Rodent Antibody Humanization Service

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Creative Biolabs has extensive experience to offer the antibody humanization service for therapeutic and diagnostic development. We have successfully performed 15 mouse/rat humanization projects during the past decade with at least one humanized antibody entered the clinical trials. We also provide humanization service to antibodies derived from other species, such as non-human primate (NHP), rabbit, dog, chicken, llama and etc.

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Background

Rodent monoclonal antibodies, primarily derived from mice or rats, play a foundational role in immunology and therapeutic research. These antibodies are generated through immunization protocols that stimulate the rodent immune system to produce targeted responses against specific antigens. Since the development of the hybridoma technology, a large number of rodent monoclonal antibodies with specificity for antigens of therapeutic interest have been generated and characterized. Rodent antibodies are highly immunogenic in humans, which limits their clinical applications, especially when repeated administration is required. Importantly, they are rapidly removed from circulation and can cause systemic inflammatory effects as well. Rodent antibody humanization is a critical biotechnological process aimed at modifying rodent-derived monoclonal antibodies to make them more compatible with the human immune system. This process is instrumental in developing therapeutic antibodies used in preclinical and research settings. By replacing specific rodent antibody regions with their human antibody equivalents, immunogenicity can be minimized while retaining the antibody's specificity and binding affinity.

Fig. 1 Antibodies of different humanization degrees.

Our Rodent Antibody Humanization Approaches

As a means of solving high immunogenicity of rodent antibodies in humans, we have developed three antibody humanization strategies that can preserve the specificity and affinity of the antibody toward the antigen whereas significantly or completely eliminate the immunogenicity of the antibody in humans.

CDR Grafting & SDR Grafting

Rodent-derived antibodies are often recognized as foreign by the human immune system, leading to immune responses that can limit their effectiveness and safety. To address this, techniques such as CDR (Complementarity-Determining Region) Grafting and SDR (Specificity-Determining Residue) Grafting have been developed to modify rodent antibodies while retaining their original antigen-binding specificity and reducing their immunogenicity.

	CDR Grafting	SDR Grafting
Application	Grafting the CDRs from a rodent antibody into human antibody frameworks, retaining the antigen-binding specificity.	Further reducing murine components by minimizing the SDRs in the CDRs, thus reducing immunogenicity.
Process	Identify CDR Loops: Isolate the CDR loops from the rodent antibody Graft CDRs: Transfer the rodent CDRs into corresponding human antibody frameworks Randomize Framework Residues: Randomize a set of framework residues using phage display and computer modeling to optimize binding affinity Screen and Select: Screen the phage display library for humanized antibodies with the best affinity and specificity	Identify SDRs: Identify the variable positions in the CDRs that directly interact with the antigen Graft SDRs: Transfer the SDRs along with the conserved residues that maintain the CDR conformation into the human antibody template Evaluate Immunogenicity
Advantages	Retains Original Specificity Improved Binding Affinity Partial Humanization: Reduces murine components, lowering immunogenicity	Further Reduced Immunogenicity Retains Binding Specificity Enhanced Clinical Compatibility: Lead to a more human-like antibody

Both CDR grafting and SDR grafting play critical roles in the humanization of rodent antibodies, making them more suitable for therapeutic use in humans. CDR grafting retains the antigen-binding specificity of the rodent antibody while randomizing framework residues to improve binding affinity. SDR grafting, on the other hand, further reduces immunogenicity by minimizing murine sequences within the CDRs, ensuring a more human-like antibody. Together, these techniques enable the development of highly specific, effective, and less immunogenic humanized antibodies, advancing the field of therapeutic antibody engineering.

Fig. 2 CDR grafting and SDR grafting.

Chain Shuffling

Chain shuffling is a humanization strategy that focuses on constructing humanized antibodies through the replacement of both the light and heavy chains of a rodent antibody with human antibody chains. This strategy helps create antibodies with higher affinity and improved human compatibility while retaining the specificity of the original rodent antibody.

