AU2006202590A1 - Methods for treating autoimmune and inflammatory conditions using antagonists of CD30 or CD30L - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: Immunex Corporation Actual Inventor(s): Kendall M. Mohler, Dauphine S. Barone, Jacques J. Peschon, Mary K. Kennedy, John D.

Pluenneke Address for Service and Correspondence: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: 9 METHODS FOR TREATING AUTOI UN NFLAMMATORY CONDITIONS USING ANTAGONISTS OF CD30 OR Our Ref: 775755 POF Code: 461115/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): 1 8006q METHODS FOR TREATING AUTOIMMUNE AND INFLAMMATORY CONDITIONS USING ANTAGONISTS OF CD30 OR The present application is a divisional application from Australian patent application number 2001281163 the entire disclosure of which is incorporated herein by reference.

FIELD OF THE INVENTION This invention relates generally to methods for treating autoimmune and inflammatory disorders that involve administering agents that block the interaction between CD30 and or IL-1 and IL-lR1, and also provides an animal model for testing the ability of a compound to treat inflammatory conditions that respond poorly to treatment with TNFcc inhibitors.

BACKGROUND OF THE INVENTION and its ligand, CD30L, are membrane proteins of the TNFR and TNF ligand superfamilies, respectively, and are expressed on various lymphoid and myeloid cells. was first described as an antigen on Hodgkin's disease cells, and presently is widely used as a clinical marker for a number of hematologic malignancies (for review, see Horie and Watanabe, Immunol 10:457-470 (1998)). A naturally-occurring soluble form of the protein is found in human serum, and levels of this protein are elevated in a variety of pathological conditions including viral infection, allergic and autoimmune conditions and neoplastic diseases.

and CD30L are expressed on T cells, and appear to be involved in regulation of the immune system. Their expression on T cells is activation-dependent. CD30 has been reported to be a specific marker of TH2 type T cells (Romagnani, J Leukocyte Biol 57:726 (1995); Romagnani, J Clin Immunol 15:121-129 (1995)). The THI and TH2 subsets of CD4' T cells can be distinguished based on which cytokines they predominantly express. Though individual T cells may actually secrete mixtures of these two groups of cytokines, chronic immune reactions are often dominated by one type or the other of CD4 T cells. T cells that produce TH 2 cytokines, which include IL-4, generally are considered to be mediators of allergic reactions. It has been suggested that the detection of circulating CD30 T cells could serve as a marker for TH2-dominated allergic conditions such as atopic dermatitis (Yamamoto et al., Allergy 55:1011-18 (2000)); however, the correlation between CD30 expression and

TH

2 phenotype has not held up over time (see, for example, Bengtsson et al, J Leukocyte Biol 58:683 (1996); Hamann et al., JImmunol 156:1387-91 (1996)). Based on experiments using a mouse model of diabetes, it has been proposed also that CD30-mediated signaling may protect against autoimmune disease (Kurts et al., Nature 398:341-344 (1999)). Others have Sreported that IL-4 upregulates, whereas IFNy downregulates, the expression of CD30 on 0 Sactivated T cells (Nakamura et al., J Immunol 158:2090-98 (1997); Gilfillan et al., JImmunol 160:2180-87 (1998)).

;The naturally-occurring ligand for CD30, CD30L, is a type II membrane 0 5 glycoprotein that binds specifically with CD30, thus triggering CD30 to transmit a signal via its cytoplasmic domain. The isolation of mouse and human cDNAs encoding CD30L is described in US5,480,981. In addition to being expressed on activated T cells, CD30L is expressed on monocytes/macrophages, granulocytes and a subset of B cells (see, for example, US5,480,981). CD30L has been reported to induce murine B cell differentiation and the proliferation of activated T cells in the presence of an anti-CD3 co-stimulus (see, for example, Smith et al., Cell 73:1349-1360 (1993)). Moreover, it has been reported that exhibits "reverse signaling," that is, the cell surface CD30L that is expressed on neutrophils and peripheral blood T cells can be activated by cross-linking to stimulate metabolic activities in those cells (Wiley et al., Jlmmunol 157:3235- 39 (1996)).

There is a need to better understand the biological activities of CD30 and and to exploit this knowledge in the treatment of human disease.

A reference herein to a patent document or other matter which is given as prior art is not to be taken as an admission that that document or matter was known or that the information it contains was part of the common general knowledge as at the priority date of any of the claims.

Throughout the description and the claims of this specification the word "comprise" and variations of the word, such as "comprising" and "comprises" is not intended to exclude other additives, components, integers or steps.

SUMMARY OF THE INVENTION In accord with this invention, an agent capable of inhibiting the binding of to is used for treating an autoimmune or chronic inflammatory condition, the method comprising administering the agent to the patient according to a regimen of dose and frequency of administration that is adequate to induce a sustained improvement in at least one indicator that reflects the severity of the patient's condition. The improvement is considered to be sustained if the patient exhibits the improvement on at least two occasions separated by at least one day. The agent may be formulated into a physiologically acceptable pharmaceutical preparation, which may be packaged with a written matter describing the foregoing use. Moreover, an inhibitor of the interaction according to this invention may be administered concurrently with other treatments being used to treat the same disorder. In a preferred embodiment, the patient is a human.

WO 02/11767 PCT/US01/24783 In one of the embodiments of the invention, preferred agents for use in treating an autoimmune or inflammatory condition include an antibody that is specific for CD30L, a non-agonistic antibody that is specific for CD30, and a soluble CD30 polypeptide that comprises all or part of the extracellular region of human CD30. The nucleotide and amino acid sequences of human CD30 are shown in SEQ ID NO:6. Suitable polypeptides for use as therapeutic agents include proteins that comprise amino acids 19- 390 of SEQ ID NO:6, or a fragment thereof that retains the ability to bind including polypeptides having an at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5% sequence identity with amino acids 19-390 of SEQ ID NO:6. Such polypeptides if administered in vivo are expected to bind with endogenous CD30L thereby interfering with the interaction between endogenous CD30 and endogenous CD30L. Such polypeptides may if desired be conjugated with another moiety, usually another protein, that promotes oligomerization. A moiety suitable for this purpose is the Fc region from an immunoglobulin molecule. Agents that antagonize the CD30/CD30L interaction may be used to prepare a pharmaceutical preparation to be administered in accord with the methods of the invention, either alone or concurrently with other treatments. Such pharmaceutical preparations may be packaged with a written matter describing the aforedescribed uses.

The hereindescribed therapeutic agents that inhibit the CD30/CD30L interaction may be used to treat a variety of diseases, including various arthritic conditions. For example, rheumatoid arthritis may be treated with the CD30/CD30L antagonists that are described above.

In one aspect of the invention, an agent capable of inhibiting the interaction of CD30 and CD30L is used concurrently with a second agent that is capable of antagonizing TNFa, IL-la, EL-13 or IL-4. Medical disorders expected to be especially responsive to these combination treatments include multiple sclerosis, systemic sclerosis, acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acute motor sensory axonal neuropathy, Fisher syndrome and systemic lupus erythematosus.

As an example, the foregoing medical disorders may be treated with an antibody specific for CD30L used together with an antagonist of IL-4. The anti-CD30L antibody and the IL-4 antagonist are used for formulating pharmaceutical preparations for this purpose, and may be packaged separately or together in one package with a written matter describing WO 02/11767 PCT/US01/24783 this use. Suitable IL-4 antagonists for use in this method of treatment include but are not limited to an antibody specific for IL-4, an antibody specific for IL-4R and a soluble IL-4 receptor comprising amino acids 1-207 or 2-207 of SEQ ID NO:16. In one of the preferred embodiments of the invention, a patient who is suffering from systemic lupus erythematosus, scleroderma or pemphigus vulgaris is treated concurrently with an inhibitor of the CD30/CD30L interaction and an antibody specific for IL-4, an antibody specific for IL-4R or a soluble IL-4 receptor, wherein the receptor comprises amino acids 1-207 or amino acids 2-207 of SEQ ID NO: 16.

In other aspects of the invention, provided are compounds, pharmaceutical preparations and methods of treatment for treating an autoimmune or chronic inflammatory condition that is resistant to treatment with an inhibitor of TNFa. This method comprises administering to a patient in need thereof an agent that is capable of inhibiting the binding of CD30 to CD30L or the binding of IL-la or IL-113 to IL-R1, thereby blocking signal transduction by CD30 or IL-1. The agent is administered according to a regimen of dose and frequency of administration that is adequate to induce a sustained improvement in at least one indicator that reflects the severity of the patient's condition, the improvement being considered sustained if the patient exhibits the improvement on at least two occasions separated by at least one day. Agents suitable for use in such methods include an antibody that is specific for CD30, CD30L, IL-la, IL-103 or IL-1R1, wherein the antibody may be a polyclonal antibody, a monoclonal antibody, a humanized antibody or a human antibody. Agents to be used for this purpose may be formulated into pharmaceutical preparations, which may be packaged with a written matter describing such use. One exemplary agent for this purpose is a soluble fragment of IL-1R2 that includes amino acids 1-333 of SEQ ID NO:8, or a subportion thereof that is capable of binding with IL-la or IL-113, thereby blocking signal transduction by IL-la or IL-10. Another agent suitable for treating such diseases is a soluble CD30 polypeptide that binds CD30L, such as a polypeptide comprising amino acids 19-390 of SEQ ID NO:6 or a CD30L-binding fragment thereof.

In yet another aspect of the invention, provided also is an animal model for screening therapeutic agents, the animal model being characterized by carrying genetic modifications that inactivate its p55 and p 7 5 TNFa receptor proteins, and also by being genetically susceptible to experimentally-induced arthritis. In a preferred embodiment, the animal model is genetically susceptible is collagen-induced arthritis. For example, WO 02/11767 PCT/US01/24783 inactivation of the p55 and p75 TNFa proteins can be introduced into wild-type DBA/1, BUB or B10.Q mice or into DA, BB-DR or LEW rats, all of which are susceptible to collagen-induced arthritis. In a preferred embodiment, the animal is a DBA/1 mouse that has been genetically modified so that it has double-null mutations in both its p55 and TNFa receptor genes.

The invention also provides methods for using the aforedescribed animal model to screen a candidate therapeutic agent to determine its efficacy in treating an autoimmune or chronic inflammatory condition that is resistant to treatment with a TNFa inhibitor.

This method comprises assays in which one induces arthritis in an animal in which the p55 and p75 TNF receptors have been inactivated, administers the candidate therapeutic agent to the animal, and determines that the agent is efficacious if the severity of the animal's arthritis is reduced after the candidate agent has been administered. In a preferred embodiment, the screening assays employ a strain of mouse or rat that is susceptible to collagen-induced arthritis and in which the p55 and p75 proteins have been inactivated by genetic modification. In such mice, arthritis is induced by injecting collagen, the candidate therapeutic agent is administered, and then the severity of the arthritis is assessed by observing the amount of erythema and edema in the animals paws.

In a preferred embodiment, the screening assays employ a DBA/1 mouse, a BUB mouse, a B10.Q mouse, a DA rat, a BB-DR rat or a LEW rat. A particularly suitable animal for use in these assays is a DBA/1 mouse carrying double-null mutations in its p55 and TNFa receptor genes.

DETAILED DESCRIPTION OF THE INVENTION The instant invention discloses methods and compounds for treating or preventing autoimmune and chronic inflammatory diseases, including conditions resistant to treatment with TNFa inhibitors, as well as a model system for screening agents for their efficacy in treating autoimmune or inflammatory conditions that are refractory to treatment with TNF( inhibitors. Patients treated in accord with the invention include those whose condition is continuous or intermittent. Diseases treatable by the subject methods include, for example, diseases such as arthritis, systemic lupus erythematosus and degenerative conditions of the nervous system, such as multiple sclerosis.

Preferably, the patient undergoing treatment is a mammal, and more preferably is a human. The subject methods are applicable to domestic animals, including pets and WO 02/11767 PCT/US01/24783 farm animals. Provided also herein are methods for using inhibitors that block CD30 or IL-1 signal transduction to treat autoimmune or chronic inflammatory diseases that are resistant to treatment with inhibitors of TNFa. In addition, methods are provided that involve treating the autoimmune or chronic inflammatory diseases described herein concurrently with two or more inhibitors selected from an IL-4 inhibitor, a TNFa inhibitor and an inhibitor of the CD30/CD30L interaction. For purposes of this disclosure, the terms "illness," "disease," "medical condition," "abnormal condition," "malady," "medical disorder", "disorder" and the like are used interchangeably.

Autoimmune conditions are characterized by the production of antibodies or effector T cells that react with native host molecules. Most B cell responses depend on helper T cells, thus the presence of autoantibodies generally involves some dysregulation of T cell function. In some cases, however, autoantibodies arise from normal T and B cell responses to foreign proteins that share antigenic epitopes with the host's own tissues.

For example, autoantibodies may be elicited by a pathogenic bacteria that expresses an antigen that resembles a host molecule. In some instances, arthritic syndromes may originate in a woman due to her exposure to fetal cells that have escaped into her circulation during pregnancy. The phrase "chronic inflammatory condition" as used herein refers to chronic disorders whose ongoing symptoms do not appear to be caused by a viral or bacterial infection, even though these diseases in some instances may have been triggered by an infection that no longer is present. Such diseases are "inflammatory" in that they are characterized by the release of inflammatory cytokines such as tumor necrosis factor (TNFa), lymphotoxin a and/or interleukin-1 and they may also involve autoimmunity. In some cases, genetic predispositions play a role in autoimmune or chronic inflammatory diseases that are treatable by the subject methods. For such patients, the subject therapies may be administered prophylactically if desired.

In one aspect of the invention, autoimmune and chronic inflammatory diseases are treated by administering an agent that inhibits signal transduction by CD30. An agent's ability to inhibit CD30 signal transduction can be demonstrated using a biological assay, such as an assay that involves determining that the agent interferes with a biological activity manifested by CD30' cells that otherwise would occur when the cells are contacted with CD30L. Alternatively, a CD30 antagonist may be identified by determining its ability to prevent CD30L from binding CD30 that is expressed on the surface of cultured cells. Therapeutic agents of the invention include but are not limited WO 02/11767 PCT/US01/24783 to agents that antagonize the specific binding of CD30 to CD30L. The terms "antagonist" and "inhibitor" are used interchangeably herein.

The term "CD30-ligand" (CD30L) refers to a human or murine protein as disclosed in Smith et al. (1993) and U.S. Patent No. 5,480,981, including CD30-binding muteins thereof. Nucleotide and amino acid sequences for human are shown in SEQ ID NOS:1 and 2, and those for mouse CD30L are shown in SEQ ID NOS:3 and 4. The extracellular region of human CD30L corresponds to amino acids 1-215 of SEQ ID NO:2. For forming soluble human CD30L molecules that can bind polypeptides comprising amino acids 44, 45, 46 or 47 through amino acid 215 of SEQ ID NO:2 may be used. The extracellular region of murine CD30L corresponds to amino acids 1-220 of SEQ ID NO:4. For forming soluble murine CD30L molecules that can bind CD30, polypeptides comprising amino acids 49-220 of SEQ ID NO:4 may be used.

As used herein, the phrase "fragment of CD30L" refers to a portion of a fulllength CD30L polypeptide that retains the ability to bind to CD30, or that is capable of eliciting an antibody that binds specifically with a CD30L polypeptide of SEQ ID NO:2 or 4 or a subportion thereof. Such fragments preferably will contain at least a portion of 'the extracellular region of The term "CD30" as used herein refers to the 595 amino acid human polypeptide encoded by the nucleotide sequence of SEQ ID NO:5, and whose amino acid sequence is shown in SEQ ID NO:6. The cloning of CD30 is described in Durkop et al.

(Cell 68:421 (1992)). The extracellular portion of human CD30 corresponds to amino acids 1-390 of SEQ ID NO:6, or if the signal peptide is removed, to amino acids 19-390 of SEQ ID NO:6. The phrase "soluble CD30" (sCD30) refers to soluble molecules that comprise all or part of the extracellular domain of the CD30 protein, and that retain the capacity to bind specifically with CD30L. Soluble CD30 polypeptides of the invention encompass recombinant sCD30 and naturally-occurring sCD30 proteins in highly purified form. If desired, the sCD30 may be linked to polyethylene glycol pegylated) to prolong its half-life in the patient's body, or may be linked to another protein moiety that promotes oligomerization.

As used herein, the phrase "fragment of CD30" refers to a portion of a full-length polypeptide that retains the ability to bind to CD30L, or that is capable of eliciting an antibody that binds specifically with a CD30 polypeptide having the amino acid WO 02/11767 PCT/US01/24783 0sequence SEQ ID NO:6 or subportion thereof or to a portion of full-length CD30 that is capable of transmitting a biological signal such as activation of NF-kB.

Soluble CD30 polypeptides according to the invention also include polypeptides that are at least 60%, or at least 70%, or at least 80%, and most preferably at least 90% of the length of the extracellular region of the human CD30 molecule, as shown in amino acids 1-390 of SEQ ID NO:6. Further included as therapeutic agents are proteins comprising a soluble CD30 polypeptide having at least 80%, at least 85%, at least 90%, at least 95%, at least 97.5%, or at least 99%, and most preferably at least 99.5% sequence c.1 identity with amino acids 1-390 of SEQ ID NO:6, where sequence identity is determined 0 10 by comparing the amino acid sequences of the two polypeptides when aligned so as to C,1 maximize overlap and identity while minimizing sequence gaps. The value for percent identity in such comparisons can be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two amino acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters to be used for the GAP program in making these comparisons include: a unary comparison matrix (containing a value of 1 for identities and 0 for nonidentities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Polypeptide Sequence and Structure, National Biomedical Research Foundation, pp. 353- 358, 1979; a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps. Other programs used by those skilled in the art of sequence comparison may also be used, such as, for example, the BLASTN program version 2.0.9, available for use via the National Library of Medicine website, or the UW-BLAST 2.0 algorithm, using standard default parameter settings described at the blast-wustl website. In addition, the BLAST algorithm uses the BLOSUM62 amino acid scoring matrix, and optional parameters that may be used are as follows: inclusion of a filter to mask segments of the query sequence that have low compositional complexity (as determined by the SEG program of Wootton Federhen (Computers and Chemistry, 1993); also see Wootton JC and Federhen S, 1996, Analysis of compositionally biased regions in sequence databases, Methods Enzynol. 266: 554-71) or segments consisting of short-periodicity internal repeats (as determined by the XNU WO 02/11767 PCT/US01/24783 program of Claverie States (Computers and Chemistry, 1993)), and a statistical significance threshold for reporting matches against database sequences, or E-score (the expected probability of matches being found merely by chance, according to the stochastic model of Karlin and Altschul (1990); if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported.); preferred Escore threshold values are 0.5, or in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, le-5, le-10, le-15, le-20, le-25, le-30, le-40, le-50, le-75, or le-100.

The phrase "CD30/CD30L interaction" as used herein refers to the specific binding of CD30 to CD30L, resulting in signal transduction by CD30. This includes instances in which at least one binding partner is a fragment of either CD30 or that is, the term may refer to the binding interaction of a CD30 fragment to CD30L, to a CD30L fragment, or a CD30 fragment to a CD30L fragment. In addition, a CD30/CD30L interaction can involve an analog of CD30 (such as an allelic variant or mutein) that is capable of binding specifically to CD30L, or may involve an analog of CD30L (such as an allelic variant or mutein) that can bind specifically with Moreover, a CD30/CD30L interaction can involve either endogenous CD30 or proteins or may involve recombinant CD30 or CD30L expressed by a cell transfected with a nucleic acid encoding the recombinant protein. Similarly, the phrase "IL-1/IL-1R interaction" refers to the specific binding between IL-1 and IL-1R, including instances where at least one of the binding partners is a fragment of the full-length polypeptide, and including instances where one of the binding partners is an allelic variant or mutein that retains the ability to bind specifically with its binding partner.

The term "IL-1" as used herein includes both IL-la and IL-10. IL-la and are two distinct proteins that both are instrumental in inflammation, and both of which bind the same two cell surface receptors. The cell surface receptors to which IL-la and both bind are known as types I and II IL-1 receptors, or "IL-1R1" and "IL-1R2." Signal transduction by IL-1 is mediated primarily by IL-1R1, while IL-1R2 is considered to be a "decoy" receptor whose function is to downregulate IL-1 biological activity.

Human IL-1R2 is described in U.S. Patent No. 5,350,683. A nucleic acid sequence encoding the IL-1R2 protein is shown in SEQ ID NO:7, and the amino acid sequence is shown in SEQ ID NO:8.

