CA2243531A1 - Therapeutic applications of t-bam (cd40-l) technology to treat inflammatory kidney diseases______________________________________ - Google Patents

Abstract

Activation by CD40 ligand of renal cells bearing CD40 on the cell surface is inhibited, both in vivo and ex vivo, with an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells. Inflammatory kidney diseases are treated.

Description

CA 02243531 1998-07-15 ~ ~ ~ 9 - - O 6 6 8 IP~A~US 1 2 MAR 1998 Dkt. 4~s58-B-pcT/Jpw/JM~ -THERAPEUTIC APPLICATIONS OF T-BAM (CD40-L) TECHNOLOG'f TO
TREAT INFLAMMATORY KIDNEY DISEASES HAVING INITIATION POINTS
_ OTHER THAN AUTOANTIR~DY DEPOSITION
This application claims the priority of U.S. Serial No.
08/641,473, filed May 1, 1996 and U.S. Serial No.
08/587,334, filed January 16, 1996, the contents of which are hereby incorporated by reference.

The in~ention disclosed herein was made with Government support under NIH Grant Nos. K0~-AR-01904, ROl-CAS5713, ROl-AI-28367, ROl-AI-14969, HL21006, H~42~33, HL50629, IS and ROl-AI-14969 from the Department of Health and Human Ser~ices. Accordingly, the U.S. Government has certain rights in thi~ in~ention. ~~ ~-Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into thi~ application to more fully describe the state of the art to which this invention pertains.
Full bibliographic citation for these references may be 2S found in the text.

~ o~ ~ ~ s-.v~ t ~ ~..
Immune complex deposition is known to play important role~ in mediating the imm~"op~thogenesis of a ~ariety of renal ~ ~o~e5, including the glomerulonephritis a~ociated with sy~temic lupu8 erythematosu~. However, infiltrating renal interstitial leukocytes, predominately -,. T cell~ and monocytes, are often seen in lupus nephritis and other in~lammatory renal diseaqes. The precise role of infiltrating T cells in the inflammatory renal process that ultimately may re~ult in renal scarring and end-organ damage is currently unknown. It is of interest that the extent of mo~n-~clear cell infiltrate correlates with progression to renal failure. Some evidence ~EN~ED ~

CA 02243~3l l998-07-l~
W097l26000 PCT~S97/00668 suggests that interstitial T cells play direct immunopathogenic roles in the initiation and/or propagation of inflammatory renal diseases, including lupus nephritis.
CD40 is a cell surface molecule expressed on a variety of cells and interacts with a 30-33 kDa activation-indùced CD4+ T cell counterreceptor termed CD40L. CD40L-CD40 interactions have been extensively studied in T cell-B
cell interactions and are essential for T cell dependent B cell differentiation and IgG, IgA and IgE production.
CD40 is also expressed on monocytes, dendritic cells, epithelial cells, endothelial cells and fibroblasts.
CD40 expression on these cells is upregulated in vitro by cytokines, most notably IFN-y. In vivo studies have demonstrated markedly upregulated CD40 expression in inflammatory sites, such as rheumatoid arthritis synovial membrane or psoriatic plaques. In vitro studies utilizing anti-CD40 mAb or CD40L+ cells demonstrate that CD40 is functionally expressed on monocytes, dendritic cells, epithelial cells, endothelial cells and fibroblasts.

~ arlier disclosure of treating idiopathic autoimmune diseases, including drug-induced lupus, such as International Patent Pu~lication No. WO 93/09812 (published May 27, 1993) was based on the finding that CD40 is expressed on the surface of B cells. The initiation point of lupus is the deposition of autoanti~odies in the kidney, which then attracts cells involved in destruction of kidney tissue. The finding, disc~ e~ below, that CD40 is expressed on kidney tubule cells provides the basis for treating inflammatory kidney diseases having initiation points other than autoantibody deposition.

CA 02243531 1998-07-15 P~71J 9 7/ 00 66 8 ~ANS 12 MAR1!b~

8um~arY o f the Invention This invention provides a method of inh~hiting activation by CD40 ligand of renal cells bearing CD40 on the surface of the cells, comprising contacting the cells with an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an amount effective to inhibit activation of the cells.

This invention provides a method of inhibiting activation by CD40 ligand of renal cells bearing CD40 on the surface of the cells, in a subject, comprising administering to the subject an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an amount effective to inhibit activation of the cells in the subject.

This invention provides a method of treating, in a subject, an inflammatory kidney disease, comprising administering to the subject an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an amount effective to inhibit activation of the cells in the subject and thereby treat the inflammatory kidney disease/having initiation points other than autoantibody doposition.
-W097/26~0 PCT~S97100668 De#cription o~ the Fi~ures Figur~s lA-Y: Atomic coordinates of crystal structure of soluble extracellular fragment of human CD40L cont~;ning residues Glyll6-Leu261 (in Brookhaven Protein Data Bank format).
(SEQ ID N0:1).

Figures 2A-C: Expression of CD40 in normal kidney.
Shown are frozen sections of normal kidney stained with ~..~lol mouse IgG (Figure 2A, magnification 25x) or anti-CD40 mAb G28.5 ~Figures 2B and 2C, magnification 40x).
Distal tubules and interstitial 1~ capillaries express CD40 while proximal tubules are CD40- (Figure 2B). Glomerular cells and epithelial cells of Bowmans capsule express CD40 (Figure 2C).

Figures 3A-C: Expression of CD40 in diffuse proliferative lupus nephritis. Shown are frozen sections of a kidney biopsy from a patient with Class IV lupus nephritis stained with control mouse IgG (Figure 3A, magnification 25x) or anti-CD40 mAb G28.5 (Figures 3B and 3C, magnification 40x).
Figure 38 shows intense CD40 staining of distal and proximal tubules. Figure 3C
shows increased and diffuse CD40 expression in the glomerulus. Figure 3C
also shows that the epithelial derived crescent is CD4 Ot .

Figure 4A: CD40L expression on interstitial mononuclear cells in class IV lupus glomerulonephritis. Shown is a frozen section obtained from a renal biopsy ~ CA 02243~31 1998-07-~ 9 7 ~ nn ~

~ ~ L ~ ~r'~A ~ lg~ 8 -S-specimen stained with anti-CD40L mAb 5c8.
Bound antibody was visualized with the Vectastain~ABC Elite kit followed by ~ne chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~. CD40L immunoreactivity is noted as staining of mononuclear cells.