Chain shuffling in antibody humanization. (Creative Biolabs Original) Fig. 3 Chain shuffling.

Light Chain Replacement: The light chain of the rodent antibody is replaced with light chains from a well-established human antibody library.
Initial Screening: The hybrid antibody library is screened using phage display technology by panning against the target antigen.
Heavy Chain Replacement: The selected hybrid antibodies with human light chains were subjected to a second round of chain shuffling, in which their heavy chains were replaced with those from the human antibody repertoire.
Secondary Screening: The newly created chimeric antibody library is screened again by phage display to select antibodies with the best affinity and specificity.
Final Humanized Antibodies: After a second round of screening, fully humanized antibodies were obtained that retained the epitope specificity of the original rodent antibody.

Higher Affinity

Chain shuffling mimics the natural selection and evolution of antibodies, which can result in humanized antibodies with superior affinity compared to the original rodent antibody.

Multiple Humanized Variants

The process generates multiple versions of humanized antibodies with different sequences but retaining the same epitope specificity. This diversity is beneficial in developing therapeutic regimens where long-term antibody treatment is required.

Avoidance of Anti-Idiotypic Responses

The production of several humanized antibody variants reduces the risk of anti-idiotypic responses, which could occur with the prolonged use of a single antibody.

Complete Humanization

The method involves replacing the light and heavy chains with those of human antibodies, so the final antibody is fully humanized and less likely to be recognized as foreign by the human immune system.

As elaborated above, the "chain shuffling" method based on chimeric phage display library construction and screening allows full, i.e. 100% humanization of a mouse antibody. We would like to propose using our HuScL-2TM Phage Display Naive Human scFv Library with a complexity of 1.42×10⁹ transformants as the backbone of the chimeric libraries and the donor of human V_L and V_H chains.

Humanized IgG Library Screening

Mammalian cell surface display technology and IgG library screening provide allows the construction and selection of full-size humanized IgG antibodies. This method leverages the power of mammalian cell display and FACS (Fluorescence-Activated Cell Sorting) to select high-affinity humanized antibodies that retain or improve upon the affinity of the original rodent antibodies. It also avoids the issues related to bacterial-based systems and enables the direct selection of functional, full-size IgG antibodies. The technology also enables the rapid screening of large antibody libraries to select antibodies with optimal properties for therapeutic use.

Library Construction: The light chain of the rodent antibody is first replaced with light chains from a well-established human antibody library. The resulting hybrid antibody library is then subjected to phage display and screened using panning against the target antigen to identify antibodies with the desired antigen-binding specificity.
Heavy Chain Replacement: High affinity hybrid antibodies with human light chains are selected and the heavy chains of these antibodies are replaced with heavy chains from the same or different human antibody library. This secondary library containing humanized heavy and light chains is further screened using phage display or mammalian cell surface display to identify the antibodies with the highest affinity.
Mammalian Cell Surface Display Library: Functional full-size humanized IgG variants were displayed on the surface of mammalian cells by transfecting cDNAs of the heavy and light chains. Antibodies displayed on the cell surface were screened using fluorescence activated cell sorting (FACS) to identify the highest affinity antibodies.
Affinity Ranking and Final Selection: After FACS selection, antibodies with the highest binding affinity are tested using surface plasmon resonance (SPR) or Biacore. Selected clones are advanced to further validation and potential therapeutic development.

Related Services

Antibody 1. (Creative Biolabs Authorized)

What Types of Antibody Humanization Services Can We Provide for You?

What Types of Antibody Conversion Services Can We Provide for You?

Antibody Camelization (Convert to Single Domain Ab)
Antibody Caninization (Convert to Dog Ab)
Antibody Simianization (Convert to NHP Ab)
Antibody Murinization (Convert to Mouse Ab)
Antibody Felinization (Convert to Cat Ab)

Antibody 2. (Creative Biolabs Authorized)

Why Choose Creative Biolabs?

