Monoclonal antibodies – all you need to know about antibody generation

Antibody generation raises a number of questions… what is the difference between their classes, forms and types? And how do recombinant monoclonal antibodies (rAbs) overcome the drawbacks of classical monoclonal antibodies (mAbs)?

Monoclonal antibodies are ubiquitous in biomedical research and medicine. They are used to fight, diagnose and research disease and to develop and test new drugs. The antibodies are divided in 5 classes or isotypes, several subtypes and forms and can be generated in vivo or in vitro.

In this short article, my aim is to define what an antibody is, to highlight the differences between a hybridoma monoclonal antibody and a recombinant monoclonal antibody and to point out how rAbs bring solutions to the classical drawbacks of mAbs.

Brief presentation of Antibodies

Antibodies (Abs) are blood glycoproteins belonging to the immunoglobulin superfamily and constitute most of the gamma globulin fraction of the blood protein. Nevertheless, they also can be found in other body fluids. They are secreted by B cells or membrane bound of B cells via their Fc region to B-cell receptor (BCR) in response to foreign organisms or antigen presence (bacteria, viruses…). Each antibody contains a Fab’s variable region containing at its extremity a paratope that is specific for one particular epitope on an antigen. When these 2 structures bound together, the Ab is able to tag the foreign organism or the infected cells for directly neutralizing them or to permit other cells of the immune system to attack them. The production of Abs is the main function of the humoral immune system.

Antibody classes or isotypes and functions

There are 5 classes of antibodies with different functions: IgG, IgM, IgA, IgE and IgD (see Fig. 2).

IgG antibodies are the most common and the most important. They circulate in the blood and other body fluids, defending against invading bacteria and viruses. The binding of IgG antibodies with bacterial or viral antigens activates other immune cells that engulf and destroy the antigens. As the smallest of the antibodies, IgG moves easily across cell membranes.

IgM is present in the blood and is the largest of the antibodies, combining five Y-shaped units. It functions similarly to IgG in defending against antigens but cannot cross membranes because of its size. IgM is the main antibody produced in an initial attack by a specific bacterial or viral antigen, while IgG is usually produced in later infections caused by the same agent.

IgA antibodies are present in tears, saliva, and mucus, as well as in secretions of the respiratory, reproductive, digestive, and urinary tracts. IgA functions to neutralize bacteria and viruses and prevent them from entering the body or reaching the internal organs.

IgE is only been found in mammals. IgE is the least abundant isotype and is synthesized by plasma cells. IgE also has an essential role in type I hypersensitivity, which manifests in various allergic diseases, such as allergic asthma, food allergies, specific types of chronic urticaria and atopic dermatitis. IgE also plays a role in responses to allergens.

IgD is present in species from cartilaginous fish to human. IgD’s function is to signal the B cells to be activated. When activated, B cells are ready to defend the body. During B cell differentiation, IgM is the exclusive isotype expressed by immature B cells. IgD starts to be expressed when the B cell exits the bone marrow to populate peripheral lymphoid tissues. The mature B cell co-expresses both IgM and IgD. IgD may have some role in allergic reactions. IgD is also able to bind to basophils and mast cells and activate these cells to produce antimicrobial factors to participate in respiratory immune defense in humans.

Antibody sub-types

For example, IgG corresponds to 70-85% of the total immunoglobulin pool antibody in normal human serum. IgG consists of 4 human subclasses (IgG1, IgG2, IgG3 and IgG4) each containing a different heavy chain. They are highly homologous and differ mainly in the hinge region and the extent to which they activate the host immune system. IgG1 and IgG4 contain two inter-chain di-sulphide bonds in the hinge region, IgG2 has 4 and IgG3 has 11. In mice, the IgG class is divided into 5 sub-classes (IgG1, IgG2A, IgG2B, IgG2C and IgG3) and in rat there are 4 (IgG1, IgG2A, IgG2B, IgG2C). Sub-class nomenclature has arisen independently for each species and so there is no general relationship between the sub-classes from each species.

Antibody forms

Polyclonal antibodies (pAbs), monoclonal antibodies (mAbs) and recombinant antibodies (rAbs) represent a collection of invaluable tools for life science research and numerous applications (WB, IHC, IF, FCM, ELISA…). To have access to the full collection of monoclonal and polyclonal antibodies available at tebu-bio, please follow these links: Primary antibodies – Secondary antibodies.

