MXPA98004342A - Therapeutic applications for the monoclonal antibody 5c8 anti-t-bam (cd40 - Google Patents

Abstract

The present invention relates to the activation of cells carrying CD40 on its cell surface by the CD40 ligand is inhibited by contacting the cells with an agent capable of inhibiting the interaction between the CD40 ligand and the cells, in an effective amount to inhibit the activation of cells. Activation of the cells carrying CD40 on its surface by the CD40 ligand in a subject is inhibited by administering to the subject an agent capable of inhibiting the interaction between the CD40 ligand and the cells, in an amount effective to inhibit the activation of the cells. cells The conditions dependent on the activation induced by the CD40 ligand of the cells carrying CD40 are treated

Description

THERAPEUTIC APPLICATIONS FOR THE MONOCLONAL ANTIBODY 5c8 ANTI-T-BAM (CD40-L) This application claims the priority of United States Patent Serial No. 08 / 567,391, filed December 1, 1995, and of United States Patent Serial No. 08 / 566,258, filed December 1, 1995. 1995 and from United States Patent Serial No. 08 / 637,323, filed April 22, 1996, the contents of which are incorporated herein by reference in the present and solie.i. your D. The invention described herein is made with Government support under the NIH Concession Nos. K08-AR-01904, RQ1 -CA5 713, ROI-? 2836-7, ROI-AI-14969, HL21006, HL42833, HL50629, and ROl-AI-14969 of the Department of Health and Human Services. Therefore, the Government of the United States has certain rights in this invention. Throughout this application, various references are mentioned within parentheses. The descriptions of these claims in their entirety are incorporated herein by reference in this application to more fully describe the state of the art to which this invention pertains. The complete bibliographic citation for these references can be found in the text or at the end of this application, preceding the sequence listing and the claims.

BACKGROUND OF THE INVENTION CD40 is a 50 kDa cell surface molecule originally described as expressed on B cells and some epithelial carcinomas (1, 2).

'CD40 interacts with CD40L (T-BAM, gp39, TRAP), a 30 kDa cell surface molecule expressed transiently on activated CD4 + T cells (3-8). The CD40L-CD40 interactions have been extensively studied in the context of T-cell B cell interactions. The binding of CD40 plays key roles in B cell activation, proliferation, differentiation, production and recovery of Ig from apoptotic signals (9-11). The critical role in vivo of the binding of CD40 in the differentiation of dr-B cell is highlighted by the hyper-IgM syndrome, a humoral immunodeficiency due to mutations in the gene coding for CD40L (12-16). The "knockouts" of murine CD40 (17) or CD40L (18) have phenotypes similar to those of patients with hyper-IgM syndrome. Interestingly, recent studies indicate that CD40 expression has a broader cellular distribution than that described 'originally. It has been shown that CD40 is expressed on monocytes (19), dendritic cells (22), epithelium (23, 21), basophils (24), and Hodgkin tumor cells (25). In addition, various cytokines can regulate the expression of CD40 on non-B cells. The expression of CD40 on cells The epithelial tissue is overridden by IL-la, TNF-a or INF-? (2J) INF- ?, in addition to IL-3 or GM-CSF, similarly upregulates the expression of CD40 on monocytes (19). The binding of CD40 in the presence of INF-? and IL-la stimulates the production of GM-CSF by thymic epithelial cells (21). In addition, 'Transfectants that express CD40L induce tumoricidal activity by monocytes and, in the presence of INF- ?, GM-CSF or IL-3, stimulate the onocites to secrete TNF-a, IL-6 or IL-8 (19). CD40 is also expressed on cells found within the synovial membrane (MS) in patients afflicted with rheumatoid arthritis (RA). A histological study of cell surface molecules expressed in SM of RA found that CD40 is expressed in a variety of cell types, including cells with morphology such as fibroblasts (26). In this report, it is shown by FACS analysis that CD40 is expressed on cultured synovial membrane (SM) fibroblasts, isolated from patients with RA, inflammatory arthritis that is not RA (IA) or osteoart itis (OA). In addition, dermal fibroblasts isolated from normal donors also express CD40. In addition, the binding of CD40 by CD40L + cells induces the activation and proliferation of fibroblasts. Endothelial cells express surface molecules, such as CD54 (ICAM-1), CD62E (E-selectin) and CD106 (VCAM-1), which mediate the adhesive interactions with leukocytes (27-35). The expression of adhesion molecules to the endothelial cell surface orchestrates the recruitment of leukocytes to inflammation sites, and is therefore subject to a fairly narrow regulation (27, 28). Resting endothelial cells express low levels of CD54 and very little or no CD62E or CD106. Following activation with IL-1, TNFa, or LPS, endothelial cells rapidly upregulate the expression of CD54, CD62E, and CD106 (27, 28). CD4 + T cells can contribute to upregulation of endothelial cell surface adhesion molecules by inducing endothelial cells or other target cells to secrete IL-1 or TNFa (36). However, the molecular details involved in the interactions of the CD4 + T cell - endothelial cell that induce endothelial cell activation, have not been completely delineated. At present it can be reported that normal human endothelial cells also express CD40 in si u and CD40L-CD40 interactions induce endothelial cell activation in vitro. Frozen sections of normal spleen, thyroid, skin, muscle, kidney, lung or umbilical cord were studied to determine the expression of CD40 by immunohistochemistry. The endothelial cells of all tissues studied express CD40 in situ. In addition, endothelial cells of the human umbilical vein (HUVEC) express CD40 in vitro and rIFN-? induces upregulation of CD40 in HUVEC. The expression of CD40 on HUVEC is functionally significant because the Jurkat CD40L + T cells upregulate the expression of CD54 (ICAM-1), CD62E (E-selectin) and CD106 (VCAM-1) in HUVEC in vitro in a manner inhibited by the mAb 5C8 anti-CD40L. Additionally, the 293 kidney cell transfectants that express CD40L, but not the control transfectants, also up-regulate the expression of CD54, CD62E and CD106 on HUVEC. These results demonstrate that CD40L-CD40 interactions induce endothelial cell activation in vit ro. It is shown for the first time that CD40L expressed on the surface of T cells induces the activation of CD40 + endorheic cells and that this activation is inhibited by an anti-CD40L monoclonal antibody. In addition, these results demonstrate a mechanism by which activated CD4 + T cells increase inflammatory responses in vivo by upregulating the expression of adhesion molecules to the endothelial cell surface.

BRIEF DESCRIPTION OF THE INVENTION This invention provides a method for inhibiting the activation by the CD40 ligand of cells carrying CD40 on the cell surface, comprising contacting the cells with an agent capable of inhibiting the interaction between the CD40 ligand and the cells, n a effective amount to inhibit the activation of cells. This invention provides a method for inhibiting the activation by the CD40 ligand of cells carrying CD40 on the cell surface, in a subject, comprising administering to the subject an agent capable of inhibiting the interaction between the CD40 ligand and the cells, in a effective amount to inhibit the activation of cells in the subject.

DESCRIPTION OF THE FIGURES Figure 1. Expression of CD40 on SM fibroblasts. FACS analyzes of the expression of CD40, CD14, CD45 or MHC Class II are shown, as indicated, on representative adherent SM cells of RA or OA, following the first passage in vitro. The X axis represents the average fluorescence intensity (MFI) and the axis? represents the number of cells. For RA cells, the MFI of CD40 expression or isotype control mAb was 21 and 9, respectively. For OA cells, the MFI of CD40 expression or isotype control mAb was 33 and 9, respectively.

Figure 2. Expression of CD40 on dermal fibroblasts at rest or stimulated with rINF- ?. FACS analyzes of CD40, CD54 or staining control mAb are shown, as indicated, on 3 lines of dermal fibroblasts. The cells were cultured in the presence or absence of rINF-? (1000 U / ml) for 24 hours. SK.l and SK.2 were studied following the second passage and CCD 965 SK was studied following the third passage in the culture. The X axis represents the average fluorescence intensity (MFI) and the Y axis represents the number of cells. The number in the upper right corner of each graph indicates CD40 MFI (subtracted background), Figure 3. Cytokine regulation of CD40 expression in SM fibroblasts. A bar graph is shown that represents the average fluorescence intensity of CD40 (MFI) on a 'line of fibroblasts of SM (0A.3) following the ceculture with rINF-? (1000 U / ml), rIL-la (10 pg / ml), rTNF-a (200 U / ml) or combinations of cytokines, as indicated. The expression of CD40 was determined by FACS analysis and background staining with a control mAb is subtracted from each value. The experiment shown is representative of 3 similar experiments performed.

Figure 4. Effect of the CD40L-CD40 interactions on the expression of CD54 (ICAM-1) of SM fibroblasts. Contour graphics of two colors showing the expression of CD13 are shown (X axis) or the expression of CD54 (Y axis) on fibroblasts IA.l of SM cultured 24 hours with the medium, rINF-? (1000 U / ml), Jurkat B2.7 cells CD40L "or Di.l cells of Jurkat CD40L + in the presence or absence of SC8 anti-CD40L mAb or control mAb P1.17 The number in the upper right corner of each graph represents the average fluorescence intensity of CD54 (MFI). The background MFI of an isotype control mAb is subtracted from each value.

The experiment shown is representative of 3 similar experiments performed.

Figure 5. Transfection of CD40L confers the ability to upregulate CD54 expression of SM fibroblasts (ICAM-1) and CD106 (VCAM-1). Bar graphs indicating the MFI of CD54 or CD106 on SM fibroblasts are shown following a culture for 24 with the medium, Dl.l CD40L + cells, B2.7 CD40L "or B2.7 CD40L + transfectants, as indicated. The expression CD54 and CD106 was determined by two-color FACS analysis as in Figure 4. The background MFI of an isotype control mAb is subtracted from each value.The experiment shown is representative of 2 similar experiments performed.

Figure 6A. Effect of 'CD40L-CD40 interactions on the secretion of IL-6 from fibroblasts. Bar graphs indicating the incorporation of 3H-thymidine by the B9 line of IL-6 indicator cells are shown following the additions of supernatants (final dilution 1:60) of SM fibroblasts grown with medium alone., ID1 cells of CD40L1"in the presence of absence of SC8 anti-CD40L mAb or control mAb P1.17, cells B2.7 CD40L" or transfectants B2.7 CD4QL +. The proliferative responses of B9 cells cultured with control supernatants of Dl.l cells, B2.7 cells or transfectants B2.7 CD40L + were 1136 cpm (± 113), 2398 cpm (± 263) and 1131 cpm (± 56). Similar results were obtained with 3 additional SM lines.

Figure 6B. Proliferation of B9 in response to rIL-6. In an experiment parallel to that shown in Figure 6A, B9 cells were cultured with varying concentrations of rIL6.

Figure 7. Effect of the binding of CD40 on the proliferation of SM fibroblasts. Bar graphs from 2 separate experiments showing the incorporation of 3H-thymidine by SM fibroblasts following co-culture in 1% of 'FM with B2.7 cells of Jurkat CD40L' treated with mitomycin-C or transfectant.es B2.7 of Jurkat CD40L + for 48 hours, where indicated, the transfectants B2.7 of Jurkat CD40L + were pretreated with anti-CD40L 5C8 mAb (5 μg / ml) or P1.17 control mAb (5 μg / ml) before the addition of the fibroblasts. At The experiment that studies the proliferation of RA.5, the proliferation of B2.7 cells of Jurkat CD40L "or transfectants B2.7 of Jurkat CD40L was 51 ± 7 cpm and 39 ± 3 cpm, respectively. the proliferation of OA.6, the proliferation of B2.7 cells of Jurkat CD40L "or 'Transfectants B2.7 of Jurkat CD40L + was 243 ± cpm and 453 ± 95 cpm, respectively. Background proliferation was subtracted in the co-culture experiments. The proliferative responses of the fibroblasts following the culture in 1% FM or 10% FM are also shown. Similar results were obtained in 3 additional experiments. The error bars show the observed error.

