Engineering t cell receptors

Inactive Publication Date: 2015-07-09
THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A major problem with each of these TCR-engineering approaches is that they require a different TCR isolated from a T cell clone with reactivity towards a specific peptide antigen in order to develop a higher affinity TCR mutant specific for the peptide antigen (cognate antigen), or structurally similar variants thereof.

Method used

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  • Engineering t cell receptors
  • Engineering t cell receptors
  • Engineering t cell receptors

Examples

Experimental program
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Effect test

example 1

Selection of a Model TCR

[0237]TCRs all adopt a similar Ig-fold and docking angle, and TCR recognition of pepMHC is mediated entirely by specific residues on CDR loops (Garcia et al. (2009) Nat Immunol, 10, 143-7; Marrack et al. (2008) Annu Rev Immunol, 26, 171-203; Rudolph et al. (2006) Annu Rev Immunol, 24, 419-66)). Hence, according to the present invention, a single TCR with known structure provides a scaffold for in vitro engineering with specificity and high affinity against non-cognate peptides displayed on MHC. That is, by generating mutants with degenerate residues within CDR loop residues that are most likely to directly contact peptide, libraries of mutants within a single TCR were generated in order to provide TCRs that can be developed having high affinity against non-cognate peptide-MHC antigens.

[0238]The general strategy used to discover, or generate, novel TCRs against non-cognate antigens from a single scaffold is shown in FIG. 1. The process involves: selecting a si...

example 2

Analysis of the Human TCR A6 in Complex with Tax:HLA.A2 as a Scaffold for TCR Engineering

[0242]For illustrative purposes, the A6 TCR was selected as the single TCR having a known structure. The structure of the A6:Tax peptide:HLA-A2 complex (PDB: 1AO7) (Garboczi et al. (1996) Nature, 384, 134-141), was published in 1996. The side view of the complex showed that the ends of the variable domains that contained the six CDRs docked onto the Tax:HLA.A2 molecule, with the central region of the binding site positioned over the peptide Tax (FIG. 2A) The top down view of the Tax:HLA.A2 complex, with the TCR “removed”, except for the six CDR loops. This view shows that the TCR adopts a diagonal position over the peptide-MHC, a finding which has now been observed for all TCR:peptide-MHC structures. In this orientation, the two CDR3 loops are positioned over the peptide, while there are various residues from CDR1 and CDR2 loops that interact predominantly with the helices of the MHC molecule. T...

example 3

Analysis of the CDR Loop Residues Most Likely to Contribute to Peptide Binding and Specificity

[0244]In order to identify potential contact and specificity-determining residues, various approaches were used to determine which residues of the A6 CDR loops would be most likely to accommodate, and provide binding energy to, a wide array of peptides in the HLA.A2 peptide-binding groove. First, a panel of other HLA.A2 restricted peptides was modeled into the A6 crystal structure (FIG. 3). Using the A6:Tax peptide:HLA.A2 crystal structure (PDB:1AO7) as a starting point (Garboczi et al. (1996) Nature, 384, 134-141), the Rosetta Backrub modeling program was used to model the HLA.A2 restricted peptides (i.e., Tax, Mart1-9 mer, Mart1-10 mer, SL9 HIV, WT1, and Survivin) into the HLA.A2 groove using Rosetta Backrub flexible backbone modeling algorithms (FIG. 3) ((Lauck et al. (2010) Nucleic Acids Res, 38, W569-75); kortemmelab.ucsf.edu / backrub / ). The peptides that were modeled into the binding g...

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Abstract

The use of model T cell receptors (TCRs) as scaffolds for in vitro engineering of novel specificities is provided. TCRs with de novo binding to a specific peptide-major histocompatibility complex (MHC) product can be isolated by: 1) mutagenizing a T cell receptor protein coding sequence to generate a variegated population of mutants (a library), 2) selection of the library of TCR mutants with the specific peptide-MHC, using a process of directed evolution and a “display” methodology (e.g., yeast, phage, mammalian cell) and the peptide-MHC ligand. The process can be repeated to identify TCR variants with improved affinity for the selecting peptide-MHC ligand.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Patent Application No. 61 / 676,373 filed Jul. 27, 2012, and this provisional application is incorporated herein by reference in its entirety.STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT[0002]This disclosure was made with U.S. Government support under Grant numbers R01 GM55767 and T32 GM070421, awarded by the National Institutes of Health. The U.S. Government has certain rights in the disclosure.FIELD OF THE INVENTION[0003]The disclosure relates to T cell receptor (TCR) scaffolds and TCR libraries, as well as methods of producing modified TCRs and single chain TCRs and the corresponding use of the TCRs for therapeutic, diagnostic, and imaging methods.STATEMENT REGARDING SEQUENCE LISTING[0004]The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C07K14/705
CPCC07K14/70503C07K14/7051C12N15/1037A61P35/00C40B40/02C12N15/85
Inventor SMITH, SHEENA N.KRANZ, DAVID M.
Owner THE BOARD OF TRUSTEES OF THE UNIV OF ILLINOIS
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