JP2008536477A - Molecule and its chimeric molecule - Google Patents

Abstract

The present invention relates generally to the fields of protein, diagnosis, therapy, and nutrition. More particularly, the present invention relates to IFN-a2B, IFN-, which has a profile of measurable physiochemical parameters that are indicative of, related to, or form the basis of one or more pharmacological traits. b1, an isolated protein molecule containing four dimeric helix bundles such as IFN-g, IL-10, its receptor such as IFNAR2, IL-10Ra, or IFN-a2B-Fc, IFN-b1- Chimeric molecules thereof comprising at least part of protein molecules such as Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc are provided. The present invention further contemplates the use of isolated proteins or chimeric molecules thereof in a range of diagnostic, prophylactic, therapeutic, nutritional and / or research applications.

Description

The present invention relates generally to the fields of protein, diagnosis, therapy, and nutrition. More particularly, the invention relates to IFN-a2B, IFN-b1 having a profile of measurable physiochemical parameters that are indicative of, related to, or form the basis of one or more pharmacological traits. An isolated protein comprising a four-helix bundle such as IFN-g, IL-10, its receptor such as IFNAR2, IL-10Ra, or IFN-a2B-Fc, IFN-b1-Fc, A chimeric molecule thereof comprising at least a portion of a protein molecule such as IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc is provided. The present invention further contemplates using the isolated protein or chimeric molecule thereof for a range of diagnostic, prophylactic, therapeutic, nutritional and / or research applications.

DESCRIPTION OF THE PRIOR ART References to any prior art in this specification are not admitted that they form part of the common general knowledge or do not imply any form of interpretation and are to be interpreted as such. Should not.

  Human interferons (IFNs) are pleiotropic cytokines that promote both innate and adaptive immune responses. They regulate immune responses against viral and bacterial infections and have a role in anti-tumor responses. Furthermore, these proteins are immunoregulatory and regulate cell growth, differentiation and proliferation in a number of ways. These molecules include IFNa-2b, IFN-b1, IFN-g, and its receptor IFN-aR2. Structurally, IFN-aR2 is a class II cytokine as well as a receptor for IL-10. Importantly, interferons (IFN-a2B, IFN-b1, and IFN-g) and IL-10 exhibit a common structure, a dimeric 4-helix bundle structure.

  IFN-a contains a structurally related protein family encoded by 14 non-allelic IFN-a genes, one of which encodes IFN-a2. IFN-a2 is a monomer having an α-helix structure and is O-glycosylated. The IFN-a2b polymorphism is characterized by a K → R at amino acid position 46 of the precursor. IFN-a is produced primarily by monocytes / macrophages and to a lesser extent by natural killer (NK) cells, T cells, dendritic cells, and plasmacytoid dendritic cells. IFN-a expression is induced by viral or bacterial infections, as well as by components of infectious agents such as LPS, bacterial DNA and double stranded RNA. The main function of IFN-a is to mediate non-viral microbial responses as well as mediate anti-viral and anti-tumor responses. The action induced by IFN-a is due to binding to common IFN cell surface receptors present on target cells, macrophages, or DCs. Treatment of DC with IFN-a promotes maturation and upregulation of costimulatory molecules such as CD40, CD80, CD86, and MHC class II.

  Human IFN-b1 is produced as a precursor protein of 187 amino acids, has an N-linked glycosylation site at Asn 80, and exists as a zinc-mediated dimer. IFN-b1 expression is primarily derived from fibroblasts, but expression has been detected in epithelial cells and lymphoblastoid cells in response to viral infection or exposure to double-stranded RNA. IFN-b1 is a pleiotropic cytokine that has numerous effects on various cells of the immune system and affects both innate and adaptive immune responses. IFN-b1 has also been shown to induce MHC II expression and antigen presentation, thereby enhancing cellular immunity. In addition, IFN-b1 exhibits anti-proliferative characteristics, inhibits tumor cell growth, and induces apoptosis of lymphoid cell lines. It is an attractive treatment for both illness and many malignancies.

Human IFN-g is produced as a 166 amino acid peptide and exists as a homodimer that associates non-covalently. IFN-g is a pleiotropic cytokine that promotes both innate and adaptive immune responses. IFN-g is induced by the pro-inflammatory cytokines IL-12 and TNF-α and non-specifically kills a variety of intracellular and extracellular pathogens such as bacteria, viruses, fungi, protozoa, helminths and tumor cells Can mediate macrophage activation. IFN-g-dependent IL-12 expression promotes TH ₁ cell-mediated immune responses for elimination of viral infections and bacterial and tumor rejection.

  IFN-g has been shown to enhance proliferation of antigen-stimulated B cells and promote Ig isotype switching from IgM to IgG2, thereby promoting antibody-dependent cytotoxicity by the NK IgG receptor .

  IFNAR2 is synthesized as a 515 amino acid transmembrane protein containing a 26 amino acid signal peptide sequence and 5 potential N-linked glycosylation sites. The short form of IFNAR2 consisting of its extracellular ligand binding domain has the ability to act as a type 1 IFN agonist or as either an antagonist or potentiator of IFN action. In the latter case, the circulating half-life of IFNα, β, ω, τ, κ, or ζ can be significantly extended by co-administration of the extracellular domain of the IFN receptor. Cleaved IFNAR2 may act as a type 1 interferon agonist through interaction and activation with cell membrane-bound IFN receptors. This may occur with or without ligand binding to truncated IFNAR2. In this case, IFNAR2 may be a useful treatment for replacing type 1 interferon or for use in combination with them. Alternatively, by binding type I interferon, truncated IFNAR2 may act as an antagonist or inhibitor, and may be useful in the treatment of diseases resulting from overproduction of type 1 interferon. For example, abnormal production of IFN-a has been found in autoimmune diseases, and such diseases including autoimmune hepatitis, lupus, systemic sclerosis, and diabetes mellitus are persistent treatments with IFN-a. It can be observed later. IFN-a may also be involved in the pathogenesis of allogeneic rejection in bone marrow transplantation, but IFNκ antagonists can be used to treat psoriasis and atopic dermatitis and to prevent type 1 diabetes .

Interleukin 10 (IL-10) is a 37 kDa homodimer that is expressed primarily by macrophages but is also produced by activated T cells, B cells, mast cells, monocytes, and keratinocytes. IL-10 is a pleiotropic cytokine that regulates multiple immune responses through action on T cells, B cells, macrophages / monocytes, and antigen-presenting cells (APCs), generally transducing immune responses from T _H1 to T Bend to _H2 . IL-10 also inhibits the synthesis of cytokines IL-1α, IL-1β, IL-6, IL-8, TNFα, GM-CSF, and G-CSF in activated monocytes and activated macrophages. IL-10 also suppresses IFNγ production by NK cells. IL-10-treated immature dendritic cells are unable to mature, exhibit a reduced ability to stimulate CD4 + T cells, and the Langerhans cell antigen presentation function is suppressed by IL-10. By inhibiting the maturation of antigen-presenting cells (APC), IL-10 retains the ability to take up antigens while suppressing migration to draining lymph nodes, a process that is critical for the innate immune response against pathogens. It has been suggested that it may constitute an ingredient. Overexpression of IL-10 has been reported in a number of different malignancies including melanoma, carcinoma, and lymphoma. In contrast, many diseases are characterized by type I cytokine patterns associated with relative deficiencies in IL-10 levels, including psoriasis and inflammatory bowel disease.

  The biological effects of IL-10 are mediated through binding to the IL-10 receptor (IL-10R) complex. This is a heterodimer comprising interleukin 10 receptor alpha chain (IL-10Rα) and interleukin 10 receptor beta chain (IL-10Rβ) and relates to IL-22, IL-28, and IL-29 Shared by cytokine receptors. IL-10Rα is a polypeptide that is diversely glycosylated at at least one of six potential glycosylation sites. IL-10R is expressed on a number of leukocytes including T cells, NK cells, macrophages / monocytes, B cells, neutrophils, and dendritic cells.

  Biological effector functions exerted by the interaction of proteins with their respective receptors are interferons and related proteins such as IL-10 and their respective receptors as therapeutic agents for modulating physiological processes. It means that it can have significant potential. However, minor changes to molecules such as primary, secondary, tertiary or quaternary structures, and translational or post-translational modification patterns have a significant effect on protein activity, secretion, antigenicity, and clearance. Can be affected. Thus, it is possible that proteins with specific primary, secondary, tertiary, or quaternary structure, or with a translated or post-translated structure or configuration that impart unique or particularly useful properties can be generated. . Consequently, it is necessary to evaluate the physiochemical properties of proteins in different production conditions in order to determine whether they have useful physiochemical characteristics or other pharmacological properties.

  To date, the problem is that the production of commercially available proteins occurs in cells derived from species that are evolutionarily distant from humans such as bacteria, yeast, fungi, and insects. These cells express proteins that are deficient in glycosylation or that exhibit a different glycosylation repertoire than human cells, which has a substantial impact on their clinical utility. For example, proteins expressed in yeast or fungal systems such as Aspergillus possess a high density of mannose, which renders the protein therapeutically ineffective (Herscovics et al. FASEB J 7: 540- 550, 1993 (Non-Patent Document 1)).

  In non-human mammalian expression systems, such as Chinese hamster ovary (CHO) cells, significant changes in glycosylation patterns have been reported compared to human cell patterns. For example, most mammals, including rodents, express the enzyme (α1,3) galactotransferase that produces Gal (α1,3) -Gal (β1,4) -GlcNAc oligosaccharides in glycoproteins. However, in humans, monkeys, and Old World monkeys, the expression of this enzyme has been inactivated through a frameshift mutation in the gene (Larsen et al. J Biol Chem 265: 7055-7061, 1990). )). Most of the CHO cell lines used in recombinant protein synthesis such as Dux-B11 inactivated genes expressing (α1,3) galactotransferase, but they are still present in human cells ( It lacks a functional (α2,6) sialyltransferase for the synthesis of α2,6) -linked terminal sialic acid. Furthermore, sialic acid motifs present on CHO cell-expressed glycoproteins are often degraded by CHO cell endogenous sialidase (Gramer et al. Biotechnology 13 (7): 692-8, 1995). )).

  As a result, proteins produced from these non-human expression systems exhibit physiochemical and pharmacological characteristics such as half-life, antigenicity, stability, and functional potency that differ from human cell-derived proteins. I think that the.

  Recent advances in stem cell technology have substantially increased the possibility of using stem cells for applications such as transplantation therapy, drug screening, toxicity testing, and functional genomics. However, stem cells are routinely maintained in culture media containing non-human proteins and are therefore not suitable for clinical applications due to the potential for contamination with non-human infectious materials. Furthermore, non-human carbohydrate moieties may be incorporated by culturing stem cells in non-human-derived media, which again impairs transplantation applications (Martin et al. Nature Medicine 11 (2): 228-232, 2005 ( Non-patent document 4)). Thus, the use of specific human derived proteins in stem cell maintenance and / or differentiation would improve the uptake of heterologous proteins and enhance the clinical utility of stem cells.

  Accordingly, there is a need to develop proteins and their receptors that have particularly desirable physiochemical and pharmacological properties for use in diagnostic, prophylactic, therapeutic, and / or nutritional research applications. And a protein comprising a dimeric four helix bundle and its receptor for research applications.

Herscovics et al. FASEB J 7: 540-550, 1993 Larsen et al. J Biol Chem 265: 7055-7061,1990 Gramer et al. Biotechnology 13 (7): 692-8, 1995 Martin et al. Nature Medicine 11 (2): 228-232, 2005

Throughout this specification, unless the text requires otherwise, the term `` comprise '' or variations such as `` comprises '' or `` comprising '' It is understood that the elements or integers described are included, or include groups of elements or integers, but do not exclude any other element or integer, or group of elements or integers.

  Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO :). SEQ ID NO: corresponds to the sequence identifier <400> 1 (SEQ ID NO: 1) <400> 2 (SEQ ID NO: 2) and the like numerically. A summary of the sequence identifier is provided in Table 1. The sequence listing is provided after the claims.

The present invention generally includes a dimeric four-helix bundle that includes a profile of physiochemical parameters that exhibit, relate to, or form the basis of one or more unique pharmacological attributes. Alternatively, the present invention relates to an isolated protein containing the receptor or a chimeric molecule thereof. More particularly, the present invention includes a physiochemical profile comprising a number of measurable physiochemical parameters, namely {[P _x ] ₁ , [P _x ] ₂ , ... [P _x ] _n }. IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, Providing an isolated protein or chimeric molecule thereof selected from the list of IL-10Ra-Fc, wherein P _x represents a measurable physiochemical parameter, and “n” is an integer of ≧ 1; Each parameter between and including [P _x ] ₁ to [P _x ] _n is a different measurable physiochemical parameter, of any one or more measurable physiochemical characteristics. The value indicates or is associated with a specific pharmacological characteristic T _y or a series of specific pharmacological characteristics {[T _y ] ₁ , [T _y ] ₂ , .... [T _y ] _m } or to form the basis, wherein T _y is a distinctive pharmacological traits And, m is an integer ≧ 1, each of _{[T y] 1 ~ [T} y] m, a different pharmacological trait.

As used herein, the term “unique” with respect to the pharmacological properties of a protein of the invention or chimeric molecule thereof is one of the proteins or chimeric molecules thereof that are unique with respect to a particular physiochemical profile, or Refers to multiple pharmacological attributes. In certain embodiments, one or more pharmacological attributes of the isolated protein or chimeric molecule thereof are the same protein or chimeric molecule thereof produced in a prokaryotic or lower eukaryotic cell or a non-human species higher eukaryotic cell. It is different or unique compared to the type. In another embodiment, the pharmacological properties of the subject isolated protein or chimeric molecule thereof contribute to the desired functional outcome. As used herein, the term “measurable physiochemical parameter”, or Px, refers to one or more measurable characteristics of an isolated protein or chimeric molecule thereof. In certain embodiments of the invention, the measurable physiochemical parameter of the subject isolated protein or chimeric molecule thereof contributes to or is otherwise responsible for the induced pharmacological property T _y .

The isolated protein or chimeric molecule thereof of the present invention, when viewed as a whole, includes physiochemical parameters (P _x ) that define the protein molecule or chimeric molecule. The physiochemical parameters are: apparent molecular weight (P ₁ ), isoelectric point (pI) (P ₂ ), number of isoforms (P ₃ ), relative intensity of different numbers of isoforms (P ₄ ), carbohydrate Weight percentage (P ₅ ), molecular weight observed after deglycosylation of N-linked oligosaccharides (P ₆ ), molecular weight observed after deglycosylation of N-linked and O-linked oligosaccharides (P ₇ ), Acid monosaccharide content percentage (P ₈ ), monosaccharide content (P ₉ ), sialic acid content (P ₁₀ ), sulfate and phosphate content (P ₁₁ ), serine / threonine: GalNAc ratio (P ₁₂ ), neutral N-linked oligosaccharide content percentage (P ₁₃ ), acidic N-linked oligosaccharide content percentage (P ₁₄ ), neutral O-linked oligosaccharide content percentage (P ₁₅ ), O-linked oligosaccharide content percentage (P ₁₆ ), N-linked oligosaccharide ratio (P ₁₇ ), O-linked oligosaccharide ratio (P ₁₈ ), N-linked oligosaccharide fraction structure (P _19), O- linked oligosaccharide picture structure (P _20), N- linked oligosaccharides Position and configuration of (P _21), O-linked positions oligosaccharides and configuration (P _22), the translation time modification (P _23), post-translational modification (P _24), acylation (P _25), acetylation ( P ₂₆ ), amidation (P ₂₇ ), deamidation (P ₂₈ ), biotinylation (P ₂₉ ), carbamylation or carbamoylation (P ₃₀ ), carboxylation (P ₃₁ ), decarboxylation (P ₃₂ ) , Disulfide bond formation (P ₃₃ ), fatty acid acylation (P ₃₄ ), myristoylation (P ₃₅ ), palmitoylation (P ₃₆ ), stearoylation (P ₃₇ ), formylation (P ₃₈ ), saccharification (P ₃₉ ) ), Glycosylation (P ₄₀ ), glycophosphatidylinositol anchor (P ₄₁ ), hydroxylation (P ₄₂ ), selenocysteine incorporation (P ₄₃ ), lipidation (P ₄₄ ), lipoic acid addition (P ₄₅ ), methyl (P ₄₆ ), N- or C-terminal blocking (P ₄₇ ), N-terminal or C-terminal removal (P ₄₈ ), nitration (P ₄₉ ), methionine oxidation (P ₅₀ ), phosphorylation (P ₅₁ ), Proteolytic cleavage (P ₅₂ ), prenylation (P ₅₃ ), farnesylation (P ₅₄ ), geranylgeranylation (P ₅₅ ), pyridoxal phosphate addition (P ₅₆ ), sialylation (P ₅₇ ), desialylation ( P _58), sulfated (P _59), ubiquitinylation or ubiquitination (P _60), addition of ubiquitin-like molecules (P _61), the primary structure (P _62), the secondary structure (P _63), tertiary structure (P ₆₄ ), quaternary structure (P ₆₅ ), chemical stability (P ₆₆ ), and thermal stability (P ₆₇ ). A list of these parameters is summarized in Table 2.

In one embodiment, the IFN-a2b of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 kDa, such as about 1-250, and in one embodiment 13-24 kDa Apparent molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4.5 to 7 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2-100 isoforms (P ₃ ), and in one embodiment 2-22 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) such as about 0-99%, such as%, and in one embodiment 0-20%;
-Monosaccharide (P ₉ ) and sialic acid (P ₁₀ ) content when normalized to GalNAc: 1 to 0-3 fucose, 1 to 0-3 GlcNAc, 1 to 0-6 galactose, 1 to 0- 3 mannose and 1 to 0-5 NeuNAc, and in one embodiment 1 to 0-1 fucose, 1 to 0-1 GlcNAc, 1 to 1-4 galactose, 1 to 0-1 mannose and 1 to 0-2 NeuNAc;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 Sialic acid content (P ₁₀ ), expressed as a percentage of monosaccharide content of IFNα2b of the invention, about 0-50%, such as 50%, and in certain embodiments 0-10%;
-60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, about 60-100% such as 100%, and in one embodiment 80-100 % Neutral O-linked oligosaccharide percentage (P ₁₅ );
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40%, such as 40%, and in one embodiment 0-20 The percentage of acidic O-linked oligosaccharides (P ₁₆ ), such as%; and
- different biological activity as human IFN-a2b expressed in a nonhuman cell lines, and in one embodiment, the ability of IFN-a2b of the present invention to inhibit the GM-CSF induced proliferation of TF-1 cells (T ₃₂₎ Is 250-600 times more potent than human IFN-a2b expressed in E. coli cells.

In one embodiment, IFN-b1 of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment 15-40 kDa apparent Molecular weight of (P ₁ );
A pI (P ₂ ) range of about 2 to about 14 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 1 to 50 isoforms; and
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-50%.

In one embodiment, the IFN-g of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment 15-30 kDa apparent Molecular weight (P ₁ );
- at about 2 to about 14, and an embodiment such as 2,3,4,5,6,7,8,9,10,11,12,13,14 4-14 of pI (P ₂₎ ranges ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 4 to 16 isoforms; and
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 61, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 % 0-45% carbohydrate weight percentage in a about 0 to 99%, and an embodiment as (P _5);
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 10-25 kDa, and in one embodiment 12-20 kDa (P ₆ );
The observed molecular weight of the molecule after removal of N-linked and O-linked oligosaccharides of about 10-25 kDa, and in one embodiment 12-20 kDa, (P ₇ );
- including PNG-ase treatment after N-48 and N-120 was identified by PMF (numbering from the start of the signal sequence) N- glycosylation site (P _21); and
-A biological activity different from human IFN-g expressed in non-human cell lines, and in one embodiment, the ability of the IFN-g of the invention to inhibit the growth of HT-29 cells in the presence of TNF-a ( T ₃₂ ) is 11-17 times more potent than human IFN-g expressed in E. coli cells.

In one embodiment, the IFNAR2-Fc of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, in one embodiment, 50-105 kDa apparent Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4 to 7 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 10 to 25 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-50%;
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 40-100 kDa, and in one embodiment 45-95 kDa, (P ₆ ); and
- about 40 to 90 kDa, and in one embodiment of the 45 to 80 kDa, observed molecular weight of the molecule of after the N- linked and O- linked oligosaccharides are removed (P _7).

In one embodiment, IL-10 of the invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment, 10-23 kDa in appearance Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 6 to 10 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 4 to 20 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-20%;
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 8-23 kDa, and in one embodiment 10-23 kDa, (P ₆ );
The observed molecular weight of the molecule after removal of the N-linked and O-linked oligosaccharides of about 8-23 kDa, and in one embodiment 10-23 kDa, (P ₇ );
-A different immunoreactivity profile (T ₁₃ ) than human IL-10 expressed in non-human cell lines, and in one embodiment, the protein concentration of IL-10 of the present invention is expressed in human IL-10 expressed in E. coli cells Underestimated when assayed using an ELISA kit containing -10; and
-A biological activity different from human IL-10 expressed in non-human cell lines, and in one embodiment, the ability of the IL-10 of the invention to induce proliferation of MC-9 cells in the presence of IL-4 ( T ₃₂ ) is 10-25 times more potent than human IL-10 expressed in E. coli cells.

In one embodiment, an IL-10Ra-Fc of the invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, in one embodiment 50-100 kDa apparent Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4.5 to 9.5 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 10 to 21 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-49%;
An observed molecular weight (P ₆ ) of the molecule after removal of the N-linked oligosaccharide of about 35-95 kDa, and in one embodiment 40-85 kDa;
The observed molecular weight of the molecule after removal of N-linked and O-linked oligosaccharides of about 30-95 kDa, and in one embodiment 36-85 kDa (P ₇ );
-Monosaccharide (P ₉ ) and sialic acid (P ₁₀ ) content when normalized to GalNAc: 1 to 0.1-4 fucose, 1 to 2-34 GlcNAc, 1 to 0.5-8 galactose, 1 to 1 13 mannose and 1 to 0-3 NeuNAc; 3 to 0.1-2 fucose, 3 to 0.01-3 GalNAc, 3 to 1-30 GlcNAc, 3 to 0.1-4 galactose when normalized to 3 times the amount of mannose 3 to 0-3 NeuNAc;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 Sialic acid content (P ₁₀ ) expressed as a monosaccharide content percentage of IL-10Rα-Fc of 0-50%, such as 50%, and in one embodiment 0-10%;
-Sulfate and phosphate content when normalized to GalNac (P ₁₁ ): 1 to 0-3 sulfate, and in one embodiment 1 to 0-1.5 sulfate; 3 times the amount of mannose Standardized 3 to 0-1 sulfate, and in one embodiment 3 to 0-0.6 sulfate;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 0-50%, such as 50%, and in one embodiment of 0-3%, sulfation was expressed as a percentage of the monosaccharide content of the molecule (P _59);
-40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 About 40-85, such as 65, 66, 61, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85% %, And in one embodiment 55-75% neutral N-linked oligosaccharide percentage (P ₁₃ );
-15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 About 15-60 like 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60% %, And in one embodiment 25-45% acidic N-linked oligosaccharide percentage (P ₁₄ );
- N-110 was identified by PMF after PNG-ase treatment, N-154, N-177 and N-323 N-glycosylation site containing (numbering from the start of the signal sequence) (P _21); and
-A biological activity different from human IL-10Ra-Fc expressed in a non-human cell line, and in one embodiment the IL of MC / 9 cells in the presence of IL-4 of the IL-10Ra-Fc of the invention The ability to neutralize -10-induced proliferation (T ₃₂ ) is 18-150 times more potent than soluble human IL-10Ra molecules expressed in E. coli cells.

In certain embodiments, the invention provides an isolated form of a protein comprising a four-helix bundle such as IFN-a2B, IFN-b1, IFN-g, IL-10, such as IFNAR2, IL-10Ra The receptor, or a chimeric molecule such as IFN-a2B-Fc, IFN-bl-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc, is contemplated. The isolated protein or chimeric molecule of the present invention has therapeutic efficacy (T ₁ ), effective therapeutic dose (TCID ₅₀ ) (T ₂ ), bioavailability (T ₃ ), dosing interval to maintain therapeutic levels ( T ₄ ), absorption rate (T ₅ ), excretion rate (T ₆ ), specific activity (T ₇ ), thermal stability (T ₈ ), freeze-drying stability (T ₉ ), serum / plasma stability (T ₁₀ ), serum half-life (T ₁₁ ), solubility in the bloodstream (T ₁₂ ), immunoreactivity profile (T ₁₃ ), immunogenicity (T ₁₄ ), inhibition by neutralizing antibodies (T _14A ), side effects ( T ₁₅ ), receptor / ligand binding affinity (T ₁₆ ), receptor / ligand activation (T ₁₇ ), tissue or cell type specificity (T ₁₈ ), biological membranes or barriers (ie, intestine, lung, blood) (Brain barrier, skin, etc.) passage ability (T ₁₉ ), angiogenesis ability (T _19A ), tissue uptake (T ₂₀ ), stability against degradation (T ₂₁ ), stability against freeze-thaw (T ₂₂ ), against protease stability (T _23), safe for ubiquitination Sex (T _24), ease of administration (T _25), mode of administration (T _26), compatibility with other pharmaceutical excipients or carriers (T _27), persistence in organism or environment (T ₂₈₎ Specific pharmacological properties selected from the group comprising, or comprising, storage stability (T ₂₉ ), toxicity in organisms and the environment (T ₃₀ ), and the like.

Furthermore, the protein or chimeric molecule of the present invention includes lymphocytes, erythrocytes, retinal cells, hepatocytes, neurons, keratinocytes, endothelial cells, endoderm cells, ectoderm cells, mesoderm cells, epithelial cells, kidney cells, hepatocytes, Bone cells, bone marrow cells, lymph node cells, epidermis cells, fibroblasts, T-cells, B-cells, plasma cells, natural killer cells, macrophages, granulocytes, neutrophils, Langerhans cells, dendritic cells, eosinic acid Spheres, basophils, breast cells, lobule cells, prostate cells, lung cells, esophageal cells, pancreatic cells, beta cells (insulin secreting cells), hemangioblasts, myocytes, elliptical cells (hepatocytes), mesenchymal cells, Various stem cells, including brain microvascular endothelial cells, astrocytes, glial cells, adult and fetal stem cells, human primary cultured cells such as various progenitor cells, and other human immortalized, transformed, or Including but cell lines but are not limited to, it may have a different altered biological effects on cell type (T _31).

Biological effects on cells include proliferation (T ₃₂ ), differentiation (T ₃₃ ), apoptosis (T ₃₄ ), cell size growth (T ₃₅ ), cytokine adhesion (T ₃₆ ), cell adhesion (T ₃₇ ), Cell spreading (T ₃₈ ), cell motility (T ₃₉ ), migration and invasion (T ₄₀ ), chemotaxis (T ₄₁ ), cell entrapment (T ₄₂ ), signal transduction (T ₄₃ ), Recruitment of proteins to receptors / ligands (T ₄₄ ), activation of JAK / STAT pathway (T ₄₅ ), activation of Ras-erk pathway (T ₄₆ ), activation of AKT pathway (T ₄₇ ), PKC pathway Activation (T ₄₈ ), PKA pathway activation (T ₄₉ ), src activation (T ₅₀ ), fas activation (T ₅₁ ), TNFR activation (T ₅₂ ), NFkB activation (T _53), activation of p38MAPK (T _54), activation of c-fos (T _55), secretion (T _56), internalization of the receptor (T _57), the crosstalk of the receptor (T _58), Surface marker up or down regulation (T ₅₉ ), FACS forward / side scatter profile change (T ₆₀ ), subgroup ratio change ( _T61 ), differential gene expression ( _T62 ), cell necrosis ( _T63 ), cell clumping ( _T64 ), cell repulsion ( _T65 ), heparin sulfate Binding (T ₆₆ ), binding to glycosylated structures (T ₆₇ ), binding to chondroitin sulfate (T ₆₈ ), binding to extracellular matrix (like collagen, fibronectin) (T ₆₉ ), artificial materials (like scaffolds) ) Binding (T ₇₀ ), carrier binding (T ₇₁ ), cofactor binding (T ₇₂ ), stem cell proliferation, differentiation, and / or self-renewal effects alone or in combination with other proteins ( T ₇₃ ) and the like are included. These are summarized in Table 3.

  The invention further includes an isolated protein, or fragment thereof, such as the extracellular domain of a membrane bound protein, linked to the constant region (Fc) or framework region of a human immunoglobulin by one or more protein linkers. Chimeric molecules are provided. Such chimeric molecules are also referred to herein as protein-Fc. Examples of such protein-Fc contemplated by the present invention include IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc Is included.

  Such protein-Fc has a profile of measurable physiochemical parameters that exhibit or are associated with one or more unique pharmacological properties of the isolated protein-Fc. Other chimeric molecules contemplated by the present invention include proteins, protein-Fc, or fragments thereof linked to a lipid moiety, such as a polyunsaturated fatty acid molecule. Such lipid moieties may be linked to amino acid residues in the backbone of the molecule or to the side chains of such amino acid residues.

  The invention further relates to an isolated protein, or an extracellular domain of a membrane-bound protein linked to a mammalian immunoglobulin constant region (Fc) or framework region through one or more protein linkers. Chimeric molecules comprising the fragments are provided. In another aspect, mammalian immunoglobulin Fc or framework regions are primates, livestock animals (e.g., cattle, sheep, pigs, horses, donkeys), laboratory animals (including humans, marmosets, orangutans, and gorillas). For example, a mammal selected from the group consisting of mice, rats, guinea pigs, hamsters, rabbits, companion animals (eg, cats, dogs), and captured wild animals (eg, rodents, foxes, deer, kangaroos) In another embodiment, the Fc or framework region is a human immunoglobulin, In a particular embodiment, the mammal is a human, and such chimeric molecules are also referred to herein as protein-Fc. Other chimeric molecules contemplated by the present invention include lipid moieties such as polyunsaturated cellular acid molecules. A protein, protein-Fc, or fragment thereof linked to is included, such a lipid moiety may be linked to an amino acid residue in the backbone of the molecule, or to the side chain of such an amine draft residue. -a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc It has a profile of measurable physiochemical parameters that exhibit or are associated with multiple unique pharmacological attributes.

  Accordingly, the present invention relates to SEQ ID NOs: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, A list consisting of 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, at least one of the arrangements of the foregoing list and at least An isolation encoded by a nucleotide sequence having about 65% identity, or a nucleotide sequence selected from nucleotide sequences that can hybridize with low-stringency conditions to any one of the foregoing sequences or its complementary form A polypeptide is provided.

  Another aspect of the invention provides an isolated polypeptide encoded by a nucleotide sequence selected from the list consisting of SEQ ID NOs: 151, 152, 153, 154 after splicing of its respective mRNA species by a cellular process To do.

  Yet another aspect of the present invention is SEQ ID NO: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, An amino acid sequence selected from the list consisting of 129, 132, 135, 138, 141, 144, 147, 150, or an amino acid sequence having at least about 65% similarity to one or more of the foregoing sequences A released polypeptide is provided.

  The present invention further contemplates a pharmaceutical composition comprising at least a portion of a protein or chimeric molecule thereof together with a pharmaceutically acceptable carrier, cofactor, and / or diluent.

  Regarding primary structure, the present invention relates to SEQ ID NOs: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, Nucleotide sequence selected from the list consisting of 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, the aforementioned sequence A nucleotide sequence having at least about 60% identity with any one of the above, or a nucleotide sequence capable of hybridizing under low stringency conditions to any one of the aforementioned sequences or their complementary forms. A deproteinized protein, chimeric molecule thereof, or fragment thereof is provided.

  Yet another aspect of the invention is SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, At least 60% selected from the list consisting of 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 Similar nucleotide sequences, or after optimal alignment and / or SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, or their complementary types One or more and low Comprising a nucleotide sequence capable of hybridizing with Rinjenshi conditions, proteins, provides an isolated nucleic acid molecule encoding the chimeric molecule or a functional part,.

  In certain embodiments, the present invention provides SEQ ID NOs: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129 , 132, 135, 138, 141, 144, 147, 150, an amino acid sequence substantially as described in one or more, or SEQ ID NO: 28, 30, 32, 34, 36, 40, after alignment, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150 and at least about 60% Proteins with similar amino acid sequences, including dimeric four helix bundles such as IFN-a2B, IFN-bl, IFN-g, IL-10, such as IFNAR2, IL-10Ra Receptor or directed to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a fragment thereof.

  In another aspect, the invention provides a human immunoglobulin substantially as described in one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. SEQ ID NO: 29 linked directly to a nucleotide sequence encoding the constant region (Fc) or framework region of or linked by one or more nucleotide sequences encoding a protein linker known in the prior art, IFN comprising a nucleotide sequence selected from the group consisting of 31, 41, 43, 45, 47, 61, 63, 65, 67, 81, 83, 101, 103, 115, 116, 118, 119, 121, 122 -a2B, IFN-bl, IFN-g, a protein containing a 4-dimer helix bundle such as IL-10, its receptor such as IFNAR2, IL-10Ra, or IFN-a2B-Fc, IFN- a chimeric molecule thereof such as bl-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc, or a fragment thereof Provides an isolated nucleic acid molecule that over de. In certain embodiments, the nucleotide sequence encoding the protein linker comprises a nucleotide sequence selected from IP, GSSNT, TRA or VDGIQWIP.

  In another aspect, the invention provides a human immunoglobulin constant substantially as described in one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. SEQ ID NOs: 30, 32, 42, 44, 46, 48, 62 linked directly to a region (Fc) or framework region, or linked by one or more protein linkers known in the prior art Two, such as IFN-a2B, IFN-bl, IFN-g, IL-10, comprising an amino acid sequence selected from the group consisting of 64, 66, 68, 82, 84, 102, 104, 117, 120, 123 Isolated protein containing a four-mer helix bundle, its receptor such as IFNAR2, IL-10Ra, or IFN-a2B-Fc, IFN-bl-Fc, IFN-g-Fc, IFNAR2-Fc, IL- 10-Fc, a chimeric molecule thereof, such as IL-10Ra-Fc, or a fragment thereof is provided.

  In one embodiment, a chimeric IFN-a2B molecule, such as an IFN-a2B or IFN-a2B-Fc of the invention, is a SEQ ID NO selected from SEQ ID NOs: 32 and 36, wherein “n” is 24 ± 5 Of the amino acid sequence starting from the nth amino acid.

  In one embodiment, a chimeric IFN-bl molecule, such as an IFN-b1 or IFN-b1-Fc of the invention, is from SEQ ID NOs: 46, 48, 54, and 56, where “n” is 22 ± 5 It includes an amino acid sequence starting from the nth amino acid of the selected SEQ ID NO.

  In one embodiment, a chimeric IFN-g molecule, such as an IFN-g or IFN-g-Fc of the invention, is from SEQ ID NOs: 66, 68, 74, and 76, where “n” is 21 ± 5 It includes an amino acid sequence starting from the nth amino acid of the selected SEQ ID NO.

  In one embodiment, a chimeric IFNAR2 molecule, such as IFNAR2 or IFNAR2-Fc of the invention, is of SEQ ID NO: selected from SEQ ID NOs: 84, 92, 94, and 96, where “n” is 27 ± 5 Contains the amino acid sequence starting from the nth amino acid.

  In one embodiment, a chimeric IL-10 molecule, such as IL-10 or IL10-Fc of the invention, has an n of SEQ ID NO: selected from SEQ ID NOs: 104 and 108, wherein “n” is 19 ± 5 Contains the amino acid sequence starting at the amino acid number.

  In one embodiment, a chimeric IL-10Ra molecule, such as IL-10Ra or IL-10Ra-Fc of the invention, has an “n” of 22 ± 5 or 32 ± 5, SEQ ID NO: 123, 144, 147 , And an amino acid sequence starting from the nth amino acid of SEQ ID NO: selected from 150.

  The invention is further extended to the use of isolated proteins, chimeric molecules thereof, or nucleic acid molecules encoding them in diagnostic, prophylactic, therapeutic, nutritional and / or research applications. More particularly, the present invention treats or prevents a condition in an animal subject or ameliorates a symptom of the condition in an animal subject, comprising administering to the animal subject an effective amount of an isolated protein or chimeric molecule thereof. Expanded to how to do.

  Furthermore, the present invention extends to the use of a protein or chimeric molecule thereof for screening small molecules that may have a variety of diagnostic, preventive, therapeutic, nutritional and / or research applications.

  The present invention further contemplates using an isolated protein or chimeric molecule thereof as an immunogen to produce antibodies for therapeutic or diagnostic applications.

  The present invention further contemplates using the isolated protein or chimeric molecule thereof in a culture medium for stem cells used in stem cells or related therapies.

  The invention also provides for the use of proteins or chimeric molecules in the manufacture of formulations for diagnostic, prophylactic, therapeutic, nutritional and / or research applications.

  The present invention also provides a human-derived protein or chimeric molecule thereof for use as a standard protein in immunoassays and kits thereof. The invention also extends to a method for determining the level of human cell expressed human protein or chimeric molecule thereof in a biological preparation.

(Table 1) Sequence identifier

(Table 2) List of physiochemical parameters

(Table 3) List of pharmacological traits

A list of abbreviations commonly used herein is provided in Tables 4 and 5.
(Table 4) Abbreviations and substitute names

Table 5: Abbreviations for amino acids

(Table 6) List of stem cells

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Unless otherwise stated, the invention is limited to specific formulations, manufacturing methods, diagnostic methods, assay protocols, nutrition protocols, or research protocols, etc., which may vary. It should be understood that not. Similarly, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not to be construed as limiting.

  As used herein, the singular forms “a”, “an” and “the” unless the text indicates otherwise. Note that multiple aspects are involved. Thus, for example, reference to “protein”, “cytokine”, “chimeric molecule”, or “receptor” includes two proteins or more with one protein, cytokine, receptor, or chimeric molecule, Cytokines, receptors, or chimeric molecules are included; “physiochemical parameters” include two or more parameters with one parameter.

  The terms “compound”, “active substance”, “chemical substance”, “pharmacologically active substance”, “drug”, “active” and “drug” are particularly desirable to refer to chemical compounds herein. Used interchangeably to refer to a protein or chimeric molecule thereof that induces a pharmacological and / or physiological effect. The term also includes the pharmaceuticals of those active substances specifically mentioned herein, including but not limited to salts, esters, amides, prodrugs, active metabolites, analogs, and the like. Containing pharmacologically active ingredients that are pharmaceutically acceptable. Where the terms “compound”, “active substance”, “chemical substance”, “pharmacologically active substance”, “drug”, “active” and “drug” are used, this term is pharmaceutically associated with the active substance itself. It should be understood that acceptable pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs and the like are included.

  References to “compound”, “active substance”, “chemical substance”, “pharmacologically active substance”, “drug”, “active” and “drug” include two, such as two or more cytokines. Or more active agent combinations are included. “Combination” also includes multiple parts, such as a two-part composition, in which the substances are provided individually and given or dispensed, or thoroughly mixed together prior to dosing.

  For example, a multi-part pharmaceutical pack can be a separately maintained four-helix bundle such as IFN-a2B, IFN-b1, IFN-g, IL-10, or IFN-a2B, IL-10Ra Its respective receptors like, or their chimeric molecules like IFN-a2B-Fc, IFN-b1-Fc, IFN-g-Fc, IFNAR2-Fc, IL-10-Fc, IL-10Ra-Fc You may have two or more proteins including

  As used herein, the terms “effective amount” and “therapeutically effective amount” are used alone or in combination with other substances to provide the desired therapeutic effect, physiological effect or outcome. Mean sufficient amount of protein or chimeric molecule thereof. Undesirable effects, such as side effects, sometimes appear with the desired therapeutic effect. Thus, when determining what is an appropriate “effective amount”, the physician will consider possible benefits against possible risks. The exact amount required will vary from subject to subject, depending on the subject's species, age and general condition, mode of administration, and the like. Thus, it may not be possible to specify an exact “effective amount”. However, an appropriate “effective amount” in any individual case may be determined by one of ordinary skill in the art using only routine experimentation.

  “Pharmaceutically acceptable” carrier, excipient, or diluent means a pharmaceutical medium that contains a material that is not biologically or otherwise undesirable, ie, the material can be any or substantially May be administered to a subject with a selected active agent without causing any adverse reactions. The carrier may include excipients and other additives such as diluents, detergents, colorants, wetting or emulsifying agents, pH buffering agents, preservatives and the like.

  Similarly, a “pharmaceutically acceptable” salt, ester, amide, prodrug, or derivative of a compound provided herein is a salt, ester, amide that is not biologically or otherwise undesirable. , Prodrugs, or derivatives.

  As used herein, the terms “treat” and “treatment” refer to a reduction in the severity and / or number of symptoms of the condition being treated, elimination of symptoms and / or underlying causes, symptoms of the condition And / or prevention of the occurrence of the underlying cause, as well as amelioration, treatment, or recovery of damage after the condition.

  “Treating” a subject may treat a clinically symptomatic individual by ameliorating the symptoms of the condition, as well as preventing the condition or other adverse physiological event in the susceptible individual.

  As used herein, a “subject” refers to an animal, in a particular embodiment, a mammal, and in a further embodiment, a human who can benefit from the pharmaceutical formulations and methods of the invention. There are no restrictions on the types of animals that could benefit from the pharmaceutical formulations and methods described in this invention. A subject, whether a human or non-human animal, is called an individual, patient, animal, host, or recipient. The compounds and methods of the present invention have applications in human and veterinary medicine, generally in conjunction with livestock or wildlife management.

  As indicated above, in certain embodiments, animals are humans or other primates such as orangutans, gorillas, marmosets, farm animals, laboratory animals, companion animals, or captured wild animals, and birds. .

  Examples of laboratory animals include mice, rats, rabbits, guinea pigs, and hamsters. Rabbits and rodents such as rats and mice provide a convenient test system or animal model. Livestock animals include sheep, cows, pigs, goats, horses, and donkeys. Non-mammalian, prokaryotic and non-mammalian eukaryotic cells such as birds, fish and amphibians including Xenopus species.

  The term “cytokine” is used in its most general sense, which includes regulating the immune system, modulating the functional activity of individual cells and / or tissues, and / or a series of physiological responses Any of a variety of proteins that are secreted by the cell are included. As used herein, the term “cytokine” includes one or more amino acid additions, deletions, or substitutions thereof, but fragments thereof that substantially retain the biological activity of the complete cytokine, It should be understood to refer to “complete” cytokines along with derivatives, homologues or chimeras.

  A “cytokine receptor” is a cell membrane associated or soluble portion of a cytokine receptor involved in cytokine signaling or regulation. As used herein, the term “cytokine receptor” includes one or more amino acid additions, deletions, or substitutions, but substantially retains the biological activity of the complete cytokine receptor. It should be understood that it refers to a “perfect” cytokine receptor together with its fragments, derivatives, homologues or chimeras.

  The term “protein” is used in its broadest sense and includes cytokines and cytokine receptors. As used herein, the term “protein” includes one or more amino acid additions, deletions, or substitutions thereof, but fragments thereof that substantially retain the biological activity of the complete protein, It should be understood to refer to a “perfect” protein together with a derivative, homologue or chimera.

The present invention provides a profile of measurable physiochemical parameters (P _x ) that exhibits, is related to or forms the basis of one or more unique pharmacological attributes (T _y ). Contemplated is an isolated protein or chimeric molecule thereof. Isolated proteins or chimeric molecules are IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL- A protein comprising a four-dimer helix bundle or a receptor thereof selected from the group comprising 10-Fc, IL-10Ra, and IL-10Ra-Fc. As used herein, IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL The terms -10-Fc, IL-10Ra, IL-10Ra-Fc include references to the entire polypeptide as well as fragments thereof.

More particularly, the present invention provides a measurable physiochemical parameter, ie a physiochemical profile comprising an array of {[P _x ] ₁ , [P _x ] ₂ ,... [P _x ] _n }. An isolated protein or chimeric molecule thereof, wherein P _x represents a measurable physiochemical parameter, “n” is an integer of ≧ 1, and [P _x ] ₁ to [P _x ] Each of _n is a different measurable physiochemical parameter, and the value of any one or more measurable physiochemical features can be a unique pharmacological characteristic _Ty or a number of unique pharmacological parameters. The properties {[T _y ] ₁ , [T _y ] ₂ , .... [T _y ] _m } indicate, are related to or form the basis of, and T _y has a specific pharmacological characteristic. M is an integer of ≧ 1, and each of [T _y ] ₁ to [T _y ] _m is a different pharmacological characteristic.

As used herein, the term “measurable physiochemical parameter” (P _x ) refers to one or more measurable characteristics of an isolated protein or chimeric molecule thereof. Examples of “specific physiochemical parameters” include apparent molecular weight (P ₁ ), isoelectric point (pI) (P ₂ ), number of isoforms (P ₃ ), relative intensity of different numbers of isoforms (P ₄ ), weight percentage of carbohydrates (P ₅ ), molecular weight observed after deglycosylation of N-linked oligosaccharides (P ₆ ), deglycosylation of N-linked and O-linked oligosaccharides Later observed molecular weight (P ₇ ), percentage content of acidic monosaccharide (P ₈ ), monosaccharide content (P ₉ ), sialic acid content (P ₁₀ ), sulfate and phosphate content (P ₁₁ ), Serine / threonine: GalNAc ratio (P ₁₂ ), neutral N-linked oligosaccharide content percentage (P ₁₃ ), acidic N-linked oligosaccharide content percentage (P ₁₄ ), neutral O-linked type Oligosaccharide content percentage (P ₁₅ ), acidic O-linked oligosaccharide content percentage (P ₁₆ ), N-linked oligosaccharide ratio (P ₁₇ ), O-linked oligosaccharide ratio (P ₁₈ ), N - linked oligosaccharide picture structure (P _19), O- linked oligosaccharide fractions of structure (P _20), the position and configuration of the N- linked oligosaccharides (P _21), the position and configuration of O- linked oligosaccharides (P _22), the translation time modification (P _23), post-translational modification (P ₂₄₎ Acylation (P ₂₅ ), acetylation (P ₂₆ ), amidation (P ₂₇ ), deamidation (P ₂₈ ), biotinylation (P ₂₉ ), carbamylation or carbamoylation (P ₃₀ ), carboxylation ( P _31), decarboxylation (P _32), disulfide bond formation (P _33), fatty acid acylation (P _34), myristoylated (P _35), palmitoyl (P _36), stearoylation (P _37), formylation (P _38), glycated (P _39), glycosylation (P _40), glycophosphatidylinositol anchor (P _41), hydroxylated (P _42), incorporation of selenocysteine (P _43), lipidated (P ₄₄ ), Lipoic acid addition (P ₄₅ ), Methylation (P ₄₆ ), N-terminal or C-terminal blocking (P ₄₇ ), N-terminal or C-terminal removal (P ₄₈ ), Nitration (P ₄₉ ), Methionine Oxidation (P ₅₀ ), phosphorylation (P ₅₁ ), proteolytic cleavage (P ₅₂ ), prenylation (P ₅₃ ), farnesylation (P ₅₄ ), geranylgeranylation (P ₅₅ ), pyridoxal phosphate addition (P ₅₆ ) , Sialylated (P ₅₇ ), desialylated (P ₅₈ ), sulfated (P ₅₉ ), ubiquitinated or ubiquitinated (P ₆₀ ), addition of ubiquitin-like molecules (P ₆₁ ), primary structure (P ₆₂ ), Secondary structure (P ₆₃ ), tertiary structure (P ₆₄ ), quaternary structure (P ₆₅ ), chemical stability (P ₆₆ ), thermal stability (P ₆₇ ) are included, but are not limited to these Absent. A summary of these parameters is provided in Table 2.

It will be readily appreciated by those skilled in the art that the term “specific pharmacological attributes” includes any pharmacologically or clinically relevant property of the protein or chimeric molecule of the present invention. Examples of `` pharmacological attributes '' that never limit the present invention include therapeutic efficacy (T ₁ ), effective therapeutic dose (TCID ₅₀ ) (T ₂ ), bioavailability (T ₃ ), therapeutic level. Dosing interval to maintain (T ₄ ), absorption rate (T ₅ ), excretion rate (T ₆ ), specific activity (T ₇ ), thermal stability (T ₈ ), freeze-drying stability (T ₉ ), Serum / plasma stability (T ₁₀ ), serum half-life (T ₁₁ ), solubility in bloodstream (T ₁₂ ), immunoreactivity profile (T ₁₃ ), immunogenicity (T ₁₄ ), inhibition by neutralizing antibodies (T _14A ), side effects (T ₁₅ ), receptor / ligand binding affinity (T ₁₆ ), receptor / ligand activation (T ₁₇ ), tissue or cell type specificity (T ₁₈ ), biological membranes or barriers ( That is, intestinal tract, lung, blood-brain barrier, skin, etc.) passage ability (T ₁₉ ), angiogenesis ability (T _19A ), tissue uptake (T ₂₀ ), stability against degradation (T ₂₁ ), stability against freezing and thawing (T _22), stability to proteases (T _23), ubiquitin Stability against reduction (T _24), ease of administration (T _25), mode of administration (T _26), compatibility with other pharmaceutical excipients or carriers (T _27), persistence in organism or environment ( T ₂₈ ), storage stability (T ₂₉ ), biological and environmental toxicity (T ₃₀ ) and the like.

Furthermore, the protein or chimeric molecule of the present invention includes lymphocytes, erythrocytes, retinal cells, hepatocytes, neurons, keratinocytes, endothelial cells, endoderm cells, ectoderm cells, mesoderm cells, epithelial cells, kidney cells, hepatocytes, bones Cells, bone marrow cells, lymph node cells, epidermal cells, fibroblasts, T-cells, B-cells, plasma cells, natural killer cells, macrophages, neutrophils, granulocytes, Langerhans cells, dendritic cells, eosinophils , Basophils, breast cells, lobule cells, prostate cells, lung cells, esophageal cells, pancreatic cells, beta cells (insulin secreting cells), hemangioblasts, myocytes, elliptical cells (hepatocytes), mesenchymal cells, brain Various stem cells, including microvascular endothelial cells, astrocytes, glial cells, adult and fetal stem cells, human primary cultured cells such as various progenitor cells, and other human immortalized, transformed, or cancer Including but胞株but are not limited to, may have a different altered biological effects on cell type (T _31). Biological effects on cells include proliferation (T ₃₂ ), differentiation (T ₃₃ ), apoptosis (T ₃₄ ), cell size growth (T ₃₅ ), cytokine adhesion (T ₃₆ ), cell adhesion (T _37), cell spreading (T _38), cell motility (T _39), migration and invasion (T _40), chemotaxis (T _41), wrapping of the cells (T _42), signaling (T ₄₃₎ , Protein mobilization to receptors / ligands (T ₄₄ ), JAK / STAT pathway activation (T ₄₅ ), Ras-erk pathway activation (T ₄₆ ), AKT pathway activation (T ₄₇ ), PKC pathway Activation (T ₄₈ ), PKA pathway activation (T ₄₉ ), src activation (T ₅₀ ), fas activation (T ₅₁ ), TNFR activation (T ₅₂ ), NFkB activation ( T _53), activation of p38MAPK (T _54), activation of c-fos (T _55), secretion (T _56), internalization of the receptor (T _57), the crosstalk of the receptor (T ₅₈₎ Surface marker up or down regulation (T ₅₉ ), FACS forward / side scatter profile change (T ₆₀ ), subgroup ratio change ( _T61 ), differential gene expression ( _T62 ), cell necrosis ( _T63 ), cell clumping ( _T64 ), cell repulsion ( _T65 ), heparin sulfate Binding (T ₆₆ ), binding to glycosylated structures (T ₆₇ ), binding to chondroitin sulfate (T ₆₈ ), binding to extracellular matrix (like collagen, fibronectin) (T ₆₉ ), artificial materials (like scaffolds) ) Binding (T ₇₀ ), carrier binding (T ₇₁ ), cofactor binding (T ₇₂ ), stem cell proliferation, differentiation, and / or self-renewal effects alone or in combination with other proteins ( T ₇₃ ) and the like are included. A summary of these attributes is provided in Table 3.

  As used herein, the term “unique” with respect to the pharmacological properties of a protein or chimeric molecule of the present invention refers to one or more of the protein or chimeric molecule thereof that is unique with respect to a particular physiochemical profile. Refers to the pharmacological characteristics of In certain embodiments, one or more pharmacological attributes of the isolated protein or chimeric molecule thereof are the same protein or chimeric molecule produced in prokaryotic or lower eukaryotic cells, or higher non-human eukaryotic cells. Compared to the type of, it is different or unique. In certain embodiments, the pharmacological characteristics of the subject isolated protein or chimeric molecule thereof are substantially similar or functionally equivalent to the naturally occurring protein.

  As used herein, the term “prokaryotic cell” refers to any prokaryotic cell, including any bacterial cell (including actinobacterial cells) or archaeal cells. As used herein, the meaning of the term “non-mammalian eukaryotic cell” is self-evident. However, for clarity, this term specifically includes yeasts such as Saccharomyces species, Pichea species; other fungi; insects including Drosophila species, and insect cells Cultures; fish including Danio species; amphibians including Xenopus species; any non-mammalian eukaryotic cells including plants and plant cell cultures.

  Reference to “stem cells” includes fetal and adult stem cells, including the stem cells listed in Table 6. The protein or chimeric molecule of the present invention may be used alone or in a cocktail of proteins to induce one or more stem cell proliferation, differentiation, or self-renewal.

  The primary structure of a protein or chimeric molecule thereof may be measured as an amino acid sequence. Secondary structure may be measured as the number and / or relative position of one or more protein secondary structures such as α-helices, parallel β-sheets, antiparallel β-sheets or turns. Tertiary structure describes the folding of a polypeptide chain to build different secondary structural elements in a specific configuration. When the helix and sheet are units of secondary structure, the domain is a unit of tertiary structure. In multidomain proteins, tertiary structure includes the arrangement of domains relative to each other. Thus, tertiary structure may be measured as the presence, absence, number, and / or relative position of one or more protein “domains”. Examples of domains that in no way limit the invention include isolated helix, helix-turn-helix domain, 4 helix bundle, DNA binding domain, 3 helix bundle, Greek key helix bundle, helix -Helix Packing Domain, β-Sandwich, Aligned β Sandwich, Orthogonal β Sandwich, β Barrel, Upside Down β Sheet, Greek Key Topology Domain, Jelly Roll Topology Domain, β Propeller, β Trefoil, β Helix, Rothman Fold , Α / β horseshoe, α / β barrel, α + β topology, disulfide rich fold, serine protease inhibitor domain, sea anemone toxin domain, EGF-like domain, complement C-module domain, wheat plant toxin protein, cobra (cobra) God Poison domain, Green Mamba anti-cholinesterase domain, kringle domains, mucin-like area, globular domain, include the spacer region. Quaternary structure is described as the arrangement of different polypeptide chains within the protein structure, with each chain carrying individual primary, secondary, and tertiary structural elements. Examples include either homo- or hetero-oligomer polymerization (eg, dimer formation or trimer formation). In one embodiment, the molecule of the invention is an IFN-a2B, IFN-a2B-Fc, IFN-bl, IFN-bl-Fc, IFN-g present as a homo- or heterodimer, trimer, or oligomer. , IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL-10-Fc, IL-10Ra, and IL-10Ra-Fc.

  Regarding the primary structure, the present invention relates to SEQ ID NOs: 27, 29, 31, 33, 35, 39. 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149, any one of the arrangements in the above list A nucleotide sequence having at least about 60% identity, or a nucleotide sequence capable of hybridizing under low stringency conditions to any one of the aforementioned sequences or a complementary form thereof. Isolated protein, chimeric molecule, or fragment thereof.

  Another aspect of the invention provides an isolated polypeptide encoded by a nucleotide sequence selected from the list consisting of SEQ ID NOs: 151, 152, 153, 154 after splicing of its respective mRNA species by a cellular process. provide.

  Yet another aspect of the invention is SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, At least 60% selected from the list consisting of 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 Similar nucleotide sequences, or after optimal alignment and / or SEQ ID NOs: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116 118,119,121,122,124,125,127,128,130,131,133,134,136,137,139,140,142,143,145,146,148,149 or its complementary type One or more and low stream Comprising a nucleotide sequence capable of hybridizing with Regency conditions, proteins, provides an isolated nucleic acid molecule encoding the chimeric molecule or a functional part,.

  In certain embodiments, the invention provides that the amino acid sequence is SEQ ID NO: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, substantially as described in SEQ ID NO: 28, 30, 32, 34, 36, after optimal alignment 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150 and at least about 60 Is directed to an isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein, chimeric molecule thereof, or fragment thereof, having a percent similarity.

  In another aspect, the invention provides a human immunoglobulin substantially as described in one or more of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19. SEQ ID NO: 29 linked directly to a nucleotide sequence encoding the constant region (Fc) or framework region of or linked by one or more nucleotide sequences encoding a protein linker known in the prior art, A protein comprising a nucleotide sequence selected from the group consisting of 31, 41, 43, 45, 47, 61, 63, 65, 67, 81, 83, 101, 103, 115, 116, 118, 119, 121, 122 An isolated nucleic acid molecule encoding the molecule, or fragment thereof, is provided. In certain embodiments, the nucleotide sequence encoding the protein linker comprises a nucleotide sequence selected from IP, GSSNT, TRA or VDGIQWIP.

  In another aspect, the present invention relates to human immunity substantially as described in one or more of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20. SEQ ID NOs: 30, 32, 42, 44 linked directly to a globulin constant region (Fc) or framework region, or linked by one or more protein linkers known in the art An isolated protein molecule or fragment thereof comprising an amino acid sequence selected from the group consisting of 46, 48, 62, 64, 66, 68, 82, 84, 102, 104, 117, 120, 123 is provided.

  In one embodiment, a chimeric IFN-a2B molecule, such as an IFN-a2B or IFN-a2B-Fc of the invention, is a SEQ ID NO selected from SEQ ID NOs: 32 and 36, wherein “n” is 24 ± 5 Of the amino acid sequence starting from the nth amino acid.

  In one embodiment, a chimeric IFN-bl molecule, such as an IFN-b1 or IFN-b1-Fc of the invention, is from SEQ ID NOs: 46, 48, 54, and 56, where “n” is 22 ± 5 It includes an amino acid sequence starting from the nth amino acid of the selected SEQ ID NO.

  In one embodiment, a chimeric IFN-g molecule, such as an IFN-g or IFN-g-Fc of the invention, is from SEQ ID NOs: 66, 68, 74, and 76, where “n” is 21 ± 5 It includes an amino acid sequence starting from the nth amino acid of the selected SEQ ID NO.

  In one embodiment, a chimeric IFNAR2 molecule, such as IFNAR2 or IFNAR2-Fc of the invention, is of SEQ ID NO: selected from SEQ ID NOs: 84, 92, 94, and 96, where “n” is 27 ± 5 Contains the amino acid sequence starting from the nth amino acid.

  In one embodiment, a chimeric IL-10 molecule, such as IL-10 or IL10-Fc of the invention, has an n of SEQ ID NO: selected from SEQ ID NOs: 104 and 108, wherein “n” is 19 ± 5 Contains the amino acid sequence starting at the amino acid number.

  In one embodiment, a chimeric IL-10Ra molecule, such as IL-10Ra or IL-10Ra-Fc of the invention, has an “n” of 22 ± 5 or 32 ± 5, SEQ ID NO: 123, 144, 147 , And an amino acid sequence starting from the nth amino acid of SEQ ID NO: selected from 150.

  Another aspect of the invention is SEQ ID NO: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129 , 132, 135, 138, 141, 144, 147, 150, or an amino acid sequence having at least about 65% similarity to one or more of the foregoing sequences, An isolated protein, chimeric molecule thereof, or fragment thereof is provided.

  In certain embodiments, the amino acid similarity (%) or nucleotide identity level includes at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least About 67%, at least about 68%, at least about 69%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least About 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least About 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, low It includes at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% similarity or identity.

  A “derivative” of a polypeptide of the invention also includes a portion or portion of a full-length parent polypeptide that retains the partial transcriptional activity of the parent polypeptide, including variants. Such “biologically active fragments” include deletion mutants and small peptides, eg, at least 10, in certain embodiments at least 20, and in further embodiments at least 30 contiguous amino acids that exhibit the essential activity. Is included. This type of peptide may be obtained by application of standard recombinant nucleic acid techniques or may be synthesized using conventional liquid phase or solid phase synthesis techniques. For example, see the solution or solid phase synthesis described in Chapter 9 entitled “Peptide Synthesis” by Atherton and Shephard, which is included in a publication entitled “Synthetic Vaccines” edited by Nicholson published by Blackwell Scientific Publications. Also good. Alternatively, peptides can be produced by digesting the amino acid sequences of the present invention with proteinases such as endoLys-C, endoArg-C, endoGlu-C and staphylococcus V8 protease. The digested fragment can be purified, for example, by high performance liquid chromatography (HPLC) techniques. It is understood that any such fragment is included in the term “derivative” as used herein, regardless of the means by which it is produced.

  Thus, the term “variant” refers to a nucleotide sequence that exhibits substantial sequence identity with a reference nucleotide sequence or polynucleotide that hybridizes with the reference sequence under stringency conditions as defined later herein. The terms “nucleotide sequence”, “polynucleotide” and “nucleic acid molecule” may be used interchangeably herein, where one or more nucleotides are added, deleted, or replaced with different nucleotides. A polynucleotide. In this regard, certain changes, including mutations, additions, deletions, and substitutions, can be made to the reference nucleotide sequence, whereby the altered polynucleotide is an organism of the reference polynucleotide or encoded polypeptide. Retain function or activity. The term “variant” also includes naturally occurring allelic variants.

  The nucleic acid molecules of the present invention may be in the form of a vector or other nucleic acid construct.

  In one embodiment, the vector is DNA and may optionally contain a selectable marker.

  Examples of selectable markers include genes that confer resistance to compounds such as antibiotics, genes that confer growth on selected substrates, and genes that encode proteins that produce a detectable signal such as luminescence Is included. A variety of such markers are known and available, including for example antibiotic resistance genes such as the neomycin resistance gene (neo) and the hygromycin resistance gene (hyg). Selectable markers also include specific media substrates, such as the tk gene (thymidine kinase) or hprt gene (hypoxanthine phosphoribosyltransferase) that confer growth potential on HAT media (hypoxanthine, aminopterin, and thymidine). And a bacterial gpt gene (guanine / xanthine phosphoribosyltransferase) that allows growth in MAX medium (mycophenolic acid, adenine, and xanthine). Other selectable markers for use in mammalian cells and plasmids with a variety of selectable markers are described in Sambrook et al. Molecular Cloning-A Laboratory Manual, Cold Spring Harbour, New York, USA, 1990.

  The selectable marker may depend on its own promoter for expression, and the marker gene may be derived from an organism that is very different from the targeted organism (e.g., used in target mammalian cells). Prokaryotic marker gene). However, it is preferred to replace the original promoter with a transcriptional mechanism that is known to function in recipient cells. A number of transcription initiation regions are available for such purposes, including, for example, metallothionein promoter, thymidine kinase promoter, β-actin promoter, immunoglobulin promoter, SV40 promoter, and human cytomegalovirus promoter. A widely used example is the pSV2-neo plasmid that has a bacterial neomycin phosphotransferase gene under the control of the SV40 early promoter and confers resistance to G418 (neomycin-related antibiotic) in mammalian cells. Numerous other changes may be used to enhance the expression of the selectable marker in animal cells, such as the addition of poly (A) sequences and the addition of synthetic translation initiation sequences. Both constitutive and inducible promoters may be used.

  The genetic constructs of the present invention may also include 3 ′ untranslated sequences. A 3 ′ untranslated sequence refers to that portion of a gene comprising a DNA segment that contains a polyadenylation signal and any other regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is characterized by affecting the addition of polyadenylate tubes to the 3 ′ end of the mRNA precursor. Polyadenylation signals are generally recognized by the presence of homology to the canonical form 5 ′ AATAAA-3 ′, but variants are not uncommon.

  Thus, a genetic construct comprising a nucleic acid molecule of the present invention operably linked to a promoter may be a cell that is incompletely dysfunctional, dysfunctional, or absent in order to correct and / or provide appropriate regulation. It may be cloned in a suitable vector for delivery to tissue. Vectors containing appropriate gene constructs may be delivered to target eukaryotic cells by a number of different means well known to those skilled in molecular biology.

  As used herein, the term “similarity” includes exact identity between sequences compared at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences that nevertheless result in different amino acids related to each other at the structural, functional, biochemical, and / or conformational levels. It is. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical, and / or conformational level. In certain embodiments, nucleotide and sequence comparisons are made at the level of identity rather than similarity.