Figure 8. Effect of rINF-? on the proliferation of fibroblasts of SM mediated by CD40L. Bar graphs demonstrating the incorporation of 3H-1imidine by SM fibroblasts following a co-culture in 1% FM are shown with B2.7 cells of Jurkat CD40L * treated with mitomycin-C or transfectants B2.7 of Jurkat CD40L + for 48 hours. Where indicated, SM fibroblasts were pretreated for 18 hours with rINF-? (1000 U / ml) before the addition of B2.7 CD40L cells "treated with mitomycin-C or transfectants B2.7 CD40L +. The proliferation of SM fibroblasts was determined as described in Materials and Methods for the First Series of Experiments The background proliferation of Jurkat B2.7 cells of CD40L "and transfectants B2, 7 of Jurkat CD40L + was 185 ± 66 cpm and 65 ± 5 cpm, respectively. Background proliferation was used in the co-culture experiments. The proliferative responses of the fibroblasts are also shown following the culture in 1% FM or 10% FM. Similar results were obtained in two additional experiments. The error bars show the observed error.

Figures 9A-D. Endothelial cells in the skin express CD40 in situ. Immunohistological studies of frozen sections showing the expression of: (a) CD40, skin are shown (magnification 40x), (b) CD34, skin (magnification 40X), (c) CD21, skin (magnification 40x) and (d) 'mouse IgG control, skin (40x magnification).

Figures 10A-D, Endothelial cells in the muscle express CD40 in situ. Immunohistological studies of frozen sections showing the expression of: (a) CD40, muscle (40x magnification), (fo) CD34, muscle (40x magnification), (c) CD21, muscle (40x magnification) and (d) control are shown , Mouse IgG, muscle (40x magnification).

Figure 11. Endothelial cells in the spleen express CD40 in situ. Immunohistological studies of frozen sections showing the expression of: (a) CD40, spleen (magnification 10x) and (b) control, mouse IgG, spleen (magnification 10x).

Figure 12. Expression of CD40 on HUVEC cells in vitro. FACS analyzes that overlap the expression of CD14, CD40, CD45 or isotype control on HUVEC are shown following the first pass. The average fluorescence intensity of the expression of CD14, CD40, CD45 or isotype control is 7, 24, 5 and 9, respectively. A representative of the expression of CD40 on HUVEC isolated from 15 umbilical cords is shown.

Figure 13. Effect of the interactions of CD40L-CD40 on the expression of CD54 (ICAM-1) on HUVEC. Contour plots of two colors are shown demonstrating the effects of CD54 expression on HUVEC following culture with the medium, Jurkat CD40L + Dl.l cells or B2.7 cells.

Jurkat CD40L "for 6 hours Where indicated, Dl.l CD40L + cells were pretreated with isotype control mAb 5C8 anti-CD40L or P1.17 mAb.The X axis demonstrates CD13 expression, and the Y axis demonstrates expression CD54 The numbers in the upper right corner of each graph indicate the percentage of CD13 + cells expressing CD54 (the background staining of the control mAb is subtracted for each value.) It shows a representative of 3 similar experiments with different lines of HUVEC.

Figure 14. Effect of the CD40L-CD40 interactions on the expression of CD54 (ICAM-1), CD62E '(E-selectin) and CD106 (VCAM-1) of HUVEC. HE • show bar graphs representing the percentage of HUVEC expressing CD54, CD62E or CD106 following culture for 6 hours with the medium, rIL-la, Jurkat CD40L + Dl.l cells or Jurkat CD40L B2.7 cells. " it was indicated, the Dl.l CD40L + cells were pretreated with anti-CD40L mAb 5C8 or mAb 'P1.17 isotype control. The expression of CD54, CD62E and CD106 of HUVEC was determined by analysis of Two-color FACS as shown in Figure 3. The background staining of the control mAb was subtracted for each value. A representative of 3 similar experiments is shown with different lines of 'HUVEC.

Figure 15. Effect of transfectants of 293 kidney cells expressing CD40L on the expression of CD54, CD62E and CD106 of HUVEC. Contour plots of two colors are shown demonstrating the effects on the expression of CD54, CD62E and CD106 in HUVEC following culture with the medium, Jurkat CD40L + Dl.l cells, transfectants of 293 CD8 + kidney cells or transfectants of Kidney cells 293 CD40L + for 6 hours. The X axis demonstrates the expression of UEA-1 and the Y axis demonstrates the expression of CD54 (left panel), CD106 (middle panel) or CD62E (right panel). The numbers in the upper right corner of each graph indicate the percentage of UEA-1 + cells expressing CD54, CD106 or CD62E, as indicated (the background staining of the control mAb was subtracted for each value). A representative of 3 similar experiments with different hUVEC lines is shown.

Figure 16A. Kinetic analysis of CD34, CD62E and CD106 up-regulation of HUVEC induced by CD40L. The percentages of HUVEC expressing CD54, CD62E, or CD106 are shown following culture with Jurkat CD40L + Dl.l cells for 6 or 24 hours. The percentage of HUVEC expressing CD54, CD62E or CD106 was determined by FACS analysis of two colors (the d staining of the control mAb was subtracted for each value). A representative of 3 similar experiments with different HUVEC lines is shown.

Figure 16B. Same as in Figure 16A except that HUVEC were cultured with B2.7 cells of Jurkat CD40L. " Figures 17A-Y: Atomic coordinates of the crystal structure of the soluble extracellular fragment of human CD40L containing the residues Gly116-Leu261 (in a Brookhaven Protein Data Bank format). (SEC DE IDENT NO: 1).

DETAILED DESCRIPTION This invention provides a method for inhibiting the activation by the CD40 ligand of cells carrying CD40 on the cell surface, comprising contacting the cells with an agent capable of inhibiting the interaction between the CD40 ligand and the cells, in a effective amount to inhibit the activation of cells.

In one embodiment, the cells carrying CD40 on the cell surface are different cells of the B cells. In another mode, they are cells Plasma, which include differentiated plasma cells such as myeloma cells. This method can be used to inhibit the activation of cells carrying CD40 either in vivo or ex vivo. "Interaction between the ligand of CD40 and CD40 on cells" refers to one or more 'functional or structural aspects, of an interrelation of CD40-CD40 ligand. Therefore, in one embodiment, an agent that inhibits the interaction can competitively bind to the CD40 ligand in such a way that it blocks or decreases the binding of the CD40 ligand to the cell CD40. In another embodiment, an agent that inhibits the interaction may be associated with CD40 or the CD40 ligand in a manner that does not inhibit the binding of the CD40 ligand to the cell CD40, but which influences the cellular response to the binding of CD40, such as by altering the rate of change of the cellular CD40 or the CD40-agent complex, altering the binding kinetics of CD40 are the CD40 ligand, or altering the rate or degree of cellular activation in response to the binding of CD40.

In specific embodiments of this invention, non-B cells, CD40-bearing cells are fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, or dendritic cells. In a more specific modality, the epithelial cells are keratinocytes. In another embodiment, macrophages are foam cells (lipid-laden macrophages). Foam cells play a role in autoimmune diseases, for example rheumatoid arthritis and atherosclerosis. In one embodiment of this invention the agent inhibits the binding of the CD40 to CD40 ligand on the cells. In an embodiment of this method, the agent • it is a protein. In a more specific embodiment, the protein comprises an antibody or portion thereof, for example a Fab, F (ab ') 2, light and / or heavy chain complementarity determining region (CDR), variable chain antibody region light and / or heavy, or a portion thereof capable of specifically binding to the CD40 ligand or the cell surface receptor of the CD40 ligand. The antibody can be a monoclonal or polyclonal antibody. In the embodiments of this invention, the monoclonal antibody is a chimeric antibody, a humanized antibody, or a primatized antibody. In another embodiment, the antibody portion comprises an individual chain antibody. An individual chain antibody is composed of variable regions joined by protein spacers in a single protein chain. In one embodiment of the method described in the foregoing, the agent specifically binds to the antigen to which the monoclonal antibody 5c8 specifically binds. In a specific embodiment, the agent is the monoclonal 5c8 antibody. The monoclonal antibody 5c8 is produced by a hybridoma cell that was deposited on November 14, 1991 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 20852, U.S.A. under the provisions of the Budapest Treaty for the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure. It was agreed that the hybridoma had the Accession number of ATCC HB 10916, In another embodiment, the antibody binds .specifically to CD40, One example of an anti-CD40 antibody is monoclonal mouse anti-human CD40, available from Genzyme Customer Service (Product 80- 3702-01, Cambridge, MA). In other embodiments, the monoclonal antibody is a chimeric antibody, a primed antibody, a humanized antibody, or • an antibody that includes a CDR region of a first human and an antibody scaffold of a second human. In one embodiment of this invention, the protein is soluble, monomeric CD40-L protein, which comprises all or part of the extracellular region of • CD40-L, or variants of it. The extracellular region of CD40-L contains the domain that binds to CD40. Thus, soluble CD40-L can inhibit the interaction between CD40L and the cell that carries the CD40. This invention contemplates that sCD40-L may constitute the complete extracellular region of CD40-L, or a fragment or derivative containing the domain that binds to CD40. The meaning of "chimeric" antibody, "primed" and "humanized" and the methods for producing them are well known to those skilled in the art. See, for example, PCT International Publication No. WO 90/07861, published July 26, 1990 (Queen, et al.); and Queen, et al. Proc. Nat'l Acad. Sci. -USA (1989) 86: 10029). Methods for making primatized antibodies are described, for example, in PCT International Publication No. WO / 02108, corresponding to International Application No. PCT / US92 / 06194 (Idee Pharmaceuticals); and in New an, et al., Biotechnology (1992) 10: 1455-1460, which are incorporated herein by reference in this application. In general, a humanized antibody is an antibody comprising one or more complementarity determining regions (CDRs) of a non-human antibody operably linked to segments of human backbone region. Additional residues associated with the non-human antibody may optionally be present. Typically, at least one heavy chain or one light chain comprises non-human CDRs. Typically, non-human CDRs are mouse CDRs. In general, a primatized anti-ruther is an antibody comprising one or more complementary determining regions (CDRs) of an antibody from a different species of a non-human primate, functionally linked to segments of the main structure region of a non-human primate. . Additional residues associated with the species, from which the CDR is derived may optionally be present. Typically, at least one heavy chain or light chain comprises the CDRs of the species that is not a non-human primate. Typically, CDRs are human CDRs. In general, a chimeric antibody is an antibody whose light and / or heavy chains contain regions of different species. For example, one or more segments of the variable region (V) of a species may be linked to one or more segments of constant region (C) of another species. Typically, a chimeric antibody contains variable region segments from a mouse linked to human constant region segments, although other mammalian species may be used. In another embodiment of this invention, the protein is soluble CD40 protein (sCD40), which comprises the extracellular region of CD40, or a portion thereof, or a variant thereof. sCD40 inhibits the interaction between CD40L and cells carrying CD40, sCD40 may be in monomeric or oligomeric form. The variants may differ from CD40 or the CD40 ligand that exist naturally in the amino acid sequence, or in forms that do not involve the sequence, or both. Variants in the amino acid sequence occur when one or more amino acids in the CD40 or the naturally occurring CD40 ligand are replaced with a different natural amino acid, an amino acid derivative or an unnatural amino acid. Particularly preferred variants include CD40 or naturally occurring CD40 ligand, or biologically active fragments of CD40 or the naturally occurring CD40 ligand, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically they have minimal influence on the secondary structure and hydrophobic nature of the protein or peptide. The variants may also have sequences that differ by one or more substitutions, deletions or non-conservative insertions of amino acids, which do not suppress the biological activity of CD40 or the CD40 ligand. Conservative substitutions (substituents) typically include the substitution of one amino acid for another with similar characteristics such as substitutions within 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. The amino acids-positively charged (basic) include arginine, lysine and histidine. The negatively charged amino acids (acids) include aspartic acid and glutamic acid. Other conservative substitutions can be taken from Table 4, and still others are described by Dayhoff in the Atlas of Protein Sequence and Structure (1988).

