What Antibody fusion proteins are being developed?

Introduction to Antibody Fusion Proteins

Antibody fusion proteins are a rapidly evolving class of engineered biomolecules that combine the exquisite selectivity of antibodies with additional functional protein domains. These chimeric constructs are designed to improve the targeting, pharmacokinetic, therapeutic, and diagnostic properties of traditional antibodies. By genetically fusing antibody fragments with toxins, cytokines, enzymes, peptides, alternative binding domains, or half-life extending moieties, researchers aim to create multifunctional agents that maintain high specificity while gaining an additional beneficial function. These molecules have emerged as promising candidates in cancer therapy, infectious disease treatment, immunomodulation, and molecular imaging, among other applications.

Definition and Basic Concepts

Antibody fusion proteins are defined as recombinant proteins that combine an antibody or antibody fragment (such as an scFv, Fab, or Fc domain) with another protein domain that imparts novel functionality. The fusion partner may serve various roles, such as localizing active payloads (e.g., toxins, cytokines, enzymes) specifically to the target tissue, modulating immune responses, or extending in vivo half-life. Fundamentally, these engineered fusion proteins leverage the high affinity and selectivity of antibodies and then add beneficial properties through the second domain. The design can involve either direct genetic fusion—resulting in a single polypeptide—or assembly through post-translational conjugation methods that promote site-specific labeling.

Historical Development

The concept of fusing antibodies with other proteins has evolved alongside advances in recombinant DNA technology. In the early days of antibody therapeutics, monoclonal antibodies emerged via hybridoma technology; however, the limitation regarding their large size and systemic toxicity led to the exploration of miniaturized formats and new fusion strategies. Early fusion proteins were based on linking Fc domains to small effectors, a concept that was later extended to include toxins (thus creating immunotoxins) and cytokines (yielding immunocytokines). Over the past 30–35 years, antibody fusion proteins have transitioned from exploratory laboratory tools to advanced clinical candidates, driven by improved molecular engineering, novel expression systems, and innovative conjugation methods. Modern developments now feature highly engineered bispecific and multispecific formats, the use of alternative binding domains such as VNARs and anticalins, and site-specific conjugation techniques that minimize heterogeneity.

Types and Mechanisms

Antibody fusion proteins come in various types depending on the nature of the fused partner and the intended functional outcome. Their mechanisms of action depend on the payload they carry and the biological role that payload is designed to execute.

Common Types of Antibody Fusion Proteins

1. Immunotoxins and Toxin Fusions
These fusion proteins fuse antibody fragments or full-length antibodies with cytotoxic toxins. One prominent example is moxetumomab pasudotox, an anti-CD22 recombinant immunotoxin that fuses the Fv fragment of an antibody with a fragment of Pseudomonas exotoxin A. Immunotoxins function by delivering the toxin selectively into tumor cells, resulting in the inhibition of protein synthesis and subsequent cell death. Similar constructs focus on targeting cancers that express specific antigens, such as those found on B cells in hematological malignancies.

2. Immunocytokines (Cytokine Fusion Proteins)
In immunocytokines, antibodies are fused with cytokines such as IL-2, IL-12, IL-15, IL-21, or interferons. These constructs aim to selectively deliver the cytokine to the tumor microenvironment, thereby stimulating an immune response while minimizing systemic toxicities associated with free cytokines. An example is the engineered human cytokine/antibody fusion protein that selectively expands regulatory T cells and offers protection in autoimmune diseases. Immunocytokines can also enhance lymphocyte infiltration and activate antitumor responses.

3. Antibody–Enzyme or Lysosomal Enzyme Fusions
Such fusion proteins link an antibody to an effector enzyme, often a lysosomal enzyme, to facilitate targeted enzyme replacement therapy or intracellular payload delivery. A method for producing an antibody fusion protein comprising an antibody and a lysosomal enzyme has been disclosed, emphasizing serum-free mammalian cell culture for high-yield production and purification by chromatography.

4. Antibody–Peptide and Bispecific Fusions
Modern designs include antibody–peptide fusion proteins that combine a targeting antibody fragment with a peptide ligand. For instance, a novel antibody-peptide bispecific fusion protein against MERS-CoV was engineered by fusing an anti-MERS-CoV scFv with a fusion inhibitory peptide (HR2P), resulting in improved antiviral inhibitory activity. Such bispecific formats can engage multiple targets or combine therapeutic functions from both antibody and peptide arms.

