Nucleotide modifications enable rational design of TLR7-selective ligands by blocking RNase cleavage.

Toll-like receptors 7 (TLR7) and 8 (TLR8) each sense single-stranded RNA (ssRNA), but their activation results in different immune activation profiles. Attempts to selectively target either TLR7 or TLR8 have been hindered by their high degree of homology. However, recent studies revealed that TLR7 and TLR8 bind different ligands resulting from the processing of ssRNA by endolysosomal RNases. We demonstrate that by introducing precise 2' sugar-modified bases into oligoribonucleotides (ORNs) containing known TLR7 and TLR8 binding motifs, we could prevent RNase-mediated degradation into the monomeric uridine required for TLR8 activation while preserving TLR7 activation. Furthermore, a novel, optimized protocol for CRISPR-Cas9 knockout in primary human plasmacytoid dendritic cells showed that TLR7 activation is dependent on RNase processing of ORNs and revealed a previously undescribed role for RNase 6 in degrading ORNs into TLR ligands. Finally, 2' sugar-modified ORNs demonstrated robust innate immune activation in mice. Altogether, we identified a strategy for creating tunable TLR7-selective agonists.

Progress in vaccine development for infectious diseases-a Keystone Symposia report.

The COVID-19 pandemic has taught us many things, among the most important of which is that vaccines are one of the cornerstones of public health that help make modern longevity possible. While several different vaccines have been successful at stemming the morbidity and mortality associated with various infectious diseases, many pathogens/diseases remain recalcitrant to the development of effective vaccination. Recent advances in vaccine technology, immunology, structural biology, and other fields may yet yield insight that will address these diseases; they may also help improve societies' preparedness for future pandemics. On June 1-4, 2022, experts in vaccinology from academia, industry, and government convened for the Keystone symposium "Progress in Vaccine Development for Infectious Diseases" to discuss state-of-the-art technologies, recent advancements in understanding vaccine-mediated immunity, and new aspects of antigen design to aid vaccine effectiveness.

IL-1 and IL-1ra are key regulators of the inflammatory response to RNA vaccines.

The use of lipid-formulated RNA vaccines for cancer or COVID-19 is associated with dose-limiting systemic inflammatory responses in humans that were not predicted from preclinical studies. Here, we show that the 'interleukin 1 (IL-1)-interleukin 1 receptor antagonist (IL-1ra)' axis regulates vaccine-mediated systemic inflammation in a host-specific manner. In human immune cells, RNA vaccines induce production of IL-1 cytokines, predominantly IL-1ß, which is dependent on both the RNA and lipid formulation. IL-1 in turn triggers the induction of the broad spectrum of pro-inflammatory cytokines (including IL-6). Unlike humans, murine leukocytes respond to RNA vaccines by upregulating anti-inflammatory IL-1ra relative to IL-1 (predominantly IL-1α), protecting mice from cytokine-mediated toxicities at >1,000-fold higher vaccine doses. Thus, the IL-1 pathway plays a key role in triggering RNA vaccine-associated innate signaling, an effect that was unexpectedly amplified by certain lipids used in vaccine formulations incorporating N1-methyl-pseudouridine-modified RNA to reduce activation of Toll-like receptor signaling.

Oncolytic adenovirus decreases the proportion of TIM-3+ subset of tumor-infiltrating CD8+ T cells with correlation to improved survival in patients with cancer.

