News
Release date:2023/10/25 16:10:20

Nanobodies are the recombinant variable domains of heavy-chain-only antibodies (HcAbs) naturally found in Camelids and Sharks. Nanobodies have a molecular weight of only 15 kDa, about 1/10 the molecular weight of conventional antibodies, making them the natural antibodies with the smallest known molecular weight. Nanobodies are structurally different from traditional antibodies, showing higher affinity and stability, microbial expression, low immunogenicity, good water solubility, and strong tissue penetration, etc., and have attracted much attention in the fields of disease treatment, diagnosis, and substance detection.

Nanobodies Structural Features

Camelid HCAb consists of a fragment crystallizable region (Fc), directly linked to a Fab fragment consisting of a single VHH domain. Due to the absence of light chains as well as the CH1 region the molecular weight of the nanobody is reduced by 90 kDa compared to that of conventional monoclonal antibodies. The VHH fragment of HCAb, with a size of about 2.5*4 nm and a molecular weight of about 15 kDa, binds a broad antigen repertoire. Similar to the variable region (VH) of conventional antibodies, nanobodies (Nbs) consist of four conserved framework regions (FR) and three hypervariable complementarity-determining regions (CDR) responsible for determining antigenic specificity.

At the same time, there are a series of differences between the nanobodies and the VH fragments of monoclonal antibodies. Firstly, the nanobodies bind to the antigen through three CDR regions due to the lack of light chain, whereas monoclonal antibodies usually require six CDR regions to bind to the antigen. Structurally, the structural variation of CDR1 and the increased length of CDR3 of the nanobodies greatly contribute to their antigen-binding versatility, despite the absence of the light chain. In addition, the FR2 of the VH domain of conventional mAb mainly consists of hydrophobic amino acid residues, i.e., V37/G44/L45/W47, whereas that of nanobodies usually consists of hydrophilic amino acid residues, i.e., F37/E44/R45/G47, which also allows them to exist in the form of soluble monomers.

Diagrammatic representation of the structure of conventional m(IgG)Ab, HCAb, and VHH (Nb)

Figure 1. Diagrammatic representation of the structure of conventional m(IgG)Ab, HCAb, and VHH (Nb) [1]

Nanobodies: Advantages & Disadvantages

The unique structure of nanobodies offers several advantages over traditional monoclonal antibodies.

Higher Tissue Penetration: The larger size of mAbs and the presence of the Fc region improves pharmacokinetic properties but also affects their tissue penetration ability. Smaller sizes of Nbs achieve higher tissue penetration and greater cell killing in vivo.

Enhanced antigen recognition: Of all the complementary decision regions, CDR3 accounts for 60-80% of the antigen recognition specificity.The long and extended CDR3 loop confers better antigen recognition specificity and affinity to VHH, which enhances the recognition of hidden tumor antigenic epitopes.

Strong Stability: Compared to mAbs, Nbs have significantly higher thermal stability, Tm values and reversible thermal denaturation. Besides, Nbs are resistant to the denaturing effects of proteases, extreme pH and hydrophobic agents.

Ability to tandem into multispecific or multivalent configurations: the small size of Nbs compared to mAbs facilitates tandeming with multiple functionally distinct antibody domains for multifunctional drug delivery, enhancing affinity while avoiding rapid renal clearance and increasing drug ratios.

Although nanobodies have many desirable properties, certain limitations also exist. Since the threshold for glomerular filtration is 50-60 kDa, the small size of 15 kDa results in low serum duration or rapid renal clearance, thus presenting a disadvantage in diagnostic screening and therapeutic applications. One solution strategy is to couple nanobodies to polyethylene glycol (PEG) or albumin. Secondly, nanobodies lack the Fc region and therefore cannot perform the effector function associated with this part. However, this problem can be solved by coupling to the Fc region to increase the therapeutic capacity.
Biopharma PEG, as a leading PEG supplier, provides you high purity PEG derivatives for your nanobody drug development.

nanobodies-features

Figure 2. Advantages of nanobody drugs [2]

Current Status of Nanobodies

Compared to the hotly researched mAb drugs, nanobodies are currently receiving less attention. Currently, only four nanobody-based therapies have been approved worldwide.

