Method establishment for characterization of anti-drug ...
1
Method establishment for characterization of anti-drug antibody responses against a Pegylated- G-CSF drug in human patients with breast cancer © 2015 CIRION BioPharma Research Introduction XM22 is a glycol-PEGylated recombinant human granulocyte colony-stimulating factor (G-CSF) that has been developed for the prevention of chemotherapeutic- induced neutropenia. A cascade of validated assays based on a risk-based approach is described for the evaluation of XM22 immunogenicity in human serum samples from breast cancer patients. Due to the nature of XM22, characterization of the binding specificity and neutralization of the drug and/or the endogenous counterpart (G-CSF) caused by the immunological response were performed. Methods Assay Cascade for Anti-XM22 Antibodies (ADA) Analysis Screening for ADA Confirmation Titer Confirmation The immunogenicity cascade begins with ADA detection using ADA binding assays, where screening positive samples are submitted to a confirmatory assay for XM22 specificity verification. Antibody titer in the confirmed-positive samples is then determined, along with a characterization of their binding specificity to protein and PEG moieties of XM22, and endogenous G-CSF. Confirmed-positive samples from the binding assays are also submitted to cell-based neutralizing antibody (NAb) assays to determine if they neutralize cell proliferation induced by XM22 or endogenous G-CSF; NAb-screening positive samples are tested in a confirmatory assay using an alternative inducer. ADA Binding Assays NAb Cell-Based Assays The binding assays use the ECL bridging format, where the bivalent property of anti-XM22 antibodies allow for simultaneous binding of a capture reagent (biotin-labeled XM22) and a detection reagent (TAG-labeled XM22). An electrical current causes the captured TAG to emit measureable light. To confirm antibody specificity to XM22 and characterize binding to drug moieties and/or the endogenous counterpart, unlabeled competitors (XM22, recombinant G-CSF, PEG and endogenous G-CSF) are added, causing a reduction in assay signal when competition is present. To compare relative ADA levels between samples, antibody titers are determined for every ADA confirmed-positive sample using serial dilutions. The cell based assays assess if samples confirmed-positive for ADAs contain NAbs that neutralize NFS-60 cell-growth induced by XM22 or endogenous G-CSF (WST-1 detection reagent). To be reported as NAb positive, a sample must show: Absence of cell growth induction when tested without inducer (assay suitability). Absence of inhibition of cell proliferation stimulated by an alternative inducer (mIL-3). NAb Results ADA Binding Assays Protein moiety of XM22 PEG moiety of XM22 Endogenous G-CSF Binding characterization Screening for NAb XM22 induction neutralization GCSF induction neutralization No induction (assay suitability) ADA ADA Assay Cut-Points Cut-points (positivity thresholds) were statistically determined using naïve serum samples to obtain pre-defined false-positive rates for each assay of the multi-tiered approach. Cut-point calculations were performed based on Shankar et al. 1 and Devanarayan & Tovey 2 . A summary of the various fixed/floating cut-points is presented in Table 1. Assay type Assay subtype False positive rate # batches, # samples Competitor Cut-point (%inhibition) Correction factor* Screening ---- 5% 4, 50 ---- ---- 14 ECL counts Confirmation ---- 1% 2, 54 XM22 28.5% ---- Characterization Drug G-CSF moiety 1% 2, 36 Filgrastim 22.0% ---- Drug PEG moiety 1% 2, 36 PEG 19.4% ---- Endogenous counterpart 1% 2, 36 G-CSF 17.5% ---- Table 1 Binding Assay Cut-Points * Floating cut-point is calculated by adding the correction factor to the mean run NC reading 1. Shankar et al. 2008. J. Pharm. Biomed. Anal. 48: 1267 – 1281. 