Implementation of the Next Generation Effector Function Assays for Comparability Assessments

Transcription

1 Implementation of the Next Generation Effector Function Assays for Comparability Assessments March 25, 2014 Poonam Aggarwal Bioassays 2014: Scientific Approaches & Regulatory Strategies to be held March 24 25,

2 Presentation Overview Overview of Fcg functionality Strategy for analysis of Fc effector function of mabs: What stage and when related to ADCC/CDC Summary

3 Effector FunctionTesting Requirements Guideline on Development, production, characterization and specifications for monoclonal antibodies and related products EMEA/CHMP/BWP/157653/2007, recommends that the ability for complement binding and activation, and/or other effector functions should be evaluated, even if the intended biological activity does not require such functions.

4 Background Immunoglobulins have the potential to mediate a variety of effector functions. Such effector functions include: 1. Fixation of complement - This results in lysis of cells and release of biologically active molecules 2. Binding to various cell types via Fc receptors. This binding can activate the cells to perform some function. Ag binding

5 Methods Target Binding ELISA SPR Functional Cell-based Assays MOA(s) 14 methods may be applied based on MOA Functional Cell-based Assays MOA(s) Fc Effector Functions SPR FcgRI FcgRIIa FcgRIIb FcgRIIIa (F/V) FcgRIIIb C1q FcRn Functional Assays ADCC CDC Adapted from Stephen L. Sazinsky, PNAS 2008,105, 51,

6 Different Fcg Receptors Clin Invest. 2008; 118(8):2677

7 What is the Role of Fcg Receptors? Phagocytosis Endocytosis ADCC (Antibody Dependent Cell-Mediated Cytotoxicity) FcγRIIIa (158V and 158F) Cytokine release during inflammation Activation of B-cells

8 Different IgG Subclasses Bind Differentially to Fc Receptors IgG 1 IgG 3 bind with higher affinity IgG 2 and IgG 4 bind with lower affinity The specific carbohydrates on the FcgR play a role in binding

9 Why We Characterize Effector Function Safety - Nonspecific effects Efficacy - MOA involving cell loss Oncology Characterization - Forced degradation, structure function and comparability studies Quality control - Biological activity assays 9

10 Effector Function Classification Jiang, X., et al., Advances in the assessment and control of the effector functions of therapeutic antibodies Nature Reviews Drug Discovery, 2011 (10),

11 Effector Function Classification Class I Class 2 Class 3 Cell-bound antigen. MOA involving Fc effector functions Cell-bound antigen. MOA not involving Fc effector functions Soluble antigen. MOA not involving Fc effector functions IgG1 and IgG3 High Moderate Low IgG1 and IgG3 with enhanced Fc functions IgG1 and IgG3 with Fc mutations to reduce Fc functions IgG2 and IgG4 IgG2 and IgG4 with Fc mutations to reduce Fc functions ADC Novel mab-like molecule High N/A N/A N/A Low Low Follow guidelines for unconjugated mabs Follow guidelines for mabs, based on mab molecular and target 11

12 Strategy for Assessment of a Molecule with Class 1 MOA (High Effector Function) Test Method GMP Assay for Lot Release and Stability Comparability** Characterization, Structure Function, & Forced Degradation Studies Release Assay based on MOA* Bioassays ADCC CDC Additional Properties Additional assays based on MOA Fcg Receptors (I, IIIa158F/V, IIIb, IIa131H/R, IIb), FcRn Binding by SPR Target Binding by SPR C1q binding 12 * Goal is single release assay, even for products with multiple MOAs; ** Methods included for comparability to be determined based on scope of process change/risk assessment.

13 Development of a Molecule with Class 1 MOA (High Effector Function) IND POC BLA Research Development Phase I Phase II Phase III Initial evaluation of MOA Collaborate with Discovery Develop & qualify a release assay based on MOA Awareness of glycan profile File release assay data in IND/IMPD Further development of functional bioassays Proliferation, RGA, ADCC, CDC, etc. Further development of binding assays FcgR, C1q, Target Apply appropriate panel of assays for Comparability Bridge phase 1 and functional release assays Validate release assay, may include ADCC or CDC Apply appropriate panel of assays for characterization, structure-function, and forced degradation studies Planning and assessment of CQA & specification strategy 13

14 Assay Testing Options Tier One: Fcg receptor and C1q binding assays Immunoassays SPR technology Tier Two: ADCC and CDC Cell based assays

15 Binding to Fcg Receptors Via SPR Case study for high effector function Example : IgG1 binding to Fcg RIIIa (158V) using a BiacoreT200. Response Units FcRIIIa RU Time Sec Method Anti-histidine antibody is immobilized onto the control and test sensor surfaces as a capture reagent.his tagged Fcg IIIa receptor is, then flowed over as a ligand Test antibody is then injected at increasing concentrations ( nm) at 1:3 dilutions series, data were fit with 1:1 Langmuir binding

16 C1q Binding Immunoassay Coat titer plate with Ab, O/N block, 1 hr at RT Add C1q protein, 1.5 hr at RT Add Antic1q Sulpho tagged, 1 hr RT Read plate Ab (test sample) Blocking Protein C1Q Protein anti-c1q mab sulpho tagged Case study for high effector fuction Case study showing an IgG1 with C1q binding activity and a negative control (null for effector function)

