Treating cancer by blocking the interaction of tim-3 and its ligand
A TIM-3, cancer technology, applied in the direction of antibody medical components, antibodies, pharmaceutical formulations, etc., can solve the problems of data inconsistency and unreliability
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0224] Example 1. Generation of Cell Lines Overexpressing GAl3
[0225] The mouse B-lymphoma cell line A20 obtained from the American Tissue and Cell Culture Collection (ATCC, Manassas, VA) was encoded with either a Flag-tagged human Gal3 protein or a Flag-tagged human PDL1 protein. Transfection of nucleic acid constructs. The construct additionally contains a marker for antibiotic resistance. Transformed cells were selected based on antibiotic resistance to form A20 cells stably expressing Flag-tagged human Gal3 protein (A20 Gal3 cells) or A20 cells stably expressing Flag-tagged human PDL1 protein (A20 hPDL1 cells).
Embodiment 2
[0226] Example 2.GA13 specifically binds to TIM-3
[0227] This example describes various assays that have been performed to assess the interaction between Gal3 and TIM-3.
[0228] Binding assay--co-immunoprecipitation
[0229] Co-immunoprecipitation experiments were performed to test whether TIM-3 specifically interacts with Gal3. 293T cells were co-transfected with plasmids encoding HA-tagged TIM-3 and plasmids encoding Flag-tagged Gal3, Flag-tagged Gal9 or Flag-tagged CEACAM1. Transfections were performed using Lipofectamine 3000 (Waltham, MA) following the manufacturer's protocol. Transfected cells were grown overnight, then washed and lysed in 1 ml lysis buffer. The lysed cells were centrifuged and the supernatant (lysate) collected. The lysates were prepared and separated on SDS PAGE, and were analyzed with anti-HA antibody ( Figure 1A ) and anti-Flag antibody ( Figure 1B ) to detect. Both anti-Flag antibody and anti-HA antibody were purchased from Sigma. Fi...
Embodiment 3
[0243] Example 3. Overexpression of Gal3 inhibits T cell activation
[0244] This example describes experiments performed to assess the functional properties of Gal3 overexpression in A20 cells.
[0245] A20 clones #41, #31 and #15 stably overexpressing hGal3 were generated as described above. Figure 6A Results of flow cytometry analysis showing hGal3 expression levels in these clones are shown. Cells of A20 or A20 Gal3 clones were mixed with mouse DO11.10 T cells. The mixture was placed in each well of a 96-well plate, and OVA323-339 peptide (Invivogen, San Diego, CA) was added to the plate. After overnight incubation, supernatants were used to measure IL-2 production by T cells by ELISA (ThermoFisher Scientific). Such as Figure 6B shows that IL-2 production by mouse DO11.10 T cells was significantly reduced when mixed with any of the three mouse A20 cell clones compared to when T cells were mixed with parental A20 cells ( Figure 6B ).
PUM
| Property | Measurement | Unit |
|---|---|---|
| diameter | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More 


