News & Blog: Article

How to Make a Great Antibody

Contributed by Will Fry, Ph.D.
(Dr. Fry, President of Antibodies Incorporated, helped build our Custom Antibody Development service.)

We expect a lot out of our antibodies.

They are critical reagents in scientific research, allowing us to study protein localization, regulation, and abundance. They are central to the diagnostics industry. And little over three decades after the first therapeutic monoclonal antibody was approved by the FDA, antibody-based therapeutics now dominate the pharmaceutical market.

So how do we go about making a good one?

First and foremost, begin with the end in mind. You must know exactly what you expect from your antibody before trying to make it. Does your antibody need to distinguish between several highly homologous protein family members? Or perhaps it must bind to a multi-pass membrane protein in intact cells. Whatever the requirement, it’s important to first know a little bit about how the immune system makes antibodies.

How the immune system makes antibodies

Lymphocytes called B cells present B cell receptors ("BCRs") on their cell surface. Each BCR consists of three components: (1) the classic Y-shaped antibody molecule that faces out into the extracellular space; (2) a transmembrane domain that tethers the antibody region to the plasma membrane; and (3) the non-covalently associated Igα/Igβ (CD79a/CD79b) heterodimers which transduce signals from the BCR binding to the cellular interior.

Each B cell produces a single B cell receptor and presents many copies on its surface. Since there are greater than 107 unique B cells that circulate in the blood, there consequently is a vast diversity of unique BCRs.

So what are all these BCRs for? BCRs bind to antigen molecules (antigen stands for antibody generating). Antigens can be any molecule capable of binding to BCRs including lipids, carbohydrates, nucleic acids, proteins, small molecules among others. Incidentally, "self-antigens" (i.e. molecules found in our own body) are typically not recognized by BCRs, as this would lead to autoimmunity. These BCRs are eliminated from the immune system during development through a process called immune tolerance.

BCR-displaying B cells constantly come into contact with foreign molecules. When a specific BCR encounters a foreign antigen for which it has high affinity, the B cell (on which the BCR is presenting) is stimulated to survive and proliferate. B cells do not act alone in this response, however. A variety of different lymphocytes, including dendritic cells, macrophages, T cells, etc., cooperate with the B cell to mount an effective immune response.

The antigen will determine if a robust immune response is elicited.

So what makes a good antigen?

Antigens have two critical determinants:
  1. a structural feature called a "B cell epitope" that can be recognized by a BCR. This can be a small molecule, peptide, or protein but does not need to be protein-based; and
  2. a "T-cell epitope" that constitutes a linear peptide and must be displayed by B cells to elicit help from T cells.
These determinants act together thus:
  • A B cell epitope binds with some affinity to the BCR and stimulates proliferation of the B-cell.
  • Upon binding of the antigen to the BCR, the antigen/BCR complex is internalized and degraded within the B cell.
  • Proteolytically cleaved fragments of the antigen are then displayed on the surface of the B-cell as T cell epitopes.
  • These are recognized by helper T cells, which then further stimulate the proliferation and maturation of these specific B cells.
  • The mature B cells then begin releasing the BCR in a soluble format, without the transmembrane domain, as antibodies.

Without T cell help, the B cells cannot mature to produce high-affinity antibodies.



This file is licensed under the Creative Commons Attribution 4.0 International license.

Above: B cell maturation.
Binding of a naive B cell BCR to a bacterium-derived foreign antigen leads to internalization of the antigen, degradation, and display of small protein fragments (T cell epitopes) on the surface of the naïve B cell. A T helper cell with a T cell receptor that is specific for the displayed fragment (T cell epitope) binds to the presented fragment, thereby connecting the B cell and T cell. This stimulates the T cell to release cytokines that promote differentiation of the B cell into memory cells and antibody producing plasma cells.


A good antigen must have both a B cell and a T cell epitope. The B and T cell epitopes do not need to both be present on the same molecule (i.e. could be present on separate molecules in a multi-subunit complex), but both need to be physically connected, such that when the antigen is bound by the BCR, the T cell epitope is internalized into the B cell and displayed on the B cell surface. In some cases, the B cell epitope and the T cell epitope are the same (this is true for certain immunogenic peptides); however, in most cases the B cell epitope is a structural feature on one part of the antigen, while the T cell epitope is a peptide sequence that binds specifically to a T cell receptor.

