CN105175553B

CN105175553B - Domain-immunoglobulin fusion proteins

Info

Publication number: CN105175553B
Application number: CN201510683410.5A
Authority: CN
Inventors: 成永喆; 梁世焕
Original assignee: Puxiang University Of Technology School-Industry-University Cooperative Force; Genexine Co Ltd
Current assignee: Puxiang University Of Technology School-Industry-University Cooperative Force; Genexine Co Ltd
Priority date: 2007-05-30
Filing date: 2008-05-30
Publication date: 2019-11-22
Anticipated expiration: 2028-05-30
Also published as: CN105175553A

Abstract

The present invention discloses the fusion protein including bioactive molecule He immunoglobulin (Ig) the Fc structural domain for being connected to bioactive molecule.The Fc structural domain is (i) IgG1, IgG2 or IgG4, or hybridization people's Fc structural domain of (ii) IgG4 and IgD.The hybrid Fc is used as the carrier of bioactive molecule.

Description

Domain-immunoglobulin fusion proteins

The application be the applying date be on May 30th, 2008, application No. is 200880016313.9, entitled " immunoglobulins The divisional application of the patent application of fusion protein ".

Technical field

The present invention relates to hybridization people Fc and domain-immunoglobulin fusion proteins, hybridize people Fc wherein and are connected to bioactivity point Son.Specifically, it is related to hybridizing people Fc, derived from the combination of immunoglobulin G (IgG) subclass or the group of people IgD and IgG It closes；And fusion protein, this Fc is coupled to bioactive molecule through covalent bond wherein.

Background technique

Bioactive molecule can have very big advantage in the treatment.But they may have as therapeutic agent it is scarce Point, because they have lower internal stability.They have shorter circulating half-life or serum half-life, because they By in vivo various enzymic digestions.Therefore, there has long been a desire for improve the circulating half-life of bioactive molecule.

It is known: increase protein size, by prevent through kidney go isolating protein can increase it half-life period (Knauf etc., J.Biol.Chem.1988.263:15064-15070).For example, it has been reported that can by the way that activated protein is coupled to human albumin Increase protein stability (Kinstler etc., Pharm.Res.1995.12:1883-1888).But extremely due to activated protein The coupling of human albumin is only slightly increased its demurrage, therefore develops having containing the activated protein for being coupled to human albumin Imitating pharmaceutical preparation is not effective ways.

The method of other reports is to adjust the glycosylation of protein.Increase glycosylation on protein and introduces sialic acid Protein can prevent the degradation of protein in liver.But the increase of protein glycosylation also results in the reduction of protein active.

For stable protein and the removing through kidney is prevented, protein is connected to polyethylene glycol (PEG).It is covalently attached to PEG has been widely used to deliver the drug (Delgado etc., 1992.9:249-304) with extended half-life period.But It has been reported that PEG is connected to cell factor or hormone leads to the reduction of receptor binding affinity, reason is caused by the connection Steric hindrance.

Recently, research and development using immunoglobulin (immunoglobulin, Ig) preparation fusion protein. Ig is the main component of blood.People Ig (human Ig, hIg) includes variety classes, such as IgG, IgM, IgA, IgD and IgE (Roitt etc., " Immunology " 1989, Gower Medical Publishing, London, U.K.；New York, N.Y.).Human IgG s can be further divided into various hypotypes, be known as human IgG1 (hIgG1), human IgG2 (hIgG2), human IgG 3 (hIgG3) and human IgG 4 (hIgG4).

Immunoglobulin is made of four polypeptide chains, two heavy chains and two light chains, they are poly- by disulfide bond formation four Body.Every chain is made of variable region and constant region.Based on isotype (isotypes), the constant region of heavy chain is further divided into three The area Ge Huosige (CH1, CH2, CH3 and CH4).Based on Ig isotype, the part the Fc of heavy chain constant region include hinge, CH2, CH3 and/or CH4 structural domain.

About serum half-life, IgG1, IgG2 and IgG4 have 21 days long half-lifts, and other immunoglobulins have Relatively short half-life period less than one week.The chimeric protein for being fused to the part Fc of IgG shows increased stability and increase Serum half-life (Capon etc., Nature 1989.337:525-531).Biological activity protein has merged the N- in the region CH1 The end C- at end, the end N- in the region Fc or the region IgG CH3.

In the phase of beginning, with cell surface receptor such as CD4 (Capon etc., Nature 1989.337:525-531), TNFR (Mohler etc., J.Immunology 1993.151:1548-1561), CTLA4 (Linsley etc., J.Exp.Med.1991.173:721-730), CD86 (Morton etc., J.Immunology 1996.156:1047-1054) Extracellular domain generates IgG fusion protein.It concurrently there are several cell factors for being fused to IgG structural domain and growth hormone. But it is different from the fusion of the extracellular domain with cell surface receptor, compared with non-fused cell factor or growth factor, use Soluble protein, which is fused to IgG, leads to the reduction of bioactivity.Chimeric protein as dimer exist, cause due to Their target molecule similar to receptor interaction and steric hindrance, reason is be in close proximity to each other two kinds of reactive proteins In the presence of.Therefore, this problem should be overcome to generate effective fusion protein.

Other limitations of Fc integration technology are that there are undesirable immune responses.The Fc structural domain of immunoglobulin has simultaneously There is cell-mediated cytotoxicity (the antibody dependent cell-mediated of effector function such as antibody-dependant Cytotoxicity, ADCC) or complement dependent cytotoxicity (complement-dependent cytotoxicity, CDC). The region Fc that this effector function generally passes through Ig takes in conjunction with the interaction of FcRs on effector cell or process complement .Therefore, it should which carrying out the blocking of the effector function of Fc, for example cell kills, cell factor is released to reduce undesirable reaction It puts or inflammation.

Disclosure of the invention

Technical problem

Generally speaking, have to biologically active least disadvantage and the minimum risk with undesirable immune response Improve the demand of Fc fusion protein.

Technical solution

The present invention provides hybrid Fc (heterozygosis Fc), derived from the combination of subclass of human IgG or the combination of people IgD and IgG.When miscellaneous When Fc being handed over to be connected to bioactive molecule, hybrid Fc effectively increases the serum half-life of bioactive molecule, and when coding When the nucleotide of Fc- polypeptide amalgamation protein is expressed, the expression of polypeptide is increased.

Present invention simultaneously provides the hybrid Fc fused polypeptides that bioactive molecule is connected in wherein hybrid Fc.The fusion egg White sometimes referred to as " bioactive molecule-Fc fusion protein " or referred to as " fusion protein ".The fusion protein can have Fc and Connector between bioactive molecule.Fc can be coupled to the end C- of bioactive molecule at its end N-.

Fusion protein can be generated as follows: building coding and the nucleotide for capableing of expressed fusion protein；In host It is expressed in cell；With harvest fusion protein.Optionally, which can encode the nucleotide of Fc by expressing, and with Usual manner connects it to bioactive molecule and generates.

