Immunomodulatory proteins
阅读说明：本技术 免疫调节蛋白 (Immunomodulatory proteins ) 是由 理查德·约翰·普利斯 于 2012-10-17 设计创作，主要内容包括：本申请涉及免疫调节蛋白。公开了一种用于治疗哺乳动物受试者的自身免疫性疾病或炎性疾病的方法,所述方法包括：向哺乳动物受试者施用有效量的多聚蛋白,所述多聚蛋白包含5个、6个或7个多肽单体单元；其中每个多肽单体单元包含Fc受体结合部分,所述Fc受体结合部分包含2个免疫球蛋白G重链恒定区；其中每个免疫球蛋白G重链恒定区包含半胱氨酸残基,所述半胱氨酸残基通过二硫键连接到毗邻多肽单体单元的免疫球蛋白G重链恒定区的半胱氨酸残基；其中所述多聚蛋白不包含另外的免疫调节部分,或当施用到哺乳动物受试者时引起抗原特异性免疫抑制的抗原部分。(The present application relates to immunomodulatory proteins. Disclosed is a method for treating an autoimmune disease or an inflammatory disease in a mammalian subject, the method comprising: administering to a mammalian subject an effective amount of a polymeric protein comprising 5, 6, or 7 polypeptide monomer units; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising 2 immunoglobulin G heavy chain constant regions; wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit; wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject.)
1. A method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein comprising 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit;
wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject.
2. The method of claim 1, wherein each polypeptide monomeric unit comprises a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions; wherein the tail segment region of each polypeptide monomer unit facilitates assembly of the 5, 6, or 7 polypeptide monomer units into a polymer.
3. The method of claim 2, wherein the tail segment region is an IgM or IgA tail segment, or a fragment or variant thereof.
4. The method of claim 1, wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence having at least 90% sequence identity to a native human immunoglobulin G1 heavy chain constant region.
5. The method of claim 1, wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence comprising a cysteine residue at position 309, and preferably further comprising a leucine residue at position 310, according to the EU numbering system.
6. The method of claim 1, wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence that is modified compared to the amino acid sequence of a native immunoglobulin G heavy chain constant region to modify the affinity of the Fc receptor binding portion for at least one Fc receptor.
7. The method of claim 1, wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence that is modified compared to the amino acid sequence of a native immunoglobulin G heavy chain constant region to suitably increase the in vivo half-life of the polymeric protein by increasing the affinity of the Fc receptor binding portion for a neonatal Fc receptor.
8. The method of claim 1, wherein the autoimmune or inflammatory disease is treatable with intravenous immunoglobulin (IVIG).
9. The method of claim 1, wherein the autoimmune or inflammatory disease is autoimmune cytopenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, asthma, kawasaki disease, guillain-barre syndrome, stevens-johnson syndrome, crohn's colitis, diabetes, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, anti-factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis or alzheimer's disease.
10. A method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein consisting of 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc; and is
Wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
Technical Field
The present invention relates to engineered proteins with immunomodulatory properties, and their medical use for the treatment of autoimmune or inflammatory diseases. In particular, the engineered proteins may be used as a replacement for intravenous immunoglobulin (IVIG).
Background
Autoimmune and inflammatory diseases are the cause of high morbidity and mortality. In 2003, autoimmune disease was the root cause of the 6 th most common death in all age groups under 75 years of age. An important therapeutic modality is intravenous immunoglobulin (IVIG) or IgG. Initially, immunoglobulin products from human plasma were developed to treat immunodeficiency. However, 70% of the prescribed IVIG is currently used for the treatment of autoimmune or inflammatory conditions. IVIG consumption worldwide has increased from 300kg per year in 1980 to 100 metric tons per year in 2010. IVIG was derived from plasma pooled from approximately 3,000 anonymous donors according to a lengthy and expensive manufacturing process. The need for extensive screening of viruses for donors and donated plasma generates high costs. In view of the increased demand, and the tight regulation of IVIG production, a shortage of IVIG may occur. IVIG preparations may be subject to inadequate sterility, the presence of impurities and batch-to-batch variations. They can vary greatly in their immunoglobulin a content, and IgA can cause allergic or anaphylactic reactions in IgA-deficient recipients. Making them unsuitable for certain patients. Very large doses of IVIG, typically 2 grams per kilogram body weight, have to be administered to patients, and this may cause adverse effects in some patients. In view of these limitations, defined alternatives for IVIG are needed.
Although the mechanism of action of IVIG is not clear, Fc fragments derived from IVIG can cure children suffering from Idiopathic Thrombotic Purpura (ITP) (debrre M et al, 1993). Although the exact receptor involved is highly controversial (Samuelsson a et al, 2001; Bazin R et al, 2006; crow.a.r et al, 2003; leontiev D et al, 2012; siragam.v. et al, 2006) and can vary as a result of the disease for which IVIG is used (Araujo L.M et al, 2011; Anthony r.m. et al, 2011), it is believed that the interaction between the Fc portion of IgG and both inhibitory and activated Fc γ rs found on monocytes and macrophages is important. Inhibitory Fc receptors Fc γ RIIb have been shown to be essential for the protective role of IVIG in the ITP mouse model (Samuelsson et al, 2001). The role of the activating Fc receptor Fc γ RIIIa has also been demonstrated in a variety of diseases (reviewed by Mekhaiel et al, 2011 b). Although recent work has demonstrated that FcRn is not involved in improving ITP in a mouse model (Crow et al, 2011), it has also been hypothesized that FcRn is responsible for maintaining the long half-life of Fc in plasma (Roopenian and akiesh, 2007). The Fc portion interacts not only with Fc receptors, but also with certain lectins. IVIG is known to bind to CD22 lectin on B lymphocytes via the terminal sialic acid of Fc-glycans (Siglec-2) (Seite J.F et al, 2010), and recent studies demonstrate that sialylated Fc is responsible for IVIG anti-inflammatory effects in mouse arthritis models due to its interaction with the human lectin receptor DC-SIGN on DC (Anthony r.m et al, 2011).
IVIG may also function by a multistep model in which injected IVIG first forms a class of immune complexes in patients (Clynes et al, 2005; Siragam et al, 2005,2006; Machino et al, 2012). After these immune complexes are formed, they interact with these receptors to mediate anti-inflammatory effects that contribute to a reduction in the severity of autoimmune diseases or inflammatory states (silagam et al, 2006). Multimers of Fc are formed in IVIG by anti-idiotype interactions (Machino et al, 2010; Machino et al, 2012; Roux and Tankersley, 1990; Teeling et al, 2001) or by covalent interactions of Fc (Yoo et al, 2003). It is hypothesized that IgG multimers show a higher avidity of binding to the above receptors and induce protective signals that cannot be induced by Fc monomers by the property of cross-linking the receptors. This is supported by Immune Complexes (IC) reversing ITP in mice (Bazin et al, 2006) and promoting the reduction of lupus by observing that IC enhances tolerogenicity of immature dendritic cells by Fc γ RIIb (Zhang et al, 2011). The proportion of multimeric IgG and/or sialylated IgG in commercially available IVIG is extremely low (< 1% and 5%, respectively), which can be the reason for the large number of administrations required (Nimmerjahn and ravatch, 2007).
Other proposed mechanisms of IVIG action are the repair of idiotypic-anti-idiotypic networks; inhibition or neutralization of cytokines by specific antibodies in IVIG; blocking the binding of adhesion molecules on leukocytes to the vascular endothelium; inhibiting complement uptake in the target tissue; neutralizing microbial toxins; blocking Fas ligand-mediated apoptosis by anti-Fas antibodies in IVIG; anti-Fas antibody induced apoptosis at high concentration IVIG; apoptosis of neutrophils by anti-Siglec-9 antibodies in IVIG; saturating the FcRn receptor to enhance the elimination of autoantibodies; induction of inhibitory Fc γ RIIb receptors on effector macrophages; growth factors that neutralize B cells, such as B cell activating factor; inhibiting a T cell proliferative response; expansion, activation, or both of a population of Treg cells; inhibiting differentiation and maturation of dendritic cells; enhancing differentiation and maturation of "primed" dendritic cells (reviewed in balloon, 2011; Mekhaiel et al, 2011 b).
Recombinant proteins have been proposed for use in the treatment of autoimmune diseases and/or as IVIG replacement compounds. US 2011/0081345(Moore) discloses single chain Fc (scfc) proteins having one Fc unit per molecule, comprising two linked Fc domain amino acid chains, which may be useful in the treatment of autoimmune diseases. US 2004/0062763 (simple University; Mosser) discloses the use of multivalent Abs or portions thereof to link Fc γ RI to upregulate IL-10 production for the treatment of autoimmune disorders. The agent may be two or more Fc fragments coupled together or provided in a single recombinant peptide. US 2008/0206246(The Rockofeller University; ravatch) discloses polypeptides comprising at least one sialylated IgG Fc region, and their use as IVIG replacement compounds. Although the compounds disclosed in these documents may target Fc receptors, compounds comprising Fc monomers or dimers may not bind Fc receptors with sufficient avidity to be effective or fully effective as IVIG replacement compounds. Therefore, they are not suitable biomimetics of the multimeric fraction of IVIG. Higher order multimers are not disclosed in these documents. Nor does it disclose any way of engineering higher order multimers that are active as IVIG replacement compounds.
US2010/0239633 (University of Maryland, Baltimore; Strome) discloses IVIG replacement compounds comprising multiple linked Fc moieties. It is envisaged that a star arrangement of C μ 4 domains and J chains of IgM is used to achieve polymerisation. However, no working examples are described and the computer simulations reported here indicate that the exemplary molecules will not polymerize efficiently and/or that the Fc portion will not be aligned for efficient binding to Fc or other receptors. Furthermore, biological complexes comprising more than one different polypeptide chain can be more difficult to manufacture uniformly, as not all polypeptide subunits can interact in a stable and predictable manner. For example, antibody heavy and light chain-expressing cell lines that produce active whole antibodies comprising the correct ratio of light and heavy chains can also produce therapeutically inactive heavy chain dimer or hemi-mer (halfmer) molecules that do not contain light chain linkers.
Linear structures are also proposed in US2010/0239633, in which individual amino acid chains are dimerised by pairing between identical Fc domain amino acid chains, thus creating an Fc region. For example, the hinge region may form an interchain disulfide bond between two individual amino acid chains. However, as illustrated in fig. 11 and 12 of US2010/0239633, where multiple Fc amino acid chains are connected in series, there may be a number of different ways in which the chains may pair. Thus, consistent products with reliable and predictable properties would not be expected. It is proposed to include a terminal heavy chain domain from IgE to prevent this "zipper" effect. However, binding of molecules to IgE receptors can have undesirable consequences, including the risk of anaphylactic shock or other allergic reactions.