5. Antibody Fusion with Alternative Binding Domains (VNARs, Anticalins, etc.)
To overcome limitations of conventional immunoglobulins, novel antibody fusion proteins incorporate non-traditional binding domains. For example, fusion proteins incorporating variable new antigen receptor (VNAR) domains, derived from shark antibodies, have been designed to harness their robust thermostability and unique epitope accessibility. Additionally, antibody–anticalin fusions, which combine an antibody fragment with an engineered lipocalin (anticalin) that binds small molecules (e.g., fluorescein derivatives), are being developed for pretargeting applications in molecular imaging.

6. Fc and Albumin Fusion Proteins
To enhance half-life and improve pharmacokinetics, antibodies can be fused with the Fc domain or albumin-binding moieties. Methods for expression technology that incorporate a hybrid isotype antibody moiety have been patented to yield products with improved expression, assembly rate, and additional functional properties. These fusions may also be designed to bind to the neonatal Fc receptor (FcRn) to extend serum half-life without compromising efficacy.

7. Site-specific Antibody Conjugates and ADC Platforms
Beyond simple genetic fusion, advanced methods utilize chemical and enzymatic techniques for post-translational conjugation to achieve homogeneous antibody-drug conjugates (ADCs) with controlled drug-to-antibody ratios (DAR). Approaches such as engineered double cysteine residues via the THIOMAB™ method allow site-specific conjugation that can yield ADCs with DARs greater than two for delivering potent cytotoxic agents.

8. Plant-Produced and Recombinant Protein A Fusions
Novel approaches include in-solution antibody harvesting using plant-produced fusion proteins. For example, a hydrophobin–Protein A fusion protein engineered in Nicotiana benthamiana is used to capture antibodies in a scalable, low-cost manner, coupling the antibody’s binding capacity with the amphipathic properties of hydrophobins.

Mechanisms of Action

The mechanisms by which antibody fusion proteins exert their effects combine the targeting specificity of antibodies and the biological activity of the fusion partner:

- Targeted Payload Delivery: In immunotoxins and ADCs, the antibody component binds specifically to an antigen expressed on the target cell (e.g., CD22 on cancer cells), thereby internalizing the toxin or drug payload to induce cytotoxicity.
- Immune Modulation: In immunocytokines, the delivered cytokine acts locally at the tumor site to stimulate innate and adaptive immune responses, promoting processes such as T cell and natural killer (NK) cell activation while reducing systemic side effects.
- Enhanced Pharmacokinetics: Fc fusion proteins and albumin fusions interact with FcRn or albumin receptors, respectively, leading to prolonged circulation half-life, reduced renal clearance, and improved biodistribution.
- Bispecific Engagement and Synergy: Antibody–peptide bispecific fusions can simultaneously engage virus particles and host cell receptors to block viral entry, as seen in antiviral approaches against MERS-CoV. Similarly, antibody–anticalin fusions in pretargeting strategies can bind both tumor antigens and small imaging ligands to enable dual diagnostics and therapy.
- Site-specific Conjugation for Controlled Activity: Chemical and enzymatic conjugation methods, such as the introduction of engineered cysteines, provide metered delivery of cytotoxic drugs via ADCs, ensuring that each antibody molecule carries a defined payload and thus a predictable therapeutic index.

Applications in Medicine

By integrating additional functional domains, antibody fusion proteins have become powerful tools in both therapeutic and diagnostic settings, offering solutions to several limitations of conventional treatment modalities.

Therapeutic Applications

Antibody fusion proteins are being developed for a wide range of therapeutic applications:

- Cancer Therapy:
A major focus has been on creating fusion proteins for targeted cancer treatment. Immunotoxins like moxetumomab pasudotox have shown significant promise in treating hematologic malignancies by directly delivering toxins to cancer cells. Besides immunotoxins, antibody–cytokine fusion proteins (immunocytokines) are being developed to enhance local immune responses within tumors, with several candidates in clinical trials that deliver IL-2, IL-12, or IL-15 conjugated to tumor-targeting antibodies. ADCs, which use site-specific conjugation to bind potent cytotoxins, represent another advanced modality that is progressing through different stages of development. Additionally, BBB-penetrating fusion proteins, such as JR-141 which combines an anti-transferrin receptor antibody with human IDS, are being engineered to cross the blood-brain barrier and target central nervous system (CNS) diseases.

