Antibody

Abstract

It has been demonstrated that certain compounds bind to TNF and stabilise a conformation of trimeric TNF that binds to the TNF receptor. Antibodies which selectively bind to complexes of such compounds with TNF superfamily members are disclosed. These antibodies may be used to detect further compounds with the same activity, and as target engagement biomarker.

1 . An antibody that selectively binds to a complex comprising (i) a trimeric protein that is a TNF superfamily member and (ii) a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling induced by the trimer through the receptor. 
     
     
         2 . The antibody of  claim 1 , wherein the compound antagonises the signalling induced by the trimer through the receptor. 
     
     
         3 . The antibody of  claim 2 , wherein the compound increases the stability of the trimeric form of the TNF superfamily member compared to the stability of the trimeric form of the TNF superfamily member in the absence of the compound. 
     
     
         4 . The antibody of  claim 3 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the trimeric form of the TNF superfamily member of at least 1° C. 
     
     
         5 . The antibody of  claim 4 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the trimeric form of the TNF superfamily member of at least 10° C. 
     
     
         6 . The antibody of  claim 5 , wherein the increase in the T m  of the trimeric form of the TNF superfamily member is between 10° C. and 20° C. 
     
     
         7 . The antibody of any one of the preceding claims, wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor compared to the binding affinity of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         8 . The antibody of  claim 7 , wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (k on-r ) and/or decreasing the off rate (k off-r ) compared to the k on-r  and k off-r  values for binding of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         9 . The antibody of  claim 8 , wherein the compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (k on-r ) compared to the k on-r  value for binding of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         10 . The antibody of any one of  claims 7  to  9 , wherein the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor by at least 10 times compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 10 nM. 
 
     
     
         11 . The antibody of any one of  claims 7  to  9 , wherein the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor by at least 4 times compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 600 pM. 
 
     
     
         12 . The antibody of  claim 11 , wherein the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 200 pM. 
     
     
         13 . The antibody of any one of the preceding claims, wherein said compound has an IC 50  value of 500 nM or less. 
     
     
         14 . The antibody of any one of the preceding claims, wherein the compound is selected from the group consisting of compounds (1)-(6), or salts or solvates thereof: 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         15 . The antibody of any one of the preceding claims, wherein the antibody binds selectively to the trimer-compound complex compared with binding to the compound in the absence of the TNF superfamily member trimer and/or binding to the TNF superfamily member trimer in the absence of the compound. 
     
     
         16 . The antibody of  claim 15 , wherein the antibody binds to the trimer-compound complex with a K D-ab  that is at least 100 times lower than the K D-ab  for binding to the trimeric TNF superfamily member in the absence of compound and/or for binding to the compound in the absence of the TNF superfamily member. 
     
     
         17 . The antibody of  claim 16 , wherein the antibody binds to the trimer-compound complex with a K D-ab  that is at least 200 times lower than the K D-ab  for binding to the trimeric TNF superfamily member in the absence of compound and/or for binding to the compound in the absence of the TNF superfamily member. 
     
     
         18 . The antibody of any one of the preceding claims, wherein the TNF superfamily member is TNFα and the receptor is the TNF receptor. 
     
     
         19 . The antibody of  claim 18 , wherein the receptor is TNFR1. 
     
     
         20 . An antibody that selectively binds to a complex comprising (i) a human TNFα and (ii) a compound selected from the group consisting of compounds (1)-(6), or salts or solvates thereof. 
     
     
         21 . The antibody of  claim 20 , wherein the TNFα is TNFα s . 
     
     
         22 . The antibody of  claim 21 , wherein the TNFα s  comprises the sequence of SEQ ID NO: 35 or SEQ ID NO: 36, or a variant thereof. 
     
     
         23 . The antibody of any one of  claims 20  to  22 , wherein the TNFα is trimeric. 
     
     
         24 . The antibody of any one of  claims 20  to  23 , wherein the antibody binds selectively to the TNFα-compound complex compared with binding to the compound in the absence of the TNFα and/or binding to the TNFα in the absence of the compound. 
     
     
         25 . The antibody of  claim 24 , wherein the antibody binds to the TNFα-compound complex with a K D-ab  that is at least 100 times lower than the K D-ab  for binding to the TNFα in the absence of compound and/or for binding to the compound in the absence of the TNFα. 
     