BACKGROUND: Oncolytic viruses are a potent form of active immunotherapy, capable of invoking antitumor T-cell responses. Meanwhile, less is known about their effects on immune checkpoints, the main targets for passive immunotherapy of cancer. T-cell immunoglobulin and mucin domain-3 (TIM-3) is a coinhibitory checkpoint driving T-cell exhaustion in cancer. Here we investigated the effects of oncolytic adenovirus on the TIM-3 checkpoint on tumor-infiltrating immune cells and clinical impact in patients with cancer receiving oncolytic immunotherapy. METHODS: Modulation of TIM-3 expression on tumor-infiltrating immune cells was studied preclinically in B16 melanoma following intratumoral treatment with Ad5/3∆24-granulocyte-macrophage colony-stimulating factor oncolytic adenovirus. We conducted a retrospective longitudinal analysis of 15 patients with advanced-stage cancer with tumor-site biopsies before and after oncolytic immunotherapy, treated in the Advanced Therapy Access Program (ISRCTN10141600, April 5, 2011). Following patient stratification with regard to TIM-3 (increase vs decrease in tumors), overall survival and imaging/marker responses were evaluated by log-rank and Fisher's test, while coinhibitory receptors/ligands, transcriptomic changes and tumor-reactive and tumor-infltrating immune cells in biopsies and blood samples were studied by microarray rank-based statistics and immunoassays. RESULTS: Preclinically, TIM-3+ tumor-infiltrating lymphocytes (TILs) in B16 melanoma showed an exhausted phenotype, whereas oncolytic adenovirus treatment significantly reduced the proportion of TIM-3+ TIL subset through recruitment of less-exhausted CD8+ TIL. Decrease of TIM-3 was observed in 60% of patients, which was associated with improved overall survival over TIM-3 increase patients (p=0.004), together with evidence of clinical benefit by imaging and blood analyses. Coinhibitory T-cell receptors and ligands were consistently associated with TIM-3 changes in gene expression data, while core transcriptional exhaustion programs and T-cell dysfunction were enriched in patients with TIM-3 increase, thus identifying patients potentially benefiting from checkpoint blockade. In striking contrast, patients with TIM-3 decrease displayed an acute inflammatory signature, redistribution of tumor-reactive CD8+ lymphocytes and higher influx of CD8+ TIL into tumors, which were associated with the longest overall survival, suggesting benefit from active immunotherapy. CONCLUSIONS: Our results indicate a key role for the TIM-3 immune checkpoint in oncolytic adenoviral immunotherapy. Moreover, our results identify TIM-3 as a potential biomarker for oncolytic adenoviruses and create rationale for combination with passive immunotherapy for a subset of patients.

Dexamethasone premedication suppresses vaccine-induced immune responses against cancer.

Glucocorticosteroids (GCS) have an established role in oncology and are administered to cancer patients in routine clinical care and in drug development trials as co-medication. Given their strong immune-suppressive activity, GCS may interfere with immune-oncology drugs. We are developing a therapeutic cancer vaccine, which is based on a liposomal formulation of tumor-antigen encoding RNA (RNA-LPX) and induces a strong T-cell response both in mice as well as in humans. In this study, we investigated in vivo in mice and in human PBMCs the effect of the commonly used long-acting GCS Dexamethasone (Dexa) on the efficacy of this vaccine format, with a particular focus on antigen-specific T-cell immune responses. We show that Dexa, when used as premedication, substantially blunts RNA-LPX vaccine-mediated immune effects. Premedication with Dexa inhibits vaccine-dependent induction of serum cytokines and chemokines and reduces both the number and activation of splenic conventional dendritic cells (cDC) expressing vaccine-encoded antigens. Consequently, priming of functional effector T cells and therapeutic activity is significantly impaired. Interestingly, responses are less impacted when Dexa is administered post-vaccination. Consistent with this observation, although many inflammatory cytokines are reduced, IFNα, a key cytokine in T-cell priming, is less impacted and antigen expression by cDCs is intact. These findings warrant special caution when combining GCS with immune therapies relying on priming and activation of antigen-specific T cells and suggest that careful sequencing of these treatments may preserve T-cell induction.

Exploiting Preexisting Immunity to Enhance Oncolytic Cancer Immunotherapy.

Because of the high coverage of international vaccination programs, most people worldwide have been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T-cell repertoire. Although CD8+ antitumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T-cell help is also required for an optimal antitumor immune response to occur. Hence, we investigated whether the pathogen-related CD4+ T-cell memory populations could be reengaged to support the CTLs, converting a weak primary antitumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of an oncolytic adenovirus coated with MHC-I-restricted tumor-specific peptides and developed it further by introducing pathogen-specific MHC-II-restricted peptides. Mice preimmunized with tetanus vaccine were challenged with B16.OVA tumors and treated with the newly developed hybrid TT-OVA-PeptiCRAd containing both tetanus toxoid- and tumor-specific peptides. Treatment with the hybrid PeptiCRAd significantly enhanced antitumor efficacy and induced TT-specific, CD40 ligand-expressing CD4+ T helper cells and maturation of antigen-presenting cells. Importantly, this approach could be extended to naturally occurring tumor peptides (both tumor-associated antigens and neoantigens), as well as to other pathogens beyond tetanus, highlighting the usefulness of this technique to take full advantage of CD4+ memory T-cell repertoires when designing immunotherapeutic treatment regimens. Finally, the antitumor effect was even more prominent when combined with the immune checkpoint inhibitor anti-PD-1, strengthening the rationale behind combination therapy with oncolytic viruses. SIGNIFICANCE: These findings establish a novel technology that enhances oncolytic cancer immunotherapy by capitalizing on pre-acquired immunity to pathogens to convert a weak antitumor immune response into a much stronger one.

