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Engineering antibodies beyond affinity: correlation of key biophysical characteristics with antibody specificity and pharmacokinetic properties
Tris Vaughan
Senior Director,
Antibody Discovery and Protein Engineering
WCBP 2015
27th-29th January 2015
2
Overview
1 Why antibody developability is important
2 Engineering beyond affinity: anti-NGF mAb case study
3
Overview
1 Why antibody developability is important
2 Engineering beyond affinity: anti-NGF mAb case study
4
Kd for targetantigen
Target specificity, species
cross-reactivity
Target clones
In vitro / in vivo potency
Lead Selection with Functional Focus
5
ChemicalStability
SolutionProperties
Physical Stability
Target clones
Lead Selection with Developability Focus
6
ChemicalStability
SolutionProperties
Physical Stability
• Conformational stability• Colloidal stability• pI • Aggregation
• Reversible self-association
• Solubility• Viscosity
• Fragmentation • Deamidation • Oxidation• Asp isomerization• Sequence variants• O-glycosylation• Glycation
Target clones
Lead Selection with Developability Focus
7
ChemicalStability
SolutionProperties
Physical Stability
Target clones
Lead Selection with Developability Focus
In Silico aggregationprediction tool
StarGazer 384-wellAggregation analysis
(Tagg)
Nanopro HT cIEF
45oC, T=2wks
T=0
T=0
Size exclusion Chromatography (SEC)
8
Overview
1 Why antibody developability is important
2 Engineering beyond affinity: anti-NGF mAb case study
9
Anti-NGF Antibody – Medi578
� Target
– Nerve growth factor
� Mechanism of action
– Selectively antagonises binding of NGF to its receptors TrkA and p75
� Characteristics
– Human, derived from phage display library– Selective over other neurotrophins– Cross-reactivity with cyno and rat NGF– KD = 69pM– Active in NGF driven in vitro assays
� Modelling suggested that a higher affinity/potency antibody would suppress serum NGF in vivo >90%
Goal to improve the affinity and potency of Antibody 10-foldGoal to improve the affinity and potency of Antibody 10-fold
Anti-NGF suppression of serum NGF (KD = 6.9pM)
X-ray co-crystal solved
MEDI578 MEDI578
NGF
Affinity Maturation of MEDI578 → MEDI1912
10
MEDI-578 Epitope competition assay
0 1 2 3 40
50
100
150
MEDI-1912MEDI-578
log antibody conc pM
surv
ival
% o
f max
Anti-NGF antibody MEDI1912
� Most improved variant, MEDI1912, exhibited ~10 fold improvement
� MEDI1912 contains 12 mutations (compared to MEDI578)
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10-fold improved cellular potency
VHCDR1 VHCDR2 VHCDR3 VLCDR1 VLCDR2 VLCDR3
Antibody 1 GGTFSTYGIS GIIP-----IFDTGNSAQSFQG SSRIYDYAGGDHYYYDM------DV SGSSSN------IGNNYVS DN-----NKRPS GTWDS S LSA-------WV
MEDI578 GGTFSTYGIS GIIP-----IFDTGNSAQSFQG SSRIYDLNPSLTAYYDM------DV SGSSSN------IGNNYVS DN-----NKRPS GTWDS S LSA-------WV
MEDI1912 GGTFWFGAFT GIIP-----IFGLTNLAQNFQG SSRIYDLNPSLTAYYDM------DV SGSSSD------IGNNYVS DN-----NKRPS GTWDS S LSA-------WV
Kon (1/Ms) Koff (1/s) KD (pM)
2.0 x 107 (20 – 3 x 10-5) (1.6 – 9.8)
0 1 2 3 40
50
100
150MEDI-578MEDI-1912
log antibody conc pM
surv
ival
% o
f m
axDorsal root ganglion cell survival PC12 cell survival
MEDI1912 is specific for NGF
12
NGF epitope competition assay
MEDI1912 reduces mechanical hypersensitivity in a mouse intra-plantar FCA model
13
MFCA032: Effect of MEDI1912 on Mechanical HypersensitivityIpsilateral/Contralateral Ratio
NaivePre-dose 4h 1 2 3 70
20
40
60
80
100
120
PBS i.v. Isotype control i.v.
***
MEDI1912 3 mg/kg i.v.
MEDI1912 0.3 mg/kg i.v.
MEDI1912 1 mg/kg i.v.N=12 per group. Data analysed using 2 w ay ANOVA w ith time and treatment as dependant factors.
