Exploiting Nanobodies' Singular Traits.

The unique class of heavy chain-only antibodies, present in Camelidae, can be shrunk to just the variable region of the heavy chain to yield VHHs, also called nanobodies. About one-tenth the size of their full-size counterparts, nanobodies can serve in applications similar to those for conventional antibodies, but they come with a number of signature advantages that find increasing application in biology. They not only function as crystallization chaperones but also can be expressed inside cells as such, or fused to other proteins to perturb the function of their targets, for example, by enforcing their localization or degradation. Their small size also affords advantages when applied in vivo, for example, in imaging applications. Here we review such applications, with particular emphasis on those areas where conventional antibodies would face a more challenging environment.

An adjuvant strategy enabled by modulation of the physical properties of microbial ligands expands antigen immunogenicity.

Activation of the innate immune system via pattern recognition receptors (PRRs) is key to generate lasting adaptive immunity. PRRs detect unique chemical patterns associated with invading microorganisms, but whether and how the physical properties of PRR ligands influence the development of the immune response remains unknown. Through the study of fungal mannans, we show that the physical form of PRR ligands dictates the immune response. Soluble mannans are immunosilent in the periphery but elicit a potent pro-inflammatory response in the draining lymph node (dLN). By modulating the physical form of mannans, we developed a formulation that targets both the periphery and the dLN. When combined with viral glycoprotein antigens, this mannan formulation broadens epitope recognition, elicits potent antigen-specific neutralizing antibodies, and confers protection against viral infections of the lung. Thus, the physical properties of microbial ligands determine the outcome of the immune response and can be harnessed for vaccine development.

Trained Immunity-Promoting Nanobiologic Therapy Suppresses Tumor Growth and Potentiates Checkpoint Inhibition.

Trained immunity, a functional state of myeloid cells, has been proposed as a compelling immune-oncological target. Its efficient induction requires direct engagement of myeloid progenitors in the bone marrow. For this purpose, we developed a bone marrow-avid nanobiologic platform designed specifically to induce trained immunity. We established the potent anti-tumor capabilities of our lead candidate MTP10-HDL in a B16F10 mouse melanoma model. These anti-tumor effects result from trained immunity-induced myelopoiesis caused by epigenetic rewiring of multipotent progenitors in the bone marrow, which overcomes the immunosuppressive tumor microenvironment. Furthermore, MTP10-HDL nanotherapy potentiates checkpoint inhibition in this melanoma model refractory to anti-PD-1 and anti-CTLA-4 therapy. Finally, we determined MTP10-HDL's favorable biodistribution and safety profile in non-human primates. In conclusion, we show that rationally designed nanobiologics can promote trained immunity and elicit a durable anti-tumor response either as a monotherapy or in combination with checkpoint inhibitor drugs.

Sortase A: A Model for Transpeptidation and Its Biological Applications.

Molecular biologists and chemists alike have long sought to modify proteins with substituents that cannot be installed by standard or even advanced genetic approaches. We here describe the use of transpeptidases to achieve these goals. Living systems encode a variety of transpeptidases and peptide ligases that allow for the enzyme-catalyzed formation of peptide bonds, and protein engineers have used directed evolution to enhance these enzymes for biological applications. We focus primarily on the transpeptidase sortase A, which has become popular over the past few years for its ability to perform a remarkably wide variety of protein modifications, both in vitro and in living cells.

Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation.

While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

Ubiquitin-like proteins.

The eukaryotic ubiquitin family encompasses nearly 20 proteins that are involved in the posttranslational modification of various macromolecules. The ubiquitin-like proteins (UBLs) that are part of this family adopt the ß-grasp fold that is characteristic of its founding member ubiquitin (Ub). Although structurally related, UBLs regulate a strikingly diverse set of cellular processes, including nuclear transport, proteolysis, translation, autophagy, and antiviral pathways. New UBL substrates continue to be identified and further expand the functional diversity of UBL pathways in cellular homeostasis and physiology. Here, we review recent findings on such novel substrates, mechanisms, and functions of UBLs.

