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Human papilloma virus (HPV) infection is a causative agent for several cancers types (genital, anal and head and neck region). The HPV E6 and E7 proteins are oncogenic drivers and thus are ideal candidates for therapeutic vaccination. We recently reported that a novel ribonucleic acid lipoplex (RNA-LPX)-based HPV16 vaccine, E7 RNA-LPX, mediates regression of mouse HPV16+ tumors and establishes protective T cell memory. An HPV16 E6/E7 RNA-LPX vaccine is currently being investigated in two phase I and II clinical trials in various HPV-driven cancer types; however, it remains a high unmet medical need for treatments for patients with radiosensitive HPV16+ tumors. Therefore, we set out to investigate the therapeutic efficacy of E7 RNA-LPX vaccine combined with standard-of-care local radiotherapy (LRT). We demonstrate that E7 RNA-LPX synergizes with LRT in HPV16+ mouse tumors, with potent therapeutic effects exceeding those of either monotherapy. Mode of action studies revealed that the E7 RNA-LPX vaccine induced high numbers of intratumoral-E7-specific CD8+T cells, rendering cold tumors immunologically hot, whereas LRT primarily acted as a cytotoxic therapy, reducing tumor mass and intratumor hypoxia by predisposing tumor cells to antigen-specific T cell-mediated killing. Overall, LRT enhanced the effector function of E7 RNA-LPX-primed T cell responses. The therapeutic synergy was dependent on total radiation dose, rather than radiation dose-fractionation. Together, these results show that LRT synergizes with E7 RNA-LPX and enhances its anti-tumor activity against HPV16+ cancer models. This work paves into a new translational therapy for HPV16+ cancer patients.

In many solid cancers, tumor-associated macrophages (TAM) represent the predominant myeloid cell population. Antigen (Ag) cross-presentation leading to tumor Ag-directed cytotoxic CD8+ T cell responses is crucial for anti-tumor immunity. However, the role of recruited monocyte-derived macrophages, including TAM, as potential cross-presenting cells is not well understood. Here, we show that primary human as well as mouse CD206+ macrophages are effective in functional cross-presentation of soluble self and non-self Ag, including tumor-associated Ag (TAA) as well as viral Ag. To confirm the presence of cross-presenting TAM in vivo, we performed phenotypic and functional analysis of TAM from B16-F10 and CT26 syngeneic tumor models and have identified CD11b+F4/80hiCD206+ TAM to effectively cross-present TAA. We show that CD11b+CD206+ TAM represent the dominant tumor-infiltrating myeloid cell population, expressing a unique cell surface repertoire, promoting Ag cross-presentation and Ag-specific CD8+ T cell activation comparable to cross-presenting CLEC9A+ dendritic cells (cDC1). The presence of cross-presenting CD206+ TAM is associated with reduced tumor burden in mouse syngeneic tumor models and with improved overall survival in cutaneous melanoma patients. Therefore, the demonstration of effective Ag cross-presentation capabilities of CD206+ TAM, including their clinical relevance, expands our understanding of TAM phenotypic diversity and functional versatility.

An amendment to this paper has been published and can be accessed via the original article.

TLR7 agonists are considered promising drugs for cancer therapy. The currently available compounds are not well tolerated when administered intravenously and therefore are restricted to disease settings amenable for topical application. Here we present the preclinical characterization of SC1, a novel synthetic agonist with exquisite specificity for TLR7. We found that intravenously administered SC1 mediates systemic release of type I interferon, but not of proinflammatory cytokines such as TNFα and IL6, and results in activation of circulating immune cells. Tumors of SC1-treated mice have brisk immune cell infiltrates and are polarized towards a Th1 type signature. Intratumoral CD8(+) T cells and CD11b(+) conventional dendritic cells (cDCs) are significantly increased, plasmacytoid dendritic cells (pDCs) are strongly activated and macrophages are M1 phenotype polarized, whereas myeloid-derived suppressor cells (MDSCs) are decreased. We further show that treatment of mice with SC1 profoundly inhibits the growth of established syngeneic tumors and results in significantly prolonged survival. Mice, which are tumor-free after SC1 treatment are protected from subsequent tumor rechallenge. The antitumor effect of SC1 depends on antigen-specific CD8(+) T cells, which we found to be strongly enriched in the tumors of SC1-treated mice. In conclusion, this study shows that systemically administered SC1 mobilizes innate and adaptive immunity and is highly potent as single agent in mice and thereby provides a rationale for clinical testing of this compound.

