Protease-Cleavable Linkers Modulate the Anticancer Activity of Noninternalizing Antibody-Drug Conjugates

Abstract

Antibody-drug conjugates (ADCs) represent an attractive class of biopharmaceutical agents, with the potential to selectively deliver potent cytotoxic agents to tumors. It is generally assumed that ADC products should preferably bind and internalize into cancer cells in order to liberate their toxic payload, but a growing body of evidence indicates that also ADCs based on noninternalizing antibodies may be potently active. In this Communication, we investigated dipeptide-based linkers (frequently used for internalizing ADC products) in the context of the noninternalizing F16 antibody, specific to a splice isoform of tenascin-C. Using monomethyl auristatin E (MMAE) as potent cytotoxic drug, we observed that a single amino acid substitution of the Val-Cit dipeptide linker can substantially modulate the in vivo stability of the corresponding ADC products, as well as the anticancer activity in mice bearing the human epidermoid A431 carcinoma. In these settings, the linker based on the Val-Ala dipeptide exhibited better performances, compared to Val-Cit, Val-Lys, and Val-Arg analogues. Mass spectrometric analysis revealed that the four linkers displayed not only different stability in vivo but also differences in cleavage sites. Moreover, the absence of anticancer activity for a F16-MMAE conjugate featuring a noncleavable linker indicated that drug release modalities, based on proteolytic degradation of the immunoglobulin moiety, cannot be exploited with noninternalizing antibodies. ADC products based on the noninternalizing F16 antibody may be useful for the treatment of several human malignancies, as the cognate antigen is abundantly expressed in the extracellular matrix of several tumors, while being virtually undetectable in most normal adult tissues.

Figure 1

Schematic representation of the molecular structure and coupling strategy used for four F16-MMAE ADCs, incorporating the Val-Cit (1), Val-Arg (2), Val-Lys (3) and Val-Ala (4) peptide linkers.

Figure 2

Synthesis and characterization of F16-MMAE ADCs 1-4. A) Schematic representation of the conjugation protocol. TCEP: tris(2-carboxyethyl)phosphine. B) size-exclusion chromatography and (C) SDS-page profiles of the products in either reducing (R) and non-reducing (NR) conditions; D) deconvoluted MS spectra: antibody light chains (LC) are functionalized with Mc-Linker-MMAE modules, while the heavy chain (HC) is glycosylated, resulting in 3 main peaks of ca. 50 KDa mass (calcd. HC mass: 48489.58). Calcd. mass of the light chains of ADCs 1-4 are 24023.90, 24022.92, 23994.90 and 23937.81, respectively (4 Cys residues are considered reduced in the calculation; calcd. mass of non-conjugated LC: 22707.25). Attribution of common extra-peaks of MS spectra is indicated in ADC 1 spectrum (deconvolution artifacts *: LC/2; **: HC/4; ***: HC/3; ****: HC/2, *****: LC·2; u: trace of non-functionalized LC). Full MS spectra (raw and deconvoluted, including non-conjugated F16 antibody) are shown in the Supporting Information.

Figure 3

Biological evaluation of F16-MMAE conjugates 1-4, featuring different cleavable dipeptide linkers: A) dose escalation study (BALB/c nude mice, n = 1 per group); B) Therapeutic activity of F16-MMAE ADCs against A431 human epidermoid carcinoma xenografted in BALB/c nude mice, after 4 administrations of ADCs (3 mg/kg every 3 days, as indicated by the arrows) and vehicle (PBS). A graph showing the percentage of body weight changes during the experiment is included in the Supporting Information. Data points represent mean tumor volume ± SEM, n = 4 per group.** indicates p < 0.01; * indicates p < 0.05.

Figure 4

A) Synthesis of the linker-drug module Mc-NC-MMAE (7). Reagents and Conditions: a) [1] 4-aminobenzyl alcohol, EDC·HCl, iPr₂Net, CH₂Cl₂, r.t. overnight; [2] 4-nitrophenyl chloroformate, pyridine, THF, r.t. 4 h; b) MMAE·TFA, HOAt, iPr₂Net, DMF, r.t. 48 h. B) size-exclusion chromatography and (C) SDS-page profiles of the F16-NC-MMAE ADC (8) in either reducing (R) and non-reducing (NR) conditions.

Figure 5

Schematic representation of the linker fragments observed by MS spectroscopy and presentation of their relative abundance after 24 and 48 h post injections in tumor-bearing mice (n = 2 mice/ADC). Data correspond to the ratio between the MS peak intensity of individual fragments and the sum of MS signal intensities of all detected peaks (100%). MS data of ADCs 2, 4 and 8 are shown in Figure S1. LC = light chain of F16 mAb.

Figure 6

A) Therapeutic activity of F16-Val-Cit-MMAE (1), F16-Val-Ala-MMAE (4) and F16-NC-MMAE (8) against A431 human epidermoid carcinoma xenografted in BALB/c nude mice, after 4 administrations of ADCs (7 mg/kg every 3 days, as indicated by the arrows) and vehicle (PBS); B) body weight changes (%) of the different treatment groups during the therapy experiment. Data points represent mean tumor volume ± SEM, n = 5 per group.

Scheme 1

Proposed proteolytic pathways of ADCs 2 and 3, bearing amino acids with basic side chains in the linker module.