Sera of camelids contain both conventional heterotetrameric antibodies and unique functional heavy (H)-chain antibodies (HCAbs). The H chain of these homodimeric antibodies consists of one antigen-binding domain, the VHH, and two constant domains. HCAbs fail to incorporate light (L) chains owing to the deletion of the first constant domain and a reshaped surface at the VHH side, which normally associates with L chains in conventional antibodies. The genetic elements composing HCAbs have been identified, but the in vivo generation of these antibodies from their dedicated genes into antigen-specific and affinity-matured bona fide antibodies remains largely underinvestigated. However, the facile identification of antigen-specific VHHs and their beneficial biochemical and economic properties (size, affinity, specificity, stability, production cost) supported by multiple crystal structures have encouraged antibody engineering of these single-domain antibodies for use as a research tool and in biotechnology and medicine.

Nanobodies: Natural Single-Domain Antibodies

Since the first PEGylated product was approved by the Food and Drug Administration in 1990, PEGylation has been widely used as a post-production modification methodology for improving biomedical efficacy and physicochemical properties of therapeutic proteins. Applicability and safety of this technology have been proven by use of various PEGylated pharmaceuticals for many years. It is expected that PEGylation, as the most established technology for extension of drug residence in the body, will play an important role in the next generation therapeutics, such as peptides, protein nanobodies and scaffolds, which due to their diminished molecular size need half-life extension. This review focuses on several factors important in the production of PEGylated biopharmaceuticals enabling efficient preparation of highly purified PEG-protein conjugates that have to meet stringent regulatory criteria for their use in human therapy. Areas addressed are PEG properties, the specificity of PEGylation reactions, separation and large-scale purification, the availability and analysis of PEG reagents, analysis of PEG-protein conjugates, the consistency of products and processes and approaches used for rapid screening of pharmacokinetic properties of PEG-protein conjugates.

https://www.creativepegworks.com/PEGylation%20of%20therapeutic%20proteins%20review%20by%20Jevsevar.pdf

PEGylation of therapeutic proteins.

Epidermal growth factor receptor targeting in cancer: a review of trends and strategies.

Monoclonal antibodies have revolutionized cancer therapy. However, delivery to tumor cells in vivo is hampered by the large size (150 kDa) of conventional antibodies. The minimal target recognition module of a conventional antibody is composed of two non-covalently associated variable domains (VH and VL). The proper orientation of these domains is mediated by their hydrophobic interface and is stabilized by their linkage to disulfide-linked constant domains (CH1 and CL). VH and VL domains can be fused via a genetic linker into a single chain variable fragment (scFv). scFv modules in turn can be fused to one another, e.g. to generate a bispecific T cell engager (BiTE), or they can be fused in various orientations to antibody hinge and Fc domains to generate bi- and multispecific antibodies. However, the inherent hydrophobic interaction of VH and VL domains limits the stability and solubility of engineered antibodies, often causing aggregation and/or mispairing of V-domains. Nanobodies (15 kDa) and nanobody-based human heavy chain antibodies (75 kDa) can overcome these limitations. Camelids naturally produce antibodies composed only of heavy chains in which the target recognition module is composed of a single variable domain (VHH or nanobody). Advantageous features of nanobodies include their small size, high solubility, high stability, and excellent tissue penetration in vivo. Nanobodies can readily be linked genetically to Fc-domains, other nanobodies, peptide tags, or toxins and can be conjugated chemically at a specific site to drugs, radionuclides, photosensitizers, and nanoparticles. These properties make them particularly suited for specific and efficient targeting of tumors in vivo. Chimeric nanobody-heavy chain antibodies combine advantageous features of nanobodies and human Fc domains in about half the size of a conventional antibody. In this review, we discuss recent developments and perspectives for applications of nanobodies and nanobody-based human heavy chain antibodies as anti-tumor therapeutics.

/pdf/nanobodies-and-nanobody-based-human-heavy-chain-antibodies-4wlgf9gqiz.pdf

Nanobodies and Nanobody-Based Human Heavy Chain Antibodies As Antitumor Therapeutics.

Iron oxide nanoparticles for targeted cancer imaging and diagnostics

Antibodies are important tools for experimental research and medical applications. Most antibodies are composed of two heavy and two light chains. Both chains contribute to the antigen-binding site which is usually flat or concave. In addition to these conventional antibodies, llamas, other camelids, and sharks also produce antibodies composed only of heavy chains. The antigen-binding site of these unusual heavy chain antibodies (hcAbs) is formed only by a single domain, designated VHH in camelid hcAbs and VNAR in shark hcAbs. VHH and VNAR are easily produced as recombinant proteins, designated single domain antibodies (sdAbs) or nanobodies. The CDR3 region of these sdAbs possesses the extraordinary capacity to form long fingerlike extensions that can extend into cavities on antigens, e.g., the active site crevice of enzymes. Other advantageous features of nanobodies include their small size, high solubility, thermal stability, refolding capacity, and good tissue penetration in vivo. Here we review the results of several recent proof-of-principle studies that open the exciting perspective of using sdAbs for modulating immune functions and for targeting toxins and microbes.

