TW201623328A - EPSIGAM fusion protein - Google Patents

Abstract

Epsi-gam provides a novel fusion protein with the ability to cross-link either of the Fc[epsilon]RI or Fc[epsilon]RII cell surface receptors with an Fc[gamma]RIIb cell surface receptor in order to block IgE-mediated biological responses.

Description

EPSIGAM fusion protein Cross reference

The present application claims the benefit of U.S. Provisional Application No. 14/243,750, filed on Apr. 2, 2014, to U.S. Patent No. 8,961,992, and U.S. Provisional Application No. 62/103,495, filed on Jan. 14, 2015. Both are incorporated herein by reference in their entirety.

Affirmation of federally sponsored research

Epsi-gam was carried out under government support under the authorization number AI092914 awarded by the National Institutes of Health. The government has certain rights in the invention.

Sequence table

This application contains a Sequence Listing which has been filed in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy was created on March 24, 2014 and is named 36249701201SEQ.txt and is 7,463 bytes in size.

The fusion protein epsi-gam provides an improved method for managing IgE-mediated allergic diseases and other IgE receptor-mediated disorders by cross-linking the FcεRI or FcεRII receptors to the FcγRIIb receptor.

Asthma is a disease associated with immunoglobulin E (IgE) and IgE cell surface receptors. The World Health Organization estimates that there are 235 million worldwide. People suffer from asthma. Mild to moderate asthma is usually treated with existing inhalants and corticosteroids, but more severe asthma is currently not well treated with existing therapies.

Allergic diseases are associated with IgE and IgE cell surface receptors. In general, allergic diseases are widespread and increasing worldwide. Allergic diseases are usually treated by vaccination or exposure to allergens in successively smaller doses. However, this therapy is expensive, time consuming and in some cases dangerous.

Epsi-gam is a novel fusion protein configured to crosslink any of the Fc[epsilon]RI or Fc[epsilon]RII (CD23) cell surface receptors with Fc[gamma]RIIb cell surface receptors.

FcεRI and FcγRIIb cell surface receptors are expressed together in, for example, basophilic blood cells and mast cells. Crosslinking of the Fc[epsilon]RI and Fc[gamma]RIIb receptors delivers an inhibitory signal to these cells that inhibits the release of histamine and can provide therapeutic effects against diseases such as asthma and allergic diseases.

Basophilic hematopoietic cells and mast cells are cell types that release mediators that cause asthma and allergic diseases, and inhibit histamine and are responsible for the cross-linking of FcεRI and FcγRIIb on eosinophils and mast cells by epsi-gam. Release of other mediators of disease manifestations of asthma and allergic diseases. Epsi-gam provides a therapy for asthma and allergic diseases by cross-linking FcεRI and FcγRIIb and thereby inhibiting the release of mediators causing asthma and allergic diseases.

FcεRII and FcγRIIb are expressed together on, for example, B lymphocytes (B cells), and certain types of immunoglobulins released by B cells are inhibited by cross-linking of FcεRII and FcγRIIb cell surface receptors on B cells by epsi-gam ( The production of such as IgE), thereby reducing the concentration of IgE in the cycle.

Epsi-gam further treats diseases such as asthma and allergic diseases by cross-linking FcεRII and FcγRIIb and thereby inhibiting the production of immunoglobulins (such as IgE) associated with asthma and allergic diseases released by B cells.

In one aspect, epsi-gam is an isolated fusion protein comprising an Fcε fragment functionally linked to an Fcγ1 fragment at its carboxy terminus.

In another embodiment, epsi-gam is a fusion protein comprising the polypeptide sequence CHε2-CHε3-CHε4-γ hinge-CHγ2CHγ3.

In another embodiment, epsi-gam is a fusion protein comprising the polypeptide sequence of SEQ ID NO: 2.

In another embodiment, epsi-gam is substantially encoded by the nucleotide sequence of SEQ ID NO: 1.

In yet another embodiment, epsi-gam comprises a homodimer of two linked polypeptides, both polypeptides comprising the sequence SEQ ID NO:2.

In yet another embodiment, epsi-gam comprises a heterodimer of two linked polypeptides, wherein one of the linked polypeptides comprises the sequence SEQ ID NO:2.

Described herein is a method for making a therapeutic fusion protein epsi-gam comprising synthesizing a polynucleotide encoding epsi-gam, integrating the polynucleotide into a performance vector, transfecting the eukaryotic cell with the expression vector, and The fusion protein containing epsi-gam was isolated.

Described herein is a method for treating or preventing the development of an allergic response in an individual comprising administering to the individual 0.01 mg/kg of the fusion protein epsi-gam, wherein the epsi-gam comprises the sequence of SEQ ID:1. In one embodiment, a method for treating or preventing the development of an allergic response in an individual comprises administering to the individual 0.1 mg/kg of the fusion protein epsi-gam, wherein the epsi-gam comprises the sequence of SEQ ID:1. In one embodiment, a method for treating or preventing the development of an allergic response in an individual comprises administering to the individual 0.3 mg/kg of the fusion protein epsi-gam, wherein epsi-gam comprises the sequence SEQ ID:1. In one embodiment, a method for treating or preventing the development of an allergic response in an individual comprises administering to the individual 1.0 mg/kg of the fusion protein epsi-gam, wherein the epsi-gam comprises the sequence of SEQ ID:1. In one embodiment, a method for treating or preventing the development of an allergic response in an individual comprises administering to the individual 3.0 mg/kg of the fusion protein epsi-gam, wherein epsi-gam comprises Sequence SEQ ID: 1. In one embodiment, a method for treating or preventing the development of an allergic response in an individual comprises administering to the individual 10.0 mg/kg of the fusion protein epsi-gam, wherein epsi-gam comprises the sequence of SEQ ID:1.

Described herein is a method for treating or preventing the development of mast cell activation syndrome in a subject, the method comprising administering to the individual a fusion protein comprising the sequence of SEQ ID: 1. In one embodiment, the mast cell activation syndrome comprises cutaneous mastocytosis, systemic mastocytosis, or invasive systemic mastocytosis.

Described herein is a method of administering rush immunotherapy prophylaxis to an individual comprising administering to the individual a fusion protein comprising the sequence of SEQ ID: 1 at the time of administration of the emergency immunotherapy prevention. In one embodiment, the fusion protein is administered to the individual before, during or after the emergency immunotherapy prophylaxis, or during one or more of these periods. In one embodiment, the fusion protein is administered monthly and the individual receives at least one administration of the fusion protein prior to the administration of the emergency immunotherapy prevention.

These and other aspects will be apparent from the following description of the embodiments and the accompanying drawings.

The novel features of epsi-gam will be specifically set forth in the scope of the accompanying claims. A better understanding of the features and advantages of epsi-gam will be obtained with reference to the following [Embodiment], which illustrates the exemplary embodiment (where the principle of epsi-gam is employed) and its accompanying drawings: Figure 1 is illustrated by A data sheet for the production of epsi-gam and similar fusion proteins produced by the method of manufacture.

2 is an example of a typical result of protein A affinity chromatography analysis of an expressed fusion protein similar to epsi-gam produced by the described manufacturing method.

Figure 3 is an illustration of typical results of protein A affinity chromatography analysis of epsi-gam produced by the described manufacturing method.

Figure 4 is the amino acid sequence of the fusion protein epsi-gam (SEQ ID NO: 2).

Figure 5 is an example of a graph showing typical results of the efficacy of epsi-gam in blocking histamine release from different mast cell types.

Figure 6 is an example of a nucleotide sequence substantially encoding a nucleotide of the fusion protein epsi-gam (SEQ ID NO: 1).

Figure 7 shows the results of an experiment in which 3 mg kg of epsi-gam was administered to rhesus macaques in an allergic asthma model.

