Method for determining potency of antigenic presenting cell based vaccines
View Patent ↗The present invention discloses methods useful in the preparation of immunostimulatory vaccines which include as one of their components, activated antigen presenting cells which are characterized by having an ability to stimulate a therapeutic T cells response in vivo.
1. A method for determining that antigen uptake has occurred for antigen presenting cell (APC) precursors and cell potency is increased to a therapeutically effective level, comprising:
(a) prior to activating the APC precursors, measuring the mean fluorescence intensity (MFI) of the surface marker CD54;
(b) incubating the precursors ex vivo with a conjugate preparation of an antigen and GM-CSF effective to activate the development of the APC precursors to antigen presenting cells (APC's), which is characterized by an ability to stimulate a therapeutic T cell response in vivo;
(c) following the activation, measuring the MFI of the same cell surface marker in activated APC's; and
(d) if the measured MFI of the surface marker following activation increases by an amount that is at least three times the standard deviation of the MFI measured prior to activation, identifying the cells as having been activated to a desired therapeutically effective level.
2. The method according to claim 1 in which the measuring of the MFI of the surface marker in both step (a) and (c) comprises:
(i) staining the cells with an antibody to the surface marker under conditions that allow specific binding; and
(ii) quantifying the MFI of the surface marker.
3. The method according to claim 2 in which the quantifying is achieved by flow cytometry.
4. The method of claim 1 in which the antigen presenting cells are dendritic cells.
5. The method of claim 4 wherein precursors of the dendritic cells are isolated from human peripheral blood, bone marrow, Langerhans cells or tissue dendritic cells.
6. The method of claim 1 wherein the increase in the MFI of CD54 is approximately between 1.5 and 22.0.
7. The method of claim 1 wherein the increase in the MFI of CD54 is approximately 1.75.
8. A method for clinical lot release testing of dendritic cell-based vaccines, comprising:
(a) measuring a first mean fluorescence intensity (MFI) of the cell surface marker CD54 in a sample of dendritic-precursor cells;
(b) exposing the dendritic-precursor cells to a conjugate preparation of an antigen and GM-CSF effective to induce the development of said dendritic-precursor cells;
(c) measuring a second MFI of the same surface marker; and
(d) releasing the lot of dendritic cells if the second MFI of the surface marker increases by an amount that is at least three times the standard deviation of the first measured MFI.
9. The method of claim 8 wherein the antigen is prostatic acid phosphatase.
10. The method of claim 8 wherein the antigen is autologous serum from a patient.