Antibodies (or immunoglobulins) are essential proteins for human immunological functioning. Antibodies are expressed on the membrane of B cells, a type of white blood cell essential for the adaptive immune response. After B cells are activated, they can differentiate into plasma cells that produce the same antibody in large quantities and expel it through exocytosis. Abzymes are these same molecules but with the addition of catalytic activity, a property rarely seen in antibodies naturally, and are currently seeing exciting development in various therapeutic fields.
Enzymes catalyze reactions that would otherwise be too slow for a biological time scale, and do this by having the following roles:
- Stabilization of transition state
- Provides reactive groups
- Localizes the substrate
- Exclusion of solvent
- Provides correct dynamics of active site residues
Antibodies are most likely to be able to replicate the first three roles, but not the last two. This partial catalytic activity is reflected in the catalytic rate of abzymes, which is significantly lower than their enzyme counterparts.
How To Make An Abzyme
Enzymes catalyze reactions by stabilizing the high-energy transition state, thereby reducing the energy required and causing the energy barrier of the reaction to be lowered. The most successful way of producing abzymes to date is by raising antibodies against transition state analogs. It is thought that if they have a high affinity for these analogs, they could reduce the energy barrier of a reaction therefore acting as an enzyme.
The other, less mainstream approach is to use an idiotypic pathway to produce an abzyme. This means raising an antibody that is complementary to the active site of the enzyme you want to mimic, then raising a second antibody to the variable region of the first antibody. The theory is that the first antibody would have the complementary properties to the active site of the enzyme, therefore the secondary antibody raised against this should have the same properties of the original enzyme’s active site (Figure 1) (Padiolleau-Lefevre, 2014).
A remarkable example of the transition state analog pathway was demonstrated by G. Wayne Zhou et al 1994. They were able to crystallize a catalytic antibody (17E8) that was raised to a norleucine phosphonate transition state analog, as part of a peptide hydrolysis reaction. 17E8 contained some of the same active residues as a class of proteases knows as serine proteases, which have a reactive triad responsible for their activity (serine-histidine-aspartic acid) – 17E8 contained the serine and histidine of this triad. The antibody also has a lysine strategically placed to stabilize to stabilize oxyanion formation and a small pocket for hydrophobic for side chains (Zhou 1994).
Remarkably, when both structures of trypsin (a serine protease that also catalyses a peptide hydrolysis reaction) and 17E8 containing the analog are superimposed, the active residues are in the exact same position relative to the substrate and to each other (Figure 2). Astonishingly, the immune system has created in about a week that evolution has taken many millennia to perfect.
Potential Clinical Applications Of Abzymes
Antibodies have exceptional specificity and high affinity, so combining this with catalytic activity seems like a perfect combination. As a large protein drug they would lack cell permeability, and therefore the focus has been on extracellular targets such as pathogens, toxins, hormones, cytokines and membrane proteins. A variety of abzyme drugs have been and are being developed and trialled.
Abzymes have been developed to hydrolyze the superantigenic region of gp102, the CD4 binding site of the HIV virus. This would prevent it from infecting its target cells, thus making the virus inert. (Planque, 2008).
Tumor necrosis factor alpha (TNFα) is a cytokine that has an important role in inflammation, and consequently dysregulation of TNFα is a common factor in autoimmune diseases such as rheumatoid arthritis and inflammatory bowel disease. Currently the most effective and useful inhibitors of TNFα are monoclonal antibodies that simply sequester TNFα, e.g. Infliximab (Remicade). The production of a catalytic antibody in this case would hopefully allow for much lower dosage to be needed as one abzyme could inactivate many more molecules of TNFα and be more cost effective in comparison. Recent research in this area has yielded ETNF-6 mAb, a catalytic antibody produced for exactly this purpose (Hifumi, 2010).
Each antibody has two variable regions, a feature that could be exploited in the development of new cancer treatments. Researchers are trying to develop antibodies where one variable region binds to a cancer associated antigen and the other has abzyme activity that actives a pro-drug (Goswami, 2009). The problem with this approach is lies in the heterogeneity of cancers, meaning cancer cells in a single patient may express different antigens.
Like in the development of any new therapeutic tools, the advancement of abzymes has not been without caveats and limitations. However, it is important to remember that the progress in this novel field is on-going and what has been yielded so far certainly seems promising; it does not seem far-fetched to predict that with their versatility, abzymes could be used to treat a plethora of different diseases in the future.
Blog written by James Noble
Zhou G.W, Guio J, Huang W, Fletterick R.J, Scanlan T.S (1994) Crystal Structure of a Catalytic Antibody with a Serine Protease Science, 265. 5175:1059-1064
Goswami RK, Huang ZZ, Forsyth JS, Felding-Habermann B, Sinha SC (2009) Multiple catalytic aldolase antibodies suitable for chemical programming. Bioorg Med Chem Lett 19:3821–3824
Hifumi E, Higashi K, Uda T (2010) Catalytic digestion of human tumor necrosis factor-a by antibody heavy chain. FEBS J 277:3823–3832
Planque S, Nishiyama Y, Taguchi H, Salas M, Hanson C, Paul S (2008) Catalytic antibodies to HIV: physiological role and potential clinical utility. Autoimmun Rev 7:473–479
Padiolleau-Lefevre S, Naya R. B., Shahsavarian M. A, Friboulet A, Avalle B (2014) Catalytic antibodies and their applications in biotechnology: state of the art. Biotechnol Lett 36:1369–1379