Fusion proteins comprising IgG2 hinge domains

专利摘要:
The present invention relates to biologically active fusion proteins containing the IgG2 hinge as a multimerization domain capable of multimerizing proteins, peptides and small molecules which are active or more active in multimeric form; compositions comprising such fusion proteins; and methods of making and using such fusion proteins.
公开号:AU2013211824A1
申请号:U2013211824
申请日:2013-01-28
公开日:2014-08-28
发明作者:David Block；Henrik Olsen；Scott Strome
申请人:University of Maryland at Baltimore；Gliknik Inc；
IPC主号:C07K19-00

专利说明:
WO 2013/112986 PCT/US2013/023404 1 FUSION PROTEINS COMPRISING IgG2 HINGE DOMAINS CROSS REFERENCE TO RELATED APPLICATIONS 100011 This application claims priority to U.S. Provisional Application No. 61/591,615, filed January 27, 2012, the contents of which are herein incorporated by reference in their entirety, EIELD OF TilE INVENTION [00021 This invention relates generally to the fields of immunology, autoimmunity, inflammation, and tumor immunology. More specifically, the present invention relates to biologically active fusion proteins containing the IgG2 hinge as a multimerization domain capable of multimerizing proteins, peptides and small molecules which are active or more active in multimeric fbrn; compositions comprising such fusion proteins; and methods of making and using such fusion proteins. SUMMARY OF TH E ]N' ENTION [00031 There is a need for an alternative to currently existing methods for multimerizing therapeutic proteins and small molecules for use in immunotherapy that solves the problem of achieving the desired level of multimerization of the proteins, peptides, small molecules and carbohydrates/sugars while not causing an adverse reaction in the patient to a multimerization domain that is foreign to the patient's immune system. The present invention relates to biologically active fusion proteins comprising one or more naturally occurring multimerization domains and one or more peptides, proteins, small molecules, nucleic acids, fatty acids, or carbohydrates/sugars, compositions comprising the same and methods of using the same. These fusion proteins have broad application for treating a wide range of immunological, endocrinologic, inflammatory, infectious, and cancer disorders including, but not limited to autoimmune disease. Additionally, certain of these fusion proteins also have WO 2013/112986 PCT/US2013/023404 2 utility as laboratory reagents, such as for use in assays where biotin-streptavidin is currently used to make multimers, as an imaging agent, or as a clinical diagnostic agent. [00041 In one embodiment, the present invention relates to a fusion protein comprising one or more IgG2 hinge domains of SEQ ID NO:I and one or more peptides, proteins, small molecules, nucleic acids, fatty acids, or carbohydrates/sugars. In a further embodiment, the one or more IlgG2 hinge domains multimerizes the one or more peptides, proteins, small molecules, nucleic acids, fatty acids, or carbohydrates/sugars into dimers or higher order multimers. In still a further embodiment, the one or more peptides, proteins or small molecules is any peptide, protein, small molecule, nucleic acid, fatty acid, or carbohydrate whose activity is improved by multimerization. In still a further embodiment, the observed improvement in activity is through increased affinity or avidity of binding of the multimerized compound to a protein. In one embodiment, the IgG2 domain of SEQ ID NO: I is fused to the C terminus of the one or more peptides or proteins. In another embodiment, the IgG2 domain of SEQ ID NO: I is fused to the N terminus of the one or more peptides or proteins. In another embodiment the lgG2 domain is fused to small molecules, nucleic acids, fatty acids, or carbohydrates/sugars. In yet another embodiment the the IgG2 domain is fused to small molecules, nucleic acids, fatty acids, or carbohydrates/sugars through a linker protein such as the Fc domain. 100051 In a specific embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one or more proteins selected from cytokines, chemokines, hormones, cell surface receptors, cell surface receptor ligands, or monoclonal antibodies. In another embodiment the IgG2 hinge is fused to the extracellular domain of one or more proteins selected from cytokines, chemokines, hormones, cell surface receptors, or cell surface receptor ligands. In a particular embodiment, the IgG2 hinge fused to the extracellular domain of a cell surface receptor forms a soluble receptor. In one embodiment the soluble receptor is from the family of TNF receptors binding members of the TNF superfamily. In a particular embodiment, the one or more protein is one or more of P[-1, PI)-l L, CTLA4, WO 2013/112986 PCT/US2013/023404 3 IL12, IL12RA, or major histocompatibility complex. In a particular embodiment, the IgG2 hinge fused to a hormone presents multimerized hormone to a hormone receptor. In one embodiment the multimerized hormone is insulin, human growth hormone, Glucagon-Like Peptide- 1, leptin, orexin, ghrelin, or sex hormones. In another embodiment, the fusion protein comprises one or more lgG2 hinge domains of SEQ ID NO: I and one or more of the external domain of PD-1, CTLA4, the p40 subunit of IL12, or human parathyroid hormone. In another embodiment the IgG2 hinge is fused to synthetic peptides that bind to the extracellular domain of one or more proteins selected from cytokines, chemokines, hormones, cell surface receptors, cell surface receptor ligands. [00061 In one embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one or more PD-1 proteins. In one embodiment, the PD-I protein comprises in extracellular domain of PD-I. In one embodiment the IgG 2 hinge is fused to the C terminus of the PD-1 peptide. In another embodiment, the IgG2 hinge is fused to the N terminus of the PD-I peptide. In still a further embodiment, the fusion protein also comprises an Fe domain in addition to the IgG2 hinge and PD-1 peptide. In still another embodiment, the fusion protein comprises a linker between the PD-i peptide and the IgG2 hinge. In a further embodiment, the fusion protein is SEQ ID NO: 5, SEQ ID NO: 6, SEQ I) NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10. 100071 In another embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one or more peptides capable of binding SIRPa. In one embodiment, the peptide that binds SIRPa is a CD47 molecule. In yet another embodiment, the peptide that binds SIPRa is a CERVIG synthetic peptide. In a further embodiment, the CERVIG peptide that binds SIRPa comprises SEQ ID NO: 11. In one embodiment the IgG2 hinge is fused to the C terminus of the CERVIG peptide. In another embodiment, the IgG2 hinge is fused to the N terminus of the CERVIG peptide. In still a further embodiment, the fusion protein also comprises an Fe domain in addition to the IgG2 hinge and CERVIG peptide. In still another embodiment, the fusion protein comprises a linker between the WO 2013/112986 PCT/US2013/023404 4 CERVIG peptide and the IgG2 hinge. In a further embodiment, the fusion protein comprises SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17. [00081 In yet another embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one or more major histocompatibility complex proteins. In one embodiment, the one or more major histocompatibility complex proteins is a class I major histocompatibility complex. In another embodiment, the one or more major histocompatibility complex is a class II major histocompatibility complex. In still a further embodiment, the fusion protein comprising one or more major histocompatibility complex proteins further comprises a label. In one embodiment the label is a dye. In another embodiment, the label is a fluorophore. In one embodiment, the fusion protein comprising one or more IgG2 hinge domains and one or more major histocomp atibility complex proteins is useful in tetramer assays, for example in detecting the presence of antigen specific T cells by flow cytometry. 100091 In one embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one ore more cell surface receptors. In one embodiment, the cell surface receptor is a G-protein coupled receptor. In a particular embodiment, the G; protein coupled receptor is a chemokine receptor. In further embodiment, the chemokine receptor is CCR5, CXCRI, or CXCR2. In another embodiment, the cell surface receptor is a T cell receptor. In still another embodiment, the cell surface receptor is a B cell receptor. In still another embodiment, the cell surface receptor is a TNF super family receptor such as CD 137, BAFF R, BCMA, CD27, CD30, CD40, DcR3, DcTRAIL, DR3, DR6, EDAR, Fas, GITR, FIVEM, lyphotoxin beta R, NGF R, osteoprotegerin, OX40, RANK, RELT, TACI, TRAIL R, TROY, TWEAK R. 100101 In another embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: I and one or more cell surface receptor ligands. In one embodiment, the cell surface receptor ligand is a TNF superfamily receptor. In a further embodiment the TNF superfamily receptor ligand is TNFa or BLyS. In a further embodiment, the cell surface WO 2013/112986 PCT/US2013/023404 5 receptor ligand is a ligand to a cell surface glycoprotein. In a further embodiment, the cell surface glycoprotein is a CD4, CD123, CD303, or a CD304 ligand. [00111 In still a further embodiment, the fusion protein comprises one or more IgG2 hinge domains of SEQ ID NO: 1 and one or more of a chemotherapeutic agent, a cytotoxic molecule, a dye and/or a fluorophore. In yet a further embodiment, the fusion protein comprises one or more lgG2 hinge domains of SEQ I) NO: I and one or more of monosaccharides, disaccharides. oligosaccharides, polysaccharides, neoglycoproteis, glycoters, glycoolymers, monodsperse nanosructures trmed glycodendrimers, sugar alcohols, and sugar-rods. [00121 In another embodiment, the present invention relates to a fusion protein comprising one or more IgG2 hinge domains of SEQ ID NTO: 1, one or more peptides, proteins, small molecules, nucleic acids, fatty acids, or carbohydrates/sugars and one or more immunoglobulin Fe domain monomers. In a further embodiment, the fusion protein contains one or more immunoglobulin Fe domains that is selected for low affinity binding to Fe gamma receptors. In a particular embodiment, the immunoglobulin Fe domain that is selected for low affinity binding to Fe gamma receptors is an IgG1, IgG2, IgG3 or IgG4 Fe domain. In a particular embodiment, the immunoglobulin Fe domain is mutated to bind poorly to Fe gamma receptors. In still a further embodiment, the Fe domain is imitated at positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414, to bind poorly to Fe gamma receptors. In yet a further embodiment, the immunoglobulin Fe domain is modified, such as by one or more glycosylation changes relative to native human irmmunoglobulin Fe, to bind poorly to Fe gamma receptors. In a particular embodiment, the immunoglobulin Fe domain is modified by hyperfucosylation, demannosylation or hemi-glycosyiation. thereby decreasing Fc receptor binding. [00131 In another embodiment, the current invention relates to a fusion protein comprising one or more immunoglobulin Fe domains that have been engineered to comprise an antigen binding site and an IgG2 hinge that multimerizes the one or more Fe domains that have been WO 2013/112986 PCT/US2013/023404 6 engineered to comprise an antigen binding site. In a further embodiment, the igG2 hinge is fused to the N terminus of the one or more Fe domains that have been engineered to comprise an antigen binding site. In another embodiment, the IgG2 hinge is fused to the C terminus of the one or more Fe domains that have been engineered to comprise an antigen binding site. In a further embodiment, the fusion protein further comprises a linker linking the IgG2 hinge with the Fe domain engineered to comprise an antigen binding site. In still a further embodiment the fusion protein comprises an addition Fec domain that has not been engineered to comprise an antigen binding site. In one embodiment, the one or more Fe domains that have been engineered to comprise an antigen binding site has been engineered to bind Her2/neu and comprises SEQ ID NO: 18. In a further embodiment, the fusion protein comprises SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21. [001-4] In another embodiment, the current invention relates to a fusion protein comprising one or more one or more antigen binding antibody variable domains, fragments or variants thereof and one or more IgG2 hinge monomers wherein the IgG2 hinge domains multimerize the one or more antigen binding antibody variable domains, fragments or variables thereof. In one embodiment, the one or more antigen binding antibody variable domains, fragments or variants thereof is a variable heavy chain linked to a variable light chain, In a further embodiment, the variable heavy chain linked to the variable light chain forms an epitope binding site. In a further embodiment, the variable heavy chain is linked to the variable light chain with a linker. In a further embodiment, the one or more one or more antigen binding antibody variable domains, fragments or variants thereof is a Fab fragment of a monovalent antibody, scFv, a diabody, a triabody, a minibody, a single-domain antibody, a nanobody or a single chain antibody. In a further embodiment, the IgG2 hinge is fused to the C terminus of the one or more one or more antigen binding antibody variable domains, fragments or variants thereof. In a further embodiment the IgG2 hinge is fused to the N terminus of the one or more one or more antigen binding antibody variable domains, fragments or variants thereof. In still a further embodiment, the fusion protein also comprises an Fe domain in addition to the IgG2 hinge and the one or more one or more antigen binding antibody variable WO 2013/112986 PCT/US2013/023404 7 domains, fragments or variants thereof. In still another embodiment, the fusion protein comprises a linker between the one or more one or more antigen binding antibody variable domains, fragments or variants thereof peptide and the IgG2 hinge. 100151 In another embodiment, the current invention relates to a pharmaceutical formulation comprising a fusion protein containing one or more IgG2 hinge domains and one or more peptides, proteins, nucleic acids, fatty acids, carbohydrates or small molecules and optionally one or more immunoglobulin Fe domains and pharmaceutically acceptable excipients. In a further embodiment, the pharmaceutical formulation comprising the fusion protein is administered to a patient in need thereof intravenously, subcutaneously, orally, intraperitoneally, sublingually, opithalmno logically, buccally, intranasally, rectally, transdermally, by subdermal implant, or intramuscularly. In a further embodiment, the fusion protein is administered before, during or after administration with an additional pharmaceutically active agent. In a further embodiment the additional pharmaceutically active agent comprises a steroid; a biologic anti-autoimmune drug such as a monoclonal antibody, a fusion protein, or an anti-cytokine; a non-biologic anti-autoimmune drug; an immunosuppressant; an antibiotic; an antifungal; an anti-viral agent; a cytokine; or an agent otherwise capable of acting as an immune-modulator. In still a further embodiment, the steroid is prednisone, prednisolone, cortisone, dexamethasone, mometesone testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclamnethasone, albuterol, or levalbuterol. In still a further embodiment, the monoclonal antibody is infliximab, adalimumab, rituximab, tocilizumab, golimumnab, ofatumumab, LY2127399, belimumab, veltuzumab, or certolizumab. In still a further embodiment, the fusion protein is etanercept or abatacept. In still a further embodiment, the anti-cytokine biologic is anakinra. In still a further embodiment, the anti-rheumatic non-biologic drug is cyclophosphamide, methotrexate, azathioprine, hydroxychloroquine, leflunomide, minocycline, organic gold compounds, fostamatinib, tofacitinib, etoricoxib, or sulfasalazine. In still a further embodiment, the immunosuppressant is cyclosporine A, tacrolimus, sirolimus, mycophenolate mofetil, everolimus, OKT3, antithymocyte globulin, basiliximab, daclizumnumab, or alemntuzumab. In WO 2013/112986 PCT/US2013/023404 8 still a further embodiment, the fusion protein is administered before, during or after administration of a chemotherapeutic agent. In still a further embodiment, the fusion protein and the additional therapeutic agent display therapeutic synergy when administered together. In one embodiment, the fusion protein is administered prior to the administration of the additional therapeutic agent. In another embodiment, the fusion protein is administered at the same time as the administration of the additional therapeutic agent. In still another embodiment, the fusion protein is administered after the administration of the additional therapeutic agent. 100161 In another embodiment, the current invention relates to a method of treating a patient in need thereof with an effective amount of a fusion protein containing one or more IgG2. hinge domains, one or more peptides, proteins, or small molecules and, optionally, one or more immunoglobulin Fe domains. In a further embodiment, the patient in need thereof has an inflammatory, infectious, neoplastic, hormonal, or autoimmune disease. In still a further embodiment, the inflammatory disease is selected from the group consisting of coronary artery disease, Alzheimer's Disease, Irritable Bowel Syndrome, and Non-alcoholic steatohepatitis. In still a further embodiment, the infectious disease is selected from among bacterial, viral, fungal, or prion infection. In a particular embodiment, the patient has sepsis. In yet a further embodiment, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, type I or type II diabetes mellitus, autoimmune thyroiditis, idiopathic thrombocytopenia purpura, autoimmune anemia, chronic inflannnatory demyelinating polyneuropathy, multifocal motor neuropathy, scleroderma, systemic lupus erythematosus, psoriasis, inflammatory bowel disease including Crohn's Disease and U cerative Colitis, autoimmune uveitis, ANCA positive vasculitis, celiac disease, penphigus, dermatopolymyositis, Goodpasture's Disease, Myasthenia gravis, Grave's Disease, Kawasaki Disease, sickle cell crisis, idiopathic pulmonary fibrosis, vitiligo, and atopic dermatitis. In yet a further embodiment, the autoimmune disease is associated with the transplantation of an organ from a donor to a recipient. In yet a further embodiment, the autoimmune disease is a disease that is not classically characterized as an autoimmune disease but in which cells of WO 2013/112986 PCT/US2013/023404 9 the immune system play an important role such as Alzheimer's disease, Parkinson's disease, Huntingdon's disease, osteopenia, and osteoporosis. In a particular embodiment, the hormonal disease is selected from diabetes, obesity, Addison's disease, Cushing's syndrome, acromegaly, polycystic ovary syndrome, hyperpara thyroidism, hyperthyroidism, hypothyroidisi, and osteoporosis. [0017] In another embodiment, the fusion protein is administered to treat humans, non human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, bovines, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish and reptiles with species-specific or chimeric fusion proteins. In yet another embodiment, the human is an adult or a child. In still another embodiment, the fusion protein is administered to prevent autoimmune disease. In a further embodiment the fusion protein is administered to prevent vaccine-associated autoimmune conditions in companion animals and livestock. DETAILED D:)E SC RI PTI[ON OF THEI INVENTION 100181 The approach to multimerize peptides, proteins, small molecules, nucleic acids, and fatty acids that are active, or more active, as dimers and higher order multimers using the naturally occurring IgG2 hinge domain includes recombinant and/or biochemical creation of immunologically active fusion proteins which are surprisingly more efficient at miultiierization than molecules multimerized using traditional imultimerization techniques. The fusion proteins described herein have utility for treating, for example, autoimmune diseases, inflammatory diseases, endocrinologic diseases, cancer and infectious diseases including sepsis. Each embodiment is described in detail below along with specific exemplary embodiments. [00191 As used herein, the use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one." WO 2013/112986 PCT/US2013/023404 10 [00201 As used herein, "fusion protein" refers to a contiguous polypeptide or molecule containing multiple domains fused or joined together to form a novel protein or protein small molecule/carbohydrate compound. For example, an exemplary "fusion protein" contains the IgG2 hinge domain of SEQ ID NO: 1, fused/joined to an immunoglobulin Fe domain or a modified immunoglobulin Fe domain, and further fused/joined to the external domain of CTLA4, to form the fusion protein. The term "fusion protein" also encompasses the fusion of an IgG2 hinge domain of SEQ ID NO: I to a non-protein or peptide such as a small molecule or carbohydrate. [00211 By "directly linked" is meant two sequences connected to each other without intervening or extraneous sequences, for example, restriction enzyme recognition sites or cloning fragments. One of ordinary skill in the art will understand that "directly linked" encompasses the addition or removal of amino acids so long as the multimerization capacity is substantially unaffected. In certain embodiments, the IgG2 hinge is directly linked to a peptide, protein, nucleic acid, fatty acid, or small molecule. In other embodiments, the IgG2 hinge is directly linked to an immunoglobulin Fe domain monomer which is in turn directly linked to a peptide, protein, nucleic acid, fatty acid,, or small molecule. In other embodiments, the peptide, protein, nucleic acid, fatty acid, or small molecule that is directly linked to the IgG2 hinge is in turn directly linked to an immunoglobulin Fe domain monomer. 