Alessandro Ascione

Selection, affinity maturation, and characterization of a human scFv antibody against CEA protein

BackgroundCEA is a tumor-associated antigen abundantly expressed on several cancer types, including those naturally refractory to chemotherapy. The selection and characterization of human anti-CEA single-chain antibody fragments (scFv) is a first step toward the construction of new anticancer monoclonal antibodies designed for optimal blood clearance and tumor penetration.MethodsThe human MA39 scFv, selected for its ability to recognize a CEA epitope expressed on human colon carcinomas, was first isolated from a large semi-synthetic ETH-2 antibody phage library, panned on human purified CEA protein. Subsequently, by in vitro mutagenesis of a gene encoding for the scFv MA39, a new library was established, and new scFv antibodies with improved affinity towards the CEA cognate epitope were selected and characterized.ResultsThe scFv MA39 antibody was affinity-maturated by in vitro mutagenesis and the new scFv clone, E8, was isolated, typed for CEA family member recognition and its CEACAM1, 3 and 5 shared epitope characterized for expression in a large panel of human normal and tumor tissues and cells.ConclusionThe binding affinity of the scFv E8 is in a range for efficient, in vivo, antigen capture in tumor cells expressing a shared epitope of the CEACAM1, 3 and 5 proteins. This new immunoreagent meets all criteria for a potential anticancer compound: it is human, hence poorly or not at all immunogenic, and it binds selectively and with good affinity to the CEA epitope expressed by metastatic melanoma and colon and lung carcinomas. Furthermore, its small molecular size should provide for efficient tissue penetration, yet give rapid plasma clearance.

The glutathione S-transferase inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol overcomes the MDR1-P-glycoprotein and MRP1-mediated multidrug resistance in acute myeloid leukemia cells.

PurposeThere has been an ever growing interest in the search for new anti-tumor compounds that do not interact with MDR1-Pgp and MRP1 drug transporters and so circumvent the effect of these proteins conferring multidrug resistance (MDR) and poor prognosis in AML patients. We have investigated the cytotoxic activity of the strong glutathione S-transferase (GST) inhibitor 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX) on AML (HL60) cell lines.MethodsFunctional drug efflux studies and cell proliferation assays were performed on both sensitive and MDR AML (HL60) cells after incubation with NBDHEX. Moreover, the mode of cell death (apoptosis vs. necrosis) as well as the correlation between NBDHEX susceptibility and GST activity or Bcl-2 expression was investigated.ResultsNBDHEX is not a substrate of either MDR1-Pgp or MRP1 efflux pumps; in fact, it is not only cytotoxic toward the parental HL60 cell line, but also overcomes the MDR phenotype of its HL60/DNR and HL60/ADR variants.ConclusionsThe data herein reported show that NBDHEX mediates efficient killing of both MDR1-Pgp and MRP1 over-expressing AML cells. Therefore, this drug can potentially be used as an effective agent for treating MDR in AML patients.

Generation of human single-chain antibody to the CD99 cell surface determinant specifically recognizing Ewing's sarcoma tumor cells.

The survival of pediatric patients with cancer entities including osteosarcoma and Ewings sarcoma (ES), remains extremely low hence novel treatment approaches are urgently needed. Therefore, based on the concept of targeted therapy, numerous potential targets for the treatment of these cancers have been evaluated pre-clinically or in some cases even clinically during the last decade. In ES the CD99 protein is an attractive target antigen. In this respect, a new entry site for therapeutic intervention may derive from specific human antibodies against CD99. Human scFvC7 was isolated from a semi-synthetic ETH-2 antibody phage library panned on the extracellular portion of recombinant human CD99 protein. The scFvC7 was genetically sequenced, tested for CD99 recognition on an array of recombinant CD99 fragments and measured for binding affinity by ELISA. Finally, it was tested for staining CD99 antigen on a large panel of tumor and normal cells and tissues by cytofluorimetric and immunohistochemical assays. The new antibody scFvC7 recognizes the CD99 extracellular domain included between residues 50 and 74 with a binding affinity of 2.4 x 10(-8) M. In contrast with all other antibodies to CD99 so far isolated, scFvC7 shows a unique specificity in cancer cell recognition: It stained prevalently ES cells while no or weak reactivity was observed on the majority of the other tumor and normal cells and tissues. Thanks to its properties the new anti-CD99 antibody here described represents the first step towards the construction of new selective ES therapeutics.

