Page 106 - Science

RESEARCH

IMMUNE ENGINEERING to wild-type IL-2 (Fig. 1, C to E). The IL-2Rb hot-
spot residues His 134 and Tyr 135 make numerous
Selective targeting of engineered contacts with IL-2 that contribute a majority of
the binding free energy between IL-2 and IL-2Rb
(6) (Fig. 1E). A double mutant IL-2Rb [His 134 →
T cells using orthogonal IL-2 Asp (H134D) and Tyr 135 → Phe (Y135F)], referred
to herein as orthoIL-2Rb, lacked detectable bind-
cytokine-receptor complexes ing to IL-2 (Fig. 1D), even in the presence of CD25
(fig. S1) (7, 9).
Next, we used yeast display-based evolution to
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Jonathan T. Sockolosky, 1,2 Eleonora Trotta, * Giulia Parisi, * Lora Picton, Leon L. Su, 1 mutate, and thus remodel, the wild-type IL-2
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Alan C. Le, Akanksha Chhabra, Stephanie L. Silveria, Benson M. George, 2,5,6 interface region that was opposing (or facing
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Indigo C. King, Matthew R. Tiffany, Kevin Jude, Leah V. Sibener, 1,9 the site of) the IL-2Rb mutations in the crystal
structure, in order to create a molecule that bound
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David Baker, Judith A. Shizuru, Antoni Ribas, 4,10 to orthoIL-2Rb but not to wild-type IL-2Rb.IL-2
Jeffrey A. Bluestone, 3,10 K. Christopher Garcia 1,2,10,11 †
residues in proximity to the orthoIL-2Rb binding
interfacewererandomlymutated andwerechosen
Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and
on the basis of a homology model of the mouse
function, especially for engineered T cells in adoptive cell immunotherapy, but its
IL-2/IL-2Rb complex (Fig. 1E) derived from the
pleiotropy leads to simultaneous stimulation and suppression of immune responses as well
crystal structure of the human IL-2 receptor com-
as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor 8
plex (6). A library of ~10 unique IL-2 mutants
orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but
was displayed on the surface of yeast (fig. S2) and
do not interact with their natural cytokine and receptor counterparts. Introduction of
subjectedtomultipleroundsofbothpositive
orthoIL-2Rb into T cells enabled the selective cellular targeting of orthoIL-2 to engineered (against orthoIL-2Rb) and negative (against IL-
+
+
CD4 and CD8 T cells in vitro and in vivo, with limited off-target effects and negligible 2Rb) selection (figs. S2 and S3). This collection of Downloaded from
toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive yeast-displayed IL-2 mutants bound the orthoIL-
cell therapy and may therefore represent a synthetic approach to achieving selective 2Rb,but not wild-type IL-2Rb,and retained CD25
potentiation of engineered cells.
binding (Fig. 1D). Sequencing of yeast clones from
the evolved IL-2 libraries revealed a consensus
doptive transfer of tumor-reactive T cells immune stimulatory and suppressive T cell re- set of mutations at IL-2 positions in close struc-
has evolved into a clinically useful therapy sponses as well as potentially severe toxicities (5). tural proximity to the orthoIL-2Rb mutations
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capable of inducing antitumor immunity This is governed by the interaction between IL-2 (fig. S4). Interestingly, a Gln → Asn (Q30N) mu-
in patients (1, 2). However, the broad ap- and the IL-2 receptor (IL-2R), which consists of tation was highly conserved across three inde-
A plication of adoptive T cell transfer (ACT) a, b,and g subunits (6). IL-2Rb and the common pendent mutant IL-2 yeast libraries, whereas http://science.sciencemag.org/
therapies to treat cancer has several limitations, g-chain (IL-2Rg) together form the signaling dimer all other IL-2 positions used a restricted but not
including the production of sufficient quantities and bind IL-2 with moderate affinity, whereas specific mutational signature. We found that
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of cells for infusion and the failure of transferred IL-2Ra (CD25) does not signal but increases the IL-2 mutations Q30N, Met → Val (M33V), and
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T cells to persist and remain functional in vivo. affinity of IL-2 for the binary (bg) IL-2 receptor Asp → Leu or Met (D34L/M) appear to form a
In the clinic, the concomitant administration of to sensitize T cells to low concentrations of IL-2. small nonpolar pocket to compensate for the
the T cell growth factor interleukin-2 (IL-2) im- The activity of IL-2 as an adjuvant to ACT is IL-2Rb Y135F mutation, whereas Gln 36 → Thr,
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proves the survival, function, and antitumor ac- dependent on the balance between activation of Ser, Lys, or Glu (Q36T/S/K/E) and Glu → Tyr or
tivity of transplanted T cells (3, 4). However, the transplanted and endogenous T cell subsets bear- His (E37Y/H) mutations present a polar or charged on March 1, 2018
use of IL-2 to potentiate ACT is complicated by ing natural IL-2 receptors, as wellas hostresponses surface to compensate for the IL-2Rb H134D mu-
the pleiotropic nature of IL-2, which induces both that cause dose-limiting toxicities. Strategies to tation (Fig. 1F).
