WO2014028374A1

WO2014028374A1 - Biomarkers predictive of rheumatoid arthritis or systemic lupus erythematosus response to anti-baff antibody therapy

Info

Publication number: WO2014028374A1
Application number: PCT/US2013/054499
Authority: WO
Inventors: Robert Warren HOFFMAN; Ernst Russell DOW
Original assignee: Eli Lilly and Co
Current assignee: Eli Lilly and Co
Priority date: 2012-08-17
Filing date: 2013-08-12
Publication date: 2014-02-20
Anticipated expiration: 2015-02-17

Description

BIOMARKERS PREDICTIVE OF RHEUMATOID ARTHRITIS OR SYSTEMIC LUPUS ERYTHEMATOSUS RESPONSE TO ANTI-BAFF

ANTIBODY THERAPY

The present invention relates to methods of using C-type lectin domain family 4, member C (CLEC4C) as a biomarker in patients having autoimmune diseases such as rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE), to determine which patients may benefit from treatment with B-cell antagonists such as tabalumab.

The pathogenesis of RA and SLE is not yet fully characterized. However, it is known that elevated levels of BAFF have been detected in the serum of patients with RA and SLE. BAFF is a member of the TNF family that specifically binds B -lymphocytes, co-stimulating their proliferation and promoting their survival. Elevated levels of BAFF in serum suggest that deregulated BAFF expression can play a role in RA and/or SLE disease pathogenesis.

Tabalumab is a monoclonal antibody that binds to and inhibits BAFF biological activity. Recently, tabalumab demonstrated efficacy of BAFF blockade in RA and is undergoing testing for the treatment of SLE. See Genovese M, et al "Effects on B cells, safety and efficacy of LY2127399, a novel anti-BAFF mab, in patients with active rheumatoid arthritis" Ann Rheum Dis 2010; 69: 69.

Previously, different panels of markers have been identified which may define distinct molecular subtypes of rheumatoid arthritis. See, US 2011/0052488 Al. In US 2011/0052488 Al, the particular marker that is the subject of the present invention is listed among hundreds of other markers some of which may be involved in the identification of certain subtypes of rheumatoid arthritis wherein such subtypes may respond to therapy using a B-cell antagonist. The preferred list of potential markers does not include the marker that is the subject of the present application nor is tabalumab mentioned as one of the medicines useful to treat rheumatoid arthritis.

Others have reported plasmacytoid dendritic cells that stained positive for CD303 (another name for CLEC4C) may decrease in patients with active RA but are then restored in patients in remission. See Kavousanaki M, et al "Novel Role of Plasmacytoid Dendritic Cells in Humans" Arthritis Rheum 2010; 62: 53-63. Although this report suggests modulation of this cell type might provide novel immune-based therapies, it does not suggest CD303 as a predictive biomarker prior to starting therapy for RA patients that may or may not respond to existing therapies.

Therefore, a need exists to identify markers to aid physicians in predicting whether a particular patient diagnosed with RA or SLE has an increased probability of responding to therapies with B-cells antagonists such as tabalumab . Patients can feel more confident in investing resources in effective treatment, and physicians can more readily tailor optimal treatment strategies for patients who have rheumatoid arthritis or systemic lupus erythematosus.

The present invention provides a method of treating rheumatoid arthritis in a patient, comprising administering an effective amount of tabalumab to the patient, provided that a sample of the patient's blood contains a level of CLEC4C mRNA less than about 8.0 dCt.

The present invention further provides a method of treating rheumatoid arthritis in a patient, comprising administering an effective amount of tabalumab to the patient, provided that the patient is selected for treatment on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt.

The present invention also provides a method of treating rheumatoid arthritis in a patient, comprising assaying a sample of blood of the patient's blood for a level of CLEC4C mRNA prior to administering tabalumab, and

administering to the patient an effective amount of tabalumab if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

The present invention further provides a method of predicting the response of a rheumatoid arthritis patient to treatment with tabalumab, comprising assaying a sample of the patient's blood to determine a level of CLEC4C mRNA in the sample, wherein a level of CLEC4C mRNA less than about 8.0 dCt is predictive of the patient's effective response to tabalumab. The present invention also provides a therapeutic regimen for treating rheumatoid arthritis, comprising:

a. selecting a patient having rheumatoid arthritis on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt;

b. optionally, parenterally administering to the patient an initial loading dose of between about 120 mg to about 300 mg of tabalumab;

c. parenterally administering to the patient one or two doses of between about 20 mg to about 150 mg of tabalumab every two weeks or every four weeks.

The present invention also provides an improved method of treating a patient having rheumatoid arthritis with tabalumab, the improvement comprising determining whether the level of CLEC4C mRNA expression in a sample of the patient's blood is less than about 8.0 dCt, and wherein the level is determined prior to administration of an effective amount of tabalumab. Preferably, the level of CLEC4C mRNA expression in a sample of the patient's blood is determined prior to the administration of tabalumab.

The present invention further provides an in vitro method of predicting the response of a rheumatoid arthritis patient to the administration of tabalumab, comprising performing qPCR on a sample of the patient's blood, wherein a level of CLEC4C mRNA less than about 8.0 dCt indicates an increased likelihood that the patient will effectively respond to the administration of tabalumab.

Preferably, qPCR is performed on a sample of the patient's blood prior to the administration of tabalumab.

The present invention also provides an in vitro method of selecting a patient having rheumatoid arthritis for treatment with a therapeutically effective amount of tabalumab, comprising assaying the level of CLEC4C mRNA in a sample of the patient's blood, wherein the patient is selected for treatment with a therapeutically effective amount of tabalumab if the level of CLEC4C mRNA is less than about 8.0 dCt. Preferably, the level of CLEC4C mRNA in a sample of the patient's blood is assayed prior to the administration of tabalumab. The present invention further provides a method of identifying rheumatoid arthritis patients eligible for treatment with tabalumab, comprising assaying for a level of CLEC4C mRNA expression by qPCR in a sample of the patient's blood, wherein the patient is eligible for treatment with tabalumab if the level of CLEC4C mRNA is less than about 8.0 dCt. Preferably, the level of CLEC4C mRNA expression in a sample of the patient's blood is assayed by qPCR prior to the administration tabalumab.

