Sirna against cbl-b and optionally il-2 and il-12 for use in the treatment of cancer
a technology of cbl-b and il-2, which is applied in the field of innate immune system activation therapy, can solve the problems of increased autoimmunity, loss of mhc i complex on the cell surface, and lack of clinically available treatment approaches, so as to improve the immune response efficiency
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Benefits of technology
Problems solved by technology
Method used
Image
Examples
example 2
CBL-B Inhibition in Nk Cells
[0060]Using CPT reaction tubes (Vacutainer) whole blood was obtained from a donor and the PBMCs were separated therefrom by centrifugation. NK cells were isolated from PBMCs (including the CD8+CD3− fraction) and subsequently CD8 and CD4 T cells were isolated by means of magnetic selection. FACS was used to verify that the purity of the corresponding cell populations was at least 90%. Both the PBMCs and the resulting isolated cell fractions were transfected with siRNA Cbl-b using an Amaxa transfection device and stimulated overnight with recombinant human IL-2 (50 ng / ml) and IL-12 (10 ng / ml) in Xvivol5 medium (FIG. 1). The IFN-gamma secretion in the cells treated in this manner was then measured in an ELISA. The result clearly indicates that the greatly enhanced IFN-gamma production of Cbl-b-inhibited PBMCs after the stimulation with IL-2 and IL-12 can be unambiguously assigned to the reaction of the NK cells.
example 3
CBL-B Inhibition in Nk Cells and Co-Stimulation
[0061]One possibility of co-stimulating NK cells is via tumor cell lines whose aberrant surface marker expression is no longer able to maintain the appropriate balance of inhibiting and activating NK receptors, thus leading to the activation of NK cells, for example the tumor cell line K562. PBMCs as well as CD8 and NK cells were isolated as described above and transfected with Cbl-b siRNA. 1×10̂5 of these transfected cells were then incubated in Xvivo medium overnight, either alone (unstim) or with 6×10̂4 of K562 tumor cells, and the secretion of IFN-gamma was determined as described above. The incubation of Cbl-b-inhibited PBMCs with this tumor cell line again resulted in a strong IFN-gamma production, which in turn could clearly be attributed to the contribution of the NK cells (FIG. 2). Furthermore, both stimulation methods resulted in an increase in the TNF-alpha production in NK cells upon inhibition by Cbl-b (FIG. 3).
example 4
Tumor Cytotoxicity by Cbl-B Inhibition in Nk Cells
[0062]One of the main functions of NK cells in the context of tumor development is the direct destruction of tumor cells. It was therefore tested whether Cbl-b-inhibited PBMCs are better suitable for the destruction of tumor cells. The Her2-positive breast carcinoma line SKBR3 was used as a target cell line since in this context it is also possible to test the antibody directed against Her2 (trastuzumab or Herceptin) which is employed in tumor therapy. PBMCs were again isolated as described above, transfected with Cbl-b siRNA and incubated for 4 hours either alone or with 4 x 10̂4 SKBR3 cells in Xvivo medium. In addition, 10 μg / ml of Herceptin antibody were added under the conditions indicated as Herceptin and in the conditions indicated as IL-2 IL-12 the stimulation was carried out as described above. The cytotoxicity of the PBMCs against the SKBR3 tumor cells was then determined by colorimetric measurement of the release of the enz...
PUM
| Property | Measurement | Unit |
|---|---|---|
| anti-tumor reactivity | aaaaa | aaaaa |
| reactivity | aaaaa | aaaaa |
| length | aaaaa | aaaaa |
Abstract
Description
Claims
Application Information
Login to View More