  Terms used to describe the sequence relationship between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “sequence” "Percent similarity", "percent sequence identity", "substantially similar" and "substantial identity" are included. A “reference sequence” is a unit comprising nucleotides and amino acid residues of at least 12, often 15-18, and more often such as at least 25 or 30 monomer units. Each of the two polynucleotides may contain (1) a sequence that is similar between the two polynucleotides (i.e., only part of the complete polynucleotide sequence), and (2) a sequence that differs from each other between the two polynucleotides. Because of the nature of sequence comparisons between two (or more) polynucleotides, the sequence of two polynucleotides is typically “comparison windows” to identify and compare local regions of sequence similarity. Is performed by comparing to "." A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may contain about 20% or less additions or deletions (ie, gaps) compared to a reference sequence (not including additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences to align comparison windows is accomplished by computer-implementation of algorithms (GAS3, BESTFIT, FASTA, and TFASTA from Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA). Or by best alignment generated by inspection and any of a variety of selected methods (ie, obtaining the highest percent homology to the comparison window). For example, reference may be made to the BLAST program family disclosed by Altschul et al. (Nucl Acids Res 25: 389, 1997). A detailed discussion of sequence analysis can be found in Ausubel et al., Unit 19.3 (In: Current Protocols in Molecular Biology, John Wiley & Sons Inc. 1994-1998).

  As used herein, the terms “sequence similarity” and “sequence identity” are the same or functional based on nucleotide to nucleotide or amino acid to amino acid relative to the comparison window. Or structurally similar to some extent. Thus, the “percent sequence identity” is, for example, comparing two optimally aligned sequences against a comparison window, the same nucleobase (eg, A, T, C, G, I). Or identical amino acid residues (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Determining the number of positions that occur in both sequences and yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window (i.e., the size of the window), and the result Calculated by the step multiplied by 100 to give a percentage of sequence identity. For the purposes of the present invention, “sequence identity” is the DNASIS computer program (Windows version 2.5, Hitachi Software Engineering Co., Ltd., South, using standard defaults used in the reference manual attached to the software. It will be understood to mean "Match Percentage" calculated by San Francisco, California, USA). Similar annotations apply with respect to sequence similarity.

Reference herein to low stringency includes at least about 0 to at least about 15% v / v formamide, and at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about washing conditions. Contains and includes about 2 M salt. Generally, low stringency is about 25-26, such as 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 and 42 ° C. 30 ° C to about 42 ° C. The temperature may be varied, and higher temperatures may be used to replace the formamide and / or create alternative stringency conditions. If necessary, at least about 16% v / v to at least about 16, such as 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and 30% for hybridization 30% v / v formamide, and at least about 0.5 M to at least about 0.9 M salt, such as 0.5, 0.6, 0.7, 0.8, or 0.9 M, and at least about 0.5, such as 0.5, 0.6, 0.7, 0.8, or 0.9 M with respect to wash conditions For moderate stringency conditions comprising or including M to at least about 0.9 M salt, or for hybridization, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 44, 45, 46, 47, 48, 49 and 50%, such as at least about 31% v / v to at least about 50% v / v formamide and 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, At least about 0.01 M to at least about 0.15 M salt, such as 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M, And at least about 0.01 M to at least about 0.15 M salt, such as 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.11, 0.12, 0.13, 0.14 and 0.15 M with respect to cleaning conditions and Alternative stringency conditions may be applied, such as high stringency conditions that encompass these. In general, washing is performed at T _m = 69.3 + 0.41 (G + C)% (Marmur and Doty, J Mol Biol 5: 109, 1962). However, the T _m of double-stranded DNA decreases by 1 ° C. for every 1% increase in the number of mismatched base pairs (Bonner and Laskey, Eur J Biochem 46:83, 1974). Formamide is optional in these hybridization conditions. Thus, in certain embodiments, the level of stringency is defined as follows: low stringency conditions are 6 × SSC buffer, 0.1% w / v SDS at 25-42 ° C .; moderate stringency is Temperature range 20-65 ° C. with 2 × SSC buffer, 0.1% w / v SDS; high stringency conditions are at least 65 ° C. with 0.1 × SSC buffer, 0.1% w / v SDS.

  As used herein, the term “translational or post-translational modification” refers to a covalent modification that occurs during or after translation of a peptide chain. Exemplary translational or post-translational modifications include acylation (including acetylation), amidation or deamidation, biotinylation, carbamylation (or carbamoylation), carboxylation or decarboxylation, disulfide bond formation, Fatty acid acylation (including myristoylation, palmitoylation, and stearoylation), formylation, glycation, glycosylation, hydroxylation, selenocysteine incorporation, lipidation, lipoic acid addition, methylation, N-terminal or C-terminal blocking , N-terminal or C-terminal removal, oxidation of methionine, phosphorylation, proteolytic cleavage, prenylation (including farnesylation, geranylgeranylation), pyridoxal phosphate addition, sialylation or desialylation, sulfation, ubiquitination (or ubiquitin) Or ubiquitin-like tongue Including but addition of click quality but are not limited to.

  Acylation entails hydrolysis of the N-terminal initiation methionine and addition of an acetyl group to the new N-terminal amino acid. Acetyl Co-A is an acetyl donor for acylation.

  Amidation is the covalent attachment of the amide group to the carboxy terminus of the peptide, which is often necessary for the biological activity and stability of the protein. Deamidation is the removal of the amide group by hydrolysis. Deamidation of amides containing amino acid residues is a rare modification, which is performed by organisms to rearrange the 3D structure and to change the charge ratio / pi.

  Biotinylation is a technique by which biotinyl groups are incorporated into molecules, catalyzed by halocarboxylase synthetase during enzyme biosynthesis, or a variety of substrates that are sensitive to biochemical assays. A technique performed in vitro to visualize them by incubating with a biotin-labeled probe and avidin or streptavidin linked to any substrate.

  Carbamylation (or carbamoylation) is the transfer of carbamoyl from a carbamoyl-containing molecule (eg, carbamoyl phosphate) to an acceptor moiety, such as an amino group.

  Carboxylation of glutamic acid residues is a vitamin K-dependent reaction that results in the formation of gamma carboxyglutamic acid (Gla residues). Gla residues in some proteins of the blood coagulation cascade are necessary for the physiological function of the protein. Carboxylation can also occur on aspartic acid residues.

  A disulfide bond is a covalent bond that forms when the thiol group of two cysteine residues is oxidized to a disulfide. Many mammalian proteins contain disulfide bonds, which are crucial for creating and maintaining protein tertiary structure and thus biological activity.

  Protein synthesis in bacteria entails formylation and deformylation of N-terminal methionine. This formylation / deformylation cycle does not occur in the cytoplasm of eukaryotic cells, but is a unique feature of bacterial cells. In addition to hydroxylation that occurs at glycine residues as part of the amidation process, hydroxylation can also occur at proline and lysine residues catalyzed by prolyl and lysyl hydroxylases (Kivirikko et al. FASEB Journal 3: 1609-1617, 1989).

  Glycation is the uncontrolled non-enzymatic addition of glucose or other sugars to the amino backbone of a protein.

  Glycosylation is the addition of sugar units to the polypeptide backbone and is described in detail later in this specification.

  Hydroxylation is a reaction dependent on vitamin C as a cofactor. An increase in the importance of hydroxylation as a post-translational modification is that hydroxy-lysine acts as an attachment site for glycosylation.

  A selenoprotein is a protein containing selenium as a trace element by incorporating selenocysteine, which is a unique amino acid during translation. The selenocysteine tRNA is charged by serine and then enzymatically selenylated to produce selenocysteinyl-tRNA. The anticodon of selenocysteinyl-tRNA interacts with a stop codon (UGA) in mRNA instead of a serine codon. Elements in the 3′-untranslated region (UTR) of selenoprotein mRNA determine whether UGA is read as a termination codon or a selenocysteine codon.

  Lipidation is a general term that includes covalent attachment of lipids to proteins, including fatty acid acylation and prenylation.

  Fatty acid acylation necessitates covalent attachment of fatty acids such as 14 carbon myristic acid (myristoylated), 16 carbon palmitic acid (palmitoylated) and 18 carbon stearic acid (stearoylated). Accompanying. Fatty acids may be linked to proteins in the pre-Golgi compartment to regulate protein targeting to the membrane (Blenis and Resh Curr Opin Cell Biol 5 (6): 984-9, 1993). Thus, acylation of fatty acids is important in the functional activity of proteins (Bernstein Methods Mol Biol 237: 195-204, 2004).

  Prenylation entails the addition of a prenyl group, ie, a 15 carbon farnesyl or 20 carbon geranylgeranyl group, to an acceptor protein. Isoprenoid compounds including farnesyl diphosphate or geranylgeranyl diphosphate are derived from the cholesterol biosynthetic pathway. The isoprenoid group is attached by thioether linkage to a cysteine residue in the consensus sequence CAAX (A is any aliphatic amino acid except alanine) present at the carboxy terminus of the protein. Prenylation enhances the ability of proteins to associate with lipid membranes, and all known GTP-bound and hydroxylated proteins (G proteins) are thus modified, so prenylation is very important for signal transduction (Rando Biochim Biophys Acta 1300 (l): 5-l6, 1996; Gelb et al. Curr Opin Chem Biol 2 (1): 40-8, 1998).

  Lipoic acid is a vitamin-like antioxidant that acts as a free radical scavenger. Lipoyl-lysine is formed by attachment of lipoic acid to lysine through an amide bond by lipoate protein ligase.

  Protein methylation is a common modification that can regulate the activity of proteins or create new types of amino acids. Protein methyltransferases transfer the methyl group from S-adenosyl-L-methionine to a nucleophilic oxygen, nitrogen, or sulfur atom on the protein. The effects of methylation can be divided into two general categories. First, the relative levels of methyltransferase and methylesterase can control the degree of methylation at a particular carboxyl group, which in turn modulates the activity of the protein. This type of methylation is reversible. The second group of protein methylation reactions entails irreversible modification of sulfur or nitrogen atoms in the protein. These reactions generate new amino acids with altered biochemical properties that alter the activity of the protein (Clarke Curr Opin Cell Biol 5: 977 983, 1993).

  Protein nitration is a significant post-translational modification that acts as a nitric oxide signaling agent. Protein nitration regulates catalytic activity, cell signaling, and cytoskeletal organization.

  Phosphorylation refers to the addition of a phosphate group by a protein kinase. Serine, threonine, and tyrosine residues are amino acids that undergo phosphorylation. Phosphorylation is a very important mechanism that regulates the biological activity of proteins.

  Many proteins are similarly modified by proteolytic cleavage. This may simply entail removal of the starting methionine. Other proteins are synthesized as inactive precursors (proproteins) that are activated by limited or specific proteolysis. A secreted fate or membrane-associated protein (preprotein) with a signal sequence of 12 to 36 amino acids that is predominantly hydrophobic is synthesized, which is cleaved after passage through the ER membrane.

  Pyridoxalphosphate is a coenzyme derivative of vitamin B6 and is involved in transamination, decarboxylation, racemization, and various modifications of amino acid side chains. All enzymes that require pyridoxal phosphate act through the formation of a Schiff base between the amino acid and the coenzyme. Most enzymes involved in the attachment of pyridoxal phosphate groups to lysine residues are self-activating.

  Sialylation refers to attachment of sialic acid to the terminal portion of a glycoprotein through various sialyltransferase enzymes, and desialylation refers to removal of sialic acid. Sialic acids include, but are not limited to, N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). Sialyl structures resulting from sialylation of glycoproteins include sialyl Lewis structures such as sialyl Lewis a and sialyl Lewis x, and sialyl T structures such as sialyl-TF and sialyl Tn.

  Sulfation occurs at tyrosine residues and is catalyzed by the enzyme tyrosyl protein sulfotransferase that occurs in the trans-Golgi network. It has been determined that 1 in 20 proteins secreted by HepG2 cells and 1 in 3 proteins secreted by fibroblasts contain at least one tyrosine sulfate residue. Sulfation has been shown to affect the biological activity of proteins. Of particular importance is that CCR5, the major HIV co-receptor, has been shown to be tyrosine sulfated, as well as sulfation of one or more tyrosine residues in the N-terminal extracellular domain of CCR5. Is required for optimal binding of MIP-Iα / CCL3, MIP-1β / CCL4, and RANTES / CCL5, and for optimal HIV co-receptor function (Moore J Biol Chem 278 (27) / 24243 -24246, 2003). Sulfation can also occur in sugars. Furthermore, sulfation of the carbohydrate portion of a glycoprotein can occur by the action of a glycosulfotransferase such as GalNAc (βl-4) GlcNAc (βl-2) Manα4 sulfotransferase.

  Post-translational modifications can include protein-protein linkages. Ubiquitin is a 76 amino acid protein that self-assembles and covalently attaches to other proteins in mammalian cells. Attachment occurs by peptide bonds between the C-terminus of ubiquitin and the amino group of lysine residues in other proteins. The attachment of a chain of ubiquitin molecules to a protein target makes the protein a target for proteasomal proteolysis, an important mechanism for regulating the steady state level of regulatory proteins, for example, with respect to the cell cycle (Wilkinson Annu Rev Nutr 15: 161-89, 1995). In contrast, mono-ubiquitination can play a role in the direct regulation of protein function. Ubiquitin-like proteins that can be covalently attached to proteins to affect their function and turnover include NEDD-8, SUMO-1, and Apg12.

Glycosylation is the addition of sugar residues in the polypeptide backbone. Sugar residues such as monosaccharides, disaccharides, and oligosaccharides include fucose (Fuc), galactose (Glc), glucose (Glc), galactosamine (CalNAC), glucosamine (GlcNAc), mannose (Man), N- Examples include, but are not limited to, acetyl-lactosamine (lacNAc), N, N′-diacetyl lactose diamine (lacdiNAc). These sugar units can be attached to the polypeptide in at least seven ways:
(1) Consensus sequence Asn-X-Ser; Asn-X-Thr; or by N-glycosidic linkage with the R-group of asparagine residues in Asn-X-Cys (N-glycosylation).
(2) by O-glycosidic linkage to the R-group of serine, threonine, hydroxyproline, tyrosine, or hydroxylysine (O-glycosylation).
(3) By the R-group of tyrosine in C-linked mannose.
(4) As a glycophosphatidylinositol anchor used to fix some proteins to the cell membrane.
(5) As a monosaccharide attachment of GlcNAc to the R-group of serine or threonine. This linkage is often reversibly related to the attachment of inorganic phosphate (Yin-o-Yang).
(6) Linear polysaccharide attachment (proteoglycan) to serine, threonine, or asparagine.
(7) By S-glycoside linkage with R-group of cysteine.

  The glycosylation structure can include one or more of the following carbohydrate antigenic determinants in Table 7.

(Table 7) List of carbohydrate antigenic determinants

  Carbohydrates are also believed to contain several antenna structures including mono, di, tri, and tetra extraneous structures.

  Glycosylation includes N-linked glycosylation, O-linked glycosylation, C-linked mannose structure, and glycophosphatidylinositol anchor, carbohydrate weight percentage, Ser / Thr-GalNAc ratio, monosaccharide, disaccharide, It may also be measured by the presence or pattern of trisaccharide and tetrasaccharide structure ratios, or lectin or antibody binding.

  Protein sialylation may be measured by the immunoreactivity of antibodies and proteins directed to a specific sialyl structure. For example, a Lewis x specific antibody reacts with CEACAM1 expressed from granulocytes but not with recombinant human CEACAM1 expressed in 293 cells (Lucka et al Glycobiology 15 (1): 87-100, 2005). Alternatively, the presence of sialyl structures on the protein can be detected by a combination of suitable measurement techniques such as mass spectrometry (MS), high performance liquid chromatography (HPLC), or glycomass fingerprinting (GMF) after glycosidase treatment. Good.

の見かけの分子量を有する。タンパク質の分子量または分子質量は、電気泳動、質量分析、勾配超遠心のような任意の通常の手段によって決定してもよい。 The apparent molecular weight of a protein includes all elements of the protein complex (cofactors and noncovalent binding domains), and all translational or posttranslational modifications (addition of covalently bound groups to the protein and removal from the protein) Is included. Apparent molecular weight is often affected by translational or post-translational modifications. The apparent molecular weight of a protein may be determined by SDS-PAGE (sodium dodecyl sulfate polyacrylamide electrophoresis), which is also the second in its two-dimensional counterpart, 2D-PAGE (two-dimensional polyacrylamide electrophoresis). It is also a dimension. However, this is more precisely a matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) MS that produces charged molecular ions, or a relatively sensitive electrospray ionization (ESI) that produces many charged peaks. ) May be more accurately determined by mass spectrometry (MS) by any of the MS. The apparent molecular weight of the protein or chimeric molecule thereof may be in the range of 1-1000 kDa. Therefore, the isolated protein of the present invention or a chimeric molecule thereof is

Has an apparent molecular weight of The molecular weight or molecular mass of a protein may be determined by any conventional means such as electrophoresis, mass spectrometry, gradient ultracentrifugation.

  The isoelectric point (or pI) of a protein is the pH at which the protein has no true charge. This attribute may be determined by isoelectric focusing (IEF), which is also the first dimension of 2D-PAGE. The experimentally determined pI value is affected by a series of translational or post-translational modifications, so the difference between experimental and theoretical pI may be as high as 5 units. Accordingly, the isolated protein or chimeric molecule of the present invention is 0, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7. , 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2 , 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7 , 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2 , 10.3, 10.4, 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7 , 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.6, 13.7, 13.8, 13.9, or 14.0.

  As used herein, the term “isoform” refers to multiple molecular forms of a given protein, including (1) primary structure (alternative splicing or polymorphism); Includes proteins with different levels of secondary structure (such as by different translational or post-translational modifications); and / or (3) tertiary or quaternary structure (such as by different subunit interactions, homo- or hetero-oligomer polymerization) It is. In particular, the term “isoform” includes a protein or chimeric molecule thereof having a certain primary structure but differing at the level of secondary or tertiary structure, or at the time of translational or post-translational modification, such as different glycosylated forms. Glycoforms containing are included.

  The chemical stability of a protein may be measured as the “half-life” of the protein in a particular solvent or environment. Typically, proteins with a molecular weight of less than 50 kDa have a half-life of about 5-20 minutes. The protein or chimeric molecule of the present invention has a half-life of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 , 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95 , 96, 97, 98, 99 or 100 hours are contemplated. Another particularly convenient measure of chemical stability is the resistance of a protein or chimeric molecule thereof to protease digestion such as trypsin or chymotrypsin digestion.

  The binding affinity of a protein or chimeric molecule thereof for its ligand or receptor may be measured as an equilibrium dissociation constant (Kd) or a functionally equivalent measurement.

  Protein solubility may be measured as the amount of protein dissolved in a given solvent or as the rate at which the protein dissolves. Furthermore, the dissolution rate and / or level of dissolution of a protein or chimeric molecule in solvents of different properties such as polarity, pH, temperature, etc. may also provide measurable physiochemical characteristics of the protein or chimeric molecule thereof. There is.

  Any “measurable physiochemical parameter” may be determined, measured, quantified, or qualitative using any method known to those of skill in the art. The following describes a set of methodologies that may be useful for determining, measuring, quantifying, or qualifying one or more measurable physiochemical parameters of an isolated protein or chimeric molecule thereof. However, it is to be understood that the present invention is in no way limited to the particular methods described or measurable physiochemical parameters that can be measured using these methods.

A glycoprotein can be said to have two basic components that interact with each other to create the entire molecule: an amino acid sequence and a carbohydrate or sugar side chain. The carbohydrate component of the molecule exists in the form of a monosaccharide or oligosaccharide side chain attached by an N- or O-linkage to the amine side chain of Asn of the amino acid backbone or the hydroxyl side chain of the Ser / Thr residue, respectively. Monosaccharides are considered to be sugars, have the basic formula (CH ₂ O) _n and most often form a cyclic structure of 5 or 6 atoms (5 and 6 carbon sugars, respectively). A term given to the smallest unit of quality. Oligosaccharides may be linear or branched, but are generally combinations of monosaccharides that do not have a long chain of tandem repeat units (as is the case with polysaccharides) to form structures of diverse complexity. . The level of branching and branching substitution, as well as the level of branching that the oligonucleotide contains, dramatically affects the overall properties of the glycoprotein and plays an important role in the biological function of the molecule. Oligosaccharides are produced in the endoplasmic reticulum (ER) and Golgi apparatus of cells and attached to the amino acid backbone. Different organisms and cell types have different ratios of glycotransferase, endoglycosidase and exoglycosidase, thus producing different oligosaccharide structures. One of the body's basic defense mechanisms is the detection and destruction of abnormal isoforms, which not only increases the effectiveness, but also reduces the detection by neutralizing antibodies. It is important to have correct glycosylation.

  Glycan chains are often expressed in a branched fashion, and even when they are linear, such chains are often subject to various modifications. Thus, complete sequencing of oligosaccharides is difficult to perform in one way and therefore requires an iterative combination of physical and chemical approaches that ultimately result in the structural details under test.

  Determination of the glycosylation pattern of a protein can be performed using a number of different systems, such as SDS-PAGE. This technique is due to the fact that glycosylated proteins often migrate as bands spread by SDS-PAGE. Different isoforms are distinguished by treating the protein with a series of substances. For example, a significant decrease in bandwidth and change in migration position after digestion with peptide-N4- (N-acetyl-β-D-glucosaminyl) asparagine amidase (PNGase) is considered a diagnosis of N-linked glycosylation. It is.

  To determine the composition of N-linked glycosylation, N-linked oligosaccharides were removed from the protein by PNGase cloned from Flavobacterium meningosepticum, and E. coli To express. Removed N-linked oligosaccharides are recovered from an Alltech Carbograph SPE Carbon column (Deerfield, Illinois, USA) as described by Packer et al. Glycoconj J 5 (8): 737-47, 1998. Also good. Next, samples are taken for monosaccharide analysis, sialic acid analysis, or sulfate analysis in a Dionex system equipped with a GP50 pump ED50 pulse amperometric or conductivity detector and various pH anion exchange columns be able to.

  The degree of O-linked glycosylation may be determined by first removing the O-linked oligosaccharide from the target protein by β loss. Removed O-linked oligosaccharides may be recovered from Alltech Carbograph SPE Carbon columns (Deerfield, Illinois, USA) as described by Packer et al (1998, supra). Next, samples are taken for monosaccharide analysis, sialic acid analysis, or sulfate analysis in a Dionex system equipped with a GP50 pump ED50 pulse amperometric or conductivity detector and various pH anion exchange columns be able to.

  Monosaccharide subunits of oligosaccharides have a variety of sensitivities to acids, and thus are targeted from target proteins by mild trifluoroacetic acid (TFA) conditions, moderate TFA conditions, and strong hydrochloric acid (HCl) conditions. Can be released. The monosaccharide mixture is then separated by high pH anion exchange chromatography (HPAEC) using various column media and detected using pulsed amperometric electrochemical detection (PAD).

  High pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) is widely used to determine the composition of monosaccharides. Fluorescent-based labeling methods have been introduced and many are available in kit form. The clear advantage of the fluorescence method is an increase in sensitivity (about 50 times). One possible disadvantage is that different monosaccharides may exhibit different selectivity for the fluorophore during the coupling reaction in either the hydrolyzate or the external standard mixture. . However, this approach can be made more sensitive using laser-induced fluorescence, along with increased sensitivity and the ability to identify which monosaccharides are present from a small amount of the total available glycoproteins. Attractive. Furthermore, the sulfate and phosphate composition may be determined using a conductivity detector. By using a standard, the peak area for the total amount of each monosaccharide present can be calculated. These data can indicate N- and O-linked glycosylation levels, degree of sialylation, and weight percent glycosylation in combination with amino acid composition, weight percent acidic glycoprotein.

  Small amount monosaccharide composition analysis of proteins is best done with PVDF (PSQ) membranes after electroblotting or when analyzing smaller amounts in dot blots. PVDF is an ideal matrix for carbohydrate analysis because neither monosaccharides nor oligosaccharides bind to the membrane once released by acid or enzymatic hydrolysis.

  The determination of the oligosaccharide content of a target molecule is performed by a number of techniques. The sugar is first removed from the amino acid backbone by enzymatic (such as digestion with PNGase) or chemical (such as β elimination by hydroxide) means. The sugar may be stabilized by reduction or may be labeled with a fluorophore to facilitate detection. The resulting free oligosaccharides can then be used with known standards to determine various structure ratios and sialylation levels, high pH anion exchange with pulsed amperometric electrochemical detection Separation is performed either by chromatography (HPAEC-PAD) or phosphor-assisted carbohydrate electrophoresis (FACE), a process similar to SDS-PAGE separation of proteins. In this process, oligosaccharides are labeled with a fluorophore that imparts electrophoretic mobility. They are separated in a high percentage of polyacrylamide gel and the resulting band pattern provides a profile of the oligosaccharide content of the target molecule. By using a standard, it is possible to obtain some information about the actual structure present, or a band can be cut out and analyzed using mass spectrometry to determine each of its structures.

  Fluorophore-assisted carbohydrate electrophoresis (FACE) is a polyacrylamide electrophoresis system designed to separate individual oligosaccharides released from a sugar conjugate. The oligosaccharide is removed from the sample protein by chemical or enzymatic means in such a way as to retain the reducing end. The oligosaccharide is then digested to a monosaccharide or labeled with a fluorophore (either charged or uncharged) intact. High percentage polyacrylamide gels and various buffer systems are used to move oligosaccharides / monosaccharides that migrate relative to their size / composition in exactly the same way as proteins. The sugar can be visualized by density measurement and the relative amount of sugar can be determined by fluorophore detection. This process is compatible with MALDI-TOF MS, so the method can be used to elucidate the actual structure.

  Quartz crystal microbalance and surface plasmon resonance (QCM and SPR, respectively) are two ways to obtain biological information through the physiochemical properties of molecules. Both indirectly measure protein-protein interactions through changes caused by the interactions in the physical characteristics of prefabricated chips. In QCM, a single crystal quartz wafer is treated with a receptor / antibody or the like that interacts with a target ligand. This chip is oscillated by microbalance and the frequency of the chip is recorded. The target protein is passed over the chip and the interaction with the bound molecules changes the frequency of the wafer. By changing the conditions under which the ligand interacts with the chip, it is possible to determine the binding characteristics of the target molecule.

  Apparent molecular weight is also a physiochemical property that can be used to determine similarity properties between a protein or chimeric molecule of the present invention and a protein or chimeric molecule produced using surrogate means.

  As used herein, the term “molecular weight” is defined as the sum of the atomic weights of the constituent atoms in a molecule and is sometimes referred to as “molecular mass” (Mr). Molecular weight can be determined theoretically by summing the atomic masses of the constituent atoms in the molecule. The term “apparent molecular weight” is defined as the molecular weight determined by one or more analytical techniques such as SDS page or ultracentrifugation and depends on the relationship between the molecule and the detection system. The apparent molecular weight of the protein or chimeric molecule thereof can be determined using any one of a series of experimental methods. Analytical methods for determining the molecular weight of proteins include, but are not limited to, size exclusion chromatography (SEC), gel electrophoresis, Rayleigh light scattering, analytical ultracentrifugation, and to some extent, time-of-flight mass spectrometry. I don't mean.

  Gel electrophoresis is a process that determines some of the physiochemical properties (especially apparent molecular weight and pI) of proteins, and in the case of two-dimensional electrophoresis to separate molecules into isoforms, thereby Provides information on post-translational modifications. Specifically, electrophoresis is the process of moving charged molecules (such as proteins or DNA) through a gel matrix (most commonly polyacrylamide or agarose) by applying an electrical potential across the gel matrix. . The most common types of electrophoresis used for proteins are isoelectric focusing, non-denaturing, and SDS polyacrylamide gel electrophoresis. In isoelectric focusing, the protein is placed in a polyacrylamide gel with a pH gradient across its length. The protein is thought to move through the gel to a point with a true charge of zero, thereby giving its isoelectric point.

  Glycomass fingerprinting (GMF) is a process whereby a single oligosaccharide profile of a protein or its isoform is identified by a specific mass spectrometry technique after electrophoresis. Sample proteins are purified either by 1D SDS-PAGE to determine the total profile or 2D gel electrophoresis for specific isoform characterization. Protein bands / spots were excised from the gel and destained to remove contaminants. Sugar is released by chemical or enzymatic means and desalted / separated using a nanoflow LC system and a graphitized carbon column. The LC stream is injected directly into the electrospray mass spectrometer, which is used to determine the mass and then the identity of the oligosaccharides present in the sample. This provides a profile or fingerprint for each isoform, which can be combined with a quantitative technique such as Dionex analysis to determine the total composition of the molecules being tested.

  Primary structure can be evaluated in determining the physiochemical properties of the protein or chimeric molecule of the present invention.

  The primary structure of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  Information regarding the primary structure of a protein or chimeric molecule thereof can be determined using a combination of mass spectrometry (MS), DNA sequencing, amino acid composition, protein sequencing, and peptide mass fingerprinting.

  N-terminal chemical sequencing, tandem mass spectrometry sequencing, or a combination of both are used to determine the sequence of the amino acid backbone. N-terminal chemical sequencing utilizes Edman chemistry (Edman P. “Sequence determination” Mol Biol Biochem Biophys 8: 211-55, 1970), which is between the N-terminal amino acid of the protein and the amino acid at position 2. States that the peptide bond is weaker than all other peptide bonds in the sequence. By using moderately acidic conditions, the N-terminal amino acid is removed and derivatized with a fluorophore (FITC) to determine the retention time on a reverse phase HPLC column and compared to a standard, Identify what the amino acids are. This method is thought to determine the actual primary structure of the molecule, but is not quantitative. Alternatively, tandem mass spectrometry may be used with nanoflow liquid chromatography (LC-MS / MS). In this process, proteins are digested into peptides using a specific endoprotease to determine the molecular weight of the peptide. Using a high energy collision gas such as nitrogen or argon, the peptide bond is cleaved and the mass of the resulting peptide is measured. By calculating the change in the mass of the peptide, it is possible to determine the sequence of each of the peptides (each amino acid has its own mass). By using different proteases, the peptides may be superimposed and their order determined, thus determining the entire sequence of the protein.

  Clearly, the combination of enzymatic digestion, chemical derivatization, liquid chromatography (LC) / MS and tandem MS provides a very powerful tool for AA sequence analysis. For example, the detailed structure of the recombinant soluble CD4 receptor has been characterized by a combination of methods that confirms more than 95% of the primary sequence of this 369 amino acid glycoprotein, N- and C-terminal The complete nature of both, the position of glycan attachment, the structure of glycans and the correct assignment of disulfide bridges are shown (Carr et al. J Biol Chem 264 (35): 21286-21295, 1989).

  Mass spectrometry (MS) is a process that measures the mass of a molecule through extrapolation of its behavior in a charged environment under vacuum. MS is very useful in stability testing and quality control. The method first requires digestion of the sample with proteolytic enzymes (trypsin, V8 protease, chymotrypsin, subtilisin and clostripain) (Franks et al. Characterization of proteins, Humana Press, Clifton, NJ, 1988; Hearn et al. al. Methods in Enzymol 104: 190-212, 1984), followed by separation of the digested sample by reverse phase chromatography (RPC). When trypsin digestion in combination with LC-MS, peptide maps are used to monitor gene stability, production lot uniformity, and protein stability during fermentation, purification, dosage form manufacture and storage be able to.

  Prior to mass spectrometry, HPLC is interfaced to the mass spectrometer using several methods: 1) direct liquid injection; 2) surface liquid; 3) moving belt system; 4) thermospray. The HPLC / MS interface used in Caprioli's study used a fused silica capillary column to transport the eluate from the column to the tip of the sample probe in the ionization chamber of the mass spectrometer. The tip of the probe is continuously bombarded by energetic Xe atoms, and when this emerges from the tip of the capillary, it causes sputtering of the sample solution. The mass is then analyzed by instrument (Caprioli et al. Biochem Biophys Res Commun 146: 291-299, 1987).

  MS / MS and LC / MS interfaces expand the potential applications of MS. MS / MS can directly identify complete sequences from partial sequences for peptides up to 25 amino acids, and can identify deamidation and isomerization sites (Carr et al. Anal Chem 63: 2802). -2824, 1991). When coupled to RPC or capillary electrophoresis (CE), MS can perform very sensitive protein analysis (Figeys and Aebersold, Electrophoresis 19: 885-892, 1998; Nguyen et al. J Chromatogr A 705 : 21-45, 1995). LC / MS allows LC methodologies to separate peptides before entering MS, such as continuous flow FAB interfaced to microbore HPLC (Caprioli et al. 1987, supra). The latter “interface” makes it possible to sequence individual peptides from a complex mixture: after fragmentation of the peptides selected by the first MS, ions in the collision cell: CID (collision-induced dissociation) Pass through the clouds. Collisions generate a characteristic set of fragments, from which the sequence may be deduced, even if other information such as the cDNA sequence is unknown. In a single MS experiment, an unfractionated peptide mixture (eg from an enzyme digest) is injected and the mass of the major ions is compared to the mass predicted from the cDNA sequence. The sequence of recombinant human interleukin-2 was confirmed by fast atom collision (FAB) -MS analysis of CNBr and proteolytic digests (Fukuhara et al. J Biol Chem 260: 10487-10494, 1985).

  Electrospray ionization MS (ESI-MS) is introduced into a needle under high pressure using a solution protein aerosol to generate a series of charged peaks of the same molecule with various charges. Since each peak generated from a different charged species produces an estimate of molecular weight, these estimates can be combined to increase the overall accuracy of the molecular weight estimate. Matrix-assisted laser desorption / ionization MS (MALDI-MS) uses a high concentration of chromophore. Higher intensity laser pulses are absorbed by the matrix, and the absorbed energy evaporates a portion of the matrix and carries the protein sample with it almost completely into the vapor phase. Next, the obtained ions are analyzed by time-of-flight MS. Mild ionization may increase the tolerance that the method provides quaternary structure information. MALDI-MS can be performed rapidly in less than 15 minutes. This does not require fragmentation of the molecule and the results are easy to interpret as a density measurement scan of an SDS-PAGE gel up to a mass range of 100 kDa.

  The amino acid sequence can be predicted by sequencing DNA encoding the protein or chimeric molecule thereof. However, sometimes the actual protein sequence may be different. Traditionally, DNA sequencing reactions are just like PCR reactions involving replicating DNA (DNA denaturation, replication). By DNA cloning techniques, the gene is cloned and the nucleotide sequence is determined.

  The amino acid sequence of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  A full sequence description of a protein or chimeric molecule thereof usually requires a description of the product. Amino acid sequencing includes trypsin digestion of gels, fractionation of digested peptides by RPC-HPLC, screening of peptide peaks with the most symmetrical absorbance profile by MALDI-TOF MS, and the first peptide (N-terminal) Screening by Edman degradation of. Edman degradation chemically derived primary sequence data is a classic method for identifying proteins at the molecular level. MALDI-TOF MS can be used for N-terminal sequence analysis. However, it is recommended that all enzyme digests for HPLC and peptide sequencing are first subjected to MS or MS / MS protein identification to reduce time and cost. The internal amino acid sequence of the SDS-PAGE separated protein is obtained by elution of the peptide by HPLC separation after trypsin or lysyl endopeptidase digestion in situ in a gel matrix.

  It is recommended that standard peptide internal sequencing be performed on the analytical sample to maintain the instrument at peak performance. More than 80% of higher eukaryotic proteins are reported to have a blocked amino terminus that interferes with direct amino acid sequencing. If a blocked eukaryotic protein is encountered, the presence of an internal standard array proves that the instrument is operating correctly.

  Edman degradation can be used for direct N-terminal sequencing by chemical techniques that derivatize the N-terminal amino acid to release the amino acid and expose the amino terminus of the next amino acid. For Edman sequencing: 1) Repeat N-terminal sequence analysis by Edbo chemical cycle by microbore HPLC. One amino acid can be identified for each Edman chemistry cycle. 2) After HPLC separation of peptides obtained in the gel or after digestion of PVDF binding protein, internal protein sequence analysis is performed by Edman degradation chemistry.

  Microbore HPLC and capillary HPLC are used to analyze and purify peptide mixtures using RPC-HPLC. In-gel samples and PVDF samples are purified using different columns. MALDI-TOF MS analysis can be used for N-terminal analysis after HPLC fractionation. The selection criteria are 1) the apparent purity of the HPLC fraction, 2) the mass of the peptide and the length thus estimated. Peptide mass information is useful for confirming Edman sequencing amino acid assignments, as well as for detections that may have post-translational or post-translational modifications.

  In-gel digests are suitable for purification on more sensitive HPLC systems. Internal sequence analysis is first enzymatically digested by SDS-PAGE. Proteins in SDS-PAGE minigels can only be reliably digested in gels with trypsin. Peptide fragments are purified by RPC-HPLC and then analyzed by MALDI-TOF MS and screened for peptides suitable for Edman sequence analysis. Proteins in the gel can only be analyzed by internal sequencing analysis, but very accurate peptide masses can be obtained, which provides additional information useful in both amino acid assignments and database searches.

  PVDF binding proteins are suitable for both N-terminal and internal Edman sequencing analysis. PVDF-binding proteins are suitable enzymes (trypsin, endoproteinase Lys-C, endoproteinase Glu-C, clostripain, endoproteinase Asp-N, thermolysin) and non-hydrogenated tritons such as X-100. Digested by ionic detergent. In PVDF binding proteins, detergents used to release digested peptides from the membrane can interfere with MALDI-TOF MS analysis. Before adding the enzyme, Cys is reduced with DTT and alkylated with iodoacetamide to produce carboxyamidomethyl Cys, which can be identified during N-terminal sequence analysis.

  To determine the amino acid composition of the protein or chimeric molecule thereof, the sample is hydrolyzed using phenol catalyzed strong hydrochloric acid (HCl) acidic conditions in the gas phase. After hydrolysis, the generated amino acid is derivatized with a fluorescent compound that imparts reverse phase characteristics specific to the complex molecule. Derivatized amino acids are separated using reverse phase high performance liquid chromatography (RP-HPLC) and detected by a fluorescence detector. By using external and internal standards, it is possible to calculate the amount of each amino acid present in the sample from the observed peak area. This information can be used to determine the identity of the sample and to quantify the amount of protein present in the sample. For example, the difference between theoretical and actual results can be used to first identify the possibility of a deamidation site. In combination with monosaccharide analysis, the composition can be determined by weight percent glycosylation and weight percent acidic glycoprotein. While this method is limited to the information that can be provided in the actual sequence of the backbone, there are inherent variations due to environmental contaminants and sometimes amino acid destruction. For example, with this method it is not possible to detect point mutations in the sequence.

  Peptide mass fingerprinting (PMF) is another method by which the identity of a protein or chimeric molecule thereof can be determined. The technique entails initial separation of the sample by electrophoretic means (one or two dimensions), excision of spots / bands from the gel, and digestion with a specific endoprotease (typically porcine trypsin). Peptides are eluted from the gel fragments and analyzed by mass spectrometry to determine the peptide mass present. The resulting peptide mass is then compared to a theoretical mass fragment database for all reported proteins (or in the case of constructs, with respect to the theoretical peptide mass of the designed sequence). The technique is due to the fact that the “fingerprint” of the protein (ie its combination of peptide masses) is unique. Identity can be determined reliably (over 90% accuracy) with as little as 4 peptides and 30% sequence coverage. Modifications such as lipid moieties and deamidation can be identified during the PMF stage of analysis. Peaks that do not correspond to the identified protein peaks are further analyzed by tandem mass spectrometry (MS-MS), which uses the energy created by the impact of collision gas to break the weaker bonds of PTM. To do. The newly released molecule and the original peptide are reanalyzed with respect to mass to identify post-translational modifications and the peptide fragments to which it is attached.

  HPLC is classified in different ways depending on size, charge, hydrophobicity, function, or specific content of the target biomolecule. In general, proteins are purified using two or more chromatographic methods. When choosing a chromatography format, it is most important to consider both the protein and sample solvent characteristics.

  The secondary structure of the protein or chimeric molecule of the present invention can also be evaluated when characterizing its properties.

  The secondary structure of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  In order to examine the secondary structure of proteins, the most common should be some spectroscopy applied and compared. Electromagnetic energy can be defined as a continuous waveform of radiation, depending on the magnitude and shape of the wave. Different spectroscopy uses different electromagnetic energy.

The wavelength is the degree of one wave of radiation (the distance between two consecutive maxima of the wave). As the radiant energy increases, the wavelength decreases. The relationship between frequency and wave number is as follows.
Wave number (cm ^-1 ) = frequency (s ^-1 ) / speed of light (cm / s)

  Absorption of electromagnetic radiation by molecules includes vibrational and rotational transitions and electronic transitions. Infrared (IR) and Raman spectroscopy are most commonly used to measure the vibrational energy of molecules to determine secondary structure. However, they differ in their approach for determining molecular absorbance.

  The energy of the scattered radiation is less than the incident radiation on the Stokes line. The energy of the scattered radiation is greater than the incident radiation for the anti-Stokes line. The increase or decrease in energy from excitation is related to the vibrational energy space in the ground electronic state of the molecule. Thus, the wave number of Stokes and anti-Stokes lines is a direct measurement of the vibrational energy of the molecule.

  Only the Stokes shift is observed in the Raman spectrum. The Stokes line has a smaller wave number (or larger wavelength) than the excitation light. A high power excitation source such as a laser must be used to enhance the Raman dispersion efficiency. Since we are interested in the difference in energy (wavenumber) between the excitation and Stokes lines, the excitation source must be monochromatic.

  In order for vibrational motion to be IR active, the dipole moment of the molecule must change. Thus, the symmetric branch in carbon dioxide is not IR active because there is no change in the dipole moment. Asymmetric branches are IR active due to changes in the dipole moment. In order for vibration to be Raman active, the polarity of the molecule must be changed by vibrational motion. Symmetric branches in carbon dioxide are Raman active because the polarity of the molecule changes. Thus, Raman spectroscopy complements IR spectroscopy (Herzberg et al. Infrared and Raman Spectra of Polyatomic Molecules, Van Nostrand Reinhold, New York, NY, 1945). For example, IR cannot detect homonuclear diatomic molecules due to the lack of dipole moment, but Raman spectroscopy changes the polarity of the molecules due to stretching of the bonds and further interacts between electrons and nuclei. Since this changes, this can be detected.

  For highly symmetric polyatomic molecules with symmetry centers (such as benzene), active bands in the IR spectrum are likely not active in the Raman spectrum or vice versa. For molecules with little or no symmetry, modes can be active in both infrared and Raman spectroscopy.

  IR spectroscopy measures the wavelength and intensity of infrared light absorption by a sample. Infrared light is very high energy and can excite molecular vibrations at higher energy levels. Both infrared and Raman spectroscopy measure bond length and angular oscillations.

  IR characterizes vibrations in molecules by measuring the absorption of light of a specific energy corresponding to vibrational excitation of molecules in the v = 0 to v = 1 (or higher) state. There is a selection rule governing whether a molecule can be detected by infrared spectroscopy, and not all normal vibration modes can be excited by infrared radiation (Herzberg et al. 1945, supra).

IR spectra can provide qualitative and quantitative information of protein secondary structure, such as α-helix, β-sheet, β-turn and disordered structure. The most informative IR bands for protein analysis are amide I (1620-1700 cm ⁻¹ ), amide II (1520-1580 cm ⁻¹ ), and amide III (1220-1350 cm ⁻¹ ). Amide I is the strongest absorption band in proteins. This consists of contraction vibrations of C = O (70-85%) and CN groups (10-20%). The exact band position is dictated by the backbone conformation and hydrogen bonding pattern. Amide II is more complex than amide I. Amide II is dominated by in-plane NH bending vibration (40-60%), CN (18-40%) and CC (10%) stretching vibrations. The amide III band is not very useful (Krimm and Bandekar, Adv Protein Chem 35: 181-364, 1986). Most of the β-sheet structure of the FTIR amide I band is usually present at about 1629 cm ⁻¹ with a minimum value of 1615 cm ⁻¹ and a maximum value of 1637 cm ⁻¹ ; the minor component is 1696 cm ^{− 1} (minimum value 1685 cm ⁻¹ ) may show a surrounding peak, α-helix is mainly found at 1652 cm ⁻¹ . Absorption near 1680 cm ^-1 is now assigned to the β-turn.

  The principle of Raman scattering is different from the principle of infrared absorption. Raman spectroscopy measures the wavelength and intensity of inelastically scattered light from molecules. Raman scattered light occurs at a wavelength shifted from incident light by the energy of molecular vibrations.

  In order for a vibration that is scattered inelastically to be Raman-active, a change in polarity between vibrations is essential. In contrasting branches, the strength of electronic bonds varies between the minimum or maximum internuclear distance. Therefore, the polarity changes during vibration, this vibration mode scatters Raman light, and the vibration is Raman active. In an asymmetric branch, electrons are more easily polarized with expanding bonds, but are less easily polarized with compressive bonds. There is no overall change in polarity and asymmetric branches are Raman inactive (Herzberg et al. 1945, supra).

  Circular dichroism can be used to detect any asymmetric structure, such as a protein. Optically active chromophores absorb different amounts of left and right polarized light, and this difference in absorbance results in a positive or negative absorption spectrum (usually the right polarization spectrum is subtracted from the left polarization spectrum). In general, the far UV or amide region (190-250 nm) is contributed primarily from peptide bonds, providing information about the environment of the carbonyl group of the amide bond, followed by the secondary structure of the protein. The α helix usually shows two negative peaks at 208 and 222 nm (Holzwarth et al J Am Chem Soc 178: 350, 1965), the β sheet shows one negative peak at 218 nm, and the random coil Has a negative peak at 196 nm. The near UV peak (250-350 nm) is contributed from the environment of aromatic chromophores (Phe, Tyr, Trp). The disulfide bond produces a minor CD band around 250 nm.

  Strong dichroism is generally associated with a side chain structure that is firmly maintained in a highly folded three-dimensional structure. Protein denaturation almost releases steric hindrance, and a weaker CD spectrum is obtained with increasing degree of denaturation. For example, the side chain CD spectrum of hGH is quite sensitive to partial denaturation by adding denaturants. Some reversible chemical alterations of the molecule, such as disulfide bond reduction or alkaline titration, may alter the side chain CD spectrum. These spectral differences for hGH can be caused by complete removal of the chromophore or by affecting changes in the partial chromophore CD response, but not by significant denaturation or conformational changes. (Aloj et al. J Biol Chem 247: 1146-1151, 1971).

  UV absorption spectroscopy is one of the most important methods for determining protein properties. This can provide information about the protein concentration and the environment immediately surrounding the chromophore. Protein functional groups such as amino, alcohol (or phenol), hydroxyl, carbonyl, carboxyl, or thiol can be converted to strong chromophores. Two types of chromophores are monitored using visible and near UV spectroscopy: metalloproteins (greater than 400 nm) and proteins containing Phe, Trp, Tyr residues (260-280 nm). The change in UV or fluorescence signal can be negative or positive depending on the protein sequence and solution properties.

  Fluorescence measures the radiant energy after a molecule is irradiated into an excited state. Many proteins emit fluorescence in the range of 300-400 nm from their aromatic amino acids when excited at 250-300 nm. Only proteins with Phe, Trp, Tyr residues can be measured with the intensity order Trp >> Tyr >> Phe. The fluorescence spectrum can reflect microenvironmental information that is affected by protein folding. For example, buried Trp is usually present in a hydrophobic environment and fluoresces in the range of up to 325-330 nm, while exposed residues or free amino acids fluoresce at about 350-355 nm. A material often used to explore protein unfolding is Bis-ANS. The fluorescence of bis-ANS is pH dependent. Even if the signal is weak in water, it can be significantly increased by binding to hydrophobic sites exposed by unfolding in the protein (James and Bottomley Arch Biochem Biophy 356: 296-300, 1998) ).

  Effective quenching of Tyr and Trp in the folded protein causes a significant increase in signal upon unfolding. Simple solutes can cause changes as well. For maximum detection sensitivity. Signal ratio can be used. For example, the ratio of fluorescence intensity at 350 nm to 330 nm was used in the rFXIII unfolding test (Kurochkin et al. J Mol Biol 248: 414-430, 1995). Because the transfer efficiency depends on the distance between the two chromophores (Honroe et al. Biochem J 258: 199-204, 1989), the conformational change is due to the excitation energy transfer between the fluorescent donor and the absorbing acceptor. You may investigate. Exploring the conformation of α-antitrypsin using fluorescence (Kwon and Yu, Biophim Biophys Acta 1335: 265-212, 1997) and determining the Tm of HSA (Farruggia et al. Int J Biol Macromol 20: 43- 51, 1997), and MerP unfolding interactions were detected (Aronsson et al. FEBS Lett. 411: 359-364, 1997).

  At neutral pH, the intensity of the fluorescence emission spectrum is in the order of Trp> Tyr. At acidic pH, fluorescence from Tyr is predominant over Typ due to conformational changes that disrupt energy transfer. Fluorescence studies also confirm the presence of intermediates in the guanidine-induced unfolding transition.

  The tertiary and quaternary structure of the physiological chemical form of the protein of the present invention or a chimeric molecule thereof is also important for confirming its function.

  The physiochemical tertiary and quaternary structure of the protein or chimeric molecule of the present invention can be assayed using one or more of the following systems.

  NMR and X-ray crystallography are the most frequently used techniques for examining the three-dimensional structure of proteins. Other less detailed methods for exploring protein tertiary structure include CD in the near UV region, a secondary derivative of UV spectroscopy (Ackland et al. J Chromatogr 540: 187-198, 1991) , And fluorescence.

  NMR is one of the main experimental methods for molecular structure and intermolecular interactions in structural biology. In addition to examining protein structure, NMR can also be used to examine the carbohydrate structure of the protein or chimeric molecule of the present invention. NMR spectroscopy is routinely used by chemists to examine chemical structures using simple one-dimensional techniques. More complex molecular structures can be determined by two-dimensional techniques as well. Time domain NMR is used to explore molecular dynamics in solution. Solid state NMR is used to determine the molecular structure of a solid. NMR can be used to examine a variety of low molecular weight compounds in proteins, nucleic acids, biological, pharmacological, and medical subjects. However, not all nuclei possess the correct properties because they are read by NMR, that is, not all nuclei possess the necessary spin for NMR. Spins cause the nucleus to produce an NMR signal and function as a small magnetic field.

  The crystal structure of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  X-ray crystallography is an experimental technique that applies the fact that X-rays are diffracted by crystals. X-rays have a suitable wavelength (angstrom range, ˜10 −8 cm) scattered by a cloud of electrons of comparable size. The electron density can be reconstructed based on diffraction patterns obtained from x-rays that scatter periodic assemblies of molecules or atoms in the crystal. To complete the reconstruction, further phase information should be obtained from the diffraction data or from the supplement of the diffraction experiment. The model is then progressively built to experimental electron density and refined to the data, resulting in a very accurate molecular structure.

  X-ray diffraction is developed to examine the structure of all states of matter by any beam, such as ions, electrons, neutrons, and protons, that have a wavelength similar to the distance of the atomic or intermolecular structure of interest. ing.

  Light scattering spectroscopy is based on the simple principle that larger particles scatter more light than smaller particles. The gradient baseline in the 310-400 nm region is derived from light scattering when large particles such as aggregates are present in the solution (Schmid et al. Protein structure, a practical approach, Creighton Ed., IRI Press , Oxford, England, 1989).

  Light scattering spectroscopy can be used to estimate the molecular weight of a protein and is a simple tool for monitoring protein quaternary structure or protein aggregation. The degree of protein aggregation can be indicated by simple turbidity measurements. Since most aggregated proteins are present as cloudy or opalescent, the final product pharmaceutical solution is subjected to lightness inspection. Quasi-elastic light scattering spectroscopy (QELSS), sometimes called photon correlation spectroscopy (PCS), or dynamic light scattering (DLS), is a macromolecule (protein, polysaccharide, synthetic polymer, micelle, colloidal particle, and aggregate. ) Is a non-invasive method for examining diffusion in complex liquids. In most cases, light scattering spectroscopy directly produces the mutual diffusion coefficient of the scattered species. When applied to dilute monodisperse solutions, the diffusion coefficient obtained by QELSS can be estimated in magnitude. For polydisperse systems this estimates the width of the molecular weight distribution. For accurate measurement, a laser power of 200 to 500 mW is essential, and a normal Ar + / Kr + gas laser is widely used (Phillies Anal Chem 62: 1049A-1057 A, 1990). Protein aggregation was detected by human relaxin (Li et al. Biochemistry 34: 5762-5772, 1995).

  The stability of a protein or its chimeric molecule is also an important determinant of function. Methods for analyzing such features include DSC, TGA, and freeze-dry chilled stage microscopy, freeze-thaw resistance analysis, and protease resistance.

  The protein or chimeric molecule of the present invention may be more stable to lyophilization (freeze drying). Lyophilization is used to enhance product stability and / or expiration dates because it is stored in powder rather than in liquid form. The process entails removing the liquid by evaporation under vacuum after the sample is first frozen. The net result is a dry “cake” of proteins and excipients (other substances used in the formulation). The hardness of the resulting cake is crucial for successful dissolution. The lyophilization process can cause changes in the protein, especially aggregate formation through cross-linking, deamidation and other modifications. They can reduce efficacy by loss, reduced activity, or induction of an immune response against aggregates. To test lyophilization stability, the protein can be formulated for lyophilization using standard stabilizers (eg, mannitol, trehalose, Tween 80, human serum albumin, etc.). After lyophilization, the amount of recovered protein can be assayed by ELISA and its activity can be assayed by a suitable bioassay. Protein aggregation can be detected by HPLC or Western blot analysis.

  Prior to lyophilization, the Tg or Te of the formulation (defining Tg or Te) should be determined in order to set the maximum allowable temperature of the product during the initial drying. Similarly, information about the crystalline or amorphous nature of the formulation will help to design a more lyophilized cycle. Product information regarding these thermal parameters can be obtained by using differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), or freeze-dry cooling stage microscope.

  Differential scanning calorimetry (DSC) is a physical thermal analysis method for measuring, characterizing, and analyzing the thermal properties of materials, and for determining heat capacity, melting enthalpy and transition point accordingly. DSC scans the temperature range at a linear rate. Individual heaters inside the instrument provide heat separately to the sample and reference pan based on the “input compensated zero balance” principle. Absorption or generation of energy during the physical transition causes an imbalance in the amount of energy provided to the sample holder. Depending on the various thermal behaviors of the sample, energy is obtained or diffused from the sample and the temperature difference is sensed as an electrical signal to the computer. As a result, the temperature of the sample holder becomes the same as that of the reference holder by automatic adjustment of the heater. The electrical force required for compensation is equivalent to the calorimetric effect.

  The purity of the organic material can be estimated by DSC based on the shape and temperature of the DSC melting endotherm. The input compensated DSC provides very high resolution compared to the heat flux DSC under the same conditions. In the case of lower resolution heat flux DSCs, the partial melting region does not “blur” over narrow temperature intervals, so a more well-defined and more accurate partial melting region is input compensated. Can be generated from type DSC. Input-compensated DSC results in an inherently better partial melting region and thus a better purity analysis. With the aid of staged DSCs, input compensated DSCs can provide direct heat capacity measurements using conventional time proof means without the need for deconvolution or extraction of sinusoidal amplitudes.

  Thermogravimetric analysis (TGA) measures the mass loss and weight loss rate of a sample as a function of temperature or time.

  As a DSC, a freeze-dry cooling stage can quickly reach a wide temperature range. Currently, as a pre-formulation and prescription testing tool, mimicking a lyophilization cycle in a freeze-dry cooling stage provides the best platform for testing thermal parameters of protein formulations on a reduced scale. Freeze-drying microscopy can predict the effects of recipes and process factors on freezing and drying. The cooling stage test only requires 2-3 mL of sample, so this technique is a valuable tool for testing small quantities of drugs that are difficult to obtain. This is a good tool for investigating the effects of freezing rate, drying rate, and thawing rate on a lyophilization cycle. Annealing studies may be advanced by testing from freeze-dry cooling stage microscopes. Due to the widespread application of lyophilization technology and the greater demand for stabilizing very expensive drugs (such as proteins and gene therapy), in-process microscope monitors will eventually be realized in the pharmaceutical industry. is there.

  The freeze-thaw resistance of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  In-translational or post-translational modifications such as glycosylation may protect the protein from repeated freeze / thaw cycles. To determine this, the protein or chimeric molecule of the present invention can be compared to a carrier-free E. coli acidic counterpart. After diluting the protein or its chimeric molecule in a suitable medium (e.g., cell growth medium, PBS, etc.), various methods such as flash freezing in liquid nitrogen, slow freezing by placing at -70 ° C, Or freeze by quick freezing with dry ice. The sample is then rapidly melted at room temperature or gradually melted at 4 ° C. Several samples are refrozen and the process is repeated many cycles. The amount of protein present can be measured by ELISA and the activity measured in a suitable bioassay selected by one skilled in the art. The amount of remaining activity / protein is compared to the starting material to determine resistance to many freeze / thaw cycles.

  The protein or chimeric molecule of the present invention may have altered thermal stability in solution. The thermal stability of the present invention may be determined in vitro as follows.

  The protein or chimeric molecule of the present invention is mixed in a buffer, for example a phosphate buffered saline containing a carrier protein, such as human serum albumin, and incubated at a specific temperature for a specific time (eg, 37 ° C. for 7 days). can do. The amount of protein or chimeric molecule thereof remaining after this treatment can be determined by ELISA and compared to material stored at -70 ° C. The biological activity of the remaining protein or chimeric molecule thereof is determined by performing a suitable bioassay selected by those skilled in the art.

  The protease resistance of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  To compare protease resistance, a solution containing a protein or chimeric molecule of the present invention, as well as a solution containing an E. coli expression equivalent, together with a selected protease (e.g., non-purified serum protease, purified protease, recombinant protease) It can be incubated for different periods. The amount of remaining protein is measured by an appropriate ELISA (e.g., an ELISA in which the epitope recognized by the capture and detection antibody is separated by a protease cleavage site) and the activity of the remaining protein or chimeric molecule thereof is selected by one skilled in the art As determined by the appropriate bioassay.

  The bioavailability of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  Bioavailability is the extent to which a drug or other substance is available to the target tissue after administration. Bioavailability may depend on the drug's half-life or ability to reach the target tissue.

  A composition comprising a protein or chimeric molecule of the present invention is injected subcutaneously or intramuscularly. The level of a protein or chimeric molecule thereof can be measured in blood by ELISA or radioactivity counting. Alternatively, blood samples can be assayed for protein activity by a suitable bioassay selected by those skilled in the art, such as by stimulating growth of a particular target cell population. If the sample is derived from plasma or serum, there can be many other molecules that can cause output activity. This can be controlled by using neutralizing antibodies against the protein being tested. Thus, the remaining biological activity is due to other serum components.

  The stability or half-life of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  The protein or chimeric molecule of the present invention may have an altered half-life in serum or plasma. The half-life of the present invention may be determined in vitro as follows: A composition comprising a protein or chimeric molecule of the present invention is mixed with human serum / plasma at a specific temperature for a specific time (e.g., Incubate at 37 ° C for 4 hours, 12 hours, etc.). The amount of protein or its chimeric molecule remaining after this treatment can be determined by ELISA. The biological activity of the remaining protein or chimeric molecule thereof is determined by performing a suitable bioassay selected by those skilled in the art. The selected sera may be derived from various human serogroups (eg, A, B, AB, O, etc.).

  The half-life of a protein or chimeric molecule thereof can also be determined in vivo. A composition comprising a protein or chimeric molecule thereof that may be labeled by a radioactive tracer or other means is administered to a species selected for testing, e.g., mouse, rat, pig, primate, human intravenous, subcutaneous. Can be injected into the retro-orbital, tail vein, intramuscularly, or intraperitoneally. Blood samples are taken at various times after injection and assayed for the presence of the protein or chimeric molecule thereof (either by ELISA or TCA-precipitated radioactivity count). A comparative composition consisting of E. coli or CHO produced protein or chimeric molecule thereof can be assayed as a control.

  To determine the half-life of a protein or chimeric molecule of the present invention in vivo, male Wag / Rij rats or other suitable animals can be injected intravenously with the protein or chimeric molecule thereof.

  Immediately prior to administration to the substrate, 200 μl of EDTA blood is taken as a negative control. At various times after injection, 200 μl of EDTA blood can be collected from the animal using the same technique. After the last blood collection, the animals are sacrificed. Centrifuge the specimen for 15 minutes at RT within 30 minutes of collection. Plasma samples are tested in a specific ELISA to determine the concentration of the protein or chimeric molecule of the present invention in each sample.

  The protein or chimeric molecule of the present invention may cross the blood brain barrier.

  In vitro assays for determining whether a protein or chimeric molecule of the present invention binds to human brain endothelial cells can be tested using the following assay.

The radiolabeled protein or chimeric molecule of the present invention can be tested for its ability to bind to human brain capillary endothelial cells. The isolated protein or chimeric molecule of the present invention can be custom-coupled to the radiolabel to specific activity by methods known in the art, for example by ¹²⁵ I by the chloramine T method and by ³ H to specific activity.

  Primary cultures of human brain endothelial cells can be grown in flat-bottom 96-well plates up to 5 days after confluence and then lightly fixed with acetone. Lyse cells and transfer to glass fiber membrane. The radiolabeled protein or chimeric molecule of the present invention can be detected using a liquid scintillation counter.

  In vivo assays for determining binding of proteins or chimeric molecules of the present invention to human brain endothelial cells can be tested using the following assays.

The human-specific protein or chimeric molecule of the present invention was tested for binding to human brain capillaries using freshly frozen (non-fixed) sections of human brain tissue, sectioned in a cryostat and placed on a glass slide. Fix in acetone. The binding of ³ H-protein or chimeric molecule of the present invention is examined in brain sections using quantitative autoradiography.

  In vivo assays can be used to determine the tissue distribution and blood clearance of the human-specific protein or chimeric molecule of the present invention in a primate system.

The protein or chimeric molecule of the present invention is used to determine the tissue distribution and blood clearance of the ¹⁴ C-labeled protein or chimeric molecule of the present invention in 2 male cynomolgus monkeys or other suitable primates. The protein or chimeric molecule of the present invention is administered to the animal at the same time as the ³ H-labeled control protein by an intravenous catheter. During the test, a blood sample is taken to determine the clearance of the protein from circulation. Twenty-four hours after injection, animals are euthanized and selected organs and representative tissues are harvested for determination of isotope distribution and clearance by combustion. In addition, capillary depletion experiments are performed on samples from different regions of the brain according to Triguero, et al. J of Neurochemistry 54: 1882-1888, 1990. This method removes more than 90% of blood vessels from brain homogenates (Triguero et al, cited above).

  The time-dependent redistribution of the radiolabeled protein or chimeric molecule of the present invention from the capillary fraction to the substantial fraction is consistent with the time-dependent movement of the protein or chimeric molecule of the present invention across the blood brain barrier.

  The protein or chimeric molecule of the present invention may promote or inhibit angiogenesis.

  The angiogenic ability of the protein or chimeric molecule of the present invention may be evaluated by methods known in the art. For example, the degree of angiogenesis may be measured by microvascular sprouting in an angiogenesis model. In this assay, rat adipose microvascular fragments (RFMF) are isolated as described in Shepherd et al. Arterioscler Thromb Vase Biol 24: 898-904, 2004. Epididymal fat beds are harvested from euthanized animals, minced and digested with collagenase. RFMF and single cells are separated from lipids and adipocytes by centrifugation and suspended in 0.1% BSA in PBS. The RFMF suspension is continuously filtered to remove tissue debris, single cells, and red blood cells from the fragments. RFMF is suspended in cold pH-neutral rat tail type 1 collagen at 15,000 RFMF / ml and seeded in wells of a 48-well culture plate (eg, 0.25 ml / well). After collagen polymerization, an equal volume of DMEM containing 10% FBS is added to each gel. After the gel is formed, vascular elongation characteristic of angiogenic germination appears by the fourth day of culture. These germinations are easily distinguished from parent vessel fragments by the absence of jagged smooth muscle related appearance. RFMF 3D cultures can be treated with a protein or chimeric molecule of the present invention and the length of vascular sprouting can be measured on days 5 and 6 of culture.

