1 to PDZ to a greater extent than it inhibits the fixation of PL1 to PDZ2 or PL2 to PDZ2). It is important, and it will be appreciated that, based on the data provided in TABLE 2 and described herein, (and additional data that can be generated using the methods described herein) inhibitors with different specificities can be easily designed.

The PDZ binding moieties and PDZ protein-PL protein binding antagonists of the invention are used to modulate biological activities or functions of the cells (eg, hematopoietic cells, such as T cells and B and analogous cells), endothelial cells, and other cells of the immune system, as described herein, and for the treatment of diseases and conditions in humans and non-human animals (eg, experimental models). Illustrative biological activities have been listed above.

When administered to patients, the compounds (eg, PL-PDZ interaction inhibitors) are useful for the treatment (improves symptoms of) a variety of diseases and conditions, including diseases characterized by inflammatory and humoral immune responses, eg, inflammation, allergy (eg, systemic anaphylaxis, hypersensitivity responses, drug allergies, allergic to insect bites; inensayoinal inflammatory diseases, ulcerative colitis, ileitis and enteritis; inflammatory psoriasis and dermatosis, scleroderma; allergic respiratory diseases such as asthma, allergic colitis , pulmonary hypersensitivity diseases, and analogous vasculitis, Rh incompatibility, transfusion reactions, drug sensitivities, PIH, atopic dermatitis, eczema, rhinitis; autoimmune diseases, such as arthritis (rheumatoid and psoriatic, multiple sclerosis, systemic lupus erythematosus insulin dependent diabetes ina, glomerulonephritis, scleroderma, MCTD, IDDM, Hashimoto's thyroiditis, Goodpasture syndrome, psoriasis and the like, osteoarthritis, polyarthritis, graft rejection (eg, allograft rejection, eg, renal allograft rejection, reverse rejection disease, transplants (heart, kidney, lung, liver, thin inensayoin, cornea, pancreas, corpse, autologous, bone marrow, and xenotransplantation)), atherosclerosis, angiogenesis-dependent disorders, cancers (eg, melanomas and breast cancer, prostate cancer) , leukemias, lymphomas, metastatic disease), infectious diseases (eg, viral infection, such as HIV, measles, parainfluenza, virus-mediated cell fusion), ischemia (eg, post-myocardial infarction complications, joint injury, kidney, scleroderma ).

The PL proteins and PDZ proteins listed in TABLE 2 are well characterized, and an experienced person, aided by this description (including the discovery of the interactions between PL proteins and PDZ proteins described herein), will recognize many uses for modulators (eg, enhancers or inhibitors) of PDZ-PL interactions such as those described in TABLE 2. To further assist the reader, an exposition of the characteristics of selected PL proteins (and their function) is provided below. It will be recognized that this description is not exhaustive and that it is not intended to limit the invention in any way. Additionally, nothing in this section should be construed as intended by the inventors to limit themselves to a particular mechanism of action.

A. CD6

As indicated above, CD6 fixes PDZ 41.8 protein. CD6 is expressed in thymocytes, T cells, and chronic lymphocytic leukemia of B cells. CD6 plays a role in costimulation of T cells, and CD6 negative T cells are less self-reactive than CD6 positive T cells. It is predicted that inhibition of CD6 and CDE6 / 41.8 interactions will reduce the symptoms of reverse rejection disease (GVHD) or psoriasis. Thus, in one aspect of the description, GVD is reduced in a patient receiving bone marrow cells from the donor by pretreating the cells with an effective amount of an antagonist. In combination with post-transplant immunosuppressive therapy such as FK606, Cellcept or cyclosporine, CD6-PDZ interaction inhibitors will improve the overall survival of transplant patients (e.g., leukemia patients).

B. CD49e (ALFA-4)

As shown by the experiments reported herein, the C-terminal end of CD49e is fixed to the 41.8 kD protein that contains the PDZ domain. CD49e is a 110 kD membrane transmembrane protein of the alpha-integrin family (alpha-integrin 5). Paired with the integrin beta-1 subunit, form VLA-5. VLA-5 is predominantly expressed in cells of the hematopoietic and lymphoid lineages that include monocytes, basophils, T cells, and activated B cells. VLA-5 is the receptor for the ubiquitously expressed fibronectin adhesion molecule. A tissue lesion such as myocardial infarction releases soluble fragments of fibronectin. The binding of these soluble fragments to VLA-5 results in chemotaxis of immune cells that include monocytes to the source of fibronectin, as well as decreasing modulation of VLA-5 expression in these cells. Such ligand-induced decreasing regulation is a common and required characteristic of chemotactic receptors. Once immune cells fully migrate to the source of fibronectin, adhesion to the fibronectin surface is enhanced by the fibronectin-VLA-5 interaction. Without intending to be bound by a particular mechanism, it is believed that the 41.8 / CD49e interaction is necessary for proper distribution in the CD49e membrane and / or for the recycling of CD49e such that when it is altered, migration and adhesion to surfaces containing fibronectin are altered analogously, resulting in an inability of immune system cells to migrate effectively to a source of fibronectin and adhere to surfaces containing fibronectin. Such alteration would therefore result in desirable reduced inflammatory processes that include reduced inflammation after myocardial infarction. Other diseases to be treated include, but are not limited to, joint inflammation, psoriasis, contact allergy, Crohn's disease, inensayoinal inflammatory disease, eczema, and atopic dermatitis.

C. CD49F (VLA-6α subunit)

As indicated above, CD59f binds to PDZ 41.8 protein. CD49f is known as an integrin subunit that pairs with the β1 integrin subunit (CD29), forming VLA-6, or with CD104 (β4 integrin subunit). The integrin family of supergenes consists of a number of αβ heterodimers of the cell surface important for many different physiological processes, including embryogenesis, thrombosis, wound healing, tumorigenesis and immune responses. Each β chain can be paired with various α chains. Both VLA-6 and CD49f / CD104 are widely expressed in epithelia in non-lymphoid tissues. VLA-6 is also expressed in platelets, monocytes, thymocytes and T lymphocytes, with increased expression in activated and resting memory T cells.

The inhibition of interactions between VLA-6 and 41.8 has a number of therapeutic functions such as the prevention and treatment of metastatic cancers, and the treatment of immunity by hyperactivity. For example, VLA-6 is associated with the invasion of prostate carcinoma and plays a role in breast cancer metastasis. Blocking the function of VLA-6 combined with conventional treatment for prostate cancer would be a more effective treatment for prevention of metastatic disease (see, Cress et al., 1995, Cancer Metastasis R). CD49f blocking by interaction with PDZ can also treat Rh incompatibility due to weakening of the memory response or in the treatment of keloids.

D. CD138 (Syndecan-1)

CD138 is a transmembrane proteoglycan receptor whose extracellular domain functions as a ligand binding domain for various components of the extracellular matrix whose intracellular portion functions to alter the cytoskeleton and transduce extracellular signals. CD138 is also fixed to FGF2 and can be a coreceptor for the FGF receptor (Yayon et al., 1991).

As previously shown, CD138 interacts with the 41.8 kD protein and with TIAM1. It has also been reported that the C term of CD138 is fixed to the PDZ domains of sintenin and human CASK (Cohen et al., 1998, J. Cell. Biol. 142: 129-138; Grootjans et al., 1997, PNAS 94 : 13683-13688; Hsueh et al., 1998, J. Cell. Biol. 142: 139-151). CD138 is expressed in pre-B cells, immature B cells, plasma cells, neural cells, the basolateral surface of epithelial cells, embryonic mesenchymal cells, vascular smooth muscle cells, endothelial cells and neural cells but not circulating B cells mature It is believed that the interaction between CD138 and the PDZ domains of the 41.8 kD protein and the TIAM1 proteins is necessary for the proper distribution of CD138 on the cell surface. The disruption of the interaction by administration of an effective amount of an antagonist is expected to interfere with the migration and adhesion of the cells to the extracellular matrix, resulting in reduced inflammatory and humoral immune responses. CD138 inhibition can be used to treat without limitation diseases such as inflammatory deterioration after myocardial infarction, joint injury, rheumatoid arthritis, vasculitis, drug reaction, scleroderma, SLE, Hashimoto's thyroiditis, Goodpasture's syndrome, juvenile dependent diabetes of insulin, and psoriasis.

E. CD98

As shown above, CD98 interacts with MPP2. CD98 is expressed at high levels in monocytes and at low levels in T cells, B cells, splenocytes, NK cells, and granulocytes. CD98 plays roles in adhesion, fusion and is a type L amino acid transporter. CD98 is also involved in virus-mediated cell fusion (eg paramyxovirus: parainfluenza type 2 virus, Newcastle disease virus, and rubulavirus) and CD98 function antagonism is expected to treat viral infections and limit the spread of the virus. CD98 inhibitors can be an antiviral agent for, but not limited to, paramyxovirus, parainfluenza, Newcastle disease and rubella. Other treatments include the treatment of acute leukemia.

F. CLASP-1

As shown above, CLASP-1 interacts with DLG1, PSD95, and NeDLG. CLASP-1 is a member of a superfamily of proteins associated with immune cells with similar motives (see PCT / US99 / 22996 published as WO 00/20434), CLASP-1 works in the maintenance of the immune system. The CLASP-1 transcript is present in lymphoid organs and neural tissue, and the protein is expressed by T and B lymphocytes and macrophages in the MOMA-1 subregion of the marginal area of the spleen, an area known to be important in the T cell interaction: B. CLASP-1 staining of individual T and B cells exhibits a pre-activation structural polarity, which is organized as a "ball" or "capsule" structure in B cells, and forming a "ring," ball "structure. or "capsule" in T cells. The location of these structures is adjacent to the microtubule organizing center ("MTOC"). CLASP-1 antibody staining indicates that CLASP-1 is at the interface of cell conjugates TB that are fully assigned to differentiation Antibodies to the extracellular domain of CLASP-1 also block the formation of TB cell conjugates and the activation of T cells.

The antagonism of the CLASP-1 function is expected to interfere with immune responses (e.g., activation of T and B cells), signal transduction, cell-cell interactions, and membrane organization. Diseases to be treated by CLASP-1 agonists / antagonists include, but are not limited to, rheumatoid arthritis, juvenile diabetes, organ rejection, reverse rejection disease, scleroderma and multiple sclerosis.

G. CLASP-4

As previously demonstrated, CLASP-4 interacts with DLG1, PSD95, NeDLG, LDP, AF6, 41.8, and MINT1. CLASP-4 is a member of a superfamily of proteins associated with immune cells with similar motives (see U.S. Patent Application 60/196527, also being processed, filed April 11, 2000). The CLASP-4 protein is expressed primarily in peripheral blood lymphocytes. Inhibition of the interaction of CLASP-4 and PDZ domains will interfere with immune responses (e.g., activation of T and B cells), signal transduction, cell-cell interactions, and membrane organization. Diseases to be treated by CLASP-4 agonists and antagonists include, but are not limited to, rheumatoid arthritis, juvenile diabetes, organ rejection, reverse rejection disease, scleroderma, multiple sclerosis, acute leukemia, blast leukemia crisis, post-inflammation. heart attack (cardiac, etc.), and atherosclerosis.

H. VCAM1

Cellular vascular adhesion molecule-1 (VCAM-1, CD106) is predominantly expressed by the vascular endothelium (i.e. endothelial cells) but has been detected in macrophages, dendritic cells, bone marrow derived cells, fibroblasts, cells cortical epithelial thymus), smooth vascular muscle cells, myoblasts and myotubes. VCAM-1 mediates adhesion by interaction with an integrin ligand, VLA-4, which is expressed by lymphocytes, monocytes and eosinophils. The interaction between VCAM-1 and VLA-4 is important for activation, crushing and extravasation of cells expressing VLA-4 when the endothelium itself has been activated due to inflammation or injury (Salomon etal., 1997, Blood 89: 2461 -2471; St-Pierre et al., 1996, Eur. J. Immunol. 26: 2050-2055; Bell et al., 1995, Int. Immunol. 7: 1861-1871).

As discussed above, the C-terminal region of VCAM-1 is a ligand for the PDZ domains of MPP1, DLG1, NeDLG1, LDP, protein 41.8, TIAM1 and K303. It is believed that these interactions mediate the function of endothelial cell interactions with leukocytes expressing integrins. When PDZ-PL interactions are altered, the adhesion of leukocytes to the endothelium will be altered analogously, resulting in, e.g., reduction of inflammation. Thus, inhibition of VCAM-1 binding to PDZ proteins is useful for reducing abnormal VCAM-1 inflammatory responses and associated pathologies such as (but not limited to), renal allograft rejection, insulin dependent diabetes, rheumatoid arthritis, complications after myocardial infarction and systemic lupus erythematosus (Pasloske et al., 1994, Ann Rev Med 45: 283; Ockenhouse et al., 1992, J. Exp. Med. 176: 1183; Solezk et al., 1997, Kidney Int. 51: 1476; Tedla, et al., 1999, Clin. Exp. Immunol. 117: 92-99; Kusterer et al., 1999, Exp Clin Endocrinol Diabetes 107: S102-107; Bonomini et al., 1998, Nephron 79: 399; Suassuna et al., 1994, Kidney Int. 46: 443; Ferri et al., 1999, Hypertension 34: 568).

I. CLASP-2

As shown above, CLASP-2 interacts with PSD-95, NeDLG, and DLG1. CLASP-2 is a member of a superfamily of proteins associated with immune cells with similar motifs (see US Patent 09/547276, also being processed, filed on April 11, 2000; WO 00/10158 filed on April 11, 2000 ; WO 00/10156 filed on April 11, 2000). The CLASP-2 transcript is very strongly present in the placenta followed by lung, kidney and heart, and the protein is expressed in T and B cells, as well as in kidney epithelial cells.

The inhibition of the interaction of CLASP-2 and PDZ domains will interfere with the function of CLASP-2 resulting in interference with the function of T and B cells (eg, activation of T and B cells), signal transduction, interactions cell-cell, and membrane organization. Additionally, since CLASP-2 is present in the heart, blocking the function or expression of CLASP-2 can selectively block immune responses in the heart (for example, to selectively stop the immune response in the heart compartment, eg after rejection of heart transplantation or after MI inflammation, without compromising immunity anywhere else). Other diseases to be treated by CLASP-1 agonists / antagonists include, but are not limited to, rheumatoid arthritis, juvenile diabetes, organ rejection, reverse rejection disease, scleroderma, and multiple sclerosis.

J. CD95 (Apo-1 / Fas)

CDE95 (Fas / Apo-1) and the Fas ligand (FasL) are a receptor-ligand pair involved in lymphocyte homeostasis and peripheral tolerance. Fas binding by its ligand results in apoptotic cell death, an important major mechanism for the safe clearance of undesirable cells during resolution of the acute inflammatory response. As shown above, CD95 is set to PDZ domains DLG1. PSD-95, NeDLG, and 41.8. Inhibition of the interaction of CD95 and PDZ domains.

K. KV1.3 (Potassium Shaker Channel Type Kv1.3)

As shown above, Kv1.3 is set to DLG1, PSD-95, NeDLG, LIMK, 41.8, RGS12, DVL1, and MINT1. Kv1.3 is a shaker-related channel protein that is involved in modulating the membrane potential of T lymphocytes (Lewis and Cahalan, 1995, Ann. Rev. Immunol. 13: 623). Inhibition of the Kv1.3 channel chronically depolarizes the T-cell membrane, reduces calcium entry through the calcium-activated calcium release channels in the plasma membrane, and consequently inhibits the calcium signaling pathway essential for activation. of lymphocytes. Hanada et al. Reported that Kv1.3 is associated with DLG1 and PSD95 in Jurkat T cells (J. Biol. Chem. 1997, 272: 26899). The administration of Kv1.3-PDZ protein agonists / antagonists will alter the signaling of T cells and may be a useful therapeutic drug to treat, but not limited to, organ transplantation, reverse rejection disease, Crohn's disease, colitis ulcerative, inensayoinal inflammatory disease, rheumatoid arthritis, osteoarthritis, multiple sclerosis, scleroderma, and mixed connective tissue disease.

