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.

Imaging the immune sequelae of infection with SARS-CoV-2 in nonhuman primates by using two nanobody PET-tracers.

Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) impacts multiple anatomical sites. Whether this is due to the virus itself or is a secondary effect caused by the influx and activation of immune cells is not known. Positron emission tomography (PET) with immunoglobulins can provide insights into which sites and cells are activated in a living animal. Our aim is to use two nanobodies as tools to monitor (1) the distribution of antigen presenting cells (APC) by virtue of their Mafa-DR expression profile, (2) virus-infected cells and viral particles using a nanobody against the SARS-CoV-2 spike protein. Two [89Zr]-labeled nanobodies that target the SARS-CoV-2 spike protein and major histocompatability complex (MHC) class II antigens (Mafa-DR), respectively, are used to monitor their distribution during an experimental SARS-CoV-2 infection in a nonhuman primate model. Scans are obtained before infection and on Day 3 and 10 post infection (pi) in two macaques each. The [89Zr]anti-SARS-CoV-2 spike nanobody localized to SARS-CoV-2-associated lung lesions and the nasal mucosa, while the [89Zr]anti-human leukocyte antigen (HLA)-DR nanobody was predominantly found in non-affected lung tissue after infection. We also detected, pi, upregulation of the Mafa-DR signal, indicative of recruitment of professional APCs, in the superior sagittal sinus. [89Zr]-labeled nanobodies show recruitment of macrophages/monocytes in non-lesional lung tissue in cynomolgus macaques after experimental infection with SARS-CoV-2, as well as accumulation of the spike protein in both lung lesions and the nasal mucosa during infection. These results show the possibility of in vivo monitoring the quality and quantity of immune responses during the initial stages of an infection.

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.

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.

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.

Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase.

Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.

One-Pot Dual Labeling of IgG 1 and Preparation of C-to-C Fusion Proteins Through a Combination of Sortase A and Butelase 1.

Site-specific chemical modification of proteins can assist in the study of their function. Furthermore, these methods are essential to develop biologicals for diagnostic and therapeutic use. Standard protein engineering protocols and recombinant expression enable the production of proteins with short peptide tags recognized by enzymes capable of site-specific modification. We report here the application of two enzymes of orthogonal specificity, sortase A and butelase 1, to prepare non-natural C-to-C fusion proteins. Using these enzymes, we further demonstrate site-selective installation of different chemical moieties at two sites in a full-size antibody molecule.

Improved synthesis of (S)-N-Boc-5-oxaproline for protein synthesis with the α-ketoacid-hydroxylamine (KAHA) ligation.

We describe a new route for the synthesis of (S)-N-Boc-5-oxaproline. This building block is a key element for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA) ligation. The new synthetic pathway to the enantiopure oxaproline is based on a chiral amine mediated enantioselective conjugate addition of a hydroxylamine to trans-4-oxo-2-butenoate. This route is practical, scalable and economical and provides decagram amounts of material for protein synthesis and conversion to other protected forms of (S)-oxaproline.

Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants.

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.

A guide to antigen processing and presentation.

Antigen processing and presentation are the cornerstones of adaptive immunity. B cells cannot generate high-affinity antibodies without T cell help. CD4+ T cells, which provide such help, use antigen-specific receptors that recognize major histocompatibility complex (MHC) molecules in complex with peptide cargo. Similarly, eradication of virus-infected cells often depends on cytotoxic CD8+ T cells, which rely on the recognition of peptide-MHC complexes for their action. The two major classes of glycoproteins entrusted with antigen presentation are the MHC class I and class II molecules, which present antigenic peptides to CD8+ T cells and CD4+ T cells, respectively. This Review describes the essentials of antigen processing and presentation. These pathways are divided into six discrete steps that allow a comparison of the various means by which antigens destined for presentation are acquired and how the source proteins for these antigens are tagged for degradation, destroyed and ultimately displayed as peptides in complex with MHC molecules for T cell recognition.

Nanobodies as in vivo, non-invasive, imaging agents.

In vivo imaging has become in recent years an incredible tool to study biological events and has found critical applications in diagnostic medicine. Although a lot of efforts and applications have been achieved using monoclonal antibodies, other types of delivery agents are being developed. Among them, VHHs, antigen binding fragments derived from camelid heavy chain-only antibodies, also known as nanobodies, have particularly attracted attention. Indeed, their stability, fast clearance, good tissue penetration, high solubility, simple cloning and recombinant production make them attractive targeting agents for imaging modalities such as PET, SPECT or Infra-Red. In this review, we discuss the pioneering work that has been carried out using VHHs and summarize the recent developments that have been made using nanobodies for in vivo, non-invasive, imaging.

Asparaginyl Ligase-Catalyzed One-Step Cell Surface Modification of Red Blood Cells.

