SK499A3 - Therapeutic applications of t-bam (cd40l) technology to treat diseases involving smooth muscle cells - Google Patents

Abstract

Activation by CD40 ligand (CD40L) of smooth muscle cells bearing CD40 on the surface of the cells is inhibited in vivo and ex vivo by contacting the cells with an agent capable of inhibiting interaction between CD40L and CD40 on the cells. In vivo inhibition of CD40-bearing smooth muscle cells is used to treat smooth muscle cell-dependent diseases.

Description

Use of an agent that inhibits the interaction of CD40 and a ligand for the preparation of a medicament and a method for selecting such an agent

Technical field

The invention relates to a method of inhibiting the activation of smooth muscle cells bearing CD40 on their surface with a CD40 ligand.

BACKGROUND OF THE INVENTION

CD40 is a cell surface molecule that is expressed on a variety of cells and interacts with an appropriate CD4 ⁺ T cell counter-receptor called CD40L (a CD40 ligand) of 30-33 kD that is induced by activation. CD40L-CD40 interactions have been extensively studied in T and B cell cell interactions and are essential for T cell-dependent B cell differentiation as well as for IgG, IgA and IgE production. CD40 is also expressed on monocytes, endothelial cells and fibroblasts. CD40 expression on these cells is activated by in vitro cytokines, most notably IFN-γ. Interestingly, in vivo studies have shown a marked upregulation of CD40 expression at inflammatory sites such as e.g. Synovial membrane in rheumatoid arthritis or psoriatic foci. In vitro studies using anti-CD40 monoclonal antibodies (mAbs) or CD40L ⁺ cells demonstrate that CD40 is functionally expressed on monocytes, dendritic cells, epithelial cells, endothelial cells, and fibroblasts.

For example, CD40L-CD40 interactions induce monocytes to secrete the pro-inflammatory cytokines IL-1α, IL-β, IL-6 and TNF-α and dendritic cells to secrete TNF-α. CD40L-CD40 interactions also support monocytes and dendritic cells to secrete the chemokines IL-8 and ΜΙΡΙα. In addition, CD40 binding increases IL-1 production mediated by GM-CSF thymus epithelial cells. In addition, CD40L-mediated signals induce monocytes to secrete

IL-10 and nitric oxide and enhance the production of IL-6 by fibroblasts. Fibroblasts also proliferate after CD40L-CD40 interactions. Finally, endothelial cells and fibroblasts activate intercellular adhesion molecules upon CD40 binding.

Vascular diseases such as atherosclerosis are treated with a variety of drugs, including cholesterol lowering drugs, beta-blockers, calcium channel blockers, and anticoagulant drugs. Smooth muscle cells are now shown to be able to express CD40. This provides the basis for the treatment of vascular diseases by inhibiting the interaction between CD40 and CD40 ligand (also known as T-BAM, 5c8 Ag, gp39 and TRAP). Other smooth muscle disorders are also treated by inhibiting CD40-CD40L interactions.

SUMMARY OF THE INVENTION

This application claims priority to U.S. Patent Application Ser. 08/677 730, filed July 8, 1996, the contents of which are hereby incorporated by reference.

The present invention has been developed with the support of government grants NIH no. K08-AR-01904, RO1-AI-28367, RO1-AI-14969, HL21006, HL42833, HL50629, and RO1-AI-14969 (Department of Health and Human Services). Thus, the United States government has certain rights to this invention.

Various references are given in brackets in the application. The contribution of these publications is included in the application to more fully describe the prior art to which the application relates. Full bibliographic data on these cited publications can be found either directly in the text or listed at the end of the description in the list under the appropriate number.

The present invention provides a method of inhibiting smooth muscle cell activation, which comprises contacting cells with a composition capable of inhibiting the interaction between CD40 ligand and CD40 on cells when the composition is present in an amount that effectively inhibits cell activation.

The present invention further provides a method of inhibiting smooth muscle cell activation in a patient that carries CD40 ligand CD40 on its surface, the method comprising administering a composition capable of inhibiting the interaction between CD40 ligand and CD40 on the cells, the patient and the composition administered in a patient. an amount effective to inhibit patient cell activation.

The present invention also provides a method of treating a patient with a smooth muscle cell-dependent disease, the method comprising administering to the patient a composition capable of inhibiting the interaction between CD40 and CD40 ligand on the cells in an amount effective to inhibit cell activation in the patient. on smooth muscle cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of inhibiting CD40 ligand activation of CD40-bearing smooth muscle cells on its surface. The method comprises contacting the cells with a composition capable of inhibiting the interaction between CD40 ligand and CD40 on the cells, wherein the composition is present in an amount effective to inhibit cell activation. In one embodiment of the present invention, the composition is capable of inhibiting any interaction between CD40 ligand and CD40. The interaction between CD40 ligand and CD40 on cells relates to one or more aspects, functional or structural, of the CD40-CD40 ligand relationship. Therefore, in one embodiment, an agent that inhibits interaction can competitively bind to a CD40 ligand by blocking or reducing the binding of CD40 ligand to cellular CD40. In another embodiment, an agent that inhibits interaction can be coupled to CD40 or CD40 ligand in a manner that does not inhibit CD40 ligand binding to cellular CD40, but which affects cellular response to CD40 binding, such as altering the metabolic circulation rate of cell CD40 or CD40 complex. by altering the binding kinetics of CD40 with a CD40 ligand, or by changing the rate or extent of cellular activation in response to CD40 binding.

In a specific embodiment of the present invention, CD40-bearing smooth muscle cells are bladder smooth muscle cells, vascular smooth muscle cells, bronchial smooth muscle cells, aortic smooth muscle cells, pulmonary vein smooth muscle cells or gastrointestinal tract smooth muscle cells. In more specific embodiments, the gastrointestinal tract smooth muscle cells are the smuggler, stomach or intestine smooth muscle cells, including the small or large intestine smooth muscle cells.

In an embodiment of the present invention, the composition inhibits the binding of CD40 ligand to CD40 on cells.

In an embodiment of the present invention, the composition is a protein.

In another embodiment of the present invention, the composition is of a non-protein nature. The term non-protein is used herein to include any compounds or compositions that contain elements other than single or conjugated polypeptide chains. This includes elements such as amino acids having non-peptide bonds, non-protein amino acids such as β, γ or δ amino acids, amino acids in the D configuration or other non-protein (not being a common part of proteins) amino acids including homocysteine, homoserine, citrulline, ornithine , γ-aminobutyric acid, canavalin, dececolic acid or β-cyanolanine, monosaccharides, polysaccharides or other groups of sugars, fatty acids or groups of lipids, nucleotides, mineral groups or other non-protein elements.

In another embodiment of the present invention, the composition is a peptidomimetic compound. The peptidomimetic compound may be at least partially unnatural. The peptidomimetic compound may be a small molecule mimetic. The compound may have increased stability, efficacy, potential, and bioavailability given by the mimetics. Further, the compound may have increased permeability to the intestinal mucosa. The compound can be prepared synthetically. The compound of the present invention may include L-, or non-naturally occurring amino acids, alpha-, alpha-substituted amino acids, N-alkylamino acids, lactic acid (an alanine isoelectric analog). The peptide backbone of the compound may have at least one PSI- [CH = CH] -substituted bond (Kempf et al., Intl. J. Peptide and Prot. Res. 38, 273-241, 1991). The compound may further include trifluorotyrosine, p-Cl-phenylalanine, p-Br-phenylalanine, poly-L-propargylglycine, poly-D, Lallylglycine or poly-L-allylglycine. In another embodiment of the present invention, a peptidomimetic compound that has biological activity to inhibit the interaction between CD40 ligand and CD40 on cells may have a bond, peptide backbone or amino acid component replaced by a suitable mimetic. Examples of non-naturally occurring amino acids that may be suitable amino acid mimetics include β-alanine, L-aaminobutyric acid, Ly-aminobutyric acid, L-aminoisobutyric acid, Le-aminocaproic acid, 7 aminoheptanoic acid, L-aspartic acid, L-aspartic acid, L-aspartic acid , cysteine (acetamidomethyl), Ν-ε-Boc-Na-CBZ-L-lysine, Ν-εBoc-Na-Fmoc-L-lysine, L-methionine sulfone, L-norleucine, L-norvaline, Na-Boc-N- ÔCBZ-L-ornithine, Ν-δ-Boc-Na-CBZ-L-ornithine, Boc-p-nitroL-phenylalanine, Boc-hydroxyproline, Boc-L-thioproline (Blondelle,

S.E. et al., Antimicrobial Agents and Chemotherapy 38, 22802286 (1994), Pinilla, C. et al., Peptide Science 37, 221-240, 1995).

In a specific embodiment of the present invention, the protein is an antibody or portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells. The antibody is a monoclonal or polyclonal antibody. In a more specific embodiment, the monoclonal antibody specifically binds to an epitope to which the monoclonal antibody 5c8 specifically binds (ATCC Accession No. HB 10916). An example of such a monoclonal antibody is monoclonal antibody 5c8 (ATCC Accession No. HB

10916). In another embodiment, the antibody specifically binds to CD40. An example of an anti-CD40 antibody is a monoclonal mouse anti-human CD40, available from Genzyme Customer Service (Product No. 80-3701-01, Cambridge, MA, USA). In other embodiments of the invention, the monoclonal antibody, chimeric antibody, primatized antibody, humanized antibody, or antibody that comprises one human CDR and the other human antibody backbone.

The terms chimeric, primatized, and humanized antibody, and methods for making such antibodies, are well known to those skilled in the art. See e.g. WO 90/07861, published Jul. 26, 1990 (Queen et al.) And Queen et al., Proc. Natl. Acad. Sci. USA 86: 10029 (1989). Methods for preparing primatized antibodies are described, for example, in PCT International Application No. 2002/5 / EC. WO. / 02108, corresponding to international application no. PCT / US92 / 06194 (Idec Pharmaceuticals) and Newman et al. (Biotechnology 10, 1455-1460, 1992), which are incorporated herein by reference.

Generally, a humanized antibody is an antibody comprising one or more complementarity determining regions (CDRs) from a non-human antibody that are operably linked to human antibody framework (framework) segments. Optionally, additional residues associated with an antibody of non-human origin may be present. Typically, non-human CDRs are murine CDRs. Generally, a primatized antibody is an antibody comprising one or more complementarity determining regions (CDRs) from an antibody of a non-primate species that is operably linked to a non-human primate framework antibody segment. Optionally, additional residues associated with the species of CDR may be present. Typically, the at least one heavy chain or one antibody light chain comprises CDRs of a species that is not a non-human primate. Typically, the CDRs are human CDRs. Generally, a chimeric antibody is an antibody whose light or heavy chains contain regions of antibodies from different species. For example, one or more variable (V) region segments of one species may be linked to one or more constant (C) region segments of other species. Typically, the chimeric antibody comprises mouse variable region segments linked to human constant region segments, although antibodies from other mammalian species may also be used.

Monoclonal antibody 5c8 is produced by hybridoma cells that were deposited on November 14, 1991 at the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, USA under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose patent management. The hybridoma was assigned ATCC accession number HB 10916.

In a specific embodiment, the antibody portion comprises a complementarity determining region or a light or heavy chain variable region. In another specific embodiment, the antibody portion comprises a Fab or a single chain antibody. A single chain antibody is made up of variable regions linked by protein linkers into a single protein chain.

In another embodiment of the present invention, the protein comprises a soluble extracellular region of CD40 ligand, or a portion thereof, capable of inhibiting the interaction between CD40 ligand and CD40 on cells, or a soluble extracellular region of CD40 or a portion or variant thereof, capable of inhibiting the interaction between CD40 ligand and CD40 on cells. In a specific embodiment of the invention, the soluble extracellular region of the CD40 ligand or CD40 is a monomer. In another embodiment, the soluble extracellular region of the CD40 oligomer is.

