Development of a Syrian hamster anti-PD-L1 monoclonal antibody enables oncolytic adenoviral immunotherapy modelling in an immunocompetent virus replication permissive setting.

Introduction: Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of cancer, but preclinical testing of hypotheses such as combination therapies has been complicated, in part due to species incompatibility issues. For example, one of few known permissive animal models for oncolytic adenoviruses is the Syrian hamster, for which an ICI, mainly an anti-PD-L1 monoclonal antibody (mAb) was not previously available. In this study, we developed an anti-Syrian hamster PD-L1 mAb to enable the evaluation of safety and efficacy, when combining anti-PD-L1 with an oncolytic adenovirus encoding tumour necrosis factor alpha (TNFα) and interleukin-2 (IL-2) (Ad5/3-E2F-D24-hTNFα-IRES-hIL-2 or TILT-123). Methods: Recombinant Syrian hamster PD-L1 was expressed and mice immunized for mAb formation using hybridoma technology. Clonal selection through binding and functional studies in vitro, in silico and in vivo identified anti-PD-L1 clone 11B12-1 as the primary mAb candidate for immunotherapy modelling. The oncolytic virus (OV) and ICI combination approach was then evaluated using 11B12-1 and TILT-123 in a Syrian hamster model of pancreatic ductal adenocarcinoma (PDAC). Results: Supernatants from hybridoma parent subclone 11B12B4 provided the highest positive PD-L1 signal, on Syrian hamster PBMCs and three cancer cell lines (HT100, HapT1 and HCPC1). In vitro co-cultures revealed superior immune modulated profiles of cell line matched HT100 tumour infiltrating lymphocytes when using subclones of 7G2, 11B12 and 12F1. Epitope binning and epitope prediction using AlphaFold2 and ColabFold revealed two distinct functional epitopes for clone 11B12-1 and 12F1-1. Treatment of Syrian hamsters bearing HapT1 tumours, with 11B12-1 induced significantly better (p<0.05) tumour growth control than isotype control by day 12. 12F1-1 did not induce significant tumour growth control. The combination of 11B12-1 with oncolytic adenovirus TILT-123 improved tumour growth control further, when compared to monotherapy (p<0.05) by day 26. Conclusions: Novel Syrian hamster anti-PD-L1 clone 11B12-1 induces tumour growth control in a hamster model of PDAC. Combining 11B12-1 with oncolytic adenovirus TILT-123 improves tumour growth control further and demonstrates good safety and toxicity profiles.

Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers.

Ovarian cancer (OvCa) is one of the most common gynecological cancers and has the highest mortality in this category. Tumors are often detected late, and unfortunately over 70% of OvCa patients experience relapse after first-line treatments. OvCa has shown low response rates to immune checkpoint inhibitor (ICI) treatments, thus leaving room for improvement. We have shown that oncolytic adenoviral therapy with Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (aka. TILT-123) is promising for single-agent treatment of cancer, but also for sensitizing tumors for T-cell dependent immunotherapy approaches, such as ICI treatments. Therefore, this study set out to determine the effect of inhibition of the immune checkpoint inhibitors (ICI), in the context of TILT-123 therapy of OvCa. We show that simultaneous treatment of patient derived samples with TILT-123 and ICIs anti-PD-1 or anti-PD-L1 efficiently reduced overall viability. The combinations induced T cell activation, T cells expressed activation markers more often, and the treatment caused positive microenvironment changes, measured by flow cytometric assays. Furthermore, in an immunocompetent in vivo C57BL/6NHsda mouse model, tumor growth was hindered, when treated with TILT-123, ICI or both. Taken together, this study provides a rationale for using TILT-123 virotherapy in combination with TILT-123 and immune checkpoint inhibitors together in an ovarian cancer OvCa clinical trial.

Adenovirus Encoding Tumor Necrosis Factor Alpha and Interleukin 2 Induces a Tertiary Lymphoid Structure Signature in Immune Checkpoint Inhibitor Refractory Head and Neck Cancer.

Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNFα and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNFα and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naïve and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNFα and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3+ tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45+ tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naïve and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNFα and mIL-2 compared to monotherapies. This observation was verified by striking CD3+ TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNFα and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNFα and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.

Oncolytic Adenovirus ORCA-010 Activates Proinflammatory Myeloid Cells and Facilitates T Cell Recruitment and Activation by PD-1 Blockade in Melanoma.

