A phase I clinical study of intratumorally administered VB4-845, an anti-epithelial cell adhesion molecule recombinant fusion protein, in patients with squamous cell carcinoma of the head and neck.

VB4-845 is a novel recombinant fusion protein that targets the epithelial cellular adhesion molecule (EpCAM). This initial clinical trial was conducted to determine the maximum tolerated dose of intratumoral injections in patients with advanced squamous cell carcinoma of the head and neck and to assess pharmacokinetics and immunogenicity. Twenty-four patients with advanced, recurrent squamous cell carcinoma of the head and neck received two cycles of five daily intratumoral VB4-845 injections of 20, 40, 80, 130, 200, or 280 microg. The maximum tolerated dose was established to be 280 microg administered daily for 5 days. Common adverse events were pain due to intratumoral injection and reversibly elevated liver enzymes. Of the 24 patients, 15 had detectable blood levels with a mean drug half-life of 4.0 +/- 0.3 h. VB4-845 reduced or stabilized tumors in 71.4% of epithelial cell adhesion molecule-positive patients. VB4-845 intratumoral injection therapy was well tolerated and feasible.

Fine specificities of murine anti-Mg monoclonal antibodies.

The specificities of two murine anti-Mg monoclonal IgG1 antibodies, 3B10 and 2D5, were determined by pepscan analysis. The peptides which correspond to various fragments of amino-terminal portions of glycophorin A of group M (GPA-M), N (GPA-N) and Mg (GPA-Mg), and replacement analogues of some of these peptides, were synthesized on plastic pins and tested for binding of the antibodies. Both antibodies bound strongly to the N-terminal Mg octapeptide 1LSTNEVAM8, but they showed different subspecificities. The essential fragment of the epitope 2D5 are amino acid residues 2STNEV6. Replacement of any of these amino acid residues by Ala, and replacement of Glu5 residue by Gly, abolished or strongly reduced the antibody binding, but replacement of Asn4 by Thr gave only a moderate decrease of peptide activity. In contrast, the Leu1 and Asn4 residues were most essential components of the epitope 3B10, while Ser2, Thr3 and Glu5 seemed to be less important. Our present results and earlier ones on the specificity of human anti-Mg alloantibodies and monoclonal anti-M/Mg antibodies showed that antibodies reacting with Mg antigen recognize different fragments and/or different amino acid residues of the amino- terminal nonglycosylated domain of GPA-Mg. The knowledge of fine specificities of antibodies reacting with Mg antigen is interesting in view of the presence of anti-Mg alloantibodies in 1-2% of human sera.

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Immunochemical characterisation of monoclonal antibodies directed to glycophorins A and/or B.

Among sixty-nine monoclonal antibodies submitted to the workshop, 28 antibodies directed to glycophorins A and/or B but without blood group specificity were investigated by a series of methods involving agglutination, flow cytometry with CHO transfected cells expressing glycophorin A, ELISA with a carbohydrate-free peptide (residues 1-72) of glycophorin A, and immunoblotting. These MAbs were subdivided in several groups according to their specificity: N-terminal portion of GPA and GPB; N-terminal trypsin-sensitive portion of GPA; extracellular ficin-sensitive portion of GPA; intracellular domain of GPA; undetermined. Both flow cytometry with transfectant cells and ELISA with the synthetic peptide prove to be of value in order to determine subspecificities within these groups.

Fine characterization of a series of new monoclonal antibodies directed against glycophorin A.

OBJECTIVES: Glycophorins A (GPA) and B (GPB) are the major sialoglycoproteins of the human erythrocyte (RBC) membrane. To prepare tools for the analysis of GPA and GPB, we produced a series of new monoclonal antibodies (mAbs) that identified epitopes of GPA. METHODS: Seven murine monoclonal antibodies directed to glycophorin A (GPA) were fully characterized by agglutination of untreated and enzyme-treated human erythrocytes, inhibition of agglutination using chemically modified glycophorins and peptides from GPA, immunoblotting, and binding to synthetic peptides on plastic pins. RESULTS: The antibodies identify epitopes located on four different portions of GPA. (1) NaM13-6D2 binds to the N-terminal portion of GPA and GPB carrying the N blood group antigen; (2) NaM26-3F4 recognizes the homologous portion of GPA and GPB corresponding to their amino acids 6-26; (3) NaM10-2H12, NaM16-IB10 and NaM10-6G4 are specific for the amino acid sequence 38-45 of GPA; and (4) NaM37-5F4 and NaM13-4E4 bind to the amino acid residues 119-124 located on the intracellular ponion of GPA. CONCLUSION: These antibodies represent precise tools to investigate GPA and related molecules in different cells and tissues.