A novel human immunoglobulin Fcγ–Fcε bifunctional fusion protein inhibits FcεRI-mediated degranulation

Allergen aggregation of IgE bound to FcεRI induces release of preformed mediators and synthesis of later acting leukotrienes and chemokines from mast cells and basophils⁵. This process has a major role in many of human diseases such as asthma, allergic rhinitis, chronic urticaria, angioedema and anaphylaxis. FcεRI aggregation induces release of preformed mediators and synthesis of later-acting leukotrienes, chemokines and cytokines⁵. The FcεRI is a heterotetramer consisting of a single IgE-binding α-subunit, a β-subunit and two disulfide-linked γ-subunits. The β and γ-subunit cytoplasmic tails each contain a conserved immunoreceptor tyrosine-based activation motif (ITAM). Cross-linking FcεRI via IgE bound to multivalent antigen activates tyrosine phosphorylation of ITAMs, thereby initiating downstream signaling⁶. Mast cells and basophils co-express FcγRIIb, which contains two extracellular immunoglobulin-like loops and a single conserved immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail⁷. FcγRIIb may co-aggregate with FcεRI under physiologic conditions. It is hypothesized that when FcγRIIb aggregates, it induces inhibitory signaling via SH2-domain-containing inositol 5-phosphatase (SHIP) and phosphorylation of FcγRIIb requires the co-aggregation with FcεRI (refs. ^8,9). Aggregating FcγRII to FcεRI leads to the rapid tyrosine phosphorylation of the FcγRIIb ITIM tyrosine by FcεRI-associated Lyn and inhibition of FcεRI signaling^10–13. To take advantage of this FcγRIIb-negative signaling in human basophils and mast cells, we constructed a single chimeric human bifunctional protein (GE2) engineered by fusing the human Fcγ1 and Fcε. Here we show that this chimeric protein is able to inhibit IgE-mediated in vitro activation of human basophils and mast cells and in vivo activation of human FcεRIα-bearing mast cells in FcεRIα transgenic mice. This approach using chimeric Fcγ proteins provides a platform for non-specific as well as future antigen-specific inhibition of IgE–FcεRI-mediated reactivity in a host of human diseases.

Human genomic DNA encoding the IgG γ1 constant region extends from the hinge through the CH3 domain. This sequence was initially cloned into a mammalian expression vector with a cytomegalovirus promoter and a murine immunoglobulin κ-chain leader sequence. Genomic DNA of the human ε heavy chain CH2 through CH4 domains was placed after the CHγ3. Between Fcγ1 and Fcε, we placed a 15 amino-acid linker (Gly₄Ser)₃, which has been used in single-chain Fv fragment expression¹⁴. This flexible peptide linker facilitates chain pairing and minimizes refolding and aggregation problems encountered when the two chains are expressed individually. The fusion protein contained the binding sites for FcεRI (CHε2–CHε3) and for FcγRII (CHγ2–CHγ3)^15,16. The first constant region domains (CHγ1 or CHε1) were deleted as this domain associates with BiP (immunoglobulin heavy-chain binding protein) and a fusion protein containing these regions was not expressed¹⁷. SDS–PAGE demonstrated the Fcγ1–Fcε fusion protein was expressed as the predicted ~140-kD dimer. Western-blot analysis and ELISA testing demonstrated that the GE2 protein was recognized by antibodies specific for human ε and γ chains.

Binding of GE2 to both the human FcεRI and FcγRII was demonstrated using CHO3D10, a human FcεRIα-transfected cell line and HMC-1, which expresses human FcγRII but not FcεR (ref. ¹⁸). GE2 protein bound to both FcεRI and FcγRII in a fashion equivalent to human IgE and IgG, respectively, as assessed by flow cytometry (data not shown). These results demonstrate that expressed GE2 protein is properly folded so as to preserve bifunctional heterotypic FcR binding.

