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    2019-10-30

    
    The PD-1/PD-L1 pathway plays a dominant role in tumor immune evasion. PD-L1 is overexpressed in a variety of cancer types and tumor-associated antigen-presenting cells.5–8 Overexpression of PD-L1 in the tumor microenvironment and peripheral blood is associ-ated with a poor prognosis for cancer patients.5,9–11 When PD-1 on activated T Spectinomycin interacts with PD-L1 on tumor cells, tumor cells are protected from cytotoxic lysis, whereas T cells are functionally impaired and lapse into anergy, exhaustion, or apoptosis.12–15 Monoclonal anti-bodies (mAbs) targeting PD-L1 or PD-1 have been approved for
    clinical treatment of several cancer types, particularly advanced solid tumors.15–17
    However, therapeutic mAbs have their limits for clinical application because of the requirement of frequent administration and high cost. In this study, we tried to address these issues by taking an active immunization approach. We designed a vaccine targeting PD-L1 and assessed its immunogenicity and anti-tumor efficacy in mice. Our data showed that the PD-L1 vaccine reversed the suppressive func-tions of the PD-1/PD-L1 pathway in the tumor microenvironment and provided tumor control in both preventive and therapeutic mouse melanoma models. Therefore, the PD-L1 vaccine is promising as an alternative strategy for anti-PD-L1 mAb-based cancer therapy.
    RESULTS
    The Design and Purification of the DPDL1E Antigen
    The tertiary structure of PD-L1 is composed of an extracellular
    domain (ECD), a transmembrane domain, and an intracellular re-gion.18,19 Because the ECD is responsible for binding of PD-1, we
    have chosen this domain as an immunogen. Because of central tolerance, the ECD is not immunogenic by itself. To break immune
    self-tolerance, we fused the ECD to the C-terminal of a carrier pro-tein, diphtheria toxin (DTT) (Figure 1A and 1B).20,21 The fusion
    molecule is designated DPDL1E. The protein was expressed in E. coli with a glutathione S-transferase (GST) fusion tag and purified by GST affinity chromatography. After removing the GST tag with
    Correspondence: Rongxiu Li, State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, 800 Dong Chuan Road, Shanghai 200240, China. E-mail: [email protected]
    This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
    www.moleculartherapy.org
    PreScission protease (PSP), the molecular weight of the protein was 43.5 kDa (Figure 1C). The protein was further purified to reduce the level of endotoxin contamination to less than 0.1 endotoxin units (EU)/mL.
    DPDL1E Immunization Induced PD-L1-Specific Humoral Immune Responses
    To examine the immunogenicity of DPDL1E, we first measured the antibody responses by ELISA with the sera of both C57BL/6 and BALB/c mice immunized with either DPDL1E or DTT. Indeed, anti-PD-L1 antibodies were induced in all DPDL1E mice, whereas no PD-L1-specific antibodies were found in DTT-immunized mice (Fig-ure 2A). Furthermore, we found that the immunoglobulin G (IgG) sub-classes were composed of IgG1, IgG2a, IgG2b, and IgG3 (Figure 2B) and that the level of IgG2 was higher than those of IgG1 and IgG3, indi-cating that the immune responses were biased toward the Th1 type. The DTT-specific IgG1 was higher than the other antibody subclasses in DPDL1E-immunized mice (Figure 2B), indicating that anti-DTT immune responses were biased toward the Th2 type. To test the func-tion of anti-PD-L1 antibodies, we performed a binding assay in vitro and found that the DPDL1E antisera could efficiently block PD-L1 and PD-1 interaction (Figure 2C) and that the degree of inhibition was correlated with the antibody titers (Figure 2D). In a parallel exper-iment, we used a PD-L1 mAb (10F.9G2) in in vitro binding assays. We found that the antibody concentration required to achieve the same level of inhibition was 11.25 mg/mL (Figure 2E).