  The angiogenic potential of the protein or chimeric molecule of the present invention may also be assessed by the in vivo angiogenesis assay described in Guedez et al, Am J Pathol 162: 1431-1439, 2003. This assay consists of implanting a semi-closed silicone cylinder (angioreactor) subcutaneously in nude mice. An angioreactor is filled with an extracellular matrix premixed or unmixed with the protein or chimeric molecule of the present invention. Angiogenesis in the angioreactor is quantified by fluorescence spectroscopy after quantification by intravenous injection of fluorescein isothiocyanate (FITC) -dextran prior to recovery. Angioreactors examined by immunofluorescence can show cells at different stages of development and infiltrating angiogenic blood vessels.

  A protein or chimeric molecule of the present invention may have a well-defined immunoreactivity profile determined by immunoassay techniques that entail the interaction of the molecule with one or more antibodies directed against the molecule. Good. Examples of immunoassay techniques include enzyme-linked immunosorbent assays (ELISA), dot blots, and immunochromatographic assays such as side flow or strip tests.

  The level of a protein or chimeric molecule thereof may be measured using immunoassay techniques such as commercially available ELISA kits. The protein or chimeric molecule of the present invention has a different immunoreactivity profile relative to the non-human cell expressed protein or chimeric molecule thereof, depending on the specificity of the antibody provided in the immunoassay kit. For example, the immunoassay capture and / or detection antibody may be an antibody specifically directed against a non-human cell expressed human protein or chimeric molecule thereof.

  Furthermore, incorrect folding of the non-human cell expressed human protein or chimeric molecule thereof may result in exposure of antigenic epitopes that are not exposed in the correctly folded human cell expressed human protein or chimeric molecule thereof. Inaccurate folding can occur, for example, through overproduction of heterologous proteins in the cytoplasm of non-human cells, such as E. coli (Baneyx Current Opinion in Biotechnology, 10: 411-421, 1999). Furthermore, the non-human cell expressed human protein or chimeric molecule thereof may have a post-translational modification pattern that is different from the pattern of the protein or chimeric molecule of the present invention. For example, a non-human cell expressed human protein or chimeric molecule thereof may exhibit an abnormal amount and / or type of carbohydrate structure, phosphate, sulfate, lipid, or other residue. This may result in exposure of antigenic epitopes that are not exposed on the protein or chimeric molecule of the present invention. Conversely, the altered pattern of post-translational modifications may result in the absence of antigenic epitopes on the protein or chimeric molecule of the present invention exposed in the non-human cell expressed human protein or chimeric molecule thereof.

By any one or combination of the above factors,
(a) a human protein naturally present in an experimental sample or human tissue, or
(b) A human cell-expressing recombinant human protein or its chimeric molecule in an experimental sample, human tissue, or human embryonic stem cell (hES) culture medium may be measured incorrectly.

  An immunoreactivity profile of a human cell expressed human protein or chimeric molecule thereof determined using a suitable immunoassay can provide an indication of the immunogenicity of the protein in humans, as described herein below. There is sex.

  Most biological products elicit a certain level of antibody response against them. Antibody reactions can possibly cause serious side effects and / or loss of efficacy. For example, some patients treated with recombinant proteins expressed from non-human cells or chimeric molecules thereof can generate neutralizing antibodies, particularly during long-term therapeutic use, thereby reducing the potency of the protein. And / or may contribute to side effects. Proteins or chimeric protein molecules expressed from human cells are less likely to produce neutralizing antibodies, thus increasing therapeutic efficacy compared to non-human cell expressed proteins or chimeric molecules thereof.

  The immunogenicity of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  Most biological products elicit a certain level of antibody response against them. Antibody reactions can possibly cause serious side effects and / or loss of efficacy. For example, some patients treated with recombinant EPO appear to produce neutralizing antibodies that cross-react with the patient's own EPO. In this case, the patient develops pure erythropoiesis and becomes resistant to EPO treatment, thereby constantly requiring dialysis.

  Immunogenicity is a property that can elicit an immune response in an organism. Immunogenicity depends in part on the size of the substance in question and in part on how similar it is to the host molecule. A protein or chimeric molecule thereof may have altered immunogenicity due to its novel physiochemical characteristics. For example, the glycosylation structure of a protein or chimeric molecule thereof may hide or obscure the epitope recognized by the antibody and thus prevent or reduce binding of the antibody to the protein or chimeric molecule thereof. . Alternatively, some antibodies may recognize glycopeptide epitopes that are not present in the non-glycosylated form of the protein.

  The ability of a patient sample to recognize a protein or chimeric molecule thereof having a unique physiochemical shape can be determined by various immunoassays as described herein. A properly designed immunoassay entails consideration for proper detection, quantification, and characterization of the antibody response. Numerous recommendations for immunoassay design and optimization are reviewed in Mire-Sluis et al. J Immunol Methods 289 (l-2): l-l6, 2004, which is incorporated herein by reference.

  The use of a protein or chimeric molecule thereof for therapeutic implants can be assayed using one or more of the following systems.

  The present invention extends to using proteins or chimeric molecules thereof to manipulate stem cells. The main therapeutic use of stem cells is tissue, cartilage, or bone regeneration. In one embodiment, the cells can be introduced into the body in a biocompatible three-dimensional matrix. Implants are thought to consist of a mixture of cells such as biodegradable polymers, proteoglycans, scaffolds, growth factors and accessory components. Incorporation of proteins or chimeric molecules thereof into these matrices during their construction is proposed to modulate cell behavior. Such implants may be used for bone formation, neuronal growth from progenitor cells, chondrocyte transplantation for cartilage replacement, and other applications. Human cell-derived proteins may reduce the amount and / or diversity of heterologous proteins from stem cell culture conditions, thereby reducing the risk of infection by non-human pathogens.

  The protein or chimeric molecule of the present invention may interact differently from the matrix used to form the implant and regulate the cells incorporated within the implant. The combination of the protein or chimeric molecule of the present invention and an implant component allows higher proliferation, enhanced differentiation, maintenance of the desired differentiation state, greater differentiation lineage specificity, enhanced secretion of matrix components, better tertiary Original structure formation, enhanced signaling, better structural performance, reduced toxicity, reduced side effects, reduced inflammation, reduced immune cell infiltrate, reduced rejection, longer duration of implants One of the following pharmacological attributes, such as longer term function of the implant, better cell stimulation around the implant, better tissue regeneration, better organ function, or better tissue remodeling: It is expected that multiple will be obtained.

  The effect of a protein or chimeric molecule thereof on differential gene expression can be assayed using one or more of the following systems.

  Differences in gene expression can be analyzed in cells exposed to the protein or chimeric molecule thereof.

  Microarray technology allows the simultaneous determination of mRNA expression of almost all genes in the genome of an organism. This method utilizes a gene “chip” in which oligonucleotides corresponding to different gene sequences are attached to a solid support. Labeled cDNA derived from mRNA isolated from the cell or tissue of interest is incubated with the chip to hybridize the attached complementary sequence to the cDNA. A control is used as well, comparing the signal from both after hybridization and washing. Dedicated software is used to determine which genes are up- or down-regulated or which gene expression has not changed. Thousands of genes can be analyzed on each chip. For example, using Affymetrix technology, the Human Genome U133 (HG-U133) set consisting of two GeneChip® arrays represents more than 39,000 transcripts from about 33,000 well-proven human genes Includes approximately 45,000 probe sets. GeneChip® mouse genome 430 2.0 contains more than 39,000 transcripts on one array.

  This type of analysis reveals changes in the overall mRNA expression pattern, and thus may reveal differences in gene expression that are not known to be controlled by specific stimuli. This technique is therefore suitable for analyzing the induced gene expression associated with the protein or chimeric molecule of the present invention.

  Definitions of known and novel genes that are regulated by specific stimuli are believed to help identify biochemical pathways that are important in the biological activity of a particular protein or chimeric molecule of the present invention. This information would be useful in identifying new therapeutic targets.

  It is believed that the system can also be used to examine differences in gene expression induced by the protein or chimeric molecule of the present invention compared to commercial products.

  The effect of a protein or chimeric molecule thereof on binding ability can be assayed using one or more of the following systems.

  The binding ability of the protein or chimeric molecule of the present invention to various substances including extracellular matrix, artificial material, heparin sulfate, carrier, or cofactor can be examined.

  The effect of a protein or chimeric molecule thereof on the ability of a particular protein to bind to the extracellular matrix can be determined using the following assay.

  The surface is coated with extracellular matrix proteins including but not limited to collagen, vitronectin, fibronectin, laminin in a suitable buffer. Unbound sites can be blocked by methods known in the art, such as incubation with BSA solution. After the surface has been washed, for example with a PBS solution, a solution containing the protein to be tested, for example a protein of the invention or a chimeric molecule, is added to the surface. After coating, the surface is washed and incubated with an antibody that recognizes the protein or chimeric molecule thereof. Next, the bound antibody is detected by, for example, an enzyme-linked secondary antibody that recognizes the primary antibody. The bound antibody is visualized by incubating with the appropriate substrate and observing the color change response. Glycosylated proteins may adhere more strongly to extracellular matrix proteins than non-glycosylated proteins.

  Alternatively, after incubating with matrix-coated wells a protein or chimeric molecule of the invention, or equivalent amount of a counterpart protein expressed by a non-human cell or its chimeric molecule (specified by ELISA concentration or bioassay activity unit) After washing the wells, the amount of binding is determined by ELISA. The determined amount can be measured indirectly by a decrease in ELISA reactivity after incubating the sample with the coated surface.

  The binding ability of a protein or chimeric molecule thereof to an artificial material can be assayed using one or more of the following systems.

  In order to determine the ability of a protein or chimeric molecule thereof to bind to an artificial material, the surface is coated with an artificial material, including but not limited to metals, scaffolds in a suitable buffer. After the surface has been washed, for example with a PBS solution, a solution containing the protein to be tested, for example a protein of the invention or a chimeric molecule, is added to the surface. After coating, the surface is washed and incubated with an antibody that recognizes the protein or chimeric molecule thereof. The bound antibody is then detected by, for example, an enzyme-linked secondary antibody that recognizes the primary antibody. The bound antibody is visualized by incubating with an appropriate substrate and observing the color change response.

  Alternatively, wells coated with an artificial material of the protein or chimeric molecule of the present invention and an equivalent amount (specified by ELISA concentration or bioassay activity unit) of a counterpart protein or chimeric molecule expressed by a non-human cell. After incubation with, the wells are washed and the amount bound is determined by ELISA. The amount of binding can be measured indirectly by the decrease in ELISA reactivity after incubating the sample with the coated surface.

  The ability to bind to artificial surfaces can have biological consequences, for example in stent coatings. Alternatively, cells are seeded thereon using a scaffold coated with the protein or chimeric finger of the present invention. Cell growth and differentiation is then monitored and compared to an uncoated or differently coated scaffold.

  The ability of a protein or chimeric molecule thereof to bind to heparin sulfate can be assayed by one or more of the following systems.

  The protein or chimeric molecule of the present invention is expected to interact differently with heparin sulfate because of its physiochemical form. These differences are expected to be evident in experimental models such as cell proliferation, differentiation, and migration. A combination of a protein or chimeric molecule thereof and heparin sulfate is expected to have unique pharmacological properties for a given treatment. This may be increased serum half-life, bioavailability, reduced immune-related clearance, greater efficacy, reduced dose, fewer side effects, and related advantages.

  The ability of a protein or chimeric molecule thereof to bind to a carrier or cofactor can be assayed by one or more of the following systems.

  Proteins or chimeric molecules thereof are likely to bind to other molecules when they are present in plasma. These molecules are called “carriers” or “cofactors” and are thought to affect factors such as biological half-life or serum half-life.

  Incubating the purified form of the protein in plasma and analyzing the resulting solution by size exclusion chromatography can determine the interaction of the protein or chimeric molecule of the invention with its binding partner. If the protein or chimeric molecule thereof binds to the cofactor, the resulting complex will have a higher molecular weight, thereby changing the elution time. Complexes can be compared for biological activity, in vitro or in vivo half-life and bioavailability.

  The effect of a protein or chimeric molecule thereof on a bioassay can be assayed using one or more of the following systems.

  Cell proliferation, cell differentiation, cell apoptosis, cell size, cytokine / cytokine receptor adhesion, cell adhesion, cell spreading, cell motility, migration and invasion, chemotaxis, ligand-receptor binding, receptor activation A variety of bioassays can be performed to test the activity of the protein or chimeric molecule of the present invention, including assays for signal transduction, and changes in subgroup ratios.

  The effect of a protein or chimeric molecule thereof on cell proliferation can be assayed using one or more of the following systems.

  Cells, in particular embodiments, exponentially growing cells are incubated in the presence of the protein or chimeric molecule of the present invention in growth medium. This can be done in flasks or 96 well plates. After the cells have been grown for a period of time, the cell number is determined by either a direct method (eg cell counting) or an indirect method (MTT, MTS, tritiated thymidine). Increase or decrease in proliferation is determined by comparison with a media-only control assay. Different concentrations of the protein or chimeric molecule thereof can be used in parallel series of experiments to obtain a dose response profile. This can be used to determine the ED50 and ED100 (dose required to effectively produce half-maximal and maximal responses).

  The effect of a protein or chimeric molecule thereof on cell differentiation or maintenance of cells in an undifferentiated state can be assayed using one or more of the following systems.

  The cells are incubated in growth medium in the presence of the protein or chimeric molecule of the present invention. After a suitable period, the cells are assayed for an indication of differentiation. This may be a specific marker on the cell surface, expression of a cytoplasmic marker, alteration of cell dimensions, shape or cytoplasmic characteristics. Markers may include proteins, sugar structures (eg, glycosaminoglycans such as heparin sulfate, chondroitin sulfate, etc.), lipids (glycosphingolipids, or lipid bilayer components). These changes can be assayed by a number of techniques including microscopy, Western blot, FACS staining or forward / side scatter profiles.

  The effect of a protein or chimeric molecule thereof on cell apoptosis can be assayed using one or more of the following systems.

  Apoptosis is defined as programmed cell death and is different from other methods of cell death such as necrosis. This is characterized by defined changes in the cell, such as activation of signal transduction pathways (eg, Fas, TNFR) which result in the activation of a subset of proteases known as caspases. Activation of initiator caspases results in activation of executive caspases that cleave a variety of cellular proteins, resulting in nuclear fragmentation, nuclear layer cleavage, cytoplasmic blister formation, and cell destruction. Apoptosis can be triggered by protein ligands such as FasL, TNFa and lymphotoxin, or by signals such as substances that cause UV light and DNA damage.

  The cells are incubated in growth medium in the presence of the protein or chimeric molecule thereof and / or other substances suitable for the assay. For example, the presence of a substance capable of inhibiting transcription (actinomycin D) or translation (cycloheximide) may be necessary. After incubation for an appropriate period, the cell number is determined by a suitable method. If the cell number decreases, it may indicate apoptosis. Other indicators of apoptosis may be obtained by observing cell staining, such as annexin staining, or the characteristic ladder formation pattern of DNA. Further evidence to confirm apoptosis may be obtained by assaying for apoptosis markers after preventing apoptosis marker expression by incubating with a cell-permeable caspase inhibitor (e.g., z-VAD FMK). is there.

  The protein or chimeric molecule of the present invention may prevent apoptosis by providing a survival signal through a cell survival pathway such as the Bcl2 or Akt pathway. Activation of these pathways can be confirmed by Western blotting for increased intracellular Bcl2 expression or for increased activated (phosphorylated) forms of Akt using phospho-specific antibodies against Akt.

  For this assay, cells are incubated in the presence or absence of survival factors (eg, IL-3 and certain immune cells). The proportion of cells incubated in the absence of survival factor will die by apoptosis with long-term culture, but cells incubated in a sufficient amount of survival factor will survive or proliferate. Activation of cellular pathways responsible for these effects can be determined by Western blotting, immunocytochemistry and FACS analysis.

  The effect of a protein or chimeric molecule thereof on inhibition of apoptosis can be assayed using one or more of the following systems.

The protein or chimeric molecule of the present invention is tested for in vitro activity to protect rat, mouse, and human cortical neurons from cell death due to hypoxia and glucose deprivation. For this, neuronal cell cultures are prepared from rat embryos. To evaluate the effect of the protein or chimeric molecule of the present invention, in a modular incubator chamber in a cell-covered incubator, wet 95% air / 5% CO ₂ (normoxic) in serum-free medium with 30 mM glucose. Or in wet 95% N ₂ /5% CO ₂ (hypoxia and glucose depletion) in serum-free medium without glucose at 37 ° C. in the absence or presence of the protein or chimeric molecule of the present invention. Keep up to time. The cell culture is exposed to hypoxia and glucose depletion for less than 24 hours and then returned to normoxia for the remaining 24 hours. Cytotoxicity is analyzed by Alamar blue fluorescence, which reports cell viability as a function of metabolic activity.

  In another method, neuronal cell cultures are treated with 1 mM L-glutamate or a-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) at various concentrations of the protein of the invention or Exposure to normoxia in the absence or presence of chimeric molecules for 24 hours. Cytotoxicity is analyzed by Alamar blue fluorescence, which reports cell viability as a function of metabolic activity.

  A protein or chimeric molecule thereof can affect the growth, apoptosis, development, or differentiation of a variety of cells. These changes can be reflected, among other measurable parameters, by changes in cell size and changes in cytoplasmic complexity due to intracellular organelle development. For example, keratinocytes induced to differentiate by suspension culture exhibit downregulation of surface markers such as β1 integrin, increased cell size and cell complexity. The effect of a protein or chimeric molecule thereof on cell size or cytoplasmic complexity can be assayed using one or more of the following systems.

  FACS measures the amount of light scattered by a cell when a laser beam is incident on it. Argon lasers that provide light with a wavelength of 488 nm are often used. The larger the cell size, the greater the destruction of the light beam in the forward direction, so the forward scatter level corresponds to the cell size. To measure changes in cell size, cells treated with a protein or chimeric molecule of the present invention are diluted in sheath fluid and injected into a flow cytometer (FACSVantage SE, Becton Dickinson). Untreated cells serve as controls. The cell passes through the light beam, and the amount of forward scattering of light corresponds to the size of the cell.

  Changes in intracellular organelle growth and development (cytoplasmic complexity) can also be measured by FACS. The intracellular organelles of the cell scatter light to the side. Thus, changes in cytoplasmic complexity can be measured by the amount of side scatter of light by the cell by the method described above, and the level of intracellular organelle complexity and the level of granularity of the cell are determined by the light given by the cell Can be estimated by measuring the side scatter level.

  The effect of a protein or its chimeric molecule on cell size or cytoplasmic complexity is to use FACS to compare the profile given by 20,000 treated cells with the signal emitted by the same number of untreated cells. Can be evaluated. By comparing signals from different treatment populations of cells, relative changes in cell size and cytoplasmic complexity can be determined.

  The effect of a protein or chimeric molecule thereof on cell growth, apoptosis, development, or differentiation can be assessed using one or more of the following systems.

Protein-induced apoptosis and changes in cell growth or cycle can be assessed by labeling the DNA of treated cells with a dye such as propidium iodide having an excitation wavelength in the range of 488 nm and an emission wavelength of 620 nm. . Cells undergoing apoptosis have concentrated DNA and different sizes and granularity. These factors give a fluorescent signal along with a specific forward and size scatter profile, thus distinguishing the cell population undergoing apoptosis from normal cells. The amount of DNA in the cell also reflects the state of the cell cycle in which the cell enters. For example, cells in the G ₂ phase will have twice the amount of DNA as the G ₀ phase of the cell. This will be reflected by the fluorescent signal given off by a cell in G ₂ phase is doubled. The effect of a protein or chimeric molecule thereof can be assessed using FACS, for example, to compare the fluorescent signal provided by 20,000 treated cells with the signal emitted by the same number of untreated cells.

  The protein of the present invention or chimeric molecule thereof may also alter the expression of various proteins. The effect of a protein or chimeric molecule thereof on protein expression by a cell can be assayed using one or more of the following systems.

  To assess the increase and decrease in protein expression throughout the cell, the cell is fixed and permeabilized and then incubated with a fluorescent-conjugated antibody that targets an epitope of the protein of interest. A wide variety of fluorescent labels can be used with the Argon laser system. Fluorescent molecules such as FITC, Alexa Fluor 488, cyanine 2, cyanine 3 are commonly used for this experiment. This method can also be used to estimate changes in expression of surface markers and proteins by labeling non-permeable cells that can only be labeled with antibodies to epitopes exposed on the cell surface. The effect of a protein or chimeric molecule thereof can be assessed using FACS, for example, to compare the fluorescent signal provided by 20,000 treated cells with the signal emitted by the same number of untreated cells.

  The effect of a protein or chimeric molecule thereof on ligand / receptor adhesion can be assayed using one or more of the following systems.

  The protein or chimeric molecule of the present invention may or may not be more adhesive to the substrate compared to the already known physiological chemical types. The interaction may be an interaction with a protein receptor in the case of a sugar structure (e.g. a selectin such as L-selectin and P-selectin), an extracellular matrix such as fibronectin, collagen, vitronectin, and laminin It may be an interaction with a component or with a non-protein component such as a sugar molecule (heparin sulfate, other glycosaminoglycans).

  A protein or chimeric molecule thereof may also interact differently with non-biogenic materials such as tissue culture plastic, medical device components (eg, stents or other implants), or dental materials. In the case of medical devices, this can change the rate of establishment, ie the interaction between the implant with a particular class of cell types or types of connections formed in the body.

  Any suitable assay for protein adhesion can be used. For example, a solution comprising a protein or chimeric molecule of the present invention is incubated with a binding partner on a fixed surface in certain embodiments. After incubation, the amount of protein or chimeric molecule present in the solution is assayed by ELISA and the difference between the amount of remaining material and the starting material is the amount bound to the binding partner. For example, the interaction of a protein or chimeric molecule with an extracellular matrix protein could be determined by first coating the wells of a 96 well plate with an ECM protein (eg, fibronectin). Next, non-specific binding is blocked by incubating with a BSA solution. After washing, a known concentration of protein or chimeric molecule solution is added for a certain period. The solution is then removed and assayed for the amount of protein or chimeric molecule thereof remaining in the solution. The amount bound to the ECM protein is determined by the appropriate system (by a labeled secondary antibody or by a biotin-avidin enzyme complex as used for ELISA) after incubating the well with an antibody against the protein or chimeric molecule thereof. It can be determined by the detecting step.

  Methods for determining the amount bound to other surfaces necessarily involve measuring the amino acids present in the solution after hydrolyzing the protein or chimeric molecule thereof from the inert implant surface.

  The effect of a protein or chimeric molecule thereof on cell adhesion can be assayed using one or more of the following systems.

  Cell adhesion to a matrix (eg, extracellular matrix components such as fibronectin, vitronectin, collagen, laminin, etc.) is at least partially mediated by integrin molecules. Integrin molecules consist of α and β subunits, and certain combinations of α and β subunits produce binding specificity for specific ligands (eg, a2b1 integrin binds to collagen, a5bl binds to fibronectin, etc.). The integrin subunit has a large extracellular domain responsible for ligand binding and a shorter cytoplasmic domain responsible for interaction with the cytoskeleton. In the presence of the ligand, the cytoplasmic domain is responsible for the induction of signaling events (“signaling from outside to inside”). The affinity of an integrin for its ligand is regulated by an extracellular signaling event, which in turn alters the integrin's cytoplasmic tail ("signaling from inside to outside").

  Incubation with the protein or chimeric molecule of the present invention can possibly alter cell adhesion in many ways. First, it can qualitatively alter the expression of certain integrin subunits, thereby causing a change in binding capacity. Second, the amount of a particular integrin that is expressed can change, altering cell binding to its target matrix. Third, the affinity of a particular integrin can be altered without altering its surface expression (internal to external signaling). All of these changes can alter the binding of cells to a range of ligands, or the binding to specific ligands.

The protein or chimeric molecule of the present invention can be tested in a Cell-ECM adhesion assay, typically performed in 96-well plates. After coating the well with matrix, unbound sites in the well are blocked with BSA. After incubating a predetermined number of cells with the coating wells, unbound cells are washed away and the bound cells are incubated in the presence or absence of the protein or chimeric molecule thereof. The cell number is determined by an indirect method such as MTT / MTS. Alternatively, the cells are radioactively labeled (eg ⁵¹ Cr) and a known amount of radioactivity (ie cells) is added to each well. The amount of bound radioactivity is determined and calculated as a percentage of the amount loaded.

  Cells also adhere to other cells, for example, one cell population adheres to a monolayer of another type of cell. To assay in this regard, floating cells added to monolayer cells are labeled with radioactivity. The cells are then incubated in the presence or absence of the protein or chimeric molecule thereof. Unbound cells can be washed and the remaining mixed cell population lysed and assayed for the amount of radioactivity present.

  The effect of a protein or chimeric molecule thereof on cell spreading can be assayed using one or more of the following systems.

  The protein or chimeric molecule of the present invention may have altered effects on cell spreading. Initiation of cell spreading is an important step in cell motility and invasive behavior. Cell spreading can be initiated in vitro in a number of ways. When cell suspension is seeded on ECM components, adhesion and ligand binding by integrin receptors occur. This initiates a signaling event that results in activation of the Cdc42, Rac, and Rho small GTPase families. Activation of these proteins results in actin polymerization and membranous extension, thereby gradually flattening the cell and bringing more integrins into contact with the receptor. Eventually, the cells are completely flattened to form a focal adhesive (large structure containing integrins and signaling proteins). Cell spreading can also be initiated by stimulation of adherent cells with growth factors, again resulting in Cdc42 / Rac / Rho protein activation and membranous pseudopod formation.

  Cell spreading can be quantified by examining a large number of cells at different times after stimulation with the protein or chimeric molecule thereof. The area of each cell can be determined using an image analysis program, and the extent of cell extension along with the percentage of cell extension can be compared over time. More rapid extension may be initiated by higher activation of the Cdc42 / Rac / Rho pathway, or the temporal, qualitative and quantitative differences in their activation may be due to the protein or chimera of the invention May be observed by molecules. This in turn may reflect differences in signaling events induced by the protein or chimeric molecule of the present invention.

  The effect of a protein or chimeric molecule thereof on cell motility, migration, and invasion can be assayed using one or more of the following systems.

  Cells that adhere to a tissue culture dish do not adhere to a single spot and remain stationary, but rather are constantly elongating and contracting their cell body parts. When viewed in time-lapse photography, the cells can be observed to move around the dish as isolated single cells or as cell colonies. This movement may be either “random walking” (ie, not going in a specific direction) or directional walking. Either type of exercise can be increased by adding growth factors. Time-lapse photographs can be used to quantify the overall distance and overall direction covered by the cells over a given period of time.

  In the case of directional migration, cells are thought to move towards a chemoattractant source by sensing a chemical gradient and directing its migration mechanism in that direction. In many cases, the chemoattractant is a growth factor. Directional migration can be quantified by providing a chemoattractant source (eg, with a fine pipette) followed by time-lapse photography of cells moving towards it.

  Another system for determining directional movement is the Boyden chamber assay. In this assay, cells are placed in an upper chamber connected to the lower chamber by a small hole in the distribution membrane. If growth medium is placed in both chambers and the chemoattractant is added only to the lower chamber, a diffusion gradient is created between the two chambers. Cells are attracted to the growth factor source and travel through the holes in the separation membrane to the lower surface of the membrane. After sufficient time, the membrane is removed and the number of cells that have migrated to the lower surface of the membrane is determined.

  The cell invasion process utilizes many of the same components as migration. Cell infiltration can be modeled using a layer of extracellular matrix into which cells infiltrate. For example, Matrigel is a mixture of basement membrane components (ECM components, growth factors, etc.) that are liquid at 4 ° C. but rapidly solidify to form a gel at 37 ° C. This can be used to coat the upper surface of the Boyden chamber and add a chemoattractant to the lower layer. In order for the cells to pass to the lower surface of the membrane, they must break down Matrigel with enzymes such as collagenase and matrix metalloproteinase (MMP) and move in a direction toward the chemoattractant . This assay mimics the various processes required for cell invasion.

  The effect on chemotaxis of a protein or chimeric molecule thereof can be assayed using one or more of the following systems.

  Cell migration to chemoattractants can be measured in a Boyden chamber in vitro. The protein or chimeric molecule of the present invention is placed in the lower chamber and the appropriate target cell population is placed in the upper chamber. To mimic the in vitro process of immune cells that migrate from the blood to the site of inflammation, migration through the cell layer may be measured. Coating the upper surface of the Boyden chamber well with a confluent sheet of cells, such as epithelial cells, endothelial cells, or fibroblasts, appears to prevent direct migration of immune cells through the well holes. Instead, the cells will need to adhere to the monolayer and migrate towards the protein being tested. If cells are present on the lower surface of the Boyden chamber, or only in wells treated with the protein or chimeric molecule thereof, the presence of cells in the medium of the lower well indicates the chemotaxis performance of the protein or chimeric molecule . To show that this effect is specific for the protein or chimeric molecule thereof, neutralizing antibodies can be incubated with the protein in the lower chamber.

  Alternatively, to test the chemotaxis prevention ability of a substrate (chemical, protein, sugar), the substance can be treated with a known chemotaxis performance (this can be a specific chemokine, a conditioned medium from a cellular source, or a series of chemokines. With the solution containing the secreting cells) in the lower chamber of the Boyden chamber. A sensitive target cell population is added to the upper chamber and the assay is performed as described above.

  The effect of a protein or chimeric molecule thereof on ligand receptor binding can be assayed using one or more of the following systems.

The protein or chimeric molecule of the present invention may have different ligand-receptor binding capabilities. Ligand-receptor binding can be measured by various parameters such as dissociation constant (Kd), dissociation rate constant (off rate) (k ⁻ ), binding rate constant (on rate) (k ⁺ ). Differences in ligand receptor binding can correlate with various timing and signaling activations, leading to different biological outcomes.

  Ligand receptor binding can be measured and analyzed by Scatchard plot or other means such as Biacore.

In the case of Scatchard analysis, for example, a protein or chimeric molecule thereof labeled with a radioactive label (e.g., ¹²⁵ I) is reacted with the corresponding ligand or the presence of different amounts of cold competitor molecules of the protein or chimeric molecule Incubate with cells expressing the receptor or extracts thereof. The amount of specifically bound labeled protein or chimeric molecule thereof is determined and binding parameters are calculated.

  In the case of Biacore, the corresponding recombinant ligand or receptor of the protein or chimeric molecule thereof is conjugated to the detection unit. A solution containing the protein or chimeric molecule thereof is passed over the detection cell and binding is determined by changes in the properties of the detection unit. The binding rate constant can be determined by passing a solution containing the protein or chimeric molecule over the detection cell unit until a fixed reading is recorded (occupying all available sites). A solution without protein or chimeric molecule is passed over the cells to dissociate the protein from the corresponding ligand or receptor to obtain a dissociation rate constant.

  The effect of a protein or chimeric molecule thereof on receptor activation can be assayed using one or more of the following systems.

  The interaction of a protein or its chimeric molecule with its corresponding ligand or receptor may be comparable to the difference in signaling events derived from the cell's endogenous protein. The timing of interaction can be characteristic of the protein or its chimeric molecule, clearly as a binding / dissociation rate constant or dissociation constant.

  Activating receptors are often internalized by cells. The receptor / ligand receptor complex can then be dissociated (eg, by lowering the pH in the cell vesicle and releasing the ligand), allowing the receptor to recirculate to the cell surface. . Alternatively, the complex may be a target for destruction. In this case, the receptors cannot be effectively down-regulated to produce more signal, but when they are recycled, they can repeat the signaling process. Differential receptor binding or activation can cause a switch from receptor destruction to a recirculation pathway, which can result in a stronger biological response.

  The effect of a protein or chimeric molecule thereof on signal transduction can be assayed using one or more of the following systems.

  Ligand or receptor binding to the protein or chimeric molecule thereof may initiate signal transduction, which may include reverse signaling through various cytoplasmic proteins. Reverse signaling occurs when the membrane-bound form of the ligand transmits a signal after binding by the soluble or membrane-bound form of its receptor. Reverse signaling can also occur after binding of a membrane bound ligand by an antibody. These signaling events (including reverse signaling events) cause changes in gene or protein expression. Therefore, the protein or chimeric molecule of the present invention can be used to activate the JAK / STAT pathway, Ras-erk pathway, AKT pathway, PKC, PKA, Src, Fas, TNFR, NFkB, p38MAPK, c-Fos activation, Different signaling can be induced or inhibited in various pathways or other signaling events, such as receptor mobilization, receptor phosphorylation, receptor internalization, receptor crosstalk or secretion.

  A ligand or receptor recruited to a protein or chimeric molecule thereof may be unique to the protein or chimeric molecule of the present invention due to the different conformations of the derived ligand or receptor. One way to assay for these differences is to immunoprecipitate ligands or receptors using antibodies cross-linked to Sepharose beads. After immunoprecipitation and washing, proteins are loaded onto 2D gels and analyzed for equivalent spot patterns. Different spots can be cut out and identified by mass spectrometer.

  The effect of a protein or chimeric molecule thereof on the up- and down-regulation of surface markers can be assayed using one or more of the following systems.

  Cells can have a variety of responses to the protein or chimeric molecule of the present invention. There are a series of proteins on the cell surface that are responsible for communication between the cell and the extracellular environment. Through the regulation of endocytosis and exocytosis processes, various proteins are transported to and from the cell surface. Typical proteins found on the cell surface include receptors, binding proteins, regulatory proteins, and signaling molecules. Changes in protein expression and degradation rates also alter protein levels on the cell surface. Some proteins are also stored in intracellular reservoirs where specific signals can induce this storage and protein traffic between cell membranes.

  Cells can be incubated for an appropriate time in a medium containing the protein or chimeric molecule of the present invention and the reaction compared to cells exposed to the same medium that does not contain the protein or chimeric molecule of the present invention. Proteins on the cell membrane are lysed and separated from the cells by centrifugation. Specific protein expression levels can be measured by Western blotting. Cells can also be labeled with fluorescent conjugated antibodies and visualized with a confocal microscope system or counted by fluorescence activated cell sorting (FACS). This is believed to detect any change in protein expression and distribution in the cell. By using multiple antibodies, changes in protein interactions can be examined by confocal microscopy and immunoprecipitation as well. Similarly, these experiments can be extended to in vivo animal models. Cells from specific parts of animals treated with the protein or chimeric molecule of the present invention may be extracted and examined by the same methodology.

  Cells that have been induced to differentiate in vitro or in vivo by adding a protein or chimeric molecule of the present invention express differentiated cells that distinguish them from intact cells. Some cells, such as progenitor cells or stem cells, can differentiate into many subpopulations that can be distinguished by their surface markers. The protein or chimeric molecule of the present invention may stimulate progenitor cells to differentiate into subgroups at a specific ratio.

  The protein of the present invention and its chimeric molecule may have an effect on cell repulsion.

  The effect of a protein or chimeric molecule thereof on the regulation of cell and neuron growth and induction is a simple assay for cell repulsion.

  Disruption of the interaction between the protein subunit and other components provides a method for inhibiting the biological effect of the protein or chimeric molecule thereof. Compounds that inhibit such biological effects are identified in a number of ways.

  High throughput screening programs use small molecule chemical entities (chemicals or peptides) to generate lead compounds for clinical development. A number of assays are used to screen library compounds for whether they affect biologically relevant endpoints. Each potential compound in the library is treated with a specific assay in one well to determine whether the compound affects the assay. Some examples of assays are provided below.

  For this assay, cells are seeded in microtiter plates (96 plates, 384 plates, etc.). The cell appears to have a reading mechanism for activation of the protein or chimeric molecule thereof. This includes assays for cell growth, assays for stimulation of specific pathways (e.g., FRET-based techniques), assays for reporter gene induction (e.g., CAT, β-galactosidase, fluorescent proteins), assays for apoptosis and assays for differentiation. There is an inevitable possibility. The cells are then exposed to the protein or chimeric molecule of the present invention in the presence or absence of certain small molecules. The drug can be added before, after, or during the addition of the protein or chimeric molecule thereof. After an appropriate period, individual wells are read using appropriate methods (eg, fluorescence for FRET, induction of fluorescent protein, number of cells by MTT, β-galactosidase activity, etc.). Control wells without any drug or cytokine serve as a comparison. Any molecule that can inhibit the receptor / cytokine complex will produce a different reading in the control well. Further experiments may be necessary to show the specificity of inhibition. Alternatively, it is believed that the drug can affect the detection method (false positive) by a non-cytokine non-receptor mechanism.

  The ligand or receptor of the protein or chimeric molecule of the present invention is immobilized on a solid surface. Next, the protein or chimeric molecule thereof and the compound to be tested are added. This can be done by adding the compound next to the protein or chimeric molecule thereof; the compound can be added first and then the protein or chimeric molecule thereof; or the compound and the protein or chimeric molecule thereof can be added simultaneously. it can. The bound protein or chimeric molecule thereof is detected with a suitable detection antibody. The detection antibody can be labeled with an enzyme (eg, alkaline phosphatase for colorimetric detection, or horseradish peroxidase), or can be labeled with a fluorescent tag for fluorescence detection. Alternatively, the protein or chimeric molecule thereof can be labeled (e.g., biotin label, radiolabel) and appropriate techniques (e.g., streptavidin can be linked to a colorimetric detection system for biotin labeling, complex can be used for radiolabeling. It can be detected by solubilization and counting). Inhibition of protein binding is measured by a decrease in reading compared to control wells.

  A soluble ligand or receptor of the protein or chimeric molecule thereof is bound to the beads. This binding reaction can be an adsorption process, or it can necessarily involve chemically linking them to a plate. The beads are incubated with the protein or chimeric molecule and the candidate compound in appropriate wells. This can be done by first adding the protein or chimeric molecule followed by the compound; adding the compound first followed by the protein or chimeric molecule; or adding the compound and the protein or chimeric molecule simultaneously. A fluorescently labeled detection antibody that recognizes the protein or chimeric molecule thereof is then added. Unbound antibody is removed and the beads are passed through FACS. If the compound inhibits the interaction between the protein or its chimeric molecule and its receptor, the amount of fluorescence detected will decrease.

  The ability of the FACS instrument to analyze the scatter profile is used to allow screening of multiple interactions of a protein with its corresponding ligand / receptor for a single inhibitor compound. Larger diameter beads have a different scattering profile than smaller beads, which can be separated for analysis (“gating”).

  Many different proteins, one of which is a protein of the invention or a chimeric molecule, are each linked to a bead of a specific diameter. The ligand / receptor mixture for the protein is then added to the bead mixture in the presence of one candidate compound. Bound ligand / receptor is detected using a specific secondary antibody that is fluorescently labeled. All antibodies can be labeled with the same detection fluorophore. The ability of the compound to prevent protein binding to its ligand / receptor is then determined by passing the sample through a FACS instrument and setting a gate for each known bead size. Individual join results are analyzed separately. The main benefit of this assay is that each compound screen can be tested on a large number of proteins simultaneously, and the screening time is proportionally reduced.

  A protein or chimeric molecule thereof may also be characterized by its crystal structure. The physiochemical form of the protein or chimeric molecule thereof may provide a unique 3D crystal structure. Furthermore, the crystal structure of the protein-ligand / receptor complex may also be made using the protein or chimeric molecule of the present invention. Since the present invention provides a protein or chimeric molecule thereof that is substantially similar to the naturally occurring form of human, the complex more reflects the in vivo structure of the naturally occurring protein-ligand / receptor complex. It may be representative. After the crystal structure is obtained, the interaction of the protein or chimeric molecule thereof with a compound that may inhibit such interaction can be identified.

  Once potential compounds are identified by high-throughput screening or from the crystal structure of the protein-ligand / receptor complex, a rational drug design process can be initiated.

  There are several steps commonly taken in the design of a mimetic from a compound having a given desired property. First, the particular parts of the compound that are critical and / or important to determine the desired property are determined. In the case of peptides, this can be done by systematically changing amino acid residues in the peptide, for example by substituting each residue in turn. Alanine scans of peptides are commonly used to refine such peptide motifs. These parts or residues constituting the active region of the compound are known as the “pharmacophore”.

  After finding a pharmacophore, data from a series of sources, such as spectrophotometric techniques, x-ray diffraction data, and data from NMR, according to its physical properties, such as stereochemistry, bonds, size and / or charge To model that structure. Computer analysis, similarity mapping (which models pharmacophore charge and / or volume rather than interatomic bonds) and other techniques can be used in this modeling process.

  In a variant of this approach, the three-dimensional structure of the ligand and its binding partner is modeled. This is particularly useful when ligands and / or binding partners change conformation upon binding, and the model takes this into account when designing mimetics. Modeling can be used to generate inhibitors that interact with linear arrays or three-dimensional configurations.

  A template molecule is then selected onto which chemical groups that mimic the pharmacophore can be grafted. The template molecule and the chemical groups implanted on it do not degrade in vivo so that the mimetic can be easily synthesized, so that the mimetic is pharmacologically acceptable, and retains the biological activity of the lead compound. In addition, it can be easily selected. Alternatively, if the mimetic is based on a peptide, additional stability can be obtained by cyclizing the peptide and increasing its hardness. The mimetic or mimetics found by this approach can then be screened to see if they have the target property or to determine the extent to which they exhibit the target property. Further optimization or modification can then be performed to arrive at one or more final mimetics for in vivo or clinical testing.

  The purpose of rational drug design is, for example, to form a drug that is a more active or more stable form of the polypeptide, or to form a drug that, for example, enhances or interferes with the function of the polypeptide in vivo. Producing structural analogs of a biologically active polypeptide of interest or of small molecules (eg, agonists, antagonists, inhibitors, or enhancers) with which they interact. See, for example, Hodgson (Bio / Technology 9: 19-21, 1991). In one approach, the three-dimensional structure of the protein of interest is first determined by X-ray crystallography, by computer modeling, or most typically by a combination of approaches. Useful information regarding the structure of a polypeptide may also be obtained by modeling based on the structure of homologous proteins. An example of rational drug design is the development of HIV protease inhibitors (Erickson et al. Science 249: 521-533, 1990). In addition, target molecules may be analyzed by alanine scanning (Wells, Methods Enzymol 202: 2699-2705, 1991). In this technique, an amino acid residue is substituted with alanine to determine its effect on the activity of the peptide. Each of the amino acid residues of the peptide is analyzed in this way to determine key regions of the peptide.

  Similarly, it is possible to isolate a target-specific antibody selected by a functional assay and then elucidate its crystal structure. In principle, this approach results in a pharma core on which subsequent drug design can be based. By creating anti-idiotype antibodies (anti-ids) against functional pharmacologically active antibodies, it is possible to bypass protein crystallography completely. As a mirror image of the mirror image, the anti-ids binding site would be expected to be an analog of the original receptor. The anti-id can then be used to identify and isolate the peptide from a chemically or biologically produced bank of peptides. The selected peptide is believed to act as a pharmacore.

  In one aspect, the protein or chimeric molecule of the present invention is used as an immunogen to generate antibodies. The physiochemical form of the protein or chimeric molecule of the present invention includes a protein or chimeric molecule; a glycopeptide specific for the protein or chimeric molecule of the present invention; or another translation time in the protein or chimeric molecule thereof or It appears to generate antibodies against post-translationally modified peptides.

  The physiochemical form of the protein or chimeric molecule of the present invention may represent an epitope that is not normally accessible (but possibly present) in vivo. For example, there may be a region in the receptor domain that normally contacts another component of the heteropolymerized receptor. These epitopes may be used to produce monoclonal antibodies that cross-react with endogenous receptors. Such antibodies may block the interaction of one receptor component with another and thus prevent signal transduction. This may be therapeutically useful in the case of cytokine or receptor overexpression. Antibodies may also be useful therapeutically in diseases in which the receptor is overexpressed and signals are transmitted without the need for a ligand.

  The antibodies are also useful for detecting the level of a protein or chimeric molecule thereof (eg, serum levels for half-life determination) during treatment of a disease. In addition, antibodies are useful as diagnostic agents for determining the presence of a protein or chimeric molecule of the present invention in a particular sample.

Reference to “antibody” or “multiple antibodies” includes, for example, complete antibodies, including monoclonal antibodies (eg, having an intact Fc region); eg, antigens comprising Fv, Fab, Fab ′ and F (ab ′) ₂ Binding antibody fragments; humanized antibodies; human antibodies (eg, produced in transgenic animals or through phage display); and immunoglobulin-derived polypeptides produced through genetic engineering techniques. References to all different types of antibodies are included. Unless otherwise indicated, the term “antibody” or “multiple antibodies” includes both intact antibodies and antigen-binding fragments thereof as used herein.

  Unless otherwise stated, specificity for an antibody of the invention is intended to mean that the antibody binds substantially only to its target antigen without recognizable binding to an irrelevant protein. . However, antibodies can be designed or selected to bind to two or more related proteins. Related proteins include different splice variants or fragments of the same protein or homologous proteins from different species. Such antibodies are still considered specific for those proteins and are included in the present invention. The term “substantially” means in this context that there is no detectable binding to non-target antigens above the basal level, ie non-specific level.

  The antibodies of the present invention may be prepared by well-known techniques. For example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (Eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Lane (eds.), Cold Spring Harbor See Laboratory Press, Cold Spring Harbor, NY, (1988).

  One method for producing the antibodies of the present invention involves non-human animals, such as mice or transgenic mice, of the protein or chimeric molecule of the present invention, or an immunogen thereof, such as a peptide comprising a receptor binding domain. Immunizing with a sex moiety, whereby antibodies to the protein or chimeric molecule thereof, or a polypeptide of the immunogenic moiety thereof are produced in an animal. Various means for increasing the antigenicity of a particular protein or chimeric molecule thereof are well known to those skilled in the art, such as administering an adjuvant or conjugated antigen containing an antigen against which an antibody response is desired and another component. Yes, you may use it. Immunization typically entails a series of boosters after the initial immunization. Blood is drawn from the animals and serum is assayed for antibody titer. The animal may be boosted until the titer reaches equilibrium. Conjugates may be made in recombinant cell culture as protein fusions. Similarly, aggregating agents such as alum are also suitable for use to enhance the immune response.

  Both polyclonal and monoclonal antibodies can be produced by this method. Methods for obtaining both types of antibodies are well known in the art. Polyclonal antibodies are less preferred, but by injecting an appropriate animal with an effective amount of the protein or chimeric molecule of the invention, or immunogenic portion thereof, collecting serum from the animal, and any known immunoadsorption technique. It is relatively easily prepared by isolating specific antibodies against the protein or chimeric molecule thereof. Antibodies produced by this technique are generally less preferred because the products can be heterogeneous.

  The use of monoclonal antibodies is particularly advantageous because they can be produced in large quantities and the product is uniform. Monoclonal antibodies may be produced by conventional techniques.

  As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous population of antibodies, ie, a naturally occurring molecule in which individual antibodies, including the population, may be present in trace amounts. It refers to being identical except for existing mutations. Monoclonal antibodies are very specific and are directed against a single antigenic site. Furthermore, in contrast to polyclonal preparations that typically include different antibodies to different determinants (epitopes), each monoclonal antibody is directed against a single antigenic determinant on the antigen. The modifier “monoclonal” indicates the character of the antibody as being derived from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al. Nature 256: 495 (1975) or by recombinant DNA methods (see, e.g., U.S. Pat.No. 4,816,567). (See). “Monoclonal antibodies” can also be isolated from phage antibody libraries using the techniques described, for example, in Clackson et al. Nature 352: 624-628, 1991 and Marks et al. J Mol Biol 222: 581-597, 1991. Also good.

  The invention comprises immunizing a non-human animal such as a mouse or transgenic mouse with the protein or chimeric molecule of the invention, collecting spleen cells from the immunized animal; fusing the collected spleen cells with a myeloma cell line And producing hybridoma cells; and identifying a hybridoma cell line producing a monoclonal antibody that binds to the protein or chimeric molecule thereof. A method for producing a hybridoma cell line is contemplated.

  Such hybridoma cell lines and the monoclonal antibodies produced by them are encompassed by the present invention. Monoclonal antibodies secreted by the hybridoma cell line are purified by conventional techniques. Hybridomas or monoclonal antibodies produced by them may be further screened to identify monoclonal antibodies with particularly desirable properties, such as the ability to inhibit cytokine signaling through their receptors.

  Proteins or chimeric molecules thereof, or immunogenic portions thereof that may be used to immunize animals in the early stages of antibody production of the invention must be of human expression origin.

Antigen-binding fragments of the antibodies of the invention may be produced by conventional techniques. Examples of such fragments include, but are not limited to, Fv fragments, including Fab, Fab, F (ab ′) ₂ , and single chain Fv fragments (named sFv or scFv). Absent. Antibody fragments and derivatives produced by genetic engineering techniques, such as disulfide stabilized Fv fragments (dsFv), single chain variable region domain (Abs) molecules, minibodies, and diabodies are also for use in accordance with the present invention. Is contemplated.

  Such fragments and derivatives of monoclonal antibodies against the protein or chimeric molecule thereof may be prepared by known techniques including the assays described herein and screened for desirable properties. The assay is for identifying fragments and derivatives that retain the activity of inhibiting signaling by the protein or chimeric molecule thereof, as well as means for identifying fragments and derivatives of the antibodies of the invention that bind to the protein or chimeric molecule thereof. Provide a means. Certain techniques entail isolating DNA encoding the polypeptide chain (or part thereof) of the subject mAb and manipulating the DNA through recombinant DNA techniques. DNA may be fused to another DNA of interest, or modified to add, delete, or replace one or more amino acid residues (e.g., by mutagenesis or other conventional techniques). May be.

  DNA encoding an antibody polypeptide (eg, heavy or light chain, variable region only or full length) may be isolated from B-cells of mice immunized with a protein or chimeric molecule of the present invention. DNA may be isolated using conventional techniques. Phage display is another example of a known technique by which derivatives of antibodies can be prepared. In one approach, a polypeptide that is a component of a subject antibody is expressed in any suitable recombinant expression system and the expressed polypeptide is constructed to form an antibody molecule.

  Single chain antibodies may be formed by linking heavy and light chain variable region (Fv region) fragments through an amino acid bridge (short peptide linker), thereby yielding a single chain polypeptide. Such single chain antibodies Fvs (scFv) have been prepared by fusing DNA encoding a peptide linker between DNAs encoding two variable region polypeptides (VL and VH). The resulting antibody fragments can form dimers or trimers depending on the length of the flexible linker between the two variable domains (Kortt et al. Protein Engineering 10: 423, 1997). Techniques developed for the production of single chain antibodies include US Pat. No. 4,946,778; Bird (Science 242: 423, 1988), Huston et al. (Proc Natl Acad Sci USA 85: 5879, 1988) and Ward. Techniques described in et al (Nature 334: 544, 1989) are included. Single chain antibodies derived from the antibodies provided herein are included in the invention.

  In one embodiment, the present invention provides an antibody fragment, chimeric, recombinant or synthetic form of an antibody that binds to a protein or chimeric molecule of the present invention and inhibits signal transduction by the protein or chimeric molecule thereof.

  Techniques for deriving antibodies of different subclasses or isotypes from a subject antibody, ie subclass switching, are known. Thus, an IgG1 or IgG4 monoclonal antibody may be derived from an IgM monoclonal antibody and vice versa. By such techniques, new antibodies can be prepared that retain the antigen binding properties of a given antibody (parent antibody), but exhibit biological properties associated with an antibody isotype or subclass that is different from the parent antibody. Recombinant DNA technology may be used. Cloned DNA encoding a particular antibody polypeptide, such as DNA encoding the constant region of an antibody of the desired isotype, may be used in such techniques.

  The monoclonal antibody production process described above may be used in animals, such as mice, to produce monoclonal antibodies. It is known that normal antibodies derived from such animals, such as mouse antibodies, are generally not suitable for administration to humans because they can cause an immune response. Accordingly, such antibodies may need to be modified to provide antibodies suitable for human administration. Processes for preparing chimeric and / or humanized antibodies are well known in the art and are described in further detail below.

  Monoclonal antibodies herein include heavy and / or light chain variable domains that are identical or homologous to the corresponding sequences in antibodies from non-human species (eg, mice), as long as they exhibit the desired biological activity. , The remainder of the chain includes fragments of such antibodies, along with “chimeric” antibodies that are identical or homologous to the corresponding sequences in antibodies derived from humans (US Pat. No. 4,816,567 and Morrison et al. Proc Natl). Acad Sci USA 81: 6851-6855, 1984).

  “Humanized” forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies will have non-human species (donors) such as mice, rats, rabbits, or non-human primates whose recipient complementarity-determining regions (CDRs) have the desired properties, such as properties and affinity. Antibody) is a human immunoglobulin (recipient antibody) that is replaced by the corresponding CDR. In some examples, framework region residues of the human immunoglobulin are replaced with corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or donor antibody. These modifications are made to further refine antibody performance. Generally, a humanized antibody has all or substantially all complementarity determining regions corresponding to regions of a non-human immunoglobulin, and all or substantially all framework region residues are human immunoglobulin sequences. It appears to contain substantially all of at least one, and typically two variable domains, corresponding to these regions. A humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically at least a portion of a human immunoglobulin. For further details, see Jones et al. Nature 321: 522-525, 1986; Reichmann et al. Nature 332: 323-329, 1988; Presta, Curr Op Struct Biol 2: 593-596, 1992; Liu et al. Proc See Natl Acad Sci USA 84: 3439, 1987; Larrick et al. Bio / Technology 7: 934, 1989; and Winter and Harris, TIPS 14: 139, 1993.

  In a further aspect, the present invention provides an immunoassay kit having the ability to assay human protein levels expressed in human cells present in a biological preparation, including biological preparations comprising naturally occurring human proteins. To do.

  Biological preparations that can be assayed using the immunoassay kits of the present invention include, but are not limited to, experimental samples, cells, tissues, blood, serum, plasma, urine, stool, saliva and sputum. Do not mean.

Immunoassay kits of the present invention include, but are not limited to, membranes, dipsticks, beads, gels, tubes, or multiwell, flat bottom, round bottom, or v-bottom microplates, such as 96 well microplates. No solid support matrix; preparation of antibody against target human protein (capture antibody); preparation of blocking solution (eg BSA or casein); similarly suitable detection molecule (eg alkaline phosphatase) against target human protein Secondary antibody preparation conjugated to (detection antibody); chromogenic substrate solution (eg, nitroblue tetrazolium); additional substrate (eg, 5-bromo-4-chloro-3-indolyl phosphate); substrate buffer Stock solutions (e.g., 0.1M Tris-HCL (pH 7.5 ) and _{0.1M NaCl, 50 mM MgCl 2)} ; known concentration protein of the present invention Other preparations of the chimeric molecule (standard); and a use instructions.

  From materials such as gold colloids conjugated to enzymes, dyes, fluorescent molecules, chemiluminescence, isotopes, or molecules including but not limited to staphylococcal protein A or staphylococcal protein G A suitable detection molecule may be selected from the list.

  In certain embodiments, the capture and detection antibody is a monoclonal antibody, the production of which is hereinbefore described after the step of immunizing a non-human animal such as a mouse or transgenic mouse with a protein or chimeric molecule of the present invention. Including performing standard methods. A monoclonal antibody may alternatively be produced by the recombinant methods described herein above and may comprise a human or chimeric antibody portion or domain.

  In another embodiment, the capture and detection antibody is a polyclonal antibody and its production is performed after the step of immunizing a non-human animal such as a mouse, rabbit, goat, or horse with the protein or chimeric molecule of the present invention. Performing the standard methods described above in the specification.

  The components of the immunoassay kit are provided in a predetermined ratio, and the relative amounts of the various reagents are varied appropriately to provide a concentration in the solution of reagents that substantially maximizes the sensitivity of the assay. In particular, the reagent is a dry powder, usually a dry powder containing excipients, usually frozen, which when dissolved will provide the respective reagent solution with the appropriate concentration for combination with the biological preparation being tested. It may be provided as a dry powder.

The instructions for use will detail the method using the immunoassay kit of the invention. For example, the instructions for use may describe a method for coating a solid support matrix with a prepared capture antibody solution at suitable conditions, eg, overnight at 4 ° C. Instructions for use further block non-specific protein binding sites with the prepared blocking solution; add serially diluted samples containing the protein of the invention or chimeric protein, and at suitable conditions, eg, 1 hour at 37 ° C., or Following the step of incubating at room temperature for 2 hours, the steps of performing a series of washes with a suitable buffer known in the art, for example, 0.05% Tween 20 in 0.1 M PBS (pH 7.2) will be described in detail. It is. In addition, the instructions may provide for a further series of washes after the incubation, after applying the detection antibody preparation at suitable conditions, eg, 1 hour at 37 ° C. or 2 hours at room temperature. A working solution of detection buffer is prepared from the supplied detection substrate and substrate buffer and then added to each well under suitable conditions for 5 minutes at room temperature to 1 hour at 37 ° C. The chromogenic reaction may be stopped by adding 1N NaOH or 2N H ₂ SO ₄ .

  In another embodiment, the instructions for use may provide for the simultaneous addition of any or all combinations of any of the above components added in a predetermined ratio, the relative amounts of the various reagents being complex formation. Appropriately varying to provide a concentration in the reagent solution that substantially maximizes the formation of a measurable signal.

  Measure the level of fluorescence, chemiluminescence, radioactivity, or other signal produced by the binding, conjugate detection reagent, or fluorescence, chemiluminescence, radioactivity, or other signal at an appropriate optical density (OD) using an ELISA plate reader or spectrophotometer Can be measured as synchrotron radiation using a spectrophotometer, fluorometer, or flow cytometer at an appropriate wavelength, can be measured in an appropriate energy spectrum using a radioactivity counter, and density It can be measured with the naked eye by means of a meter or by comparison with a chart or guide. A serial dilution of the standard preparation is assayed simultaneously with the sample. A standard curve or chart can be generated and the level of the protein or chimeric molecule of the present invention in the sample can be interpolated from the standard curve or chart.

  The present invention also provides a human-derived protein or chimeric molecule thereof for use as a standard protein in an immunoassay. The invention further comprises (a) mixing one or more antibodies against a human protein or chimeric molecule thereof and a biological preparation, (b) each antibody against a human protein or chimeric molecule in the biological preparation. (C) mixing a standard human protein sample or chimeric molecule sample with one or more antibodies to the human protein or chimeric molecule, (d) a standard human protein sample or Determining the level of binding of each antibody to the chimeric molecule sample; (e) determining the level of each antibody bound to the human protein or chimeric molecule in the biological preparation to a standard human protein sample or chimeric molecule sample; Suitable for measuring human proteins or chimeric molecules, including comparing to the level of each antibody bound Comprises an assay, also extends to a method for determining the level of human cell expressed human protein or chimeric molecule thereof in a biological preparation.

  In particular, a standard human protein sample or chimeric molecule sample is a preparation comprising a protein or chimeric molecule of the present invention.

  Biological preparations include, but are not limited to, laboratory samples, cells, tissues, blood, serum, plasma, urine, stool, saliva, and sputum. The biological preparation binds to one or more capture antibodies as described herein above or by methods known in the art. For example, a solid support matrix is first coated with a capture antibody preparation under suitable conditions (e.g., overnight at 4 ° C.), and then non-specific protein binding sites are blocked with the prepared blocking solution, followed by the present invention. After a step of adding a serially diluted sample containing the protein or chimeric molecule and incubating under suitable conditions (e.g., 1 hour at 37 ° C or 2 hours at room temperature), a suitable buffer known in the art (e.g., A series of washes with 0.05% Tween 20 in 0.1 M PBS (pH 7.2).

  The biological preparation is mixed with one or more detection antibodies conjugated to a suitable detection molecule as described herein. For example, after applying the detection antibody preparation, it is incubated under suitable conditions (eg, 1 hour at 37 ° C. or 2 hours at room temperature) followed by a further series of washes.

The determination of the binding level may be performed as previously described herein or by methods known in the art. For example, a working solution of detection buffer is prepared from detection substrate and substrate buffer and added to each well under suitable conditions from 5 minutes at room temperature to 1 hour at 37 ° C. The chromogenic reaction may be stopped by adding 1N NaOH or 2N H ₂ SO ₄ .

  In certain embodiments, the present invention contemplates an isolated protein or chimeric molecule as previously described herein.

In one embodiment, the IFN-a2b of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 kDa, such as about 1-250, and in one embodiment 13-24 kDa Apparent molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4.5 to 7 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2-100 isoforms (P ₃ ), and in one embodiment 2-22 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) such as about 0-99%, such as%, and in one embodiment 0-20%;
-Monosaccharide (P ₉ ) and sialic acid (P ₁₀ ) content when normalized to GalNAc: 1 to 0-3 fucose, 1 to 0-3 GlcNAc, 1 to 0-6 galactose, 1 to 0- 3 mannose and 1 to 0-5 NeuNAc, and in one embodiment 1 to 0-1 fucose, 1 to 0-1 GlcNAc, 1 to 1-4 galactose, 1 to 0-1 mannose and 1 to 0-2 NeuNAc;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 Sialic acid content (P ₁₀ ), expressed as a percentage of monosaccharide content of IFNα2b of the invention, about 0-50%, such as 50%, and in certain embodiments 0-10%;
-60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, about 60-100% such as 100%, and in one embodiment 80-100 % Neutral O-linked oligosaccharide percentage (P ₁₅ );
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40%, such as 40%, and in one embodiment 0-20 The percentage of acidic O-linked oligosaccharides (P ₁₆ ), such as%; and
- different biological activity as human IFN-a2b expressed in a nonhuman cell lines, and in one embodiment, the ability of IFN-a2b of the present invention to inhibit the GM-CSF induced proliferation of TF-1 cells (T ₃₂₎ Is 250-600 times more potent than human IFN-a2b expressed in E. coli cells.

In one embodiment, IFN-b1 of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment 15-40 kDa apparent Molecular weight of (P ₁ );
A pI (P ₂ ) range of about 2 to about 14 such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 1 to 50 isoforms; and
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-50%.

In one embodiment, the IFN-g of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment 15-30 kDa apparent Molecular weight (P ₁ );
- at about 2 to about 14, and an embodiment such as 2,3,4,5,6,7,8,9,10,11,12,13,14 4-14 of pI (P ₂₎ ranges ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 4 to 16 isoforms; and
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-45%;
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 10-25 kDa, and in one embodiment 12-20 kDa (P ₆ );
The observed molecular weight of the molecule after removal of N-linked and O-linked oligosaccharides of about 10-25 kDa, and in one embodiment 12-20 kDa, (P ₇ );
- including PNG-ase treatment after N-48 and N-120 was identified by PMF (numbering from the start of the signal sequence) N- glycosylation site (P _21); and
-A biological activity different from human IFN-g expressed in non-human cell lines, and in one embodiment, the ability of the IFN-g of the invention to inhibit the growth of HT-29 cells in the presence of TNF-a ( T ₃₂ ) is 11-17 times more potent than human IFN-g expressed in E. coli cells.

In one embodiment, the IFNAR2-Fc of the present invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, in one embodiment, 50-105 kDa apparent Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4 to 7 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 10 to 25 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-50%;
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 40-100 kDa, and in one embodiment 45-95 kDa, (P ₆ ); and
- about 40 to 90 kDa, and in one embodiment of the 45 to 80 kDa, observed molecular weight of the molecule of after the N- linked and O- linked oligosaccharides are removed (P _7).

In one embodiment, IL-10 of the invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 , 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, and in one embodiment, 10-23 kDa in appearance Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 6 to 10 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 4 to 20 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-20%;
The observed molecular weight of the molecule after removal of the N-linked oligosaccharide of about 8-23 kDa, and in one embodiment 10-23 kDa, (P ₆ );
The observed molecular weight of the molecule after removal of the N-linked and O-linked oligosaccharides of about 8-23 kDa, and in one embodiment 10-23 kDa, (P ₇ );
-A different immunoreactivity profile (T ₁₃ ) than human IL-10 expressed in non-human cell lines, and in one embodiment, the protein concentration of IL-10 of the present invention is expressed in human IL-10 expressed in E. coli cells Underestimated when assayed using an ELISA kit containing -10; and
-A biological activity different from human IL-10 expressed in non-human cell lines, and in one embodiment, the ability of the IL-10 of the invention to induce proliferation of MC / 9 cells in the presence of IL-4 ( T ₃₂ ) is 10-25 times more potent than human IL-10 expressed in E. coli cells.