Table 4: Amino Acid Preservative Replacements Other variants within the invention are those with modifications that increase the stability of the peptide. Such variants may contain, for example, one or more non-peptide linkages (which replace the peptide linkages) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring amino acids or synthetic amino acids such as beta or gamma amino acids and cyclic variants.

The incorporation of the D-amino acids instead of the L-amino acids in the polypeptide can increase its resistance to proteases. See, for example, U.S. Patent 5,219,990. The peptides of this invention can also be modified by various changes such as insertions, deletions and substitutions, either • conservative or non-conservative where such changes can provide certain advantages in their use. In other embodiments, variants with amino acid substitutions that are less conservative may also result in desired derivatives, for example, causing changes in the - Loading, conformation and other biological properties. Such substitutions would include, for example, substitution of a hydrophilic residue with a hydrophobic residue, replacement of a cysteine or proline with another residue, replacement of a residue having a small side chain with a 'waste having a bulky side chain or the substitution of a waste having a net positive charge for a waste having a net negative charge. When the result of a given substitution can not be predicted with certainty, the derivatives can be easily tested according to the 'methods described herein to determine the presence or absence of the desired characteristics.

Variants within the scope of the invention include proteins and peptides with amino acid sequences that have at least eighty percent homology to the extracellular region of CD40 or the extracellular region of the CD40 ligand. More preferably, the sequence homology is at least ninety percent or at least ninety-five percent. Only when it is possible to replace scaffold substituents, it is also possible to replace functional groups that decorate the scaffold with groups characterized by similar characteristics. These substitutions will initially be conservative, that is, the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequence modifications may include, for example, chemical derivatization in vivo or in vitro of portions of CD40 or naturally occurring CD40 ligand, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation. In additional mode, the protein, which includes the extracellular region of the CD40 and CD40 ligand, is modified by chemical molasses in which the activity is preserved. For example, proteins can be amidated, sulfated, 'halogenated in a unique way or in a multiple way, alkylated, carboxy ladated, or phosphorylated. The protein may also be acetylated in a single or multiple manner, such as with a group with a farnesyl moiety, or with a fatty acid, which may be monounsaturated or polyunsaturated saturated. The fatty acid may also be fluorinated in a single or multiple manner. The invention also includes methionine analogues of the protein, for example methionine sulfone and methionine sulfoxide analogues. The invention also includes salts of the proteins, such as ammonium salts, including alkyl or arylammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, et Isulfate and salts of benzenesulfonate. The soluble and monomeric CD40-L protein can comprise all or part of the extracellular region of CD40-L. The extracellular region of CD40-L contains the domain that binds to CD40. Thus, soluble CD40-L can inhibit the interaction between CD40-L and the cell carrying CD40. This invention contemplates that sCD40-L can constitute the entire extracellular region of CD40-L, or a fragment or derivative that contains the domain that binds to CD40. In another embodiment of this invention, the protein comprising soluble extracellular region of CD40 or the portion thereof additionally 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 the protein? or to the G protein. In another embodiment the Fc region comprises IgG, IgGi, IgG2, IgG3, IgG4, IgA, IgAj, JgA2f IgM, IgD, or IgE. In another embodiment of this invention, SCD40 comprises the CD40 / Fc fusion protein. The fusion protein can be prepared using 'conventional techniques of cutting with enzymes and linking the fragments of the desired sequences. The Fc regioins suitable for the fusion protein are the Fc regions that can bind protein A or protein G, or that are capable of recognition by an antibody that can be used in the purification or detection of a fusion protein. which comprises the region of Fc. For example, the Fc region may include the Fc region of human IgGi or murine IgG]. This invention also provides a nucleic acid molecule that encodes the CD40 / Fc fusion protein. The method for creating soluble forms of membrane molecules by recombinant means is well known, in which the sequences encoding the transmembrane and cytoplasmic domains are suppressed. See generally Hammonds et al., U.S. Patent No. 5,057,417. In addition, methods for preparing sCD40 and the CD40 / Fc fusion protein are well known. See, for example, PCT International Publication No. WO 93/08207; Fanslow et al., "Soluble Forms of CD40 Inhibit Biologic Responses of Human B Cells," J. Immunol. , vol. 149, pp.655.60 (July 1992). In one embodiment of this invention, the agent is a small molecule. As used herein, a small molecule is a compound having a molecular weight between 20 Da and IxlO 6 Da, preferably from 50 Da to 2 kDa. In one embodiment of this invention, the agent is selected by a selection method. In a specific embodiment, the small molecule or other agent is selected by a method of sleeping comprising, isolating a sample of cells, for example a sample of a biological fluid (eg, blood) from an animal; culturing the sample under conditions that allow the activation of the cells carrying CD40 contained therein; contacting the sample with a number of cells expressing a protein that is specifically recognized by the antibody monoclonal 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with a protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the access number of ATCC HB 10916, effective to activate the cells carrying CD40; • contacting the sample with a quantity of a small molecule (or other pharmaceutical compound or agent) effective to inhibit the activation of CD40-bearing cells if the small molecule is capable of inhibiting the activation of CD40-bearing cells; and determine if the cells that express • the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Access number of ATCC HB 10916 activates the cells carrying CD40 in the presence of the small molecule (or other compound or pharmaceutical agent). The cell sample may be isolated from various tissues, including cell lines in culture, or cells isolated from an animal, such as cells dispersed from a solid tissue, cells derived from a bone marrow biopsy, or cells isolated from a body fluid. such as blood or lymphatic fluid. In another specific embodiment the agent (molecule) is selected based on a three-dimensional structure of the extracellular soluble region of the CD40 ligand or portion thereof capable of inhibiting the interaction between the CD40 ligand and CD40 on the cells. The agent can be selected from a library of known agents, modified from a known agent based on the three-dimensional structure, or designed and synthesized de novo based on the three-dimensional structure. In specific embodiments the agent (molecule) is designed by optimizing the structure of a guiding inhibitory agent, based on a three-dimensional structure of a complex of the soluble extracellular region of the CD40 ligand or portion thereof with the guiding inhibitory agent. A guiding inhibitory agent is a molecule that has been identified which, when contacted with the CD40 ligand or portion thereof, binds to and complexes with the soluble extracellular region of the CD40 ligand, CD40, or portion thereof, thereby decreasing the capacity of the complexed CD40 ligand or bound or the portion of the CD40 ligand to activate the cells carrying CD40. In another embodiment, a guiding inhibitory agent can act by interacting with either the extracellular region of the CD40 ligand, CD40, or in a tertiary complex with both a portion of the ligand of CD40 and CD40, decreasing the capacity of the CD40-CD40 ligand complexed to activate cells that carry CD40. In the methods of the invention, the CD40 ligand can be either soluble or bound to cells, such as activated T cells, and can be either the full-length native CD40 ligand or portions thereof. The decreased ability to activate cells carrying CD40 can be measured in different ways. One way in which it can be measured is by showing that the CD40 ligand, in the presence of inhibitor, causes a. lower degree of activation of CD40-bearing cells, compared to treatment of cells with a similar amount of CD40 ligand without inhibitor under similar conditions. The decreased ability to activate cells carrying CD40 may also be indicated by a higher concentration of CD40 inhibitor-ligand complex that is required to produce a similar degree of activation of CD40-bearing cells under similar conditions, compared to the non-CD40 ligand. United. In the extreme, the CD40 ligand contacted with the inhibitor may be unable to activate CD40-bearing cells at concentrations and under conditions that allow activation of these cells by the unbound CD40 ligand or a given portion thereof. The agent (small molecule) can be selected by a computational selection method using the crystal structure of a soluble fragment of the extracellular domain of human CD40L containing the residues Glyll6-Leu261 (sCD40L (116-261)). The crystal structure to be used with the selection method can be determined at a resolution of 2 A by the molecular replacement method. Briefly, a soluble fragment of the extracellular domain of the human CD40 ligand containing the amino acid residues Gly 116 for the C-terminal residue Leu 261 is first produced in soluble form, then purified and crystallized. The crystals can be tested to determine their diffraction capacity on the X-ray beam of an Elliot GX-13 generator. Molecular replacement and refining can be done with the XPLOR and QUANTA program package (Molecular Simulations, Inc.) Software. In particular, a three-dimensional model of the human SCD40L can be constructed using the murine CD40L model using the QUANTA protein homology modeling suite. This model can then be used as a probe for molecular replacement and refining calculations using XPLOR. This method of determining the crystal structure of sCD40L is described in more detail in Karpusas et al., "2A crystal structure of an extracellular fragment of human CD40 ligand", Structure (October 1995) 3 (10): 1031-103. The atomic coordinates of sCD401 (116-261) are provided in Figures 17A-Y. The selection method for selecting an agent includes computational drug design and iterative optimization of structures, as described in the following. The agent can be a selected small molecule inhibitor using computational drug design. Using this method, the coordinates of the crystal structure of sCD40L are used as an input to a computer program, such as DOCK, which produces a list of small molecule structures that are expected to bind to CD40L. The use of such computer programs is well known. See, for example, Kuntz, "Structure- Based Strategies for drug design and discovery", Science, vol. 257, p. 1078 (1992). The list of small molecule structures can then be selected by biochemical assays to determine binding to CD40L. Type biochemical tests Competition, which are well known, can be used. See, for example, Bajorath et al., _ "Identification of residues of CD40 and its ligand which are critical for receptor-ligand interaction", Biochemistry, 34, p. 1833 (1995). The structures found to bind to CD40L can thus be used as agents for the present invention. The agent can also be a modified small molecule, determined by interactive structure optimization cycles. Using this approach, a small molecule inhibitor of CD40L found using the previous computational approximation or other approximation can be co-crystallized with SCD40L, and the crystal structure of the complex resolved by molecular replacement. The revealed evolution through molecular replacement can be used to optimize the structure of small molecule inhibitors, clarifying how molecules interact with CD40L. The small molecule can be modified to improve its physicochemical properties, including specificity and affinity for CD40L. In one embodiment of this invention, the agent binds specifically to CD40 on the cell surface. In a specific embodiment the agent is a protein, for example an antibody or the extracellular region of the CD40 ligand. The antibody can be a polyclonal or monoclonal antibody. It is preferred that the monoclonal antibody be chimeric or humanized. It can also be primatized.

In vivo use This invention provides a method for inhibiting the activation by the CD40 ligand of cells carrying CD40 on the cell surface, in a subject, comprising administering to the subject an agent capable of inhibiting the interaction between the CD40 ligand and the cells, in an amount effective to inhibit the activation of the cells in the subject. In one embodiment, the cells carrying CD40 on the cell surface are different cells from B cells. In another embodiment, they are plasma cells, including differentiated plasma cells such as myeloma cells. In specific embodiments of this invention, the cells that they are not B, cells carrying CD40 are fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, or dendritic cells. In a more specific modality, the epithelial cells are keratinocytes. In another embodiment, macrophages are foam cells (lipid-laden macrophages). Foam cells play a role in autoimmune diseases, for example, rheumatoid arthritis and atherosclerosis. In one embodiment of this method, the agent is a protein. In a more specific embodiment, the protein comprises an antibody or portion thereof, for example, a Fab, F (ab ') 2, light chain and / or heavy chain complementarity determining region (CDR), variable region of antibody of light and / or heavy chain, or a portion thereof capable of specifically binding to the CD40 ligand or to the cell surface receptor of the CD40 ligand, or to CD40. An example of an anti-CD40 antibody is the monoclonal mouse anti-human CD40, available from Genzyme, Customer Service (Product 80- 3702-01, Cambridge, MA). The antibody can be a monoclonal or polyclonal antibody. In embodiments of this invention, the monoclonal antibody is a chimeric antibody, a humanized antibody, or a primatized antibody. In another embodiment, the antibody portion comprises an individual chain antibody. An individual chain antibody is composed of variable regions linked by protein spacers in an individual protein chain. In one embodiment of the method described in the foregoing, the agent- specifically binds to the antigen to which the monoclonal antibody 5c8 specifically binds (ATCC Accession Number HB 10916). In a specific embodiment, the agent is monoclonal antibody 5c8 (Accession number of ATCC HB 10916).

