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A desired goal of targeted cancer treatments is to achieve high tumor specificity with minimal side effects. Despite recent advances, this remains difficult to achieve in practice as most approaches rely on biomarkers or physiological differences between malignant and healthy tissue, and thus benefit only a subset of patients in need of treatment. To address this unmet need, we introduced a Click Activated Protodrugs Against Cancer (CAPAC) platform that enables targeted activation of drugs at a specific site in the body, i.e., a tumor. In contrast to antibodies (mAbs, ADCs) and other targeted approaches, the mechanism of action is based on in vivo click chemistry, and is thus independent of tumor biomarker expression or factors such as enzymatic activity, pH, or oxygen levels. The CAPAC platform consists of a tetrazine-modified sodium hyaluronate-based biopolymer injected at a tumor site, followed by one or more doses of a trans-cyclooctene (TCO)-modified cytotoxic protodrug with attenuated activity administered systemically. The protodrug is captured locally by the biopolymer through an inverse electron-demand Diels–Alder reaction between tetrazine and TCO, followed by conversion to the active drug directly at the tumor site, thereby overcoming the systemic limitations of conventional chemotherapy or the need for specific biomarkers of traditional targeted therapies. Here, TCO-modified protodrugs of four prominent cytotoxics (doxorubicin, paclitaxel, etoposide and gemcitabine) are used, highlighting the modularity of the CAPAC platform. In vitro evaluation of cytotoxicity, solubility, stability and activation rendered the protodrug of doxorubicin, SQP33, as the most promising candidate for in vivo studies. In mice, the maximum tolerated dose (MTD) of SQP33 in combination with locally injected tetrazine-modified biopolymer (SQL70) was determined to be 19.1-times the MTD of conventional doxorubicin. Pharmacokinetics studies in rats show that a single injection of SQL70 efficiently captures multiple SQP33 protodrug doses given cumulatively at 10.8-times the MTD of conventional doxorubicin with greatly reduced systemic toxicity. Finally, combined treatment with SQL70 and SQP33 (together called SQ3370) showed antitumor activity in a syngeneic tumor model in mice., The Click Activated Protodrugs Against Cancer (CAPAC) platform uses click chemistry to activate cytotoxic drugs directly at a target site with minimal toxicity, overcoming limitations of conventional chemotherapy and traditional targeted therapies.

BackgroundImmunotherapies have enabled unprecedented durable responses in solid tumors. However, they benefit only a subset of patients and have varying response rates across tumor types. Conversely, conventional chemotherapies are effective in a larger patient population, but lack specificity and result in severe dose-limiting systemic toxicities. While anthracyclines such as doxorubicin (Dox) may provide added benefit by inducing anti-tumor immune activation,1 their overall effect is limited by cumulative dose cardiotoxicity.2 Here, we present the Click Activated Protodrugs Against Cancer (CAPAC) platform that activates cytotoxic protodrugs at the tumor using click chemistry. The CAPAC platform is agnostic to tumor characteristics such as biomarker expression and can be applied in a variety of tumor types. SQ3370 (lead candidate of CAPAC) consists of an intratumorally-injected biopolymer that can activate multiple doses of a systemic protodrug to release active Dox at the tumor. SQ3370 enables a 19-fold increase over the conventional Dox dose in mice with minimal systemic toxicity.3 In tumor-bearing mice, SQ3370 improved overall survival, T-cell infiltration, and induced a robust anti-tumor response against both biopolymer-injected and non-injected lesions,4 suggesting that SQ3370 promotes anti-tumor immune activation. This makes SQ3370 an ideal candidate for combination approaches with immunotherapies.MethodsSQ3370 treatment is described in figure 1. Immunocompetent mice were inoculated with MC38 tumors. SQL70 was given intratumorally; SQP33 was given intravenously as five daily doses. Immune adjuvants such as toll-like receptor (TLR) agonists or STING agonist were coadministered with SQ3370. Saline and conventional Dox served as controls.ResultsCombining SQ3370 with endosomal TLR agonists such as poly I:C (TLR3a), imiquimod (TLR7a) or CpG ODN (TLR9a) resulted in improved tumor growth inhibition and survival in MC38 tumor-bearing mice compared with monotherapy. Combination with STING agonist, ADU-S100, also enhanced antitumor efficacy. Body weight data suggests minimal impact of the monotherapy or combination therapy on systemic toxicity.ConclusionsCAPAC represents a new therapeutic modality to treat solid tumors by expanding the pharmacological capabilities of cancer drugs with known efficacy and experimental drugs. SQ3370, CAPAC's lead candidate, improves safety and efficacy as compared to conventional Dox, and combination of SQ3370 with immunotherapy shows enhanced benefit. SQ3370 is being evaluated in a Phase I study in advanced solid tumors (NCT04106492).AcknowledgementsThis work was supported by Shasqi and the National Institutes of Health.ReferencesMattarollo SR, Loi S, Duret H, Ma Y, Zitvogel L, Smyth MJ. Pivotal role of innate and adaptive immunity in anthracycline chemotherapy of established tumors. Cancer Res 2011;71:4809–20.Quintana RA, Banchs J, Gupta R, Lin HY, Raj SD, Conley A, Ravi V, Araujo D, Benjamin RS, Patel S, Vadhan-Raj S. Early evidence of cardiotoxicity and tumor response in patients with sarcomas after high cumulative dose doxorubicin given as a continuous infusion. Sarcoma 2017;7495914.Wu K, Yee NA, Srinivasan S, Mahmoodi A, Zakharian M, Oneto JM, Royzen M. Click activated protodrugs against cancer increase the therapeutic potential of chemotherapy through local capture and activation. Chem Sci 2021;12:1259–71.Srinivasan S, Yee NA, Wu K, Zakharian M, Mahmoodi A, Royzen M, Mejía Oneto JM. SQ3370 activates cytotoxic drug via click chemistry at tumor and elicits sustained responses in injected and non-injected lesions. Adv Ther 2021;4:2000243.Ethics ApprovalThis study, project number: SSQ-FFS-ON-20210225-01V3_1, 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.Abstract 695 Figure 1Illustration and Molecular Description of SQ3370. SQ3370 consists of 2 components and utilizes Shasqi's proprietary click chemistry-based CAPAC platform. First, SQL70, a tetrazine-modified sodium hyaluronate biopolymer, is injected at the tumor site. Then, SQP33, a trans-cyclooctene (TCO)-modified protodrug of doxorubicin (Dox), is given systemically as 5 daily doses. SQP33 protodrug has attenuated toxicity and is converted to active Dox by SQL70 biopolymer through an efficient covalent reaction between tetrazine and TCO moieties

