Spike mutation resilient scFv76 antibody counteracts SARS-CoV-2 lung damage upon aerosol delivery.

The uneven worldwide vaccination coverage against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and emergence of variants escaping immunity call for broadly effective and easily deployable therapeutic agents. We have previously described the human single-chain scFv76 antibody, which recognizes SARS-CoV-2 Alpha, Beta, Gamma and Delta variants. We now show that scFv76 also neutralizes the infectivity and fusogenic activity of the Omicron BA.

Human inhalable antibody fragments neutralizing SARS-CoV-2 variants for COVID-19 therapy.

As of December 2021, coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), remains a global emergency, and novel therapeutics are urgently needed. Here we describe human single-chain variable fragment (scFv) antibodies (76clAbs) that block an epitope of the SARS-CoV-2 spike protein essential for ACE2-mediated entry into cells. 76clAbs neutralize the Delta variant and other variants being monitored (VBMs) and inhibit spike-mediated pulmonary cell-cell fusion, a critical feature of COVID-19 pathology.

Growth inhibition of human ovarian carcinoma by a novel AvidinOX-anchored biotinylated camptothecin derivative.

Oxidized form of avidin, named AvidinOX, provides stable fixation of biotinylated molecules in tissues thus representing a breakthrough in topical treatment of cancer. AvidinOX proved to be a stable receptor for radiolabeled biotin, biotinylated antibodies and cells. In order to expand applicability of the AvidinOX-based delivery platform, in the present study we investigated the possibility to hold biotinylated chemotherapeutics in AvidinOX-treated sites.

Development of single chain variable fragment (scFv) antibodies against surface proteins of 'Ca. Liberibacter asiaticus'.

'Candidatus Liberibacter asiaticus' is the causal agent of citrus huanglongbing, the most serious disease of citrus worldwide. We have developed and applied immunization and affinity screening methods to develop a primary library of recombinant single chain variable fragment (scFv) antibodies in an M13 vector, pKM19. The antibody population is enriched for antibodies that bind antigens of 'Ca.

Optimized selection of anti-tumor recombinant antibodies from phage libraries on intact cells.

Generation of human recombinant antibody libraries displayed on the surface of the filamentous phage and selection of specific antibodies against desirable targets allows production of fully human antibodies usable for repeated administration in humans. Various lymphoid tissues from immunized donors, such as lymph nodes or peripheral blood lymphocytes from individuals with tumor or lymphocytes infiltrating tumor masses may serve as a source of specific anti-tumor antibody repertoire for generation of tumor-focused phage display libraries. In the case of lack of tumor-associated antigens in the purified form, high affinity anti-tumor antibodies can be isolated through library panning on whole cells expressing these antigens.

Simultaneous display of two large proteins on the head and tail of bacteriophage lambda.

Background: Consistent progress in the development of bacteriophage lambda display platform as an alternative to filamentous phage display system was achieved in the recent years. The lambda phage has been engineered to display efficiently multiple copies of peptides or even large protein domains providing a powerful tool for screening libraries of peptides, proteins and cDNA.

Results: In the present work we describe an original method for dual display of large proteins on the surface of lambda particles.

Evaluation of T7 and lambda phage display systems for survey of autoantibody profiles in cancer patients.

In the current study we attempted to evaluate the suitability of T7 Select 10-3b and lambdaKM8 phage display systems for the identification of antigens eliciting B cell responses in cancer patients and the production of phage-displayed antigen microarrays that could be exploited for the monitoring of autoantibody profiles. Members of 15 tumour-associated antigen (TAA) families were cloned into both phage display vectors and the TAA mini-libraries were immunoscreened with 22 melanoma patients' sera resulting in the detection of reactivity against members of 5 antigen families in both systems, yet with variable sensitivity. T7 phage display system showed greater sensitivity for the detection of antibodies against members of CTAG, MAGEA and GAGE families, both systems showed equal performance in detecting the reactivity against MAGEC and SSX2 while only lambdaKM8 allowed the detection of anti-CTAGE5 antibodies.

Tumor-infiltrating B lymphocytes as an efficient source of highly specific immunoglobulins recognizing tumor cells.

Background: There is much evidence that tumor cells elicit a humoral immune response in patients. In most cases, the presence of antibodies in peripheral blood is detected only in small proportion of patients with tumors overexpressing the corresponding antigen. In the present study, we analyzed the significance of local humoral response provided by tumor-infiltrating lymphocytes in breast cancer patients.

New display vector reduces biological bias for expression of antibodies in E. coli.

We report the development of a novel phagemid vector, pKM19, for display of recombinant antibodies in single-chain format (scFv) on the surface of filamentous phage. This new vector improves efficacy of selection and reduces the biological bias against antibodies that can be harmful to host bacteria. It is useful for generation of large new antibody libraries, and for the subsequent maturation of antibody fragments.

A novel approach for identification of tumor-associated antigens expressed on the surface of tumor cells.

To improve tumor targeting in a subset of patients, where tumor cells do not express the well-known tumor antigens widely used in immunotherapy, we have developed a novel biotechnological tool. It is useful for tumors of various origins for the identification of tumor-associated proteins, which are differentially expressed in tumor cells with respect to normal tissue, and exposed on the cell surface. For this purpose, a combination of techniques, such as "suppression subtractive hybridization" and "transmembrane trapping," was employed.

A study of the humoral immune response of breast cancer patients to a panel of human tumor antigens identified by phage display.

Objective: In this article we provide evidence of a significant spontaneous humoral response in cancer patients.

Methods: A panel of tumor-associated antigens, previously identified through serological screening of phage-displayed cDNA libraries from solid human tumors, breast carcinoma cell lines and human testis by employing breast cancer patient sera, was used in this study to survey sera from 182 patients with known disease histories and clinical stages.

