TW201714894A - Immunoglobulin fusion proteins - Google Patents

Immunoglobulin fusion proteins Download PDF

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TW201714894A
TW201714894A TW104135389A TW104135389A TW201714894A TW 201714894 A TW201714894 A TW 201714894A TW 104135389 A TW104135389 A TW 104135389A TW 104135389 A TW104135389 A TW 104135389A TW 201714894 A TW201714894 A TW 201714894A
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amino acid
polypeptide
hfc
human
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TW104135389A
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成永喆
梁世涣
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格納西尼有限公司
浦項工科大學校產學協力團
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Abstract

Disclosed are fusion proteins comprising a biologically active molecule and an immunoglobulin (Ig) Fc domain which is linked to the biologically active molecule. The Fc domain is a hybrid human Fc domain of (i) IgG1, IgG2 or IgG4 or (ii) IgG4 and IgD. The hybrid Fc is useful as a carrier of biologically active molecules.

Description

免疫球蛋白融合蛋白質 Immunoglobulin fusion protein

本發明係關於雜合人類Fc及免疫球蛋白融合蛋白質，其中雜合人類Fc係與生物活性分子連接。特別地，本發明係關於雜合人類Fc，其係衍生自人類免疫球蛋白G(IgG)亞類的組合或人類IgD及IgG的組合，及融合蛋白質，其中此種Fc經由共價鍵與生物活性分子偶合。 The present invention relates to hybrid human Fc and immunoglobulin fusion proteins in which a hybrid human Fc line is linked to a biologically active molecule. In particular, the present invention relates to a heterozygous human Fc derived from a combination of human immunoglobulin G (IgG) subclasses or a combination of human IgD and IgG, and a fusion protein wherein such Fc via covalent bonds and organisms Active molecule coupling.

生物活性分子在治療上可能有重大意義。然而，因為彼等在體內的穩定性低，所以可能不利於作為治療劑。因為在活體內受到各種酵素的分解，所以彼等之循環半衰期或血清半衰期短。因此，一直希望能改善生物活性分子的循環半衰期。 Bioactive molecules may be of great significance in therapy. However, because of their low stability in the body, they may be disadvantageous as a therapeutic agent. Because of the decomposition of various enzymes in the living body, their circulating half-life or serum half-life is short. Therefore, it has been desired to improve the circulating half-life of biologically active molecules.

已知增加蛋白質的尺寸可防止該蛋白質被腎臟除去而增長其半衰期(Knauf et al.,J.Biol.Chem.1988.263：15064-15070)。例如，已報告經由偶合活性蛋白質與人類白蛋白而增加蛋白質穩定性(Kinstler et al.,Pharm.Res.1995.12：1883-1888)。然而，因為偶合活性蛋白質與人類白蛋白只稍微增加其滯留時間，所以並非是發展含有與人類白蛋白偶合之活性蛋白質之有效藥物配方的有效方 法。 It is known that increasing the size of a protein prevents the protein from being removed by the kidney and increases its half-life (Knauf et al., J. Biol. Chem. 1988. 263: 15064-15070). For example, it has been reported to increase protein stability via coupling of active proteins with human albumin (Kinstler et al., Pharm. Res. 1995. 12: 1883-1888). However, because the coupled active protein and human albumin only slightly increase their residence time, it is not an effective way to develop an effective pharmaceutical formula containing the active protein coupled to human albumin. law.

其他報告的方法為調控蛋白質的糖基化。蛋白質的附加糖基化及導入唾液酸至蛋白質以防止肝中蛋白質的降解。但是，增加蛋白質的糖基化亦導致蛋白質生物活性的降低。 Other reported methods are to regulate glycosylation of proteins. Additional glycosylation of proteins and introduction of sialic acid to proteins to prevent degradation of proteins in the liver. However, increasing the glycosylation of proteins also leads to a decrease in the biological activity of the protein.

為了穩定蛋白質及防止腎臟清除作用，使蛋白質與聚乙二醇(PEG)共軛。與PEG共價共軛一直廣泛用於使藥物有延長的半衰期(Delgado et al.,1992.9：249-304)。然而，有報告指出PEG與細胞介素或激素共軛時由於共軛造成的立體阻礙而導致受體結合親和力降低。 In order to stabilize the protein and prevent renal clearance, the protein is conjugated to polyethylene glycol (PEG). Covalent conjugation with PEG has been widely used to give drugs a prolonged half-life (Delgado et al., 1992.9: 249-304). However, it has been reported that conjugated affinity of PEG is reduced due to steric hindrance caused by conjugation when PEG is conjugated to interleukin or hormone.

近來，一直在研究及發展使用免疫球蛋白(Ig)製造的融合蛋白質。Ig為血液的主要成分。人類Ig(hIg)包含各種類別諸如IgG、IgM、IgA、IgD及IgE(Roitt et al.,"Immunology" 1989,Gower Medical Publishing,London,U.K.；New York,N.Y.)。人類IgGs可進一步分類成稱為人類IgG1(hIgG1)、人類IgG2(hIgG2)、人類IgG3(hIgG3)及人類IgG4(hIgG4)的各種亞型。 Recently, fusion proteins made using immunoglobulin (Ig) have been researched and developed. Ig is the main component of blood. Human Ig (hIg) contains various classes such as IgG, IgM, IgA, IgD, and IgE (Roitt et al., "Immunology" 1989, Gower Medical Publishing, London, U.K.; New York, N.Y.). Human IgGs can be further classified into various subtypes called human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG3 (hIgG3), and human IgG4 (hIgG4).

免疫球蛋白由4個多肽鏈組成，2個重鏈及2個輕鏈，其等係經由二硫鍵結合而形成四聚體。各鏈係由可變區及恆定區組成。重鏈恆定區取決於同型而進一步分為3個或4個區(CH1、CH2、CH3及CH4)。重鏈恆定區的Fc部分，取決於Ig同型，包含鉸鏈、CH2、CH3及/或CH4結構域。 The immunoglobulin consists of four polypeptide chains, two heavy chains and two light chains, which are combined to form a tetramer via a disulfide bond. Each chain consists of a variable region and a constant region. The heavy chain constant region is further divided into three or four regions (CH1, CH2, CH3, and CH4) depending on the isotype. The Fc portion of the heavy chain constant region, depending on the Ig isotype, comprises the hinge, CH2, CH3 and/or CH4 domains.

就血清半衰期而言，IgG1、IgG2及IgG4具 有21天的長半衰期，而其他免疫球蛋白則具有少於1星期相對較短的半衰期。與IgG的Fc部分融合的嵌合蛋白質顯示增加的穩定性及增長的血清半衰期(Capon et al.,Nature 1989.337：525-531)。生物活性蛋白質與IgGs CH1區的N-端、Fc區的N-端或CH3區的C-端融合。 In terms of serum half-life, IgG1, IgG2 and IgG4 have There is a long half-life of 21 days, while other immunoglobulins have a relatively short half-life of less than 1 week. Chimeric proteins fused to the Fc portion of IgG show increased stability and increased serum half-life (Capon et al., Nature 1989. 337: 525-531). The biologically active protein is fused to the N-terminus of the IgGs CH1 region, the N-terminus of the Fc region, or the C-terminus of the CH3 region.

初期，以細胞表面受體諸如CD4(Capon et al.,Nature 1989.327：525-531)、TNFR(Mohler et al.,J.Immunology 1993.151：1548-1561)、CTLA4(Linsley et al.,J Exp.Med.1991.173：721-730)、CD86(Morton et al.,J.Immunology 1996.156：1047-1054)的胞外結構域而創造IgG融合蛋白質。並且，一些細胞介素及生長激素係與IgG結構域融合。然而，與細胞表面受體之胞外結構域的融合不同，相較於非融合細胞介素或生長激素，融合可溶性蛋白質與IgGs導致生物活性降低。嵌合蛋白質以二聚體存在，由於存在兩個彼此緊靠的活性蛋白質，而導致與彼等之目標分子如受體間因相互作用所致的立體阻礙。因此，必須克服此問題而製造有效的融合蛋白質。 Initially, cell surface receptors such as CD4 (Capon et al., Nature 1989. 327: 525-531), TNFR (Mohler et al., J. Immunology 1993. 151:1548-1561), CTLA4 (Linsley et al., J Exp. The extracellular domain of Med. 1991. 173: 721-730), CD86 (Morton et al., J. Immunology 1996. 156: 1047-1054) creates an IgG fusion protein. Also, some interleukin and growth hormone lines are fused to the IgG domain. However, unlike fusion of the extracellular domain of cell surface receptors, fusion of soluble proteins with IgGs results in reduced biological activity compared to non-fused interleukins or growth hormone. Chimeric proteins exist as dimers, resulting in steric hindrance due to interactions with their target molecules, such as receptors, due to the presence of two active proteins that are in close proximity to one another. Therefore, it is necessary to overcome this problem to produce an effective fusion protein.

Fc融合技術的其他限制為不良免疫反應之存在。免疫球蛋白的Fc結構域亦具有應用功能，諸如抗體依賴型細胞介導的細胞毒性作用(ADCC)或補體依賴型細胞毒性作用(CDC)。一般透過Ig的Fc區與效應細胞的FcRs間的相互作用或透過補體結合而獲得這種效應功能。因此，必須進行Fc效應功能阻斷而降低不良反應諸如細胞殺傷、細胞介素釋放或發炎。 Other limitations of Fc fusion technology are the presence of adverse immune responses. The Fc domain of an immunoglobulin also has an application function, such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). This effector function is typically obtained by interaction between the Fc region of Ig and the FcRs of effector cells or by complement binding. Therefore, Fc effector function blockade must be performed to reduce adverse reactions such as cell killing, interleukin release or inflammation.

總而言之，仍需要有最少生物活性損失及較少不良免疫反應風險之改良Fc融合蛋白質。 In summary, there is still a need for improved Fc fusion proteins with minimal loss of biological activity and less risk of adverse immune reactions.

本發明提供雜合Fc，其係衍生自人類IgG亞類的組合或人類IgD及IgG的組合。與生物活性分子連接時，雜合Fc對增長生物活性分子的血清半衰期以及在表現編碼Fc-多肽融合蛋白質之核苷酸時，對增加多肽的表現水平有效。 The invention provides hybrid Fc that is derived from a combination of human IgG subclasses or a combination of human IgD and IgG. When linked to a biologically active molecule, the hybrid Fc is effective for increasing the serum half-life of the biologically active molecule and for expressing the level of expression of the polypeptide when expressing the nucleotide encoding the Fc-polypeptide fusion protein.

本發明亦提供雜合Fc融合多肽其中該雜合Fc係與生物活性分子連接。融合蛋白質有時稱為“生物活性分子-Fc融合蛋白質”或簡稱“融合蛋白質”。融合蛋白質在Fc及生物活性分子間可具有連接物。Fc可在其N-端與生物活性分子的C-端偶合。 The invention also provides a hybrid Fc fusion polypeptide wherein the hybrid Fc line is linked to a biologically active molecule. Fusion proteins are sometimes referred to as "bioactive molecule-Fc fusion proteins" or simply "fusion proteins." The fusion protein may have a linker between the Fc and the biologically active molecule. Fc can be coupled at its N-terminus to the C-terminus of the biologically active molecule.

經由製造編碼及能夠表現融合蛋白質的核苷酸建構，在寄主細胞中表現該建構，然後收獲融合蛋白質即可生產融合蛋白質。或者，可用常用方式表現編碼Fc的核苷酸並使其與生物活性分子偶合而生產融合蛋白質。 The fusion protein can be produced by producing the coding and expression of the nucleotide capable of expressing the fusion protein, expressing the construct in the host cell, and then harvesting the fusion protein. Alternatively, the Fc-encoding nucleotide can be expressed in a conventional manner and coupled to a biologically active molecule to produce a fusion protein.

下列式可表示根據本發明一個具體例的多肽：N'-(Z1)p-Y-Z2-Z3-Z4-C' The following formula may represent a polypeptide according to a specific example of the present invention: N'-(Z1) p -Y-Z2-Z3-Z4-C'

其中N'為多肽的N-端及C'為多肽的C-端；Z1表明胺基酸序列，該胺基酸序列包含至少SEQ ID NO：7位置90至98之胺基酸殘基C-端的一部分；Y表明胺基酸序列，該胺基酸序列包含至少SEQ ID NO：7位置99至162之胺基酸殘基C-端的一部分；Z2表明胺基酸序列，該胺基酸序列包含至少SEQ ID NO：7位置163至199之胺基酸殘基N-端的一部分；Z3表明胺基酸序列，該胺基酸序列包含至少SEQ ID NO：6位置115至220之胺基酸殘基C-端的一部分；Z4表明胺基酸序列，該胺基酸序列包含至少SEQ ID NO：6位置221至327之胺基酸殘基N-端的一部分；及p為0或1之整數，其中Z2及Z3的胺基酸殘基總數在80及140間，包含80及140。 Wherein N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; Z1 represents the amino acid sequence, and the amino acid sequence comprises at least the SEQ ID NO: a portion of the C-terminus of the amino acid residue at position 7 to position 90 to 98; Y represents an amino acid sequence comprising at least the C-terminus of the amino acid residue at positions 99 to 162 of SEQ ID NO: 7. a portion; Z2 represents an amino acid sequence comprising at least a portion of the N-terminus of the amino acid residue at positions 163 to 199 of SEQ ID NO: 7; Z3 represents an amino acid sequence, the amino acid sequence comprising at least SEQ ID NO: 6 is a portion of the C-terminus of the amino acid residue at positions 115 to 220; Z4 represents an amino acid sequence comprising at least the amino acid residue N at positions 221 to 327 of SEQ ID NO: 6. a portion of the terminal; and p is an integer of 0 or 1, wherein the total number of amino acid residues of Z2 and Z3 is between 80 and 140, inclusive.

另一個具體例中，Z1為包含SEQ ID NO：7位置90至98之胺基酸殘基的胺基酸序列。Z1可為由SEQ ID NO：7位置90至98之5至9個胺基酸殘基組成的胺基酸序列。Z1亦可為由SEQ ID NO：7位置90至98之胺基酸殘基組成的胺基酸序列。 In another embodiment, Z1 is an amino acid sequence comprising an amino acid residue at positions 90 to 98 of SEQ ID NO: 7. Z1 may be an amino acid sequence consisting of 5 to 9 amino acid residues at positions 90 to 98 of SEQ ID NO: 7. Z1 may also be an amino acid sequence consisting of amino acid residues at positions 90 to 98 of SEQ ID NO: 7.

Y可為包含SEQ ID NO：7位置99至162之胺基酸殘基C-端的5或更多個，或10或更多個連續(consecutive)胺基酸殘基的胺基酸序列。某些具體例中，Y可為包含SEQ ID NO：7位置158至162之胺基酸殘基、SEQ ID NO：7位置153至162之胺基酸殘基、SEQ ID NO：7位置143至162之胺基酸殘基、SEQ ID NO：7位置133至162之胺基酸殘基或SEQ ID NO：7位置99至162之胺基 酸殘基的胺基酸序列。 Y may be an amino acid sequence comprising 5 or more, or 10 or more, consecutive amino acid residues at the C-terminus of the amino acid residues at positions 99 to 162 of SEQ ID NO: 7. In certain embodiments, Y can be an amino acid residue comprising positions 158 to 162 of SEQ ID NO: 7, an amino acid residue at positions 153 to 162 of SEQ ID NO: 7, and position 143 of SEQ ID NO: 7 to An amino acid residue of 162, an amino acid residue at positions 133 to 162 of SEQ ID NO: 7 or an amino group at positions 99 to 162 of SEQ ID NO: 7. The amino acid sequence of the acid residue.

Z2可為包含SEQ ID NO：7位置163至199之胺基酸殘基N-端的4至37個，或6至30個連續胺基酸殘基的胺基酸序列(hIgD)。某些具體例中，Z2可為人類IgG2 CH2結構域的6個N-端胺基酸殘基或人類IgD CH2結構域的8個N-端胺基酸殘基。 Z2 may be an amino acid sequence (hIgD) comprising 4 to 37, or 6 to 30, contiguous amino acid residues at the N-terminus of the amino acid residues at positions 163 to 199 of SEQ ID NO: 7. In certain embodiments, Z2 can be the six N-terminal amino acid residues of the human IgG2 CH2 domain or the eight N-terminal amino acid residues of the human IgD CH2 domain.

Z2及Z3的胺基酸殘基總數可在80及140間。具體例中，Z2及Z3的胺基酸殘基總數在90及120間，包含90及120。另一個具體例中，Z2及Z3的胺基酸殘基總數在105及115間，包含105及115。具體例中，Z2及Z3的胺基酸殘基總數為108。再另一個具體例中，Z2及Z3的胺基酸殘基總數為109。 The total number of amino acid residues of Z2 and Z3 may be between 80 and 140. In a specific example, the total number of amino acid residues of Z2 and Z3 is between 90 and 120, including 90 and 120. In another embodiment, the total number of amino acid residues of Z2 and Z3 is between 105 and 115, inclusive. In a specific example, the total number of amino acid residues of Z2 and Z3 is 108. In still another specific example, the total number of amino acid residues of Z2 and Z3 is 109.

Z4可為包含SEQ ID NO：6位置221至327的90或更多個，或100或更多個連續胺基酸殘基的胺基酸序列(hIgG4)。Z4可為SEQ ID NO：6位置221至327之胺基酸殘基的胺基酸序列。 Z4 may be an amino acid sequence (hIgG4) comprising 90 or more, or 100 or more contiguous amino acid residues at positions 221 to 327 of SEQ ID NO: 6. Z4 may be the amino acid sequence of the amino acid residue at positions 221 to 327 of SEQ ID NO: 6.

根據具體例，Z3-Z4為選自SEQ ID NO：6位置115至220之胺基酸殘基的C-端部分及SEQ ID NO：6位置221至327之胺基酸殘基的N-端部分之連續胺基酸序列的胺基酸序列。 According to a specific example, Z3-Z4 is a C-terminal portion of an amino acid residue selected from positions 115 to 220 of SEQ ID NO: 6, and an N-terminal of an amino acid residue at positions 221 to 327 of SEQ ID NO: 6. A portion of the amino acid sequence of the contiguous amino acid sequence.

根據本發明具體例的多肽之胺基酸殘基總數為154至288。 The total number of amino acid residues of the polypeptide according to a specific example of the present invention is 154 to 288.

於一個具體例中，Y可為包含至少SEQ ID NO：7位置99至162之胺基酸殘基的一部分的胺基酸序 列，p可為1或0(零)，Z2可為包含至少SEQ ID NO：7位置163至199之胺基酸殘基的一部分的胺基酸序列，及Z3可為包含至少SEQ ID NO：6位置121至220之胺基酸殘基的一部分的胺基酸序列。此具體例中，p為1時，Z1可為包含SEQ ID NO：7位置90至98之胺基酸殘基的胺基酸序列。 In one embodiment, Y can be an amino acid sequence comprising at least a portion of an amino acid residue at positions 99 to 162 of SEQ ID NO: 7. Column, p may be 1 or 0 (zero), Z2 may be an amino acid sequence comprising at least a portion of the amino acid residues at positions 163 to 199 of SEQ ID NO: 7, and Z3 may comprise at least SEQ ID NO: The amino acid sequence of a portion of the 6 amino acid residues at positions 121 to 220. In this embodiment, when p is 1, Z1 may be an amino acid sequence comprising an amino acid residue at positions 90 to 98 of SEQ ID NO: 7.

另一個具體例中，Y可為SEQ ID NO：7位置99至162之胺基酸殘基C-端的20或更多個連續胺基酸殘基、30或更多個連續胺基酸殘基、40或更多個連續胺基酸殘基、50或更多個連續胺基酸殘基或60或更多個連續胺基酸殘基。Z2可為SEQ ID NO：7位置163至170之胺基酸殘基，Z3可包括SEQ ID NO：6位置121至220之胺基酸殘基C-端的71至100個連續胺基酸殘基。Z2及Z3的胺基酸殘基總數可為108。 In another embodiment, Y can be 20 or more contiguous amino acid residues, 30 or more contiguous amino acid residues at the C-terminus of the amino acid residue at positions 99 to 162 of SEQ ID NO: 7. 40 or more contiguous amino acid residues, 50 or more contiguous amino acid residues or 60 or more contiguous amino acid residues. Z2 may be the amino acid residue at positions 163 to 170 of SEQ ID NO: 7, and Z3 may include 71 to 100 contiguous amino acid residues at the C-terminus of the amino acid residue at positions 121 to 220 of SEQ ID NO: 6. . The total number of amino acid residues of Z2 and Z3 may be 108.

