Kinetically guided radical-based synthesis of C(sp3)-C(sp3) linkages on DNA.

DNA-encoded libraries (DEL)-based discovery platforms have recently been widely adopted in the pharmaceutical industry, mainly due to their powerful diversity and incredible number of molecules. In the two decades since their disclosure, great strides have been made to expand the toolbox of reaction modes that are compatible with the idiosyncratic aqueous, dilute, and DNA-sensitive parameters of this system. However, construction of highly important C(sp3)-C(sp3) linkages on DNA through cross-coupling remains unexplored. In this article, we describe a systematic approach to translating standard organic reactions to a DEL setting through the tactical combination of kinetic analysis and empirical screening with information captured from data mining. To exemplify this model, implementation of the Giese addition to forge high value C-C bonds on DNA was studied, which represents a radical-based synthesis in DEL.

Cycloisomerization of Olefins in Water.

Preparative reactions that occur efficiently under dilute, buffered, aqueous conditions in the presence of biomolecules find application in ligation, peptide synthesis, and polynucleotide synthesis and sequencing. However, the identification of functional groups or reagents that are mutually reactive with one another, but unreactive with biopolymers and water, is challenging. Shown here are cobalt catalysts that react with alkenes under dilute, aqueous, buffered conditions and promote efficient cycloisomerization and formal Friedel-Crafts reactions. The constraining conditions of bioorthogonal chemistry are beneficial for reaction efficiency as superior conversion at low catalyst concentration is obtained and competent rates in dilute conditions are maintained. Efficiency at high dilution in the presence of buffer and nucleobases suggests that these reaction conditions may find broad application.

Novel PIKK inhibitor antibody-drug conjugates: Synthesis and anti-tumor activity.

Novel neolymphostin-based antibody-drug conjugate (ADC) precursors were synthesized either through amide couplings between both cleavable and non-cleavable linkers and neolymphostin derivatives, or through Cu(I)-catalyzed acetylene-azide click cycloadditon between non-cleavable linkers and neolymphostin acetal derivatives. These precursors were site-specifically conjugated to cysteine mutant trastuzumab-A114C to provide neolymphostin-based ADCs. Preliminary in vitro data indicated that the corresponding ADCs were active against HER2-expressing tumor cell lines, thus providing a proof-of-concept for using neolymphostin as ADC-based anticancer agents.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

A general approach to site-specific antibody drug conjugates.

Using an expanded genetic code, antibodies with site-specifically incorporated nonnative amino acids were produced in stable cell lines derived from a CHO cell line with titers over 1 g/L. Using anti-5T4 and anti-Her2 antibodies as model systems, site-specific antibody drug conjugates (NDCs) were produced, via oxime bond formation between ketones on the side chain of the incorporated nonnative amino acid and hydroxylamine functionalized monomethyl auristatin D with either protease-cleavable or noncleavable linkers. When noncleavable linkers were used, these conjugates were highly stable and displayed improved in vitro efficacy as well as in vivo efficacy and pharmacokinetic stability in rodent models relative to conventional antibody drug conjugates conjugated through either engineered surface-exposed or reduced interchain disulfide bond cysteine residues. The advantages of the oxime-bonded, site-specific NDCs were even more apparent when low-antigen-expressing (2+) target cell lines were used in the comparative studies. NDCs generated with protease-cleavable linkers demonstrated that the site of conjugation had a significant impact on the stability of these rationally designed prodrug linkers. In a single-dose rat toxicology study, a site-specific anti-Her2 NDC was well tolerated at dose levels up to 90 mg/kg. These experiments support the notion that chemically defined antibody conjugates can be synthesized in commercially relevant yields and can lead to antibody drug conjugates with improved properties relative to the heterogeneous conjugates formed by nonspecific chemical modification.

Effect of attachment site on stability of cleavable antibody drug conjugates.

The systemic stability of the antibody-drug linker is crucial for delivery of an intact antibody-drug conjugate (ADC) to target-expressing tumors. Linkers stable in circulation but readily processed in the target cell are necessary for both safety and potency of the delivered conjugate. Here, we report a range of stabilities for an auristatin-based payload site-specifically attached through a cleavable valine-citrulline-p-aminobenzylcarbamate (VC-PABC) linker across various sites on an antibody. We demonstrate that the conjugation site plays an important role in determining VC-PABC linker stability in mouse plasma, and that the stability of the linker positively correlates with ADC cytotoxic potency both in vitro and in vivo. Furthermore, we show that the VC-PABC cleavage in mouse plasma is not mediated by Cathepsin B, the protease thought to be primarily responsible for linker processing in the lysosomal degradation pathway. Although the VC-PABC cleavage is not detected in primate plasma in vitro, linker stabilization in the mouse is an essential prerequisite for designing successful efficacy and safety studies in rodents during preclinical stages of ADC programs. The divergence of linker metabolism in mouse plasma and its intracellular cleavage offers an opportunity for linker optimization in the circulation without compromising its efficient payload release in the target cell.