The present invention further provides tabalumab for use in treating rheumatoid arthritis in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample, and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than 8.0 dCt.

In addition, the present invention provides tabalumab for use in treating rheumatoid arthritis in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than about 8.0 dCt, as measured by qPCR.

The present invention also provides tabalumab for use in treating rheumatoid arthritis in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than 8.0 dCt, wherein tabalumab is administered in one or two doses of between about 20 mg to about 150 mg every two weeks or every four weeks.

The present invention further provides tabalumab for use in treating rheumatoid arthritis in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than about 8.0 dCt, as measured by qPCR, wherein tabalumab is administered in one or two doses of between about 20 mg to about 150 mg every two weeks or every four weeks . The present invention also provides a method of treating SLE in a patient, comprising administering an effective amount of tabalumab to the patient, provided that the patient is selected for treatment on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt.

The present invention also provides a method of treating SLE in a patient, comprising assaying a sample of blood of the patient's blood for a level of CLEC4C mRNA prior to administering tabalumab, and administering to the patient an effective amount of tabalumab if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

The present invention further provides a method of predicting the response of a SLE patient to treatment with tabalumab, comprising assaying a sample of the patient's blood to determine a level of CLEC4C mRNA in the sample, wherein a level of CLEC4C mRNA less than about 8.0 dCt is predictive of the patient's effective response to tabalumab.

The present invention also provides a therapeutic regimen for treating

SLE, comprising:

a. selecting a patient having SLE on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt;

The present invention also provides an improved method of treating a patient having SLE with tabalumab, the improvement comprising determining whether the level of CLEC4C mRNA expression in a sample of the patient's blood is less than about 8.0 dCt, and wherein the level is determined prior to administration of an effective amount of tabalumab. Preferably, the level of CLEC4C mRNA expression in a sample of the patient's blood is determined prior to the administration of tabalumab. The present invention further provides an in vitro method of predicting the response of a SLE patient to the administration of tabalumab, comprising performing qPCR on a sample of the patient's blood, wherein a level of CLEC4C mRNA less than about 8.0 dCt indicates an increased likelihood that the patient will effectively respond to the administration of tabalumab. Preferably, qPCR is performed on a sample of the patient's blood prior to the administration of tabalumab.

The present invention also provides an in vitro method of selecting a patient having SLE for treatment with a therapeutically effective amount of tabalumab, comprising assaying the level of CLEC4C mRNA in a sample of the patient's blood, wherein the patient is selected for treatment with a therapeutically effective amount of tabalumab if the level of CLEC4C mRNA is less than about 8.0 dCt. Preferably, the level of CLEC4C mRNA in a sample of the patient's blood is assayed prior to the administration of tabalumab.

The present invention further provides a method of identifying SLE patients eligible for treatment with tabalumab, comprising assaying for a level of CLEC4C mRNA expression by qPCR in a sample of the patient's blood, wherein the patient is eligible for treatment with tabalumab if the level of CLEC4C mRNA is less than about 8.0 dCt. Preferably, the level of CLEC4C mRNA expression in a sample of the patient's blood is assayed by qPCR prior to the administration tabalumab.

The present invention further provides tabalumab for use in treating SLE in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample, and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than 8.0 dCt.

In addition, the present invention provides tabalumab for use in treating SLE in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than about 8.0 dCt, as measured by qPCR. The present invention also provides tabalumab for use in treating SLE in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than 8.0 dCt, wherein tabalumab is administered in one or two doses of between about 20 mg to about 150 mg every two weeks or every four weeks.

The present invention further provides tabalumab for use in treating SLE in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA is less than about 8.0 dCt, as measured by qPCR, wherein tabalumab is administered in one or two doses of between about 20 mg to about 150 mg every two weeks or every four weeks.

A person of skill in the art would understand that the present invention further provides a method of treating SLE in a patient, comprising administering an effective amount of a B -cell antagonist to the patient, provided that the patient is selected for treatment on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt. In a preferred embodiment the B-cell antagonist inhibits the proliferation and/or survival of B-cells. In another preferred embodiment the B-cell antagonist is a large protein molecule such as a peptibody or antibody. In a more preferred embodiment the B-cell antagonist inhibits the binding of BAFF, APRIL, BAFF and APRIL, CD20 or CD22 to its receptor. In a more preferred embodiment the B-cell antagonist is an anti-BAFF antibody. In a most preferred embodiment the B-cell antagonist is selected from a group consisting of Belimumab, Abatacept, Rituximab, Blisibimod, Ocrelizumab, Ataticept and Epratuzumab.FIG. 1 shows the CLEC4C mRNA expression level of responders and non-responders to tabalumab therapy, as measured by microarray fluorescence.

FIG. 2 shows the CLEC4C expression level of responders and non- responders to tabalumab therapy, as measured by qPCR. The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies. The term "anti-BAFF antibody" as used herein is understood to mean an antibody that blocks the function of BAFF, for example by neutralizing BAFF and preventing it from interacting with its receptor.

As used herein, a "therapeutically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of the anti-BAFF antibody tabalumab may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-BAFF antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-BAFF antibody are outweighed by the therapeutically beneficial effects. The term "therapeutically effective amount" is used interchangeably herein with "an effective amount".

