Immunologic Effects of Stereotactic Body Radiotherapy in Dogs with Spontaneous Tumors and the Impact of Intratumoral OX40/TLR Agonist Immunotherapy

Stereotactic body radiotherapy (SBRT) is known to induce important immunologic changes within the tumor microenvironment (TME). However, little is known regarding the early immune responses within the TME in the first few weeks following SBRT. Therefore, we used the canine spontaneous tumor model to investigate TME responses to SBRT, and how local injection of immune modulatory antibodies to OX40 and TLR 3/9 agonists might modify those responses. Pet dogs with spontaneous cancers (melanoma, carcinoma, sarcoma, n = 6 per group) were randomized to treatment with either SBRT or SBRT combined with local immunotherapy. Serial tumor biopsies and serum samples were analyzed for immunologic responses. SBRT alone resulted at two weeks after treatment in increased tumor densities of CD3+ T cells, FoxP3+ Tregs, and CD204+ macrophages, and increased expression of genes associated with immunosuppression. The addition of OX40/TLR3/9 immunotherapy to SBRT resulted in local depletion of Tregs and tumor macrophages and reduced Treg-associated gene expression (FoxP3), suppressed macrophage-associated gene expression (IL-8), and suppressed exhausted T cell-associated gene expression (CTLA4). Increased concentrations of IL-7, IL-15, and IL-18 were observed in serum of animals treated with SBRT and immunotherapy, compared to animals treated with SBRT. A paradoxical decrease in the density of effector CD3+ T cells was observed in tumor tissues that received combined SBRT and immunotherapy as compared to animals treated with SBRT only. In summary, these results obtained in a spontaneous large animal cancer model indicate that addition of OX40/TLR immunotherapy to SBRT modifies important immunological effects both locally and systemically.

Acquired Drug Resistance Enhances Imidazoquinoline Efflux by P-Glycoprotein

Multidrug-Resistant (MDR) cancers attenuate chemotherapeutic efficacy through drug efflux, a process that transports drugs from within a cell to the extracellular space via ABC (ATP-Binding Cassette) transporters, including P-glycoprotein 1 (P-gp or ABCB1/MDR1). Conversely, Toll-Like Receptor (TLR) agonist immunotherapies modulate activity of tumor-infiltrating immune cells in local proximity to cancer cells and could, therefore, benefit from the enhanced drug efflux in MDR cancers. However, the effect of acquired drug resistance on TLR agonist efflux is largely unknown. We begin to address this by investigating P-gp mediated efflux of TLR 7/8 agonists. First, we used functionalized liposomes to determine that imidazoquinoline TLR agonists Imiquimod, Resiquimod, and Gardiquimod are substrates for P-gp. Interestingly, the least potent imidazoquinoline (Imiquimod) was the best P-gp substrate. Next, we compared imidazoquinoline efflux in MDR cancer cell lines with enhanced P-gp expression relative to parent cancer cell lines. Using P-gp competitive substrates and inhibitors, we observed that imidazoquinoline efflux occurs through P-gp and, for Imiquimod, is enhanced as a consequence of acquired drug resistance. This suggests that enhancing efflux susceptibility could be an important consideration in the rational design of next generation immunotherapies that modulate activity of tumor-infiltrating immune cells.

Increasing the potency of dendritic cell based vaccines for the treatment of cancer

