Radiation Augments the Local Anti-Tumor Effect of In Situ Vaccine With CpG-Oligodeoxynucleotides and Anti-OX40 in Immunologically Cold Tumor Models

Abstract

IntroductionCombining CpG oligodeoxynucleotides with anti-OX40 agonist antibody (CpG+OX40) is able to generate an effective in situ vaccine in some tumor models, including the A20 lymphoma model. Immunologically “cold” tumors, which are typically less responsive to immunotherapy, are characterized by few tumor infiltrating lymphocytes (TILs), low mutation burden, and limited neoantigen expression. Radiation therapy (RT) can change the tumor microenvironment (TME) of an immunologically “cold” tumor. This study investigated the effect of combining RT with the in situ vaccine CpG+OX40 in immunologically “cold” tumor models.MethodsMice bearing flank tumors (A20 lymphoma, B78 melanoma or 4T1 breast cancer) were treated with combinations of local RT, CpG, and/or OX40, and response to treatment was monitored. Flow cytometry and quantitative polymerase chain reaction (qPCR) experiments were conducted to study differences in the TME, secondary lymphoid organs, and immune activation after treatment.ResultsAn in situ vaccine regimen of CpG+OX40, which was effective in the A20 model, did not significantly improve tumor response or survival in the “cold” B78 and 4T1 models, as tested here. In both models, treatment with RT prior to CpG+OX40 enabled a local response to this in situ vaccine, significantly improving the anti-tumor response and survival compared to RT alone or CpG+OX40 alone. RT increased OX40 expression on tumor infiltrating CD4+ non-regulatory T cells. RT+CpG+OX40 increased the ratio of tumor-infiltrating effector T cells to T regulatory cells and significantly increased CD4+ and CD8+ T cell activation in the tumor draining lymph node (TDLN) and spleen.ConclusionRT significantly improves the local anti-tumor effect of the in situ vaccine CpG+OX40 in immunologically “cold”, solid, murine tumor models where RT or CpG+OX40 alone fail to stimulate tumor regression.

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“…OX40 agonist in combination with PD-1 monoclonal antibody showed an increase in effector CD4 + and CD8 + T cells and a decrease in the number of Treg cells and MDSCs because of the formation of a local immunostimulatory microenvironment [40] . Similarly, several preclinical studies have been conducted on the combination of OX40 agonist with radiotherapy, which demonstrated that this combination can effectively inhibit local tumor growth and improve survival rate, with increased activation of CD4 + and CD8 + T cells, depletion of Treg cells, and decreased expression of FOXP3 in TDLNs [44,45] . In a mouse model of anti-PD-1 resistant lung tumors, intratumoral injection of OX40 agonist antibody after radiotherapy effectively inhibited local tumor growth, limited lung metastasis, and improved survival [46] .…”

Factors impacting the efficacy of the in-situ vaccine with CpG and OX40 agonist

“…A20 lymphoma was obtained from Dr. Stephen Gillies in 2017. To achieve consistent tumor engraftment, A20 cells were passaged in vivo as previously described [ 25 ]. B78-D14 (B78) melanoma was obtained from Dr. Ralph Reisfeld in 2002.…”

“…OX40 activation on T regulatory cells (Tregs) can lead to Treg apoptosis, decreased production of interleukin (IL)-10, and decreased FoxP3 expression [ 22 – 24 ]. In the A20 model, we and others have shown IT injections of CpG can increase OX40 expression on CD4 + effector and CD8 + T cells in the tumor microenvironment (TME) [ 17 , 25 ].…”

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“…Tumor antigen-specific cytotoxic T cells (CTL, CD8 + IFN-γ + ) which could bind to antigen MHCI complex, directly eradicate tumor cells [52]. 4T1 tumor has been regarded as immune-deserted ones [53], and immunofluorescent staining demonstrated that infiltration of CD8 + T cells was lacking in PBStreated tumor (Fig. 7A).…”