Fanyan Meng

CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients

Strategies that enhance the function of T cells are critical for immunotherapy. One negative regulator of T-cell activity is ligand PD-L1, which is expressed on dentritic cells (DCs) or some tumor cells, and functions through binding of programmed death-1 (PD-1) receptor on activated T cells. Here we described for the first time a non-viral mediated approach to reprogram primary human T cells by disruption of PD-1. We showed that the gene knockout of PD-1 by electroporation of plasmids encoding sgRNA and Cas9 was technically feasible. The disruption of inhibitory checkpoint gene PD-1 resulted in significant reduction of PD-1 expression but didn’t affect the viability of primary human T cells during the prolonged in vitro culture. Cellular immune response of the gene modified T cells was characterized by up-regulated IFN-γ production and enhanced cytotoxicity. These results suggest that we have demonstrated an approach for efficient checkpoint inhibitor disruption in T cells, providing a new strategy for targeting checkpoint inhibitors, which could potentialy be useful to improve the efficacy of T-cell based adoptive therapies.

CRISPR-Cas9-mediated disruption of PD-1 on human T cells for adoptive cellular therapies of EBV positive gastric cancer

ABSTRACT The successful use of immune cell checkpoint inhibitors PD-1 and PD-L1, over the past 5 y has raised the concern of using immunotherapy to treat various cancers. Epstein-Barr virus-associated gastric cancer (EBVaGC) exhibits high infiltration of lymphocytes and high amplification of immune-related genes including PD-L1 as distinguished from Epstein-Barr virus-non-associated gastric cancer (EBVnGC). Here, we presume that this PD-1/PD-L1 pathway may hinder the efficacy of adoptive T cell therapy toward EBVaGC. These studies reveal possibility of generating PD-1-disrupted CTL by CRISPR-Cas9 system and demonstrate enhanced immune response of these PD-1-disrupted CTLs to the EBV-LMP2A antigen and superior cytotoxicity to the EBV-positive gastric cancer cell. In addition, when combined with low-dose radiotherapy, these PD-1-disrupted CTLs mediated an impressive antitumor effect in a xenograft mouse model of EBVaGC. Taken together, these studies illustrate PD-1/PD-L1-mediated immune tolerance of EBVaGC and provide a new strategy for targeting immune checkpoints to break the tolerance for the T cell-based adoptive therapy.

MHC class II restricted neoantigen: A promising target in tumor immunotherapy

Neoantigen is a patient-specific tumor antigen resulted from mutations during oncogenesis. Emerging data suggested that immune responsiveness against neoantigens correlated with the success of clinical tumor immunotherapies. Nowadays, the majority of studies on neoantigens have focused on MHC class I restricted antigens recognized by CD8+ T cells. With improved understanding of the underlying principles of tumor biology and immunology, increasing emphasis has been put on CD4+ T cells and MHC class II restricted antigens. MHC class II restricted neoantigen has the potential to be a promising target of tumor immunotherapy, although the limited comprehension and technical difficulties need to be overcome before being applied into clinical practice. This review discussed the immunologic mechanism, screening technique, clinical application, limitations and prospectives of MHC class II restricted neoantigens in tumor immunotherapy.

Phase I clinical study of personalized peptide vaccination combined with radiotherapy for advanced hepatocellular carcinoma

AIM To assess the efficacy and safety of a new treatment modality, cellular immune therapy based on personalized peptide vaccination (PPV-DC-CTL) combined with radiotherapy, for treating advanced hepatocellular carcinoma (HCC). METHODS A total of nine patients with advanced HCC were enrolled. Multidisciplinary consultation confirmed that all the patients definitely had no opportunity of surgery, because four patients had multiple liver metastases (the number of liver lesions > 3), one patient had liver metastases and portal vein tumor thrombosis, one patient had lung and bone metastases, two patients had liver and lung metastases and one patient had liver metastasis and peritoneal metastasis. Patients with metastasis were treated with precise radiotherapy combined with PPV-DC-CTL. RESULTS Following radiotherapy and one to three cycles of PPV-DC-CTL treatment, AFP levels were significantly decreased in six patients and imaging assessment of the lesions showed a partial response (PR) in three patients and stable disease in the other three patients. The response rate was 33% and disease control rate was 66%. This regimen was found to be safe and well tolerated. None of the patients developed liver or kidney side effects. Only one patient developed grade II bone marrow suppression and the remaining patients had no significant hematological side effects. CONCLUSION Radiotherapy combined with PPV-DC-CTL provides a new therapeutic strategy for patients with advanced HCC, which is well tolerated, safe, feasible and effective.

Tumor‑penetrating peptide fused EGFR single‑domain antibody enhances radiation responses following EGFR inhibition in gastric cancer

Radiotherapy has been the primary method for the local control of several types of unresectable tumor, including gastric cancer. Patients with gastric cancer frequently express high levels of epidermal growth factor receptor (EGFR), which have been found to increase following radiotherapy treatment. This provides a basis for the combination of antibodies targeting EGFR and radiotherapy. In our previous study, a protein (anti‑EGFR‑iRGD) with bispecific targets and high permeability was constructed, and its effects on inhibiting the proliferation of gastric cancer cells was investigated. In the present study, the capacity of anti‑EGFR‑iRGD to modulate a radiation response was investigated and the specific mechanisms underlying these interactions were evaluated in gastric cancer cell lines and xenografts exhibiting high levels of EGFR. The radioenhancement of anti‑EGFR‑iRGD was associated with inhibited radiation‑induced upregulation of EGFR, inhibited cell proliferation and promotion of cell apoptosis. In addition, anti‑EGFR‑iRGD appeared to permeate more into the tumor tissue following radiation. These findings indicated that the recombinant protein anti‑EGFR‑iRGD was a selective and effective radiosensitizer in EGFR‑overexpressing gastric cancer cells and xenografts. These results further suggested that anti‑EGFR‑iRGD is a potential superior EGFR‑targeted therapy combined with radiotherapy. Overall, the present study suggested that anti‑EGFR‑iRGD may be a promising candidate for preclinical and clinical use.