In addition, each form has advantages and disadvantages when compared with their counterparts.

a. Polyclonal antibodies (pAbs)

pAbs display multi-epitope binding properties which make these reagents ideally suited for many applications. Their clonal and biophysical diversity allows greater sensitivity and utility in certain types of applications and in life science research. However, the finite supply of pAbs limits their appeal and the use of controls and standards in experiments is essential to ensure the reproducibility.

b. Hybridoma monoclonal antibodies (mAbs)

mAbs are the product of spenocyte and myeloma cell fusions, which are produced using standard protocols. This process starts by injecting a mouse, or other mammal, with an antigen that induces an immune response. A type of white blood cell, the B cell, produces antibodies that bind to the injected antigen. These antibodies produced are then harvested from the mouse. These isolated B cells are in turn fused with immortal B cell cancer cells to produce a hybrid cell line called a hybridoma. This hybridoma has both the antibody-producing ability of the B-cell and the high longevity of the myeloma. The hybridomas can be grown in culture, each culture starting with one hybridoma cell which produce one antibody per culture (monoclonal). The monoclonal antibodies produced by each hybridoma line are all chemically identical. Nevertheless, it requires considerable time, money and expertise to produce, screen and bank mAbs.

c. Recombinant monoclonal antibodies (rAbs)

rAbs are constructed in vitro, outside the constraints of the immune system, using recombinant DNA technologies. The antibody genes are isolated and then incorporated into plasmid DNA vectors, and the resulting plasmids are transformed or transfected into expression hosts such as bacteria, yeast, or mammalian cell lines (similar process to classical recombinant protein production). rAbs offer the specificity and the reproducibility of mAbs with the advantage of recombinant modifications readily available. Thus, they can be used in all applications where classical mAbs are used. They exist in various forms from IgG to dAb, by passing by ScFv, Fab, Diabody forms as well as mono,-, bi- and tri-variants (see Fig. 3). The role and the engineering of rAbs will expand as this technology continues to develop.

Advantages of rAbs compared to mAbs

As mentioned above, producing rAbs is cheaper than generating mAbs. Indeed, rAbs required less purified antigen to produce than classical mAbs. Moreover, the production time is shorter (weeks vs. months).

Mass production of rAbs does not required the use of animals (overcomes ethical concerns).

rAbs can be produced in several formats like Fab fragments, single-chain variable region fragments (ScFv), diabodies (Dimeric ScFvs) and in several hosts (from bacteria to human cells) (see Fig. 4). We can switch the antibodies of species (from mouse to human), of classes (from IgG to IgE) and of substypes (from an entire IgG form to ScFv) with no immeasurable effort.

rAbs can easily be fused with drugs and toxins and, thus, be used as therapeutic molecules such as Antibody Drug Conjugates or ADCs (see previous posts related to ADCs metabolism by Lysosomes or linkers)

rAbs, unlike mAbs, are reliable and highly reproducible because they are defined by their sequences that encode them. They also can be easily optimized in order to, for ex, improve their affinity to their target (by using bioinformatic analysis, Directed mutagenesis experiments and High throughput expression and binding studies).

Nowadays, a growing number of innovative mAb therapeutics are on the global market including mAbs and rAbs.

Generic name of therapeutic antibodies

To finish, the names of the antibodies are not abstract concepts and must follow certain rules which make it possible to quickly identify their origin and their application.

Each mAb generated follows a rule determined by the WHO International Nonproprietary Names (INN) system of recombinant monoclonal antibodies which fixes the end of the name by a target infix and a source infix and the suffix of mab (see Tab. 1 and Fig. 5 below). This system is saturated and clinical considerations argue for an abandon of this system. Nevertheless, it really helps you to rapidly know  which type of antibody you have in hand.

Conclusion

To conclude, rAbs are the future of therapeutic antibodies and enable total control of quality, reproducibility, costs and time devoted to production, to easily convert the isotype and to eliminate the numerous ethical and animal welfare concerns commonly associated with traditional monoclonal antibody production.

Production and purification of rAbs requires genuine know-how, to be able to select the right sequence, expression system and purification strategy (including beads and buffers).

At our labs, we offer help base on our real knowledge acquired over the past years of how to produce quality proteins efficiently by taking care of their intrinsic biophysical characteristics. With our services, you get…

• Free in silico analysis of the protein sequence wished to determine the best way to proceed for the production and the purification of your antibody of interest including bioinformatics analysis of protein solubility, stability, but also the study of the impact of post-translational modifications, and the identification of metal ion binding sites and disorder zones …
• Cloning step in different expression vectors for production in prokaryotic and eukaryotic cells
• Amplification of the expression vector(s) by using endotoxin free process (Macherey Nagel’s kits) from mL to L(s)
• Multiplex production for variant expression or for buffer impact analysis
• Antibody / Protein production in small (few mL) to large batches (5L or more) in a plethora bacterial strains or in our human cell line (Hek 293 6E licensed by Yves Durocher specially optimized for high yield protein production)
• Antibody / Protein purification by using Automatized, gravity flow or magnetic systems and standard or innovative chromatography / approaches (Protein A / G, Mab select, NHS activated beads, Hydrophobic, Ion exchange and multimodal chromatography…) from one purification step to multiple purification steps depending on the purity required (up to >99% purity).
• Re-batches easily programmable
• Our Protocols can be easily transposable (when defined at the beginning of the study).
• Entire experiments and Quality controls totally customizable
• Delivery of all the protein produced and a production report including the strategy and the results obtained

Interested in learning more about recombinant monoclonal antibodies vs. monoclonal antibodies?
Don’t hesitate to contact us, or to learn how we can help you with successful antibody production!

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