The success of chimeric antigen receptor T cell therapies targeting solid tumors is limited by the immunosuppressive tumor microenvironment. We demonstrate that endowing CAR T cells with ectopic interleukin-9 (IL-9) signaling by co-expressing an IL-9 receptor, rewires CAR T cell fate under antigen stress to enhance anti-tumor efficacy. In preclinical solid tumor models, IL-9-signaling CAR T cells exhibit increased expansion, persistence, and tumor infiltration, resulting in superior tumor control at significantly lower doses than conventional products. Trajectory and RNA velocity analyses of single-cell RNA sequencing data reveal that IL-9 signaling alters CAR T cell differentiation under antigen stress away from dysfunction, favoring a multipotent transition toward CD8+ cell memory and effector states, and promoting a CD4+ cell proliferative state. Interrogation of transcription factor pathways indicates that IL-9-mediated activation of STAT1 and STAT4 drives the superior phenotype of IL-9-signaling CAR T cells, providing a promising therapeutic strategy for targeting solid cancers.
Abstract Synthetic receptor signalling has the potential to endow adoptively transferred T cells with new functions that overcome major barriers in the treatment of solid tumours, including the need for conditioning chemotherapy 1,2 . Here we designed chimeric receptors that have an orthogonal IL-2 receptor extracellular domain (ECD) fused with the intracellular domain (ICD) of receptors for common γ-chain (γ c ) cytokines IL-4, IL-7, IL-9 and IL-21 such that the orthogonal IL-2 cytokine elicits the corresponding γ c cytokine signal. Of these, T cells that signal through the chimeric orthogonal IL-2Rβ-ECD–IL-9R-ICD (o9R) are distinguished by the concomitant activation of STAT1, STAT3 and STAT5 and assume characteristics of stem cell memory and effector T cells. Compared to o2R T cells, o9R T cells have superior anti-tumour efficacy in two recalcitrant syngeneic mouse solid tumour models of melanoma and pancreatic cancer and are effective even in the absence of conditioning lymphodepletion. Therefore, by repurposing IL-9R signalling using a chimeric orthogonal cytokine receptor, T cells gain new functions, and this results in improved anti-tumour activity for hard-to-treat solid tumours.
Adoptive T cell therapy (ACT) using genetically modified T cells has shown exceptional efficacy in the treatment of CD19+ hematological cancers. However, far less impressive results have been achieved in the treatment of solid tumors due to local immunosuppression, rendering tumor-infiltrating T cells (TILs) hypofunctional. We have previously shown that adenovirus (Ad) infection can enhance the efficacy of ACT (Tähtinen et al, CIR 2015) and that intratumoral administration of immunostimulatory cytokines can result in favorable alteration of tumor microenvironment (Tähtinen et al, PLOS One 2015). To combine the benefits of both approaches, we studied if replication-deficient Ad5-vectors coding for interleukin-2 (IL-2) and tumor necrosis factor alpha (TNF-a) would affect the activity of adoptively transferred, TCR-transgenic TRP-2(180-188) specific T cells in vivo. To gain clinically relevant mechanism-of-action data, we chose to use ret transgenic mouse model that develops spontaneous malignant skin melanoma which metastasizes into distant organs. Following ACT and intratumoral virus injection, a significant increase in activated PD-1+ CD8+ T cells was seen in both cutaneous lesions and in metastatic lymph nodes. Interestingly, a reverse correlation between tumor weight and the number of tumor-reactive PD-1+ TILs (p=0.0015) was observed, indicating that T-cell hypofunction was overcome and successful tumor lysis was achieved. Local expression of cytokines did not affect the levels of immunosuppressive immune cell subsets such as myeloid-derived suppressor cells (MDSCs) or T regulatory cells (Tregs), latter of which has previously been associated with systemic IL-2 therapy (Ahmadzadeh and Rosenberg, Blood 2006). Instead, Ad5-IL2 treatment induced upregulation of IL-2 receptor α-chain (CD25) in conventional CD4+CD25+Foxp3-cells, suggesting that these helper T cells contributed to CD8+ TIL activation. Finally, beneficial ratios between tumor-reactive PD-1+ CD8+ TILs and Tregs was observed in primary and secondary tumor sites, indicating that IL-2 and TNF-a coding adenoviruses can modify the cellular composition of the tumor microenvironment in favor of adoptively transferred T cells. In conclusion, IL-2 and TNF-a coding adenoviruses can break tumor-associated immunotolerance and significantly increase the levels of active, tumor-reactive T-cells both in injected cutaneous lesions and in non-injected metastatic lymph nodes. Importantly, this triple modality may represent an efficient approach to achieve "CD19-like" clinical responses in the treatment of solid, metastatic cancers currently incurable by standard therapies.