L. DNAM-1

As shown above, DNAM-1 binds to several PDZ proteins, which include MPP2, DLG1, PSD95, NeDLG, LIM, AF6, 41.8 and RGS12. DNAM-1 is constitutively associated with Fyn, but requires the presence of pervanadate (a tyrosine phosphatase inhibitor) (see Shibuya, et al. 1999, Immunity 11: 615-623). After stimulation with anti-CD3 (or DNAM-1), DNAM-1 is phosphorylated in serine 329 (Shibuya, et al. 1998, J. Immun. 561: 1671-1676) and is associated with LFA-1. Additionally, Fyn becomes associated with DNAM-1 regardless of pervanadate. Fyn phosphorylates DNAM-1 on Y322, but does not require that Y322 continue to be set to DNAM-1.

Since DNAM-1 does not in itself have an SH3 binding domain but only has the Src phosphorylation site at Y322, an adapter molecule for bridging DNAM-1 and Fyn must be present. DLG1 has been described in the literature as present in T cells (Hanada, et al. 1997. J. Biol. Chem. 272: 26899), but is not fixed to Fyn. PSD95 does not have an SH3 fixation site. However, several of the other PDZ proteins do have SH3 binding domains that include, but are not limited to, NeDLG, RGS12, MPP2 and can perform this function. This adapter PDZ listed above is set to DNAM-1 by its PDZ domain and simultaneously it is set to the F3 SH3 domain for its proline-rich sequences precisely N-terminals to the PDZ domain. The Y139 site is a candidate phosphorylation site to control the association of Fyn to DNAM-1 and the PDZ adapter.

Based on this analysis, inhibition of the association of PDZ with DNAM-1 using the reagents described herein will inhibit the association of Fyn with DNAM-1 and subsequent phosphorylation of Y322 and activation of cytotoxic T cells. Diseases that can be treated include, but are not limited to Crohn's disease, multiple sclerosis, ulcerative colitis, inensayoinal inflammatory disease, reverse rejection disease, juvenile diabetes, and Hashimoto's disease.

M. CD83 (HB15)

CD83 is a transmembrane glycoprotein, predominantly expressed in activated dendritic cells (DCs), Langerhans cells in the skin, with some weak expression detected in activated peripheral lymphocytes, and interdigital reticulum cells within the T cell areas of lymphoid organs (Zhou and Tedder 1995, J. Immunol.

154: 3821-3835; Zhou et al., 1992, J. Immunol. 149: 735-742). CDE83 is regulated again in increasing sense after activation of an immature DCs, and is the main and characteristic discriminant marker for activated mature CDs (Czerniecki et al., 1997, J. Immunol. 159: 3823-3837). DCs function as antigen presenting cells (APSc). The increasing regulation and expression of CD83 seems to be therefore necessary for DCs to mature and function as APCS.

As demonstrated by experiments described above, CD83 binds to the PDZ domains of DLG1, PSD95, and NeDLG. These interactions between CD83 and PDZ domains, and between CD83 and DLG1, PSD95 and NeDLG are believed to be important for the proper distribution and recycling of CD83. The alteration of the CD83 and PDZ proteins with agonists and antagonists can be used to treat, but not limited to, psoriasis, cancers, allergies, autoimmune diseases such as multiple sclerosis, and systemic lupus erythematosus.

N. CD44 (phagocytic glycoprotein 1, lymphocyte return receptor, p85 and HCAM)

CD44 is a single-step transmembrane protein that has several different isoforms due to alternative remodeling. It has a broad expression pattern that is detected in both hematopoietic and non-hematopoietic cells, including epithelial, endothelial, mesenchymal and neuronal cells. CD44H is a major isoform that is expressed in lymphoid, myeloid and erythroid cells (reviewed in Barclay et al., 1987, The Leukocyte Antigen Facts Book, 2nd edition, Academic Press). CD44 is a hyaluronate (HA) receptor, which is a constituent of the extracellular matrix (ECM). In the immune system, CD44 functions as an adhesion molecule on the surface of leukocytes and erythrocytes that fixes HA polymers in the ECM, and can also act as a signaling receptor when HA becomes soluble during inflammatory reactions or tissue deterioration. The cytoplasmic region of CD44 has been shown to be fixed or associated with the actin cytoskeleton by interactions with spectrin and members of the ERM family (ezrina, radixin, and meosin) (reviewed in Lesley et al., 1993; Bajorath, 2000 , Proteins 39: 103-111). Additionally, CD44 is associated with p56Lck non-receptor tyrosine kinase (Taher et al., 1996, J. Biol. Chem. 271: 2863-2867). CD44 has been shown to be a co-stimulating molecule with CD3 / TCR coupling to activate T cells (reviewed in Aruffo, 1996, J. Clin. Invest. 98: 2191-2192).

As described above by experiments reported herein, the C term of CD44 is a ligand for the PDZ domain contained in MPP1, prIL-16 and MINT1. It is believed that the interactions of CD44 with PDZ domains, and between CD44 with MPP1, prIL-16 and MINT1 function in the maintenance of leukocyte structure and in the signaling of leukocytes. Thus, when a CD44-PDZ interaction is disturbed, CD44 will stop transducing appropriate intracellular signals, and will maintain an appropriate distribution of CD44 on the surface, which will prevent leukocyte adhesion to the endothelium during inflammation and tissue deterioration. The administration of agonists / antagonists of this interaction will therefore result, but not limited to, reduced inflammatory responses during tissue ischemia and cell lysis (eg rhabdomyosis), vascular insufficiencies (eg freezing necrosis), psoriasis, eczema, disease Reverse rejection, granuloma annulare, and scleroderma.

O. CD97 (CD55)

As stated above, CD97 is set to the PDZ domains of DLG1 and 41.8. CD97 is a 7-section transmembrane protein with 79.7 kD expressed in granulocytes and monocytes and at low levels in resting T cells and B cells. After activation of T or B cells, CD97 expression levels in T cells and B cells increase rapidly (Eichler et al., 1994, Scand. J. Immunol. 39, 111-115; Pickl et al. , 1995, Leukocyte Typing V: 1151-1153). When expressed in COS cells, CD97 confers adhesion to lymphocytes and erythrocytes.

In accordance with the present description, the interaction of CD97 with DLG1 and the 41.8 kD protein may be altered to interfere with the appropriate membrane distribution of CD97 and / or the recycling of CD97. Such modulation will affect the CD97-dependent adhesion of cells with therapeutic benefit. Without limitation, agonists and antagonists of the interaction of CD97-PDZ proteins can be used to treat rheumatoid arthritis, osteoarthritis, Crohn's disease, ulcerative colitis, and psoriasis.

P. Glycoforin C (GC)

As demonstrated above, the C-terminus of glycoforin C (GC) interacts with the PDZ domains of human DLG, PSD95, NeDLG, MMP2, AF6, 41.8 and MINT1 (with Mint-1 previously described). Glycoforin C is an integral membrane protein expressed in erythroid, thymus, stomach, breast, adduct and fetal liver cells, monocytes, T and B cells (Le Van Kim et al., 1989, J. Biol. Chem. 264: 20407- 20414, and is known for its role in human erythrocytes, where it interacts with MPP1 and protein 4.1 to regulate the shape, integrity and mechanical stability of red blood cells (Marfatia et al., 1997, J. Biol. Chem. 272: 24191-24197; Reid et al., 1987, Blood 69: 1068-1072).

The interactions between glycophorin C and the PDZ DLG, PSD95, NeDLG, MMP2, AF6, 41.8 and MINT1 proteins are considered necessary for the maintenance of the physical integrity of the cells in which they are expressed. Modulation of GC-PDZ interactions will alter their function and can be used to treat, but not limited to, polycythemia vera, and spherocytosis.

Q. CDw128A (IL8RA)

As described above, CDW128A binds to the PDZ domains of DLG1 and NeDLG. There are two forms for the IL-8 receptor, IL-8RA (CDw128A) and IL-8RB (CDw128B), both of which are members of the G protein-coupled receptor superfamily and the chemokine receptor branch of the rhodopsin family (CDw128A and CDw128B both fix IL-8 with the same affinity, but only CDw128B fixes another 3 CXC chemokines related to IL-8: melanoma growth stimulating activity (GRO / MGSA), neutrophil activating peptide 2 (NAP) -2) and ENA-78. See, eg, Ahuja, SK and Murphy, PM. 1996, J. Biol. Chem. 271: 20545-50.

CDw128A is expressed in all granulocytes, a subset of T cells, monocytes, endothelial cells, keratinocytes, erythrocytes, and melanoma cells. IL-8 induces chemotaxis of neutrophils, basophils, and T lymphocytes and increases adhesion of neutrophils and monocytes to endothelial cells. The binding of IL-8 to IL-8RA induces a transient increase in intracellular calcium levels, activation of phospholipase D, a respiratory burst of neutrophils and chemotaxis. This pro-inflammatory response is effective in normal immune responses. CDw128A inhibitors are useful for the treatment of psoriasis, rheumatoid arthritis, polyarthritis, and for the control of angiogenesis-dependent disorders such as melanomas and breast cancer.

(R) CD3-eta (η)

CD3-η is a remodeling variant of CD3 zeta and a component of the CD3 / TCR complex, which is required for antigen recognition, signal transduction and T cell activation (Weiss and Littman, 1994, Cell 76: 263-274 .). See, Barclay et al., 1997, The Leucocyte Antigen Facts Book. 2nd edition, Academic Press. As demonstrated by experiments reported herein, the C-terminal region of CD3-η is a ligand for the PDZ domains of MINT1, protein 41.8, DNG1 and PSD95. The interactions of CD3-η with PDZ domains, and CD3-η with MINT1, protein 41.8, DLG1 and PSD95 are believed to be important in the activation of T cells, which is required for all cellular immune responses. Modulation of this interaction by agonists and antagonists can be used to treat, but not limited to, acute and chronic allograft rejection, multiple sclerosis, reverse rejection disease, and rheumatoid arthritis.

(S) LPAP (CD45-AP, LSM-1)

LPAP is a transmembrane protein expressed in resting and activated T and B cells. LPAP has been shown to bind to CD45, a protein that is part of the T cell receptor complex and has been found to co-localize with CD4, CD2 and Thy-1. LPAP has also been precipitated by co-immunization with p56 (lck) and ZAP-70. The actual function of LPAP is unknown, but it has been suggested that it is an assembly molecule for the CD45 complex.

As demonstrated above, LPAP is set to DLG-1 and MINT-1. Notably, DLG-1 and MINT-1 both are expressed in T cells. DLG-1 has also been shown to coprecipitate with p56 (lck) in T cells. The assays described herein have also shown that DLG-1 is fixed to CD95 and KV1.3, and fixing MINT-1 to KV1.3. All these molecules are involved in signaling by the TCR. It is believed that LPAP works by organizing CD45 signaling in the activation of T cells, possibly by recruiting p56 (lck) as a substrate for CD45. It has been shown that blocking the CD45 function severely impairs the T-cell response. It is expected that inhibition of the interaction between the LPAP and PDZ proteins will alter the CD45-mediated pathway from the rest of the immune response. PL-PDZ fixation agonists and antagonists can be used to treat, but not limited to, rheumatoid arthritis, transplant rejection, multiple sclerosis, scleroderma and reverse rejection disease.

(T) CD46 (Complement membrane cofactor protein (MCP))

CD46 is a membrane protein that is expressed in all nucleated cells, but not in erythrocytes. CD46 is a member of the complement activation protein family regulator. Its primary function is the protection of cells against complement attack by deactivation of the C3b / C4b complement deposited on the membrane (Liszewski, et al., 1999, Adv. Immunol. 61: 201-283). CD46 exists in more than 8 isoforms that are generated by differential remodeling, with molecular weights ranging from 45 to 70 kD. In addition to the above function, CD46 also serves as the receptor for measles virus and other pathogenic microorganisms

(e.g. Streptococcus pyogenes) (Manchester et al., 1994, Proc. Natl. Acad. Sci. USA 91: 2161; Okada et al., 1995, Proc. Natl. Acad. Sci. USA 92: 2489-2493.). CD46 also appears to be overexpressed in certain tumors (Jurianz et al., 1999, Mol Immunol 36: 929-939), thus making tumor cells insensitive to complement action. See, Barclay et al., (1997). The Leucocyte antigen facts book, 2nd edition, Academic Press.

As indicated above, CD46 is set to DLG1, PSD95 and Ne-DLG. It is believed that this interaction is necessary for proper distribution in CD46 membranes and / or CD46 recycling. Altering the CD46-PDZ interaction can reduce the ability of measles virus and other pathogens to enter cells, and make tumors expressing CD46 sensitive to complement attack. The administration of agonists and antagonists of the CD46-PDZ interaction is useful for the treatment, but not limited to, of viral infectious diseases and cancers.

(U) CDw128B

As described above, CDw128B binds to the PDZ domains of DLG1, NeDLG, PSD95, and 41.8 in the tests described above. There are two forms for the IL-8 receptor, IL-8RA (CDw128A) and IL-8RB (CDw128B), both of which are members of the G protein-coupled receptor superfamily and the chemokine receptor branch of the family of Rhodopsin CDw128A and CDw128B both fix IL-8 with equal affinity, but only CDw128B also fixes three other CXC chemokines related to IL-8: melanoma growth stimulating activity (GRO / MGSA), neutrophil activating peptide 2 (NAP- 2) and ENA-78. See, e.g., Ahuja, SK and Murphy, PM. 1996. J. Biol. Chem. 271: 20545-50.

CDw128B is expressed in all granulocytes, a subset of T cells, monocytes, endothelial cells, keratinocytes, erythrocytes, and melanoma cells. IL-8 induces chemotaxis of neutrophils, basophils, and T lymphocytes but reduced relative to IL8RA and increases the adhesion of neutrophils and monocytes to endothelial cells. The binding of IL-8A to its receptor induces a transient increase in intracellular calcium levels and granule release, but does not induce activation of phospholipase D or a respiratory burst in neutrophils. This pro-inflammatory response is effective in normal immune responses. CDw128B inhibitors are useful for the treatment of psoriasis, rheumatoid arthritis, polyarthritis, and for the control of angiogenesis-dependent disorders such as melanomas and breast cancer.

(V) DOCK2

The DOCK family are a group of transmembrane morphogenetic proteins that interact with the cytoskeleton to affect changes in cell shape. Members of this new family include Drosophila myoblast city (mbc), DOCK180, DOCK2, DOCK3, CED5, KIAA0209 and CLASP. The prototypic molecule, DOCK1 or DOCK180 is the human homologue of the C. elegans gene, CED5 that is involved in the entrapment and phagocytosis of apoptotic cells by macrophages. DOCK2 is found in peripheral blood lymphocytes and can convert a crushed cell into a rounded morphology after transfection (Nagase, et. Al. 1996. DNA Res 3: 321-329, Nishihara, H. 1999. Hokkaido Igaku Zasshi 74: 157).

As previously shown, DOCK2 is a PL and binds to PDZ proteins. Using PDZ proteins as an adapter, DOCK2 is complexed with A, B, C to control the shape of the cell in preparation for the transit of lymphocytes for vascular circulation. The modulation of DOCK2 by agonists and antagonists of their interaction with PDZ proteins can be used to treat, but not limited to, acute leukemia, blast crisis, post-myocardial infarction inflammation, and post-traumatic inflammation.