In one embodiment, an IL-10Ra-Fc of the invention is characterized by one or more profiles of the following physiochemical parameters (P _x ) and pharmacological traits (T _y ), including:
-1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 , 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 , 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, about 1-250, such as 250 kDa, in one embodiment 50-100 kDa apparent Molecular weight (P ₁ );
A pI (P ₂ ) range of about 2 to about 14, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, and in one embodiment 4.5 to 9.5 ;
-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 isoforms About 2 to 100 isoforms (P ₃ ), and in one embodiment 10 to 21 isoforms;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 , 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 Carbohydrate weight percentage (P ₅ ) of about 0-99%, such as%, and in one embodiment 0-49%;
An observed molecular weight (P ₆ ) of the molecule after removal of the N-linked oligosaccharide of about 35-95 kDa, and in one embodiment 40-85 kDa;
The observed molecular weight of the molecule after removal of N-linked and O-linked oligosaccharides of about 30-95 kDa, and in one embodiment 36-85 kDa (P ₇ );
-Monosaccharide (P ₉ ) and sialic acid (P ₁₀ ) content when normalized to GalNAc: 1 to 0.1-4 fucose, 1 to 2-34 GlcNAc, 1 to 0.5-8 galactose, 1 to 1 13 mannose and 1 to 0-3 NeuNAc; 3 to 0.1-2 fucose, 3 to 0.01-3 GalNAc, 3 to 1-30 GlcNAc, 3 to 0.1-4 galactose when normalized to 3 times the amount of mannose 3 to 0-3 NeuNAc;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 Sialic acid content (P ₁₀ ) expressed as a monosaccharide content percentage of IL-10Rα-Fc of 0-10%, such as 50%, and in one embodiment 0-10%;
-Sulfate and phosphate content when normalized to GalNac (P ₁₁ ): 1 to 0-3 sulfate, and in one embodiment 1 to 0-1.5 sulfate; 3 times the amount of mannose Standardized 3 to 0-1 sulfate, and in one embodiment 3 to 0-0.6 sulfate;
-0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 , 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 Sulfation (P ₅₉ ), expressed as a percentage of the monosaccharide content of the molecule, 0-50%, such as 50%, and in one embodiment 0-3%;
-40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64 , 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85% like about 40-85 %, And in one embodiment 55-75% neutral N-linked oligosaccharide percentage (P ₁₃ );
-15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 About 15-60 like 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60% %, And in one embodiment 25-45% acidic N-linked oligosaccharide percentage (P ₁₄ );
-An N-glycosylation site (P ₂₁ ) comprising N-110, N-154, N-177 and N-323 (numbering from the start of the signal sequence) identified by PMF after PNGase treatment; and
-A biological activity different from human IL-10Ra-Fc expressed in a non-human cell line, and in one embodiment the IL of MC / 9 cells in the presence of IL-4 of the IL-10Ra-Fc of the invention The ability to neutralize -10-induced proliferation (T ₃₂ ) is 18-150 times more potent than soluble human IL-10Ra molecules expressed in E. coli cells.

In one embodiment, the protein or chimeric molecule of the present invention comprises at least one of the following structures in the N-linked fraction (P ₁₉ ): In these notations, “u” or “?” Represents that the anomeric configuration is a or b and / or the position of the linkage is 2, 3, 4 and / or 6.

In one embodiment, the protein or chimeric molecule of the present invention comprises at least one of the following structures in the O-linked fraction (P ₂₀ ). In these notations, “u” or “?” Represents that the anomeric configuration is a or b and / or the position of the linkage is 2, 3, 4 and / or 6.

  A physiochemical form of a protein or chimeric molecule of the present invention includes introducing into a host cell one or more transgenes encoding an enzyme or enzymes that will produce the desired physiochemical form. May be obtained by modifying host cells by a variety of methods known in the art, including but not limited to these. Such transgenes include ST3Gall, ST3Gal2, ST3Gal3, ST3Gal4, ST3Gal5, ST3Gal6, ST6Gal1, ST6Gal2, ST6GalNAc1, ST6GalNAc2, ST6GalNAc3, ST6GalNAc4, ST6GalNAc5, ST6GalNA, ST6Gal4 Types of sialyltransferases; galactosyltransferases such as GalT1, GalT2; fucosyltransferases such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11; sulfotransferases; GNT1, GNT2, GNT3, GlcNAc transferases such as GNT4, GNT5; antenna cleaving enzymes and endoglycosidases.

  For example, insufficient terminal sialylation of an N-glycan structure that results in a reduction in serum half-life of an expressed protein such as recombinant human AchE results in the rat β-galactoside α2,6-sialyltransferase transgene being expressed in HEK 293 Can be improved by adding to cells (J Biochem 336: 647-658, 1998; J Biochem 363: 619-631, 2002).

  Similarly, inadequate formation of certain Lewis x groups, such as sialyl Lewis x structures in N-glycan structures, resulting in reduced ligand binding of expressed proteins such as recombinant human PSGL-1, is a fucosyltransferase Improvement can be made by adding a transgene to HEK293 cells (Fritz et al. PNAS 95: 12283-12288, 1998).

  In one embodiment, the protein or chimeric molecule thereof is obtained by either α-2,3- or α-2,6-sialyltransferase or by α-2,3-sialyltransferase and α-2,6-sialyltransferase. And a human cell line transformed with both ("sialylated protein"). Examples of sialylated proteins include sialylated-IFN-a2B, sialylated-IFN-a2B-Fc, sialylated-IFN-b1, sialylated-IFN-b1-Fc, sialylated-IFN-g, sialylated- IFN-g-Fc, sialylated-IFNAR2, sialylated-IFNAR2-Fc, sialylated-IL-10, sialylated-IL-10-Fc, sialylated-IL-10Ra, sialylated-IL-10Ra-Fc included.

In particular, sialylated proteins are characterized by a profile of physiochemical parameters (P _x ) that includes one or more physiochemical parameters. The monosaccharide (P ₉ ) and sialic acid content (P ₁₀ ) of the sialylated protein are 1 to 0.1-100 NeuNAc when normalized to GalNAc, and 3 times the amount of mannose when normalized to 3 vs. 0.1-100 NeuNAc. The percentage of neutral N-linked oligosaccharides of sialylated proteins (P ₁₃ ) is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, _{13, 14} , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 0-99%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. The percentage of acidic N-linked oligosaccharides of sialylated proteins (P ₁₄ ) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, _{13, 14, 15, 16} , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%, such as 1-100%. The percentage of neutral O-linked oligosaccharides of sialylated proteins (P ₁₅ ) is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, _{13, 14} , 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 0-99%, such as 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%. The percentage of acidic O-linked oligosaccharides of the sialylated protein (P ₁₆ ) is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, _{13, 14, 15, 16} , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100%, such as 1-100%. The in vivo half-life (T ₁₁ ) of the sialylated protein is increased compared to the half-life of the protein or chimeric molecule of the present invention expressed without the transgene.

  In one embodiment, the sialylated protein has at least one of the structural formulas described herein, or wherein one or more NeuNAc linkages are α-2,6-linkages in the N-linked fraction. Contains at least one of the structural formulas described in the book.

  In one embodiment, the sialylated protein has at least one of the structural formulas described herein, or wherein one or more NeuNAc linkages are α-2,6-linked in an O-linked fraction. Contains at least one of the structural formulas described in the book.

  In one embodiment, a protein of the invention or chimeric molecule thereof is produced using a human cell line transformed with FUT3 (“fucosylated protein”). Examples of fucosylated proteins include fucosylated-IFN-a2B, fucosylated-IFN-a2B-Fc, fucosylated-IFN-b1, fucosylated-IFN-b1-Fc, fucosylated-IFN-g, fucosylated- IFN-g-Fc, fucosylated-IFNAR2, fucosylated-IFNAR2-Fc, fucosylated-IL-10, fucosylated-IL-10-Fc, fucosylated-IL-10Ra, fucosylated-IL-10Ra-Fc included.

In particular, fucosylated proteins are characterized by a profile of physiochemical parameters (P _x ) that includes one or more physiochemical parameters. The monosaccharide (P ₉ ) and sialic acid content (P ₁₀ ) of fucosylated proteins are 1 to 0.1-100 NeuNAc when normalized to GalNAc, and 3 times the amount of mannose when normalized to 3 vs. 0.1-100 NeuNAc.

  In one embodiment, the fucosylated protein has a higher proportion of structures, including a Lewis structure (Lewis a, Lewis b, Lewis x or Lewis y) or a sialyl Lewis structure (such as sialyl Lewis a or sialyl Lewis x).

  In one embodiment, the fucosylated protein has an altered binding affinity for the ligand as compared to the binding affinity of the protein or chimeric molecule of the invention expressed without the transgene.

  Using the respective forward and reverse primers for protein molecules selected from IFN-a2B, IFN-b1, IFN-g, IFNAR2, IL-10, and IL-10Ra, the DNA encoding the relevant protein is used in the art. Amplified from ESTs according to the method of PCR Super Mix High Fidelity (Catalog No: 10790-020) by Invitrogen by the polymerase chain reaction (PCR) by methods known in the art. Digest the amplicon and use appropriate vectors such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1 / GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1 / Hygro (+), pcDNA3.1 / Hygro (-), Ligate to the corresponding restriction enzyme site of pEF6 / V5-His. The ligated vector is transformed into an appropriate E. coli host cell such as XLGold, supercompetent cell (Strategene), XL-Blue, DH5α, DH10B and the like.

  To produce chimeric molecules, DNA sequences of immunoglobulin Fc domains such as IgGl, IgG2, IgG3, IgG4, IgGAl, IgGA2, IgGM, IgGE, IgGD are ESTs by PCR using appropriate forward and reverse primers. Amplify from. An amplicon is a suitable vector such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1 / GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1 / Hygro (+), pcDNA3.1 / Hygro (-), Clone into the corresponding restriction enzyme site of pEF6 / V5-His. The DNA sequence of the relevant protein is amplified and cloned in-frame with the Fc in the corresponding restriction enzyme site of each Fc vector.

  In certain embodiments, the Fc receptor binding region or complement activation region of the Fc region is recombined to include one or more amino acid insertions, deletions, or substitutions relative to the amino acid sequence of the Fc region. It may be modified. In addition, the receptor binding region or complement activation region of the Fc region can be altered to its glycosylation pattern, addition or removal of carbohydrate moieties, polyunsaturated fatty acid moieties or other lipid based amino acid backbones or any May be chemically modified by addition to the relevant translational or post-translational entity. The Fc region may also be truncated due to cleavage by enzymes including papain, pepsin, or any other site-specific protease. The Fc region may promote spontaneous formation by a dimeric, trimeric, or higher multimeric chimeric protein that can bind better to its corresponding ligand or receptor.

  In order to identify / isolate bacterial colonies containing vectors with the correct gene, diagnostic digests with appropriate restriction enzymes may be performed. Positive colonies are isolated and stored as a glycerol stock at -70 ° C. The clones are then expanded into 750 ml of sterile LB broth containing ampicillin (100 μg / ml) with shaking at 37 ° C. for 16 hours. Plasmids are prepared according to methods known in the art, preferably according to the Qiagen Endofree Plasmid Mega Kit (Qiagen Mega Prep Kit # 12381).

  Suitable human host cells for the introduction of cloned DNA sequences containing the protein or chimeric molecule of the present invention include HEK 293 and any derivative thereof, HEK 293 cl8, HEK 293-T, HEK 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD-293 (Stratagene), 293A (Invitrogen), HeLa cells and any derivatives thereof, HepG2, PA-1 Jurkat, THP-1, HL-60, H9, HuT 78, Hep-2 , Hep G2, MRC-5, PER.C6, SKO-007, U266, Y2 (Apollo), WI-38, and WI-L2, but are not limited thereto.

  The physiochemical form of the protein or chimeric molecule of the present invention includes, but is not limited to, introducing into the host cell an enzyme that produces the desired physiochemical form or a transgene encoding a plurality of enzymes. This may be done by modifying the host cell by various methods known in the art. The introduction of specific DNA sequences can be used to optimize the integration of the cloned DNA sequence into the host cell genome, with various types of integration including site-specific, targeted, direct or enzyme-mediated integration. Including, but not limited to.

  Protein or chimeric molecule DNA can be obtained by various transfection methods known in the art, for example using chemical reagents such as DEAE-dextran, calcium phosphate, artificial liposomes, or by direct microinjection, electroporation, biolistic It can be introduced into a suitable host cell by particle delivery or infection or transfection with a viral construct described below.

  DEAE-dextran is a cationic polymer that associates with negatively charged nucleic acids. The excess positive charge contributed by the polymer in the DNA / polymer complex causes the complex to more closely associate with the negatively charged cell membrane. Complex uptake probably occurs by endocytosis. Other synthetic cationic polymers including polybrene, polyethyleneimine, and dendrimers are also used for transfection.

  Calcium phosphate coprecipitation can be used for transient and stable transfection of various types of cells. The DNA is mixed with calcium phosphate in a controlled manner and added to the buffered saline / phosphate solution and the mixture is incubated at room temperature. A precipitate is formed, which is taken up by the cells by endocytosis or phagocytosis.

The most commonly used synthetic lipid component of liposomes for liposome-mediated gene delivery is the component that has a true positive overall charge at physiological pH. Often, cationic lipids are mixed with neutral lipids such as L-dioleoylphosphatidylethanolamine (DOPE). The cationic portion of the lipid molecule associates with the negatively charged nucleic acid, thereby causing nucleic acid compression in the liposome / nucleic acid complex.
Complex uptake occurs by endocytosis.

  Although direct microinjection of DNA into cultured cells or nucleic acids is a tedious technique, it is effective but is not appropriate when a large number of transfected cells are required.

  Electroporation uses electrical pulses to create pores that allow nucleic acids to pass through cells. This technique requires fine tuning and optimization for pulse duration and intensity for each type of cell used. Commercially available electroporation devices include Amaxa Biosystems' Nucleofector Kits (Amaxa Biosystems, Germany).

  This method relies on rapid delivery of nucleic acids on microprojectiles to recipient cells.

  Infection or transfection with a virus or retroviral construct includes using a retrovirus, such as a lentivirus, or a DNA virus, such as an adenovirus. The process involves using a viral or retroviral vector to transfer the foreign gene into the host cell.

  In some embodiments, the protein or chimeric molecule thereof is produced by a transient method or from a stably transfected cell line. Transient transfection is performed using either adherent or suspension cell lines. For adherent cell lines, cells are grown in serum-containing media (2-10% serum) and in media such as DMEM, DMEM / F12 (JRH). The serum used can be fetal calf serum (FCS), donor calf serum (DCS), newborn calf serum (NBCS) and the like. Plasmid vectors are introduced into cells by standard methods known in the art. In certain embodiments, the DNA of the protein or chimeric molecule thereof is transfected using DEAE dextran or calcium phosphate precipitation. Following transfection, the cells are switched to an appropriate collection medium (eg, serum-free DMEM / F12) to recover the expressed protein or chimeric molecule thereof.

  Transient expression of a protein or its chimeric molecule from suspension cells can be performed by introducing a plasmid vector using the method outlined above. Suspension cells can be grown in either serum-containing medium or serum-free medium (eg, Freestyle medium (Invitrogen), CD293 medium (Invitrogen), Excell medium (JRH), etc.). Transfection can be performed in the absence of serum by a suitable transfection method, a suitable transfection method, for example by transfecting a suitable medium with Lipofectamine in OptiMEM medium.

  Transient expression usually results in a peak expression 2-3 days after transfection. Episomal vectors replicate in cells to produce sustained expression. Therefore, to obtain large quantities of product, episomal expression vectors are transfected into cells and the cells are expanded. The protein or chimeric molecule thereof is expressed in the medium and the cells are expanded for several weeks and recovered. The expression medium can be a medium with or without serum, and the cells can be adapted to either adhere or float.

  After obtaining a stable clone by transfection of the expression vector into cells, it is selected with an appropriate substance such as phleomycin, hygromycin, puromycin, neomycin G418, methotrexate and the like. Since plasmids contain resistance genes in addition to genes encoding proteins or chimeric molecules, stable clones are believed to survive selection. One to two days after gene transfer, selection is initiated on either the whole cell population (stable pool) or cells seeded at clonal density. Non-transfected cell populations are also selected to determine cell killing efficacy with the selection agent. In the case of adherent cells, the cells are grown in tissue culture plates until visible individual clones are obtained. They are then removed from the plate by trypsinization or by physical removal and placed in tissue culture wells (eg, one clone per well of a 96 well plate). For suspension cells, perform limiting dilution cloning after selection. The clones are then expanded before being characterized and / or subjected to a further round of limiting dilution analysis.

  Stable clones that grow in serum-containing media detach after adapting to a gradual reduction in serum levels and grow in suspension under low serum. Serum levels are further reduced until a serum free state is obtained. Some growth media allow more rapid adaptation (eg, direct exchange from serum-containing adhesion conditions to serum-free suspension growth), an example being Invitrogen's CD293 media.

  After growth in serum-free medium, the clone can begin medium optimization. Clones are tested for integrated viable cell counts in a number of different growth media until production characteristics such as optimal formulation or multiple formulations are obtained. This may depend on the product production method. For example, cells may be expanded in one medium and then additives that enhance expression may be added prior to product recovery.

  Overexpressed proteins or chimeric molecules can accumulate in the host cell. NP40, Triton X-100, Triton X-114, Sodium dodecyl sulfate (SDS), Sodium cholate, Sodium deoxycholate, CHAPS, CHAPSO, Brij-35, Brij-58, Tween-20 Inevitably involves treating the host cell with a lysis buffer including, but not limited to, a buffer comprising, Tween-80, octyl glucoside, and octyl thioglucoside. Another method of host cell lysis may include sonication, homogenization, French press treatment, repeated freeze-thaw cycles, and treatment of cells with hypotonic solutions.

  The final product can be produced in many different types of bioreactors including, as non-limiting examples, stirred tanks, airlifts, packed bed perfusion, microcarriers, hollow fibers, bag technology, cell factories. The method may be continuous culture, batch, fed-batch, or induction. To obtain an increase in volumetric protein production, peptone may be added to the low serum culture.

  The protein or chimeric molecule of the present invention is purified using a purification strategy specifically tailored for the protein or chimeric molecule of the present invention. Purification methods include tangential flow filtration (TFF), ammonium sulfate precipitation, size exclusion chromatography (SEC), gel filtration chromatography (GFC), affinity chromatography (AFC), protein A affinity purification, receptor-mediated ligand chromatography ( RMLC), dye ligand chromatography (DLC), ion exchange chromatography (IEC) including anion or cation exchange chromatography (AEC or CEC), reverse phase chromatography (RPC), hydrophobic interaction chromatography (HIC) ), Metal chelation chromatography (MCC), but is not limited thereto.

  TFF is a rapid and efficient method for separating biomolecules and is used for sample concentration, desalting, or fractionation. TFF can concentrate samples as small as 10 ml from samples as large as several hundred liters. Along with suitable molecular weight cut-off membranes, TFF can separate and isolate biomolecules of different sizes and molecular weights (nominal molecular weight cut-off (NMWC) 5 KDa, 10 KDa, 30 KDa, 100 KDa). The sample buffer can be desalted or exchanged using a diafiltration process that entails reconcentration following sample dilution.

  Salting out or ammonium sulfate precipitation is useful for concentrating diluted solutions of proteins. This is also useful for fractionating a mixture of proteins. Increasing the ionic strength of a solution containing protein causes a reduction in the repulsive effect of similar charges between protein molecules. Similarly, the ability to retain the solvation shell around the protein molecule is reduced. If these forces are sufficiently reduced, the protein appears to precipitate; hydrophobic proteins precipitate at lower salt concentrations than hydrophilic proteins. Fractionation of the protein mixture by centrifugation after a gradual increase in ionic strength can be a very effective way to partially purify the protein.

  SEC separates proteins by size based on sample flow within a porous matrix. SEC has the same principle as GFC when used to separate molecules in aqueous systems. In SEC, molecules larger than the fill pore elute first with the solvent tip and are completely excluded. Molecules of intermediate size between completely excluded molecules and retained molecules pass through the pores of the matrix according to their size. Small molecules that freely enter and exit the pores are retained. Thus, different sized proteins have different elution volumes and retention times. For structurally similar molecules, they elute earlier if the molecular size is larger. Before eluting any sample, a standard curve must be established to determine working limits and reference retention times.

  If the protein shape is the same, the molecular weight can be rapidly screened in the eluate from the column by UV absorption, fluorescence, or light scattering, depending on the packing material of various pore sizes in the column. Photon correlation spectroscopy (PCS) is usually performed on static samples and for liquid chromatography detection. Low angle laser light scattering is also coupled to chromatographic detection to directly detect molecular weight independent of protein shape (Carr et al. Anal Biochem 175: 492-499, 1988). SEC-HPLC was used to detect hGH degradation and aggregation (Pikal et al. Pharm Res 8: 427-436, 1991). This was also used for the estimation of contaminants when testing β-galactosidase (Yoshioka et al. Pharm Res 10: 103-108, 1993).

  AFC purifies biomolecules according to specific interactions between their chemical structure and a suitable affinity ligand. The target molecule is specifically and reversibly adsorbed by a complementary immobilized ligand. The ligand can be an inhibitor, substrate, analog, or cofactor, or an antibody that can specifically recognize the target molecule. The adsorbed molecules are then eluted by competitive displacement or by a conformational change due to pH or ionic strength shifts.

  Protein A affinity purification is an example of affinity purification that generally utilizes the affinity of a particular bacterial protein that binds to the antibody, regardless of the specificity of the antibody for the antigen. Protein A, Protein G and Protein L are three proteins with well-characterized antibody binding properties. These proteins are produced recombinantly and are routinely used for affinity purification of important antibody types from various species. A genetically engineered recombinant form of protein A and G called protein A / G is also available. These antibody binding proteins are immobilized to support the matrix. This method is modified to purify a recombinant protein in which the protein A binding region (Fc region) of the antibody is linked to the target protein. Binding to the immobilized protein A molecule occurs at physiological conditions and is eluted by changes in pH or ionic strength.

  RMLC is a special type of AFC that takes advantage of the receptor's inherent affinity for its cognate target molecule. The acceptor molecule is immobilized on a suitable chromatographic support matrix through reactive amine, reactive hydrogen, carbonyl, carboxyl, or sulfhydryl groups. In one example of RMLC, a receptor-Fc chimeric molecule is immobilized on protein A sepharose beads through the affinity of the Fc portion of the receptor for protein A. This method has the advantage of immobilizing the receptor in a direction that exposes its ligand binding site to its cognate cytokine. Adsorption of the target molecule to the receptor is performed under physiological conditions, and elution is performed by changing the pH or ionic strength.

  DLC is a type of ALC that takes advantage of the selective and reversible binding ability of reactive dyes to proteins. The dye is generally a monochlorotriazine compound. Reactive chloro groups can easily fix triazine dyes to support matrices such as sepharose or agarose, and more recently to nylon membranes.

  The first discovery that these dyes can bind to proteins is that blue dextran (conjugate of Cibacron Blue FG-3 A) used as a void volume marker in gel filtration columns can delay elution of certain proteins Derived from the observation. Much research has been done on the specificity of dyes for specific proteins, most of which use the original Cibacron Blue dye. The dye appears to be most effective in binding proteins and enzymes that utilize nucleotide cofactors such as kinases and dehydrogenases, but other proteins such as serum albumin bind as well. It has been proposed that the aromatic triazine dye structure is similar to the nucleotide structure of nicotinamide adenosine dinucleotide (NAD) and that the dye interacts with the dinucleotide cage in these proteins. In many cases, the binding protein can be eluted from the column competitively by the substrate or nucleotide cofactor, and the dye has been shown to compete with the substrate-binding site in free solution. Similarly, it appears that these dyes may be able to bind to proteins by electrostatic and hydrophobic interactions and by more specific “pseudo-affinity” interactions with ligand binding sites. It is. Enhancing the specificity of dye ligands to be more similar to ligands (biomimetic dyes) by modification has been successful in the purification of many dehydrogenases and proteases (McGettrick et al. Methods Mol Biol 244: 151-7 , 2004).

  Ion exchange chromatography (IEC) purifies proteins using protein retention on the column due to electrostatic interactions between the ion exchange column matrix and the protein. If the mobile phase pH is above the pI of the target protein, the protein appears to be negatively charged and interact with the anion exchange column (AEC). If the pH of the mobile phase is lower than the pI of the target protein, the protein should be positively charged and a cation exchange column (CEC) should be used. The target protein is eluted by increasing the concentration of counterion having the same charge as the target molecule.

  RPC separates biomolecules according to the hydrophobic interaction between the molecule and the chromatographic support matrix. An ionizable compound is best analyzed in its neutral form by controlling the pH of the separation. Mobile phase additives such as trifluoroacetic acid increase the hydrophobicity of the protein by forming ion pairs that strongly adsorb to the stationary phase. Biomolecules are eluted from the chromatographic support by changing the polarity of the mobile phase.

  HIC is similar to RPC but has a larger nominal pore size. In HIC, the elution solvent uses an aqueous salt solution instead of the aqueous or organic mobile phase used in RPC. Similarly, the elution order of samples is opposite to that obtained by RPC. The surface of a protein consists of hydrophilic residues and hydrophobic “spots” that are usually present inside the protein folded to stabilize the protein. When hydrophobic mottles are exposed to an aqueous environment, they destroy the normal solvation properties of the protein, which is a thermodynamic disadvantage. In aqueous mobile phases, the higher the inorganic salt concentration (e.g. ammonium sulfate), the higher the surface tension, thereby increasing the strength of the hydrophobic interaction between the hydrophobic group of the HIC resin and the protein and adsorption Is done. However, while the salt concentration gradient is reduced, the surface tension of the aqueous mobile phase is reduced, thus reducing hydrophobic interactions, thereby causing protein desorption from the hydrophobic groups of the column.

MCC is a technique in which proteins are separated based on their affinity for chelating metal ions. A variety of metal ions, including but not limited to Cu ²⁺ , Co ²⁺ , Zn ²⁺ , Mn ²⁺ , Mg ²⁺ , or Ni ^2+, are covalently bound chelating ligands (e.g. , Iminodiacetic acid) to the stationary phase of the chromatography support. Metal ion free binding sites are used to bind to different proteins and peptides. Elution can occur by replacing the protein with a competitive molecule or changing the pH. For example, lowering the pH of the buffer causes a reduction in protein metal ion complex binding affinity and protein desorption. Alternatively, the bound protein can be eluted from the column using a falling pH gradient in the form of a step gradient or as a linear gradient.

  The physiochemical form of the protein or chimeric molecule of the present invention may be obtained by chemical and / or enzymatic modification to molecules expressed in a variety of ways known in the art.

  The present invention contemplates chemical or enzymatic coupling of carbohydrates to the peptide chain of a protein or chimeric molecule after the protein or chimeric molecule has been expressed and purified. Chemical and / or enzymatic coupling techniques may be used to modify, increase or decrease the number or profile of carbohydrate substituents. Depending on the coupling mode used, the sugar (s) can be (a) an amide group of arginine, (b) a free carboxyl group, (c) a sulfhydryl group such as a cysteine group, (d) serine, threonine, hydroxyllysine, Or hydroxyl groups such as hydroxyproline, (e) aromatic residues such as phenylalanine, tyrosine, or tryptophan residues, (f) glutamine amide groups, or (g) histidine, arginine, or lysine groups. You may attach to such an amino group. The addition can be done chemically or enzymatically. For example, sequential addition of sugar units to a protein or chimeric molecule thereof can be performed using a suitable recombinant glycosyltransferase. Glycosyltransferases can also be used to add sugars with covalently attached substituents. For example, sialic acid covalently attached to polyethylene glycol (PEG) can be transferred to the terminal galactosyl group by sialyltransferase to increase molecular size and serum half-life.

  The carbohydrate or side chain of a protein or chimeric molecule can also be chemically or enzymatically modified to incorporate a variety of functional groups including phosphate, sulfate, hydroxyl, carboxylate, O-sulfate and N-acetyl groups. .

  Carbohydrates present on the protein or chimeric molecule thereof may also be removed chemically or enzymatically. Trifluoromethanesulfonic acid or an equivalent compound can be used for chemical deglycosylation. This treatment can result in cleavage of most or all sugars except the linking sugar, but the polypeptide remains intact. Individual sugars or entire chains can likewise be removed from a protein or chimeric molecule thereof by various endoglycosidases and exoglycosidases.

  The glycan component of a protein or chimeric molecule can be treated with sialidase or mild acid treatment to remove any residual sialic acid, to trim the antenna of N-linked oligosaccharides shortly, or O-linked oligosaccharides It may be modified synthetically by treatment with exo or endoglycosidase to shorten it. Similarly, treatment with fucosidase or sulfatase may be used to remove side chains such as fucose and sulfate. Pseudoglycan structures such as polyethylene glycol or dextran may be chemically added to the amino acid backbone, or glycosyltransferase cocktails may be used with a sugar-dUDP precursor to add sugar subunits to glycans synthetically. Good.

  The present invention contemplates a protein or chimeric molecule thereof chemically or enzymatically coupled to a radionuclide. Such proteins or chimeric molecules include IFN-a2B, IFN-a2B-Fc, IFN-b1, IFN-b1-Fc, IFN-g, IFN-g-Fc, IFNAR2, IFNAR2-Fc, IL-10, IL You may select from the list including -10-Fc, IL-10Ra, and IL-10Ra-Fc.

Using an iodination technique, an iodine isotope (eg, ¹²³ I) may be attached to the peptide chain of the protein or chimeric molecule thereof. In particular, isotopes may be attached to (a) the phenol ring of tyrosine, or (b) the imidazole ring of histidine on the peptide chain of the protein or chimeric molecule thereof. Iodination may be performed using chloramine-T, iodine monochloride, triiodide, electrolyte, enzyme, conjugate, metal removal, iodogen, or iodobead methods.

Technetium labeling techniques may be performed using methods known in the art, such as reduction of ^99m TcO ₄ ^- with a reducing agent (e.g. stannous chloride) followed by a bifunctional chelator such as diethylenetriaminepentaacetic acid (DTPA). by performing the ^99m Tc labeling of the protein or chimeric molecule by, may be used to the protein or chimeric molecule of the present invention is attached to ^99m Tc.

  The present invention contemplates a protein or chimeric molecule thereof chemically or enzymatically coupled to a chemotherapeutic agent. A suitable substance (eg, zoledronic acid) may be conjugated to the protein or chimeric molecule thereof using methods known in the art, eg, by N-hydroxysulfosuccinimide enhanced calcium carbodiimide mediated coupling reaction.

  The present invention contemplates a protein or chimeric molecule thereof chemically or enzymatically coupled to a toxin. Suitable toxins, including melittin, snake venom, truncated pseudomonas endotoxin, ricin, gelonin, and diphtheria toxin, using methods known in the art, for example using maleimide or carbodiimide coupling chemistry, It may be conjugated to a protein or chimeric molecule thereof.

  The isolated protein or chimeric molecule thereof described herein may be delivered to the subject by any means that produces contact between the target receptor or ligand in the subject and the isolated protein or chimeric molecule. In certain embodiments, the protein or chimeric molecule thereof is delivered to the subject as a “pharmaceutical composition”.

  In another aspect, the present invention contemplates a pharmaceutical composition comprising one or more of the aforementioned isolated protein or chimeric protein molecules together with a pharmaceutically acceptable carrier or diluent.

  Composition types suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable in manufacturing and storage conditions and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium containing, for example, water, ethanol, polyol (for example, glycerol, polyethylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. For example, appropriate fluidity can be maintained by using a surfactant. Prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be advantageous to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of injectable compositions can be brought about by using absorption delaying agents, such as aluminum monostearate, and gelatin in the composition.

  Suitable injectable solutions are prepared by incorporating the required amount of the active compound in an appropriate solvent, together with the active ingredient and any other active ingredients as required, followed by filter sterilization or other suitable sterilization means. Is done. In the case of sterile powders for the preparation of sterile injectable solutions, suitable preparation methods include vacuum drying and freeze-drying techniques that yield a powder of the active ingredient plus any additional desired ingredients.

  If the active ingredient is suitably protected, it may be administered orally, for example with an inert diluent or an assimilable edible carrier, enclosed in hard or soft gelatin capsules, compressed into tablets Often, it may be incorporated directly by a daily diet or administered through breast milk. For oral therapeutic administration, the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active ingredient. The percentage of compositions and preparations can of course be conveniently from about 5 to about 80% of the unit weight. The amount of active ingredient in such therapeutically useful compositions is such that a suitable dosage will be obtained. In certain embodiments, compositions or preparations according to the present invention are prepared such that an oral dosage unit form contains from about 0.1 μg to 200 mg of a modulator. Another dosage includes about 1 μg to about 1000 mg and about 10 μg to about 500 mg. These doses may be per individual or per kg body weight. Administration may be hourly, daily, weekly, monthly or yearly.

  Tablets, troches, pills, capsules, etc. may also contain the ingredients described hereinafter. Binders such as gum, acacia, corn starch, or gelatin; excipients such as calcium phosphate; disintegrants such as corn starch, potato starch, alginic acid, etc .; lubricants such as magnesium stearate; and sucrose, lactose, or Sweeteners such as saccharin may be added, or flavorings such as peppermint, winter green oil, or cherry flavorings may be added. When the unit dosage form is a capsule, it may contain a liquid carrier in addition to the types of materials described above. Various other materials may be present as coatings or otherwise to modify the physical shape of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound and may contain sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used to prepare any unit dosage form must be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations.

  The present invention also contemplates topical formulations. In topical formulations, the active substance is suspended in a cream, lotion, wax or other liquid solution so that the active ingredient is introduced onto the body surface of the subject by topical application of a cream, lotion, wax or liquid solution. May be. The term “biological surface” as used herein contemplates any surface on or within an organism. In contrast, “biological surfaces” to which the topical compositions of the present invention may be applied include any epithelial surface such as skin, respiratory tract, gastrointestinal tract and urinary tract.

  In addition to conventional cream, emulsion, patch, or spray formulations, the substances of the present invention may also be delivered topically and / or transdermally using iontophoresis or perforation based methodologies.

  “Iontophoresis” is based on the ability of current to move charged particles. Adjacent electrode pairs placed on the skin cause an electrical potential between the skin and the underlying capillary. At the anode, positively charged drug molecules are driven from the skin surface to the capillaries. Conversely, negatively charged drug molecules appear to be transferred from the skin to the cathode. Because electrophoretic delivery can be switched on and off at all, and the current can be modified, iontophoretic delivery allows for rapid onset and termination, and drug delivery is highly controllable and programmable.

  Perforation technology uses various types of radio frequency pulses (such as radio frequency radiation, laser, heat, or sound) to temporarily destroy the stratum corneum, the layer of skin that stops the passage of many drug molecules into the bloodstream. Energy). It is important to note that, unlike iontophoresis, the energy used in the perforation technique is not used to transport drugs across the skin, but promotes its movement. The perforations provide a “window” through which drug substance can pass more easily and more quickly than usual.

  Pharmaceutically acceptable carriers and / or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and substances for pharmaceutically active substances is well known in the art, and unless any normal medium or substance is incompatible with the modulator, its medium in the pharmaceutical composition. Use is contemplated. Supplementary active compounds can also be incorporated into the compositions.