US2010/0239633 also proposes making cluster molecules in which a multimerization region, such as an IgG2a hinge or isoleucine zipper, causes the amino acid chains to dimerize or multimerize, thus joining together pairs of Fc domain amino acid chains to form a functional Fc unit. In subsequent studies, Jain et al (2012) described the preparation of recombinant proteins in which human IgG2 hinge sequence or isoleucine zipper was fused to mouse IgG2 a. The authors report that either of the multimerization regions causes the formation of homodimers (i.e., a single functional Fc unit referred to herein as a monomer) and multimers. The multimeric fractions of these proteins have some efficacy in treating a mouse model of ITP or collagen-induced arthritis. However, most of the prepared proteins comprising the IgG2 hinge are monomeric or exist as dimers or trimers. Higher order oligomers with unknown quaternary structure account for only a small fraction. In proteins containing isoleucine zipper, the proportion of higher order multimers is lower. Higher order multimers do not behave uniformly in size and their structure, including the nature of the glycan attached to N297, is unknown. Given the low proportion of some or all of these multimers in the protein produced, isolating them for therapeutic use would make substantial waste inevitable and appear unlikely to be commercially viable.
For the treatment of autoimmune and inflammatory diseases, there is a need for structurally defined compounds that effectively target the appropriate mechanisms underlying the biological activity of IVIG.
The listing or discussion of a prior-published document in this specification should not be taken as an admission that the document is part of the state of the art or is common general knowledge.
Summary of The Invention
A first aspect of the invention provides a method for treating an autoimmune disease or an inflammatory disease in a mammalian subject, the method comprising:
administering to a mammalian subject an effective amount of a polymeric protein comprising 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising 2 immunoglobulin G heavy chain constant regions;
wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit;
wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject.
A second aspect of the invention provides a method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to a mammalian subject an effective amount of a polymeric protein consisting of 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker (linker) connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc; and is
Wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
A third aspect of the invention provides a method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to a mammalian subject an effective amount of a polymeric protein consisting of 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion and a tail segment region (tailpiece region);
wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc;
wherein each modified human immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of a modified human immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit; and is
Wherein the tail segment region is fused to each of the two modified human immunoglobulin G heavy chain constant regions of the polypeptide monomer unit and facilitates assembly of the monomer units into a polymer.
In a fourth aspect, the invention provides a multimeric protein comprising 5, 6 or 7 polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit;
wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject;
wherein each polypeptide monomer unit does not comprise a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions.
In a fifth aspect, the invention provides a multimeric protein consisting of 5, 6 or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc; and is
Wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
A sixth aspect of the invention provides a nucleic acid molecule comprising a coding portion encoding a monomeric unit of a polypeptide of a polyprotein as defined according to the fourth or fifth aspects of the invention.
Further aspects of the invention are an expression vector comprising a nucleic acid molecule of the sixth aspect of the invention, a host cell comprising an expression vector, and a therapeutic composition comprising a polyprotein of the fourth or fifth aspect of the invention.
Medical uses of the treatment methods corresponding to the first to third aspects of the invention are also envisaged.
The present application provides the following:
1) a method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein comprising 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit;
wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject.
2) The method of 1), wherein each polypeptide monomer unit comprises a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions; wherein the tail segment region of each polypeptide monomer unit facilitates assembly of the 5, 6, or 7 polypeptide monomer units into a polymer.
3) The method of claim 2), wherein the tail segment region is an IgM or IgA tail segment, or a fragment or variant thereof.
4) The method of 1), wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence having at least 90% sequence identity to a native human immunoglobulin G1 heavy chain constant region.
5) The method of claim 1), wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence comprising a cysteine residue at position 309, and preferably further comprising a leucine residue at position 310, according to the EU numbering system.
6) The method of claim 1), wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence that is modified compared to the amino acid sequence of a native immunoglobulin G heavy chain constant region to modify the affinity of the Fc receptor binding portion for at least one Fc receptor.
7) The method of claim 1), wherein each of the two immunoglobulin G heavy chain constant regions comprises an amino acid sequence that is modified compared to the amino acid sequence of a native immunoglobulin G heavy chain constant region to suitably increase the in vivo half-life of the polymeric protein by increasing the affinity of the Fc receptor binding portion for a neonatal Fc receptor.
8) The method of 1), wherein the autoimmune or inflammatory disease is treatable with intravenous immunoglobulin (IVIG).
9) The method of claim 1), wherein the autoimmune or inflammatory disease is autoimmune cytopenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, asthma, kawasaki disease, Guillain-Barre syndrome, Stephens-Johnson syndrome, Crohn's colitis, diabetes, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, anti-factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis or Alzheimer's disease.
10) A method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein consisting of 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc; and is
Wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
11) A method for treating an autoimmune or inflammatory disease in a mammalian subject, the method comprising:
administering to the mammalian subject an effective amount of a polymeric protein consisting of 5, 6, or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion and a tail segment region;
wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc;
wherein each modified human immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of a modified human immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit; and is
Wherein the tail segment region is fused to each of the two modified human immunoglobulin G heavy chain constant regions of each polypeptide monomer unit and facilitates assembly of the 5, 6, or 7 polypeptide monomer units into a polymer.
12) The method of 11), wherein the tail segment region is an IgM or IgA tail segment, or a fragment or variant thereof.
13) A polymeric protein comprising 5, 6 or 7 polypeptide monomer units;
wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions;
wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit;
wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject;
wherein each polypeptide monomer unit does not comprise a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions.
14) A polymeric protein consisting of 5, 6 or 7 polypeptide monomer units;
wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions and optionally a polypeptide linker connecting the two immunoglobulin G heavy chain constant regions as a single chain Fc; and is
Wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
15) A nucleic acid molecule comprising a coding portion encoding a polypeptide monomer unit of a polyprotein as defined in 13) or 14).
Brief Description of Drawings
FIGS. 1A and 1B: hexameric Fc and structural characterization. Figure 1a. hexamer Fc model (left panel) showing Cys309 and Cys360 (tail fragment) disulfide bonds (disulphide linkage) formed between one monomer and its adjacent monomer. Tapping mode atomic force microscopy images (right hand panel) revealed barrel-shaped, six-fold symmetric complexes consistent with hexamers. At smaller scan sizes (lower panels of the two right-hand panels), these complexes are shown to be about 20nm in diameter (scale bar 50nm in lower panels). Figure 1b hexameric Fc generated from mammalian cell lines was detected as a molecule of about 312kDa by size exclusion chromatography (highlighted trace hexamers). A small proportion of dimers was also detected.
FIG. 2: binding of hexameric Fc to human and mouse Fc γ receptors (Fc γ R). Titrated amounts (50-0.3nM) of Fc protein were coated onto ELISA wells. Binding by human or mouse glutathione-S-transferase (GST) -fused Fc γ R as shown was visualized using horseradish peroxidase (HRP) conjugated anti-GST antibody. Values represent triplicate determinations. The error bars are the Standard Error (SE) around the mean. As described in Mekhaiel et al, 2011a, antigen-fused Fc proteins bind little to Fc receptors.
FIG. 3: binding of hexameric Fc to human CD19+ human B lymphocytes. The characterized flow cytometry plots show different populations of human leukocytes represented by their forward and side scatter patterns (left). Single CD19+ B lymphocytes stained with anti-human CD19-FITC (boxed, middle panel) were gated and investigated for binding to hexameric Fc (right panel). Binding of 50. mu.g of hexa-Fc to CD19+ B cells is indicated by the furthest marked line on the right (hexa-Fc indicated by the arrow). Prior incubation of cells with FcRL 5-specific monoclonal antibody 509F6 (hexa-Fc + mAb 509F6 blocking FcRL5 as indicated by the arrow) shed hexameric Fc binding, indicating that FcRL5 is part of the reason hexameric Fc binds B cells.
FIG. 4: complement fixation assay for Fc proteins. Deposition of Fc fusions by C1q (left panel) and C5-9 (right panel) as determined by ELISA. Each dot represents the average optical density (+/-SD) for each mouse duplicate within a given group. Data from one of three replicate experiments is shown.
FIG. 5: hexameric Fc proteins protect against platelet loss in a mouse model of ITP. Balb/C mice were injected intraperitoneally (i.p.) with hexa-Fc, IVIG (Gamma Gard) or PBS. After one hour, ITP was induced in all mice. Platelets were calculated at the time points shown after treatment.
Detailed description of the preferred embodiments of the invention
According to a first aspect of the invention, there is provided a method for treating an autoimmune disease or an inflammatory disease in a mammalian subject. Treating a subject by administering a multimeric protein comprising 5, 6, or 7 polypeptide monomer units; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions.
The term "immunoglobulin G heavy chain constant region" means a native immunoglobulin G heavy chain region or a variant or fragment thereof. The Fc receptor binding portion typically comprises the Fc portion of immunoglobulin G, or a fragment or variant thereof. The term "Fc portion" includes fragments of IgG molecules obtained by limited proteolysis of the enzyme papain, which functions at the hinge region of IgG. The Fc portion obtained in this way comprises two identical disulfide-linked peptides comprising the heavy chain CH2 and CH3 domains of IgG, also referred to as
Typically, each immunoglobulin G heavy chain constant region comprises the amino acid sequence of a mammalian heavy chain constant region, preferably a human heavy chain constant region, or a variant thereof. A suitable human IgG subtype is
The Fc receptor binding portion may comprise more Fc portions than immunoglobulins. For example, it may include the hinge region of an immunoglobulin that occurs between the CH1 and CH2 domains in a native immunoglobulin. For certain immunoglobulins, the hinge region is necessary for binding to an Fc receptor. Preferably, the Fc receptor binding portion lacks the CH1 domain and the heavy chain variable region domain (VH). The Fc receptor binding portion may be truncated at the C-terminus and/or N-terminus compared to the Fc portion of the corresponding immunoglobulin. Thus, this Fc receptor binding portion is a "fragment" of the Fc portion.
Suitably, each immunoglobulin heavy chain constant region or variant thereof is an IgG heavy chain constant region comprising an amino acid sequence comprising a cysteine residue linked by a disulfide bond to a cysteine residue of an immunoglobulin G heavy chain constant region adjacent to a monomeric unit of a polypeptide, which is more similar to the corresponding part of IgM or IgA, as IgM and IgA are naturally polymeric and IgG is naturally monomeric, the ability to form a polymer based on the monomeric unit of an IgG heavy chain constant region may be increased by modifying part of the IgG heavy chain constant region to be more similar to the corresponding part of IgM or IgA. suitably, each immunoglobulin heavy chain constant region or variant thereof is an IgG heavy chain constant region comprising an amino acid sequence comprising a cysteine residue at position 309 according to the EU numbering system, and preferably also comprising a leucine residue at position 310. the sequence of Kabat EA et al, 1983Sequences of proteins of immunological interest. USDepartment of Health and Human Services, National institute of Health, Washington DC describes the numbering system for IgG (Sonsc et al, J28156) and the homologous mutation of IgG residues in
Suitably, each polypeptide monomer unit comprises a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions, wherein the tail segment region of each polypeptide monomer unit facilitates assembly of the monomer units into a polymer.
