Development of a p-Hydroxybenzyl-Alcohol-Linked Glutamate Prodrug for Activation by Pseudomonas Carboxypeptidase G2.

Directed enzyme-prodrug therapies used for targeted drug delivery require prodrugs that are chemically stable and processed efficiently by the activating enzyme. We recently reported the development of AMS-6-Glu (2), a glutamate-masked version of the cytotoxic natural product 5'-O-sulfamoyladenosine (AMS, 1) that can be activated by Pseudomonas carboxypeptidase G2 (CPG2). Herein, we report the development of a second-generation prodrug, AMS-5'-PHOBA-Glu (5), that undergoes cleavage by CPG2 with >160-fold higher efficiency. Use of a p-hydroxybenzyl alcohol (PHOBA) self-immolative linker overcame unexpected chemical instability observed with a conventional p-aminobenzyl alchohol (PABA) linker.

Host Interactions with Engineered T-cell Micropharmacies.

Genetically engineered, cytotoxic, adoptively transferred T cells localize to antigen-positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with a killing mechanism orthogonal to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Herein, we expanded the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with T-cell receptor (TCR)-engineered T cells. We demonstrate that SEAKER cells localized specifically to tumors, and activated bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells were efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.

Host-cell Interactions of Engineered T cell Micropharmacies.

Genetically engineered, cytotoxic, adoptive T cells localize to antigen positive cancer cells inside patients, but tumor heterogeneity and multiple immune escape mechanisms have prevented the eradication of most solid tumor types. More effective, multifunctional engineered T cells are in development to overcome the barriers to the treatment of solid tumors, but the interactions of these highly modified cells with the host are poorly understood. We previously engineered prodrug-activating enzymatic functions into chimeric antigen receptor (CAR) T cells, endowing them with an orthogonal killing mechanism to conventional T-cell cytotoxicity. These drug-delivering cells, termed Synthetic Enzyme-Armed KillER (SEAKER) cells, demonstrated efficacy in mouse lymphoma xenograft models. However, the interactions of an immunocompromised xenograft with such complex engineered T cells are distinct from those in an immunocompetent host, precluding an understanding of how these physiologic processes may affect the therapy. Here, we also expand the repertoire of SEAKER cells to target solid-tumor melanomas in syngeneic mouse models using specific targeting with TCR-engineered T cells. We demonstrate that SEAKER cells localize specifically to tumors, and activate bioactive prodrugs, despite host immune responses. We additionally show that TCR-engineered SEAKER cells are efficacious in immunocompetent hosts, demonstrating that the SEAKER platform is applicable to many adoptive cell therapies.

Chemoenzymatic Synthesis of Novel Cytotoxic Epoxyketones Using the Eponemycin Biosynthetic Enzyme EpnF.

Eponemycin is an α,ß-epoxyketone natural product that inhibits the proteasome via covalent interaction of the epoxyketone warhead with catalytic N-terminal threonine residues. The epoxyketone warhead is biosynthesized from a ß-ketoacid substrate by EpnF, a recently identified flavin-dependent acyl-CoA dehydrogenase-like enyzme. Herein, we report biochemical characterization of EpnF kinetics and substrate scope using a series of synthetic ß-ketoacid substrates. These studies indicate that epoxide formation likely occurs prior to other tailoring reactions in the biosynthetic pathway, and have led to the identification of novel epoxyketone analogues with potent anticancer activity.

Engineering CAR-T cells to activate small-molecule drugs in situ.

Chimeric antigen receptor (CAR)-T cells represent a major breakthrough in cancer therapy, wherein a patient's own T cells are engineered to recognize a tumor antigen, resulting in activation of a local cytotoxic immune response. However, CAR-T cell therapies are currently limited to the treatment of B cell cancers and their effectiveness is hindered by resistance from antigen-negative tumor cells, immunosuppression in the tumor microenvironment, eventual exhaustion of T cell immunologic functions and frequent severe toxicities. To overcome these problems, we have developed a novel class of CAR-T cells engineered to express an enzyme that activates a systemically administered small-molecule prodrug in situ at a tumor site. We show that these synthetic enzyme-armed killer (SEAKER) cells exhibit enhanced anticancer activity with small-molecule prodrugs, both in vitro and in vivo in mouse tumor models. This modular platform enables combined targeting of cellular and small-molecule therapies to treat cancers and potentially a variety of other diseases.

