Studies on anti-component 5 antibodies in animals infected with Trypanosoma cruzi

A competitive antibody enzyme immunoassay, using a monoclonal antibody against the species-specific Trypanosoma crnzi antigen 5, was used to investigate the presence of anti-component 5 antibodies in sera of opossums, dogs, rabbits and rats infected with this parasite. The sera from 51 Venezuelan patients with Chagas’disease were also tested. About 90% of the infected subjects showed significant levels of anti-component 5 antibodies. Nevertheless, these antibodies were not detected in the sera of dogs, rats and opossums infected with T. cruzl Some sera from infected rabbits presented significant results but close to the limit ofpositivity ofthe test. These findings suggest that the immune response in animals naturally or experimentally infected with T. cruzi is different from that observed in human Chagas’disease.

Prevention of respiratory syncytial virus infections

Respiratory syncytial virus is the most important cause of viral lower respiratory illness in infants and children worldwide. By the age of 2 years, nearly every child has become infected with respiratory syncytial virus and re-infections are common throughout life. Most infections are mild and can be managed at home, but this virus causes serious diseases in preterm children, especially those with bronchopulmonary dysplasia. Respiratory syncytial virus has also been recognized as an important pathogen in people with immunossupressive and other underlying medical problems and institutionalizated elderly, causing thousands of hospitalizations and deaths every year. The burden of these infections makes the development of vaccines for respiratory syncytial virus highly desirable, but the insuccess of a respiratory syncytial virus formalin-inactivated vaccine hampered the progress in this field. To date, there is no vaccine available for preventing respiratory syncytial virus infections, however, in the last years, there has been much progress in the understanding of immunology and immunopathologic mechanisms of respiratory syncytial virus diseases, which has allowed the development of new strategies for passive and active prophylaxis. In this article, the author presents a review about novel approaches to the prevention of respiratory syncytial virus infections, such as: passive immunization with human polyclonal intravenous immune globulin and humanized monoclonal antibodies (both already licensed for use in premature infants and children with bronchopulmonary dysplasia), and many different vaccines that are potential candidates for active immunization against respiratory syncytial virus.

Evaluation of a monoclonal antibody affinity purified antigen for zymodeme specific serological diagnosis of Trypanosoma cruzi infection

Theoretically, serological assays with affinity purified marker antigens can allow strain-specific diagnosis even when parasites cannot be retrieved from and infected host. A Trypanosoma cruzi antigen was purified by affinity chromatography using a zymodeme (Z) 2 specific monoclonal antibody (2E2C11). An indirect enzyme-linked immunosorbent assay (ELISA) based on the purified antigen could discriminate between sera from rabbits immunized with T. cruzi zymodeme clones but could not discriminate between sera from mice infected with different zymodemes.

A novel anti-CD19 monoclonal antibody (GBR 401) with high killing activity against B cell malignancies.

BACKGROUND: CD19 is a B cell lineage specific surface receptor whose broad expression, from pro-B cells to early plasma cells, makes it an attractive target for the immunotherapy of B cell malignancies. In this study we present the generation of a novel humanized anti-CD19 monoclonal antibody (mAb), GBR 401, and investigate its therapeutic potential on human B cell malignancies. METHODS: GBR 401 was partially defucosylated in order to enhance its cytotoxic function. We analyzed the in vitro depleting effects of GBR 401 against B cell lines and primary malignant B cells from patients in the presence or in absence of purified NK cells isolated from healthy donors. In vivo, the antibody dependent cellular cytotoxicity (ADCC) efficacy of GBR 401 was assessed in a B cell depletion model consisting of SCID mice injected with healthy human donor PBMC, and a malignant B cell depletion model where SCID mice are xenografted with both primary human B-CLL tumors and heterologous human NK cells. Furthermore, the anti-tumor activity of GBR 401 was also evaluated in a xenochimeric mouse model of human Burkitt lymphoma using mice xenografted intravenously with Raji cells. Pharmacological inhibition tests were used to characterize the mechanism of the cell death induced by GBR 401. RESULTS: GBR 401 exerts a potent in vitro and in vivo cytotoxic activity against primary samples from patients representing various B-cell malignancies. GBR 401 elicits a markedly higher level of ADCC on primary malignant B cells when compared to fucosylated similar mAb and to Rituximab, the current anti-CD20 mAb standard immunotherapeutic treatment for B cell malignancies, showing killing at 500 times lower concentrations. Of interest, GBR 401 also exhibits a potent direct killing effect in different malignant B cell lines that involves homotypic aggregation mediated by actin relocalization. CONCLUSION: These results contribute to consolidate clinical interest in developing GBR 401 for treatment of hematopoietic B cell malignancies, particularly for patients refractory to anti-CD20 mAb therapies.

