ARD Effector Function Roadmap_Feb2013

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, 2014
1
Presentation Overview
• Overview of Fcg functionality
• Strategy for analysis of Fc effector
function of mAbs: What stage and when
related to ADCC/CDC
• Summary
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’.
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
Ag binding
2. Binding to various cell
types via Fc receptors.
This binding can activate
the cells to perform some
function.
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, 20167–20172
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Different Fcg Receptors
Clin Invest. 2008; 118(8):2677
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
Different IgG Subclasses Bind Differentially to Fc Receptors
 IgG1 IgG3 bind with higher affinity
 IgG2 and IgG4 bind with lower affinity
 The specific carbohydrates on the FcgR
play a role in binding
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
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), 101-110
10
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
High
N/A
N/A
N/A
Low
Low
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
Follow guidelines for unconjugated mAbs
Follow guidelines for mAbs, based on mAb molecular and
target
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Strategy for Assessment of a Molecule with Class 1 MOA
(High Effector Function)
Bioassays
Additional
Properties
Test Method
GMP Assay for Lot
Release and
Stability
Comparability**
Characterization,
Structure Function, &
Forced Degradation
Studies
Release Assay based on
MOA*
√
√
√
ADCC
√
√
CDC
√
√
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
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determined based on scope of process change/risk assessment.
Development of a Molecule with Class 1 MOA
(High Effector Function)
IND
Research
Development
• 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
POC
Phase I
Phase II
• 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
BLA
Phase III
• Apply appropriate
panel of assays for
characterization,
structure-function,
and forced
degradation studies
• Planning and
assessment of CQA
& specification
strategy
13
Assay Testing Options
• Tier One: Fcg receptor and C1q binding
assays
• Immunoassays
• SPR technology
• Tier Two: ADCC and CDC Cell based
assays
Binding to Fcg Receptors Via SPR
Case study for high effector function
Example : IgG1 binding to Fcg RIIIa (158V) using a BiacoreT200.
FcRIIIa
RU
2500
Response Units
2000
1500
1000
500
0
-500
-100
0
100
200
300
Time
400
500
600
700
800
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 (12.3-1000
nM) at 1:3 dilutions series,
data were fit with 1:1
Langmuir binding
C1q Binding Immunoassay
Case study for high effector fuction
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 showing an IgG1 with C1q binding
activity and a negative control (null for effector
function)
ADCC: Antibody-Dependent Cell-mediated Cytotoxicity
Case study for high effector fuction
NK Cell
NK Cell
Target
Antigen
target
cell
1. mab binds to the
target cell through
specific antigen
Cytokines,
Granules
CD16 or
FcγRIII
target
cell
2. Natural killer
cells (NK) bind to
mab Fc through it’s
Fc receptors
target
cell
target
cell
3. NK cell secretes
apoptosis-inducing
agents after binding
4. Target cell death
through apoptosis
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
ADCC: Antibody-Dependent Cell-mediated Cytotoxicity
Samples prepared at different concentrations to evaluate assay sensitivity. Sample is
diluted from 25-200% and tested for ADCC response
Case study for high effector fuction
Effector
engineered
Jurkat cell
Target Cell
= NFAT-RE-luc
35000
y = 7823.9x + 17024
R² = 0.9771
33000
31000
29000
RLU
27000
25000
23000
21000
19000
17000
15000
0%
50%
100%
150%
200%
Target Mock Potency Values
250%
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 45’@
37°C/ 5 %CO2
Dilute human serum complement in
assay medium and add to assay plates
Incubate assay plates for 2 hours @ 37°C/
5 %CO2
Transfer solution to white plate
Develop plates read on
Luminescent-capable plate reader.
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Strategy for Assessment of a Molecule with Class 2 MOA
(Low or Moderate Effector Function)
Bioassays
Additional
Properties
Test Method
GMP Assay for
Lot Release
and Stability
Comparability**
Characterization,
Structure Function, &
Forced Degradation
Studies
Release Assay based
on MOA*
√
√
√
ADCC
TBD
√
CDC
TBD
√
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
Development of a Molecule with Class 2 MOA
(Low or Moderate Effector Function)
IND
Research
Development
• 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
POC
Phase I
Phase II
• 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
BLA
Phase III
• Apply appropriate
panel of assays for
characterization,
structure-function,
and forced
degradation studies
• Planning and
assessment of
Critical quality
attributes &
specification
strategy
22
Strategy for Assessment of a Molecule with Class 3 MOA
(Low Effector Function)
Bioassays
Additional
Properties
Test Method
GMP Assay for
Lot Release
and Stability
Comparability**
Characterization,
Structure Function,
& Forced
Degradation Studies
Release Assay based
on MOA*
√
√
√
ADCC
TBD
TBD
CDC
TBD
TBD
Additional assays
based on MOA
√
√
Fcg Receptors
(I, IIIa158F/V, IIIb,
IIa131H/R, IIb), FcRn
Binding by SPR
TBD
TBD
Target Binding by SPR
√
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
Comparing ADCC Activity in Class 1 versus Class 2
Target
Cell
Effector
engineer
ed Jurkat
cell
Case study for moderate effector function
= NFAT-REluc
Development of a Molecule with Class 3 MOA
(Low Effector Function)
IND
Research
Development
• 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
POC
Phase I
Phase II
• 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
BLA
Phase III
• Apply appropriate
panel of assays for
characterization,
structure-function,
and forced
degradation studies
• Planning and
assessment of CQA
& specification
strategy
25
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
Additional Resources
• GUIDELINE ON DEVELOPMENT, PRODUCTION,
CHARACTERISATION AND SPECIFICATIONS FOR
MONOCLONAL ANTIBODIES AND RELATED PRODUCTS
– EMEA/CHMP/BWP/157653/2007
– www.ema.europa.eu/ema/pages/includes/document/open_document.js
p?webContentId=WC500003073
• “Advances in the assessment and control of the effector
functions of therapeutic antibodies”. Jiang, X., et al., Nature
Reviews Drug Discovery, 2011 (10), 101-110
– http://ecfmp.pfizer.com/btxps/ARDBIOIT/Projects/Effector%20Function/
Effector-Function_Whitepaper_Bergelson.pdf
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