Manufacturing NKG2D CAR-T cells with piggyBac transposon vectors and K562 artificial antigen-presenting cells.

Non-viral platforms can be applied rapidly and cost-effectively for chimeric antigen receptor (CAR)-T cell manufacturing. In the present paper, we describe in detail a clinically relevant manufacturing process for NKG2D CAR-T cells through electroporation of CAR-encoding piggyBac transposon plasmids and in vitro expansion with K562 artificial antigen-presenting cells. With an optimized protocol, we generated the final cell therapy products with 89.2% ± 10.2% NKG2D CAR-positive cells and achieved the corresponding antigen-dependent expansion between 50,000 and 60,000 folds within 4 weeks. To facilitate repeated CAR-T cell infusions, we evaluated the practicality of cryopreservation followed by post-thaw expansion and an extended manufacturing process for up to 9 rounds of weekly K562 cell stimulation. We found that neither compromised the in vitro anti-tumor activity of NKG2D CAR-T cells. Interestingly, the expression of T cell exhaustion markers TIGIT, TIM3, and LAG3 was reduced with extended manufacturing. To enhance the safety profile of the NKG2D CAR-T cells, we incorporated a full-length CD20 transgene in tandem with the CAR construct and demonstrated that autologous NK cells could mediate efficient antibody-dependent cell-mediated cytotoxicity to remove these CAR-T cells. Collectively, our study illustrates a protocol that generates large numbers of efficacious NKG2D CAR-T cells suitable for multiple rounds of infusions.

Adoptive Transfer of NKG2D CAR mRNA-Engineered Natural Killer Cells in Colorectal Cancer Patients.

By fusing the extracellular domain of the natural killer (NK) cell receptor NKG2D to DAP12, we constructed a chimeric antigen receptor (CAR) to improve NK cell tumor responses. An RNA electroporation approach that provides transient expression of the CAR was adopted as a risk mitigation strategy. Expression of the NKG2D RNA CAR significantly augmented the cytolytic activity of NK cells against several solid tumor cell lines in vitro and provided a clear therapeutic benefit to mice with established solid tumors. Three patients with metastatic colorectal cancer were then treated with local infusion of the CAR-NK cells. Reduction of ascites generation and a marked decrease in number of tumor cells in ascites samples were observed in the first two patients treated with intraperitoneal infusion of low doses of the CAR-NK cells. The third patient with metastatic tumor sites in the liver was treated with ultrasound-guided percutaneous injection, followed by intraperitoneal infusion of the CAR-NK cells. Rapid tumor regression in the liver region was observed with Doppler ultrasound imaging and complete metabolic response in the treated liver lesions was confirmed by positron emission tomography (PET)- computed tomographic (CT) scanning. Our results highlight a promising therapeutic potential of using RNA CAR-modified NK cells to treat metastatic colorectal cancer.

Intraperitoneal immunotherapy with T cells stably and transiently expressing anti-EpCAM CAR in xenograft models of peritoneal carcinomatosis.

The epithelial cell adhesion molecule (EpCAM) is overexpressed in a wide variety of tumor types, including peritoneal carcinomatosis (PC) from gastrointestinal and gynecological malignancies. To develop a chimeric antigen receptor T (CART) cell therapy approach to treat patients with end-stage PC, we constructed third generation CARs specific to EpCAM using the 4D5MOC-B single chain variable fragment. CART cells were generated with lentiviral transduction and exhibited specific in vitro killing activity against EpCAM-positive human ovarian and colorectal cancer cells. A single intraperitoneal injection of the CART cells eradicated established ovarian xenografts and resulted in significantly prolonged animal survival. Since EpCAM is also expressed on normal epithelium, anti-EpCAM CART cells were generated by mRNA electroporation that display a controlled cytolytic activity with a limited CAR expression duration. Multiple repeated infusions of these RNA CAR-modified T cells delayed disease progression in immunodeficient mice bearing well-established peritoneal ovarian and colorectal xenografts. Thus, our study demonstrates the effectiveness of using anti-EpCAM CAR-expressing T cells for local treatment of PC in mice. The possibility of using this approach for clinical treatment of EpCAM-positive gastrointestinal and gynecological malignancies warrants further validation.

Co-Expansion of Cytokine-Induced Killer Cells and Vγ9Vδ2 T Cells for CAR T-Cell Therapy.

Gamma delta (γδ) T cells and cytokine-induced killer (CIK) cells, which are a heterogeneous population of T lymphocytes and natural killer T (NKT) cells, have been separately expanded ex vivo and shown to be capable of targeting and mediating cytotoxicity against various tumor cells in a major histocompatibility complex-unrestricted manner. However, the co-expansion and co-administration of these immune cells have not been explored. In this study we describe an efficient method to expand simultaneously both CIK and Vγ9Vδ2 T cells, termed as CIKZ cells, from human peripheral blood mononuclear cells (PBMCs) using Zometa, interferon-gamma (IFN-γ), interleukin 2 (IL-2), anti-CD3 antibody and engineered K562 feeder cells expressing CD64, CD137L and CD86. A 21-day culture of PBMCs with this method yielded nearly 20,000-fold expansion of CIKZ cells with γδ T cells making up over 20% of the expanded population. The expanded CIKZ cells exhibited antitumor cytotoxicity and could be modified to express anti-CD19 chimeric antigen receptor (CAR), anti-CEA CAR, and anti-HER2 CAR to enhance their specificity and cytotoxicity against CD19-, CEA-, or HER2-positive tumor cells. The tumor inhibitory activity of anti-CD19 CAR-modified CIKZ cells was further demonstrated in vivo in a Raji tumor mouse model. The findings herein substantiate the feasibility of co-expanding CIK and γδ cells for adoptive cellular immunotherapy applications such as CAR T-cell therapy against cancer.