2. Devanarayan & Tovey. 2011. Chapter 16 (289 – 308) in Detection and Quantification of Antibodies to Biopharmaceuticals. Wiley, Singapore, 404 pages. Positive Control Samples Positive control (PC) antibodies for the screening assay were surrogate affinity purified polyclonal anti-XM22 antibodies produced in rabbits. Titration of these antibodies in pooled normal human serum revealed a lack of hook effect at concentrations up to 60 000.0 ng/mL, and allowed for estimation of the assay LOD at 8.8 ng/mL (concentration at screening cut-point, Figure 1). The assay LOD was then confirmed by fortifying anti-XM22 antibodies in 15 breast cancer matrix lots at concentrations in the vicinity of the estimated LOD; the LOD was established as the lowest tested concentration with readings ≥ screening cut-point in at least 95% of qualified lots (to be qualified, naïve lots had to have a signal < screening cut-point). Figure 2 illustrates results for 3 tested concentrations, with a final LOD of 10.0 ng/mL. Assay type Assay subtype PC antibody Unit PC samples (ng/mL) Inter-run CV LPC MPC HPC Screening ---- Anti-XM22 ECL counts 468.8 3750.0 6000.0 7.4 - 8.8% Confirmation ---- Anti-XM22 %inhibition 500.0 ---- 6000.0 0.9 - 9.4% Characterization Drug G-CSF moiety Anti-XM22 %inhibition 500.0 ---- 6000.0 0.4 - 2.3% Drug PEG moiety Anti-PEG %inhibition 50.0 ---- 150.0 12.2 - 21.4% Endogenous counterpart Anti-XM22 %inhibition 500.0 ---- 6000.0 0.9 - 9.4% Table 2 Precision of PC Samples in Binding Assays NAb Cell-Based Assays NAb Assay Cut-Points Since these assays are based on cell proliferation, false-negative results could be obtained if the samples themselves induced cell-proliferation. This type of non-specific response was identified using a suitability assay, for which a “no-inducer” cut-point was determined. Samples inducing cell growth were therefore labeled as having an “inconclusive” result in the neutralizing assay. All determined cut-points are summarized in Table 4. Assay type False positive rate # batches, # samples Cut-point Correction factor* XM22 inducer 1% 3, 54 Ratio to VC of 0.824 ---- GCSF inducer 1% 3, 54 Ratio to VC of 0.821 ---- No inducer 0.1% 3, 54 ---- 1.402 Table 4 Functional Cell-Based Assay Cut-Points The confirmatory neutralizing cell-based assay utilizes the ability of murine interleukin-3 (mIL-3) to stimulate NFS-60 cell growth independent of the G-CSF receptor. Since the ADA samples are from breast cancer patients who received cytotoxic agent for chemotherapy, this step is critical to differentiate non-specific cell growth inhibition mediated from cytotoxic agents from NAb-inhibited G-CSF-dependent cell growth. To statistically evaluate this inhibition, cells induced with mIL-3 in absence or in presence of samples were tested in 6 replicates each and compared with a one-tailed t-test; a significant p-value from this t-test (p < 0.01) would indicate that the inhibition from samples is non-specific. During validation, this approach was tested by adding naïve matrix (VC), matrix containing non-specific cytotoxic agents (Doxorubicin/Docetaxel) and matrix containing elevated levels of anti-XM22 antibodies to mIL-3-induced cells (Figure 3). Confirmatory NAb Assay With a fit for purpose approach, the characterization of detected ADAs was considered important, mainly due to the presence of an endogenous counterpart (G-CSF). This characterization included determination of the moieties targeted by the ADAs as well as an evaluation of their neutralizing activity (NAb). * Floating cut-point is calculated by multiplying the mean run NC reading with the correction factor. Inconclusive Linglong Zou 1 , Martin Roberge 2 , Chantale Lapierre 2 , Marie-Noëlle Lepage 2 , Yann Fortin 2 , Loan Nguyen 2 , Nicole Rousseau 2 , Suzie Larocque 2 , Mathilde Yu 2,3 , Patrick Liu 1 ( 1 Global Bioassays and Technology, Teva Pharmaceuticals, 2 CIRION BioPharma Services Inc., 3 corresponding author) 0 1 2 3 4 Cells + mIL-3 Cells + mIL-3 + cytotoxic control Cells + mIL-3 + anti-XM22 HPC sample OD t-test p-value Cells + mIL-3 vs Cells + mIL-3 + cytotoxic control < 0.0001 Cells + mIL-3 vs Cells + mIL-3 + anti-XM22 HPC sample 0.4435 Specificity of surrogate anti-XM22 antibodies was confirmed since they did not cause a significant reduction in cell proliferation induced by mIL-3. Conclusion/Discussion Figure 3 Functional Cell-Based Assay Specificity Evaluation The rabbit anti-XM22 antibodies were also used as PC samples in the confirmation and characterization assays (drug and endogenous G-CSF specificity). However, PC antibodies for the PEG-binding characterization assay were different; since the anti-XM22 antibody/XM22 drug binding interaction could not be displaced with a PEG competitor, a commercial rabbit polyclonal anti-PEG antibody was used instead. Precision of PC samples for the various assays are summarized in Table 2 (pre-set acceptance criteria required CVs ≤ 25.0% for all assays). naïve 5.0 10.0 15.0 0 50 100 150 200 Screening cut-point Anti-XM22 antibody fortification (ng/mL) ECL counts Positive Control Samples (continued) Selectivity/Interference No interference of highly lipemic or severely hemolyzed serum was observed in the assay as 5 lots from each serum type fortified at the LOD level had signals remaining above the screening cut-point (Figure 3). No interference of concomitant drugs was observed either as pooled serum lots fortified at 3 PC antibody levels did not have significantly different signals with or without presence of concomitant drugs at low and high concentrations (Figure 4). Tolerance of the assay to the drug (XM22) was evaluated at > 10 ng/mL, which is more than what is expected in the immunogenicity sampling time points (data not shown). 0.0 10.0 0.0 10.0 0 50 100 150 200 Screening cut-point Lipemic serum Hemolyzed serum Anti-XM22 antibody fortification (ng/mL) ECL counts Inducer Titrations and Assay Sensitivity To determine assay sensitivity of the cell-based assay, the first step was to determine inducer concentrations which would lie within the linear portion of the dose-response curve (Figure 5). With these EC50 concentrations determined, titrations of neutralizing antibodies (Figure 6) were then used to evaluate assay sensitivity (interpolation of the cut-points (see Table 4) on the curves). Figure 2 Assay Sensitivity (LOD Confirmation) Figure 1 Anti-XM22 Antibody Titration in Screening Assay 0.1 1 10 100 1000 10000 100000 10 100 1000 10000 100000 1000000 Screening cut-point Anti-XM22 antibody concentration (ng/mL) ECL counts 1 10 100 0 25 50 75 100 125 150 XM22 G-CSF G-CSF EC50 = 15.0 pg/mL XM22 EC50 = 30.0 pg/mL Inducer (pg/mL) %viability 0.01 0.1 1 10 0.0 0.5 1.0 XM22 inducer G-CSF inducer Cut-points Anti-XM22 antibody (µg/mL) Ratio to VC Figure 6 Neutralizing Antibody Titrations Figure 5 Inducer Titrations Inducer Assay sensitivity XM22 1.1 µg/mL GCSF 1.6 µg/mL Low High Low High -40 -20 0 20 40 Docetaxel/Doxorubicin concentration Etoposide/Cisplatin concentration LPC MPC HPC %difference in ECL counts (with drug / without drug) Figure 4 Concomitant Drug Interference Figure 3 Lipemic/Hemolyzed Serum Interference
Transcript of Method establishment for characterization of anti-drug ...
