Overview of Fcab

Background of Fcab

Antibodies have emerged as promising therapeutic agents for various diseases, such as cancer, autoimmune disorders, and infectious diseases. While the conventional immunoglobulin G (IgG) format has been widely used, it does come with certain limitations, such as large size, high production cost, immunogenicity, and potential toxicity. To address these challenges and enhance the efficacy and safety of antibody-based therapeutics, alternative antibody formats including single-chain variable fragments (scFvs), diabodies, single-domain antibodies (sdAbs), bispecific antibodies (bsAbs), and antibody-drug conjugates (ADCs) have been developed. One such innovative formats is the Fc fragment with antigen-binding (Fcab), a novel construct introduced by Obinger and colleagues in 2010.

Fcab is a cutting-edge antibody format that comprises a homodimeric Fc region engineered with an antigen-binding site. This unique combination allows Fcabs to harness Fc domain-mediated functions and antigen-binding capabilities simultaneously. Notably, Fcab is one-third smaller than full-length antibodies, enabling higher tissue penetration, which is especially advantageous for the treatment of solid tumors. Additionally, Fcab can serve as an excellent foundation for creating targeted antibody-drug conjugates (ADCs) that achieve precise drug delivery by specifically coupling cytotoxic drugs to Fcab.

Structural Features of Fcab

At its core, Fcab consists of a homodimeric Fc region with a strategically engineered antigen-binding site located on each CH3 domain. The Fc region originates from human IgG1, retaining the ability to bind to Fc receptors (FcRs) and neonatal Fc receptor (FcRn). These interactions mediate the effector functions and serum half-life of Fcab, respectively. The antigen-binding site is carefully crafted by introducing mutations into the C-terminal loops of the CH3 domain, specifically the AB-loop, the CD-loop, and the EF-loop. By modifying=these loops, different antigen-binding residues can be accommodated without compromising the integrity of the Fc structure.

Engineering the antigen-binding site can be accomplished through various methods, such as targeted mutagenesis, directed evolution, yeast display, or phage display. The selection and affinity maturation of Fcab clones can be performed by screening large libraries of Fcab variants for specific binding to target antigens. For example, Obinger and colleagues generated a high-affinity Fcab against CD16A by performing targeted mutagenesis of the AB-loop and CD-loop of the CH3 domain. Similarly, Wozniak-Knopp and colleagues generated a low-nanomolar affinity Fcab against VEGF through directed evolution and yeast display of all three C-terminal loops and the C-terminus of the CH3 domain.

The structural and stability aspects of Fcab have been thoroughly investigated using several biophysical techniques, including X-ray crystallography, nuclear magnetic resonance (NMR), circular dichroism (CD), differential scanning calorimetry (DSC), and molecular dynamics (MD) simulations. For instance, the crystal structure of an Fcab bound to HER2 revealed an antigen-binding site formed by a protruding CD-loop interacting with two HER2 molecules in a negative cooperative manner. Furthermore, NMR and CD studies showed that Fcab retains a similar secondary structure and thermal stability as wild-type Fc. Moreover, DSC analysis indicated that Fcab exhibits a higher melting temperature than Fab. Finally, MD simulations highlighted the presence of a flexible CD-loop in Fcab, capable of adopting diverse conformations in solution.

Generation Methods of Fcab

Fcab can be synthesized using various sophisticated techniques, including targeted mutagenesis, directed evolution, yeast display, and phage display. These methods effectively introduce mutations into the C-terminal loops of the CH3 domain, namely the AB-loop, the CD-loop, and the EF-loop, to facilitate the creation of an antigen-binding site. The process of selecting and refining Fcab clones involves screening large libraries of Fcab variants to identify those that exhibit specific binding to the desired target antigens.

Targeted mutagenesis involves the deliberate introduction of pre-defined mutations into specific residues or regions of the CH3 domain. For example, Obinger and his team successfully generated a high-affinity Fcab targeting CD16A by employing targeted mutagenesis on the AB-loop and CD-loop of the CH3 domain. They adopted a rational design strategy based on structural modeling and sequence alignment to pinpoint potential antigen-binding residues. Subsequently, they constructed a comprehensive Fcab variant library, incorporating different combinations of mutations, which was then subjected to screening via flow cytometry to identify the variants displaying binding to CD16A-expressing cells.

Directed evolution introduces random mutations throughout the entire CH3 domain or selected regions via error-prone PCR or DNA shuffling. For instance, Wozniak-Knopp and his colleagues successfully generated a low-nanomolar affinity Fcab targeting VEGF using directed evolution and yeast display techniques on all three C-terminal loops and the C-terminus of the CH3 domain. They used error-prone PCR to generate diverse Fcab variant library, with an average mutation rate of 1.5 mutations per clone. Subsequently, they displayed the Fcab variants on the surface of yeast cells and conducted screening through fluorescence-activated cell sorting (FACS) to identify the variants exhibiting binding to VEGF.

