Creative Biolabs is proud to introduce a unique therapeutic antibody discovery service: Humanization of Bovine Ultralong CDR3 Antibodies.
By merging the ultralong CDR3s discovered in bovine with human IgG scaffolds, novel therapeutic antibodies against challenging hot antigen families, including GPCRs and other intra-membrane targets on the cell surface are possible. Humanized IgG scaffolds that accommodate ultralong CDR3s may target small proteins with desirable therapeutic properties, which are challenging for conventional antibodies.
Bovine antibodies with ultralong complementarity determining region 3 (CDR3) represent a class of antibodies with unique structures and unparalleled ability to target complex antigens. These antibodies are characterized by an exceptionally long CDR3 in the heavy chain, which can extend to 50-70 amino acids, much longer than the 6-20 amino acids of conventional antibodies. This significant extension forms a unique "stalk and knob" structure:
The high cysteine content in the ultralong CDR3 promotes the formation of complex disulfide-bonding minidomains, which in turn leads to the remarkable diversity and specificity of this class of antibodies. These structural features enable bovine antibodies to target challenging antigens, including GPCRs and other transmembrane proteins that are usually inaccessible to conventional antibodies.
Despite the promise of bovine antibodies, there is a risk of immunogenicity when used directly in humans due to their xenogeneic origin. Humanization, the process of grafting bovine antigen-binding regions (e.g., ultralong CDR3) onto a human antibody scaffold, addresses this challenge. This transformative technology preserves the unique binding capabilities of bovine antibodies while ensuring compatibility with the human immune system, thereby reducing immunogenicity and enhancing therapeutic potential.
Fig. 1 Design principles for the development of bovine and human antibodies with ultra-long CDRs.1, 3
Humanization involves a series of precise steps to ensure the functional and structural integrity of the bovine ultralong CDR3 while adapting it to human frameworks.
The humanization of bovine antibodies begins with a detailed sequence analysis, focusing particularly on the CDR3 region. Creative Biolabs utilizes bioinformatics tools to identify the unique features of the ultralong CDR3 and the conserved structural elements in the bovine antibody. Framework regions are selected based on their similarity to known human antibody sequences, ensuring that the non-CDR regions of the bovine antibody are modified to resemble human immunoglobulins as closely as possible.
The key challenge in humanizing bovine antibodies lies in retaining the functional characteristics of the ultralong CDR3. Unlike traditional humanization approaches, where CDR regions are grafted onto a human framework, bovine antibody humanization must account for the unique structural dependencies of the ultralong CDR3. The grafting process involves incorporating the CDR regions, particularly the ultralong CDR3, onto a human scaffold while preserving the essential interactions that maintain its structural integrity.
The ultralong CDR3 stalk and knob must be preserved in a way that does not compromise the antibody's affinity or specificity. This often requires advanced molecular modeling techniques, which Creative Biolabs employs to predict the impact of humanization on the structure and binding capability of the antibody.
After the initial grafting of the CDRs, the humanized antibody is subjected to optimization. Affinity maturation may be necessary to restore or enhance the binding properties of the humanized antibody, especially if modifications during humanization have altered the interactions between the ultralong CDR3 and the antigen. Directed evolution techniques, such as phage display or yeast display, are commonly employed to introduce mutations that enhance the antibody's affinity and maintain its functionality.
1. Access to Cryptic Epitopes
The ultralong CDR3 loop found in bovine antibodies provides access to cryptic or deeply buried epitopes that are often inaccessible to traditional antibodies. By humanizing these antibodies, Creative Biolabs facilitates their use in therapeutic applications targeting pathogens or disease markers that have thus far eluded conventional antibody therapies.
2. Unique Structural Capabilities
Bovine antibodies exhibit unique structural elements that enable binding to diverse and difficult targets. Humanizing these antibodies allows for the retention of these unique features while reducing immunogenicity, providing a therapeutic advantage when targeting viral proteins or in targeting complex cancer antigens.
3. Broadening the Therapeutic Antibody Repertoire
Humanization of bovine antibodies helps to expand the diversity of available therapeutic antibodies, especially for use in fields where conventional human or rodent-derived antibodies have failed. This broadens the repertoire of antibodies that can be used in cancer immunotherapy, infectious disease treatment, and neutralization of complex toxins.
The ultralong CDR3 of bovine antibodies can access deeply buried viral epitopes and neutralize complex pathogens. Therefore, it has broad prospects in the fight against HIV, influenza, and coronavirus that traditional antibodies do not have.
Humanized bovine antibodies are able to target tumor-associated antigens that standard monoclonal antibodies cannot access. The ultralong CDR3 can accurately navigate through the complex tumor microenvironment, enhancing its ability to bind to cryptic epitopes expressed by cancer cells.
The structural diversity and specificity of the ultralong CDR3 domain enable humanized bovine antibodies to effectively neutralize toxins. They can precisely target unique conformational epitopes found in venoms, bacterial toxins, and other harmful substances, providing therapeutic potential for toxin-related emergencies.
G protein-coupled receptors (GPCRs) are important drug targets. Due to their complex transmembrane structure, they have always been a challenge for antibody-based therapies. However, humanized bovine antibodies with ultralong CDR3s are able to bind with high affinity to conformational epitopes within GPCRs. This unique ability opens the way for the development of GPCR-related therapy and the discovery of anti-GPCR antibody.
