Screening Methods

Abstract

The present invention provides polypeptide binding agents, e.g. antibodies, that exhibit the ability to kinetically modulate the binding and signaling of biological signaling complexes, e.g., receptor-ligand complexes; methods of identifying such polypeptide binding agents, methods of making such polypeptide binding agents, compositions comprising such polypeptide binding agents, and methods of using such polypeptide binding agents.

1 . A method of identifying a candidate kinetic modulating antibody that modulates binding between first and second components of a signaling complex, comprising the steps of:
 (a) measuring a binding affinity or binding rate parameter of said first component for said second component, in the presence of a test antibody,   (b) measuring a binding affinity or binding rate parameter of said first component for said second component in the absence of said test antibody; and   (c) identifying said test antibody as a candidate kinetic modulating antibody when said test antibody exhibits a 1.5-fold to 1000-fold difference in the binding affinity or binding rate parameters measured in steps (a) and (b).   
     
     
         2 . The method of  claim 1  wherein the test antibody is identified as a candidate positive modulating antibody if the test antibody strengthens the binding affinity or binding rate parameter between said first component and said second component by about 1.5-fold to 1000-fold. 
     
     
         3 . The method of  claim 2  wherein the test antibody strengthens the binding affinity or binding rate parameter between said first component and said second component by about 2-fold to 200-fold. 
     
     
         4 . The method of  claim 1  wherein the test antibody is identified as a candidate negative modulating antibody if the test antibody weakens the binding affinity or binding rate parameter between said first component and said second component by about 1.5-fold to 1000-fold. 
     
     
         5 . The method of  claim 4  wherein the test antibody weakens the binding affinity or binding rate parameter between said first component and said second component by about 2-fold to 200-fold. 
     
     
         6 - 43 . (canceled) 
     
     
         45 . The method of  claim 2  wherein
 i) the antigen to which the test antibody binds is the first component and the test antibody is at a saturating concentration compared to the concentration of the first component; or 
 ii) the antigen to which the test antibody binds is the second component and the test antibody is at a saturating concentration compared to the concentration of the second component. 
 
     
     
         45 . (canceled) 
     
     
         47 . The method of  claim 45  wherein the concentration of the test antibody is greater than or equal to the K D  of the test antibody for a complex comprising the first component and the second component. 
     
     
         48 . The method of  claim 47  wherein the concentration of the second component is less than the K D  of the test antibody for the first component. 
     
     
         49 . The method of  claim 48  wherein the concentration of the first component is at a subsaturating concentration for the binding of first component to second component. 
     
     
         50 . The method of  claim 49  wherein the concentration of the first component is within the range of about EC 20  to EC 80  for the interaction of the first component with the second component. 
     
     
         51 . The method of  claim 2  wherein
 i) one or more concentrations of the test antibody is contacted with multiple different concentrations of the first component in the presence of one or more concentrations of the second component; or 
 ii) one or more concentrations of the test antibody is contacted with multiple different concentrations of the second component in the presence of one or more concentrations of the first component. 
 
     
     
         51 - 67 . (canceled) 
     
     
         69 . The method of  claim 1  further comprising, prior to step (a), assaying a plurality of test antibodies for binding affinity to a complex comprising said first component and second component, optionally with an equilibrium dissociation constant K D  of 10 −5 M or stronger binding affinity. 
     
     
         70 . The method of  claim 1  further comprising, prior to step (a),
 i) assaying a plurality of test antibodies for binding affinity to said first component, optionally with an equilibrium dissociation constant K D  of 10 −5 M or stronger binding affinity; or 
 ii) assaying a plurality of test antibodies for binding affinity to said second component, optionally with an equilibrium dissociation constant K D  of 10 −5 M or stronger binding affinity. 
 
     
     
         70 - 71 . (canceled) 
     
     
         73 . The method of  claim 1  further comprising measuring a binding affinity or binding rate parameter of said first component for a binding partner, wherein the binding partner is not said second component, in the presence and absence of said test antibody. 
     
     
         73 . (canceled) 
     
     
         75 . The method of  claim 73  comprising identifying a test antibody that does not significantly change the binding affinity or binding rate parameter of said first component for said binding partner. 
     
     
         76 . The method of  claim 1  wherein said test antibody is selected from the group consisting of antibody fragments, scFv, Fab, CDRs, rodent antibodies, mammalian antibodies, human antibodies, chimeric antibodies, monoclonal antibodies and humanized antibodies. 
     
