Ssx-2 peptides presented by hla class II molecules

A method for selectively enriching a population of T lymphocytes with CD4+ T lymphocytes specific for a SSX-2 HLA class II-binding peptide comprising:

• contacting an isolated population of T lymphocytes with an agent presenting a complex of the SSX-2 HLA class II-binding peptide and an HLA class II molecule in an amount sufficient to selectively enrich the isolated population of T lymphocytes with the CD4+ T lymphocytes,
• wherein the SSX-2 HLA class II-binding peptide comprises an amino acid sequence set forth as SEQ ID NO:8, or a functional variant thereof comprising 1-5 amino acid substitutions,
• optionally wherein the SSX-2 HLA class II-binding peptide comprises, or preferably consists of an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7,
• optionally wherein the HLA class II molecule is an HLA-DP molecule
• optionally wherein the SSX-2 HLA class II binding peptide comprises an endosomal targeting portion of human invariant chain Ii.

A method for identifying functional variants of a SSX-2 HLA class II-binding peptide, comprising

• selecting a SSX-2 HLA class II-binding peptide which comprises an amino acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7, an HLA class II-binding molecule which binds the SSX-2 HLA class II-binding peptide, and a T cell which is stimulated by the SSX-2 HLA class II-binding peptide presented by the HLA class II-binding molecule;
• mutating a first amino acid residue of the SSX-2 HLA class II-binding peptide to prepare a variant peptide;
• and
• determining the binding of the variant peptide to HLA class II-binding molecule and the stimulation of the T cell, wherein binding of the variant peptide to the HLA class II-binding molecule and stimulation of the T cell by the variant peptide presented by the HLA class II-binding molecule indicates that the variant peptide is a functional variant,
• optionally further comprising a step of comparing the stimulation of the T cell by the SSX-2 HLA class II-binding peptide and the stimulation of the T cell by the functional variant as a determination of the effectiveness of the stimulation of the T cell by the functional variant.

A method for identification of HLA class II-binding epitopes of a protein, comprising:

• obtaining a peptide library of peptides that span the amino acid sequence of the protein;
• and
• contacting a population of cells containing CD4+ T lymphocytes with the peptide library in the presence of antigen presenting cells to stimulate proliferation and/or cytokine production by CD4+ T lymphocytes that selectively bind a peptide in the peptide library;
• wherein the stimulation of CD4+ T lymphocytes indicates that a peptide in the library contains at least one HLA class II epitope,
• optionally wherein the peptides are at least about 12 amino acids in length, preferably wherein the peptides are between about 14 and about 50 amino acids in length, more preferably wherein the peptides are between about 20 and about 22 amino acids in length,
• optionally wherein the peptides overlap each other by at least about 4 amino acids, preferably wherein the peptides overlap each other by at least about 10 amino acids,
• optionally wherein the antigen presenting cells are autologous peripheral blood mononuclear cells,
• optionally further comprising screening the isolated CD4+ T lymphocytes with submixtures or single peptides,
• optionally further comprising isolating the stimulated CD4+ T lymphocytes, preferably wherein the isolation of the stimulated CD4+ T lymphocytes is carried out by cytokine guided flow cytometry cell sorting,
• optionally further comprising clonally expanding the stimulated CD4+ T lymphocytes by periodic stimulation with the selected peptide, and
• optionally wherein the population of cells containing CD4+ T lymphocytes further comprises CD8+ T lymphocytes, and wherein the stimulation of both CD4+ and CD8+ T lymphocytes indicates that a peptide in the synthetic library contains both HLA class I and HLA class II epitopes.