A practical strategy to reconstitute the Fc functions of nanobody was developed by nanobody C-terminal dinitrophenylation. The Fc functions are successfully reinstated as proved by the potent ADCC and CDC in vitro and anti-tumor efficacies in vivo.
Cancer immunotherapy based on natural killer (NK) cells is demonstrated to be a promising strategy. However, NK cells are deficient in ligands that target specific tumors, resulting in limited antitumor efficacy. Here, a glycoengineering approach to imitate the chimeric antigen receptor strategy and decorate NK cells with nanobodies to promote NK‐based immunotherapy in solid tumors is proposed. Nanobody 7D12, which specifically recognizes the human epidermal growth factor receptor (EGFR) that is overexpressed on many solid tumors, is coupled to the chemically synthesized DBCO‐PEG4‐GGG‐NH2 by sortase A‐mediated ligation to generate DBCO‐7D12. The NK92MI cells bearing azide groups are then equipped with DBCO‐7D12 via bioorthogonal click chemistry. The resultant 7D12‐NK92MI cells exhibit high specificity and affinity for EGFR‐overexpressing tumor cells in vitro and in vivo by the 7D12‐EGFR interaction, causing increased cytokine secretion to more effectively kill EGFR‐positive tumor cells, but not EGFR‐negative cancer cells. Importantly, the 7D12‐NK92MI cells also show a wide anticancer spectrum and extensive tumor penetration. Furthermore, mouse experiments reveal that 7D12‐NK92MI treatment achieves excellent therapeutic efficacy and outstanding safety. The authors’ works provide a cell modification strategy using specific protein ligands without genetic manipulation and present a potential novel method for cancer‐targeted immunotherapy by NK cells.
BackgroundSortase A (SrtA) is a transpeptidase found in Staphylococcus aureus, which is widely used in site-specific protein modification. However, SrtA was expressed in Escherichia coli (E. coli) in rather low level (ranging from several milligrams to 76.9 mg/L at most). The present study aims to optimize fermentation conditions for improving SrtA expression in E. coli.ResultsUnder the optimized media (0.48 g/L glycerol, 1.37 g/L tryptone, 0.51 g/L yeast extract, MOPS 0.5 g/L, PBS buffer 180 mL/L) and condition (30 °C for 8 h) in a 7-L fermentor, the enzyme activity and the yield of SrtA reached 2458.4 ± 115.9 U/mg DCW and 232.4 ± 21.1 mg/L, respectively, which were higher by 5.8- and 4.5-folds compared with initial conditions, respectively. The yield of SrtA also represented threefold increase than the previously reported maximal level. In addition, the enzymatic characterizations of SrtA (optimal temperature, optimal pH, the influence of metal irons, and tolerance to water-soluble organic solvents) were determined.ConclusionsEnhanced expression of SrtA was achieved by optimization of medium and condition. This result will have potential application for production levels of SrtA on an industry scale. Moreover, the detailed enzymatic characterizations of SrtA were examined, which will provide a useful guide for its future application.Electronic supplementary materialThe online version of this article (doi:10.1186/s40643-017-0143-y) contains supplementary material, which is available to authorized users.
Developing monoclonal antibodies (mAbs) for cancer immunotherapy is expensive and complicated. Nanobodies are small antibodies possessing favorable pharmacological properties compared with mAbs, but have limited anticancer efficacy due to the...
Rhamnose and sTn antigen were co-conjugated to bovine serum albumin (BSA), a weakly immunogenic carrier protein, for cancer vaccine development. The immune responses against sTn have been significantly augmented with...
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