100221 By "homologous" is meant identity over the entire sequence of a given nucleic acid or amino acid sequence. For example, by "80% homologous" is meant that a given sequence shares about 80% identity with the claimed sequence and can include insertions, deletions, substitutions, and frame shifts. One of ordinary skill in the art will understand that sequence alignments can be done to take into account insertions and deletions to determine identity over the entire length of a sequence. [00231 The following paragraphs define the building blocks of the fusion proteins of the present invention, both structurally and functionally, and then define the fusion proteins themselves. However, it is first helpful to note that, as indicated above, each of the fusion WO 2013/112986 PCT/US2013/023404 11 proteins of the present invention has at least one IgG2 hinge domain. The lgG2 hinge domain interacts with an IgG 2 hinge domain on another fusion protein to create multimers. Therefore, the most functional form of the fusion proteins and discussed herein generally exist in a dimeric (or multimeric) form. The monomers of the fusion proteins discussed herein are the single chains that must associate with at least a second chain to form a functional homodimeric structure and multimeric structure. 4 gG2 hinge [00241 As is known in the art, the human IgG2 can forn covalent dimers through the hinge region (Yoo, E.M. et al. J. Immunol. 170, 3134-3138 (2003); Salfeld Nature Biotech. 25, 1369-1372 (2007)). The dimer formation of lgG2 is potentially mediated through the IgG2 hinge structure by C-C bonds (Yoo et al 2003), suggesting that the hinge structure alone can mediate dimer formation. The amount of IgG2 dimers found in human serum, however, is limited. It can be estimated from an SDS-PAGE gel that the amount of IgG2 existing as a dimer of the homodimer is less than 10% of the total IgG2 (Yoo et al. 2003). Furthermore, there is no quantitative evidence of the multimerization domain of IgG2 beyond the dimer of the homodimer. (Yoo et al. 2003). That is, native IgG2 has not been found to form higher order multimers in human serum. Therefore, the results presented herein are surprising in the degree of multimerization relative to native IgG2 and particularly surprising in that the IgG2 hinge-containing fusion proteins may be present in high order multimers. The amino acid sequence of the human IgG2 hinge monomer is as follows: ERKCCVECPPCP (SEQ ID NO: 1). We have demonstrated that mutation of any one of the 4 cysteines in SEQ ID NO: I may be associated with greatly diminished multimerization of the fusion protein. There are two C-X-X-C portions of the lgG2 hinge monomer referred to herein as "amino acid cores." Thus, fusion protein monomers of the present invention may comprise either the complete 12 amino acid sequence of the IgG2 hinge monomer, or either or both of the four amino acid cores along with Fec domain monomers. While the X-X of the amino acid core structures can be any amino acid, in a preferred embodiment the X-X sequence is V-F or P-1 P. The skilled WO 2013/112986 PCT/US2013/023404 12 artisan willI understand that the IgG2 hinge monomer may be comprised of any portion of the hinge sequence in addition to the core four amino acid structure, including all of the IgG2 hinge sequence. Thus, the IgG2. hinge sequence may comprise all 12 amino acids of SEQ ID NO: 1, or any combination of amino acids while maintaining the two C-X-X-C motifs, so long as the IgG2 hinge maintains its ability to multimerize the protein, peptide, nucleic acid, fatty acid, or small molecule. For example, the IgG2 hinge may comprise C-X-X-C-X-X-C alone or with any combination of amino acids flanking the core structure. Without being bound by theory, the IgG2 hinge of one fusion protein may bind the IgG2 hinge of another fusion protein, thereby forming a dimer of the homodimer, or higher order multimers while retaining increased activity such as functional binding to receptors compared to the unmultimerized proteins. Alternatively, the C-X-X-C motifs in the lgG 2 hinge may forrn C-C bridges with C's in other protein domains thereby forming a dimer of the homodimer, or higher order multimers while retaining increased functional binding to receptors compared to the unmultimerized proteins. Without being bound by theory, the multimers formed through C-C bridges are stable and comprise covalent bonds on disulfide bonding analysis. Peptides, Proteins, Nucleic acids, Fatty acids, and Small Vlolecules The peptides, proteins, nucleic acids, fatty acids, and small molecules useful in the present invention are those that show improved or increased activity when m ultimerized. Examples of proteins whose activity is improved by multimerization include, for example, monoclonal antibodies, bispecific antibodies, members of the TNFR superfamily (such as 4-1BB, APRIL, BAFF, TRAIL, BLyS, LIGHT, Lymphotoxin, Lymphotoxin beta, TRANCE, TWEAK, TN F alpha, TNF-beta, CD27 ligand, CD30 ligand, CD40 ligand, EDA, EDA-Al, EDA-A2, FAS ligand, GITR ligand, OX40 ligand, and TL1A), an interferon (such as IFNA1 (Interferon alI), IFNA2 IFNA4, IFNA5, IFNA8, IFNB1, IFNG (Interferon y), and IFNK), an interleukin (such as II10, IL 11, ILI2A, L1213, IL13, TXLNA, 1L15, 1L16, IL17A, IL17B, ILI7C, IL25 (IL17E), IL18, IL19, ILIA, ILIB, ILIFI, IL36RN., IL36A, IL37, IL36B, IL36G, IL2, IL20, 1L21, HL22, IL24, 1L3, IL4, IL5, 1L6, IL7, IL8, 1L9), a chemokine (such as MCP-1, MIP-la, WO 2013/112986 PCT/US2013/023404 13 MIP-lb, RANTES, cotaxin, MPF-I, CXCL-17, CXCL-10, CXC3) a bone morphogenic proteins and TGF- family member (such as BMPi, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7. BMP8B, GDF1O (BMP3B), GDFII (BMPI1), GDF2 (BMP9), GDF3, GDF5, MSTN, GDF9. INHA, INHBA,. NODAL, TGFA, TGFB1, TGFB2, and TGFB3), any PDGF/VXEIGF Family (such as FIGF (VEGFD) and PDGFA), other cytokines (such as CSFI, (MCSF), FAM3B,. LEFTY2), a soluble receptor (such as any of the 109 soluble receptors listed in the R&D Systems Hematopoietic Array and Common Analytes Array), a cell surface receptor including any Cluster of Differentiation, or a G-protein coupled receptor such as chemokine receptors (such as CCR5, CXCRI, and CXR2), TNF Super family receptors (such as CD137, BAFF R, BCMA, CD2'7, CD30, CD40, DcR3, DcTRAIL, DR3, DR6, EDAR, Fas, GITR, HVEM, lyphotoxin beta R, NGF R, osteoprotegerin, OX40, RANK, RELT, TACI, TRAIL R1, TRAIL R2, TRAIL R3, TRAIL R4, TROY, or TWEAK R), ligands to a cell surface receptor including any cluster of differentiation (CD4, CD303 or CD304), a naturally occurring hormone (such as ca-NISH, GLP-1, insulin, human growth hormone, glucagon, insulin-like growth factor-1, leptin, erythropoietin, thyroid stimulating hormone, follicle stimulating hormone, prolactin, leutinizing hormone, vasopressin, oxytocin, adrenocorticotropic hormone, thyrotropin releasing hormone, gonadotropin releasing hormone, growth hormone releasing hormone, corticotropin releasing hormone, somatostatin, melatonin thyroxine, calcitonin, parathyroid hormone, phosphatonin, osteocalcin, glucocorticoids such as cortisol, mineralocorticoids such as aldosterone, androgens such as testosterone and DH EA, estrogens such as estradiol, progestins such as progesterone, amylin, human chorionic gonadotropin, calcitriol, calciferol, gastrin, secretin, atrial naturetic peptide, cholecvstokinin, incretins, fibroblast growth factor 19, neuropeptide Y, ghrelin, PYY 3-36, angiotensinogen, thrombopoeitin, hepcidin, retinol binding protein 4, and adiponectin), neurotransmitters (such as epinephrine, norepinephrine, serotonin, acetylcholine, glutamate, glycine, aspartate, GABA, nitric oxide, histamine, dopamine, trace amines that bind to TAAR receptors, GHB that binds to GABAb receptor, hypocretin, niacin, endocannabinoids such as anandamine, 2-AC, noladin ether, NADA, and OAD, and endogenous opioids such as WO 2013/112986 PCT/US2013/023404 14 enkephalin, beta-endorphin, dynorphin, endomorphin, nociceptin, opiorphin, and morphine), growth factors (such as granulocyte macrophage colony stimulating factor, epidermal growth factor, fibroblast growth factor, and platelet derived growth factor), a fragment of any thereof, a functional analogue of any thereof, a functional analogue of a fragment of any thereof, and any combination thereof. Examples of peptides whose activity is improved by multimerization include, for example, the external domain of CTLA4, the p40 subunit of 1L12/23, and human parathyroid hormone. Examples of small molecules whose activity is improved by multimerization include, for example, chemotherapeutic agents, cytotoxic molecules, dyes, and fluorophores. Specific examples of chemotherapeutic agents include mechlorethamine, chlorambucil, melphalan, daunorubicin, doxorubicin, epirubicin, idarubicin, mnitoxantrone, valrubicin, paclitaxel, docitaxel, epothilones, etopiside, teniposide, tafluposide, azacitidine, azathioprine, capecitabine, cytarabine, doxifluridine, fluorouracil, gemcitabine, mercaptopurine, methotrexate, tioguanine, bleomycin, carboplatin, cisplatin, oxaliplatin, all-trans retinoic acid, vinblastine, vincristine, vindesine, and vinorelbine. Specific examples of cytotoxic molecules include methotrexate, cyclophosphamide, or azathioprine, mycophenolate, and cyclosporine A. 100251 CTLA-4, also known as C1)152, is a member of the immunoglobulin superfamily that is expressed on T cells and transmits inhibitory signals to T cells. Like CD28, CTLA4 binds B7.1 and B7.2 (CD80 and CD86, respectively) on antigen presenting cells (APCs). However, unlike CD28, CTLA-4 transmits an inhibitory signal to the T cell instead of a co stimulatory signal and binds with a higher affinity than does CD28. The increased affinity of CTLA-4 for the B7 molecules allows CTLA-4 to sequester B7 ligands from CD28 and antagonize CD28-dependant costimulation. CTLA-4 contains an extracellular domain, a transmembrane domain and a cytoplasmic tail. In accordance with the present invention, the entire CTLA-4 molecule may be included in the fusion protein or just the extracellular domain of CTLA-4 may be included in the fusion protein.
WO 2013/112986 PCT/US2013/023404 15 [00261 CTLA-4 has been an attractive pharmaceutical target. Since CTLA-4 binds with higher affinity to B7.1 and B7.2 molecules on APCs than does CD28, it has been investigated extensively as a potential therapy for autoimmune diseases. Fusion proteins of CTLA-4 and antibodies (CTLA4-Ig, abatacept) are commercially available for treatment of rheumatoid arthritis. Additionally, betacept, a second generation CTLA4-lg fusion has recently been approved by the FDA for renal transplantation patients that are sensitized to Epstein Barr Virus. 100271 Conversely, CTLA-4 antagonists also show great promise in the treatment of cancer as inhibitors of immune system tolerance. Antagonistic antibodies against CTLA-4 (ipilmumnab) are being developed for this clinical indication. [00281 CTLA-4 dimerization appears to be essential for biological activity. CTLA-4 does not undergo any detectable conformational change upon B7.2 binding (Schwartz, et al. (2001) "Structural basis for co-stimulation of the human CTLA-4/B7-2 complex," Nature, 410(6828):604-8), and before ligation to B7 it exists as a nonfunctional covalent homodimer (Lindsey, et al. (1995) "Binding Stoichiometry of the Cytotoxic T Lymphocyte-associated Molecule-4 (CTLA-4)" J. Biol. Chem., 270(25):15417-15424). It has been shown that CTLA-4 dimerization/multimerization is hierarchically regulated by intermolecular disulfide bonding, N-linked glycosylation, and B7 ligand-driven dimerization. (Darlington, et al. (2005) "Hierarchical Regulation of CTLA-4 dimer-based lattice fonnation and its biological relevance for T cell inactivation," J. Immunol., 175:996-1004). Therefore, the nultimers of the current invention are particularly useful in enhancing the biological activity of CTLA-4 by creating dimers and higher ordered nmitimers of CTLA-4 proteins and peptides. [00291 Programmed cell death-1(PD-1) is an immunoreceptor belonging to the CD28/CTLA-4 family. PD-1 negatively regulates the antigen receptor signaling by recruiting protein tyrosine phosphatase, SHP-2, upon interaction with either of its two ligands, PDL-1 or PDL-2. (Okazaki and Honjo (2007) "PD-i and PD-I ligands: from discovery to clinical application," International Immunol., 19(7):813-824), WO 2013/112986 PCT/US2013/023404 16 [00301 PD-1 is a type I transmembrane glycoprotein comprising an IgV-type extracellular domain which shares homology with CTLA-4, CD28 and ICOS. PD-i is expressed on peripheral T and B cells upon activation. PD-I ligands PDL- I and PDL-2 are also type I transmembrane glycoproteins composed of IgC and IgV-type extracellular domains, however, unlike P'D-I, PDL-I and PDL-2, comprise no intracellular signaling domains. (Okazaki and Honjo). [00311 PD-1 dysregulation is associated with human autoimmune diseases such as lupus, rheumatoid arthritis, type I diabetes, multiple sclerosis, ankylosing spondylitis, myocardial infarction and allergy, while aberrant PDL-I and PDL-2 are associated with many infectious diseases such as HIV, [ICV, HBV, .pylori infections. Antagonists of PD-I are useful in the treatment of cancer and infectious disease, while agonists of PD-I are useful in the treatment of autoimnunity, allergy and transplant rejection. Without being bound by theory, the PD-i, extracellular domain containing multimers of the present invention are useful in binding PDL-1 ligands without sending an intrcellular signal, thereby acting as a PDL-1/PDL-2 sink and blocking PD-i signaling. Therefore, the multimers of the current invention are particularly useful in blocking the biological activity of PD-1 by creating dimers and higher ordered multimers of PD-1 proteins and peptides which bind to PDL-1/PDL-2 without the associated cellular signaling. [00321 IL-12 is a cytokine that is produced by dendritic cells, macrophages and certain B cells in response to antigenic stimulation. IL-12 functions in the diffIerentiation of naive T cells into ThO cells that will eventually develop into Th1 cells in the continued presence of IL- 2 since IL-12 also stimulates production of interferon gamma (IFNy), and tumor necrosis factor alpha (TNFa), while decreasing IL-4 production, thus leading to differentiation of T cells into Th1 cells. [00331 IL-23 is a cytokine produced by dendritic cells and macrophages in response to danger signals, including cell debris. I L-23 functions in directing memory T cells toward the WO 2013/112986 PCT/US2013/023404 17 Thl7 phenotype. Therefore, while IL-12 mainly acts on naive T cells, IL-23 acts predominantly on memory T cells. [00341 Interleukin-12 (IL-12) is a heterodimer of the IL-12 p35 (IL-12a) and common p40 (IL-12P) subunits, while IL-23 is a heterodimer of the IL-23 p19 (IL-23a) and common p40 (IL-12p) subunits. Both heterodimeric IL-12 and IL-23 are agonistic for the IL-12 and IL-23 receptors, which in turn activate the transcription activator STAT4 to stimulate the production of I FNacj. The naturally occurring homodimer of the common p40 subunit, on the other hand, is antagonistic of the IL-12 and IL-23 receptors. Therefore, while pharmaceutical companies have developed antibodies which are aimed at decreasing IL-12/23 receptor activity, these antibodies may also bind and remove from circulation, the naturally antagonistic p40/p40 homodimers. The present invention aims to increase the presence of p40/p40 homodimers and higher order multimers of p40/p40 in an effort to decrease pro inflammatory IL-11/3 receptor signaling and dampen the Thi immune response, which is particularly useful in patients with certain autoimmune diseases such as rheumatoid arthritis, type I diabetes and multiple sclerosis. [00351 CD47 is a cell surface transmembrane Ig superfamily member and is an extracellular ligand for signal regulatory protein (SIRPa). Interactions between SIRPa and CD47 regulate immune cell functions such as neutrophil transmigration in response to inflammatory stimuli. (Liu, et al., (2004) "Peptide-Mediated Inhibition of Neutrophil Transmigration by Blocking cD47 Interactions with Signal Regulatory Protein U," .1. Immunol., 172:2278-2585). [00361 Liu, et al., identified a novel function blocking peptide, CERVIGTGWWVRC (SEQ I) NO: 11) that mimics an epitope on CD47 and binds to SIRPU and is capable of inhibiting neutrophil migration into inflammatory sites. These peptides are referred to herein as "CERVIG peptides." By CERVIG peptide is meant, any peptide containing the CERVIG residues and capable of binding SIRPa. SEQ ID NO: 11 is one example of a CERVIG peptide. Therefore, the nultimers of the current invention, comprising CERVIG peptides, WO 2013/112986 PCT/US2013/023404 18 IgG2 hinge domains and optionally Fe domains are particularly useful in blocking the biological activity of CD47 by creating dimers and higher ordered multimers of the CERVIG peptides. [00371 Antibodies including monoclonal antibodies can also be multimerized by the present invention. Examples of monoclonal antibodies include 3F8, 8H9, abagovomab, abciximab, adalimumab, adecaturnurnab, afelimomab, afutuzumab, alaciz umab pegol, ALD5 18, alemtuzumab, altumomab pentetate, amatuximab, anatumomab mafenatox, anrukinzumab (IMA-638), apolizumab, arciturnornab, aselizurnab, atinurnab, atlizumab (tocilizumab), atorolimumab, bapineuzumab, basiliximab, bavituximab, bectumomab, belirnurnab, benralizumab, bertilirnurnab, besilesomab, bevacizumab, biciroiab, bivatuzunab mertansine, blinaturmormab, blosozumab, brentuximab vedotin, briakinumab, brodalunab, canakinumab, cantuzurnab mertansine, cantuzunab ravtansine, capromab pendetide, cariurmab, caturmaxomab, CC49, cedelizumab, certolizumab pegol, cetuximab, Ch.14.18, citatuzunab bogatox, cixutumumab, clenoliximab., clivatuzumab tetraxetan, con atumumab, crenezumab, CR6261, dacetuzumab, daclizurnab, dalotuzumab, daratumumab, denosumab. detumomab, dorlimomab aritox, drozitumab., ecromeximab, eculizunab, edobacornab, edrecolomab, efalizumab, efungumab, elotuzurnab, elsili mornab, enavatuzurnab, enlimormab pegol, enokizurmab, ensituximab, epitumomab cituxetan, epratuzunab, erlizumab, ertumaxoiab, etaracizunab, etrolizumab, exbivirumnab, fanolesomab, faralimomab, farletuzumab, FBTA05, felvizumab, fezakinumab, ficlatuzurnab, figitunumab, flanvotumab, fontolizumab, foraluiab, foravirunab, fresolirmurnab, fulranumab, galiximab, ganitumab, gantenerurnab, gavilimomab, gerntuzunab ozogamicin, gevokizumab, girentuximab, glermbatunumab vedotin, golirnunab, gomiliximab, GS6624, ibalizumab, ibritumomab tiuxetan, icrucumab, igovomab, imeirornab, indatuxinab ravtansine. infliximab, intetumumab., inolinonab, inotuzunab ozogamicin, ipilimumab, iratumurnab, itolizumab, ixekizumiab, keliximrab, labetuzurnab, lebrikizurnab, lemalesonab, lerdelimunab, lexatumumab, libivirumab, lintuzumab., lorvotuzumab