Generation of human scFvs antibodies recognizing a prion protein epitope expressed on the surface of human lymphoblastoid cells

BackgroundA hallmark of prion disease is the transformation of normal cellular prion protein (PrPc) into an infectious disease-associated isoform, (PrPsc). Anti-prion protein monoclonal antibodies are invaluable for structure-function studies of PrP molecules. Furthermore recent in vitro and in vivo studies indicate that anti-PrP monoclonal antibodies can prevent the incorporation of PrPc into propagating prions.In the present article, we show two new human phage antibodies, isolated on recombinant hamster prion protein (rHaPrP).ResultsWe adopted an antibody phage display strategy to isolate specific human antibodies directed towards rHaPrP which has been used as a bait for panning the synthetic ETH-2 antibody phage library. Two phage antibodies clones named MA3.B4 and MA3.G3 were isolated and characterized under genetic biochemical and immunocytochemical aspects. The clones were found to recognize the prion protein in ELISA studies. In flow-cytometry studies, these human single chain Fragment variable (scFv) phage-antibodies show a well defined pattern of reactivity on human lymphoblastoid and myeloid cells.ConclusionSequence analysis of the gene encoding for the antibody fragments and antigen recognition patterns determined by flow-cytometry analysis indicate that the isolated scFvs recognize novel epitopes in the PrPc molecule. These new anti PrPc human antibodies are unique reagents for prion protein detection and may represent a biologic platform to develop new reagents to treat PrPsc associated disease.

Generation of human antibody fragments recognizing distinct epitopes of the nucleocapsid (N) SARS-CoV protein using a phage display approach

BackgroundSevere acute respiratory syndrome (SARS)-CoV is a newly emerging virus that causes SARS with high mortality rate in infected people. Successful control of the global SARS epidemic will require rapid and sensitive diagnostic tests to monitor its spread, as well as, the development of vaccines and new antiviral compounds including neutralizing antibodies that effectively prevent or treat this disease.MethodsThe human synthetic single-chain fragment variable (scFv) ETH-2 phage antibody library was used for the isolation of scFvs against the nucleocapsid (N) protein of SARS-CoV using a bio panning-based strategy. The selected scFvs were characterized under genetics-molecular aspects and for SARS-CoV N protein detection in ELISA, western blotting and immunocytochemistry.ResultsHuman scFv antibodies to N protein of SARS-CoV can be easily isolated by selecting the ETH-2 phage library on immunotubes coated with antigen. These in vitro selected human scFvs specifically recognize in ELISA and western blotting studies distinct epitopes in N protein domains and detect in immunohistochemistry investigations SARS-CoV particles in infected Vero cells.ConclusionThe human scFv antibodies isolated and described in this study represent useful reagents for rapid detection of N SARS-CoV protein and SARS virus particles in infected target cells.

Generation and characterization of a human single-chain fragment variable (scFv) antibody against cytosine deaminase from Yeast

BackgroundThe ability of cytosine deaminase (CD) to convert the antifungal agent 5-fluorocytosine (5-FC) into one of the most potent and largely used anticancer compound such as 5-fluorouracil (5-FU) raised considerable interest in this enzyme to model gene or antibody – directed enzyme-prodrug therapy (GDEPT/ADEPT) aiming to improve the therapeutic ratio (benefit versus toxic side-effects) of cancer chemotherapy. The selection and characterization of a human monoclonal antibody in single chain fragment (scFv) format represents a powerful reagent to allow in in vitro and in vivo detection of CD expression in GDEPT/ADEPT studies.ResultsAn enzymatic active recombinant CD from yeast (yCD) was expressed in E. coli system and used as antigen for biopanning approach of the large semi-synthetic ETH-2 antibody phage library. Several scFvs were isolated and specificity towards yCD was confirmed by Western blot and ELISA. Further, biochemical and functional investigations demonstrated that the binding of specific scFv with yCD did not interfere with the activity of the enzyme in converting 5-FC into 5-FU.ConclusionThe construction of libraries of recombinant antibody fragments that are displayed on the surface of filamentous phage, and the selection of phage antibodies against target antigens, have become an important biotechnological tool in generating new monoclonal antibodies for research and clinical applications. The scFvH5 generated by this method is the first human antibody which is able to detect yCD in routinary laboratory techniques without interfering with its enzymatic function.

Human monoclonal antibodies in single chain fragment variable format with potent neutralization activity against influenza virus H5N1

Effective diagnostic and therapeutic strategies are needed to control and combat the highly pathogenic avian influenza virus (AIV) subtype H5N1. To this end, we developed human monoclonal antibodies (mAbs) in single chain fragment variable (scFv) format towards the H5N1 avian influenza virus to gain new insights for the development of immunotherapy against human cases of H5N1. Using a biopanning based approach a large array of scFvs against H5N1 virus were isolated from the human semi-synthetic ETH-2 phage antibody library. H5N1 ELISA-positive scFvs with unique variable heavy (VH) and light (VL) chain gene sequences showed different biochemical properties and neutralization activity across H5N1 viral strains. In particular, the scFv clones AV.D1 and AV.C4 exerted a significant inhibition of the H5N1 A/Vietnam/1194/2004 virus infection in a pseudotype-based neutralization assay. Interestingly, these two scFvs displayed a cross-clade neutralizing activity versus A/whooping swan/Mongolia/244/2005 and A/Indonesia/5/2005 strains. These studies provide proof of the concept that human mAbs in scFv format with well-defined H5N1 recognition patterns and in vitro neutralizing activity can be easily and rapidly isolated by biopanning selection of an entirely artificial antibody repertoire using inactivated H5N1 virus as a bait.

