Happy to share our new study in @natBME describing our “Zip-sort” strategy to purify dual-transduced cells with larger transgene payloads. Thanks to all our collaborators. @Parkerici @Cityofhope @MSKCancerCenter @Bushmanlab @VardhanaLab @KlebanoffLab 1/.

RT @KlebanoffLab: 🚨Today, in @NatureCancer, we show that CAR-T cells and CAR-NK cells contain the seeds of their own self-destruction. Disr….

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RT @KlebanoffLab: 🚨Today in @Nature, we show that mRNA mis-splicing creates clonal public neoantigens in solid cancers. An amazing collabor….

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In summary, our proof of principle study demonstrates that Zip-sorted T cells co-expressing multiple CARs and switch receptors can eliminate tumor populations with high target antigen diversity while resisting exhaustion and inhibitory ligand-mediated suppression. 14/

However, co-expression of switch receptors promoted T cell expansion in vivo and improved anti-leukemia activity of quad-CAR T cells. 13/

To attempt to overcome greater antigen heterogeneity, we engineered T cells co-expressing four CARs (quad-CAR). While quad-CAR T cells lysed antigen-expressing targets in vitro, in vivo activity against a mixture of four antigen-expressing leukemia populations was limited. 12/.

Dual-CAR triple-Switch T cells retained enhanced cytokine secretion and lytic capacity following serial encounter with leukemia cells heterogeneously expressing target antigens and inhibitory ligands. 11/

Co-expression of multiple switch receptors attenuated transcriptomic signatures of exhaustion observed in dual-CAR T cells and promoted expression of gene pathways associated with T cell proliferation and metabolism. 10/

Multi-CAR multi-Switch receptor T cells eliminated mixtures of leukemia cells heterogeneously expressing target antigens and inhibitory ligands in vivo. 9/

The enhanced packaging capacity of two vectors allowed us to co-express multiple CARs and switch receptors to engineer T cells to receive positive stimulation from multiple tumor-associated antigens and inhibitory ligands. 8/

Our primary goal was to use Zip-sort to co-express multiple transgenes in T cells to simultaneously overcome multiple CAR T cell failure modes (antigen loss escape, T cell exhaustion, inhibitory receptor signaling). 7/

In collaboration with @Bushmanlab, we found that while Zip-sorted T cells showed about two-fold increased vector integration compared with single-transduced cells, there was no increased clonal expansion or overall differences in vector integration patterns. 6/

Zip-sorting facilitates engineering of cells incorporating multiple transgenes, uses clinically translatable immunomagnetic sorting principles, and can purify cells across a range of initial transduction efficiencies. 5/

However, incorporation of a linked “blocking zipper” inhibited extracellular zipper pairing and restored selective purification of dual-transduced cells. 4/

While our initial Zip-sort configuration enabled enrichment of dual-transduced cells using capture-zippers with short extracellular spacers, when we incorporated larger spacers we observed non-selective extracellular zipper pairing. 3/

By co-expressing a heterodimerizing leucine zipper pair comprising a membrane-bound capture-zipper and an affinity-tagged secreted-zipper (each on one of two vectors), we can achieve selective, single-step immunomagnetic purification of dual-transduced cells. 2/