Discovery of a macrocyclic o-aminobenzamide Hsp90 inhibitor with heterocyclic tether that shows extended biomarker activity and in vivo efficacy in a mouse xenograft model.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research, heterocycle-containing tethers were explored with the intent to further improve potency and minimize hERG liabilities. This effort culminated in the discovery of compound 10, which efficiently suppressed proliferation of HCT116 and U87 cells. This compound showed prolonged Hsp90-inhibitory activity at least 24h post-administration consistent with elevated and prolonged exposure in the tumor. When studied in a xenograft model, the compound demonstrated significant suppression of tumor growth.

Macrocyclic lactams as potent Hsp90 inhibitors with excellent tumor exposure and extended biomarker activity.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. In continuation of our research in this area, macrocyclic amides and lactams were explored to reduce the risk of hERG liabilities. This effort culminated in the discovery of compound 38, which showed a favorable in vitro profile, and efficiently suppressed proliferation of several relevant cell lines. This compound showed prolonged Hsp90-inhibitory activity at least 24 h post-administration, consistent with elevated and prolonged exposure in the tumor.

Discovery of a stable macrocyclic o-aminobenzamide Hsp90 inhibitor which significantly decreases tumor volume in a mouse xenograft model.

An extension of our previously reported series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. Addition of a second methyl group to the tether provided analogs that show increased potency in binding as well as cell-proliferation assays and, more importantly, are stable toward microsomes. We wish to disclose the discovery of a macrocycle which showed impressive biomarker activity 24-h post dosing and which demonstrated prolonged exposure in tumors. When studied in a lung cancer xenograft model, the compound demonstrated significant tumor size reduction.

Design and SAR of macrocyclic Hsp90 inhibitors with increased metabolic stability and potent cell-proliferation activity.

A novel series of macrocyclic ortho-aminobenzamide Hsp90 inhibitors is reported. A basic nitrogen within the tether linking the aniline nitrogen atom to a tetrahydroindolone moiety allowed access to compounds with good physical properties. Important structure-activity relationship information was obtained from this series which led to the discovery of a soluble and stable compound which is potent in an Hsp90 binding and cell-proliferation assay.

Development of Highly Optimized Antibody-Drug Conjugates against CD33 and CD123 for Acute Myeloid Leukemia.

PURPOSE: Mortality due to acute myeloid leukemia (AML) remains high, and the management of relapsed or refractory AML continues to be therapeutically challenging. The reapproval of Mylotarg, an anti-CD33-calicheamicin antibody-drug conjugate (ADC), has provided a proof of concept for an ADC-based therapeutic for AML. Several other ADCs have since entered clinical development of AML, but have met with limited success. We sought to develop a next-generation ADC for AML with a wide therapeutic index (TI) that overcomes the shortcomings of previous generations of ADCs. EXPERIMENTAL DESIGN: We compared the TI of our novel CD33-targeted ADC platform with other currently available CD33-targeted ADCs in preclinical models of AML. Next, using this next-generation ADC platform, we performed a head-to-head comparison of two attractive AML antigens, CD33 and CD123. RESULTS: Our novel ADC platform offered improved safety and TI when compared with certain currently available ADC platforms in preclinical models of AML. Differentiation between the CD33- and CD123-targeted ADCs was observed in safety studies conducted in cynomolgus monkeys. The CD33-targeted ADC produced severe hematologic toxicity, whereas minimal hematologic toxicity was observed with the CD123-targeted ADC at the same doses and exposures. The improved toxicity profile of an ADC targeting CD123 over CD33 was consistent with the more restricted expression of CD123 in normal tissues. CONCLUSIONS: We optimized all components of ADC design (i.e., leukemia antigen, antibody, and linker-payload) to develop an ADC that has the potential to translate into an effective new therapy against AML.

Synthesis and PKCtheta inhibitory activity of a series of 4-indolylamino-5-phenyl-3-pyridinecarbonitriles.

A series of 4-indolylamino-5-phenyl-3-pyridinecarbonitrile inhibitors of PKCtheta were synthesized as potential anti-inflammatory agents. The effects of specific substitution on the 5-phenyl moiety and variations of the positional isomers of the 4-indolylamino substituent were explored. This study led to the discovery of compound 12d, which had an IC(50) value of 18nM for the inhibition of PKCtheta.

Benzo[c][2,7]naphthyridines as inhibitors of PDK-1.

A series of substituted benzo[c][2,7]-naphthyridines were prepared and showed good potency in inhibiting PDK-1. The synthesis and SAR of this series of compounds are presented as well as the X-ray crystal structure of one of these analogs in a complex with PDK-1.