Where a region is described as being fused to the C-terminus of another region, the preceding region may be fused directly to the C-terminus of the following region, or it may be fused to an intermediate amino acid sequence which is itself fused to the C-terminus of the following region. N-terminal fusions can be similarly understood.
An intermediate amino acid sequence may be provided between the heavy chain constant region and the tail segment, or the tail segment may be fused directly to the C-terminus of the heavy chain constant region. For example, a short linker sequence may be provided between the tail segment region and the immunoglobulin heavy chain constant region. Typical linker sequences are between 1 and 20 amino acids in length, typically 2, 3, 4, 5, 6 or up to 8, 10, 12, or 16 amino acids in length. A suitable linker, included between the heavy chain region and the tail segment region, encodes Leu-Val-Leu-Gly.
A preferred tail region is that of human IgM which is PTLYNVSLVMSDTAGTCY (Rabbits TH et al, 1981.Nucleic Acids Res.9(18), 4509-. Suitably, this tail fragment may be modified at the N-terminus by substitution of the initial Thr to Pro, thus giving rise to sequence PPLYNVSLVMSDTAGTCY. This does not affect the ability of the tail segment to promote polymerization of the monomer. Further suitable variants of the human IgM tail fragment are described in Sorensen et al (1996) JImmunol 156: 2858-. An additional IgM tail fragment sequence is GKFTLYNVSLIMSDTGGTCY from rodents (Abbas and Lichtman, Cellular and molecular immunology, Elsevier Saunders,5 th edition, 2005). An alternative preferred tail fragment region is that of human IgA, which is PTHVNVSVVMAQVDGTCY (Putnam FW et al, 1979, J.biol.chem 254: 2865-2874). Other suitable tail fragments of IgM or IgA from other species, or even synthetic sequences that facilitate assembly of monomeric units into polymers, may be used. It is not necessary to use immunoglobulin tail fragments from the same species from which the constant region of an immunoglobulin heavy chain is derived, although it is preferred to do so.
"variants" and "fragments" are defined in relation to the heavy chain constant region. Variants of IgM tail fragments typically have an amino acid sequence identical to 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 of the 18 amino acid positions of PPLYNVSLVMSDTAGTCY. Variants of the IgA tail typically have an amino acid sequence that is identical to 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 of the 18 amino acid positions of PTHVNVSVVMAQVDGTCY. These fragments of the IgM or IgA tail fragments typically comprise 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17 amino acids. Fragments of the variants are also contemplated. Typically, fragments and variants of the IgM or IgA tail fragment retain the penultimate cysteine residue, as it is believed that it forms a disulfide bond between two monomeric units in a polyprotein.
The ability of a given tail segment region to facilitate assembly of monomeric units into polymers can be tested by comparing the proportion of proteins having a high molecular size when formed from monomeric units lacking the tail segment with the proportion of proteins having a high molecular size when formed from monomeric units comprising the tail segment. The latter may form higher proportions of high molecular size polymers under natural conditions. Native molecular weight can be determined by size exclusion chromatography, for example on a Sephadex-200 column of AKTA FPLC (Amersham). Alternatively, non-reducing gel electrophoresis may be used, as described in Smith et al (supra) or Sorensen et al (supra).
When the monomeric units have been assembled into a polymer, the Fc receptor binding moieties are arranged in a planar polymeric structure with their spatial orientation allowing each Fc receptor binding moiety to bind to an Fc receptor. IgM is naturally a pentamer or hexamer, and IgA naturally forms dimers, trimers or tetramers. These properties are determined at least in part by the ability of the tail segment to cause monomers to link into aggregates. Pentameric IgM is formed when IgM is linked to J chain, although it is typically hexameric in the absence of J chain. J-chain may or may not be included as an additional component of the polymeric fusion protein of the present invention. Preparation is simplified by omitting the J chain polypeptide, which is not required in any case for the polymerization of IgG (Ghumra et al, 2008). Secreted IgM or IgA found on mucosal surfaces contain Secretory Components (SC), and partially polymeric Ig receptors are used to alter their position from blood to secretions. SC may or may not be included as an additional component of the polymeric protein of the present invention. The preparation is simplified by omitting the SC, which is not required for polymerization in any case.
According to a first aspect of the invention a polyprotein comprising 5, 6 or 7 polypeptide monomer units is used. The exemplary Fc proteins described herein comprising the human IgG1 heavy chain constant region and the human IgM tail fragment form hexamers and dimers. Other IgG heavy chain constant region-based variants, including different or no tail fragments, can form aggregates with varying numbers of monomers. In particular, they may be pentamers or heptamers rather than hexamers. In the case where the Fc protein is naturally linked as a polymer having a different number of monomer units, the polymer having the desired number of monomer units can be separated according to molecular size, for example, by gel filtration. Mixtures in which at least a proportion of the proteins are in the pentameric, hexameric and/or heptameric form may also be used. In the absence of proteins having other numbers of monomeric units, pentamers, hexamers and/or heptamers are suitably used.
In the multimeric protein for use according to the first aspect of the invention, the Fc receptor binding portion of each monomeric unit is capable of binding to an Fc receptor. It will be understood that an Fc receptor binding portion comprising an Fc portion of a particular immunoglobulin will bind to a different Fc receptor depending on the binding specificity of the particular immunoglobulin. Typically, the Fc receptor binding moiety will have an affinity (affinity) for a given Fc receptor that is at least comparable to that of a native monomeric immunoglobulin molecule. However, lower affinity may be acceptable, as the polymeric protein comprises a plurality of such Fc receptor binding moieties, and will therefore bind Fc receptors with higher affinity. Thus, the Fc receptor binding portion will typically have an affinity that is at least one tenth, suitably at least one fifth and most suitably at least one half of the affinity of the corresponding native monomeric immunoglobulin molecule to which a given Fc receptor binds.
The affinity constant can be readily determined by surface plasmon resonance analysis (Biacore). The Fc receptor binding moiety can be passed to the Fc receptor via a flow cell from an amine-coupled CM5 sensor chip. An equimolar concentration of Fc receptor binding moiety or intact monomeric antibody can be injected at each Fc receptor and linkage and dissociation observed in real time. Data from BIAcore X or 3000 instruments can be analyzed using BIAevaluation 3.0 software to determine accurate affinity constants.
There are three classes of human Fc gamma receptors (Gessner et al (1998) Ann Hematol 76: 231-48; Raghavan and Bjorkman (1996) Ann Rev Cell Dev Biol 12: 181-220). Fc γ RI (CD64) binds monomeric IgG with high affinity. Fc γ RII (CD32) and Fc γ RIII (CD16) are low affinity receptors for Fc and can only interact with high affinity with antibodies presented to the immune system as Immune Complexes (IC). Although larger (>350kDa) poly-iggs and circulating ICs were almost totally removed in preparations of IVIG, these were common in healthy individuals where they could contribute as much as 10% of the total plasma Ab concentration, suggesting a physiological role in maintaining immune homeostasis in these healthy individuals (Nezlin R (2009) Immunol Lett 122,141-4).
Fc γ RII and Fc γ RIII are closely related to structural aspects of their ligand binding domains. Of the three individual human genes Fc γ RIIA, Fc γ RIIB and Fc γ RIIC, two give rise to alternatively spliced variants encoding Fc γ RII. Fc γ RIIa delivers an activation signal, while Fc γ RIIb delivers an inhibition signal. The functional basis for the different signals arises from signal transduction motifs located within the cytoplasmic tail of the receptor. The immunoreceptor tyrosine-based inhibitor motif (ITIM) located at the cytoplasmic tail of Fc γ RIIb is involved in negative receptor signaling. The ITIM motif is a unique feature of the Fc γ RIIb receptor, as it does not appear significantly in any other Fc γ receptor class. In contrast, the activating immunoreceptor tyrosine-based activation motif or ITAM is located at the cytoplasmic tail of Fc γ RIIa. The ITAM motif switches the activation signal. They can also be found in the FcRy chain, which is identical to the gamma chain of the high affinity IgE receptor (fceri). Although Fc γ RIIa and Fc γ RIIb are widely expressed in myeloid cells and some T cell subsets, they are significantly absent in NK cells. In humans there are two alleles of the Fc γ RIIa receptor, termed His131(H131) and Arg131 (R131). The Fc γ RIIa-Arg131 allele is associated with increased susceptibility to infection by podded bacteria such as Haemophilus influenzae (Haemophilus influenzae), Streptococcus pneumoniae (Streptococcus pneumoniae) and neisseria meningitidis (neisseria meningitidis), which elicits an IgG2 response (Pleass RJ and Woof JM, 2001). The Fc receptor encoded by this allele was unable to bind IgG2 and therefore was unable to trigger clearance of IgG 2-coated bacteria. However, both variants bound
Human Fc γ RIII also exists in multiple subtypes derived from two different genes (Fc γ RIIIA and Fc γ RIIIB). Fc γ RIIIb is unique in its attachment to the cell membrane via a glycosyl phosphatidyl anchor. Expression of Fc γ RIIIb is restricted to neutrophils, while Fc γ RIIIa is expressed by macrophages and NK cells. Fc γ RIIIa is also expressed by some subpopulations of T cells and certain monocytes. Fc γ RIIIa requires the presence of the FcR γ chain or CD3 zeta chain for cell surface expression and signaling. The FcR gamma chain or CD3 zeta chain is dimeric and possesses an ITAM motif. Fc γ RIIIa forms multimeric complexes with these subunits and transduces signaling through them. Thus, there is considerable Fc γ R receptor heterogeneity and multiple expression.
The binding sites for Fc γ RII and Fc γ RIII are located at the hinge and proximal regions of the CH2 domain of IgG, which was originally identified for Fc γ RI (Duncan et al (1988) Nature 332: 563-4; Morgan et al (1995) Immunol 86: 319-324; Lund et al (1991) J Immunol 147: 2657-2662).
