Background
Since the first therapeutic antibody was approved by the FAD, antibody products have developed rapidly in the drug market. Building on the success of monoclonal antibodies, recent studies have utilized the good specificity and pharmacokinetic properties of antibodies to create new antibody fusion proteins that can deliver therapeutic payloads in a targeted manner.
On October 25, 2021, Aliyah B Silver's team published an article titled "Engineered antibody fusion proteins for targeted disease therapy" in Trends Pharmacol Sci. The article summarizes the latest engineering and translational advances in therapeutic approaches that fuse antibodies with disease-related payload genes, including cytokines, toxins, enzymes, neuroprotectants, and soluble factor traps. Currently, many antibody fusion proteins are in clinical trials, and other innovative molecules are about to follow. These potent and multifunctional drug candidates are expected to become major players in the field of therapeutic development in the coming years.
Preclinical and clinical progress of payload immunocytokines
Payload immunocytokines are therapeutic fusion proteins containing cytokines fused to antibodies or antibody fragments specific for disease-associated antigens or other cell markers. Antibody fusion proteins have been diversified by linking various payloads to different antibody components, including full-length immunoglobulins, Fc domains, single-chain variable fragments, single-domain antibodies, and antigen-binding fragments.
Payload immunocytokines in cancer
Immunocytokine payloads have been the most extensively studied as cancer therapeutics. In recent formulations, antibodies have been fused to cytokine payloads, including interleukins IL-4, IL-15, TNFα, IL-2, IL-12, IL-21, IFN-α, and IFN-β. These cytokines may exert immunostimulatory, immunosuppressive, or pleiotropic effects in homeostasis.
Cytokine payloads are delivered to the tumor microenvironment (TME) by fusing cytokines to recombinant F8 monoclonal antibody fragments. Several F8 cytokine fusion proteins have demonstrated success in preclinical and clinical cancer therapy. For example, an SCFV-based fusion molecule containing IL-4, F8-IL4-F8, was shown to localize to tumors and inhibit cancer growth in mice.
The ECM-targeting antibody L19 has been incorporated into recently developed immunocytokines. It binds to the alternatively spliced EDB of fibronectin, enabling selective localization to tumor-associated blood vessels. Fusions of L19 scFv with TNFα, IL-12, or IL-2 have demonstrated antitumor effects. They are currently in various stages of human clinical trials as monotherapy and/or in combination with other immunocytokines and established cancer therapies.
Another tumor-targeting strategy exploits free DNA fragments found in necrotic tumor regions. Specifically, the histone-binding human IgG1 antibody NHS76 has been conjugated to an IL-12 heterodimer (IL-12p70) to enhance tumor localization (NHS-IL12). Fusion to the full-length antibody increases the plasma half-life and antitumor efficacy of IL-12.
Alternatively, affinity-impaired cytokines can be used to ensure that the antibody targeting domain, rather than the cytokine payload, directs the localization of the fusion protein. Fusion of a PD-1-specific full IgG antibody to an affinity-impaired IL-21 variant delivered IL-21 to PD-1-expressing T cells while increasing IL-21's serum half-life and minimizing off-target effects on local antigen-presenting cells. The resulting anti-PD-1-IL-21 immunocytokine provided protection in a mouse model of anti-PD-1-refractory humanized melanoma.
Potent immune cytokines in chronic inflammatory and autoimmune diseases
In chronic inflammatory and autoimmune diseases, antibodies targeting the ECM are also being used to direct anti-inflammatory cytokine activity to sites of inflammation. Immunocytokines that inhibit inflammation by binding to F8, L19, and other antibodies have shown preclinical promise in arthritis. Novel immunocytokines that bind IL-4, IL-9, IFN-α, and IL-10 have been explored for the treatment of chronic inflammatory and autoimmune diseases. In a mouse model, F8-IL4, combined with dexamethasone, induced a complete cure of collagen-induced arthritis. In another study, a fusion protein composed of an anti-CD86 scFv fused to the Fc domain and monomeric IL-10 with impaired affinity induced the differentiation of CD86+ tolerogenic DCs and could be used to treat graft-versus-host disease (GVHD).
Emerging approaches for signal-biasing immunocytokines
Advances in structure-guided molecular engineering have facilitated the design of biased payload immunocytokines that modulate the activity of the incorporated cytokines. Among these, IL-2 is currently the most actively studied and advanced cytokine in immunocytokine development.
Biasing cytokine activity toward anti-cytokine antibodies by fusion:
The systemic toxicity, short serum half-life, and activation of immune-stimulatory cells by IL-2 limit its clinical application. IL-2 signals through heterodimeric receptors composed of IL-2Rβ and γC chains or heterotrimeric receptors composed of IL-2Rβ, γC, and IL-2Rα. The design of IL-2 immunocytokines has favored cytokine activity toward specific immune cell subsets, thereby improving safety and increasing IL-2 half-life . A promising approach, first proposed by Boyman et al., is the development of anti-IL-2 antibodies that selectively direct IL-2 activity toward effector cells or Tregs . The S4B6 antibody biases IL-2 activity toward the expansion of effector cells, and the IL-2/S4B6 complex produces potent antitumor activity in mice without causing systemic toxicity. The JES6-1 antibody induces preferential expansion of Tregs, and the IL-2/JES6-1 complex protects against diabetes and other autoimmune diseases in mice. Both S4B6 and JES6-1 were genetically fused to IL-2, resulting in intramolecularly assembled immunocytokines that are more potent than the individual complexes.
