Introduction Antibody-drug conjugates (ADCs) are heterogeneous molecules composed of: An antibody (whole mAb or fragment) assuring the specificity A cytotoxic drug conferring the toxicity A stable chemical linker ADCs are becoming increasingly important in oncology due to their specificity for tumor-associated antigens. With their heterogeneous composition, they pose novel challenges in their development. For example, the neo-epitopes formed in the mAb as a result of the conjugation may lead to a significant immune response in the patient population. Since immunogenicity assessment is a requirement from regulatory agencies in the safety profile evaluation of biopharmaceuticals, it is critical to develop and validate adequate assays. Here we show the challenges/solutions and special considerations when analyzing the immunogenicity of ADCs in Cancer Studies. Analytical Methods For immunogenicity assessment of therapeutic protein products, guidance documents (US FDA 2016) and publications (Shankar, 2008) describe strategies and development as well as validation of assays. A tiered approach often applied to monoclonal antibodies (mAB) could be adapted for ADC immunogenicity appreciation, with the following steps: Screening Confirmatory Titer Characterization assay Neutralizing assay Additional assays could complete the immunogenicity evaluation such as Isotyping and Relative affinity assessment. The stepwise immunogenicity assessment procedure is illustrated in Figure 1. Screening, confirmatory and characterization assays are presented in Figures 2A, 2B and 2C-2E respectively. The screening assay presented is a bridging assay, with pre-incubation in presence of conjugates for capture and detection. In the confirmatory assay, exogenous ADC added to samples competes for binding to conjugates used for capture and detection. Variation of percent inhibitions in a therapeutic-naïve population is taken into consideration for confirming a positive response. With the characterization assay, domain specificity is assessed by using ADC components (whole mAB; cytotoxic drug or linker) as inhibitors. Neo-epitopes formed in the mAb as a result of the conjugation are in this case not depicted. Molar ratio as well as drug-to- antibody ratio (DAR) should be considered to set up the concentrations of inhibitors. Figure 1: Tiered approach for Immunogenicity Assessment Immunogenicity Assessment of Antibody Drug Conjugates in Cancer Studies M. Montjovent, H. Faust, P. Brennecke and P. Struwe Celerion Switzerland AG, 8320 Fehraltorf, Switzerland Discussion Immunogenicity Ligand Binding Assays for ADCs are more complex than for mAB and therefore require specific considerations which comprise: Choice of reference items Choice of inhibitors Labeling conditions Immunogenic reactivity of key reagents Linker chemistry Outlier(s) elimination for cut point determination Conclusion Ligand binding assays for ADCs present specific bioanalytical challenges. They are addressed successfully by optimizing the following steps: Definition of the approach Selection of the Assay format Assay development The Immunogenicity and Pharmacokinetic (PK) assays developed at Celerion Switzerland AG are outstanding tools for early clinical development of ADCs with improved reliability, maximal reproducibility and robustness. References Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products. Guidance for Industry. Draft Guidance. US FDA 2016 Shankar G et al., Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal. 2008 Dec 15;48(5):1267-81 Case Study 2: Neutralizing Assay Challenge: During the early feasibility phase of a method transfer to detect neutralizing antibodies using a competitive ligand binding assay, a poor free drug tolerance was assessed. The initial format required the spiking of each sample with a defined concentration of ADC (Figure 4A). Solution: The format was reconsidered and neutralizing antibodies were captured on streptavidin plates by biotinylated ADC. The free drug tolerance was improved. Moreover, a specific tuning was performed to optimize the labeling conditions of the target used for detection (Figure 4B). Figure 4: Neutralizing Antibody Assay Optimization Screening Assay Negative for anti-ADC Positive for anti-ADC ConfirmatoryAssay Negative for anti-ADC Positive for anti-ADC Characterization Assay Neutralizing Assay Isotyping Assay Relative Affinity Assessment Titer Assay Figure 2: Assay Formats Acidification Neutralization Pre-incubation Capture on ECL plate Detection A Detection A Acidification Neutralization Pre-incubation Capture on ECL plate Detection Poor Free Drug Tolerance B Detection Acidification Neutralization Capture on ELISA plate Incubation with Detection Reagent Incubation with Biotinylated conjugate Acidification Neutralization Capture on ECL plate Detection B Acidification Neutralization Pre-incubation Capture on ECL plate D Detection Acidification Neutralization Pre-incubation Capture on ECL plate E Detection Coating Capture on E C Poor Free Drug T A Acidification Neutralization Capture on B Anti-Cytotoxin Target Streptavidin ECL plate Legend: ADC CL plate Detection Tolerance n ECL plate Detection SulfoTAG-label Neutralizing Anti-ADC Biotin-label Acidification Neutralization Pre-incubation Capture on ECL plate C Detection Analytical Challenges and Solutions Case Study 1: Screening Assay Challenge: During the early feasibility phase of a method development to detect anti-ADC antibodies including a pre-incubation with conjugates on a deep-well plate, a poor free drug tolerance was found (Figure 3A). Solution: The format was reconsidered and neutralized samples were directly incubated on ADC coated plates. The free drug tolerance was improved (Figure 3B). Figure 3: Screening Assay Optimization ELISA plate Legend: ADC Streptavidin-HRP Anti-ADC Biotin-label Streptavidin ECL plate SulfoTAG-label Streptavidin ECL plate Legend: Anti-ADC ADC Biotin-label Cytotoxin mAb (ADC without Cytotoxin) Unrelated mAb
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Introduction
Antibody-drug conjugates (ADCs) are heterogeneous molecules composed of:
An antibody (whole mAb or fragment) assuring the specificity
A cytotoxic drug conferring the toxicity
A stable chemical linker
ADCs are becoming increasingly important in oncology due to their specificity for tumor-associated antigens. With their heterogeneous composition, they pose novel challenges in their development. For example, the neo-epitopes formed in the mAb as a result of the conjugation may lead to a significant immune response in the patient population.
