Study Of Strcture-Activity Relationship (SAR) Of CpG ODNs As ISS And Related Anti-tumor Activities In Mice

CpG oligonucleotides (CpG ODNs) are known to stimulate innate and adaptive immunity because of their interesting immunostimulatory properties in a number of vertebrate, such as stimulating B-cell proliferation, enhancing expression and synthesis of cytokines, and promoting NK cell cytotoxicity. Immunostimulatory activities of CpG ODNs depend on their structural and chemical characteristics. Though a number of researchers studied the relationship between the CpG ODNs structure and their immunostimulatory properties, more detailed and systematic structure-activity relationship (SAR) studies of CpG ODNs were still lacked. Recently CpG ODNs have been explored for cancer therapy, due to their immunostimulatory properties, such as inducing the release of large amounts of type I IFN, inhibitors of the synthesis of Th2 cytokines, inducers of Th1 cytokine synthesis, and the activation of NK cytotoxicity or CTL precursors into antitumor CTLs.In this study, we tried to reveal some changes of the immunostimulatory properties of CpG ODNs induced by the modifications of ODN structure, for instance, additions and deletions of the functional CpG motifs, modification of the distance and the sites of CpG motifs, addition of self- complementary sequences, etc. We recruited CpG 1826, a confessedly effective immnostimulatory B-type CpG ODN for murine, as the positive control and the template of the SAR study. Various modification of the primary structure were performed on the basis of the ODN 1826, and immunostimulatory activities of the structurally-modified CpG ODNs were comprehensively investigated, including the ability to stimulate mouse B cells proliferation, ability to induce cytokines secretion (IL-6, IL-12 and IFN-α, etc.), and to induce NK cell killing activity, and the changes of expression of various lymphocyte surface molecules. Several excellent CpG ODNs with good immunostimulatory activities were screened; best of whom, 0DN4, 0DN7, ODN10 and ODN11 were comprehensively evaluated, including the immunostimulatory activity and the anti-tumor activity in vitro and in vivo. Furthermore, the molecular mechanism of CpG ODN as ISS was partly investigated.1. To reveal the Structure-Activity Relationship (SAR) study of the immunostimulatory CpG ODN for murine, a series of CpG ODNs with different primary structures were synthesized. Their capabilities to stimulate mouse spleen cells proliferation were determined by [³H] thymidine incorporation assay, cytokine secretion (IL-6, IL-12 and IFN-α) spectra induced by CpG ODNs were assessed by ELISA, abilities to induce NK cell killing activity of CpG ODNs were evaluated by standard 4-h ⁵¹Cr-release assay. An effective CpG ODN for murine, ODN1826 was set as the template of modification and positive control. Results showed that 1) the optimal stimulatory hexameric CpG motif for murine was 5'-TCGACGTT-3', changed the hexameric motif from "GACGTT" to "GTCGTT" weakened the immunostimulatory activity of the ODN. 2) The CpG island without flank dinulcleotide seemed no immunostimulatory efficiency, for instance, the ODN15. 3) The number of CpG motif was not a vital factor for immunostimulatory activity, simply add a hexameric CpG motif (ODN9) contributed no significance to immunostimulatory activity, and in the ODN11, the number of hexameric CpG motif was decreased to one, but the activity of this ODN improved, of course, the improvement of the activity might be derived from the 3'-end palindrome tail. 4) Owning to lack of a TpC dinucleotide on the 5' end, the immune-stimulatory effects of the ODN6 and ODN8 were weakened. 5) The optimal distance between two hexameric CpG motifs seemed to be three nucleotides, modification the distance to a shorter (ODN5, the two motifs were connective) or a longer (ODN6, the two motifs spaced by an octanucleotides) one both weakened the comprehensive immunostimulatory activity. 6) Palindrome was essential to the immunostimulatory potency of a CpG ODNs. Results showed that, all ODNs with best general immunostimulatory activities were that with a self-complementary palindrome sequence, such as ODN4, ODN7, ODN10, and ODN11. However, 7) the palindrome structures must to be at the 3'-end of the sequence, once the palindrome moved to the 5'-end, for instance, the ODN3, ODN12 and ODN15, the immunostimulatory activity were then almost totally lost. ODN10, one of the most potent ODNs involved in the study, possessed a holistic self-complementary palindrome structure (5'-TCCATGACGTTTTAAAACGTCATGGA-3'). In theory, the sequence would form a big hairpin structure, so it was hard to keep a free 5'-end. But the comprehensive immunostimulatory activity of ODN10 was distinguished. Furthermore, in another study, a cyclic CpG ODNs without any free end behaved potent immunostimulatory efficiency (data not shown), this result challenged the hypothesis that the free 5' end was essential to keep the immunostimulatory activity for a CpG ODN. Undoubtedly, more investigations still needed to be carried out to elucidate it.2. The analysis of cytokines secretion was a traditional method for SAR of CpG ODN. Generally, a certain cytokine be secreted by several immune cells, for example IFN-αsecretion by pDC or cells of mononuclear phagocyte system (MPS). So we could identify which cells were influenced by CpG ODN using by flow cytometry analysis. Activation of B cell is characterized by up-regulation of cell-surface molecules such as costimulatory