Subsequent statistical signif icnace obtained using Bonferroni's Post Hoc test
Time Post Dose
Ipsi
/Co
ntra
Rat
io
MEDI1912 exhibited significant problematic biophysical and DMPK characteristics
14
HPLC-SEC Analytical Ultracentrifugation
Visible OpalescenceReversible self-association
Monomer-dimer-trimer equilibrium Adsorption to surfaces
e.g. SEC column matrix, filter membranesLow purification yieldPoor solubilityPhase-separation & opalescence issuesHigh viscosity (when >30mg/mL)Poor PK in both rat (t½ = 4 days) & NHP
MEDI1912 exhibited significant problematic biophysical and DMPK characteristics
15
Visible OpalescenceReversible self-association
Monomer-dimer-trimer equilibrium Adsorption to surfaces
e.g. SEC column matrix, filter membranesLow purification yieldPoor solubilityPhase-separation & opalescence issuesHigh viscosity (when >30mg/mL)Poor PK in both rat (t½ = 4 days) & NHP
Rat PK NHP PK
16
In silico prediction of protein aggregation propensity
� Spatial aggregation propensity (SAP) algorithm developed by Professor Bernhardt Trout, MIT
– Predicts aggregation propensity based upon atomistic analysis
� SAP algorithm incorporated into Accelrys Discovery Studio
Chennamsetty et al., 2009
Engineering MEDI1912 biophysical properties via a structure-based methodology
� Spatial aggregation propensity (SAP) software used to predict aggregation prone regions on IgG surface
– Three potential positions of interest identified: W30S, F31T (both in VH CDR1) and L56T (VH CDR2)
17
MEDI578 MEDI1912
Engineering MEDI1912 biophysical properties via a structure-based methodology
� Spatial aggregation propensity (SAP) software used to predict aggregation prone regions on IgG surface
– Three potential positions of interest identified: W30S, F31T (both in VH CDR1) and L56T (VH CDR2)
18
MEDI578 MEDI1912
W30
F31
L56
VH CDR2
VH CDR1
Creation & screening of variant IgGs by HPLC-SEC
19
ControlsControls
MEDI1912
MEDI578
Reference WFT
WTL
WTT
SFL
SFT
STL
STT
Panel of variants
Mutant STT ameliorates aberrant biophysical properties
20
Analytical Ultracentrifugation
0.000 0.002 0.004 0.0061e-007
2e-007
3e-007
4e-007
5e-007
Conc (g/ml)
D (
cm2 /s
)
Dynamic Light Scattering
min2 4 6 8 10 12 14 16 18
mAU
0
50
100
150
200
250
VWD1 A, Wavelength=280 nm (APR2014\CL MICA AND TS 22NDAPR 2014 2014-04-22 10-33-53\MEDI1578.D)
8.6
89
VWD1 A, Wavelength=280 nm (APR2014\CL MICA AND TS 22NDAPR 2014 2014-04-22 10-33-53\MEDI1912.D)
Area : 5046 .48
15
.047
MEDI1912_STT
HPLC-SEC
AUC
AUC
Mutant STT ameliorates aggregation of MEDI1912
21
MEDI1912 MEDI1912 - STT
Mutant STT retains affinity and potency of MEDI1912
� STT demonstrates identical affinity for NGF and equipotency in relevant cellular assays
– The three mutations were not associated with decreased affinity
Affinity (BIAcore) for NGF
MEDI1912 1.6 - 9.8 pM
STT 1.8 – 8.3 pM
pERK Potency Assay
-12 -11 -10 -9 -8
0
50
100
NIP228 IgG1TM YTE
MED1912 IgG1TM YTE
MEDI1912_STT IgG1TM
IgG Log [M]
% N
GF
-in
du
ce
d p
ER
K
Mutant STT has reinstated favourable DMPK properties
MEDI1912 STTIsotype
LUNG
LIVER
KIDNEY
STT shows no discernible non-specific binding by IHC
Summary
� With higher affinity mAbs can come other challenges
– MEDI1912 exhibited aberrant CMC/manufacturability and non-linear, impaired PK profile that endangered product development
� In silico prediction can be used to enhance developability characteristics
� Mutant STT retains all of the affinity / potency gains of MEDI1912, but with optimal biophysical characteristics and PK profile
� Clear link between biophysical – DMPK – tissue specificity
� Generic engineering approach could be used to improve the biophysical properties of any therapeutic protein
24
Acknowledgements
� MEDI578 and MEDI1912 project teams
� Claire Dobson
� Andrew Buchanan
� Bojana Popovic
� Chris Lloyd
� Daniel Higazi
� Arthur Lewis
� David Lowe
� Tristan Vaughan
� Biopharmaceutical Development– Chris van der Walle
– David Hayes
– Catherine Galy
– Leanne Amery
– Sofia Ekizoglou
– Richard Turner
� BSU650
� DMPK Team– Jo Goodman
� AstraZeneca Discovery Sciences:– Lise-Lotte Olsson
– Anna aagaard
– Linda Cederblad
– Niek Dekker
– Paul Wan
– Tomas Akerud
� Biologics Expression Team– Neil Brikett
– Anna Czyz
– Sonia Raithatha
– Melanie Medcalf
– Carolina Casado
– Nathan Hudson
– Richard Porter
– Nicola Forrest-Owen
– Robin Butler
25