Figur- 4B: Isotype control staining of interstitial mononuclear cells in class IV lupus glomerulonephritis. Shown is a frozen section obtained from the same patient studied in Figure 4A and stained with a~ -IgG2a isotype control mAb. Bound antibody was visualized with the Vectastain~ ABC
Elite kit followed by the chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~. Note the lack of immunoreactivity ( st~ i n i ng).

Figur- 5s CD40L expression on interstitial mononuclear cells in class IV lupus glomerulonephritis. Shown is a frozen section obtained from a renal biopsy specimen stained with anti-CD4OL mAb 5c8.
This specimen was obtained from a different patient than shown in Figure 4A.
Bound antibody was visualized with the Vectastain~ABC Elite kit followed by the chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~. CD40L immunoreactivity is noted as staining of mononuclear cells.

~7'~', CA 02243~31 1998-07-1~
PCT~US 9 ~ 6 6 g -6- ~ 'r'~1A~g98 Staininq with an isotype control mAb was negative (not shown).

F~gur- 6: Renal CD40 expression in focal segmental glomerulocclerosis (FSGS). Shown is a frozen section obtained from a renal biopsy specimen stained with anti-CD40 mAb G28.S. Bound antibody was visualized with the Vectastain ABC Elite kit followed by the chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~. Note the intense CD40 staining.
Staining with an isotype control mAb was~-negative (not shown).

F$gur- 7: CD40L expression on interstitial mononuclear cells in focal segmental glomerulosclerosis. Shown is a frozen section obtained from the same patient as studied in Figure 6 stained with anti-CD40L mAb 5c8. Bound antibody was visualized with the Vectastain ABC Elite kit followed by the chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma). CD40L
immunoreactivity is noted as staining of mononuclear cells. Staining with an isotype control mAb was negative (not shown).

Figur- 8: Renal CD40 expression in IgA nephropathy.
Shown is a frozen section obtained from a renal biopsy specimen stained with anti-CD40 mAb G28.5. Bound antibody was _ visualized with the Vectastain~ABC Elite A~,3~-t ~-.E ~

,,~ CA 02243531 1998-07-15 , PC~IU~ 97/î n6 -7- !P~ 1 2 MAR 19~8 kit followed by the chromogen 3-amino-s-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma2l. Note the intense S CD40 staining. Staining with an isotype control mAb was negative (not shown).

Figure 9: CD40L expression on interstitial mononuclear cells in IgA nephropathy.
Shown is a frozen section obtained from the same patient as studied in Figure 8 stained with anti-CD40L mAb 5c8. Bound antibody was visualized with the Vectastain~ABC Elite kit followed by th~
chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~. CD40L immunoreactivity is noted as staining of mononuclear cells.
Staining with as isotype control mAb was negative (not shown).

CA 02243~31 1998-07-1~
WO97/26000 PCT~S97/00668 ~et~iled Descri~tion This invention provides a method of inhibiting activation by CD40 ligand of renal cells bearing CD40 on the cell surface, comprising contacting the cells with an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an amount effective to inhibit activation of the cells. In one embodiment of this invention the agent is capable of inhibiting any interaction between CD40 ligand and CD40.
"Interaction between CD40 ligand and CD40 on the cells"
refers to one or more aspects, functional or structural, of a CD40-CD40 ligand interrelationship. Therefore, in one embodiment, an agent which inhibits interaction may competitively bind to CD40 ligand in such a way to block or ~; ;n;~h the binding of CD40 ligand to cellular CD40.
In another embo~ nt an agent which inhibits interaction may associate with CD40 or CD40 ligand in a manner which does not inhibit binding of CD40 ligand to cellular CD40, but which influences the cellular response to the CD40 ligation, such as by altering the turnover rate of the cellular CD40 or the CD40-agent complex, by altering binding kinetics of CD40 with CD40 ligand, or by altering the rate or extent of cellular activation in-response to CD40 ligation.

In specific embodiments the CD40-bearing renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, prn~;~l tubule cells, parietal epithelial cells, visceral epithelial cells, cells of a Henle loop or limb thereof, and interstitial inflammatory cells. In a more specific embodiment the parietal epithelial cells are crescent parietal epithelial cells.
In an embodiment of this invention the agent inhibits binding of CD40 ligand to CD40 on the cells.

CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 _g_ In an embodiment of this invention the agent is a protein.

In another embodiment of this invention the agent is a nonprotein. As used herein the term nonprotein includes any and all compounds or agents which encompass elements other than simple or coniugated polypeptide ch~in~. This includes elements such as amino acids having non-peptide linkages; no,l~roLein amino acids such as ~, y, or ~ amino acids, amino acids in D configuration, or other nonprotein amino acids including homocysteine, homoserine, citrulline, ornithine, y-aminobutyric acid, canavanine, djenkolic acid, or ~-cyanoalanine;
monosaccharides, polysaccharides, or carbohydrate moieties; fatty acids or lipid moieties; nucleotide moieties, mineral moieties; or other nonprotein elements.

In a specific embodiment the protein comprises an antibody or portion thereof capable of inhibiting interaction between CD40 ligand and CD40 on the cells.
The antibody is a monoclonal or po-yclonal antibody. In a more specific embodiment the monoclonal antibody specifically binds to the epitope to which monoclonal 25 antibody 5c8 (ATCC Accession No. HB 10916) specifically binds. An example of such a monoclonal antibody is monoclonal antibody 5c8 (ATCC Accession No. HB 10916).
In another ~ nt, the antibody specifically binds to CD40. One example of an anti-CD40 antibody is the monoclonal mouse anti-human CD40, available from Genzyme Customer Service (Product 80-3702-01, Cambridge, MA). In other embodiments the monoclonal antibody is a ch;m~ric antibody, a primatized antibody, a humanized antibody, or an antibody which includes a CDR region from a first human and an antibody scaffold from a second human.