A therapeutically effective amount of tabalumab may be between about 20 mg to about 150 mg, administered parenterally, preferably, subcutaneously, once or twice every two or every four weeks. For example, a therapeutically effective amount of tabalumab may be about 120 mg, about 90 mg, about 60 mg, or about 30 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every 4 weeks; or about 120 mg, about 90 mg, about 60 mg, or about 30 mg administered parenterally, preferably, subcutataneously, to a RA or SLE patient once every 2 weeks; or about 120 mg or about 90 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every 4 weeks; or about 120 or about 90 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every 2 weeks; or about 120 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every four weeks; or about 90 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every four weeks; or about 120 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every two weeks; or about 90 mg administered parenterally, preferably, subcutaneously, to a RA or SLE patient once every two weeks. Further, when treated with tabalumab for the first time, a RA or SLE patient may be administered a higher loading dose of tabalumab of between about 120 mg to about 300 mg which will be followed by administration of a lower dose thereafter. Preferably, the loading dose is twice the subsequent regular weekly or monthly dose.

Tabalumab is described in the art (see, for example, U.S. Patent No.

7,317,089). Tabalumab is a human monoclonal antibody designed for the treatment of autoimmune diseases and B cell malignancies. Tabalumab specifically binds to BAFF polypeptides. BAFF is also known as TNFSF13b, BLys, TALL-1, THANK, neutrokine-a, and zTNF. Tabalumab has a high affinity for BAFF (e.g., ¾ = 10^"8 M or less), a slow off rate for BAFF dissociation (e.g.,

3 1

Koff = 10^" sec^" or less) and neutralizes BAFF activity in vitro and in vivo.

Tabalumab also inhibits BAFF- induced proliferation in an in vitro neutralization assay with an IC50 of lxlO^"8 M or less.

Tabalumab comprises a heavy chain having an amino acid sequence as shown below (SEQ ID NO: 1):

Gin Val Gin Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu

Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie

Gly Glu He Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala

Arg Gly Tyr Tyr Asp He Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp Tyr

Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly

Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser

Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val

Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe

Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val

Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val

Asp His Lys Pro Ser Gin Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gin Glu

Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg

Val Val Ser Val Leu Thr Val Leu His Gin Asp Tip Leu Asn Gly Lys

Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser lie Glu

Lys Thr lie Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr

Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp lie Ala Val Glu Trp

Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His

Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu

Gly Lys

Tabalumab further comprises a light chain having an amino acid sequence as shown below (SEQ ID NO: 2):

Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Arg Tyr

Tyr Asp Ala Ser Asn Arg Ala Thr Gly He Pro Ala Arg Phe Ser Gly

Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr He Ser Ser Leu Glu Pro

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Arg

Thr Phe Gly Gin Gly Thr Lys Val Glu He Lys Arg Thr Val Ala Ala

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

Phe Asn Arg Gly Glu Cys

The heavy chain variable region of tabalumab has an amino acid sequence as shown below (SEQ ID NO: 3):

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNY NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARGYYDILTGYYYYFDYWGQGTLV TVSS

The light chain variable region of tabalumab has an amino acid sequence as shown below (SEQ ID NO: 4):

EIVLTQSPATLSLSPGERATLSCRASQSVSRYLAWYQQKPGQAPRLLIYDASNRATGIPARF SGSGSGTDSTLTISSLEPEDFAVYYCQQRSNWPRTFGQGTKVEIKRT

B-cell antagonists such as Belimumab, Abatacept, Rituximab, Blisibimod, Ocrelizumab, Ataticept and/or Epratuzumab are also well described in the art. For example, the variable regions of Belimumab (also know as Benylsta) are described in US patent No. 7,138,501 (Seq ID No. 327 or Clone ID II 16A01).

This disclosure describes a biomarker, CLEC4C, that is predictive of response in RA or SLE for treatment with tabalumab as well as other B-cell antagonists. The CLEC4C gene encodes a member of the C-type lectin/C-type lectin-like domain (CTL/CTLD) superfamily, namely C-type lectin domain family 4, member C. Alternate names include blood dendritic cell antigen 2 protein (BDCA2), C-type lectin superfamily member 7 (CLECSF7), C-type lection superfamily member 11 (CLECSF11), clusters of differentiation 303 (CD303), HECL protein, dendritic cell lectin (DLEC), and dendritic lectin. Members of this family share a common protein fold and have diverse functions, such as cell adhesion, cell-cell signaling, glycoprotein turnover, and roles in inflammation and immune response. Members of this family may play a role in dendritic cell function.

Messenger RNA expression encoding CLEC4C correlates with response to tabalumab therapy as well as other B- cell antagonists. Statistical analyses at baseline identified CLEC4C probe sets as having the largest margin when comparing responder and non-responder group outcomes. A CLEC4C profile determination according to the present invention may include the measurement of gene expression in a biological sample (i.e., whole blood) isolated from a patient. Gene expression can be detected as, for example, protein or mRNA expression of a target gene(s). That is, the presence or expression level (amount) of a gene can be determined by detecting and/or measuring the level (amount) of mRNA or protein expression of the gene.

A variety of suitable methods can be employed to detect and/or measure the level and/or amount of mRNA expression of a gene. For example, mRNA expression can be determined using Northern blot or dot blot analysis, reverse transcriptase PCR (RT-PCR), quantitative real time PCR (qPCR), in situ hybridization (e.g., quantitative in situ hybridization), nucleic acid array (e.g., oligonucleotide arrays or gene chips) analysis, and combinations thereof. Details of such methods are well known.

The presence or level (amount) of one or more discrete mRNA populations in a biological sample can be determined by isolating total mRNA from the biological sample and subjecting the isolated mRNA to agarose gel

electrophoresis to separate the mRNA by size. The size-separated mRNAs are then transferred (e.g., by diffusion) to a solid support such as a nitrocellulose membrane. The presence or level (amount) of one or more mRNA populations in the biological sample can then be determined using one or more detectably- labeled polynucleotide probes, complementary to the mRNA sequence of interest, which bind to and thus render detectable their corresponding mRNA populations. Detectable labels are well known in the art and include, but are not limited to, fluorescent (e.g., umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, allophycocyanin (APC), phycoerythrin, and the like), luminescent (e.g., europium, terbium, Qdot™

125 131 35 32 33 3

nanoparticles, and the like), radiological (e.g., I, I, S, P, P, H, and the like), and enzymatic (horseradish peroxidase, alkaline phosphatase, beta- galactosidase, acetylcholinesterase, and the like) labels.