<p>Tumours can be eradicated by T cells that recognise unique tumour-associated antigens. These T cells are initially stimulated by dendritic cells (DCs) that have acquired antigens from tumour tissue. Vaccination strategies that increase the frequencies of tumour-specific T cells by enhancing the activity of DCs are being evaluated in the clinic as novel cancer therapies. Our hypothesis is that existing DC-based vaccination strategies can be improved by stimulating toll-like receptor (TLR) signalling in the DCs, and also by encouraging interactions with iNKT cells, as these two activities are known to enhance DC function. It was also hypothesised that superior T cell responses could be induced by combining these two activities together. We used the TLR 4 agonist monophosphoryl lipid A (MPL) alone and in combination with other TLR agonists to achieve effective activation of bone marrow-derived DCs (BM-DCs) cultured in-vitro, which was characterised by upregulated expression of maturation markers on the cell surface, and enhanced release of pro-inflammatory cytokines. Some TLR agonist combinations provided significantly enhanced activities in this regard, notably the combination of MPL with either the TLR 2 agonist Pam3Cys, or the TLR 7/8 agonist Resiquimod. Although in-vitro activated BM-DCs were unable to induce stronger antigen-specific CD8+ T cell responses after intravenous injection when compared to BMDCs without TLR stimulation, enhanced CD8+ T cell responses were achieved in-vivo with the co-administration of TLR ligands, implying that TLR stimulation needed to act on cells of the host. Further studies identified the langerin-expressing CD8ɑ+ splenic DC subset in the spleen as recipients of antigen that was transferred from injected cells, and that these recipients were participants in the cross-presentation and T cell priming activities driving the CD8+ T cell response after vaccination. Antigen-loaded BM-DCs carrying the NKT cell ligand ɑ-galactosylceramide (ɑ-GalCer) were found to consistently increase antigen-specific CD8+ T cell responses in-vivo, and also cytotoxic responses as seen in cytotoxic killing assays. Again, langerin-expressing CD8ɑ+ splenic DCs were shown to be involved in this response by acquiring antigen and ɑ-GalCer from the injected vaccine BM-DCs. Finally, it was possible to achieve even greater CD8+ T cell responses in-vivo by injecting BM-DCs carrying antigen and ɑ-GalCer, together with timely co-administration of the TLR agonist. These results suggest a reassessment of the activities of DC-based vaccines to include the important role of “courier” to DCs already resident in the host that can be exploited to improve vaccination outcomes.</p>

Increasing the potency of dendritic cell based vaccines for the treatment of cancer

<p>Tumours can be eradicated by T cells that recognise unique tumour-associated antigens. These T cells are initially stimulated by dendritic cells (DCs) that have acquired antigens from tumour tissue. Vaccination strategies that increase the frequencies of tumour-specific T cells by enhancing the activity of DCs are being evaluated in the clinic as novel cancer therapies. Our hypothesis is that existing DC-based vaccination strategies can be improved by stimulating toll-like receptor (TLR) signalling in the DCs, and also by encouraging interactions with iNKT cells, as these two activities are known to enhance DC function. It was also hypothesised that superior T cell responses could be induced by combining these two activities together. We used the TLR 4 agonist monophosphoryl lipid A (MPL) alone and in combination with other TLR agonists to achieve effective activation of bone marrow-derived DCs (BM-DCs) cultured in-vitro, which was characterised by upregulated expression of maturation markers on the cell surface, and enhanced release of pro-inflammatory cytokines. Some TLR agonist combinations provided significantly enhanced activities in this regard, notably the combination of MPL with either the TLR 2 agonist Pam3Cys, or the TLR 7/8 agonist Resiquimod. Although in-vitro activated BM-DCs were unable to induce stronger antigen-specific CD8+ T cell responses after intravenous injection when compared to BMDCs without TLR stimulation, enhanced CD8+ T cell responses were achieved in-vivo with the co-administration of TLR ligands, implying that TLR stimulation needed to act on cells of the host. Further studies identified the langerin-expressing CD8ɑ+ splenic DC subset in the spleen as recipients of antigen that was transferred from injected cells, and that these recipients were participants in the cross-presentation and T cell priming activities driving the CD8+ T cell response after vaccination. Antigen-loaded BM-DCs carrying the NKT cell ligand ɑ-galactosylceramide (ɑ-GalCer) were found to consistently increase antigen-specific CD8+ T cell responses in-vivo, and also cytotoxic responses as seen in cytotoxic killing assays. Again, langerin-expressing CD8ɑ+ splenic DCs were shown to be involved in this response by acquiring antigen and ɑ-GalCer from the injected vaccine BM-DCs. Finally, it was possible to achieve even greater CD8+ T cell responses in-vivo by injecting BM-DCs carrying antigen and ɑ-GalCer, together with timely co-administration of the TLR agonist. These results suggest a reassessment of the activities of DC-based vaccines to include the important role of “courier” to DCs already resident in the host that can be exploited to improve vaccination outcomes.</p>

