Creative Biolabs has built up a novel intrabody generation platform, Super™ Intrabody Platform, based on an innovative combination of Yeast Two-hybrid System with antibody library technology.

Background

Intrabodies are antibodies that work within cells to bind to intracellular antigens. They are directed against intracellular target molecules and expressed within a specific subcellular compartment as directed by the intracellular localization signals genetically fused to N- or C-terminus. For this reason, intrabodies are defined as antibodies that have been modified for intracellular localization. As antibodies ordinarily are designed to be secreted from cells, intrabodies require special alterations, including modification of sequences for hyperstability, selection of antibodies resistant to the more reducing intracellular environment, or expression as a fusion protein with maltose binding protein or other stable intracellular proteins. Intrabodies have wide applications in dissecting target protein function, in target validation and functional genomics, as well as acting as potential therapeutic reagents. Intrabodies have promising applications against hepatitis B, avian influenza, prion diseases,inflammation, Parkinson's disease, and Huntington's disease.

Super™ Intrabody Platform

Super™ Intrabody Platform develops intrabodies in the form of single domain antibody, although human antibodies in scFv format have been developed as first generation of intrabodies. Single domain antibodies (also called as sdAbs, V_HH antibodies or domain antibodies) are antibody fragments consisting of a single monomeric variable antibody domain. With a molecular weight of only 12–15 kDa, single domain antibodies are much smaller than common antibodies (150–160 kDa), and even smaller than Fab fragments (~50 kDa) and single-chain variable fragments (~25 kDa).

We have created a large single domain llama antibody library using Matchmaker™ Gold Yeast Two-hybrid System (Clontech), a GAL4-based two-hybrid assay, in which an antigen protein is expressed as a fusion to the Gal4 DNA-binding domain (DNA-BD), while single domain antibodies engineered from heavy-chain antibodies found in camelids are expressed as fusions to the Gal4 activation domain (AD). As a result, when antigen (bait) and antibody library (prey) fusion proteins interact, the DNA-BD and AD are brought into proximity to activate transcription of reporter genes, making the discovery of high affinity single domain antibodies specific for the antigen.

Since the antibodies discovered on this platform are expressed and selected inside of yeast cells, there are the best intrabodies in the first place that do not require further sequence modification. Also, this platform does not require antigen in a purified protein form to raise antibodies. Instead, the gene of the antigen is expressed into an intracellular protein to capture its antibodies in the yeast cells. As a result, intrabodies discovered from this platform usually recognize the native conformation of the antigen and are of immediate therapeutic potential.

Published Data

• ER Intrabody-Mediated Inhibition of Interferon α Secretion

Fig. 2 Subcellular colocalization and co-immunoprecipitation of the intrabody and IFNα4 upon transient transfection of HEK293T cells. (Konrad Büssow, 2019)

Interferon α (IFNα) protects against viral infection by activating various IFNα stimulating genes. It is reported that an scFv intrabody holds back IFNα isoforms in the endoplasmic reticulum (ER) and thus counteracts IFNα secretion. This intrabody is constructed from the variable domain of the anti-mouse IFNα rat monoclonal antibody 4EA1 and can recognize five isomers (IFNα1, IFNα2, IFNα4, IFNα5, and IFNα6). Studies have shown that anti-IFNα antibodies significantly inhibit the secretion of recombinant IFNα4 by HEK293T cells. In HEK293T cells, antibodies can co-locate with IFNα4 and ER marker calcitonin, indicating the formation of antibodies and IFNα4 complexes in the ER. Through the co-immunoprecipitation assay, the researchers confirmed the intracellular binding of antibodies and antigens.

• A Specific Mini-Intrabody Mediates the Lysosome Degradation of Mutant Huntingtin

Fig. 3 Effects of different intrabody fragments on mHTT. (Caijuan Li, 2023)

Misfolding and accumulation of misfolded proteins can lead to many neurodegenerative diseases that can be treated by reducing or removing mutant proteins. Huntington's disease (HD) is mainly caused by the accumulation of mutant Huntington protein (mHTT) in cells. This protein can dissolve and accumulate in the central nervous system and lead to neuronal damage and death. Here, the researchers produced an intrabody fragment that specifically binds to mHTT and connects to lysosomes for degradation. The results show that mini-intrabody peptides linked to lysosomes can effectively reduce mutant proteins and may be used to treat neurodegenerative diseases caused by the accumulation of mutant proteins.

FAQ

1. What is an intrabody and how does it differ from traditional antibodies?

   An intrabody is a type of antibody that is engineered to function inside cells, whereas traditional antibodies generally operate outside cells in the extracellular space. Intrabodies are typically single-chain variable fragments (scFv) of antibodies or single domain antibodies (sdAbs) that can be expressed within the cytoplasm or other cellular compartments. They are designed to specifically bind to intracellular target proteins, thereby modulating their function directly within the cellular environment. This ability makes intrabodies useful tools for therapeutic interventions, particularly in cases where external accessibility to a target protein is restricted.

