Overview of Fab-arm Exchange Technology

What is Fab-arm Exchange Technology

Bispecific antibodies (bsAbs) are a class of antibodies that can simultaneously recognize two different targets or epitopes, with a wide range of clinical applications. Compared with traditional monospecific antibodies, bispecific antibodies can achieve various therapeutic effects, such as blocking two signaling pathways, redirecting immune cells to kill tumor cells, enhancing antigen presentation or facilitating drug delivery. Currently, several bispecific antibody technologies have been developed, such as heavy chain heterodimerization, single chain variable fragment fusion, and cross-linking. However, these technologies require complex engineering modifications of antibodies, or involve the expression and assembly of multiple peptide chains, which increase the production cost and difficulty.

Fig.1 Characteristics of IgG4

Fab-arm exchange is a technology that uses the natural phenomenon of half-antibody exchange of IgG4 antibodies to generate bispecific antibodies. IgG4 has low effector function and mainly exerts its effect by blocking or modulating targets. Fab-arm exchange technology only requires simply mixing two IgG4 antibodies with different target specificities and incubating them under mild reducing conditions for a certain time to achieve efficient generation of bispecific antibodies. This technology has the advantages of high efficiency, universality, flexibility and controllability, and has been widely used in various targets and disease areas.

Structural Features of Fab-arm Exchange

The antibody types involved in Fab-arm exchange are mainly IgG4. IgG4 is a subtype of human immunoglobulin G (IgG), accounting for 3%-6% of the total IgG in human serum. IgG4 differs from other IgG subtypes in that it has a reversible reduction site (S228P) at the inter-heavy chain disulfide bond, causing the inter-heavy chain disulfide bond to be temporarily reduced to half-antibodies. Half-antibodies can re-form intact antibodies with other IgG4 half-antibodies, resulting in different light chain pairing or bispecificity. This phenomenon is called Fab-arm exchange or heavy chain exchange.

IgG4 is structurally similar to IgG1. It consists of two heavy chains and two light chains, each heavy chain and light chain are connected by a disulfide bond, forming a Fab fragment. Two Fab fragments are connected by a hinge region, forming a F(ab')2 fragment. The hinge region contains two pairs of inter-chain disulfide bonds that connect the two heavy chains together, forming an Fc fragment. The end of the Fc fragment contains a variable region (CH3) for binding to Fc receptors or complement. The peculiarity of IgG4 is that its S228P mutation makes it balance between inter-heavy chain and intra-heavy chain disulfide bonds. Under mild reducing conditions, the inter-heavy chain disulfide bonds can be opened, releasing half-antibodies. Half-antibodies can recombine with other sources of half-antibodies, forming new intact antibodies. In this way, it is possible to have two Fab fragments with different target specificities connected to the same Fc fragment, forming bispecific antibodies. IgG4 has low effector function and mainly exerts its effect by blocking or modulating targets. It binds weakly to FcγRs other than FcγRI (CD64), and cannot induce complement-dependent cytotoxicity (CDC) and natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but can induce macrophage-mediated antibody-dependent cellular phagocytosis (ADCP). These properties make IgG4 exhibit a relatively "non-inflammatory" characteristic, more suitable for therapeutic monoclonal antibody production.

Generation Methods of Fab-arm Exchange

Fab-arm exchange is a technology that uses the natural phenomenon of half-antibody exchange of IgG4 antibodies to generate bispecific antibodies. This technology only requires simply mixing two IgG4 antibodies with different target specificities and incubating them under mild reducing conditions for a certain time to achieve efficient generation of bispecific antibodies. This technology has the advantages of high efficiency, universality, flexibility and controllability, and has been widely used in various targets and disease areas. The specific steps of Fab-arm exchange technology are as follows:

• Express and purify two IgG4 antibodies with different target specificities separately, one containing the K409R mutation and the other containing the F405L mutation (European numbering), which are located in the CH3 region of the heavy chain and can promote pairing between half-antibodies.
• Mix the two IgG4 antibodies in a certain ratio and add an appropriate amount of reducing agent (such as DTT or TCEP), incubate at 37°C for a certain time (usually 2-24 hours), so that the inter-heavy chain disulfide bonds are opened, releasing half-antibodies.
• Remove the reducing agent and incubate under oxidizing conditions for a certain time (usually 2-24 hours), so that half-antibodies re-form disulfide bonds, generating bispecific antibodies.
• Analyze the exchange efficiency and the purity, stability and functionality of the final product by chromatography or mass spectrometry methods.

Fab-arm exchange technology can be scaled from laboratory scale (micrograms to milligrams) to industrial scale (milligrams to grams) or even production scale (grams to kilograms), using similar exchange conditions and standard antibody processing techniques. Starting from high-quality purified proteins, exchange efficiencies of ≥95% can routinely be obtained within 2–3 days (including quality control).