A kind of polypeptide of embodiment according to the present invention can be indicated with following formula:

N′-(Z1)_p-Y-Z2-Z3-Z4-C′

Wherein N ' be polypeptide the end N- and C ' be polypeptide the end C-；

Z1 indicates amino acid sequence comprising in at least C- of the amino acid residue of 90 to 98 positions of SEQ ID NO:11 At least part of end part or the amino acid residue in the position 90-98 of SEQ ID NO:14；

Y indicates amino acid sequence comprising in at least C- of the amino acid residue of 99 to 113 positions of SEQ ID NO:11 At least part of end part or the amino acid residue in 99 to 162 positions of SEQ ID NO:14；

Z2 indicate amino acid sequence comprising 111 to 147 positions of SEQ ID NO:12 amino acid residue at least At least part of N- end part or the amino acid residue in 163 to 199 positions of SEQ ID NO:14；

Z3 indicates amino acid sequence comprising the 118-223 of SEQ ID NO:11, SEQ ID NO:12 114-219, At least C- end part of the amino acid residue of 115 to 220 positions of the 165-270 or SEQ ID NO:13 of SEQ ID NO:24；

Z4 indicates amino acid sequence comprising the 224-330 of SEQ ID NO:11, SEQ ID NO:12 220-326, At least N- end part of the amino acid residue of the position 221-327 of the 271-377 or SEQ ID NO:13 of SEQ ID NO:24 with And

P is 0 or 1 integer,

Wherein Z2 and Z3 total amino acid residues are between 80 and 140, and both ends numerical value is included.

Z1 can be amino acid sequence comprising the amino acid residue C- from the position 90-98 of SEQ ID NO:11 5 to 9 continuous amino acid residues of end side, or the end amino acid residue C- from the position 90-98 of SEQ ID NO:14 5-9 continuous amino acid residue of side.In some embodiments, Z1 can be IgG1 CH1 structural domain (SEQ ID NO:11) Or 5,6,7,8 or 9 C-terminal amino acid residues of IgD CH1 structural domain (SEQ ID NO:14).

In another embodiment, Z1 is amino acid sequence comprising the amino of the position 90-98 of SEQ ID NO:11 The amino acid residue of the position 90-98 of sour residue or SEQ ID NO:14.Z1 can be the position of the 90-98 by SEQ ID NO:11 Set or 5 to 9 amino acid residues in position of the 90-98 of SEQ ID NO:14 composition amino acid sequence.Z1 is also possible to by SEQ The amino acid sequence of 90 to the 98 amino acid residue composition of 90 to 98 amino acid residues or SEQ ID NO:14 of ID NO:11.

Y can be amino acid sequence comprising the amino acid residue from 99 to 113 positions of SEQ ID NO:11 5 or more of the end side C- or 10 or more continuous amino acid residues, or arrived from the 99 of SEQ ID NO:14 10 or more continuous amino acid residues of the end side amino acid residue C- of 162 positions.In some embodiments, Y can be with It is amino acid sequence comprising the amino acid residue of 99 to 113 positions of SEQ ID NO:11, the 158 of SEQ ID NO:14 are arrived The amino acid residue of 162 positions, the amino acid residue of 153 to 162 positions of SEQ ID NO:14, SEQ ID NO:14 143 To the amino acid residues of 162 positions, the amino acid residue of 133 to 162 positions of SEQ ID NO:14 or SEQ ID NO:14 The amino acid residue of 99 to 162 positions.

Z2 can be amino acid sequence comprising the ammonia from 111 to 147 positions of SEQ ID NO:12 (hIgG2) 4 to 37 or 6-30 continuous amino acid residue of the end side N- of base acid residue, or from SEQ ID NO:14's (hIgD) 6-30 continuous amino acid residue of the end side N- of the amino acid residue of 163 to 199 positions.In some embodiments, Z2 can To be 6 n terminal amino acid residues of human IgG2's CH2 structural domain or 8 n terminal amino acid residues of people's IgD CH2 structural domain.

The total amino acid residues of Z2 and Z3 can be between 80 and 140.In one embodiment, the amino of Z2 and Z3 Sour total number of residues can be between 90 and 120, and both ends numerical value is included.In another embodiment, the amino of Z2 and Z3 Sour total number of residues can be between 105 and 115, and both ends numerical value is included.In one embodiment, the amino acid of Z2 and Z3 Total number of residues is 108.In a more embodiment, the total amino acid residues of Z2 and Z3 are 109.

Z4 can be amino acid sequence comprising from 224-330, SEQ ID of SEQ ID NO:11 (hIgG1) The 221 of the 220-326 of NO:12 (hIgG2), 271-377 the or SEQ ID NO:13 (hIgG4) of SEQ ID NO:24 (hIgG3) To 90 or more of the amino acid residue of 327 positions or 100 or more continuous amino acid residues.Z4 can be SEQ ID The 221 of the 271-377 or SEQ ID NO:13 of the 224-330 of NO:11, the 220-326 of SEQ ID NO:12, SEQ ID NO:24 To the amino acid sequence of the amino acid residue of 327 positions.

According to embodiment, Z3-Z4 is amino acid sequence selected from the following: (i) is by 118 to the 223 of SEQ ID NO:11 The N- end part of the amino acid residue of 224 to 330 positions of the C- end part and SEQ ID NO:11 of the amino acid residue of position The continuous amino acid sequence of composition, (ii) by SEQ ID NO:12 114 to 219 positions amino acid residue C- end part and The continuous amino acid sequence that the end N- of the amino acid residue of 220 to 326 positions of SEQ ID NO:12 is grouped as, (iii) by The C- end part of the amino acid residue of 165 to 270 positions of SEQ ID NO:24 and 271 to 377 positions of SEQ ID NO:24 Amino acid residue the continuous amino acid sequence that is grouped as of the end N-, and (iv) by 115 to 220 positions of SEQ ID NO:13 The C- end part of amino acid residue and the end N- of amino acid residue of 221 to 327 positions of SEQ ID NO:13 be grouped as Continuous amino acid sequence.

According to embodiment of the present invention, the total amino acid residues of polypeptide are from 154 to 288.

In one embodiment, Y can be amino acid sequence comprising the ammonia of the position 99-113 of SEQ ID NO:11 At least part of base acid residue, p can be 0 or 1；Z2 can be amino acid sequence comprising the 111- of SEQ ID NO:12 At least part of the amino acid residue of 147 positions；It can be amino acid sequence with Z3 comprising the 118- of SEQ ID NO:11 223,115 to 220 positions of the 165-270 or SEQ ID NO:13 of the 114-219 of SEQ ID NO:12, SEQ ID NO:24 At least part of amino acid residue.In this embodiment, when p is 1, Z1 can be amino acid sequence comprising SEQ At least part of the amino acid residue of 90 to 98 positions of ID NO:11.

In further embodiment, Z3 can be the 114- of the 118-223 of SEQ ID NO:11, SEQ ID NO:12 219,73 to 106 continuous amino acids of the position 115-220 of the 165-270 or SEQ ID NO:13 of SEQ ID NO:24 are residual Base, and the total amino acid residues of Z2 and Z3 can be 110.Z2 can be the ammonia of the position 111-116 of SEQ ID NO:12 Base acid residue and Z3 can be the 120-223 of SEQ ID NO:11, the 116-219 of SEQ ID NO:12, SEQ ID NO:24 The amino acid sequence of 118 to 220 positions of 167-270 or SEQ ID NO:13.

In another embodiment, Y can be amino acid sequence comprising 99 to 162 positions of SEQ ID NO:14 Amino acid residue at least part, p can be 1 or 0 (zero)；Z2 can be amino acid sequence comprising SEQ ID NO: At least part of the amino acid residue of 14 position 163-199；It can be amino acid sequence with Z3 comprising SEQ ID NO: At least part of the amino acid residue of 13 121 to 220 positions.In this embodiment, when p is 1, Z1 can be with amino Acid sequence comprising at least part of the amino acid residue of 90 to 98 positions of SEQ ID NO:14.

In further embodiment, Y can be the C- of the amino acid residue of the position 99-162 of SEQ ID NO:14 20 continuous amino acid residues of end side or more, 30 continuous amino acid residues or more, 40 continuous amino acids are residual Base or more, 50 continuous amino acid residues or more or 60 continuous amino acid residues or more.Z2 can be The amino acid residue of 163 to 170 positions of SEQ ID NO:14, Z3 may include 124-223, SEQ ID of SEQ ID NO:11 The 120-219 of NO:12, the amino acid residue of the position 121-220 of the 171-270 or SEQ ID NO:13 of SEQ ID NO:24 71-100 continuous amino acid residue of the end side C-.The total amino acid residues of Z2 and Z3 can be 108.