於一個具體例中，可經由SEQ ID NO：1組成的核苷酸序列編碼多肽。該多肽為由SEQ ID NO：11組成的胺基酸序列。 In one embodiment, the polypeptide can be encoded via the nucleotide sequence consisting of SEQ ID NO: 1. The polypeptide is an amino acid sequence consisting of SEQ ID NO:11.

於一個具體例中，使多肽的N-端與生物活性分子融合，相較於所述生物活性分子天然形式的循環半衰期，顯示增長的循環半衰期。生物活性分子可為多肽、蛋白質或肽。生物活性分子可為多肽、肽或蛋白質藥物。生物活性分子可為可溶性蛋白質諸如，但不限於，激素、細胞介素、生長因子、共激分子，激素受體、細胞介素受體、生長因子受體或短肽。生物活性分子可為EPO或其變 異體/片段、p40或其變異體/片段(如，含有Asn303Gln取代的p40變異體)、G-CSF或其變異體/片段、TNF受體、GM-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-10、IL-10受體、TGF-β、TGF-β受體、IL-17、IL-17受體、VII因子、CXCL-11、FSH、人類生長激素、骨形成蛋白-1(BMP-1)、CTLA4、PD-1、GLP-1、β細胞素、OPG、RNAK、α干擾素、β干擾素或彼等之變異體/片段。生物活性分子可為分泌性蛋白質，其可為成熟的形式。 In one embodiment, the N-terminus of the polypeptide is fused to a biologically active molecule, which exhibits an increased circulating half-life compared to the circulating half-life of the native form of the biologically active molecule. The biologically active molecule can be a polypeptide, protein or peptide. The biologically active molecule can be a polypeptide, peptide or protein drug. The biologically active molecule can be a soluble protein such as, but not limited to, a hormone, an interleukin, a growth factor, a co-stimulatory molecule, a hormone receptor, a cellular receptor, a growth factor receptor, or a short peptide. The bioactive molecule can be EPO or its variant Allogeneic/fragment, p40 or variants/fragments thereof (eg, containing the pn variant of Asn303Gln substitution), G-CSF or variants/fragments thereof, TNF receptor, GM-CSF, IL-1, IL-2, IL -3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-10 receptor, TGF-β, TGF-β receptor, IL-17, IL-17 Receptor, factor VII, CXCL-11, FSH, human growth hormone, bone morphogenetic protein-1 (BMP-1), CTLA4, PD-1, GLP-1, beta cytokines, OPG, RNAK, alpha interferon, beta Interferons or their variants/fragments. The biologically active molecule can be a secreted protein, which can be in a mature form.

於一個具體例中，提供製造根據申請專利範圍第1項中所述之多肽的方法，其中該方法包括步驟：(i)將編碼多肽的DNA分子導入哺乳動物寄主細胞、(ii)使該細胞在可表現多肽的條件下於生長培養基中生長、(iii)收獲經表現的多肽。哺乳動物寄主細胞可為CHO、COS或BHK細胞。 In a specific embodiment, there is provided a method of producing a polypeptide according to claim 1, wherein the method comprises the steps of: (i) introducing a DNA molecule encoding the polypeptide into a mammalian host cell, and (ii) causing the cell The growth medium is grown under conditions in which the polypeptide can be expressed, and (iii) the expressed polypeptide is harvested. The mammalian host cell can be a CHO, COS or BHK cell.

另一個具體例中，提供(i)降低自體免疫疾病的症狀、預防或治療自體免疫疾病、(ii)抑制移植排斥反應或(iii)治療或預防內毒素誘發之休克的方法，該方法包含給藥治療有效量的上述多肽，其中該多肽係與生物活性分子融合。 In another embodiment, the method of (i) reducing symptoms of an autoimmune disease, preventing or treating an autoimmune disease, (ii) inhibiting transplant rejection, or (iii) treating or preventing endotoxin-induced shock is provided. A therapeutically effective amount of the above polypeptide is administered, wherein the polypeptide is fused to a biologically active molecule.

於一個具體例中，提供編碼根據本發明具體例所述之多肽的單離核酸分子。多肽可具有由SEQ ID NO：11組成的胺基酸序列。核酸分子可具有如SEQ ID NO：1所示之核苷酸序列。核酸分子可進一步包含訊號序列或先導序列。 In one embodiment, an isolated nucleic acid molecule encoding a polypeptide according to a particular embodiment of the invention is provided. The polypeptide may have an amino acid sequence consisting of SEQ ID NO:11. The nucleic acid molecule can have a nucleotide sequence as shown in SEQ ID NO: 1. The nucleic acid molecule can further comprise a signal sequence or a leader sequence.

根據本發明所述之具體例，提供包含核酸分子的表現載體(vector)及含有該載體之寄主細胞。表現載體的實例可包含，而不限於，pAD11 EPO-hFc-1、pAD11G-CSF-hFc-1、pAD11 p40N303Q-hFc-1、pAD11 EPO-hFc-6、pAD11 G-CSF-hFc-6、pAD11 p40N303Q-hFc-6、pAD11 EPO-hFc-5、pAD11 G-CSF-hFc-5、pAD11 p40N303Q-hFc-5及pAD11 TNFR-hFc-5。 According to a specific example of the present invention, a vector comprising a nucleic acid molecule and a host cell containing the vector are provided. Examples of expression vectors can include, without limitation, pAD11 EPO-hFc-1, pAD11G-CSF-hFc-1, pAD11 p40N303Q-hFc-1, pAD11 EPO-hFc-6, pAD11 G-CSF-hFc-6, pAD11 p40N303Q-hFc-6, pAD11 EPO-hFc-5, pAD11 G-CSF-hFc-5, pAD11 p40N303Q-hFc-5 and pAD11 TNFR-hFc-5.

於一個具體例中，提供將生物活性分子投予哺乳動物的方法，該方法包含將核酸分子給藥有需要的哺乳動物之步驟。 In one embodiment, a method of administering a biologically active molecule to a mammal is provided, the method comprising the step of administering a nucleic acid molecule to a mammal in need thereof.

另一個具體例中，多肽包含Fc結構域而該Fc結構域以N-端至C-端的方向由鉸鏈區、CH2結構域及CH3結構域組成，其中所述鉸鏈區包含至少人類IgD鉸鏈區或人類IgG1鉸鏈區之胺基酸殘基的一部分；所述CH2結構域包含至少人類IgG4 CH2結構域之胺基酸殘基的一部分，其中人類IgG4 CH2結構域N-端的4至37個連續胺基酸殘基經至少人類IgG2 CH2結構域N-端區或人類IgD CH2結構域N-端區之胺基酸殘基的一部分置換，所述CH3結構域包含至少人類IgG4 CH3結構域之胺基酸殘基的一部分。 In another embodiment, the polypeptide comprises an Fc domain and the Fc domain consists of a hinge region, a CH2 domain, and a CH3 domain in an N-terminal to C-terminal orientation, wherein the hinge region comprises at least a human IgD hinge region or a portion of an amino acid residue of the human IgG1 hinge region; the CH2 domain comprising at least a portion of an amino acid residue of a human IgG4 CH2 domain, wherein the human IgG4 CH2 domain has 4 to 37 contiguous amine groups at the N-terminus The acid residue is replaced by at least a portion of an amino acid residue of the N-terminal region of the human IgG2 CH2 domain or the N-terminal region of the human IgD CH2 domain, the CH3 domain comprising at least the amino acid of the human IgG4 CH3 domain Part of the residue.

鉸鏈區可包含至少人類IgG1鉸鏈區之胺基酸殘基的一部分，所述CH2結構域包含至少人類IgG4 CH2結構域之胺基酸殘基的一部分，其中人類IgG4 CH2結構域N-端的4至37個胺基酸殘基至少經人類IgG2 CH2結構域 N-端區之胺基酸殘基的一部分置換。 The hinge region may comprise at least a portion of an amino acid residue of a human IgG1 hinge region comprising at least a portion of an amino acid residue of a human IgG4 CH2 domain, wherein the human IgG4 CH2 domain is N-terminal to 4 37 amino acid residues at least via the human IgG2 CH2 domain Part of the amino acid residue of the N-terminal region is replaced.

鉸鏈區可包含至少人類IgD鉸鏈區之胺基酸殘基的一部分，所述CH2結構域包含至少人類IgG4 CH2結構域之胺基酸殘基的一部分，其中人類IgG4 CH2結構域N-端的4至37個胺基酸殘基至少經人類IgD CH2結構域N-端區之胺基酸殘基的一部分置換。 The hinge region may comprise at least a portion of an amino acid residue of a human IgD hinge region comprising at least a portion of an amino acid residue of a human IgG4 CH2 domain, wherein the human IgG4 CH2 domain is N-terminal 4 to The 37 amino acid residues are at least replaced with a portion of the amino acid residues of the N-terminal region of the human IgD CH2 domain.

多肽可進一步包含CH1結構域，其中所述CH1結構域包含至少人類IgG1 CH1結構域之胺基酸殘基的一部分，及其中所述CH1結構域係與所述鉸鏈區之N-端偶合。多肽可進一步包含CH1結構域，其中所述CH1結構域包含至少人類IgD CH1結構域之胺基酸殘基的一部分，及其中所述CH1結構域係與所述鉸鏈區之N-端偶合。多肽可進一步包含與所述鉸鏈區之N-端偶合的第二多肽，其中該第二多肽為生物活性非免疫球蛋白多肽。多肽可進一步包含經由連接物與所述CH1結構域之N-端或與所述CH4結構域之C-端偶合的生物活性分子，其中所述生物活性分子並非免疫球蛋白多肽。多肽及生物活性分子可經由連接物彼此偶合。連接物分子為白蛋白連接物或合成連接物。白蛋白連接物包括胺基酸序列EMP、ENDEMPAD、EENDEMPADLPS、CIAEVENDEMPADLPSLA、SHCIAEVENDEMPADLPSLA或PLLEKSHCIAEVENDEMPADLPSLAADFVESKD。合成連接物可為由Gly及Ser殘基組成的10至20個胺基酸殘基的肽。於一個具體例中，這種Gly-Ser連接物為 GGGGSGGGGSGGGSG。 The polypeptide may further comprise a CH1 domain, wherein the CH1 domain comprises at least a portion of an amino acid residue of a human IgG1 CH1 domain, and wherein the CH1 domain is coupled to the N-terminus of the hinge region. The polypeptide may further comprise a CH1 domain, wherein the CH1 domain comprises at least a portion of an amino acid residue of a human IgD CH1 domain, and wherein the CH1 domain is coupled to the N-terminus of the hinge region. The polypeptide may further comprise a second polypeptide coupled to the N-terminus of the hinge region, wherein the second polypeptide is a biologically active non-immunoglobulin polypeptide. The polypeptide may further comprise a biologically active molecule coupled via a linker to the N-terminus of the CH1 domain or to the C-terminus of the CH4 domain, wherein the bioactive molecule is not an immunoglobulin polypeptide. The polypeptide and biologically active molecules can be coupled to one another via a linker. The linker molecule is an albumin linker or a synthetic linker. The albumin linker comprises the amino acid sequence EMP, ENDEPDAD, EENDEMPADLPS, CIAEVENDEMPADLPSLA, SHCIAEVENDEMPADLPSLA or PLLEKSHCIAEVENDEMPADLPSLAADFVESKD. The synthetic linker can be a peptide of 10 to 20 amino acid residues consisting of Gly and Ser residues. In one embodiment, the Gly-Ser linker is GGGGSGGGGSGGGSG.

本發明亦包含包括重組Fc區的抗體分子，該重組Fc區如上文所說明。 The invention also encompasses antibody molecules comprising a recombinant Fc region as described above.

第1圖顯示雜合Fcs(hFcs)的示意圖，該Fcs可使用作為指定為“X”之生物活性分子的載體蛋白質。 Figure 1 shows a schematic representation of hybrid Fcs (hFcs) which can be used as a carrier protein designated as a biologically active molecule designated "X".

第2圖顯示hFcs的圖示隨後詳細說明衍生自IgG1、IgG2、IgG4及IgD的胺基酸位置。除非另有指明，於整篇申請書中，應用相同規則指示多肽中胺基酸的位置。 Figure 2 shows a schematic representation of hFcs followed by a detailed description of the amino acid positions derived from IgGl, IgG2, IgG4 and IgD. Unless otherwise indicated, the same rules are applied throughout the application to indicate the position of the amino acid in the polypeptide.

第3圖顯示hFcs的圖示，各hFcs經由指示為“AL”的白蛋白連接肽在C-端與稱為“X”的生物活性分子共軛。 Figure 3 shows a graphical representation of hFcs, each of which is conjugated to a biologically active molecule designated "X" at the C-terminus via an albumin linker designated "AL".

第4圖顯示與連接物共軛之hFcs的圖示隨後詳細說明衍生自人類白蛋白之白蛋白連接物的胺基酸位置。 Figure 4 shows a graphical representation of hFcs conjugated to a linker followed by a detailed description of the amino acid position of the albumin linker derived from human albumin.

第5圖顯示hFc-6之疏水性作圖結果。 Figure 5 shows the results of hydrophobic mapping of hFc-6.

第6(a)圖顯示使用特定ELISA檢測之MabThera®(利妥昔(Rituximab))、hIgG1、Enbrel®(恩博(etanercept))、EPO-hFc-5、G-CSF-hFc-5、p40N303Q-hFc-5之Fc γ RI結合活性的結果；第6(b)圖顯示使用特定ELISA檢測之MabThera®(利妥昔)、hIgG1、Enbrel®(恩博)、EPO-hFc-5、G-CSF-hFc-5、p40N303Q-hFc-5之C1q結合活性的結果。 Figure 6(a) shows MabThera® (Rituximab), hIgG1, Enbrel® (etanercept), EPO-hFc-5, G-CSF-hFc-5, p40N303Q using specific ELISA assays Results of Fc γ RI binding activity of -hFc-5; Figure 6(b) shows MabThera® (rituximab), hIgG1, Enbrel®, EPO-hFc-5, G- using specific ELISA assays Results of C1q binding activity of CSF-hFc-5, p40N303Q-hFc-5.

第7(a)圖顯示與人類F36E細胞株中EPO比較的EPO-IgG1 Fc、EPO-hFc-1、EPO-hFc-5、EPO-hFc-6及Aranesp® (阿法達貝泊汀(darbepoetin alfa))之生物活性的結果；第7(b)圖顯示小鼠造血細胞株(NFS-60)中Neulasta®(培非格司亭(pegfilgrastim))及G-CSF-hFc-5之體外生物活性的結果；第7(c)圖顯示人類PBMCs中p40及p40N303Q-hFc-5之體外生物活性的結果；第7(d)圖顯示鼠類L929細胞中Enbrel®(恩博)及TNFR-hFc-5之體外生物活性的結果；及第7(e)圖顯示人類WISH細胞中thFc-1-AL(0)-IFN-β及thFc-1-AL(3)-IFN-β之體外生物活性的結果。 Figure 7(a) shows EPO-IgG1 Fc, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6 and Aranesp® compared to EPO in human F36E cell lines (Results of biological activity of darbepoetin alfa); Figure 7(b) shows Neulasta® (pegfilgrastim) and G in mouse hematopoietic cell line (NFS-60) -In vitro biological activity of CSF-hFc-5; Figure 7(c) shows the in vitro biological activity of p40 and p40N303Q-hFc-5 in human PBMCs; Figure 7(d) shows Enbrel in murine L929 cells Results of in vitro biological activity of ® (Enbo) and TNFR-hFc-5; and Figure 7(e) shows thFc-1-AL(0)-IFN-β and thFc-1-AL in human WISH cells (3) - Results of in vitro biological activity of IFN-β.

第8(a)圖顯示經由SC途徑(左框面)及IV途徑(右框面)給藥石蟹獼猴之Aranesp®(α達貝泊汀(darbepoetin alfa))、EPO-hFc-1或EPO-hFc-5之體內半衰期的結果；第8(b)圖顯示經由SC途徑(左框面)及IV途徑(右框面)給藥Sprague Dawley大鼠之LEUCOSTIM®(惠爾血添(filgrastim))及G-CSF-hFc-1之藥物動力學的結果；第8(c)圖顯示經由SC途徑給藥石蟹獼猴之p40N303Q-hFc-5及Enbrel®(恩博)之藥物動力學的結果；第8(d)圖顯示經由SC途徑給藥Sprague Dawley大鼠之TNFR-hFc-5及Enbrel®(恩博)之藥物動力學的結果。 Figure 8(a) shows Aranesp® (darbepoetin alfa), EPO-hFc-1 or EPO- administered to the stone crab macaque via the SC route (left frame) and IV route (right frame). Results of in vivo half-life of hFc-5; Figure 8(b) shows LEUCOSTIM® (filgrastim) administered to Sprague Dawley rats via the SC route (left frame) and IV route (right frame) And the pharmacokinetic results of G-CSF-hFc-1; Figure 8(c) shows the results of pharmacokinetics of p40N303Q-hFc-5 and Enbrel® administered by the SC route; Figure 8(d) shows the results of pharmacokinetics of TNFR-hFc-5 and Enbrel® administered to Sprague Dawley rats via the SC route.

第9(a)圖顯示經由SC途徑(上框面)及IV途徑(下框面)給藥石蟹獼猴之Aranesp®(α達貝泊汀)及EPO-hFc-5之體內生物活性的結果及第9(b)圖顯示經由SC途徑(上框面)及IV途徑(下框面)給藥Sprague Dawley大鼠之LEUCOSTIM®(惠爾血添)及G-CSF-hFc-1之體內生物活性的結果。 Figure 9(a) shows the results of in vivo bioactivity of Aranesp® (alpha dabepoetin) and EPO-hFc-5 administered to the stone crab macaque via the SC route (upper frame) and the IV route (lower frame). Figure 9(b) shows the in vivo biological activity of LEUCOSTIM® (WHIL) and G-CSF-hFc-1 administered to Sprague Dawley rats via the SC route (upper frame) and IV route (lower frame). the result of.

本發明提供雜合人類免疫球蛋白Fc片段，其包含從N-端至C-端方向之鉸鏈區、CH2結構域及CH3結構域，其中鉸鏈區為至少人類IgD鉸鏈區或人類IgG1鉸鏈區的部分胺基酸序列；及CH2結構域為人類IgG4 CH2結構域，其一部分，在N-端區，經人類IgG2 CH2或人類IgD CH2結構域N-端區的4至37個胺基酸殘基置換。此種雜合Fc片段，與生物活性分子，諸如生物活性分子連接時，產生Fc融合蛋白質，最小化Fc融合蛋白質的非特異性免疫反應、延長生物活性分子的血清半衰期及最優化生物活性分子的活性。 The invention provides a hybrid human immunoglobulin Fc fragment comprising a hinge region, a CH2 domain and a CH3 domain from the N-terminus to the C-terminus, wherein the hinge region is at least a human IgD hinge region or a human IgG1 hinge region The partial amino acid sequence; and the CH2 domain is a human IgG4 CH2 domain, a portion thereof, in the N-terminal region, 4 to 37 amino acid residues in the N-terminal region of human IgG2 CH2 or human IgD CH2 domain Replacement. Such a hybrid Fc fragment, when linked to a biologically active molecule, such as a biologically active molecule, produces an Fc fusion protein that minimizes the non-specific immune response of the Fc fusion protein, prolongs the serum half-life of the biologically active molecule, and optimizes the biologically active molecule. active.

