Hla-based methods and compositions and uses thereof

Abstract

Compositions and methods for isolating HLA-peptides from cells. A universal platform and methods for profiling the HLA-peptidome, enabling identification of endogenously presented HLA-peptides from cell lines expressing any possible class I or II construct.

1 - 21 . (canceled) 
     
     
         22 . A method for training an MHC-peptide presentation prediction algorithm implemented in a computer processor, comprising:
 (a) contacting a plurality of training peptides with a plurality of training cells in vitro, wherein each training cell of the plurality of training cells expresses a recombinant MHC protein, and wherein the recombinant MHC protein incorporates into a cell membrane of the training cell, and
 (i) wherein the training cell does not express an endogenous MHC protein, or 
 (ii) wherein the recombinant MHC protein is linked to an affinity acceptor peptide sequence; 
   (b) immunoprecipitating the plurality of training cells with an affinity molecule that binds to the affinity acceptor peptide sequence, thereby obtaining immunoprecipitated training cells;   (c) identifying training peptides bound to the recombinant MHC protein of the immunoprecipitated training cells by mass spectrometry; and   (d) inputting as a variable information on the training peptides identified by mass spectrometry and information on the recombinant MHC protein into the presentation prediction algorithm, thereby training the MHC-peptide presentation prediction algorithm.   
     
     
         23 . The method of  claim 22 , wherein the affinity acceptor peptide sequence is operably linked to an extracellular portion of the recombinant MHC protein. 
     
     
         24 . The method of  claim 22 , wherein the affinity acceptor peptide is biotin acceptor peptide (BAP). 
     
     
         25 . The method of  claim 24 , wherein the affinity acceptor peptide specific binding molecule is biotin or an antibody specific to the affinity acceptor peptide. 
     
     
         26 . The method of  claim 24 , wherein the affinity molecule is streptavidin, Neutravidin, or a derivative thereof. 
     
     
         27 . The method of  claim 22 , wherein the MHC protein is an MHC class II protein. 
     
     
         28 . The method of  claim 22 , wherein the plurality of training cells expresses a single recombinant MHC protein. 
     
     
         29 . The method of  claim 22 , wherein the plurality of training cells expresses:
 (a) a first recombinant MHC protein comprising a first MHC molecule and a first affinity acceptor peptide; and   (b) a second recombinant MHC protein comprising a second MHC molecule and a second affinity acceptor peptide,   wherein the first MHC molecule and the second MHC molecule are non-identical; and   wherein the first affinity acceptor peptide and the second affinity acceptor peptide are non-identical.   
     
     
         30 . The method of  claim 22 , wherein the training cell is an antigen presenting cell line. 
     
     
         31 . The method of  claim 22 , wherein inputting the variable further comprises inputting one or more variables selected from the group consisting of peptide sequence, amino acid physical properties, peptide physical properties, expression level of the source protein of the peptide within a cell, protein stability, protein translation rate, ubiquitination sites, protein degradation rate, translational efficiencies from ribosomal profiling, protein cleavability, protein localization, motifs of host protein that facilitate TAP transport, host protein is subject to autophagy, motifs that favor ribosomal stalling, and features that favor non-sense mediated degradation (NMD). 
     
     
         32 . The method of  claim 31 , wherein the motifs that favor ribosomal stalling comprises polyproline or polylysine stretches. 
     
     
         33 . The method of  claim 31 , wherein the features that favor NMD are selected from the group consisting of a long 3′ UTR and a stop codon greater than 50 nucleic acids upstream of a last exon:exon junction. 
     
     
         34 . The method of  claim 22 , wherein the plurality of training peptides comprises endogenous peptides of the plurality of training cells. 
     
     
         35 . The method of  claim 22 , wherein the recombinant MEW protein comprises at least 10 different recombinant MEW proteins. 
     
     
         36 . The method of  claim 22 , wherein identifying further comprises performing a sequencing analysis, a biochemical analysis or a combination thereof. 
     
     
         37 . The method of  claim 22 , further comprising generating an HLA-allele specific binding peptide sequence database for a plurality of HLA alleles. 
     
     
         38 . The method of  claim 22 , further comprising generating a peptide library comprising peptides associated with a disease prior to immunoprecipitating. 
     
     
         39 . The method of  claim 22 , further comprising, prior to (b), lysing the plurality of training cells. 
     
     
         40 . The method of  claim 22 , wherein the recombinant MHC protein is linked to an affinity acceptor peptide sequence by a cleavable linker. 
     
     
         41 . A library of cells, each cell of the library expressing a different recombinant MHC protein encoded by an expression vector, wherein each recombinant MEW protein incorporates into a cell membrane of the cells; and wherein:
 (i) each cell of the library does not express an endogenous MHC protein, or   (ii) an extracellular portion of each recombinant MEW protein is linked to an affinity acceptor peptide sequence.