The -~n; ng of "chimeric", "primatized" and "humanized"

CA 02243531 1998-07-15 _ ~
' 7 / 0 0 6 6 8 antibody and methods of producing them are well known to those of skill in the art. See, for exa-~le, PCT
International Publication No. W0 90/07861, published July 26, 1990 (Queen, et al.); and Queen, et al. Proc. Nat'l 5 Acad. Sci.-USA (1989) 86: 10029). Methods of making primatized antibodies are disclosed, for example, in PCT
International publication No. W0/02108, corresponding to International Application No. PCT/US92/06194 ( IDEC
Pharmaceuticals); and in Newman, et al., Biotechnoloqy (1992) 10:1455-1460, which are hereby incorporated by reference into this application.

Generally, a humanized antibody is an antibody comprisin~
one or more complementarity determining regions (CDRs) of a non-human antibody functionally joined to human framework region segments. Additional residues associated with the non-human antibody can optionally be present. Typically, at least one heavy chain or one light chain comprises non-human CDRs. Typically, the non-human CDRs are mouse CDRs. Generally, a primatized antibody is an antibody comprising one or more complementarity determining regions (CDRs) of an antibody of a species other than a non-human primate, functionally joined to framework region segments of a non-human pr~m~te. Additional residues associated with the species from which the CDR is derived can optionally be present.
Typically, at least one heavy chain or one light chain comprises CDRs of the species which is not a nonhuman primate. Typically, the CDRs are human CDRs. Generally, a chimeric antibody is an antibody whose light and/or heavy ~hAinc contain regions from different species. For example one or more variable (V) region segments of one species may be joined to one or more constant (C) region segments of another species. Typically, a chimeric antibody contains variable region segments of a mouse joined to human constant region segments, although o~her mammalian species may be used.

CA 02243~31 1998-07-1~
W097/26000 PCT~S97tO0668 Monoclonal antibody 5c8 is produced by a hybridoma cell which was deposited on November 14, 1991 with the American Type Culture Collection (ATCC), 12301 Parklawn Drive, Rockville, Maryland 208S2, 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. The hybridoma was accorded ATCC Accession Number HB 10916.

In a specific embodiment the portion of the antibody comprises a complementarity determining region or variable region of a light or heavy chain. In another specific embo~im~nt the portion of the antibody comprises a complementarity determining region or a variable 1~ region. In another specific embodiment the portion of the antibody comprises a Fab or a single chain antibody.
A single chain antibody is made up of variable regions linked by protein spacers in a single protein chain.

In another em~odiment the protein comprises soluble extracellular region of CD40 ligand, or portion thereof, or variant thereof, capable of inhibiting any interaction between CD40 ligand and CD40 on the cells; or soluble extracellular region of CD40, or portion thereof, or variant thereof, capable of inhibiting any interaction between CD40 ligand and CD40 on the cells. In a specific embodiment the soluble extracellular region of CD40 ligand or CD40 is a monomer. In another embodiment the soluble extracellular region of CD40 is an oligomer Variants can differ from naturally occurring CD40 or CD40 ligand in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring CD40 or CD40 ligand is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. Particularly CA 02243~31 1998-07-1~
W097/26000 PCT~S97100668 preferred variants include naturally occurring CD40 or CD40 ligand, or biologically active fragments of naturally occurring CD40 or CD40 ligand, whose sequences differ from the wild type sequence by one or more conservative amino acid substitutions, which typically have ; n;~l influence on the secondary structure and hydrophobic nature of the protein or peptide. Variants may also have sequences which differ by one or more non-conservative amino acid substitutions, deletions or insertions which do not abolish the CD40 or CD40 ligand biological activity. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine;
1~ valine, isoleucine; aspartic acid,glutamic acid;
asparagine,glutamine; serine,threonine;lysine, arginine;
and phenylalanine,tyrosine. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine.
The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Other conservative substitutions can be taken from Table 1, and yet others are described by Dayhoff in the Atlas of Protein Sequence and Structure (1988).

CA 02243~3l l998-07-l~
W097/260~0 PCT~S97/00668 Table l: Conservative Amino Acid Replacements For Amino Acid Code Replace with any of Alanine A D-Ala, Gly,beta-ALa, L-Cys,D-Cys ~ Arginine R D-Arg, Lys,homo-Arg, D-homo-Arg, Met,D-Met, Ile, D-Ile, orn, D-Orn Asparagine N D-Asn,Asp,D-Asp,Glu,D-Glu, Gln,D-Gln Aspartic Acid D D-Asp,D-Asn,Asn, Glu,D-Glu, Gln, D-Gln Cysteine C D-Cys, S-Me-Cys,Met,D-Met,Thr, D-Thr Glutamine Q D-Gln,Asn, D-Asn,Glu,D-Glu,Asp, D-Asp Glutamic Acid E D-Glu,D-Asp,Asp, Asn, D-Asn, Gln, D-Gln Glycine G Ala, D-Ala,Pro, D-Pro, Beta-Ala, Acp Isoleucine I D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine L D-Leu, Val, D-Val, Met, D-Met Lysine K D-~ys,Arg, D-Arg, homo-Arg, D-homo-Arg, Met, D-Met, Ile, D-Ile, Orn, D-Orn Methionine M D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val, Norleu 15 Phenylalanine F D-Phe,Tyr, D-Thr,L-Dopa,His,D-His, Trp, D-Trp, Trans 3,4 or 5-phenylproline, cis 3,4 or 5 . phenylproline Proline P D-Pro, L-I-thioazolidine-4-carboxylic acid, D- or L-l-oxazolidine-4-carboxylic acid CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 Serine S D-Ser, Thr, D-Thr, allo-Thr, Met, D-Met, Met(O), D-Met(O), Val, D-Val Threonine T D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Met(O) D-Met(O), Val, D-Val Tyrosine Y D-Tyr,Phe, D-Phe, L-Dopa, His,D-His Valine V D-Val, Leu,D-Leu,Ile,D-Ile, Met, D-Met Other variants within the invention are those with modifications which increase peptide stability. Such variants may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) 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 or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Patent 5,219,990.

The peptides of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or nonconservative where such changes might provide for certain advantages in their use.

In other embodiments, variants with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties.
Such substitutions would include for example, substitution of hydrophilic residue ~or a hydrophobic CA 02243~3l l998-07-l~
W097/26000 PCT~S97/~0668 residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed 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 having at least eighty percent homology with the extracellular region of CD40 or the extracellular region of CD40 ligand. More preferably the sequence homology is at least ninety percent, or at least ninety-five percent.