W. CD34

As shown above, CD34 is set to DLG1, PSD95, and NeDLG. CD34 is expressed in a small subpopulation of bone marrow cells that includes hematopoietic stem cells. CD34 is also present in stomatal cells of the bone marrow, and in endothelial cells. The CD62L (selectin-L) and CD62E (selectin-E) selectins are fixed to CD34. CD34 mediates ligation and / or leukocyte curl. The properties of CD34 hematopoietic stem cells include myeloid differentiation from stem cells. Modulation of the CD34-PDZ interaction with agonists and / or antagonists can be used to treat, but not limited to, myelodysplasia, leukemias, post-traumatic inflammation, and inflammation following myocardial infarction.

X. Fc epsilon receptor beta I chain (FcεRβI)

The high affinity receptor for human IgE, FcεRI, is composed of a homodimer α, β, and γ disulfide bound. The α chain fixes the Fc portion of IgE, while the β chain serves to amplify the signals that are transduced by the γ chain homodimer. There are both complexes (αβγ2 tetramer and αγ2 trimer), but the β chain amplifies the signal 5 to 7 times, as measured by Syk activation and calcium mobilization. Additionally, FcεRβI is a PDZ ligand and is a member of the CD20 / FcεRβI receptor family. As shown above, FcεRβI is set to MINT1.

As the high affinity receptor for IgE, FcβRI in basophils and mast cells plays an essential role in the initiation of allergic responses. FcβRI signaling begins by crosslinking a multivalent allergen bound to IgE. The result is vesicular degranulation, release of histamine, leukotrienes and pro-inflammatory cytokines (IL-6 and TNFα), factors responsible for the symptoms of immediate hypersensitivity. The alteration of the signaling by addressing the PL / PDZ interaction with agonists and antagonists can be used to treat, but not limited to, asthma, atopic dermatitis, eczema, drug reaction, mastocytosis, urticaria, eosinophilia-myalgia syndrome (Turner, H ., et al., 1999, Nature 402 SUPP: B24).

Y. Ligand FAS (FasL)

CD95 (Fas / Apo-I) and the Fas ligand (FasL) are a receptor-ligand pair critically involved in lymphocyte homeostasis and peripheral tolerance. Fas fixation by its ligand results in apoptotic cell death, an important major mechanism for the safe clearance of undesirable cells during resolution of the acute inflammatory response. FasL is primarily restricted to activated T lymphocytes and is rapidly induced. The Fas ligand is frequently regulated increasingly in breast cancer, compared to normal breast epithelial cells and benign breast disease. As shown above, FasL is set to the PDZ domain KIAA0561. PDZ-PL modifiers are useful for the treatment, but not limited to, of tumors, e.g. tumors not sensitive to conventional chemotherapy.

Z. CDW125 (IL5R)

As shown above, CDW125 is fixed to PTN-4 and RGS12. CDW125 is an IL-5 receptor expressed in eosinophils and basophils. IL-5 promotes the growth and differentiation of eosinophil precursors and activates mature eosinophils (Takatsu et al. 1994, Adv. Immunol. 57: 145-190). The secreted form of CDw125 has antagonistic properties and is capable of inhibiting the proliferation and differentiation of eosinophils induced by IL-5. Modulation of the binding of CDW125 to the PDZ domains can be used to treat, but not limited to, asthma, atopic dermatitis, eczema, drug reaction, hives, mastocytosis, and eosinophilia.

AA Burkitt Lymphoma Receptor 1 (BLR-1; CXCR5)

BLR-1 is a transmembrane receptor detected primarily in B cells and has been shown to be increasingly regulated in stimulated T cells (Dobner et al., 1992, Eur J. Immunol. 22: 2795-2799 .; Flynn et al., 1998, J. Exp. Med. 188: 297-304). BLR-1 works in the chemotaxis of B and T cells in the follicles of secondary lymphoid organs (eg, spleen) for proper development and selection against antigens (Forster et al., 1996, Cell 87: 1037-1047. A ligand is the chemoattractant of B lymphocytes (BLC), which is strongly expressed in the follicles of Peyer's patches, spleen, and lymph nodes (Gunn et al., 1998, Nature

391: 799-803). Consistent with its chemotactic role is the demonstration that the expression of BLR-1 is regulated in decreasing sense in activated and developed B cells (plasma cells) to prevent them from being retained in the follicles (Forster et al., 1994, Cell Mol Biol 40: 381-387), and that blr-1 B cells fail to migrate to B cell follicles (Forster et al., 1996).

As demonstrated by experiments reported herein, the C-terminal end of BLR-1 is fixed to the MINT1 protein containing PDZ domain. Without intending to be bound by a particular mechanism, the interaction between BLR-1 and MINT1, and between BLR-1 and PDZ domains is necessary for proper distribution and signaling of BLR-1 on the cell surface. When this interaction is broken, the chemotactic abilities of the lymphoid cells that express BLR-1 break down analogously. This breakage results in a reduced immune response, interference with the ability of lymphocytes to circulate properly and develop responses to the antigen. The agonists and antagonists of this interaction can be used for the treatment, but not limited to, of systemic lupus erythematosus, and scleroderma and other autoimmune diseases.

BB CD4

CD4 is a co-receptor with the T-cell receptor (TCR) involved in antigen recognition. Both CD4 and TCR belong to the family of immunoglobulin supergenes. The activation of T cells is enhanced by increased avidity of T cells for effector cells and target cells. The cytoplasmic domain is involved in signal transduction and association with tyrosine kinase p56lck. CD4 is expressed in most thymocytes, two thirds of peripheral blood T lymphocytes, monocytes and macrophages.

Type 1 human immunodeficiency virus (HIV-1) infects cells by membrane fusion mediated by their envelope glycoproteins (gp120-gp41) and is triggered by the interaction of CD4 and a chemokine co-receptor, CCR5 or CXCR4. Modulation of CD4-PDZ inhibitors with agonists and antagonists can be used to treat, but not limited to, HIV infection immediately after exposure to HIV, rheumatoid arthritis, multiple sclerosis, scleroderma, systemic lupus erythematosus, and psoriasis. .

6.5 Agonists and Antagonists of PDZ-PL Interactions

As described herein, the interactions between PDZ proteins and PL proteins in cells (e.g., hematopoietic cells, e.g., T cells and B cells) can be altered or inhibited by the administration of inhibitors.

or antagonists. Inhibitors can be identified using screening assays described herein. The reasons described herein are used to designate inhibitors. In some aspects of the description, the antagonists have a structure (eg, peptide sequence) based on the C-terminal residues of the PL domain proteins listed in TABLE 2. In some aspects of the description, the antagonists have a structure ( eg, peptide sequence) based on a PL motif described herein.

PDZ / PL agonists and antagonists can be any of a wide variety of compounds, both naturally occurring and synthetic, organic and inorganic, and including polymers (eg, oligopeptides, polypeptides, oligonucleotides, and polynucleotides), small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide analogs, analogs of naturally existing structures (eg, peptidomimetics, nucleic acid analogs, etc.), and numerous other compounds. Although, for convenience, the present disclosure relates primarily to antagonists of PDZ-PL interactions, it will be recognized that PDZ-PL interaction agonists can also be used in the methods described herein.

In one aspect, peptides and peptidomimetics or analogs contain an amino acid sequence that binds to a PDZ domain in hematopoietic cells such as T cells and B cells, or otherwise inhibits the association of PL proteins and PDZ proteins. In one embodiment, the antagonists comprise a peptide having a sequence corresponding to the carboxy-terminal sequence of a PL protein listed in TABLE 2, eg, a peptide listed in TABLE 4. Typically, the peptide comprises at least all four ( 4) Certain residues of the PL protein, and often the inhibitory peptide comprises more than four residues (eg, at least five, six, seven, eight, nine, ten, twelve or fifteen residues) of the C-terminus of the PL protein. See, e.g., section 6.5.1, below. In addition, the C-terminal domains of specific surface receptors expressed by the hematopoietic system and endothelial cells can themselves be used as inhibitors, and can be used as the basis for the rational design of non-peptide inhibitors. See section 6.6, below.

In some aspects, the antagonist is a fusion protein that comprises such a sequence. Fusion proteins that contain a transmembrane transporter amino acid sequence are particularly useful. See, e.g. Section 6.5.2, below.

In some aspects, the inhibitor is a conserved variant of the C-terminal PL protein sequence that has inhibitory activity. (See, e.g. section 6.5.3, below).

In some aspects, the antagonist is a peptidomimetic of a C-terminal PL sequence. See, e.g. Section 6.5.4, below.

In some aspects, the inhibitor is a small molecule (i.e., it has a molecular weight less than 1 kD). See, e.g. Section 6.5.5, later.

6.5.1 Peptide Antagonists

In one aspect, the antagonists comprise a peptide having a sequence of a carboxy terminus of PL protein listed in TABLE 2. The peptide comprises at least the two (2) C-terminal residues of the PL protein, and typically, the peptide inhibitor comprises more than two residues (eg at least three, four, five, six, seven, eight, nine, ten, twelve or fifteen residues), of the C-terminus of the PL protein. In most cases, residues shared with the inhibitor peptide with the PL protein are found in the C-terminus of the peptide. However, in some aspects, the sequence is internal. Similarly, in some cases, the inhibitory peptide comprises residues of a PL sequence that is near the C-terminus, but not in the C-terminus of a PL protein (see, Gee et al., 1998, J. Biological Chem. 273: 21980-87).

For example, the "core sequence of the PDZ domain binding motif" of a surface receptor of hematopoietic cells at its C-terminus contains the last four amino acids, this sequence being used to direct PDZ domains in hematopoietic cells. The four amino acid core of a PDZ domain binding motif sequence may contain additional amino acids in its amino terminus to further increase its binding affinity and / or stability. In one embodiment, the peptide of the PDZ domain binding motif sequence can be from four amino acids to 15 amino acids. It is preferred that the length of the sequence be 6-10 amino acids. More preferably, the PDZ domain binding motif sequence contains 8 amino acids. Additional amino acids at the amino terminal end of the core sequence can be derived from the natural sequence in each surface receptor of the hematopoietic cells or a synthetic linker. Additional amino acids may also be conservatively substituted. When the third residue of the term C is S, T or Y, this residue may be phosphorylated before the use of the peptide.

In some aspects, peptide and non-peptide inhibitors of ... are small, e.g., they are less than 10 amino acid residues in length if it is a peptide. Additionally, it has been reported that a limited number of amino acid ligands are directly in contact with the PDZ domain (generally less than 8) (Kozlov et al., 2000, Biochemistry 39, 2572; Doyle et al., 1996, Cell 85, 1067 ) and that peptides as short as the 3 amino acids of terminal C often retain binding properties similar to longer peptides (> 15 amino acids) (Yanagisawa et al., 1997, J. Biol. Chem. 272, 8539).

FIGURES 3A-H show the use of peptides to inhibit PL-PDZ interactions using the G test described above. In FIGURES 3A and B, inhibition assays were carried out using GST fusion proteins containing PDZ domains of DLG1 or PSD95 (see above and TABLE 3). Biotinylated PL peptide binding for Clasp2, CD46, Fas, or KV1.3 (as listed in TABLE 4) was determined in the presence of various competing peptides (at a concentration of 100 μM) or in the absence of a competitor (matched 100% fixation). The competing peptides were 8-mer peptides having the sequence of the C-term of Clasp2 (MTSSSSVV), CD46 (REVKFTSL), or Fas (RNEIQSLV), an unmarked 19-grouper that had the C-sequence of KV1.3 ( that is, non-biotinylated AA33L as listed in TABLE 3), or a peptide having the sequence of residues 64-76 of hemoglobin (Vidal et al., 1999, J. Immunol. 163, 4811), that is An unrelated competitor. Biotinylated peptide binding (10 μM for Fas and KV1.3, 20 μM for Clasp2 and CD46) to GST alone was subtracted from binding to fusion proteins to obtain the net signal for each experimental condition. This net signal was then normalized by dividing by the signal in the absence of competing peptide, and the data was plotted. Error bars indicated the standard deviation of duplicate measurements. Specific inhibition of Clasp2 PL-DLG PDZ binding with Clasp-2 8-grouper, CD46 8-grouper, Fas-8-grouper and KV13 peptide was observed, but not in the absence of a peptide or using a peptide unrelated

FIGURES 3C-F show similar assays that use shorter peptides for inhibition (e.g., a 3-grouper and a 5-grouper). FIGURES 3C-E show the binding of biotinylated PL peptides for Clasp-2, CD46, Fas, or KV1.3, at the indicated concentration (as listed in TABLE 3) to GST fusion proteins containing PDZ domains of NeDLG , DLG1 or PSD95 in the absence or presence of a 1 mM 3-mer peptide having the sequence of the C-terminus of Clasp-2 (SVV). (Table 3). FIGURE 3F shows the effect on the binding of a 5-mer CD49E peptide (ATSDA) to GST fusion proteins containing a 41.8 kD PDZ domain.

6.5.2 Peptide Variants

Having identified PDZ binding peptides and PDZ-PL interaction inhibitor sequences, variations of these sequences can be made and the resulting peptide variants can be tested for PDZ domain binding or PDZ-PL inhibitory activity. In additional aspects, the variants have the same or different binding capacity of a PDZ domain as the parental peptide. Typically, such amino acid substitutions are conservative, that is, amino acid residues are replaced with other amino acid residues that have physical and / or chemical properties similar to the residues they replace. Preferably, conservative amino acid substitutions are those in which one amino acid is replaced with another amino acid within the same designated class.

6.5.3 Peptidomimetics

Once the PDZ binding peptides and PDZ-PL interaction inhibitor sequences have been identified, peptide mimetics can be prepared using routine methods, and the mimetic inhibitory activity can be confirmed using the assays of the invention. Thus, in some additional aspects, the antagonist is a peptide mimetic of a C-terminal sequence of PL. The skilled professional will recognize that individual synthetic residues and polypeptides incorporating mimetics can be sintered using a variety of procedures and methodologies, all of which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman et al. (Eds) John Wiley & Sons, Inc., NY. Polypeptides incorporating mimetics can also be produced using solid phase synthesis procedures, as described, e.g. by Di Marchi, et al., U.S. Pat. No. 5,422,426. Mimetics can also be synthesized using combinatorial methodologies. Various techniques for generating peptide and peptidomimetic libraries are well known, and include, e.g., Multipin, tea bag techniques, and mixed coupling-coupling technique; see, e.g., Al-Obeidi (1998) Mol. Biotechnol 9: 205-223; Hruby (1997) Curr. Opin. Chem. Biol. 1: 114

119; Ostergaard (1997) Mol. Divers 3: 17-27; Ostresh (1996) Methods Enzymol. 267: 220-234.

6.5.4 Small Molecules

In some aspects, the inhibitor is a small molecule (i.e., it has a molecular weight less than 1 kD). Methods for screening small molecules are well known in the art and include those described above in section 6.4.

6.6. Cellular Surface Receivers and PDZ Domain Fixation Sequences

The following sections describe specific surface receptors expressed by different types of cells in the hematopoietic system and the immune response. The C terms of these receptors are used as inhibitors, or serve as the basis for the design of peptides of PDZ domain binding motif sequences, variants, fusion proteins, peptidomimetics, and small molecules for use in the inhibition of PDZ interactions. -PL. In a preferred aspect, the peptides are tested in an assay for inhibitory or modulating activity (see also, TABLE 4, and the discussion above).

6.6.1 Sequences of the Reason for Fixing the PDZ Domain of T Cell Surface Receivers

A number of surface receptors expressed by T cells contain a sequence of motifs for the PDZ domain (PL sequence). These molecules include CD3η, CD4, CD6, CD38, CD49e, CD49f, CD53, CD83, CD90, CD95, CD97, CD98, CDw137 (41BB), CD166, CDwl28 (IL8 R), DNAM-1, Fas ligand (FasL) and LPAP (Barclay

et al., 1997, The Leucocyte Antigen Facts Book, second edition, Academic Press), CLASP-1, CLASP-2, CLASP-4, KV 1.3, and DOCK2.

