Synthetic Antibodies

Abstract

The present invention provides methods for synthetic antibodies, methods for making synthetic antibodies, methods for identifying ligands, and related methods and reagents.

1 . A method for identifying affinity elements to a target of interest, comprising
 (a) contacting a substrate surface comprising an array of between 10 2  and 10 7  different test compounds of known composition with a target of interest under conditions suitable for moderate affinity binding of the target to target affinity elements if present on the substrate, wherein the target is not an Fv portion of an antibody, and wherein the different test compounds are not derived from the target; and   (b) identifying test compounds that bind to the target with at least moderate affinity, wherein such compounds comprise target affinity elements.   
     
     
         2 . The method of  claim 1  wherein the substrate surface is addressable. 
     
     
         3 . The method of  claim 1  or  2 , further comprising
 (c) identifying test compounds that do not bind to the target with at least moderate affinity. 
 
     
     
         4 . The method of any one of  claims 1 - 3 , wherein the test compounds have a molecular weight of between 1000 Daltons and 10,000 Daltons. 
     
     
         5 . The method of any one of  claims 1 - 4 , wherein the test compounds are polypeptides. 
     
     
         6 . The method of any one of  claims 1 - 5 , further comprising contacting the same substrate surface or a separate substrate surface with competitor, and determining a ratio of test compound binding to target versus test compound binding to competitor. 
     
     
         7 . The method of any one of  claims 1 - 6  further comprising identifying combinations of target affinity elements that bind to different sites on the same target. 
     
     
         8 . The method of  7 , further comprising determining an appropriate spacing between the target affinity elements in an affinity element combination to increases a binding affinity and/or specificity for the target of the affinity element combination relative to a binding affinity and/or specificity of the target affinity elements alone for the target. 
     
     
         9 . The method of  claim 7  or  8 , further comprising linking a combination of affinity elements, wherein the linker provides a spacing of between about 0.5 nm and about 30 nm between a first affinity element and a second affinity element. 
     
     
         10 . The method of any one of  claims 1 - 9  wherein one or both of the first affinity element and the second affinity element have a dissociation constant for binding to the first target of between about 1 μM and 500 μM. 
     
     
         11 . The method of any one of  claims 8 - 10 , further comprising optimizing binding affinity of one or both of the first affinity element and the second affinity element to the target. 
     
     
         12 . A synthetic antibody made by the method of any one of  claims 9 - 11 . 
     
     
         13 . A synthetic antibody comprising:
 (a) a first affinity element that can bind a first target;   (b) a second affinity element that can bind the first target, and which can bind to the first target in the presence of the first affinity element bound to the first target; and   (c) a linker connecting the first affinity element and the second affinity element,   wherein one or both of the first affinity element and the second affinity element have a molecular weight of at least 1000 Daltons;   wherein at least one of the first affinity element and the second affinity element are not derived from the first target;   wherein the synthetic antibody has an increased binding affinity and/or specificity for the first target relative to a binding affinity and/or specificity of the first affinity element for the first target and relative to a binding affinity and/or specificity of the second affinity element for the target; and   wherein the first target is not the Fv of an antibody.   
     
     
         14 . The synthetic antibody of  claim 13  wherein both the first affinity element and the second affinity element have a molecular weight of between about 1000 Daltons and 10,000 Daltons. 
     
     
         15 . The synthetic antibody of  claim 13  or  14 , wherein the linker provides a spacing of between about 0.5 nm and about 30 nm between the first affinity element and the second affinity element. 
     
     
         16 . The synthetic antibody of any one of  claims 13 - 15  wherein neither the first affinity element nor the second affinity element are derived from an Fv region of an antibody. 
     
     
         17 . The synthetic antibody of any one of  claims 13 - 16 , wherein neither the first affinity element nor the second affinity element are derived from the first target. 
     
     
         18 . The synthetic antibody of any one of  claims 13 - 17 , wherein the first affinity element and the second affinity element comprise polypeptides. 
     
     
         19 . The synthetic antibody of any one of  claims 13 - 17 , wherein the first affinity element and the second affinity element comprise nucleic acids. 
     
     
         20 . The synthetic antibody of  claim 18 , wherein a net charge of the synthetic antibody at a pH 7 is between +2 and −2. 
     
     
         21 . The synthetic antibody of any one of  claims 13 - 20  wherein at least one of the first and second affinity elements is a non-naturally occurring compound. 
     
     
         22 . The synthetic antibody of any one of  claims 13 - 21 , wherein the linker is an amino acid linker. 
     
     
         23 . The synthetic antibody of any one of  claims 13 - 21  wherein the linker is a nucleic acid linker. 
     
     
         24 . The synthetic antibody of  claim 23 , wherein one or both of the first affinity element and the second affinity element are not nucleic acids. 
     
     
         25 . The synthetic antibody of any one of  claims 13 - 24  wherein the first affinity element and the second affinity element are different and bind to separate regions of the first target. 
     
     
         26 . The synthetic antibody of any one of  claims 13 - 25  further comprising a third affinity element connected to the first affinity element and the second affinity element. 
     
     
         27 . The synthetic antibody of  claim 26  wherein the third affinity element can bind to a second target different than the first target. 
     