overcome these limitations could improve T cell Because of the affinity-enhancing effects of
1 Departments of Molecular and Cellular Physiology and immunotherapy (7, 8). Recognizing the need for CD25 expressiononthe interactionofIL-2with
Structural Biology, Stanford University School of Medicine, new approaches that afford precise targeting of the binary (bg)IL-2receptor(10), IL-2 mutants
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Stanford, CA 94305, USA. Stanford Cancer Institute, IL-2–dependent functions to a specific cell type with negligible binding to IL-2Rb alone may still
Stanford University School of Medicine, Stanford, CA 94305, of interest, we devised a strategy to redirect the form a functional signaling complex on cells that
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USA. Diabetes Center and Department of Medicine,
University of California, San Francisco, CA 94143, USA. specificity of IL-2 toward adoptively transferred also express CD25 (8). Therefore, we used a yeast-
4 Division of Hematology-Oncology, Department of Medicine, T cells. This method, based on receptor-ligand based functional screen to further triage IL-2 mu-
David Geffen School of Medicine, and Jonsson Comprehensive orthogonalization, uses a mutant IL-2 cytokine tants that bound specifically to the orthoIL-2Rb
Cancer Center, University of California, Los Angeles, CA and mutant IL-2 receptor that bind specifically and signaled selectively on T cells that express the
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90095, USA. Department of Blood and Marrow Transplantation,
Institute for Stem Cell Biology and Regenerative Medicine, to one another but not to their wild-type counter- orthoIL-2Rb (Fig. 1G and fig. S5), and produced
and Ludwig Center for Cancer Stem Cell Research and Medicine, parts (Fig. 1A). recombinantformsofselectIL-2mutants(orthoIL-2)
Stanford University School of Medicine, Stanford, CA 94305, We focused on the murine IL-2/IL-2Rb inter- for characterization (figs. S6 to S8).
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USA. Stanford Medical Scientist Training Program, Stanford action to enable in vivo characterization in syn- We focused our efforts on two orthoIL-2 mu-
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University, Stanford, CA 94305, USA. Department of
Biochemistry, Howard Hughes Medical Institute, and Institute for geneic mouse models. The IL-2Rb chain was tants, 1G12 and 3A10. OrthoIL-2 1G12 and 3A10
Protein Design, University of Washington, Seattle, WA 98195, chosen as the mutant receptor because the b share the consensus Q30N, M33V, and D34L
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USA. Department of Pediatrics and Genetics, Stanford chain is required for signal transduction and mutations but differ at positions Glu , Gln ,
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University School of Medicine, Stanford, CA 94305, USA. 37 41
9 Immunology Graduate Program, Stanford University School can bind IL-2 independently. We devised a two- Glu , and Arg (Fig. 1I). OrthoIL-2 1G12 and
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of Medicine, Stanford, CA 94305, USA. Parker Institute for step approach to engineer orthogonal IL-2/IL- 3A10 bound the orthoIL-2Rb with an affinity
Cancer Immunotherapy, 1 Letterman Drive, Suite D3500, San 2Rb pairs informed by the crystal structure of the comparable to that of the wild-type IL-2/IL-2Rb
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Francisco, CA 94129, USA. Howard Hughes Medical Institute, IL-2 high-affinity receptor complex (6)(Fig. 1B). interaction and displayed little to no detectable
Stanford University School of Medicine, Stanford, CA 94305, First, point mutations of the IL-2Rb chain were binding to wild-type IL-2Rb (Fig. 1H and figs. S7
USA.
*These authors contributed equally to this work. identified from inspection of the interface be- and S8) but differed in their ability to activate
†Corresponding author. Email: kcgarcia@stanford.edu tween IL-2 and IL-2Rb that abrogated binding IL-2Rb signaling in CD25-positive wild-type
Sockolosky et al., Science 359, 1037–1042 (2018) 2 March 2018 1of6