Design and Use of a Phage Display Library

Pini, Alessandro; Viti, Francesca; Santucci, Annalisa; Bárbara, Cristina; Zardi, Luciano; Neri, Paolo; Neri, Dario

doi:10.1074/jbc.273.34.21769

Cited by 399 publications

(115 citation statements)

References 54 publications

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“…The resulting L19-UG and L19-UG-L19 constructs (Fig. 2a) were then cloned into the vector pcDNA3.1 and used to transfect CHO cells grown in ProCHO5 animal protein-free media (Lonza, Verviers, Belgium) to produce 5-10 mg/liter recombinant protein that can be efficiently purified either on ED-B (the antigen of L19) or protein-A affinity chromatography because the variable heavy region of immunoglobulin chain of L19 belongs to the subgroup III and thus contains protein A-binding sites (7,17). In SDS-PAGE both purified proteins migrate as homodimers in nonreducing conditions and as monomers in reducing conditions, showing apparent sizes of about 63 and 35 kDa, respectively, for the divalent format and of 124 and 62 kDa, respectively, for the tetravalent format.…”

Section: Resultsmentioning

confidence: 99%

“…We describe the use of UG for the production of a bivalent and tetravalent format of L19, an scFv specific for the angiogenesis-associated extra domain B (ED-B) of fibronectin (FN) (7), of an immunocytokine composed of IL2 and L19, and of a tetravalent dual specificity antibody composed of L19 and the scFv D2E7, a human antibody able to neutralize TNF-␣ activity (8). We report and discuss the characterization, properties, and the biological activity, both in vitro and in vivo, of these molecules.…”

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confidence: 99%

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Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

Ventura

Sassi

Fossati

et al. 2009

Journal of Biological Chemistry

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We report a novel strategy to engineer and express stable and soluble human recombinant polyvalent/polyspecific fusion proteins. The procedure is based on the use of a central skeleton of uteroglobin, a small and very soluble covalently linked homodimeric protein that is very resistant to proteolytic enzymes and to pH variations. Using a human recombinant antibody (scFv) specific for the angiogenesis marker domain B of fibronectin, interleukin 2, and an scFv able to neutralize tumor necrosis factor-␣, we expressed various biologically active uteroglobin fusion proteins. The results demonstrate the possibility to generate monospecific divalent and tetravalent antibodies, immunocytokines, and dual specificity tetravalent antibodies. Furthermore, compared with similar fusion proteins in which uteroglobin was not used, the use of uteroglobin improved properties of solubility and stability. Indeed, in the reported cases it was possible to vacuum dry and reconstitute the proteins without any aggregation or loss in protein and biological activity.The generation of recombinant polyvalent and/or polyspecific fusion proteins for use as components of novel drugs is still hindered by factors that limit their production, storage, and use, chief of which are issues related to instability and/or inadequate solubility. Here we describe a novel approach based on the use of uteroglobin (UG) 3 as a skeleton for the generation of polyvalent/polyspecific recombinant proteins. Human UG is a small (15.8 kDa) globular, nonglycosylated, and homodimeric secreted protein that was discovered independently by two groups in the 1960s in rabbit uterus (1, 2), and it is the first member of a new superfamily of proteins, the so-called Secretoglobins (Scgb) (3). UG is present in the blood at a concentration of about 15 g/ml and is found in urine and in other body fluids. The UG monomer is composed of about 70 amino acids, depending on the species, and is organized in a four ␣-helix secondary structure; the two subunits are joined in an antiparallel fashion by disulfide bridges established between two highly conserved cysteine residues in amino-and carboxyl-terminal positions (4) (see Fig. 1). The exact functions of UG are not yet clear, but the protein has been reported to have antiinflammatory properties due to its ability to inhibit the soluble phospholipase A 2 . Moreover, UG contains a central hydrophobic cavity able to accommodate hydrophobic molecules such as progesterone, retinol, and prostaglandin D 2 . Theoretically, this cavity could be loaded with different types of therapeutic hydrophobic substances and delivered to targets (for exhaustive reviews on UG, see Refs. 5, 6and references therein).The high solubility and stability of UG to pH and temperature variations, its resistance to proteases, and its homodimeric structure prompted us to consider the protein as a candidate linker for the generation of polyvalent and polyspecific recombinant proteins. We demonstrate here that the use of UG as a linker could provide a general method for...

