Synthetic Immunology Research Articles

Bottom-up synthetic immunology.

Infectious diseases and cancer evade immune surveillance using similar mechanisms. Targeting immune mechanisms using common strategies thus represents a promising avenue to improve prevention and treatment. Synthetic immunology can provide such strategies by applying engineering principles from synthetic biology to immunology. Synthetic biologists engineer cells by top-down genetic manipulation or bottom-up assembly from nanoscale building blocks. Recent successes in treating advanced tumours and diseases using genetically engineered immune cells highlight the power of the top-down synthetic immunology approach. However, genetic immune engineering is mostly limited to ex vivo applications and is subject to complex counter-regulation inherent to immune functions. Bottom-up synthetic biology can harness the rich nanotechnology toolbox to engineer molecular and cellular systems from scratch and equip them with desired functions. These are beginning to be tailored to perform targeted immune functions and should hence allow intervention strategies by rational design. In this Perspective we conceptualize bottom-up synthetic immunology as a new frontier field that uses nanotechnology for crucial innovations in therapy and the prevention of infectious diseases and cancer.

Synthetic Immunology—Building Immunity from the Bottom‐Up with Synthetic Cells

Synthetic cells can advance immunotherapy, offering innovative approaches to understanding and enhancing immune responses. This review article delves into the advancements and potential of synthetic cell technologies in immunology, emphasizing their role in understanding and manipulating immune functions. Recent progress in understanding vertebrate immune systems and the challenges posed by diseases highlight the need for innovative research methods, complementing the analysis of multidimensional datasets and genetic engineering. Synthetic immune cell engineering aims to simplify the complexity of immunological systems by reconstructing them in a controlled setting. This approach, alongside high‐throughput strategies, facilitates systematic investigations into immunity and the development of novel treatments. The article reviews synthetic cell technologies, focusing on their alignment with the three laws of immunity: universality, tolerance, and appropriateness. It explores the integration of synthetic cell modules to mimic processes such as controlled T‐cell activation, bacteria engulfment and elimination, or cellular maturation into desirable phenotypes. Together, such advancements expand the toolbox for understanding and manipulating immune functions. Synthetic cell technologies stand at the innovation crossroads in immunology, promising to illuminate fundamental immune system principles and open new avenues for research and therapy.

Engineering B cells with customized therapeutic responses using a synthetic circuit

The expansion of genetic engineering has brought a new dimension for synthetic immunology. Immune cells are perfect candidates because of their ability to patrol the body, interact with many cell types, proliferate upon activation, and differentiate in memory cells. This study aimed at implementing a new synthetic circuit in B cells, allowing the expression of therapeutic molecules in a temporally and spatially restricted manner that is induced by the presence of specific antigens. This should enhance endogenous B cell functions in terms of recognition and effector properties. We developed a synthetic circuit encoding a sensor (a membrane-anchored B cell receptor targeting a model antigen), a transducer (a minimal promoter induced by the activated sensor), and effector molecules. We isolated a 734-bp-long fragment of the NR4A1 promoter, specifically activated by the sensor signaling cascade in a fully reversible manner. We demonstrate full antigen-specific circuit activation as its recognition by the sensor induced the activation of the NR4A1 promoter and the expression of the effector. Overall, such novel synthetic circuits offer huge possibilities for the treatment of many pathologies, as they are completely programmable; thus, the signal-specific sensors and effector molecules can be adapted to each disease.

Bottom-up assembly of target-specific cytotoxic synthetic cells

Immune vigilance ensures body integrity by eliminating malignant cells through the complex but coordinated cooperation of highly diversified lymphocytes populations. The sheer complexity of the immune system has slowed development of immunotherapies based on top-down genetic engineering of lymphocytes. In contrast, bottom-up assembly of synthetic cell compartments has contributed novel engineering strategies to reverse engineer and understand cellular phenomena as molecularly defined systems. Towards reducing the complexity of immunological systems, herein, a bottom-up approach for controlled assembly of fully-synthetic immune-inspired cells from predefined molecular components based on giant unilamellar vesicles is described. For construction of target-specific cytotoxic immune cells, the Fas-ligand-based apoptosis-inducing immune cell module is combined with an antibody-mediated cellular cytotoxicity-inspired system. The designed immune cells identify leukemia cells by specific surface antigens. Subsequently, they form stable attachments sites and eliminate their targets by induction of apoptosis. A structural and functional characterization of the synthetic immune cells by means of microfluidics, live cell, confocal and electron microscopy, dynamic light scattering as well as flow cytometry is presented. This study demonstrates the bioinspired construction of effector immune cells from defined molecular building blocks, enabling learning-by-building approaches in synthetic immunology.

