Overview of Orthogonal Fab

Background of Orthogonal Fab

Bispecific antibodies (BsAbs) have emerged as promising therapeutic molecules, capable of simultaneously targeting two different antigens or epitopes. These unique antibodies combine the specificities and properties of two monoclonal antibodies (mAbs) into a single molecule, offering synergistic activities and novel mechanisms of action for disease intervention. BsAbs can facilitate co-clustering of signaling receptors, enable immune cells to kill cancer cells, and block multiple pathways involved in inflammation or tumor escape. While several BsAb formats exist, such as diabodies, IgG-scFv, and CrossMab, they often come with drawbacks, including altered native antibody geometry, complex production processes, and bivalent antigen recognition-induced receptor activation.

Orthogonal Fab presents a revolutionary approach to overcome the limitations of existing BsAb formats. It is a novel format of bispecific IgG antibodies, developed through structure-based design of an orthogonal Fab interface. By modifying the Fab interface of each parental mAb and co-expressing them in the same cell, Orthogonal Fab achieves correct heavy chain-light chain pairing. Crucially, Orthogonal Fab retains the stability and solubility of native IgG, while binding to target antigens monovalently to avoid receptor activation. This format also lends itself to generating trispecific antibodies (TsAbs), denoted as OrthoTsAbs, capable of binding to three different antigens or epitopes by combining two orthogonal Fabs with distinct geometries and mechanisms of action.

Structure Features of Orthogonal Fab

The core of Orthogonal Fab's design lies in its ability to self-assemble from two different antibodies through Fab interface modification. The Fab interface is the region where the heavy chain and the light chain interact with each other. Through computational and rational design approaches, the Fab interface is engineered to establish orthogonal heavy chain-light chain pairs, ensuring that each light chain binds with higher affinity to its cognate heavy chain than the noncognate heavy chain. This strategic engineering mitigates the light chain mispairing issue and ensures the correct assembly of bispecific IgG. Orthogonal Fab possesses a native-like IgG structure, preserving its well-known stability and solubility. However, its distinct feature lies in the monovalent antigen recognition capability due to each Fab arm's unique specificity, making it highly desirable for certain clinical applications where receptor activation needs to be avoided. Furthermore. Orthogonal Fab can be expanded to generate OrthoTsAbs by introducing a third specificity to one of the Fc domains.

Fig.1 Schematic Diagrams of Various OrthoTsAbs (Xiufeng Wu, 2018)

Generation Method of Orthogonal Fab

The generation of Orthogonal Fab involves generated by co-expressing two parental monoclonal antibodies, each engineered with orthogonal Fab interfaces. These interfaces are meticulously designed using computational methods and the X-ray crystal structures of the parental antibodies as a foundation. The design strategy aims to create mutations at the heavy chain-light chain interface that enhance the affinity and specificity of each light chain for its cognate heavy chain while reducing these properties for the noncognate heavy chain. Experimental validation of the orthogonal Fab interfaces is carried out using techniques such as surface plasmon resonance (SPR), analytical ultracentrifugation (AUC), size-exclusion chromatography (SEC), and electrospray ionization mass spectrometry (ESI-MS). The co-expression of orthogonal Fab antibodies can be performed in mammalian cells (e.g., Chinese hamster ovary cells), or bacterial cells (e.g., Escherichia coli cells). To obtain purified orthogonal Fab antibodies, protein A affinity chromatography followed by ion exchange chromatography or hydrophobic interaction chromatography is employed. Comprehensive characterization of the orthogonal Fab antibodies involves techniques like SDS-PAGE, SEC, ESI-MS, SPR, ELISA, and cell-based assays. Further optimization of orthogonal Fab antibodies is achieved through directed evolution methods, such as phage display or yeast display, enhancing their affinity, stability, solubility, and functionality.

