Abstract 2526: Optimal cleavable linker for antibody-SN-38 conjugates for cancer therapy: Impact of linker's stability on efficacy
Authors: Govindan Serengulam V. , Cardillo Thomas M. , Tat Fatma , Arrojo Roberto , Sharkey Robert M. , Goldenberg David M. 
Published: August 2012

We reported therapeutic results of antibody (MAb) conjugates of the topo I inhibitor, SN-38, in human tumor xenografts in nude mice. These involved SN-38 derivatives, CL2-SN-38 (maleimido-[x]-Phe-Lys-PABOCO-20-O-SN-38) and its ‘Phe’-deleted variant, CL2A-SN-38. Conjugates of these derivatives showed similar serum stabilities and equivalent therapeutic efficacies. A more stably linked, cathepsin-B sensitive, derivative, CL2E-SN-38 (maleimido-[x]-Phe-Lys-PABOCO-N(Me)-(CH2)2-N(Me)-CO-10-O-SN-38) was prepared to determine the relative advantage of CL2E vs. CL2A linkers for MAb-SN-38 conjugates using MAbs with a spectrum of internalizing rates in hematological and solid tumor cell lines. Initial studies found these linkers differed in the drug-release rate from the MAb conjugates in human serum, with half-lives (t½) of ∼ 1 day for CL2A- and &gt;10 days for CL2E-conjugates. The t½ for cathepsin-B cleavage at the lysosomal pH (pH 5) for CL2A- and CL2E-SN-38 derivatives were 10 h and 0.5 h, respectively; the CL2A linker cleavage rate at this pH was the same with or without the enzyme, indicating a pH-mediated, but cathepsin-B insensitive, drug release. The release of SN-38 from CL2E-SN-38, requiring both the cathepsin-B cleavage and an intramolecular cyclization, proceeded with an overall t½ of 10 h at pH 5, very similar to that for CL2A-SN-38, suggesting that the corresponding MAb conjugates would liberate free SN-38 at a similar rate in the lysosome. For a slowly internalizing anti-CEACAM5 MAb, hMN-14 (labetuzumab), SN-38 conjugate with the CL2A linker was superior to that with CL2E in the LS174T colonic tumor cell line and xenograft model. With a more rapidly internalizing anti-TROP-2 MAb, hRS7, IC50s for CL2A- and CL2E-SN-38 conjugates were 9 nM and 132 nM, and 20 nM and 242 nM, in the pancreatic tumor cell line, Capan-1, and the lung adenocarcinoma cell line, Calu-3, respectively. CL2A-SN-38 and CL2E-SN-38 conjugates of the fast internalizing anti-CD22 MAb, hLL2 (epratuzumab), exhibited EC50 values of 3.2 nM and 135.8 nM, respectively, in the Raji lymphoma cell line. With an anti-CD74 MAb, hLL1 (milatuzumab), which internalizes exceptionally quickly with rapid CD74 re-cycling, CL2A- and CL2E-SN-38 conjugates showed IC50 values of 5 nM and 34 nM, respectively, in the CD74-positive melanoma cell line, A-375. The IC50 for free SN-38 ranged from 0.5 nM to 7 nM in these cell lines. While specific antitumor effects against human tumor xenografts were found using CL2A-SN-38 conjugates, specificity could not be shown in a 4-day in vitro MTS assay, most likely because of the 1-day half-life for drug release. However, with hLL1-CL2E-SN-38 conjugate in the A-375 cell line, specificity was established vs. non-targeting anti-CD22 conjugate, hLL2-CL2E-SN-38. The significance of these findings will be presented, together with the data from ongoing therapy studies in xenograft models.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2526. doi:1538-7445.AM2012-2526