Doxorubicin Prodrug Research Articles

Cathepsin B-Cleavable Cyclopeptidic Chemotherapeutic Prodrugs.

Cyclopeptidic chemotherapeutic prodrugs (cPCPs) are macromolecular protease-sensitive doxorubicin (DOX) prodrugs synthesized from a cyclodecapeptidic scaffold, termed Regioselectively Addressable Functionalized Template (RAFT). In order to increase the chemotherapeutic potential of DOX and limit its toxicity, we used a Cathepsin B (Cat B)-sensitive prodrug concept for its targeted release since this enzyme is frequently overexpressed in cancer cells. Copper-free “click” chemistry was used to synthesize cPCPs containing up to four DOX moieties tethered to the upper face of the scaffold through a Cat B-cleavable peptidic linker (GAGRRAAG). On the lower part, PEG 5, 10 and 20 kDa and a fifth peptidyl DOX moiety were grafted in order to improve the solubility, bioavailability and pharmacokinetic profiles of the compound. In vitro results on HT1080 human fibrosarcoma cells showed that cPCPs display a delayed action that consists of a cell cycle arrest in the G2 phase comparable to DOX alone, and increased cell membrane permeability.

Epidermal growth factor (EGF)-based activatable probe for predicting therapeutic outcome of an EGF-based doxorubicin prodrug

One of the most promising approaches for the treatment of colorectal cancer is targeting epidermal growth factor receptor (EGFR). Comprehensive research has led to significant clinical outcomes using EGFR-targeted anticancer drugs; however, the response to these drugs still largely varies among individuals. The current diagnostic platform provides limited information that does not enable successful prediction of the anticancer performance of EGFR-targeted drugs. Here, we developed a EGFR-targeted activatable probe for predicting therapeutic efficacy of EGFR-targeted doxorubicin prodrug in colorectal cancer therapy. The EGF-conjugated fluorescence-activatable probe (EGF-probe) and EGF-conjugated doxorubicin prodrug (EGF-prodrug) were both fabricated using peptide substrates that can be dissociated by lysosomal enzymes, and thus share an intracellular mechanism of action. We demonstrated that after EGFR-mediated endocytosis, lysosomal enzymes de-quench the fluorescence of EGF-probe and activate the cytotoxicity of EGF-prodrug. When evaluated in vivo, EGF-probe yielded an outstanding cancer-specific imaging ability with reduced background signals. EGF-prodrug also successfully targeted the tumor and promoted cancer cell death. We tested different colorectal cancer cell types to investigate the correlation between the fluorescence recovery efficiency of EGF-probe and the cytotoxicity of EGF-prodrug. Strong correlations were observed both in vitro and in vivo. The actions of EGF-probe and EGF-prodrug were dependent on the inherent lysosomal activity of the cell type rather than its EGFR expression level. Our proposed approach using EGF-probe and EGF-prodrug may overcome the major drawback of the conventional theranostic platform and provide great opportunity for successful personalized cancer therapy.

Abstract 6263: Hypoxia-targeting prodrug approach for multimodal chemotherapy and immunogenic cell death in aggressive triple negative breast cancer

