Methods

Abstract

The present invention relates to methods of diagnosing, prognosing, preventing or delaying onset of or treating rheumatoid arthritis, methods of distinguishing between different types or stages of rheumatoid arthritis, of identifying an individual at risk of developing rheumatoid arthritis, and of monitoring efficacy of a treatment regime in an individual being treated for rheumatoid arthritis, using a citrullinated enolase peptide to detect or capture antibodies associated with rheumatoid arthritis.

1 - 35 . (canceled) 
     
     
         36 . A citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof, for use in medicine. 
     
     
         37 . A method selected from the group consisting of aiding in diagnosing rheumatoid arthritis in an individual, aiding in distinguishing between different types or stages of rheumatoid arthritis in an individual, aiding in prognosing rheumatoid arthritis in an individual, and aiding in identifying an individual at risk of developing rheumatoid arthritis, comprising:
 a) exposing a citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof, to a sample from said individual; and   b) detecting antibody which has bound to the peptide.   
     
     
         38 . The method of  claim 37 , further comprising exposing a control peptide to a sample from the same individual and detecting antibody which has bound to the control peptide, wherein the control peptide is identical to the citrullinated enolase peptide except that:
 (a) the residue corresponding to human α-enolase amino acid 15 is replaced by a residue other than citrulline or an analogue thereof; and   (b) where the citrullinated enolase peptide comprises a residue corresponding to amino acid 9 of human α-enolase, which residue is citrulline or an analogue thereof, the residue corresponding to amino acid 9 of human α-enolase in the control peptide is citrulline or an analogue thereof, or another residue.   
     
     
         39 . The method of  claim 38  wherein one or both amino acid residues of the control peptide corresponding to amino acid residues 15 or 9 of human α-enolase is arginine. 
     
     
         40 . The method of  claim 37  wherein:
 (a) the citrullinated enolase peptide comprises KIHAXEIFDSXGNPTVE (SEQ ID NO. 7) or a variant thereof and the control peptide comprises KIHAXEIFDSRGNPTVE (SEQ ID NO. 13) or a variant thereof or KIHAREIFDSRGNPTVE (SEQ ID NO. 14) or a variant thereof, wherein X is citrulline or an analogue thereof; or   (b) the citrullinated enolase peptide comprises or consists of KIHAREIFDSXGNPTVE (SEQ ID NO. 8) or a variant thereof and the control peptide comprises or consists of KIHAREIFDSRGNPTVE (SEQ ID NO. 12) or a variant thereof, wherein X is citrulline or an analogue thereof.   
     
     
         41 . The method of  claim 37 , further comprising testing a sample from the same individual for antibodies which bind to cyclic citrullinated peptides which peptides are not citrullinated enolase peptides or variants thereof as defined in  claim 36 . 
     
     
         42 . The method of  claim 37 , further comprising exposing the citrullinated enolase peptide to a control sample and detecting antibody which has bound to the peptide. 
     
     
         43 . The method of  claim 42 , wherein the control sample is from one or more individuals not suffering from RA. 
     
     
         44 . The method of  claim 37 , wherein the individual is a human. 
     
     
         45 . The method of  claim 37 , wherein the sample is serum. 
     
     
         46 . A method of treating rheumatoid arthritis, comprising the steps of:
 (i) exposing a citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof, to a sample from said individual and detecting antibody which has bound to the peptide; and   (ii) administering an anti-TNF therapy or antibiotic to said individual.   
     
     
         47 . The method of  claim 46 , wherein the antibiotic is doxycycline. 
     
     
         48 . The method of  claim 46 , further comprising exposing a control peptide to a sample from the same individual and detecting antibody which has bound to the control peptide, wherein the control peptide is identical to the citrullinated enolase peptide except that:
 (a) the residue corresponding to human α-enolase amino acid 15 is replaced by a residue other than citrulline or an analogue thereof; and   (b) where the citrullinated enolase peptide comprises a residue corresponding to amino acid 9 of human α-enolase, which residue is citrulline or an analogue thereof, the residue corresponding to amino acid 9 of human α-enolase in the control peptide is citrulline or an analogue thereof, or another residue.   
     
     
         49 . The method of  claim 48  wherein one or both amino acid residues of the control peptide corresponding to amino acid residues 15 or 9 of human α-enolase is arginine. 
     
     
         50 . The method of  claim 46  wherein:
 (a) the citrullinated enolase peptide comprises or consists of KIIIAXEIFDSXGNPTVE (SEQ ID NO. 7) or a variant thereof and the control peptide comprises or consists of KIHAXEIFDSRGNPTVE (SEQ ID NO. 13) or a variant thereof or KIHAREIFDSRGNPTVE (SEQ ID NO. 14) or a variant thereof, wherein X is citrulline or an analogue thereof; or   (b) the citrullinated enolase peptide comprises or consists of KIHAREIFDSXGNPTVE (SEQ ID NO. 8) or a variant thereof and the control peptide comprises or consists of KIHAREIFDSRGNPTVE (SEQ ID NO. 14) or a variant thereof, wherein X is citrulline or an analogue thereof.   
     
     
         51 . The method of  claim 46 , further comprising testing a sample from the same individual for antibodies which bind to cyclic citrullinated peptides which peptides are not citrullinated enolase peptides or variants thereof as defined in  claim 36 . 
     
     
         52 . The method of  claim 46 , further comprising exposing the citrullinated enolase peptide to a control sample and detecting antibody which has bound to the peptide. 
     
     
         53 . The method of  claim 52 , wherein the control sample is from one or more individuals not suffering from RA. 
     
     
         54 . The method of  claim 46 , wherein the individual is a human. 
     
     
         55 . The method of  claim 46 , wherein the sample is serum. 
     
