Adaptive antitumor immune response stimulated by bio-nanoparticle based vaccine and checkpoint blockade

Abstract

Background: Interactions between tumor and microenvironment determine individual response to immunotherapy. Triple negative breast cancer (TNBC) and hepatocellular carcinoma (HCC) have exhibited suboptimal responses to immune checkpoint inhibitors (ICIs). Aspartate β-hydroxylase (ASPH), an oncofetal protein and tumor associated antigen (TAA), is a potential target for immunotherapy.

Methods: Subcutaneous HCC and orthotopic TNBC murine models were established in immunocompetent BALB/c mice with injection of BNL-T3 and 4 T1 cells, respectively. Immunohistochemistry, immunofluorescence, H&E, flow cytometry, ELISA and in vitro cytotoxicity assays were performed.

Results: The ASPH-MYC signaling cascade upregulates PD-L1 expression on breast and liver tumor cells. A bio-nanoparticle based λ phage vaccine targeting ASPH was administrated to mice harboring syngeneic HCC or TNBC tumors, either alone or in combination with PD-1 blockade. In control, autocrine chemokine ligand 13 (CXCL13)-C-X-C chemokine receptor type 5 (CXCR5) axis promoted tumor development and progression in HCC and TNBC. Interactions between PD-L1⁺ cancer cells and PD-1⁺ T cells resulted in T cell exhaustion and apoptosis, causing immune evasion of cancer cells. In contrast, combination therapy (Vaccine+PD-1 inhibitor) significantly suppressed primary hepatic or mammary tumor growth (with distant pulmonary metastases in TNBC). Adaptive immune responses were attributed to expansion of activated CD4⁺ T helper type 1 (Th1)/CD8⁺ cytotoxic T cells (CTLs) that displayed enhanced effector functions, and maturation of plasma cells that secreted high titers of ASPH-specific antibody. Combination therapy significantly reduced tumor infiltration of immunosuppressive CD4⁺/CD25⁺/FOXP3⁺ Tregs. When the PD-1/PD-L1 signal was inhibited, CXCL13 produced by ASPH⁺ cancer cells recruited CXCR5⁺/CD8⁺ T lymphocytes to tertiary lymphoid structures (TLSs), comprising effector and memory CTLs, T follicular helper cells, B cell germinal center, and follicular dendritic cells. TLSs facilitate activation and maturation of DCs and actively recruit immune subsets to tumor microenvironment. These CTLs secreted CXCL13 to recruit more CXCR5⁺ immune cells and to lyse CXCR5⁺ cancer cells. Upon combination treatment, formation of TLSs predicts sensitivity to ICI blockade. Combination therapy substantially prolonged overall survival of mice with HCC or TNBC.

Conclusions: Synergistic antitumor efficacy attributable to a λ phage vaccine specifically targeting ASPH, an ideal TAA, combined with ICIs, inhibits tumor growth and progression of TNBC and HCC.

Keywords: Aspartate β-hydroxylase (ASPH); Hepatocellular carcinoma; Immune checkpoint inhibitor; Lambda phage vaccine; Metastasis; Tertiary lymphoid structures; Triple negative breast cancer.