     
         76 - 84 . (canceled) 
     
     
         86 . The method of  claim 1  wherein i) said first component is a soluble ligand and said second component is a membrane-bound receptor; or ii) said first component is a membrane-bound receptor and said second component is a soluble ligand; or iii) said first component is a membrane-bound ligand and said second component is a membrane-bound receptor. 
     
     
         86 - 94 . (canceled) 
     
     
         96 . The method of  claim 1 , further comprising measuring a binding affinity or binding rate parameter of said first component and/or said second component for a third component, in the presence and absence of the test antibody. 
     
     
         96 - 107 . (canceled) 
     
     
         109 . The method of  claim 1  further comprising measuring the level of signaling mediated by said signaling complex in the presence and absence of the test antibody. 
     
     
         110 . The method of  claim 109  wherein the level of signaling mediated by the signaling complex is measured in a phosphorylation assay, ion flux assay, molecular transport assay, or gene expression assay. 
     
     
         111 . The method of  claim 109  wherein said test antibody increases or decreases the EC 50  of the first component of said signaling complex by about 1.5-fold to about 1000-fold. 
     
     
         112 . The method of  claim 109  wherein said test antibody
 i) does not significantly change the maximal agonist response of the signaling produced by said first component; or 
 ii) reduces the maximal agonist response of the signaling produced by said signaling complex by about 1.5-fold to 1000-fold; or 
 iii) increases the maximal agonist response of the signaling produced by said first component by at least 10%. 
 
     
     
         112 - 120 . (canceled) 
     
     
         122 . A positive modulating antibody (M) that strengthens the binding of a first component (C1) to a second component (C2) of a signaling complex, said antibody characterized by the following equilibrium dissociation constant K D  binding properties: (i) said antibody binds with an equilibrium dissociation constant K D  of 10 −5 M or less to any one of C1, C2, or a complex comprising C1 and C2 (C1C2), and (ii) any one or more of K [C1C2]M , K [MC2]C1 , or K [MC1]C2  is about 1.5-fold to 1000-fold lower than any one or more of K MC2  or K MC1 , wherein C1 or C2 is the secreted protein of any of SEQ ID NOS: 1-88. 
     
     
         122 - 123 . (canceled) 
     
     
         125 . A negative modulating antibody (M) that weakens the binding of a first component (C1) to a second component (C2) of a signaling complex, said antibody characterized by the following equilibrium dissociation constant K D  binding properties: (i) said antibody binds with an equilibrium dissociation constant K D  of 10 −5 M or less to any one of C1, C2, or a complex comprising C1 and C2 (C1C2), and (ii) any one or more of K MC2  or K MC1  is about 1.5-fold to 1000-fold lower than any one or more of K [C1C2]M , K [MC2]C1 , or K [MC1]C2 , wherein C1 or C2 is the secreted protein of any of SEQ ID NOS: 1-88. 
     
     
         125 - 136 . (canceled) 
     
     
         138 . The antibody of claim  117  which is a purified monoclonal antibody. 
     
     
         139 . A polynucleotide encoding an antibody of claim  117 . 
     
     
         139 - 140 . (canceled) 
     
     
         142 . A host cell comprising the polynucleotide of  claim 139 . 
     
     
         142 . (canceled) 
     
     
         144 . A method of producing an antibody comprising culturing the host cell of  claim 142  in culture medium under suitable conditions, and isolating the antibody from the host cell or culture medium. 
     
     
         144 - 145 . (canceled) 
     
     
         147 . A sterile composition comprising an antibody of claim  117  and a sterile pharmaceutically acceptable diluent. 
     
     
         148 . A method of administering a composition to a mammal comprising administering the sterile composition of  claim 147 . 
     
     
         148 - 149 . (canceled) 
     
     
         151 . The method of  claim 73  wherein
 i) the first component is IL-1 beta, the second component is IL-1R1, and the binding partner is either IL-1R2 or IL-1 accessory protein; or 
 ii) the first component is TNF alpha, the second component is TNFR1, and the binding partner is TNFR2; or 
 iii) the first component is TNF alpha, the second component is TNFR2, and the binding partner is TNFR1. 
 
     
     
         151 - 152 . (canceled)