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The development and validation of an ultra-sensitive double antigen ELISA method for the determination of bevacizumab (Avastin®) in human serumJamil Hantash and Tony Joaquim

PurposeBevacizumab (Avastin®) is a recombinant human IgG1 monoclonal antibody specifi c for all human vascular endothelial growth factor-A (VEGF-A) isoforms (Figure 1). In 1997, the humanization of the murine anti-VEGF Mab A.4.6.1. was reported.1 Like its murine counterpart, bevacizumab binds to and neutralizes all human VEGF-A isoforms and bioactive proteolytic fragments, but not mouse or rat VEGF.1 However, bevacizumab was observed to inhibit the growth of human tumor cell lines in nude mice. In addition, studies have demonstrated that bevacizumab, in combination with chemotherapy, resulted in increased survival in patients with previously untreated metastatic colorectal cancer relative to chemotherapy alone, leading to FDA approval of the fi rst anti-angiogenic agent.2 Anti-VEGF monoclonal antibodies and other VEGF inhibitors block the growth of several tumor cell lines in nude mice.

Clinical trials with VEGF inhibitors in a variety of malignancies are ongoing.3 The humanized anti-VEGF monoclonal antibody, bevacizumab, has been approved by the FDA as a fi rst-line treatment for metastatic colorectal cancer in combination with chemotherapy. Furthermore, VEGF is implicated in intraocular neovascularization associated with diabetic retinopathy and age-related macular degeneration. The pharmacokinetic properties of bevacizumab in several species have been previously described and are consistent with a typical humanized monoclonal antibody. In 1997, Phase I clinical trials with bevacizumab were initiated. These Phase I studies showed that the antibody as a single agent was relatively non-toxic and that adding it to standard chemotherapy regimens did not signifi cantly exacerbate chemotherapy-associated toxicities.4 In 1998, several Phase II studies were initiated with bevacizumab in different tumor types, either as a single agent or in combination with chemotherapy.4

There is growing evidence that VEGF and VEGF receptors are expressed by a variety of leukemias and other hematologic malignancies, suggesting that inhibition of VEGF or VEGFR signaling may have a role in the treatment of such conditions. Several clinical trials are currently testing these hypotheses. Although bevacizumab was generally well tolerated, some serious and unusual toxicities were noted. Some open-label Phase I and II clinical trials had identifi ed a number of adverse events, including thrombosis and bleeding as potential bevacizumab-related toxicities. In addition, most common adverse reactions are epistaxis, headache, hypertension, rhinitis, proteinuria, taste alteration, dry skin, rectal hemorrhage, lacrimation disorder, back pain and exfoliative dermatitis. Bevacizumab is dosed and administered up to 15 mg/kg (non- squamous non-small cell lung cancer: 15 mg/kg IV every three weeks with carboplatin/paclitaxel) in patients without evidence of dose-limiting toxicities.4 However, in case of overdose, it is recommended that the patient be monitored for any signs or symptoms of adverse reactions or effects and appropriate treatment instituted immediately. Serum through levels might be related to predict some clinical outcome during maintenance therapy. It was also possible that the surveillance of circulating concentration during maintenance therapy represents a direct and/or indirect factor for some other side effects. In this context, identifi cation of biomarkers for (non) response and risk factors for adverse drug reactions that might be related to serum drug levels and maintaining the effective minimum concentration in order to potentially avoid some side effects with a reliable method might be benefi cial.5

In this poster, we report on the development and validation of an ultra-sensitive method for assessment of the presence of this molecule, which may be used to support clinical trials.

Results continued

Results

Conclusion

References

The validated assay showed that it had suitable performance features necessary for supporting high-throughput, clinical trial pharmacokinetic end-point sample analysis.

1) Presta LG, Chen H, O’Connor SJ, Chisholm V, Meng YG, Krummen L, Winkler M, and Ferrara N. Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders. Cancer Res 1997 Oct 15; 57(20):4593-9.

2) Ohtsu A, Shah MA, Van Cutsem E, Rha SY, Sawaki A, Park SR, Lim HY, Yamada Y, Wu J, Langer B, Starnawski M and Kang YK. Bevacizumab in combination with chemotherapy as fi rst-line therapy in advanced gastric cancer: a randomized, double-blind, placebo-controlled phase III study. J Clin Oncol 2011 Oct 20; 29(30):3968-76.

3) Ferrara N. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 2004 Aug; 25(4):581-611.

4) Ribatti D. History of Research on Tumor Angiogenesis. Springer Netherlands (2009).

5) http://www.antibodies-online.com/kit/1540255/Bevacizumab+ELISA/

6) Clarke JM and Hurwitz HI. Understanding and targeting resistance to anti-angiogenic therapies. Journal of Gastrointestinal Oncology (Sept 2013): 4(3).

