Selective high-performance liquid chromatographic purification of bispecific monoclonal antibodies.

The recent development of improved production techniques for bispecific monoclonal antibodies (biMAbs) has significantly increased interest in specific purification procedures. In this investigation, a general high-performance liquid chromatographic (HPLC) purification method is proposed that allows highly purified biMAbs to be obtained from mouse ascites fluid containing a mixture of different antibodies, i.e., parental MAbs, active biMAb and a mixture of randomly assembled heavy and light chains. Proteins from ascites fluid were precipitated with ammonium sulphate and applied to a high-performance protein A column to separate the total immunoglobulin fraction. BiMAbs were isolated from other immunoglobulins by two subsequent passages through a high-performance hydroxyapatite (HPHT) column. This purification protocol combines specificity of protein A for immunoglobulin G (IgG) and high selectivity of hydroxyapatite for different IgG idiotypes. All purification steps were performed rapidly and reliably by HPLC. This method was applied to the purification of six different biMAbs with consistently high yields, purity and homogeneity. This general purification method may prove extremely valuable when highly pure preparation of biMAbs is required, as for in vivo use.

CD38: a multi-lineage cell activation molecule with a split personality.

This review reports the characteristics of the human surface molecule CD38, a structure not linked to a definite line and predominantly expressed in early and activated phenotypes. The CD38 molecule consists of a single chain of 46 kDa, spanning the membrane and with the carboxyl terminus located in the extracellular compartment. The CD38 molecule is also involved in the transduction of activation and proliferation signals, which are line unrestricted. The gene coding for the CD38 antigen has been cloned and used for the construction of simian and mouse transfectants expressing the human molecule. These cell models are used for the analysis of several unanswered issues, mainly concerning the in vivo function of CD38, the existence of a natural ligand and of polymorphism in the population.

Retrovirus-derived shuttle vectors in the generation of murine bispecific MoAbs to be used in ex vivo and in vivo immunotherapy.

Mouse monoclonal antibodies specific for CD3, FcR and a melanoma associated antigen have been produced. Drug resistence of such hibrydomas has been obtained tranfecting them with plasmids containing genes conferring specific resistence. Retrovirus derived shuttle vectors with high transfection efficiency have been used for transfection. Hybrydomas were than fused and bispecific antibody producing cells selected. Purification was performed by HPLC. Such bispecific antibodies can be used in ex vivo and in vivo immunotherapy.

Gene transfer by retrovirus-derived shuttle vectors in the generation of murine bispecific monoclonal antibodies.

The present study reports on the use of gene transfer by retrovirus-derived shuttle vectors in the generation of hybrid hybridomas secreting bispecific monoclonal antibodies. neo- and dhfr- genes were infected into distinct murine hybridomas, thus conferring a dominant resistance trait to geneticin (G418) and to methotrexate. The vectors employed were replication-deficient and dependent on complementation by a helper virus provided by the irradiated packaging lines. After cocultivation with the relevant packaging cell lines, stable hybridoma lines expressing the selectable markers were easily obtained and were then suitable for conventional somatic fusion. This high-efficiency method was used to generate two bispecific monoclonal antibodies simultaneously targeting molecules expressed on cytotoxic cells (i.e., T lymphocytes and natural killer cells) against a human melanoma-associated antigen.