Just as it is possible to replace substituents of the scaffold, it is also possible to substitute functional groups which decorate the scaffold with groups characterized by similar features. These substitutions will initially be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequence modifications may include, for example, in vivoor in vitro chemical derivatization of portions of naturally occurring CD40 or CD40 ligand, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycolsylation.
3û
ln a further embodiment the protein, including the extracellular region of CD4û ligand and CD40, is modified ~y chemical modifications in which activity is preserved.
For example, the proteins may be amidated, sulfated, singly or multiply halogenated, alkylated, carboxylated, or phosphorylated. The protein may also be singly or multiply acylated, such as with an acetyl group, with a CA 02243~31 1998-07-1~
W097/Z6000 PCT~S97/00668 farnesyl moiety, or with a fatty acid, which may be saturated, monounsaturated or polyunsaturated. The fatty acid may also be singly or multiply fluorinated. The invention also includes methionine analogs of the protein, for example the methionine sulfone and methionine sulfoxide analogs. The invention also includes salts of the proteins, such as ammonium salts, including alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and benzensulfonate salts.

The soluble, -nom~ic CD40-L protein can comprise all or part of the extracellular region of CD40-L. The extracellular region of C~40-L contains the domain that binds to CD40. Thus, soluble CD40-L can inhibit the interaction between CD4OL and the CD40-bearing cell.
This invention contemplates that sCD40-L may constitute the entire extracellular region of CD40-L, or a fragment or derivative containing the domain that binds to CD40.

Soluble CD40 protein (sCD40) comprises the extracellular region of CD40. sCD40 inhibits the interaction between 25 CD40L and CD40-bearing cells. sCD40 may be in monomeric or oligomeric form.

In another embodiment of this invention the protein comprising soluble extracellular region of CD40 or portion thereof further comprises an Fc region fused to the extracellular region of CD40 or portion ~hereof. In a specific : hnAim~nt the Fc region is capable o~ binding to protein A or protein G. In another embodiment the Fc region comprises IgG, IgGl, IgG2, IgG3, IgG4, IgA, IgA1, IgA2, IgM, IgD, or IgE.

The soluble CD40/Fc fusion protein can be prepared using CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 conventional techniques of enzymes cutting and ligation of fragments from desired sequences. Suitable ~c regions for the fusion protein are Fc regions that can bind to protein A or protein G, or that are capable of recognition by an antibody that can be used in purification or detection o~ a fusion protein comprising the Fc region. For example, the Fc region may include the Fc region of human IgGl or murine IgG1. This invention also provides a nucleic acid molecule which encodes the CD40/Fc fusion protein.

The method of creating soluble forms of membrane molecules by recombinant means, in which sequences encoding the tra~! - hrane and cytoplasmic domains are deleted, is well known. See generally Hammonds et al., U.S. Patent No. 5,057,417. In addition, methods of preparing sCD40 and CD40/Fc fusion protein are well-known. See, e.g., PCT International Publication No. WO
93/08207; Fanslow et al., "Soluble Forms of C~40 Inhibit Biologic Responses of Human B Cells, "J. Immunol., vol.
149, pp.655-60 (July 1992).

In an embodiment of this invention, the agent is selected by a screening method.
In a specific embodiment the agent is selected by a screening method, which comprises isolating a sample of cells; culturing the sample under conditions permitting activation of CD40-bearing cells; contacting the sample with cells expressing a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. HB 10916, or with a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC
3~ Accession no. ~B 10916, effective to activate the CD40-bearing cells; contacting the sample with an amount of the agent effective to inhibit activation of the CD40-CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 bearing cells if the agent is capable of inhibiting activation of the CD40-bearing cells; and determining whether the cells expressing the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. HB
10916, or with the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. ~B 10916, activate the CD40-bearing cells in the presence of the agent. The cell sample may be isolated from diverse tissues, including cell lines in culture or cells isolated from an animal, such as dispersed cells 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-dimensiQnal structure of soluble extracellular region of CD40 ligand or portion thereof capable of inhibiting interaction between CD40 ligand and CD40 on the cells. The agent may 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-~; -n~ional structure. In specific embodiments the agent (molecule) is designed by structure optimization of a lead inhibitory agent based on a three-dimensional structure of a complex of the soluble extracellular region of CD40 ligand or portion thereof with the lead inhibitory agent. A lead inhibitory agent is a molecule which has been identified which, when it is contacted with CD40 ligand, binds to and complexes with the soluble extracellular region of CD40 ligand, CD40, or portion thereof, thereby decreasing the ability of the complexed or bound CD40 ligand or CD40 ligand portion to activate CD40-bearing cells. In another embodiment, a lead inhibitory agent may act by interacting with either the CA 02243531 1998-07-1~ ~1 - PCT/~S 97/0066&

19 IPEA/US ~ ~ ~AR-1998 extracellular region of CD40 ligand, CD40, or in a tertiary complex with both a portion of CD40 ligand and CD40, decreasing the ability of the complexed CD40 ligand-CD40 to activate the CD40-bearing cells. In the S methods of the invention, the CD40 ligand may be either soluble or bound to cells such as activated T cells, and may be either full length native CD40 ligand or portions thereof. Decreased ability to activate CD40-bearing cells may be measured in different ways. One way it may be measured is by showing that CD40 ligand, in the presence of inhibitor, causes a lesser degree of activation of CD40-bearing cells, as compared to treatment of the cells with a similar amount of CD40 ligand without inhibitor under similar condition~ -Decreased ability to activate CD40-bearing cells may also be indicated by a higher concentration of inhibitor-CD40 ligand complex being required to produce a similar degree of activation of CD40-bearing cells under similar conditions, as c~ ~red to unbound CD40 ligand. At the extreme, the inhibitor-contacted CD40 ligand may be unable to activate CD40-bearing cells at concentrations and under conditions which allow activation of these cell~ by unbound CD40 ligand or a given portion thereof.

