Blocking Programmed Death-1 Ligand-PD-1 Interactions by Local Gene Therapy Results in Enhancement of Antitumor Effect of Secondary Lymphoid Tissue Chemokine

Abstract

The negative signal provided by interactions of programmed death-1 (PD-1) and its ligands, costimulatory molecules PD-L1 (also B7-H1) and PD-L2 (also B7-DC), is involved in the mechanisms of tumor immune evasion. In this study, we found that this negative signal was also involved in immune evasion in tumor immunotherapy. When we used different doses of a constructed eukaryotic expression plasmid, pSLC, which expresses functional murine secondary lymphoid tissue chemokine (SLC, CCL21), to treat BALB/c mice inoculated with H22 murine hepatoma cells, the inhibitory effect was enhanced along with the increase of pSLC dosage. Unexpectedly, however, the best complete inhibition rate of tumor was reached when pSLC was used at the dosage of 50 μg but not 100 or 200 μg. RT-PCR and real-time PCR revealed that both PD-L1 and PD-L2 genes were expressed in tumor and vicinal muscle tissues of tumor-bearing mice and the expression level was significantly increased if a higher dosage of pSLC was administered. We then constructed a eukaryotic expression plasmid (pPD-1A) that expresses the extracellular domain of murine PD-1 (sPD-1). sPD-1 could bind PD-1 ligands, block PD-Ls-PD-1 interactions, and enhance the cytotoxicity of tumor-specific CTL. Local gene transfer by injection of pPD-1A mediated antitumor effect and improved SLC-mediated antitumor immunity. The combined gene therapy with SLC plus sPD-1 did not induce remarkable autoimmune manifestations. Our findings provide a potent method of improving the antitumor effects of SLC and possibly other immunotherapeutic methods by local blockade of negative costimulatory molecules.

Tumor cell expression of programmed cell death‐1 ligand 1 is a prognostic factor for malignant melanoma

“…Many groups of investigators have reported that blockade of the PD-1/PD-L1 interaction promotes tumor immunity. [35][36][37] Conversely, there remains a problem regarding PD-1/PD-L1 blockade because of the possible expansion of poorly immunogenic cells. 38 Further studies may be required to clarify the therapeutic effect of PD-1/PD-L1 blockade in malignant melanoma models and clinical trials.…”

HSP70 vaccine in combination with gene therapy with plasmid DNA encoding sPD‐1 overcomes immune resistance and suppresses the progression of pulmonary metastatic melanoma

“…The expression of B7-H1 on melanoma cells were examined by FACS analysis as described previously. 25 …”

“…25 The expression plasmid pMSH70 of human HSP70 was kindly provided by Dr. Richard Morimoto and Ji-zhong Cheng (The University of Texas Medical Branch). Plasmid DNA was prepared by selective compaction with spermine (Sigma) and endotoxin levels were less than 3 pg/lg of plasmid DNA as determined by Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD).…”

“…25,26 Briefly, melanoma cells at a concentration of 5 3 10 7 / ml subjected to 2 cycles of freeze-thaw, and mixed with a 2-fold volume of distilled water and centrifugated to remove cell debris. The supernatant was incubated in a boiling water bath for 10 min, then in an ice bath for 30 min and centrifugated to remove the denatured high MW proteins.…”

Reconstructed Adeno-Associated Virus with the Extracellular Domain of Murine PD-1 Induces Antitumor Immunity

“…Immunohistochemistry was performed with the method of SP, as described (Wiendl et al, 2002;He et al, 2004). Goat anti-mouse PD-1 antibody (R&D systems Minneapolis, CA) was used to detect PD-1 expression at a dilution of 1: 200.…”