In one embodiment, the subject invention provides methods of treating autoimmune or chronic inflammatory conditions that are resistant to treatment with drugs WO 02/11767 PCT/US01/24783 that inhibit TNFca. In such patients, the lack of response to TNFa inhibition may be partial or complete. TNFc inhibitors to which these diseases are unresponsive may include one or more of the following: etanercept (p75 TNFR:Fc, sold as ENBREL®; Immunex Corporation); LENERCEPT® (p55 TNFR-Ig fusion protein; Roche); or antibodies against TNFoa, including humanized antibodies such as infliximab (REMICADE®; Centocor), D2E7 (BASF Pharma) or CDP571 (HIUMICADE®; Celltech).

Arthritis that is resistant to one of these or to another TNF inhibitor is referred to as "TNFca-independent arthritis." Diseases resistant to treatment with TNFac inhibitors may be treated by administering one or more agents that inhibit signal transduction by or IL-1, such as an inhibitor of the CD30/CD30L interaction and/or of the IL-1/IL-1R interaction. In addition, diseases that are less than fully responsive to TNFa inhibitors are treated with a combination of a TNFa inhibitor administered concurrently with an inhibitor of the CD30/CD30L interaction and/or with an inhibitor of an IL-1/IL-1R interaction.

Therapeutic agents Any physiologically acceptable agent capable of blocking signal transduction by may be used as a therapeutic agent in the disclosed methods of treatment, including but not limited to: antibodies that bind specifically to CD30L and thereby inhibit its binding to CD30; non-agonistic antibodies that bind specifically to CD30 and inhibit its ability to transmit a biological signal; muteins or analogs such as allelic variants of or fragments thereof that bind to CD30 but that do not stimulate transduction of a biological signal; sCD30 molecules that contain the extracellular domain of CD30 or a portion thereof that is capable of binding CD30L; and small organic molecules that block signal transduction by CD30 or that interfere with the biological functioning of Soluble CD30 molecules used as therapeutic agents as described herein comprise the extracellular region of CD30. An exemplary CD30 protein is shwon in SEQ ID NO:6, in which amino acids 1-390 of SEQ ID NO:6 correspond to the extracellular region. If less than the entire extracellular domain is used, the fragment must retain the ability to bind CD30L, which ability may be ascertained using any convenient binding assay format. The presence of the signal peptide in the sCD30 is optional. Suitable assays include testing for the ability of the sCD30 to competitively block binding of labeled CD30L to cells expressing surface CD30 or to block binding of isolated or cells expressing surface CD30L to cell-surface CD30 or CD30 that is anchored to a WO 02/11767 PCT/US01/24783 solid support such as an ELISA plate or a chromatography matrix, such as agarose beads.

When used therapeutically, these sCD30s block the binding of membrane-bound CD30 to cells expressing useful as CD30 antagonists for the disclosed therapeutic methods include oligomeric sCD30 polypeptides (such as dimers or trimers), as well as monomers.

Oligomers may be linked by disulfide bonds formed between cysteine residues on different sCD30 polypeptides. In one embodiment of the invention, the therapeutic agent is a sCD30 that is created by fusing the extracellular domain of CD30 (or a binding portion thereof) to the PFc region of an antibody, preferably a human antibody, in a manner that does not interfere with binding of the CD30 moiety to CD30L. The resulting fusion protein is a CD30:Fc that will dimerize by the spontaneous formation of disulfide bonds between the Fc moieties on two of the fusion protein chains. Native Fc region polypeptides or muteins thereof may be employed in making these constructs.

Suitable CD30:Fc's will reduce or abolish the. ability of agonistic anti-CD30 antibodies to stimulate CD30, or will competitively inhibit the binding of CD30L to membrane-bound An example of a suitable CD30:Fc protein that may be used in the subject methods is one that is constructed as described in U.S. 5,677,430.

Other suitable oligomeric sCD30 polypeptides may be prepared by fusing the extracellular domain of CD30 (or a fragment thereof) to a "leucine zipper," which is a peptide that promotes oligomerization of the proteins in which they are present. Leucine zippers are motifs found in several DNA-binding proteins (see, for example, Landschulz et al., Science 240:1759 (1988)), and naturally-occurring leucine zipper peptides and derivatives thereof can promote the formation of dimers or trimers of protein chains in which they are present. Examples of leucine zipper domains useful for producing soluble oligomeric proteins are described in WO 94/10308.

Another method of linking multiple copies of soluble CD30 polypeptides is by fusing monomers together with suitable peptide linkers. A fusion protein comprising two or more copies of sCD30 separated by peptide linkers may be produced by recombinant DNA technology. Such peptide linkers optimally are from 5 to 100 amino acids in length, preferably comprising amino acids selected from the group consisting of glycine, asparagine, serine, threonine, and alanine. The production of recombinant fusion proteins comprising peptide linkers is illustrated in U.S. 5,073,627.

In yet other embodiments, antagonists can be designed to reduce the level of endogenous CD30 or CD30L gene expression, for example, by using well-known 11 WO 02/11767 PCT/US01/24783 antisense or ribozyme approaches to inhibit or prevent translation of CD30 or mRNAs; triple helix approaches to inhibit transcription of CD30 or CD30L genes; or targeted homologous recombination to inactivate or "knock out" the CD30 or genes or their endogenous promoters or enhancer elements. Such antisense, ribozyme, and triple helix antagonists may be designed to reduce or inhibit either unimpaired, or if appropriate, mutant CD30 or CD30L gene activity. Techniques for the production and use of such molecules are well known to those of skill in the art.

Antisense RNA and DNA molecules can act to directly block the translation of mRNA by hybridizing to targeted endogenous mRNA thereby preventing translation.

Alternatively, antisense RNA or DNA can inhibit or prevent transcription of the target gene. The antisense approach involves designing oligonucleotides (either DNA or RNA) that are complementary to a CD30 or CD30L mRNA, or complementary to a portion of the target gene, such as a regulatory element that controls transcription of the gene.

Antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.

In one embodiment of the invention, ribozyme molecules designed to catalytically cleave CD30 or CD30L mRNA transcripts are used to prevent translation of CD30 or mRNA and expression of CD30 or CD30L polypeptides. (See, e.g., WO 90/11364, or U.S. Patent No. 5,824,519).

Provided herein are therapies for treating autoimmune and chronic inflammatory conditions that are resistant to treatment with an inhibitor of TNFca. Therapies according to this embodiment of the invention are administered to a patient who has already been observed to be partially or fully unresponsive to treatment with a TNFa inhibitor. To treat a patient whose autoimmune or chronic inflammatory condition is resistant to a TNFa inhibitor, the patient is administered an effective amount of an agent that is capable of blocking the binding of CD30 to CD30L, such as one of the inhibitors described above.

Alternatively, the patient is administered an EL-1 antagonist that is capable of inhibiting signal transduction by IL-1, such as by reducing the amount of IL-1 produced in the patient's body, or by interfering with the binding of IL-1 to IL-1R1. In the case of patients who are partially responsive to treatment with a TNFa inhibitor, treatments according to this embodiment of the invention may be administered concomitantly with a TNFa inhibitor. The therapeutic agents of this embodiment of the invention generally are administered in the form of a physiologically acceptable composition.

WO 02/11767 PCT/US01/24783 In a further embodiment of the invention, patients are treated with an inhibitor of the CD30/CD30L interaction administered concurrently with an antagonist of IL-4. IL-4 is a cytokine with a broad range of activities, and is expressed by CD30 T cells. The cell surface receptors to which IL-4 binds are referred to as the "IL-4 receptor" or "IL-4R." (see for example, U.S. Patent No. 5,599,905). The interaction between IL-4 and its receptor can be inhibited by administering a soluble IL-4 receptor (sIL-4R), such as the sIL-4Rs described in U.S. Patent No. 5,599,905. Preventing this interaction will hinder or prevent IL-4-mediated biological activities. IL-4 can induce a chronic inflammatory effect in some diseases and can exacerbate some autoimmune disorders. In such diseases, the infiltration and proliferation of TH2 cells is fueled by IL-4 and CD30 signal transduction. These cells cause the overproduction of IL-4. Accordingly, diseases where this occurs, including atopic dermatitis, asthma, systemic lupus erythematosus, scleroderma or pemphigus vulgaris, are effectively treated by administering an agent that inhibits IL-4 concurrently with an agent that inhibits the interaction between CD30 and CD30L. The latter preferably is an antibody that is specific for Methods of using IL-4 inhibitors to treat immune or inflammatory responses are illustrated, for example, in U.S. Patent No. 5,767,065. IL-4 antagonists that may be administered in combination with inhibitors of the CD30/CD3-L interaction include, but are not limited to, IL-4 receptors (EL-4R) and other EL-4-binding molecules, IL-4 muteins and antibodies that bind specifically with L-4 or 1L-4 receptors thereby blocking signal transduction, as well as antisense oligonucleotides and ribozymes targeted to IL-4 or IL-4R. Polyclonal or monoclonal antibodies specific for EL-4 or IL-4 receptor may be prepared using standard procedures. Among the IL-4 receptors suitable for use as described herein are soluble fragments of human IL-4R that retain the ability to bind EL-4.

Such fragments retain all or part of the IL-4R extracellular region and are capable of binding EL-4. In a preferred embodiment of the invention, the patient is treated concurrently with sIL-4R and an antibody specifically for IL-4 receptors are described in U.S. Patent 5,599,905; Idzerda et al., J. Exp. Med.

171:861-873, March 1990 (human IL-4R); and Mosley et al., Cell 59:335-348, 1989 (murine IL-4R), each of which is hereby incorporated by reference in its entirety. The protein described in those three references is sometimes referred to in the scientific literature as 1L-4Ra. Unless otherwise specified, the terms "EL-4R" and "1L-4 receptor" as used herein encompass this protein in various forms that are capable of functioning as WO 02/11767 PCT/US01/24783 IL-4 antagonists, including but not limited to soluble fragments, fusion proteins, oligomers, and variants that are capable of binding IL-4. Suitable IL-4Rs include variants in which valine replaces isoleucine at position 50 (see Idzerda et al., 1990), and include slow-release formulations, and PEGylated derivatives (modified with polyethylene glycol) are contemplated, as well as recombinant fusion proteins comprising heterologous polypeptides fused to the N-terminus or C-terminus of an IL-4R polypeptide, including signal peptides, immunoglobulin Fc regions, poly-His tags or the FLAG® polypeptide described in Hopp et al., Biol/Technology 6:1204, 1988, and U.S. Patent 5,011,912, as well as fusions of IL-4 receptors with oligomer-promoting leucine zipper moieties.

Soluble recombinant fusion proteins comprising an IL-4R and immunoglobulin constant regions are described, for example, in EP 464,533. A nucleotide sequence encoding human IL-4 receptor is shown in SEQ ID NO:15 and amino acid sequence for human IL-4 receptor is shown in SEQ ID NO: 16. In a preferred embodiment, the IL-4 antagonist to be used in combination with an inhibitor of the CD30/CD30L interaction is a soluble human IL-4 receptor comprising amino acids 1 to 207 of SEQ ID NO:16 and in another preferred embodiment, the IL-4 antagonist comprises amino acids 2 to 207 of SEQ ID NO:16. IL-4 antagonists useful for the hereindescribed methods of treatment also include molecules that selectively block the synthesis of endogenous IL-4 or ILA-4R, such as antisense nucleic acids or ribozymes targeted against either of these molecules.

Various IL-4 antagonists that may be used for the hereindescribed methods of treatment can be identified, for example, by their ability to inhibit 3 H-thymidine incorporation in cells that normally proliferate in response to IL-4, or by their ability to inhibit binding of IL-4 to cells that express IL-4R. In one assay for detecting IL-4 antagonists, one measures the ability of a putative antagonist to block the IL-4-induced enhancement of the expression of CD23 on the surfaces of human B cells. For example, B cells isolated from human peripheral blood are incubated in microtiter wells in the presence of EL-4 and the putative antagonist. Following the incubation, washed cells are then incubated with labelled monoclonal antibody against CD23 (available from Pharmingen) to determine the level of CD23 expression. An anti-huIL-4R murine mAb (R&D Systems) previously shown to block the binding and function of both hlL-4 and hEL-13 may used as a positive control for neutralization of CD23 induction by IL-4.

Alternatively, suitable IL-4 antagonists may be identified by determining their ability to prevent or reduce the impaired the barrier function of epithelium that results when IL-4 is WO 02/11767 PCT/US01/24783 incubated with the epithelium. For this purpose, one may use confluent monolayers of human epithelial cell lines such as Calu-3 (lung) or T84 (intestinal epithelium).

Incubation of such monolayers with IL-4 causes significant damage to their barrier function within about 48 hours. To assay IL-4 antagonists, monolayers may be tested for their permeability, for example, by adding radiolabeled mannitol to cells incubated with IL-4 in the presence or absence of an antagonist. Alternatively, transepithelial resistance (indicating an intact barrier) may be determined using a voltmeter.

In other embodiments of the invention, antagonists of the CD30/CD30L interaction are administered concurrently with an IL-1 antagonist. Alternatively, IL-1 antagonists may be administered alone for treating autoimmune or inflammatory conditions that are resistant to treatment with TNFa inhibitors. Suitable agents for inhibiting signal transduction by IL-1 include antibodies specific for II-1 or IL-1R1, particularly humanized antibodies. Other suitable IL-1 antagonists include: IL-1 receptor antagonist (IL-ira); receptor-binding fragments of IL-1 that block native EL-1 from binding IL-1Rl; antibodies directed against IL-1 or IL-1R1; and recombinant proteins comprising all or part of a receptor for EL-1 or modified variants thereof, including genetically-modified muteins, multimeric forms and sustained-release formulations.

IL-Ira is a naturally-occurring endogenous antagonist of IL-1 and binds both IL-1R1 and IL-1Rs. Preferred IL-1 antagonists include soluble IL-1R2 molecules that are capable of binding IL-1 and that comprise all or part of the extracellular domain of IL-1R2. These soluble IL-1R2 molecules block IL-1 from interacting with membrane-bound IL-1Rl.

Other useful IL-1 antagonists include soluble forms of IL-IR1 that are capable of competitively inhibiting the interaction of IL-1 with IL-IR1 and further include IL-1 converting enzyme (ICE) inhibitors, which generally are small organic molecules. A preferred IL-1 antagonist is a soluble fragment of IL-1R2 that includes amino acids 1-333 of SEQ ID NO:8, or a subportion thereof that is capable of binding specifically with IL-lx or IL-13. Soluble L-1R2s to be used in accord with the invention include, for example, analogs or fragments of native IL-1R2 having at least 20 amino acids, that lack the transmembrane region of the native molecule, and that are capable of binding IL-1.

The ability of soluble IL-1R2 to bind IL-1 (including binding to fragments of IL-la or can be assayed using ELISA or any other convenient assay.

Other suitable IL-1 antagonists are chimeric proteins in which one of the IL-1 antagonists described above is fused with another polypeptide that promotes the WO 02/11767 PCT/US01/24783 spontaneous formation of a dimer, trimer or higher order multimer. A suitable polypeptide moiety for promoting dimerization is the Fc region of a human immunoglobulin. For example, soluble IL-1R2 polypeptides or fragments thereof may be fused the Fe region of an immunoglobulin to form a chimeric protein that is capable of dimerizing. Any of the foregoing IL-1 antagonists, other than ICE inhibitors, may be covalently linked to polyethylene glycol (pegylated) to prolong their half-life in the body.

It is understood that while IL-la and IL-10 are the IL-Is most commonly associated with inflammation, other forms of IL-1 exist, and the invention encompasses the use of therapeutic agents targeting these other forms for treating autoimmune and chronic inflammatory diseases that are resistant to treatment with TNFa inhibitors.

Preferred agents for use in the subject therapeutic methods include antibodies that block the CD30/CD30L, IL-1/IL-1R or IL-4/IL-4R interactions. Such antibodies are specifically immunoreactive with their target, that is, they bind to the target protein via the antigen-binding site of the antibodies and do not bind unrelated proteins to a significant degree. Antibodies specific for CD30L will bind endogenous CD30L, thus reducing the amount of endogenous CD30L available for binding to cell surface Also suitable for use as a therapeutic agent of the subject invention are biologically active fragments of antibodies. For example, a biologically active fragment of an antibody is an antibody protein that is truncated relative to the intact antibody, but that retains the ability to specifically bind CD30L and to block its interaction with Antigen-binding fragments of antibodies, include, but are not limited to, Fab and F(ab') 2 fragments, and may be produced by conventional procedures.

Antibodies that antagonize the CD30, IL-1 or IL-4 signal transduction can be identified in any suitable assay, including assays based on biological function or assays based on the detection of physical binding. Examples of such assays are disclosed, for example, in U.S. 5,677,430. One preferred assay tests an antibody's ability to block the binding of cell surface CD30L to cell surface CD30. Cell lines suitable for such assays include the CD30+ HDLM-2 or L540 cell lines, or may use activated T cells expressing An assay based on biological function, for example, could entail determining whether an antibody can antagonize the ability of membrane-bound CD30L to stimulate proliferation of CD30' cells that are responsive to such stimulation. Yet another assay employs the CD30 K299 human cell line. It has been observed that proliferation of these cells is inhibited by contact with CD30L-transfected cells. An antibody specific for WO 02/11767 PCT/US01/24783 (or other antagonist that blocks CD30L) could suppress this effect of CD30L and thus enhance the proliferation of these cells in this assay. In other instances, the antibody is tested for its ability to block the binding of labeled recombinant CD30L to cell surface In other instances, the assays employ cells transfected with DNA encoding human CD30L, IL-1R1, IL-1R2 or IL-4R. For example, the ability of a monoclonal antibody against human CD30L to block CD30/CD30L interactions can be assessed by determining whether it can block the binding of human CD30:Fc to either cells transfected with human CD30L or to activated human T cell blasts as assessed by FACS analysis. Similarly, the specificity of an antibody for IL-4R could be tested by determining if the antibody blocks binding. of labeled IL-4 to IL-4R.

Other suitable assays utilize soluble forms of both binding partners, for example, and sCD30L. For example, sCD30 may be bound to a solid phase, such as a column chromatography matrix or to an ELISA plate. For an ELISA assay using and sCD30L, a sCD30, such as CD30:Fc, is fixed to an ELISA plate, and an antibody raised against CD30L is tested to see if it is an antagonist by checking its ability to inhibit the binding of a soluble CD30L leucine zipper construct to the anchored sCD30. In this assay, binding of the leucine zipper construct or other soluble CD30L to the ELISA plate is measured using a biotinylated non-neutralizing anti-CD30L monoclonal antibody, or alternatively, by using an antibody to the leucine zipper end of the CD30L construct.

Other assays test the ability of an antibody to block the binding of IL-1 to cell surface IL-1R1.

Therapeutic agents suitable for use in the subject methods include non-agonistic antibodies against human CD30. Such antibodies can block the CD30/CD30L interaction. Monoclonal antibodies against human CD30 can be prepared, for example, as described in U.S. 5,677,430, then tested to determine if they are agonistic or nonagonistic. A non-agonistic antibody against CD30 may be distinguished from an agonistic antibody by testing its effect on CD30 in a suitable biological assay, such as the assays described U.S. 5,677,430. In one such assay, an antibody specific for CD30 is tested to determine whether it can induce proliferation of activated T cells prepared from peripheral blood or whether it can induce proliferation of the Hodgkin's disease-derived cell lines HDLM-2 or L-540. An agonistic antibody, will induce such proliferation. In contrast, a non-agonistic antibody against CD30 will bind specifically with CD30 but will not induce proliferation of target cells in these or other assays that rely on signal transduction by WO 02/11767 PCT/US01/24783 Therapeutic agents according to this invention may be administered concurrently with one or more additional therapeutic molecules to treat autoimmune or chronic inflammatory diseases. As used herein, "concurrently" includes instances where the drugs in a combination treatment are administered over the same time period or are alternated. This includes simultaneous and sequential administration, and the different drugs may be present in the same or separate pharmaceutical compositions. The frequency and route of administration for the different drugs in such combinations may be the same or different. Therapeutic agents that may be used in such combinations include, for example, antagonists of CD30, IL-1 or IL-4 as described above, and also include nonsteroidal anti-inflammatory drugs (NSAIDs), corticosteroids, analgesics, cytokine suppressive drugs, disease-modifying anti-rheumatic drugs (DMARDs), methotrexate, and so on. As an example, antagonists of CD30 or IL-1 may be combined with each other or with antagonists of TNFc, IL-2, IL-6, RANK or other cytokines that may contribute to autoimmunity or chronic inflammation. In some preferred embodiments, the additional therapeutic agents target a cytokine. An antagonist that targets a cytokine may comprise a soluble receptor against the cytokine, and usually includes part or all of the extracellular domain of a receptor for the cytokine. The soluble cytokine receptor may be used as a monomer, or as a dimer or higher multimer.(for example, as a fusion molecule wherein the soluble receptor is attached to the dimer-promoting Fc portion of human immunoglobulin). In other embodiments, the soluble cytokine receptor is pegylated to increase its serum half-life. In some embodiments, the soluble cytokine antagonist comprises a soluble TNF receptor (type I or II). Small organic molecules that inhibit inflammatory cytokines may also be used in combination with the subject therapeutic agents. More than one antagonist of CD30 signal transduction may be administered concurrently for treating autoimmune or chronic inflammatory diseases, and may be administered alone or together with other drugs that are effective against the same autoimmune or chronic inflammatory condition or that are being administered to treat a different condition in the same patient.