- Infectious Diseases and Antiviral Strategies:
Fusion proteins that target viral pathogens are receiving considerable attention. An antibody–peptide bispecific fusion protein against MERS-CoV, for instance, has been engineered to combine the neutralizing capacity of an antibody with the direct antiviral capacity of a fusion inhibitory peptide. Such constructs offer a dual mechanism to prevent viral entry and replication by disrupting virus–host cell interactions. Furthermore, immunotoxins and antibody fusions are investigated for their capability to treat infections by specifically eliminating infected cells, thereby reducing viral load.

- Autoimmune Diseases and Immunomodulation:
In the context of autoimmune diseases, antibody-cytokine fusion proteins that preferentially stimulate regulatory T cells while avoiding systemic activation of effector immune cells are being developed. This approach provides a strategy to recalibrate dysfunctional immune responses with potentially fewer systemic side effects. By fusing cytokines to antibodies that target specific cell populations, these immunocytokines promise to deliver immunomodulatory effects in a highly controlled manner.

- Enzyme Replacement and Metabolic Disorders:
Antibody fusion proteins that deliver enzymes to specific cellular compartments are particularly relevant for treating metabolic and lysosomal storage disorders. The fusion of antibodies with lysosomal enzymes, as described in some patents, allows for efficient targeting and uptake while reducing systemic clearance, thereby offering a targeted enzyme replacement therapy.

- Drug Delivery and Targeted Therapy:
Several fusion proteins are designed to serve as vehicles for intracellular drug delivery. These “penetration system” fusion proteins facilitate the transport of therapeutic peptides or small-molecule drugs across biological barriers, such as the cell membrane or nuclear envelope, ensuring that the payload reaches the intended intracellular target with high specificity.

Diagnostic Applications

Antibody fusion proteins also hold substantial promise in the field of diagnostics:

- Molecular Imaging and Radiopharmaceuticals:
Antibody fusion proteins are being developed as imaging agents for cancer and other diseases. For instance, fusion proteins that combine an antibody with a cytokine or imaging label (via conjugation of radioactive isotopes or near-infrared fluorophores) are used in nuclear medicine to visualize tumor lesions in vivo. Moreover, the development of fusion proteins that incorporate anticalin domains has advanced pretargeting strategies, where the antibody binds the tumor antigen and subsequently recruits a small imaging ligand with high affinity, resulting in excellent tumor-to-background contrast.

- Biosensors and Immunoassays:
In diagnostic assays, antibody fusion proteins can enhance detection sensitivity and specificity. One example involves the fusion of Protein A with hydrophobin, produced in plants, to capture antibodies from solution, thereby reducing the costs and improving scalability in antibody harvesting. Another innovative approach has been the development of bioluminescent fusion proteins that combine the ZZ domain of protein A with firefly luciferase, providing sensitive detection in Western blotting and dot blot assays with sustained luminescence that rivals conventional HRP-based detection methods.

- Antibody Harvesting and Purification:
New fusion protein designs are also improving the processes used to purify antibodies. By fusing Protein A with hydrophobin tags, researchers have achieved more efficient partitioning and recovery of antibodies from complex biological mixtures, streamlining the purification process for analytical and diagnostic purposes.

Current Development and Research

In recent years, antibody fusion proteins have advanced rapidly through preclinical investigations and multiple clinical development pipelines. Research conducted at various institutions and companies is broadening our understanding of their potential, and novel engineering strategies are constantly under evaluation.

Development Stages and Pipeline

Antibody fusion protein candidates are at various stages of development, from early proof-of-concept studies and preclinical investigations to Phase 1/2 clinical trials. Key development highlights include:

- Approved and Late-Stage Candidates:
Some fusion proteins, such as moxetumomab pasudotox, have progressed to late-stage clinical trials—particularly in the context of hematologic malignancies. These later-stage agents underscore the translational potential of fusion protein technology.