In 2018, the world's first nanobody Caplacizumab (Cablivi ) was approved in the EU for the treatment of acquired thrombotic thrombocytopenic purpura. And subsequently, in 2019, it was approved by the FDA. It is a genetic fusion of two identical nanobodies against the van Willebrandt factor linked via a peptide linker.

In 2021, Envafolimab (KN-035), developed by Alphamab Oncology, was approved in China for adult patients with microsatellite instability-high (MSI-H) or mismatch repair deficient (dMMR) advanced solid tumors. It is the first and only globally approved subcutaneously injectable PD-L1 antibody. 

In 2022, Ozoralizumab (Nanozora), a trivalent anti-tumor necrosis factor alpha (TNFα) NANOBODY compound, was approved in Japan for the treatment of rheumatoid arthritis.

On Feb 28, 2022, Ciltacabtagene autoleucel (cilta-cel),  a nanobody-based CAR-T cell therapy developed by Legend Biotech and Janssen, was approved by FDA for the treatment of relapsed/refractory multiple myeloma.

approved-nanobody-drugs

Figure 3. Approved Nanobody based therapies

Besides, there are more than 20 nanobody drugs in clinical trials. Ablynx, a giant in nanobody drug discovery and development, has as many as 40+ drug candidates with applications in many different areas such as cancer, autoimmune diseases, respiratory diseases, and hematologic diseases.

Product Name Disease and/or Condition Targeted Target Antigen Clinical Trial Status Manufacturer
Calplacizumab Acquired thrombotic thrombocytopenic purpura Von Willebrand factor Approved
(USA, EU)
Ablynx, Ghent, Belgium
Envafolimab MSI-H or dMMR advanced solid tumors PD-L1 Approved
(China)
Alphamab Oncology
Ozoralizumab Rheumatoid arthritis Tumor necrosis factor-alpha Approved
(Japan)
Taisho Pharmaceuticals, Tokyo, Japan
Ciltacabtagene autoleucel, LCAR-B38M Refractory/relapsed multiple myeloma B-cell maturation antigen Approved
(USA) 
Janssen Research & Development, LLC, Raritan, United States
Gefurulimab (ALXN1720) Myasenthia Gravis Autoantibodies against acetylcholine receptors Phase 3 Alexion Pharmaceuticals, Boston, United States
Vobarilizumab (ALX-0061) Rheumatoid arthritis, systemic lupus erythematosus Interleukin-6 Phase 2 Ablynx, Ghent, Belgium
68-GaNOTA-Anti-HER2 VHH1 1 Breast carcinoma HER2 Phase 2 Universitair Ziekenhuis Brussel, Brussels, Belgium
Sonelokimab (M1095) Psoriasis Interleukin-17A/F Phase 2 Bond Avillion 2 Development LP, London, England
Nb V565 Crohn’s Disease Tumor necrosis factor Phase 2 VHsquared Ltd., Copenhagen, Denmark
ARP1, VHH batch 203027 Diarrhea Rotavirus Phase 2 International Centre for Diarrhoeal Disease Research, Dhaka, Bangladesh
ALX-0171 Lower respiratory tract infection Respiratory syncytial virus Phase 2 Ablynx, Ghent, Belgium
LMN-101 Campylobacteriosis Campylobacter jejuni Phase 2 Lumen Bioscience, Inc., Seattle, United States
M6495 Osteoarthritis A Disintegrin and Metalloproteinase with Thrombospondin Motifs-5 Phase 1 Merck KGaA, Darmstadt, Germany
131I-GMIB-Anti-HER2-VHH1 Breast carcinoma HER2 Phase 1 Precirix, Brussels, Belgium

Table. List of nanobody drugs in clinical trials [1]

Applications of Nanobodies

1. Nanobodies for Diagnostic Applications

Nanobody-based immunoassays can be fully utilized to detect and identify targets as well as foreign pathogens or toxins that are more difficult to detect clinically. For example, EGFR is overexpressed in many tumors and is an attractive target for tumor drug targeting. Nanobodies targeting EGFR have been developed and successfully applied to the diagnosis of breast, ovarian, and prostate cancers. In addition, nanobodies are used in molecular imaging techniques such as positron emission tomography (PET), single photon emission computed tomography (SPECT), near-infrared fluorescence (NIR), and ultrasound molecular imaging for disease diagnosis.