Abstract Immune checkpoint blockade molecules, including antibodies against PD-1 and PD-L1 have gained much clinical recognition in the past few years. These strategies include harnessing the host's immune system to invade cancer cells by inhibiting the interaction between PD-1 and its ligand, PD-L1, thereby facilitating the activation of CD8+ T cells to mount an attack on cancer cells. Triple Negative Breast Cancer (TNBC) accounts for 10-20% of the total breast carcinoma cases. There is no FDA-approved targeted therapy for TNBC due to lack of the major biomarkers: estrogen, progesterone and HER-2. TNBC is difficult to treat, with a high rate of metastasis to nearby organs. We have identified the role of a hypoxia biomarker, carbonic anhydrase IX (CAIX) in proliferation and metastasis of TNBC. Based on this observation, we developed a CAIX-targeted Doxorubicin (Dox) prodrug nanoparticle, abbreviated as CAIX-Dox-NP. CAIX-Dox-NP can deliver the chemotherapeutic, Dox payload selectively in hypoxic tumor microenvironment, thus reducing Dox-associated cardiotoxicity. Most of the advanced stages TNBC solid tumors present themselves with oxygen-deficient, vascularized and matrix-rich core which is impermeable to most of the chemotherapy drugs. CAIX-Dox-NP has the ability to penetrate deep into the TNBC and kill epithelial tumor cells as well as tumor-growth promoting T-cells. CAIX-Dox-NP showed greater penetration in the core of hypoxic TNBC spheroid. Recent studies showed that Immunogenic Cell Death (ICD) has vital role in resurrecting the CD8+ T-cells mediated tumor killing. Combination of anti-PD-1 with inducers of ICD like Dox accentuate the ability of CD8+ T cells in killing of cancer cells. Our approach is to utilize CAIX-Dox for achieving CAIX-mediated targeted delivery and multimodal chemotherapy in addition to induction of ICD. Preliminary studies with CAIX-Dox has profound effect of killing TNBC cells, 4T1, through induction of early phase apoptosis. The Western Blot analysis in 4T1 treated with CAIX-Dox has proved the significant upregulation of ICD biomarkers, HMGB1 and calreticulin, by greater than 3-fold compared to control. To improve the bioavailability of CAIX-Dox, we developed CAIX-Dox-NP that can hitchhike on the circulating plasma albumin. The nanoparticles travel to the tumor and deliver the CAIX-Dox at the hypoxic core of TNBC, resulting in induction of apoptosis and ICD. Inspired from this outcome, we are developing a model for coculture of cancer and T-cells for evaluating if CAIX-Dox treatment can reeducate T-cells in killing cancer cells through ICD pathway. Citation Format: Ketki Bhise, Samaresh Sau, Mohd Ahmar Rauf, Arun K. Rishi, Arun K. Iyer. Hypoxia-targeting prodrug approach for multimodal chemotherapy and immunogenic cell death in aggressive triple negative breast cancer [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 6263.

Abstract 6245: SQ3370 enhances the safety and therapeutic index of cytotoxic agents via local activation therapy with improved local and systemic immune response

Abstract 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.

Tetrazine Carbon Nanotubes for Pretargeted In Vivo "Click-to-Release" Bioorthogonal Tumour Imaging.

The bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.

Tetrazine Carbon Nanotubes for Pretargeted In Vivo “Click‐to‐Release” Bioorthogonal Tumour Imaging

AbstractThe bioorthogonal inverse‐electron‐demand Diels–Alder (IEDDA) cleavage reaction between tetrazine and trans‐cyclooctene (TCO) is a powerful way to control the release of bioactive agents and imaging probes. In this study, a pretargeted activation strategy using single‐walled carbon nanotubes (SWCNTs) that bear tetrazines (TZ@SWCNTs) and a TCO‐caged molecule was used to deliver active effector molecules. To optimize a turn‐on signal by using in vivo fluorescence imaging, we developed a new fluorogenic near‐infrared probe that can be activated by bioorthogonal chemistry and image tumours in mice by caging hemicyanine with TCO (tHCA). With our pretargeting strategy, we have shown selective doxorubicin prodrug activation and instantaneous fluorescence imaging in living cells. By combining a tHCA probe and a pretargeted bioorthogonal approach, real‐time, non‐invasive tumour visualization with a high target‐to‐background ratio was achieved in a xenograft mice tumour model. The combined advantages of enhanced stability, kinetics and biocompatibility, and the superior pharmacokinetics of tetrazine‐functionalised SWCNTs could allow application of targeted bioorthogonal decaging approaches with minimal off‐site activation of fluorophore/drug.