- Preclinical Advancements:
Numerous studies have demonstrated the utility of antibody fusion proteins in various models. For example, antibody–cytokine fusion proteins have been tested in vivo, showing favorable pharmacodynamics and biodistribution profiles. Research in animal models has also successfully showcased the antiviral efficacy of antibody–peptide fusions in neutralizing MERS-CoV.

- Innovative Expression and Conjugation Techniques:
The development of hybrid isotype antibody fusion proteins that combine sequences from multiple antibody types is supported by advancements in expression technology. These innovations aim to improve expression yields, assembly rates, and overall functionality. Moreover, site-specific conjugation methods, employing engineered cysteine residues, have allowed for homogeneous ADCs with defined drug-to-antibody ratios, increasing clinical predictability and safety.

Notable Companies and Research Institutions

Global pharmaceutical and biotechnology companies, along with academic research institutions, are actively driving the development of antibody fusion proteins. Notable players include:

- ImmunityBio, Inc.:
Their focus on antibody cytokine fusion proteins—such as N-803—is designed to boost lymphocyte activity for cancer therapy, and the company’s annual report highlights its pipeline and next-generation platforms incorporating such molecules.

- Fusion Pharmaceuticals:
This company is working on next-generation radiopharmaceuticals that incorporate antibody fusion proteins for targeted alpha therapy. Their clinical trials exploring pre-administration of “cold” antibody prior to imaging agent administration are paving the way for improved dosimetry and tumor uptake in solid tumors.

- Academic and Collaborative Research Institutions:
Numerous academic laboratories are engaged in developing novel antibody fusion formats. Research groups are exploring antibody–anticalin fusions, antibody–peptide bispecific constructs, and innovative fusion strategies employing VNARs. Collaborative efforts between academia and industry are catalyzing improvements in both production platforms and application strategies.

- Patented Technologies:
Several patents document proprietary methods for creating and expressing antibody fusion proteins with enhanced therapeutic properties. These patents not only underscore the commercial interest in the technology but also provide insight into the variety of fusion constructs being pursued.

Challenges and Future Directions

Despite the promising advances in antibody fusion proteins, several challenges remain that researchers are actively addressing. The future development of this field will likely depend on overcoming technical hurdles and further enhancing therapeutic efficacy.

Technical and Production Challenges

1. Expression and Folding:
Recombinant production of fusion proteins can be challenging due to the need for correct folding, assembly, and post-translational modifications. The complexity of multi-domain proteins, particularly those that fuse antibodies with enzymatic or bulky effector domains, may lead to issues such as aggregation or low yield. New expression technologies in mammalian cells, yeast, and even plant systems are being developed to address these issues.

2. Purification and Heterogeneity:
The development of homogeneous products is paramount for clinical applications. Traditional conjugation methods that target lysine or cysteine residues often result in heterogeneous mixtures with variable drug-to-antibody ratios (DARs), affecting safety and efficacy. Innovations such as site-specific conjugation through engineered cysteines (THIOMAB™ technology) are being used to achieve consistent and predictable products.

3. Stability and Pharmacokinetics:
Fusion proteins must maintain their biological activity in vivo for extended periods. While strategies like Fc or albumin fusion improve half-life, ensuring stability without compromising the targeting functionality of the antibody remains an ongoing challenge. Engineering modifications to reduce aggregation and improve thermal stability are crucial areas of current research.

4. Immunogenicity:
Any recombinant protein administered therapeutically can potentially induce an immune response. Even when humanized or fully human antibody fragments are used, the fusion partner might increase immunogenic risk. Careful design and screening for immunogenic epitopes are essential, as is the development of robust preclinical models to predict clinical immunogenicity.

5. Production Scale-Up and Regulatory Considerations:
The complexity of fusion protein manufacture and the need for precise quality control impose challenges on scale-up and standardization. Regulatory agencies require detailed characterization and consistency in product quality, which can be difficult when dealing with multi-component molecules with varied post-translational modifications.

Future Prospects and Research Directions

Looking forward, several promising trends are emerging in the antibody fusion protein arena:

1. Integration of Multispecific and Bispecific Approaches:
Future fusion proteins are likely to take advantage of further developments in multispecificity. By designing antibodies that combine multiple binding sites—targeting both tumor antigens and immune effector receptors, for example—researchers can create synergistic therapies that more effectively eradicate disease while reducing side effects.