In one embodiment, the pharmaceutical composition of the present invention comprises A-β-lipoproteinemia, AV, A-β-2-microglobulin amyloidosis, AT, A1AD, A1AT, Argenaes, Aarskog syndrome, Aarskog-Scott syndrome , Arce-Smith Syndrome, Ars Syndrome, AAT, Abdelhalden-Kaufmann-Lignac Syndrome, Abdominal Muscle Defect Syndrome, Abdominal Wall Defect, Abdominal Epilepsy, Abdominal Migraine, Abductor Spastic Disorders, Abercrombie Syndrome, Eyelid-Large Syndrome, ABS , HPRT deficiency, Synzel-type corpus callosum defect, limb scalp cranial defect, menarche deficiency, HGPRT deficiency, absorptive oxalate, Abt-Lettler-Siewe disease, ACADL, ACADM deficiency, ACADM, ACADS, spiny erythrocytosis- Neuropathy, Spinous erythrocytosis, Bullous spinal lysis, Black epidermopathy, Bullous acanthosis, Black epidermosis with type A insulin resistance, Type B insulin Anti-melanocytic dermatosis, epidermal hypertrophic nevus, acatalaseemia, acatalaseemia, ACC, accessory atrioventricular route, accessory atrioventricular route, anencephalopathy, ACF with heart defect, achalasia, ashar-thiere Syndrome, ACHARD (Marfan mutant), Aschar disease, abile uric jaundice, cartilage free, type IV cartilage free, type III cartilage free, achondroplasia, late cartilage dysplasia, cartilage dysfunction Dwarfism, Achu Syndrome, Color Blindness, Achromatope, All Color Blind (Achromatopic), All Color Blindness (Achromatopsia), Achromatic Nevi, Acid Ceramidase Deficiency, Acid Maltase Deficiency, Acid β-Glucosidase Deficiency, Methylmalon Acidemia , Propionic acidemia, acidemia with interstitial ataxia and weakness, acidosis, tarsal epiphyseal tissue coupling, ACM, acoustic schwannoma, acoustic neuroma, ACPS with dysplasia, ACPS II, ACPS IV, ACPS I II, Acquired aphasia with convulsions, Acquired Brown syndrome, Acquired epilepsy aphasia, Acquired factor XIII deficiency, Acquired type ACC (caused by infection in utero), Acquired hyperoxaluria Disease, acquired hypogammaglobulinemia, acquired immune deficiency syndrome (AIDS), acquired iron overload, acquired lipodystrophy, acquired partial lipodystrophy, acquired migratory spleen, ACR, facial and genital abnormalities Accompanied apical dysplasia, pneumoconiosis, scintzel-type apical corpus callosum syndrome, cuspid digitosis, type I cuspid digitosis, type I subtype I cuspid digitosis, type II cuspid polydactyly , Type III polydactyly, type IV polydactyly, cusp polydactyly V (ACS5 or ACS V) subtype I, cephalic asymmetric and mild syndactyly, cusp, tip Cartilage hyperplasia, enteric extremity dermatitis, apical dysplasia, apical dysplasia, acrodulotrophy neuropathy, nager limb face Dysplasia, posterior limb facial bone dysplasia, genie-wiedep limb facial bone dysplasia, familial acromegaly, acromegaly, hereditary acromegaly, intermediate limb shortening dysplasia, Distal mesotrophic dwarfism, small tip skeletal dysplasia, osteoporosis and tip osteolysis with changes in the cranial mandible, tip osteolysis, tip abnormal sensation, ACS I, Type II ACS, Type III ACS, ACS3 , ACTH deficiency, myoclonus action, acute brachial neuritis syndrome, acute brachial radiculitis syndrome, acute cerebral Gaucher disease, acute cholangitis, acute disseminated cerebrospinal radiculopathy, acute disseminated histiocytosis-X, acute bleeding Gray leukoencephalitis, acute idiopathic polyneuritis, acute immune polyneuritis, acute infantile Pelizaeus-Merzbacher sclerosis, acute intermittent porphyria, acute porphyria, acute sarcoidosis, acute shoulder neuritis, acute toxic epidermis Exfoliation, long chain acyl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase deficiency, acyl-CoA dihydroxyacetone acyltransferase, acyl coenzyme A oxidase deficiency, ADA, ADA deficiency, Adam complex, enamel Behcet syndrome, adamantinome , Adams-Oliver syndrome, adaptive colitis, combined ADD, ADD, Addison's disease with brain sclerosis, Addison anemia, Addison's disease, Addison-Bielmer anemia, Addison-Schilder's disease, Addison's malignant anemia, thumb adduction- Mental retardation, adductor spastic dysphonia, adductor spastic dysphonia, adenoma-related masculinization in older women, colorectal nematosis, colonic nematode polyposis, familial nematode polyposis, adenosine deaminase deficiency, Adenyl succinase deficiency, mainly hyperactivity-impulsive ADHD, mainly careless ADHD, ADHD, adhesive arachnoiditis, Dee syndrome, Addie syndrome, Addie tension pupil, Addie pupil, steatosis retinitis polydactyly, reproductive pigment retinitis syndrome, painful fat, painful steatosis, genital dystrophy, Adolescent cystinosis, ADPKD, adrenal cortex, adrenal disease, adrenal hyperfunction caused by pituitary ACTH excess, adrenal hypoplasia, adrenal insufficiency, adrenal neoplasia, adrenal masculinization, adrenal-pigment retinitis- Multifinger syndrome, adrenocortical insufficiency, adrenocortical hypofunction, isolated adrenocorticotropic hormone deficiency, adrenal genital syndrome, adrenal cerebral leukodystrophy, adrenal spinal neuropathy, adrenal-pigment retinitis-multifinger syndrome, adult cystine accumulation , Adult dermatomyositis, adult hypophosphatemia, adult macular retinal degeneration, adult-onset ALD, adult-onset ceroidosis, adult-onset renal medullary cystosis, adult-onset malignant poor Adult-onset Schindler disease, adult-onset subacute necrotizing encephalomyelopathy, adult-type polycystic kidney disease, adult-onset renal medullary cystosis, Adynlosuccinate lyase deficiency, AE, AEC syndrome, AFD, anfibrinogenemia, African siderosis, AGA, ganglion cell-deficient giant colon, age-related macular degeneration, no cerebral complications, no corpus callosum, no corpus callosum-infantile spasticity-eye abnormalities, Callosal-free choroidal retinal abnormalities, corpus callosal-free choroidal retinal abnormalities, aggressive mastocytosis, primary agnosia, AGR triad, AGU, brainless gyrus, brainless gyrus-thickening of the brain gyrus-band spectrum, AHC , AHD, AHDS, AHF deficiency, AHG deficiency, AHO, Ahumada Del Castillo, Ecardi syndrome, AIED, AIMP, AIP, AIS, ataxia, ALA-D porphyria, lactose degrading enzyme deficiency , Aladil syndrome, Aland Island disease (X-linked), Alanine Disease, Albers-Schonberg disease, albinism, albinoidism, allbright hereditary bone dysplasia, alkaptonuria, alcohol-related birth defects, alcoholic fetal disorder, alcoholic cirrhosis, Ald, ALD , ALD, aldosterone, aldosteronism with normal blood pressure, Aldrich syndrome, Alexander disease, Alexander disease, pain dystrophy, pain neurodystrophy, alkaptonuria, alkaptonuria tissue browning, alkyl DHAP synthase deficiency, Alan-Hellundon- Dudley Syndrome, Alan-Herundon Syndrome, Alan-Herundon-Dudley Mental Retardation, Allergic Granulomatous Antitis (Clonkite-Canadian Allergic Granulomatous Vasculitis, Anobar Total Proencephalopathy, Alopecia areata, Celsus Alopecia, Scarring hair loss , Localized alopecia, alopecia-white hair-uveitis-vitiligo-hearing loss-skin-uvea-O, semi-systemic alopecia, complete alopecia, systemic alopecia, Alpers disease, cirrhosis with Alpers diffuse brain gray Denatured, alpaz progressive infant polydystrophy, α-1-antitrypsin deficiency, α-1,4-glucosidase deficiency, α-galactosidase A deficiency, α-galactosidase B deficiency, α high-density lipoprotein deficiency, α-L- Fucosidase-deficient type 3 fucosidosis, Schindler-type α-GalNAc deficiency, α-lipoproteinemia, α-Mannosidosis, Schindler-type α-N-acetylgalactosaminidase deficiency, Schindler-type α-NAGA deficiency, α-neuraminidase deficiency Atypical alpha-thalassemia / mental retardation syndrome, alpha lipoproteinemia, Alport syndrome, ALS, Alstrem syndrome, Alstreem, Alstrem syndrome Hemiplegic syndrome, childhood alternation hemiplegia, Alzheimer's disease, familial cataract idiopathic, adult familial cataract idiopathic, infant familial cataract idiopathic, vulvar dysplasia, AMC, AMD, enamel epithelioma, enamel Dysplasia, non-partum amenorrhea-milk leak, amenorrhea-milk leak-FSH syndrome, amenorrhea, amino acid disorder, amino aciduria-osteomalacia-hyperphosphatemic syndrome, AMN, amniocentesis, amnion Band, amniotic band syndrome, amniotic band disruption complex, amniotic band sequelae, amnion rupture sequelae, congenital amputation, AMS, Dranger Amsterdam dwarf syndrome, amylo-1 6-glucosidase deficiency, amyloid arthropathy in chronic hemodialysis , Amyloid corneal dystrophy, amyloid polyneuropathy, amyloidosis, familial Mediterranean fever amyloidosis, amylopectinosis, congenital myogenesis dysfunction, amyotrophic lateral sclerosis, amyotrophic lateral stiffness Dermatosis, amyotrophic lateral sclerosis-polyglucosan body, AN, AN 1, AN 2, anal closure, anal membrane, anorectal congenital anomaly, anal stenosis, analine 60 amyloidosis, a-alpha lipoproteinemia, anorectal, Anorectal, Anaplastic astrocytoma, Andersen disease, Anderson-Fabry disease, Andersen glycogenosis, Anderson-Warburg syndrome, Andre syndrome, Type II Andre syndrome, Male hormone refractory, Partial male hormone refractory syndrome, Partial Androgen refractory syndrome, androgen steroids, autoimmune hemolytic anemia, blackfan-diamond anemia, congenital anemia, thumb and finger joint syndrome, hemolytic cold antibody anemia, hemolytic anemia with PGK deficiency, malignant Anemia, anencephalopathy, Angelman syndrome, vascular-bone hypertrophy syndrome, vascular follicular lymph node proliferation, vascular hemophilia, angulated hemangioma, diffuse Keratinized hemangioma, diffuse keratoangioma, retinal hemangiomatosis, hemangioma-like lymphoid, hereditary angioneuric edema, non-sweat ectodermal dysplasia, non-sweat X-linked ectodermal dysplasia , Ataxia, alumina-vulva dysplasia- mental retardation, anorexia associated with mental retardation, ataxia-cerebellar ataxia- mental retardation, partial aroma-cerebellar ataxia- mental retardation, partial Ataxia-Cerebellar ataxia-mental retardation, type I ataxia, type II ataxia, alumina-Wilms tumor-related, achroma-Wilms tumor-gonadal adenoma, eyelid adhesion-ectodermal defect-lips / Cleft palate, ankylosing spondylitis, annular groove, edentulous, anodontia vera, abnormal trichromatic color, dysplasia of teeth, coronary dentin dysplasia, noun aphasia, anophthalmia, anorectal Anorectal congenital anomalies, anolfactory sensation, anterior curvature of the leg with dwarfism, anterior corneal dystrophy, anticonvulsant syndrome, anti-eps Invar virus nuclear antigen (EBNA) antibody deficiency, antibody deficiency, antibody deficiency with almost normal immunoglobulin, antihemophilic factor deficiency, antihemophilic globulin deficiency, antiphospholipid syndrome, antiphospholipid antibody syndrome, Antithrombin III deficiency, classic (type I) antithrombin III deficiency, antitrypsin deficiency, Antrei-Bixler syndrome, Antoni paralysis, tibial anxiety, aortic arch syndrome, aortic mitral valve closure with left ventricular hypoplasia syndrome, aorta Stenosis, Aparoschisis, APC, APECED syndrome, Apele syndrome, Apele, aphasia, congenital extracranial dysplasia, congenital skin dysplasia, congenital skin dysplasia with limb transverse leg defect, aplastic anemia Aplastic anemia with congenital anomalies, APLS, apnea, appalachian amyloidosis, apple peel syndrome, apraxia, cheek facial apraxia Apraxia, ideal apraxia, ideal ataxia, ideal ataxia, ataxia, ocular ataxia, APS, arachnoiditis, contracture virus spider fingers, spider fingers, arachnoid cyst, ossification Arachnoiditis, arachnoiditis, alan-duchene, alan-duchene muscle atrophy, aplastic anemia, arginase deficiency, arginineemia, arginosuccinase deficiency, arginosuccinase deficiency, arginosuccinate lyase deficiency, Arginosuccinate lyase-ASL, Arginosuccinate synthase deficiency, Arginosuccinic aciduria, Argonz-Delcastillo syndrome, Olfactory encephalopathy, Armenia syndrome, Arnort-Chiari malformation, Arnort-Chiari syndrome, ARPKD, Arrhythmia myoclonus, Arrhythmogenic right ventricular formation Abnormality, arterial liver dysplasia, arteriovenous malformation, brain arteriovenous malformation, giant cell arteritis, arthritis, arthritic urethritis Joint-tooth-bone dysplasia, joint-eye disorders, congenital multiple joint relaxation, congenital multiple joint contracture, distal type IIA congenital multiple joint contracture, ARVD, arylsulfatase-B deficiency, AS, ASA Deficiency, ascending paralysis, ASD, atrial septal defect, ASH, Ascherman syndrome, Ashkenazi amyloidosis, ASL deficiency, aspartylglucosamineuria, aspartylglucosamineuria, Asperger syndrome, Asperger's autism, asphyxia Dysplasia, asplenia syndrome, ASS deficiency, asthma, stage I astrocytoma (benign), stage II astrocytoma (benign), asymmetric crying with heart defect, asymmetric septal hypertrophy, asymptomatic corpus callosum Development, AT, AT III deficiency, ATIII mutant IA, AT III mutant Ib, AT 3, ataxia, telangiectasia ataxia, ataxia with type II lactic acidosis, ataxic cerebral palsy, ataxia , Ataxic aphasia
, ATD, athetoid cerebral palsy, atopic eczema, esophageal closure with or without tracheoesophageal fistula, atrial septal defect, first atrial septal defect, atrial septal small ventricular septal defect, atrial flutter, atrial fibrillation Movement, auricular dysplasia, atrial septal defect, atrioventricular block, atrioventricular duct defect, atrioventricular septal defect, hereditary medullary atrophy, atrophic leg air, olive cerebellar atrophy, attention deficit disorder, attention deficit hyperactivity disorder, Attenuating colonic necromatous polyposis, atypical amyloidosis, atypical hyperphenylalaninemia, ear canal closure, auricular temporal syndrome, autism, Asperger-type autism, autism dementia ataxia and deliberate hand Cannot be used, infant autism autism, autoimmune Addison disease, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune multiple endocrine adenopathy-candidiasis, autoimmune type I Adenopathy, autosome Albinism, autosomal dominant Helioophthalmic Outburst syndrome, late-onset autosomal dominant desmin distal myopathy, autosomal dominant EDS, autosomal dominant Emery-Dreis muscular dystrophy Chromosome dominant keratoconus, autosomal dominant Perizeus-Merzbacher brain sclerosis, autosomal dominant polycystic kidney disease, autosomal dominant spinocerebellar degeneration, autosomal recessive agammaglobulinemia, autosomal recessive central myopathy, autosomal recessive conrady Hünnerman syndrome, autosomal recessive EDS, autosomal recessive emery-Dreifys muscular dystrophy, autosomal recessive ophthalmopathy, autosomal recessive hereditary corpus callosum, autosomal recessive keratoconus, autosomal recessive polycystic kidney, autosomal recessive Severe combined immune deficiency, AV, AVM, AVSD, AWTA, axillary abscess, giant axis Neuropathy, Azorea Neuropathy, BK Mol Syndrome, Babinsky-Frelich Syndrome, BADS, Bayerje Syndrome, Balkan Disease, Barrer-Jerold Syndrome, Balloon Mitral Valve, Barlow Disease Concentric Sclerosis, Baltic Myoclonus Epilepsy, Bannayan-Zonana Syndrome (BZS), Bannayan-Riley-Le Barcava Syndrome, Banti Syndrome, Valdey-Beedle Syndrome, Lymphocyte Syndrome, Barlow Syndrome, Barackel-Simmons Disease, Barrett's Esophagus, Barrett Ulcer, Barth Syndrome, Barter Syndrome, Basal Cell Nevus Syndrome, Basedow's disease, Bassen-Kornzweig syndrome, Batten's disease, Batten-Meow syndrome, Batten-Spielmeier-Forked syndrome, Batten-Turner syndrome, Batten-Turner congenital myopathy, Batten-Forked syndrome, BBB syndrome, BBB syndrome (Opitz), BBB syndrome, BBBG syndrome, BCKD deficiency, BD, BDLS, BE, virus syndrome, virus-heuch syndrome, bean syndrome, BEB, bechteref syndrome, becker disease, becker muscular dystrophy, becker nevus, Beckwith-Wiedemann Syndrome, Beckwith Syndrome, Begues-Cessar Syndrome, Behcet Syndrome, Behcet's Disease, Veil 1, Veil 2, Bell Palsy, Benign Melanoderma, Benign Astrocytoma, Benign Cranial Tumor, Benign Cystine Accumulation , Benign essential blepharospasm, benign essential tremor, benign familial hematuria, benign focal muscle atrophy, ALS benign focal muscle atrophy, benign hydrocephalus, benign hyperkinetic syndrome, benign keratosis, benign paroxysmal peritonitis, Benign recurrent hematuria, benign recurrent intrahepatic cholestasis, benign spinal muscle atrophy with carved hypertrophy, benign symmetric lipomatosis, central nervous system Benign tumor, belardineri-seep syndrome, berger disease, beriberi, berman syndrome, bernard-hornel syndrome, bernard-surrier syndrome, besniew urticaria, vest disease, beta-alanine pyruvate aminotransferase, beta-galactosidase deficiency, morchio syndrome, beta -Glucuronidase deficiency, beta oxidation deficiency, beta thalassemia major, beta thalassemia minor, beta lipoprotein deficiency, Bethlem myopathy, Buren syndrome, BH4 deficiency, Biber-Haab-Dimmer corneal dystrophy, bivalve Plastic aortic valve, beadle-valde, bifid skull, bifunctional enzyme deficiency, bilateral acoustic neurofibromatosis, bilateral acoustic neuroma, bilateral right sequelae, bilateral renal aplasia, bilateral temporal lobe disorder, biliary Seizures, type I bilirubin glucuronosyltransferase deficiency, Binda (Binder) syndrome, Binswanger disease, Binswanger encephalopathy, biotinidase deficiency, Sexkel-type avian cranial dwarfism, birth defect, nevus, bilateral cranial forceps mark syndrome, biventricular fibrosis, Bjornstad syndrome , BK mol syndrome, sensory nerve type Black Rocks-Shiroko-Hearing Impairment (BADS), Black Fan-Diamond Anemia, Pyorrhea idiopathic arthritis, arthritis, eyelid contraction, drooping, reverse internal angle keratoderma syndrome, blepharospasm, benign Essential blepharospasm, blepharospasm of oral mandibular ataxia, Bressy cyst, BLFS, blindness, Bloch-Siemens pigmented melanocytosis, Cutis linearis, Bloch-Siemens-Salzburger syndrome, Bloch-Salzburger syndrome, blood Type A blood, type B blood, type AB blood, type O blood, Bloom syndrome, Bloom-Tre-Mackacek syndrome, blue gum -Like nevus, blue child, blue diaper syndrome, BMD, BOD, BOFS, bone tumor-epithelial cyst-polyps, Wyvern-Mason syndrome, Bonneby-Ullrich syndrome, Buch syndrome, BOR syndrome, BORJ, Beleson syndrome, Beleson -Forsman-Lehman Syndrome, Bowen Syndrome, Bowen-Conrady Syndrome, Bowen-Conrady Hütterite, Bowen-Conrady-type Hütterite Syndrome, Bowman, BPEI, BPES, brachial neuritis, brachial neuritis syndrome, brachial plexus Neuritis, brachial plexus neuropathy, brachial ischemia, Brachmann-Dranger syndrome, short head, congenital short form, bradycardia, brain damage due to perinatal asphyxia, brain tumor, benign brain tumor, malignant brain tumor, minute Branched α-keto acid dehydrogenase deficiency, type I branched chain ketonuria, branching deficiency, dwarf ophthalmology syndrome, erosive otorenal dysplasia, fertile ear Syndrome, erosive ophthalmology syndrome, erosive ear syndrome, blunt syndrome, brandy wine type dentinal dysplasia, brandy wine type dentinal dysplasia, breast cancer, BRIC syndrome, fragile bone disease, type III familial hyperlipoproteinemia , Broad thumb syndrome, wide thumb and big toe characteristic facial mental retardation, broad thumb-toe thumb, broca aphasia, block-During disease, bronze diabetic, bronze sylder disease, brown albinism, heredity Brown enamel, Brown-Sequard syndrome, Brown syndrome, BRRS, Brueghel syndrome, Breton total agammaglobulinemia, BS, BSS, Buchanan syndrome, Budd syndrome, Bad-Chiari syndrome, Burger-Glutz (Gruetz) syndrome, X-linked medullary spinal muscular atrophy, Bulldog syndrome, hereditary blister, blister-like CIE, congenital bullous ichthyosis erythroderma, bullous ichthyosis, bullous pemphigoid, -Kit lymphoma, African Burkitt lymphoma, non-African Burkitt lymphoma, BWS, Bayer's disease, C syndrome, C1 esterase inhibitor dysfunction type II angioedema, C1-INH, C1 esterase inhibitor deficient type I angioedema, C1NH, Kutch-Rich disease, CAD, CADASIL, CAH, rib valgus, hallux valgus, calcium pyrophosphate dihydrate deposition, no occurrence of corpus callosum and ocular abnormalities, spine muscle atrophy carved hypertrophy, flexion dysplasia , Dwarfism dwarfism, flexion syndrome, flexor-palate cleft clubfoot, flexor-restricted jaw range of movement, dwarfism, dwarf syndrome, long-finger flexor syndrome, Kamrach-Engelmann disease, Canada- Cannavan white matter dystrophy, cancer, Lynch cancer familial syndrome, Cantrel syndrome, Cantrel-Haller-Ravich syndrome, five cantrel syndrome, including Cronkite disease, Canavan disease, Canavan disease Carbamyl phosphate synthetase deficiency, carbohydrate deficient glycoprotein syndrome, type Ia carbohydrate deficient glycoprotein syndrome, carbohydrate-induced hyperlipidemia, glucose galactose carbohydrate intolerance, carbon dioxide acidosis, multiple carboxylase deficiency, heart-limb syndrome , Cardiac hearing syndrome, Gerver and Lange-Nielsen's cardiac hearing syndrome, cardiac skin syndrome, cardiac-facial-skin syndrome, Kayler type cardiac facial syndrome, diffuse glycogenic cardiac hypertrophy, cardiomyogenic melanosis, cardiac myopathy, Cardiac myopathy associated with desmin storage myopathy, cardiac myopathy due to desmin deficiency, cardiac myopathy-neutropenia syndrome, cardiac myopathy-neutropenia syndrome, fatal infant heart myopathy, cardiopathic amyloidosis, cardia spasm, cardocardiac (Cardocardiac) syndrome, creatinine-acylcarnitine translo Case deficiency, carnitine deficiency and disorders, primary carnitine deficiency, secondary carnitine deficiency, carnitine deficiency secondary to MCAD, carnitine deficiency syndrome, carnitine palmitoyltransferase I & II (CPT I & II), carnitine palmitoyl transferase deficiency, 1 Type carnitine palmitoyltransferase deficiency, type 2 carnitine palmitoyltransferase deficiency, including benign classical muscle type, including severe infant type, carnitine transport deficiency (primary carnitine deficiency), carnosinase deficiency, carnosinemia, calorie disease, carpenter syndrome, Carpenters, cartilage-hair dysplasia, Castleman's disease, hyaline vascular Castleman's disease, plasma cell Castleman's disease, Castleman's tumor, cat eye syndrome, cat cry syndrome, catalase deficiency, white Cataract-tooth syndrome, cataract X-linked to Hutchinson's teeth, catecholamine hormone, Catel-Manzke syndrome, Katel-Manzke-type palatal syndrome, tail dysplasia, tail dysplasia, tail regression syndrome, Major causalgia syndrome, sea surface tumor, cavernous hemangioma, spongiform hemangioma, spongiform lymphangioma, spongiform malformation, Kayler syndrome, Cazenave vitiligo, CBGD, CBPS, CCA, CCD, CCHS, CCM syndrome, CCMS, CCO, CD, CDG1a, CDGlA, type Ia CDGS, CDGS, CDI, CdLS, celiac disease, celiac sprue, celiac sprue-dermatitis, cellular immune deficiency with purine nucleoside reoside phosphorylase deficiency, Celsus vitiligo, central Apnea, central core disease, central diabetes insipidus, central neurofibromatosis, central hypoventilation, central sleep apnea, efferent lipodystrophy, central myopathy, CEP, Aneurysm, craniothoracic lipodystrophy, ceramide trihexosidase deficiency, cerebellar agenesis, cerebellar dysplasia, cerebellar unilateral development, cerebellar dysplasia, cerebellar dysplasia, cerebellar dysgenesis-hypernea Eye movement-ataxia-delay, cerebellar syndrome, cerebellar parenchymal disorder IV, cerebellar medullary malformation syndrome, cerebellum-ocular cutaneous telangiectasia IV, familial cerebellar parenchymal disorder IV, cerebellar pontine tumor, cerebral arachnoiditis, subcortical Autosomal dominant cerebral arteropathy with infarction and leukodystrophies, cerebral beriberi, cerebral palsy, cerebral giantosis, cerebral ischemia, cerebrovascular malformation, cerebral palsy, brain-ocular kidney dystrophy, brain-eye-face-skeletal syndrome, Brain-radius-mandibular syndrome, cerebral liver and kidney syndrome, macular degeneration, Fukuyama-type cerebral muscular dystrophy, cerebral ocular dysplasia, muscular dystrophy syndrome, cerebral ophthalmofacial skeletal syndrome, cerebral retinal arteriovenous aneurysm, cerebrosidoripi Dorsis, cerebral tendon xanthosis, cerebrovascular iron calcium deposition, adult ceroid lipofuscinosis, cervical dystonia, cervical dystonia, cervical ocular auditory syndrome, cervical stenosis, cervical fusion, CES, CF, CFC syndrome, CFIDS, CFND, CGD, CGF, systemic flaccid skin, Chanarin-Doffman disease, Chanarin-Doffman syndrome, Chanarin-Doffman ichthyosis syndrome, Chandler syndrome, Charcot disease, Charcot-Marie-Tooth, Charcot-Marie-Tooth disease, variant Charcot- Marie-Tooth disease, Charcot-Marie-Tooth-Lucy-Levy disease, CHARGE Union, Charge syndrome, CHARGE syndrome, Chaund ectodermal dysplasia, Chediak-East syndrome, Chediak-Steinbrink-East syndrome, granulation Cholecystitis, cleft lip, Chemke syndrome, Cheney syndrome, cherry red spot myo Clonus syndrome, CHF, CHH, Chiari disease, Chiari malformation I, Chiari malformation, type I Chiari (chiari malformation I), type II Chiari (chiari malformation II), Chiari I syndrome, Chiari-Bud syndrome, Chiari-Fronmel syndrome, Chiari Malformation II, CHILD syndrome, CHILD ichthyosis syndrome, CHILD syndrome ichthyosis, childhood adrenal cerebral leukodystrophy, childhood dermatomyositis, childhood dystonia, childhood periodic vomiting, childhood giant axonal neuropathy, childhood hypophosphatemia, childhood muscular dystrophy , CHN, cholestasis, Norwegian hereditary cholestasis, intrahepatic cholestasis, neonatal cholestasis, cholestasis of oral contraceptive users, cholestasis with peripheral pulmonary stenosis, cholestasis during pregnancy , Cholesterol desmolase deficiency, punctate cartilage dysplasia, congenital chondrogenic dyscalcia, fetal cartilage dysplasia Usually chondrogenic dysplasia
Otony, chondrogenesis, chondrogenesis with clubfoot, epiphyseal chondrogenesis, hyperplastic chondrogenesis, cartilage ectodermal dysplasia, chondrogenesis dysfunction, chondrodystrophia, osteochondral dystrophy, Chorea-spiny erythrocytosis, chorionic villus collection, chorioretinal abnormalities, choroidal retinal abnormalities with ACC, choroidal retinal colobom-Joubert syndrome, Christo-Siemens-Touren syndrome, Christmas disease, Christmas tree syndrome, chromosome 3 distant 3p deletion, chromosome 3 distal 3p monosomy, chromosome 3 distal 3q2 duplication, chromosome 3 distal 3q2 trisomy, chromosome 3 monosomy 3p2, chromosome 3q partial duplication syndrome, chromosome 3q, partial trisomy syndrome, Chromosome 3-Trisomy 3q2, Chromosome 4 deletion 4q31-qter syndrome, Chromosome 4 deletion 4q32-qter syndrome, Chromosome 4 deletion 4q33-qter syndrome, Chromosome 4 long arm deletion, Chromosome 4 long arm deletion, chromosome 4 monosomy 4q, chromosome 4-monosomy 4q, chromosome 4 monosomy distal 4q, chromosome 4 partial deletion 4p, chromosome 4, partial short arm deletion 4 partial chromosome 4 distal monosomy, chromosome 4 partial monosomy 4p, chromosome 4 partial trisomy 4 (q25-qter), chromosome 4 partial trisomy (q26 or q27-qter), chromosome 4 partial Trisomy 4 (q31 or 32-qter), chromosome 4 partial trisomy 4p, chromosome 4 partial trisomy 4q2 and 4q3, chromosome 4 partial trisomy distal 4, chromosome 4 circular, chromosome 4q end deletion Syndrome, chromosome 4q-syndrome, chromosome 4q-syndrome, chromosome 4 trisomy 4, chromosome 4 trisomy 4p, chromosome 4 XY / 47 XXY (mosaic), chromosome 5 monosomy 5p, chromosome 5, partial short arm Syndrome, chromosome 5 trisomy 5p, chromosome 5 trisomy 5p complete (5p11-pter), Chromosome 5 trisomy 5p partial (5p13 or 14-pter), chromosome 5p-syndrome, chromosome 6 partial trisomy 6q, chromosome 6 circular, chromosome 6 trisomy 6q2, chromosome 7p2, partial chromosome 7 short arm partial Deletion (7p2-), Chromosome 7p deletion [del (7) (p21-p22)], Chromosome 8 monosomy 8p2, Chromosome 8 monosomy 8p21-pter, Chromosome 8 partial deletion (short arm ), Chromosome 8 partial monosomy 8p2, Chromosome 9 full trisomy 9P, Chromosome 9 short arm partial deletion, Chromosome 9 partial monosomy 9p, Chromosome 9 partial monosomy 9p22, Chromosome 9 partial monosomy 9p22-pter, including chromosome 9 partial trisomy 9P, chromosome 9 circular, chromosome 9 tetrasomy 9p, chromosome 9 tetrasomy 9P mosaic phenomenon, chromosome 9 trisomy 9p (multiple variants), chromosome 9 trisomy 9 Chromosome 9 trisomy mosaic, including chromosome 9 (pter-p21 or q32) Risomy mosaic, chromosome 10 distal trisomy 10q, chromosome 10 monosomy, chromosome 10 monosomy 10p, chromosome 10, partial deletion (short arm), chromosome 10p-partial, chromosome 10 partial trisomy 10q24-qter, chromosome 10 trisomy 10q2, chromosome 11 long arm partial monosomy, chromosome 11 partial monosomy 11q, chromosome 11 partial trisomy, chromosome 11 partial trisomy 11q13-qter, chromosome 11 partial Trisomy 11q21-qter, chromosome 11 partial trisomy 11q23-qter, chromosome 11q, partial trisomy, chromosome 12 equimolar chromosome 12p mosaic, chromosome 13 partial monosomy 13q, chromosome 13, partial monosomy of long arm, Chromosome 14 circular, chromosome 14 trisomy, chromosome 15 distal trisomy 15q, chromosome r15, chromosome 15 circular, chromosome 15 trisomy 15q2, chromosome 15q, partial duplication syndrome, interchromosome 17 18p long-arm deletion syndrome, chromosome 18 monosomy 18p, chromosome 18 monosomy 18Q, chromosome 18 circular, chromosome 18 tetrasomy 18p, chromosome 18q-syndrome, chromosome 21 mosaic 21 syndrome, Chromosome 21 circular, chromosome 21 translocation 21 syndrome, chromosome 22 reverse duplication (22pter-22q11), chromosome 22 partial trisomy, chromosome 22 circular, chromosome 22 trisomy mosaic, chromosome 48 XXYY, chromosome 48 XXXY, chromosome r15, chromosome triplet, chromosome triplet, chromosome triploid syndrome, X chromosome, XXY chromosome, chronic abile urinary jaundice, chronic adhesive arachnoiditis, chronic adrenocortical insufficiency, chronic cavernositis, chronic Congenital aplastic anemia, chronic phagocytic dysfunction, chronic familial granulomatosis, chronic familial jaundice, chronic fatigue immune dysfunction syndrome (CFIDS), chronic granulomatosis, chronic Giant-Barre syndrome, chronic idiopathic Jaundice, chronic idiopathic polygod Transinflammatory (CIP), chronic inflammatory demyelinating polyneuropathy, chronic inflammatory demyelinating polyradiculoneuropathy, chronic motor tics, chronic mucocutaneous candidiasis, chronic multiple tics, chronic non-specific ulcerative colon Inflammation, chronic obstructive cholangitis, chronic peptic ulcer esophagitis syndrome, chronic progressive chorea, chronic progressive extraocular palsy syndrome, chronic progressive extraocular palsy and myopathy, chronic progressive with red muscle fiber excitement Extraocular muscle palsy, chronic relapsing polyneuropathy, chronic sarcoidosis, chronic spastic dysphonia, childhood chronic vomiting, CHS, Churg-Strauss syndrome, scar pemphigoid, CIP, congenital pigmented cirrhosis, cirrhosis, cystinuria Citrullinemia, CJD, classic Schindler disease, classic Pfeiffer syndrome, classic maple syrup urine disease, classic hemophilia, classic type I cocaine syndrome (type A), classic Leigh disease, classic phenylke Urinary dysfunction, classic X-linked Pelizeus-Merzbacher encephalopathy, CLE, cleft lip / palate mucous cyst lower lip PP distal genital abnormality, cleft lip-palatosis eyelid reduction eyelid sequestration, abnormal thumb and microcephaly Cleft lip / palatitis, cleft palate-joint contracture-dandy walker malformation, cleft palate cleft lip, clavicular cranial dysplasia with jaw disease, thumb loss & distal fingerless, clavicular cranial dysplasia, clavicular skull formation Abnormal, Click Noise Syndrome, CLN1, Chronic Spastic Crowston Syndrome, Clubfoot, CMDI, CMM, CMT, CMTC, CMTX, COA Syndrome, Aortic Constriction, Coat's Disease, Cobblestone Dysfunction, Coach Jewish Disorder, Cocaine Syndrome , COD-MD syndrome, COD, Coffin-Lorley syndrome, Coffin syndrome, Coffin-Siriz syndrome, COFS syndrome, Kogan corneal dystrophy, Kogan-Rees syndrome, Cohen syndrome, cold agglutinin disease, cold Somatic disease, cold antibody hemolytic anemia, ulcerative colitis, severe colitis, ulcerative colitis chronic non-specific ulcerative colitis, collodion children, post-cardiac deficit nostril closed growth genital development retardation abnormal ear abnormalities, colobom , Colonic neuropathy, color blindness, color blindness, Colpocephaly, columnar esophagus, complex cone rod degeneration, complex immune deficiency with immunoglobulin, complex intermediate ectoderm dysplasia, unclassifiable hypogammaglobulinemia , Unclassifiable immunodeficiency, single ventricle, hydrocephalus, complete deficiency of hypoxanthine-guanine phosphoribosyltransferase, complete atrioventricular septal defect, complement component 1 inhibitor deficiency, complement component C1 regulatory component deficiency, complete Heart block, complex carbohydrate intolerance, complex local pain syndrome, complex V ATP synthase deficiency, complex I, complex I NADH dehydrogenase deficiency, complex II, complex II succinate dehydrogenase Case deficiency, complex III, complex III ubiquinone-cytochrome c oxidoreductase deficiency, complex IV, complex IV cytochrome c oxidase deficiency, complex IV deficiency, complex V, concussion brain injury, cone-rod Degeneration, progressive cone-rod degeneration, rod dystrophy, cone-rod dystrophy, confluent reticulopapillomatosis, congenital abscesses with low PK motility, congenital athymic parathyroid deficits , Congenital depigmentation, congenital Addison disease, congenital adrenal hyperplasia, congenital adrenal hyperplasia, congenital anfibrinogenemia, congenital alveolar hypoventilation, congenital neonatal anemia, congenital bilateral persylvia ( Persylvian syndrome, congenital brown syndrome, congenital new blood vessel defect, congenital central hypoventilation syndrome, congenital cerebral palsy, congenital cervical osteosynthesis, congenital saddle thumb with mental retardation, congenital contracture Sex spider finger Congenital multiple contractures with thumb, congenital cyanosis, congenital cranial scalp defect, congenital intrahepatic bile duct dilation, congenital demyelinating neuropathy, congenital phagocytic dysfunction, congenital dysplasia vasodilation, Congenital hematopoietic porphyria, congenital factor XIII deficiency, congenital autonomic respiratory depression, type I congenital familial non-hemolytic jaundice, congenital familial delayed diarrhea, type II congenital cocaine syndrome (type B ), Congenital systemic fibromatosis, congenital rubella, congenital giant axonal neuropathy, congenital heart block, congenital heart defect, erythrodermal ichthyosis and limb defect, congenital Hemolytic jaundice, congenital hemolytic anemia, congenital hepatic fibrosis, congenital hereditary corneal dystrophy, congenital hereditary lymphedema, congenital hyperchondrogenesis, congenital myelinogenic polyneuropathy, congenital myelin Hypoplastic neuropathy, congenital myelin Reduced formation, congenital myelin formation (bulb) polyneuropathy, congenital ichthyosis-like erythroderma, congenital keratoconus, congenital lactic acidosis, congenital lactose intolerance, congenital lipodystrophy, congenital cirrhosis, Congenital lobar emphysema, congenital focal emphysema, congenital macroglossia, congenital medullary stenosis, congenital giant colon, congenital pheochromocytoma nevus, congenital mesoderm dysmorphic dystrophy, congenital mesoderm dystrophy, congenital Microvilli atrophy, congenital polyarticular contracture, congenital myotonic dystrophy, congenital neuropathy caused by reduced myelination, congenital pancytopenia, congenital pernicious anemia, congenital due to intrinsic factor deficiency Pernicious anemia, congenital pernicious anemia due to intrinsic factor deficiency, congenital pigmented cirrhosis, congenital porphyria, congenital proximal myopathy associated with desmin storage myopathy, a prior Emphysema, congenital erythroblastic fistula, congenital erythroblastic fistula, congenital retinal blindness, congenital retinal cyst, congenital retinitis pigmentosa, congenital retinal segregation, congenital rod disease, congenital rubella syndrome , Congenital scalp defects with distal limb loss, congenital sensory neuropathy, congenital SMA with joint contractures, congenital spherocytic anemia, congenital spinal epiphyseal dysplasia, congenital tethered spinal cord syndrome, congenital Tyrosinosis, congenital varicella syndrome, congenital vascular cavernous malformation, congenital retinal vascular capsule, congenital letter blindness, congenital migratory spleen (children), congenital myocardial disorder, keratoconus, conjugated hyperbilirubinemia, Conjunctivitis, woody conjunctivitis, conjunctivitis-urethral-synovial syndrome, Kong syndrome, connective tissue disease, Konrady disease, Konrady-Hünermann syndrome, constitutional aplastic anemia, constitutional erythropoiesis, constitutional eczema, constitutional liver function Disorder, constitutional platelet disorder , Congenital constriction ring, infarct epicarditis with dwarfism, continuous muscle fiber activity syndrome, contractile spider fingers, leg muscle atrophy contracture eye movement aphasia, convulsions, Cooley anemia, copper transport disease, Hepatic coproporphyrin porphyria, Sanbo heart, left tribo chamber, trichamber, double chamber, Cory disease, corneal dystrophy, corneal amyloidosis, corneal turbidity-relaxing skin-mental retardation, corneal dystrophy, Cornelia-Drague syndrome , Corneal dentin dysplasia, corneal artery disease, coronary heart disease, no corpus callosum, cortex-basal ganglion degeneration (CBGD), cortical deformation, type I corticosterone methyl oxidase deficiency, type II corticosterone methyl oxidase Deficiency, cortisol, costello syndrome, cot death, COVESDEM syndrome, COX, COX deficiency, COX deficiency, French-Canadian type COX deficiency, COX deficiency infant mitochondrial myopathy detoni-funko Benign infantile mitochondrial myopathy COX deficiency, including needle play, CP, CPEO, CPEO with myopathy, CPEO with red muscle fiber excitation, familial CPPD, CPT deficiency, CPTD, cranial arteritis, cranial meninges Cerebral aneurysm, cranial-mouth-finger syndrome, cranial carpal dystrophy, cranial aneurysm, Scott-type cranial-finger syndrome-mental retardation, craniofacial dysplasia, craniofacial dysplasia-PD artery-hyperlipa-lip dysplasia, skull Facial nose dysplasia, skull metaphyseal malformation, cranio-oral syndrome, type II cranio-oral syndrome, cruzon-type ankle, scapulocephalus, skull fusion-posterior nostril closure-radial humeral fusion, skull fusion-rich hair- Facial and other abnormalities, skull fusion, central dysplasia of the face, primary skull fusion, skull fusion-radial dysplasia syndrome, skull fusion with rib defects, bipartite skull, CREST syndrome, Creutzfeldt-Jakob disease, cat cry syndrome , Sleeper death, I Crigler-Nager syndrome, Crohn's disease, Cronkite-Canadian syndrome, Cross syndrome, Cross syndrome, Cross-McZic-Brine syndrome, Cruzon, Cruzon syndrome, Cruzon craniofacial osteogenesis, essential mixed cryoglobulinemia, latent eyeball Syndromed syndrome, latent testis-dwarf-subnormal spirit, Schneider's crystal corneal dystrophy, CS, CSD, CSID, CSO, CST syndrome, curly hair-blind adhesion-nail dysplasia, Cushman-Batten-Steinert syndrome, Curse-McClin Type porcupine ichthyosis, curse mcklin, gassing, gassing syndrome, gassing iii, hereditary cutaneous malignant melanoma, cutaneous porphyria, flaccid skin-growth syndrome, congenital marble-like telangiectasia, CVI, CVID, CVS, periodic vomiting syndrome,
Renal medullary cyst disease, cystic synovial edema, cystic fibrosis, cystic lymphangioma, cysteine-lysine-arginine-ornithineuria, cystine accumulation disease, cystine accumulation disease, cystineuria, dibasic amino acid urine Cystinuria with infectious disease, type I cystinuria, type II cystinuria, type III cystinuria, congenital renal medullary cyst, cytochrome C oxidase deficiency, DC, tear saliva adenopathy, tear salivary gland disease, Dalpro ( Dalpro), Dalton, Congenital color blindness, Danbolt-Cross syndrome, Butoh-butoh syndrome, Dandy-Walker syndrome, Dandy-Walker cyst, Dandy-Walker deformity, Dandy-Walker malformation, Danish heart amyloidosis (III Type), Darier's disease, Davidson's disease, David's disease, DBA, DBS, DC, DD, De Barsy syndrome, Dobarsh-Moens -Dierck syndrome, Drange syndrome, Domorsier syndrome, Dosanctis-Cacchione syndrome, Dotoni-Fanconi syndrome, congenital deafness-functional heart disease, deafness-dwarfism-retinal atrophy, deafness-functional heart disease, Hearing loss nail dystrophy Bone dysplasia and mental retardation, Hearing loss and torsional Bjorn stud type, sensory nerve deafness with anal closure and hypoplastic thumb, debranching enzyme deficiency, shedding skin, enterocyte intrinsic factor receptor deficiency, Natural killer lymphocyte deficiency, carnitine renal reabsorption deficiency, glycoprotein neuraminidase deficiency, mitochondrial respiratory chain complex IV deficiency, platelet glycoprotein Ib deficiency, von Willebrand factor receptor deficiency, short chain acyl-CoA dehydrogenase (ACADS), medium Deformation with blastomere dwarfism, degenerative chorea, degenerative lumbar stenosis, Dogo's disease, Dogo-Colemai Ard's disease, Dogo syndrome, DEH, Dégerine-Lucy syndrome, Dégerine-Sotta disease, partial deletion 9p syndrome, partial deletion 11q syndrome, partial deletion 13q syndrome, Delleman-Oorthuys syndrome, Derman Syndrome, dementia with leaf atrophy and neurocytoplasmic inclusions, demyelinating disease, DeMyer syndrome, coronary dentin dysplasia, root dentin dysplasia, type I dentin dysplasia, type II dentin dysplasia, brandy Wine-type dentin hypoplasia, Shields-type dentin hypoplasia, Type III dentin hypoplasia, Dentin-eye-bone dysplasia, Dentin ocular skin syndrome, Donnie-Drash syndrome, Depaken, Depaken (TM) exposure, Depacote , Spraying of depacote, depigmentation-gingival fibromatosis-microphthalmia, Durham disease, atopic dermatitis, epidermolytic dermatitis, herpetic dermatitis, polymorphic dermatitis, generalized skin relaxation , Systemic skin relaxation, dermatophytosis, dermatomyositis sign (sine) myositis, dermatomyositis, skin fragility, Stevens-Johnson type stomatitis, Desbuquois syndrome, desmin accumulation myopathy, neonatal desquamation, second-color weakness, developmental Dyslexia, developmental Gerstman syndrome, Devergie disease, Dovic disease, Dovic syndrome, right thoracic-bronchial dilatation-sinusitis, visceral-inverted concomitant right thoracic heart, DGS, DGSX Golabi-Rosen ) Syndrome, including DH, DHAP alkyltransferase deficiency, DHBS deficiency, DHOF, DHPR deficiency, diabetes insipidus, diabetes insipidus diabetes mellitus eye atrophy and deafness, pituitary diabetes insipidus, insulin-dependent diabetes mellitus, diabetes mellitus, intrinsic Diabetic Addison Myxedema, Diabetic Acidosis, Diabetic Beard Syndrome, Diabetic Neuropathy, Diamond-Blackfan Anemia, Diaphragmatic Apnea Osteopathic tissue connection, deformed dwarfism, bent bone dysplasia, bent dwarf syndrome, dicarboxylic acid amino aciduria, dicarboxylate calciumuria caused by fatty acid β-oxidation deficiency, dicarboxylic due to fatty acid β-oxidation deficiency Calcium aciduria, dicarboxylic aciduria due to MCADH deficiency, two-color type color blindness, Dicker-Opitz, DIDMOAD, diencephalic syndrome, pediatric diencephalon syndrome, diabetic diencephalopathy, dienoyl-CoA reductase deficiency, infant generalized brain Degeneration, generalized brain degenerative disease, generalized idiopathic osteoproliferative disorder, diffuse glycopeptideuria, DiGeorge syndrome, finger-mouth cranial syndrome, finger-ear palat syndrome, type I finger-ear palat syndrome, type II finger ear Palatal syndrome, dihydrobiopterin synthetase deficiency, dihydropteridine reductase deficiency, dihydroxyacetone phosphate synthase, dilated (congestive) cardiac myopa Seed, Dimitri disease, bilateral cerebral palsy, diploid Y syndrome, disaccharide oxidase deficiency, disaccharide intolerance I, discoid lupus, discoid lupus erythematosus, DISH, keratosis disorder, type I Cornification disorder, cornification disorder 4, cornification disorder 6, keratinization disorder 8, netherton keratinization disorder 9, phytanic acid type cornification disorder 11, cornification disorder 12 (neutral fat accumulation type), cornification disorder 13 Keratosis disorder 14, keratosis disorder cornification disorder 14, cornification disorder 15 (keratitis deafness type), keratinization disorder 16, mutant erythematous keratoderma type keratosis disorder 18, keratinization disorder 19, keratosis disorder 20, keratosis disorder 24, spleen deviation, disseminated lupus erythematosus, disseminated neurodermatitis, disseminated sclerosis, distal 11q monosomy, distal 11q-syndrome, type IIA congenital distal poly joint contracture, Type IIA congenital distal multiple joint contracture, Type IIA distal joint contracture, Type 2A distal joint contracture, Distal duplication 6q, Distal duplication 10q, Duplicate (10q) syndrome, Distal duplication 15q, Distal Monosomy 9p, distal trisomy 6q, Trisomy 10q syndrome, distal trisomy 11q, divalproic acid, DJS, DKC, DLE, DLPIII, DM, DMC syndrome, DMC disease, DMD, hereditary DNS, DOC 1, DOC 2, DOC 4, DOC 6 (Harlequin type) , Curs McClin type DOC 8, Phytanic acid type DOC 11, DOC 12 (neutral fat storage type), DOC 13, DOC 14, schizophrenia type DOC 14, DOC 15 (keratitis hearing loss type), DOC 16, one side Sexual dysplasia DOC 16, DOC 18, DOC 19, DOC 20, DOC 24, Dore body myelopathy, long spine dysplasia, spider limb, spider limb syndrome, dominant Kenny-Cafe syndrome, dominant congenital myotony , Donahue syndrome, Donato Landsteiner hemolytic anemia, Donato Landsteiner syndrome, DOOR syndrome, DOORS syndrome, dopa responsive ataxia (DRD), Dorfman Chanarin syndrome, Dowling-Meara syndrome, Down syndrome, DR syndrome, Drash syndrome DRD Dreis-emery muscular dystrophy with contraction, dresser syndrome, migratory spleen, drug-induced melanosis, drug-induced lupus erythematosus, drug-related adrenal insufficiency, dramand syndrome, dry leg, dry eye, DTD, duane regression syndrome, duane Syndrome, Type 1A, 1B and 1C Duane Syndrome, Type 2A, 2B and 2C Duane Regression Syndrome, Type 3A, 3B and 3C Duane Syndrome, Dubin-Johnson Syndrome, Dubovitz Syndrome, Duchenne, Duchenne Muscular Dystrophy, Duchenne Paralysis, Duhring's Disease, Duncan disease, Duncan disease, duodenal obstruction, duodenal stenosis, duodenal inflammation, double 4p syndrome, partial duplication 6q, dupuis syndrome, dupuytren contracture, dutch-kennedy syndrome, dwarfism, anteflex dwarfism, tubular bone Cortical thickening and transient low calcium Dwarfism with leprosy, Lewy dwarfism, retrospective dwarfism, dwarfism nail dysplasia, dwarfism pericarditis, dwarfism with renal atrophy and hearing loss, dwarfism with rickets , DWM, Diguev-Merckio-Clausen syndrome, familial autonomic neuropathy, familial dysbetalipoproteinemia, chondrogenesis with hemangiomas, dyschondrosthesis, hereditary universal dyschromia, visceral cyst brain malformation Congenital abnormal keratinization, autosomal recessive keratinization, Scoggins type congenital abnormal keratinization, congenital abnormal keratinization syndrome, nutritional vesicular abnormal keratinization, dyslexia, abnormal myeloid leukodystrophy, abnormal Myeloid leukodystrophies-giant erythroblasts, convulsive dysphonia, spotted epiphyseal malformation, unilateral epiphyseal malformation, nail malformation with insufficient number of teeth, clavicular cranial malformation, fibrous dysplasia, X -Linkage dysplasia giant syndrome, bone-like dentin dysplasia, dysplastic mother Syndrome, nevus dysplasia, cerebellar myoclonic dyskinesia, esophageal dyskinesia, ataxia, ectopic ectopic dystrophy, corneal dystrophy, mesoderm dystrophy, epidermolysis bullous dystrophy, dystrophy, asphyxic chest, Myotonic dystrophy, ED syndrome, Eagle-Barrett syndrome, Eale's retinopathy, Eales disease, ear abnormalities with posterior scoliosis-contracture-bone dysplasia, ear palate short stature syndrome, initial restraint defect, small fairy face Early hypercalcemia syndrome, early onset ataxia, Eaton-Lambert syndrome, EB, Ebstein abnormality, EBV sensitivity (EBVS), EBVS, ECD, ECPSG, ectodermal dysplasia, cleft lip and palate Germinal dysplasia, ectoderm dysplasia-exocrine pancreatic insufficiency, rap-Hodgkin ectoderm dysplasia, congenital ectoderm mesoderm dysplasia, bone involvement Ectodermal mesoderm dysplasia, multi-orbital erosive ectoderm, lens translocation, vesical valgus, ectopic ACTH syndrome, ectopic corticotropin syndrome, ectopic anus, finger missing, Missing finger, missing finger-ectodermal dysplasia syndrome, missing finger ectodermal dysplasia syndrome, missing finger ectodermal labial cleft / palatosis, eczema, eczema-thrombocytopenia-immunodeficiency syndrome, EDA, EDMD, EDS, arterial ecchymosis EDS, EDS joint relaxation, classical severe EDS, dysfibronectinemia EDS, Gravis EDS, EDS hyperactivity, EDS dorsal scoliosis, EDS dorsal scoliosis, mild EDS, EDS Ocular scoliosis, EDS progeria, EDS periodontitis, vascular EDS, EEC syndrome, EFE, EHBA, EHK, Ehrers-Danlos syndrome, Ehrers-Danlos syndrome, Ehrers-Danlos IX, Eisenmenger complex, Eisenmenger complex , Eisenmenger disease, Eisenmenger reaction, Zenmenger Syndrome, Ekbum Syndrome, Ekman-Ropestein Disease, Ektrodactyly in the Hand, EKV, Elastin Fiber Disorder, Systemic Elastic Fiber Rupture, Elastic Fiber Dystrophy Syndrome, Selective Silence (Obsolete), Selective Silence Disease, electrocardiogram (ECG or EKG), electron transfer flavin protein (ETF) dehydrogenase deficiency (GAII & MADD), electrophysiological research (EPS), birth elephant nail, congenital hemangiomatous elephantiasis, vasodilatory hypertrophy, high calcium Little Fairy Face with Bloodemia, Ellis-Funkrefeld Syndrome, Ellis-Funkrefeld Syndrome, Renal Germoma, Renal Embryosyosarcoma, Renal Embryon Carcinosarcoma, Renal Embryo Mixed Tumor, EMC, Emery-Dreis Muscular Dystrophy, Emery -Dreifis muscular dystrophy, Emery-Dreifth syndrome, EMF, EMG syndrome, Turkish vagina syndrome, diffuse periaxial encephalitis Pericardial axonal encephalitis, cerebral hernia, craniofacial hemangiomatosis, encephalopathy, cerebral trigeminal hemangiomatosis, endochondromatosis with multiple cavernous hemangioma, endemic polyneuritis, intracardiac uplift defect , Endocardial bulging defect, endocardial dysplasia, endocardial fibroelastosis (EFE), endogenous hypertriglyceridemia, endolymphatic hydrops, endometrial proliferation, endometriosis, endocardial myocardial fibrosis Congenital endothelial corneal dystrophy, endothelial epithelial corneal dystrophy, endothelium, Engelmann's disease, hypertonic tongue, enteritis, intestinal cobalamin malabsorption, eosinophilic syndrome, eosinophilic cellulitis, eosinophilic fasciitis, Eosinophilic granulomas, eosinophil syndrome, epidermal nevus syndrome, epidermolysis bullosa, acquired epidermolysis bullosa, hereditary epidermolysis bullosa, lethal epidermolysis bullosa, hereditary late epidermolysis, epidermolysis Keratosis, exfoliative keratosis (bullous CIE), preeclamptic epilepsy, epilepsy, epinephrine Phosphorus, epiphyseal changes and intensity myopia, benign epiphyseal osteochondroma, epiphyseal limb dysplasia, interstitial abnormal eye movement, epithelial basement corneal dystrophy, juvenile Miesmann epithelial corneal dystrophy, multiple epithelium with nevus , Epithelium, Epival, EPS, Male Epstein-Barr virus-induced lymphoproliferative disease, Elp-Goldfram syndrome, Eertheim chest disease, Exudative polymorphic erythema, Stevens Johnson polymorphic erythema, Fetus Erythroblastic fistula, fetal erythroblastosis, neonatal erythroblastosis, childhood erythroblastic anemia, erythrocyte phosphoglycerate kinase deficiency, erythropoiesis dysplasia, symmetric progressive erythema keratoderma, symmetric progression Erythematous keratoderma, erythematous keratoderma, variant erythematous keratoderma, winter erythematous epidermis detachment, hematopoietic porphyria, hematopoietic porphyria, Escobar syndrome, food Esophageal closure, esophageal peristalsis, esophagitis-peptic ulcer, esophageal closure and / or tracheoesophageal fistula, essential familial hyperlipidemia, essential fruit diabetes, essential hematuria, essential hemorrhagic thrombocythemia, essential Mixed mixed cryoglobulinemia, essential Moskowitz disease, essential thrombocythemia,
Essential thrombocytopenia, Essential thrombocytosis, Essential tremor, Esterase inhibitor deficiency, Fanconi anemia, Estren-Dameshek variant, Estrogen-related cholestasis, ET, ETF, Ethylmalonic acid Adipic aciduria, Eulenburg disease, pc, EVCS, exaggerated startle response, brain hernia, exogenous hypertriglyceridemia, umbilicus-big tongue-giant syndrome, ocular protruding goiter, enlarged rubella syndrome, bladder valgus, EXT, External chondroma syndrome, extrahepatic bile duct closure, extramedullary plasmacytoma, exudative retinitis, ocular regression syndrome, FA1, FAA, Fabry disease, FAC, FACB, FCAD, FACE, FAF, FAG, FACH, facial nerve palsy, Facial paralysis, facial ectodermal dysplasia, facial ectodermal dysplasia, face-scapula-humerus dystrophy, face-ear-vertebral spectrum, face-heart-skin syndrome, face-anterior-nasal dysplasia, facial skin skeleton Syndrome, face Genital syndrome, facial genital dysplasia, facial genital popliteal syndrome, facial palatal syndrome, type II facial palatal syndrome, facial scapulohumeral muscular dystrophy, artificial hypoglycemia, factor VIII deficiency, factor IX deficiency, factor Factor XI deficiency, factor XII deficiency, factor XIII deficiency, Farle disease, Farle disease, gastric intrinsic factor secretion deficiency, Fairbank disease, tetralogy of Fallot, familial acromegaly, familial microtip disease, familial line Colonic polyposis, familial adenoma polyposis with onset of extraintestinal symptoms, familial adipogenic forebrain, familial alpha-lipoprotein deficiency, familial amyotrophic chorea with spiny erythrocytosis, familial arrhythmia Myoclonus, familial articular cartilage calcification, familial atypical mol-malignant melanoma syndrome, type III familial hyperlipoproteinemia, familial calc gout, familial calcium pyrophosphate arthropathy, familial chronic obstructive pulmonary disease Familial continuous dermabrasion, familial cutaneous amyloidosis, familial dysproteinemia, familial emphysema, familial microvillous enteropathy, familial foveal retinopathy, familial hibernation syndrome, familial high cholesterol, family Familial hemochromatosis, familial blood high cholesterol, familial high density lipoprotein deficiency, familial serum high cholesterol, familial hyperlipidemia, familial hypoproteinemia with lymphangiectopathy, familial jaundice , Familial juvenile renal fistula-related eye abnormalities, familial lichenoid amyloidosis (type IX), familial lumbar stenosis, familial primary lymphedema, familial Mediterranean fever, familial polyposis, familial segmental blisters Familial paroxysmal polyseritis, familial colorectal polyposis, familial primary pulmonary hypertension, familial renal diabetes, familial splenic anemia, familial startle disease, familial visceral amyloidosis (V Type III), FAMMM, FANCA, FANCB, FANCC, FANCD, FANCE, Fanconi panmyelopathy, Fanconi pancytopenia, Fanconi II, Fanconi anemia, Fanconi anemia, Fanconi anemia complementation group, Fanconi Anemia complementation group A, Fanconi anemia complementation group B, Fanconi anemia complementation group C, Fanconi anemia complementation group D, Fanconi anemia complementation group E, Fanconi anemia complementation group F, Fanconi anemia complementation group G, Fanconi anemia Complement group H, Estren-Dameshek mutant Fanconi anemia, FANF, FANG, FANH, FAP, FAPG, Farber disease, Faber lipogranulomatosis, FAS, fasting hypoglycemia, fat-induced hyperlipidemia, childhood lethality Granulomatous disease, fatty acid disorder, fatty liver with encephalopathy, FAV, FCH, FCMD, FCS syndrome, FD, FDH, Stevens-Johnson type febrile mucocutaneous syndrome, acute febrile neutrophilic skin disease, onset Seizures, Feinberg Syndrome, Feissinger-Leroy-Reiter Syndrome, Female Pseudo Turner Syndrome, Bilateral Thigh Developmental Abnormality-Robin Abnormality, Bilateral Thigh Developmental Insufficiency, Thigh Facial Syndrome, Thigh Developmental Deficiency-Abnormal Facial Syndrome, Fetal Alcohol Syndrome, Fetal Anticonvulsant Syndrome, Fetal Hydrocele Cyst, Alcoholic Fetal Effect, Varicella Fetal Effect, Thalidomide Fetal Effect, Varicella-Zoster Virus Fetal Effect, Fetal Endocardial Myocardial Fibrosis Fetal facial syndrome, fetal photoirrititis syndrome, fetal transfusion syndrome, fetal valproate syndrome, fetal valproate exposure syndrome, fetal varicella infection, fetal varicella-zoster syndrome, type II FFDD, FG syndrome, FGDY, FHS, fibrin Stabilizing factor deficiency, fibrinase deficiency, fibrin-like degeneration of astrocytes, fibrin-like leukodystrophy, fibrinoli Case deficiency, perineural fibrinoblastoma, pancreatic fibrocystic disease, progressive ossifying fibrosis, fibroelastic endocarditis, fibromyalgia, fibromyalgia-fibromyositis, fibromyositis, fibrosis Cholangitis, connective histitis, polyarticular fibrosis, fibrocavernositis, fibrotic dysplasia, penile fibrosis, penile fibrosis sclerosis, Fickler-Winkler type, Feeder disease, fifth finger syndrome , Filippi syndrome, Finnish amyloidosis (type V), 1st congenital heart block, first and second bowel syndrome, Fischer syndrome, fish odor syndrome, fissure tongue, squamous tumor syndrome, Fratau-Schilder Disease, flavin-containing monooxygenase 2, floating β disease, floating harbor syndrome, migratory spleen, hypotonia syndrome, valve tightness syndrome, fluent aphasia, FMD, FMF, adult liver type FMO, FMO2, FND, focal brain Ischemia, focal skin Dysplastic syndrome, focal hypodermis, focal phalangeal dysplasia, focal ataxia, focal epilepsy, type II focal facial skin dysplasia, focal neuromuscular tone, FODH, foil syndrome, von (Fong ) Disease, FOP, Forbes disease, Forbes-Allbright syndrome, Forrestil disease, Forsius-Ericsson syndrome (X-linked), Fothergill disease, fountain syndrome, progressive foveal dystrophy, type II FPO syndrome, FPO, Fraccaro cartilage Asymptomatic (Type IB), Fragile X Syndrome, French Shetty-Salen-Crane Syndrome, Francois Craniofacial Malformation Syndrome, Francois-Neatens Spotted Dystrophy, Spotted Corneal Dystrophy, Fraser Syndrome, FRAXA, FRDA, Fredrickson Type I Hyperlipoproteinemia, Freeman-Sheldon syndrome, Freire-Mia syndrome, Fly syndrome, Free Dry Ataxia, Friedreich's disease, Friedreich's fist, FRNS, Frehlic syndrome, Fronmel-Chiari syndrome, Fronmel-Chiari syndrome lactation uterine atrophy, front finger syndrome, anterior facial nasal bone dysplasia, anterior nasal dysplasia, anterior nasal dysplasia Dysplasia with coronal skull fusion, fructose-1-phosphate aldolase deficiency, fructoseemia, fructosis, Fryns syndrome, FSH, FSHD, FSS, Fuchs Dystrophy, type 1 fucose accumulation disease, 2 Type fucose accumulation disease, type 3 fucose accumulation disease, fukuhara syndrome, fukuyama disease, fukuyama type muscular dystrophy, fumaryl acetoacetase deficiency, grooved tongue, G syndrome, G6PD deficiency, G6PD, GA I, GA IIB, GA IIA, GA II, GAII & MADD, non-partum milk leak-amenorrhea syndrome, non-pregnant milk leak-amenorrhea syndrome, galactosamine-6-sulfatase deficiency, galactose-1-phosphate Gyltransferase deficiency, galactosemia, GALB deficiency, Galloway-Mowat syndrome, Galloway syndrome, GALT deficiency, gamma globulin deficiency, GAN, ganglioside neuraminidase deficiency, ganglioside sialidase deficiency, type 1 gangliosidesis GM1, type 2 gangliosidesis GM2, Gangliosidosis β-hexosaminidase B deficiency, Gardner syndrome, multiple osteogenesis imperfecta, Garies-Mason syndrome, Gasser syndrome, gastric endogenous secretory factor deficiency, enterocyte cobalamin, gastrinoma, gastritis, gastroesophageal laceration -Bleeding, gastrointestinal polyposis ectoderm change, gastrointestinal ulcer, gastric wall rupture, Gaucher disease, Gaucher-Schlagenhaufer, Gaet-Wernicke syndrome, GBS, GCA, GCM syndrome, GCPS, Gee-Harter Herter disease, Gee-Thaysen disease, Cook's disease, Gerlinau syndrome, Ginny-Wiedemann syndrome, systemic ataxia, systemic familial neuromuscular tone, systemic fibromatosis, systemic flexion epilepsy, systemic glycogenopathy, systemic hyperhidrosis, systemic lipofuscin Disease, systemic myasthenia gravis, systemic muscle tone, systemic sporadic neuromuscular tone, genetic disease, genital defect, genital ureteral defect, Gerstman syndrome, Gerstman quadruple, GHBP, GHD, GHR, giant axon disease , Giant axonal neuropathy, giant benign lymphoma, giant cell glioblastoma, astrocytoma, giant cell arteritis, giant cell liver disease, giant cell hepatitis, neonatal giant cell cirrhosis, giant retinal cyst, giant lymph node hyperplasia Formation, hereditary giant platelet syndrome, giant tongue, gic macular dystrophy, Gilbert's disease, Gilbert's syndrome, Gilbert-Dreifth syndrome, Gilbert-Lereboulet syndrome, Guildford syndrome, Jill・ La Tourette Syndrome, Gillespie Syndrome, Gingival Fibroma-Abnormal Fingernail Nasal Ear Splenomegaly, GLA deficiency, GLA, GLB1, glaucoma, retinal glioma, complete aphasia, spherical cell leukodystrophy, hypoglossoid mandibular disease Cleft palate, glucocerebrosidase deficiency, glucocerebrosidosis, glucose-6-phosphate dehydrogenase deficiency, glucose-6-phosphate transport deficiency, glucose-6-phosphate translocase deficiency, glucose-G-phosphatase deficiency, glucose -Galactose malabsorption, glucosylceramide lipidosis, glutaric aciduria I, glutaric acidemia I, glutaric acidemia II, glutaric aciduria II, type II glutaric aciduria, type III glutaric aciduria, glutaric acidemia Disease I, glutaric acidemia II, glutaric aciduria I, glutaric aciduria II, type IIA glutaric aciduria, type IIB glutaric aciduria, glutaryl-CoA dehydride Genase deficiency, glutarate-aspartate transport deficiency, gluten-sensitive enteropathy, type VII muscle glycogen disease, glycogen storage disease I, glycogen storage disease III, glycogen storage disease IV, type V glycogen storage disease VI, glycogen storage disease VI, glycogen storage disease VII, glycogen storage disease VIII, type II glycogen storage disease, type II glycogen storage disease, glycogenosis, type I glycogenosis, type IA glycogenosis, type IB glycogenosis, type II glycogenosis, type II sugar Primary disease, type III glycogenosis, type IV glycogenosis, type V glycogenosis, type VI glycogenosis, type VII glycogenosis, type VIII glycogenosis, glycolic aciduria, glycolipid lipidosis, type 1 GM2 gangliosidosis, type 1 GM2 gangliosidosis, GNPTA, goiter-like autoimmune thyroiditis, golddonal syndrome, golddonal-gorin syndrome, goldscheider disease, gorz syndrome, gorz-gorin syndrome, gonadal development Dysgenesis 45X, gonadal dysgenesis XO, corner development failure-insufficient teeth, Goodman syndrome, Goodman, Goodpasture syndrome, Gordon syndrome, Gorin syndrome, Gorin-Shordley-Moss syndrome, congenital progressive symmetric Gottron Erythematous keratoderma, Gottron syndrome, Gujrou-Carto syndrome, epileptic seizures, granular corneal dystrophy, granulomatous arteritis, granulomatous colitis, granulomatous dermatitis with eosinophilia, granulomatous Ileitis, Graves' disease, Graves' hyperthyroidism, Graves' disease, Greig's cranial multifinger syndrome, Greno type I corneal dystrophy, Greno type II corneal dystrophy, Glenblad-Strandberry syndrome, Groton syndrome, growth hormone receptor deficiency, Growth hormone binding protein deficiency, growth hormone deficiency, Mayer (Myhre) Growth-mental deficiency syndrome, growth retardation-Rieger abnormality, GRS, Gruber syndrome, GS, GSD6, GSD8, GTS, guanosine triphosphate-cyclohydrolase deficiency, guanosine triphosphate-cyclohydrolase Deficiency, Guenther porphyria, Guerin-Stern syndrome, Gearan-Barre, Gearan-Barre syndrome, Gunter disease, H disease, H. Grotton syndrome, habitual spasticity, HAE, Hageman factor deficiency, Hageman factor, Heim- Haim-Munk syndrome, Hajdu-Cheney syndrome, Haju-Cheney, HAL deficiency, Hall-Pallister syndrome, Hallemann-Strife-François syndrome, Hallemann-Strife syndrome, Halleforfor Den-Spatz disease, Hallerfolden-Spatz syndrome, allopo-Siemens disease, double thumb postaxial polydactyly, callosal defect, Hull -Si-Behcet Syndrome, lymphoma hamartoma, hand-Schuller-Christian syndrome, HANE, Hanhart syndrome, happy doll syndrome, Harada disease, HARD +/- E syndrome, HARD syndrome, hare lips, Harlequin fetus, Harlequin DOC 6 , Harlequin ichthyosis, Harley syndrome, Harrington syndrome, Heart syndrome, Hartnup disease, Hartnup disorder, Hartnup syndrome, Hashimoto disease, Hashimoto-Plitzker syndrome, Hashimoto syndrome, Hashimoto thyroiditis, Hashimoto-Plitzker syndrome, Hay-Wells syndrome, Hay-Wells syndrome with ectodermal dysplasia, HCMM, HCP, HCTD, HD, heart-hand syndrome (Halt-Aurum type), heart disease, Hecht syndrome, HED, Halefort-Waldenstrom Lofgren's syndrome, heglin disease, Heinrichsbauer syndrome
, Hemangioma, familial hemangioma, hemangioma-thrombocytopenia syndrome, hemangiomatosis chondrogenesis, fistula hemangioma cleft lip syndrome, hemifacial dwarfism, hemilateral giant brain, unilateral cerebral palsy, half Lateral cerebral palsy, spinal resection, hemochromatosis, hemochromatosis syndrome, hemodialysis-related amyloidosis, hemoglobin repo syndrome, newborn hemolytic anemia, hemolytic cold antibody anemia, newborn hemolytic disease, hemolytic-uremic syndrome, Hemophilia, hemophilia A, hemophilia B, hemophilia B factor IX, hemophilia C, hemorrhagic dystrophic thrombocytopenia, hemorrhagic anemia, hemosiderin deposition, liver fructokinase deficiency , Liver phosphorylase kinase deficiency, hepatic porphyria, hepatic porphyria, hepatic vein-occlusion disease, hepatitis C, hepatorenal syndrome, hepatic lens nuclear degeneration, hepatic phosphorylase deficiency, hepatic renal glycogenosis, hepatorenal syndrome, hepatic renal tyrosineemia, Hereditary acromegaly, hereditary alkaptonuria, hereditary amyloidosis, hereditary angioedema, hereditary nonreflective standing, hereditary polyneurotic ataxia, hereditary ataxia, Friedreich's hereditary ataxia, Hereditary benign black epidermoma, hereditary cerebellar ataxia, hereditary chorea, hereditary chronic progressive chorea, hereditary connective tissue disorder, hereditary coproporphyria, hereditary coproporphyrin porphyria, hereditary skin malignancy Melanoma, hereditary hearing loss-retinitis pigmentosa, hereditary zinc deficiency disorder, hereditary DNS, hereditary ectopic lipidosis, hereditary emphysema, hereditary fructose intolerance, hereditary hemorrhagic telangiectasia, type I Hereditary hemorrhagic telangiectasia, type II hereditary hemorrhagic telangiectasia, type III hereditary hemorrhagic telangiectasia, hereditary uric acid hypertension and choreoathetosis syndrome, hereditary major non-red blood cell Disease, hereditary minor non-erythrocytosis, hereditary lymphedema, hereditary late lymphedema, type I hereditary lymphedema, type II hereditary lymphedema, hereditary motor sensory neuropathy, hereditary motor sensory neuropathy I, hereditary motor sensory neuropathy III, hereditary nephritis, hereditary nephritis and deafness, hereditary nephropathy amyloidosis, hereditary nephropathy and hearing loss, hereditary nonpolyposis colorectal cancer, hereditary nonpolyposis colon Rectal cancer, hereditary nonspherical cell hemolytic anemia, hereditary nail dysplasia, hereditary optic neuroretinopathy, hereditary colorectal polyposis, type I hereditary sensory autonomic disorder, type II hereditary sensory autonomic disorder, type III Hereditary sensory autonomic neuropathy, hereditary sensorimotor neuropathy, type I hereditary sensory neuropathy, type I hereditary sensory neuropathy, type II hereditary sensory neuropathy, type III hereditary sensory neuropathy, type I heritability Sensory radiculopathy, type I remains Hereditary sensory radiculopathy, type II hereditary sensory radiculopathy, hereditary site-specific cancer, hereditary spherocytic hemolytic anemia, hereditary spherocytosis, type 1 hereditary tyrosineemia, hereditary connection Tissue disorders, Herlitz syndrome, Hermans-Herzberg nevus, Hermannsky-Padlac syndrome, half-yin, herpes zoster, Stevens-Johnson iris herpes, Yale disease, heterozygous β thalassemia, hexoaminidase α subunit deficiency ( Mutant B), hexoaminidase α-subunit deficiency (mutant B), HFA, HFM, HGPS, HH, HHHO, HHRH, HHT, Galloway hiatal hernia-small head-nephrosis, sweat gland abscess, axillary adenitis, Sweat gland abscess, hyperhidrotic ectoderm malformation, HIE syndrome, severe anal obstruction, high potassium, high scapula, HIM, Hirschsprung's disease, acquired Hirschsprung's disease, Hirschs Purung's disease Ulnar foot multifinger and VSD, Hirschsprung's disease with D-shaped short finger, hirsutism, HIS deficiency, histidine ammonia lyase (HAL) deficiency, histidase deficiency, histidineemia, histiocytosis, Histiocytosis X, HLHS, type II HLP, HMG, HMI, HMSN I, HNHA, HOCM, Hodgkin's disease, Hodgkin's disease, Hodgkin's lymphoma, Hollander-Jimons disease, Holmes-Ardy syndrome, holocarboxylase synthetase deficiency, global forebrain disease , Total forebrain malformation complex, all forebrain sequelae, Holt-Aurum syndrome, Holt-Aurum heart-hand syndrome, homocystinemia, homocystinuria, homogentisic acid oxidase deficiency, homogentisic aciduria, homozygous alpha-1 Antitrypsin deficiency, HOOD, Horner syndrome, Houghton disease, HOS, HOS1, Huston-Harris type cartilage development (type IA), HPS, HRS, HS, type I HSAN, type II HSAN, HSAN-III, HSMN, type III HSMN, HSN I, HSN-III, Hübner-Harter disease, Hanner's piece, Hanner's ulcer, Hunter syndrome, Hunter-Thompson-type distal middle limb shortening dysplasia, Huntington's disease, Huntington's disease, Fuller's disease, Fuller's syndrome, Fuller-Scheier syndrome, HUS, Hutchinson-Gilford premature syndrome, Hutchinson-Gilford syndrome, Hutchinson-Weber-Poitz syndrome, Bowen-Conrady-type footerite syndrome, hyaline pannerv Disorder, hydroencephalopathy, hydrocephalus, hydrocephalus retinal dysplasia, internal dandy-walker hydrocephalus, non-traffic dandy-walker hydrocephalus, hydrocephalus, hydronephrosis with unusual facial expression, Hydroxylase deficiency, cervical hygroma, high IgE syndrome, high IgM syndrome, hyperaldosteronism, low potassium alk Hyperaldosteronism with cissis, hyperaldosteronism without hypertension, hyperammonemia, hyperammonemia due to carbamylphosphatase synthetase deficiency, hyperammonemia due to ornithine transcarbamidase deficiency, type II hyperammonemia , Hyper-β carnosineemia, hyperbilirubinemia I, hyperbilirubinemia II, familial hypercalcemia with nephrocalcinosis and indicanuria, hypercalcemia-valvular aortic stenosis, hypercalciuria Rickets, respiratory acidosis, hypercatabolic protein-losing enteropathy, hyperchloremic acidosis, hypercholesterolemia, type IV hypercholesterolemia, hypermilky lipidemia, hypercystinuria, startle hyperemia , Hyperextension joints, hyperglobulinemia purpura, ketoacidosis and propionic acid type lactic acidosis Concomitant hyperglycinemia, non-ketotic hyperglycinemia, hypergonadotropic hypogonadism, hyperimmunoglobulin E syndrome, hyperimmunoglobulin E-recurrent infection syndrome, hyperimmunoglobulinemia E-staphylococci, high potassium Hyperemia syndrome, hyperlipidemia retinitis, hyperlipidemia I, hyperlipidemia IV, type I hyperlipoproteinemia, type III hyperlipoproteinemia, type IV hyperlipoproteinemia, Hyperoxaluria, phalangeal hyperplasia of the index finger with Pierre-Robin syndrome, hyperphenylalaninemia, hyperplastic epidermolysis bullosa, hyperventilation, hyperkalemia, high pre-beta lipoproteinemia, Type I hyperprolinemia, Type II hyperprolineemia, hypersplenism, isolation with esophageal abnormalities and hypospadias, isolation-hypospadias syndrome, hypertrophic cardiomyopathy, hypertrophic interstitial neuropathy , Hypertrophic interstitial neuritis, hypertrophic interstitial radiculopathy Harm, refsum hypertrophic neuropathy, hypertrophic obstructive cardiomyopathy, hyperuricemia chorea athetosis self-multiplication syndrome, hyperuricemia-mental retardation, hypervaline, hypocalcification (mineralization) type, Hypochondrosis, hypochondrogenesis, hypogammaglobulinemia, infant transient hypogammaglobulinemia, genital hypoplasia dystrophy with diabetic tendency, sublingual-finger defect syndrome, hypoglycemia, extrinsic hypoglycemia Hypoglycemia with macrotonia, type 1a hypoglycosylation syndrome, type 1a hypoglycosylation syndrome, hypogonadism with olfactory sensation, pituitary dysfunction hypogonadism and olfactory sensation, no sweating Ectodermal dysplasia, autosomal dominant anhidrotic ectodermal dysplasia, autosomal recessive anhidrotic ectodermal dysplasia, hypokalemia, hypokalemia alkalosis with hypercalciuria, Hypokalemia syndrome, c Hypolactasia, hypomature (Yukihat-like teeth), Ito's melaninopathy, limb dysplasia-oligo-facial hemangioma syndrome, hypomyelination neuropathy, hypoparathyroidism, hypophosphatasia Hypophosphatemic rickets with hypercalcemia, depigmentation, depigmented macular lesions, subangular antimuscular dysplasia with heart defects, dysplastic anemia, congenital dysplastic anemia, Dysplastic cartilage dysplasia, dysplastic enamel-nail detachment-hypohidrosis, dysplasia (dysplasia-explastic) type, dysplastic left ventricular syndrome, dysplasia-thumb thumbarthrosis , Hypokalemia syndrome, hypospadias-dysphagia syndrome, hyposmia, hypothalamic hamartoma pituitary hypoanalysis polydactyly, hypothalamic childhood-obesity, hypothyroidism, hypotension-intelligence Decline-hypogonadism-obesity syndrome, hypoxanth -Guanine phosphoribosyltransferase deficiency (complete deficiency), I-cell disease, iatrogenic hypoglycemia, IBGC, IBIDS syndrome, IBM, IBS, IC, I-cell disease, ICD, Kogan-Lease ICE syndrome, ice Land type amyloidosis (type VI), I-cell disease, ichthyosis-like erythroderma cornea-related hearing loss, ichthyosis-like erythroderma hair overgrowth and male, ichthyosis-like erythroderma with leukocyte vacuolation, ichthyosis, Congenital ichthyosis, congenital ichthyosis with folliculosis, porcupine ichthyosis, pregnant porcupine ichthyosis, convoluted linear ichthyosis, simple ichthyosis, ichthyosis-tay syndrome, vulgaris ichthyosis, ichthyosis Septic triglycerides, jaundice leptospirosis, jaundice hemorrhagic fever leptospirosis, jaundice (chronic familial), neonatal pregnancy jaundice, juvenile intermittent jaundice, idiopathic hypoalveolar ventilation, idiopathic amyloidosis, idiopathic Takayasu arteritis, idiopathic basal calcification (IBGC), Idiopathic brachial plexus neuropathy, idiopathic cervical ataxia, idiopathic pulmonary dilatation, idiopathic facial paralysis, idiopathic familial hyperlipidemia, idiopathic hypertrophic subaortic stenosis, idiopathic hypoproteinemia, idiopathic Immunoglobulin deficiency, idiopathic neonatal hepatitis, idiopathic nonspecific ulcerative colitis, idiopathic peripheral periphalitis, idiopathic pulmonary fibrosis, idiopathic refractory ironblastic anemia, idiopathic renal hematuria, idiopathic Fatty stool, idiopathic thrombocythemia, idiopathic thrombocytopenic purpura, idiopathic thrombocytopenic purpura (ITP), IDPA, IgA nephropathy, IHSS, ileitis, ileocolitis, illinois amyloidosis, ILS, IM , IMD2, IMD5, immunodeficiency due to lack of thymus, paroxysmal cold immunohemolytic anemia, immunodeficiency with telangiectasia ataxia, cellular immunodeficiency with abnormal immunoglobulin synthesis, unclassifiable mutant immunodeficiency, Immunodeficiency with high IgM, immunity with leukopenia Insufficiency, immunodeficiency-2, immunodeficiency-5 (IMD5), immunoglobulin deficiency, anal obstruction, anal obstruction with abnormalities in limbs, nasolacrimal duct obstruction, premature aging syndrome, incompetent neutrophil syndrome, mouth Short finger flexor muscle that cannot be completely opened, INAD, arginase-type congenital urea synthesis abnormality, arginosuccinate-type congenital urea synthesis abnormality, carbamyl phosphate-type congenital urea synthesis abnormality, citrullinemia type congenital urea synthesis abnormality, glutamate Synthetase-type congenital urea synthesis abnormality, INCL, inclusion body myositis, incomplete atrioventricular septal defect, incomplete testicular feminization, dyschromia, depigmentation, loss of index finger with Pierre-Robin syndrome, Indiana amyloidosis ( Type II), painless systemic mastocytoma, infantile acquired aphasia, autosomal recessive polycystic kidney disease, infantile beriberi, infantile brain ganglioside, infantile cerebral palsy, infantile system Chin accumulation disease, infantile epilepsy, infantile Fanconi syndrome with cystinosis, infant Finnish neuronal ceroid lipofuscinosis, infant Gaucher disease, infantile hypoglycemia, infantile hypophosphatasia, infantile lobar Emphysema, infantile myoclonic encephalopathy, infantile myoclonic encephalopathy polyclonic convulsions, infantile myofibromatosis, infantile necrotic encephalopathy, infantile neuroceroid lipofuscinosis, infantile neuroaxonal dystrophy, infantile onset type Schindler's disease, infant phytanic acid accumulation disease, infant refsum disease (IRD), infantile sypoidosis GM-2 gangliosidosis (type S), infant sleep apnea, temporal spasm, infant spinal muscular atrophy (all types) Infantile spinal muscular atrophy, type I infant spinal muscular atrophy, infantile neuronal ceroid lipofuscinosis, infectious jaundice, inflammatory bowel disease, inflammatory breast cancer, inflammatory linear nevus With fat syndrome, occipital foramen prolapse, insulin-resistant black epidermopathy, insulin-induced lipodystrophy, insulin-dependent diabetes mellitus, intentional myoclonus, intermediate cystine accumulation disease, intermediate maple syrup urine disease, pyruvate dehydrogenase deficiency Intermediate ataxia, intermittent maple syrup urine disease, endohydrocephalus, interstitial cystitis, 4q interstitial loss, intestinal lipodystrophy, intestinal lipophagocytic granulomatosis, intestinal lymphangiectasia , Intestinal polyposis I, intestinal polyposis II, intestinal polyposis III, intestinal polyposis-cutaneous pigmentation syndrome, intestinal pseudo-obstruction with extraocular muscle paralysis, intracranial neoplasia, intracranial tumor, intracranial vascular malformation, intrauterine dwarfism , Intrauterine Cinecia, Reverse-smile-invisible neuropathic bladder, Iowa-type amyloidosis (Type IV), IP, IPA, Iris corneal endothelial syndrome, Corgan-Reese-type iris cornea Skin (ICE) syndrome, iris corner occurs failure with an abnormality in the body, iris atrophy associated with corneal edema glaucoma, iris nevus syndrome, iron
Excessive anemia, iron overload disease, irritable bowel syndrome, irritable colon syndrome, Isaac syndrome, Isaac-Merten syndrome, ischemic heart myopathy, isolated circulatory defect sequelae, isoleucine 33 amyloidosis, isovaleric acid CoA Dehydrogenase deficiency, isovaleric acidemia, isovaleric acidemia, isovaleryl-CoA carboxylase deficiency, ITO melaninopathy, ITO, ITP, IVA, Ivemark syndrome, Ivanov cyst, Jackknife spasm, Jackson-Vice skull fusion, Jackson-Vice Syndrome, Jackson epilepsy, Jacobsen syndrome, Jadassohn-Lewandov syndrome, Jaffe-Liechtenstein disease, Jacob disease, Jacob-Kreuzfeld disease, Janeway I, Janeway hypogammaglobulinemia, Janzen metaphyseal osteogenesis imperfecta, Janzen metaphyseal cartilage Dysplasia, Jarcho-Levin syndrome, Joe Winking, JBS, JDMS, Jegger's syndrome, jejunum closure, jejunitis, jejunitis, Gerver-Langer-Nielsen syndrome, Junu syndrome, JMS, Job syndrome, Job-Buckley syndrome, Johansson-Blizzard syndrome, John-Dalton, Johnson-Stevens syndrome, Johnston hair loss, Joseph's disease, type I Joseph's disease, type II Joseph's disease, type III Joseph's disease, Joubert syndrome, Joubert-Bolthauser syndrome, JRA, Juberg-Hayward Syndrome, Juberg-Marsidi Syndrome, Juberg-Marcidi Mental Retardation Syndrome, Jumping French, Main Jumping French, Juvenile Arthritis, Juvenile Autosomal Recessive Polycystic Kidney, Juvenile Cystic Accumulation, Young Sexual (pediatric) dermatomyositis (JDMS), juvenile diabetes , Juvenile Gaucher disease, juvenile gout chorea athetosis mental retardation syndrome, juvenile vitamin B12 intestinal malabsorption, juvenile vitamin B12 intestinal malabsorption, juvenile macular degeneration, juvenile pernicious anemia, juvenile malignant anemia, juvenile rheumatoid arthritis Osteoarthritis, including juvenile spinal muscular atrophy, including juvenile spinal muscular atrophy ALS, type III juvenile spinal muscular atrophy, paraarticular pain adiposity, paraglomerular hyperplasia, Kabuki make-up syndrome, Kohler (Kahler) disease, Kalman syndrome, Kanner syndrome, Kanzaki disease, Kaposi disease (not Kaposi sarcoma), kappa light chain deficiency, Karsch-Neugebauer syndrome, Cartagener syndrome-chronic sinus bronchial disease and right chest heart , Cartagener Triad, Casabach-Merit Syndrome, Caste Syndrome, Kawasaki Disease, Kawasaki Syndrome, KBG Syndrome KD, Keynes-Saier Disease, Keynes-Saier Syndrome , Kennedy disease, Kennedy syndrome, Kennedy spinal medullary atrophy, Kennedy-Stephanis disease, Kenny disease, Kenny syndrome, Kenny-type tubular stenosis, Kenny-Caffe syndrome, congenital palmokeratoderma Spider finger, keratitis ichthyosis deafness syndrome, keratoconus, posterior keratoconus, keratolysis, congenital exfoliative epidermis exfoliation, keratolytic winter erythema, corneal softening, follicular keratosis, spiny hair loss Follicular keratosis, spiny alopecia folliculosis ichthyosis, keratokeratosis, palmokeratosis with periodontal onychomycosis, congenital palmokeratoderma flatfoot nail fistula Periodontal spider finger, congenital palmokeratoderma, flatfoot, nail fistula, periodontitis, spider finger, acrostomy, keratosis, Rubra Figurata, seborrheic horn Keratosis, keto acid decarboxylase deficiency, ketoaciduria, ketogenic glycinemia, KFS, KID syndrome No kidney development, Cystic kidney-retinal dysplasia Joubert syndrome, Kyrian syndrome, Kyrian / Texler-Nikola syndrome, Killo-Nevin syndrome III, hair loss, Kinsbourne syndrome, Clover leaf cranial deformity, Kleine-Levin syndrome, Kleine -Levin hibernation syndrome, Kleinfelder, Klipel-Feuille syndrome, Type I Klipel-Feuille syndrome, Type II Kliper-Faille syndrome, Type III Klipel-Faille syndrome, Klipel-Tornonnay syndrome, Klipel-Tornonnay-Weber syndrome, Clewer-Beauty Krabbe white matter dystrophy including syndrome, KMS, Kneist dysplasia, Kneist syndrome, Kebner's disease, Koebberling-Dunnigan syndrome, Kohler-Dunnigan syndrome, Cook's disease, Korsakov psychosis, Korsakov syndrome, Krabbe disease -Kramer syndrome, KSS, KTS, KTW syndrome, Kuchs disease, Kugelberg-Welander disease, Kugelberg-Welander syndrome, Kushmar-Laundry paralysis, KWS, L-3-hydroxy-acyl-CoA dehydrogenase (LCHAD) deficiency Syndrome, Labhart-Willi syndrome, labyrinth syndrome, labyrinth hydrops, lacrimal-auricular-tooth-finger syndrome, lactase isolation intolerance, lactase deficiency, milk-uterus atrophy, lactic acidosis laver hereditary optic nerve Lactic acid and pyruvate with disability, sugar sensitivity, lactate and pyruvate with intercalated ataxia and frailty, lactate and pyruvate, lactic acidosis, lactose intolerance in adults, lactose intolerance, childhood Lactose intolerance, LADD syndrome, LADD, Lafora disease, including Lafora disease, Lake-Lorland factor deficiency, LAM, Lumber Ichthyosis, Lambert-Eaton syndrome, Lambert-Eaton myasthenia syndrome, phyllodes recessive ichthyosis, lenticular ichthyosis, Lancereaux-Mathieu-Weil spirochetosis, Landau-Kreffner syndrome, Landgie-DeJerine muscular dystrophy, laundry ascending paralysis , Langer-Saridino type cartilage dysplasia (type II), Langer-Gyzion syndrome, Langerhans cell granulomatosis, Langerhans cell histiocytosis (LCH), large atrial ventricular defect, Laron dwarfism, Laron type pituitary dwarf , Larsen syndrome, pharyngeal ataxia, Larter (observed in Malaysia), late infantile neuroaxonal dystrophy, late infantile neuroaxonal dystrophy, type III late cocaine syndrome (type C), late onset Ataxia, late-type immunoglobulin deficiency, late-onset Pelizaeus-Merzbacher sclerosis Lattice corneal dystrophy, Lattice dystrophy, Ronois-Bansade, Ronois-Cleret syndrome, Lawrence syndrome, Lawrence-Moon syndrome, Lawrence-Moon / Valday-Bedle, Lawrence-Saipe syndrome, LCA, LCAD deficiency, LCAD, LCAD, LCADH deficiency , LCH, LCHAD, LCPD, Lejnu syndrome, Leband syndrome, Leber's cataract, Leber's congenital cataract, congenital deletion of cone rod, Leber's congenital wall plate retinal degeneration, Leber's congenital wall plate retinal dysplasia , Leber's disease, Leber's optic atrophy, Leber's optic neuropathy, left ventricular fibrosis, leg ulcer, Leg-Calve-Perthes disease, Lee's disease, Lee's syndrome, Lee's syndrome (subacute necrotizing cerebrospinal disorder), Lee's necrotic cerebrospinal Disorder, Lennox-Gastaut syndrome, Kuroko-polydipsia-digestion syndrome, renunciation dysplasia syndrome, renunciation dysplasia, Nile microphthalmia, Renshi syndrome, LEOPARD syndrome, Fairy disease, leptomeningeal hemangiomatosis, Leptospira jaundice, Lully-Wel disease, Lully-Wel chondrogenic dysplasia, Lully-Wel syndrome, Lermoye syndrome, Leroy disease, Lesch-Nyhan syndrome , Lethal infant cardiomyopathy, lethal neonatal dwarfism, lethal osteochondrodysplasia, letterer-zieve disease, leukocytic leukoderma, leukocyte inclusions with platelet abnormalities, leukodystrophy, rosental fibers White matter dystrophy, concentric axial leukoencephalitis, Levine-Crickery syndrome, diabetes, Levy-Hollister syndrome, LGMD, LGS, LHON, LIC, cuspid red lichen, lichen planus, lichen amyloidosis, lichen planus , Psoriatic lichen, lignac-drepley-fanconi syndrome, lignac-fanconi syndrome, ligneous conjunctivitis, limb girdle muscular dystrophy Fee, limb malformation-tooth-finger syndrome, terminal dextrin formation, linear nevus melaninopathy, linear nevus sebaceous gland syndrome, linear scleroderma, linear sebaceous nevus onset, linear sebaceous nevus syndrome , Tongue cleft, fold tongue, scrotum tongue, lingual facial dyskinesia, lip fake-angiomatous groin cyst syndrome, lipid granulomatosis, lipid histiocytosis, kelasin lipid, lipid storage disease, lipid associated with SCAD deficiency Storage myopathy, infantile ganglioside lipidosis, lipotrophic diabetes mellitus, lipodystrophies, lipoid corneal dystrophy, lipoid hyperplasia-male pseudohemiyang, congenital pancreatic lipomatosis, type I lipomucopolysaccharidosis, fatty meningocele , Familial lipoprotein lipase deficiency, LIS, LIS1, cerebral gyrus deficiency, type I gyrus deficiency, cerebral gyrus deficiency with no callosal cerebellar dysplasia or other abnormalities, Little disease, liver phosphorylase deficiency, LKS , LM syndrome, leaf wilt , Cerebral lobe atrophy, lobar total proencephalopathy, infantile leaf tension emphysema, Robstein's disease (type I), lobster claws deformity, localized epidermolysis bullosa, localized lipodystrophy, localized neuritis of the shoulder band, Lefler disease, Lefler endocardial fibrosis with eosinophilia, Lefler fibrosis wall endocarditis, Loken syndrome, Loken-senior syndrome, long chain 3-hydroxyacyl-CoA dehydrogenase (LCHAD) ), Long-chain acyl-CoA dehydrogenase deficiency, long-chain acyl-CoA dehydrogenase (ACADL), long-chain acyl-CoA dehydrogenase deficiency, QT prolongation syndrome without hearing loss, Lou Gehrig's disease, Lou Gehrig's disease Syndrome, hypoglycemia, low density beta lipoprotein deficiency, anal occlusion failure, low potassium syndrome, low syndrome, low syndrome, low-bickel syndrome, low-terry-macraclan syndrome, lower Headache, LS, LTD, Lubs syndrome, raft disease, lumbar canal stenosis, lumbar stenosis, lumbosacral stenosis, Lundberg-Unferricht disease, Lundberg-Unferricht disease, lupus, lupus, erythematous Lupus, Roshka-Majandi foramen closure, Reyer syndrome, Reyer syndrome, Lymph node-like goiter, Lymphatic dilated protein-losing enteropathy, Lymphatic leiomyosarcoma, Lymphatic leiomyosarcoma, Lymphangioma, Lymphatic malformation , Lynch syndrome, Lynch syndrome I, Lynch syndrome II, Schindler lysosomal α-N-acetylgalactosaminidase deficiency, lysosomal glycoamino acid storage disease-diffuse keratoangioma, lysosomal glucosidase deficiency, MAA, Machado disease, Machado-Joseph Disease, macroencephalopathy, macrocephaly, macrocephaly hemihypertrophy, macrocephaly with multiple lipoma and hemangioma, pseudopapillary edema and Macrocephaly with multiple hemangiomas, macroglobulinemia, macroglossia, megalingual-umbilical hernia-viscosity syndrome, large blind eyelid syndrome, familial Bernard-Sulier type giant thrombocytopenia, macular degeneration, macular amyloidosis, macular degeneration , Discoid macular degeneration, senile macular degeneration, macular dystrophy, macular corneal dystrophy, MAD, Madelung's disease, mahucci syndrome, epileptic seizure, malabsorption-ectodermal dysplasia-nasal dysplasia, Roger's disease, Tic disease, malaria, male extremity and renal malformation, male Turner syndrome, malignant epidermis hyperplasia, malignant melanosis, malignant astrocytoma, malignant atrophic papulosis, malignant fever, malignant hyperphenylalaninemia, malignant hyperthermia , Malignant hyperthermia, malignant melanoma, malignant tumor of central nervous system, Mallory-Vais laceration, Mallory-Vais laceration, Mallory-Va Syndrome, Paget's disease of the breast, mandibular enamel epithelioma, mandibular facial dysplasia, mannosidosis, map-dot-fingerprint corneal dystrophy, maple syrup urine disease, marble-like bone, Marquia-Farva-Micheli syndrome, Marcus -Ganjo Winking disease, Marcus-Gann phenomenon, Marcus-Gun droop with Jowinking syndrome, Marcus-Gann syndrome, Marcus-Gann (Jawwinking) syndrome, Marcus-Gun droop (with Jowinking), Marden-Walker type connective tissue disorder, Marfan inability to live, Marfan-Achar syndrome, Marfan syndrome, Marfan syndrome I, Marfan mutant, Marfan syndrome-like hyperkinetic syndrome, limbic dystrophy , Marie ataxia, Marie disease, Marie-Sandton disease, Ma --- Strulumper disease, Marie-Strumpel spondylitis, Marinesco-Sjogren's syndrome, Marinesco-Sjogren-Gorland syndrome, Marker X syndrome, Maroto-Ramy syndrome, Maloto-type limb end mesothelial malformation, non-Marshall with visual hearing loss Germinal dysplasia, Marshall-Smith syndrome, Marshall syndrome, Marshall-type deafness-myopia-cataract-vomeronas, Martin-Albright syndrome, Martin-Bell syndrome, Martell syndrome, MASA syndrome, large clonic muscle spasm, mast cell Leukemia, mastocytosis, mastocytosis with associated bleeding disorder, Maumenee corneal dystrophy, maxillary enamel epithelioma, maxillofacial bone dysplasia, maxillary nasal bone dysplasia, binder-type maxillary nasal bone dysplasia, maxillary blepharoplasty, May-Heglin abnormality, MCAD deficiency, MCAD, McCardle disease, Mac Kukuon-All Bright, MCD, McKujik
Metaphyseal cartilage dysplasia, MCR, MCTD, Meckel syndrome, Meckel-Gruber syndrome, midline face syndrome, Mediterranean anemia, medium chain acyl-CoA dehydrogenase (ACADM), medium chain acyl-CoA dehydrogenase (MCAD) deficiency , Medium chain acyl-CoA dehydrogenase deficiency, medullary cyst disease, medullary canine kidney, MEF, giant esophagus, encephalopathy, megacephalopathy with hyaline inclusions, encephalopathy with hyaline panneuropathy, megaloblastic anemia, pregnancy Megaloblastic anemia, giant cornea-mental retardation syndrome, Mayer-Gorin syndrome, meige lymphedema, meige syndrome, black skin leukodystrophy, black spot-intestinal polyposis, black spot-intestinal polyposis, MELAS syndrome, MELAS , Mercerson syndrome, Melnic-Fraser syndrome, Melnic-Niddles osteodysplasia, Melnic-Niddles syndrome, membranous lipodystrophy, Mendes Dacosta syndrome, Meniere's disease, Meniere's disease, meningiocapillary hemangiomatosis, Menkes disease, Menkes disease I, mental retardation aphasia dragged walking addendum (MASA), mental retardation-deafness-skeletal abnormalities-rough with complete lips Face, mental retardation with fifth toe nail dysplasia, mental retardation with osteochondral abnormality, growth retardation-hearing loss-microgenital disease and X-linked mental retardation, Menzel OPCA, Mermaid syndrome, MERRF, MERRF Syndrome, Merten-Singleton syndrome, MES, mesangial IGA nephropathy, mesenteric lipodystrophies, mid-tooth-cataract syndrome, mesoderm dystrophy, mesoblastic dwarfism-Merderung deformity, metabolic acidosis, irrigation White matter dystrophy, metatarsal club, retroactive dwarf syndrome, retroactive dysplasia, retroactive dysplasia I, retroactive dysplasia II, methylmalonic acidemia, methylmalonic aciduria, moilengrahat disease, MFD1 MG, MH, MHA, cerebellar disease, microcephaly dwarfism I, microcephaly, microcephaly-hiatal hernia-Galloway nephrosis, microcephaly-hiatal hernia-nephrotic syndrome, microcystic corneal dystrophy, microerythrocyte , Cerebellar gyrus defect, microphthalmia, microphthalmia or anophthalmia with associated abnormalities, cerebellar gyrus excess with muscular dystrophy, microtia patella deficient jaw syndrome, microvillous inclusion disease, MID, middle systolic-click- Late systolic noise syndrome, Mischer type I syndrome, Micrich syndrome, Micrich-Laddeck syndrome, Micrich-Sjogren syndrome, mild autosomal recessive, mild intermediate maple syrup urine disease, mild maple syrup urine disease, Miller syndrome, Miller-Dieker syndrome , Miller-Fischer syndrome, Milroy's disease, Minkowski-Schofar syndrome, epileptic seizures, Minnow-vonvilleb Endopathic disease, mirror image right chest heart, mitochondrial β-oxidation disorder, mitochondria and cytosol, mitochondrial cytotoxicity, mitochondrial cytotoxicity, Keynes-Saier type, mitochondrial encephalopathy, mitochondrial brain myopathy lactic acidosis and seizure-like episodes, mitochondrial PEPCK deficiency, mitral Valve prolapse, mixed apnea, mixed connective tissue disease, mixed liver porphyria, mixed nonadhesive aphasia, mixed sleep apnea, mixed tonic clonic torticollis, MJD, MKS, ML I, ML II, ML III, ML IV, Type I ML disorder, Type II ML disorder, Type III ML disorder, Type IV ML disorder, MLNS, MMR syndrome, MND, MNGIE, MNS, Mobits I, Mobits II, Mobits syndrome, Mobius syndrome, Mersch- Voltmann's syndrome, Morr's syndrome, brachial hair, monophasic visual amnesia, polyneuropathy, peripheral mononeuritis, peripheral mononeuropathy, monosomy 3p2, partial monosomy 9p, partial monosomy 11q, partial monosomy 13q, monosomy 18q syndrome, monosomy X, single fibrotic dysplasia, Morgany-Turner-Allbright syndrome, localized scleroderma, Morquio disease, Morquio syndrome , Morquio syndrome A, Morquio syndrome B, Morquio-Brelsford syndrome, Morvan disease, mosaic tetrasomy 9p, motoneuron disease, motoneuron syndrome, motoneuron disease, motoneuron disease, motoneuron disease (local and slow), hazy Disease, moyamoya disease, MPS, MPS I, MPS IH, MPS 1 H / S Halar / Scheier Syndrome, MPS IS Shiye Syndrome, MPS II, MPS IIA, MPS IIB, MPS II-AR Autosomal Recessive Hunter Syndrome, MPS II- XR, severe autosomal recessive MPS II-XR, MPS III, MPS III ABC and D Sanfiroppo A, MPS IV, MPS IV A and B Morquio A, MPS V, MPS VI, MPS VI Severe intermediate mild maloto-ramie, MPS VII, MPS VII Sly syndrome, MPS VIII, MPS disorder MPS disorder I, MPS disorder II, MPS disorder III, MPS disorder VI, type VII MPS disorder, MRS, MS, MSA, MSD, MSL, MSS, MSUD, MSUD, type Ib MSUD, type II MSUD, mucocutaneous lymph node syndrome, mucolipidosis I, mucolipidosis II, mucolipidosis III, mucolipidosis IV, mucopolysaccharidosis, mucopolysaccharidosis IH , Mucopolysaccharidosis IS, mucopolysaccharidosis II, mucopolysaccharidosis III, mucopolysaccharidosis IV, mucopolysaccharidosis VI, mucopolysaccharidosis VII, type I mucopolysaccharidosis, type II mucopolysaccharidosis, type III mucopolysaccharidosis, VII Type mucopolysaccharidosis, mucosis, mucosulfatosis, mucinous colitis, mucobisidesis, myocardial cerebral eye deficiency dwarfism, myoliver cerebral ocular deficiency dwarfism, Muellerian tube dysplasia-renal growth dysfunction-cervical chest Mueller with somite abnormalities, Muellerian tube-renal-cervical chest-upper limb defect, upper limb and rib abnormalities No kidney development, Muellerian tube-renal-cervical thoracic abnormalities, Binswanger type multiple infarct dementia, multicentric Castleman disease, multifocal eosinophil granuloma, multiple acyl-CoA dehydrogenase deficiency, multiple acyl -CoA dehydrogenase deficiency / type II glutaric aciduria, multiple hemangioma and endochondroma, multiple carboxylase deficiency, multiple cartilage endochondrosis, multiple cartilage exogenesis, multiple endochondromatosis, type II Multiple endocrine deficiency syndrome, multiple epiphyseal dysplasia, multiple exostosis, multiple external bone syndrome, multiple familial polyposis, multiple black syndrome, multiple myeloma, polyneuritis of the shoulder band, multiple osteochondral Tumor, multiple peripheral neuritis, multiple colon polyposis, multiple pterygium syndrome, multiple sclerosis, multiple sulfatase deficiency, multiple symmetric lipomatosis, multiple system atrophy, multiple bone union osteogenesis , Long bone Multiple osteosynthesis with fold failure, Mulvihill-Smith syndrome, MURCS-related, Murk-Yanzen metaphyseal chondrogenesis, muscle carnitine deficiency, muscle core disease, muscle phosphofructokinase deficiency , Myocardial core disease, muscular dystrophy, classic X-linked recessive muscular dystrophy, congenital muscular dystrophy with central nervous system involvement, congenital progressive muscular dystrophy with mental retardation, facial scapulohumeral muscular dystrophy, rheumatoid arthritis, muscle stiffness-progression Spasticity, musculoskeletal pain syndrome, multiple extremity disorders, speechlessness, mvp, MVP, MWS, myasthenia gravis, myasthenia gravis pseudoparaplegia, Lambert-Eaton myasthenia syndrome, myelin-disruptive diffuse sclerosis Disease, myeloma, Myhre syndrome, myoclonic seizures with epilepsy, myoclonic ataxia, infant myoclonic encephalopathy, myoclon -Nus epilepsy, Hartung-type myoclonic epilepsy, myoclonic epilepsy associated with red muscle fiber excitement, myoclonic epilepsy and red muscle fibrosis, myogenic clonic familial epilepsy, myogenic clonic familial epilepsy, muscle Clonic seizures, myoclonus, myoclonic epilepsy, myoencephalopathy red muscle fibrosis, myofibromatosis, congenital myofibromatosis, myogenic face-scapulo-radial syndrome, myo-gastrointestinal disorders and encephalopathy, multiple Congenital myopathy joint contracture, myopathic carnitine deficiency, middle myocardial fibril myopathy, congenital non-progressive myopathy, congenital non-progressive myopathy with central axis, myopathy with carnitine palmitoyltransferase deficiency, myopathy-Marinesco-Sjogren Syndrome, Myopathy-Metabolic Carnitine Palmitoyl Transfer -Deficiency, myopathy mitochondria-encephalopathy-lactic acidosis-seizure, myopathy with myoplasm and intermediate filament, myophosphorylase deficiency, progressive ossifying myositis, atrophic muscle tone, congenital muscle tone, congenital intermittent muscle Tension, myotonic dystrophy, myotonic myopathy dwarfism chondrogenic anomaly ophthalmofacial abnormality, myotubular myopathy, X-linked myotubular myopathy, Myproic Acid, Millie Atit (observed in Siberia), myxedema , N-acetylglucosamine-1-phosphotransferase deficiency, N-acetylglutamate synthase deficiency, NADH-CoQ reductase deficiency, Negeri ectodermal dysplasia, Nager syndrome, Nager apical facial bone dysplasia, Nager syndrome, NAGS deficiency, nail dystrophy Deafness syndrome, nail growth deficiency, insufficient teeth, nail-patella syndrome, na -Holans syndrome, microcephaly dwarfism, microcephaly, microphthalmia, narcolepsy, narcolepsy syndrome, NARP, nasal-frontal-face dysplasia, nasopharyngeal hypothyroidism pancreatic enzyme deficiency congenital deafness, Nasal maxillary dystrophy, nasal fat dystrophy, NBIAl, ND, NDI, NDP, Lee's necrotizing cerebrospinal disorder, necrotizing respiratory granulomatosis, nail-Dingwall syndrome, Nelson syndrome, nemarin myopathy, Neonatal adrenal white matter dystrophy, neonatal adrenal white matter dystrophy (NALD), neonatal adrenal white matter dystrophy (ALD), neonatal autosomal recessive polycystic kidney disease, neonatal dwarfism, neonatal hepatitis, neonatal hypoglycemia, neonatal lactose intolerance, Neonatal lymphoedema due to exudative enteropathy, neonatal necrotizing enterocolitis, neonatal progeria syndrome, Wiedemann-Lautenstrauch neonatal pseudo-hydrocephalic progeria syndrome, neoplastic spider Inflammation, nephroblastoma, nephrogenic diabetes insipidus, familial juvenile nephronophthesis, nephrotic cystinosis, nephropathy-pseudoyin-yang-Wilms tumor, nephrotic-small head syndrome, nephrosis-neural vagus Syndrome, nephrotic-diabetes-dwarfism-rickets-hypophosphatase blood syndrome syndrome, Netherton disease, Netherton syndrome, Netherton syndrome ichthyosis, Nettleship-Falls syndrome (X-linked), New-Laxova syndrome , Neuhausel syndrome, neural tube defects, neuralgic muscle atrophy, neuraminidase deficiency, neurocutaneous melanosis, vestibular schwannomas, schwannomas, neurospinous erythrocytosis, Schindler type neuroaxonal dystrophy, with type 1 brain iron accumulation Neurodegeneration (NBIA1), acoustic neurofibromas, multiple congenital neurogenic joint contractures, optic neuromyelitis, neuromuscular tone, focal neuromuscular tone, systemic familial neuromuscular Tension, systemic diffuse neuromuscular tone, Schindler type neuroaxonal dystrophy, adult type neuronal ceroid lipofuscinosis, juvenile type neuronal ceroid lipofuscinosis, type 1 neuronal ceroid lipofuscinosis, neurocytopathic acute Gaucher disease, nerve Impaired amyloidosis, neuropathic beriberi, neuropathic ataxia retinitis, brachial plexus neuropathy syndrome, type I hereditary sensory neuropathy, type II hereditary sensory neuropathy, neuropsychiatric porphyria, nerve fat storage Disease, nevus, nevus-like basal cell carcinoma syndrome, nevus, spongiform nevus, comed nevus, depigmented nevus, Yadassorn sebaceous nevus, nezerofu syndrome, no nezerofu thymus, nezerofu severe combined immunodeficiency , NF, NFl, NF2, NF-1, NF-2, NHS, Niemann-Pick disease, A-type Niemann-Pick disease (acute neuropathy), B-type Niemann-Pick disease, C-type Niemann-Pi Bock disease (chronic neuropathy), D-type Niemann-Pick disease (Nova-Scotia variant), E-type Niemann-Pick disease, F-type Niemann-Pick disease (blue-blue histiocytosis), night blindness, black spine teeth Degeneration, Niikawa Kuroki syndrome, NLS, NM, type I Noack syndrome, nocturnal myoclonic hereditary essential myoclonus, nodular corneal degeneration, non-bullous congenital ichthyosis-like erythroderma, non-traffic hydrocephalus , Non-deficient α-thalassemia / mental retardation syndrome, type I non-ketotic hyperglycinemia (NKHI), non-ketotic hyperglycinemia, non-lipid reticuloendotheliosis, non-neuropathic chronic adult Gaucher disease Carnitine deficiency due to non-scarring epidermolysis bullosa, non-arteriosclerotic brain calcium deposition, non-articular rheumatism, non-cerebral juvenile Gaucher disease, non-diabetic diabetes, non-ischemic cardiomyopathy, non-ketotic hypoglycemia and MCAD deficiency Acyl-CoA dehydrogener Non-ketotic hypoglycemia, non-ketotic glycinemia, nonne syndrome, nonne-milroy-meige syndrome, non-paque opalescent dentin, non-partum milk leak-amenorrhea, non-secretory bone marrow Tumor, nonspherical erythrocytic hemolytic anemia, non-tropical sprue, Noonan syndrome, norepinephrine, normotensive hydrocephalus, Norman-Roberts syndrome, Norrbottnian Gaucher disease, Norrie disease, Norwegian hereditary cholestasis, NPD, NPS, NS, NSA, nuchal dementia syndrome, nutritional neuropathy, Naihan disease, OAV spectrum, obstructive apnea, obstructive hydrocephalus, obstructive sleep apnea, OCC syndrome, obstructive thrombotic aorta , OCCS, occult intracranial vascular malformation, occult spinal fusion failure sequelae, Ochoa syndrome, tissue browning, tissue brown degeneration arthritis, OCR, OCRL,
Octocephaly, eye pallor, eye herpes, myasthenia gravis, eye-ear-spine dysplasia, eye-ear-spine spectrum, eye-cheek-genital syndrome, ocular brain syndrome with depigmentation , Eye brain skin syndrome, eye-brain-kidney, eye brain kidney dystrophy, eye brain kidney syndrome, ocular cranial syndrome (obsolete), eye skin whitening syndrome, eye skin whitening, Chediak-east type eye skin whitening, Eye-tooth-finger dysplasia, eye-finger syndrome, eye-tooth-bone dysplasia, eye-gastrointestinal muscular dystrophy, ocular gastrointestinal muscular dystrophy, ocular mandibular cranial disorder with poor hair, mandibular facial syndrome, congenital detention Eye movement with contraction and muscle atrophy, ocular sympathetic nerve palsy, ODD syndrome, ODOD, odontogenic tumor, tooth hair syndrome, OFD, OFD syndrome, Ohio amyloidosis (type VII), OI, congenital OI, late onset OI, Oldfield Syndrome, Amniotic Fluid Hypomaniasis, Mental Retardation Microphthalmia, Incomplete polydystrophy, olive bridge cerebellar atrophy, olive bridge cerebellar atrophy with dementia and extrapyramidal signs, olive bridge cerebellar atrophy with retinal degeneration, olive bridge cerebellar atrophy I, olive bridge cerebellar atrophy II, olive bridge cerebellar atrophy III , Olive Bridge cerebellar atrophy IV, Olive Bridge cerebellar atrophy V, Orie disease, Orie osteochondromatosis, Umbilical hernia-visceral giant-big tongue syndrome, Ondine cursing, Ryukyu neuropathy, Ryukyu polyneuropathy, nail dysplasia , Dysplasia of the nail with neutropenia, OPCA, OPCA I, OPCA II, OPCA III, OPCA IV, OPCA V, OPD syndrome, type I OPD syndrome, type II OPD syndrome, OPD I syndrome, OPD II syndrome , Ocular arthropathy, ocular palsy-intestinal pseudo-occlusion, ocular palsy, retinitis pigmentosa and cardiac myopathy, ocular palsy plus syndrome, ocular palsy syndrome, Opitz BBB syndrome, Opitz-Frias syndrome, Opitz G Symptoms , Opitz G / BBB compound syndrome, Opitz isolation-hypourethral syndrome, Opitz-Kaveggia syndrome, Opitz ocular pharyngeal syndrome, Opitz triangular skull syndrome, Opitz syndrome, ocular clonus, ocular clonus-myoclonus, ocular spinal cord Inflammation, optic atrophy polyneuropathy and deafness, optic craniomyelopathy, optic neuromyelitis, optic neuromyelitis, chiasm arachnoiditis, orofacial cleft, orofacial dyskinesia, orofacial ataxia, mouth-face-finger syndrome, type I Mouth-face-finger syndrome, mouth-face-finger syndrome I, mouth-face-finger syndrome II, mouth-face-finger syndrome III, mouth-face-finger syndrome IV, orbital cyst with brain and focal skin malformations, Ornithine carbamyltransferase deficiency, ornithine transcarbamylase deficiency, cranio-finger syndrome, orofacial finger syndrome, oral mandibular ataxia, orthostatic hypotension, Austler-Weber-Rendu disease, osteocular shape Abnormalities, Osteophthalmic dysplasia, Degenerative osteomyelitis, Degenerative osteochondral dystrophy, Osteochondrosis, Melnick &Niddle's dysplasia, Osteogenic dysplasia, Osteogenic dysgenesis, Congenital dysplasia, Late bone formation Insufficiency, bone hypertrophic nevus, multiple infantile hyperosseous sclerotic bone disorder, multiple infantile hyperosseous osteosclerotic bone disorder, osteopathyosis, marble bone disease, autosomal dominant adult Type osteomyelopathy, autosomal recessive malignant infant type osteomyelopathy, mild autosomal recessive intermediate type osteomyelopathy, fragile systemic osteosclerosis, osteosclerotic myeloma, primary mouth defect (including intracardiac elevation defect) , Secondary mouth deficit, OTC deficiency, ear palate syndrome, type I ear-palate-finger syndrome, type II ear-palate-finger syndrome, ear tooth dysplasia, ear palate syndrome, type II ear palate syndrome, Oathorn Skin, Turner ovarian dwarfism, Turner ovarian dysplasia, OWR, Oxalic acid, oxidase deficiency, cephalus, cephalus-cephalus, PV, PA, PAC, ichthyosis-like nail thickening, congenital nail thickening with birth teeth, congenital nail thickening, congenital nail thickening, Disseminated focal (follicular) keratosis, Yardassson-Lewandowsky type congenital nail thickening, PAF with MSA, Paget's disease, bone Paget's disease, breast Paget's disease, nipple paget's disease, nipple areola Paget's disease, Pagon syndrome, painful ophthalmoplegia, PAIS, palatal myoclonus, palate-ear-finger syndrome, type I palate-ear-finger syndrome, type II palate-ear-finger syndrome, Parister syndrome, Paristar-Hall syndrome, Paristar-Killian mosaic syndrome, Paristar mosaic aneuploidy, Paristar-mosaic syndrome, Paristar mosaic syndrome tetrasomy 12p, Paristar-W syndrome, palmokeratosis and alopecia, paralysis, pancreas Fibrosis Pancreatic insufficiency bone marrow dysfunction, pancreatic ulcerative tumor syndrome, panmyelogenous fistula, panmyelosis, pantothenate kinase-related neurodegeneration (PKAN), papillon-lefèvre syndrome, ecdyseptic pseudospinal fistula, myotonic periodic paralysis , Paralytic beriberi, paralytic brachial neuritis, paramedian paralipotomy fossa-Pyerygium syndrome, median paramesencephalic syndrome, paramedullary tissue hyperplasia, multiple paramyoclonus, congenital paramyotonia, von Eulenburg Congenital paramyotonia, Parkinson's disease, paroxysmal atrial tachycardia, paroxysmal cold hemoglobinuria, paroxysmal ataxia bile athetosis, paroxysmal motility ataxia, paroxysmal nocturnal hemochromatosis, paroxysmal normoglobinuria, seizure Sexual Sleep, Parrot Syndrome, Parry Disease, Parry-Lomberg Syndrome, Parsonage-Turner Syndrome, Partial Androgen Insensitive Syndrome, Chromosome 4 Short Arm Partial deletion, partial deletion of short arm of chromosome 5, partial deletion of short arm of chromosome 9, partial duplication 3q syndrome, partial 15q syndrome, partial facial palsy with urinary abnormalities, partial limb partial giantism -Nevus -Hemitrophic -Diacrosis, Partial lipodystrophy, Chromosome 11 long arm partial monosomy, Chromosome 13 long arm partial monosomy, Partial spinal cord perception syndrome, Partial trisomy 11q, Partonton syndrome, PAT Patency of arterial duct, pathological myoclonus, juvenile arthritis with arthritis, Paulitis, PBC, PBS, PC deficiency, group A PC deficiency, group B PC deficiency, PC, Eulenburg disease, PCC deficiency, PCH, PCLD, PCT, PD, PDA, PDH deficiency, Poisson syndrome pyruvate carboxylase deficiency, childhood obstructive sleep apnea, skin detachment syndrome, Pelizaeus-Merzbacher sclerosis, pellagra-cerebellar ataxia-renal amino aciduria syndrome , Pelvic pain symptoms , Pemphigus vulgaris, penaschoel II syndrome, penaschoel II syndrome, penile fibromatosis, penile fibrosis, penile sclerosis, pentax syndrome, cantrel pentagonal, pentagonal syndrome, pentasomy x, PEPCK deficiency, pepper syndrome, perhenentupa ) Syndrome, periarticular fibrosis, pericardial contracture with growth failure, pericollagenous amyloidosis, perinatal polycystic kidney disease, perineal anus, periodic amyloid syndrome, periodic peritonitis syndrome, periodic somnolence and morbid hunger, Periodic syndrome, peripheral cyst degeneration of the retina, peripheral bone dysplasia-nasal dysplasia-mental retardation, peripheral neuritis, peripheral neuropathy, peritoneal pericardial diaphragmatic hernia, pernicious anemia, limb disease with micrognathia , Radial muscle atrophy, radial nerve palsy, Peroutka sneeze, peroxisomal acyl-CoA oxidase, peroxisomal β-oxidation disorder, peroxy Somal bifunctional enzyme, peroxisomal thiolase, peroxisomal thiolase deficiency, arterial tube remnant, perthes disease, epileptic seizure, small seizure variant, Poitz-Jegers syndrome, Poitz-Touraine syndrome, Peyronie's disease, Pfeiffer, type I puffifer Syndrome, PGA I, PGA II, PGA III, PGK, type I PH, type I PH, pharyngeal sac syndrome, PHD short chain acyl-CoA dehydrogenase deficiency, phenylalanine hydroxylase deficiency, phenylalaninemia, phenylketonuria, phenylpyrubin Acid mental retardation, seal limb disease, seal limb syndrome, phosphoenolpyruvate carboxykinase deficiency, phosphofructokinase deficiency, phosphoglycerate kinase deficiency, phosphoglycerokinase, phosphorylase 6 kinase deficiency, phosphorylase deficiency glycogen storage disease, Phosphorylase kinase deficiency, light sneezing reflex, light sneezing, phototherapeutic keratotomy, PHS, physicist John Dalton, phytanic acid storage disease, Pi phenotype ZZ, PI, brain Pick disease, Pick disease, Pickwick syndrome , Pierre-Robin anomaly, Pierre-Roban complex, Pierre-Robin sequelae, Pierre-Robin syndrome, Pierre-Robin syndrome with finger joint excess and thumb, Pierre-Marie disease, Red blood cell pigmentation Degeneration, torsion and nerve deafness, torsion hair-sensory deafness, pituitary dwarfism II, pituitary tumor after adrenalectomy, pore eruption, pore erythema, PJS, PKAN, PKD , PKDl, PKD2, PKD3, PKU, PKUl, tonsillar, plasma cell myeloma, plasma cell leukemia, plasma thromboplastin component deficiency, plasma transglutaminase deficiency, cavernous sclerosis, penile Plastic sclerosis, PLD, saddle tongue, PLS, PMD, pulmonary kidney syndrome, PNH, PNM, PNP deficiency, POD, POH, polymorphic skin atrophy and cataract, congenital polymorphic skin atrophy, Polish abnormality, Polish sequelae, Polish Syndactyly, Polish Syndrome, Progressive Polyodystrophy, Intestinal Polyarthritis, Nodular Polyarteritis, Type I Multiarticular Onset Juvenile Arthritis, Type II Multiarticular Onset Juvenile Arthritis, Type I and Type II Joint-onset juvenile arthritis, polychondritis, polycystic kidney disease, medullary polycystic kidney disease, polycystic liver, polycystic ovary disease, polycystic kidney disease, multifinger-Jubert syndrome, polydysplastic epidermolysis bullosa, mental Weak polydystrophy, polydystrophy dwarfism, type III multigland autoimmune syndrome, type II multigland autoimmune syndrome, type I multigland autoimmune syndrome, type II multigland autoimmune syndrome, type II multigland Deficiency syndrome, multi-glandular syndrome, polymorphic macular alteration , Polymorphic macular degeneration, polymorphism of platelet glycoprotein Ib, hereditary polymorphic corneal dystrophy, rheumatic polymyalgia, polymyositis and dermatomyositis, primary agammaglobulinemia, peripheral polyneuritis, Polyneuropathy-deafness-optic atrophy, peripheral polyneuropathy, polyneuropathy polyradiculoneuropathy, polyosseous fibrosis, polyosseous sclerosing histiocytosis, familial polyposis, Gardner polyposis, Hamartoma intestinal polyposis, polyposis-osteoma-epidermoid cyst syndrome, pigmented polyposis epilation fingernail change, polyp and plaque syndrome, relapsing polyserumitis, polysomy Y, polydactyly with unique cranial shape, Polydactyly-Greig's facial cranial dysplasia, Pompu disease, Pamp disease, Popliteal pterygium syndrome, Porcupine male, Cerebrospinal disease, Cerebrospinal disease, Porphobilinogen deaminator (PBG-D), porphyria, acute intermittent porphyria, porphyria ALA-D, late cutaneous porphyria, hereditary late cutaneous porphyria, symptomatic late cutaneous porphyria, hepatic atypical porphyria , Swedish porphyria, Atypical porphyria, Acute intermittent porphyria, Porphyrin, Alopecia acne, Port wine discoloration, Portuguese amyloidosis, Postinfection polyneuritis, Posthypoxic intentional myoclonus, Postaxial limb facial bone formation Failure, postaxial polydactyly, post-encephalitis intentional myoclonus, hereditary retrocorneal dystrophy, retrothalamic syndrome, postmyelographic arachnoiditis, postnatal cerebral palsy, postoperative cholestasis, postpartum milk leakage-amenorrhea syndrome, Postpartum hypopituitarism, postpartum panhypophyseal syndrome, postpartum panhypophyseal hypofunction, postpartum pituitary necrosis, postural low Pressure, potassium loss nephritis, potassium loss syndrome, Potter type I infant polycystic kidney disease, Potter type III polycystic kidney disease, PPH, PPS, Prader-Villi syndrome, Prader-Rabhard-Villifancorn syndrome, prealbumin Tyr-77 Amyloidosis, abnormal preexcitability syndrome, pregnenolone deficiency, premature atrial contraction, premature premature aging syndrome, supraventricular premature contraction, premature contraction ventricular waveform, postnatal or congenital neuroaxonal dystrophy, premature dementia, premature Macular degeneration, primary adrenal insufficiency, primary agammaglobulinemia, primary aldosteronism, primary alveolar hypoventilation, primary amyloidosis, primary anemia, primary limbs, primary bile, primary biliary cirrhosis, Primary Brown syndrome, primary carnitine deficiency, primary central hypoventilation syndrome, cartagener type primary ciliary dyskinesia, primary Cutaneous amyloidosis, primary ataxia, primary adrenal cortex dysfunction, primary familial dysplasia of the maxilla, primary hemochromatosis, primary hyperhidrosis, primary hyperoxaluria [type I], type 1 Primary hyperoxaluria (PH1), type I primary hyperoxaluria, type II primary hyperoxaluria, type III primary hyperoxaluria, primary hypogonadism, primary Intestinal lymphangiectasia, primary lateral sclerosis, primary non-hereditary amyloidosis, primary obstructive pulmonary vascular disease, primary progressive multiple sclerosis, primary pulmonary hypertension, primary dyslexia, primary Renal diabetes, primary sclerosing cholangitis, primary thrombocythemia, primary central nervous system tumor, primary visual agnosia, idiopathic colitis, idiopathic rectal colitis, adult progeria, childhood premature aging syndrome, Premature dwarfism, premature short stature with pigmented nevus, dobarge progeria syndrome, multisystem wilt Progressive autonomic failure with, progressive bulbar paralysis,
Progressive cardiomyopathy, including progressive medullary palsy, progressive familial cerebellar ataxia, progressive cerebral polydystrophy, progressive choroidal atrophy, progressive dysplasia, progressive unilateral facial atrophy, progressive familial Myoclonic epilepsy, progressive unilateral facial atrophy, progressive hypoerythemia, progressive infant polydystrophy, progressive lens degeneration, progressive lipodystrophy, childhood progressive muscular dystrophy, progressive myoclonic epilepsy, progressive osteodysplasia, progression Degenerative decubitus syndrome, progressive spinal medullary atrophy, progressive supranuclear palsy, progressive systemic sclerosis, progressive wall plate chorionic dystrophy, proline oxidase deficiency, propionic acidemia, type I propionic acidemia ( PCCA deficiency), type II propionic acidemia (PCCB deficiency), propionyl CoA carboxylase deficiency, first color weakness, first color blindness, tamper Loss of quality enteropathy, congestive heart failure secondary protein loss enteropathy, Proteus syndrome, including 4q proximal deletion, PRP, PRS, Pruneberry syndrome, PS, pseudo-fuller polydystrophy, pseudo -Polydystrophy, pseudo melanosis, pseudochondrogenic dysplasia, pseudocholinesterase deficiency, familial pseudogout, pseudohemophilic, pseudosemiyinyang, pseudosemiyinyang-nephron disorder-Wilms tumor, pseudohypertrophy Muscular dystrophy, pseudohypoparathyroidism, pseudohypophosphatasia, pseudopolydystrophy, pseudothalidomide syndrome, pseudoelastic fibroma xanthoma, psoriasis psospermatous follicular hyperplasia, PSP, PSS, psychomotor spasm , Psychomotor convulsions, psychomotor equivalent epilepsy, PTC deficiency, pterygium, pterygium syndrome, universal pterygium, pterygial lymphatic dilatation, pulmonary valve closure, pulmonary artery lymphangiomyoma, pulmonary stenosis , Pulmonary artery stenosis-ventricular septal defect, pulp stone, dental pulp dysplasia, pulseless disease, pure alymphocytosis, pure cutaneous histiocytosis, purine nucleoside phosphorylase deficiency, hemorrhagic purpura, Purtilo syndrome, PXE, Dominant PXE, Recessive PXE, Concentrated dysplasia, Concentrated dysostosis, Pycnopia, Pyroglutamic aciduria, Calcium pyroglutamate, Pyrroline carboxylase dehydrogenase deficiency, Pyruvate carboxylase deficiency, Group A pyruvate Carboxylase deficiency, group B pyruvate carboxylase deficiency, pyruvate decarboxylase deficiency, pyruvate kinase deficiency, q25-qter, q26, q27-qter, q31 or 32-qter, QT prolongation with extracellular hypocalcemia, congenital QT prolongation without deafness, QT prolongation with congenital deafness, cerebral limb failure paralysis, cerebral limb paralysis, Quantal Squander Quantal Squander, r4, r6, rl4, r18, r21, r22, dorsal spine, rib dysplasia-amegakaryocyte thrombocytopenia, rib dysplasia-thrombocytopenia, radial nerve palsy, sensory nerve root neuropathy, Recessive sensory root neuropathy, tooth root dysplasia, rapid onset ataxia- tremor, lap-Hodgkin syndrome, lap-Hodgkin ectodermal dysplasia syndrome, wrap-Hodgkin hypohidrosis ectoderm malformation, Rare hereditary ataxia with polyneuropathy changes and deafness caused by phytanate hydroxylase deficiency, Rautenstrauch-Wiedemann syndrome, Lautenstrauch-Wiedemann neonatal progeria, Lehno phenomenon, RDP, reactive functional hypoglycemia, reactive hypoglycemia secondary to mild diabetes, recessive Kenny-Café syndrome, reclin recessive congenital myotony, re Klinhausen's disease, rectal perineal fistula, recurrent vomiting, reflex neurovascular dystrophy, reflex sympathetic dystrophy syndrome, refractive error, refractory anemia, cold paralysis, refsum disease, refsum disease, localized enteritis, Reed-Barlow syndrome Leifenstein Syndrome, Leger Abnormal-Mental Retardation, Leger Syndrome, Lyman Cyclic Disease, Lyman Syndrome, Rice-Bucklers Corneal Dystrophy, Reiter Syndrome, Recurrent Giant-Barre Syndrome, Relapsing-Remitting Multiple Sclerosis, No kidney development, hereditary renal dysplasia-blind, Loken-Senior type renal dysplasia-renal dysplasia, renal diabetes, type A renal diabetes, type B renal diabetes, type O renal diabetes , Kidney-eye brain dystrophy, renal-retinal dysplasia with medullary cystic disease, familial kidney-retinal dystrophy, kidney-retinal syndrome, Randu-Austler-U -Barr syndrome, respiratory acidosis, respiratory chain disorder, respiratory myoclonus, restless leg syndrome, localized cardiomyopathy, stagnation hyperlipidemia, Rethore syndrome (discontinued word), reticulohypergenesis, retinal dysplasia-cyst Kidney-jubert syndrome, retinal cone degeneration, retinal cone dystrophy, retinal cone-rod dystrophy, retinitis pigmentosa, congenital deafness of retinitis pigmentosa, retinoblastoma, retinol deficiency, retina segregation, juvenile retina Separation, regression syndrome, retroocular neuropathy, retrophak syndrome, Rett syndrome, reverse aortic constriction, Reye syndrome, Reye syndrome, RGS, Rh blood factor, Rh disease, Rh factor incompatibility, Rh incompatibility, Rh factor incompatibility, rheumatic fever , Rheumatoid arthritis, rheumatoid myositis, nasal sinus cerebral arachnoiditis, limb root punctate cartilage dysplasia (RCDP), catalaseism, classic refsum disease, RHS, rhythmic myoclonus Rib-gap defect with micrognathia, Ribbing disease (obsolete), living disease, Richner-Hanhardt's disease, Rieger syndrome, Rieter syndrome, right ventricular fibrosis, Riley-Day syndrome, Riley- Smith syndrome, circular chromosome 14, circular chromosome 18, circular 4, circular chromosome 4, circular 6, circular chromosome 6, circular 9, circular chromosome 9 R9, circular 14, circular 15, circular chromosome 15 (mosaic pattern) , Ring 18, ring chromosome 18, ring 21, ring 21, ring 22, ring 22, chromosome, Ritter disease, Ritter-Rayer syndrome, RLS, RMSS, Roberts SC- seal limb syndrome, Roberts syndrome, Roberts limb seal Limb disease syndrome, Robertson's ectodermal dysplasia, Robin's malformation syndrome, Robin's sequelae, Robin's syndrome, Robinau dwarfism, Robinau's syndrome, dominant robinau syndrome, recessive robinau syndrome Somatic myopathy, Roger's disease, Robitansky's disease, Romano-Ward syndrome, Romberg syndrome, edentulous root teeth, Rosenberg-Chutorian syndrome, Rosewater syndrome, Roseelli-Gulienatti syndrome, Rothmund -Thomson syndrome, Lucy-Levy syndrome, RP, RS X-linkage, RS, RSDS, RSH syndrome, RSS, RSTS, RTS, congenital rubella, Rubinstein syndrome, Rubinstein-Thebi syndrome, Rubinstein-Thebi broad thumb -Toe syndrome, red dwarfism, rule syndrome, Russell's intercerebral cachexia, Russell syndrome, Russell syndrome, Russell-Silver dwarfism, Russell-Silver syndrome, X-linked Russell-Silver syndrome, Rubarkava-Myr (Ruvalcaba-Myhre) -Smith syndrome (RMSS), Rubarkava syndrome, Lubarka with mental retardation Type bone dysplasia, sacral retraction, no congenital sacrum, SAE, Sessre syndrome, Sakati, Sakachi syndrome, Sakachi-Nyhan syndrome, point spasm, salivary sweat gland paresis syndrome, Salzmann's nodular corneal dystrophy, Zandhoff disease , Sanfilipo Syndrome, Type A Sanfilipo, Type B Sanfilipo, Santavuori disease, Santavuori-Haltia disease, Beck sarcoid, sarcoidosis, sestle, Saturday night paralysis, SBMA, SC seal limb syndrome, SC syndrome, SCA 3, SCAD deficiency, adult-onset local SCAD deficiency, congenital systemic SCAD deficiency, SCAD, SCADH deficiency, burn-like skin syndrome, congenital scalp deficiency, ship-like skull, higher scapula, scapula myopathy, scapula Peroneal muscular dystrophy, myopathy scapula syndrome, scarring blisters, SCHAD, Schaumann's disease, Cheyre syndrome, Schereshevk ii) -Turner syndrome, Schilder's disease, Schilder's encephalitis, Schilder's disease, type I Schindler disease (infant onset type), infant onset type Schindler disease, Schindler disease, type II Schindler disease (adult onset type), Schinzel syndrome, Schinzel-Gedione Syndrome, synzel apical corpus callosum syndrome, synzel-geizion midfacial regressive syndrome, Schizencephaly, schizophrenia, Schmidt metaphyseal cartilage malformation, Schmidt metaphyseal osteogenesis, Schmidt-Fracaro syndrome, Schmidt syndrome , Schopf-Scharz-Passarge syndrome, Schuler-Christian disease, Schut-Haymaker, Schwarz-Jumper-Arberfeld syndrome, 1A and 1B Schwarz-Jumper syndrome, Schwarz-Jumper syndrome , Type 2 Schwarz-Jumpel Symptom group, SCID, scleroderma, familial progressive systemic sclerosis, diffuse familial encephalopathy, sciatic nerve contusion, Scott skull syndrome with mental retardation, scrotal tongue, SCS, SD, SDS, SDYS, Seasonal conjunctivitis, sebaceous nevus syndrome, sebaceous nevus, seborrheic keratosis, seborrheic wart, Zeckel syndrome, Zeckel dwarfism, second-degree congenital heart block, secondary amyloidosis, secondary Blepharospasm, secondary non-tropical sprue, secondary brown syndrome, secondary limbs, secondary systemic amyloidosis, secondary ataxia, secretory component deficiency, secretory IgA deficiency, late-onset SED, congenital Sex SED, SEDC, segmental linear colorless nevus, segmental ataxia, segmental myoclonus, Saipe syndrome, Zeiterberger disease, seizure, selective lack of IgG subclass, selective silence, selective lack of IgG subclass, selective IgM deficiency, selective mute, selective IgA deficiency, self Healing histiocytosis, hemilobular forebrain, vas deferens, senile retinal segregation, senile warts, senior-loken syndrome, type I hereditary sensory neuropathy, type II hereditary sensory neuropathy, type I heritability Sensory neuropathy, sensory root neuropathy, recessive sensory root neuropathy, septic progressive granulomatosis, septal-eye dysplasia, serous localized meningitis, serum protease inhibitor deficiency, serum carnosinase deficiency, setreis (Setleis) syndrome, severe combined immunodeficiency, severe combined immunodeficiency with adenosine deaminase deficiency, severe combined immunodeficiency (SCID), gender reversal, sexual childhood, SGB syndrome, Sheehan syndrome, Shields-type dentin formation Insufficiency, herpes zoster, varicella-zoster virus, sailor's beriberi, SHORT syndrome, short arm 18 deletion syndrome, short chain acyl-CoA dehydrogenase deficiency, short chain acyl-CoA dehydrogenase (SCAD) deficiency, short stature Dilated facial telangiectasia, short stature / skeletal abnormalities-delay-giant teeth, short stature-hyperextension-Leger anomaly-delayed dentition, short stature-nail dysplasia, telangiectatic erythema on short stature, SHORT syndrome , Shoshin beriberi, upper limb syndrome, Sprizen-Goldberg syndrome, Scharmann syndrome, Schwachmann-Baudian syndrome, Schwachmann-Diamond syndrome, Schwachmann syndrome, Schwachmann-Diamond-Oski syndrome, Schwachmann syndrome , Shy-Drager syndrome, Shy-Maggie syndrome, SI deficiency, sialidase deficiency, type I juvenile sialidosis, type II infantile sialidosis, sialidosis, sialolipidosis, sinus dysfunction syndrome, sickle cell anemia, sickle cell disease, sickle disease Erythrocyte-hemoglobin C disease, sickle cell-hemoglobin D disease, sickle cell-thalassemia Disease, sickle cell formation tendency, ironblastic anemia, ironblastic anemia, ironblast syndrome, SIDS, Siegel-Cattan-Mamou syndrome, Siemens-Bloch-type pigmented dermatitis, Siemens syndrome, Siewerling-Creutsfeld disease, Siewert syndrome, Silver syndrome, Silver-Russell dwarfism, Silver-Russell syndrome, Simmons disease, Simmons syndrome, simple epidermolysis bullosa, Simpson dysmorphic syndrome , Simpson-Gorabi-Bemel syndrome, Sinding-Larsen-Johanson syndrome, monofetal-Melten syndrome, sinus arrhythmia, venous sinus, sinus tachycardia, mermaid malformation sequelae, mermaid malformation, inverted bronchiectasis and Sinusitis, SJA syndrome, Sjogren-Larson syndrome ichthyosis, Sjogren syndrome, Sjogren syndrome, SJS, skeletal dysplasia, Weiss -Netter-stool (Stuhl) skeletal dysplasia, cutaneous detachment syndrome, skin neoplasia, cranial asymmetry and mild lag, cranial asymmetry and mild digitosis, SLE, sleep epilepsy, sleep apnea, SLO, Sly syndrome SMA, Infant Acute SMA, SMA I, SMA III, Type SMA, Type II SMA, Type III SMA, SMA3, SMAX1, SMCR, Smith-Lemli-Opitz Syndrome, Smith-Magenis Syndrome, Smith- Magenis chromosome region, Smith-McCort dwarfism, Smith-Opitz congenital syndrome, Smith disease, Smoldering myeloma, SMS, SNE, light exposure sneeze, valproate sodium, bone-isolated plasma cells Tumor, Sorsby disease, Sotos syndrome, Souques-Charcot syndrome, South African hereditary porphyria, spastic vocalization
Disability, spastic torticollis, spastic torticollis, spastic cerebral palsy, spastic colon, spastic vocal disorder, spastic paraplegia, SPD calcification, specific antibody deficiency with normal immunoglobulin, specific reading disorder, SPH2 , Spherocytic anemia, spherocytosis, spherical lens-short syndrome, sphingomyelin lipidosis, sphingomyelinase deficiency, spider finger, spielmeier-forked disease, spielmeier-forked-batten syndrome, spina bifida, spina bifida, spinal cord Arachnoiditis, Spinal arteriovenous malformation, Hereditary familial spinal ataxia, Spinal medullary muscular dystrophy, Spinal cord contusion, Spinal diffuse idiopathic skeletal hyperostosis, Spinal cord DISH, Spinal muscular atrophy, All types of spinal muscle Atrophy, ALS type spinal muscular atrophy, carve spinal muscular atrophy-overnutrition, type I spinal muscular atrophy, type III spinal muscular atrophy, type 3 spinal muscular atrophy, carve spinal muscle Constriction-Overnutrition, Spinal ossification arachnoiditis, Spinal stenosis, Spinocerebellar ataxia, Type I spinocerebellar atrophy, Type I spinocerebellar ataxia (SCA1), Type II spinocerebellar ataxia (SCAII), Type III Spinocerebellar ataxia (SCAIII), Type III spinocerebellar ataxia (SCA 3), Type IV spinocerebellar ataxia (SCAIV), Type V spinocerebellar ataxia (SCA V), Type VI spinocerebellar ataxia (SCA VI), type VII spinocerebellar ataxia (SCAVII), spirochete jaundice, splenic hypoplasia syndrome, splenic spleen, splenic sphenoid, tear deformity-mandibular facial bone dysplasia, split hand malformation, spondyloarthritis, vertebral rib malformation -I, late vertebral epiphyseal malformation, vertebral thoracic dysplasia, spinal caudal radiculopathy, spongy kidney, pleomorphic cavernoblastoma, spontaneous hypoglycemia, Sprengel deformity, spring catarrh, SRS, ST, fish rot odor syndrome, staphylococcal burn skin syndrome, Stargardt disease, startle disease, epilepsy status, steel- Richardson-Olszewsky syndrome, bristle disease, Stein-Levensall syndrome, Steinert disease, Stengel syndrome, Stengel-Batten-Meow-Spiele-Meier-Fork disease, stenotic cholangitis, lumbar canal stenosis, stenosis, Steroid sulfatase deficiency, Stevanovic ectodermal dysplasia, Stevens-Johnson syndrome, STGD, Stickler syndrome, Stiff-Man syndrome, Stiff-Person syndrome, Still's disease, Stilling-Churck-Duen syndrome, Stiris (Stillis) disease, stimulus-sensitive myoclonus, Stone-Man syndrome, Stoneman, Streator abnormality, autosomal dominant striatal nigra degeneration, striatal acne bulb dentin calcification, stroma, desmede membrane, Interstitial corneal dystrophy, lymphoma thyroid , Sturge-Carlishire-Weber syndrome, Sturge-Weber syndrome, Sturge-Weber nevus, subacute necrotizing encephalomyelopathy, subacute spongiform encephalopathy, subacute necrotizing encephalopathy, subacute sarcoidosis, subacute neuropathic aorta Inferior stenosis, subcortical atherosclerotic encephalopathy, subendocardial sclerosis, succinylcholine sensitivity, congenital sucrase-isomaltase deficiency, congenital sucrose-isomaltose malabsorption, congenital sucrose intolerance, sudan-favorable leukodystrophy ADL , Pelizaeus-Merzbacher Sudan-favored leukodystrophies, Sudan-favorable leukodystrophies, Sudden infant death syndrome, Zudeck atrophy, Sugi-Kajii syndrome, Summerskill syndrome, Summit cuspid fingers, Schmidt Apical syndactyly, Schmidt syndrome, superior oblique tendon sheath syndrome, suprarenal Gland, supravalvular aortic stenosis, supraventricular tachycardia, dementia-heart syndrome, dementia-heart syndrome, SVT, sweat gland abscess, sweat gland taste syndrome, sweet syndrome, Swiss cheese cartilage syndrome, synaptic cephalopathy, microcephaly Type I digitosis with psychiatric retardation, symptomatic hepatic tubule dysplasia, syringomyelia, systemic white blood reticuloendotheliosis, systemic amyloidosis, systemic carnitine deficiency, systemic elastic fiber rupture, systemic erythema Lupus, systemic mastocytosis, systemic mastocytosis, systemic onset juvenile arthritis, systemic sclerosis, Systopic spleen, T-lymphocyte deficiency, rapid nutritional hypoglycemia, tachycardia, plateau Syndrome, Takayasu disease, Takayasu arteritis, footpad, adductor foot, cusp, adductor foot, clubfoot, tandem spinal stenosis, tanger disease, wall plate retinal degeneration, TAR syndrome, late ataxia, late onset Muscular dystrophy, late-onset dyskinesia, late-onset oral dystrophy Neszy, late ataxia, delayed ulnar nerve palsy, target cell anemia, tarsal hypertrophy, Talli disease, TAS midline deficit, TAS midline deficit, tysax sphingolipidosis, tysax disease, tay syndrome ichthyosis, Tesax sphingolipidosis, Tay syndrome ichthyosis, type I Tabi syndrome, Tabe syndrome, TCD, TCOF1, TCS, TD, TDO syndrome, TDO-I, TDO-II, TDO-III, telangiectasia, with associated abnormalities Ocular sequestration, Ocular sequestration-hypospadias syndrome, temporal lobe epilepsy, temporal arteritis / giant cell arteritis, temporal arteritis, TEN, superior oblique tendon sheath adhesion, tonic myalgia, 4q end deletion Including Terien corneal dystrophy, Teschler-Nikola-Killian syndrome, tethered spinal cord syndrome, tethered spinal cord malformation, tethered spinal cord syndrome, tethered cervical spinal cord syndrome, tetrahydrobiopterin deficiency, tetrahydrobiopte Deficiency, tetralogy of Fallot, limb seal limb-thrombocytopenia syndrome, chromosome 9 short arm tetrasomy, tetrasomy 9p, chromosome 18 short arm tetrasomy, thalamus syndrome, thalamic pain syndrome, thalamic hypersensitivity perception, intermediate thalassemia , Thalassemia minor, thalassemia major, thiamine deficiency, thiamine responsive maple syrup urine disease, thin basement membrane neuropathy, thiolase deficiency, RCDP, acyl-CoA dihydroxyacetone phosphate acyltransferase, third and fourth pharyngeal sac syndrome, third Congenital (complete) heart block, Tomsen's disease, thoracic pelvic-phalangeal dystrophy, thoracic spinal canal, thoracoabdominal syndrome, thoracoabdominal shift heart syndrome, 3M syndrome, 3M microosteopathia, Grantsman and Negeri's platelets Asthenia, essential thrombocythemia, thrombocytopenia-rib deficiency syndrome, thrombocytopenia -Hemangioma syndrome, thrombocytopenia-rib deficiency syndrome, hereditary thrombosis tendency due to AT III, thrombotic thrombocytopenic purpura, thromboulcerative colitis, thymic malformation with normal immunoglobulin, no thymus, di George type thymic dysplasia, thymic hypoplasia including primary agammaglobulinemia, DiGeorge type thymic dysgenesis, congenital thymic dysplasia, painful tic, tic, Tinel syndrome, Tolosa-Hunt syndrome, ankylosing spasticity Neck, tension pupil syndrome, tooth-nail syndrome, torch infection, TORCH syndrome, torsion ataxia, torticollis, total lipodystrophy, pulmonary venous connection abnormalities, Touraine aphthosis, Tourette syndrome, Tourette disorder, Towns- Brox syndrome, Towns syndrome, toxic paralytic anemia, toxic epidermal necrosis, diaphyseal tibia-fibula toxic hypertrophic ossification, toxic hypertrophic ossification, other substance-like Cytomegalovirus herpes simplex virus toxoplasmosis, tracheoesophageal fistula with or without esophageal closure, tracheoesophageal fistula, transient neonatal myasthenia gravis, transient atrioventricular septal defect, aortic inversion, transtelephonic Monitoring, transthyretin methionine-30 amyloidosis (type I), rhomboid encephalopathy-multiple bone fusion syndrome, trecher-collins syndrome, trecher-collins-french shetty syndrome 1, trebol disease, sanbo heart, hair teeth Bone syndrome, dentate syndrome, gray hair dystrophy, hair nose and phalanx syndrome, hair nose and phalanx syndrome, tricuspid valve closure, trifunctional protein deficiency, trigeminal neuralgia, triglyceride storage disease long chain fatty acid oxidative disorder, triangularitis , Triangular skull disease, triangular skull syndrome, triangular skull “C” syndrome, trimethylamineuria, thumb Finger dystrophy-stunted distal phalange-nail dystrophy, thumb triode syndrome, Behcet's trisymptom syndrome, triple X syndrome, triplo X syndrome, triploid syndrome, triploid, triploid syndrome, opening Disorder-Pseudo flexion syndrome, trisomy, trisomy G syndrome, trisomy X, partial trisomy 6q, partial trisomy 6q syndrome, trisomy 9 mosaic, trisomy 9P syndrome (partial), partial trisomy 11q, trisomy 14 mosaic, trisomy 14 Mosaic syndrome, Trisomy 21 syndrome, Trisomy 22 mosaic, Trisomy 22 mosaic syndrome, TRPS, TRPS1, TRPS2, TRPS3, True semi-yin-yang, Common arterial trunk, Tryptophan malabsorption, Tryptophan pyrolase deficiency, TS, TTP, TTTS Tuberculosis, tubule dilatation, Turcot syndrome, Turner syndrome, Turner-Kieser syndrome, Turner phenotype with normal chromosomes (karyotype), Turner-Burney syndrome, tower skull, twin-to-twin transfusion syndrome, twin-to-twin transfusion syndrome, type A, type B, AB, type O, type I diabetes, type I family Sexually Incomplete Male, Type I Familial Incomplete Male False Semi-Yin Yang, Type I Gaucher Disease, Type I (PCCA Deficiency), Type I Tyrosineemia, Type II Gaucher Disease, Type II Histiosphere Syndrome, Type II (PCCB Deficiency) ), Type II tyrosineemia, multiple congenital type IIA distal joint contractures, type III Gaucher disease, type III tyrosineemia, type III dentinogenesis, typical retinal sequestration, tyrosinase-negative albinism ( Type I), tyrosinase-positive white skin syndrome (type II), type 1 acute tyrosinemia, type 1 chronic tyrosinemia, tyrosine disease, UCE, ulcerative colitis, chronic non-specific ulcerative colitis, ulna -Breast syndrome, Parister ulna-Breast syndrome, Ulnar nerve palsy, UMS, Unclassified FOD, Uncoupled benign bilirubinemia, accessory Hypogonadism, unilateral ichthyosis-like erythroderma with unilateral leg malformation, unilateral chondromatosis, unilateral deficiency of the pectoralis muscle and hand fingers, unilateral hemigenesis, unilateral macrocephaly Fat dystrophy, unilateral renal failure, unstable colon, Umbrellicht disease, Umfelricht-Lundborg disease, Umfelricht-Lundborg-Rough disease, Umfellich syndrome, upper extremity-cardiovascular syndrome (Halt-Aurum type) ), Upper motor neuron disease, upper respiratory apnea, urea cycle deficiency or disorder, arginase urea cycle disorder, arginosuccinase urea cycle disorder, carbamyl phosphate synthetase urea cycle disorder, citrullinemia urea cycle disorder, N-acetylglutamate synthetase type urea cycle disorder, OTC type urea cycle disorder, urethral syndrome, urethral-eye-joint syndrome, severe Differentiated type I uridine diphosphate glucuronosyltransferase, urinary tract defect, urofacial syndrome, uroporphyrinogen III cosynthase, pigmented urticaria, Usher syndrome, type I asher, type II asher, type III asher, type IV Usher, uterine adhesion, uroporphyrinogen I-synthase, uveitis, uve meningitis syndrome, V-CJD, VACTEL-related, VACTERL-related, VACTERL syndrome, rib valgus, valine transaminase deficiency, valineemia, Valproic acid exposure, Valproic acid exposure, Valproic acid, Van Buren's disease, Van der Huve-Habertsma-Wardenburch-Gauldi syndrome, Mutant immunoglobulin deficient hypogammaglobulinemia, Mutant Creutzfeldt -Jakob disease (V-CJD), varicella embryo disorder, atypical porphyria, Binswanger type vascularity Diagnosis, Vasodilatory tumor, Vascular hemophilia, Vascular malformation, Cerebral vascular malformation, Vasculitis, Vasomotor ataxia, Vasopressin-resistant diabetes insipidus, Vasopressin-sensitive diabetes insipidus, VATER-related, Vcf syndrome, Vcf , Palatal and facial syndrome, palatal and facial syndrome, gender-related arthritis, venous malformation, ventricular fibrillation, ventricular septal defect, congenital ventricular defect, ventricular septal defect, ventricular tachycardia, venous malformation, VEOHD, insect Stunted dysfunction, cerebellar insufficiency, spring catarrh, verruca, vertebral anal tracheoesophageal esophageal rib, vertebral ankylosing osteoproliferative disease, early early stage Huntington's disease, very long chain acyl-CoA dehydrogenase (VLCAD) deficiency, vestibular nerve sheath Tumor, vestibular schwannoma neurofibromatosis, vestibular cerebellar, phyllophyllocephalus, visceral xanthogranulomatosis, visceral xanthogranulomatosis, visceral myopathy-extraocular muscle palsy, visceral giant disease-umbilical hernia- Big tongue syndrome, visual amnesia, vitamin A deficiency Group, vitamin B-1 deficiency, yolk-neck macular dystrophy, vitiligo, head vitiligo, vitreous retinal dystrophy, VKC, VKH syndrome, VLCAD, forked syndrome, forked brain syndactyly, forked-Koyanagiharada syndrome, von Bechteref- Strumpel syndrome, congenital von Eulenburg paramyotonia, von Frey syndrome, von Gierke disease, von Hippel-Lindau syndrome, von Michlic syndrome, von Recklinghausen disease, von Willebrand disease, VP, Floric disease (Type II), VSD, vulgaris keratosis, ichthyosis vulgaris, W syndrome, Waardenburg syndrome, Waardenburg-Crane syndrome, Waardenburg syndrome type I (WS1), Waardenburg syndrome type II (WS2), Waardenburg type IIA Syndrome (WS2A), type IIB Waldenburg syndrome (WS2B), I Type II Waldenburg syndrome (WS3),
Type IV Waldenburg syndrome (WS4), Weelsch syndrome, WAGR complex, WAGR syndrome, Waldenstrom macroglobulinemia, Waldenstrom purpura, Waldenstrom syndrome, Waldman disease, Walker -Walburg syndrome, migratory spleen, Wallburg syndrome, warm antibody hemolytic anemia, warm reaction antibody disease, Wartenberg syndrome, WAS, brain water, Watson syndrome, Watson-Arazil syndrome, Waterhouse-Friedersensen syndrome, low Degenerative Disease, WBS, Weaver Syndrome, Weaver-Smith Syndrome, Weber-Cokane Syndrome, Wegener's Granulomatosis, Weil's Disease, Weil Syndrome, Weil-Marquesani, Weil-Marquesani Syndrome, Weil-Rey Syndrome, Weissmann-Netter-Stool Syndrome, weissenbach Weissenbacher-Zweymuller Syndrome, Wells Syndrome, Wenkebach, Werdnig-Hoffmann Disease, Werdnich-Hoffmann Paralysis, Werhlhof Disease, Werner Syndrome, Wernicke (C) I Syndrome, Wernicke Aphasia, Wernicke-Korsakoff Syndrome, West Syndrome, Wet Legs, WHCR, Whipple's Disease, Whipple's Disease, Whistling Facial Syndrome, Whistling Facial-Wind Pane Syndrome, White-Darier's Disease, Whitnal-Norman Syndrome, Spiral Nevus-Like Hypermelanosis, WHS, Wiercker syndrome, Wierker syndrome, Wierker-Wolf syndrome, Wiedmann-Beckwith syndrome, Wiedemann-Rautenstrauch syndrome, Wildervank syndrome, Willebrand-Juergens disease, Villi-Prder syndrome Williams Syndrome, Williams-Buren Syndrome, Wilms Tumor, Wilms Tumor-Iris-Gonadoblastoma-Mental Retardation Syndrome, Wilms Tumor-Iris Urogenital Abnormality-Mental Retardation, Wilms Tumor-Iris-Gonioblastoma-Mental Retardation Syndrome, Wilms tumor pseudo-half yin-yang-nephropathy, Wilms tumor and pseudo-half yin-yang, Wilms tumor-pseudo-yin-yang-glomeropathy, Wilson disease, Winchester syndrome, Winchester-Grossman syndrome, Viscot-Oldrich syndrome, Viscot-Oldrich Type immunodeficiency, baldness ectodermal dysgenesis, baldness tooth-nail syndrome, witmark-ekbum syndrome, WM syndrome, WMS, WNS, wolfard's disease, wolfalt-kugelberg-verandel disease, wolf syndrome, wolf-hirschhorn (Hirschhorn) Chromosome region (WHCR), Wolf Rushhorn syndrome, Wolf-Parkinson-White syndrome, Wolfram syndrome, Wolman disease (lysosomal lipase deficiency), Woody Guthrie disease, WPW syndrome, writer's cramp, WS, WSS, WWS, Wyvern-Mason syndrome, X-linked Addison disease, X-linked adrenal white matter dystrophy (X-ALD), X-linked adult spinal medullary muscular atrophy, X-linked adult spinal muscular atrophy, X-linked agammaglobulinemia with growth hormone deficiency, X-linked gamma globulinemia, lymphoproliferative X-linked syndrome, X-linked cardiomyopathy and neutropenia, X-linked core myopathy, X-linked copper deficiency, X-linked copper malabsorption, X-linked dominant Conrady -Hünermann syndrome, X-linked dominant hereditary corpus callosum, X-linked ataxia- tremor, X-linked ichthyosis, X-linked infantile agammaglobulinemia, X-linked infant necrotic encephalopathy, X- Linked juvenile retinal separation, X-linked brain Times deficit, X-linked lymphoproliferative syndrome, X-linked mental retardation-sick thumb syndrome, X-linked mental retardation with hypotension, X-linked mental retardation and testicular testis, X-linked progressive complex variant Immunodeficiency, X-linked recessive Conrady-Hünermann syndrome, X-linked recessive severe combined immunodeficiency, X-linked retinal segregation, X-linked spinal epiphyseal dysfunction, xanthine oxidase deficiency (hereditary xanthineuria deficiency), hereditary Xanthine urine deficiency (xanthine oxidase deficiency), systemic xanthogranulomatosis, nodular xanthoma, xeroderma pigmentosum, dominant type xeroderma pigmentosum, AI XPA classic xeroderma pigmentosum, B II XPB type pigment Xeroderma pigmentosum, EV XPE type xeroderma pigmentosum, C III XPC type xeroderma pigmentosum, D IV XPD type xeroderma pigmentosum, F VI XPF type xeroderma pigmentosum, G VII XPG type pigmented xerosis Xeroderma, XP-V variant xeroderma pigmentosum, xerosis-clubfoot and enamel deficiency, xeroderma idiopathic, xerophthalmia, dryness Keratitis, XLP, XO syndrome, XP, XX male syndrome, gender reversal, XXXXX syndrome, XXY syndrome, XYY syndrome, XYY chromosome pattern, yellow mutated albinism, yellow onychoderma, YKL, arteritis in young women Yunis-Varon syndrome, YY syndrome, ZE syndrome, Z- and protease inhibitor deficiency, Zellweger syndrome, Zellweger cerebral liver and kidney syndrome, ZES, Chien-Oppenheim disease (twisted ataxia), Escherichia coli, yeast to treat alone or in combination with another drug for conditions including Timmermann-Levant syndrome, congenital zinc deficiency, Jinser-Cole-Engman syndrome, ZLS, Zollinger-Ellison syndrome Or a protein or chimeric molecule expressed by a non-human cell line, such as a protein or chimeric molecule expressed by CHO It can be used alone or in combination with other drugs or therapy.