Figure 8 shows the results of an experiment in which 3 h/kg or 10 mg/kg of epsi-gam was administered to rhesus monkeys in an allergic asthma model.

Figure 9 shows the results of an experiment in which methotrexate sensitivity was measured after administration of 3 mg/kg of epsi-gam to rhesus monkeys in an allergic asthma model.

Figure 10 shows the experimental results of epsi-gam inhibiting the relative expression of IgE transcripts in human B cells.

Unless otherwise defined, the technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

I. Problem analysis

Fusion proteins similar to epsi-gam have been shown to treat asthma and allergic diseases in the early stages of therapeutic research, however, such fusion molecules cannot be produced in amounts sufficient to provide an actual clinical treatment regimen. The inability to generate large amounts of fusion protein molecules or scale-up of these molecules is a problem that hinders the translation of such fusion proteins into clinical therapies. Fusion proteins similar to epsi-gam have been shown to be highly prone to aggregation, posing a risk to human clinical therapies. However, epsi-gam production causes very few protein aggregates.

Epsi-gam differs from similar fusion protein molecules in that epsi-gam can be scaled very efficiently and is therefore feasible to translate into viable clinical therapies. The exceptional scale of epsi-gam is an unexpected result that translates epsi-gam into clinical therapy. Unable to scale Other similar fusion molecules cannot be translated into viable clinical therapies.

Therapy development usually begins with the creation and testing of novel molecules in the laboratory, and is often followed by the development and optimization of scaled procedures. The scale of the molecule is to produce a molecule that is sufficient for molecular applications, such as for use as a clinical therapy. The development of scaled procedures involves a number of parameters, such as selection of expression vectors, cell lines, and purification methods. For example, a first order sequence of a protein of interest can be modified to confer properties required for scale, such as high yield and low aggregation.

The development of large-scale programs is a complex process, and in most cases, the scale of a particular fusion protein has proven to be infeasible due to unpredictable and immutable scale problems. Thus, unforeseen scale issues hinder the translation of molecules into therapeutics for treating patients, and the steps in scaled design involve, inter alia, designing fusion proteins that will produce high yields and low aggregate levels.

The scale problems that are common in the development of novel protein therapies include, for example, high aggregation rates of proteins in tissue culture and low production rates.

Protein aggregation is a relatively common problem in the treatment of protein production. However, the extent to which some proteins aggregate may not only make them difficult to manufacture, but also present clinical safety risks. If the protein aggregates are present after storage or after administration to a patient, they can cause immunogenicity, which is an anti-drug response that can cause drug deactivation or other unfavorable clinical phenomena.

The low yield of therapeutic recombinant proteins can make manufacturing economically or physically infeasible.

When scaled up, fusion proteins that display promise in the early stages of development may unpredictably have high levels of aggregation, low yield, or high levels of aggregation and low yields, making these fusion proteins untranslated into clinical therapies.

In the early stages of therapeutic development, certain fusion proteins have been shown to be effective means of inhibiting IgE-mediated biological responses associated with asthma and allergic diseases, but these molecules have not been scaled up due to high aggregation rates and low yields. Quantity.

One such protein (eg, epsi-gam, fused to the Fc portion of IgE and the Fc portion of IgG) is E2G, as described in US 7,488,804. Another such molecule is GE2, which is described in US 7265208 along with related molecules.

The large-scale manufacturing of E2G, GE2 and related molecules is considered to be infeasible due to the extremely large accumulation during scale up and the low yield of therapeutic proteins. These scale issues have hampered GMP manufacturing of E2G, GE2 and related molecules.

However, epsi-gam differs from E2G, GE2 and related molecules in that epsi-gam produces very high yields at scale and has a very low degree of aggregation.

The exceptionally high yield of epsi-gam during scale-up is an unexpected result that translates epsi-gam into clinical therapies, while other similar fusion molecules that cannot be scaled are not feasible to translate into viable clinical therapies.

Figure 1 is an example of a data sheet showing typical yields of two other fusion proteins in the E2G, GE2, and GE2 families, all of which are produced by a manufacturing method substantially identical to the epsi-gam that produces high yields and described herein. . When comparing the yield of epsi-gam to the yield of two other fusion proteins in the GE2 and GE2 families, the yield of epsi-gam is typically about 22 times higher. When comparing the yield of epsi-gam to the yield of E2G, the yield of epsi-gam is typically significantly higher by about 22,000 times. The degree of aggregation of GE2 and two GE2-related molecules is 4-5 times that of epsi-gam, and E2G aggregates at a substantially higher degree, estimated to be as much as 10 times the concentration of epsi-gam.

Figure 2 shows a typical result of protein A affinity chromatography analysis of GE2, which is an expression fusion protein similar to epsi-gam produced by the described manufacturing method. As evidenced by the chromatographic results, the total aggregation of GE2 was about 28%, which is a typical result of a fusion protein similar to epsi-gam. However, it should be noted that depending on the manufacturing factors such as the cells used, the type of medium, and other relevant factors, a more typical value of the aggregate formation percentage of GE2 and similar proteins may range from 30% aggregation to 60% aggregation.

Figure 3 shows the protein A pro-expressed epsi-gam produced by the described manufacturing method And typical results of chromatographic analysis. As evidenced by the chromatographic results, the total aggregation rate of epsi-gam is approximately 6%, which is typical for epsi-gam made via the described method. From a standpoint of aggregation, a typical aggregation rate of about 6% enables epsi-gam to be a highly advantageous candidate for mass production and commercialization via the described manufacturing methods. Due to the infeasible high aggregation results of similar fusion proteins in scale, the extremely low aggregation of epsi-gam is an unexpected beneficial result of producing epsi-gam via the described manufacturing methods.

The substantially higher yield and relatively low aggregation rate of epsi-gam is not only a substantial result, but also an unexpected result when compared to four other similar fusion proteins, including similar E2G. Of the five fusion molecules that show promise in the treatment of asthma and allergic diseases, only epsi-gam produces substantially sufficient yield and a sufficiently low aggregation rate to allow the translation of novel epsi-gam fusion proteins into viable clinical therapies.

II. Definition

The term "IgG inhibitory receptor" is used to define one of the inhibitory receptor superfamilies (IRS) now known or hereinafter found, and includes the IgG receptor FcyRIIb. The FcγRIIb receptor cross-linked to the FcεRI or FcεRII receptor is capable of inhibiting the FcεR-mediated response, whether the reaction is via IgE acting by means of a high-affinity IgE receptor (such as FcεRI) or a low-affinity IgE receptor (such as FcεRII) or It is mediated by another mechanism, such as an autoantibody against FcεRI.

The term "FcγRIIb" is used to refer to the FcγRIIb receptor of any species, including any mammalian species found in nature. In one embodiment, the mammal is a human.

FcγRIIb is an isoform of a low affinity IgG receptor FcγRII containing an immunoreceptor tyrosine-based inhibitory element (ITIM). The FcγRIIb receptor is found, for example, on basophilic blood cells, mast cells, B cells, and dendritic cells. FcγRIIb has three alternative splicing forms, termed FcγRIIb1, FcγRIIb1' and FcγRIIb2, which differ only in their cytoplasmic sequence. All three alternative splicing isoforms contain two extracellular Ig-like loops and a single conserved ITIM motif in their cytoplasmic tail, and are specifically included in the definition of FcyRIIb and other splice variants that may be found in the future.

The term "FcεRI" refers to the FcεRI receptor of any species, including any mammalian species found in nature. Fc-εRI is a member of the multiple subunit immune response receptor (MIRR) family of cell surface receptors. Receptors in the MIRR family of cell surface receptors are typically capable of transducing intracellular signals via association with cytoplasmic tyrosine kinases.