Bacille Calmette-Guérin vaccine reprograms human neonatal lipid metabolism in vitro and in vivo

SummaryVaccines have generally been developed with limited insight into their molecular impact. While systems vaccinology, including metabolomics, enables new characterization of vaccine mechanisms of action, these tools have yet to be applied to infants at high risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) protects infants against disseminated tuberculosis (TB) and TB-unrelated infections via incompletely understood mechanisms. We employed mass spectrometry-based metabolomics of blood plasma to profile BCG-induced infant responses in Guinea Bissau in vivo and the U.S. in vitro. BCG selectively altered plasma lipid pathways, including lysophospholipids. BCG-induced lysophosphatidylcholines (LPCs) correlated with both TLR agonist- and purified protein derivative (PPD, mycobacterial antigen)-induced blood cytokine production in vitro, raising the possibility that LPCs contribute to BCG immunogenicity. Analysis of an independent newborn cohort from The Gambia demonstrated shared vaccine-induced metabolites such as phospholipids and sphingolipids. BCG-induced changes to the plasma lipidome and LPCs may contribute to its immunogenicity and inform the discovery and development of early life vaccines.HighlightsNeonatal BCG immunization generates distinct metabolic shifts in vivo and in vitro across multiple independent cohorts.BCG induces prominent changes in concentrations of plasma lysophospholipids (LPLs)BCG induced changes in plasma lysophosphatidylcholines (LPCs) correlate with BCG effects on TLR agonist- and mycobacterial antigen-induced cytokine responses.Characterization of vaccine-induced changes in metabolism may define predictive signatures of vaccine responses and inform early life vaccine development.Abstract FigureGraphical abstract:BCG vaccination perturbs metabolic pathways in vivo and in vitro.Vaccines have traditionally been developed empirically, with limited insight into their impact on molecular pathways. Metabolomics provides a new approach to characterizing vaccine mechanisms but has not yet been applied to human newborns, who are at the highest risk of infection and receive the most vaccines. Bacille Calmette-Guérin (BCG) prevents disseminated mycobacterial disease in children and can induce broad protection to reduce mortality due to non-TB infections. Underlying mechanisms are incompletely characterized. Employing mass spectrometry-based metabolomics, we demonstrate that early BCG administration alters the human neonatal plasma metabolome, especially lipid metabolic pathways such as lysophosphatidylcholines (LPCs), both in vivo and in vitro. Plasma LPCs correlated with both innate TLR-mediated and PPD antigen-induced cytokine responses suggesting that BCG-induced lipids might contribute to the immunogenicity of this vaccine. Vaccine-induced metabolic changes may provide fresh insights into vaccine immunogenicity and inform the discovery and development of early life vaccines.

860 The anti-tumor activity of HSP-90 therapeutic cancer vaccine (AST-021p) combine with TLR2/3 agonist in a MMTV-neu transgenic model