Advantages and Disadvantages of Fab-arm Exchange

Fab-arm exchange technology is a technology that uses the natural phenomenon of half-antibody exchange of IgG4 antibodies to generate bispecific antibodies. This technology has the following advantages:

• Simple process: Only two mutation sites need to be introduced in the CH3 region of two IgG1 antibodies, which can realize the pairing and recombination of half-antibodies.
• Natural structure: The generated bispecific antibodies retain the Fc fragment and Fab fragment of IgG1, and have a structure, function and pharmacokinetics similar to natural IgG1.
• High purity: Bispecific antibodies can be easily separated and purified by chromatography or mass spectrometry methods, and the exchange efficiency can usually reach more than 95%.
• High flexibility: Different IgG1 antibodies can be selected for mixing and exchange according to different targets and disease areas, generating various combinations of bispecific antibodies.

Fab-arm exchange technology also has the following disadvantages:

• Low stability: Because the inter-heavy chain disulfide bonds can be opened by reducing agents, bispecific antibodies may dissociate or re-exchange under reducing conditions, leading to changes in structure and function.
• Low effector function: Because IgG4 has low effector function, bispecific antibodies cannot effectively induce complement-dependent cytotoxicity (CDC) and natural killer cell-mediated antibody-dependent cellular cytotoxicity (ADCC), and can only exert their effects by blocking or modulating targets.
• High immunogenicity: Because bispecific antibodies contain different light chain pairing or bispecificity, they may be recognized as foreign by the human immune system, causing immune reactions or neutralizing antibody production.

Fab-Arm Exchange in Clinical Applications

Fab-arm exchange technology has been used to develop various bispecific antibodies targeting different diseases and antigens. Currently, there are some Fab-arm exchange-based bispecific antibodies undergoing clinical trials or already on the market. For example, amivantamab (JNJ-61186372) is a bispecific antibody targeting EGFR and cMET for the treatment of non-small cell lung cancer (NSCLC). The antibody was generated by Fab-arm exchange technology and has efficient Fc-mediated effects, including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). The antibody was granted accelerated approval by the US FDA in December 2020 for the treatment of EGFR exon 20 insertion-positive advanced or metastatic NSCLC. Moreover, GEN3009 (DuoHexaBody-CD37) is a bispecific antibody targeting CD37 for the treatment of B-cell malignancies. The antibody was generated by Fab-arm exchange technology and introduced the E430G mutation to enhance IgG hexamerization and thus CDC activity. The antibody has entered clinical phase I/IIa trials for the treatment of relapsed or refractory chronic lymphocytic leukemia (CLL) and non-Hodgkin lymphoma (NHL). Another example is GEN1046 (DuoBody-PD-L1×4-1BB), a bispecific antibody targeting PD-L1 and 4-1BB for the treatment of solid tumors. The antibody was generated by Fab-arm exchange technology and has dual immunomodulatory effects: it can block the interaction between PD-L1 and PD-1, thereby relieving the inhibition of tumor cells on immune cells, and it can activate the 4-1BB signaling pathway, thereby enhancing the proliferation and effector function of immune cells. The antibody has entered clinical phase I/II trials for the treatment of various solid tumors.

Conclusion

Fab-arm exchange is a new method for generating bispecific antibodies with advantages such as simplicity, efficiency, and stability. Fab-arm exchange technology can utilize existing IgG production and purification technologies, without the need for additional engineering or fusion proteins. Fab-arm exchange technology can flexibly choose two different targets, without considering affinity or pairing issues. Fab-arm exchange technology can generate bispecific antibodies with normal IgG structure and function, including Fc effects, half-life, stability, etc. Fab-arm exchange technology has been used to develop various bispecific antibodies targeting different diseases and antigens. Currently, there are some Fab-arm exchange-based bispecific antibodies undergoing clinical trials or already on the market, showing good therapeutic efficacy and safety.

However, Fab-arm exchange technology also has some limitations and challenges. For example, it requires point mutations in the two parental antibodies, which may affect their affinity or stability. It requires Fab arm exchange under reducing conditions, which may cause partial denaturation or aggregation of the antibodies. It requires strict quality control of the final product to ensure exchange efficiency and purity. In addition, Fab-arm exchange technology still needs to prove its long-term safety and efficacy in larger-scale clinical trials, as well as its relative advantages over other bispecific antibody formats.

Therefore, Fab-arm exchange technology as a new method for generating bispecific antibodies has great potential and prospects, but also needs further optimization and validation. In the future, Fab-arm exchange technology may be combined with other technologies to generate more diversified and functionalized multispecific antibodies, providing more options and possibilities for immunotherapy.

References

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