Results: This analysis reveals a statistically significant association between tumor disease and presence in peripheral blood of IgG antibodies against four autoantigens.

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

Background: CEA 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.

Methods: The 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.

Efficient display of scFv antibodies on bacteriophage lambda.

In the present work we demonstrate the efficient display of functional scFv antibodies on the bacteriophage lambda capsid. A single-chain (scFv) anti-CEA antibody gene was cloned in two different vectors to obtain fusion of the scFv antibody to the N- or C-terminus of the bacteriophage lambda capsid protein D (gpD). Lambda bacteriophage assembly occurs in the reducing environment of the cytoplasm; despite this the lambda-displayed anti-CEA antibody fragments retain the capacity to recognize the antigen, indicating correct single-chain antibody folding.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity.

Display libraries on bacteriophage lambda capsid.

Phage display is an established technology that has been successfully applied, in the last fifteen years, to projects aimed at deciphering biological processes and/or at the isolation of molecules of practical value in several diverse applications. Bacteriophage lambda, representing a molecular cloning and expression tool widely utilized since decades, has also been exploited to develop vectors for the display of libraries on its capsid. In the last few years, lambda display approach has been consistently offering new enthralling perspectives of technological application, such as domain mapping, antigen discovery, and protein interaction studies or, more generally, in functional genomics.

Identification of a panel of tumor-associated antigens from breast carcinoma cell lines, solid tumors and testis cDNA libraries displayed on lambda phage.

Background: Tumor-associated antigens recognized by humoral effectors of the immune system are a very attractive target for human cancer diagnostics and therapy. Recent advances in molecular techniques have led to molecular definition of immunogenic tumor proteins based on their reactivity with autologous patient sera (SEREX).

Methods: Several high complexity phage-displayed cDNA libraries from breast carcinomas, human testis and breast carcinoma cell lines MCF-7, MDA-MB-468 were constructed.

Use of an immunoglobulin G avidity assay based on recombinant antigens for diagnosis of primary Toxoplasma gondii infection during pregnancy.

The objective of this work was to develop an antibody-specific immunoglobulin G (IgG) avidity assay to discriminate between acute and latent phases of Toxoplasma gondii infection by using recombinant antigens. One hundred twenty-one serum samples from women who developed IgG antibodies against Toxoplasma during pregnancy were used. The IgG avidities of antibodies directed against epitopes carried by fragments of GRA3, GRA7, MIC3, and SAG1 antigens were measured by performing parallel enzyme immunoassays.

Identification of tumor-associated antigens by screening phage-displayed human cDNA libraries with sera from tumor patients.

Screening cDNA libraries from solid human tumors with sera of autologous patients (SEREX) has proven to be a powerful approach to identifying tumor antigens recognized by the humoral arm of the immune system. In many cases, application of this methodology has led to the discovery of novel tumor antigens as unknown gene products. We tried to improve the potency of the SEREX approach by combining it with phage-display technology.

Molecular dissection of the human B-cell response against Toxoplasma gondii infection by lambda display of cDNA libraries.

The disorders generated by Toxoplasma gondii infection are closely associated with the competence of the host immune system and both humoral and cell mediated immunity are involved in response to parasite invasion. To identify antigens implicated in human B-cell responses, we screened a phage-display library of T. gondii cDNA fragments with sera of infected individuals.

Identification of a human immunodominant B-cell epitope within the GRA1 antigen of Toxoplasma gondii by phage display of cDNA libraries.

Excreted secreted antigens of the protozoan parasite Toxoplasma gondii play a key role in stimulating the host immune system during acute and chronic infection. With the aim of identifying the immunodominant epitopes of T. gondii antigens involved in the human B-cell response against the parasite, we employed a novel immunological approach.

ADAM-HCV, a new-concept diagnostic assay for antibodies to hepatitis C virus in serum.

We screened phage libraries using sera from noninfected individuals and patients infected by hepatitis C virus (HCV). By applying different selection and maturation strategies, we identified a wide collection of efficient phage-borne ligands for HCV-specific antibodies. The selected ligands retained their antigenic properties when expressed as multimeric synthetic peptides.

'In vitro evolution' of ligands for HCV-specific serum antibodies.

We developed a strategy to improve the properties of ligands selected from phage-displayed random peptide libraries. A site-directed mutagenesis protocol that introduces mutations and extends the size of a target sequence has been set up to generate diversity in a single or in a population of clones. The pool of mutants thus created is screened to identify variants with the desired properties.

The SH3 domains of endophilin and amphiphysin bind to the proline-rich region of synaptojanin 1 at distinct sites that display an unconventional binding specificity.

The proline-rich domain of synaptojanin 1, a synaptic protein with phosphatidylinositol phosphatase activity, binds to amphiphysin and to a family of recently discovered proteins known as the SH3p4/8/13, the SH3-GL, or the endophilin family. These interactions are mediated by SH3 domains and are believed to play a regulatory role in synaptic vesicle recycling. We have precisely mapped the target peptides on human synaptojanin that are recognized by the SH3 domains of endophilins and amphiphysin and proven that they are distinct.

Binding specificity and in vivo targets of the EH domain, a novel protein-protein interaction module.

EH is a recently identified protein-protein interaction domain found in the signal transducers Eps15 and Eps15R and several other proteins of yeast nematode. We show that EH domains from Eps15 and Eps15R bind in vitro to peptides containing an asparagine-proline-phenylalanine (NPF) motif. Direct screening of expression libraries with EH domains yielded a number of putative EH interactors, all of which possessed NPF motifs that were shown to be responsible for the interaction.