Article

https://doi.org/10.1038/s41586-018-0830-7

De novo design of potent and selective
mimics of IL-2 and IL-15

Daniel-Adriano Silva1,2,14*, Shawn Yu1,3,14, Umut Y. Ulge1,3,14, Jamie B. Spangler4,5,14, Kevin M. Jude5, Carlos Labão-Almeida6,
Lestat R. Ali7, Alfredo Quijano-Rubio1,2,3, Mikel Ruterbusch8, Isabel Leung9, Tamara Biary7, Stephanie J. Crowley7, Enrique Marcos1,2,10,
Carl D. Walkey1,2, Brian D. Weitzner1,2, Fátima Pardo-Avila11, Javier Castellanos1,2, Lauren Carter1, Lance Stewart1,
Stanley R. Riddell9, Marion Pepper8, Gonçalo J. L. Bernardes6,12, Michael Dougan7, K. Christopher Garcia5,13* & David Baker1,2,13*

We describe a de novo computational approach for designing proteins that recapitulate the binding sites of natural
cytokines, but are otherwise unrelated in topology or amino acid sequence. We use this strategy to design mimics of thecentral immune cytokine interleukin-2 (IL-2) that bind to the IL-2 receptor βγc heterodimer (IL-2Rβγc) but have nobinding site for IL-2Rα (also called CD25) or IL-15Rα (also known as CD215). The designs are hyper-stable, bind humanand mouse IL-2Rβγc with higher affinity than the natural cytokines, and elicit downstream cell signalling independentlyof IL-2Rα and IL-15Rα. Crystal structures of the optimized design neoleukin-2/15 (Neo-2/15), both alone and in complex
with IL-2Rβγc, are very similar to the designed model. Neo-2/15 has superior therapeutic activity to IL-2 in mouse modelsof melanoma and colon cancer, with reduced toxicity and undetectable immunogenicity. Our strategy for building hyperstable de novo mimetics could be applied generally to signalling proteins, enabling the creation of superior therapeuticcandidates.

The considerable potential of the central immune cytokine interleukin-2(IL-2) for cancer treatment1–3 has sparked numerous efforts to improve
its therapeutic properties by mutation and/or chemical modification4–11. Such efforts have sought to simplify manufacturing, extend
half-life, and modulate interactions with receptors12–14. However, there
are inherent challenges to the development of a new therapeutic agent
when starting with a naturally occurring bioactive protein. First, mostnatural proteins are only marginally stable15–17; hence, amino acidsubstitutions aimed at increasing efficacy can decrease expression orcause aggregation, making manufacture and storage difficult. Moresubstantial changes, such as deletion or fusion of functional or targetingdomains, are often unworkable and can markedly alter pharmacokinetic properties and tissue penetration13. Second, any immune response
against the engineered variant may cross-react with the endogenous
molecule18–24 with potentially dangerous consequences. Third, thetarget receptor-subunit interaction profile can be difficult to reprogram. The clinical use of IL-2 has been mainly limited by toxicity25–27
which, although incompletely understood in humans, is considerably
reduced in animals deficient in the IL-2Rα chain (CD25−). Previous
efforts to ablate the interaction between CD25 and IL-2 by either mutation5,8,28,29 (as in super-2) or PEGylation (for example, NKTR-21411),
have resulted in markedly reduced stability, binding, and/or potencyof these reengineered cytokines, while failing to completely eliminate
the interaction with CD25. Here we describe a computational design
approach to generate mimetics of natural proteins with improved therapeutic properties that circumvent these challenges, allowing the engineering of hyper-stable de novo cytokine mimics that display specificreceptor-binding interfaces that are optimal for treating disease.

IL-2/IL-15 mimics that bind and activate IL-2Rβγc

Cytokines interact with multiple receptor subunits30–33, and likemost naturally occurring proteins, contain non-ideal structural features that compromise their stability but are important for function.
We developed a computational protein design method in which thestructural elements that interact with the desired receptor subunit(s)are fixed in space (Fig. 1a), and an idealized de novo globular protein
structure is built to support these elements. De novo design has beenused previously to support short linear epitopes34–37; here we support
more complex binding interfaces by using a parametric constructionof disembodied helices coupled with knowledge-based loop closure38(Fig. 1b, c). We tested our approach by attempting de novo design ofstable idealized proteins with interaction surfaces mimicking thoseof IL-2 and IL-15 for IL-2Rβγc32,39, but entirely lacking the CD25interaction surface.
Native IL-2 comprises four helices (Fig. 1a) connected by long irregular loops. The N-terminal helix (H1) interacts with both the β- andγ-subunits, the third helix (H3) interacts with the β-subunit, and the
C-terminal helix (H4) interacts with the γ-subunit. The α-subunit(CD25) interacting surface is formed by the irregular second helix(H2) and two long loops, one connecting H1 to H2 and the otherconnecting H3 to H4. We aimed to build an idealized protein that recapitulates the interface formed by H1, H3 and H4 with β and γ-subunits
and to replace H2 with a helix that provides better packing and does
not interact with the α-subunit. Two generations of designed mimics
were made. In a first generation, we used all helices (H1, H2, H3 andH4) from IL-2 (Fig. 1a) as starting points for structure idealization by(independently) rebuilding each disembodied helix using commonly1Institute for Protein Design, University of Washington, Seattle, WA, USA. 2Department of Biochemistry, University of Washington, Seattle, WA, USA. 3Department of Bioengineering, University ofWashington, Seattle, WA, USA. 4Departments of Biomedical Engineering and Chemical and Biomolecular Engineering, Johns Hopkins University, Baltimore, MD, USA. 5Departments of Molecular
and Cellular Physiology and Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. 6Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa,
Lisbon, Portugal. 7Division of Gastroenterology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA. 8Department of Immunology, University of Washington School of
Medicine, Seattle, WA, USA. 9Fred Hutchinson Cancer Research Center, Clinical Research Division, Seattle, WA, USA. 10Institute for Research in Biomedicine (IRB Barcelona), The Barcelona
Institute of Science and Technology, Barcelona, Spain. 11Department of Structural Biology, Stanford University School of Medicine, Stanford, CA, USA. 12Department of Chemistry, University ofCambridge, Cambridge, UK. 13Howard Hughes Medical Institute, Chevy Chase, MD, USA. 14These authors contributed equally: Daniel-Adriano Silva, Shawn Yu, Umut Y. Ulge, Jamie B. Spangler.
*e-mail: dadriano@uw.edu; kcgarcia@stanford.edu; dabaker@uw.edu
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Article RESEARCHab

H1→H2→H3→H4c

H2'

IL-2Rα

H3

H1→H3

Gen1

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H1

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. a.

H2′→H4eTarget IL-2Rβγc
Generation 2 (human)
Generation 2 (mouse)

pSTAT5 EC50 (M)dGeneration 1 (human)

H1→H3→H2′→H4→ 3→→
H2′→H4→H1→H3→ → →H3

Sequence design

10–8

10–9

H3→H2′→H4→H1
H3→H2→H4→H1 H2
H2′→H3→H1→H4→H3→H1→H4

10–10
Neoleukin-2/15

10

–8

10
10
IL-2Rβγc Kd (M)–7–6Filtering
Score / residuefForward folding

–2.2
–2.8
–3.4

0

2
4
6
8
R.m.s.d. to design

Fig. 1 | Computational design of de novo cytokine mimics. a, Structureof human IL-2 (HsIL-2) (cartoon representation) in complex with its
receptor IL-2Rαβγc (surface representation) (Protein Data Bank (PDB)
ID: 2B5I). b, The designed mimics have four helices; three (blue, yellow
and red) mimic IL-2 interactions with IL-2Rβγc, whereas the fourth(green) holds the first three in place. Top, first generation (gen 1): each of
the core elements of IL-2 (helices H1–H4) were independently idealized
by assembly of four residue clustered protein fragments. Bottom, second
generation (gen 2): the core elements were built using parametricequations that recapitulate the shape of each disembodied helix, allowingchanges in the length of each helix by up to ±8 amino acids. c, Pairs
of helices were reconnected using ideal loop fragments; representative
examples are shown with the newly built elements connecting each pairof helices in magenta. d, Combination of helix hairpins in c to generate
fully connected protein backbones. e, Rosetta flexible backbone sequence
design. f, Binding and activity of selected designs (solid symbols), thegreen arrow originates at the parent of the best optimized design Neo-2/15.
(see Extended Data Table 1).

occurring protein fragments (see Methods) and connecting helices withfragment-derived loops (Fig. 1c) to generate fully connected backbones(Fig. 1d). Rosetta combinatorial flexible backbone sequence design was
carried out40–42 for each backbone (in complex with human IL-2Rβγc),
resulting in a considerably more regular structure for H2 (H2′) thanin IL-2 (Fig. 1b, top; see Methods). The four lowest-energy designs
and eight single-disulfide-stapled variants (Supplementary Table 1)
were selected for experimental characterization by yeast display(see Methods). Eight designs bound fluorescently-tagged βγ-chimeric
IL-2 receptor at low-nanomolar concentrations (Supplementary Fig. 1),
and the highest affinity non-disulfide design (G1_neo2_40) was subjected to site-saturation mutagenesis (Supplementary Table 5) andselection for increased affinity to human IL-2Rβγc. A second roundof selection was then carried out on a library combining the bestindividual affinity-enhancing substitutions (combinatorial library,
Supplementary Fig. 2, Supplementary Table 7). The highest-affinity
variants (Supplementary Fig. 4, Supplementary Table 2) were expressed
recombinantly in Escherichia coli and found to elicit pSTAT5 signalling in vitro on IL-2-responsive mouse cells at low-nanomolar or evenpicomolar concentrations (Extended Data Table 1), but had relatively
low thermal stability (melting temperature, Tm < 45 °C, SupplementaryFigs. 3, 5). To improve stability, in a second generation of designs, werepeated the computational design protocol starting from the backboneof the highest affinity first-round design (G1_neo2_40_1F, topology:
H1→H4→H2′→H3), but this time coupling the loop-building processwith parametric variation of the helix lengths (up to ±8 amino acids,
Fig. 1b, bottom). This second-generation approach improved the quality of the models by enabling the exploration of a substantially larger
number of combinations of high-quality loops connecting each pairof helices. The 14 second-generation designs with highest predictedaffinity and stability, along with 27 Rosetta-sequence redesigns of
G1_neo2_40_1F (Supplementary Table 3), were experimentally characterized. All but one of the designs were found to bind the human IL-2
receptor at low-nanomolar concentrations (Fig. 1f, Extended Data
Table 1, Supplementary Fig. 6). The three designs with highest affinity
and stability (one sequence redesign and two new mimetics) were
subjected to site-saturation mutagenesis (Supplementary Table 6),
followed by selection on combinatorial libraries of substitutions
to increase affinity for mouse IL-2Rβγc (Supplementary Figs. 8–10,
Supplementary Tables 6, 8), which yielded higher-affinity hyper-stablevariants of the de novo mimics (Extended Data Table 1, Supplementary
Tables 4, 8, Supplementary Figs. 11–13). The second-generation
optimized design with highest overall affinity for both human andmouse IL-2Rβγc, Neo-2/15, is a 100-residue protein with a topology
and sequence that is quite different from human or mouse IL-2
(BLASTP sequence identity to human and mouse IL-2 of 14% and 24%,
respectively; MICAN structure-based sequence identity to human andmouse IL-2 of 29% and 16% respectively, see Methods and Extended
Data Table 1).

Functional characterization of Neo-2/15

Neo-2/15 binds with high affinity to human and mouse IL-2Rβγc (withdissociation constants, Kd ≈ 19 nM and Kd ≈ 38 nM, respectively), butdoes not interact with IL-2Rα (Fig. 2a). The affinities of Neo-2/15 forthe human and mouse IL-2 receptors (IL-2Rβ and IL-2Rβγc) are higher
than those of the corresponding native IL-2 cytokines (Extended Data
Table 1). Neo-2/15 activates IL-2Rα− human YT-1 cells more potently
than native human IL-2 (half-maximal response dose, EC50 = 49 pM
versus 410 pM for the native human IL-2). Similarly, Neo-2/15 activates IL-2Rα− mouse primary T cells more potently than native mouse
1 0 J A N U A R Y 2 0 1 9 | V O L 5 6 5 | N A T U RE | 1 8 7© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article

10–6

E

J

CD25–

0
120

10–12

10–9

10–6

80

10–8

10–6

HsIL-2RβHsIL-2Rγ

1.0

10–10
10–8
10–6
Concentration (M)

10–6

10–10

10–8

E

10–6

MmIL-2Rβ
MmIL-2Rγ

+

0.0

D

120

J

CD25+

E

J

CD25–

10–12
10–9
10–6
Concentration (M)

80
40
0
120

10–12

10–9

10–6

80
40
0

10–10
10–8
10–6
Concentration (M)

20
0

10–12
10–9
10–6
Concentration (M)5

10 15 20 25 30
Time at 80 ºC (min)90 120Ex vivo, murine splenocytes (5-day survival)
MmIL-2Neoleukin-2/15
Super-2
No heat incubation

0.0

10–8

MmIL-2Rγ

1.0

+

0.0

10–10

After 1 h at 95 °C

10–10

0.0

Normalized pSTAT5 (MFI %)

1.0

10–6

HsIL-2Rγ

1.0

0.0

10–8

Ex vivo, mouse primary
C57BL/6 CD4+ T cells

10–10

60

40
0

0.0

Super-2

4×105
2×105
1×104
100
10
1
0.1

10–8

MmIL-2Rβ40

HsIL-2

100

100
10
1
0.1

1.0

10–10

80

100
10
1
0.1

0.0

CD25+

Binding
Neoleukin-2/15

100
10
1
0.1

10–6

J

Protein thermal stability

MmIL-2

100
10
1
0.1

10–8

HsIL-2RβD

HsIL-2
Super-2
120

100
10
1
0.1

1.0

10–10

c

Cell signalling
Neoleukin-2/15

E

MmIL-2Rα

1.0

In vitro, human NK (YT) cells

0.0

Mouse

HsIL-2Rα

1.0

MmIL-2

Normalized response (%)

Super-2

Cell relative luminescence on day 5

HsIL-2

Human

Normalized responsebBinding
Neoleukin-2/15

Normalized pSTAT5 (MFI %)

a

4×105
2×105
1×104

Concentration (ng ml–1)

Fig. 2 | Characterization of Neo-2/15. a, Receptor subunit binding
assessed by surface plasmon resonance (SPR). Neo-2/15 (red) does notbind IL-2Rα (top row), but binds more tightly than IL-2 (light blue) andsuper-2 (dark blue) to IL-2Rβ (second row) and IL-2Rβγc (bottom row)
(Kd values in Extended Data Table 1; experiments performed 3 times withsimilar results). b, Neo-2/15 stimulates STAT5 phosphorylation (pSTAT5)
more potently than IL-2 in cells expressing IL-2Rβγc but lacking IL-2Rα(CD25−) in both (in vitro) human NK cells (YT cell line; n = 3;experiments performed 3 times with similar results) and mouse primary

CD4+ T cells (n = 4; data from a pool of 3 independent experiments).
c, Top, Neo-2/15 can be incubated for 2 h at 80 °C without loss of binding
to human IL-2Rβγc, whereas human IL-2 and super-2 quickly lose
activity (immobilized IL-2Rγc with soluble IL-2Rβ at 500 nM; experiment
performed once). Bottom, following incubation at 95 °C for 1 h, Neo-2/15
still drives cell survival effectively (with ~70% luminescence remaining
at 10 ng ml−1 relative to cells treated with non-heat incubated neo 2/15),
whereas mouse IL-2 (MmIL-2) and super-2 are inactivated (n = 3;experiment performed 3 times with similar results). Data are mean ± s.d.