The humanization of bovine antibodies with ultralong CDR3 regions represents a paradigm shift in antibody engineering. By merging the exceptional antigen-binding capabilities of bovine ultralong CDR3s with human antibody frameworks, Creative Biolabs provides innovative solutions for developing next-generation therapeutics. These humanized antibodies hold the promise of revolutionizing treatments for complex diseases, particularly those involving elusive or challenging antigens.
Fig. 4 Overview about the generation of EGFR-specific ultralong CDR-H3 antibodies and Knobbodies.2, 3
Here, the researchers combined bovine immune technology with yeast surface display technology to produce epidermal growth factor receptor (EGFR) specific bovine ultralong CDR-H3 antibodies. After immunization, ultra-long CDR-H3 regions were specifically amplified and transplanted into IGHV1-7 scaffolds by homologous recombination to promote Fab display. Antigen-specific clones were obtained by FACS and reformatted into chimeric antibodies. The results show that bovine chimeric antibodies can effectively induce antibody-dependent cell-mediated cytotoxicity (ADCC) and resist tumor cells with overexpression of EGFR. In addition, most antibodies can significantly inhibit EGFR-mediated downstream signal transduction. Scientists have also shown that a minor fraction of CDR-H3 knobs derived from the generated antibodies can act independently as complementary sites to promote EGFR binding when grafted into the Fc part of human IgG1.
Humanization of bovine ultralong CDR3 antibodies involves modifying the antibody derived from cattle so that it is less likely to be recognized as foreign by the human immune system. This process is essential for therapeutic applications, as it reduces the risk of immune responses that can diminish the efficacy of the antibody or lead to adverse reactions in humans. Bovine ultralong CDR3 antibodies are particularly valued for their unique ability to bind to antigenic targets that might be inaccessible to human antibodies, making them promising candidates for addressing complex diseases such as cancer and autoimmune disorders.
The humanization of bovine ultralong CDR3 antibodies is typically achieved through CDR grafting. This method involves transplanting the Complementarity-Determining Regions (CDRs) from the bovine antibody into a human antibody framework. These CDRs are the parts of the antibody that directly bind to antigens. By grafting them onto a human antibody framework, researchers can maintain the unique antigen-binding properties of bovine antibodies while significantly reducing their immunogenicity in humans. Additional refinements, such as modifying certain framework residues to better support the bovine CDRs, can further enhance stability and binding efficiency.
One of the primary challenges in humanizing bovine ultralong CDR3 antibodies is maintaining the original high specificity and affinity for antigens after modification. Bovine ultralong CDR3 regions often adopt unique and complex structures that are crucial for their ability to recognize and bind to specific targets. Transferring these regions into a human antibody framework without losing functional integrity requires precise engineering. Additionally, ensuring that the humanized antibodies do not elicit an immune response in humans adds another layer of complexity, as even minor discrepancies in the protein structure can trigger immunogenicity.
Recent advances in computational biology and protein engineering have significantly improved the success rates of humanizing bovine ultralong CDR3 antibodies. Techniques such as molecular modeling and in silico simulations are now commonly used to predict the structural compatibility of bovine CDRs with human frameworks, allowing for more accurate design of humanized antibodies. Furthermore, advancements in gene editing technologies, such as CRISPR/Cas9, enable precise modifications at the DNA level, facilitating the insertion of bovine CDR sequences into human antibody genes. These technological developments have streamlined the humanization process, increasing the feasibility of producing effective and safe bovine-derived therapeutic antibodies for clinical use.
Humanized bovine ultralong CDR3 antibodies are especially effective in applications where high specificity and affinity are critical, such as in targeting hidden or conformationally complex epitopes that are not easily accessed by human antibodies. These unique characteristics make them particularly useful in oncology, where they can be used to target cancer cells expressing specific, often cryptic, tumor antigens without affecting healthy cells. Additionally, due to their high specificity, these antibodies are also valuable in research and diagnostic settings, enabling precise detection and study of biomarkers associated with various diseases.
Bovine ultralong CDR3 regions are distinguished by their exceptional length and complexity compared to their human counterparts. Typically, CDR3 regions in human antibodies consist of about 12 to 15 amino acids, whereas in bovine antibodies, these regions can extend up to 60 amino acids. This extraordinary length allows bovine ultralong CDR3s to form unique structural motifs such as extended loops or even knob-like structures that can penetrate deep into antigenic sites that are generally inaccessible to human antibodies. This structural uniqueness is what makes bovine ultralong CDR3s highly desirable for targeting complex antigens, though it also presents challenges in maintaining structural integrity and function when transplanted into a human antibody scaffold during humanization.
High-throughput sequencing and advanced bioinformatics tools allow for a more precise understanding and selection of bovine antibodies with desirable properties for humanization. Structural biology technologies, such as X-ray crystallography and cryo-electron microscopy, provide detailed insights into the three-dimensional structures of these antibodies, facilitating more accurate modeling and engineering. Additionally, automated synthetic biology platforms are increasingly used to construct and test multiple variants of humanized antibodies rapidly, significantly accelerating the development process and improving the chances of retaining the original antibody's efficacy and specificity in the humanized version.
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