How to make bad antigens good

It only makes sense that a viral protein should be immunogenic. After all, the immune system evolved to fight off infectious disease and pathogens. Viral proteins are naturally foreign to the host they invade, and so these antigens possess both a B cell epitope and a T cell epitope and elicit vigorous antibody-based immune responses.

Antibodies can also be made against small molecules such as drugs of abuse and steroid hormones. How are these made? Small molecules that are not protein-based often have a B cell epitope but not a T cell epitope. Therefore, these small molecules cannot, by themselves, elicit a robust immune response. To provide the missing T cell epitope, we chemically couple (i.e., conjugate) these small molecules to an immunogenic carrier protein that provides the needed material. When introduced to the immune system, the small molecule portion of these conjugates is bound by the B cell receptor. The conjugate is then internalized into the B cell, proteolytically degraded, and then the peptide fragments derived from the carrier protein are displayed on the B cell surface as T cell epitopes to elicit help from T helper cells. Similarly, peptides that encode a discrete region of a protein of interest are also conjugated to immunogenic carrier proteins to create proper antigens.

As biologists, we generally want to make antibodies against proteins. Often these proteins are highly conserved, and this presents some challenges. For example, if we try to make a mouse monoclonal antibody against a mouse protein or a highly-conserved protein, it is likely that the B cells of the mouse will not bind with high affinity to the antigen due to immune tolerance (i.e. these B cells may have been eliminated during development). Similarly, even if the B cells bind and internalize the antigen, it is unlikely that the mouse protein (or highly-conserved protein) will have T cell epitopes that will be recognized by T helper cells once presented on the B cell surface.

How to make good antibodies against conserved proteins

To overcome this challenge, we have two approaches at our disposal:
  1. alter the antigen; and
  2. alter the host animal.

These two approaches are frequently used together in order to optimize the immune response.

There are many examples of mouse monoclonal antibodies being generated against mouse proteins. If a recombinant mouse protein is being used as the immunogen, it can be conjugated to a carrier protein or expressed as a fusion protein. Either can be designed to contain the necessary T cell epitopes.

As an example of the second approach, the specialized mouse strain NZB/W autoimmune mouse model has impaired immune tolerance. By using such a mouse model along with an antigen engineered to contain T cell epitopes, even a mouse protein can become a potent immunogen. Of course, selection of an alternative host, such as chicken, rabbit, or goat, can also help ensure that the protein antigen appears more foreign to the host immune system and meets the two criteria above for optimizing immunogenicity of the antigen.

Other Key Pieces of the Antibody Puzzle

Adjuvants

A necessary component of any antibody project is a carefully selected adjuvant. Co-injected into the host animal along with the antigen, the adjuvant performs two key tasks that ultimately stimulate the immune response:
  1. concentrates the antigen near the injection site and prevent its rapid dispersal and degradation; and
  2. stimulates the innate immune response leading to increased phagocytosis (uptake) of the antigen into immune cells such as macrophages and dendritic cells.

There are a variety of adjuvant options, and selection depends upon the nature of the antigen. For example, native proteins sensitive to denaturation may require one type of adjuvant, whereas small molecule conjugates may perform best with another type. Similarly, multi-pass membrane proteins present in a membrane fraction should not be mixed with adjuvants that could solubilize the membrane and alter the protein topology.

Antibody Screening

Particularly for monoclonal antibodies, where one can choose from hundreds of possible clones, a carefully considered and robust screening methodology is essential to success. As an example, if you are looking for an antibody that must recognize the native conformation of your protein of interest, then a screening assay must be set up to identify "hits" that fit this criteria. By contrast, if you are looking for an antibody that recognizes your protein of interest but not highly-related family members, then counter-screening, or a depletion strategy should be introduced as early as possible into the project.

Conclusion

It has been said that antibody development is more art than science. Perhaps closer to the truth is that, as with the crafting of anything of intricacy, producing an antibody of outstanding quality requires an intimate understanding of the science underlying key factors like those described above, and a technical proficiency at applying that understanding that can only come from decades of experience at the bench. We at Antibodies, Aves and PhosphoSolutions certainly think so!

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