The Fc γ receptor (Fc γ R) triggers an activating and/or inhibitory signaling pathway that sets a threshold for cell activation and ends up with a well-balanced immune response (Nimmerjahn F and ravatch JV (2008) nat. rev. immunol.8: 34-47). Activating and inhibitory fcrs are widely expressed in the hematopoietic system, but are expressed particularly on professional Antigen Presenting Cells (APC) (Nimmerjahn F and ravatch JV (2008) supra). In humans, for example, Fc γ RI is constitutively expressed by blood bone marrow Dendritic Cells (DCs) and Fc γ RII has been detected in every subset of DCs examined to date, whereas expression of Fc γ RI, Fc γ RIIB and Fc γ RIII is predominantly on mouse DCs (ravech JV (2003) in Fundamental Immunology (compiled by Paul WE) 685-700 (Lippincott-Raven, Philidelphia); Bajtay Z et al (2006) immunol. Lett.104: 46-52). Fc γ R also plays an important role in antigen presentation and immune complex-mediated maturation of Dendritic Cells (DCs), as well as in regulation of B cell activation and survival of plasma cells (ravech JV (2003) supra; Bajtay Z et al (2006) supra). Furthermore, by modulating DC activity, Fc γ R regulates whether an immunogenic or tolerogenic response is elicited after recognition of antigenic peptides presented on the surface of DCs into cytotoxic T cells, helper T cells and regulatory T cells. Fc γ R also cooperates with Toll-like receptors (TLRs) to control the levels of important regulatory cytokines IL-12 and IL-10 (Polumuri SK,2007, J.Immunol 179: 236-. Therefore, Fc γ rs are involved in modulating innate and adaptive immune responses, which makes them attractive targets for the development of novel immunotherapeutic approaches (Nimmerjahn F and ravatch JV (2008) supra).
Inhibitory Fc γ RIIB is known to control the strength of immune responses, as DCs derived from Fc γ RIIB knockout mice produce stronger and longer lasting immune responses in vitro and in vivo (Bergtold a, Desai DD et al (2005) Immunity23: 503-514; Kalergis a M and ravatch JV. (2002) j.exp.med.195: 1653-1659). More importantly, Fc γ IIB deficient DCs or DCs incubated with mAbs that block the binding of immune complexes to Fc γ RIIB showed spontaneous maturation (Boruchov AM, et al (2005) J. Clin. invest.115: 2914-. This suggests that inhibitory Fc γ R not only regulates the intensity of cell activation but also actively prevents spontaneous DC maturation under non-inflammatory steady state conditions. In fact, low levels of immune complexes can be seen in the sera of healthy donors, emphasizing the importance of regulatory mechanisms to prevent unwanted DC activation (DhodapkarKM, et al (2005) Proc. Natl Acad. Sci. USA 102: 2910-. Loss of Fc γ RIIB also resulted in the priming of more antigen-specific T cells (Kalergis A M and Ravetch JV. (2002) J.exp.Med.195: 1653-1659). Thus, binding of the polymeric Fc protein to multiple copies of fcyriib induces a negative response from cells expressing this receptor.
FcRL5 is a recently described Fc receptor capable of inhibiting signaling that is expressed on B cells and binds to aggregated IgG but not monomeric IgG (Wilson et al, 2012). These authors suggested that IgG could be synergistically bound to FcRL5 and Fc γ RIIb expressed on B cells (bind cooperativity). As described in example 3, exemplary hexameric proteins can bind FcRL5 independent of Fc γ RIIb.
Comprising human I in the Fc receptor binding portionIn the case of an immunoglobulin heavy chain constant region of the gG isotype or variant thereof, it will typically bind to human Fc γ receptors (Fc γ RI, Fc γ RII and Fc γ RIII) and/or
The Fc receptor binding moiety also typically binds to lectins, also known as glycan receptors, particularly lectins that bind to sialic acid. Exemplary lectins are human DC-SIGN (or mouse homolog SIGN-R1) and CD22, which contribute to the therapeutic properties of IVIG (reviewed in Mekhaiel,2011 b). As explained in example 3, the polymeric protein is predicted to interact with these receptors. SIGN-R1 binding can be determined by surface plasmon resonance, as described in Jain et al, 2012. Similar methods can be used to determine binding to CD22 or DC-SIGN.
As described above, appropriate limits and determinations of affinity constants for Fc receptor binding portions or fragments of Fc portions comprising variants of native immunoglobulin heavy chain constant regions are made.
"variant" refers to a protein in which an amino acid insertion, deletion or substitution, whether conservative or non-conservative, has been made at one or more positions.
A "variant" may have modified amino acids. Suitable modifications include acetylation, glycosylation, hydroxylation, methylation, nucleotidylation, phosphorylation, ADP-ribosylation, and other modifications known in the art. Such modifications may occur post-transcriptionally in the case of peptides prepared by recombinant techniques. Otherwise, the synthetic peptide may be modified using techniques known in the art.
Modifications may be included prior to incorporating the amino acid into the peptide. The carboxylic acid groups may be esterified or may be converted to amides and the amino groups may be alkylated, e.g. methylated. Variants may also be post-transcriptionally modified, for example by removal of carbohydrate side chains or single sugar moieties such as sialic acid groups or by addition of sialic acid groups.
"conservative substitutions" refer to groups such as Val, Ile, Leu, Ala, Met; asp and Glu; asn, Gln; ser, Thr, Gly, Ala; lys, Arg, His; and combinations of Phe, Tyr, Trp. Preferred conservative substitutions include Gly, Ala; val, Ile, Leu; asp and Glu; asn, Gln; ser, Thr; lys, Arg; and Phe, Tyr.
Typical variants of an immunoglobulin G heavy chain constant region will have an amino acid sequence that is at least 70%, typically at least 80%, at least 90%, at least 95%, at least 99%, or at least 99.5% identical to the corresponding immunoglobulin G heavy chain constant region of a native immunoglobulin. Suitably, the variant is a variant of the human immunoglobulin G1 heavy chain constant region and has an amino acid sequence at least 90%, at least 95%, at least 99% or at least 99.5% identical to the latter.
"fragment" refers to a protein in which there is a deletion at one or more positions. Typically a fragment of the Fc portion comprises at least 60%, more typically at least 70%, 80%, 90%, 95% or up to 99% of the complete sequence of the Fc portion. Fragments of the variants are also included.
The percent sequence identity between two polypeptides may be determined using a suitable computer program, such as the GAP program of the University of Wisconsin genetic computing group and will be understood to be calculated with respect to polypeptides for which the sequences have been optimally aligned.
Alternatively, alignment may be performed using the Clustal W program (Thompson et al, (1994) Nucleic acids sRs, 22(22), 4673-80). The parameters that can be used are as follows:
fast pairwise alignment parameters: k-tuple (K-tuple) (character) size, 1; window size, 5; interval penalty, 3; number of upper diagonals, 5; the scoring method comprises the following steps: x percent.
Multiple alignment parameters: gap opening penalty, 10; gap extension penalty, 0.05.
The scoring matrix: BLOSUM.
Variants may be natural or prepared using protein engineering methods and site-directed mutagenesis methods well known in the art.
A "peptide" typically comprises up to 10, 20, 50 or 100 amino acids. Peptides and polypeptides may conveniently be blocked at the N-or C-terminus so as to help reduce susceptibility to exoproteolytic digestion. Peptides and polypeptides can be produced by recombinant protein expression or in vitro translation systems (Sambrook et al, "Molecular cloning: A Laboratory Manual",2001, third edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). Peptides can be synthesized by solid phase peptide synthesis in the Fmoc polyamide model as disclosed by Lu et al (1981) J.org.chem.46,3433 and references therein.
Suitably, in the Fc protein for use according to the first aspect of the invention, each immunoglobulin G heavy chain constant region comprises an amino acid sequence that is modified compared to the amino acid sequence of a native immunoglobulin G heavy chain constant region to modify the affinity of the Fc receptor binding portion for at least one Fc receptor. The affinity of the Fc receptor binding portion for low affinity inhibitory and/or activating Fc γ receptors typically can be increased.
The interaction between IgG and Fc receptors has been analyzed in biochemical and structural studies using wild-type and mutant Fc. One consensus suggests that some regions for binding Fc receptors are located in the hinge region closest to the CH2 domain and at the amino terminus of the CH2 domain adjacent to the hinge, including for example residues 233 to 239 (Glu-Leu-Gly-Pro-Ser). Mutations in this region can result in altered binding to Fc receptors. This region appears to be responsible for direct interaction with Fc receptors (Woof JM and Burton D, Nature Reviews Immunology 2004,4: 89-99). Further into the CH2 domain and away from the hinge are other residues that may contribute to Fc receptor binding under at least certain circumstances, including, for example, Pro-329(EU numbering) which appears as a unique site for human IgG1 involved in direct contact with the Fc receptor and Asn-297 which appears as a unique site for N-linked glycosylation within the Fc region of
The activating Fc γ receptor is as described above; in humans, low affinity activating Fc γ receptors are Fc γ RIIA/C and Fc γ RIIIA. Fc γ RI is a high affinity activating receptor. Fc γ RIIB is an inhibitory human Fc receptor. Since the ligand binding properties of Fc γ RIIB and Fc γ RIIA/C are the same, it is not possible to increase the affinity to Fc γ RIIA/C while simultaneously decreasing the affinity to Fc γ RIIB. Lazar et al (2006) PNAS 103:4005-10 describe mutations in the Fc portion of human IgG that affect binding affinity to different Fc receptors. Wild type IgG binding to Fc γ RIIIa has a K of 252nMD(ii) a K of I332E mutantDK of 30nM and S239D/I332E mutantDWas 2 nM. The combination of the a330L mutation with S239D/I332E increased Fc γ RIIIa affinity and decreased Fc γ RIIb affinity. Shields RL et al (2001) J.biol.chem.276: 6591-Busy 6604 describe mutations in the Fc portion of human IgG1 that affect binding affinity to different Fc receptors. The S298A mutation increased affinity for Fc γ RIIIa and decreased affinity for Fc γ RIIA; the E333A mutation increased affinity for Fc γ RIIIa and decreased affinity for Fc γ RIIA; mutation K334A increased the affinity for Fc γ RIIIa. Any or all of the above mutations may be used alone or in combination. Other suitable mutations may be identified by conventional methods.
The affinity of the Fc receptor binding moiety for CD22 or DC-SIGN/SIGN-R1 can be increased by increasing the amount of sialic acid bound on the Fc receptor binding moiety. Typically, this is achieved by increasing the amount or proportion of terminal sialic acid residues on the N297 glycan, which can be added post-translationally to the Fc peptide in the endoplasmic reticulum.
