Single Domain Antibody (SdAb) Specificity Optimization Solution

Novel Solutions Development Process Highlight Case Study Published Data Associated Services FAQ Resources

Creative Biolabs is a well-recognized expert in VHH discovery and development. With our rich experience in phage display mutant library technology and in silico modeling, Creative Biolabs is now offering a novel service to optimize the specificity of the VHH of interest, which can solve your trouble from unwanted cross-reactions.

Remove Cross-Reaction or Enhance Target Specificity of VHH Candidates

VHHs usually exhibit strong specificity toward their target proteins but encounter specificity issues when the target proteins share high homology with other proteins, leading to cross-reactions. VHH fragment sequence analysis combined with computer modeling acts as a solution to refine VHH binding specificity and remove unwanted cross-reactions to prevent numerous side effects from off-target interference.

Typical Pathway for VHH Specificity Optimization

Phase I Discovery

Sequence Analysis
Sequence Analysis Starting with in silico sequence of primary VHH and targets, our scientists can handle the intact design and prediction processes to fit the demands for VHH specificity optimization.
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In Silico Modeling
In Silico Modeling Our scientists can use in silico modeling to generate a 3D structure for mutation design for the best-fit candidate sites.

Phase II SdAb Discovery

B-Cell Sorting Based V<sub>H</sub>H Discovery
B-Cell Sorting Based VHH Discovery To bring our customers impeccable service of VHH discovery, our seasoned scientific team has developed integrated B-cell sorting platform to maximize our customers' success in discovering valuable sdAbs.

Phase III Characterization

VHH Specificity Verification
VHH Specificity Verification Creative Biolabs can verify the specificity of selected VHH upon to your specific demands.
V<sub>H</sub>H Affinity Measurement
VHH Affinity Measurement A series of technologies are available to measure the affinity of specific sdAbs with the most advanced systems (e.g. Biacore, ProteOn, Octet).
V<sub>H</sub>H Stability Evaluation
VHH Stability Evaluation A series of custom analytical approaches are available to assess the stability of specific sdAbs upon your request, including the evaluation of aggregation and stability under extreme condition.
Functional V<sub>H</sub>H Screening
Functional VHH Screening A series of assays are available to further analysis and screening VHH candidates with desired functions, including but not limited to binding assays, immunomodulation assessments (activation or blocking), neutralizing assay, Fc profiling (ADCC, ADCP, CDC, etc.), endocytosis & internalization assays, cell proliferation assays.
VHH Paratope Mapping
VHH Paratope Mapping Paratope, also named as an antigen-binding site, is a part of the antibody which allows the recognition and binding to an antigen. Paratope mapping is a powerful technology to understand the antigen-binding site well and plays an important role in vaccines and biotherapeutics discovery.
VHH Epitope Mapping
VHH Epitope Mapping Epitopes consist of groups of amino acids which interact with antibodies and therefore determine the antigenicity. Now we can provide the epitope mapping services with 100% accuracy and efficiency.

Phase IV Development

Multivalent V<sub>H</sub>H Development
Multivalent VHH Development The development of multivalent VHH provides various alternative formats which can endow sdAbs with specific properties, such as extended half-life, increased avidity, and the recognition of different targets.
V<sub>H</sub>H Affinity Maturation
VHH Affinity Maturation Elaborate procedures can be tailored to develop sdAbs with increased affinity against the target of interest.
Multivalent V<sub>H</sub>H Development
VHH Humanization Creative Biolabs provides elegant sdAb humanization strategy, which is an efficient approach to reduce or eliminate the immunogenicity of sdAb drug candidates.

Phase V Manufacture

V<sub>H</sub>H Scale-up Production
VHH Scale-up Production Transient transfection method can be employed for custom VHH production via either E. coli or mammalian system.
V<sub>H</sub>H Stable Cell Line Construction
VHH Stable Cell Line Construction Our scientists are professional in constructing stable cell lines with high yield to meet your demands of long-term VHH production.

Features of One-Stop VHH Specificity Optimization

In terms of our advanced technical platforms, scientists at Creative Biolabs are confident in offering high-quality VHH specificity optimization solutions to assist with your valuable projects. A series of VHH candidates can be rapidly optimized with desired specificity to meet your particular application requirements. Should you be interested in this novel specificity optimization service, please feel free to contact us for more details.

Case Study

This project aims to create humanized VHH antibody sequences. Through computational bioinformatics analysis, researchers modeled the variable regions and obtained a series of single-domain antibody variants. Throughin silico post-translational modification (PTM) and aggregation assessments of a particular VHH, we determined that the antibody faced deamidation risk. The research team conducted T-cell epitope analysis alongside B-cell epitope analysis and MHC II epitope analysis for this VHH and discovered that the predicted epitope demonstrated potential immunogenic properties.