Since immunogenicity assessment is a requirement from regulatory agencies in the safety profile evaluation of biopharmaceuticals, it is critical to develop and validate adequate assays.
Here we show the challenges/solutions and special considerations when analyzing the immunogenicity of ADCs in Cancer Studies.
Analytical Methods
For immunogenicity assessment of therapeutic protein products, guidance documents (US FDA 2016) and publications (Shankar, 2008) describe strategies and development as well as validation of assays. A tiered approach often applied to monoclonal antibodies (mAB) could be adapted for ADC immunogenicity appreciation, with the following steps:
Screening
Confirmatory
Titer
Characterization assay
Neutralizing assay
Additional assays could complete the immunogenicity evaluation such as Isotyping and Relative affinity assessment.
The stepwise immunogenicity assessment procedure is illustrated in Figure 1. Screening, confirmatory and characterization assays are presented in Figures 2A, 2B and 2C-2E respectively.
The screening assay presented is a bridging assay, with pre-incubation in presence of conjugates for capture and detection.
In the confirmatory assay, exogenous ADC added to samples competes for binding to conjugates used for capture and detection. Variation of percent inhibitions in a therapeutic-naïve population is taken into consideration for confirming a positive response.
With the characterization assay, domain specificity is assessed by using ADC components (whole mAB; cytotoxic drug or linker) as inhibitors. Neo-epitopes formed in the mAb as a result of the conjugation are in this case not depicted. Molar ratio as well as drug-to-antibody ratio (DAR) should be considered to set up the concentrations of inhibitors.
Figure 1: Tiered approach for Immunogenicity Assessment
Immunogenicity Assessment of Antibody Drug Conjugates in Cancer StudiesM. Montjovent, H. Faust, P. Brennecke and P. StruweCelerion Switzerland AG, 8320 Fehraltorf, Switzerland
Discussion
Immunogenicity Ligand Binding Assays for ADCs are more complex than for mAB and therefore require specific considerations which comprise:
Choice of reference items
Choice of inhibitors
Labeling conditions
Immunogenic reactivity of key reagents
Linker chemistry
Outlier(s) elimination for cut point determination
Conclusion
Ligand binding assays for ADCs present specific bioanalytical challenges. They are addressed successfully by optimizing the following steps:
Definition of the approach
Selection of the Assay format
Assay development
The Immunogenicity and Pharmacokinetic (PK) assays developed at Celerion Switzerland AG are outstanding tools for early clinical development of ADCs with improved reliability, maximal reproducibility and robustness.
References
Assay Development and Validation for Immunogenicity Testing of Therapeutic Protein Products. Guidance for Industry. Draft Guidance. US FDA 2016
Shankar G et al., Recommendations for the validation of immunoassays used for detection of host antibodies against biotechnology products. J Pharm Biomed Anal. 2008 Dec 15;48(5):1267-81
Case Study 2: Neutralizing Assay
Challenge: During the early feasibility phase of a method transfer to detect neutralizing antibodies using a competitive ligand binding assay, a poor free drug tolerance was assessed. The initial format required the spiking of each sample with a defined concentration of ADC (Figure 4A).
Solution: The format was reconsidered and neutralizing antibodies were captured on streptavidin plates by biotinylated ADC. The free drug tolerance was improved. Moreover, a specific tuning was performed to optimize the labeling conditions of the target used for detection (Figure 4B).
Figure 4: Neutralizing Antibody Assay Optimization
1
Screening Assay
Negative for anti-ADC Positive for anti-ADC
Con�rmatoryAssay
Negative for anti-ADC Positive for anti-ADC
CharacterizationAssay
NeutralizingAssay
IsotypingAssay
Relative A�nityAssessment
TiterAssay
Figure 2: Assay Formats
Acidi�cation NeutralizationPre-incubation
Capture on ECL plate
Detection
A
Detection
AAcidi�cation Neutralization
Pre-incubationCapture on ECL plate
Detection
Poor Free Drug Tolerance
B
Detection
Acidi�cation NeutralizationCapture on ELISA plate
Incubation with DetectionReagent
Incubation withBiotinylated conjugate
Acidi�cation Neutralization Capture on ECL plate Detection
B
Acidi�cation NeutralizationPre-incubation
Capture on ECL plateD
Detection
Acidi�cation NeutralizationPre-incubation
Capture on ECL plateE
Detection
Coating Capture on EC
Poor Free Drug T
A
Acidi�cation Neutralization Capture on
B
Anti-Cytotoxin
Target
Streptavidin ECL plateLegend:
ADC
CL plate Detection
Tolerance
n ECL plate Detection
SulfoTAG-label
NeutralizingAnti-ADC
Biotin-label
Acidi�cation NeutralizationPre-incubation
Capture on ECL plateC
Detection
Analytical Challenges and Solutions
Case Study 1: Screening Assay
Challenge: During the early feasibility phase of a method development to detect anti-ADC antibodies including a pre-incubation with conjugates on a deep-well plate, a poor free drug tolerance was found (Figure 3A).
Solution: The format was reconsidered and neutralized samples were directly incubated on ADC coated plates. The free drug tolerance was improved (Figure 3B).
Figure 3: Screening Assay Optimization
ELISA plateLegend:
ADC Streptavidin-HRP
Anti-ADC
Biotin-labelStreptavidin ECL plate
SulfoTAG-label
Streptavidin ECL plateLegend: Anti-ADC
ADC
Biotin-labelCytotoxin
mAb(ADC without Cytotoxin)
Unrelated mAb
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