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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

Overview

Linus Carl Pauling

Nature of Chemical Bond

To Elucidate Complex Structure

Nobel Prize Awarded: 1954

Frederick Sanger

Structure of Protein (Insulin)

Max Ferdinand Perutz

John Cowdery Kendrew

Structure of Globular Protein

(Myoglobin & Hemoglobin)

Dorothy Mary Hodgkin

Advanced X-ray Crystallography

For 3-D Structures of Biomolecules

Derek H.R. Barton

Odel Hassel Developed Concept of

Conformation and

Application to HOS

Derek H.R. Barton

Odel Hassel Developed Concept of

Conformation and

Application to HOS

David C. Phillips

First Structure of

Enzyme Lysozyme

1969-1972

WOLF Prize Awarded: 1987

Christian B. Anfinsen

Relationship between

Amino Acid Sequence and

Biologically Active HOS

Stanford Moore

William H. Stein Connection between Chemical

Structure and Catalytic Activity

(Ribonuclease)

Aaron Klug

X-ray Diffraction Crystallographic

EM of Complex Structures

Johann Deisenhofer

Robert Huber

Hartmut Michel 3D Structure of Photosynthetic

Reaction Centre

Johann Deisenhofer

Robert Huber

Hartmut Michel 3D Structure of Photosynthetic

Reaction Centre

John E. Walker

Paul D. Boyer

Elucidation of the Enzyme Mechanism

Underlying ATP Synthesis

Anthony A. Kossiakoff

Human Growth Hormone and

Receptor at 2.8 Å Resolution

Kurt Wüthrich

NMR Elucidation of 3D Structures of

Biomolecules in Solution

Peter Agre

Roderick Machinnon

Structure and Function

of Potassium Channel

Venkatraman Ramakrishnan

Thomas A. Steitz

Ada E. Yonath

Structure and Function of the Ribosome

Brian K. Kobilka

Robert J. Lefkowitz

Structure and Function of

G-protein Coupled Receptors

Brian K. Kobilka

Robert J. Lefkowitz

Structure and Function of

G-protein Coupled Receptors

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)

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

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

How can changes in HOS affect clinical

performance?

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

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

Structure-function relationship study

Log Fraction of Intact Susceptible Residues

vs.Time

0 1 3 24

t-BHP Oxidation Time (Hour)

0 1 3 24

0 4 8 12 16 20 24

-1.100

-0.900

-0.700

-0.500

-0.300

-0.100

0 1 2 3 7

Irradiation Time (Day)

-1.100

-0.900

-0.700

-0.500

-0.300

-0.100

0 1 2 3 7

0 1 2 3 4 5 6 7

-1.100

-0.900

-0.700

-0.500

-0.300

-0.100

0 1 2 3 7

-1.100

-0.900

-0.700

-0.500

-0.300

-0.100

0 1 2 3 7

0 1 2 3 4 5 6 7

■ Met-101

Trp-105 ▲ Met-255

Met-361

Ο Met-431

activity

0 1 3 24

0 4 8 12 16 20 24

MAb A MAb B

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

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

Case Study 1 Summary

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

• 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.

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

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.

Overview

▪ Amylin is a 37 amino acid peptide, co-

secreted with insulin from pancreatic beta

▪ 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

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

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

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

Amylin in Disease Pathophysiology: Rationale for Target

Amylin

aggregates form

pores in the

membrane: MOA

for amylin fibrils

induced cell

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

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

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

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.

binding

site‡

• 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

Theoretical 2xG0F mass

53530 Da

53380 Da

53450 Da

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

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-YTE

Fc-TM+ YTE

11+ 12+ 13+ 14+

G0F G1F

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

• 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

Increase in observed hydrogen-exchange

rates with Fc mutations

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

R2 all ≥0.99

Exchangeable

amides

WT TM YTE TM/YTE

k1 (slow) 0.01 0.01 0.03 0.01

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

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

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.

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

• 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

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)

Acknowledgments

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it from your system and note that you must not copy, distribute or take any action in reliance on it. Any unauthorized use or disclosure of the

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F: +44 (0)20 7604 8151, www.astrazeneca.com

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