     
         26 . The antibody of  claim 25 , wherein the antibody binds to the TNFα-compound complex with a K D-ab  that is at least 200 times lower than the K D-ab  for binding to the TNFα in the absence of compound and/or for binding to the compound in the absence of the TNFα. 
     
     
         27 . The antibody of any one of the preceding claims, which comprises at least one heavy chain complementarity determining region (HCDR) sequence selected from SEQ ID NOs: 4-6 and 19-21 and/or at least one light chain complementarity determining region (LCDR) sequence selected from SEQ ID NOs: 1-3, 17 and 18. 
     
     
         28 . The antibody of  claim 27 , which comprises a HCDR3 sequence of SEQ ID NO: 6 or SEQ ID NO: 21. 
     
     
         29 . The antibody of  claim 27  or  28 , which comprises HCDR1, HCDR2 and HCDR3 sequences and LCDR1, LCDR2, and LCDR3 sequences contained within a heavy chain variable region (HCVR) and light chain variable region (LCVR) pair of SEQ ID NOs: 8/7 or SEQ ID NOs: 23/22. 
     
     
         30 . The antibody of  claim 29 , wherein the HCDR1/HCDR2/HCDR3 sequence combination is selected from SEQ ID NOs: 4/5/6 and SEQ ID NOs: 19/20/21, and/or the LCDR1/LCDR2/LCDR3 sequence combination is selected from SEQ ID NOs: 1/2/3 and SEQ ID NOs: 1/17/18. 
     
     
         31 . The antibody of any one of  claims 27  to  30 , which comprises a HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequence combination of SEQ ID NOs: 4/5/6/1/2/3 or SEQ ID NOs: 19/20/21/1/17/18. 
     
     
         32 . The antibody of any one of  claims 27  to  31 , which comprises a heavy chain variable region (HCVR) sequence of SEQ ID NO: 8 or 23 and/or a light chain variable region (LCVR) sequence of SEQ ID NO: 7 or 22, or sequences which are at least 95% identical thereto. 
     
     
         33 . The antibody of  claim 32 , which comprises a HCVR and LCVR sequence pair of SEQ ID NOs: 8/7 or SEQ ID NOs: 23/22, or sequences which are at least 95% identical thereto. 
     
     
         34 . The antibody of  claim 33 , wherein:
 (a) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 4/5/6/1/2/3 and the remainder of the HCVR and LCVR comprise at least 95% identity to SEQ ID NOs: 8 and 7 respectively; or   (b) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 19/20/21/1/17/18 and the the remainder of the HCVR and LCVR comprise at least 95% identity to SEQ ID NOs: 23 and 22 respectively.   
     
     
         35 . The antibody of  claim 32 , which comprises a heavy chain of SEQ ID NO: 12, 13, 27 or 28 and/or a light chain of SEQ ID NO: 11 or 26, or sequences which are at least 95% identical thereto. 
     
     
         36 . The antibody of  claim 35 , which comprises a heavy and light chain pair of SEQ ID NOs: 12/11, 13/11, 27/26 or 28/26, or sequences which are at least 95% identical thereto. 
     
     
         37 . The antibody of  claim 36 , wherein:
 (a) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 4/5/6/1/2/3 and the remainder of the heavy and light chains comprise at least 95% identity to SEQ ID NOs: 12 and 11 respectively;   (b) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 4/5/6/1/2/3 and the remainder of the heavy and light chains comprise at least 95% identity to SEQ ID NOs: 13 and 11 respectively;   (c) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 19/20/21/1/17/18 and the remainder of the heavy and light chains comprise at least 95% identity to SEQ ID NOs: 27 and 26 respectively; or   (d) the HCDR1/HCDR2/HCDR3/LCDR1/LCDR2/LCDR3 sequences consist of SEQ ID NOs: 19/20/21/1/17/18 and the remainder of the heavy and light chains comprise at least 95% identity to SEQ ID NOs: 28 and 26 respectively.   
     
     
         38 . An antibody which competes for binding to TNFα with, or binds to the same epitope on TNFα as, an antibody as defined in any one of  claims 27  to  37 . 
     
     
         39 . An antibody according to any one of the preceding claims, which is a humanised antibody. 
     
     
         40 . An antibody according to any one of the preceding claims, which is a Fab, modified Fab, Fab′, modified Fab′, F(ab′) 2 , Fv, single domain antibody or an scFv. 
     
     
         41 . An isolated polynucleotide encoding an antibody as defined in any one of  claims 1  to  40 . 
     
