ABSTRACT
Antibody-drug conjugates utilize the targetting potential of antibodies to improve the potential of cytostatic or cytocidal drugs. One such murine monoclonal antibody, CTM01 (mCTM01), which recognizes an epitope on breast epithelial mucin, has potential for the treatment of breast and ovarian cancers. We examine in this paper the comparative properties of mCTM01 against a number of other anti-mucin antibodies. We then describe the humanization and high level re-expression of humanized CTM01 (hCTM01), a process designed to avoid the immune response to administered murine antibodies in human patients and to produce sufficient material for clinical studies. We show that the humanized form has properties superior to mCTM01 in terms of binding affinity to antigen presented on tumour cells.
Subject(s)
Antibodies, Monoclonal/chemistry , Antigens, Neoplasm/immunology , Breast Neoplasms/immunology , Immunotherapy , Membrane Glycoproteins/immunology , Mucins/immunology , Ovarian Neoplasms/immunology , Protein Engineering , Recombinant Fusion Proteins/chemistry , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , Antibody Affinity , Breast Neoplasms/therapy , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mice , Minisatellite Repeats , Molecular Sequence Data , Mucin-1 , Neoplasm Proteins/immunology , Ovarian Neoplasms/therapy , Peptide Fragments/immunology , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/therapeutic useABSTRACT
An equimolar solution of aldoxime and tetramethylguanidine at 70 degrees C removes both the base and phosphate protection from oligonucleotides prepared by solid phase phosphate triester technology. The rate of cleavage from the succinyl linkage commonly used for solid phase synthesis is also increased. The method is simpler, faster and more easily automated than existing methods.
Subject(s)
DNA/chemical synthesis , Oligodeoxyribonucleotides/chemical synthesis , Oligonucleotides/chemical synthesis , Chemical Phenomena , Chemistry , Chromatography, High Pressure Liquid , Chromatography, Ion Exchange , Indicators and Reagents , Kinetics , OrganophosphatesABSTRACT
A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.
Subject(s)
Oligodeoxyribonucleotides/chemical synthesis , Oligonucleotides/chemical synthesis , Benzenesulfonates , Indicators and Reagents , Kinetics , Methylene Chloride , Solvents , Temperature , Trityl CompoundsABSTRACT
DNA complementary to calf stomach mRNA has been synthesised and inserted into the Pst1 site of pAT153 by G-C tailing. Clones containing sequences coding for prochymosin were recognised by colony hybridisation with cDNA extended from a chemically synthesised oligodeoxynucleotide primer, the sequence of which was predicted from the published amino acid sequence of calf prochymosin. Two clones were identified which together contained a complete copy of prochymosin mRNA. The nucleotide sequence is in substantial agreement with the reported amino acid sequence of prochymosin and shows that this protein has a mol.wt. of 40431 and chymosin a mol.wt. of 35612. The sequence also indicates that prochymosin is synthesised as a precursor molecule, preprochymosin, having a 16 amino acid hydrophobic leader sequence analogous to that reported for other secreted proteins.
