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 anilino maytansinoid conjugates (AaMCs) have been prepared in which a maytansinoid bearing an aniline group was linked through the aniline amine to a dipeptide, which in turn was covalently attached to a desired monoclonal antibody. Several such conjugates were prepared utilizing different dipeptides in the linkage including Gly-Gly, l-Val-l-Cit, and all four stereoisomers of the Ala-Ala dipeptide. The properties of AaMCs could be altered by the choice of dipeptide in the linker. Each of the AaMCs, except the AaMC bearing a d-Ala-d-Ala peptide linker, displayed more bystander killing in vitro than maytansinoid ADCs that utilize disulfide linkers. In mouse models, the anti-CanAg AaMC bearing a d-Ala-l-Ala dipeptide in the linker was shown to be more efficacious against heterogeneous HT-29 xenografts than maytansinoid ADCs that utilize disulfide linkers, while both types of the conjugates displayed similar tolerabilities.
Several antibody-maytansinoid conjugates (AMCs) are in clinical trials for the treatment of various cancers. Each of these conjugates can be metabolized by tumor cells to give cytotoxic maytansinoid metabolites that can kill targeted cells. In preclinical studies in mice, the cytotoxic metabolites initially formed in vivo are further processed in the mouse liver to give several oxidized metabolic species. In this work, the primary AMC metabolites were synthesized and incubated with human liver microsomes (HLMs) to determine if human liver would likely give the same metabolites as those formed in mouse liver. The results of these HLM metabolism studies as well as the subsequent syntheses of the resulting HLM oxidation products are presented. Syntheses of the minor impurities formed during the conjugation of AMCs were also conducted to determine their cytotoxicities and to establish how these impurities would be metabolized by HLM.
A new type of antibody–drug conjugate (ADC) has been prepared that contains a sulfur-bearing maytansinoid attached to an antibody via a highly stable tripeptide linker. Once internalized by cells, proteases in catabolic vesicles cleave the peptide of the ADC’s linker causing self-immolation that releases a thiol-bearing metabolite, which is then S-methylated. Conjugates were prepared with peptide linkers containing only alanyl residues, which were all l isomers or had a single d residue in one of the three positions. A d-alanyl residue in the linker did not significantly impair a conjugate’s cytotoxicity or bystander killing unless it was directly attached to the immolative moiety. Increasing the number of methylene units in the maytansinoid side chain of a conjugate did not typically affect an ADC’s cytotoxicity to targeted cells but did increase bystander killing activity. ADCs with the highest in vitro bystander killing were then evaluated in vivo in mice, where they displayed improved efficacy compared to previously described types of maytansinoid conjugates.
Antibody–drug conjugates (ADCs) that incorporate the exatecan derivative DXd in their payload are showing promising clinical results in solid tumor indications. The payload has an F-ring that also contains a second chiral center, both of which complicate its synthesis and derivatization. Here we report on new camptothecin-ADCs that do not have an F-ring in their payloads yet behave similarly to DXd-bearing conjugates in vitro and in vivo. This simplification allows easier derivatization of camptothecin A and B rings for structure–activity relationship studies and payload optimization. ADCs having different degrees of bystander killing and the ability to release hydroxyl or thiol-bearing metabolites following peptide linker cleavage were investigated.
Six antibody-maytansinoid conjugates (AMCs) consisting of the cytotoxic maytansinoid, DM1 or DM4, linked to a tumor-targeting monoclonal antibody are undergoing clinical evaluation, with the most advanced in Phase III testing. In these AMCs, the maytansinoid is attached through its C3 side chain to the antibody via a thioether or disulfide bond. The antibody moiety of the conjugate binds specifically to a tumor cell after which the conjugate is internalized and processed to release one or more maytansinoid metabolites (MMs) which kill the cell. Some of these MMs are hydrophobic and highly potent against both the forming cell and neighboring dividing cells, while others are charged or polar compounds that are highly toxic to the cell that intracellularly released them, but have a relatively low toxicity to adjacent cells due to poor membrane permeability. Similar MMs are formed from AMCs inside cells of the reticulo-endothelial system (RES), the primary route of elimination of proteinaceous therapeutics such as AMCs. We have previously reported, from studies with radiolabeled MMs in mice, that the primary elimination route of RES-formed MMs is via the liver, and entails biliary excretion of relatively low potency charged or polar maytansinoids. It would be desirable to know if MMs generated from AMCs in the RES systems of patients can be inactivated by the oxidative processes of the human liver. To model this, MMs and other maytansinoids were synthesized and then exposed in vitro to human liver microsomes. The resulting microsomal metabolites were identified by HPLC/MS and their cytotoxicities were determined. In general, the major metabolites formed from microsome exposure occurred via oxidation on the C3 side chain, with only minimal microsome-dependent alterations noted in the macrocycle. Non-charged hydrophobic maytansinoids containing at least one sulfur atom were oxidized on the sulfur atom(s) to yield one or more products which were significantly less cytotoxic than the substrate. For example, the highly cytotoxic tumor cell metabolite, S-methyl DM4, was converted to its sulfoxide and sulfone analogues, which were more than 20-fold less potent than S-methyl DM4. Charged maytansinoids containing an amino acid or a carboxylic acid were poorly metabolized by the human liver microsomes even when these maytansinoids contained one or more sulfur atoms. These data suggest that the highly potent, non-charged MMs formed in the tumor cells or the RES systems of patients would be rendered less toxic through oxidation in the liver prior to biliary elimination. Furthermore, the less potent, charged MMs likely would not be further processed by liver in a way that would increase their systemic toxicity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2668.
