Antibody Humanization Techniques

First Things First: Understanding Antibody Structure

Before delving into humanization techniques, let’s quickly review the basic structure of an antibody. Antibodies, also known as immunoglobulins, are Y-shaped proteins consisting of two identical heavy chains and two identical light chains. These chains contain regions crucial for antigen recognition called the variable regions, which are further divided into variable heavy (VH) and variable light (VL) chains. 

In each variable domain (VL and VH), three non-adjacent loops exist, known as complementarity-determining regions (CDRs) or hypervariable regions. These loops directly engage with an antigen. Therefore, the combination of CDRs forms the antigen-binding site, enabling antibodies to recognize and bind to specific targets, such as pathogens, toxins, allergens, or cancer cells.

Antibodies also contain constant regions, which vary based only on antibody class (IgG, IgA, IgM, IgE, and IgD), not between individual antibodies (like variable regions). These regions provide the antibody with unique effector functions such as binding specific receptors on immune cells, activating complement proteins, or facilitating the destruction of pathogens.

All antibody classes have one constant region on each light chain designated as CL. However, there are multiple constant regions for each heavy chain. For instance, the antibody classes IgG, IgA, and IgD have three constant regions on each heavy chain designated as CH1, CH2, and CH3, while IgM and IgE have four constant regions designated as CH1, CH2, CH3, and CH4. Together, the variable and constant regions assemble into tiny yet mighty tailor-made warriors that combat disease with precision and grace

The Need for Humanized Antibodies: Why Is Humanization of Antibodies Important in Monoclonal Antibody Engineering?

Monoclonal antibodies used to be exclusively made by immunizing animals such as mice. However, due to their non-human origin, murine antibodies have limited efficacy and safety as human therapeutic antibodies because they elicit immune responses in humans. However, making human monoclonal antibodies by immunizing humans is prohibitive. 

To overcome this limitation, scientists developed humanization techniques to make the murine antibodies “look” more like human antibodies while retaining their therapeutic efficacy. Humanized antibodies maintain the antigen-binding specificity of the parental antibody while minimizing the risk of immune rejection. 

This transformative approach mitigates the risk of adverse immune reactions, offering a safer passage for therapeutic antibodies into the human body. 

• Reduced Immunogenicity: Non-human antibodies can be recognized as foreign by the human immune system, leading to an immune response that can neutralize the therapeutic effects of the antibody and potentially cause adverse reactions. Incorporating human constant regions into the non-human antibodies minimizes the risk of triggering an immune response when administered to patients.
• Improved Pharmacokinetics: Humanized antibodies exhibit longer half-lives in circulation, enhancing efficacy and reducing dosing frequency.
• Enhanced Safety Profile: Humanized antibodies have decreased immunogenicity and are associated with fewer adverse reactions. This improves clinical safety and makes them suitable for a wide range of patients, including those with sensitive or compromised immune systems.
• Facilitating Regulatory Approval: Humanized therapeutic antibodies are more likely to be approved by regulatory agencies because they are considered safer and more effective than non-humanized antibodies.
• Broadening Therapeutic Applications: Humanized antibodies expand the potential impact of antibody therapies as they can be used in a variety of therapeutic areas, including oncology, autoimmune diseases, and infectious diseases.

How Do You Humanize Antibodies? The Humanization Process Explained

By meticulously modifying antibody structures to mirror those naturally occurring in humans, researchers are not just altering genetic codes; they’re sculpting precision medicine. Antibody humanization modifies non-human antibodies to make them more similar to human antibodies.

There are varying degrees of antibody humanization, leading to differences in the extent to which antibodies resemble human antibodies. For example, mouse antibodies can be humanized by simply swapping the mouse constant regions for human constant regions, or the mouse complementarity determining regions (CDRs) can be transferred to a human antibody scaffold (90% human). 

The former is more likely to preserve the overall structure and function of the antibody. This process reduces the risk of immune reactions when these antibodies are used in humans for therapeutic purposes.

What Is the Difference Between Human and Humanized Antibodies?

The difference between human and humanized antibodies lies in their origin and the extent of genetic modification. Human antibodies are produced by the human immune system or by transgenic mice genetically engineered to contain the human immunoglobulin gene locus. Humanized antibodies, on the other hand, are originally generated from non-human sources, such as mice, and then modified.

So, the key distinction is that human antibodies are entirely derived from human gene sequences, whereas humanized antibodies are initially non-human but are modified to closely resemble human antibodies, with the goal of reducing the risk of immune reactions when used in therapeutic applications. 

Each method has its advantages and disadvantages, often balancing between maintaining a high affinity for the target antigen and reducing immunogenicity.

