Granzyme B PET Imaging in Response to In Situ Vaccine Therapy Combined with αPD1 in a Murine Colon Cancer Model

Immune checkpoint inhibitors (ICIs) block checkpoint receptors that tumours use for immune evasion, allowing immune cells to target and destroy cancer cells. Despite rapid advancements in immunotherapy, durable response rates to ICIs remains low. To address this, combination clinical trials are underway assessing whether adjuvants can enhance responsiveness by increasing tumour immunogenicity. CpG-oligodeoxynucleotides (CpG-ODN) are synthetic DNA fragments containing an unmethylated cysteine-guanosine motif that stimulate the innate and adaptive immune systems by engaging Toll-like receptor 9 (TLR9) present on the plasmacytoid dendritic cells (pDCs) and B cells. Here, we have assessed the ability of AlF-mNOTA-GZP, a peptide tracer targeting granzyme B, to serve as a PET imaging biomarker in response to CpG-ODN 1585 in situ vaccine therapy delivered intratumourally (IT) or intraperitoneally (IP) either as monotherapy or in combination with αPD1. [18F]AlF-mNOTA-GZP was able to differentiate treatment responders from non-responders based on tumour uptake. Furthermore, [18F]AlF-mNOTA-GZP showed positive associations with changes in tumour-associated lymphocytes expressing GZB, namely GZB+ CD8+ T cells, and decreases in suppressive F4/80+ cells. [18F]AlF-mNOTA-GZP tumour uptake was mediated by GZB expressing CD8+ cells and successfully stratifies therapy responders from non-responders, potentially acting as a non-invasive biomarker for ICIs and combination therapy evaluation in a clinical setting.

CpG Oligodeoxynucleotides for Anticancer Monotherapy from Preclinical Stages to Clinical Trials

CpG oligodeoxynucleotides (CpG ODNs), the artificial versions of unmethylated CpG motifs that were originally discovered in bacterial DNA, are demonstrated not only as potent immunoadjuvants but also as anticancer agents by triggering toll-like receptor 9 (TLR9) activation in immune cells. TLR9 activation triggered by CpG ODN has been shown to activate plasmacytoid dendritic cells (pDCs) and cytotoxic T lymphocytes (CTLs), enhancing T cell-mediated antitumor immunity. However, the extent of antitumor immunity carried by TLR agonists has not been optimized individually or in combinations with cancer vaccines, resulting in a decreased preference for TLR agonists as adjuvants in clinical trials. Although various combination therapies involving CpG ODNs have been applied in clinical trials, none of the CpG ODN-based drugs have been approved by the FDA, owing to the short half-life of CpG ODNs in serum that leads to low activation of natural killer cells (NK cells) and CTLs, along with increases of pro-inflammatory cytokine productions. This review summarized the current innovation on CpG ODNs that are under clinical investigation and explored the future direction for CpG ODN-based nanomedicine as an anticancer monotherapy.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

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

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.

A Novel CpG Oligodeoxynucleotide Enhanced the Therapeutic Effect of Epirubicin on Bladder Tumors in Rats

Background CpG oligodeoxynucleotides, which boast anti-inflammatory, anti-infectious, and chemotherapeutic activities, are promising immunomodulators. Recently, some preclinical studies have highlighted the potent immunostimulatory and anti-tumor effects of CpG oligodeoxynucleotides, which has aroused interest in their potential clinical effects on human cancers. Methods In this study, we evaluated the therapeutic effect of a new type of CpG oligodeoxynucleotide whose sequence (5’-AACGTTGTCGTCGACGTCGTCGTCAGGCCTGACGTTATCGATGGCGTTGTCGTCAACGTTGTCGTTAACGTT-3’) was designed by our laboratory in combination with epirubicin in a bladder tumor rat model induced by N-methyl-N-nitrosourea instillation. Moreover, we explored the safety of the novel CpG oligodeoxynucleotide for bladder tumor therapy by observing the degree of cystolith in the bladder and comparing the results against those of Bacillus Calmette–Guérin therapy, which is a gold-standard treatment for bladder tumor. Results All results showed that CpG oligodeoxynucleotide combined with epirubicin significantly inhibited the growth of bladder tumors and reduced the pathological grading. As compared with bladder cells or cytokines observed under the positive control or epirubicin-alone treatment conditions, all indexes including histopathological grading, Mutation P53 gene protein expression, and Interleukin-2 (IL-2) level were significantly optimized by instillation of CpG oligodeoxynucleotide. Immunohistochemical examination indicated that CpG oligodeoxynucleotide reduced the expression of Mutation P53 gene protein in the bladder tumor rat model. Specifically, the level of IL-2 in rat serum was increased by more than 30% CpG oligodeoxynucleotide treatment combined with epirubicin. Also, in comparison with the degree of cystolith observed in the Bacillus Calmette–Guérin group, no obvious side effects were caused by CpG oligodeoxynucleotide. Conclusions CpG oligodeoxynucleotide as an immunomodulator can enhance the efficacy of epirubicin and presents higher safety than Bacillus Calmette–Guérin in treating bladder cancer.