Genetic construction, expression, and characterization of a single chain anti-CEA antibody fused to cytosine deaminase from yeast

We report the genetic construction and expression of a fusion protein between a single chain fragment variable (scFv) human antibody (E8) specific for CEA cell surface antigen and yeast cytosine deaminase (yCD). Sequences encoding for the scFvE8 human monoclonal antibody recognizing an epitope shared by CEACAM1, CEACAM3 and CEACAM5 isoforms were assembled with a monomer of yCD. The construct was placed under the transcriptional regulation of the lac promoter, and in frame with 6xHis tag for protein purification. After transformation and induction of E. coli, the protein was recovered from cell lysates and processed for purification. The scFvE8:yCD fusion protein possessed the binding specificity for melanoma (Mel P5) and colon carcinoma (LoVo) cell lines similar to its cognate human scFv antibody. The scFv8:yCD system showed the ability to render tumor cells susceptible to the far less toxic substrate 5-fluorocytosine (5-FC) by its enzymatic conversion into 5-fluorouracil (5-FU). In vitro pre-treatment of Mel P5 and LoVo cell lines with scFvE8:yCD followed by cell washing and incubation with 5-FC, resulted in significant cell killing supporting the utility of this fusion protein as an agent for tumor-selective prodrug activation. This study shows the feasibility of constructing fusion proteins in a prokaryotic cell based system consisting of a human scFv antibody and yCD to convert the antifungal agent 5-FC to 5-FU, one of the widely used anticancer agent.

CEACAM1 is a Privileged Cell Surface Antigen to Design Novel ScFv Mediated-Immunotherapies of Melanoma, Lung Cancer and Other Types of Tumors

Carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1) is a cell surface glycoprotein involved in intercellular binding, belonging to the immunoglobulin superfamily. It is involved in cell-cell recognition and modulates cellular processes that range from vascular angiogenesis to the regulation of insulin homeostasis and T-cell proliferation. Aberrant expression of CEACAM1 is often associated with progression and metastatic potential in melanoma, lung carcinoma and other types of tumor. Tumor-specific antigens such as CEACAM1 are ideal targets for cancer immunotherapy because they are over-expressed by the cancer cell and not on non-malignant tissues, minimizing the risk of autoimmune destruction. Many of the limitations of therapeutic use of rodent monoclonal antibodies (mAbs) can now be overcome by exploiting the use of recombinant antibody fragments and the advances in antibody engineering methods to improve tumor retention, reduce immunogenicity and modulate pharmacokinetics. In addition, a novel effective model of immunotherapeutic treatments of tumors includes antibody drug conjugates (ADCs) that combine specific mAbs and antibody fragments with cytotoxic drugs, proteins, enzymes, radionuclides and nanoparticles. This review aims to describe how these antibody engineering approaches can meet the challenges for generating new and effective antibody constructs for diagnosis and therapy of CEACAM1 expressing malignancies.

Identification by phage display of the linear continuous MRPr1 epitope in the multidrug resistance-associated protein (MRP1).

Abstract In order to study the structure of the multidrug resistance-associated protein (MRP1), which is one of the most important members of the ATP-binding cassette (ABC) protein family acting as drug-efflux systems, we have developed an epitope mapping-based strategy. By means of the mAb MRPr1, we have immunoselected clones from two distinct random peptide libraries displayed on phages and have identified several peptide sequences mimicking the internal conformation of this 190 kDa multidrug transporter protein. Phage clones able to block the immunolabeling of the MRPr1 antibody to MRP1-overexpressing multidrug resistance (MDR) H69/AR cells were isolated and, after sequencing the corresponding inserts, their amino acid sequence was compared to that of MRP1. This analysis led to the identification of the consensus sequence L.SLNWED, corresponding to the MRP1 segment LWSLNKED (residues 241–248). This MRP1sequence is partially overlapping with the MRPr1 epitope GSDL WSLNKE (residues 238–247) previously mapped using peptide scanning techniques. These results demonstrate the high reliability of phage display technology to study not only the topography of complex integral membrane proteins such as MRP1, but also to help identify critical residues participating in the formation of the epitope structure.