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PROGRESS IN UNDERSTANDING
RELATIONSHIPS BETWEEN HIGHER ORDER
STRUCTURE AND CLINICAL PERFORMANCE--
IS DE NOVO PROTEIN DESIGN FEASIBLE?
April 13-15, 2015
CASSS HOS conference
VP, Analytical Biotechnology
Mark Schenerman, Ph.D.
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
2
Overview
3
4
Linus Carl Pauling
Nature of Chemical Bond
To Elucidate Complex Structure
Nobel Prize Awarded: 1954
4
5
Frederick Sanger
Structure of Protein (Insulin)
Nobel Prize Awarded: 1958
6
Max Ferdinand Perutz
John Cowdery Kendrew
Structure of Globular Protein
(Myoglobin & Hemoglobin)
Nobel Prize Awarded: 1962
7
Dorothy Mary Hodgkin
Advanced X-ray Crystallography
For 3-D Structures of Biomolecules
Nobel Prize Awarded: 1964
8
Derek H.R. Barton
Odel Hassel Developed Concept of
Conformation and
Application to HOS
Nobel Prize Awarded: 1969
9
Derek H.R. Barton
Odel Hassel Developed Concept of
Conformation and
Application to HOS
Nobel Prize Awarded: 1969
9
10
David C. Phillips
First Structure of
Enzyme Lysozyme
1969-1972
WOLF Prize Awarded: 1987
11
Christian B. Anfinsen
Relationship between
Amino Acid Sequence and
Biologically Active HOS
Nobel Prize Awarded: 1972
12
Stanford Moore
William H. Stein Connection between Chemical
Structure and Catalytic Activity
(Ribonuclease)
Nobel Prize Awarded: 1972
13
Aaron Klug
X-ray Diffraction Crystallographic
EM of Complex Structures
Nobel Prize Awarded: 1982
14
Johann Deisenhofer
Robert Huber
Hartmut Michel 3D Structure of Photosynthetic
Reaction Centre
Nobel Prize Awarded: 1988
15
Johann Deisenhofer
Robert Huber
Hartmut Michel 3D Structure of Photosynthetic
Reaction Centre
Nobel Prize Awarded: 1988
16
John E. Walker
Paul D. Boyer
Elucidation of the Enzyme Mechanism
Underlying ATP Synthesis
Nobel Prize Awarded: 1997
16
17
Anthony A. Kossiakoff
Human Growth Hormone and
Receptor at 2.8 Å Resolution
18
Kurt Wüthrich
NMR Elucidation of 3D Structures of
Biomolecules in Solution
Nobel Prize Awarded: 2002
19
Peter Agre
Roderick Machinnon
Structure and Function
of Potassium Channel
Nobel Prize Awarded: 2003
20
Venkatraman Ramakrishnan
Thomas A. Steitz
Ada E. Yonath
Structure and Function of the Ribosome
Nobel Prize Awarded: 2009
21
Brian K. Kobilka
Robert J. Lefkowitz
Structure and Function of
G-protein Coupled Receptors
Nobel Prize Awarded: 2012
22
Brian K. Kobilka
Robert J. Lefkowitz
Structure and Function of
G-protein Coupled Receptors
Nobel Prize Awarded: 2012
Characterisation of Biotherapeutics
Includes vaccines, soluble receptors, growth factors, cytokines, gene therapy
vectors, hormones, and monoclonal antibodies
Complex, unique properties & processes
Purity, Stability, Structure
Integrity
(e.g. physical and/or chemical degradation)
Primary Sequence/Identity
(correct translation and processing
e.g. C-terminal lysine variants)
Post-translational Modifications
Intracellular (e.g. glycosylation)
Extracellular/chemical (e.g. deamidation)
Higher Order Structure
(e.g. disulfide bonds, -helices, -sheets, 3D structure)
Jefferis et al. Expert Opin. Biol. Ther. (2007) 7(9):1401-1413
Conventional Low Resolution: • Raman (amide III and I; sidechains) • FTIR (amide I) • Circular Dichroism (Near and Far-UV) • Fluorescence • Ion Mobility Spectrometry-MS
Moderate to High Resolution: • HDX-MS (sequence or moderate resolution) • X-Ray Diffraction (atomic resolution, crystallography) • NMR (atomic resolution, solution phase structure, dynamics) • Cryo-TEM (large molecules, 2.8 A resolution possibilities) • Epitope-specific monoclonal antibody binding
Future? • X-ray structure technology advances (X-ray free-electron lasers; single-
molecule diffraction) • In-cell NMR structure technology advances (high throughput, Protein
Structure Initiative, expansion to larger size molecules) • Cryo-TEM technology advances (Phase plate preventing aberrations from
defocusing, automation to routinely define atomic resolution structures) • Integration of multiple structural methodologies (e.g., SAXS with NMR)
24
Low to High Resolution HOS Techniques
Proteins have dynamic processes that we want
to understand at a molecular level
(Un)Folding Synthesis
Endosome
Chain pairing
Conformational
switching CDR binding entropy (Kd)
Domain translation/rotation
Allostery Antigen allosteric effects
Non-paratope mAb residues
(Dis)Association mAb:Ag complex
mAb self-association
Aggregation
Chemical
degradation Deamidation
Oxidation
Cytochrome C
F1/F0 ATPase
25
Better understanding of drug and target
structural dynamics
After S. Berger (Waters Corp.) 26
Where does the
antibody bind
to its target?