Immune checkpoint inhibitors have advanced the treatment of melanoma. Nevertheless, a majority of patients are resistant, or develop resistance, to immune checkpoint blockade, which may be related to prevailing immune suppression by myeloid regulatory cells in the tumor microenvironment (TME). ORCA-010 is a novel oncolytic adenovirus that selectively replicates in, and lyses, cancer cells. We previously showed that ORCA-010 can activate melanoma-exposed conventional dendritic cells (cDCs). To study the effect of ORCA-010 on melanoma-conditioned macrophage development, we used an in vitro co-culture model of human monocytes with melanoma cell lines. We observed a selective survival and polarization of monocytes into M2-like macrophages (CD14+CD80-CD163+) in co-cultures with cell lines that expressed macrophage colony-stimulating factor. Oncolysis of these melanoma cell lines, effected by ORCA-010, activated the resulting macrophages and converted them to a more proinflammatory state, evidenced by higher levels of PD-L1, CD80, and CD86 and an enhanced capacity to prime allogenic T cells and induce a type-1 T cell response. To assess the effect of ORCA-010 on myeloid subset distribution and activation in vivo, ORCA-010 was intratumorally injected and tested for T cell activation and recruitment in the human adenovirus nonpermissive B16-OVA mouse melanoma model. While systemic PD-1 blockade in this model in itself did not modulate myeloid or T cell subset distribution and activation, when it was preceded by i.t. injection of ORCA-010, this induced an increased rate and activation state of CD8α+ cDC1, both in the TME and in the spleen. Observed increased rates of activated CD8+ T cells, expressing CD69 and PD-1, were related to both increased CD8α+ cDC1 rates and M1/M2 shifts in tumor and spleen. In conclusion, the myeloid modulatory properties of ORCA-010 in melanoma, resulting in recruitment and activation of T cells, could enhance the antitumor efficacy of PD-1 blockade.

Ad5/3 is able to avoid neutralization by binding to erythrocytes and lymphocytes.

Oncolytic adenoviruses are promising cancer therapeutic agents. Clinical data have shown adenoviruses' ability to transduce tumors after systemic delivery in human cancer patients, despite antibodies. In the present work, we have focused on the interaction of a chimeric adenovirus Ad5/3 with human lymphocytes and human erythrocytes. Ad5/3 binding with human lymphocytes and erythrocytes was observed to occur in a reversible manner, which allowed viral transduction of tumors, and oncolytic potency of Ad5/3 in vitro and in vivo, with or without neutralizing antibodies. Immunodeficient mice bearing xenograft tumors showed enhanced tumor transduction following systemic administration, when Ad5/3 virus was bound to lymphocytes or erythrocytes (P < 0.05). In conclusion, our findings reveal that chimeric Ad5/3 adenovirus reaches non-injected tumors in the presence of neutralizing antibodies: it occurs through reversible binding to lymphocytes and erythrocytes.

Tumor microenvironment remodeling by an engineered oncolytic adenovirus results in improved outcome from PD-L1 inhibition.

Checkpoint inhibitors have revolutionized cancer therapy and validated immunotherapy as an approach. Unfortunately, responses are seen in a minority of patients. Our objective is to use engineered adenoviruses designed to increase lymphocyte trafficking and cytokine production at the tumor, to assess if they increase the response rate to checkpoint inhibition, as these features have been regarded as predictive for the responses. When Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (an oncolytic adenovirus coding for TNFa and IL-2, also known as TILT-123) and checkpoint inhibitors were used together in fresh urological tumor histocultures, a significant shift toward immune activity (not only tumor necrosis alpha and interleukin-2 but also interferon gamma and granzyme B) and increased T-cell trafficking signals (CXCL10) was observed. In vivo, our viruses enabled an anti-PD-L1 (a checkpoint inhibitor) delivering complete responses in all the treated animals (hazard ratios versus anti-PD-L1 alone 0.057 [0.007; 0.451] or virotherapy alone 0.067 [0.011; 0.415]). To conclude, when an engineered oncolytic adenovirus was utilized to modify the tumor microenvironment towards what meta-analyses have pointed as predictive markers for checkpoint inhibitory therapy, the response to them increased synergistically. Of note, key findings were confirmed in fresh patient-derived tumor explants.

Oncolytic viruses for cancer immunotherapy.