Combinations used to treat multiple sclerosis include inhibitors of administered in conjunction with other drugs used to treat this condition, including but not limited to mitoxantrone (NOVANTRONE®; Immunex Corporation), interferon -la (AVONEX®; Ares-Sorono Group), interferon 1-lb (BETASERONO; Berlex WO 02/11767 PCT/US01/24783 Laboratories, Inc.) and/or glatiramer acetate (COPAXONE®; Teva Pharmaceuticals). IL- 4 antagonists may be added to any of the foregoing combination.

For treating various rheumatic conditions, including rheumatoid arthritis, an agent capable of inhibiting CD30 signal transduction may be administered alone or concurrently with inhibitors of TNF, such as antibodies against TNFa (for example, humanized antibodies such as REMICADE® (Centocor), D2E7 (BASF Pharma), or HUMICADE® (Celltech)); soluble forms of the TNF receptor (such as ENBREL® (Immunex Corporation) or LENERCEPT® (Roche) or pegylated soluble TNF receptors.

Preparation of Therapeutic Antibodies CD30L polypeptides suitable for use as an immunogen in producing therapeutic antibodies include but are not limited to full length CD30L (recombinant or prepared from a naturally-occurring source) or immunogenic fragments thereof, particularly fragments containing all or part of the extracellular domain of CD30L. To be effective as an immunogen, a fragment of CD30L need not retain the capacity to bind CD30, but must be large enough to be antigenic. To be antigenic, a peptide generally must contain at least 20 amino acids. The amino acid sequence of full-length human is shown in SEQ ID NO:2 and mouse CD30L in SEQ ID NO:4; peptides corresponding to at least 20 contiguous amino acids of either of these proteins may be used as an immunogen to prepare therapeutic agents for use as described herein.

CD30 polypeptides suitable for use as immunogens in producing non-agonistic antibodies include but are not limited to full length CD30 proteins (recombinant or prepared from a naturally-occurring source) or immunogenic fragments thereof, particularly fragments comprising all or part of the extracellular domain as defined by amino acids 1-390 of SEQ ID NO:6. Immunogens for raising antibodies preferably contain at least 20 contiguous amino acids of the protein shown in SEQ ID NO:6.

IL-1 or IL-1R polypeptides suitable for use as immunogens in producing antagonistic antibodies include but are not limited to full length proteins (recombinant or prepared from a naturally-occurring source) or immunogenic fragments thereof, particularly fragments comprising all or part of the extracellular domain of human IL-1R2 as shown in amino acids 1-333 of SEQ ID NO:8. Preferred immunogens contain at least contiguous amino acids of the protein shown in SEQ ID NO:8.

WO 02/11767 PCT/US01/24783 Immunogens for raising therapeutic antibodies ^against TNFa, TNFR, IL-4 or IL4-R will consist of an at least 20 amino acid segment of the target protein. Suitable antigens for raising therapeutic antibodies also include any of the aforementioned immunogens fused to another protein, including proteins fused to an N-terminal "flag" (see, for example, Hopp et al., Bio/Technology 6:1204 (1988); U.S. Patent No.

5,011,912), or fused to the Fc portion of an immunoglobulin molecule, preferably a human immunoglobulin. Preferred therapeutic agents include both polyclonal and monoclonal antibodies (MABs), either of which may be generated using the abovedescribed polypeptides as immunogens.

Polyclonal antibodies may be generated according to standard protocols using a variety of warm-blooded animals such as horses, cows, rabbits, mice, rats, or various species of fowl. In general, the animal is immunized with CD30L, CD30, IL-1, IL-1R1, TNFa, TNFR1 or TNFR2, or an immunogen derived therefrom, through intraperitoneal, intramuscular, or subcutaneous injections. The immunogenicity of an immunogenic polypeptide usually is increased through the co-administration of an adjuvant such as RIBI (Corixa), or Freund's complete or incomplete adjuvant, or other suitable adjuvant.

After several booster immunizations, serum samples are collected and tested for specific reactivity with the target polypeptide by any suitable method, such as ELISA, antibodycapture or modified ABC assays, or a dot blot assay. Once the titer of antibody has reached a plateau in terms of reactivity to the target, the polyclonal antiserum is harvested.

For an ABC assay, a plastic dish, such as an ELISA plate, is coated with an antibody that is specifically reactive with the Fc portion of immunoglobulin from for the same species of animal that was used to raise the antibody against the target polypeptide.

For example, if a target protein is was injected into a rabbit and the resulting anti-target polyclonal antibody is to be evaluated for specificity, the plates are coated with antibody specifically reactive with the Fc portion of rabbit IgG. In the next step, a sample of antibody from the immunized rabbit is incubated in the dish under conditions that promote binding between the rabbit IgG and the anchored anti-rabbit antibody. Next, labeled target protein is added, and the dish is incubated under conditions that promote antibody binding. If the sample of antibody being tested is specific for the target protein, then the labeled target protein will become bound to the captured rabbit antibody, and thereafter can be detected after the plate is washed. For example, if the target protein is WO 02/11767 PCT/US01/24783 labeled with biotin, target protein that has become bound can be detected by using a streptavidin-tagged enzyme that is capable of generating a colored product.

Suitable procedures for generating monoclonal antibodies include several methods described in the art (see, for example, U.S. Patent 4,411,993; Kennett et al. (eds.), Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York (1980); and Harlow and Lane Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988)). Mice or rats generally are used for the initial immunizations, which are performed as described above for raising polyclonals. After immunization, the immunized animal's spleen cells are harvested and fused according to standard procedures with a myeloma cell line to yield immortalized hybridoma cells that produce monoclonal antibodies. Many myeloma lines suitable for hybridomas are known, including many that are available from the American Type Culture Collection (ATCC), Rockville, Maryland (see Catalog "of Cell Lines Hybridomas, ATCC). Individual hybridoma cells are isolated after the fusion step to be screened to identify those producing monoclonal antibodies having the desired specific immunoreactivity. For therapeutic use, high affinity antibodies are preferred.

Hybridomas with the desired specification are identified by assays such as ELISA, ABC, modified ABC, or dot blot assays, then are isolated and propagated. The monoclonal antibodies are harvested and purified using standard methods.

Monoclonal antibodies against CD30L may be raised and screened for specific reactivity according to the methods described in U.S. 5,677,430 or by any of the various methods known in the art for raising monoclonals. Screening may involve determining the capacity of the monoclonals to antagonize the binding of cell-surface CD30L to cellsurface CD30 or to antagonize signal transduction by cell-surface Antibodies useful for the subject therapeutic methods also include chimeric antibodies and antibodies produced or modified via protein engineering or recombinant DNA technology (see, for example, Alting-Mees et al., Strategies in Molecular Biology 3:1 (January, 1990)). Production of chimeric and otherwise engineered antibodies is described in the prior art (see, for example, Reichmann et al., Nature 332:323 (1988); Liu et al., PNAS 84:3439 (1987); Larrick et al., Bio/Technology 7:934 (1989); and Winter and Harris, TIPS 14:139 (1993)).

When an antibody is to be administered as a therapeutic agent to a human patient in accord with the subject methods, a humanized antibody is preferred. Even more preferred are human antibodies. Humanized antibodies comprise an antigen binding WO 02/11767 PCT/US01/24783 domain derived from a non-human antibody (for example, a rat or mouse), and may contain the entire variable region of the non-human antibody, or may contain only that portion of the non-human variable region that includes the antigen-binding site. In a preferred embodiment of the invention, the only non-human portion of the humanized antibody is the hypervariable region. Procedures for preparing humanized antibodies are known, and include techniques that involve recombinant DNA technology. Production of humanized antibodies is described, for example, in Winter and Harris, 1993. To create a humanized antibody, DNA encoding the antigen-binding sites of a monoclonal antibody directed against the target may be isolated and inserted directly into the genome of a cell that is producing human antibodies (see Reichmann et al. (1988)). For example, this method could be used for antibodies against CD30, CD30L, IL,-1, IL-1R1, TNFa, TNFR-1 or TNRF-2, EIL-4 or IL-4R.

In one procedure for preparing humanized antibodies, cDNA is prepared on a mRNA template derived from a hybridoma cell line that produces a non-human monoclonal antibody having the desired specificity. In essence, a fragment of the cDNA that encodes the variable region of the monoclonal antibody (or a fragment thereof containing the antigen binding site) is isolated, and this cDNA fragment is fused to DNA encoding the human counterpart of the remainder of the human antibody molecule, thereby reconstructing a functional antibody molecule. Host cells are transfected with an expression vector containing the fused gene, and are cultured to produce the desired recombinant fusion protein. To isolate DNA encoding the variable region of an immunoglobulin chain, one may employ, for example, the method of Larrick et al.

(1989), which involves the polymerase chain reaction (PCR), using a mixture of upstream primers corresponding to the leader sequence, and a downstream primer based on the conserved sequence of the constant region. PCR primers for amplifying the variable region from mouse or human hybridoma cell immunoglobulin mRNA are commercially available (for example, from Stratacyte, La Jolla, California). PCR primers may be used to amplify heavy or light chain variable region DNA, and the resulting amplified DNA may be inserted into vectors such as ImmunoZAP* H or ImmunoZAP* L (Stratacyte), respectively, for expression in E. coli.

To produce a humanized antibody for use as a therapeutic agent in accord with the invention, variable region DNA is isolated from a murine or rat hybridoma cell that expresses an antibody with the desired specificity, and this DNA is fused with a constant region DNA amplified from a cDNA encoding a human antibody. These or similar 22 WO 02/11767 PCT/US01/24783 techniques may be used, for example, to produce a single-chain antigen-binding protein containing a VL domain fused to a VH domain through a peptide linker (see Bird et al., Science 242:423 (1988)). Further genetic manipulations may be performed to replace all but the hypervariable regions of the antibody with human sequences.

Human antibodies may be generated using methods involving non-human animals. For example, DNA encoding one or more entire human immunoglobulin chains may be introduced into a mouse to produce a transgenic animal, and then the antibody is isolated from cultured cells derived from the mice. The endogenous immunoglobulin genes in the recipient mouse may be inactivated by various means, and human immunoglobulin genes introduced into the mouse to replace the inactivated genes. These genetic manipulations will result in human immunoglobulin polypeptide chains replacing endogenous immunoglobulin chains in at least some instances, and in such mice some or most of the antibodies produced upon immunization will be human antibodies. Examples of techniques for the production and use of such transgenic animals are described in U.S.

Patent Nos. 5,814,318, 5,569,825, and 5,545,806.

Animal Model for Diseases Resistant to Treatment with TNFa Inhibitors TNF inhibition has demonstrated beneficial effects in a significant percentage of rheumatoid arthritis patients and patients with other kinds of arthritis or other chronic inflammatory diseases. However, a subset of rheumatoid arthritis patients respond poorly or not at all to treatment with TNF inhibitors (TNF-independent rheumatoid arthritis). To provide a tool for identifying effective treatments for patients with autoimmune or chronic inflammatory conditions that show little or no improvement in response to treatment with TNFax inhibitors, provided herein is an animal model that is useful for screening candidate therapeutic agents -that may be effective for such diseases. The model animal is characterized by carrying genetic modifications that inactivate its and p75 TNFa receptor proteins and by being genetically susceptible to experimentallyinduced arthritis. The animal model is created by genetically modifying a strain of animal that already is known to be genetically susceptible to experimentally-induced arthritis. Provided also are methods for screening candidate therapeutic agents to determine their effectiveness in treating a medical disorder that is resistant to treatment with a TNFa inhibitor. Such medical disorders include rheumatoid arthritis and other kinds of arthritis.

WO 02/11767 PCT/US01/24783 The subject animal model is created by introducing genetic modifications into a strain of animal, usually a mammal, that prior to modification is genetically susceptible to experimentally-induced arthritis. The genetic modifications result in the inactivation of the animal's p55 and p75 TNFcc receptor (TNFR) proteins. The p55 and p75 TNFRs are also called the types I and II TNFRs, respectively.

A model animal according to the subject invention may be deficient in its p55 and TNFR proteins as the result of any of several different types of mutation strategies.

For example, the deficiency may result from mutations that inhibit the transcription of a translatable TNFR messenger RNA or that result in production of defective TNFR proteins that do not bind TNFa. Cells from the subject animal model will bind no detectable TNFa as compared with animals that express at least one of these TNFR proteins in wild-type form. The ability of cells from a subject animal model to bind TNFa may be assayed, for example, as described in Peschon et al. (1998) or by other suitable methods. For example, cells taken from a genetically modified animal may be tested for expression of functional TNFR I and II by being incubated with labeled biotinylated TNFa and streptavidin-conjugated phycoerythrin, then analyzed by flow cytometry to determine if the phycoerythrin was captured on the cells. Various cells may be isolated from the test animal to be used in such a binding assay, including conconavalin A-stimulated thymocytes, thioglycolate-elicited peritoneal exudate cells and bronchoalveolar lavage cells collected after intranasal administration of lipopolysaccharide (Peschon et al., 1998). If such cells from the mutated animal fail to bind labeled TNFa, this indicates that both types I and II TNFR are suitably inactivated.

In a preferred embodiment of the invention, the animal in which the p55 and proteins are inactivated is a rodent strain that is susceptible to experimental collageninduced arthritis (CIA). In a preferred embodiment, mutations are introduced into this rodent's genome that result in inactivation of the genes encoding the p55 and p75 TNFa receptors. In one preferred embodiment, the rodent is a strain of mouse or rat. Mice susceptible to CIA include mice that carry the H-2 q MHC haplotype or the H-2r MHC haplotype. Exemplary strains of CIA-susceptible mice include the DBA/1, BUB and B10.Q strains, and exemplary strains of CIA-susceptible rats include the DA, BB-DR and LEW strains (see, for example, Joe and Wilder, Mol Med Today 5:367-369 (1999) and Anthony and Haqqi, Clin Exp Rheumatol 17:240-244 (1999)).

WO 02/11767 PCT/US01/24783 An exemplary animal model according to the invention is a DBA/1 mouse carrying double-null mutations in its p55 and p75 TNFR genes, that is, a p55-'p75-" DBA/1 mouse. As used herein, a "null" mutation means that the gene is sufficiently changed relative to the wild-type gene such that it does not give rise to a protein that is recognizable by antibodies specific for the corresponding wild-type receptor protein.

This may be accomplished by introducing a deletion or insertion into the wild-type gene, or by other means. Once established in a strain of mouse, a null mutation may be transferred to a different strain by appropriate genetic manipulations. A double-null mutation means that both alleles of that gene carry a null mutation.

In other embodiments, the genetic modifications may be introduced into rodent strains that are susceptible to forms of arthritis other than CIA, such as, for example, one of the arthritis-susceptible strains that are described in Joe and Wilder (1999).

Provided herein are methods that employ the subject animal model for screening assays to determine whether a candidate therapeutic agent is effective for treating autoimmune or chronic inflammatory conditions that are resistant to treatment with a TNFa inhibitor. TNFx inhibitors to which these diseases are unresponsive may include receptor-based TNFa inhibitors such as ENBREL® (Immunex Corporation) and LENERCEPT® (Roche) or other drugs that incorporate a soluble TNFR, humanized antibodies against TNFa such as REMICADE® (Centocor), D2E7 (BASF Pharma) or CDP571 (HUMICADE®; Celltech) or small molecules whose pharmacologic effectiveness may be based on reducing endogenous TNFa (such as, for example, pentoxifylline, thalidomide or others).

Candidate therapeutic agents that are tested in the subject assays are determined to be effective if when administered to the subject animal model the agent brings about a reduction in the severity of arthritis that has been induced in the animal. The severity of arthritis in the animal may be assessed by any desired method, which may be based, for example, on assigning to each animal a numerical score that reflects the degree of swelling or stiffness of the animal's limbs. Efficacy of a test agent in reducing the severity of disease is determined by comparing this score averaged over a group of arthritic animals that receive the test agent with the score for a group that receives a placebo. The test agent and placebo generally are administered over a period of at least one week, but may be administered for a longer period, for example, over a period of 2, 3, WO 02/11767 PCT/US01/24783 4, 5, 6, 7 or 8 or more weeks. Alternatively, the effects of a single dose may be assessed using this model.

If DBA/1 p55'"p75 mice with CIA are used in the assays, an efficacious therapeutic agent will partially or fully ameliorate the symptoms of CIA in the mice. A reduction in severity of arthritis is determined to be present in DBA/1 p55"/"p75-' mice if the average clinical score (determined as described below) for an agent-treated group of mice is one or more clinical score units lower than the average clinical score for a group of negative control mice that are given a placebo. Preferably, the average clinical score in the agent-treated DBA/1 p55-/-p75' mice will be at least 2 clinical score units lower than the placebo-treated mice, and more preferably it will be at least 5 units lower.

To determine clinical score for individual DBA/1 p55"/"p75 mice in which CIA .has been induced, the following index may be used in which each paw is assigned a score in clinical units based on the following: 0 normal appearance 1 erythema/edema in 1-2 digits 2 erythema/edema in >2 digits, or mild swelling in ankle/wrist joint 3 erythema/edema in entire paw 4 massive erythema/edema of entire paw extending into proximal joints; ankylosis, loss of function Paw scores are combined for each mouse to determine a final score for that mouse, and then final scores for all animals in the test agent group are averaged and compared with the average score for the placebo-treated group. Generally, each test group will contain between 5 and 30 animals, though smaller or larger numbers of animals may be used. This or similar clinical scoring systems are suitable for evaluating CIA in DBA/1 p55-'p75'" mice, but also are suitable for other species and for animal models involving types of experimental arthritis other than CIA. If desired, other kinds of arthritis scoring systems may be used, such as, for example, a system based on levels in the blood or other tissues of molecules that reflect the degree of inflammation or molecules that are specific disease markers.

When DBA/1 p55-/-p75 mice are used, the CIA is induced according to the procedure given in Example 2, or using a similar protocol. In one preferred screening assay, a test agent is administered to a DBA/1 p55'"p75 mouse that is subjected to CIA induction, with the first dose of the agent being administered on the day the collagen boost is given, or the first dose may be delayed until the onset of symptoms, which 26 WO 02/11767 PCT/US01/24783 generally take 15-60 days to appear in these mice. In this assay, negative control DBA/1 p75" mice are treated with a placebo, such as rat or mouse IgG or a physiologically acceptable saline solution. If desired, an agent known to be efficacious against CIA in these mice can be included in the assay as a positive control; for example, antibody against CD30L or against II-IR1 can be used as a positive control (see Example 4), although this is optional. Doses of the test agent and control agent(s) may be administered daily, every two days, every three days, two times per week, one time per week, or less often if desired. The duration of the testing period is variable, and may continue, for example, for three days, one week, two weeks, three or for four or more weeks.

In the subject assays, the test agent and placebo may be administered by any suitable route, including orally administered liquid or solid forms, topical application, aerosol inhalation, transfection of host cells by recombinant DNA expressing the test agent, or by injection, including intraperitoneal, intravenous, subcutaneous or intramuscular injection. If desired, the test agent may be administered via a slow-release implant. When the test agent is incorporated into a slow-release formulation, very few doses are required, and a single dose may be used. If the test agent is an antibody and the subject is a mouse, the candidate therapeutic test agent may be administered at a dose of 20-75 ug/mouse, and is administered every day, every two to four days, or once a week.

If the test agent is a small molecule, such as an organic molecule, suitable doses for testing are from 0.5-1000 pg/mouse, and the agent may be administered once every one to ten days. If a larger species of model animal is used, dose is adjusted upward in proportion to average body weight for the larger species.