IL-2 (EC50 = 130 pM versus 30 nM). These results are consistent withthe higher binding affinity of Neo-2/15 for the IL-2 receptors (Fig. 2b,
Supplementary Table 9). Neo-2/15 is also more active than super-2, avariant of IL-2 engineered for higher affinity for IL-2Rβ8, on IL-2Rα−mouse primary T cells (EC50 = 130 pM versus 660 pM) and less active
than super-2 on IL-2Rα+ cells (EC50 = 24 pM versus 1.2 pM), presumably owing to its complete lack of IL-2Rα binding (Fig. 2b). Neo-2/15 is
hyper-stable (Supplementary Fig. 13) and does not lose binding affinity
for the IL-2Rβγc following incubation at 80 °C for 2 h, whereas humanIL-2 and super-2 are inactivated after 10 min (half-inactivation
time ≈ 4.2 min and 2.6 min, respectively, Fig. 2c, top). In ex vivo primary cell cultures, Neo-2/15 drives T cell survival effectively after
60 min of boiling at 95 °C, whereas these conditions inactivated both
IL-2 and super-2 (Fig. 2c, bottom). This unprecedented stability for acytokine-like molecule, as well as elimination of the requirement forcold chain storage, suggests a robustness to mutations (Extended Data
Fig. 8), genetic fusions, and chemical modification (Supplementary
Fig. 14) greatly exceeding that of native IL-2, which could contribute to
the development of improved or new therapeutic properties (Extended
Data Figs. 3, 4).

H3, and with γc via the H1 and H4 helices (Fig. 3); these regions alignclosely with both the computational design model (Fig. 3a) and themonomeric crystal structure (Fig. 3c). A shift of about 4.0 Å for helix
H4 (see Fig. 3c) in the mouse complex may reflect the optimization
for high-affinity binding to both the mouse and human receptors. The
design of Neo-2/15 was guided by the human complex structure, andsimulations suggest that there is little or no helix shift in this complex (Extended Data Fig. 7). Consistent with this, the helices of apo
Neo-2/15 superimpose closely on those of human IL-2 in complex
with its receptor (Fig. 3e, f), despite the different topologies of thetwo proteins (Fig. 3d).

Structure of Neo-2/15 and complex with MmIL-2Rβγc

The X-ray crystal structure of Neo-2/15 is very similar to the computational design model (root mean square deviation of Cα atoms,
r.m.s.d. = 1.1–1.3 Å for the 6 copies in the asymmetric unit, Fig. 3a).
We determined the structure of mouse IL-2Rβγc in complex with Neo2/15 and found that it aligns very closely to the previously rep
orted human IL-2 receptor complex43 (Fig. 3b, Extended Data Table 2).
The Neo-2/15 design model and the unbound crystal structure alignwith the mouse ternary complex structure with r.m.s.d. of 1.27 and1.29 Å, respectively (Fig. 3c). The order of helices in Neo-2/15 (using
IL-2 numbering) is H1→H3→H2′→H4 (Figs. 1a, 3a, d). The H1–H3
loop is disordered in the ternary complex, but helix H3 is in close agreement with the predicted structure; there is also an outward movementof helix H4 and the H2′–H4 loop compared to the monomeric structure(Fig. 3c). Neo-2/15 interacts with mouse IL-2Rβ via helices H1 andTherapeutic applications of Neo-2/15

The inherent low stability of IL-2 and its tightly evolved dependence on
CD25 have been barriers to the clinical translation of re-engineered IL-2
compounds. Other efforts have focused on IL-1544, because it elicits
similar signalling to IL-2 by dimerizing IL-2Rβγc, but has no affinity
for CD25. However, IL-15 activity is dependent on trans presentation of IL-15Rα, which is displayed primarily on antigen-presenting
cells and NK cells. The low stability of native IL-15 and its dependence on trans presentation have also presented substantial barriers to
re-engineering efforts44,45.
Dose-escalation studies on naive mice show that Neo-2/15 causes
lower expansion of immunosuppressive T regulatory (Treg) cells thanmouse IL-2 (Fig. 4a, left), leading to a higher CD8+ killer T cell:Treg
ratio with Neo-2/15 (Fig. 4a, right). The increased expansion of Treg
cells by mouse IL-2 is expected, because it binds preferentially to CD25+
cells35,46,47. The higher CD8+ T cell:Treg ratios achieved with Neo-2/15
are generally associated with better tumour killing8,11,29; this functional advantage of Neo-2/15 is likely to stem from its higher affinity
for IL-2Rβγc and lack of bias towards CD25+ cells. Similarly, in amouse model of airway inflammation that normally induces a small
percentage of tissue-resident CD8+ T cells (Thy1.2−CD44+CD8+),
Neo-2/15 elicits an increase in the population of tissue-resident CD8+
T cells without increasing the population of antigen-specific Treg
(CD4+Foxp3+; Fig. 4b).

1 8 8 | N A T U RE | V O L 5 6 5 | 1 0 J A N U A R Y 2 0 1 9© 2019 Springer Nature Limited. All rights reserved.

Article RESEARCH
a

c

b

H3

H3

H1

H1

H4

H2′

H4

Interface helices

H3

d

Mmγc

MmIL-2RβH1

MmIL-2RβH4IL-2Rβinterfacee
IL-2Rβinterfaceγcinterface

HsIL-2

Neo-2/15

γcinterfaceIL
L-2Rα
intterfacee

H3 H1 H2H4

H3

H2′
Neo-2/15

H4
Y37
L85

H8
Q13

HsIL-2

Mmγc

Y14
L19

H1

Y14
L19

H1
K33
R81

MmIL-2Rβ

f

Mmγc

L17
Q22
F99
S130

L43
V91

H3 H1H2′H4Fig. 3 | Structure of Neo-2/15 and its ternary complex with mouse
IL-2Rβγc. a, Top, structural alignment of Neo-2/15 chain A (orange) withthe design model (grey, r.m.s.d. ≈ 1.11 Å, for 100 Cα atoms). Bottom,
detail of interface helices H1, H3 and H4 (numbered according to HsIL-2,
Fig. 1). The interface side chains are shown in sticks. b, Crystal structureof the ternary complex of Neo-2/15 (red) with mouse IL-2Rβ (wheat)and IL-2Rγc (pink), aligned to the design model against human IL-2Rβγc(grey, r.m.s.d. ≈ 1.27 Å, for the 93 modelled Cα atoms of Neo-2/15 in the
ternary complex). c, Structural alignment of monomeric Neo-2/15 (chain A,
orange) with Neo-2/15 in the ternary complex (red, r.m.s.d. ≈ 1.71 Å
for the 93 modelled Cα atoms in the ternary complex), highlighting a~4.0 Å shift of helix H4 in the ternary-complex structure compared to themonomeric crystal structure (green double-headed arrow). d, Comparisonof the crystallographic structures of human IL-2 (cartoon representationin blue) (left) and Neo-2/15 from the ternary complex in b (cartoon
representation in red) (right). The regions that interact with IL-2Rβ andγc are indicated. Topology diagrams are shown below the structures. The
loop-rich region from IL-2 that interacts with the IL-2Rα does not exist in
the de novo mimic Neo-2/15. e, f, Comparison of the binding interfacesof Neo-2/15 and human IL-2. Interfacial amino acids are shown as sticks,
and those that differ between human IL-2 and Neo-2/15 are denotedwith labels.

To test whether Neo-2/15 is immunogenic, naive and tumour-bearing
mice were treated with Neo-2/15 daily (over a period of 4 weeks and2 weeks, respectively). Little or no immunogenicity was observed ineither case (Fig. 4c, Extended Data Fig. 5). A similar lack of immune
response has been observed for other de novo-designed therapeutic candidates35 (probably owing to their small size and high stability).
Polyclonal antibodies against Neo-2/15 were generated by vaccinating
mice with an inactive Neo-2/15 mutant—Neo-2/15(Y14D/F99D), also
known as KO Neo-2/15—in complete Freund’s adjuvant. These polyclonal Neo-2/15 antibodies do not cross-react with human or mouseIL-2 (Fig. 4c, Extended Data Fig. 5), suggesting that even if there is an
immune response to Neo-2/15 in a therapeutic setting, this response
is unlikely to cross-react with endogenous IL-2. This is consistent withthe low sequence identity between Neo-2/15 and human or mouseIL-2 (Extended Data Table 1), which makes an autoimmune response
against host IL-2 much less likely with Neo-2/15 than with previously
engineered human IL-2 variants (for example, super-2 or PEGylatedIL-2), which differ from endogenous IL-2 by only a few mutations (theBLASTP sequence identities of Neo-2/15 and super-2 to human IL-2
are 14% and 95%, respectively).
We tested the therapeutic efficacy of Neo-2/15 in B16F10 (melanoma) and CT26 (colon cancer) mouse models. Single-agent treatment with Neo-2/15 led to dose-dependent delays in tumour growthin both cancer models. In CT26 colon cancer, single-agent treatment showed improved efficacy compared to mouse IL-2 (Fig. 4d,
Extended Data Fig. 1). In B16F10 melanoma, previous studies haveshown that single-agent treatment with IL-2 is only partially effective, and co-treatment with the anti-melanoma cell antibody TA99
(anti-TRP1 monoclonal antibody) is synergistic with IL-25,12,14 andIL-15 (superagonist complex ALT-803)48. In long-term survival

experiments (8 weeks), Neo-2/15 in combination with TA99 showed
substantially reduced toxicity and an overall superior therapeutic effect compared to mouse IL-2 (Fig. 4e, Extended Data Fig. 2),
whereas treatment with TA99 alone had little effect. Mice treatedwith both mouse IL-2 and TA99 steadily lost weight and their overall
health declined to the point of requiring euthanasia, whereas little
decline was observed with the combination of Neo-2/15 and TA99
(Fig. 4e). Consistent with a therapeutic benefit, Neo-2/15 treatment
led to significantly increased intratumoural CD8:Treg ratios (Fig. 4f,
Extended Data Fig. 1), which are known to correlate with effective
antitumour immune responses49. The increases of CD8:Treg ratioswith Neo-2/15 treatment are dose- and antigen-dependent (Fig. 4f),
which is in agreement with the enhanced therapeutic effects observed
at higher doses and in combination with TA99 (Extended Data Fig. 2).
Collectively, these data show that Neo-2/15 exhibits the predicted
homeostatic benefit derived from its IL-2-like immunostimulatory
activity, but without the adverse effects associated with preferential
binding to CD25+ cells. These enhanced properties and low toxicity could enable routine use of Neo-2/15 for indications for which
IL-2 is not broadly used, such as to enhance CAR-T cell therapies(Extended Data Fig. 4). The activation of pSTAT5 signalling in naive
mouse peripheral blood lymphocytes (CD8+ T and B cells) observed
an hour after exposure to Neo-2/15 was markedly reduced after threehours (Extended Data Fig. 6), suggesting that the efficacy of Neo-2/15
can be increased using standard approaches for extending circulation
half-life, such as PEGylation50.
De novo design of protein mimetics has the potential to transformthe field of protein-based therapeutics, enabling the development ofmolecules that improve on biology by enhancing therapeutic properties and reducing side effects, not only for cytokines, but for almost
1 0 J A N U A R Y 2 0 1 9 | V O L 5 6 5 | N A T U RE | 1 8 9© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article

0.5

f

P = 0.004
21

5 7
14
Days after tumour cell implantation

20

0

42

0
56
4 tumour-freemice
5

n = 10

1,000

14

28

CD8+ : Treg
42

0

No tx

VA
O

2
sI
LH

KO MmIL-2 NeoNeo-2/15
2/15

40
NS
20
0

NS

KO
MmIL-2
Neo-2/15

Neo2/15

1 μg day–1

1

n
ss
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rde
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igh our bu umou
uro
T
m
m
Tu
Tu

0
28
56
14
42
Days after tumour cell implantation

20

3

We

0

56

P = 0.012
NS

10 μg day–1

P = 0.036–6

CD8+ : Treg

28

40

P = 0.015

Reason foreuthanization

SurvivingCD8+ : Treg

P = 0.043

2,000

14

CD8+ : Treg ratio (melanoma tumour)

60 Weight change (week-7)

–12

0

M
m
IL
-

N

Target protein (ELISA coating)

100

1,000

0

Tumour size (mm3)n=5n = 10Survival (%)

2,000

2

0.0

eo
-

N
o
tx
M
m
IL
-2
N
eo
-2
/1
5

0

P = 0.003
P = 0.003
P = 0.002
Anti-Neo-2/15 pAb(–) Control
Neo-2/15
Treated micesera
MmIL-2

1.0

2/
15

20

Absorbance at 450 nm

40

Immunogenicity

Melanoma

21

60
20

60

% of initial
weight0100

0Number of mice

Tumour size (mm3)

600

14

5

P = 0.047
P = 0.02c

MmIL-2 (13 μg day–1) + TA99 mAb (150 μg, 2× week)
Neo-2/15 (10 μg day–1) + TA99 mAb (2× week)

n=5

5 7

10

e

Colon cancer
No tx
MmIL-2 (10 μg day–1)
Neo-2/15 (10 μg day–1)

1,200

1
1
10
0.
1
1
10

tx
o
N

0.

