An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly

Wörn, Arne; Plückthun, Andreas

doi:10.1016/s0014-5793(98)00463-3

Cited by 114 publications

(82 citation statements)

References 39 publications

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“…Total RNA was extracted from the CD1d-transfected mouse RMA.S cell line using the RNeasy Mini Kit (QIAGEN). For the anti-HER2 antibody part, the plasmid pIG6-4D5, containing the scFv fragment derived from the mouse anti-HER2 antibody 4D5, was used as template (27) (kind gift from A. Pluckthun, University of Zurich, Zurich, Switzerland). Briefly, the entire mouse β2m was amplified with a Hind III site at the N-terminus for subsequent cloning in the PEAK 8 expression vector (EdgeBioSystems) and an Nhe I site at its C-terminus for its ligation to the N-terminal sequence of the a1 domain of CD1d, with the insertion of a sequence encoding a flexible glycine/serine-rich peptide linker (GGGGSGGSGSGGG).…”

Section: Methodsmentioning

confidence: 99%

“…In addition, to confer the tumor localization properties of the αGalCer/ sCD1d, we fused the β 2 m-CD1d molecule to an antitumor antibody fragment in view of our previous experience with tumor targeting of MHC class I coupled to different antitumor Fab′ fragments (25,26). To do so, a second (G 10 S 3 ) linker was inserted at the C-terminus of CD1d before ligation to the cDNA of the anti-HER2 4D5 scFv (27), followed by the same small linker (G 3 S 3 ) and His tag ( Figure 1A). Transient transfection of HEK293 EBNA cells adapted to grow in suspension in serum-free medium (28) gave similar yields for both recombinant CD1d proteins, alone or fused to the anti-HER2 scFv (up to 10 mg/l supernatant).…”

Section: Construction Expression and Purification Of Recombinant Momentioning

confidence: 99%

“…Here we developed a similar strategy applied to CD1d fused to the anti-HER2 scFv 4D5 with the aim to redirect iNKT cells to HER2-expressing tumor cells. This high-affinity antibody, scFv, has been derived from the mouse anti-HER2 mAb 4D5 (27), which is at the origin of the humanized version Herceptin (trastuzumab) used in the clinic (39). The tyrosine kinase receptor HER2 is overexpressed in 25%-30% of human breast cancers, primary tumors, and metastatic sites, and this parameter is associated with poor prognosis.…”

Section: Figurementioning

confidence: 99%

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Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Stirnemann¹,

Romero²,

Baldi³

et al. 2008

J. Clin. Invest.

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Invariant NKT (iNKT) cells are potent activators of DCs, NK cells, and T cells, and their antitumor activity has been well demonstrated.A single injection of the high-affinity CD1d ligand α-galactosylceramide (αGalCer) leads to short-lived iNKT cell activation followed, however, by long-term anergy, limiting its therapeutic use. In contrast, we demonstrated here that when αGalCer was loaded on a recombinant soluble CD1d molecule (αGalCer/sCD1d), repeated injections led to sustained iNKT and NK cell activation associated with IFN-γ secretion as well as DC maturation in mice. Most importantly, when αGalCer/sCD1d was fused to a HER2-specific scFv antibody fragment, potent inhibition of experimental lung metastasis and established s.c. tumors was obtained when systemic treatment was started 2-7 days after the injection of HER2-expressing B16 melanoma cells. In contrast, administration of free αGalCer at this time had no effect. The antitumor activity of the CD1d-anti-HER2 fusion protein was associated with HER2-specific tumor localization and accumulation of iNKT, NK, and T cells at the tumor site. Targeting iNKT cells to the tumor site thus may activate a combined innate and adaptive immune response that may prove to be effective in cancer immunotherapy.

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Section: Methodsmentioning

confidence: 99%

Section: Construction Expression and Purification Of Recombinant Momentioning

confidence: 99%

Section: Figurementioning

confidence: 99%

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Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Stirnemann¹,

Romero²,

Baldi³

et al. 2008

J. Clin. Invest.

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“…So far, only two artificial pairs (Val:Ala and Ala:Tyr) have been found to be functional replacements for the S-S bonds in Ig fold domains (3,40,43,44). It has also been reported that one of the Cys residues involved in an S-S bond can be replaced by Tyr, Ser, or Val (41,45,46).…”

Section: Discussionmentioning

confidence: 99%

Cellular Quality Control Screening to Identify Amino Acid Pairs for Substituting the Disulfide Bonds in Immunoglobulin Fold Domains