USP16 is an ISG15 cross-reactive deubiquitinase that targets pro-ISG15 and ISGylated proteins involved in metabolism.

Interferon-induced ubiquitin (Ub)-like modifier ISG15 covalently modifies host and viral proteins to restrict viral infections. Its function is counteracted by the canonical deISGylase USP18 or Ub-specific protease 18. Notwithstanding indications for the existence of other ISG15 cross-reactive proteases, these remain to be identified. Here, we identify deubiquitinase USP16 as an ISG15 cross-reactive protease by means of ISG15 activity-based profiling. Recombinant USP16 cleaved pro-ISG15 and ISG15 isopeptide-linked model substrates in vitro, as well as ISGylated substrates from cell lysates. Moreover, interferon-induced stimulation of ISGylation was increased by depletion of USP16. The USP16-dependent ISG15 interactome indicated that the deISGylating function of USP16 may regulate metabolic pathways. Targeted enzymes include malate dehydrogenase, cytoplasmic superoxide dismutase 1, fructose-bisphosphate aldolase A, and cytoplasmic glutamic-oxaloacetic transaminase 1. USP16 may thus contribute to the regulation of a subset of metabolism-related proteins during type-I interferon responses.

The Angelman Syndrome protein Ube3A regulates synapse development by ubiquitinating arc.

Angelman Syndrome is a debilitating neurological disorder caused by mutation of the E3 ubiquitin ligase Ube3A, a gene whose mutation has also recently been associated with autism spectrum disorders (ASDs). The function of Ube3A during nervous system development and how Ube3A mutations give rise to cognitive impairment in individuals with Angleman Syndrome and ASDs are not clear. We report here that experience-driven neuronal activity induces Ube3A transcription and that Ube3A then regulates excitatory synapse development by controlling the degradation of Arc, a synaptic protein that promotes the internalization of the AMPA subtype of glutamate receptors. We find that disruption of Ube3A function in neurons leads to an increase in Arc expression and a concomitant decrease in the number of AMPA receptors at excitatory synapses. We propose that this deregulation of AMPA receptor expression at synapses may contribute to the cognitive dysfunction that occurs in Angelman Syndrome and possibly other ASDs.

Nanobodies in cancer.

For treatment and diagnosis of cancer, antibodies have proven their value and now serve as a first line of therapy for certain cancers. A unique class of antibody fragments called nanobodies, derived from camelid heavy chain-only antibodies, are gaining increasing acceptance as diagnostic tools and are considered also as building blocks for chimeric antigen receptors as well as for targeted drug delivery. The small size of nanobodies (∼15 kDa), their stability, ease of manufacture and modification for diverse formats, short circulatory half-life, and high tissue penetration, coupled with excellent specificity and affinity, account for their attractiveness. Here we review applications of nanobodies in the sphere of tumor biology.

Targeted delivery of an anti-inflammatory corticosteroid to Ly6C/G-positive cells abates severity of influenza A symptoms.