Variants may differ from the naturally occurring CD40 or CD40 ligand in amino acid sequence or non-sequence-related methods or both. Variants in an amino acid sequence are formed when one or more amino acids in a naturally occurring CD40 or CD40 ligand are replaced by a different natural amino acid, amino acid derivative, or non-naturally occurring amino acid. Particularly preferred variants include, in particular, naturally occurring CD40 or CD40 ligand, or biologically active fragments of naturally occurring CD40 or CD40 ligand, whose sequences differ from the wild-type sequence by substitution of one or more conserved amino acids, which typically have minimal effect on secondary structure and hydrophobic the nature of the protein or peptide. Variants may also have sequences that differ by substitution of one or more non-conservative amino acids, deletions, or insertions that do not interfere with the biological activity of CD40 or CD40 ligand. Conservative substitutions typically include substitutions of one amino acid by another with similar properties to those in the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. Non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. Positively charged (basic) amino acids include arginine, lysine and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Other conservative substitutions that can be used are shown in Table 2. 1 and others are described by Dayhoff in Atlas of Protein Sequence and Structure (1988).

Table 1

Replacements of conservative amino acids

amino acid code can be replaced by any of: alanine A D-Ala, Gly, beta-Ala, L-Cys, and D-Cys arginine R D-Arg, Lys, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn asparagine A D-Asn, Asp, D-Asp, Glu, D-Glu, Gln, D-Gln Aspartic acid D D-Asp, D-Asn, Asn, Glu, D-Glu, Gln, D-Gln cysteine C D-Cys, S-Me-Cys, Met, D-Met, Thr, DThr glutamine Q D-Gln, Asn D-Asn, Glu, D-Glu, Asp, DAsp Glutamic acid E D-Glu, D-Asp, Asp. Asn, D-Asn, Gln, D-Gln glycine G Ala, D-Ala, Pro, D-Pro, Beta-Ala, Acp isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met leucine L D-Leu, Val, D-Val, Met, D-Met lysine The D-Lys, Arg, D-Arg, homo-Arg, D-homoArg, Met, D-Met, Ile, D-Ile, Orn, DOrn methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, Dleu, Val, D-Val, Norleu phenylalanine F D-Phe, Tyr, D-Thr, L-Dopa, His, DHis, Trp, D-Trp, Trans 3,4 or 5-phenylproline, Cis 3, 4 or 5- -phenylproline proline P D-Pro, L-1-thioazolidine-4-carboxylic acid, D- or L-1-0xazolidine-4-carboxylic acid serine WITH D-Ser, Thr, D-Thr. Allo-Thr, Met, DMet. Met (O), D-Met (0), Val, D-Val threonine T D-Thr, D-Ser, Al-Thr, Met, DMet, Met (O), D-Met (0), Val, D-Val tyrosine Y D-Tyr, Phe, D-Phe, L-Dopa, His, DH 18 valine in D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

Other variants of the compound of the invention are those modifications that enhance peptide stability. These variants may contain, for example, one or more non-peptide bonds (which replace peptide bonds) in the peptide sequence. Also included are variants that contain residues other than naturally occurring L-amino acids, such as Damino acids or non-naturally occurring or synthetic amino acids, such as β- or γ-amino acids, and cyclic variants. Incorporation of the D-site and L-amino acids into a polypeptide may increase its resistance to proteases. See e.g. U.S. Patent 5,219,990.

The peptides of the invention may also be modified by various changes, such as insertions, deletions, and substitutions, either conservatively or non-conservatively, where such changes may provide certain advantages in use.

In other embodiments, variants with amino acid substitutions that are less conservative can also cause the desired derivatives, e.g. causing changes in charge, conformation, and other biological properties. Such substitutions would include, for example, substitution of a hydrophilic residue by a hydrophobic, substitution of a cysteine or proline residue by another residue, substitution of a residue having a small side chain by a residue having a large side chain, or substitution of a residue having a net positive charge by a residue having a net negative charge. If the outcome of a given substitution cannot be predicted with certainty, the derivatives can be easily tested according to the methods described in this application to determine whether or not they have the desired properties.

Variants within the scope of the invention include proteins and peptides with an amino acid sequence having at least 80% homology and extracellular regions of CD40 or extracellular regions of CD40 ligand. More preferably, the sequence homology is at least 90% or even more preferably at least 95%.

As the backbone substituents can be replaced, functional groups that extend the scaffold can also be substituted, in particular by groups characterized by similar properties. These substitutions will initially be conservative, i. the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequential modifications may include, for example, in vivo or in vitro chemical derivation of a portion of naturally occurring CD40 or CD40 ligand, as well as changes in acetylation, methylation, phosphorylation, carboxylation, or glycosylation.

In another embodiment, the protein comprising the extracellular region of CD40 and CD40 ligand is modified by chemical modifications in which its activity is maintained. For example, proteins may be amidated, sulfated, singly or multiply halogenated, alkylated, carboxylated, or phosphorylated. The protein may also be singly or multiply acylated, such as with an acetyl group, a farnesyl group, or a fatty acid, which may be saturated or mono- or polyunsaturated. The fatty acid may also be fluorinated singly or multiply. The invention also encompasses methionine analogs of the protein, such as methionine sulfone and sulfoxide analogs. The invention also encompasses protein salts such as ammonium salts, including alkylammonium or arylammonium salts, and salts such as sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, etonate, mesylate.

The soluble monomeric CD40-L protein may comprise all or part of the extracellular region of CD40-L. The extra-cellular region of CD40-L contains a domain that binds to CD40. Thus, soluble CD40-L can inhibit the interaction between CD40L and the CD40-bearing cell. The present invention contemplates that SCD40-L (soluble CD40-L) may comprise the entire extracellular region of CD40-L or a fragment or derivative comprising a domain that binds CD40.

The soluble CD40 protein (sCD40) contains the extracellular region of CD40. sCD40 inhibits the interaction between CD40L and CD40-bearing cells. The sCD40 may be in monomeric or oligomeric form.

In another embodiment of the present invention, the protein comprising the soluble extracellular region of CD40 or portion thereof further comprises an Fc region fused to the extracellular region of CD40 or portion thereof. In a specific embodiment, the Fc region is capable of binding to protein A or protein G. In another embodiment, the Fc region comprises IgG, IgG 1, IgG ₂ , IgG 3, IgG ₄ , IgA, AgA 1, IgA ₂ , IgM, IgD, or IgE.

A soluble CD40 / Fc fusion protein can be prepared using conventional enzymatic cleavage and ligation techniques with the desired sequences. Suitable Fc regions for the fusion protein are Fc regions that can bind to protein A or protein G, or are capable of being recognized by an antibody that can be used in the purification or detection of a fusion protein comprising an Fc region. For example, the Fc region may comprise a human IgGx or murine IgG 1 region. The present invention also provides a nucleic acid molecule that encodes a CD40 / Fc fusion protein.

A method for preparing soluble forms of membrane molecules by recombinant techniques in which sequences encoding the transmembrane and cytoplasmic domains are deleted is well known. See generally Hammonds et al., U.S. Pat. No. 5,057,417. In addition, methods for preparing the sCD40 and CD40 / Fc fusion proteins are known. See e.g. PCT International Application no. WO93 / 08207 a

also Fanslow et al Soluble I ľorms o f CD40 Inhibit biology Responses of Human Cells, J. Immunol. volume 149, ss. 655- 60, July 1992. In an embodiment present invention is a agent selected test method. In the specific embodiments invention is a agent selected

an assay method comprising isolating a sample of cells, culturing the sample under conditions permitting activation of CD40-bearing cells, contacting the sample with cells expressing a protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having accession no. ATCC HB 10916 or with a protein that is specifically recognized by monoclonal antibody 5c8 produced by a hybridoma having ATCC HB 10916 accession number, effective to activate CD40-bearing cells, contacting the sample with an amount of the composition effective to inhibit CD40-bearing cell activation if the composition is capable of inhibiting CD40-bearing cells and determining whether cells expressing a protein that is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having accession no. ATCC HB10916 or with a protein that is specifically recognized by monoclonal antibody 5c8 produced by a hybridoma having accession no. ATCC HB 10916, activate CD40-bearing cells in the presence of the preparation. The cell pattern can be isolated from various tissues, including cell lines in tissue cultures, or cells isolated from an animal, such as scattered solid tissue cells, cells derived from a bone marrow biopsy, or cells isolated from a body fluid such as blood or lymphatic cells. fluid.

In another specific embodiment of the invention, the composition (molecule) is selected based on the three-dimensional structure of the soluble extracellular region of CD40 ligand or a portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells. The composition may be selected from a library of known compositions, modified from a known composition based on a three-dimensional structure, or designed and synthesized de novo based on a three-dimensional structure In specific embodiments of the invention, the composition (molecule) is designed by structurally optimizing the base inhibitor leader based on the three-dimensional structure of the complex a soluble extracellular region of CD40 ligand, or portions thereof, with a basic inhibitor. The basic inhibitory preparation is a molecule that is known to bind and form a complex with the soluble extracellular region of CD40 ligand, CD40, or a portion thereof, when in contact with the CD40 ligand, thereby reducing the ability of the complex or bound ligand or portion of CD40 ligand to activate purified. diffractive cells carrying CD40. In another embodiment of the invention, the base inhibitory agent may act through interaction with either the extracellular region of CD40 ligand, CD40 or in a tertiary complex with both a portion of CD40 ligand and CD40 thereby reducing the ability of the CD40-CD40 ligand complex to activate CD40-bearing cells. In the methods of the present invention, the CD40 ligand may be either soluble or cell bound, such as activated T cells, and may be either a full-length native CD40 ligand or portions thereof. The decreased ability to activate CD40-bearing cells can be measured in various ways. One way that can be measured is to demonstrate that CD40 ligand in the presence of an inhibitor causes a lesser degree of activation of CD40-bearing cells as compared to the effect of the amount of CD40 ligand on non-inhibitor cells under similar conditions. A reduced ability to activate CD40-bearing cells may also be indicated by a higher concentration of the CD440 ligand inhibitor complex that is required to produce a similar degree of activation of CD40-bearing cells as compared to unbound CD40 ligand under similar conditions. As a last resort, the CD40 ligand may be contacted by an inhibitor unable to activate CD40-bearing cells at concentrations and conditions that allow the activation of these cells not bound by the ligand or a portion thereof.

The composition (molecule) can be selected by a computational assay method based on the crystalline structure of the soluble fragment of the extracellular domain of human CD40L containing residues Glyn6L®U26i (SEQ ID NO .: 1) (sCD40L 116-261).

The crystalline structure suitable for use in testing was determined by a 2 Ä resolution by the molecular replacement method. In short, the point is a soluble fragment of the extracellular domain of human CD40 ligand containing amino acid residues Glyue to the residue Leu 6 R ₂ at the C-terminal portion, was first produced in soluble form, and then crystallized. The crystals were used to collect the data. Molecular replacement and refinement were performed with the XPLOR and QUANTA programs (Molecular Simulations, Inc.). Specifically, a three-dimensional model of human SCD40L was constructed using a QUANTA program modeling protein homology. This model was used as a probe for calculating crystallographic analysis and refined using

XPLOR. This method of determining the crystalline structure of SCD40L is described in more detail in Karpusas et al., 2 A crystal structure of an extracellular fragment of human CD40 ligand, Structure, 3 (10), 1031-1039, October 1995. The atomic coordinates of sCD40L (116216) are in 2A-Y. The test method for selecting the formulation includes computational design of the formulation and repeated optimization of the structure as described below.

