Discrepant Immunostimulatory Activities And Antitumor Efficacies In Response To Different Classes Of CpG Oligodeoxynucleotides

BackgroundOligodeoxynucleotides(ODNs)containing unmethylated cytosine-phosphate-guanine dinucleotide(CpG),as ligands of toll-like receptor 9(TLR9),directly activate plasmacytoid dendritic cells(pDCs)and B cells to induce humoral and cellular immunity.Based on the sequences,structures,and in vitro activities,CpG ODNs were mainly divided into three classes:A(D,CpG-A),B(K,CpG-B),and C(CpG-C).CpG-A ODNs contain a central CpG-containing palindromic sequence,capped at the 3?and 5?-end by phosphorothioate-modified poly G tails.CpG-A ODNs stimulate pDCs to produce large amounts of interferon(IFN)-?.CpG-B ODNs contain a full phosphorothioate backbone and encode one or more CpG motifs,without poly G tails.CpG-B ODNs stimulate B cells.CpG-C ODNs have the characteristics of both CpG-A ODNs and CpG-B ODNs,and contain a full phosphorothioate backbone and a palindromic CpG-containing motif at the 3?-end.CpG-C ODNs induce pDCs to secret IFN-?and stimulate B cells.CpG-C ODNs exhibit intermediate immunostimulatory efficacies between CpG-A ODNs and CpG-B ODNs.In addition,CpG ODNs have been demonstrated to stimulate type-I helper T cells(Th1)-biased immune responses,including promoting the production of antigen-specific antibodies and the cytotoxicity of CD8~+T cells.In tumor microenvironments,CpG ODNs activate immune system and break immunosuppression state,thus inhibiting tumor growth.In preclinical and clinical studies,three classes of CpG ODNs have shown antitumor activities.In recent years,CpG ODNs in combination with other drugs,especially immune checkpoint inhibitors such as programmed cell death-1(PD-1)inhibitors,have received extensive attention for the treatment of tumors.In preclinical studies,three classes of CpG ODNs combined with PD-1 inhibitors synergistically inhibit mouse tumor models of colon cancer,melanoma,lymphoma,breast cancer,bladder cancer,among other cancers.In clinical studies,only CpG-A ODNs and CpG-C ODNs in combination with PD-1 inhibitors are currently undergoing phase I/II clinical trials,while no clinical trials of CpG-B ODNs combined with PD-1 inhibitors are implemented.However,specific differences among three classes of CpG ODNs in antitumor effects or synergistic antitumor effect with anti-PD-1antibody are still unknown.Thus,it is urgent to compare immunostimulatory activities and antitumor effects of three classes of CpG ODNs,so as to provide a theoretical basis for the selection of clinical treatment for cancer patients.MethodsThree typical classes of CpG ODNs were selected:ODN 1585(CpG-A)?ODN 1826(CpG-B)and ODN 2395(CpG-C).The stimulatory effects of the three classes of CpG ODNs on cytokine secretion,pDC activation,and B-cell activation and proliferation in mouse splenocytes were verified using enzyme-linked immunosorbent assay(ELISA),cytometric bead array(CBA)and flow cytometry.The activation of TLR9-nuclear factor(NF)-?B pathway in mice induced by the three classes of CpG ODNs were determined using TLR9-overexpressing HEK-Blue?-mTLR9 cells and the negative control HEK-Blue?Null1 cells.The immunostimulatory effects of the three classes of CpG ODNs on pDCs and B cells were compared at transcriptome level by RNA sequencing.Then,the antitumor effects of the three classes of CpG ODNs with different doses,as well as the antitumor effects of low-dose and high-dose CpG ODNs combined with the anti-PD-1 antibody in a CT26 subcutaneous mouse tumor model,were assayed.The expression of PD-L1,the ligands of PD-1,on multiple host immune cells,and the proportion and function of T cells in the tumor microenvironments were determined by flow cytometry.Finally,we designed a novel CpG-C ODNs—HP06T07 with cooperating organization.The immunostimulatory effects of HP06T07 on cytokine secretion,B-cell activation and proliferation in mouse splenocytes and human peripheral blood mononuclear cells(PBMCs)were verified by ELISA,CBA and flow cytometry.The TLR9 target by HP06T07 was investigated using HEK-Blue?