Antibody Proteins

What are the structures and functions of antibody proteins?

• The adaptive immune system produces antibodies in response to foreign material the body has come into contact with.

• Antibody proteins, also called immunoglobulins, consist of two copies each of heavy and light chains and form a Y-shaped structure. The light-chain protein has two domains, a variable domain and a constant domain, while the heavy-chain protein has four domains, a variable domain and three constant domains. Each domain has a similar overall structural fold known as the immunoglobulin fold.

• Mild treatment with protease can cleave an antibody into Fab and Fc fragments.

• The variable domains located at the N termini of the heavy and light chains form the epitope binding site of the antibody.

• There are two classes of immunoglobulin light chains and five classes of immunoglobulin heavy chains.

• Recombination of immunoglobulin genes has the capacity to generate billions of different antibodies through the process of V(D)J recombination and junctional diversification.

How are antibodies used as biochemical reagents to detect and purify specific proteins?

• Protein-specific antibodies are useful reagents in protein biochemistry because they identify target proteins with high affinity and specificity.

• Polyclonal antibodies are generated by injecting host animals with an antigenic protein or oligopeptide to induce an immune reaction and stimulate antibody production. Polyclonal antibodies are isolated from blood serum and represent a collection of antigen-selective antibodies that recognize multiple epitopes and are often affinity purified to remove nonspecific antibodies.

• Monoclonal antibodies are produced by injecting host animals with an antigenic protein or oligopeptide to induce an immune reaction and stimulate antibody production. Spleen cells are isolated from the immunized animal and fused with an immortalized tumor cell line to generate immortalized mouse hybridoma cells, which produce a single antibody that recognizes a particular epitope.

• Western blotting is a technique that identifies a specific protein in a sample after gel electrophoresis by using antibody–antigen recognition coupled with a colorimetric indicator.

• Immunofluorescence techniques use an antibody tagged with a fluorescent marker to detect a protein of interest, often using microscopy to visualize the fluorescence within a cell.

• The enzyme-linked immunosorbent assay (ELISA) is a biochemical tool to detect a specific protein in a mixed sample. In a sandwich ELISA, an antibody specific to the antigenic protein is linked to a 96-well plate, then the sample is applied. After washing to remove unbound material, two more antibodies are added to provide a visual cue that the epitope is present in a particular sample.

• Immunoprecipitation uses an antibody linked to a carbohydrate bead to specifically separate one protein (or complex containing that protein) from a mixture of material.

• SARS-CoV-2 at-home antigen tests use lateral flow assays to detect antigens from the virus in a liquid sample. These assays contain antibodies specific to a viral antigen (usually the nucleocapsid protein) as well as secondary antibodies that enable detection of a color change in the test and/or control lines.

How are therapeutic antibodies used to treat human disease?

• Therapeutic antibodies utilize antibodies that recognize specific antigens to target or alter a cellular or physiologic process.

• The first antibody therapy approved for use in humans was Orthoclone, a mouse monoclonal antibody used to reduce acute organ rejection after transplant. Orthoclone targeted the CD3 protein on T cells and blocked T-cell signaling, leading to T-cell apoptosis and decreased immune response. However, Orthoclone itself was immunogenic because it was derived from a mouse antibody and was discontinued from use in 2010.

• Herceptin is a monoclonal antibody that targets HER2+ breast cancer cells and reduces signaling, thereby reducing cell growth. Herceptin is a “humanized” monoclonal antibody that contains variable regions from the mouse fused to constant regions from a human, thereby reducing immunogenicity.

• Antibody–drug conjugates are compounds containing an antibody covalently attached to a drug. The antibody can be used to target a particular cell and deliver the drug to that cell.

• Bispecific antibodies can target two antigens or epitopes simultaneously. In some cases they can function like two separate monospecific antibodies and often work as inhibitors. Hemlibra is a bispecific antibody that functions as a replacement for the activated factor VIII (VIIIa) that is missing or defective in individuals with hemophilia A. It brings together components in the blood clotting cascade to promote coagulation and reduce the frequency of uncontrolled bleeds.

How do mRNA vaccines drive the production of antigens to stimulate immunity?

• mRNA vaccines contain an mRNA that encodes a portion of a protein antigen and is encased in a lipid nanoparticle. Once the vaccine enters a cell, the mRNA is translated by host cell ribosomes to produce an antigen. This antigen is displayed on the surface of dendritic cells for recognition by other cells of the immune system, so antibodies can be produced in the event of future exposure.

• The mRNA component of an mRNA vaccine has several modifications that increase its stability and translation efficiency. These modifications include a 5′ cap, a 3′ poly(A) tail, UTRs that flank the coding region, and N1-methylpseudouridine residues in place of uridine residues.

• The lipid nanoparticles of mRNA vaccines sequester mRNA inside the particle to reduce immune responses to the mRNA, protect mRNA from degradation, and enhance uptake into cells. There are five components of the lipid nanoparticles in use in the SARS-CoV-2 vaccines used during the pandemic: charged and neutral ionizable lipids, phosphatidylcholine, cholesterol, and polyethylene glycol–modified lipids.