High-throughput sequencing to identify miRNA biomarkers in colorectal cancer patients.

The altered expression of microRNAs (miRNAs) is associated with a number of cancer types. The study of the association between the miRNA profile and cancer may be useful to identify potential biomarkers of certain types of cancer. In the present study, 19 miRNAs were identified by high-throughput sequencing in the serum of colorectal cancer (CRC) patients. A network analysis was performed based on a computational approach to identify associations between CRC and miRNAs. The present study may be useful to identify cancer-specific signatures and potentially useful biomarkers for the diagnosis of CRC. The network analysis of miRNA-target genes may aid in identifying altered miRNA regulatory networks that are involved in tumor pathogenesis.

Identification of miRNAs differentially expressed in clinical stages of human colorectal carcinoma-an investigation in Guangzhou, China.

Aberrant expression of microRNAs (miRNAs) has been implicated in human cancer, including colorectal cancer (CRC). Such dysregulated miRNAs may have potential as diagnostic markers or therapeutic targets. However, the nature of an association between these miRNAs and clinical stages of CRC is still not clear. To this end, we performed a miRNA profiling of 1547 distinct human miRNAs using 31 samples of tumor and paired normal mucosa obtained from 31 CRC patients. Based on statistical analyses of profiling data, we identified 569 miRNAs that were significantly dysregulated in CRC relative to normal tissues (P<0.05). Among the 569 dysregulated miRNAs, downregulation of 17 was associated with stages II, III, and IV colon and rectal cancers (separate or combined), according to our criteria. We also assessed the potential of these dysregulated miRNAs as diagnostic biomarkers for CRC patients who were without metastasis, and the value of the dysregulated miRNAs for predicting metastasis, lymph node and distant. Their distinct expression patterns in colon and rectal cancers were also examined. Although our findings cannot be immediately applied toward clinical diagnosis, our new study model for determining and assessing the biomarker potential of dysregulated miRNAs should be useful in further research in detection of human CRC.

[Influence of phosphatidylinositol-3-kinases P85α silence by RNA interference on cell cycle and apoptosis of colorectal cancer cells in vitro].

OBJECTIVE: To investigate the effect of phosphatidylinositol- 3-kinases(PI3K) P85α silence on cell cycle and apoptosis of colorectal cancer cells. METHODS: Four shRNA vectors(shRNA/89, 324, 1073 and 1123) and one negative control vector were designed and stably transfected into SW480 cells. Western blotting was used to determine the expression level of P85α. Flow cytometry was used to determine the PI-labeled cycle after stable transfection. Annexin V-FITC kit was used to determine the apoptosis. RESULTS: Western blotting analysis showed that the expression level of PI3K P85α protein was significantly decreased in cells transfected with shRNA/324 vector. The inhibition rate was 90%. The group was selected for the following experiments. G1 phase cells in the interference group and the control group were (62.4±2.7)% and (51.2±3.5)%, respectively. S phase cells in the interference group and the control group were (23.9±1.7)% and (34.1±3.4)%, respectively. Apoptosis cells induced by 5-FU of interference group and control group were(11.1±3.7)% and (1.4±0.6)%, respectively. The differences were all significant (P<0.05). CONCLUSIONS: Depletion of PI3K P85α can significantly induce SW480 cell cycle arrest and sensitize SW480 cells to 5-FU induced apoptosis. PI3K P85α may be a new therapeutic target for colorectal cancer cells.

[Cloning of hsa-miR-148a and construction of its retroviral expression vector].

OBJECTIVE: To clone hsa-miR-148a and construct its retroviral expression vector. METHODS: The pre-miR-148a amplified by PCR was inserted to pMSCV to construct the recombinant retroviral expression plasmid pMSCV-miR-148a, which was confirmed by restriction endonuclease analysis and DNA sequencing. The retroviral expression vector pMSCV-miR-148a and PIK packaging plasmid were cotransfected into 293FT packaging cells by calcium phosphate-mediated transfection to produce the retrovirus, and the retrovirus titer was measured by infection of NIH3T3 cells. RESULTS: Restriction enzyme digestion and DNA sequencing demonstrated that the retroviral vector pMSCV-miR-148a was constructed successfully, and the virus titer was 5x10(8) CFU/ml after infection of NIH3T3 cells. CONCLUSION: The successful construction of the retroviral expression vector MSCV-miR-148a allows the production of high-titer retrovirus to facilitate further study of the molecular functions of miR-148a.