The use of supercytokines, immunocytokines, engager cytokines, and other synthetic cytokines in immunotherapy.

Cytokines exert powerful immunomodulatory effects that are critical to physiology and pathology in humans. The application of natural cytokines in clinical studies has not been clearly established, and there are often problems associated with toxicity or lack of efficacy. The key reasons can be attributed to the pleiotropy of cytokine receptors and undesired activation of off-target cells. With a deeper understanding of the structural principles and functional signals of cytokine-receptor interactions, artificial modification of cytokine signaling through protein engineering and synthetic immunology has become an increasingly feasible and powerful approach. Engineered cytokines are designed to selectively target cells. Herein, the theoretical and experimental evidence of cytokine engineering is reviewed, and the "supercytokines" resulting from structural enhancement and the "immunocytokines" generated by antibody fusion are described. Finally, the "engager cytokines" formed by the crosslinking of cytokines and bispecific immune engagers and other synthetic cytokines formed by nonnatural analogs are also discussed.

The Evolution and Recent Progress in the Field of Synthetic Immunology

Synthetic immunology is a field in which researchers design constructs that will help immune cells battle pathogens, most commonly cancer cells. This is particularly crucial for human health due to the considerable number of ways that invaders (to the body) possess to minimize the effectiveness of the immune system. Frequently, these changes take place in the form of developing more advanced synthetic receptors for better recognition of pathogens so that T-cells can execute more precise functions in the body. Other changes are also made to give researchers more control over the advancements that have been inserted into the body, heightening the level of safety for the patients who receive them. Considering the newfound research that has been conducted, this paper focuses on the significance of upgrading various parts of the immune system in terms of the way that they can help protect the body. It also highlights the extensive potential this field has in the future considering the adaptability and functionality of the current, newly-designed systems in place.

Building on Synthetic Immunology and T Cell Engineering: A Brief Journey Through the History of Chimeric Antigen Receptors.

Advancement in our understanding of immune cell recognition and emerging cellular engineering technologies during the last decades made active manipulation of the T cell response possible. Synthetic immunology is providing us with an expanding set of composite receptor molecules capable to reprogram immune cell function in a predefined fashion. Since the first prototypes in the late 1980s, the design of chimeric antigen receptors (CARs; T-bodies, immunoreceptors), has followed a clear line of stepwise improvements from antigen-redirected targeting to designed "living factories" delivering transgenic products on demand. Building on basic research and creative clinical exploration, CAR T cell therapy has been achieving spectacular success in the treatment of hematologic malignancies, now beginning to improve the outcome of cancer patients. In this study, we briefly review the history of CARs and outline how the progress in the basic understanding of T cell recognition and of cell engineering technologies made novel therapies possible.

CAR T cells: Building on the CD19 paradigm.

Spearheaded by the therapeutic use of chimeric antigen receptors (CARs) targeting CD19, synthetic immunology has entered the clinical arena. CARs are recombinant receptors for antigen that engage cell surface molecules through the variable region of an antibody and signal through arrayed T-cell activating and costimulatory domains. CARs allow redirection of T-cell cytotoxicity against any antigen of choice, independent of MHC expression. Patient T cells engineered to express CARs specific for CD19 have yielded remarkable outcomes in subjects with relapsed/refractory B- cell malignancies, setting off unprecedented interest in T-cell engineering and cell-based cancer immunotherapy. In this review, we present the challenges to extend the use of CAR T cells to solid tumors and other pathologies. We further highlight progress in CAR design, cell manufacturing, and genome editing, which in aggregate hold the promise of generating safer and more effective genetically instructed immunity. Novel engineered cell types, including innate T-cell types, natural killer (NK) cells, macrophages, and induced pluripotent stem cell-derived immune cells, are on the horizon, as are applications of CAR T cells to treat autoimmunity, severe infections, and senescence-associated pathologies.

Design of Smart Antibody Mimetics with Photosensitive Switches.