The core C-terminal sequence of CD3 is SSQL (SEQ ID NO: 4). When naturally existing residues are added to the core sequence, SSSQL (SEQ ID NO: 5), SSSSQL (SEQ ID NO: 6), PSSSSQL (SEQ ID NO: 7), and PPSSSSQL (SEQ ID NO: 8) can also be used to target a protein that contains PDZ domain in T cells.

The C-terminal core sequence of CD4 is CSPI (SEQ ID NO: 9). When naturally existing residues are added to the core sequence, TCSPI (SEQ ID NO: 10), KTCSPI can also be used; (SEQ ID NO: 11), QKTCSPI (SEQ ID NO: 12), and FQKTCSPI (SEQ ID NO: 13) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CD6 is ISAA (SEQ ID NO: 14). When naturally existing residues are added to the core sequence, DISAA (SEQ ID NO: 15), DDISAA (SEQ ID NO: 16), YDDISAA (SEQ ID NO: 17), and DYDDISAA (SEQ ID NO: 18 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD38 is TSEI (SEQ ID NO: 19). When naturally existing residues are added to the core sequence, CTSEI (SEQ ID NO: 20), SCTSEI (SEQ ID NO: 21), SSCTSEI (SEQ ID NO: 22), and DSSCTSEI (SEQ ID NO: 23 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD49e and s TSDA (SEQ ID NO: 24). When naturally existing residues are added to the core sequence, ATSDA (SEQ ID NO: 25), PATSDA (SEQ ID NO: 26), PPATSDA (SEQ ID NO: 27), and KPPATSDA (SEQ ID NO: 28 can also be used. ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD49f e s TSDA (SEQ ID NO: 29). When naturally existing residues are added to the core sequence, LTSDA (SEQ ID NO: 30), RLTSDA (SEQ ID NO: 31), ERLTSDA (SEQ ID NO: 32) can also be used, and

KERLTSDA (SEQ ID NO: 33) to address a protein that contains PDZ domain in T cells.

The C-terminal core sequence of CD53 is TIGL (SEQ ID NO: 34). When naturally existing residues are added to the core sequence, QTIGL (SEQ ID NO: 35), SQTIGL (SEQ ID NO: 36), TSQTIGL (SEQ ID NO: 37), and KTSQTIGL (SEQ ID NO: 38 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD83 and s TELV (SEQ. ID. NO: 248). When naturally existing residues are added to the core sequence, KTELV (SEQ. ID. NO: 249), HKTELV (SEQ. ID. NO: 250), PHKTELV (SEQ. ID. NO: 251), and TPHKTELV can also be used (SEQ. ID. NO: 252) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD90 is FMSL (SEQ ID NO: 39). When naturally existing residues are added to the core sequence, DFMSL (SEQ ID NO: 40), TDFMSL (SEQ ID NO: 41), ATDFMSL (SEQ ID NO: 42), and QATDFMSL (SEQ ID NO: 43 can also be used ) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CD95 is QSLV (SEQ ID NO: 44). When naturally existing residues are added to the core sequence, IQSLV (SEQ ID NO: 45), EIQSLV (SEQ ID NO: 46), NEIQSLV (SEQ ID NO: 47), and RNEIQSLV (SEQ ID NO: 48 can also be used. ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD97 is ESGI (SEQ ID NO: 49). When naturally existing residues are added to the core sequence, SESGI (SEQ ID NO: 50), ASESGI (SEQ ID NO: 51), RASESGI (SEQ ID NO: 52), and LRASESGI (SEQ ID NO: 53 can also be used ) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CD98 is PYAA (SEQ ID NO: 54). When naturally existing residues are added to the core sequence, FPYAA (SEQ ID NO: 55), RFPYAA (SEQ ID NO: 56), LRFPYAA (SEQ ID NO: 57), and LLRFPYAA (SEQ ID NO: 58 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CDw137 e s GCEL (SEQ ID NO: 59). When naturally existing residues are added to the core sequence, GGCEL (SEQ ID NO: 60), EGGCEL (SEQ ID NO: 61), EEGGCEL (SEQ ID NO: 62), and EEEGGCEL (SEQ ID NO: 63) can also be used. ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CD166 e s KTEA (SEQ ID NO: 64). When naturally existing residues are added to the core sequence, HKTEA (SEQ ID NO: 65), NHKTEA (SEQ ID NO: 66), NNHKTEA (SEQ ID NO: 67), and ENNHKTEA (SEQ ID NO: 68 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of CDw128 and SSNL (SEQ ID NO: 69). When naturally existing residues are added to the core sequence, VSSNL (SEQ ID NO: 70), NVSSNL (SEQ ID NO: 71), VNVSSNL (SEQ ID NO: 72), and SVNVSSNL (SEQ ID NO: 73 can also be used ) to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of DNAM-1 is KTRV (SEQ ID NO: 74). When naturally existing residues are added to the core sequence, PKTRV (SEQ ID NO: 75), RPKTRV (SEQ ID NO: 76), RRPKTRV (SEQ ID NO: 77), and SRRPKTRV (SEQ ID NO: 78 can also be used ) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of FasL is LYKL (SEQ ID NO: 79). When naturally existing residues are added to the core sequence, GLYKL (SEQ ID NO: 80), FGLYKL (SEQ ID NO: 81), FFGLYKL (SEQ ID NO: 82), and TFFGLYKL (SEQ ID NO: 83 can also be used ) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of LPAP is VTAL (SEQ ID NO: 84). When naturally existing residues are added to the core sequence, HVTAL (SEQ ID NO: 85), LHVTAL (SEQ ID NO: 86), GLHVTAL (SEQ ID NO: 87), and QGLHVTAL (SEQ ID NO: 88 can also be used ) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CLASP-1 and SAQV (SEQ. ID. NO: 218). When naturally existing residues are added to the core sequence, SSAQV (SEQ. ID. NO: 219), SSSAQV (SEQ. ID. NO: 220), ISSSAQV (SEQ. ID. NO: 221), and SISSSAQV can also be used (SEQ. ID. NO: 222) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CLASP-2 and SSVV (SEQ. ID. NO: 223). When naturally existing residues are added to the core sequence, SSSVV (SEQ. ID. NO: 224), SSSSVV (SEQ. ID. NO: 225), TSSSSVV (SEQ. ID. NO: 226), and MTSSSSVV can also be used (SEQ. ID. NO: 227) to address a protein containing PDZ domain in T cells.

The core C-terminal sequence of CLASP-4 and YAEV (SEQ. ID. NO: 228). When naturally existing residues RYAEV (SEQ. ID. NO: 229), PRYAEV (SEQ. ID. NO: 230), SPRYAEV (SEQ. ID. NO:) are added to the core sequence.

231), and GSPRYAEV (SEQ. ID. NO: 232) can also be used to address a protein containing PDZ domain in T cells.

The C-terminal core sequence of KV1.3 is FTDV (SEQ. ID. NO: 238). When naturally existing residues are added to the core sequence, IFTDV (SEQ. ID. NO: 239), KIFTDV (SEQ. ID. NO: 240), KKIFTDV (SEQ. ID. NO: 241), and IKKIFTDV can also be used (SEQ. ID. NO: 242) to address a protein containing PDZ domain in cells

T.

The C-terminal core sequence of DOCK2 is STDL (SEQ. ID. NO: 243). When naturally existing residues are added to the core sequence, LSTDL (SEQ. ID. NO: 244), SLSTDL (SEQ. ID. NO: 245), DSLSTDL (SEQ. ID. NO: 246), and PDSLSTDL can also be used (SEQ. ID. NO: 247) to address a protein containing PDZ domain in T cells.

6.6.2 Sequences of the Reason for Setting the PDZ Domain of B Cell Surface Receivers

A number of surface receptors expressed by B cells contain a PDZ domain motif sequence. These molecules include, but are not limited to, CD38, CD53, CD95, CD97, CD98, CDw137, CD138, CDw125 (IL5R), DNAM-1, LPAP, Syndecan-2 (Barclay ct al., 1997, The Leucocyte Antigen Facts Book , second edition, Academic Press) and BLR-1. The specific motif sequences of CD38, CD53, CD83, CD95, CD97, CD98, CDw137, DNA-1, DOCK2, LPAP, CLASP-1, CLASP-2 and CLASP-4 have been described in the previous paragraphs.

The C-terminal core sequence of CD138 is EFYA (SEQ ID NO: 89). When naturally existing residues are added to the core sequence, EEFYA (SEQ ID NO: 90), QEEFYA (SEQ ID NO: 91), KQEEFYA (SEQ ID NO: 92), and TKQEEFYA (SEQ ID NO: 93) can also be used. to target a protein that contains PDZ domain in B cells.

The core C-terminal sequence of CDw125 and s DSVF (SEQ ID NO: 94). When naturally existing residues are added to the core sequence, EDSVF (SEQ ID NO: 95), LEDSVF (SEQ ID NO: 96), TLEDSVF (SEQ ID NO: 97), and ETLEDSVF (SEQ ID NO: 98) can also be used to target a protein that contains PDZ domain in B cells.

The core C-terminal sequence of Syndecan-2 is EFYA (SEQ. ID. NO: 258). When naturally existing residues are added to the core sequence, KEFYA (SEQ. ID. NO: 259), TKEFYA (SEQ. ID. NO: 260), PTKEFYA (SEQ. ID. NO: 261), and APTKEFYA ( SEQ ID NO: 262) PDZ to address a protein containing PDZ domain in B cells.

The core C-terminal sequence of BLR-1 is LTTF (SEQ. ID. NO: 253). When naturally existing residues are added to the core sequence, SLTTF (SEQ. ID. NO: 254), TSLTTF (SEQ. ID. NO: 255), ATSLTTF (SEQ. ID. NO: 256), and NATSLTTF ( SEQ ID NO: 257) to address a protein containing PDZ domain in B cells.

6.6.3 Sequence Motifs for the Fixation of Surface Receptors of Natural Aggressive Cells

A number of surface receptors expressed by NK cells contain a PDZ domain motif sequence. These molecules include, but are not limited to, CD38, CD56, CD98 and DNAM-1. The specific motif sequences of CD38, CD98 and DNAM-1 have been described in the previous paragraphs.

The C-terminal core sequence of CD56 is ESKA (SEQ ID NO: 99). When naturally existing residues are added to the core sequence, NESKA (SEQ ID NO: 100), ENESKA (SEQ ID NO: 101), KENESKA (SEQ ID NO: 102), and TKE-NESKA (SEQ ID N0: 103) to address a protein containing PDZ domain in NK cells.

6.6.4 Sequence Motifs of the PDZ Domain of Monocyte Surface Receivers

A number of surface receptors expressed by cells of the monocytic lineage (monocytes and macrophages) contain a motive sequence of the PDZ domain. These molecules include, but are not limited to, CD38, CD44, CD46, CD49e, CD49f, CD53, CD61, CD95, CD97, CD98, CD148, CDw128, CDw137, Ly-6, DNAM-1 and

Fcε RIβ. The specific motif sequences of CD38, CD49e, CD49f, CD53, CD95, CD97, CD98, CDw128, CDw137, DNAM-1, Galectin 3 (Mac-2) and the mannose receptor have been described in the previous paragraphs.

The C-terminal core sequence of CD44 is KIGV (SEQ ID NO: 104). When naturally existing residues are added to the core sequence, MKIGV (SEQ ID NO: 105), DMKIGV (SEQ ID NO: 106), VDMKIGV (SEQ ID NO: 107) and NVDMKIGV (SEQ ID NO: 108) can also be used for target a protein that contains PDZ domain in monocytes.

The C-terminal core sequence of CD46 e s FTSL (SEQ ID NO: 109). When naturally existing residues are added to the core sequence, KFTSL (SEQ ID NO: 110), VKFTSL (SEQ ID NO: 111), EVKFTSL (SEQ ID NO: 112) and REVKFTSL (SEQ ID NO: 113) can also be used for target a protein that contains PDZ domain in monocytes.

The core C-terminal sequence of CD61 e s KSLV (SEQ ID NO: 114). When naturally existing residues are added to the core sequence, LKSLV (SEQ ID NO: 115), FLKSLV (SEQ ID NO: 116), RFLKSLV (SEQ ID NO: 117) and GRFLKSLV (SEQ ID NO: 118) can also be used for target a protein that contains PDZ domain in monocytes.

The C-terminal core sequence of CD148 is GYIA (SEQ ID NO: 119). When naturally existing residues are added to the core sequence, NGYIA (SEQ ID NO: 120), TNGYIA (SEQ ID NO: 121), KTNGYIA (SEQ ID NO: 122) and GKTNGYIA (SEQ ID NO: 123) can also be used to target a protein that contains PDZ domain in monocytes.

The C-terminal core sequence of Ly-6 is QTLL (SEQ ID NO: 124). When naturally existing residues are added to the core sequence, LQTLL (SEQ ID NO: 125), LLQTLL (SEQ ID NO: 126), VLLQTLL (SEQ ID NO: 127) and SVLLQTLL (SEQ ID NO: 128) can also be used for target a protein that contains PDZ domain in monocytes.

The C-terminal core sequence of Fcε RIβ is PIDL (SEQ ID NO: 129). When naturally existing residues are added to the core sequence, PPIDL (SEQ ID NO: 130), SPPIDL (SEQ ID NO: 131), MSPPIDL (SEQ ID NO: 132) and EMSPPIDL (SEQ ID NO: 133) can also be used for target a protein that contains PDZ domain in monocytes.

The core C-terminal sequence of Galectin 3 is YTMI (SEQ ID NO: 134). When naturally existing residues are added to the core sequence, SYTMI (SEQ ID NO: 135), ASYTMI (SEQ ID NO: 136), SASYTMI (SEQ ID NO: 137) and TSASYTMI (SEQ ID NO: 138) can also be used for target a protein that contains PDZ domain in monocytes.

The C-terminal core sequence of the mannose receptor is HSVI (SEQ ID NO: 139). When naturally existing residues are added to the core sequence, EHSVI (SEQ ID NO: 140), NEHSVI (SEQ ID NO: 141), QNEHSVI (SEQ ID NO: 142) and EQNEHSVI (SEQ ID NO: 143) can also be used for target a protein that contains PDZ domain in monocytes.

6.6.5. Sequences Reason for Setting the PDZ Domain of Granulocyte Surface Receptors

A number of surface receptors expressed by granulocytes contain a motive sequence of the PDZ domain. These molecules include, but are not limited to, CD53, CD95, CD97, CD98, CD148, CDw125, CDw128,

Fcε RIβ and G-CSFR. The specific motif sequences of most of these molecules have been described in the previous paragraphs.

The G-CSFR core C-terminal sequence is TSVL (SEQ ID NO: 144). When naturally existing residues are added to the core sequence, ITSVL (SEQ ID NO: 145), PITSVL (SEQ ID NO: 146), FPITSVL (SEQ ID NO: 147) and LFPITSVL (SEQ ID NO: 148) can also be used for target a protein that contains the PDZ domain in monocytes.

6.6.6 Sequence Reasons for the PDZ Domain of Endothelial Cell Surface Receptors

Although endothelial cells are not hematopoietic cells, they interact intimately with the hematopoietic system since they form the lining of blood vessels. As such, endothelial cells come into contact with the cells of the hematopoietic system. Thus, the ability to regulate the function of endothelial cells provides indirect regulation of hematopoietic cells.