molecules CD80, and CD19 is the characteristic surface marker of B cell. CpG ODN can also activate pDC and promote its maturation, and one of the mature markers is up-regulation of MHC II molecules (Ia^b) expression on pDC, and CD11c is the characteristic surface marker of pDC. In the study, we determine the effect of CpG ODN on different immune cells activity by flow cytometry analysis on the basis of these marker molecules. Results showed that all CpG ODNs, except ODN3, up-regulated the level of CD80 expression on CD19-positive cells, in which ODN1826, 0DN4, 0DN7, ODN10 and ODN11 were especially efficient. As to Ia^b expression on CD11c-positive cells and CD94 expression on NK cells, the status were similar, the most potent CpG ODNs were still the ODN4, ODN7, ODN10 and ODN11. These results were consistent with previous reports.3. To evaluate the anti-tumor activity of CpG ODNs, we detected the lytic activity of mouse spleen cells induced by CpG ODNs against B16.F1 melanoma cells in vitro. All groups treated with CpG ODNs (ODN1-11) showed anti-tumor activiy when compared to the non-CpG ODN (P < 0.05), and the activity of ODN4, ODN7, ODN10 and ODN11 were more potent, when compared to ODN1826 (P < 0.05).4. Therapeutic effects of CpG ODN (ODN1826, ODN4, ODN7, ODN10, and ODN11) as mono-therapeutical agents in murine melanoma were measured in vivo. All the CpG ODNs could significantly prolonged survival of mice with B16 melanoma when compared to the PBS treatment (P < 0.01), and the ODN10 group showed more potential (P< 0.001). In addition, both ODN10 and ODN11 groups showed effectively compared wtih ODN 1826 (P < 0.01). ODN1826 inhibited the tumor growth by 17.8 %. In contrast, ODN10 and ODN11 inhibited the tumor growth by 55.2 % and 40.7 % on day 20, respectively. Histopathological analysis showed that CpG ODN treatment could make the tumor cell exhibit extensive cellular polymorphism including cavitate nuclei, swollen nuclei, and apoptosis. In the absence of CpG ODN treatment, tumor-bearing mice appeared large-scale necroses and vasal infarct by metastasis melanoma mass in spleen, and metastasis melanoma in vessel of oxter lymph nodes. Both spleen and oxter lymph nodes were normal in CpG ODN treatment group. Immunohistochemistry analysis showed that proliferating cell nuclear antigen (PCNA) was expresses exclusively in tumor tissues with melanoma mice, and CpG ODN treatment could decrease its expression. Expression of CD63, a melanoma metastasis-associated antigen, was similar to PCNA. The results suggest that CpG ODNs can inhibit B16 melanoma growth and metastasis in vivo, and novel ODN10 presented more potent activity.5. Immunologic effects of CpG ODN (ODN 1826, ODN4, ODN7, ODN10, and ODN11) in tumor-bearing mice were measured in vivo. In tumor-bearing mice, proliferation of B cells and T cells, NK cytotoxicity of splenocytes, phagocytic ability of peritoneal macrophage, and IL-12 secretion level were lower, and the IgE levels were higher than those in normal mice (P<0.05). Above immune activities from CpG ODN-treated tumor-bearing mice dramatically increased compared with the PBS-treated ones (P<0.01), and those of ODN10 group was more significantly than ODN 1826 group (P<0.05). Immunohistochemistry analysis showed that CpG ODN could induce CD80 expression in tumor tissues with melanoma mice. The present study suggested that CpG ODN could activate the host anti-tumor immune response to inhibit the growth of melanoma tumor in mice.6. Further and deeper investigations on the immunostimulatory mechanism of CpG ODNs showed that the different structural CpG ODN could increase the different transcription activity of NF-κB and AP1, and presented different binding activity with TLR9. It has been indicated that a receptor, TLR9, is engaged in the recognition of CpG ODN and subsequent initiation of the signaling cascade leading to the activation of the transcription factor NF-κB or AP1. Electrophoretic Mobility Shift Assay (EMSA) results showed that CpG ODNs could increase the expression of NF-κB and AP1. In the concrete, ODN10 could induce NF-κB expression, and its activity was lower than 1826 or 0DN11. On the other hand, ODN10 could induce AP1 expression, and its activity was higher than 1826 or ODN11. Maybe ODN10 promoting high level of IL-12 secretion was associated with the AP1 over expression. In addition, previous studies showed that TLR9 reads CpG DNA sequence from 5'- to the 3'-end, and modifying by 5'-end block abrogate immunostimulation. We measured the binding activity between TLR9 and CpG ODNs (three types: ODN1826, ODN10 and ODN11), and first found that three types CpG ODN were able to integrate with TLR9, though they were labeled and blocked by biotin at their 5'-end. Namely, biotin conjugation at the 5'-end did not effect the binding activity between TLR9 and CpG ODNs. It suggested that the binding of TLR9 and CpG ODNs is not associated with the free 5'-end of the CpG ODN sequence. We also found that different type CpG ODN was in different states: ODN1826 was in a single linear structure; ODN10 possessed three structures: linear, dimeric and holistic self-complementary palindrome structure, namely, a big hairpin structure; ODN11 also possessed three type structures: linear, dimeric and a partly palindrome one. It showed that all the different structures of CpG ODNs could bind with TLR9. Both ODN10 and ODN11 possessed significant characters of C-class CpG ODN, viz. strongly stimulate B cells as well as type I IFN and IL-12 secretion, maybe due to their structural multiformity.