There are two features in our antibody humanization services that single out us from our peers. First of all, antibody affinity maturation is an integrated step in our humanization procedure, thus there is no need to improve the affinity after humanization. Secondly, in addition to the common computational and biochemical methods, we have developed a proprietary in vivo approach to evaluate the immunogenicity of the humanized antibodies in primates. The immunogenicity measured in primates is the closest one that may mimic the true immunogenicity of the humanized antibodies in humans.

Talk to Our Antibody Expert

FAQ

What is antibody humanization and why is it important?
Antibody humanization refers to the process of modifying non-human antibodies, typically from mice, to make them more similar to human antibodies in structure and function. This is crucial in the development of therapeutic antibodies to minimize the immune response against the antibody when used in human patients. Humanized antibodies are less likely to be recognized as foreign by the human immune system, reducing the risk of adverse reactions and increasing the therapeutic efficacy of the antibody in treating diseases.
What are the main challenges in antibody humanization?
One of the primary challenges is maintaining the high affinity and specificity of the antibody towards its target antigen after modification. The process involves grafting animal-derived complementarity-determining regions (CDRs) into a human antibody framework, which can potentially alter the antibody's original binding properties. Additionally, achieving optimal expression and solubility of the humanized antibody in clinical applications can be difficult. Ensuring that the humanized antibody does not elicit an immune response while retaining its functional efficacy also poses a significant challenge.
How do humanized antibodies differ from chimeric and fully human antibodies?
Humanized, chimeric, and fully human antibodies represent different strategies in antibody engineering for therapeutic use. Chimeric antibodies are produced by fusing the variable regions of a mouse antibody (which bind to the antigen) with the constant regions of a human antibody. This reduces the immunogenicity compared to fully mouse antibodies but is more immunogenic than humanized antibodies. Humanized antibodies, as previously described, involve transplanting only the antigen-specific CDRs from a non-human species into a human antibody framework, minimizing the non-human content. Fully human antibodies are generated either through phage display techniques or from mice genetically engineered to produce human antibodies, containing no non-human antibody sequences, which makes them the least likely to be recognized as foreign by the human immune system.
What are the clinical implications of using humanized antibodies?
Humanized antibodies have significant clinical implications, particularly in the fields of oncology, autoimmunity, and infectious diseases. By reducing the immunogenicity of therapeutic antibodies, humanization helps prevent immune reactions such as the production of anti-drug antibodies (ADAs), which can neutralize therapeutic effects and lead to treatment failures. Humanized antibodies thus have a better safety profile and potentially longer duration of action in patients. They are integral to treatments for conditions like rheumatoid arthritis, various cancers, and chronic inflammatory diseases, offering targeted therapy with fewer side effects compared to traditional treatments.
What factors influence the selection of a human framework for antibody humanization?
The selection of a human framework for antibody humanization is influenced by several factors aimed at optimizing the therapeutic efficacy and minimizing the immunogenicity of the antibody. These factors include the similarity of the human framework to the original non-human antibody, particularly in terms of the framework regions that interact with the CDRs. The stability and solubility of the framework are also critical considerations, as these properties affect the expression levels and the functional integrity of the antibody. Additionally, the framework's own immunogenic potential is assessed to ensure that it does not elicit an immune response in patients.
How is the success of antibody humanization evaluated?
The success of antibody humanization is evaluated through a combination of biochemical, structural, and functional assays. Biochemically, researchers assess the binding affinity and specificity of the humanized antibody compared to the original non-human antibody to ensure that the key antigen-binding properties are retained. Structural analyses, often involving techniques like X-ray crystallography or cryo-electron microscopy, are used to examine the conformation of the CDRs within the human framework. Functionally, the antibody's ability to elicit the desired immune response or therapeutic effect is tested in vitro and in vivo. Immunogenicity tests are also conducted to determine the extent of immune response that the humanized antibody might provoke in a clinical setting. These comprehensive evaluations help in refining the humanization process to develop effective and safe therapeutic antibodies.