607 Regional delivery of a TLR9 agonist to boost checkpoint inhibitor responsiveness in liver metastases

BackgroundClass C TLR9 agonists, CpG oligodeoxynucleotides (ODNs) enhance responsiveness to anti-PD1 therapy in solid tumors through favorable modulation of the tumor microenvironment (TME) [1]. Recently, we reported that regional delivery of a TLR9 agonist eliminated myeloid derived suppressor cells (MDSC) and promoted pro-inflammatory/anti-tumorigenic M1 macrophage programming in the TME of liver metastases (LM) [2]. Further, we found enhanced TLR9 activation in LM following regional TLR9 agonist infusion compared to the systemic treatment. We hypothesize that regional delivery of a TLR9A into LM will enhance the responsiveness to systemically infused anti-PD1 therapy.MethodsIn this study, we treated mice with established MC38-CEA-Luc LM with ODN-2395 (30µg/mouse) regionally with or without anti PD-1 antibody (250µg/mouse) intraperitoneally.ResultsControl of LM growth (Figure 1) was significantly higher with combinatorial treatment as compared to anti-PD1 (p<0.01) or PBS treatments (p<0.05). To study the impact of TLR9 activation on human MDSC, we treated healthy donor PBMCs with ODN-2395 or SD101. We found that both reduced the hu-MDSC (CD11b+CD33+HLADR-) population in a dose-dependent manner with an increase in PD-L1 expression as determined by flow cytometry (FC) analysis (Figure 2). Moreover, by using Luminex, demonstrated that ODN-2395 and SD101 enhanced expression of IL 29, IFNα, and NFκB, along with downstream cytokines IL 6 and IL 10. To investigate the effect of SD101 in modulating the differentiation of huMDSC from huPBMC, we treated huPBMC with IL6+GM-CSF in the presence or absence of SD101. By FC analysis, we found that SD-101 blocked huMDSC development induced by IL6+GM-CSF, preferentially limited the more immunosuppressive monocytic MDSC subtype, and drove M1 macrophage polarization. Treatment of SD101 only once for 48hrs was sufficient to inhibit huMDSC differentiation for two weeks.Abstract 607 Figure 1Combinatorial treatment of CPI and ODN’s reduces tPV = portal vein; IP = intraperitoneal.Eight to twelve weeks old male C57/BL6 mice were challenged intra-splenic with 0.5e6 MC38-CEA-Luc cells for a week. Bioluminescence value was determined by IVIS on D0, and mice were randomized accordingly and treated with 30 µg/mouse ODN2395 via PV with or without 250 µg/mouse anti-PD1 antibody via IP on D0, D+3 and D+6. PBS served as the vehicle (Veh) control and administered via PV. Fold change of the tumor burden was calculated based on D0 baseline bioluminescence. Tumor progression was analyzed unpaired t test among groups. (*p <0.05).Abstract 607 Figure 2Human PBMC treated with ODN2395 and SD101 reducesCtrl = control; MDSC = ODN = oligodeoxynucleotide’ PBMC = peripheral blood monocytes.Human PBMC were isolated from the Leukoreduction system chamber. 1e6/ml PBMCs were treated with increasing concentrations (0.04–10 µM) SD101, ODN2395 along with ctrl ODN5328 (1µM) for 48 hours. Panels A and B: MDSC population and their corresponding PD–L1 expression were evaluated (n=12). Four donors with three replicates were used. Data represented as mean ± SEM.ConclusionsBoth the in vitro and in vivo findings suggest that regional TLR9 stimulation in a model of LM improves responsiveness to systemic anti-PD-1 therapy through elimination of MDSC, and the effect on huMDSC was confirmed in vitro. Increased PDL-1 expression in response to TLR9 stimulation among MDSC may further enhance the anti-PD-1 effect. Therefore, combing regional infusions of a TLR9 agonist with systemic anti-PD-1 agents may be a promising approach for liver tumor treatment.ReferencesWang, S., et al., Intratumoral injection of a CpG oligonucleotide reverts resistance to PD-1 blockade by expanding multifunctional CD8+ T cells. Proc Natl Acad Sci U S A, 2016. 113(46): p. E7240-E7249.Ghosh CC, H.K., O’Connell K, Laporte J, Guha P, Cox B, Jaroch D, Katz SC, Regional administration of class C CpG Oligodeoxynucleotides results in superior intrahepatic TLR9 activation and immunomodulation compared to systemic infusion, Abstract: AACR Annual Meeting. 2021.

