Two humanized monoclonal antibody constructs bearing the same variable regions of an anti-CD3 monoclonal antibody, whole IgG and FvFc, were expressed in CHO cells. Random and site-specific integration were used resulting in similar expression levels. The transfectants were selected with appropriate selection agent, and the surviving cells were plated in semi-solid medium for capture with FITC-conjugated anti-human IG antibody and picked with the robotic ClonePix FL. Conditioned media from selected clones were purified by affinity chromatography and characterized by SDS-PAGE, Western-blot, SEC-HPLC, and isoelectric focusing. Binding to the target present in healthy human mononuclear cells was assessed by flow cytometry, as well as by competition between the two constructs and the original murine monoclonal antibody. The humanized constructs were not able to dislodge the murine antibody while the murine anti-CD3 antibody could dislodge around 20% of the FvFc or IgG humanized versions. Further in vitro and in vivo pre-clinical analyses will be carried out to verify the ability of the humanized versions to demonstrate the immunoregulatory profile required for a humanized anti-CD3 monoclonal antibody.

Tumor necrosis factor alpha (TNFα) is a pro-inflammatory cytokine that mediates the homeostasis of immune responses; its exacerbated production is associated with the pathogenesis of autoimmune and chronic inflammatory diseases. Anti-TNFα drugs have revolutionized the treatment of inflammatory conditions such as rheumatoid arthritis and Crohn's disease. Currently, a worldwide race is on stage for the production of biosimilars moved by patent expiration of monoclonal antibodies (mAbs), such as anti-TNFα adalimumab. Our goal was to develop the first stage of an adalimumab biosimilar candidate with potential for national production, through the generation of a productive and stable cell line and assess its functionality. The robotic system ClonePix was used for screening and isolation of colonies from transfected CHO-S stable pools plated in semisolid medium. Selected clones were expanded based on growth and productivity. Purified mAbs from different clones were tested for binding and functional activity. The binding affinity of the denominated adabut clones to TNFα and FcRγ did not differ statistically when compared to reference adalimumab. One functional activity assay demonstrated the antibody neutralization capacity of the cytotoxicity induced by TNFα in L929 murine fibroblasts. A second assay confirmed adabut as an antagonist of the TNFα activity by the inhibition of the cell adhesion molecule expression in HUVEC cultures. The binding and functional activity analyses performed with selected adabut clones in comparison to reference adalimumab represent an important status of "non-inferiority," part of the process required for a biosimilar development. We generated and selected high-quality adabut clones which mAbs may be further developed as the first in-house made Brazilian biosimilar, demonstrating a success case for our incipient biotechnology industry, or also modified as biobetters, thus representing an innovative strategy for the patients' welfare.

The immobilisation of cells in a perfusion culture allows to obtain a high cell concentration and an ef®cient removal of the catabolites without cell loss. A disadvantage of this system is that the cell density cannot be directly monitored. The cellular metabolism is just followed by online measurements of pH and dissolved oxygen (DO) and off-line determinations of residual metabolites. In this article, we report a high cell density achieved by the cultivation of a hybridoma in a bubblecolumn bioreactor ®lled with hollow glass cylinders. The parameters monitored during the cultivation were pH, temperature, DO, glucose, lactate and monoclonal antibody. The glucose uptake rate was used to estimate the cell concentration along the time. The maximum cell concentration calculated for the considered cultivation time was 2.7´10 7 cell á ml A1 . The glucose concentration in the media decreased stepwise twice, causing a decrease on the speci®c growth rate, while maintaining high antibody productivity levels. Maximum monoclonal antibody productivity was 503 lg á l A1 á day A1 and speci®c productivity, considering calculated cell density, was 0.019 ng á cell A1 á day A1 . IntroductionAlthough target of a great number of studies, mammalian cell culture kinetics is still not completely understood. It depends on so many factors, including the cell line physiology, media formulation, type of bioreactor, gas supply and operating conditions. Often, the particular characteristics of a given cell line are functions of the culture conditions, including the composition of the medium, the bioreactor type, and the way it is operated [1].The most commonly used basal media for the growth of hybridomas are DME and RPMI 1640, formulated with different concentrations of glucose and glutamine, the major nutrients related to growth in conventional cell culture media, representing cell mass precursors and energy source. The other nutrients do not count for growth limitation, as they are usually supplied in excess. In a comparison between DME and RPMI media, it was concluded that DME seems to be more ef®cient for hybridoma growth, supporting greater number of cells for longer periods than RPMI, while the antibody synthesis depends only on the cell number, regardless of the medium used [2,3].Concerning the culture mode, perfusion culture of mammalian cells allows the maintenance of long-term, high-density and high-productivity cultures. Suspension cultures of hybridoma in a perfusion system makes it dif®cult to control the problems caused by the shear forces and to increase the dilution rate above the maximum speci®c growth rate, permitting to achieve high cellular viability. Several devices have been attempted to retain the cells, with more or less ef®cacy [4±6]. The immobilisation of hybridoma cells in any kind of support allows more ef®cient perfusion and confers more stability to the culture system, therefore, increasing the potential for longer cultures, higher cell density and antibody productivity [7]. The immobilisation condition protect...

Objetivos: O anticorpo monoclonal anti-CD3 tem sido empregado na prevenção e tratamento de episódios de rejeição aguda em transplante de órgãos. Neste estudo retrospectivo, relatamos a experiência de três instituições brasileiras que utilizaram o anticorpo anti-CD3 produzido pelo Instituto Butantan (São Paulo, Brasil) em pacientes transplantados renais. Métodos: Foram analisados 25 pacientes que receberam anti-CD3 para profilaxia (n=9) e tratamento de rejeição celular aguda (n=16). Resultados: Os pacientes que utilizaram o anti- CD3 profilaticamente eram sensibilizados em sua maioria (89%) e apresentaram ocorrência de rejeição aguda em 33% dos casos. O uso terapêutico foi indicado para tratamento de rejeição córtico-resistente ou rejeições de maior severidade histológica, e reverteu clinicamente 69% dos episódios tratados. Na maioria dos pacientes, o uso do anti-CD3 Butantan reduziu o número de células CD3+ a valores menores que 30 cel/mm3 no segundo dia de tratamento. O evento adverso mais freqüentemente observado foi febre e infecções bacterianas e virais, seis meses após o tratamento, que foram observadas em 13 e 10 pacientes, respectivamente. No período médio de seguimento de oito anos nenhuma ocorrência de tumor foi relatada. Conclusões: Concluímos que a utilização do anti-CD3 Butantan mostrou-se eficaz na profilaxia e tratamento de rejeição aguda em pacientes transplantados renais.

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