Fig.1 VHH model. (Creative Biolabs Original)Figure 1. VHH homology model.

By using pbd 5IMM as the model structure, we built the VHH homology.

Table 1. PTM analysis for both parental sdAb and humanized variants.

Table1 PTM analysis. (Creative Biolabs Original)

In silico post-translational modification (PTM) assessments were performed on the parental VHH and humanized VHH, and it was found that there were two high-risk isomerization sites in the CDR2 region and one deamidation site in the FR region of the humanized VHH sequence.

Fig.2 Aggregation analysis. (Creative Biolabs Original)Figure 2. Aggregation analysis of parental sdAb and HuVHHv1.

The aggregation of the parental VHH and humanized VHH was predicted. The results showed that the aggregation tendency of humanized VHH was stronger than that of the parental VHH, which was due to germline humanization.

Fig.3 SDS-PAGE result. (Creative Biolabs Original)Figure 3. SDS-PAGE result of TOP 5 humanized VHH candidates and parental sdAb

The TOP 5 humanized VHHs and parental VHHs were expressed and purified. SDS results showed that the 5 variants were of good quality and had no obvious aggregation and precipitation, and could be used for further affinity determination.

Published Data

Fig. 1 Prediction and Experimental Validation of Aggregation Tendencies in VHH Domains via HIC Analysis.1

The scientific study describes a cutting-edge methodology that uses yeast surface display (YSD), next-generation sequencing (NGS), and artificial intelligence/machine learning (AI/ML) to quickly identify de novo humanized single domain antibodies (sdAbs) with optimized VHH specificity and commendable early-stage development profiles. It shows how NGS can analyze large sequence spaces and how AI/ML can be used to design new sequences with better potency or developability. The ability of long short-term memory (LSTM) networks, a subset of recurrent neural networks, to capture intricate correlations between amino acids that dictate the structure and function of proteins is especially noteworthy. The high-affinity binding and favorable biophysical characteristics of the discovered VHHs are validated experimentally. Accurate predictions in silico are crucial for efficient sequence selection and enhanced developability profiles, thus supporting the use of AI/ML for specificity optimization. By predicting which sequences are less likely to aggregate, researchers can prioritize candidates that are more likely to maintain their structural integrity and specificity in biological systems. Besides, understanding and optimizing the biophysical properties, such as hydrophobicity and aggregation propensity, contribute to the overall developability of an antibody, affecting its manufacturability, stability, and pharmacokinetics.

Associated Services

Reference

  1. Arras, Paul, et al., "AI/ML combined with next-generation sequencing of VHH immune repertoires enables the rapid identification of de novo humanized and sequence-optimized single domain antibodies: a prospective case study." Frontiers in Molecular Biosciences 10 (2023): 1249247. Distributed under Open Access License CC BY 4.0, without modification.

FAQ

1. Why is the specificity of VHH antibodies critical in research and therapeutic applications?
Specificity is vital because it determines the ability of a VHH to bind exclusively to its target antigen without cross-reacting with other molecules. High specificity ensures accurate detection, quantification, and targeted treatment of diseases, thereby reducing off-target effects and false positives in research.
2. How does the natural diversity of camelid VHH repertoires affect specificity optimization?
The natural diversity of camelid VHH repertoires generates a wide range of distinct binding locations and frameworks. This diversity enables the selection of VHHs with high specificity and affinity for different antigens, which may then be fine-tuned using techniques such as phage display and directed evolution.
3. What role do complementarity-determining regions (CDRs) play in the specificity of VHHs?
CDRs are hypervariable sections of the VHH that interact directly with the antigen, determining the specificity and affinity of antibody binding. Optimization frequently focuses on the CDRs modifications to improve binding characteristics while retaining or increasing specificity.
4. Can you explain the importance of structural stability in VHH specificity optimization?
Structural stability is critical because it guarantees that the VHH retains its conformation and binding activity under a variety of circumstances. Enhanced stability helps to improve binding specificity by preserving the precise conformation required for specific antigen engagement, even under demanding conditions like high temperatures or fluctuating pH levels.
5. What are some challenges in optimizing VHH specificity, and how can they be addressed?
There are several challenges in VHH specificity optimization, including cross-reactivity, poor affinity, and structural constraints. The unwanted binding to non-target molecules may occur as cross-reactivity, but this can be solved by improving the selection procedure to eliminate non-specific binders. Poor affinity is often increased via affinity maturation procedures. Due to some structural constraints, it is difficult to modify the VHH without disrupting its general structure and normal functions. However, this obstacle can be handled with the help of advanced bioinformatics and structural biology technologies.

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