mertansine, lucatummainab, lumiliximab, mapaturnurnab, maslirnornab, mavrilimumab, rnazumab, WO 2013/112986 PCT/US2013/023404 19 mepolizumab, metelimurnab, milatuzumab, minretumomab, mitumomab, mogamulizumaib, morolimuniat, motavizuinab, moxetuinomab pasudotox, iuromonab-CD3, nacolomab tafenatox, namilumab, naptumornab estafenatox, narnatumab, natalizunab, nebacumab, necitumumat, nerelimomab. nimotuzumat, nofetumomab merpentan. ocrelizumab, odulimomab, ofatunumab, olaratumab, olokizumab, omali zunab, onartuzumab, oportuzumab monatox, oregovomab, otelixizumab, oxelumab, ozoralizumab, pagibaximab, palivizumal, panitumumab, panobac umab, pascolizunab, pateclizumal, pemtumoniab, pertuzumab, pexelizumab, pintumomat, ponezumat, priliximat, pritumumab, PRO 140, racotunomab, radretumab, rafiviru mab, ramucirumab, ranibizumab, raxibacumab, regavirumab, reslizumab, rilotumumab, rituximab, robatumumab, roledumab, romosozumab, rontalizuiab, rovelizumiab, ruplizumab, sanializuniab, sarilumab , satunioniat pendetide, secukinumab, sevirumab, sibrotuzumab, sifalimumab, siltuximab, siplizurnab, sirukumab, solanezumab, sonepcizumab, sontuzumab, stamulumab, sulesomab, suvizumab, tabalumab, tacatuzumab tetraxetan, tadocizumab, talizunab, tanezumab, tapiturmomab paptox, tefibazumab, telimomab aritox, tenatumomab, teneliximab, teplizumab, teprotumumat, TGN1412, ticilimumab (trenelimunab), tigatuzumab, TNX-650, tocilizumab (=:atlizumab), toralizumat, tositumomab, tralokinumab, trastuzumab, TRBS07. tregalizumab, tremelinunab, tucotuzumab celnioleukin, tuviruinab, ublituxinab, ureliimab, urtoxazumab, ustekinumab, vapaliximab, vatelizumba, vedolizurnab, veltuzumab, vepalimomab, vesencunab, visilizumab, volociximab, votunumab, zaluturnurnab, zanolirmnimab, ziralimumab, zolimom'ab aritox 100381 The antibodies to be multimerized can be bispecfic antibodies such as, for example blinaturnomab. Blinatumonab has the structure scFv-ScFv, where one Fv is anti-CD19 and the other Fv is anti CD3A, The addition of an IgG2 hinge will result in a multimeric form of the antibody with multivalent binding to both epitopes, a useful feature for targeting low expressing antigens such as low expressing tumor-specific antigens on cancer cells. Additionally, multi-specific antibodies with multivalent binding to two or more epitopes can WO 2013/112986 PCT/US2013/023404 20 be created by the addition of an IgG2 hinge to a protein construct designed to express multiple antibodies. [00391 Antigen binding domains of antibodies can be made using the variable portion of the heavy chain (VH) linked to the variable portion of the light chain (VL), forming an antigen binding site. (Holt, et al., (2003) "Domain antibodies: proteins for therapy," Trends in Biotechnology, 21(11):484-490). The variable light chain can be linked to the variable heavy chain using one of many possible linker regions and may also contain the CHI region of VH or VL. The VH or VL or both in tandem can be linked to the multimerizing IgG2 hinge domain and optionally to an Fe. Alternatively, the VH or VL may be co-expressed in the same cell and not fused as a chimeric protein. Alternatively, the VH o V 1 may be co-expressed in the same cell and each individually fused to the multimerizing igG2 hinge domain as a chimeric protein. Multimerization of these antigen binding domains, herein referred to as "domain antibodies" are useful as soluble receptors, for example, to cytokines, such as TNF c. IL-1, IL-12, IL-8. IFNa, IFN, IFNy, IL-18, IL-27, and other proinflammatory mediators. [00401 Multimerized domain antibodies are also useful as potent agonists of cell surface receptors, including, without limitation, hormonal receptors. Domain antibodies can also useful as potent antagonists of cell surface receptors, such as, for example, to a low expressing tunor receptor. Domain antibodies are also useful as diagnostic reagents, useful in methods such as, inimunohistochemistry, flow cytometry, ELISA , ELISPOT, or any other assay where an antigen binding domain of an antibody is used. Single chain antibodies generated by placing the VL domain and a linker on the N-terminus of the VH domain of a human IgG1 monoclonal antibody (VL -linker-VH-CH1-CH2-CHI3) (Wu, et al. (2001) "Multimerization of a chimeric anti-CD20 single-chain Fv-Fc fusion protein is mediated through variable domain exchange," Protein Engineering 14(12):1025-1033) can also be linked to a multimerizing IgG 2 hinge to form multimerized single chain antibodies such as: a. IgG2 hinge - VL- linker - VHI- - IgG1 CH1- - IgGI Hinge - IgGI12 -- IgGi C-13 WO 2013/112986 PCT/US2013/023404 21 b. IgG2 hinge - VL- linker - VH - IgG1 CHI - IgGl CH2 - IgG CH3 c. VL- liner - VH - IgG2 hinge - IgGI CHI - IgG1 Hinge - IgGi CH2 - IgGI CH3 d. VL- linker - VH - IgG2 hinge - IgGI CHI - IgGI CH2 - IgG1 CH3 e. VL- liner - VH - CH1 - IgG2 Hinge - IgG1 Hinge - IgGi CH2 - IgGI CH3 f, VI- liner - VH - C1 I - IgG2 Hinge- IgGI CH2 - lgGI CR3 g, VL- linker -VH - IgG 1 CH I - IgGI Hinge - IgGi CH2 - IgGI CH3 - IgG2 hinge h, VL- linker - VH - IgG 1 CHI - IgG1 CH2 -- IgGI CH13 - IgG2 hinge. [00411 In addition to the domain antibodies discussed above, other small recombinant monoclonal antibody fragments and variants, including monovalent antibody fragments, such as Fab, scFv, diabodies, triabodies, rninibodies and single domain antibodies can also be multimerized using the IgG2 hinge of the present invention. These fragments, like domain antibodies, retain the target specificity of whole monoclonal antibodies, but can be produced more echnomically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. These monoclonal antibody fragments or variants can be directly linked to an IgG2 hinge with or without an Fe domain. 100421 Additionally, the antibody-derived and non-Ig binding scaffolds summarized by Wurch, et aL, (2008) "Development of novel protein scaffolds as alternatives to whole antibodies for imaging and therapy: status on discovery research and clinical validation," Current Pharmaceutical Biotechnology, 23(9):1126-1136, can also be multimerized by the methods of the current invention. [00431 Fully functional antibodies, termed nanobodies, lacking light chains, resembling those made by camels and llamas (Deffer, et al., (2009) African Journal of Biotechnology, 8(12):2645-2652) can also be multinierized by the IgG2 hinge of the present invention.
WO 2013/112986 PCT/US2013/023404 22 These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (cC-12 and cCH3) in which the cloned and isolated V-H domain is a stable polypeptide harboring the full antigen-binding capacity of the original heavychain antibody. There is no cCH1 or light chain. This heavy chain antibody can be linked to the multimerizing IgG2 hinge domain to form a multimerizing single chain antibody as follows: a. hlgG2 hinge - VI - c-linge - c112 - cC 13 b, hIgG2 hinge - VIH [H - cCH]2 - cC3I c. VIH - hlgG2 hinge - cinge - eCH2 - cC'-13 d. VHH - hIgG2 hinge - cCH2 - eCH3 e. VHH - cHinge - cCH2 - cC13 - higG2 hinge f VHH -- cC12 - cC[13 - hIgG2 hinge [00441 Major histocompatibity complex (MIC) molecules expressed on the surface of antigen presenting cells bind antigen and present the antigen to T cells. MHC molecules, particularly MHC class I molecules, although MHC class II molecules can also be multimerized, are multimerized for use in tetramer assays. In certain embodiments, tetramer assays are used to detect the presence of antigen specific T-cells. In order for a T-celi to detect the peptide to which it is specific, it must recognize both the peptide and the MHC complex at the surface of a cell with which it comes into contact. Because the binding affinity of a T-cell receptor to MIHC complexed with a peptide is so low, this has historically been a challenging problem. An earlier solution was realized by creating a tetramer of NHC molecules each presenting an identical peptidic antigen which increases the avidity of the binding. Multimerizing the MHC --- antigen complex with the IgG2 hinge will further increase binding affinity and avidity. These compounds can be labeled for use in detection, for example with a dye or fluorophore. Similar compounds comprising the biotin-streptavidin MHC tetramers can also be used.
WO 2013/112986 PCT/US2013/023404 23 [00451 The important role of multivalent carbohydrate derivatives in glycobiology is well described. (R. Roy, Topics Curr Chem 1997,187;241-274; M. Mammen, S.K. Choi, G.tM. Whitesides, Angew. Chem 1998,110;2908-2953). 100461 Linear or branched oligonueleotide multimers are useful as amplifiers in biochemical assays US Pat No. 5124246) [00471 Higher fatty acids can form associative multimers by hydrogen binding as a result of the presence of negatively polarized oxygen atom from the carbonyl group and the positively polarized hydrogen atom from the carboxyl group (Preparative Layer Chromatography, Teresa Kowalska and Joseph Sherma). Multimers of certain fatty acids may be therapeutically useful, alone or in combination with proteins. High-density lipoprotein (HDL) is positively associated with a decreased risk of coronary heart disease (CHD). HDL is composed of 4 apolipoproteins per particle. HDL itself is heterogeneous. HDL may be composed of apo A-I and apo A-II or of apo A-I alone. HDL2 is usually made up only of apo A-I, while 1-i)L3 contains a combination of apo A-I and apo A-Il. HDL particles that are less dense than HDL2 are rich in apo E. A fusion protein of the IgG2 hinge and either a fatty acid, or combination of fatty acids, or one or more apolipoproteins will create multimers of that fatty acid or apolipoprotein(s). These multimers may be therapeutically useful, for example in reversing cholesterol transport, improving atherosclerosis, decreasing primary or secondary myocardial or stroke risk, or treating diseases of lipid-rich organs such as brain, including Alzheiner's Disease and Parkinson's Disease. [00481 Improved blood clotting is sometimes needed, for example in surgical situations. Von Willebrand factor is a protein crucial for blood clotting (Science 324, 1330-1334). Endothelial cells secrete von Willebrand Factor as very large multimers which is then cleaved quickly to smaller multimers by available metalloproteases such as ADAMTS13 (Nat. Med 15 (7); 738). Smaller multimers of von Willebrand factor are worse than the larger multimers WO 2013/112986 PCT/US2013/023404 24 at promoting blood clots. A fusion protein of the IgG2 hinge and von Willebrand factor will increase the multimer size and increase functionality. Fe Domain [00491 As used herein, "Fc domain" or "Immunoglobulin Fe domain" describes the minimum region (in the context of a larger polypeptide) or smallest protein folded structure (in the context of an isolated protein) that can bind to or be bound by an Fc receptor (FcR). In both an Fe fragment and an Fe partial fragment, the Fe domain is the minimum binding region that allows binding of the molecule to an Fe receptor. While an Fe domain can be limited to a discrete polypeptide that is bound by an Fe receptor, it will also be clear that an Fe domain can be a part or all of an Fe fragment, as well as part or all of an Fe partial fragment. When the term "Fe domains" is used in this invention it will be recognized by a skilled artisan as meaning more than one Fe domain. An Fe domain is comprised of two Fe domain monomers. As further defined herein, when two such Fe domain monomers associate to form a homodimer, the resulting Fe domain has Fe receptor binding activity. Thus an Fe domain is a homodimeric structure that can bind an Fc receptor. [00501 The specific CHI1, CH2, CH3 and CH-4 domains and hinge regions that comprise the Fc domain monomers of the fusion proteins of the present invention may be independently selected, both in terms of the immunoglobulin subclass, as well as in the organism, from which they are derived. Accordingly, the fusion proteins disclosed herein may comprise Fe domain monomers and partial Fe domain monomers that independently come from various immunoglobulin types such as human IgG1, IgG2, lgG3, IgG4, IgAl, IgA2, IgD, IgE, and IgM, mouse IgG2a, or dog IgA or IgB. Similarly each Fe domain monomer and partial Fe domain monomer may be derived from various species, preferably a mammalian species, including non-human primates (e.g., monkeys, baboons, and chimpanzees), humans. murine, rattus, bovine, equine, feline, canine, porcine, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish and reptiles to produce species-specific or chimeric fusion proteins.
WO 2013/112986 PCT/US2013/023404 25 [00511 The individual Fe domain monomers and partial Fe domain monomers may also be humanized. One of skill in the art will realize that different Fe domains and partial Fe domains will provide different types of functionalities. For example, Fe 4 Rs bind specifically to IgG immunoglobulins and not well to other classes of immunoglobulins. [00521 "Capable of specifically binding to a FeyR" as used herein refers to binding to an FeyR. Specific binding is generally defined as the amount of labeled ligand which is displaceable by a subsequent excess of unlabeled ligand in a binding assay. However, this does not exclude other means of assessing specific binding which are well established in the art (e.g., Mendel CM, Mendel DB, 'Non-specific' binding. The problem and a solution. Biochem J. 1985 May 15;228(1):269-72). Specific binding may be measured in a variety of ways well known in the art such as surface plasmon resonance (SPR) technology (commercially available through BIACORE@) or biolayer interferometry (commercially available through Fortelfio@) to characterize both association and dissociation constants of the fusion proteins (Asian K, Lakowicz JR, Geddes C. Plasmon light scattering in biology and medicine: new sensing approaches, visions and perspectives. Current Opinion in Chemical Biology 2005, 9:538-544). The fusion proteins of the present invention are designed in such a way as to include Fe domain monomers, however the Fe domain monomer to be included in the fusion protein is selected for poor binding to Fe gamma receptors. By "poor binding to Fe gamma receptors" is meant that the Fe domain binds with relatively weaker affinity than an Fe gamma receptor that binds with higher affinity. For example, 1gG2 and IgG4 naturally bind poorly to Fe gamma receptors and therefore these isotypes are particularly useful in the present invention. 100531 Alternatively, an isotype such as IgGI, which under normal circumstances binds to Fe gamma receptors with higher affinity may be mutated or otherwise modified to decrease Fe binding affinity. These mutations and modifications are described below in more detail. Without being bound by theory, it is thought that a fusion protein containing an Fe domain that does not bind well to Fe gamma receptors retains useful drug characteristics relative to WO 2013/112986 PCT/US2013/023404 26 fusion proteins not containing the Fe domain including increased serum half life and more efficient nufacturing purification. Therefore, a fusion protein whose primary sequence is comprised of an IgG2 hinge will create multimers that will have important clinical and non clinical utility, independent of their binding to Fc gamma receptors. [00541 The present invention also encompasses fusion proteins comprising Fe domains and Fc partial domains having amino acids that differ from the naturally-occurring amino acid sequences of the Fc domain. Preferred Fe domains for inclusion in the fusion proteins of the present invention have poor specific binding affinity to either a holo-Fey receptor or a soluble extracellular domain portion of an FecyR. Primary amino acid sequences and X-ray crystallography structures of numerous Fc domains and Fe domain monomers are available in the art. See. e.g., Woof JM, Burton DR. Human antibody-Fc receptor interactions illuminated by crystal structures. Nat Rev Immunol. 2004 Feb;4(2):89-99. Representative Fc domains with Fey receptor binding capacity include the Fe domains from human IgG1. These native sequences have been subjected to extensive structure-function analysis including site directed mutagenesis mapping of functional sequences. Based on these prior structure-function studies and the available crystallography data, one of skill in the art may design functional Fc domain sequence variants that diminish the Fe domain's FcyR receptor binding capacity but which retain functional utility, such as creating a more efficiently purified fusion protein or a fusion protein with a longer half-life than the same protein lacking the Fe domain or partial domain. [00551 The amino acid changes may be found throughout the sequence of the Fe domain, or be isolated to particular Fc partial domains that comprise the Fe domain. The functional variants of the Fe domain used in the fusion proteins of the present invention will have at least about 50%. 60%, 70%, 80%, 90%. 95%, 96%, 97%, 98% or 99% sequence identity to a native Fe domain. Similarly, the functional variants of the Fc partial domains used in the fusion proteins of the present invention will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a native Fc partial domain.
WO 2013/112986 PCT/US2013/023404 27 [00561 The skilled artisan will appreciate that the present invention further encompasses the use of functional variants of Fc domain monomers in the construction of Fc fragment monomers, Fe partial fragment monomers, and fusion proteins of the present invention. The functional variants of the Fe domain monomers will have at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to a native Fe domain monomer sequence. [00571 The amino acid changes decrease or prevent altogether the binding affinity of the fusion protein to the Fey receptor. Preferably such amino acid changes will be conservative amino acid substitutions, however, such changes include deletions, additions and other substitutions. Conservative amino acid substitutions typically include changes within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine, glutamine, serine and threonine; lysine, histidine and arginine; and phenylalanine and tyrosine. Additionally, the amino acid change may enhance multimerization frequency, extent, percentage, or strength, for example by the addition of cysteine residues. [00581 The amino acid changes may be naturally occurring amino acid changes resulting in Fe domain polymorphisms, or the amino acid changes may be introduced, for example by site directed mutagenesis. The amino acid changes can occur anywhere within the Fe domain so long as the Fe domain retains the desired biological activity. In a preferred embodiment, the polymorphism or mutation leads to decreases receptor binding. The polymorhisn/ilmutation preferably occurs at one or more of amino acid positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414 according to the EU index as in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991). Specific polymorphisms/mutations in these amino acid positions are well known in the art and can be found, for example in Sheilds, et al. (2001) .J. Biol. Chem. 276(9):6591-6604.
WO 2013/112986 PCT/US2013/023404 28 [00591 In a preferred embodiment, the polymorphism/mutation contains one or more amino acid substitutions of positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414 of IgGi Fc. In a further embodiment, the polymorphism/mutation contains two or more amino acid substitutions of positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414 of IgG I Fe. In a further embodiment, the polymorphism/mutation contains three or more amino acid substitutions of positions233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414 of IgGi Fe. In a further embodiment, the polynorphisrnimutation contains more than three amino acid substitutions of positions23 3, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414 of IgGI Fe. [00601 The terrn "functional variant" as used herein refers to a sequence related by homology to a reference sequence which is capable of mediating the same biological effects as the reference sequence (when a polypeptide), or which encodes a polypeptide that is capable of mediating the same biological effects as a polypeptide encoded by the reference sequence (when a polynucleotide). Functional sequence variants include both polynucleotides and polypeptides, Sequence identity is assessed generally using BLAST 2.0 (Basic Local Alignment Search Tool), operating with the default parameters: Filter-On, Scoring Matrix BLOS M62, Word Size -3, E value - 10, Gap Costs - 11,1 and Alignments -50. [00611 In addition to the amino acid sequence composition of native Fe domains, the carbohydrate content of the Fc domain is known to play an important role on Fe domain structure and binding interactions with FcyR. See, e.g., Robert L. Shields, et al. Lack of Fucose on Human IoGl N-Linked Oiigosaccharide hIproves Binding to Human FeyRIII and Antibody-dependent Cellular Toxicity. J. Biol. Chem., Jul 2002; 277: 26733 - 2674; Ann Wright and Sherie L. Morrison. Effect of C2- Associated Carbohydrate Structure on Ig Effector Function: Studies with Chimeric Mouse-Human IgGl Antibodies in Glycosylation Mutants of Chinese Hamster Ovary Cells. J. Immunol, Apr 1998; 160: 3393 - 3402.