Other mutations may suitably be made to increase the potency of the Fc receptor binding portion. Suitably, each immunoglobulin G heavy chain constant region comprises an amino acid sequence that is modified compared to the amino acid sequence of the native immunoglobulin G heavy chain constant region to suitably increase the in vivo half-life of the polyprotein by increasing the affinity of the Fc receptor binding portion for the neonatal Fc receptor. Increasing serum persistence allows for higher circulating levels, less frequent administration, and reduced doses. This may be achieved by enhancing the binding of the Fc region to neonatal fcr (fcrn). FcRn expressed on the surface of endothelial cells binds Fc in a pH-dependent manner and prevents its degradation. Although the hexameric Fc described in the examples was unable to bind human FcRn, it bound very well to mouse FcRn. Amino acid substitutions M252Y/S254T/T256E and/or H433K/N434F may be introduced into the Fc receptor binding portion to increase the in vivo half-life of IgG without unduly affecting the Fc γ R interaction (Vaccaro C, et al (2005) nat. Biotech.23: 1283-1288). Additionally or alternatively, H310 may be reintroduced.
Binding of multiple Fc receptors by polymeric Fc proteins can cause different intracellular signaling phenomena than binding of a single Fc receptor by a monomeric Fc protein or IgG. Binding of multiple Fc receptors by polymeric proteins may be more effective in blocking Fc receptor binding to other ligands, particularly in the case of low affinity Fc receptors. The effectiveness of polymeric proteins can be compared in a number of ways to the effectiveness of monomeric units that do not form polymers. In such tests, it is typical that the monomeric units of a polymeric Fc protein have the same affinity for a given Fc receptor individually, because the monomeric units do not form polymers.
The polymeric protein will have greater affinity for low affinity receptors, including Fc receptors, than for control monomeric units. They may also have greater affinity for FcRL, FcRn, CD22, DC-SIGN, or other Fc receptors. Avidity is the overall binding force of multivalent interactions. The interaction between the Fc binding region and the Fc receptor has a characteristic affinity, whereas for each interaction the affinity of the interaction is almost a fewWhich increases in steps. For low affinity Fc receptors, an increase in binding force may allow biologically relevant interactions with multimeric proteins, which may not be achieved by monomeric or dimeric units. Multivalent binding by polymeric proteins results in a considerable increase in stability as measured by the equilibrium constant (L/mol) compared to binding of control monomeric proteins. For example, a typical monovalent interaction between an Fc moiety and an Fc receptor may have about 104Equilibrium constant of L/mol. The hexavalent interaction may provide about 1011Equilibrium constant of L/mol. The equilibrium constant may vary based on the Fc portion and Fc receptor. However, hexameric proteins will typically exhibit an increase in binding energy of up to about 10 compared to control
As described above, the affinity of Fc receptors of polymeric proteins can be compared to the affinity of monomeric units that do not form polymers by surface plasmon resonance analysis (Biacore).
It has been found that the ability of a multimeric protein to bind to an Fc receptor can be reduced by fusing a bulky antigen (bulk antigen) to the multimeric protein (Mekhaiel et al, 2011 a). Suitably, the polymeric protein for use according to the first aspect of the invention does not comprise (e.g. by fusion or conjugation) a moiety that reduces binding affinity to Fc γ RII. The effect on affinity can be determined by comparing the binding affinity of a given receptor for the polymeric protein containing the moiety being tested to the affinity achieved by the polymeric protein lacking that moiety. Typically, if the affinity decreases more than 10-fold, or more than 5-fold or more than 2-fold, a polymeric protein comprising the moiety may be unsuitable. Reduced binding affinity to Fc γ RII will also indicate reduced binding affinity to other Fc receptors including Fc γ RI or Fc γ RIII. Alternatively, the binding affinity of either or both of these other receptors may be tested.
As described in example 3, the increased affinity of polymeric proteins for low affinity receptors may use the biological mechanisms underlying IVIG therapy, particularly those mediated by the low affinity receptors Fc γ RIIB, Fc γ RIIIA, CD22 and DC-SIGN. Increased affinity for the high affinity IgG receptor Fc γ RI was also observed for the exemplary hexameric protein. Fc γ RI is implicated in inflammatory autoimmune diseases (Hussein OA et al, Immunol Invest 2010,39:699-712) and Fc γ RI directed immunotoxins inhibit arthritis (Van Vuuren AJ et al, J.Immunol 2006176: 5833-8). Furthermore, microRNA-127 inhibits lung inflammation by targeting Fc γ RI (Xie T et al, J.Immunol 2012188,2437-44). Furthermore, it has been proposed in US 2004/0062763 (sample university; Mosser) that the attachment of Fc γ RI on macrophages induces the production of the anti-inflammatory cytokine IL-10. Thus, although Fc γ RI is not considered to be the basis for IVIG effects, the attachment of Fc γ RI may also have beneficial effects in the context of the present invention.
The immunomodulatory properties of the polymeric proteins result from interactions between the Fc receptor binding portion and the Fc receptor and/or other receptors and components of the immune system that interact with the Fc portion. The polymeric protein for use according to the first aspect of the invention does not comprise further immunomodulatory moieties, or antigenic moieties that cause antigen-specific immunosuppression when administered to a mammalian subject. Unlike therapeutic approaches that provide immunosuppressive agents such as TNF receptors, the polymeric proteins described herein rely on different therapeutic mechanisms, which may make them more broadly useful for therapy. Neither of the polymeric proteins is dependent on antigen-specific immunosuppression, which limits the applicability of other therapeutic approaches to specific diseases.
An "immunomodulatory moiety" is an agent that has immunomodulatory activity in a healthy or diseased mammalian subject when covalently linked to a polymeric protein described herein, or when present in the absence of the polymeric protein.
"immunomodulatory activity" refers to altering an immune response in a subject to increase or decrease an immune system component such as a cytokine or antibody; or increase or decrease immune functions such as antigen presentation. A "regulatory" moiety may be, for example, a chemokineA receptor for a daughter or chemokine, a receptor for a cytokine or cytokine, a Toll-like receptor (TLR), an acute phase protein (acute phased protein), a complement component, an immune receptor, a CD molecule, or a signaling molecule. Such agents are known to possess immunomodulatory activity in healthy or diseased mammalian subjects in the absence of polymeric proteins. Examples of such agents are described in immunological textbooks such as (Abbas and Lichtman, Cellular and Molecular Immunology, Elsevier Saunders,5thEdn,2005), and the skilled person can find additional examples in the relevant literature, therefore, the invention excludes conjugates or fusion proteins and polymeric proteins comprising any of these agents, in particular, immunomodulatory moieties found in monomeric Fc fusion proteins known as etanercept, alefacept, acacept (abatacept), beliracetam, asecept, briobacept, linacept, or aflibercept.
As mentioned above, the immunomodulatory properties of the polymeric proteins result from the interaction between the Fc receptor binding moiety and the Fc receptor and/or other receptors and components of the immune system that interact with the Fc moiety. Modifications of the Fc receptor binding moiety, such as modified glycosylation, are not considered additional immunomodulatory moieties, but rather are components of the Fc receptor binding moiety that modify binding to Fc receptors, lectins, or other immune system components. Therefore, they are not excluded from the present invention.
Suitable experimental tests for immunomodulatory moieties are to provide polymeric proteins lacking and comprising putative immunomodulatory moieties and to test the efficacy of either protein in the ITP mouse model described in example 4. If the putative immunomodulatory moiety possesses immunomodulatory activity, it may alter a parameter of the response. For example, it may alter the rate or extent of platelet recovery.
An "antigen" is a molecule that specifically binds to an antibody or TCR. Antigens that bind to antibodies include all classes of molecules and are referred to as B cell antigens. Exemplary types of molecules include peptides, polypeptides, glycoproteins, polysaccharides, gangliosides, lipids, phospholipids, DNA, RNA, fragments thereof, portions thereof, and combinations thereof. TCRs bind only peptide fragments of proteins complexed with MHC molecules; the peptide ligands and native proteins from which they are derived are referred to as T cell antigens. "epitope" refers to an antigenic determinant of a B cell or T cell antigen. Where the B cell epitope is a peptide or polypeptide, it typically comprises three or more amino acids, generally at least 5 and more usually at least 8 to 10 amino acids. The amino acids may be contiguous amino acid residues in the primary structure of the polypeptide, or may be spatially juxtaposed in the folded protein. T cell epitopes can bind to MHC class I or MHC class II molecules. Typically, MHC class I binding T cell epitopes are 8 to 10 amino acids long. Class II molecules bind peptides that can be 10 to 30 residues long or longer, with optimal lengths of 12 to 16 residues. Peptides that bind to a particular allelic form of an MHC molecule comprise amino acid residues that allow complementary interaction between the peptide and an allelic MHC molecule. The ability of putative T cell epitopes to bind to MHC molecules can be predicted and experimentally confirmed (Dimitrov I et al, Bioinformatics.2010Aug 15; 26(16): 2066-8).
The polymeric protein for use according to the first aspect of the invention does not comprise an antigenic moiety that causes antigen-specific immunosuppression when administered to a mammalian subject. By "comprising", we include an antigen covalently linked to at least one peptide monomer unit, such as by chemical conjugation or recombinant fusion. Typically, the polymeric protein does not comprise a moiety that acts as an antigen, whether by causing immunosuppression or immunostimulation, when administered to a mammalian subject.
By "antigen-specific immunosuppression", we include suppression of antibody responses and/or suppression of T cell responses. T cell responses can be inhibited by clonal deletion, anergy, or suppression of antigen-reactive T cells, such as by induction of regulatory T cells. T cell responses may also deviate from a more aggressive form to a less aggressive form, for example from a Th1 type response to a Th2 type response.
Suitable tests for antigenic moieties that cause antigen-specific immunosuppression are to provide polymeric proteins lacking the antigenic moiety and polymeric proteins comprising the antigenic moiety and test either protein in the mammalian subject to be treated for the autoimmune or inflammatory disease. The polymeric proteins lacking or comprising the antigen can be administered sequentially in the same mammalian subject, and the subject's immune response to the antigen is monitored after treatment with either protein. Alternatively, a group of similar subjects can be treated with a polyprotein lacking the antigen or a polyprotein comprising the antigen. Conventional techniques can be used to identify and monitor immune responses to antigens, whether B cell responses or T cell responses. An antigen does not cause antigen-specific immunosuppression when administered to a mammalian subject if there is no difference in the extent or type of immune response to the antigen following administration of the polyprotein lacking the antigen in the subject as compared to the polyprotein comprising the antigen. Thus, antigen-specific means of immunosuppression by the antigenic portion of the polymeric protein are excluded. This does not mean that the immune response to a particular antigen may not decrease in response to treatment. The immune response to an autoantigen involved in an autoimmune disease may be reduced in response to treatment, however this may be achieved by a polyprotein lacking the antigen and likewise by a polyprotein comprising the antigen. Similar tests can be used to identify whether a polymeric protein comprises a moiety that acts as an antigen when administered to a mammalian subject. In such tests, the dosages, formulations and characteristics of the polymeric protein administration are the same to allow meaningful comparisons.