The presence or level (amount) of discrete populations of mRNA in a biological sample can also be determined by methods including, but are not limited to, northern blotting, reverse transcription PCR, quantitative PCR ("qPCR") (e.g., endpoint quantitative PCR or real time quantitative PCR). qPCR associates the amount of amplified product to fluorescence intensity using a fluorescent reporter molecule. Suitable fluorescent reporter molecules include, but are not limited to, a dye-labeled probe (e.g., a sequence specific probe comprising an oligonucleotide labeled with a fluorescent dye plus a quencher), a double- stranded DNA binding dye (e.g., a non-specific DNA-binding dye that fluoresces when bound to double-stranded DNA), and the like.

In qPCR, RNA is extracted from cells and reverse transcribed into DNA. Typically, probes (oligonucleotides having a reporter dye attached to the 5' end and a quencher attached to the 3 ' end) are added to the transcribed DNA sample. The probes typically hybridize to an internal region of a PCR product. In an unhybridized state, the proximity of the reporter dye and quencher prevents detection of the fluorescent signal from the probe. During PCR, when the polymerase replicates a template on which the probe is bound, the 5 '-nuclease activity of the polymerase cleaves the probe, thereby decoupling the reporter dye and the quencher. Fluorescence increases with each cycle, proportional to the amount of probe cleavage. Baseline is the noise level in early cycles where there is no detectable increase in fluorescence due to PCR products. A threshold for detection of fluorescence is typically set slightly above background. The threshold cycle (Ct) is the cycle in which there is detectable significant increase in fluorescence. That is, Ct represents the number of cycles it takes for the amount of amplified DNA to exceed threshold. This value serves as a tool for quantifying the level (amount) of starting template in the sample. The lower the Ct number, the greater the level (amount) of starting template in the sample. Delta Ct (dCt or ACt) represents the Ct value after normalization with a housekeeping gene, as determined by qPCR. That is, dCt is the difference in Ct values between the gene of interest and a housekeeping gene, as determined by qPCR.

In real time PCR, the fluorescent signal is measured as each cycle of the amplification cycle progresses, allowing quantification of the initial template to be based on the fluorescent signal before environmental (e.g., limiting reagents, accumulation of inhibitors, etc.) or enzymatic factors (e.g., inactivation of polymerase) start to affect the efficiency of amplification. The first cycle at which the amplification generated fluorescence can be distinguished from the ambient background signal is called the "Ct" or threshold cycle. This Ct value (also known as CT) can be directly correlated to the starting target concentration for the sample. Relative quantification of mRNA expression can be expressed as relative fold quantitation (RFQ) compared to the Ct value.

As used herein, an "effective response" refers to an improvement in one or more generally accepted measures of responsiveness to rheumatoid arthritis or systemic lupus erythematosus therapies. More specifically, responsiveness (and conversely, non-responsiveness) of a patient suffering from RA or SLE to anti- BAFF antibody can be classified in several ways. Classification is dependent on the patient's disease, the severity of the disease, and the particular medicament the patient is administered. For example, responsiveness of a patient with rheumatoid arthritis can be classified as achieving at least about 20% (e.g., at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, or at least about 70% or more) improvement in one or more (e.g., two or more, three or more, four or more, five or more, or more than six) of a number of objective clinical indicia (e.g., American College of Rheumatology (ACR)-core set measures) such as, but not limited to, tender joint count, swollen joint count, pain, global self-assessment of improvement by a patient, global assessment of improvement of the patient by a physician, improvement of a patient's disability, or presence or amount of an acute-phase reactant (e.g., as determined by erythrocyte sedimentation rate (ESR) or presence or level of C-reactive protein). That is, a patient can be classified as responsive to a B-cell antagonist such as tabalumab if, for example, he or she exhibits about 20% improvement in tender and swollen joint counts and 20% improvement in 3 of the 5 remaining ACR-core set measures described above. For example, a patient can be classified as responsive to tabalumab using an (ACRN score) of 20% (ACR20), 50% (ACR50), or 70% (ACR70). The ACRN score is an abstract numerical score to describe improvement of disease condition by reporting percentage improvement. Such improvements can be compared to, e.g., the responsiveness of the same patient to a placebo.

The responsiveness of a patient suffering from RA to a B-cell antagonist such as tabalumab can also be classified using a Disease Activity Score (DAS or DAS28), which is a measure of the number of swollen and tender joints, and the erythrocyte sedimentation rate (ESR) or C-reactive protein (CRP) level. For example, the DAS can be measured by evaluating the number of swollen joints and/or the number of tender joints, the ESR (in mm/hr) or CRP (in mg/L) and the visual analog scale (VAS) of the general health of the patient. An effective response of a patient to a B-cell antagonist such as tabalumab may be a DAS improvement of at least about 1.2 (e.g., at least about 1.5, at least about 1.5, at least about 1.6, at least about 1.7, at least about 1.8, at least about 1.9, at least about 2.0, at least about 2.1, at least about 2.2, at least about 2.3, at least about 2.4, or at least about 2.5 or more) for at least 1 month (e.g., at least about 1.5 months, at least about 2.0 months, at least about 2.5 months, at least about 3 months, at least about 4 months, at least about 5 months, or at least about 6 months or more.) The DAS ranges from 1 to 9.

The responsiveness of a patient suffering from SLE to a B-cell antagonist such as tabalumab can also be classified by use of the Systemic Lupus

Erythematosus Responder Index (SRI), British Isles Lupus Assessment Group (BT.LAG) A/B flares. Safety of . Estrogens in Lupus Erythematosus National Assessment-Systemic Lupus Erythematosus Disease Activity Index (SELENDA- SLEDAI), and analysis of changes in levels of anti-double-straaded DNA antibodies (a biomarker indicative of lupus disease activity).