Artificially cloaked viral nanovaccine for cancer immunotherapy.

Virus-based cancer vaccines are nowadays considered an interesting approach in the field of cancer immunotherapy, despite the observation that the majority of the immune responses they elicit are against the virus and not against the tumor. In contrast, targeting tumor associated antigens is effective, however the identification of these antigens remains challenging. Here, we describe ExtraCRAd, a multi-vaccination strategy focused on an oncolytic virus artificially wrapped with tumor cancer membranes carrying tumor antigens. We demonstrate that ExtraCRAd displays increased infectivity and oncolytic effect in vitro and in vivo. We show that this nanoparticle platform controls the growth of aggressive melanoma and lung tumors in vivo both in preventive and therapeutic setting, creating a highly specific anti-cancer immune response. In conclusion, ExtraCRAd might serve as the next generation of personalized cancer vaccines with enhanced features over standard vaccination regimens, representing an alternative way to target cancer.

Abscopal Effect in Non-injected Tumors Achieved with Cytokine-Armed Oncolytic Adenovirus.

Cancer treatment with local administration of armed oncolytic viruses could potentially induce systemic antitumor effects, or the abscopal effect, as they self-amplify in tumors, induce danger signaling, and promote tumor-associated antigen presentation. In this study, oncolytic adenovirus coding for human tumor necrosis factor alpha (TNF-α) and interleukin-2 (IL-2) Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 (also known as [a.k.a.] TILT-123) provoked antitumor efficacy in tumors that were injected with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 and those that were left non-injected in the same animal. Importantly, the virus was able to travel to distant tumors. To dissect the effects of oncolysis and cytokines, we studied replication-incompetent viruses in mice. Systemic antitumor effects were similar in both models, highlighting the importance of the arming device. The cytokines induced positive changes in immune cell infiltrates and induced the expression of several immune-reaction-related genes in tumors. In addition, Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 was able to increase homing of adoptively transferred tumor-infiltrating lymphocytes into both injected and non-injected tumors, possibly mediated through chemokine expression. In summary, local treatment with Ad5/3-E2F-d24-hTNF-α-IRES-hIL-2 resulted in systemic antitumor efficacy by inducing immune cell infiltration and trafficking into both treated and untreated tumors. Moreover, the oncolytic adenovirus platform had superior systemic effects over replication-deficient vector through spreading into distant tumors.

Pancreatic cancer therapy with combined mesothelin-redirected chimeric antigen receptor T cells and cytokine-armed oncolytic adenoviruses.

Pancreatic ductal adenocarcinoma (PDA) is characterized by its highly immunosuppressive tumor microenvironment (TME) that limits T cell infiltration and induces T cell hypofunction. Mesothelin-redirected chimeric antigen receptor T cell (meso-CAR T cell) therapy has shown some efficacy in clinical trials but antitumor efficacy remains modest. We hypothesized that combined meso-CAR T cells with an oncolytic adenovirus expressing TNF-α and IL-2 (Ad5/3-E2F-D24-TNFa-IRES-IL2, or OAd-TNFa-IL2) would improve efficacy. OAd-TNFa-IL2 enhanced the antitumor efficacy of meso-CAR T cells in human-PDA-xenograft immunodeficient mice and efficacy was associated with robustly increased tumor-infiltrating lymphocytes (TILs), enhanced and prolonged T cell function. Mice treated with parental OAd combined with meso-CAR T developed tumor metastasis to the lungs even if primary tumors were controlled. However, no mice treated with combined OAd-TNFa-IL2 and meso-CAR T died of tumor metastasis. We also evaluated this approach in a syngeneic mouse tumor model by combining adenovirus expressing murine TNF-α and IL-2 (Ad-mTNFa-mIL2) and mouse CAR T cells. This approach induced significant tumor regression in mice engrafted with highly aggressive and immunosuppressive PDA tumors. Ad-mTNFa-mIL2 increased both CAR T cell and host T cell infiltration to the tumor and altered host tumor immune status with M1 polarization of macrophages and increased dendritic cell maturation. These findings indicate that combining cytokine-armed oncolytic adenovirus to enhance the efficacy of CAR T cell therapy is a promising approach to overcome the immunosuppressive TME for the treatment of PDA.

