Early detection of cancer is an important driver of increased survival, quality of life and reduced healthcare costs. Although liquid biopsy provides a new option for early cancer detection, the limitations of detecting a few molecules of blood-based biomarkers naturally shed by the cancer remain. Earli is developing a highly sensitive, orthogonal approach that uses a genetic construct that usurps dysregulated pathways and actively forces cancer cells to drive the expression of a detectable ‘synthetic’ biomarker. We previously used murine xenograft models to validate the tumor detection properties of EARLI-001, a circular DNA containing a human derived cancer-activated promoter to drive the transient expression of secreted embryonic alkaline phosphatase (SEAP), a protein normally expressed only during fetal development. Biodistribution following intravenous (IV) administration of EARLI-001 shows a broad tissue tropism, enabling the potential to monitor multiple tissues for malignant cells. A single dose of EARLI-001 administered in a pulmonary metastatic cancer mouse model resulted in a 95-fold increase in serum SEAP compared to healthy controls. Recognizing the inherent limitations of murine models, we evaluated EARLI-001 in canines diagnosed with cancer. Dog cancers share many features with human malignancies including spontaneous tumor formation, heterogeneity, growth kinetics, histology, and comparable dysregulated genetic pathways. Furthermore, the use of dogs allows the study of EARLI-001 in an immune competent setting and with substantially increased size (up to 65 kg) comparable to scaling into human subjects. EARLI-001 was first tested for safety and biodistribution in 54 purpose-bred beagles in MTD and GLP toxicology studies which reveal that it is safe and well tolerated across a broad range of clinical doses. Biodistribution analysis in 12 tissues showed nanoplasmid levels peaking in the first days post dose and rapidly decreased to background levels by 60 days. These non-tumor bearing dogs showed no serum SEAP signal at all doses. Following IACUC approval and informed owner consent, EARLI-001 was administered to companion dogs in a standard 3 x 3 dose escalation study, with an expanded mid-range cohort. These companion dogs harbored a wide variety of spontaneous tumors in different tissues of origin. In tumor bearing dogs, EARLI-001 elicited a clear and discernable cancer-activated SEAP signal in 1 of 3 dogs dosed at 0.01 mg/kg, 3 of 6 dosed at 0.03 mg/kg, 2 of 2 at 0.07 mg/kg and 8 of 8 dogs dosed at 0.1 mg/kg. Furthermore, the MTD was not reached, and no significant dose-limiting toxicity was observed in these older, disease-burdened animals. These results show the utility of the platform to interrogate tumor biology and detect the presence of malignancies in relevant in vivo models and provided the critical safety and efficacy data towards a first-in-human clinical study in lung cancer initiated in 2021. Citation Format: Regina Nieu, Emily Phenix, Jennifer Hauss, Hadley Hanson, Nga Ho, Alex Harwig, Shireen Rudina, Badri Ananthanarayanan, Hong Chang, Bijee George, Maggie C. Louie, Julie Bulman-Fleming, Michael Kent, David Suhy. Detection of cancer in dogs using a novel genetic-based synthetic biomarker platform [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3382.

Patient derived xenograft (PDX) models of pancreatic cancer provide an excellent platform for understanding human disease, characterizing tumor-initiating cells, and testing of novel therapeutics. Herein, we compared the natural pancreatic microenvironment via orthotopic implantation and the traditional approach of subcutaneous implantation for effects on PDX tumor growth, tumor morphology, cell population diversity, tumorigenic potential, metastatic potential, and response to therapeutics. Two approaches were used: (1) primary human cancer tissue fragments were implanted in orthotopic and subcutaneous environments; and, (2) PDX previously established by subcutaneous engraftment were reimplanted into either an orthotopic or a subcutaneous environment. We report that orthotopically-engrafted primary PDX more closely reflect the gross tumor morphology of the original patient than subcutaneously-derived tumors. In models derived from subcutaneous engraftment, variation of tumor morphology, tumor growth, cell population diversity, and tumorigenic potential between tumors grown in orthotopic and subcutaneous environments were dependent on PDX line; and, stromal infiltration and mucin production were more prominent in the orthotopic background for some PDX models. In addition, we observe increased metastatic potential in orthotopic tumor-bearing mice compared with