Rapid conjugation of antibodies to toxins to select candidates for the development of anticancer Antibody-Drug Conjugates (ADCs)

Abstract

Antibody-Drug Conjugates (ADCs) developed as a targeted treatment approach to deliver toxins directly to cancer cells are one of the fastest growing classes of oncology therapeutics, with eight ADCs and two immunotoxins approved for clinical use. However, selection of an optimum target and payload combination, to achieve maximal therapeutic efficacy without excessive toxicity, presents a significant challenge. We have developed a platform to facilitate rapid and cost-effective screening of antibody and toxin combinations for activity and safety, based on streptavidin-biotin conjugation. For antibody selection, we evaluated internalization by target cells using streptavidin-linked antibodies conjugated to biotinylated saporin, a toxin unable to cross cell membranes. For payload selection, we biotinylated toxins and conjugated them to antibodies linked to streptavidin to evaluate antitumour activity and pre-clinical safety. As proof of principle, we compared trastuzumab conjugated to emtansine via streptavidin-biotin (Trastuzumab-SB-DM1) to the clinically approved trastuzumab emtansine (T-DM1). We showed comparable potency in reduction of breast cancer cell survival in vitro and in growth restriction of orthotopic breast cancer xenografts in vivo. Our findings indicate efficient generation of functionally active ADCs. This approach can facilitate the study of antibody and payload combinations for selection of promising candidates for future ADC development.

Figure 1

Antibody Streptavidin linked to Biotin-Saporin as payload can be used to investigate antibody internalization (a) Schematic of Antibody-SB-Saporin conjugate generation. Streptavidin is conjugated randomly to lysine residues of the antibody with an average of two molecules of streptavidin per antibody. Streptavidin-linked antibody was conjugated to Monobiotin-Saporin at a molar ratio of 1:6. (b) Binding assay of trastuzumab, Trastuzumab-SB-Saporin and Trastuzumab-S to Her2-high SKBR-3 breast cancer cells and their concentrations at 50% of the maximum mean fluorescence intensity. (c–f) Top panels, target antigen recognition by naked antibodies, anti-Her2 trastuzumab (c), anti-CSPG4 (d), anti-EGFR Cetuximab (e), anti-FRα MOv18 (f) on cancer cell lines expressing different target antigens expression (Her2 (c), CSPG4 (d), EGFR (e), FRα (f)). Bottom panels (c–f), investigation of cell viability upon treatment with the naked antibody (black), antibody-Streptavidin-Biotin-Saporin conjugate (orange) or Saporin alone (grey). The ribosome inhibitor Saporin, unable to enter the cell alone, can be used to investigate antibody internalization by measuring viability (MTS) of antibody-SB-Saporin conjugate-treated cells. N = 1 for all binding assays and N = 3-4 independent experiments for MTS studies. All experiments were performed in triplicate, error bars represent Standard Deviation (SD).

Figure 2

Antibody-drug conjugate generation via Streptavidin-Biotin linking results in functionally active ADCs with anti-tumour potency in vitro (a) Schematic of Antibody-SB-Drug conjugate generation. Toxins containing functional groups such as thiols or amines can be directly biotinylated using commercially available kits and conjugated to streptavidin-linked antibodies for rapid Antibody-SB-Drug production. (b) Binding assay of trastuzumab, T-DM1 and Trastuzumab-SB-DM1 (molar ratio 1:3.5) to Her2-high HCC1954 breast cancer cells and their concentrations required to reach 50% of the maximum mean fluorescence intensity (MFI^max). (c) Investigation of cell viability of breast cancer cell lines with high (SKBR-3, HCC1954) and low (MDA-MB-231) Her2 expression. Tumour cells were treated with the naked antibody Trastuzumab, positive control T-DM1, trastuzumab-Streptavidin-Biotin-DM1 (Trastuzumab-SB-DM1) or DM1 toxin alone. (d) Investigation of cell viability of breast cancer cell lines with high (CAL51) and low (MDA-MB-468) Folate Receptor α expression upon treatment with the naked antibody MOv18, MOv18-Streptavidin-Biotin-A-419259 (molar ratio 1:8) (MOv18-SB-A-419259) or Src-inhibitor A-419259 alone. N = 3-4 independent experiments for all MTS studies, each condition performed in triplicate, error bars represent Standard Deviation (SD).

Figure 3

Trastuzumab-SB-DM1 internalization and mechanism of action is comparable to the clinically available T-DM1. HER2-high HCC1954 breast cancer cells were treated with naked antibody trastuzumab, control ADC T-DM1, Trastuzumab-SB-DM1 or PBS for 72 h and were stained for anti-IgG to investigate the internalization of trastuzumab antibody/ADC and anti-β-tubulin to compare tubulin disassembly (white arrowheads) upon treatment with T-DM1 and Trastuzumab-SB-DM1 ADCs. Scale bar, overview 50 μm, zoom 10 μm.

Figure 4

Trastuzumab-SB-DM1 and T-DM1 display comparable effects in restricting orthotopic mammary fat pad breast tumour growth in vivo (a) Schematic of Trastuzumab-SB-DM1 in vivo treatments. Human HCC1954 mammary fat pad xenograft-bearing mice were treated intravenously (i.v.) with one dose of Trastzumab-SB-DM1 following establishment of palpable tumours. (b) Growth curves of HCC1954 orthotopic tumours, mean ± SEM (left panel) and mouse weight mean ± SD (right panel). N = 10 mice per treatment group, treated with a single-dose of trastuzumab (4 mg/kg), T-DM1 (4 mg/kg), Trastuzumab-SB-DM1 (4 mg/kg), or PBS (right panel). ****P < 0.0001, by Dunnett’s multiple comparisons test.

Figure 5

Schematic of applications for the Streptavidin-Biotin linking approach to screen antibodies and payloads for ADC development (a) Antibody streptavidin labelling and conjugation to biotinylated Saporin as a quick tool to investigate antibody internalization. This can be applied for target selection of antibodies as well as to screen different antibody clones/isotypes/mutants to a particular target to select for the most suitable antibody for ADC development. (b) Antibody streptavidin labelling and conjugation to biotinylated payloads can be used for payload selection and preliminary in vivo studies. Commercially available biotinylation kits can be used to biotinylate toxins of interest for conjugation to streptavidin-linked antibodies to generate Antibody-SB-Drug conjugates with different drug-antibody ratios (DARs) due to the tetravalency of Streptavidin. Antibody-SB-Drug conjugates can then be used in vitro for payload selection and in vivo for preliminary studies.