A number of surface receptors expressed by endothelial cells contain a motive sequence of the PDZ domain. These molecules include, but are not limited to, CD34, CD46, CD66b, CD66c, CD105, CD106, CD62e (selectin E) and VCAM1.

The core sequence of the C-terminal of CD34 is DTEL (SEQ ID NO: 149). When naturally existing residues are added to the core sequence, ADTEL (SEQ ID NO: 150), VADTEL (SEQ ID NO: 151), VVADTEL (SEQ ID NO: 152) and HVVADTEL (SEQ ID NO: 153) can also be used for directing a protein that contains PDZ domain in endothelial cells.

The C-terminal core sequence of CD66b and CD66c is VALI (SEQ ID NO: 154). When naturally existing residues are added to the core sequence, RVALI (SEQ ID NO: 155), ARVALI (SEQ ID NO: 156), LARVALI (SEQ ID NO: 157) and VLARVALI (SEQ ID NO: 158) can also be used for directing a protein that contains PDZ domain in endothelial cells.

The C-terminal core sequence of CD105 is SSMA (SEQ ID NO: 159). When naturally existing residues are added to the core sequence, TSSMA (SEQ ID NO: 160), STSSMA (SEQ ID NO: 161), CSTSSMA (SEQ ID NO: 162) and PCSTSSMA (SEQ ID NO: 162) can also be used for directing a protein that contains PDZ domain in endothelial cells.

The C-terminal core sequence of CD106 is KSKV (SEQ ID NO: 163). When naturally existing residues are added to the core sequence, QKSKV (SEQ ID NO: 164), AQKSKV (SEQ ID NO: 165), EAQKSKV (SEQ ID NO: 166) and VEAQKSKV (SEQ ID NO: 167) can also be used for directing a protein that contains PDZ domain in endothelial cells.

The C-terminal core sequence of CD62e is SYIL (SEQ ID NO: 168). When naturally existing residues are added to the core sequence, PSYIL (SEQ ID NO: 169), KPSYIL (SEQ ID NO: 170), QKPSYIL (SEQ ID NO: 171) and YQKPSYIL (SEQ ID NO: 172) can also be used for directing a protein that contains PDZ domain in endothelial cells.

The C-terminal core sequence of VCAM1 is KSKV (SEQ. ID. NO: 233). When naturally existing residues are added to the core sequence, QKSKV (SEQ. ID. NO: 234), AQKSKV (SEQ. ID. NO: 235), EAQKSKV (SEQ. ID. NO: 236), and VEAQKSKV ( SEQ ID NO: 237) to address a protein containing PDZ domain in endothelial cells.

6.6.7 Cellular Surface Receptors of Mast Cells, Basophils and Eosinophils

FcεRIβ, CDw125, CDw128 and IL-8RB are transmembrane receptors expressed by mast cells, basophils and eosinophils. These receptors play a role in the activation of these cells resulting in degranulation and release of histamine in allergic reactions. The C-terminal core sequence of FcεRIβ is PIDL (SEQ ID NO: 4). When naturally existing residues are added to the core sequence, PPIDL (SEQ ID NO: 5), SPPIDL (SEQ ID NO: 6), MSPPIDL (SEQ ID NO: 7) and EMSPPIDL (SEQ ID NO: 8) can also be used to target a protein that contains PDZ domain in mast cells. Additionally, residue E may be substituted with G to increase its binding affinity.

The C-terminal core sequence of CDw125 and s DSVF (SEQ ID NO: 9). When naturally existing residues are added to the core sequence, EDSVF (SEQ ID NO: 10), LEDSVF (SEQ ID NO: 11), TLEDSVF (SEQ ID NO: 12), and ETLEDSVF (SEQ ID NO: 13 can also be used ) to target a protein containing PDZ domain in mast cells.

The C-terminal core sequence of CDw128 and SSNL (SEQ ID NO: 14). When naturally existing residues are added to the core sequence, VSSNL (SEQ ID NO: 15), NVSSNL (SEQ ID NO: 16), VNVSSNL (SEQ ID NO: 17), and SVNVSSNL (SEQ ID NO: 18 can also be used ) to target a protein containing PDZ domain in mast cells.

The C-terminal core sequence of IL-8RB and s STTL (SEQ ID NO: 19). When naturally existing residues are added to the core sequence, TSTTL (SEQ ID NO: 20), HTSTTL (SEQ ID NO: 21), GHTSTTL (SEQ ID NO: 22) and SGHT-STTL (SEQ ID NO:) can also be used. 23) to address a protein that contains PDZ domain in mast cells.

6.6.8. Other Sequences Reason for PDZ Domain Fixation

The core C-terminal sequence of NMDA is ESDV (SEQ ID NO: 263). When naturally existing residues are added to the core sequence, IESDV (SEQ. ID. NO: 264), SIESDV (SEQ. ID. NO: 265), PSLESDV (SEQ. ID. NO: 266), and MPSIESDV can also be used (SEQ. ID. NO: 267) to address a protein containing PDZ domain in neuronal cells.

The core C-terminal sequence of neurexin is EYYV (SEQ. ID. NO: 268). When naturally existing residues are added to the core sequence, KEYYV (SEQ. ID. NO: 269), DKEYYV (SEQ. ID. NO: 270), KDKEYYV (SEQ. ID. NO: 271), and NKDKEYYV can also be used (SEQ. ID. NO: 272) to address a protein containing PDZ domain in neuronal cells.

The core C-terminal sequence of glycoforin C is EYFI (SEQ. ID. NO: 273). When naturally existing residues are added to the core sequence, KEYFI (SEQ. ID. NO: 274), RKEYFI (SEQ. ID. NO: 275), SRKEYFI (SEQ. ID. NO: 276), and SSRKEYFI can also be used (SEQ. ID. NO: 277) to address a protein containing PDZ domain.

The C-terminal core sequence of CD148 is KTIA (SEQ. ID. NO: 278). When naturally existing residues are added to the core sequence, GKTIA (SEQ. ID. NO: 279), FGKTIA (SEQ. ID. NO: 280), TFGKTIA (SEQ. ID. NO: 281), and TTFGKTIA (SEQ. ID NO: 282) can also be used to target a protein containing PDZ domain in epithelial or myeloid cells.

6.7. Peptide Preparation

6.7.1. Chemical synthesis

The peptides of the invention or analogs thereof can be prepared using virtually any method known in the art for the preparation of peptides and peptide analogs. For example, the peptides can be prepared in a linear fashion using conventional synthesis of solution or solid phase peptides and cleaved from the resin followed by purification procedures (Creighton, 1983, Protein Structures and Molecular Principles,

W.H. Freeman and Co., N.Y.). Suitable procedures for the synthesis of the peptides described herein are well known in the art. The composition of the synthetic peptides can be confirmed by amino acid analysis or sequencing (e.g., the Edman degradation procedure and mass spectroscopy).

Additionally, analogs and derivatives of the peptides can be chemically synthesized. The link between each amino acid of the peptides of the invention can be an amide, a substituted amide or an amide isoster. Non-classical amino acids or chemical analogs of amino acids can be introduced as a substitution or addition in the sequence. Non-classical amino acids include, but are not limited to, the D isomers of the common amino acids, α-amino-isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, design amino acids such as β-methyl-amino acids, Cα-methylamino acids, Nαmethylamino acids, and amino acid analogs in general. Additionally, the amino acid can be D (dextrorotatory) or L (levorotatory).

6.7.2. Recombinant Synthesis

If the peptide is composed entirely of amino acids encoded by genes, or a portion thereof is composed in this way, the peptide or the relevant portion can also be synthesized using conventional techniques of recombinant genetic engineering. For recombinant production, a nucleotide sequence encoding a linear form of the peptide is inserted into an appropriate expression vehicle, that is, a vector containing the elements necessary for transcription and translation of the inserted coding sequence, or in the case of a viral RNA vector, the elements necessary for replication and translation. The expression vehicle is transfected into a suitable target cell that will express the peptide. Depending on the expression system used, the expressed peptide is then isolated by procedures well established in the

technique. Methods for recombinant production of proteins and peptides are well known in the art (see, eg, Maniatis et al., 1989, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, NY; and Ausubel et al., 1989, Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, NY).

A variety of host expression vector systems can be used to express the peptides described herein. These include, but are not limited to, microorganisms such as bacteria transformed with bacteriophage recombinant DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeasts or filamentous fungi transformed with recombinant expression vectors of yeasts or fungi containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (eg, cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (eg, Ti plasmid) containing a sequence coding; or animal cell systems.

The expression elements of expression systems vary in their robustness and their specificities. Depending on the host / vector system used, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used in the expression vector. For example, when cloning is carried out in bacterial systems, inducible promoters such as bacteriophage pL, plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like can be used; when cloning is performed in insect cell systems, promoters such as the baculovirus polyhedron promoter can be used; when cloning is carried out in plant cell systems, promoters derived from the plant cell genome (eg, heat shock promoters; the promoter for the small subunit of RUBISCO; the promoter for the chlorophyll binding protein a / b can be used) ) or from plant viruses (eg, the CaMV 35S RNA promoter; the TMV envelope protein promoter); when cloning is carried out in mammalian cell systems, promoters derived from the genome of mammalian cells (eg, metallothionein promoter) or mammalian virus (eg, late adenovirus promoter; 7.5K virus promoter can be used Vaccinia); when cell lines containing multiple copies of the expression product are generated, vectors based on SV40, BPV and EBV with an appropriate selectable marker can be used.

In cases where plant expression vectors are used, the expression of the sequences encoding the peptides of the invention can be driven by any of a number of promoters. For example, viral promoters such as the 35S RNA and 19S RNS promoters of CaMV can be used (Brisson et al., 1984, Nature

310: 511-514), or the TMV coat protein promoter (Takamatsu et al., 1987, EMBO J. 6: 307-311); alternatively, plant promoters such as the small subunit of RUBISCO (Coruzzi et al., 1984, EMBO J. 3: 1671-1680; Broglie et al., 1984, Science 224: 838-843) or shock promoters can be used thermal, eg, hsp17.5-E or hsp17.3-B soybean (Gurley et al., 1986, Mol. Cell. Biol. 6: 559-565). These constructs can be introduced into ‘planleukocytes’ using Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. For reviews of such techniques see, e.g., Weissbach & Weissbach, 1988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and Grierson & Corey, 1988, Plant Molecular Biology, 2nd edition, Blackie, London, chap. 7-9.

In an insect expression system that can be used to produce the peptides of the invention, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus is allowed to grow in Spodoptera frugiperda cells. A coding sequence can be cloned into non-essential regions (for example in the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (for example, the polyhedrin promoter). Successful insertion of a coding sequence will result in the deactivation of the polyhedrin gene and the production of non-occluded recombinant virus (ie, viruses lacking the proteinaceous coat encoded by the polyhedrin gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed; (e.g., see Smith et al., 1983, J. Virol. 46: 584 Smith, U.S. Patent No. 4,215,051). Additional examples of this expression system can be found in Current Protocols in Molecular Biology, Vol. 2, Ausubel et al., Eds., Greene Publish. Assoc. & Wiley Interscience.

In mammalian host cells, a number of virus-based expression systems can be used. In cases where an adenovirus is used as an expression vector, a coding sequence can be linked to an adenovirus transcription / translation control complex, e.g. the late promoter and the tripartite conductive sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. Insertion into a non-essential region of the viral genome (eg, region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the peptide in infected hosts (eg, see Logan & Shenk, 1994, Proc. Natl Acad. Sci. USA 81: 3655-3659). Alternatively, the 7.5K Vaccinia promoter can be used (see, eg, Mackett et al., 1982, Proc. Natl. Acad. Sci. USA 79: 7415-7419; Mackett et al., 1984, J. Virol. 49 : 857864; Panicali et al., 1982, Proc. Natl. Acad. Sci. USA 79: 4927-4931).

Other expression systems for producing linear peptides of the invention will be apparent to those skilled in the art.

6.7.3 Purification of Peptides and Peptide Analogs

The peptides and peptide analogs of the invention can be purified by methods known in the art such as high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, affinity chromatography and the like. The actual conditions used to purify a particular peptide or analogue will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to those skilled in the art. Purified peptides can be identified by assays based on their physical or functional properties, including radioactive labeling followed by gel electrophoresis, radioimmunoassays, ELISA, bioassays, and the like.

For purification by affinity chromatography, any antibody that specifically binds peptides or peptide analogs can be used. For the production of antibodies, various host animals can be immunized, including but not limited to rabbits, mice, rats, etc., by injection with a peptide. The peptide can be attached to a suitable carrier, such as BSA or KLH, by means of a functional side chain group or linkers attached to a functional side chain group. Various adjuvants can be used to increase the immune response, depending on the host species, including but not limited to Freund's (complete or incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic polyols, polyanions, peptides , oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants such as BCG (Calmette-Guerin bacilli) and Corynebacterium parvum.

Monoclonal antibodies to a peptide can be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique originally described by Koehler and Milstein, 1975, Nature 256: 495-497, the human B cell hybridoma technique, Kosbor et al., 1983, Immunology Today 4:72 ; Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80: 2026-2030 and the EBV hybridoma technique (Cole et al., 1985, Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Additionally, techniques developed for the production of "chimeric antibodies" can be used (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al., 1984, Nature 312: 604- 608; Takeda et al., 1985, Nature 314: 452-454) by remodeling the genes of a mouse antibody molecule of appropriate antigen specificity together with genes of a human antibody molecule of appropriate biological activity. Alternatively, techniques described for the production of single chain antibodies (U.S. Patent No. 4,946,778) can be adapted to produce single chain antibodies with peptide specificity.

Antibody fragments containing deletions of specific binding sites can be generated by known techniques. For example, such fragments include, but are not limited to, F (ab ') 2 fragments, which can be produced by pepsin digestion of the antibody molecule and Fab fragments, which can be generated by reduction of the disulfide bridges of the F fragments. (ab ') 2. Alternatively, expression libraries (Huse et al., 1989, Science 246: 1275-1281) can be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for the peptide of interest.

The antibody or antibody fragment specific for the desired peptide may be attached, for example, to agarose, and the antibody-agarose complex is used in immunochromatography to purify peptides of the invention. See, Scopes, 1984, Protein Purification: Principles and Practice, Springer-Verlag, New York, Inc., NY, Livingstone, 1974, Methods Enzymology: Immunoaffinity Chromatography of Proteins 34: 723-731.

6.8. Uses of PDZ Domain Fixation and Antagonist Compounds

In one aspect of the invention, the PDZ-PL binding compounds and inhibitors of the PDZ domain of the present invention are useful in regulating various activities of hematopoietic cells (eg, T cells and B cells) and other cells involved in the immune response.

In one aspect of the description, the compounds are used to inhibit leukocyte activation, which manifests itself in measurable events that include, but are not limited to, cytokine production, cell adhesion, cell number expansion, apoptosis and cytotoxicity. . As a corollary, the compounds can be used to treat various conditions associated with undesirable activation of leukocytes, including but not limited to acute and chronic inflammation, reverse rejection disease, transplant rejection, hypersensitivity and autoimmunity such as multiple sclerosis, arthritis rheumatoid, periodontal disease, systemic lupus erythematosus, juvenile diabetes mellitus, non-insulin dependent diabetes, and allergies, and other conditions indicated herein (see, eg, Section 6.4, above).