Method establishment for characterization of anti-drug antibody responses against a Pegylated-G-CSF drug in human patients with breast cancer
© 2015 CIRION BioPharma Research
Introduction XM22 is a glycol-PEGylated recombinant human granulocyte colony-stimulating factor (G-CSF) that has been developed for the prevention of chemotherapeutic- induced neutropenia. A cascade of validated assays based on a risk-based approach is described for the evaluation of XM22 immunogenicity in human serum samples from breast cancer patients. Due to the nature of XM22, characterization of the binding specificity and neutralization of the drug and/or the endogenous counterpart (G-CSF) caused by the immunological response were performed.
Methods Assay Cascade for Anti-XM22 Antibodies (ADA) Analysis
Screening for ADA
Confirmation
Titer
Confirmation
The immunogenicity cascade begins with ADA detection using ADA binding assays, where screening positive samples are submitted to a confirmatory assay for XM22 specificity verification. Antibody titer in the confirmed-positive samples is then determined, along with a characterization of their binding specificity to protein and PEG moieties of XM22, and endogenous G-CSF. Confirmed-positive samples from the binding assays are also submitted to cell-based neutralizing antibody (NAb) assays to determine if they neutralize cell proliferation induced by XM22 or endogenous G-CSF; NAb-screening positive samples are tested in a confirmatory assay using an alternative inducer.
ADA Binding Assays
NAb Cell-Based Assays
The binding assays use the ECL bridging format, where the bivalent property of anti-XM22 antibodies allow for simultaneous binding of a capture reagent (biotin-labeled XM22) and a detection reagent (TAG-labeled XM22). An electrical current causes the captured TAG to emit measureable light. To confirm antibody specificity to XM22 and characterize binding to drug moieties and/or the endogenous counterpart, unlabeled competitors (XM22, recombinant G-CSF, PEG and endogenous G-CSF) are added, causing a reduction in assay signal when competition is present. To compare relative ADA levels between samples, antibody titers are determined for every ADA confirmed-positive sample using serial dilutions.
The cell based assays assess if samples confirmed-positive for ADAs contain NAbs that neutralize NFS-60 cell-growth induced by XM22 or endogenous G-CSF (WST-1 detection reagent). To be reported as NAb positive, a sample must show: Absence of cell growth induction when tested
without inducer (assay suitability). Absence of inhibition of cell proliferation
stimulated by an alternative inducer (mIL-3).
NAb
Results ADA Binding Assays
Protein moiety of XM22 PEG moiety of XM22 Endogenous G-CSF
Binding characterization Screening for NAb XM22 induction neutralization GCSF induction neutralization No induction (assay suitability)
ADA
ADA Assay Cut-Points Cut-points (positivity thresholds) were statistically determined using naïve serum samples to obtain pre-defined false-positive rates for each assay of the multi-tiered approach. Cut-point calculations were performed based on Shankar et al.1 and Devanarayan & Tovey2. A summary of the various fixed/floating cut-points is presented in Table 1.
Assay type
Assay subtype
False positive rate
# batches, # samples
Competitor Cut-point (%inhibition)
Correction factor*
Screening ---- 5% 4, 50 ---- ---- 14 ECL counts
Confirmation ---- 1% 2, 54 XM22 28.5% ----
Characterization
Drug G-CSF moiety 1% 2, 36 Filgrastim 22.0% ----
Drug PEG moiety 1% 2, 36 PEG 19.4% ----
Endogenous counterpart 1% 2, 36 G-CSF 17.5% ----
Table 1 Binding Assay Cut-Points
* Floating cut-point is calculated by adding the correction factor to the mean run NC reading
1. Shankar et al. 2008. J. Pharm. Biomed. Anal. 48: 1267 – 1281. 2. Devanarayan & Tovey. 2011. Chapter 16 (289 – 308) in Detection and Quantification of Antibodies to Biopharmaceuticals. Wiley,
Singapore, 404 pages. Positive Control Samples
Positive control (PC) antibodies for the screening assay were surrogate affinity purified polyclonal anti-XM22 antibodies produced in rabbits. Titration of these antibodies in pooled normal human serum revealed a lack of hook effect at concentrations up to 60 000.0 ng/mL, and allowed for estimation of the assay LOD at 8.8 ng/mL (concentration at screening cut-point, Figure 1). The assay LOD was then confirmed by fortifying anti-XM22 antibodies in 15 breast cancer matrix lots at concentrations in the vicinity of the estimated LOD; the LOD was established as the lowest tested concentration with readings ≥ screening cut-point in at least 95% of qualified lots (to be qualified, naïve lots had to have a signal < screening cut-point). Figure 2 illustrates results for 3 tested concentrations, with a final LOD of 10.0 ng/mL.
