Biomedical subjects

R E Offord

Publications and source records attributed to R E Offord.

117 records · Page 7Linked to original sources

PubMedDate not supplied

Site-specific modification of a fragment of a chimeric monoclonal antibody using reverse proteolysis.

We propose a novel method for the site-specific labeling of antibodies under mild conditions and give as an example the modification of an F(ab')2-like fragment of the chimeric monoclonal antibody B72.3. The F(ab')2-like fragment was produced by the action of the protease lysyl endopeptidase. Reverse proteolysis, catalyzed by the same enzyme, was then used to attach carbohydrazide specifically to the carboxyl termini of the heavy chains of the fragment. Finally, a radiolabeled chelator possessing an aldehyde group was conjugated to the modified fragment through a hydrazone linkage. The resulting site-specifically labeled F(ab')2-like fragment was characterized by gel electrophoresis and by enzymic digestion. It was found to possess immunoreactivity equivalent to that of the unmodified F(ab')2-like fragment as determined by immunofluorescence and ELISA (enzyme-linked immunosorbent assay) techniques. The advantages and disadvantages of this labeling method, which appear to be of quite general applicability, are discussed.

Antibodies, Monoclonal↗

PubMedDate not supplied

Construction of protein analogues by site-specific condensation of unprotected fragments.

The extreme sensitivity to periodate of 1-amino, 2-hydroxy compounds permits the selective conversion of N-terminal serine and threonine to an aldehydic group. We have used this reaction to construct analogues of human granulocyte colony stimulating factor (G-CSF) by allowing such oxidized peptides to react with others that have had a hydrazide derivative attached to the C-terminus by reversed proteolysis. Two recombinant analogues of G-CSF were used as starting materials. Both had only a single lysine residue (at position 62 and 75, respectively) followed immediately by a serine. Digestion of each analogue by the lysine-specific protease from Achromobacter lyticus gave two fragments, one of which could be N-terminally oxidized and the other converted to the C-terminal hydrazide derivative by reversed proteolysis using the same enzyme. After preliminary studies with model peptides, we first reacted the corresponding peptide pairs together and then, in order to eliminate the 64-74 disulfide loop, fragment 1-62 from the first analogue with fragment 76-174 from the second. Reactions are efficient (up to 80% product based on the oxidized fragment) and take place under very mild conditions. The hydrazone bond can easily be stabilized by reduction with NaBH3CN. This method represents a new, reasonably general route for the construction of large protein chimeras of precisely controlled structure.

Amino Acid Sequence↗

PubMedDate not supplied

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. This form possesses a terminal aldehyde group which permits site-specific attachment by formation of a hydrazone bond to the carboxyl termini of polypeptide chains which have been modified enzymatically with carbohydrazide in a prior step. Once the carrier is linked to protein in one of the above ways, i.e. through formation of either an oxime or hydrazone bond, the Ser residues of the carrier (but not of the protein) may be oxidized by very mild periodate treatment to generate aldehyde groups. Drugs possessing a hydrazide group (e.g. methotrexate gamma-hydrazide or desacetylvincaleukoblastine hydrazide) may then be conjugated via hydrazone formation to the aldehyde groups of the carrier. A cluster of five drug molecules may thus be attached to a single site on a protein, giving a relatively homogeneous product in spite of the high drug conjugation ratio. Synthesis of the carrier, formation of a pentadrug-protein conjugate, and wider implications of the chemistry are presented.

Amino Acid Sequence↗

PubMedDate not supplied

Site-specific religation of G-CSF fragments through a thioether bond.

A new approach is described for linking, through a thioether bond, the C-terminus of one unprotected polypeptide with the N-terminus of another. Homocysteine thiolactone is attached to the C-terminus of one polypeptide by reverse proteolysis and provides through hydroxylamine treatment a free sulfhydryl group. The alpha-amino group of a second polypeptide is selectively iodoacetylated by reaction with iodoacetic anhydride at pH 6.0 or the N-hydroxysuccinimide ester derivative at pH 7.0. Coupling of the two modified fragments occurs in a spontaneous alkylation reaction under mild conditions. After preliminary experiments with small peptides, this approach was extended to large protein fragments derived from recombinant analogs of G-CSF by enzymatic digestion. This approach provides a means of making head-to-tail protein chimeras or introducing noncoded structural elements into a protein.