  In another embodiment, the pharmaceutical composition comprising isolated IFN-a2B or a chimeric molecule thereof is hairy cell leukemia, malignant melanoma, follicular lymphoma, acupuncture condylome (afflicts the outer surface of the genitals and anal area) , AIDS-related Kaposi's sarcoma, chronic hepatitis B and C, non-Hodgkin lymphoma, renal cell carcinoma, ovarian cancer, pancreatic cancer, carcinoid, glioma, chronic myelogenous leukemia, mesothelioma, head and neck cancer (hypopharyngeal cancer , Laryngeal cancer, oral and oral cavity cancer, oropharyngeal cancer), T-cell leukemia, lymphoma, T-cell lymphoma, mantle cell lymphoma, progressive multifocal diffuse small lymphocyte / marginal lymphoma, follicular nucleus Cut small cell lymphoma, follicular mixed cell lymphoma, cutaneous T-cell lymphoma (mycosis fungoides, Sezary syndrome), liver cancer, unspecified solid tumor (e.g., advanced, metastatic, unresectable) Good) Including tumors and chronic viral infections; multiple myeloma, gastrointestinal tumors, esophageal cancer, meningioma, small cell lung cancer, HIV-related cancer in children or adults, bladder cancer, lymphoma, malignant tumor after immunosuppression, chronic lymphocytes Leukemia; anal intraepithelial neoplasia (AIN) / squamous intraepithelial lesion (SIL), nephroblastoma, neuroblastoma, leukoencephalopathy, lymphomatoid granulomatosis, temporal fossa fibromatosis, Hemangioma, giant cell tumor of bone, carcinoma in situ, Zollinger-Ellison syndrome, non-B islet cell carcinoma, essential thrombocythemia, hepatitis A, hepatitis B, hepatitis C, hepatitis D, SARS, West Nile HIV, sclerosing panencephalitis, herpesvirus 8, hand-foot-and-mouth disease, Mediterranean fever; atopic disease (eg, atopic dermatitis, atopic eczema, hyperimmunoglobulin E syndrome (HIES)), asthma, allergy including nasal mucosa Allergic diseases including type 1 diabetes mellitus Autoimmune diseases such as multiple sclerosis; endometrioid cysts, systemic amyloidosis, Waldenstrom's macroglobulinemia, prevention of cognitive decline in Alzheimer's disease, Churg-Strauss syndrome, and unregulated proinflammatory cytokine responses To treat various diseases or indications, including but not limited to treatment of infections that can be harmful (eg, Japanese encephalitis); systemic lupus erythematosus, or other Biology, drugs (eg, didanosine, AZT, ribavirin, viramidine, zidovudine, dideoxycytidine, antiviral agents including statin molecules) or treatment (eg, in surgery, radiation therapy, vaccine therapy, hyperthermia, peripheral stem cell transplantation; conventional Chemotherapy, anthracycline chemotherapy treatment, cisplatin, strept Other combination chemotherapy including zocine, doxorubicin, fluorouracil, mitomycin C, busulfan, oblimersen, etoposide, vincristine, cyclophosphamide, gencitabine, paclitaxel, taxol, cytarabine; IFN-b, IFN-g, IL-2, TNF -α, IL-12, GM-CSF, sagramostim, BCG, thalidomide, imatinib mesylate, bevacizumab, filgrastim, dexamethasone, isoretinoin, treretinoin, SU011248, treatment with Gleevec) Can be used.