• Potency (Case study 1)
• Stability (Case studies 2 and 3)
• PK/PD (Case study 4)
• Immunogenicity
• Acute Safety/Toxicity
27
How can changes in HOS affect clinical
performance?
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
28
Overview
• MAb A (IgG1) lost bioactivity after UV light
exposure, while MAb B with < 15 amino acid difference did not
• Both MAbs have the same mechanism of action
29
Effect of Higher Order Structure on Product
Degradation
• Inspection of X-ray crystal structure for exposed residues
• Characterization of the MAbs after UV light exposure
• Characterization of the MAbs after Met-specific t-butyl hydroperoxide oxidation (t-BHP)
• Characterization of the MAbs after Trp-specific ozone oxidation
• Multiplicity of Trp photoproducts detected by fluorescence spectroscopy
• Protection from a peptide mimetic that bound to CDR
30
Structure-function relationship study
31
Log Fraction of Intact Susceptible Residues
vs.Time
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 3 24
t-BHP Oxidation Time (Hour)
EL
IS
A (L
og
)
ELISA
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 3 24
t-BHP Oxidation Time (Hour)
EL
IS
A (L
og
)
ELISA
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 4 8 12 16 20 24
t-BHP Oxidation Time (Hour)
Lo
g (
Fra
cti
on
al
Valu
e)
-1.100
-0.900
-0.700
-0.500
-0.300
-0.100
0.100
0 1 2 3 7
Irradiation Time (Day)
EL
IS
A (L
og
)
ELISA
(%)
-1.100
-0.900
-0.700
-0.500
-0.300
-0.100
0.100
0 1 2 3 7
Irradiation Time (Day)
EL
IS
A (L
og
)
ELISA
(%)
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 2 3 4 5 6 7
Irradiation Time (Day)
Lo
g (
Fra
cti
on
al
Valu
e)
-1.100
-0.900
-0.700
-0.500
-0.300
-0.100
0.100
0 1 2 3 7
Irradiation Time (Day)
EL
IS
A (L
og
)
ELISA
(%)
-1.100
-0.900
-0.700
-0.500
-0.300
-0.100
0.100
0 1 2 3 7
Irradiation Time (Day)
EL
IS
A (L
og
)
ELISA
(%)
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 2 3 4 5 6 7
Irradiation Time (Day)
Lo
g (
Fra
cti
on
al
Valu
e)
■ Met-101
Trp-105 ▲ Met-255
Met-361
Ο Met-431
activity
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 3 24
t-BHP Oxidation Time (Hour)
EL
IS
A (L
og
)
ELISA
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 1 3 24
t-BHP Oxidation Time (Hour)
EL
IS
A (L
og
)
ELISA
-1.1
-0.9
-0.7
-0.5
-0.3
-0.1
0.1
0 4 8 12 16 20 24
t-BHP Oxidation Time (Hour)
Lo
g (
Fra
cti
on
al
Valu
e)
MAb A MAb B
UV UV
t-BHP t-BHP
Bioactivity loss of MAb A correlated with Trp-105
oxidation, not Met oxidation
Wei, Z, et al. Anal. Chem. 2007, 79, 2797-2805
32
Trp-105 in MAb A is Exposed (Based on X-ray
Crystal Structure)
Trp-105 in HC CDR3 region in MAb A (not present in MAb B) is
the most solvent-exposed Trp residue
Trp-54/Trp-55 in HC CDR2 region have some solvent exposure
All other Trp residues are shielded from solvent
• Trp-105 in the CDR of MAb A is responsible
for loss of bioactivity upon UV light
irradiation
• Photosensitivity of Trp-105 is attributed to
higher order structure, which makes it the
most solvent-exposed Trp residue
• Degree of exposure of residue Trp-105,
influences the rate of degradation
• MAb A needs to be protected from light
exposure
• Trp-105 may be engineered out without loss
of the activity
33
Case Study 1 Summary
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
34
Overview
• G-CSF is a 14 kDa cytokine
that stimulates white blood cell
production and is used to
prevent neutropenia in
chemotherapy patients
• Methionine oxidation has been
associated with a loss in higher
order structural stability and
biological activity
35 See G-CSF PDB file 1CD9, Aritomi et al., 1999. See Lu et al., 1999; also see Reubsaet et al. 1998; Torosantucci et al., 2014
Met Oxidation of Granulocyte Colony-Stimulating Factor
(G-CSF) Results in Loss of HOS Stability and Biological
Activity
122
138
127
• Oxidation of Met-127 and Met-138 residues accounted for
most of the loss in biological activity. Oxidation of Met 1 had
little impact.