根據本發明一個具體例的Fc融合蛋白質中，IgD CH2結構域N-端與IgG4 CH2結構域剩下部分的組合係設計為所形成之融合蛋白質的2個不同Ig次單元重組的區域為疏水性。所形成之融合蛋白質的疏水性區域將位於折疊蛋白質內部，而最小化不良之非特異性免疫反應。 According to a specific example of the Fc fusion protein of the present invention, the combination of the N-terminus of the IgD CH2 domain and the remainder of the IgG4 CH2 domain is designed such that the region of the two different Ig subunits of the formed fusion protein is hydrophobic. . The hydrophobic region of the resulting fusion protein will be located inside the folded protein, minimizing undesirable non-specific immune responses.

如本文所使用，術語“Fc片段”或“Fc”意指包含免疫球蛋白之重鏈恆定區1(CH1)、重鏈恆定區2(CH2)及重鏈恆定區3(CH3)，而不包含免疫球蛋白之重鏈及輕鏈之可變區及輕鏈恆定區1(CL1)的蛋白質。Fc可進一步包含重鏈恆定區的鉸鏈區。本文中雜合Fc或雜合Fc片段有時稱為“hFc”。 The term "Fc fragment" or "Fc" as used herein means heavy chain constant region 1 (CH1), heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3) comprising immunoglobulin, without A protein comprising a variable region of a heavy chain and a light chain of an immunoglobulin and a light chain constant region 1 (CL1). The Fc may further comprise a hinge region of the heavy chain constant region. Hybrid Fc or hybrid Fc fragments herein are sometimes referred to as "hFc".

此外，本發明Fc片段可為具有天然糖鏈、比天然形式增長的糖鏈或比天然形式減短的糖鏈之形式， 或可為去糖基化形式。可用所屬領域的普通方法而達到增加、減少或移除免疫球蛋白Fc糖鏈，諸如化學方法、酵素方法及使用微生物的遺傳工程方法。從Fc片段移除糖鏈造成與第一補體成分C1的C1q部分之結合親和力的顯著減少及抗體依賴型細胞介導的細胞毒性作用(ADCC)或補體依賴型細胞毒性作用(CDC)的減少或喪失，因此不會在體內誘發不必要的免疫反應。在這方面，去糖基化或非糖基化形式的免疫球蛋白Fc片段在一些情況下可能更適合本發明作為藥物載體之目的。 Furthermore, the Fc fragment of the present invention may be in the form of a natural sugar chain, a sugar chain that grows more than the natural form, or a sugar chain that is shorter than the native form. Or it may be in a deglycosylated form. Increasing, reducing or removing immunoglobulin Fc sugar chains can be achieved by conventional methods in the art, such as chemical methods, enzyme methods, and genetic engineering methods using microorganisms. Removal of the sugar chain from the Fc fragment results in a significant decrease in binding affinity to the C1q portion of the first complement component C1 and a decrease in antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC) or Lost, so it does not induce unnecessary immune reactions in the body. In this regard, immunoglobulin Fc fragments in a deglycosylated or non-glycosylated form may in some instances be more suitable for the purposes of the present invention as a pharmaceutical carrier.

如本文所使用，術語“去糖基化”意指從Fc片段酵素性移除糖部分，而術語“非糖基化”意指Fc片段係經由原核生物，較佳為E.coli.生產之未糖基化的形式。 As used herein, the term "deglycosylation" means the removal of a sugar moiety from an Fc fragment, and the term "non-glycosylation" means that the Fc fragment is produced via a prokaryote, preferably E. coli. Unglycosylated form.

術語“雜合”，如本文所使用，意指編碼不同源的2個或更多個免疫球蛋白Fc片段的序列存在於單鏈免疫球蛋白Fc片段中。 The term "hybrid", as used herein, means that the sequences encoding two or more immunoglobulin Fc fragments of different origin are present in a single-chain immunoglobulin Fc fragment.

於一個具體例中，雜合人類Fc包含從N-端至C-端方向的鉸鏈區、CH2結構域及CH3結構域，其中鉸鏈區為至少人類IgD鉸鏈區或人類IgG1鉸鏈區的部分胺基酸序列；及CH2結構域為人類IgG4 CH2結構域，其一部分，在N-端區，經人類IgG2 CH2或人類IgD CH2結構域N-端區的4至37個胺基酸殘基置換。雜合人類Fc可經由共價鍵在其N-端與生物活性分子的C-端連合。 In one embodiment, the hybrid human Fc comprises a hinge region, a CH2 domain, and a CH3 domain from the N-terminus to the C-terminus, wherein the hinge region is at least a human IgD hinge region or a partial amine group of a human IgG1 hinge region The acid sequence; and the CH2 domain is a human IgG4 CH2 domain, a portion of which, in the N-terminal region, is replaced with 4 to 37 amino acid residues in the N-terminal region of human IgG2 CH2 or human IgD CH2 domain. A heterozygous human Fc can be conjugated to the C-terminus of a biologically active molecule at its N-terminus via a covalent bond.

另一個具體例中，生物活性分子-雜合Fc融 合多肽可以下列式表示：N'-X-(Z1)p-Y-Z2-Z3-Z4-C'，或N'-(Z1)p-Y-Z2-Z3-Z4-(連接物)q-X-C' In another embodiment, the biologically active molecule-hybrid Fc fusion polypeptide can be represented by the formula: N'-X-(Z1) p -Y-Z2-Z3-Z4-C', or N'-(Z1) p - Y-Z2-Z3-Z4-(linker) q -XC'

其中N'為多肽N-端及C'為多肽C-端；Z1表明包含至少SEQ ID NO：7位置90至98的胺基酸殘基之C-端一部分的胺基酸序列；Y表明包含至少SEQ ID NO：7位置99至162的胺基酸殘基之C-端一部分的胺基酸序列；Z2表明包含至少SEQ ID NO：7位置163至199的胺基酸殘基之N-端一部分的胺基酸序列；Z3表明包含至少SEQ ID NO：6位置115至220的胺基酸殘基之C-端一部分的胺基酸序列；Z4表明包含至少SEQ ID NO：6位置221至327的胺基酸殘基之N-端一部分的胺基酸序列；p及q各為0或1之整數，其中Z2及Z3的胺基酸殘基總數在80至140間，包含80及140。連接物為連接物分子，及X為有利的生物活性分子。 Wherein N' is the N-terminus of the polypeptide and C' is the C-terminus of the polypeptide; Z1 indicates an amino acid sequence comprising at least a portion of the C-terminus of the amino acid residue at positions 90 to 98 of SEQ ID NO: 7; Y indicates inclusion At least a portion of the amino acid sequence of the C-terminus of the amino acid residue at positions 99 to 162 of SEQ ID NO: 7; Z2 indicating the N-terminus of the amino acid residue comprising at least positions 163 to 199 of SEQ ID NO: 7. a portion of the amino acid sequence; Z3 indicates an amino acid sequence comprising at least a portion of the C-terminus of the amino acid residue at positions 115 to 220 of SEQ ID NO:6; Z4 indicates at least SEQ ID NO: 6 positions 221 to 327 The amino acid sequence of a portion of the N-terminus of the amino acid residue; p and q are each an integer of 0 or 1, wherein the total number of amino acid residues of Z2 and Z3 is between 80 and 140, inclusive. The linker is a linker molecule and X is a beneficial biologically active molecule.

於一個具體例中，Z3-Z4為由SEQ ID NO：6位置115至220之胺基酸殘基之C-端部分及SEQ ID NO：6位置221至327之胺基酸殘基之N-端部分的連續胺基酸序列組成的胺基酸序列。 In one embodiment, Z3-Z4 is the C-terminal portion of the amino acid residue at positions 115 to 220 of SEQ ID NO: 6 and the N- group of the amino acid residue at positions 221 to 327 of SEQ ID NO: 6. The amino acid sequence consisting of the contiguous amino acid sequence of the terminal moiety.

根據本發明之一個具體例，多肽的胺基酸殘基總數為154至288。 According to one embodiment of the invention, the total number of amino acid residues of the polypeptide is from 154 to 288.

給藥予個體時，相較於單獨使用生物活性分子X式N'-X-(Z1)p-Y-Z2-Z3-Z4-C'及N'-(Z1)p-Y-Z2-Z3-Z4-(連接物)q-X-C'之多肽，增長生物活性分子X的 循環半衰期。 When administered to an individual, the bioactive molecule X-form N'-X-(Z1)pY-Z2-Z3-Z4-C' and N'-(Z1)pY-Z2-Z3-Z4-( Linker) a polypeptide of qX-C' that increases the bioactive molecule X Cyclic half-life.

連接物可衍生自人類白蛋白(CAA00606)。連接物可包括胺基酸序列EMP、ENDEMPAD、EENDEMPADLPS、CIAEVENDEMPADLPSLA、SHCIAEVENDEMPADLPSLA或PLLEKSHCIAEVENDEMPADLPSLAADFVESKD。或者，連接物可為合成連接物。合成連接物可為由總共10至20個Gly及Ser殘基組成的肽。於一個具體例中，這種Gly-Ser連接物為GGGGSGGGGSGGGSG。 The linker can be derived from human albumin (CAA00606). The linker may comprise an amino acid sequence EMP, ENDEPDAD, EENDEMPADLPS, CIAEVENDEMPADLPSLA, SHCIAEVENDEMPADLPSLA or PLLEKSHCIAEVENDEMPADLPSLAADFVESKD. Alternatively, the linker can be a synthetic linker. The synthetic linker can be a peptide consisting of a total of 10 to 20 Gly and Ser residues. In one embodiment, the Gly-Ser linker is GGGGSGGGGSGGGSG.

Z1可包括至少IgD(SEQ ID NO：7)CH1結構域的一部分。Z1可包括IgD CH1結構域C-端區的5至9個或7至9個連續胺基酸殘基(SEQ ID NO：7位置90至98)。一些具體例中，Z1可為IgG1 CH1結構域或IgD CH1結構域的5、6、7、8或9個C-端胺基酸殘基。 Z1 can include at least a portion of the IgD (SEQ ID NO: 7) CH1 domain. Z1 may comprise 5 to 9 or 7 to 9 contiguous amino acid residues of the C-terminal region of the IgD CH1 domain (SEQ ID NO: 7 positions 90 to 98). In some embodiments, Z1 can be 5, 6, 7, 8, or 9 C-terminal amino acid residues of the IgG1 CH1 domain or the IgD CH1 domain.

一些具體例中，Z1為包含SEQ ID NO：7位置90至98的胺基酸殘基的胺基酸序列。Z1可為由SEQ ID NO：7位置90至98的5至9個胺基酸殘基組成的胺基酸序列。Z1亦可為由SEQ ID NO：7位置90至98的胺基酸殘基組成的胺基酸序列。 In some embodiments, Z1 is an amino acid sequence comprising an amino acid residue at positions 90 to 98 of SEQ ID NO: 7. Z1 may be an amino acid sequence consisting of 5 to 9 amino acid residues at positions 90 to 98 of SEQ ID NO: 7. Z1 may also be an amino acid sequence consisting of amino acid residues at positions 90 to 98 of SEQ ID NO: 7.

Y可包括至少人類IgG1或IgD鉸鏈區的一部分。Y可包括IgG1鉸鏈區或IgD鉸鏈區C-端的5或更多個，或10或更多個連續胺基酸殘基。某些具體例中，Y可為包含SEQ ID NO：7位置158至162之胺基酸殘基、SEQ ID NO：7位置153至162之胺基酸殘基、SEQ ID NO：7 位置143至162之胺基酸殘基、SEQ ID NO：7位置133至162之胺基酸殘基或SEQ ID NO：7位置99至162之胺基酸殘基的胺基酸序列。 Y may comprise at least a portion of a human IgGl or IgD hinge region. Y may comprise 5 or more, or 10 or more contiguous amino acid residues, of the IgGl hinge region or the C-terminus of the IgD hinge region. In certain embodiments, Y can be an amino acid residue comprising positions 158 to 162 of SEQ ID NO: 7, an amino acid residue at positions 153 to 162 of SEQ ID NO: 7, SEQ ID NO: 7. The amino acid sequence at positions 143 to 162, the amino acid residue at positions 133 to 162 of SEQ ID NO: 7, or the amino acid sequence at amino acid positions 99 to 162 of SEQ ID NO: 7.

Z2可包括人類IgG2 CH2結構域N-端區或IgD CH2結構域N-端區的4至37個、6至30個、6至12個、6至8個、8個或6個連續胺基酸殘基。某些具體例中，Z2可為人類IgD CH2結構域的8個N-端胺基酸殘基(SEQ ID NO：7位置163至170的胺基酸殘基)。 Z2 may comprise 4 to 37, 6 to 30, 6 to 12, 6 to 8, 8, or 6 consecutive amino groups of the N-terminal region of the human IgG2 CH2 domain or the N-terminal region of the IgD CH2 domain. Acid residue. In certain embodiments, Z2 can be the 8 N-terminal amino acid residues of the human IgD CH2 domain (amino acid residues at positions 163 to 170 of SEQ ID NO: 7).

Z2及Z3的胺基酸殘基總數可在90至120間，包含90及120，或105至115間，包含105及115。 The total number of amino acid residues of Z2 and Z3 may range from 90 to 120, including 90 and 120, or 105 to 115, including 105 and 115.

Z4可為包含IgG4 CH3結構域的90或更多個，或100或更多個連續胺基酸殘基(SEQ ID NO：6位置221至327之胺基酸殘基)的胺基酸序列。Z4可為大於98%或95%之人類IgG1、IgG2、IgG3或IgG4 CH3結構域胺基酸殘基的胺基酸殘基。示例之具體例中，Z4為包括完整人類IgG CH3結構域胺基酸序列的胺基酸序列。例如，Z4為人類IgG4 CH3結構域的胺基酸序列，根據EU Index,Kabat編號，相當於人類IgG4胺基酸殘基341至447(相當於SEQ ID NO：6位置221至327的胺基酸殘基)。 Z4 can be an amino acid sequence comprising 90 or more, or 100 or more contiguous amino acid residues (amino acid residues at positions 221 to 327 of SEQ ID NO: 6) comprising an IgG4 CH3 domain. Z4 can be an amino acid residue of greater than 98% or 95% of the human IgGl, IgG2, IgG3 or IgG4 CH3 domain amino acid residues. In a specific example of the example, Z4 is an amino acid sequence comprising the entire human IgG CH3 domain amino acid sequence. For example, Z4 is the amino acid sequence of the human IgG4 CH3 domain, according to the EU Index, Kabat numbering, corresponding to human IgG4 amino acid residues 341 to 447 (corresponding to the amino acid of positions 221 to 327 of SEQ ID NO: 6) Residues).

於一個具體例中，Y可為包含至少人類IgD鉸鏈區C-端區的一部分(SEQ ID NO：7位置99至162之胺基酸殘基)的胺基酸序列，p可為1或0(零)，Z2可為包含至少人類IgD CH2結構域N-端區的一部分(SEQ ID NO：7位置163至199之胺基酸殘基)的胺基酸序列，及Z3可為 包含至少人類IgG4 CH2結構域C-端區的一部分(SEQ ID NO：6位置121至220之胺基酸殘基)的胺基酸序列。例如，Y可為SEQ ID NO：7位置158至162、133至162或99至162的胺基酸殘基，Z2可為SEQ ID NO：7位置163至170的胺基酸殘基，及Z3可為SEQ ID NO：6位置121至220的胺基酸殘基。 In one embodiment, Y can be an amino acid sequence comprising at least a portion of the C-terminal region of the human IgD hinge region (amino acid residues at positions 99 to 162 of SEQ ID NO: 7), p can be 1 or 0. (Z), Z2 may be an amino acid sequence comprising at least a portion of the N-terminal region of the human IgD CH2 domain (amino acid residues at positions 163 to 199 of SEQ ID NO: 7), and Z3 may be An amino acid sequence comprising at least a portion of the C-terminal region of the human IgG4 CH2 domain (amino acid residues at positions 121 to 220 of SEQ ID NO: 6). For example, Y can be the amino acid residue at positions 158 to 162, 133 to 162 or 99 to 162 of SEQ ID NO: 7, and Z2 can be the amino acid residue at positions 163 to 170 of SEQ ID NO: 7, and Z3 It may be an amino acid residue at positions 121 to 220 of SEQ ID NO: 6.

此具體例中，p為1時，Z1可為包含人類IgD CH1結構域C-端區(SEQ ID NO：7位置90至98之胺基酸殘基)的胺基酸序列。例如，Z1可為SEQ ID NO：7位置90至98的胺基酸殘基。 In this embodiment, when p is 1, Z1 may be an amino acid sequence comprising a C-terminal region of the human IgD CH1 domain (amino acid residues at positions 90 to 98 of SEQ ID NO: 7). For example, Z1 can be an amino acid residue at positions 90 to 98 of SEQ ID NO: 7.

此具體例中，Y可為人類IgD鉸鏈區C-端之20個或更多個的連續胺基酸殘基、30個或更多個的連續胺基酸殘基、40個或更多個的連續胺基酸殘基、50個或更多個的連續胺基酸殘基或60個或更多個的連續胺基酸殘基(SEQ ID NO：7位置99至162的胺基酸殘基)。Z3可包括SEQ ID NO：6位置121至220胺基酸殘基C-端的71至100個連續胺基酸殘基。Z2及Z3的胺基酸殘基總數可為108。 In this particular example, Y can be 20 or more contiguous amino acid residues at the C-terminus of the human IgD hinge region, 30 or more contiguous amino acid residues, 40 or more Continual amino acid residue, 50 or more contiguous amino acid residues or 60 or more contiguous amino acid residues (amino acid residues at positions 99 to 162 of SEQ ID NO: 7) base). Z3 may comprise from 71 to 100 contiguous amino acid residues at the C-terminus of the amino acid residues at positions 121 to 220 of SEQ ID NO: 6. The total number of amino acid residues of Z2 and Z3 may be 108.

於一個具體例中，本發明提供雜合Fc，該雜合Fc為hFc-1、hFc-2、hFc-3、hFc-4、hFc-5或hFc-6之一，如第1圖及第2圖所示，或如第3圖及第4圖所示的thFc-1或thFc-2。雖然第1圖及第3圖描繪雙鏈Fcs，惟本發明包含單鏈雜合Fc分子。本發明亦包含編碼雜合Fc的多核苷酸分子。彼等包含，但不限於，如SEQ ID NO：1(hFc-5) 所示之多核苷酸序列。 In one embodiment, the invention provides a hybrid Fc that is one of hFc-1, hFc-2, hFc-3, hFc-4, hFc-5 or hFc-6, as shown in Figure 1 and 2 shows, or thFc-1 or thFc-2 as shown in Figs. 3 and 4. Although Figures 1 and 3 depict double-stranded Fcs, the invention encompasses single-chain hybrid Fc molecules. The invention also encompasses polynucleotide molecules encoding hybrid Fc. These include, but are not limited to, SEQ ID NO: 1 (hFc-5) The polynucleotide sequence shown.

人類免疫球蛋白的胺基酸序列為所屬領域所習知且儲存於公共可存取的儲存庫。例如，人類IgG1恆定區、人類IgG2恆定區、人類IgG3恆定區、人類IgG4恆定區及人類IgD恆定區的胺基酸序列可分別得自CAA75032、CAC20455、CAC20456、AAH25985及P01880。 The amino acid sequences of human immunoglobulins are well known in the art and are stored in publicly accessible repositories. For example, the amino acid sequences of the human IgGl constant region, the human IgG2 constant region, the human IgG3 constant region, the human IgG4 constant region, and the human IgD constant region can be obtained from CAA75032, CAC20455, CAC20456, AAH25985, and P01880, respectively.