The distribution of Ly6C/G-positive cells in response to an infection of the mouse respiratory tract with influenza A virus was followed noninvasively over time by immuno-positron emission tomography. We converted nanobodies that recognize Ly6C and Ly6G, markers of neutrophils and other myeloid cells, as well as an influenza hemagglutinin-specific nanobody, into 89Zr-labeled PEGylated positron emission tomography (PET) imaging agents. The PET images showed strong accumulation of these imaging agents in the lungs of infected mice. Immunohistochemistry of influenza virus-infected mice and control mice, injected with a biotinylated and PEGylated version of the Ly6C/G-specific nanobody, showed the presence of abundant Ly6C/G-positive myeloid cells and positivity for Ly6C/G on bronchial epithelium in influenza virus-infected mice. This is consistent with focal inflammation in the lungs, a finding that correlated well with the immuno-PET results. No such signals were detected in control mice. Having shown by PET the accumulation of the Ly6C/G-specific nanobody in infected lungs, we synthesized conjugates of Ly6C/G-specific nanobodies with dexamethasone to enable targeted delivery of this immunosuppressive corticosteroid to sites of inflammation. Such conjugates reduced the weight loss that accompanies infection, while the equivalent amount of free dexamethasone was without effect. Nanobody-drug conjugates thus enable delivery of drugs to particular cell types at the appropriate anatomic site(s). By avoiding systemic exposure to free dexamethasone, this strategy minimizes its undesirable side effects because of the much lower effective dose of the nanobody-dexamethasone conjugate. The ability to selectively target inflammatory cells may find application in the treatment of other infections or other immune-mediated diseases.

Posttranscriptional Regulation of Glycoprotein Quality Control in the Endoplasmic Reticulum Is Controlled by the E2 Ub-Conjugating Enzyme UBC6e.

ER-associated degradation (ERAD) is essential for protein quality control in the ER, not only when the ER is stressed, but also at steady state. We report a new layer of homeostatic control, in which ERAD activity itself is regulated posttranscriptionally and independently of the unfolded protein response by adjusting the endogenous levels of EDEM1, OS-9, and SEL1L (ERAD enhancers). Functional UBC6e requires its precise location in the ER to form a supramolecular complex with Derlin2. This complex targets ERAD enhancers for degradation, a function that depends on UBC6e's enzymatic activity. Ablation of UBC6e causes upregulation of active ERAD enhancers and so increases clearance not only of terminally misfolded substrates, but also of wild-type glycoproteins that fold comparatively slowly in vitro and in vivo. The levels of proteins that comprise the ERAD machinery are thus carefully tuned and adjusted to prevailing needs.

A class II MHC-targeted vaccine elicits immunity against SARS-CoV-2 and its variants.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 100 million infections and millions of deaths. Effective vaccines remain the best hope of curtailing SARS-CoV-2 transmission, morbidity, and mortality. The vaccines in current use require cold storage and sophisticated manufacturing capacity, which complicates their distribution, especially in less developed countries. We report the development of a candidate SARS-CoV-2 vaccine that is purely protein based and directly targets antigen-presenting cells. It consists of the SARS-CoV-2 Spike receptor-binding domain (SpikeRBD) fused to an alpaca-derived nanobody that recognizes class II major histocompatibility complex antigens (VHHMHCII). This vaccine elicits robust humoral and cellular immunity against SARS-CoV-2 and its variants. Both young and aged mice immunized with two doses of VHHMHCII-SpikeRBD elicit high-titer binding and neutralizing antibodies. Immunization also induces strong cellular immunity, including a robust CD8 T cell response. VHHMHCII-SpikeRBD is stable for at least 7 d at room temperature and can be lyophilized without loss of efficacy.

In vivo detection of antigen-specific CD8+ T cells by immuno-positron emission tomography.

The immune system's ability to recognize peptides on major histocompatibility molecules contributes to the eradication of cancers and pathogens. Tracking these responses in vivo could help evaluate the efficacy of immune interventions and improve mechanistic understanding of immune responses. For this purpose, we employ synTacs, which are dimeric major histocompatibility molecule scaffolds of defined composition. SynTacs, when labeled with positron-emitting isotopes, can noninvasively image antigen-specific CD8+ T cells in vivo. Using radiolabeled synTacs loaded with the appropriate peptides, we imaged human papillomavirus-specific CD8+ T cells by positron emission tomography in mice bearing human papillomavirus-positive tumors, as well as influenza A virus-specific CD8+ T cells in the lungs of influenza A virus-infected mice. It is thus possible to visualize antigen-specific CD8+ T-cell populations in vivo, which may serve prognostic and diagnostic roles.

