Antibody epitope

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 which binds to an epitope comprising at least the following residues of trimeric TNFα:
 (a) Y115 
 (b) Q149; and 
 (c) N137 or K98, or both, 
 
       wherein Y115 and Q149 are present on the C chain of trimeric TNFα, and N137 and K98 are present on the A chain of trimeric TNFα, and wherein the residue numbering is according to SEQ ID NO: 36. 
     
     
         2 . An antibody according to  claim 1 , which binds to an epitope further comprising E146 on the C chain of trimeric TNFα or P117 on the C chain of trimeric TNFα, or both E146 and P117. 
     
     
         3 . An antibody according to  claim 1  or  2 , which binds to an epitope further comprising A145, which residue is present on the C chain of trimeric TNFα. 
     
     
         4 . An antibody according to any one of  claims 1 - 3 , which binds to an epitope further comprising Q88, which residue is present on the A chain of TNFα. 
     
     
         5 . An antibody according to any one of the preceding claims, which binds to an epitope further comprising one or more residues selected from the group consisting of:
 (a) L63;   (b) L94;   (c) S95; and   (d) A96;   
       wherein L63 is present on the C chain of trimeric TNFα and L94, S95 and A96 are present on the A chain of trimeric TNFα. 
     
     
         6 . An antibody according to any one of the preceding claims, which binds to an epitope further comprising one or more residues selected from the group consisting of:
 (a) 197;   (b) L93; and   (c) S81,   
       wherein I97, L93 and S81 are all present on the A chain of trimeric TNFα. 
     
     
         7 . An antibody according to any one of the preceding claims, which binds to an epitope further comprising one or more residues selected from the group consisting of:
 (a) K65;   (b) Q67;   (c) P113;   (d) D143;   (e) T77;   (f) T79;   (g) I83;   (h) T89;   (g) K90;   (i) V91;   (j) N92;   (k) E135;   (l) I136; and   (m) R138;   
       wherein K65, Q67, P113 and D143 are present on the C chain of trimeric TNFα and T77, T79, I83, T89, K90, V91, N92, E135, I136 and R138 are present on the A chain of trimeric TNFα. 
     
     
         8 . An antibody according to any one of the preceding claims, which binds to an epitope further comprising one or more residues selected from the group consisting of:
 (a) Y119;   (b) Q47; and   (c) S133,   
       wherein all of Y119, Q47 and S133 are present on the A chain of trimeric TNFα. 
     
     
         9 . An antibody according to  claim 1 , which binds to an epitope comprising the following residues of TNFα of SEQ ID NO: 36:
 (a) T77, T79, T89, K90, V91, N92, L93, L94, S95, A96, I97, E135, I136, N137 and R138, which residues are present on the A chain of trimeric TNFα and K65, Q67, Y115, A145, E146 and Q149, which residues are present on the C chain of trimeric TNFα; 
 (b) T77, T79, 183, T89, K90, V91, N92, L93, L94, S95, A96, I97, K98, E135, I136, N137 and R138, which residues are present on the A chain of trimeric TNFα and L63, K65, Q67, P113, Y115, D143, A145, E146 and Q149, which residues are present on the C chain of trimeric TNFα; 
 (c) T77, T79, S81, 183, Q88, T89, K90, V91, N92, L93, L94, S95, A96, I97, K98, E135, I136, N137 and R138, which residues are present on the A chain of trimeric TNFα and L63, K65, Q67, P113, Y115, P117, D143, A145, E146 and Q149, which residues are present on the C chain of trimeric TNFα; or 
 (d) Q27, Q47, T77, T79, S81, 183, Q88, T89, K90, V91, N92, L93, L94, S95, A96, I97, K98, Y119, R131, S133, E135, I136, N137 and R138, which residues are present on the A chain of trimeric TNFα and L63, K65, Q67, P113, Y115, P117, D143, A145, E146 and Q149, which residues are present on the C chain of trimeric TNFα. 
 
     
     
         10 . An antibody according to any one of the preceding claims, which has an affinity (K D-ab ) of 1 nM or less. 
     
     
         11 . An antibody according to  claim 10 , which has an affinity of 200 pM or less. 
     
     
         12 . An antibody according to any one of the preceding claims, which is a humanised antibody. 
     
     
         13 . 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. 
     
     
         14 . An antibody according to any one of the preceding claims, which binds a complex of trimeric TNFα and any one of compounds (1)-(7): 
       
         
           
           
               
               
           
         
         
           
           
               
               
           
         
       
     
     
         15 . An antibody according to  claim 14 , which has an affinity (K D-ab ) of 1 nM or less for the trimer-compound complex. 
     
     
         16 . An antibody according to  claim 15 , wherein the affinity is 200 pM or less. 
     
     
         17 . An antibody which competes for binding to TNFα with an antibody as defined in any one of the preceding claims. 
     
     
         18 . An isolated polynucleotide encoding an antibody as defined in any one of  claims 1  to  17 . 
     
     
         19 . An antibody as defined in any one of  claims 1  to  17  for use in a method of treatment of the human or animal body by therapy. 
     
