Anti-ranibizumab Neutralizing Antibody Assays

Ranibizumab Neutralizing 

Antibody Assay: 

Comparison of Competitive 

Ligand-Binding vs. Bioassay 

Formats 

Mauricio Maia, Ph.D. 

Bioanalytical Sciences 

Genentech 

PSWC2010-AAPS Annual Meeting – Nov 16, 2010 

Mauricio Maia Genentech 2010 1

Outline 

• Introduction to Ranibizumab 

• Nab Assay Challenges 

• Cell-based Assay 

• Ligand-binding Assay 



Lucentis ® 

• AKA “Ranibizumab” 

• 48 kD protein, Fab fragment of a humanized mAb 

• Binds to VEGF and inhibits its activities 

• Currently approved for AMD & RVO indications 

• Patients dosed ITV once a month 

• Endpoint = visual acuity 



Visual Changes in AMD 



Anti-Ranibizumab Antibody Assay 

Strategy 

Sample 

+ 

Screening 

Assay 

- 

Confirmatory 

Assay 

- 

Report 

+ 

Characterization (titration, neutralizing antibody 

assay) 



Anti-Ranibizumab Antibody Assay 

Strategy 

Sample 

+ 

Screening 

Assay 

- 

Confirmatory 

Assay 

- 

Report 

+ 

Characterization (titration, neutralizing antibody 

assay) 



Post-marketing Commitment 

“To detect and characterize immune responses to 

ranibizumab: 

…Develop and validate an assay to detect neutralizing 

anti-ranibizumab antibodies.” 



Challenges 

• Priority: Cell-based assay 

• Assay sensitivity (NAb vs. Screening) 

• Highly-variable effect of human serum matrix 

• Preferred format related to drug MOA 

• Positive control source, type 



NAb Assays – Points to Consider 

Nab assay measures opposite effect of drug 

The larger the drug-induced signal, the higher the Nab concentration 

needed to counter-act that effect 

Thus, in general, best Nab sensitivity achieved at lowest drug concentrations 

Challenge for inhibitory mAb therapeutic drugs: 

at low drug concentrations, the degree of signal inhibition 

is reduced, as well… 

Goal – select drug-inhibited effect that gives 

highest signal-to-noise with the lowest drug concentrations … 



Pharmacological Effects of 

VEGF 

Induced by hypoxia, injury, GH, cytokines 

Increases local angiogenesis 

Increases vascular permeability 

Induces vasodilation 

Stimulates release of cytokines 

Stimulates tissue factor expression 

Induces cell migration 

Survival factor 



Signal-to-Noise in “VEGF-Based” NAb 

BioAssays 

Goal – select drug-induced effect that gives highest signal-to-noise with the lowest drug concentrations 

Max Signal 

“Drop” 

NAb+ (high) 

Dynamic 

Range 

S/N 

ratio 

Non-specific 

Serum Effect 

NAb+ (low) 

Assay 

cutpoint 

Buffer 


VEGF 

Ranibizumab 


Anti-Ranibizumab NAb Assay Formats: 

Best Candidates 

• Inhibition of VEGF-induced HUVEC 

proliferation 

• Inhibition of VEGF-induced HUVEC tissue 

factor expression 

• Inhibition of Ranibizumab binding to VEGF 

(Competitive Ligand-Binding Assay) 



Anti-Ranibizumab NAb Bioassay: 

HUVEC Proliferation-Inhibition Assay 

1. Incubate Ranibizumab , rhVEGF, serum sample and HUVEC. 

Ranibizumab 

2. Incubate cells for 4 days. 

rhVEGF 

HUVEC cells 

Serum with Anti- 

Ranibizumab 

NAb 

3. Incubate cells with 10% Alamar Blue TM dye for 6 hrs. 

4. Detect Alamar Blue TM fluorescence at 530nm ex/590nm em. 

PSWC2010-AAPS Annual Meeting 

Nov 16, 2010 


Ranibizumab Dose-Response 

5800 

Inhibition of rhVEGF Induced-HUVEC (P3) Proliferation by Lucentis 

4800 

3800 

2800 

1800 

0.01 0.1 1 10 

Ranibizumab (ng/mL) 

Final Concentration of rhuFab V2 (nM) 

Ranibizumab 

y = ( (A - D)/(1 + (x/C)^B ) ) + D: A B C D R^2 

1 (VEGF165: Concentration vs Mean Value) 5004.349 4.709 0.418 2486.57 0.997 



V10/LU25/a-CDR 1000ng/mL 

V20/LU25/a-CDR 1000ng/mL 

Mean Alamar Blue Fluorescence 

Effect of Anti-Ranibizumab CDR Ab 

on HUVEC Proliferation NAb Assay 

4000 

3000 

Assay almost 10-fold less 

sensitive than screening 

assay 

2000 

1000 



HUVEC Proliferation NAb Assay 

Summary of Parameters Tested & Optimized 

• VEGF Concentrations* 

• Ranibizumab Concentrations 

• Cell density 

• HUVEC + Sample incubation time 

• Serum Concentration 

• ± Heat-inactivation of serum 

• Sample pre-incubation time 

‣ Approximate Sensitivity = 1000 ng/mL positive control pAb 

* Optimized concentration = 10 ng/mL (well-above 

ambient levels in serum); VEGF EC50 = 20-30 ng/mL 



Other Serum Growth Factors That May 

Affect HUVEC Proliferation 

• Angiopoietin 

• Fibroblast growth factor 

• Tumor necrosis factor-alpha 

• Apelin 

• Insulin-like growth factors 



CONCLUSIONS 

• BETTER ASSAY SENSITIVITY NEEDED 

• FIND AN ALTERNATIVE APPROACH 

Competitive Ligand-Binding Assay…….. 



