PRODUCTION OF ANTIGEN-SPECIFIC T-CELLS

1 . A method for identifying an antigen-specific T cell Receptor (TCR), comprising:

magnetically enriching and expanding a heterogeneous T cell population with paramagnetic nanoparticles having an MHC-peptide antigen presenting complex on the surface of the nanoparticles,

sorting the expanded T cells with the MHC-peptide ligand, to obtain a T cell population with desired antigen specificity; and

sequencing the TCR genes or portions thereof in the T cell population.

2 - 10 . (canceled)

11 . A method for screening a T cell population for reactivity to a library of antigenic peptides, comprising:

magnetically enriching and expanding antigen-specific T cells in the population with a cocktail of paramagnetic nanoparticles, each having a surface-conjugated MHC-peptide antigen presenting complex that presents an antigenic peptide of interest,

and phenotypically evaluating the expanded T cells.

12 - 23 . (canceled)

24 . A method for expansion of T cells comprising a heterologous or engineered T cell receptor (TCR), comprising:

magnetically enriching and expanding a T cell population comprising T cells expressing a heterologous or engineered T cell receptor (TCR), with paramagnetic nanoparticles having an MHC-peptide antigen presenting complex on the surface thereof that is recognized by the heterologous or engineered T cell receptor (TCR).

25 - 27 . (canceled)

28 . A method for preparing an antigen-specific T-cell population, comprising:

providing a sample comprising T cells from a patient or a suitable donor;

contacting said sample with first nanoparticles which are paramagnetic and comprise on their surface an MHC-peptide antigen-presenting complex, wherein the MHC-peptide complex is prepared by passive loading of MHC-conjugated nanoparticles;

placing a magnetic field in proximity to the paramagnetic nanoparticles,

recovering antigen-specific T cells associated with the paramagnetic particles, and

optionally expanding the recovered T cells in the presence of a magnetic field.

29 - 45 . (canceled)

46 . A method for generating a T cell expressing a chimeric antigen receptor (CAR), comprising:

magnetically enriching and expanding a T cell population with paramagnetic nanoparticles having an MHC-peptide antigen presenting complex on the surface thereof, to thereby prepare an enriched and expanded antigen-specific T cell population; and

transforming the T cell population with a chimeric antigen receptor (CAR).

47 - 63 . (canceled)

64 . A method for expanding a T cell expressing a CAR, comprising:

providing the T cell population expressing a CAR according to claim 46 , and

magnetically expanding the T cell population in the presence of paramagnetic nanoparticles having an MHC-peptide antigen presenting complex on the surface thereof.

65 . (canceled)

66 . A method for treating a patient having cancer, comprising:

administering the CAR-T prepared according to the method of claim 46 , and

administering an artificial antigen presenting cell to the patient, presenting the antigen of interest in complex with MHC, and a lymphocyte costimulatory ligand.

67 . A method for treating a patient having hematological cancer that has relapsed after allogeneic stem cell transplantation, comprising:

providing a sample comprising T cells from a suitable donor;

contacting said sample with nanoparticles which are paramagnetic and comprise on their surface: (1) an MHC-peptide antigen-presenting complex, wherein the MHC-peptide complex is prepared by passive loading of MHC-conjugated nanoparticles (signal 1); and (2) an anti-CD28 co-stimulatory ligand (signal 2);

placing a magnetic field in proximity to the paramagnetic nanoparticles,

recovering antigen-specific T cells associated with the paramagnetic particles,

expanding the recovered T cells; and

administering expanded T cells to the patient.

68 - 76 . (canceled)

77 . A method for preparing a cytotoxic T lymphocyte (CTL) population comprising at least 10 6 CTLs having a central memory or effector memory phenotype, the method comprising:

providing CD8+ T cells isolated from a peripheral blood mononuclear cell (PBMC) sample from a patient or donor, the CD8+ cells being isolated by positive or negative selection,

contacting said sample with paramagnetic nanoparticles having a size in the range of about 10 to about 250 nm, which comprise on their surfaces MHC Class I-peptide antigen-presenting complexes and lymphocyte co-stimulatory ligands, wherein the MHC Class I-peptide antigen presenting complex ligands and the lymphocyte co-stimulatory ligands are present on the same of different nanoparticles;

placing a magnetic field in proximity to the paramagnetic nanoparticles for about 5 minutes,

separating a magnetic fraction from a non-magnetic fraction, and

expanding the magnetic fraction in culture for 2 to 3 weeks to prepare the CTL population.

78 . The method of claim 77 , wherein the MHC Class I-peptide antigen-presenting complex ligands present at least two tumor associated antigens.

79 . The method of claim 78 , wherein the tumor-associated antigens are AML-associated antigens.

80 . The method of claim 79 , wherein the AML-associated antigens include one or more selected from: Survivin, WT-1, PRAME, RHAMM, and PR3.

81 . The method of claim 77 , wherein the lymphocyte co-stimulatory ligands are B7.1 or an activating antibody against CD28.

82 . The method of claim 77 , wherein the MHC Class I-peptide antigen presenting complexes are HLA-Ig ligands.

83 . The method of claim 77 , wherein the CTL population comprises at least 10 8 CTLs.

84 . The method of claim 77 , wherein the sample comprises at least about 10 6 CD8-enriched cells.

85 . The method of claim 77 , wherein the paramagnetic nanoparticles are dextran-coated iron oxide nanoparticles.

86 . The method of claim 77 , wherein the CTL population is at least 10% specific for the peptide antigen(s).