ABOUT THE RESEARCHER

OVERVIEW

Much of the research in the Ploegh lab is in the fields of biochemistry and immunology, which started with a specific focus on the unraveling the mechanisms by which MHC molecules interact with antigens inside a cell. Later, while at the Whitehead Institute, the Ploegh lab began using a technique called ‘sortagging’ to look at the pathways through which viruses are able to avoid detection by the immune system. Ploegh has been involved in further developing therapeutic roles for sortagging. Additionally, the Ploegh lab now focuses on utilizing ‘nanobodies’, derived from the antibodies of alpaca’s. Much of the research currently done in the lab uses these nanobodies for fundamental research and therapeutic applications.

Hidde Ploegh’s laboratory is interested in the biochemistry of immune recognition, and in mechanisms by which pathogens avoid being seen by the immune system. His earlier work centered on the analysis of the biochemical pathways involved in antigen processing and presentation by the products of the Major Histocompatibility Complex (MHC), which led to studies into glycoprotein biosynthesis and trafficking more generally. The discovery that human cytomegalovirus exploits an unusual mechanism to dispose of Class I MHC products, critical for recognition by cytotoxic T cells of virus-infected cells, led to observations that illuminated new aspects of glycoprotein quality control. Ploegh has applied chemistry to develop activity-based probes to study proteasomal proteolysis and more specifically the role of ubiquitin-specific proteases, also in the context of herpesvirus infections. More recently Ploegh has combined the generation of camelid-derived antibody fragments with a protein engineering approach, based on the use of bacterial sortases in conjunction with peptide chemistry. This combination is being developed to enable the visualization, by non-invasive means, of anti-tumor and anti-virus immune responses using positron emission tomography.

BACKGROUND

The Ploegh laboratory is interested in molecular aspects of immune recognition. This pursuit is complemented by the development of new technologies. These include the application of enzymatic methods for site-specific labeling of proteins. We then apply these methods to the modification of nanobodies, the smallest antibody-derived fragments that retain the capacity to bind antigens. The past few years have seen a focus on discovery of alpaca-derived nanobodies that recognize surface antigens on lymphocytes and myeloid cells. When properly modified with fluorophores or radioisotopes, the labeled nanobodies can be used for imaging applications, both for microscopy and to perform whole animal non-invasive imaging experiments by positron emission tomography (PET). The latter approach has been useful in examining the response to checkpoint blocking antibodies We are now developing a suite of nanobodies that can be used in phenotypic screens to perturb intracellular protein-protein interactions, a concept that we have applied to the discovery of nanobodies with antiviral activity.

The enzymatic methods for protein modification are exploited also in the manipulation of immune responses. We find that attaching an antigenic payload to a nanobody that seeks out professional antigen presenting cells can induce strong T cell responses and improve antibody formation. When applied under non-inflammatory conditions, such nanobody-antigen adducts appear capable of imposing antigen-specific tolerance, an approach we first explored by attaching offending self antigens to modified red blood cells as a means of attenuating autoimmune disease. Much of the above relies on a chemistry capability, centered on the synthesis of modified peptides. In the past we have used modified peptides (peptide vinyl sulfones) as a new class of proteasome inhibitors, an approach extended later to the design and synthesis of electrophilic ubiquitin derivatives. The latter serve as activity-based probes that detect ubiquitin-specific proteases. Most of the enzymatic methods fro protein modification rely on the use of sortase A, an enzyme that recognizes an LPXTG motif at or near the C-terminus of a protein to be modified. More recently we have begun to use plant derived asparagine endopeptidases, which enable a quasi-irreversible modification of proteins with payloads of interest.

The lab makes use of a number of mouse models to study anti-tumor immunity and anti-viral responses. We have made extensive use of somatic cell nuclear transfer to create mouse models with more physiological expression of rearranged antigen receptor genes. We refer to these animals as transnuclear mice. This approach has been applied to CD4 and CD8 T cells, iNKT cells and B cells. In the case of the CD4 T cell compartment, we are particularly interested in a line of mice that recognizes an antigen derived from Parabacteroides goldsteinii, a commensal gut-resident microbe. Having identified the actual peptide antigen recognized, this model should allow us to shed light in interactions between a healthy microbiota and adaptive immunity.

Current (bio)chemistry in the lab

We apply chemistry to study problems in cell biology and immunology. We use enzymes, in combination with synthetic compounds, to perform reactions that would be difficult or impossible to achieve using chemistry or biology alone. Ligase enzymes that catalyze the site-specific formation of an amide bond (Sortase A, butelase, OaAEP1) are used to label proteins with tags of choice, including fluorophores, radiometal chelators, non-natural peptides, click chemistry handles, bioactive small molecules, etc. The selectivity of these enzymes allow us to achieve selective labeling in complex biological environments in which standard approaches fail. For example, we have demonstrated an ability to selectively label appropriately tagged proteins on the surface of live cells using Sortase A.