In one embodiment, polypeptide can by selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, The nucleotide sequence volume of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:26 and SEQ ID NO:27 Code.Polypeptide is selected from SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO: 22, the amino acid sequence of SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:29.

In one embodiment, polypeptide merges at its end N- with bioactive molecule, with the bioactive molecule The circulating half-life of native form (native form) compare, which shows increased circulating half-life.It should Bioactive molecule can be polypeptide, protein or non-peptide (apeptide).The bioactive molecule can be polypeptide, peptide or egg White matter drug.Bioactive molecule can be soluble protein, such as, but not limited to: hormone, cell factor, growth factor, Costimulatory molecules, hormone receptor, cytokine receptor, growth factor receptors or small peptide.The bioactive molecule can be EPO or Its variant/segment, p40 or its variant/segment (for example, p40 variant containing Asn303Gln displacement), G-CSF or it Variant/segment, 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 Ⅴ II, CXCL-11, FSH, human growth hormone (HGH), Bones morphology are raw At albumen -1 (bone morphogenetic protein-1, BMP-1), CTLA4, PD-1, GLP-1, β tunicin (betacellulin), OPG, RNAK, alpha interferon (interferon-alpha), beta interferon (interferon-beta) or Their variant/segment.The bioactive molecule can be secreted protein, can be mature form.

In one embodiment, the method for generating polypeptide according to claim 1 is provided, wherein this method includes following Step: the DNA molecular of coding said polypeptide is introduced mammalian host cell by (i), (ii) by cell in its culture medium The polypeptide is grown under conditions of being expressed；The polypeptide of (iii) harvest expression.Mammalian host cell can be CHO, COS or bhk cell.

In another embodiment, provide following methods: (i) reduces the symptom of autoimmune disease, prevents or control Autoimmune disease is treated, (ii) inhibits graft rejection, or (iii) to treat or prevent the shock of endotaxin induction comprising applies With the polypeptide described above of therapeutically effective amount, wherein peptide fusion to bioactive molecule.

In one embodiment, the nucleic acid molecules of separation are provided, the polypeptide of embodiment according to the present invention is encoded. Polypeptide can have amino acid sequence selected from the following: SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:28 and SEQ ID NO:29.Nucleic acid molecules can have Have a nucleotide sequence shown in following sequence: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:26 or SEQ ID NO:27.Nucleic acid molecules can further include letter Number sequence or leader sequence.

Embodiment according to the present invention provides the expression vector including the nucleic acid molecules and the place containing the carrier Chief cell.The example of expression vector includes but is not limited to 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 and pAD11 TNFR-hFc-5.

In one embodiment, provide to mammal delivering bioactive molecule method comprising to need its Mammal administration of nucleic acid molecule the step of.

In another embodiment, polypeptide is included in the end N- and is tied to C- extreme direction by hinge area, CH2 structural domain and CH3 The Fc structural domain of structure domain composition, wherein the hinge area includes the amino acid residue of people IgD hinge area or human IgG1's hinge area At least partially；The CH2 structural domain includes at least part of the amino acid residue of 4 CH2 structural domain of human IgG, wherein in people The N- end part or people IgD of 4-37 continuous amino acid residue human IgG2's CH2 structural domain at the end N- of IgG4 CH2 structural domain At least part of the amino acid residue of the N- end part of CH2 structural domain is replaced and the CH3 structural domain includes 4 CH3 of human IgG At least part of the amino acid residue of structural domain.

Hinge area may include at least part of the amino acid residue of the hinge area of human IgG1, the CH2 structural domain packet At least part of the amino acid residue of 4 CH2 structural domain of human IgG is included, wherein the 4-37 at the end N- of 4 CH2 structural domain of human IgG At least part of a continuous amino acid residue amino acid residue of the N- petiolarea of human IgG2's CH2 structural domain is replaced.

Hinge area may include at least part of the amino acid residue of the hinge area of people IgD, and the CH2 structural domain includes At least part of the amino acid residue of 4 CH2 structural domain of human IgG, wherein at 4-37 of the end N- of 4 CH2 structural domain of human IgG At least part of the continuous amino acid residue amino acid residue of the N- petiolarea of human IgG2's CH2 structural domain is replaced.

Polypeptide may further include CH1 structural domain, wherein the CH1 structural domain includes the ammonia of human IgG1's CH1 structural domain At least part of base acid residue, and wherein the CH1 structural domain is coupled to the end N- of the hinge area.Polypeptide can be into one Step includes CH1 structural domain, wherein the CH1 structural domain includes at least part of people's IgD CH1 domain amino acid residues, and And wherein the CH1 structural domain is coupled to the end N- of the hinge area.Polypeptide, which may further include, is coupled to the hinge area The end N- the second polypeptide, wherein second polypeptide is the NIg polypeptide of bioactivity.Polypeptide can be wrapped further It includes and the end N- of the CH1 structural domain is coupled to by connector or is coupled to the bioactive molecule at the CH4 domain C-end, Wherein the bioactive molecule is not immunoglobulin polypeptides.Polypeptide and bioactive molecule can connect each other through connector It connects.Connector molecule is the connector of albumin connector or synthesis.The albumin connector includes the 321 of SEQ ID NO:25 To the amino acid sequence of 323,318 to 325,316 to 328,313 to 330,311 to 333 or 306 to 338.The synthesis connector It can be the peptide for 10 to 20 amino acid residues being made of Gly and Ser residue.In one embodiment, this Gly-Ser Connector is GGGGSGGGGSGGGSG (SEQ ID NO:32).

The present invention also includes the antibody molecule in the area Fc containing recombination, and the area Fc of the recombination is as described above.

Brief description

Fig. 1 shows the schematic diagram of hybrid Fc s (hybrid Fcs, hFcs), may be used as the bioactivity indicated with " X " The carrier protein of molecule.

Fig. 2 shows the diagram of hFcs, wherein to IgG1 (SEQ ID NO:11), IgG2 (SEQ ID:12), IgG4 is derived from The amino acid position of (SEQ ID:13) and IgD (SEQ ID:14) are described in detail.Unless otherwise indicated, the application is all more The name of amino acid position uses same rule in peptide.

Fig. 3 shows hFcs diagram, wherein at the end C-, each hFc is coupled to by the albumin connector indicated with " AL " The bioactive molecule indicated with " X ".

Fig. 4 shows the diagram with the hFcs of connector coupling, wherein to derived from the white of human albumin (SEQ ID NO:25) The amino acid position of albumen connector is described in detail.

Fig. 5 shows the result of the hydrophobicity distribution (hydrophobicity plot) of hFc-6.

Fig. 6 (a) display is analyzed using specific ELISA(Rituximab)、hIgG1、 (etanercept), the Fc γ RI binding activity result of EPO-hFc-5, G-CSF-hFc-5, p40N303Q-hFc-5；Fig. 6 (b) Display is analyzed using specific ELISA(Rituximab)、hIgG1、(etanercept)、 The C1q binding activity result of EPO-hFc-5, G-CSF-hFc-5, p40N303Q-hFc-5.