In this review, we discuss the use of oncolytic viruses in cancer immunotherapy treatments in general, with a particular focus on adenoviruses. These serve as a model to elucidate how versatile viruses are, and how they can be used to complement other cancer therapies to gain optimal patient benefits. Historical reports from over a hundred years suggest treatment efficacy and safety with adenovirus and other oncolytic viruses. This is confirmed in more contemporary patient series and multiple clinical trials. Yet, while the first viruses have already been granted approval from several regulatory authorities, room for improvement remains.As good safety and tolerability have been seen, the oncolytic virus field has now moved on to increase efficacy in a wide array of approaches. Adding different immunomodulatory transgenes to the viruses is one strategy gaining momentum. Immunostimulatory molecules can thus be produced at the tumor with reduced systemic side effects. On the other hand, preclinical work suggests additive or synergistic effects with conventional treatments such as radiotherapy and chemotherapy. In addition, the newly introduced checkpoint inhibitors and other immunomodulatory drugs could make perfect companions to oncolytic viruses. Especially tumors that seem not to be recognized by the immune system can be made immunogenic by oncolytic viruses. Logically, the combination with checkpoint inhibitors is being evaluated in ongoing trials. Another promising avenue is modulating the tumor microenvironment with oncolytic viruses to allow T cell therapies to work in solid tumors.Oncolytic viruses could be the next remarkable wave in cancer immunotherapy.

Modulation of the tumor microenvironment with an oncolytic adenovirus for effective T-cell therapy and checkpoint inhibition.

Despite exciting proof-of-concept data mediated by adoptive T-cell transfer and checkpoint blockade, major challenges imposed by the tumor microenvironment restrict clinical benefits to a minority of patients with advanced or metastatic solid malignancies. While employment of toxic pre- and postconditioning regimens to circumvent the inefficacy of T-cell transfer presents a fundamental problem for heavily pretreated cancer patients, for checkpoint blockade, the main issue relates to low single-agent response rates. To overcome these hurdles, combination therapy with oncolytic adenovirus is becoming an attractive solution given multiple intrinsic modulatory effects on the intratumoral immune compartment, engineering capabilities and safety profile. Here, we provide a short overview on the tumor microenvironmental challenges in solid tumors, and how oncolytic adenoviruses can counteract these barriers. Finally, the immunotherapeutic potential of oncolytic adenoviruses will be discussed in the context of clinical experience with adoptive T-cell therapy and immune checkpoint inhibitors.

Oncolytic adenovirus shapes the ovarian tumor microenvironment for potent tumor-infiltrating lymphocyte tumor reactivity.

BACKGROUND: Ovarian cancers often contain significant numbers of tumor-infiltrating lymphocytes (TILs) that can be readily harnessed for adoptive T-cell therapy (ACT). However, the immunosuppressive ovarian tumor microenvironment and lack of tumor reactivity in TILs can limit the effectiveness of the therapy. We hypothesized that by using an oncolytic adenovirus (Ad5/3-E2F-D24-hTNFa-IRES-hIL2; TILT-123) to deliver tumor necrosis factor alpha (TNFa) and interleukin-2 (IL-2), we could counteract immunosuppression, and enhance antitumor TIL responses in ovarian cancer (OVCA). METHODS: We established ex vivo tumor cultures freshly derived from patients with advanced OVCA and evaluated the effects of Ad5/3-E2F-D24-hTNFa-IRES-hIL2 or Ad5/3-E2F-D24 (the control virus without TNFa and IL-2) on TILs, cytokine response and tumor viability. Tumor reactivity was assessed by determining interferon gamma (IFNg) response of clinically relevant TILs towards autologous T-cell-depleted ex vivo tumor cultures pretreated with or without the aforementioned oncolytic adenoviruses. RESULTS: Treatment of ex vivo tumor cultures with Ad5/3-E2F-D24-hTNFa-IRES-hIL2 caused a substantial rise in proinflammatory signals: increased secretion of IFNg, CXCL10, TNFa and IL-2, and concomitant activation of CD4+ and CD8+ TILs. Potent tumor reactivity was seen, as clinically relevant TIL secreted high levels of IFNg in response to autologous T-cell-depleted ovarian ex vivo tumor cultures treated with Ad5/3-E2F-D24-hTNFa-IRES-hIL2. This phenomenon was independent of PD-L1 expression in tumor cells, a factor that determined the variability of IFNg responses seen in different patient samples. CONCLUSIONS: Overall, oncolytic adenovirus Ad5/3-E2F-D24-hTNFa-IRES-hIL2 was able to rewire the ovarian tumor microenvironment to accommodate heightened antitumor TIL reactivity. Such effects may improve the clinical effectiveness of ACT with TILs in patients with advanced OVCA.

Constitutively active GSK3ß as a means to bolster dendritic cell functionality in the face of tumour-mediated immune suppression.

