Camel/shark source antibodies and Nanobodies
In addition to conventional antibodies, camelids and sharks also contain specific heavy chain antibodies (hcAbs) that are composed entirely of heavy chain homodimers lacking the light chain. The Fab portion of these antibodies is referred to as VHH (the variable domain of the heavy chain antibody) and is the smallest antigen binding region found in nature.
Nanobodies are VHH-derived recombinant domains and are capable of binding antigen. They are very stable and can be easily mass produced by traditional simple systems, such as bacteria (but conventional antibodies with light and heavy chains are difficult to express in bacterial systems) and are therefore promising tools for research and therapeutic applications, especially in the field of super-resolution microscopy, mass spectrometry and targeted protein degradation. Nanobodies can be delivered to living cells by covalent attachment to the polypeptide, or can be expressed and recognized directly in vivo, but conventional antibodies with both light and heavy chains cannot be used for living cells. For example, anti-GFP Nanobodies are used to develop electromagnetic control systems that study neuronal activity in vivo. When the anti-RFP or GFP Nanobody binds to the far red dye Atto, it can obtain a fluorescence signal amplification of 118 times that of GFP or RFP, which can be used to generate whole body mouse neuronal connections. They can also be used in structural studies to stabilize the active state of proteins. AAV expression vectors have been shown to produce universal influenza vaccines with linked Nanobodies against four different influenza strains. Recombinant anti-mouse and anti-rabbit IgG secondary Nanobodies have the potential to replace widely used polyclonal secondary antibodies. Nanobodies have a unique ability to cross the blood-brain barrier; however, they are often processed and cleared out of the body very quickly. Nanobodies can be used for specific purposes, such as (co)immunoprecipitation or real-time fluorescent protein tracing of intracellular targets in living cells.
Cattle Long CDR3H
Approximately 10% of bovine immunoglobulins contain a long CDR3H region with multiple cysteine residues that are believed to contribute to antibody diversity.
Applications of antibody fragments
In some applications, fragments are more advantageous than intact antibodies. This topic was recently disscussed by Nelson. One of the advantages is that the fragment is smaller than the intact antibody, and can enter the tissue in which the intact antibody cannot enter and exert therapeutic effects and immunohistochemical staining. Antibody fragments are smaller than conventional antibodies and are generally not glycosylated, allowing their products to be expressed in prokaryotic expression systems, saving time and money. However, fragments lacking the Fc domain are faster in vivo than conventional antibodies and are unable to elicit Fc-mediated cytotoxicity unless they bind to a valid original for better therapeutic purposes. While the lack of an Fc domain is advantageous for immunohistochemistry and other assays because of the reduced non-specific binding of antibodies to Fc receptors. Antibody fragments without an Fc region have the advantage of being able to reduce non-specific binding. The anti-influenza neuraminidase antibody is a ScFv that is widely used in diagnosis. The anti-epithelial cell adhesion molecule Ep-CAM antibody MOC-31 is a ScFv for cancer therapy. Bispecific antibodies, trispecific antibodies and tetraspecific antibodies have potential applications in radioimmunotherapy and in vivo imaging diagnosis.
Although various antibody fragments have certain advantages, they are generally not used in experiments. Of more than 45,000 articles surveyed, only a few articles were related to the application of antibody Fab fragments.
Fc fragment receptor
The Fc receptor (FcR) is a molecule expressed mainly on/in innate immune cells that recognizes and binds to the Fc domain of an antibody, thereby providing them with a cellular system to elicit an immune response. The multiple functions of FcR reflect the broad protection or regulation of antibodies, including mediation of targeting substrates for neutralization and clearance, and adaptive immunity. The biological function of FcR is regulated by an immunoreceptor tyrosine-based activation motif (ITAM) and an immunoreceptor tyrosine-based inhibition motif (ITIM) as receptor interfaces for activation and inhibition of signaling pathways, respectively. Thus, signaling by ITAMs can trigger cell activation, phagocytosis and endocytosis, while signaling by ITIMs has an inhibitory effect on cell activation. A description of the FcR of all classes of immunoglobulins is available, some of which are discussed below.
This family includes FcγRI, FcγRII, FcγRIII and subtypes thereof. They are responsible for antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ACDP).
Another IgG-binding receptor is the neonatal Fc receptor (FcRn), which is involved in the transfer of passive humoral immunity from the mother to the fetus. FcRn also protects IgG from degradation in vivo, which is why they have a long half-life in serum. The Fc-FcRn interaction is promoted by changes in the Fc region, which leads to the development of better therapeutic antibodies.
They include a high affinity FcεRI capable of binding to monomeric IgE and a low affinity C-type lectin FcεRII capable of preferentially interacting with the IgE complex. FcεRI mediates immediate hypersensitivity responses to many allergic reactions by stimulating cell degranulation and releasing a range of inflammatory mediators on mast cells and basophils. FcεRII can exist in both membrane-bound forms for the delivery of down-regulated IgE synthesis and in the presence of soluble fragments to produce an up-regulation effect. Its role in the endocytic process of IgE allergen complexes in human airways and intestinal epithelial cells is being actively studied and may be a potential target for the treatment of allergic airway inflammation caused by food allergies.
FcαRI is the only member of this type and is expressed only in bone marrow lineage cells. It plays a role in pro-inflammatory and anti-inflammatory responses depending on the IgA binding state. Although secretory IgA (SIgA) binding to mucosal sites has anti-inflammatory effects, including prevention of pathogen invasion, binding of serum IgA leads to an inflammatory response. FcαRI also regulates neutrophil viability based on the inflammatory microenvironment.
TRIM21 shows a very broad range of antibody specificities and can therefore be distinguished from other FcRs. It can bind IgG, IgM and IgA. In addition, it is expressed in most cells that produce tissue. TRIM2 is involved in antibody-mediated viral replication interference by targeting cytosolic virus-antibody complexes for proteasomal degradation.
Binding of the Fc domain to FcR may have negative effects in monoclonal antibody-based assays such as immunohistochemistry (IHC), flow cytometry (FACS), and chromatin immunoprecipitation (ChIP). Non-specific binding to FcR may introduce background noise, resulting in false positives. Solutions to this problem include the use of (i) an isotype control for gating, (ii) serum to compete extensively for non-specifically binding receptors, or (iii) purified IgG to specifically block Fc receptors .