Development and Evaluation of Farletuzumab-[Mal-PEG2-Val-Cit-pAB-Eribulin] ADCs (MORAb-202)

Microtubule-Targeting Agent (MTA) & Eribulin

Although microtubule-targeting agents (MTAs) have been studied extensively as payloads for antibody-drug conjugates (ADCs), in many cases the benefits of these ADCs have been limited due to lack of efficacy or significant toxicity, prompting research to continue on new MTA payloads for the next-generation ADCs. Eribulin is a synthetic analog of the macrocyclic polyether halichondrin B. Compared to other MTAs, eribulin's fewer side effects of peripheral neuropathy, potent antimitotic activity, and nonmitotic effects on tumor biology make it an interesting candidate for investigation as a MTA payload for ADCs.

Creative Biolabs has extensive experience in the field of developing ADCs by using microtubule-targeting agents (MTA) as payloads and provides high-quality, customarily tailored ADC development services.

Preparation of Farletuzumab-[Mal-PEG2-Val-Cit-pAB-eribulin] (MORAb-202)

In the study, the antibody, the linker, and the drug are respectively humanized anti-human FRα farletuzumab, Mal-PEG2-Val-Cit-pAB, and eribulin.

Fig.1. Synthetic methods of Mal-PEG2-Val-Cit-PAB-eribulin. (Cheng X, et al., 2018)

First, the farletuzumab in DPBS solution was mixed with TCEP solution in 1× DPBS containing 2 mmol/L EDTA (DPBS/EDTA) and gently mixed for 80 minutes at room temperature. Subsequently, Mal-PEG2-VCP-eribulin in DMSO solution was added to 50% propylene glycol in DPBS/EDTA, and mixed well. Farletuzumab/TCEP solution was added and mixed well. Conjugation proceeded for 4 hours at room temperature with gentle mixing. Unreacted Mal-PEG2-VCP-eribulin was removed by G-25 chromatography on an AKTA FPLC using 1× DPBS as the mobile phase. Finally, the farletuzumab-[Mal-PEG2-Val-Cit-pAB-eribulin] (MORAb-202) was purified and concentrated, then stored at -80 °C.

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MORAb-202 Characterization

The study indicated the characterization of MORAb-202 in five aspects, including DAR analysis by HIC-HPLC and LC/MS, aggregation analysis by size-exclusion chromatography, antigen-binding studies by antigen-specific ELISAs, in vitro cytotoxicity assays, and in vivo efficacy in an NCI-H2110 xenograft model and the PDX models of NSCLC and gastric cancer.

Critical Quality Attributes (CQAs) Analysis

When caproyl or short (PEG₂) spacers were used, conjugation was efficient, with a drug-to-antibody ratio of 4.0 and low aggregate levels. Additionally, the binding capacity of non-cleavable maleimide-based farletuzumab (MORAb-202) was not significantly affected by conjugation, relative to unmodified antibodies. MORAb-202 and unmodified antibodies had similar binding abilities to cell-surface antigens.

Cytotoxicity Assay

MORAb-202 potency was evaluated on a number of tumor cell lines varying in anatomical origin and FRA expression, including IGROV1, OVCAR-3, CaoV3, NCI-H2110, etc. It was found that MORAb-202 exhibited high potency (IC50 of 20 pmol/L) on the FRAhi tumor cell line IGROV1. Furthermore, MORAb-202 also demonstrated subnanomolar potency on NCI-H2110 and OVCAR-3 cells and nanomolar potency on cell lines with moderate to low FRA expression across multiple tumor cell line origins, suggesting that MORAb-202 may be useful in targeting FRA-expressing tumors regardless of tissue origin or FRA cell surface expression levels.

In Vivo Efficacy of MORAb-202

• MORAb-202 was administered in an NCI-H2110 xenograft model in CB17 SCID mice as a single dose intravenously at 1 mg/kg, 2.5 mg/kg, and 5 mg/kg to evaluate dose-dependent effects. The result showed that at 1 mg/kg, MORAb-202 had a marginal effect on tumor growth; at the 2.5 mg/kg dose, it induced prolonged tumor growth inhibition, and MORAb-202 at 5 mg/kg induced a complete response (Fig. 2A).
• MORAb-202 was administered in PDX models of NSCLC and gastric cancer as a single dose intravenously at 5 mg/kg to evaluate the effect on tumor growth inhibition. Consistent with results from the NCI-H2110 study, durable tumor growth inhibition was observed in both the LXFA-737 model and the GA0055 model, while farletuzumab at an equal dose to MORAb-202 had no effect on tumor growth in the LXFA-737 model (Fig. 2B and C).

Fig. 2. In vivo efficacy of MORAb-202. (Cheng X, et al., 2018)

Reference

1. Cheng X, Li J, Tanaka K, et al. MORAb-202, an Antibody-Drug Conjugate Utilizing Humanized Anti-human FRα Farletuzumab and the Microtubule-targeting Agent Eribulin, has Potent Antitumor Activity. Mol Cancer Ther. 2018, 17(12):2665-2675.

For Research Use Only. NOT FOR CLINICAL USE.