17 ADCC: Antibody-Dependent Cell-mediated Cytotoxicity Case study for high effector fuction NK Cell NK Cell target cell Target Antigen CD16 or FcγRIII target cell Cytokines, Granules target cell target cell 1. mab binds to the target cell through specific antigen 2. Natural killer cells (NK) bind to mab Fc through it s Fc receptors 3. NK cell secretes apoptosis-inducing agents after binding 4. Target cell death through apoptosis

18 Effector Cells: Challenges & Alternatives Limitation of using primary PBMC or primary NK cells in ADCC assay Donor to donor variation Limited number of cells can be used Cost of the primary cells Difficult to use quantitative data analysis tools FcgRIIIA (CD16)- transfected NK92 cell line Derived from natural killer cells Use the same mechanism and same assay platform Can replace primary NK cells in ADCC assay FcgRIIIA (CD16)- transfected Jurkat cell line NFAT-RE luciferase reporter gene activation Signaling pathway of effector cell activation Reporter Gene assay

19 RLU ADCC: Antibody-Dependent Cell-mediated Cytotoxicity Samples prepared at different concentrations to evaluate assay sensitivity. Sample is diluted from % and tested for ADCC response Case study for high effector fuction Target Cell Effector engineered Jurkat cell = NFAT-RE-luc y = x R² = % 50% 100% 150% 200% 250% Target Mock Potency Values

20 CDC Assay: Release Assay Case study for high effector fuction Prepare reference material and sample serial dilutions in dilution plates Add serial dilutions to assay plates Seed cells in 96-well assay plates Incubate assay plates for 37 C/ 5 %CO2 Dilute human serum complement in assay medium and add to assay plates Develop plates read on Luminescent-capable plate reader. Incubate assay plates for 2 37 C/ 5 %CO2 Transfer solution to white plate 20

21 Strategy for Assessment of a Molecule with Class 2 MOA (Low or Moderate Effector Function) Test Method GMP Assay for Lot Release and Stability Comparability** Characterization, Structure Function, & Forced Degradation Studies Release Assay based on MOA* Bioassays ADCC TBD CDC TBD Additional Properties Additional assays based on MOA Fcg Receptors (I, IIIa158F/V, IIIb, IIa131H/R, IIb), FcRn Binding by SPR Target Binding by SPR C1q binding * Goal is single release assay, even for products with multiple MOAs; ** Methods included for comparability to be determined based on scope of process change/risk assessment. 21

22 Development of a Molecule with Class 2 MOA (Low or Moderate Effector Function) IND POC BLA Research Development Phase I Phase II Phase III Initial evaluation of MOA Collaborate with Discovery Develop & qualify a release assay based on MOA (often an ELISA) Awareness of glycan profile sections of IND/IMPD File release assay data in IND/IMPD Collaborate with Discovery Further development of functional bioassays Potential Release: Proliferation, RGA, etc. Potential Characterization: ADCC, CDC Further development of binding assays FcgR, C1q, Target Apply appropriate panel of assays for Comparability Bridge phase 1 and functional release assays Validate release assay, unlikely to include ADCC or CDC Apply appropriate panel of assays for characterization, structure-function, and forced degradation studies Planning and assessment of Critical quality attributes & specification strategy 22

23 Strategy for Assessment of a Molecule with Class 3 MOA (Low Effector Function) Test Method GMP Assay for Lot Release and Stability Comparability** Characterization, Structure Function, & Forced Degradation Studies Release Assay based on MOA* Bioassays Additional Properties ADCC TBD TBD CDC TBD TBD Additional assays based on MOA Fcg Receptors (I, IIIa158F/V, IIIb, IIa131H/R, IIb), FcRn Binding by SPR Target Binding by SPR TBD TBD TBD C1q binding TBD TBD * Goal is single release assay, even for products with multiple MOAs; ** Methods included for comparability to be determined based on scope of process change/risk assessment. 23

24 Comparing ADCC Activity in Class 1 versus Class 2 Target Cell Effector engineer ed Jurkat cell = NFAT-REluc Case study for moderate effector function

25 Development of a Molecule with Class 3 MOA (Low Effector Function) IND POC BLA Research Development Phase I Phase II Phase III Initial evaluation of MOA Collaborate with Discovery Develop & Qualify a release assay based on MOA (often an ELISA) Confirm tested for lack of Effector function Further development of functional bioassays and binding assays Proliferation, RGA, SPR, etc. Typically, ADCC and CDC are not required Apply appropriate panel of assays for Comparability Bridge phase 1 and functional release assays Validate release assay Apply appropriate panel of assays for characterization, structure-function, and forced degradation studies Planning and assessment of CQA & specification strategy 25

26 Summary: Classification of mab products based on their potential for effector function Use of a tiered approach towards ADCC/CDC/Fcg functional testing Use of alternative ADCC cell based assays that are easy to use and potentially more robust and reproducible results that may be used in comparability assessment

27 Additional Resources GUIDELINE ON DEVELOPMENT, PRODUCTION, CHARACTERISATION AND SPECIFICATIONS FOR MONOCLONAL ANTIBODIES AND RELATED PRODUCTS EMEA/CHMP/BWP/157653/ p?webcontentid=wc Advances in the assessment and control of the effector functions of therapeutic antibodies. Jiang, X., et al., Nature Reviews Drug Discovery, 2011 (10), Effector-Function_Whitepaper_Bergelson.pdf 27