Interleukin 8 activity influences the efficacy of adenoviral oncolytic immunotherapy in cancer patients.

After the landmark approval of T-VEC, oncolytic viruses are finding their way to the clinics. However, response rates have still room for improvement, and unfortunately there are currently no available markers to predict responses for oncolytic immunotherapy. Interleukin 8 (IL-8) production is upregulated in many cancers and it also connects to several pathways that have been shown to impair the efficacy of adenoviral immunotherapy. We studied the role of IL-8 in 103 cancer patients treated with oncolytic adenoviruses. We found high baseline serum IL-8 concentration to be independently associated with poor prognosis (p<0.001). Further, normal baseline IL-8 was associated with improved prognostic potential of calculation of the neutrophil-to-lymphocyte ratio (p<0.001). Interestingly, a decrease in IL-8 concentration after treatment with oncolytic adenovirus predicted better overall survival (p<0.001) and higher response rate, although this difference was not significant (p=0.066). We studied the combination of adenovirus and IL-8 neutralizing antibody ex vivo in single cell suspensions and in co-cultures of tumor-associated CD15+ neutrophils and CD3+ tumor-infiltrating lymphocytes derived from fresh patient tumor samples. These results indicate a role for IL-8 as a biomarker in oncolytic virotherapy, but additionally provide a rationale for targeting IL-8 to improve treatment efficacy. In conclusion, curtailing the activity of IL-8 systemically or locally in the tumor microenvironment could improve anti-tumor immune responses resulting in enhanced efficacy of adenoviral immunotherapy of cancer.

A novel in silico framework to improve MHC-I epitopes and break the tolerance to melanoma.

Tolerance toward tumor antigens, which are shared by normal tissues, have often limited the efficacy of cancer vaccines. However, wild type epitopes can be tweaked to activate cross-reactive T-cell clones, resulting in antitumor activity. The design of these analogs (i.e., heteroclitic peptides) can be difficult and time-consuming since no automated in silico tools are available. Hereby we describe the development of an in silico framework to improve the selection of heteroclitic peptides. The Epitope Discovery and Improvement System (EDIS) was first validated by studying the model antigen SIINFEKL. Based on artificial neural network (ANN) predictions, we selected two mutant analogs that are characterized by an increased MHC-I binding affinity (SIINFAKL) or increased TCR stimulation (SIIWFEKL). Therapeutic vaccination using optimized peptides resulted in enhanced antitumor activity and against B16.OVA melanomas in vivo. The translational potential of the EDIS platform was further demonstrated by studying the melanoma-associated antigen tyrosinase related protein 2 (TRP2). Following therapeutic immunization with the EDIS-derived epitope SVYDFFAWL, a significant reduction in the growth of established B16.F10 tumors was observed, suggesting a break in the tolerance toward the wild type epitope. Finally, we tested a multi vaccine approach, demonstrating that combination of wild type and mutant epitopes targeting both TRP2 and OVA antigens increases the antitumor response. In conclusion, by taking advantage of available prediction servers and molecular dynamics simulations, we generated an innovative platform for studying the initial sequences and selecting lead candidates with improved immunological features. Taken together, EDIS is the first automated algorithm-driven platform to speed up the design of heteroclitic peptides that can be publicly queried online.

Adenoviral production of interleukin-2 at the tumor site removes the need for systemic postconditioning in adoptive cell therapy.