Thus, one aspect of the description also refers to methods of use in such compositions in the modulation of leukocyte activation as measured, for example, by cytotoxicity, cytokine production, cell proliferation, and apoptosis. Trials for activation are well known. For example, PDZ / PL interaction antagonists can be evaluated as follows: (1) cytotoxic T lymphocytes can be incubated with radioactively labeled target cells and antigen specific lysis of these target cells can be detected by the release of radioactivity, (2 ) adjuvant T lymphocytes can be incubated with antigens and antigen presenting cells, and cytokine synthesis and secretion can be measured by standard methods (Windhagen A, et al., 1995, Immunity 2 (4): 373-80), (3 ) the antigen presenting cells can be incubated with whole protein antigen and the presentation of said antigen in MHC can be detected by T lymphocyte activation assays or by biophysical methods (Harding et al., 1989, Proc. Natl. Acad. Sci. 86: 4230-4), (4) mast cells can be incubated with reagents that cross-link their Fc-epsilon receptors and histamine release measured by enzyme immunoassay (Siraganian, et al., 1983, TIPS 4: 432-437).

Similarly, the effect of PDZ / PL interaction antagonists on leukocyte activation products in a model organism (e.g., mouse) or a human patient can also be evaluated by various methods that are well known. For example, (1) antibody production in response to vaccination can be readily detected by standard methods commonly used in clinical laboratories, e.g., an ELISA; (2) Migration of immune cells to sites of inflammation can be detected by scraping the surface of the skin and placing a sterile container to capture the cells that migrate at the site of scraping (Peters et al., 1988, Blood 72: 1310-5); (3) proliferation of peripheral blood mononuclear cells in response to mitogens or mixed lymphocyte reaction can be measured using 3H-thymidine; (4) the phagocytic capacity of granulocytes, macrophages, and other phagocytes in PBMCs can be measured by location of PBMCs in wells together with labeled particles (Peters et al., 1988); and (5) the differentiation of immune system cells can be measured by labeling PBMCs with antibodies to CD molecules such as CD4 and CDE8 and measuring the fraction of PBMCs expressing these markers.

In an illustrative assay, human peripheral blood mononuclear cells (PBMC), human T cell clones (eg, Jurkat E6, ATCC TIB-152), EBV transformed B cell clones (eg, 9D10, ATCC CRL-8752), clones or antigen-specific T cell lines can be used to examine PDZ / PL interaction antagonists in vitro. Inhibition of the activation of these cells or cell lines can be used for the evaluation of potential antagonists of the PDZ / PL interaction.

Standard methods by which hematopoietic cells are stimulated to undergo the characteristic activation of an immune response are, for example,

A) Antigen specific stimulation of immune responses. Pre-immunized or naive mouse splenocytes can be generated by standard procedures. Additionally, clones and hybridomas of antigen-specific T cells (e.g., MBP-specific), and numerous B-cell lymphoma cell lines (e.g., CH27), have been previously characterized and are available for the assays described below. Splenocytes or antigen-specific B cells can be mixed with antigen-specific T cells in the presence of antigen to generate an immune response. This can be done in the presence or absence of PDZ / PL interaction antagonists to test whether the PDZ / PL interaction antagonists modulate the immune response, later.

B) Activation of nonspecific T cells. The following methods can be used to activate T cells in the absence of antigen: 1) T-cell receptor cross-linking (TCR) by the addition of antibodies against receptor activation molecules (eg, TCR, CD3, or CD2) together with antibodies against costimulant molecules, for example anti-CD28; 2) activation of cell surface receptors in a nonspecific modality using lectins such as concanavalin A (with A) and phytohemagglutinin (PHA); 3) mimetization of activation mediated by cell surface receptors using pharmacological agents that activate protein kinase C (e.g., forbol esters) and increase in cytoplasmic Ca2 + (e.g., ionomycin).

C) Activation of nonspecific B cells: 1) Application of antibodies against cell surface molecules such as IgM, CD20 or CD21. 2) Lipopolysaccharide (LPS), phorbol esters, calcium ionophores and ionomycin can also be used to prevent triggering of the receptor.

D) Mixed lymphocyte reaction (MLR). Mixing of the donor's PBMC with the recipient's PBMC to activate the lymphocytes by presenting missing tissue antigens, which occurs in all cases except in identical twins.

E) Generation of a specific clone or T cell line (a) that recognizes a particular antigen. A standard approach is to generate tetanus toxin-specific T cells from a donor that has recently been potentiated with tetanus toxin. Matching antigen presenting cells in the major histocompatibility complex (MHC-) and a source of tetanus toxin are used to maintain antigen specificity of the cell line or T cell clone (Lanzavecchia, A., et al., 1983, Eur. J. Immun 13: 733-738).

Trial Quantification

The assays described above can be quantified by a variety of well known quantification methods. For example:

(TO)

 Tyrosine Phosphorylation

Tyrosine phosphorylation of rapid response proteins such as HS1, PLC-r, ZAP-76, and Vav is an early biochemical event that follows leukocyte activation. Phosphorylated proteins in tyrosine can be detected by Western blotting using antibodies against phosphorylated tyrosine residues. Tyrosine phosphorylation of these rapid response proteins can be used as a standard assay for leukocyte activation (J. Biol. Chem., 1997, 272 (23): 14562-14570). Any change in the phosphorylation pattern of these proteins or related proteins when immune responses are generated in the presence of potential antagonists of the PDZ / PL interaction is indicative of a potential antagonist of the PDZ / PL interaction.

(B)

 Intracellular Calcium Flow

The kinetics of intracellular concentrations of Ca2 + are measured over time after stimulation of preloaded cells with a calcium sensitive dye. After fixation of Ca2 +, the indicator dye (e.g., Fluor-4 (Molecular Probes)) exhibits an increase in the level of fluorescence using flow cytometry, fluorometry in solution, and confocal microscopy. Any change in the level or synchronization of calcium flow when immune responses are generated in the presence of PDZ / PL interaction antagonists is indicative of an inhibition of this response.

(C)

 Regulation of Early Activation Markers

Increased and decreased expression / regulation of marker levels of early lymphocyte activation such as CD69, IL-2R, MHC class II, B7 and TCR are commonly measured with fluorescently labeled antibodies using flow cytometry. All antibodies are commercially available. Any change in the expression levels of lymphocyte activation markers when immune responses are generated in the presence of the PDZ / PL interaction antagonists is indicative of an inhibition of this response.

(D)

 Increased Acid / Increased Metabolic Activity

The activation of most known signal transduction pathways triggers increases in acidic metabolites. This reproducible biological event is measured as the acid release rate using a microphysiometer (Molecular Devices), and is used as an early activation marker when comparing cell treatment with potential biological therapeutic methods (McConnell, HM et al., 1992, Science 257: 1906-1912 and McConnell, HM, 1995, Proc. Natl. Acad. Sci. 92: 2750-2754). Any statistically significant increase or decrease in acid release from the sample treated with the PDZ / PL interaction antagonist, compared to a control sample (without treatment), suggests an effect of the PDZ / PL interaction antagonist on the function. biological

(AND)

 Cell Proliferation / Cell Viability Assays

(one)

 3H-thymidine incorporation

Exposure of lymphocytes to antigen or mitogen in vitro induces DNA synthesis and cell proliferation. The measurement of mitotic activity by incorporation of 3H-thymidine into freshly synthesized DNA is one of the most frequently used assays for quantitative activation of T cells. Depending on the population of cells and the form of stimulation used to activate T cells, mitotic activity can be measured within 24-72 hours in vitro, after 3H-thymidine pulses (Mishell, BB and SM Shiigi, 1980, Selected Methods in Cellular Immunology, WH Freeman and Company and Dutton, RW and Pearce, JD, 1962, Nature 194: 93). Any increase

or statistically significant decrease in CPM of the sample treated with the PDZ / PL interaction antagonist, compared to a control sample (without treatment), suggests an effect of the PDZ / PL interaction antagonist on the biological function.

(2)

MTS [5- (3-carboxymethoxyphenyl) -2- (4,5-dimethylthiazolyl) -3- (4-sulfophenyl) -tetrazolium, internal salt] is a colorimetric method for determining the number of viable cells in proliferation or cytotoxicity assays (Barltrop, JA et al., 1991, Bioorg. & Med. Chem. Lett. 1: 611). 1-5 days after lymphocyte activation, the MTS tetrazolium compound, Owen reagent, undergoes biological reduction by cells to a formazan colored product that is soluble in tissue culture medium. The color intensity is read at 490 nm minus 650 nm using a microplate reader. Any statistically significant increase or decrease in color intensity of the sample treated with the PDZ / PL interaction antagonist, as compared to a control sample (without treatment), may suggest an effect of the PDZ / PL interaction antagonist in the biological function (Mosmann, T., 1983, J. Immunol. Methods 65:55 and Barltrop, JA et al., (1991)).

(3)

 Bromodeoxyuridine (BrdU), a thymidine analog, is easily incorporated into cells that undergo DNA synthesis. Pulsed BrdU cells are labeled with an enzyme-conjugated anti-BrdU antibody (Gratzner, H.G., 1982, Science 218: 474-475). A soluble colorimetric substrate is used to visualize proliferating cells that have incorporated BrdU. The reaction is stopped with sulfuric acid and the plate is read at

450 nm using a microplate reader. Any statistically significant increase or decrease in the color intensity of the sample treated with the PDZ / PL interaction antagonist, compared to a control sample (without treatment), suggests an effect of the PDZ / PL interaction antagonist on the biological function

(F)

 Apoptosis due to Annexin V

Programmed cell death or apoptosis is an early event in a cascade of catabolic reactions that lead to cell death. A loss in the integrity of the cell membrane allows the fixation of fluorescently conjugated phosphatidylserine. Stained cells can be measured by fluorescence microscopy and flow cytometry (Vermes, I., 1995, J. Immunol. Methods. 180: 39-52). In one embodiment, any statistically significant increase or decrease in the number of apoptotic cells in the sample treated with the PDZ / PL interaction antagonist, as compared to a control sample (without treatment), suggests an effect of the interaction antagonist. PDZ / PL on biological function. For evaluation of apoptosis in situ, assays can also be used to evaluate cell death in tissue samples in in vivo studies.

(G)

 Quantification of Cytokine Production

Supernatants from cells collected after stimulation of the cells for 16-48 hours are stored at -80 ° C until assayed or directly assayed for cytokine production. Multiple cytokine assays can be performed on each sample. IL-2, IL-3, IFN-γ and other cytokine ELISA assays are available for mouse, rat, and human (Endogen, Inc. and BioSource). Cytokine production is measured using an ELISA protocol standard two-antibody sandwiches as described by the manufacturer. The presence of horseradish peroxidase is detected with 3,3 ', 5,5'-tetramethyl-benzidine (TMB) substrate and the reaction is stopped with sulfuric acid. Absorbance at 450 nm is measured using a microplate reader. Any statistically significant increase or decrease in the color intensity of the sample treated with the PDZ / PL interaction antagonist, compared to a control sample (without treatment), suggests an effect of the PDZ / PL interaction antagonist on the biological function See also Example 1, below. The detection of intracellular cytokines using anti-cytokine antibodies provides the additional advantage of cytokine measurement before mixed cell populations. This allows phenotyping the frequency of measurement of the types of cytokine-producing cells in suspension or in tissues.

(H)

 NF-AT Can be Visualized by Immunostaining

Activation of T cells requires the importation of the nuclear factor from activated T cells (NF-AT) into the nucleus. This translocation of NF-AT can be visualized by immunostaining with an anti-NF-AT antibody (Cell 1998, 93: 851861). Therefore, the nuclear translocation of NF-AT has been used to test the activation of T cells. Similarly, NF-AT / luciferase reporter assays have been used as a standard measure of T cell activation (MCB 1996, 12: 7151-7160). Any statistically significant increase or decrease in nuclear translocation of NF-AT produced by the sample treated with the PDZ / PL interaction antagonist, as compared to a control sample (without treatment), suggests an effect of the PDZ interaction antagonist. / PL on biological function. In order to optimize the use of the peptides and peptide analogs described herein in a human individual, various animal models can be used to define certain clinical parameters. For example, the compounds of the invention can be tested in different dosages, formulations and routes of administration in a mouse model of heart transplantation to optimize their ability to inhibit rejection responses to solid organ transplants (Fulmer et al., 1963, Am. J. Anat. 113: 273; Jockusch et al., 1983, Exp. Neurol. 81: 649).

In situations where inhibition of a T-cell response is desired, the compounds of the invention can be used to inhibit interactions of the PDZ domain with CD3, CD4, CD6 and CDw137. Additionally, the compounds of the invention can be used to inhibit interactions of the PDZ domain with CD53 and CD138 in B cells. In order to inhibit allergic reactions mediated by IgE, the compounds of the invention can be used to inhibit interactions of the PDZ domain with FcεRIβ, CDw125 and CDw128. Additionally, a motif sequence of the PDZ domain (PL sequence) of CD95 can be used to induce lymphoma apoptosis.

(I) Tests for the Release of Inflammatory Mediators

Tests for the release of inflammatory mediators are well known in the art in order to access the effect of compounds or IgE-mediated degranulation treatments. See, Berger et al., 1997, Measuring Cell Degranulation v.g., chap. 19.6 Immunology Method Manual. Academic Press, Ltd. 1436-1440 and Siraganian, 1983, Histamine Secretion from Mast Cells and Basophil. TIPS 4: 432-437.

6.9. Formulation and Administration Route

6.9.1 Introduction of Agonists or Antagonists (e.g., Peptides and Fusion Proteins) into Cells

In one aspect, PDZ-PL antagonists are introduced into a cell to modulate (i.e. increase or decrease) a biological function or activity of the cell. Many small organic molecules easily cross cell membranes (or they can be modified by an experienced person using routine methods to increase the ability of compounds to enter cells, eg, by load reduction or elimination, increased lipophilicity, conjugation of the molecule to a targeting moiety of a cell surface receptor such that it then interacts with the receptor). Methods for introducing major molecules are also well known, e.g. fusion peptides and proteins, including, e.g., injection, liposome-mediated fusion, application of a hydrogel, conjugation to a cell endocytosed targeting residue conjugate, electroporation, and the like).

In one aspect, the antagonist or agent is a fusion polypeptide or derivatized polypeptide. A fusion protein

or derivatized may include a targeting moiety that increases the ability of the polypeptide to cross a cell membrane or cause the polypeptide to be preferably administered to a specified cell type (eg, liver cells or tumor cells) or preferably to a cell compartment (eg , nuclear compartment). Examples of targeting molecules include lipid tails, amino acid sequences such as the antenapedia peptide or a nuclear localization signal (NLS; e.g. Xenopus nucleoplasmin, Robbins et al., 1991, Cell 64: 615).

In one aspect, a peptide sequence or a particular peptide analog that inhibits a binding PDZprotein PL domain in an assay is introduced into a cell by linking the sequence to an amino acid sequence that facilitates its transport through the plasma membrane (a "transmembrane transport sequence"). The peptides can be used directly or fused to a transmembrane transport sequence in order to facilitate their entry into the cells. In the case of such a fusion peptide, each peptide can be fused with a heterologous peptide directly at its amino terminus or by use of a flexible polylinker such as the G-G-G-G-S pentamer (SEQ ID NO: 1) repeated 1 to 3 times. Said linker has been used in the construction of single chain antibodies (scFv) by insertion between VH and VL (Bird et al., 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5979-5883). The linker is designed to enable the correct interaction between two beta sheets that form the variable region of the single chain antibody. Other linkers that can be used include Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO: 2) (Chaudhary et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1066-1070) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp ( SEQ ID NO: 3) (Bird et al., 1988, Science 242: 423-426).