The composition may be an inhibitor selected using a computational design of the composition. Using this method, the coordinates of the crystal structure of sCD40L are used as an input for a computer program such as DOCK, which outputs a list of molecular structures in which binding to CD40L is expected. The use of such computer programs is well known to those skilled in the art. See e.g. Kuntz, Structure-Based Strategies for Drug Design and Discovery, Science, volume 257, p. 1078, 1992. The list of molecular structures can then be tested by biochemical assays for CD40L binding. Competitive type biochemical assays well known in the art can be used. See e.g. Bajorath et al., Identical of residues of CD40 and its ligand which are critical for receptor-ligand interaction, Biochemistry, 34, p. Thus, structures found to bind CD40L can be used as the compositions of the present invention. The composition can also be modified or a proposed CD40L inhibitor molecule, found from using the above computational approach or another approach, co-crystallized with SCD40L, and the crystalline structure of the complex can be solved by replacing the molecules. The information obtained through molecular replacement can be used to optimize the structure of the inhibitor so as to elucidate how molecules interact with CD40L. The molecule can be modified to improve its physicochemical properties, including specificity and affinity for CD40L.

In an embodiment of the invention, the composition is a small molecule. A small molecule in this disclosure is a compound having a molecular weight of between 20 Da to 1x10 ⁶ Da, preferably from 50 Da to 2 kDa.

The present invention also provides a method for inactivating CD40-bearing smooth muscle cells on the cell surface of a CD40 ligand in a patient, the method comprising administering to the patient a composition capable of inhibiting the interaction between CD40 ligand and CD40 on cells. activation of patient cells.

In a specific embodiment of the invention, the smooth muscle cells carrying CD40 are bladder smooth muscle cells, vascular smooth muscle cells, bronchial smooth muscle cells, aortic smooth muscle cells, coronary artery smooth muscle cells, lung vein smooth muscle cells or gastrointestinal smooth muscle cells. In more specific embodiments of the invention, the smooth muscle cells, gastrointestinal tract are the esophagus, stomach or intestine smooth muscle cells, including the small or large intestine smooth muscle cells.

In an embodiment of the present invention, the composition inhibits the binding of CD40 ligand to CD40 on cells.

In an embodiment of the present invention, the composition is a protein. In another embodiment of the invention, the composition is of a non-protein nature.

In a specific embodiment, the protein comprises an antibody or portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells. The antibody is monoclonal or polyclonal. In a more specific embodiment of the invention, the monoclonal antibody specifically binds to an epitope to which the monoclonal antibody 5c8 specifically binds (ATCC Accession No. HB 10916). An example of such a monoclonal antibody is monoclonal antibody 5c8 (ATCC Accession No. HB 10916). In other embodiments, the monoclonal antibody is a chimeric or humanized antibody.

In a specific embodiment, the antibody portion comprises a complementarity determining region or a light or heavy chain variable region. In another specific embodiment, the antibody portion comprises a complementarity determining region or a variable region. In another specific embodiment, the antibody portion comprises a Fab or single chain antibody.

In another embodiment of the invention, the protein comprises a soluble extracellular region of CD40 ligand or a portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells, or a soluble extracellular region of CD40 or a portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells. In a specific embodiment of the invention, the soluble extracellular region of the CD40 ligand or CD40 monomer is. In another embodiment, the soluble extracellular region of CD40 is an oligomer.

In another embodiment of the present invention, the protein comprising the soluble extracellular region of CD40 or portion thereof further comprises an Fc region fused to the extracellular region of CD40 or portion thereof. In a specific embodiment of the invention, the Fc region is capable of binding to protein A or protein G. In another embodiment, the Fc region comprises IgG, IgG 1, IgG 2, IgG 3, IgG ₄ , IgA, IgA 1, IgA 2, IgM, IgD or IgE.

When administered, proteins are often rapidly removed from the circulation and can therefore produce only a relatively short-term pharmacological effect. Consequently, frequent injections of active proteins may be required to maintain the therapeutic effect. By the addition of water in polyethylene glycol, polypropylene glycol, relatively large doses of modified soluble polymers proteins, polyethylene glycol and carboxylmethylcellulose colloopolymers, dextran, biocovalent such as polyvinyl alcohol, polyvinylpyrrolidone or polyproline having a substantially long-term injection time proteins (Abuchowski et al., in: Enzymes as Drugs, Holcenberg et al., eds. Wiley-Interscience, New York, NY, 367-383, 1981, Anderson, WF, Human Gene Therapy, Science, 256, 808-813 , 1992, Newmark et al., J. Appl. Biochem. 4, 185-189, 1982 and Katre et al., Proc. Natl. Acad. Sci. USA 84, 1487-1491, 1987). Such modifications may also increase the solubility of the protein in aqueous solutions, eliminate aggregation, increase the physical and chemical stability of the protein, and significantly reduce the immunogenic and antigenic action of the protein. As a result, such polymeric protein additive compounds achieve the desired biological activity in vivo by less frequent administration or at lower dosages than unmodified protein.

Attachment of polyethylene glycol (PEG) to proteins is particularly useful because PEG is very toxic to mammals (Carpenter et al., 1971). For example, the PEGadenosine deaminase additive compound has been approved in the United States for use in humans for the treatment of severe combined immunodeficient syndrome. A second advantage provided by PEG conjugation is that it effectively reduces the immunogenic and antigenic action of heterologous proteins. For example, the PEG-human protein addition compound can be used to treat the disease in other mammalian species without the risk of eliciting a serious immune response. In one embodiment of the invention, the protein may be administered in a microencapsulation device to reduce the risk or prevent a host immune response against the protein. The protein may also be administered in a membrane micro-capsule such as a liposome.

Polymers such as PEG can conveniently be attached to one or more reactive amino acid residues in the protein, such as the α-amino group of the amino acid of the N-terminal moiety, ε-amino groups of lysine side chains, sulfhydryl groups of cysteine side chains, carboxyl groups of asparagyl and glutams. side chains, α-carboxyl group of the C-terminal amino acid, tyrosine side chains or activated glycosyl chain derivatives linked to certain asparagine, serine or threonine residues.

A number of activated forms of PEG suitable for direct reaction with proteins have been described. Useful PEG reagents for reaction with protein amino groups include active carboxylic acid esters or carbonate derivatives, particularly those in which the starting groups are N-hydroxylsuccinimide, pnitrophenol, imidazole or 1-hydrozy-2-nitrobenzene-4-sulfonate. PEG derivatives containing maleimide or haloacetyl groups are useful reagents for modifying protein free sulfhydryl groups. Similarly, PEG reagents containing aminohydrazine or hydrazine groups are useful for the reaction with aldehydes formed by periodate oxidation of carbonate groups in proteins.

The subject that can be treated by the methods described is an animal. Suitably, the animal is a mammal. Examples of mammals that can be treated include, but are not limited to, non-human primates, rodents (including rats, mice, hamsters, and guinea pigs), cows, horses, sheep, goats, pigs, dogs, and cats.

In an embodiment of the invention, the formulation is selected by a test method.

In a specific embodiment of the invention, the composition is selected by an assay method comprising isolating a cell sample, culturing the sample under conditions permitting activation of CD40-bearing cells, contacting the sample with cells expressing a protein that is specifically recognized by monoclonal antibody 5c8 produced by hybridoma having accession no. ATCC HB 10916 or with a protein that is specifically recognized by monoclonal antibody 5c8 produced by a hybridoma having accession no. ATCC HB 10916, efficiently activating CD40-bearing cells, contacting the sample with an amount of the composition effective to inhibit the activation of CD40-bearing cells if the composition is capable of inhibiting CD40-bearing cells activation and determining whether cells expressing a protein specifically recognized by monoclonal antibody 5c8 produced by a hybridoma no. ATCC HB 10916, activate CD40-bearing cells in the presence of the preparation. A sample of cells may be isolated from various tissues, including cell lines in tissue cultures or cells isolated from an animal, such as scattered solid tissue cells, cells derived from a bone marrow biopsy, or cells isolated from a body fluid such as blood or lymphatic fluid.

In another specific embodiment of the invention, the molecule (preparation) is selected based on the three-dimensional structure of the soluble extracellular region of CD40 ligand or a portion thereof capable of inhibiting the interaction between CD40 ligand and CD40 on cells. The molecule can be selected from a library of known molecules, modified from a known molecule based on a three-dimensional structure, or designed and synthesized de novo based on a three-dimensional structure. In specific embodiments of the invention, the composition or molecule is designed by structurally optimizing the base inhibitor composition based on the three-dimensional structure of the soluble extracellular region of the CD40 ligand soluble portion or portion thereof with the base inhibitor composition.

Method of treatment

The present invention provides a method of treating a smooth muscle cell dependent disease in a subject. The method of treatment comprises the above-described method for inhibiting CD40 ligand-bearing smooth muscle cell activation on its surface by CD40 ligand, comprising administering a composition capable of inhibiting the interaction between CD40 ligand and CD40 on cells of a subject, wherein the composition is administered in an amount effective to inhibit cell activation in the subject.

In an embodiment of the invention, the smooth muscle cell dependent disease is a vascular disease. In a specific embodiment of the invention, the vascular disease is atherosclerosis.

In another embodiment of the invention, the smooth muscle cell-dependent disease is a gastrointestinal tract disease. In a specific embodiment of the invention, the gastrointestinal tract disease is selected from the group consisting of esophageal motility disorders, inflammatory bowel disease and scleroderma.

In an embodiment of the invention, the smooth muscle cell-dependent disease is bladder disease.

The compounds of the present invention may be administered by any route that is medically acceptable. This may include parenteral injection such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intraperitoneal, intraventricular, or others, as well as oral, nasal, ophthalmic, topical, or inhalation. Sustained release administration is also specifically included in the invention by methods such as depot injections of disintegrating implants applied directly during surgery.

intratumoral, intraepidural

The compounds are administered at any dose per kg of body weight and at any dosage frequency that is medically acceptable. Acceptable dosages include the range between 0.01 to 200 mg / kg of patient body weight. A preferred dosage range is between 0.1 to 50 mg / kg. Particularly preferred is a dose of between about 1 to 30 mg / kg. Dosage is repeated at intervals ranging from every day to every other month. One preferred dosing regimen is to administer a compound of the invention daily for three days of treatment, after which the compound is administered every three weeks, each administration being intravenously at a dose of 5 or 10 mg / kg body weight. Another preferred regimen is to administer the compound of the invention intravenously daily at a dose of 5 mg / kg body weight for the first three days of treatment, after which the compound is administered subcutaneously or intra-muscularly every week at a dose of 10 mg. Another preferred regimen is to administer a single dose of a compound of the invention parenterally at a dose of 20 mg / kg body weight, followed by administering the compound to a subject subcutaneously or intraneally culinary every week at a dose of 10 mg.

The compounds of the invention may be administered as a single dose for certain indications, such as preventing an immune response to an antigen to which a subject is exposed for a short time, e.g. exogenous antigen, administered one day of treatment. Examples of antigens would include co-administration of a compound of the invention together with a gene therapy vector or therapeutic agent, such as an antigenic pharmaceutical or blood product. In indications where the antigen is present chronically, such as control of the immune response to the transplanted tissue or chronically administered antigenic drugs, the compounds of the invention are administered at intervals as long as medically indicated over a period of days or weeks to the end of life.