Alternatively, the polymeric protein can be tested in a healthy mammalian subject to determine the presence or absence of a moiety that is an antigen. Such tests are particularly useful in identifying antigen-specific immune stimuli. Suitably, antigen-specific immune stimulation does not occur. Such tests may be useful in confirming the non-immunogenicity of the polymeric protein. Components of the polymeric protein that are not naturally found in the mammalian subject to which the polymeric protein is to be administered, such as immunogenic and selected non-immunogenic components of the linker peptide, can be tested. In such tests, the polymeric protein is administered and, after an appropriate period of time, e.g., two weeks, to allow an immune response to develop against the putative antigen, the blood sample is tested using ELISA to determine the level of antibodies to the putative antigen.
Suitable animal models can also be used to test for the presence of portions of the antigen that cause antigen-specific immunosuppression or that act as an antigen when administered to a mammalian subject. For example, the ITP mouse model described in example 4 can be used. The extent and type of immune response to the antigen is then tested after administration of the polyprotein lacking the antigen or putative antigen or the polyprotein comprising the antigen or putative antigen.
Suitably, although the polyprotein for use according to the first aspect of the invention is capable of binding to C1q, it does not activate the classical pathway of complement (Mekhaiel et al, 2011 a). Complement binding and activation can be assessed by ELISA on wells coated with polyprotein or control IgG or monomeric Fc as described in Lewis M et al, mol. Immunol 2008,45:818-827 and as performed in example 3. Wells of a 96-well plate were coated overnight with 100 μ Ι protein in carbonate buffer,
Suitably, the polymeric protein used according to the first aspect of the invention is capable of binding protein G or protein a with sufficient affinity to allow either of the two substrates to be used as a capture reagent for purification of the polymeric protein. Protein G binds in the interdomain region C γ 2-C γ 3 and is used in the examples to purify hexameric and monomeric Fc proteins. Other receptors that bind to the same domain may also bind to polymeric proteins such as TRIM21(Mallery DL et al, 2010, Proc. Natl. Acad. Sci. U.S.A.107(46): 19985-. TRIM21 is believed to be important in removing and immunizing against viruses.
Suitably, the polymeric protein for use according to the first aspect of the invention has a molecular weight of from about 230kD to 400 kD. For example, it may have a molecular weight of about 230kD, 240kD, 250kD, 260kD, 270kD, 280kD, 290kD, 300kD, 310kD, 320kD, 330kD, 340kD, 350kD, 360kD, 370kD, 380kD, 390kD or 400 kD. Polymers in this size range are typically easier to synthesize and assemble for mammalian cells than molecules with larger molecular weights, like IgM of about 750kDa, or complex proteins like antibodies that require expression and assembly of two different polypeptide chains (light and heavy). Proteins with multiple different polypeptide chains, including antibodies, are more difficult to produce homogeneousness in manufacture.
Suitably, the polymeric protein for use according to the first aspect of the invention has a diameter of about 20nm, such as from 15nm to 25nm or up to 30 nm. It has been demonstrated that 20nm particles are easier to deliver to the lymphatic system than 45nm and 100nm nanoparticles, which can be important for function and tissue penetration (Reddy et al, 2007). Smaller particles are also easier to manufacture by recombinant expression techniques.
As a result of molecular size and diameter, polymeric proteins typically have a good degree of tissue penetration, which can aid their therapeutic effect (Vollmers and Brandlein, 2006). Polymers will naturally show slower penetration over time compared to small molecules or monomers, however even intact IgM molecules (750kDa) reach implanted tumors in mice and primary tumors and metastases in patients after intravenous (i.v.) or intraperitoneal (i.p.) administration (Vollmers et al, 1998a, b, Oncol Rep 5: 549-552; Oncol Rep 5: 35-40). Pentameric IgM can reach and subcutaneously shrink transplanted tumors on the back of animals when injected intraperitoneally as demonstrated by standard immunohistochemical techniques (all Vollmers references, supra). This shows that molecules as large as 750kDa can leave the peritoneal cavity, enter the circulation and reach the implanted tumor. In their way, before the molecules reach their target, they have to cross several endothelial disorders of lymphatic and blood vessels, Jain et al, 2001J.Control.Release 74: 7-25. In summary, it is predicted that the multimeric proteins used according to the invention, including the exemplified hexameric Fc proteins, will show intermediate permeation times (hours) between larger IgM (days) and smaller IgG (minutes). For conditions like ITP that require biological activity, intermediate infiltration and accumulation may be advantageous over treatment using antibodies as carriers where rapid infiltration is preferred.
Suitably, rather than being in trans, the polymeric protein used according to the first aspect of the invention has a spatial orientation that is cis with respect to the cell surface (each Fc is in the same plane parallel to the cell surface and the long axis of each Fc is perpendicular to the cell surface). The cis orientation allows for the attachment of multiple receptors on the same cell, as the receptor binding moieties are all closely opposed to the same cell. Conversely, trans orientation in which Fc is in the plane perpendicular to the cell surface may result in cross-linking of both cells by the same protein. In cis orientation, the biological effects of receptor binding are concentrated on specific cells and are therefore more likely to be productive.
According to a first aspect of the invention, the polymeric protein is intended for use in the treatment of an autoimmune disease or an inflammatory disease. "autoimmune disease" includes any disease in which the immune system attacks the body's own tissues. "inflammatory disease" includes any disease characterized by destructive inflammation, which may be recurrent or chronic and does not involve normal tissue repair. Such diseases include in particular "autoinflammatory diseases" in which the innate immune system causes inflammation which may be of unknown origin. Autoinflammatory disorders are characterized by intense episodes of inflammation that lead to such symptoms as fever, rash, or joint swelling. These diseases also carry the risk of amyloidosis, a potentially lethal accumulation of blood proteins in vital organs.
Suitable autoimmune or inflammatory diseases for treatment include those treatable with intravenous immunoglobulin (IVIG). These may be diseases which are currently treated routinely with IVIG or for which IVIG has been found to be clinically useful, such as autoimmune cytopenia, Guillain-Barre syndrome, myasthenia gravis, anti-factor VIII autoimmune disease, dermatomyositis, vasculitis and uveitis (see, van der Meche FG et al, Lancet i,406 (1984); Sultan Y et al, Lancet ii,765 (1984); Dalakas MC et al, N.Engl. J.Med.329,1993 (1993); JayneDR et al, Lancet 337,1137 (1991); Lehoang P et al, Ocul.Immunol. Inflamm.8,49 (2000)). IVIG is typically used in the treatment of Idiopathic Thrombocytopenic Purpura (ITP), Kawasaki disease, Guillain-Barre syndrome, and chronic inflammatory demyelinating polyneuropathy (Orange et al, 2006, J Allergy Clin Immunol 117: S525-53). IVIG is also increasingly being used to treat a variety of other autoimmune diseases that do not respond to mainstream therapy, including arthritis, diabetes, myositis, crohn's colitis, and systemic lupus erythematosus.
Autoimmune or inflammatory diseases suitable for treatment include autoimmune cytopenia, idiopathic thrombocytopenic purpura, rheumatoid arthritis, systemic lupus erythematosus, asthma, kawasaki disease, Guillain-Barre syndrome, Stevens-Johnson syndrome, Crohn's colitis, diabetes, chronic inflammatory demyelinating polyneuropathy, myasthenia gravis, anti-factor VIII autoimmune disease, dermatomyositis, vasculitis, uveitis, or Alzheimer's disease.
The condition to be treated may include an inflammatory disease with an imbalance in the cytokine network, an autoimmune disorder mediated by pathogenic autoantibodies or auto-aggressive T cells, or an acute or chronic phase of a chronic relapsing autoimmune, inflammatory or infectious disease or process. In addition, other medical conditions having an inflammatory component are included such as amyotrophic lateral sclerosis, huntington's disease, alzheimer's disease, parkinson's disease, myocardial infarction, stroke, hepatitis b, hepatitis c, human immunodeficiency virus-related inflammation, adrenoleukodystrophy, and epileptic disorders, particularly those believed to be associated with post-viral encephalitis including Rasmussen syndrome, West syndrome, and Lennox-Gastaut syndrome.
The condition to be treated may be a hematological immune disease, such as idiopathic thrombocytopenic purpura, alloimmune/autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, parvovirus B19-associated red cell aplasia, acquired anti-factor VIII autoimmunity, acquired von Willebrand disease, multiple myeloma and monoclonal gammopathy of undefined significance, aplastic anemia, pure red blood cell aplasia, Diamond-Blckfan anemia, neonatal hemolytic disease, immune-mediated neutropenia, platelet infusion refractoriness, post neonatal transfusion purpura, hemolytic uremic syndrome, systemic vasculitis, thrombotic thrombocytopenic purpura, or Erwinia syndrome.
Alternatively, can be used for treating a neuroimmune disorder, such as Guillain-Barre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, Paraproteinemic IgM demyelinating Polyneuropathy (Paraproteinemic IgMdemyelinating Polyneuropathy), Lambert-Eton myasthenia syndrome, myasthenia gravis, multifocal motor neuropathy, lower motor neuron syndrome associated with anti-GM 1 antibody, demyelination, multiple sclerosis and optic neuritis, stiff person syndrome, paraneoplastic cerebellar degeneration with anti-Yo antibody, paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibody, epilepsy, encephalitis, myelitis, myelopathy, particularly myelopathy associated with human T-cell lymphotropic virus-1, autoimmune diabetic neuropathy, or Acute Idiopathic autonomic neuropathy (Acute iopathic neuropathy), or Alzheimer's disease.
Can be used for treating rheumatic diseases, such as Kawasaki disease, rheumatoid arthritis, Fisher's syndrome, ANCA positive vasculitis, idiopathic polymyositis, dermatomyositis, antiphospholipid syndrome, recurrent spontaneous abortion, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's disease, CREST syndrome or uveitis.
Can be used for treating skin immune diseases such as epidermal necrolysis, gangrene, granuloma, autoimmune blistering diseases of skin including pemphigus vulgaris, bullous pemphigoid, and deciduous pemphigus, vitiligo, streptococcal toxic shock syndrome, scleroderma, systemic sclerosis including diffuse and limited systemic sclerosis of skin, atopic dermatitis, or dermosterol-dependent atopic dermatitis.
Can be used for treating musculoskeletal immune diseases, such as inclusion body myositis, necrotizing fasciitis, inflammatory myopathy, myositis, anti-decorin (BJ antigen) myopathy, paraneoplastic necrotic myopathy, X-linked vacuolar myopathy, penicillamine-induced polymyositis, atherosclerosis, coronary artery disease, or cardiomyopathy.
