Antibody fusion proteins (AFPs) are engineered biopharmaceuticals in which antibodies are genetically fused with other bioactive proteins to create multifunctional molecules with enhanced therapeutic potential. These chimeric proteins combine the high specificity of antibodies with the biological activity of cytokines, enzymes, ligands, or other functional proteins, allowing for targeted therapeutic action and improved pharmacokinetics.

While early antibody fusion constructs were prepared through chemical crosslinking methods, these approaches often resulted in heterogeneous and unstable products with large molecular size, poor tissue penetration, and elevated immunogenicity. The rapid progress of genetic engineering and recombinant antibody technology-particularly the advent of phage display libraries in the 1990s-has revolutionized the design and production of antibody fragments such as Fab and scFv. Their small molecular size, strong binding activity, high stability, and ease of genetic manipulation have made them ideal building blocks for modern antibody fusion protein construction.

Table 1. Comparison of Monoclonal Antibodies and Antibody Fusion Proteins

The design of AFPs typically involves connecting the antibody and the functional protein through a flexible peptide linker. This linker maintains the structural integrity of both domains while enabling efficient expression in prokaryotic, eukaryotic, mammalian, or even plant expression systems, supporting scalable biomanufacturing.

The length and composition of the linker play a crucial role in the folding, stability, and immunogenicity of the fusion protein. A linker that is too short may disrupt tertiary structure and interfere with function, whereas an excessively long linker could increase immunogenicity.

By combining the targeting capability of antibodies with the activity of biofunctional proteins, antibody fusion proteins have become powerful tools in immunodiagnostics, immunotherapy, targeted drug delivery, and bioassay development. Their ability to deliver therapeutic agents precisely to disease sites has positioned AFPs as next-generation "guided biotherapeutics."

Figure 1. The structure of a prototypic IgG Fc-fusion and the means by which it can be presently modified. (Czajkowsky DM, et al., 2012)

Classification of Antibody Fusion Proteins

Antibody fusion proteins can be broadly divided into two categories:

1. Fc-Containing Antibody Fusion Proteins

These molecules consist of the Fc region of IgG1 fused to functional proteins that mediate antigen binding or biological activity. The Fc fragment not only contributes to effector functions-such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)-but also extends serum half-life via interaction with neonatal Fc receptors (FcRn).

Examples include:

• Abatacept (CTLA-4-Ig): a fusion of CTLA-4 and IgG1 Fc used in rheumatoid arthritis.
• Alefacept (LFA-3-Ig): fusing lymphocyte function-associated antigen 3 with Fc for psoriasis therapy.

Fc fusion proteins can be further subclassified into:

• Cytolytic Fc-fusion proteins, which retain Fc effector functions for cell killing.
• Non-cytolytic Fc-fusion proteins, engineered with mutations to eliminate Fc receptor or complement binding, retaining only half-life extension and protein stability.

The choice of Fc domain-commonly from human IgG1 due to its long half-life and strong FcγR affinity is critical. Fc engineering strategies can tune ADCC and CDC activity to achieve the desired therapeutic effect.

2. Fab/scFv-Based Fusion Proteins

These fusions utilize antibody fragments (Fab or scFv) combined with bioactive proteins such as cytokines, toxins, enzymes, or ligands. Their small size allows efficient tissue penetration and targeted delivery, functioning as "biological missiles" that direct therapeutic payloads precisely to antigen-expressing cells.

In most designs, the functional protein is fused to the C-terminus of the antibody fragment to avoid steric interference with the antigen-binding site at the N-terminus.

Mechanisms of Action

Antibody fusion proteins act through several mechanisms:

1. Neutralization or Blocking Activity: By binding and neutralizing disease mediators (e.g., TNFα), they inhibit pathogenic signaling: such as Etanercept's inhibition of TNFα in rheumatoid arthritis.
2. Fc-Mediated Immune Effector Functions: The Fc portion interacts with Fc receptors on immune cells to trigger phagocytosis, ADCC, or immune modulation.
3. Targeted Delivery of Therapeutic Agents: Antibody moieties guide cytotoxic drugs, toxins, or radioisotopes to specific cell populations, minimizing off-target effects and enhancing efficacy.

Approved therapeutic examples include:

• Etanercept: TNF receptor-Fc fusion, treating autoimmune arthritis.
• Abatacept: CTLA-4-Fc fusion, blocking T-cell activation.
• Alefacept: LFA-3-Fc fusion, used in psoriasis.
• Recombinant human TNF receptor-Fc fusion: developed in China for rheumatoid arthritis and ankylosing spondylitis.