Acetylation↗

PubMedDate not supplied

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. The conjugate obtained retained both enzymic activity and antigen-binding capacity. The antigen-binding capacity was better than that of a similar conjugate made conventionally by random reaction with side chains.

Antibodies, Monoclonal↗

PubMedDate not supplied

High yield, site-specific coupling of N-terminally modified beta-lactamase to a proteolytically derived single-sulfhydryl murine Fab'.

The preparation of bispecific protein conjugates capable of performing diverse biological functions is an area of active investigation. Such conjugates are routinely prepared using techniques which employ random derivatization of lysine residues, but the overall utility of these methods is limited due to poor yields and heterogeneous conjugates. In this report we describe the development of site-specific linkage methodology for the chemical synthesis of a homogeneous enzyme-antibody Fab' conjugate with coupling efficiencies of at least 72%. The N-terminal threonine residue of beta-lactamase from the P99 strain of Enterobacter cloacae was oxidized to an aldehyde functional group under mild conditions with a 5-fold molar excess of sodium periodate. The murine Fab' with a single sulfhydryl at the hinge region was generated by further digestion of the peptic Fab' fragment with lysyl endopeptidase to remove a decapeptide containing two of the three cysteine residues. Coupling of the two modified proteins was accomplished through a bifunctional coupling reagent containing meleimide and aminooxy functional groups. Synthesis of the linker is described. Yields of 1:1 enzyme-Fab' were at least three times higher than for comparable random derivatization methods. Immunoreactivity and enzymatic activity were unaffected. Biodistribution studies showed a more favorable tumor to blood ratio with the site-specifically linked conjugate.

Amino Acid Sequence↗

PubMedDate not supplied

Site-specific attachment of functionalized poly(ethylene glycol) to the amino terminus of proteins.

A convenient method for the construction of site-specifically modified poly(ethylene glycol)-protein conjugates is described. This method relies on the ability to generate a reactive carbonyl group in place of the terminal amino group. If the protein has N-terminal serine or threonine, this can be done by very mild periodate oxidation and generates a glyoxylyl group. A method less restricted by the nature of the N-terminal residue, but which requires somewhat harsher conditions, is metal-catalyzed transamination, which gives a keto group. The N-terminal-introduced reactive carbonyl group specifically reacts, under mild acidic conditions, with an aminooxy-functionalized poly(ethylene glycol) to form a stable oxime bond. Using polymers of different size and shape (linear or multibranched), various conjugates of IL-8, G-CSF, and IL-1ra were constructed and further characterized with respect to their biological activity and pharmacokinetic behavior in rats. Unlike most previous methods, this approach places a single PEG chain at a defined site on the protein. It should therefore be more likely to conserve biological activity when the latter depends on interaction with another macromolecule (unlike enzymic activity which often survives multiple PEGylation).

Animals↗

PubMedDate not supplied

Enzymatic semisynthesis of insulin specifically labelled with tritium at position B-30.

We have synthesized porcine insulin labelled with tritium at position B-30 using enzyme-catalysed formation of a peptide bond. The resulting insulin derivative has the label in the expected position and is biologically active. We have tested our procedure to prepare batches up to 50 muCi of tritiated insulin at a specific radioactivity of up to 1.14 Ci/mmol.

Alanine↗

PubMedDate not supplied

Semisynthesis of human growth hormone-releasing factor by trypsin catalyzed coupling of leucine amide to a C-terminal acid precursor.

Human growth hormone-releasing factor, GRF(1-44)-NH2, was synthesized by trypsin catalyzed coupling of Leu-NH2 to Arg43 of the precursor, GRF(1-43)-OH, prepared by solid phase peptide synthesis. The semisynthetic GRF(1-44)-NH2 was fully characterized and showed full potency in the rat pituitary in vitro bioassay. Conversion to GRF(1-44)-NH2 was limited to 60-70% in both 75% v:v N,N'-dimethylacetamide and 95% v:v 1,4-butanediol due to competing transpeptidations at Arg41 and Arg38 generating [Leu42]-GRF(1-42)-NH2 and [Leu39]-GRF(1-39)-NH2 side-products, respectively. The rates of formation and yields of GRF(1-44)-NH2 versus pH, Leu-NH2 concentration, and solvent composition were also studied.

Amino Acid Sequence↗