The terms "FcεRII" and "CD23" are used interchangeably and refer to the FcεRII receptor of any species, including any mammalian species found in nature.

The term "immunoglobulin" (Ig) is used to refer to the immunologically conferring portion of serum globulin and other glycoproteins, which may not exist in nature but have the same functional characteristics. The term "immunoglobulin" or "Ig" specifically includes "antibody" (Abs). While antibodies exhibit binding specificity for a particular antigen, immunoglobulins include antibodies and other antibody-like proteins that lack antigen specificity. Native immunoglobulins are secreted by differentiated B cells called plasma cells, and immunoglobulins that do not have any known antigen specificity are produced by the immune system at lower levels and are produced by myeloma at increased levels. As used herein, the terms "immunoglobulin", "Ig" and grammatical variants thereof are used to include antibodies and Ig proteins that do not have a known antigen specificity or that do not have an antigen binding region.

Some major human antibodies or immunoglobulin classes are further divided into subclasses, such as IgG, IgA, which are not known to be subclasses of M or E. IgG is known to have four known isotypes: IgG ₁ (γ ₁ ), IgG ₂ (γ ₂ ), IgG ₃ (γ ₃ ), and IgG ₄ (γ ₄ ).

The constant region of the immunoglobulin heavy chain is further divided into a globular, structurally dispersed region comprising a heavy chain constant domain. For example, the constant region of an IgG ₁ immunoglobulin heavy chain comprises three constant domains (CH1, CH2 and CH3) and a hinge region between the CH1 domain and the CH2 domain. The IgE immunoglobulin heavy chain contains four constant domains: CH1, CH2, CH3 and CH4 and does not have a hinge region.

The term "Fcγ1" refers to the Fcγ1 sequence of any species, including any mammalian species found in nature. In one embodiment, the animal is a human.

Amino acid is represented by its common one-letter or three-letter code, as per the technology The practice in the middle. Thus, the names of the twenty naturally occurring amino acids are as follows: alanine = Ala (A); arginine = Arg (R); aspartic acid = Asp (D); aspartame = Asn (N) ); cysteine = Cys (C); glutamic acid = Glu (E); glutamic acid = Gln (O); glycine = Gly (G); histidine = His (H); Leucine = Ile (I); leucine = Leu (L); lysine = Lys (K) methionine = Met (M); phenylalanine = Phe (F); valine = Pro ( P) serine = Ser (S); threonine = Thr (T); tryptophan = Trp (W); tyrosine = Tyr (Y); valine = Val (V). Polypeptides herein include all L-amino acids, all D-amino acids, or mixtures thereof. Polypeptides that completely comprise D-amino acids are advantageous because they are expected to be resistant to proteases found naturally in the human body and may have a longer half-life.

The polynucleotide vector may be in any of several forms including, but not limited to, RNA, DNA, RNA encapsulated in a retrovirus envelope; DNA encapsulated in an adenovirus envelope; encapsulated in another virus or DNA in virus-like forms (such as herpes simplex and adeno-associated virus (AAV)); DNA encapsulated in liposomes; complexed with polylysine, complexed with synthetic polycationic proteins, bound to transferrin, and such as poly A compound of ethylene glycol (PEG) is compounded to immunologically "mask" the protein and/or increase the half-life or bind to the DNA of the non-viral protein. In one embodiment, the polynucleotide is DNA. As used herein, "DNA" includes not only bases A, T, C, and G, but also analogs thereof or any of such modified forms of such bases, such as methylated nucleotides; Internucleotide modifications, such as the use of uncharged bonds and thioate saccharide analogs; and modified and/or alternative backbone structures such as polyamido.

The term "IgE-mediated biological response" is used to refer to a condition or disease characterized by signal transduction via IgE receptors, including the high affinity IgE receptor Fc[epsilon]RI and the low affinity IgE receptor Fc[epsilon]RII. Definitions include, but are not limited to, conditions associated with allergic hypersensitivity and atopic allergy, such as asthma, allergic rhinitis, atopic dermatitis, partial food allergies, chronic rubella, and angioedema, and usually by Beetle needles, severe food allergies (such as reactions to peanuts) or severe physiological conditions of anaphylactic shock caused by drugs such as penicillin.

The term "treat/treatment" refers to both therapeutic and prophylactic or preventative measures, wherein the goal is to prevent or slow (reduce) undesired physiological changes or conditions. A subject in need of treatment includes an individual who already has a condition or disorder and who is prone to have the condition or disorder or the condition or disorder to be prevented.

The term "patient" refers to any animal, and in one embodiment a mammal, that is, an individual who is examined, treated, analyzed, or diagnosed. In one embodiment, the human is an individual. The individual or patient may or may not have a disease or other pathological condition.

The term "disease" refers to any disruption of normal bodily functions, or the appearance of any type of lesion.

III. epsi-gam

Figure 4 is the amino acid sequence of the fusion protein epsi-gam. Epsi-gam is a novel fusion protein that is configured to crosslink FcγRIIb cell surface receptors with FcεRI or FcεRII cell surface receptors in order to block certain IgE-mediated biological responses.

IgE plays an important role in many acute and chronic allergic reactions, including, for example, asthma, allergic rhinitis, atopic dermatitis, severe food allergies, chronic rubella and angioedema, and, for example, food allergies, bee stings or penicillin A serious physiological condition of anaphylactic shock caused by allergies. The Fc portion of human IgE is capable of binding to FcεRI or FcεRII cell surface receptors on various cell types such as basophils, mast cells, dendritic cells, and B lymphocytes (B cells). When an antigen binds to IgE that binds to certain cell surface receptors, for example, on basophilic blood cells and mast cells, IgE binding causes these cells to release vasoactive and proinflammatory mediators, including histamine. Mediators released when antigen-bound IgE binds to certain cell surface receptors contribute significantly to asthma as well as acute and delayed allergic reactions.

A cell surface receptor known to bind to human IgE is a high affinity cell surface receptor called FcεRI. FcεRI is located on a cell type including basophilic blood cells and mast cells. Binding of the antigen to IgE that has bound to FcεRI on the cell surface can activate the cascade that causes the release of the mediator from the cell. Released mediators such as certain allergic diseases and asthma turn off.

Another cell surface receptor known to bind to human IgE is the low affinity Fc[epsilon]RII receptor found on, for example, B cells. The binding of IgE to B cell surface receptors plays a role in determining the type of immunoglobulin produced by B cells.

It is known that the Fc portion of human IgG binds to an immunoreceptor tyrosine-based inhibitory unit (ITIM) containing a receptor called FcγRIIb. FcγRIIb is found, for example, on basophilic blood cells, mast cells, dendritic cells, and B cells. Cross-linking of the cell surface receptor FcγRIIb with the FeRI receptor inhibits the release of mediators associated with certain allergic diseases and asthma.

Epsi-gam is a fusion protein comprising the Fc portion of IgE and the Fc portion of IgG. Epsi-gam is configured to crosslink any of the Fc[epsilon]RI and Fc[epsilon]RII receptors to the inhibitory Fc[gamma]RIIb receptor.

It has been discovered that cross-linking of the FcγRIIb receptor with any of the two IgE-specific receptors on various cells has an overall inhibitory effect on their cells. When FcγRIIb and FcεRI are cross-linked on the cell surface of basophilic hematopoietic or mast cells, cross-linking results in inhibition of FcεRI-mediated activation and secretion of histamine and other mediators. When FcγRIIb is cross-linked to IgE-specific receptors on B cells, cross-linking inhibits class switching and antigen-specific IgE production.