BackgroundAST-021p, which is derived from HLA class II binding epitopes of human HSP90 protein, is an investigational therapeutic cancer vaccine for the malignant neoplasms. AST-021p is designed to demonstrate the immunologic efficacy by activating antigen-specific CD4+ Th1 cell in humans. Due to their ability to link the innate with the adaptive immune response, Toll-like receptor (TLR) agonists are highly promising as adjuvants in vaccines against life-threatening and complex diseases such as cancer, AIDS and malaria. In this study, AST-021p was investigated to evaluate the immunogenicity and tumor growth inhibitory effect under the condition of combining with various immune adjuvants derived from TLR agonists, using in-vivo model.MethodsThree different agonists of TLR (TLR-4, TLR-2/3, TLR-7/8) were assigned to investigate the immunogenicity in each group (4 FVB mice/group, total 4 groups). AST-021p was intradermally injected 3 times with different TLR-agonists and the immunogenicity was assessed from mouse splenocyte by HSP90-specific IFN-γ ELISpot method. We also examined the efficacy of AST-021p and selected TLR-agonist in MMTVneu Tg mice (4 mice/group, conducted twice and A total 8 mice was assigned to each group). The combination of AST-021p and TLR-2/3 agonist (AST-021p plus TLR-2/3 agonist) was injected 3 times every 10 days to mice followed by inoculated mouse mammary cancer cell line. The tumor volume change and immunogenicity were evaluated.ResultsThe most effective TLR-agonist as a potent immune adjuvant was a TLR-2/3 agonist (L-pampoTM, supplied by CHA Vaccine Institute). In MMTV-Neu transgenic mice, AST-021p (100 μg) plus TLR-2/3 agonist significantly enhanced immunogenicity by increasing up to 130±10 HSP-90 epitope specific T cells per 1x105 splenocytes (P<0.001). AST-021p plus TLR-2/3 agonist also showed higher tumor growth inhibitory effect (170±108 mm3) on post-implantation 35th day by suppressing mouse mammary cancer cell line (5x105)-derived tumor growth, compared with a TLR-2/3 agonist alone (1031±450 mm3).ConclusionsCombination regimen of AST-021p and TLR-2/3 agonist (as immune adjuvant) demonstrated significant immunogenicity and tumor prevention effect in in-vivo study. These data supported the clinical study of AST-021p combined with TLR-2/3 agonist as active immune adjuvant in certain tumor types, and phase 1/2 clinical program would be expected to be initiated.AcknowledgementsNot applicableTrial RegistrationNot applicableReferencesCsermely P, Schnaider T, Soti C, Prohaszka Z, Nardai G. The 90-kDa molecular chaperone family: structure, function, and clinical applications. A comprehensive review. Pharmacol Ther 1998;79,129–168.Wang H, Lu M, Yao M, Zhu W. Effects of treatment with an Hsp90 inhibitor in tumors based on 15 phase II clinical trials. Mol Clin Oncol 2016;5,326–334.Ramalingam S, Goss G, Rosell R. Schmid-Bindert G, Zaric B, Andric Z, Bondarenko I, Komov D, Ceric T, Khuri F. A randomized phase II study of ganetespib, a heat shock protein 90 inhibitor, in combination with docetaxel in second-line therapy of advanced non-small cell lung cancer (GALAXY-1). Ann Oncol Off J Eur Soc Med Oncol 2015,26,1741–1748.Ethics ApprovalAll experimental procedures involving mice were performed with the guidance protocols approved by the Institutional Animal Care and Use Committee of Korea University (IACUC, Approval number: KOREA-2019-129)ConsentIt is not an abstract containing sensitive or identifiable information.

A TLR5 mono-agonist restores inhibited immune responses to Streptococcus pneumoniae during influenza virus infection in human monocytes

