Resources>Blog>Single-Domain Antibodies in Tumor Immunotherapy: Challenges and Opportunities
Single-Domain Antibodies in Tumor Immunotherapy: Challenges and Opportunities
Biointron2025-02-24Read time: 8 mins
Single-domain antibodies (sdAbs), also known as VHH antibodies or nanobodies, have emerged as a powerful tool in tumor immunotherapy, offering advantages over conventional monoclonal antibodies due to their small size, enhanced tissue penetration, and lower immunogenicity. They have roles in immune checkpoint blockade, multivalent antibody therapies, chimeric antigen receptor T (CAR-T) cell therapy, antibody-drug conjugates (ADCs), and tumor imaging. While these advances demonstrate the promise of sdAbs in cancer treatment, challenges remain in their clinical translation, stability, and large-scale production.
Immune Checkpoint Inhibitors Based on Single-Domain Antibodies
Immune checkpoint inhibitors (ICIs) are used in cancer therapy by blocking inhibitory pathways that suppress the immune response. Standard ICIs, such as anti-PD-1, anti-PD-L1, and anti-CTLA-4 antibodies, have shown efficacy in multiple cancer types but limitations can include poor tissue penetration and immune-related adverse events.
SdAbs, derived from camelid heavy-chain antibodies, provide a promising alternative due to their high stability and small molecular size (~12–15 kDa). These properties allow for better tumor penetration and reduced off-target toxicity. SdAb-based ICIs have demonstrated enhanced anti-tumor activity and improved pharmacokinetics in preclinical models. Notably, bispecific checkpoint inhibitors combining sdAbs with other immune-modulating agents have been developed to enhance immune activation within the tumor microenvironment (TME).
However, despite these advantages, challenges remain in optimizing sdAb stability, prolonging their half-life, and reducing potential off-target effects. Engineering sdAbs with albumin-binding domains or PEGylation strategies has been explored to improve their circulation time.
Formatting and gene-based delivery of a human PD-L1 single domain antibody for immune checkpoint blockade. DOI: 10.1016/j.omtm.2021.05.017
Multivalent and Bispecific Antibody Strategies Using Single-Domain Antibodies
Multivalent antibodies enhance therapeutic efficacy by engaging multiple antigenic targets. Traditional bispecific antibodies have complex structures that complicate manufacturing and stability. SdAbs, due to their modular nature, can be easily engineered into bispecific and multivalent formats with improved functionality.
Bispecific sdAbs can simultaneously target immune checkpoints and tumor-associated antigens, improving immune activation while reducing immune escape mechanisms. For example, sdAbs targeting PD-1 and CTLA-4 have shown increased T-cell activation compared to monovalent ICIs. Additionally, sdAbs fused to T-cell engagers (TCEs) enhance T-cell-mediated tumor killing.
Multivalent sdAbs have also been designed to target tumor antigens with higher avidity, increasing their therapeutic index. Tandem sdAbs fused to Fc fragments extend their half-life and optimize immune effector functions. These innovations improve both efficacy and safety in antibody-based immunotherapies.
CAR-T cell therapy, a form of adoptive cell therapy, has demonstrated success in hematologic malignancies. Traditional CAR-T designs incorporate scFv antibody fragments to recognize tumor antigens, but these fragments can have stability issues and cause immunogenicity.
SdAb-based CAR-T cells offer several advantages over scFv-based designs. The small size of sdAbs enables more efficient antigen recognition, while their stability enhances the durability of CAR expression. Additionally, sdAbs reduce the risk of tonic signaling, a common issue in CAR-T therapies that leads to T-cell exhaustion.
Recent studies have reported promising results using sdAb-based CAR-T cells targeting CD19, BCMA, and other tumor-associated antigens. These modified CAR-T cells exhibit improved tumor infiltration, lower immunogenicity, and enhanced anti-tumor activity. SdAbs have also been integrated into dual-targeting CAR constructs, allowing for more effective tumor eradication and reduced relapse rates.
Future research aims to optimize sdAb-CAR constructs, improve their expansion in vivo, and address resistance mechanisms in solid tumors, where CAR-T therapy has traditionally faced challenges.
ADCs represent a significant advancement in targeted cancer therapy by delivering cytotoxic payloads directly to tumor cells, minimizing systemic toxicity. While conventional ADCs rely on full-length IgG antibodies, their large size limits tissue penetration and clearance. SdAb-based ADCs overcome these limitations by offering enhanced tumor penetration and rapid blood clearance, reducing off-target toxicity.
SdAbs can be conjugated with various cytotoxic agents, including microtubule inhibitors and DNA-damaging agents, to improve selectivity and potency. Additionally, novel antibody immunostimulatory conjugates (ISACs) that combine sdAbs with immune-modulating agents have shown promise in enhancing anti-tumor immune responses.
Despite their advantages, challenges remain in optimizing the stability and conjugation efficiency of sdAb-ADCs. Strategies such as site-specific conjugation techniques and linker optimization are being explored to enhance their therapeutic potential.
Structure of VHH and nADC. DOI: 10.3390/cancers16152681
Tumor Imaging and Diagnostics with Single-Domain Antibodies
Accurate tumor imaging is needed for cancer diagnosis and treatment monitoring. Traditional antibody-based imaging agents often exhibit long circulation times, leading to high background signal and delayed imaging results. SdAbs offer a promising alternative due to their rapid tissue penetration, fast clearance, and high specificity.
Radiolabeled sdAbs have been developed for positron emission tomography (PET) and single-photon emission computed tomography (SPECT) imaging of tumors. Fluorescence-labeled sdAbs have also been used in intraoperative imaging to assist in real-time tumor resection. Compared to conventional imaging agents, sdAb-based tracers provide higher contrast and improved detection sensitivity.
Efforts are ongoing to optimize sdAb half-life and enhance imaging signal retention without compromising clearance rates. Albumin-binding strategies and fusion with half-life extension domains are being investigated to refine their clinical utility.
While sdAbs offer multiple advantages in tumor immunotherapy, several challenges must be addressed for their widespread clinical application.
Stability and Half-Life: SdAbs have a relatively short half-life, necessitating modifications such as PEGylation or fusion with albumin-binding domains to enhance their pharmacokinetics.
Manufacturing and Scalability: Large-scale production of sdAbs requires optimized expression and purification strategies. Bacterial and yeast expression systems have been explored, but achieving high-yield and cost-effective production remains a focus of ongoing research.
Immunogenicity and Safety: Despite their reduced immunogenicity compared to full-length antibodies, long-term studies on sdAb safety and potential immune responses are necessary for regulatory approval.
Application in Solid Tumors: While sdAb-based therapies have shown promise in hematologic cancers, their efficacy in solid tumors remains a challenge due to the immunosuppressive TME. Combination strategies with immune-modulating agents may improve therapeutic outcomes.
The integration of sdAbs into tumor immunotherapy continues to evolve, with advancements in engineering strategies and clinical development paving the way for their broader application. Future studies will focus on optimizing their design, improving delivery mechanisms, and overcoming barriers to their widespread adoption in cancer treatment.
