Methods and compositions for determining the antigen specificity of t cells
AssigneesThe Broad Institute, Inc., The General Hospital Corporation
InventorsNir Hacohen, Matthew Bakalar
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Abstract
The present disclosure provides methods and compositions for determining the antigen specificity of T cells and in a scalable, high-throughput approach. The disclosure provides methods for producing RNA-barcoded pMHC multimers that can be decoded using single-cell RNA sequencing methods. Among these, disclosed herein are multivalent virus-like-particles bound with pMHC in E. coli cells that encapsulate an RNA barcode encoding the peptide identity.
Claims105
A method for producing complexes between an antigen peptide and a major histocompatibility complex (MHC), comprising:
- (a) providing an Escherichia coli cell under conditions suitable for expression, wherein the E. coli cell comprises one or more sequences encoding a MHC, one or more sequences encoding one or more heterologous oxidation enzymes, and a sequence encoding an antigen peptide;
- and
- (b) isolating complexes between the antigen peptide and MHC (pMHC) from the E. coli cell.
A viral-like particle (VLP) conjugated to a pMHC, comprising:
- (a) a VLP comprised of a self-assembling coat protein;
- (b) a major histocompatibility complex (MHC);
- and
- (c) an antigen peptide complexed with the MHC (pMHC),
- wherein the self-assembling coat protein and MHC are modified by fusion to a binding protein and binding peptide, respectively, and
- wherein the binding protein conjugates to the binding peptide.
A method for producing viral-like particles (VLPs) conjugated to pMHC, comprising:
- (a) providing an E. coli cell under conditions suitable for expression, wherein the E. coli cell comprises one or more sequences encoding a major histocompatibility complex (MHC) modified by fusion with a binding peptide, one or more sequences encoding one or more heterologous oxidation enzymes, a sequence encoding an antigen peptide, and a sequence encoding a self-assembling coat protein modified by fusion with a binding protein capable of conjugating to the binding peptide;
- and
- (b) isolating VLPs conjugated to complexes between the antigen peptide and MHC (pMHC) from the E. coli cell.
A method for identifying pMHC-TCR pairs, comprising:
- (a) providing an E. coli cell under conditions suitable for expression, wherein the E. coli cell comprises one or more sequences encoding a major histocompatibility complex (MHC) modified by fusion with a binding peptide, one or more sequences encoding one or more heterologous oxidation enzymes, a sequence encoding an antigen peptide, and a sequence encoding a self-assembling coat protein modified by fusion with a binding protein capable of conjugating to the binding peptide;
- (b) isolating virus-like particles (VLPs) conjugated to complexes between the antigen peptide and MHC (pMHC) from the E. coli cell;
- (c) contacting the isolated VLPs with a population of T cells;
- (d) sequencing the population of T cells;
- and
- (e) determining pMHC-T cell receptor (TCR) cognate pairs by identifying TCR-encoding sequences and antigen peptide-encoding sequences comprised by each T cell within the population of T cells.
Dependencies are inferred from the claim text.