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. 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.

While systemic immuno-oncology therapies have shown remarkable success, only a limited subset of patients benefit from them. Our Click Activated Protodrugs Against Cancer (CAPAC™) Platform is a click chemistry-based approach that activates cancer drugs at a specific tumor with minimal systemic toxicity. CAPAC Platform is agnostic to tumor characteristics that can vary across patients and hence applicable to several types of tumors. We describe the benefits of SQ3370 (lead candidate of CAPAC) to achieve systemic anti-tumor responses in mice bearing two tumors. SQ3370 consists of a biopolymer, injected in a single lesion, followed by systemic doses of an attenuated protodrug of doxorubicin (Dox). SQ3370 was well-tolerated at 5.9-times the maximum dose of conventional Dox, increased survival by 63% and induced a systemic antitumor response against injected and non-injected lesions. The sustained anti-tumor response also correlated with immune activation measured at both lesions. SQ3370 could potentially benefit patients with micro-metastatic lesions.

While systemic immuno-oncology therapies have shown remarkable success, only a limited subset of patients benefit from them. Our Click Activated Protodrugs Against Cancer (CAPAC™) Platform is a click chemistry-based approach that activates cancer drugs at a specific tumor with minimal systemic toxicity. CAPAC Platform is agnostic to tumor characteristics that can vary across patients and hence applicable to several types of tumors. We describe the benefits of SQ3370 (lead candidate of CAPAC) to achieve systemic anti-tumor responses in mice bearing two tumors. SQ3370 consists of a biopolymer, injected in a single lesion, followed by systemic doses of an attenuated protodrug™ of doxorubicin (Dox). SQ3370 was well-tolerated at 5.9-times the maximum dose of conventional Dox, increased survival by 63% and induced a systemic anti-tumor response against injected and non-injected lesions. The sustained anti-tumor response also correlated with immune activation measured at both lesions. SQ3370 could potentially benefit patients with micro-metastatic lesions.

