A novel chimeric PD1-NKG2D-41BB receptor enhances antitumor activity of NK92 cells against human lung cancer H1299 cells by triggering pyroptosis.

Chimeric antigen receptor (CAR)-modified adoptive natural killer (NK) cells represent a promising immunotherapeutic modality for cancer treatment but face many challenges in solid tumors. One major obstacle is the immune-suppressive effects induced by inhibitory receptors (IR) including PD1. To interfere with PD1 signaling to augment CAR-NK cells' activity against solid tumors, we rationally designed a novel chimeric costimulatory converting receptor (CCCR), comprising mainly the extracellular domain of PD1, transmembrane and cytoplasmic domains of NKG2D, and the cytoplasmic domain of 41BB. This NK-tailored CCCR was able to switch the negative PD1 signal to an activating signal and hence reversed the immune suppressive effects of PD1. The CCCR-modified NK92 (CCCR-NK92) cells retained typical characteristics of NK cells and exhibited enhanced antitumor activity against human lung cancer H1299 cells in vitro compared with untransduced NK92 cells. The rapid clearance of H1299 cells was caused by CCCR-NK92 cell-induced extensive pyroptosis. In a lung cancer xenograft model, CCCR-NK92 cells significantly inhibited tumor growth. Our results highlight a promising immunotherapeutic potential of using NK-tailored CCCR engineered NK92 cells to treat human lung cancer.

A novel bispecific chimeric PD1-DAP10/NKG2D receptor augments NK92-cell therapy efficacy for human gastric cancer SGC-7901 cell.

Cell-based immunotherapy continues to be a promising avenue for cancers that standard therapy has failed. Although the specificity, avidity, and efficacy of infused cells have improved, immunocytotherapy still faces substantial hurdles. To this end, we developed a structure-based rational design approach and constructed a novel Dual Targeting Chimeric Receptor (DTCR) PD1-DAP10/NKG2D comprising the truncated ectodomain of PD1 fused to a key co-stimulatory receptor DAP10, and subsequently harnessed the activating receptor NKG2D, which evaluated the capacity of solid tumor cell killing. Retroviral transduction of DTCR dramatically increased NK92 cell surface expression of PD1 and NKG2D, which boosted robust cytotoxicity against human gastric cell SGC-7901. Chimeric receptor DTCR stimulation elicited a significant increase of TNF-α and TRAIL, which can trigger apoptosis of SGC-7901 cells. More importantly, DTCR-NK92 cells had considerable antitumor activity in the solid tumor cell SGC-7901-bearing mice model. Collectively, we demonstrated that expression of DTCR markedly augmented the cytotoxic potential of NK92 cells against solid tumor cells, and this potentially promising treatment modality will facilitate clinical translation of potent NK-tailored chimeric receptor strategy for a generalized cellular therapy that may be conducive to treat a wide range of solid tumors.

Structure-based rational design of a novel chimeric PD1-NKG2D receptor for natural killer cells.

Chimeric antigen receptor (CAR)-engineered natural killer (NK) cells have the potential to provide the potential for the implementation of allogeneic "off-the-shelf" cellular therapy against cancers. Currently, most CARs are not optimized for NK cells, so new NK-tailored CARs are needed. Here, a major activating receptor of NK cells, NKG2D was harnessed to design different chimeric receptors that mediate strong NK cell signaling. In these NKG2D signaling-based chimeric receptors, the extracellular domain of inhibitory receptor PD-1 was employed to reverse the immune escape mediated by PD-1 ligands in the solid tumors. To achieve the rational design of chimeric PD1-NKG2D receptors, we developed a transmembrane protein tertiary structure prediction program (PredMP & I-TASSER) and optimized the conformation of the PD-1 ectodomain by genetically altering the sequences encoding the hinge and intracellular domain. Finally, we identified a chimeric PD1-NKG2D receptor containing NKG2D hinge region and 4-1BB co-stimulatory domain to exhibit stable surface expression and mediate in vitro cytotoxicity of NK92 cells against various tumor cells. This strategy now provides a promising approach for the computer-aided design (CAD) of potent NK cell-tailored chimeric receptors with NKG2D signaling.