Inflammatory responses are characterized by redness, swelling by temperature and pain as a consequence of capillary dilation with swelling and migration of phagocytic leukocytes. Inflammation is further defined by Gallin (Chapter 26, Fundamental Immunology, 2nd Ed., Raven Press, New York, pp. 721-733, 1989), which is incorporated by reference.

For a better understanding of the invention, a number of experimental details are set forth in the following examples. However, it will be apparent to those skilled in the art that the specific methods and results presented herein are merely illustrative of the invention, which is fully described in the claims that follow.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A: Flow cytometry (FACS) analysis of human aortic smooth muscle resting cells. The dotted line represents the isotype control antibody, the dashed line represents the anti-CD54 mAb, and the solid line represents the antiCD40 mAb. This figure shows that smooth muscle cells do not express constitutive CD40.

Figure 1B: FACS analysis of human aortic smooth muscle cells in the presence of IFN-γ (1000 U / cm ³ ) after 72 hours of tissue culture. This figure shows activation of CD40 expression by smooth muscle cells in response to IFN-γ.

Figure 1C: FACS analysis of human aortic smooth muscle cells in the presence of IL-1α (1 ng / cm ³ ) after 72 hours of tissue culture. Activation of smooth muscle cell CD40 expression was not observed.

Figure 1D: FACS analysis of human aortic smooth muscle cells in the presence of TNF-α (200 U / cm ³ ) after 72 hours of tissue culture. Activation of smooth muscle cell CD40 expression was not observed.

Figures 2A to 2Y: Coordinates of the atomic crystalline structure of the soluble extracellular fragment of human CD40L containing Glyn6-Leu26i residues (in Brookhaven Protein Data Bank format (see SEQ ID NO .: 1)).

Figures 3A and 3B: CD40 is expressed in situ on smooth muscle cells and macrophages in atherosclerosis lesions after transplantation. Shown are photomicrographs of two-color immunohistochemical studies showing CD40 expression (brown label) on smooth muscle cells (red label) in Figure 3A and macrophages (red label) in Figure 3B in a patient with atherosclerosis after transplantation.

Figures 4A and 4B: Normal coronary artery of a patient with idiopathic cardiomyopathy stained with hematoxylin and eosin (Fig. 4A) and anti-CD40 mAb (Fig. 4B).

Fig. 4A: It is worth noting the absence of thickening of the intima or inflammatory infiltrate.

Fig. 4B .: CD40 expression is restricted to endothelial cells flanking the vascular lumen. There was no reactivity with the isotype specific control mAb (not shown) (Fig. 4A, Fig. 4B x25).

Figures 5A and 5B: Fibroatheromatous plaques in the coronary artery of a patient with ischemic cardiomyopathy stained with hematoxylin and eosin (Fig. 5A) and anti-CD40 mAb (Fig. 5B).

Figures 6A to 6C: Early intimal foam cell-rich lesion in a patient with coronary artery disease (TCAD) stained with hematoxylin and eosin (Fig. 6A) and anti-CD40 mAb (Fig. 6B, Fig. 6C).

Fig. 6A: Intimal lesion of numerous foam cells, macrophages and smooth muscle cells.

Fig. 6B: CD40 is strongly expressed on many intimal cells in this early lesion in TCAD.

Fig. 6C: particular foam cells showed rich labeling on CD40 (Fig. 6A x25, Fig. 6B x50, Fig. 6C x400).

Figures 7A-7D: Inflammatory infiltrate present in the intimal lesion fibrous cover of native CA labeled anti-CD40L mAb (Figure 7A), control mAb (Figure 7B), anti CD4 mAb (Figure 7C), and anti-CD8 mAb (Figure 7A). 7D).

Fig. 7A: Characteristic immunoreactivity and cell surface immunoreactivity for CD40L that is restricted to lymphocytes.

Fig. 7B: The same lesion labeled with the control mAb of the irrelevant consensus isotype had no immunological labeling.

Fig. 7C: Virtually all lymphocytes in native CA lesions (as well as many macrophages and foam cells) were CD4 +, suggesting that CD40L ⁺ lymphocytes are CD4 ⁺ T cells.

Fig. 7D: CD8 ⁺ T cells are unique in native CA plaques (Fig. 7A, 7B x 1000, Fig. 7C, 7D x400).

Figures 8A and 8B: Deep intimal lymphoid aggregates in TCAD labeled anti-CD40L mAb (Figure 8A), control mAb (Figure 8A) and anti-CD4 mAb (Figure 8C).

Fig. 8A: Most CD40L ⁺ cells in TCAD (arrows) were found in lymphoid aggregates in the intime and far from the endothelial surface.

Fig. 8B: In these intimal lymphoid aggregates, the control mAb of the irrelevant consensus isotype showed no cell labeling.

Fig. 8C: The same intimal lymphoid aggregates as above contained almost exclusively CD4 ⁺ T cells, suggesting that CD40L is expressed on CD4 ⁺ T cells in these lesions (Fig. 8A8C x400).

Figures 9A and 9B: Endothelial inflammation site (endothelitis) in TCAD labeled anti-CD8 mAb (Fig. 9A) and anti-CD40L (Fig. 9B).

Fig. 9A: CD8 ⁺ T cells attached to lumina endothelial cells in TCAD characteristic of endothelial inflammation. Most of the CD8 ⁺ T cells were present in endothelial inflammation foci, while they were isolated in intimal lymphoid aggregates far from the endothelial surface.

Fig. 9B: inflammatory cells in the endothelial inflammation foci are CD40L ⁺ . Similarly, CD40L expression was not detected on endothelial cells (Fig. 9A-9B x400).

Figures 10A and 10B:

Fig. 10A: double immunological labeling of intimal lesion in native CA with mAb anti-CD40 (brown) and mAb anti-CD68 (red), a marker for macrophages. The central cell cluster (arrows) shows strong labeling for CD40 and CD68.

Fig. 10B: TCAD double immunostaining with antiCD40 mAb (brown) and smooth muscle actin (red) shows CD40 ⁺ smooth muscle cells (arrows). The reactivity of CD40 is restricted to intimal smooth muscle cells (arrows) while the median myocytes were CD40 (Fig. 10A-B x400).

Figures 11A-11D: Serial sections of native CA demonstrating intimal non-inurization and labeled with anti-CD34 mAb (Figure 11A), anti-CD40 (Figure 11B), anti-ICAM-1 (Figure 11C) and anti-VCAM-1 (Fig. 11D).

Fig. 11A: Endothelial cells of newly formed intimal blood vessels highlighted by CD34 labeling.

Fig. 11B: Intimal neovascularization endothelial cells strongly express CD40. Adjacent inflammatory cells are also labeled with CD40.

Fig. 11C and 11D: Neovascularization foci also show potent endothelial reactivity for ICAM-1 (Fig. 11C) and VCAM-1 (Fig. 11D) (Fig. 11A-11D x400).

Figures 12A to 12C: Double immunostaining of an actively inflamed intimal lesion of native CA with anti-CD40 mAb (brown) and anti-adhesive molecules (red) anti-ICAM-1 (Fig. 12A), anti-VCAM-1 (Fig. 12B) ) and irrelevant control mAb (Fig. 12C).

Fig. 12A: Essentially all CD40 ⁺ (brown) cells, predominantly macrophages (long arrows) and intima myocytes (short arrows) are reactive to ICAM-1 (red).

Fig. 12B: A large number of CD40 ⁺ (brown) inflammatory cells and intima myocytes (arrows) are also reactive to VCAM-1 (red).

Fig. 12C: Same intimal double-labeled CD40 (brown) and irrelevant isotype matched Ab control control replacing anti-ICAM-1 and anti-VCAM-1 mAbs (red). Only brown and not red markings can be recognized, indicating the absence of interference detection technique for sequentially applied mAbs anti-CD40 and anti-ICAM or anti-VCAM (see Material and Methods for more details) (Figs. 12A-Cx400).

Figure 13: Double immunostaining of native CA intimal lesion with anti-p65 mAb labeling activated NF-κΒ (brown) and CD40 (red). Activated NF-κΒ was recognized exclusively in the nuclei of CD40 + cells (arrows), most of which are macrophages (x400).

DETAILED DESCRIPTION OF THE INVENTION

Examples 1 and 2

Examples 1 and 2 below demonstrate that inflammatory cytokines induce CD40 expression in smooth muscle cells. In addition, they demonstrate that CD40L-mediated signals regulate smooth muscle function.

Example 1

FACS analysis was used to determine whether smooth muscle cells express CD40. Human aortic smooth muscle cells were cultured in 6-well plates in M199 nutrient medium supplemented with 25% FCS, 5% human serum, 90 pg / ml heparin, 15 gg / ml endothelial cell growth factor, and 1% penicillinstreptomycin. Media was changed every 2-3 days and when cells were near confluency, they were grown for 72 hours in the presence or absence of IFN-γ (1000 U / cm ³ ), IL-1α (1 ng / cm ³ ) or TNF-α (200 µg / cm ³ ). U / cm ³ ). Cells were harvested by trypsin-EDTA treatment and CD40 expression was determined by FACS analysis with anti-CD40 mAb G28.5. Cells were also labeled with an isotype negative control mAb and anti-CD54 mAb (ICAM-1) was used as a positive control.

Smooth muscle cells do not express constitutive CD40 as shown in Figure 1A. However, IFN-γ, in contrast to IL-γ or

TNF-α induces expression of CD40 by smooth muscle cells (Figures ΙΑ, 1B and 1C). These studies demonstrate that IFN-γ induces CD40 expression on human aortic smooth muscle cells.

Example 2

Expression of CD40 on a smooth muscle cell was examined in situ. Cells found in normal vascular medium that are morphologically similar to smooth muscle cells do not react with anti-CD40 mAb. However, cells that morphologically resemble smooth muscle cells found in inflammatory lesions in accelerated atherosclerosis after transplantation express CD40 in situ. These studies suggest that inflammatory cytokines induce smooth muscle cells to express CD40. In addition, these studies demonstrate that GD40L-mediated signals regulate smooth muscle function.

Example 3

CD40L * CD4 ⁺ cells and CD40 ⁺ target cells are present in atherosclerosis and coronary heart disease after heart transplantation.

Activated endothelial cells (EC), macrophages (Mac) and CD4 ⁺ T cells are present in lesions of coronary atherosclerosis (CA) and atherosclerosis after heart transplantation (TA). Because CD40L is an activation-induced CD4 ⁺ T cell surface molecule that transmits CD40 ⁺ contact-dependent activation signals to target cells, including EC (induced expression of ICAM, VCAM and E-selectin) and Mac (induces NO, TNF-α and IL- 1), we investigated the in situ expression of CD40L and CD40 in CA (n = 5) and TA (n = 5). CD40L and CD40 expression was determined using anti-CD40L 5c8 mAb, anti-CD40 G28.5 mAb, or appropriate control mAbs. Frozen sections of normal coronary arteries (n = 3) do not contain T cells and CD40 expression is restricted to EC. In contrast, CA and TA-associated lesions contain CD40L ⁺ CD4 ⁺ T cells as determined by immuno-tagging of serial sections. In addition, CD40 expression on frozen sections from CA and TA patients is markedly on EC, infiltrating mononuclear cells, foam cells, and intimal smooth muscle cells (SMC). Two-color immunohistochemical analysis (ie with double labeling) of paraffin-fixed tissue using specific markers for SMC (smooth muscle actin) or Mac (HAM-56) confirmed the expression of CD40 on these cells and SMC media are CD40, confirming that local inflammatory mediators induce CD40 expression on SMC in vivo. Activation of CD40 and CD40L * CD4 ⁺ T cells was detected at all stages of TA and was most visible in early lesions of CA, including lipid bands. In summary, these studies confirm that CD40L ⁺ T cells can interact with CD40 ⁺ target cells in CA and TA and contribute to the pathogenesis of these diseases by promoting the formation of anti-inflammatory molecules.