Circulating tumour DNA in patients with advanced melanoma treated with dabrafenib or dabrafenib plus trametinib: a clinical validation study.

BACKGROUND: Melanoma lacks validated blood-based biomarkers for monitoring and predicting treatment efficacy. Cell-free circulating tumour DNA (ctDNA) is a promising biomarker; however, various detection methods have been used, and, to date, no large studies have examined the association between serial changes in ctDNA and survival after BRAF, MEK, or BRAF plus MEK inhibitor therapy. We aimed to evaluate whether baseline ctDNA concentrations and kinetics could predict survival outcomes. METHODS: In this clinical validation study, we used analytically validated droplet digital PCR assays to measure BRAFV600-mutant ctDNA in pretreatment and on-treatment plasma samples from patients aged 18 years or older enrolled in two clinical trials. COMBI-d (NCT01584648) was a double-blind, randomised phase 3 study of dabrafenib plus trametinib versus dabrafenib plus placebo in previously untreated patients with BRAFV600 mutation-positive unresectable or metastatic melanoma. Patients had an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1. COMBI-MB (NCT02039947) was an open-label, non-randomised, phase 2 study evaluating dabrafenib plus trametinib in patients with BRAFV600 mutation-positive metastatic melanoma and brain metastases. Patients in cohort A of COMBI-MB had asymptomatic brain metastases, no previous local brain-directed therapy, and an ECOG performance status of 0 or 1. Biomarker analysis was a prespecified exploratory endpoint in both trials and performed in the intention-to-treat populations in COMBI-d and COMBI-MB. We investigated the association between mutant copy number (baseline or week 4 or zero conversion status) and efficacy endpoints (progression-free survival, overall survival, and best overall response). We used Cox models, Kaplan-Meier plots, and log-rank tests to explore the association of pretreatment ctDNA concentrations with progression-free survival and overall survival. The effect of additional prognostic variables such as lactate dehydrogenase was also investigated in addition to the mutant copy number. FINDINGS: In COMBI-d, pretreatment plasma samples were available from 345 (82%) of 423 patients and on-treatment (week 4) plasma samples were available from 224 (53%) of 423 patients. In cohort A of COMBI-MB, pretreatment and on-treatment samples were available from 38 (50%) of 76 patients with intracranial and extracranial metastatic melanoma. ctDNA was detected in pretreatment samples from 320 (93%) of 345 patients (COMBI-d) and 34 (89%) of 38 patients (COMBI-MB). When assessed as a continuous variable, elevated baseline BRAFV600 mutation-positive ctDNA concentration was associated with worse overall survival outcome (hazard ratio [HR] 1·13 [95% CI 1·09-1·18], p<0·0001 by univariate analysis), independent of treatment group and baseline lactate dehydrogenase concentrations (1·08 [1·03-1·13], p=0·0020), in COMBI-d. A ctDNA cutoff point of 64 copies per mL of plasma stratified patients enrolled in COMBI-d as high risk or low risk with respect to survival outcomes (HR 1·74 [95% CI 1·37-2·21], p<0·0001 for progression-free survival; 2·23 [1·73-2·87], p<0·0001 for overall survival) and was validated in the COMBI-MB cohort (3·20 [1·39-7·34], p=0·0047 for progression-free survival; 2·94 [1·18-7·32], p=0·016 for overall survival). In COMBI-d, undetectable ctDNA at week 4 was significantly associated with extended progression-free and overall survival, particularly in patients with elevated lactate dehydrogenase concentrations (HR 1·99 [95% CI 1·08-3·64], p=0·027 for progression-free survival; 2·38 [1·24-4·54], p=0·0089 for overall survival). INTERPRETATION: Pretreatment and on-treatment BRAFV600-mutant ctDNA measurements could serve as independent, predictive biomarkers of clinical outcome with targeted therapy. FUNDING: Novartis.

