Giorgio Fassina

Novel ligands for the affinity-chromatographic purification of antibodies

Affinity chromatography represents one of the most powerful fractionation techniques for the large-scale purification of biotechnological products. Despite its potential, the use of this methodology is limited by the availability of specific ligands for each target. Combinatorial chemistry and molecular modeling, often combined, have become interesting and innovative methods for generating novel ligands, tailored to specific biotechnological needs. One of the greatest area of application has been the discovery of novel ligands for the purification of antibodies, which represent an emerging but very important class of innovative therapeutic agents for the treatment of a vast array of diseases. Naturally available affinity ligands, such as Protein A or G for IgG purification or lectins for IgA and IgM purification, which are obtained from microorganisms or genetically modified bacteria through complex and expensive procedures, are not well suited for large-scale purification and require moreover time-consuming analytical controls to check for the presence of contaminants which may affect the safety of the purified antibody for clinical purposes. Recent results suggest that the application of combinatorial technologies and molecular modeling for the discovery of synthetic ligands may open new avenues for the development of more efficient, less expensive and--more importantly--safer procedures for antibody purification at the industrial level.

Immunoglobulin specificity of TG19318: a novel synthetic ligand for antibody affinity purification†

A synthetic ligand [TG19318], able to mimic protein A in the recognition of the immunoglobulin Fc portion, has been previously identified in our laboratory through the synthesis and screening of multimeric combinatorial peptide libraries. In this study we have fully characterized its applicability in affinity chromatography for the downstream processing of antibodies, examining the specificity and selectivity for polyclonal and monoclonal immunoglobulins derived from different sources. Ligand specificity was broader than protein A, since IgG deriving from human, cow, horse, pig, mouse, rat, rabbit, goat and sheep sera, IgY obtained from egg yolk, and IgM, IgA and IgE were efficiently purified on TG19318 affinity columns. Adsorbed antibodies were conveniently eluted by a buffer change to 0.1 M acetic acid or 0.1 M sodium bicarbonate pH 9, with full retention of immunological properties. Monoclonal antibodies deriving from cell culture supernatants or ascitic fluids were also conveniently purified on TG19318 affinity columns, even from very diluted samples. The affinity constant for the TG19318–IgG interaction was 0.3 µM, as determined by optical biosensor measurements. Under optimized conditions, antibody purity after affinity purification was close to 95%, as determined by densitometric scanning of SDS–PAGE gels of purified fractions, and maximal column capacity reached 25 mg Ig/ml support. In vivo toxicity studies in mice indicated a ligand oral toxicity greater than 2000 mg kg−1 while intravenous toxicity was close to 150 mg kg−1. Validation of antibody affinity purification processes for therapeutic use, a very complex, laborious and costly procedure, is going to be simplified by the use of TG19318, which could reduce considerably the presence of biological contaminants in the purified preparation, a very recurrent problem when using recombinant or extractive biomolecules as affinity ligands. Copyright

Protein A mimetic peptide ligand for affinity purification of antibodies.

A peptide mimicking protein A for its ability to recognize the Fc immunoglobulin portion has been identified through screening of a synthetic multimeric peptide library. Screening of the multimeric library, composed of randomized synthetic tripeptide tetramers, has been carried out using a very simple assay, measuring the library ability to interfere with the interaction between protein A and biotinylated immunoglobulins, monitored on solid phase using an enzyme‐linked immunosorbent assay format. The tetrameric tripeptide identified after three screening cycles was produced in larger amounts and then immobilized in high yield on preactivated solid support for the preparation of affinity columns, which proved useful for a very convenient one‐step purification of antibodies directly from crude sera. Antibody purity after affinity purification was close to 95 per cent, as determined by densitometric scanning of sodium dodecyl sulphate–polyacrylamide gel electrophoresis gels of purified fractions, and up to 2 mg of antibody could be purified from 1 ml of peptide‐derivatized affinity support. The ligand was stable to treatment with a vast array of sanitation agents, such as ethanol and 0.1 M sodium hydroxide, and to repeated use, thus making the ligand applicability extremely attractive for the purification of monoclonal antibodies for therapeutic use. Column binding selectivity was similar to that of protein A‐affinity columns, since immunoglobulin G from several sources (rabbit, goat, sheep, mouse) was conveniently purified, with no detection of leaked ligand fragments in the purified preparations.

