Methods for assaying t-cell dependent bispecific antibodies
A bispecific antibody-dependent technology, applied in chemical instruments and methods, biochemical equipment and methods, microbial determination/inspection, etc., can solve highly variable and time-consuming problems
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Embodiment 1
[0248] Example 1: T Cell Activation Assay
[0249] A T cell activation assay has been developed to determine the potency and specificity of T cell dependent bispecific (TDB) antibodies with respect to activating T cells in the presence of target cells. For an illustrative schematic see figure 2 . Since TDB is bivalent and bispecific, with one arm specific for a TCR complex subunit and the other arm specific for the target antigen, crosslinking of the TCR leading to T cell activation can only occur in the target region. Occurs when both cells and T cells are bound by TDB. TCR-mediated cross-linking by anti-CD3-containing TDBs activates T cell signaling cascades, leading to phosphorylation and nuclear localization of transcription factors, including NFAT and NFκB, resulting in target genes such as cytokines or cytocidal agents (Brown, WM, 2006, Curr Opin Investig Drugs 7:381-388; Ferran, C et al., 1993, ExpNephrol 1:83-89; Shannon, MF et al., 1995, J Leukoc Biol 57:767-773;...
Embodiment 2
[0253] Example 2: Quantitative method for detecting TDB-mediated T cell activation
[0254] A sensitive and quantitative platform TDB cell-based assay has been developed to determine the potency of anti-CD3-containing TDB by measuring the induction of T cell activation in the presence of target cells. This TDB T cell activation assay detects activation of T cells by TDB in the presence of target cells by measuring TCR crosslink-induced activation of the Rel / NFκB signaling pathway using the engineered T cell reporter gene cell line Jurkat-NFκBluc. Activated NFκB translocates to the nucleus, binds to eight NFκB-responsive elements in a synthetic promoter and drives transcription of luciferase.
[0255] In this assay, dilutions of anti-CD20 / CD3 (or anti-HER2 / CD3 or anti-CD79b / CD3) assay standards, controls, and test samples were prepared and 50 μL added to a 96-well assay plate. The ready-to-use (R-to-U) frozen or cultured cells were then used to prepare target cells (for αCD20 / ...
Embodiment 3
[0312] Example 3: Analysis of anti-FcRH5 / anti-CD3 antibodies
[0313] The potency of the anti-FcRH5 / anti-CD3 TBD was measured using the assay described in Example 2. In this assay, dilutions of anti-FcRH5 / CD3 assay standards, controls, and test samples were prepared and 50 μL was added to a 96-well assay plate. Target cells (FcRH5-expressing EJM cells) and JurkatNFkB reporter cells were then prepared, either frozen or cultured using ready-to-use (R-to-U) following assessment of frozen versus freshly cultured cells. Combine equal volume 4.0x 10 5 cells / mL target cell dilution and 1.6x 10 6 cells / mL JurkatNFkB cell dilution to prepare a cell mixture with a target:effector (T:E) cell ratio of 1:4. 50 μL of mixed target and JurkatNFkBLuc cells were added to each TDB dilution in the assay plate. Use the same T:E ratio for reference and control TDB. After 4-5 hours of assay incubation, the amount of luciferase activity induced by each sample was measured using a luminescent pla...
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