A New Triglycyl Peptide Linker for Antibody–Drug Conjugates (ADCs) with Improved Targeted Killing of Cancer Cells

Abstract

A triglycyl peptide linker (CX) was designed for use in antibody-drug conjugates (ADC), aiming to provide efficient release and lysosomal efflux of cytotoxic catabolites within targeted cancer cells. ADCs comprising anti-epithelial cell adhesion molecule (anti-EpCAM) and anti-EGFR antibodies with maytansinoid payloads were prepared using CX or a noncleavable SMCC linker (CX and SMCC ADCs). The in vitro cytotoxic activities of CX and SMCC ADCs were similar for several cancer cell lines; however, the CX ADC was more active (5-100-fold lower IC 50 ) than the SMCC ADC in other cell lines, including a multidrug-resistant line. Both CX and SMCC ADCs showed comparable MTDs and pharmacokinetics in CD-1 mice. In Calu-3 tumor xenografts, antitumor efficacy was observed with the anti-EpCAM CX ADC at a 5-fold lower dose than the corresponding SMCC ADC in vivo. Similarly, the anti-EGFR CX ADC showed improved antitumor activity over the respective SMCC conjugate in HSC-2 and H1975 tumor models; however, both exhibited similar activity against FaDu xenografts. Mechanistically, in contrast with the charged lysine-linked catabolite of SMCC ADC, a significant fraction of the carboxylic acid catabolite of CX ADC could be uncharged in the acidic lysosomes, and thus diffuse out readily into the cytosol. Upon release from tumor cells, CX catabolites are charged at extracellular pH and do not penetrate and kill neighboring cells, similar to the SMCC catabolite. Overall, these data suggest that CX represents a promising linker option for the development of ADCs with improved therapeutic properties.

Antibody–Drug Conjugates (ADCs) for Personalized Treatment of Solid Tumors: A Review

“…The linker should be sufficiently stable in circulation to allow the payload to remain attached to the antibody while in circulation as it distributes into tissues (including solid tumor tissue), yet should allow efficient release of an active cell-killing agent once the ADC is taken up into the cancer cells [5]. Linkers can be characterized as either cleavable, where a chemical bond (or bonds) between the payload and the attachment site on the antibody (usually an amino acid) can be cleaved intracellularly [16–18], or as non-cleavable, where the final active metabolite released within the cell includes the payload and all elements of the linker still attached to an amino acid residue of the antibody, typically a lysine or a cysteine residue, following complete proteolytic degradation of the ADC within the lysosome of the cell [19–21]. …”

Expanding the Reach of Antibody–Drug Conjugates

“…One such linker was recently described. 10 Linker designs that release metabolites that are potent and cell-permeable could diffuse into and kill proximal bystander cells resulting in greater efficacy against tumors that express the antigen heterogeneously. Linkers that do not rely on lysosomal cleavage to release active drug may provide the opportunity to use antibodies that do not efficiently traffic through lysosomes.…”

Antibody–drug conjugates: Recent advances in linker chemistry

“… Chemical trigger Structure Mechanism Payload Ref. Acid cleavable triggers Hydrazone trigger Linkers cleavage by low pH of tumor acidic microenvironment or lysosomes Calicheamicin 10 Carbonate trigger SN-38 27 Silyl ether trigger MMAE 28 GSH cleavable trigger Disulfide trigger Linkers cleavage by high level of GSH in cytoplasm DM1, DM3, MMAE 30 PBD 31 Fe(II) cleavable trigger 1,2,4-Trioxolane trigger Linker cleavage by elevating levels of ferrous iron MMAE 34 Cathepsin cleavable triggers Dipeptide trigger Linkers cleavage by cathepsin in lysosomes MMAE, DM1 21 Triglycyl (CX) trigger DM1 22 cBu-Cit trigger MMAE, PBD 20 Glycosidase cleavable triggers β -Glucuronide trigger Linkers cleavage by β -glucuronidase in lysosomes MMAE 35 β -Galactoside trigger Linkers cleavage by β -galactosidase in lysosomes MMAE 36 Phosphatase cleavable triggers Pyrophosphate trigger Linkers cleavage by phosphatase and pyrophosphates in lysosomes Budesonide 18 Sulfatase cleavable trigger Arylsulfate trigger Linkers cleavage by sulfatase in lysosomes MMAE 37 Photo-responsive cleavable triggers Heptamethine cyanine fluorophore trigger Linkers cleavage...…”

“…designed a triglycyl peptide linker (CX) for ADCs with maytansinoid (DM1) as the payload ( Fig. 2 B) 22 . This linker consisted of three glycyl residues and showed extremely high stability in mouse plasma.…”