It is understood that the methods of selecting and methods of predicting as described herein relate to in vitro methods. As described herein, the expression level (e.g., mRNA expression level) of CLEC4C can predict the response of a patient to tabalumab or another B-cell antagonists as described herein. As used herein, "assaying" can include, but is not limited to, comparing the expression of CLEC4C in a test sample with a known or a control expression level in a reference sample. For example, the expression level of CLEC4C in a test sample can be compared to the corresponding expression levels in a healthy subject, or an average expression level of multiple healthy patients, of the same species. The control sample can also be the expression level (or average expression level) of one or more patients who have either responded to a B-cell antagonist such as tabalumab or not responded to a B-cell antagonist such as tabalumab. Assaying can also include determining if the expression level of CLEC4C falls within a range of values predetermined as predictive of responsiveness or non- responsiveness of a patient to a B-cell antagonist such as tabalumab. Assaying can also include determining if the expression of CLEC4C falls above or below a predetermined cut-off value. A cut-off value is typically an expression level of a gene, or ratio of the expression level of a gene with the expression level of another gene, as determined by qPCR, for example, above or below which is considered predictive of responsiveness or non-responsiveness of a patient to a B-cell antagonist such as tabalumab.

Some cut-off values are not absolute in that clinical correlations can still remain significant over a range of values on either side of the cut-off; however, it is possible to select an optimal cut-off value of expression levels of genes for particular sample types. Cut-off values determined for use in the methods described herein can be compared with, for example, published ranges of expression levels but can be individualized to the methodology used and patient population. It is understood that improvements in optimal cut-off values could be determined depending on the sophistication of statistical methods used and on the number and source of samples used to determine reference level values for the genes and sample types. Therefore, established cut-off values can be adjusted up or down, on the basis of periodic re-evaluations or changes in methodology or population distribution.

The work disclosed herein is intended to illustrate but not limit the invention. The work identifies CLEC4C as a gene whose expression level is predictive of the responsiveness or non-responsiveness of a patient to tabalumab. An elevated expression level of CLEC4C can be predictive of responsiveness to tabalumab. Conversely, a decreased expression level of CLEC4C can be predictive of non- responsiveness to tabalumab. For example, a tabalumab responder can demonstrate a CLEC4C mRNA expression level of less than about 8.0 dCT, less than about 7.7 dCT, less than about 7.0 dCT, less than about 6.6 dCT or less than about 5.8 dCT, as determined by qPCR.

After predicting that a patient will respond or will not respond tabalumab according to the methods described herein, a medical practitioner can select the appropriate therapy for the patient, e.g., tabalumab or B-cell antagonists such an anti-BAFF antibody, respectively. Selecting a therapy for a patient can be, for example writing a prescription for a medicament; giving (but not necessarily administering) a medicament to a patient - e.g., giving a sample of a prescription medication to a patient while the patient is at the physician's office;

communication (e.g., verbal, written (other than a prescription), or electronic (e.g., email or post to a secure site)) to the patient of the suggested or recommended therapy (e.g., tabalumab or non-anti-BAFF antibody); or identifying a suitable therapy for a patient and disseminating the information to other medical personnel (e.g., by patient record).

It is understood that one can determine the level (amount) of CLEC4C in whole blood from a patient, e.g., a patient having RA or SLE. A whole blood sample can be further fractionated, if desired, to a fraction containing particular cell types, for example red blood cells, white blood cells (leukocytes), and/or plasmacytoid dendritic cells (pDCs) and the level (amount) of CLEC4C can then be determined.

EXAMPLES

Whole blood is collected using PAXgene® (from Becton Dickinson) or Tempus® (from Life Technologies, Inc.) blood RNA tubes at baseline (i.e., anti- BAFF naive) from 158 patients (having active RA and an inadequate response to methotrexate (MTX) therapy) enrolled in a Phase 2 randomized trial in which patients received placebo + methotrexate, 1, 3, 10, 30, 60, or 120 mg of tabalumab every 4 weeks over 24 weeks. 152 out of the 158 samples passed quality control (QC) and were subjected to genetic analysis. In addition to the 152 QC-approved samples collected from study subjects, whole blood is also obtained from 30 healthy control subjects for a total of 182 whole blood samples. The healthy control samples are also collected using PAXgene® or Tempus® blood RNA tubes.

Microarray Analysis

Affymetrix U133 Plus 2.0 expression arrays (Affymetrix, Inc., Santa Clara, CA) are used to conduct whole-genome gene expression analysis. This microarray measures the gene expression levels of more than 47,000 transcripts and variants, representing approximately 39,000 genes.

RNA is extracted from the 182 whole blood samples using RNA purification kits (RNEASY® from Qiagen). RNA is converted to complementary DNA (cDNA) according to Affymetrix expression analysis protocol. The cDNA is then labeled, quantified, hybridized, washed, stained, and scanned, according to publicly available Affymetrix expression analysis protocol. Data acquisition is performed using the GeneChip® Scanner 3000 from Affymetrix, enabled for high resolution scanning. Data analysis is performed with GeneChip® Operating Software (GCOS) from Affymetrix, the High Resolution Scanning Patch from Affymetrix, and default statistical algorithm patterns as determined by

Affymetrix.

The raw intensity data are compared after normalization using Robust Multichip Average (RMA) algorithm. See Irizarry, et al., 31 Nucleic Acids Research el 5 (2003). Statistical analyses are performed using Messina, two sample t-test, and regression modeling.