Systemic high dose interleukin-2 (IL-2) postconditioning has long been utilized in boosting the efficacy of T cells in adoptive cell therapy (ACT) of solid tumors. The resulting severe off-target toxicity of these regimens renders local production at the tumor an attractive concept with possible safety gains. We evaluated the efficacy and safety of intratumorally administered IL-2-coding adenoviruses in combination with tumor-infiltrating lymphocyte therapy in syngeneic Syrian hamsters bearing HapT1 pancreatic tumors and with T cell receptor transgenic ACT in B16.OVA melanoma bearing C57BL/6 mice. The models are complementary: hamsters are semi-permissive for human oncolytic adenovirus, whereas detailed immunological analyses are possible in mice. In both models, local production of IL-2 successfully replaced the need for systemic recombinant IL-2 (rIL-2) administration and increased the efficacy of the cell therapy. Furthermore, vectored delivery of IL-2 significantly enhanced the infiltration of CD8+ T cells, M1-like macrophages, and B-cells while systemic rIL-2 increased CD25 + FoxP3+ T cells at the tumor. In contrast with vectored delivery, histopathological analysis of systemic rIL-2-treated animals revealed significant changes in lungs, livers, hearts, spleens, and kidneys. In summary, local IL-2 production results in efficacy and safety gains in the context of ACT. These preclinical assessments provide the rationale for ongoing clinical translation.

Intravenously usable fully serotype 3 oncolytic adenovirus coding for CD40L as an enabler of dendritic cell therapy.

Vaccination with dendritic cells (DCs), the most potent professional antigen-presenting cells in the body, is a promising approach in cancer immunotherapy. However, tumors induce immunosuppression in their microenvironment that suppresses and impairs the function of DCs. Therefore, human clinical trials with DC therapy have often been disappointing. To improve the therapeutic efficacy and to overcome the major obstacles of DC therapy, we generated a novel adenovirus, Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 and expresses hCD40L for induction of antitumor immune response. The specific aim is to enhance DCs function. Data from a human cancer patient indicated that this capsid allows effective transduction of distant tumors through the intravenous route. Moreover, patient data suggested that virally produced hCD40L can activate DCs in situ. The virus was efficient in vitro and had potent antitumor activity in vivo. In a syngeneic model, tumors treated with Ad5/3-CMV-mCD40L virus plus DCs elicited greater antitumor effect as compared with either treatment alone. Moreover, virally coded CD40L induced activation of DCs, which in turn, lead to the induction of a Th1 immune response and increased tumor-specific T cells. In conclusion, Ad3-hTERT-CMV-hCD40L is promising for translation into human trials. In particular, this virus could enable successful dendritic cell therapy in cancer patients.

Oncolytic Adenoviruses Armed with Tumor Necrosis Factor Alpha and Interleukin-2 Enable Successful Adoptive Cell Therapy.

Adoptive cell therapy holds much promise in the treatment of cancer but results in solid tumors have been modest. The notable exception is tumor-infiltrating lymphocyte (TIL) therapy of melanoma, but this approach only works with high-dose preconditioning chemotherapy and systemic interleukin (IL)-2 postconditioning, both of which are associated with toxicities. To improve and broaden the applicability of adoptive cell transfer, we constructed oncolytic adenoviruses coding for human IL-2 (hIL2), tumor necrosis factor alpha (TNF-α), or both. The viruses showed potent antitumor efficacy against human tumors in immunocompromised severe combined immunodeficiency (SCID) mice. In immunocompetent Syrian hamsters, we combined the viruses with TIL transfer and were able to cure 100% of the animals. Cured animals were protected against tumor re-challenge, indicating a memory response. Arming with IL-2 and TNF-α increased the frequency of both CD4+ and CD8+ TILs in vivo and augmented splenocyte proliferation ex vivo, suggesting that the cytokines were important for T cell persistence and proliferation. Cytokine expression was limited to tumors and treatment-related signs of systemic toxicity were absent, suggesting safety. To conclude, cytokine-armed oncolytic adenoviruses enhanced adoptive cell therapy by favorable alteration of the tumor microenvironment. A clinical trial is in progress to study the utility of Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (TILT-123) in human patients with cancer.

Dasatinib Changes Immune Cell Profiles Concomitant with Reduced Tumor Growth in Several Murine Solid Tumor Models.

