CASSS 2018

Danlin Yang

IgG CooperativityLearning new tricks from old dogs

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Diebolder CA, et al. Complement is activated by IgG hexamers assembled at the cell surface. Science 2014; 343:1260–3.

Cooperativity- some rules Mechanisms of cooperativity

IgG cooperativity Experimental system Self-association and cooperativity

Functional implications

Outline

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Number of cooperative units: N𝑃𝑃 + 𝐿𝐿↔

𝐾𝐾1 𝑃𝑃𝐿𝐿 + 𝐿𝐿𝛼𝛼𝐾𝐾1 𝑃𝑃𝐿𝐿2 + 𝐿𝐿

𝛼𝛼2𝐾𝐾12 𝑃𝑃𝐿𝐿3 + 𝐿𝐿…𝑛𝑛Cooperative free energy: α

𝑃𝑃 + 𝐿𝐿↔𝐾𝐾1 𝑃𝑃𝐿𝐿 + 𝐿𝐿

𝛼𝛼𝐾𝐾1 𝑃𝑃𝐿𝐿2

Cooperativity- some rules

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1E-3 0.01 0.1 1 10 100 10000.0

0.2

0.4

0.6

0.8

1.0

Non-cooperative α = 10 α = 1000

Θ

[L]free

-3 -2 -1 0 1 2 3

0.0

0.2

0.4

0.6

0.8

1.0

Θ

Log[L]free

Non-cooperative N = 2, α = 10 N = 3, α = 10/step N = 6, α = 10/step

Intramolecular: allostery Conformational

2o or 3o structural changes Configurational- covalent change

Kinase/phosphatase PO42-

Class/Subclass swapping E.g. IgG1IgG4

Intermolecular: associative Mechanisms

Weak self-association Allostery on binding

IgG::C1q assembly Fc:FcR binding

Mechanisms of cooperativity

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Mechanisms are non-exclusive

Rayner, et al. (2012) “The Solution Structure of Rabbit IgG Accounts for Its Interactions with the Fc Receptor and Complement C1q and Its Conformational Stability” JMB 425

Experimental system 12 anti-IL13 mAbs + Protein A purified Human IgG

V-region3 Paratopes

C-region4 subclasses

mAb 1

IgG1IgG2IgG4

IgG4Pro

mAb 2

IgG1IgG2IgG4

IgG4Pro

mAb 3

IgG1IgG2IgG4

IgG4Pro

Hu p-IgG IgG1,2,4

Solvents

PBS [pH 7.4]

10 mM sodium acetate [pH 5], 50 mM NaCl

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No evidence for V-V or C-V conformational or configurational cooperativity

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Solution hydrodynamics by AUC

• mAb hydrodynamics are comparable• Slight differences between subclasses expected/observed

• Labeled IgG indistinguishable from unlabeled

Red: pH 5 acetate Blue: pH 7.4 PBS

sw = 6.43 ± 0.09

mAb 3sw = 6.37 ± 0.05

mAb 2sw = 6.37 ± 0.06

mAb 1

𝒔𝒔 =𝑴𝑴𝒃𝒃𝒇𝒇

=𝒗𝒗𝝎𝝎𝟐𝟐𝒓𝒓

Sedimentation velocity provides molecular information

Solution charge (ZDHH) by MCE

Danlin Yang

7Yang D, et al. (in press) “Weak IgG self- and hetero-association characterized by fluorescence analytical ultracentrifugation” Protein Sci.

ZDHH distribution from MCE PBSIEF

Human poly-IgG

8Yang D, et al. (in press) “Weak IgG self- and hetero-association characterized by fluorescence analytical ultracentrifugation” Protein Sci.

Fc binding FcγR Cellular binding & transport RI, RIIA,RIIB,RIIIA,RIIIB, Rn

C1q binding sites Adaptive & innate immune coupling “Self” versus “foreign”

Tolerance versus immunity

Intermolecular cooperativityIgG effector functions

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Rayner, et al. (2012) “The Solution Structure of Rabbit IgG Accounts for Its Interactions with the Fc Receptor and Complement C1q and Its Conformational Stability” JMB 425

Gaboriaud, et al. (2012) “The Human C1q Globular Domain: Structure and Recognition of Non-Immune Self Ligands” Front. Immuno. 2:92

Diebolder CA, et al. Complement is activated by IgG hexamers assembled at the cell surface. Science 2014; 343:1260–3.

Weak Ab:Ab association?Tracer IgG in different IgG backgrounds

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PMT

Dichroicmirror

Spatial filter

Fluorescent event

Collimation

Objective lens

Expansion

Lase

r

Dichroic filter

1000

2000

3000

4000

fluo

resc

ence

cou

nts

Fluo

resc

ence

inte

nsity

4000

3000

2000

1000

0

6.0 6.5 7.0

Radius (cm)

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Hydrodynamic nonideality: ks

Concentration-dependent nonideality Sedanal: fit for kS, B, Ka

ks reflects interactions between adjacent molecules ks = 9 non-interacting spheres

ks > 9 repulsion or asymmetry

ks < 9 attractive interactions

Uttinger, et. al (2017) Nanoscale, 9:17770 - 17780

Results- all show weak attraction1 mg/mLIgG1IgG4ProHu IgG

20 mg/mLIgG1IgG4ProHu IgG

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Implications for antibody functions

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Diebolder CA, et al. Complement is activated by IgG hexamers assembled at the cell surface. Science 2014; 343:1260–3.

• IgG:IgG interactions source of cooperative free energy for effector functions

• Generality of cooperative interactions allows wider array of epitope spacing to initiate complement activation

12 mAbs produced and characterized Stable cell lines available

No evidence for intramolecular cooperativity No allostery Sub-class swap had no effect on Ag binding

Charge must be measured pI of Hu poly-IgG ranges from < 4 to > 10 ZDHH Hu poly-IgG is -6±3

All IgGs exhibit weak self-association Kd ~100 – 2000 IgG4 weakest

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Summary

Danlin YangSue ChaseWalt StaffordJack Correia

Thanks toChris RobertsDavid HayesBoehringer-IngelheimBITC

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