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.
Abstract Dendritic cell (DC) therapy is considered a promising immunotherapeutic approach for treatment of advanced cancer. However, the immunosuppressive tumor microenvironment leads to DC dysfunction. Therefore, in clinical trials DC therapy has generally failed to fulfill its expectations. Oncolytic adenoviruses are well tolerated and have shown to activate antitumor immune responses. Importantly, they can convert immunosuppression locally into a proinflammatory state. To improve the proper activation of transferred DCs, we armed oncolytic adenovirus with CD40 ligand (CD40L). CD40L is known to play an important role in the regulation of immune cells through its capacity to stimulate dendritic cells that leads to the activation of cytotoxic T cells. Therefore, we generated a novel virus Ad3-hTERT-CMV-hCD40L, which is fully serotype 3 adenovirus (Ad3) for intravenous delivery. It features a human telomerase reverse transcriptase (hTERT) promoter for tumor specific replication and expresses human CD40L (hCD40L) under a cytomegalovirus (CMV) promoter for induction of antitumor efficacy. Of note, human and animal data have shown the ability of Ad3 to successfully reach tumors through the intravenous route. In syngeneic studies in an immunocompetent model, DC therapy with our murine CD40L-armed adenovirus showed significant antitumor immune response. This enhanced therapeutic effect is associated with increased tumor specific T cells and induction of T-helper type 1 immune response. This synergistic effect was further evaluated in mice humanized with human peripheral blood mononuclear cells. Treatment with hCD40L-armed adenovirus and human DCs showed 100% survival in conjunction with tumor control. To conclude, CD40L armed oncolytic adenovirus 3 improves DC therapy by favorable alteration of tumor microenvironment. These findings support clinical trials where DC therapy is enhanced with oncolytic adenovirus. Citation Format: João Manuel Santos, Akseli Hemminki, Mikko Siurala, Otto Hemminki, Riikka Havunen, Victor Cervera-Carrascon, Suvi Sorsa, Hongjie Wang, Andre Lieber, Tanja de Gruijl, Anna Kanerva, Sadia Zafar. Oncolytic adenovirus 3 coding for CD40L as an enhancer of dendritic cell therapy [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B07.
Breast cancer is a heterogeneous disease, characterized by several distinct biological subtypes, among which triple-negative breast cancer (TNBC) is one associated with a poor prognosis. Oncolytic virus replication is an immunogenic phenomenon, and viruses can be armed with immunostimulatory molecules to boost virus triggered antitumoral immune responses. Cyclophosphamide (CP) is a chemotherapy drug that is associated with cytotoxicity and immunosuppression at higher doses, whereas immunostimulatory and anti-angiogenic properties are observed at low continuous dosage. Therefore, the combination of oncolytic immuno-virotherapy with low-dose CP is an appealing approach.We investigated the potency of oncolytic adenovirus Ad5/3-D24-GMCSF on a TNBC cell line and in vivo in an orthotopic xenograft mouse model, in combination with low-dose CP or its main active metabolite 4-hydroperoxycyclophosphamide (4-HP-CP). Furthermore, we summarized the breast cancer-specific human data on this virus from the Advanced Therapy Access Program (ATAP).Low-dose CP increased the efficacy of Ad5/3-D24-GMCSF in vitro and in a TNBC mouse model. In ATAP, treatments appeared safe and well-tolerated. Thirteen out of 16 breast cancer patients treated were evaluable for possible benefits with modified RECIST 1.1 criteria: 1 patient had a minor response, 2 had stable disease (SD), and 10 had progressive disease (PD). One patient is alive at 1,771 d after treatment.Ad5/3-D24-GMCSF in combination with low-dose CP showed promising efficacy in preclinical studies and possible antitumor activity in breast cancer patients refractory to other forms of therapy. This preliminary data supports continuing the clinical development of oncolytic adenoviruses for treatment of breast cancer, including TNBC.
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