Enhancement of the Immunostimulatory Effect of Phosphodiester CpG Oligodeoxynucleotides by an Antiparallel Guanine-Quadruplex Structural Scaffold

Guanine-quadruplex-based CpG oligodeoxynucleotides (G4 CpG ODNs) have been developed as potent immunostimulatory agents with reduced sensitivity to nucleases. We designed new monomeric G4 ODNs with an antiparallel topology using antiparallel type duplex/G4 ODNs as robust scaffolds, and we characterized their topology and effects on cytokine secretion. Based on circular dichroism analysis and quantification of mRNA levels of immunostimulatory cytokines, it was found that monomeric antiparallel G4 CpG ODNs containing two CpG motifs in the first functional loop, named G2.0.0, could maintain antiparallel topology and generate a high level of immunostimulatory cytokines in RAW264 mouse macrophage-like cell lines. We also found that the flanking sequence in the CpG motif altered the immunostimulatory effects. Gc2c.0.0 and Ga2c.0.0 are monomeric antiparallel G4 CpG ODNs with one cytosine in the 3′ terminal and one cytosine/adenine in the 5′ terminal of CpG motifs that maintained the same resistance to degradation in serum as G2.0.0 and improved interleukin-6 production in RAW264 and bone marrow-derived macrophages. The immunostimulatory activity of antiparallel G4 CpG ODNs is superior to that of linear natural CpG ODNs. These results provide insights for the rational design of highly potent CpG ODNs using antiparallel G4 as a robust scaffold.

Macrophages Impair TLR9 Agonist Antitumor Activity through Interacting with the Anti-PD-1 Antibody Fc Domain

Background. A combination of TLR9 agonists and an anti-PD-1 antibody has been reported to be effective in immunocompetent mice but the role of innate immunity has not yet been completely elucidated. Therefore, we investigated the contribution of the innate immune system to this combinatorial immunotherapeutic regimens using an immunodeficient mouse model in which the effector functions of innate immunity can clearly emerge without any interference from T lymphocytes. Methods. Athymic mice xenografted with IGROV-1 human ovarian cells, reported to be sensitive to TLR9 agonist therapy, were treated with cytosine–guanine (CpG)-oligodeoxynucleotides (ODNs), an anti-PD-1 antibody or their combination. Results. We found that PD-1 blockade dampened CpG-ODN antitumor activity. In vitro studies indicated that the interaction between the anti-PD-1 antibody fragment crystallizable (Fc) domain and macrophage Fc receptors caused these immune cells to acquire an immunoregulatory phenotype, contributing to a decrease in the efficacy of CpG-ODNs. Accordingly, in vivo macrophage depletion abrogated the detrimental effect exerted by the anti-PD-1 antibody. Conclusion. Our data suggest that if TLR signaling is active in macrophages, coadministration of an anti-PD-1 antibody can reprogram these immune cells towards a polarization state able to negatively affect the immune response and eventually promote tumor growth.