Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies

Kurucz, István; Titus, Julie A.; Jost, Carolina R.; Segal, David M.

doi:10.1016/0161-5890(95)00105-0

Cited by 73 publications

(35 citation statements)

References 26 publications

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“…Using molecular genetics, two scFvs can be engineered in tandem into a single polypeptide, separated by a linker domain, called a "tandem scFv" (tascFv). TascFvs have been found to be poorly soluble 10 and require refolding 11 when produced in bacteria, or they may be manufactured in mammalian cell culture systems, which avoids refolding requirements but may result in poor yields. 12 Construction of a tascFv with genes for two different scFvs yields a "bispecific single-chain variable fragments" (bis-scFvs).…”

Section: Overview Of Fragments In Developmentmentioning

confidence: 99%

Antibody fragments

Nelson¹

2010

mAbs

398

232

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T he antibody molecule is modular and separate domains can be extracted through biochemical or genetic means. It is clear from review of the literature that a wave of novel, antigen-specific molecular forms may soon enter clinical evaluation. This report examines the developmental histories of therapeutics derived from antigen-specific fragments of antibodies produced by recombinant processes. Three general types of fragments were observed, antigen-binding fragments (Fab), single chain variable fragments (scFv) and "third generation" (3G), each representing a successive wave of antibody fragment technology. In parallel, drug developers have explored multi-specificity and conjugation with exogenous functional moieties in all three fragment types. Despite high hopes and an active pipeline, enthusiasm for differentiating performance of fragments should, perhaps, be tempered as there are yet few data that suggest these molecules have distinct clinical properties due only to their size.

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Section: Overview Of Fragments In Developmentmentioning

confidence: 99%

Antibody fragments

Nelson¹

2010

mAbs

398

232

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“…Protein expression was induced in transformed Escherichia coli strain BL21(DE3) as described (32). C3d was isolated from inclusion bodies after extensive sonication, then solubilized, oxidized, and renatured following the method of Kurucz et al (33). C3d was further purified on a Mono Q HR 5/5 FPLC column (Pharmacia Biotech) at pH 8.3.…”

Section: Isolation Of Human C3 and Recombinant Human C3d; Generation mentioning

confidence: 99%

Complement Receptor Type 1 (CD35) Mediates Inhibitory Signals in Human B Lymphocytes

Józsi¹,

Prechl

Bajtay

et al. 2002

The Journal of Immunology

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The complement system—particularly component C3—has been demonstrated to be a key link between innate and adaptive immunity. The trimolecular complex of complement receptor type 2 (CR2), CD19, and CD81 is known to promote B cell activation when coligated with the B cell Ag receptor. In the present study, we aimed to elucidate the role of human complement receptor type 1 (CR1), the other C3-receptor on B cells. As ligand, aggregated C3 and aggregated C3(H2O), i.e., multimeric “C3b-like C3”, are used, which bind to CR1, but not to CR2. In experiments studying the functional consequences of CR1-clustering, the multimeric ligand is shown to inhibit the proliferation of tonsil B cells activated with a suboptimal dose of anti-IgM F(ab′)2. Importantly, this inhibitory activity also occurs in the presence of the costimulatory cytokines IL-2 and IL-15. The anti-IgM-induced transient increase in the concentration of intracellular free Ca2+ and phosphorylation of several cytoplasmic proteins are strongly reduced in the presence of the CR1 ligand. Data presented indicate that CR1 has a negative regulatory role in the B cell Ag receptor mediated activation of human B lymphocytes.

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“…Previously, we reported these modifications (32) and the procedure is based on inclusion body solubilization in a weak detergent, SLS in the presence of metal ion catalysts which dramatically reduces the incidence of aggregate formation. Kurucz et al (37) explain that the procedure results in high yields with affinities that are similar to native sFvs because the SLS allows correct disulfide pairing and prevents intermolecular aggregation, behaving as prototypic chaperone proteins that do assist in proper refolding of polypeptide chains off the ribosome in vivo. When used with the hma modification, this procedure resulted in profound improvements in yield and purity with at least two proteins we are seeking to develop for phase 1 studies (32).…”

Section: Discussionmentioning

confidence: 99%

“…The proteins were refolded using a sodium N-lauroyl-sarcosine (SLS) air oxidation method modified from a previously reported procedure for isolating sFv (37). Inclusion bodies were dissolved at 20:1 (mg wet weight/mL) in 100 mmol/L Tris (pH 10) and 2.5% SLS (Sigma, St Louis, MO).…”

Section: Methodsmentioning

confidence: 99%

A Bispecific Recombinant Immunotoxin, DT2219, Targeting Human CD19 and CD22 Receptors in a Mouse Xenograft Model of B-Cell Leukemia/Lymphoma

Vallera

Todhunter

Kuroki

et al. 2005

Clinical Cancer Research

102

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A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT 390 ) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same singlechain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19 + CD22 + Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC 50 = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.

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Correct disulfide pairing and efficient refolding of detergent-solubilized single-chain Fv proteins from bacterial inclusion bodies

Cited by 73 publications

References 26 publications

Antibody fragments

Antibody fragments

Complement Receptor Type 1 (CD35) Mediates Inhibitory Signals in Human B Lymphocytes

A Bispecific Recombinant Immunotoxin, DT2219, Targeting Human CD19 and CD22 Receptors in a Mouse Xenograft Model of B-Cell Leukemia/Lymphoma

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