One-Stop Solution for Anti-Membrane Protein VHH Antibody Development

Creative Biolabs is a leading service provider focusing on single domain antibody (sdAb) discovery and development. SdAb is an artificially designed antibody molecule, also known as VHH antibody or camelid antibody. Based on our deep understanding of various membrane proteins, our scientists have built up one-stop solutions to generate high-quality single domain antibodies that can properly recognize the target membrane protein for our customers all over the world.

Membrane Protein: A Class of the Most Important & Challenging Targets for Antibody Development

In most cancer diseases, therapeutic targets are cell surface molecules that are predominantly comprised of membrane proteins. Membrane protein is a class of targets that are composed of a wide range of structurally and functionally diverse proteins involved in a variety of important physiological and homeostatic processes, such as:

• Membrane receptor proteins relay signals between the cell's internal and external environments.
• The molecules and ions across the membrane via transport proteins.
• Membrane proteins may present enzyme activity, such as oxidoreductase and hydrolase.
• Cell adhesion molecules allow cells to identify each other and interact.

In terms of the ability to activate/block the downstream function or promote the analysis of 3D structure, antibodies that can direct against target membrane proteins have played critical roles as either therapeutic/diagnostic agents or research tools. However, membrane protein is also one of the most challenging targets to generate high-quality antibodies, and the limitations usually include:

1) The challenge of designing and producing properly antigens that can mimic the epitopes of naïve membrane proteins;

2) Homology issues that may cause immunological tolerance;

3) Potential steric issues caused by the multi-pass membrane proteins, in which some epitopes cannot be reached by conventional antibodies.

Developing High-Quality Anti-Membrane Protein VHH Antibody at Creative Biolabs

Fortunately, Creative Biolabs has solutions to overcome all these limitations by using VHH instead of full-length IgG or other antibody fragments. VHH is a unique antibody format that consists of a single monomeric variable domain without a CH domain or light chain in the conventional Fab region. It is usually generated from camelid or cartilaginous fishes, which indicate relatively low homology for most of the membrane proteins associated with therapeutic targets. Taking advantage of the small size, high stability, and great penetrability, VHH can carry out the task of recognizing even the most cryptic epitopes. In the meantime, our scientists have also integrated our Magic™ membrane protein platform and DNA immunization strategy into the profiles, which can offer comprehensive solutions for antigen design and preparation. Therefore, the development of single domain antibodies against membrane proteins is now highly sought after for research, diagnostic, and therapeutic purposes, and our one-stop solution could be the most efficient option to achieve your purpose with high success rates.

Typical Pathway for Membrane Protein-Specific VHH Development

Phase I Target Identification

Antigen Design & Preparation Starting with in silico sequence of targets, our scientists can handle the intact design and preparation processes to fit the demands for VHH discovery. You can also provide the prepared target for further processes.

Target Expression Cell Lines Positive cell lines that express the target of interest can be generated and employed as antigen for VHH development. The corresponding negative cell lines which do not express the target will be used as a control to ensure reliability. You can also provide these cell lines for further processes.

Epitopes Mimic Peptides Peptides can be designed and synthesized properly to mimic the desired target epitopes. Carrier proteins will be conjugated for short peptides to ensure immunogenicity or promote screening processes.

Target Expression Plasmids For antigens which are difficult to be expressed or purified, the corresponding plasmids can be constructed for DNA immunization, which can permit in vivo antigen production and trigger the desired immune response.

Lipoparticle Lipoparticle is highly purified virus-like particle (VLP) that contains intact, native membrane proteins harvested from host cells. It has a much smaller size, increased stability, and higher display density than target expressing cell lines, which can be used as both antigen and screening target.

Nanodiscs Nanodisc is formed by self-assembled phospholipid and membrane scaffold protein. It is a powerful tool to keep membrane protein in a naïve form with the folded conformation and intact function.

Proteoliposome Proteoliposome can well keep membrane proteins correctly folded with their naïve structures, activities, and functions, it has been frequently used to study membrane protein for antibody development, binding tests, and functional assays.

Phase II Discovery

Conventional Immunization This strategy can elicit responses from several easily accessible sites thus contribute to the development of antibodies with high specificity and affinity.

DNA Immunization DNA immunization allows the antigen production in vivo and keeps its naïve conformation as much as possible. It is an efficient approach to elicit an immune response against challenging antigens.

