JP-2022531357-A5 -

Dates

Publication Date

20230512

Application Date

20200501

Description

A five-parameter nonlinear regression model determined that the IC50 of the denatured IMP761 antibody (74 ng/ml and 81.5 ng/ml after 10 and 20 minutes at 70°C, respectively) was higher than that of the reference IMP761 antibody stored at 4 °C (41 ng/ml). The claims at the time of filing were as follows: [Claim 1] An in vitro assay for determining the activity of lymphocyte activation gene 3 (LAG-3) agonists, A step of supplying multiple effector T cells, wherein each effector T cell expresses LAG-3 and a T cell receptor (TCR) on its surface and contains a reporter gene encoding a reporter, and the expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; An in vitro assay comprising the step of determining the activity of the agonist from the extent to which the expression of the reporter changes in the presence of the agonist compared to the expression of the reporter in the absence of the agonist. [Claim 2] The assay according to claim 1 for determining the efficacy of a preparation of LAG-3 agonist. [Claim 3] An in vitro assay for screening LAG-3 agonists, A step of supplying multiple effector T cells, wherein each effector T cell expresses LAG-3 and a T cell receptor (TCR) on its surface and contains a reporter gene encoding a reporter, and the expression of the reporter is regulated by LAG-3-mediated inhibition of TCR signaling within the effector T cell; An in vitro assay comprising the step of determining whether a candidate agonist is an agonist of LAG-3 by determining the extent to which the expression of the reporter changes in the presence of the candidate agonist compared to the expression of the reporter in the absence of the candidate agonist. [Claim 4] The assay according to claim 1 or 2, wherein the reporter is expressed at a basal level in effector T cells in the absence of the agonist, or the assay according to claim 3, wherein the reporter is expressed at a basal level in effector T cells in the absence of the candidate agonist. [Claim 5] The assay according to any one of claims 1 to 4, wherein the expression of the reporter is reduced in the presence of the agonist or candidate agonist compared to the expression of the reporter in the absence of the agonist or candidate agonist. [Claim 6] The expression of the reporter changes in each effector T cell in response to the activation of the effector T cells via the TCR. The steps include activating the effector T cells by antigen-independent, MHC class II-independent, and TCR-mediated T cell activation in the presence and absence of the agonist or candidate agonist, The assay according to any one of claims 1 to 5, further comprising the step of determining the activity of the agonist or candidate agonist from the extent to which the expression of the reporter changes in response to the activation of the effector T cells in the presence of the agonist or candidate agonist, compared to the expression of the reporter in response to the activation of the effector T cells in the absence of the agonist or candidate agonist. [Claim 7] The assay according to claim 6, wherein the expression of the reporter increases in each effector T cell in response to the activation of the effector T cell via the TCR, decreases in the presence of the agonist or candidate agonist as a result of LAG-3-mediated inhibition of TCR signaling within the effector T cell, and the activity of the agonist or candidate agonist is determined by the extent to which the expression of the reporter in response to the activation of the effector T cell decreases in the presence of the agonist or candidate agonist compared to the expression of the reporter in response to the activation of the effector T cell in the absence of the agonist or candidate agonist. [Claim 8] The assay according to claim 6 or 7, wherein the effector T cells are activated by contacting the effector T cells with an antigen-independent, MHC class II-independent T cell activator under conditions of antigen-independent, MHC class II-independent TCR-mediated activation of the effector T cells by the activator. [Claim 9] The assay according to claim 8, wherein the effector T cells are contacted with the T cell activator at a concentration of the T cell activator that causes maximum inhibition of reporter expression in the presence of an excess of LAG-3 agonist. [Claim 10] The assay according to claim 8 or 9, wherein the effector T cells are contacted with the T cell activator in the absence of the agonist or candidate agonist, at a concentration of the T cell activator lower than the concentration of the T cell activator at which maximum expression of the reporter is observed in response to activation of the effector T cells. [Claim 11] The assay according to any one of claims 8 to 10, wherein the T cell activator comprises or consists of an anti-CD3 antibody, or a fragment or derivative thereof that retains antigen-independent, MHC class II-independent, TCR-