Efficacy of the test agent in DBA/1 p551-/p75"' mice or another strain of animal is determined by comparing the percentage of animals affected with arthritis in test and negative control groups, or by comparing the percentage of animals exhibiting severe disease during the testing period, and/or by comparing the mean clinical score for control groups and test groups after administration of the test agent. Mean clinical score may be determined according to the index described in Example 2, or by other suitable means. A test agent is determined to be useful for treating disorders resistant to TNFL inhibitors if animals to whom the test agent is administered exhibit a reduced severity of disease as compared with control animals that receive placebo instead of the test agent. In one preferred embodiment, the observed improvement is statistically significant, that is, WO 02/11767 PCT/US01/24783 I,0 p<0.05. Optionally, a dose dependency of the therapeutic response is established by Sadministering the test agent at several different doses.

Candidate therapeutic agents to be screened in the subject animal model include any agent that is potentially effective for treating diseases that are resistant to treatment with TNFa inhibitors. This includes, for example, agents that target cytokines other than TNFa that are sometimes associated with inflammation (such as, for example, GM-CSF, interferon-y, lymphotoxin-a, IL-1, EL-4, EL-8, EL-15, IL-17 and IL-18). Candidate C<I therapeutic agents include soluble receptor molecules and antibodies specific for target NC- cytokines or their receptors. Small organic molecules may also be screened using the 0 10 subject animal model, including but not limited to agents with potential for interfering with CD30 or IL-1 signal transduction Therapeutic methods Disclosed herein are methods for treating a variety of autoimmune and chronic inflammatory diseases by administering to a patient in need thereof an effective amount of one of the above-described agents. Blockers of CD30 signal transduction are used for treating any of the medical disorders listed below, and blockers of CD30 or IL-1 signal transduction are used for treating disorders listed below that respond poorly or not at all to treatment with a TNFa antagonist. In some instances, a disease is generally responsive to treatment with a TNFa inhibitor, but unresponsive in certain patients. An example of such a disease is rheumatoid arthritis. Rheumatoid arthritis patients who respond poorly or not at all to TNFa inhibitors will particularly benefit from treatment with an antagonist of the CD30/CD30L or IL-1/IL-Rl interaction.

Preferably, the patient is a human, and may be either a child or an adult. In one embodiment of the invention, a condition believed to be TH2 driven or a condition characterized by high levels of expression of CD30 on activated T cells, is treated by administering a CD30 inhibitor concurrently with an IL-4 inhibitor.

For the subject therapeutic methods, the therapeutic agents preferably are administered in the form of a physiologically acceptable composition comprising a purified recombinant protein in conjunction with physiologically acceptable carriers, excipients and/or diluents. Such carriers are nontoxic to recipients at the dosages and concentrations employed. Compositions suitable for in vivo administration may be formulated according to methods well-known in the art. Components that are commonly employed in such formulations include those described in Remington's Pharmaceutical WO 02/11767 PCT/US01/24783 Sciences, 16th ed., 1980, Mack Publishing Company. Ordinarily, the preparation of such compositions entails combining the therapeutic agent with buffers, antioxidants such as ascorbic acid, low molecular weight polypeptides (such as those having fewer than amino acids), proteins, amino acids, carbohydrates such as glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline mixed with non-specific serum albumin are exemplary appropriate diluents. If desired, the therapeutic agent may be formulated as a lyophilizate using appropriate excipient solutions such as sucrose as a diluent. Appropriate dosages can be determined in standard dosing trials, and may vary according to the chosen route of administration. In accordance with appropriate industry standards, preservatives may also be added, such as benzyl alcohol.

The amount and frequency of administration may vary, depending on such factors as the nature and severity of the indication being treated, the desired response, the duration of treatment, the age, weight and condition of the patient, and so forth. The dose of a therapeutic agent may be adjusted to accommodate various routes of administration, or according to the needs of individual patients as determined by the patient's physician.

Arthritis may be treated by the methods and compositions disclosed herein. As used here, the term "arthritis" refers to chronic inflammatory conditions that primarily affect joints, or the connective tissue surrounding joints, although various body organs may also become affected. Arthritis may be autoimmune or traumatic in origin, or it may be triggered by exposure to a foreign antigen, thereafter leading to a chronic condition that is no longer dependent on the continued presence of the triggering antigen. The term "arthritis," as used herein, includes: arthritis deformans; osteoarthritis; rheumatoid arthritis (adult and juvenile); Lyme disease arthritis; reactive arthritis including Reiter's disease; psoriatic arthritis; arthritis nodosa; seronegative spondylarthropathies, including but not limited to ankylosing spondylitis. The efficacy of anti-CD30L treatment in treating arthritic disease is illustrated in Examples 2 and 4.

The subject inhibitors, compositions and combinations are useful in treating a variety of rheumatic disorders, which are defined herein as any chronic disorder involving painful and often multiple localized inflammations of the joints, muscles, nerves, tendons, skin, eyes, connective tissues or various other organ systems. These include but are not limited to: arthritis; scleroderma; gout; systemic lupus erythematosus; polymyalgia rheumatica; fibromyalgia; Still's disease; chronic uveitis; disorders resulting in inflammation of the voluntary muscle, including dermatomyositis and polymyositis, 29 WO 02/11767 PCT/US01/24783 including sporadic inclusion body myositis; and inflammatory conditions such as chronic back or neck pain and sciatica. Systemic lupus erythematosus can cause inflammation of the joints, skin, kidneys, heart, lungs, blood vessels and brain. In its advanced forms, systemic lupus erythematosus this condition can result in kidney failure. Treatment with antibody against CD30L appeared to delay the progression of kidney failure in a mouse model for this disease (see Example 6).

Provided also are methods for using the subject inhibitors, compositions or combination therapies to treat various disorders of the endocrine system, including but not limited to: juvenile or maturity onset diabetes (including autoimmune, insulindependent types of diabetes; non-insulin dependent types and obesity-mediated diabetes); idiopathic adrenal atrophy; Addison's disease; hypothyroidism; Grave's disease; autoimmune thyroiditis, such as Hashimoto's thyroiditis; and polyglandular autoimmune syndromes (types I and II).

Conditions of the gastrointestinal system also are treatable with the subject inhibitors, compositions or combination therapies, including but not limited to: autoimmune sclerosing cholangitis; coeliac disease; inflammatory bowel diseases, including Crohn's disease and ulcerative colitis; autoimmune pancreatitis, including chronic pancreatitis; idiopathic gastroparesis; and idiopathic ulcers, including gastric and duodenal ulcers.

Included also are methods for using the subject inhibitors, compositions or combination therapies for treating disorders of the genitourinary system, such as autoimmune and idiopathic glomerulonephritis; and chronic idiopathic prostatitis (nonbacterial), including benign prostatic hypertrophy.

Also provided herein are methods for using the subject inhibitors, compositions or combination therapies to treat various hematologic disorders, including but not limited to: anemias and hematologic disorders, including pernicious anemia and aplastic anemia, and Fanconi's aplastic anemia; autoimmune hemolytic anemia; idiopathic thrombocytopenic purpura (ITP); myelodysplastic syndromes (including refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation); and autoimmune lymphoproliferative syndrome

(ALPS).

In addition, the subject inhibitors, compositions and combination therapies are used to treat hereditary conditions such as Gaucher's disease, Huntington's disease, and inuscular dystrophy.

WO 02/11767 PCTIUS01/24783 The disclosed inhibitors, compositions and combination therapies are furthermore used to treat conditions that affect the liver such as autoimmune or chronic inflammatory hepatitis that is not due to viral infection.

In addition, the disclosed inhibitors, compositions and combination therapies are used to treat various autoimmune or chronic inflammatory disorders that involve hearing loss. One of these is inner ear or cochlear nerve-associated hearing loss that is thought to result from an autoimmune process, autoimmune hearing loss. This condition currently is treated with steroids, methotrexate and/or cyclophosphamide, which may be administered concurrently with an inhibitor of the CD30/CD30L interaction.

A number of inflammatory pulmonary disorders also can be treated with the disclosed inhibitors, compositions and combination therapies, including: idiopathic lymphangioleiomyomatosis; chronic obstructive pulmonary disease (COPD) associated with chronic non-infectious bronchitis or with emphysema; and fibrotic lung diseases, such as cystic fibrosis and idiopathic pulmonary fibrosis.

Disorders associated with transplantation also are treatable with the disclosed inhibitors, compositions or combination therapies, including graft-versus-host disease.

To prevent or ameliorate graft-versus-host disease, the subject inhibitors may be administered prior to, concomitantly with, or following bone marrow or solid organ transplantation, including transplantation of heart, liver, lung, skin, kidney or other organs.

The subject inhibitors and the disclosed compositions and combination therapies also are useful for treating chronic inflammatory eye diseases, including autoimmune uveitis.

The subject inhibitors and the disclosed compositions and combination therapies also are useful for treating inflammatory disorders that affect the female reproductive system, including: multiple implant failure/infertility; fetal loss syndrome or IV embryo loss (spontaneous abortion); and endometriosis.

Other medical disorders treatable with the disclosed inhibitors, compositions and combination therapies include chronic degenerative diseases of the central nervous system. This includes, for example, diseases associated with demyelination, such as multiple sclerosis, systemic sclerosis and the Guillain-Barre syndromes (including acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acute motor sensory axonal neuropathy and Fisher syndrome). In a preferred embodiment, multiple sclerosis is treated with an antagonist of the CD30/CD30L interaction (most preferably an 31 WO 02/11767 PCT/US01/24783 antibody against CD30L) alone or concurrently with a TNFcc inhibitor or an IL-4 inhibitor (most preferably sIL-4R). Multiple sclerosis is representative of a chronic, degenerative disease of the central nervous system, which besides the demyelinating conditions also include, for example, amyotrohpic lateral sclerosis (Lou Gehrig's Disease); Bell's palsy; Parkinson's disease and idiopathic chronic neuronal degeneration, all of which may be treated with an agent capable of inhibiting the interaction of and CD30. The efficacy of anti-CD30L antibody in ameliorating a multiple sclerosis-like disease is illustrated in Example Other chronic inflammatory conditions treatable with the disclosed inhibitors, compositions and combination therapies include cold agglutinin disease; Behcet's syndrome; Sjogren's syndrome; and idiopathic tenosynovitis, as well as various chronic inflammatory disorders associated with hereditary deficiencies. The subject inhibitors, compositions and combination therapies furthermore are useful for treating Bell's palsy (idiopathic facial paralysis); chronic fatigue syndrome (not associated with ongoing infection); chronic degenerative vertebral disc disease; Gulf war syndrome; and myasthenia gravis, which may be treated concurrently with corticosteroids.

Disorders involving the skin or mucous membranes also are treatable using the subject inhibitors, compositions or combination therapies. These include: acantholytic diseases, including Darier's disease, keratosis follicularis, pemphigus vulgaris and paraneoplastic pemphigus; acne rosacea; alopecia areata; bullous pemphigoid; eczema; erythema, including erythema multiforme and erythema multiforme bullosum (Stevens- Johnson syndrome); inflammatory skin disease; lichen planus; linear IgA bullous disease (chronic bullous dermatosis of childhood); loss of skin elasticity; neutrophilic dermatitis (Sweet's syndrome); pityriasis rubra pilaris; psoriasis; pyoderma gangrenosum; loss of skin elasticity; and toxic epidermal necrolysis.

Other diseases that can be treated with the disclosed compounds, compositions and combination therapies include: autoimmune-associated chronic mucocutaneous candidiasis; allergies; sarcoidosis; multicentric reticulohistiocytosis; Wegener's granulomatosis; arteritis, including giant cell arteritis; vasculitis and chronic autoimmune myocarditis.

To treat a medical disorder using the compounds and compositions provided herein, a therapeutically effective amount of a therapeutic agent according to the invention is administered to a mammal in need thereof. The agent is administered WO 02/11767 WO 0211767PCTIUS01/24783 according to a regimen of dose and frequency of administration that is adequate to induce a sustained improvement in at least one indicator that reflects the severity of the disorder.

An improvement is considered "sustained" if the patient exhibits the improvement on at least two occasions separated by at least one day, but preferably that are separated by one week, two weeks, three weeks or four or more weeks. The severity of the disorder is determined based on signs or symptoms, or may be determined by questionnaires that are administered to the patient, such as the quality-of-life questionnaires often used by physicians to assess the status of chronic disease conditions.

One or more indicators that reflect the severity of a patient's illness may be assessed for determining whether the frequency and duration of drug treatment is sufficient. The baseline value for a chosen indicator is established by examination of the patient prior to administration of the first dose of the therapeutic agent. Preferably, the baseline examination is done within about 60 days of administering the first dose.

For example, if the condition being treated is an arthritic condition, such as rheumatoid arthritis, psoriatic arthritis, osteoarthritis, one or more indicators for determining sufficiency of treatment may. be chosen from among: number of tender, painful or swollen joints; degree of joint pain or tenderness or swelling; patient selfassessment quality-of-life questionnaires), and physician assessment. For many arthritic diseases, the patient's self-assessment is a satisfactory indicator. Typical selfassessment questionnaires will reflect a patient's ability to conduct their daily activities, their perception of well-being, their level of pain and so on. The duration of treatment required to induce a measurable improvement for an arthritic or any other type of disease treatable as described herein is typically one to several weeks.

If the condition being treated is multiple sclerosis, suitable indicators for determining sufficiency of treatment include observing an improvement in one or more of the following: bladder and bowel control; fatigue; spasticity; body or hand tremors; muscle weakness; ability to walk; numbness in limbs; ability to concentrate performance on a simple memory test); and subjective level of pain. Alternatively, the indicator may consist of the patient's score on a quality of life questionnaire as described above.

If the condition being treated is systemic lupus erythematosus, the indicator for determining sufficiency of treatment may consist of an observed improvement in one of the following: fatigue; fever; ulcers of the mouth and nose; facial rash ("butterfly rash"); photosensitivity (SLE often flares up after exposure to sunlight); pleuritis; pericarditis; 33 WO 02/11767 PCT/US01/24783 Raynaud's phenomenon (reduced circulation to fingers and toes with exposure to cold); kidney function; and white blood cell count (SLE patients often have decreased numbers of white blood cells).

Improvement in a patient's condition is induced by repeatedly administering a dose of a therapeutic agent according to the invention until the patient manifests an improvement over baseline for the chosen indicator or indicators. In treating chronic conditions, a satisfactory degree of improvement usually is obtained after repeatedly administering the agent or agents over a period of at least a month or more, for one, two, or three months or longer. In some instances, improvement may occur sooner than one month, for example, after three weeks, two weeks, one week, or even after a single dose. A treatment duration of one to six weeks, or even a single dose, may be sufficient for treating occasional flare-ups in patients suffering from chronic conditions that tend to go into remission in-between flare-ups. For persistent conditions, treatment may be continued indefinitely if desired. Evan after a condition has shown improvement, maintenance therapy may be continued indefinitely at the same level or at a reduced dose or frequency of administration. If the dose or frequency of administration has been reduced, it may be resumed at the previous level if the patient's condition should worsen.

In addition, treatment with inhibitors of the subject invention may be administered prophylactically to patients who are predisposed to an autoimmune or chronic inflammatory condition.

Any efficacious route of administration may be used to therapeutically administer the subject therapeutic agents, combinations and compositions. If injected, the agents can be administered, for example, via intra-articular, intravenous, intramuscular, intralesional, intraperitoneal or subcutaneous routes. Bolus injection or continuous infusion may be used. Other suitable means of administration include sustained release from microparticles, implants or the like, aerosol inhalation, eyedrops, oral preparations, including pills, syrups, lozenges or chewing gum, and topical preparations such as lotions, gels, sprays, ointments or other suitable techniques.

Alternatively, proteinaceous agents, such as antibodies or antigen-binding fragments thereof, may be administered by implanting cultured cells that express the protein. In one embodiment, the patient's own cells are induced to produce the therapeutic agent by transfection in vivo or ex vivo with a DNA that encodes a protein that blocks the CD30/CD30L, IL-1/IL-1R, IL-4/IL-4R or TNF/TNFR interactions. This DNA can be introduced into the patient's cells, for example, by using naked DNA or liposome- 34 WO 02/11767 PCT/US01/24783 encapsulated DNA that encodes the agent, by using calcium-phosphate precipitated DNA, or by other means of transfection. Autologous cells that are transfected ex vivo are returned to the patient's body.

Regardless of the route of administration that is chosen, it is understood that the treatment regimens may be adjusted depending on the patient's needs, in accord with general principles of medicine.

When the therapeutic agent is an antibody, such as, for example, an antibody against CD30, CD30L, IL-1, IL-1R1, TNFa, IL-4 or IL-4R, preferred dose ranges for therapeutic or prophylactic purposes in humans include 0.1 to 20 mg/kg, or more preferably, 0.5-10 mg/kg. Another preferred dose range is 0.75 to 7.5 mg/kg, as exemplified in the experiments of Example 2 (adjusted for human body weight according to DeVita et al., eds., Cancer Principles Practice of Oncology, 4 th Ed., J.B.

Lippincott, 1993). A larger or smaller amount of antibody per dose may be used to accommodate differences in affinity of the antibody for the antigen. A suitable dose range for CD30:Fc is 0.01-20 mg/kg of body weight, or more preferably, 0.1-10 mg/kg of body weight. For other soluble proteins used as inhibitors in accord with the invention, suitable dose ranges include 2-500 pg/kg, 0.5-10 mg/kg and 10 to 50 mg/kg of body weight. It is understood that the skilled physician will adjust the dose and frequency of administration in accord with the needs of the patient and the nature of the disease being treated.

The following examples are offered by way of illustration, and not by way of limitation. Those skilled in the art will recognize that variations of the invention embodied in the examples can be made, especially in light of the teachings of the various references cited herein.

EXAMPLE 1 Monoclonal Antibodies Directed Against A monoclonal antibody directed against murine CD30L was produced in rats as follows. Lewis rats were repeatedly immunized by the intraperitoneal route with 10-20 x 10 6 transfected CHO cells expressing full length murine CD30L. When serum titers were detected in the rats, they were given an intravenous boost with 10 x 106 transfected CHO cells. After three days, spleen cells from the immunized rats were fused with AG8.653 mouse myeloma cells. When hybridomas were well established in 96-well plates, supernatants from each well were screened by an ELISA assay that employed WO 02/11767 PCT/US01/24783 CHO cells that were transfected with DNA encoding CD30L (CELISA). For the CELISA, CHO cells expressing the recombinant CD30L were adhered to the ELISA plates, and each supernatant was screened for its ability to react with the expressed on the CHO cells. Hybridoma cells from positive wells were expanded in 48well plates and screened by CELISA and also by fluorescence-activated cell sorting (FACS) using transfected and non-transfected CHO cells. Hybridoma cells that bound transfected but not non-transfected CHO cells were selected and screened further by FACS against mouse lymphoma cells that naturally express CD30L (EL4 cells).

Hybridoma isolates that recognized both recombinant and naturally expressed were cloned twice by limiting dilution cloning, during which the activity of the supernatants was tracked at each step by CELISA and/or FACS assays. One of these clones, the M15 clone, was chosen for further propagation. In additional experiments, it was determined that the M15 antibody specifically blocked the binding interaction -between murine CD30 and EXAMPLE 2 Treatment of collagen-induced arthritis in mice Arthritis treatments that are effective in treating collagen-induced arthritis (CIA) are also effective in treating arthritis in humans (see, for example, Anthony and Haqqi, Clin Exp Rheumatol 17:240-244 (1999)), hence the effects of antagonizing the CD30/CD30L interaction was tested in CIA mice. Collagen-induced arthritis (CIA) was elicited in male DBA/1 mice (Harlan, UK) by injecting the mice with type II chicken collagen (Sigma). Mice were injected on day 0 with 100 jg of the collagen in complete Freund's adjuvant, and boosted on day 21 with a dose of 200 Rig in incomplete Freund's adjuvant. Collagen injections were administered intradermally at the base of the tail. In this model, generally 75-100% of the mice are affected, that is, 75-100% of the mice exhibit arthritis symptoms after collagen injection. In these experiments, indicia of CIA usually appeared in affected, mice by day 23.

Mice injected with collagen as described above were injected intraperitoneally with doses ranging from 0.15-150 pg of the M15 monoclonal antibody whose preparation is described in Example 1. Four experiments were conducted, each involving 13-15 mice per test group. In each of the four experiments, positive control mice received 150 jgg of etanercept (ENBREL®, Immunex Corporation), which is known to be effective against CIA, and negative control mice received this same amount of human IgG (hulgG) or rat 36 WO 02/11767 PCT/US01/24783 IN IgG. Daily injections of M15, etanercept or IgG were initiated on day 21, the day of the second collagen injection, and were continued until day 33.