We have applied this approach, in combination with nanobodies, to produce nanobody conjugates used as cancer vaccines and as immunogens for the generation of antibodies against poorly immunogenic cargoes. Nanobody conjugates have also been used as imaging agents (immunofluorescence, PET), where the small size of nanobodies enables enhanced penetration and higher quality imaging. Site-specific labeling also enables the production of fusions, such as C terminal-to-C terminal fusions, that are not accessible by genetic means. This enables us to use nanobodies to deliver bioactive polypeptides to destinations of choice. Finally, we are addressing the shortcomings of enzymatic ligation chemistry, such as the reversibility of the ligation reaction, through the exploration of new enzymes and new chemistry.

Selected Publications

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  1. Cheloha RW, Fisher FA, Woodham AW, Daley E, Suminski N, Gardella TJ, Ploegh HL. Improved GPCR ligands from nanobody tethering. Nat Comm. 2020 11: 2087.
  2. Rehm FBH, Harmand TJ, Yap K, Durek T, Craik DJ, Ploegh HL. Site-specific sequential protein labeling catalyzed by a single recombinant ligase. J Am Chem Soc. 2019 Oct 01.
  3. Ling J, Cheloha RW, McCaul N, Sun ZJ, Wagner G, Ploegh HL. A nanobody that recognizes a 14-residue peptide epitope in the E2 ubiquitin-conjugating enzyme UBC6e modulates its activity. Mol Immunol. 2019 Oct; 114:513-523.
  4. Khan NS, Lukason DP, Feliu M, Ward RA, Lord AK, Reedy JL, Ramirez-Ortiz ZG, Tam JM, Kasperkovitz PV, Negoro PE, Vyas TD, Xu S, Brinkmann MM, Acharaya M, Artavanis-Tsakonas K, Frickel EM, Becker CE, Dagher Z, Kim YM, Latz E, Ploegh HL, Mansour MK, Miranti CK, Levitz SM, Vyas JM. CD82 controls CpG-dependent TLR9 signaling. FASEB J. 2019 Aug 13; fj201901547R.
  5. Cheloha RW, Woodham AW, Bousbaine D, Wang T, Liu S, Sidney J, Sette A, Gellman SH, Ploegh HL. Recognition of Class II MHC Peptide Ligands That Contain ß-Amino Acids. J Immunol. 2019 Sep 15; 203(6):1619-1628.
  6. Rashidian M, LaFleur MW, Verschoor VL, Dongre A, Zhang Y, Nguyen TH, Kolifrath S, Aref AR, Lau CJ, Paweletz CP, Bu X, Freeman GJ, Barrasa MI, Weinberg RA, Sharpe AH, Ploegh HL. Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade. Proc Natl Acad Sci U S A. 2019 Aug 20; 116(34):16971-16980.
  7. Cheloha RW, Li Z, Bousbaine D, Woodham AW, Perrin P, Volaric J, Ploegh HL. Internalization of Influenza Virus and Cell Surface Proteins Monitored by Site-Specific Conjugation of Protease-Sensitive Probes. ACS Chem Biol. 2019 Aug 16; 14(8):1836-1844.
  8. Jailkhani N, Ingram JR, Rashidian M, Rickelt S, Tian C, Mak H, Jiang Z, Ploegh HL, Hynes RO. Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix. Proc Natl Acad Sci U S A. 2019 Jul 09; 116(28):14181-14190.
  9. Fang T, Li R, Li Z, Cho J, Guzman JS, Kamm RD, Ploegh HL. Remodeling of the Tumor Microenvironment by a Chemokine/Anti-PD-L1 Nanobody Fusion Protein. Mol Pharm. 2019 06 03; 16(6):2838-2844.
  10. Xie YJ, Dougan M, Jailkhani N, Ingram J, Fang T, Kummer L, Momin N, Pishesha N, Rickelt S, Hynes RO, Ploegh H. Nanobody-based CAR T cells that target the tumor microenvironment inhibit the growth of solid tumors in immunocompetent mice. Proc Natl Acad Sci U S A. 2019 Apr 16; 116(16):7624-7631.
  11. Yang YS, Moynihan KD, Bekdemir A, Dichwalkar TM, Noh MM, Watson N, Melo M, Ingram J, Suh H, Ploegh H, Stellacci FR, Irvine DJ. Targeting small molecule drugs to T cells with antibody-directed cell-penetrating gold nanoparticles. Biomater Sci. 2018 Dec 18; 7(1):113-124.