1
1
10

0.

1
1
10

tx

0.

N

MmIL-2 Neo-2/15
Dose (μg day–1 per mouse)

Percentage of CD44+Thy1.2–

5

NS P < 0.001

N
o
tx
M
m
IL
-2
N
eo
-2
/1
5

Ratio

10

0

0

d

Tumour size (mm3)

Airway inflammation model%Treg (Der p1+FoxP3+), SLO% Lung resident CD8+

CD8+ : Treg

P = 0.014
P = 0.017

15

2

o

Percentage of CD45+

NS
P < 0.001
P = 0.006

4

MmIL-2 Neo-2/15
Dose (μg day–1 per mouse)

Survival (%)

b

T cells (naive mice)%Treg (CD25+FoxP3+)

Percentage of CD4+CD44+

a

40
20
0

P = 0.041
NS

KO
MmIL-2
Neo-2/15

Neo2/15

1 μg day–1 + TA99 (150 μg, 2× week)

Fig. 4 | In vivo characterization of Neo-2/15. a, Mouse IL-2 but not
Neo-2/15 enhances CD4+ Treg expansion in naive mice T cells. n = 2(no tx (no treatment); 0.1, 1 mouse IL-2; 0.1, 1 Neo-2/15); n = 3(10 mouse IL-2; 10 Neo-2/15). b, In a mouse airway inflammation
model (20 µg per day per mouse, 7 days), Neo-2/15 does not increase
the frequency of antigen-specific CD4+Foxp3+ Treg cells in the lymphoidorgans (SLO). n = 6 (no tx); n = 8 (MmIL-2; Neo-2/15). c, Neo-2/15
does not have detectable immunogenicity. Mice were immunized with
Neo-2/15 (red, n = 10) or mouse IL-2 (grey, n = 10) (14 days of daily
intraperitoneal injections, 10 μg day−1). IgG was detected by enzymelinked immunosorbent assay (ELISA). Anti-Neo-2/15 polyclonal antibody
was used as a positive control (black, n = 2) and did not cross-react withmouse or human IL-2. White, negative control (n = 2). OVA, ovalbumin.
d, Neo-2/15 is more effective than mouse IL-2 in a colorectal cancer model(CT26). Starting on day 6, mice were administered daily with mouse IL-2
or Neo-2/15 (10 μg) intraperitoneally, or were left untreated. Top, tumour

growth curves show only surviving mice and stop if a group fell below 50%of the initial n. Bottom, survival curves. Mice were euthanized if weightloss exceeded 10% or tumour size reached 1,000 mm3. e, In a melanoma
model (B16F10), combination of mAb TA99 with Neo-2/15 is more
effective than with mouse IL-2. Starting on day 1, mice were administereddaily with Neo-2/15 (10 μg) or equimolar mouse IL-2 intraperitoneally.
Twice-weekly treatment with TA99 was added on day 3. Left, tumour
growth curves. Top right, survival curve; inset shows average weightchange. Bottom right, quantification of the cause of death. f, Neo-2/15
elicits a higher CD8+:Treg ratio than mouse IL-2. Data are mean ± s.d.,
except in growth curves, where data are mean ± s.e.m. Results are analysedby one-way ANOVA (95% confidence interval), except survival curves,which are assessed using the Mantel–Cox test (95% confidence interval).
All experiments were performed twice with similar results, except b, whichshows data pooled from 3 independent experiments.

any biologically active molecule with known or accurately predictable structure. Because of the incremental nature of current traditional
engineering approaches (for example, 1–3 amino acid substitutions,chemical modification at a single site), most of the shortcomings of theparent molecule are inevitably passed on to the resulting engineered
variants, often in an exacerbated form. By building mimics completelyfrom scratch, these shortcomings can be avoided. Unlike recombinant
IL-2 and its engineered variants, Neo-2/15 is highly expressed in E.
coli (Supplementary Fig. 13), retains activity at high temperature,
does not interact with IL-2Rα, and is robust to substantial sequencechanges (Extended Data Fig. 8), which may enable the engineering of
new functions. Immunogenicity against the de novo designed proteins appears to be low, probably because of their small size and highstability35, and in contrast to incremental variants of human IL-2, any
antibody response mounted against mimetics is unlikely to cross-reactwith its natural (cytokine) counterpart. Because of their high stability and robustness, along with their tailored interaction surfaces, denovo designed protein mimetics are likely to be particularly powerfulfor developing next-generation therapeutics that combine different
protein functionalities.

Online content

Any methods, additional references, Nature Research reporting summaries, source
data, statements of data availability and associated accession codes are available athttps://doi.org/10.1038/s41586-018-0830-7.

Received: 1 June 2018; Accepted: 15 November 2018;
Published online 9 January 2019.
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Acknowledgements We thank B. Nordstrom, J. Nordstrom, P. Barrier and
J. Barrier for the IPD Fund (Budget Number: 68-0341); CONACyT SNI(Mexico), CONACyT postdoctoral fellowship (Mexico) and IPD translational
research program to D.-A.S.; NIH MSTP grant T32 GM007266 to S.Y.; JDRF
(2-SRA-2016-236-Q-R) to U.Y.U.; la Caixa Fellowship (la Caixa Banking
Foundation, Barcelona, Spain) to A.Q.-R.; FCT Portugal Ph.D. studentship to
C.L.-A.; European Research Council (ERC StG, grant agreement 676832),
FCT Investigator (IF/00624/2015), and the Royal Society (UF110046 andURF\R\180019) to G.J.L.B.; Marie Curie International Outgoing Fellowship(FP7-PEOPLE-2011-IOF 298976) to E.M.; Natural Sciences and Engineering
Research Council of Canada Postdoctoral Fellowship to C.D.W.; Washington
Research Foundation to B.D.W.; NIH grant R35GM122543 to F.P.-A.; Mentored
Clinical Scientist Development Award 1K08DK114563-01, and the American
Gastroenterological Association Research Scholars Award to M.D.; NIHRO1-AI51321, NIH-RO1-AI51321, Mathers Foundation, Younger Endowed
Chair, and Howard Hughes Medical Institute to K.C.G.; and Howard Hughes
Medical Institute and Michelson Medical Research Foundation to D.B. See
Supplementary Information for extended acknowledgements.
Reviewer information Nature thanks Y. Jones, W. Schief and the otheranonymous reviewer(s) for their contribution to the peer review of this work.
Author contributions D.-A.S., S.Y., U.Y.U., J.B.S., M.P., G.J.L.B., M.D., K.C.G. and
D.B. designed the research; D.-A.S. developed the method for designing denovo protein mimics, and designed and invented the IL-2/IL-15 mimics; S.Y.,
D.-A.S. and U.Y.U. characterized and optimized the IL-2/IL-15 mimics; J.B.S.
performed BLI binding characterization, in vitro cell signalling, and recombinant
IL-receptor production; K.M.J. performed crystallography experiments; A.Q.-R.
characterized binding and stability, directed evolution and protein expression;
L.R.A., T.B. and S.J.C. performed in vivo naive mouse T cell response, melanoma
cancer model and immunogenicity experiments; C.L.-A. performed in vivo
colorectal cancer experiments; M.R. performed ex vivo cell signalling andin vivo airway inflammation experiments; I.L. performed in vivo CAR-T cell
experiments; C.D.W. designed and characterized single-cysteine mutations;
E.M. and J.C. assisted in developing the computational design methods; B.D.W.
designed and characterized disulfide-stapled variants; F.P.-A. performed
and analysed molecular dynamics simulations; L.C. performed optimizationand production of recombinant protein; L.S. supervised and coordinatedcollaborations; S.R.R. supervised in vivo CAR-T cell experiments, M.P. supervised
research for ex vivo cell signalling and in vivo tissue residency; G.J.L.B.
supervised research for the in vivo colorectal cancer model; M.D. coordinatedresearch for in vivo naive mouse T cell response, melanoma cancer modeland immunogenicity; D.-A.S., S.Y., U.Y.U., J.B.S., M.D., K.C.G. and D.B. wrote themanuscript; D.-A.S., K.C.G. and D.B. supervised and coordinated the overall
research.
Competing interests D.-A.S., S.Y., U.Y.U., A.Q.-R., C.D.W. and D.B. are co-founders
and stockholders of Neoleukin Therapeutics, a company that aims to develop
the inventions described in this manuscript. D.-A.S., S.Y., U.Y.U., J.B.S., A.Q.-R.,
K.C.G. and D.B. are co-inventors on a US provisional patent application(no. 62/689769), which incorporates discoveries described in this manuscript.
Additional information
Extended data is available for this paper at https://doi.org/10.1038/s41586018-0830-7.
Supplementary information is available for this paper at https://doi.org/
10.1038/s41586-018-0830-7.
Reprints and permissions information is available at http://www.nature.com/
reprints.
Correspondence and requests for materials should be addressed to D.-A.S. or
K.C.G. or D.B.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional
claims in published maps and institutional affiliations.

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RESEARCH Article
Methods

No statistical methods were used to predetermine sample size. The experiments
were not randomized. The investigators were not blinded to allocation duringexperiments and outcome assessment.
Computational design of de novo cytokine mimetics. The design of de novo
cytokine mimetics began by defining the structure of human IL-2 in the quaternarycomplex with the human IL-2Rβγc receptor as the template for the design. Afterinspection, the residues composing the binding site were defined as hotspots. The
structure was fed into the new mimetic design protocol that is programmed in
PyRosetta, which can automatically detect the core secondary structure elementsthat compose the target template and produce the resulting de novo mimetic backbones. In brief, the mimetic building algorithm works as follows. For the first generation of designs, each of the core elements was idealized by reconstruction using
loops from a clustered database of highly ideal fragments (fragment size = 4 aminoacids, see Data availability). After idealization, the mimetic building protocol aims
to reconnect the idealized elements by pairs in all possible combinations. To do this,it uses combinatorial fragment assembly of sequence-agnostic fragments from thedatabase, followed by Cartesian-constrained backbone minimization for potential
solutions (that is, where the N and C ends of the built fragment are close enough tolink the two secondary structures). After minimization, the solutions are verified
to contain highly ideal fragments (that is, that every overlapping fragment thatcomposes the two connected elements is also contained within the database) andthat no backbone clashes with the target (context) receptor. Successful solutions
were then profiled using the same database of fragments in order to determine
the most probable amino acids at each position (this information was encoded
as metadata on each design). Next, solutions for pairs of connected secondary
structures were combinatorially recombined (by using graph theory-connectedcomponents) to produce fully connected backbones. Since the number of solutions
grows exponentially with each pair of elements, at each fragment combination step
we ranked the designs to favour those with shorter interconnections between pairsof secondary structure core elements (that is, effectively with shorter loops), andkept only the top solutions. Fully connected backbone solutions were profiled bylayer (interface, core, non-core surface, surface) in order to restrict the identitiesof the possible amino acids to be layer-compatible. Finally, all the information on
hotspots, compatible built-fragment amino acids and layers was combined (hotspot
has precedence to amino acid probability, and amino acid probability took precedence to layer) and output as Rosetta resfiles (specific for a given de novo mimic
backbone result). These backbones and their corresponding resfiles were thenpassed to RosettaScripts for flexible backbone design and filtering (Supplementary
Information, Appendix A). For the second generation of designs, we followed two
approaches. In the first approach, we simply executed Rosetta sequence redesignsof our best first generation optimized design (G1_neo2_40_1F, Supplementary
Information, Appendix B). In the second approach, we engineered new mimetics
using G1_neo2_40_1F as the target template. The mimetic design protocol in thissecond generation was similar to the one described for the first generation, but with
two key differences. First, the core elements (that is, those that are secondary structures) were no longer built from fragments, but instead by discovering parametricequations of repetitive phi and psi angles (omega fixed to 180°) that result in secondary structures that recapitulated each of the target helices as close as possible.
Second, a ‘pitch’ on the phi and psi angles was allowed every 3rd residue in order
to allow the helices the possibility to have curvature (final angle parameters: H1:
phi = −60.4, psi = −45.8, phi_pitch = −1.0, psi_pitch = 2.0; H2: phi = −64.5,
psi = −38.4, phi_pitch = 4.0, psi_pitch = −8.0; H3: phi = −64.6, psi = −40.6,
phi_pitch = 0.0, psi_pitch = 0.0; H4: phi = −64.3, psi = −41.7, phi_pitch = 0.0,
psi_pitch = 0.0). By using these parametric equations, the algorithm can variate thelength of each of the core-elements up to ±8 amino acids (compared to the input
template). Reductions in the size of the core elements were not allowed to remove
hotspots from the binding site. All length variations of the core elements werereconnected with loops from a clustered database of highly ideal loops (fragment
size of 7 amino acids). The rest of the design algorithm is in essence similar to theone followed in the generation one. However, the implementation of the second
generation algorithm is more convenient, because the input PDB incorporates
PDBInfoLabels metadata to define the hotspots, and subsequently, it uses thisinformation to integrate the loop amino acids-preferences, layers, and hotspots
into the final output(s) as PDBInfoLabels metadata. This information can be conveniently used for the subsequent step of sequence design with RosettaScripts(Supplementary Information, Appendix C). The Rosetta energy functions used
for sequence design were ‘talaris2013’ and ‘talaris2014’, for the first and second
generation of designs, respectively.
The databases of highly ideal fragments used for the design of the backbones for the de novo mimetics were constructed with the Rosetta application‘kcenters_clustering_of_fragments’ using an extensive database of non-redundant(publicly available) protein structures from the RCSB protein data bank, which
was comprised of 16,767 PDB structures for the 4-mer database used in the first