During these experiments, mice were assessed three times per week for clinical signs of arthritis by an independent observer blinded to the treatment groups. Disease was evaluated using an arthritis index system that has been established for this model system. For scoring, each paw was assigned a clinical score based on the index. Paw scores were combined for each animal to determine a clinical score for that animal. The index used was as follows:

O

0 normal appearance 0 10 1 erythema/ edema in 1-2 digits

O

NC 2 erythema/ edema in >2 digits, or mild swelling in ankle/wrist joint 3 erythema/ edema in entire paw 4 massive erythema/edema of entire paw extending into proximal joints; ankylosis, loss of function Improved mean clinical scores were evident in all groups that received M15 by the fifth day of M15 administration, and in one experiment by the third day of administration. Final results of the four experiments are summarized in Tables 1-4, shown below. Mean clinical scores, percent disease incidence (mice exhibiting CIA divided by total number of mice in group), and percent of affected mice exhibiting severe disease were calculated for each group of mice. Mice considered to have "severe disease" are those that had a clinical score greater than two at any time during the experiment. Statistical significance was determined for the differences in mean clinical score between the negative control group and the other groups of mice, based on scores on the last day of the experiment. Statistical significance was determined using a oneway analysis-of-variance (ANOVA) with Dunnett's method Motuisky, Analyzing Data with GraphPad Prism, 1999, GraphPad Software, Inc., San Diego, CA). One-way ANOVA compares three or more groups when the data are categorized in one way.

Dunnett's method compares control groups to treatment groups.

The first experiment tested the effects of administering to CIA mice a dose of 150 pg/day of the M15 antibody. By day 4 or 5 of this experiment, mean clinical score differences began to appear between negative control animals and those that received or etanercept. The results of this first experiment are summarized in Table 1. As was expected, the mice that received etanercept (positive control group) exhibited a lower WO 02/11767 PCT/US01/24783 incidence of disease and a reduced incidence of severe disease when compared with the negative control mice that received HulgG. The mice that received M15 also exhibited a lower disease incidence and a reduced percentage of mice with severe disease (see Table 1) as compared with negative controls. In addition, the M15 group had a lower mean clinical score than the negative controls on the last day of the experiment (day 33). For both the etanercept and the M15 groups, the last day differences in mean clinical score relative to negative controls were found to be statistically significant (p 0.05 or less).

Treatment etanercept HuIg Disease Incidence 53% 33% 87% TABLE 1 Severe Mean Clinical Score on Disease Last Day of Experiment 27% 2.53 7% 0.53 80% 6.47 A second experiment employed a similar protocol to compare doses of 50 jig and 150 gig of M15. The results are shown in Table 2. For this experiment, statistically significant improvement in mean clinical score was seen at the end of the experiment for both the etanercept group and for both doses of Treatment 150 Rg 50 jig etanercept HuIg Disease Incidence 40% 33% 47%% 87% TABLE 2 Severe Mean Clinical Score on Disease Last Day of Experiment 20% 1.2 20% 1.07 6% 0.73 80% 7.53 In a third experiment, doses of 15, 50 and 150 gig of M15 were compared, and the results are summarized in Table 3. Again, mice that received M15 experienced a lower incidence of disease, as well as a lower incidence of severe disease as compared with negative controls. A statistically significant improvement in mean clinical score vis-a-vis controls was observed at all three doses of M15 in this experiment.

38 WO 02/11767 PCT/US01/24783 TABLE 3 Disease Severe Mean Clinical Score Treatment Incidence Disease on Last Day 150 gg 42% 42% 3.29 50 jig 71% 57% 3.71 15 gg 47% 47% 3.3 etanercept 53% 20% 1.33 Hulg 87% 80% 8.4

(N

0 In a fourth experiment, 0.15, 1.5, 15 and 150 gg doses of M15 were tested, using rat IgG as the negative control. Results of this experiment are shown in Table 4. As seen in Table 4, improvement in mean clinical score exhibited a dose-dependency at the higher doses. For the 15 and 150 gg doses, the improvement in clinical score was statistically significant.

TABLE 4 Disease Severe Mean Clinical Score Treatment Incidence Disease on Last Day 150 jig 33% 26% 1.47 15 jig 80% 33% 2.67 1.5 jig 73% 67% 5.47 0.15 gg 67% 67% 5.67 etanercept 40% 13% 1 rat IgG 87% 67% 5.8 EXAMPLE 3 TNF-independent model of murine collagen-induced arthritis A novel mouse was generated by moving previously described targeted null mutations in both the p55 and the p75 TNFa receptors (Peschon et al., J Immunol 160:943-952, 1998) from the CIA-insensitive C57BL/6 genetic background to the CIA-sensitive DBA/1 genetic background.

DBA/1 mice doubly deficient in p55 and p75 receptors (DBA/1 p55-/p75 were generated by crossing C57BL/6 p55"'-p75'" mice (Peschon et al., 1998) with DBA/1 mice WO 02/11767 PCT/US01/24783 obtained from Jackson Laboratories. The resulting double heterozygotes were crossed again to DBA/I mice. The resulting progeny that were heterozygous for both mutations were further tested for homozygosity at the DBA/1 MHC complex (required for CIA sensitivity) by FACS analysis using antibodies specific for C57BL/6 and DBA/1 MHC alleles. Antibodies against the C57BL16 and DBA/1 MHC alleles were purchased from BD PharMingen.

Those progeny that were homozygous for the DBA/1 MHC were crossed to DBA/1 for another three generations to generate DBA/1 mice heterozygous for both and p75 TNF receptor mutations (DBA/1 N5 p55"'p75/-; "N5" refers to the fifth backcross generation). DBA/1I N5 p55/-p75 mice were intercrossed to establish a colony of DBA/1 mice doubly deficient in p55 and p75 TNF receptors (DBA/I N5 In order to identify mice that were homozygous for each of the null mutations, DNA from the progeny of the latter crosses was analyzed using PCR assays specific for the murine p55 and p75 TNFR genes, using DNA extracted from ear punches.

For tracking mutations in the p55 gene, the following PCR primers were used: 5'-GGATTGTCAC GGTGCCGTTG AAG-3' (SEQ ID NO:9) 5'-CCGGTGGATG TGGAATGTGT G-3' (SEQ ID 5'-TGCTGATGGG GATACATCCA TC-3' (SEQ ID NO:11) pgk5'-66: 5'-CCGGTGGATGTGGAATGTGTG-3' (SEQ ID NO: 12) Twenty-five pmole each of the four primers listed above were added to the ear punch DNA and the mixture subjected to 32 cycles of PCR for 1 minute at 94', 1 min. 65" and seconds at 72'. PCR products were resolved and visualized on 3 USB fine resolution agarose (cat. 73422) gels run in TAE buffer and stained in ethidium bromide. Expected PCR products using the above primers were 120 bp for p55 and 155 bp for p55-"1.

Heterozygous mice were expected to yield both products. Additional nonspecific products migrating at about 300-500 bp were seen occasionally in p55- mice.

The following primers were used to track the p75 mutations: 5'-AGAGCTCCAGGCACAAGGGC-3' (SEQ ID NO:13) p80i-1: 5'-AACGGGCCAGACCTCGGGT-3' (SEQ ID NO:14) pgk5'-66: 5'-CCGGTGGATGTGGAATGTGTG-3' (SEQ ID NO:12) These PCR reactions used 50 pmole p80-Kas, 100 pmole p80i-1 and 20 pmole pgk5'-66 for 32-34 cycles for 1 minute at 94°, 1 minute at 65° and 30 seconds at 72°. PCR products WO 02/11767 PCTIUS01/24783 were resolved and visualized on 3 USB fine resolution agarose gels run in TAE buffer and stained in ethidium bromide. p75'" mice were expected to yield a 275 bp product, and p75 mice to yield a 160 bp product. Heterozygotes were expected to yield both products. Additional nonspecific products were occasionally seen migrating at about 100 bp.

Homozygous DBA/1 p55'1-p75' mice were thus identified using PCR and thereafter were interbred.

EXAMPLE 4 Collagen-induced arthritis in DBA/1 p55-/'p75- mice The following experiments using the p55-'p75'1 DBA/1 mice of Example 3 demonstrate that TNFa-independent CIA can be effectively treated either by administering a test agent that inhibits signal transduction by CD30 or IL-1.

CIA was induced in p55'- 1 p75-"' DBA/1 mice by administering heterologous type H collagen according to the protocol described above in Example 2. Two experiments were conducted, each using 15 mice per test group. These experiments were designed to determine whether the inhibitors being tested could prevent the development of CIA in these mice. Mice were randomly divided into groups (n 15) at the time of the boost and were injected daily with either the test agent or the control protein for 14 days.

Compared with wild-type DBA/1 mice, the DBA/1 p55"/'p75 mice injected with collagen displayed a delayed onset and slower course of disease. However, significant clinical symptoms appeared in the mutant mice 15-60 days after the second collagen injection. As expected, no diminution of arthritis symptoms was observed in DBA/I p 5 5 mice when they were treated with p75 TNFR.Fc (ENBREL®; Immunex Corporation). In contrast, ENBREL® is highly effective in reducing arthritis symptoms in wild-type DBA/1 mice with CIA (see Example 2 above). Thus, the arthritis observed in the p55'p75 DBA/1 mice is not mediated by TNFa.

Experiments were conducted to determine whether molecules other than TNFac played a role in CIA in DBA/1 p55- p75 mice. In these experiments, agents that inhibit the CD30/CD30L or the IL-1/IL-1R1 interaction were tested to see if they would affect CIA in this animal model. Monoclonal antibodies against either IL-1R1 (M147; Immunex Corporation) or murine CD30L (M15; preparation described in Example 1) were administered by intraperitoneal injection to these mice, while control mice received rat IgG. Each experimental group consisted of 15 mice. Antibody treatment was initiated WO 02/11767 PCT/US01/24783 at day 21 (at the time of the collagen boost), and was administered for 21 days for experiment and for 28 days in experiment For M15 antibody, a dose of 50 Rxg was administered per day, and for the M147 antibody, a dose of 50 pg was administered every two days. Clinical score was determined three times per week, using the clinical scoring system described in Example 2.

As illustrated in Table 5 (experiment and Table 6 (experiment the administration of M147 or M15 significantly reduced arthritis in the DBA/1 p55"-p75' mice, with M147 resulting in an almost complete amelioration of disease.

TABLE Disease Severe Mean Clinical Score Treatment Incidence Disease on Last Day 26% 0.06% 0.75 M147 0.06% 0% 0.07 rat IgG 67% 60% 4.27 TABLE 6 Disease Severe Mean Clinical Score Treatment Incidence Disease on Last Day 60% 40% 2.7 M147 0% 0% 0 rat IgG 80% 67% 4.1 The results of these two experiments show that collagen-induced arthritis can be established in a TNFa-independent manner, and that inhibiting either the IL-1/IL-1R1 or CD30/CD30L interactions effectively reduce disease in these mice.

EXAMPLE Mouse experimental allergic encephalomyelitis model This example demonstrates the efficacy of antagonists of the CD30/CD30L interaction for treating multiple sclerosis. A mouse model for multiple sclerosis was employed for this purpose. Chronic experimental autoimmune encephalomyelitis

(EAE),

which is a well accepted experimental model for this disease, was induced in female C57BL/6 mice (Taconic Farms Inc., Germantown, NY) using a modification of the WO 02/11767 PCT/US01/24783 protocol described by Mendel et al. (Eur. J. Immunol. 25:1951-59, 1995). In brief, disease induction involved the immunization of mice with the MOG35-55 peptide derived from rat myelin oligodendrocyte glycoprotein (Mendel et al., 1995). Modifications to the disease induction protocol of Mendel et al. included the use of a lower dose of MOG35-55 for immunization (see below), no booster immunization, and the use of RIBI® adjuvant instead of complete Freund's adjuvant.

To induce EAE, groups of age and weight-matched mice (11-13 mice per group) were given a dose of 100 pg of rat MOG35-55. The MOG35-55 was emulsified in 0.2 ml RIBI adjuvant (Corixa Corporation), and injected subcutaneously at three sites distributed over the shaved flank. To induce EAE with accelerated onset, the mice in a second experiment (Experiment 2) received by intravenous injection 500 ng pertussis toxin (List Biological Laboratory Inc, Campbell, CA), which was administered 48 hours after they received their dose of MOG35-55. The mice in Experiment 1 received no pertussis toxin, thus disease onset in Experiment 2 was accelerated as compared with Experiment 1.

Administration of antibody or placebo was initiated on the day after the MOG35-55 was administered (day 1) and was continued through day 11. Each mouse was injected intraperitoneally every other day with 0.2 ml pyrogen-free phosphatebuffered saline (PBS) or 0.2 ml PBS containing one of the following: 100 pRg (ii) 100 gpg rat IgG (Sigma); or (iii) 75 gg M147 (anti-ILlR1). Endotoxin levels were <10 EU/mg of protein for all reagents. Mice were monitored daily for days (Experiment 1) or 30 days (Experiment 2) for weight loss, disease onset and severity of clinical signs of EAE by an independent observer blinded to the treatment groups.

The severity of EAE was assessed using a standard EAE index system in which is used to indicate an asymptomatic mouse and clinical scores ranging from 0.5 to 4 was used to indicate varying degrees of ascending paralysis. The severity of EAE was assessed using a slightly modified version of a commonly used EAE scoring system. In this system, were used to indicate a mouse with no evidence of disease and scores of were used to indicate varying degrees of ascending paralysis as follows: 1, tail paralysis; 2, hind limb weakness; 3, partial hind limb paralysis; 4, complete hind limb paralysis; 5, moribund or dead. The disease protocol described above induces an acute episode of disease in control mice (peak score of 2-4) from which most recover at least partially. Thus the acute episode of disease is not lethal and mice do not reach a score of The aforedescribed scale was modified to include a score of which was given to WO 02/11767 PCT/US01/24783 mice that showed the earliest signs of EAE but that did not exhibit complete paralysis of the tail. Mice given a score of 0.5 exhibited some or all of the following symptoms: overnight weight loss of 1-2 grams; noticeable tremor when held up by the tail; and weakness at the distal tip of the tail.

The median day of onset of EAE was determined by Kaplan-Meier Survival analysis. Significant differences in onset between groups were assessed using a Log- Rank comparison. Fischer's exact test was used to analyze the statistical significance of differences in the incidence of EAE among the groups of mice.

Results of these two experiments demonstrated the ameliorating effects of either of the tested antibodies on the onset, incidence and severity of the clinical course of EAE.

As shown below in Table 7, administration of either anti-CD30L (M15) or anti-IL-IR1 (M147) resulted in delayed disease onset and a reduced incidence of disease in both of the experiments.

TABLE 7 Incidence Median Day of Onset Combined Grou Expt Expt 2 Results Expt Expt2 PBS 10/11 11/13 21/24 22 16 rat IgG 9/11 12/13 21/24(88%) 21 16 (92%) anti- 6/11 8/13 14/24(58%) 35 23 (62%) anti- 5/11 9/13 14/24(58%) not IL-1R determined In Table 7, for the combined results for mice that received M15 or M147, when compared with the rat IgG group, p<0.05 for the incidence of disease. In Experiment 1, for median day of onset for the M15 mice, p=--0.067 vs Rat IgG and p<0.05 vs PBS. In Experiment 2 in Table 5, for median day of onset, p<0.005 vs rat IgG for both the and M147 groups.

For these same two experiments, Table 8 shows the mean percent change in body weights within each group over the acute course of disease. As shown in Table 8, mice that received either anti-CD30L or anti-IL-IR1 antibodies lost less weight during this time than mice that received rat IgG or PBS.

WO 02/11767 PCT/US01/24783 TABLE 8 Mean Percent Body Mean Percent Body Weight Change Weight Change Treatment SEM (All mice) SEM (Affected mice only) Expt anti-CD30L -2.0 2.4* -6.2 2.7 anti-IL-IRI -1.5 -5.4 3.2 PBS -7.3 3.1 -9.2 2.7 Rat IgG -9.7 +1.8 -11.4 +1.75 Expt 2 anti-CD30L 0.7 -5.3 anti-IL-IRI -7.7 3.5* -13.3 3.7 PBS -16.3 2.9 -19.9 1.8 Rat IgG -19.9 2.9 -21.7 +2.6 To calculate the weight data in Table 8, the baseline weight for each mouse was defined as its weight on day 12 (Experiment 1) or day 10 (Experiment 2) relative to immunization. These days were chosen as reference points because all of the mice in each experiment were weight-matched on day 0 and the mean weight of all the groups increased in a similar manner between days 0-12 (Experiment 1) or days 0-10 (Experiment The mean weights of the various groups diverged after these time points due to weight loss associated with the onset of EAE. The left-hand column of Table 8 shows the mean percent body weight change calculated for all of the mice in each treatment group, that is, both the clinically affected (clinical score of at least 0.5) and nonaffected mice were included for this calculation. The right-hand column of Table 8 shows the mean body weight change during the acute phase of the disease for only the mice that were clinically affected. Body weight change for the affected mice was calculated based on the difference between the baseline weight of each individual mouse and the minimum weight observed for that mouse after the onset of disease. For those mice that never showed clinical evidence of disease, the percent body weight change for Table 8 was calculated by comparing the baseline weight of each non-affected mouse to its weight on day 25 of the experiment.

The data in Table 8 illustrate that either anti-CD30L or anti-IL-Ri is effective in slowing the weight loss otherwise observed in mice injected with MOG35-55. The WO 02/11767 PCT/US01/24783 Student's t test was used to determine the statistical significance of these body weight differences. Numbers found to be statistically significant are marked with asterisks in Table 8, and p values were as follows: *p<0.05 vs rat IgG controls; **p<0.01 vs rat IgG controls; ***p<0.001 vs rat IgG controls.

Table 9 presents the clinical score results for Experiments 1 and 2. Group means and SEM were calculated based on the peak clinical score for each mouse (0 for nonaffected mice; 0.5 to 4 for affected mice). The mean peak clinical score for each antibody-treated group was compared with the rat IgG-treated group using the Student's t 0 test, and the p values are shown in the last column of Table 9.

TABLE 9 Mean Peak Clinical Score Treatment SEM (scale p vs Rat IgG Expt 1 anti-CD30L 1.1 0.3 0.0621 anti-IL-IRI 0.9 0.3* 0.0362 PBS 2.0 0.3 0.7402 Rat IgG 2.1 +0.4

NA

Expt2 anti-CD30L 1.3 0.4* 0.0122 anti-IL-IRI 1.3 0.3* 0.0056 PBS 2.2 0.3 0.3262 Rat IgG 2.7 0.3

NA

As shown in Table 9, significant differences in clinical score were observed between groups of mice treated with either M15 or M147, as compared with the control mice. Differences that were determined to be statistically significant (p<0.05) are marked in Table 9with an asterisk.

EXAMPLE 6 Blocking CD30L delays kidney failure in a murine model of systemic lupus erythematosus Female (NZB x NZW)pi hybrid mice (referred to hereafter as "NZB/W mice") spontaneously develop a lupus-like disease characterized by the presence of serum WO 02/11767 PCT/US01/24783 autoantibodies to double-stranded DNA (dsDNA). These mice, which eventually experience total kidney failure, are often used as a model for experimentation directed at better understanding and treating lupus in humans. Over time, this condition in NZB/W mice progresses to kidney malfunction as manifested by the appearance of proteinuria. In a trial experiment to study the onset and progression of disease in these mice, about half had serum anti-dsDNA titers and about 10% had proteinurea by 26 weeks of age. By 38 weeks of age, 20% of the mice had died, and of the remaining mice, 52% were proteinurea positive and 98% had serum anti-dsDNA titers.

Female NZB/W mice 32 weeks of age were used in an experiment to determine whether anti-CD30L could delay the development of lupus-like disease. Within a group of 32 week old mice, 98% were serum positive for antibodies against dsDNA (detected by ELISA), but only 40% had progressed to renal disease as assessed by proteinuria (detected using CHEMSTRIP®; Roche). Mice from this group that were negative for proteinuria were used in the following experiment.

Thirty-two week old NZ/B mice that did not have proteinuria were divided into two groups, and each group was treated every other day with 150 pgg of either anti- (M15) (group of 10 mice) or rat IgG as a control (group of 9 mice), administered by intraperitoneal injection. Treatments were continued for five weeks, and the mice were assessed weekly for the presence of proteinuria. Approximate values for the percentages calculated from the results of this experiment are summarized in Table below.