Fig. 7 (a) be shown in EPO-IgG1Fc, EPO-hFc-1 in people's F36E cell line compared with EPO bioactivity, EPO-hFc-5, EPO-hFc-6 andThe bioactivity result of (derbepoetin alfa)；Fig. 7 (b) is shown in small In mouse hematopoietic cell system (NFS-60)(pegfilgrastim) and the Bioactivity knot of G-CSF-hFc-5 Fruit；Fig. 7 (c) is shown in the Bioactivity result of p40 and p40N303Q-hFc-5 in human PBMC s；Fig. 7 (d) is shown in mouse In section's L929 cell(etanercept) and the Bioactivity result of TNFR-hFc-5；It is shown in Fig. 7 (e) ThFc-1-AL (0)-IFN-β and thFc-1-AL (3)-IFN-β Bioactivity result in people's WISH cell.

Fig. 8 (a) display is applied through SC approach (left figure) and IV approach (right figure) to macaque The result of the Half-life in vivo of (derbepoetin alfa), EPO-hFc-1 or EPO-hFc-5；The percutaneous bet of Fig. 8 (b) display It penetrates approach (left figure) and Intravenous administration route (right figure) applies to Sprague Dawley rat(non-lattice Take charge of pavilion) and G-CSF-hFc-1 pharmacokinetics result；Fig. 8 (c) shows what subcutaneous injection approach was applied to macaque(etanercept) result of pharmacokinetics；Fig. 8 (d) shows subcutaneous injection approach to Sprague Dawley rat application TNFR-hFc-5 and(etanercept) result of pharmacokinetics.

Fig. 9 (a) shows what subcutaneous injection approach (the picture left above) and Intravenous administration route (bottom-right graph) were applied to macaqueThe in vivo bioactivity of (derbepoetin alfa) and EPO-hFc-5 as a result, the percutaneous bet of Fig. 9 (b) display It penetrates approach (above) and Intravenous administration route (following figure) applies to Sprague Dawley rat(non-lattice Take charge of pavilion) and G-CSF-hFc-1 in vivo bioactivity result.

Realize best mode of the invention

The present invention provide hybridization human immunoglobulin(HIg) Fc segment, include from the end N- to the end C- hinge area, CH2 structural domain and CH3 structural domain, wherein the hinge area is at least partly amino acid sequence of people IgD hinge area or human IgG1's hinge area；CH2 structure Domain is 4 CH2 structural domain of human IgG, CH2 structural domain N- petiolarea a part by human IgG2 CH2 or people's IgD CH2 structural domain The 4-37 amino acid residue of N- petiolarea substitutes.When being connected to bioactive molecule, such as the bioactivity of biologically active polypeptide Molecule, when for generating Fc fusion protein, this hybrid Fc segment makes the nonspecific immunity of Fc fusion protein react minimum, prolongs The sad phase of the serum of the bioactive molecule of growth active peptides half, and make the activity of the bioactive molecule of biologically active polypeptide It optimizes.

In Fc fusion protein in a kind of embodiment according to the present invention, design IgD CH2 structural domain the end N- with The combination of the remainder of IgG4 CH2 structural domain, so that the region of the fusion protein generated at two difference Ig subunits reconstitutions It is hydrophobic.The hydrophobic region of the fusion protein of generation should be located inside folded protein, minimize undesirable non-specific exempt from Epidemic disease reaction.

As used herein, term " Fc segment " or " Fc " refer to protein, the heavy chain constant region 1 containing immunoglobulin (CH1), heavy chain constant region 2 (CH2) and heavy chain constant region 3 (CH3), and do not contain the heavy chain and light chain of the immunoglobulin Variable region and constant region of light chain 1 (CL1).It can further include the hinge area in heavy chain constant region.Hybrid Fc or Hybrid Fc segment is at sometimes herein called " hFc ".

In addition, Fc segment of the invention can be with natural sugar chain, increased sugar chain and day compared with natural sugar chain Right sugar chain compares the form of reduced sugar chain, or can be in the form of deglycosylated.By this field common methods, such as Chemical method, Enzymology method and using microorganism gene engineering method, can with the increase of adaptive immune globulin Fc sugar chain, subtract Less or remove.From Fc segment removal sugar chain cause with the binding affinity of the part Clq of the first complement component C1 fall sharply and The reduction or forfeiture of cytotoxicity (ADCC) or the complement dependent cytotoxicity effect (CDC) of antibody dependent cellular mediation, Therefore unnecessary vivo immunization reaction will not be induced.On this point, in some cases, deglycosylation or non-glycosylated (aglycosylated) immunoglobulin Fc segments of form are more likely to be appropriate for target of the invention as pharmaceutical carrier.

As used herein, term " deglycosylation " refers to that saccharide part is removed from Fc segment by enzyme and term " non-saccharide Base " (aglycosylation) means the Fc piece for generating the form that is not glycosylated by prokaryotes, preferably Escherichia coli Section.

As used herein, term " hybridization (heterozygosis) " refers to two or more immunoglobulin Fcs of coding separate sources The sequence of segment is with the appearance of single-chain immunoglobulins Fc segment.

In one embodiment, hybridization people Fc includes hinge area, CH2 structural domain and CH3 on the end N- to C- extreme direction Structural domain, wherein the hinge area is at least partly amino acid sequence of people IgD hinge area or human IgG1's hinge area；And CH2 knot Structure domain is 4 CH2 structural domain of human IgG, N- petiolarea a part by the end N- of human IgG2 CH2 or people's IgD CH2 structural domain The 4-37 amino acid residue in area substitutes.Hybridization people Fc can be connected to bioactive molecule at its end N- by covalent bond The end C-.

In another embodiment, bioactive molecule-hybrid Fc fused polypeptide can be indicated with following formula:

N′-X-(Z1)_p- Y-Z2-Z3-Z4-C ', or

N′-(Z1)_p- Y-Z2-Z3-Z4- (connector)_q-X-C′

Wherein N ' be polypeptide the end N- and C ' be polypeptide the end C-；Z1 indicates amino acid sequence comprising SEQ ID NO: The amino acid of the position 90-98 of at least C- end part or SEQ ID NO:14 of the amino acid residue of 11 90 to 98 positions is residual At least part of base；Y indicates amino acid sequence comprising the amino acid residue of 99 to 113 positions of SEQ ID NO:11 At least at least part of the amino acid residue of 99 to 162 positions of C- end part or SEQ ID NO:14；Z2 indicates amino acid Sequence comprising at least N- end part or SEQ ID NO of the amino acid residue of 111 to 147 positions of SEQ ID NO:12: At least N- end part of the amino acid residue of 14 163 to 199 positions；Z3 indicates amino acid sequence comprising SEQ ID NO: The 115 of the 165-270 or SEQ ID NO:13 of 11 118-223, the 114-219 of SEQ ID NO:12, SEQ ID NO:24 are arrived At least C- end part of the amino acid residue of 220 positions；Z4 indicates amino acid sequence comprising the 221- of SEQ ID NO:13 At least N- end part of the amino acid residue of 327 positions, and p and q are each 0 or 1 integers, the wherein amino of Z2 and Z3 Sour total number of residues can be between 80 and 140, and both ends numerical value is included, and connector is connector molecule and X is interested Bioactive molecule.

In one embodiment, Z3-Z4 is amino acid sequence selected from the following: (i) by SEQ ID NO:11 118 to The end N- of the amino acid residue of 224 to 330 positions of the C- end part and SEQ ID NO:11 of the amino acid residue of 223 positions The continuous amino acid sequence being grouped as, (ii) by SEQ ID NO:12 114 to 219 positions amino acid residue C- end part The continuous amino acid sequence being grouped as with the end N- of the amino acid residue of 220 to 326 positions of SEQ ID NO:12, (iii) By the C- end part of the amino acid residue of 165 to 270 positions of SEQ ID NO:24 and 271 to 377 of SEQ ID NO:24 The continuous amino acid sequence that the end N- for the amino acid residue set is grouped as, and (iv) by 115 to 220 of SEQ ID NO:13 The end N- of the amino acid residue of 221 to 327 positions of the C- end part and SEQ ID NO:13 for the amino acid residue set is grouped At continuous amino acid sequence.

