Creative Biolabs is one of the well-recognized experts who are professional in supporting a broad range of single domain antibody (sdAb) development projects. Along with extensive experience in developing novel VHHs, our scientists are proud to tailor and conduct the best-fit proposal to meet your specific project requirements.
The development of VHH technology has had a great impact on medicine. To further improve desired potency and properties, the development of multivalent VHH provides various alternative antibody formats with modified architectures.
Compared with conventional antibodies, VHH presents great advantages thanks to its small size ( ~12-15 kDa). However, the small size and monovalent binding also result in rapid blood clearance, fast off-rates, and poor retention times in therapy. In this case, the multivalent VHH enables the modification of avidity, specificity, size, tumor penetration, and also optimized half-life.
With beneficial pharmacologic and pharmacokinetic properties, VHHs present great potential in the treatment and diagnosis of various diseases. While the camelid or non-human origin may cause potential immunogenic issues. To overcome this risk, Creative Biolabs provides an elegant HuSdL™ humanization platform that can almost eliminate the immunogenicity of a VHH in humans while retaining its specificity and affinity. It is a novel solution to develop therapeutic VHHs for human use.
Creative Biolabs has developed various methods for the humanization of non-human VHHs, including but not limited to:
CDR (complementarity determining region) grafting combined with the back-mutation method.
Although in most cases, the VHHs isolated from gene synthesis, naïve, or immunized libraries present reasonable affinity, it is still desirable to increase their affinity. Creative Biolabs has developed a quantitative library sorting method via phage display or yeast surface display technology to improve the affinity of VHHs.
In addition, the unique DNA mutagenesis technique provided by Creative Biolabs enables a huge number of variants of the parental VHH with defined positions mutated. Combined with our advanced phage display VHH construction and screening technologies, we are proud to achieve a 10-100-fold increase in affinity for low-affinity VHHs.
Compared with conventional antibodies, VHHs present better characteristics including small size, high affinity, and simple production. However, the stability is still an important factor, especially for the VHHs derived from human VH and VL domain.
The VHH stability can be defined by two concepts, chemical stability (proteolytic stability) and physical stability (thermodynamic stability). Creative Biolabs now has developed two efficient methods to improve the stability of VHHs.
Bioconjugation is a chemical strategy process to form a stable covalent link between two molecules and results in diverse functionalities including revealing enzyme function, and specific biomarkers, determining protein biodistribution, and tracking cellular actions. Compared with conventional antibodies, VHHs present great advantages, such as small size and high affinity. It has been found that conjugated VHHs play an important role in therapeutics and diagnostics.
Compared to the conventional therapeutic approaches, monoclonal antibody-based immunotherapies show great advantages against various infectious diseases and cancers. With high specificity and affinity to the target, antibody-drug conjugates (ADCs) expand the therapeutic window of the conjugated drugs (payloads). With the small size and higher stability, VHHs further improve the efficiency and applications of ADCs.
The VHH-drug conjugates include three components, the VHHs, a cytotoxic payload, and a molecular linker. Creative Biolabs provides a one-stop VHH-drug conjugate development service to help create highly customized ADCs.
VHH-based CART Development
Chimeric antigen receptors (CARs), also named chimeric T cell receptors, chimeric immunoreceptors or artificial T cell receptors, enable immune effector cells (usually T cells or NK cells) to recognize target cells with the corresponding antigen and exercise their cytotoxic activity.
The VHH-based CART mainly consists of the VHH, a transmembrane domain, an extracellular hinge, and an internal signaling domain. With our cutting-edge platform, we can generate CART efficiently.
Creative Biolabs is a leading service provider specializing in VHH development. Equipped with advanced platforms and powerful technologies, we are confident in offering the best VHH development services for our customers all over the world. If you are interested in our service, please feel free to contact us for more details.
