Double Targeted Oligodeoxynucleotides With Both The Activation Of TLR9 And Inhibition Of TGF-β2 Or CTLA-4 And Their Immunobiological Activities

The balance of immune regulation is crucial for homeostasis.When the body is infected by external pathogens or damaged,the immunoactivation signals will be activated to protect the body,then the immunosuppression signals will be generated to avoid excessive immune response to maintain the immune balance of the body.It can be seen that immunoactivation signals and immunosuppression signals are significant to the body.When the agents that stimulate immunoactivation signals are used to treat tumor or as vaccine adjuvant,due to the generation of feedback immunosuppressive signals after immune activation,the immunoactivation signals will be inhibited and the duration of immunoactivation will be shortened,which may be the reason why the treatment of tumor or as vaccine adjuvant alone can not produce expected effects.Similarly,when we use agents that inhibit immunosuppressive signals to achieve immune activation,the premise is that the body can fully mobilize the immune response and produce immunosuppressive molecules,otherwise it can not produce the expected effect.Therefore,agents that take into account both stimulating immunoactivation signal and inhibiting immunosuppressive signal should stimulate a stronger immune response.In this study,in order to achieve stronger immune activation by taking into account both stimulating immunoactivation signal and inhibiting immunosuppressive signal,we designed double targeted ODN（Dual-ODN）to achieve the porpose of stimulating immunoactivation signal and inhibiting immunosuppressive signal on a oligodeoxynucleotides（ODN）molecule.In this process,we selected the TLR9 agonist CpG ODN as part of stimulation of immunoactivation signal of Dual-ODN,considering that immunosuppressive signals can be immunosuppressive receptors expressed on the cell surface or immunosuppressive cytokines,we selected immunosuppressive receptor CTLA-4expressed on activated T cells and immunosuppressive cytokine TGF-β2 respectively as part of inhibition of immunosuppressive signal of Dual-ODN.According to this,we designed two types Dual-ODN,and studied their structural characteristics and immunobiological activities.The results were as follows:1.Designed,analysed and identified double targeted oligodeoxynucleotides that activating TLR9 and inhibiting TGF-β2 or CTLA-4.Firstly,we selected the conserved sequences of TGF-β2 mRNA 3’UTR and CTLA-4mRNA 3’UTR of human and mouse by using bioinformatics methods and analysed the secondary structure,screened the appropriate targeted sequences of TGF-β2 mRNA 3’UTR and CTLA-4 mRNA 3’UTR,and designed oligodeoxynucleotides targeting the sequences respectively basd on the principle of antisense oligonucleotides.According to the characteristics of TLR9 agonist CpG ODN and some immune stimulation sequences,we designed two types of double targeted oligodeoxynucleotides.By changing the position of these two parts on the Dual-ODN,we designed three kinds of Dual-ODN for each type and named GB-1～3 and G4-1～3.We analyzed the secondary structures of Dual-ODN through Mfold online software.According to the structural characteristics of TLR9 agonist,the stability of Dual-ODN and the specificity of Dual-ODN targeting on TGF-β2 or CTLA-4 mRNA,and the Dual-ODN of GB series and G4 series were scored respectively.We found that in terms of the structural characteristics of TLR9 agonists,it is predicted that GB-2 and GB-3 of GB series and G4-3of G4 series have stronger immunostimulatory effect.In terms of the stability of Dual-ODN,it was predicted that the secondary structures of GB-1 of GB series and G4-1 of G4 series are more stable.In terms of the specificity of Dual-ODN targeting on TGF-β2 or CTLA-4mRNA,all Dual-ODN of GB series and G4 series have good specificity.2.The immunobiological activity of Dual-ODNIn order to determine whether the two types of Dual-ODN designed by ourselves have the expected double targeted immune activity,we mainly studied the following three parts:the first part is the immune activity of Dual-ODN as TLR9 agonist,the second is the inhibitory effect of GB series Dual-ODN on TGF-β2 and the inhibitory effect of G4 Series Dual-ODN on CTLA-4,the third is the therapeutic effect of GB-2 of GB series on subcutaneous melanoma mice.2.1 The immunologic activity of Dual-ODN as TLR9 agonistsWhen we were studying the immunologic activity of Dual-ODN as TLR9 agonists,immune cells proliferation assay,B cell activation and the anti-VSV assay were mainly used.The results showed that the two types of Dual-ODN designed by us could significantly promote the proliferation of mouse splenocytes and B cells,and significantly up-regulate the expression levels of CD80,CD86 and MHCⅡon B cells,indicating that two types of Dual-ODN have the characteristics of B-type CpG ODN.In the study of anti-VSV assay,only GB-2 of GB series was found to have antiviral activity,suggesting that GB-2 has the characteristic of C-type CpG ODN.It can be seen that the two types of Dual-ODN designed by us have the activity of activating TLR9.2.2 The immunosuppressive molecular targeting of Dual-ODNWhen we are studying the molecular targeting of Dual-ODN,the immunosuppressive molecular targeting of GB series and G4 Series Dual-ODN were studied respectively.The results showed that in the inhibition of GB series Dual-ODN on TGF-β2,GB-2 can inhibit mRNA expression level of TGF-β2 in B16 melanoma cells.In the inhibition of G4 Series Dual-ODN on CTLA-4,G4-3 can inhibit the expression of CTLA-4 on CD4⁺T cells in mouse lymph node cells.It can be seen that the GB-2 of GB series and G4-3 of G4 series designed by us can play double targeted roles as Dual-ODN,and can achieve the purpose of our expected design.2.3 The therapeutic effect of GB-2 on melanoma miceWe selected GB-2 to study whether it can play a stronger immune activation effect than single target ODN.We treated subcutaneous melanoma mice with GB-2 and observed its effect on the survival time of tumor bearing mice.The results showed GB-2 could significantly prolong the survival of tumor-bearing mice,and the therapeutic effect was significantly better than single target ODN treatment groups.In conclusion,GB-2 and G4-3 Dual-ODN designed by us have double targeted effects of activating TLR9 and inhibiting immunosuppressive molecules,and they are expected to become new powerful immune enhancers for subsequent tumor immunotherapy or as vaccine adjuvants.