The compounds of this invention can be administered in any manner that is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumoral, intraperitoneal, intraventricular, intraepidural, or other, as well as oral, nasal, ophthalmic, rectal, topical, or inhaled. Sustained-release administration is also specifically included in the invention, by means such as injections of erodible implant deposits applied directly during surgery. The compounds are administered in any dose by body weight and any frequency of dosage that is medically acceptable. For example, the acceptable dosage for the compound of this invention (especially for the antibody or antibody portion of this invention) includes a range of between about 0.01 and 200 mg / kg body weight of the subject. A dosage range is between about 0.1 and 50 mg / kg. In an even more specific embodiment, the dose is between about 1 and 30 mg / kg. The dosage is repeated in the range that is in the range from every day to every two months. A dosage regimen is to administer a compound of the invention daily for the first three 'days of treatment, after which the compound is administered every three weeks, and each administration is intravenously at five or 10 mg / kg of body weight. Another regimen is to administer a compound of the invention daily intravenously at 5 mg / kg of body weight, during the first three days of treatment, after which the compound is administered subcutaneously or intramuscularly every week at 10 minutes. mg per subject. Another regimen is to administer a single dose of the compound of the invention parenterally at 20 mg / kg body weight followed by administration of the compound subcutaneously or intramuscularly each week at 10 mg per subject. The compounds of the invention can be administered as a single dose for certain indications, such as preventing the immune response to an antigen to which the subject is exposed for a short time, such as an exogenous antigen administered in a single day of treatment. Examples of the antigen would include the co-administration of a compound of the invention together with a gene therapy vector, or a therapeutic agent such as an antigenic pharmaceutical or a blood product. In indications where the antigen is present chronically, such as in controlling the immune reaction to a transplanted tissue or to chronically administrating antigenic pharmaceutical compounds, the compounds of the invention are administered at intervals for as long as medically indicated, in the interval from days to weeks of the subject's life. This invention provides a method for inhibiting an inflammatory response in a subject, comprising the method described above inhibiting the activation by the CD40 ligand of cells, different from B cells, which carries CD40 on the cell surface (e.g. , fibroblast cells, endothelial cells, or inoculated kerat cells) in a subject. Inflammatory responses are characterized by redness, swelling, heat and pain, as a consequence of capillary dilation with edema and migration of phagocytic leukocytes. Inflammation is further defined by Gallin (Chapter 26, Fundamental Immunology, 2nd ed., Raven Press, New York, 1989, p. '721-733), which is incorporated herein by reference. This method is effective to inhibit the activation of any fibroblasts. In particular modalities, fibroblasts are synovial membrane fibroblasts, fibroblasts 'Dermal, fibroblast, lung, or liver fibroblasts. In particular embodiments, the condition dependent on the activation induced by the CD40 ligand of the fibroblast cells is selected from the group consisting of arthritis, scleroderma and fibrosis (for example fibrotic diseases of the 'liver and lung). In one embodiment of this invention, fibrotic disease of the lung is caused by rheumatoid arthritis or scleroderma. In one embodiment of this invention, arthritis is rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease or osteoarthritis. In another specific modality, fibrosis is pulmonary fibrosis, pulmonary fibrosis due to hypersensitivity or pneumoconiosis. In another specific modality, the fibrotic disease of the liver is Hepatitis-C, Hepatitis-B, Hepatitis that is not B and that is not C, cirrhosis, or cirrhosis of the liver secondary to an offensive toxin, drugs, a viral infection, or an autoimmune disease. Alcohol consumption is an example of an offensive toxic that can cause cirrhosis of the liver. An example of a drug that can cause cirrhosis of the liver is Bleomycin. Others with known in the art. Examples of viral infections that can cause fibrotic disease of the liver include, among others known in the art, Hepatitis-B, Hepatitis-C, and Hepatitis that is not B and that is not C. Examples of autoimmune diseases that can cause fibrotic disease of the liver include, among others known in the art, primary biliary cirrhosis, and Lupoidea hepatitis (autoimmune hepatitis). In specific modalities, pulmonary fibrosis is pulmonary fibrosis secondary to adult respiratory distress syndrome (ARDS), drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis; pneumoconiosis is asbestosis, silicosis, or Farmer's lung, as well as other pneumoconioses that are known in the art, with which this invention relates. This invention provides a method for treating a condition dependent on activation induced by the CD40 ligand of endothelial cells in a subject, comprising the method described above inhibiting the activation of endothelial cells by the CD40 ligand in a subject. In the modalities of this invention, the dependent condition of the activation induced by the CD40 ligand of endothelial cells is selected from the group consisting of atherosclerosis, reperfusion injury, allograft rejection, organ rejection, and autoimmune diseases. Chronic Inflammatory, In a specific modality, atherosclerosis is accelerated atherosclerosis with organ transplantation. The expression of CD40 and CD401 in SITU in accelerated atherosclerosis associated with rejection of transplants has been studied. Frozen sections of coronary arteries from 4 patients with heart transplants who required a new transplant due to accelerated atherosclerosis were analyzed by routine ohiochemistry using G28 mAb. > anti-CD40, 5C8 anti-CD40L mAb or control mAbs. Staining with H and E routinely reveals typical intimal hyperplasia, proliferation of smooth muscle cells, and infiltration of inflammatory cells associated with the disease. The CD40 was widely expressed in the lesions: endothelial cells, foam cells and inflammatory cells that were 'infiltrated, all expressed -CD40. The immunoractivity of CD40L was observed as a discrete and weak staining of the mononuclear cells that infiltrated, presumably CD4 + T cells. Together, these studies demonstrated the presence of CD40L + mononuclear cells and CD40 + endothelial cells, foam cells and inflammatory cells in situ in accelerated atherosclerosis injuries associated with the transplant. In another specific modality, the chronic inflammatory autoimmune disease is vasculitis, rheumatoid arthritis, scleroderma, or multiple sclerosis. This invention provides a method for treating a condition dependent on the activation induced by the CD40 ligand of keratinocytes in a subject, comprising the method described in the above 'to inhibit the activation of keratinocyte cells by the CD40 ligand in a subject .

In a specific embodiment, the condition dependent on the activation induced by the CD40 ligand of keratinocytes is psoriasis. This invention provides a method for treating a condition dependent on the activation induced by the CD40 ligand of macrophages in a • subject, comprising the method described above inhibiting the activation of macrophages by the CD40 ligand in a subject. In the specific modalities, the condition dependent on the activation induced by the. CD40 ligand of macrophages is atherosclerosis or arthritis • Rheumatoid The subject that can be tethered by the methods described in the above is an animal. Preferably the animal is a mammal. Examples of mammals that can be treated include, but are not limited to, humans; rodents such as 'murine animals, rats and mice, as well as rabbits, and guinea pigs; cows; horses; sheep, goats; pigs Dogs and cats. This invention also provides a method for treating a condition dependent on the activation induced by the CD40 ligand of plasma cells in a subject (including malignant plasma cells), which comprises administering to the subject an agent capable of inhibiting the interaction between the ligand of - CD40 and cells, in an amount effective to inhibit the activation of cells in the subject. Plasma cells are differentiated B cells. In a specific modality, the condition is multiple myeloma. This invention provides a method of promoting the growth of CD40-bearing cells on the cell, which comprises contacting the cells with an amount of CD40 ligand effective to promote the growth of the cells. In one embodiment, cells are cells carrying CD40 on the cell surface different from B cells. In specific embodiments, non-B cells carrying CD40 on the cell surface are endothelial cells, fibroblasts, epithelial cells, T cells, or basophils. In another embodiment, the cells are plasma cells, including differentiated plasma cells such as myeloma cells. This invention further provides a pharmaceutical composition comprising a therapeutically effective amount of the agent described herein, capable of inhibiting the interaction between the CD40 ligand and the cells carrying CD40 on the cell surface, and a pharmaceutically acceptable carrier. This invention will be better understood from the Experienced Details which follow. However, one skilled in the art will readily appreciate that the specific methods and results discussed are merely illustrative of the invention as described more fully in the claims which follow thereafter.

Experimental Details FIRST SERIES OF EXPERIMENTS Materials and Methods Patients studied All RA patients studied meet the criteria of the American College of Rheumatology for R? (19), The diagnosis of OA was established by the physicians of the patients using clinical and radiographic criteria. A patient with chronic inflammatory arthritis (IA) of unknown etiology was also studied.

Monoclonal Antibodies and T-cell Lines The murine IgG2a anti-CD40L mAb (5C8) (3) was previously generated. Anti-MHC Class I (W6 / 32), anti-MHC Class II (L243), anti-CD14 (3C10), anti-CD40 (G2R.5) and anti-CD45 (GAP 8.3) hybridomas were purchased from Amer ican 'Type Culture Collection (ATCC) (Rockville, MD). Hybridoma ascites were purified on a Protein G column (Pharmacia, Piscataway, NJ). Anti-CD13 and anti-CD54 mAbs were purchased from Biosource International (Camarillo, CA). The anti-CD106 mAb is kindly provided by Biogen (Cambridge, MA) and biotinylated as previously described (20). The isotype control mAbs used for the FACS analysis were purchased from Becton-Dickinson (San Jose, CA) or Caltag (South San Francisco, CA). P1.17 is a murine mAb of control IgG2a obtained from Biogen and used for functional studies. Dl.l is a subclone of the Jurkat T cell that constitutively expresses CD40L (3, 21). B2.7 is a subclone of Jurkat of CD40L "(3, 21) Transfectants B2.7 of Jurkat CD40L + expressing the full length CD40L protein are generated as previously reported (20).

Isolation of fibroblasts Synovial membrane was obtained from 6 patients with RA or 8 patients with OA suffering from joint replacement surgery. The MS of a patient with AI was collected in arthroscopy. The SM is cut into small pieces and grown in petri dishes for tissue culture of -100 mm (Corning, Corning, NY) or 25 cm2 flasks (Costar, Cambridge, MA) with Modified Dulbecco's Medium of Isocove (Gibco , Grand Island, NY) supplemented with 10% FCS (Summit Biotechnology, Ft. Collins, CO) and 1% penicillin-streptomycin (Sigma, St. Louis, MO) (10% FM). The synoviocytes are allowed to adhere for several days, during which time the tissue debris and non-adherent cells are eliminated. The synoviocytes are grown to confluence and are treated with 1% trypsin-EDTA (Sigma). The synoviocytes are studied between 1-6 passages in vi tro. A normal frozen dermal fibroblast line following the second passage (CCD 965SK) was purchased from ATCC. The lines of dermal fibroblasts are studied between 2-4 pasa j es.

Studies on cytosine efestos on the expression of CD40 in fibroblasts To study the effects of cytokines • on the expression of CD40 in fibroblasts, the cells are cultured in 6-well plates (Nunc, Denmark) and were cultivated near the confluence.

The medium is aspirated and the fibroblasts are then cultured with the indicated concentrations of rINF-? (Biogen), rIL-la (R &D, Minneapolis, MN), rTNF-a '(Upstate Biotechnology, Lake Placid, NY), rIL-4 (Biosource International), rGM-CSF (Immunex, Seattle, WA) or combinations of cytokines in 3 ml of FM to % At the indicated time points, the medium is aspirated, the cells are washed once with saline and 1 ml of 1% trypsin-EDTA is added to them. 'wells. After 7 minutes cold 10% FM is added to the wells, and the cells are harvested for FACS analysis.