36 See Lu et al., 1999; also see Reubsaet et al. 1998; Torosantucci et al., 2014;
Met Oxidation of G-CSF Results in Loss of HOS Stability
and Biological Activity
Thermal stability of oxidized G-CSF peaks
(monitored by the melt of CD -helix signal)
Peak 1:
3% active, Met 1, 138, 127 and 122 are
all oxidized
Peak 2:
15% active, Met 1, 138 and 127 oxidized
Peak 3:
20% active, Met 1 and Met 138 oxidized
Oxidized Peaks from RP-HPLC
• All the oxidized forms were able to bind the soluble G-
CSF receptor.
37 See Lu et al., 1999; also see Reubsaet et al. 1998; Torosantucci et al., 2014;
Met Oxidation of G-CSF Results in Loss of HOS Stability
and Biological Activity
Thermal stability of oxidized G-CSF peaks
(monitored by the melt of CD -helix signal) Oxidized Peaks from RP-HPLC
Peak 1:
3% active, Met 1, 138, 127 and 122 are
all oxidized
Peak 2:
15% active, Met 1, 138 and 127 oxidized
Peak 3:
20% active, Met 1 and Met 138 oxidized
38 See Lu et al., 1999; also see Reubsaet et al. 1998; Torosantucci et al., 2014;
Met Oxidation of G-CSF Results in Loss of HOS
Stability and Biological Activity
• Met-138 is solvent accessible and its local environment
seems to be critical for G-CSF function.
• Lu et el. speculated that oxidation of Met-138 may
induce a local conformational change near the receptor
binding region which impacts the biological activity
related to downstream intracellular signaling.
• The G-CSF form oxidized at both Met-127 (less
accessible) and Met-122 (in hydrophobic core) was
unstable and had a decreased ability to dimerize the
receptor.
• Mutation of both Met-127 and Met-138 to Leu stabilized
G-CSF such that it retained activity when exposed to
oxidative stress.
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
39
Overview
▪ Amylin is a 37 amino acid peptide, co-
secreted with insulin from pancreatic beta
cells
▪ Suppresses glucagon secretion and slows
down gastric emptying
▪ Peripheral amylin decreases food intake
and body weight, mediated by receptors in
area postrema (Hindbrain)
▪ Receptors are also found in other brain
regions
▪ Amylin enhances leptin signalling in the
hypothalamus
Roth et al, 2009
Amylin Overview
40
Diabetes Spectrum July 2004 vol. 17
no. 3 183-190
SYMLIN® (pramlintide acetate) injection APPROVED FOR TWO DISTINCT PATIENT POPULATIONS
• First-in-class therapy approved for patients with type 2 and type 1 diabetes not properly controlled with optimal mealtime insulin
• Approved dosing regimens:
– Type 1 diabetes: up to 60 mcg with major meals
– Type 2 diabetes: up to 120 mcg with major meals
• SYMLIN offers reductions in:
– Postprandial glucose levels
– Glucose fluctuations
– A1C values compared to insulin alone
– Mealtime insulin usage
– Body weight in most patients
• Nausea most frequently reported AE
• Boxed warning highlights increased risk of insulin-induced severe hypoglycemia, particularly in type 1 diabetes
41
SYMLIN® (pramlintide acetate) injection MOA
• Pramlintide is a synthetic analog of human amylin, a naturally occurring
neuroendocrine hormone synthesized by pancreatic beta cells that contributes to
glucose control during the postprandial period.
