Synthesis of Drug Linker Constructs Cleavable by Tumor Upregulated Extracellular Proteases for the Delivery of Potent Inhibitors of Tubulin Polymerization
Introduction
Development of immunoglobulin (Ig) based therapies and delivery systems has been a significant focus in cancer research since their inception in the 1990s due to their propensity for high target affinity and desirable pharmacokinetic properties. Thirty years later, therapies that utilize Ig targeting strategies commanded $46 billion in the oncology space of 2021 and have been established as critical treatment options for patients. A subset of these therapies involves the combination of immunoglobulins and cytotoxic drugs to form antibody-drug conjugates (ADCs), in which these two critical entities are typically joined by a linker that is cleavable by enzymes endemic to the targeted tissues. Selectivity is governed by the choice of targeting protein and by the identity of the cleavable linker. The peptide sequences utilized herein are conjugated with cytotoxic agents KGP18 and separately KGP156. These molecules are benzosuberene-based anticancer agents that exhibit potent growth inhibition [GI50 <10 nM against NCI-H460 (lung), DU-145 (prostate), PANC-1 (pancreatic), and MDA-MB-231 (breast)] and strong inhibition of tubulin polymerization (IC50 = 1.2 and 1.7 μM, respectively) through binding at the colchicine site. These compounds also function as promising vascular disrupting agents (VDAs) and thus offer a unique dual mechanism of action that may prove desirable as payloads in drug-linker constructs and antibody-drug conjugates (ADCs). In previous studies we synthesized drug-linker constructs bearing the Val-Cit dipeptide linker utilized in the FDA approved ADC Adcetris (Seattle Genetics) with KGP18 and KGP156 (separately) serving as payloads. An extension of these studies to include the synthesis of a series of linker constructs bearing tripeptide sequences that are cleavable by urokinase-type plasminogen activator (Gly-Gly-Arg) (uPA) or plasmin (Ile-Phe-Lys) are described herein.
Methods
The synthesis of the payloads KGP18, KGP156 as well as the synthesis of short peptide linkers followed procedures that have been established by the Pinney group. Normal or reversed phase chromatography enabled by medium pressure liquid chromatography (MPLC) was used in the purification of intermediates, and preparative high pressure liquid chromatography (HPLC) was used as necessary for additional purification and determination of purity. All intermediates were characterized by the use of high resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR, 1H and 13C nuclei) spectroscopy.
Results
The synthesis of four drug-linker constructs has been achieved, each bearing a cytotoxic payload and a short peptide sequence providing a foundation for future conjugation to various targeting proteins. Each sequence was assembled rapidly through a sequential coupling-deprotection methodology, with the N-terminus being capped by an ω-maleimide caproic acid moiety that serves as a Michael acceptor for protein conjugation. The C-terminus was affixed through an amide bond to a self-immolative para-amino benzyl alcohol (PABA) bridge. The aniline-based payload KGP156 was covalently attached to the constructs through an amide bond, while the phenolic-based payload KGP18 was covalently tethered through two carbamate bonds via a dimethylethylenediamine (DMED) bridge.
Conclusion
The synthesis of four novel drug-linker constructs incorporating tripeptide sequences to enhance targeting was achieved. Ongoing studies will evaluate the plasma stability of these constructs and determine the efficiency of enzyme-mediated payload release.