Crosslinking of inhibitory receptors (such as FcγRIIb) expressed on certain cells (such as mast cells, basophils, and B cells) with high-affinity IgE receptors (such as FcεRI) or low-affinity IgE receptors (such as FcεRII) The FcεR-mediated biological response can be inhibited. Such biological responses include allergic reactions or autoimmune responses via FcεR. Types of biological reactions include, but are not limited to, conditions associated with IgE-mediated responses, such as asthma, allergic rhinitis, food allergies, chronic rubella, and angioedema, to Hymenoptera (eg, bees and wasps) An allergic reaction to a needle or a drug such as penicillin, and includes a severe physiological response to anaphylactic shock.

Figure 5 is an example of a graph showing the typical results of epsi-gam in blocking the release of histamine from different mast cell types. From the lungs of human lungs, cord blood and skin The cells are separately isolated in culture and stimulated to release histamine. As shown in the graph, the epsi-gam-treated cells released much less histamine than the control. Treatment of mast cells with epsi-gam was effective in inhibiting their release of histamine by stimulation compared to controls. See also example 1 below.

Epsi-gam is a polypeptide comprising an Fcε fragment sequence comprising the CH2, CH3 and CH4 domains of the IgE heavy chain constant region linked at its C-terminus to the N-terminus of the IgG ₁ heavy chain constant region (CHε2-CHε3- The CHε4 sequence), the IgG ₁ heavy chain constant region includes a functionally active hinge, a CH2 and a CH3 domain (gamma hinge-CHγ2-CHγ3 sequence).

The two polypeptide sequences that form epsi-gam are functionally linked, meaning that both retain the ability to bind to their respective natural receptors. Receptors that bind to epsi-gam include, for example, a native IgG inhibitory receptor, such as a low affinity Fc[gamma]RIIb receptor, and a native high affinity IgE receptor (such as Fc[epsilon]RI) or a low affinity IgE receptor (such as Fc[epsilon]RII). Therefore, an epsi-gam fusion protein comprising a functionally linked Fcε fragment and an Fcγ fragment can crosslink the respective native receptor FcγRIIb with FcεRI, or FcγRIIb can be cross-linked with FcεRII.

In order to achieve a functional linkage between the two binding sequences within the epsi-gam fusion protein, preferably, the binding sequence remains bound to its respective binding affinity of the native immunoglobulin ligand similar to the receptor. The ability of the receptor. Receptor binding domains within the native IgG and IgE heavy chain constant region sequences have been identified, and the CH2-CH3 interface of the IgG Fc domain has been reported to contain binding sites for a variety of Fc receptors, including Fc[gamma]RIIb low affinity receptors. Based on the FcεRI binding study, six amino acid residues (Arg-408, Ser-411, Lys-415, Glu-452, Arg-465, and Met-469) located in three loops (CD, EF, and FG) The outer ridge is formed on most of the exposed side of the human IgE heavy chain CH3 domain, and is involved in binding to the high affinity receptor FcεRI mainly due to electrostatic interaction. The high affinity receptor binding site in the IgE protein includes the Pro343-Ser353 peptide sequence in the CH3 domain of the IgE heavy chain, but the sequence at the N-terminus or C-terminus of the core peptide is also required to provide for maintaining the receptor binding structure. The structural skeleton of the shape. In particular, including His in the C-terminal region of the ε chain to maintain high affinity between IgE and FcεRI Force interaction makes an important contribution. The Fc[epsilon] and Fc[gamma]l polypeptide sequences within the epsi-gam fusion protein are designed to bind to residues within the binding regions.

Epsi-gam is typically produced and acts as a homodimer or heterodimer comprising two fusion proteins covalently linked to each other as described above.

In one embodiment, the covalent linkage of two fusion proteins that form a homodimer or a heterodimer is achieved via one or more disulfide bonds. For example, an epsi-gam protein can be produced as a homodimer comprising two CHε2-CHε3-CHε4-γ ₁ hinge-CH γ ₁ 2-CH γ ₁ 3-chains linked to each other by an interchain disulfide bond. To provide an immunoglobulin-like structure. Epsi-gam can also be produced as a heterodimer in which two different fusion proteins are linked to each other by one or more covalent bonds, such as single or multiple disulfide bonds.

Figure 6 is an example of a nucleotide sequence substantially encoding a nucleotide of the fusion protein epsi-gam. Epsi-gam contains 552 amino acid long nucleotide sequences.

The epsi-gam amino acid sequence differs from the amino acid sequence of E2G in the presence of two amino acids. The amino acids that are not present in epsi-gam but are present in the polypeptide sequence of E2G are arginine and serine at positions 321 and 322, respectively, of the E2G polypeptide sequence.

The arginine and serine, which are present in the polypeptide sequence of E2G but not present in the sequence of epsi-gam, are encoded by restriction sites located on the nucleotide encoding E2G. A restriction site encoding the nucleotides of arginine and serine found in E2G was added to the nucleotide encoding E2G to facilitate colonization of the nucleotide encoding E2G.

Due at least in part to the position of arginine and serine in the E2G polypeptide sequence and the effect of these amino acids on the E2G configuration, E2G has a high degree of aggregation and low yield in scale.

The design of the fusion protein epsi-gam of arginine and serine was removed from positions 321 and 322 of E2G to unexpectedly and significantly produce improvements in aggregation and yield upon scale. As discussed above, the scale of epsi-gam produces a very low aggregation rate and a 22,000-fold higher yield compared to E2G. Among the fusion protein populations including E2G, GE2 and similar molecules, epsi-gam is the only fusion protein that is feasible to translate into clinical therapy.

Epsi-gam is a novel fusion protein that is configured to crosslink an Fc[epsilon]RI or Fc[epsilon]RII receptor with an Fc[gamma]RIIb receptor, thereby inhibiting the release of certain cellular mediators including histamine. Epsi-gam is capable of acting as a therapy for a variety of human diseases including allergic reactions and asthma. Importantly, the novel fusion protein epsi-gam overcomes some of the manufacturing-related limitations currently seen in the prior art, enabling epsi-gam to be effectively translated into a suitable mass-producing clinical therapy alone.

IV. Making epsi-gam

Epsi-gam is highly advantageous for large-scale production of fusion proteins, because when using the described manufacturing method to make epsi-gam, epsi-gam can be produced with unexpectedly high yields and very low aggregation compared to other similar fusion proteins. .

Aggregated fusion proteins are completely unusable and pose potential clinical risks. Thus, fusion proteins with high percent aggregation cannot be transitioned to a wide range of clinical uses, whereas fusion proteins with unexpectedly low aggregation rates, such as epsi-gam, are highly advantageous for transition to clinical use from a manufacturing standpoint.

When comparing the yield of epsi-gam produced by the described manufacturing method with the yield of other fusion proteins produced by the same method, the yield of epsi-gam was significantly higher than that of a similar fusion protein, and the aggregate protein concentration of epsi-gam Significantly lower.

Epsi-gam is a polypeptide in which the Fcε and Fcγ1 polypeptide sequences are directly fused. Nucleotide and amino acid sequences of native immunoglobulin constant regions (including native IgG and IgE constant region sequences) are available, for example, from Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition. Public Health Service, National Institute Of Health, Bethesda, Md. (1991).

Epsi-gam is produced, at least in part, by the following general procedure (see also Example 2 below): epsi-gam encoding DNA is obtained via methods such as gene synthesis. Subsequently, the epsi-gam encoding the cDNA is incorporated into a suitable vector. Subsequently, the eukaryotic cells are transfected with a vector containing epsi-gam encoding the cDNA, for example, via electroporation, Such as Chinese Hamster Ovary/CHO cells. Subsequently, epsi-gam was isolated from eukaryotic cells and purified.