-mTLR9 cells and human monocyte-derived dendritic cells(MoDCs)that do not express TLR9.The antitumor effects of HP06T07 with different doses and administration regimens in the CT26 subcutaneous mouse tumor model were assayed to determine the optimal dose and adminitration regimen.The infiltration of T cells in the tumor microenvironments were determined by immunohistochemistry.ResultsThe three classes of CpG ODNs stimulated mouse IFN-?,IFN-?,tumor necrosis factor(TNF)-?and interleukin(IL)-6 secretion in different dose-dependent manners.CpG-B induced weak IFN-?production,and CpG-A induced weak IL-6 production.The three classes of CpG ODNs similarly stimulated pDC and B-cell activation,but the effects of CpG-C were between CpG-A and CpG-B at low concentrations.The effects of CpG-C on B-cell proliferation were similar to CpG-B,and the effects of it on TLR9-NF-?B activation were slightly stronger than CpG-B,while CpG-A had a weaker effect on B-cell proliferation and TLR9-NF-?B activation.At low concentrations,the effects of CpG-C on B-cell proliferation and TLR9-NF-?B activation were between CpG-A and CpG-B.RNA sequencing analysis revealed that CpG-C functioned similarly to CpG-A in pDCs,whereas CpG-B showed stronger expression of a few other immune function genes such as CCL3,CXCL16,IL-10,among other genes;moreover,CpG-C exhibited nearly identical effects as CpG-B in B cells.CpG-B and CpG-C at their respective optimal concentrations possessed similar antitumor effects to significantly suppress CT26 tumor growth,while CpG-A possessed weak inhibitory effects on murine CT26 tumor.Moreover,compared with CpG-B,CpG-C in combination with anti-PD-1 antibody inhibited tumor growth more quickly and effectively,while CpG-A in combination with anti-PD-1 antibody possessed no synergistic anti-tumor effect.We found that CpG-B significantly upregulated PD-L1 expression on multiple host immune cells in the tumor microenvironments compared to CpG-A and CpG-B.Meanwhile,CpG ODNs and CpG ODNs combined with the anti-PD-1 antibody significantly increased the infiltration of CD8~+T cells,stimulated the IFN-?expression of T cells,and induced the differentiation of CD44~+CD62L~-effector memory T cells in the tumor microenvironments.Based on the powerful immunostimulatory activities and antitumor effects of CpG-C,we designed and synthesized HP06T07.Compared to ODN 2395-treated groups,HP06T07significantly induced cytokine secretion,B-cell activation and proliferation in mouse splenocytes and human PBMCs.Intratumoral injection of HP06T07 significantly inhibited the CT26 tumor growth and prolonged mouse survival.Moreover,HP06T07 had the best antitumor effects when given 9 times at 2-day intervals.HP06T07 promoted T-cell infiltration in the tumor microenvironments by immunohistochemstry.Conclusion1.Different classes of CpG ODNs have different optimal concentrations and dose-dependent manners.At their respective optimal concentrations,CpG-C functioned similarly to CpG-A in pDCs and exhibited nearly identical effects to CpG-B in B cells.2.Both CpG-B and CpG-C significantly inhibited mouse CT26 colon cancer,and both of them in combination with the anti-PD-1 antibody possessed synergistic anti-tumor effects.However,CpG-C in combination with the anti-PD-1 antibody inhibited tumor growth more quickly and effectively.CpG-B and CpG-C,together with the anti-PD-1 antibody,promoted the infiltration of effector memory T cells.3.HP06T07,as CpG-C ODNs,significantly stimulated cytokine secretion,and B-cell activation and proliferation in mouse splenocytes and human PBMCs,and inhibited tumor growth in the mouse model of CT26 colon cancer.