     
         56 . A method of monitoring efficacy of a treatment regime in an individual being treated for rheumatoid arthritis comprising:
 (i) obtaining a first sample from said individual;   (ii) obtaining a second sample from said individual after said individual has been subject to the treatment regime for a period of time;   (iii) providing a citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human-α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof;   (iv) exposing said citrullinated enolase peptide to said first sample and detecting antibody which has bound to said peptide; and   (v) exposing said citrullinated enolase peptide to said second sample and detecting antibody which has bound to said peptide.   
     
     
         57 . The method of  claim 56 , wherein the treatment regime comprises anti-TNF therapy or doxycycline therapy. 
     
     
         58 . The method of  claim 56  wherein the second sample is obtained up to 2 years after the first sample was obtained. 
     
     
         59 . The method of  claim 56 , further comprising exposing a control peptide to a sample from the same individual collected at substantially the same time as the first sample, and to a sample from the same individual collected at substantially the same time as the second sample, and, for each of the samples, detecting antibody which has bound to the control peptide, wherein the control peptide is identical to the citrullinated enolase peptide except that:
 (a) the residue corresponding to human α-enolase amino acid 15 is replaced by a residue other than citrulline or an analogue thereof; and   (b) where the citrullinated enolase peptide comprises a residue corresponding to amino acid 9 of human α-enolase, which residue is citrulline or an analogue thereof, the residue corresponding to amino acid 9 of human α-enolase in the control peptide is citrulline or an analogue thereof, or another residue.   
     
     
         60 . The method of  claim 59  wherein one or both of the amino acid residues of the control peptide corresponding to amino acid residues 15 or 9 of human α-enolase is arginine. 
     
     
         61 . The method of  claim 60  wherein:
 (a) the citrullinated enolase peptide comprises or consists of KIHAXEIFDSXGNPTVE (SEQ ID NO. 7) or a variant thereof and the control peptide comprises or consists of KIHAXEIFDSRGNPTVE (SEQ ID NO. 13) or a variant thereof or KIHAREIFDSRGNPTVE (SEQ ID NO. 14) or a variant thereof, wherein X is citrulline or an analogue thereof; or   (b) the citrullinated enolase peptide comprises or consists of KIHAREIFDSXGNPTVE (SEQ ID NO. 8) or a variant thereof and the control peptide comprises or consists of KIHAREIFDSRGNPTVE (SEQ ID NO. 14) or a variant thereof, wherein X is citrulline or an analogue thereof.   
     
     
         62 . The method of  claim 56  wherein the individual is a human. 
     
     
         63 . The method of  claim 56  wherein the sample is serum. 
     
     
         64 . A method of treating an individual with rheumatoid arthritis comprising:
 (i) providing a citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof, which peptide is immobilised to a support; and   (ii) performing immunoapheresis with the peptide immobilised to the support.   
     
     
         65 . The citrullinated enolase peptide of  claim 36 , wherein the citrullinated enolase peptide comprises KIHAXEIFDSXGNPTVE (SEQ ID NO. 7) or KIHAREIFDSXGNPTVE (SEQ ID NO. 8) or a variant thereof wherein X is citrulline or an analogue thereof. 
     
     
         66 . The citrullinated enolase peptide of  claim 36 , wherein the citrullinated enolase peptide is a cyclic peptide. 
     
     
         67 . The citrullinated enolase peptide of  claim 36 , wherein the citrullinated enolase peptide comprises or consists of CKIHAXEIFDSXGNPTVEC (SEQ ID NO. 9) or CKIHAREIFDSXGNPTVEC (SEQ ID NO. 10) wherein X is citrulline or an analogue thereof. 
     
     
         68 . A citrullinated enolase peptide with an amino acid sequence comprising the amino acid sequence of a region of human α-enolase consisting of five to 150 amino acids encompassing amino acids 14 to 16 of human α-enolase in which amino acid 15 is citrulline or an analogue thereof; or a variant thereof wherein the peptide does not consist of IHAXEEIFDSXGNPTVEVDLFTSK (SEQ ID NO. 15). 
     
     
         69 . The citrullinated enolase peptide of  claim 68 , which comprises KIHAXEIFDSXGNPTVE (SEQ ID NO. 7) or KIHAREIFDSXGNPTVE (SEQ ID NO. 8) or a variant thereof, wherein X is citrulline or an analogue thereof. 
     
     
         70 . The citrullinated enolase peptide of  claim 69 , which comprises CKIHAXEIFDSXGNPTVEC (SEQ ID NO. 9) or CKIHAREIFDSXGNPTVEC (SEQ ID NO. 10), wherein X is citrulline or an analogue thereof. 
     
     
         71 . A control peptide which is identical to the citrullinated enolase peptide of  claim 68 , except that:
 (a) the citrulline residue or analogue thereof corresponding to human α-enolase amino acid 15 is replaced by a different residue; and   (b) where the citrullinated enolase peptide comprises a residue corresponding to amino acid 9 of human α-enolase, which residue is citrulline or an analogue thereof, the residue corresponding to amino acid 9 of human α-enolase in the control peptide is citrulline or an analogue thereof, or another residue.   
     
     
         72 . The control peptide of  claim 71 , wherein one or both of the amino acid residues of the control peptide corresponding to amino acid residues 15 or 9 of human α-enolase is arginine. 
     
     
         73 . The citrullinated enolase peptide of  claim 68 , wherein the peptide is a cyclic peptide. 
     
     
         74 . A kit of parts comprising:
 (i) a citrullinated enolase peptide of  claim 36 ; and   (iii) a reagent which binds to an antibody.   
     
     
         75 . The kit of  claim 74  further comprising the control peptide of  claim 71 .