2S The agent (molecule) can be selected by a computational scr~en~ n7 method using the crystal structure of a soluble fragment of the extracellular domain of human CD40L
contAin~nq residues Glyll6-Leu261 (SEQ ID. NO: l)(SCD40L
(116-261).
- 30 The crystal structure to be used with the screening method has been determined at 2 A resolution by the method of molecular replacement. In brief, a soluble fragment of the extracellular domain of human CD40 ligand containing amino acid residues Gly 116 to the c-terminal residue Leu 261 was first produced in soluble form, then purified and crystallized. The crystals were used to collect diffraction data. Molecular replacement and CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 refinement were done with the XPLOR program package and QUANTA (Molecular Simulations, Inc.) Software. In particular, a 3-dimensional model of human sCD40L was constructed using the murine CD4OL model using QUANTA
protein homology modeling software. This model was used as a probe for crystallographic analysis calculations and refined using XPLOR. This method of determining the crystal structure of sCD4OL is described in more detail in Karpusas et al., "2 ~ crystal structure of an extracellular fragment of human CD40 ligand," Structure (October 1995) 3(10):1031-1039. The atomic coordinates of sCD40L(116-261) are provided in Figures lA-Y. The screening method for selecting an agent includes computational drug design and iterative structure optimization, as described below.

The agent may be an inhibitor selected using ~ul~.~u~atio~l drug design. Using this method, the sCD40L
crystal structure coordinates are used as an input for a computer program, such as DOCK, which outputs a list of molecular structures that are expected to bind to CD40L.
Use of such computer programs is well-known. See, e.g., Kuntz, UStructure-Based Strategies for drug design and discovery," Science, vol. 257, p. 107~ (1992~. The list of molecular structures can then be screened by biochemical assays for CD40L binding. Competition-type biochemical assays, which are well known, can be used.
See, e.g., Bajorath et al., "Identification of residues of CD40 and its ligand which are critical for the receptor-30 ligand interaction," Biochemistry, 34, p. 1833 (199~).
The structures that are found to bind to CD40L can thus be used as agents for the present invention. The agent may also be a modified or designed molecule, determined by interactive cycles of structure optimization. Using this approach, a small molecule inhibitor of CD40L found using the above computational approach or other approach can be co-crystallized with sCD40L and the crystal CA 02243~31 1998-07-1~
W097126000 PCT~S97/00668 structure of the complex solved by molecular replacement.
The information revealed through molecular replacement can be used to optimize the structure of the inhibitors ~ by clarifying how the molecules interact with CD4OL. The molecule may be modified to improve its physiochemical properties, including specificity and affinity for CD40L.

In an emboA;~ent of this invention the agent is a small molecule. As used herein a small molecule is a compound 10 having a molecular weight between 20 Da and lx106 Da, preferably from 50 Da to 2 kDa.

This invention also provides a method of inhibiting activation by CD40 ligand of renal cells bearing CD40 on the surface of the cells, in a subject, comprising administering to the subject an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an amount effective to inhibit activation of the cells in the subject.
In specific embodiments the CD40-bearing renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubules, pro~imal tubules, parietal epithelial cells, visceral epithelial cells, cells of a Henle loop or limb thereof, and interstitial inflammatory cells. In a more specific embodiment the parietal epithelial cells are crescent parietal epithelial cells.

In an embodiment of this invention the agent inhibits binding of CD40 ligand to CD40 on the cells.

In an embodiment of this invention the agent is a protein. In another embodiment of this invention the agent is a nonprotein.

In a specific embodiment the protein comprises an CA 02243~3l l998-07-l~
W097/26000 PCT~S97/00668 antibody or portion thereof capable of inhibiting any interaction between CD40 ligand and CD40 on the cells.
The antibody is a monoclonal or polyclonal antibody. In a more specific embodiment the monoclonal antibody S specifically binds to the epitope to which monoclonal antibody 5c8 (ATCC Accession No. HB 10916~ specifically binds. An example of such a monoclonal antibody is monoclonal antibody 5c8 (A~CC Accession No. HB 10916).
In other embodiments the monoclonal antibody is a chimeric antibody or a humanized antibody.

In a specific embodiment the portion of the antibody comprises a complementarity determining region or variable region of a light or heavy chain. In another lS specific embo~;m~nt the portion of the antibody comprises a complementarity determining region or a variable region. In another specific embodiment the portion of the antibody comprises a Fab or a single chain antibody.

In another embodiment the protein comprises soluble extracellular region of CD40 ligand or portion thereof capable of inhibiting any interaction between CD40 ligand and CD40 on the cells; or soluble extracellular region of CD40 or portion thereof capable of inhibiting any interaction between CD40 ligand and CD40 on the cells.
In a specific embo~; -nt the soluble extracellular region of CD40 ligand or CD40 is a monomer. In another ~ ' Ai -nt the soluble extracellular region of CD40 is an oligomer.
In another embodiment of this invention the protein comprising soluble extracellular region of CD40 or portion thereof further comprises an Fc region fused to the extracellular region of CD40 or portion thereof. In 3~ a specific em~odiment the Fc region is capable of binding to protein A or protein G. In another specific embodiment the Fc region comprises IgG, IgGl, IgGz, IgG3, CA 02243~3l l998-07-l~
W097/26000 PCT~S97/00668 IgG~, IgA, IgA1, IgA2, IgM, IgD, or IgE.

The sub~ect which can be treated by the above-described methods is an ~n; ~1. Preferably the animal is a mammal.
Examples of mammals which may be treated include, but are not limited to, humans, non-human primates, rodents (including rats, mice, hamsters and guinea pigs) cow, horse, sheep, goat, pig, dog and cat.

In an embodiment of this invention, the agent is selected by a scr~n;ng method.

In a specific embodiment the agent is selected by a screening method, which comprises isolating a sample of cells; culturing the sample under conditions permitting activation of CD40-bearing cells; contacting the sample with cells expressing a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. HB 10916, or with a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC
Accession no. HB 10916, effective to activate the CD40-bearing cells; contacting the sample with an amount of the agent effective to inhibit activation of the CD40-bearing cells if the agent is capable of inhibiting activation of the CD40-bearing cells; and determining whether the cells expressing the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. HB
30 10916, or with the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession no. HB 10916, activate the CD40-bearing cells in the presence of the agent. The cell sample may be isolated from diverse tissues, including cell lines in culture or cells isolated from an ~n;-~l, such as dispersed cells from a solid tissue, cells derived from a bone marrow biopsy, or cells CA 02243~31 1998-07-1~
W097/260~0 PCT~S97/00668 isolated from a body fluid such as blood or lymphatic fluid.