Human polymeric IgA is superior to IgG and single-chain Fv of the same monoclonal specificity to inhibit urease activity associated with Helicobacter pylori.

Helicobacter-induced gastritis is considered nowadays an epidemic, the prevalence of which is one of the highest world-wide (70%), with as much as 40% of the population in industrialized countries. Helicobacter pylori (H. pylori) antigens (Ag) capable to elicit a protective immune response in animal models have been identified, but these antigens have not been shown to be strongly immunogenic when administered to humans. Due to their stability in the gastric environment and avidity, passive administration of secretory immunoglobulin A (SIgA) antibodies (Ab) targeting protective Ag might be particularly relevant as a substitute or complement to current therapies. To this aim, we have designed expression vectors to convert a scFv polypeptide specific for H. pylori urease subunit A into human IgG, polymeric IgA (IgAp/d) and SIgA. Purified proteins show proper binding characteristics toward both the native and denatured forms of H. pylori urease. The direct comparison between different isotype and molecular forms, but of unique specificity, demonstrates that SIgA and IgAp/d are more efficient in blocking free and H. pylori-associated urease than IgG and scFv. We conclude that the expression system reported herein will represent a valuable tool to produce human SIgA Ab of multiple specificities against H. pylori antigens involved in colonization and persistence.

Pulmonary biology of anti-interleukin 5 antibodies

Interleukin 5 (IL-5) is a critical cytokine for the maturation of eosinophil precursors to eosinophils in the bone marrow and those eosinophils then accumulate in the lungs during asthma. We have studied anti IL-5 antibodies on allergic responses in mice, guinea pigs and monkeys and are extending this experiment into humans with a humanized antibody. In a monkey model of pulmonary inflammation and airway hyperreactivity, we found that the TRFK-5 antibody blocked both responses for three months following a single dose of 0.3 mg/kg, i.v. This antibody also blocked lung eosinophilia in mice by inhibiting release from the bone marrow. To facilitate multiple dosing and to reduce immunogenicity in humans, we prepared Sch 55700, a humanized antibody against IL-5. Sch 55700 was also active against lung eosinophilia in allergic monkeys and mice and against pulmonary eosinophilia and airway hyperresponsiveness in guinea pigs. Furthermore, as opposed to steroids, Sch 55700 did not cause immunosuppression in guinea pigs. Studies with this antibody in humans will be critical to establishing the therapeutic potential of IL-5 inhibition.

Human Plasma-derived Polymeric IgA and IgM Antibodies Associate with Secretory Component to Yield Biologically Active Secretory-like Antibodies.

Immunotherapy with monoclonal and polyclonal immunoglobulin is successfully applied to improve many clinical conditions, including infection, autoimmune diseases, or immunodeficiency. Most immunoglobulin products, recombinant or plasma-derived, are based on IgG antibodies, whereas to date, the use of IgA for therapeutic application has remained anecdotal. In particular, purification or production of large quantities of secretory IgA (SIgA) for potential mucosal application has not been achieved. In this work, we sought to investigate whether polymeric IgA (pIgA) recovered from human plasma is able to associate with secretory component (SC) to generate SIgA-like molecules. We found that ∼15% of plasma pIgA carried J chain and displayed selective SC binding capacity either in a mixture with monomeric IgA (mIgA) or after purification. The recombinant SC associated covalently in a 1:1 stoichiometry with pIgA and with similar efficacy as colostrum-derived SC. In comparison with pIgA, the association with SC delayed degradation of SIgA by intestinal proteases. Similar results were obtained with plasma-derived IgM. In vitro, plasma-derived IgA and SIgA neutralized Shigella flexneri used as a model pathogen, resulting in a delay of bacteria-induced damage targeted to polarized Caco-2 cell monolayers. The sum of these novel data demonstrates that association of plasma-derived IgA or IgM with recombinant/colostrum-derived SC is feasible and yields SIgA- and SIgM-like molecules with similar biochemical and functional characteristics as mucosa-derived immunoglobulins.

Production, characterization, and application of antibodies against heat-labile type-I toxin for detection of enterotoxigenic Escherichia coli

Strains of enterotoxigenic Escherichia coli (ETEC) are responsible for significant rates of morbidity and mortality among children, particularly in developing countries. The majority of clinical and public health laboratories are capable of isolating and identifying Salmonella, Shigella, Campylobacter, and Escherichia coli O157:H7 from stool samples, but ETEC cannot be identified by routine methods. The method most often used to identify ETEC is polymerase chain reaction for heat-stable and heat-labile enterotoxin genes, and subsequent serotyping, but most clinical and public health laboratories do not have the capacity or resources to perform these tests. In this study, polyclonal rabbit and monoclonal mouse IgG2b antibodies against ETEC heat-labile toxin-I (LT) were characterized and the potential applicability of a capture assay was analyzed. IgG-enriched fractions from rabbit polyclonal and the IgG2b monoclonal antibodies recognized LT in a conformational shape and they were excellent tools for detection of LT-producing strains. These findings indicate that the capture immunoassay could be used as a diagnostic assay of ETEC LT-producing strains in routine diagnosis and in epidemiological studies of diarrhea in developing countries as enzyme linked immunosorbent assay techniques remain as effective and economical choice for the detection of specific pathogen antigens in cultures.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Cross-neutralization of four paramyxoviruses by a human monoclonal antibody.

