Contribution of Linker Stability to the Activities of Anticancer Immunoconjugates

Abstract

The linker component of antibody-drug conjugates (ADC) is a key feature in developing optimized therapeutic agents that are highly active at well tolerated doses. For maximal intratumoral drug delivery, linkers are required that are highly stable in the systemic circulation, yet allow for efficient drug release at the target site. In this respect, amide bond-based technologies constitute a technological advancement, since the linker half-lives in circulation ( t 1/2 approximately 7 days) are much longer than earlier generation linkers that break down within 1-2 days. The amide linkers, some of which contain peptides, are appended to the mAb carriers through thioether/maleimide adducts. Here, we describe that use of a bromoacetamidecaproyl (bac) in place of the maleimidocaproyl (mc) increases the plasma stability of resulting thioether ADCs. One such ADC, 1F6-C4v2-bac-MMAF, which is directed against the CD70 antigen on lymphomas and renal cell carcinoma, was prepared containing a bac thioether spacer between the drug (MMAF) and the mAb carrier (1F6-C4v2). There was no measurable systemic drug release from this ADC for 2 weeks postadministration in mice. In order to assess the impact of improving linker stability beyond mc containing ADCs, a series of mc and bac-linked 1F6-MMAF conjugates were compared for tolerability, intratumoral drug delivery, and therapeutic efficacy in nude mice with renal cell carcinoma xenografts. There were no statistically significant efficacy differences between sets of mc and bac containing ADCs, although the bac linker technology led to 25% higher intratumoral drug exposure over a 7 day period compared to the corresponding mc linker. The mechanism of drug release from maleimide-adducts likely involves a retro-Michael reaction that takes place in plasma, based on in vitro studies demonstrating that some of the released drug-maleimide derivative became covalently bound to cysteine-34 of serum albumin. In summary, the data indicate that new linkers can be obtained with improved in vivo stability by replacing the maleimide with an acetamide, but the resulting ADCs had similar tolerability and activity profiles.

A general approach to site-specific antibody drug conjugates

“…In contrast, our anti-5T4 NDC had a PK that was equal to or slightly better than the WT Ab. The anti-drug ELISA also confirmed that the oxime linkage between the SMD and Ab is highly stable in vivo-a significant improvement relative to conventional maleimido linkages that have been reported to be chemically unstable in plasma (26,28).…”

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.…”

Immunogenicity of Antibody Drug Conjugates: Bioanalytical Methods and Monitoring Strategy for a Novel Therapeutic Modality

“…A variety of forms of the ADC may be generated in vivo. For example, variants derived from ADCs, based on maleimide adducts have been described (38)(39)(40). Reactivity of ADA for all three structural components of the ADC (mAb, linker, and small-molecule drug) can be determined.…”