Figure 6 is an example of a nucleotide sequence substantially encoding a nucleotide of the fusion protein epsi-gam. Nucleotide sequences encoding essentially the epsi-gam polypeptide sequences can be prepared by methods well known in the art, such as methods using recombinant DNA techniques or conventional gene synthesis. It will be understood that the nucleotide sequence shown in Figure 6 is only one example of a nucleotide encoding epsi-gam, as is well known in the art, and that different codons can encode the same amino acid. Thus, it is understood that certain nucleotide substitutions can be made to the nucleotide sequence shown in Figure 6, and the nucleotide sequence will still encode epsi-gam.

Suitable vectors for the manufacture of epsi-gam are prepared by inserting cDNA-encoding epsi-gam into a plastid (such as the Lonza GS system) using standard techniques of recombinant DNA technology, such as described in "Molecular Cloning: A Laboratory Manual, 2nd ed. (Sambrook et al., 1989); "Oligonucleotide Synthesis" (edited by MJ Gait, 1984); "Animal Cell Culture" (edited by RI Freshney, 1987); "Methods in Enzymology" (Academic Press, Inc.); "Handbook of Experimental Immunology", 4th edition (edited by DM Weir and CC Blackwell, Blackwell Science Inc., 1987); "Gene Transfer Vectors for Mammalian Cells" (JMMiller and MP Calos, eds., 1987); Current Protocols in Molecular Biology" (FMAusubel et al., ed., 1987); "PCR: The Polymerase Chain Reaction" (Mullis et al., ed., 1994); and "Current Protocols in Immunology" (J. E., et al., 1991) . The isolated plastids and DNA fragments are cleaved, tailored and joined together in a specific order to produce the desired vector. After ligation, the vector containing the gene to be expressed is transferred to a suitable host cell.

The host cell can be any eukaryotic or prokaryotic host known to be used to express a heterologous protein. Thus, polypeptides comprising epsi-gam can be expressed in eukaryotic hosts such as eukaryotic microorganisms (yeast) or cells, plants and insect cells isolated from multicellular organisms (mammalian cell cultures). Examples of mammalian cell lines suitable for the expression of heterologous polypeptides include monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell line 293S (Graham et al., J. Gen. Virol) .36:59 [1977]); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary (CHO) cells (Urlaub and Chasin, Proc. Natl. Acad. Sci. USA 77: 4216 [1980]); Monkey kidney cells (CV1-76, ATCC CCL 70); African green monkey cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34) Human lung cells (W138, ATCC CCL 75); and human liver cells (Hep G2, HB 8065). In general, myeloma cells, particularly myeloma cells that do not produce any endogenous antibodies (eg, non-immunoglobulin producing myeloma cell line SP2/0) can be used to produce the fusion proteins herein.

Saccharomyces cerevisiae is the most commonly used in low-level eukaryotic hosts. However, a variety of other genus, species, and strains are also available and suitable for use herein, such as methanol yeast (EP 183,070; Sreekrishna et al, J. Basic Microbiol. 28: 165-278 (1988)). Yeast expression systems are commercially available and are commercially available, for example, from Invitrogen (San Diego, Calif.). Other yeasts suitable for bifunctional protein expression include, but are not limited to, Kluyveromyces hosts (U.S. Patent No. 4,943,529), such as Kluyveromyces lactis ; Schizosaccharomyces pombe (Beach and Nurse, Nature 290: 140 (1981)); Aspergillus host, such as A. niger (Kelly and Hynes, EMBO J. 4: 475-479 (1985)) and Aspergillus nidulans ( A) .nidulans ) (Ballance et al, Biochem. Biophys. Res. Commun. 112:284-289 (1983)); and Hansenula host, such as Hansenula polymorpha . Yeast grows rapidly on inexpensive (minimum) media, and recombination can be readily selected by supplementation, and expression proteins can be engineered especially for cytoplasmic localization or extracellular output, and are more suitable for large-scale fermentation.

The eukaryotic expression system using an insect cell host can rely on a plastid or baculovirus expression system. A typical insect host cell is derived from Spodoptera frugiperda . For the performance of heterogeneous proteins, the Autographa californica baculovirus (Autographa californica) recombinant nuclear polyhedrosis virus infection in the form of these cells, in the form of a recombinant expression of the virus under control of the polyhedrin promoter Focus on genes. Other insects infected with this virus include the cell line known commercially as "High 5" (Invitrogen), which is derived from Brassica chinensis ( Trichoplusia ni ). Another baculovirus sometimes used is the Bombyx mori nuclear polyhedrosis virus, which infects silk worms (silkworm). Baculovirus expression system and many are commercially available, for example from ^{Invitrogen (Bac-N-Blue TM} ), Clontech (BacPAK TM Baculovirus expression system), Life Technologies (BAC-TO -BAC TM), Novagen ( ^{Bac Ventor System TM), Pharmingen and} Quantum Biotechnologies. Another insect cell host is a common fruit fly, Drosophila melanogaster (Drosophila melanogaster), by Invitrogen (The DES ^TM system) provide transient or stable commercially mass-based transfection kits.

Prokaryotes can be the host for the initial colonization step and are suitable for rapid generation of large amounts of DNA, suitable for generating a single sequence for mutation induction, for simultaneous screening of many mutants and for DNA sequencing of the resulting mutants. Strand DNA template. Escherichia coli ( E. coli ) strains suitable for producing epsi-gam peptides include, for example, BL21 carrying the inducible T7 RNA polymerase gene (Studier et al., Methods Enzymol . 185: 60-98 (1990)); AD494 (DE3) EB105; and CB (E. coli B) and its derivatives; K12 strain 214 (ATCC 31,446); W3110 (ATCC 27,325); X1776 (ATCC 31,537); HB101 (ATCC 33,694); JM101 (ATCC 33,876); NM522 ( ATCC 47,000); NM538 (ATCC 35,638); NM539 (ATCC 35,639), and the like. Many other species and genus of prokaryotes can also be used. The epsi-gam peptide of the present invention can be readily produced in large quantities by using a recombinant protein in bacteria in which the peptide is fused to a cleavable ligand for affinity purification.

Suitable promoters, vectors and other components for expression in a variety of host cells are well known in the art.

Transfection of suitable cells with a vector containing a nucleotide sequence encoding epsi-gam is achieved, for example, using electroporation techniques.

Whether a particular cell or cell strain is suitable for producing a polypeptide in a functionally active form herein can be determined by empirical analysis. For example, a construct construct comprising a coding sequence for a desired protein can be used to transfect a candidate cell line. Subsequently, the transfected cells are grown in the culture. The transfected cells secrete epsi-gam into the cell culture medium.

Isolation of the fusion protein epsi-gam can be achieved by, for example, collecting in a medium in which the transfected cells are grown, analyzing the presence of secreted epsi-gam and purifying the secreted epsi-gam. Subsequently, epsi-gam can be quantified by methods known in the art, such as by ELISA in which the antibody specifically binds to the IgG, IgE portion of the protein.

V. The nature of targeted diseases

The allergic reaction is classified according to the type of immune response induced and the resulting tissue damage resulting from the reactivity of the antigen. Type I (synaptic hypersensitivity) occurs when antigens (in this case called allergens) entering the body encounter mast cells or basophilic blood cells, which are due to IgE It is sensitive to antigens linked to its high affinity receptor (FcεRI). Upon reaching sensitive mast cells, the allergen cross-links with IgE bound to FcεRI, resulting in an increase in intracellular calcium (Ca ²⁺ ), which triggers pre-formed mediators (such as histamine and protease) and newly synthesized lipid-derived mediators ( Releases such as leukotrienes and prostaglandins. These endocrine products produce acute clinical symptoms of allergic hypersensitivity reactions. Stimulating basophilic blood cells and mast cells will also produce and release pro-inflammatory mediators that are involved in the acute and delayed phases of allergic reactions.

Various allergens have been identified to date, and new allergens are regularly identified, colonized, and sequenced. Allergens enter the body in different forms and are associated with different diseases and disease processes.