Advances in Engineered Targeted Antibody Fusion Proteins

1. Tumor-Targeted Cytokine Antibody Fusion Proteins

Cytokine-antibody fusions are an emerging field in cancer immunotherapy. By fusing cytokines (e.g., IL-2, IL-12, IFN-α) with antibodies that target tumor-associated extracellular matrix (ECM) components, researchers can deliver immune-stimulatory molecules directly into the tumor microenvironment (TME).

Key strategies include:

• F8 antibody targeting fibronectin extra domain A (EDA), overexpressed in tumor vasculature but absent in healthy tissue.
• L19 antibody targeting fibronectin extra domain B (EDB), used in fusion constructs with TNFα, IL-12, and IL-2 currently in clinical trials.
• NHS-IL12 fusion, which binds histones in necrotic tumor areas to enhance localized IL-12 delivery and synergize with radiotherapy and checkpoint blockade.

These targeted cytokine fusions enhance antitumor efficacy while minimizing systemic toxicity.

Figure 2. Recent payload immunocytokine formats. (Silver AB, et al., 2021)

2. Inflammation-Targeted Cytokine Fusion Proteins

Similar ECM-targeting approaches have been applied to autoimmune and inflammatory diseases. Fusions such as F8-IL4, F8-IL9, and F8-IL10 localize anti-inflammatory cytokines to diseased tissues, improving therapeutic outcomes in arthritis, pulmonary hypertension, and graft-versus-host disease (GVHD) models.

Additionally, anti-CD86 scFv-IL10 and Clec9A-targeted IFNα constructs have demonstrated selective immunomodulation with reduced systemic side effects.

3. Signal-Biased Cytokine Antibody Fusion Proteins

Advances in structural immunology have enabled biased cytokine signaling, where engineered cytokine variants preferentially activate desired receptor subunits.

• IL-2/S4B6 complexes enhance effector T-cell expansion without systemic toxicity.
• CEA-IL2v and EGFR-IL2v fusions employ IL-2 mutants with reduced IL-2Rα affinity to selectively stimulate antitumor responses.
• Masked IL-2 fusions, where IL-2 is sterically shielded by IL-2Rβ until released by tumor-associated proteases, further refine spatial control of cytokine activation.

Figure 3. Cytokine incorporation in biased cytokine-antibody fusions. (Silver AB, et al., 2021)

4. Recombinant Immunotoxins (RITs)

RITs consist of antibody fragments fused with potent toxins, combining antibody specificity with intracellular cytotoxicity.

Common toxin payloads include:

• Pseudomonas exotoxin A (PE)
• Diphtheria toxin (DT)
• Shiga toxin 1 (Stx1)

Examples such as Moxetumomab pasudotox and Oportuzumab monatox are FDA-approved RITs for hematologic malignancies. However, challenges remain-particularly immunogenicity and poor tumor penetration. Emerging RITs explore endogenous human enzymes or superantigen fusions (SAgs) to overcome these limitations.

Figure 4. Mechanisms of action for RITs, ADEPTs, targeted enzyme replacement, neuroprotective, and soluble factor trap fusion proteins. (Silver AB, et al., 2021)

Applications and Therapeutic Impact

Antibody fusion proteins are driving innovation across multiple therapeutic areas:

• Autoimmune diseases: Targeted cytokine and receptor-Fc fusions modulate overactive immune responses.
• Cancer immunotherapy: Cytokine, toxin, and checkpoint fusion proteins deliver immune activation directly to tumor sites.
• Infectious diseases: Fc-fusions enhance antiviral antibody durability and effector responses.
• Enzyme replacement therapy: Fc- or antibody-based fusions improve pharmacokinetics and tissue targeting of deficient enzymes.

Their versatility, modular design, and genetic scalability make AFPs central to next-generation biologic development.

Creative Biogene Antibody Fusion Protein Services

Creative Biogene provides end-to-end antibody fusion protein development services, supporting researchers and biopharma partners across discovery, optimization, and preclinical production. We combine flexible platform technologies with project-specific engineering strategies, enabling rapid iteration without sacrificing molecular rigor. Whether you are exploring a novel AFP concept or advancing a lead candidate toward preclinical validation, Creative Biogene delivers technically robust, application-ready antibody fusion protein solutions-designed to de-risk development and accelerate your biologics pipeline.