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Background: Metastatic breast cancer (MBC) which causes significant morbidity and mortality worldwide is in need of more effective treatment regimens. In combination with chemotherapy, anti-PD1 antibody pembrolizumab has been shown to prolong progression-free survival (PFS) of patients with triple-negative subtype MBC (TN-MBC), however, its efficacy remains low for the other 80% of patients with MBC. MBC’s heterogenous pattern of immune infiltration and expression make it challenging to treat with single immunotherapeutic agents such as pembrolizumab and successful immunotherapy must therefore target multiple pathways. To augment antitumor host immune responses during treatment, studies have examined adjunct agents such as “oncolytic” adenovirus (OAds), which are vectors that preferentially replicate in and lyse tumor cells leading to the activation of host immunity. OAds have been tested in a myriad of clinical trials with the hope to enhance host immune activation but those trials have shown limited successes. Methods: To overcome the multiple immunogenic barriers in solid tumors, our group developed a binary oncolytic/helper-dependent adeno-immunotherapy (CAdVEC). The first generation CAdVEC (CAdTrio) contains an OAd and a “helper-dependent” adenovirus (HDAd) that produces immunostimulant molecules including interleukin (IL)-12p70 and anti-PD-L1 antibody. Based on successful results using animal models, a first-in-human Phase 1 study with CAdTrio was conducted among patients with all solid tumors (NCT03740256). Four patients with MBC were enrolled in the virus dose-escalation phase of the trial and received an intra-tumor injection of CAdTrio. Given the novelty of this binary agent, starting dose of CAdTrio was more than 2-logs lower than that used in other OAd trials. Three patients received dose level (DL) 1 and one patient received DL2. All patients also received pembrolizumab 6 weeks after the virus injection. The primary endpoint for this phase I dose escalation was dose-limiting toxicities (DLT). Secondary outcomes included overall response rates (ORR), disease control rate (DCR), PFS, overall survival (OS), and treatment-related adverse events. Results: No patients developed DLT. The most common toxicities were fever, fatigue and pain around the injection site, but none were greater than grade 2. No significant elevation in liver enzymes were observed. Three of the four patients had partial response (PR). One patient progressed after ten weeks of stable disease and passed away. The three patients with PR received pembrolizumab within 7 weeks of CAdVEC injection. Analysis of injected tissues prior to pembrolizumab treatment showed that CAdTrio repolarized the tumor microenvironment toward immune activity by increasing the number of infiltrating Th1 immune cells, leading to responses in some treated tumors and even in one distant metastasis, demonstrating the potent systemic immune response to local CAdTrio treatment in patients with MBC. Conclusions: Our study demonstrated that intra-tumor injection with CAdTrio was safe and effective in patients with MBC but the significance of the results was limited by the small sample size. An MBC dedicated phase II trial is planned to be conducted to fully evaluate the efficacy and safety of CAdVEC treatment and to further elucidate mechanisms of resistance/sensitivity among patients with MBC. Citation Format: Natalie Chen, Daniel Wang, Caroline E. Porter, Amanda Rosewell Shaw, Catherine R. Robertson, Mae L. Woods, Ya Xu, Greyson Biegert, Alphi Kuriakose, Tao Wang, Bambi J. Grilley, Helen Heslop, Malcolm K. Brenner, Masataka Suzuki, Bora Lim. Treatment of Metastatic Breast Cancer with Multipotent Oncolytic/Helper Adenovirus CAdVEC [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-06-04.

We show that a binary oncolytic/helper-dependent adenovirus (CAdVEC) that both lyses tumor cells and locally expresses the proinflammatory cytokine IL-12 and PD-L1 blocking antibody has potent antitumor activity in humanized mouse models. On the basis of these preclinical studies, we treated four patients with a single intratumoral injection of an ultralow dose of CAdVEC (NCT03740256), representing a dose of oncolytic adenovirus more than 100-fold lower than used in previous trials. While CAdVEC caused no significant toxicities, it repolarized the tumor microenvironment with increased infiltration of CD8 T cells. A single administration of CAdVEC was associated with both locoregional and abscopal effects on metastases and, in combination with systemic administration of immune checkpoint antibodies, induced sustained antitumor responses, including one complete and two partial responses. Hence, in both preclinical and clinical studies, CAdVEC is safe and even at extremely low doses is sufficiently potent to induce significant tumor control through oncolysis and immune repolarization.