Precision therapy for immune tolerance Autoimmune diseases, such as multiple sclerosis (MS), result from a breach of immunological self-tolerance and tissue damage by autoreactive T lymphocytes. Current treatments can cause systemic immune suppression and side effects such as increased risk of infections. Krienke et al. designed a messenger RNA vaccine strategy that lacks adjuvant activity and delivers MS autoantigens into lymphoid dendritic cells. This approach expands a distinct type of antigen-specific effector regulatory T cell that suppresses autoreactivity against targeted autoantigens and promotes bystander suppression of autoreactive T cells against other myelin-specific autoantigens. In mouse models of MS, the vaccine delayed the onset and reduced the severity of established disease without showing overt symptoms of general immune suppression. Science , this issue p. 145

Genetic engineering of T cells with a T cell receptor (TCR) targeting tumor antigen is a promising strategy for cancer immunotherapy. Inefficient expression of the introduced TCR due to TCR mispairing may limit the efficacy and adversely affect the safety of TCR gene therapy. Here, we evaluated the safety and therapeutic efficiency of an optimized single-chain TCR (scTCR) specific for an HLA-A2.1-restricted (non-mutated) p53(264–272) peptide in adoptive T cell transfer (ACT) models using our unique transgenic mice expressing human p53 and HLA-A2.1 that closely mimic the human setting. Specifically, we showed that adoptive transfer of optimized scTCR-redirected T cells does not induce on-target and off-target autoimmunity. Furthermore, ACT resulted in full tumor protection and led to a long-lived effective, antigen-specific memory T cell response in syngeneic and xenograft models. Taken together, the study demonstrated that our scTCR specific for the broadly expressed tumor-associated antigen p53(264–272) can eradicate p53(+)A2.1(+) tumor cells without inducing off-target or self-directed toxicities in mouse models of ACT. These data strongly support the improved safety and therapeutic efficacy of high-affinity p53scTCR for TCR-based immunotherapy of p53-associated malignancies.

The 13th Annual Meeting of the Association for Cancer Immunotherapy (CIMT) brought together more than 800 scientists in Mainz, Germany, from May 11–13, 2015, to present and discuss current research...

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

Intranodal immunization with antigen-encoding naked mRNA has proven to be an efficacious and safe approach to induce antitumor immunity. Thanks to its unique characteristics, mRNA can act not only as a source for antigen but also as an adjuvant for activation of the immune system. The search for additional adjuvants that can be combined with mRNA to further improve the potency of the immunization revealed Fms-like tyrosine kinase 3 (FLT3) ligand as a potent candidate. Systemic administration of the dendritic cell-activating FLT3 ligand prior to or along with mRNA immunization-enhanced priming and expansion of antigen-specific CD8(+) T cells in lymphoid organs, T-cell homing into melanoma tumors, and therapeutic activity of the intranodally administered mRNA. Both compounds demonstrate a successful combination in terms of boosting the immune response. This chapter describes methods for intranodal immunization with naked mRNA by co-administration of FLT3 ligand, which leads to strong synergistic effects.

Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

The aim of this study was to investigate the occurrence of subclinical Mycobacterium avium spp. paratuberculosis (MAP) infections at slaughter by testing ileocaecal lymph nodes with a semi-nested IS900 PCR. Tissue samples were available within the framework of a parallel study investigating BSE-susceptibility factors in members of BSE-cohorts in the German Federal State of Lower Saxony. Ileocaecal lymph nodes were collected over a 2-year sampling period from 99 slaughter cattle of a mean age of 6.5 years (5.5-7.5 years). A recently developed IS900 semi-nested polymerase chain reaction (snPCR) assay offering a sensitivity of 1 genome equivalent was used for the detection of MAP-DNA. Based on this snPCR, 17 out of the 99 samples gave positive results, indicating a MAP occurrence of 17.17% in the random sample. All PCR products were sequenced for screening of polymorphisms. Nucleotide homologies of 98.5-100% were found with respect to the MAP K10 reference sequence IS900 (GenBank: AE16958). PCR analysis of ileocaecal lymph nodes collected from slaughter cattle proved to be a suitable technique to determine MAP occurrence in the local cattle population.

The adoptive transfer of tumor-reactive cells is a promising approach in the treatment of human malignancies, but the challenge of isolating T cells with high-avidity for tumor antigens in each patient has limited its widespread application. Using HLA-A2.1 transgenic mice, we have demonstrated the feasibility of T-cell receptor (TCR) gene transfer into T cells to circumvent self-tolerance to the widely expressed human p53(264-272) tumor-associated antigen and developed approaches to generate high-affinity CD8-independent TCR. However, a safety concern of TCR gene transfer is the risk of pairing between introduced and the naturally expressed endogenous TCR chains, resulting in the generation of self-reactive T cells (off-target autoimmunity). We first genetically modified p53TCR constructs to minimize mispairing and improve correct pairing of the introduced TCR. We and others have shown that, cysteine modifications are able to increase the expression of the introduced TCR but fail to prevent mispairing formation in mouse and human T cells. To further enhance preferential TCR pairing, cell surface expression and TCR function, we introduced additional cysteine residues into the TCR α and β chain constant domains along with codon-optimization of the TCR sequences and cloning of the TCR constructs into one single 2A-based retroviral vector. To overcome TCR mispairing formation, we designed a single chain (sc) TCR by connecting the variable TCRa domain to the TCRb chain via a short peptide linker co-expressed with a truncated constant TCR a domain. Beside off-target toxicity, adoptive transfer of high-avidity T cells may potentially cause severe on-target toxicity for normal cells expressing low level of antigens. In this respect, pre-clinical in vivo studies are still missing. Here, we evaluated the safety issues raised by the risk of p53TCR gene transfer-associated on/off-target toxicities in relevant mouse models of adoptive transfer. In vitro studies showed that, scTCR-modified CD4+ and CD8+ T cells displayed similar high-avidity compared to the full-length TCR, as determined by peptide titration in cytotoxicity assays and were able to mediate specific lysis of p53 mutant A2.1+ tumor cells. Though, genetic modifications preserved the antigen specificity of these TCRs, the full-length version of the TCR could not prevent mispairing-mediated lethal off-target autoimmunity in vivo. In sharp contrast, T cells engrafted with the modified scTCR did not induce graft-versus-host disease (GVHD) following adoptive transfer. We next assessed the potential of scTCR-modified T cells to cause on-target autoimmunity in vivo, using mice which express the human wild type p53 and A2.1Kb (Hupki-A2.1Kb). We found that lymphodepleting preconditioning regimens plus vaccination-induced expansion of transferred TCR-specific T cells did not result in a depletion of hematopoietic cells, as mice recovered normal white blood cell counts, including lymphocytes and monocytes and survived without any sign of toxicity. Importantly, our study demonstrated that high-avidity scTCR-engineered human T cells were able to eradicate established tumors and persist for more than 6 months after infusion without inducing xeno-GVHD in NodScid IL-2R gamma chain-null mice. Taken together, our study provided evidence that an optimized high-affinity scTCR-specific for the broadly expressed tumor-associated antigen p53(264-272) can eradicate p53+A2.1+ tumor cells in vivo without inducing off-target or self-directed toxicities in humanized mouse models of adoptive T-cell transfer. These data strongly support the improved safety and therapeutic efficacy of high-affinity scp53TCR for TCR-based immunotherapy of p53-associated malignancies. Disclosures: No relevant conflicts of interest to declare.