Flow based assays for therapeutics

Abstract

This invention provides methods to evaluate therapeutic efficacy of therapeutic monoclonal antibodies.

What is claimed is: 
     
         1 . A method of analyzing a response to therapeutic antibodies, the method comprising:
 exposing a first aliquot of whole blood sample to a first therapeutic antibody and a second aliquot of whole blood sample to a second therapeutic antibody;   combining exposed blood cells from the whole blood sample with a panel of labeled reporters, the panel of labeled reporters comprising at least two labeled reporters selected from the group consisting of a labeled reporter directed against a target cell depletion surface marker, a labeled reporter directed against a target cell activation surface marker expressed on white blood cell populations, and a labeled reporter directed against cytokine production by white blood cells to thereby simultaneously produce assay signals generated by each for the at least two labeled reporters;   subjecting the whole blood cell sample mixtures to flow cytometry and measuring signals generated by each of the plurality of labeled cells of each aliquot in a flow cytometer; and   combining the measured signals into an assay output for flow cytometric multiparameter analysis to determine a cellular response to the first therapeutic antibody and to the second therapeutic antibody and provide simultaneous differentiation between signals associated with one or more of target cell depletion, activation of white cell subpopulations, and cytokine production by white cells.   
     
     
         2 . The method of  claim 1 , wherein at least one of the first therapeutic antibody and the second therapeutic antibody are in dry form. 
     
     
         3 . The method of  claim 1 , wherein at least one of the panel of labeled reporters are in dry form. 
     
     
         4 . The method of  claim 1 , wherein the target cell is a B cell. 
     
     
         5 . The method of  claim 1 , wherein the first therapeutic antibody and the second therapeutic antibody independently binds to at least one antigen selected from the group consisting of CD20, CD38, CD19, PDL1, and PD1. 
     
     
         6 . The method of  claim 1 , wherein the white cell subpopulations include NK cells. 
     
     
         7 . The method of  claim 1 , wherein the therapeutic antibody binds a cytokine. 
     
     
         8 . The method of  claim 7 , wherein the cytokine is TNFα 
     
     
         9 . The method of  claim 1 , wherein the plurality of labeled reporters includes a cell surface marker and a cytokine. 
     
     
         10 . The method of  claim 1 , wherein the plurality of labeled reporters includes antibodies to CD137 and to CD69. 
     
     
         11 . The method of  claim 1 , wherein the plurality of labeled reporters includes antibodies to INF-7 and to IL8. 
     
     
         12 . The method of  claim 1 , wherein the plurality of labeled reporters includes antibodies to CD66b and CD11c. 
     
     
         13 . The method of  claim 1 , wherein the plurality of labeled reporters includes antibodies to CD203c, CD63, CD3, CRTH2, and CD45. 
     
     
         14 . The method of  claim 1 , wherein:
 the therapeutic antibody binds to at least one antigen to generate a signal associated with target cell depletion, the at least one antigen comprising CD19, CD38, tumor necrosis factor alpha (TNFα), PD-1, PD-L1, CRTH2, or a mixture thereof,   the therapeutic antibody binds to at least one antigen to generate a signal associated with activation of white blood cell subpopulations, the at least one antigen comprising CD69, CD107a, CD54, CD137, CD66b, CD11b, CD11c, TNFα, CD203c, CD16, CRTH2, or a mixture thereof;   the therapeutic antibody binds to at least one antigen to generate a signal associated with cytokine production by white blood cells, the at least one antigen comprising IFNγ, TNFα, IL-6, IL-8, IL-10, or a mixture thereof, or   a mixture thereof.   
     
     
         15 . The method of  claim 1 , wherein the at least two labeled reporters are independently in wet or dry form.