Methods of making and using soluble mhc molecules

Abstract

Provided herein are methods of making a detectably-labeled, soluble MHC molecule that can be used in a novel K on -rate assay and an improved TCR ligand k off -rate assay.

What is claimed is: 
     
         1 . A method of identifying a TCR that associates with a MHC-peptide complex, the method comprising:
 (a) providing a multimer complex comprising a backbone reversibly associated with a plurality of detectably-labeled, soluble MHC monomers, each loaded with a peptide of interest;   (b) contacting the multimer complex with a TCR under conditions that allow the formation of a MHC-peptide-TCR complex;   (c) disrupting the multimer complexes; and   (d) determining the MHC-peptide-TCR K off , wherein the K off  indicates a degree to which a TCR associates with the MHC-peptide complex.   
     
     
         2 . The method of  claim 1 , wherein the method further comprises first forming the multimer complex by performing:
 providing a detectably-labeled, soluble MHC monomer loaded with a cleavable peptide;   exchanging the cleavable peptide with the peptide of interest, thereby generating a detectably-labeled, soluble MHC monomer loaded with the peptide of interest; and   forming the multimer complex by reversibly associating the backbone with a plurality of the detectably-labeled, soluble MHC monomers, each loaded with the peptide of interest.   
     
     
         3 . The method of  claim 1 , wherein the peptide of interest comprises a cysteine residue. 
     
     
         4 . The method of  claim 1 , wherein a K off  of about 20 seconds or longer indicates that the TCR associates with the MHC-peptide complex. 
     
     
         5 . The method of  claim 1 , wherein a K off  of about 15 to about 500 seconds or longer indicates that the TCR associates with the MHC-peptide complex. 
     
     
         6 . The method of  claim 1 , wherein the TCR is in a library comprising a plurality of T cells, each T cell presenting a complete TCR, and one or both of a CD4 molecule and a CD8 molecule. 
     
     
         7 . The method of  claim 1 , further comprising transducing a T cell with a nucleic acid encoding the alpha and beta chains of the TCR. 
     
     
         8 . The method of  claim 1 , wherein the method is performed at a temperature of between about 4° C. and about 37° C. 
     
     
         9 . The method of  claim 8 , wherein the temperature is about 20° C. 
     
     
         10 . The method of  claim 1 , wherein the peptide of interest comprises a neo antigen. 
     
     
         11 . A method of selecting a T cell suitable for adoptive transfer, the method comprising:
 (a) providing a multimer complex comprising a backbone reversibly associated with a plurality of a detectably-labeled, soluble MHC monomer loaded with a peptide of interest;   (b) contacting the multimer complex with a TCR library comprising a plurality of T cells expressing a plurality of TCRs under conditions that allow the formation of a MHC-peptide-TCR complex;   (c) disrupting the multimer complexes;   (d) determining K off  of a MHC-peptide-TCR complex; and   (e) selecting a T cell for adoptive therapy, wherein the T cell expresses a TCR having a K off  for the MHC-peptide complex of about 15 seconds to about 500 seconds.   
     
     
         12 . The method of  claim 11 , wherein the method further comprises first forming the multimer complex by performing:
 providing a detectably-labeled, soluble MHC monomer loaded with a cleavable peptide;   exchanging the cleavable peptide with the peptide of interest, thereby generating a detectably-labeled, soluble MHC monomer loaded with the peptide of interest; and   forming the multimer complex by reversibly associating the backbone with a plurality of the detectably-labeled, soluble MHC monomer loaded with the peptide of interest.   
     
     
         13 . The method of  claim 11 , wherein the TCR library comprises a plurality of T cells, each T cell presenting a complete TCR, and one of a CD4 molecule and a CD8 molecule. 
     
     
         14 . The method of  claim 11 , further comprising:
 (f) expanding the T cell selected in (e) to a population of at least about 1×10 2  T cells; and   (g) administering a desired number of T cells to a subject.   
     
     
         15 . The method of  claim 12 , wherein at least about 1×10 6  T cells are administered to the subject. 
     
     
         16 . The method of  claim 11 , further comprising isolating the TCR genes from the T cell selected in (e). 
     
     
         17 . The method of  claim 12 , wherein the label is selected from the group consisting of a radiolabel, fluorescent agents, chromogenic agents, chemiluminescent agents and magnetic particles. 
     
     
         18 . The method of  claim 17 , wherein the label is a fluorescent dye selected from the group consisting of an Atto dye, an Alexafluor dye, quantum dots, Hydroxycoumarin, Aminocoumarin, Methoxycoumarin, Cascade Blue, Pacific Blue, Pacific Orange, Lucifer yellow, NBD, R-Phycoerythrin (PE), PE-Cy5 conjugates, PE-Cy7 conjugates, Red 613, PerCP, TruRed, FluorX, Fluorescein, BODIPY-FL, Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, TRITC, X-Rhodamine, Lissamine Rhodamine B, Texas Red, Allophycocyanin (APC), APC-Cy7 conjugates, Indo-1, Fluo-3, Fluo-4, DCFH, DHR, SNARF, GFP (Y66H mutation), GFP (Y66F mutation), EBFP, EBFP2, Azurite, GFPuv, T-Sapphire, Cerulean, mCFP, mTurquoise2, ECFP, CyPet, GFP (Y66W mutation), mKeima-Red, TagCFP, AmCyan1, mTFP1, GFP (S65A mutation), Midoriishi Cyan, Wild Type GFP, GFP (S65C mutation), TurboGFP, TagGFP, GFP (S65L mutation), Emerald, GFP (S65T mutation), EGFP, Azami Green, ZsGreen1, TagYFP, EYFP, Topaz, Venus, mCitrine, YPet, TurboYFP, ZsYellow1, Kusabira Orange, mOrange, Allophycocyanin (APC), mKO, TurboRFP, tdTomato, TagRFP, DsRed monomer, DsRed2 (“RFP”), mStrawberry, TurboFP602, AsRed2, mRFP1, J-Red, R-phycoerythrin (RPE), B-phycoerythrin (BPE), mCherry, HcRed1, Katusha, P3, Peridinin Chlorophyll (PerCP), mKate (TagFP635), TurboFP635, mPlum, and mRaspberry. 
     
     
         19 . The method of  claim 11 , wherein the method is performed at a temperature of between about 4° C. and about 37° C. 
     
     
         20 . A method of identifying member of a binding pair of interest, the method comprising:
 (a) forming a labeling mixture comprising:
 (1) TCEP; 
 (2) a maleimide-conjugated detectable label; and 
 (3) a first member of a binding pair; 
   (b) incubating the labeling mixture at a desired incubation temperature a desired incubation period;   (c) removing unbound maleimide-conjugated detectable label from the labeling mixture;   (d) contacting the one member of a binding pair with a screening library comprising a known or suspected second member of the binding pair under conditions that allow the formation of an association complex; and   (e) detecting the formation of an association complex.