Human telomerase reverse transcriptase (hTERT) has been found to be closely related to tumor transformation, growth, and metastasis. Thus, the delivery of hTERT small interfering RNA (siRNA) is an important approach for cancer gene therapy. However, the single anticancer effect of gene silencing is often limited by poor specificity or low efficiency in siRNA delivery and release. In this work, we present small and thin black phosphorus (BP) nanosheets as a biodegradable delivery system for hTERT siRNA. The BP nanosheets prepared with poly(ethylene glycol) (PEG) and polyethylenimine (PEI) modification (PPBP), exhibited high siRNA loading capacity and robust cell uptake. The PPBP nanosheets also exhibited potent photodynamic therapy/photothermal therapy (PDT/PTT) activities when exposed to different wavelengths of laser irradiation. More importantly, PPBP nanosheets underwent a gradual degradation when presented in a mixture of low pH and reactive oxygen species (ROS)-rich environment. The degradation of PPBP was strengthened especially after local and minimal invasive PDT treatment, because of excessive ROS production. Further delivery and release of siRNA to the cytoplasm for gene silencing was achieved by PEI-aided escape from the acidic lysosome. Thus, PPBP-siRNA efficiently inhibited tumor growth and metastasis by specific delivery of hTERT siRNA and a synergistic combination of targeted gene therapy, PTT and PDT.

The B7 family ligand HERV-H LTR–associating protein 2 (HHLA2) is an attractive target for cancer immunotherapy because of its coinhibitory function, overexpression in human cancers, and association with poor prognoses. However, the knowledge of the HHLA2 pathway is incomplete. HHLA2 has an established positive receptor transmembrane and immunoglobulin (Ig) domain containing 2 (TMIGD2) but a poorly characterized negative receptor human killer cell Ig-like receptor, three Ig domains, and long cytoplasmic tail (KIR3DL3). Here, KIR3DL3 and TMIGD2 simultaneously bound to different sites of HHLA2. KIR3DL3 was mainly expressed on CD56dim NK and terminally differentiated effector memory CD8+ T (CD8+ TEMRA) cells. KIR3DL3+ CD8+ TEMRA acquired an NK-like phenotype and function. HHLA2 engagement recruited KIR3DL3 to the immunological synapse and coinhibited CD8+ T and NK cell function and killing, inducing immune-evasive HHLA2+ tumors. KIR3DL3 recruited SHP-1 and SHP-2 to attenuate Vav1, ERK1/2, AKT, and NF-κB signaling. HHLA2+ tumors from human kidney, lung, gallbladder, and stomach were infiltrated by KIR3DL3+ immune cells. KIR3DL3 blockade inhibited tumor growth in multiple humanized mouse models. Thus, our findings elucidated the molecular and cellular basis for the inhibitory function of KIR3DL3, demonstrating that the KIR3DL3-HHLA2 pathway is a potential immunotherapeutic target for cancer.

The identification of CTL epitopes from tumor antigens is very important for the development of peptide-based, cancerspecific immunotherapy. Heparanase is broadly expressed in various advanced tumors and can serve as a universal tumorassociated antigen. Although several epitopes of heparanase antigen are known in humans, the corresponding knowledge in mice is still rather limited. The present study was designed to predict and identify the CTL epitopes in the mouse heparanase protein. For this purpose, H-2K b -restricted CTL epitopes were identified by using the following four-step procedure: (a) a computer-based epitope prediction from the amino acid sequence of mouse heparanase, (b) a peptidebinding assay to determine the affinity of the predicted epitopes with the H-2K b molecule, (c) the testing of the induction of CTLs toward various carcinoma cells expressing heparanase antigens and H-2K b , and (d) the induction of immunoprotection and immunotherapy in vivo. The results showed that, of the tested peptides, effectors induced by peptides of mouse heparanase at residue positions 398 to 405 (LSLLFKKL; mHpa398) and 519 to 526 (FSYGFFVI; mHpa519) lysed three kinds of carcinoma cells expressing both heparanase and H-2K b (B16 melanoma cells, EL-4 lymphoma cells, and Lewis lung cancer cells). In vivo experiments indicated that mHpa398 and mHpa519 peptides offered the possibility of not only immunizing against tumors but also treating tumorbearing hosts successfully. Our results suggest that the mHpa398 and mHpa519 peptides are novel H-2K b -restricted CTL epitopes capable of inducing heparanase-specific CTLs in vitro and in vivo. These epitopes may serve as valuable tools for the preclinical evaluation of vaccination strategies. [Cancer Res 2008;68(5):1529-37]