Example 4

CD40 is expressed on smooth muscle cells and macrophages in lesions of atherosclerosis after transplantation

In situ expression of CD40 in native atherosclerosis or atherosclerosis after transplantation was studied by two-color immunohistochemical analysis. Double immunohistochemical labeling studies were performed on coronary arteries that were fixed in 10% buffered formalin and paraffin-embedded. Paraffin was removed from the sections by xylene, the sections were hydrated, and the endogenous peroxidase was quenched with 1.5% H 2 O 2 in 80% alcohol. The sections were then treated with 0.01% pepsin in HCl (pH 1.5) for 15 minutes at 37 ° C. Then, sections were rinsed with PBS and incubated with 10% horse serum for 20 minutes to block non-specific labeling. Anti-CD40 labeling was then detected using the Vector ABC Elite Kit (Vector) using sequentially a biotinylated secondary antibody, avidin peroxidase complex and as a development to 3,3'-diaminobenzidine. The occurrence of CD40 was observed as a brown label. Then the sections were rinsed with PBS and again blocked with 10% horse serum. Sections were then incubated for 1 hour with mAb specific for smooth muscle cells (smooth muscle actin) or for macrophages (HAM 56). Primary antibodies were then conjugated to alkaline phosphatase using the avidin-biotin system (Vector). The Red vector (Vector) was used to detect alkaline phosphatase activity and the reaction gave a red label. Therefore, double-labeled cells had brown (CD40) and red (smooth muscle cells or macrophages) labeling. To control interference between the two immunohistohemical procedures used for double-label analysis, serial sections of each sample were also labeled either with CD40 smooth muscle actin or HAM 56. See Figures 3A and 3B. Control sections had the same immunoreactivity distribution for each of the primary mAbs as the double-labeled sections.

Example 5

Distribution of CD40L and CD40 in native coronary atherosclerosis and coronary artery disease after heart transplantation: correlation of CD40 expression with the occurrence of intercellular adhesion molecules, activated NF-κΒ and the presence of T lymphocytes.

T cells play a role in the pathogenesis of native coronary atherosclerosis (CA) and coronary artery disease (TCAD), but the mechanism by which T cells interact with other cells in these lesions is not fully understood. CD40L is an activation-induced CD4 ⁺ T cell surface molecule that interacts with CD40 + target cells, including macrophages and endothelial cells, and induces proinflammatory molecule formation, including ICAM-1 and VCAM-1. In addition, CD40 binding is known to activate the transcription factor NF-κΒ. To determine whether CD40L-CD40 interactions can play a role in the pathogenesis of CA or TCAD, an immunohistochemical study of CD40L and CD40 expression was performed on frozen sections of coronary arteries obtained from recipients of cardiac allograft with CA (n = 10) or TCAD (n = 9). . Using two different anti-CD40L mAbs, it was found that CD40L expression was restricted to infiltrating lymphocytes in CA and TCAD. CD40 expression was significantly activated on intimal endothelial cells, foam cells, macrophages and smooth muscle cells in both diseases. Double immunostaining demonstrated that many CD40 ⁺ cells co-expressed ICAM-1, VCAM-1, or an activated form of NF-κΒ. The extent of expression of CD40, ICAM-1 and VCAM-1 had a statistically significant correlation with disease severity and intimal lymphocyte counts. These studies collectively demonstrate the presence of activated CD40L * and CD40 ⁺ cells in both lesions, CA and TCAD, and suggest that CD40L mediated interaction with CD40 ⁺ macrophages, foam cells, smooth muscle cells, or or

endothelial cells may contribute to the pathogenesis of these diseases.

Several lines of evidence suggest that cell-mediated immune mechanisms contribute to inflammatory lesions (1-4) characteristic of native coronary atherosclerosis (CA) (510) and ischemic heart disease after heart transplantation (TCAD (11-13)). express activation markers such as CD25 and MHC class II molecules are present early in the development of vascular lesions in both diseases (5, 14) Activated macrophages are generally found in lesions in both diseases, as well as cytokines associated with a> immune-dependent response on T cells, including IFN-γ, IL-1 and

TNF-α (5-17). As further evidence that T cells can play a pathogenic role in CA, CD4 ⁺ T cell clones were isolated from human fibroateromatous plaques in CA that proliferate and secrete IFN-γ when brought into contact with oxidized LDL (18), mainly component of lesions in both diseases, native CA and TCAD (1, 19, 20). In addition, hyperlipidemia-induced atherosclerotic lesions are reduced in mice treated with anti-CD4 mAb (21). Similarly, vascular lesions in TCAD are significantly improved when allografts are transplanted or treated with anti-CD413 or anti-IFN-γ mAbs by a strain of T-cell genetically deficient mice (13) (22). Taken together, these data strongly suggest that T cells as well as T cell-derived effector molecules are involved in the pathogenesis of these diseases (9, 23, 24).

CD40L is a surface molecule with a molecular weight of 30-33 kDa expressed on activated CD4 ⁺ T cells, which delivers contact-dependent signals to CD40 ⁺ target cells when B cells (25-29). CD40L mediated signals are critically important in the development of T cell-dependent humoral immune responses in vitro and in vivo (30). It is not known whether CD40L-CD40 interactions also play a role in cell-mediated in vitro and in vivo immune responses (31, 32). Interestingly, macrophages and endothelial cells, cell types known to be involved in the pathogenesis of CA and TCAD, also express CD40 (33-37). In addition, binding of CD40 to macrophages and endothelial cells in vitro induces the production of molecules that enhance immune responses or have pro-inflammatory effects. For example, CD40L-CD40 interactions induce expression of an MHC II molecule. class and common stimulating molecules CD86 on in vitro macrophages (.38). In addition, binding of CD40 to macrophages induces the production of cytokines (TNF-α, IL-β, IL-12), chemokines (IL-8, β-la), nitric oxide (NO) via induction of NO-synthase 2, procoagulant protein tissue factor and intercellular mass metalloproteinases (33, 34, 39-42). CD40L-CD40 interactions activate intercellular adhesion molecules CD54 (ICAM-1), CD106 (VCAM-1) and CD62E (E-selectin) on endothelial cells (35-37). Many of the effects of CD40 binding depend on activation of the transcription factor NF-? B (43-45).

These findings suggest that CD40 binding to a variety of target cells may potentiate CD4 ⁺ T cells in vivo. This hypothesis in the kidneys of nephropathy patients

IgA and inflammatory cells with a mediated inflammatory response promote activation of CD40 expression by lupoid glomerulonephritis, glomerulonephritis ANCA ⁺ Tav skin of patients with renal disease (46). Since the interaction of T by macrophages, endothelial cells and possibly other cells plays a role in the pathogenesis of CA and TCAD, the present study investigates the expression of CD40L and CD40 in these two diseases using immunohistochemical methods. CD401 is expressed on T cells and CD40 expression is activated on endothelial cells, smooth muscle cells, macrophages, and foam cells in intimal lesions in both diseases. In addition, using double labeling, it was found that many CD40 + cells in these lesions co-express CD54, CD106, and an activated form of NF-κB.

Description of the methods used

Human coronary arteries

Segments of the left coronary artery or the proximal part of the racket interventricularis anterior were obtained from the explanted hearts of 23 allograft recipients. Nine patients underwent retransplantation because they developed severe coronary heart disease after transplantation of the heart (TCAD). The survival of the first allograft in these patients ranged between 38 and 103 months. Ten patients received cardiac allografts because they developed severe ischemic heart disease and ischemic cardiomyopathy. Control coronary arteries without atherosclerotic changes were obtained from explanted hearts of 4 patients, 3 had idiopathic cardiomyopathy and one heart sarcoma. Portions of each vessel were snap frozen in isopentane at 80 ° C and serial sections 4 mm thick were cut on a cryostate (Reichert Histostat). Sections were mounted on sialin-coated slides, air dried, fixed for 1 minute in cold acetone, for additional 7 minutes in cold acetone / chloroform 1: 1 and stored at -80 ° C. One section from each coronary artery was fixed in 10% formalin and stained with hematoxylin and eosin for histological evaluation.

Primary antibodies

Anti-CD40 from G28.5 hybridoma (IgG1) was obtained from the American Type Culture Collection, Rockville, MD. The anti-CD40L 5c8 mAb (IgG2a) was prepared as described (28). Both mAbs G28.5 5c8 were purified from ascites using a protein g column (Pharmacia, Piscataway, NJ). Another anti-CD40L mAb (IgG1) was obtained from Calbiochem (San Diego, CA). An IgM anti-CD40 mAb was obtained from Caltag (Burlingame, CA) and a double immuno-labeled study was used. Monoclonal Ab against CD3, CD4, CD8, CD34, CD68 (Novocastra, Burlingham, CA, all IgG1) and smooth muscle cell actin (SMA) (DAK, Carpinteria, CA, IgG2a) were used to distinguish different cell types in intimal plaques , including T cells (CD3, CD4 or CD8), endothelial cells (CD34), macrophages (CD68) and smooth muscle cells (SMA). Anti-ICAM-1 (IgG1) and anti-VCAM-1 (IgG1) mAbs were obtained from CHEMICON _™ (Temecula, CA). Distribution of activated NF-κΒ was detected by mAb p65 (IgG3 (BOEHRINGER MANNHEIM ™), which binds to an epitope of the p65 subunit NF-κΒ blocked by IκB, this epitope is only accessible if NF-κΒ is activated by dissociation of IκB (47). control mAb (Mopec 21, 22) was obtained from SIGMA ™ (St. Louis, MO).

Immunohistochemical method

The frozen sections were washed in phosphate buffered saline (PBS) and the endogenous peroxidase was quenched with 0.5% hydrogen peroxide. Sections were blocked with 10% goat and aggregated human Ig (80 mg / ml) in PBS and then incubated for one hour with the indicated primary mAb or the corresponding control mAb. Frozen tonsil sections with follicular hyperplasia were used as positive controls to determine the optimal dilution of each mAb. The primary mAb bound to the target gene was linked to a biotin labeled, isotype specific goat anti-mouse IgG1, IgG2a, IgG3 or IgM (Fisher Scientific, Pittsburg, PA). Peroxidase activity was detected with chromogen (red) with 3-amino-9-ethylcarbazole (AEC, VECTOR ™, Burlingham, CA) and sections were counterstained with Mayer's hematoxylin (SIGMA ™, St. Louis, MO).

Double-labeled immunohistochemistry was used to identify CD40-expressing cell types and to analyze CD40 distribution in relation to ICAM-1, VCAM-1 or activated NF-κ-in atherosclerotic lesions. All sections were immunologically labeled with anti-CD40 mAb IgM first. Secondary Ab was biotinylated with goat anti-mouse IgM, which was then conjugated to an avidin-biotin-peroxidase complex. The chromogen used to detect the occurrence of anti-CD40 IgM mAbs was 3,3 'diaminobenzidine (brown). Sections were then washed extensively and incubated with a second primary mAb that labeled either a cell-specific smooth muscle cell marker (SMA) or macrophages (CD68), leukocyte adhesion molecules (ICAM-1, VCAM-1), or an activated form of NF-κΒ. . All of these second primary mAbs were isotype IgG1, IgG2a or IgG3. Appropriate isotype specific biotinylated secondary antibody and conjugated to avidin-biotin-alkaline phosphatase complex (VECTOR ™, Burlingham, CA) was applied. Intereference between sequentially applied labeling procedures was avoided by using various immunoenzyme techniques (peroxidase and alkaline phosphatase) and an isotype specific secondary Ab for each cannon antigen. In addition, double-labeled control sections were prepared in which one of the two primary mAbs was replaced with an isotype-matched control mAb.