WO 2013/112986 PCT/US2013/023404 29 Carbohydrate content may be controlled using, for example, particular protein expression systems including particular cell lines or in vitro enzymatic modification. Thus, the present invention includes fusion proteins comprising Fe domains with the native carbohydrate content of holo-antibody from which the domains were obtained, as well as those fusion proteins with an altered carbohydrate content. In another embodiment, multimer components of the fusion protein are characterized by a different glycosylation pattern compared with the homodimer component of the same fusion protein. In a preferred embodiment, the fusion protein is enriched for homodimers and multimers comprising a glycosylation pattern that decreases Fc receptor binding. In a particular embodiment, the Fe domain is hyperfucosylated, demannosylated or heii-glycosylated, thus resulting in decreased binding to Fc receptors (Yamime-Ohnuki and Sato (2009) "Production of Therapeutic Antibodies with Controlled Fucosylation," mAbs, 1:3, 230-236). 100621 The present invention also encompasses fusion proteins comprising Fe domains which comprise antigen binding-sites in the structural loops of the Fe domains, such as those described by Wozniak-Knopp, et al. (2010) "Introducing anti gen-binding sites in structural loops of immunoglobin constant domains: Fe fragments with engineered HER2/neu-binding sites and antibody properties. In these constructs, antigen binding sites are introduced in the loop regions, particularly the loop regions located at the C-terminal tip of the Fc CH3 domain which includes loops AB, CD and EF. These engineered Fes can not only bind to antigen through the engineered antigen binding site, but also retain the ability to elicit effector functions via binding to Fey receptors, complement and FcRn. These engineered Fe can comprise antigen binding domains that bind any antigen. For example, the Fe can be engineered to comprise an antigen binding site that binds the same antigens as the monoclonal antibodies discussed above. In one embodiment, the Fe is engineered to express a Her2/neu binding site. In a further embodiment, the Fc engineered to express a Her2/neu binding site (SEQ ID NO: 18) is fused to an IgG2 hinge to mediate multimerization of the engineered Fe. The IgG2 hinge can be fused to C terminus of the engineered Fc (SEQ ID NO: 19) or the N terminus of the engineered Fe (SEQ ID NO: 20). Additionally, the fusion WO 2013/112986 PCT/US2013/023404 30 protein may comprise an Fc CH2 and CH3 domain between the IgG2 hinge and the engineered FC (IgG2 hinge-C[12-CH3-engineered Fc) (SEQ ID NO: 21). These multimerized compounds will have a high avidity for both the antigen, e.g. Her2/neu as well as FcyRIla which will increase the tumor killing potency of the compounds. Fusion Proteins [00631 As used herein, the term "fusion protein" means any single, contiguous peptide molecule that contains at least the IgG2 hinge of SEQ ID NO: I and a peptide, protein, nucleic acid, fatty acid, or small molecule whose activity is increased by multimerization. The fusion protein of the present invention may also include an immunoglobulin Fe domain monomer which binds with decreased affinity, or not at all, to Fe receptors. Furthermore, the fusion proteins of the present invention also refers to a protein that, when associated with at least a second fusion protein, forms a dimeric or multimeric structure comprising at least two peptides, proteins, nucleic acids, fatty acids, or small molecules whose activity is increased by multimerization. 100641 The regions of the fusion proteins, the IgG2 hinge, the peptide, protein, nucleic acids, fatty acids, or small molecule to be multimerized and the immunoglobulin Fe domain monomer, may be arranged from amino terminal to carboxy terminal of successive regions of the fusion protein. The regions may be directly linked to each other or linked using small regions of amino acid linker residues. Fusion proteins of the present invention comprise the amino terminus of the peptide or protein, linked to the carboxy terminus of the IgG2 hinge or the amino terminus of the IgG2 hinge linked to the carboxy terminus of the peptide, protein. With respect to small molecules, nucleic acids and fatty acids, the fusion protein may comprise a small molecule, nucleic acid or fatty acid linked to the carboxy terminus of the IgG2 hinge. Conversely, the fusion protein may comprise a small molecule, nucleic acid or fatty acid linked to the amino terminus of the IgG2 hinge. For example. the fusion proteins of the present invention may include: WO 2013/112986 PCT/US2013/023404 31 Protein/peptide/small molecule/nucleic acid/fatty acid-IgG2 hinge IgG2 hinge-protein/peptide/ small molecule/nucleic acid/fat Fusion proteins of the present invention also comprise small molecules or fatty acids linked to the IgG2 hinge. For example, the small molecule, fatty acid, or nucleic acid may be Small molecule/nucleic acid/fatty acid-IgG2 hinge IgG2 hinge- small molecule/nucleic acid/fatty acid Alternatively the small molecule, nucleic acid, or fatty acid may be linked to the IgG2 hinge through a linker or through a sugar bound to the IlgG2 hinge. [00651 In one embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a IL-12 p40 subunit. In another embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a CTLA4 protein or CTLA-4 extracellular domain peptide. In a further embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a PD-1 protein or PD-I extracellular domain peptide. In a further embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a CERVIG peptide. In a further embodiment the exemplified fusion proteins contain a CTLA4 protein or CTLA4 extracellular domain peptide and also contain a 17 molecule. Examples of such molecules include: CT LA4-IgG2 hinge IgG2 hinge-CTLA4 17- IgG2 hinge IgG2 hinge-B7 IgG2 hinge-CTLA4-B7 B7-CTLA4-1gG2 hinge WO 2013/112986 PCT/US2013/023404 32 CTLA4-B7-IgG2 hinge IgG2 hinge-B7-CTLA4 IgG2 hinge-CTLA4-B7 [00661 In one embodiment the B7 is B7.1. In another embodiment, the B7 is B7.2. [00671 Additional fusion proteins of the present invention comprise the amino terminus of the peptide, protein, small molecule, nucleic acid or fatty acid linked to the carboxy terminus of the IgG2 hinge which is in turn linked via the amino terminus of the IgG2 hinge to the carboxy terminus of an immunoglobulin Fc domain monomer, for example, Fc-IgG2 hinge peptide protein small molecule/nucleic acid/fatty acid [00681 The fusion proteins of the present invention may also include: Protein/peptide/smalI molecule/nucleic acid/fatty acid -IgG2 hinge- Fe Fc-Protein/peptid e/small molecule/nucleic acid/fatty acid -IgG2 hinge IgG2 hinge-Fec-peptide/protein/smaIl molecule/nucleic acid/fatty acid Protein/peptide/small molecule/nucleic acid/fatty acid -Fc-IgG2 hinge I1gG2 hinge-peptide/proteinlsmall molecule/nucleic acid/fatty acid -Fc [00691 In one embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a IL-12 p40 subunit. In another embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a CTLA4 protein or CTLA-4 extracellular domain peptide. In a further embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a PD-1 protein or PD-1 extracellular domain peptide. In a further embodiment, the peptide/proteinl/small molecule/nucleic acid/fatty acid is a CERVIG peptide SEQ ID NO: I In a further WO 2013/112986 PCT/US2013/023404 33 embodiment the exemplified fusion proteins contain a CTLA4 protein or peptide and also contain a B7 molecule. Examples of fusion proteins containing B7 molecules include: CTLA4-IgG2 hinge- Fc CTLA4-Fec-IgG2 hinge Fc-CTLA4 -IgG2 hinge IgG2 hinge- CTLA4-Fc Fc-IgG2 hinge- CTLA4 IgG2 hinge-Fe-CTLA4 B7-IgG2 hinge- Fe B7-Fe-IgG 2 hinge Fc-B7 -IgG2 hinge IgG2 hinge- B7-Fe Fc-IgG2 hinge - B7 IgG12 hinge-Fe-B7 B17-C"I'LA4-I gG12 hinge- Fe Ec-B7-CTLA4-IgG2 hinge B7-CI'LA4-Fc-IgGI2 hinge CT LA4-B7-IgG2 hinge-Fe Fc-CTLA4-B7-IgG2 hinge WO 2013/112986 PCT/US2013/023404 34 CT LA4-B7-Fc-IgG2 hinge IgG2 hinge-B7-CTLA4-Fe Fc-IgG2 hinge-B7-CTLA4 lgG2 hinge-Fe-B7-CTLA4 IgG12 hinge-(C'LA4-37-Fe Fc-IgG2 hinge-CTLA4-37 IgG2 hinge-Fe-CTLA4-B7 [00701 In one embodiment the [B7 is 137.1, In another embodiment, the 137 is 137.2. [00711 In another embodiment, the peptide/protein/sma Il molecule/nucleic acid/fatty acid is a IL-12 p40 subunit. IgG2 hinge - IL-12 p40 IL-12 p40 - IgG2 hinge IgG2 hinge - Fe domain - IL- 12 p40 IgG2 hinge - IL-12 p 4 0 - Fe domain Fe domain - IL-12 p40 - IgG2 hinge IL-12 p40 - Fe domain - lgG2 hinge Fe domain -IgG2 hinge - IL-12 p40 IL-12 p 4 0 -IgG2 hinge - Fe domain WO 2013/112986 PCT/US2013/023404 35 [00721 In a further embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a PD-I protein or PD-i extracellular domain peptide. IgG2 hinge - PD-i PD-1 - IgG2 hinge IgG2 hinge - Fe domain - PD-I IgG2 hinge - PD-I - Fc domain Fc domain - PD-I - IgG2 hinge PD-I - Fe domain - IgG2 hinge Fe domain -IgG2 hinge - PD-i PD-i -IgG2 hinge - Fe domain [00731 In a further embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is a CERVIG peptide. IgG2 hinge - CERVIG CERVIG - IgG2 hinge IgG2 hinge - Fe domain - CERVIG IgG2 hinge - CERVIG - Fe domain Fe domain - CERVIG - IgG2 hinge CERVIG - Fe domain - IgG2 hinge WO 2013/112986 PCT/US2013/023404 36 Fc domain -IgG2 hinge - CERVIG CERVIG -lgG2 hinge - Fc domain [00741 In another embodiment, the peptide/protein/small molecule/nucleic acid/fatty acid is Von Willebrand factor. Examples of fusion proteins containing Von Willebrand Factor molecules include: IgG2 hinge - VWE VWF - IgG2 hinge IgG2 hinge - Fc domain --- VWF IgG2 hinge - VWF - Fe domain Fe domain - VWF - IgG2 hinge VWF - Fe domain - IgG2 hinge Fe domain -IgG2 hinge - VWF VWF -1gG2 hinge - Fdomain [00751 In another embodiment, the the peptide/protein/small molecule/nucleic acid/fatty acid is an Ml-IC molecule. In this embodiment, MIC molecules are multimerized to form multimers such as MHC tetramers. Examples of fusion proteins containing MHC molecules include: fluorescent tag - M-IC - antigen - IgG2 hinge fluorescent tag - IgG2 hinge - MHC - antigen MHC - antigen - IgG2 hinge IgG2 hinge - MHC - antigen WO 2013/112986 PCT/US2013/023404 37 IgG2hinge - M-HC - antigen - biotinylated tetramer unit [00761 In another embodiment, small peptides are fused to the IgG2 hinge, with or without an Fc, in order to form multiners that increase the affinity and avidity of binding of the small peptides to their target because of multivalent binding. Small peptides often have very short clinical half lives which can be improved through the addition of Fe. In one embodiment of this approach, a 21 mer peptide with the sequence LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) is a peptide antagonist of CXCR4 (Zhou et al Biochemistry 2000 39(13) pp 3782). The peptide has been demonstrated to prevent CXCR4 signaling and CXCR4-mediated entry of HIV into CD4 cells. By adding the IgG2 hinge, the peptide will multimerize and will bind with greater affinity and avidity. By adding the Fc, the half life of the multimerized compound is increased. Therapeutic uses include treatment of HIV disease and immune disorders. Examples of fusion proteins containing the LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) peptide include: LGASWHRP[)KC( I CGYQKRPLP (SEQ I) NO: 2) - IgG2 hinge - IgG4 Fe LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) - IgG4 Fe - - IgG2 hinge IgG4 Fe - LGASWHR-PDKCCLGYQKRPLP (SEQ ID NO: 2) - IgG2 hinge IgG4 Fe - IgG2 hinge - LGASW[JRPDK-CCLGYQKRPLP (SEQ ID NO: 2) IgG2 hinge - IgG 4 Fe - LGASWI-RPDK-CCLGYQKRPLP (SEQ ID NO: 2) IgG2 hinge - LGASWHRPDKCCL.GYQKRPLP (SEQ I) NO: 2) - IgG4 Fe LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) - IgG2 hinge - IgG2 CH2 IgG2 CH3 LGASWHRP[)KCCLGYQIKRPLP (SEQ I) NO: 2) - IgG2 CH2 - IgG2 CH3- IgG2 hinge IgG2 CH2 - IgG2 CH3- LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) - IgG2 hinge WO 2013/112986 PCT/US2013/023404 38 IgG2 CH2 - IgG2 CH3- IgG2 hinge - LGASWHRPDKCCLGYQKRPLP (SEQ ID N0:2) IgG2 hinge - IlgG2 (112 - IgG2 CH3 - LGASW-iRPDIKCCLGYQKRPLP (SEQ ID NO: 2) IgG2 hinge - LGASWHRPDKCCLGYQKRPLP (SEQ ID NO: 2) - IgG2 CH2 IgG2 CH3 [00771 In another similar embodiment, Nakamura et al (Journal of Biotechnology 2005;116 (3);21 1-219) have identified a peptide which miics EGF binding to EGFR and inhibits mitogenesis and EGFR signaling. This small peptide can be linked to IgG2 hinge and optionally to Fe as described above. This will be useful as an anticancer agent. [00781 In another embodiment, Maruta et al (Cancer Gene Ther. 2002. 9(6);543-52) have identified the small peptide NQLPLAT (SEQ I) NO: 3) that binds FGF receptor-expressing cells. MQLPLAT (SEQ ID NO: 3) binds to and accumulates in cancer cells relative to normal. They are not characterized as antagonist or agonist. This small peptide can be linked to IgG2 hinge and optionally to Fe as described above. This will be useful as an anticancer agent and could be used for specific delivery of a toxic compound to target cells by a compound. Examples of fusion proteins containing the MQLPLAT (SEQ ID NO: 3) peptide include: IgG2 hinge - IgG4 Fc - FGF binding peptide-peptide toxin IgG2 hinge - IgG4 Fc - FGF binding peptide - small molecule toxin IgG4 Fe - FGF binding peptide-peptide toxin - IgG2 hinge IgG4 Fe - FGF binding peptide - small molecule toxin - IgG2 hinge FGF binding peptide - peptide toxin - 1gG2 hinge - IgG4 Fe FGF binding peptide - small molecule toxin - IgG2 hinge - IgG4 Fe WO 2013/112986 PCT/US2013/023404 39 IgG2 hinge - IgG2 CH2 - IgG2 CH3 - FGF binding peptide-peptide toxin IgG2 hinge - IgG2 CH2 - IgG2 CH3 - FGF binding peptide - small molecule toxin IgG2 CH2 - IgG2 CH3 - FGF binding peptide-peptide toxin - IgG2 hinge IgG2 CH2 - IgG2 CH3 - FGF binding peptide - small molecule toxin - IgG2 hinge FOF binding peptide - peptide toxin - IgG2 hinge - IgG2 (12 - IgG2 C1-1 FOF binding peptide - small molecule toxin - lgG2 hinge - gG2 C12 - lgG2 C113 [00791 In another embodiment, Ruff et al (FEBS letters 1987: 211 (1);i 7-22) have identified an octa peptide ASTTTNYT (SEQ ID NO: 4) that blocks CD4 receptor binding by HIV. This small peptide can be linked to IgG2 hinge and optionally to Fe as described above. This will be useful as an antiviral. 100801 In another embodiment, Noberini et al (PlosOne. 2011. 6(12) e2861 1) have identified a 15 mer peptide called TNYL-RAW that targets the EphB4 receptor and inhibits angiogenesis through disruption of EphB4- ephrin -B12 interactions. This small peptide can be linked to IgG2 hinge and optionally to Fe as described above. This will be useful as an oncolytic agent.In another embodiment, Holt et al,, (Trends in Biotechnology. 21(11)(2003) and others have described the ability to generate binding domains using the variable portion of the heavy chain (V-Il) linked to the variable portion of the light chain (VL), forming an epitope binding site. The variable light chain can be linked to the variable heavy chain using one of many possible linker regions and may contain the CH1I region of VH or VL. The VH or VL or both can be linked to the multimerizing IgG2 hinge domain and optionally to Fe as described above. Alternatively the VH or VL miay not be directly fused as a chimeric protein but may instead be co-expressed in the same cell. In a further embodiment the co-expressed VL is also linked to an IgG2 hinge region. In a further embodiment, the VH is not linked to a CH I so that the co-expressed peptides comprise VH -- IgG2 hinge and VL, WO 2013/112986 PCT/US2013/023404 40 [00811 Holliger and Hudson (Nat Biotechnol. 2005 Sep;23(9):1126-36) summarize a range of small recombinant fragments that are variants of a monoclonal antibody, including monovalent antibody fragments such as Fab, scFv and engineered variants; diabodies, triabodies, minibodies and single-domain antibodies. These fragments retain the targeting specificity of whole monoclonal antibodies but can be produced more economically and possess other unique and superior properties for a range of diagnostic and therapeutic applications. Each of these can similarly be multimerized by inclusion of the IgG2 hinge domain as disclosed above. 100821 Wurch et al (Current Pharmaceutical Biotechnology, 2008, 9, 502-509) summarize about fifty different antibody-derived and non-Ig scaffolds that have been discovered and documented, including scaffolds of the IgG superfamily, loop-containing or highly structured protein scaffolds that provide a rigid core structure suitable for grafting loops, oligomeric protein scaffolds allowing the incorporation of variable loops in a favorable 3D configuration, and carrier proteins that display a single binding interface. Each of these can similarly be multimerized by inclusion of the IgG2 hinge domain as disclosed above. [00831 In another embodiment, Wu et al 2001 Protein Engineering Vol 14 no 12 pp 1025 1033 have demonstrated the ability to generate single chain antibodies by placing the V. domain and a linker on the N-terniinus of the VHi domain of a human Ig G1 monoclonal antibody (V- linker - VH7- CHI - CH2 -- CH3). 100841 In another embodiment, Deffar et al (African Journal of Biotechnology Vol. 8 (12), pp. 2645-2652, 17 June, 2009) have demonstrated that camels and llamas possess fully functional antibodies that lack light chains. These heavy-chain antibodies contain a single variable domain (VHH) and two constant domains (cCH2 and cC13) in which the cloned and isolated VH-I domain is a stable polypeptide harboring the full antigen-binding capacity of the original heavy-chain antibody. There is no cCH1 or light chain. The authors call these heavy chain antibodies nanobodies. This heavy chain antibody can be linked to the multimerizing IgG2 hinge domain to form a multimerizing single chain antibody as follows: WO 2013/112986 PCT/US2013/023404 41 hIgG2 hinge - VHH --- cHinge - cCH2 - cCH3 hIgG2 hinge - VHH --- cCH2 - cCH3 VHI - hIgG2 hinge - cHinge cC[H2 - cCH3 VHI - hIgG2 hinge - cCH2 - cCH3 VIH - cringe - cCH2 -- cC3 - higG2 hinge VIH -cCI2 - cCH3 - hIgG2 hinge [00851 The immunoglobulin Fe domain is any Fe domain or fragment that binds with lower affinity to Fc gamma receptors than does native IgG1, but which also retains other beneficial features including, the retained ability to bind to Protein A or Protein G affinity columns or preferred binding to the neonatal FeN receptor. The Fe domains may be naturally occurring, may be comprised of naturally occurring components in non-natural combination, or may be comprised of naturally occurring and non-natural components. Examples of such Fe domains include: IgG2 hinge-IgG2 CH2-IgG2 CH3 IgG2 hinge-IgG3 CH2-IgG3 CH3 IgG2 hinge-IgG3 CH2-IgG2 CH3 IgG2 hinge-IgG2 CH2-IgG3 CH3 IgG4 hinge-IgG4 CH2-IgG4 CH3 IgG2 CH2- IgG2 CH3 IgG4 CH2-IgG4 CH3 WO 2013/112986 PCT/US2013/023404 42 IgG4 CH2-IgG2 CH-3 IgG2 CH2-IgG4 CH3 Hemiglycosylated IgGi hinge-IgGi CH2-IgG1 CH3 Hemiglycosylated IgG1 CH2-IgG1 CH3 Hemiglycosylated IgG3 hinge-IgG 3 C[12-IgG3 CH3 Hemiglycosylated lIG3 CH-2-IgG3 CI 3 IgG I hinge- lgG2 CH2-IgG3 C113 IgG I hinge-IgG1 (12 N297A-lgGI CH3 IgGI hinge-IgGI (12 D265A-lgGi C13 [00861 Alternatively, the immunoglobulin Fe domain is an Fc domain or fragment that has been engineered to contain an antigen binding site. These fusion proteins are useftnl in the treatment of inflammatory disease, autoimmune disease and cancer. For example, an Fc engineered to express a Her2/neu antigen binding site will be useful in the treatment of breast cancer. Examples of fusion proteins comprising such Fe domains are: Engineered Fc-IgG2 hinge IgG2 hinge-Engineered Fe IgG2 hinge-CH2-CH3-Engineered Fe Pharmaceutical Compositions [00871 Administration of the fusion protein compositions described herein will be via any common or uncommon route, orally, parenterally, or topically. Exemplary routes include, but WO 2013/112986 PCT/US2013/023404 43 are not limited to oral, nasal, buccal, rectal, vaginal, ophthalmic, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intratumoral, spinal, intrathecal, intra-articular, intra-arterial, sub-arachnoid, sublingual, oral mucosal, bronchial, lymphatic, intra-uterine, subcutaneous,. intratumor, integrated on an implantable device such as a suture or in an implantable device such as an implantable polymer, intradural, intracortical, or dermal. Such compositions would normally be administered as pharmaceutically acceptable compositions as described herein. In a preferred embodiment the isolated fusion protein is administered intravenously or subcutaneously. 