7) http://www.epitomics.com/products/product_info/6117-1

FIGURE 1 VEGF-axis dependent and non-VEGF mediated

mechanisms of resistance to anti-angiogenic

therapies. General mechanisms of action for

bevacizumab activity of RTK inhibitors are

depicted. Novel targeted therapies include

TRC105, PF-03446962, LY2157299, demcizumab,

MEDI0639, and RGN-421 inhibiting TGF-β and

Dll4-notch signaling.6

TABLE 3Dilution Integrity

Nominal concentration (ng/mL)

Dil QC Back calculated

concentration (ng/mL)

Dil QC Back calculated

concentration after multiplying with the DF (ng/mL)

1000 900.0 90000.0

800 759.5 94937.5

600 615.5 102583.3

400 425.8 106450.0

300 308.9 102966.7

150 165.0 110000.0

Mean concentration found (ng/mL)

- 101156.3

Inter-run SD - 7411.9

Inter-run %CV - 7.3%

Inter-run %Bias - 1.2%

n - 6

TABLE 1Standards Performance

Run STD7 50.0

ng/mL

STD6 100

ng/mL

STD5 250

ng/mL

STD4 500

ng/mL

STD3 750

ng/mL

STD2 900

ng/mL

STD1 1000

ng/mL

1 49.7 100.2 255.0 499.1 640.8 790.2 1008.12 45.2 83.0 240.0 485.5 680.1 1015.2 1010.53 48.6 85.6 260.0 515.2 749.2 958.2 1020.04 47.5 84.0 255.8 518.2 850.9 999.1 1018.55 48.2 86.4 245.6 505.9 846.2 1035.0 1001.36 46.5 99.5 249.5 480.9 860.1 1034.2 1014.9

Mean Concentration Found (ng/mL)

47.6 89.8 251.0 500.8 771.2 972.0 1012.2

Inter-run SD 1.6 7.9 7.4 15.3 95.6 93.5 7.0Inter-run %CV 3.4% 8.8% 2.9% 3.1% 12.4% 9.6% 0.7%Inter-run %Bias -4.8% -10.2% 0.4% 0.2% 2.8% 8.0% 1.2%n 6 6 6 6 6 6 6

TABLE 2 Precision and accuracy

Run LLOQ QC

100.0 ng/mL

Low QC 200

ng/mL

Mid QC 400

ng/mL

High QC 800

ng/mL

ULOQ QC 1000

ng/mL

1 (mean of n = 3) 85.2 180.5 335.8 800.4 1100.02 (mean of n = 3) 95.4 190.5 338.1 801.5 1149.53 (mean of n = 3) 80.2 189.5 338.1 803.5 1140.54 (mean of n = 3) 80.5 200.3 402.5 804.9 1125.85 (mean of n = 3) 80.6 210.8 415.4 800.4 1000.56 (mean of n = 3) 95.2 205.8 445.2 800.7 935.0

Mean Concentration Found (ng/mL)

86.2 196.2 379.2 801.9 1075.2

Inter-run SD 7.3 11.4 47.9 1.9 87.4Inter-run %CV 8.5% 5.8% 12.6% 0.2% 8.1%Inter-run %Bias -13.8% -1.9% -5.2% 0.2% 7.5%

n 18 18 18 18 18

FIGURE 4 Quality control sample performance

FIGURE 3 Representative standard curve 5PL regression

FIGURE 5 Dilution linearity performance

FIGURE 6 Spiked selectivity lots performance

MethodsSolid phase enzyme-linked immunosorbent assay (ELISA) based on the double antigen assay principle. Diluted standards, quality controls and human sera samples were incubated in microtiter plates coated with human vascular endothelial growth factor (VEGF). After incubation, the wells are washed. A biotin-conjugated human VEGF was then added and allowed to bind to bevacizumab (Avastin®) captured by the reactant on the surface of the wells. Following incubation, wells are washed and then streptavidine–horse radish peroxidase (HRP) was added and incubated; then, the wells were washed and the bound enzymatic activity was detected by addition of chromogen-substrate. The color that developed was proportional to the amount of bevacizumab in the sample, quality control sample, or standard.

FIGURE 2 Sandwich ELISA Illustration or the Assay Protocol Steps7

The assay was validated under Tandem Labs standard operating procedures and in compliance with the FDA draft guidance document. The pooled human sera were spiked with bevacizumab at 50, 100, 250, 500, 750, 900 and 1000 ng/mL. The validated assay had a detection limit of 100 ng/mL. The linear calibration curve ranged from 100 to 1000 ng/mL with bias ranging from -10.2% to 8.0%, using a 5PL regression. The bevacizumab quality control (QC) samples were prepared in pooled human sera at 100, 200, 400, 800 and 1000 ng/mL and analyzed in six different assays (n = 3 at each level). The QC samples had CVs ranging from 0.20 to 8.1% and bias ranging from -5.2 to 8.50%.

Dilution linearity QC spiked with bevacizumab at 100,000 ng/mL was diluted using negative pooled sera. The diluted QC samples had CVs ranging from 0.57 to 4.6% and bias ranging from -8.13 to 6.27%. Selectivity samples (ten independent human sera lots) spiked with bevacizumab at 100 ng/mL showed bias ranging from -10.2 to 12.6% with all ten lot recovering at within ±20% of nominal concentration.

Figure obtained from http://www.antibodies-online.com/kit/1540255/Bevacizumab+ELISA/