     
         42 . An antibody as defined in any one of  claims 1  to  40  for use in a method of treatment of the human or animal body by therapy. 
     
     
         43 . A pharmaceutical composition comprising an antibody as defined in any one of  claims 1  to  40  and a pharmaceutically acceptable adjuvant and/or carrier. 
     
     
         44 . Use of an antibody as defined in any one of  claims 1  to  40  as a target engagement biomarker for the detection of a compound-trimer complex in a sample obtained from a subject; wherein said antibody is detectable and said complex comprises a trimeric protein that is a TNF superfamily member and a compound that is capable of binding to a trimeric protein that is a TNF superfamily member, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling induced by the trimer through the receptor. 
     
     
         45 . A method of detecting target engagement of a compound to a trimeric TNF superfamily member, whereby the compound-trimer complex binds to the requisite receptor and modulates the signalling induced by the trimer through the receptor, said method comprising:
 (a) obtaining a sample from a subject administered said compound;   (b) contacting an antibody as defined in any one of  claims 1  to  40  to said sample and a control sample, wherein said antibody is detectable;   (c) determining the amount of binding of said detectable antibody to said sample and said control sample,   
       wherein binding of said detectable antibody to said sample greater than binding of said detectable antibody to said control sample indicates target engagement of said compound to said trimeric TNF superfamily member. 
     
     
         46 . Use of an antibody as defined in any one of  claims 1  to  40  in screening for a compound that elicits a conformational change in a trimeric TNF superfamily member, wherein said conformational change modulates the signalling of the requisite TNF superfamily receptor on binding of the trimeric TNF superfamily member. 
     
     
         47 . A complex comprising a trimeric protein that is a TNF superfamily member and a compound that is bound thereto, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling induced by the trimer through the receptor, wherein said complex binds to an antibody as defined in any one of  claims 1  to  40  with a K D-ab  of 1 nM or less. 
     
     
         48 . The complex of  claim 47 , wherein the TNF superfamily member is TNFα. 
     
     
         49 . A TNFα trimer, said TNFα trimer being able to bind TNFR1, but wherein signalling from said bound TNFR1 is attenuated or antagonised, wherein said TNFα trimer binds to either or both of the following antibodies with a a K D-ab  of 1 nM or less:
 (i) an antibody with a heavy chain of SEQ ID NO: 27 and a light chain of SEQ ID NO: 26; or 
 (ii) an antibody with a heavy chain of SEQ ID NO: 12 and a light chain of SEQ ID NO: 11. 
 
     
     
         50 . A TNFα trimer according to  claim 49 , wherein the TNFα subunits comprise the amino acid sequence of SEQ ID NO: 36, or a corresponding sequence. 
     
     
         51 . A compound that is capable of binding to a trimeric protein that is a TNF superfamily member to form a complex, whereby the compound-trimer complex binds to the requisite TNF superfamily receptor and modulates the signalling induced by the trimer through the receptor, wherein the compound-trimer complex binds to an antibody as defined in any one of  claims 1  to  40  with a K D-ab  of 1 nM or less. 
     
     
         52 . The compound of  claim 51 , wherein the TNF superfamily member is TNFα. 
     
     
         53 . A complex according to  claim 47  or  48 , a trimer according to  claim 49  or  50 , or a compound according to  claim 51  or  52  for use in a method of therapy practised on the human or animal body. 
     
     
         54 . The complex, trimer or compound for use according to  claim 53 , for use in the treatment and/or prevention of one or more of autoimmune and inflammatory disorders; neurological and neurodegenerative disorders; pain and nociceptive disorders; and cardiovascular disorders. 
     
     
         55 . The complex, trimer or compound for use according to  claim 54 , for use in the treatment and/or prevention of one or more of rheumatoid arthritis, Crohn's disease, psoriasis, systemic lupus erythematosus, Alzheimer's disease, Parkinson's disease and epilepsy. 
     
     
         56 . A method of treating and/or preventing one of more of autoimmune and inflammatory disorders; neurological and neurodegenerative disorders; pain and nociceptive disorders; and cardiovascular disorders, by directly or indirectly administering to a patient in need thereof a complex according to  claim 47  or  48 , a trimer according to  claim 49  or  50 , or a compound according to  claim 51  or  52 . 
     