As two prominent examples of intracellular single-domain antibodies or antibody mimetics derived from synthetic protein scaffolds, monobodies and nanobodies are gaining wide applications in cell biology, structural biology, synthetic immunology, and theranostics. Herein, a generally applicable method to engineer light-controllable monobodies and nanobodies, designated as moonbody and sunbody, respectively, is introduced. These engineered antibody-like modular domains enable rapid and reversible antibody-antigen recognition by utilizing light. By the paralleled insertion of two light-oxygen-voltage domain 2 modules into a single sunbody and the use of bivalent sunbodies, the range of dynamic changes of photoswitchable sunbodies is substantially enhanced. Furthermore, the use of moonbodies or sunbodies to precisely control protein degradation, gene transcription, and base editing by harnessing the power of light is demonstrated.

Engineered Th17 Cell Differentiation Using a Photoactivatable Immune Modulator.

T helper 17 (Th17) cells, an important subset of CD4+ T cells, help to eliminate extracellular infectious pathogens that have invaded our tissues. Despite the critical roles of Th17 cells in immunity, how the immune system regulates the production and maintenance of this cell type remains poorly understood. In particular, the plasticity of these cells or their dynamic ability to trans-differentiate into other CD4+ T cell subsets remains mostly uncharacterized. Here, we report a synthetic immunology approach using a photoactivatable immune modulator (PIM) to increase Th17 cell differentiation on demand with spatial and temporal precision to help elucidate this important and dynamic process. In this chemical strategy, we developed a latent agonist that upon photochemical activation releases a small-molecule ligand that targets the aryl hydrocarbon receptor (AhR) and ultimately induces Th17 cell differentiation. We used this chemical tool to control AhR activation with spatiotemporal precision within cells and to modulate Th17 cell differentiation on demand using UV light illumination. We envision that this approach will enable an understanding of the dynamic functions and behaviors of Th17 cells in vivo during immune responses and in mouse models of inflammatory disease.

Abstract 6248: Engineered probiotics for local delivery of checkpoint blockade nanobodies

Abstract Checkpoint inhibitors have revolutionized cancer therapy but only work in a subset of patients and can lead to a multitude of toxicities, suggesting the need for more targeted delivery systems. Because of their preferential colonization of tumors, microbes are a natural platform for the local delivery of cancer therapeutics. Here, we engineer a probiotic bacteria system for the controlled production and intratumoral release of nanobodies targeting programmed cell death-ligand 1 (PD-L1) and cytotoxic T lymphocyte-associated protein-4 (CTLA-4) using a stabilized lysing release mechanism. We used computational modeling coupled with experimental validation of lysis circuit dynamics to determine the optimal genetic circuit parameters for maximal therapeutic efficacy. A single injection of this engineered system demonstrated an enhanced therapeutic response compared to analogous clinically relevant antibodies, resulting in tumor regression in syngeneic mouse models. Supporting the potentiation of a systemic immune response, we observed a relative increase in activated T cells, an abscopal effect, and corresponding increases in systemic T cell memory populations in mice treated with probiotically delivered checkpoint inhibitors. Last, we leveraged the modularity of our platform to achieve enhanced therapeutic efficacy in a poorly immunogenic syngeneic mouse model through effective combinations with a probiotically produced cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF). Together, these results demonstrate that our engineered probiotic system bridges synthetic biology and immunology to improve upon checkpoint blockade delivery. Citation Format: Candice Gurbatri, Ioana Lia, Rosa Vincent, Courtney Coker, Samuel Castro, Piper Treuting, Taylor Hinchliffe, Nicholas Arpaia, Tal Danino. Engineered probiotics for local delivery of checkpoint blockade nanobodies [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6248.

Ethical and Biosafety Oversight of New CAR T-Cell Products

Genetic Engineering & Biotechnology NewsVol. 39, No. 5 OpinionEthical and Biosafety Oversight of New CAR T-Cell ProductsDevelopment of synthetic immunology challenges us to protect study participants and the publicDaniel Kavanagh, PhDDaniel Kavanagh, PhDDaniel Kavanagh, PhD, is senior scientific advisor, gene therapy at WCG (WIRB-Copernicus Group). Previously, he was on the faculty at Harvard Medical School, where he conducted gene therapy research and was vice chair of an Institutional Biosafety Committee overseeing human gene transfer studies.Search for more papers by this authorPublished Online:27 Apr 2019https://doi.org/10.1089/gen.39.05.05AboutSectionsView articleView Full TextPDF/EPUB Permissions & CitationsPermissionsDownload CitationsTrack CitationsAdd to favorites Back To Publication ShareShare onFacebookTwitterLinked InRedditEmail View articleFiguresReferencesRelatedDetails Volume 39Issue 5May 2019 InformationCopyright © GEN PublishingTo cite this article:Daniel Kavanagh, PhD.Ethical and Biosafety Oversight of New CAR T-Cell Products.Genetic Engineering & Biotechnology News.May 2019.12-13.http://doi.org/10.1089/gen.39.05.05Published in Volume: 39 Issue 5: April 27, 2019PDF download