Broadly neutralizing antibodies reactive against most and even all variants of the same viral species have been described for influenza and HIV-1 (ref. 1). However, whether a neutralizing antibody could have the breadth of range to target different viral species was unknown. Human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) are common pathogens that cause severe disease in premature newborns, hospitalized children and immune-compromised patients, and play a role in asthma exacerbations. Although antisera generated against either HRSV or HMPV are not cross-neutralizing, we speculated that, because of the repeated exposure to these viruses, cross-neutralizing antibodies may be selected in some individuals. Here we describe a human monoclonal antibody (MPE8) that potently cross-neutralizes HRSV and HMPV as well as two animal paramyxoviruses: bovine RSV (BRSV) and pneumonia virus of mice (PVM). In its germline configuration, MPE8 is HRSV-specific and its breadth is achieved by somatic mutations in the light chain variable region. MPE8 did not result in the selection of viral escape mutants that evaded antibody targeting and showed potent prophylactic efficacy in animal models of HRSV and HMPV infection, as well as prophylactic and therapeutic efficacy in the more relevant model of lethal PVM infection. The core epitope of MPE8 was mapped on two highly conserved anti-parallel β-strands on the pre-fusion viral F protein, which are rearranged in the post-fusion F protein conformation. Twenty-six out of the thirty HRSV-specific neutralizing antibodies isolated were also found to be specific for the pre-fusion F protein. Taken together, these results indicate that MPE8 might be used for the prophylaxis and therapy of severe HRSV and HMPV infections and identify the pre-fusion F protein as a candidate HRSV vaccine.

Current state of vaccine therapies in non-small-cell lung cancer.

A large variety of cancer vaccines have undergone extensive testing in early-phase clinical trials. A limited number have also been tested in randomized phase II clinical trials. Encouraging trends toward increased survival in the vaccine arms have been recently observed for 2 vaccine candidates in patients with non-small-cell lung cancer. These have provided the impetus for the initiation of phase III trials in large groups of patients with lung cancer. These vaccines target 2 antigens widely expressed in lung carcinomas: melanoma-associated antigen 3, a cancer testis antigen; and mucin 1, an antigen overexpressed in a largely deglycosylated form in advanced tumors. Therapeutic cancer vaccines aim at inducing strong CD8 and CD4 T-cell responses. The majority of vaccines recently tested in phase I clinical trials show efficacy in terms of induction of specific tumor antigen immunity. However, clinical efficacy remains to be determined but appears limited. Efforts are thus aimed at understanding the basis for this apparent lack of effect on tumors. Two major factors are involved. On one hand, current vaccines are suboptimal. Strong adjuvant agents and appropriate tumor antigens are needed. Moreover, dose, route, and schedule also need optimization. On the other hand, it is now clear that large tumors often present a tolerogenic microenvironment that hampers effective antitumor immunity. The partial understanding of the molecular pathways leading to functional inactivation of T cells at tumor sites has provided new targets for intervention. In this regard, blockade of cytotoxic T-lymphocyte antigen-4 and programmed death-1 with humanized monoclonal antibodies has reached the clinical testing stage. In the future, more potent cancer vaccines will benefit from intense research in antigen discovery and adjuvant agents. Furthermore, it is likely that vaccines need to be combined with compounds that reverse major tolerogenic pathways that are constitutively active at the tumor site. Developing these combined approaches to vaccination in cancer promises new, exciting findings and, at the same time, poses important challenges to academic research institutions and the pharmaceutical industry.

Nanomedicines for active targeting: physico-chemical characterization of paclitaxel-loaded anti-HER2 immunonanoparticles and in vitro functional studies on target cells.