Anti-Ranibizumab NAb Assay: 

Competitive Ligand-Binding Assay 

LIGHT 

X 

Sample: Anti- 

Ranibizumab 

antibody 

* 

BV-TAG- 

Ranibizumab 

* 

* 

Biotinylated-rhVEGF 

Streptavidin coated bead 



Assay Specificity 

Specific Antibodies 

2000 

Cyno anti-Ranibizumab CDR 

Rabbit anti-Ranibizumab 

Cyno anti-Bevacizumab CDR 

1500 

1000 

Non-Specific Antibodies 

Cyno antibodies to anti-CD11a mAb CDR 

Cyno antibodies to anti-CD20 mAb whole 

Goat antibodies to anti-CD20 mAb CDR 

Goat anti-huIgG Fab 

500 

0 

0.01 0.1 1 10 100 

Conc (nM) in NHS 

Interfering Molecules 

Ranibizumab (Unlabeled) 

rhVEGF (Unlabeled) 



However….. 

2000 

Specific Antibodies 

Cyno anti-Ranibizumab CDR 

Rabbit anti-Ranibizumab 

Cyno anti-Bevacizumab CDR 

1500 

1000 

500 

0 

0.01 0.1 1 10 100 

Interfering Molecules 

Ranibizumab (Unlabeled) 

rhVEGF (Unlabeled) 

Conc (nM) in NHS 

Data suggests that endogenous VEGF gives false(+) result in the NAb assay 



Therefore, need to first do….. 

VEGF removal may potentially remove all ranibizumab bound to VEGF in the samples, 

while not reducing NAb concentration, thereby improving assay’s drug tolerance 



ECLU Units 

Cutpoint Determination 

Sera from AMD Patients 

3500 

3000 

2500 

2000 

1500 

Cutpoint 

1000 

500 

0 

0 5 10 15 20 25 30 35 

AMD Samples 



ECLU Units 

Cutpoint Determination 

Sera from AMD Patients 

3500 

3000 

2500 

2000 

1500 

Cutpoint 

1000 

500 

0 

0 5 10 15 20 25 30 35 

AMD Samples 

Negative Serum - Avg 2419 

Average (panel): 1858 

SD 234.4 

1.65 SD 387 

CP Factor 0.608 

Cutpoint: 1472 

False Positive 

Rate: 6.7% (2 out of 30) 



Assay Reproducibility 

Mean ECLU 

Exp. Date Cutpoint Neg 

Control 

LC 

(1/6k dil.) 

HC 

(1/2k dil.) 

1 Day1 2019 3322 1771 889 

2 Day2 1465 2410 947 549 

3 Day2 1513 2488 1127 460 

4 Day2 1673 2751 1131 539 

5 Day3 1538 2530 1037 513 

6 Day3 1531 2518 1032 530 

7 Day4 1593 2620 993 504 

8 Day4 1706 2807 1070 607 

Mean 1630 2681 1139 574 

SD 177 292 263 134 

% CV 11 11 23 23 

N 8 8 8 8 



INTERFERENCE 

Effect of Ranibizumab on Anti-Ranibizumab NAb 

Detection 

Serum concentrations after monthly intravitreal injections of ranibizumab were in the range 

of 0.3ng/ml to 2.36 ng/mL, measured one day post-injection 



INTERFERENCE 

Effect of VEGF on Anti-Ranibizumab NAb Detection 

- Concentrations tested based on range of endogenous serum VEGF reported in literature 

- VEGF not expected to accumulate in serum via binding to Ranibizumab 

3000 

2500 

2000 

1500 

1000 

Assay Diluent (AD) 

1 ng/mL VEGF 

10 ng/mL VEGF 

100 ng/mL VEGF 

Cutpoint 

500 

0 

Negative Control 

+ 400 ng/mL Anti-CDR Ab 

Assay can tolerate close to 100 ng/mL of circulating VEGF 



Mean ECLU 

Matrix Interference: 

Sera from Individual Patients 

4000 

3500 

Nab spiked into sera from individual patients 

Sample alone 

400 ng/mL NAb 

3000 

2500 

2000 

1500 

Cutpoint 

1000 

500 

0 5 10 15 20 

Serum Sample of AMD Patient 



Summary of Assay 

Parameters 

Assay Parameter CLB Assay Bioassay 

Endpoint 

Inhibition of VEGF-A binding to 

drug 

Inhibition of Proliferation 

Minimal Required Dilution No dilution required 1:5 dilution in assay medium 

Sensitivity (ng/mL) ~ 200 ~ 1000 

Specificity 

No interference from endogenous 

VEGF-A 

Endogenous VEGF-A interference 

Drug Tolerance (ng/mL) 50 Not Determined 

Intra-assay Precision Not Determined Not Determined 

Inter-assay Precision

Fulfilling the PMC: 

Nab Data from 3 Pivotal AMD Trials 



Acknowledgements 

Genentech Bioanalytical Sciences 

John Lowe 

Eric Wakshull 

Valerie Quarmby 

An Song 

Jeriza Rusit 

Marco Palencia 

Patricia Siguenza 

Also: 

Ann Kearns 

Scot Collins 

Lisa Damico

Anti-ranibizumab Neutralizing Antibody Assays

Create successful ePaper yourself

Delete template?

Save as template?