Published Data
The Impact of Subcellular Localization of EGFR-targeted VHH Photosensitizer Conjugates on Efficiency
Fig. 1 Cellular Interaction Dynamics and Internalization Patterns of 7D12 Conjugates.1,2
This article examines the influence of subcellular localization on the efficacy of epidermal growth factor receptor (EGFR)-targeted VHH photosensitizer conjugates in photoimmunotherapy (PIT). The researchers employed VHH 7D12 and conjugated it with the photosensitizer IRDye700DX, thereby further enhancing its cellular internalization through conjugation with a cell-penetrating peptide (CPP). The experiment depicted in Fig. 1 used confocal microscopy to observe the uptake of 7D12 conjugates in different cells, with results demonstrating that VHH[PS] conjugates exhibited a primary binding affinity for the cell surface, whereas VHH[PS]-CPP conjugates demonstrated a higher degree of intracellular uptake. This suggested that cell surface binding is a critical determinant of PIT efficiency and internalization may potentially diminish therapeutic efficacy. The subsequent results indicated that VHH[PS] conjugates were more effective in killing EGFR-positive cancer cells upon near-infrared light illumination compared to VHH[PS]-CPP. These findings provide important insights for optimizing PIT strategies and improving treatment outcomes.
References
van Lith, Sanne AM, et al. "The effect of subcellular localization on the efficiency of EGFR-targeted VHH photosensitizer conjugates." European Journal of Pharmaceutics and Biopharmaceutics 124 (2018): 63-72.
under Open Access license CC BY 4.0, without modification.
FAQ
1. How can VHHs be engineered for improved therapeutic efficacy?
By modifying their pharmacokinetic profiles, decreasing potential immunogenicity, and achieving affinity maturation, VHHs can be engineered to improve their therapeutic efficacy. Their binding affinity and specificity can be increased by a variety of methods, including site-directed mutagenesis, directed evolution, and rational design. PEGylation, fusion with additional medicinal compounds, or integration into multivalent forms can increase their stability and prolong their half-lives. Humanization techniques are also employed to lessen the possibility of inducing aberrant immunological reactions in patients.
2. What methods are used to humanize VHHs, and why is humanization important?
Modifying camelid sequences to make them similar to human antibody sequences is commonly used to humanize VHHs while reducing immunogenicity. One common approach is called complementarity-determining region (CDR) grafting, which means the antigen-binding CDRs of the VHH are transplanted onto a human antibody framework. Alongside, computer-based methods and designs help in predicting immune system responses to make them less strong while keeping the ability to bind antigens. Humanization is crucial for clinical applications to ensure patient safety and efficacy while minimizing adverse immune reactions.
3. What is the significance of creating multivalent VHH?
Multivalent VHH with increased binding avidity has better therapeutic efficacy and potency due to the ability to target multiple epitopes simultaneously. These multivalent constructs can improve antigen clustering and promote stronger immune responses, showing promising potential in dealing with diseases like cancer or infections. Therefore, generating multivalent VHH is of great importance in promoting the development of clinical treatment.
4. How are VHHs linked to drugs to form VHH-drug conjugates?
Chemical conjugation processes use stable covalent bonds to connect VHHs with medications. Peptide, thioether, and disulfide linkers are also frequently used to join the drug with the VHH while preserving bioactivity. To guarantee that the medication attaches to particular locations on the VHH, preserving antigen binding capacity and enhancing pharmacokinetics, site-specific conjugation techniques are applied. For VHH-drug conjugates to have increased therapeutic efficacy and stability, these conjugation techniques are essential.
5. What are some challenges in developing VHH-drug conjugates?
The challenges in developing VHH-drug conjugates include maintaining conjugate stability, achieving high specificity and affinity for target cells, and limiting potential immunogenicity. Another difficulty is optimizing the manufacturing process to create conjugates in large quantities while retaining consistency and activity. Navigating the difficulties of linker design is also required to enable accurate medication release to the target without premature degradation. Addressing these difficulties would necessitate advanced biotechnological technologies and extensive testing to ensure clinical efficacy and safety.
6. What role does computational modeling play in VHH stability improvement?
Computational modeling plays a critical role in simulating the effects of mutations on VHH structure and stability before actual laboratory work. This in silico approach allows researchers to prioritize mutations that are most likely to result in increased stability by computing changes in free energy and structural conformation. It helps streamline the experimental process by focusing resources on the most promising candidates, thus accelerating the development of more stable VHHs.
Fig. 1 Cellular Interaction Dynamics and Internalization Patterns of 7D12 Conjugates.1,2