Overexpression of squamous cell carcinoma antigen variants in hepatocellular carcinoma

Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (score⩽1) was associated to lower MIB-1 labelling index, compared to cases with SCCA score ⩾2 (mean±s.d.: 2%±2.4 vs 7.5%±10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G351 to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.Pathogenetic mechanisms of hepatocellular carcinoma (HCC) are still unclear and new tools for diagnostic and therapeutic purposes are ongoing. We have assessed whether squamous cell carcinoma antigen (SCCA), a serpin overexpressed in neoplastic cells of epithelial origin, is also expressed in liver cancer. Squamous cell carcinoma antigen was evaluated by immunohistochemistry in 65 HCCs of different aetiology and in 20 normal livers. Proliferative activity was assessed using MIB-1 antibody. In 18 surgical samples, tumour and nontumour liver tissue was available for SCCA cDNA amplification and sequencing. Squamous cell carcinoma antigen was detected in 55 out of 65 (85%) tumour specimens, but in none of the 20 controls. In the majority of the cases, the positive signal was found in the cytoplasm of more than 50% of the hepatocytes. Low or undetectable SCCA (score⩽1) was associated to lower MIB-1 labelling index, compared to cases with SCCA score ⩾2 (mean±s.d.: 2%±2.4 vs 7.5%±10.3, P<0.05). Squamous cell carcinoma antigen mRNA could be directly sequenced in 14 out of 18 liver tumours but in none of the corresponding nontumour samples. From sequence alignment, a novel SCCA1 variant (G351 to A) was identified in five cases, while SCCA1 was revealed in six cases and SCCA2 in three cases. In conclusion, SCCA variants are overexpressed in HCC, independently of tumour aetiology. A novel SCCA1 variant has been identified in one third of liver tumours.

Affinity purification of immunoglobulins from chicken egg yolk using a new synthetic ligand

Due to the peculiar composition of the egg yolk and the lack of specific affinity ligands, Y immunoglobulins are normally purified using complex and time consuming procedures involving a combination of precipitation and chromatographic steps first to extract and capture and then to polish IgY. In this study, we have examined the applicability for IgY affinity purification of TG19318, a synthetic ligand for immunoglobulin, obtained from the screening of combinatorial libraries, and already characterized for its capability to purify immunoglobulins of class G, M, E and A. Soluble proteins were separated from the lipidic fraction of egg yolk by the water dilution method and loaded on to TG19318 affinity columns prepared by immobilizing the ligand on the commercially available support Emphaze. In a single chromatographic step TG19318 affinity columns led to an efficient capture of IgY directly from crude samples, and with a purity degree higher than 90%, as determined by densitometric scanning of SDS-PAGE analysis of bound fractions, and with full recovery of antibody activity, as determined by ELISA assay. Higher recovery and purity of IgY was obtained by using loading buffers at pH close to 6.5. Column capacity, determined by applying 4x excess IgY to 1 ml bed volume column, and eluting the retained immunoglobulins, was close to 65 mg of IgY per ml of resin. Chemical and chromatographic stability of TG19318/Emphaze was tested before and after various treatments. The derivatized matrix was found to be very stable, in terms of ligand leakage and maintenance of IgY binding capacity, under conditions of normal column usage, cleaning and storage.

Prevention of systemic lupus erythematosus in MRL/lpr mice by administration of an immunoglobulin-binding peptide.

Systemic lupus erythematosus (SLE) is a multisystem chronic inflammatory disease of unknown etiology that affects many organs, including the kidney. The presence of multiple autoantibodies and other immunological abnormalities point to basic defects in immunoregulatory controls that normally maintain self-tolerance. The deposition on kidney tissue of autoantibodies as immune complexes (ICs) through the interaction with Fc-receptor γ-chains is thought to trigger an inflammatory response typical of SLE, leading to glomerulonephritis. Using combinatorial chemistry approaches, we have identified a peptide able to bind to immunoglobulins and to interfere with Fcγ-receptor recognition. Administration of this peptide to MRL/lpr mice, an animal model used to study SLE, resulted in a remarkable enhancement of the survival rate (80%) compared to placebo-treated animals (10%). Consistent with this was a significant reduction of proteinuria, a clinical sign of SLE. Kidney histological examination of treated animals confirmed the preservation of tissue integrity and a remarkable reduction in IC deposition. These results support the role of Fcγ receptors in SLE pathogenesis and open new avenues for the development of drugs to treat autoimmune disorders.

Affinity purification of immunoglobulin M using a novel synthetic ligand.

