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.1 and BA.2 variants. Cryoelectron microscopy (cryo-EM) analysis reveals that scFv76 binds to a well-conserved SARS-CoV-2 spike epitope, providing the structural basis for its broad-spectrum activity. We demonstrate that nebulized scFv76 has therapeutic efficacy in a severe hACE2 transgenic mouse model of coronavirus disease 2019 (COVID-19) pneumonia, as shown by body weight and pulmonary viral load data. Counteraction of infection correlates with inhibition of lung inflammation, as observed by histopathology and expression of inflammatory cytokines and chemokines. Biomarkers of pulmonary endothelial damage were also significantly reduced in scFv76-treated mice. The results support use of nebulized scFv76 for COVID-19 induced by any SARS-CoV-2 variants that have emerged so far.

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. In two independent animal models, intranasal administration counteracted the infection. Because of their high efficiency, remarkable stability, resilience to nebulization, and low cost of production, 76clAbs may become a relevant tool for rapid, self-administrable early intervention in SARS-CoV-2-infected subjects independently of their immune status.

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. A novel biotinylated gimatecan-derived camptothecin, coded ST8161AA1, was injected at suboptimal doses into human tumors xenografted in mice alone or pre-complexed to AvidinOX. Significantly higher growth inhibition was observed when the drug was anchored to AvidinOX suggesting the potential utility of this delivery modality for the local treatment of inoperable tumors.

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. Liberibacter asiaticus'. The primary library has more than 10(7) unique antibodies and the genes that encode them. We have screened this library for antibodies that bind to specifically-chosen proteins that are present on the surface of 'Ca. Liberibacter asiaticus'. These proteins were used as targets for affinity-based selection of scFvs that bind to the major outer membrane protein, OmpA; the polysaccharide capsule protein KpsF; a protein component of the type IV pilus (CapF); and, two flagellar proteins FlhA and FlgI. These scFvs have been used in ELISA and dot blot assays against purified protein antigens and 'Ca. Liberibacter asiaticus' infected plant extracts. We have also recloned many of these scFvs into a plasmid expression vector designed for the production of scFvs. Screening of these scFvs was more efficient when phage-bound, rather than soluble scFvs, were used. We have demonstrated a technology to produce antibodies at will and against any protein target encoded by 'Ca. Liberibacter asiaticus'. Applications could include advanced diagnostic methods for huanglongbing and the development of immune labeling reagents for in planta applications.

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. However, affinity selection against cell surface specific antigens within highly heterogeneous population of molecules is not a very efficient process that often results in the selection of unspecific antibodies or antibodies against intracellular antigens that are generally useless for targeted immunotherapy. In this work, we developed a new cell-based antibody selection protocol that, by eliminating the contamination of dead cells from the cell suspension, dramatically improves the selection frequency of anti-tumor antibodies recognizing cell surface 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. An anti-CEA single-chain antibody fragment and green fluorescent protein or alkaline phosphatase were simultaneously displayed by engineering both gpD and gpV lambda proteins. CONCLUSIONS: Here we show that such modified phage particles can be used for the detection of target molecules in vitro and in vivo. Dual expression of functional moieties on the surface of the lambda phage might open the way to generation of a new class of diagnostic and therapeutic targeted nanoparticles.

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. The biological properties of both phages turned out to be equally suitable for the production of antigen microarrays however in line with the plaque assay the sensitivity for the detection of various autoantibodies differed between the vectors. However, presumably due to the higher variability of the background signals in the microarray assay, it turned out to have comparable, in some cases even slightly lower sensitivity than the plaque assay. Next, we explored the repertoire of antigens that could be identified by screening T7 phage-displayed testis cDNA library with sera from melanoma patients. From the 243 antigens identified, only 24 represented known genes translated in their natural reading frame and included known TAAs like Annexin XI-A and a novel potential CT antigen SPAG8. Another 12 were uncharacterised genes but the remaining clones contained DNA fragments in non-natural reading frames that most likely represent mimotopes, nevertheless, they may turn out to be valid biomarkers.

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. METHODS: The ability of a patient's immune system to produce specific antibodies inside tumor tissue, capable of recognizing tumor cells, was explored through analysis of the oligoclonality of antibodies derived from tumor-infiltrating lymphocytes and construction of a series of recombinant antibody libraries in scFv format, derived from breast tumor-infiltrating B lymphocytes. These libraries and one from peripheral blood lymphocytes of a single breast cancer patient were panned against three purified surface tumor antigens, such as CEA, MUC1 and ED-B domain, and against intact MCF7 breast carcinoma cells. RESULTS: Application of novel display vector, pKM19, allowed isolation of a large panel of breast cancer-specific antibodies against known tumor antigens, as well as against breast carcinoma cells. Reactivity of novel scFvs was confirmed by ELISA, immunohistochemistry, fluorescence staining and flow cytometry. We demonstrated that seven of ten primary breast tumor specimens, obtained using discarded surgical material, could be exploited as an appropriate source for generation of phage display libraries, giving highly specific antitumor antibodies which recognize heterologous tumor cells. CONCLUSION: Local humoral immune response within tumor tissue in breast cancer patients frequently has an oligoclonal character. Efficient selection of specific antitumor antibodies from recombinant antibody libraries, derived from such oligoclonal tumor-infiltrated B lymphocytes, indicates the presence of natural immune response against tumor antigens in these patients. The described method is very promising for development of antitumor antibodies, potentially useful for diagnostic and therapeutic approaches.

