To Be Humanized or Not to Be? That Is the Question for Camelid VHH Antibodies

This blog post was written by AvantGen, a pioneering biotechnology CRO dedicated to accelerating antibody-based therapeutics for their scientific partners by using next-gen and clinically validated antibody discovery and optimization platforms. For more information about AvantGen and its innovative work, please visit www.avantgen.com. Their services are available on the Scientist.com marketplace.

Antibody therapeutics have become a cornerstone in the treatment of various diseases, ranging from cancers to autoimmune disorders to infectious diseases. While traditional mammalian antibodies, primarily derived from mice, have been at the forefront of these advances, camelid antibodies have emerged as an exciting alternative. Known for their unique structural features and highly developable properties, camelid antibodies — specifically those derived from camels, llamas and alpacas — offer several distinct advantages. However, like any emerging therapeutic drug aiming to treat patients, both safety and efficacy are critical to navigate the clinical path. Antibody humanization, an engineering process to modify a non-human antibody to be more human-like to reduce immunogenicity, has been an established method for IgG mAbs for decades. The process to humanize camelid antibodies and even whether it is required, on the other hand, remains to be seen.

This article delves into the significance of camelid antibody humanization, the pros and cons and how to go about it.

What Are Camelid Antibodies?

Camelid antibodies, specifically the heavy chain only antibodies from the taxonomy family Camelidae, are structurally distinct from conventional mammalian antibodies. Unlike conventional IgG antibodies, which are composed of two heavy chains and two light chains, camelid antibodies lack a light chain and can even be recombinantly produced without the heavy chain Fc, creating a single variable domain of the heavy chain, also known as VHH, nanobody and single domain antibody (sdAb).

These antibodies have several desirable properties:
• Small size: At only about 15 kDa, VHH are significantly smaller than conventional antibodies (which are around 150 kDa). This small size allows them to penetrate tissues more easily and reach targets with unique epitopes that larger antibodies may struggle to access.
• Stability: Some VHH are generally stable at extreme pH levels, high temperatures and in the presence of denaturants. This stability makes them attractive candidates for drug development, particularly for therapeutic use in challenging environments.
• High affinity: Despite their small size, camelid antibodies often exhibit high specificity and affinity for their target antigens, making them effective tools for diagnostic and therapeutic applications.

The Need for Humanization

While camelid antibodies have impressive characteristics, their use in human therapies, as with all non-human biologics, may be hindered by one potential and complex issue: immunogenicity. When introduced into the human body, foreign proteins, especially those derived from non-human species like camels, can trigger an immune response. This response may reduce the efficacy of the therapeutic antibody and lead to adverse effects, including immunogenic reactions or the production of anti-drug antibodies (ADAs) against the biologic drug.

To mitigate this, camelid antibodies are often humanized before use in humans. Humanization is the process of modifying the camelid antibody to make it more similar to human antibodies, thus reducing its potential immunogenicity while preserving its unique therapeutic properties.

To Be Humanized?

1. Reduced Immunogenicity: Simply put, the main motivation of humanizing VHH antibodies is the reduction of potential immune responses when administered to human patients. By replacing portions of the VHH with human sequences, the body is less likely to recognize it as a foreign protein.
2. Efficient Production and Optimization: VHH can be produced in a variety of systems, including bacteria, yeast and mammalian cells. VHH humanization can also generate more optimal versions with improved expression, stability and even affinity.
3. High Target Affinity and Specificity: When done correctly, VHH retain their high affinity and specificity even after humanization. This ensures that the therapeutic effect remains potent and that they can be used for targeted therapies.
4. Cost-Effectiveness: Due to their small size, VHH humanization requires less exploration of sequences than IgG, allowing for the possibility of humanizing and even affinity maturing multiple VHH simultaneously with an affordable budget.

Or Not to Be?

