Human cytomegalovirus evades antibody-mediated immunity through endoplasmic reticulum-associated degradation of the FcRn receptor

Partial funding for Open Access provided by the UMD Libraries' Open Access Publishing Fund.Human cytomegalovirus (HCMV) can persistently infect humans, but how HCMV avoids humoral immunity is not clear. The neonatal Fc receptor (FcRn) controls IgG transport from the mother to the fetus and prolongs IgG half-life. Here we show that US11 inhibits the assembly of FcRn with β2m and retains FcRn in the endoplasmic reticulum (ER), consequently blocking FcRn trafficking to the endosome. Furthermore, US11 recruits the ubiquitin enzymes Derlin-1, TMEM129 and UbE2J2 to engage FcRn, consequently initiating the dislocation of FcRn from the ER to the cytosol and facilitating its degradation. Importantly, US11 inhibits IgGFcRn binding, resulting in a reduction of IgG transcytosis across intestinal or placental epithelial cells and IgG degradation in endothelial cells. Hence, these results identify the mechanism by which HCMV infection exploits an ER-associated degradation pathway through US11 to disable FcRn functions. These results have implications for vaccine development and immune surveillance

Next Generation Receptors for the Enhanced Control of Cell-based Immunotherapies

Many potential immunotherapies are hindered by poor efficacies and unacceptable toxicities, such as on-target, off-tumor toxicity. In contrast to constitutive CAR expression, inducing a T cell to only express a CAR in the presence of a second tumor antigen adds additional regulation over the cytotoxic response. The ability to tune the level and duration of an induced cell-based immunotherapeutic would also further improve on the safety of cell-based immunotherapies and provide a platform for robust tuning of an immunotherapeutic regimen. In pursuit of this goal, I have engineered a set of SyNthetic Intramembrane Proteolysis Receptors (SNIPRs), a series of Type I transmembrane proteins which, upon binding to their designated target, activate an orthogonal transcriptional program. Compared to current synthetic receptor designs, SNIPRs are smaller, more well-expressed, more sensitive, and more easily tunable in response to surface antigen and certain soluble antigens. I have demonstrated that SNIPRs, when expressed in human primary T-cells, can respond to tumor cells both in vitro and in vivo by outputting regulatable levels of a CAR or cytokine. I have also demonstrated in vivo that a SNIPR-CAR circuit can specifically clear a dual positive tumor and a SNIPR-superIL2 circuit can improve tumor clearance at lower T cell dosages. Together, these data demonstrate the potential for SNIPRs in the development of safer and more effective cell-based immunotherapeutics

Structural mechanism of serum amyloid A-mediated inflammatory amyloidosis

Serum amyloid A (SAA) represents an evolutionarily conserved family of inflammatory acute-phase proteins. It is also a major constituent of secondary amyloidosis. To understand its function and structural transition to amyloid, we determined a structure of human SAA1.1 in two crystal forms, representing a prototypic member of the family. Native SAA1.1 exists as a hexamer, with subunits displaying a unique four-helix bundle fold stabilized by its long C-terminal tail. Structure-based mutational studies revealed two positive-charge clusters, near the center and apex of the hexamer, that are involved in SAA association with heparin. The binding of high-density lipoprotein involves only the apex region of SAA and can be inhibited by heparin. Peptide amyloid formation assays identified the N-terminal helices 1 and 3 as amyloidogenic peptides of SAA1.1. Both peptides are secluded in the hexameric structure of SAA1.1, suggesting that the native SAA is nonpathogenic. Furthermore, dissociation of the SAA hexamer appears insufficient to initiate amyloidogenic transition, and proteolytic cleavage or removal of the C-terminal tail of SAA resulted in formation of various-sized structural aggregates containing ∼5-nm regular repeating protofibril-like units. The combined structural and functional studies provide mechanistic insights into the pathogenic contribution of glycosaminoglycan in SAA1.1-mediated AA amyloid formation

Design and modular assembly of synthetic intramembrane proteolysis receptors for custom gene regulation in therapeutic cells

[Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors we call SyNthetic Intramembrane Proteolysis Receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the potential transformative utility of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation.]https://www.biorxiv.org/content/10.1101/2021.05.21.445218v1Published versio

Modular design of synthetic receptors for programmed gene regulation in cell therapies

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation

Modular design of synthetic receptors for programmed gene regulation in cell therapies

Synthetic biology has established powerful tools to precisely control cell function. Engineering these systems to meet clinical requirements has enormous medical implications. Here, we adopted a clinically driven design process to build receptors for the autonomous control of therapeutic cells. We examined the function of key domains involved in regulated intramembrane proteolysis and showed that systematic modular engineering can generate a class of receptors that we call synthetic intramembrane proteolysis receptors (SNIPRs) that have tunable sensing and transcriptional response abilities. We demonstrate the therapeutic potential of the receptor platform by engineering human primary T cells for multi-antigen recognition and production of dosed, bioactive payloads relevant to the treatment of disease. Our design framework enables the development of fully humanized and customizable transcriptional receptors for the programming of therapeutic cells suitable for clinical translation

Antigen-dependent inducible T cell reporter system for PET imaging of breast cancer and glioblastoma.

For the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII- animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression

Antigen-dependent inducible T cell reporter system for PET imaging of breast cancer and glioblastoma

AbstractFor the past several decades, chimeric antigen receptor T cell (CAR T) therapies have shown promise in the treatment of cancers. These treatments would greatly benefit from companion imaging biomarkers to follow the trafficking of T cells in vivo. Using synthetic biology, we engineered T cells with a chimeric receptor SyNthetic Intramembrane Proteolysis Receptor (SNIPR) that induces overexpression of an exogenous reporter gene cassette upon recognition of specific tumor markers. We then applied a SNIPR-based positron emission tomography (PET) reporter system to two cancer-relevant antigens, human epidermal growth factor receptor 2 (HER2) and epidermal growth factor receptor variant III (EGFRvIII), commonly expressed in breast and glial tumors respectively. Antigen-specific reporter induction of the SNIPR-PET T cells was confirmed in vitro using GFP fluorescence, luciferase luminescence, and the HSV-TK PET reporter with [18F]FHBG. T cells associated with their target antigens were successfully imaged using PET in dual xenograft HER2+/HER2- and EGFRvIII+/EGFRvIII-animal models, with > 10-fold higher [18F]FHBG signals seen in antigen-expressing tumors versus the corresponding controls. The main innovation described is therefore PET detection of T cells via specific antigen-induced signals, in contrast to reporter systems relying on constitutive gene expression

Reproducibility of fluorescent expression from engineered biological constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.Peer reviewe