The field of synthetic cytokine biology continues
The field of synthetic cytokine biology continues to grow through molecular, physiological and pathophysiological insights, as well as via new methodical advances. The approval of chimeric antigen receptor (CAR) T cell immunotherapy by the US Food and Drug administration (FDA) in August 2017 for relapsed/refractory B cell precursor acute lymphoblastic leukemia in patients up to 25 years of age  is based on a combination of synthetic biology and genetic engineering in a therapeutic setting. The FDA-approved CAR T ep4 are composed of an extracellular single chain Fv (scFv) antibody fragment directed against the B cell-specific antigen CD19 (higher expression on tumorigenic B cells), the transmembrane domain and intracellular T cell receptor (TCR) activation and co-stimulatory domains, (e.g., typically the activation domain from CD3 in combination with one or two additional co-stimulatory domains from 4-1BB, CD27, CD28, ICOS, or OX40) 5, 6, 7, 8, 9. The patients’ T cells are isolated, genetically modified by the addition of the CAR cDNA, expanded, and infused back to the patient. Separation of T cell subsets on the level of CD4/CD8 may be performed. Once injected, CAR T cells can identify tumor cells via specific contacts between a CAR T cell and a tumor antigen. After CAR clustering, the CAR T cell is activated and can kill the tumor cell via cytotoxicity, which can eventually result in tumor remission. Despite a number of significant side effects, this type of cellular therapy has led to groundbreaking success in a number of malignancies and is currently being tested for a variety of others. Apart from this remarkable advance in synthetic-biology-based therapeutic approaches, many challenges currently remain with regard to efficacy, and to the important side effects that have to be overcome . This review discusses the most recent developments in synthetic cytokine biology, focusing on novel/engineered cytokines derived from natural cytokines or unrelated binding domains and structurally modified ligand-dependent and independent cytokine receptors. Current CAR T cell technology might be combined with these additional synthetic cytokine systems, in order to improve the outcomes of T cell immunotherapies.
Improved Synthetic Cytokines Assemble Novel Receptor Complexes Recombinant forms of many cytokines are successful drugs, including human growth hormone (hGH), EPO, TPO, G-CSF, IFNs, IL-2, and IL-11 . For all of the previous classes of cytokine, conjugates, fusion proteins, or deletion variants have been generated in order to obtain mimetics with improved biological signaling activity, stability, or novel functions 11, 12, such as mutated inhibitory IL-4 variants, tumor targeting cytokines, and selectively neuroprotective EPO lacking hematopoietic effects 11, 12, 13, 14, 15. Cytokines contain at least two receptor-binding sites to accomplish receptor dimerization. Dominant negative cytokine variants have been generated for many cytokines by mutation of one receptor-binding site, including super-binder mutations in the remaining receptor-binding site to increase antagonistic properties . Hence, the dominant negative cytokine variant Anakinra was clinically approved by the FDA for the treatment of rheumatoid arthritis and other conditions. Anakinra is a modified version of the IL-1 receptor antagonist . Fusion of two dominant negative cytokine variants in which each variant binds only one receptor subunit generates the so-called synthekines. Synthekines recruit two receptor subunits. The resulting receptor dimerization induces signal transduction. Synthekines can be designed to model signal transduction via known receptor complexes, or in a way that nonnatural receptor combinations are forced to dimerize. Thus, the selection of cytokines incorporated into synthekines can determine the receptor combination that is activated. In this way, nonnatural unique and programmable signaling outputs can be achieved (Figure 1) . The design of synthetic ligands causes corresponding receptor homo- or heterodimerization. One study fused dominant negative (DN) cytokines of IL-4, IL-2, and IFNω. The resulting synthekines, IFNDN-IL-4DN and IL-2DN-IL-4DN, activated nonnatural receptor pairs of IFNAR2-IL-4Rα and IL-2Rβ-IL-4Rα. This was demonstrated by phospho-flow cytometric analysis of 120 components of different signaling pathways. Cultured human T cells stimulated with natural ligands or engineered synthekines displayed in vitro activation of different transcription factors or even completely different signaling pathways. This suggested novel signaling patterns executed by nonnatural receptor pairs . However, when using synthekines in vivo, signaling intensity will not be simply dictated by the receptor composition but will also be dependent on the expression of receptors and intracellular signaling molecules in target cells. Single cell analysis of immune cells in quiescent and stimulated cellular states may thus be of great help in estimating the activatability and outcome intensity of target cells .