Advantages and Disadvantages of Orthogonal Fab

Orthogonal Fab is a bispecific or trispecific IgG antibody with a native-like structure and monovalent antigen recognition capability. The production process is simplified by co-expressing two parental monoclonal antibodies with orthogonal Fab interfaces, eliminating the need for extensive engineering of individual antibodies. Additionally, Orthogonal Fab can be easily optimized using directed evolution methods to improve its performance.

However, Orthogonal Fab does have certain limitations. The engineering of orthogonal Fab interfaces requires computational and rational design approaches, which may not be universally applicable for all antibody pairs and could potentially introduce unwanted mutations that affect antigen binding or folding. There may also be some degree of light chain mispairing or aggregation, leading to reduced purity, yield, or efficacy. Furthermore, Orthogonal Fab may exhibit lower avidity compared to other bispecific or trispecific antibody formats that bind to antigens bivalently or multivalently, potentially impacting its potency or pharmacokinetics.

From the table above, it becomes evident that Orthogonal Fab technology holds distinct advantages over other technologies, especially in maintaining the native IgG structure and properties while enabling ability to monovalent recognition of multiple antigens. This feature helps avoid unnecessary side effects such as receptor activation, cell aggregation or cross-linking. However, Orthogonal Fab technology also has certain drawbacks, primarily the requirement for Fab interface modification, which may affect antibody affinity or stability. Hence, careful consideration and corresponding optimization are necessary during  the design and screening of Orthogonal Fabs.

The Application of Orthogonal Fab Technology in Clinical Trials

To date no Orthogonal Fab-based bispecific antibody has received approval. Nevertheless, several candidates are undergoing clinical trials for various indications.

A brief overview of these bispecific antibodies is as follows:

• LY3164530: Binds to HER3 and EGFR, two receptor tyrosine kinases involved in tumor growth and survival. May inhibit tumor signaling and induce antibody-dependent cellular cytotoxicity (ADCC) against tumor cells by blocking both receptors.
• LY3434172: Binds to PD-L1 and TGF-βRII, molecules implicated in immune suppression in the tumor microenvironment. By blocking PD-L1, LY3434172 may enhance T cell activation and anti-tumor immunity. It may also inhibit TGF-β signaling to prevent tumor invasion, metastasis, and immune evasion.
• LY3502970: Binds to PD-L1 and 4-1BB, molecules involved in T cell regulation. By blocking PD-L1, LY3502970 enhances T cell activation and anti-tumor immunity. It also stimulates 4-1BB to promote T cell proliferation, survival, and effector function.
• LY3415244: Binds to CD3 and CD33, molecules expressed on T cells and AML cells, respectively. By bringing T cells and AML cells into close proximity, LY3415244 activates T cells and induces T cell-mediated cytotoxicity against AML cells.
• LY3321367: Binds to TIM-3 and PD-L1, two molecules involved in immune exhaustion in the tumor microenvironment. By blocking both molecules, LY3321367 may restore T cell function and anti-tumor immunity.
• LY3437940: Binds to CD3 and BCMA, two molecules expressed on T cells and MM cells, respectively. By bringing T cells and MM cells into close proximity, LY3437940 activates T cells and induces T cell-mediated cytotoxicity against MM cells.

Conclusion

Orthogonal Fab technology offers a promising approach to generating of bispecific or trispecific IgG antibodies with monovalent antigen recognition. Its advantages lie in the native-like structure, stability, solubility, and simplified production and optimization process. However, some limitations, such as the need for computational and rational design, the possibility of light chain mispairing or aggregation, and the lower avidity compared to bivalent or multivalent formats, should be carefully considered when applying Orthogonal Fab in different clinical applications. Future research should focus on enhancing the design and engineering of orthogonal Fab interfaces, reducing light chain mispairing or aggregation, and improving the potency and pharmacokinetics of Orthogonal Fab antibodies. Additionally, more clinical data are required to evaluate the safety and efficacy of Orthogonal Fab antibodies in different indications and patient populations.

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