Melvin Silberklang

Cloning and expression of cDNA encoding antistasin, a leech-derived protein having anti-coagulant and anti-metastatic properties

As a factor Xa inhibitor, antistasin is a potent anti-coagulant and anti-metastatic agent that is found in the salivary gland of the Mexican leech Haementaria officinalis. cDNA clones that encode antistasin have been isolated. Subsequent sequence analysis and comparison with the amino acid sequence of the mature protein indicates that antistasin is produced as a pre-protein containing a 17-amino acid signal peptide. Antistasin exists as at least two variants. By sequence analysis of multiple cDNA clones, we found two additional sites for amino acid substitutions, confirming variants that differ from each other by amino acid changes at a minimum of four residues. These sequence variations appear to be the result of allelic variation rather than gene duplication as deduced from DNA blot analyses. Sequence data suggest that antistasin may have evolved from a smaller ancestral gene by a duplication event giving rise to a two-fold structural homology between the N- and C-terminal halves of the molecule. Insect cells transfected with a recombinant baculovirus expressed antistasin which was biologically active and had an electrophoretic mobility identical to that of the native molecule.

Antigenic analysis of the epstein-barr virus major membrane antigen (gp350/220) expressed in yeast and mammalian cells: Implications for the development of a subunit vaccine

The Epstein-Barr virus (EBV) major surface membrane antigen, gp350/220, was expressed in recombinant yeast cells and in several recombinant mammalian cell lines. Each of the expressed proteins was analyzed for its ability to bind to a panel of anti-gp350/220 monoclonal antibodies and to a series of anti-EBV positive human sera. The antigens also were used as immunogens for the immunization of rabbits. Each expressed protein was found to be unique both in its pattern of reactivity to the various antibodies and in the spectrum of antibody induced following animal immunization. These results suggest that cell-specific post-translational modifications critically influence the antigenic presentation of the expressed proteins. Nonetheless, all of the mammalian cell-derived versions of the membrane antigen were found capable of inducing EBV-specific neutralizing antibodies.

Use of Lipid Emulsions as Nutritional Supplements in Mammalian Cell Culture

The requirement of many mammalian cells grown in vitro for an exogenous source of lipids is usually met by the addition of protein-complexed lipids, such as those contained in serum, partially purified serum fractions, and lipid-rich serum albumin.’ We have been developing high productivity processes for the production of recombinant therapeutic monoclonal antibodies by NSO cells. This cell line, like the parental NS1 cell line? requires lipids and, in particular, cholesterol for optimal growth. In contrast to serum-derived lipid components, lipid emulsions can be readily customized to meet the nutritional needs of different cell lines, either as supplements or complete replacements for lipoproteins. In addition, emulsions offer the advantages of obviating any concern about unknown adventitious contaminants, which might be present in serum-derived components, as well as considerably reducing media costs. For application to commercial large-scale cell culture operations, suitable lipid emulsions should be compatible with filter sterilization and should be capable of formulation by a readily scalable method. However, many of the current methods for manufacture of nutritional lipid emulsions for cell culture rely on extensive vortexing3 or s~nication.~ In search of a more scalable emulsification technology, we investigated the use of a high pressure homogenizer, the Microfluidics Microflui d i ~ e r . ~ . ~ We have found this instrument convenient to generate stable lipid emulsions that could be filter sterilized and could substitute for lipoproteins in serum-free cell culture media.

Expression and characterization of the N-terminal half of antistasin, an anticoagulant protein derived from the leech Haementeria officinalis

Antistasin, a 15-kDa anticoagulant protein isolated from the salivary glands of the Mexican leech Haementeria officinalis, has been shown to be a potent inhibitor of factor Xa in the blood coagulation cascade. Antistasin possesses a twofold internal homology between the N- and C-terminal halves of the molecule, suggesting a gene duplication event in the evolution of the antistasin gene. This structural feature also suggests that either or both halves of the protein may possess biological activity if expressed as separate domains. Because the N-terminal domain contains a factor Xa P1-reactive site, we chose to express this domain in an insect cell baculovirus expression system. Characterization of this recombinant half antistasin molecule reveals that the N-terminal domain inhibits factor Xa in vitro, with a K(i) of 1.7 nM.

Derivation of Therapeutically Active Humanized and Veneered Anti-CD18 Antibodies

The identification and production of murine monoclonal antibodies (MAbs) has led to numerous therapeutic applications of these exquisitely specific molecules to human disease. The technologies of molecular biology have further expanded the utility of many antibodies by allowing the creation of class-switched molecules, whose functionality has been improved by the acquisition or loss of complement fixation. The size of the bioactive molecule may also be reduced so as to increase the tissue target availability of the antibody, either by changing the class from an IgM to an IgG, removing most of the heavy chain constant region in the creation of a F(ab′)2, or severely truncating both heavy and light chain constant regions with the formation of an Fv antibody. Common to all of these potentially therapeutic forms of antibody are the requisite CDRs (complementarity-determining regions), which guide the molecule to its ligand, and the framework residues (FRs), which support the latter structures and dictate the disposition of the CDRs relative to one another.