Selective targeting of engineered T cells using orthogonal IL-2 cytokine-receptor complexes

Abstract

Interleukin-2 (IL-2) is a cytokine required for effector T cell expansion, survival, and function, especially for engineered T cells in adoptive cell immunotherapy, but its pleiotropy leads to simultaneous stimulation and suppression of immune responses as well as systemic toxicity, limiting its therapeutic use. We engineered IL-2 cytokine-receptor orthogonal (ortho) pairs that interact with one another, transmitting native IL-2 signals, but do not interact with their natural cytokine and receptor counterparts. Introduction of orthoIL-2Rβ into T cells enabled the selective cellular targeting of orthoIL-2 to engineered CD4⁺ and CD8⁺ T cells in vitro and in vivo, with limited off-target effects and negligible toxicity. OrthoIL-2 pairs were efficacious in a preclinical mouse cancer model of adoptive cell therapy and may therefore represent a synthetic approach to achieving selective potentiation of engineered cells.

Fig. 1. Engineering and characterization of orthogonal IL-2 and IL-2R pairs

(A) Schematic overview of orthogonal IL-2/IL-2R pairs, consisting of a mutant IL-2 cytokine and mutant IL-2R that interact specifically with each other but do not cross-react with their wild-type counterparts. (B) Strategy used to engineer orthogonal IL-2/IL-2Rβ pairs. (C) Wild-type and mutant IL-2Rβ tetramer binding to wild-type IL-2 displayed on yeast by fluorescence-activated cell sorting. MFI, mean fluorescence intensity. Data are representative of two independent experiments. (D) Histograms of wild-type IL-2Rβ (blue), orthoIL-2Rβ (red), or CD25 (purple) binding to yeast-displayed wild-type IL-2, the naïve mutant IL-2 yeast library, or mutant IL-2 yeast clones after in vitro evolution. In vitro evolution of three independent mutant IL-2 yeast libraries (fig. S4) yielded similar results. (E) Homology model of the mouse IL-2/IL-2Rβ structure and the site I interface of IL-2 (gray) and contacts with IL-2Rβ His¹³⁴ and Tyr¹³⁵ (teal). Dashed lines indicate potential polar contacts. (F) Model of the orthoIL-2/orthoIL-2Rβ interactions. (G) Off-yeast pSTAT5 functional screen of IL-2 mutant activity on wild-type and orthoIL-2Rβ CTLL-2 T cells. (H) Representative surface plasmon resonance (SPR) sensograms of wild-type and orthoIL-2 binding to wild-type IL-2Rβ or orthoIL-2Rβ. Data are representative of two independent experiments. K_D, dissociation constant. (I) Sequences of wild-type (WT) IL-2, orthoIL-2 1G12, and orthoIL-2 3A10 and corresponding in vitro bioactivity (pSTAT5 EC₅₀) on wild-type and orthoIL-2Rβ CTLL-2 Tcells. Amino acid codes: A, Ala; D, Asp; E, Glu; F, Phe; H, His; K, Lys; L, Leu; M, Met; N, Asn; Q, Gln; T, Thr; V, Val; Y, Tyr.

Fig. 2. OrthoIL-2 signals through the orthoIL-2R expressed in primary mouse lymphocyte subsets, resulting in specific expansion of CD4 and CD8 T cells in vitro

(A) Flow cytometry data of mouse T cells transduced with the orthoIL-2Rβ and a YFP reporter (top panels) and associated cell surface levels of CD25, IL-2Rβ, and IL-2Rγ. (B to F) Dose-response curves of (B) STAT5 phosphorylation after 20 min of stimulation and (C) proliferation of wild-type (open circles) and orthoIL-2Rβ (solid circles) CD8⁺ Tcells cultured for 4 days in IL-2 or orthoIL-2; (D) table of respective pSTAT5 and proliferation EC₅₀ from data in (B) and (C). Representative histograms of (E) STAT5 phosphorylation and (F) scatterplots of CD8⁺ wild-type (YFP⁻) and orthoIL-2Rβ (YFP⁺) T cells expanded in IL-2. Data are means ± SD (n = 3 biological replicates). Dashed lines represent curves fit to a log (agonist) versus response (three parameters) model in Prism. (G) Dose-response curves of STAT5 phosphorylation (left) and proliferation (right) of wild-type and orthoIL-2Rβ CD4⁺ T_regs cultured in IL-2 or orthoIL-2. Data are means ± SD (n = 3 biological replicates). (H) Representative histograms of primary mouse B cells transduced with the orthoIL-2Rβ and stimulated with the indicated cytokines for quantification of intracellular pSTAT5 as in fig. S9.