Converting an Anti-Mouse CD4 Monoclonal Antibody into an scFv Positron Emission Tomography Imaging Agent for Longitudinal Monitoring of CD4+ T Cells.

Immuno-positron emission tomography (PET), a noninvasive imaging modality, can provide a dynamic approach for longitudinal assessment of cell populations of interest. Transformation of mAbs into single-chain variable fragment (scFv)-based PET imaging agents would allow noninvasive tracking in vivo of a wide range of possible targets. We used sortase-mediated enzymatic labeling in combination with PEGylation to develop an anti-mouse CD4 scFv-based PET imaging agent constructed from an anti-mouse CD4 mAb. This anti-CD4 scFv can monitor the in vivo distribution of CD4+ T cells by immuno-PET. We tracked CD4+ and CD8+ T cells in wild-type mice, in immunodeficient recipients reconstituted with monoclonal populations of OT-II and OT-I T cells, and in a B16 melanoma model. Anti-CD4 and -CD8 immuno-PET showed that the persistence of both CD4+ and CD8+ T cells transferred into immunodeficient mice improved when recipients were immunized with OVA in CFA. In tumor-bearing animals, infiltration of both CD4+ and CD8+ T cells increased as the tumor grew. The approach described in this study should be readily applicable to convert clinically useful Abs into the corresponding scFv PET imaging agents.

Display of Native Antigen on cDC1 That Have Spatial Access to Both T and B Cells Underlies Efficient Humoral Vaccination.

Follicular dendritic cells and macrophages have been strongly implicated in presentation of native Ag to B cells. This property has also occasionally been attributed to conventional dendritic cells (cDC) but is generally masked by their essential role in T cell priming. cDC can be divided into two main subsets, cDC1 and cDC2, with recent evidence suggesting that cDC2 are primarily responsible for initiating B cell and T follicular helper responses. This conclusion is, however, at odds with evidence that targeting Ag to Clec9A (DNGR1), expressed by cDC1, induces strong humoral responses. In this study, we reveal that murine cDC1 interact extensively with B cells at the border of B cell follicles and, when Ag is targeted to Clec9A, can display native Ag for B cell activation. This leads to efficient induction of humoral immunity. Our findings indicate that surface display of native Ag on cDC with access to both T and B cells is key to efficient humoral vaccination.

Noninvasive imaging of tumor progression, metastasis, and fibrosis using a nanobody targeting the extracellular matrix.

Extracellular matrix (ECM) deposition is a hallmark of many diseases, including cancer and fibroses. To exploit the ECM as an imaging and therapeutic target, we developed alpaca-derived libraries of "nanobodies" against disease-associated ECM proteins. We describe here one such nanobody, NJB2, specific for an alternatively spliced domain of fibronectin expressed in disease ECM and neovasculature. We showed by noninvasive in vivo immuno-PET/CT imaging that NJB2 detects primary tumors and metastatic sites with excellent specificity in multiple models of breast cancer, including human and mouse triple-negative breast cancer, and in melanoma. We also imaged mice with pancreatic ductal adenocarcinoma (PDAC) in which NJB2 was able to detect not only PDAC tumors but also early pancreatic lesions called pancreatic intraepithelial neoplasias, which are challenging to detect by any current imaging modalities, with excellent clarity and signal-to-noise ratios that outperformed conventional 2-fluorodeoxyglucose PET/CT imaging. NJB2 also detected pulmonary fibrosis in a bleomycin-induced fibrosis model. We propose NJB2 and similar anti-ECM nanobodies as powerful tools for noninvasive detection of tumors, metastatic lesions, and fibroses. Furthermore, the selective recognition of disease tissues makes NJB2 a promising candidate for nanobody-based therapeutic applications.

Immuno-PET identifies the myeloid compartment as a key contributor to the outcome of the antitumor response under PD-1 blockade.