A number of peptide sequences capable of facilitating the entry of a peptide linked to these sequences into a cell through the plasma membrane have been described in the art (Derossi et al., 1998, Trends in Cell Biol. 8: 84). For the purpose of this invention, such peptides are collectively referred to as transmembrane transporter peptides. Examples of these peptides include, but are not limited to, HIV-derived tat (Vives et al., 1997, J. Biol. Chem. 272: 16010; Nagahara et al., 1998, Natl. Med. 4: 1449), antenapedia of Drosophila (Derossi et al., 1994, J. Biol. Chem. 261: 10444), VP22 of the herpes simplex virus (Elliot and D'Hare, 1997, Cell 88: 223-233), the determining regions of complementarity (CDR) 2 and 3 anti-DNA antibodies (Avrarneas et al., 1998, Proc. Natl. Acad. Sci. USA, 95: 5601-5606), the 70 kDa protein from thermal shock (Fujihara, 1999, EMBO J. 18: 411-419) and transportana (Pooda et al., 1998, FASEB J. 12: 67-77). In a preferred embodiment of the invention, a truncated HIV HIV peptide having the sequence of GYGRKKRRQRRRG SEQ ID NO: 173) is used.

It is preferred that a transmembrane transporter sequence be fused to a carboxy-terminal sequence of a hematopoietic cell surface receptor at its amino terminus with or without a linker. Generally, the C term of a motif sequence of the PDZ domain (PL sequence) has to be free in order to interact with a PDZ domain. The transmembrane transporter sequence can be used in whole or in part as long as it is capable of facilitating the entry of the peptide into a cell.

An alternative aspect of the description, a single C-terminal sequence of a hematopoietic cell surface receptor can be used when administered in a manner that allows entry into cells in the absence of a transmembrane transporter sequence. For example, the peptide can be administered in a liposome formulation or using a gene therapy approach by administering a coding sequence for the purpose of fixing the PDZ domain alone or as a fusion molecule in a target cell.

The compounds can also be administered via liposomes, which serve to direct the conjugates to a particular tissue, such as a lymphoid tissue, or selectively target infected cells, as well as increase the half-life of the peptide composition. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, laminar layers and the like. In these preparations, the peptide to be administered is incorporated as part of a liposome, alone or in association with a molecule that is fixed, eg, to a prevailing receptor between lymphoid cells, such as monoclonal antibodies that bind to the CD45 antigen, or with other therapeutic or immunogenic compositions. Thus, the liposomes

loaded with a peptide or conjugate of the desired invention can be directed to the site of lymphoid cells, where the liposomes then administer the selected inhibitor compositions. Liposomes for use in the invention are formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by considerations, e.g., of liposome size, lability in acid medium and stability of liposomes in the bloodstream. A variety of methods for preparing liposomes are available, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng 9: 467 (1980), U.S. Pat. No. 4,235,871, 4,501,728 and 4,837,028.

Liposome addressing using a variety of targeting agents is well known in the art (see, e.g., U.S. Patent Nos. 4,957,773 and 4,603,044). For targeting the immune cells, a ligand to be incorporated into the liposome may include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells. A liposome suspension containing a peptide or conjugate can be administered intravenously, locally, topically, etc. in a dose that varies according to, among other things, in administration mode, the conjugate being administered, and the phase of the disease being treated.

In order to specifically administer a PDZ domain binding sequence (PL sequence) peptide sequence to a specific cell type, the peptide can be linked to a specific cell targeting moiety, including, but not limited to, ligands for various leukocyte surface molecules such as growth factors, hormones and cytokines, as well as antibodies or antigen binding fragments thereof. Since a large number of cell surface receptors have been identified in leukocytes, ligands or antibodies specific for these receptors can be used as cell-specific targeting moieties. For example, interleukin-2, B7-1 (CD80), B7-2 (CD86) and CD40 or peptide fragments thereof can be used to specifically target activated T cells (The Leucocyte Antigen Facts Book, 1997, Barclay et al. ( editors), Academic Press). CD28, CTLA-4 and CDE40L or peptide fragments thereof can be used to specifically target B cells. Additionally, Fc domains can be used to address certain receptor expressing cells such as monocytes.

Antibodies are the most versatile cell-specific targeting residues because they can be generated against any antigen on the cell surface. Monoclonal antibodies against specific markers of leukocyte lineage such as certain CD antigens have been generated. Genes from the variable region of an antibody can be easily isolated from hybridoma cells by methods well known in the art. However, since the antibodies are assembled between two heavy chains and two light chains, it is preferred to use a scFv as a cell-specific targeting moiety. Such scFv are constituted by VH and VL domains linked to a single polypeptide chain by a flexible linker peptide.

The sequence of the PDZ domain binding moiety (PL sequence) may be linked to a transmembrane transporter sequence and a cell-specific targeting moiety to produce a tri-fusion molecule. This molecule can be attached to a molecule on the surface of leukocytes, passes through the membrane and directs the PDZ domains. Alternatively, a motif sequence of the PDZ domain (PL sequence) can be linked to a specific targeting moiety of a cell that is fixed to a surface molecule that internalizes the fusion peptide.

In another approach, artificial polymer microspheres of mixed amino acids (proteinoids) have been used to administer pharmaceutical products. For example, U.S. Patent No. 4,925,673 describes carriers of drug-containing proteinoid microspheres as well as methods for their preparation and use. These proteinoid microspheres are useful for the administration of a number of active agents. See also U.S. Pat. No. 5,907,030 and 6,033,884.

6.9.2 Introduction of Polynucleotides in Cells

A polynucleotide encoding a C-terminal surface receptor peptide may be useful in the treatment of various abnormal conditions associated with the activation of leukocytes. By introducing gene sequences into cells, gene therapy can be used to treat conditions in which leukocytes are activated resulting in deleterious consequences. In one aspect, a polynucleotide encoding a PL sequence peptide of the invention is introduced into a cell in which it is expressed. The expressed peptide then inhibits the interaction of PDZ proteins and PL proteins in the cell.

Thus, in one aspect, the polypeptides of the invention are expressed in a cell by introduction of a nucleic acid (e.g., a DNA or mRNA expression vector) that encodes the desired protein or peptide in the cell. Expression can be constitutive or inducible, depending on the vector and the choice of promoter. Methods for introduction and expression of nucleic acids in the cell are well known in the art and are described herein.

In a specific aspect, nucleic acids comprising a sequence encoding a peptide described herein are administered to a human individual, are administered to a human individual. In this aspect, the nucleic acid produces its encoded product that mediates a therapeutic effect by inhibiting the activation of

leukocytes Any of the methods for gene therapy available in the art can be used. Illustrative methods are described below.

For general reviews of gene therapy methods, see Goldspiel et al., 1993, Clinical Pharmacy 12: 488-505; Wu and Wu, 1991, Biotherapy 3: 87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol. Toxicol 32: 573-596; Mulligan, 1993, Science 260: 926-932; and Morgan and Anderson, 1993, Ann. Rev. Biochem. 62: 191-217; May, 1993, TIBTECH 11 (5): 155-215. The methods commonly known in the art of recombinant DNA technology that can be used are described in Ausubel et al., (Editors), 1993, Current Protocols in Molecular Biology, John Wiley & Sons, NY; Kriegler, 1990, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY.

In a preferred aspect, the therapeutic composition comprises a coding sequence that is part of an expression vector. In particular, such a nucleic acid has a promoter operably linked to the coding sequence, said promoter being inducible or constitutive, and, optionally, tissue specific. In another specific embodiment, a nucleic acid molecule is used in which the coding sequence and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing intrachromosomal expression of the nucleic acid (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).

The administration of nucleic acid to a patient can be direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid carrier vector, or indirectly, in which case, the cells are first transformed with the nucleic acid in vitro and then transplant the patient. These two approaches are known, respectively, as gene therapy in vivo or ex vivo.

In a specific aspect, the nucleic acid is administered directly in vivo, where it is expressed to produce the encoded product. This can be done by any methods known in the art, eg, by construction thereof as part of an appropriate nucleic acid expression vector and administration thereto, such that it becomes intracellular, eg, by infection using a retroviral vector. defective or attenuated or other viral vector (see US Patent No. 4,980,286), by direct injection of naked DNA, by the use of microparticle bombardment (eg, a gene cannon; Biolistic, Dupont), by lipid coating or cell surface receptors or transfectant agents, by encapsulation in liposomes, microparticles, or microcapsules, by administration thereof in conjunction with a peptide known to penetrate the nucleus, or by administration thereof bound to a ligand subjected to receptor-mediated endocytosis (see eg, Wu and Wu, 1987, J. Biol. Chem. 262: 4429-4432) which can then be used to address cell types that specifically express the receptors. In another aspect, a nucleic acid-ligand complex can be formed in which the ligand comprises a fusogenic viral peptide to break endosomes, making it possible for the nucleic acid to prevent lysosomal degradation. In a further aspect, the nucleic acid can be directed in vivo for specific absorption and expression by the cell, by addressing a specific receptor (see, eg PCT Publications WO 92/06180 dated April 16, 1992; WO 92/22635 dated December 23, 1992; WO92 / 20316 dated November 26, 1992; WO93 / 14188 dated July 22, 1993; WO 93/20221 dated October 14, 1993). Alternatively, the nucleic acid can be introduced intracellularly and incorporated into the host cell's DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86: 8932-8935; Zijlstra et al., 1989, Nature 342: 435-438).

In a preferred aspect, adenovirus can be used as viral vectors in gene therapy. Adenoviruses have the advantage of being able to infect cells that are not in a state of division (Kozarsky and Wilson, 1993, Current Opinion in Genetics and Development 3: 499-503). Other cases of the use of adenovirus in gene therapy can be found in Rosenfeld et al., 1991, Science 252: 431-434; Rosenfeld et al., 1992, Cell 68: 143-155; and Mastrangeli et al., 1993, J. Clin. Invest. 91: 225-234. Additionally, adenoviral vectors with modified tropism can be used for specific cell targeting (WO 98/40508). An adeno-associated virus (AAV) has also been proposed for use in gene therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.

204: 289-300).

Additionally, retroviral vectors (see Miller et al., 1993, Meth. Enzymol. 217: 581-599) have been modified to remove retroviral sequences that are not necessary for viral genome packaging and integration into the host cell's DNA. The coding sequence to be used in gene therapy is cloned into the vector, which facilitates the administration of the gene to a patient. Additional details about retroviral vectors can be found in Boesen et al., 1994, Biotherapy 6: 291-302, which describes the use of a retroviral vector to administer the mdr1 gene to hematopoietic stem cells in order to make stem cells more resistant to Chemotherapy Other references illustrating the use of retroviral vectors in gene therapy are Clowes et al., 1994, J. Clin. Invest. 93: 644-651; Kiem et al., 1994, Blood 83: 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129-141; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 110-114.

Another approach to gene therapy involves transferring a gene to cells in tissue culture. Usually, the transfer method includes the transfer of a selectable marker to the cells. The cells are then placed under selection in order to isolate those cells that have absorbed and are expressing the transferred gene. These cells are then administered to a patient.

In this aspect, the nucleic acid is introduced into a cell before in vivo administration of the cell.

resulting recombinant. Such introduction can be performed by any method known in the art, including, but not limited to, transfection, electroporation, lipofection, microinjection, infection with a viral vector or bacteriophage vector containing the nucleic acid, cell fusion, transfer sequences. of chromosome-mediated genes, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous methods are known in the art for the introduction of foreign genes into cells. See, e.g., Loeffler and Behr, 1993, Meth. Enzymol 217: 599-618; Cohen et al., 1993, Meth. Enzymol 217: 618-644; Clina, 1985, Pharmac. Ther. 29: 69-92) and can be used in accordance with the present invention, provided that the necessary developmental and physiological functions of the recipient cells are not altered. The method should provide stable transfer of the nucleic acid into the cell, so that the nucleic acid can be expressed by the cell and is preferably inheritable and expressible by its cell line. In a preferred embodiment, the cell used for gene therapy is autologous to the patient.

In a specific aspect, the nucleic acid to be introduced for gene therapy purposes comprises an inducible promoter operably linked to the coding sequence, such that the expression of the nucleic acid can be controlled by control of the presence or absence of the appropriate transcription inducer. .

Oligonucleotides such as RNA molecules and antisense DNA, and ribozymes that function to inhibit the translation of a leukocyte surface receptor mRNA, especially its C-terminus, are also within the scope of the invention. The RNA and antisense DNA molecules act to directly block mRNA translation by binding to the target mRNA and preventing protein translation. In relation to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between the -10 and +10 regions of a nucleotide sequence are preferred.

The antisense oligonucleotide may comprise at least one modified base moiety that is selected from the group that includes, but is not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5 - (carboxyhydroxymethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylkeosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methyl-dimene-2, 2-dimethylanine-2, 2-dimethylaminin-2, 2-dimethylanine-2, 2-dimethyl-dimethyl, 2-dimethylanine-2, 2-dimethyl-dimethyl, 2-dimethyl-dimethyl, 2-dimethylanine-2, 2-dimethyl-dimethyl, 2-dimethylanine, 2-2-dimethyl-dimethyl, 2-dimethylanine, 2-2-dimethylanine, 2-2-dimethylanine, 2-2-dimethylanine, 2-2-dimethylanine, 2-2-dimethyl-dimethyl-2-dimethyl, 2-dimethylanine methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylkeosine, 5'methoxycarboxymethyluracil, 5-methoxyuracyl, 2-methylthio-n-6-methylthio-n-6-methylthio-n-6-methylthio-n-6-methyl-thione uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic-methyl ester, acid uracil-5-oxyacetic (v), 5-methyl-2-thiouracil, 3- (3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves specific hybridization of the sequence of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Within the scope of the invention are ribozyme molecules with an engine-modified hammerhead motif that specifically and efficiently catalyze the endonucleolytic cleavage of leukocyte surface receptor RNA sequences.

Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites that include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene that contains the cleavage site can be evaluated for predicted structural features such as secondary structure that may make the oligonucleotide sequence inappropriate. The suitability of candidate targets can also be assessed by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays.

RNA and antisense DNA and ribozyme molecules can be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemical synthesis of oligodeoxyribonucleotides well known in the art such as for example chemical synthesis of solid phase phosphoramidites. Alternatively, RNA molecules can be generated by in vitro and in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors containing suitable RNA polymerase promoters such as promoters of T7 or SP6 polymerases. Alternatively, they can be stably introduced into antisense cDNA construct cell lines that synthesize constitutively or inducibly antisense RNA, depending on the promoter used.

Various modifications can be made to the DNA molecules as a means of increasing intracellular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences of ribo- or deoxynucleotides to the 5 'and / or 3' ends of the molecule or the use of phosphorothioate or 2'-Omethyl instead of phosphodiesterase bonds within the Oligodeoxyribonucleotide main chain.

6.9.3. Other Pharmaceutical Compositions

The compounds described herein can be administered to an individual per se or in the form of a sterile composition or a pharmaceutical composition. Pharmaceutical compositions comprising the compounds of the invention can be manufactured by means of conventional processes of mixing, dissolution, granulation, manufacture of dragees, levigation, emulsification, encapsulation, entrapment or lyophilization. The

Pharmaceutical compositions may be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or adjuvants that facilitate the processing of peptides or analogues of active peptides in preparations that can be used pharmaceutically. The appropriate formulation depends on the route of administration selected.

For topical administration, the compounds described herein can be formulated as solutions, gels, ointments, creams, suspensions, etc. as they are well known in the art.

Systemic formulations include those that are designed for administration by injection, e.g., subcutaneous, intravenous, intramuscular, intrathecal or intraperitoneal injection, as well as those designed for transdermal, transmucosal, oral or pulmonary administration.

For injection, the compounds described herein can be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline buffer. The solution may contain formulating agents such as suspending, stabilizing and / or dispersing agents.

Alternatively, the compounds can be found in powder form for constitution with a suitable vehicle, e.g. sterile pyrogen-free water, before use.

For transmucosal administration, penetrants suitable for the permeate barrier are used in the formulation. Such penetrants are generally known in the art. This route of administration can be used to administer the compounds to the nasal cavity.

