Asparaginyl endopeptidases (AEP) are ideal for peptide and protein labeling. Its pairing with a simple chemical reaction significantly lowers the amount of label needed for effective bioconjugation.
In conclusion, IL-2 and TNF-a coding adenoviruses can break tumor-associated immunotolerance and significantly increase the levels of active, tumor-reactive T-cells both in injected cutaneous lesions and in non-injected metastatic lymph nodes. Importantly, this triple modality may represent an efficient approach to achieve "CD19-like" clinical responses in the treatment of solid, metastatic cancers currently incurable by standard therapies.
The synthesis of proteins by solid-phase chemical ligation (SPCL) suffers from paucity of linkers that can be cleaved under mild conditions. Here, we deployed the spontaneous nickel-assisted cleavage (SNAC) tag,...
S254in vivo. We therefore characterized the CD19CAR T cells after ex vivo expansion. We found that 40% of AKT-inhibited CD19CAR T cells expressed CD62L and co-expressed CD28. In contrast, only 10% of control untreated CD19CAR T cells expressed CD62L and they were CD28 negative, indicating that AKT-inhibited CD19CAR T cells may have superior anti-tumor activity following adoptive transfer. To test the potency of the AKT inhibitor treated CAR T cells, 0.5x10 6 CD19+ acute lymphoid leukemic cells (SupB15) that were engineered to express firely lucifierase were inoculated intravenously into NOD/Scid IL-2RgammaCnull (NSG) mice. Five days post tumor engraftment, 2x10 6 CD8+ CD19CAR T cells were intravenously injected into tumor bearing mice. Control mice received either no T cells, non-transduced T cells (Mock), or CD19CAR T cells that were not treated with AKT inhibitor during in vitro expansion. Tumor signals post T cells infusion were monitored by biophotonic imaging. In contrast to the untreated CD19CAR T cells, which exhibited lower and transient anti-tumor activity, AKT-inhibited CD19CAR T cells completely eradicated the CD19 + tumor in all mice (Figure 1) 21 days post CD19CAR T cell infusion. In conclusion, inhibition of AKT signaling during the ex vivo priming and expansion gives rise to a CD19CAR T cell population that possesses superior antitumor activity. These findings suggest that therapeutic modulation of AKT might be a strategy to augment antitumor immunity for adoptive CAR T cell therapy, which could easily be transitioned into the clinic with the availability of pharmaceutical grade AKT inhibitor.
Transpeptidases are ideal biocatalysts for site-specific peptide and protein labeling, whereas reactions that target N-terminus cysteine with commercially available reagents have become common practice. However, a versatile approach that allows bioconjugation at the terminus of choice (N or C), while avoiding the use of backbone-modified substrates (<i>e.g.</i> depsipeptide) or large excess of reagent, is highly desirable. Aiming to meet these benchmarks, we have combined the advantages of asparaginyl endopeptidase (AEP) catalysis with a N-terminal cysteine trapping reaction and created a chemo-enzymatic labeling system. In this approach, polypeptide with a Asn-Cys-Leu recognition sequence are ligated with a counterpart possessing an N-terminal Gly-Leu by AEP; the byproduct Cys-Leu is subsequently trapped by a stable and inexpensive scavenger, 2-formyl phenylboronic acid (FPBA), to yield an inert thiazolidine derivative, thereby driving the reaction forward to product formation. By carefully screening the reaction conditions for optimal compatibility and minimal hydrolysis, conversion to the ligated product in the model reaction resulted in excellent yields. The versatility of this AEP ligation/FPBA coupling system was further demonstrated by site-specific labeling the N- or C-termini of various proteins.
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