生物活性分子X可為可溶性蛋白質。其可包含，但不限於，激素、細胞介素、生長因子、共激分子，激素受體、細胞介素受體、生長因子受體或短肽。例如，X可為EPO、p40、G-CSF、TNF受體或其變異體/片段。X可為GM-CSF、IL-1、IL-2、IL-3、IL-4、IL-5、IL-6、IL-7、IL-8、IL-10、IL-10受體、TGF-β、TGF-β受體、IL-17、IL-17受體、VII因子、CSCL-11、FSH、人類生長激素、骨形成蛋白-1、CTLA4、PD-1、GLP-1、β細胞素、OPG、RNAK、α干擾素、β干擾素或彼等之變異體/片段。亦可包含，但不限於，抗體之Fab區。生物活性分子亦可為分泌性蛋白質。於一個具體例中，生物活性分子不屬於免疫球蛋白家族。 The biologically active molecule X can be a soluble protein. It may include, but is not limited to, hormones, interleukins, growth factors, co-stimulatory molecules, hormone receptors, interleukin receptors, growth factor receptors or short peptides. For example, X can be EPO, p40, G-CSF, TNF receptor or variants/fragments thereof. X can be GM-CSF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-10 receptor, TGF -β, TGF-β receptor, IL-17, IL-17 receptor, factor VII, CSCL-11, FSH, human growth hormone, bone morphogenetic protein-1, CTLA4, PD-1, GLP-1, β cells , OPG, RNAK, alpha interferon, beta interferon or variants/fragments thereof. Also included, but not limited to, the Fab region of the antibody. The biologically active molecule can also be a secreted protein. In one embodiment, the biologically active molecule does not belong to the immunoglobulin family.

術語“變異體”意指與參考核酸或多肽不同但保有其基本性質的多核苷酸或核酸。一般，變異體總體非常類似，而且，在許多區域與參考核酸或多肽相同。而且，術語“變異體”意指生物活性分子藥物的生物活性部分，其保有至少一種如本文或者所屬領域所習知的功能及/或治療性質。一般，變異體總體非常類似，而且，在許 多區域與有利的生物活性多肽的胺基酸序列相同。 The term "variant" means a polynucleotide or nucleic acid that differs from the reference nucleic acid or polypeptide but retains its essential properties. In general, variants are very similar overall and, in many regions, are identical to a reference nucleic acid or polypeptide. Moreover, the term "variant" means a biologically active portion of a biologically active molecule drug that retains at least one of the functional and/or therapeutic properties as is known herein or in the art. In general, the variants are very similar overall, and, in Xu The multi-region is identical to the amino acid sequence of the advantageous biologically active polypeptide.

本發明亦提供包括或者由至少具有，例如，與多肽的胺基酸序列有80%、85%、90%、95%、96%、97%、98%、99%或100%一致性的胺基酸序列所組成的蛋白質。本發明亦提供這些多肽的片段。本發明包含的另外多肽為多核苷酸編碼的多肽，該多核苷酸在下列條件下與編碼本發明多肽的核酸分子補體雜合：嚴格雜合條件(如，與和濾膜結合的DNA於約45℃，在6x氯化鈉/檸檬酸鈉(SSC)中雜合，隨後於約50至65℃，在0.2xSSC，0.1%SDS中洗滌1次或多次)、高度嚴格條件(如，與和濾膜結合的DNA於約45℃，在6x氯化鈉/檸檬酸鈉(SSC)中雜合，隨後於約68℃，在0.1xSSC，0.2% SDS中洗滌1次或多次)或所屬領域熟練之技術人員習知的其他嚴格雜合條件(參見，例如，Ausubel,F.M.et al.,eds.,1989 Current protocol in Molecular Biology,Green publishing associates,Inc.，及John Wiley & Sons Inc.,New York,at pages 6.3.1 6.3.6 and 2.10.3)。本發明亦包含編碼這些多肽的多核苷酸。 The invention also provides an amine comprising or consisting of at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence of the polypeptide. A protein consisting of a base acid sequence. Fragments of these polypeptides are also provided by the invention. Further polypeptides encompassed by the invention are polypeptides encoded by polynucleotides which are hybridized to a complement of a nucleic acid molecule encoding a polypeptide of the invention under stringent conditions: for example, with DNA conjugated to a filter. Hybrid in 6x sodium chloride/sodium citrate (SSC) at 45 ° C, followed by washing one or more times in 0.2xSSC, 0.1% SDS at about 50 to 65 ° C, highly stringent conditions (eg, with The DNA bound to the filter is hybridized in 6x sodium chloride/sodium citrate (SSC) at about 45 ° C, followed by one or more washes in 0.1 x SSC, 0.2% SDS at about 68 ° C or Other stringent heterozygous conditions well known to those skilled in the art (see, for example, Ausubel, FM et al., eds., 1989 Current protocol in Molecular Biology, Green Publishing associates, Inc., and John Wiley & Sons Inc., New York, at pages 6.3.1 6.3.6 and 2.10.3). The invention also encompasses polynucleotides encoding these polypeptides.

具有與查詢(query)胺基酸序列至少，例如，95%“一致性(identical)”的胺基酸序列的多肽，意指目的多肽序列可包含查詢胺基酸序列的每100個胺基酸各有達5個胺基酸變化，除此之外與查詢胺基酸序列一致。換言之，為得到具有與查詢胺基酸序列至少95%一致性的胺基酸序列之多肽，可用另外的胺基酸***、缺失或取代目的序列中達5%之胺基酸殘基。這些參考序列的改變可發生於 參考胺基酸序列的胺基或羧基端位置或這些端點位置間之任何地方，在參考序列的殘基中各自地或呈一個或多個鄰接群組而散布在參考序列中。 A polypeptide having an amino acid sequence at least, for example, 95% "identical" to a query amino acid sequence, meaning that the polypeptide sequence of interest may comprise every 100 amino acids of the amino acid sequence of the query. There are up to 5 amino acid changes, each of which is consistent with the query amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least 95% identical to the amino acid sequence of the query, an additional amino acid can be used to insert, delete or replace up to 5% of the amino acid residue in the sequence of interest. Changes to these reference sequences can occur in The amino or carboxy terminal position of the reference amino acid sequence or anywhere between these endpoint positions is interspersed in the reference sequence either individually or in one or more contiguous groups in the residues of the reference sequence.

作為一個實際問題，任何特別之多肽是否與，例如，本發明白蛋白融合蛋白質或其片段之胺基酸序列至少80%、85%、90%、95%、96%、97%、98%或99%相同，通常可用習知之電腦程式測定。測定查詢序列(本發明序列)及目的序列之間最佳整體配對的較佳方法，亦稱為全局序列比對，可使用以Brutlag et al.(Comp.App.Biosci.6：237 245(1990))計算法為基礎的FASTDB電腦程式測定。序列比對中查詢及目的序列兩者皆為核苷酸序列或皆為胺基酸序列。全局序列比對結果以一致性百分比表示。使用於FASTDB胺基酸比對的較佳參數為：矩陣(Matrix)=PAM 0、k-元組(k-tuple)=2、失配罰值(Mismatch Penalty)=1、連結罰值(Joining Penalty)=20、隨機化群組長度(Randomization Group Length)=0、截止評分(Cutoff Score)=1、窗口尺寸(Window Size)=序列長度、缺口罰值(Gap Penalty)=5、缺口尺寸罰值(Gap Size Penalty)=0.05、窗口尺寸=500或任何一個較短的目的胺基酸序列長度。 As a practical matter, is any particular polypeptide at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or, for example, the amino acid sequence of the albumin fusion protein or fragment thereof of the invention or 99% of the same, usually measured by a computer program of the prior art. A preferred method for determining the optimal overall pairing between a query sequence (a sequence of the invention) and a sequence of interest, also known as a global sequence alignment, can be used in Brutlag et al. (Comp. App. Biosci. 6:237 245 (1990). )) Calculation method based FASTDB computer program. Both the query and the sequence of interest in the sequence alignment are nucleotide sequences or all are amino acid sequences. Global sequence alignment results are expressed as percent identity. The preferred parameters for the FASTDB amino acid alignment are: Matrix = PAM 0, k-tuple = 2, Mismatch Penalty = 1, Link Penalty (Joining) Penalty)=20, Randomization Group Length=0, Cutoff Score=1, Window Size=Sequence Length, Gap Penalty=5, Gap Penalty Gap Size Penalty = 0.05, window size = 500 or any shorter length of the amino acid sequence of interest.

變異體通常具有與該變異體有相同長度之一段正常HA或治療性蛋白質至少75%(較佳至少約80%、90%、95%或99%)之序列一致性。使用特製用於序列相同性搜索的blastp、blastn、blastx、tblastn及tblastx程式採用的計算法(Karlin et al.,Proc.Natl.Acad.Sci.USA 87： 2264 2268(1990)及Altschul,J.Mol.Evol.36：290 300(1993)，完全納入參考)，經BLAST(本地序列基本搜索工具(Basic Local Alignment Search Tool))分析測定核苷酸或胺基酸序列級別的同源性或一致性。 A variant typically has a sequence identity of at least 75% (preferably at least about 80%, 90%, 95% or 99%) of a portion of normal HA or therapeutic protein of the same length as the variant. Calculations using blastp, blastn, blastx, tblastn, and tblastx programs tailored for sequence identity searches (Karlin et al., Proc. Natl. Acad. Sci. USA 87: 2264 2268 (1990) and Altschul, J. Mol. Evol. 36: 290 300 (1993), fully incorporated by reference), determination of nucleotides or amines by BLAST (Basic Local Alignment Search Tool) analysis Sequence homology or identity at the base acid level.

本發明多核苷酸變異體可在編碼區、非編碼區或兩者中含有變化。特別佳為含有產生沉默性取代、添加或缺失但不會改變編碼多肽的性質或活性之改變的多核苷酸變異體。以由於遺傳密碼子簡併而經由沉默性取代產生的核苷酸變異體為較佳。再者，多肽變異體其中少於50、少於40、少於30、少於20、少於10個，或5至50、5至25、5至10、1至5或1至2個胺基酸以任何組合經取代、缺失、添加者亦為較佳。可因種種理由而產生多核苷酸變異體，如，為特別的寄主而優化密碼子表現(改變人類mRNA密碼子成為細菌寄主，諸如，酵母菌或E.coli偏好的密碼子)。 Polynucleotide variants of the invention may contain variations in the coding region, the non-coding region, or both. Particularly preferred are polynucleotide variants that contain silent substitutions, additions or deletions that do not alter the property or activity of the encoded polypeptide. Nucleotide variants produced by silent substitution due to degeneracy of the genetic code are preferred. Furthermore, the polypeptide variants are less than 50, less than 40, less than 30, less than 20, less than 10, or 5 to 50, 5 to 25, 5 to 10, 1 to 5 or 1 to 2 amines. It is also preferred that the base acid is substituted, deleted or added in any combination. Polynucleotide variants can be produced for a variety of reasons, such as optimizing codon expression for a particular host (changing human mRNA codons to bacterial hosts, such as yeast or E. coli preferred codons).

為了建構各種Fc融合蛋白質諸如EPO-Fc融合建構、G-CSF-Fc融合建構或人類p40-Fc融合建構，而分別從NP_000790(SEQ ID NO：8)、CAA27291(SEQ ID NO：9)、AAG32620(SEQ ID NO：10)及NP_001057(SEQ ID NO：13)得到人類EPO、人類G-CSF、人類p40及人類TNF受體的胺基酸序列。於一個具體例中，位置303之胺基酸殘基Asn經Gln置換的修飾人類p40係與多肽連接。 To construct various Fc fusion proteins such as EPO-Fc fusion constructs, G-CSF-Fc fusion constructs or human p40-Fc fusion constructs, from NP_000790 (SEQ ID NO: 8), CAA27291 (SEQ ID NO: 9), AAG32620, respectively. (SEQ ID NO: 10) and NP_001057 (SEQ ID NO: 13) give amino acid sequences of human EPO, human G-CSF, human p40 and human TNF receptor. In one embodiment, the modified human p40 line substituted with Gln at the amino acid residue Asn at position 303 is linked to the polypeptide.

根據本發明的另一方面，提供含有改造的Fc區之全抗體。 According to another aspect of the invention, a whole antibody comprising an engineered Fc region is provided.

根據一個具體例，本發明提供製造融合蛋白質的方法，該方法包括：(i)將編碼融合蛋白質的DNA分子導入哺乳動物寄主細胞、(ii)使該細胞在表現融合蛋白質的生長培養基條件下生長、(iii)收獲產生的融合蛋白質。 According to a specific embodiment, the present invention provides a method of producing a fusion protein, the method comprising: (i) introducing a DNA molecule encoding a fusion protein into a mammalian host cell, and (ii) growing the cell under growth medium conditions expressing the fusion protein; And (iii) harvesting the resulting fusion protein.

另一個示例之具體例中，提供包括上述融合蛋白質或抗體分子或抗體片段的醫藥組成物。亦提供經給予該醫藥組成物而治療或預防某些症狀的方法。例如，提供一種方法，其係(i)降低自體免疫疾病的症狀/預防或治療自體免疫疾病、(ii)抑制移植排斥反應、(iii)治療/預防內毒素誘發之休克，包括給予治療有效量的雜合Fc及p40蛋白質或其變異體/片段的融合蛋白質。 In another specific example, a pharmaceutical composition comprising the above fusion protein or antibody molecule or antibody fragment is provided. Methods of treating or preventing certain symptoms by administering the pharmaceutical composition are also provided. For example, a method is provided which (i) reduces symptoms of autoimmune diseases/prevention or treatment of autoimmune diseases, (ii) inhibits transplant rejection, (iii) treats/prevents endotoxin-induced shock, including administration of treatment An effective amount of a fusion protein of a hybrid Fc and p40 protein or a variant/fragment thereof.

組成物可包括藥物載劑(carrier)。藥物載劑可為任何適合將抗體投遞予患者的可相容、無毒物質。載劑中可包含無菌水、酒精、脂肪、蠟及惰性固體。藥學上可接受之輔劑(緩衝劑、分散劑)亦可納入醫藥組成物中。 The composition can include a drug carrier. The pharmaceutical carrier can be any compatible, non-toxic substance suitable for delivering the antibody to a patient. Sterile water, alcohol, fat, wax, and inert solids may be included in the carrier. Pharmaceutically acceptable adjuvants (buffers, dispersing agents) can also be included in the pharmaceutical compositions.

可用多種方式將抗體組成物給予個體。例如，可經腸外給藥醫藥組成物，如，皮下、肌肉內或靜脈。可依慣用、熟知的滅菌技術將這些組成物滅菌。如需要使接近生理條件，組成物可含有藥學上可接受之輔助物質，諸如pH調節及緩衝劑、毒性調節劑等，例如乙酸鈉、氯化鈉、氯化鉀、氯化鈣、乳酸鈉等。這些配方中融合蛋白質、抗體或抗體片段的濃度可有很大變化，如，以重量計，從少於約0.5%，通常約1%或至少約1%，到多達15或20%， 且主要依照所選擇的特定給藥模式基於流體體積、黏度等而選擇。 The antibody composition can be administered to an individual in a variety of ways. For example, a pharmaceutical composition can be administered parenterally, such as subcutaneously, intramuscularly or intravenously. These compositions can be sterilized according to conventional and well known sterilization techniques. The composition may contain pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like, if necessary to approximate physiological conditions. The concentration of the fusion protein, antibody or antibody fragment in these formulations can vary widely, such as from less than about 0.5%, typically from about 1% or at least about 1%, up to 15 or 20% by weight, And is selected based primarily on fluid volume, viscosity, etc., depending on the particular mode of administration selected.

本發明亦提供編碼融合蛋白質的單離核酸分子及承載該核酸分子的表現載體。此種核酸可直接投予至需要由該核酸編碼之多肽的對象。或者，在培養基中表現該核酸而製造多肽然後給予個體。 The invention also provides an isolated nucleic acid molecule encoding a fusion protein and an expression vector carrying the nucleic acid molecule. Such a nucleic acid can be administered directly to a subject in need of a polypeptide encoded by the nucleic acid. Alternatively, the nucleic acid is expressed in a culture medium to produce a polypeptide which is then administered to an individual.

術語“肽”、“多肽”或“蛋白質”意指有2至40個胺基酸的分子，較佳為有3至20個胺基酸者及最佳為有6至15個胺基酸者。例示之肽可隨機經由上文引證之任何方法而產生、從肽庫中取得(如，噬菌體展示庫)或經由蛋白質分解而衍生。 The term "peptide", "polypeptide" or "protein" means a molecule having from 2 to 40 amino acids, preferably from 3 to 20 amino acids and most preferably from 6 to 15 amino acids. . The exemplified peptides can be generated randomly, by any method cited above, taken from a peptide library (eg, a phage display library), or derivatized via proteolysis.

術語“生理活性多肽”、“生物活性分子”、“生理活性蛋白質”、“活性多肽”、“多肽藥物”及“蛋白質藥物”，如本文所使用，彼等之意義是可以互換的，且特點為彼等以生理活性形式展現各種體內生理功能。 The terms "physiologically active polypeptide", "biologically active molecule", "physiologically active protein", "active polypeptide", "polypeptide drug" and "protein drug", as used herein, are meant to be interchangeable and characteristic. They exhibit various physiological functions in vivo in a physiologically active form.

由於具有容易被體內之蛋白質分解酵素變性或降解的性質，多肽具有不能長時間持續生理作用的缺點。然而，當多肽藥物與根據本發明具體例的免疫球蛋白Fc片段連接(或偶合)而形成融合蛋白質時，該藥物增加結構穩定性及血清半衰期。而且，相較於其他已知多肽藥物配方，連接Fc片段的多肽具有低很多的生理活性降低。因此，相較於傳統多肽藥物的體內生物可利用性，根據本發明包括多肽藥物及Fc片段或多肽藥物及Fc片段的共軛物 之融合多肽具有明顯改善體內生物可利用性的特點。亦在本發明具體例中清楚說明這點。即，與本發明Fc片段連接時，相較於彼等之天然形式或其他傳統融合形式，IFN-α、G-CSF、EPO、p40、TNF受體及其他蛋白質藥物展現增加之體內生物可利用性。 Polypeptides have the disadvantage of not being able to sustain physiological effects for a long period of time due to their property of being easily denatured or degraded by proteolytic enzymes in the body. However, when a polypeptide drug is linked (or coupled) to an immunoglobulin Fc fragment according to a specific example of the present invention to form a fusion protein, the drug increases structural stability and serum half-life. Moreover, the polypeptide linked to the Fc fragment has a much lower physiological activity reduction than other known polypeptide drug formulations. Thus, conjugates of polypeptide drugs and Fc fragments or polypeptide drugs and Fc fragments are included in accordance with the present invention, as compared to the in vivo bioavailability of conventional polypeptide drugs. The fusion polypeptide has the characteristics of significantly improving the bioavailability in vivo. This is also clearly explained in the specific example of the present invention. That is, when linked to the Fc fragment of the present invention, IFN-α, G-CSF, EPO, p40, TNF receptors, and other proteinaceous drugs exhibit increased in vivo bioavailability compared to their native forms or other conventional fusion forms. Sex.

應瞭解本發明係將傳統重組DNA方法學用於產生Fc融合蛋白質、含有根據本發明之改造Fc區的抗體及在實踐本發明中有效用的抗體片段。Fc融合建構較佳產生在DNA級別，將形成的DNAs整合至表現載體，並表現而製造本發明之融合蛋白質、抗體或抗體片段。 It will be appreciated that the present invention utilizes conventional recombinant DNA methodology for the production of Fc fusion proteins, antibodies comprising the engineered Fc regions according to the invention, and antibody fragments useful in the practice of the invention. The Fc fusion construct preferably results in the integration of the formed DNAs into the expression vector at the DNA level and is expressed to produce the fusion protein, antibody or antibody fragment of the invention.

如本文所使用，瞭解術語“載體”意指任何核酸，該核酸包含能夠納入至寄主細胞並與寄主細胞基因體重組及整合，或如游離基因體(episome)自主複製的核苷酸序列。此種載體包含線狀核酸、質體、噬菌質體、粘質體、RNA載體、病毒載體等。病毒載體的非限制實例包含反轉錄病毒、腺病毒及腺相關病毒。如本文所使用，瞭解術語“基因表現”或目標蛋白質“表現”意指DNA序列轉錄、mRNA轉錄本的轉譯及Fc融合蛋白質產物或抗體或抗體片段的分泌。 As used herein, the term "vector" is understood to mean any nucleic acid comprising a nucleotide sequence that can be incorporated into a host cell and recombined and integrated with the host cell genome, or autonomously replicated as an episome. Such vectors include linear nucleic acids, plastids, phage, plastids, RNA vectors, viral vectors, and the like. Non-limiting examples of viral vectors include retroviruses, adenoviruses, and adeno-associated viruses. As used herein, the term "gene expression" or "expression" of a target protein is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of an Fc fusion protein product or antibody or antibody fragment.