Studies on the functional consequences of CD40 binding of fibroblasts To determine the effect of the binding of CD40 on the expression of fibroblast cell surface molecules, fibroblasts are cultured in 6-well plates as described above. When the fibroblasts are near the confluence, 1 × 10 6 Jurkat CD40L + Dl.l cells, Jurkat CD40L "B2.7 cells or Jurkat CD40L + B2.7 transfectants. Where indicated • Dl cells are added. l are pretreated with anti-CD40L mAb 5C8 (10 μg / ml) or isotype control mAb P1..17 (10 μg / ml) before addition to the fibroblasts.After 24 hours, cells are harvested by trypsinization and two-color FACS analyzes are performed.For studies that determine the effect of CD40 binding on fibroblast proliferation, approximately 5 x 10 3 cells are added to 96-well flat bottom (Nunc) wells in 10 wells. % of FM After 18 hours the medium is changed to 1% of FM and rINF-? 1000 U / ml is added to the indicated cells. After 18 additional hours, 1 x 10 n transfectants are added to the fibroblasts. B2.7 of Jurkat CD40L + treated with mitomycin-C (Sigma) or B2.7 cells of Jurkat CD40L in 1% FM Some anti-CD40L (5 μg / ml) or control P1.17 mAb (5 μg / ml) are also added to some wells as indicated. 10% FM is added to some cells as a control for the proliferation induction of SM fibroblasts. The cultures are maintained for an additional 48 hours and pulsed with 1 μCi of 3 H ~ thymidine for at least 18 hours of the experiment. Following the initiation, the incorporation of H-thymidine is determined by harvesting on glass fiber filter strips (Cambridge Technologies, Watertown, MA) and scintillation counting (BetaCounter, Pharmacia). To determine the effect of the binding of CD40 on the production of IL-6, a bioassay using the B9 line of murine B cells sensitive to IL-6 (22) was performed. Equal numbers of fibroblas are sown in 10% FM in 96-well plates as mentioned in the previous one. After they stick during the night, 1 × 10 ^ Jurkat CD40L + Dl.l cells treated with mitom cyna-C, Jurkat CD40L B2.7 cells or Jurkat CD40L + B2.7 transfectants are added to the fibroblasts. Where indicated, the Dl.l st cells pretreat with anti-CD40L 5C8 mAb (10 μg / ml) or control mAb P1.17 (10 μg / ml). The control wells consist of Jurkat cells grown alone. After 48 hours, serial dilutions of control fibroblasts or supernatants or rIL-6 are added to 7.5 x 10 3 B9 cells in 96-well plates. B9 cells are maintained in culture for 96 hours, pulsed with 1 μCi of 3 H-thymidine for at least 18 hours and harvested as mentioned above.

Cytofluorographic analysis The methods used for the cytofluorographic analysis have been previously described (21). In all experiments, the cells are first treated with aggregated human immunoglobulin (Enzyme International, Fallbrook, CA) to block non-specific Ig binding. For single-color FACS analysis, cells are stained with saturating concentrations of primary antibody for 30-60 minutes at 4 ° C. Following the washing, goat anti-mouse IgG of conjugated F (ab) 2 is added 'with FITC (Cappel, Co.chranville, PA) for 30-60 minutes at 4 ° C. The cells are washed and fixed with 1% formaldehyde before the FACS analysis. For the analysis of two-color FACS, the cells are stained simultaneously with the mAbs conjugated with FITC or PE indicated for 30-60 minutes at 4 ° C. The intensity of the fluorescence is measured on a FACScan cytofluorograph with the Consort-30 software (Becton-Dickinson, Moun tainview, CA). The mean fluorescence intensity (MFI) refers to the values normalized to the logarithm scale as calculated by the Becton-Dickinson C30 software.

Results Expression of CD40 on SM or cultured dermal fibroblasts To determine whether SM fibroblasts express CD40, the SM derived from 6 patients with RA, 1 patient with IA or 8 patients with OA are first shredded and placed in culture, after which discard the non-adherent cells. As expected, the primary cultures of adherent cells were pleomorphic with respect to morphology and phenotype. A minority of cells assume a stellate morphology or a rounded appearance characteristic of macrophages. However, the majority of cells in the primary cultures have a morphology and phenotype such as fibroblasts, ie CD45"CD14" MHC Class II (Figure 1) . Virtually all cells have a morphology and phenotype such as fibroblasts following 2-3 passages in vitro. Five fibroblast lines of RA to determine the expression of CD40 following the first and second passages in vitro, and have CD40 + by FACS analysis (figure 1). A line of IA fibroblasts similarly expresses CD40 (table 1). A line of RA fibroblasts has been in culture for 2 months before the analysis, and had CD40"(data not shown) Eight OA fibroblast lines are studied to determine the expression of CD40 following the first or second passages in vitro, and all have CD40 + (figure 1).

To determine if the expression of CD40 in fibroblasts is restricted to SM fibroblasts, normal dermal fibroblasts are analyzed to determine the expression of CD40 following 2-4 passages in vitro. For varying degrees, the 3 lines of dermal fibroblasts studied also express the CD40 molecules on the cell surface (Figure 2). However, the expression of CD40 on the synovial membrane or the dermal fibroblasts decreases with the increase of the time in culture, in such a way that some lines of fibroblasts become CD40"after 3-4 passages (data not shown). Studies show that dermal fibroblasts or SM fibroblasts isolated from patients with various arthritis can express CD40 in vitro, Effect of cytokines on the expression of CD40 in ibroblasts It is known that interferon-? (INF-?) Upregulates the expression of CD40 on B cells (23), macrophages (12), and thymic epithelial cells (15). In addition, IL-la or TNF-α upregulates the expression of CD40 in thymic epithelial cells (15). Therefore, it is later questioned whether rINF- ?, rIL-la or rTNF-a regulates the expression of CD40 in cultured SM fibroblasts. The cells are cultured with the indicated cytokines and the expression of CD40 is determined by FACS analysis. As a control for the effects of these cytokines on the expression of molecules on the cell surface of the SM fibroblasts, the expression of CD54 (ICAM-1) is also determined (24). rINF-? upregulates the expression of CD40 in SM fibroblasts. (table 1 and figure 3). By contrast, rIL-la and rTNF-a have a minimal effect on the expression of CD40 in SM fibroblasts (table 1 and figure 3). However, either rlL- la or rTNF-a increase the effect of rINF-? on the expression of CD40 in SM fibroblasts (figure 3). rINF-? it also induces the expression of CD40 on SM fibroblasts that have lost the expression of CD40 during serial passages in culture (data not shown). Also, rINF-? upregulates the expression of CD40 on dermal fibroblasts (Figure 2). rIL-4 or rGM-CSF upregulate the expression of CD40 on B cells (25) or monocytes (12), respectively. However, rIL-4 or rGM-CSF does not . have effects on the expression of CD40 in SM fibroblasts (data not shown). Together, these studies show that rINF-? it induces and upregulates the expression of CD40 in fibroblasts and the addition of rIL-la or rTNF-a increases this effect.

• Efesto of the CD40L-CD40 interactions on the expression of CD54 (ICAM-1) and CD106 (VCAM-1) in SM fibroblasts Because it is known that the activation of CD40 upregulates a variety of cell surface molecules on B cells, including • adhesion molecules (26), s < - determined whether the binding of CD40 upregulates the expression of CD54 or CD106 on SM fibroblasts. Fibroblasts from SM with Jurkat CD40L1 Dl.l cells in the presence or absence of 5C8 anri ~ CD40L mAb or control mAb.

SM fibroblasts are also cultured with cells • B2.7 of Jurkat CD40L "or transfectants B2.7 of Jurkat CD40L +, After the period of time indicated in culture, the expression of CD54 or CD106 in fibroblasts of SM is determined by FACS analysis of two colors. is used to discriminate SM fibroblasts from Jurkat T cells (27) .Dl.l CD40L + cells, but not cells . B2.7 CD40L "control, induce a 2-4 fold increase in the expression of CD54 in SM fibroblasts (Figures 4 and 5) in a manner that specifically inhibited by mAb 5C8 but not by the control mAb (Figure 4 In addition, Di, 1 CD40L + and CD40L + transfectants from Jurkat, but not B2.7 cells.

• Control CD40L, similarly upregulates the expression of CD1.06 in fibroblasts of SM (Figure 5) Together, these results demonstrate that CD40L-CD40 interactions upregulate the expression of CD54 and CD106 in SM fibroblasts. .

• Efesto of the binding of CD40 on the secretion of IL-6 in SM fibroblasts. The binding of CD40 induces B cells (28) and monocytes (12) to produce IL-6. Interestingly, SM fibroblasts produce IL-6 in vivo (29, 30) and in vitro (31). The next series • of experiments questions whether the CD40L-CD40 interactions effect the secretion of IL-β by SM fibroblasts. Therefore, they are grown • SM fibroblasts with Jurkat CD40L + Dl.l cells treated with mitomycin C in the presence or absence of anti-CD40L 5C8 mAb or control mAb. Additionally, SM fibroblasts are cultured with B2.7 cells from Jurkat CD40L "or transfectants B2.7 of Jurkat CD40L +.

Supernatants of fibroblasts or supernatants Control cells, from Jurkat cultured alone are harvested after 48 hours and dilutions are added to line B9 of murine B cells responsive to IL-6. Dl.l cells and transfectants B2.7 CD40L +, but not B2.7 cells CD40L, increase the secretion of IL-6 from fibroblasts 'of SM (figure 6). Additionally, anti-CD40L 5C8 mAb, but not the control mAb, inhibits this effect of Dl.l cells. Control supernatants harvested from Jurkat cells grown alone do not induce B9 proliferation (See description of the Figure 6). These studies indicate that the binding of CD40 on SM fibroblasts increases the secretion of IL-6.

Effect of CD40L-CD40 interactions on the proliferation of SM fibroblasts Because the binding of CD40 induces the proliferation of B cells (5, 21), it was later questioned whether CD40L + cells induce the proliferation of SM fibroblasts. Therefore, SM fibroblasts are grown overnight in 1% FM to stop growth, as previously described (32), and other additions are made to the cells in 1% FM, unless Indicate otherwise. B2.7 CD40L + transfectants treated with mitomycin C or B2.7 CD40L "cells are then added to the SM fibroblasts, where indicated, the co-culture experiments also include anti-CD40L mAb 5C8 or control isotope mAb P1.17. In some experiments, SM fibroblasts are pretreated overnight with rINF-? Before the addition of B2 transfectants, 7 CD40L +. Because it is known that fibroblasts proliferate in the presence of a medium containing 10% FCS ((32)), each experiment includes control fibroblasts cultured in 10% F. The incorporation of 3H-thymidine is determined after 48 hours. The transfectants B2.7 CD40L +, in contrast to the parental B2.7 CD40L cells, induce the proliferation of SM fibroblasts (Figure 7) Additionally, the anti-CD4CL mAb 5C8 specifically inhibits the capacity of transfectants B2.7 CD40L + to induce the proliferation of fibroblasts (Figure 7). • pretreatment of SM fibroblasts with rINF-? increases the capacity of transfectants B2.7 CD40L + to induce the proliferation of SM fibroblasts (figure 8). Together, these data demonstrate that signals mediated by CD40L induce the proliferation of SM fibroblasts in vitro, and this 'effect is increased by rINE'- ?.