Inhalation of airborne allergens causes allergic rhinitis and allergic asthma, depending on the nature of the exposure However, it can be acute or chronic. Exposure of the eye to airborne allergens causes allergic conjunctivitis. Common airborne allergens include pollen, animal scales, dust mites, and other insect proteins and mold spores that contain most of the most common seasonal hay fever and allergic asthma.

Ingestion of allergens leads to gastrointestinal and systemic allergic reactions. The most common food allergens involved are peanuts, shellfish, milk, fish, soybeans, wheat, eggs, and tree nuts (such as walnuts). In susceptible populations, these foods can trigger a variety of allergic symptoms such as nausea, vomiting, diarrhea, rubella, angioedema, asthma and severe systemic allergies.

Skin exposure to allergens (such as natural rubber latex proteins found in latex gloves) can cause local allergic reactions, and the location of contact with allergens is urticaria (rubella).

Systemic exposure to allergens (such as bee stings, penicillin injections, or the use of natural rubber latex (NRL) gloves) that occurs inside the patient during surgery can cause skin, gastrointestinal, and respiratory reactions, including respiratory blockage and severe systemic allergies. reaction. Hymenoptera insects are usually insects that cause an allergic reaction and usually cause anaphylactic shock. Examples include various bees including bees, wasps, bumblebees, wasps, and white-faced wasps. Some ants known as fire ants ( Solenopsis invicta ) are an increasing cause of allergies in the United States because they expand their range in this country. Protein in NRL gloves has become an increasing concern for health care workers and patients, and currently there is no successful form of therapy for this problem.

Therapeutic use of VI. epsi-gam

Epsi-gam provides a novel therapeutic strategy for the treatment of diseases mediated via high affinity IgE receptors.

In particular, epsi-gam is provided for the treatment and prevention of, for example, but not limited to, asthma, allergic rhinitis, atopic dermatitis, severe food allergies, chronic rubella and angioedema, as well as by, for example, a bee sting needle or A compound of severe physiological condition of anaphylactic shock caused by penicillin allergy.

Epsi-gam can be further used for acute or chronic inhibition of IgE-mediated responses to severe environmental and occupational allergens.

Epsi-gam can be used to provide protection against allergy vaccination or immunotherapy, and to induce non-allergic responsive conditions during treatment for a particular allergen.

When administered to an individual at risk, epsi-gam can also prevent allergic diseases by preventing allergic sensitization of environmental and occupational allergens. For example, individuals at risk for asthma genes and individuals exposed to occupational allergens at work.

Epsi-gam can be used acutely to desensitize patients so that the administration of therapeutic agents such as penicillin can be safely provided. Similarly, epsi-gam can be used to desensitize patients, allowing standard allergen vaccination to be provided with greater safety.

Epsi-gam can also be used as a chronic therapy to prevent clinical responsiveness, prevent environmental allergens such as food or inhaled allergens.

In addition, epsi-gam has great promise for the treatment of chronic rubella and angioedema. Chronic rubella and angioedema usually co-exist, but they also appear alone. These conditions are common and, once present for more than six months, they typically last for ten or more years. The letter epsi-gam forms the basis for novel and effective treatment of these diseases by safely blocking access to FcεRI.

In addition, depending on the role of mast cells and basophilic blood cells in the disease, epsi-gam can be used to treat inflammatory arthritis, such as rheumatoid arthritis or other autoimmune conditions.

In addition, epsi-gam can be used to treat chronic idiopathic rubella and a group of disorders known as mast cell activation syndrome.

Chronic idiopathic rubella (CIU) is a type of rubella affecting approximately 150,000 individuals in the United States. In the case of rubella, the individual suffers from a rubella block (a raised erythema lesion) that can occur anywhere on the surface of the skin and can cause itching. Individuals with chronic forms of rubella will generally have long-term non-dissipative lesions, such as lesions that do not dissipate for six or more weeks. Idiopathic Chronic type rubella is a chronic rubella with an unknown cause (hence, it is idiopathic). Many individuals with CIU have a certain type of autoimmune cause for their CIU.

Of the approximately 150,000 individuals with CIU in the United States, approximately 60,000 individuals or 40% of CIU patients have autoantibodies to their high-affinity IgE receptors on mast cells and basophilic blood cells. Salty autoantibody binding can trigger CIU in such individuals. Traditional treatments for such individuals include antihistamines and steroids associated with severe side effects. Epsi-gam is an excellent treatment for CIU in these individuals.

When administering epsi-gam to an individual with CIU, epsi-gam crosslinks mast cells with IgE and IgG receptors on basophilic blood cells, thereby inhibiting inflammatory mediators (such as histamine) from such cells The release of). Inhibition of mast cells and basophilic blood cells by epsi-gam can treat and prevent the development of CIU, for example by blocking the binding of autoantibodies to IgE type receptors and, for example, by generally downregulating allergic responses.

Mast cell activation syndrome or mast cell activation disease (MCAD) is a disease class associated with the accumulation of mast cells in any potential organ or tissue of an individual. Mast cells accumulated in MCAD are associated with abnormal release of different types of mast cell mediators. The subgroup of MCAD includes cutaneous mastocytosis affecting approximately 150,000 individuals in the United States, systemic mastocytosis affecting approximately 15,000 individuals, and invasive systemic mastocytosis affecting approximately 1,000 individuals in the United States. Skin mastocytosis causes severe itching and rubella and usually affects children. Systemic mastocytosis causes a variety of symptoms associated with systemic mediator release, including itching, rubella, syncope, headache, blushing, angioedema, abdominal pain, diarrhea, nausea, and vomiting. Invasive systemic mastocytosis is a more aggressive form of systemic mastocytosis, which may include bone marrow invasion. Epsi-gam is an effective treatment for MCAD.

When epsi-gam is administered to an individual with MCAD, epsi-gam crosslinks IgE and IgG receptors on mast cells, thus inhibiting the release of inflammatory mediators (such as histamine) from such cells. Inhibition of media by, for example, blocking by inhibition of epsi-gam mast cells To treat and prevent the development of MCAD.

Additionally, epsi-gam can be used to provide effective adjuvant therapies in individuals receiving immunotherapy prophylaxis, such as emergency immunotherapy prophylaxis.

Rapid administration of emergency immunotherapy prevention to make individuals sensitive to allergens. In some cases, an individual may need to be sensitive to an allergen via a rapid method, such as receiving repeated low dose exposures for a short period of time. Emergency immunotherapy prevention can be administered, for example, in the event that someone with a severe or life threatening allergy will experience exposure or will soon be exposed to an allergen. For example, individuals with severe insect venom allergies can receive emergency immunotherapy prevention in a series of injections shortly before the local insect season begins. The combination of epsi-gam and emergency immunotherapy prevention ensures safe delivery of prevention, provides sustained tolerance and allows for faster delivery of emergency immunotherapy prevention protocols.

Epi-gam crosslinks IgE and IgG receptors on cells involved in allergic reactions when administered to epsi-gam to individuals who will undergo emergency immunotherapy prevention or are undergoing emergency immunotherapy prevention or recent experience with emergency immunotherapy prevention Thus, inhibition of the release of inflammatory mediators, such as histamine, from such cells. Inhibition of cells involved in allergic reactions allows individuals with severe allergies to allergens to be safely and rapidly sensitized to their allergens. Sensitization with epsi-gam via an emergency immunotherapy prevention protocol also produces more effective sensitization, which produces fewer symptoms, less severe symptoms, and longer sustained tolerance.