Preclinical Efficacy and Safety Comparison of CD3 Bispecific and ADC Modalities Targeting BCMA for the Treatment of Multiple Myeloma.

The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.

Caveolae-Mediated Endocytosis as a Novel Mechanism of Resistance to Trastuzumab Emtansine (T-DM1).

Trastuzumab emtansine (T-DM1) is an antibody-drug conjugate (ADC) that has demonstrated clinical benefit for patients with HER2+ metastatic breast cancer; however, its clinical activity is limited by inherent or acquired drug resistance. The molecular mechanisms that drive clinical resistance to T-DM1, especially in HER2+ tumors, are not well understood. We used HER2+ cell lines to develop models of T-DM1 resistance using a cyclical dosing schema in which cells received T-DM1 in an "on-off" routine until a T-DM1-resistant population was generated. T-DM1-resistant N87 cells (N87-TM) were cross-resistant to a panel of trastuzumab-ADCs (T-ADCs) with non-cleavable-linked auristatins. N87-TM cells do not have a decrease in HER2 protein levels or an increase in drug transporter protein (e.g., MDR1) expression compared with parental N87 cells. Intriguingly, T-ADCs using auristatin payloads attached via an enzymatically cleavable linker overcome T-DM1 resistance in N87-TM cells. Importantly, N87-TM cells implanted into athymic mice formed T-DM1 refractory tumors that remain sensitive to T-ADCs with cleavable-linked auristatin payloads. Comparative proteomic profiling suggested enrichment in proteins that mediate caveolae formation and endocytosis in the N87-TM cells. Indeed, N87-TM cells internalize T-ADCs into intracellular caveolin-1 (CAV1)-positive puncta and alter their trafficking to the lysosome compared with N87 cells. T-DM1 colocalization into intracellular CAV1-positive puncta correlated with reduced response to T-DM1 in a panel of HER2+ cell lines. Together, these data suggest that caveolae-mediated endocytosis of T-DM1 may serve as a novel predictive biomarker for patient response to T-DM1. Mol Cancer Ther; 17(1); 243-53. ©2017 AACR.

A PTK7-targeted antibody-drug conjugate reduces tumor-initiating cells and induces sustained tumor regressions.

Disease relapse after treatment is common in triple-negative breast cancer (TNBC), ovarian cancer (OVCA), and non-small cell lung cancer (NSCLC). Therapies that target tumor-initiating cells (TICs) should improve patient survival by eliminating the cells that can drive tumor recurrence and metastasis. We demonstrate that protein tyrosine kinase 7 (PTK7), a highly conserved but catalytically inactive receptor tyrosine kinase in the Wnt signaling pathway, is enriched on TICs in low-passage TNBC, OVCA, and NSCLC patient-derived xenografts (PDXs). To deliver a potent anticancer drug to PTK7-expressing TICs, we generated a targeted antibody-drug conjugate (ADC) composed of a humanized anti-PTK7 monoclonal antibody, a cleavable valine-citrulline-based linker, and Aur0101, an auristatin microtubule inhibitor. The PTK7-targeted ADC induced sustained tumor regressions and outperformed standard-of-care chemotherapy. Moreover, the ADC specifically reduced the frequency of TICs, as determined by serial transplantation experiments. In addition to reducing the TIC frequency, the PTK7-targeted ADC may have additional antitumor mechanisms of action, including the inhibition of angiogenesis and the stimulation of immune cells. Together, these preclinical data demonstrate the potential for the PTK7-targeted ADC to improve the long-term survival of cancer patients.

Molecular Basis of Valine-Citrulline-PABC Linker Instability in Site-Specific ADCs and Its Mitigation by Linker Design.

The degree of stability of antibody-drug linkers in systemic circulation, and the rate of their intracellular processing within target cancer cells are among the key factors determining the efficacy of antibody-drug conjugates (ADC) in vivo Previous studies demonstrated the susceptibility of cleavable linkers, as well as auristatin-based payloads, to enzymatic cleavage in rodent plasma. Here, we identify Carboxylesterase 1C as the enzyme responsible for the extracellular hydrolysis of valine-citrulline-p-aminocarbamate (VC-PABC)-based linkers in mouse plasma. We further show that the activity of Carboxylesterase 1C towards VC-PABC-based linkers, and consequently the stability of ADCs in mouse plasma, can be effectively modulated by small chemical modifications to the linker. While the introduced modifications can protect the VC-PABC-based linkers from extracellular cleavage, they do not significantly alter the intracellular linker processing by the lysosomal protease Cathepsin B. The distinct substrate preference of the serum Carboxylesterase 1C offers the opportunity to modulate the extracellular stability of cleavable ADCs without diminishing the intracellular payload release required for ADC efficacy. Mol Cancer Ther; 15(5); 958-70. ©2016 AACR.

