A Novel Redox Method for Rapid Production of Functional Bi-Specific Antibodies For Use in Early Pilot Studies

We demonstrate here a rapid alternative method for the production of functional bi-specific antibodies using the mild reducing agent 2-mercaptoethanesulfonic acid sodium salt (MESNA). Following reduction of a mixture of two monoclonal antibodies with MESNA to break inter heavy chain bonds, this solution is dialysed under oxidising conditions and antibodies are allowed to reform. During this reaction a mixture of antibodies is formed, including parental antibodies and bi-specific antibody. Bi-specific antibodies are purified over two sequential affinity columns. Following purification, bi-specificity of antibodies is determined in enzyme-linked immunosorbent assays and by flow cytometry. Using this redox method we have been successful in producing hybrid and same-species bi-specific antibodies in a time frame of 6–10 working days, making this production method a time saving alternative to the time-consuming traditional heterohybridoma technology for the production of bi-specific antibodies for use in early pilot studies. The use of both rat and mouse IgG antibodies forming a rat/mouse bi-specific antibody as well as producing a pure mouse bi-specific antibody and a pure rat bi-specific antibody demonstrates the flexibility of this production method

gp100/pmel17 and tyrosinase encode multiple epitopes recognized by Th1-type CD4+T cells

CD4+ T cells modulate the magnitude and durability of CTL responses in vivo, and may serve as effector cells in the tumour microenvironment. In order to identify the tumour epitopes recognized by tumour-reactive human CD4+ T cells, we combined the use of an HLA-DR4/peptide binding algorithm with an IFN-γ ELISPOT assay. Two known and three novel CD4+ T cell epitopes derived from the gp 100/pmel17 and tyrosinase melanocyte-associated antigens were confirmed or identified. Of major interest, we determined that freshly-isolated PBMC frequencies of Th1-type CD4+ T recognizing these peptides are frequently elevated in HLA-DR4+ melanoma patients (but not normal donors) that are currently disease-free as a result of therapeutic intervention. Epitope-specific CD4+ T cells from normal DR4+ donors could be induced, however, after in vitro stimulation with autologous dendritic cell pulsed with antigens (peptides or antigen-positive melanoma lysates) or infected with recombinant vaccinia virus encoding the relevant antigen. Peptide-reactive CD4+ T cells also recognized HLA-DR4+ melanoma cell lines that constitutively express the relevant antigen. Based on these data, these epitopes may serve as potent vaccine components to promote clinically-relevant Th1-type CD4+ T cell effector function in situ. http://www.bjcancer.com © 2001 Cancer Research Campaig

Collaboration - a competitor's tool: The story of Centocor, an entrepreneurial biotechnology company

Biotechnology companies have relied on alliances for survival and growth since their inception. This history of Centocor illustrates the pivotal role collaborations played for pioneers in the industry. Five years after its founding Centocor had become a competitive and profitable diagnostics company based on partnerships with research institutes and larger health care companies. In 1992, however, Centocor faced collapse, brought on by a departure from collaboration and going it alone in the development and marketing of the company's first therapeutic. What saved the company and enabled it to prosper in therapeutics was a reversion to the old strategy of collaboration.alliances, biotechnology, technology transfer, pharmaceutical, diagnostics,