  In another embodiment, the pharmaceutical composition comprising isolated IFN-b1 or a chimeric molecule thereof is a head and neck squamous cell carcinoma, breast cervical cancer, malignant melanoma, primitive neuroectodermal tumor, hepatocellular carcinoma, carcinomatosis , Lung cancer, brain metastasis, renal cell carcinoma, glioma, pleural mesothelioma or malignant pleural effusion, condylopodia, chronic active hepatitis B virus (HBV), hepatitis C virus (HCV), encephalitis virus Infectious diseases (e.g., West Nile virus), multiple sclerosis (MS), including relapsing-remitting MS, cytomegalovirus CMV, hand-foot-and-mouth disease, progressive leishmaniasis, SARS, viral infections that cause myocarditis, Yuze, herpesvirus 6, acute attack, ulcerative colitis, HIV, AIDS-related Kaposi's sarcoma, HTLV-1-related myelopathy (HAM), adrenoleukodystrophy, chronic inflammatory demyelinating polyradiculoneuropathy (CIDP) ), Bone marrow failure, optic neuritis, malnutrition , And Guillain - alone in the treatment of Barre syndrome, or other biological, can be used in combination drug, or treatment with. In another embodiment, a pharmaceutical composition comprising isolated IFN-b1 or a chimeric molecule thereof can be used in combination with EPO for the treatment of MS.

  In another embodiment, the pharmaceutical composition comprising isolated IFN-g or a chimeric molecule thereof comprises pulmonary fibrosis, including idiopathic pulmonary fibrosis; asthma; marble bone disease; systemic fungal infection, tuberculosis, leprosy, Mycobacterium avium, human papilloma virus (HPV), chronic hepatitis C (HCV), hepatitis B virus (HBV), chronic hepatitis B virus fibrosis, HIV, AIDS-related Kaposi's sarcoma-related acute crypt Cox meningitis, chronic granulomatous disease (CGD) and related bacterial and fungal diseases, aspergillosis or other fungal fungal infections, cryptococcal meningitis, chlamydial infections, bacteria including leishmaniasis, fungi And viral infections; atopic dermatitis, cystic fibrosis, airway sequelae after chronic bronchitis, Churg-Strauss syndrome (CSS), multiple sclerosis, rheumatoid arthritis, small cell lung cancer, malignant pleural mesothelioma (MP M), melanoma, gastric cancer, ovarian cancer, peritoneal cancer, renal cell carcinoma, colorectal cancer, non-Hodgkin lymphoma and other solid tumors, unexplained pulmonary fibrosis, granulomatous loose skin multiple myeloma and leukocyte adhesion It can be used alone or in combination with other biology, drugs (eg, statin molecules) or therapy in the treatment of diseases such as deficiency syndrome.

  In yet another embodiment, a pharmaceutical composition comprising isolated IFN-g or a chimeric molecule thereof is used alone or in other treatments for the treatment of surgical patients with reduced cellular immunity after various injuries. It can be used in combination with biology, drugs, or treatment.

  In another embodiment, the pharmaceutical composition comprising the chimeric molecule such as isolated IFNAR2 or IFNAR2-Fc is a chronic viral infection, hairy cell leukemia, malignant melanoma, follicular lymphoma, AIDS-related Kaposi's sarcoma, or To enhance the therapeutic effect of IFNα in the treatment of chronic hepatitis B and C; treatment of viral infections such as chronic active hepatitis B, hepatitis C virus, cytomegalovirus, and HIV, and the head and neck IFNβ in the treatment of squamous cell carcinoma, breast and cervical cancer, malignant melanoma, primitive neuroectodermal tumor, hepatocellular carcinoma, carcinomatosis, lung cancer, brain metastasis, renal cell carcinoma, glioma, and pleural mesothelioma To enhance therapeutic efficacy; in the treatment of infections with hepatitis C virus, parvovirus, and other viruses, and to treat ovarian cancer, melanoma, myeloid leukemia, and other cancers To enhance the therapeutic effect of IFNω as an anti-tumor agent to treat; treatment of IFNτ in the treatment of HIV-1 and papillomavirus, autoimmune diseases such as multiple sclerosis, and to prevent allergic sensitization To enhance the effect; to enhance the therapeutic effect of IFNκ in the treatment of infection with encephalomyocarditis and other viruses; or in the treatment of infection with hepatitis virus, herpes simplex virus, encephalomyocarditis virus, and To enhance the therapeutic effect of IFNζ in the treatment of certain cancers such as renal cell carcinoma and myeloid leukemia, it can be used alone or in combination with other biology, drugs or therapies.

  In yet another embodiment, a pharmaceutical composition comprising isolated IFNAR2 or a chimeric molecule thereof is used in place of a type 1 interferon or in combination with a type 1 interferon in the diseases described herein. For use, it can be used as a type 1 interferon agonist alone or in combination with other biology, drugs, or treatments.

  In yet another embodiment, a pharmaceutical composition comprising isolated IFNAR2 or a chimeric molecule thereof is type 1 with treatment of autoimmune hepatitis, lupus, systemic sclerosis, psoriasis, atopic dermatitis, and diabetes mellitus. To prevent allogeneic rejection after diabetes and bone marrow transplantation, it can be used as an antagonist or inhibitor of type 1 interferon alone or in combination with other biology, drugs or treatments.

  In another embodiment, the pharmaceutical composition comprising isolated IL-10 or chimeric molecule thereof is used in the treatment of diseases including cancer, autoimmunity and chronic inflammation, e.g., the therapeutic composition is persistent against antigens and stimulators. And preventing excessive immune responses; autoimmune diseases (including thyroiditis, type 1 diabetes, inflammatory bowel disease (eg Crohn's disease), rheumatoid arthritis and psoriasis), allergic contact dermatitis, transplantation Rejection, GVHD, HCV infection, ulcerative colitis, viral infections including HIV; Wegener's granulomatosis; pancreatitis; acute pancreatitis due to endoscopic retrograde cholangiopancreatography (ERCP); vascular injury; CNS disease, multiple It can be used alone or in combination with other biology, drugs, or therapies in the treatment of seizure symptoms in multiple sclerosis, Alzheimer's disease, and meningitis. Furthermore, the therapeutic compositions of the present invention are born to mothers with intrauterine infections to inhibit mast cells, to protect against acute myocarditis, to promote the survival of neurons and glial cells. To provide neuroprotection to infants, to prevent autoimmune diseases including thyroiditis and type 1 diabetes, as an antithrombotic agent, and to prevent necrosis and fibrotic liver injury, alone or It can be used in combination with other drugs or treatments.

  In another embodiment, the pharmaceutical composition comprising isolated IL-10Ra or a chimeric molecule thereof comprises various types of cancer (such as melanoma, carcinoma, lymphoma); eosinophilia, Sezary syndrome; erythema Lupus, systemic lupus erythematosus; systemic sclerosis; bullous disease; rheumatoid arthritis; atopic dermatitis; viral infections; and in the treatment of diseases including trauma, alone or other biology, drugs Or it can be used in combination with treatment.

  In yet another embodiment, a pharmaceutical composition comprising isolated IL-10Ra or a chimeric molecule thereof is mediated by cytokines to initiate antitumor effects in cancers that overexpress IL-10, such as B-cell lymphoma. To initiate reversal of tumor-induced monocyte deactivation, to reduce susceptibility to secondary bacterial pneumonia due to excessive IL10 production and reduced neutrophil function in the lung, self in graft-versus-host disease Can be used alone or in combination with other biology, drugs or treatments to prevent immune-mediated liver lesions, to enhance delayed-type hypersensitivity, It can be a useful strategy to promote effectiveness.

  However, the pharmaceutical compositions of the present invention have higher pharmacological efficacy, increased thermal stability, increased serum half-life, or compared to a protein or chimeric molecule thereof expressed in a non-human cell line, or Has higher solubility in the bloodstream. The present invention also shows a reduction in the risk of immune related clearance or related side effects. Because of these improved properties, the compositions of the invention can be administered fewer times than proteins or chimeric molecules expressed in non-human cell lines. A reduction in the number of doses is expected to increase patient compliance and thereby improve treatment outcome. The patient's quality of life increases as well.

  Thus, in one embodiment, a pharmaceutical composition of the invention can be administered to a patient in a therapeutically effective amount in the same manner that a protein or chimeric molecule expressed in a non-human cell line is administered. A therapeutic amount is that amount of the composition necessary for the desired in vivo activity. The exact amount of composition administered is a matter of selectivity affected by factors such as the exact type of condition being treated, the condition of the patient being treated and other ingredients in the composition. A pharmaceutical composition comprising an isoform or chimeric molecule of the protein of the invention is formulated with efficacy effective for administration by various means to a human patient experiencing one or more of the above disease states. Also good. The average therapeutically effective amount of the composition can vary. Effective amounts are expected to be in the range of 0.1 ng / kg body weight to 20 μg / kg body weight, or based on the recommendations and prescriptions of a qualified physician.

  The invention further extends to the use of isolated proteins or chimeric molecules comprising at least a portion of the protein or chimeric molecule thereof, and compositions comprising them in a variety of therapeutic and / or diagnostic applications.

  More particularly, the present invention involves administering to a mammalian subject an effective amount of an isolated protein, protein, fragment thereof, chimeric molecule thereof comprising an extracellular domain, or composition comprising an isolated protein or chimeric molecule. To a method of treating or preventing a condition in a mammalian subject that can be improved by increasing the amount or activity of a protein or chimeric molecule of the present invention.

  The invention is further illustrated by the following non-limiting examples.

Examples Example 1 Preparation of vector-Fc constructs
(a) Preparation of DNA construct that expresses Fc 22) was amplified from an EST cDNA library (clone ID 6277773, Invitrogen) by polymerase chain reaction (PCR). This amplicon was cloned into the corresponding enzyme site of pIRESbleo3 (Catalog Number 6989-1, BD Biosciences) to create the construct pIRESbleo3-Fc. Digestion of pIRESbleo3-Fc with BamH1 and BstX1 released an insert of the expected size of 780 bp as determined by gel electrophoresis.

(b) Preparation of DNA construct expressing protein A DNA sequence encoding a protein was amplified from an EST cDNA library by PCR using a forward primer and a reverse primer incorporating restriction enzyme sites described in Table 8. After amplification, the amplicon was digested with appropriate restriction enzymes and cloned into the expression vectors listed in Table 8 to make a vector-protein construct. Digestion of the vector containing the DNA sequence encoding the protein with the appropriate restriction enzymes released the expected size fragments shown in Table 8. The vector-protein construct was sequenced to confirm the integrity of the cloning procedure described herein.

(c) Megaprep vector-protein preparation Sterile LB broth containing ampicillin (100 μg / ml) was inoculated with 750 μl of an overnight culture of vector-protein transformed E. coli. The culture was incubated for 16 hours at 37 ° C. with infiltration. Plasmids were prepared according to the Qiagen Endofree Plasmid Mega Kit (Qiagen Mega Prep Kit # 12381).

Table 8: Protein-Fc and related cloning information

  Alternatively, a restriction site that allows the DNA sequence encoding the protein to be cloned upstream of the Fc nucleotide sequence in the vector-Fc so that the nucleotide sequence of the protein and Fc is fused directly or in-frame with a linker. The incorporated primer can also be used to amplify the nucleotide sequence of a protein cloned into a vector (such as pIRESbleo3 or pCEP4).

Example 2
(a) Production, isolation and purification of IFN-a2b of the present invention
(i) Production of IFN-a2b of the present invention
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells are seeded in 90 ml Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) per plate, and the medium contains 10% (v / v) heat-inactivated calf fetal serum ( FCS, JRH Biosciences), 10 mM HEPES (Sigma), 4 mM L-glutamine (Amresco), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, streptomycin sulfate 5000 μg / ml) (JRH Biosciences) was added. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was washed with fresh DMEM / with 10% (v / v) heat inactivated FCS, 10 mM HEPES, 4 mM L-glutamine and 1% (v / v) penicillin-streptomycin. Exchanged to 120 ml F12. The calcium phosphate / DNA precipitate is prepared so that 1200 μg of pIRESbleo3 (Invitrogen) plasmid DNA carrying the gene of human IFN-a2b and 3720 μl of CaCl ₂ (2.5 M) to a final volume of 30 ml (solution A) in sterile H ₂ O Prepared by adding. Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 20 minutes and then mixed with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml of DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine, 4.1 g / L mannose (Sigma) , 15 mM HEPES, 1% (v / v) penicillin-streptomycin and ITS solution (5 mg / L bovine insulin, 5 mg / L partially iron-saturated human transferrin, and 5 μg / ml selenium) (Sigma) fresh 100 ml of serum-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 4.1 g / L mannose, 15 mM HEPES, 1% (v / v) penicillin-streptomycin And 100 ml of fresh serum-free DMEM / F12 medium supplemented with ITS solution was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

  On day 4, cell culture supernatant was collected. After adding 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) to the collected cell culture supernatant and combining with the collected material on the third day, 0.45 μm low molecule Fine particles were removed using a protein binding filter (Durapore, Millipore). The mixture was stored at -70 ° C or used immediately.

(ii) Isolation and purification of IFN-a2b of the present invention A dye-ligand chromatography (DLC) process was used as the first step in IFN-a2b purification. A library of immobilized reactive dyes was used to screen IFN-a2b for efficient binding and release in a batch purification microtiter format. Appropriate dye-protein and pH combinations were tested in a small column format.

For bulk scale, DLC reactive dye # 18 High (Zymatrix) was selected as the reactive dye with the best binding and elution properties of IFN-a2b. Filtered cell culture supernatant is applied to 4.0 ml or 8.0 ml column bodies (Alltech, Extract Clean Filter columns) with 3 ml or 6 ml DLC resin pre-equilibrated to pH 6 with 50 mM MES / 5 mM MgCl ₂ , respectively. Passed by gravity flow. Bulk flow-through samples were stored at 4 ° C. until the ELISA results confirmed that the purification was successful. The column was washed with buffer A (20 mM MES / 5 mM MgCl ₂ , pH 6) until the fractions appeared clear. Three elution buffers were used in the following order to elute IFN-a2b.
Eluent 1: Buffer C (50 mM Tris-Cl / 10 mM EDTA pH 8)
Eluent 2: EN 1.0 (50 mM Tris-Cl / 10 mM EDTA / 1.0 M NaCl pH 8)
Eluent 3: EN 2.0 (50 mM Tris-Cl / 10 mM EDTA / 2.0 M NaCl pH 8)

  Eluted fractions were assayed by silver-stained SDS PAGE using NuPAGE MES SDS running buffer (Invitrogen) and by anti-IFN-a2b ELISA (Bender MedSystems) using 4-12% NuPAGE Bistris gels. IFN a2b was found to bind to the reactive dye 18 High and was found to elute in buffer EN1.0 and buffer EN2.0. DLC fractions containing IFN-a2b were pooled for size exclusion chromatography.

  Size exclusion chromatography was performed on the combined DLC fractions using a Superdex 75 grade 16/70 column (Pharmacia, Uppsala, Sweden). An isocratic flow of 50 mM MES buffer (pH 6.5) was used at a flow rate of 1.5 ml / min. Total run time was 115 minutes and the peak eluted between 20 and 105 minutes. Eluted fractions were assayed using 4-12% NuPAGE Bistris gels by silver stained SDS PAGE using NuPAGE MES SDS running buffer (Invitrogen) and by anti-IFN-a2b ELISA (Bender MedSystems). The peak eluting at about 89-103 minutes was found to contain IFN-a2b.

  The resulting fractions were analyzed for apparent molecular weight and purity levels by 1D SDS PAGE using an ELISA and 4-12% NuPAGE Bistris gel using NuPAGE MES SDS running buffer (Invitrogen) to determine anti-IFN-a2b ELISA Quantified by (Bender MedSystems). Fractions containing cytokines were desalted in PBS using HiPrep 26/10 high speed desalting columns (Pharmacia).

  Purified IFN-a2b was found to have an apparent molecular weight of 18-24 kDa and was found to be at least 99% pure as assessed by silver-stained SDS-PAGE. The final concentration of IFN-a2b was found to be at least 115 μg / ml as assessed by anti-IFN-a2b ELISA (Bender MedSystems).

(b) Production, isolation and purification of IFN-b1 of the present invention
(i) Production of IFN-b1 of the present invention
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells were treated with 10% (v / v) donor calf serum (DCS, JRH Biosciences), 4 mM L-glutamine (Amresco), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, sulfate Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) 90 ml / plate supplemented with streptomycin 5 mg / ml) (JRH Biosciences). Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was replaced with 120 ml fresh DMEM / F12 supplemented with 10% (v / v) DCS, 4 mM L-glutamine and 1% (v / v) penicillin-streptomycin. To the calcium phosphate / DNA precipitate, add 1200 μg of pIRESbleo3 (Invitrogen) plasmid DNA carrying the gene of human IFN-b1 and 3720 μl of 2.5 M CaCl ₂ to a final volume of 30 ml (solution A) in sterile H ₂ O. It was prepared by. Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 20 minutes and then mixed with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. After 4 hours, DMEM / with a final pH of 7 was added with 10% (v / v) DCS, 4 mM L-glutamine, 1% (v / v) penicillin-streptomycin, and a final concentration of 3.5 mM HCl. 100 ml of F12 was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml of DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine, 4.1 g / L mannose (Sigma) , And 100 ml of fresh serum-free phenol red-free DMEM / F12 medium supplemented with 1% (v / v) penicillin-streptomycin was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 4.1 g / L mannose, and 1% (v / v) penicillin-streptomycin added 100 ml of fresh serum-free phenol red-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) was added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

  On day 4, cell culture supernatant was collected. After adding 100 mM PMSF (1% (v / v)) to the collected cell culture supernatant and combining it with the collected product on the third day, fine particles were collected using a 0.45 micron low molecular protein binding filter (Durapore, Millipore). Removed. The mixture was stored at -70 ° C or used immediately.

(ii) Expression of highly sialylated IFN-b1 The protocol described in Example 2 (b) (i) above was repeated using co-transfection of pIRESbleo3-IFN-bl and pCEP4-a2,6ST. pCEP4-a2,6ST was constructed as described in Example 8. pCEP4-a2,6ST and pIRESbleo3-IFN-bl are mixed at a ratio of 1: 3, 1200 μg of the mixture is added to 3720 μl of 2.5 M CaCl ₂ in sterile H ₂ O and the final volume is 30 ml (solution A). did.

(iii) Identification of IFN-b1 using Western blot analysis. -Run for 1 hour at 200 V in glycine SDS running buffer (Invitrogen).

  After transferring the protein on the gel to a nitrocellulose membrane, it was blocked with 1% bovine serum albumin containing 0.05% Tween-20 (TBS-T) to prevent non-specific binding. Membranes were incubated with monoclonal anti-IFN-b1 antibody (R & D Systems) for 1 hour at room temperature with gentle agitation. After washing with TBS-T to eliminate unbound primary antibody, the membrane was incubated with anti-mouse alkaline phosphatase (AP) conjugated secondary antibody (Promega) solution for 1 hour at room temperature. The membrane was washed again to eliminate unbound secondary antibody. Specific binding of the anti-IFN-b1 antibody was visualized by AP color development kit (Bio-Rad) according to the manufacturer's instructions.

  The IFN-b1 of the present invention was found to have an apparent molecular weight of 20-35 kDa.

(iv) Purification of IFN-b1 of the present invention using ion exchange chromatography and metal chelate chromatography The supernatant recovered from Example 2 (b) (i) and 2 (b) (ii) was treated with anion or cation. Purify individually using ion exchange resin (Amersham Biosciences Q or SP sepharose FF). Bound IFN-b1 is eluted from the column with 1 M NaCl. The resulting fractions are analyzed for apparent molecular weight and purity level using 4-20% gradient trisglycine gel (Invitrogen) by 1D SDS PAGE and quantified by anti-IFN-b1 ELISA (PBL Biomedical Laboratories).

Further purification of the IFN-b1 of the present invention can be performed by metal chelate chromatography (MCC) using a HiTrap Chelating HP column (1 ml; Amersham Biosciences). A HiTrap Chelating HP column is packed with Zn ²⁺ ions and equilibrated according to the manufacturer's recommendations before use. The IEC fraction containing IFN-b1 was applied to the column using a syringe and unbound protein was removed with binding buffer (0.02 M sodium phosphate, pH 7). Elute IFN-b1 using the three elution buffers in the following order.
Eluent 1: 0.02 M sodium phosphate 0.5 M NaCl pH 7
Eluent 2: 0.02 M sodium phosphate 1 M NaCl pH 7
Eluent 3: 0.02 M sodium phosphate 1 M NaCl pH 3.5

Eluted fractions are assayed by silver-stained SDS PAGE using 4-20% Trisglycine gel (Invitrogen) and by anti-IFN-1b ELISA (PBL Biomedical Laboratories). IFN-b1 binds to a HiTrap Chelating HP column packed with Zn ²⁺ ions and elutes in 0.02 M sodium phosphate 1 M NaCl pH 3.5.

  Alternatively, the purification of IFN-b1 can also be performed by using MCC as the primary purification step followed by a secondary purification step using IEC.

(c) Production, isolation and purification of IFN-g of the present invention
(i) Production of IFN-g of the present invention
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells are seeded in 90 ml Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) per plate, and the medium contains 10% (v / v) heat-inactivated calf fetal serum ( FCS, JRH Biosciences), 4 mM L-glutamine (Amresco), 10 mM HEPES (Sigma), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, streptomycin sulfate 5 mg / ml) (JRH Biosciences) was added. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was treated with fresh DMEM supplemented with 10% (v / v) heat-inactivated FCS, 4 mM L-glutamine, 10 mM HEPES, and 1% (v / v) penicillin-streptomycin. / F12 was replaced with 120 ml. To the calcium phosphate / DNA precipitate, add 1200 μg of pIRESbleo3 (Invitrogen) plasmid DNA with human IFN-g gene and 3720 μl of 2.5 M CaCl ₂ to a final volume of 30 ml (solution A) in sterile H ₂ O. It was prepared by. Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 20 minutes and then mixed with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine, 15 mM HEPES, 4.1 g / L Fresh with mannose (Sigma), 1% (v / v) penicillin-streptomycin and ITS solution (5 mg / L bovine insulin, 5 mg / L partially iron-saturated human transferrin, and 5 μg / ml selenium) (Sigma) 100 ml of serum-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 15 mM HEPES, 4.1 g / L mannose, 1% (v / v) penicillin-streptomycin And 100 ml of fresh serum-free DMEM / F12 medium supplemented with ITS solution was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

On day 4, cell culture supernatant was collected. 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant and combined with the collected product on the third day. The pH of the combined collection was adjusted to 6 by adding one-tenth volume of 200 mM MES (Sigma) / 50 mM MgCl ₂ (Sigma) pH 6 and then 0.45 micron small protein binding filter (Durapore , Millipore). The mixture was stored at -70 ° C or used immediately.

(ii) Isolation and purification of IFN-g of the present invention The dye-ligand chromatography (DLC) process was used as the first step of IFN-g purification. A library of immobilized reactive dyes was used to screen IFN-g for efficient binding and release in a batch purification microtiter format. The appropriate dye-protein combination was then tested in a small column format.

  For small scale purification, a 5 ml sample of thawed cell culture supernatant was passed through a 0.5 ml dye-ligand column at either pH 6 or 7. During this optimization phase, the optimal reactive dye-cytokine and pH combination was chosen to maximize recovery in fractions due to scale up in bulk DLC.

For the bulk scale, DLC reactive dye # 18 High (Zymatrix) was selected as the reactive dye with the best binding and elution properties of IFN-g. Filtered cell culture supernatant is applied to 4.0 ml or 8.0 ml column bodies (Alltech, Extract Clean Filter columns) with 3 ml or 6 ml DLC resin pre-equilibrated to pH 6 with 50 mM MES / 5 mM MgCl ₂ , respectively. Passed by gravity flow. The column was washed with buffer A (20 mM MES / 5 mM MgCl ₂ , pH 6) until the fractions appeared clear (not pale yellow). Three elution buffers were used in the following order to elute IFN-g.
Eluent 1: Buffer C (50 mM Tris-Cl / 10 mM EDTA pH 8)
Eluent 2: EN 1.0 (50 mM Tris-Cl / 10 mM EDTA / 1.0 M NaCl pH 8)
Eluent 3: EN 2.0 (50 mM Tris-Cl / 10 mM EDTA / 2.0 M NaCl pH 8)

  Eluted fractions were assayed by silver-stained SDS PAGE using NuPAGE MES SDS running buffer (Invitrogen) using 4-12% NuPAGE Bistris gel and by anti-IFN-g ELISA (R & D Systems). IFN g eluted in buffer EN1.0 and fractions containing IFN-g were pooled for size exclusion chromatography.

  Size exclusion chromatography was performed on the combined DLC fractions using a Superdex 75 grade 16/70 column or a Sephadex 200 grade (Pharmacia, Uppsala, Sweden) column. An isocratic flow of 50 mM MES buffer (pH 6.5) was used at a flow rate of 1.5 ml / min. The total effluent time was 120 minutes and the peak eluted between 20 and 100 minutes. Eluted fractions were assayed by silver-stained SDS PAGE using NuPAGE MES SDS running buffer and by anti-IFN-g ELISA using 4-12% NuPAGE Bistris gels. IFN-g was found to elute at the 42-55 minute peak of the efflux time.

  Further purification was performed by passing selected fractions from the SEC column through a cation exchange column (Bio-Rad Laboratories, Uno S12) pre-equilibrated with 50 mM MES pH 5.6. Bound IFN-g was eluted from the column with a gradient of 50 mM MES pH 5.6 containing 50 mM MES pH 5.6 to 1 M NaCl. The resulting fractions were analyzed for apparent molecular weight and purity levels by ELISA and 1D SDS PAGE using NuPAGE MES SDS running buffer (Invitrogen) using 4-12% NuPAGE Bistris gels, and anti-IFN-g ELISA Quantified by

  Purified IFN-g was found to have an apparent molecular weight of approximately 18-28 kDa and was found to be at least 95% pure as assessed by Coomassie Brilliant Blue staining. The final concentration of IFN-g was found to be 55 μg / ml as assessed by anti-IFN-g ELISA.

(d) Production and purification of IFNAR2-Fc of the present invention
(i) Production of IFNAR2-Fc of the present invention
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells are seeded in 90 ml Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) per plate, and the medium contains 10% (v / v) heat-inactivated calf fetal serum ( FCS, JRH Biosciences), 4 mM L-glutamine (Amresco), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, streptomycin sulfate 5 mg / ml) (Gibco) were added. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was washed with 120 ml fresh DMEM / F12 supplemented with 10% (v / v) heat-inactivated FCS, 4 mM L-glutamine, and 1% (v / v) penicillin-streptomycin. Was replaced. To the calcium phosphate / DNA precipitate, add 1200 μg of pIRESbleo3 (Invitrogen) plasmid DNA carrying the gene for human IFNAR2-Fc and 3 ml of 2.5 M CaCl ₂ to a final volume of 30 ml (solution A) in sterile H ₂ O. It was prepared by. Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 30 minutes and then mixed for several seconds with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. After 4 hours, add 10% (v / v) heat inactivated FCS, 4 mM L-glutamine, 1% (v / v) penicillin-streptomycin, and a final concentration of 3.5 mM HCl, final pH 7. 100 ml of DMEM / F12 with was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine, 0.5 g / L mannose (Sigma) And 100 ml of fresh serum-free DMEM / F12 medium supplemented with 1% (v / v) penicillin-streptomycin was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 0.5 g / L mannose, and 1% (v / v) penicillin-streptomycin added 100 ml of fresh serum-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

  On day 4, cell culture supernatant was collected. 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant and combined with the collected product on the third day. After adjusting the pH of the combined collection to 8 by adding 2 M Tris-HCl pH 8 (Sigma) to a final concentration of 15 mM, a 0.45 micron small molecule protein binding filter (Durapore, Millipore) is added. Used to remove particulates. The mixture was stored at -70 ° C or used immediately.

(ii) Purification of IFNAR2-Fc of the present invention
1 L of pH adjusted medium containing IFNAR2-Fc was passed by gravity flow through a 1 ml bed volume Protein A Sepharose column (Pharmacia) pre-equilibrated to pH 8 with 100 mM Tris-HCl pH 8 (Sigma). After washing with 20 column volumes of buffer (100 mM Tris-HCl pH 8), IFNAR2-Fc was eluted with 0.1 M citric acid (Sigma) pH 4 in 1 ml fractions, then 2 in each fraction. Immediately neutralized to pH 8 by adding 200 μl M Tris-HCl pH 8. Fractions were analyzed by silver stained SDS PAGE using 4-20% gradient trisglycine gel (Invitrogen) and by measuring absorbance at 280 nm using bovine serum albumin standards (New England Biolabs). Quantified by spectrophotometry.

  Purified IFNAR2-Fc was found to have an apparent molecular weight of approximately 50-80 kDa as judged by silver stained SDS PAGE using a 4-20% gradient trisglycine gel. The final concentration of IFNAR2-Fc was found to be 145 μg / ml as estimated by spectrophotometry.

(e) Production, isolation and purification of IL-10 of the present invention
(i) Production of IL-10 of the present invention
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells are seeded in 90 ml Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) per plate, the medium containing 10% (v / v) donor calf serum (DCS, JRH Biosciences), 4 mM L-glutamine (Amresco), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, streptomycin sulfate 5000 μg / ml) (JRH Biosciences) were added.

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was replaced with 120 ml fresh DMEM / F12 supplemented with 10% (v / v) DCS, 4 mM L-glutamine and 1% (v / v) penicillin-streptomycin. Calcium phosphate / DNA precipitate is prepared so that pIRESbleo3 (Invitrogen) plasmid DNA carrying human IL-10 gene 1200 μg and CaCl ₂ (2.5 M) 3720 μl in sterile H ₂ O to a final volume of 30 ml (solution A) Prepared by adding. Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 20 minutes and then mixed with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. After 4 hours, DMEM / with a final pH of 7 was added with 10% (v / v) DCS, 4 mM L-glutamine, 1% (v / v) penicillin-streptomycin, and a final concentration of 3.5 mM HCl. 100 ml of F12 was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml of DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine, 4.1 g / L mannose (Sigma) , And 100 ml of fresh serum-free phenol red-free DMEM / F12 medium supplemented with 1% (v / v) penicillin-streptomycin was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 4.1 g / L mannose, and 1% (v / v) penicillin-streptomycin added 100 ml of fresh serum-free phenol red-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

On day 4, cell culture supernatant was collected. 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant and combined with the collected product on the third day. Adjust the pH of the combined collection to 6 by adding one-tenth volume of 200 mM MES / 50 mM MgCl ₂ pH 6 and then microparticles using a 0.45 micron small molecule protein binding filter (Durapore, Millipore) Was removed. The mixture was stored at -70 ° C or used immediately.

(ii) Isolation and purification of IL-10 of the present invention A dye-ligand chromatography (DLC) process was used as the first step of IL-10 purification. IL-10 was screened for efficient binding and release in a batch purified microtiter format using a library of immobilized reactive dyes. Appropriate dye-protein combinations were tested in a small column format.

  For small scale purification, a 5 ml sample of thawed cell culture supernatant was passed through a 0.5 ml dye-ligand column at pH 6 or 7.3. In this optimization step, the optimal dye bead-cytokine and pH combination was selected to maximize recovery in fractions for scale up in bulk DLC.

For the bulk scale, DLC reactive dye No. 18 High (Zymatrix) was selected as the reactive dye with the best binding and elution properties of IL-10. Filtered cell culture supernatant is applied to 4.0 ml or 8.0 ml column bodies (Alltech, Extract Clean Filter columns) with 3 ml or 6 ml DLC resin pre-equilibrated to pH 6 with 50 mM MES / 5 mM MgCl ₂ , respectively. Passed by gravity flow. Bulk flow-through samples were stored at 4 ° C. until the ELISA results confirmed that the purification was successful. The column was washed with buffer A (20 mM MES / 5 mM MgCl ₂ , pH 6) until the fractions appeared clear. Three elution buffers were used in the following order to elute IL-10.
Eluent 1: Buffer C (50 mM Tris-Cl / 10 mM EDTA pH 8)
Eluent 2: EN 1.0 (50 mM Tris-Cl / 10 mM EDTA / 1.0 M NaCl pH 8)
Eluent 3: EN 2.0 (50 mM Tris-Cl / 10 mM EDTA / 2.0 M NaCl pH 8)

  Eluted fractions were assayed using silver stained SDS PAGE using 4-20% trisglycine gel (Invitrogen) and by IL-10 ELISA (R & D Systems). IL-10 was found to bind to the reactive dye 8 High and was found to elute in buffer EN1.0 and buffer EN2.0. DLC fractions containing IL-10 were pooled for fast desalting and the buffer was changed to 50 mM MES pH 5.6 using a HiPrep 26/10 desalting column (Amersham Biosciences).

  Further purification by passing selected fractions from the desalting column through a strong cation exchange column (Bio-Rad Laboratories, Macro-Prep High Support) pre-equilibrated with 50 mM MES pH 5.6 (Sigma) Went. Bound IL-10 was eluted from the column with a linear gradient of 50 mM MES pH 6.5 containing 50 mM MES pH 6.5-1 M NaCl. The resulting fractions were analyzed for apparent molecular weight and purity levels by ELISA and 1D SDS PAGE using 4-20% gradient trisglycine gel (Invitrogen) and quantified by IL-10 ELISA.

  Purified IL-10 was found to have an apparent molecular weight of 16-23 kDa and was at least 99% pure as assessed by 1D SDS PAGE using a 4-20% gradient trisglycine gel (Invitrogen). Was found. The final concentration of IL-10 was found to be 22.9 μg / ml as assessed by IL-10 ELISA.

(f) Production and purification of human cell expressed IL-10Ra-Fc
(i) Production of IL-10Ra-Fc expressed in human cells
On day 0, in 500 cm ² tissue culture dishes (Corning), transform fetal human kidney cell lines such as HEK 293, HEK 293 c18, HEK 293T, 293 CEN4, HEK 293F, HEK 293FT, HEK 293E, AD- 3 × 10 ⁷ 293 (Stratagene) or 293A (Invitrogen) were seeded. Cells are seeded in 90 ml Dulbecco's Modified Eagle Medium / Ham Nutrient Mixed Medium F12 (DMEM / F12) (JRH Biosciences) per plate, and the medium contains 10% (v / v) heat-inactivated calf fetal serum ( FCS, JRH Biosciences), 4 mM L-glutamine (Amresco), and 1% (v / v) penicillin-streptomycin (penicillin G 5000 U / ml, streptomycin sulfate 5000 μg / ml) (JRH Biosciences) were added.

On the first day, transfection was performed using calcium phosphate. Prior to transfection, the medium in each plate was transferred to 120 ml fresh DMEM / F12 supplemented with 10% (v / v) heat-inactivated FCS, 4 mM L-glutamine and 1% (v / v) penicillin-streptomycin. Exchanged. The calcium phosphate / DNA precipitate should be 1200 μg of pIRESbleo3 (Clonetech, BD Biosciences) plasmid DNA with human IL-10Ra-Fc gene and 3720 μl of CaCl ₂ in sterile H ₂ O to a final volume of 30 ml (solution A). It was prepared by adding to Solution A was added dropwise to 30 ml of 2 × HEPES buffered saline (HBS) (solution B) with a 10 ml pipette. During the dropwise addition, bubbles were sprayed lightly on the solution B. The mixture was incubated at 25 ° C. for 20 minutes and then mixed with a vortex mixer. 12 ml of the mixture was added dropwise to each plate. After 4 hours, add 10% (v / v) heat inactivated FCS, 4 mM L-glutamine, 1% (v / v) penicillin-streptomycin, and a final concentration of 3.5 mM HCl, final pH 7. 100 ml of DMEM / F12 with was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On the second day, the cell culture supernatant was discarded. The contents of the plate were washed twice with 50 ml of DMEM / F12 medium per plate, 40 mM N-acetyl-D-mannosamine (New Zealand Pharmaceuticals), 10 mM L-glutamine (Amresco), 4.1 g / L mannose (Sigma) and 100 ml of fresh serum-free DMEM / F12 medium supplemented with 1% (v / v) penicillin-streptomycin was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ .

On day 3, cell culture supernatant was collected and 40 mM N-acetyl-D-mannosamine, 10 mM L-glutamine, 4.1 g / L mannose, and 1% (v / v) penicillin-streptomycin added 100 ml of fresh serum-free DMEM / F12 medium was added to each plate. Plates were incubated overnight at 37 ° C. and 5% CO ₂ . 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant, and the mixture was stored at 4 ° C.

  On day 4, cell culture supernatant was collected. 100 mM PMSF (1% (v / v)) and 500 mM EDTA (1% (v / v)) were added to the collected cell culture supernatant and combined with the collected product on the third day. Adjust the pH of the combined harvest to 8 by adding 2 M Tris-HCl pH 8 (Sigma) to a final concentration of 100 mM, then use a 0.45 micron small molecule protein filter (Durapore, Millipore). Fine particles were removed. The mixture was stored at -70 ° C or used immediately.

(ii) Purification of human cell expressed IL-10Ra-Fc
1 L of pH adjusted medium containing IL-10Ra-Fc was passed by gravity flow through a 1 ml bed volume protein A sepharose column (Pharmacia) pre-equilibrated to pH 8 with 100 mM Tris-HCl. After washing with 20 column volumes of buffer (100 mM Tris-HCl pH 8), IL-10Ra-Fc was eluted with 0.1 M citric acid (Sigma) pH 2.2 into 1 ml fractions. Was immediately neutralized to pH 8 by adding 300 μl of 2 M Tris-HCl pH 8. Fractions were analyzed by silver-stained SDS PAGE using a 4-20% gradient trisglycine gel (Invitrogen) and spectrophotometrically determined by measuring absorbance at 280 nm using a bovine serum albumin standard (Pierce). Quantified by the method. Pure fractions containing IL-10Ra-Fc were pooled and dialyzed for 4 days against 2 changes per day against PBS pH 7.4 1L.

  Purified IL-10Ra-Fc was found to have an apparent molecular weight of 65-80 kDa and be at least 95% pure as judged by silver-stained SDS PAGE using a 4-20% gradient trisglycine gel. The final concentration of IL-10Ra-Fc was estimated to be 100 μg / ml as estimated by spectrophotometry.

Example 3
(a) Characterization of IFN-a2b of the present invention
(i) Two-dimensional polyacrylamide electrophoresis The sample collected from Example 2 was re-purified (18 MOhm) with a dialysis or desalting column (Pharmacia HR 10/10 Fast Desalting Column), and the buffer was exchanged with a SpeedVac concentrator. Used to dry. Alternatively, the sample was subjected to sedimentation, eg, TCA sedimentation, using methods known in the art. The dried sample was then MSD buffer (5 M urea, 2 M thiourea, 65 mM DTT, 2% (w / v) CHAPS, 2% (w / v) sulfobetaine 3-10, 0.2% (v / v ) Amphoteric carrier, 40 mM Tris, 0.002% (w / v) bromophenol blue, water) Redissolved in 240 μl and centrifuged at 15000 g for 8 minutes.

  Isoelectric focusing (IEF) was performed using precast 11 cm or precast 17 cm gel pH 3-10 immobilized pH gradient IEF strips (BioRad). In a sealed tube, the IEF strip was rehydrated in the sample at room temperature for at least 6 hours. The IEF strip was placed in the electrophoresis chamber and covered with paraffin oil. IEF at 100 V for 1 hour, 200 V for 1 hour, 600 V for 2 hours, 1000 V for 2 hours, 2000 V for 2 hours, 3500 V for 12 hours, and 100 V for 11 cm strips Up to 12 hours or in the case of 17 cm strips (using the same V increase procedure) was done for 85 kV hours.

  After isoelectric focusing, the strip was reduced and alkylated before being subjected to a second dimensional gel. Strips in 1 x Tris / HCl pH 8.8, 6 M urea, 2% (w / v) SDS, 2% (v / v) glycerol, 5 mM tributylphosphine (TBP), 2.5% (v / v) acrylamide solution Incubated for at least 20 minutes.

  Criterion pre-dispensing (11 x 8 cm 1 mm thick) 11 cm strips were separated in the second dimension by 10-20% Tris glycine gradient gel (BioRad). 17 cm strips were separated on a 17 × 17 cm, 1.5 mm thick, self-dispensing 10-20% Tris glycine gradient gel. Gels were also provided with Precision or Kaleidoscope molecular weight markers (BioRad). Strips were placed in place using 0.5% agarose containing bromophenol blue as a tracking dye.

  SDS-PAGE was performed using a Criterion or Protean II electrophoresis system (BioRad) (for an 11 cm gel, 200 V for 1 hour (just before the buffer front just flows out of the gel edge), and 17 (In the case of cm gel, 21 hours at a constant current of 15 mA per gel) The buffer used was based on 192 mM glycine, 0.1% (w / v) SDS, 24.8 mM Tris, pH 8.3.

The completed second dimension gel was fixed in 10% methanol (MeOH) and 7% acetic acid (Hac) for 30 minutes to overnight. The gel was then stained with Sypro Ruby gel stain (BioRad) for at least 3 hours and destained in 10% MeOH and 7% HAc for at least 30 minutes. Alternatively, after fixation, the gel was stained with Deep Purple fluorescent stain. Gels were incubated twice for 30 minutes in 300 mM Na ₂ CO ₃ , 35 mM NaHCO ₃ and then in the dark for at least 1 hour in 1: 200 diluted Deep Purple stain. The gel was then destained by incubating twice for 10 minutes in 10% MeOH, 7% HAc. In each case, the gel was imaged using an FX laser densitometer (BioRad) and appropriate filters.

  The software ImageJ (http://rsb.info.nih.gov/ij/) was used to analyze the relative intensity of protein spots on the gel. Concentration measurements were performed on spots in selected areas of the gel and background subtraction was performed using appropriate areas of the gel lacking protein spots. Volume integration was performed for each protein spot of interest, and the center of mass of the spot was calculated therefrom. The relative intensity rate was calculated for each protein spot, and the intensity of each protein spot relative to the other spots in the gel was determined by standardizing the sum of all spot intensities to 100%.

  The molecular weight of each spot was determined by measuring the respective distance of the spot from the baseline of the gel and comparing it to the distance indicated by the Precision or Kaleidoscope molecular weight markers also applied to the gel. An exponential function having a fourth-order polynomial was fitted to a precise marker, and each protein spot position was interpolated. In this way, the molecular weight of each spot could be accurately determined.

  The charge of the isoform (pKa value) was determined by measuring the distance of each of the spots from the left side of the gel using ImageJ. Since the relationship between the pi value of the strip and the physical distance of the gel is linear, pi values corresponding to the various pKa values of the isoform spots were easily determined.

  The major protein spot in the resulting gel corresponds to IFN-a2b. Low intensity spots may be IFN-a2b or low level contaminants, but these cannot be confirmed by PMF due to low intensity. Examination of the gel revealed that IFN-a2b of the present invention contained 2 to 22 isoforms. Tables 9 and 10 show the important properties of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% of actual value, or overall ± 2) %, Whichever is greater). The values listed correspond to strong centers in the selected area of the gel containing the spot and thus reflect the pI and molecular weight of the protein at one specific reading within the selected area of the gel. It ’s just that. Taking into account the inherent variation in the size and location of protein spots within the 2D gel, the pI values of the IFN-a2b of the present invention range from about 4.5-7 based on the values listed in Tables 9 and 10 And the apparent molecular weight of the IFN-a2b of the present invention was determined to be in the range of 13-24 kDa based on the values listed in Tables 9 and 10.

Table 9: Molecular weight and pI value of IFN-a2b isoform

Table 10: Molecular weight and pI value of IFN-a2b isoform

(ii) One-dimensional polyacrylamide gel electrophoresis After drying the sample collected from Example 2 (a), it was added to 60 μl of 1D sample buffer (10% glycerol, 0.1% SDS, 10 mM DTT, 63 mM Tris-HCl). Dissolve and heat at 100 ° C. for 5 minutes. For PNGase F treatment, a small 30 μl sample was taken and NP40 was added to a final concentration of 0.5%. 5 μl of PNGase F is added and the sample is incubated at 37 ° C. for 3 hours. For sample glycosidase cocktail treatment, a small volume is taken and NP40 is added to a final concentration of 0.5%. Add 1 μl of PNGase F, and 1 μl each of sialidase A (neuraminidase), O-glycanase, β (1-4) -galactosidase, and β-N-acetylglucosaminidase. Treated and untreated samples are incubated at 37 ° C. for 3 hours. Treated and untreated samples are run on a shaped Tris gel, such as Tris 4-20% gradient gel (BioRad) or Tris hydrochloride gradient gel (Invitrogen). A molecular weight marker for accuracy (BioRad catalog number 161-0363) is applied to the gel as well. Criterion 4-20% or 18% gels are used for 1D SDS PAGE (BioRad catalog number 345-0033 or 345-0024). SDS-PAGE is performed for about 1 hour at 200 V using either a Mini Protean II or a Criterion electrophoresis system (BioRad), or until the buffer tip is almost drained from the gel tip. The buffer used was 192 mM glycine, 0.1% (w / v) SDS, 24.8 mM Tris base pH 8.3. Fix the finished gel in 10% MeOH and 7% HAc for at least 30 minutes. The gel is stained with Sypro Ruby gel dye (BioRad) for at least 3 hours and destained with 10% MeOH and 7% HAc for at least 30 minutes. Alternatively, the gel is stained with Deep Purple (Amersham) according to the manufacturer's instructions. The gel is imaged using an FX laser density meter (BioRad) and an appropriate filter.

  IFN- of the present invention after release of N-linked oligosaccharide (by PNAase treatment) and after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) Determine the apparent molecular weight of a2b (observed by SDS-PAGE).

(iii) N-terminal sequencing of protein The protein band was excised from the gel prepared as described above (two-dimensional gel) and placed in a 0.5 ml test tube and 100 ml extraction buffer (100 mM sodium acetate, 0.1% SDS, 50 mM DTT, pH 5.5) was added. Gel slices were incubated at 37 ° C. with shaking for 16 hours. The supernatant was applied to ProSorb membrane (ABI) according to the manufacturer's instructions and sequenced using an automated 494 protein sequencer (Applied Biosystems) according to the manufacturer's instructions. The obtained sequence (CDLPQTH (S / F) LG) confirmed that the protein identity was human IFN-a2b.

(iv) Peptide mass fingerprinting Cut out the protein band from the gel prepared as above (either from the two-dimensional gel or the one-dimensional gel) and wash buffer (50% acetonitrile in 50 mM NH ₄ HCO ₃ ) 25 μl Washed with. Gel pieces were placed at room temperature for at least 1 hour and dried by vacuum centrifugation for 30 minutes. 12 μl of gel fragment and trypsin solution (20 μg trypsin, 1200 μl NH ₄ HCO ₃ ) were placed in each sample well and incubated at 4 ° C. for 1 hour. The remaining trypsin solution was removed and 20 μl of 50 mM NH ₄ HCO ₃ was added. The mixture was incubated overnight at 37 ° C. with gentle infiltration. Peptide samples were concentrated and desalted using prefabricated microcolumns containing C18 Zip-Tip (Millipore, Bedford, Mass.) Or Poros R2 (Perseptive Biosystems, Framingham, Mass.) Chromatography resin. The bound peptide was eluted directly onto the target plate with 0.8 μl of matrix solution (α-cyano-4-hydroxycinnamic acid (Sigma) in 70% acetonitrile / 1% formic acid, 8 mg / ml). Peptide mass fingerprints of tryptic peptides were generated by matrix-assisted laser desorption / ionization time-of-flight mass spectrometry (MALDI-TOF MS) using Perseptive Biosystems Voyager DE-STR. Spectra were acquired in reflectron mode using an acceleration voltage of 20 kV. Mass calibration was performed using trypsin autolysis peaks, 2211.11 Da and 842.51 Da as internal standards. Data generated from peptide mass fingerprinting (PMF) was used to confirm protein identity. The program PeptIdent (www.expasy.ch/tools/peptident.html) was used to search in databases such as SWISS-PROT and TrEMBL (mainly for human sapien (human) and mammalian entries). . Identification parameters included 0.1 Da peptide mass tolerance, missed up to 1 trypsin cleavage per peptide, and methionine sulfoxide and cysteine-acrylamide modifications. Identification was based on the number of matching peptide masses and the total percentage of amino acid sequences covered by such peptides compared to other database entries. In general, peptide matches with at least 30% total sequence coverage are required for confidence in identification, but very low and high mass proteins, and such proteins due to protein fragmentation are not necessarily This criterion may not be met and therefore requires further identification.

  If MALDI-TOF PMF provides indeterminate or no protein identification, subject the same spot excised from the remaining peptide mixture or duplicate gel to trypsin digestion and electrospray ionization tandem MS (ESI-MS / MS) was used for analysis. For ESI-MS / MS, the peptide was eluted directly from the Poros R2 microcolumn with 1-2 μl of 70% acetonitrile, 1% formic acid into a borosilicate nanoelectrospray needle (Micromass, Manchester, UK). Tandem MS was performed using a Q-Tof hybrid quadrupole / direct acceleration TOF mass spectrometer (Micromass). A nanoelectrospray needle containing the sample was attached to the source and a stable flow was obtained with a capillary voltage of 900-1200 V. Precursor ion scanning was performed to detect the mass to charge ratio (m / z) value of the peptides in the mixture. The m / z of individual precursor ions was selected for fragmentation and collided with argon gas using a collision energy of 18-30 eV. Fragment ions (corresponding to the loss of amino acids from the precursor peptide) were recorded and processed using MassLynx Version 3.4 (Micromass). The amino acid sequence was estimated from the mass difference in the y-ion or b-ion “ladder” system using the program MassSeq (Micromass) and confirmed by manual interpretation. The peptide sequences were then used to search the NCBI and TrEMBL databases using the program BLASTP “short nearly exact matches”. A minimum of two matching peptides was required to provide confidence in a given identification.

  The identity of the gel spot was confirmed to be IFN-a2b.

(b) Characterization of IFN-b1 of the present invention
(i) Two-dimensional polyacrylamide gel electrophoresis Samples taken from Example 2 (b) are treated and analyzed as described above in Example 3 (a) (i). Spots corresponding to the unique isoform of IFN-b1 of the present invention are identified.

(ii) One-dimensional polyacrylamide gel electrophoresis Samples recovered from Example 2 (b) (ii), 2 (b) (iii), or 2 (b) (iv) were used in Example 3 (a) (ii) Treat as previously described. IFN- of the present invention after release of N-linked oligosaccharide (by PNGase treatment) and after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) Determine the apparent molecular weight of b1 (observed by SDS-PAGE).

(iii) N-terminal sequencing of proteins N-terminal sequencing of IFN-b1 of the present invention is performed as described above in Example 3 (a) (iii).

(iv) Peptide mass fingerprinting
Peptide mass fingerprinting of IFN-b1 is performed as previously described in Example 3 (a) (iv).

  The identity of the gel spot was confirmed to be IFN-b1.

(c) Characterization of IFN-g of the present invention
(ii) Two-dimensional polyacrylamide gel electrophoresis Samples collected from Example 2 (c) were treated and analyzed as described above in Example 3 (a) (i).

  The major protein spots in the resulting gel correspond to the IFN-g isoform. The low intensity spots may be IFN-g or low level contaminants, but these are not confirmed by PMF due to low intensity. Examination of the gel revealed that IFN-g of the present invention contained 4 to 16 isoforms. Tables 11 and 12 show the important properties of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% of actual value, or ± 2 of total) %, Whichever is greater). The values listed correspond to strong centers in the selected area of the gel containing the spot and thus reflect the pI and molecular weight of the protein at one specific reading within the selected area of the gel. It ’s just that. Taking into account the inherent variation in the size and position of protein spots within the 2D gel, the pI values of the IFN-g of the present invention range from about 4 to 14 based on the values listed in Tables 11 and And the apparent molecular weight of the IFN-g of the present invention was determined to be in the range of 15-30 kDa based on the values listed in Tables 11 and 12.

Table 11: Molecular weight and pI value of IFN-g isoform

Table 12: Molecular weight and pI value of IFN-g isoform

(ii) One-dimensional polyacrylamide gel electrophoresis The sample recovered from Example 2 (c) was treated as described above in Example 3 (a) (ii). The apparent molecular weight (as observed by SDS-PAGE) of IFN-g of the present invention after release of N-linked oligosaccharides (by PNGase treatment) was 12-20 kDa. The apparent molecular weight (observed by SDS-PAGE) of the IFN-g of the present invention after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) is 12-20 kDa.

(iii) N-terminal sequencing of proteins N-terminal sequencing of the IFN-g of the present invention is performed as previously described in Example 3 (a) (iii).

(iv) Peptide mass fingerprinting
Peptide mass fingerprinting of IFN-g is performed as previously described in Example 3 (a) (iv).

  The identity of the gel spot was confirmed to be IFN-g.

  In addition, the 1 Da shift observed in the mass of tryptic peptides is due to the asparagine residue (N) of the two NX (S / T / C) motifs observed in the theoretical amino acid sequence of human IFN-g. (D) shows that it has been modified, which is consistent with the fact that PNGase F is known to be able to induce D residue modification from N upon removal of associated N-linked oligosaccharides. To do. Thus, the two confirmed N-glycosylation sites of IFN-g of the present invention were found to be N-48 and N-120 (numbering from the start of the signal sequence).

(d) Characterization of IFNAR2-Fc of the present invention
(i) Two-dimensional polyacrylamide gel electrophoresis Samples recovered from Example 2 (d) were treated and analyzed as described above in Example 3 (a) (i).

  The major protein spot in the resulting gel corresponds to the isoform of IFNAR2-Fc. The low intensity spots may be IFNAR2-Fc or low level contaminants, but these cannot be confirmed by PMF due to low intensity. Examination of the gel revealed that IFNAR2-Fc of the present invention contains 10 to 25 isoforms. Table 13 shows the important properties of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% of actual value, or ± 2% of total, Whichever is greater). The values listed correspond to strong centers in the selected area of the gel containing the spot and thus reflect the pI and molecular weight of the protein at one specific reading within the selected area of the gel. It ’s just that. Taking into account the inherent variation in the size and position of the protein spots within the 2D gel, the pI values of the IFNAR2-Fc of the present invention range from about 4-7 based on the values listed in Table 13 As well as the apparent molecular weight of IFNAR2-Fc of the present invention was determined to be in the range of 50-105 kDa based on the values listed in Table 13.

Table 13: Molecular weight and pI value of IFNAR2-Fc isoform

(ii) One-dimensional polyacrylamide gel electrophoresis Samples recovered from Example 2 (d) were treated as described previously in Example 3 (a) (ii). The apparent molecular weight (as observed by SDS-PAGE) of IFNAR2-Fc of the present invention after release of N-linked oligosaccharides (by PNGase treatment) was 45-95 kDa. The apparent molecular weight (observed by SDS-PAGE) of IFNAR2-Fc of the present invention after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) is 45-80 kDa.

(iii) N-terminal sequencing of proteins N-terminal sequencing of IFNAR2-Fc of the present invention is performed as previously described in Example 3 (a) (iii).

(iv) Peptide mass fingerprinting
Peptide mass fingerprinting of IFNAR2-Fc is performed as previously described in Example 3 (a) (iv).

  The identity of the gel spot was confirmed to be IFNAR2-Fc.

(e) Characterization of IL-10 of the present invention
(i) Two-dimensional polyacrylamide gel electrophoresis Samples taken from Example 2 (e) were treated and analyzed as previously described in Example 3 (a) (i).

