Enhanced opsonisation of Rhesus D-positive human red blood cells by recombinant polymeric immunoglobulin G anti-G antibodies.

BACKGROUND: Anti-RhD antibodies (anti-D) are important in the prophylaxis of haemolytic disease of the foetus and newborn (HDFN) due to RhD incompatibility. Current preparations of anti-D are sourced from hyperimmune human plasma, so its production carries a risk of disease and is dependent on donor availability. Despite the efforts to develop a monoclonal preparation with similar prophylactic properties to the plasma-derived anti-D, no such antibody is yet available. Here we studied the agglutinating, opsonic and haemolytic activities of two recombinant polymeric immunoglobulins (Ig) against the G antigen of the Rh complex. MATERIALS AND METHODS: Recombinant polymeric anti-G IgG1 (IgG1µtp) and IgG3 (IgG3µtp) were produced in vitro, purified by protein G-affinity chromatography, and analysed by gel electrophoresis. Their agglutinating, opsonic and haemolytic activities were evaluated using haemagglutination, erythrophagocytosis, and complement activation assays. RESULTS: The recombinant IgG1µtp and IgG3µtp anti-G antibodies ranged from 150,000 to 1,000,000 Da in molecular weight, indicating the formation of polymeric IgG. No complement activation or haemolytic activity was detected upon incubation of RhD-positive red-blood cells with the polymeric anti-G IgG. Both polymers were better opsonins than a prophylactic preparation of plasma-derived anti-D. DISCUSSION: The enhanced opsonic properties of the polymeric anti-G IgG1µtp and IgG3µtp could allow them to mediate the clearance of RhD-positive red blood cells from circulation more efficiently than natural or other synthetic prophylactic anti-D options. Their inability to induce complement-mediated haemolysis would be prophylactically convenient and is comparable in vitro to that of the available plasma-derived polyclonal anti-D preparations. The described properties suggest that polymeric antibodies like these (but with anti-D specificity) may be testable candidates for prophylaxis of HDFN caused by anti-D.

CTLA4Fcε, a novel soluble fusion protein that binds B7 molecules and the IgE receptors, and reduces human in vitro soluble CD23 production and lymphocyte proliferation.

Immunoglobulin E-mediated allergy and certain autoimmune diseases are characterized by the presence of a T helper type 2 (Th2) immune response and allergen-specific or self-reactive IgE. Soluble CD23 (sCD23) is a B-cell factor that fosters IgE class-switching and synthesis, suggesting that sCD23 may be a therapeutic target for these pathologies. We produced a recombinant protein, CTLA4Fcε, by fusing the ectodomain of the immunoregulatory molecule cytotoxic T-lymphocyte antigen 4 (CTLA-4) with a fragment of the IgE H-chain constant region. In SDS-PAGE/inmunoblot analyses, CTLA4Fcε appeared as a 70,000 MW polypeptide that forms homodimers. Flow cytometry showed that CTLA4Fcε binds to IgE receptors FcεRI and FcεRII/CD23, as well as to CTLA-4 counter-receptors CD80 and CD86. Binding of CTLA4Fcε to FcεRII/CD23 appeared stronger than that of IgE. Since the cells used to study CD23 binding express CD80 and CD86, simultaneous binding of CTLA4Fcε to CD23 and CD80/CD86 seems to occur and would explain this difference. As measured by a human CD23-specific ELISA, CTLA4Fcε - but not IgE - induced a concentration-dependent reduction of sCD23 in culture supernatants of RPMI-8866 cells. Our results suggest that the simultaneous binding of CTLA4Fcɛ to CD23-CD80/CD86 may cause the formation of multi-molecular complexes that are either internalized or pose a steric hindrance to enzymatic proteolysis, so blocking sCD23 generation. CTLA4Fcε caused a concentration-dependent reduction of lymphocyte proliferation in human peripheral blood mononuclear cell samples stimulated in vitro with concanavalin A. The ability to bind IgE receptors on effector cells, to regulate the production of sCD23 and to inhibit lymphocyte proliferation suggests that CTLA4Fcɛ has immunomodulatory properties on human Th2 responses.