Fresh human basophils expressing both FcεRI and FcγRII can be passively sensitized with chimeric human IgE specific for NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl) and then histamine release induced by cross-linking the anti-NIP IgE–FcεRI complex with NIP-BSA (ref. ¹⁹). We sensitized basophils with 1 μg/ml human anti-NIP IgE, plus doses of GE2 ranging from 0.01 to 10 μg/ml for 1 hour before activation with 50 ng/ml NIP-BSA (Fig. 1a). Myeloma IgE (PS myeloma) was used as a control. We found that 1 μg GE2 at 1 μg per ml inhibited almost half of histamine release, whereas at 10 μg/ml GE2 gave an average of 84% inhibition. 10 μg of non-specific PS IgE only decreased histamine release by 19%. Adding the GE2 at the same time as the IgE anti-NIP gave optimal inhibition. The longer the delay in GE2 addition following sensitization with IgE anti-NIP, the less the inhibition (Fig. 1b). These results show that the inhibition of antigen-driven histamine release induced by GE2 is dependent on time and dosage. We obtained similar results with cultured human mast cells; a dose of 1 μg caused 50% inhibition of IgE-mediated degranulation (data not shown). Cross-linking of GE2 by adding an anti-IgG antibody in vitro along with GE2 caused enhanced inhibition of IgE-mediated release (data not shown). In performing this experiment, we sought to mimic what may occur in vivo should subjects given GE2 make an antibody response against it. The actual fate of antibody bound GE2, should it occur in vivo, is not possible to predict.

Tyrosine phosphorylation of Syk is a critical step in human mast cell and basophil mediator release¹⁹. Cross-linking FcεRI on human basophils with IgE directed to NIP and NIP-BSA induces substantial tyrosine phosphorylation of Syk which was markedly reduced in cells pre-incubated with GE2 (Fig. 2). Thus, GE2 co-aggregation of FcεRI and FcγRII inhibits IgE-mediated Syk phosphorylation, which may contribute to the inhibition of histamine release.

Transgenic mice altered to express the human FcεRIα and with the murine FcεRIα chain knocked out demonstrate allergic reactivity mediated by passively administered human IgE antibody along with antigen^20,21. The human FcεRIα chain is coupled with the mouse β and γ chains to form a functional chimeric human/mouse FcεRI. This model takes advantage of the fact that murine mast cells also express FcγRIIb that can interact well with human IgG. This is in contrast to the lack of interaction between the murine FcεRI and human IgE. We used passive cutaneous anaphylaxis (PCA) to test the GE2 protein’s ability to block IgE-driven FcεRI-mediated mast-cell release. We intradermally primed transgenic mice with 250 ng of chimeric human anti-NP (4-hydroxy-3-nitrophenylacetyl) IgE and simultaneously injected individual sites with saline, GE2 or IgE myeloma protein. Four hours later, mice were given a systemic challenge with 0.5 mg NP-BSA plus 1% Evans Blue intravenously. The intensity of the PCA at each site was assessed by the size of the skin bluing after 30 min. The size and color intensity of the reaction at the sites of GE2 injection were decreased compared with sites injected with an equivalent amount of human IgE myeloma (Fig. 3). Similar results were obtained using genetically engineered human IgE anti-dansyl antibody and dansyl-BSA as an antigen. A total of 30 mice were tested and the GE2 was 2–4 times more potent than purified control human IgE in its ability to block PCA.

Our results demonstrate that a single chimeric molecular human protein consisting of Fcγ1 plus Fcε directly inhibits in vitro histamine release from human basophils and also inhibits humanized FcεRI-mediated mast-cell degranulation in transgenic mice. GE2 was more potent than control human IgE in all assays. Even if the GE2 protein did not have a 15 amino-acid linker between the two segments of ‘normal’ human Fcγ1 and Fcε, it would likely be recognized as foreign and induce an antibody response. With the 15 amino-acid linker, it is highly likely that antibody against GE2 would be expressed and will be primarily directed at this linker region. In fact, such an antibody response to GE2 may well prove advantageous as antibodies against GE2 will lead to increased cross-linking of Fcγ and Fcε receptors and should enhance inhibition of basophil and mast-cell function as we have observed in vitro using an anti-IgG antibody. This truncated Fcγ–Fcε fusion protein, GE2, provides a novel approach for the immunotherapy of diseases mediated via the FcεR such as allergic asthma, allergic rhinitis, chronic urticaria, angioedema and anaphylaxis. Replacement of the Fcε sequence in our construct with specific allergen genes will result in chimeric bifunctional proteins. We predict that these proteins will have the ability to inhibit mast-cell and basophil reactivity in an antigen-specific manner rather than in the global fashion of GE2. Such novel antigen-specific reagents may be useful in allergen immunotherapy.