Fig. 3. OrthoIL-2 promotes the specific expansion of orthoIL-2Rβ–modified T cells in mice with negligible toxicity

(A) Schematic of the adoptive CD8⁺ T cell transplant mouse model. (B) Quantification of donor wild-type and ortho CD8⁺ T cells in the spleen of recipient mice treated twice daily with phosphate-buffered saline (PBS), IL-2 (250,000 IU/dose), orthoIL-2 1G12 (250,000 IU/dose), or orthoIL-2 3A10 (2,500,000 IU/dose). (C) Representative flow cytometry data quantified in (B) depicting donor (Thy1.1⁺) wild-type (YFP⁻) and orthoIL-2Rβ (YFP⁺) CD8⁺ T cells in the spleen of recipient mice. (D) Spleen weight of mice treated in (B) normalized to total body weight on day of killing. (E to G) Quantification of exogenous cytokine administration on host (E) CD8⁺ memory phenotype T cell (MP, CD44⁺CD62L⁺), (F) CD4⁺ T_reg (CD25⁺Foxp3⁺), and (G) natural killer (NK) cell (CD3-NK1.1⁺CD49b⁺) numbers in the spleen of mice treated in (A). (H) Representative flow cytometry data as quantified in (F) and (G). Data in (B) to (H) are means ± SD (n = 5 mice per group). *P < 0.05, ****P < 0.0001 [analysis of variance (ANOVA)]; ns, not significant. (I) Quantification of donor wild-type and orthoIL-2Rβ CD4⁺ T_eff in the spleen of recipient mice treated once daily with PBS, IL-2 (250,000 IU/dose), or orthoIL-2 1G12 (1,000,000 IU/dose). Data are means ± SD and are representative of two independent experiments (n = 4 mice per group). *P < 0.05, ***P < 0.001 (ANOVA). (J) Survival of mice that received a mixture of wild-type and orthoIL-2Rβ CD8⁺ T cells followed by daily administration of IL-2 or orthoIL-2 fused to MSA. All mice received a total of 250,000 IU/day of the respective MSA fusion protein on an IL-2 basis for 5 days. (K) Mouse body weight over time normalized to the group average on day 0 as treated in (J). (L) Platelet counts in peripheral blood on day 4 as treated in (J). Data in (J) to (L) are means ± SD (n = 5 mice per group). ****P < 0.0001 (ANOVA). (M to O) Quantification of cytokine administration on host (M) CD8⁺ and (N) CD4⁺ T cell production of IFN-γ upon ex vivo restimulation with phorbol 12-myristate 13-acetate (PMA) and ionomycin. (O) Representative flow cytometry data as quantified in (M) and (N). (P and Q) Serum (P) IFN-γ and (Q) IL-5 concentrations on day 7 in mice treated daily with PBS or with MSA–IL-2, MSA-1G12, or MSA-3A10 (each 25,000 IU/dose) for 7 days. Data are means ± SD (n = 5 mice per group). ****P < 0.0001 (ANOVA).

Fig. 4. OrthoIL-2–expanded T cells retain effector function and promote an antitumor response against syngeneic B16-F10 tumors in mice

(A) Quantification of total number of IFN-γ–positive wild-type or orthoIL-2Rβ CD8⁺ T cells recovered from the spleen as treated in Fig. 3 (left) and representative flow cytometry data (right). (B) Cell surface expression levels of PD-1 (left) and TIM-3 (right) on wild-type and orthoIL-2Rβ CD8⁺ T cells in the spleen after administration of the indicated cytokines. Data are means ± SD (n = 5 mice per group). *P < 0.05, ****P < 0.0001 (ANOVA). (C) gp100 pMHC tetramer staining of orthoIL-2Rβ–transduced pmel-1 transgenic CD8⁺ T cells. (D) In vitro cytotoxicity of orthoIL-2Rβ pmel-1 transgenic T cells against antigen-positive (B16-F10) but not antigen-negative (MC38) tumor cells at a 20:1 (E:T) ratio. Data are means ± SD (n = 3 biological replicates). **P < 0.01 (Student t test). (E and F) Tumor growth (E) and survival (F) of C57BL/6J mice bearing subcutaneous B16-F10 tumors treated with wild-type (wt T) or orthoIL-2Rβ pmel-1 transgenic CD8⁺ T cells (ortho T) and IL-2 or orthoIL-2 1G12. Data are means ± SEM (n = 5 mice per group). ****P < 0.0001 (two-way ANOVA) (E); **P < 0.01 (log-rank test) (F). (G and H) Tumor growth (G) and survival (H) of C57BL/6J mice bearing subcutaneous B16-F10 tumors treated with wild-type (wt T) or orthoIL-2Rβ pmel-1 transgenic CD8⁺ T cells (ortho T) and IL-2 or orthoIL-2 3A10 fused to MSA. Data are means ± SEM (n = 4 mice per group). ****P < 0.0001 (two-way ANOVA) (G); **P < 0.01 (log-rank test) (H).