Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Völkel, Tina; Korn, Tina; Bach, Miriam; Müller, Rolf; Kontermann, Roland E.

doi:10.1093/protein/14.10.815

Cited by 58 publications

(34 citation statements)

References 30 publications

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“…The diabody preparation contained a contaminant of approximately 35 kDa, similar to that observed by others. 15 Fast protein liquid chromatography (FPLC) analysis (not shown), indicated the diabody to have a solution size of about 50 kDa to 60 kDa, in line with the predicted molecular mass of 54 kDa, and implying that the diabody runs aberrantly on SDS-PAGE. Immunoblotting showed that the diabody bound the MSP1 19 fusion protein ( Figure 1C).…”

Section: Characterization Of Msp1 19 -Specific Human Abs and A Diabodmentioning

confidence: 72%

“…First, the VH and VL regions of B10 and M22 were amplified. The 5Ј VH primers incorporated a 5Ј BssHII site in B10 VH and a sequence encoding the C-terminal-half of a 19-residue-"long" linker (RGSGGGGSGGRAGSGGGGS) 15 in M22 VH. The 3Ј primer for both VHs incorporated a sequence encoding a GGGGS linker, 16 whereas the 5Ј VL primers incorporated overlapping GGGGS linkers.…”

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

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Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Pleass

Ogun

McGuinness

et al. 2003

Blood

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Parasite drug resistance and difficulties in developing effective vaccines have precipitated the search for alternative therapies for malaria. The success of passive immunization suggests that immunoglobulin (Ig)-based therapies are effective. To further explore the mechanism(s) by which antibody mediates its protective effect, we generated human chimeric IgG1 IntroductionMalaria continues to kill approximately 2 million people each year. 1 The success of passive immunization in humans and animals suggests that immunoglobulin (Ig)-based therapies could be effective. 2,3 Manipulating antibody (Ab) genes allows the design of Ig with defined class and specificity, targeting protective epitopes on the parasite surface. An appropriate target is the 19-kDa C-terminal region of merozoite surface protein 1 (MSP1 19 ). This polypeptide displays limited sequence polymorphism, 4 is expressed by all vertebrate life-cycle stages, 5 and acts as a major target of the erythrocyte invasion-inhibitory Ab response in individuals immune to Plasmodium falciparum malaria. 6 The mechanisms whereby Ig mediates protective immunity in malaria remain unclear. 7 A primary role of Ab appears to involve blocking of merozoite invasion of erythrocytes. 8 However, studies using human Ab suggest that the protective effects are mediated through interaction with specific receptors for the Ig Fc region (FcR) expressed on various effector cells. 9,10 Using Plasmodium yoelii in a murine model to address these possibilities, we generated human chimeric IgG1 and IgA1 Abs recognizing an epitope on P yoelii MSP1 19 . We have investigated the potential of these intact Abs, and a novel diabody with specificity for both MSP1 19 and a human receptor for IgG Fc, Fc␥RI, as novel malaria therapeutics. Materials and methods Construction of expression vectorsVariable heavy (VH) and light (VL) complementary DNAs (cDNAs) were cloned from mouse hybridomas B10 (anti-MSP1 19 ) and M22 (anti-human Fc␥RI) by reverse transcriptase-polymerase chain reaction (RT-PCR) using degenerate primers (5Ј-SARGTNMARCTGCAGSAGTC-3Ј and 5Ј-TGAGGAGACGGTGACCGTGGTYCCTTGGCCCC-3Ј for VH and 5Ј-GAYATTGAGCTCACMCARWCTMCA-3Ј and 5Ј-CCGTTTBAKCTC-GAGCTTKGTSCC-3Ј for VL). 11,12 An alternative primer incorporating a BssHII restriction enzyme site was used to reamplify B10 VH to facilitate subcloning. The IgA1 heavy-chain constant region was subcloned as a BamHI-SalI fragment from an ␣ chain gene-containing plasmid 13 into pAD-Gal4-2.1 (Stratagene, La Jolla, CA) and then as a BamHI-XbaI fragment into pVHExpress (a kind gift from Andrew Bradbury, Los Alamos National Laboratory, Los Alamos, NM), 14 replacing the ␥1 constant domains to yield pVHExpress ␣1H. The VH genes were inserted into pVHExpress and pVHExpress ␣1H, upstream of the ␥1 or ␣1 constant regions, respectively, and the VL genes into pVKExpress upstream of the human gene. 14 To generate the Fc␥RI x MSP1 19 diabody, an overlapping PCR-based strategy was used. First, the VH and VL regions of B10 and M22 were amplified. The 5Ј VH primers inco...

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Section: Characterization Of Msp1 19 -Specific Human Abs and A Diabodmentioning

confidence: 72%

Section: Construction Of Expression Vectorsmentioning

confidence: 99%

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Pleass

Ogun

McGuinness

et al. 2003

Blood

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“…Previous studies suggest that linkers shorter than 12 amino acids can result in multimeric complexes (diabody, triabody, etc.) due to "domain swapping" and that transition between monovalent and divalent scFv can be somewhat controlled by linker length (Atwell et al, 1999;Völkel et al, 2001). Considering these findings, it was our intention to design a predominately monomeric scFv.…”

Section: Discussionmentioning

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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“…due to ''domain swapping,'' and that transition between monovalent and divalent scFv can be somewhat controlled by the linker length. 31,32 Considering these findings, and with the intention of producing a predominately monomeric scFv, we used a 15 amino acid linker to connect the two chains. 10 Source …”

Section: Expression Of Scfv6h4mentioning

confidence: 99%

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4‐methylenedioxymethamphetamine

Celikel¹,

Peterson²,

Owens³

et al. 2009

Protein Science

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Methamphetamine (METH) is a major drug threat in the United States and worldwide. Monoclonal antibody (mAb) therapy for treating METH abuse is showing exciting promise and the understanding of how mAb structure relates to function will be essential for future development of these important therapies. We have determined crystal structures of a high affinity anti-(1)-METH therapeutic single chain antibody fragment (scFv6H4, K D 5 10 nM) derived from one of our candidate mAb in complex with METH and the (1) stereoisomer of another abused drug, 3,4-methylenedioxymethamphetamine (MDMA), known by the street name ''ecstasy.'' The crystal structures revealed that scFv6H4 binds to METH and MDMA in a deep pocket that almost completely encases the drugs mostly through aromatic interactions. In addition, the cationic nitrogen of METH and MDMA forms a salt bridge with the carboxylate group of a glutamic acid residue and a hydrogen bond with a histidine side chain. Interestingly, there are two water molecules in the binding pocket and one of them is positioned for a CAHÁ Á ÁO interaction with the aromatic ring of METH. These first crystal structures of a high affinity therapeutic antibody fragment against METH and MDMA (resolution 5 1.9 Å , and 2.4 Å , respectively) provide a structural basis for designing the next generation of higher affinity antibodies and also for carrying out rational humanization.

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Optimized linker sequences for the expression of monomeric and dimeric bispecific single-chain diabodies

Cited by 58 publications

References 30 publications

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

Novel antimalarial antibodies highlight the importance of the antibody Fc region in mediating protection

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

Crystal structures of a therapeutic single chain antibody in complex with two drugs of abuse—Methamphetamine and 3,4‐methylenedioxymethamphetamine

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