Future Directions for VHH Antibody Research and Development

VHH antibodies, also known as nanobodies, have emerged as a transformative class of biologics with applications spanning oncology, neurology, and targeted drug delivery. Their small size, high stability, and unique binding properties allow them to overcome many limitations of conventional monoclonal antibodies (mAbs). Advances in bioinformatics, artificial intelligence (AI), screening technologies, and delivery strategies are accelerating Nb research, with promising implications for precision medicine and intracellular targeting.

Leveraging AI and Bioinformatics for VHH Antibody Discovery

The integration of AI and bioinformatics into VHH antibody discovery is revolutionizing antibody screening and design. Traditional selection methods rely on phage or yeast display libraries, but next-generation sequencing (NGS) combined with high-throughput sequencing enables a different approach.

• PBMC-based screening: Peripheral blood mononuclear cells (PBMCs) can be collected at peak immune response after antigen immunization. Direct deep sequencing of PBMC repertoires allows for rapid identification without the need for labor-intensive library construction.

• AI-driven structural modeling: AI-based protein modeling tools, such as AlphaFold and Rosetta, have demonstrated superior accuracy in predicting antibody structures. These models enable in silico affinity maturation, optimizing antibody binding affinity and specificity while reducing wet-lab experimental requirements.

• Sequence-activity relationship analysis: AI-assisted analysis of NGS data identifies dominant clones with desirable biophysical properties. Machine learning algorithms can optimize combinatorial mutagenesis libraries, as demonstrated in studies on antibodies targeting kynurenine.¹

Related: Generative AI and the Antibody Sector

Intrabodies and the Potential for Intracellular Targeting

Unlike conventional antibodies, which primarily target extracellular proteins, VHH antibodies can function as intracellular antibodies (intrabodies or iDAbs). Their small size and stability allow them to penetrate cells and target previously "undruggable" intracellular proteins, including transcription factors, signaling molecules, and misfolded proteins.

• TCR-like nanobodies (Nbs) for MHC-peptide recognition: One of the major challenges in immunotherapy is targeting intracellular tumor antigens presented by major histocompatibility complex (MHC) molecules. T-cell receptor (TCR)-like Nbs offer a promising approach for targeting MHC-peptide complexes with high specificity. However, achieving TCR-level affinity remains a technical hurdle requiring further optimization.

• Gene therapy synergy: iDAbs can be delivered via viral vectors (e.g., adeno-associated viruses, AAVs) or mRNA technology, expanding their therapeutic potential for treating neurodegenerative disorders and genetic diseases.

• Functional warheads for intracellular modulation: Nbs can be engineered with functional domains to modulate protein activity, degrade targets via PROTAC-like mechanisms, or interfere with disease-relevant protein interactions.

With these advancements, iDAbs could play a role in developing next-generation cancer immunotherapies and neurological disease treatments.

Related: VHH Antibody Discovery

Drug Delivery Across Biological Barriers

One of the major advantages of VHH antibodies is their ability to cross biological barriers, including the blood-brain barrier (BBB), enabling targeted central nervous system (CNS) therapies. Recent breakthroughs highlight their potential in neurodegenerative disease treatment.

• Nb-mediated transport across the BBB: Nb188, an anti-human transferrin receptor (TfR) VHH, facilitates BBB penetration when fused to therapeutic cargo. For example, Nb188-neurotensin fusion successfully delivered neuropeptides to the CNS, overcoming natural transport limitations.²

• Heterodimeric Nbs for Alzheimer's therapy: Nb-based therapeutics targeting β-secretase 1 (BACE1) have shown promise in reducing amyloid-β (Aβ) levels. Fusion proteins combining Nb62 or Nb188 with anti-BACE1 Fabs effectively reduced Aβ accumulation in preclinical models.

• Inhalable Nb formulations: Companies such as AdAlta are developing inhalable Nbs (e.g., AD-214) for pulmonary diseases, enhancing bioavailability and reducing systemic exposure.

Related: Applications of VHH Antibodies

Imaging and Theranostics in Oncology

VHH antibodies are increasingly used in cancer diagnostics and theranostics, providing high-affinity tumor targeting with minimal background signal. Their rapid clearance and deep tissue penetration make them ideal for molecular imaging and targeted therapy.

• Surgical tracers for precision oncology: Nb-based tracers enhance tumor visualization during surgery, improving resection accuracy. Ongoing research is exploring fluorophore- or radionuclide-labeled Nbs for real-time imaging.

• Radiolabeled Nbs for PET imaging: ^68Ga-labeled Nbs targeting HER2, EGFR, and PD-L1 have demonstrated high specificity and rapid tumor accumulation, enabling noninvasive imaging of cancer biomarkers.

• Targeted Nb-drug conjugates (NbDCs): Similar to ADCs, NbDCs can be conjugated with cytotoxic payloads or radionuclides for selective tumor killing while minimizing systemic toxicity.

Next-Generation Platforms and Emerging Modalities

Beyond camelid-derived VHHs, alternative single-domain antibody (sdAb) platforms are gaining attention. Shark-derived variable new antigen receptors (VNARs) also exhibit high stability and unique binding properties.

• VNARs in clinical development: AD-214, an anti-CXCR4 i-body, is entering clinical trials for idiopathic pulmonary fibrosis (IPF), with potential applications in oncology and inflammatory diseases.

• Multi-specific Nbs: Bispecific and trispecific Nb formats are being explored to enhance immune cell recruitment and tumor specificity.

• Nb-based cell therapies: Nb-CAR T and Nb-CAR NK cells are being investigated for solid tumor immunotherapy, potentially offering improved tumor infiltration compared to traditional CAR-T cells.

Related: Conventional Antibodies, Heavy Chain-Only Antibodies, and VHH Antibodies

The field of VHH antibody research is rapidly evolving, driven by advances in AI-assisted antibody discovery, intracellular targeting strategies, improved delivery mechanisms, and novel imaging applications. As these therapeutics progress through clinical trials, their potential to transform cancer therapy, neurological disease treatment, and targeted drug delivery continues to grow.

At Biointron, we are dedicated to accelerating antibody discovery, optimization, and production. Our team of experts can provide customized solutions that meet your specific research needs, including VHH Antibody Discovery. Contact us to learn more about our services and how we can help accelerate your research and drug development projects.

References:

1. Alexander, E., & Leong, K. W. (2024). Discovery of nanobodies: a comprehensive review of their applications and potential over the past five years. Journal of Nanobiotechnology, 22(1). https://doi.org/10.1186/s12951-024-02900-y

2. Wouters, Y., Jaspers, T., Rué, L., Lutgarde Serneels, Strooper, B. D., & Dewilde, M. (2022). VHHs as tools for therapeutic protein delivery to the central nervous system. Fluids and Barriers of the CNS, 19(1). https://doi.org/10.1186/s12987-022-00374-4