Self-cleaving ribozymes: substrate specificity and synthetic biology applications.

Various self-cleaving ribozymes appearing in nature catalyze the sequence-specific intramolecular cleavage of RNA and can be engineered to catalyze cleavage of appropriate substrates in an intermolecular fashion, thus acting as true catalysts. The mechanisms of the small, self-cleaving ribozymes have been extensively studied and reviewed previously. Self-cleaving ribozymes can possess high catalytic activity and high substrate specificity; however, substrate specificity is also engineerable within the constraints of the ribozyme structure. While these ribozymes share a common fundamental catalytic mechanism, each ribozyme family has a unique overall architecture and active site organization, indicating that several distinct structures yield this chemical activity. The multitude of catalytic structures, combined with some flexibility in substrate specificity within each family, suggests that such catalytic RNAs, taken together, could access a wide variety of substrates. Here, we give an overview of 10 classes of self-cleaving ribozymes and capture what is understood about their substrate specificity and synthetic applications. Evolution of these ribozymes in an RNA world might be characterized by the emergence of a new ribozyme family followed by rapid adaptation or diversification for specific substrates.

Protection of nuclear DNA by lifespan-extending compounds in the yeast Saccharomyces cerevisiae.

DNA damage has been hypothesized to be a driving force of the aging process. At the same time, there exists multiple compounds that can extend lifespan in model organisms, such as yeast, worms, flies, and mice. One possible mechanism of action for these compounds is a protective effect against DNA damage. We investigated whether five of these lifespan-extending compounds, dinitrophenol, metformin, rapamycin, resveratrol, and spermidine, could protect nuclear DNA in the yeast Saccharomyces cerevisiae at the same doses under which they confer lifespan extension. We found that rapamycin and spermidine were able to decrease the spontaneous mutation rate at the CAN1 locus, whereas dinitrophenol, metformin, and resveratrol were able to protect yeast against CAN1 mutations induced by ethyl methanesulfonate (EMS). We also tested whether these compounds could enhance survival against EMS, ultraviolet (UV) light, or hydrogen peroxide (H2O2) insult. All five compounds conferred a protective effect against EMS, while metformin and spermidine protected yeast against UV light. Somewhat surprisingly, none of the compounds were able to afford a significant protection against H2O2, with spermidine dramatically sensitizing cells. We also examined the ability of these compounds to increase lifespan when growth-arrested by hydroxyurea; only spermidine was found to have a positive effect. Overall, our results suggest that lifespan-extending compounds may act in part by protecting nuclear DNA.

Anti-Lymphocyte Antigen 6 Complex, Locus E-Seco-Cyclopropabenzindol-4-One-Dimer Antibody-Drug Conjugate That Forms Adduct with α1-Microglobulin Demonstrates Slower Systemic Antibody Clearance and Reduced Tumor Distribution in Animals.

Anti-Ly6E-seco-cyclopropabenzindol-4-one dimer antibody-drug conjugate (ADC) has been reported to form an adduct with α1-microglobulin (A1M) in animal plasma, but with unknown impact on ADC PK and tissue distribution. In this study, we compared the PK and tissue distribution of anti-Ly6E ADC with unconjugated anti-Ly6E mAb in rodents and monkeys. For PK studies, animals received an intravenous administration of anti-Ly6E ADC or unconjugated anti-Ly6E mAb. Plasma samples were analyzed for total antibody (Tab) levels and A1M adduct formation. PK parameters were generated from dose-normalized plasma concentrations. Tissue distribution was determined in tumor-bearing mice after a single intravenous dosing of radiolabeled ADC or mAb. Tissue radioactivity levels were analyzed using a gamma counter. The impact of A1M adduct formation on target cell binding was assessed in an in vitro cell binding assay. The results show that ADC Tab clearance was slower than that of mAb in mice and rats but faster than mAb in monkeys. Correspondingly, the formation of A1M adduct appeared to be faster and higher in mice, followed by rats, and slowest in monkeys. Although ADC tended to show an overall lower distribution to normal tissues, it had a strikingly reduced distribution to tumors compared with mAb, likely due to A1M adduct formation interfering with target binding, as demonstrated by the in vitro cell binding assay. Together, these data 1) demonstrate that anti-Ly6E ADC that forms A1M adduct had slower systemic clearance with strikingly reduced tumor distribution and 2) highlight the importance of selecting an appropriate linker-drug for successful ADC development. SIGNIFICANCE STATEMENT: Anti-lymphocyte antigen 6 complex, locus E, ADC with seco-cyclopropabenzindol-4-one-dimer payload formed adduct with A1M, which led to a decrease in systemic clearance but also attenuated tumor distribution. These findings demonstrate the importance of selecting an appropriate linker-drug for ADC development and also highlight the value of a mechanistic understanding of ADC biotransformation, which could provide insight into ADC molecule design, optimization, and selection.

Antibody Conjugation of a Chimeric BET Degrader Enables in†vivo Activity.

