Introduction to Antibodies

An antibody is a Y-shaped protein/glycoprotein produced by the immune system against pathogenic invaders such as bacteria, viruses and even fungi. Antibodies, also known as immunoglobulins, are produced by specialized cells of the adaptive immune system.

Plasma cells, which are differentiated B cells, produce antibodies after the activation of the immune system in response to antigens. Below is a high-level sequence of biological events that leads to the production of antibodies.

• Alien substances or molecules (i.e., antigens) enter our bodies.
• The immune system recognizes the antigens as non-self.
• B cells bind to the antigens using receptor proteins on their cell surfaces, divide, then form mature clones (a group of identical cells). Alternatively, antigen presenting cells (APCs) can engulf the antigen and present fragments to the B cell for activation which triggers the previous events.
• Plasma cells (mature B cells) release countless antibodies into the bloodstream that start circulating in the body, recognize antigens, bind them through their epitope, and neutralize their potentially harmful actions in our bodies.

Antibodies can be found in bodily fluids, secretions, circulating in the bloodstream and on the surface of B-cells. The tip of the antibody contains a paratope that identifies and binds to the specific epitope present on a corresponding antigen.

An antibody has multiple variable regions (antigen binding sites) on both tips of its Y-branch, enabling our body to recognize a plethora of antigens entering our body. An antibody also contains a constant region where the fragment crystallizable (Fc) region is found. The constant region of the antibody is used to identify the different isotypes of immunoglobulins, such as IgA, IDA, IgE, IgM and IgG.

Various analytical techniques are used to study the structure of antibodies in labs, such as X-ray crystallography, chromatography, mass spectrometry, peptide mapping and protein sequencing. These techniques also inform on the isotype (antibody class) and provide data for their intended application.

In addition to natural antibodies produced by our body, researchers can now design several antibodies (like monoclonal antibodies) in labs to treat life-threatening diseases, such as cancer and autoimmune disorders.

There are also polyspecific monoclonal antibodies, such as bi-specific and tri-specific antibodies, that are designed to target multiple epitopes simultaneously in tumor microenvironments. However, their complex structures and potential for immunogenicity make their development, manufacturing and approval challenging.

While developing bi-specific or tri-specific antibodies can be challenging, there are solutions available to help ease the burden for error prone and resource intensive processes, such as for stable cell line development.

Here is a resource to learn more about the effective and efficient monoclonal antibody development steps.

This article provides deeper insights into the structure and functions of the five classes of antibodies, their applications in medical and biotech research settings, and the challenges in producing each antibody type for clinical applications.

Basic Structure of Antibodies

The Y-shaped structure of a human antibody molecule consists of four polypeptides, two light chains, and two heavy chains, both containing constant and variable regions. The heavy chain has four domains, while the light chain has two domains that are involved in identifying and binding different antigens.

Two types of light chains found in antibodies include lambda (λ) and kappa (κ), whereas the heavy chain has five isotypes, which include μ, δ, γ, α, and ε, respectively.

Here are some structural features of an antibody:

• Antigen-binding site: The antigen-binding site can be found in the variable fragment region or Fv region. It is a subset of the Fab (fragment antigen-binding) region, characterized by three beta-strand loops that interact with the structure of an antigen. These loops, referred to as complementarity-determining regions (CDRs), play a crucial role in determining the antibody's ability to bind to a specific antigen.

• Fc (fragment crystallizable) region: It contains constant heavy chain domains. The regions have conserved glycosylation sites that influence the interaction of antibodies with effector molecules. The effector molecules bind to the Fc regions to activate the complement system to fight pathogens and microbes.

• Protein structure: The antibody structure has two terminals, the N-terminal (the tip of the antibody) and the C-terminal, the end of the trunk of the antibody. The Y-shaped structure consists of 7 to 9 strands arranged in two beta sheets. These sheets are held together by a disulfide bond to form the immunoglobulin fold.

• Antibody complexes: Antibodies can exist in different forms, such as single units or larger complexes. Some antibodies are monomers resembling a Y shape. Nevertheless, certain classes of antibodies can assemble as dimers (consisting of two units, as seen in IgA), tetramers (composed of four units, observed in teleost fish IgM), or pentamers (composed of five units, as seen in mammalian IgM).

Antibody Isotypes & Their Structure

The structural differences in the constant region of antibodies determines their class and divides them into the following five categories:

1. IgG Antibody

IgG has a tetrameric quaternary structure consisting of four polypeptide chains. IgG contains two light chains of 25 kDa and two γ (gamma) heavy chains of 50 kDa. It’s involved in various immune responses, such as triggering the complement system, neutralizing bacterial toxins, and can cross the placenta.

It’s one of the most predominant antibodies in our body, with a molecular weight of 150,000 Da (or 150 kDa). Around 80% of serum Immunoglobulin is IgG. It also has the longest half-life of 23 days, compared to other antibody classes.

IgG structure has 2 gamma (γ) heavy chains (H-chains) and 2 lambda (λ) or 2 kappa (k) light chains, thus having two identical antigen-binding sites. The antibody molecule is further categorized into four subclasses, IgG1, IgG2, IgG3, and IgG4, based on the small differences in their H-chains.

The IgG molecule can cross the placenta to provide immunity to the developing fetus. Further, this molecule has a role in initiating secondary immune responses and fighting diseases and infections such as HIV.

2. IgM Antibody

IgM has a pentameric (and often hexameric) structure, consisting of five basic Y-shaped units bonded together by disulfide bonds and a joining (J) chain. This creates 10 antigen-binding sites that are especially effective in binding antigenic determinants present on the outer surface of the bacteria.