Can be used for treating gastrointestinal immune diseases, such as pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac disease, dermatitis herpetiformis, cryptogenic cirrhosis, reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, and sclerosing cholangitis.
The disease may be, for example, inflammation following infection, asthma,
An exemplary disease for treatment is Idiopathic Thrombocytopenic Purpura (ITP).
The polymeric proteins used in a given species typically include the Fc portion of an IgG from that species, and may also include the tail fragment portion of an IgA or IgM from that species. The mammalian subject to be treated is typically a human, although other mammals may be treated and indeed birds, amphibians and reptiles may be treated.
Typically, the polymeric protein is provided as a suitably formulated therapeutic composition. Suitably, they are provided as injectables (injectables) as liquid solutions or suspensions; solid forms suitable for solution in liquid, or suspension prior to injection, may also be prepared. The formulation may also be emulsified. In addition, minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents may be included, if desired.
The carrier may preferably be a liquid formulation and is preferably a buffered isotonic aqueous solution. Suitably, the therapeutic composition has a physiological or near physiological pH. Suitably, it has physiological or near physiological permeability and salinity and/or is sterile and endotoxin free. It may comprise sodium chloride and/or sodium acetate. The pharmaceutically acceptable carrier may also include excipients such as diluents and the like and additives such as stabilizers, preservatives, solubilizers and the like. As used herein, the term "pharmaceutically acceptable" refers to approved by a regulatory agency of the united states or europe or other government or listed in the U.S. pharmacopeia or other generally recognized pharmacopeia for use in humans.
The pharmaceutical composition may be formulated for administration in any suitable manner, including, for example, topical (e.g., transdermal or ocular), oral, buccal, nasal, vaginal, rectal, or parenteral administration. The term parenteral as used herein includes subcutaneous, intradermal, intravascular (e.g., intravenous), intramuscular, spinal, intracranial, intrathecal, intraocular, periocular, intraorbital, intrasynovial and intraperitoneal injections, as well as any similar injection or infusion technique. Forms suitable for oral use include, for example, tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. The compositions provided herein can be formulated as a freeze-dried product. Typically, the compositions are formulated for intravenous administration.
Aqueous suspensions contain the active ingredient in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include suspending agents (e.g., sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia); and dispersing or wetting agents (e.g., naturally occurring phospholipids such as lecithin, condensation products of alkylene oxides with fatty acids such as polyoxyethylene stearate, condensation products of ethylene oxide with long chain aliphatic alcohols such as heptadecaethyleneoxycetanol, condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides such as polyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives, such as ethyl, or n-propyl, p-hydroxybenzoate; one or more colorants; one or more flavoring agents; and one or more sweetening agents, such as sucrose or saccharin.
The formulations may be for topical or topical application, such as for topical application to the skin, a wound, or a mucous membrane such as the eye. Formulations for topical administration typically comprise a topical vehicle in combination with an active agent, with or without additional optional components. Suitable topical vehicles and additional components are well known in the art, and the choice of vehicle will be apparent depending on the particular physical form and mode of delivery. The body surface vehicle comprises water; organic solvents such as alcohols (e.g., ethanol or isopropanol) or glycerol; glycols (e.g., butanediol, isoprene glycol, or propylene glycol); fatty alcohols (e.g., lanolin); mixtures of water and organic solvents and mixtures of organic solvents such as alcohols and glycerol; lipid-based materials such as fatty acids, acylglycerols (including oils, such as mineral oils, and fats of natural or synthetic origin); phosphoglycerides, sphingolipids and waxes; protein-based materials, such as collagen and gelatin; silicone-based materials (including non-volatile and volatile); and hydrocarbon-based materials such as microsponges and polymer matrices.
The pharmaceutical compositions may be formulated as an inhalation formulation, including a spray, mist or aerosol. For inhalation formulations, the compounds provided herein can be delivered by any inhalation method known to those skilled in the art. Such inhalation methods and devices include, but are not limited to, metered dose inhalers with propellants such as CFC or HFA or physiologically and environmentally acceptable propellants. Other suitable devices are pneumatic inhalers (breath operated inhalers), multi-dose dry powder inhalers and aerosol nebulizers. The aerosol formulations used in the methods typically comprise a propellant, a surfactant and a co-solvent and can be packaged in conventional aerosol containers sealed by a suitable metering valve.
The inhalation composition may comprise a liquid or powder composition suitable for nebulisation and intrabronchial use containing the active ingredient, or an aerosol composition for dispensing metered dose administration by an aerosol unit. Suitable liquid compositions comprise the active ingredient in an aqueous, pharmaceutically acceptable inhalation solvent, such as isotonic saline or bacteriostatic water. The solution is administered by means of a pump or squeeze-actuated nebulized spray dispenser, or by any other conventional means that causes or causes the necessary dosage amount of the liquid composition to be inhaled into the lungs of the patient. Suitable dosage forms for administration in which the carrier is liquid, e.g. nasal spray or nasal drops, include aqueous or oily solutions of the active ingredient.
Formulations or compositions suitable for nasal administration in which the carrier is a solid include coarse powders having a particle size, for example in the
The actual dosage amount of the composition to be administered to a mammalian subject can be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic intervention, patient's characteristics, and the route of administration. In any event, the physician responsible for administration will determine the concentration of the active ingredient in the composition and the appropriate dosage for the individual subject.
The pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, for example, the active compound may constitute between about 2% to about 75%, or between about 25% to about 60%, and any range derivable therein, by weight of the unit. In other non-limiting examples, the dose can further comprise from about 1 mg/kg/body weight, about 5 mg/kg/body weight, about 10 mg/kg/body weight, about 50 mg/kg/body weight, about 100 mg/kg/body weight, and any range derivable therein per administration. The effective amount of polyprotein is generally from about 1% to about 20% of the effective amount of IVIG. Depending on the condition being treated, an effective IVIG dose may generally range from about 100mg/Kg to about 2 g/Kg.
The suitability of the polymeric protein and therapeutic composition can be tested in animal models prior to administration to a human patient. For suitable animal models, it is important that the Fc receptor in the animal is capable of binding to the Fc receptor binding portion of the polyprotein. Human immunoglobulins are known to bind to mouse Fc receptors. For example, human IgM binds to mouse Fc μ receptors. Pleass RJ,2009Parasite Immunology 31: 529-reservoir 538 reports which Fc receptor can bind which antibody from which species. However, in case the polyprotein comprises Fc binding portions derived from human immunoglobulin heavy chain sequences, it is advantageous to use transgenic mice expressing human Fc receptors. Suitable transgenic mice express human Fc γ RI receptor (CD64) that binds to human IgG1 and IgG3 (Heijnen IA et al, J Clin invest.1996Jan 15; 97(2): 331-8). Transgenic mice expressing low affinity fcrs, such as Fc γ RIIA (CD32) (McKenzie SE 2002, Blood Rev 16:3-5) are also available.
A suitable mouse model is the ITP mouse model described in example 4. Other suitable models include collagen-induced arthritis in mice (Jain et al, 2012) or non-obese diabetic (NOD) mouse models (Inoue, Y. et al (2007) J.Immunol.179, 764-774).
According to the second and third aspects of the invention, there is provided a method for treating an autoimmune disease or an inflammatory disease in a mammalian subject.
According to a second aspect, the subject is treated by administering a polyprotein comprising 5, 6, or 7 polypeptide monomer units; wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions; and optionally, a polypeptide linker that links the two immunoglobulin G heavy chain constant regions to a single chain Fc; wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
According to a third aspect, the subject is treated by administering a polyprotein comprising 5, 6, or 7 polypeptide monomer units; wherein each polypeptide monomer unit consists of an Fc receptor binding portion and a tail segment region; wherein the Fc receptor binding portion consists of two immunoglobulin G heavy chain constant regions; and optionally a polypeptide linker linking the two immunoglobulin G heavy chain constant regions to a single chain Fc; wherein each modified human immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of a modified human immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit; and wherein the tail segment region is fused to each of the two modified human immunoglobulin G heavy chain constant regions of the polypeptide monomer unit and facilitates assembly of the monomer units into a polymer.
For either of the second or third aspects, the term "immunoglobulin G heavy chain constant region" is as described in relation to the first aspect of the invention. Immunoglobulin G heavy chain constant regions are typically associated as monomeric units by disulfide bonds, such as occur in natural antibodies. Alternatively, the two constant regions may be produced as single chains of amino acids with intermediate linker regions, i.e. as single chains fc (scfc).
The polyprotein used according to the second aspect of the invention is formed by virtue of each immunoglobulin G heavy chain constant region comprising a cysteine residue that is disulfide-bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomer unit. The tail fragment is not included. In contrast, the polyprotein used according to the third aspect of the invention includes tail fragments that facilitate assembly of monomeric units into polymers. The tail fragments are as described in relation to the first aspect of the invention. A short linker sequence may be provided between the tail segment region and the immunoglobulin heavy chain constant region.
With respect to the second and third aspects, when the monomeric units have been assembled into a polymer, the Fc receptor binding moieties are arranged in a multimeric structure that is spatially oriented to allow each Fc receptor binding moiety to bind to an Fc receptor. The Fc receptor binding portion of each monomeric unit is as described in relation to the first aspect of the invention. Where the Fc receptor binding portion comprises an immunoglobulin heavy chain constant region of a human IgG isotype or variant thereof, it will typically bind to human Fc γ receptors (Fc γ RI, Fc γ RII and Fc γ RIII) and/or human FcRL5 and/or CD22 and/or DC-SIGN. Polymeric proteins will have greater affinity for Fc receptors than control monomeric units (which do not polymerize because they lack the necessary cysteine residues).
The immunomodulatory properties of the polymeric proteins used according to the second or third aspect of the invention result from the interaction between the Fc receptor binding moiety and the Fc receptor and/or other receptors and components of the immune system that interact with the Fc moiety. In particular, no additional immunomodulatory moieties are required; and does not require an antigenic moiety that causes antigen-specific immunosuppression when administered to a mammalian subject. Typically, there is no moiety that is an antigen when administered to a mammalian subject.
Further features of the polymeric protein for use according to the second or third aspect of the invention are described in relation to the first aspect of the invention. Typically, although the polyprotein binds to C1q, it actually activates the classical pathway of complement; typically, it can bind protein G or protein a; typically it has a molecular weight of from about 230 to 400 kDa; typically, it has a diameter of about 20 nm; typically, it has good tissue penetration; and typically it has a spatial orientation that is cis with respect to the cell surface.
As described in relation to the first aspect of the invention, the polyprotein used according to the second or third aspect of the invention is expected to treat autoimmune or inflammatory diseases.