In another specific em~odiment the molecule (agent) is S selected based on a three-dimensional structure of soluble extracellular region of CD40 ligand or portion thereof capable of inhibiting any interaction between CD40 ligand and CD40 on the cells. The molecule may be selected from a library of known molecules, modified from a known molecule based on the three~ n~ional structure, or designed and synthesized de novo based on the three-~i -~ional structure. In specific embodiments the agent or molecule is designed by structure optimization of a lead inhibitory agent based on a three-dimensional structure of a complex of the solubleextracellular region of CD40 ligand or portion thereof with the lead inhibitory agent.

Method of Treatment This invention provides a method of treating, in a subject, an inflammatory kidney disease, comprising the above-described method of inhibiting activation by CD40 ligand of renal cells bearing CD40 on the surface of the cells, which comprises administering to the subject an agent capable of inhibiting interaction between CD40 ligand and CD40 on the cells, the agent being present in an ~-L effective to inhibit activation of the cells in the subject, thereby treating the inflammatory kidney disease.

The inflammatory kidney disease may be one which is initiated by autoantibody deposition in kidney, or one which is not initiated by autoantibody deposition in kidney. ~any kidney diseases for which the methods of the invention are useful include ones which have multifactorial etiology.

CA 02243~31 1998-07-1~

In an embodiment of this invention the kidney disease is selected from the group consisting of: membranous glomerulonephritis, m;nim-l change disease/acute tubular - necrosis; pauci-immune glomerulonephritis; focal segmental glomerulosclerosis; interstitial nephritis;
antitissue antibody-induced glomerular injury, such as anti-basement membrane antibody disease; circulating immune-complex disease; glomerulopathies associated with multisystem diseases; drug-induced glomerular disease;
renal transplant rejection; rapidly progressive glomerulonephritis; and post-streptococcal glomerulonephritis. Circulating immune-complex diseases include infective endocarditis, leprosy, syphilis, hepatitis B, malaria, and diseases of endogenous antigens 1~ such as DNA, thyroglobulin, autologous immunoglobulins, erythrocyte stroma, renal tubule antigens, and tumor-specific or tumor-associated antigens. Glomerulopathies associated with multisystem diseases include diabetic nephropathy, systemic lupus erythematosus, Goodpasture's disease, vasculitis, multiple myeloma, Waldenstrom's macroglobulinemia, and amyloidosis. In specific embodiments the vasculitis is Henoch-Schonlein purpura, polyarteritis nodosa (sometimes called polyarteritis), Wegener's granulomatosis, cryoglobulinemia (sometimes 2~ called cryoimmunoglobulinemia). The kidney disease may also be one which affects the renal tubules, such as toxins, neoplasias, hypersensitivity nephropathy, Sjogren's syndrome, and AIDS. In a specific embodiment the pauci-immune glomerulonephritis is ANCA+ pauci-immune glomerulonephritis, or Wegener's granulomatosis. In another specific embo~;m~nt the interstitial nephritis is drug-induced interstitial nephritis.

The compounds of this invention may be administered in any ~n~er which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, CA 02243~31 1998-07-1~ -W097/26000 PCT~S97/00668 intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophth~l ic, rectal, topical, or inhaled.
Sustained release administration is also specifically included in the invention, by such means as depot in;ections of erodible implants directly applied during surgery.

The compounds are A~; n;~tered at any dose per body weight and any dosage fre~uency which is medically acceptable. Acceptable dosage includes a range of between about 0.01 and 200 mg/kg subject body weight. A
preferred dosage range is between about 0.1 and 50 mg/kg.
Particularly preferred is a dose of between about 1 and 30 mg/kg. The dosage is repeated at intervals ranging from each day to every other month. One preferred dosing regimen is to administer a compound of the invention daily for the first three days of treatment, after which the compound is administered every 3 weeks, with each ~i n; ctration being intravenously at 5 or 10 mg/kg body weight. Another preferred regime is to administer a compound of the invention daily intravenously at 5 mg/kg body weight for the first three days of treatment, after which the compound is administered subcutaneously or intramuscularly every week at lO mg per subject. Another preferred regime is to administer a single dose of the compound of the invention parenterally at 20 mg/kg body weight, followed by a~ ;n;~tration of the compound subcutaneously or int~ cularly every week at 10 mg per subject.

The compounds of the invention may be administered as a single dosage for certain indications such as preventing immune response to an antigen to which a subject is exposed for a brief time, such as an exogenous antigen a~;n;ctered on a single day of treatment. Examples of such an antigen would include coadministration of a ~7 ~ R ~g~
compound of the invention along with a gene therapy vector, or a therapeutic agent such as an antigenic pharmaceutical or a blood product. In indications where antigen is chronically present, such as in controlling S immune reaction to transplanted tissue or to chronically administered antigenic pharmaceuticals, the compounds of the invention are administered at intervals for as long a time as medically indicated, ranging from days or weeks to the life of the subject.
Inflammatory responses are characterized by redness, swelling, heat and pain, as consequences of capillary dilation with edema and migration of phagocytic leukocytes. Inflammation is further defined by Galli~ ~
(Chapter 26, Fun~amental Immunology, 2d Ed., Raven Press, New York, 1989, pp. 72l-733), which is herein incorporated by reference.

This invention will be better understood from the Experimental 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.
~p-ri~ ~t~l Details CD40 expression in normal kidney and in renal biopsy specimens obtained from patients with systemic lupus erythematosus and other kidney diseases was examined.

and M-tho~s ~..~hi ~toch-~i~try Immunohistochemical analyses of frozen sections were performe 7 with a Vectastain~ Elite Kit (Vector, '~END~

CA 0 2 2 4 3 5 3 1 19 9 8 - 0 7 - 15 ~ ,~ 9 7 -28- ~PEA/VS i2~1A~l~B
Burlingame, CA) as previously described. Briefly, the tissue was first blocked with PBS containing horse serum and 1% BSA and additional blocking was obtained utilizing an Avidin/8iotin Blocking Kit also purchased from Vector~
The sections were then stained with 1:1000 dilutions of anti-CD40 mAb G28.5 or an isotype control mAb in PBS
followed by biotinylated horse anti-mouse IgG.
Endogenous peroxidase activity was blocked with 1:400 dilution of H202. Bound antibody was visualized with the Vectastain~ABC reagent followed by the chromogen 3-amino-9-ethylcarbazole (Vector Laboratories). The tissue was counterstained with Mayer's hematoxylin (Sigma~.