     
         57 . The method of  claim 56 , wherein one or more of rheumatoid arthritis, Crohn's disease, psoriasis, systemic lupus erythematosus, Alzheimer's disease, Parkinson's disease and epilepsy are treated and/or prevented. 
     
     
         58 . The complex, trimer or compound for use according to any one of  claims 53 - 55 , or the method of  claim 56  or  57 , wherein the therapy is on the human body or the patient is a human. 
     
     
         59 . A method of identifying a compound that is capable of binding to a trimeric protein that is a TNF superfamily member and modulating signalling of the trimeric protein through the receptor, comprising the steps of:
 (a) performing a binding assay to measure the binding affinity of a test compound-trimer complex comprising a trimeric protein that is a TNF superfamily member and a test compound to an antibody that selectively binds to said complex;   (b) comparing the binding affinity as measured in step (a) with the binding affinity of a different compound-trimer complex known to bind with high affinity to the antibody referred to in step (a); and   (c) selecting the compound present in the compound-trimer complex of step (a) if its measured binding affinity is acceptable when considered in the light of the comparison referred to in step (b).   
     
     
         60 . The method of  claim 59 , wherein the antibody binds selectively to the TNF trimer-compound complex compared with binding to the compound in the absence of the TNF superfamily member trimer and/or binding to the TNF superfamily member trimer in the absence of the compound. 
     
     
         61 . The method of  claim 60 , wherein the antibody binds to the TNF superfamily member trimer-compound complex with a K D-ab  that is at least 100 times lower than the K D-ab  for binding to the TNF superfamily member trimer in the absence of compound and/or for binding to the compound in the absence of the TNF superfamily member trimer. 
     
     
         62 . The method of  claim 61 , wherein the antibody binds to the TNF superfamily member trimer-compound complex with a K D-ab  that is at least 200 times lower than the K D-ab  for binding to the TNF superfamily member trimer in the absence of compound and/or for binding to the compound in the absence of the TNF superfamily member trimer. 
     
     
         63 . The method of any one of  claims 59  to  62 , wherein the antibody is as defined in any one of  claims 1  to  40 . 
     
     
         64 . The method of any one of  claims 59  to  63 , which is a high throughput assay. 
     
     
         65 . The method of any one of  claims 59  to  64 , wherein the test compound increases the stability of the trimeric form of the TNF superfamily member compared to the stability of the trimeric form of the TNF superfamily member in the absence of the compound. 
     
     
         66 . The method of  claim 65 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the trimeric form of the TNF superfamily member of at least 1° C. 
     
     
         67 . The method of  claim 66 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the trimeric form of the TNF superfamily member of at least 10° C. 
     
     
         68 . The method of  claim 67 , wherein the increase in the T m  of the trimeric form of the TNF superfamily member is between 10° C. and 20° C. 
     
     
         69 . The method of any one  claims 59  to  68 , wherein the test compound increases the binding affinity of the TNF superfamily member to the requisite receptor compared to the binding affinity of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         70 . The method of  claim 69 , wherein the test compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (k on-r ) and/or decreasing the off rate (k off-r ) compared to the k on-r  and k off-r  values for binding of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         71 . The method of  claim 69 , wherein the test compound increases the binding affinity of the TNF superfamily member to the requisite receptor by increasing the on rate (k on-r ) compared to the k on-r  value for binding of the TNF superfamily member to its receptor in the absence of the compound. 
     
     
         72 . The method of  claim 69 ,  70  or  71 , wherein the test compound decreases the K D-r  of the TNF superfamily member to the requisite receptor compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor by at least 10 times compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 10 nM. 
 
     
     
         73 . The method of  claim 69 ,  70  or  71 , wherein the test compound decreases the K D-r  of the TNF superfamily member to the requisite receptor compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNF superfamily member to the requisite receptor by at least 4 times compared to the K D-r  of the TNF superfamily member to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 600 pM. 
 
     
     
         74 . The method of  claim 73 , wherein the K D-r  value of the TNF superfamily member for binding to the requisite receptor in the presence of the compound is less than 200 pM. 
     
     
         75 . The method of any one  claims 59  to  74 , wherein said test compound has an IC 50  value of 500 nM or less. 
     
     
         76 . The method of any one of  claims 59  to  75 , wherein the compound in step (b) is selected from the group consisting of compounds (1)-(6), or salts or solvates thereof. 
     
     
         77 . The method of any one of  claims 59  to  76 , wherein the TNF superfamily member is TNFα and the receptor is the TNF receptor.