[3] Protein engineering of single-chain Fv analogs and fusion proteins

Huston, James S.; Mudgett-Hunter, Meredith; Tai, Mei-Sheng; McCartney, John E.; Warren, Frederick; Haber, Edgar; Oppermann, Hermann

doi:10.1016/0076-6879(91)03005-2

Cited by 198 publications

(95 citation statements)

References 73 publications

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“…16,25 The scFv can be constructed with either sequence, VHLnVL or VLLnVH. 25 The VHVL construct typically exhibits Euclidean distances between Ln termini of 29 ±35 A Ê , whereas the VL ± VH orientation generally exhibits distances that are 5 ±10 A Ê longer for the same Fv. 26 Thus, differential orientation of a domain may distort the native Fv conformation.…”

Section: Discussionmentioning

confidence: 99%

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2

Zeytin

Tripathi

Bhattacharya‐Chatterjee

et al. 2000

Cancer Gene Ther

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Anti -idiotype antibody, 1A7, functionally mimics the tumor -associated antigen disialoganglioside GD2, which is overexpressed on the surface of a number of neuroectodermal tumors such as melanoma, neuroblastoma, soft tissue sarcoma, and small cell carcinoma of the lung. Immunization of mice with 1A7 generated the production of anti -GD2 antibodies. In a phase I clinical trial, immunization of patients with 1A7, mixed with the adjuvant QS21, demonstrated that 1A7 could act as a surrogate antigen for GD2 and induce strong humoral immune responses in advanced stage melanoma patients. DNA vaccines have recently been shown to invoke humoral as well as cellular responses in injected hosts against the transgene product. To evaluate the efficiency of DNA vaccines encoding anti -idiotype antibodies, we constructed expression plasmids encoding the variable heavy ( VH ) and variable light ( VL ) chains of 1A7. The plasmids were made in two configurations, expressing either the VH ( pc1A7VHLnVL ) or the VL ( pc1A7VLLnVH ) chain of 1A7 at the amino terminus, linked together by a 15 -amino acid linker ( Ln ) . In vitro transcription / translation assays and transfection of CHO -K1 cells with the plasmids demonstrated that a $30 -kDa protein was expressed by both configurations of the single -chain variable fragment. This protein can be specifically precipitated by monoclonal anti -GD2 antibody, 14G2a. Following intramuscular injection in mice, the plasmids were detectable in the injected tissues for at least 3 months and the injected plasmids actively transcribed the single -chain variable fragment 1A7 gene at the injected site. A single, intramuscular immunization of a group of C57BL / 6 mice with pc1A7VLLnVH in phosphate -buffered saline induced humoral immune responses against 1A7 as well as GD2, the nominal antigen. Multiple immunizations, however, were required to elicit stronger immune responses.

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Section: Discussionmentioning

confidence: 99%

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2

Zeytin

Tripathi

Bhattacharya‐Chatterjee

et al. 2000

Cancer Gene Ther

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“…Moreover, the uptake of scFv is significantly faster than that of Fab. 15) These previous observations suggest that the immunogene utilizing scFv and PEI has favorable features for rapid uptake and expression in A431 cells.…”

Section: Discussionmentioning

confidence: 99%

Targeted gene delivery using humanized single‐chain antibody with negatively charged oligopeptide tail

2004

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e have been developing a non-viral cell-specific gene delivery system named immunoporter, using a modified monoclonal antibody.1-6) The original immunoporter was a conjugate between a monoclonal antibody and a cationic peptide poly-L-lysine (pLys).1) The Fab fragment of antibody was also conjugated with pLys.3) These whole-antibody or Fab-fragment immunoporters readily bound to DNA and the resulting complex, named an immunogene, delivered a reporter gene or therapeutic gene(s) to target cells in vitro and in vivo.5) Most importantly, the Fab immunoporter carrying herpes simplex virus (HSV) thymidine kinase (TK) gene together with ganciclovir treatment was effective in suppression of the growth of epidermal growth factor (EGF) receptor-hyperproducing tumor cells in nude mice. 5) Thus, the immunoporter/immunogene approach was considered as a promising alternative to viral vectors for targeted gene delivery. However, the chemical conjugation of monoclonal antibody or Fab fragment is laborious and unsuitable for large-scale preparation, which would be required for future clinical use. To overcome this potential problem, we exploited recombinant single-chain antibody (scFv) technology.7) The scFv is regarded as the minimal structural component of an antibody required for antigen-binding activity, 7) and can be produced in high quantity by means of gene expression in Escherichia coli, 8) yeast 9) or mammalian cells. 10)Recently, we were able to construct several modified scFv genes for use as immunoporter/immunogene systems. 11) One of these recombinant immunoporters consisting of scFv and a negatively charged oligopeptide tail was capable of delivering genes to A431 tumor cells through the EGF receptor. 11)In this paper, we studied various conditions necessary for the formation of proper recombinant immunogenes. Moreover, we present evidence that recombinant immunogenes made with humanized scFv can be potent gene transfer vehicles to target particular tumor cells. Materials and MethodsCell culture. Human squamous carcinoma A431 cells over-expressing EGF receptors were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum at 37°C and 5% CO 2 .B4G7 monoclonal antibody. B4G7 antibody is an IgG2b class murine monoclonal antibody and is specific to human EGF receptor. 20,21) Plasmid preparation. pSRK-GL3 encodes luciferase (Luc) gene and pSRD-TK encodes HSV-TK gene.11) These recombinant plasmids were purified with Endfree QIAGEN Plasmid Purification Kit (QIAGEN).Purification of recombinant immunoporters. The murine type recombinant immunoporter (mBBD20) and humanized immunoporter (hCaCD20) were purified as previously described. 11)In brief, the immunoporters secreted by yeast Pichia pastris into the medium were purified by successive use of anion-exchange chromatography on "STREAMLINE" Q-XL (Amersham Pharmacia Biotech), immobilized metal ion-affinity chromatography and gel filtration. Finally, the immunoporters were dialyzed against 600 mM NaCl. The yields of mBBD20 and hCaCD20 wer...