Immunotherapy using checkpoint-blocking antibodies against PD-1 has produced impressive results in a wide range of cancers. However, the response remains heterogeneous among patients. We used noninvasive immuno-positron emission tomography (PET), using 89Zr-labeled PEGylated single-domain antibody fragments (nanobodies or VHHs), to explore the dynamics and distribution of intratumoral CD8+ T cells and CD11b+ myeloid cells in response to anti-PD-1 treatment in the MC38 colorectal mouse adenocarcinoma model. Responding and nonresponding tumors showed consistent differences in the distribution of CD8+ and CD11b+ cells. Anti-PD-1 treatment mobilized CD8+ T cells from the tumor periphery to a more central location. Only those tumors fully infiltrated by CD8+ T cells went on to complete resolution. All tumors contained CD11b+ myeloid cells from the outset of treatment, with later recruitment of additional CD11b+ cells. As tumors grew, the distribution of intratumoral CD11b+ cells became more heterogeneous. Shrinkage of tumors in responders correlated with an increase in the CD11b+ population in the center of the tumors. The changes in distribution of CD8+ and CD11b+ cells, as assessed by PET, served as biomarkers to gauge the efficacy of anti-PD-1 treatment. Single-cell RNA sequencing of RNA from intratumoral CD45+ cells showed that CD11b+ cells in responders and nonresponders were markedly different. The responders exhibited a dominant population of macrophages with an M1-like signature, while the CD45+ population in the nonresponders displayed an M2-like transcriptional signature. Thus, by using immuno-PET and single-cell RNA sequencing, we show that anti-PD-1 treatment not only affects interactions of CD8+ T cells with the tumor but also impacts the intratumoral myeloid compartment.

Exploring cellular biochemistry with nanobodies.

Reagents that bind tightly and specifically to biomolecules of interest remain essential in the exploration of biology and in their ultimate application to medicine. Besides ligands for receptors of known specificity, agents commonly used for this purpose are monoclonal antibodies derived from mice, rabbits, and other animals. However, such antibodies can be expensive to produce, challenging to engineer, and are not necessarily stable in the context of the cellular cytoplasm, a reducing environment. Heavy chain-only antibodies, discovered in camelids, have been truncated to yield single-domain antibody fragments (VHHs or nanobodies) that overcome many of these shortcomings. Whereas they are known as crystallization chaperones for membrane proteins or as simple alternatives to conventional antibodies, nanobodies have been applied in settings where the use of standard antibodies or their derivatives would be impractical or impossible. We review recent examples in which the unique properties of nanobodies have been combined with complementary methods, such as chemical functionalization, to provide tools with unique and useful properties.

Turnip yellow mosaic virus protease binds ubiquitin suboptimally to fine-tune its deubiquitinase activity.

Single-stranded, positive-sense RNA viruses assemble their replication complexes in infected cells from a multidomain replication polyprotein. This polyprotein usually contains at least one protease, the primary function of which is to process the polyprotein into mature proteins. Such proteases also may have other functions in the replication cycle. For instance, cysteine proteases (PRO) frequently double up as ubiquitin hydrolases (DUB), thus interfering with cellular processes critical for virus replication. We previously reported the crystal structures of such a PRO/DUB from Turnip yellow mosaic virus (TYMV) and of its complex with one of its PRO substrates. Here we report the crystal structure of TYMV PRO/DUB in complex with ubiquitin. We find that PRO/DUB recognizes ubiquitin in an unorthodox way: It interacts with the body of ubiquitin through a split recognition motif engaging both the major and the secondary recognition patches of ubiquitin (Ile44 patch and Ile36 patch, respectively, including Leu8, which is part of the two patches). However, the contacts are suboptimal on both sides. Introducing a single-point mutation in TYMV PRO/DUB aimed at improving ubiquitin-binding led to a much more active DUB. Comparison with other PRO/DUBs from other viral families, particularly coronaviruses, suggests that low DUB activities of viral PRO/DUBs may generally be fine-tuned features of interaction with host factors.