Epsi-gam can be administered to an individual, for example, one month or more before the start of emergency immunotherapy prevention. Epsi-gam can be administered to an individual, for example, one week or more prior to the initiation of emergency immunotherapy prevention. Epsi-gam can be administered to an individual, for example, one day or more before the start of emergency immunotherapy prevention. Epsi-gam can be administered to an individual, for example, one hour or more before the initiation of emergency immunotherapy prevention. Epsi-gam can be administered to an individual, for example, just prior to the onset of emergency immunotherapy prevention. Epsi-gam can be administered to an individual at any time during the initiation of emergency immunotherapy prevention, for example. Epsi-gam can be administered to an individual at any time, for example, after initiation of emergency immunotherapy prevention.

Epsi-gam can similarly be used as any and all food-specific subcutaneous immunotherapy (SCIT), sublingual immunotherapy, peptide-based method (Circassia), oral immunotherapy for food, endolymph (such as In the case of emergency immunotherapy, the ipivision of the same way of epsi-gam is administered as an adjuvant therapy.

VII. Compositions and formulations

For therapeutic use including prophylaxis, epsi-gam can be formulated as a pharmaceutical composition in admixture with a pharmaceutically acceptable carrier or diluent. Methods of making pharmaceutical formulations are well known in the art.

The pharmaceutical composition of epsi-gam may comprise a fusion molecule as well as a conventional carrier and other ingredients as appropriate.

Suitable forms are determined depending on the use or route of entry (eg oral, transdermal, inhalation or injection). Such forms should allow the agent or composition to reach the target cell, whether or not the target cell is present in a multicellular host or culture. For example, a pharmacological agent or composition that is injected into the bloodstream should be soluble. Other factors are known in the art and include considerations such as the toxicity and form of the barrier agent or composition to exert its effect.

Carriers or excipients can also be used to facilitate the administration of the compound. Examples of carriers and excipients include calcium carbonate; calcium phosphate; various sugars such as lactose, glucose or sucrose; or starch type; cellulose derivatives; gelatin; vegetable oils; polyethylene glycol; and physiologically compatible Solvent.

The compositions or pharmaceutical compositions can be administered by a variety of routes including, but not limited to, oral, intravenous, intraarterial, intraperitoneal, subcutaneous, intranasal or intrapulmonary routes.

In one embodiment, epsi-gam is administered intravenously to an individual. In one embodiment, the intravenous dose of epsi-gam can comprise, for example, 0.01 mg/kg. In one embodiment, the intravenous dose of epsi-gam can comprise, for example, 0.1 mg/kg. In one embodiment, the intravenous dose of epsi-gam can comprise, for example, 0.3 mg/kg. In one embodiment, the intravenous dose of epsi-gam can comprise, for example, 1.0 mg/kg. In one embodiment, epsi-gam An intravenous dose can, for example, comprise 3.0 mg/kg. In one embodiment, the intravenous dose of epsi-gam can comprise, for example, 10.0 mg/kg. It will be understood that examples of epsi-gam intravenous doses are non-limiting and that other doses are suitable for intravenous administration to an individual.

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation.

One route of administration of a compound of the invention can be an inhalation for intranasal and/or intrapulmonary delivery. One example of a method of intrapulmonary delivery is an exhalation-actuated metered dose inhaler that operates to provide an automatically metered dose in response to a patient's inspiratory action.

For topical administration, the compounds of the invention are formulated into ointments, salves, gels or creams, as generally known in the art.

The compounds of the invention may be administered in combination with one or more other therapeutic agents that treat an IgE-mediated allergic disease or condition. Such other therapeutic agents include, but are not limited to, corticosteroids, beta antagonists, theophylline, leukotriene inhibitors, allergen vaccination, and bioreactive modifiers (such as soluble recombinant human soluble IL-4 receptor ( Immunogen)) and therapy for targeting Toll-like receptors. (See, for example, Barnes, The New England Journal of Medicine 341: 2006-2008 (1999)). Thus, the compounds of the invention are useful as supplements to traditional allergy therapies, such as corticosteroid therapy performed with inhaled or oral corticosteroids.

VIII. Products

The invention also provides articles comprising epsi-gam. The article comprises a container and a label or package insert on or associated with the container.

Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials such as glass or plastic.

The container holds a composition that is effective to treat the condition and can have a sterile inlet.

The container may also be an inhaler device such as those described above.

At least one active agent in the composition is epsi-gam.

The label or package insert indicates that the composition is used to treat a selected condition, such as allergies A condition, such as asthma or any of the IgE-mediated allergies as described above.

Further details are illustrated by the following non-limiting examples.

Instance Example 1: Mast cell efficacy

Examples of how to obtain mast cell efficacy results are provided below.

Mast cells are sensitive by incubating with allergic human serum as a source of allergen-specific IgE for 3-5 days at 37 ° C, 5% CO ₂

Thereafter, the cells are typically exposed to epsi-gam for 2 hours, but may be as long as 24 hours, after which the cells are exposed to the allergen for 45 minutes to thresh the histamine.

Histamine and cells released into the supernatant were collected and histamine content was quantified using histamine EIA.

Example 2: Making epsi-gam

An example of how to make epsi-gam is provided below.

The nucleotide sequence SEQ ID NO: 1 is generated via gene synthesis, which essentially encodes the amino acid sequence of epsi-gam. After gene synthesis, the sequence was inserted into the selection vector pUC57. A restriction site is created on either side of the performance cassette so that the gene can be excised for selection into the expression vector.

The selection vector containing epsi-gam was further impregnated with HindIII and EcoRI restriction enzymes for selection into expression vectors (such as the Lonza GS system pXC-17.4 plastid) for expression of a single unit of protein. This expression of the plastid associates the performance of the foreign protein with the branylamine synthase (GS) gene.

The GS Chinese Hamster Ovary (CHO) cell line has mutations in two copies of the GS gene, so in the absence of branic acid (the amino acid necessary for growth) it requires GS to behave on the plastid to survive.

Once the epsi-gam insert was cloned into this vector, individual bacterial colonies were selected for sequence analysis. Use the primer outside the ORF of epsi-gam for the plastid The plastids are verified when the sequence and the appropriate orientation. Based on this information, a single selection line was selected for scale and to produce a sufficient number of plastids transfected with CHO cells.

Bacterial colonies were grown in animal-free, soy-trypsin-based medium and the plastids were purified using a low endotoxin kit.

In the absence of selection, GS CHO cells are incorporated into a suitable medium formulation; that is, contain L-glutamic acid. CHO cells were maintained at 5% CO ₂ in an incubator at 37 °C.

CHO cell transfection by the expression vector was performed using electroporation. The cells were electroporated by delivering a single pulse of 300 V, 900 μF, with the resistance set to infinity. Immediately after electroporation, each batch of cells was added to a flask containing the appropriate medium. The cells were gradually mixed and incubated overnight in a cell culture incubator. The next day, the cells were centrifuged to remove the medium containing the plastids.

When the number of cells reached about 0.6 x 10 ⁶ cells per ml, the cells were expanded into selection medium. After two weeks of selection, individual colonies of CHO cells were selected based on growth on the transwell plate. The CHO cell secretes a fusion protein having the sequence of SEQ ID NO: 2.

Individual colonies were evaluated based on growth and yield characteristics via scale.

Subsequently, the amount and integrity of the product were analyzed by protein A affinity chromatography followed by AU280 nm; ELISA and SDS-PAGE analysis.

Example 3: Emergency Immunotherapy Prevention

At the appropriate dose, 3 times a month (12 weeks) to participate in one of the two dose groups (20 experiments, 10 placebo / group), with a bee licking needle to record allergies (IgE, skin test, symptoms) and willing Sixty individuals who experienced a one-month emergency IT agreement in the hospital voted for epsi-gam. Based on the document of drug activity (PD biomarker), emergency immunotherapy prevention was initiated after 1-2 times of epsi-gam administration.

