Avidity-Mediated Enhancement of In vivo Tumor Targeting by Single-Chain Fv Dimers

Abstract

Radiolabeled single-chain Fv (sFv) molecules display highly specific tumor retention in the severe combined immunodeficient (SCID) mouse model; however, the absolute quantity of sFv retained in the tumors is diminished by the rapid renal elimination resulting from the small size of the sFv molecules (M r 27,000) and by dissociation of the monovalent sFv from tumor-associated antigen. We previously reported significant improvement in tumor retention without a loss of targeting specificity on converting monovalent sFv into divalent [(sFvV) 2 ] dimers, linked by a disulfide bond between COOH-terminal cysteinyl peptides engineered into the sFvV . However, our data for enhanced dimer localization in tumors could not distinguish between the contributions of enhanced avidity and increased systemic retention associated with the larger size of 54 kDa [(sFvV) 2 ] dimers relative to 27-kDa sFv. In this investigation, we have compared tumor targeting of divalent antic-erbB-2/HER2/neu 741F8-1 (sFvV) 2 homodimers with monovalent 741F8/26-10 (sFvV) 2 heterodimers (M r 54,000) and 741F8 sFv monomers (741F8 sFv has binding specificity for erbB-2/ HER2/neu and 26-10 sFv specificity for digoxin and related cardiac glycosides). These studies allowed us to distinguish the dominant effect of valency over molecular weight in accounting for the superior tumor retention of 741F8-1 (sFvV) 2 homodimers. Each of the radioiodinated species was administered i.v. to SCID mice bearing SK-OV-3 human tumor xenografts and tumor localization at 24 hours post i.v. injection was determined for 125 I-741F8-1 (sFvV) 2 (3.57 %ID/g), 125 I-741F8/26-10 (sFvV) 2 (1.13 %ID/g), and 125 I-741F8-1sFv (1.25 %ID/g). These findings substantiate that the improved tumor retention of (sFvV) 2 homodimers over sFv monomers results from the availability of dual binding sites rather than from the slower systemic clearance of homodimers.Antibody engineering permits the design of new antibodybased proteins that can potentially address the shortcomings of intact antibodies as cancer therapeutics (1). One approach has been to produce the minimal antibody binding site in the form of a single-chain Fv (sFv), comprising the heavy and light chain variable domains of an antibody, joined by an appropriate linker peptide (2, 3). These 26-to 27-kDa sFv antibody species can be engineered from monoclonal antibodies or selected from phage-antibody libraries to obtain monovalent sFv species with high affinity and specificity for a target of choice. We have previously investigated the advantage of targeting tumors with sFv dimers, prepared as disulfide-linked (sFvV) 2 (4) or noncovalent diabodies (5), and in both cases dimeric/divalent species showed significant improvement over monomers. This investigation addresses the specific contributions of valence and molecular size to the in vitro binding characteristics and in vivo tumor targeting of sFv monomers and dimers.This investigation was based on our preparation of 741F8-1 sFv monomers, dimers, and bispecific heterodimers (4...

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