Since the first report of the development of monoclonal antibodies (mAbs) using hybridoma cell lines in 1975, and the approval of the first murine monoclonal antibody for human therapy in 1986, the subsequent recognition that mAbs could be engineered to improve their efficacy has inspired the construction of a series of novel antibodies: chimeric antibodies, humanized antibodies, and fully human antibodies produced in transgenic mice (to reduce the immunogenicity of rodent antibodies). The focus has subsequently expanded to include enhancing effector function, controlling half-life, tumor and tissue accessibility, enhancing antibody physical properties (such as stability), and more efficient (lower cost) engineering production.
(Moraes J. Z, et al. Current Research in Immunology. 2021)
One of the advantages of recombinant antibodies is the ability to engineer antibodies. With the primary amino acid sequence of the antibody, researchers can use the original sequence to create a variety of antibodies.
Chimeric antibodies
Most mouse antibodies have limited therapeutic applications due to their short serum half-life, inability to trigger human antibody effector functions, and immune rejection. To reduce the immunogenicity of mouse antibodies, chimeric antibodies containing human constant domains and mouse variable domains are genetically engineered to retain antibody specificity. Furthermore, to address cross-reactivity issues associated with detection antibodies due to species origin, the constant regions of antibodies derived from different species can be replaced to alter the antibody's species identity (mouse, rabbit, or human).
Based on the antibody engineering platform, Mabnus Bio has undergone extensive screening and evaluation, including Fc receptor binding (SPR) and C1q binding (ELISA), and can tailor chimeric antibodies with different subtypes and antibody backbones according to the specific antibody application requirements of academic research or industry.
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Humanized antibodies
Antibody humanization is an essential technique for reducing the potential immunogenicity risks associated with animal-derived antibodies and has been applied to the majority of therapeutic antibodies on the market. However, other properties must also be considered in the current humanization of antibodies (including binding affinity, physicochemical stability, host cell expression and pharmacokinetics, as well as the basic antibody engineering methods involved).
(Hebditch M, et al. PeerJ. 2019)
Antibody humanization involves transplanting amino acids from the complementarity-determining regions (CDRs) onto a human germline framework while retaining key non-human amino acid backmutations. This is a balance between introducing as much human sequence as possible to reduce the risk of immunogenicity while retaining the core amino acids of the parent antibody to maintain its original binding activity, with additional consideration given to the antibody's developability throughout the process.
Our antibody humanization platform combines the advantages of both rational and empirical approaches to antibody humanization. Humanized antibodies are designed using our proprietary humanization and optimization algorithms and are produced through gene synthesis and fusion of humanized antibody variable regions with a human antibody backbone. After a comprehensive analysis of the humanized parental monoclonal antibody sequence and structural information, multiple frameworks are selected from homologous human mature antibody germline sequences to avoid potential limitations imposed by specific frameworks during design. 3D structural modeling is used to identify positions in the human sequence requiring back mutations to restore CDR conformation and optimal antigen binding. Structural algorithms generate multiple possibilities for each position, identifying the optimal antibody sequence for maximum activity and specificity.
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Bi/multispecific antibodies
Although natural antibodies are monospecific, multispecific antibodies, which can recognize different epitopes on the same or different antigens, are gaining increasing attention for diagnostic and therapeutic applications. Initially, therapeutic applications focused on retargeting effector cells for cancer treatment, including T cells that cannot be recruited to tumor cells by normal antibodies.
(Labrijn AF, et al. Nat Rev Drug Discov. 2019)
Over the past decade, researchers have established numerous other therapeutic strategies based on bispecific antibodies. In addition to retargeting effector molecules, cells, and genetic vectors, they have also explored dual targeting and pretargeting strategies, half-life extension, and delivery across biological barriers such as the blood-brain barrier. MysBio's extensive experience in multispecific antibody projects enables clients to design and express diverse multispecific antibody formats. Starting with a hybridoma cell line or antibody sequence, we can deliver custom multispecific antibodies in milligram to gram quantities.
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Fragment Antibodies
In many applications of antibodies, Fc-mediated effects are not required: antibodies are only used to block signaling molecules or receptors, and a common solution is to use antibody fragments that lack the Fc domain: solid tumors often prevent antibodies from penetrating to the center and lead to reduced therapeutic efficacy. The use of smaller fragments can achieve deeper penetration; the long serum half-life mediated by the interaction of Fc with the FcRn receptor leads to poor contrast. Fragment antibodies can also be used for radiolabeled imaging and cancer treatment. Rapid clearance from the circulation through the kidneys is also beneficial for reducing long-term radiation exposure; when targeting multiple disease-associated antigens, multimeric antibody fragment design is also a candidate strategy.
Based on our antibody engineering platform, Mabnus Bio can optimize and synthesize antibody fragments. Using our proprietary antibody fragment expression vectors, we can significantly increase soluble expression yields, retain the specificity of the original immunoglobulin, and in some applications outperform intact IgG in terms of construction speed, production yield, and engineering flexibility.
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Fc transformation
For general detection antibodies, we mainly consider their specificity and affinity. However, for functional antibodies, in addition to these two indicators, we also need to consider antibody-mediated humoral immunity and innate immune activity. The pharmacological properties of antibodies largely depend on their Fc region.
Complement-dependent cytotoxicity (CDC): After an antibody binds to its corresponding antigen, a conformational change exposes the complement binding site of the Fc region, activating the complement system and forming a membrane attack complex (MAC) on the surface of target cells, mediating target cell lysis.
Antibody-dependent cell-mediated cytotoxicity (ADCC): The antibody Fc region binds to the FcR on the surface of killer cells, mediating the release of cytokines and cytotoxic granules from killer cells to directly kill target cells. NK cells are the primary cells mediating ADCC.
Antibody-dependent cell-mediated phagocytosis (ADCP): The Fc region of an antibody binds to the FcR of macrophages or neutrophils, promoting phagocytosis by phagocytes through the IgG bridge function.
Extending the half-life of IgG: The binding of the antibody Fc region to the neonatal Fc receptor FcRn is pH-dependent. It binds to FcRn with high affinity under acidic conditions within the cell and rapidly dissociates from FcRn under slightly alkaline conditions in the blood. This receptor-mediated recycling mechanism prolongs the half-life of IgG.
For years, scientists have studied the structures involved in antibody Fc binding to various ligands, aiming to modify the natural properties of antibodies. Currently, a large number of different Fc mutants have been developed to enhance or silence Fc effector functions. Based on its antibody engineering platform, Mabnus Bio can generate functionally differentiated recombinant antibodies by targeting core amino acid sites on the Fc to address various antibody-mediated functions (enhancing effector function, reducing effector function, and extending serum half-life).
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