Semiquantitative analysis of lesions

The extent of atherosclerotic lesions was quantified in each section by the degree of vascular lumen narrowing in the range of 0 to 4, in which 0 meant no narrowing, 1 less than 25%, 2 less than 50%, 3 less than 90% and 4 over 90% . Each coronary artery lesion was also evaluated for macrophage, smooth muscle cells, endothelial cells (neovascularization) (48) indicating the absence of the respective cell type, 1 isolated cells, 2 small sets of cells, 3 occurrence of bearing densities, and 4 densities present in the densities. the whole plate.

intimal foam cells, and T cells when 0

Similarly, the incidence of CD40, ICAM-1 and VCAM-1 was scored in the range of 0 to 4 when 0 was the absence of the molecule, 1 was found on isolated cells, 2 was less than 50%, 3 was less than 90% and 4 was more than 90% of all cells (49). Since the expression of CD40L in positive samples was limited to isolated cells, its incidence was not available for quantitative evaluation.

Statistical analysis

Differences in histological evaluation between groups of samples were analyzed using a non-parametric Kruskal-Wallis test. The dependence between variables was determined using Spearman correlation.

The results

Normal coronary arteries

Coronary artery segments of control patients showed no intimal thickening or inflammation as evidenced by hematoxylin and eosin staining (Figures 4A-4B). Specifically, macrophages, smooth muscle cells, foam cells or lymphocytes were not present in the intime and the cells were not immunoreactive with either of the anti-CD40L mAbs used in this study. CD40 immunoreactivity was present and confined to endothelial cells flanking the lumen of control arteries (Fig. 4B). Control vessel cells did not express VCAM-1 or activated NF-κΒ, and rarely vascular endothelial cells weakly expressed ICAM-1.

Histology of native CA and TCAD

In 7 out of 10 patients with CA, protruding fibroatheromatous plaques with eccentric constriction, a cell-free lipid-rich center, cholesterol slots, and an overlapping fibrous cover were found in the coronary artery segments.

The lesion cell lesion was greatest in the shoulder regions containing macrophages and lymphocytes (Fig. 5A). Smooth muscle cells, macrophages, foam cells, and foci of neovascularization were also disseminated in intimal lesions. Plates from 3 patients with mild, early vascular lesions were eccentric, small, rich in macrophages, foam cells and lymphocytes.

Coronary artery lesions in 9 patients had intimal circumferential thickening with marked luminal constriction (see Table 2).

Table 2

Semi-quantitative (range 0-4) evaluation of cell composition of intimal lesion in native coronary atherosclerosis (CA) and coronary artery disease after cardiac transplantation (TCAD) and immunoreactivity for CD40, ICAM-1 and VCAM-1. Values are expressed as mean + standard deviation.

1 Intimate plate inspection (N = 4) CA (n = 9) TCAD (n = 8) thickness 0.3 ± 0.5 2.1 ± 0.9 * 3.1 ± 0.8 * CD4 ⁺ lymphocytes 0 1.3 ± 0.9 * 3.2 ± 0.8 * CD8 ⁺ lymphocytes 0 0.3 ± 0.5 2.6 ± 1.1 * Macrophages (CD68) 0.5 ± 0.6 2.1 ± 0.8 * 3.8 ± 0.4 * Foam cells 0 1.2 ± 0.8 * 2.4 ± 1.3 * Smooth cells muscle 0.8 ± 1 1.7 ± 0.7 2.9 ± 0.8 * neovascularization 0 1.8 ± 0.7 * 2.6 ± 0.9 * CD40 0.5 ± 0.6 2.2 ± 0.7 * 3.3 ± 0.9 * 1ICAM-1 0.5 ± 0.6 2.3 ± 1.7 * 3.6 ± 0.7 * 1VCAM-1 0.3 ± 0.5 1.7 ± 0.7 * 2.9 ± 0.9 *

* p 0.05 for CA or TCAD versus control Kruskal-Wallis tests

The lesions were composed of concentric layers of smooth muscle cells and intercellular matter and there was excessive infiltration of macrophages and lymphocytes along with areas of neovascularization. In 4 coronary arteries, atheromatous lipid-rich lesions and foam cells were recognized beside the concentric layers of smooth muscle cells (Figs. 6A-C). Subendothelial lymphocyte accumulation (endothelitis) and lymphocyte aggregates in adventitions were also characteristic features of TCAD lesions.

Immunohistochemical analysis of CA and TCAD expression

A significant difference from normal coronary arteries that are free of infiltrating lymphocytes or CD40L expressing cells is that both lesions, CA and TCAD, contain CD40L ⁺ cells. In native atherosclerosis, CD40L-positive immuno-labeling was restricted to a minority of intima lymphocytes. CD40L labeling was usually weak and was observed either in small cytoplasmic granules or on the cell surface (Fig. 7A-D). In native CA, most intimal lymphocytes were CD4 ⁺ T cells, CD8 ⁺ T cells were rarely present (Fig. 7A-D). Analysis of serial sections labeled with anti-CD4 or anti-CD8 mAb demonstrates that CD40L ⁺ lymphocytes were primarily CD4 ⁺ T cells. Endothelial cells, smooth muscle cells, macrophages, and foam cells did not react with any anti-CD40L mAb used in this study. No labeling was observed for isotype control mAbs.

In TCAD lesions, positive immunostaining for CD40L was also associated exclusively with lymphocytes (Figs. 8A-C). In contrast to CA, both CD8 ⁺ and CD4 ⁺ T cells were present in TCAD lesions. However, CD8 + T cells were found predominantly in subendothelial regions of endothelitis (Figs. 8A-C), while CD4 + T cells were localized in aggregates deeper in the intima adjacent to the inner elastic membrane (Figs. 8A-C) and adventitia of coronary arteries. CD40L expression correlated spatially with CD4 ⁺ T cells in intime and adventitia of coronary arteries in TCAD. The number of CD40L ⁺ T cells was higher in TCAD lesions than in native CA lesions. Similar to CA, endothelial cells, smooth muscle cells, macrophages, or foam cells in TCAD lesions did not react with any antiCD40L mAb used in this study (Figs. 9A-B). These data suggest that CD40L express cells, presumably CD4 ⁺ T cells, are present in lesions of native CA and TCAD.

Immunohistochemical analysis of CD40 expression in CA and TCAD

In contrast to poor expression of CD40 restricted to luminal endothelial cells in normal coronary arteries (Figs. 4A-B), CD40 immunoreactivity was activated and widespread in lesions of native CA (Figs. 5A-B). CD40 expression was recorded in endothelial cells, smooth muscle cells, macrophages and foam cells. In intimal lesions of native CA, the mean number of CD40 positive cells was significantly higher than in control arteries (2.2 + 0.7 versus 0.5 + 0.6, Table 2). Double immunostaining with specific markers for macrophages or smooth muscle cells confirmed that these cells and foam cells of both lines express CD40 (Fig. 10AB). Interestingly, CD40 ⁺ smooth muscle cells were present in the intime near inflammatory infiltrates until smooth muscle cells in the artery medium had positive immunoreactivity for CD40 (Figs. 10A-B). Analysis of serial sections labeled with CD40 or endothelial marker CD34 suggests that endothelial cells lining newly formed vessels of intima and vassa vasorum adventicie were also strongly CD40 ⁺ (Figs. 11A-D).

In the arteries of TACD patients, the model of CD40 expression distribution was similar to that of native CA. However, the average CD40 immunoreactivity score was significantly higher in TCAD than in native CA or control arteries (Table 2). Double immuno-labeling showed that intimal smooth muscle cells and macrophages express CD40 (Figs. 10A-B). In addition, the foam cells (Fig. 6AB) and endothelial cells flanking the vascular lumen, the newly formed vessels of the intima and your adventitial vasorum were markedly CD40 ⁺ . Together, these data demonstrate that endothelial cells, smooth muscle cells, and macrophages express CD40 in both native CA and TCAD.

Relationship between CD40 expression and intercellular adhesive molecules and NF-κΒ activation in CA and TCAD lesions.

Macrophages and endothelial cells in CA and TCAD express intercellular adhesive molecules that regulate the pathway of leukocytes into lesions. Since CD40 binding induces the activation of intercellular adhesive molecules and activation of NF-κΒ on cells in vitro, the question was whether CD40 expression was associated with co-expression of intercellular adhesive molecules or NF-κΒ in CA or TCAD lesions. Initially, native CA showed that lumina endothelial cells exhibit foci positive immunological labeling for ICAM-1 with isolated endothelial cells expressing VCAM-1. In contrast, endothelial cells delimit the newly formed vessels of the intima and your adventicia vasorum was strongly positive for ICAM-1 and VCAM-1 (Fig. 11A-D). Intimal smooth muscle cells, macrophages, and foam cells were also mild to strong positive for ICAM-1 and VCAM-1 (Figs. 12A-C). There was a significant correlation (p <0.05) between CD40 and ICAM-1 (r = 0.85) and VCAM-1 (r = 0.72). Intimal lymphocyte counts correlated significantly with the scores for CD40 and leukocyte adhesion molecules (see Table 3).

Table 3

Correlation of scores (0-4) for different cell types of intimal lesions in CA (n = 10) or TCAD (n = 9) with scores (04) for expression of CD40 and adhesive molecules (ICAM-1, VCAM-1). Values are expressed as a Spearman correlation coefficient (range -1 to 1, with 0 being no correlation and -1 or 1 perfect correlation).

Cell type Group CD40 ICAM-1 VCAM-1 T lymphocytes (CD4 ⁺ CD8 ⁺ ) CA 0.78 * 0.77 * 0.83 ** TCAD 0.79 * 0.87 * 0.77 * Macrophages (CD68 ⁺ ) CA 0.93 *** 0.84 ** 0.77 * TCAD 0.81 ** 0, 68 * 0.55 Foam cells CA 0.81 ** 0.68 * 0.36 TCAD 0.44 0.33 0.26 Smooth Muscle Cells (SMA ⁺ ) CA 0.72 * 0.81 ** 0.56 TCAD 0.12 0.38 0.02 Newly Opened Vessels (CD34 ⁺ ) CA 0.69 * 0.72 * 0.53 TCAD 0.85 ** 0.87 ** 0.77 * J

* ρ <0.05, ** ρ <0.01 and *** ρ <0.001 - significance level for

Spearman correlation

Of all these cell types, only intimal lymphocyte assessments significantly correlated with CD40 expression, and the extent of ICAM-1 and VCAM-1 in intimal plaques in both CA and TCAD indicated that lymphocytes are involved in the induction of CD40 and adhesion molecules in both diseases. . Macrophages and neovascularization also showed significant correlation with CD40 expression in CA and TCAD.

Double immunostaining of CA lesions with anti-CD40 and anti-ICAM-1 or anti-VCAM-1 mAbs showed that CD40 was localized along with these adhesion molecules on many cells (Figs. 12A-C). In addition, activated NF-κΒ was observed in nuclei of neointimal endothelial cells, macrophages, and smooth muscle cells (Fig. 13) and double immuno-labeling showed that many CD40 + cells also expressed activated NFκB.