Example 4: Epsi-gam improves early and late clinical response in a rhesus monkey model of asthma

Figures 7-9 show the results of an experiment in which epsi-gam was administered to 12 rhesus monkeys. Prior to the provision of epsi-gam, 12 rhesus monkeys were pre-sensitive to house dust mite, following Van Scott, M. et al., Dust mite-induced asthma in cynomolgus monkeys. J Appl Physiol 96: 1433-1444, The protocol described in 2004 develops asthma. Twelve rhesus monkeys showed an increase in lung tolerance (R _lung ) and a decrease in dynamic compliance (C _dynamics ) in response to lung stimulation.

For the IgE content specific for house dust mites (HDM), approximately 150 rhesus monkeys were screened to select animals that had been exposed to and were allergic to HDM. Under a recognized agreement (Van Scott, JA Physiol, 2004), the macaques were sensitized subcutaneously and aerosolized with HDM every two weeks to induce respiratory symptoms in allergic asthma. The pathology and physiology of this model and its association with human disease have been established in previous publications (Van Scott, JA Physiol, 2004). Nine of the twenty animals developed tolerance to aerosolized HDM (R _lung ) response, and three other animals exhibited increased respiratory rate and reduced dynamic compliance (C _dynamics ), but R _lungs after HDM challenge No significant changes. Bronchoalveolar lavage differentiated cell counts confirmed that the respiratory tract in the tolerant responder was infiltrated by eosinophils. For 12 HDM-sensitive animals, see the test with epsi-gam. Targeted responses to aerosolized allergens that demonstrate effective sensitization include a 100% increase in lung tolerance (R _lung ), a 40% reduction in dynamic compliance (C _dynamics ), and an HDM aerosol concentration that induces these targeted responses. reduce.

Twelve HDM-sensitive animals were divided into three groups, the tolerant responders and non-responders were equally divided, and four animals in each group were initially provided with vehicle control (PBS) IV infusion over a 30 minute period. After using the instrument for light anesthesia, the baseline measurements of lung tolerance (R _lung ) and dynamic compliance (C _dynamics ) after challenge with one dose of aerosolized HDM (individualized for each animal) are obtained, yielding at least 100 % R _lung increased and 40% C decreased _dynamically . The same dose of aerosolized HDM was administered every two weeks to establish the underlying response. As illustrated in Figure 7, a consistent base reaction (circle) was obtained in all groups over a 6 week period.

Subsequently, four animals of each group were treated by IV infusion of 1 mg/kg, 3 mg/kg or 10 mg/kg of epsi-gam over 30 minutes. Similarly, R _lung and C _dynamic measurements were obtained after two weeks of aerosolized HDM challenge using the same dose of HDM used in each animal of the study vehicle group. After treatment, animals receiving the 3 mg/kg dose of epsi-gam exhibited smaller HDM-induced changes in R _lung and C _dynamics for up to 8 weeks (Figure 7). No consistent effects were observed in the 1 mg/kg group and similar reactions, but slightly more changes were found in the 10 mg/kg group (Figure 8).

Animals were tested for sensitivity to methotrexate 24 hours after nebulized HDM challenge. Increasing doses of methotrexate were delivered to the respiratory tract until an increase of 100% in R _lungs and/or a 50% decrease in C _{dynamics was} observed. For animals receiving 3 mg/kg of epsi-gam, the induced concentration of methotrexate (defined as the dose that causes 100% maximal response to R _lung ) is plotted in Figure 9.

Example 5: epsi-gam suppression category conversion

Immunoglobulins consist of an antigen recognition domain (VDJ) and an effector domain, regulated by various constant domains. The five constant regions (Cμ, Cδ, Cγ, Cε, and Cα) are downstream of the VDJ region and their performance is controlled by individual promoters. Homotypic transformation occurs through a multi-step process involving recombination of germline transcription and class switching. Class switching recombination is thought to occur only in the germinal centers of lymph nodes and spleen. However, germline transcripts have been observed in nasal and bronchial biopsies of individuals with rhinitis and asthma after allergen challenge. These studies have suggested that local IgE class switching can significantly promote the possibility of disease etiology.

Two signals are required in human B cells to produce IgE class switching: participating in CD40L and IL-4 or IL-13 on B cells. Human B cells exhibit "low affinity" (nM) IgE receptor (CD23) and FcγRIIb. It has been previously shown ( Reference ) that the involvement of the FcyRIIb receptor results in inhibition of IgE production via inhibition of class switching recombination, and this effect is mediated via the ITIM domain within FcyRIIb. To determine whether epsi-gam is involved in CD23 and FcγRIIb, it can lead to a decrease in IgE production, and human tonsil primary B cells are isolated via tissue digestion followed by magnetic cell separation. B cells were cultured in the presence of anti-CD40 antibody (0.5 μg/mL), IL-4 (20 ng/mL), and IL-13 (200 ng/mL) to induce class switching recombination. Increasing concentrations of epsi-gam (0-80 nM) were added during this period, and the amount of IgE transcript obtained was quantified by quantitative PCR on day 7 after stimulation. Two reference genes were used to normalize the expression of IgE, large ribosomal protein (RPLO) and beta actin. Standardization was performed by the method of Vandesompele, et al. ( Reference ). Figure 10 shows that epsi-gam inhibits the relative expression of IgE transcripts in human B cells.

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Claims (19)

A fusion protein comprising the sequence of SEQ ID NO: 2. The fusion protein of claim 1, further comprising: a homodimer comprising two covalently linked polypeptides, the two covalently linked polypeptides comprising the sequence of SEQ ID NO: 2. The fusion protein of claim 1, further comprising: a heterodimer comprising two covalently linked polypeptides, one of the two covalently linked polypeptides comprising the sequence of SEQ ID NO: 2. A method for producing a fusion protein, comprising: integrating a polynucleotide encoding the fusion protein comprising the sequence of SEQ ID NO: 2 into a performance vector; transfecting the eukaryotic cell with the expression vector; The eukaryotic cell isolates the fusion protein comprising the sequence of SEQ ID NO: 2. The method of claim 4, wherein the eukaryotic cell is a Chinese Hamster Ovary/CHO cell. A method for treating or preventing asthma comprising administering to a subject a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing an allergic response comprising administering to a subject a fusion protein comprising the sequence of SEQ ID NO: 2. The method of claim 7, wherein the allergic reaction comprises at least one of: allergic rhinitis, atopic dermatitis, severe food allergy, chronic rubella, angioedema, and IgE-mediated response to a drug. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 0.01 mg/kg of a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 0.1 mg/kg of a fusion comprising the SEQ ID NO: 2 protein. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 0.3 mg/kg of a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 1.0 mg/kg of a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 3.0 mg/kg of a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing an allergic reaction in an individual, the method comprising intravenously administering to the individual 10.0 mg/kg of a fusion protein comprising the sequence of SEQ ID NO: 2. A method for treating or preventing development of mast cell activation syndrome in an individual, the method comprising administering to the individual a fusion protein comprising the sequence of SEQ ID NO: 2. The method of claim 15, wherein the mast cell activation syndrome comprises cutaneous mastocytosis, systemic mastocytosis or invasive systemic mastocytosis. A method of administering an emergency immunotherapy to an individual, the method comprising administering to the individual a fusion protein comprising the sequence of SEQ ID NO: 2 at the time of administration of the emergency immunotherapy prevention. The method of claim 17, wherein the fusion protein is one of the administration of the emergency immunotherapy prevention, the administration of the emergency immunotherapy prevention, or the administration of the emergency immunotherapy prevention. The individual is administered for a plurality of time periods. The method of claim 17, wherein the fusion protein is administered monthly and the individual is The administration of the emergency immunotherapy prevention has been administered at least once before the administration of the fusion protein.