適用的表現載體為RcCMV(Invitrogen，Carlsbad)或其變異體。適用的表現載體應攜帶人類巨細胞病毒(CMV)啟動子而啟動建構哺乳動物細胞中目的基因的轉錄及攜帶牛生長激素多腺苷酸化訊號序列而增加轉錄後RNA的穩定狀態程度。本發明的具體例中，表現載體為 pAD11，其係RcCMV的修飾載體。攜帶編碼生物活性分子藥物的核苷酸序列之表現載體的實例可包含，而不限於，pAD11 EPO-hFc-1、pAD11 G-CSF-hFc-1、pAD11 p40N303Q-hFc-1、pAD11 EPO-hFc-6、pAD11 G-CSF-hFc-6、pAD11 p40N303Q-hFc-6、pAD11 EPO-hFc-5、pAD11 G-CSF-hFc-5、pAD11 p40N303Q-hFc-5或pAD11 TNFR-hFc-5，如實例中更詳細說明。 A suitable expression vector is RcCMV (Invitrogen, Carlsbad) or variants thereof. A suitable expression vector should carry the human cytomegalovirus (CMV) promoter to initiate transcription of the gene of interest in the mammalian cell and to carry the bovine growth hormone polyadenylation signal sequence to increase the degree of stability of the post-transcriptional RNA. In a specific example of the present invention, the performance carrier is pAD11, which is a modified vector of RcCMV. Examples of expression vectors carrying a nucleotide sequence encoding a bioactive molecule drug may include, without limitation, pAD11 EPO-hFc-1, pAD11 G-CSF-hFc-1, pAD11 p40N303Q-hFc-1, pAD11 EPO-hFc -6, pAD11 G-CSF-hFc-6, pAD11 p40N303Q-hFc-6, pAD11 EPO-hFc-5, pAD11 G-CSF-hFc-5, pAD11 p40N303Q-hFc-5 or pAD11 TNFR-hFc-5, More detailed in the examples.

可用本發明DNA序列轉化或轉染適當的寄主細胞，並利用於表現及/或分泌目標蛋白質。目前使用於本發明的較佳寄主細胞包含不死融合瘤細胞、NS/0骨髓瘤細胞、293細胞、中國倉鼠卵巢細胞、HeLa細胞及COS細胞。 Suitable host cells can be transformed or transfected with the DNA sequences of the invention and utilized to express and/or secrete the protein of interest. Preferred host cells currently used in the present invention comprise immortal fusion tumor cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells, HeLa cells, and COS cells.

一直使用於哺乳動物細胞中製造高程度表現的融合蛋白質或抗體或抗體片段之表現系統為DNA建構體，該DNA建構體從5'至3'之方向編碼分泌盒，包含訊號序列及免疫球蛋白Fc區，及目標蛋白質諸如p40、EPO、G-CSF、TNF受體。在這樣的系統中成功地表現數種目標蛋白質包含，例如，IL2、CD26、Tat、Rev、OSF-2、ss；IG-H3、IgE受體、PSMA及gp120。這些表現建構體揭露於Lo等人之U.S.Patent Nos.5,541,087及5,726,044，其內容以引用方式併入本文中。 The expression system that has been used in mammalian cells to produce highly expressed fusion proteins or antibodies or antibody fragments is a DNA construct that encodes a secretion cassette from the 5' to the 3' direction, including signal sequences and immunoglobulins. The Fc region, and target proteins such as p40, EPO, G-CSF, TNF receptors. Several target proteins are successfully expressed in such systems, for example, IL2, CD26, Tat, Rev, OSF-2, ss; IG-H3, IgE receptor, PSMA, and gp120. </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt; </ RTI> <RTIgt;

本發明之融合蛋白質或抗體分子或抗體片段在表現時可能或可能不包含訊號序列。如本文所使用，瞭解術語“訊號序列”意指指導生物活性分子藥物分泌的 節段；融合蛋白質在寄主細胞中轉譯後裂解。發明之訊號序列為多核苷酸，該多核苷酸編碼起動傳輸蛋白質穿過內質網膜的胺基酸序列。適用於本發明的訊號序列包含抗體輕鏈訊號序列，如，抗體14.18(Gillies et al.,J.Immunol.Meth.1989.125：191-202)，抗體重鏈訊號序列，如，MOPC141抗體重鏈訊號序列(Sakano et al.,Nature 1980.286：676-683)，及任何其他所屬領域習知的訊號序列(參見，如，Watson et al.,Nucleic Acids Research 1984.12：5145-5164)。 The fusion protein or antibody molecule or antibody fragment of the invention may or may not contain a signal sequence when expressed. As used herein, the term "signal sequence" is understood to mean the secretion of a drug from a biologically active molecule. Segment; the fusion protein is cleaved after translation in the host cell. The signal sequence of the invention is a polynucleotide encoding an amino acid sequence that initiates transport of the protein across the endoplasmic reticulum membrane. The signal sequence suitable for use in the present invention comprises an antibody light chain signal sequence, eg, antibody 14.18 (Gillies et al., J. Immunol. Meth. 1989. 125: 191-202), antibody heavy chain signal sequence, eg, MOPC141 antibody heavy chain signal Sequence (Sakano et al., Nature 1980. 286: 676-683), and any other signal sequence known in the art (see, e.g., Watson et al., Nucleic Acids Research 1984. 12: 5145-5164).

如所屬領域熟練之技術人員所明白的，使用於分泌盒之特別訊號序列的合適性需要一些常規試驗。 As will be appreciated by those skilled in the art, the suitability of the particular signal sequence for use in a secretion cassette requires some routine experimentation.

免疫球蛋白Fcy1基因的Fcy1區較佳包含至少免疫球蛋白鉸鏈結構域的一部分及至少CH3結構域，或更佳地至少鉸鏈結構域的一部分、CH2結構域及CH3結構域。如本文所使用，瞭解免疫球蛋白鉸鏈區的“部分”意指含有至少1個，較佳2個能夠形成鍵間二硫鍵的半胱胺酸之免疫球蛋白鉸鏈的一部分。編碼分泌盒的DNA可為其基因體組態或其cDNA組態。某些情況下，從人類免疫球蛋白Fcy2重鏈序列製造Fc區為有利。雖然基於人類免疫球蛋白y1及y2序列的Fc融合在小鼠中作用相似，但是基於y2序列的Fc融合可在人類中展現優異藥物動力學。 The Fcy1 region of the immunoglobulin Fcy1 gene preferably comprises at least a portion of an immunoglobulin hinge domain and at least a CH3 domain, or more preferably at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. As used herein, "part" of an immunoglobulin hinge region is understood to mean a portion of an immunoglobulin hinge containing at least 1, preferably 2, cysteine capable of forming an inter-bond disulfide bond. The DNA encoding the secretion cassette can be configured for its genome or its cDNA configuration. In some cases, it is advantageous to make an Fc region from a human immunoglobulin Fcy2 heavy chain sequence. Although Fc fusion based on human immunoglobulin y1 and y2 sequences functions similarly in mice, Fc fusion based on the y2 sequence can exhibit superior pharmacokinetics in humans.

再者，這些恆定區建構的取代或缺失，其中恆定區結構域的一個或多個胺基酸殘基經取代或缺失亦為有效用。實例之一是在上CH2區導入胺基酸取代而創建對Fc受體親和力減低的Fc變異體(Cole et al.(1997)J. Immunol.159：3613)。所屬領域具普通熟練之人員可使用熟知的分子生物技術而製備這些建構。 Furthermore, substitutions or deletions of these constant region constructs in which one or more amino acid residues of the constant region domain are substituted or deleted are also effective. One example is the introduction of an amino acid substitution in the upper CH2 region to create an Fc variant with reduced affinity for the Fc receptor (Cole et al. (1997) J. Immunol. 159: 3613). Those skilled in the art can prepare these configurations using well-known molecular biotechnology.

能夠與本發明免疫球蛋白Fc片段共軛的蛋白質藥物的非限制實例包含人類生長激素、骨形成蛋白-1、生長激素釋放激素、生長激素釋放肽、干擾素及干擾素受體、顆粒細胞群落刺激因子(G-CSF)、顆粒細胞-巨噬細胞群落刺激因子(GM-CSF)、類昇糖素胜肽、G蛋白偶聯受體、介白素及介白素受體、酵素、T細胞因子、紅血球生成素、高糖基化紅血球生成素、血管生成素、VII因子、VIIa因子、VIII因子、IX因子、XIII因子、胞漿素原活化因子、血纖維蛋白結合肽、脲激酶、上皮細胞生長因子、表皮細胞生長因子、濾泡刺激素、黃體激素、黃體激素釋放激素、神經生長因子、細胞表面抗原、病毒疫苗抗原、單株抗體、多株抗體、抗體片段。特別地，較佳作為生物活性分子係在治療或預防疾病而給藥予身體時需要頻繁用藥者，其包含人類生長激素、干擾素(α-、β-及γ-干擾素等)、顆粒細胞群落刺激因子(G-CSF)、紅血球生成素(EPO)、TFN受體、p40及抗體片段。此外，某些衍生物只要相較於天然生物活性分子彼等實質上具有相同或改進的功能、結構、活性或穩定性者亦包含在本發明生物活性分子的範疇內。本發明中，最佳多肽藥物為α-干擾素。 Non-limiting examples of protein drugs that can be conjugated to the immunoglobulin Fc fragments of the invention include human growth hormone, bone morphogenetic protein-1, growth hormone releasing hormone, growth hormone releasing peptide, interferon and interferon receptor, granulosa cell population Stimulating factor (G-CSF), granulosa cell-macrophage community stimulating factor (GM-CSF), glucagon-like peptide, G-protein coupled receptor, interleukin and interleukin receptor, enzyme, T Cytokines, erythropoietin, hyperglycosylated erythropoietin, angiopoietin, factor VII, factor VIIa, factor VIII, factor IX, factor XIII, plasminogen activator, fibrin binding peptide, urea kinase, Epithelial cell growth factor, epidermal growth factor, follicle stimulating hormone, luteinizing hormone, progesterone releasing hormone, nerve growth factor, cell surface antigen, viral vaccine antigen, monoclonal antibody, polyclonal antibody, antibody fragment. In particular, it is preferred that the bioactive molecule system is administered to the body in the treatment or prevention of diseases, and includes frequent administration of human growth hormone, interferon (α-, β-, and γ-interferon, etc.), granulosa cells. Community stimulating factor (G-CSF), erythropoietin (EPO), TFN receptor, p40 and antibody fragments. In addition, certain derivatives are also included within the scope of the biologically active molecules of the present invention as long as they have substantially the same or improved function, structure, activity or stability as compared to the natural biologically active molecule. In the present invention, the optimal polypeptide drug is alpha-interferon.

本發明的另一方面，IgG-Fc及IgG-CH融合蛋白質，例如，係合成可組合形成二聚體之單體。通常，在IgG鉸鏈區藉由二硫鍵連接二聚體。分泌IgG融合蛋白 質之細胞的條件化培養基可含有IgG融合蛋白質單體及二聚體的混合物。作為人類治療之用途，最好使用IgG融合蛋白質之單體或者二聚體的同質群體，而非2種形式的混合物。 In another aspect of the invention, IgG-Fc and IgG-CH fusion proteins, for example, are monomers that can be combined to form a dimer. Typically, the dimer is linked by a disulfide bond in the IgG hinge region. Secreted IgG fusion protein The conditioned medium of the cytoplasmic cells may contain a mixture of IgG fusion protein monomers and dimers. For human therapeutic use, it is preferred to use a homogenous population of monomers or dimers of the IgG fusion protein rather than a mixture of the two forms.

本發明亦提供得到本質上為純製劑的二聚體活性多肽-IgG融合蛋白質的方法。該方法一般經由得到能夠表現IgG融合蛋白質的寄主細胞、收集條件化培養基及經由管柱層析程序從單體融合蛋白質、聚集體及汙染蛋白質而純化二聚體融合蛋白質而完成。表現IgG融合蛋白質的適當寄主細胞包含酵母菌、昆蟲、哺乳動物或其他真核細胞。具體例中，寄主細胞可為哺乳動物細胞，特別是COS、CHO或BHK細胞。 The invention also provides a method of obtaining a dimeric active polypeptide-IgG fusion protein that is essentially a pure preparation. This method is generally accomplished by obtaining a host cell capable of expressing an IgG fusion protein, collecting a conditioned medium, and purifying the dimeric fusion protein from a monomer fusion protein, aggregate, and contaminating protein via a column chromatography program. Suitable host cells that express an IgG fusion protein comprise yeast, insect, mammalian or other eukaryotic cells. In particular embodiments, the host cell can be a mammalian cell, particularly a COS, CHO or BHK cell.

本發明亦提供多肽藥物及Fc片段的新穎融合蛋白質。於一個具體例中，多肽藥物諸如EPO、p40、G-CSF或TNF受體不經介於其間的肽連接物而直接與雜合Fc片段連接。另一個具體例中，多肽藥物經由1至50個胺基酸的肽連接物而彼此連接，及更佳為經由1至7個胺基酸的肽連接物。為此目的，特別適用的連接物包含由Gly及Ser殘基(如，Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser)組成或由衍生自人類白蛋白的胺基酸組成的免疫非活性肽。 The invention also provides novel fusion proteins of polypeptide drugs and Fc fragments. In one embodiment, the polypeptide drug, such as EPO, p40, G-CSF, or TNF receptor, is directly linked to the hybrid Fc fragment without intervening peptide linkers. In another embodiment, the polypeptide drug is linked to each other via a peptide linker of 1 to 50 amino acids, and more preferably a peptide linker via 1 to 7 amino acids. For this purpose, particularly suitable linkers comprise a Gly and Ser residue (eg, Gly Gly Ser Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Gly Ser) or consist of an amino acid derived from human albumin. Immunologically inactive peptides.

使用連接物的情況，可以特定方向製作連接物及多肽藥物。即，可使連接物與雜合Fc片段的N-端、C-端或游離基連接，亦可連接多肽藥物的N-端、C-端或游 離基。當連接物為肽連接物時，連結可發生於特定連接位點。 In the case of a linker, the linker and the polypeptide drug can be made in a specific direction. That is, the linker can be linked to the N-terminus, C-terminus or free radical of the hybrid Fc fragment, or can be linked to the N-terminus, C-terminus or tour of the polypeptide drug. Off the base. When the linker is a peptide linker, the linker can occur at a particular linker site.

本發明亦提供製造多肽藥物-雜合Fc片段的方法。 The invention also provides methods of making a polypeptide drug-hybrid Fc fragment.

本發明亦提供經由給藥多肽藥物而減緩症狀的治療方法。這些方法包含將有效量的本發明多肽給藥予具有症狀的哺乳動物，而該症狀可能或可能不是直接與目的疾病有關。例如，可將編碼所需多肽藥物-雜合Fc片段融合蛋白質的核酸，諸如DNA或RNA，給藥予個體，較佳為哺乳動物，作為治療劑。此外，可將含有編碼多肽藥物-雜合Fc片段融合蛋白質的核酸之細胞給藥予個體，較佳為哺乳動物，作為治療劑。再者，可將多肽藥物-雜合Fc片段融合建構給藥予個體，較佳為哺乳動物，作為治療劑。這種嵌合多肽可經靜脈、皮下、口服、口腔、舌下、鼻內、腸外、直腸、***或經肺部途徑而給藥。 The invention also provides methods of treatment for alleviating symptoms via administration of a polypeptide drug. These methods comprise administering an effective amount of a polypeptide of the invention to a symptomatic mammal, and the condition may or may not be directly related to the disease of interest. For example, a nucleic acid encoding a desired polypeptide drug-hybrid Fc fragment fusion protein, such as DNA or RNA, can be administered to an individual, preferably a mammal, as a therapeutic agent. Further, a cell containing a nucleic acid encoding a polypeptide drug-hybrid Fc fragment fusion protein can be administered to an individual, preferably a mammal, as a therapeutic agent. Further, the polypeptide drug-hybrid Fc fragment can be fused to an individual, preferably a mammal, as a therapeutic agent. Such chimeric polypeptides can be administered by intravenous, subcutaneous, oral, buccal, sublingual, intranasal, parenteral, rectal, vaginal or pulmonary routes.

顆粒細胞群落刺激因子(G-CSF)為對顆粒細胞的增殖及分化很重要的蛋白質，特別是中性粒細胞。顆粒細胞吞噬微生物侵入者及細胞碎片及因此對感染回應至關重要。化學療法破壞顆粒細胞及/或降低顆粒細胞之產生。因此，本發明G-CSF(包含其變異體/片段)-fc融合蛋白質可適用於治療/預防/改善化學療法誘導之骨髓移植後的嗜中性白血球缺乏骨髓抑制症、急性白血病、再生不良性貧血、骨髓造血不良症候群、嚴重慢性嗜中性白血球缺乏症或移植之周邊血液先驅細胞移動的症狀。 Granulocyte community stimulating factor (G-CSF) is a protein important for the proliferation and differentiation of granulosa cells, especially neutrophils. Granulocyte phagocytosis of microbial invaders and cellular debris and thus response to infection is critical. Chemotherapy destroys granulosa cells and/or reduces the production of granulosa cells. Therefore, the G-CSF (including variants/fragments thereof)-fc fusion protein of the present invention can be used for treating/preventing/improving chemotherapeutic-induced bone marrow transplantation, neutrophilic leukemia deficiency, acute leukemia, dysplasia Symptoms of anemia, bone marrow hematopoietic syndrome, severe chronic neutrophil deficiency, or migration of peripheral blood precursor cells.

本發明之融合蛋白質不僅適用作為治療劑，而且所屬領域之熟練技術人員認知該融合蛋白質適用於製造用於診斷的抗體。同樣地，使用本發明的方法亦包含為此用途而適當地給藥DNA或RNA，如，以載體或其他投遞系統。 The fusion protein of the present invention is not only useful as a therapeutic agent, but those skilled in the art recognize that the fusion protein is suitable for use in the manufacture of antibodies for diagnosis. Likewise, the use of the methods of the invention also encompasses the proper administration of DNA or RNA for such use, e.g., in a carrier or other delivery system.

再者，預期本發明融合蛋白質亦可與多數不同生物活性分子一起給予有意的接受者。然亦預期，可由所屬領域熟練技術人員技術水平內的常規實驗測定融合蛋白質與其他分子的最優組合、給藥模式、劑量。 Furthermore, it is contemplated that the fusion proteins of the invention may also be administered to an intended recipient along with a plurality of different biologically active molecules. It is also contemplated that the optimal combination, mode of administration, dosage of the fusion protein with other molecules can be determined by routine experimentation within the skill of the art to those skilled in the art.

[實施例] [Examples]

經由下列非限制實例進一步說明本發明。 The invention is further illustrated by the following non-limiting examples.