For oral administration, the compounds can be easily formulated by combining the active peptides or peptide analogs with pharmaceutically acceptable barriers well known in the art. Such carriers make it possible for the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, sludges, suspensions and the like, for oral ingestion by a patient to be treated. For oral solid formulations such as, for example, powders, capsules and tablets, suitable excipients include fillers such as sugars, such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose, and / or polyvinylpyrrolidone (PVP); granulation agents; and fixing agents. If desired, disintegrating agents may be added, such as cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

If desired, solid dosage forms can be coated with sugar or enteric coated using standard techniques.

For liquid oral preparations such as, for example, suspensions, elixirs and suitable solutions, carriers, excipients or diluents include water, glycols, oils, alcohols, etc. Additionally, flavoring agents, preservatives, coloring agents and the like can be added.

For oral administration, the compounds may be in the form of tablets, pills, etc. formulated in a conventional manner.

For administration by inhalation, the compounds for use according to the present description are conveniently administered in the form of an aerosol spray from pressurized containers or a nebulizer, with the use of a suitable propellant, eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, dioxide Carbon or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve for administering a measured amount. Capsules and cartridges can be formulated, e.g. of gelatin for use in an inhaler or insufflator, containing a powder mixture of the compound and a suitable powder base such as lactose or starch.

The compounds may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described above, the compounds can also be formulated as a depot type preparation. Such long acting formulations can be administered by implantation (for example subcutaneous or intramuscular) or by intramuscular injection. Thus, for example, the compounds can be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil)

or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.

Alternatively, other pharmaceutical administration systems may be employed. Liposomes and emulsions are well known examples of delivery vehicles that can be used to administer peptides and peptide analogs. Certain organic solvents such as dimethylsulfoxide can also be used, although usually at the cost of increased toxicity. Additionally, the compounds can be administered using a

Sustained release system, such as semipermeable matrices of solid polymers containing the therapeutic agent. Various sustained release materials have been established and are well known to those skilled in the art. Sustained-release capsules may, depending on their chemical nature, release the compounds for a few weeks to more than 100 days. Depending on the chemical nature and biological stability of the therapeutic reagent, additional strategies for protein stabilization may be employed.

Since all the compounds described herein may contain charged side chains or ends, they may be included in any of the formulations described above as free acids or bases or as pharmaceutically acceptable salts. Pharmaceutically acceptable salts are those salts that substantially retain the biological activity of free bases and that are prepared by reaction with inorganic acids. Pharmaceutical salts tend to be more soluble in aqueous solvents and other protic solvents than the corresponding free base forms.

6.10. Effective Dosages

The compounds described herein will generally be used in an amount effective to achieve the desired purpose. For use in order to inhibit disorders associated with the activation of leukocytes, the compounds of the invention or pharmaceutical compositions thereof are administered or applied in a therapeutically effective amount. Therapeutically effective amount is an amount effective to improve or prevent symptoms, or prolong the survival of the patient being treated. The determination of a therapeutically effective amount is fully within the capabilities of those skilled in the art, especially in light of the detailed description provided herein. An "inhibitory amount" or "inhibitory concentration" of a PL-PDZ binding inhibitor is an amount that reduces the fixation by at least about 40%, preferably at least about 50%, often at least about 70%, and even as much as at least about 90%. Fixation can be measured in vitro (e.g., in a trial A

or test G) or in situ.

For systemic administration, a therapeutically effective amount can be estimated initially from in vitro assays. For example, a dose can be formulated in animal models to achieve a circulating concentration range that includes the IC50 value as determined in cell culture (i.e., the concentration of the test compound that inhibits 50% of the receptor interactions of Leukocyte-protein surface containing the PDZ domain). Such information can be used to more accurately determine useful doses in humans.

Initial dosages can also be estimated from in vivo data, e.g., animal models, using methods that are well known in the art. A person with ordinary experience in the art could easily optimize administration to humans based on animal data.

The amount and dosage ranges can be adjusted individually to provide plasma levels of the compounds that are sufficient to maintain the therapeutic effect. The usual patient doses for administration by injection vary from about 0.1 to 5 mg / kg / day, preferably from about 0.5 to 1 mg / kg / day. Therapeutically effective serum levels can be achieved by administration of multiple doses each day.

In cases of local administration or selective absorption, the effective local concentration of the compounds may not be related to the plasma concentration. A person with experience in the art will be able to optimize therapeutically effective local doses without excessive experimentation.

The amount of compound administered will of course depend on the individual being treated, the weight of the individual, the severity of the affliction, the manner of administration and the criteria of the prescribing physician.

The therapy can be repeated intermittently while the symptoms are detectable or even when they are not detectable. The therapy can be provided alone or in combination with other drugs. In the case of conditions associated with the activation of leukocytes such as transplant rejection and autoimmunity, drugs that can be used in combination with the compounds of the invention include, but are not limited to, steroidal and non-steroidal anti-inflammatory agents. .

6.10.1 Toxicity

Preferably, a therapeutically effective dose of the compounds described herein will provide therapeutic benefit without causing substantial toxicity.

The toxicity of the compounds described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g. by determination of the LD50 value (the lethal dose for 50% of the population) or the DL100 value (the lethal dose for 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Compounds that exhibit high therapeutic indices are preferred. Data obtained from these cell culture assays and animal studies can

used in the formulation of a dosage range that is non-toxic for use in humans. The dosage of the compounds described herein is preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage may vary within this range depending on the dosage form employed and the route of administration used. The exact formulation, route of administration and dosage can be selected by the individual physician taking into account the patient's condition. (See, e.g., Fingl et al., 1975, in: The Pharmacological Basis of Therapeutics, Cap. 1, page 1).

6.11. EXAMPLE 1: ACTIVATION OF THE T-CELLS INHIBITED BY THE TAT FUSION PEPTIDES OF THE CARBOXYL TERM OF THE SURFACE RECEIVER OF THE T-CELLS

6.11.1. Materials and methods

6.11.1.1 Peptide Synthesis

All peptides were chemically synthesized by standard procedures. The fusion peptide of the term carboxyl Tat-CD3 (GYGRKKRRQRRRGPPSSSSGL, SEQ ID NO: 174); the fusion peptide of the term carboxyl Tat-CLASP1 (GY-GRKKRRQRRRGSISSSAEV, SEQ ID NO: 175); the fusion peptide of the carboxyl term Tat-CLASP2 (GYGRKKRRQRRRG-MTSSSSVV, SEQ ID NO: 176); and Tat peptide (GYGRKKRRQRRRG, SEQ ID NO: _); they were dissolved at 1 mM in PBS, pH 7, or dH2O. Stock peptide MBPAc1-16 (AcASQKRPSQRHGSKYLA) was dissolved at 5 mM. All peptides were divided into aliquots and stored at -80 ° C until tested.

6.11.1.2 Cell Cultures

The cells were maintained and tested in RPMI 1640 medium supplemented with 10% fetal calf serum (HyClone), 2 mM glutamine, 10 mM Hepes, 100 U / ml penicillin, 100 μg / ml streptomycin, non-essential amino acids 0 , 1 mM, 1 mM sodium pyruvate, and 50 μM beta-mercaptoethanol.

6.11.1.3 T cell stimulation test

Supernatants were tested for cytokine production after activation of T cell lines. Mouse T cell lines were stimulated using two different methods, or with antigen and antigen presenting cells or with anti-mouse CD3.

Antigen-specific mouse T cells, BR4.2, were activated with the 16 N-terminal amino acid sequences of the myelin basic protein (MBPAc1-16) and splenocytes of syngeneic mice in 96-well plates. Antigen presenting cells treated with mitomycin C, 2 x 105 B10.BR, were added to each row of serially diluted MBPAc1-16 from 0 to 200 µM. Next, 10 µM Tat peptides or medium alone were added to each row. Finally, 2 x 104 MBPAc-specific T16 cells, pre-loaded with 10 µM Tat peptides (see above) were added to all wells (see above) (Rabinowitz et al., 1997, Proc. Natl. Acad. Sci. USA, 94: 8702-8707). The cells were activated by night incubation at 5% CO2, 37 ° C. The cell supernatant was collected and stored at 80 ° C until assayed for cytokine production. The final volume was 200 μl / well.

Mouse anti-CD3 antibody (Pharmingen # 145-2C11) was coated overnight at 4 ° C using flat-bottom ELISA plates with 96 wells at a final concentration of 0.5 μg / ml, diluted in PBS. Immediately before use, the plates were washed 3 times with 200 μl PBS / well to remove excess anti-CD3. To ensure that the cells had sufficient time to transduce the Tat peptides before activation, T cells (5 x 105 cells / ml) were pretreated with or without 10 μM Tat peptides for 2 hours at 5% CO2, 37 ° C, and They were then diluted in medium with or without 10 μM Tat peptides at a final concentration of 2 x 104 cells per well in a final volume of 200 μl. The cells were then treated as described above.

6.11.1.4 Cytokine ELISA

IFNγ of the cell supernatants, described above, was measured at room temperature using Endogen protocol 3 ELISA, Inc .. Briefly, 96-well, flat-bottomed ELISA plates and high fixation were pre-incubated overnight with antibody of coating (MM700). The plates were washed with 50 mM TRIS, 0.2% Tween 20, pH 8 and then blocked for one hour with PBS plus 2% BSA. The washed plates were then incubated for one hour with 25 μl of cell supernatant and 25 μl of blocking buffer, or with 50 μl of standard IFNγ. The presence of IFNγ was detected with a biotin-labeled mouse anti-IFNγ monoclonal antibody (MM700B, Endogen, Inc.). Quantitative amounts of streptavidin detection antibody conjugated to horseradish peroxidase are disclosed. The enzyme and colored substrate for HRP, tetramethylbenzidine (TMB), was developed for up to 30 minutes and stopped with 1.0 M H2SO4. Absorbance at 450 nm was measured using a microtiter plate reader (Thermo Max, Molecular Devices) and the unknown IFNγ concentration of cell supernatants was calculated from a standard curve generated by Softmax Pro software (Molecular Devices).

6.11.2 Results

Peptides containing Tat transporter sequences linked to C-terminal sequences of various PLs were tested for their ability to inhibit T cell activation. FIGURE 4A shows that Tat-CD3 fusion peptide inhibits T cell activation. Peptide mediated: MHC compared to Tat peptide controls alone or without peptide. FIGURE 4B shows that the fusion peptide of the term carboxyl Tat-CLASP-2 inhibited the activation of monoclonal anti-CD3-mediated T cells compared to Tat peptide alone. The Tat-CLASP 1 fusion peptide did not inhibit the activation of T cells in this experiment. These results indicate that peptides containing potential inhibitory sequences can be transported in T cells through transporter peptide such as Tat to alter the organization of surface receptors mediated by PDZ proteins. Alteration of the organization of surface receptors mediated by PDZ leads to blockage of T cell activation in response to the antigen.

6.12. EXAMPLE 2. DESIGN OF AN INHIBITOR OF THE BINDING FIXATION DLG1 WITH POWER GREATER THAN 100 Um

A GST / DLG1 fusion protein (see TABLE 3) and a biotin-labeled peptide corresponding to the 20 amino acids of the C-terminal of the CLASP-2 protein, AA2L peptide (see TABLE 4), were synthesized and purified by well-known standard methods in the art as described above. It was then demonstrated that this combination of PDZ ligands is specifically fixed using both "A" and "G" tests (see TABLE 2). Once the specific binding was demonstrated, the apparent affinity of the binding interaction was determined using Approach 1 of the section entitled "Measurement of the Affinity of Ligand PDZ Fixation" (see FIGURE 2A). The apparent affinity measured was 21 μM. This implies that the labeled 21 µM AA2L CLASP-2 peptide occupied 50% of the binding sites for CLASP-2 in DLG1. Therefore, the unlabeled CLASP-2 21 μM peptide should be able to block the binding of a given ligand for DLG1 by approximately 50%, assuming that the given ligand (1) is fixed to the same or the same DLG1 sites as Qasp ~ 2 and (2) is not added at sufficient concentration to significantly reduce the fixation of the CLASP-2 peptide (i.e. it cannot exceed the CLASP-2 polypeptide).

To detect such inhibition it was necessary to synthesize an analogue of the CLASP-2 AA2L peptide that (1) retained similar binding properties of DLG1 and (2) did not generate a signal in the selected assay by itself to measure the inhibition. Since most of the molecular interactions between PDZ proteins and their ligands involve only the 6 amino acids of the C-terminal of the ligand, it was anticipated that an eight-amino acid variant of the CLASP-2 peptide, MTSSSSVV, would retain DLG1 binding properties similar to those of the CLASP-2 AA2L peptide of 20 amino acids. Therefore, this eight amino acid CLASP-2 peptide (which lacked a functional marker) was synthesized and purified by standard techniques as described above. When 100 µM of the eight amino acid CLASP-2 peptide (functionally unlabeled) and 20 µM of the biotin-labeled CLASP-2 AA2L peptide were added simultaneously to DLG1 in a variant of the "G" assay (described above), the peptide binding CLASP-2 AA2L labeled was, as predicted, inhibited by more than 50% (FIGURE 3A). An analogous experiment in which the labeled CLASP-2 AA2L peptide was replaced with another labeled DLG1 ligand, the labeled Fas AA13L peptide demonstrated a similar inhibition by the eight amino acid CLASP-2 peptide (FIGURE 3A). Therefore, an effective inhibitor of the binding of DLG1 ligand (ie the CLASP-2 peptide of eight amino acids MTSSSSVV) with a known power range (order of magnitude 21 μM) based on the affinity knowledge, 21 μM was designed , with which a particular labeled ligand, the CLASP-2 AA2L peptide, was fixed to DLG1.

The present invention should not be considered limited in scope by the illustrated embodiments proposed only as illustrations of simple aspects of the invention, and any sequences that are functionally equivalent are within the scope of the invention. In fact, various modifications of the invention in addition to those already indicated and described herein will be apparent to those skilled in the art from the foregoing description and the accompanying drawings. Such modifications should be considered within the scope of the appended claims.

Claims (8)

1. A fusion peptide or peptide analog thereof for modulating the binding of a PDZ protein and a PL protein comprising:

(i)

 a transmembrane transporter amino acid sequence capable of facilitating the entry of a peptide bound to it into a cell through the plasma membrane,

(ii)

 an optional flexible polylinker peptide sequence, and

(iii) a peptide sequence of the carboxyl terminal comprising a PDZ domain binding motif in terminal C, the sequence of the PDZ domain binding motif comprising X * -S / T-D / E-V / I / L, where X * is any non-aromatic amino acid.

2.

The fusion peptide or peptide analog of claim 1, wherein the modulation activity functions to inhibit the binding of the PDZ protein to the PL protein.

3.

The peptide or peptide fusion analog of claim 2, wherein the C-terminal sequence comprises any of the E-S-D-V or E-T-D-V sequences.

Four.

The peptide or peptide fusion analog of any one of claims 1 to 3, wherein the sequence of the carboxyl terminal peptide comprises 4 to 15 amino acids.

5.

The peptide or peptide fusion analog of any one of claims 1 to 3, wherein the amino acid sequence of the transmembrane transporter is selected from (a) HIV-derived tat, (b) Drosophila aerial therapy, and (c) VP22 from herpes simple.

6.

The peptide or peptide fusion analog of claim 5, wherein the carboxyl terminal peptide sequence comprises 4 to 15 amino acids.

7.

The peptide or peptide fusion analog of any one of claims 1 to 3, wherein the amino acid sequence of the transmembrane transporter is a tat peptide comprising the sequence GYGRKKRRQRRRG.

8.

The peptide or peptide fusion analog of claim 3, wherein the amino acid sequence of the transmembrane transporter is a tat sequence derived from HIV, and wherein the carboxyl terminal peptide sequence contains 4 to 15 amino acids.