Assay type Assay subtype PC
antibody Unit
PC samples (ng/mL) Inter-run CV LPC MPC HPC
Screening ---- Anti-XM22 ECL counts 468.8 3750.0 6000.0 7.4 - 8.8%
Confirmation ---- Anti-XM22 %inhibition 500.0 ---- 6000.0 0.9 - 9.4%
Characterization
Drug G-CSF moiety Anti-XM22 %inhibition 500.0 ---- 6000.0 0.4 - 2.3%
Drug PEG moiety Anti-PEG %inhibition 50.0 ---- 150.0 12.2 - 21.4%
Endogenous counterpart Anti-XM22 %inhibition 500.0 ---- 6000.0 0.9 - 9.4%
Table 2 Precision of PC Samples in Binding Assays
NAb Cell-Based Assays
NAb Assay Cut-Points Since these assays are based on cell proliferation, false-negative results could be obtained if the samples themselves induced cell-proliferation. This type of non-specific response was identified using a suitability assay, for which a “no-inducer” cut-point was determined. Samples inducing cell growth were therefore labeled as having an “inconclusive” result in the neutralizing assay. All determined cut-points are summarized in Table 4.
Assay type
False positive rate
# batches, # samples
Cut-point Correction factor*
XM22 inducer 1% 3, 54 Ratio to VC of 0.824 ----
GCSF inducer 1% 3, 54 Ratio to VC of 0.821 ----
No inducer 0.1% 3, 54 ---- 1.402
Table 4 Functional Cell-Based Assay Cut-Points
The confirmatory neutralizing cell-based assay utilizes the ability of murine interleukin-3 (mIL-3) to stimulate NFS-60 cell growth independent of the G-CSF receptor. Since the ADA samples are from breast cancer patients who received cytotoxic agent for chemotherapy, this step is critical to differentiate non-specific cell growth inhibition mediated from cytotoxic agents from NAb-inhibited G-CSF-dependent cell growth. To statistically evaluate this inhibition, cells induced with mIL-3 in absence or in presence of samples were tested in 6 replicates each and compared with a one-tailed t-test; a significant p-value from this t-test (p < 0.01) would indicate that the inhibition from samples is non-specific. During validation, this approach was tested by adding naïve matrix (VC), matrix containing non-specific cytotoxic agents (Doxorubicin/Docetaxel) and matrix containing elevated levels of anti-XM22 antibodies to mIL-3-induced cells (Figure 3).
Confirmatory NAb Assay
With a fit for purpose approach, the characterization of detected ADAs was considered important, mainly due to the presence of an endogenous counterpart (G-CSF). This characterization included determination of the moieties targeted by the ADAs as well as an evaluation of their neutralizing activity (NAb).
* Floating cut-point is calculated by multiplying the mean run NC reading with the correction factor.
Inconclusive
Linglong Zou1, Martin Roberge2, Chantale Lapierre2, Marie-Noëlle Lepage2, Yann Fortin2, Loan Nguyen2, Nicole Rousseau2, Suzie Larocque2, Mathilde Yu2,3, Patrick Liu1 (1Global Bioassays and Technology, Teva Pharmaceuticals, 2CIRION BioPharma Services Inc., 3corresponding author)
0
1
2
3
4
Cells + mIL-3 Cells + mIL-3 +cytotoxic control
Cells + mIL-3 +anti-XM22 HPC sample
OD
t-test p-value
Cells + mIL-3 vs Cells + mIL-3 + cytotoxic control < 0.0001
Cells + mIL-3 vs Cells + mIL-3 +
anti-XM22 HPC sample 0.4435
Specificity of surrogate anti-XM22 antibodies was confirmed since they did not cause a significant reduction in cell proliferation induced by mIL-3.