• Pramlintide is provided as an acetate salt of the synthetic 37-amino acid
polypeptide, which differs in amino acid sequence from human amylin by
replacement with proline at positions 25 (alanine), 28 (serine), and 29 (serine).
Pramlintide: Pharmacokinetic Profile
Upon Subcutaneous (SC) Injection
Half Life ~48 min
Pramlintide is administered by SC
injection prior to meals
Pramlintide
K C N T A T C A T Q R L A N F L V H S S N N F G P I L P P T N V G S N T Y
Human
K C N T A T C A T Q R L A N F L V H S S N N F G A I L S S T N V G S N T Y
43
Amylin in Disease Pathophysiology: Rationale for Target
Amylin
aggregates form
pores in the
membrane: MOA
for amylin fibrils
induced cell
death
Pancreatic Islets from a 54
year old T2D patient
Immunostained with anti-
human amylin antibody
(brown) Acc. Chem. Res. (2012); 45(3); 454-462, Biochimie (2011); 93; 793-805, Kajava et al. Mol. Biol. (2005) 348-247
and Amylin & Related Proteins: Physiology & Pathophysiology. Cooper, G.J.S. (2011)
Amyloid (Thioflavin S)
Insulin +
Thioflavin S
IAPP +
ThioflavinS
Normal T2D
Diabetes. 1999 Feb;48(2):241-53.
Amylin in Disease Pathophysiology: Amyloid aggregates
Normal T2D
Insu
lin/A
myl
oid
Exp Gerontol. 2003 Apr;38(4):347-51.
• Islet amyloid is found in up to 90% of patients with type 2 diabetes at autopsy
• Degree of amylin oligomerization correlates with severity of the disease in humans.
Kahn et al., 1999 Westermark, 1994
Amyloidogenesis
Nelson et al. Nature 2005
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
47
Overview
Fc region can be utilised as a motif
in therapeutic proteins
• Fc region is constant across
antibodies that target unique
antigens.
• Fc-fusions can enhance
production and half-life of non-
antibody drugs (e.g. Enbrel /
Etanercept). IgG1
48
TM Variant*: Engineered for decreased ADCC activity
L12F, L13E & P109S
YTE Variant#:
Engineered for increased serum half-life
M30Y, S32T & T34E
Engineered Fc variants modulate effector
function and in vivo half-life
FcγRIII
binding site†
† Sondermann et al. Nature 2000, 406: 267-273
‡ Burmeister, Huber & Bjorkman Nature 1994, 372: 379-383
* Oganesyan V et al. Acta Crystallogr D Biol Crystallogr. 2008 Jun;64(Pt 6):700-4
# Oganesyan et al. Mol Immunol. 2009 May;46(8-9):1750-5.
FcRn
binding
site‡
49
• All observed masses agree with
theoretical values to within 2 Da.
• Glycoform pattern is consistent
between mutant variants,
indicating functional/thermal
stability differences are not as a
result of glycosylation differences.
Deconvoluted MS spectra for Fc
fragments of WT, TM, YTE and
TM-YTE engineered human IgG1.
Glycoforms are consistent between variants
50
Theoretical 2xG0F mass
53530 Da
Theoretical 2xG0F mass
53380 Da
Theoretical 2xG0F mass
53450 Da
Theoretical 2xG0F mass
53298 Da
• TM-YTE double mutant showed lower thermal stability than the single mutants in the CH2 domain.
• Are the effects additive or co-operative?
Fc engineered variants also modulate
thermodynamic stability
Fc Construct Tm1 mean
(˚C)
n
Wild type 70.1 12
TM 64.1 21
YTE 62.2 6
TM-YTE 58.1 5
Differential Scanning
Calorimetry: First transition
(assigned to CH2)
Fc-WT
Fc-TM
Fc-YTE
Fc-TM+ YTE
11+ 12+ 13+ 14+
G0F G1F
G2F
G0F
G1F
G2F
G0F
G1F
G2F
G0F
G1F
G2F
Global Fc conformation is unchanged
Native mass spectrum Deconvoluted
• 11+ to 14+ charge states are observed by Native MS, with partial
resolution of the glycan profile.
