Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.
We describe here the first in vivo targeting of tumors with a single-chain antigen-binding protein. The molecule, which was constructed and expressed in Escherichia coli, is a novel recombinant protein composed of a variable light-chain (VL), amino acid sequence of an immunoglobulin tethered to a variable heavy-chain (VH) sequence by a designed peptide. We show that this protein, derived from the DNA sequence of the variable regions of the antitumor monoclonal antibody B6.2, has the same in vitro antigen-binding properties as the B6.2 Fab' fragment. Comparative pharmacokinetic studies in athymic mice demonstrate much more rapid alpha and beta phases of plasma clearance for the single-chain antigen-binding protein than for the Fab' fragment, as well as an extremely rapid whole-body clearance. Half-life values for alpha and beta phases of single-chain antigen-binding protein clearance were 2.4 minutes and 2.8 hours, respectively, versus 14.8 minutes and 7.5 hours for Fab'. Furthermore, the single-chain antigen-binding protein molecule did not show accumulation in the kidney as did the Fab' molecule or, as previously shown, the F(ab')2 molecule. Despite its rapid clearance, the single-chain antigen-binding protein showed uptake in a human tumor xenograft comparable to that of the Fab' fragment, resulting in tumor to normal tissue ratios comparable to or greater than those obtained with the Fab' fragment. These studies thus demonstrate the in vivo stability of recombinant single-chain antigen-binding proteins and their potential in some diagnostic and therapeutic clinical applications in cancer and other diseases.
We have studied the patterns of expression of various genes during maturation of normal human adult erythroid precursors cultured in a two-phase liquid culture method. In the first phase, peripheral blood mononuclear cells are cultured for one week in the presence of a combination of growth factors, but not erythropoietin (Epo). In Phase II, Epo is included in the medium. Cell samples were taken throughout phase II, and expression of globins, transcription factors, and cytokine receptors was assayed by RT-PCR and quantified by phosphor imaging. We have divided phase II into stages: early (days 0-5), intermediate (days 6-10) and late (days 11-15) and measured maximum expression of each gene. During early phase II, gamma-globin, Spl, and GATA-2 mRNAs were expressed at their highest levels. As the cells matured during the intermediate period, GATA-2 levels remained high, and then declined, while the transcription factors GATA-1, EKLF, NF-E2, and the Epo receptor (EpoR) reached maximum expression. In late phase II, beta-globin increased and reached its maximum level of expression. This erythroid culture system appears to recapitulate normal adult erythropoiesis at the molecular level, and thus may be a suitable model to examine the molecular basis of severe congenital or acquired disorders of erythropoiesis.
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