Innate Immune Signaling Organelles Display Natural and Programmable Signaling Flexibility

The signaling organelles of the innate immune system consist of oligomeric protein complexes known as supramolecular organizing centers (SMOCs). Examples of SMOCs include myddosomes and inflammasomes, which respectively induce transcription-dependent and -independent inflammatory responses. The common use of oligomeric structures as signaling platforms suggests multifunctionality, but each SMOC has a singular biochemically defined function. Here, we report that the myddosome is a multifunctional organizing center. In addition to promoting inflammatory transcription factor activation, the myddosome drives the rapid induction of glycolysis. We identify the kinase TBK1 as a myddosome component that promotes glycolysis, but not nuclear factor κB (NF-κB) activation. Synthetic immunology approaches further diversified SMOC activities, aswe created interferon- or necroptosis-inducing myddosomes, inflammasomes that induce interferon responses instead of pyroptosis, and a SMOC-like nanomachine that induces interferon expression in response to a chemical ligand. These discoveries demonstrate the flexibility of immune signaling organelles, which permits the design of user-defined innate immune responses.

Synthetic Biology Approaches in Immunology.

Breakthroughs in gene synthesis has allowed synthetic biologists the ability to design any DNA sequence of interest, enabling the possibility to create complex systems inside cells with novel functions to tackle problems in immunology. Synthetic immunology of mammalian cells expressing natural or synthetic genes can guide and induce immune responses in patients. Through recent developments in engineering chimeric receptors, it is now feasible to customize control over engineered cells to target the disease sites with specificity. These cells can avoid immune rejection if derived from expandable cell types (e.g., stem cells or T cells) and then can be grown in abundance before implantation. However, safety concerns of engineered cells in circulation necessitates the development of a wide range of mechanisms to kill cells after their therapeutic life ends. This therapeutic effect is still predominantly the secretion of therapeutic proteins, but novel therapeutic interventions have been explored by synthetic biologists. In the pursuit of engineering new cell functions for synthetic immunology, it is possible that many problems previously thought intractable may actually be possible.

Remote Control of Mammalian Cells with Heat-Triggered Gene Switches and Photothermal Pulse Trains.

Engineered T cells are transforming broad fields in biomedicine, yet our ability to control cellular activity at specific anatomical sites remains limited. Here we engineer thermal gene switches to allow spatial and remote control of transcriptional activity using pulses of heat. These gene switches are constructed from the heat shock protein HSP70B' (HSPA6) promoter, show negligible basal transcriptional activity, and activate within an elevated temperature window of 40-45 °C. Using engineered Jurkat T cells implanted in vivo, we use plasmonic photothermal heating to trigger gene expression at specific sites to levels greater than 200-fold. We show that delivery of heat as thermal pulse trains significantly increase cellular thermal tolerance compared to continuous heating curves with identical area-under-the-curve (AUC), enabling long-term control of gene expression in Jurkat T cells. This approach expands the toolkit of remotely controlled genetic devices for basic and translational applications in synthetic immunology.

Synthetic Immunology: Hacking Immune Cells to Expand Their Therapeutic Capabilities.

The ability of immune cells to survey tissues and sense pathologic insults and deviations makes them a unique platform for interfacing with the body and disease. With the rapid advancement of synthetic biology, we can now engineer and equip immune cells with new sensors and controllable therapeutic response programs to sense and treat diseases that our natural immune system cannot normally handle. Here we review the current state of engineered immune cell therapeutics and their unique capabilities compared to small molecules and biologics. We then discuss how engineered immune cells are being designed to combat cancer, focusing on how new synthetic biology tools are providing potential ways to overcome the major roadblocks for treatment. Finally, we give a long-term vision for the use of synthetic biology to engineer immune cells as a general sensor-response platform to precisely detect disease, to remodel disease microenvironments, and to treat a potentially wide range of challenging diseases.