TABLE Treatment Percent Incidence Progression to Proteinuria Week 1 Week 2 Week 3 Week 4 Week 10% 10% 10% 10% Rat IgG 0% 10% 20% 30% The data in Table 10 shows a trend toward decreased incidence of proteinuria in mice treated with anti-CD30L as compared with control mice. A scale from 1-5 was used to assess the degree of proteinurea among these mice. At five weeks, the mean proteinurea index for control mice in this experiment was 1.7, whereas M15-treated mice had a mean proteinurea index of 0.6. This result suggests that humans suffering from WO 02/11767 PCT/US01/24783 systemic lupus erythematosus might benefit from treatment with antibodies against or with other antagonists of the CD30/CD30L interaction.

To confirm the result presented above, a second group of female NZB/W mice are randomly assigned to treatment groups as antibodies against dsDNA first appear in their serum. This experiment is designed to test the effects of treatment on the progression of anti-dsDNA titers and the progression of proteinurea. One group of mice is treated with 150 gpg of anti-CD30L (M15), and the other group is treated with rat IgG as a control.

Treatments are administered three times per week by intraperitoneal injection for a period of three weeks. Mice are monitored weekly for titers of serum antibodies to dsDNA and for the appearance of proteinuria.

In other experiments, the efficacy of anti-CD30L treatment will be tested in a model of chemically induced lupus. In this model, administration of the isoprenoid alkane pristane (2,6,10,14 tetramethylpentadecane) induces autoantibody production and immune complex mediated glomerulonephritis. One advantage of this model is that it is not restricted to a particular mouse strain. Initial experiments are underway in normal BALB/c and C57BL/6 strains of mice.

Claims (13)

1. A method of treating an autoimmune or chronic inflammatory condition in a patient, said method comprising administering to the patient an agent that is capable of inhibiting the binding of CD30 to CD30L, wherein the agent is administered according to a regimen of dose and frequency of administration that is adequate to induce a sustained improvement in at least one indicator that reflects the severity of the patient's condition, the improvement being considered sustained if the patient exhibits the improvement on at least two occasions separated by at least one day.

2. A method according to claim 1, wherein the patient is a human.

3. A method according to claim 1 or 2, wherein the agent is selected from the group consisting of: an antibody that is specific for a non-agonistic antibody that is specific for CD30; and a soluble CD30 polypeptide comprising an amino acid sequence selected from the group consisting of: amino acids 19-390 of the human CD30 polypeptide of SEQ ID NO:6; (ii) a fragment of the sequence of wherein said fragment is capable of binding CD30L; and (iii) a CD30-binding polypeptide having at least 90% identity to amino acids 19- 390 of SEQ ID NO:6.

4. A method according to claim 2, wherein the agent is a soluble human polypeptide comprising amino acids 19-390 of SEQ ID NO:6 or a fragment thereof, said polypeptide further comprising an Fc region of a human immunoglobulin.

5. A method according to claim 2, 3 or 4, wherein the condition is arthritis.

6. A method according to claim 5, wherein the condition is rheumatoid arthritis.

7. A method according to claim 6, wherein the agent is selected from among the group consisting of an antibody that is specific for CD30L, a soluble human polypeptide comprising amino acids 19-390 of SEQ ID NO:6, a fragment of amino acids 19-390 of SEQ ID NO:6, a fusion protein comprising amino acids 19-390 of SEQ ID NO:6 and an Fc region of a human immunoglobulin protein; and a fusion protein comprising a CD30L-binding fragment of amino acids

19-390 of SEQ ID NO:6 and an Fc region of a human immunoglobulin protein. 8. A method according to any one of claims 1 to 3, wherein the agent is administered concurrently with a second agent that is an antagonist of TNFa, IL-Ia, IL-10 or IL-4. 9. A method according to claim 2, wherein the patient's condition is selected from the group consisting of multiple sclerosis, systemic sclerosis, acute inflammatory demyelinating polyneuropathy, acute motor axonal neuropathy, acute motor sensory axonal neuropathy and Fisher syndrome. A method according to claim 2, wherein the patient's condition is systemic lupus erythematosus. 11. A method according to claim 8, wherein the agent capable of blocking the CD30/CD30L interaction is an antibody specific for CD30L and the second agent is an antagonist of IL-4. 12. A method according to claim 11, wherein the IL-4 antagonist is selected from the group consisting of an antibody specific for IL-4, an antibody specific for IL-4R and a soluble IL-4 receptor comprising amino acids 1-207 or 2-207 of SEQ ID NO:16. 13. A method according to claim 12, wherein the patient's condition is selected from the group consisting of systemic lupus erythematosus, scleroderma and pemphigus vulgaris. 14. A method of treating an autoimmune or chronic inflammatory condition that is resistant to treatment with an inhibitor of TNFo, said method comprising administering to a patient in need thereof an agent that is capable of inhibiting the binding of CD30 to CD30L or the binding of IL-la or IL-10 to IL-R1, thereby blocking signal transduction by CD30 or IL-1 and wherein said agent is administered according to a regimen of dose and frequency of administration that is adequate to induce a sustained improvement in at least one indicator that reflects the severity of the patient's condition, the improvement being considered sustained if the patient exhibits the improvement on at least two occasions separated by at least one day. A method according to claim 14, wherein the agent is an antibody specific for CD30L, IL-la, IL-lp or IL-1RI, and further wherein said antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody and a human antibody. 16. A method according to claim 14, wherein the agent is a soluble fragment of IL-1R2 that includes amino acids 1-333 of SEQ ID NO:8, or a subportion thereof that is capable of binding with IL-la or IL-Ip. 17. A method according to claim 14, wherein the agent is a soluble CD30 polypeptide comprising amino acids 19-390 of SEQ ID NO:6 or a CD30L-binding fragment thereof. 18. An animal model for screening therapeutic agents, wherein said animal model: carries genetic modifications that inactivate its p55 and p75 receptor proteins; and is genetically susceptible to experimentally-induced arthritis. 19. An animal model according to claim 18, wherein the arthritis to which the animal model is genetically susceptible is collagen-induced arthritis. An animal model according to claim 18 or 19, wherein the animal model is a strain of mouse that is selected from the group consisting of DBA/1, BUB and B10.Q or a strain of rat that is selected from the group consisting of DA, BB-DR and LEW.

21. An animal model according to claim 20, wherein the animal model is a strain of mouse and the strain of mouse is DBA/1, and further wherein the strain of mouse has double-null mutations in its p55 and p75 TNFa receptor genes.

22. A method for using the animal model of claim 18 to screen a candidate therapeutic agent to determine its efficacy in treating an autoimmune or chronic inflammatory condition that is resistant to treatment with a TNFc inhibitor, said method comprising inducing arthritis in the animal model of claim 18, administering the candidate therapeutic agent to said animal, and determining that the agent is efficacious if the severity of said animal's arthritis is reduced after the candidate agent has been administered.

23. A method according to claim 22 wherein the animal model is a strain of mouse or rat that is susceptible to collagen-induced arthritis, wherein the arthritis is induced by injecting collagen, and further wherein after administering the candidate therapeutic agent the severity of the arthritis is assessed by observing the amount of erythema and edema in the animal's paws.