The ability to selectively degrade proteins with bifunctional small molecules has the potential to fundamentally alter therapy in a variety of diseases. However, the relatively large size of these chimeric molecules often results in challenging physico-chemical properties (e. g., low aqueous solubility) and poor pharmacokinetics which may complicate their in vivo applications. We recently discovered an exquisitely potent chimeric BET degrader (GNE-987) which exhibited picomolar cell potencies but also demonstrated low in vivo exposures. In an effort to improve the pharmacokinetic properties of this molecule, we discovered the first degrader-antibody conjugate by attaching GNE-987 to an anti-CLL1 antibody via a novel linker. A single IV dose of the conjugate afforded sustained in vivo exposures that resulted in antigen-specific tumor regressions. Enhancement of a chimeric protein degrader with poor in vivo properties through antibody conjugation thereby expands the utility of directed protein degradation as both a biological tool and a therapeutic possibility.

Prediction of Human Pharmacokinetics of Antibody-Drug Conjugates From Nonclinical Data.

Prediction of human pharmacokinetics (PK) based on preclinical information for antibody-drug conjugates (ADCs) provide important insight into first-in-human (FIH) study design. This retrospective analysis was conducted to identify an appropriate scaling method to predict human PK for ADCs from animal PK data in the linear range. Different methods for projecting human clearance (CL) from animal PK data for 11 ADCs exhibiting linear PK over the tested dose ranges were examined: multiple species allometric scaling (CL vs. body weight), allometric scaling with correction factors, allometric scaling based on rule of exponent, and scaling from only cynomolgus monkey PK data. Two analytes of interest for ADCs, namely total antibody and conjugate (measured as conjugated drug or conjugated antibody), were assessed. Percentage prediction errors (PEs) and residual sum of squares (RSS) were compared across methods. Human CL was best estimated using cynomolgus monkey PK data alone and an allometric scaling exponent of 1.0 for CL. This was consistently observed for both conjugate and total antibody analytes. Other scaling methods either underestimated or overestimated human CL, or produced larger average absolute PEs and RSS. Human concentration-time profiles were also reasonably predicted from the cynomolgus monkey data using species-invariant time method with a fixed exponent of 1.0 for CL and 1.0 for volume of distribution. In conclusion, results from this retrospective analysis of 11 ADCs indicate that allometric scaling of CL with an exponent of 1.0 using cynomolgus monkey PK data alone can successfully project human PK profiles of an ADC within linear range.

Preclinical pharmacokinetics and pharmacodynamics of DCLL9718A: An antibody-drug conjugate for the treatment of acute myeloid leukemia.

Few treatment options are available for acute myeloid leukemia (AML) patients. DCLL9718A is an antibody-drug conjugate that targets C-type lectin-like molecule-1 (CLL-1). This receptor is prevalent on monocytes, neutrophils, and AML blast cells, and unlike CD33, is not expressed on hematopoietic stem cells, thus providing possible hematopoietic recovery. DCLL9718A comprises an anti-CLL-1 IgG1 antibody (MCLL0517A) linked to a pyrrolobenzodiazepine (PBD) dimer payload, via a cleavable disulfide-labile linker. Here, we characterize the in vitro and in vivo stability, the pharmacokinetics (PK) and pharmacodynamics (PD) of DCLL9718A and MCLL0517A in rodents and cynomolgus monkeys. Three key PK analytes were measured in these studies: total antibody, antibody-conjugated PBD dimer and unconjugated PBD dimer. In vitro, DCLL9718A, was stable with most (> 80%) of the PBD dimer payload remaining conjugated to the antibody over 96 hours. This was recapitulated in vivo with antibody-conjugated PBD dimer clearance estimates similar to DCLL9718A total antibody clearance. Both DCLL9718A and MCLL0517A showed linear PK in the non-binding rodent species, and non-linear PK in cynomolgus monkeys, a binding species. The PK data indicated minimal impact of conjugation on the disposition of DCLL9718A total antibody. Finally, in cynomolgus monkey, MCLL0517A showed target engagement at all doses tested (0.5 and 20 mg/kg) as measured by receptor occupancy, and DCLL9718A (at doses of 0.05, 0.1 and 0.2 mg/kg) showed strong PD activity as evidenced by notable reduction in monocytes and neutrophils.

Exploration of Pyrrolobenzodiazepine (PBD)-Dimers Containing Disulfide-Based Prodrugs as Payloads for Antibody-Drug Conjugates.

A number of cytotoxic pyrrolobenzodiazepine (PBD) monomers containing various disulfide-based prodrugs were evaluated for their ability to undergo activation (disulfide cleavage) in vitro in the presence of either glutathione (GSH) or cysteine (Cys). A good correlation was observed between in vitro GSH stability and in vitro cytotoxicity toward tumor cell lines. The prodrug-containing compounds were typically more potent against cells with relatively high intracellular GSH levels (e.g., KPL-4 cells). Several antibody-drug conjugates (ADCs) were subsequently constructed from PBD dimers that incorporated selected disulfide-based prodrugs. Such HER2 conjugates exhibited potent antiproliferation activity against KPL-4 cells in vitro in an antigen-dependent manner. However, the disulfide prodrugs contained in the majority of such entities were surprisingly unstable toward whole blood from various species. One HER2-targeting conjugate that contained a thiophenol-derived disulfide prodrug was an exception to this stability trend. It exhibited potent activity in a KPL-4 in vivo efficacy model that was approximately three-fold weaker than that displayed by the corresponding parent ADC. The same prodrug-containing conjugate demonstrated a three-fold improvement in mouse tolerability properties in vivo relative to the parent ADC, which did not contain the prodrug.