100881 The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, hydrogels, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. [00891 The fusion protein compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. 100901 Sterile injectable solutions are prepared by incorporating the fusion proteins in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, fol lowed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the WO 2013/112986 PCT/US2013/023404 44 basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. [00911 Further, one embodiment is a fusion protein composition suitable for oral administration and is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable or edible and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a fusion protein preparation contained therein, its use in an orally administrable a fusion protein composition for use in practicing the methods of the present invention is appropriate. Examples of carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof. The term "oral administration" as used herein includes oral, buccal, enteral or intragastric administration. [00921 In one embodiment, the fusion protein composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, microencapsulation, absorption and the like. Such procedures are routine for those skilled in the art. 100931 In a specific embodiment, the fusion protein composition in powder form is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can be carried out in any convenient manner such as grinding. Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity through, i.e., denaturation in the stomach. Examples of stabilizers for use in an orally administrable composition include buffers, antagonists to the secretion of stomach acids, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, etc., proteolytic enzyme inhibitors, and the like. More WO 2013/112986 PCT/US2013/023404 45 preferably, for an orally administered composition, the stabilizer can also include antagonists to the secretion of stomach acids. [00941 Further, the fusion protein composition for oral administration which is combined with a semi-solid or solid carrier can be further formulated into hard or soft shell gelatin capsules, tablets, or pills. More preferably, gelatin capsules, tablets, or pills are enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, i.e., U.S. Pat. No. 5,629,001. Upon reaching the small intestines, the basic p-i therein dissolves the coating and permits the composition to be released to interact with intestinal cells, e.g., Peyer's patch M cells. 100951 In another embodiment, the fusion protein composition in powder form is combined or mixed thoroughly with materials that create a nanoparticle encapsulating the fusion protein or to which the fusion protein is attached. Each nanoparticle will have a size of less than or equal to 100 microns. The nanoparticle may have mucoadhesive properties that allow for gastrointestinal absorption of a fusion protein that would otherwise not be orally bioavailable. [00961 In another embodiment, a powdered composition is combined with a liquid carrier such as, i.e., water or a saline solution, with or without a stabilizing agent. [00971 A specific fusion protein formulation that may be used is a solution of fusion protein in a hypotonic phosphate based buffer that is free of potassium where the composition of the buffer is as follows: 6 m M sodium phosphate monobasic monohydrate, 9 mM sodium phosphate dibasic heptahydrate, 50 nM sodium chloride, pH 7.0.-/- 0. 1. The concentration of fusion protein in a hypotonic buffer may range from 10 microgram/ml to 100 milligram/ml. This formulation may be administered via any route of administration, for example, but not limited to intravenous administration. [00981 Further, a fusion protein composition for topical administration which is combined with a semi-solid carrier can be further formulated into a cream or gel ointment. A preferred WO 2013/112986 PCT/US2013/023404 46 carrier for the formation of a gel ointment is a gel polymer. Preferred polymers that are used to manufacture a gel composition of the present invention include, but are not limited to carbopol, carboxymethyl-cellulose, and pluronic polymers. Specifically, a powdered fusion protein is combined with an aqueous gel containing an polymerization agent such as Carbopol 980 at strengths between 0.5% and 5% wi/volume for application to the skin for treatment of disease on or beneath the skin. The term "topical administration" as used herein includes application to a dermal, epidermal, subcutaneous or mucosal surface. 100991 Further, a fusion protein composition can be formulated into a polymer for subcutaneous or subdermal implantation. A preferred formulation for the implantable drug infused polymer is an agent Generally Regarded as Safe and may include, for example, cross linked dextran (Samantha Hart, Master of Science Thesis, "Elution of Antibiotics from a Novel Cross-Linked Dextran Gel: Quantification" Virginia Polytechnic Institute and State University, June 8, 2009) dextran-tyramine (Jin, et al. (2010) Tissue Eng. Part A. 16(8):2429-40), dextran-polyethylene glycol (Jukes, et al. (2010) Tissue Eng. Part A., 16(2):565-73), or dextran-gluteraldehyde (Brondsted, et al, (1998) J. Controlled Release, 53:7-13). One skilled in the art will know that many similar polymers and hydrogels can be formed incorporating the fusion protein fixed within the polymer or hydrogel and controlling the pore size to the desired diameter. 1001001 Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The formulations are easily administered in a variety of dosage forms such as ingestible solutions, drug release capsules and the like. Some variation in dosage can occur depending on the condition of the subject being treated. The person responsible for administration can, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations meet sterility, general safety and purity standards as required by FDA Center for Biologics Evaluation and Research standards.
WO 2013/112986 PCT/US2013/023404 47 [001011 The route of administration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, transdermal, subderinal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal. intraperitoneal, intratumoral, perfusion, lavage, direct injection, and oral administration. The term "parenteral administration" as used herein includes any form of administration in which the compound is absorbed into the subject without involving absorption via the intestines. Exemplary parenteral administrations that are used in the present invention include, but are not limited to intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, nasal or intraarticular administration. Therapeutic Applications of Fusion Proteins [00102] Based on rational design the fusion proteins of the present invention will serve as important biopharmaceuticals for treating autoimmune diseases and for modulating immune function in a variety of other contexts such as bioimnunotherapy for cancer and inflammatory diseases. Conditions included among those that may be effectively treated by the compounds that are the subject of this invention include an inflammatory disease with an imbalance in cytokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic relapsing autoimmune, inflammatory, or infectious disease or process. [001031 The general approach to therapy using the isolated fusion proteins described herein is to administer to a subject having a disease or condition, a therapeutically effective amount of the isolated imnnunologically active fusion protein to effect a treatment. In some embodiments, diseases or conditions may be broadly categorized as inflammatory diseases with an imbalance in cvtokine networks, an autoimmune disorder mediated by pathogenic autoantibodies or WO 2013/112986 PCT/US2013/023404 48 [001041 The term "treating" and "treatment" as used herein refers to administering to a subject a therapeutically effective amount of a fusion protein of the present invention so that the subject has an improvement in a disease or condition, or a symptom of the disease or condition. The improvement is any improvement or remediation of the disease or condition, or symptom of the disease or condition. The improvement is an observable or measurable improvement, or may be an improvement in the general feeling of well-being of the subject. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not be a complete cure for the disease. Specifically, improvements in subjects may include one or more of: decreased inflammation; decreased inflammatory laboratory markers such as C-reactive protein; decreased autoimmunity as evidenced by one or more of: improvements in autoinmune markers such as autoantibodies or in platelet count, white cell count, or red cell count, decreased rash or purpura, decrease in weakness, numbness, or tingling, increased glucose levels in patients with hyperglycemia, decreased joint pain, inflammation, swelling, or degradation, decrease in cramping and diarrhea frequency and volume, decreased angina, decreased tissue inflammation, or decrease in seizure frequency; decreases in cancer tumor burden, increased time to tumor progression, decreased cancer pain, increased survival or improvements in the quality of life; or delay of progression or improvement of osteoporosis. [00105] The term "therapeutically effective amount" as used herein refers to an amount that results in an improvement or remediation of the symptoms of the disease or condition. [001061 As used herein, "prophylaxis" can mean complete prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the severity of subsequently developed disease symptoms. 1001071 The term "subject" as used herein, is taken to mean any mammalian subject to which fusion proteins of the present invention are administered according to the methods described herein. In a specific embodiment, the methods of the present disclosure are employed to treat a human subject. The methods of the present disclosure may also be WO 2013/112986 PCT/US2013/023404 49 employed to treat non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, bovines, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish and reptiles to produce species specific or chimeric fusion protein molecules. [001081 In particular, the fusion proteins of the present invention may be used to treat conditions including but not limited to congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other conditions of the myocardium, systemic lupus erythematosus, diabetes, spondylopathies, synovial fibroblasts, and bone marrow stroma; bone loss; Paget's disease, osteoclastoma; multiple myeloma; breast cancer; disuse osteopenia; malnutrition, periodontal disease, Gaucher's disease, Langerhans' cell histiocytosis, spinal cord injury, acute septic arthritis, osteomalacia, Cushing's syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, periodontal reconstruction, and bone fractures; sarcoidosis; osteolytic bone cancers, lung cancer, kidney cancer and rectal cancer; bone metastasis. bone pain management, and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; transplantation rejection, viral infections,. hematologic neoplasias and neoplastic-like conditions for example, Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lyrnphoma, marginal zone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells, including B-cell acute lymphoblastic leukemia/l lymphoma, and T-cell acute lymphoblastic leukemia/lymphoma, thymoma, tumors of the mature T and NK cells, including peripheral T cell leukemias, adult T-cell leukemia/I-cell lymphomas and large granular lymphocytic leukemia, Langerhans cell histiocytosis, myeloid neoplasias such as acute myelogenous leukemias, including AML with maturation, AML without differentiation, acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemias, myelodysplastic syndromes. and chronic myieloproliferative disorders, including chronic myielogenous leukemia, tumors of the central nervous system, e.g., brain tumors (glioma, WO 2013/112986 PCT/US2013/023404 50 neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancers, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma)) or other cancer. [001091 "Cancer" herein refers to or describes the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but are not limited to carcinoma, lymphoma, blastoma, sarcoma (including liposarcoma, osteogenic sarcoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, iymphangioendotheiiosa rcomna, leiornyosarcoma., rhabdomyosarcoma, fibrosarcoma, myxosarcoma, chondrosarcoma), neuroendocrine tumors, mesothelioma, chordoma, synovioma, schwanorna, meningioma, adenocarcinoma., melanoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, small cell lung carcinoma, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, testicular cancer, esophageal cancer, tumors of the biliary tract, Ewing's tumor, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, testicular tumor, lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniophairngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendrogliomna, meningioma, melanoma, neuroblastoma, retinoblastoma, WO 2013/112986 PCT/US2013/023404 51 leukemia, lymphoma, multiple myeloma, Waldenstrom's macroglobulinemia, rnyelodysplastic disease, heavy chain disease, neuroendocrine tumors, Schwanoma, and other carcinomas, as well as head and neck cancer. 1001101 The fusion proteins of the present invention may be used to treat autoimmune diseases. The term "autoimmune disease" as used herein refers to a varied group of more than 80 diseases and conditions. In all of these diseases and conditions, the underlying problem is that the body's immune system attacks the body itself. Autoimmune diseases affect all major body systems including connective tissue, nerves, muscles, the endocrine system, skin, blood, and the respiratory and gastrointestinal systems. Autoimmune diseases include, for example, systemic lupus erythematosu s, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and type I diabetes. [00111] The disease or condition treatable using the compositions and methods of the present invention may be a hematoimnunological process, including but not limited to Idiopathic Thrombocytopenic Purpura, alloimmune/autoimmune thrombocytopenia, Acquired immune thrombocytopenia, Autoimmune neutropenia, Autoimmune hemolytic anemia, Parvovirus 1319-associated red cell aplasia, Acquired antifactor VIll autoimmunity, acquired von Willebrand disease, Multiple MyCloma and Monoclonal Gammopathy of Unknown Significance, Sepsis, Aplastic anemia, pure red cell aplasia, Diamond-Blackfan anemia, hemolytic disease of the newborn, Immune-mediated neutropenia, refractoriness to platelet transfiusion, neonatal, post-transfusion purpura, hemolytic urernmic syndrome, systemic Vasculitis, Thrombotic thrombocytopenic purpura, or Evan's syndrome. [001121 The disease or condition may also be a neuroimmunological process, including but not limited to Guillain-Barre syndrome, Chronic Inflammatory Demyelinating Polyradiculoneuropathy, Paraproteinemic IgM demyelinating Polyneuropathy, Lambert Eaton myasthenic syndrome, Myasthenia gravis, Multifocal Motor Neuropathy, Lower Motor Neuron Syndrome associated with anti-/GM], Demyelination, Multiple Sclerosis and optic neuritis, Stiff Man Syndrome, Paraneoplastic cerebellar degeneration with anti-Yo antibodies, WO 2013/112986 PCT/US2013/023404 52 paraneoplastic encephalomyelitis, sensory neuropathy with anti-Hu antibodies, epilepsy, Encephalitis, Myelitis, Myelopathy especially associated with Human T-cell lymphotropic virus- 1, Autoimmune Diabetic Neuropathy, Alzheimer's disease, Parkinson's disease, Huntingdon's disease, or Acute Idiopathic Dysautonomic Neuropathy. [001131 The disease or condition may also be a Rheumatic disease process, including but not limited to Kawasaki's disease, Rheumatoid arthritis, Felty's syndrome, ANCA-positive Vasculitis, Spontaneous Polymyositis, Dermatomyositis, Antiphospholipid syndromes, Recurrent spontaneous abortions, Systemic Lupus Erythematosus, Juvenile idiopathic arthritis, Raynaud's, CREST syndrome, or Uveitis. 1001141 The disease or condition may also be a dermatoimmunological disease process, including but not limited to Toxic Epidermal Necrolysis, Gangrene, Granuloma, Autoimmune skin blistering diseases including Pemphigus vulgaris, Bullous Pemphigoid, Pemphigus foliaceus, Vitiligo, Streptococcal toxic shock syndrome, Scleroderma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, or Atopic dermatitis (especially steroid dependent). [001151 The disease or condition may also be a musculoskeletal immunological disease process, including but not limited to Inclusion Body Myositis, Necrotizing fasciitis, Inflammatory [Mlyopathies, Myositis, Anti-Decorin (BJ antigen) Myopathy, Paraneoplastic Necrotic Myopathy, X-linked Vacuolated Myopathy, Penacilla mine-induced PolymyosItis, Atherosclerosis, Coronary Artery Disease, or Cardiomyopathy. 1001161 The disease or condition may also be a gastrointestinal immunological disease process, including but not limited to 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, or sclerosing cholangitis.