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“…Accordingly, in vitro protein refolding is indispensable to obtain functional scFv, but this remains a formidable problem. 19) In this study, several refolding methods were attempted without success, the results suggesting that refolding under reducing conditions is not suitable for 3C10 recombinant scFv. Finally, relatively efficient production of immunoreactive 3C10 scFv was achieved with a procedure based primarily on disulfide restricted refolding.…”

Section: Discussionmentioning

confidence: 99%

Production of a Single‐chain Variable Fragment Antibody Recognizing Type III Mutant Epidermal Growth Factor Receptor

Nakayashiki

Yoshikawa

Nakamura

et al. 2000

Japanese Journal of Cancer Research

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The type III deletion mutant of the epidermal growth factor receptor (EGFR) is a potential target in diagnostic and therapeutic approaches for those glioblastomas characterized by its expression. We previously raised a mouse monoclonal antibody, 3C10 (IgG2b) specifically recognizing this mutant EGFR. In this study, a single-chain variable fragment (scFv) antibody was produced. Partial determination of its N-terminal amino acid sequence and preparation of adequate primers for variable heavy chain (V H ) and variable light chain (V L ) genes were performed to allow cloning by means of reverse transcriptase-polymerase chain reaction. The genes cloned were assembled with a linker, (Gly 4 Ser) 3 , and ligated into a bacterial expression vector to express the scFv as cytoplasmic inclusion bodies. After appropriate refolding, the antibody activity of the V H -V L scFv was examined in an enzyme-linked immunosorbent assay. 3C10 scFv showed a selective reactivity with the mutant peptide, similarly to the parental 3C10 antibody. A mouse transfectant expressing the type III mutant EGFR and a glioblastoma with type III deletion-mutant EGFR were positively stained by immunofluorescence. By Biacore analysis, the affinity (K A ) of the parental 3C10 for the mutant peptide was 9.7 × × × ×10, while that of 3C10 scFv was 2.45-2.48× × × ×10, being approximately 4-fold weaker. The results together suggested that the scFv antibody retained the appropriate structure to recognize a conformational epitope of the mutant receptor, similarly to the parental antibody.Key words: scFv -Epidermal growth factor receptor -Type III deletion mutant -GlioblastomaThe epidermal growth factor receptor (EGFR) gene is amplified and overexpressed in about 40% of glioblastoma cases. This amplification is frequently related to structural rearrangement, resulting in in-frame deletion mutations in the extracellular domain. Such deletions in EGFR have been classified into three types, based on size and location.1-7) Type III has been identified in about 17% of glioblastomas, and is characterized by an 801 bp in-frame deletion, which creates a unique sequence with a glycine residue at the fusion junction between amino acid residues 5 and 274. Since the sequence around the fusion junction is expressed only in glioblastoma cells, it is a potential target for diagnostic and therapeutic approaches. Accordingly, several laboratories including our own [8][9][10][11] have attempted to produce mouse monoclonal antibodies with specificity for the type III mutant. Wikstrand et al. 8) and Hills et al. 9) obtained antibodies by immunization with a synthetic peptide, named Pep3, covering the fusion junction of the type III deletion mutant which had been used by Humphrey et al. 10) for production of polyclonal rabbit anti-serum. Using the same peptide, we also succeeded in producing an antibody, 3C10, showing specificity for type III mutant EGFR. 11)With the recent advances in technology involving cloning of immunoglobulin (Ig) genes, generation of recombinant/chimeri...

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[3] Protein engineering of single-chain Fv analogs and fusion proteins

Cited by 198 publications

References 73 publications

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2

Construction and characterization of DNA vaccines encoding the single-chain variable fragment of the anti-idiotype antibody 1A7 mimicking the tumor-associated antigen disialoganglioside GD2

Targeted gene delivery using humanized single‐chain antibody with negatively charged oligopeptide tail

Production of a Single‐chain Variable Fragment Antibody Recognizing Type III Mutant Epidermal Growth Factor Receptor

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