Abstract

Anti-CD30 diabodies were engineered with two cysteine mutations for site-specific drug conjugation in each chain of these homodimeric antibody fragments. Diabodies were conjugated with approximately 4 equivalents of the anti-tubulin drugs, monomethyl auristatin E or F, via a protease-cleavable dipeptide linker, to create the conjugates, diabody-vcE4 and diabody-vcF4, respectively. Diabody conjugation had only minor (<3-fold) effects on antigen binding. Diabody-vcF4 was potently cytotoxic against the antigen-positive cell lines, Karpas-299 (34 pmol/L IC(50)) and L540cy (22 pmol/L IC(50)), and was 8- and 21-fold more active than diabody-vcE4 against these cell lines, respectively. Clearance of diabody-vcF4 (99-134 mL/d/kg) was 5-fold slower than for the nonconjugated diabody in naive severe combined immunodeficient mice. Diabody-vcF4 had potent and dose-dependent antitumor activity against established Karpas-299 xenografts and gave durable complete responses at well-tolerated doses. Biodistribution experiments with diabody-[(3)H]-vcF4 (0.72-7.2 mg/kg) in tumor-bearing mice showed a dose-dependent increase in total auristatin accumulation in tumors (< or =520 nmol/L) and decrease in relative auristatin accumulation (< or =8.1 %ID/g), with peak localization at 4 to 24 h after dosing. Diabody-vcF4 had approximately 4-fold lower cytotoxic activity than the corresponding IgG1-vcF4 conjugate in vitro. A similar potency difference was observed in vivo despite 25- to 34-fold faster clearance of diabody-vcF4 than IgG1-vcF4. This may reflect that dose-escalated diabody-vcF4 can surpass IgG1-vcF4 in auristatin delivery to tumors, albeit with higher auristatin exposure to some organs including kidney and liver. Diabody-drug conjugates can have potent antitumor activity at well-tolerated doses and warrant further optimization for cancer therapy.

Highlights

  • Twelve antibody-based drugs have been approved for anticancer therapy including 9 by the U.S Food and Drug Administration [1, 2]

  • Diabodies have previously been successfully engineered for site-specific conjugation at two sites per diabody using a COOH-terminal cysteine residue or a linker cysteine [30, 31]

  • An anti-CD30 diabody was engineered for efficient site-specific drug conjugation at four sites per diabody with minimal affect on antigen binding

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Summary

Introduction

Twelve antibody-based drugs have been approved for anticancer therapy including 9 by the U.S Food and Drug Administration [1, 2]. These antibody therapeutics provide significant benefit to some patients but are seldom curative. Ten ADCs are in clinical trials as of May 2008, including Mylotarg [5]. This progress has encouraged further improvements to enhance the clinical potential of ADCs (3 – 5), including engineering of the IgG delivery vehicle [7], and the use of alternative antibody formats as described here

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