David T.W. Fei

Biological activity of bevacizumab, a humanized anti-VEGF antibody in vitro

Bevacizumab (Avastin™, Genentech) is a humanized monoclonal antibody targeting vascular endothelial growth factor (VEGF), a critical angiogenic factor involved in both physiological and pathological conditions. It has been recently approved by the US FDA as a first-line therapy for widespread metastatic colorectal cancer. This report is a detailed biological characterization of bevacizumab in a variety of in vitro models. It is shown that bevacizumab potently neutralizes VEGF and blocks its signal transduction through both the VEGFR-1 and VEGFR-2 receptors, as demonstrated by the inhibition of VEGF-induced cell proliferation, survival, permeability, nitric oxide production, as well as migration and tissue factor production. Although bevacizumab retains the ability to bind to human Fcγ receptors and complement protein C1q, it does not demonstrate cell or complement-mediated cytotoxicity in either VEGF producing or targeting cells. Thus the mechanism of anti-tumor activity of bevacizumab is most likely due to its anti-angiogenesis effect through binding and neutralization of secreted VEGF.

Cyclic AMP response to recombinant human relaxin by cultured human endometrial cells--a specific and high throughput in vitro bioassay.

A specific and high throughput 96-well format bioassay for recombinant human relaxin (rhRLX) has been developed using human endometrial cells (NHE cells). rhRLX caused a time- and dose-dependent stimulation of cyclic AMP (cAMP) with 1/2 maximal activity of 3.56 +/- 0.65 ng/ml (n = 30). The range of the standard curve was 0.39 to 25 ng/ml with interplate precision of 17 and 22% CV for high and low controls respectively. The cAMP response requires forskolin and 3-isobutyl-1-methylxanthine, and is enhanced by prostaglandin E2 and F2 alpha. The NHE cells do not respond to A or B chains of rhRLX, or a whole array of hormones. Preincubation of rhRLX with specific monoclonal antibody completely abolished the cAMP response. This bioassay has been used to determine the biological activity of several manufactured lots of recombinant human relaxin.

A sensitive fluorometric enzyme-linked immunosorbent assay that measures vascular endothelial growth factor165 in human plasma

A specific and sensitive fluorometric enzyme-linked immunosorbent assay (ELISA) was developed to measure endogenous levels of vascular endothelial growth factor (VEGF165) in human plasma. The ELISA can be performed in 10% human EDTA plasma, yielding a neat plasma sensitivity of 10 pg/ml or 0.2 pM. The recovery of recombinant human VEGF (rhVEGF) added to human plasma ranges from 89 to 100%. The capture antibody depletes the endogenous signal in normal human plasma, suggesting that the signal is specific for VEGF. The inter-assay and intra-assay coefficients of variation (CV) for the ELISA ranges from 5 to 14% and 8 to 18%, respectively. Characterization of the ELISA using plasmin derived VEGF variants suggests the assay is specific for the VEGF165 isoform. The heterodimer, VEGF(165/110) quantitates similar to that of the intact VEGF165 homodimer, however, the homodimers VEGF121, VEGF110 and the carboxy terminal domain (residues 111-165) are not detected in the assay. Circulating endogenous VEGF levels measured in 50 normal healthy individuals range from 20 to 141 pg/ml, with a mean of 42 +/- 22 pg/ml. There were no significant differences in VEGF levels between males and females. Circulating endogenous VEGF levels in cancer patients ranged from 32 to 418 pg/ml, averaging 129 +/- 17 pg/ml.

Allergen-induced histamine release in rat mast cells transfected with the α subunits of FcεRI

A rat mast cell histamine assay (RMCHA) has been developed to quantitate the biological activity of a recombinant humanized, monoclonal anti-IgE antibody (rhuMAbE25). Rat mast cells (RBL 48), transfected with the α subunit of the high affinity human IgE receptor (FceRI), were presensitized for 2 h with human plasma containing IgE specific for ragweed and challenged with ragweed allergen in the presence of 50% D2O. Histamine release plateaus at 0.1 μg/ml of ragweed. The release of histamine was time, temperature and Ca2+ dependent. This ragweed-induced histamine release could be inhibited by rhuMAbE25 in a dose-dependent fashion with an IC50 of 1.19 ± 0.31 μg/ml (n = 25). Other humanized MAbs and recombinant human growth factors neither trigger histamine release nor inhibit ragweed-induced histamine release. This RMCHA correlates well with the human basophil histamine assay (HBHA) (Fei et al., 1994) with a correlation coefficient of 0.93 (n = 59, p < 0.0001). Histamine was also released when the cells were presensitized with human plasma containing the respective allergen-specific IgE and then challenged with standardized mite, D. farinae, house dust mix, standardized cat pelt, or Alternaria tenuis. Comparison of allergen-induced histamine release showed a good correlation between RMCHA and HBHA with a correlation coefficient of 0.69 (n = 37, p = 0.0001). We conclude that RMCHA provides a useful tool to confirm allergen-specific IgE in allergic patients and can be used to evaluate the biological activity of any anti-IgE monoclonal antibody. Moreover, RMCHA provides an unique opportunity to study the mechanism of IgE-mediated histamine release in the absence of interfering proteins and growth factors normally present in whole blood.