Yeast display involves expressing Fcab variants as fusion proteins with an anchor protein on the surface of yeast cells. These Fcab variants can be displayed as either secreted proteins in the culture supernatant or as membrane-bound proteins on the yeast cell surface. The latter option nables efficient screening and selection of Fcab clones using flow cytometry or FACS. In contrast, phage display entails expressing Fcab variants as fusion proteins with a coat protein on the surface of bacteriophages. The Fcab variants can be presented as either monovalent or multivalent formats on different types of phages, such as M13, fd, or T7. To enrich for specific binders, the phage display library can be subjected to panning against immobilized target antigens or antigen-expressing cells.

Advantages and Disadvantages of Fcab

Fcab offers several advantages over IgG and other alternative antibody formats. One of its key benefits is the ability to combine Fc-mediated functions, such as long serum half-life, effector cell activation, and complement-dependent cytotoxicity, while being the antigen-binding function at only one-third of the size of IgG. This unique feature enhances the pharmacokinetics and pharmacodynamics of Fcab in vivo. Moreover, Fcab demonstrates superior tissue penetration capabilities, making it highly attractive for the treatment of solid tumors. Additionally, Fcab serves as an excellent scaffold for creating antibody-drug conjugates (ADCs), where cytotoxic drugs can be site-specifically linked to Fcab. Fcab-ADCs retain the FcRn and antigen binding properties of the parent Fcab, exhibiting selective cell killing and serum stability. Furthermore, Fcab-ADCs show enhanced penetration in tumor spheroids compared to IgG-ADCs. Fcab generation can be accomplished using various methods, such as targeted mutagenesis, directed evolution, yeast display, or phage display, enabling the introduction of antigen-binding sites into the C-terminal loops of the CH3 domain. The selection and affinity maturation of Fcab clones can be carried out through the screening of extensive Fcab variant libraries, specifically selecting those with binding specificity to target antigens.

Fig.1 Conceptual Representation of Potential Advantages of Fcab-Drug Conjugates (Jäger S, 2021)

However, Fcab is not without its drawbacks. Some of its disadvantages include lower avidity, higher immunogenicity, lower expression or production yield, and lower stability compared to IgG. One of the main concerns is that Fcab may exhibit lower avidity than IgG due to its monovalent binding mode. This could potentially impact its potency and efficacy in certain applications, particularly when the target antigen has low density or affinity. Additionally, Fcab may display higher immunogenicity compared to IgG, as the introduction of foreign residues or loops into the CH3 domain may lead to unwanted immune responses or anti-drug antibodies in human patients. Fcab might also have lower expression or production yield compared to IgG, owing the engineering of the CH3 domain, potentially affecting the cost and scalability of Fcab manufacturing, requiring additional optimization or purification steps. Furthermore, Fcab may demonstrate lower stability than IgG, attributed to the flexibility or aggregation of the C-terminal loops of the CH3 domain necessitating more stringent storage conditions or formulations.

Clinical Data of Fcab

Fcab has undergone evaluation in clinical trials for various diseases, such as cancer, autoimmune disorders, and infectious diseases. For instance, efgartigimod (efgartigimod alfa-fcab, Vyvgart™) developed by argenx, is a first-in-class neonatal Fc receptor antagonist designed to treat autoimmune diseases. Efgartigimod contains an Fc region with an engineered antigen-binding site, which binds to FcRn and inhibits its interaction with IgG, thereby reducing serum levels of pathogenic IgG antibodies and alleviating autoimmune disease symptoms. Efgartigimod has been approved in the USA and Japan for treating generalized myasthenia gravis in adults who are anti-acetylcholine receptor (AChR) antibody positive. It is also undergoing clinical trials for other autoimmune diseases, such as bullous pemphigoid, chronic inflammatory demyelinating polyradiculoneuropathy, immune thrombocytopenia, autoimmune myositis and pemphigus. Furthermore, HER2-Fcab (HER2-binding Fcab) is a high-affinity Fcab developed by F-star Therapeutics to target HER2-positive cancers. HER2-Fcab induces HER2 degradation and apoptosis. Currently, HER2-Fcab is in phase I clinical trials for patients with advanced solid tumors that are refractory to standard therapies. Additionally, LAG-3-Fcab (LAG-3-binding Fcab) binds to LAG-3 and blocks its interaction with MHC class II. Merck KGaA has developed LAG-3-Fcab for the treatment of cancer and it can be used as a building block for novel bispecific antibody therapeutics targeting LAG-3 and other antigens. LAG-3-Fcab is currently in preclinical development.

References

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