A kind of embodiment according to the present invention, the total amino acid residues of the polypeptide are from 154 to 288.

When being administered to target person, compared with individual X, formula N '-X- (Z1)_p- Y-Z2-Z3-Z4-C ' and N '-(Z1)_p- Y-Z2-Z3-Z4- (connector)_qThe polypeptide of-X-C ' increases the circulating half-life of bioactive molecule X.

Connector can be derived from human albumin (CAA00606SEQ ID NO:25).The connector may include SEQ ID 321 to 323,318 to 325,316 to 328,313 to 330,311 to 333 or 306 to 338 amino acid sequences of NO:25. Optionally, connector can be the connector of synthesis.The synthesis connector can be by 10-20 Gly and Ser amino acid in total The peptide of residue composition.In one embodiment, which is GGGGSGGGGSGGGSG (SEQ ID NO:32).

Z1 may include at least the one of the CH1 structural domain of human IgG1 (SEQ ID NO:11) or IgD (SEQ ID NO:14) Part.Z1 may include the C- petiolarea (position of the 90-98 of SEQ ID NO:11) or IgD CH1 structure of IgG1 CH1 structural domain 5 to 9 or 7 to 9 continuous amino acid residues of the C- petiolarea (position of the 90-98 of SEQ ID NO:14) in domain.In some implementations In mode, Z1 can be 5,6,7,8 or 9 C-terminal amino acid residues of IgG1 CH1 structural domain or IgD CH1 structural domain.

In some embodiments, Z1 is amino acid sequence comprising the amino of 90 to 98 positions of SEQ ID NO:11 The amino acid residue of 90 to 98 positions of sour residue or SEQ ID NO:14.Z1 can be by the 90 to 98 of SEQ ID NO:11 The amino acid sequence of 5 to 9 amino acid residues composition of the position of the 90-98 of position or SEQ ID NO:14.Z1 is also possible to The amino being made of 90 to 98 amino acid residues of SEQ ID NO:11 or 90 to 98 amino acid residue of SEQ ID NO:14 Acid sequence.

Y may include at least part of the hinge area of human IgG1 or IgD.Y may include IgG1 hinge area (SEQ ID The amino acid of 99 to 113 positions of NO:11) or IgD hinge area (amino acid of 99 to 162 positions of SEQ ID NO:14) 5 or more of the end C- or 10 or more continuous amino acid residues.In some embodiments, Y can be amino Acid sequence comprising 158 to 162 positions of the amino acid residues of 99 to 113 positions of SEQ ID NO:11, SEQ ID NO:14 Amino acid residue, the amino acid residue of 153 to 162 positions of SEQ ID NO:14,143 to 162 of SEQ ID NO:14 99 to the 162 of the amino acid residues of 133 to 162 positions of the amino acid residue, SEQ ID NO:14 set or SEQ ID NO:14 The amino acid residue of position.

Z2 may include that (amino acid of 111 to 147 positions of SEQ ID NO:12 is residual for the end N- of human IgG2's CH2 structural domain Base) or the end N- (amino acid residues of 163 to 199 positions of SEQ ID NO:14) of IgD CH2 structural domain 4 to 37,6 arrive 30,6 to 12,6 to 8,8 or 6 continuous amino acid residues.In some embodiments, Z2 can be human IgG2's CH2 structural domain 6 n terminal amino acid residues (amino acid residue of the position 111-116 of SEQ ID NO:12) or people's IgD CH2 structural domain 8 n terminal amino acid residues (amino acid residue of the position 163-170 of SEQ ID NO:14).

The total amino acid residues of Z2 and Z3 can be between 90 and 120, and both ends numerical value is included；Or 105 and 115 it Between, both ends numerical value is included.

Z4 can be amino acid sequence comprising IgG4 CH3 structural domain is (in 224-330, SEQ of SEQ ID NO:11 220-326, the amino acid of 221 to 327 positions of the 271-377 or SEQ ID NO:13 of SEQ ID NO:24 of ID NO:12 is residual Base) 90 or more or 100 or more continuous amino acid residues.Z4 may be greater than human IgG1, IgG2, IgG3 or 98% or 95% amino acid residue of IgG4 CH3 structural domain.In an exemplary embodiment, Z4 be include human IgG The amino acid sequence of CH3 structural domain whole amino acid sequence.For example, Z4 is the amino acid sequence of 4 CH3 structural domain of human IgG, it is right Should be in the amino acid residue 341-447 of human IgG 4, such as according to EU Index, (it is corresponding to SEQ ID NO:13's for Kabat number The amino acid residue of the position 221-327).

In one embodiment, Y can be amino acid sequence comprising the C-terminal amino acid residue of human IgG1's hinge area At least part of (99 to 113 positions of SEQ ID NO:11), p can be 1 or 0；Z2 can be amino acid sequence comprising At least part of the N- petiolarea (amino acid residue is in the position 111-147 of SEQ ID NO:12) of human IgG2 CH2；It can with Z3 To be amino acid sequence comprising any one C- petiolarea (118- of the amino acid residue in SEQ ID NO:11 of subclass of human IgG 223,115 to 220 positions of 165-270 the or SEQ ID NO:13 of the 114-219 of SEQ ID NO:12, SEQ ID NO:24) At least part.In this embodiment, when p is 1, Z1 can be the C- petiolarea (SEQ including human IgG1's CH1 structural domain The amino acid residue of 90 to 98 positions of ID NO:11) at least part of amino acid sequence.For example, Z1 can be SEQ ID 90 to 98 amino acid residues of NO:11.

In further embodiment, Z3 can be 4 CH2 structural domain of human IgG (in the 115-220 of SEQ ID NO:13 Position), human IgG1 CH2 structural domain (in the position 118-223 of SEQ ID NO:11), human IgG2 CH2 structural domain is (in SEQ ID The position 114-219 of NO:12), the C- petiolarea of 3 CH2 structural domain of human IgG (in the position 165-270 of SEQ ID NO:24) 73 To 106 continuous amino acid residues, and the total amino acid residues of Z2 and Z3 can be 110.For example, Z2 can be in SEQ The amino acid sequence and Z3 of the amino acid residue of the position 111-116 of ID NO:12 can be the 117 of SEQ ID NO:13 To the amino acid sequence of the amino acid residue of 220 positions.

In another embodiment, Y can be the C- petiolarea including people's IgD hinge area (the 99 of SEQ ID NO:14 arrived The amino acid residue of 162 positions) at least part of amino acid sequence, p can be 1 or 0 (zero)；Z2 can be including people At least part of ammonia of the N- petiolarea (amino acid residues of 163 to 199 positions of SEQ ID NO:14) of IgD CH2 structural domain Base acid sequence and Z3 can be the C- petiolarea including 4 CH2 structural domain of human IgG (121 to 220 positions of SEQ ID NO:13 Amino acid residue) at least part of amino acid sequence.For example, Y can be 158 to 162, the 133 of SEQ ID NO:14 To the amino acid residue of 162 or 99 to 162 positions, Z2 can be the amino acid in 163 to 170 positions of SEQ ID NO:14 Residue and Z3 can be the amino acid residue in the position 121-220 of SEQ ID NO:13.

In this embodiment, when p is 1, Z1 can be the C-terminal area including people's IgD CH1 structural domain, and (amino acid is residual Base is in 90 to 98 positions of SEQ ID NO:14) amino acid sequence.For example, Z1 can be 90 to 98 of SEQ ID NO:14 Amino acid residue.