WO 2013/112986 PCT/US2013/023404 53 [001171 The disease or condition may also be Graft Versus Host Disease, Antibody mediated rejection of the graft, Post-bone marrow transplant rejection, Post-infectious disease inflammation, Lymphoma, Leukemia, Neoplasia, Asthma, Type 1 Diabetes mellitus with anti-beta cell antibodies, Sjogren's syndrome., Mixed Connective Tissue Disease, Addison's disease, Vogt-Koyan agi--I arada Syndrome, Membranoproliferative glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolyarterits, Churg-Strauss syndrome, Polyarteritis nodosa or Multisystem organ failure. 1001181 The fusion proteins disclosed herein may also be readily applied to alter immune system responses in a variety of contexts to affect specific changes in immune response profiles. Altering or modulating an immune response in a subject refers to increasing, decreasing or changing the ratio or components of an immune response. For example, cytokine production or secretion levels may be increased or decreased as desired by targeting the appropriate combination of cytokine receptors with a fusion protein designed to interact with those receptors. The immune response may also be an effector function of an immune cell expressing a receptor of the multimerized protein, peptide or small molecule, including increased or decreased phagocytic potential of monocyte macrophage derived cells, increased or decreased osteoclast function, increased or decreased antigen presentation by antigen presenting cells (e.g. DCs), increased or decreased NK cell function, increased or decreased B-cell function, as compared to an immune response which is not modulated by a fusion protein disclosed herein. The fusion proteins described herein may be used to modulate expression of co-stimulatory molecules from an immune cell, including a dendritic cell, a macrophage, an osteoclast, a monocyte, or an NK cell or to inhibit in these same immune cells' differentiation, maturation, or of decreasing cytokine secretion, including interleukin-12 (IL- 12), or interleukin-23 (IL 23) or of increasing cytokine secretion, including interleukin-10 (IL- 10), or interleukin-6 (IL-6). A skilled artisan may also validate the efficacy of a fusion protein by exposing an WO 2013/112986 PCT/US2013/023404 54 immune cell to the fusion protein and measuring modulation of the immune cell function, wherein the immune cell is a dendritic cell, a macrophage, an osteoclast, an NK cell, or a monocyte. In one embodiment the immune cell is exposed to the fusion protein in vitro and further comprising the step of determining an amount of a cell surface receptor or of a cytokine production, wherein a change in the amount of the cell surface receptor or the cytokine production indicates a modulation of the immune cell function. In another embodiment the immune cell is exposed to the fusion protein in vivo in a model animal for an autoimmune disease further comprising a step of assessing a degree of improvement in the autoirnmnune disease. EXAMPLES: Exam~pj_1_ - Construct Design _of _imunologi calv_ Active _Ig2 hinge ing EI IP 9j f -n binge -2 in e multimers A. CTLA-4 [001191 The 2-hinge CTLA fusion construct is engineered using PCR. A cDNA clone containing the CTLA4 extracellular domain is obtained either from a commercial vendor of which there are many (Origen catalog #SC303605 encoding Homo sapiens cytotoxic T lymphocyte-associated protein 4 (CTLA4), transcript variant I NM 005214.3) or by synthesizing the cDNA at a commercial vendor of which there are many (DNA2.0 Menlo Park California). Alternatively the cDNA can be obtained by PCR from a cDNA library of which there are many commercial vendors (Invitrogen cat# 10425-0 15 Supercrt HNua SlerecDNALirary), Primers complementary to the human IgG2 hinge sequence and additionally containing DNA sequences complementary to the CTLA cDNA domain are used by PCR to generate a fusion PCR product encoding the human IgG2 hinge domain and the sequence encoding the CTLA4 extracellular domain. The PCR fragment is then cloned into one of many commercial available expression vectors (pcDNAT33-TOPO@ vector, Invitrogen). Alternatively restriction enzyme recognition sites can be added to the PCR WO 2013/112986 PCT/US2013/023404 55 primers to facilitate further manipulation and subeloning of the insert. A stop codon is added before the restriction site of the C terminal primer to prevent read through of flanking sequences for this construct. Generating the DNA fragment encoding the IgG2 hinge fusion by PCR allows for placing the 2-hinge either N-terminal or C-terminal to the fusion partner or by using a two-step PCR method using overlapping primers to position the 2-hinge internally in the fusion partner or between two separate fusion partners. It also allows for incorporating leader peptides facilitating the secretion of fusion proteins by incorporating DNA sequences encoding leader peptides in the 5-prime PCR primers. 1001201 The above example uses DNA sequences encoding the extracellular domain of the CTLA4. Alternatively we will use the complete CTLA4 sequence or we will use sequences encoding other proteins where the receptor binding and or biological function can be improved by multimerization. As mentioned in the previous example we can use the complete coding sequence, the sequence encoding extracellular domains or smaller sequences encoding receptor binding peptide domains allowing for generating smaller multivalent binding fusion proteins. [001211 The 2-hinge CTLA construct is similarly made and contains the IgG2 hinge and the extracellular domain of the CTLA4 as described above but also contained two epitope tags added to the C terminus of the construct. These epitope tags are used for identification or purification of the protein. In this second construct the two epitope tags, V5 and His tag, are present in frame prior to the stop codon but can also be present at the N-terminal. The purification tags can be one or several of the many protein tags used for purification and identification including the GST, myc, His and V5 tags. B. PD-i [001221 The 2-hinge PD-I fusion construct is engineered using PCR. A cDNA clone containing the PD-1 extracellular domain is obtained either from a commercial vendor or by synthesizing the cDNA at a commercial vendor of which there are many (DNA2.0 Menlo WO 2013/112986 PCT/US2013/023404 56 Park California). Alternatively the cDNA can be obtained by PCR from a cDNA library of which there are many commercial vendors. Primers complementary to the human IgG2 hinge sequence and additionally containing DNA sequences complementary to the PD-1 cDNA domain are used by PCR to generate a fusion PCR product encoding the human IgG2 hinge domain and the sequence encoding the PD-1 extracellular domain. The PCR fragment is then cloned into one of many commercially available expression vectors (cDNA 3 TOPO@K vector, Invitrogen). Alternatively restriction enzyme recognition sites can be added to the PCR primers to facilitate further manipulation and subeloning of the insert. A stop codon is added before the restriction site of the C terminal primer to prevent read through of flanking sequences for this construct. Generating the DNA fragment encoding the igG2 hinge fusion by PCR allows for placing the 2-hinge either N-terrninal or C-terminal to the fusion partner or by using a two step PCR method using overlapping primers to position the 2-hinge internally in the fusion partner or between two separate fusion partners. It also allows for incorporating leader peptides facilitating the secretion of fusion proteins by incorporating DNA sequences encoding leader peptides in the 5-prime PCR primers. [001231 The above example uses DNA sequences encoding the extracellular domain of the PD-1. Alternatively we will use the complete PD-i sequence or we will use sequences encoding other proteins where the receptor binding and or biological function can be improved by multimerization. As mentioned in the previous example we can use the complete coding sequence, the sequence encoding extracellular domains or smaller sequences encoding receptor binding peptide domains allowing for generating smaller multivalent binding fusion proteins. 1001241 The 2-hinge PD-1 construct is similarly made and contains the IgG2 hinge and the extracellular domain of the PD-1 as described above but also contained two epitope tags added to the C terminus of the construct. These epitope tags are used for identification or purification of the protein. In this second construct the two epitope tags, V5 and His tag, are present in frame prior to the stop codon but can also be present at the N-terminal. The WO 2013/112986 PCT/US2013/023404 57 purification tags can be one or several of the many protein tags used for purification and identification including the GST, myc, His and V5 tags. C. CERVIG PEPTIDES [001251 The 2-hinge CERVIG fusion construct is engineered using PCR. A cDNA clone containing the CERVIG is obtained by synthesizing the cDNA at a commercial vendor of which there are many (DNA2.0 Menlo Park California). Alternatively the cDNA can be obtained by PCR. Primers complementary to the human IgG2 hinge sequence and additionally containing DNA sequences complementary to the CERVIG DNA domain are used by PCR to generate a fusion PCR product encoding the human IgG2 hinge domain. The PCR fragment is then cloned into one of many commercially available expression vectors (pcDNA* 3O @ eco, Invitrogen). Alternatively restriction enzye recognition sie can be added to the PCR primers to facilitate further manipulation and subeloning of the insert. A stop codon is added before the restriction site of the C terminal primer to prevent read through of flanking sequences for this construct. Generating the DNA fragment encoding the IgG2 hinge fusion by PCR allows for placing the 2-hinge either N-terminal or C-terminal to the fusion partner or by using a two step PCR method using overlapping primers to position the 2-hinge internally in the fusion partner or between two separate fusion partners. It also allows for incorporating leader peptides facilitating the secretion of fusion proteins by incorporating DNA sequences encoding leader peptides in the 5-prime PCR primers. These fusion proteins produce high molecule weight multimers compared to constructs with no IgG2. hinge multimers. These multimers were visualized by non-reducing SDS-PAGE. D. FCs ENGINEERED TO HAVE AN ANTIGEN BINDING SITE [001261 The 2-hinge Fc/Her2neu fusion construct is engineered using PCR. A cDNA clone containing the Fe domain engineered to have a Her2/neu binding site (SEQ ID NO: 18) is obtained by synthesizing the cDNA at a commercial vendor of which there are many WO 2013/112986 PCT/US2013/023404 58 (DNA2.O Menlo Park California). Alternatively the cDNA can be obtained by PCR. Primers complementary to the hurnan IgG2 hinge sequence and additionally containing DN A sequences complementary to the Fc/Her2neu domain are used by PCR to generate a fusion PCR product encoding the human IgG2 hinge domain. The PCR fragment is then cloned into one of many commercially available expression vectors (pcDNATM3.3~TO ector, Invitrogen). Alternatively restriction enzyme recognition sites can be added to the PCR primers to facilitate further manipulation and subeloning of the insert. A stop codon is added before the restriction site of the C terminal primer to prevent read through of flanking sequences for this construct. Generating the DNA fragment encoding the IgG2 hinge fusion by PCR allows for placing the 2-hinge either N-terminal or C-terminal to the fusion partner or by using a two step PCR method using overlapping primers to position the 2-hinge internally in the fusion partner or between two separate fusion partners. It also allows for incorporating leader peptides facilitating the secretion of fusion proteins by incorporating DNA sequences encoding leader peptides in the 5-prime PCR primers. These fusion proteins produce high molecule weight multimers compared to constructs with no IgG2 hinge multimers. These multimers were visualized by non-reducing SDS-PAGE. Example 2 - Expression of Recombinant Proteins [001271 Numerous expression systems exist that are suitable for use in producing the compositions discussed above. Eukaryote-based systems in particular can be employed to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Many such systems are commercially and widely available. [001281 In a particular embodiment, the 2-hinge multimers described herein are produced using Chinese Hanster Ovary (CHO) cells which are well established for the recombinant production of immunoglobulin proteins following standardized protocols. Alternatively, for example, transgenic animals may be utilized to produce the human 2-hinge multimers described herein, generally by expression into the milk of the animal using well established transgenic animal techniques. Lonberg N. Human antibodies from transgenic animals. Nat WO 2013/112986 PCT/US2013/023404 59 Biotechnol. 2005 Sep;23(9):1117-25; Kiprivanov SM, Le Gall F. Generation and production of engineered antibodies. Mol Biotechnol. 2004 Jan;26(1):39-60; See also Ko K, Koprowski H. Plant biopharming of monoclonal antibodies. Virus Res. 2005 Jul;111(l):93-100. 1001291 The insect cell/baculovirus system can produce a high level of protein expression of a heterologous nucleic acid segment, such as described in U.S. Patent No. 5,871,986, 4,879,236, both incorporated herein by reference in their entirety, and which can be bought, for example, under the name MAXBAC@ 2.0 from INVITROGEN@ and BACPACK T M BAJCILOVIRU S EXPRESSION SYST EM FROM CLONTECH®. [001301 Other examples of expression systems include STRATAGENE@'s COMPLETE CONTROLIM Inducible Mammalian Expression System, which utilizes a synthetic ecdysone-inducible receptor. Another example of an inducible expression system is available from INVITROGEN®, which carries the T-REX
T
" (tetracycline-regulated expression) System, an inducible mammalian expression system that uses the full-length CMV promoter. INVITROGIEN@) also provides a yeast expression system called the Pichia methanolica Expression System, which is designed for high-level production of recombinant proteins in the methylotrophic yeast Pichia methanolica. One of skill in the art would know how to express vectors such as an expression construct described herein, to produce its encoded nucleic acid sequence or its cognate polypeptide, protein, or peptide. See, generally, Recombinant Gene Expression Protocols By Rocky S. Tuan, Humana Press (1997), ISBN 0896033333; Advanced Technologies for Biopharrnaceutical Processing By Roshni L. Dutton, Jeno M. Scharer, Blackwell Publishing (2007), ISBN 0813805171; Recombinant Protein Production With Prokaryotic and Eukaryotic Cells By Otto-Wilhelm Merten, Contributor European Federation of Biotechnology, Section on Microbial Physiology Staff, Springer (2001), ISBN 0792371372.
WO 2013/112986 PCT/US2013/023404 60 Example 3 - Expression and Purification of Immunologically Active 2-hinge fusion proteins [001311 Nucleic acid constructs described in Examples I and 2 are transfected into cell lines that do not naturally express the 2hinge recombinant chimerics. The encoded polypeptides are expressed as secreted proteins due to their secretory leader sequences, which generally are removed by endogenous proteases during transport out of the cells or may be subsequently cleaved and removed by techniques well known in the art. These secreted immunologically active biomimetics are purified using Protein A or protein G affinity chromatographic columns in case they are engineered to contain an Fe domain. Protein A and protein G purification is well known in the art and multiple commercial vendors exist. In case the IgG2 multimeric fusion protein contains alternative protein tags like His, myc or V5 tags these tags can be utilized for purification. Aternatively for non-tagged IgG2 fusion multimeric proteins purification can be achieved by traditional purification methods like ion exchange, gel-filtration and hydrophobic interaction column chromatography. His tag chromatographic approaches and other affinity chromatographic purification methods are well known in the art. Size and purity of the purified 2-hinge fusion protein is verified by reducing and/or non-reducing SDS PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis). Example 4 - Expression and Purification of Immunologically Active IgG2 hinge uhtimers for Large Scale Production [00132] While various systems can be used to produce large amounts of a specific protein including bacteria, insect cells or yeast, expression in mammalian cells can minimize problems due to altered glycosylation of the proteins. Mammalian cells like CHO cells have been used to overproduce various proteins fused to an Ig backbone. In case the IgG2 hinge fusion protein contains a Fe domain in the construct the Fe domain becomes a tag that permits subsequent purification from the cell supernatant using protein affinity column purification (Harris, CL, DM Lublin and BP Morgan Efficient generation of monoclonal WO 2013/112986 PCT/US2013/023404 61 antibodies for specific protein domains using recombinant immunoglobulin fusion proteins: pitfalls and solutions., J. Immunol. Methods 268:245-258, 2002). Many fusion proteins are directly cloned in frame with the constant region of Ig, specifically the CH2 and CH3 partial Fe domain monomers. A specific example of expression of interferon gamma receptor extracellular domain being expressed with Ig has been used to produce large amounts of the protein with functional activity (Fountoulakis, M, C. Mesa, G. Schmid, R. Gentz, M. Manneberg, M. Zulauf, Z. Dembic and G. Garotta, Interferon gamma receptor extracellular domain expressed as IgG fusion protein in Chinese hamster ovary cells: Purification, biochemical, characterization and stoichiornetry of binding, J. Biol. Chem.. 270:3958-3964, 1995). Example 5 - Therapeutic Utility in Mouse Model of Arthritis [001331 The therapeutic murine Collagen-Induced Arthritis ("CIA") model is a well established and predictive model for the efficacy of therapeutic compounds in rheumatoid arthritis. This model is well suited to assess compounds containing multimerized CTLA4 2hinge as a therapeutic drug ("Example 5 Test Article"). In this model, collagen is injected and thereby arthritis is induced in the mouse. Drugs can be assessed for the ability to ameliorate or reverse worsening arthritis. At day 0 DBA1/J mice, with the exception of one negative control group, will be injected with bovine Type If collagen solution in a 1:1 mixture with Complete Freund's Adiuvant. At day 20 the collagen-immunization will be repeated except for one control group of 10 animals that will receive no collagen and are expected not to develop arthritis. From day 22-27 the mice that will have received collagen injections will be scored every other day for development of arthritis. Each paw will receive a score as follows: 0 = no visible effects of arthritis; 1 = edema and/or erythema of I digit; 2 = edema and/or erythema of 2 digits; 3 = edema and/or erythema of more than 2 digits; 4 = severe arthritis of entire paw and digits. A calculated Arthritic Index (Al) score will be obtained by addition of individual paw scores and recorded at each measurement with a maximum Al = 16. Mice will be selected into groups for treatment when they have an Al WO 2013/112986 PCT/US2013/023404 62 score of 3 and grouped for treatment. On the day when a group with an Al = 3 is formed, testing with compound will begin in that group. A second control group will receive no therapeutic treatment. In this experiment, groups will be individually treated with: a) Example 5 Test Article 10 mg/Kg administered IV every 4 days, b) Example 5 Test Article 30 mg/Kg administered IV every 4 days, c) Example 5 Test Article 50 ng/Kg administered IV every 4 days, d) Example 5 Test Article 100 mg/Kg administered IV every 4 days, and e) etanercept 10 mg/Kg administered IV every 2 days. Each group will be treated for 4 doses at treatment days 0, 4, 8, and 12 and the mice observed through day 21. The mice receiving Example 5 Test Article will have Al scores significantly lower than the no treatment control group and comparable or better in comparison with the etanercept-treated group and will demonstrate a dose-response relationship. Example 6 - Therapeutic Utility in Mouse Model of Arthritis [001341 The CIA experiment of Example 5 will be repeated with compounds containing multimerized p40-IgG2 hinge as a therapeutic drug ("Example 6 Test Article"). In this experiment, groups will be individually treated with: a) Example 6 Test Article 10 mg/Kg administered IV every 4 days, b) Example 6 Test Article 30 mg/Kg administered IV every 4 days, c) Example 6 Test Article 50 mg/Kg administered IV every 4 days, d) Example 6 Test Article 100 mg/Kg administered IV every 4 days, and e) prednisolone 10 mg administered orally daily. Each Test Article group will be treated for 4 doses at treatment days 0, 4, 8, and 12 and the mice observed through day 21. The mice receiving Example 6 Test Article will have AI scores significantly lower than the no treatment control group and comparable or better in comparison with the steroid-treated group and will demonstrate a dose-response relationship. Example 7 - Diagnostic Utility in Flow Cvtometry 1001351 Flow cytometry is a technique for counting and examining microscopic particles, such as cells, by suspending them in a stream of fluid and passing them by an electronic WO 2013/112986 PCT/US2013/023404 63 detector. It allows simultaneous multiparametric analysis of the physical and/or chemical characteristics of up to thousands of particles per second. Flow cytometry is routinely used in the diagnosis of health disorders but has many other applications in both research and clinical practice. A common research use is to physically sort particles based on their properties, such as a cell surface marker, so as to purity populations of interest. [001361 As MHC tetramer, which is frequently created via the use of biotin-streptavidin affinity among the MHC units, is a useful reagent in performing flow cytometry, especially for T-cells, so multimerized MHC fused to the IgG2 hinge will be of even greater utility. A peptide from the antigen of interest will be attached to the MHC - IgG2 hinge fusion protein and the protein will be fluorescently labeled. Just as each MHC tetramer must be custom made for each antigen-specific T-cell that one desires to detect, so each IgG2 hinge- MHC multimer must similarly be custom made. The fluorescent MIC - antigen - IgG2 hinoe multimer will bind only the specific T-cells that respond to that peptide. The multimerized IgG2 hinge - MHC - antigen will be able to be detected by flow cytometry via the fluorescent label at sensitivities as great as, or greater than, a tetramer comprised of the same M HC and combined by biotin - Streptavidin binding.