Novel antibody-based proteins for cancer immunotherapy.

The relative success of monoclonal antibodies in cancer immunotherapy and the vast manipulation potential of recombinant antibody technology have encouraged the development of novel antibody-based antitumor proteins. Many insightful reagents have been produced, mainly guided by studies on the mechanisms of action associated with complete and durable remissions, results from experimental animal models, and our current knowledge of the human immune system. Strikingly, only a small percent of these new reagents has demonstrated clinical value. Tumor burden, immune evasion, physiological resemblance, and cell plasticity are among the challenges that cancer therapy faces, and a number of antibody-based proteins are already available to deal with many of them. Some of these novel reagents have been shown to specifically increase apoptosis/cell death of tumor cells, recruit and activate immune effectors, and reveal synergistic effects not previously envisioned. In this review, we look into different approaches that have been followed during the past few years to produce these biologics and analyze their relative success, mainly in terms of their clinical performance. The use of antibody-based antitumor proteins, in combination with standard or novel therapies, is showing significant improvements in objective responses, suggesting that these reagents will become important components of the antineoplastic protocols of the future.

Decreased survival of human breast cancer cells expressing HER2/neu on in vitro incubation with an anti-HER2/neu antibody fused to C5a or C5a desArg.

Treatment of human epidermal growth factor receptor 2 (HER2/neu)-expressing breast cancer patients with a monoclonal antibody (mAb) directed against HER2/neu improves the outcome of chemotherapy. In cases in which remission is observed, antibody-dependent cell-mediated cytotoxicity (ADCC) seems to be one of the main mechanisms of anti-HER2/neu mAb action, implicating Fc gamma receptors (Fc gamma Rs) in this tumoricidal activity. In vitro and in vivo studies have revealed that anti-HER2/neu-mediated ADCC is mainly accomplished by polymorphonuclear granulocytes (PMN). C5a, a cleavage product of the complement component C5, modulates Fc gamma R expression via upregulation of activating and downregulation of inhibitory Fc gamma Rs. C5a also recruits PMNs to sites of inflammation and increases PMN survival. To enhance the recruitment and activation of C5a receptor-bearing cells into the tumor microenvironment, we developed antibody fusion proteins composed of a human IgG3 anti-HER2/neu antibody genetically fused to C5a [anti-HER2/neu IgG3-(C5a)] or to its derivative, C5a(desArg) [anti-HER2/neu IgG3-(C5a(desArg))]. Both fusion proteins were expressed, properly assembled, and secreted by murine myeloma cells, and displayed chemotactic activity on human PMN. Under comparable conditions, anti-HER2/neu IgG3-(C5a(desArg)) increased the survival of PMN more efficiently than anti-HER2/neu IgG3-(C5a) or C5a(desArg). Surprisingly, incubation of the fusion proteins with breast cancer cells that overexpress HER2/neu (SK-BR-3) induced cell death at a dose at which the anti-HER2/neu IgG3 antibody was innocuous. In the presence of human peripheral blood leukocytes as effector cells, both fusion proteins induced tumor cell death more efficiently than anti-HER2/neu IgG3. These data suggest that anti-HER2/neu IgG3-(C5a) and anti-HER2/neu IgG3-(C5a(desArg)) fusion proteins possess novel properties that could be useful in cancer immunotherapy.

Recombinant polymeric IgG anti-Rh: a novel strategy for development of direct agglutinating reagents.

