Fusion proteins are a new type of multi-domain artificial protein produced by fusing a biologically active functional protein molecule with other natural proteins (fusion partners) using genetic engineering, chemical modification and other techniques. This can optimize protein performance and even produce new functions. Functional protein molecules are generally endogenous ligands or their receptors, including cytokines, growth factors, hormones, enzymes or peptides and other active substances. Common fusion partners include immunoglobulin (Ig), albumin, transferrin, etc. Among them, fusion proteins based on the fragment crystallizable (Fc) are most widely used.
Fusion proteins can interact with multiple targets, so they can simultaneously act on two or more pathways in the development of diseases at the same time. The construction of bi- or multi-functional specific fusion proteins has become the focus of current market research and development. According to an analysis of fusion protein bi-specific targets registered by the Chemical Abstracts Service (CAS), the most extensively studied bi-specific targets are CD19 and CD3, IL-1α and IL-1β, and EGFR and CD3.
Main Fusion Protein Technology
There are many natural active substances in the human body, but only a few dozen substances are truly applied in the therapeutic field, and most of them are greatly limited in clinical application due to poor stability and short half-life. By fusing biologically active functional proteins with proteins that extend the half-life, the metabolic time in the body can be significantly extended. Therapeutic fusion proteins are classified based on their extended half-life, mainly consisting of:
- Fused with Fc of Antibody. The Fc of the antibody contained in a Fc fusion protein can’t engage in antigen-antibody reactions. However, the Fc can mediate various biological functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) and can choose fusion partners according to the cytotoxicity differences mediated by the four subtypes. Relying on a long-term mechanism for fusion partners can be achieved by neonatal Fc receptors (FcRn), the mediated circulatory pathway prolonging the drug’s half-life in the plasma and increasing the stability of the fusion protein. The first Fc fusion protein in the World, Etanercept, is a “Star” therapeutic product. Currently, many biosimilars have been approved for marketing.
- Fused with Human Serum Albumin. Human serum albumin (HSA), the most abundant soluble protein in plasma, is an important natural drug carrier with non-toxicity, non-immunogenicity, and broad tissue distribution advantages. Since HSA can be abundantly expressed in yeast at a low cost, it is often used as a fusion carrier for fusion protein drugs such as GLP-1 receptor agonist Albiglutide (Tanzeum), coagulation factor IX albumin fusion protein (Idelvion), etc.
- Fused with PEG or XTEN polymers. Polyethylene glycol (PEG), the first polymer used to improve half-life in vivo, and several other PEG-modified drugs have been approved for marketing, e.g., Esperoct, a long-acting recombinant coagulation factor VIII modified by PEG, approved by U.S. regulatory authorities in 2019 for the treatment of Hemophilia A. However, PEG is not biodegradable. Although PEG is generally considered to have low immunogenicity, reports have identified antibodies that recognize and bind to PEG. Therefore, the advantages and limitations of PEG have stimulated the development of alternative degradable fillers, such as the recently emerged extended recombinant polypeptide (XTEN) technology. XTEN polymers are non-immunogenic polypeptides composed of six hydrophilic, chemically stable amino acids: A, E, G, P, S and T. They can be biodegraded and extend the half-life of peptides and proteins. Other extenders include transferrin, carboxy-terminal peptides, elastin-like peptides, etc.
Quantitative Analysis of Drug Concentration in Blood
Ligand binding methods (LBA) enzyme-linked immunosorbent assay (ELISA) platforms are mostly used for the detection of fusion protein drug concentration. In preclinical ELISA methods, the solid phase carrier surface is usually coated with capture reagents, such as target antigens or anti-idiotype antibodies (Anti-ID Abs) as a capture reagent. The detection reagents are horseradish peroxidase-labeled Goat anti-human IgG Fc secondary antibodies (Goat anti-human IgG Fc-HRP) or custom-labeled anti-idiotype antibodies or antigens. However, in clinical trials, Goat anti-human IgG Fc-HRP can bind with immunoglobulins in serum, so it is generally not applicable. More specific antibody pairs are selected for analysis. The MSD platform can also achieve higher sensitivity and a wider linear range.
Based on some characteristics of fusion protein drugs, in addition to conventional administration methods, some drugs can be designed for intraocular delivery, and some drugs can even cross the blood-brain barrier. The drug is a growth factor fusion protein in a preclinical project for intraocular administration.
Immunogenicity Analysis
Like conventional monoclonal antibodies, fusion protein drugs carry unnatural human protein sequences and have the potential to induce immunogenicity, so comprehensive risk assessments, strategies, and analyses are required to detect and characterize the immunogenicity of fusion protein drugs to predict and understand potential clinical effects.