权利要求:
Claims (151)
[1] 1. A fusion protein comprising: one or more lgG2 hinge monomers; and one or more peptides, proteins, carbohydrates/sugars or small molecules wherein said one or more lgG2 hinge domains multimerizes said one or more peptides, proteins or small molecules into dimers or higher order multimers.
[2] 2. The fusion protein of claim 1, wherein said one or more peptides, proteins or small molecules is any peptide, protein, small molecule or carbohydrate/sugar whose activity is improved by multimerization.
[3] 3. The fusion protein of claim 2, wherein said protein or peptide is selected from the group consisting of cytokines, chemokines, hormones, monoclonal antibodies and antibody like compounds, cell surface receptors, cell surface receptor ligands and fragments thereof.
[4] 4. The fusion protein of claim 2 wherein the protein is selected from the group consisting of PD-1, PDL-IL, CERVIG peptide, CTLA4, IL12, ILI2RA, major histocompatibility complex and insulin.
[5] 5. The fusion protein of claim 2, wherein said peptide is selected from the group consisting of the external domain of PD-1, CTLA4, the p40 subunit of IL12, and human parathyroid hormone
[6] 6, The fusion protein of claim 2, wherein said small I molecule is selected from the group consisting of chemotherapeutic agents, cytotoxic molecules, dyes and flurophores.
[7] 7. The fusion protein of claim 2, wherein the carbohydrate/sugar is selected from the group consisting of monosaccharides, disaccharides, o ligosaccharides, polysaccharides, WO 2013/112986 PCT/US2013/023404 65 neoglycoproteins, glycoclusters, glycopolymers, monodisperse nanostructures termed glycodendrimers, sugar alcohols, and sugar-rods.
[8] 8. The fusion protein of claim 1, wherein the IgG2 hinge is at least 80% homologous to SEQ ID NO:1.
[9] 9. The fusion protein of claim 1, wherein the IgG2 hinge is at least 90% homologous to SEQ ID NO:1.
[10] 10. The fusion protein of claim 1, wherein the IgG2 hinge is at least 95% homologous to SEQ ID NO:1.
[11] 11. The fusion protein of claim 1, wherein the IgG2 hinge is 100% homologous to SEQ ID NO: 1.
[12] 12. The fusion protein of claim 1, wherein the IgG2 hinge comprises at least one C-X-X C motifs.
[13] 13. The fusion protein of claim 1, wherein the IgG2 hinge comprises at least two C-X-X C motifs.
[14] 14. The fusion protein of claim 12 or 13, wherein the X-X in the said C-X-X-C motif comprises V-E or P-P.
[15] 15. The fusion protein of claim 1, further comprising an immunoglobulin Fe domain.
[16] 16. The fusion protein of claim 15, wherein said immunoglobulin Fe domain is selected for poor binding to Fe gamma receptors.
[17] 17. The fusion protein of claim 16, wherein the Fe domain is an IgG1, IgG2, IgG3 or IgG4 Fe domain. WO 2013/112986 PCT/US2013/023404 66
[18] 18. The fusion protein of claim 15, wherein said immunoglobulin Fe domain is mutated to bind poorly to Fc gamma receptors.
[19] 19. The fusion protein of claim 18, wherein said Fc domain is mutated at one or more of positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414.
[20] 20. The fusion protein of claim 15,. wherein said Fe domain is modified to bind poorly to an Fc gamma receptor.
[21] 21. The fusion protein of claim 20, wherein said Fe domain is modified by hyperficosylation, demannosylation or hemi-glycosylation.
[22] 22. A pharmaceutical formulation comprising the fusion protein of claim 1, and pharma ceutically acceptable excipients.
[23] 23. A method of treating a patient in need thereof with the pharmaceutical forrmulation of claim 22.
[24] 24. The method of claim 23, wherein said patient has an inflammatory disease.
[25] 25. The method of claim 24, wherein the inflammatory disease is an autoimmune disease.
[26] 26. The method of claim 25, wherein the autoimmune disease is arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis, idiopathic thrombocytopenic purpura, chronic inflammatory polvneuropathy, multifocal motor neuropathy, scleroderma, autoimmune uveitis, systemic lupus erythmatosis, myasthenia gravis, rheumatoid arthritis, Crohn's disease, and atopic derm'natitis.
[27] 27. The method of claim 25, wherein the autoimmune disease is associated with the transplantation of an organ from a donor to a recipient. WO 2013/112986 PCT/US2013/023404 67
[28] 28. The method of claim 24. wherein the inflammatory disease is an infectious disease.
[29] 29. The method of claim 28, wherein the infectious disease is a bacterial or fungal infection.
[30] 30. The method of claim 28, wherein the infectious disease is a viral infection.
[31] 31. The method of claim 23, wherein the fusion protein is administered to the patient intravenously, subcutaneously, orally, intraperitoneally, sublingually, bucally, transdermally, by subdermal implant, or intramuscularly.
[32] 32. The method of claim 31, wherein the fusion protein is administered intravenously.
[33] 33. The method of claim 23, further comprising administering an additional pharmaceutically active agent.
[34] 34. The method of claim 33, wherein the additional pharmaceutically active agent comprises a steroid, a monoclonal antibody, an antibiotic, an anti-viral agent, a cytokine, or an agent otherwise capable of acting as an immune modulator.
[35] 35. The method of claim 35, wherein the steroid is prednisolone, cortisone, mometesone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclamethasone, albuterol, or levalbuterol.
[36] 36. A fusion protein comprising: one or more IgG2 hinge monomers; and one or more Fe domains, wherein said one or more Fe domains has been engineered to comprise an antigen binding site; WO 2013/112986 PCT/US2013/023404 68 wherein said one or more IgG2 hinge domains multimerizes said one or more peptides, proteins or small molecules into dimers or higher order multimers.
[37] 37. The fusion protein of claim 36 wherein said fusion protein is capable of binding: a) Fe yR, complement, or FeRn; and b) the antigen for which the Fe domain has been engineered to bind.
[38] 38. The fusion protein of claim 36 wherein the antigen for which the Fe domain has been engineered to bind is [Her2/neu.
[39] 39. The fusion protein of claim 36, wherein the IgG2 hinge is at least 80% homologous to SEQ ID NO:1.
[40] 40. The fusion protein of claim 36, wherein the IgG2. hinge is at least 90% homologous to SEQ ID NO:1.
[41] 41. The fusion protein of claim 36, wherein the IgG2 hinge is at least 95% homologous to SEQ ID NO:I.
[42] 42. The fusion protein of claim 36, wherein the IgG2 hinge is 100% homologous to SEQ ID NO: 1.
[43] 43. The fusion protein of claim 36, wherein the IgG2 hinge comprises at least one C-X-X C motifs.
[44] 44. The fusion protein of claim 36, wherein the IgG2 hinge comprises at least two C-X X-C motifs.
[45] 45. The fusion protein of claim 43 or 44, wherein the X-X in the said C-X-X-C motif comprises V-E or P-P. WO 2013/112986 PCT/US2013/023404 69
[46] 46. A pharmaceutical formulation comprising the fusion protein of claim 36, and pharma ceutically acceptable excipients.
[47] 47. A method of treating a patient in need thereof with the pharmaceutical formulation of claim 22.
[48] 48. The method of claim 47, wherein said patient has an inflammatory disease.
[49] 49. The method of claim 48, wherein the inflammatory disease is an autoimmune disease.
[50] 50. The method of claim 49, wherein the autoinmune disease is arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis, idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, scleroderma, autoimmune uveitis, systemic lupus erythmatosis, myasthenia gravis, and atopic dermatitis.
[51] 51. The method of claim 49, wherein the autoimmune disease is associated with the transplantation of an organ from a donor to a recipient.
[52] 52. The method of claim 48, wherein the inflammatory disease is an infectious disease.
[53] 53. The method of claim 52, wherein the infectious disease is a bacterial infection or a viral infection.
[54] 54. The method of claim 52, wherein said patient has cancer.
[55] 55. The method of claim 47, wherein the fusion protein is administered to the patient intravenously, subcutaneously, orally, intraperitoneally, sublingually, bucally, transdermally, by subdermal implant, or intramuscularly.
[56] 56. The method of claim 55, wherein the fusion protein is administered intravenously. WO 2013/112986 PCT/US2013/023404 70
[57] 57. The method of claim 47, further comprising administering an additional pharmaceutically active agent.
[58] 58. The method of claim 57, wherein the additional pharmaceutically active agent comprises a steroid, a monoclonal antibody, an antibiotic, an anti-viral agent, a cytokine, or an agent otherwise capable of acting as an immune modulator.
[59] 59. The method of claim 58, wherein the steroid is prednisolone, cortisone, mometesone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclamethasone, albuterol, or levalbuterol.
[60] 60. A fusion protein comprising: one or more IgG2 hinge monomers; and one or more antigen binding antibody variable domains, fragments or variants thereof; wherein said one or more 1gG2 hinge domains multimerizes said one or more antigen binding antibody variable domains, fragments or variants thereof into diners or higher order multimers.
[61] 61. The fusion protein of claim 60 comprising a variable heavy chain (VH) linked to a variable light chain (VL).
[62] 62. The fusion protein of claim 61 wherein the fusion of the VH to the VL forms an epitope binding site.
[63] 63. The fusion protein of claim 61 wherein the VH 1 is linked to the VL with a linker.
[64] 64. The fusion protein of claim 63 further com:iprising the CHI region of VH or VL.
[65] 65. The fusion protein of claim I wherein the VHI and VL are co-expressed in the same cell. WO 2013/112986 PCT/US2013/023404 71
[66] 66. The fusion protein of claim 60, wherein the one or more antigen binding antibody variable domains, fragments or variants thereof is a Fab fragment, scFv, a diabody, a triabody, a minibody, a single-domain antibody, a nanobody or a single chain antibody.
[67] 67. The fusion protein of claim 60, wherein the IgG2. hinge is at least 80% homologous to SEQ ID NO:I.
[68] 68. The fusion protein of claim 60, wherein the 1gG2 hinge is at least 90% homologous to S EQ ID NO:1.
[69] 69. The fusion protein of claim 60, wherein the lgG2 hinge is at least 95% homologous to SEQ ID NO:I.
[70] 70. The fusion protein of claim 60, wherein the IgG2 hinge is 100% homologous to SEQ ID NO: 1.
[71] 71. The fusion protein of claim 60, wherein the IgG2 hinge comprises at least one C-X-X C motifs.
[72] 72. The fusion protein of claim 60, wherein the lgG2 hinge comprisies at least two C-X X-C motifs.
[73] 73. The fusion protein of claim 71 or 72, wherein the X-X in the said C-X-X-C motif comprises V-E or P-P.
[74] 74. The fusion protein of claim 1, further comprising an immunoglobulin Fe domain.
[75] 75. The fusion protein of claim 74, wherein said immunoglobulin Fe domain is selected for poor binding to Fe gamma receptors.
[76] 76. The fusion protein of claim 75, wherein the Fe domain is an IgG1, IgG2, IgG3 or IgG4 Fe domain. WO 2013/112986 PCT/US2013/023404 72
[77] 77. The fusion protein of claim 74, wherein said immunoglobulin Fe domain is mutated to bind poorly to Fc gamma receptors.
[78] 78. The fusion protein of claim 77, wherein said Fc domain is mutated at one or more of positions 233, 234, 235, 236, 238, 239, 265, 269, 270, 292, 293, 295, 296, 297, 303, 327, 329, 338, 376, and/or 414.
[79] 79. The fusion protein of claim 74, wherein said Fe domain is modified to bind poorly to an Fc gamma receptor.
[80] 80. The fusion protein of claim 79, wherein said Fe domain is modified by hyperficosylation, demannosylation or hemi-glycosylation.
[81] 81. A pharmaceutical formulation comprising the fusion protein of claim 60, and pharma ceutically acceptable excipients.
[82] A method of treating a patient in need thereof with the pharmaceutical formulation of claim 81.
[83] 83. The method of claim 82, wherein said patient has an inflammatory disease.
[84] 84. The method of claim 83, wherein the inflammatory disease is an autoimmune disease.
[85] 85. The method of claim 84, wherein the autoimmune disease is arthritis, multiple sclerosis, type I diabetes, autoimmune thyroiditis, idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, scleroderma, autoimmune uiveitis, systemic lupus erythmatosis, myasthenia gravis, and atopic dermatitis.
[86] 86. The method of claim 84, wherein the autoimmune disease is associated with the transplantation of an organ from a donor to a recipient.
[87] 87. The method of claim 83, wherein the inflammatory disease is an infectious disease. WO 2013/112986 PCT/US2013/023404 73
[88] 88. The method of claim 87. wherein the infectious disease is a bacterial infection.
[89] 89. The method of claim 87, wherein the infectious disease is a viral infection.
[90] 90. The method of claim 82., wherein the fusion protein is administered to the patient intravenously, subcutaneously, orally, intraperitoneally, sublingually, bucally, transdenally, by subdermal implant, or intramuscularly.
[91] 91. The method of claim 31, wherein the fusion protein is administered intravenously.
[92] 92. The method of claim 23, further comprising administering an additional pharmaceutically active agent.
[93] 93. The method of claim 33, wherein the additional pharmaceutically active agent comprises a steroid, a monoclonal antibody, an antibiotic, an anti-viral agent, a cytokine, or an agent otherwise capable of acting as an immune modulator.
[94] 94. The method of claim 35, wherein the steroid is prednisolone, cortisone, mometesone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclamethasone, albuterol, or levalbuterol.
[95] 95. The fusion protein of claim 1, wherein the one or more peptides, proteins, carbohydrates/sugars or small molecule is fused to the N terminus of the IgG2 hinge.
[96] 96. The fusion protein of claim 1, wherein the one or more peptides, proteins, carbohydrates/sugars or small molecule is fused to the C terminus of the IgG2 hinge.
[97] 97. The fusion protein of claim 95 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is CTLA-4 or an extracellular domain thereof.
[98] 98. The fusion protein of claim 97, further comprising a B7. I or B7.2 protein. WO 2013/112986 PCT/US2013/023404 74 98. The fusion protein of claim 95 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is PD-1.
[99] 99. The fusion protein of claim 98 wherein the PD-i is the extracellular domain of PD-1.
[100] 100. The fusion protein of claim 99 comprising SEQ ID NO: 6.
[101] 101. The fusion protein of claim 95 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is a CERVIG peptide.
[102] 102. The fusion protein of claim 101 wherein the CERVIG comprises SEQ ID NO: 11,
[103] 103. The fusion protein of claim 102 comprising SEQ ID NO: 16,
[104] 104. The fusion protein of claim 96 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is CTLA-4 or an extracellular domain thereof.
[105] 105. The fusion protein of claim 104, further comprising a B7.1 or B7.2 protein.
[106] 106. The fusion protein of claim 96 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is PD-i.
[107] 107. The fusion protein of claim 106 wherein the PD-1 is the extracellular domain of PD-1.
[108] 108. The fusion protein of claim 107 comprising SEQ ID NO: 5.
[109] 109. The fusion protein of claim 96 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is a CERVIG peptide.
[110] 110. The fusion protein of claim 109 wherein the CERVIG comprises SEQ I) NO: 11.
[111] 111. The fusion protein of clain 36 wherein the lgG2 hinge is fused to the C terminus of the one or more Fe domains engineered to comprise an antigen binding site. WO 2013/112986 PCT/US2013/023404 75
[112] 112. The fusion protein of claim 36 wherein the IgG2 hinge is fused to the N terminus of the one or more Fc domains engineered to comprise an antigen binding site.
[113] 113. The fusion protein of claim 1II wherein the antigen to which the Fc domain is engineered to bind is Her2/neu of SEQ ID NO: 18.
[114] 114. The fusion protein of claim 113 comprising SEQ I) NO: 19
[115] 115. The fusion protein of claim 112 wherein the antigen to which the Fe domain is engineered to bind is Her2/neu of SEQ ID NO: 18.
[116] 116. The fusion protein of claim 115 comprising SEQ ID NO: 20.
[117] 117. The fusion protein of claim 60 wherein the lgG2 hinge is fused to the C terminus of the one or more antigen binding antibody variable domains, fragments or variants thereof.
[118] 118. The fusion protein of claim 60 wherein the IgG2 hinge is fused to the N terminus of the one or more antigen binding antibody variable domains, fragments or variants thereof.
[119] 119. The fusion protein of claim 15 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is PD-1.
[120] 120. The fusion protein of claim 119 wherein the PD-i is the extracellular domain of PD-1.
[121] 121. The fusion protein of claim 120 comprising SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO:9, or SEQ 11) NO:10.
[122] 122 ) The fusion protein of claim 15 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is a CERVIG peptide.
[123] 123. The fusion protein of claim 122 wherein the CERVIG peptide comprises SEQ ID NO: 11. WO 2013/112986 PCT/US2013/023404 76
[124] 124. The fusion protein of claim 123 comprising SEQ ID NO: 13, SEQ ID NO: 14 or SEQ ID NO: 15.
[125] 125. The fusion protein of claim I further comprising a linker between the one or more peptides, proteins, carbohydrates/sugars or small molecules and one or more IgG2 hinge monomers.
[126] 126. The fusion protein of claim 125 wherein the one or more peptides, proteins, carbohydrates/sugars or small molecules is a CE RVI(i peptide.
[127] 127. The fusion protein of claim 126 wherein the CERVIG peptide comprises SEQ ID NO: 11.
[128] 128. The fusion protein of claim 127 comprising SEQ ID NO: 17.
[129] 129. The fusion protein of claim 36 further comprising one or more additional Fe domains.
[130] 130. The fusion protein of claim 129 wherein the Fe domain that has been engineered to comprise an antigen binding site has been engineered to bind Her2/neu.
[131] 131. The fusion protein of claim 130 wherein the Fe domain that has been engineered to bind Her2/neu comprises SEQ I) NO: 18.
[132] 132. The fusion protein of claim 131 comprising SEQ ID NO: 21,
[133] 133. The fusion protein of claim 4 wherein the protein is a major histocompatibility complex.
[134] 134. The fusion protein of claim 133 wherein the major histocompatibility complex is a class I major histocompatibility complex. WO 2013/112986 PCT/US2013/023404 77
[135] 135. The fusion protein of claim 133 wherein the major histocompatibility complex is a class II major histocompatibility complex.
[136] 136. The fusion protein of claim 133 further comprising a dye or a fluorophore.
[137] 137. The use of the fusion protein of claim 136 in flow cytometry to detect antigen specific T cells.
[138] 138. The fusion protein of claim 3 wherein the protein or peptide is a cell surface receptor.
[139] 139. The fusion protein of claim 138 wherein the cell surface receptor is a G-protein coupled receptor.
[140] 140. The fusion protein of claim 139 wherein the G-protein coupled receptor is a chemokine receptor.
[141] 141. The fusion protein of claim 140 wherein the chemokine receptor is CCR5, CXCR1 or CXCR2.
[142] 142. The fusion protein of claim 138 wherein the cell surface receptor is a B cell receptor.
[143] 143. The fusion protein of claim 138 wherein the cell surface receptor is a T cell receptor.