Dissusion This study extends the current knowledge of the expression and function of CD40, demonstrating specifically that: 1) dermal fibroblasts or 'of cultured SMs express CD40 molecules on the cell surface, determined by analysis of FACS, 2) rINF-? upregulates the expression of CD40 of fibroblasts, and this effect is increased by rIL-la or rTNF-a, 3) interactions of CD40L-CD40 upregulates the expression of CD54 and CD106 in 'SM fibroblasts, 4.) the binding of CD40 increases the production of IL-6 in SM fibroblasts, and 5)? induces the proliferation of SM fibroblasts. Together, these data demonstrate that CD40L-CD40 interactions functionally activate fibroblasts in vitro. Several lines of evidence suggest that T cells modulate fibroblast functions in vivo. This is important because fibroblasts play repairing roles following tissue injury, producing extracellular matrix proteins. In addition, lymphocytes, macrophages and fibroblasts are the predominant cell types in inflammatory granulomatous reactions characteristic of certain infections. In addition, T cells directly or indirectly mediate the fibroblast activation and collagen deposition seen in diseases such as scleroderrnia or chronic graft-versus-host disease (33-35). Animal models demonstrate that T cells modulate the function of the cells. fibroblasts during host responses to tissue injury. In this regard, studies of wound healing show that wound strength and hydroxyproline content are significantly decreased by treating mice with cyclosporin A (36) or anti-Thy mAb 1.2 which decreases the T cell (37). T cells also modulate the result in several models • Fibrosy animals. For example, bleomycin-induced pulmonary fibrosis is significantly attenuated in athymic mice relative to control euthymic mice (38). In addition, inflammatory reactions of the joints or liver and collagen deposition are also -reduced significantly in athymic rats following the intraperitoneal injection of cell wall extracts is retococcal (39, 40). One study suggests that human fibroblasts can express CD40 in vivo. Potocnik and colleagues studied the expression and distribution 'of various cell surface molecules, including CD40, on PBL, SF and SM of RA (18). For immunohistochemistry they noted the expression of CD40 in a variety of MS cells from RA, including cells with spindle-shaped morphology suggestive of fibroblasts. SM fibroblasts are a predominant cellular component of the rheumatoid cloth. Producing collagenase, PGE2, IL-6 and other mediators, it is thought that synovial fibroblasts are important contributors to the destruction of the characteristic RA joints (30, 41-43). While electron microscopy studies have shown direct fibroblast T contact in the rheumatoid synovium (449), most studies have suggested that cytokines derived from macrophages, such as IL-1 or TNF-a, activate fibroblasts (30) These studies suggest that direct contact mediated by CD40L-CD40 interactions also provides activation and proliferative signals to SM fibroblasts.The mechanism by which signals mediated by CD40L increase the proliferation of SM fibroblasts is Currently unknown, it is possible that CD40L-CD40 interactions induce the secretion of cytokines, such as IL-1, GM-CSF and FGF, which can stimulate the proliferation of SM fibroblasts in an autocrine or paracrine fashion (31). The binding of CD40 also induces B cells to express c-myc (45) a proto-oncogene associated with proliferative cells. Immunohistological studies show that synoviocytes as SM fibroblasts of RA express c-myc in situ (46). Therefore, it will be of interest to determine specifically if the binding of CD40 also induces the expression of c-myc in SM fibroblasts.

Similar to the binding of CD40 on B cells (26), the interactions of CD40L-CD40 increases CD54 expression of fibroblasts. In addition, the CD40L-CD40 interactions upregulate the expression of CD106 from fibroblasts. CD54 and CD106 play a key role in recruiting immune cells to sites of inflammation, interacting with CDlla / CD18 (LFA-1) or CD49d (VLA-4), respectively, expressed on leukocytes (24). There is also evidence that these ligand-counter-ligand interactions increase proliferative signals to T cells (47). It is known that CD54 and CD106 are expressed on synoviocytes as RA fibroblasts in vivo ((48-50)) and various cytokines upregulate the expression of CD54 and CD106 in synovial fibroblasts in vitro (49, 51, 52). In addition, the adhesion of T cells to SM fibroblasts in vitro is partially mediated by the CDlla / CD18-CD54 interactions (53) and the CD49d-CD106 interactions (49). Therefore, upregulation of CD54 and CD106 on SM fibroblasts by CD40L + T cells may represent a mechanism to increase the recruitment / retention of cytokine-mediated inflammatory cells to SM. Additionally, upregulation of CD54 and CD106 in SM fibroblasts mediated by CD40L can play direct signaling roles to T cells through interactions with their counter-receptors. Interestingly, the in vivo administration of an anti-murine hamster mAb (MRl) CD40L prevents the induction of collagen-induced arthritis, a murine model of .RA '(54). The fact that MRl blocks the production of anti-collagen autoantibodies is probably related to the known role of CD40L-CD40 interactions in humoral immune responses of the T cell (9-11). In addition, MRl prevents the development of cellular thickening of the synovial lining and the inflammatory cellular infiltration of SM characteristic of collagen-induced arthritis (54). These studies suggest that CD40L-CD40 interactions of T-cell fibroblasts play roles in mediating the inflammatory reactions seen in collagen-induced arthritis, and also play immunopathogenic roles in human fibrotic diseases such as RA or scleroderma, mediated in part by T-cell-dependent fibroblast activation. In addition, this study provides new rational motives for blocking CD40L-CD40 interactions as therapy for human diseases mediated by fibroblast activation induced by CD4 + T cells.

TABLE 1 Legend of Table 1. The regulation of cytokines in 'the expression of CD40 in SM fibroblasts. The expression of CD40 'average fluorescence intensity is shown as determined by FACS analysis on the indicated SM fibroblasts lines, following coculture with medium, rINF-? (1000 U / ml), rIL-a (10 pg / ml) or rTNF-a (200 U / ml). The background staining (MFI) of a control mAb was subtracted for each value.

SECOND SERIES OF EXPERIMENTS Materials and Methods Monoslonal Antibodies, Lestinas and T-cell Lines The murine anti-CD40L mAb (5C8) of IgG2a is previously generated (20). Hybridomas 6/32 (anti-MHC Class I), L243 (anti-MHC Class II), 3C10 (anti-CD14), THB.5 (anti-CD21), G28.5 (anti-CD40) and GAP 8.3 ( anti-CD45) were purchased from the American Type Culture Collection (ATCC) (Rockville, MD). Hybridoma ascites are purified on a Protein G column (Pharmacia, Piscataway, NJ). Anti-CD13 MaBs conjugated with FITC, anti-CD19 conjugated to FITC and anti-CD54 conjugated to PE were purchased from Biosource International (Camarillo, CA) and the anti-CD34 mAb was obtained from Biogenex (San Ramón, CA). An additional anti-CD54 mAb, as well as anti-CD62E and anti-CD106 mAbs, are kindly provided by Biogen (Cambridge, MA). L243 and mABs provided by Biogen are biotinylated as previously described '(37). Anti-CD80 mAbs conjugated with PE and biotinylated anti-CD86 were purchased from Becton Dickinson (San José, CA) and PharMingen (San Diego, CA), respectively. The isotype control mAbs used for the FACS analysis were purchased from Becton Díckinson or Caltag Laboratories (South San Francisco, CA). P1.17 is a murine mAb of irrelevant IgG2a control (Biogen) used for functional studies. UEA-1 conjugated to FITC was obtained from Sigma (St. Louis, MO). Dl.l is a T cell subclone of Jurkat that constitutively expresses CD40L (20, 42). B2.7 is a Jurkat CD40L T cell sucblone (20, 42) .293 CD40L + kidney cells stably transfected or 293 CD8 + kidney cells are generated as previously reported (37). of Ramos respond to signals mediated by CD40L (38, 39) and were obtained from ATCC.

. Endothelialis cell cultures The endothelial cells of the human umbilical vein (HUVEC) were isolated as previously reported (40, 41). HUVEC was grown in M199 medium (Gibco, Grand Island, NY) supplemented with % of FCS (Summit Biotechnology, St. Collins, CO), • 5% human serum (Gemini, Calabasas, CA), heparin 90 μg / ml (Sigma), endothelial cell growth factor 15 μg / ml. (Collaborative Research, Bedford, 'MA) and 1% penicillin-streptomycin (Sigma) (complete medium M199). HUVEC were treated for 3 minutes with 1% Trypsin-EDTA (Sigma).

All experiments with HUVEC were performed in complete M199 medium following 1-3 passages.

Studies on cytokine efestos on CD40 expression in HUVEC To study the effects of cytokines on CD40 expression, HUVEC was cultured in 6-well plates (Nunc, Denmark) and cultured near confluence. The medium is aspirated and the 'HUVEC are then incubated with rIFN-? 1000 U / ml (Biogen), rIL-la 10 pg / ml (R &D, Minneapolis, MN) or rTNF-a 200 U / ml (Upstate Biotechnology, Lake Placid, NY) in 3 ml of M199 complete medium. At the indicated times, the medium is aspirated, the cells are washed once with saline and 1 ml of 1% trypsin-EDTA is added to the wells. It is added to the wells Dulbecco's Medium Modified from Isocove Cold (Gibco) containing 10% FCS (Summit) after 3 minutes and the cells were collected for FACS analysis.

Studies on the funsional consequences of the binding of CD40 in HUVEC. To study the effect of CD40 binding on the expression of cell surface molecules of HUVEC, cells were cultured in 6-well plates as described above. When HUVEC was near the confluence they are added to the crop 1 x 106 . Jurkat CD40L + Dl.l cells, Jurkat B2.7 cells CD40L "transfectants of kidney cells 293 CD40L + or transfectants of CD8 kidney cells. Where indicated, CD40L 'cells are pretreated with anti-CD40L mAb 5C8 (10 μg / ml) or isotype control mAb P1.17 (10 μg / ml) before addition to HUVEC.

. After the indicated time in culture, the cells are harvested by trinization and the two-color FACS analyzes are performed.

Functional studies of the binding of CD40 on Ramos 266 cells. , Experiments to control the binding of CD40 on Ramos 2G6 cells are carried out by cultivating 2 x 105 Ramos 2G6 cells with 1 x 105 cells Dl.lo control cells for 24 hours in 96-well plates containing 200 μg of Medium of Dulbecco Modified by Isocove (Gibco) containing 10% FCS (Summit) and 1% penicillin-streptomycin (Sigma).

Sitofluorographic analysis The methods used for cytofluorographic analyzes have been previously described (20, 42). In all experiments, the cells are first treated with added human immunoglobulin (Enzyme International, Fallbrook, CA) to block the non-specific Ig binding. For the analysis of single-color FACS, the cells are stained with saturation concentrations of primary antibody for 30-60 minutes at 4 ° C. Following washing, goat anti-mouse IgG of F (ab) 2 conjugated with FITC is added (Jackson Immunoresearch Laboratires, West 'Grove, PA) for 30 -60 minutes at 4 ° C. The cells are washed and fixed with 1% formaldehyde before the FACS analysis. For the analysis of two-color FACS, the cells are first stained with the indicated biotinylated mAbs. Following washing, the cells are then stained with streptavidin-PE (Calbiochem, La Jolla, -CA) and anti-CD13 mAb conjugated with FITC or UEA-1 conjugated with FITC, as indicated. HUVEC are distinguished from Jurkat cells in the analysis of two-color FACS by positive staining with anti-CD13 mAb or UEA-1, a lectin that binds selectively to endothelial cells (43). The intensity of the fluorescence is measured in a FACScan cytofluorograph with the Consort-30 software (Becton-Dickinson, Mountainview, CA). The average fluorescence intensity (MFI) refers to values normalized to the logarithm scale calculated by the Consort 30 software.

Characterization of the expression of CD40 in endothelial cells in itself We studied frozen sections of spleen, thyroid, skin, muscle, kidney, lung or umbilical cord normal to determine the expression of CD40, as previously described (38). The immunohistological analysis is performed with the indicated mAbs, and the reactivity is detected using the Vector ABC Elite and 3-amino-9-ethecarbazol (AEC) kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions. The frozen control sections were stained with appropriate concentrations of mouse IgG (Sigma).