Paclitaxel (Tx)-loaded anti-HER2 immunonanoparticles (NPs-Tx-HER) were prepared by the covalent coupling of humanized monoclonal anti-HER2 antibodies (trastuzumab, Herceptin) to Tx-loaded poly (dl-lactic acid) nanoparticles (NPs-Tx) for the active targeting of tumor cells that overexpress HER2 receptors. The physico-chemical properties of NPs-Tx-HER were compared to unloaded immunonanoparticles (NPs-HER) to assess the influence of the drug on anti-HER2 coupling to the NP surface. The immunoreactivity of sulfo-MBS activated anti-HER2 mAbs and the in vitro efficacy of NPs-Tx-HER were tested on SKOV-3 ovarian cancer cells that overexpress HER2 antigens. Tx-loaded nanoparticles (NPs-Tx) obtained by a salting-out method had a size of 171+/-22 nm (P.I.=0.1) and an encapsulation efficiency of about of 78+/-10%, which corresponded to a drug loading of 7.8+/-0.8% (w/w). NPs-Tx were then thiolated and conjugated to activated anti-HER2 mAbs to obtain immunonanoparticles of 237+/-43 nm (P.I.=0.2). The influence of the activation step on the immunoreactivity of the mAbs was tested on SKOV-3 cells using 125I-radiolabeled mAbs, and the activity of the anti-HER2 mAbs was minimally affected after sulfo-MBS functionalization. Approximately 270 molecules of anti-HER2 mAbs were bound per nanoparticle. NPs-Tx-HER exhibited a zeta potential of 0.2+/-0.1 mV. The physico-chemical properties of the Tx-loaded immunonanoparticles were very similar to unloaded immunonanoparticles, suggesting that the encapsulation of the drug did not influence the coupling of the mAbs to the NPs. No drug loss was observed during the preparation process. DSC analysis showed that encapsulated Tx is in an amorphous or disordered-crystalline phase. These results suggest that Tx is entrapped in the polymeric matrix and not adsorbed to the surface of the NPs. In vitro studies on SKOV-3 ovarian cancer cells demonstrated the greater cytotoxic effect of NPs-Tx-HER compared to other Tx formulations. The results showed that at 1 ng Tx/ml, the viability of cells incubated with drug encapsulated in NP-Tx-HER was lower (77.32+/-5.48%) than the viability of cells incubated in NPs-Tx (97.4+/-12%), immunonanoparticles coated with Mabthera, as irrelevant mAb (NPs-Tx-RIT) (93.8+/-12%) or free drug (92.3+/-9.3%).

Therapeutic targeting of tumor growth and angiogenesis with a novel anti-S100A4 monoclonal antibody

S100A4, a member of the S100 calcium-binding protein family secreted by tumor and stromal cells, supports tumorigenesis by stimulating angiogenesis. We demonstrated that S100A4 synergizes with vascular endothelial growth factor (VEGF), via the RAGE receptor, in promoting endothelial cell migration by increasing KDR expression and MMP-9 activity. In vivo overexpression of S100A4 led to a significant increase in tumor growth and vascularization in a human melanoma xenograft M21 model. Conversely, when silencing S100A4 by shRNA technology, a dramatic decrease in tumor development of the pancreatic MiaPACA-2 cell line was observed. Based on these results we developed 5C3, a neutralizing monoclonal antibody against S100A4. This antibody abolished endothelial cell migration, tumor growth and angiogenesis in immunodeficient mouse xenograft models of MiaPACA-2 and M21-S100A4 cells. It is concluded that extracellular S100A4 inhibition is an attractive approach for the treatment of human cancer.

A phase IIa randomised clinical study of GNbAC1, a humanised monoclonal antibody against the envelope protein of multiple sclerosis-associated endogenous retrovirus in multiple sclerosis patients.

BACKGROUND: GNbAC1 is an immunoglobulin (IgG4) humanised monoclonal antibody against multiple sclerosis-associated retrovirus (MSRV)-Env, a protein of endogenous retroviral origin, expressed in multiple sclerosis (MS) lesions, which is pro-inflammatory and inhibits oligodendrocyte precursor cell differentiation. OBJECTIVE: This is a randomised, double-blind placebo-controlled dose-escalation study followed by a six-month open-label phase to test GNbAC1 in MS patients. The primary objective was to assess GNbAC1 safety in MS patients, and the other objectives were pharmacokinetic and pharmacodynamic assessments. METHODS: Ten MS patients were randomised into two cohorts to receive a single intravenous infusion of GNbAC1/placebo at doses of 2 or 6 mg/kg. Then all patients received five infusions of GNbAC1 at 2 or 6 mg/kg at four-week intervals in an open-label setting. Safety, brain magnetic resonance imaging (MRI), pharmacokinetics, immunogenicity, cytokines and MSRV RNA expression were studied. RESULTS: All patients completed the study. GNbAC1 was well tolerated in all patients. GNbAC1 pharmacokinetics is dose-linear with mean elimination half-life of 27-37 d. Anti-GNbAC1 antibodies were not detected. Cytokine analysis did not indicate an adverse effect. MSRV-transcripts showed a decline after the start of treatment. Nine patients had stable brain lesions at MRI. CONCLUSION: The safety, pharmacokinetic profile, and pharmacodynamic responses to GNbAC1 are favourable in MS patients over a six-month treatment period.