The LBA method is usually used for immunogenicity detection of fusion protein drugs. In contrast, the bridging method based on the electrochemiluminescence (Meso Scale Discovery, MSD) platform is often used for semiquantitative analysis of anti-drug antibodies. The capture reagent is a biotin-labeled drug, and the detection reagent is a ruthenium-labeled drug. The detection can also be performed using an analysis method based on the ELISA platform. Typically, the drug is coated on the solid phase carrier surface, the capture reagent is a biotin-labeled drug or a digoxin-labeled drug, and the detection reagent is streptavidin labeled with horseradish peroxidase (SA-HRP) or anti-digoxin (Anti-digoxin-HRP). For the positive control antibody, typically a polyclonal antibody produced from immunized animals or a combination of one or more monoclonal antibodies is used as an alternative. However, the prepared positive control antibodies are not ideal in the anti-drug antibodies (ADA) detection methods for a few fusion protein drugs. In a preclinical project, the drug is a type of fusion protein composed of an antibody fused with a peptide. Due to the unique nature of the drug’s structure and target, many optimization ways are attempted, but the drug tolerance can’t meet the requirements of the toxicology experiment. A method that involves the purification of ADA using magnetic beads is used. The purified ADA is directly coated onto high-binding plates, then the detection reagent is added, and response signals are obtained. This method demonstrates high sensitivity, and the drug tolerance meets the requirements of the toxicology experiment.
As fusion protein drugs belong to multi-domain therapeutic products, ADAs generated against different epitopes, e.g., antibody fusion protein drugs, can be against the antibody parts, linkers, proteins, or their complexes. Therefore, the immunogenicity analysis of fusion protein drugs should respond to all their structural domains. Especially in the clinical, there is a need for a comprehensive and in-depth analysis of the sites where ADA is produced. This can provide more data analysis for Pharmacokinetics (PK), Pharmacodynamics (PD), and even some adverse reaction events that may occur in the clinical. The competitive method is often used to identify immunogenicity analysis of multi-domain therapeutic products. Generally, the first step is to conduct preliminary screening and confirmatory assay for the total fusion protein drug and then perform domain-specific analysis for the samples confirmed to be positive. Suppose the inhibition after adding the antibody is higher than the cut-off value set by the method. In that case, the generated ADA is considered specific to the antibody part and if the linker protein is added. Inhibition is higher than the cut-off value, which is considered the linker-protein part.
Before carrying out the PK or ADA analytical method, it is necessary to prepare key reagents used in the method, such as monoclonal antibodies or polyclonal antibodies.
Immunotoxicity
Immunotoxicity analysis is recommended for fusion protein drugs. Studying aspects such as immune phenotypes and cytokines can help better observe changes in immune cells, states, functions, and disease progression in the body.
Immunophenotyping Analysis
The unique ability of flow cytometry is used to simultaneously analyze multiple parameters of mixed cell populations. The most basic form is to use fluorescently conjugated antibodies to specifically bind to the antigens corresponding to the cell surface and use flow cytometry to analyze the cells according to the fluorescence on the cell surface. It can analyze T, B, and NK cells in peripheral blood to identify the influenced cell population and perform in-depth analysis of each cell population, such as identifying helper T cells, cytotoxic T cells, regulatory T cells, etc. In addition, it can detect changes in the expression of specific proteins or receptors and provide useful information on changes in biomarkers.
Cytokine Analysis Multiple platforms, such as MSD, CBA/FCM, Luminex, ELISA, etc., can be used to simultaneously detect multiple cytokines in the serum of experimental animals or humans. A comparison of cytokine detection parameters across various platforms is shown in Table 1.
Table 1: Comparison of cytokine detection parameters across various platforms
ELISA is the earliest technology used for cytokine detection, and it is easy to operate, cost-effective, and suitable for serum, plasma, and tissue fluid samples. The lowest detectable concentration can reach ng per milliliter. However, ELISA can only be used for single cytokine detection, with a narrow linear range and relatively limited sensitivity.
The intracellular staining and cytometric bead array (CBA) technique is commonly used to detect cytokines. It can detect the expression of specific intracellular cytokines and a variety of cytokines secreted to the extracellular space, such as IL-2, IL-4, IL-5, IL-6, IL-13, TNF-α and IFN-γ. Th1/Th2-related cytokines are frequently used as detection indicators in experiments for serum from species such as non-human primates, rats, and mice.
Cytokine analysis based on the MSD platform can obtain data results with better sensitivity and wider linear range, with the lowest detectable cytokine at fg per milliliter, and can simultaneously quantify up to 10 types of cytokines. The test samples include conventional serum, plasma, tissue fluid, cerebrospinal fluid, urine, saliva and aqueous humor of the eye.
The Luminex platform is also widely used in cytokine analysis. The new generation of multiplex detection capabilities based on xMAP technology offers high-throughput, faster result acquisition, and automation capabilities with Bio-Plex 3D Suspension chip systems. In addition to the quantitative detection of cytokines, it has excellent applications in measuring hormone levels, pathogen detection, and tumor marker detection. In preclinical cytokine applications, the test species cover mice, rats, dogs, non-human primates, etc., and the test matrices include serum and plasma.
Other immunodetection platforms, such as Gyros and EliSpot, can all be used to determine cytokines.
A Final Word
Currently, the primary indications of therapeutic fusion drugs focus on autoimmune diseases, eye diseases and anti-anemia treatment. Fusion proteins can be used as drugs and show substantial advantages in vaccines and other advanced therapies such as the hot chimeric antigen receptors T cell (CAR-T) therapy. Therefore, more innovative achievements using fusion proteins will likely emerge in the coming years. New fusion protein technologies are also driving fusion protein drugs to play a bigger role.
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