Enhanced Anti-Brain Metastasis from Non-Small Cell Lung Cancer of Osimertinib and Doxorubicin Co-Delivery Targeted Nanocarrier.

PurposeCurrently, the treatment of brain metastases from non-small cell lung cancer (NSCLC) is rather difficult in the clinic. A combination of small molecule-targeted drug and chemo-drug is a promising therapeutic strategy for the treatment of NSCLC brain metastases. But the efficacy of this combination therapy is not satisfactory due to the blood–brain barrier (BBB). Therefore, it is urgent to develop a drug delivery system to enhance the synergistic therapeutic effects of small molecule–targeted drug and chemo-drug for the treatment of NSCLC brain metastases.MethodsT7 peptide installed and osimertinib (AZD9291) loaded intracellular glutathione (GSH) responsive doxorubicin prodrug self-assembly nanocarriers (T7-DSNPs/9291) have been developed as a targeted co-delivery system to enhance the combined therapeutic effect on brain metastases from NSCLC. In vitro cell experiments, including intracellular uptake assay, in vitro BBB transportation, and MTT assay were used to demonstrate the efficacy of T7-DSNPs/9291 in NSCLC brain metastasis in vitro. Real-time fluorescence imaging analysis, magnetic resonance imaging analysis, and Kaplan–Meier survival curves were used to study the effect of T7-DSNPs/9291 on an animal model in vivo.ResultsT7-DSNPs/9291 could significantly enhance BBB penetration of AZD9291 and doxorubicin via transferrin receptor-mediated transcytosis. Moreover, T7-DSNPs/9291 showed significant anti-NSCLC brain metastasis effect and prolonged median survival of an intracranial NSCLC brain metastasis animal model.ConclusionT7-DSNPs/9291 is a potential drug delivery system for the combined therapy of brain metastasis from NSCLC.

Redox-responsive dasatinib-containing hyaluronic acid prodrug and co-delivery of doxorubicin for cancer therapy

Redox-responsive prodrugs were synthesized by conjugating dasatinib (Das)/cholesterol (Chol) to hyaluronic acid (HA) via the cystamine dihydrochloride (Cys), and hexamethylene diamine (Hda) linkers. In a redox environment (10 mM dithiothreitol, DTT), a substantial amount of the grafted Das was released and faster than that observed under physiological conditions. The cytotoxicity of redox-sensitive HA-Cys-P(SSDas/CCChol) was greater than that of redox-insensitive HA-Hda-PCCDas. Flow cytometry showed that the uptake of HA-targeted DOX–encapsulated micelles was faster than that of free DOX.

Mineralization of pH-Sensitive Doxorubicin Prodrug in ZIF-8 to Enable Targeted Delivery to Solid Tumors.

The zeolitic imidazolate framework (ZIF-8), composed of zinc ion and dimethylimidazole, is widely used in drug delivery because of the easy fabrication process and the good biosafety. However, ZIF-8 suffers from low affinity to nonelectric-rich drugs and does not have surface functional groups. Here, to deliver doxorubicin (DOX) with ZIF-8 to specific target sites, DOX was first modified with a pH-sensitive linker containing two carboxyl groups to form the inactive prodrug CAD and subsequently seeded inside ZIF-8 by a 5 min mineralization process. CAD has high affinity to ZIF-8 because of the carboxyl groups and can anchor to the ZIF-8 surface to enable the surface modification with folic acid for tumor targeting. Moreover, the DOX release is precisely controlled by three steps of acidic pH response, with the dissociation of the FA layer, the breakdown of the ZIF-8 structure, and the cleavage of the pH-sensitive linker in prodrug. This novel “prodrug-ZIF-8” strategy has opened a new horizon in drug delivery.