High expression levels of human epidermal growth factor receptor 2 (HER2) have been associated with poor prognosis in patients with pancreatic adenocarcinoma (PDAC). However, HER2-targeting immunotherapies have been unsuccessful to date. Here we increase the breadth, potency, and duration of anti-PDAC HER2-specific CAR T-cell (HER2.CART) activity with an oncolytic adeno-immunotherapy that produces cytokine, immune checkpoint blockade, and a safety switch (CAdTrio). Combination treatment with CAdTrio and HER2.CARTs cured tumors in two PDAC xenograft models and produced durable tumor responses in humanized mice. Modifications to the tumor immune microenvironment contributed to the antitumor activity of our combination immunotherapy, as intratumoral CAdTrio treatment induced chemotaxis to enable HER2.CART migration to the tumor site. Using an advanced PDAC model in humanized mice, we found that local CAdTrio treatment of primary tumor stimulated systemic host immune responses that repolarized distant tumor microenvironments, improving HER2.CART anti-tumor activity. Overall, our data demonstrate that CAdTrio and HER2.CARTs provide complementary activities to eradicate metastatic PDAC and may represent a promising co-operative therapy for PDAC patients., Rosewell Shaw et al. show that a previously developed immunotherapy strategy, coupling oncolytic adenoviral immunotherapy with clinically tested HER2-specific CAR T-cells, is effective against pancreatic ductal adenocarcinoma (PDAC). This combination therapy produces a curative response in both PDAC xenografts and humanized mouse models.

No single cancer immunotherapy will likely defeat all evasion mechanisms of solid tumors, including plasticity of tumor antigen expression and active immune suppression by the tumor environment. In this study, we increase the breadth, potency, and duration of anti-tumor activity of chimeric antigen receptor (CAR) T cells using an oncolytic virus (OV) that produces cytokine, checkpoint blockade, and a bispecific tumor-targeted T cell engager (BiTE) molecule. First, we constructed a BiTE molecule specific for CD44 variant 6 (CD44v6), since CD44v6 is widely expressed on tumor but not normal tissue, and a CD44v6 antibody has been safely administered to cancer patients. We then incorporated this BiTE sequence into an oncolytic-helper binary adenovirus (CAdDuo) encoding an immunostimulatory cytokine (interleukin [IL]-12) and an immune checkpoint blocker (PD-L1Ab) to form CAdTrio. CD44v6 BiTE from CAdTrio enabled HER2-specific CAR T cells to kill multiple CD44v6+ cancer cell lines and to produce more rapid and sustained disease control of orthotopic HER2+ and HER2−/− CD44v6+ tumors than any component alone. Thus, the combination of CAdTrio with HER2.CAR T cells ensures dual targeting of two tumor antigens by engagement of distinct classes of receptor (CAR and native T cell receptor [TCR]), and significantly improves tumor control and survival.

In solid tumors, chimeric antigen receptor (CAR)-modified T cells must overcome the challenges of the immunosuppressive tumor microenvironment. We hypothesized that pre-treating tumors with our binary oncolytic adenovirus (CAd), which produces local oncolysis and expresses immunostimulatory molecules, would enhance the antitumor activity of HER2-specific CAR T cells, which alone are insufficient to cure solid tumors. We tested multiple cytokines in conjunction with PD-L1-blocking antibody and found that Ad-derived IL-12p70 prevents the loss of HER2.CAR-expressing T cells at the tumor site. Accordingly, we created a construct encoding the PD-L1-blocking antibody and IL-12p70 (CAd12_PDL1). In head and neck squamous cell carcinoma (HNSCC) xenograft models, combining local treatment with CAd12_PDL1 and systemic HER2.CAR T cell infusion improved survival to >100 days compared with approximately 25 days with either approach alone. This combination also controlled both primary and metastasized tumors in an orthotopic model of HNSCC. Overall, our data show that CAd12_PDL1 augments the anti-tumor effects of HER2.CAR T cells, thus controlling the growth of both primary and metastasized tumors.