their subcutaneous counterparts. Tumor metastasis occurred in distal filtering organs, including the lung, liver, lymph nodes, and peritoneal cavity, among others. This result is consistent with the observation of increased circulating tumor cells in the blood of orthotopic tumor bearing mice. Comparison of therapeutic efficacy with gemcitabine chemotherapy and antibody-drug conjugate (ADC) therapy in orthotopic and subcutaneous tumors showed therapeutic response was dependent on PDX line. Interestingly, for PDX lines that exhibited morphological differences between orthotopic and subcutaneous backgrounds, treatment was less effective in the orthotopic microenvironment suggesting the microenvironment alters the ability of therapeutics to impede tumor growth. Reduced therapeutic response was also found to be consistent with reduced ADC uptake and vascularity in orthotopic tumors. Taken together, the orthotopic PDX model serves as a more accurate representation of human pancreatic cancer by displaying stromal-rich morphology, exhibiting metastatic cell behavior, and recapitulating therapeutic response challenges observed in patients. Citation Format: Sarah L. Fong, Marybeth A. Pysz, Kristen D. McKnight, Nicole Taylor, Regina Nieu, Juliet Markeson, Hanna Rammoth, Archana Dilip, Kayla Fasano, Emily Janke, Erica Anderson, Holger Karsunky, Scott Dylla. Comparison of orthotopic and subcutaneous implantation of pancreatic cancer patient-derived xenograft models for drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4937. doi:10.1158/1538-7445.AM2017-4937

The expression of synthetic biomarkers from genetic constructs represents a new paradigm of molecular diagnostic tools for early cancer detection and localization. One of the limitations of liquid biopsy approaches is the low abundance of endogenous biomarkers shed in early-stage disease. To overcome this, Earli’s platform instead uses genetic constructs to force tumors to produce cancer-activated synthetic biomarkers. Thus, technologies that deliver DNA to tumor cells in multiple tissues need to be developed. While non-viral delivery systems such as lipid nanoparticles (LNPs) have achieved clinical success for RNA, the delivery of DNA to tumor cells remains a challenge. We have engineered two classes of nanoparticles using biodegradable and ionizable cationic materials. The efficacy of these nanoparticles to deliver a DNA nanoplasmid containing a cancer-activated promoter to drive expression of a secreted embryonic alkaline phosphatase (SEAP) was evaluated in multiple cell-derived xenograft (CDX) models. The first class of nanoparticles was comprised of poly-beta-amino-ester polymers (PBAEs). Systematic screening of a library of PBAEs and optimization of PEG-lipid content produced nanoparticles approximately 80-100 nm in diameter with slightly positive zeta potential, enabling biodistribution to mouse lung tissues. Testing of initial nanoparticle compositions identified safe and effective formulations that produced 5-fold higher SEAP expression levels in lung tumor-bearing animals versus naïve mice. Further enhancements produced SEAP levels up to 250-fold higher in tumor-bearing versus control mice but resulted in significant increases in liver enzymes, suggesting tolerability issues that are currently being addressed by improving the polymer and formulation composition. The second class of nanoparticles was composed of ionizable lipids such as DLin-MC3-DMA that have been clinically validated for RNA delivery to the liver. Optimized compositions with helper lipid and PEG-lipid variants identified formulations that could successfully deliver DNA to the liver. These formulations produced 30-fold higher SEAP expression in tumor-bearing animals compared to naïve controls in an H1299 CDX model. A 7-fold difference in SEAP levels was demonstrated in an orthotopic Hep3B CDX model between tumor versus non-tumor bearing animals. Strikingly, LNP delivery of a cancer-activated Renilla luciferase construct produced tumor-specific bioluminescence, demonstrating the ability of the LNPs to localize liver tumors. LNP formulations were well tolerated, producing transient, mild to moderate elevations in liver enzymes one day after administration that subsequently resolved. These potent and safe nanoparticle delivery systems are promising agents for enabling future clinical translation of our synthetic biomarker platform. Citation Format: Badri Ananthanarayanan, Regina Nieu, Weihang Ji, Blaine McCarthy, Shengshuang Zhu, Alex Harwig, Maggie C. Louie, David Suhy. Nanoparticle delivery of cancer-activated DNA enables the detection and localization of tumors in mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1732.