While monoclonal antibodies of the G class can be conveniently purified by affinity chromatography using immobilized protein A or G, even on a large scale, scaling up IgM purification still presents several problems, since specific and cost-effective ligands for IgM are not available. A synthetic peptide (TG19318), deduced from the screening of a combinatorial peptide library, was characterized previously by our group for its binding properties for immunoglobulins of the G class and its applicability as a synthetic ligand for polyclonal and monoclonal IgG purification, from sera or cell culture supernatants. In this study, we have examined the ligand recognition properties for IgM, immobilizing the synthetic peptide on different affinity supports and examining its ability to purify IgMs from serum, ascitic fluid and cell culture supernatants. TG19318 affinity columns proved useful for a very convenient one-step purification of monoclonal IgMs directly from crude sources, loading the samples on the columns equilibrated with saline buffers at pH values ranging from 5 to 7, and eluting adsorbed IgM by a buffer change to 0.1 M acetic acid or 0.05-0.1 M sodium bicarbonate, pH 9.0. Antibody purity after affinity purification was very high, close to 85-95%, as determined by densitometric scanning of sodium dodecyl sulfate-polyacrylamide gels of purified fractions, and by gel permeation analysis. Antibody activity was fully recovered after purification, as determined by immunoassays. Column capacity was related to the type of support used for ligand immobilization, and ranged from 2 to 8 mg of IgM/ml of support.

SERPINB3 induces epithelial–mesenchymal transition†

Epithelial–mesenchymal transition is believed to facilitate invasion and metastasis formation of epithelial tumour cells. SERPINB3 is a serine protease inhibitor, physiologically found in normal squamous epithelium but over‐expressed in epithelial tumours and known to inhibit apoptosis. We tested the hypothesis that SERPINB3 has a role in invasion by modulating the epithelial–mesenchymal transition programme, using morphological, molecular and cell biology techniques on HepG2 cell clones transfected with the human SERPINB3 gene. The paracrine effect of this serpin was determined by the addition of exogenous recombinant SERPINB3 protein to HepG2 and MDCK cell line. SERPINB3 expression leads to changes in transfected cells morphology, characterized by clusters of loosely connected cells with elongated shape. Ultrastructural analysis confirmed the decrease of desmosomal junctions and widening of intercellular spaces. These alterations were associated with a reduction of E‐cadherin and an increase of β‐catenin, with a parallel increase of cell proliferation. SERPINB3 clones, untransfected HepG2 and MDCK cells treated with exogenous SERPINB3 expressed vimentin, undetectable in controls. SERPINB3 induced significant cell scattering, migration and invasiveness in untransfected cells. These effects were not dependent on the anti‐protease activity of the protein, as documented by the results obtained with an active loop‐deleted recombinant SERPINB3 protein. Scatter activity was inhibited by an anti‐SERPINB3 antibody in a dose‐dependent manner and SERPINB3‐transfected cells formed a significantly higher number of colonies on soft agar than controls. In conclusion, the observed results indicate that SERPINB3 induces deregulation of adhesion processes and increases the invasiveness potential supported by features of epithelial–mesenchymal transition, acting at both the autocrine and the paracrine level. Copyright

Affinity purification of mouse monoclonal IgE using a protein A mimetic ligand (TG19318) immobilized on solid supports.

A synthetic ligand (TG19318), deduced from the screening of a combinatorial peptide library, has been previously characterized by our group for its applicability in affinity chromatography for polyclonal and monoclonal IgG purification from crude sources. In this study we have extended the characterization of its recognition properties for other immunoglobulin classes, evaluating its ability to purify mouse monoclonal IgE from ascitic fluid. TG19318 affinity columns proved useful for a very convenient one‐step purification of IgE directly from crude ascites, by loading the samples on the columns equilibrated with 50 mM sodium phosphate at pH 7 and eluting the adsorbed IgE by a buffer change to 0.1 M acetic acid. Antibody purity after affinity purification was very high and no albumin traces were detected, as determined by SDS– PAGE analysis. Antibody activity was fully recovered after purification, as determined by immunoassays on antigen‐coated plates, and up to 5 mg of IgEs could be purified on a 1 ml column in a single run. Copyright

De novo design of peptides targeted to the EF hands of calmodulin.

This report describes the use of the concept of inversion of hydropathy patterns to the de novo design of peptides targeted to a predetermined site on a protein. Eight- and 12-residue peptides were constructed with the EF hands or Ca2+-coordinating sites of calmodulin as their anticipated points of interaction. These peptides, but not unrelated peptides nor those with the same amino acid composition but a scrambled sequence, interacted with the two carboxyl-terminal Ca2+-binding sites of calmodulin as well as the EF hands of troponin C. The interactions resulted in a conformational change whereby the 8-mer peptide-calmodulin complex could activate phosphodiesterase in the absence of Ca2+. In contrast, the 12-mer peptide-calmodulin complex did not activate phosphodiesterase but rather inhibited activation by Ca2+. This inhibition could be overcome by high levels of Ca2+. Thus, it would appear that the aforementioned concept can be used to make peptide agonists and antagonists that are targeted to predetermined sites on proteins such as calmodulin.