Staining was evaluated visually. In the following tables ~0~ indicatas no staining; 1+ indicates minimal staining, 2+ indicates moderate staining; and 3+ indicates intense staining.

Re~ult~
~-1~5i~ of C~0 ~~pr-ssion in norm-l ki~n-Y

Initial studies of renal CD40 expression were prompted by the observation that CD40 is normally expressed on endothelial cells in a variety of tissues. Consistent with this finding, it was found that renal interstitial capillaries and larger vessels express CD40. CD40 was also found to be expressed on other renal parenchymal cells, such as glomerular endothelial cells, glomerular mesangial cells and parietal epithelial cells of Bowman's capsule. Glomerular visceral epithelial cells do not express CD40. Distal tubules are strongly immunoreactive for CD40 and staining was most intense along the basolateral membrane. In contrast, proximal tubules are not immunoreactive with anti-CD40 mAb. An isotype control mAb did not stain renal specimens. The --- immunoreactivity noted with anti-CD40 mAb G28.5 is most L ~

CA 02243~31 1998-07-1~
W097/26000 PCT~S97/00668 likely specific and does not represent cross-reactivity because similar staining was noted with an additional anti-CD40 mAb. Thus it is concluded that renal parenchymal cells differentially express CD40.
s ~nalY8is of renal CD40 expression in 8yStQmiC 1UPUS
erYthematosu~

Whether renal CD40 expression is upregulated in lupu5 glomerulonephritis was analyzed. Frozen sections obt~;ne~ from patient biopsy specimens were stained with anti-CD40 mAb G28.5 or an isotype control mAb.

1~ Renal CD40 expression in systemic lupus erythematosus was analyzed. Patients with Class III and IV lupus nephritis tended to have increased CD40 expression on glomerular endothelial cells, mesangial cells and distal tubules.
In addition, proximal tubules are CD40+ in patients with Class III and IV lupus nephritis. Also, there is striking CD40 expression on parietal epithelial cells in patients with crescent formation. CD40 is also present on interstitial inflammatory cells. The distribution and intensity of renal CD40 expression in patients with pure Class V disease was similar to that seen in normal kidney.

Whether renal CD40 upregulation was unique to systemic lupus erythematosus was investigated. To do so, CD40 expression was investigated in patients with the following renal diseases: membranous glomerulonephritis, m;n;~l change disease/acute tubular necrosis, ANCA+
pauci-immune glomerulonephritis, focal segmental glomerulosclerosis and IgA nephropathy. Proximal tubule CD40 expression was upregulated in ANCA+ pauci-immune glomerulonephri~is, focal segmental glomerulosclerosis and IgA nephropathy. In contrast, there was little W097/26000 PCT~S97/00668 proximal tubule CD40 immunoreactivity in membranous glomerulonephritis or mi n; ~1 change disease/acute tu'bular necrosis. Crescent parietal epithelial cells in IgA nephropathy are also striking CD40+. Interstitial inflammatory cells, when present, also express CD40.
These findings demonstrate that CD40 expression is upregulated in a variety of inflammatory renal diseases.
Moreover., these studies indicate that CD40L mediated interactions with renal parenchymal cells play roles in normal renal physiology and augment inflammatory responses in renal diseases.

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-W097/26000 PCT~S97/00668 Analysis of re~l CD40-ligand expression in inflammatory r~nal di~eases In situ CD40L expression was studied in renal biopsy specimens from patients with SLE GN (n=18), as well as in normal kidney and biopsy specimens from patients with IgA
nephropathy, focal segmental glomerulosclerosis, mi";~l change ~ic~e~ idiopathic membranous &N and ANCA+ pauci-immune GN. T~lnoh; ~tochemical studies were performed on frozen sections utilizing anti-CD40L mAb 5C8 or controls mAbs. Upregulation of CD40L expression is observed in class IV lupus glomerulonephritis (Figures 4A, 4B and 5), focal segmental glomerulosclerosis (Figure 7 ) and Iga nephropathy (Figure 9). CD40L expression is noted as dim, discrete st~;n;ng of some infiltrating mononuclear cells.
These results provide further evidence that CD4OL
mediated signals play a role in the immunopathogenesis of inflammatory glomerular or tubulointerstitial diseases by interacting with CD40+ target cells in the kidney.

W097/26000 PCT~S97/00668 SEQUENCE LISTING

(1~ GENERAL INFORMATION:
(i) APPLICANTS: Yellin, Michael J.
- Lederman, Seth ~ Chess, Leonard - Karpusas, Mihail N.
Thomas, David W.
(ii) TITLE OF lNv~N~ ON: THERAPEUTIC APPLICATIONS OF T-BAM
(CD40-L) TECHNOLOGY TO TREAT
INFLAMMATORY KIDNEY DISEASES
(iii) NUMBER OF SEQUENCES: 1 (iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Cooper ~ Dunham LLP
(B) STREET: 1185 Avenue of the Americas (C) CITY: New York (D) STATE: New York (E) COUNTRY: USA
(F) ZIP: 10036 ( V ) COM~U'l'~ A n~ RT ~F~ FORM:
(A) MEDIUM TYPE: Floppy disk (B) COM~U~l~: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.30 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: Not Yet Known (B) FILING DATE: Herewith (C) CLASSIFICATION:
(vii) PREVIOUS APPLICATION DATA:
(A) APPLICATION NUMBER: US 08/587,334 (B) FILING DATE: 16-JAN-1996 (C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: White Esq., John P.
(B) REGISTRATION NUMBER: 28,678 (C) REFERENCE/DOCKET NUMBER: 48558-B
(ix) TELECOMMUNICATION INFORMATION:
- (A) TELEPHONE: (212)278 0400 (B) TELEFAX: (212)391 0525 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LEN&TH: 146 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein CA 0224353l l998-07-l5 W097/26000 PCT~S97/00668 -3~-(iii) HYP~~ CAL: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Gly Asp Gln Asn Pro Gln Ile Ala Ala His Val Ile Ser Glu Ala Ser ~er Lys Thr Thr Ser Val Leu Gln Trp Ala Glu Lys Gly Tyr Tyr Thr Met Ser Asn Asn Leu Val Thr Leu Glu Asn Gly Lys Gln Leu Thr Val Lys Arg Gln Gly Leu Tyr Tyr Ile Tyr Ala Gln Val Thr Phe Cys Ser Asn Arg Glu Ala Ser Ser Gln Ala Pro Phe Ile Ala Ser Leu Cys Leu ~ys Ser Pro Gly Arg Phe Glu Arg Ile Leu Leu Arg Ala Ala Asn Thr ~is Ser Ser Ala Lys Pro Cys Gly Gln Gln Ser Ile His Leu Gly Gly lOO 105 llO