Red blood cells (RBCs) can serve as vascular carriers for drugs, proteins, peptides, and nanoparticles. Human RBCs remain in the circulation for ∼120 days, are biocompatible, and are immunologically largely inert. RBCs are cleared by the reticuloendothelial system and can induce immune tolerance to foreign components attached to the RBC surface. RBC conjugates have been pursued in clinical trials to treat cancers and autoimmune diseases and to correct genetic disorders. Still, most methods used to modify RBCs require multiple steps, are resource-intensive and time-consuming, and increase the risk of inflicting damage to the RBCs. Here, we describe direct conjugation of peptides and proteins onto the surface of RBCs in a single step, catalyzed by a highly efficient, recombinant asparaginyl ligase under mild, physiological conditions. In mice, the modified RBCs remain intact in the circulation, display a normal circulatory half-life, and retain their immune tolerance-inducing properties, as shown for protection against an accelerated model for type 1 diabetes. We conjugated different nanobodies to RBCs with retention of their binding properties, and these modified RBCs can target cancer cells in vitro. This approach provides an appealing alternative to current methods of RBC engineering. It provides ready access to more complex RBC constructs and highlights the general utility of asparaginyl ligases for the modification of native cell surfaces.

Induction of antigen-specific tolerance by nanobody-antigen adducts that target class-II major histocompatibility complexes.

The association of autoimmune diseases with particular allellic products of the class-II major histocompatibility complex (MHCII) region implicates the presentation of the offending self-antigens to T cells. Because antigen-presenting cells are tolerogenic when they encounter an antigen under non-inflammatory conditions, the manipulation of antigen presentation may induce antigen-specific tolerance. Here, we show that, in mouse models of experimental autoimmune encephalomyelitis, type 1 diabetes and rheumatoid arthritis, the systemic administration of a single dose of nanobodies that recognize MHCII molecules and conjugated to the relevant self-antigen under non-inflammatory conditions confers long-lasting protection against these diseases. Moreover, co-administration of a nanobody-antigen adduct and the glucocorticoid dexamethasone, conjugated to the nanobody via a cleavable linker, halted the progression of established experimental autoimmune encephalomyelitis in symptomatic mice and alleviated their symptoms. This approach may represent a means of treating autoimmune conditions.

Chemical Synthesis of the Highly Hydrophobic Antiviral Membrane-Associated Protein IFITM3 and Modified Variants.

Interferon-induced transmembrane protein 3 (IFITM3) is an antiviral transmembrane protein that is thought to serve as the primary factor for inhibiting the replication of a large number of viruses, including West Nile virus, Dengue virus, Ebola virus, and Zika virus. Production of this 14.5 kDa, 133-residue transmembrane protein, especially with essential posttranslational modifications, by recombinant expression is challenging. In this report, we document the chemical synthesis of IFTIM3 in multi-milligram quantities (>15 mg) and the preparation of phosphorylated and fluorescent variants. The synthesis was accomplished by using KAHA ligations, which operate under acidic aqueous/organic mixtures that excel at solubilizing even the exceptionally hydrophobic C-terminal region of IFITM3. The synthetic material is readily incorporated into model vesicles and forms the basis for using synthetic, homogenous IFITM3 and its derivatives for further studying its structure and biological mode of action.

Protein chemical synthesis by α-ketoacid-hydroxylamine ligation.

Total chemical synthesis of proteins allows researchers to custom design proteins without the complex molecular biology that is required to insert non-natural amino acids or the biocontamination that arises from methods relying on overexpression in cells. We describe a detailed procedure for the chemical synthesis of proteins with the α-ketoacid-hydroxylamine (KAHA ligation), using (S)-5-oxaproline (Opr) as a key building block. This protocol comprises two main parts: (i) the synthesis of peptide fragments by standard fluorenylmethoxycarbonyl (Fmoc) chemistry and (ii) the KAHA ligation between fragments containing Opr and a C-terminal peptide α-ketoacid. This procedure provides an alternative to native chemical ligation (NCL) that could be valuable for the synthesis of proteins, particularly targets that do not contain cysteine residues. The ligation conditions-acidic DMSO/H2O or N-methyl-2-pyrrolidinone (NMP)/H2O-are ideally suited for solubilizing peptide segments, including many hydrophobic examples. The utility and efficiency of the protocol is demonstrated by the total chemical synthesis of the mature betatrophin (also called ANGPTL8), a 177-residue protein that contains no cysteine residues. With this protocol, the total synthesis of the betatrophin protein has been achieved in around 35 working days on a multimilligram scale.

New chemistries for chemoselective peptide ligations and the total synthesis of proteins.

The identification of fast, chemoselective bond-forming reactions is one of the major contemporary challenges in chemistry. The requirements of the native chemical ligation - an N-terminal cysteine and C-terminal thioesters - have encouraged a search for alternative amide-forming ligation reactions. Among successful alternatives to native chemical ligation, are the α-ketoacid-hydroxylamine ligation with 5-oxaproline and, serine/threonine ligation, and potassium acyltrifluoroborate (KAT) ligation. In addition, the KAT ligation, along with the non-amide forming alkyne-azide ligation, is very useful for synthetic conjugations. All of these recent ligation methods were applied to synthesize different proteins, and have allowed chemists to incorporate unnatural amino acids, or to modify the peptide backbone.