| CPC G01N 33/6848 (2013.01) [G01N 33/56977 (2013.01); G01N 33/6842 (2013.01); G01N 33/6878 (2013.01); G01N 2333/70539 (2013.01); G01N 2458/15 (2013.01); G01N 2496/00 (2013.01); G01N 2560/00 (2013.01); G01N 2570/00 (2013.01)] | 16 Claims |
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1. A method of inducing a T cell response in an individual, comprising
obtaining a biological sample comprising target cells over-presenting, overexpressing, and/or selectively presenting at least one MHC peptide ligand on the cell surface from the individual,
quantifying the copy number of the at least one MHC peptide ligand per target cell from the sample,
wherein the quantifying comprises
a) preparing a cell lysate from the target cells over-presenting, overexpressing, and/or selectively presenting said at least one MHC peptide ligand from the biological sample to obtain a preparation A,
b) determining a cell count of said preparation A,
c) adding a pre-determined amount of said at least one MHC peptide ligand labelled with a first isotope in an MHC complex to be quantified to said preparation A (“spiking I”) to obtain a preparation B,
d) isolating the at least one MHC peptide ligand and the at least one MHC peptide ligand labelled with a first isotope from said preparation B to obtain a peptide eluate,
e) adding a pre-determined amount of the at least one MHC peptide ligand labelled with a second isotope to be quantified to said peptide eluate (“spiking II”) to obtain a preparation C,
wherein the first isotope is different from the second isotope,
f) performing a mass spectrometry analysis on said at least one MHC peptide ligand in the preparation C to generate at least one
fa) signal for said at least one MHC peptide ligand labelled with the first isotope isolated from the preparation B to calculate efficiency of said isolating,
fb) signal for the pre-determined amount of said at least one MHC peptide ligand labelled with the second isotope as added to the preparation C, and
fc) signal for said at least one MHC peptide ligand from said preparation A,
f1) performing a mass spectrometry analysis on serially titrated amounts of said at least one MHC peptide ligand labelled with a third isotope and the pre-determined amount of said at least one MHC peptide ligand labelled with a fourth isotope as an internal standard to generate
f1a) signal for the serially titrated amounts of said at least one MHC peptide ligand labelled with a third isotope, and
f1b) signal for the pre-determined amount of said at least one MHC peptide ligand labelled with a fourth isotope,
wherein the third isotope is different from the fourth isotope,
f2) plotting a ratio of the signal of f1a)/the signal of f1b) to generate a calibration curve, wherein a lower limit of quantitation (LLOQ) is determined when the ratio of the signal of f1a)/the signal of f1b) deviates from linearity in the calibration curve, and;
g) quantifying said at least one MHC peptide ligand in the preparation A based on a comparison of the signals as obtained in f) with
ga) the cell count as obtained,
gb) the pre-determined amount of said at least one peptide-MHC ligand labelled with the first isotope and/or peptide-MHC ligand complex labelled with the first isotope to be quantified as added to the preparation B, and
gc) the pre-determined amount of said at least one MHC peptide ligand labelled with a second isotope to be quantified as added to the preparation C,
wherein the quantifying in g) comprises selecting a ratio of the signal of fc)/the signal of fb) at or above the LLOQ and calculating the copy number of the at least one MEW peptide ligand per target cell based on the selected ratio in the calibration curve,
selecting the at least one MHC peptide ligand that has the calculated copy number from 10 to 30000 copies per target cell,
preparing a composition comprising a T cell, wherein the T cell binds to the selected at least one MEW peptide ligand in a complex with an MHC molecule, and
administering the composition to the individual.
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