A diabody that dissociates to monomer forms at low concentration: effects on binding activity and tumor targeting

Baocheng, Huang; Foote, Linda J.; Lankford, Trish K.; Davern, Sandra; McKeown, Catherine K.; Kennel, Stephen J.

doi:10.1016/j.bbrc.2004.12.114

Cited by 19 publications

(13 citation statements)

References 19 publications

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“…Reducing linker length leads to autoassembly of scFv monomers to form functional divalent dimmers (diabodies; refs. [24][25][26]. When the linker is completely removed, scFv molecules exist as trimers (tribodies), which can be either trivalent or nonfunctional (noncognate V H /V L pair; refs.…”

Section: Discussionmentioning

confidence: 99%

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Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

Shi

Liu²,

Zheng³

et al. 2006

Cancer Research

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The clinical use of the single-chain fixed-variable (scFv) fragments of recombinant monoclonal antibodies as credible alternatives for classic therapeutic antibodies has two limitations: rapid blood clearance and inefficient local expression of functional molecules. In attempt to address these issues, we have developed a novel gene therapy protocol in which the anti-death receptor 5 (DR5) scFv fragments were either in vitro expressed in several tumor cell lines, or in vivo expressed in mice, using recombinant adeno-associated virus (rAAV)-mediated gene transfer. Viral transduction using the rAAV-S3C construct, which encodes a scFv molecule (S3C scFv) specific to DR5, led to stable expression in tumor cell lines and showed apoptosis-inducing activity in vitro, which could be inhibited by recombinant DR5 but not by DR4. A single i.m. injection of rAAV-S3C virus in nude mice resulted in stable expression of DR5-binding S3C scFv proteins in mouse sera for at least 240 days. Moreover, the expression of S3C scFv was associated with significant suppression of tumor growth and the increase of tumor cell apoptosis in previously established s.c. human lung LTEP-sml and liver Hep3B tumor xenografts.

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Section: Discussionmentioning

confidence: 99%

“…The length of the linker peptide seems to play an important role in affecting the function of scFv by determining whether the scFv molecules exist as monovalent monomers, or autoassemble into divalent dimmers (diabodies) and trivalent trimers (tribodies; refs. [22][23][24][25][26][27][28][29].…”

Section: Introductionmentioning

confidence: 99%

Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

Shi

Liu²,

Zheng³

et al. 2006

Cancer Research

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“…Amino acid sequences linking the heavy and light chains that are Ͼ12 residues yield a predominance of monomers, whereas shorter linkers yield noncovalently bound multivalent scFv proteins. The in vitro ratio of monomer to multimers is often dynamic and dependent on protein concentration and buffer conditions (Lee et al, 2002;Huang et al, 2005).…”

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confidence: 99%

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Peterson¹,

Laurenzana²,

Atchley³

et al. 2008

J Pharmacol Exp Ther

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Chronic or excessive (ϩ)-methamphetamine (METH) use often leads to addiction and toxicity to critical organs like the brain. With medical treatment as a goal, a novel single-chain variable fragment (scFv) against METH was engineered from anti-METH monoclonal antibody mAb6H4 (IgG, light chain, K d ϭ 11 nM) and found to have similar ligand affinity (K d ϭ 10 nM) and specificity as mAb6H4. The anti-METH scFv (scFv6H4) was cloned, expressed in yeast, purified, and formulated as a naturally occurring mixture of monomer (ϳ75%) and dimer (ϳ25%). To test the in vivo efficacy of the scFv6H4, male Sprague-Dawley rats (n ϭ 5) were implanted with 3-day s.c. osmotic pumps delivering 3.2 mg/kg/day METH. After reaching steady-state METH concentrations, an i.v. dose of scFv6H4 (36.5 mg/kg, equimolar to the METH body burden) was administered along with a [ 3 H]scFv6H4 tracer. Serum pharmacokinetic analysis of METH and [3 H]scFv6H4 showed that the scFv6H4 caused an immediate 65-fold increase in the METH concentrations and a 12-fold increase in the serum METH area under the concentration-time curve from 0 to 480 min after scFv6H4 administration. The scFv6H4 monomer was quickly cleared or converted to multivalent forms with an apparent t 1/2z of 5.8 min. In contrast, the larger scFv6H4 multivalent forms (dimers, trimers, etc.) showed a much longer t 1/2z (228 min), and the significantly increased METH serum molar concentrations correlated directly with scFv6H4 serum molar concentrations. Considered together, these data suggested that the scFv6H4 multimers (and not the monomer) were responsible for the prolonged redistribution of METH into the serum.There are currently nearly 20 monoclonal antibody (mAb) medications approved by the United States Food and Drug Administration and over 20 more in early clinical or preclinical trials (Holliger and Hudson, 2005). These medications include full-length IgG mAbs, along with five mAb fragments as Fab, F(abЈ) 2 (antigen binding fragments of IgG), or singlechain variable fragment (scFv) proteins.IgG mAbs are typically chimeric, humanized, or fully human proteins and are administered to patients requiring a long-acting antagonist with minimal extravascular penetration (Bazin-Redureau et al., 1997). IgG mAb is best for this purpose because it has a terminal elimination half-life (t 1/2z ) ranging from approximately 3 to 26 days. The longest t 1/2z values are usually achieved when the antibody does not bind to tissue sites and is not prematurely cleared due to antigenicity. When a short duration of action and greater extravascular penetration are needed, a significantly smaller fragment like Fab (t 1/2z ranging from 0.5-21 h; Trang, 1992) or scFv (t 1/2z ranging from minutes to hours; Goel et al., 2000) is used. In particular, rat pharmacokinetic studies of an antianthrax toxin scFv report a t 1/2␣ of ϳ5 min (Maynard et al., 2002). In addition, PCKN studies of a scFv in mice report t 1/2␣ values of 2.7 and 1 min Willuda et al., 2001). It is possible that a short-acting scFv could be used to

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“…These results suggest that one "induced fit" upon binding to Cn2 might stabilize the antibody packing of the light chain and heavy chain of each monomer, thus increasing the direct interactions by hydrogen bonds and reducing the surface cavities in the binding site. In the absence of toxin, the non-covalent diabodies could dissociate at a concentration of 10 nM resulting in monomers with no binding activity [69]. In conclusion, to the best of our knowledge, this is the first report in which the expression of different formats of antibody fragments are compared and contrasted in terms of thermodynamic properties and neutralization capacity.…”

Section: Discussionmentioning

confidence: 91%

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

et al. 2012

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A diabody that dissociates to monomer forms at low concentration: effects on binding activity and tumor targeting

Cited by 19 publications

References 19 publications

Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

Development and Preclinical Testing of a High-Affinity Single-Chain Antibody against (+)-Methamphetamine

Evaluation of three different formats of a neutralizing single chain human antibody against toxin Cn2: Neutralization capacity versus thermodynamic stability

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