generation of designs, and of 7,062 PDB structures for the 7-mer database used
for the second generation designs (see Code availability). The computational algorithm for designing the second generation de novo mimics is available (see Codeavailability).
Yeast display. Yeast were transformed with genes encoding the proteins to be
displayed together with a linearized pETcon3 vector. The vector was linearized by
100-fold overdigestion by NdeI and XhoI (New England Biolabs) and then purified
by gel extraction (Qiagen). The genes included 50 bases of overlap with the vector
on both the 5′ and 3′ ends, such that homologous recombination would place thegenes in frame between the AGA2 gene and the Myc tag on the vector. Yeast wasgrown in C-Trp-Ura medium before induction in SGCAA medium as previously
described34,35,51. After induction for 12–18 h, cells were washed in chilled display buffer (50 mM NaPO4 pH 8, 20 mM NaCl, 0.5% BSA) and incubated withvarying concentrations of biotinylated receptor (either human or mouse IL-2Rα,IL-2Rβ or γc) while being agitated at 4 °C. After approximately 30 min, cells were
washed again in a chilled buffer and then incubated on ice for 5 min with a FITCconjugated Myc antibody (1 μl per 3 × 106 cells) and streptavidin–phycoerythrin(1 μl per 100 μl volume of yeast). Yeast was then washed and counted by flow
cytometry (Accuri C6) or sorted by fluorescence-activated cell sorting (FACS)(Sony SH800).
Mutagenesis and affinity maturation. Site-saturation mutagenesis (SSM) libraries
were constructed from synthetic DNA from Genscript. For each amino acid on
each design template, forward primers and reverse primers were designed such that
PCR amplification would result in a 5′ PCR product with a degenerate NNK codon
and a 3′ PCR product, respectively. Amplification of ‘left’ and ‘right’ products by
COF and COR primers yielded a series of template products each consisting ofa degenerate NNK codon at a different residue position. For each design, theseproducts were pooled to yield the SSM library. SSM libraries were transformedby electroporation into conditioned Saccharomyces cerevisiae strain EBY100 cells,
along with linearized pETcon3 vector, using the previously described protocol52.
For details of the primers used in the creation of SSM libraries, see Supplementary
Tables 5, 6.
Combinatorial libraries were constructed from synthetic DNA from Genscript
containing ambiguous nucleotides and similarly transformed into a linearizedpETcon3 vector. For details of the primers used in the creation of combinatorial
libraries see Supplementary Tables 7, 8.
Protein expression. Genes encoding the designed protein sequences weresynthesized and cloned into pET-28b(+) E. coli plasmid expression vectors (GenScript, N-terminal 6× His-tagged followed by a thrombin cleavage
site. For all the designed proteins, the sequence of the N-terminal tag used is
MGSSHHHHHHSSGLVPRGSHM (unless otherwise noted), which is followed
immediately by the sequence of the designed protein. Plasmids were then transformed into chemically competent E. coli Lemo21 cells (NEB). Protein expression was performed using terrific broth and M salts, cultures were grown at 37 °C
until A600 nm reached approximately 0.8, then expression was induced with 1 mM
isopropyl β-d-thiogalactopyranoside (IPTG), and the temperature was lowered
to 18 °C. After expression for approximately 18 h, cells were harvested and lysedwith a Microfluidics M110P microfluidizer at 18,000 psi, then the soluble fraction
was clarified by centrifugation at 24,000g for 20 min. The soluble fraction was
purified by Immobilized Metal Affinity Chromatography (Qiagen) followed by
FPLC size-exclusion chromatography (Superdex 75 10/300 GL, GE Healthcare).
The purified Neo-2/15 was characterized by mass spectrum verification of themolecular weight of the species in solution (Thermo Scientific), size-exclusion
chromatography–multi-angle laser light scattering (SEC-MALLS) in order to verify
monomeric state and molecular weight (Agilent, Wyatt), SDS–PAGE, and endotoxin levels (Charles River).
Human and mouse IL-2 complex components including human IL-2 (residues 1–133), human IL-2Rα (residues 1–217), human IL-2Rβ (residues 1–214),
human IL-2Rγc (residues 1–232), mouse IL-2 (residues 1–149), mouse IL-2Rα
ectodomain (residues 1–213), mouse IL-2Rβ ectodomain (residues 1–215), andmouse γc ectodomain (residues 1–233) were secreted and purified using a baculovirus expression system, as previously described39,43. For the zippered human
IL-2Rβγc heterodimer, the aforementioned extracellular domain residues for thehuman/mouse IL-2Rβ and human/mouse IL-2Rγc were separately cloned into
baculovirus expression constructs containing 3C protease-cleavable basic andacidic leucine zippers, respectively, for a high-fidelity pairing of the receptor subunits, as described previously53. The IL-2Rβ and IL-2Rγc constructs were transfected independently and their corresponding viruses were co-titrated to determine
optimal infection ratios for equivalent expression of the two chains. Insect cellsecretion and purification proceeded as described for IL-2 cytokine and receptor
subunits. All proteins were purified to >98% homogeneity with a Superdex 200
sizing column (GE Healthcare) equilibrated in Hepes-buffered saline (HBS). Purity
was verified by SDS–PAGE analysis. For expression of biotinylated human IL-2 andmouse IL-2 receptor subunits, proteins containing a C-terminal biotin acceptor© 2019 Springer Nature Limited. All rights reserved.

Article RESEARCH
peptide (BAP, LNDIFEAQKIEWHE) were expressed and purified as described by
Ni-NTA affinity chromatography and then biotinylated with the soluble BirA ligase
enzyme in 0.5 mM bicine pH 8.3, 100 mM ATP, 100 mM magnesium acetate, and
500 mM biotin (Sigma). Excess biotin was removed by size-exclusion chromatography on a Superdex 200 column equilibrated in HBS.
Neo-2/15 crystal and co-crystal structures. C-terminally 6× His-tagged endoglycosidase H (endoH) and mouse IL-2Rβ and IL-2Rγc were expressed separately inHigh Five cells using a baculovirus system as previously described52. Cells expressing IL-2Rγc were grown in the presence of 5 µM kifunensine. After approximately
72 h, the secreted proteins were purified from the medium by passing over a
Ni-NTA agarose column and eluted with 200 mM imidazole in HBS buffer(150 mM NaCl, 10 mM HEPES pH 7.3). EndoH was exchanged into HBS buffer
by diafiltration. Mouse IL-2Rγc was deglycosylated by overnight incubation with
1:75 (w/w) endoH. Mouse IL-2Rβ and IL-2Rγc were further purified and buffer
was exchanged by FPLC using an S200 column (GE Life Sciences).
Monomeric Neo-2/15 was concentrated to 12 mg/ml and crystallized by vapour
diffusion from 2.4 M sodium malonate pH 7.0, and crystals were harvested andflash frozen without further cryoprotection. Crystals diffracted to 2.0 Å resolution
at Stanford Synchrotron Radiation Laboratory beamline 12-2 and were indexed
and integrated using XDS54. The space group was assigned with Pointless55, andscaling was performed with Aimless56 from the CCP4 suite57. Our predicted model
was used as a search ensemble to solve the structure by molecular replacement inPhaser58, with six protomers located in the asymmetric unit. After initial rebuilding with Autobuild59, iterative cycles of manual rebuilding and refinement were
performed using Coot60 and PHENIX61.
To crystallize the ternary Neo-2/15–mouse IL-2Rβ–mouse IL-2Rγc complex,
the three proteins were combined in equimolar ratios, digested overnight with
1:100 (w/w) carboxypeptidases A and B to remove purification tags, and purified
by FPLC using an S200 column; fractions containing all three proteins were pooled
and concentrated to 20 mg/ml. Initial needle-like microcrystals were formed byvapour diffusion from 0.1 M imidazole pH 8.0, 1 M sodium citrate and used toprepare a microseed stock for subsequent use in microseed matrix screening62(MMS). After a single iteration of MMS, crystals grown in the same precipitantwere cryoprotected with 30% ethylene glycol, harvested and diffracted anisotropically to 3.4 Å × 3.8 Å × 4.1 Å resolution at Advanced Photon Source beamline
23ID-B. The structure was solved by molecular replacement in Phaser using thehuman IL-2Rβ and IL-2Rγc structures (PDB ID: 2B5I) as search ensembles. This
produced an electron density map into which two poly-alanine α-helices could
be manually built. Following rigid-body refinement in PHENIX, electron density
for the two unmodelled alpha helices, along with the BC loop and some aromaticside chains, became visible, allowing docking of the monomeric Neo-2/15. Twofurther iterations of MMS and use of an additive screen (Hampton Research) produced crystals grown by vapour diffusion using 150 nl protein, 125 nl well solution
containing 0.1 M Tris pH 7.5, 5% dextran sulfate, 2.1 M ammonium sulfate and25 nl microseed stock containing 1.3 M ammonium sulfate, 50 mM Tris pH 7.5,
50 mM imidazole pH 8.0, 300 mM sodium citrate. Crystals cryoprotected with
3 M sodium malonate were flash frozen and diffracted anisotropically to 2.5 Å ×
3.7 Å × 3.8 Å at Advanced Light Source beamline 5.0.1. After processing the datawith XDS, an elliptical resolution limit was applied using the STARANISO server(http://staraniso.globalphasing.org/) as described63. Rapid convergence of themodel was obtained by refinement against these reflections using TLS and target
restraints to the higher resolution human receptor (PDB ID: 2B5I) and Neo-2/15
structures in Buster v.2.10.364, with manual rebuilding in Coot, followed by a final
round of refinement in PHENIX with no target restraints. Structure figures wereprepared with PyMOL65. Software used in this project was installed and configured
by SBGrid66.
Cell lines. Unmodified YT-167 and IL-2Rα+ YT-1 human NK cells68 were cultured
in RPMI complete medium (RPMI 1640 medium supplemented with 10% fetal
bovine serum (FBS), 2 mM l-glutamine, minimum non-essential amino acids,
sodium pyruvate, 25 mM HEPES, and penicillin–streptomycin (Gibco)). CTLL-2
cells purchased from ATCC were cultured in RPMI complete with 10% T-STIM
culture supplement with ConA (Corning). Twenty-four hours before signallingstudies, CTLL-2 cells were resuspended in RPMI lacking T-STIM culture supplement for IL-2 starvation. Viability (>95%) of CTLL-2 cells was verified by trypan
blue exclusion (counted in a haemocytometer) immediately before performing thesignalling assays. All cells were maintained at 37 °C in a humidified atmospherewith 5% CO2. The subpopulation of YT-1 cells expressing IL-2Rα was purified
via magnetic selection as described previously39. Enrichment and persistence of
IL-2Rα expression were monitored by analysis of phycoerythrin (PE)-conjugatedanti-human IL-2Rα (BioLegend) antibody binding on an Accuri C6 flow cytometer(BD Biosciences). Cell lines were regularly tested via PCR to ensure the absence of
mycoplasma contamination.
Circular dichroism. Far-ultraviolet circular dichroism measurements were carriedout with an AVIV spectrometer model 420 in PBS (pH 7.4) in a 1 mm path length