nanobody-Diagnos

Figure 4. Nanobodies for Diagnostic Applications [3]

2. Nanobodies for Therapeutic Applications

The high affinity and specificity of nanobodies make it easier to target binding to receptors on cancer cells at the site of obstruction and penetrate into less vascularized tissues. Meanwhile, given the low immunogenicity of the nanobody, repeated administration of the nanobody to mice was detected to have not induced any humoral and cellular immune response, which is important for the treatment of the disease.

A. Nanobody-based drug delivery

Modification of nanobodies onto nanodrug carriers (e.g., liposomes, micelles, albumin- and ferritin-based nanoparticles, and polymer-based multimers) enables active targeting of nanobody-based anticancer drugs to tumor tissues. Nanobodies with tumor-specific receptors can be used as carriers for the delivery of toxins or drugs to the tumor site, enabling the targeted release of drugs or toxins, reducing damage to normal cells and reducing side effects.

B. Nanobody-Drug Conjugates (NDC)

Antibody-drug conjugates (ADCs) combine the potent killing effect of small-molecule drugs with the highly targeted nature of mAb. The large size of mAb (~150 kDa) and the presence of the Fc region help to improve the pharmacokinetic properties of mAb, but also affect their tissue penetration capabilities. Nanobodies (Nb),  as novel smaller, highly selective antibody derivatives or fragments, have received much attention for developing the next generation of targeted drug conjugates.  Nanobody drug conjugates (NDCs) have advantages over larger ADCs because they offer rapid systemic clearance, enhanced stability, and increased tumor penetration rate.

C. Nanobody-based CAR-T Cell therapies

Typically, single-chain variable fragment (scFv) of monoclonal antibodies is used as the antigen-targeting structural domain of CARs. However, the use of scFv as CAR targeting domain has some limitations. In recent years, researchers have been focusing on other types of CAR structural domains, including nanobodies, peptides or ligands. Among them, nanobodies have shown particular advantages as alternative CAR-targeting structural domains, including the low immunogenicity, high stability, strong specificity and high affinity of nanobodies, as well as a simple and feasible development process. The results of many studies have confirmed that nanobody-based CAR-Ts can perform the same functions as scFv-based CAR-Ts in both preclinical and clinical settings.

Conclusion

Overall, nanobodies play an important role in the diagnosis and therapeutic treatment of diseases, and their properties are fundamentally different from those of conventional antibodies with double-chain structures. The small molecular weight, binding antigen specificity, high affinity, and high stability of nanobodies enable them to successfully target tumors, tumor microenvironments, and antigens of viral infections, etc. Nanobodies are increasingly being used as diagnostic tools for molecular imaging techniques, as evidenced by successful early clinical trials. As therapeutic agents, nanobodies can aid in the delivery of drugs and can be used in CAR-T cell therapy and viral infection treatment. The full range of possible applications for nanobodies remains to be explored, but as a novel type of antibody, they will continue to shine in the field.

References:
[1] Jin, B.-k.; Odongo, S.; Radwanska, M.; Magez, S. Nanobodies: A Review of Generation, Diagnostics and Therapeutics. Int. J. Mol. Sci. 2023, 24, 5994. https://doi.org/10.3390/ijms24065994
[2] Salvador, JP., Vilaplana, L. & Marco, MP. Nanobody: outstanding features for diagnostic and therapeutic applications. Anal Bioanal Chem 411, 1703–1713 (2019). https://doi.org/10.1007/s00216-019-01633-4
[3] Elisha R. Verhaar, Andrew W. Woodham, Hidde L. Ploegh, Nanobodies in cancer, Seminars in Immunology, Volume 52, 2021, 101425, ISSN 1044-5323, https://doi.org/10.1016/j.smim.2020.101425

Previous:Targeted Therapies for Multiple Myeloma (MM) Next:Peptide Lipidation:  A Breakthrough Strategy