Minibody-Based and scFv-Based Antibody Fragment-Drug Conjugates Selectively Eliminate GD2-Positive Tumor Cells

Abstract

Ganglioside GD2 is a well-established target expressed on multiple solid tumors, many of which are characterized by low treatment efficiency. Antibody-drug conjugates (ADCs) have demonstrated marked success in a number of solid tumors, and GD2-directed drug conjugates may also hold strong therapeutic potential. In a recent study, we showed that ADCs based on the approved antibody dinutuximab and the drugs monomethyl auristatin E (MMAE) or F (MMAF) manifested potent and selective cytotoxicity in a panel of tumor cell lines and strongly inhibited solid tumor growth in GD2-positive mouse cancer models. Here, we employed two different GD2-binding moieties–minibodies and scFv fragments that carry variable antibody domains identical to those of dinutuximab, and site-directly conjugated them to MMAE or MMAF by thiol-maleimide chemistry with drug-to-antibody ratios (DAR) of 2 and 1, respectively. Specific binding of the antibody fragment-drug conjugates (FDCs) to GD2 was confirmed in direct ELISA, flow cytometry, and confocal microscopy. Selective cytotoxic and cytostatic effects of the conjugates were observed in GD2-positive but not GD2-negative neuroblastoma and melanoma cell lines. Minibody-based FDCs demonstrated more pronounced cytotoxic effects and stronger antigen binding compared to scFv-based FDCs. The developed molecules may offer considerable practical benefit, since antibody fragment-drug conjugates are capable of enhancing therapeutic efficacy of ADCs by improving their pharmacokinetic characteristics and reducing side effects.

A humanized trivalent Nectin-4-targeting nanobody drug conjugate displays potent antitumor activity in gastric cancer

“…The successful preclinical trials of Padcev for treating urothelial carcinoma have demonstrated the efficacy of targeting Nectin-4 in cancer drug development [ 42 ], concurrently propelling the advancement of Nectin-4 ADCs. Given the high production costs, limited tissue penetration, binding site barriers, and off-target toxicity associated with monoclonal antibodies (mAb)-based ADCs, small-sized carriers, such as antigen-binding peptides [ 11 ], scFv [ 38 , 43 ], Fab [ 44 , 45 ], and Nb [ 16 , 21 ], have been developed to address the narrow therapeutic window of ADCs. Among these, Nb stands out for its advantages, including a small molecular size, high stability, low immunogenicity, and cost-effectiveness.…”

“…The average DAR of trivalent Nectin-4 Nb conjugated with vcMMAE was determined using ultraviolet-visible (UV-VIS) spectroscopy [ 38 , 39 ]. The Nano300 full-wavelength ultramicro spectrophotometer (Allsheng) was employed to detect and record the absorbance values of huNb26/Nb26-Nbh, vcMMAE, and Nectin-4 NDC at 254 nm and 280 nm.…”

Aminooxy Click Modification of a Periodate-Oxidized Immunoglobulin G: A General Approach to Antibody–Drug Conjugates with Dye-Mediated Expeditious Stoichiometry Control

“…Cell viability was analyzed by the MTT assay as described previously [ 59 ]. Briefly, cells were cultured in 96-well tissue culture plates (3 × 10 3 cells/plate) with serial dilutions of ADCs 14 and 31 , the 6H8 antibody, doxorubicin, MMAE, and conjugates 10 and 30 under standard conditions for 72 h. After that, a solution of MTT was added, and incubation was performed for 2 h, after which the medium was discarded and the formazan precipitate was dissolved in DMSO.…”

Reforming solid tumor treatment: the emerging potential of smaller format antibody-drug conjugate

“…Except for scFv formats, minibodies (scFv-CH3 dimers) have also been developed as targeted vectors. Recently, two different Ganglioside GD2-binding minibodies and scFv fragments identical to those of dinutuximab were site-specifically conjugated to MMAE or MMAF using thiol-maleimide chemistry with DAR of 2 and 1, respectively [ 35 ]. Both formats elicited selective cytotoxic and cytostatic effects exclusively in GD2-positive neuroblastoma and melanoma cell lines, with IC 50 values in the nanomolar range.…”