cuvette with a protein concentration of ~0.20 mg/ml (unless otherwise mentioned
in the text). Temperature melts were performed from 25 to 95 °C and monitored
absorption signal at 222 nm (steps of 2 °C/min, 30 s of equilibration by step).
Wavelength scans (195–260 nm) were collected at 25 °C and 95 °C, and again at
25 °C after fast refolding (~5 min).
Binding studies. Surface plasmon resonance (SPR): For IL-2 receptor affinity titration studies, biotinylated human or mouse IL-2Rα, IL-2Rβ, and IL-2Rγc receptors were immobilized to streptavidin-coated chips for analysis on a Biacore T100
instrument (GE Healthcare). An irrelevant biotinylated protein was immobilized
in the reference channel to subtract non-specific binding. Less than 100 responseunits (RU) of each ligand was immobilized to minimize mass transfer effects.
Threefold serial dilutions of human IL-2, mouse IL-2, super-2, or engineered IL-2
mimetics, were flowed over the immobilized ligands for 60 s and dissociation
was measured for 240 s. For IL-2Rβγc binding studies, saturating concentrationsof human IL-2Rβ (3 μM) or mouse IL-2Rβ (5 μM) were added to the indicatedconcentrations of human IL-2 or mouse IL-2, respectively. Surface regeneration
for all interactions was conducted using 15 s exposure to 1 M MgCl2 in 10 mM
sodium acetate pH 5.5. SPR experiments were carried out in HBS-P+ buffer (GEHealthcare) supplemented with 0.2% bovine serum albumin (BSA) at 25 °C, andall binding studies were performed at a flow rate of 50 l min−1 to prevent analyterebinding. Data was visualized and processed using the Biacore T100 evaluation
software version 2.0 (GE Healthcare). Equilibrium titration curve fitting and equilibrium binding dissociation (Kd) value determination was implemented using
GraphPad Prism assuming all binding interactions to be first order. SPR experiments were reproduced three times with similar results. Biolayer interferometry:binding data were collected in a Octet RED96 (ForteBio) and processed using the
instrument’s integrated software using a 1:1 binding model. Biotinylated target
receptors, either human or mouse IL-2Rα, IL-2Rβ or γc were functionalized to
streptavidin-coated biosensors (SA ForteBio) at 1 µg/ml in binding buffer (10 mM
HEPES (pH 7.4), 150 mM NaCl, 3 mM EDTA, 0.05% surfactant P20, 0.5% non-fat
dry milk) for 300 s. Analyte proteins were diluted from concentrated stocks into
binding buffer. After baseline measurement in binding buffer alone, the binding
kinetics were monitored by dipping the biosensors in wells containing the target
protein at the indicated concentration (association step) and then dipping the sensors back into baseline/buffer (dissociation). For heterodimeric receptor binding
experiments for IL-2Rβγc, γc was bound to the sensor while IL-2Rβ was in solution
at saturating concentrations (that is, at least ~2.5 fold molar excess over the Kd).
STAT5 phosphorylation studies. In vitro studies: Approximately 2 × 105 YT-1,
IL-2Rα+ YT-1, or starved CTLL-2 cells were plated in each well of a 96-well plateand re-suspended in RPMI complete medium containing serial dilutions of human
IL-2, mouse IL-2, super-2, or engineered IL-2 mimetics. Cells were stimulated for
15 min at 37 °C and immediately fixed by addition of formaldehyde to 1.5% and
10 min incubation at room temperature. Permeabilization of cells was achievedby resuspension in ice-cold 100% methanol for 30 min at 4 °C. Fixed and permeabilized cells were washed twice with FACS buffer (PBS pH 7.2 containing
0.1% bovine serum albumin) and incubated with Alexa Fluor 647-conjugatedanti-STAT5 pY694 (BD Biosciences) diluted 1:50 in FACS buffer for 2 h at room
temperature. Cells were then washed twice in FACS buffer and mean fluorescence intensity (MFI) was determined on a CytoFLEX flow cytometer (BeckmanCoulter). Dose–response curves were fitted to a logistic model and half-maximal
effective concentration (EC50 values) and corresponding 95% confidence intervals
were calculated using GraphPad Prism data analysis software after subtraction of
the MFI of unstimulated cells and normalization to the maximum signal intensity.
Experiments were conducted in triplicate and performed three times with similar
results. Ex vivo studies: spleens and lymph nodes were collected from wild-type
C57BL/6J or B6;129S4-Il2ratm1Dw (CD25KO) mice purchased from The JacksonLaboratory and made into a single cell suspension in sort buffer (2% fetal calf
serum in PBS pH 7.2). CD4+ T cells were enriched through negative selection
by staining the cell suspension with biotin-conjugated anti-B220, CD8, NK1.1,
CD11b, CD11c, Ter119, and CD19 antibodies at 1:100 for 30 min on ice. Following
a wash with sort buffer, anti-biotin MicroBeads (Miltenyi Biotec) were added tothe cell suspension at 20 μl per 107 total cells and incubated on ice for 20 min. Cells
were washed and then resuspended. Negative selection was then performed using
EasySep Magnets (STEMCELL Technologies). Approximately 1 × 105 enrichedcells were added to each well of a 96-well plate in RPMI complete medium with
5% FCS with tenfold serial dilutions of mouse IL-2, super-2, or Neo-2/15. Cells
were stimulated for 20 min at 37 °C in 5% CO2, fixed with 4% PFA and incubatedfor 30 min at 4 °C. Following fixation, cells were harvested and washed twice withsort buffer and again fixed in 500 μl 90% ice-cold methanol in dH2O for 30 min
on ice for permeabilization. Cells were washed twice with Perm/Wash Buffer (BDBiosciences) and stained with anti-CD4-PerCP in Perm/Wash buffer (1:300),
anti-CD44–Alexa Fluor 700 (1:200), anti-CD25–PE-Cy7 (1:200), and 5 μl per
sample of anti-pSTAT5-PE pY694 for 45 min at room temperature in the dark.
Cells were washed with Perm/Wash and re-suspended in sort buffer for analysis© 2019 Springer Nature Limited. All rights reserved.

RESEARCH Article
on a BD LSR II flow cytometer (BD Biosciences). Dose–response curves were fitted
to a logistic model and EC50 values and corresponding 95% confidence intervals
were determined using GraphPad Prism data analysis software after subtractionof the MFI of untreated cells and normalization to the maximum signal intensity.
Experiments were performed in triplicate and repeated three times with similar
results.
In vivo mouse airway inflammation experiments. Mice (C57BL/6J, The JacksonLaboratory) were inoculated intranasally with 20 μl whole house dust mite antigen(Greer) resuspended in PBS to a total of 23 μg Derp1 per mouse. From days 1–7,
mice were given a daily intraperitoneal injection of 20 μg mouse IL-2 in sterile PBS(pH 7.2), a molar equivalent of Neo-2/15 in sterile PBS, or no injection. On Day 8,
circulating T cells were intravascularly labelled and tetramer-positive cells wereenriched from lymph nodes and spleen or lung as previously described69. Both thecolumn flow-through and bound fractions were saved for flow cytometry analysis. Cells were surface stained with antibodies and analysed on a BD LSR II flow
cytometer with BD FACSDiva software (BD Biosciences). Antibodies used: FITC
anti-Ki67, clone SolA15, PerCP-Cy5.5 anti-CD25, clone PC61, eFluor 450 antiFoxp3, clone FJK-16S, BV510 anti-CD8, clone 53-6.7, BV605 anti-PD-1, clone J43,
BV711 anti-CD4, clone RM4-5, BV786 anti-CD62L, clone MEL-14, PE anti-CD69,
clone H1.2F3, PE-CF594 anti-B220, clone RA3-6B2, PE-Cy7 anti-CXCR5, clone
2G8 and BUV395 anti-Thy1.2, clone 53-2.1. All flow cytometry files were analysed using FlowJo 9.9.4 and statistical analysis was performed using Prism 7. All
experiments were performed in accordance with the University of Washington
Institutional Care and Use Committee guidelines.
Colorectal carcinoma in vivo mice experiments. CT26 cells were sourced fromthe research group of J. Demengeot at Instituto Gulbenkian de Ciência, Portugal.
On day 0, 5 × 105 cells were injected subcutaneously into the flanks of BALB/c micepurchased from Charles River with 50 μl of a 1:1 mixture of Dulbecco’s modified
Eagle medium (Gibco) with Matrigel (Corning). Starting on day 6, when tumour
volume reached around 100 mm3, Neo-2/15 and mouse IL-2 (Peprotech) wereadministered daily by intraperitoneal (i.p.) injection in 50 μl PBS (Gibco). Mice
were euthanized when tumour volume reached 1,300 mm3. BALB/c mice werepurchased from Charles River. Flow cytometry: all reagents were purchased from
Gibco by Life Technologies (Thermo Fisher Scientific) unless stated otherwise.
Excised tumours were minced and digested using a mix of collagenase I, collagenase IV (Worthington) and DNase I (Roche) in a shaker for 20 min, 250 r.p.m.
at 37 °C. After digestion, samples were passed through a 100-μm cell strainer, andresuspended in cold complete RPMI 1640 medium, supplemented with 10 mM
HEPES buffer, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol, 100 U/ml
penicillin and 100 µg/ml streptomycin and complemented with 1% non-essential
amino acids (NEAA), 1% GlutaMAX supplement and 10% heat-inactivated FBS(HI FBS). The cell suspensions from the spleens and the inguinal lymph nodes
were obtained through the smashing of the tissues against the filter of a 100-μm
cell strainer. Cells were resuspended in PBS with 2% FBS and 1 mM EDTA andstained for extracellular markers for 45 min at 4 °C. Cell suspensions were thenfixed, permeabilized and stained for intracellular markers using the eBioscienceFoxp3 Transcription Factor Staining Buffer Set from ThermoFisher Scientific.
Samples were analysed in a BD LSRFortessa flow cytometer equipped with a BD
FACSDiva software and data were analysed in FlowJo v.10 software and the statistical analysis was performed using Prism 5. Antibodies (BioLegend) used incolon carcinoma experiments were: CD45-BV510 (30-F11), CD3-BV711 (17A2),
CD49b-FITC (DX5), CD4-BV605 (RM4-5), CD8-PECy7 (53-6.7), and Foxp3-APC(FJK-16 s; eBioscience). Fixable Viability Dye eFluor 780 (eBioscience) was used to
exclude dead cells. Animals were maintained according to protocols approved by
the Direção Geral de Veterinária and iMM Lisboa ethical committee.
Melanoma in vivo experiments. B16F10 cells were purchased from ATCC.
On day 0, 5 × 105 cells were inoculated into the mice (C57BL/6J purchasedfrom Jackson) by subcutaneous injection in 500 μl Hank’s balanced salt solution (Gibco). Starting on the specified day, Neo-2/15 or mouse IL-2 (Peprotech)treatments were administered daily by intraperitoneal injection in 200 μl LPS-free
PBS (Teknova). Treatment with TA99 (a gift from N. Momin and D. Wittrup,
Massachusetts Institute of Technology) at 150 μg per mouse was added lateron the day indicated in the text. Mice were euthanized when tumour volume
reached 2,000 mm3. Flow cytometry: excised tumours were minced, enzymaticallydigested (Miltenyi Biotec), and passed through a 40-μm filter. Cells from spleens
and tumour-draining lymph nodes were dispersed into PBS through a 40-μm
cell strainer using the back of a 1-mL syringe plunger. All cell suspensions were
washed once with PBS, and the cell pellet was resuspended in 2% inactivated fetal
calf serum containing fluorophore-conjugated antibodies. Cells were incubatedfor 15 min at 4 °C then fixed, permeabilized, and stained using a BioLegend FoxP3
staining kit. Samples were analysed on a BD Fortessa flow cytometer. Antibodies(BioLegend) used in melanoma experiments were: CD45-BV711 (clone
30-F11), CD8-BV650 (53-6.7), CD4-BV421 (GK1.5), TCRβ-BV510 (H57-597),
CD25-AF488 (PC61), FoxP3-PE (MF-14). Animals were maintained according toprotocols approved by Dana–Farber Cancer Institute (DFCI) Institutional Animal
Care and Use Committee.
Generation of anti-Neo-2/15 polyclonal antibody. Mice (C57BL/6J purchasedfrom Jackson) were injected intraperitoneally with 500 μg of KO Neo-2/15 in 200 μlof a 1:1 emulsion of PBS and complete Freund’s adjuvant. Mice were boosted ondays 7 and 15 with 500 μg of KO Neo-2/15 in 200 μl of a 1:1 emulsion of PBS andincomplete Freund’s adjuvant. On day 20, serum was collected and recognitionof Neo-2/15 was confirmed by ELISA. For the ELISA, plates were coated with
Neo-2/15, KO Neo-2/15, or mouse IL-2 mixed with ovalbumin for a total of 100 ng/
well in carbonate buffer. Coated plates were incubated with mouse serum diluted
1:1,000 in PBS. Binding was detected using anti-mouse IgG conjugated to horseradish peroxidase (HRP) and developed with 3,3′,5,5-tetramethylbenzidine (TMB).
Results were quantified using absorption at 450 nm.
Enzyme-linked immunosorbent assay. High-binding 96-well plates (Corning)were coated overnight at 4 °C with 100 ng/ml of Neo-2/15, mouse IL-2 (Peprotech),
HsIL-2 (Peprotech) or ovalbumin (Sigma-Aldrich) in carbonate buffer. Antibody
binding to target proteins was detected using HRP-conjugated sheep anti-mouse
IgG (GE Healthcare) at 75 ng/ml. Plates were developed with tetramethylbenzidineand HCl. Absorbance was measured at 450 nm with an EnVision Multimode Plate
Reader (PerkinElmer).
T cell proliferation assay. Cells were isolated from mice (C57BL/6J purchased from Jackson) spleens using the EasySep T Cell Isolation Kit (StemCell
Technologies). Cells were plated in RPMI in 96-well culture plates at a density of
10,000 cells/well. Medium was supplemented with regular or heat-treated Neo2/15, mouse IL-2, or super-2 (as indicated). After 5 days of incubation at 37 °C,
cell survival and proliferation were measured by CellTiter-Glo Luminescent Cell
Viability Assay (Promega).
In vivo experiments. For Treg expansion experiments (Fig. 4a), naive C57BL/6
mice were treated daily with Neo-2/15 or mouse IL-2 at the indicated concentrations (n = 2–3 per group). After 14 days, spleens were harvested and analysed byflow cytometry using the indicated markers. For immunogenicity experiments(Fig. 4c), C57BL/6 mice were inoculated with 5 × 105 B16F10 cells by subcutaneous injection. Starting on day 1, mice were treated daily with Neo-2/15 (10 μg)
or equimolar mouse IL-2 by intraperitoneal injection (n = 10 for each group).
After 14 days, serum (antiserum) was collected and IgG was detected by ELISA
in plates coated with 10% fetal bovine serum (negative control), Neo-2/15, mouse
IL-2, human IL-2, or ovalbumin as a negative control. Polyclonal mouse IgG against
Neo-2/15 (anti-Neo-2/15 polyclonal antibody) was generated using complete
Freund’s adjuvant in conjunction with KO Neo-2/15, an inactive double point
mutant of Neo-2/15: Y14D, F99D. For colorectal cancer experiments (Fig. 4d),
BALB/C mice were inoculated with CT26 tumours. Starting on day 6, mice weretreated daily by intraperitoneal injection of mouse IL-2 or Neo-2/15 (10 μg), or left
untreated (n = 5 per group); For melanoma experiments (Fig. 4e), C57BL/6 mice
were inoculated with B16 tumours. Starting on day 1, mice were treated daily byintraperitoneal injection of Neo-2/15 (10 μg) or equimolar mouse IL-2 (n = 10 pergroup). Twice-weekly treatment with TA99 was added on day 3. Mice were euthanized when weight loss exceeded 10% of initial weight or when tumour size reached2,000 mm3; For CD8+: Treg ratio in melanoma experiments (Fig. 4f), C57BL/6 mice
were inoculated with B16 tumours and treated by daily intraperitoneal injection as
indicated. Treatment with TA99 was started on day 5 and continued twice-weekly.
Tumours were collected from mice when they reached 2,000 mm3, and were analysed by flow cytometry. The CD8+:Treg cell ratio was calculated by dividing thepercentage of CD45+TCRβ+ cells that were CD8+ by the percentage that were
CD4+CD25+FoxP3+.
CAR-T cell in vivo experiments. In vitro T cell proliferation assay. Primary
human T cells were obtained from healthy donors, who provided written informed
consent for research protocols approved by the Institutional Review Board of theFHCRC. Peripheral blood mononuclear cells (PBMC) were isolated by centrifugation over Ficoll-Hypaque (Sigma). T cells were isolated using EasySep CD8 or
CD4 negative isolation kits (Stemcell Technologies). To stimulate T cells, T cells
were thawed and incubated with anti-CD3/CD28 Dynabeads (Gibco) at 1:1 ratio
in medium supplemented with 50 IU/ml (3.1 ng/ml) IL-2. Beads were removed
after four days of incubation. Stimulated or freshly thawed unstimulated T cells
were plated at 30,000 or 50,000 cells/well, respectively, in 96-well format andcultured in indicated concentrations of IL-2 or Neo-2/15 in triplicate. Three days
later, proliferation was measured using CellTiter-Glo 2.0. (Promega). In vivo RAJI
experiment: the FHCRC Institutional Animal Care and Use Committee approved
all mouse experiments. Six-to-eight-week-old NSG mice were obtained from theJackson Laboratory. RAJI tumour cells (0.5 × 106) transduced with (ffLuc)-eGFP
were injected into the tail vein of NSG mice. Seven days after tumour injection,
lentiviral transduced anti-CD19 CAR T cells (0.4 × 106 CD4+, 0.4 × 106 CD8+)
prepared as described70 were infused intravenously into mice. Human IL-2 or
Neo-2/15 at 20 µg/mouse was injected intraperitoneally from day 8 to 16 after
tumour injection.

