Science - USA (2020-09-25)

GSDMD was essential for cell infiltration to
the CNS during EAE. Mice receiving DMF
demonstrated reduced infiltration of myeloid
cells, CD4+and CD8+T cells (Fig. 4, E and F,
and fig. S13A). DMF reduced T helper 1 (TH1)
[interferon-g+(IFN-g+)] and TH17 (IL-17A+) cell
numbers in the CNS (Fig. 4, G and H, and fig.
S13B). Post mortem brain tissue from MS
patients stained positive for GSDMD-N (Fig.
4I and table S4). Patients with MS had eleva-
ted levels of IL-1band GSDMD-N in peripheral
blood mononuclear cells (PBMCs) (Fig. 4, J to
L, and table S3). Both IL-1band GSDMD-N
were reduced in patients taking DMF (Tecfidera)
(Fig. 4, J to L, and table S3). Thus, DMF re-
duces GSDMD-driven responses in EAE, which
supports a model in which elevated GSDMD
contributes to MS. GSDMD has also been
linked to familial Mediterranean fever (FMF).
FMF results from constitutive activation of the
pyrin inflammasome, and mice that harbor
theMefvV726/726allele exhibit features of the
human disease.Gsdmd-deficient mice are re-
scued from disease in this model ( 19 ). Ad-
ministration of DMF alleviated weight loss,
splenomegaly, IL-1bsecretion, GSDMD-N
formation, and liver pathology in theMefvV726/V726
mouse model (fig. S14, A to E).
Thesedatacollectivelyindicatethatfuma-
rate mitigates pyroptosis. Succination of GSDMD
on Cys^192 prevents its processing and oligomer-
ization, which limits pore formation, cytokine
release, and cell death (fig. S15). Additional
studies reinforce the importance of Cys^191 /
Cys^192 as a target of other GSDMD-targeting
drugs ( 15 , 20 , 21 ). Our study provides mecha-
nistic insight into the immunomodulatory ac-
tivity of Tecfidera (DMF) used for MS and
underscores the importance of GSDMD as a
driver of chronic inflammation. This work also
highlights the potential for the treatment of
chronic inflammatory diseases with inhibitors
of GSDMD.

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ACKNOWLEDGMENTS

Funding:This work is supported by pilot grants from the

Worcester Foundation for Biomedical Research and the Dan and

Diane Riccio Fund for Neuroscience from UMASS Medical School.

F.H. is a GSK postdoctoral fellow. N.K.-C. is a Cancer Research

Irvington Fellow, and L.S.-G. is an EMBO long-term fellow and a

Crohn’s and Colitis Foundation fellow.Author contributions:

F.H. conceived the study, developed the concept, performed

experiments, analyzed data, and wrote the manuscript. L.S.-G.,

N.K.-C., R.W., and Z.J. performed experiments and analyzed data.

S.L., B.W., and S.Y. performed and analyzed EAE experiments.

S.P. provided experimental advice. C.I., R.D., and F.K. provided

clinical expertise and PBMCs from consenting MS patients through

the University of Massachussetts MS Center biorepository. S.A.S.

and K.M. designed and performed mass spectrometry

experiments. V.V.M. and P.R.T. synthesized the MMF-Yne probe.

K.A.F. conceived the study, developed the concept, supervised the

research, and wrote the manuscript.Competing interests:K.A.F.

serves on the scientific advisory board of Quench Bio and

NodThera. The University of Massachusetts Medical School have

filed a provisional patent application on succination of GSDMD

described in this study, listing F.H., P.T., and K.A.F. as inventors.

Data and materials availability:All data in this paper are

presented in the main text and supplementary materials.

SUPPLEMENTARY MATERIALS

science.sciencemag.org/content/369/6511/1633/suppl/DC1

Materials and Methods

Figs. S1 to S15

Tables S1 to S4

References ( 22 – 24 )

MDAR Reproducibility Checklist

View/request a protocol for this paper fromBio-protocol.