Utilizing VEGF165b mutant as an effective immunization adjunct to augment antitumor immune response.

Compelling evidence has shown that blocking VEGF via monoclonal antibodies may be beneficial in that it not only inhibits tumor angiogenesis but also reduces immune suppression and promotes T cell infiltration into tumors. Herein, we determined whether our recently generated VEGF165b mutant could be used as a co-immunization adjunct to augment the peptide cancer-vaccine- induced immune response in a mouse model of breast cancer. When co-immunized mVEGF165b with the peptide-based cancer vaccine (MUC1, a T-cell epitope dominant peptide vaccine from Mucin1), the VEGF antibody titers increased approximately 600,000-fold in mice. Moreover, the anti-VEGF antibody also reduced the frequency of regulatory T cells (Tregs) in both preventive and therapeutic scenarios. Mechanistically, the decrease of the Tregs population was associated with a remarkably increased MUC-1-specific IFN-γ-producing CD8+ T cells and anti-MUC1 humoral response. Finally, this combination co-immunization produced a superior antitumor response and significantly prolonged survival of tumor-bearing mice. In conclusion, our findings suggest that mVEGF165b may be an ideal immunization adjunct to enhance the immune efficacy of peptide-based tumor vaccines by overcoming immune tolerance.

VEGF165b mutant with a prolonged half-life and enhanced anti-tumor potency in a mouse model.

VEGF165b has been shown to be an effective anti-cancer agent; however, its short half-life limits further application in the clinical field. The development of a mutant VEGF165b with a prolonged half-life is urgently needed for its future application. A mutant VEGF165b was generated by inactivation of its plasmin cleavage site. The mutant and native VEGF165b proteins without purification tags were expressed via the Pichia pastoris expression system followed by purification with a HiTrap heparin affinity chromatography column through optimization of the purification conditions. Furthermore, its binding affinity with VEGF Receptors and its functions in vitro and in vivo were examined. Results showed that the half-life of mutant VEGF165b increased to approximately 10 times (Intravenous), 9.1 times (Intraperitoneal) and 5.4 times (Subcutaneous) greater than that of VEGF165b, and the mutation did not cause significant alteration of VEGFR1 and VEGFR2 binding affinity. Mutant VEGF165b inhibited the proliferation and migration of HUVECs in vitro, similar to the native VEGF165b. In a mouse melanoma model, mutant VEGF165b exhibited stronger anti-tumor activity in comparison with its native counterpart. These results indicate that the mutant VEGF165b had a prolonged half-life and retained the anti-angiogenic activity of the native VEGF165b, suggesting that this novel mutant VEGF165b may be a stronger anti-cancer agent.

[Over-expressed HER2 enhances proliferation, migration and invasion of melanoma B16 cells].

Objective To establish a melanoma B16 cell line stably over-expressing the HER2 (ErbB2) gene and study its effect on the proliferation, migration and invasion of melanoma cells. Methods The recombinant plasmid pCMV3-ErbB2 was transfected into B16 melanoma cell line by cationic liposome. The positive clones were screened with hygromycin B. Real-time quantitative PCR (qRT-PCR) was utilized to determine the expression of ErbB2 mRNA. Immunofluorescence was used to evaluate the expression of ErbB2 protein. MTT assay was applied to detect the cell proliferation ability. The cell migration ability was examined by a scratch assay. TranswellTM assay was performed to determine the effect of HER2 over-expression on the capability of B16s invasion. Results The cell line over-expressing HER2 was successfully established as showed by qRT-PCR. Over-expression of HER2 remarkably enhanced the proliferation, invasion and migration of B16 cells. Conclusion Over-expression of HER2 can enhance the growth and invasion of B16 cells.