Anti-Rh alloantibodies are used in research and clinic laboratories to define the Rh antigenic profile of human blood samples. IgM anti-Rh antibodies directly agglutinate Rh-positive RBCs. Anti-Rh antibodies of the IgG isotype bind to Rh antigens with a higher intrinsic affinity than IgM and sensitize RBCs, but do not induce direct hemagglutination. The aim of this work was to produce IgG anti-Rh possessing direct hemagglutinating properties of IgM. To achieve this goal, recombinant antibody technology was used to construct genes encoding Ig light and heavy chains that will form polymers with anti-Rh specificity. Expression vectors and liposome-mediated DNA transfer were used to generate transfectomas secreting human recombinant IgG3 anti-Rh. ELISA, SDS-PAGE, and hemagglutination were used to identify and characterize the recombinant antibody produced. Thus, a recombinant polymeric IgM-like IgG3 anti-Rh antibody was produced that directly agglutinates RBCs with specificity identical to that of the parent non-agglutinating IgG. The results obtained suggest that the technology used here to generate polymeric IgM-like IgG3 anti-Rh antibodies can be applied to produce Rh blood typing reagents. This approach might also be used to develop reagents for which cell surface antigen binding and agglutination or aggregation is required.

Liver sinusoidal endothelial cells as possible vehicles for gene therapy: a comparison between plasmid-based and lentiviral gene transfer techniques.

UNLABELLED: Liver sinusoidal endothelial cells (LSECs) constitute an attractive target for gene therapy of several liver and systemic diseases. However, there are few reports showing an efficient plasmid-based or viral methodology to deliver recombinant genes into these cells. In the present study, the authors evaluated in vitro gene transfer efficiency of standard plasmid-based techniques (i.e., electroporation, lipofection, and calcium phosphate) and lentiviral-mediated gene transduction into primary murine LSECs, using reporter genes. The results show that electroporation is the most effective in vitro plasmid-gene transfer method to deliver GFP into LSECs (31%), as compared with lipofection and calcium phosphate transfection (6% and 4%, respectively). However, lentiviral transduction resulted in higher, efficient, and stable gene transfer (70%) as compared with plasmid-based techniques. CONCLUSIONS: The highly efficient gene expression obtained by lentiviral transduction and electroporation shows that these methodologies are highly reliable systems for gene transfer into LSECs.

Risk of adverse post-transplant events after kidney allograft transplantation as predicted by CTLA-4 +49 and TNF-alpha -308 single nucleotide polymorphisms: a preliminary study.

Genetic differences between donor and recipient HLA haplotypes are of major importance for transplant rejection. Other genetic variations occurring in genes encoding cytokines and costimulatory molecules also appear to exert an influence on the manner the host immune system recognizes the allograft. The aims of this work were: 1) to study selected single nucleotide polymorphisms (SNPs) at the loci encoding the T-cell regulatory molecule CTLA-4 (CD152), and the cytokines interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, IL-10, and transforming growth factor (TGF)-beta1 in a sample of healthy volunteers and a group of kidney-transplanted patients; and 2) to investigate whether an association exists between any of the SNPs studied and acute or chronic rejection, or non-responsiveness to steroid treatment during episodes of acute rejection (AR) after kidney allograft transplantation. When healthy volunteers were compared with transplanted patients, no significant differences were found in the distribution of genetic frequencies for any of the SNPs analyzed. However, in transplanted patients who received a kidney from a living related donor (KdTxL), a statistically significant association was found between carrying the CTLA-4 +49 A/A genotype and protection from experiencing acute rejection. No such association was found in the group of transplanted patients who received a kidney from a cadaveric non-related donor (KdTxCad). In both, KdTxL and KdTxCad patients, responsiveness to steroid treatment during acute rejection was also in association with the CTLA-4 (+49A/G) SNP. The CTLA-4 +49G allele was found at a very low frequency among steroid-resistant compared with steroid-sensitive patients. Finally, a statistically significant association was found between the presence of the TNF-alpha -308A allele and protection to suffer from chronic rejection. The genetic differences found may serve as risk predictors of adverse post-transplant events.

High efficiency and long-term foreign gene expression in cultured liver sinusoidal endothelial cells by retroviral transduction.