Recombinant porcine prorelaxin produced in Chinese hamster ovary cells is biologically active

Although prorelaxin has a similar structure as proinsulin, the posttranslational processing of prorelaxin seems to be quite different from that of proinsulin. There are no pairs of basic residues flanking the relaxin moiety in most prorelaxins studied so far. Instead, the prorelaxins of many species contains a tetrabasic sequence (Arg-Lys-Lys-Arg) between the connecting peptide and the A-chain. This is the recognition sequence of furin. In order to study this possible processing by furin, we express the recombinant porcine prorelaxin in Chinese hamster ovary cells. The expected 19 kDa recombinant porcine prorelaxin was found to be constitutively secreted into the medium at a level of approximately 250 ng/ml. No conversion of the 19 kDa prorelaxin into the 6 kDa relaxin was observed. Unlike most prohormones which are biologically inactive, the recombinant prorelaxin was found to be biologically active in an in vitro bioassay.

Purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli

In this report we describe the purification and characterization of recombinant porcine prorelaxin expressed in Escherichia coli. Nucleotide sequence encoding porcine prorelaxin was inserted into an E. coli expression vector, pOTS, and the recombinant plasmid was transformed into the E. coli host (AR120). Upon induction with nalidixic acid, the 19-kDa recombinant porcine prorelaxin was produced at a level of approximately 8% of the total accumulated cell protein. The recombinant prorelaxin was purified to homogeneity by CM-cellulose chromatography and reversed-phase HPLC, after refolding in the presence of reduced and oxidized glutathione and a low concentration of guanidine-HCl. The identity of the recombinant prorelaxin was confirmed by the correct size, immunoreactivity with antibodies against native porcine relaxin, and direct amino-terminal sequence analysis. Furthermore, the purified recombinant prorelaxin could be converted to the 6-kDa relaxin by limited digestion with trypsin. Trypsin was shown to cleave at the carboxyl side of Arg29 and Arg137 residues of the recombinant prorelaxin, producing the des-ArgA1-B29-relaxin, and degrade the 13-kDa connecting peptide into small peptides. Both the recombinant prorelaxin and converted relaxin were found to be biologically active in an in vitro bioassay for relaxin.

A novel bioactivity assay for monoclonal antibodies directed against IgE

A novel bioactivity assay has been developed to quantitate the biological activity of a humanized, monoclonal anti-IgE antibody (rhuMAbE25) in human whole blood. Heparinized blood specimens from prescreened healthy donors were sensitized for 2 h with a constant amount of human plasma containing IgE specific for ragweed and then challenged with ragweed allergen. Histamine was released in a dose-dependent fashion and reached plateau levels after 30 min. As expected, the release of histamine by ragweed allergen was time, temperature and Ca2+ dependent, and could be enhanced by the presence of 33% deuterium oxide. Allergen-triggered release could be inhibited by rhuMAbE25 with an effective dose range from 0.1 to 1 microgram/ml. Preincubation with other humanized MAbs, which exhibit 95% homology to rhuMAbE25 but differ in epitope specificity, failed to inhibit the ragweed-induced histamine release. Overall, this bioactivity assay has a low interassay variability (%CV) of 17% (n = 23) and can be readily modified to determine if rhuMAbE25 or other anti-allergy therapeutics are capable of blocking histamine release elicited by other allergens. Moreover, the assay can be used to confirm IgE-mediated allergic responses and to provide early information regarding safety and potential efficacy of therapeutics aimed at blocking IgE dependent responses.

Multiplexed Serum Measurement of IgG, IgA, IgM, and IgE Antibody Responses to Therapeutic Biologicals

Analytical methods characterizing the immunogenicity of therapeutic proteins are useful for monitoring, characterizing and predicting reactions to biopharmaceuticals. A multiplexed assay capable of isotyping the specific IgG, IgA, IgM and IgE (IgGAME) antibody responses against a biotherapeutic was demonstrated in a hyper-immunized cynomolgus monkey, over a 15-month period. The quantitative range of the antibody measurements was determined to be 15 ng/ml to 50 ng/ml in 10% serum. By the use of any biotinylated or fluorescently tagged therapeutic as a detector, this multiplexed isotyping assay can be broadly applied to human and non-human primate IgG, IgA, IgM and IgE immunogenicity studies.