The microarray fluorescence results are shown in FIG. 1. As shown in

FIG. 1, patients who responded to tabalumab had high CLEC4C levels at baseline, corresponding to high fluorescence numbers. Results are further discussed below. qPCR Analsysis

RNA is extracted from the 182 whole blood samples using RNeasy kits

(from Qiagen). The quality of RNA is assessed and the amount present quantified to assure both the purity of the sample and to normalize the quantity present for comparison to Universal Human Reference (UHR) RNA. RNA is converted to complementary DNA (cDNA) which is then amplified using sequence specific primers for CLEC4C (purchased from Life Technologies, Inc.) and control primers that yield a product of distinct molecular mass, which is used as an internal control for the complete amplification along with one or more housekeeping gene(s) for calculation of dCt (dCT) relative amplification units.

As controls and for potential normalization, the assay is carried out for three constitutive genes that are transcribed at a relative constant level and should not differ between samples (GAPDH, IGSF6, and SELL). The use of these control genes corrects for variation in RNA content, variation in reverse- transcription efficiency, possible RNA degradation or presence of inhibitors in the RNA sample, variation in nucleic acid recovery and differences in sample handling. UHR RNA is used to demonstrate the analytic performance of the qPCR assays. Assay performance metrics include qPCR amplification efficiency and intermediate precision.

The UHR is a mixture of RNA obtained from multiple cell lines where the exact copy of any specific gene is not known. This control is used to determine the relationship between amplification and the total RNA mass used for the amplification and not specific gene copy number. The UHR analysis, including a reverse transcription (RT) step, is performed on a UHR stock titrated from 50 ng to 0.5 pg per PCR reaction. The amplification efficiency and R-squared value is determined from the standard titration curve.

To assess the functionality of the qPCR gene expression assay, whole blood extracted RNA is tested against plasmids containing the target region of interest for CLEC4C that the assay is designed to measure. To assess assay

7

performance, titration curves of plasmids are prepared from 1.0 xlO copies per

o

reaction to 1.0 x 10 copies per reaction. Copies present are determined by taking the initial mass of cDNA divided by the molecular weight (as determined by the length of the plasmid) and multiplying by Avogadro's number, as there was one copy of the gene per molecule of plasmid. Plasmids cDNA are reconstituted in water and aliquotted. Based on mass and plasmid size, plasmids are diluted to specific numbers of copies per reaction. Seven-point titrations of each plasmid are prepared to provide PCR reactions with 1.0 x 10⁷ to 1.0 xl0° copies of the gene per PCR reaction by performing a series of 10-fold dilutions. Plasmid titration curves demonstrate the relationship between amplification and the copies of genes in the reaction, and not mass of DNA.

Each qPCR assay is tested against its counterpart plasmid at each titration point, in quadruplicate. From this data, a titration curve is produced. Each assay is also tested against a non-template control. From the dilutional curve, the amplification efficiency and R-squared value is determined.

Four (4) qPCR replication wells per reverse transcriptase are run at each plasmid and UHR concentration. Plate validity control samples are used to monitor amplification of potential nucleic-acid contamination in assay materials and assess reverse transcriptase (RT) and qPCR reagent performance. Controls used to assess the validity of the qPCR amplification plate are: no reverse transcriptase control; no-template negative control; positive control gene(s) in UHR (UHR at 5 ng/PCR reaction and 0.005 ng/PCR reaction).

Assay efficiency, assay sensitivity/selectivity, and linear dynamic range are estimated. Assay efficiency is defined as the rate at which a PCR amplicon is generated. The slope of a standard curve is commonly used to estimate the PCR amplification efficiency of a real-time PCR reaction. A real-time PCR standard curve is graphically represented as a semi-log regression line plot of Ct value vs. log of input nucleic acid. Calculation for estimating the efficiency (E) of a realtime PCR assay is: E=(10^A(-l/slope)-l)*100. Slope, y-intercept, r², and efficiency are averages for 3 separate determinations.

Controls without reverse transcriptase are performed on all UHR samples for all assays. cDNA synthesis reactions without reverse transcriptase enzyme ("no-RT Controls") are used to assess background amplification due to genomic DNA (gDNA) contamination. Plate validity control samples are used to monitor amplification of potential nucleic-acid contamination in assay materials and assess reverse transcriptase (RT) and qPCR reagent performance monitor qPCR reagent performance. GAPDH is used as a positive control gene based on historical data from several qPCR validation projects. Input masses of 5 ng and 0.005 ng total RNA per qPCR reaction for GAPDH in UHR are chosen to approximate low and high Ct mean values and to have reasonably reliable qualification.

The raw intensity data are compared after normalization using Robust

Multichip Average (RMA) algorithm. See Irizarry, et al., 31 Nucleic Acids Research el 5 (2003). Statistical analyses are performed using Messina, two sample t-test, and regression modeling.

Equipment included ABI 7900HT with a 384-well block; 7900HT Fast Real-Time PCR System with 384-Well Block Upgrade, Part number:

4329001/4329002; BioMek Liquid Handling System; BioMek Liquid Handling System FX; NanoDrop ND1000; and a Thermalcycler, BioRad. Materials included Taqman Universal PCR Mastermix (Life Technologies/Applied Biosystems (Cat. # 4304437); Water, Molecular Biology Grade, Hyclone/Fisher Scientific (Cat. # SH30538.02); Universal Human Reference Stratagene Cat. # 740000); High capacity cDNA Reverse Transcription Kit, Life

Technologies/Applied Biosystems (Cat. # 4368814); RNase Inhibitor, Life Technologies/Applied Biosystems (Cat. # N808-0119). Applied Biosystems /Life Technologies Gene Expression Assays, Catalog # 4331182 gene materials and Origene plasmid DNA control catalog numbers are given below.

The qPCR results are shown in FIG. 2, plotted against future ACRN scores. As shown in FIG. 2, patients who responded to tabalumab had high CLEC4C levels at baseline, corresponding to low dCt numbers. Results are further discussed below.

