Rodent Antibody Humanization Service

Background CDR & SDR Grafting Chain Shuffling Humanized IgG Library Screening Highlights Published Data FAQ Resources

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.

Background

Humanization is important for reducing the immunogenicity of monoclonal antibodies derived from xenogeneic sources (commonly rodent) and for improving their activation of the human immune system. 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. As a means of circumventing these problems, 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. The first approach is CDR grafting and the second is chain shuffling. These two methods are all based on phage display of humanized scFv variants and selection of high-affinity humanized binders through bio-panning. The third method, humanized IgG library screening, is somehow unique. We will make a library of humanized whole IgG to be displayed on the surface of mammalian cells and then high affinity binders will be sorted by FACS.

CDR Grafting & SDR Grafting

We have established a CDR (complementarity-determining region) grafting platform, which is featured with randomization of a small set of framework residues using phage display technology and computer modeling. In this platform, six CDR loops comprising the antigen-binding site are grafted into corresponding human framework regions. Unfortunately, simple grafting of the rodent CDRs into human frameworks does not always reconstitute the binding affinity and specificity of the original antibody because framework residues are involved in antigen binding, either indirectly, by supporting the conformation of the CDR loops, or directly, by contacting the antigen. For this reason, we have developed a computer modeling method to randomize certain framework residues in addition to CDR grafting. The grafted CDRs together with the randomized residues are cloned into a phage display library and the humanized antibodies with the best affinity are selected by screening of the library. This approach allows the epitope specificity of the original antibody to be retained. Of note, humanization by this approach is not 100% since the CDR regions are still of a rodent origin.

Humanization CDR SDR Grafting

To reduce the immunogenicity of CDR-grafted humanized antibodies, the murine content in the CDR-grafted humanized antibodies is minimized through SDR grafting. Within each CDR, there are more variable positions that are directly involved in the interaction with antigen, i.e., specificity-determining residues (SDRs), whereas there are more conserved residues that maintain the conformations of CDRs loops. SDRs may be identified from the 3D structure of the antigen antibody complex and/or the mutational analysis of the CDRs. An SDR-grafted humanized antibody is constructed by grafting the SDRs and the residues maintaining the conformations of the CDRs onto human template, and its immunogenic potential is evaluated by measuring the reactivity to the sera from patients who had been immunized with the parental antibody.

Chain Shuffling

We have also optimized a chain shuffling strategy that is an entirely selective humanization strategy based on construction and screening of two chimeric phage display libraries. In this approach, the light chain of the rodent antibody is first replaced by light chains in one of our well-tested human antibody libraries; the resulting hybrid antibody library is then screened by panning against the particular antigen. After that, the heavy chain of the selected hybrid antibodies is replaced by heavy chains of the human antibody library. Subsequent screening of this secondary chimeric library will produce fully humanized antibodies. Since phage display library screening mimics in vivo antibody selection and evolution procedure, chain shuffling can result in humanized antibodies whose affinities are higher than that of the original antibody. Also, this sequential chain shuffling procedure can generate several versions of humanized antibodies with different sequences. The production of multiple humanized antibodies retaining the same epitope specificity is important in therapeutic regimens that call for long-term treatment with antibodies in which anti-idiotypic responses might be avoided by administration of alternative antibodies.

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-2^TM 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

In this method, a mammalian cell surface display library is made to display full-size humanized IgG variants that are further selected using FACS. First, we will select an acceptor human V_H and a receptor V_L from a subgroup of human antibodies based on consensus sequences. Next, CDR grafting is conducted as described above. In order to increase (or keep) the affinity of the humanized antibodies, a mammalian cell surface display IgG library is created to display all possible variants of the humanized IgG. Since the size limit for the mammalian cell surface display IgG library, decisions must be made as to which amino acids to diversify and to what extent so that there are fewer nonsense antibody mutants that waste the capacity of the library. Back mutations in framework regions are designed based on computer modeling or antigen/antibody structure information. Also, we will distinguish between residues with solvent accessible side chains from those with buried side chains. We have confirmed that randomization of residues with buried side chains is a waste of library sequence space. Also, we do not mutate glycines and tryptophans since changes in these residues usually abolish binding. Sometime, we study the AA sequence of the parent antibody to find out conserved framework positions (in comparison with germ-line and antibody subfamily sequences). We may then introduce mutations to the positions in the frame work regions that are not conserved. Supposedly, these regions will be antigen-specific and change in these regions may increase affinity but not immunogenicity. In order to create a largest possible library, trimer codon technology is employed to randomize those defined framework residues on both humanized heavy-chain and humanized light-chain.

Finally, the humanized IgG library is created, in which the cDNA of the heavy chain variants (with positions to be randomized using trimer codons) are cloned into a mammalian cell surface display vector, while the cDNA of the light chain variants are inserted into a separate mammalian expression vector to be expressed as free (not membrane-anchored proteins). The cDNA of the heavy-chain library and the light-chain library is then mixed and transfected into 293 cells for surface display of functional whole size humanized IgG variants. Top binders are then sorted by FACS. After stringent selections, a fraction of ELISA positive mutant binders will have a higher affinity than the parent antibody. The affinity of the positive clones will be ranked using the Surface Plasmon Resonance instrument Biacore. Top 3-5 clones will then be expressed and purified to measure the affinity.

This method allows selection of humanized antibodies in a full-size IgG format that retain or increase the original affinity of the mouse antibody. Also, in comparison with the above elaborated bacterial phage display based methods, this mammalian cell surface display approach allows selection of high affinity humanized antibodies in a dimeric IgG format; selected IgG are immediately good for further downstream applications—avoiding time consuming conversion of scFv to IgG and codon optimization of converted IgG (for expression in mammalian cells), which sometime deselect some humanized scFvs. Also, some good antibodies have sequences that prevent protein synthesis and phage display in bacterial cells; these problems can be well overcome in this exclusively mammalian cells based system.

Humanization CDR SDR Grafting

Features of Our Humanization Services

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.

Other optional Human or Humanized Antibody Services:

Published Data

Humanization of Murine Monoclonal neutralizing Antibody Against Human Adenovirus 7

Fig. 3 Amino acid sequences of the VH and VL chains of murine antibody 10G12 and humanized antibodies. (Lei Chen, 2023)

Human adenovirus type 7 (HAdV7) is usually associated with febrile acute respiratory disease (ARD) outbreaks. 10G12, a humanized drug candidate, is a mouse monoclonal antibody (mAb) that specifically recognizes and neutralizes HAdV7. Here, the researchers designed six variants of 10G12, which increased the degree of humanization and studied their biological activities. Humanized monoclonal antibody 10G12-M2 has been proven to have high binding affinity, specificity, and strong virus inhibition of parent antibodies, and can recognize conformational neutralizing epitopes of Hexon proteins. Through molecular docking simulation experiments, the results show that there are several interactions between Hexo protein and 10G12-M2, including hydrogen bond and salt bridge interactions. Generally speaking, 10G12-M2 has excellent biological activity after humanization and has the potential to be used to prevent or treat HAdV7.

Reference

Chen, Lei, et al. "Humanization and characterization of a murine monoclonal neutralizing antibody against human adenovirus 7." Virology 583 (2023): 36-44.

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.