In this embodiment, Y can be the end side C- of people's IgD hinge area (amino acid residue be in SEQ ID NO:14 The position 99-162) 20 continuous amino acid residues or more, 30 continuous amino acid residues or more, 40 it is continuous Amino acid residue or more, 50 continuous amino acid residues or more or 60 continuous amino acid residues or more.Z3 It may include 71 to 100 continuous amino acid residues of the end side amino acid residue C- of the position 121-220 of SEQ ID NO:13. The total amino acid residues of Z2 and Z3 can be 108.

Table 1 is shown in human IgG1 useful in the hFcs of building embodiment according to the present invention, IgG2, IgG3 and IgD The amino acid sequence of segment.

Table 1

* EU index is in " Sequences of Proteins of Immunological Interest, 5th Description in Edition, United States Department of Health and Human Services. ".

Underlined region in every amino acid sequence of * indicates the most short-movie section of acceptable amino acid residue range.

In one embodiment, the present invention provides hybrid Fc, is hFc-1, hFc-2, hFc- as illustrated in figs. 1 and 2 3, hFc-4, hFc-5 or hFc-6 or one of thFc-1 or thFc-2 such as the display of Fig. 3 and 4.Although Fig. 1 and 3 describes double-strand Fcs, but the present invention includes single-stranded hybrid Fc molecule.The amino acid sequence of hFc-1 to hFc-6 is respectively displayed on SEQ ID In NOs:18-23 and Fc-1 and thFc-2 amino acid sequence is respectively displayed in SEQ ID NO:28 and SEQ ID NO:29. The present invention also includes the polynucleotide molecule for encoding hybrid Fc.Their including but not limited to such as SEQ ID NO:1 (hFc-1), SEQ ID NO:2 (hFc-2), SEQ ID NO:3 (hFc-3), SEQ ID NO:4 (hFc-4), SEQ ID NO:5 (hFc-5), Polynucleotide shown in SEQ ID NO:6 (hFc-6), SEQ ID NO:26 (thFc-1) and SEQ ID NO:27 (thFc-2) Sequence.

Human immunoglobulin(HIg) amino acid sequence is known in the art, and they deposit in publicly accessible storage Place.For example, the ammonia of human IgG1's constant region, human IgG2's constant region, 3 constant region of human IgG, 4 constant region of human IgG and people's IgD constant region Base acid sequence is obtained from CAA75032, CAC20455, CAC20456, AAH25985 and P01880 respectively.These sequences respectively become SEQ ID NO:11,12,24,13 and 14 (reproduced as).

Bioactive molecule X can be soluble protein.It may include but unlimited hormone, cell factor, growth because Son, costimulatory molecules, hormone receptor, cytokine receptor, growth factor receptors or small peptide.For example, X can be EPO, p40, G- CSF, TNF receptor or its variant/segment.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 Ⅴ II, CXCL-11, FSH, life are long Hormone, bone morphogenetic protein -1, CTLA4, PD-1, GLP-1, β tunicin, OPG, RNAK, alpha interferon, beta interferon or Their variant/segment.It also can include but is not limited to the area Fab of antibody.Bioactive molecule is also possible to secretory egg White matter.In one embodiment, which is not belonging to immunoglobulin class.

Term " variant " refers to different from reference nucleic acid or polypeptide but retains the polynucleotide or core of its fundamental property Acid.In general, variant is generally very much like, and identical as reference nucleic acid or polypeptide in many regions.Meanwhile term " variant " refers to the biologically-active moiety of bioactive molecule drug, and retain its as described in otherwise herein or this field its Its known at least one function and/or therapeutic properties.In general, variant is generally closely similar, and in many regions It is identical as the amino acid sequence of interested biologically active polypeptide.

The present invention also provides protein comprising the ammonia with such as polypeptide shown in SEQ ID NOs:18-23 and 28-29 Base acid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical amino acid sequence or Optionally it is made of it.The present invention also provides the segment of these polypeptides.Further, polypeptide by the invention is by such The polypeptide of polymerized nucleoside acid encoding, (for example, at about 45 DEG C in 6 × sodium chloride/sodium citrate under stringent hybridisation conditions (SSC) it with the filter hybridization of combination DNA in, is then cleaned in 0.2 × SSC, 0.1%SDS at about 50-65 DEG C primary or more It is secondary), under high high stringency conditions (for example, about 45 DEG C in 6 × sodium chloride/sodium citrate (SSC) with combine the filter membrane of DNA miscellaneous Hand over, then cleaned in 0.2 × SSC, 0.1%SDS at about 68 DEG C one or many) or those of ordinary skill in the art known to Other high stringency conditions under (see, e.g., Ausubel, F.M. etc., eds., 1989Current protocol in Molecular Biology, Green publishing associates, Inc., and John Wiley&Sons Inc., New York, 6.3.16.3.6 and 2.10.3 pages), the polynucleotides are complementary with the nucleic acid molecules that encode polypeptide of the invention Chain (complement) hybridization.The present invention also includes the polynucleotide for encoding these polypeptides.

It is " same with inquiry amino acid sequence (a query amino acid sequence) at least such as 95% for having The polypeptide of the amino acid sequence of one property " is intended that the amino acid sequence and inquiry amino acid sequence of target (subject) polypeptide It is identical, in addition in every 100 amino acid of inquiry amino acid sequence, subject polypeptide sequence may include most 5 amino acid Change.In other words, to obtain the polypeptide having with the identity of inquiry amino acid sequence at least 95%, in target sequence most More 5% amino acid residue can be inserted into, pick out or with another amino acid replacement.The change of these reference sequences can be with The amino of reference amino acid sequence or the position of c-terminus or any position between this end positions are appeared in, it is single Ground is dispersed in the residue in adjacent sets either one or more in reference sequences in the residue of reference sequences.

In fact, any specific polypeptide whether with the amino acid sequence of albumin fusion protein for example of the invention or its Segment at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% are identical usually to can be used known computer Program determination.Best total matched preferred method between search sequence (sequence of the invention) and target sequence is measured, also referred to as The algorithm (Comp.App.Biosci.6:237245 (1990)) based on Brutlag etc. can be used in global sequence alignment The measurement of FASTDB computer program.In sequence alignment, inquiry and target sequence be or both be nucleotide sequence or It is both amino acid sequence.The result of global sequence alignment is indicated with homogeneity percentage.In FASTDB amino acid alignment The preferred parameter used is: tuple=2 matrix=PAM 0, k-, Mismatch Penalty=1, connection point penalty=20, random groups length= 0, cut off score=1, window size=sequence length, gap penalty=5, vacancy size point penalty=0.05, window size=500 Or the length of subject amino acid sequence, it is whichsoever shorter.

Variant and standard HA or treatment albumen usually have at least 75% (preferably at least about 80%, 90%, 95% or 99%) sequence identity, the standard HA or treatment albumen and variant have equal length.In nucleotide or amino acid sequence water Flat homology or identity using be designed to search program blastp, blastn of sequence similarity, blastx, Tblastn and tblastx (Karlin etc., Proc.Natl.Acad.Sci.USA 87:22642268 (1990) and Altschul, J.Mol.Evol.36:290300 (1993) is fully incorporated by reference) used by algorithm by BLAST (Basic Local Alignment Search Tool) analysis be measured.

Polynucleotides variant of the invention can contain the variation in code area, noncoding region or both.Particularly preferably Be polynucleotides variant, contain and generate silent substitutions, increase or missing but the property or active for not changing coding polypeptide Variation.As gene-code degeneracy and the nucleotide variants as caused by silent substitutions are preferred.Moreover, small wherein In 50, less than 40, less than 30, less than 20, less than 10 or 5-50,5-25,5-10,1-5 or 1-2 amino acid are by with any group Conjunction displacement, missing, increased polypeptide variants are also preferred.Polynucleotides variant can be generated due to many reasons, such as (codon in people mRNA is changed into bacterial host such as yeast or large intestine for optimizing the codon expression of specific host Bacillus (E.coli) preferred codon).