     
         20 . A pharmaceutical composition comprising an antibody as defined in any one of  claims 1  to  17  and a pharmaceutically acceptable adjuvant and/or carrier. 
     
     
         21 . Use of an antibody as defined in any one of  claims 1  to  17  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 trimeric TNFα and a compound that is capable of binding to trimeric TNFα, whereby the compound-trimer complex binds to the requisite receptor and modulates the signalling induced by the trimer through the receptor. 
     
     
         22 . A method of detecting target engagement of a compound to trimeric TNFα, 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  17  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 TNFα. 
     
     
         23 . Use of an antibody as defined in any one of  claims 1  to  17  in screening for a compound that elicits a conformational change in a TNFα trimer, wherein said conformational change modulates the signalling of the requisite receptor on binding of the trimeric TNFα. 
     
     
         24 . A complex comprising a TNFα trimer and a compound that is bound thereto, whereby the compound-trimer complex binds to the requisite 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  17  with a K D-ab  of 1 nM or less. 
     
     
         25 . A compound that is capable of binding to a TNFα trimer to form a complex, whereby the compound-trimer complex binds to the requisite 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  17  with a K D-ab  of 1 nM or less. 
     
     
         26 . A method of identifying a compound that is capable of binding to a TNFα trimer and modulating signalling of the trimer through the requisite receptor, comprising the steps of:
 (a) performing a binding assay to measure the binding affinity of a test compound-trimer complex comprising a TNFα trimer and a test compound to an antibody as defined in any one of  claims 1  to  17  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). 
 
     
     
         27 . A method according to  claim 26 , wherein the antibody binds selectively to the TNFα trimer-compound complex compared with binding to the compound in the absence of the TNFα trimer and/or binding to the TNFα trimer in the absence of the compound. 
     
     
         28 . A method according to  claim 27 , wherein the antibody binds to the TNFα trimer-compound complex with a K D-ab  that is at least 100 times lower than the K D-ab  for binding to the TNFα trimer in the absence of compound and/or for binding to the compound in the absence of the TNFα trimer. 
     
     
         29 . A method according to  claim 28 , wherein the antibody binds to the TNFα trimer-compound complex with a K D-ab  that is at least 200 times lower than the K D-ab  for binding to the TNFα trimer in the absence of compound and/or for binding to the compound in the absence of the TNFα trimer. 
     
     
         30 . A method according to any one of  claims 26  to  29 , which is a high throughput assay. 
     
     
         31 . A method according to any one of  claims 26  to  30 , wherein the test compound increases the stability of the TNFα trimer compared to the stability of the TNFα trimer in the absence of the compound. 
     
     
         32 . A method according to  claim 31 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the TNFα trimer of at least 1° C. 
     
     
         33 . A method according to  claim 32 , wherein the increase in stability results in an increase in the thermal transition midpoint (T m ) of the TNFα trimer of at least 10° C. 
     
     
         34 . A method according to  claim 33 , wherein the increase in the T m  of the TNFα trimer is between 10° C. and 20° C. 
     
     
         35 . A method according to any one of  claims 26  to  34 , wherein the test compound increases the binding affinity of the TNFα trimer to the requisite receptor compared to the binding affinity of the TNFα trimer to its receptor in the absence of the compound. 
     
     
         36 . A method according to  claim 35 , wherein the test compound increases the binding affinity of the TNFα trimer 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 TNFα trimer to its receptor in the absence of the compound. 
     
     
         37 . A method according to  claim 35 , wherein the test compound increases the binding affinity of the TNFα trimer to the requisite receptor by increasing the on rate (k on-r ) compared to the k on-r  value for binding of the TNFα trimer to its receptor in the absence of the compound. 
     
     
         38 . A method according to  claim 35 ,  36  or  37 , wherein the test compound decreases the K D-r  of the TNFα trimer to the requisite receptor compared to the K D-r  of the TNFα trimer to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNFα trimer to the requisite receptor by at least 10 times compared to the K D-r  of the TNFα trimer to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNFα trimer for binding to the requisite receptor in the presence of the compound is less than 10 nM. 
 
     
     
         39 . A method according to  claim 35 ,  36  or  37 , wherein the test compound decreases the K D-r  of the TNFα trimer to the requisite receptor compared to the K D-r  of the TNFα trimer to its receptor in the absence of the compound, wherein:
 a) the compound decreases the K D-r  of the TNFα trimer to the requisite receptor by at least 4 times compared to the K D-r  of the TNFα trimer to its receptor in the absence of the compound; 
 b) the K D-r  value of the TNFα trimer for binding to the requisite receptor in the presence of the compound is less than 600 pM. 
 
     
     
         40 . A method according to  claim 39 , wherein the K D-r  value of the TNFα trimer for binding to the requisite receptor in the presence of the compound is less than 200 pM. 
     
     
         41 . A method according to any one of  claims 26  to  40 , wherein said test compound has an IC 50  value of 500 nM or less. 
     
     
         42 . A method according to any one of  claims 26  to  41 , wherein the compound in step (b) is selected from the group consisting of compounds (1)-(7), or salts or solvates thereof. 
     
     
         43 . A method according to any one of  claims 26  to  42 , wherein the receptor is the TNF receptor.