Ultralow-dose binary oncolytic/helper-dependent adenovirus promotes antitumor activity in preclinical and clinical studies

Abstract

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.

Fig. 1.. ULCA controls primary tumor growth in breast cancer xenograft mouse models.

(A) ffLuc-labeled MCF-7 and SUM-159 cells were orthotopically transplanted into the mammary fat pad of NSG female mice (n = 5 animals per condition). After tumor volume reached >100 mm³, we injected a total of 1 × 10⁶ vp of CAdVEC (OAd:HD = 1:1) intratumorally. Control mice received vehicle (PBS) alone. We monitored tumor bioluminescence at the indicated time points. Data are presented as means ± SD. Two MCF-7 mice treated with CAdVEC were euthanized at 44 days after CAdVEC injection due to coronavirus disease (COVID) animal experiment restrictions. (B) Primary tumor volumes were monitored at different time points. Tumor volumes shown here are from 42 days (MCF-7) and 24 days (SUM-159) after injection of CAdVEC; individual data points are represented in violin plot. (C) We collected serum samples from mice at 0, 3, 7, 21, and 42 days after injection of CAdVEC and measured IFN-γ and IL-12p70 levels by enzyme-linked immunosorbent assay (ELISA). Data are presented as means ± SD.

Fig. 2.. ULCA controls tumor growth in humanized mouse models.

(A) ffLuc-labeled MCF-7 and SUM-159 cells were orthotopically transplanted into the mammary fat pad of humanized female mice (n = 5 animals per condition). After tumor volume reached >100 mm³, we intrathecally injected a total of 1 × 10⁶ vp of CAdVEC (OAd:HD = 1:1). Control mice received vehicle (PBS) alone. We monitored tumor bioluminescence at the indicated time points. Data are presented as means ± SD. (B) Primary tumor volumes were monitored at different time points. Tumor volumes shown here are from 72 days (MCF-7) and 24 days (SUM-159) after injection of CAdVEC; individual data points are represented in violin plot. (C) We collected serum samples from mice at 0, 3, 7, and 21 days after injection of CAdVEC and measured human T_H1 and T_H2 cytokine levels by Multiplex. IL-1β was undetectable level. Data are presented as means ± SD.

Fig. 3.. ULCA repolarizes the TME and induces adaptive immune responses against cancer cells in humanized mouse models.

MCF-7 and SUM-159 cells were orthotopically transplanted into the mammary fat pad of humanized female mice. After tumor volume reached >100 mm³, a total of 1 × 10⁶ vp of CAdVEC (OAd:HD = 1:1) were injected intrathecally. Tumor, spleen, and blood samples were collected at 24 days after CAdVEC injection. (A) Because of limited human immune cells in MCF-7 tumors, we pooled immune cells from four to five MCF-7 tumors and stained for flow cytometry and phenotyping. Immune cells from repeated experiment were stained with T cell markers. Single-stained t-distributed stochastic neighbor embedding (tSNE) plots are in fig. S5. (B) Immune cells from SUM-159 tumors were stained for flow cytometry and phenotyped. Immune cells from repeated experiment were stained with T cell markers. Data are presented as means ± SD. Single-stained tSNE plots are in fig. S5. (C) CD8⁺ T cells were isolated from spleen samples, and we performed an IFN-γ ELISPOT assay with irradiated cancer cells (E:T = 10:1). Data are presented as means ± SD.

Fig. 4.. ULCA treatment is safe in patients.

A total of 5 × 10⁹ vp of CAdVEC (OAd:HD = 1:1) in 500 μl were intratumorally injected. (A) We measured liver enzymes aspartate aminotransferase (AST) and alanine transaminase (ALT) at the indicated time points. (B) We measured human T_H1 and T_H2 cytokine levels in patient plasma samples at the indicated time points by Multiplex. (C) We extracted DNA from 1 week after CAdVEC biopsy samples and determined the copy number of each Ad vector by quantitative polymerase chain reaction (PCR). Serum, urine, and buccal swab were collected at the indicated time points. DNA was extracted from these samples, and the copy number of OAd was determined by quantitative PCR. Ad neutralizing IgG in 1:1000 diluted serum was determined at the indicated time points. Neutralization was calculated on the basis of negative and positive controls.

Fig. 5.. ULCA treatment changes circulating immune cell profile.

(A) Lymphocyte numbers in PBMCs were counted at the indicated time points. (B) We phenotyped freshly isolated PBMCs with different immune cell markers (detailed in fig. S8) at the indicated time points. Flowsom data are representative data from patient #1. (C) We phenotyped freshly isolated PBMCs with different T cell markers (detailed in fig. S8) at the indicated time points. Flowsom data are representative data from patient #1. (D) We measured human CXCL10 and CCL5 levels in plasma samples by ELISA. We phenotyped freshly isolated CXCR3-positive PBMCs from patients before CAdVEC treatment (detailed in fig. S8). Flowsom data are representative data from patient #1. (E) Matched pre- and post-tumor biopsies from patients #3 and #4 were stained with human CD56 and CD8 IgG for immunohistochemistry (IHC). (F) We performed Visium spatial gene expression with pre- and post-CAdVEC tumor biopsy samples from patient #4. Immune cell signatures were performed using spaceranger count.

Fig. 6.. ULCA treatment stimulates antitumor activity.

(A) Swimmers plot of CAdVEC injected tumor in each patient after ULCA treatment. (B) Each patient’s tumor images at the indicated time points. Some patients also had scans at the indicated time points.