Single-chain Fv-streptavidin substantially improved therapeutic index in multistep targeting directed at disialoganglioside GD2

Department of Pediatrics, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA.
Journal of Nuclear Medicine (Impact Factor: 6.16). 06/2004; 45(5):867-77.
Source: PubMed


Multistep targeting can improve the therapeutic index of antibody-based targeting, particularly relevant to pediatric tumors where acute toxicity and late effects of treatment are major concerns. Neuroblastoma is uniquely suited for such investigations because of its abundance of surface ganglioside GD2.
5F11scFv (scFv = single-chain variable fragment) was constructed from the variable regions of the heavy (V(H)) and kappa-light (V(L)) chain complementary DNA (cDNA) of anti-GD2 IgM hybridoma 5F11 and ligated to full-length streptavidin cDNA for expression in Escherichia coli. Purified 5F11-scFv-streptavidin (5F11-scFv-SA) was a homotetramer and showed comparable avidity to 5F11 IgM and a 30-fold improvement over monomeric scFv. Biodistribution of 5F11-scFv-SA was studied in nude mice xenografted with neuroblastoma LAN-1. Twenty-four hours after intravenous injection of 300-900 microg 5F11-scFv-SA, 150-450 microg of a thiogalactoside-containing clearing agent, (Gal-NAc)(16)-alpha-S-C(5)H(10)-NH-LC-N-Me-biotin (molecular weight, 8652), were administered intravenously, followed by approximately 2.5 microg (1.85-3.7 MBq) (111)In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-biotin ((111)In-DOTA-biotin) intravenously 4 h later and clocked as time 0.
Tumor uptake (percentage of injected dose per gram [%ID/g]) at 2 h was 7 %ID/g and decayed with a half-life of 72 h, whereas blood %ID/g rapidly decreased to <1/500 of that of tumor after the first 24 h. The tumor-to-nontumor (T/NT) ratio at 72 h was high (median, 106; range, 3.4 [kidney] to 1660 [blood]). When the area under the radioactivity curve was computed, the T/NT organ ratio was favorable (4.8 for kidney and 162 for blood). When human and murine tumors were surveyed, the T/NT ratio of (111)In-DOTA-biotin uptake correlated with their levels of GD2 expression as assayed by flow cytometry. Biotinylated polypeptides (bovine serum albumin and vasointestinal peptides) achieved selective tumor targeting when the multistep strategy was applied.
Improvement in the T/NT ratio using pretargeting strategy may increase the efficacy and safety of scFv-based approaches in cancer therapy. Additionally, since biotinylated polypeptides can be rendered tumor selective, a large repertoire of agents can potentially be explored.

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Available from: John Chung, Mar 03, 2014
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    • "A recent phase III randomized trial showed that ch14.18 when combined with GM-CSF and interleukin-2, was associated with a significantly improved survival in patients with high-risk neuroblastoma [3]. Other forms of antibody based strategies directed at GD2 have also been explored, including immunotoxins [14], immunoliposomes [15], multistep targeting [16] and chimeric immune receptors to retarget T-cells [17]. "
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    ABSTRACT: Ganglioside GD2 is a cell surface glycolipid that is highly expressed on cancer cells of neuroectodermal origin, including neuroblastoma, retinoblastoma, melanoma, sarcomas, brain tumors and small cell lung cancer. Monoclonal antibodies (MoAb) that target GD2 have shown clinical efficacy in the treatment of GD2 expressing tumors, and are expected to be the new standard of care for the treatment of pediatric neuroblastoma. In this study, the crystal structure of anti-GD2 murine MoAb 3F8 was solved to 1.65 Å resolution and used as a template for molecular docking simulations of its antigen, the penta-saccharide head group of GD2. Molecular docking revealed a binding motif composed of 12 key interacting amino acid side-chains, involving an extensive network of interactions involving main-chain and side-chain hydrogen bonding, two Pi - CH interactions, and an important charged interaction between Arg95 of the H3 loop with the penultimate sialic acid residue of GD2. Based on in silico scanning mutagenesis of the 12 interacting amino acids from the docked 3F8:GD2 model, a single point mutation (Heavy Chain: Gly54Ile) was engineered into a humanized 3F8 (hu3F8) MoAb and found to have a 6-9 fold enhancement in antibody-dependent cell-mediated cytotoxicity of neuroblastoma and melanoma cell lines. With enhanced tumor-killing properties, the re-engineered hu3F8 has the potential be a more effective antibody for the treatment of GD2-positive tumors.
    PLoS ONE 05/2013; 8(5):e63359. DOI:10.1371/journal.pone.0063359 · 3.23 Impact Factor
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    • "m3F8 was a mouse IgG3 antibody with kappa light chain,15 its anti-NB activity has been previously described.15,46,47 It was produced as ascites and purified by affinity chromatography: protein A (GE Healthcare) with > 90% pure by SDS-PAGE. "
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    ABSTRACT: Murine IgG3 anti-GD2 antibody m3F8 has shown anti-neuroblastoma activity in Phase I/II studies, where antibody-dependent cell-mediated cytotoxicity (ADCC) played a key role. Humanization of m3F8 should circumvent human anti-mouse antibody (HAMA) response and enhance its ADCC properties to reduce dosing and pain side effect. Chimeric 3F8 (ch3F8) and humanized 3F8 (hu3F8-IgG1 and hu3F8-IgG4) were produced and purified by protein A affinity chromatography. In vitro comparison was made with m3F8 and other anti-GD2 antibodies in binding, cytotoxicity, and cross-reactivity assays. In GD2 binding studies by SPR, ch3F8 and hu3F8 maintained K(D) comparable to m3F8. Unlike other anti-GD2 antibodies, m3F8, ch3F8 and hu3F8 had substantially slower k(off.). Similar to m3F8, both ch3F8 and hu3F8 inhibited tumor cell growth in vitro, while cross-reactivity with other gangliosides was comparable to that of m3F8. Both peripheral blood mononuclear cell (PBMC)-ADCC and polymorphonuclear leukocytes (PMN)-ADCC of ch3F8 and hu3F8-IgG1 were more potent than m3F8. This superiority was consistently observed in ADCC assays, irrespective of donors or NK-92MI-transfected human CD16 or CD32, whereas complement mediated cytotoxicity (CMC) was reduced. As expected, hu3F8-IgG4 had near absent PBMC-ADCC and CMC. Hu3F8 and m3F8 had similar tumor-to-non tumor ratios in biodistribution studies. Anti-tumor effect against neuroblastoma xenografts was better with hu3F8-IgG1 than m3F8. In conclusion, humanizing m3F8 produced next generation anti-GD2 antibodies with substantially more potent ADCC in vitro and anti-tumor activity in vivo. By leveraging ADCC over CMC, they may be clinically more effective, while minimizing pain and HAMA side effects. A Phase I trial using hu3F8-IgG1 is ongoing.
    OncoImmunology 07/2012; 1(4):477-486. DOI:10.4161/onci.19864 · 6.27 Impact Factor
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