Whole Cell Immunization Whole cell immunization benefits from higher immunogenicity, naïve antigen structure, simple purification process, which can elicit the immune response against the antigen overexpressing cell line.

Immune Library-Based Discovery Our scientists are confident in generating high-quality immune library and performing comprehensive library screening and validation strategies to select the best-fit VHH for your targets.

Premade Library Based VHH Discovery Creative Biolabs has owned a series of high-quality in-house VHH libraries (originated from camels, llamas as well as humans) which are great resources available for your VHH discovery project.

Phase III Characterization

Functional VHH Screening A series of assays are available to further analysis and screening sdAb 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 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).

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.

De Novo Sequencing

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 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.

More Creative Biolabs offers a variety of sdAb characterization services for your project. Our scientists can also provide customized solutions to meet your specific demands.

Phase IV 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.

VHH Affinity Maturation Elaborate procedures can be tailored to develop VHH with increased affinity against the target of interest.

VHH Conjugation A variety of custom VHH Conjugations are available of your choice.

VHH Affinity Maturation Elaborate procedures can be tailored to develop VHH with increased affinity against the target of interest.

VHH Humanization Creative Biolabs provides elegant sdAb humanization strategy, which is an efficient approach to reduce or eliminate the immunogenicity of sdAb drug candidates.

More Creative Biolabs offers a variety of sdAb development services for your project. Our scientists can also provide customized solutions to meet your specific demands.

Phase V Manufacture

VHH Scale-up Production Transient transfection method can be employed for custom sdAb production via either E. coli or mammalian system.

VHH Stable Cell Line Construction Our scientists are professional in constructing stable cell lines with high yield to meet your demands of long-term sdAb production.

More Creative Biolabs offers a variety of VHH manufacture services for your project. Our scientists can also provide customized solutions to meet your specific demands.

Features of One-Stop Anti-Membrane Protein VHH Development

• Comprehensive membrane protein preparation and immunization strategies (e.g., lipoparticle, cell line construction, and DNA immunization)
• The scientific group with extensive experience in dealing with membrane proteins, who can tailor the most suitable solutions for your projects
• Advanced platforms to ensure the best achievement for every single step (from the membrane protein preparation to the VHH candidates' validation)
• Multiple pathways to enhance the success rate
• Reliable lab report with timely update
• Affordable price with the best quality

Creative Biolabs has developed a variety of VHH antibodies against membrane proteins with years of experience. We are always pleased to offer the best service and the most qualified outcomes to meet your specific requirements. If you are interested in developing novel VHH antibodies against the membrane protein of interest, please do not hesitate to contact us for more details.

Published Data

• Anti-Membrane Type 1-Matrix Metalloproteinase VHH Has Specificity against MT1-MMP and Potential to Be Applied in PET Imaging

Fig. 1 SdAbs from Different Clones Demonstrate Specific Reactivity against MT1-MMP.^{1, 2}

Fig. 2 Biodistribution and Pharmacokinetics of [⁶⁸Ga] Ga-NOTA-sdAbs.^{1, 2}

The article presents a comprehensive study on the development of anti-membrane type 1-matrix metalloproteinase (MT1-MMP) sdAbs for positron emission tomography (PET) imaging in triple-negative breast cancer (TNBC), underscoring the aggressive nature of TNBC and the significance of identifying biomarkers for improved patient outcomes. The article describes how a llama-derived sdAb library was generated and screened against the catalytic domain of MT1-MMP, resulting in the discovery of particular sdAbs. Following a comprehensive analysis, two of these sdAbs were tagged with gallium-68 (⁶⁸Ga) and tested in a TNBC mouse model. The immunoPET imaging exhibited precise in vivo tumor targeting with high signal-to-background ratios, with (⁶⁸Ga) Ga-NOTA-3CMP75 exhibiting more tumor uptake than (⁶⁸Ga) Ga-NOTA-3TPA14. The imaging data was shown to be perfectly correlated with immunohistochemical staining results, demonstrating the sdAbs' selectivity for MT1-MMP. The results show that anti-membrane protein sdAbs have great specificity against MT1-MMP, making them intriguing candidates for PET imaging in TNBC and warranting further exploration as diagnostic tools.

References

1. Mulero, Francisca, et al., "Development of anti-membrane type 1-matrix metalloproteinase nanobodies as immunoPET probes for triple negative breast cancer imaging." Frontiers in Medicine 9 (2022): 1058455.
2. Under Open Access License CC BY 4.0, without modification.

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