Neoantigen Dendritic Cell Vaccination Combined with Anti-CD38 and CpG Elicits Anti-Tumor Immunity against the Immune Checkpoint Therapy-Resistant Murine Lung Cancer Cell Line LLC1.

Changbo Sun,Kazuhiro Kakimi,Koji Nagaoka,Jun Nakajima,Hidewaki Nakagawa,Yukari Kobayashi

doi:10.3390/cancers13215508

Abstract

Simple SummaryDespite the substantial achievements to date, a significant proportion of patients still fail to benefit from immune-checkpoint therapies (ICT). The absence of T cell infiltration and insufficient immune recognition may account for the primary resistance to immune checkpoint therapy. The present study compared the ICT response of two murine lung cancer cell line models, ASB-XIV and LLC1. ASB-XIV tumors are inflamed and are sensitive to ICT, while non-inflamed LLC1 tumors are resistant. We employed in-depth tumor analysis, including whole-exome sequencing, RNA-sequencing, and flow cytometry, to reveal the molecular mechanisms of resistance to ICT, and sought strategies to promote inflammatory/immunogenic pathway activation and inhibit immunosuppressive factors present in LLC1 tumors. An appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant “cold” tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells. Thus, the future direction of ICT is combination immunotherapy.An important factor associated with primary resistance to immune-checkpoint therapies (ICT) is a “cold” tumor microenvironment (TME), characterized by the absence of T cell infiltration and a non-inflammatory milieu. Whole-exome and RNA sequencing to predict neoantigen expression was performed on the LLC1 cell line which forms “cold” tumors in mice. Dendritic cell (DC)-based vaccination strategies were developed using candidate neoantigen long peptides (LPs). A total of 2536 missense mutations were identified in LLC1 and of 132 candidate neoantigen short peptides, 25 were found to induce CD8+ T cell responses. However, they failed to inhibit LLC1 growth when incorporated into a cancer vaccine. In contrast, DCs pulsed with LPs induced CD4+ and CD8+ T cell responses and one of them, designated L82, delayed LLC1 growth in vivo. By RNA-Seq, CD38 was highly expressed by LLC1 tumor cells and, therefore, anti-CD38 antibody treatment was combined with L82-pulsed DC vaccination. This combination effectively suppressed tumor growth via a mechanism relying on decreased regulatory T cells in the tumor. This study demonstrated that an appropriate vaccination strategy combining neoantigen peptide-pulsed DC with anti-CD38 antibody can render an ICT-resistant “cold” tumor susceptible to immune rejection via a mechanism involving neutralization of regulatory T cells.

Highlights

Cancer treatment, including for lung cancer, has been radically altered by the advent of immune checkpoint therapy (ICT) over the last few years [1]
LLC1 Tumors Are Resistant to Immune Checkpoint Blockade
BALB/c and C57BL/6 mice were subcutaneously inoculated with 1 × 106 cells of the lung cancer lines ASB-XIV or LLC1, respectively, and received anti-PD-1, anti-CTLA-4, or a combination of both antibodies. Both cell lines generated tumors growing progressively in untreated mice (Figure 1A), whereas treatment with anti-PD-1 or anti-CTLA-4 antibodies inhibited ASB-XIV tumor growth

Summary

Introduction

Cancer treatment, including for lung cancer, has been radically altered by the advent of immune checkpoint therapy (ICT) over the last few years [1]. Lung cancer is the leading cause of cancer death, with an estimated 1.8 million deaths worldwide [2]. ICT was first approved for second-line or later therapy for advanced disease, for the firstline treatment for patients whose tumors expressed PD-L1 on at least 50% of the tumor cells [3–5]. Combination ICT-chemotherapy is approved for first-line treatment of patients with either squamous or non-squamous non-small cell lung cancer, regardless of PD-L1 expression [6]. Many patients fail to respond to ICT, or eventually progress after initially responding [7,8]. An unmet medical need to understand why some lung cancers are resistant to ICT There is. an unmet medical need to understand why some lung cancers are resistant to ICT

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