Early detection and localization of primary cancers has been shown to improve clinical outcomes and overall survival. While liquid biopsy may indicate the presence of cancer and possibly provide the tissue of origin, it cannot pinpoint the exact location of cancer and its metastases. Furthermore, current imaging techniques lack the sensitivity to locate early-stage tumors, diminishing the ability for early clinical intervention. Current imaging approaches using 18F-FDG rely on increased metabolic rates of tumors vs. surrounding non-tumor tissue, often resulting in high background and inability to detect low-metabolic tumors. Earli is developing a highly sensitive, orthogonal approach that uses genetic constructs to usurp dysregulated cancer pathways to force the tumor to produce a synthetic biomarker used to detect and localize the malignancy. We previously demonstrated the utility of cancer-activated expression of blood-based biomarkers in preclinical cancer models, which are currently in an ongoing clinical study. EARLI-201 is novel genetic imaging probe comprised of a DNA construct containing a cancer-activated promoter to drive the expression of a PET Reporter Gene (PRG). The HSV-sr39TK PRG product specifically sequesters the 18F-FHBG tracer inside the cell, enabling detection by PET imaging. Initial studies for sensitivity combined H1299 lung cancer cells engineered with only two copies of the PRG were implanted subcutaneously into immunocompromised mice followed by the administration of the 18F-FHBG tracer. Results demonstrate a robust signal from as few as 62,000 injected cells. Given that a 1 mm3 tumor contains ~1,000,000 cells, suggests that we can localize tumors as small as 6.25mm3 with only a 1% transfection efficiency. EARLI-201 was tested in a variety of murine models including the metastatic “Lung Trap” and spontaneous HCC-MYC/Twist1, which produce discrete tumor nodules in the lungs or liver, respectively. A single intravenous injection of EARLI-201 given at Day 1 results in cancer-activated expression and accumulation of the PRG in tumors. 18F-FHBG PET imaging three days post dosing revealed distinct tumor foci that co-register with tumors detected by CT. Additional ex vivo techniques confirmed PRG expression only in tumor-containing tissues and not in healthy tissue. Imaging the same animals with 18F-FDG failed to produce any appreciable signal in the same tumors, likely because they are of low metabolic nature. These pre-clinical results clearly demonstrate the potential of Earli’s approach to detect and localize very small tumors currently outside the sensitivity of existing imaging modalities. Additional experiments to evaluate EARLI-201 in companion dogs diagnosed with cancer are currently in progress and provides the ability to validate the platform in larger body masses with spontaneously formed tumors. Citation Format: Alex Harwig, Morgan Wang, Ling Tong, Regina Nieu, Vy Nguyen, Dean Felsher, David Suhy. A more sensitive approach to cancer imaging using cancer-activated PET reporters [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2480.

Early detection of cancer is a powerful driver of increased survival rate, quality of life and reduced healthcare costs. ‘Liquid biopsies' that rely upon chemistry for the detection of blood-based endogenous cancer biomarkers such as proteins, circulating tumor DNA or RNA show only limited sensitivity for certain early stage cancers where the tumor mass shedding those biomarkers is smaller. Rather than searching for elusive natural biomarkers shed into the bloodstream, we are employing a promising alternate approach rooted in biology to usurp the highly dysregulated transcriptional pathways that give rise to malignancies to force cancer cells to produce easily detectable ‘synthetic' biomarkers that do not naturally occur. EARLI-001 is a DNA nanoplasmid comprised of a cancer-activated promoter to drive expression of Secreted Embryonic Alkaline Phosphatase (SEAP). The survivin promoter was initially selected for proof of concept because the corresponding anti-apoptotic gene product is widely overexpressed in many cancers, including melanoma, liver, breast, lung, colon and ovarian cancer, while not being expressed in normal adult tissues. The synthetic biomarker SEAP is a secreted variant of human placental alkaline phosphatase, which is only expressed during fetal development and thus has near-zero background in adult blood. EARLI-001 is formulated using a non-viral linear polyethyleneimine and administered intravenously to enable broad tissue distribution and transient transfection of multiple tissues. The sensitivity and specificity of EARLI-001 for tumor detection were established using a variety of cell-derived xenograft models. A single administration of EARLI-001 in immunocompromised mice bearing a modest burden of lung metastases drove cancer-activated SEAP expression that was at least 100-fold higher than the corresponding expression in non-tumor bearing animals. In an immune-competent syngeneic model, EARLI-001 produced 10-fold higher SEAP expression in mice bearing lung metastases of 4T1 mammary tumor cells than naïve mice. EARLI-001 could also discriminate mice bearing orthotopic Hep3B liver xenograft tumors from naïve control mice. Finally, the sensitivity of EARLI-001 in detecting small tumors was demonstrated in a longitudinal study, in which robust cancer-specific SEAP expression occurred even when the lung tumor burden was too low to result in significant changes in organ weight. The toxicology, pharmacokinetics, and biodistribution of EARLI-001 were evaluated in several GLP studies in mice as well as canines, enabling translation to a first-in-man clinical study. This Phase 1 dose escalation study will evaluate the safety, tolerability and PK/PD of EARLI-001 in subjects with locally advanced or metastatic lung cancer and enable further clinical development of the cancer-activated synthetic biomarker platform. Citation Format: Badriprasad Ananthanarayanan, Regina Nieu, Evan Bishop, Shireen Rudina, Alex Harwig, Bijee George, David Suhy. Preclinical development of EARLI-001, a genetic platform producing cancer-activated synthetic biomarkers for the early detection of malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2548.