In order to construct various Fc fusion proteins such as EPO-Fc fusion constructs, G-CSF-Fc fusion constructs or people p40- The amino acid sequence of Fc fusion constructs, people EPO, human G-CSF, people p40 and people's TNF receptor can be respectively from NP_000790 (SEQ ID NO:15), CAA27291 (SEQ ID NO:16), AAG32620 (SEQ ID NO:17) and NP_001057 (SEQ ID NO: 31) it obtains.In one embodiment, the wherein 303 amino acid residue Asn people p40 being modified that Gln is replaced is connected It is connected to polypeptide.

According to another aspect of the present invention, the whole antibody in the area Fc containing transformation is provided.As used herein, term " antibody " Including whole antibody and comprising in CH1, hinge area, CH2 or CH3 at least two antibody fragment.Full monoclonal antibody is preferred. The heavy chain variable region of antibody is selected for its binding specificity and can be any type, such as, such as inhuman, source of people (fully human) change or complete people.When the heavy chain variable region of antibody be inhuman (such as, such as mouse) and with basis When the area the Fc recombination ground combination of the transformation of the disclosure, generated recombinant antibodies are referred to as chimeric antibody.When the heavy chain of antibody can Become area be humanization and when being combined with recombinating with the area Fc according to the transformation of the disclosure, generated recombinant antibodies are referred to as Humanized antibody.When antibody heavy chain variable region is people's and when combining with the area the Fc recombination according to the transformation of the disclosure, institute The recombinant antibodies of generation are referred to as fully human antibodies.For example, heavy chain variable region be humanization and including human skeleton area and non- People (being in this case mouse) complementary determining region (complementary determining regions, CDRs).It should be understood that For skeleton area can be originated from a kind of source or more than one sources and CDRs can be originated from a kind of source or more than one come Source.The method of antibody humanization is known to the skilled person and is known in the art.

The light chain of antibody can be people, inhuman or humanization.In the embodiment that Fig. 1 is shown, light chain is people Source and including human skeleton area, inhuman (being in this case mouse) CDRs and human constant region.It is construed as, skeleton area can A kind of source or more than one sources can be originated to be originated from a kind of source or more than one sources and CDRs.

The antibody in the area Fc containing transformation is selected based on its ability for being integrated to cell surface molecule or shla molecule It selects, the shla molecule is integrated to cell surface molecule.Thus, for example antibody can be based on it in conjunction with these cell surfaces point Son ability selected, cytokine receptor (for example, IL-2R, TNF-aR, IL-15R etc.), adhesion molecule (for example, E-Selectin, palatelet-selectin, L-selectin, VCAM, ICAM etc.), cell differentiation or active antigen (for example, CD3, CD4, CD8, CD20, CD25, CD40 etc.) and it is other.Optionally, antibody can combine the ability of shla molecule to be selected based on it, The shla molecule is integrated to cell surface molecule.These shla molecules include, but are not limited to: cell factor and and chemotactic The factor (for example, interleukin 1 (IL-1), IL-2, IL-3, IL-5, IL-6 etc.), growth factor (for example, EGF, PGDF, GM-CSF, HGF, IGF, BMP-1 etc.), the molecule (for example, EPO, TPO, SCF, PTN etc.) of Cell differentiation inducing activity and other.

In general, antibody disclosed herein is constructed by using operation generally acknowledged employed in technique for gene engineering It completes.For example, these technologies are well known in the art: separation DNA, preparation and selection express the carrier of DNA, purify and divide It analyses nucleic acid, the specific method for manufacturing recombinant vector DNA, shear DNA, connection DNA with restriction enzyme, by stable or instantaneous method By the DNA comprising carrier DNA import host cell, cultivate in selective or non-selective culture medium host cell to select and Maintain the cell of expression DNA.

Many institute's weeks in hybridoma method as known in the art or this field can be used in monoclonal antibody disclosed herein The method for the other recombinant DNAs known is derived.In hybridoma method, mouse or other suitable is immunized with DNA, peptide or protein matter The host animal of conjunction, the DNA, peptide or protein matter cause lymphocyte to generate antibody.

Optionally, lymphocyte can be immunized in vitro.Then, it with suitable fusion agent such as polyethylene glycol, will answer The lymphocyte for answering antigen generation is merged with myeloma cell, to form hybridoma.Then, the hybridoma is connect It plants and is grown in suitable culture medium, the culture medium preferably includes one or more of parental generation marrow for inhibiting not merge Tumor cell growth or the substance of existence.Preferred myeloma cell is those myeloma cells: they effectively merge, support choosing Generate to the cytotostatic for the generation antibody selected antibody and to culture medium such as HAT culture medium (Sigma Chemical Company, St.Louis, Mo., Catalog No.H-0262) it is insensitive.

The antibody in the area Fc containing transformation is also used as the composition being administered alone, and the composition combined treatment agent is given Out.For diagnostic purpose, the antibody can be labeled or not be labeled.

Unlabelled antibody can be described other marked with other marked antibody (secondary antibody) in conjunction with using Antibody and transformation antibody response, antibody such as special to human immunoglobulin(HIg) constant region.Optionally, antibody can be direct It is labeled.Can be used many kinds of labels, such as radionuclide, fluorescer, dark, zymolyte, enzyme cofactor, enzyme inhibitor, Ligand (especially haptens) etc..It measures using panimmunity and is well-known to those skilled in the art.

According to one embodiment, the present invention provides the method for generating fusion protein, this method comprises: (i) melting coding The DNA molecular of hop protein introduces mammalian host cell, and (ii) makes cell in its growth under conditions of expressed fusion protein It is grown in culture medium；And the fusion protein that (iii) harvest generates.

In the embodiment of other examples, the medicine comprising above-mentioned fusion protein or antibody molecule or antibody fragment is provided Compositions.The method for treating or preventing certain symptoms by applying described pharmaceutical composition is also provided.For example, providing such as lower section Method: (i) reduces the symptom of autoimmune disease, preventing/treating autoimmune disease, and (ii) inhibits graft rejection, (iii) Treatment/prevention endotoxin induced shock comprising upper a effective amount of hybrid Fc and p40 albumen or its variant/piece are treated in application The fusion protein of section.

Composition may include pharmaceutical carriers.Pharmaceutical carriers can be any compatible, nontoxic substance, be suitable for resist Body is delivered to patient.Sterile water, ethyl alcohol, fat, wax and inert solid may include in the carrier.Pharmaceutically acceptable adjuvant (buffer, dispersing agent) can also mix in pharmaceutical composition.

Antibody compositions can be administered to object in various ways.For example, pharmaceutical composition can with parenteral, for example, subcutaneously, Muscle or intravenous administration.These compositions can be sterilized by common, known sterilization technology.Composition may include Pharmaceutically acceptable auxiliary substance, the auxiliary substance are needed for obtaining approximate physiological condition, and such as pH adjusts gentle Electuary, toxicity modifiers etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate etc..Fusion protein, antibody or The concentration of antibody fragment in these formulations can be extensively varied, for example, by weight from be less than about 0.5%, usually or At least about 1% up to 15 or 20% and its mainly according to selected specific method of application be based on liquid volume, viscosity etc. into Row selection.

The present invention also provides the isolated nucleic acid molecules of encoding fusion protein, and carry the expression vector of nucleic acid molecules. The nucleic acid can directly be delivered to the object for needing the polypeptide of the nucleic acid encode.Optionally, by expressing in the medium Nucleic acid and generate polynucleotides, be then administered to object.