Val Phe Glu Leu Gln Pro Gly Ala Ser Val Phe Val Asn Val Thr Asp Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu Lys Leu

Claims (42)

What is claimed:

1. A method of treating, in a subject, an inflammatory kidney disease having initiation points other than autoantibody deposition, comprising the step of administering to the subject an agent capable of inhibiting activation by CD40 ligand of renal cells bearing CD40 on the surface of the cells, in an amount effective to inhibit activation of the renal cells in the subject.

2. The method of claim 1, wherein the kidney disease is selected from the group consisting of:
minimal change disease;
acute tubular necrosis;
interstitial nephritis; and amyloidosis.

3. The method of claim 1, wherein the kidney disease affects renal tubules.

4. The method of claim 3, wherein the kidney disease which affects renal tubules is selected from the group of:
a kidney disease associated with a toxin and a neoplasia.

5. The method of claim 1, wherein the renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, proximal tubule cells, parietal epithelial cells, visceral epithelial cells, cells of a Henle limb, and interstitial inflammatory cells.

6. The method of claim 5, wherein the parietal epithelial cells are crescent parietal epithelial cells.

7. The method of claim 1, wherein the subject is a mammal.

8. The method of claim 7, wherein the mammal is a human.

9. The method of claim 7, wherein the mammal is a rodent.

10. The method of claim 1, wherein the agent is capable of inhibiting binding of CD40 ligand to CD40 on the cells.

11. The method of claim 1, wherein the agent specifically binds the antigen to which monoclonal antibody 5c8 (ATCC Accession No. HB 10916) specifically binds.

12. The method of claim 1, wherein the agent is selected from a library of known reagents.

13. The method of claim 1, wherein the agent is modified from a known agent.

14. The method of claim 13, wherein the modified agent is designed by structure optimization of a lead inhibitor based on a three-dimensional structure of a complex of soluble extracellular region of CD40 ligand or portion thereof with the lead inhibitor.

15. The method of claim 1, wherein the agent is a protein.

16. The method of claim 15, wherein the protein comprises an antibody or portion thereof.

17. The method of claim 16, wherein the antibody is a monoclonal antibody.

18. The method of claim 16, wherein the portion of the antibody comprises a complimentarity determining region or variable region of a light or heavy chain.

19. The method of claim 16, wherein the portion of the antibody comprises a complimentarity determining region or a variable region.

20. The method of claim 19, wherein the portion of the antibody comprises a Fab or a single chain antibody.

21. The method of claim 17, wherein the monoclonal antibody specifically binds to the epitope to which monoclonal antibody 5c8 (ATCC Accession No. HB
10916) specifically binds.

22. The method of claim 21, wherein the monoclonal antibody is monoclonal antibody 5c8 (ATCC
Accession No. HB 10916).

23. The method of claim 17, wherein the monoclonal antibody a chimeric antibody.

24. The method of claim 17, wherein the monoclonal antibody is a humanized antibody.

25. The method of claim 17, wherein the monoclonal antibody specifically binds to CD40.

26. The method of claim 25, wherein the antibody is humanized, chimeric, or primatized.

27. The method of claim 1, wherein the agent comprises a soluble extracellular region of CD40 ligand, or variant thereof including conservative substituents, or portion thereof; or soluble extracellular region of CD40, or variant thereof including conservative substituents, or portion thereof.

28. The method of claim 27, wherein the soluble extracellular region of CD40 ligand or CD40 is a monomer.

29. The method of claim 27, wherein the soluble extracellular region of CD40 is an oligomer.

30. The method of claim 27, wherein the soluble extracellular region of CD40 or portion thereof or CD40 ligand or portion thereof further comprises an Fc region fused to the extracellular region of CD40 or portion thereof or CD40 ligand or portion thereof.

31. The method of claim 30, wherein the Fc region is capable of binding to protein A or protein G.

32. The method of claim 31, wherein the Fc region comprises IgG, IgA, IgM, IgD or IgE, or subclasses thereof.

33. The method of claim 32, wherein the IgG is IgG1, IgG2, IgG3 or IgG4; or the IgA is IgA1 or IgA2.

34. The method of claim 1, wherein the agent is a nonprotein.

35. The method of claim 1, wherein the agent is a small molecule.

36. The method of claim 1, wherein the agent is administered to the subject with a gene therapy vector, a therapeutic agent or a blood product.

37. The method of claim 36, wherein the therapeutic agent is an antigenic pharmaceutical.

38. The method of claim 1, wherein the agent is selected by a screening method, comprising the steps of:
(a) isolating a sample of cells;
(b) culturing the sample under conditions permitting activation of CD40-bearing cells;
(c) contacting the sample with cells expressing a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession No. HB 10916, or with a protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession No. HB
10916, effective to activate the CD40-bearing cells;

(d) contacting the sample with an amount of the agent effective to inhibit activation of the CD40-bearing cells if the agent is capable of inhibiting activation of the CD40-bearing cells; and (e) determining whether the cells expressing the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession No. HB 10916, or with the protein which is specifically recognized by monoclonal antibody 5c8 produced by the hybridoma having ATCC Accession No.
HB 10916, activate the CD40-bearing cells in the presence of the agent.

39. The method of claim 38, wherein the sample of cells is isolated from tissues.

40. The method of claim 38, wherein the sample of cells is selected from the group consisting of:
cell lines in culture;
cells isolated from an animal; and cells isolated from a body fluid.

41. The method of claim 38, wherein the agent is selected from a library of known reagents.

42. The method of claim 41, wherein the known agents are nonprotein agents.