  The major protein spot in the resulting gel corresponds to the isoform of IL-10. The low intensity spots can be IL-10 or low level contaminants, but these are not confirmed by PMF due to their low intensity. Examination of the gel revealed that IL-10 of the present invention contained 4 to 28 isoforms. Tables 14 and 15 show the important properties of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% of actual value, or ± 2 of total) %, Whichever is greater). The values listed correspond to strong centers in the selected area of the gel containing the spot and thus reflect the pI and molecular weight of the protein at one specific reading within the selected area of the gel. It ’s just that. Taking into account the inherent variation in the size and location of protein spots within the 2D gel, the pI values of IL-10 of the present invention range from about 6 to 10 based on the values listed in Tables 14 and 15 The apparent molecular weight of IL-10 of the present invention was determined to be in the range of 10-23 kDa based on the values listed in Tables 14 and 15.

Table 14: Molecular weight and pI value of IL-10 isoform

Table 15: Molecular weight and pI value of IL-10 isoform

(ii) One-dimensional polyacrylamide gel electrophoresis The sample recovered from Example 2 (e) was treated as described above in Example 3 (a) (ii). The apparent molecular weight (as observed by SDS-PAGE) of IL-10 of the present invention after release of N-linked oligosaccharides (by PNGase treatment) was 10-23 kDa. The apparent molecular weight of IL-10 of the present invention (observed by SDS-PAGE) after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) is 10-23. kDa.

(iii) N-terminal sequencing of proteins The N-terminal sequencing of IL-10 of the present invention is performed as previously described in Example 3 (a) (iii).

(iv) Peptide mass fingerprinting
Peptide mass fingerprinting of IL-10 is performed as previously described in Example 3 (a) (iv).

  The identity of the gel spot was confirmed to be IL-10.

(f) Characterization of IL-10Ra-Fc of the present invention
(i) Two-dimensional polyacrylamide gel electrophoresis Samples collected from Example 2 (f) were treated and analyzed as previously described in Example 3 (a) (i).

  The major protein spots in the resulting gel correspond to the IL-10Ra-Fc isoform. The low intensity spots may be IL-10Ra-Fc or low level contaminants, but these are low intensity and cannot be confirmed by PMF. Examination of the gel revealed that IL-10Ra-Fc of the present invention contained 10 to 21 isoforms. Table 16 shows the important properties of these isoforms: pI value (± 1.0), apparent molecular weight (± 20%), and relative intensity (± 20% of actual value, or ± 2% of total, Whichever is greater). The values listed correspond to strong centers in the selected area of the gel containing the spot and thus reflect the pI and molecular weight of the protein at one specific reading within the selected area of the gel. It ’s just that. Taking into account the inherent variation in the size and position of protein spots within the 2D gel, the pI values of IL-10Ra-Fc of the present invention range from about 4.5 to 9.5 based on the values listed in Table 16. The apparent molecular weight of the IL-10Ra-Fc of the present invention was determined to be in the range of 50-100 kDa based on the values listed in Table 16.

Table 16: Molecular weight and pI value of IL-10Ra-Fc isoform

(ii) One-dimensional polyacrylamide gel electrophoresis The sample recovered from Example 2 (f) was treated as described above in Example 3 (a) (ii). The apparent molecular weight (observed by SDS-PAGE) of IL-10Ra-Fc of the present invention after release of N-linked oligosaccharide (by PNGase treatment) was 40-85 kDa. The apparent molecular weight (observed by SDS-PAGE) of IL-10Ra-Fc of the present invention after release of N-linked oligosaccharide (by PNGase treatment) and O-linked oligosaccharide (by glycosidase cocktail) is 36 It was ˜85 kDa.

(iii) N-terminal sequencing of proteins N-terminal sequencing of IL-10Ra-Fc of the present invention is performed as described above in Example 3 (a) (iii).

(iv) Peptide mass fingerprinting
Peptide mass fingerprinting of IL-10Ra-Fc is performed as previously described in Example 3 (a) (iv).

  The identity of the gel spot was confirmed to be IL-10Ra-Fc.

  Furthermore, the 1 Da shift observed in the mass of tryptic peptides is due to the asparagine residues (N) of the four NX (S / T / C) motifs observed in the theoretical amino acid sequence of human IL-10Ra-Fc. This indicates that PNGase F can induce N-to-D residue modifications upon removal of associated N-linked oligosaccharides. Is consistent with Thus, the four confirmed N-glycosylation sites of IL-10Ra-Fc of the present invention are N-110, N-154, N-177 and N-323 (numbering from the start of the signal sequence) I found something.

Example 4
Characterization of the protein of the present invention
(a) Analysis of amino acid, monosaccharide, oligosaccharide, phosphate, sulfate, and isoform composition of IFN-a2b of the present invention
(i) Preparation of samples for amino acid, monosaccharide, oligosaccharide, phosphate, sulfate, and isoform analysis Hydrolysis for the characterization of monosaccharide and oligosaccharide glycosylation and phosphate and sulfate post-translational modifications The sugars were first removed from the polypeptide backbone by degradation or enzymatic means. Prior to starting the analysis, sample buffer components were also removed and replaced with water to avoid hydrolysis and inhibition of the enzyme reaction. A solution of purified IFN-a2b in PBS is used with a regenerated cellulose dialysis membrane (Spectrapore) with a nominal molecular weight cut-off (NMWC) of 5 KDa for 1 liter of deionized ultrafiltered water (18 MOhm). It was dialyzed thoroughly for 4 days while changing twice a day. After dialysis, the solution was dried using Savant Speed Vac (New York, USA). The dried sample was then resuspended in 2 ml of deionized ultrafiltered water (18 MOhm) and aliquoted for various analyses.

(ii) Analysis of amino acid composition by gas phase hydrolysis The amino acids in the samples were analyzed using precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC). Stable fluorescent amino acid derivatives were separated and quantified by reverse phase (C18) HPLC. The procedure used was based on the Waters AccQTag amino acid analysis method.

  Three copies of 100 μl sample of IFN-a2b preparation were collected and dried with Speed Vac. The dried sample was then hydrolyzed at 110 ° C. for 24 hours. After hydrolysis, the sample was dried again and then derivatized as follows. The dried sample was resuspended in 10 μl of internal amino acid standard solution (α-aminobutyric acid, AABA), 35 μl borate buffer was added followed by 15 μl AQC derivatization reagent. The reaction mixture was heated in a heating block at 50 ° C. for 12 minutes. The derivatized amino acid sample was transferred in turn to an autosampler of an HPLC system consisting of a Waters Alliance 2695 Separation Module, Waters 474 Fluorescence Detector, and Waters 2487 Dual λ Absorbance Detector. The control and analysis software was a Waters Empower Pro Module (Waters Corporation, Milford, Mass., USA). Samples were passed through a Waters AccQTag column (15 cm × 3.9 mm ID) using chromatographic parameters known in the art (ie, appropriate eluent and gradient flow).

(iii) Analysis of neutral and amino monosaccharide composition
Two 100 μl samples of the IFN-a2b preparation were taken and processed in two different ways to liberate monosaccharides. Each treatment was performed in triplicate as described below.
1. Hydrolyze with 2 M trifluoroacetic acid (TFA) heated to 100 ° C. for 4 hours to liberate neutral sugars (galactose, glucose, fucose, and mannose).
2. Hydrolyze with 4 M HCl heated to 100 ° C. for 4 hours to liberate amino sugars (N-acetyl-galactosamine, N-acetyl-glucosamine).

  All hydrolysates were lyophilized using a Speed Vac system and redissolved in 200 μl water containing 0.8 nmol internal standard. The internal standard for neutral and amino sugars was 2-deoxy-glucose. The sample was then centrifuged at 10,000 g for 30 minutes to remove protein debris. The supernatant was transferred to a new tube and analyzed by high pH anion exchange chromatography using a Dionex LC 50 system (Dionex Ltd) equipped with a GP50 pump and an ED50 pulse amperometric detector. Neutral and amino sugars were analyzed using a Dionex CarboPac PA-20 column. Elution was performed at a uniform hydroxide concentration of 10 mM for 20 minutes. This was achieved with a Dionex EG50 eluent generation system.

(iv) Analysis of acidic monosaccharide composition
A 100 μl sample of the IFN-a2b preparation was taken and processed in the following manner to release sialic acid monosaccharides. Processing was done in triplicate.

  Samples were hydrolyzed with 0.1 M TFA at 80 ° C. for 40 minutes to liberate N-acetyl and N-glycosylneuraminic acid. The hydrolyzate was lyophilized using Speed Vac and redissolved in 200 μl of water containing 0.8 nmol of internal standard. The internal standard for sialic acid analysis was lactobionic acid. The sample was then centrifuged at 10,000 g for 30 minutes to remove protein debris. The supernatant was transferred to a new tube and analyzed by high pH anion exchange chromatography using a Dionex LC 50 system equipped with a GP50 pump and an ED50 pulse amperometric detector. Analysis of sialic acid was performed with Dionex CarboPac PA1 using chromatographic parameters known in the art (ie, appropriate eluent and gradient flow).

(v) Analysis of oligosaccharide composition To analyze the oligosaccharide composition, 300 μl samples of IFN-a2b preparation were collected in triplicate and processed in one of the following ways.

  1. Release of N-linked oligosaccharides is achieved by the enzyme peptide-N4- (N-acetyl-β-D-glucosaminyl) asparagine amidase (PNGase). First, 1/5 volume of denaturing solution (2% SDS (Sigma) / 1 M β-mercaptoethanol (Sigma)) is added to the sample. Heat the sample at 100 ° C. for 5 minutes. 1/10 volume of 15% Triton X-100 (Sigma) is added to the sample. Mix sample gently and cool to room temperature. Add 25 units of PNGase (Sigma) and incubate at 37 ° C.

  2. Release of O-linked oligosaccharides is achieved by a β-elimination step. First, 1/2 volume of 4 M borohydride (prepared as needed) (Sigma) solution is added to the sample. Add 1/2 volume of 0.4 M NaOH (BDH, HPLC grade) to the sample. Samples are incubated at 50 ° C. for 16 hours. The sample is cooled on ice and 1/2 volume of 0.4 M acetic acid (Sigma) is added to the sample.

  Both N-bound and O-bound samples are further processed using a Carbo Pac graphitized carbon SPE column to remove buffer components. The column equilibration and elution conditions are as follows.

First, the column is pre-equilibrated with 1 column volume of 80% acetonitrile (Sigma) and then 2 column volumes of H ₂ O. Samples are added under gravity flow and the column is washed with 2 column volumes of H ₂ O. To elute neutral oligosaccharides, apply 2 ml of 50% acetonitrile to the column. To elute acidic oligosaccharides, apply 2 ml of 50% acetonitrile / 0.1% formic acid to the column. The remaining oligosaccharide is eluted by adding 2 ml of 80% acetonitrile / 0.1% formic acid.

  Individual fractions from the SPE column containing neutral or acidic N-linked oligosaccharides and neutral or acidic O-linked oligosaccharides are completely dried using Speed Vac. Samples are redissolved in 200 μl water and analyzed by high pH anion exchange column chromatography using a Dionex LC 20 system equipped with a GP50 pump and an ED50 pulse amperometric detector. Analysis of neutral and acidic oligosaccharides is performed using a CarboPac PA100 column and parameters known in the art (ie, appropriate eluent and gradient flow).

(vi) Analysis of Sulfuric Acid and Phosphate Composition Sulfuric acid / phosphate analysis was performed essentially by the method described by Harrison and Packer (Harrison and Packer Methods Mol Biol 125: 211-216, 2000). A 100 μl sample of the IFN-a2b preparation was taken for sulfuric acid and phosphate analysis and hydrolyzed in 4 M HCl at 100 ° C. for 4 hours. HCl was removed by drying the sample on a Speed Vac system. The sample was then redissolved in 200 μl of H ₂ O. A 24 μl sample was injected into a Dionex LC 50 system equipped with a GP50 pump and an ED50 conductivity detector. Separations were performed on a Dionex IonPac IS11 anion exchange column using chromatographic parameters known in the art (ie, appropriate eluent and gradient flow).

(vii) Further separation of protein isoforms
Further separation of the IFN-a2b isoform is performed using a thin film anion exchange column. An appropriate amount, for example 24 μl, of the sample is separated on a ProPac SAX-10 column (Dionex Ltd) using a Dionex SUMMIT system (Dionex Ltd) equipped with a UV-Vis detector. Separation is performed using a suitable eluent and gradient flow known in the art. It can be seen that the TNF-a isoform is eluted with a different peak pattern.

(viii) Results Amino acid composition
IFN-a2b was hydrolyzed and derivatized and analyzed by the described reverse phase high performance liquid chromatography to yield the following amino acid composition (Table 17). Results are expressed as the weight and percentage of occurrence of each amino acid in the sequence (including standard deviation (SD)). Glycine is a known contaminant in amino acid analysis that can artificially change the amino acid composition. Taking this into account, the results are comparable with theoretical values. The percentage of various other amino acids is also slightly different from the expected theoretical level. This may indicate the presence of low level contaminants.

(Table 17)

Monosaccharide Results Individual monosaccharides were hydrolyzed from the amino acid backbone of IFN-a2b and analyzed by high pH anion exchange chromatography (HP AEC) as described, resulting in the following compositional analysis. Glucose is a common contaminant and is not usually found in N- or O-linked oligosaccharides. Results from the samples are normalized to GalNAc (Tables 18-20). Table 21 is a summary of the results from the three samples.

(表１９)

(表２０)

(表２１)

(Table 18)

(Table 19)

(Table 20)

(Table 21)

  Taking into account the inherent variations in the chromatographic techniques described above, the monosaccharide and sialic acid content of the IFN-a2b of the present invention, when normalized to GalNAc, is about 1 to 0-3 fucose, 1 to 0- 3 GlcNAc, 1 to 0-6 galactose, 1 to 0-3 mannose and 1 to 0-5 NeuNAc, and in one embodiment, 1 to 0-1 fucose, 1 to 0-1 GlcNAc, 1 pair It is determined to be 1-4 galactose, 1 to 0-1 mannose and 1 to 0-2 NeuNAc.

  In view of the inherent diversity of the chromatographic techniques described above, sialic acid as a percentage of monosaccharide content of IFN-a2b of the present invention was determined to be in the range of about 0-10%, and IFN- The percentage of a2b acidic O-linked oligosaccharides is determined to be in the range of about 0-20%.

(b) Composition analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms of IFN-b1 of the present invention
(i) Sample Preparation for Analysis of Amino Acids, Monosaccharides, Oligosaccharides, Phosphate, Sulfates, and Isoforms Purified IFN-b1 in PBS was described previously in Example 4 (a) (i) Treat as described.

(ii) Analysis of amino acid composition by gas phase hydrolysis
A sample of the IFN-b1 preparation is treated as described above in Example 4 (a) (ii).

(iii) Neutral and amino monosaccharide composition analysis
Samples of the IFN-b1 preparation are treated as described above in Example 4 (a) (iii).

(iv) Composition analysis of acidic monosaccharides
Samples of the IFN-b1 preparation are treated as previously described in Example 4 (a) (iv).

(v) Oligosaccharide composition analysis
Samples of the IFN-b1 preparation are treated as described above in Example 4 (a) (v).

(vi) Sulfate and phosphate composition analysis
Samples of the IFN-b1 preparation are treated as described above in Example 4 (a) (vi).

(vii) Further separation of protein isoforms
A sample of the IFN-b1 preparation is treated as described above in Example 4 (a) (vii). The IFN-b1 isoform is found to elute with a distinct peak pattern.

(c) Composition analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms of IFN-g of the present invention
(i) Sample Preparation for Analysis of Amino Acids, Monosaccharides, Oligosaccharides, Phosphate, Sulfates, and Isoforms Purified IFN-g in PBS was described previously in Example 4 (a) (i). Treat as described.

(ii) Analysis of amino acid composition by gas phase hydrolysis
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (ii).

(iii) Neutral and amino monosaccharide composition analysis
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (iii).

(iv) Composition analysis of acidic monosaccharides
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (iv).

(v) Oligosaccharide composition analysis
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (v).

(vi) Sulfate and phosphate composition analysis
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (vi).

(vii) Further separation of protein isoforms
Samples of the IFN-g preparation are treated as described above in Example 4 (a) (vii). The IFN-g isoform is found to elute with a distinct peak pattern.

(d) Composition analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms of IFNAR2-Fc of the present invention
(i) Preparation of samples for analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms A solution of purified IFNAR2-Fc in PBS was described previously in Example 4 (a) (i). Treat as described.

(ii) Analysis of amino acid composition by gas phase hydrolysis
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (ii).

(iii) Neutral and amino monosaccharide composition analysis
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (iii).

(iv) Composition analysis of acidic monosaccharides
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (iv).

(v) Oligosaccharide composition analysis
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (v).

(vi) Sulfate and phosphate composition analysis
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (vi).

(vii) Further separation of protein isoforms
Samples of the IFNAR2-Fc preparation are treated as described above in Example 4 (a) (vii). The IFNAR2-Fc isoform is found to elute with a distinct peak pattern.

(e) Composition analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms of IL-10 of the present invention
(i) Sample Preparation for Analysis of Amino Acids, Monosaccharides, Oligosaccharides, Phosphate, Sulfates, and Isoforms Purified IL-10 in PBS was described previously in Example 4 (a) (i). Treat as described.

(ii) Analysis of amino acid composition by gas phase hydrolysis
Samples of IL-10 preparation are treated as described above in Example 4 (a) (ii).

(iii) Neutral and amino monosaccharide composition analysis
Samples of IL-10 preparation are treated as previously described in Example 4 (a) (iii).

(iv) Composition analysis of acidic monosaccharides
Samples of IL-10 preparation are treated as described above in Example 4 (a) (iv).

(v) Oligosaccharide composition analysis
Samples of IL-10 preparation are treated as described above in Example 4 (a) (v).

(vi) Sulfate and phosphate composition analysis
Samples of IL-10 preparation are treated as described above in Example 4 (a) (vi).

(vii) Further separation of protein isoforms
Samples of IL-10 preparation are treated as described above in Example 4 (a) (vii). The IL-10 isoform is found to elute with a distinct peak pattern.

(f) Composition analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms of IL-10Ra-Fc of the present invention
(i) Preparation of samples for analysis of amino acids, monosaccharides, oligosaccharides, phosphates, sulfates, and isoforms. Treated as described in

(ii) Analysis of amino acid composition by gas phase hydrolysis
Samples of IL-10Ra-Fc preparation were treated as described above in Example 4 (a) (ii).

(iii) Neutral and amino monosaccharide composition analysis
Samples of IL-10Ra-Fc preparation were treated as described above in Example 4 (a) (iii).

(iv) Composition analysis of acidic monosaccharides
Samples of IL-10Ra-Fc preparation were treated as described above in Example 4 (a) (iv).

(v) Oligosaccharide composition analysis
Samples of IL-10Ra-Fc preparation are treated as described above in Example 4 (a) (v).

(vi) Sulfate and phosphate composition analysis
Samples of IL-10Ra-Fc preparation were treated as described above in Example 4 (a) (vi).

(vii) Further separation of protein isoforms
Samples of the IL-10Ra-Fc preparation are treated as described above in Example 4 (a) (vii). The IL-10Ra-Fc isoform is found to elute with a distinct peak pattern.

(vii) Results Amino acid composition
IL-10Ra-Fc was hydrolyzed and derivatized and analyzed by the described reverse phase high performance liquid chromatography to yield the following amino acid composition (Table 22). Results are expressed as the weight and percentage of occurrence of each amino acid in the sequence, including standard deviation (SD). Glycine is a known contaminant in amino acid analysis that can artificially change the amino acid composition. Taking this into account, the results are comparable with theoretical values.

(Table 22)

Monosaccharides and sulfates Individual monosaccharides and sulfates were hydrolyzed from the amino acid backbone of IL-10Ra-Fc and analyzed by high pH anion exchange chromatography (HP AEC) as described to yield the following composition: Got the analysis. The results from the samples are normalized to GalNAc and 3 times the amount of mannose, respectively (Tables xxx-xxx). Table xxx summarizes the results from the three samples. Glucose is a common contaminant and is usually absent from N- or O-linked oligosaccharides. GlcNAc levels are high, which may be due to contaminants co-eluting with GlcNAc in the analysis.

(表２４)

(表２５)

(表２６)

(Table 23)

(Table 24)

(Table 25)

(Table 26)

  Taking into account the inherent variation of the chromatographic techniques described above, the IL-10Ra-Fc monosaccharide, sialic acid, and sulfate content of the present invention, when normalized to GalNAc, is about 1 to 0.1-4. Fucose, 1 to 2-34 GlcNAc, 1 to 0.5-8 galactose, 1 to 1-13 mannose, 1 to 0-3 NeuNAc, and 1 to 0-1.5 sulfate, 3 times the amount of mannose Normalized to about 3 to 0.1-2 fucose, 3 to 0.01-3 GlcNAc, 3 to 1-30 GlcNAc, 3 to 0.1-4 galactose, 3 to 0-3 NeuNAc, and 3 to 0-0.6 sulfate Determined to be.

  Amino acid composition data was combined with monosaccharide and sulfate data to provide various species content (Table 27).

(Table 27)

  In view of the inherent variations in the chromatographic techniques described above, sialic acid as a percentage of monosaccharide content of the IL-10Ra-Fc of the present invention was determined to be in the range of about 0-10%, and the IL of the present invention Sulfation as a percentage of monosaccharide content of -10Ra-Fc was determined to be in the range of about 0-3%, and the percentage of acidic N-linked oligosaccharides of IL-10Ra-Fc of the present invention was about 25 Determined to be ˜45%.

Example 5 Sugar Mass Fingerprinting Proteins of the invention are separated using 2D gel electrophoresis techniques as in Example 3 and blotted onto polyvinyldifluoroethane (PVDF) membranes. Spots are stained with one of a standard series of protein stains (Colloid Coomassie Blue, Sypro Ruby, or Deep Purple) and the relative isoforms are quantified using a densitometry algorithm. Individual spots are excised and treated appropriately with a series of deglycosylation enzymes and / or chemical means to remove the existing oligosaccharides according to the methods described herein. Once the oligosaccharide has been removed, it is separated and analyzed with a liquid chromatography-electrospray mass spectrometry system (LC-MS) using a graphitized carbon column and an organic solvent (MeCN) gradient elution system. The peak profile created is a “fingerprint” of the oligosaccharides present in the isoform. Furthermore, the mass spectrometric system simultaneously obtains information about the mass of each sugar present in the sample and uses this to identify their structure by pattern matching with the GlycoSuite database. In addition, individual mass peaks can be fragmented multiple times to obtain MS ⁿ spectra. These fragments allow structure prediction using methods known in the art, for example, using the GlycosidIQ software package.

  When the above separation, deglycosylation and analysis techniques are repeated with the corresponding proteins expressed in non-human cell lines such as E. coli, yeast, or CHO cells, each glycomass fingerprint will be significantly different. Is found. At the structural level, such results indicate that the pattern of glycan structures present in the proteins of the invention and the corresponding non-human cell expressed proteins are different.

Example 6 Fluorophore-assisted carbohydrate electrophoresis A fluorophore-assisted carbohydrate electrophoresis procedure (FACE procedure) is used to derive an oligosaccharide profile of a target molecule. Oligosaccharides derived from the target cytokine are hydrolyzed from the amino acid backbone using ammonium hydroxide and subsequently labeled using fluorophore 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS). Polyacrylamide gel electrophoresis is used to separate the standards and the standards used to identify oligosaccharide profiles typical of target molecules. In addition, oligosaccharides are identified by matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF), depending on the fluorophore and the specific matrix for ionizing each sugar. Determine the mass of each sugar and identify possible structures using the GlycoSuite database. The possible sugar structures are further characterized by tandem mass spectrometry techniques, allowing partial or complete characterization of the existing oligosaccharides and their relative amounts. In addition, this process is repeated using isoforms identified by 2D gel electrophoresis, creating a profile of oligosaccharides present in each isolated isoform.

Example 7 QCM and SPR
The binding properties and activity of the target molecule are determined using the quartz crystal microbalance method (QCM) or the surface plasmon resonance method (SPR). In either case, the appropriate receptor for the molecule is bound to the wafer using the chemistry described by the manufacturer. The target molecule is dissolved in an appropriate biological buffer and the target molecule interacts with it by passing the buffer through a receptor on the chip. The change in total protein mass on the wafer surface is measured by the change in oscillation frequency (in the case of QCM) or the change in light scattering properties of the chip (in the case of SPR). The chip is then treated with only biological buffer to observe the release of the target molecule into solution. The target molecule binding curve is then calculated using the rate at which the receptor reaches saturation and complete separation.

Example 8 Production of Transgenic Host Cell Line
(a) Transgenic host cell strain having α-2,6-sialyltransferase A cDNA encoding α-2,6-sialyltransferase (α2,6ST) is amplified from poly (A) -primed cDNA by PCR. The PCR product is ligated into an appropriate vector, such as pIRESpuro4 or pCEP4, to generate the α2,6ST plasmid. The cloned cDNA is sequenced and its identity is confirmed by comparison with the published α-2,6ST cDNA sequence. DNA sequencing is performed using known methods.

  Mammalian host cells containing the same lineage of cell clones that express high levels of the target molecule (cell line-target molecule) are transfected with an α2,6ST plasmid that also has an antibiotic resistance marker. Selection of stably transfected cells is done by incubating the cells in the presence of antibiotics; colonies of antibiotic-resistant cells that emerge after transfection are pooled and tested for intracellular α 2,6ST activity . In order to isolate individual cell clones expressing α2,6ST, the cell pool is cloned by a limiting dilution step as described by Kronman (Gene 121: 295-304, 1992). Individual cell clones are selected at random, the cells are expanded, and the clones are tested for α2,6ST activity.

  The cell pellet is washed, resuspended in lysis buffer, placed on ice and then sonicated. The cell lysate is centrifuged and the clear supernatant is assayed for protein concentration (by known methods) and sialyltransferase activity. Sialyltransferase activity is assayed by known methods, such as those detailed by Datta et al. (J Biol Chem 270: 1497-1500, 1995).

  Highly expressed α2,6ST cell line—Target molecule From the cells, the expressed target molecule is purified and subjected to in vitro and / or in vivo half-life bioassays (see Example 10). Target molecules derived from high-expressing α 2,6ST cells are in vitro and / or compared to target molecules derived from the same parent cell line or other cell lines that have not undergone subsequent transgene manipulation. 1 shows an extended in vivo half-life.

(b) Transgenic host cell line with fucosyltransferase
A cDNA encoding a fucosyltransferase (FT) such as FUT1, FUT2, FUT3, FUT4, FUT5, FUT6, FUT7, FUT8, FUT9, FUT10, FUT11, etc. is amplified from the poly (A) -primed cDNA by PCR. The PCR product is ligated into an appropriate vector, such as pIRESpuro4 or pCEP4, to generate the α2,6ST plasmid. The cloned cDNA is sequenced and its identity is confirmed by comparison with the published FT cDNA sequence. DNA sequencing is performed using known methods.

  Human host cells containing the same lineage of cell clones (cell line-target molecule) expressing high levels of the target molecule are transfected with an FT plasmid that also has an antibiotic resistance marker. The selection of stably transfected cells is done by incubating the cells in the presence of antibiotics; antibiotic resistant cell colonies that appear after transfection are pooled and tested for intracellular FT activity. To isolate individual cell clones that express FT, the cell pool is cloned by a limiting dilution step as described by Kronman (1992, supra); individual cell clones are randomly selected and the cells are Expand and test clones for FT activity.

  The cell pellet is washed, resuspended in lysis buffer, placed on ice and then sonicated. The cell lysate is centrifuged and the clear supernatant is assayed for protein concentration (by known methods) and FT activity. FT activity is assayed by known methods, such as those detailed by Mas et al. (Glycobiology 8 (6): 605-13, 1998).

  Highly expressed FT cell line—Target molecule is purified from the target cell. For example, Lewis x-specific antibodies such as L5 and KM93, HECA493, 2H5, or CSLEX according to methods known in the art as detailed in Lucca et al. (Glycobiology 15 (1): 87, 2005). Are tested for the presence of a Lewis x structure or a sialyl Lewis x structure. Alternatively, the presence of Lewis x structure or sialyl Lewis x structure can be determined by treating the sample with an appropriate glycosidase and detecting the effect of glycosidase on parameters such as mass using MS or retention time using HPLC. It can also be determined. Sugar mass fingerprinting as described in Example 5 can also be used to predict the presence of a Lewis x structure or a sialyl Lewis x structure.

Example 9 Differential Gene Expression Differences in gene expression can be analyzed using target cell lines of target molecules. Target cells are cultured to the appropriate density and treated with a range of concentrations of target molecule or buffer control for a time such as 72 hours.

  At various time points RNA is recovered, purified and reverse transcribed according to Affymetrix procedures. A labeled cRNA (eg, biotin label) is then prepared and hybridized to an expression array such as U133 GeneChip. After washing and signal amplification, the GeneChip is scanned using a GeneChip scanner (Affymetrix) and hybridization intensity and fold change information at various time points is obtained using GeneChip software (Affymetrix).

  The target molecule induces unique gene expression, resulting in a different mRNA profile as compared to profiles induced by cytokines or receptors produced from various sources such as E. coli, yeast, or CHO cells.

Example 10 Determination of the half-life of a target molecule of the invention The half-life of a target molecule is determined in an in vitro system. The composition containing the target molecule is mixed with human serum / plasma and incubated at a specific temperature for a specific time (eg, 4 hours at 37 ° C., 12 hours, etc.). The amount of target molecule remaining after this treatment is determined by ELISA or dot blot methods known in the art. The biological activity of the remaining target molecule is determined by performing an appropriate bioassay selected by those skilled in the art. The selected serum can be selected from various human blood types (eg, type A, type B, type AB, type O, etc.).

  The half-life of the target molecule is also determined in an in vivo system. The composition comprising the target molecule is labeled with a radioactive tracer (or other means) and is suitable for the species of test, e.g. mouse, rat, pig, primate, or human intravenous, subcutaneous, retro-orbital, intramuscular, or Inject intraperitoneally. Blood samples are taken at time points after injection and assayed for the presence of the target molecule (either by ELISA, dot blot, or by a trichloroacetic acid (TCA) precipitable label such as a radioactive count). Comparative compositions consisting of target molecules produced from other sources such as E. coli, yeast, or CHO cells can be run as controls.

Example 11
(a) In Vivo Studies Using the Target Molecules of the Invention Individual subjects of the in vivo studies described herein are warm-blooded vertebrates, including humans.

  Clinical trials are subject to strict controls to ensure that individuals are not unnecessarily endangered and that individuals are well informed about their role in the study.

  The test is preferably done in a double-blind manner to account for the psychological effects of receiving treatment. Volunteers are randomly assigned to placebo or target molecule treatment groups. In addition, the relevant clinician is not informed of the plan made for a given subject in order to avoid biasing findings after treatment. Using this randomized approach, each volunteer has the same opportunity to be given a new treatment or placebo.

  Volunteers are given the target molecule or placebo for an appropriate period of time, and the biological parameters associated with the indicated condition or condition are determined at the first time point (baseline measurement before treatment), at the end (after the last treatment), and study Measure regularly during the period. Such measurements include target molecule levels in body fluids, tissues, or organs compared to pre-treatment levels. Other measures include, but are not limited to, an indication of the condition or condition being treated, body weight, blood pressure, serum titer of a pharmacological indicator of a disease such as a particular disease indicator, or toxicity, and ADME (absorption, Measurements of distribution, metabolism, and excretion) are included.

  Information recorded for each patient includes age (years), gender, height (cm), family history (yes / no) of the condition or condition, motivation assessment (little / moderate / high), and the disease or condition indicated This includes the number and type of previous treatment plans for.

  Volunteers participating in this study are adults aged 18 to 65 years, and approximately the same number of men and women participate in the study. Volunteers with certain characteristics are equally distributed for placebo and targeted molecule treatment. In general, volunteers treated with placebo have little response to treatment, whereas volunteers treated with the target molecule show a favorable trend in disease state or condition indicators at the end of the study.

(b) Treatment of chronic hepatitis using IFN-a2b of the present invention The individual subjects of the in vivo studies described herein are warm-blooded vertebrates, including humans.

  Clinical trials are subject to rigorous control so that individuals are not unnecessarily at risk and that individuals are fully informed about their role in the trial.

  In certain embodiments, trials are performed in a double-blind manner to compensate for the psychological effects of receiving treatment. Volunteers are randomly assigned to treatment groups of recombinant interferon α2-b (IFN-a2b) expressed in E. coli or IFN-a2b of the present invention. In addition, to prevent prejudice in the physician's treatment, physicians are informed whether the drug they administer is IFN-a2b (expressed in E. coli) or IFN-a2b of the present invention. Absent. With this randomized approach, each volunteer has the same opportunity to receive either an existing treatment or a new treatment.

  Volunteers are given either IFN-a2b (expressed in E. coli) or IFN-a2b of the present invention for a period of time to determine the biological parameters associated with chronic hepatitis C at the start of the study (before any treatment is performed). Baseline measurement), at the end (after the last treatment), and at regular intervals during the study period. Such measurements include IFN-a2b levels in body fluids, tissues, or organs and neutralizing antibody levels against IFN-a2b compared to pre-treatment levels. Other measurements include ADME such as serum half-life and solubility, along with serum titers of pharmacological indicators of chronic hepatitis C such as body weight, blood pressure, specific liver enzymes (e.g. alanine aminotransferase) or toxicity. (Absorption, distribution, metabolism and excretion) measurements include but are not limited to.

  Information recorded for each patient includes age (years), gender, height (cm), family disease status or history of condition (yes / no), motivational scoring (slight / moderate / pretty), and This includes the number and type of previous treatment regimens for chronic hepatitis C.

  Volunteers who participated in this study are adults aged 18 to 65 years, and almost the same number of men and women are participating in the study. Volunteers with certain characteristics are equally distributed for IFN-a2b (expressed in E. coli) or IFN-a2b treatment of the invention. In general, volunteers treated with IFN-a2b of the present invention show increased liver function at the end of the study compared to volunteers treated with commercially available IFN-a2b (expressed in E. coli).

(c) Determining the efficacy of IFN-b1 of the invention in combination with recombinant human erythropoietin (rhEPO) therapy in the treatment of multiple sclerosis Individual subjects in the in vivo studies described herein are warm-blooded vertebrae An animal, including humans.

  Clinical trials are subject to rigorous control so that an individual is fully informed about its role in the trial so that the individual is not unnecessarily at risk.

  In certain embodiments, trials are performed in a double-blind manner to compensate for the psychological effects of receiving treatment. A subject is randomly administered IFN-b1 of the present invention in combination with optimal background (OB) therapy or placebo (OB therapy with EPO therapy alone) for recombinant human erythropoietin (EPO) therapy. With this randomized approach, each volunteer has the same opportunity to receive either a new treatment or a placebo.

  The subject for the trial is a human who exhibits an acute demyelination event consistent with multiple sclerosis. Information recorded for each patient includes age (years), gender, height (cm), family disease status or history of condition (yes / no), motivational scoring (slight / moderate / pretty), and This includes the number and type of previous treatment therapies for multiple sclerosis.

  The route of administration of IFN-b1 of the present invention in combination with OB therapy or placebo for EPO treatment is (but is not limited to) intradermal, intramuscular, or subcutaneous injection.

  Subjects are administered IFN-b1 of the present invention for an appropriate period in conjunction with EPO-treated OB therapy or placebo to study biological parameters associated with acute demyelination events consistent with multiple sclerosis Measure at regular intervals at the beginning (baseline measurement before any treatment), at the end (after the last treatment), and during the study period. Such measurements include IFN-b1 and EPO levels in body fluids, tissues, or organs compared to pre-treatment levels. Other measures are treated, such as multiple sclerosis functional composite score (MSFC), body weight, blood pressure, serum anti-IFN-β titer, and anti-EPO neutralizing antibody (NAbs), or toxicity This includes, but is not limited to, ADME (absorption, distribution, metabolism and excretion) measurements, and health-related quality of life indicators as well as disease state or condition indicators.

  Patients receiving IFN-b1 treatment of the present invention include lower MSFC scores, lower serum anti-IFN-β and anti-EPO NAb titers, longer survival times and better overall general health One or more indications of lower multiple sclerosis are shown without limitation.

Example 12
(a) Comparison of biological activity of IFN-a2b of the present invention and IFN-a2b expressed in non-human cell lines
IFN-a2b is cytotoxic to the human erythroleukemia TF-1 cell line. TF-1 cells proliferate when treated with GM-CSF, but IFN-a2b inhibits this proliferation leading to cell death.

  In a 96-well plate, 20,000 TF-1 cells / well of 0.2 ng / ml GM-CSF (Peprotech catalog number 300-03 or R & D Systems catalog number 215-GM-010), and 0-100 ng / ml Treatment with IFN-a2b of the present invention at 37 ° C. for 68 hours. Cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices).

  The above assay was repeated using IFN-a2b produced in E. coli (WHO 95/566). Each ED50 value was calculated using a logarithmic plot of absorbance versus IFN-a2b concentration and a 4-parameter equation.

  The ED50 of IFN-a2b of the present invention (0.011-0.017 ng / ml) was significantly different from the ED50 of human IFN-a2b produced in E. coli (4.4-6.6 ng / ml; see FIG. 2) . Thus, IFN-a2b of the present invention was 250-600 times more potent than human IFN-a2b expressed in E. coli in inhibiting GM-CSF-induced proliferation of TF-1 cells.

(b) Comparison of the biological activity of IFN-b1 of the present invention against IFN-b1 expressed using a non-human system
IFN-b1 is cytotoxic to the human erythroleukemia TF-1 cell line. TF-1 cells proliferate when treated with GM-CSF, but IFN-b1 inhibits this proliferation leading to cell death.

  In a 96-well plate, 20,000 TF-1 cells / well of 0.2 ng / ml GM-CSF (Peprotech catalog number 300-03 or R & D Systems catalog number 215-GM-010), and 0-100 ng / ml Treatment with IFN-b1 of the present invention at 37 ° C. for 68 hours. Cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices). The ED50 was calculated after fitting the absorbance curve, and the value of EPO concentration was calculated using a 4-parameter equation.

  The ED50 of IFN-b1 of the present invention was 35 to 53 ng / ml (FIG. 3).

  When the above assay is repeated using human IFN-b1 expressed in non-human cell lines such as E. coli, yeast, or CHO cells, the ED50 is found to be significantly different.

(c) Comparison of the activity of IFN-g of the present invention and IFN-g expressed using a non-human system
IFN-g is known to inhibit the growth of human HT-29 cells in the presence of TNF-a. In 96 well plates, 4000 HT-29 cells / well with 1 ng / ml TNF-a (Peprotech catalog number 300-01A or R & D Systems catalog number 210-TA-010) and 0-20 ng / ml of the present invention Of IFN-g for 88 hours at 37 ° C. Next, the cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices). The ED50 was calculated after fitting the absorbance curve, and the value of IFN-g concentration was calculated using a 4-parameter equation.

  The above assay was repeated with human IFN-g (Peprotech catalog number 300-02) expressed in E. coli cells.

  The ED50 of IFN-g of the present invention (0.04-0.06 ng / ml) was significantly different from the ED50 of human IFN-g produced in E. coli (0.44-0.66 ng / ml; see FIG. 4) . Thus, IFN-g of the present invention was 11 to 17 times more potent than human IFN-g expressed in E. coli in inhibiting the growth of HT-29 cells in the presence of TNF-a.

(d) Comparative bioassay of IFNAR2-Fc of the present invention and soluble IFNAR2 molecules expressed in non-human cells
IFN-a2B is cytotoxic to the human erythroleukemia TF-1 cell line. TF-1 cells proliferate when treated with GM-CSF, but IFN-a2B inhibits this proliferation leading to cell death. Addition of IFNAR2 neutralizes the action of IFN-a2B, resulting in GM-CSF-induced proliferation of TF-1 cells.

  In a 96-well plate, 20000 TF-1 cells / well at 0.2 ng / ml GM-CSF (Peprotech catalog number 300-03 or R & D Systems catalog number 215-GM-010), and 0-5000 ng / ml Treated with IFNAR2-Fc of the present invention at 37 ° C. for 24-48 hours. Cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices).

  After fitting the absorbance curve, the ED50 was calculated and the IFNAR2-Fc concentration value of the present invention was calculated using a 4-parameter equation. The ED50 of IFNAR2-Fc of the present invention was 0.14 to 0.18 ng / ml (FIG. 5).

(e) Comparison of IL-10 activity of the present invention and IL-10 expressed in non-human systems
IL-10 induces the growth of the murine mast cell line MC / 9 in the presence of IL-4.

  In 96-well plates, 21,000 MC / 9 cells / well were treated with 0-50 ng / ml IL-10 for 90 hours at 37 ° C. with 200 pg / ml IL-4 (Peprotech catalog number 200-04). Cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices). Each ED50 was calculated using a logarithmic plot of absorbance versus IL-10 concentration and a 4-parameter equation.

  The above assay was repeated with human IL-10 expressed in E. coli (Peprotech catalog number 1-9276).

  The ED50 of IL-10 of the present invention (0.012-0.018 ng / ml) was significantly different from the ED50 of human IL-10 produced in E. coli (0.19-0.29 ng / ml; see FIG. 6) . Thus, IL-10 of the present invention was 10-25 times more potent than human IL-10 expressed in E. coli in the growth of MC / 9 cells in the presence of IL-4.

(f) Comparison of the biological activity of IL-10Rα-Fc of the present invention and IL-10Rα-Fc expressed using a non-human system The mouse mast cell line MC / 9 responds to a series of cytokines. IL-10 has been reported to induce proliferation in MC / 9 cells in the presence of IL-4. IL-10Rα-Fc blocks IL-10 activity by binding to IL-10 and competitively inhibits the binding of these molecules to its cellular IL-10 receptor site. Is biologically inert. Thus, incubation of IL-10 with IL-10Rα-Fc neutralizes the proliferative effect of IL-10 on MC / 9 cells.

  In a 96-well plate, 1 ng / ml IL-10 (Peprotech catalog number 1-9276) was incubated with 0-2500 ng / ml IL-10Ra-Fc of the present invention for 1 hour at 37 ° C. 21,000 cells / well of MC / 9 cells with 200 pg / ml IL-4 (Peprotech catalog number 200-04) were added at 37 ° C. for 90 hours. Cell number was measured using CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega). In this assay, the tetrazolium compound MTS (3- (4,5-dimethylruthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2 in the presence of an electronic coupling reagent (phenazine methosulfate). -(4-sulfophenyl) -2H-tetrazolium) is bioreduced by the cells into the formazan product. The formazan concentration was determined by reading the absorbance of the resulting solution at 490 nm with a spectrophotometer (E max precision microplate reader, Molecular Devices).

  The above assay was repeated with soluble IL-10Rα molecules (R & D Systems catalog number 274-R1) expressed in E. coli cells.

  Each neutralization dose (ND50) was calculated using a logarithmic plot of absorbance versus IL-10Ra-Fc concentration and a 4-parameter equation.

  In the presence of 0.5 ng / ml IL-10, the ND50 of IL-10Ra-Fc of the present invention (0.8-1.0 ng / ml) is equivalent to that of soluble human IL-10Ra produced in E. coli (18-121 ng / ml). ml; significantly different from FIG.

  Thus, IL-10Ra-Fc of the present invention was 18-150 times more potent than soluble human IL-10Ra expressed in E. coli in inhibiting IL-10-induced proliferation of MC / 9 cells.

Example 13
(a) In vitro comparison of immunoreactivity profiles of IFN-a2b of the present invention and human IFN-a2b expressed using a non-human system Protein estimation of IFN-a2b of the present invention is based on standard protein assay techniques. Using, for example, the calculated absorbance count (ε) and the molecular mass measured based on SDS-PAGE analysis, by A280 absorbance method, or Bradford protein assay (Bradford Anal Biochem 72: 248-254, 1976) Determined by.

  The IFN-a2b of the present invention, normalized using standard protein assay results, can be obtained in a suitable commercially available IFN-a2b quantitative immunoassay supplied with non-human cell expressed IFN-a2b standards, e.g., anti-IFN-a2b In an ELISA kit, it is diluted and tested according to the manufacturer's instructions.

  Alternatively, IFN-a2b levels of the present invention are determined using quantitative immunoassay techniques developed using components available from commercial sources. For example, anti-IFN-a2b ELISA uses human IFN-a Mab, eg, using R & D Systems' IFN-a Mab (Cat # 21112-1) as a capture antibody; human IFN-a Pab, eg, the art A suitable detection molecule, for example using R & D Systems' IFN-a Pab (Catalog No. 331100-1) tagged with biotin as the detection antibody, and using recombinants expressed in E. coli cells Type human IFN-a2b, for example R & D Systems recombinant human IFN-a2b (Cat. No. 11105-1) is used as a protein standard. The protein concentration of IFN-a2b of the present invention, standardized using standard protein assay results, is assayed by the reagents described above using ELISA methods known in the art.

  The protein concentration of the IFN-a2b of the present invention determined by a quantitative immunoassay developed using a commercially available ELISA kit or component of origin is determined by the capture and / or detection antibody used in the commercially available ELISA kit or immunoassay technique. However, it will be different from the concentration determined by standard protein assays because it is made against non-human cell expressed human IFN-a2b protein.

  At the structural level, such results will show different immunoreactivity profiles of IFN-a2b and non-human cell expressed human IFN-a2b molecules of the invention.

(b) In vitro comparison of immunoreactivity profiles of IFN-b1 of the present invention and human IFN-b1 expressed using a non-human system The protein estimation of IFN-b1 of the present invention is based on standard protein assay techniques. Using, for example, determined by the A280 absorbance method using a calculated absorbance count (ε) and molecular mass measured based on SDS-PAGE analysis, or by the Bradford protein assay (Bradford 1976, supra).

  The IFN-bl of the present invention, standardized using standard protein assay results, is diluted in a commercial ELISA kit, for example, in the R & D Systems human IFN-bl ELISA kit (Cat. # 41400-1) according to the manufacturer's instructions. And tested. The above ELISA kit uses human IFN-b1 expressed in E. coli cells as a standard substance.

  The protein concentration of the IFN-b1 of the present invention determined by a commercially available ELISA kit indicates that the capture and / or detection antibodies used in the commercially available ELISA kit are made against non-human cell expressed human IFN-b1 protein This will be different from the concentration determined by standard protein assays. At the structural level, such results will show different immunoreactivity profiles of IFN-b1 of the present invention and non-human cell expressed human IFN-b1 molecules.

(c) In vitro comparison of immunoreactivity profiles of IFN-g of the present invention and human IFN-g expressed using a non-human system Protein estimation of IFN-g of the present invention uses standard protein assay techniques As determined by the A280 absorbance method using, for example, the calculated absorbance count (ε) and molecular mass measured based on SDS-PAGE analysis, or by the Bradford protein assay (Bradford 1976, supra).

  Standardized using standard protein assay results, the IFN-g of the present invention is a commercially available ELISA kit, such as the R & D Systems human IFN-γDuoSet® ELISA kit (catalog number DY285), or the R & D Systems human IFN-γQuantiKine. Diluted and tested in a ® ELISA kit (Catalog Number DIF50) according to manufacturer's instructions. The above ELISA kit uses human IFN-g expressed in E. coli as a standard substance.

  The protein concentration of the IFN-g of the present invention determined by a commercially available ELISA kit indicates that the capture and / or detection antibodies used in the commercially available ELISA kit are made against non-human cell expressed human IFN-g protein This will be different from the concentration determined by standard protein assays.

  At the structural level, such results will show different immunoreactivity profiles of the IFN-g of the invention and non-human cell expressed human IFN-g molecules.

(d) In vitro comparison of immunoreactivity profiles of IL-10 of the present invention and human IL-10 expressed using a non-human system.Protein estimation of IL-10 of the present invention was performed using the R & D Systems human IL-10 DuoSet ( Determined according to manufacturer's instructions using a registered trademark ELISA kit (catalog number DY217B).

  IL-10 of the present invention, normalized using the above assay results, was diluted and tested using the R & D Systems human IL-10 DuoSet® ELISA kit (Cat. No. DY217B) according to the manufacturer's instructions. The above ELISA kit uses human IL-10 expressed in E. coli as a standard substance.

  The results of the R & D Systems DuoSet® human IL-10 ELISA kit resulted in a standard curve of IL-10 expressed in E. coli with a concentration curve of the IL-10 of the present invention at OD450 nm (FIG. 8). . Analysis of these data by log-log curve fitting performed by Molecular Devices curve fitting software yielded further results as well (Table 28).

Table 28: Values derived from IL-10 ELISA fit curve (log (y) = A + B ^* log (x))

  These results use E. coli expressed human IL-10 standards and antibodies to E. coli expressed human IL-10 that have been used to quantify the level of native human expressed IL-10 in research and human patient samples The R & D Systems human IL-10 DuoSet® ELISA kit, which is a commercially available kit, underestimates the IL-10 concentration of the present invention. Values derived from the fitted curves indicate that the correlation coefficient is high for both curves (Table 28). However, the curve slope of IL-10 of the present invention was different from that for human IL-10 expressed in E. coli (Table 28). Furthermore, the IL-10 of the present invention and the y-section of human IL-10 expressed in E. coli are different (Table 28), suggesting that there are inherent differences in the immunoreactivity of these two proteins. .

  In summary, these results show different immunoreactivity profiles of IL-10 of the present invention and non-human cell expressed IL-10 molecules.

(e) In vitro comparison of immunoreactivity profiles of IL-10Ra-Fc of the present invention and IL-10Ra-Fc expressed using a non-human system. IL-10Ra-Fc of the present invention is a suitable protein assay. For example, it was determined using the Raleigh protein estimation method using human IgG as a standard substance.

  The IL-10Ra-Fc of the present invention, standardized using standard protein assay results, is subjected to quantitative immunoassay techniques developed using components available from commercial sources. For example, anti-IL-10Ra-Fc ELISA uses human IL-10Ra-Fc Mab (R & D Systems catalog number MAB274) as a capture antibody and biotinylated human IL-10Ra-Fc Pab (R & D Systems catalog number BAF874) as a detection antibody. , And recombinant human IL-10Ra-Fc (R & D Systems catalog number 874-RB-100) expressed in Sf 21 insect cells, as a protein standard. The protein concentration of the IL-10Ra-Fc of the present invention, standardized using standard protein assay results, is assayed by the reagents described above using ELISA methods known in the art.

  The protein concentration (as a monomer) of the IL-10Ra-Fc of the present invention, determined by a quantitative immunoassay developed with the source component, is the capture and / or detection antibody used in the immunoassay technique is non-human. Concentrations determined by standard protein assays will be different because they are made against cell-expressed human chimeric IL-10Ra-Fc protein. It should be noted that the IL-10Ra-Fc of the present invention is expressed as a homodimer.

  At the structural level, such results will show different immunoreactivity profiles of IL-10Ra-Fc of the present invention and non-human cell expressed human chimeric IL-10Ra-Fc molecules.

Example 14 Further Purification of Target Molecules of the Invention and Peptide Mass Fingerprinting by ESI-MS / MS In addition to the purification procedure described in Example 2, the purification of target molecules of the invention can be performed using commercially available columns. Perform by HPLC. The eluted protein is monitored by absorbance at 215 nm or 280 nm and recovered with correction for delay due to tube volume between flow cell and collection port.

  As described in Example 3, gel fragments containing protein samples from 1D or 2D gels are digested in trypsin solution. Alternatively, the solution containing the protein sample is digested with trypsin in ammonium bicarbonate buffer (10-25 mM, pH 7.5-9). Incubate the solution at 37 ° C. overnight. The reaction is then stopped by adding acetic acid until the pH is in the range of 4-5. Peptide samples were prepared using prefabricated microcolumns containing C18 Zip-Tip (Millipore, Bedford, Mass.) Or Poros R2 chromatography resin (Perspetive Biosystems, Framingham, Mass.) As described in Example 3. Concentrate and desalinate.

The protein sample (2-5 μl) is injected onto a micro C18 pre-column, washed with 0.1% formic acid at 30 μl / min, concentrated and desalted. After washing for 3 minutes, the precolumn is switched to an analytical column (Atlantis, 75 μm × 100 mm, Waters Corporation) containing C18 RP silica. Using a linear solvent gradient from H ₂ O: CH ₃ CN (95: 5; + 0.1% formic acid) to H ₂ O: CH ₃ CN (20:80; + 0.1% formic acid), 200 nl / The peptide is eluted from the column over 40 minutes. The LC eluent is subjected to cation nanoflow electrospray analysis on a Micromass QTOF Ultima mass spectrometer (Micromass, Manchester, UK).

  Tandem MS is performed using a Q-Tof hybrid quadrupole / direct acceleration TOF mass spectrometer (Micromass). QTOF operates in data-dependent collection mode (DDA). TOFMS search scans were collected (m / z 400-2000, 1.0 s) and the three largest multivalent ions (count> 15) in the search scan were sequentially subjected to MS / MS analysis. MS / MS spectra were accumulated for 8 seconds (m / z 50-2000).

  Search LC / MS / MS data using Mascot (Matrix Science, London, UK) and Protein Lynx Global Server ("PLGS") (Micromass). The protein sample is expected to be the target molecule.

Example 15
(a) Immunogenicity in non-human animals
(i) Immunization of animals with a target protein Immunize the expressed protein. One month after immunization, the animals are subjected to secondary immunization. Prior to immunization, the protein is emulsified in an adjuvant, eg, complete Freund's adjuvant for primary immunization and incomplete Freund's adjuvant for secondary immunization.

(ii) Detection of antibody to target protein In order to detect antibody response, blood is collected from the tail of each group of animals and the serum is pooled. Protein-specific antibodies are detected by solid phase ELISA using 50 ng / well of the protein of the invention. Various immunoglobulin isotypes are detected using labeled detection antibodies produced against IgG1, IgG2, IgG2b, IgG3, IgM, IgA, and IgD. Alternatively, the antibody response is measured against a protein of the invention blotted on a membrane as a dot blot or a Western blot. Detection of various immunoglobulin isotypes is detected as described above. It is expected that the proteins of the invention will elicit an antibody response that is different from antibodies of proteins expressed in non-human cells.

(iii) T cell proliferation assay Euthanized animals are euthanized and spleen cells are prepared. An appropriate number of spleen cells, eg, 5 × 10 ⁵ cells, derived from an animal immunized with the protein of the present invention are cultured with various concentrations of the protein of the present invention, while simultaneously expressing the protein expressed in non-human cells Equivalent numbers of spleen cells from the immunized animal are cultured with proteins expressed in various concentrations of non-human cells. To perform a T cell proliferation assay, spleen cells are cultured for 96 hours and treated with 1 μCi [ ³ H] thymidine (6-7 μCi / umol) for the last 16 hours. Cells are harvested on filter strips and [ ³ H] thymidine incorporation is measured using standard methods. It is expected that the proteins of the invention will elicit a different proliferative response compared to proteins expressed in non-human cells.

(iv) IFNγ assay
To perform an IFNγ assay, the culture supernatant of spleen cells incubated with the protein of the invention or expressed in non-human cells is collected at 96 hours and sandwich ELISA, eg R & D Systems anti-IFNγ Quantikine® Detect with ELISA kit (catalog number DIF50) according to manufacturer's instructions. It is expected that IFNγ production of culture supernatants derived from cells incubated with the proteins of the invention will be different compared to culture supernatants derived from cells incubated with proteins expressed in non-human cells.

(b) In vitro human immunogenicity assay
(i) Human T cell response assay
Human dendritic cells and CD4 ⁺ T cells are prepared from human blood as described in Stickler et al. Toxicological Sciences 77: 280-289, 2004. Co-cultures of dendritic cells and CD4 ⁺ T cells containing 2 x 10 ⁴ dendritic cells and 2 x 10 ⁵ CD4 ⁺ T cells are plated in 96 well plates. The protein of the present invention and the protein expressed in non-human cells can be converted into peptides using a suitable enzyme determined by cleavage site prediction software such as Peptide Cutter ( http://au.expasy.org/tools/peptidecutter ). Enzymatic digestion into fragments. The resulting peptide fragment is purified by a suitable technique such as liquid chromatography and added to the co-culture to a final concentration of 5 ug / ml. Cultures are incubated for 5 days, then 0.5 uCi ³ H thymidine is added to each culture. Cells are harvested on filter strips and cell proliferation is measured by [ ³ H] thymidine incorporation.

  It is expected that peptides derived from the proteins of the present invention will elicit a weaker proliferative response compared to peptides derived from proteins expressed in non-human cells.

(ii) Human antibody response assay Blood is collected from a human donor who has been treated with a protein expressed in non-human cells and serum is prepared. Protein-specific antibodies are detected by solid phase ELISA against 50 ng / well of the protein of the invention and proteins expressed in non-human cells. Various immunoglobulin isotypes are detected using labeled detection antibodies produced against human IgG1, IgG2, IgG3, IgG4, IgM, IgA, and IgD.

  Alternatively, antibody responses are measured against proteins of the invention and proteins expressed on non-human cells blotted on membranes as dot blots or Western blots. Detection of various immunoglobulin isotypes is detected as described above.

  Immunoglobulins present in the serum of persons treated with proteins expressed in non-human cells bind to proteins expressed in non-human cells but bind weakly or not to the proteins of the present invention. Is predicted.

Example 16
Preparation of proteins of the invention from recombinant genomic constructs Genomic sequences encoding IFN-a2b, IFN-bl, IFN-g or IL-10 of the invention (SEQ ID NOs: 139, 140, 141, 142, respectively) ) By PCR and suitable expression vectors such as pIRESbleo3, pCMV-SPORT6, pUMCV3, pORF, pORF9, pcDNA3.1 / GS, pCEP4, pIRESpuro3, pIRESpuro4, pcDNA3.1 / Hygro (+), pcDNA3. Cloning into 1 / Hygro (-), pEF6 / V5-His. These recombinant constructs are then prepared for human cell transformation as previously described in Example 1 (c). Production and purification of the IFN-a2b, IFN-bl, IFN-g or IL-10 of the present invention from recombinant DNA constructs is performed as described above in Example 2.

  Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. The present invention should be understood to include all such changes and modifications. The invention also includes all of the steps, features, compositions, and compounds cited or shown herein, individually or collectively, and any and all combinations of any two or more of such steps or features. including.

References

1 is a schematic representation of a cloning process for inserting a cDNA encoding a protein of the invention into a pIRESbleo3 or pIRESbleo3-Fc vector. It is a graph which shows the cytotoxic effect of IFN-a2b (circle) of this invention and human IFN-a2b (square) expressed in E. coli on GM-CSF-induced proliferation of TF-1 cells. It is a graph which shows the cytotoxic effect of IFN-b1 (circle) of the present invention on GM-CSF-induced proliferation of TF-1 cells. It is a graph which shows the inhibitory effect of human IFN-g (circle) of this invention and the human IFN-g (square) expressed in E. coli cells on the growth of human HT-29 cells in the presence of TNF-a. 2 is a graph showing the neutralizing effect of IFNAR2-Fc of the present invention on IFN-a2b-mediated cytotoxicity of GM-CSF-induced TF-1 cell proliferation. FIG. 6 is a graph showing the effect of IL-10 (circle) of the present invention and human IL-10 (square) expressed in E. coli on the proliferation of MC / 9 cells in the presence of IL-4. Neutralization of IL-10R-Fc (circles) of the present invention and soluble human IL-10R molecules (squares) expressed in E. coli cells on IL-10-mediated proliferation of MC / 9 cells in the presence of IL-4 It is a graph which shows an effect. FIG. 5 is a graph showing an in vitro comparison of immunoreactivity profiles of IL-10 (squares) of the present invention and human IL-10 (diamonds) expressed in E. coli cells. Error bars represent the standard error of the mean value.

Claims (16)

An isolated protein comprising a profile of measurable physiochemical parameters, wherein the profile exhibits, is related to or forms the basis of one or more unique pharmacological traits, The isolated protein contains a physiochemical profile that includes a number of measurable physiochemical parameters {[P _x ] ₁ , [P _x ] ₂ ,... [P _x ] _n }, _where P _x is Represents a measurable physiochemical parameter, where “n” is an integer of > 1, and each of [P _x ] ₁ to [P _x ] _n is a different measurable physiochemical parameter and is measurable Any one value of a physiochemical characteristic, or a series of values of a plurality of measurable physiochemical characteristics, can be represented by a unique pharmacological trait T _y or a series of distinct physiochemical traits {[T _y ] ₁ , [T _{y] 2,} .... it shows the [T _{y] m},} associated with it, or to form the basis, wherein, T _y is Represents a chromatic pharmacological trait, m is> an integer 1, are [T _y] respectively ₁ ~ [T _{y] m,} a different pharmacological trait, isolated protein is IFN-a2B, IFN An isolated protein selected from the group comprising bl, IFN-g, IFNAR2, IL-10 and IL-10Ra-Fc.   2. The isolated protein of claim 1 comprising one or more measurable physiochemical parameters described in Table 2.   2. The isolated protein of claim 1 comprising one or more unique pharmacological traits described in Table 3.   A chimeric molecule comprising isolated IFN-a2B, IFN-bl, IFN-g, IFNAR2 or IL-10, or a fragment thereof, according to claim 1, fused to one or more peptides, polypeptides, or protein moieties. .   5. The chimeric molecule of claim 4, wherein the peptide, polypeptide, or protein portion comprises a human immunoglobulin constant region (Fc) or framework region.   5. The chimeric molecule of claim 4, selected from the group comprising IFN-a2B-Fc, IFN-bl-Fc, IFN-g-Fc, IFNAR2-Fc and IL-10-Fc.   A pharmaceutical composition comprising the isolated protein or chimeric molecule of any one of claims 1-6.   An effective amount of the isolated protein according to any one of claims 1 to 3, the chimeric molecule according to any one of claims 4 to 6, or the pharmaceutical composition according to claim 7 to a mammalian subject. A method of treating or preventing a condition in a mammalian subject, wherein the condition can be ameliorated by increasing the amount or activity of the protein comprising the step of:   SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 or at least about 90% identical to any one of the sequences in the previous list Or a nucleotide sequence selected from nucleotide sequences capable of hybridizing under high stringency conditions to any one of the aforementioned sequences or their complementary forms.   SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 or at least about 90% identical to any one of the sequences in the previous list Or an isolated protein or chimeric molecule encoded by a nucleotide sequence selected from nucleotide sequences that have the ability to hybridize under high stringency conditions with any one of the aforementioned sequences or their complementary forms .   SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 nucleotide sequence with at least 90% similarity, or after optimal alignment and / or SEQ ID NO: 27, 29, 31, 33, 35, 39, 41, 43, 45, 47, 49, 51, 53, 55, 59, 61, 63, 65, 67, 69, 71, 73, 75, 79, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105, 107, 111, 113, 115, 116, 118, 119, 121, 122, 124, 125, 127, Hybridize under high stringency conditions with one or more of 128, 130, 131, 133, 134, 136, 137, 139, 140, 142, 143, 145, 146, 148, 149 or their complements Can Including Kureochido sequence encodes a protein, the chimeric molecule or a functional portion thereof, an isolated nucleic acid molecule.   SEQ ID NO: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50, 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106, 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, Amino acid sequences substantially as set forth in one or more of 144, 147, 150, or after optimal alignment, SEQ ID NO: 28, 30, 32, 34, 36, 40, 42, 44, 46, 48, 50 , 52, 54, 56, 60, 62, 64, 66, 68, 70, 72, 74, 76, 80, 82, 84, 86, 88, 90, 92, 94, 96, 100, 102, 104, 106 , 108, 112, 114, 117, 120, 123, 126, 129, 132, 135, 138, 141, 144, 147, 150, having an amino acid sequence having at least about 90% similarity, An isolated nucleic acid molecule comprising a nucleotide sequence encoding a protein or chimeric molecule.   (a) a solid support matrix; (b) one or more antibodies to the human protein according to any one of claims 1 to 3 or the chimeric molecule according to any one of claims 4 to 6; c) a blocking solution; (d) one or more substrate storage solutions; (e) a substrate buffer solution; (f) a standard human protein or chimeric molecule sample; and (g) a biological preparation containing instructions for use. A kit for determining the level of a human protein or chimeric molecule present and expressed in human cells.   14. Kit according to claim 13, wherein the standard human protein or chimeric molecule sample is an isolated protein according to claim 2 or 3 or a preparation of the chimeric molecule according to claim 4.   15. A kit according to claim 13 or 14, wherein each antibody is derived from immunization of a mammal with a preparation comprising the isolated protein of claim 2 or 3 or the chimeric molecule of claim 4.   16. A human protein expressed in a human cell is a naturally occurring human IFN-a2B, IFN-bl, IFN-g, IFNAR2, IL-10, or IL-10Ra, any one of claims 13-15. The described kit.

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