Hagihara

Matsuda

Yumoto

2005

Journal of Biological Chemistry

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We are interested in determining which amino acid pairs can be substituted for the disulfide (S-S) bonds in proteins without disrupting their native structures under physiological conditions. In this study, we focused on the intradomain S-S bonds in Ig fold domains and aimed to determine a simple rule for replacement of their S-S bonds. The cysteines of four different Ig fold domains were mutated randomly, and the amino acid pairs substituted for the S-S bonds were screened by the method utilizing a cellular quality control system. Among the 36 selected mutants, 31 were natively folded without S-S bonds, as judged from the cooperativity of thermal unfolding. In addition, the selected mutant llama heavy chain antibodies retained antigen-binding affinity. At least two of the pairs Ala:Ala, Ala:Val, Val: Ala, and Val:Val were found in the selected mutants for all four different Ig fold domains, and they were stably folded at 30°C. This suggests that examination of these four pairs could be enough to obtain natively folded Ig fold domains without S-S bonds. Native disulfide (S-S)1 bonds in proteins stabilize their functional structures, and the removal of an S-S bond results in marked destabilization of these proteins (1-8). It is of interest to determine which amino acid pairs can be substituted for the S-S bonds without disrupting their native structures under physiological conditions. Many S-S-bonded proteins are often regarded as targets for industrial and pharmaceutical uses. For practical applications, efficient production of these proteins with recombinant technology is essential, and the in vitro formation of correct S-S bonds is one of the most challenging problems for the preparation of functional recombinant proteins. Eliminating even one S-S bond critically decreases the possible S-S-bonding combinations, which results in higher yields of active proteins.In this study, we focused on the replacement of intradomain S-S bonds in Ig fold domains. Four typical Ig fold domains were used, and amino acid pairs substituted for S-S bonds were selected by cellular quality control screening. Then, the selected mutants were examined for their ability to form the native structures using CD and functional assay. Two of the four Ig fold domains tested here were variable fragments, i.e. the variable region of the heavy chain of camelid heavy chain antibody (VHH) and an engineered mouse V L domain ( graft). The other two were ␤2-microglobulin (␤2-m) and the constant domains of the human light chain (C L fragment). The VHH used here recognizes human chorionic gonadotropin (hCG) as an antigen (9). The graft is based on the sequence of the humanized 4D5-derived V L domain artificially designed to change the antigen specificity and interdomain interactions (10 -12).Our screening method, which utilizes a cellular quality control system for the secretory pathway in Saccharomyces cerevisiae and is referred to as "cellular quality control screening," was developed to screen for sequences that can fold into the native structures u...

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“…Many scFv cannot tolerate the loss of these disulfide bonds [107], and only a very few scFv constructs have been shown to fold correctly and retain their activity in the absence of intra-domain disulfide bonds [108,109]. Proba et al demonstrated that, although the stable ABPC48 scFv derived from its wild-type parental mAb is naturally missing a disulfide bond in the V H chain, restoration of the disulfide bond by point mutation can increase the stability of the scFv above that of the unmodified ABPC48 scFv [106].…”

Section: Stability Of Scfv Constructsmentioning

confidence: 99%

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

Cheng

Allen

2010

Expert Opinion on Drug Delivery

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Importance of the field-Targeted liposomal drugs represent the next evolution of liposomal drug delivery in cancer treatment. In various preclinical cancer models, antibody-targeted PEGylated liposomal drugs have demonstrated superior therapeutic effects over their non-targeted counterparts. Single chain Fv (scFv) has gained popularity in recent years as the targeting agent of choice over traditional targeting agents such as monoclonal antibodies (mAb) and antibody fragments (e.g., Fab′).Areas covered in this review-This review is focused mainly on advances in scFv-targeted liposomal drug delivery for the treatment of cancers, based on a survey of the recent literature, and on experiments done in a murine model of human B-lymphoma, using anti-CD19 targeted liposomes targeted with whole mAb, Fab′ fragments and scFv fragments.What the reader will gain-This review examines the recent advances in PEGylated immunoliposomal drug delivery, focusing on scFv fragments as targeting agents, in comparison with Fab′ and mAb.Take home message-For clinical development, scFv are potentially preferred targeting agents for PEGylated liposomes over mAb and Fab′, owing to factors such as decreased immunogenicity, and pharmacokinetics/biodistribution profiles that are similar to non-targeted PEGylated (Stealth ® ) liposomes.

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An intrinsically stable antibody scFv fragment can tolerate the loss of both disulfide bonds and fold correctly

Cited by 114 publications

References 39 publications

Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Sustained activation and tumor targeting of NKT cells using a CD1d–anti-HER2–scFv fusion protein induce antitumor effects in mice

Cellular Quality Control Screening to Identify Amino Acid Pairs for Substituting the Disulfide Bonds in Immunoglobulin Fold Domains

The use of single chain Fv as targeting agents for immunoliposomes: an update on immunoliposomal drugs for cancer treatment

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