Dasatinib, a broad-range tyrosine kinase inhibitor, induces rapid mobilization of lymphocytes and clonal expansion of cytotoxic cells in leukemia patients. Here, we investigated whether dasatinib could induce beneficial immunomodulatory effects in solid tumor models. The effects on tumor growth and on the immune system were studied in four different syngeneic mouse models (B16.OVA melanoma, 1956 sarcoma, MC38 colon, and 4T1 breast carcinoma). Both peripheral blood (PB) and tumor samples were immunophenotyped during treatment. Although in vitro dasatinib displayed no direct cytotoxicity to B16 melanoma cells, a significant decrease in tumor growth was observed in dasatinib-treated mice compared with vehicle-treated group. Further, dasatinib-treated melanoma-bearing mice had an increased proportion of CD8+ T cells in PB, together with a higher amount of tumor-infiltrating CD8+ T cells. Dasatinib-mediated antitumor efficacy was abolished when CD4+ and CD8+ T cells were depleted with antibodies. Results were confirmed in sarcoma, colon, and breast cancer models, and in all cases mice treated daily with dasatinib had a significant decrease in tumor growth. Detailed immunophenotyping of tumor tissues with CyTOF indicated that dasatinib had reduced the number of intratumoral regulatory T cells in all tumor types. To conclude, dasatinib is able to slow down the tumor growth of various solid tumor models, which is associated with the favorable blood/tumor T-cell immunomodulation. The assessment of synergistic combinatorial therapies with other immunomodulatory drugs or targeted small-molecule oncokinase inhibitors is warranted in future clinical trials. Cancer Immunol Res; 5(2); 157-69. ©2017 AACR.

T-Cell Therapy Enabling Adenoviruses Coding for IL2 and TNFα Induce Systemic Immunomodulation in Mice With Spontaneous Melanoma.

The immunosuppressive microenvironment of solid tumors renders adoptively transferred T cells hypofunctional. However, adenoviral delivery of immunostimulatory cytokines IL2 and TNFα can significantly improve the efficacy of adoptive T-cell therapy. Using ret transgenic mice that spontaneously develop skin malignant melanoma, we analyzed the mechanism of action of adenoviruses coding for IL2 and TNFα in combination with adoptive transfer of TCR-transgenic TRP-2-specific T cells. Following T-cell therapy and intratumoral virus injection, a significant increase in antigen-experienced, tumor-reactive PD-1 CD8 T cells was seen in both cutaneous lesions and in metastatic lymph nodes. A reverse correlation between tumor weight and the number of tumor-reactive PD-1 tumor-infiltrating lymphocytes (TILs) was observed, suggesting that these T cells could target and kill tumor cells. It is interesting to note that, local expression of cytokines did not affect intratumoral levels of T-regulatory cells (Tregs), which had previously been associated with systemic IL2 therapy. Instead, Ad5-IL2 induced upregulation of IL2 receptor α-chain (CD25) on conventional CD4CD25Foxp3 cells, indicating that these CD4 T cells may contribute to CD8 T-cell activation and/or homing. Signs of therapy-induced resistance were also observed as the expression of PD-L1 on tumor-infiltrating granulocytic myeloid-derived suppressor cells was upregulated as a reaction to PD-1+ TILs. Finally, beneficial ratios between tumor-reactive PD-1 CD8 TILs and immunosuppressive cell subsets (Tregs and nitric oxide-producing myeloid-derived suppressor cells) were observed in primary and secondary tumor sites, indicating that local delivery of IL2 and TNFα coding adenoviruses can systemically modify the cellular composition of the tumor microenvironment in favor of adoptively transferred T cells.

Oncolytic Adenovirus Expressing Monoclonal Antibody Trastuzumab for Treatment of HER2-Positive Cancer.

Monoclonal anti-HER2 antibody trastuzumab has significantly improved the survival of patients with HER2-overexpressing tumors. Nevertheless, systemic antibody therapy is expensive, limited in efficacy due to physical tumor barriers, and carries the risk of severe side effects such as cardiomyopathy. Oncolytic viruses mediate cancer-selective transgene expression, kill infected cancer cells while mounting antitumor immune responses, and have recently demonstrated promising efficacy in combination treatments. Here, we armed an oncolytic adenovirus with full-length trastuzumab to achieve effective in situ antibody production coupled with progressive oncolytic cancer cell killing. We constructed an infectivity-enhanced serotype 5 oncolytic adenovirus, Ad5/3-Δ24-tras, coding for human trastuzumab antibody heavy- and light-chain genes, connected by an internal ribosome entry site. Infected cancer cells were able to assemble full-length functional antibody, as confirmed by Western blot, ELISA, and antibody-dependent cell-mediated cytotoxicity assay. Importantly, oncolysis was required for release of the antibody into tumors, providing additional spatial selectivity. Ad5/3-Δ24-tras showed potent in vitro cytotoxicity and enhanced antitumor efficacy over oncolytic control virus Ad5/3-Δ24 or commercial trastuzumab in HER2-positive cancer models in vivo (both P < 0.05). Furthermore, Ad5/3-Δ24-tras resulted in significantly higher tumor-to-systemic antibody concentrations (P < 0.001) over conventional delivery. Immunological analyses revealed dendritic cell activation and natural killer cell accumulation in tumor-draining lymph nodes. Thus, Ad5/3-Δ24-tras is an attractive anticancer approach combining oncolytic immunotherapy with local trastuzumab production, resulting in improved in vivo efficacy and immune cell activation in HER2-positive cancer. Moreover, the finding that tumor cells can produce functional antibody as directed by oncolytic virus could lead to many valuable antitumor approaches. Mol Cancer Ther; 15(9); 2259-69. ©2016 AACR.