Bias-loaded immune cytokine design:
Mutagenesis-biased cytokines (called muteins) can be fused to antibodies targeting disease antigens to generate biased payload immunocytokines. Fusion of an IL-2 mutant with an anti-carcinoembryonic antigen (CEA) antibody (CEA-IL2v) inhibited tumor growth and improved survival in CEA+ tumor-bearing mice. CEA-IL2v also synergized with other antibody immunotherapies to reduce tumor burden in mouse models of CEA-expressing breast and colon cancer. Another fusion protein replaced one Fab of an anti-epidermal growth factor receptor (EGFR) antibody with a hypermutated form of IL-2 (sumIL-2), which reduces IL-2Rα binding activity. The resulting molecule inhibited tumor growth in a mouse model of melanoma.
IL-5 can also serve as a biased payload immunocytokine. IL-15 shares the IL-2Rβ and γC chains of IL-2R and also binds to the high-affinity IL-15Rα chain. A fusion protein linking the IL-5 receptor linker (RLI) to an anti-CD20 antibody prolonged survival in a mouse model of lymphoma and also significantly extended the circulating half-life.
Biasing cytokine signaling through “masking” approaches:
The effector cell-biased IL-2 variant and the IL-2Rβ extracellular domain (ECD) are separately fused to the Fc domain, such that the IL-2Rβ chain binds and "masks" the fused IL-2, which is then linked to the respective Fc domain via a cleavable linker. In the presence of tumor-associated matrix metalloproteinases, the hidden IL-2 payload is selectively released, resulting in the molecule exhibiting superior efficacy to IL-2 and potent synergy with checkpoint blockade therapy.
Innovative antibody fusion protein strategy
Antibody fusions involving non-cytokine payloads are under development, such as recombinant immunotoxins (RITs), antibody-directed enzyme prodrug therapy (ADEPT), targeted enzyme replacement fusion proteins, neuroprotective payload fusion proteins, and soluble factor trap fusion proteins.
Recombinant immunotoxins (RITs)
Recombinant immunotoxins are formed by the fusion of antibody fragments and toxic groups. RITs are mainly used as cancer treatments and can be divided into pore-forming toxins (PFTs); ribosome-inactivating proteins (RIPs); and microtubule-disrupting proteins (MDPs), which can prevent mitosis and vesicle transport, thereby inducing apoptosis.
RIPs are the most diverse and well-developed class of toxins used for RIT, while MDPs are the least. Both classes require the antibody portion of the RIT to facilitate endocytosis to achieve cell killing. In contrast, most PFTs integrate directly into the cell membrane and therefore do not require antibody-mediated endocytosis to exert their cytolytic function. The integration of PFTs into the cell membrane is often sensitive to lipids synthesized in excess by certain tumor cells, which limits toxicity to only those target cells.
RITs currently in clinical trials or approved by the FDA use one of three bacterial toxins, all of which are RIPs. PE has been extensively modified for use in RITs to increase cytotoxicity, reduce immunogenicity, and increase resistance to protease degradation.
One of the major challenges facing RITs is that many patients develop anti-drug antibodies against the toxic moiety, which can accelerate clearance and/or neutralize the toxic moiety, significantly hindering therapeutic efficacy. The size of immunotoxins limits their clinical application in oncology to the treatment of hematologic cancers, as most RITs are unable to adequately penetrate solid tumors. Furthermore, the inefficient cytosolic delivery of RITs requires cytotoxic payloads with exceptional potency.
Current immunotoxins primarily focus on exogenous toxins, but endogenous RIP toxins, such as granzyme B and granzyme, have recently been developed in the clinical stage because they are not immunogenic. Another RIT approach, modified superantigens (SAgs) targeting tumors, differ from other immunotoxins in that modified Staphylococcal enterotoxin A, such as the one used in naptumomab estafenatox, recruit and activate T cells to kill target cells.
Antibody-enzyme fusion proteins and other emerging therapeutic approaches
Antibody-directed enzyme prodrug therapy (ADEPT) is a strategy that sequentially administers a cancer-targeting antibody-enzyme fusion protein and an enzyme-activated prodrug. Unlike most RITs, the antibody in the ADEPT fusion protein should not trigger receptor-mediated endocytosis, as the enzyme must remain extracellular to access the prodrug. The fusion protein MFECP1, composed of a CEA-specific antibody fragment and bacterial carboxypeptidase G2 (CPG2), demonstrated a favorable safety profile and localization to patient tumors in early clinical trials, but was limited by immunogenicity.
Antibody-enzyme fusion proteins for targeted enzyme replacement therapy represent another class of molecules that have entered clinical trials. The antibody in these fusion proteins facilitates transport of the enzymatic payload to lysosomes, thereby replacing the function of the mutant enzyme. In addition to promoting endocytosis via the human insulin receptor (HIR), HIRMAb-enzyme fusion proteins also facilitate transport across the blood-brain barrier. HIRMAb fusion proteins have also been expanded to deliver non-enzymatic agents. For example, HIRMAbs fused to neuroprotective payloads such as glial cell line-derived neurotrophic factor (GDNF) and erythropoietin (EPO) have shown preclinical promise as potential treatments for acute ischemic stroke and traumatic brain injury.
Summarize
Antibody fusion proteins play a crucial role in the development of antibody-based therapeutics. Various formats have been developed, integrating cytokines, enzymes, toxins, neuroprotectants, and soluble factor-trapping payloads. Targeting payloads to specific cells or tissues can significantly improve their efficacy, allowing for lower doses and preventing adverse side effects. These fusion proteins are versatile and can address a wide range of disease indications. Despite challenges such as immunogenicity, delivery, and tumor penetration, the development of antibody fusion proteins holds great promise and is expected to provide new strategies and approaches for the treatment of a variety of diseases. The development of antibody fusion proteins will undoubtedly benefit from and advance combination therapy approaches that incorporate other immunotherapy modalities.