With systemic chemotherapy, only 1-2% of the administered dose reaches a localized tumor, while the remaining leads to adverse off-target toxicities, including immunosuppression. Hence, there is a critical need to locally deliver cytotoxics directly to the tumor. Our patented approach (SQ3370) consists of: 1. SQL70 - a drug-activating biomaterial carrying no payload 2. SQP33 - a chemically-modified prodrug of doxorubicin (Dox) with attenuated activity. SQL70 is injected at the tumor site followed by SQP33 administered systemically. The prodrug is first captured by the biomaterial due to their complementary chemical reactivities. The active drug is then spontaneously released, providing local delivery directly to the tumor region while reducing systemic side effects. Previously, we have shown that the greater safety of SQP33 allows it to be given at over 38 times the dose of standard Dox in SQL70-injected mice. Pharmacokinetic and biodistribution studies in SQ3370-treated rodents and dogs show highly efficient capture and activation of the prodrug, likely due to the rapid reaction of SQP33 with SQL70. Without SQL70, SQP33 shows minimal spontaneous conversion to activated Dox. Additionally, after SQL70 injection, increased dosing of SQP33 led to higher active drug levels at the injection site but not to other tissues. These studies indicate that a single injection of SQL70 can selectively activate multiple doses of SQP33 at the target site, maximizing the local therapeutic index. Recent studies demonstrate that SQ3370 treatment enhances therapeutic response and survival in tumor-bearing mice: In a syngeneic MC38 colorectal cancer model, SQ3370 treatment showed significantly improved anti-tumor response and overall survival compared to conventional Dox therapy. In mice bearing two tumors, we observed that SQL70 injection at one tumor site followed by systemic SQP33 treatment induced an anti-tumor response in both the injected and non-injected tumors. Furthermore, we found that SQ3370 increased CD4 and CD8 TILs and decreased regulatory T-cells in both injected and non-injected tumors. These findings suggest that SQ3370 is capable of inducing an immune-mediated anti-tumor effect, thus expanding potential treatment options in the clinic and highlighting the advantages of immune-sparing cytotoxic therapy. Collectively, our results demonstrate that SQ3370 enables delivery of cytotoxic drugs to a target site while limiting exposure in off-target tissues in small and large animals, leading to improvements in both safety and efficacy. Citation Format: Nathan Yee, Sangeetha Srinivasan, Amir Mahmoodi, Michael Zakharian, Maksim Royzen, Jose Mejia Oneto. SQ3370 enhances the safety and therapeutic index of cytotoxic agents via local activation therapy with improved local and systemic immune response [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6245.

e15525 Background: With systemic chemotherapy, only 1-2% of the dose given reaches a local tumor, while the remaining leads to off-target toxicities. Hence, there is a critical need to locally deliver cytotoxics directly to the tumor. Our approach (SQ3370) consists of: 1. SQL70 - drug-activating biomaterial carrying 2. SQP33 - chemically-modified prodrug of doxorubicin (Dox) with attenuated activity. Methods: SQL70 is injected at the tumor site followed by SQP33 given systemically. The prodrug is captured by the biomaterial due to their complementary chemical reactivities. The active drug is then spontaneously released, providing local delivery directly to the tumor region while reducing systemic side effects. Results: We have shown that the greater safety of SQ3370 allows significantly higher doses to be administered compared to conventional Dox. PK studies in rodents and dogs show highly efficient capture and activation of the prodrug, likely due to the rapid reaction of SQP33 with SQL70. Without SQL70, SQP33 shows minimal spontaneous conversion to active Dox. A single SQL70 injection selectively activated multiple doses of SQP33 at the target site, maximizing the local therapeutic index. Additionally, SQP33 was capable of being activated by SQL70 injected in various regions in the body, including peri/intratumoral, intraperitoneal, subcutaneous and intramuscular sites. SQ3370 treatment also enhances efficacy in tumor-bearing mice: In a syngeneic MC38 colorectal cancer model, SQ3370 significantly improved anti-tumor response and overall survival compared to conventional Dox. In mice bearing two tumors, SQL70 injection at one tumor site followed by systemic SQP33 treatment induced an anti-tumor response in both the injected and non-injected tumors. This technology also enabled a sustained anti-tumor response upon rechallenge with MC38 tumor cells, without additional treatment. Furthermore, SQ3370 increased CD4 and CD8 TILs and decreased regulatory T-cells in both injected and non-injected tumors, suggesting that SQ3370 can induce an immune-mediated anti-tumor effect, thus expanding potential treatment options in the clinic and highlighting the advantages of immune-sparing cytotoxic therapy. Conclusions: SQ3370 enables delivery of cytotoxic drugs to multiple target sites while limiting off-target exposure, leading to improved safety and efficacy. A multicenter, first-in-human, Phase 1 dose-escalation clinical trial (NCT04106492) in patients with advanced solid tumor malignancies is planned to commence enrollment in April, 2020.