Statistical Analyses

For responder and non-responder classification, the patients given 60 mg and 120 mg of tabalumab are combined for analysis. Responders are patients with an ACRN of > 20 and a low DAS28 score at V8 (Week 16). Non-responders are patients with an ACRN of < 20 at V8 (Week 16). This produced 22 responders and 13 non-responders, as shown in Table 1 below.

Table 1. Baseline CLEC4C mRNA Levels by Microarray Expression and qPCR with Response at Week 16

Future CLEC4C

Subject CLEC4C Non- ACRN Microarray Responder

ID dCt Responder

scores Fluorescence

1 76.9231 8.98859 4.5775 Y

2 25 4.43047 8.5107 Y

3 79.1667 8.46328 5.4345 Y

4 45.7143 3.81375 9.1427 Y

5 12.5 3.81982 8.4567 Y

6 45.4098 7.51596 5.7104 Y

7 62.5 8.31462 5.7917 Y

8 42.8571 6.91341 6.6638 Y

9 32 4.6449 7.2426 Y

10 21.4286 4.5866 8.4813 Y

11 66.6667 4.33686 8.9569 Y

12 19.4872 4.81402 8.2545 Y

13

10.5263 4.0169 9.9862 Y

14 20 4.20632 8.3098 Y

15 -20 3.27717 10.2952 Y

16

27.2727 4.45572 8.3299 Y

17

13.3333 4.27248 8.0429 Y

18 46.1538 6.09865 5.6215 Y 19 70 6.86648 5.8712 Y

20 25.3521 6.85753 5.7171 Y

21 45.4545 8.35015 5.2179 Υ

22 9.52381 4.18435 8.8685 Υ

23

16.6667 4.5852 9.8193 Υ

24 33.5385 5.99391 6.8464 Υ

25 -3.125 4.64365 7.8557 Υ

26 63.6364 3.45885 9.3427 Υ

27 9.09091 4.34398 8.0726 Υ

28 64.3836 5.15437 7.6947 Υ

29 40 4.17801 9.0571 Υ

30 24.5283 7.07545 6.6031 Υ

31 36.7647 7.42856 6.7842 Υ

32 71.6418 4.8732 7.7915 Υ

33

71.4286 4.62936 8.0388 Υ

34 48.6486 7.2624 6.5079 Υ

35 36.9231 4.58566 8.3308 Υ

The mean level of CLEC4C gene expression (based on two CLEC4C probe sets) measured by Affymetrix was found to be lower in patients than in controls. Mean expression after normalization for patients (n=152) was 5.79 with a range of 3.18 to 9.69. Mean expression for control healthy blood donors (n=30) was 6.88 with a range of 4.36 to 9.02.

Also, observed was a bimodal distribution of CLEC4C in the RA patients which remained stable over the treatment period. Patients that responded to tabalumab had consistent expression levels of CLEC4C (which was higher than non-responders) throughout the treatment period and did not increase. Similarly, patients that did not respond to tabalumab had consistent expression levels of CLEC4C throughout the treatment period. The Table 2 below represents averages of expression levels as measured by microarray analysis of the responders and non-responders at various timepoints in the treatment period.

Table 2. Averages Baseline Week l Week 2 Week 16

Responders 6.16 6.16 5.86 6.04

Non- Responders 4.3 4.46 4.49 4.49

Among patients, those with higher CLEC4C gene expression were more likely to respond to tabalumab. Messina analysis at baseline identified both CLEC4C probe sets as having the largest margin when comparing responder and non-responder group outcome at V8 (Week 16) using a combined ACRN/DAS28 endpoint. A large margin means that there is a wide range of values that will give good separation between responders and non-responders. A two-sample t-test on the same data was significant (p<0.00047), as shown in FIG. 1. As shown in Table 1, patients with a CLEC4C mRNA expression level of 4.83 or higher (as measured by microarray fluorescence) at V2 (baseline) showed significant clinical response to tabalumab as subsequently measured by ACRN scores at V8 (Week 16). These microarray expression findings are validated using qPCR. As shown in FIG. 2, for CLEC4C, the change in threshold cycle versus ACRN was statistically significant (p<0.00045) after correction for multiple comparisons using False Discovery Rate (FDR) and Bonferroni techniques (FDR). Thus, the subgroup of RA patients with higher levels of CLEC4C mRNA expression at baseline was more likely to respond to treatment with tabalumab.

SEQUENCE LISTING

SEQ ID NO: 1 (Tabalumab heavy chain):

Gin Val Gin Leu Gin Gin Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr

Tyr Trp Ser Trp lie Arg Gin Pro Pro Gly Lys Gly Leu Glu Trp lie

Gly Glu He Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu Lys

Ser Arg Val Thr lie Ser Val Asp Thr Ser Lys Asn Gin Phe Ser Leu

Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Gly Tyr Tyr Asp He Leu Thr Gly Tyr Tyr Tyr Tyr Phe Asp Tyr Trp Gly Gin Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gin Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Gin Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gin Glu Asp Pro Glu Val Gin Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gin Asp Tip Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser He Glu Lys Thr He Ser Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro Pro Ser Gin Glu Glu Met Thr Lys Asn Gin Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp He Ala Val Glu Trp Glu Ser Asn Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp

Lys Ser Arg Trp Gin Glu Gly Asn Val Phe Ser Cys Ser Val Met His

Glu Ala Leu His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Leu

Gly Lys.

SEQ ID NO: 2 (Tabalumab light chain):

Glu lie Val Leu Thr Gin Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gin Ser Val Ser Arg Tyr

Tyr Asp Ala Ser Asn Arg Ala Thr Gly He Pro Ala Arg Phe Ser Gly

Ser Gly Ser Gly Thr Asp Ser Thr Leu Thr lie Ser Ser Leu Glu Pro

Glu Asp Phe Ala Val Tyr Tyr Cys Gin Gin Arg Ser Asn Trp Pro Arg

Thr Phe Gly Gin Gly Thr Lys Val Glu lie Lys Arg Thr Val Ala Ala

Pro Ser Val Phe He Phe Pro Pro Ser Asp Glu Gin Leu Lys Ser Gly

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

Lys Val Gin Trp Lys Val Asp Asn Ala Leu Gin Ser Gly Asn Ser Gin

Glu Ser Val Thr Glu Gin Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

Asn Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

Ala Cys Glu Val Thr His Gin Gly Leu Ser Ser Pro Val Thr Lys Ser

Phe Asn Arg Gly Glu Cys.