Clinical-stage ADCs with a maytansinoid cytotoxic moiety (AMCs) currently use either a cleavable, hindered disulfide linker or the non-cleavable, thioether linker, SMCC. Both types of linkers have demonstrated comparative advantages pre-clinically for different cancer targets. Pre-clinically, benefits of the thioether linker can include greater AMC tolerability, while benefits of a hindered disulfide linker can include enhanced AMC activity. A goal of continued linker research is to create additional linker options to achieve the maximal therapeutic window for each AMC developed. We thus designed a new, tri-glycyl peptide linker (CX) which, like the SMCC linker, utilizes lysosomal cleavage of the AMC's antibody component. However, the CX linker provides an additional cleavage site to release a maytansinoid catabolite (MAY) bearing a carboxylic acid that should be largely uncharged at lysosomal pH (4.5) and should more readily diffuse out of the lysosome. To assess if AMCs with the CX linker were more active than those with the SMCC linker, we prepared AMCs (DM1 and 3H-DM1) with each of these linkers (CX-AMC, SMCC-AMC) with an anti-EpCAM and a non-targeting antibody, and compared their pharmacokinetics (PK), in vitro cytotoxic activity, cellular processing, and in vivo efficacy in EpCAM+ cell lines and xenografts. Two types of in vitro cytotoxicity patterns were observed: in one set of cell lines (HT29, COLO 205, OVCAR-3, LOVO) both AMCs were similarly active, whereas in another set (Calu3, A431, CAL27, SW2) the CX-AMC was more active (>5-fold lower IC50) than the SMCC-AMC. In cell lines where the CX-AMC was more active, a 3-fold greater amount of CX MAY was found in medium. This was due to better lysosomal processing (∼10-35% higher) and CX MAY export out of the lysosome, followed by its ultimate export into the medium. CX MAY, negatively charged at physiological pH, was 1000-fold less cytotoxic than a neutral maytansinoid when added extracellularly, suggesting that the CX-AMC would not have bystander killing activity on neighboring cancer cells. The CX-AMC was more cytotoxic against multidrug resistant cells (HCT-15, LOVODOX) than SMCC-AMC. A CX-AMC was prepared with a high load of 8 average DM1 per antibody, which was more cytotoxic than a typical AMC with 4 DM1 per antibody against low-antigen cells in vitro. We compared the efficacy of anti-EpCAM-CX-AMC and SMCC-AMC (4 DM1/antibody) against a Calu3 lung cancer xenograft model in SCID mice. The SMCC-AMC was inactive at the highest tested dose of 15 mg/kg conjugate. In contrast, the CX-AMC was active even at the lowest tested dose of 3 mg/kg, which was about 1/60th of the maximum tolerated dose (MTD). The MTD of CX-AMC was similar to that of SMCC-AMC in mice. Similar PK profiles were observed for CX-AMC and SMCC-AMC in CD-1 mice, indicating that the CX linker is highly stable in circulation. In conclusion, the CX peptide is a promising linker option for AMCs in cancer therapy. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C164. Citation Format: Rajeeva Singh, Nathan Fishkin, Yelena Kovtun, Gregory Jones, Jose Ponte, Hans Erickson, Erica Hong, Yulius Setiady, Andre Dandeneau, Katharine Lai, Jennifer Coccia, Leanne Lanieri, Juliet Bouchard, Karen Veale, Ravi Chari, Wayne Widdison. New tri-glycyl peptide linker offers advantages for maytansinoid antibody-drug conjugates (ADCs). [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C164.
<p>Supplementary Figures (S1-S4). Supplementary Figure S1: In vitro cytotoxic activities of tetraglycyl (Gly4), triglycyl (Gly3), diglycyl (Gly2), and valine-citrulline-glycine (VCG)-linked anti-EGFR ADCs and a triglycyl-linked non-binding antibody ADC in PC-9, Ca9-22, HSC-2, H1975, A-431, and OSC-19 cells (3-4 DAR ADCs). Supplementary Figure S2: In vitro cytotoxic activities of anti-EpCAM CX and SMCC ADCs toward a low EpCAM antigen-expressing cell line, RPMI 8226 (~50,000 EpCAM per cell). The CX ADCs tested included a conjugate with a typical payload number (3.9 maytansinoid molecules per antibody molecule; 3.9 DAR) and conjugates with high payload numbers (8 and 9.6 DAR). The SMCC ADC had a typical payload number (4.3 DAR). Supplementary Figure S3: Binding-competition ELISA of catabolites of CX ADC (DM-CX1 and DM-CX2) and SMCC ADC (lysine-SMCC-DM1): inhibition by catabolites toward binding of anti-maytansine antibody to immobilized BSA-maytansinoid conjugate. Supplementary Figure S4: Test of bystander cytotoxic activity of anti-EGFR-CX-DM1 or SPDB-DM4 conjugate in mixed culture of EGFR-positive (Ca9-22) and EGFR-negative (Ramos) cells.</p>
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