• All four samples show a similar charge distribution centred around the
12+ charge state, suggesting there is no global change in protein
conformation associated with either TM, YTE or combination TM-YTE
engineered variants. 52
• Cross sections suggest the four variants have similar conformational ensembles.
• TM and YTE samples show a reduced intensity of the ~3800 Å2 species at 14+
versus WT. This difference becomes more pronounced with TM-YTE.
• TM-YTE > TM > YTE > WT propensity to adopt an extended gas-phase
conformation: suggests a lower energy barrier for gas phase unfolding.
Native Ion-Mobility Mass Spectrometry of Fc
Native ion mobility mass spectra (IM-MS) for the 11-14+ charge states, with
estimated collisional cross-section (CCS) values for each sub-population
modal arrival time TM TM-YTE
WT YTE
53
• Four overlapping peptides (each
observed as multiple ions).
• Deuterium incorporation pattern
(TM-YTE > YTE >> TM ≥ WT)
indicates a possible site of co-
operative change in
conformational dynamics.
TM and YTE mutations have a co-operative
effect on local structure and dynamics
WT TM YTE TM/YTE
54
Increase in observed hydrogen-exchange
rates with Fc mutations
WT TM YTE TM/YTE
WT TM YTE TM/YTE
k1 (slow) 0.01 0.01 0.03 0.01
k2 (intermediate) - - - 0.09
k3 (fast) 3.55 3.31 3.38 4.27 fast
inter
slow
R2 all ≥0.99
55
Exchangeable
amides
WT TM YTE TM/YTE
k1 (slow) 0.01 0.01 0.03 0.01
k2 (intermediate) - - - 0.09
k3 (fast) 3.55 3.31 3.38 4.27
WT TM YTE TM/YTE
k1 (slow) 0.01 0.01 0.03 0.01
k2 (intermediate) - - - 0.09
k3 (fast) 3.55 3.31 3.38 4.27
Deprotected
relative to WT
Protected
relative to WT
Summary
Global conformational dynamics
Global conformation is preserved between the Fc variants
Protein folding
Native ion-mobility MS indicates a lower energy barrier on the gas-phase
unfolding pathway
Local conformational dynamics
Hydrogen/deuterium-exchange MS indicates a co-operative local change to
Fc structure and dynamics when both TM and YTE mutations are present
together
Data correlate with observed thermodynamics data (DSC) for Fc regions in 44
different IgG1s
56
Molecular mechanisms of therapeutic protein
instability
This analytical approach may be applied to inform on molecular
mechanisms of therapeutic protein degradation in response to
production and storage stresses
(e.g. shear stress, thermal fluctuations).
57 Tavakoli-Keshe, R., Phillips, J. J., Turner, R. and Bracewell, D. G.
(2014), J. Pharm. Sci., 103: 437–444.
Impact
Coupling of structural MS analysis to
biopharmaceutical development studies
Broader knowledge of antibody characteristics earlier in the
drug project lifecycle
Candidate selection for stability through the primary recovery
and downstream processing stages of production
Therapeutic protein format
• Inform on choice of molecular format early in drug project.
• Insight to guide future molecular engineering.
History and importance of HOS
Case study 1: Potency loss
Case study 2: Stability loss
Case study 3: Aggregation
Case study 4: Fc engineering enhances PK
Summary—future directions
58
Overview
• How close are we to de novo protein design?
Good understanding of dynamic behavior of domains
Some success in predicting dynamics based on putting
multiple domains together
Next 2-5 years will continue to show advances in
designing more complex larger proteins
Merging in silico design and screening methodologies to routinely
design favored attributes into a molecule
Within 10 years we may be able to ask a computer to
design a protein for us from the ground up
59
Summary—future directions
• Increased processing power and capacity of
computing clouds (or whatever succeeds
clouds)
• Increased understanding of the molecular forces
that underpin protein dynamics
• Increased ability (through better, more complex
and therefore realistic algorithms) to simulate
these molecular dynamics in complex
environments and systems
60
What will it take to get to this 10 year vision?
• Rick Remmele
• Ziping Wei (Novavax)
• Li Peng
• Joe Grimsby
• Ruchi Gupta
• J.J. Phillips (Cambridge University)
• Jared Bee
• Dan Higazi
• Alistair Kippen
• David Lowe
• Amy Rosenberg (FDA)
• Sepideh Farshadi (graphics artist)
61
Acknowledgments
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62