/pdf/single-domain-antibodies-promising-experimental-and-4ohmb6x5r7.pdf

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

Nanobodies are soluble, versatile, single-domain binding modules derived from the VHH variable domain of heavy chain antibodies naturally occurring in camelids. Nanobodies hold huge promise as novel therapeutic biologics. Membrane proteins are among the most interesting targets for therapeutic nanobodies because they are accessible to systemically injected biologics. In order to be effective, therapeutic nanobodies must recognize their target membrane protein in native conformation. However, raising nanobodies against membrane proteins in native conformation can pose a formidable challenge since membrane proteins typically contain one or more hydrophobic transmembrane regions and therefore are difficult to purify in native conformation. Here we describe a highly efficient genetic immunization strategy that circumvents these difficulties by driving expression of the target membrane protein in native conformation by cells of the immunized camelid. The strategy encompasses ballistic transfection of skin cells with cDNA expression plasmids encoding one or more orthologs of the membrane protein of interest and, optionally, other co-stimulatory proteins. The plasmid is coated onto 1 µm gold particles which are then injected into the shaved and depilated skin of the camelid. A gene gun delivers a helium pulse that accelerates the DNA coated particles to a velocity sufficient to penetrate through multiple layers of cells in the skin. This results in the exposure of the extracellular domains of the membrane protein on the cell surface of transfected cells. Repeated immunization drives somatic hypermutation and affinity maturation of target-specific heavy chain antibodies. The VHH/nanobody coding region is PCR-amplified from B cells obtained from peripheral blood or a lymph node biopsy. Specific nanobodies are selected by phage display or by screening of nanobody-based heavy chain antibodies expressed as secretory proteins in transfected HEK cells. Using this strategy, we have successfully generated agonistic and antagonistic nanobodies against several cell surface ecto-enzymes and ligand-gated ion channels.

/pdf/a-cdna-immunization-strategy-to-generate-nanobodies-against-3xikhr47iw.pdf

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

The CD8αβ coreceptor is crucial for effective peptide: MHC-I recognition by the TCR of CD8(+) T cells. Adenosine diphosphate ribosyl transferase 2.2 (ART2.2) utilizes extracellular NAD(+) to transfer ADP-ribose to arginine residues of extracellular domains of surface proteins. Here, we show that in the presence of extracellular NAD(+) , ART2.2 caused ADP-ribosylation of CD8-β on murine CD8(+) T cells in vitro and in vivo. Treatment with NAD(+) prevented binding of anti-CD8-β mAb YTS156.7.7 but not of mAb H35-17.2, indicating that NAD(+) caused modification of certain epitopes and not a general loss of CD8-β. Loss of antibody binding was strictly dependent on ART2.2, because it was not observed on ART2-deficient T cells or in the presence of inhibitory anti-ART2.2 single-domain antibodies. ADP-ribosylation of CD8-β occurred during cell isolation, particularly when cells were isolated from CD38-deficient mice. Incubation of ART2-expressing, but not of ART2-deficient, OVA-specific CD8(+) T cells with NAD(+) interfered with binding of OVA257-264 :MHC-I tetramers. In line with this result, treatment of WT mice with NAD(+) resulted in reduced CD8(+) T-cell mediated cytotoxicity in vivo. We propose that ADP-ribosylation of CD8-β can regulate the coreceptor function of CD8 in the presence of elevated levels of extracellular NAD(+) .

https://onlinelibrary.wiley.com/doi/pdf/10.1002/eji.201243231

CD8-β ADP-ribosylation affects CD8(+) T-cell function

The present invention contemplates isolated polynucleotide for the production of a VHH-containing heavy chain antibody in a mammal and vectors comprising said isolated polynucleotide. Moreover, the invention relates to a transgenic mammal comprising the vector for the production of a VHH-containing heavy chain antibody. Further, the invention relates to VHH-containing heavy chain antibodies as well as methods for the production and cloning of VHH-containing heavy chain antibodies.

Janusz Wesolowski

Papers

Single domain antibodies: promising experimental and therapeutic tools in infection and immunity.

A cDNA Immunization Strategy to Generate Nanobodies against Membrane Proteins in Native Conformation

CD8-β ADP-ribosylation affects CD8(+) T-cell function

Vhh-containing heavy chain antibody and production thereof