Bicistronic chimeric antigen receptors and their uses

[0300] This example demonstrates the production of CAR constructs, lentiviral vectors encoding the CAR constructs, and CAR-expressing T cells according to embodiments of the invention, as well as the production of other CARs for comparison.

[0301] The CAR construct was synthesized by GENEWIZ (South Plainfield, NJ, USA) and then subcloned into the lentiviral plasmid backbone between the NhE1 site and the HincII site.

[0302] Lentiviral vectors encoding CAR constructs were generated by transient transfection of the 293T cell line. Briefly, 293T cells were seeded into poly-D-lysine-coated 15 cm plates (BD Biosciences, San Jose, CA, USA). The next day, plasmids encoding the CAR constructs together with packaging and envelope vectors (pMDLg / pRRE, pMD-2G, and pRSV-Rev) were transfected into 293T using lipofectamine 3000 (Life Technologies, Carlsbad, CA, USA). cell. Lentiviral supernatants were collected 48-72 hours after transfection, centrifuged at 3000 RPM for 10 minutes to r...

[0311] This example demonstrates surface expression on human T cells of a CAR cleaved from a CAR construct according to an embodiment of the invention compared to other CARs.

[0312] The surface expression of anti-CD19 CAR and anti-CD22 CAR on V1-transduced T cells was about 15%, while the expression of anti-CD19 CAR from vectors encoding only a single anti-CD19 CAR was 61%, and that from vectors encoding only a single anti-CD22 The expression of the anti-CD22 CAR of the CAR vector was 56% ( Figures 2A-2C ).

[0313] Human PBMC from healthy donors were activated for 24 hours with CD3 / CD28 microbeads. Activated T cells were then transduced with the vector alone or co-transduced with both a single anti-CD19 CAR and a single anti-CD22 CAR vector. Surface expression of anti-CD19 CAR and anti-CD22 CAR was analyzed on day 8. Co-transduced T cells had much lower expression of both anti-CD19 and anti-CD22 CARs compared to the bispecific cyclic CAR6. Anti-CD19 and anti-CD22 CARs ...

[0316] This example demonstrates the in vitro activity of CAR constructs according to embodiments of the present invention compared to other CARs based on cytokine production.

[0317] CAR-transduced T cells (1E5) were washed 3 times with 1×PBS, and then co-incubated with an equal amount of target cells in 200ml RPMI medium in a 96-well plate in a 37°C incubator for 15 to 20 hours. Target cells were K562 expressing CD19 or CD22 or both CD19 and CD22. K562 cells were used as a negative control. Cytokines levels of IL2 and IFNγ in culture supernatants were measured with ELISA kits (R&D Systems, Minneapolis, MN, USA). All tests were set up in triplicate. V1 CAR T cells produced large amounts of IL2 and IFNg when co-cultured with CD22-expressing target cells, but produced only low levels of IL2 and IFNg when co-cultured with CD19-expressing target cells ( Figure 5A and 5B ).

[0318] The CML cell line K562 was artificially transduced with CD19 or CD22 or both to express the ...