Preparation of a trivalent antigen-binding construct using polyoxime chemistry: improved biodistribution and potential for therapeutic application.

In an attempt to improve the pharmacokinetic behavior of an antitumor radioimmunoconjugate, we have prepared a trivalent antigen-binding construct formed from three Fab' fragments derived from the parent murine monoclonal antibody (MAb) 35 directed against the carcinoembryonic antigen. The construct was generated by a novel approach using polyoxime chemistry. This approach leads to a homogeneous construct, as judged by SDS-PAGE and by mass spectrometry, which was found to retain full immunoreactivity.

In vitro and in vivo comparison of a randomly coupled antibody fragment-enzyme conjugate with a site-specific conjugate.

Two antibody fragment-enzyme conjugates, one obtained by random coupling of the two protein component, the other by site-specific ligation of the same component, were compared in vitro and in vivo for their usefulness in antibody directed enzyme prodrug therapy (ADEPT). The in vitro studies have shown that the site-specific conjugate has a higher antigen binding capacity, while both conjugates had similar specific enzymic activities. In vivo, the site-specific conjugate was cleared more rapidly.

Preparation and characterization of novel substrates of insulin proteinase (EC 3.4.99.45).

The specificity of insulin proteinase (EC 3.4.99.

Site-specific conjugation of an enzyme and an antibody fragment.

A site-specific immunoconjugate was prepared between an F(ab')2-like fragment of the monoclonal anti-CEA murine IgG1 A5B7 and a mutant of the dimeric enzyme carboxypeptidase G2 possessing an N-terminal Thr in place of Ala. First an aldehyde was introduced at the N-terminus of the enzyme by mild periodate oxidation and a residue of carbohydrazide was specifically introduced at the C-terminus of the truncated heavy chain of the F(ab')2-like fragment by reverse proteolysis. Then the two modified proteins were conjugated by the formation of a hydrazone bond between the hydrazide and the aldehyde groups.

Protein conjugates of defined structure: synthesis and use of a new carrier molecule.

A new carrier molecule, NH2OCH2CO-(Gly)3-[Lys(H-Ser-)]5-Gly-OH, has been synthesized to facilitate the preparation of protein conjugates of defined structure. Special features are as follows: (i) (aminooxy)-acetyl as a terminal group, which reacts specifically to form an oxime bond under very mild conditions with an aldehyde group placed on a protein in a prior step; (ii) a spacer group of three Gly residues; and (iii) a set of five Lys residues, each of which is acylated with a Ser residue. A second form of the carrier molecule, HCO-m-C6H4CH = NOCH2CO-(Gly)3-[Lys(H-Ser)]5-Gly-OH, was also prepared.