Disappearance of bone metastases in chemotherapy‑resistant gastric cancer treated with antigen peptide‑pulsed dendritic cell‑activated cytotoxic T lymphocyte immunotherapy: A case report

The adoptive transfer of cytotoxic T lymphocytes (CTLs) stimulated by specific tumor antigen peptide-pulsed dendritic cells (DCs) is one of the most promising immunotherapeutic strategies currently available for patients with gastric cancer (GC). The present case report describes a patient with chemotherapy-resistant stage IV GC with multiple bone metastases, who had been treated with antigen peptide-pulsed DC-CTLs. DCs and CTLs were transfused into the patient subcutaneously and intravenously with simultaneous oral administration of low-dose cyclophosphamide. Following 3 cycles of combination therapy, marked remission regarding the number of metastatic bone lesions was achieved, confirmed by the use of enhanced computerized tomography, computerized tomography and magnetic resonance imaging. After 1 year, 8 cycles of adoptive immunotherapy were administered, and a further decrease in the number of metastatic bone lesions was observed in addition to a marked improvement in the patients quality of life. Therefore, personalized antigen peptide-pulsed DC-CTLs combined with oral administration of low-dose cyclophosphamide may serve as a promising anticancer therapy to eradicate tumor cells, and therefore this approach is recommended for future cases of a similar nature.

Lipid insertion enables targeted functionalization of paclitaxel-loaded erythrocyte membrane nanosystem by tumor-penetrating bispecific recombinant protein

Background There is currently much interest in cancer cell targeting and tumor penetrating for research and therapeutic purposes. Purpose To improve targeting delivery of antitumor drugs to gastric cancer, in this study, a tumor-targeting biocompatible drug delivery system derived from erythrocyte membrane for delivering paclitaxel (PTX) was constructed. Methods Erythrocyte membrane of human red blood cells (RBCs) were used for preparing of erythrocyte membrane-derived vesicles. 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-(maleimide[polyethylene glycol]-3400) (DSPE-PEG-MAL), a phospholipid derivative, was used to insert tumor-targeting molecular into erythrocyte membrane-derived vesicles. A lipid insertion method was used to functionalize these vesicles without the need for direct chemical conjugation. Furthermore, a tumor-penetrating bispecific recombinant protein named anti-EGFR-iRGD was used for the first time in this work to enable nanosystem to target and penetrate efficiently into the tumor site. Results Paclitaxel (PTX)-loaded anti-EGFR-iRGD-modified erythrocyte membrane nano-system (anti-EGFR-iRGD-RBCm-PTX, abbreviated to PRP) were manufactured. PRP was spheroid, uniformly size, about 171.7±4.7 nm in average, could be stable in vitro for 8 days, and released PTX in a biphasic pattern. PRP showed comparable cytotoxicity toward human gastric cancer cells in vitro. In vivo studies showed that, PRP accumulated in tumor site within 2 h of administration, lasted longer than 48 h, and the tumor volume was reduced 61% by PRP treatment in Balb/c nude mice, without causing severe side effects. Conclusion PRP has potential applications in cancer treatment and as an adjunct for other anticancer strategies.

Personalised peptide vaccination combined with radiotherapy for patients with advanced hepatocellular carcinoma: a phase 1 study

Abstract Background We aimed to investigate a new treatment method for the treatment of advanced hepatocellular carcinoma. The method was a cellular immune therapy based on personalised peptide vaccination (PPV-DC-CTL) combined with radiotherapy. Methods We did a phase 1 study in which patients with advanced hepatocellular carcinoma who were not eligible for surgery, as confirmed by multidisciplinary consultation, were treated with precise radiotherapy combined with PPV-DC-CTL in the Comprehensive Cancer Centre of Nanjing Drum Tower Hospital, Nanjing, China. To personalise the vaccine, the peptide candidate library, including mutated peptides and highly expressed peptides, was established according to the gene mutation and expression spectra of hepatocellular carcinoma and previous studies about PPV. Peptides for vaccination of every patient were selected from the peptide candidate library, with the consideration of the pre-existing immunity of the host before vaccination. The assay of peptide-specific IFN-γ production was used to define pre-existing immunity. Side-effects were measured with a haemogram. This study is registered with the Chinese Clinical Trial Registry, number ChiCTR-OIC-16010025. Findings A total of nine patients with advanced hepatocellular carcinoma were admitted. Four patients had multiple liver metastases (liver lesions more than three pieces), one patient had liver metastasis and portal vein tumour thrombosis, one patient had lung and bone metastases, two patients had liver and lung metastases, and one patient had liver metastasis and peritoneal metastasis. Following radiotherapy and 1–3 cycles of PPV-DC-CTL treatment, AFP concentrations were significantly reduced in six patients compared with baseline concentrations, and imaging assessment of the lesions showed a partial response in three of the patients and stable disease in the other three patients. An objective response was achieved in 33% and disease control in 66%. This regimen was found to be safe and well tolerated because none of the patients developed liver or kidney side-effects. Only one patient developed grade 2 bone marrow suppression, and the other patients had no significant side-effects. Interpretation Radiotherapy combined with PPV-DC-CTL provides a new therapeutic strategy for patients with advanced hepatocellular carcinoma, which is well tolerated, safe, feasible, and effective. Funding National Natural Science Foundation of China, Jiangsu Provincial Medical Youth Talent, and the Key Medical Science and Technology Development Project of Nanjing.