31 March 2020; accepted 5 August 2020

Published online 20 August 2020

10.1126/science.abb9818

PROTEIN DESIGN

Designed protein logic to target cells with precise

combinations of surface antigens

Marc J. Lajoie1,2*†‡, Scott E. Boyken1,2*‡, Alexander I. Salter3,4*, Jilliane Bruffey1,2,5,

Anusha Rajan3,4, Robert A. Langan1,2‡, Audrey Olshefsky1,6, Vishaka Muhunthan3,4,

Matthew J. Bick1,2‡, Mesfin Gewe^4 , Alfredo Quijano-Rubio1,2,6, JayLee Johnson^1 , Garreck Lenz^1 ,

Alisha Nguyen^1 , Suzie Pun6,7, Colin E. Correnti^4 , Stanley R. Riddell3,4,8, David Baker1,2,9†

Precise cell targeting is challenging because most mammalian cell types lack a single surface

marker that distinguishes them from other cells. A solution would be to target cells using specific

combinations of proteins present on their surfaces. In this study, we design colocalization-

dependent protein switches (Co-LOCKR) that perform AND, OR, and NOT Boolean logic operations.

These switches activate through a conformational change only whenall conditions are met,

generating rapid, transcription-independent responses at single-cell resolution within complex cell

populations. We implement AND gates to redirect T cell specificity against tumor cells expressing

two surface antigens while avoiding off-target recognition of single-antigen cells, and three-input

switches that add NOT or OR logic to avoid or include cells expressing a third antigen. Thus, de novo

designed proteins can perform computations on the surface of cells, integrating multiple distinct

binding interactions into a single output.

B

iological systems are complex; there-

fore, interventions that perturb these

systems must achieve specific target-

ing in mixed populations of closely re-

lated cells. Cells displaying a specific

surface marker can be targeted with anti-

bodies, but a single marker is rarely sufficient

to identify specific cell types. Bispecific anti-

bodies can achieve some selectivity by simul-

taneously engaging two targets ( 1 , 2 ), but

this strategy requires delicate tuning of the

individual binding affinities to reduce inter-

actions with cells expressing just one of the

targets. A generalized approach for distin-

guishing cells using combinations of sur-

face markers is needed. Toward this end, we

sought to develop a modular protein system

capable of taking multiple binding events as

input, computing combinations of Boolean

logic operations (AND, OR, and NOT) with-

out requiring cellular machinery for signal

integration, and producing a single output

(Fig. 1A).

How does one design a system that activates

only on the surface of a cell and not in solu-

tion? Given that antigen binding at the cell

surface increases the local concentration of

the bound protein, such a system potentially

could be constructed from an actuator that

SCIENCEsciencemag.org 25 SEPTEMBER 2020•VOL 369 ISSUE 6511 1637

(^1) Institute for Protein Design, University of Washington,
Seattle, WA, USA.^2 Department of Biochemistry, University of
Washington, Seattle, WA, USA.^3 Immunotherapy Integrated
Research Center, Fred Hutchinson Cancer Research Center,
Seattle, WA, USA.^4 Clinical Research Division, Fred
Hutchinson Cancer Research Center, Seattle, WA, USA.
(^5) Graduate Program in Molecular and Cellular Biology,
University of Washington, Seattle, WA, USA.^6 Department of
Bioengineering, University of Washington, Seattle, WA, USA.
(^7) Molecular Engineering and Sciences Institute, University of
Washington, Seattle, WA, USA.^8 Department of Medicine,
University of Washington School of Medicine, Seattle, WA,
USA.^9 Howard Hughes Medical Institute, University of
Washington, Seattle, WA, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: [email protected] (M.J.L.);
[email protected] (D.B.)‡Present address: Lyell Immunopharma,
Inc., Seattle, WA, USA.
RESEARCH | REPORTS