SEQ ID NO: 3 (Tabalumab heavy chain variable region):

SEQ ID NO: 4 (Tabalumab light chain variable region):

Claims

WE CLAIM:

1. A method of treating rheumatoid arthritis in a patient, comprising administering a therapeutically effective amount of tabalumab to the patient, provided that a sample of the patient's blood contains a level of CLEC4C mRNA less than about 8.0 dCt.

2. A method of treating rheumatoid arthritis in a patient, comprising administering a therapeutically effective amount of tabalumab to the patient, provided that the patient is selected for treatment on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt.

3. A method of treating rheumatoid arthritis in a patient, comprising assaying a sample of the patient's blood for a level of CLEC4C mRNA prior to administering tabalumab, and administering to the patient a therapeutically effective amount of tabalumab if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

4. An in vitro method of selecting a patient having rheumatoid arthritis for treatment with a therapeutically effective amount of tabalumab, comprising assaying the level of CLEC4C mRNA in a sample of the patient's blood, wherein the patient is selected for treatment with a therapeutically effective amount of tabalumab if the level of CLEC4C mRNA is less than about 8.0 dCt.

5. A method of identifying rheumatoid arthritis patients eligible for treatment with tabalumab, comprising assaying for a level of CLEC4C mRNA expression by qPCR in a sample of the patient's blood prior to the administration of a therapeutically effective amount of tabalumab, wherein the patient is eligible for treatment with tabalumab if the CLEC4C mRNA level in the sample is less than about 8.0 dCt.

6. An improved method of treating a patient having rheumatoid arthritis with tabalumab, the improvement comprising determining whether the level of CLEC4C mRNA expression in a sample of the patient's blood is less than about 8.0 dCt, and wherein the level is determined prior to administration of a therapeutically effective amount of tabalumab.

7. The method of any one of claims 1-6, wherein the therapeutically effective amount of tabalumab is one dose of between about 20 mg to about 150 mg administered parenterally every two weeks or every four weeks.

8. The method of claim 6, further comprising parenterally administering to the patient an initial loading dose of between about 120 mg to about 300 mg of tabalumab.

9. A method of predicting the response of a rheumatoid arthritis patient to treatment with tabalumab, comprising assaying a sample of the patient's blood to determine a level of CLEC4C mRNA in the sample, wherein a level of CLEC4C mRNA less than about 8.0 dCt is predictive of the patient's effective response to tabalumab.

10. An in vitro method of predicting the response of a rheumatoid arthritis patient to the administration of tabalumab, comprising performing qPCR on a sample of the patient's blood, wherein a level of CLEC4C mRNA less than about 8.0 dCt indicates an increased likelihood that the patient will effectively respond to the administration of tabalumab.

11. The method of any one of claims 1-10, wherein the sample of the patient's blood is a sample of whole blood.

12. A therapeutic regimen for treating rheumatoid arthritis, comprising: a. selecting a patient having rheumatoid arthritis on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt; and

b. parenterally administering to the patient one dose of between about 20 mg to about 150 mg of tabalumab every two weeks or every four weeks.

13. The therapeutic regimen for treating rheumatoid arthritis of claim 12, further comprising parenterally administering to the patient an initial loading dose of between about 120 mg to about 300 mg of tabalumab.

14. The therapeutic regimen of claim 12 or 13, wherein the sample of the patient's blood is a sample of whole blood.

15. Tabalumab for use in treating rheumatoid arthritis in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample, and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

16. Tabalumab for use according to claim 15 wherein tabalumab is administered to the patient if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt, as measured by qPCR.

17. Tabalumab for use according to claim 15 or 16 wherein tabalumab is administered parenterally to the patient in one dose of between about 20 mg to about 150 mg every two weeks or every 4 weeks.

18. Tabalumab for use according to any one of claims 15-17 wherein tabalumab is administered parenterally to the patient in an initial loading dose of between about 120 mg to about 300 mg.

19. Tabalumab for use according to any one of claims 15-18, wherein the sample of the patient's blood is a sample of whole blood.

20. A method of treating systemic lupus erythematosus in a patient, comprising administering a therapeutically effective amount of tabalumab to the patient, provided that a sample of the patient's blood contains a level of CLEC4C mRNA less than about 8.0 dCt.

21. A method of treating Systemic lupus erythematosus in a patient, comprising administering a therapeutically effective amount of tabalumab to the patient, provided that the patient is selected for treatment on the basis of a sample of the patient's blood having a level of CLEC4C mRNA less than about 8.0 dCt.

22. A method of treating Systemic lupus erythematosus in a patient, comprising assaying a sample of the patient's blood for a level of CLEC4C mRNA prior to administering tabalumab, and administering to the patient a therapeutically effective amount of tabalumab if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

23. Tabalumab for use in treating Systemic lupus erythematosus in a patient, comprising in vitro assaying a sample of the patient's blood, determining the level of CLEC4C mRNA in the sample, and administering a therapeutically effective amount of tabalumab to the patient if the level of CLEC4C mRNA in the sample is less than about 8.0 dCt.

24. Tabalumab for use according to claim 23 wherein tabalumab is administered parenterally to the patient in one dose of between about 20 mg to about 150 mg every two weeks or every 4 weeks.

25. Tabalumab for use according to any one of claims 23-24 wherein tabalumab is administered parenterally to the patient in an initial loading dose of between about 120 mg to about 300 mg.

26. Tabalumab for use according to any one of claims 25, wherein the sample of the patient's blood is a sample of whole blood