US2023304190A1PendingUtilityA1

Methods of screening antigen-binding molecules by normalizing for the concentration of antigen-binding molecule

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Assignee: MESO SCALE TECHNOLOGIES LLCPriority: Feb 23, 2018Filed: May 9, 2023Published: Sep 28, 2023
Est. expiryFeb 23, 2038(~11.6 yrs left)· nominal 20-yr term from priority
Inventors:Ka Y. KwongIlia Davydov
C40B 30/04C07K 16/005C07K 16/26C12N 15/1037G01N 33/54306
59
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Claims

Abstract

The invention provides singleplex and multiplex assays for screening of antigen-binding molecules for their affinity to antigens by normalizing for the concentration of the antigen-binding molecule.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         1 . A method of screening a candidate antigen-binding molecule for affinity to an antigen, comprising:
 a. contacting the candidate antigen-binding molecule with a substrate, the substrate comprising (i) an antigen immobilized on the substrate, and (ii) a normalization binding molecule immobilized on the substrate;   b. detecting a binding of the candidate antigen-binding molecule with the antigen on the substrate;   c. detecting a binding of the candidate antigen-binding molecule from (b) to the immobilized normalization binding molecule to determine a concentration of the candidate antigen-binding molecule; and   d. determining the affinity of the candidate antigen-binding molecule from the binding of the candidate antigen molecule determined in (b) and the binding of the antigen-binding molecule to the normalization binding molecule determined in (c).   
     
     
         2 . The method of  claim 1 , wherein the candidate antigen-binding molecule is selected from the group consisting of an antibody (Ab), an immunoglobulin G (IgG), an antigen-binding fragment (Fab), variable fragment (Fv), a single-chain fragment variable (scFv), an antibody with one V-gene domain, a bivalent diabody, and combinations thereof. 
     
     
         3 . The method of  claim 2 , wherein the candidate antigen-binding molecule is an Ab or Fab. 
     
     
         4 . The method of any of  claims 1 - 3 , wherein the candidate antigen-binding molecule is displayed on a bacteriophage (phage). 
     
     
         5 . The method of  claim 4 , wherein the phage infects a bacterium selected from the group consisting of  Escherichia coli, Bacillus pumilus, Bacillus subtilis, Cellulosimicrobium cellulans, Oerskovia turbata, Pseudomonas aeruginosa, Pseudomonas syringae, Salmonella enterica  and  Thermus thermophilus.    
     
     
         6 . The method of  claim 5 , wherein the phage infects  Escherichia coli  ( E. coli ). 
     
     
         7 . The method of  claim 6 , wherein the phage is an  E. coli  filamentous bacteriophage. 
     
     
         8 . The method of  claim 6 , wherein the phage is selected from the group consisting of k, M13, P1, T4, f1, fd and Mu. 
     
     
         9 . The method of  claim 8 , wherein the phage is M13 or T4. 
     
     
         10 . The method of any of  claims 1 - 3 , wherein the candidate antigen-binding molecule is derived from one or more hybridoma supernatants. 
     
     
         11 . The method of  claim 10 , wherein the candidate antigen-binding molecule is a monoclonal antibody. 
     
     
         12 . The method of any of  claims 1 - 11 , wherein the substrate comprises a plurality of immobilized antigens. 
     
     
         13 . The method of  claim 12 , wherein the substrate is a multi-well plate or a microparticle. 
     
     
         14 . The method of  claim 13 , wherein the substrate is a multi-well plate. 
     
     
         15 . The method of  claim 14 , wherein the immobilized antigen and the immobilized normalization binding molecule are located within one or more single wells of the multi-well plate. 
     
     
         16 . The method of  claim 13 , wherein the substrate is a microparticle. 
     
     
         17 . The method of  claim 16 , wherein the microparticle comprises polypropylene, latex, polystyrene, polyacrylamide, silica, alumina, a magnetic material, or combinations thereof. 
     
     
         18 . The method of  claim 16 , wherein the substrate comprises a plurality of microparticles, wherein the microparticles are coded to discriminate between microparticles comprising an antigen and a normalization molecule. 
     
     
         19 . The method of any of  claims 1 - 18 , wherein the antigen and normalization binding molecule are immobilized on the substrate surface via at least one linker molecule. 
     
     
         20 . The method of  claim 19 , wherein the at least one linker molecule is an oligonucleotide. 
     
     
         21 . The method of  19 , wherein the at least one linker molecule is biotin complexed with streptavidin. 
     
     
         22 . The method of any of  claims 1 - 18 , wherein the antigen is immobilized on the substrate surface via a targeting agent bound to the surface and a targeting agent complement indirectly bound to the antigen. 
     