Results Characterization in si tu and in vi txo of the expression of CD40 in endothelial cells. The first series of experiments was performed to determine if normal endothelial cells express CD40 in si tu. Therefore, sections Frozen obtained from normal spleen, thyroid, skin, muscle, kidney, lung or umbilical cord were stained with anti-CD40 mAb or control mouse IgG and endothelial cell reactivity was scored. Additional controls include staining with anti-CD34 mAb (reagent with hematopoietic stem cells and 'endothelial cells (44)) or anti-CD21 mAb (reactive with B cell and epithelial cells (17)). The endothelial cells of all tissues studied express CD40 in situ. Figures 9-11 demonstrate the innce of CD40 representative of endothelial cells in normal skin (Figure 9), 'muscle (figure 10) and. spleen (figure 11). The pattern of endothelial reactivity was similar to that seen with anti-CD34 mAb (Figures 9 and 10). In contrast, endothelial cells do not react with anti-CD21 mAb (Figures 9 and 10) or mouse IgG (Figures 9-11). To further explore the expression of CD40 in endothelial cells and its in vitro function, it is subsequently questioned whether cultured human umbilical vein endothelial cells (HUVEC) also express CD40. HUVEC were isolated, cultured at confluence and CD40 expression determined by FACS analysis following trypsinization. The cells morphologically resemble endothelial cells, and phenotypic analysis showed that the cells were CD13 and reactive with UEA-1, a lectin that selectively binds to endothelial cells (43). In addition, the cells were CD14"CD45" MHC Class II "by FACS analysis.Therefore, these cultures did not contain significant numbers of contaminating non-endothelial cells.HUVEC constitutively express CD40 in vitro (Figure 12), Similar results were obtained of HUVEC isolated from 15 individuals To determine whether pro-inflammatory cytokines regulate CD40 expression in endothelial cells, as has been demonstrated for B cells (45), monocytes (14), thymic epithelial cells (18) and fibroblasts ( 19), HUVEC were cultured with rIFN- ?, rIL-la, or rTNF-a for 48 hours, rINF- ?, in contrast to rlL-la or rTNF-a, induces a 2-3-fold increase in the expression of CD40 in HUVEC (Table 2) Together, these studies demonstrate that endothelial cells from normal tissues express CD40 in situ and in vitro, and that rIFN-α up-regulates CD40 expression in endothelial cells in vitro.

Efesto of interassiones CD40L-CD40 on the expression of CD54, CD62E and CD1 6 in HUVEC. Active endothelial cells express cell surface molecules, such as CD54, CD62E and CD106, which play important roles in the mediation of intercellular adhesive interactions (1, 2). Interestingly, the binding of CD40 on B cells (46) or fibroblasts (19) induces upregulation of adhesion molecule. Therefore, it is later questioned whether the CD40L-CD40 interactions affect the expression of 'the expression of CD54, CD62E or CD106 in HUVEC in vitro determined by the analysis of two-color FACS.

HUVEC were cultured with Jurkat CD40L + Dl.l cells or Jurkat CD40L B2.7 cells. "Where indicated, Jurkar's Dl.l cells were pretreated with anti-CD40L 5C8 mAb or control mAb prior to treatment. 'addition to HUVEC. As a positive control, HUVEC was also cultivated with rlL-la. Jurkat's Dl.l cells CD40L +, but not B2.7 cells of Jurkat CD40L ", induce the upregulation of CD54, CD62E and CD106 on HUVEC (Figures 13 and 14;) This effect of Dl.l cells is inhibited by the anti-CD40L 5C8 mAb but not by an isotype control mAb (Figures 13 and 14) These studies strongly suggest that CD40L-CD40 interactions upregulate the expression of CD54, CD62E and CD106 'on HUVEC.

Ephesus of the 293 CD40L + kidney cell transducers on the expression of CD54, CD62E and CD106 on HUVEC. To determine whether signals mediated by CD40L were sufficient, in the absence of additional specific lymphoid interactions, to regulate endothelial cell adhesion molecules, HUVEC were cultured with 293 CD40L + kidney cells stably transfected or control 293 CD8 + transfectants. As a positive control, HÚVEC was also cultured with Dl.l CD40L + cells. Similar to Dl.l CD40L + cells, 293 CD40L + kidney cell transfectants upregulate the expression of CD54, CD62E and CD106 on HUVEC (FIG. 15). The control CD8 293 transfectants have no effect on the expression of CD54, CD62E or CD106 on HUVEC. Together, these studies demonstrate that CD40L-CD40 interactions are sufficient for. upregulate these adhesion molecules on HUVEC in vitro.

Analysis of the cethnic of the upregulation of CD54, CD62E and CD106 in HUVEC mediated by CD40L. Kinetics of up-regulation of CD54, . CD62E or CD106 by rlL-la or rTNF-a in vitro has been well established (1, 2). CD54 and CD106 are up-regulated 6 hours following activation and the expression persists for more than 24 hours. In contrast, the expression of CD62E reaches a maximum of 6 hours following activation, and return to the • baseline (no expression) for 24 hours. In the next series of experiments, the kinetics of the upregulation of CD54, CD62E or CD106 in HUVEC induced by CD40L. They were grown HUVEC with Dl.l CD40L + cells or B2.7 CD40L cells and analyzed at various time points to • determine the expression of CD54, CD62E or CD106. Following the culture with Dl.l CD40L + cells, the expression of CD54 or CD106 in HUVEC is up-regulated for 6 hours and persists in expression for more than 24 hours (figure 16). In contrast, the expression of CD62E induced by CD40L reaches a maximum for 6 hours and returns to baseline for 24 hours (FIG. 16). Therefore, the kinetics of upregulation mediated by CD40L, rTNF-a or rlL-that of CD54, CD62E or CD106- in HUVEC are similar.

To determine if the CD40L-CD40 interactions upregulate the expression of CD80, CD86 or MHC Class II on HUVEC. Activated endothelial cells are competent to express MHC Class II molecules and provide co-tidal signals to T cells (10, 47-49). The binding of CD40 on B cells or dendritic cells scr regulates MHC expression Class II, as well as, the expression of co-stimulatory molecules CD80 and CD86 (36, 37, 50-52). Therefore, the following series of experiments determines whether the CD40L-CD40 interactions similarly upregulate MHC Class II, CD80 or CD86 expression on HUVEC. HUVEC were cultured with Dl.l CD40L + cells or B2.7 CD40L cells for 24 or 48 hours and the expression of CD80, CD86 and MHC Class II was determined by two-color FACS analysis as a positive control for the effect of the binding of CD40 in HUVEC, the expression of CD54 is also determined.In addition, HUVEC with rIFN-γ was also cultured as a control for upregulation of MHC Class II As a positive control for upregulation of CD80, CD86 and MHC Class As measured by CD40L, Dl.l cells were cultured with Bamos 2G6 B cells (38-39) In contrast to the effects of CD40 binding on B cells or dendritic cells, the CD40L-CD40 interactions do not upregulate the expression of MHC Class II, Cd80 or CD86 on HUVEC (table 3).

Dissolution CD40 is a cell surface molecule constitutively expressed in a variety of cells, including B cells (12, 13), monocytes (14), dendritic cells (15), epithelial cells (17, 18), basophils (16). ) and fibroblasts . (19) The counter-receptor for CD40 is CD40L, a surface molecule of CD4 + T cells expressed transiently, induced by activation, of -33 kDa (20-25), It is shown that endothelial cells in the spleen, thyroid, skin, muscle, kidney, lung or umbilical cord express CD40 i_n_ • if you . This finding is consistent with a previous report that the endothelial cells in the synovial membrane of rheumatoid arthritis express CD40 (11). In addition, endothelial cells of the human umbilical vein (HUVEC) express CD40 in vitro. More importantly, the expression of CD40 on endothelial cells is functionally significant, because Jurkat T cells. CD40L + or 293 CD40L + kidney cell transfectants, but not the control cells, upregulate the expression of intercellular adhesion molecules CD54 (ICAM-1), CD62E (E-selectin) and CD106 (VCAM-1) on HUVEC . The results described herein demonstrate that endothelial cells express CD40 and CD40L-CD40 interactions induce the activation of endothelial cells i.n. Endothelial cells play central roles in inflammatory responses, in part by expressing CD54, CD62E and CD106 (1, 2). These adhesion molecules interact with specific cell surface receptors on leukocytes and promote the transmigration of inflammatory cells through the barrier of endothelial cells. The expression of these particular endothelial cell surface molecules are regulated very tightly (1, 2). Resting endothelial cells express low levels of CD54 and very little or none of CD62E or CD1-06. However, endothelial cells upregulate the expression of CD54, CD62E and CD106 following activation with IL-1 or TNF. These findings demonstrate a means by which activated CD4 + T cells upregulate adhesion molecules of endothelial cells by direct cell-cell contact. Because the expression of CD40L is also tightly regulated, it is likely that CD40L-CD40 interactions occur during immune responses driven by Ag. In this regard, in vitro studies show that resting CD4 + T cells do not express detectable CD40L ( 20-22, 25, 53). However, Cd40L is transiently expressed on activated CD4 + T cells in vitro; the maximum expression is seen 6 hours following activation and the levels return to baseline (no expression) for 24-48 hours (20, 21, 53). CD40L is also rapidly down-modulated by cells expressing CD40 in a process that is at least partially due to endocytosis mediated by the receptor (54). In vivo, CD40L expression is normally restricted to CD4 + T cells in secondary lymphoid tissue (38), the site of Ag-specific T-B interactions restricted by MHC. However, immunohistological studies of the synovial membrane of rheumatoid arthritis or psoriatic plaques demonstrate the presence of CD40L + CD4 + T cells. These studies suggest that APCs at sites of inflammation induce infiltrating CD4 + T cells to express CD40L. CD40L + CD4 + cells then play roles in increasing the inflammatory process by interacting with CD40 + endothelial cells. The functional consequences of this interaction allow additional adhesion and transmigration of immune cells at sites of inflammation. The fact that the binding of CD40 regulates the expression of endothelial cell surface adhesion molecules is consistent with a general role for CD40 signaling in the regulation of the expression and / or function of adhesion molecules on a variety of cells . In this respect, it has been shown that signals mediated by CD40L induce the upregulation of CD54 and CD106 on cultured synovial membrane fibroblasts (19). The binding of CD4Q also upregulates the expression of CD54 on B cells (46) and induces the CD54-dependent homoaggregation of B cells (55). Interestingly, pretreatment of B cells with anti-CD40 mAb increases the heterotypic interactions of B cells are endothelial cells activated in vitro, of a sea-dependent interaction of CD49d (VLA-4) / CD106 (56) . Because CD40 binding does not over-regulate the expression of CD49d in B cells, we hypothesized that. CD40 mediates the activation of CD49d-induced signals. The binding of CD40 on B cells or dendritic cells also upregulates MHC expression Class II, as well as, costimulatory molecules CD80 and CD86 (36, 37, 50-52). From Interestingly, the endothelial cells stimulated with rIFN-? they are competent to express MHC Class II in vitro (57), and endothelial cells - in situ within the inflammatory tissue can express MHC Class II (10, 58-60). In addition, the endothelial cells are competent to present • Ag to T cells in vitro, and provide costimulatory signals appropriate to T cells required for the production and proliferation of IL-2 (10, 47-49). However, it is shown here that the CD40L-CD40 interactions do not upregulate the • MHC Class II, CD80 or CD86 expression on HUVEC i_n vitro. This finding is consistent with previous studies suggesting that human endothelial cells do not express CD80 (47, 61). Costimulatory molecules expressed on endothelial cells are not precisely known. The work by Pber and colleagues demonstrates that blocking the CD2-CD54 (LFA-3) interactions inhibits the ability of endothelial cells to • proliferation of allogeneic T cells (47, 48), However, it is not clear if the CD2-CD58 interactions improve intercellular adhesion and / or provide costimulatory signals to T cells. It will be of interest to determine if the signals mediated by CD40L modulate the capacity of cells Endothelial cells activate T cells. Finally, endothelial cells are activated in a variety of diseases mediated by CD4 + T cells. For example, adhesion molecules of the endothelial cell surface are upregulated in rheumatoid arthritis (62), 'scleroderma (63) and in. the rejection of transplant (64). In addition, CD4 + T cells play roles in atherosclerosis (65) and accelerated atherosclerosis associated with transplantation (60). The precise mechanistic role of the interactions mediated by CD40L with endothelial cells in these diseases is not known. However, an antibody to CD40L, MRl, inhibits murine models of diseases mediated by CD4 + T cells and / or cell infiltrates • inflammatory. For example, MRl prevents cellular hypertrophy of the synovial lining and cellular infiltrate associated with collagen-induced atritis, a murine model of rheumatoid arthritis (66). In addition, MRl inhibits a murine model of multiple sclerosis (EAE) and inhibits the rejection of • allografts (67). Blocking the CD40L-dependent interactions with endothelial cells and / or fibroblasts mediates, in part, these MRl effects. The results described here suggest that CD40L-CD40 interactions on the surface of endothelial cells play roles 'immunopathogenic in inflammatory diseases.