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. In comparison with commonly used plasmids, this vector is designed to have relatively low expression levels of cloned scFv antibodies due to the amber codon positioned in a sequence encoding for the PhoA leader peptide. Moreover, fusion of antibodies to the carboxy terminal part only of the gene III protein improves display of scFv on bacteriophage surface in this system. Despite the lower antibody expression, the functional test performed with a new scFv library derived from human peripheral blood lymphocytes demonstrates that specific antibodies can be easily isolated from the library, even after the second selection round. The use of the pKM19 vector for maturation of an anti-CEA antibody significantly improves the final results. In our previous work, an analogous selection through the use of a phagemid vector, with antibody expression under the control of a lacP promoter, led to isolation of anti-CEA phage antibodies with improved affinities, which were not producible in soluble form. Probably due to the toxicity for E. coli of that particular anti-CEA antibody, 70% of maturated clones contained suppressed stop codons, acquired during various selection/amplification rounds. The pKM19 plasmid facilitates an efficient maturation process, resulting in selection of antibodies with improved affinity without any stop codons.

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. In applying this novel approach to breast cancer, we identified a large panel of cDNA fragments encoding for the well-known tumor-associated surface antigens, such as erb-B2, erbB3 and the urokinase receptor and, more importantly, for several clones overexpressed in breast cancer, whose cDNA fragments match the sequences of hypothetical transmembrane proteins with unknown function. The latter may represent novel tumor-specific targets.

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. One of these antigens (D7-1) is particularly interesting in that the antibody response against it grows with cancer progression from stages I through IV, with an incidence of 13.2, 13.5, 18.2 and 27%, respectively. The significance of this stage-dependent increase in the incidence is confirmed by the Mantel-Haenszel Chi-squared test (P=0.001). CONCLUSIONS: Our data confirm association between breast cancer diagnosis of patients and presence in their peripheral blood of antibodies against several autoantigens identified by phage display.

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. 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. RESULTS: The 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. CONCLUSION: The 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.

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. Efficient production of functional scFv exposed on lambda capsid with viable antigen binding specificity allowed us to study and compare the capacity of display, the stability of recombinant antibody expression and the assembly efficiency of bacteriophage particles decorated with recombinant antibody fused to the amino- or carboxy-terminus of lambda D protein.

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. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.

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. The cDNAs were expressed in the libraries as fusion to bacteriophage lambda protein D. Lambda-displayed libraries were efficiently screened with sera from patients with breast cancer. RESULTS: A panel of 21 clones representing 18 different antigens, including eight proteins of unknown function, was identified. Three of these antigens (T7-1, T11-3 and T11-9) were found to be overexpressed in tumors as compared to normal breast. A serological analysis of the 21 different antigens revealed a strong cancer-related profile for at least five clones (T6-2, T6-7, T7-1, T9-21 and T9-27). CONCLUSIONS: Preliminary results indicate that patient serum reactivity against five of the antigens is associated with tumor disease. The novel T7-1 antigen, which is overexpressed in breast tumors and recognized specifically by breast cancer patient sera, is potentially useful in cancer diagnosis.

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. The avidity index for Toxoplasma-specific antibodies against a homogeneous mixture of recombinant GRA3, GRA7, MIC3, and SAG1 antigens correlated closely with the IgG avidity of antibodies against lysed whole-cell T. gondii antigen. The avidity assay performed with the recombinant MIC3 antigen highlighted the presence of avidity low-antibodies IgG exclusively in sera collected within 2 months after primary infection. The presence of T. gondii-specific, low-avidity IgG antibodies against recombinant MIC3 antigen can be used to determine the point of infection with T. gondii within a 2-month time frame after infection.

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. We designed a new lambda vector to express protein fragments as N-terminal fusions to the D capsid protein and generated high-complexity cDNA libraries from human breast carcinoma cell lines and solid tumors. Screening these phage-displayed libraries required limited amounts of sera from patients and efficiently identified several tumor antigens specifically reacting with sera from breast cancer patients.

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. This approach identified a panel of recombinant phage clones carrying B-cell epitopes. All the peptide sequences selected by this procedure are regions of T. gondii gene products. These regions contain epitopes of the T. gondii antigens SAG1, GRA1, GRA7, GRA8 and MIC5, which are recognised by human immunoglobulins. Moreover, we report the isolation and characterisation of two additional immunodominant regions encoded by GRA3 and MIC3 genes, whose products have never been described as antigens of the human B-cell response against T. gondii infection. These results demonstrate potential of lambda-display technology for antigen discovery and for the study of the human antibody response against infectious agents.