Emerging principles of cytokine pharmacology and therapeutics

Abstract

Cytokines are secreted signalling proteins that play essential roles in the initiation, maintenance and resolution of immune responses. Although the unique ability of cytokines to control immune function has garnered clinical interest in the context of cancer, autoimmunity and infectious disease, the use of cytokine-based therapeutics has been limited. This is due, in part, to the ability of cytokines to act on many cell types and impact diverse biological functions, resulting in dose-limiting toxicity or lack of efficacy. Recent studies combining structural biology, protein engineering and receptor pharmacology have unlocked new insights into the mechanisms of cytokine receptor activation, demonstrating that many aspects of cytokine function are highly tunable. Here, we discuss the pharmacological principles underlying these efforts to overcome cytokine pleiotropy and enhance the therapeutic potential of this important class of signalling molecules.

Fig. 1 |. Mechanism of cytokine signalling and sources of pleiotropy.

a | Model of the two-step activation mechanism common among cytokine receptors, in which the soluble cytokine ligand (yellow) first engages a high-affinity receptor subunit (cyan) via site 1, enabling recruitment of the low-affinity receptor subunit (green) from elsewhere on the cell surface. Formation of the ternary cytokine receptor complex enables the activation and transphosphorylation of the intracellular Janus kinases (JAKs) (green, purple), triggering the phosphorylation and nuclear translocation of the signal transducer and activator of transcription (STAT) transcription factors. b | Many cytokines exhibit functional and/or cellular pleiotropy due to the activation of multiple downstream transcription factors or due to their ability to signal on diverse cell types such as macrophages (cyan), T cells (lilac), B cells (red) or natural killer cells (white). c | Modulation of cytokine receptor affinity at site 1 alters the dose sensitivity (EC₅₀) of STAT activation, whereas modulation of site 2 alters the maximal strength (Emax). Graphs show the extent of STAT phosphorylation (pSTAT) as a function of cytokine concentration.

Fig. 2 |. Tunable parameters in cytokine receptor pharmacology.

a | Model highlighting the three parameters of cytokine receptor signalling that can be controlled through cytokine engineering to influence cytokine function, including receptor affinity, receptor geometry/orientation and receptor subunit composition. b | Cytokine partial agonists, which exhibit a lower signalling maximal strength (E_max) relative to their natural cytokine counterparts, can overcome cytokine pleiotropy by exploiting differential signalling response thresholds across cell populations. For example, interleukin-2 (IL-2) partial agonists can selectively exert immunosuppressive activity by preferentially expanding CD4⁺ regulatory T cells (T_reg cells) (blue) over effector CD8⁺ T cells or natural killer cells (red). JAK, Janus kinase; pSTAT, signal transducer and activator of transcription (STAT) phosphorylation.

Fig. 3 |. Tuning cytokine function by controlling receptor affinity.

a | Crystal structure of the interleukin-2 (IL-2) receptor complex [PDB:2B5I] showing IL-2 (yellow), IL-2Rα (pink), IL-2Rβ (cyan) and common γ-chain (γ_c) (green). Protein engineering strategies that increase the affinity of IL-2 for IL-2Rβ yielded super-2, a more potent IL-2 variant with reduced dose sensitivity (EC₅₀). By contrast, reducing the affinity of IL-2 for γ_c yielded partial agonists, such as H9T, which preferentially polarize CD8⁺ T cells towards a TCF1⁺ stem-like state. b | Cryo-electron microscopy structure of the IL-10 receptor complex [PDB:6×93] showing IL-10 (green), IL-10Rα (blue) and IL-10Rβ (purple). Structure-based design of IL-10 variants with reduced affinity for IL-10Rβ yielded myeloid selective variants, such as 10-DE, which suppress pro-inflammatory myeloid activity but no longer stimulate IFNγ production by T cells. c | Cryo-electron microscopy model of the IL-12 receptor complex [EMD:21645] showing IL-12p35 (green), IL-12p40 (yellow), IL-12Rβ1 (blue) and IL-12Rβ2 (purple). Structure-based design of IL-12 variants with reduced affinity for IL-12Rβ1 retains activity on CD8⁺ effector T cells without stimulating natural killer cell functions. d | Crystal structure of the IL-22 receptor complex [PDB:6WEO] showing IL-22 (cyan), IL-22Rα (purple) and IL-10Rβ (orange). Structure-based design of IL-22 variants with reduced affinity for IL-10Rβ, such as 22-B3, yielded biased agonists that selectively activate STAT3 but not STAT1 or STAT5 in epithelial cells. pSTAT, signal transducer and activator of transcription (STAT) phosphorylation; T_reg cells, regulatory T cells; WT, wild type.

Fig. 4 |. Tuning cytokine function by controlling receptor geometry.

a | Crystal structures of the erythropoietin (EPO) receptor (EpoR) in complex with EPO [PDB:1CN4] (left), a diabody [PDB:4Y5Y] (middle) and a homodimeric DARPin [PDB:6MOI] (right). b | Modulation of receptor distance, such as through diabodies, can tune the strength of downstream signal transducer and activator of transcription (STAT) activation. c | Modulation of receptor angle, such as through dimeric DARPins, yields varying degrees of partial agonism which can differentially impact downstream biological functions. pSTAT, STAT phosphorylation.

Fig. 5 |. Nanobody-based surrogate cytokines.

a | Single-domain antibody fragments (nanobodies) can be raised against various distinct epitopes on cytokine receptors, such as IL-2Rβ (cyan) and common γ-chain (γ_c) (green). b | Nanobodies targeting different cytokine receptors can be paired in a combinatorial manner, providing a panel of potential surrogate cytokines with various receptor binding sites and orientations. c | Different nanobody pairings can elicit distinct signalling profiles, such as varying levels of partial or biased agonism. d | Structural model of a heterodimeric nanobody pairing capable of engaging the interleukin-2 (IL-2) receptor and initiating signal transduction, with IL-2Rβ (cyan), γ_c (green) and nanobodies (grey and red; respectively [PDB:7S2S] and [PDB:7S2R]). ECD, extracellular domain; JAK, Janus kinase; pSTAT, signal transducer and activator of transcription (STAT) phosphorylation; VHH, variable region of heavy chain only antibody.

Fig. 6 |. Tuning cytokine function by controlling receptor composition.

a | A ‘synthekine’ comprising dominant negative (DN) versions of interleukin-2 (IL-2) (yellow) and IL-4 (purple) linked together through a flexible linker can recruit an unnatural receptor dimer comprising IL-2Rβ (cyan) and IL-4Rα (orange), to elicit a signal transducer and activator of transcription (STAT) activation signal distinct from that of the IL-2 receptor [PDB:2B5I] (left) or the IL-4 receptor [PDB:3BPL] (right) alone. b | Synthekines that pair different cytokine receptor subunits in non-natural combinations can enable the selective activation of a wide range of STAT pairings, theoretically enabling the customized activation of distinct transcriptional programmes. γ_c, common γ-chain; JAK, Janus kinase.