Production of an anti-TNFα antibody in murine myeloma cells by perfusion culture.

Infliximab is a mouse/human chimeric IgG1 monoclonal antibody which recognizes the proinflammatory cytokine, tumor necrosis factor α (TNFα), and inhibits receptor interactions, thereby decreasing inflammation and autoimmune response in patients. This monoclonal antibody has been successfully used to treat rheumatoid arthritis, ankylosing spondylitis, and psoriatic arthritis. However, the high treatment cost limits patient access to this biotherapy. One alternative to this problem is the use of biosimilars. In this work, we describe the stable expression and physicochemical characterization of an anti-TNFα antibody. While infliximab is produced in recombinant murine SP2/0 cells, our anti-TNFα IgG antibody was expressed in recombinant murine NS0 myeloma cells. The best anti-TNFα antibody-expressing clone was selected from three clone candidates based on the stability of IgG expression levels, specific productivity as well as TNFα-binding activity compared to commercial infliximab. Our results indicate that the selected cell clone, culture medium, and fermentation mode allowed for the production of an anti-TNFα antibody with similar characteristics to the reference commercially available product. An optimization of the selected culture medium by metabolomics may increase the volumetric productivity of the process to satisfy the demand for this product. Further experiments should be performed to evaluate the biological properties of this anti-TNFα antibody. KEY POINTS: • An anti-TNFα antibody was produced in NS0 cells using perfusion culture. • A proprietary chemically defined culture medium was used to replace commercially available protein-free medium. • The purified anti-TNFα antibody was comparable to the reference marketed product.

Immunogenicity of autologous immunoglobulins: principles and practices.

The immunogenicity of immunoglobulin idiotypes in syngeneic systems is a rather rare phenomenon. Very few studies have attempted to determine the mechanisms underlying the anti-idiotypic response that certain autologous idiotypes can elicit. Furthermore, the studies addressing a possible physiological role for such behaviors are even less. In the present article, the results of the characterization of a highly immunogenic idiotype are reviewed and compared with some related works on the subject. We finally propose a possible immunoregulatory role for idiotypic immunogenicity.

Gangliosides, Ab1 and Ab2 antibodies IV. Dominance of VH domain in the induction of anti-idiotypic antibodies by gene gun immunization.

The heavy chain of anti-N-glycolyl-ganglioside P3 mAb plays the main role in its binding properties. At least one hybrid idiotype consisting on the P3 VH and an unrelated VL domain retains antigen recognition. Moreover, the unusual immunogenic properties of P3 idiotype could be modified by single mutations of H-CDR residues. Here, we show that DNA gene gun immunization with the P3 VH combined with an unrelated VL domain or with itself (VH dimer, VHD) is enough for inducing anti-idiotypic antibodies, independently of antigen recognition by the resulting molecule. The scFv fragment of P3 mAb was also able to induce an anti-idiotypic response. For both the P3 and the P3 anti-idiotypic 1E10 mAbs, heavy chains dominate the induction of antibodies against the respective idiotypes.

Gangliosides, Ab1 and Ab2 antibodies III. The idiotype of anti-ganglioside mAb P3 is immunogenic in a T cell-dependent manner.

P3 mAb is an IgM monoclonal antibody specific for N-glycolyl-containing gangliosides. The immunogenicity of the P3 idiotype has been previously described by immunizing syngeneic BALB/c mice with the purified murine IgM or the mouse-human chimeric IgG antibody. In the present work we study the antibody response against the idiotype of P3 mAb through immunization with DNA. We used small immune proteins (SIP) consisting on the idiotype in the scFv format, covalently linked to gamma1CH3, the self-dimerizing domain of murine IgG1. SIPs were previously shown to be appropriate to induce specific anti-idiotypic responses. By gene gun immunization, a polyspecific response was occasionally generated, particularly with the P3 idiotype. A single shot of DNA was sufficient to induce a strong and long-lasting anti-P3 idiotype response. In addition, by delivery of the same DNA construct with a recombinant adeno-associated virus the unique immunogenicity of the P3 idiotype was demonstrated. The requirement of T cells in the anti-P3 idiotype response was indicated by the lack of P3-specific anti-idiotypic antibodies following immunization of both, allogeneic C57BL/6 and athymic BALB/c mice.

Insights into the immunogenetic basis of two ganglioside-associated idiotypic networks.

The heavy-chain variable regions (VH) from 14F7 MAb, an IgG1 antibody specific for GM3(NeuGc) ganglioside, and its anti-idiotype, the 4G9 MAb, were cloned and sequenced. Comparison with previously reported sequences showed that VH 14F7 belongs to the J558(VHI) gene family and that it is highly mutated. VH 4G9 belongs to the Q52(VHII) gene family. The HCDR3 14F7 sequence contains three basic residues that could be involved in the binding to 4G9 MAb, which bears acidic residues in its HCDR3. Studies performed in the syngeneic model showed that 14F7 MAb requires both coupling to KLH and the use of Freund's adjuvant to induce an effective anti-idiotypic IgG (Ab2) response. In contrast, P3 MAb, a germline gene-encoded Ab1 that also recognizes the GM3(NeuGc) ganglioside through a basic motif in its H-CDRs, has been reported to be immunogenic in syngeneic mice, even when injected in saline. In addition, when Leghorn chickens were immunized with 14F7 or P3 MAbs emulsified in Freund's adjuvant, only P3-immunized animals were able to develop antibodies that recognized NeuGc-containing gangliosides, antigens which are not present in the normal tissues of this animal species. This phenomenon could be due to the lack of idiotypic connectivity of 14F7MAb.