<實施例1>製備hFc-1、hFc-2、hFc-3、hFc-4、hFc-5及hFc-6融合蛋白質的表現載體<Example 1> Expression vector for preparation of hFc-1, hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 fusion proteins

hFc-1包含IgG1 CH1區C-端的9個胺基酸(90至98)、IgG1鉸鏈區(99至113)、IgG2 CH2區N-端的6個胺基酸(111至116)、IgG4 CH2區的103個胺基酸(118至220)及IgG4 CH3區的107個胺基酸(221至327)(第1及2圖)。為得到各自編碼人類EPO(SEQ ID NO：2)、人類G-CSF(SEQ ID NO：3)及人類p40N303Q(衍生自人類p40次單元第303個胺基酸之Asn經Gln取代的突變體)(顯示p40N303Q之核苷酸序列為SEQ ID NO：4，及顯示人類p40之胺基酸序列為SEQ ID NO：10)的密碼子優化核苷酸，分別地，經由TOP Gene Technologies(Quebec,Canada)(www.topgenetech.com)的客製化服務合成這些核苷酸分 子。優化基因的密碼子使用對增加蛋白質表現水平是很有幫助的。密碼子使用模式在生物體間是不同的。一些密碼子在一種生物體內使用很頻繁但是在另一種生物體內卻很少使用。這種密碼使用的偏差歸因於轉譯效率，生物體合成編碼蛋白質的能力。在EPO、G-CSF及p40N303Q之ATG序列的5'端造出EcoR I位點及在hFc-1之末端密碼子的3'端造出Xba I位點，而將各融合基因***表現載體pAD11(SEQ ID NO：5)。從RcCMV骨架(得自Invitrogen,Carlsbad)得到表現載體pAD11。pAD11包含衍生自巨細胞病毒(CMV)的啟動子、衍生自牛生長激素的多腺核苷酸化(poly(A))序列、衍生自兔β球蛋白的球蛋白間插序列(gIVS)(Mol Cell Biol,1988 8：4395)等。亦有從RcCMV載體(Invitrogen)的數個修飾而製作pAD11載體。用Xho I酵素處理而移除新黴素抗性區及在CMV啟動子區的3'添加gIVS。此外，在CMV啟動子的5'添加小鼠二氫葉酸還原酶(DHFR)基因(Pubmed,NM 010049)。組合包含上述數種要素的許多表現試驗之後研製成pAD11載體。吾等未發表的結果中，相較於RcCMV載體(Invitrogen)，pAD11載體顯示增加約12倍表現水平。在EPO、G-CSF及p40N303Q編碼序列的3'端及hFc-1編碼序列的5'端造出Nhe I位點而製作框內EPO、G-CSF及p40N303Q的3'端及hFc-1的5'端間的連接位點。使用各自限制酶位點而次選殖後，產生hFc-1融合EPO、G-CSF或p40N303Q的最後表現載體，然後分別指明為pAD11 EPO-hFc-1、pAD11 G-CSF-hFc-1及pAD11 p40N303Q-hFc-1。 hFc-1 contains 9 amino acids (90 to 98) at the C-terminus of the IgG1 CH1 region, IgG1 hinge region (99 to 113), 6 amino acids (111 to 116) at the N-terminus of the IgG2 CH2 region, and IgG4 CH2 region. 103 amino acids (118 to 220) and 107 amino acids (221 to 327) in the IgG4 CH3 region (Figs. 1 and 2). To obtain respective coding human EPO (SEQ ID NO: 2), human G-CSF (SEQ ID NO: 3) and human p40N303Q (a mutant derived from Gln substituted by Asn derived from the 303th amino acid of human p40 subunit) (The nucleotide sequence showing p40N303Q is SEQ ID NO: 4, and the codon-optimized nucleotide showing the amino acid sequence of human p40 is SEQ ID NO: 10), respectively, via TOP Gene Technologies (Quebec, Canada) ) (www.topgenetech.com)'s customized service to synthesize these nucleotides child. Optimizing codon usage of genes is helpful in increasing protein performance levels. The codon usage pattern is different between organisms. Some codons are used frequently in one organism but rarely in another. This bias in the use of passwords is attributed to the efficiency of translation, the ability of organisms to synthesize proteins. The EcoR I site was generated at the 5' end of the ATG sequence of EPO, G-CSF and p40N303Q, and the Xba I site was generated at the 3' end of the end codon of hFc-1, and each fusion gene was inserted into the expression vector pAD11. (SEQ ID NO: 5). The expression vector pAD11 was obtained from the RcCMV backbone (available from Invitrogen, Carlsbad). pAD11 comprises a promoter derived from cytomegalovirus (CMV), a polyadenylation (poly(A)) sequence derived from bovine growth hormone, and a globulin intervening sequence (gIVS) derived from rabbit beta globulin (Mol) Cell Biol, 1988 8:4395) et al. The pAD11 vector was also produced by several modifications from the RcCMV vector (Invitrogen). The neomycin resistance zone was removed by treatment with Xho I enzyme and gIVS was added 3' to the CMV promoter region. In addition, the mouse dihydrofolate reductase (DHFR) gene (Pubmed, NM 010049) was added 5' to the CMV promoter. A number of performance assays containing several of the above elements were combined to develop a pAD11 vector. In our unpublished results, the pAD11 vector showed an approximately 12-fold increase in performance levels compared to the RcCMV vector (Invitrogen). The 3' end of the EPO, G-CSF and p40N303Q coding sequences and the 5' end of the hFc-1 coding sequence were used to create the Nhe I site to make the 3' end of the EPO, G-CSF and p40N303Q and hFc-1. The junction site between the 5' ends. The final expression vector of hFc-1 fusion EPO, G-CSF or p40N303Q was generated using the respective restriction enzyme sites and subcloned, and then designated as pAD11 EPO-hFc-1, pAD11 G-CSF-hFc-1 and pAD11, respectively. p40N303Q-hFc-1.

hFc-6包含IgD CH1結構域C-端的9個胺基酸(90至98)、IgD鉸鏈區的64個胺基酸(99至162)、IgD CH2結構域N-端的8個胺基酸(SHTQPLGV；163至170)、IgG4 CH2結構域的100個胺基酸(121至220)及IgG4 CH3結構域的107個胺基酸(221至327)(第1及2圖)。為得到編碼hFc-6的密碼子優化核苷酸分子，而由TOP Gene Technologies(www.topgenetech.com)的客製化服務合成該基因。使用包含於hFc-6編碼區N-端(90及91胺基酸)的Nhe I位點(gctagc：Ala-Ser)而製作框內EPO、G-CSF或p40N303Q的3'端及hFc-6的5'端間的融合。而且，在hFc-6基因的3'端造出Xba I位點而將各hFc-6融合基因***pAD11載體。使用各自限制酶位點而次選殖後，產生hFc-6融合EPO、G-CSF及p40N303Q的最後表現載體，然後分別指定為pAD11 EPO-hFc-6、pAD11 G-CSF-hFc-6及pAD11 p40N303Q-hFc-6。hFc-2、hFc-3、hFc-4及hFc-5具有相同的CH2及CH3區，但具有不同大小的IgD鉸鏈(第1及2圖)。hFc-2、hFc-3、hFc-4及hFc-5分別包含IgD鉸鏈C-端的5個胺基酸(158至162)、10個胺基酸(153至162)、20個胺基酸(143至162)、30個胺基酸(133-162)(第1及2圖)。由TOP Gene Technologies(www.topgenetech.com)的客製化服務合成融合基因總尺寸的最小基因片段而製作EPO、G-CSF、p40N303Q或TNFR(腫瘤壞死因子受體II)(SEQ ID NO：12)及編碼這些hFcs的核酸分子間的融合基因。與編碼鉸鏈及各hFc-2、 hFc-3、hFc-4或hFc-5 CH2區N-端的核苷酸分子融合的各EPO、G-CSF、p40N303Q或TNFR合成片段包含從全部EPO、G-CSF、p40N303Q或TNFR序列至相同酵素位點，位於IgG4(SEQ ID NO：6)CH2區第138至140胺基酸殘基的BstE II位點(GGTGACC)的序列。分別用位於5'端及3'端的EcoR I及BstE II切割包含數個基因片段的次選殖載體，然後接合hFc-6的CH2-CH3區。最後，用EcoR I及Xba I位點次選殖各融合基因至pAD11，然後分別指定為pAD11 EPO-hFc-2、pAD11 EPO-hFc-3、pAD11 EPO-hFc-4、pAD11 EPO-hFc-5、pAD11 G-CSF-hFc-2、pAD11 G-CSF-hFc-3、pAD11 G-CSF-hFc-4、pAD11 G-CSF-hFc-5、pAD11 p40N303Q-hFc-2、pAD11 p40N303Q-hFc-3、pAD11 p40N303Q-hFc-4、pAD11 p40N303Q-hFc-5及pAD11 TNFR-hFc-5。 hFc-6 comprises 9 amino acids (90 to 98) at the C-terminus of the IgD CH1 domain, 64 amino acids (99 to 162) in the IgD hinge region, and 8 amino acids at the N-terminus of the IgD CH2 domain ( SHTQPLGV; 163 to 170), 100 amino acids (121 to 220) of the IgG4 CH2 domain and 107 amino acids (221 to 327) of the IgG4 CH3 domain (Figs. 1 and 2). To obtain a codon-optimized nucleotide molecule encoding hFc-6, the gene was synthesized by a customization service of TOP Gene Technologies (www.topgenetech.com). The 3' end of the EPO, G-CSF or p40N303Q and hFc-6 were made using the Nhe I site (gctagc: Ala-Ser) contained in the N-terminus (90 and 91 amino acids) of the hFc-6 coding region. The fusion between the 5' ends. Furthermore, an Xba I site was created at the 3' end of the hFc-6 gene and each hFc-6 fusion gene was inserted into the pAD11 vector. After sub-selection using the respective restriction enzyme sites, the final expression vector of hFc-6 fusion EPO, G-CSF and p40N303Q was generated, and then designated as pAD11 EPO-hFc-6, pAD11 G-CSF-hFc-6 and pAD11, respectively. p40N303Q-hFc-6. hFc-2, hFc-3, hFc-4 and hFc-5 have the same CH2 and CH3 regions, but have different sizes of IgD hinges (Figs. 1 and 2). hFc-2, hFc-3, hFc-4 and hFc-5 comprise 5 amino acids (158 to 162) at the C-terminus of the IgD hinge, 10 amino acids (153 to 162), and 20 amino acids ( 143 to 162), 30 amino acids (133-162) (Figs. 1 and 2). EPO, G-CSF, p40N303Q or TNFR (tumor necrosis factor receptor II) was produced by the TOP Gene Technologies (www.topgenetech.com) customization service to synthesize the smallest gene fragment of the total size of the fusion gene (SEQ ID NO: 12) And a fusion gene between nucleic acid molecules encoding these hFcs. With coding hinges and each hFc-2, Each EPO, G-CSF, p40N303Q or TNFR synthetic fragment fused to the N-terminal nucleotide molecule of the hFc-3, hFc-4 or hFc-5 CH2 region comprises from all EPO, G-CSF, p40N303Q or TNFR sequences to the same enzyme Site, the sequence of the BstE II site (GGTGACC) at amino acid residues 138 to 140 of the IgG4 (SEQ ID NO: 6) CH2 region. The secondary selection vector containing several gene fragments was cleaved with EcoR I and BstE II at the 5' end and the 3' end, respectively, and then the CH2-CH3 region of hFc-6 was ligated. Finally, each fusion gene was selected from the EcoR I and Xba I sites to pAD11, and then designated as pAD11 EPO-hFc-2, pAD11 EPO-hFc-3, pAD11 EPO-hFc-4, pAD11 EPO-hFc-5, respectively. , pAD11 G-CSF-hFc-2, pAD11 G-CSF-hFc-3, pAD11 G-CSF-hFc-4, pAD11 G-CSF-hFc-5, pAD11 p40N303Q-hFc-2, pAD11 p40N303Q-hFc-3 , pAD11 p40N303Q-hFc-4, pAD11 p40N303Q-hFc-5 and pAD11 TNFR-hFc-5.

<實施例2>製備thFc-1及thFc-2偶合至IFN-β的表現載體<Example 2> Preparation of expression vector for coupling of thFc-1 and thFc-2 to IFN-β

thFc-1包含人類組織血漿素原活化因子(tPA)訊號序列的23個胺基酸(MDAMLRGLCCVLLLCGAVFVSPS)、IgG1鉸鏈區的15個胺基酸(99至113)、IgG2 CH2區N-端的6個胺基酸(111至116)、IgG4 CH2區的103個胺基酸(118至220)及IgG4 CH3區的107個胺基酸(221至327)(第3圖)。thFc-2包含tPA訊號序列的23個胺基酸(MDAMLRGLCCVLLLCGAVFVSPS)、IgD鉸鏈區的15個胺基酸(148至162)、IgD CH2區N-端的8個胺基酸(163至 170)、IgG4 CH2區的100個胺基酸(121至220)及IgG4 CH3區的107個胺基酸(221至327)(第3圖)。為得到編碼與缺失訊號序列的人類IFN-β之N-端偶合的thFc-1或thFc-2的密碼子優化核苷酸，而由TOP Gene Technologies(Quebec,Canada)(www.topgenetech.com)的客製化服務合成這些核苷酸分子。在thFc-1或thFc-2的5'端造出EcoR I位點及在IFN-β末端密碼子的3'端造出Not I位點而將各融合基因***表現載體pAD11(SEQ ID NO：5)。使用各自限制酶位點而次選殖後，分別指定最後表現載體為pAD11 thFc-1-AL(0)-IFN-β及pAD11 thFc-2-AL(0)-IFN-β。 thFc-1 contains 23 amino acids (MDAMLRGLCCVLLLCGAVFVSPS) of the human tissue plasminogen activator (tPA) signal sequence, 15 amino acids (99 to 113) in the IgG1 hinge region, and 6 amines at the N-terminus of the IgG2 CH2 region. 103 acids (111 to 116), 103 amino acids (118 to 220) in the IgG4 CH2 region, and 107 amino acids (221 to 327) in the IgG4 CH3 region (Fig. 3). thFc-2 contains 23 amino acids of the tPA signal sequence (MDAMLRGLCCVLLLCGAVFVSPS), 15 amino acids of the IgD hinge region (148 to 162), and 8 amino acids of the N-terminus of the IgD CH2 region (163 to 170), 100 amino acids (121 to 220) in the IgG4 CH2 region and 107 amino acids (221 to 327) in the IgG4 CH3 region (Fig. 3). To obtain codon-optimized nucleotides encoding thFc-1 or thFc-2 encoding the N-terminus of human IFN-β with a missing signal sequence, by TOP Gene Technologies (Quebec, Canada) (www.topgenetech.com) The customization service synthesizes these nucleotide molecules. The EcoR I site was created at the 5' end of thFc-1 or thFc-2 and the Not I site was generated at the 3' end of the IFN-β terminal codon. Each fusion gene was inserted into the expression vector pAD11 (SEQ ID NO: 5). After sub-selection using the respective restriction enzyme sites, the final expression vectors were designated as pAD11 thFc-1-AL(0)-IFN-β and pAD11 thFc-2-AL(0)-IFN-β, respectively.

由TOP Gene Technologies(www.topgenetech.com)的客製化服務合成從thFc-1 CH3區Pst I位點經由不同尺寸的白蛋白連接物(3aa、8aa、13aa、18aa、23aa及33aa)或Gly-Ser連接物(15aa)而與缺失訊號序列的IFN-β偶合之基因片段(第4圖)，藉此製作經由不同尺寸的白蛋白連接物或Gly-Ser連接物而與IFN-β偶合的thFc。為了將7個不同的基因片段***表現載體pAD11 thFc-1-AL(0)-IFN-β及pAD11 thFc-2-AL(0)-IFN-β，而在彼等之5'端造出Pst I位點及在IFN-β末端密碼子的3'端造出Not I位點。使用各自限制酶位點而次選殖後，指定最後表現載體為pAD11 thFc-1-AL(1)-IFN-β、pAD11 thFc-1-AL(2)-IFN-β、pAD11 thFc-1-AL(3)-IFN-β、pAD11 thFc-1-AL(4)-IFN-β、pAD11 thFc-1-AL(5)-IFN-β、pAD11 thFc-1-AL(6)-IFN-β、pAD11 thFc-1-GS-IFN-β、pAD11 thFc-2-AL(1)-IFN-β、pAD11 thFc-2-AL(2)-IFN-β、pAD11 thFc-2-AL(3)-IFN-β、pAD11 thFc-2-AL(4)-IFN-β、pAD11 thFc-2-AL(5)-IFN-β、pAD11 thFc-2-AL(6)-IFN-β及pAD11 thFc-2-GS-IFN-β。 Synthetic services from TOP Gene Technologies (www.topgenetech.com) synthesize from the Pst I site of the thFc-1 CH3 region via different size albumin connectors (3aa, 8aa, 13aa, 18aa, 23aa and 33aa) or Gly -Ser linker (15aa) and a gene fragment coupled to the IFN-β of the missing signal sequence (Fig. 4), thereby making coupling with IFN-β via different size albumin conjugates or Gly-Ser conjugates thFc. In order to insert 7 different gene fragments into the expression vectors pAD11 thFc-1-AL(0)-IFN-β and pAD11 thFc-2-AL(0)-IFN-β, Pst was produced at the 5' end of them. The I site and the Not I site were created at the 3' end of the IFN-β terminal codon. After sub-selection using the respective restriction enzyme sites, the final expression vector was designated as pAD11 thFc-1-AL(1)-IFN-β, pAD11 thFc-1-AL(2)-IFN-β, pAD11 thFc-1- AL(3)-IFN-β, pAD11 thFc-1-AL(4)-IFN-β, pAD11 thFc-1-AL(5)-IFN-β, pAD11 thFc-1-AL(6)-IFN-β , pAD11 thFc-1-GS-IFN-β, pAD11 thFc-2-AL(1)-IFN-β, pAD11 thFc-2-AL(2)-IFN-β, pAD11 thFc-2-AL(3)-IFN-β, pAD11 thFc-2-AL(4)-IFN-β, pAD11 thFc-2-AL(5) - IFN-β, pAD11 thFc-2-AL(6)-IFN-β and pAD11 thFc-2-GS-IFN-β.

<實施例3>表現人類EPO-hFcs、人類G-CSF-hFcs、人類p40N303Q-hFcs、人類TNFR-hFc-5及thFcs-IFN-β蛋白質<Example 3> Expression of human EPO-hFcs, human G-CSF-hFcs, human p40N303Q-hFcs, human TNFR-hFc-5 and thFcs-IFN-β protein

使用COS-7細胞做進行表現試驗及使用補充10%胎牛血清(Hyclone,South Logan)及抗生素(Invitrogen,Carlsbad)的DMEM培養基(Invitrogen,Carlsbad)培養。使用常規電穿孔法將編碼EPO-hFcs、G-CSF-hFcs、p40N303Q-hFcs、TNFR-hFc-5、thFcs-IFN-β的載體轉染至5X106 COS-7細胞。轉染後48小時，收取上清液及細胞。用數種試劑盒以所有樣本做ELISA檢測(EPO用R&D system,Minneapolis,#DEP00；G-CSF用Biosource,Camarillo,#KHC2032；p40N303Q用R&D system,Minneapolis,#DY1240；TNFR用R&D system,Minneapolis,#DRT200；IFN-β用PBL Biomedical Laboratories,#41410-1A)及用抗人類IgG抗體(Santa Cruz Biotechnology,Santa Cruz)做西方墨點分析，而檢查各載體融合蛋白質的表現。結果，上清液及細胞溶解產物中的所有載體均顯示正確表現模式(數據未顯示)。 COS-7 cells were used for performance testing and cultured in DMEM medium (Invitrogen, Carlsbad) supplemented with 10% fetal bovine serum (Hyclone, South Logan) and antibiotics (Invitrogen, Carlsbad). Vectors encoding EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5, thFcs-IFN-β were transfected into 5× 10 6 COS-7 cells using conventional electroporation. 48 hours after transfection, supernatant and cells were collected. ELISA was performed on all samples using several kits (R&D system for EPO, Minneapolis, #DEP00; Biosource for C-CSF, Camarillo, #KHC2032; R&D system for p40N303Q, Minneapolis, #DY1240; R&D system for TNFR, Minneapolis, #DRT200; IFN-β was assayed for Western blot analysis using PBL Biomedical Laboratories, #41410-1A) and anti-human IgG antibody (Santa Cruz Biotechnology, Santa Cruz). As a result, all the vectors in the supernatant and cell lysate showed the correct expression pattern (data not shown).