Adoptive immunotherapy utilizing cancer antigen-specific T-cell receptors.

Synthetic immunology based on rapidly-advancing gene-engineering and immunobiology has made novel anticancer adoptive immunotherapies, using gene-modified T lymphocytes to express cancer antigen-specific receptors, a reality. Various technological innovations have overcome recent difficulties and achieved clear and long-lasting clinical efficacy against tumors, while seeking more powerful effector gene-modified T cells has yielded serious treatment-related adverse events. In this article, along with introducing our clinical trial for a novel anti-leukemia adoptive immunotherapy regimen using gene-modified autologous lymphocytes to express leukemia antigen Wilms Tumor 1(WT1)-specific T cell receptor (TCR) against refractory acute myeloid leukemia (AML) and high-risk myelodysplastic syndrome (MDS), we provide an overview of the current status of this emerging treatment option and discuss its future form in the context of neoantigens encoded by mutated genes in cancer cells and immune checkpoint inhibitors.

D-Amino Acid Probes for Penicillin Binding Protein-based Bacterial Surface Labeling

Peptidoglycan is an essential and highly conserved mesh structure that surrounds bacterial cells. It plays a critical role in retaining a defined cell shape, and, in the case of pathogenic Gram-positive bacteria, it lies at the interface between bacterial cells and the host organism. Intriguingly, bacteria can metabolically incorporate unnatural D-amino acids into the peptidoglycan stem peptide directly from the surrounding medium, a process mediated by penicillin binding proteins (PBPs). Metabolic peptidoglycan remodeling via unnatural D-amino acids has provided unique insights into peptidoglycan biosynthesis of live bacteria and has also served as the basis of a synthetic immunology strategy with potential therapeutic implications. A striking feature of this process is the vast promiscuity displayed by PBPs in tolerating entirely unnatural side chains. However, the chemical space and physical features of this side chain promiscuity have not been determined systematically. In this report, we designed and synthesized a library of variants displaying diverse side chains to comprehensively establish the tolerability of unnatural D-amino acids by PBPs in both Gram-positive and Gram-negative organisms. In addition, nine Bacillus subtilis PBP-null mutants were evaluated with the goal of identifying a potential primary PBP responsible for unnatural D-amino acid incorporation and gaining insights into the temporal control of PBP activity. We empirically established the scope of physical parameters that govern the metabolic incorporation of unnatural D-amino acids into bacterial peptidoglycan.

Adoptive immunotherapy for hematological malignancies using T cells gene-modified to express tumor antigen-specific receptors.

Accumulating clinical evidence suggests that adoptive T-cell immunotherapy could be a promising option for control of cancer; evident examples include the graft-vs-leukemia effect mediated by donor lymphocyte infusion (DLI) and therapeutic infusion of ex vivo-expanded tumor-infiltrating lymphocytes (TIL) for melanoma. Currently, along with advances in synthetic immunology, gene-modified T cells retargeted to defined tumor antigens have been introduced as “cellular drugs”. As the functional properties of the adoptive immune response mediated by T lymphocytes are decisively regulated by their T-cell receptors (TCRs), transfer of genes encoding target antigen-specific receptors should enable polyclonal T cells to be uniformly redirected toward cancer cells. Clinically, anticancer adoptive immunotherapy using genetically engineered T cells has an impressive track record. Notable examples include the dramatic benefit of chimeric antigen receptor (CAR) gene-modified T cells redirected towards CD19 in patients with B-cell malignancy, and the encouraging results obtained with TCR gene-modified T cells redirected towards NY-ESO-1, a cancer-testis antigen, in patients with advanced melanoma and synovial cell sarcoma. This article overviews the current status of this treatment option, and discusses challenging issues that still restrain the full effectiveness of this strategy, especially in the context of hematological malignancy.

A chemically programmed antibody is a long-lasting and potent inhibitor of influenza neuraminidase.

Programming an anti-flu strategy: A new and potent neuraminidase inhibitor that maintains long-term systemic exposure of an antibody and the therapeutic activity of the neuraminadase inhibitor zanamivir has been created. This strategy could provide a promising new class of influenza A drugs for therapy and prophylaxis, and validates enzyme inhibitors as programming agents in synthetic immunology.