Immunopotentiating properties of a multispecific α-anti-idiotype antibody.

Multispecificity is not a well-understood property of some antibodies. Different functions have been attributed to multispecific natural antibodies, commonly associated with the neutralization and clearance of antigens. Much less is known about the role of antibodies like these, based on their idiotypic connectivity. B7Y33 is a chimeric IgG1 version of a polyreactive α anti-idiotype antibody that is able to interact with different immunoglobulin and non-immunoglobulin antigens. Here we report the capacity of this antibody to enhance the immunogenicity of several autologous IgMs in adjuvant-free conditions. Our results suggest that the formation of immune complexes seems to be necessary, but not sufficient, to this activity. The potential involvement of the interaction of B7Y33 with the FcγRIIb is discussed.

Expression and biological characterization of an anti-CD20 biosimilar candidate antibody: a case study.

The CD20 molecule is a non-glycosylated protein expressed mainly on the surface of B lymphocytes. In some pathogenic B cells, it shows an increased expression, thus becoming an attractive target for diagnosis and therapy. Rituximab is a chimeric antibody that specifically recognizes the human CD20 molecule. This antibody is indicated for the treatment of non-Hodgkin lymphomas and autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus. In this work, we describe the stable expression and biological evaluation of an anti-CD20 biosimilar antibody. While rituximab is produced in fed-batch culture of recombinant Chinese hamster ovary (CHO) cells, our biosimilar antibody expression process consists of continuous culture of recombinant murine NS0 myeloma cells. The ability of the purified biosimilar antibody to recognize the CD20 molecule on human tumor cell lines, as well as on peripheral blood mononuclear cells from humans and primates, was demonstrated by flow cytometry. The biosimilar antibody induced complement-dependent cytotoxicity, antibody-dependent cell-mediated cytotoxicity and apoptosis on human cell lines with high expression of CD20. In addition, this antibody depleted CD20-positive B lymphocytes from peripheral blood in monkeys. These results indicate that the biological properties of the biosimilar antibody compare favorably with those of the innovator product, and that it should be evaluated in future clinical trials.

Chimeric anti-N-glycolyl-ganglioside and its anti-idiotypic MAbs: immunodominance of their variable regions.

P3 monoclonal antibody (MAb) is a murine IgM that specifically recognizes N-glycolyl (NeuGc)-gangliosides and sulfatides. It also reacts with antigens expressed in human breast tumors and melanoma. In syngeneic model, P3 MAb is able to elicit a strong anti-idiotypic (Ab2) antibody response, even in the absence of adjuvants or carrier proteins. 1E10 MAb is an anti-idiotypic antibody specific for P3 MAb that has demonstrated anti-tumoral effects in syngeneic and allogeneic animals. Here we report the construction of the human IgG(1) chimeric P3 and 1E10 antibodies, and the evaluation of the maintenance of the main properties of the murine MAbs. Chimeric P3 antibody specifically reacted with GM3(NeuGc) and GM2(NeuGc) gangliosides, and not with their acetylated variants. Also, it strongly recognized the anti-idiotypic 1E10 MAb. Chimeric 1E10 antibody specifically reacted with P3 MAb. Upon immunization of Balb/c mice with both chimeric antibodies, we were able to demonstrate the immunodominance of their variable regions. The anti-idiotypic response induced by both antibodies was strong and in most of the mice was even significantly higher than the anti-isotypic response, despite the fact that 70% of the chimeric molecule is xenogenic with respect to the animal model.

Purificación de una toxina a partir de la anemona de mar Stoichactis Helianthus / Purification of toxin from sea anemone Stoichactis Helianthus

La obtención de sustancias tóxicas de origen natural, tanto para células completas como para membranas, ha ocupado a muchos investigadores con el objetivo de utilizarlas para el tratamiento de las neoplasias. Una fuente de estas toxinas lo constituyen los invertebrados marinos. Se describe el aislamiento y purificación de una toxina hemolítica obtenida de la anémona de mar Stoichactis helianthus. Se determinó su peso molecular, termoestabilidad, así como su grado de pureza y se estableció un esquema para la prrificación de esta toxina que resulta ser fuertemente hemolítica frente a eritrocitos humanos. Se obtuvo aproximadamente 1 g de toxina por cada 200 g del animal completo, con un 0,5 % de rendimiento. La obtención de dicha toxina constituye uno de los pasos para su posterior utilización en la construcción de inmunotoxinas, mediante su unión covalente a anticuerpos monoclonales que servirán para su posible tratamiento de las neoplasias malignas

Preparación de una inmunotoxina por acoplamiento de un anticuerpo monoclonal contra linfocitos T y una toxina hemolítica de origen marino / Preparation of an immune toxin by coupling a monoclonal antibody to T lymphocytes and a hemolytic toxin of marine etiology

En la actualidad la oncología orienta las investigaciones hacia la búsqueda de tratamientos del cáncer más eficaces y específicos que los citostáticos clásicos. Se presenta en este artículo la primera experiencia en Cuba de formación de moléculas híbridas por acoplamiento químico entre anticuerpos monoclonales y toxina. Los resultados de los experimentos demuestran que puede obtenerse una molécula híbrida entre el anticuerpo monoclonal IOR-T1 y la toxina hemolítica aislada a partir del Stoichactis Helianthus que es toxina para linfocitos de sangre periférica a concentraciones equivalentes a las concentraciones tóxicas de la toxina no acoplada