Carrier-free nanoassembly of doxorubicin prodrug and siRNA for combinationally inducing immunogenic cell death and reversing immunosuppression

Abstract Doxorubicin (DOX) can elicit antitumor immunity responses by inducing immunogenic cell death (ICD) but also triggers upregulated expression of various immunosuppressive genes to counteract the ICD effect. To resolve this conflict, a carrier-free nanoassembly of acid-activatable DOX prodrug and small interfering RNA (siRNA) was developed to combinationally induce ICD and reverse immunosuppression. The carrier-free nanoassembly with rather high drug contents (4.13 % for siRNA and 21.67 % for DOX) was formed via cooperative π–π stacking and electrostatic interactions. The formed nanoassembly, termed as PEG@D:siRNA, possessed a well-defined nanostructure: a core consisting of DOX plus siRNA and a shell consisting of polyethylene glycol (PEG). It has been demonstrated that this carrier-free nanoassembly carrying siRNA targeting PD-L1 can significantly increase tumor-infiltrating T lymphocytes, improve interferon-γ (IFN-γ) expression, and ultimately strengthen the ICD effect of the DOX prodrug, resulting in a significantly enhanced anticancer immune response and superior tumor growth inhibition. In addition, carrier-free nanoassembly PEG@D:siRNA can also be conveniently extended as a general strategy to combine chemotherapy and immunotherapy, providing a facile avenue for improving cancer chemoimmunotherapy.

Prodrug-Based Nanoreactors with Tumor-Specific In Situ Activation for Multisynergistic Cancer Therapy

Efficient drug delivery into tumor cells while bypassing many biological barriers is still a challenge for cancer therapy. By taking advantage of the palladium (Pd)-mediated in situ activation of a prodrug and the glucose oxidase (GOD)-based β-d-glucose oxidation reaction, we developed a multisynergistic cancer therapeutic platform that combined doxorubicin (DOX)-induced chemotherapy with GOD-mediated cancer-orchestrated oxidation therapy and cancer starvation therapy. In the present work, we first synthesized DOX prodrugs (pDOXs) and temporarily assembled them with β-cyclodextrins to reduce their toxic side effects. Then, a nanoreactor was constructed by synthesizing Pd0 nanoparticles in situ within the pores of mesoporous silica nanoparticles for the conversion of pDOX into the active anticancer drug. Furthermore, GOD was introduced to decrease the pH of the tumor microenvironment and induce cancer-orchestrated oxidation/starvation therapy by catalyzing β-d-glucose oxidation to form hydrogen peroxide (H2O2) and gluconic acid. Our study provides a new strategy that employs a cascade chemical reaction to achieve combined orchestrated oxidation/starvation/chemotherapy for the synergistic killing of cancer cells and the suppression of tumor growth.

Ultrasound Combined with Core Cross-Linked Nanosystem for Enhancing Penetration of Doxorubicin Prodrug/Beta-Lapachone into Tumors.

BackgroundNanosized drug delivery systems (NDDSs) have shown excellent prospects in tumor therapy. However, insufficient penetration of NDDSs has significantly impeded their development due to physiological instability and low passive penetration efficiency.MethodsHerein, we prepared a core cross-linked pullulan-modified nanosized system, fabricated by visible-light-induced diselenide bond cross-linked method for transporting β-Lapachone and doxorubicin prodrug (boronate-DOX, BDOX), to improve the physiological stability of the NDDSs for efficient passive accumulation in tumor blood vessels (β-Lapachone/BDOX-CCS). Additionally, ultrasound (US) was utilized to transfer β-Lapachone/BDOX-CCS around the tumor vessel in a relay style to penetrate the tumor interstitium. Subsequently, β-Lapachone enhanced ROS levels by overexpressing NQO1, resulting in the transformation of BDOX into DOX. DOX, together with abundant levels of ROS, achieved synergistic tumor therapy.ResultsIn vivo experiments demonstrated that ultrasound (US) + cross-linked nanosized drug delivery systems (β-Lapachone/BDOX-CCS) group showed ten times higher DOX accumulation in the tumor interstitium than the non-cross-linked (β-Lapachone/BDOX-NCS) group.ConclusionThus, this strategy could be a promising method to achieve deep penetration of NDDSs into the tumor.

