Potent Anticarcinoma Activity of the Humanized Anti-CD70 Antibody h1F6 Conjugated to the Tubulin Inhibitor Auristatin via an Uncleavable Linker

Abstract

Purpose: The antitubulin agent monomethyl auristatin F (MMAF) induces potent antitumor effects when conjugated via protease cleavable linkers to antibodies targeting internalizing, tumor-specific cell surface antigens. Humanized 1F6 (h1F6) is a humanized monoclonal antibody targeting CD70, a member of the tumor necrosis factor family that is expressed on hematologic malignancies and carcinomas. Here, we tested h1F6^maleimidocaproyl (mc) MMAF conjugates, consisting of an uncleavable mc linker, for their ability to interfere with the growth of CD70-positive carcinomas. Experimental Design: To evaluate the optimal drug per antibody ratio, we conjugated either four or eight MMAF molecules to the cysteines that comprise the interchain disulfides of h1F6 and determined antitumor activities in vitro and in xenografted mice. The tumor types tested included glioblastoma, patient-derived renal cell carcinoma (RCC) cell isolates, and standard RCC tumor cell lines. Results: All h1F6-mcMMAF conjugates potently interfered with the growth of all carcinomas in vitro and resulted in complete responses of RCC tumors implanted orthotopically or s.c. in mice. In vitro, h1F6-mcMMAF(8) was generally more potent than h1F6-mcMMAF(4). However, h1F6-mcMMAF(4) displayed equal or better efficacy than h1F6-mcMMAF(8) when administered to tumor-bearing mice. Conclusions: We showed that h1F6-mcMMAF conjugates inhibited the growth of human carcinomas and that increased drug loading, while improving potency in vitro, did not substantially affect the pharmacodynamic and pharmacokinetic properties in vivo. Based on these findings, h1F6-mcMMAF(4), designated SGN-75, has been identified as a potential antibody-drug conjugate for clinical development.

Novel anti–B-cell maturation antigen antibody-drug conjugate (GSK2857916) selectively induces killing of multiple myeloma

“…These data are consistent with previously published studies with the other mcMMAF ADC targeting CD70. 30,31,[33][34][35] Both ADCs induced apoptosis of CD138 1 patient MM cells following 3 days' treatment, and greater dose-dependent killing was seen by J6M0-mcMMAF than J6M0-vcMMAE ( Figure 2C). Percentages of annexin V/propidium iodide (PI) cells combined were 45.8% 6 0.5% and 74.5% 6 1.2% at 0.01 mg/mL and 1 mg/mL J6M0-mcMMAF, respectively.…”

“…These data are consistent with previously published studies with the other mcMMAF ADC targeting CD70. 30,31,[33][34][35] Both ADCs induced apoptosis of CD138…”

Synthesis of site-specific antibody-drug conjugates using unnatural amino acids

“…We chose to use auristatin, a highly potent tubulin inhibitor, because it is well characterized and can be modified for facile coupling to an antibody (10,30). Although the first generation of auristatin-linker conjugates were coupled to the N terminus of the peptide and contained a cathepsin B protease cleavage site, Doronina et al synthesized C-terminally linked derivatives that were reported to be more cytotoxic and have improved pharmacological profiles (30 (31), presumably because of degradation of the ADC in the lysosome and release of drug. Therefore, we first chose to investigate ADCs containing a noncleavable linker (nAF) coupled to the C terminus of auristatin F (AF) (Fig.…”

Intracellular Released Payload Influences Potency and Bystander-Killing Effects of Antibody-Drug Conjugates in Preclinical Models

“…The monoclonal antibodies used to measure CD30, CD70, and CD71 surface expression were BerH2 (BD Biosciences), h1F6 (14), and chimeric OKT9. Chimeric OKT9 was developed from mouse hybridoma cell line OKT9 (ATCC).…”