Note: Descriptions are shown in the official language in which they were submitted.
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
--2--
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.
-
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W097/26~0 PCT~S97100668
-4-
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
-8-
~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~
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_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
-10- -
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
12
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).
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-13-
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
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W097/26000 PCT~S97/00668
-14-
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
-15-
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
-16-
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
-17-
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
-18-
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
-20-
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
-21-
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
-23-
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
-24-
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~
WO 97/26000 PCT/US97/00668
-25-
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
-26-
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
CA 02243531 1998-07-15
~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
-29-
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
CA 02243531 1998-07-15
W097/26000 PCT~S97/00668
-30-
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.
CA 02243531 1998-07-15
WO 97/26000 PCT/US97/00668
--3i--
r + +
a c~
c
a a a,
SU ~L ~
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,_, a V u u
c ~ I r
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a ~r~ u
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CA 02243531 1998-07-15
WO 97/26000 PCTWS97/00668
--32--
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a) ~ o++++++++++++++ a~+
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a~
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V rn ~ ~
t~') H H ~ I H H H H H H H H H
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CA 02243531 1998-07-15
WO 97126000 PCT/US97/00668
--33--
o U~+ +
v +, + + + + + + +,a) + ~, o +i S-~
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,, ~
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H ~--.i
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a, v
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U~ ~
V H ~ H H H H H H ~ H
-- ~ H
~ L~) o c\i o I ~
U~ o ~ o
CA 02243531 1998-07-15
WO 97/26000 PCTIUS97/00668
--34--
~ + + +
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o~3 ~ ++1 '-~+1 +
a
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C o V O O ~ O +
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V ' t-'
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t~ ~ I ~ I t
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U'~ o
CA 02243531 1998-07-15
WO 97126000 PCT/US97/00668
--35--
~ + + + + +
U~
-
U~ -~ + o +
X ~ ~ C~
o
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-
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~U~
s~
a~
o~ + + + +
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U~
Z ~
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++~ + + +
C p~
.,
aJ
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V ~ ~ Z
E~
,~,, o a~ c~l o ~I
a) ~ ~ ~ ~ t~l
~u V V ~
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-
CA 02243531 1998-07-15
W097/26000 PCT~S97/00668
-36-
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.
CA 02243531 1998-07-15
W097/26000 PCT~S97/00668
-37-
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
1 5 10 15
~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
115 120 125
Pro Ser Gln Val Ser His Gly Thr Gly Phe Thr Ser Phe Gly Leu Leu
130 135 140
Lys Leu
145