TERT, BRAF, and NRAS Mutational Heterogeneity between Paired Primary and Metastatic Melanoma Tumors.

Mutational heterogeneity can contribute to therapeutic resistance in solid cancers. In melanoma, the frequencies of intertumoral and intratumoral heterogeneity are controversial. We examined mutational heterogeneity within individual patients with melanoma using multiplatform analysis of commonly mutated driver and nonpassenger genes. We analyzed paired primary and metastatic tumors from 60 patients and multiple metastatic tumors from 39 patients whose primary tumors were unavailable (n = 271 tumors). We used a combination of multiplex SNaPshot assays, Sanger sequencing, mutation-specific PCR, or droplet digital PCR to determine the presence of BRAFV600, NRASQ61, TERT-124C>T, and TERT-146C>T mutations. Mutations were detected in BRAF (39%), NRAS (21%), and/or TERT (78%). Thirteen patients had TERTmutant discordant tumors; seven of these had a single tumor with both TERT-124C>T and TERT-146C>T mutations present at different allele frequencies. Two patients had both BRAF and NRAS mutations; one had different tumors and the other had a single tumor with both mutations. One patient with a BRAFmutant primary lacked mutant BRAF in at least one of their metastases. Overall, we identified mutational heterogeneity in 18 of 99 patients (18%). These results suggest that some primary melanomas may be composed of subclones with differing mutational profiles. Such heterogeneity may be relevant to treatment responses and survival outcomes.

Validation of Circulating Tumor DNA Assays for Detection of Metastatic Melanoma.

The detection of cell-free, circulating tumor DNA (ctDNA) in the blood of patients with solid tumors is often referred to as "liquid biopsy." ctDNA is particularly attractive as a candidate biomarker in the blood. It is relatively stable after blood collection, can be easily purified, and can be quantitatively measured with high sensitivity and specificity using advanced technologies. Current liquid biopsy research has focused on detecting and quantifying ctDNA to (1) diagnose and characterize mutations in a patient's cancer to help select the appropriate treatment; (2) predict clinical outcomes associated with different treatments; and (3) monitor the response and/or progression of a patient's disease. The diagnostic use of liquid biopsies is probably greatest in tumors where the difficulty and/or risk of obtaining a tissue specimen for molecular diagnostics is high (e.g., lung, colon). In metastatic melanoma, however, obtaining a tissue sample for molecular diagnostics is not typically a major obstacle to patient care plans; rather predicting treatment outcomes and monitoring a patient's disease course during therapy are considered the current priorities for this cancer type. In this chapter we describe an approach to the validation of ctDNA detection assays for melanoma, focusing primarily on analytical validation, and provide methods to guide the use of droplet digital PCR assays for measuring ctDNA levels in plasma samples.

Development of Novel Mutation-Specific Droplet Digital PCR Assays Detecting TERT Promoter Mutations in Tumor and Plasma Samples.

Detecting mutations in the plasma of patients with solid tumors is becoming a valuable method of diagnosing and monitoring cancer. The TERT promoter is mutated at high frequencies in multiple cancer types, most commonly at positions -124 and -146 (designated C228T and C250T, respectively). Detection of these mutations has been challenging because of the high GC content of this region (approximately 80%). We describe development of novel probe-based droplet digital PCR assays that specifically detect and quantify these two mutations, along with the less common 242-243 CC>TT mutation, and demonstrate their application using human tumor and plasma samples from melanoma patients. Assay designs and running conditions were optimized using cancer cell line genomic DNAs with the C228T or C250T mutations. The limits of detection were 0.062% and 0.051% mutant allele fraction for the C228T and C250T assays, respectively. Concordance of 100% was observed between droplet digital PCR and sequencing-based orthogonal methods in the detection of TERT mutant DNA in 32 formalin-fixed, paraffin-embedded melanoma tumors. TERTmutant DNA was also identified in 21 of 27 plasma samples (78%) from patients with TERTmutant tumors, with plasma mutant allele fractions ranging from 0.06% to 15.3%. There were no false positives in plasma. These data demonstrate the potential of these assays to specifically detect and quantify TERTmutant DNA in tumors and plasma of cancer patients.