<實施例4>hFc-融合蛋白質的純化<Example 4> Purification of hFc-fusion protein

用α-MEM(Invitrgen,Carlsbad)、10%透析胎牛血清(JRH Biosciences,Kansas)、HT添加物(Invitrogen,Carlsbad)及抗生素(Invitrogen,Carlsbad)培養CHO/DHFR-/- 細胞(中國倉鼠卵巢細胞，DG44,ATCC)。依照常規CaPO4共沉澱法將表現載體轉染至CHO細胞。轉染後48小時，使CHO細胞從盤中脫離並稀釋數倍(1/2、1/5、1/20、1/50、1/100、1/200、1/500)。將稀釋細胞舖佈於100mm培養皿並以無HT添加物的培養基培養。篩選過程中，不用繼代而補充不含HT添加物的新鮮培養基至細胞。舖佈後2至3週產生群落並將個別的群落移至48孔盤。ELISA檢測後篩選陽性群落做EPO、G-CSF、p40N303Q及TNFR檢測。將顯示最高表現的各群落使用無血清的培養基(JRH Biosciences,Kansas)做大規模(5L)培養。使用收取的無血清上清液做各融合蛋白質的純化。於純化，用20mM磷酸鈉緩衝液(pH 7.0)平衡HiTrap重組蛋白質A FF(Amersham biosciences,Piscataway)管柱。將過濾的上清液加入管柱中並用0.1M檸檬酸鈉(pH 3.0)洗提。用膜(MWCO 12-14K,Spectrapor,Rancho Dominguez)透析超過3次後，最後得到洗提蛋白質。所有蛋白質樣本濃度均用BCA試劑盒(Pierce Biotechnology,Rockford)做總蛋白質的測量及用ELISA試劑盒做EPO-hFcs、G-CSF-hFcs、p40N303Q-hFcs、TNFR-hFc-5及thFcs-IFN-beta的測量。 CHO/DHFR -/- cells (Chinese hamster ovary) were cultured with α-MEM (Invitrgen, Carlsbad), 10% dialyzed fetal bovine serum (JRH Biosciences, Kansas), HT supplement (Invitrogen, Carlsbad) and antibiotics (Invitrogen, Carlsbad) Cells, DG44, ATCC). The expression vector was transfected into CHO cells according to conventional CaPO 4 coprecipitation. 48 hours after transfection, CHO cells were detached from the dish and diluted several times (1/2, 1/5, 1/20, 1/50, 1/100, 1/200, 1/500). The diluted cells were spread on a 100 mm culture dish and cultured in a medium without HT additive. During the screening process, fresh medium without HT supplements was added to the cells without subculture. Communities were generated 2 to 3 weeks after spreading and individual colonies were moved to 48-well plates. After the ELISA, positive colonies were screened for EPO, G-CSF, p40N303Q and TNFR detection. Each of the communities showing the highest performance was cultured in a large scale (5 L) using serum-free medium (JRH Biosciences, Kansas). Purification of each fusion protein was performed using the collected serum-free supernatant. For purification, the HiTrap recombinant protein A FF (Amersham biosciences, Piscataway) column was equilibrated with 20 mM sodium phosphate buffer (pH 7.0). The filtered supernatant was added to a column and eluted with 0.1 M sodium citrate (pH 3.0). After dialysis for more than 3 times with a membrane (MWCO 12-14K, Spectrapor, Rancho Dominguez), the eluted protein was finally obtained. All protein sample concentrations were measured using the BCA kit (Pierce Biotechnology, Rockford) for total protein and EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5 and thFcs-IFN- using ELISA kits. Beta measurement.

<實施例5>Fc γ RI及C1q結合檢測<Example 5> Fc γ RI and C1q binding assay

探討hFc-5-融合蛋白質是否結合Fc γ RI及C1q，而將MabThera®(利妥昔，Roche)、hIgG1(Calbiochem,Cat#,400120)、Enbrel®(恩博，Amgen)、EPO-hFc-5、G-CSF-hFc-5及p40N303Q-hFc-5系列稀釋(2倍，從2ug/ml 到16ng/ml)並塗布於8孔條帶(COSTAR,New York)於4℃過夜。製作標準曲線，亦將Fc γ RI(R&D,cat# BAF1257)或C1q(AbD serotech,Cat#.2221-5504)系列稀釋(2倍，從2ug/ml到32ng/ml)並塗布於8孔條帶(COSTAR,New York)於4℃過夜。用洗滌緩衝液(含0.05% Tween的PBS)洗滌各條帶之樣本並於RT用10% FBS的PBS溶液阻斷1小時後，將2ug/ml Fc γ RI或C1q加入各孔中隨後在室溫(RT)培養2小時。用洗滌緩衝液洗滌所有條帶。於C1q結合試驗，將2.5ug/mlHRP共軛抗C1q(AbD serotech,cat#.2221-5004P)加入各孔中隨後在黑暗條件下於RT培養30分鐘。於Fc γ RI結合試驗，將2ug/ml生物素標記抗Fc γ RI(R&D,cat#.1257-FC)加入各孔中隨後在RT培養1小時。用洗滌緩衝液洗滌後，將稀釋3,000倍的鏈親和素-HRP(BD,cat#.554066)加至各條帶隨後在黑暗條件下於RT培養30分鐘。洗滌條帶後，添加TMB溶液(TMB過氧化酶基質及過氧化酶基質溶液B的1：1混合物，KPL,cat#.50-76-01,cat#,50-65-00)而顯色並添加2N H2SO4終止顯色。如第6(a)及6(b)圖所示，MabThera®、Enbrel®及hIgG1與Fc γ RI及C1q結合良好，但EPO-hFc-5、G-CSF-hFc-5及p40N303Q-hFc-5則否。 To investigate whether hFc-5-fusion protein binds to Fc γ RI and C1q, while MabThera® (Ritux, Roche), hIgG1 (Calbiochem, Cat#, 400120), Enbrel® (Enbo, Amgen), EPO-hFc- 5. G-CSF-hFc-5 and p40N303Q-hFc-5 serial dilutions (2 fold from 2 ug/ml to 16 ng/ml) and plated in 8-well strips (COSTAR, New York) overnight at 4 °C. A standard curve was prepared, and Fc γ RI (R&D, cat# BAF1257) or C1q (AbD serotech, Cat#.2221-5504) was also serially diluted (2 times, from 2 ug/ml to 32 ng/ml) and coated on 8-well strips. The band (COSTAR, New York) was left overnight at 4 °C. The samples of each band were washed with washing buffer (PBS containing 0.05% Tween) and blocked with 10% FBS in PBS for 1 hour after RT, and 2 ug/ml Fc γ RI or C1q was added to each well and then in the chamber. Incubate for 2 hours at warm (RT). All strips were washed with wash buffer. In the C1q binding assay, 2.5 ug/ml HRP conjugated anti-C1q (AbD serotech, cat #.2221-5004P) was added to each well followed by incubation at RT for 30 minutes in the dark. In the Fc γ RI binding assay, 2 ug/ml biotinylated anti-Fc γ RI (R&D, cat #.1257-FC) was added to each well followed by incubation at RT for 1 hour. After washing with washing buffer, 3,000-fold diluted streptavidin-HRP (BD, cat #.554066) was added to each band and then incubated at RT for 30 minutes under dark conditions. After washing the strip, add TMB solution (1:1 mixture of TMB peroxidase substrate and peroxidase matrix solution B, KPL, cat#.50-76-01, cat#, 50-65-00) to develop color The color development was terminated by the addition of 2N H 2 SO 4 . As shown in Figures 6(a) and 6(b), MabThera®, Enbrel®, and hIgG1 bind well to Fc γ RI and C1q, but EPO-hFc-5, G-CSF-hFc-5, and p40N303Q-hFc- 5 No.

<實施例6>純化hFc-融合蛋白質的體外生物活性<Example 6> In vitro biological activity of purified hFc-fusion protein

探討EPO-hFc蛋白質的體外生物活性，將人類F35E細胞株培養於補充10%FBS、抗生素及5IU/ml重組人類EPO(DongA,Republic of Korea)的RPMI1640培養基(Cambrex,Charles City)。將2X104細胞接種到96孔細胞 培養盤(Corning,Netherlands)的測試孔而建立生物測定。將系列稀釋(5倍，從0、0.064mIU/ml到25IU/ml)的EPO、EPO-hFc-1、EPO-hFc-5、EPO-hFc-6、EPO-IgG1 Fc或Aranesp®(α達貝泊汀，Amgen)樣本加到這些孔中然後將孔盤在加濕的5%CO2培養箱中於37℃培養72小時。根據製造商規程，使用細胞生長比色檢測試劑盒(Sigma-Aldrich.Korea)進行MTT檢測。人類F35E細胞株對rEPO顯示強烈增殖反應，在細胞數目及吸光值的劑量依賴性方式上得到證明。如第7(a)圖所示，相較於EPO蛋白質，偶合IgG1 Fc或hFcs的Aranesp®及EPO蛋白質顯示失去生物活性。然而，EPO-hFc-1、EPO-hFc-5及EPO-hFc-6明顯顯示比EPO-IgG1 Fc高的生物活性。此外，EPO-hFc-5及EPO-hFc-6比Aranesp®顯示稍高的生物活性，指明這些hFc-融合蛋白質就維持EPO蛋白質而言顯現較Aranesp®為佳。 To investigate the in vitro biological activity of EPO-hFc protein, human F35E cell line was cultured in RPMI1640 medium (Cambrex, Charles City) supplemented with 10% FBS, antibiotics and 5 IU/ml recombinant human EPO (DongA, Republic of Korea). Bioassays were established by inoculating 2X10 4 cells into test wells of 96-well cell culture dishes (Corning, Netherlands). Serial dilutions (5x, from 0, 0.064mIU/ml to 25IU/ml) of EPO, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6, EPO-IgG1 Fc or Aranesp® (alpha A sample of bepoetin, Amgen) was added to the wells and the wells were incubated at 37 ° C for 72 hours in a humidified 5% CO 2 incubator. MTT assays were performed using a cell growth colorimetric assay kit (Sigma-Aldrich. Korea) according to the manufacturer's protocol. The human F35E cell line showed a strong proliferative response to rEPO and was demonstrated in a dose-dependent manner in cell number and absorbance. As shown in Figure 7(a), Aranesp® and EPO proteins coupled to IgG1 Fc or hFcs showed loss of biological activity compared to EPO proteins. However, EPO-hFc-1, EPO-hFc-5 and EPO-hFc-6 clearly showed higher biological activity than EPO-IgG1 Fc. In addition, EPO-hFc-5 and EPO-hFc-6 showed slightly higher biological activity than Aranesp®, indicating that these hFc-fusion proteins are better than Aranesp® in maintaining EPO protein.

探討G-CSF-hFc蛋白質的體外生物活性，將小鼠造血細胞株NFS-60培養於補充10%FBS、抗生素及100units/ml重組小鼠IL-3(R&D system,Minneapolis)的RPMI1640培養基(Cambrex,Charles City)。將2X104細胞接種到96孔細胞培養盤(Corning,Netherlands)的孔中而建立生物測定。將系列稀釋(3倍，從0到10,000pg/ml)的G-CSF-hFc-5及Neulasta®(培非格司亭，Amgen)樣本加到這些孔中然後將孔盤在加濕的5%CO2培養箱中於37℃培養72小時。在3重複孔中檢測蛋白質樣本並將實驗重複進行5次。培養後72小時，根據製造商規程，使用細胞生長比 色檢測試劑盒(Sigma-Aldrich.Korea)進行MTT檢測。如第7(b)圖中說明，G-CSF-hFc-5比Neulasta®顯示稍高的體外生物活性。 To investigate the in vitro biological activity of G-CSF-hFc protein, mouse hematopoietic cell line NFS-60 was cultured in RPMI1640 medium (Cambrex supplemented with 10% FBS, antibiotics and 100 units/ml recombinant mouse IL-3 (R&D system, Minneapolis). , Charles City). Bioassays were established by seeding 2X10 4 cells into wells of 96-well cell culture dishes (Corning, Netherlands). Serial dilutions (3x, from 0 to 10,000 pg/ml) of G-CSF-hFc-5 and Neulasta® (pefestin, Amgen) samples were added to the wells and the wells were then humidified 5 Incubate at 37 ° C for 72 hours in a % CO 2 incubator. Protein samples were detected in 3 replicate wells and the experiment was repeated 5 times. At 72 hours after the culture, MTT assay was performed using a cell growth colorimetric detection kit (Sigma-Aldrich. Korea) according to the manufacturer's protocol. As illustrated in Figure 7(b), G-CSF-hFc-5 showed slightly higher in vitro biological activity than Neulasta®.

探討p40N303Q-hFc蛋白質的體外生物活性，在補充10%FBS及抗生素的RPMI1640培養基(Cambrex,Charles City)中培養類風溼性關節炎患者的人周邊血液單核細胞(PBMCs)與有或無10ng/ml人類p40(R&D system)或p40N303Q-hFc-5之2ug/ml抗人類CD3抗體(R&D system,# MAB100)。6天後，經FACS分析測量CD4及IL-17陽性的細胞。如第7(c)圖所示，p40N303Q-hFc-5顯示比p40蛋白質較強烈的CD4+/IL-17+細胞產生抑制效應，指明p40N303Q-hFc-5對Th17極化的抑制功能。 To investigate the in vitro biological activity of p40N303Q-hFc protein, human peripheral blood mononuclear cells (PBMCs) of rheumatoid arthritis patients with or without 10ng/ in RPMI1640 medium (Cambrex, Charles City) supplemented with 10% FBS and antibiotics. 2 human p40 (R&D system) or 2 ug/ml anti-human CD3 antibody (R&D system, #MAB100) of p40N303Q-hFc-5. After 6 days, CD4 and IL-17 positive cells were measured by FACS analysis. As shown in Figure 7(c), p40N303Q-hFc-5 showed a potent inhibitory effect on CD4 + /IL-17 + cells than p40 protein, indicating the inhibitory function of p40N303Q-hFc-5 on Th17 polarization.

探討TNFR-hFc蛋白質的體外生物活性，在補充10% FBS及抗生素的RPMI1640培養基(Cambrex,Charles City)中培養鼠L929細胞。將3X104細胞接種到96孔細胞培養盤(Corning,Netherlands)的孔中，然後用1ng/ml的TNF-α處理而建立細胞病變抑制檢測。將系列稀釋(2倍，從15.6到1,000ng/ml)的TNFR-hFc-5及Enbrel®(恩博，Amgen)樣本加到這些孔中然後將孔盤在加濕的5%CO2培養箱中於37℃培養48小時。培養後，根據製造商規程，使用細胞生長比色檢測試劑盒(Sigma-Aldrich.Korea)進行MTT檢測。如第7(d)圖中說明，TNFR-hFc-5較Enbrel®顯示稍高的體外生物活性。 To investigate the in vitro biological activity of TNFR-hFc protein, murine L929 cells were cultured in RPMI1640 medium (Cambrex, Charles City) supplemented with 10% FBS and antibiotics. 3× 10 4 cells were seeded into wells of a 96-well cell culture dish (Corning, Netherlands), and then treated with 1 ng/ml of TNF-α to establish a cytopathic inhibition assay. Serial dilutions (2x, from 15.6 to 1,000 ng/ml) of TNFR-hFc-5 and Enbrel® (Amgen, Amgen) samples were added to the wells and the wells were placed in a humidified 5% CO 2 incubator. The medium was cultured at 37 ° C for 48 hours. After the incubation, MTT assay was performed using a cell growth colorimetric detection kit (Sigma-Aldrich. Korea) according to the manufacturer's protocol. As illustrated in Figure 7(d), TNFR-hFc-5 showed slightly higher in vitro biological activity than Enbrel®.

探討thFc-1-AL(0)-IFN-β及thFc-1-AL(3)- IFN-β蛋白質的體外生物活性，將WISH細胞(ATCC,CCL-25)培養於補充10%FBS及抗生素的DMEM/F12(Cambrex,Charles City)。將3X104細胞接種到96孔細胞培養盤(Corning,Netherlands)的孔中，然後用1,500PFU/孔的VSV(ATCC,VR-158)處理而建立細胞病變抑制檢測。將系列稀釋(2倍，從40IU/ml)的重組IFN-β(WHO standard,NIBSC 00/572)、thFc-1-AL(0)-IFN-β及thFc-1-AL(3)-IFN-β蛋白質樣本加到這些孔中然後將孔盤在加濕的5%CO2培養箱中於37℃培養48小時。培養後，根據製造商規程，使用細胞生長比色檢測試劑盒(Sigma-Aldrich.Korea)進行MTT檢測。如第7(e)圖中說明，thFc-1-AL(3)-IFN-β比thFc-1-AL(0)-IFN-β顯示20倍高的體外生物活性，指明白蛋白連接物在維持INF-β融合Fc之生物活性上的重要性。 To investigate the in vitro biological activities of thFc-1-AL(0)-IFN-β and thFc-1-AL(3)-IFN-β protein, and to culture WISH cells (ATCC, CCL-25) supplemented with 10% FBS and antibiotics. DMEM/F12 (Cambrex, Charles City). 3X10 4 cells were seeded into wells of a 96-well cell culture dish (Corning, Netherlands) and then treated with 1,500 PFU/well of VSV (ATCC, VR-158) to establish a cytopathic inhibition assay. Serial dilution (2x from 40 IU/ml) of recombinant IFN-β (WHO standard, NIBSC 00/572), thFc-1-AL(0)-IFN-β and thFc-1-AL(3)-IFN A beta protein sample was added to the wells and the wells were incubated for 48 hours at 37 ° C in a humidified 5% CO 2 incubator. After the incubation, MTT assay was performed using a cell growth colorimetric detection kit (Sigma-Aldrich. Korea) according to the manufacturer's protocol. As illustrated in Figure 7(e), thFc-1-AL(3)-IFN-β showed 20-fold higher in vitro biological activity than thFc-1-AL(0)-IFN-β, indicating that the protein linker is The importance of maintaining the biological activity of INF-β fusion Fc.

<實施例7>純化hFc-融合蛋白質的體內半衰期<Example 7> Purification of in vivo half-life of hFc-fusion protein

比較EPO-hFc-1、EPO-hFc-5及Aranesp®的半衰期，用這些蛋白質以2,400IU/kg劑量經由單次皮下(SC)注射或單次靜脈(IV)注射處理15隻石蟹獼猴。在注射前及注射後1、3、6、12、24、30、48、54、72、78、96、120、168、336、504及672h取得各獼猴的血液樣本。將血液樣本於室溫培養30min使凝結。於3000rpm離心10min後，取得各樣本的血清並貯存於低溫冷凍箱。用EPO ELISA試劑盒(R&D,cat #.DEP00)檢試得自各點之所有樣本以定量EPO。如第8(a)圖中所示，經由SC或IV途徑注射EPO-hFc-1或EPO-hFc-5的所有個別獼猴比經由SC或IV途徑注射 Aranesp®的個別獼猴顯示較長半衰期。 The half-lives of EPO-hFc-1, EPO-hFc-5 and Aranesp® were compared and 15 stone crab macaques were treated with these proteins at a dose of 2,400 IU/kg via a single subcutaneous (SC) injection or a single intravenous (IV) injection. Blood samples of each macaque were obtained before injection and at 1, 3, 6, 12, 24, 30, 48, 54, 72, 78, 96, 120, 168, 336, 504 and 672 h after injection. Blood samples were incubated for 30 min at room temperature to allow coagulation. After centrifugation at 3000 rpm for 10 min, serum of each sample was taken and stored in a low temperature freezer. All samples from each point were tested with EPO ELISA kit (R&D, cat #.DEP00) to quantify EPO. As shown in Figure 8(a), all individual macaques injected via EPO-hFc-1 or EPO-hFc-5 via the SC or IV route were injected via the SC or IV route. Individual macaques of Aranesp® show a longer half-life.