1. Similarity to Human Antibodies: Interestingly, while the evolutionary divergence between humans, mice, rabbits and Camelidae family is estimated to have occurred around the same time of ~90 million years ago, most VHH have high amino acid sequence identity with human heavy chain VH3 family, and the VH3-23 subfamily is the most frequently used heavy chain in FDA-approved antibody therapeutics. The preexisting similarity between VHH and human VH is such that VHH is just as human-like in terms of sequence identity as a humanized murine VH sequence.
2. The Jury is still Out: Decades of drug treatment with humanized antibodies originating from mice have proven the significant benefit of reducing patient immunogenicity, especially when compared to older murine or chimeric antibodies. In contrast, there are only four approved VHH based drugs currently (all are humanized), and at least 35 in clinical trial with some non-humanized. Importantly, most VHH trials have revealed no or minimal immunogenicity, humanized or not.¹
3. Loss of Unique Properties: While each VHH is sequence-dependent, sometimes the amino acid residues that are not human-like, are also the key feature of its drug property. By modifying the sequence to help reduce immunogenicity, there is a risk that the therapeutic efficacy or developability of camelid antibodies may be reduced in the process. Of particular importance of VHH uniqueness is the framework-2 region, where a series of hydrophilic amino acids are present and different from human, and supports solubility of VHH in the region where a human VH normally interfaces with a light chain. Mutational analysis at each of these residues have shown different VHH will tolerate, and some will even be more stable from certain humanization mutations, while others lose their key therapeutic properties.²
4. Pharmacokinetics: How long a drug stays in a patient is an important feature of drug design, as longer is not always better for certain applications. While IgG based therapeutics typically have a half-life of several weeks, VHH range from hours to days due to their smaller size. This half-life exposure time is ideal for imaging and radiopharmaceuticals, and by limiting the drug presence, there is less potential for immunogenicity.
5. Additional Resources: VHH humanization requires additional time and cost, extending the drug development life cycle.

Methods of Humanizing Camelid Antibodies

There are several techniques used to humanize camelid antibodies, each with its own advantages and challenges:

1. Complementarity-Determining Region (CDR) Grafting using Structural and Residue Analysis: This conventional method carried over from humanizing other antibody species involves transferring the CDRs of the camelid antibody onto the closest matching human antibody framework by both sequence and structure. Each residue is then analyzed individually to determine if it should be retained as camelid, known as back mutations.
2. In vitro Display Technologies: Not mutually exclusive with the above method, in vitro display technologies with either phage or yeast can be employed for rapidly screening many variations of humanized sequences to select antibodies. Phage display has several disadvantages in therapeutic development compared to yeast display, which has been already addressed here. Libraries can be designed to also include changes in the CDRs to simultaneously affinity mature VHH, even combining multiple VHHs.
3. AI/ML and other In-Silico Prediction and Design Tools: Advances in computational tools and deep learning algorithms have enabled scientists with a plethora of tools to aid in VHH humanization, from basic sequence-based humanness and immunogenicity scoring to de novo antibody design. The accuracy of these models varies. One advantage of using deep learning models is they can potentially find higher order patterns and key features in human and camelid antibodies that are not on a residue-to-residue basis.

AvantGen uses a combination of all three above to rapidly screen in a therapeutic-relevant and novel yeast display system. Multiple VHHs can be simultaneously humanized and affinity matured in bulk, with consistent and significant affinity and development improvements.

Alternatively, you can avoid the humanization process altogether by using a humanized VHH discovery library. To learn more about AvantGen’s AvantGeneer and AvantFoil VHH discovery and optimization platforms and services, please contact partner@avantgen.com or connect via the Scientist.com link below.

Connect with AvantGen on Scientist.com

References

1. Rossotti, M.A.; Bélanger, K.; Henry, K.A.; Tanha, J. Immunogenicity and Humanization of Single-domain Antibodies. FEBS J. 2022, 289, 4304 – 4327.
2. Vincke, Cécile et al. General Strategy to Humanize a Camelid Single-domain Antibody and Identification of a Universal Humanized Nanobody Scaffold. Journal of Biological Chemistry, Volume 284, Issue 5, 3273 – 3284.