[144] 144. The fusion protein of claim 138 wherein the cell surface receptor is a TNF superfamily receptor.
[145] 145. The fusion protein of claim 144 wherein the TNF superfamily receptor is CD 137, BAFF R, BCMA, CD27, CD30, (D40, DR3, DcTRAIL, DR3, DR6, EDAR, Fas, GITR, HVEM, lyphotoxin beta R, NGF R, osteoprotegerin, OX40, RANK, RELT, TACI, TRAIL R, TROY, or TWEAK R. WO 2013/112986 PCT/US2013/023404 78
[146] 146. The fusion protein of claim 145 wherein the TRAIL R is TRAIL RI, TRAIL R2, TRAIL R3 or TRAIL R4.
[147] 147. The fusion protein of claim 3 wherein the protein or peptide is a cell surface rector ligand.
[148] 148. The fusion protein of claim 147 wherein the cell surface receptor ligand is a ligand to a TNF superfamily receptor.
[149] 149. The fusion protein of claim 148 wherein the ligand to a TNF super family receptor is TNFc or BLyS.
[150] 150. The fusion protein of claim 147 wherein the cell surface receptor ligand is a ligand to a cell surface glycoprotein.
[151] 151. The fusion protein of claim 150 wherein the ligand binding to a cell surface glycoprotein is a CD4, CD123, CD303, or A CD304 ligand. GLIK_008_01WO_SeqList_ST25.txt SEQUENCE LISTING <110> GLIKNIK INC. Block, David Olsen, Henrick Strome, Scott <120> FUSION PROTEINS COMPRISING IgG2 HINGE DOMAINS <130> GLIK-008/01WO <150> US 61/591,615 <151> 2012-01-27 <160> 21 <170> PatentIn version 3.5 <210> 1 <211> 12 <212> PRT <213> Homo sapiens <400> 1 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 1 5 10 <210> 2 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Peptide antagonist of CXCR4 <400> 2 Leu Gly Ala Ser Trp His Arg Pro Asp Lys Cys Cys Leu Gly Tyr Gln 1 5 10 15 Lys Arg Pro Leu Pro 20 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> Small peptide that binds FGF receptor-expressing cells <400> 3 Met Gln Leu Pro Leu Ala Thr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> Octa peptide that blocks CD4 receptor binding by HIV Page 1 GLIK_008_01WO_SeqList_ST25.txt <400> 4 Ala Ser Thr Thr Thr Asn Tyr Thr 1 5 <210> 5 <211> 182 <212> PRT <213> Artificial Sequence <220> <223> G131 (2hinge-PD-1) PD-1 PD1L pathway interacting construct <400> 5 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr 35 40 45 Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe 50 55 60 Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr 65 70 75 80 Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu 85 90 95 Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu 100 105 110 Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn 115 120 125 Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala 130 135 140 Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg 145 150 155 160 Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly 165 170 175 Gln Phe Gln Thr Leu Val 180 <210> 6 <211> 182 Page 2 GLIK_008_01WO_SeqList_ST25.txt <212> PRT <213> Artificial Sequence <220> <223> G132 (PD-1-2hinge) PD-1 PD1L pathway interacting construct <400> 6 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Glu Arg Lys Cys Cys Val 165 170 175 Glu Cys Pro Pro Cys Pro 180 <210> 7 <211> 414 <212> PRT <213> Artificial Sequence <220> <223> G133 (2hinge-Fc-PD-1) G019-PD-1 PD-1 PD1L pathway interacting construct <400> 7 Page 3 GLIK_008_01WO_SeqList_ST25.txt Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 35 40 45 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 50 55 60 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 65 70 75 80 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 85 90 95 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 130 135 140 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 145 150 155 160 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 165 170 175 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 225 230 235 240 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255 Ser Leu Ser Leu Ser Pro Gly Lys Pro Gly Trp Phe Leu Asp Ser Pro 260 265 270 Page 4 GLIK_008_01WO_SeqList_ST25.txt Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val 275 280 285 Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser 290 295 300 Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr 305 310 315 320 Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp 325 330 335 Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met 340 345 350 Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly 355 360 365 Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala 370 375 380 Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro 385 390 395 400 Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val 405 410 <210> 8 <211> 399 <212> PRT <213> Artificial Sequence <220> <223> 2hinge-CH2-CH3-PD-1 (G051-PD1) PD-1 PD1L pathway interacting construct <400> 8 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 65 70 75 80 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Page 5 GLIK_008_01WO_SeqList_ST25.txt 85 90 95 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 100 105 110 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 115 120 125 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 130 135 140 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 145 150 155 160 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200 205 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 210 215 220 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 225 230 235 240 Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Gly Trp Phe Leu Asp Ser 245 250 255 Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val 260 265 270 Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr 275 280 285 Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln 290 295 300 Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln 305 310 315 320 Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His 325 330 335 Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys 340 345 350 Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Page 6 GLIK_008_01WO_SeqList_ST25.txt 355 360 365 Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His 370 375 380 Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr Leu Val 385 390 395 <210> 9 <211> 414 <212> PRT <213> Artificial Sequence <220> <223> PD-1-Fc-2hinge (PD-1- G045) PD-1 PD1L pathway interacting construct <400> 9 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp 20 25 30 Asn Pro Pro Thr Phe Ser Pro Ala Leu Leu Val Val Thr Glu Gly Asp 35 40 45 Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val 50 55 60 Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala 65 70 75 80 Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg 85 90 95 Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg 100 105 110 Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu 115 120 125 Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val 130 135 140 Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro 145 150 155 160 Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Glu Pro Lys Ser Cys Asp 165 170 175 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 180 185 190 Page 7 GLIK_008_01WO_SeqList_ST25.txt Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 195 200 205 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 210 215 220 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 225 230 235 240 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 245 250 255 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 260 265 270 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu 275 280 285 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 290 295 300 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu 305 310 315 320 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 325 330 335 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 340 345 350 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 355 360 365 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 370 375 380 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 385 390 395 400 Gly Lys Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 405 410 <210> 10 <211> 414 <212> PRT <213> Artificial Sequence <220> <223> Fc-PD-1-2h construct PD-1 PD1L pathway interacting construct <400> 10 Page 8 GLIK_008_01WO_SeqList_ST25.txt Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 20 25 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 145 150 155 160 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 225 230 235 240 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Pro Gly Trp Phe 245 250 255 Leu Asp Ser Pro Asp Arg Pro Trp Asn Pro Pro Thr Phe Ser Pro Ala 260 265 270 Page 9 GLIK_008_01WO_SeqList_ST25.txt Leu Leu Val Val Thr Glu Gly Asp Asn Ala Thr Phe Thr Cys Ser Phe 275 280 285 Ser Asn Thr Ser Glu Ser Phe Val Leu Asn Trp Tyr Arg Met Ser Pro 290 295 300 Ser Asn Gln Thr Asp Lys Leu Ala Ala Phe Pro Glu Asp Arg Ser Gln 305 310 315 320 Pro Gly Gln Asp Cys Arg Phe Arg Val Thr Gln Leu Pro Asn Gly Arg 325 330 335 Asp Phe His Met Ser Val Val Arg Ala Arg Arg Asn Asp Ser Gly Thr 340 345 350 Tyr Leu Cys Gly Ala Ile Ser Leu Ala Pro Lys Ala Gln Ile Lys Glu 355 360 365 Ser Leu Arg Ala Glu Leu Arg Val Thr Glu Arg Arg Ala Glu Val Pro 370 375 380 Thr Ala His Pro Ser Pro Ser Pro Arg Pro Ala Gly Gln Phe Gln Thr 385 390 395 400 Leu Val Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 405 410 <210> 11 <211> 12 <212> PRT <213> Unknown <220> <223> CERVIG peptide for interaction with CD47/SIRPa pathway <400> 11 Cys Glu Arg Val Ile Gly Thr Gly Trp Val Arg Cys 1 5 10 <210> 12 <211> 264 <212> PRT <213> Artificial Sequence <220> <223> G126 (Fc-CERVIG peptide) - G001-CERVIGTGWVRC (without multimerization domain) CD47/SIRPa pathway contruct <400> 12 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 20 25 30 Page 10 GLIK_008_01WO_SeqList_ST25.txt Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 145 150 155 160 Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln 210 215 220 Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His 225 230 235 240 Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys Cys Glu Arg Val 245 250 255 Ile Gly Thr Gly Trp Val Arg Cys 260 <210> 13 <211> 276 <212> PRT <213> Artificial Sequence <220> Page 11 GLIK_008_01WO_SeqList_ST25.txt <223> G122 (CERVIG peptide-Fc-2hinge) - CERVIGTGWVRC-G045 CD47/SIRPa pathway contruct <400> 13 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Cys Glu Arg Val Ile Gly Thr Gly Trp Val Arg Cys 20 25 30 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 35 40 45 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 50 55 60 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 65 70 75 80 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 85 90 95 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110 Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 130 135 140 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 145 150 155 160 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 165 170 175 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 225 230 235 240 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255 Page 12 GLIK_008_01WO_SeqList_ST25.txt Ser Leu Ser Leu Ser Pro Gly Lys Glu Arg Lys Cys Cys Val Glu Cys 260 265 270 Pro Pro Cys Pro 275 <210> 14 <211> 276 <212> PRT <213> Artificial Sequence <220> <223> G123 ( 2hinge-Fc-"CERVIG" peptide) - G019- CERVIGTGWVRC CD47/SIRPa pathway contruct <400> 14 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 35 40 45 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 50 55 60 Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 65 70 75 80 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 85 90 95 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 130 135 140 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 145 150 155 160 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr 165 170 175 Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190 Page 13 GLIK_008_01WO_SeqList_ST25.txt Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220 Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe 225 230 235 240 Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys 245 250 255 Ser Leu Ser Leu Ser Pro Gly Lys Cys Glu Arg Val Ile Gly Thr Gly 260 265 270 Trp Val Arg Cys 275 <210> 15 <211> 261 <212> PRT <213> Artificial Sequence <220> <223> G124 (2hinge-FcCH2-FcCH30"CERVIG"peptide) -G051 CERVIGTGWVRC CD47/SIRPa pathway contruct <400> 15 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 65 70 75 80 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 85 90 95 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 100 105 110 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 115 120 125 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Page 14 GLIK_008_01WO_SeqList_ST25.txt 130 135 140 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met 145 150 155 160 Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200 205 Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val 210 215 220 Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln 225 230 235 240 Lys Ser Leu Ser Leu Ser Pro Gly Lys Cys Glu Arg Val Ile Gly Thr 245 250 255 Gly Trp Val Arg Cys 260 <210> 16 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> CERVIG-2h CD47/SIRPa pathway contruct <400> 16 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Cys Glu Arg Val Ile Gly Thr Gly Trp Val Arg Cys 20 25 30 Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 35 40 <210> 17 <211> 69 <212> PRT <213> Artificial Sequence <220> <223> CERVIG-linker-2h CD47/SIRPa pathway contruct <400> 17 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro Page 15 GLIK_008_01WO_SeqList_ST25.txt 1 5 10 15 Gly Ser Thr Gly Cys Glu Arg Val Ile Gly Thr Gly Trp Val Arg Cys 20 25 30 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 35 40 45 Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Arg Lys Cys Cys Val Glu 50 55 60 Cys Pro Pro Cys Pro 65 <210> 18 <211> 257 <212> PRT <213> Artificial Sequence <220> <223> GB6500 (without multimerization domain) Her2/neu and FcGRIIIA interacting construct <400> 18 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 20 25 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 145 150 155 160 Page 16 GLIK_008_01WO_SeqList_ST25.txt Asp Glu Tyr Leu Tyr Gly Asp Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Arg His Ser Ala Arg Met 210 215 220 Trp Arg Trp Ala His Gly Asn Val Phe Ser Cys Ser Val Met His Glu 225 230 235 240 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 245 250 255 Lys <210> 19 <211> 257 <212> PRT <213> Artificial Sequence <220> <223> GB6545 (Modified Fc -2hinge) Her2/neu and FcGRIIIA interacting construct <400> 19 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro 20 25 30 Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe 35 40 45 Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val 50 55 60 Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe 65 70 75 80 Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro 85 90 95 Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr 100 105 110 Page 17 GLIK_008_01WO_SeqList_ST25.txt Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val 115 120 125 Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala 130 135 140 Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg 145 150 155 160 Asp Glu Tyr Leu Tyr Gly Asp Val Ser Leu Thr Cys Leu Val Lys Gly 165 170 175 Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro 180 185 190 Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser 195 200 205 Phe Phe Leu Tyr Ser Lys Leu Thr Val Pro Arg His Ser Ala Arg Met 210 215 220 Trp Arg Trp Ala His Gly Asn Val Phe Ser Cys Ser Val Met His Glu 225 230 235 240 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 245 250 255 Lys <210> 20 <211> 269 <212> PRT <213> Artificial Sequence <220> <223> GB6519 ( 2hinge - Modified Fc) Her2/neu and FcGRIIIA interacting construct <400> 20 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro 20 25 30 Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala 35 40 45 Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro 50 55 60 Page 18 GLIK_008_01WO_SeqList_ST25.txt Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val 65 70 75 80 Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val 85 90 95 Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln 100 105 110 Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln 115 120 125 Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 130 135 140 Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 145 150 155 160 Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr Leu 165 170 175 Tyr Gly Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser 180 185 190 Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr 195 200 205 Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr 210 215 220 Ser Lys Leu Thr Val Pro Arg His Ser Ala Arg Met Trp Arg Trp Ala 225 230 235 240 His Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 245 250 255 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 260 265 <210> 21 <211> 254 <212> PRT <213> Artificial Sequence <220> <223> GB6551 (2hinge-CH2-CH3 modified Fc) Her2/neu and FcGRIIIA interacting construct <400> 21 Met Glu Thr Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15 Gly Ser Thr Gly Glu Arg Lys Cys Cys Val Glu Cys Pro Pro Cys Pro Page 19 GLIK_008_01WO_SeqList_ST25.txt 20 25 30 Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 35 40 45 Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 50 55 60 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr 65 70 75 80 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu 85 90 95 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His 100 105 110 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys 115 120 125 Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln 130 135 140 Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Tyr 145 150 155 160 Leu Tyr Gly Asp Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro 165 170 175 Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn 180 185 190 Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu 195 200 205 Tyr Ser Lys Leu Thr Val Pro Arg His Ser Ala Arg Met Trp Arg Trp 210 215 220 Ala His Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 225 230 235 240 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 245 250 Page 20

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同族专利:

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AU2013211824A8|2014-11-06|

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EP2807194A4|2015-12-02|

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JP2015506372A|2015-03-02|

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EP2807194A1|2014-12-03|

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法律状态:
2014-11-06| TH| Corrigenda|Free format text: IN VOL 28 , NO 34 , PAGE(S) 4606 UNDER THE HEADING PCT APPLICATIONS THAT HAVE ENTERED THE NATIONAL PHASE - NAME INDEX UNDER THE NAME UNIVERSITY OF MARYLAND, BALTIMORE; GLIKNIK INC., APPLICATION NO. 2013211824, UNDER INID (72) ADD CO-INVENTOR STROME, SCOTT |

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2017-09-28| FGA| Letters patent sealed or granted (standard patent)|

优先权:

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申请号 | 申请日 | 专利标题

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US201261591615P| true| 2012-01-27|2012-01-27||

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US61/591,615||2012-01-27||

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PCT/US2013/023404|WO2013112986A1|2012-01-27|2013-01-28|Fusion proteins comprising igg2 hinge domains|

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