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Section: Resultsmentioning

confidence: 99%

mentioning

confidence: 99%

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

Ventura

Sassi

Fossati

et al. 2009

Journal of Biological Chemistry

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“…Indeed, it may be anticipated that once the most relevant, accessible biomarkers specific for a patient's disease are identified, high-affinity ligands such as recombinant human antibodies and their fragments, can be prepared to assess the precise localization of the pathologic lesions and to selectively destroy them [37]. For instance, the human antibody L19, a specific ligand of the extracellular B domain of fibronectin, a biomarker of several cancer types [38,39], is currently being tested in clinical trials, both as an imaging tool (conjugated to radioactive iodine [40]) and as a therapeutic agent (fused with human interleukin-2 [41,42]). We believe that our innovative approach will promote the development of targeted therapies and may thus represent a significant step toward a clean and effective fight against diseases and particularly cancer.…”

Section: Discussionmentioning

confidence: 99%

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Castronovo¹,

Kischel²,

Guilhot³

et al. 2007

Proteomics

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Targeting of tumoral tissues is one of the most promising approaches to improve both the efficacy and safety of anticancer treatments. The identification of valid targets, including proteins specifically and abundantly expressed in cancer lesions, is of utmost importance. Despite stateof-the-art technologies, the discovery of cancer-associated target proteins still faces the limitation, in human tissues, of antigen accessibility to suitable high-affinity ligands such as human mAb bound to bioactive molecules. Terminal perfusion of tumor-bearing mice or ex vivo perfusion of human cancer-bearing organs with a reactive biotin ester solution has successfully led to the identification of novel accessible biomarkers. This methodology is however restricted to perfusable organs, and excludes most of the tissues of interest to targeted therapies, e.g. primary breast cancer and metastases. Herein, we report on the development of a new chemical proteomic method that bypasses the perfusion step and thus offers the potential to identify accessible molecular targets in virtually all types of animal and human tissues. We have validated our new procedure by identifying biomarkers selectively expressed in human breast carcinoma. Overall, this powerful technology may lay the ground not only for custom-made therapies in cancer, but also for the development of therapies that need to be selectively delivered in a specific tissue.

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“…The diversity is introduced by PCRs with DNA -oligonucleotides having degenerated codons at desired positions. This procedure has the advantage that variations are only allowed in positions essential for antigen binding and by choosing wellexpressed and well-folding frameworks (Pini et al, 1998;Knappik et al, 2000;Sö derlind et al, 2000). Both naïve and semi-synthetic antibody libraries have yielded antibodies with KD's down to the sub-nanomolar range, so both types of libraries -or combinations thereof -can be considered as sources for the selection of antibodies for functional genomics.…”

Section: Phage Antibody Libraries and Formatsmentioning

confidence: 99%

Perspectives for systematic in vitro antibody generation

Konthur

Hust

Dübel

2005

Gene

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After the completion and refinement of the human genome, the characterization of individual gene products in respect of their functions, their modifications, their cellular localization and regulation in both space and time has generated an increased demand for antibodies for their analysis. Taking into account that the human genome contains¨25,000 genes, and that their products are found in different splice variants and produce proteins with post-translational modifications, it can be estimated that at least 100,000 different protein products have to be investigated to gain a complete picture of what's going on in the proteome of a cell.Antibodies are preferred tools helping with the characterization and detection of proteins as well as with elucidating their individual functions. The generation of antibodies to all available human protein products by immunization and/or the hybridoma technology is not only logistically and financially enduring, but may prove to be a difficult task, as quite a number of interesting targets may evade the immune response of experimental animals, for example, allosteric variants dependent on fragile interactions to cofactors, highly conserved antigens etc. For this reason, alternative methods for the generation of antibodies have to supplement these approaches. In vitro methods for antibody generation are seen to offer this capability. In addition, they may provide a cost effective and large scale production alternative for detection reagents for the research community in their own right. Among in vitro techniques, phage display has been evolved as the most efficient option for tackling this problem and approaches optimised for automation are emerging.Maximum benefit for proteomic research could be generated by judicious and preferably international coordination of the ongoing efforts to combine the strengths of the well established animal based approaches and the novel opportunities offered by in vitro methods. D

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Design and Use of a Phage Display Library

Cited by 399 publications

References 54 publications

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

Use of Uteroglobin for the Engineering of Polyvalent, Polyspecific Fusion Proteins

Identification of specific reachable molecular targets in human breast cancer using a versatile ex vivo proteomic method

Perspectives for systematic in vitro antibody generation

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