24. A method according to claim 23, wherein the animal model is selected from the group consisting of a DBA/1 mouse, a BUB mouse, a B10.Q mouse, a DA rat, a BB-DR rat and a LEW rat. A method according to claim 24, wherein the animal model is a DBA/1 mouse carrying double-null mutations in its p55 and p75 TNFT receptor genes. DATED: 12 June 2006 Phillips Ormonde Fitzpatrick Attorneys for: IMMUNEX CORPORATION WO002/11767 PCTIUS01/24783 SEQUENCE LISTING <110> IMMUNEX CORPORATION Mohler, Kendall M. Barone, Dauphine S. Peschon, Jacques J. Kennedy, Mary K. Pluenneke, John D. <120> METHODS FOR TREATING AUTOIMMUNE AND CHRONIC INFLAMMATORY CONDITIONS USING ANTAGONISTS OF CD30 OR <130> 2959-Wo <140> to be assigned-- <141> 2001-08-06 <150> 60/224,079 <151> 2000-08-08 <160> 16 <170> Patentln version 3.1 <210> <211> <212> <213> <220> <221> <222> <223> 1 705 DNA Homo sapiens ODS .(705) <400> 1 atg gac cca ggg ctg Met Asp Pro Gly Leu 1 5 cag caa gca ctc aac gga atg gcc cct Gin Gin Ala Leu Asn Gly Met Ala Pro 10 cct gga Pro Gly gac aca gcc Asp Thr Ala acc acg agc Thr Thr Ser atg Met cat gtg ccg gcg His Val Pro Ala ggc Gly 25 tcc gtg gcc agc Ser Val Ala Ser cac ctg ggg His Leu Gly ctg gct ctg Leu Ala Leu cgc agc tat ttc Arg Ser Tyr Phe ttg acc aca gcc Leu. Thr Thr Ala act Thr tgc ctt Cys Leu gtc ttc acg gtg Val Phe Thr Val gcc Ala 55 act att atg gtg Thr Ile Met Val ttg Leu gtc gtt cag agg Val Val Gin Arg acg Thr aat Asn gac tcc att ccc Asp Ser Ile Pro tgc tca gaa gac Cys Ser Glu Asp tca cct gac aac Ser Pro Asp Asn gtc Val 75 ccc ctc aaa gga Pro Leu Lys Gly gga Gly 192 240 288 ctc tta tgt atc Leu Leu Cys Ile ctg Leu 90 aaa aga gct cca Lys Arg Ala Pro ttc aag Phe LYS aag tca tgg gcc tac ctc caa gtg gca aag cat cta aac aaa acc aag WO 02/11767 WO 0211767PCTIUS01/24783 Lys Ser Trp ttg tct tgg Leu Ser Trp 115 Ala Tyr 100 Leu Gin Val.Ala Lys His Leu Asn.Lys Thr Lys 105 110 aac aaa gat ggc Asn Lys Asp Gly ctc cat gga gtc Leu His Gly Val aga Arg 125 tat cag gat Tyr Gin Asp ggg aat Gly Asn 130 ctg gtg atc caa Leu Vai Ile Gin ttc cct Phe Pro 135 ggt ttg tac Gly Leu Tyr atc att tgc caa Ile Ile Cys Gin 432 480 ctg Leu 145 cag ttt ctt gta Gin Phe Leu Val caa Gin 150 tgc cca aat aat Cys Pro Asn Asn tct Ser 155 gtc gat ctg aag Val Asp Leu Lys ttg Leu 160 gag ctt ctc atc Giu Leu Leu Ile aac Asn 165 aag cat atc aaa Lys His Ile Lys cag gcc ctg gtg Gin Ala Leu Val aca gtg Thr Vai 175 tgt gag tct Cys Glu Ser ttc ttg ctg Phe Leu Leu 195 gga Gly 180 atg caa acg aaa Met Gin Thr Lys cac His 185 gta tac cag aat Val Tyr Gin Asn ctc tct caa Leu Ser Gin 190 gtc aat gtg Val Asn Val gat tac ctg cag Asp Tyr Leu Gin gtc Val 200 aac acc acc ata Asn Thr Thr Ile tca Ser 205 gat aca Asp Thr 210 ttc cag tac ata Phe Gin Tyr Ile gat Asp 215 aca agc acc ttt Thr Ser Thr Phe ctt gag aat gtg Leu Glu Asn Val t tg Leu 225 tcc atc ttc tta Ser Ile Phe Leu tac Tyr 230 agt aat tca gac tga Ser Asn Ser Asp <210> 2 <211> 234 <212> PRT <213> Homo sapiens <400> 2 Met Asp Pro Gly Leu .1 5 Gin Gin Ala Leu Asn 10 Gly Met Ala Pro Pro Giy Asp Thr Ala Thr Thr Ser Met His Val Pro Ala Ser Vai Ala Ser His Leu Giy Leu Ala Leu Arg Ser Tyr Phe Tyr 40 Leu Thr Thr Ala Thr Cys Leu Val Phe Thr Val Ala Thr Ile Met Val Leu Val Val Gin Arg Thr Asp Ser Ile Pro Asn Ser Pro Asp Asn Vai Pro Leu Lys Gly Gly WO 02/11767 WO 0211767PCTIUS01/24783 Asn Cys Ser Giu Asp Leu Leu Cys Ile Leu 90 Lys Arg Ala Pro Phe Lys Lys Ser Trp Leu Ser Trp 115 Ala 100 Tyr Leu Gin Vai Lys His Leu Asn Lys Thr Lys 110 Tyr Gin Asp Asn Lys Asp Gly Ile 120 Leu His Giy Vai Gly Asn 130 Leu Val Ile Gin Pro Gly Leu Tyr Phe 140 Ile Ile Cys Gin Leu 145 Gin Phe Leu Val Gin 150 Cys Pro Asn Asn Vai Asp Leu Lys Leu 160 Giu Leu Leu Ile Asn 165 Lys His Ile Lys Lys 170 Gin Ala Leu Val Thr Vai 175 Cys Giu Ser Phe Leu Leu 195 Met Gin Thr Lys Val Tyr Gin Asn Leu Ser Gin 190 Val Asn Val Asp Tyr Leu Gin Val 200 Asn Thr Thr Ile Ser 205 Asp Thr 210 Phe'Gin Tyr Ile Thr Ser Thr Phe Pro 220 Leu Glu Asn Val Leu *225 Ser Ile Phe Leu Tyr 230 Ser Asn Ser Asp <210> 3 <211> 720 <212> DNA <213> Mus sp. <220> <221> CDS <222> <223> <400> 3 atg gag cca ggg ctg Met Glu Pro Gly Leu 1 5 gac cca gcc atg cag Asp Pro Aia Met Gin caa caa gca ggc Gin Gin Ala Gly agc Ser 10 tgt ggg gct cct Cys Gly Ala Pro tcc cct Ser Pro gtg cag ccc Val Gin Pro ggc Gly 25 tcg gta gcc agc Ser Val Ala Ser ccc tgg aga Pro T-p, Arg WO 02/11767 WO 0211767PCTIUS01124783 ag'c acg agg Ser Thr Arg ccc tgg aga agc aca agt cgc agc tac Pro Trp Arg Ser Thr Ser Arg Ser Tyr tac ctc ago Tyr Leu Ser acc acc Thr Thr gca ctg gtg tgc Ala Leu Val Cys ctt Leu 55 gtt gtg gca gtg Val Val Ala Val gcg Ala atc att ctg gta Ile Ile Leu Val c tg Leu gta gtc cag aaa Val Val Gin Lys aag Lys 70 gac tcc act cca Asp Ser Thr Pro aat Asn 75 aca act gag aag Thr Thr Giu Lys gc Ala 144 192 240 288 336 ccc ott aaa gga Pro Leu Lys Gly gga Gly aat tgc toa gag Asn Cys Ser Giu gat Asp 90 otc ttc tgt acc Leu Phe Cys Thr otg aaa Leu Lys agt act coa Ser Thr Pro aag aag tca tgg Lys Lys Ser Trp tac ctc caa gtg tca aag cat Tyr Leu Gin Vai Ser Lys His 110 ctc aac aat acc aaa ctg tca Leu Asn Asn Thr Lys Leu Ser tgg Trp 120 aac gaa gat ggc Asn Glu Asp Gly acc Thr 125 atc cac gga Ile His Gly ctc ata Leu Ile 130 tao cag gac ggg Tyr Gin Asp Giy aac Asn 135 otg ata gtc caa Leu Ile Val Gin ttc Phe 140 cot ggc ttg tac Pro Gly Leu Tyr 384 432 480 528 atc gtt tgc caa Ile Val Cys Gin ctg Leu 150 cag ttc ctc gtg Gin Phe Leu Val tgc tca aat cat Cys Ser Asn His tct Ser 160 gtg gac ctg aca Val Asp Leu Thr ttg Leu 165 cag cto ctc atc Gin Leu Leu Ile aat Asn 170 tcc aag ato aaa Ser Lys Ile Lys aag cag Lys Gin 175 acg ttg gta Thr Leu Val cag aat ctc Gin Asn Leu 195 aca Thr 180 gtg tgt gag tct Vai Cys Giu Ser gtt cag agt aag Val Gin Ser Lys aac atc tac Asn Ile Tyr 190 aac tot acc Asn Ser Thr tct cag ttt ttg ctg cat tao tta cag Ser Gin Phe Leu Leu His Tyr Leu Gin gtc Val 205 ata tca Ile Ser 210 gtc agg gtg gat Vai Arg Val Asp aat Asn 215 ttc cag tat gtg Phe Gin Tyr Vai gat Asp 220 aca aac act ttc Thr Asn Thr Phe 624 672 720 cct ctt gat aat gtg cta Pro Leu Asp Asn Val Leu 225 230 too gtc ttc tta Ser Val Phe Leu tat Tyr 235 agt ago tca gac tga Ser Ser Ser Asp <210> 4 <211> 239 <212> PRT <213> Mus sp. <400> 4 WO 02/11767 WO 0211767PCTIUS01/24783 Met Glu Pro Gly Leu Gin Gin Ala Gly Cys Gly Ala Pro Ser Pro Asp Pro Ala Met Gin Val Gin Pro Gly 25 Ser Val Ala Ser Pro Trp Arg Tyr Leu Ser Ser Thr Arq Pro Trp Arg Ser Thr Ser 40 Arg Ser Tyr Phe Thr Thr Ala Leu Val Cys Leu Val Val Ala Val Ala Ile Ile Leu Val Leu Val Val Gin Lys Lys 70 Asp Ser Thr Pro Asn 75 Thr Thr Giu Lys Ala Pro Leu Lys Gly Gly Asn Cys Ser Giu Asp 90 Leu Phe Cys Thr Leu Lys Ser Thr Pro Ser 100 Lys Lys Ser Trp Tyr Leu Gin Vai Ser Lys Hi-s 110 Ile His Gly Leu Asn Asn Thr Lys Leu Ser Trp 120 Asn Giu Asp Gly Thr 125 Leu Ile 130 Tyr Gin Asp Gly Leu Ile Val Gin Phe 140 Pro Gly Leu Tyr Ile Val Cys Gin Leu 150 Gin Gin Phe Leu Vai Leu Leu Ile Asn 170 Cys Ser Asn His Ser 160 Val Asp Leu Thr Leu 165 Ser Lys Ile Lys Lys Gin 175 Thr Leu Val Vai Cys Giu Ser Val Gin Ser Lys Asn Ile Tyr 190 Asn Ser Thr Gin Asn Leu 195 Ser Gin Phe Leu Leu His Ty'r Leu Gin 200 Val 205 Ile Ser 210 Val Arg Val Asp Asn 215 Phe Gin Tyr Vai Asp 220 Thr Asn Thr Phe Pro 225 Leu Asp Asn Val Leu 230 Ser Val Phe Leu Tyr 235 Ser Ser Ser Asp <210> WO 02/11767 WO 02/ 1767PCTLJS01124783 <211> 1788 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(1788) <223> <400> atg cgc gtc ctc ctc gcc gcg ctg gga Met Arg Val Leu Leu Ala Ala Leu Gly 1 5 ctg Leu 10 ctg ttc ctg ggg Leu Phe Leu Gly gcg cta Ala Leu cga gcc ttc Arg Ala Phe ccc agc cac Pro Ser His cca Pro cag gat cga ccc Gin Asp Arg Pro t tc Phe 25 gag gac acc tgt Giu Asp Thr Cys cat gga aac His Gly Asn tac cgc tgc Tyr Arg Cys tac tat gac aag Tyr Tyr Asp Lys gtc agg agg tgc Val Arg Arg Cys ccc atg Pro Met ggg ctg ttc ccg aca cag cag tgc cca Gly Leu Phe Pro Thr Gin Gin Cys Pro 55 cag Gin agg cct act gac Arg Pro Thr Asp tgc Cys agg aag cag tgt Arg Lys Gin Cys gag Giu 70 cct gac tac tac Pro Asp Tyr Tyr gat gag gcc gac Asp Giu Ala Asp cgc Arg tgt aca gcc tgc Cys Thr Ala Cys gtg Val act tgt tct cga Thr Cys Ser Arg ga t Asp 90 gac ctc gtg gag Asp Leu Val Giu aag acg Lys Thr ccg tgt gca Pro Cys Ala ttc tgt tcc Phe Cys Ser 115 tgg Trp 100 aac tcc tcc cgt Asn Ser Ser Arg tgc gaa tgt cga Cys Giu Cys Arg ccc ggc atg Pro Giy Met 110 ttc ttc cat Phe Phe His 336 384 acg tct gcc gtc Thr Ser Ala Val aac Asn 120 tcc tgt gcc cgc Ser Cys Ala Arg tct gtc Ser Vai 130 tgt ccg gca ggg Cys Pro Ala Gly atg Met 135 att gtc aag ttc Ile Val Lys Phe cca Pro 140 ggc acg gcg cag Gly Thr Ala Gin aag Lys 145 aac acg gtc tgt Asn Thr Vai Cys gag Giu 150 ccg gct tcc cca Pro Ala Ser Pro gtc agc cct gcc Val Ser Pro Ala tgt Cys 160 gcc agc cca gag aac tgc aag gaa ccc tcc agt ggc acc atc Ala Ser Pro Giu Asn Cys Lys Giu Pro Ser Ser Gly Thr Ile 165 170 ccc cag Pro Gin 175 gcc aag ccc Ala Lys Pro acc Thr 180 ccg gtg tcc cca Pro Val Ser Pro gca acc Ala Thr 185 tcc agt gcc Ser Ser Ala agc acc atg Ser Thr Met 190 cct gta aga ggg ggc acc cgc ctc gcc cag gaa gct gct tct aaa ctg Pro Val Arg Gly Gly Thr Arg Leu Ala Gin Giu Ala Ala Ser Lys Leu 58 WO 02/11767 WO 0211767PCT/US01/24783 195 acg agg Thr Arg 210 gct ccc gac tct Ala Pro Asp Ser ccc Pro 215 tcc tct gtg gga Ser Ser Vai Gly agg Arg 220 cct agt tca gat Pro Ser Ser Asp cca Pro 225 ggt ctg tcc cca. Gly Leu Ser Pro aca Thr 230 cag cca tgc cca Gin Pro Cys Pro gag Glu 235 ggg tct ggt gat Gly Ser Gly Asp aga aag cag tgt Arg Lys Gin Cys aca gcc tgc gtg Thr Ala Cys Val 260 tgt gca tgg aac Cys Ala Trp Asn 275 gag Giu 245 ccc gac tac tac Pro Asp Tyr Tyr ctg Leu 250 gac gag gcc ggc Asp Glu Ala Gly cgc tgc Arg Cys 255 768 agc tgt tct cga Ser Cys Ser Arg gat Asp 265 gac ctt gtg gag Asp Leu Val Giu aag acg cca Lys Thr Pro 270 ggc atg atc Gly Met Ile 816 864 tcc tcc cgc Ser Ser Arg acc Thr 280 tgc gaa tgt cga Cys Glu Cys Arg tgt gcc Cys Ala 290 aca tca gcc acc Thr Ser Ala Thr aac Asn 295 tcc tgt gcc Cgc Ser Cys Ala Arg tgt Cys 300 gtc ccc tac cca Val Pro Tyr Pro atc Ile 305 gac Asp tgt gca gga gag Cys Ala Gly Giu acc acc ttt gag Thr Thr Phe Giu 325 acg Thr 310 gtc acc aag ccc Val Thr Lys Pro gat atg gct gag Asp Met Ala Giu 912 960 1008 gcg cca ccc ctg Ala Pro Pro Leu ggg Gly 330 acc cag ccg gac Thr Gin Pro Asp tgc aac Cys Asn 335 ccc acc cca Pro Thr Pro agc ttg ctg Ser Leu Leu 355 gag Giu 340 aat ggc gag gcg ASn Gly Giu Ala gcc agc acc agc Ala Ser Thr Ser ccc act cag Pro Thr Gin 350 atc cca acc Ile Pro Thr 1056 1104 gtg gac tcc cag Val Asp Ser Gin gcc Ala 360 agt aag acg ctg Ser Lys Thr Leu agc gct Ser Ala 370 ccc gtc gct ctc Pro Val Ala Leu tcc acg ggg aag Ser Thr Gly Lys ccc Pro 380 gtt ctg gat gca Val Leu Asp Ala ggg Gly 385 cca gtg ctc ttc Pro Val Leu Phe tgg Trp 390 gtg atc ctg gtg Val Ile Leu Val ttg Leu 395 gtt gtg gtg gtc Val Val Val Val ggc Gly 400 tcc agc gcc ttc Ser Ser Ala Phe ctc Leu 405 ctg tgc cac cgg Leu Cys His Arg agg Arg 410 gcc tgc agg aag Ala Cys Arg Lys cga att Arg Ile 415 1152 1200 1248 1296 1344 cgg cag aag Arg Gin Lys cta gag ctt Leu Giu Leu 435 ctc Leu 420 cac ctg tgc tac His Leu Cys Tyr ccg Pro 425 gtc cag acc tcc cag ccc aag Val Gin Thr Ser Gin Pro Lys gtg gat tcc aga ccc agg agg agc tca Val Asp Ser Arg Pro Arg Arg Ser Ser 440 acg Thr 445 cag ctg agg Gin Leu Arg WOO02/11767 PCT/US01/24783 agt ggt Ser Gly 450 gcg tcg gtg aca Ala Ser Val Thr gaa Glu 455 ccc gtc gcg gaa Pro Val Ala Glu cga ggg tta atg Arg Gly Leu Met agc Ser 465 cag cca ctg atg Gin Pro Leu Met gag Glu 470 acc tgc cac agc Thr Cys His Ser gtg Val 475 ggg gca gcc tac Gly Ala Ala Tyr ctg Leu 480 1392 1440 1488 gag agc ctg ccg Glu Ser Leu Pro ctg Leu 485 cag gat gcc agc Gin Asp Ala Ser ccg Pro 490 gcc ggg ggc ccc Ala Gly Gly Pro tcg tcc Ser Ser 495 ccc agg gac Pro Arg Asp aag att gag Lys Ile Glu 515 ctt Leu 500 cct gag ccc cgg Pro Glu Pro Arg gtg Val 505 tcc acg gag cac Ser Thr Glu His acc aat aac Thr Asn Asn 510 atc gtg ggg Ile Val Gly 1536 1584 aaa atc tac atc Lys Ile Tyr Ile aag gct gac acc Lys Ala Asp Thr gtg Val 525 acc gtg Thr Val 530 aag gct gag dtg Lys Ala Glu Leu ccg Pro 535 gag ggc cgg ggc Glu Gly Arg Gly gcg ggg cca gca Ala Gly Pro Ala .gag Glu 545 ccc gag ttg gag Pro Glu Leu Glu gag Glu 550 gag ctg gag gcg Glu Leu Glu Ala gac Asp 555 cat acc ccc cac His Thr Pro His tac Tyr 560 1632 1680 1728 ccc gag cag gag Pro Glu Gin Glu gaa ccg cct ctg Glu Pro Pro Leu agc tgc agc gat Ser Cys Ser Asp gtc atg Val Met 575 ctc tca gtg Leu Ser Val gaa Glu 580 gag gaa ggg aaa Glu Glu Gly Lys gaa Glu 585 gac ccc ttg ccc Asp Pro Leu Pro aca gct gcc Thr Ala Ala 590 1776 tct gga aag tga Ser Gly Lys 595 <210> 6 <211> 595 <212> PRT <213> Homo sapiens <400> 6 Met Arg Val Leu Leu 1 5 1788 Ala Ala Leu Gly Leu 10 Leu Phe Leu Gly Ala Leu Arg Ala Phe Pro Ser His Pro Gin Asp Arg Pro Phe 25 Glu Asp Thr Cys His Gly Asn Tyr Tyr Asp Lys Ala Val Arg Arg Cys Cys Tyr Arg Cys WOO02/11767 PCT/USOI/24783 Pro Met Gly Leu Phe Pro Thr Gin Gin Cys Pro Gin Arg 55 Pro Thr Asp Arg Lys Gin Cys Glu Pro Asp Tyr Tyr Leu 75 Asp Glu Ala Asp Arg Cys Thr Ala Cys Val Thr Cys Ser Arg Asp 90 Asp Leu Val Glu Lys Thr Pro Cys Ala Phe Cys Ser 115 Asn Ser Ser Arg Cys Glu Cys Arg Pro Gly Met 110 Phe Phe His Thr Ser Ala Val Ser Cys Ala Arg Cys 125 Ser Val 130 Cys Pro Ala Gly Ile Val Lys Phe Pro 140 Gly Thr Ala Gin Lys 145 Asn Thr Val Cys Glu 150 Pro Ala Ser Pro Val Ser Pro Ala Cys 160 Ala Ser Pro Glu Asn 165 Cys Lys Glu Pro Ser 170 Ser Gly Thr Ile Pro Gin 175 Ala Lys Pro Pro Val Arg 195 Pro Val Ser Pro Ala 185 Thr Ser Ser Ala Ser Thr Met 190 Ser Lys Leu Gly Gly Thr Arg Ala Gin Glu Ala Thr Arg 210 Ala Pro Asp Ser Pro 215 Ser Ser Val Gly Arg Pro Ser Ser 220 Gly Ser Gly Asp Asp Pro 225 Gly Leu Ser Pro Thr 230 Gin Pro Cys Pro Glu 235 Cys 240 Arg Lys Gin Cys Thr Ala Cys Val 260 Glu 245 Pro Asp Tyr Tyr Leu 250 Asp Glu Ala Gly Arg Cys 255 Ser Cys Ser Arg Asp 265 Asp Leu Val Glu Lys Thr Pro 270 Gly Met Ile Cys Ala Trp Asn Ser Ser Arg Thr 275 280 Cys Glu Cys Arg Pro 285 Cys Ala Thr Ser Ala Thr Asn Ser Cys Ala Arg Cys Val Pro Tyr Pro WO 02/11767 PCT/US01/24783 290 295 Ile 305 Cys Ala Gly Glu Thr 310 Val Thr Lys Pro Gin 315 Asp Met Ala Glu Lys 320 Asp Thr Thr Phe Glu 325 Ala Pro Pro Leu Thr Gln Pro Asp Cys Asn 335 Pro Thr Pro Ser Leu Leu 355 Glu 340 Asn Gly Glu Ala Pro 345 Ala Ser Thr Ser Pro Thr Gin 350 Ile Pro Thr Val Asp Ser Gin Ser Lys Thr Leu Pro 365 Ser Ala 370 Pro Val Ala Leu Ser 375 Ser Thr Gly Lys Val Leu Asp Ala Gly 385 Pro Val Leu Phe Trp 390 Val Ile Leu Val Leu Val Val Val Val 395 Gly 400 Ser Ser Ala Phe Leu 405 Leu Cys His Arg Ala Cys Arg Lys Arg Ile 415 Arg Gln Lys Leu Glu Leu 435 Leu 420 His Leu Cys Tyr Val Gin Thr Ser Gin Pro Lys 430 Gin Leu Arg Val Asp Ser Arg Pro 440 Arg Arg Ser Ser Thr 445 Ser Gly 450 Ala Ser Val Thr Glu 455 Pro Val Ala Glu Glu Arg Gly Leu 460 Gly Ala Ala Tyr Met Ser 465 Gin Pro Leu Met Glu 470 Thr Cys His Ser Val 475 Leu 480 Glu Ser Leu Pro Leu 485 Gin Asp Ala Ser Pro 490 Ala Gly Gly Pro Ser Ser 495 Pro Arg Asp Leu 500 Lys Ile Glu Lys 515 Pro Glu Pro Arg Ser Thr Glu His Thr Asn Asn 510 Ile Val Gly Ile Tyr Ile Met 520 Lys Ala Asp Thr Val 525 Thr Val 530 Lys Ala Glu Leu Pro 535 Glu Gly Arg Gly Leu 540 Ala Gly Pro Ala WO 02/11767 WO 0211767PCTJUS01/24783 Glu Pro Glu 545 Pro GJlu Gin Leu Ser Val Ser Gly Lys 595 Leu Glu Glu Glu Leu Glu Ala Asp His Thr Pro His Tyr 550 555 560 Glu Thr Glu Pro Pro Leu Gly Ser Cys Ser Asp Val Met 565 570 575 Glu Glu Glu Gly Lys Glu Asp Pro Leu Pro Thr Ala Ala 580 585 590 <210> <211> <212> <213> <220> <221> <222> <223> <220> <221> <222> <223> <220> <221> <222> <223> 7 1357 DNA Homo sapiens matpeptide CDS (154) (1347) sig-peptide (154)..(192) <400> 7 ctggaaaata cattctgcta ctcttaaaaa ctagtgacgc tcatacaaat caacagaaag agcttctgaa ggaagacttt aaagctgctt ctgccacgtg ctgctgggtc tcagtcctcc acttcccgtg tcctctggaa gttgtcagga gca atg ttg cgc ttg tac gtg ttg Met Leu Arg Leu Tyr Val Leu gta atg gga gtt tct gcc ttc acc ctt cag cct gcg gca cac aca ggg Val Met Gly Val. Ser Ala Phe Thr Leu Gin Pro Ala Ala His Thr Gly -1 11 5 gct gcc aga agc tgc cgg ttt cgt ggg agg cat tac aag cgg gag ttc Ala Ala Arg Ser Cys Arg Phe Arg Gly Arg His Tyr Lys Arg Glu Phe 20 120 174 222 270 agg ctg gaa ggg gag cct gta gcc ctg agg Arg Leu Glu Gly Glu Pro Val Ala Leu Arg 35 tgc ccc cag gtg ccc tac Cys Pro Gin Val Pro Tyr 318 WO 02/11767 WO 0211767PCTIUSOI/24783 tgg ttg tgg gcc Trp Leu Trp Ala tct gtc agc Ser Val Ser cgc atc aac ctg aca tgg cat aaa Arg Ile Asn Leu Thr Trp His Lys aat gac Asn Asp tct gct agg acg Ser Ala Arg Thr gtc Val 65 cca gga gaa gaa Pro Gly Glu Giu gag aca cgg atg Giu Thr Arg Met ttg cag gag gac Leu Gin Giu Asp tgg Trp tct Ser gcc Ala cag gac ggt gct Gin Asp Gly Ala ctg Leu 80 tgg ctt ctg cca gcc Trp Leu Leu Pro Ala 85 414 462 510 ggc acc tac gtc Gly Thr Tyr Vai tgc Cys act act aga aat Thr Thr Arg Asn gct Ala 100 tct tac tgt gac Ser Tyr Cys Asp aaa atg Lys Met 105 tcc att gag Ser Ile Giu atc tca tac Ile Ser Tyr 125 ctc Leu 110 aga gtt ttt gag Arg Val Phe Giu aat Asn 115 aca gat gct ttc Thr Asp Ala Phe ctg ccg ttc Leu Pro Phe 120 gta tta gta Val Leu Val ccg caa att tta Pro Gin Ile Leu acc Thr 130 ttg tca acc tct Leu Ser Thr Ser ggg Gly 135 tgc cct Cys Pro 140 gac ctg agt gaa Asp Leu Ser Giu ttc Phe 145 acc cgt gac aaa Thr Arg Asp Lys act Thr 150 gac gtg aag att Asp Val Lys Ile caa Gin 155 tgg tac aag gat Trp, Tyr Lys Asp ctt ctt ttg gat Leu Leu Leu Asp gac aat gag aaa Asp Asn Glu Lys 654 702 750 cta agt gtg agg Leu Ser Vai Arg ggg Gly 175 acc act cac tta Thr Thr His Leu c tc Leu 180 gta cac gat gtg Vai His Asp Vai gcc ctg Ala Leu 185 gaa gat gct Giu Asp Ala cag caa tac Gin Gin Tyr 205 ggc Gly 190 tat tac cgc tgt Tyr Tyr Arg Cys Ctg aca ttt gcc Leu Thr Phe Ala cat gaa ggc His Giu Gly 200 aag aaa aaa Lys Lys Lys aac atc act agg Asn Ile Thr Arg agt Ser 210 att gag cta cgc Ile Glu Leu Arg aaa gaa Lys Giu 220 gag acc att cct Giu Thr Ile Pro atc att tcc ccc Ile Ile Ser Pro ctc Leu 230 aag acc ata tca Lys Thr Ile Ser gc t Ala 235 tct ctg ggg tca Ser Leu Gly Ser aga Arg 240 ctg aca atc ccg Leu Thr Ile Pro tgt Cys 245 aag gtg ttt ctg Lys Vai Phe Leu gga Gly 250 acc ggc aca ccc Thr Gly Thr Pro tta Leu 255 acc acc atg ctg Thr Thr Met Leu tgg Trp 260 tgg acg gcc aat Trp Thr Ala Asn gac acc Asp Thr 265 cac ata gag His Ile Glu agc Ser 270 gcc tac ccg gga Ala Tyr Pro Gly cgc gtg acc gag Arg Val Thr Glu ggg cca cgc Gly Pro Arg 280 1038 WO 02/11767 WO 0211767PCTIUS01/24783 cag gaa tat Gin Glu Tyr 285 tca gaa aat aat gag aac tac att gaa Ser Giu Asn Asn Giu Asn Tyr Ile Giu 290 gtg cca ttg att Val Pro Leu Ile 295 ttt aaa tgt gtt Phe Lys Cys Val ttt gat Phe Asp 300 cct gtc aca aga Pro Val Thr Arg gag Glu 305 gat ttg cac atg Asp Leu His Met gat Asp 310 1086 1134 1182 1230 gtc Val 315 cat aat acc ctg His Asn Thr Leu agt Ser 320 ttt cag aca cta Phe Gin Thr Leu cgc Arg 325 acc aca gtc aag Thr Thr Vai Lys gaa Giu 330 gcc tcc tcc acg Ala Ser Ser Thr tcc tgg ggc att Ser Trp Giy Ile gtg Val 340 ctg gcc cca ctt Leu Ala Pro Leu tca ctg Ser Leu 345 gcc ttc ttg Ala Phe Leu aga act gga Arg Thr Gly 365 gtt Val 350 ttg ggg gga ata Leu Gly Gly Ile atg cac aga cgg Met His Arg Arg tgc aaa cac Cys Lys His 360 cat cat caa His His Gin 1278 1326 aaa gca gat ggt Lys Ala Asp Gly ctg Leu 370 act gtg cta tgg Thr Val Leu Trp cct Pro 375 gac ttt Asp Phe 380 caa tcc tat ccc Gin Ser Tyr Pro tgaaataaat 1357 <210> 8 <211> 398 <212> PRT <213> Homo sapiens <400> 8 Met Leu Arg Leu Tyr Val Leu Val Giy Val Ser Ala Phe Thr Leu -1 1 Arg Phe Arg Gly Gin Pro Ala Ala His Thr Gly Ala Ala Arg Ser Arg His Tyr Lys Arg Giu 25 Phe Arg Leu Glu Gly 30 Glu Pro Val Ala Leu Arg Cys Pro Gin Pro Tyr Trp Leu Ala Ser Val Ser Pro Arg Ile Asn Leu Glu Glu Giu Thr Trp His Lys Asn Asp Ser Ala Arg Thr 60 Val Pro Gly Trp Leu Leu Thr Arg Met Trp Al a 75 Gin Asp Gly Ala Leu Pro Ala Leu Gin Glu Asp Ser Gly Thr Tyr Val Cys Thr Thr Arg Asn WO 02/11767 WO 0211767PCTIUSOI/24783 Ala Ser Tyr Cys Asp 100 Thr Asp Ala Phe Leu 120 Met Ser Ile Giu Leu 110 Arg Vai Phe Giu Asn 115 Pro Phe Ile Ser Tyr 125 Pro Gin Ile Leu Thr Leu 130 Ser Thr Ser Asp Lys Thr 150 Giy 135 Val Leu Val Cys Asp Leu Sex Glu Phe Thr Arg 145 Leu Leu Leu Asp Val Lys Ile Gin 155 Trp Tyr Lys Asp Asp Lys 165 Asp Asn Giu Lys Leu Ser Vai Arg Giy 175 Thr Thr His Leu Leu 180 Val His Asp Val Ala 185 Leu Glu Asp Ala Gly 190 Tyr Tyr Arg Cys Leu Thr Phe Ala His 200 Giu Gly Gin Gin Tyr 205 Asn Ile Thr Arg Ser Ile 210 Giu Leu Arg Ser Pro Leu 230 Ile 215 Lys Lys Lys Lys Giu .220 Giu Thr Ile Pro Val Ile Ile 225 Leu Thr Ile Lys Thr Ile Ser Aia 235 Ser Leu Gly Ser Pro Cys 245 Lys Val Phe Leu Gly 250 Thr Giy Thr Pro Leu 255 Thr Thr Met Leu Trp 260 Trp, Thr Aia.Asn Asp 265 Thr His Ile Giu Ser 270 Ala Tyr Pro Giy Arg Vai Thr Giu Giy 280 Pro Arg Gin Giu Tyr 285 Ser Giu Asn Asn Giu Asn 290 Tyr Ile Giu His Met Asp 310 Vai Pro Leu Ile Phe Asp 295 300 Pro Val Thr Arg Giu Asp Leu 305 Phe Gin Thr Phe Lys Cys Val Vai His 315 Asn Thr Leu Ser 320 Leu Arg 325 Thr Thr Val Lys Giu 330 Ala Ser Ser Thr Phe 335 Ser Trp Giy Ile WO 02/11767 PCT/US01/24783 S Val Leu Ala Pro Leu Ser Leu Ala Phe Leu Val Leu Gly Gly Ile Trp 340 345 350 355 Met His Arg Arg Cys Lys His Arg Thr Gly Lys Ala Asp Gly Leu Thr S360 365 370 Val Leu Trp Pro His His Gln Asp Phe Gln Ser Tyr Pro Lys 375 380 385 ci 0 <210> 9 c <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 9 ggattgtcac ggtgccgttg aag 23 <210> <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> ccggtggatg tggaatgtgt g 21 <210> 11 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 11 tgctgatggg gatacatcca tc 22 <210> 12 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 12 ccggtggatg tggaatgtgt g 21 WO 02/11767 WO 0211767PCTIUS01/24783 <210> 13 KI <211> <212> DNA <213> Artificial Sequence S <220> <223> Synthetic primer <400> 13 S agagctccag gcacaagggc <210> 14 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> Synthetic primer <400> 14 aacgggccag acctcgggt <210> <211> 2478 <212> DNA <213> Homo sapiens <220> <221> CDS <222> (1)..(2475) <223> <220> <221> matpeptide <222> <223> <400> atg ggg tgg ctt tgc tct Met Gly Trp Leu Cys Ser -20 ggg ctc ctg ttc Gly Leu Leu Phe gtg agc tgc ctg gtc Val Ser Cys Leu Val ctg ctg cag gtg Leu Leu Gin Val acc tgc gtc tcc Thr Cys Val Ser aat ggt ccc acc Asn Gly Pro Thr agc tct ggg Ser Ser Gly aac atg Asn Met -1 1 aag gtc ttg Lys Val Leu. cag gag ccc Gin Glu Pro tgg aag atg Trp Lys Met gac tac atg Asp Tyr Met agc Ser 15 atc tct act tgc Ile Ser Thr Cys gag Giu 96 144 192 aat tgc Asn Cys agc Ser 30 acc gag ctc cgc Thr Glu Leu Arg ctg Leu ttg tac cag ctg Leu. Tyr Gin Leu gtt ttt ctg ctc tcc gaa gcc cac acg tgt atc cct gag aac aac gga Val Phe Leu Leu Ser Glu Ala His Thr Cys Ile Pro Glu Asn Asn Gly 240 WO 02/11767 WO 0211767PCTIUS01/24783 ggo gog ggg tgo Gly Ala Gly Cys tgc cac ctg ctc Cys His Leu Leu gat gac gtg gtc Asp Asp Val Val agt gcg Ser Ala gat aac tat Asp Asn Tyr ggc tcc ttc S Gly Ser Phe aca Thr ctg gao ctg Leu Asp Leu tgg got Trp Ala 80 ggg cag cag ctg Gly Gin Gin Leu ctg tgg aag Leu Trp Lys cca gga aac Pro Gly Asn aag ccc agc gag Lys Pro Ser Glu gtg aaa ccc agg Val Lys Pro Arg gc Ala 100 ctg aca Leu Thr 105 gtt cac acc aat Val His Thr Asn gtc Val 110 tcc gac act ctg ctg ctg acc tgg agc Ser Asp Thr Leu Leu Leu Thr Trp Ser 115 432 aac Asn 120 cog tat ccc cct Pro Tyr Pro Pro gac aat tac ctg tat Asp Asn Tyr Leu Tyr