探討G-CSF-hFc-1的藥物動力學，將100ug/kg作為對照組的LEUCOSTIM®(惠爾血添，DongA,Republic of Korea)及G-CSF-hFc-1經SC或IV途徑給藥每組2隻的雄性Sprague Dawley大鼠(Charles River Laboratories,Wilmington)。在注射前及注射後1、2、3、4、8、12、24、48、72、96、120及192h取得血液。於室溫培養30min後於3000rpm離心10min，取得血清並貯存於低溫冷凍箱。將樣本稀釋數種倍數諸如1/2、1/5、1/50、1/250、1/500，使用G-CSF試劑盒(Biosource,Camarillo,#KHC2032)定量。如第8(b)圖中所示，經由SC或IV途徑注射的G-CSF-hFc-1比LEUCOSTIM®顯示較長半衰期。G-CSF-hFc-1及G-CSF分別具有於SC給藥後的8.76h及2.36h體內t1/2及IV給藥後的10.42h及1.78h。因此，與LEUCOSTIM®比較，G-CSF-hFc-1顯示於SC注射後增強3.7倍及於IV注射後增強5.9倍。 To investigate the pharmacokinetics of G-CSF-hFc-1, 100 ug/kg of LEUCOSTIM® (DongA, Republic of Korea) and G-CSF-hFc-1 as a control group were administered by SC or IV route. Two male Sprague Dawley rats (Charles River Laboratories, Wilmington) were placed in each group. Blood was obtained before injection and at 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120 and 192 h after injection. After incubating at room temperature for 30 min, it was centrifuged at 3000 rpm for 10 min to obtain serum and stored in a low temperature freezer. Samples were diluted several times such as 1/2, 1/5, 1/50, 1/250, 1/500, quantified using the G-CSF kit (Biosource, Camarillo, #KHC2032). As shown in Figure 8(b), G-CSF-hFc-1 injected via the SC or IV route showed a longer half-life than LEUCOSTIM®. G-CSF-hFc-1 and G-CSF had 10.42 h and 2.36 h after SC administration, respectively, and 10.42 h and 1.78 h after t 1/2 and IV administration. Therefore, compared with LEUCOSTIM®, G-CSF-hFc-1 showed a 3.7-fold increase after SC injection and a 5.9-fold increase after IV injection.

探討p40N303Q-hFc-5及Enbrel®的藥物動力學，以100ug/kg劑量單次SC注射處理每組3隻的石蟹獼猴。在注射前及注射後8、24、48、72、96、120、168、336、504及672h取得各獼猴的血液樣本。將血液樣本於室溫培養30min使凝結。於3000rpm離心10min後，取得各樣本的血清並貯存於低溫冷凍箱。用ELISA試劑盒(分別為R&D system,Minneapolis,#DY1240及#DRT200)測試得自各點之所有樣本的人類p40及人類TNFR II定量。如第8(c) 圖中所示，雖然p40N303Q-hFc-5的Cmax值比Enbrel®低(平均3ng/ml對7ng/ml)，p40N303Q-hFc-5顯示比Enbrel®長的半衰期(平均199h對127h)。 To explore the pharmacokinetics of p40N303Q-hFc-5 and Enbrel®, three groups of stone crab macaques were treated with a single SC injection at a dose of 100 ug/kg. Blood samples of each macaque were obtained before injection and at 8, 24, 48, 72, 96, 120, 168, 336, 504 and 672 h after injection. Blood samples were incubated for 30 min at room temperature to allow coagulation. After centrifugation at 3000 rpm for 10 min, serum of each sample was taken and stored in a low temperature freezer. Human p40 and human TNFR II were quantified from all samples at each point using ELISA kits (R&D system, Minneapolis, #DY1240 and #DRT200, respectively). As in 8(c) As shown, although the Cmax value of p40N303Q-hFc-5 was lower than that of Enbrel® (average 3 ng/ml vs. 7 ng/ml), p40N303Q-hFc-5 showed a longer half-life than Enbrel® (mean 199 h vs. 127 h).

探討TNFR-hFc-5及Enbrel®的藥物動力學，以500ug/kg劑量單次SC注射處理每組3隻的雄性Sprague Dawley大鼠(Charles River Laboratories,Wilmington)。在注射前及注射後2、4、8、12、24、30、48、72及120h取得各大鼠的血液樣本。將血液樣本於室溫培養30min使凝結。於3000rpm離心10min後，取得各樣本的血清並貯存於低溫冷凍箱。用ELISA試劑盒(R&D system,Minneapolis,#DRT200)測試得自各點之所有樣本的人類TNFR II定量。如第8(d)圖中所示，雖然TNFR-hFc-5顯示與Enbrel®有同樣的半衰期(平均28.6h對29.4h)，惟TNFR-hFc-5顯示比Enbrel®稍高的AUC水平(平均198.1對172.9ug*h/ml)。 The pharmacokinetics of TNFR-hFc-5 and Enbrel® were investigated and 3 male Sprague Dawley rats (Charles River Laboratories, Wilmington) were treated with a single SC injection at a dose of 500 ug/kg. Blood samples from each rat were obtained before injection and at 2, 4, 8, 12, 24, 30, 48, 72 and 120 hours after injection. Blood samples were incubated for 30 min at room temperature to allow coagulation. After centrifugation at 3000 rpm for 10 min, serum of each sample was taken and stored in a low temperature freezer. Human TNFR II quantification of all samples from each spot was tested using an ELISA kit (R&D system, Minneapolis, #DRT200). As shown in Figure 8(d), although TNFR-hFc-5 showed the same half-life as Enbrel® (average 28.6 h vs. 29.4 h), TNFR-hFc-5 showed slightly higher AUC levels than Enbrel® ( The average is 198.1 versus 172.9ug*h/ml).

<實施例8>純化hFc-融合蛋白質的體內生物活性<Example 8> In vivo biological activity of purified hFc-fusion protein

比較EPO-hFc-5及Aranesp®的體內生物活性，以2,400IU/kg劑量用SC注射或單次IV注射處理每組3隻的石蟹獼猴。在注射前及注射後1、3、6、12、24、30、48、54、72、78、96、120、168、336、504及672h取得各獼猴的血液樣本。測量包含網狀紅血球的各種血液細胞數目而評估EPO-hFc-5及Aranesp®的體內生物活性。如第9(a)圖中所示，就獼猴網狀紅血球的增加而言，以SC及IV途徑EPO-hFc-5均顯示比Aranesp®稍高的體內效力。 The in vivo biological activities of EPO-hFc-5 and Aranesp® were compared, and each group of 3 stone crab macaques was treated with SC injection or single IV injection at a dose of 2,400 IU/kg. Blood samples of each macaque were obtained before injection and at 1, 3, 6, 12, 24, 30, 48, 54, 72, 78, 96, 120, 168, 336, 504 and 672 h after injection. The in vivo biological activities of EPO-hFc-5 and Aranesp® were evaluated by measuring the number of various blood cells including reticulocytes. As shown in Figure 9(a), EPO-hFc-5 showed slightly higher in vivo potency than Aranesp® in terms of SC and IV pathways in terms of the increase in rhesus reticular red blood cells.

探討G-CSF-hFc-1的體內生物活性，以100ug/kg劑量將作為對照組的LEUCOSTIM®(惠爾血添，DongA,Republic of Korea)及G-CSF-hFc-1經SC或IV途徑給藥每組2隻的雄性Sprague Dawley大鼠(Charles River Laboratories,Wilmington)。在注射前及注射後1、2、3、4、8、12、24、48、72、96、120及192h用EDTA管取得血液。用RBC溶解緩衝液(BD Bioscience,Korea)處理各血液樣本4分鐘並使用血球計3次重複計數用FACS緩衝液稀釋的總WBCs(白血球)數。使用流式細胞儀(FACS caliber)用FSC(前散射光)測定細胞大小及用SSC(側散射光)測定顆粒而測量顆粒細胞數目。如第9(b)圖中所示，經SC或IV途徑處理的LEUCOSTIM®導致WBC及顆粒細胞的數目高峰在注射後24小時，而G-CFS-hFc-1則導致WBC及顆粒細胞的數目高峰在SC注射後72小時及IV注射後48小時。從注射後24h至120h，相較於LEUCOSTIM®，G-CSF-hFc-1具有更持續的體內生物活性。 To investigate the in vivo biological activity of G-CSF-hFc-1, and to use LEUCOSTIM® (DongA, Republic of Korea) and G-CSF-hFc-1 as a control group at a dose of 100 ug/kg via SC or IV route. Two male Sprague Dawley rats (Charles River Laboratories, Wilmington) were administered per group. Blood was obtained with an EDTA tube before injection and at 1, 2, 3, 4, 8, 12, 24, 48, 72, 96, 120 and 192 h after injection. Each blood sample was treated with RBC lysis buffer (BD Bioscience, Korea) for 4 minutes and the total number of WBCs (white blood cells) diluted with FACS buffer was counted three times using a hemocytometer. The number of granulosa cells was measured by measuring the cell size with FSC (front scattered light) using a flow cytometer (FACS caliber) and measuring the particles with SSC (side scattered light). As shown in Figure 9(b), LEUCOSTIM® treated with the SC or IV route resulted in a peak in the number of WBCs and granulosa cells 24 hours after injection, whereas G-CFS-hFc-1 resulted in the number of WBCs and granulosa cells. The peak was 72 hours after SC injection and 48 hours after IV injection. From 24 h to 120 h after injection, G-CSF-hFc-1 has a more sustained in vivo biological activity than LEUCOSTIM®.

雖然參照示例之具體例特別地顯示並說明本發明，但所屬領域一般熟練技術人員應瞭解，在不脫離由下列申請專利範圍所限定的本發明精神及範圍的情況下，可以做形式和細節上的各種變化。 While the invention has been particularly shown and described with reference to the specific embodiments of the embodiments of the invention, Various changes.

<110> 格納西尼有限公司(GENEXINE,INC.) 浦項工科大學校產學協力團(POSTECH ACADEMY-INDUSTRY FOUNDATION) <110> GENEXINE, INC. POSTECH ACADEMY-INDUSTRY FOUNDATION

<120> 免疫球蛋白融合蛋白質(IMMUNOGLOBULIN FUSION PROTEINS) <120> Immunoglobulin fusion protein (IMMUNOGLOBULIN FUSION PROTEINS)

<160> 32 <160> 32

<170> KopatentIn 1.71 <170> KopatentIn 1.71

<210> 1 <210> 1

<211> 735 <211> 735

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 雜合Fc片段5(hFC-5) <223> Hybrid Fc fragment 5 (hFC-5)

<400> 1 <400> 1

<210> 2 <210> 2

<211> 579 <211> 579

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 根據密碼子優化合成之人類EPO基因 <223> Human EPO gene synthesized according to codon optimization

<400> 2 <400> 2

<210> 3 <210> 3

<211> 612 <211> 612

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 根據密碼子優化合成之人類G-CSF基因 <223> Human G-CSF gene synthesized by codon optimization

<400> 3 <400> 3

<210> 4 <210> 4

<211> 984 <211> 984

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 根據密碼子優化合成之人類p40基因 <223> Human p40 gene synthesized by codon optimization

<400> 4 <400> 4

<210> 5 <210> 5

<211> 5698 <211> 5698

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> pAD11 <223> pAD11

<400> 5 <400> 5

<210> 6 <210> 6

<211> 327 <211> 327

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> PEPTIDE <221> PEPTIDE

<222> (1)..(327) <222> (1)..(327)

<223> 人類IgG4恆定區部分(Genbank登錄號No. AAH25985) <223> Human IgG4 constant region portion (Genbank accession No. AAH25985)

<400> 6 <400> 6

<210> 7 <210> 7

<211> 383 <211> 383

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> PEPTIDE <221> PEPTIDE

<222> (1)..(383) <222> (1)..(383)

<223> 人類IgD恆定區(Genbank登錄號No.P01880) <223> Human IgD constant region (Genbank Accession No. P01880)

<400> 7 <400> 7

<210> 8 <210> 8

<211> 193 <211> 193

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> PEPTIDE <221> PEPTIDE

<222> (1)..(193) <222> (1)..(193)

<223> 人類EPO前驅物(Genbank登錄號No.NP_000790) <223> Human EPO precursor (Genbank accession No. NP_000790)

<400> 8 <400> 8

<210> 9 <210> 9

<211> 207 <211> 207

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> PEPTIDE <221> PEPTIDE

<222> (1)..(207) <222> (1)..(207)

<223> 人類G-CSF(Genbank登錄號No.CAA27291) <223> Human G-CSF (Genbank Accession No. CAA27291)

<400> 9 <400> 9

<210> 10 <210> 10

<211> 328 <211> 328

<212> PRT <212> PRT

<213> 智人 <213> Homo sapiens

<220> <220>

<221> PEPTIDE <221> PEPTIDE

<222> (1)..(328) <222> (1)..(328)

<223> 人類IL12 p40次單元(Genbank登錄號No.AAG32620) <223> Human IL12 p40 subunit (Genbank accession No. AAG32620)

<400> 10 <400> 10

<210> 11 <210> 11

<211> 245 <211> 245

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> hFc-5之胺基酸序列 <223> Amino acid sequence of hFc-5

<400> 11 <400> 11

<210> 12 <210> 12

<211> 771 <211> 771

<212> DNA <212> DNA

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 根據密碼子優化合成之人類可溶性TNF受體2基因 <223> Human soluble TNF receptor 2 gene synthesized by codon optimization

<400> 12 <400> 12

<210> 13 <210> 13

<211> 257 <211> 257

<212> PRT <212> PRT

<213> 人工序列 <213> Artificial sequence

<220> <220>

<223> 由根據密碼子優化合成之基因編碼之人類可溶性TNF受體2 <223> Human soluble TNF receptor 2 encoded by a gene encoded by codon optimization

<400> 13 <400> 13

由於本案的圖僅為說明用，並非本案的代表圖。故本案無指定代表圖。 Since the figure in this case is for illustrative purposes only, it is not a representative figure of this case. Therefore, there is no designated representative map in this case.

Claims (12)

一種嵌合多肽，其係包括：雜合人類Fc多肽；及與雜合人類Fc多肽偶合的生物活性多肽、其變異體或片段，其中上述雜合人類Fc多肽係以下列式表示：N'-(Z1)P-Y-Z2-Z3-Z4-C’其中：N'為雜合人類Fc多肽的N-端及C'為雜合人類Fc多肽的C-端；Z1為由SEQ ID NO：7位置90至98序列的C-端開始的5個或更多個連續胺基酸殘基組成的胺基酸序列；Y為由SEQ ID NO：7位置99至162序列的C-端開始的5個或更多個連續胺基酸殘基組成的胺基酸序列；Z2為由SEQ ID NO：7位置163至199序列的N-端開始的4個或更多個連續胺基酸殘基組成的胺基酸序列；Z3為由SEQ ID NO：6位置115至220序列的C-端開始的71個或更多個連續胺基酸殘基組成的胺基酸序列；Z4為由SEQ ID NO：6位置221至327序列的N-端開始的80個或更多個連續胺基酸殘基組成的胺基酸 序列；及p為0或1之整數，其中生物活性多肽、其變異體或片段係在雜合人類Fc多肽的N-端或C-端融合，其中與上述雜合人類Fc多肽融合的上述生物活性多肽、其變異體或片段相較於未與雜合人類Fc多肽融合的上述生物活性多肽、其變異體或片段，顯示增長的循環半衰期。 A chimeric polypeptide comprising: a hybrid human Fc polypeptide; and a biologically active polypeptide, variant or fragment thereof, coupled to a hybrid human Fc polypeptide, wherein said hybrid human Fc polypeptide is represented by the formula: N'- (Z1) P -Y-Z2-Z3-Z4-C' wherein: N' is the N-terminus of the heterozygous human Fc polypeptide and C' is the C-terminus of the hybrid human Fc polypeptide; Z1 is represented by SEQ ID NO: An amino acid sequence consisting of 5 or more contiguous amino acid residues starting at the C-terminus of the 7-position 90 to 98 sequence; Y is the C-terminus of the sequence 99 to 162 of SEQ ID NO: 7 An amino acid sequence consisting of 5 or more contiguous amino acid residues; Z2 is 4 or more contiguous amino acid residues starting from the N-terminus of the sequence 163 to 199 of SEQ ID NO:7 Amino acid sequence consisting of; Z3 is an amino acid sequence consisting of 71 or more contiguous amino acid residues starting from the C-terminus of position 115 to 220 of SEQ ID NO: 6; Z4 is SEQ ID NO: an amino acid sequence consisting of 80 or more contiguous amino acid residues starting at the N-terminus of the 221 to 327 sequence; and p is an integer of 0 or 1, wherein the biologically active polypeptide, a variant thereof Or a fragment in a heterozygous person An N-terminal or C-terminal fusion of an Fc-like polypeptide, wherein the biologically active polypeptide, variant or fragment thereof fused to the above-described hybrid human Fc polypeptide is compared to the above biologically active polypeptide not fused to a hybrid human Fc polypeptide, Its variant or fragment shows a cyclic half-life of growth. 如申請專利範圍第1項所述之嵌合多肽，其中該生物活性多肽為人類生長激素(“hGH”)或顆粒細胞群落刺激因子(“G-CSF”)。 The chimeric polypeptide of claim 1, wherein the biologically active polypeptide is human growth hormone ("hGH") or granulosa cell community stimulating factor ("G-CSF"). 如申請專利範圍第1項所述之嵌合多肽，其中該雜合人類Fc多肽係經由核苷酸序列SEQ ID NO：1編碼。 The chimeric polypeptide of claim 1, wherein the hybrid human Fc polypeptide is encoded by the nucleotide sequence of SEQ ID NO: 1. 如申請專利範圍第1項所述之嵌合多肽，其中該雜合人類Fc多肽具有胺基酸序列SEQ ID NO：11。 The chimeric polypeptide of claim 1, wherein the hybrid human Fc polypeptide has the amino acid sequence SEQ ID NO: 11. 如申請專利範圍第1項所述之嵌合多肽，其中p為0。 The chimeric polypeptide of claim 1, wherein p is 0. 如申請專利範圍第1項所述之嵌合多肽，其中該雜合人類Fc多肽與生物活性多肽、其變異體或片段係經由選自白蛋白連接物及合成連接物所組成之群組的連接物而彼此偶合。 The chimeric polypeptide of claim 1, wherein the hybrid human Fc polypeptide and the biologically active polypeptide, variants or fragments thereof are linked via a group selected from the group consisting of an albumin linker and a synthetic linker. Coupled with each other. 如申請專利範圍第6項所述之嵌合多肽，其中該白蛋白連接物包括胺基酸序列EMP、ENDEMPAD、EENDEMPADLPS、CIAEVENDEMPADLPSLA、SHCIAEVENDEMPADLPSLA或 PLLEKSHCIAEVENDEMPADLPSLAADFVESKD。 The chimeric polypeptide of claim 6, wherein the albumin linker comprises an amino acid sequence EMP, ENDEPDAD, EENDEMPADLPS, CIAEVENDEMPADLPSLA, SHCIAEVENDEMPADLPSLA or PLLEKSHCIAEVENDEMPADLPSLAADFVESKD. 如申請專利範圍第6項所述之嵌合多肽，其中該合成連接物為10至20個胺基酸殘基的肽，其中該肽係由Gly及Ser殘基構成。 The chimeric polypeptide of claim 6, wherein the synthetic linker is a peptide of 10 to 20 amino acid residues, wherein the peptide is composed of Gly and Ser residues. 一種核酸分子，係編碼申請專利範圍第1項所述之嵌合多肽者。 A nucleic acid molecule which encodes the chimeric polypeptide of claim 1 of the patent application. 一種表現載體，該表現載體包括申請專利範圍第9項所述之核酸分子。 A performance vector comprising the nucleic acid molecule of claim 9 of the patent application. 一種製造申請專利範圍第1項所述之嵌合多肽的方法，其中該方法包括步驟：(i)將編碼申請專利範圍第1項所述之嵌合多肽的核酸分子導入哺乳動物寄主細胞、(ii)使該細胞在可表現多肽之條件下的培養基中生長、(iii)從該細胞或該培養基收獲表現的嵌合多肽。 A method for producing the chimeric polypeptide of claim 1, wherein the method comprises the steps of: (i) introducing a nucleic acid molecule encoding the chimeric polypeptide of claim 1 of the patent application into a mammalian host cell, ( Ii) growing the cell in a medium under conditions which express the polypeptide, and (iii) harvesting the expressed chimeric polypeptide from the cell or the medium. 一種增長生物活性多肽、其變異體或片段之循環半衰期的方法，包括將申請專利範圍第1項所述之嵌合多肽給藥予個體。 A method of increasing the circulating half-life of a biologically active polypeptide, variant or fragment thereof, comprising administering a chimeric polypeptide according to claim 1 of the patent application to an individual.
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