© 2019 Springer Nature Limited. All rights reserved.

Article RESEARCH
Disulfide-stapling Neo-2/15 to increase its stability and binding potency. Neo-2/15
is highly modular; this makes it possible to further tune its properties. As proof
of concept, we designed stability-enhancing disulfide staples that preserve thefunction of the protein71. Two computational design strategies were tested. First,
we designed internal disulfide bridges for all pairs of positions with favourable
geometrical arrangements inside of Neo-2/15. The four best disulfide-stapled
designs (that is, with the most favourable energy and minimal geometric distortion) were recombinantly expressed in E. coli. A design that bridges residues
38–75 (stabilizing helices H3→H2′) was confirmed to be monomeric by sizeexclusion chromatography followed by multi-angle light scattering (SEC–MALS).
In the second approach, we remodelled the N- and C- termini of Neo-2/15 toallow the introduction of a single-disulfide staple encompassing the entire
protein. We generated a total of 330 models that were then filtered based on fragment quality and disulfide-bond geometry. Finally, the designs were manuallyinspected and six were selected (representing a diversity of insertion lengths)
and experimentally characterized as described above. One design, replacing theterminal residues P and S with the amino acid sequences CNSN and NFQC at
the N and C termini, respectively (Extended Data Fig. 3) was confirmed to be
monomeric by SEC–MALS. The designs from both disulfide-stapling strategies
successfully increased the stability of Neo-2/15 (Tm > 95 °C) and its binding
potency (Extended Data Fig. 3).
Pharmacodynamics and pharmacokinetics of Neo-2/15 in mice. We assessed thein vivo duration of pSTAT5 signalling response to Neo-2/15 in peripheral blood
lymphocytes of naive mice (CD8 and B cells, Extended Data Fig. 6). As expected,
Neo-2/15 has a substantial effect (similar to mouse IL-2) in CD8 cell signalling 1 h
after administration, but as expected from the small size of Neo-2/15, the signalling effect decreases greatly after 3 h (Extended Data Fig. 6) and is undetectable
after 8 h (data not shown). This suggests that future engineering of Neo-2/15 toextend half-life may be possible; there are a number of approaches such as Fc
fusions, site-specific PEGylation (for example, through engineered cysteines, suchas those demonstrated in Supplementary Fig. 14), fusions to targeting domains (forexample, mAbs, sdAbs or VHHs12,72, DARPins72, or de novo designed binding
proteins35,73,74) that can be used to extend its half-life and would likely translate
into improved pharmacokinetics.
Molecular dynamics simulations of apo Neo-2/15 and holo Neo-2/15. Molecular
dynamics simulations in explicit water solvent initiated from the computationalmodel of apo Neo-2/15 recapitulated the crystallographic structure of (monomeric)apo Neo-2/15 (average r.m.s.d. to crystal structure = 1.9 Å; Extended Data Fig. 7a).
For instance, molecular dynamics simulations initiated from the ternary complexof Neo-2/15 with the mouse IL-2Rβγc were more likely to sample the crystallographic structure observed for Neo-2/15 in the ternary complex with mouse
IL-2Rβγc, including the outward movement of helices H2′–H4 (Neo-2/15 averager.m.s.d. to crystal structure = 1.4 Å; Extended Data Fig. 7c). The conformation of
Neo-2/15 seems to be stabilized in the ternary complexes (either with the mouseor human receptors; Extended Data Fig. 7c, d). Molecular dynamics simulations
were performed using GROMACS 2018.175,76 with the Amber99SB-ILDN force
field77. Each system consisted of the protein in a solvated dodecahedron box (minimum initial distance from the protein to the boundary = 1 nm) filled with explicit
TIP3P waters78 and neutralized with Cl− or Na+ ions. The solvated systems wereenergy-minimized using the steepest descent minimization method, followed byequilibration for 200 ps under the NPT ensemble with position restraints (1,000 kJ
mol−1 nm−1, applied on all the heavy atoms of the protein). Pressure couplingto 1 atm was performed with the Berendsen barostat79, and the temperature wascoupled to 310 K using the velocity-rescaling thermostat. The equilibrated systems
were used as starting conformations for production runs. In the case of the monomers, we performed 5 independent production simulations of 100 ns each, andfor the complexes bound to any of the IL-2 receptors, we performed 5 independent
simulations of 90 ns each. The production simulations were conducted under the
NPT ensemble, with the Parrinello–Rahman barostat80 for pressure coupling to
1 atm. The cut-off for van der Waals and short-range electrostatic interactions was
set to 1 nm. Long-range electrostatic interactions were treated with the particlemesh Ewald (PME) summation method81, and the Verlet cut-off scheme wasused82. The LINCS algorithm was used to constrain all chemical bonds and allow
an integration time-step of 2 fs. The simulation trajectories were recorded every
20 ps and were analysed using GROMACS.
Statistical and power analyses. For statistical test a P value of less than 0.05 considered significant unless otherwise noted. For comparison of fitted curves in cellularphospho-STAT5 signalling assays, differences in EC50 values were considered statistically significant if their 95% confidence intervals did not overlap. In vivo airway
inflammation experiments; comparison of cell populations were performed using
a two-tailed t-test. In vivo mouse colon cancer experiments; comparisons of thesurvival of tumour-bearing mice were performed using the log-rank Mantel–Cox
test (95% confidence interval). Comparisons of weight loss in tumour-bearing mice
were performed using a two-tailed t-test. In vivo mouse melanoma experiments:

comparisons of the survival of tumour-bearing mice were performed using the
log-rank Mantel–Cox test (95% confidence interval). Comparisons of weight loss
in tumour-bearing mice were performed using a two-tailed t-test. The minimumgroup size was determined using G*Power for an expected large effect size (Cohen’sd = 1.75). For all the bar plots, the whiskers represent ±s.d. and individual data
points are shown (as dots) for experiments where the n < 5. Unless otherwise
noted, results were analysed by one-way ANOVA, if significant (95% confidence
interval), post hoc t-tests were performed comparing groups, and P values were
adjusted for multiple comparisons are reported.
Software. The design of de novo protein mimics was performed using the custom
Python code ‘protein mimic designer’ (https://www.python.org), IPython83, andusing the scientific high-performance modules: PyRosetta42, numpy and scipy84,85,
matplotlib86, sklearn87, cython88 and pandas89. Data analyses were performed withcustom code in Python and IPhyton. Protein sequence design was performed
with Rosetta40,41 and RosettaScripts40. Protein visualization was performed
using PyMOL56. Simple protein–protein sequence alignments were performed
using BLASTP, and structural based sequence comparisons were performed using
MICAN90. Sequence logos were generated with WebLogo91.
Reporting summary. Further information on research design is available in
the Nature Research Reporting Summary linked to this paper.
Code availability. The Python implementation of the de novo protein mimetic
design algorithm (generation 2) and the clustered fragments database used fordesigning the de novo protein mimetics are available as part of the Rosetta distribution (https://www.rosettacommons.org) under the submodule ‘pyrosetta_
scripts/apps/deNovoProteinDesign/deNovoProteinMimeticsGen2/’. Other code isavailable upon request.

Data availability

Structures for Neo-2/15 monomer and its ternary complex with mouse IL-2Rβγchave been deposited in the Protein Data Bank with accession numbers 6DG6 and
6DG5, respectively. Diffraction images have been deposited in the SBGrid Data
Bank with accession numbers 587 and 588, respectively, and validation reports areincluded in the Supplementary Information. Other data and materials are available
upon request from the corresponding authors.
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Extended Data Fig. 1 | Therapeutic effect of Neo-2/15 on colon cancer.
a, BALB/C mice were inoculated with CT26 tumours. Starting on day 9
and ending on day 14, mice were treated daily with intraperitoneal
injection of mouse IL-2 or Neo-2/15 at the specified concentrations (n = 4
per group), or were left untreated (n = 6 per group). Top, tumour growth
curves show data only for surviving mice and stop if the number of micein a group fell below 50% of the initial number. Bottom, survival curves.
Mice were euthanized when weight loss exceeded 10% of initial weight orwhen tumour size reached 1,300 mm3. The experiments were performed
twice with similar results. b–d, Bar plots comparing the T cell populations
in BALB/C mice (n = 3 per group) that were inoculated with CT26

tumours and treated, starting from day 6, by daily intraperitoneal injectionof 10 μg Neo-2/15, 10 μg mouse IL-2 or no treatment (no tx). On day 14
the percentage of Treg cells (CD4+CD45+FoxP3+, top) and CD8:Treg cellratio (CD45+CD3+CD8+ cells:Treg cells; bottom) were assessed intumours (b), neighbouring inguinal lymph node (LN) (c), and spleen (d).
Data are mean ± s.d., except in growth curves, where data aremean ± s.e.m. Results were analysed by one-way ANOVA (95%
confidence interval), except for survival curves that were assessed using
the Mantel–Cox test (95% confidence interval). Experiments were
performed twice with similar results.

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RESEARCH Article

Extended Data Fig. 2 | Therapeutic effect of Neo-2/15 on melanoma.
Survival curves (top) and tumour growth curves (bottom) for C57BL/6
mice that were inoculated with B16 tumours (as in Fig. 4a) and treatedwith low (1 μg per mouse per day) or high (10 μg per mouse per day) dosesof Neo-2/15. a, Starting on day 1, mice (n = 5 per group) were treated daily
with intraperitoneal injection of single agent Neo-2/15 at 1 μg per mouseor equimolar mouse IL-2 (left), or the same treatments in combination
with a twice-weekly treatment with TA99 (started on day 5) (right). Mice
were euthanized when tumour size reached 2,000 mm3. Tumour growth
curves show data only for surviving mice and stop if the number of micein a group fell below 50% of the initial number. The experiments were
performed twice with similar results. b, Similar to a, but starting on day 4.
Mice (n = 5 per group) were treated daily with intraperitoneal injection of
single agent Neo-2/15 at 10 μg per mouse or equimolar mouse IL-2 (left),or the same treatments in combination with a twice-weekly treatment
with TA99 (started on day 4) (right). Mice were euthanized when tumour
size reached 1,000 mm3. The therapeutic effect of Neo-2/15 is dose-

dependent (higher doses have a stronger effect) and is potentiated in thepresence of the antibody TA99. Tumour growth curves show data only forsurviving mice and stop if the number of mice in a group fell below 50%of the initial number. The experiments were performed twice with similar
results. c, C57BL/6 mice were immunized with 500 μg KO Neo-2/15 incomplete Freund’s adjuvant and boosted on days 7 and 15 with 500 μg
KO Neo-2/15 in incomplete Freund’s adjuvant. Reactivity against KO
Neo-2/15 and native Neo-2/15, as well as cross-reactivity with mouse IL-2
were determined by incubation of serum (diluted 1:1,000 in PBS) withplate-bound KO Neo-2/15, Neo-2/15 or mouse IL-2 as indicated. Serumbinding was detected using an anti-mouse secondary antibody conjugatedto HRP followed by incubation with TMB. Data are reported as opticaldensity at 450 nm. Top, naive mouse serum; bottom, immunized mouseserum. The experiments were performed once. In all the growth curves,
data are mean ± s.e.m. Results were analysed by one-way ANOVA (95%
confidence interval), except for survival curves that were assessed using
the Mantel–Cox test (95% confidence interval).

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Extended Data Fig. 3 | Single disulfide-stapled variants of Neo-2/15
with higher thermal stability. a, b, Structural models of disulfidestabilized variants of Neo-2/15 (grey) are shown superposed on the ternary
crystal structure of Neo-2/15 (red) with mutated residues highlighted inmagenta and the disulfide bond shown in gold. Two strategies were used
to generate the disulfide stapled variants. a, Top, internal placement of thedisulfide linking residues 38 and 75. Bottom, experimental CD spectra of
the design at 25 °C, 95 °C and then cooled back to 25 °C, showing complete
recovery of ellipticity spectrum (full reversibility) upon cooling. b, Top,for the terminal disulfide variant, three residues were added to eachterminus in order to allow the disulfide to be formed without distorting
the Neo-2/15 structure. Bottom, experimental CD spectra of the design at
25 °C, 95 °C and then cooled back to 25 °C, showing complete recovery of
ellipticity spectrum (full reversibility) upon cooling. c, Thermal meltingof each disulfide variant in a and b between 25 °C and 95 °C (heating
rate ≈ 2 °C min−1) was monitored using circular dichroism at 222 nm.
Each of the disulfide-stapled variants shows improved stability relative
to native Neo-2/15. d, Binding strength of each disulfide variant wasmeasured by biolayer interferometry, showing that the introduction ofdisulfide bonds does not disrupt binding. Furthermore, both disulfide
variants exhibit improved binding of IL-2Rβγc (Kd ≈ 1.3 ± 0.49 nM and
1.8 ± 0.26 nM for the internal and external disulfide staples, respectively),compared to Neo-2/15 (Kd ≈ 6.9 ± 0.61 nM) under the same experimental
conditions. These results are consistent with the expected effect of
disulfide-induced stabilization on a de novo protein binding site71.
Thermal denaturation experiments were performed 3 times with similar
results; binding experiments were performed once.