In a fourth aspect, the invention provides a multimeric protein comprising 5, 6 or 7 polypeptide monomer units; wherein each polypeptide monomer unit comprises an Fc receptor binding portion comprising two immunoglobulin G heavy chain constant regions; wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit; wherein the polymeric protein does not comprise an additional immunomodulatory portion, or an antigenic portion that causes antigen-specific immunosuppression when administered to a mammalian subject; wherein each polypeptide monomer unit does not comprise a tail segment region fused to each of the two immunoglobulin G heavy chain constant regions.
The polyprotein of the fourth aspect is as described for the polyprotein used according to the first aspect, except that it is expressly excluded that each polypeptide monomeric unit comprises a tail segment region fused to each of two immunoglobulin G heavy chain constant regions. Typically, the monomer unit does not comprise a polypeptide region. The polyprotein does not contain the tail segment region.
In a fifth aspect, the invention provides a multimeric protein consisting of 5, 6 or 7 polypeptide monomer units; wherein each polypeptide monomer unit consists of an Fc receptor binding portion consisting of two immunoglobulin G heavy chain constant regions; and optionally, a polypeptide linker that links the two immunoglobulin G heavy chain constant regions to a single chain Fc; and wherein each immunoglobulin G heavy chain constant region comprises a cysteine residue that is disulfide bonded to a cysteine residue of an immunoglobulin G heavy chain constant region of an adjacent polypeptide monomeric unit.
The polymeric protein of the fifth aspect is as described for the polymeric protein for use according to the third aspect of the invention.
A sixth aspect of the invention provides a nucleic acid molecule comprising a coding portion encoding a polypeptide monomer unit of a polyprotein as defined according to the fourth or fifth aspects of the invention. Nucleic acid molecules encoding polypeptide monomer units of multimeric proteins are described in relation to the first, second and third aspects of the invention.
Conventional recombinant DNA methodology can be used to produce multimeric proteins as described, for example, in (Sambrook et al, "Molecular cloning: A laboratory Manual",2001, third edition, Cold Spring Harbor laboratory Press, Cold Spring Harbor, N.Y.). The monomeric unit structure is preferably produced at the DNA level, and the resulting DNA is integrated into an expression vector and expressed to produce monomeric units that assemble to form the polymeric protein.
The nucleic acid molecule of the sixth aspect of the invention comprises a coding portion comprising a coding sequence for an immunoglobulin G heavy chain constant region or single chain fc (scfc) in a 5 'to 3' direction. In suitable embodiments, the latter is fused in frame with the coding sequence of the tail segment region. The DNA encoding the coding sequence may be in its genomic configuration or in its cDNA configuration. It will be appreciated that additional coding sequences may be provided between the heavy chain and tailpiece coding sequences to allow these components to be separated from each other in the expressed protein by the linker sequence. Nucleic acids encoding linker sequences may be included, for example to allow incorporation of useful restriction sites and/or to allow in-frame transcription of heavy and tail segment coding regions. Suitable coding regions can be amplified by PCR and manipulated using standard techniques (Sambrook et al, supra). Mutations compared to the native nucleic acid sequence can be made by overlap PCR (SOEing PCR) or site-directed mutagenesis.
Suitably, the coding portion of the nucleic acid molecule encodes a signal peptide which is adjacent to a monomeric unit of the polypeptide. This facilitates isolation of the expressed monomeric unit from the host cell. Thus, a nucleic acid molecule will comprise a coding portion comprising a signal sequence fused in frame to the coding sequence of a monomeric unit in the 5 'to 3' direction. The portion of DNA encoding the signal sequence preferably encodes a peptide segment that directs secretion of the monomeric unit and is thereafter cleaved from the remainder of the monomeric unit. The signal sequence is a polynucleotide encoding an amino acid sequence that initiates transport of the protein across the membrane of the endoplasmic reticulum. Useful signal sequences include antibody light chain signal sequences, such as antibody 14.18(Gillies et al (1989) J. OF IMMUNOL. METH.,125:191), antibody heavy chain signal sequences, such as MOPC141 antibody heavy chain signal sequence (Sakano et al (1980) NATURE 286:5774), and any other signal sequence known in the art (see, e.g., Watson (1984) NUCLEIC ACIDS RESEARCH 12: 5145).
Further aspects of the invention are an expression vector comprising a nucleic acid molecule of the sixth aspect of the invention; a host cell comprising the expression vector; and a therapeutic composition comprising a multimeric protein of the fourth or fifth aspect of the invention. The therapeutic composition is as described for the polymeric protein for use according to the first aspect of the invention.
The nucleic acid molecule as described herein may generally be part of an expression vector. As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence capable of being incorporated into and recombined with and integrated into a host cell genome, or autonomously replicated as an episome. Such vectors include linear nucleic acids, plasmids, phages, cosmids, RNA vectors, viral vectors, and the like. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses. As used herein, the term "gene expression" or "expression" of a monomeric unit is understood to mean the transcription of a DNA sequence, the translation of an mRNA transcript, and optionally also the secretion of the monomeric unit.
Typically, a host cell for expression of the expression vector is provided. The cell may be a mammalian, avian, insect, reptile, bacterial, plant or fungal cell. Examples of mammalian cells include, but are not limited to, human, rabbit, chicken, rodent (e.g., mouse, rat) cells. Typical mammalian cells include myeloma cells, Sp2/0 cells, CHO cells, L cells, COS cells, fibroblasts, MDCK cells, HT29 cells, HEK cells, or T84 cells. A preferred host cell is CHO-K1. The expression vector can be introduced into the host cell using standard techniques, including calcium phosphate transfection, nuclear microinjection, DEAE-dextran transfection, bacterial protoplast fusion, and electroporation.
A polymeric protein can be prepared by a method comprising: (1) preparing a vector comprising a nucleic acid molecule encoding a monomeric unit; (2) transfecting a host cell with the vector; (3) culturing the host cell to provide expression; and (4) recovering the polymeric protein.
The polymeric proteins can be recovered as products of different molecular sizes. Typically, the desired polymeric protein comprising 5, 6 or 7 monomeric units represents at least 40% by weight of the total protein including monomeric units. Suitably, it constitutes at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by weight of the total protein including monomeric units. A high proportion of monomeric units that can be recovered as the desired polymeric protein contributes to production efficiency.
If the polyprotein is secreted by the host cell, it can be conveniently recovered by affinity chromatography using its affinity for Fc binding agents, such as protein a or protein G, suitably a protein G HiTrap (GE healthcare) column. Proteins can be eluted from such columns into neutral buffer by low pH. Dialysis can then be performed with buffer exchange. If the host cell does not secrete the polyprotein, it can be recovered by affinity chromatography after lysis of the cells.
The present invention will be further described in detail in the following examples, but is not limited thereto.
Example 1: production of recombinant multimeric Fc proteins
A DNA construct was prepared as follows. A commercially available pFUSE-hIgG1-Fc2 expression vector was obtained from InvivoGen, from Autogen Bioclear, Wiltshire, UK. The expression vector contained the coding sequence for the signal sequence from IL2 and downstream thereof the coding sequence for the Fc portion from
To insert the IgM tail sequence into a commercially available vector, primers were designed that when annealed together would form a double stranded sequence with overhanging bases encoding the Nhe1 restriction site to allow for C-terminal subcloning into Fc. To maintain the reading frame of the protein encoded by the plasmid, the existing stop codon is removed, and to allow for convenient restriction sites, additional DNA bases are inserted. Preceding the IgM tail fragment is a short 5' linker encoding the four amino acids Leu-Val-Leu-Gly; the linker does not affect the function of the IgM tail fragments.
The pFUSE vector was then digested with the restriction enzyme Nhe1 and the above IgM tail insert was ligated to generate an intermediate plasmid. To allow translation of the IgM tail fragment after the Fc region, the stop codon present was mutated in a subsequent step by site-directed mutagenesis using the Quick Change II kit (Stratagene, LaJolla, Calif., USA). Primers 3 and 4(SEQ ID No.3 and 4) were designed to remove this stop codon and create an AvrII restriction enzyme site between the Fc region and the IgM tail segment. This resulted in a plasmid called pFUSE-hIgG 1-Fc-TP.
In human IgM, the cysteine at position 309 participates in the formation of a disulfide bridge between two monomers of IgM in a pentamer. In order to better mimic the protein sequence of human IgM,
A control plasmid encoding a monomer of human IgG1 Fc lacking the 309/310 mutation and the tail fragment was also prepared.
The nucleic acid encoding sequence of the monomeric unit assembled into a polymer is SEQ ID No. 7.
The coding sequence has the following regions:
coding sequence of 1-60 IL2 signal peptide
Coding sequence of Fc region of 61-753 human IgG1
325-330 cys-309 and leu-310 mutations
754-810 IgM tail fragment coding sequence (including linker region and stop codon)
The amino acid sequence of the monomer unit assembled into the polymer is SEQ ID No. 8.
The amino acid sequence has the following regions:
1-20 IL2 Signal peptide
21-247 Fc region of human IgG1
109-110 cys-309 and leu-310 mutations
248-2514 amino acid linker
252-substituted 269 IgM tail fragment
During expression, the IL2 signal peptide is cleaved and thus the final protein product has 249 amino acids.
Polymeric and control monomeric Fc proteins were prepared as follows. CHO-K1 cells (European Collection of Cell Cultures) were transfected with the selected plasmids and positive clones by electroporation. At 37 deg.C/5% CO2Next, cells were grown in FCS supplemented with 10% ultra-low (ultra-low) bovine IgG, 100IU/ml penicillin, and 100. mu.g ml-1DMEM complete medium of streptomycin (PAA). In a container containing 400 μ g ml-1Stable transfectants were selected from the medium of Zeocin (Invivogen). Clones secreting Fc fusion proteins were detected by a sandwich enzyme-linked immunosorbent assay (ELISA) using goat anti-human IgG-Fc (Sigma-Aldrich: A0170). Fc fusion proteins were purified on protein G-Sepharose (GE Healthcare, Little Chalfont, Bucks, UK) from large scale cultures in DMEM supplemented with fbs (gibco) containing ultra-low IgG. Eluted fractions from affinity purification were pooled and AKTA was usedFPLC(GE Healthcare) was separated by size exclusion chromatography on a high performance Superdex-20010/300 GL column. The eluted fractions are coagulated with a known high MWGel filtration standards (Biorad) were compared. The integrity of the proteins was verified by SDS-PAGE on native 6% Tris-glycine gels for prestained molecular weight markers (Novex-Invitrogen) against
The polyprotein is expressed as a complex of about 312kD and about 100kD, consistent with expression as a hexameric and dimeric entity. As illustrated in FIG. 1B, the ratio is about 90% hexamer to 10% dimer (w/w) according to size exclusion chromatography.