     
         28 . The synthetic antibody of  claim 27 , wherein the second affinity element also binds to the second target, and wherein a spatial arrangement of the first affinity element, the second affinity element, and the third affinity element permits only one of the first target and the second target to be bound by the synthetic antibody. 
     
     
         29 . The synthetic antibody of  claim 26 , wherein the synthetic antibody further comprises a fourth affinity element connected to the first affinity element, the second affinity element, and the third affinity element, wherein the third and fourth affinity elements are spatially arranged relative to the first affinity element and the second affinity element to provide binding of a further target in the presence of the first target bound to the synthetic antibody. 
     
     
         30 . The synthetic antibody of any one of  claims 13 - 29  further comprises a first signaling element and a second signaling element, wherein a spatial relationship of the first signaling element and the second signaling element are altered to produce a detectable signal upon target binding to the synthetic antibody. 
     
     
         31 . The synthetic antibody of any one of  claims 13 - 30 , bound to a substrate. 
     
     
         32 . A substrate comprising:
 (a) a surface; and   (b) a plurality of synthetic antibodies according to any one of  claims 13 - 30  attached to the surface.   
     
     
         33 . The substrate of  claim 32  wherein the plurality of synthetic antibodies comprises a plurality of different synthetic antibodies. 
     
     
         34 . A method for making a synthetic antibody, comprising connecting at least a first affinity element and a second affinity element for a given target via a linker;
 wherein one or both of the first affinity element and the second affinity element have a molecular weight of at least 1000 Daltons;   wherein at least one of the first affinity element and the second affinity element are not derived from the first target;   wherein the synthetic antibody has an increased binding affinity and/or specificity for the first target relative to a binding affinity and/or specificity of the first affinity element for the first target and relative to a binding affinity and/or specificity of the second affinity element for the target; and   wherein the first target is not the Fv of an antibody.   
     
     
         35 . The method of  claim 34  wherein both the first affinity element and the second affinity element have a molecular weight of between 1000 Daltons and 10,000 Daltons. 
     
     
         36 . The method of  claim 34  or  35 , wherein the linker provides a spacing of between about 0.5 nm and about 30 nm between the first affinity element and the second affinity element. 
     
     
         37 . The method of any one of  claims 34 - 36  wherein neither the first affinity element nor the second affinity element are Fv regions of an antibody. 
     
     
         38 . The method of any one of  claims 34 - 37 , wherein at least one of the first affinity element and the second affinity element comprises a nucleic acid. 
     
     
         39 . The method of any one of  claims 34 - 37 , wherein at least one of the first affinity element and the second affinity element comprises a polypeptide. 
     
     
         40 . The method of  claim 39 , wherein a net charge of the synthetic antibody at pH 7 is between +2 and −2. 
     
     
         41 . A method for ligand identification, comprising:
 (a) contacting a substrate surface comprising a target array with one or more potential ligands, wherein the contacting is done under conditions suitable for moderate to high affinity binding of the one or more ligands to suitable targets present on the substrate; and   (b) identifying targets that bind to one or more of the ligands with at least moderate affinity.   
     
     
         42 . The method of  claim 41 , wherein the one or more potential ligands are selected from the group consisting of antibodies and synthetic antibodies according to any one of  claims 13 - 31 . 
     
     
         43 . The method of  claim 41  or  42 , wherein the array of targets is mounted in a flow chamber, wherein
 (i) a first buffer comprising the one or more potential ligands is flowed over the addressable array, 
 (ii) wherein identifying targets that bind to one or more of the ligands with at least moderate affinity comprises analyzing real-time affinity data gathered by an array reader; 
 (iii) the first buffer flow over the addressable array is stopped after at least moderate binding to the array is detected; 
 (iv) repeating steps (i)-(iii) a desired number of times using a further buffer comprising one or more further potential ligands. 
 
     
     
         44 . A method for identifying a synthetic antibody profile for a test sample of interest, comprising contacting a substrate comprising a plurality of synthetic antibodies according to the present invention with a test sample and comparing synthetic antibody binding to the test sample with synthetic antibody binding to a control sample, wherein synthetic antibodies that differentially bind to targets in the test sample relative to the control sample comprise a synthetic antibody profile for the test sample 
     
     
         45 . The method of  claim 44 , wherein the control sample is contacted with the same substrate as the test sample. 
     
     
         46 . The method of  claim 44  or  45 , wherein the test sample is a disease state test sample. 
     
     
         47 . The method of  claim 44  or  45 , wherein the test sample is a research test sample. 
     
     
         48 . A composition, comprising:
 (a) a first affinity element bound to a template nucleic acid strand;   (b) a second affinity element bound to a complementary nucleic acid strand, wherein the first affinity element and the second affinity element non-competitively bind to a common target;   wherein the template nucleic acid strand and the complementary nucleic acid strand are annealed via base pairing to form an assembly;   wherein the first affinity element and the second affinity element are separated in the assembly; and   wherein either the template nucleic acid strand, the complementary nucleic acid strand, or both, are bound to a surface of a substrate.   
     
     
         49 . An array, comprising a plurality of the compositions of  claim 48  bound to a substrate surface, wherein the plurality of compositions comprises one or both of:
 (a) a plurality of compositions wherein the first ligand and the second ligand are the same for each composition, but wherein the separation of the first ligand from the second ligand in the assembly differs; and 
 (b) a plurality of compositions wherein the first ligand and/or the second ligand are different for each composition.