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RESEARCH Article

Extended Data Fig. 4 | The stimulatory effect of Neo-2/15 on human
CAR-T cells. a, b, Human primary CD4 (top) or CD8 (bottom) T cells
stimulated with CD3/CD28 antibodies (a) or unstimulated (b) were
cultured in indicated concentrations of human IL-2 or Neo-2/15. T cellproliferation was measured as fold change over T cells cultured without

IL-2 supplement. Experiments were performed 3 times with similar
results. Data are mean ± s.d. c, NSG mice inoculated with 0.5 × 106
RAJI tumour cells were treated with 0.8 × 106 anti-CD19 CAR-T cells
7 days post-tumour inoculation. Tumour growth was analysed bybioluminescence imaging. The experiment was performed once.

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Article RESEARCH

Extended Data Fig. 5 | Immunogenicity of Neo-2/15 in healthy naive
mice. a, Naive C57BL/6 mice were treated daily with Neo-2/15 (n = 10),KO Neo-2/15 (n = 5), mouse IL-2 (n = 5) or left untreated (n = 5).
Blood was collected after 28 days and the serum was diluted 1:100 andanalysed for IgG against Neo-2/15, mouse IL-2, human IL-2, KO Neo2/15 and ovalbumin using ELISA. FBS (10%) was used as a negative
control. Polyclonal antibody against Neo-2/15 was used as a positive
control. All statistical comparisons between sera from treated mice andnegative control serum were not significant (two-way ANOVA with a 95%
confidence interval). All statistical comparisons between Neo-2/15 andmouse IL-2 treated mice serum were not significant (two-way ANOVA
with a 95% confidence interval). The experiments were performed once.
b, After 14 days, immune cell populations in the blood of treated mice
were quantified by flow cytometry. B cell:T cell ratio (top right) was
calculated by dividing the percentage of B220+ cells by the percentage of
CD3+ cells. CD8+ cell:CD4+ cell ratio (top left) was calculated by dividing
the percentage of CD3+CD8+ cells by the percentage of CD3+CD4+
cells. NK cells (bottom left) were identified by their expression of NK1.1.
Results were analysed by one-way ANOVA (95% confidence interval). The
experiments were performed once. In all cases, data are mean ± s.d.

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RESEARCH Article

Extended Data Fig. 6 | Kinetics of STAT5 phosphorylation with Neo2/15 treatment. Naive C57BL/6 mice were treated once with 13 µg mouseIL-2 (n = 5) or 10 µg Neo-2/15 (n = 5), or were left untreated (n = 5).
Phosphorylation of STAT5 was measured in peripheral blood at theindicated time points by flow cytometry using an anti-pSTAT5 antibody.

Mean fluorescence intensity (MFI) is shown at each time point for
TCRβ+CD8+ cells (top) and TCRβ−B220+ cells (bottom). Data aremean ± s.d. Results were analysed by one-way ANOVA (75% confidence
interval). The experiments were performed once.

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Article RESEARCH

Extended Data Fig. 7 | See next page for caption.

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RESEARCH Article
Extended Data Fig. 7 | Conformational flexibility of Neo-2/15 inmolecular dynamics simulations. a, Molecular dynamics simulations
started from the computational model of Neo-2/15 (top) converged into
structures similar to the crystal conformation. Apo Neo-2/15 is shown in
red thick tubes (chain A from PDB ID: 6GD6) and 45 (randomly selected)
molecular dynamics conformations from 5 independent simulations
are shown in thin grey tubes. Bottom, the plot shows the r.m.s.d. along
5 independent simulations (average r.m.s.d. = 1.93 Å). b, Similar toa, but for (control) molecular dynamics simulations started from thecrystallographic structure of human IL-2. Top, crystal conformationof human IL-2 (chain A from PDB ID: 2B5I) is shown in blue thicktubes and 45 (randomly selected) conformations from 5 independent
molecular dynamics simulations are shown in thin grey tubes (averager.m.s.d. = 2.02 Å). c, Top, similar to a and b, but showing molecular
dynamics structures for simulations started from the computationalmodel of Neo-2/15 bound to human IL-2Rβγc. The plot shows the r.m.s.d.
along 5 independent molecular dynamics simulations (average r.m.s.d.
to apo Neo-2/15 (model) = 1.28 Å). The lower structure shows thenearest conformation (to the apo Neo-2/15 computational model) thatwas sampled on each of the 5 independent simulations (structures fromthe first 50 ns of molecular dynamics simulations were not considered).
Bottom, a 2D scatter plot (and the underlying density plot, in which
yellow, blue, green and purple represent decreasing densities) comparing
the r.m.s.d. (after discarding the first 50 ns of each simulation) for apo
Neo-2/15 (computational model) versus the r.m.s.d. for the holo crystal

structure of Neo-2/15 (in complex with the mouse receptor). The
conformations sampled by Neo-2/15 when in complex with human
IL-2Rβγc are more similar to the apo Neo-2/15 structure (computationalmodel) than to the Neo-2/15 conformation observed in complex withmouse IL-2Rβγc. d, As in c, but for molecular dynamics simulations
started from the computational model of apo Neo-2/15 in complex
with the crystallographic structure of mouse IL-2Rβγc. The model of
apo Neo-2/15 was generated by aligning (using TMalign) the ternarycomputational model of Neo-2/15 with human IL-2Rβγc (from c) into
our crystallographic structure of mouse IL-2Rβγc (PDB ID: 6GD5)(average r.m.s.d. to holo Neo-2/15 (mouse) = 1.43 Å). Bottom, 2D scatterplot (and the underlying density plot, in which yellow, blue, green andpurple represent decreasing densities) comparing the r.m.s.d. (after
discarding the first 50 ns of molecular dynamics simulation) for apo
Neo-2/15 (computational model) versus the r.m.s.d. for the holo crystal
structure of Neo-2/15 (in complex with the mouse receptor). Unlike inc, the conformations sampled by Neo-2/15 when in complex with mouse
IL-2Rβγc are more similar to the Neo-2/15 conformation observed in
the crystallographic structure of the ternary complex of Neo-2/15 withmouse IL-2Rβγc (Fig. 3). For clarity, all the r.m.s.d. plots were filtered(running average filter, 5 frames = 100 ps), and points in the 2D scatterplots were subsampled every 25 conformations (that is, every 500 ps);
however, the density plot corresponds to all the analysed conformations(that is, the last 40 ns of 5 molecular dynamics simulations that wereanalysed, and conformations were recorded each 20 ps).

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Article RESEARCH

Extended Data Fig. 8 | Overall sequence conservation in binding
residues for each of the four common helices, combining informationfrom the three different de novo-designed IL-2 mimics. Sequence logos
were generated using combined data from binding experiments (using theheterodimeric mouse IL-2Rβγc, see Methods) from 3 independent SSM
mutagenesis libraries for G2_neo2_40_1F_seq27, G2_neo2_40_1F_seq29
and G2_neo2_40_1F_seq36 (Supplementary Figs. 8–10). All of these
proteins are functional high-affinity mimetics of mouse and human IL-2
(see Supplementary Figs. 6–11), some having topologies that differ fromthat of Neo-2/15, but all containing the four Helices H1, H3, H2′ and H4.
The logos show the combined information for each helix independently.

Below each logo, a line graph shows the probability score (higher means
more conserved) for each amino acid in the Neo-2/15 sequence. The redline highlights positions where the Neo-2/15 amino acid has a probability
score ≥30% (that is, these amino acids contribute more generally to
receptor binding as they are globally enriched in the binding populations
across all of the de novo IL-2 mimics tested). The topology of each helix
in Neo-2/15 is shown left of each logo. The sequences of the Neo-2/15
helices and those of the corresponding helices (structurally aligned)in human IL-2 and IL-15 are shown below the graphs, highlighting thedistinctiveness of the Neo-2/15 helices and binding interfaces.

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RESEARCH Article
Extended Data Table 1 | Characterization of several de novo designed mimics of IL-2/IL-15

The table shows experimental and structural properties for several de novo mimics of IL-2/IL-15 mimics. Human IL-2, mouse IL-2, and super-2 are shown as references. The sequence similarity wascalculated by structural alignment against human IL-2 (PDB ID: 2B5I) or mouse IL-2 (PDB ID: 4YQX). The EC50 field refers to pSTAT5 cell signalling measured across 6 independent experiments(denoted by a–f in parentheses). N/S, not significant; N/A, not available. The binding and signalling experiments were performed 3 times with similar results.

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Article RESEARCH
Extended Data Table 2 | Crystallographic data table for monomeric Neo-2/15 and the quaternary complex of Neo-2/15 with mouse IL-2RβγcaStatistics are for data that were truncated by STARANISO to remove poorly measured reflections affected by anisotropy.
The resolution limits for three directions in reciprocal space are indicated here. To calculate this, STARANISO computed an ellipsoid post-fitted by least squares to the cut-off surface, removing points
where the fit was poor. Note that the cut-off surface is unlikely to be perfectly ellipsoidal, so this is only an estimate.
c
The anisotropic completeness was obtained by least-squares fitting an ellipsoid to the reciprocal lattice points at the cut-off surface defined by a local mean l /σl threshold of 1.0, rejecting outliers in
the fit owing to spurious deviations (including any cusp), and calculating the fraction of observed data lying inside the ellipsoid so defined. Note that the cut-off surface is unlikely to be perfectly
ellipsoidal, so this is only an estimate.
dValues in parentheses are for the highest-resolution shell.
b© 2019 Springer Nature Limited. All rights reserved.

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in reporting. For further information on Nature Research policies, see Authors & Referees and the Editorial Policy Checklist.

Statistical parameters

nature research | reporting summary

Daniel-Adriano Silva, K. Christopher Garcia &Corresponding author(s): David Baker

When statistical analyses are reported, confirm that the following items are present in the relevant location (e.g. figure legend, table legend, main
text, or Methods section).
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Only common tests should be described solely by name; describe more complex techniques in the Methods section.

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variation (e.g. standard deviation) or associated estimates of uncertainty (e.g. confidence intervals)
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Give P values as exact values whenever suitable.

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For hierarchical and complex designs, identification of the appropriate level for tests and full reporting of outcomes
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State explicitly what error bars represent (e.g. SD, SE, CI)
Our web collection on statistics for biologists may be useful.

Software and code
Policy information about availability of computer code
Data collection

The design of protein mimics was performed using custom code programmed in python and ipython, in combination with the scientific/
high-performance modules: pyrosetta, numpy and scipy, matplotlib, sklearn, cython and pandas. Protein sequence design was
performed using Rosetta and RosettaScripts. The computer code and examples are provided along with SI.

Data analysis

Data analysis was performed using custom code programmed in python and ipython and Rosetta, in combination with the scientific/highperformance modules: pyrosetta, numpy and scipy, matplotlib, sklearn, cython and pandas. Protein visualization was performed using
PyMOL (PyMOL | pymol.org ).

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Policy information about availability of data
All manuscripts must include a data availability statement. This statement should provide the following information, where applicable:- Accession codes, unique identifiers, or web links for publicly available datasets- A list of figures that have associated raw data
- A description of any restrictions on data availability
PDBs for Neoleukin-2/15 monomer and for its ternary complex with mIL-2Rβgc have been deposited in the RCSB protein data bank (PDB IDs: 6DG6 and 6DG5,
respectively), diffraction images have been deposited in the SBGrid Data Bank (IDs: 587 and 588, respectively) and validation reports for each of the PDBs are part ofthe supplementary information. The databases of clustered fragments and the algorithms used for designing de novo protein mimetics (programmed as python/pyrosetta scripts) as described in this manuscript are available in the online repository Zenodo (ID: “to be provided with the final manuscript”) .Other data andmaterials related to this manuscript are available upon request to the corresponding authors.

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Life sciences study design
All studies must disclose on these points even when the disclosure is negative.
Sample size

Yeast display: Sample size was based on historical data which revealed statistical significance in previous experiments.
In vitro Stat5 experiments: Sample size was based on historical data which revealed statistical significance in previous experiments.
Ex vivo Stat5 experiments: Sample size was based on historical data which revealed statistical significance in previous experiments.
In vivo Airway inflammation experiments: Sample size was based on historical data which revealed statistical significance in previous
experiments.
Colorectal carcinoma in vivo experiments: Sample size was based on historical data for similar colorectal cancer experiments, .no statisticalmethods were used to pre-determine sample size.
Melanoma in vivo experiments: The minimum group size was determined using G*Power for an expected large effect size (Cohen’s d = 1.75).
Car-T cells in vivo experiments: Sample size was based on historical data for similar CAR-T cell experiment which revealed statisticalsignificance in previous experiments.

Data exclusions

Yeast display: No data were excluded from the analyses.
In vitro Stat5 experiments: No data were excluded from the analyses for STAT5 phosphorylation studies and surface plasmon resonancestudies. Cell populations were identified based on forward/side scatter profiles and singlets were discriminated using forward scatter area
versus height plots.
Ex vivo Stat5 experiments: No data were excluded from the analyses.
In vivo Airway inflammation experiments: Two samples were excluded from analysis of % lung resident CD8+ T cells in Figure 4b due to failureof intravascular labeling (Thy1.2-BUV395) prior to sacrifice, as determined by the absence of Thy1.2+ cells in the collected sample.
Colorectal carcinoma in vivo experiments: No data were excluded from the analysis
Melanoma in vivo experiments: For melanoma tumor model experiments, no data points were excluded from analysis.
Car-T cells in vivo experiments: No data were excluded from the analysis

Yeast display: All experiments were repeated at least once and all attempts of replication were successful.

April 2018

Replication

In vitro Stat5 experiments: All STAT5 phosphorylation studies were performed in triplicate and iterated at least 3 times to ensure
reproducibility. Surface plasmon resonance studies were performed at least twice to ensure reproducibility.
Ex vivo Stat5 experiments: All experiments were repeated and all attempts of replication were successful.

2

In vivo Airway inflammation experiments: All experiments were repeated and all attempts of replication were successful.