     
         23 . The method of any of  claims 1 - 18  and  22 , wherein the normalization binding molecule is immobilized on the substrate surface via a targeting agent bound to the surface and a targeting agent complement indirectly bound to the normalization binding molecule. 
     
     
         24 . The method of  claim 22 , wherein the targeting agent complement is additionally bound to a linking agent, and the antigen is bound to a supplemental linking agent, wherein the linking agent and the supplemental linking agent are binding partners. 
     
     
         25 . The method of  claim 23  or  24 , wherein the targeting agent complement is additionally bound to a linking agent, and the normalization binding molecule is bound to a supplemental linking agent, wherein the linking agent and the supplemental linking agent are binding partners. 
     
     
         26 . The method of  claim 24  or  25 , further comprising a bridging agent, wherein the bridging agent has a first binding site for the linking agent, and a second binding site for the supplemental linking agent. 
     
     
         27 . The method of any of  claims 1 - 26 , wherein the normalization binding molecule is an antibody or antigen binding fragment thereof that binds an epitope on the candidate antigen-binding molecule. 
     
     
         28 . The method of any one of  claims 4 - 9 , wherein the normalization binding molecule is an antibody or antigen binding fragment thereof that binds an epitope on the candidate antigen-binding molecule, or an antibody or antigen-binding fragment thereof that binds a phage coat protein. 
     
     
         29 . The method of  claim 27  or  28 , wherein the epitope on the candidate antigen-binding molecule is a constant domain or fragment thereof of an antibody light chain. 
     
     
         30 . The method of  claim 29 , wherein the epitope on the candidate antigen-binding molecules is a constant region or fragment thereof on a kappa light chain. 
     
     
         31 . The method of  claim 29 , wherein the epitope on the candidate antigen-binding molecules is a constant region or fragment thereof on a lambda light chain. 
     
     
         32 . The method of  claim 27  or  28 , wherein the epitope on the candidate antigen-binding is a constant domain or fragment thereof of an antibody heavy chain. 
     
     
         33 . The method of  claim 28 , wherein the normalization binding molecule is an antibody or antigen-binding fragment that specifically binds a phage coat protein. 
     
     
         34 . The method of  claim 33 , wherein the phage coat protein is pIII, pVIII, pVII or pIX. 
     
     
         35 . The method of  claim 33 , wherein the phage coat protein is a fusion protein. 
     
     
         36 . The method of  claim 33 , wherein the phage comprises a capsid-attached tag. 
     
     
         37 . The method of  claim 36 , wherein the normalization binding molecule specifically binds the capsid-attached tag. 
     
     
         38 . The method of  claim 37 , wherein the tag is Strep II tag (STAG), FLAG-tag and His-tag. 
     
     
         39 . The method of any of  claims 1 - 38 , wherein the detecting in (b) comprises measuring optical absorbance, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, light scattering, magnetism, or a combination thereof. 
     
     
         40 . The method of any of  claims 1 - 38 , wherein the detecting in (c) comprises measuring optical absorbance, fluorescence, phosphorescence, chemiluminescence, electrochemiluminescence, light scattering, magnetism, or a combination thereof. 
     
     
         41 . A method of screening a candidate antigen-binding molecule for affinity to an antigen, comprising:
 a. contacting (1) a control compound at a plurality of concentrations, and (2) a candidate antigen-binding molecule with a substrate, the substrate comprising (i) an antigen immobilized on the substrate, and (ii) a normalization binding molecule immobilized on the substrate;   b. detecting a binding of the candidate antigen-binding molecule with an antigen on the substrate;   c. detecting a binding of the candidate antigen-binding molecule from (b) to the immobilized normalization binding molecule;   d. detecting a binding of the control compound at the plurality of concentrations to the immobilized normalization binding molecule, wherein the detecting produces a plurality of detection signals;   e. generating a standard calibration curve utilizing the plurality of detection signals;   f. determining a first and a second concentration for each candidate antigen-binding molecule, wherein the first concentration is an apparent concentration determined from binding of the antigen-binding molecule to the immobilized antigen, and the second concentration is an actual concentration determined from the binding to the immobilized normalization binding molecule, wherein the actual concentration of each candidate antigen-binding molecule binding is determined using the calibration curve; and   g. calculating a ratio of the first and second concentrations for each candidate molecule, wherein the ratio reflects the relative affinity of the antigen-binding molecule.   
     
     
         42 . The method of  claim 41 , wherein the control compound is a bacteriophage and the candidate antigen-binding molecules are displayed on bacteriophages. 
     
     
         43 . The method of  claim 41 , wherein the control is an antibody, and the candidate antigen-binding molecules are mutated antibodies.

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