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Abstract
Background Cancer immunotherapies are dependent on endogenous biomarker expression and other biological factors that often result in varying response rates across tumor types and benefit only a subset of patients. Conversely, conventional cytotoxics, the first-line treatment against solid tumors, are effective in a large patient population, but lack specificity, and often result in dose-limiting systemic toxicity. Here, we present SQ3370, a modular approach that activates doxorubicin (Dox) directly at the tumor site with reduced toxicity and potentially activates an immune response against tumor. The technology is independent of biomarkers, enzymatic activity, pH or oxygen levels and is hence expected to be effective in a wider group of patients. SQ3370 consists of a local intratumoral injection of a prodrug-capturing biomaterial (SQL70) followed by 5 daily infusions of SQP33, an attenuated prodrug of Dox. Complementary click chemistry groups in both components allow active Dox release at the tumor site (figure 1). SQP33 prodrug is ~82-fold less cytotoxic than Dox in vitro. We safely administered SQ3370 in dogs at 8.95-times the veterinary clinical dose of Dox, thus widening the therapeutic window, and showed minimal side effects including cardiotoxicity and immunosuppression. We hypothesize that releasing Dox at a local site with SQ3370 may also promote immune activation against the tumor. We evaluated this in a dual-tumor model of syngeneic MC38 tumors.
Methods Immunocompetent mice were inoculated with MC38 tumor cells in two subcutaneous flanks. One tumor was intratumorally injected with the biomaterial, SQL70. SQP33 prodrug, control Dox, or saline was administered intravenously as per treatment schedule (figure 2A). Tumors harvested from a subset of mice at 2 weeks were assessed for immune biomarkers.
Results In mice bearing two tumors, SQ3370 significantly increased overall survival and the anti-tumor response against injected tumors (figure 2B,C). Surprisingly, SQ3370 also induced regression of the non-injected tumors (figure 2D). Assessment of tumor-Infiltrating immune cells showed an increase in CD3+, CD4+, and CD8+ T cells and a decrease in regulatory T cells in both injected and non-injected lesions (figure 3). The T-cell response correlated with the anti-tumor efficacy data, supporting the immune activation hypothesis.
SQ3370 Components and administrationSQ3370 consists of a local intratumoral injection of a prodrug-capturing biomaterial (named SQL70; shown in step 1) followed by 5 daily systemic infusions of SQP33, an attenuated prodrug of doxorubicin (step 2). Complementary chemical groups in the two components allow the local capture of the prodrug (step 3) and trigger release of active doxorubicin at the tumor site (step 4).
Systemic anti-tumor response and improved survivalImmunocompetent C57BL/6 mice were inoculated with mouse MC38 tumors. Tumors were implanted on Day 0. Treatments started on Day 7 with local injection of SQL70 biomaterial at ‘injected’ tumor, followed by systemic administration of saline, Dox, or SQP33 prodrug (A). Median overall survival (B) was significantly higher with SQ3370 as compared to Dox, as determined by Logrank test. Injected tumor response (C) was also significantly better with SQ3370, as assessed by corrected t tests, and non-injected tumors (D) showed a favorable response only with SQ3370. Injected-tumor growth curves show mean ± SEM, and growth curves for non-injected tumors show the percent change in tumor size from initial measurement. Curves stopped when a mouse died, or its tumor volume reached 2000 mm3.
Tumor-infiltrating immune cells in both tumorsTumor samples were stained with antibodies and analyzed by multicolor flow cytometry. Dead cells were excluded from analysis. Results show mean ± SEM (n = 3–5 per group) as a percentage of total or marker-gated (CD4 or CD8) cells obtained from the tumor sample. Statistical significance was assessed using a corrected t test.
Conclusions SQ3370 is a proof of concept example for a novel modular approach that addresses limitations of current immuno- and cytotoxic therapies for patients with solid tumors. Local release of Dox with SQ3370 expands the therapeutic window of Dox, minimizes toxicities and leads to a robust anti-tumor response that potentially also causes immune activation against the tumors.
Acknowledgements The authors would like to thank the National Institutes of Health (NIH), the National Science Foundation (NSF), and Y Combinator.
Ethics Approval This study, project number: SQI-FFS-ON-20181119_04_v4, was approved by the Institutional Animal Care and Use Committee (IACUC) of the vendor, following the guidance of Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC), accreditation number 001516.
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