Chemo-immunotherapy with doxorubicin prodrug and erythrocyte membrane-enveloped polymer nano-vaccine enhances antitumor activity

There are plenty of evidences to show that combining of chemotherapy and immunotherapy should be a very potent anti-cancer therapeutic method. In this research, we evaluated the anti-tumor activity of doxorubicin prodrug combined with erythrocyte membrane-enveloped polymer nano-vaccine against tumor in vitro and in vivo. We also analyzed the immune cell populations to investigate the effect in the tumor microenvironment and explored the inhibitory of lung metastasis. Our study showed that chemo-immunotherapy could inhibit the growth of melanoma tumor in mice. This combination approach promoted a stronger immune response by up-regulating the expression of dendritic cells and cytotoxic T cells in lymph nodes and increased the secretion of cytokines. It also restricted the immunosuppressive environment through inhibiting expression of regulatory T cells. It suggested that combination of prodrug and nano-vaccine achieved better anti-tumor effect than monotherapy, which should be widely valued and further studied to establish in a more economical and efficient way, expected to apply in the future clinical practice of cancer treatment.

Binary Prodrug of Dichloroacetic Acid and Doxorubicin with Enhanced Anticancer Activity.

The inevitable challenge in conventional chemotherapy is to deliver the anticancer drugs to the dense population of tumors cells while minimizing the drug-associated side effects on the normal cells. Cancer cells' preference for glycolysis for energy production is well recognized. Intuitively, taking advantage of such cancer-associated metabolism would be a promising strategy for anticancer drug delivery with minimal side effects. In this investigation, we have designed a binary prodrug PDOX as a sequential drug delivery regimens to realize the combination therapy for cancer. As cancer cells exhibit abrupt metabolism with elevated pyruvate dehydrogenase kinase (PDK) activity, dichloroacetic acid (DCA, a well-known PDK inhibitor) was used in combination with anticancer drug doxorubicin (DOX). The designed molecular prodrug was activated selectively by cancer-associated esterase to deliver DCA and DOX, respectively, and induced synergetic effects. Hence, sequential targeted delivery of molecular prodrug PDOX offers a promising approach to overcome the offside drug toxicity, pharmacokinetics, and biodistribution of individuals and provide an alternative option for cancer treatment.

Redox-Sensitive Hyaluronic Acid Polymer Prodrug Nanoparticles for Enhancing Intracellular Drug Self-Delivery and Targeted Cancer Therapy.

Currently, available nanoscale anticancer drug delivery systems have low targeting and release efficiency, limiting their therapeutic effects. Thus, tumor-targeting nanocarriers for self-assembly of amphiphilic polymer-drug conjugates are urgently needed to improve drug targeting and treatment efficacy. Here, we report the construction of a stable, reduction-sensitive prodrug conjugate based on hyaluronic acid-grafted pH-sensitive doxorubicin (DOX). The amphiphilic prodrug copolymer self-assembled into spherical nanoparticles in aqueous solution and exhibited an average diameter of 150 nm. Prodrug micelles were stable in a normal physiological environment and achieve selective and rapid release under acidic pH and/or high reduction conditions. Cell Counting Kit-8, flow cytometry, and live cell imaging assays showed that the prodrug had high targeting and antitumor activity against CD44 receptors. Moreover, in vivo pharmacokinetics and biodistribution studies showed that the prodrug had a longer circulation time in BALB/c mice and higher accumulation in 4T1 tumors. Interestingly, the prodrug could effectively treat tumors with few side effects. These results showed that the DOX prodrug micelles developed in this study may have great potential in targeted therapy.