125. aat Asn 130 cat ctc acc tat His Leu Thr Tyr gca Ala 135 gtc aac att tgg Val Asn Ile Trp agt Ser 140 gaa aac gac cog Giu Asn Asp Pro goa Ala 145 gat ttc aga ato Asp Phe Arg Ile tat aac Tyr Asn 150 gtg aco tao Vai Thr Tyr tot ggg att Ser Gly Ile 170 ota Leu 155 gaa ccc too oto Glu Pro Ser Leu ogo Arg 160 ato goa goc ago Ile Ala Ala Ser aco otg aag Thr Leu Lys 165 cag tgo tat Gin Cys Tyr too tao agg gca Ser Tyr Arg Ala cgg Arg 175 gtg agg goc tgg Val Arg Ala Trp aao aco Asn Thr 185 aco tgg agt gag Thr Trp Ser Giu tgg Trp 190 ago coo ago acc Ser Pro Ser Thr aag Lys 195 tgg cac aac too Trp His Asn Ser tao Tyr 200 agg gag ccc ttc Arg Giu Pro Phe gag Glu 205 cag cac oto otg Gin His Leu Leu ggo gtc ago gtt Gly Vai Ser Val 672 720 768 tgc att gtc ato Cys Ile Val Ile otg Leu 220 goc gto tgo ctg Ala Val Cys Leu t tg Leu 225 tgo tat gtc ago Cys Tyr Val Ser ato aco Ile Thr 230 aag att aag Lys Ile Lys ogo oto gtg Arg Leu Vai 250 aaa Lys 235 gaa tgg tgg gat Glu Trp Trp Asp att coo aac oca Ile Pro Asn Pro goc ogo ago Ala Arg Ser 245 cag tgg gag Gin Trp Giu 816 864 got ata ata ato Ala Ile Ile Ile cag Gin 255 gat got cag ggg Asp Ala Gin Gly toa Ser 260 aag cgg Lys Arg 265 too oga ggo cag Ser Arg Gly Gin gaa Glu 270 oca goc aag tgo Pro Ala Lys Cys oca Pro 275 cac tgg aag aat His Trp Lys Asn ott aco aag oto Leu Thr Lys Leu ttg Leu 285 coo tgt ttt otg Pro Cys Phe Leu gag cac aac atg aaa agg Glu His Asn Met Lys Arg 290 295 WO 02/11767 WO 0211767PCTIUSOI/24783 gat gaa gat cct Asp Giu Asp Pro aag gct gcc aaa Lys Ala Ala Lys gag Giu 305 atg cct ttc cag Met Pro Phe Gin ggc tct Gly Ser 310 1008 gga aaa tca Gly Lys Ser cca gag agc Pro Glu Ser 330 gca Ala 315 tgg tgc cca gtg Trp CYS Pro Val gag Giu 320 atc agc aag aca Ile Ser Lys Thr gtc ctc tgg Val Leu Trp 325 gag gcc ccg Glu Ala Pro 1056 1104 atc agc gtg gtg Ile Ser Val Val tgL gtg gag ttg Cys Vai Giu Leu ttt Phe 340 gtg gag Val Giu 345 tgt gag gag gag Cys Giu Giu Giu gag Giu 350 gag gta gag gaa Giu Val Giu Giu gaa Giu 355 aaa ggg agc ttc Lys Gly Ser Phe tgt Cys 360 gca tcg cct gag Ala Ser Pro Glu agc Ser 365 agc agg gat gac Ser Arg Asp Asp Ltc Phe 370 cag gag gga agg Gin Giu Giy Arg 1152 1200 1248 ggc att gtg gcc Gly Ile Val Ala cgg Arg 380 cta aca gag agc Leu Thr Glu Ser ctg Leu 385 ttc ctg gac Ctg Phe Leu Asp Leu ctc gga Leu Gly 390 gag gag aat Giu Glu Asn ctt cca cct Leu Pro Pro 410 ggg Gly 395 ggc ttt tgc cag Gly Phe Cys Gin cag Gin 400 gac atg ggg gag Asp Met Gly Glu Lca tgc ctt Ser Cys Leu 405 gat gag ttc Asp Glu Phe 1296 1344 tcg gga agt acg Ser Gly Ser Thr agt Ser 415 gct cac atg CCC Ala His Met Pro tgg Trp 420 cca agt Pro Ser 425 gca ggg CCC aag Ala Gly Pro Lys gag Giu 430 gca CCL CCC tgg Ala Pro Pro Trp ggc Gly 435 aag gag cag CCL Lys Giu Gin Pro ctc Leu 440 aac Asn CaC Ctg gag CCa His Leu Giu Pro Ctg act tgC aca Leu Thr Cys Thr 460 agt Ser 445 CCL cct gcc agc Pro Pro Ala Ser ccg Pro 450 acc cag agt cca Thr Gin Ser Pro gag acg CCC CLC Glu Thr Pro Leu gtc Val 465 atc gca ggc aac Ile Ala Gly Asn CCL gCt Pro Ala 470 1392 1440 1488 1536 1584 taC CgC agc 'Tyr Arg Ser ctg ggt cca Leu Giy Pro 490 agc aac LCC ctg Ser Asn Ser Leu agc Ser 480 cag tca cag Lgt Gin Ser Pro Cys CCC aga gag Pro Arg Glu 485 gta gaa CCC Val Giu Pro gac cca Ctg Ctg Asp Pro Leu Leu gcc Ala 495 aga cac ctg gag Arg His Leu Giu gaa Glu 500 gag atg Giu Met 505 CCC Lgt gLc CCC Pro Cys Val Pro cag Gin 510 CLC tCt gag cca Leu Ser Giu Pro acc act gtg CCC Thr Thr Val Pro 515 cga aat gtc ctc Arg Asn Val Leu caa Gin cag Gin 535 1632 1680 ccL Pro 520 gag cca gaa acc Giu Pro Giu Thr Lgg Trp 525 gag Cag atc CLC Giu Gin Ile Leu cgc Arg 530 WO 02/11767 WO 0211767PCT/USOI/24783 cat ggg gca gct His Gly Ala Ala gca Ala 540 gcc ccc gtc tcg Ala Pro Val Ser gcc Ala 545 ccc acc agt ggc Pro Thr Ser Gly tat cag Tyr Gin 550 1728 gag ttt gta Glu Phe Val gtg ggc ttg Val Gly Leu 570 gcg gtg gag cag Ala Val Giu Gin ggt Gly 560 ggc acc cag gcc Gly Thr Gin Ala agt gcg gtg Ser Ala Val 565 ttc tca agc Phe Ser Ser 1776 1824 ggt ccc cca gga Gly Pro Pro Gly gag Glu 575 gct ggt tac aag Ala Gly Tyr Lys ctg ctt Leu Leu 585 gcc agc agt gct Ala Ser Ser Ala tcc cca gag aaa Ser Pro Glu Lys tgt Cys 595 ggg ttt ggg gct Gly Phe Gly Ala agc agt ggg gaa gag Ser Ser Gly Giu Glu 600 tgc cct ggg gac cct Cys Pro Gly Asp Pro 620 ggg Gly 605 tat aag cct ttc Tyr Lys Pro Phe gac ctc att cct Asp Leu Ile Pro ggc Gly 615 1872 1920 1968 gcc cca gtc cct Ala Pro Val Pro gtc Val 625 ccc ttg ttc acc Pro Leu Phe Thr ttt gga Phe Gly 630 ctg gac agg Leu Asp Arg agc tcc cca Ser Ser Pro 650 cca cct cgc agt Pro Pro Arg Ser cag agc tca cat Gin Ser Ser His ctc cca agc Leu Pro Ser 645 gta gag gac Val Glu Asp 2016 2064 gag cac ctg ggt Glu His Leu Gly ctg Leu 655 gag ccg ggg gaa Glu Pro Gly Glu atg cca Met Pro 665 aag ccc cca ctt Lys Pro Pro Leu cag gag cag gcc Gin Glu Gin Ala aca Thr 675 gac ccc ctt gtg Asp Pro Leu Val gac Asp 680 agc ctg ggc agt Ser Leu Gly Ser ggc Gly 685 att gtc tac tca Ile Val Tyr Ser ctt acc tgc cac Leu Thr Cys His c tg Leu 695 2112 2160 2208 tgc ggc cac ctg Cys Gly His Leu aaa Lys 700 cag tgt cat ggc Gin Cys His Gly cag Gin 705 gag gat ggt ggc Glu Asp Gly Gly cag acc Gin Thr 710 cct gtc atg *Pro Val Met tcg ccc cct Ser Pro Pro 730 gcc Ala 715 agt cct tgc tgt Ser Pro Cys Cys ggc Gly 720 tgc tgc tgt gga Cys Cys Cys Gly gac agg tcc Asp Arg Ser 725 oca ggt ggg Pro Gly Gly 2256 2304 aca acc Ccc ctg Thr Thr Pro Leu agg Arg 735 gcc cca gac ccc Ala Pro Asp Pro gtt cca Val Pro 745 ctg gag gcc agt Leu Glu Ala Ser tgt ccg gcc tcc Cys Pro Ala Ser ctg Leu 755 gca ccc tcg ggc Ala Pro Ser Gly 2352 2400 atc Ie 760 tca gag aag agt Ser Glu Lys Ser aaa Lys 765 tcc tca tca tcc Ser Ser Ser Ser ttc Phe 770 cat cct gcc cct His Pro Ala Pro ggc Gly 775 aat gct cag agc tca ago cag acc ccc aaa atc gtg aac ttt gtc tcc 71 2448 WO 02/11767 WO 0211767PCTIUS01/24783 Asn Ala Gin gtg gga ccc Val Gly Pro Ser Ser 780 aca tac Thr Tyr 795 Ser Gin Thr Pro Lys Ile Val Asn Phe Val Ser 785 790 atg agg gtc tct tat Met Arg Val Ser 2478 <210> 16 <211> 825 <212> PRT <213> HOMO sapiens <400> 16 Met Gly Trp Leu Cys Ser Gly Leu Leu Phe Pro Val Ser Cys Leu Leu Leu Gin Val Ala Ser Ser Gly Asn Met -1 1 Lys Val Leu Gin Glu Pro Trp Lys Met Thr Cys Val Ser Asp Tyr Met Ser 15 Ile Ser Thr Cys Giu Asn Gly Pro Thr Asn Cys Thr Giu Leu Arg Leu Tyr Gin Leu Val Phe Leu Leu Ser Glu 45 Ala His Thr Cys Ile Pro Glu Asn Asn Gly Ala Gly Cys Cys His Leu Leu Asp Asp Val Val Ser Ala Asp Asn Tyr Gly Ser Phe Thr Leu Asp Leu Trp Ala 80 Gly Gin Gin Leu Leu Trp Lys Pro Gly Asn Lys Pro Ser Glu His 95 Vai Lys Pro Arg Ala 100 Leu Thr 105 Val His Thr Asn Tyr Pro Pro Asp 125 Val Ser Asp Thr Leu 110 Asn Tyr Leu Tyr -Asn 130 Leu 115 Leu Thr Trp Ser Asn 120 Pro His Leu Thr Tyr Vai Asn Ie Trp Ser Glu Asn Asp Pro 140 Ala 145 Asp Phe Arg Ile Tyr Asn 150 Val Thr Tyr Leu Glu Pro Ser Leu Arg 160 Ie Ala Ala Ser Thr Leu Lys 165 WOO02/11767 PCT/US01/24783 4 Ser Gly Ile .170 Ser Tyr Arg Ala Arg 175 Val Arg Ala Trp Ala 180 Gin Cys Tyr Asn Thr 185 Thr Trp Ser Glu Trp 190 Ser Pro Ser Thr Lys 195 Trp His Asn Ser Tyr 200 Arg Glu Pro Phe Glu 205 Gin His Leu Leu Leu 210 Gly Val Ser Val Cys Ile Val Ile Leu 220 Ala Val Cys Leu Cys Tyr Val Ser Ile Thr 230 Lys Ile Lys Arg Leu Val 250 Lys 235 Glu Trp Trp Asp Gin 240 Ile Pro Asn Pro Ala Arg Ser 245 Gin Trp Glu Ala Ile Ile Ile Asp Ala Gin Gly Ser 260 Lys Arg 265 Ser Arg Gly Gln Glu 270 Pro Ala Lys Cys His Trp Lys Asn Cys 280 Leu Thr Lys Leu Leu 285 Pro Cys Phe Leu Glu 290 His Asn Met Lys Asp Glu Asp Pro His 300 Lys Ala Ala Lys Met Pro Phe Gin Gly Ser 310 Gly Lys Ser Ala 315 Trp Cys Pro Val Glu 320 Ile Ser Lys Thr Val Leu Trp 325 Pro Glu Ser 330 Ile Ser Val Val Cys Val Glu Leu Phe Glu Ala Pro 340 Val Glu 345 Cys Glu Glu Glu Glu 350 Glu Val Glu Glu Glu 355 Lys Gly Ser Phe Cys 360 Ala Ser Pro Glu Ser 365 Ser Arg Asp Asp Phe 370 Gin Glu Gly Arg Glu 375 Gly Ile Val Ala Arg 380 Leu Thr Glu Ser Leu 385 Phe Leu Asp Leu Leu Gly 390 Glu Glu Asn Gly Gly Phe Cys Gin Gln Asp Met Gly Glu 395 400 Ser Cys Leu 405 WO 02/11767 WO 02/ 1767PCTIUS01/24783 Leu Pro Pro 410 Ser Gly Ser Thr Ser 415 Ala His Met Pro Trp 420 Asp Glu Phe Pro Ser 425 Ala Gly Pro Lys Giu 430 Ala Pro Pro Trp Gly 435 Lys Giu Gin Pro Leu 440 His Leu Glu Pro Ser 445 Pro Pro Ala Ser Thr Gin Ser Pro Asp 455 Asn Leu Thr Cys Thr 460 Giu Thr Pro Leu Val 465 Ile Ala Gly Asn Pro Ala 470 Tyr Arg Ser Leu Gly Pro 490 Ser Asn Ser Leu Gin Ser Pro Asp Pro Leu Leu Ala 495 Arg His Leu Giu Cys Pro Arg Giu 485 Glu Val Giu Pro 500 Thr Val Pro Gin Giu Met 505 Pro Cys Val Pro Gin 510 Leu Ser Giu Pro Thr 515 Pro 520 Glu Pro Giu Thr Trp 525 Giu Gin Ile Leu Arg Asn Val Leu Gin 535 His Gly Ala Ala Ala 540 Ala Pro Val Ser Ala 545 Pro Thr Ser Gly Tyr Gin 550 Giu Phe Val Val Gly Leu 570 His 555 Ala Val Glu Gin Gly 560 Gly Thr Gin Ala Ser Ala Val 565 Phe Ser Ser Gly Pro Pro Gly Glu 575 Ala Gly Tyr Lys Leu Leu 585 Ala Ser Ser Ala Ser Pro Giu Lys Gly Phe Gly Ala Ser 600 Ser Gly Giu Glu Gly 605 Tyr Lys Pro Phe Asp Leu Ile Pro Gly 615 Cys Pro Gly Asp Leu Asp Arg Giu 635 Pro Ala 620 Pro Val Pro Val 625 Pro Leu Phe Thr Phe Gly 630 Pro Pro Arg Ser Pro 640 Gin Ser Ser His Leu Pro Ser 645 WO 02/11767 WO 0211767PCT/US01/24783 Ser Ser Pro 650 Glu His Leu Giy Giu Pro Gly Giu Lys Val Giu Asp 660 Asp Pro Leu Val Met Pro Lys Pro Pro Leu 665 Pro Gin Giu Gin 670 Ile Val Tyr Ser Ala Thr 675 Asp 680 Ser Leu Gly Ser Gly 685 Leu Thr Cys His Leu 695 Cys Gly His Leu Gin Cys His Gly Gin 705 Giu Asp Giy Giy Gin Thr 710 Pro Vai Met Ser Pro Pro 730 Aia 715 Ser Pro Cys Cys Gly 720 Cys Cys Cys Giy Asp Arg Ser 725 Pro Gly Gly Thr Thr Pro Leu Arg 735 Ala Pro Asp Pro Ser 740 Val Pro 745 Leu Giu Ala Ser Leu 750 Cys Pro Ala Ser Leu 755 Ala Pro Ser Gly Ile 760 Ser Giu Lys Ser Lys 765 Ser Ser Ser Ser Phe 770 His Pro Aia Pro Asn Ala Gin Ser Ser 780 Ser Gin Thr Pro Lys 785 Ile Vai Asn Phe Vai Ser 790 Val Gly Pro Thr 795 Tyr Met Arg Vai