The liver sinusoidal endothelial cells (LSECs) constitute a very specialized endothelium. Due to their multiple functions and privileged location in the liver, these cells constitute an excellent target for gene therapy. In this work, the authors investigate the efficiency of retroviral gene transduction as a method for in vitro gene delivery into murine LSECs. Gene transduction into murine LSECs was performed using the PCMMP-eGFP/pIK-MLVgp retrovirus pseudotyped with the vesicular stomatitis virus G glycoprotein (VSV-g), containing eGFP as a reporter gene. Retroviral transduction resulted in a high efficiency of gene transfer (99%) and stable expression of eGFP in LSECs. The retroviral transduction protocol did not affect the morphology or expression of endothelial cell markers or the biological functions of LSECs. The authors have developed conditions for high-efficiency and stable retroviral gene transduction of LSECs. These results raise the possibility of liver gene therapy using LSECs as vehicle for the delivery of therapeutic proteins by means of retroviral vectors.

Producción de anticuerpos monoclonales con especificidad por los grupos sanguíneos A y B: evaluación de su uso como reactivos hemoclasificadores / A and B blood group-specific monoclonal antibodies: production and evaluation as ABO-blood typing reagents

El desarrollo de reactivos hemoclasificadores mediante la aplicación de la tecnología para la producción de anticuerpos monoclonales (AcMo) ha sido exitoso y ello ha permitido reducir los costos asociados a su producción. En Venezuela el consumo de estos reactivos depende principalmente de la importación, con el consecuente gasto de divisas. Con el propósito de ayudar a solventar esta situación el presente trabajo se planteó como. 1) Generar hibridomas productores de AcMo con especificidad anti-A y anti-B, 2) Caracterizar y producir a mediana escala los AcMo obtenidos, 3) Realizar estudios de campo, con el fin de lograr su certificación como reactivos hemoclasificadores. El producto de este trabajo fue la obtención de 22 hibridomas, 11 productores de AcMo anti-A y 11 productores de anti-B. Cuatro AcMo fueron caracterizados y estudiados: Au18Kt3F, MG3 (ambos IgM anti-A), SS4.5 (IgG anti-B) y BB2-3 (IgM anti-B). Para la producción de estos AcMo a mayor escala se emplearon los bio-reactores comerciales “miniPerm” y “Tecnomouse”, lográndose una concentración elevada de los mismos. Los valores de parámetros funcionales como avidez, potencia y especificidad de los AcMo producidos resultaron aceptables al compararse con hemoclasificadores comerciales, lo que hace viable su utilización como reactivos hemoclasificadores

Expression of human soluble complement receptor 1 by a pig endothelial cell line inhibits lysis by human serum.

The importance of complement activation and naturally occurring anti-pig antibodies in the hyperacute rejection (HAR) observed in models of pig-to-human xenotransplantation is well established. To overcome this, much effort has been dedicated to preparing transgenic pigs by knocking out Galalpha(1-3)Gal expression in these animals, or knocking in the expression of human complement regulatory proteins (CRPs), such as CD59 or decay accelerating factor. A soluble form of another membrane CRP, complement receptor type 1 (CR1), has also been shown to inhibit complement activation. Here, we show that transfection of a pig endothelial cell line with a truncated form of human soluble complement receptor 1 (sCR1) almost completely protected these cells from complement-mediated lysis by human AB serum. Pigs genetically manipulated to express human sCR1 may represent an additional strategy to inhibit HAR of pig-to-human transplanted organs.

[A and B blood group-specific monoclonal antibodies. Production and evaluation as ABO-blood typing reagents]. / Producción de anticuerpos monoclonales con especificidad por los grupos sanguíneos A y B. Evaluación de su uso como reactivos hemoclasificadores.

The Monoclonal Antibody (MoAb) technology has been successfully applied to develop reagents for human blood group classification. There is no production of this kind of reagents in Venezuela, and the local demand (blood banks and clinical laboratories) is mainly supplied with imported material. Considering this we decided to apply MoAb techniques to generate murine hybridomas secreting anti-A or anti-B specific MoAb. MoAb obtained were characterized and produced in enough quantity to perform validation studies as blood typing reagents. Out of 22 hybridomas that were initially selected, 11 were anti-A secretors and 11 were anti-B secretors. Four MoAb were further characterized: Au18Kt3F, MG3 (both IgM anti-A), SS4.5 (IgG1 anti-B) and BB2-3 (IgM anti-B). Conditions were also established for growing the hybridomas Au18Kt3F and BB2-3 in the bioreactors "miniPerm" and "Tecnomouse", allowing for scale-up production of these MoAb. Avidity and specificity were estimated for each one, and the results were comparable to those obtained from commercially available reagents, making feasible its use as blood typing reagents.