Nanoparticle delivery of a pH-sensitive prodrug of doxorubicin and a mitochondrial targeting VES-H8R8 synergistically kill multi-drug resistant breast cancer cells

Multi-drug resistance (MDR) remains a major obstacle in cancer treatment while being heavily dependent on mitochondrial activity and drug efflux. We previously demonstrated that cationic lipids, such as the vitamin E succinate modified octahistidine-octaarginine (VES-H8R8) conjugate, target mitochondria, resulting in depolarized mitochondria and inhibited drug efflux in MDR breast cancer cells. We hypothesized that the effective cell uptake, efflux inhibition, and mitochondrial depolarization properties of VES-H8R8 would synergistically enhance the toxicity of a pH-sensitive prodrug of doxorubicin (pDox) when co-encapsulated in nanoparticles (NPs). pDox was successfully synthesized and validated for pH-sensitive release from NPs under lysosome-mimicking, acidic conditions. The synergistic effect of VES-H8R8 and pDox was confirmed against MDR breast cancer cells in vitro. Importantly, synergism was only observed when VES-H8R8 and pDox were co-encapsulated in a single nanoparticulate system. The synergistic mechanism was investigated, confirming superior pDox uptake and retention, Pgp efflux inhibition, mitochondrial depolarization, and enhanced induction of ROS, and apoptosis. This work demonstrates the translational potential of doubly-loaded NPs co-encapsulating pDox with VES-H8R8 to synergistically kill MDR breast cancer cells.

Effect of local activation via SQ3370 on the safety of chemotherapy with concomitant anti-tumor immune response.

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.

Switchable Fluorescence of Doxorubicin for Label-Free Imaging of Bioorthogonal Drug Release.

Monitoring the release and activation of prodrug formulations provides essential information about the outcome of a therapy. While the prodrug delivery can be confirmed by using different imaging techniques, confirming the release of active payload by using imaging is a challenge. Here, we have discovered that the switchable fluorescence of doxorubicin can validate drug release upon its uncaging reaction with a highly specific chemical partner. We have observed that the conjugation of doxorubicin with a trans-cyclooctene (TCO) diminishes its fluorescence at 595 nm. This quenched fluorescence of the doxorubicin prodrug is recovered upon its bond-cleaving reaction with tetrazine. Clinically assessed iron oxide nanoparticles were used to formulate a doxorubicin nanodrug. The release of doxorubicin from the nanodrug was studied under various experimental conditions. A fivefold increase in doxorubicin fluorescence is observed after complete release. The studies were carried out in vitro in MDA-MB-231 breast cancer cells. An increase in Dox signal was observed upon tetrazine administration. This switchable fluorescence mechanism of Dox could be employed for fundamental studies, that is, the reactivity of various tetrazine and TCO linker types under different experimental conditions. In addition, the system could be instrumental for translational research where the release and activation of doxorubicin prodrug payloads can be monitored by using optical imaging systems.

Hypoxia Targeting Nanoparticles for Immunogenic Cell Death of Triple Negative Breast Cancer