Influenza A virus (IAV) predisposes individuals to often more severe secondary bacterial infections with Streptococcus pneumonia (S. pneumoniae). The outcomes of these infections may be made worse with the increase in antimicrobial resistance and a lack of new treatments to combat this. Th17 responses are crucial in clearing S. pneumoniae from the lung. We previously demonstrated that early IAV infection of human monocytes significantly reduced levels of S. pneumoniae-driven cytokines involved in the Th17 response. Here, we have further identified that IAV targets specific TLRs (TLR2, TLR4, TLR9) involved in sensing S. pneumoniae infection resulting, in a reduction in TLR agonist-induced IL-23 and TGF-β. The effect of IAV is more profound on the TLR2 and TLR9 pathways. We have established that IAV-mediated inhibition of TLR9-induction is related to a downregulation of RORC, a Th17 specific transcription factor. Other studies using mouse models demonstrated that TLR5 agonism improved the efficacy of antibiotics in the treatment of IAV/S. pneumoniae co-infections. Therefore, we investigated if TLR5 agonism could restore inhibited Th17 responses in human monocytes. Levels of pneumococcus-driven cytokines, which had previously been inhibited by IAV were not reduced in the presence of the TLR5 mono-agonist, suggesting that such treatment may overcome IAV inhibition of Th17 responses. The importance of our research is in demonstrating the IAV directly targets S. pneumoniae-associated TLR pathways. Additionally, the IAV-inhibition of Th17 responses can be restored by TLR5 agonism, which indicates that there may be a different Th17 signalling pathway which is not affected by IAV infection.

P09.03 Hyaluronic acid as a new immunologic adjuvant in cancer: design of effective preventive and therapeutic vaccination strategies for HER2/neu-positive breast tumors

BackgroundThe use of proteins as immunogens is attractive for the development of vaccines, but requires efficient adjuvants to overcome their weak immunogenicity. Recently, we investigated the potential of the TLR2/4 agonist hyaluronan (HA) as an immunological adjuvant for protein-based vaccines.1 2 Conjugation of HA to antigens strongly increased their immunogenicity and promoted their rapid translocation to draining lymph nodes, resulting in robust and long-lasting humoral responses.1 On these bases, we investigated the potentiality of HA-based technology in the design of cancer vaccines. To this aim, HA was conjugated to the extracellular domain of rat HER2/neu (rHER2/neu) and validated in the preventive and therapeutic vaccination settings.Materials and MethodsFemale BALB/c or BALB-neuT mice were immunized with rHER2/neu-HA. In vivo depletion of CD4+, CD8+ T and B cells was performed, and sera and spleens were collected to characterized antigen-specific humoral and cellular responses. Vaccinated BALB/c mice were challenged and re-challenged with rHER2/neu-overexpressing TUBO cells to assess the protective or therapeutic activity of rHER2/neu-HA vaccination strategy, as well as immunological memory.ResultsHA performed efficiently as robust and long-lasting humoral (IgG1, IgG2a, and IgG2b) and cellular responses were detected using very low antigen doses and number of boosters. Outstandingly, at 1-year post-vaccination, anti-rHER2/neu specific antibodies showed even improved effector functions (maturation of affinity for the receptor and increased complement-derived cytotoxicity functions). HA vaccination turned out effective in both the prophylactic (100% mice survived) and therapeutic (tumor regression in 2/12 mice) settings, and broke tolerance against rHER2/neu, delaying spontaneous tumor growth in BALB-neuT mice. Both humoral and cellular responses contributed to the success of HA-based vaccination, but CD8+ T cells played only a marginal role.ConclusionsCancer vaccines have not yet achieved significant clinical efficacy due to their poor immunogenicity, and the validation of more effective adjuvants occurred sometimes at the expense of safety. HA combines the unique immunomodulatory features of a TLR agonist with the tolerability of a fully natural polymer, proving to be a promising adjuvant for the creation of effective and safe cancer vaccines with the potential for rapid clinical translation.ReferencesDalla Pietà A, Carpanese D, et al. Hyaluronan is a natural and effective immunological adjuvant for protein-based vaccines. Cell Mol Immunol 2021;18(5):1197–1210.Rosato A, Montagner IM, Carpanese D, Dalla Pietà A. Hyaluronic acid as a natural adjuvant for protein and peptide-based vaccines. 30.04.2020. WO/2020/084558, PCT/IB2019/059122.Disclosure InformationD. Carpanese: None. I. Montagner: None. A. Dalla Pietà: None. V. Rossi: None. A. Penna: None. G. Zuccolotto: None. G. Pasut: None. A. Grigoletto: None. A. Rosato: None.