Melanoma in vivo experiments: Findings were conserved across a minimum of two experiments.
Car-T cells in vivo experiments: Experiments were not repeated, but multiple subjects were included in each experimental group.

Randomization

Yeast display: Not used.
In vitro Stat5 experiments: Not used.
Ex vivo Stat5 experiments: Not used. Mice used in the in vivo experiments were purchased from The Jackson Laboratory and randomly
allocated into experimental groups upon receipt.
In vivo Airway inflammation experiments: Mice used in the in vivo experiments were purchased from The Jackson Laboratory and randomly
allocated into experimental groups upon receipt.

nature research | reporting summary

Colorectal carcinoma in vivo experiments: Experiments were repeated with similar results that are included in the extended information.

Colorectal carcinoma in vivo experiments: Mice were randomly assigned into each experimental group according to their tumor volumes andin order to attain an even tumor volume average across experimental groups.
Melanoma in vivo experiments: The mice were given tumors and then randomized after tumor injection to specific treatments. In someinstances, mice were further randomized if a treatment was started later in the experiments. This is specifically stated in the methods or
figure legend when that was the case.
Car-T cells in vivo experiments: Mice were randomly allocated into experimental groups.

Blinding

Yeast display: Investigators were not blinded to the treatment groups.
In vitro Stat5 experiments: Investigators were not blinded to the treatment groups.
Ex vivo Stat5 experiments: Investigators were not blinded to the treatment groups.
In vivo Airway inflammation experiments: Investigators were not blinded to the treatment groups. Blinding was not relevant as mice were notscored throughout treatment.
Colorectal carcinoma in vivo experiments: Investigators were not blinded to the treatment groups.
Melanoma in vivo experiments: Investigators were not blinded to the treatment groups.
Car-T cells in vivo experiments: Investigators were not blinded to the treatment groups.

Reporting for specific materials, systems and methods
Materials & experimental systems

Methods

n/a Involved in the study

n/a Involved in the study

Unique biological materials

ChIP-seq

Antibodies

Flow cytometry

Eukaryotic cell lines

MRI-based neuroimaging

Palaeontology
Animals and other organisms
Human research participants

Policy information about availability of materials
Obtaining unique materials

Materials related to this manuscript are available upon request to the corresponding authors.

April 2018

Unique biological materials

3Antibodies used

Yeast display: FITC-conjugated anti-c-Myc antibody
In vitro Stat5 experiments:- Alexa Fluor® 647-conjugated mouse anti-STAT5 pY694 (BD Biosciences, 612599, Clone 47/Stat5(pY694))- AlexaFluor 700 anti-CD44, clone IM7
Ex vivo Stat5 experiments:- PE anti-pSTAT5 (pY694), clone 47- PE-Cy7 anti-CD25, clone PC61
- PerCP anti-CD4, clone RM4-5- AF700 anti-CD44, clone IM7
In vivo Airway inflammation experiments:- FITC anti-Ki67, clone SolA15- PerCP-Cy5.5 anti-CD25, clone PC61
- eFluor 450 anti-Foxp3, clone FJK-16S- BV510 anti-CD8, clone 53-6.7- BV605 anti-PD-1, clone J43- BV711 anti-CD4, clone RM4-5- BV786 anti-CD62L, clone MEL-14- PE anti-CD69, clone H1.2F3- PE-CF594 anti-B220, clone RA3-6B2- PE-Cy7 anti-CXCR5, clone 2G8- BUV395 anti-Thy1.2, clone 53-2.1

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Antibodies

Colorectal carcinoma in vivo experiments: (BioLegend antibodies):
Brilliant Violet 510™ anti-mouse CD45 Antibody, 103138, 30-F11, B235434;
Brilliant Violet 711™ anti-mouse CD3 Antibody, 100241, 17A2, B245637;
FITC anti-mouse CD49b (pan-NK cells) Antibody, 108906, DX5, B159570;
Brilliant Violet 605™ anti-mouse CD4 Antibody , 100548, RM4-5, B244808;
PE/Cy7 anti-mouse CD8a Antibody, 100721, 53-6.7, B239089;
FOXP3 Monoclonal Antibody (FJK-16s), APC, eBioscience™, 17-5773-82, FJK-16s, 4303649.
Melanoma in vivo experiments: (BioLegend antibodies):- CD45-BV711 (clone 30-F11, catalog 103147, lot number B243834),- CD8-BV650 (53-6.7, 100742, B249326),- CD4-BV421 (GK1.5, 100443, B244726),- TCRβ-BV510 (H57-597, 109233, B245719),- CD25-AF488 (PC61, 102017, B220118),- FoxP3-PE (MF-14, 126404, B254089).

Validation

Yeast display: Positive and negative controls of specific-binding (for each of the florescent labeled targets) were included in eachexperiment.
In vitro Stat5 experiments: Antibodies were used at the dilutions recommended by the manufacturer and validated using
appropriate positive (cytokine-stimulated cells) and negative (unstimulated cells) controls.
Ex vivo Stat5 experiments: Positive controls of specific-binding (for each of the florescent labeled targets) were included in eachexperiment.
In vivo Airway inflammation experiments: N/A
Colorectal carcinoma in vivo experiments: Every antibody was previously validated by the manufacturer. All antibody stainings
were previously titrated and tested by single-staining. Numerous references were cited in the product's web-page.
Melanoma in vivo experiments: antibodies were validated by BioLegend; citations are available on the manufacturer’s website.

Eukaryotic cell lines
Cell line source(s)In vitro Stat5 experiments: CTLL-2 cells were purchased from ATCC.
Colorectal carcinoma in vivo experiments: CT26.WT (ATCC® CRL-2638™) .
Melanoma in vivo experiments: B16 cells were obtained from the ATCC.

Authentication

In vitro Stat5 experiments: ATCC provided authentication for cell lines.
Colorectal carcinoma in vivo experiments: no authentication was performed.

April 2018

Policy information about cell lines

4Melanoma in vivo experiments: no authentication was performed.
In vitro Stat5 experiments: All cells tested negative for mycoplasma contamination via PCR assay.
Colorectal carcinoma in vivo experiments: Cell line was tested negative for mycoplasma using the MycoAlert™ Mycoplasma
Detection Kit from Lonza.
Melanoma in vivo experiments: testing for mycoplasma was done every 3-6 months.

Commonly misidentified lines

None.

(See ICLAC register)Animals and other organisms
Policy information about studies involving animals; ARRIVE guidelines recommended for reporting animal researchLaboratory animals

Ex vivo Stat5 experiments: Female wild-type C57BL/6J and CD25KO mice aged 6-8 weeks were purchased from The JacksonLaboratory and used in the experiments.
In vivo Airway inflammation experiments: Female wild-type C57BL/6J and CD25KO mice aged 6-8 weeks were purchased fromThe Jackson Laboratory and used in the experiments.

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Mycoplasma contamination

Colorectal carcinoma in vivo experiments: Mus musculus, BALB/c, female and male, age between 10-12 weeks of age.
Melanoma in vivo experiments: Female C57BL/6 mice between 6 and 8 weeks old were used.
Car-T cells in vivo experiments: NSG mice were obtained from the Jackson Laboratory

Wild animals

None

Field-collected samples

None

Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation

Yeast display: Yeast were grown in C-Trp-Ura media prior to induction in SGCAA media as previously described. 12-18 hours after
induction, cells were washed in chilled display buffer (50mM NaPO4 pH 8, 20mM NaCl, 0.5% BSA) and incubated with varying
concentrations of biotinylated receptor (either human or murine IL-2Rα or IL-2Rβ, Ɣc) while being agitated at 4°C. Afterapproximately 30 minutes, cells were washed again in chilled buffer, and then incubated on ice for 5 minutes with FITCconjugated anti-c-Myc antibody (1 uL per 3x106 cells) and streptavidin-phycoerythrin (1 uL per 100 uL volume of yeast). Yeast
were then washed and counted by flow cytometry (Accuri C6) or sorted by FACS (Sony SH800).
In-vitro Stat5 experiments: Cells were stimulated for 15 min at 37ºC and immediately fixed by addition of formaldehyde to 1.5%
and 10 min incubation at room temperature. Permeabilization of cells was achieved by resuspension in ice-cold 100% methanol
for 30 min at 4ºC. Fixed and permeabilized cells were washed twice with FACS buffer (phosphate-buffered saline [PBS] pH 7.2
containing 0.1% bovine serum albumin) and incubated with Alexa Fluor® 647-conjugated anti-STAT5 pY694 (BD Biosciences)
diluted 1:50 in FACS buffer for 2 hr at room temperature. Cells were then washed twice in FACS buffer and MFI was determinedon a CytoFLEX flow cytometer (Beckman-Coulter).

April 2018

Ex vivo Stat5 experiments: Spleens and lymph nodes were harvested from wild-type C57BL/6J or B6;129S4-Il2ratm1Dw(CD25KO) mice purchased from The Jackson Laboratory and made into a single cell suspension in sort buffer (2% Fetal Calf
Serum in pH 7.2 phosphate-buffered saline). CD4+ T cells were enriched through negative selection by staining the cellsuspension with biotin-conjugated anti-B220, CD8, NK1.1, CD11b, CD11c, Ter119, and CD19 antibodies at 1:100 for 30 min on
ice. Following a wash with sort buffer, anti-biotin MicroBeads (Miltenyi Biotec) were added to the cell suspension at 20 μL per
107 total cells and incubated on ice for 20 minutes. Cells were washed, resuspended and negative selection was then performed
using EasySep Magnets (STEMCELL Technologies). Approximately 1 x105 enriched cells were added to each well of a 96-wellplate in RPMI complete medium with 5% FCS with 10-fold serial dilutions of mIL-2, Super-2, or Neoleukin-2/15. Cells were
stimulated for 20 min at 37ºC in 5% CO2, fixed with 4% PFA and incubated for 30 minutes at 4ºC. Following fixation, cells were
harvested and washed twice with sort buffer and again fixed in 500 μL 90% ice-cold methanol in dH2O for 30 min on ice forpermeabilization. Cells were washed twice with Perm/Wash Buffer (BD Biosciences) and stained with anti-CD4-PerCP in Perm/
Wash buffer (1:300), anti-CD44-Alexa Fluor 700 (1:200), anti-CD25-PE-Cy7 (1:200), and 5 μL per sample of anti-pSTAT5-PE pY694
for 45 min at room temperature in the dark. Cells were washed with Perm/Wash and re-suspended in sort buffer for analysis on
a BD LSR II flow cytometer (BD Biosciences).

5Colorectal carcinoma in vivo experiments: Mice tumours were minced, digested using a mix of collagenase I, collagenase IV and
DNase I for 20 minutes at 37ºC. After digestion the samples were passed through a 100uM cell strainer, and resuspended in cold
complete DMEM medium. The cell suspensions from the spleens and the inguinal lymph nodes were obtained trough thesmashing of the tissues against the filter of a 100uM cell strainer.
Melanoma in vivo experiments: Cell suspensions from spleens, tumors, and tumor-draining lymph nodes were washed once with
PBS, and the cell pellet was resuspended in 2% inactivated fetal calf serum then stained.

Instrument

Yeast display: Sony SH800, Accuri C6nature research | reporting summary

In vivo Airway inflammation experiments: Circulating T cells were intravascularly labeled and tetramer positive cells wereenriched from lymph nodes and spleen or lung as previously described (Hondowicz et al., Immunity, 2016). Both the column
flow-trhough and bound fractions were saved for flow cytometry analysis. Cells were surface stained and fixed/permeabilized(eBioscience) according to manufacturer's directions prior to intracellular staining for Foxp3 or Ki67. Samples were then analyzed
on a BD LSR II.

In-vitro Stat5 experiments: CytoFLEX, Accuri C6
Ex vivo Stat5 experiments: BD LSR II
In vivo Airway inflammation experiments: BD LSR II
Colorectal carcinoma in vivo experiments: BD LSRFortessa™
Melanoma in vivo experiments: special order BD Biosciences LSRFortessa.

SoftwareYeast display: CytExpert Software for CytoFLEX and Flowjo
In-vitro Stat5 experiments: CytExpert Software for CytoFLEX and Flowjo
Ex vivo Stat5 experiments: BD FACSDiva was used for flow cytometry sample collection, and FlowJo 9.9.4 was used for analysis of
the flow samples.
In vivo Airway inflammation experiments: BD FACSDiva was used for flow cytometry sample collection, and FlowJo 9.9.4 wasused for analysis of the flow samples.
Colorectal carcinoma in vivo experiments: BD FACSDiva software™ for data collection, parameterization and compensation;
FlowJo, LCC for data analysis
Melanoma in vivo experiments: analysis was performed on FlowJo 10.4.2.

Cell population abundance

Yeast display: No cell sorting was performed for binding screening assays.
In-vitro Stat5 experiments: No cell sorting was performed.
Ex vivo Stat5 experiments: No cell sorting was performed.
In vivo Airway inflammation experiments: No cell sorting was performed.
Colorectal carcinoma in vivo experiments: No cell sorting was performed.
Melanoma in vivo experiments: No cell sorting was performed.

Gating strategy

In-vitro Stat5 experiments: Cell populations were identified based on forward/side scatter profiles and singlets werediscriminated using forward scatter area versus height plots. Gating examples are shown in the supplementary information
figures.

In vivo Airway inflammation experiments: Gating strategies are shown in the supplementary information figures.

April 2018

Ex vivo Stat5 experiments: Gating strategies are shown in the supplementary information figures.

Colorectal carcinoma in vivo experiments: FSC and SSC gating was performed in order to specifically select lymphocytes,
according to what is defined as the gating strategy for lymphocytes in lymphoid organs (small size, low complexity). Boundaries
between positive and negative were defined according to the unstained sample, single stainings and fluorescence minus one
controls in order to prevent the selection of unspecific signal, autofluorescence or parasitic fluorescence from otherfluorophores. Gating strategies are shown in the supplementary information figures.

6

Tick this box to confirm that a figure exemplifying the gating strategy is provided in the Supplementary Information.

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Melanoma in vivo experiments: Gating strategies are shown in the supplementary information figures.

April 20187