TABLE 2 'Legend of Table 2 .. Effect of cytokines on the expression of CD40 in HUVEC. The average fluorescence intensity (MFI) of the expression of CD40 or CD54 on HUVEC cultured in the presence or absence of rIFN-α is shown. (1000 U / ml), rJL-la (10 pg / ml) or rTNF-a (200 U / ml) for 48 hours. The MFI of CD40 or CD54 was determined by. FAC analysis? and the background staining of the control mAb was subtracted from each value. Similar results were obtained in 2 additional experiments with different HUVEC lines.

TABLE 3 Legend of Table 3. Effect of CD40L-CD40 interactions on the expression of CD80 and CD86 of MHC Class II in HUVEC. The average fluorescence intensity of CD54, CD80, CD86 or MHC Class II expression in HUVEC is shown following the culture with the medium, rIFN-? (1000 U / ml), Jurkat CD40L + Dl.l cells or B2.sup.40 CD40L cells for 48 hours. In a parallel experiment, the Ramos sensitive 2G6 B cell line to CD40L (38-39) was cultured with Jurkat CD40L + Dl.l cells or B2.7 cells.

CD40L for 24 hours. The expression of CD54, CD80 and CD86 of MHC Class II or 2G6 of Ramos in HUVEC, was determined by FACS analysis of two colors. The background staining of the control mAb was subtracted for each value. A representative of 3 similar experiments with different HUVEC lines is shown. ND = not finished.

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LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTS: yellin, Michael J. Lederman, Seth Chess, Leonard Karpusas, Mihail N. Thomas, David W. (ii) TITLE OF THE INVENTION: THERAPEUTIC APPLICATIONS FOR THE MONOCLONAL ANTIBODY 5c! ANTI-T-BAM (CD40-L) (iii) SEQUENCE NUMBER: 1 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Cooper & Dunham LLP (B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA (F) ZIP (ZIP): 10036 (v) LEGIBLE COMPUTER FORM: ( A) TYPE OF MEDIUM: Soft disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0. Version # 1.30 (vi) CURRENT APPLICATION DATE: (A) APPLICATION NUMBER: Not yet known (B) DATE OF SUBMISSION: Same (C) CLASSIFICATION (vii) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 566,258 (B) DATE OF SUBMISSION: 01-DEC-l 995 (C) CLASSIFICATION (vii) DATE OF PREVIOUS APPLICATION: (A) APPLICATION NUMBER: US 08 / 567,391 (B) DATE OF SUBMISSION: 01-DEC- 1995 (C) CLASSIFICATION (viii) EMPLOYEE / AGENT INFORMATION: (A) NAME: White Esq., John P. (B) REGISTRATION NUMBER: 28,678 (C) REFERENCE NUMBER / FILE: 47279-B (ix) INFORMATION OF TELECOMMUNICATION: (A) TELEPHONE: (212) 278 0400 (B) TELEFAX: (212) 391 0525 (2) INFORMATION FOR THE IDENT SEC NO: l (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (iii) HYPOTHETICAL :. NONE (xi) DESCRIPTION OF THE SEQUENCE: SEC DE IDENT NO: l: Gly Asp Gln Asn Pro Gln He Wing Wing His Val He Ser Glu 1 5 10 Wing Being Ser Lys Thr Thr Ser Val Leu Gln Trp Wing Glu Lys 20 25 Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn 30 35 40 Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr He 45 50 55 Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Gl Ala Ser Ser 60 65 70 Gln Ala Pro Phe He Wing Ser Leu Cys Leu Lys Ser Pro Gly 75 80 Arg Phe Glu Arg He Leu Leu Arg Wing Wing Asn Thr His Ser 85 90 95 Wing Wing Pro Cys Gly Gln Gln Ser He His Leu Gly Gly 100 105 110 val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val 115 120 125 Thr Asp Pro Ser Gln Val Ser Kis Gly Thr Gly Phe Thr Ser 130 135 140 Phe Gly Leu Leu Lys Leu 145

Claims (35)

1. A method for inhibiting the activation, by the CD40 ligand, of cells carrying CD40 on the cell surface, different from B cells, characterized in that it comprises the step of contacting the cells in vitro with an agent capable of inhibiting the interaction between CD40 and the CD40 ligand

2. The method, in accordance with the 'claim 1, characterized in that the cells carrying CD40 are selected from the group consisting of: fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Ste inberg cells, keratinocytes and dendritic cells,

3. The method according to claim 1, characterized in that the agent binds to the antigen to which monoclonal antibody 5c8 binds specifically (ATCC Accession Number HB 10916).

4. The method, according to claim 1, characterized in that the agent binds specifically to CD40.

5. The method according to claim 1, characterized in that the agent is selected from the group consisting of: proteins, without proteins and small molecules.

6. The method, according to claim 5, characterized in that the protein comprises the soluble extracellular region of the CD40 ligand, or variants thereof including conservative substitutes, or portion thereof; or the soluble extracellular region of CD40, or variants thereof which includes conservative substituents, or portion thereof.

7. The method, according to claim 5, c acterized because the protein is an antibody or portion thereof.

8. The method according to claim 7, characterized in that the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies.

9. The method, according to claim 7, characterized in that the antibody is the monoclonal antibody 5c8 (Accession number of ATCC HB 10916).

10. The method, according to claim 1, characterized in that the agent is selected by opt imi. structure of a guiding inhibitory agent, based on a three-dimensional structure of a complex of the extracellular CD 0 soluble region or portion thereof with the guiding inhibitory agent.

11. The method, according to claim 1, characterized in that the agent specifically inhibits the activation induced by 'CD40 of endothelial or fibroblast cells that drive CD40 on the cell surface.

12. The method according to claim 11, characterized in that the fibroblasts are selected from the group consisting of: synovial membrane fibroblasts, dermal fibroblasts, lung fibroblasts and liver fibroblasts.

13. The use, according to claim 1, wherein the agent is selected by means of a selection method, characterized in that it comprises the steps of: isolating a sample of cells; cultivate the -sample under conditions that allow the activation of the cells that drive CD40; contacting the sample with cells expressing a protein, which is specifically recognized by a monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with a protein which is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, effective to activate cells carrying CD40; and contacting the sample with an amount of the effective agent to inhibit the activation of CD40-bearing cells if the agent is capable of inhibiting the activation of CD40-bearing cells; and determining whether the cells expressing the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession Number of ATCC HB 10916 activates the cells carrying CD40 in the presence of the agent.

14: The use of an agent capable of inhibiting the interaction between the ligand CD40 and CD40 in the preparation of a medicament for inhibiting the activation, by the CD40 ligand, of the cells bearing CD40 on the cell surface, characterized in that the cells that carry CD40 are distinct from B cells.

15. The use, according to claim 14, characterized in that the cells carrying CD40 are selected from the group consisting of -of: fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, keratinocytes and dendritic cells.

16. The use, according to claim 14, characterized in that the agent binds to the antigen to which the monoclonal antibody specifically binds • 5c8 (Accession number of ATCC HB 10916).

17. The use, according to claim 1, characterized in that the agent binds specifically to CD40.

18. The use, according to claim 14, characterized in that the agent is selected from the group consisting of: proteins, without proteins and small molecules.

19. The method used in accordance with claim 18, characterized in that the protein comprises the soluble estradiol region of the CD40 ligand, or variants thereof, which includes conservative substituents, or portions thereof; or a soluble extracellular region of CD40, or variants thereof which includes conserved substituents, or portions thereof.

20. The use, in accordance with the rei indication 18, characterized in that the protein is an antibody or portion thereof.

21. The use according to claim 20, characterized in that the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies, humanized antibodies and primed antibodies.

22. The use, according to claim 21, characterized in that the antibody is the monoclonal antibody 5c8 (Accession number of ATCC HB 10916).

23. The use, according to claim 14, characterized in that the agent is selected by optimizing the structure of a guiding inhibitory agent, based on a three-dimensional structure of a complex of the extracellular CD40 soluble region or portion thereof with the inhibitory agent guide.

24. The use, according to claim 15, characterized in that the agent specifically inhibits the activation induced by 'CD40 of endothelial or fibroblast cells that drive CD40 on the cell surface.

25. The use according to claim 24, characterized in that the fibroblast cells are selected from the group consisting of: fibroblasts of the synovial membrane, dermal fibroblasts, lung fibroblasts and liver fibroblasts.

26. The use according to claim 14, characterized in that the agent specifically inhibits the cellular activation by the CD40 ligand of the CD40-bearing cells which are involved in an inflammatory response.

27, The use, according to claim 26, characterized in that the inflammatory response is selected from the group consisting of: arthritis, scleroderma and fibrosis.

28. The use, according to claim 27, characterized in that the arthritis is selected from the group consisting of: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Ly disease, and osteoarthritis

29. The use, according to claim 27, characterized in that the fibrosis is selected from the group consisting of: pulmonary fibrosis, pulmonary fibrosis due to hypersensitivity and pneumoconiosis,

30. The use according to claim 29, characterized in that the pulmonary fibrosis is selected from the group consisting of: pulmonary fibrosis secondary to the adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis and hypersensitivity pneumonitis.

31, The use, according to claim 29, characterized in that the pneumoconiosis is selected from the group consisting of: asbestosis, silicosis and Farmer's lung. '42

32. The use according to claim 27, characterized in that the fibrosis is a fibrotic disease of the liver or lung.

33. The use, according to claim 32, characterized in that the fibrotic disease of the liver is selected from the group consisting of: Hepatitis-C; Hepatitis B; cirrhosis; cirrhosis of the liver secondary to an offensive toxic; cirrhosis of the liver secondary to drugs; cirrhosis of the liver secondary to a viral infection; cirrhosis of the liver secondary to an autoimmune disease.

34. The use, according to claim 14, wherein the agent is selected by means of a selection method, characterized in that it comprises the steps of: isolating a sample of cells; cultivate the sample under conditions that allow the activation of those cells in the sample, which san cells that drive CD40; contacting the sample with cells expressing a protein, which is specifically recognized by a monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with a protein which is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, effective to activate cells carrying CD40; contacting the sample with an amount of the agent effective to inhibit the activation of CD40-bearing cells if the agent is capable of inhibiting the activation of CD40-bearing cells; and determining whether the cells expressing the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the Hybridoma having the Accession Number of ATCC HB 10916 activates the cells carrying CD40 in the presence of the agent.

35. A method for selecting an agent by means of a selection method, characterized in that it comprises the steps of: isolating a sample of cells; cultivate the sample under conditions that allow the activation of those cells in the sample, which are cells that drive CD40; contacting the sample with cells expressing a protein, which is specifically recognized by a monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with a protein which is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, effective to activate cells carrying CD40; contacting the sample with an amount of the agent effective to inhibit the activation of CD40-bearing cells if the agent is capable of inhibiting the activation of CD40-bearing cells; and determining whether the cells expressing the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession number of ATCC HB 10916, or with the protein that is specifically recognized by the monoclonal antibody 5c8 produced by the hybridoma having the Accession Number of ATCC HB 10916 activates the cells carrying CD40 in the presence of the agent.