Conclusion/Discussion
Figure 3 Functional Cell-Based Assay Specificity Evaluation
The rabbit anti-XM22 antibodies were also used as PC samples in the confirmation and characterization assays (drug and endogenous G-CSF specificity). However, PC antibodies for the PEG-binding characterization assay were different; since the anti-XM22 antibody/XM22 drug binding interaction could not be displaced with a PEG competitor, a commercial rabbit polyclonal anti-PEG antibody was used instead. Precision of PC samples for the various assays are summarized in Table 2 (pre-set acceptance criteria required CVs ≤ 25.0% for all assays).
naïve 5.0 10.0 15.00
50
100
150
200
Screening cut-point
Anti-XM22 antibody fortification (ng/mL)
ECL
coun
ts
Positive Control Samples (continued)
Selectivity/Interference No interference of highly lipemic or severely hemolyzed serum was observed in the assay as 5 lots from each serum type fortified at the LOD level had signals remaining above the screening cut-point (Figure 3). No interference of concomitant drugs was observed either as pooled serum lots fortified at 3 PC antibody levels did not have significantly different signals with or without presence of concomitant drugs at low and high concentrations (Figure 4). Tolerance of the assay to the drug (XM22) was evaluated at > 10 ng/mL, which is more than what is expected in the immunogenicity sampling time points (data not shown).
0.0 10.0 0.0 10.00
50
100
150
200
Screeningcut-point
Lipemic serum Hemolyzed serum
Anti-XM22 antibody fortification (ng/mL)
ECL
coun
ts
Inducer Titrations and Assay Sensitivity To determine assay sensitivity of the cell-based assay, the first step was to determine inducer concentrations which would lie within the linear portion of the dose-response curve (Figure 5). With these EC50 concentrations determined, titrations of neutralizing antibodies (Figure 6) were then used to evaluate assay sensitivity (interpolation of the cut-points (see Table 4) on the curves).
Figure 2 Assay Sensitivity (LOD Confirmation)
Figure 1 Anti-XM22 Antibody Titration in Screening Assay
0.1 1 10 100 1000 10000 10000010
100
1000
10000
100000
1000000
Screening cut-point
Anti-XM22 antibody concentration (ng/mL)
ECL
coun
ts
1 10 1000
25
50
75
100
125
150
XM22G-CSF
G-CSFEC50 =
15.0 pg/mL
XM22EC50 =
30.0 pg/mL
Inducer (pg/mL)
%vi
abili
ty
0.01 0.1 1 100.0
0.5
1.0
XM22 inducerG-CSF inducer
Cut-points
Anti-XM22 antibody (µg/mL)
Rat
io to
VC
Figure 6 Neutralizing Antibody Titrations Figure 5 Inducer Titrations
Inducer Assay sensitivity
XM22 1.1 µg/mL
GCSF 1.6 µg/mL
Low High Low High-40
-20
0
20
40
Docetaxel/Doxorubicinconcentration
Etoposide/Cisplatinconcentration
LPCMPCHPC
%di
ffere
nce
in E
CL
coun
ts(w
ith
drug
/ w
itho
ut d
rug)
Figure 4 Concomitant Drug Interference Figure 3 Lipemic/Hemolyzed Serum Interference
![[Amir Abedi] Anti-Political Establishment Parties(BookFi.org)](https://static.fdocuments.in/doc/165x107/545e02b2af795930708b45fb/amir-abedi-anti-political-establishment-partiesbookfiorg.jpg)