Adenoviral Delivery of Tumor Necrosis Factor-α and Interleukin-2 Enables Successful Adoptive Cell Therapy of Immunosuppressive Melanoma.

Adoptive T-cell transfer is a promising treatment approach for metastatic cancer, but efficacy in solid tumors has only been achieved with toxic pre- and postconditioning regimens. Thus, adoptive T-cell therapies would benefit from complementary modalities that enable their full potential without excessive toxicity. We aimed to improve the efficacy and safety of adoptive T-cell transfer by using adenoviral vectors for direct delivery of immunomodulatory murine cytokines into B16.OVA melanoma tumors with concomitant T-cell receptor transgenic OT-I T-cell transfer. Armed adenoviruses expressed high local and low systemic levels of cytokine when injected into B16.OVA tumors, suggesting safety of virus-mediated cytokine delivery. Antitumor efficacy was significantly enhanced with adenoviruses coding for murine interleukin-2 (mIL-2) and tumor necrosis factor-α (mTNFα) when compared with T-cell transfer alone or viruses alone. Further improvement in efficacy was achieved with a triple combination of mIL-2, mTNFα, and OT-I T-cells. Mechanistic studies suggest that mIL-2 has an important role in activating T-cells at the tumor, while mTNFα induces chemokine expression. Furthermore, adenovirus treatments enhanced tumor-infiltration of OT-I T-cells as demonstrated by SPECT/CT imaging of (111)In-labeled cells. Our results suggest the utility of cytokine-coding adenoviruses for improving the efficacy of adoptive T-cell therapies.

Syngeneic syrian hamster tumors feature tumor-infiltrating lymphocytes allowing adoptive cell therapy enhanced by oncolytic adenovirus in a replication permissive setting.

Adoptive transfer of tumor-infiltrating lymphocytes (TIL) has shown promising yet sometimes suboptimal results in clinical trials for advanced cancer, underscoring the need for approaches improving efficacy and safety. Six implantable syngeneic tumor cell lines of the Syrian hamster were used to initiate TIL cultures. TIL generated from tumor fragments cultured in human interleukin-2 (IL-2) for 10 d were adoptively transferred into tumor-bearing hamsters with concomitant intratumoral injections of oncolytic adenovirus (Ad5-D24) for the assessment of antitumor efficacy. Pancreatic cancer (HapT1) and melanoma (RPMI 1846) TIL exhibited potent and tumor-specific cytotoxicity in effector-to-target (E/T) assays. MHC Class I blocking abrogated the cell killing of RPMI 1846 TIL, indicating cytotoxic CD8(+) T-cell activity. When TIL were combined with Ad5-D24 in vitro, HapT1 tumor cell killing was significantly enhanced over single agents. In vivo, the intratumoral administration of HapT1 TIL and Ad5-D24 resulted in improved tumor growth control compared with either treatment alone. Additionally, splenocytes derived from animals treated with the combination of Ad5-D24 and TIL killed autologous tumor cells more efficiently than monotherapy-derived splenocytes, suggesting that systemic antitumor immunity was induced. For the first time, TIL of the Syrian hamster have been cultured, characterized and used therapeutically together with oncolytic adenovirus for enhancing the efficacy of TIL therapy. Our results support human translation of oncolytic adenovirus as an enabling technology for adoptive T-cell therapy of solid tumors.