1. Purification and Characterization of Fibroblast Growth Factor-like Antibodies

2. Introduction: bFGF  Basic fibroblast growth factor (bFGF), is a single-chain polypeptide composed of 146 amino acids.  bFGF is a normal component of bone extracellular matrix as well as a constituent of various normal and tumor tissues that does not normally circulate in the serum of healthy non-pregnant adult humans.  Previous studies have determined that bFGF has potent mitogenic and neurotrophic functions, meaning it promotes the growth and proliferation of certain types of cells (i.e. endothelial cells) as well as survival and neurite outgrowth of brain neurons in vitro and in vivo. (Zimering, M. B. et al. Circulating Fibroblast Growth Factor-Like Autoantibodies in Two Patients with Multiple Endocrine Neoplasia Type 1 and Prolactinoma. Journal of Clinical Endocrinology and Metabolism 79.6 (1994) 1546- 5152. )

3. Introduction: bFGF-like Antibodies  A bFGF-like substance has been identified by prior research in the plasma of patients with various cancers of the endocrine system.  This bFGF-like substance is an immunoglobulin G, which may have been created as an antibody against anti-bFGF antibodies.  The creation of such antiidiotype antibodies is not uncommon for the immune system.  In prior experiments, strong evidence has been gathered that the bFGF- like antibody mimics the growth-promoting effects of bFGF.  An antibody remains in circulation much longer than bFGF and is much more gradually degraded in plasma, thus making bFGF-like antibody of considerable interest in procedures that require the growth- promoting effects of bFGF. (Zimering, M. B. et al. Circulating Fibroblast Growth Factor-Like Autoantibodies in Two Patients with Multiple Endocrine Neoplasia Type 1 and Prolactinoma. Journal of Clinical Endocrinology and Metabolism 79.6 (1994) 1546-5152. )

4. Primary Hypothesis  Purification of bFGF-like antibody samples enhances the biological activity (i.e. growth- promoting effects in endothelial cells) of the these samples.

5. Purpose of Study To purify and characterize the biochemical properties of the bFGF-like antibody. To answer questions about the activity of the antibody at various dilutions so as to determine peak biological activity. Ultimately, purification of the antibody is a means at deriving its amino acid sequence, at which point it might be synthesized in vitro.

6. Methods & Materials: Methods of Purification  Protein-A affinity chromatography: To separate the immunoglobulin G (IgG) fraction from plasma, 0.5-mL aliquots of plasma were applied to a 1-mL column of packed protein-A beads. The column was washed with 5 mL 10 mM Tris (flow-through fraction), and then eluted stepwise with 5 x 1.0 mL.1 M (mol/L) citric acid, pH 3.0. The second and third eluate fractions were pooled, and the rest were discarded  Hydroxyapatite chromatography: To further purify the IgG fraction from plasma, 0.5 mL aliquots of protein-A-eluted fractions were applied to a hydroxyapatite column. The column was washed with 5 mL of 10 mM sodium phosphate (flow- through fraction). Subsequently, the column was eluted with a linear gradient of sodium phosphate (50-400 mM). The eluates were collected as separate 1 mL fractions.  Antihuman IgG affinity chromatography: To further purify the IgG fractions obtained from hydroxyapatite chromatography, 0.5 mL aliquots of the 400 mM hydroxyapatite fraction were applied to an immunoaffinity column. The column was washed with 10 mL of 10mM Tris and eluted with a pH gradient of citric acid (3.3 - 2.3 pH). The eluate were collected as separate 1.5 mL fractions of different pH.

7. Methods & Materials: Bioactivity Assays, Protein Determination, & Mass Spec.  Protein determination: Dye was added to minute amounts of the samples as well as to protein standards of known concentrations. Upon comparing the absorbance readings of the known and the unknown proteins, the protein concentrations of the laboratory samples were determined.  Mass spec: Modern time-of-flight mass spectrometry was used to determine the precise molecular weights of multiple proteins from various laboratory samples (e.g. hydroxyapatite fractions and protein-A eluate).  Colorimetric assay: After being plated into 96-well plates along with test fractions, endothelial cells were allowed to incubate for 4 days. Then, cell-associated bioactivity was determined by colorimetry in an MR 700 plate reader (Dyna-Tech). Growth-promoting activity is expressed as a percentage of the control cell number for cells grown in medium 199 and 10% fetal calf serum without added test fractions. In the graphic representation of the results, each point represents the mean of readings from 6 wells.

8. Results  Stepwise purification of the IgG fraction from plasma resulted in a diminishing concentration of protein but a gradually rising percentage of bioactivity among the fractions.

9. The optimum bioactivity of the protein-A eluate was 128%. The protein concentration of the eluate was determined to be.5466 mg/mL.

10.  The 400 mM hydroxyapatite fraction eluted from a sample of the eluate showed more bioactivity (149%) than the protein-A eluate itself, while having a protein concentration of.2516 mg/mL. The 400mM fraction displayed more bioactivity with a smaller amount of protein than the eluate.

11.  In the next purification step, a 400mM fraction of the eluate was applied to an immunoaffinity column. The fraction eluted at a pH of 3.3 displayed 153% bioactivity with a protein concentration of.0129 mg/mL. This fraction displayed more bioactivity with a substantially smaller amount of protein.

12. To further characterize the antibody, a separate protein-A eluate was taken out to different dilutions. The resulting dose-proliferation curves of bioactivity in endothelial cells showed a sharp rise in activity at a 1:1600 dilution, and a subsequent drop at higher dilutions. The activity of the antibody reached its peak at a dilution of 1:1600.

13. Results When an older eluate (patient 89) was examined with mass spec, the result was an extremely sharp peak, which is an indicator of substantial purity. The peptide which induced this peak was of a molecular weight of approximately 13 kDa. This peptide was subsequently sequenced and the first 12 amino acids of it’s amino acid sequence were determined.

14. Discussion  As the IgG fraction was further purified the concentration of protein in the purified samples was reduced as their bioactivity rose.  This suggests that the methods use in conducting the purification were successful in getting rid of proteins with inhibitory effects, thus allowing the protein in the sample to be more active despite being at a lower concentration.  It is likely that this is because the inhibitory effects of other proteins mask the biological activity of the bFGF-like antibody that is the subject of the current research.  Hence, samples that are less pure exhibit less bioactivity than purer samples despite having higher protein concentrations.

15. Discussion  The further purification of bFGF-like substances is an end toward which the current study strives. Once the antibody of interest can be isolated in a relatively pure state, it's amino acid sequence might be determined just as some of the amino acid sequence of the older eluate was.  Determining the sequence of the peptide in the older eluate offers strong evidence that the bioactivity of the fraction and others like it that were used in laboratory experiments is indeed the result of an IgG.  The derived amino acid sequence is probably the degraded part of the variable region of one of the light chains from an IgG.  Prior to this discovery, it was not unlikely that contamination of samples by a small growth factor was the cause of bioactivity in laboratory samples.

16. Acknowledgements  I thank Dr. Mark B. Zimering, MD PhD, for his intellect and good humor in the lab as well as providing the opportunity to conduct this research.  I thank Mrs. Jeanette Kim for her diligence in making SRTP enjoyable.  I thank Dr. Robert Donnelly, PhD for running our laboratory samples over mass spectroscopy.  I thank the VA for providing the laboratory facility.