ABSTRACT
The recombinant humanized antibody (rhuMAb) VEGF has a high affinity for vascular endothelial growth factor and is currently being evaluated in clinical trials as a cancer therapeutic. Under acidic pH and low ionic strength conditions, the antibody was predominantly present as monomer. Under physiological conditions, the appearance of significant amounts of a noncovalent, reversible dimer were observed by size-exclusion chromatography. The kinetics and thermodynamics of the reversible self-association for rhuMAb VEGF monomer were investigated as a function of pH, temperature, and ionic strength by size-exclusion chromatography using the concentration jump method. The rate constant for dimer formation ranged 23-112 M(-)(1) min(-)(1) under the conditions studied, values that are significantly lower than those reported in the literature for other proteins that self-associate. The rate constant for dissociation ranged 0.0039-0.021 min(-)(1). Gibbs' free energies, enthalpies, entropies, and activation energies were determined and revealed that dimer formation is optimal at pH 7.5-8.0, which may be reflective of charge shielding occurring near the pI of the protein. There was a negative change in entropy for dissociation (values from -18.1 to -12.8 cal/mol K). In the presence of D(2)O or 1 M NaCl, dimerization was enhanced. The results of the kinetic and thermodynamic analysis of this study indicate that rhuMAb VEGF dimerization occurs primarily through hydrophobic interactions.
Subject(s)
Antibodies, Monoclonal/chemistry , Antibodies, Monoclonal/metabolism , Endothelial Growth Factors/immunology , Lymphokines/immunology , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Animals , Antibodies, Monoclonal/genetics , CHO Cells , Calcium Chloride/chemistry , Chromatography, Gel , Cricetinae , Dimerization , Humans , Hydrogen-Ion Concentration , Kinetics , Light , Models, Chemical , Osmolar Concentration , Scattering, Radiation , Sodium Chloride/chemistry , Temperature , Thermodynamics , Vascular Endothelial Growth Factor A , Vascular Endothelial Growth FactorsABSTRACT
To develop a candidate vaccine for human immunodeficiency virus, type 1 (HIV-1), chimeric proteins were constructed by inserting sequences derived from the V3 loop of gp120 into a nontoxic form of Pseudomonas exotoxin (PE). Inserts of 14 or 26 amino acids, constrained by a disulfide bond, were introduced between domains II and III of PE. V3 loop-toxin proteins expressed in Escherichia coli and corresponding to either MN (subtype B) or Thai (subtype E) strains, were recognized by strain-specific monoclonal anti-gp120 antibodies. When loop sequences were introduced into an enzymatically active form of the toxin, there was no loss of toxin-mediated cell killing, suggesting that these sequences were co-transported to the cytosol. Sera from rabbits injected with nontoxic PE-V3 loop chimeras were reactive for strain-specific gp120s in Western blots, immunocapture assays, enzyme-linked immunosorbent assays, and neutralized HIV-1 infectivity. Since toxin vectors were designed to receive oligonucleotide duplexes encoding any V3 loop sequence, this approach should allow for the production of V3 loop-toxin chimeras corresponding to multiple HIV isolates.
Subject(s)
ADP Ribose Transferases , AIDS Vaccines , Bacterial Toxins , Exotoxins/toxicity , HIV Envelope Protein gp120/toxicity , HIV-1/drug effects , Pseudomonas aeruginosa , Vaccines, Synthetic , Virulence Factors , Acquired Immunodeficiency Syndrome/blood , Acquired Immunodeficiency Syndrome/immunology , Amino Acid Sequence , Animals , Carcinoma, Squamous Cell , Cell Survival/drug effects , Enzyme-Linked Immunosorbent Assay , Escherichia coli , Exotoxins/chemistry , Exotoxins/immunology , HIV Antibodies/blood , HIV Antibodies/immunology , HIV Envelope Protein gp120/chemistry , HIV Envelope Protein gp120/immunology , HIV-1/isolation & purification , Humans , Molecular Sequence Data , Neutralization Tests , Protein Conformation , Rabbits , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/toxicity , Tumor Cells, Cultured , Pseudomonas aeruginosa Exotoxin AABSTRACT
Confluent monolayers of Caco-2 cells, a human colonic carcinoma cell line, have been used extensively to predict intestinal absorption. A direct comparison of uptake characteristics, however, between cell monolayers and human tissue is missing in the literature. We have determined the flux for a series of small organic molecules, peptide and protein therapeutics, across Caco-2 monolayers and normal human colonic and rectal tissue in vitro to assess whether or not a predictive correlation of transport exists. Caco-2 cells were grown to confluency of Snapwells, and human tissue was obtained from patients undergoing surgery for localized tumors. Mucosa-serosa fluxes were measured by HPLC for small molecules and peptides, and proteins were analyzed by ELISA or RIA. Permeability coefficients were calculated from flux data and compared with previously published coefficients where possible. The permeability coefficients for the examined molecules were of a similar magnitude across Caco-2 cell monolayers and human tissues, ranging from 10(-7) to 10(-5) cm/s. A best-fit analysis of a log-log plot of transport measurements obtained in these two systems gave a good correlation (R2 = 0.991). From this limited data set it appears that uptake characteristics for human colon and rectum are similar to those of Caco-2 cell monolayers. Thus, flux measurements across Caco-2 monolayers may be predictive for permeabilities of human colon and rectum for different classes of therapeutics.
