Engineering IL2-Fc for cancer immunotherapy Daniel Christ Garvan Institute of Medical Research Sydney, Australia

Rosenberg (2014)

High dose IL-2 therapy

•  Vascular leak syndrome (VLS): capillary leak leading to fluid accumulation

•  Pulmonary oedema •  Reduced organ perfusion (hypoxia of vital organs) •  Intensive patient management required

Interleukin-2 biology

•  Pleiotropic

-  Expansion of CD8+ T-cells and NK cells -  Expansion of regulatory T-cells (Tregs)

Boyman and Sprent (2012)

IL-2/anti-IL-2 antibody complexes

•  Enhanced proliferation of CD8+ T-cells and NK cells

•  Enhanced antitumour properties Boyman et al. 2006 Science, Krieg et al. 2010 PNAS

IL-2 receptor structure

Boder 2012 Nature

IL-2

CD122 CD132 CD25

Half-life extension via FcRn recycling

Steric blockade of CD25 binding site

IL-2/anti-IL-2 antibody complexes

Boyman et al. 2006 Science, Krieg et al. 2010 PNAS

Reduced renal clearance (> 50–60 kDa)

Can we re-capitulate the effects of IL-2/mAb complexes within a single polypeptide chain?

Mutant IL-2-Fc fusion proteins with superior activity and selectivity

IL-2/anti-IL-2 antibody complexes

•  CD8+ T-cells and NK cell proliferation ✔ •  Antitumour properties ç Boyman et al. 2006 Science, Krieg et al. 2010 PNAS

B16F10

IL-2WTFc displays high and unexpected anti-tumour activity

Influence of immune effector functions

IL-2WTFc targets Tregs for FcγR-dependent depletion

Interactions with FcγR and CD25 are required for potent antitumour activity

B16F10

CT26

Interactions with FcγR and CD25 are required for potent antitumour activity

IL-2WTFc mediates Treg depletion in tumour tissue

B16F10

PBS

IL-2W

T Fc

IL-23X Fc

0

10

20

30

40

50

ns ***

Treg

s(%

of C

D4)

Spleen dLN Tumour

PBS

IL-2W

T Fc

IL-23X Fc

0

10

20

30

40ns ***

PBS

IL-2W

T Fc

IL-23X Fc

0

20

40

60

80*** ***

0

10

20

30

40

50Tr

egs

(% o

f CD

4)*** ****

Spleen

0

10

20

30

40 ns ****

dLN

0

20

40

60

80 **** ****

Tumour

PBS Fc Fcnil PBS Fc Fcnil PBS Fc Fcnil

B16F10

IL-2WTFc / antibody combination therapy results in superior efficacy

Summary

1.  Interleukin-2 (IL-2) is a pleiotropic cytokine with proven track record as a human therapeutic

2.  IL-2/mAb complexes increase IL-2 in vivo activity

3.  Engineered IL-2-Fc fusion proteins exceed the efficacy of IL-2/mAb complexes

4.  Intriguingly, Treg depletion, rather than cytotoxic cell expansion, dominant mechanism of antitumour activity of IL-2-Fc

5.  Future developments will require carefully balancing of effects on CD8+/NK cells and Tregs to achieve optimal therapeutic outcomes

Rodrigo Vazquez-Lombardi, Claudia Loetsch, Daniela Zinkl, Jenny Jackson, Peter Schofield, Elissa K. Deenick, Cecile King, Tri Giang Phan, Kylie E. Webster, Jonathan Sprent and Daniel Christ (2017) Nature Communications 8:15373 Rodrigo Vazquez-Lombardi, Damien Nevoltris, Ansha Luthra, Peter Schofield, Carsten Zimmermann and Daniel Christ (2018) Nature Protocols accepted

Acknowledgements Rodrigo Vazquez-Lombardi

Jonathan Sprent

Christ Lab Peter Schofield Jenny Jackson

David Langley Romain Rouet

Mahdi Zeraati Mahmoud Abdelatti

Ansha Malik Damien Nevoltris

Sprent Lab Kylie Webster Tri Giang Phan Claudia Loetsch Theresa Corpuz Joanna Warren