Method for determining the in vivo interaction mode

Abstract

Herein is reported a method for determining the binding interaction with a multimeric antigen of an antibody of the human IgG1 subclass that has at least two binding sites specifically binding to the antigen comprising the steps of 1) determining the binding affinity of the antibody for the multimeric antigen, and 2) incubating a mixture comprising the antibody and a polypeptide that is derived from lysine-gingipain of porphyromonas gingivalis under conditions and for a time sufficient to cleave the antibody into Fabs and Fc-region, and determining the binding affinity of the Fabs of the antibody for the multimeric, whereby the binding affinity of the antibody to the multimeric antigen to be affinity-driven if the binding affinity determined in both steps are comparable and to be avidity-driven if the binding affinity determined in both steps are different.

1 . A method for determining the binding interaction with a multimeric antigen of an antibody of the human IgG1 subclass comprising the following steps:
 1) determining the binding affinity of the antibody for the multimeric antigen,   2) incubating a mixture comprising the antibody, the antigen and lysine-gingipain of  porphyromonas gingivalis  or an enzymatically active fragment thereof at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, whereby the concentration of the antibody is higher than the concentration of the antigen, and determining the binding affinity of the Fabs of the antibody for the multimeric antigen,   and   3) determining the binding affinity of the antibody to the multimeric antigen to be affinity-driven if the binding affinity determined in both steps is comparable and to be avidity-driven if the binding affinity determined in both steps is different.   
     
     
         2 . A method for selecting the assay format for determining the binding interaction of an antibody of the human IgG1 subclass with a multimeric antigen comprising the following steps:
 1) determining the binding affinity of the antibody for the multimeric antigen using a surface plasmon resonance method,   2) incubating a mixture comprising the antibody, the antigen and lysine-gingipain of  porphyromonas gingivalis  or an enzymatically active fragment thereof at a pH of 7.5 to 8.5, in the presence of a reducing agent, at a temperature of 30° C. to 42° C., for a time of 10 min. to 240 min. to cleave the antibody into Fabs and Fc-region, whereby the concentration of the antibody is higher than the concentration of the antigen, and determining the binding affinity of the Fabs of the antibody for their antigen using surface plasmon resonance by directly applying the incubated reaction mixture obtained in the previous step in the surface plasmon resonance method,   
       whereby the binding affinity of the antibody to the multimeric antigen is i) affinity-driven if the binding affinity determined in both steps is comparable, or ii) avidity-driven if the binding affinity determined in both steps is different,
 and 
 selecting 
 i) in case of an affinity-driven interaction with a soluble multimeric antigen a solution assay, 
 ii) in case of an avidity-driven interaction with a soluble multimeric antigen a solution or a surface assay, 
 iii) in case of an affinity-driven interaction with a surface bound antigen a solution assay, or 
 iv) in case of an avidity-driven interaction with a surface bound antigen a surface assay 
 for determining the binding interaction of the antibody of the human IgG1 subclass with the multimeric antigen. 
 
     
     
         3 . The method according to  claim 1 , wherein the binding affinity is determined in solution using an ELISA or a surface plasmon resonance method. 
     
     
         4 . The method according to  claim 1 , wherein the binding affinity is determined using a cellular assay using FACS or a cellular effect. 
     
     
         5 . The method according to  claim 1 , wherein the binding affinities determined in step 1) and 2) of the antibody to the multimeric antigen are comparable if the binding affinities determined in both steps differ by a factor of 2 or less (the smaller value is used as basis for the calculation) and is different if the binding affinities determined in both steps differ by more than a factor of 2 (the smaller value is set to 100%). 
     
     
         6 . The method according to  claim 1 , wherein the binding affinity is determined with an antibody: multimeric antigen ratio of 10 or more. 
     
     
         7 . The method according to  claim 1 , wherein the polypeptide that is derived from lysine-gingipain of  porphyromonas gingivalis  is the lysine-gingipain of  porphyromonas gingivalis.    
     
     
         8 . The method according to  claim 7 , wherein the polypeptide that is derived from lysine-gingipain of  porphyromonas gingivalis  comprises the amino acid sequence of SEQ ID NO: 02 or SEQ ID NO: 03 or SEQ ID NO: 04 or a functional variant thereof 
     
     
         9 . The method according to  claim 7 , wherein the polypeptide that is derived from lysine-gingipain of  porphyromonas gingivalis  has an amino acid sequence that comprises at least residues 230 to 739 of SEQ ID NO: 01. 
     
     
         10 . The method according to  claim 1 , wherein the reducing agent is selected from the group consisting of 2-mercaptoethanol, cysteine, and dithiothreitol. 
     
     
         11 . The method according to  claim 10 , wherein the reducing agent is cysteine. 
     
     
         12 . The method according to  claim 10 , wherein the reducing agent is cysteine at a concentration of from 0.5 mM to 10 mM. 
     
     
         13 . The method according to  claim 1 , wherein the pH value is about pH 8. 
     
     
         14 . The method according to  claim 1 , wherein the temperature is of from 35° C. to 38° C. 
     
     
         15 . The method according to  claim 1 , wherein the incubating is for a time of about 60 min. 
     
     
         16 . The method according to  claim 1 , wherein the incubated mixture is used for the determination of the binding affinity without intermediate purification. 
     
     
         17 . The method according to  claim 1 , wherein the determining of the binding affinity is by surface plasmon resonance.