It’s the first antibody produced by the mature B cell after the initiation of an immune response. IgM antibody molecules play a role in neutralizing and eliminating pathogens and initiating inflammatory responses through the complement pathway.

IgM constitutes about 5-10% of the total serum Immunoglobulin, with an average concentration of 1.5mg/dl.

3. IgA Antibody

The IgA antibody molecule has an alpha H-type chain with IgA1 and IgA2 subclasses. IgA is typically present as a monomer in serum; however, it can also form dimers through disulfide bonds and a J-chain. It also includes a secretory component connected to the dimer’s second constant domains through disulfide bonds.

It is a predominant immunoglobulin protein in external secretions, such as saliva, tears, digestive tract and colostrum (lactating mother’s first milk). Besides providing a first-line defense against pathogens, the antibody also limits inflammation.

IgA plays a crucial role in protecting mucosal surfaces from toxins, bacteria, and viruses by directly neutralizing them or preventing their binding to the mucosal surface.

4. IgD Antibody

This antibody is a monomer consisting of two delta (δ) heavy chains and two light chains. It’s either present in free form in serum, constituting less than 1% of total serum immunoglobulins, or bound to B cell through the Fc receptor. It has a crucial role in the induction of B cell antibody production, activates the immune system’s pro-inflammatory infections and preventing respiratory tract infections.

5. IgE Antibody

The IgE antibody molecule is structurally a monomer with two heavy chains (ε chain) and two light chains. Despite its low serum concentration and short half-life, the IgE isotype is a highly potent immunoglobulin. Like the IgG molecule, each IgE molecule is composed of a four-chain unit, providing two antigen-binding sites. However, what sets IgE apart is the presence of an additional constant domain (CH4) on each of its heavy chains. This unique feature enables IgE to attach themselves to the surface of basophils and mast cells specifically.

With its distinctive structure and diverse physiological functions, IgE is involved in Type I hypersensitivity reactions, defense against parasitic infections, autoimmune processes and venom protection.

Antibody Glycosylation

Antibodies undergo modifications after translation, and one of the most common modifications is antibody glycosylation. Glycosylation happens at multiple sites and residues on the antibody with each impacting various antibody mediated functions.

Antibody glycosylation influences:

• The binding of the antibody with effector molecules
• Antibody-dependent cell-mediated cytotoxicity (ADCC) functions
• Complement-dependent cytotoxicity (CDC)

By changing the glycoforms attached to antibodies, scientists can create antibodies with specific characteristics required to achieve therapeutic efficacy.

Clinical Antibody Applications

Therapeutic antibody technology is a dominant force in the biologics market, driving significant interest in developing novel and improved antibody treatment strategies. Here are a few applications of antibodies extensively used in clinical settings:

• Current therapeutic monoclonal antibodies in clinics are primarily of the IgG isotype. IgG antibodies can induce cancer cell killing through various mechanisms: apoptosis induction, growth inhibition, complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC) by NK cells, and antibody-dependent cellular phagocytosis (ADCP) by monocytes/macrophages.

• IgA antibodies are touted for high effectiveness in tumor eradication. It’s been actively tested in clinical settings for anti-cancer therapeutic applications.

• In the field of clinical immunology, analyzing a patient's antibody profile helps diagnose certain diseases, such as an elevated IgM level indicating viral hepatitis.

• Utilizing monoclonal antibodies also enables scientists to bind and purify specific proteins that exhibit a singular antigenic determinant.

• Radioimmunotherapy is a treatment method that uses special antibodies combined with radioactive materials to target and destroy specific cells in the body. The radioactive materials, like iodine-131 and yttrium-90, emit a type of radiation that can harm both the targeted cells and nearby cells that the antibodies cannot reach directly. This approach helps attack cancer cells effectively, even those deep within solid tumors.

Antibody Isotype Production Challenges

Therapeutic approaches utilizing monoclonal antibodies have experienced notable progress. The modification of their format, production techniques, and functionalization has significantly enhanced their effectiveness, especially in the field of oncology.

IgG antibodies have different isotypes approved as a monoclonal antibody (mAb) therapy. The commonly used isotypes include IgG 1, IgG 2, IgG 4, or hybrid 2/4. The short lifespan of IgG3 poses challenges while the longer hinge region increases complexities involved in bioprocessing.

Growing evidence supports the potential of IgA or anti-FcαRI as promising therapeutic approaches due to superior tumor killing capabilities compared to IgG isotypes. However, they suffer several limitations, such as:

• A high heterogeneous glycosylation profile
• The relatively short serum half-life
• Production and purification challenges

Similarly, glycosylation challenges with producing IgE antibodies have prevented their use in the clinic. Selecting an appropriate cell line expression system and minimizing the addition of foreign glycans are important steps to mitigate potential immunogenicity risks.

Scientists are further exploring other antibody isotypes for their efficient production and therapeutic applications.

Future Perspectives

Despite significant advancements in antibody engineering methods over the past two decades, there is a continued demand for new approaches.

Computational methods to predict point mutations that enhance affinity and other desirable product quality attributes continues to get lots of attention. Ongoing efforts focus on developing novel strategies to create antibody-based molecules with superior potency, specificity, localization and safety.

Modifications to the binding arms, Fc regions, and development of bi-specific, multispecifics, and antibody-drug conjugates will continue to experience rapid growth. However, process improvements to address their unique production challenges will be needed, including the development of analytical assays and release testing criteria.

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