Conventional antibodies or fully sized antibodies are glycoproteins called immunoglobulins that are produced by the reaction of plasma cells with foreign molecules or antigens. The most basic function of antibodies is to bind specific antigens and stimulate immune responses to protect the body from infection. Antibodies include several subtypes, and here IgG and IgM subtypes are mainly described. Antibodies to IgG and IgM subtypes are widely used in research, diagnosis and treatment.
- Structure of conventional antibodies
The basic structure of an intact antibody consists of four peptide chains, including two heavy chains and two light chains, joined together by disulfide bonds. The antibody is shaped like a letter Y, and the hinge region of the Y structure is elastic. Each peptide chain has a constant region (very conserved across all antibodies) and a variable region (specific in antibody). The symbol of the light chain variable region is VL, and the symbol for the light chain constant region is CL (Fig. 1 left). Similarly, the variable and constant regions of the heavy chain are designated as (VH) and (CH), respectively. Carbohydrates usually bind to the CH2 region of the heavy chain. The Fc segment includes only the constant region (CH) of the heavy chain, but the Fab segment (Fab) that binds to the antigen includes a variable region of the constant region and the heavy chain and a variable region (VH and VL) of the light chain. The Fv region (variable fragment) contains only two variable regions.
Fig. 1 The basic structure of a complete conventional antibody (left) and usual antibody fragment (right)
- Application of conventional antibodies
Conventional antibodies have been used in research to detect target proteins by Western blotting, immunohistochemistry, and enzyme-linked immunosorbent assays (ELISA) for decades. Full-size antibodies are also used in clinical tests, such as pregnancy tests and the detection of HIV in the blood by ELISA. In addition, conventional intact antibodies are also used in the treatment of diseases. For example, infliximab is an antibody that recognizes tumor necrosis factor and is used to treat intestinal diseases and rheumatoid arthritis. Trastuzumab or Herceptin is an antibody that binds to epithelial growth factor II and is used to treat metastatic breast cancer. In addition, there are many antibodies, including Muromomab, that are used in basic therapies after organ transplantation to prevent graft rejection.
Advantages of using conventional antibodies include the ability of the Fc region to activate the body's immune response and bind to the target molecule to destroy it. Disadvantages of using intact antibodies include the inability to penetrate into certain tissues due to their large size. The ability of the Fc segment to activate some immune responses that are harmful to the patient is a disadvantage of clinically applied intact antibody therapy. The Fc region usually causes some non-specific binding and is detrimental to the application of antibody detection.
- Antibody fragment
A fragment of an antibody can be obtained by chemical reagents and genetic engineering methods. The fragments obtained by chemical reagent are achieved by disrupting the disulfide bond in the hinge region or by digesting the antibody with proteases, including pepsin and papain. Genetically engineered fragments provide a large number of fragments, each with specific binding regions and functional traits.
Fab, Fab', (Fab') 2, and Fv
An antigen-binding fragment (Fab) can be obtained by chemical treatment and protease digestion, which is derived from the variable regions of the IgG and IgM. The antibody portion from which the Fabs fragment is removed is the Fc fragment and consists of the constant region of the heavy chain. Antigen-binding fragments include Fab, Fab', (Fab') 2, and Fv. These fragments are capable of binding antigen, but they lack the Fc segment, which includes constant regions 2 and 3 of the heavy chain. When the antibody was digested with papain, two separate F(ab) fragments were isolated from the Fc region. However, after digestion with pepsin, a F(ab')2 fragment with a small portion of the Fc hinge region was isolated from the antibody. Although the separation of antibody fragments by chemical means can produce many useful diagnostic and therapeutic tools, it is very time consuming and requires a large amount of antibody as a raw material.
The monovalent F(ab) fragment has only one antigen-binding region, whereas the multivalent F(ab')2 fragment has two antigen-binding regions that are joined together by disulfide bonds. The F(ab')2 fragment produces two monovalent Fab' fragments and a free thio group that can be used for the binding of other molecules.
The Fv fragment is the smallest fragment of the product after enzymatic analysis of IgG and IgM type antibodies. Fv fragment antigen binding region, which consists of VH and VC regions, but they lack the CH1 and CL regions (see Figure 1 right panel). VH and VL are combined in the Fv fragment by non-covalent bonds.
ScFv, bispecific antibody, trispecific antibody, tetraspecific antibody, double-scFv, mimi antibody, Fab2, Fab3
The genetic engineering method is capable of producing a single-chain variable region (ScFv), which is an Fv-type fragment that includes VH and VL regions linked together by a flexible polypeptide (see Fig. 1 right panel). If the binding region has a length of at least 12 residues, the ScFv fragment is the monoclonal antibody. Different forms of Fv molecules can be created by manipulating the length of the V-domain and the hinge region. The resulting scFv molecule whose linker is a 3-11 residue cannot be folded into a functional Fv domain. Together with other scFv molecules, these molecules create a bivalent, bispecific antibody. If the length of the linker is less than 3 residues, the scFv molecules can interact to produce a trispecific or tetraspecific antibody. Multivalent scFvs have a stronger affinity for antigen binding than corresponding monovalent antibodies. The Mini antibody is a scFv-CH3 fusion protein that is loaded into a bivalent dimer. Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can be produced by two different variable regions, allowing these Bis-scFv molecules to simultaneously bind two non-existing epitopes. Genetic methods can be used to generate bispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). These antibody fragments are capable of binding to 2 (Fab2) or 3 (Fab3) different antibodies at the same time.