2. What are the potential applications of intrabodies in medical research and treatment?

   In research, they can be used to study protein functions within cells by either inhibiting or stabilizing specific proteins. For therapeutic applications, intrabodies can be employed to interfere with the progression of diseases at a cellular level, including neurodegenerative diseases like Alzheimer's and Parkinson's, where they can prevent the aggregation of disease-causing proteins. Additionally, they are being explored in cancer therapy to block interactions that lead to tumor growth and metastasis.

3. How are intrabodies delivered into cells for therapeutic purposes?

   Delivering intrabodies into cells can be accomplished through several methods, including viral vectors, lipid nanoparticles, and cell-penetrating peptides. Viral vectors, such as lentiviruses or adeno-associated viruses, are commonly used due to their high efficiency in transducing a variety of cell types. Lipid nanoparticles can encapsulate intrabody-encoding RNA or DNA, facilitating entry into cells without the use of viruses. Cell-penetrating peptides offer another strategy by directly translocating intrabody proteins into cells.

4. What challenges must be overcome for the effective use of intrabodies in clinical settings?

   These include ensuring the stability and solubility of intrabodies within the cellular environment, achieving specific and efficient delivery to target cells, and avoiding immune responses that could neutralize their therapeutic effects. Additionally, rigorous testing is necessary to confirm that intrabodies do not interfere with essential cellular functions, which could lead to unintended side effects.

5. What are the advantages of using intrabodies over small molecule inhibitors?

   Due to their ability to be engineered to bind specific epitopes, intrabodies can target proteins with high precision, reducing the likelihood of off-target effects that are often associated with small molecules. Additionally, intrabodies can be designed to interfere with protein-protein interactions that are typically challenging to target with small molecules. This makes intrabodies powerful tools for modulating complex biological pathways and processes that are involved in diseases.

6. Can intrabodies be used in combination with other therapies?

   Intrabodies can be used in combination with other therapeutic approaches to enhance treatment efficacy and potentially reduce side effects. For example, in cancer therapy, intrabodies can be used alongside chemotherapy or immune checkpoint inhibitors to target tumor cells more effectively and to help overcome resistance to other treatments. In neurodegenerative diseases, combining intrabodies with therapies that address other aspects of disease pathology, such as inflammation or oxidative stress, may provide a more comprehensive approach to treatment.

7. How do intrabodies contribute to the study of protein function in live cells?

   By binding to specific proteins, intrabodies can modulate their activity in real-time, providing insights into protein dynamics, interactions, and roles in cellular processes. This ability to interfere with or stabilize proteins in their native context allows researchers to dissect complex biological pathways and understand the implications of protein functions under physiological conditions. Moreover, fluorescently tagged intrabodies can be used to visualize the location and movement of proteins within live cells, enhancing our understanding of cellular organization and dynamics.

8. What technological advancements are driving the development of intrabodies?

   Recent advancements in protein engineering and gene delivery technologies are significantly driving the development of intrabodies. Techniques such as phage display and yeast display have enabled the rapid identification and optimization of intrabodies that have high affinity and specificity for target proteins. Additionally, improvements in gene editing tools like CRISPR/Cas9 allow for precise insertion of intrabody genes into specific loci in the genome, enhancing the stability and expression of intrabodies in therapeutic contexts. Advances in delivery mechanisms, particularly non-viral methods such as nanoparticle-based systems, are also crucial in facilitating the safe and effective delivery of intrabody genes to target cells in vivo.

9. How are intrabodies being used in diagnostic applications?

   Intrabodies can be used to detect the presence of specific proteins associated with diseases. Because of their high specificity and ability to bind to unique epitopes, intrabodies can be engineered to recognize disease-specific markers within cells or in complex biological samples. This capability is particularly valuable in conditions where early detection of cellular biomarkers can significantly influence the outcome, such as in cancer or infectious diseases. By tagging intrabodies with fluorescent markers or other reporters, they can be used in various imaging and diagnostic platforms to provide rapid and accurate disease detection.

10. What role do intrabodies play in targeted cancer therapy?

    Intrabodies can enable the specific inhibition or modification of oncogenic proteins within cancer cells. By targeting these proteins, which are often inaccessible to conventional antibodies due to their intracellular location, intrabodies can interfere with cancer cell growth and survival directly at the molecular level. This targeted approach helps to minimize damage to normal cells, reducing the side effects typically associated with broader-acting cancer treatments like chemotherapy. Intrabodies can also be engineered to disrupt specific protein-protein interactions that are critical for cancer cell proliferation and metastasis, offering a highly targeted therapeutic strategy that is currently being explored in various stages of research and clinical trials.

11. How is the specificity of intrabodies achieved and verified?

    The specificity of intrabodies is achieved through their design and engineering process, which involves selecting antibody fragments that bind exclusively to a target protein or a specific part of a protein. This is typically accomplished using display technologies such as phage display, where vast libraries of antibody fragments are screened to identify those that bind with high specificity and affinity to the target. Once potential intrabodies are identified, their specificity is verified through a series of in vitro and in vivo experiments. These tests include binding assays to confirm interaction with the target protein and not with other proteins, and functional assays to ensure that the intrabody effectively alters the activity of the target protein as intended. This rigorous verification process is essential to confirm that the intrabody will perform its desired function without unintended interactions, ensuring its safety and efficacy for therapeutic use.

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