Methods for assaying t-cell dependent bispecific antibodies

Abstract

The present invention provides a cell-based assay for measuring T cell activation mediated by a T cell-dependent bispecific antibody (TDB). In some aspects, the assay is useful for detecting a TDB in a composition, quantitating the amount of TDB in a composition, determining the potency and/or specificity of a TDB, or determining if a population of cells expresses a target antigen. Compositions and kits are also contemplated.

1 .- 78 . (canceled) 
     
     
         79 . Method for determining the specificity of a T cell dependent bispecific antibody (TDB), wherein the TDB comprises a target antigen binding fragment and a CD3 binding fragment, the method comprising
 a) contacting a population of T cells and test cells with the TDB, wherein the T cells comprise nucleic acid encoding a reporter operably linked to a response element that is responsive to T cell activation, and wherein the test cells do not express the target antigen;   b) contacting a population of T cells and test cells with the TDB, wherein the T cells comprise nucleic acid encoding a reporter operably linked to a response element that is responsive to T cell activation, and wherein the test cells do not express the target antigen;   comparing expression of the reporter in the presence of the test cell in part a) with expression of the reporter in the presence of target cells in part b), wherein the ratio of expression of the reporter of the test cells to the target cells is indicative of the specificity of the TDB.   
     
     
         80 . The method of  claim 79 , wherein the reporter is a luciferase, a fluorescent protein, an alkaline phosphatase, beta lactamase, or a beta galactosidase. 
     
     
         81 . The method of  claim 80 , wherein the luciferase is a firefly luciferase, a  Renilla  luciferase, or a nanoluciferase. 
     
     
         82 . The method of  claim 79 , wherein the response element that is responsive to T cell activation is an NFAT promoter, an AP-1 promoter, an NFκB promoter, a FOXO promoter, a STAT3 promoter, a STAT5 promoter or an IRF promoter. 
     
     
         83 . The method of  claim 82 , wherein the response element that is responsive to T cell activation comprises T cell activation responsive elements from any one or more of NFAT, AP-1, NFκB, FOXO, STAT3, STAT5 and IRF. 
     
     
         84 . The method of  claim 79 , wherein the population of T cells is population of CD4 +  T cells or CD8 +  T cells. 
     
     
         85 . The method of  claim 79 , wherein the population of T cells is population of Jurkat T cells or CTLL-2 T cells. 
     
     
         86 . The method of  claim 79 , wherein the target antigen is expressed on the surface of the target cell. 
     
     
         87 . The method of  claim 79 , wherein the target antigen is CD4, CD8, CD18, CD19, CD11a, CD11b, CD20, CD22, CD34, CD40, CD79α (CD79a), CD79β (CD79b), EGF receptor, HER2 receptor, HER3 receptor, HER4 receptor, FcRH5, CLL1, LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, αv/β3 integrin, VEGF, flk2/flt3 receptor; obesity (OB) receptor; mp1 receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7, Tenb2, STEAP, or transmembrane tumor-associated antigens (TAA). 
     
     
         88 . The method of  claim 79 , wherein
 a) the target antigen is HER2 receptor and the target cell is a BT-474 cell,   b) the target antigen is HER2 receptor and the target cell is a SKBR3 cell,   c) the target antigen is CD20 and the target cell is a Wil2-S cell, or   d) the target antigen is CD79b and the target cell is a BJAB cell.   
     
     
         89 . The method of  claim 79 , wherein the ratio of T cells to test cells in the population of cells of step a) and/or the ratio of T cells to target cells in the population of cells of step b) is about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9 or about 1:10. 
     
     
         90 . The method of  claim 79 , wherein the ratio of T cells to test cells in the population of cells of step a) and/or the ratio of T cells to target cells in the population of cells or step b) is about 1:4. 
     
     
         91 . The method of  claim 79 , wherein the population of cells of steps a) and/or b) ranges from about 1×10 3  to about 1×10 6 . 
     
     
         92 . The method of  claim 79 , wherein the population of cells of steps a) and/or b) ranges from about 1×10 4  to about 5×10 4 . 
     
     
         93 . The method of  claim 79 , wherein population of T cells and test cells of step a) and the population of T cells and target cells of step b) are contacted with a composition comprising the TDB at a concentration ranging from about 0.01 ng/mL to about 100 ng/mL. 
     
     
         94 . The method of  claim 79 , wherein the reporter is detected after any one or more of 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 20 or 24 hours after contacting the cells with the composition.