RN927C, a Site-Specific Trop-2 Antibody-Drug Conjugate (ADC) with Enhanced Stability, Is Highly Efficacious in Preclinical Solid Tumor Models.

Trop-2, also known as TACSTD2, EGP-1, GA733-1, and M1S1, is frequently expressed on a variety of human carcinomas, and its expression is often associated with poor prognosis of the diseases. However, it is also present on the epithelium of several normal tissues. A comprehensively designed Trop-2-targeting antibody-drug conjugate (ADC), balancing both efficacy and toxicity, is therefore necessary to achieve clinical utility. To this end, we developed a cleavable Trop-2 ADC (RN927C) using a site-specific transglutaminase-mediated conjugation method and a proprietary microtubule inhibitor (MTI) linker-payload, PF-06380101. Robust in vitro cytotoxicity of RN927C was observed on a panel of Trop-2-expressing tumor cell lines, with IC50 generally in the subnanomolar range. As expected for an MTI-containing ADC, RN927C readily induced mitotic arrest of treated cells in vitro and in vivo, followed by subsequent cell death. The in vivo efficacy of RN927C was tested in multiple cell line and patient-derived xenograft tumor models, including pancreatic, lung, ovarian, and triple-negative breast tumor types. Single-dose administration of RN927C at 0.75 to 3 mg/kg was generally sufficient to induce sustained regression of Trop-2-expressing tumors and showed superior efficacy over standard treatment with paclitaxel or gemcitabine. Administration of RN927C in nonhuman primate toxicity studies resulted in target-mediated effects in skin and oral mucosa, consistent with Trop-2 expression in these epithelial tissues with minimal, non-dose limiting off-target toxicities. On the basis of the combined efficacy and safety results, RN927C is postulated to have a favorable therapeutic index for treatment of solid tumors. Mol Cancer Ther; 15(11); 2698-708. ©2016 AACR.

2-(Quinazolin-4-ylamino)-[1,4]benzoquinones as covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2.

A series of 2-(quinazolin-4-ylamino)-[1,4] benzoquinone derivatives that function as potent covalent-binding, irreversible inhibitors of the kinase domain of vascular endothelial growth factor receptor-2 (VEGFR-2) has been prepared by ceric ammonium nitrate oxidation of substituted (2,5-dimethoxyphenyl)(6,7-disubstituted-quinazolin-4-yl)amines and by displacement of the chlorine atom of substituted 2-chloro-5-(6,7-disubstituted-quinazolin-4-ylamino)-[1,4]benzoquinones with various amines, anilines, phenols, and alcohols. Enzyme studies were conducted in the absence and presence of glutathione and plasma. Several of the compounds inhibit VEGF-stimulated autophosphorylation in intact cells. Kinetic experiments were performed to study the reactivity of selected inhibitors toward glutathione. Reactivities correlated with LUMO energies calculated as averages of those of individual conformers weighted by the Boltzmann distribution. These results and molecular modeling were used to rationalize the biological observations. The compounds behave as non-ATP-competitive inhibitors. Unequivocal evidence, from mass spectral studies, indicates that these inhibitors form a covalent interaction with Cys-1045. One member of this series displays antitumor activity in an in vivo model.

Site-Dependent Degradation of a Non-Cleavable Auristatin-Based Linker-Payload in Rodent Plasma and Its Effect on ADC Efficacy.

The efficacy of an antibody-drug conjugate (ADC) is dependent on the properties of its linker-payload which must remain stable while in systemic circulation but undergo efficient processing upon internalization into target cells. Here, we examine the stability of a non-cleavable Amino-PEG6-based linker bearing the monomethyl auristatin D (MMAD) payload site-specifically conjugated at multiple positions on an antibody. Enzymatic conjugation with transglutaminase allows us to create a stable amide linkage that remains intact across all tested conjugation sites on the antibody, and provides us with an opportunity to examine the stability of the auristatin payload itself. We report a position-dependent degradation of the C terminus of MMAD in rodent plasma that has a detrimental effect on its potency. The MMAD cleavage can be eliminated by either modifying the C terminus of the toxin, or by selection of conjugation site. Both approaches result in improved stability and potency in vitro and in vivo. Furthermore, we show that the MMAD metabolism in mouse plasma is likely mediated by a serine-based hydrolase, appears much less pronounced in rat, and was not detected in cynomolgus monkey or human plasma. Clarifying these species differences and controlling toxin degradation to optimize ADC stability in rodents is essential to make the best ADC selection from preclinical models. The data presented here demonstrate that site selection and toxin susceptibility to mouse plasma degradation are important considerations in the design of non-cleavable ADCs, and further highlight the benefits of site-specific conjugation methods.