Triple Negative Breast Cancer (TNBC) accounts for 10–20% mortality amongs all breast carcinoma cases. There is no targeted therapy approved for TNBC owing to absence of major biomarkers: estrogen, progesterone and Her‐2. Advanced stage TNBC tumors present themselves with a hypoxic, vascularized drug‐resistant core, which is rich in cancer stem cells and tumor‐associated growth factors. Earlier reports from our lab have identified the role of hypoxia biomarker, carbonic anhydrase IX (CAIX) in tumor proliferation and metastasis. Based on this, we have developed a CAIX‐targeted prodrug of Doxorubicin (CAIX‐Dox). Further, we have encapsulated the prodrug in long circulating, serum albumin binding lipid nanoparticles (abbreviated CAIX‐Dox‐NP) that can selectively deliver Dox to the hypoxic regions of the tumor, thus reducing Dox‐associated toxicity. CAIX‐Dox‐NP were synthesized using high pressure homogenization for achieving nanosized particles with a narrow polydispersity index. Nanoparticles with size ~200 nm and CAIX‐Dox loading of 13–19% were obtained, which were stable over 48 h at 10% serum and for over 3 months at refrigerated conditions. CAIX‐Dox‐NP were optimized based on lipid composition and ratio of aqueous to organic phases. CAIX‐Dox exhibited a sustained release pattern at physiological and mildly acidic conditions in vitro. Recent studies have shown that immunogenic cell death (ICD) plays an important role in resurrecting CD8+ T cells mediated tumor killing. ICD inducers like Dox, when presented as hypoxia targeting prodrug in the form of CAIX‐Dox, has the ability to penetrate deep into the tumor tissues, including hypoxic regions, killing epithelial cells as well as tumor promoting T cells. CAIX‐Dox has shown killing of syngeneic 4T1 TNBC cells via ICD pathway as demonstrated by Western blot analysis through upregulation of ICD markers like HMGB1, calreticulin and induction of early phase apoptosis on treatment over 72 h, by a factor greater than 3‐fold as compared to control. Thus far, CAIX‐Dox portend to be a new avenue for the treating TNBC, warranting further investigations.

Abstract B19: Robust antitumor effect of doxorubicin prodrug combined with anti-PD-1 in murine squamous cell cancer model

Abstract Background: Combination therapy of anti-PD-1 agents with other class of chemotherapeutic agents to improve treatment outcome is being attempted. Anthracycline-derived chemotherapeutic agents are known to activate immunogenic cell-death mechanism. Previously, we developed caspase-3 specific prodrug, MPD-1 containing doxorubicin. It is linked to peptide moiety cleavable by caspase-3 produced during apoptosis induced by radiation therapy (RT). We hypothesized that anti-PD-1 alone may activate MPD-1 by producing caspase-3 and reinforce antitumor efficacy of MPD-1 and anti-PD-1 effect. This study was designed to evaluate the antitumor effects of combined treatment of anti-PD-1 and MPD-1 in murine squamous cell carcinoma models. Methods: Murine SCC VII (squamous cell carcinoma) cells were inoculated to flank of mice, female C3H/J. We divided 6 treatment groups; control, RT, anti-PD-1, anti-PD-1 with MPD-1, RT with MPD-1, and triple therapy (anti-PD-1 + RT + MPD-1) (n = 5/each group). After tumor grew up to 100-200 mm3, treatment was initiated. Tumor size and mice weight were measured. Expression of caspase-3 was evaluated in cell lines and tumor tissues after each treatment. Distribution of CD8+, CD4+, and FoxP3+ cells was analyzed. Results: RT with MPD-1, anti-PD-1 with MPD-1, and triple treatment enhanced the synergistic antitumor activity compared to single treatment in SCC VII bearing mice. Anti-PD-1 with MPD-1 presented slightly better antitumor effect than RT with MPD-1. Caspase-3 was produced in tumor tissue of anti-PD-1 treatment group, which enhanced antitumor effect of activated MPD-1, while cell after anti-PD-1 did not enhance caspase-3 expression. Triple treatment suppressed tumor growth mostly, which was sustained more than 2 weeks even after cessation of treatment. Proportion of CD45+CD8+ T-cells in triple treatment group was increased mostly among tumor-infiltrating lymphocytes. Body weight of each treatment group was not significantly different. Conclusion: Systemic anti-PD-1 initiates apoptosis and produces caspase-3. This process activates MPD-1 and induces sequential mediated by tumor-infiltrating lymphocytes. Combined anti-PD-1 and MPD-1 treatment will be effective for the distant metastatic lesions. Citation Format: Yoon Se Lee, Minsu Kwon, In Kyung Son, Youngro Byun, Sang Yoon Kim. Robust antitumor effect of doxorubicin prodrug combined with anti-PD-1 in murine squamous cell cancer model [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 B19.