Production of human monoclonal antibodies against Galalpha(1-3)Gal.

The hyperacute rejection observed in models of pig-to-human xenotransplantation is mainly because of the presence of natural antibodies in human blood with specificity for the Galalpha(1-3)Gal (Gal) carbohydrate moiety present on the surface of porcine endothelial cells. Human monoclonal anti-Gal antibodies could be of use both in the study of the basic mechanisms of hyperacute rejection as well as in its clinical prevention. In the present study we prepared 42 heterohybridomas (human-mouse) secreting antibodies with specificity for the Gal epitope. All of the antibodies produced were of the IgM isotype, according to a dot-blot assay. Twenty-seven antibodies were further characterized, and shown to be specific for Gal by different methods, including an enzyme-linked immunosorbent assay, in which the plates were sensitized with mouse laminin as a source of Gal. Specificity was also confirmed using purified Gal carbohydrate in a hemagglutination inhibition assay. The antibodies were shown to mediate lysis of Gal-expressing rabbit erythrocytes in the presence of complement. However, the heterohybridomas themselves were shown to express Gal, a result of the mouse P3x63Ag8.653 hybridoma cells used during hybridoma generation. The presence of this epitope on the surface of anti-Gal-producing cells, and on the antibody itself, represents a limitation to the production of high affinity anti-Gal antibodies.

Influence of the isotype of the light chain on the properties of IgG.

It is widely appreciated that the isotype of the H chain of the Ab molecule influences its functional properties. We have now investigated the contribution of the isotype of the L chain to the structural and functional properties of the Ab molecule. In these studies, the L chain variable region of a murine anti-dansyl Ab was joined to either human kappa or lambda constant region domains and expressed with mouse-human chimeric H chains of the four human IgG isotypes. The resulting Abs were secreted as fully assembled molecules although, as has been previously observed, IgG4 with either kappa or lambda L chains was also secreted as HL half-molecules. However, the isotype of the L chain can influence the kinetics of intracellular assembly with IgG1lambda, IgG2lambda, and IgG4lambda assembling more slowly than their kappa counterparts. The isotype of the L chain also influenced the susceptibility of the interchain disulfide bonds to attack by reducing agents with variable effects, depending on the isotype of the H chains. For IgG2, but not for IgG1, -3, and -4, the isotype of the L chain influenced the rate of clearance in mice, with IgG2lambda having a shorter in vivo half-life than IgG2kappa. Only slight differences were also observed between lambda and kappa molecules in their kinetics of binding to and dissociation from the hapten dansyl. These studies demonstrate that the isotype of the L chain has only a slight impact on the structural and functional properties of variable region identical Abs.

Anticuerpos monoclonales y su aplicación en hematología / Monoclonal antibodies and its applications in hematology

El advenimiento de los anticuerpos monoclonales ha sido, en el ámbito de las ciencias biomédicas, uno de los desarrollos metodológicos más importantes de los últimos 20 años. Su utilización parece no tener más límites que aquellos que se imponga al usuario, como bien lo ha ilustrado la generación de los llamados anticuerpos monoclonales catalíticos. El detallado conocimiento de la organización de los genes que codifican estas proteínas, en conjunto con las avanzadas tecnologías para la manipulación genética de las que se dispone en la actualidad ha permitido el desarrollo de técnicas para la elaboración de genotecas combinatorias tanto de orígen humano como múrido las cuales anticipa sean de gran utilidad en el entendimiento de enfermedades autoinmunes relacionadas con la respuesta inmune humoral y en la elaboración de reactivos terapéuticos aplicables a una variedad de patologías