Antibody Humanization Methods & Techniques

Humanization of antibodies is not a one-size-fits-all approach but rather a meticulous process often involving mutational analysis and iterative refinement. Scientists evaluate various humanization protocols and strategies based on factors such as humanness scores, which assess the similarity of the humanized antibody to natural human antibodies, and immunogenicity profiles.

This trial-and-error process aims to minimize immunogenicity while maximizing therapeutic efficacy. Over the years, several methods and approaches have been developed to achieve this, each with its unique approach to reducing immunogenicity and enhancing compatibility with the human immune system.

1. Chimeric Antibodies

The initial strategy to decrease human antigenicity involved creating chimeric antibodies. This process involves fusing the variable region of mouse antibodies with the constant region of human antibodies. The result? An antibody is predominantly human in composition, with about 70% of its structure derived from human amino acid sequences.

These chimeric antibodies maintain the original mouse antibody’s specificity and binding strength while substantially lowering the likelihood of a human anti-mouse antibody (HAMA) reaction. Although some chimeric antibodies are in clinical use, it’s crucial to remember that their variable regions, originating from mice, could still elicit HAMA responses.

2. CDR-Homology-Based Humanization

This antibody humanization technique involves grafting CDRs (parts of the antibody that bind to the antigen) from a non-human antibody (such as mouse CDRs) into human antibody framework regions (FR) that have been selected for their homology to the original non-human framework. However, this method may occasionally reduce the antibody’s binding strength or its selectivity towards the intended antigen, thus more research is necessary.

3. Germline Humanization

Here, we dive into the genetic ancestry of humans, selecting antibody frameworks from human germline sequences. In this approach, the variable regions of a non-human antibody are modified to closely resemble human antibody variable regions by using human germline sequences. It’s like choosing an heirloom that’s been passed down through generations, ensuring compatibility and heritage.

4. Specificity Determining Residues (SDR) Grafting

The SDR is the crucial CDR residue. Thus, SDR grafting involves identifying and transferring only the key residues that dictate the antigen-binding specificity from the non-human antibody to a human framework. It’s like pinpointing the exact brush strokes that define a masterpiece, ensuring the antibody maintains its unique ability to identify and neutralize specific pathogens.

5. Resurfacing Approach

The resurfacing method strategically alters the surface amino acid residues of a non-human antibody to mirror those found on human antibodies. This approach is predicated on the insight that a protein’s immunogenicity is largely attributed to its surface residues, so it focuses on the regions that are most likely to be recognized by the human immune system.

By selectively modifying these exterior residues to resemble human variants, the antibody’s potential to elicit an immune response is diminished while its antigen-binding function remains intact, ensuring that its affinity for the antigen is not compromised. 

6. Transgenic Mouse

By introducing human immunoglobulin genes into the mouse genome, researchers can create transgenic mice that produce antibodies with human-like properties. These mice, known as “humanized” or “HumAb” mice, are capable of mounting an immune response that generates fully human antibodies.

The technique involves sophisticated genetic engineering, where the mouse’s own antibody-producing genes are replaced with human versions. When immunized with a target antigen, the mice produce a diverse repertoire of human antibodies. The antibodies produced can then be harvested and further refined for therapeutic use.

This method has been instrumental in the development of monoclonal antibody therapies, providing a more efficient and ethically favorable alternative to traditional methods of antibody production.

What Are the Applications of Humanized Antibodies?

Humanized antibodies have found widespread clinical application across diverse therapeutic areas, including oncology, autoimmune diseases, infectious diseases, and beyond. They have demonstrated remarkable success in targeting specific molecules involved in disease pathogenesis, offering precision treatment options with reduced side effects compared to traditional therapies in a range of applications.

Therapeutic antibody development has surged since the first licensed immunotherapy in 1986. The FDA’s increasing approval rate for monoclonal antibody drugs, particularly humanized and fully humanized variants, underscores a pivotal shift in the market.

With all monoclonal antibodies approved in 2021 originating from human sources, it’s evident that future clinical trials and antibody technology will be predominantly shaped by these safer and more efficacious antibody formats as therapeutic agents.

Harnessing Antibody Humanization for Tomorrow’s Therapies

Humanization techniques are truly a groundbreaking advancement in the field of therapeutic antibodies, unlocking the full potential of monoclonal antibodies in treating various diseases. By bridging the gap between murine and human immune systems, humanized antibodies offer a promising avenue for precision medicine, providing targeted therapies with improved safety and efficacy profiles.

As research in antibody engineering continues to evolve, humanized antibodies will undoubtedly remain at the forefront of therapeutic innovation, offering hope to patients worldwide. At ProteoGenix, we recognize the pivotal role this step plays in realizing your therapeutic ambitions. With our monoclonal antibody humanization services, a team of recognized experts in the field and over two decades of proven track record, we’re here to help. 

Book a call now – together, we’ll navigate the complexities of antibody humanization and work on bringing your vision to life.