In patients with cancer, the functionality of Dendritic Cells (DC) is hampered by high levels of tumor-derived suppressive cytokines, which interfere with DC development and maturation. Poor DC development can limit the efficacy of immune checkpoint blockade and in vivo vaccination approaches. Interference in intracellular signaling cascades downstream from the receptors of major tumor-associated suppressive cytokines like IL-10 and IL-6, might improve DC development and activation, and thus enhance immunotherapy efficacy. We performed exploratory functional screens on arrays consisting of >1000 human kinase peptide substrates to identify pathways involved in DC development and its inhibition by IL-10 or IL-6. The resulting alterations in phosphorylation of the kinome substrate profile pointed to glycogen-synthase kinase-3ß (GSK3ß) as a pivotal kinase in both DC development and suppression. GSK3ß inhibition blocked human DC differentiation in vitro, which was accompanied by decreased levels of IL-12p70 secretion, and a reduced capacity for T cell priming. More importantly, adenoviral transduction of monocytes with a constitutively active form of GSK3ß induced resistance to the suppressive effects of IL-10 and melanoma-derived supernatants alike, resulting in improved DC development, accompanied by up-regulation of co-stimulatory markers, an increase in CD83 expression levels in mature DC, and diminished release of IL-10. Moreover, adenovirus-mediated intratumoral manipulation of this pathway in an in vivo melanoma model resulted in DC activation and recruitment, and in improved immune surveillance and tumor control. We propose the induction of constitutive GSK3ß activity as a novel therapeutic means to bolster DC functionality in the tumor microenvironment.

CD40L coding oncolytic adenovirus allows long-term survival of humanized mice receiving dendritic cell therapy.

Dendritic cells (DCs) are crucial players in promoting immune responses. Logically, adoptive DC therapy is a promising approach in cancer immunotherapy. One of the major obstacles in cancer immunotherapy in general is the immunosuppressive tumor microenvironment, which hampers the maturation and activation of DCs. Therefore, human clinical outcomes with DC therapy alone have been disappointing. In this study, we use fully serotype 3 oncolytic adenovirus Ad3-hTERT-CMV-hCD40L, expressing human CD40L, to modulate the tumor microenvironment with subsequently improved function of DCs. We evaluated the synergistic effects of Ad3-hTERT-CMV-hCD40L and DCs in the presence of human peripheral blood mononuclear cells ex vivo and in vivo. Tumors treated with Ad3-hTERT-CMV-hCD40L and DCs featured greater antitumor effect compared with unarmed virus or either treatment alone. 100% of humanized mice survived to the end of the experiment, while mice in all other groups died by day 88. Moreover, adenovirally-delivered CD40L induced activation of DCs, leading to induction of Th1 immune responses. These results support clinical trials with Ad3-hTERT-CMV-hCD40L in patients receiving DC therapy.

Adenovirus Coding for Interleukin-2 and Tumor Necrosis Factor Alpha Replaces Lymphodepleting Chemotherapy in Adoptive T Cell Therapy.

Lymphodepleting preconditioning with high-dose chemotherapy is commonly used to increase the clinical efficacy of adoptive T cell therapy (ACT) strategies, however, with severe toxicity for patients. Conversely, oncolytic adenoviruses are safe and, when engineered to express interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-α), they can achieve antitumor immunomodulatory effects similar to lymphodepletion. Therefore, we compare the safety and efficacy of such adenoviruses with a cyclophosphamide- and fludarabine-containing lymphodepleting regimen in the setting of ACT. Human adenovirus (Ad5/3-E2F-D24-hTNF-α-IRES-hIL-2; TILT-123) replication was studied using a Syrian hamster pancreatic tumor model (HapT1) infused with tumor-infiltrating lymphocytes (TILs). Using the oncolytic virus instead of lymphodepletion resulted in superior efficacy and survival. Immune cells responsive to TNF-α IL-2 were studied using an immunocompetent mouse melanoma model (B16.OVA) infused with ovalbumin-specific T (OT-I) cells. Here, the adenovirus approach improved tumor control together with increased intratumoral Th1 cytokine levels and infiltration of CD8+ T cells and CD86+ dendritic cells. Similar to humans, lymphodepleting preconditioning caused severe cytopenias, systemic inflammation, and damage to vital organs. Toxicity was minimal in adenovirus- and OT-I-treated mice. These findings demonstrate that ACT can be effectively facilitated by cytokine-coding adenovirus without requiring lymphodepletion, a rationale being clinically investigated.

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.

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.