In TCAD, a strong positive immunostaining for ICAM-1 and VCAM-1 was present in lumina endothelial cells, particularly those close to the endothelial foci. The endothelial cells of your intimal newly formed vasculature of your adventitia vasorum were strongly immunoreactive to ICAM-1 and VCAM-1. The evaluation of immunologic labeling of adhesive molecules in TCAD was higher than in CA or normal coronary arteries (Table 2). Significant correlation (p <0.05) was between CD40 and ICAM-1 (r = 0.82) and VCAM-1 (r = 0.89). Intimal lymphocyte counts also correlated significantly with the expression of CD40, ICAM-1, VCAM-1 (Table 3). Similar to CA, two-color immunohistochemical studies have shown that many CD40 ⁺ cells co-express ICAM-1 or VCAM-1 in TCAD (Fig. 12A-C). Immunologic labeling for the activated nuclear form of NF-κΒ was more widely spread in TCAD than in native CA. NF-κΒ positive macrophages and smooth muscle cells were also CD40 ⁺ (Fig. 13). Together, these studies demonstrate that in lesions of both native CA and TCAD, CD40 is co-expressed on many cells with intercellular adhesion molecules or NF-κB.

Discussion

Native atherosclerosis (CA) and transplant atherosclerosis (TCAD) are inflammatory diseases mediated by complex interactions between activated T cells, endothelial cells, macrophages, and smooth muscle cells (2, 8, 12, 13, 17). T cells are thought to play a role in the pathogenesis of CA and TCAD, but the mechanism by which they are involved in these processes is not fully understood (5, 9, 50). Studies have shown that CD40L, a CD4 + T cell surface molecule induced by activation, provides contact-dependent activation signals to endothelial cells and macrophages expressing CD40, resulting in the formation of pro-inflammatory molecules such as intercellular adhesion molecules ICAM-1 and VCAM-1 (31, 32, 35-37) and activation of transcriptional activating factor NF-κΒ (43-45, in vitro). It is interesting that

TCAD in mouse models is at least partially dependent on CD40L-CD40 interactions (51). In a study by Larson et al. anti-CD40L mAb treatment significantly inhibited allogeneic heterotopic transplant rejection and partially blocked associated vasculopathy. In addition, TCAD in this model was almost completely prevented by administration of a combination of anti-CD40L mAb and CTLA4-Ig fusion protein, a molecule that blocks co-stimulating T cell pathways (51). It is possible that CD40LCD40 interactions may be involved in the pathogenesis of CA or TCAD in humans.

To further investigate this hypothesis, other immunohistochemical techniques were used for normal and atherosclerotic coronary arteries to study the expression and cellular distribution of CD40L and CD40. Normal coronary arteries do not contain CD40L expressing cells and CD40 immunoreactivity in these vessels has been restricted to lumina endothelial cells. In contrast, CD40L is expressed on lymphocytes in lesions of both native CA and TCAD. CA lesions were found to contain few CD8 ⁺ T cells, while TCAD lesions contained CD8 ⁺ T cells in close proximity to the lumina endothelium (endothelitis) and CD4 ⁺ T cells deeper in intime and adventure. By localizing and labeling serial sections of anti-CD4 or anti-CD8 mAb, it was concluded that CD40L ⁺ lymphocytes are the most likely CD4 ⁺ T cells in lesions in both diseases. Using two different anti-CD40L mAbs, it was found that CD40L immunoreactivity was weak and was not associated with either granules or cytoplasm or cell surface. A similar model of CD40L immunoreactivity was observed in a study of CD40L and CD40 expression in glomerulonephritis (46). The weak and often cytoplasmic labeling pattern of CD40 expression in inflammatory tissues may relate to the transient nature of CD40 expression on activated T cells (27-29) and the fact that CD40L occupation on target cells induces rapid attenuation of CD40L by receptor-mediated endocytosis ( 52) and receptor depletion (53). These regulatory mechanisms are likely to direct CD40L-mediated signals to the respective target cells recognized.

CD40 expression was found to be markedly activated on many cells in lesions in both diseases. Macrophages and foam cells expressing CD40 were particularly noticeable in the inflammatory infiltrate of the lipid-rich plaque region of the plaques known to contain dense inflammatory infiltrates (54, 55). CD40 expression was also activated on lumina endothelial cells in both diseases, which was particularly noticeable in TCAD. The endothelial cells of the newly formed intimal blood vessels and your adventitial vasorum were strongly CD40 ⁺ in both diseases. Smooth muscle cells expressing CD40 occurred in the intime of both CA and TCAD, usually in close proximity to inflammatory infiltrates. Interestingly, smooth muscle cells in the same vessel medium were evaluated as CD40. IFN-γ induced CD40 expression on many cells in vitro (33, 35-37, 56), including smooth muscle cells, and this effect is enhanced by cytokines such as IL-β and TNF-α (36). Therefore, marked activation of CD40 expression on many cell types in these lesions can cause cytokine release by T cells, macrophages, and other lesion cells. Double immunostaining showed that many CD40 + cells also simultaneously express intercellular adhesion molecules ICAM-1 and VCAM-1 as well as an activated form of NF-κΒ. The present study also demonstrates the presence of CD40L ⁺ T cells and activated CD40 ⁺ target cells in vascular lesions of native CA and TCAD.

Previous studies have shown that CD40 was expressed on some epithelial cell and B cell tumors (57, 58).

Later, it has been reported that CD40 is constitutively expressed or inducible on many cell types in vitro (33-37, 56). Furthermore, it is increasingly apparent that CD40L-CD40 interactions play a key role in cell-mediated inflammatory responses in vivo (31, 32). In this regard, recent publications demonstrate in situ CD40L or CD40 expression in human inflammatory diseases (35, 46, 59). For example, CD40 expression is activated on the brain infiltrating macrophages of patients with multiple sclerosis (59), on skin endothelial cells and keratinocytes in psoriasis (35), and on many kidney cells of inflammatory glomerulonephritis patients (46). In addition, inflammatory infiltrates in the brains of multiple sclerosis patients (59) and in the kidneys of patients with inflammatory glomerulonephritis (46) contain CD40L * T cells. It is therefore likely that CD40 expression is activated in many inflammatory diseases and is a molecular mechanism that allows T cells to transmit pro-inflammatory signals to a variety of target cells. In this regard, the findings presented herein mean that CD40 expression is activated in CA and TCAD and that CD40L ⁺ infiltrating T cells are found in lesions, serve as evidence of the hypothesis that immune-mediated inflammatory responses play a role in the pathogenesis of these diseases (5-7, 9, 18, 21, 23, 50).

Observations regarding CD40L-mediated activation of endothelial cells and macrophages in vitro and studies of CD40L-CD40 interactions in the pathogenesis of TCAD in murine models suggest a possible pathogenic role of CD40L-CD40 interactions in CA and TCAD. For example, CD40L mediated signals activate the expression of ICAM-1 and VCAM-1 on endothelial cells, in vitro (35-37). These intercellular adhesion molecules that regulate leukocyte departure and retention at inflammatory sites are activated on endothelial cells of the newly formed intimal vessel and your vasorum (49, 60). Therefore, it is interesting that many CD40 + cells, and in particular endothelial cells of the intima and your vasorum, that express ICAM-1 or VCAM-1 at the same time, have been found in CA and TCAD lesions. The activation of ICAM-1 and VCAM-1 is known to be dependent on NF-κB activation (61). The present study also demonstrated that CD40 ⁺ intimal macrophages, smooth muscle cells, and endothelial cells express an activated form of NF-κΒ. These studies suggest that CD40L ⁺ CD4 ⁺ T cells can induce the activation of intercellular adhesive molecules on CD40 + target cells in CA and TCAD, in part possible by NF-κB activation.

CD40L-mediated signals also induce IL-6 and IL-8 secretion in endothelial cells (62) and induce a procoagulant surface by activating tissue factor and attenuating thrombodulin expression. Regarding macrophages, CD40LCD40 interactions induce secretion of pro-inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α), chemokines, intercellular mass metalloproteinases in these cells and express tissue factor in vitro (33, 34, 38, 41 , 42). All of these pro-inflammatory molecules are likely to play a role in the pathogenesis of CA and TCAD (10, 17, 63-66).

Binding of CD40 to macrophages also induces NO formation (39, 40). Interestingly, blocking CD40L-CD40 interactions in mouse TCAD models is associated with attenuation of iNOS expression and reduction of TCAD lesions (51). INOS has been shown to be expressed in lesions of CA (67, 68), cardiac allograft rejection (69, 70) and TCAD (71, 68),

72). CD40L mediated signals may be involved in promoting the generation of any of these molecules in CA or TCAD. CD40L-CD40 interactions have clearly pro-inflammatory effects in TCAD mouse models (51) as well as collagen-induced arthritis (73), lupus-like glomerulonephritis (74) and experimental allergic encephalomyelitis (59).

The expression of CD40L and CD40 (62) in carotid atherosclerosis has also been studied in humans. CD40 was found to be activated in the lesions and had extensive cellular distribution. CD40L has been reported to be widely expressed on smooth muscle cells, endothelial cells and macrophages in atherosclerotic lesions, while in the present study using two different anti-CD40L mAbs. CD40L expression was restricted to T cells. No disease was observed in situ expression of CD40L on macrophages, endothelial cells or smooth muscle cells. Similarly, CD40L immunoreactivity has also been found to be restricted to T cells in other inflammatory diseases, including glomerulonephritis (46), rheumatoid arthritis and chronic sinusitis. In addition, Gerrits et al. reported that CD40L expression was restricted to CD4 + T cells in multiple sclerosis lesions (59). The discrepancies between the results presented herein and those of Mach et al. are currently unclear, but may relate to subtle variations in immunohistochemical techniques or in the nature of lesions.

List of references cited

1. Nilsson, J. 1993. Transplant Proc. 25: 2063-2064.

2. Munro, J., and R. Cotran. 1988. Lab. Invesco. 58: 249-261.

3. Cramer, D., et al. 1992. J. Keart Lung Transplant. 11: 458-466.

4. Eillingham, M. , 1992. 11: S38-S44. 5. Zhou, X., et al. 1996th 6. Wick, G., et al. 1995th 7. Stemme, S. et al.

Thrombc. 12: 206-211.

J. Heart Lung Transplant.

Am. J. Páchol. 149: 359-365. Immunol. Today. 16: 27-33. 1992. Arteriosclerosis and

8. Ross, R. 1993. Nature. 362: 801-809.

9. Lichtman, A., et al. 1996. Am. J. Pathol. 149: 351-357.

10. Kishikawa, H., et al. 1993. Virchows Arrch. 423: 433-442.

11th Krensky, A. 1994. Kidney Int. 45: 5QS-56S. 12. Salomon, R. et al. 1,991th Am. J. Pathol. 138: 791 -798. 13. Shi, C., , et al. 1996th Proc. Natl. Acad. Sci. USA.

: 4051-4056.

14. Jonasson, L., et al. 1985th J Clin Invest 76: 125-131. 15 Dec Russell, P.S., et al. 1994th Am. J. Pathol 144 : 260-274. 16. Moyer, C., et al. 1992. J Pathol. 138: 951-960. 17th Libby, P. and Z. Gallis . 1995th Ann NY Acad. Sci

748: 158-168.

18. Stemme, S., et al. 1995. Proc. Natl. Acad. Sci., USA, 52: 3893-3897.

19.. Witzxum. J. and D. Steinberg. 1991. J. Clin. Invest., 8S: 1785-1792.

20. de Loraeril, M.

125 -.974-980.

et al.

1993. Am. Heart

Zl. Emescn, E., et al. 1995. Am J Pachcl. 149: 57 = -585.