2. Advancements in Conjugation Chemistry:
Continued progress in chemical and enzymatic conjugation techniques will enable the production of more defined and homogenous ADCs. Novel methods that allow dual-site labeling or incorporate multiple payloads with precise dosing control hold great promise for next-generation therapeutics.

3. Exploration of Alternative Binding Scaffolds:
The incorporation of non-conventional binding domains such as VNARs, anticalins, and other alternative scaffolds is expected to expand the diversity of antibody fusion proteins. These domains confer improved tissue penetration, stability, and unique epitope recognition capabilities, potentially opening up new therapeutic niches including CNS disorders and infectious diseases.

4. Immunomodulation and Personalized Medicine:
As our understanding of the tumor microenvironment and immune regulation deepens, fusion proteins designed to locally modulate immune responses will become more sophisticated. Tailoring immunocytokines to selectively activate or inhibit specific immune subsets in a patient-specific manner is a key future goal. Personalized fusion protein therapies, possibly guided by diagnostic imaging technologies, could allow patients to receive customized treatments with optimal efficacy.

5. Innovative Production Platforms:
Novel expression systems (e.g., plant-based expression as demonstrated with hydrophobin–Protein A fusions) could significantly reduce production costs and improve scalability. Such systems may also be tailored to generate fusion proteins with enhanced post-translational modifications that mimic human glycosylation patterns, further enhancing product safety and efficacy.

6. Combination Therapies and Synergistic Modalities:
The trend toward combination therapies—linking antibody fusion proteins with other therapeutic agents such as immune checkpoint inhibitors or traditional chemotherapies—will likely continue. These combinations offer the opportunity to exploit multiple mechanisms of action simultaneously, potentially overcoming resistance mechanisms and leading to improved clinical outcomes. Recent developments in antibody combinations for breast cancer and multiple myeloma underscore the potential for these synergistic approaches.

7. Emerging Clinical Data and Regulatory Approvals:
As more clinical data become available, regulatory pathways for antibody fusion proteins will continue to evolve. Early-phase successes have demonstrated promising efficacy and favorable safety profiles in various indications, which is expected to accelerate the clinical translation of new candidates. The ongoing clinical trials and published results are laying the groundwork for broader regulatory approvals in the near future.

Conclusion

Antibody fusion proteins represent a versatile and rapidly developing platform in modern biopharmaceutical research. The various types engineered—from immunotoxins and immunocytokines to bispecific antibody–peptide fusions and antibody–anticalin constructs—offer multiple avenues to address significant unmet medical needs. Their mechanisms of action, which rely on the precise targeting abilities of antibodies combined with the potent pharmacological effects of the fusion moieties, allow for the tailored treatment of cancers, infectious diseases, autoimmune disorders, and beyond.

Historically, antibody fusion proteins have stemmed from the need to overcome limitations inherent in conventional antibodies, and innovations over the past few decades have continuously improved their design, expression, and purification. Today, researchers are addressing production challenges through advanced expression systems (including mammalian, yeast, and plant-based systems), precise site-specific conjugation methods, and sophisticated engineering techniques that enhance stability and reduce immunogenicity. Pioneering companies such as ImmunityBio, Fusion Pharmaceuticals, and numerous academic research institutions are at the forefront of this technology, translating promising preclinical findings into early clinical evaluations.

Despite the technical challenges—ranging from expression and folding difficulties to issues with product heterogeneity and immunogenicity—the prospects for antibody fusion proteins remain very bright. Future directions include the development of multispecific and bispecific constructs, improved chemical and enzymatic conjugation techniques, and the use of alternative scaffolds to further enhance targeting and tissue penetration. Moreover, innovative production platforms are set to reduce costs and improve scalability, while combination therapy approaches promise synergistic improvements in patient outcomes.

In summary, antibody fusion proteins are being developed for a broad array of therapeutic and diagnostic applications. They integrate the high specificity of antibody-based targeting with the added functionality of diverse payloads, creating next-generation treatments that are more potent, safer, and tailored to individual clinical needs. As research continues to advance and new clinical data emerge, the future of antibody fusion protein therapeutics appears exceptionally promising, heralding a new era in precision medicine and targeted therapy.