HER2, commonly referred to as ErbB2, is a receptor tyrosine kinase that, along with EGFR, makes up one of the four members of the ErbB family of proteins. These ErbB proteins are expressed in most epithelial cell layers and play a key role in cell differentiation. HER2 has been found to be overexpressed in a number of human cancers, including breast and gastric carcinomas. Overexpression of the HER2 receptor represents a potential target for chimeric antigen receptor (CAR) T cell therapy. CAR T clinical trials in leukemia and lymphoma have demonstrated durable remission of the disease or even cure, but application of CAR T cells to solid tumors such as head and neck squamous cell carcinoma (HNSCC) lags behind. To evaluate HER2 expression in HNSCC, we prepared 4 different tumor microarrays (TMAs) of patient tumors based on their location (larynx, salivary gland, oropharynx or oral cavity) for IHC staining. These TMAs were then stained and scored by two different methods: with an anti-HER2 monoclonal antibody (CB11) and scoring based on breast cancer guidelines, or with the FDA-approved HER2 HercepTest IHC procedure. Based on the level of HER2 expression determined by TMA staining, we identify HNSCC as a candidate tumor type for preclinical testing of HER2-specific CAR T therapy. We utilize the chick embryo chorioallantoic membrane (CAM) tumor model, a naturally immune-deficient platform for growth of vascularized, three-dimensional tumors, as a rapid and inexpensive system to assess CAR T efficacy against human HNSCC tumors in vivo. To demonstrate tumor killing efficacy of HER2-specific CAR T cells (HER2.CAR Ts) on the CAM, we engrafted tumors derived from the HER2-positive (+) HNSCC cell line FaDu and HER2-negative (–) breast adenocarcinoma cell line MDA-MB-468 onto the CAM of day 7 fertilized chicken eggs. Both cell lines had been previously genetically engineered to express firefly luciferase (ffLuc). On day 10, established tumors were treated with HER2.CAR Ts generated by retroviral transduction of primary activated human T cells with a second-generation CAR T construct incorporating a CD28.ζ signaling domain. Four days after CAR T treatment, tumors were excised from the CAM, homogenized, and lysed; the luminescence of the resulting cell lysates (ffLuc activity) was used to quantify the relative number of viable tumor cells. While CAR T treatment resulted in an average 56% decrease in tumor size in the HER2+ FaDu tumors, there was no significant change in HER2– MDA-MB-468 tumor size. These results suggest that HER2-expressing HNSCC can be effectively targeted by HER2-directed CAR T calls and demonstrate the potential of the CAM tumor model as a cost-effective tool for rapid preclinical assessment of CAR T cell therapy. Citation Format: Emilie A. Warren, Hsuan-Chen Liu, Caroline E. Porter, Kershena S. Liao, Meenakshi Hegde, Wendong Yu, Patricia D. Castro, Vlad Sandulache, Nabil Ahmed, Masataka Suzuki, Andrew Sikora. Overexpression of HER2 in head and neck cancer represents a potential target for T cell immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 574.

Chimeric antigen receptor–modified T cells (CAR T cells) produce proinflammatory cytokines that increase expression of T-cell checkpoint signals such as PD-L1, which may inhibit their functionality against solid tumors. In this study, we evaluated in human tumor xenograft models the proinflammatory properties of an oncolytic adenovirus (Onc.Ad) with a helper-dependent Ad (HDAd) that expresses a PD-L1 blocking mini-antibody (mini-body; HDPDL1) as a strategy to enhance CAR T-cell killing. Coadministration of these agents (CAd-VECPDL1) exhibited oncolytic effects with production of PD-L1 mini-body locally at the tumor site. On their own, HDPDL1 exhibited no antitumor effect and CAd-VECPDL1 alone reduced tumors only to volumes comparable to Onc.Ad treatment. However, combining CAd-VECPDL1 with HER2.CAR T cells enhanced antitumor activity compared with treatment with either HER2.CAR T cells alone or HER2.CAR T cells plus Onc.Ad. The benefits of locally produced PD-L1 mini-body by CAd-VECPDL1 could not be replicated by infusion of anti-PD-L1 IgG plus HER2.CAR T cells and coadministration of Onc.Ad in an HER2+ prostate cancer xenograft model. Overall, our data document the superiority of local production of PD-L1 mini-body by CAd-VECPDL1 combined with administration of tumor-directed CAR T cells to control the growth of solid tumors. Cancer Res; 77(8); 2040–51. ©2017 AACR.