22. Russeii, P-, et al. 1994. Transplar.cacicn. 57: 1357-1371.

23. Russell; M., et al. 1996. J Clin Invesc. 97: 833-838.

24. Hancock, W., et al. 1996. Proc Nad Acad Sci. 93: 13967-13972.

25. Graf, D., et al. Eur J Immunol. 22: 3191-3194.

26. Armicage, R.J., et al. 1992. Nacure. 357 (€ 373) -80-2.

27. Lane, P., et al. Eur J Immunol. 22: 2573-2578.

28. Lederman, S., (4): 1091-101. ec al. , 1992. J Exp Med. 175 29. Noelle, R. ec al. 1992 . Why NaCl Acad Sci USA. 89: 6550-6554. Banchereau, J. . ec al. 1994. Annu. Rev. Immunol.

12: 881-922.

31. Noelle, R. 1995. Immunicy. 4: 415-419.

32. Scouc, R., and J. Succles. 1996. Immunol Today.

17: 487-492.

33. Alderson, M.R., et al. 1993. J. Exp. Med. 178

12): 669-74.

34. Caux, C., et al. 1994. J. Exp. Med. 180: 1263-1272. 3Ξ. Hcllenbaugh, D., et al. 1995. J. Exp. Med. 182 -.33-40.

35. Karmann, K., et al. 199Ξ. Proc Natl Acad Sci, USA. 92: 4342-4346.

37. Yellin, M.J., et al. 1995. 'J. Exp. Med.

182: 1857-1864.

38. Kiener, et al. 1995. J. Immunol. 155: 4917-4925. 35. Scouc, R., et al. 1996. J. Immunol. 156: 5-11.

40. Tian, L., et al. 1995. Eur J. Immunol. 25: 306-305.

41. Pradier, 0th , et al. 1996 FROM · M · 3048-3054. 42. Malik, N. , et al. 1996. J Immu: 43. T I., et al. 1 = 3 : 4357-4366. 44. Hess et al. 1555. J lacnun: • 45. Karmanr. X .. et al. 1555. ΞΞ · 45. Yellin, M .. et al. 1557th • 40: 124-134. 47th Br and K. . et al. 1996. J Clin

1994 xmmunoj.

Immunol.

-4555.

48. Kuamamoto, M., et al. 1995. Human Path. 26: 450-456.

49. O'Brien, K., et al. 1996. Circulation. 93: 672-682.

50. Haraoka, S., et al. 1995. Virchows Arrch. 426: 307-315.

.

Larsen, C., et al. 1996. Nature. 381: 434-438.

52nd Yellin, M. J .. et al. 1994. J Imrnunol (2) : 598-608 .. 53. Graf, D., et al. 1995th Eur J Imrnunol. 25: 1745 54. Bjoerkerud. with., and B. Bjoerkerud. 1996th Pathol. 149: 367-380. 55. van der Wal, A., et al . 1994. Circulation. 89: 56. Yellin, M. J., et al. 1995. J. Leuk. 58: 209-216.

57. Pauli, S., et al. 1985. Cancer Imrnunol. Immunother. 20: 23-28.

58. Clark, E.A., and J.A. Ledbetter. 1986. Proc. Natl. Acad. Sci. USA. 83: 4494-4498.

59. Gerritse, K., et al. 1996. Proc. Natl. Acad. Sci. 93 -.2495-2504.

60. Davies, M., et al. 1993. J Pichol. 171: 223-229.

Collins, T., et al. 1995. FASZB J. 9: 899-909.

Mach. F., et al. 1997. Proc Natl Acad Sci, USA.

SI € Z

94: 1921-1936.

63. Berliner, J., et al.

91: 2488-2495.

64. Libbv, et al. 1995. J. Cardiovasc Pharx. 25 Suppl 2: S9-12.

1995.

Circulaticn.

Li, Z., et al. 1996. Am J Park. 148: 121-126.

Galis, Z., et al. 1994. J Clin Invest. 94: 2493-2503. Buttery, L., et al. 1996. Lafc. Invest. 75: 77-85. Aji, W., et al. 1997. Circulation. 95: 430-427. Yang, X., et al. 1994. J Clir. Invest. 94: 714-721. Wcrrali, N., et al. 1995. J Exp Med. 181: 63-70. Russell, M., et al. 1995. Circulation. 92: 457-464.

5.

.

.

.

.

.

71st

72. Akyurek,

149: 1891-1990.

.

.

et al

1996.

am

Pathol.

Durie, F. E., et al. 1993. Science. 261: 1328-1330. Mohan, C., et al. J Immunol. 154: 1470-1480.

Sequence Listing (2) Sequence Information SEQ ID NO: 1:

(i) sequence characteristics:

(A) length: 146 amino acids (B) type: amino acid (D) topology: linear (ii) molecule type: protein (iii) hypothetical: no (xi) sequence description SEQ ID NO .: 1:

ramparts Asp gin same time for 5 GLR. ill Ala Ala Kis 1C wall ill Ser Glu Ala 15 Ser Ser Lys Thr Thr 20 Ser wall Leu GLR. Tra * 25 Ala Glu Lys Gly Tyr 3 * 0 Tyr Thr Met Ser same time 35 same time Leu wall Thr Leu 40 Glu same time Gly Lys gin 45 Leu Thr wall Lys Arg 50 gin Gly Leu Tyr Tyr 55 Ile Tyr Ala gin wall 60 Thr Phe cys Ser same time 65 Arg Glu Ala Ser Ser 70 GLR. Ala for Phe Ile 75 Ala Ser Leu cys Leu 80 Lys Ser for Gly Arg 85 Phe Glu Arg Ile Leu 90 Leu Arg Ala Ala same time 95 Thr His Ser Ser Ala 100 Lys for Cys Gly gin 105 GLR. Ser Ile His Leu 110 Gly Gly wall PNE Glu 115 Leu gin for Gly Ala 120 Ser wall Phe wall same time 125 wall Thr Asp for Ser 130 gin wall Ser His Gly 135 Thr Gly Phe Thr Ser 140 Phe Gly Leu Leu

Lvs Leu

Claims (21)

Use of an agent that inhibits the interaction between CD40 ligand and CD40, for the preparation of a medicament for inhibiting CD40 cell surface-mediated activation of CD40-bearing cells, wherein the CD40-bearing cells are bladder smooth muscle cells, vascular smooth muscle cells, aortic smooth muscle cells , coronary artery smooth muscle cells, pulmonary vein smooth muscle cells, or gastrointestinal smooth muscle cells. Use according to claim 1, wherein the gastrointestinal smooth muscle cells are esophageal smooth muscle cells, stomach smooth muscle cells, intestinal smooth muscle cells or small intestine smooth muscle cells. The use of claim 1, wherein the agent specifically inhibits binding of CD40 ligand to CD40 smooth muscle cells. The use of claim 1, wherein the agent specifically binds to an epitope to which the monoclonal antibody 5c8 produced by hybridoma ATCC No. 1 binds specifically. HB 10916. The use of claim 1, wherein the agent specifically binds to CD40. The use of claim 1, wherein the agent is selected from the group consisting of proteins, non-proteins and peptidomimetic compounds. The use of claim 6, wherein the protein is an antibody or a portion thereof. The use of claim 7, wherein the antibody is selected from the group consisting of monoclonal antibodies, polyclonal antibodies, chimeric antibodies, humanized antibodies, primatized antibodies, and such antibodies that comprise one human CDR and another human antibody framework. The use of claim 8, wherein the monoclonal antibody is antibody 5c8 produced by the hybridoma ATCC # 1. HB 10916. The use of claim 6, wherein the protein comprises a ‘soluble extracellular region of CD40 ligand or variants thereof including conservative substituents or a portion thereof, or comprises a soluble extracellular region of CD40 or variants thereof including conservative substituents, or a portion thereof. The use of claim 10, wherein the soluble extracellular region of the CD40 or CD40 ligand is a monomer or an oligomer. The use of claim 10, wherein the soluble extracellular region of CD40 or portion thereof further comprises an Fc region fused to the extracellular portion of CD40 or portion or ligand of CD40 or portion thereof. The use of claim 1, wherein the agent is selected or designed by optimizing the structure of the parent inhibitory agent based on the three-dimensional structure of the soluble extracellular region of CD40 or a portion thereof with the parent inhibitory agent. The use of claim 1, wherein the agent specifically inhibits cellular activation by a CD40 ligand of CD40-bearing smooth muscle cells involved in a smooth muscle cell-dependent disease. Use according to claim 14, wherein the smooth muscle cell-dependent disease belongs to the group comprising vascular diseases, bladder diseases and gastrointestinal tract diseases. Use according to claim 15, wherein the gastrointestinal tract disease belongs to the group comprising esophageal motility disorders, inflammatory bowel disease and scleroderma. The use of claim 15, wherein the vascular disease is atherosclerosis. The use according to claim 1, wherein the reagent is selected by a test method comprising performing the following steps: (a) a sample of smooth muscle cells is isolated, which are bladder smooth muscle cells, vascular smooth muscle cells, aortic smooth muscle cells, coronary artery smooth muscle cells, pulmonary vein smooth muscle cells or gastrointestinal smooth muscle cells; (b) the sample is cultured under conditions that allow activation of those smooth muscle cells in the sample carrying CD40; c) contacting the sample with cells expressing a protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma ATCC no. HB 10916, or the sample is contacted with a protein that is specifically recognized by the monoclonal antibody 5c8 produced by hybridoma ATCC No. 5, &lt; tb &gt; HB 10916, wherein said cells or protein effectively activate smooth muscle cells bearing CD40, (d) contacting the sample with an amount of an agent that effectively inhibits CD40-bearing smooth muscle cell activation when said agent inhibits CD40-CD40L dependent activation of CD40-bearing smooth muscle cells; and (e) determining whether cells expressing a protein which is specifically recognized by monoclonal antibody 5c8 produced by hybridoma ATCC no. HB 10916, or whether a protein that is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma ATCC HB no. 10916, activate CD40-bearing smooth muscle cells in the presence of the agent. 19. A method for selecting an agent that inhibits CD40 ligand interaction of smooth muscle cells comprising bladder smooth muscle cells, vascular smooth muscle cells, aortic smooth muscle cells, coronary artery smooth muscle cells, lung vein smooth muscle cells or gastrointestinal smooth muscle cells which carry CD40 on their cell surface characterized in that it consists of steps when it is (a) isolate a sample of smooth muscle cells which are bladder smooth muscle cells, vascular smooth muscle cells, aortic smooth muscle cells, coronary artery smooth muscle cells, pulmonary vein smooth muscle cells or gastrointestinal tract smooth muscle cells; (b) the sample is cultured under conditions that allow the activation of smooth muscle cells in the sample carrying CD40; c) contacting the sample with cells expressing a protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma ATCC no. HB 10916, or the sample is contacted with a protein that is specifically recognized by the monoclonal antibody 5c8 produced by hybridoma ATCC no. HB 10916, wherein said cells or protein effectively activate smooth muscle cells bearing CD40, (d) contacting the sample with an amount of an agent that effectively inhibits the activation of CD40-bearing smooth muscle cells when said agent inhibits CD40-CD40L dependent activation of the CD40-bearing smooth muscle cells; and (e) determine whether cells expressing a protein that is specifically recognized by monoclonal antibody 5c8 produced by hybridoma ATCC no. HB 10916; HB 10916, activate smooth muscle cells bearing CD40 in the presence of the agent. Use according to claim 18, wherein the sample of smooth muscle cells is selected from the group consisting of: cell lines in culture, cells isolated from an animal, cells isolated from solid tissue, cells obtained from a bone marrow biopsy and cells isolated from body fluids. 21. The method of claim 19, wherein the sample of smooth muscle cells is selected from the group consisting of: cell lines in culture, cells isolated from an animal, cells isolated from a solid tissue, cells obtained from a bone marrow biopsy and cells isolated from body fluids.