Term " peptide ", " polypeptide " or " protein " refers to the molecule of 2 to 40 amino acid, and preferably 3 to 20 amino The molecule of acid and the molecule of most preferably 6 to 15 amino acid.Illustrative peptide can be random by any means of above-mentioned reference It generates, is carried in peptide library (for example, phage display library), or derived by digesting protein.

As used herein, term " drug " refers to the substance that therapeutic activity is shown when being administered to human or animal, drug Example includes, but are not limited to: polypeptide, compound, extract and nucleic acid.Preferably polypeptide drugs.

As used herein, term " physiological active polypeptide ", " bioactive molecule ", " physiologically active protein matter ", " activity it is more Peptide ", " polypeptide drugs " and " pharmaceutical grade protein " their meaning can be interchanged, and it is characterized in that they with physiological activity Form shows various body physiological functions.

Polypeptide drugs have the shortcomings that physiological action a rapid lapse of time cannot be maintained, this is because it is easy to be deposited in vivo Proteolysis enzyme denaturation or degradation property.But when polypeptide drugs are connected (or coupling) to implementing according to the present invention When immunoglobulin Fc segments described in mode form fusion protein, the drug has increased structural stability and serum half It declines the phase.In addition, the polypeptide for being connected to Fc segment has the physiological activity than other known polypeptide drugs formulation much less It reduces.Therefore, compared with the vivo biodistribution availability of traditional polypeptide drugs, the fusion comprising polypeptide drugs and Fc segment it is more Peptide or polypeptide drugs and the conjugate of Fc segment of the present invention are characterized by having that the vivo biodistribution significantly improved utilizes Degree.This is explicitly described also by embodiments of the present invention.That is, when being connected to Fc segment of the invention When, IFN-α, G-CSF, EPO, p40, TNF receptor and other oroteins drug are shown to be merged with its native form or other tradition Form compares increased vivo biodistribution availability.

It is to be understood that the present invention has developed traditional recombinant DNA method, to generate Fc fusion protein, comprising basis The Fc domain antibodies of transformation of the invention and the antibody fragment practiced for the present invention.Fc fusion constructs are preferably in DNA level It generates and generated DNA is integrated into expression vector, and express to generate fusion protein of the invention, antibody or resist Body segment.

As used herein, term " carrier " is understood to be meant that any nucleic acid comprising such nucleotide sequence, institute Host can be mixed and recombinate with host cell gene group and be integrated into host cell gene group or energy by stating nucleotide sequence Enough independently replicated as episome.These carriers include linear nucleic acid, plasmid, antibiotic, clay, RNA carrier, viral vectors etc. Deng.The non-limiting example of viral vectors includes retrovirus, adenovirus and the relevant virus of gland-.As used herein, term " gene expression " or " expression " of target protein is understood to be meant that transcription of DNA sequences, translation mRNA transcript, and secretion Fc fusion protein product or antibody or antibody fragment.

Useful expression vector is RcCMV (Invitrogen, Carlsbad) or its variant.Useful expression vector should be taken Band human cytomegalovirus (cytomegalovirus, CMV) promoter, to promote interested gene in mammalian cell Composing type transcription, and bovine growth hormone polyadenylation signal sequence is carried, with the stable state water of RNA after increase transcription It is flat.In embodiments of the present invention, expression vector is pAD11, is the modification carrier of RcCMV.Carry encoding bioactive The example of the expression vector of the nucleotide sequence of molecular drug may include being not limited to: 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, as being more fully described in embodiment.

Suitable host cell can be converted or be transfected with DNA sequence dna of the invention, and for the expression of target protein And/or secretion.It include that the hybridoma of infinite multiplication, NS/0 myeloma are thin currently used for preferred host cell of the invention Born of the same parents, 293 cells, Chinese hamster ovary cell, HeLa cell and COS cell.

Have been used to the one of the fusion protein for generating high level expression in mammalian cells or antibody or antibody fragment A expression system is the DNA construction that secreting, expressing box is encoded on 5 ' to 3 ' directions comprising signal sequence and immune globulin The white area Fc and target protein, such as p40, EPO, G-CSF, TNF receptor.Several target proteins succeed in this system Express and include, for example: IL2, CD26, Tat, Rev, OSF-2, ss in ground；IG-H3, IgE receptor, PSMA and gp120.These Expression construct is disclosed in U.S. Patent No. 5,541, No. 087 and the 5th, 726, No. 044 of Lo et al., and content passes through reference It is incorporated herein.

Fusion protein or antibody molecule or antibody fragment of the invention may include or do not include signal sequence in expression. As used herein, term " signal sequence " is understood to be meant that guidance bioactive molecule drug；The secretion of fusion protein is simultaneously And the segment being sheared after being translated in host cell thereafter.Signal sequence of the invention is the multicore glycosides of encoding amino acid sequence Acid, the amino acid sequence start transhipment of the protein across endoplasmic reticulum.It include antibody light chain for signal sequence of the invention Signal sequence, such as antibody 14.18 (Gillies etc., J.Immunol.Meth.1989.125:191-202), heavy chain of antibody letter Number sequence, such as MOPC141 antibody heavy chain signal sequence (Sakano etc., Nature 1980.286:676-683) and ability Any other signal sequence known to domain (see, e.g. Watson etc., NucleicAcids Research 1984.12: 5145-5164)。

Signal sequence in the art by sufficiently it is qualitative and it is known include 16 to 30 amino acid residues, Yi Jike To include more or fewer amino acid residues.Typical signal peptide by three district's groups at: alkaline N- petiolarea, central hydrophobic region and More polar C- petiolarea.Central hydrophobic region includes 4 to 12 hydrophobic residues, transit period of the hydrophobic residue in nascent polypeptide Between across membrane lipid bilayer anchor signal peptide.After actuation, signal peptide is usually known as the thin of signal peptidase endoplasmic reticulum is intracavitary The shearing of born of the same parents' enzyme.The potential shearing site of signal peptide generally follows " (- 3, -1) rule ".Therefore, typical signal peptide is -1 and -3 There is small, neutral amino acid residue in position and lack proline residue in this zone.

Signal peptidase shears this signal peptide between -1 and+1 amino acid.Therefore, signal sequence can during secretion To be sheared from the amino terminal of fusion protein.This leads to the Fc fusion protein being made of immunoglobulin fc region and target protein Secretion.Being discussed in detail for signal peptide sequence is provided by von Heijne (1986) NucleicAcids Res.14:4683A.

As it will be apparent to those skilled in the art that, the adaptability of the specific signal sequence for secreting, expressing box It may need some routine experiments.

Such experiment includes the ability of measurement signal sequence guidance Fc fusion protein secretion, also measures sequence ready for use Optimum configuration (configuration), genome or cDNA to reach effective secretion of Fc fusion protein.In addition, this field Technical staff can be according to the signal peptide for the rule preparation synthesis that von Heijne (1986) provide, and pass through conventional reality It tests and the effect of this synthetic signal sequence is tested.Signal sequence can also be known as " signal peptide ", " leader sequence " or " preceding Lead peptide ".

The fusion of signal sequence and immunoglobulin fc region is sometimes referred to as secreting, expressing box.Show for what the present invention practiced The secreting, expressing box of example property is polynucleotides, polynucleotides light chain encoding immunoglobulin gene on 5 ' to 3 ' directions The area Fcy1 of signal sequence and human immunoglobulin(HIg) y1 gene.The area Fcy1 of immunoglobulin Fc y1 gene preferably includes at least A part of immunoglobulin hinge structural domain and at least CH3 structural domain, or more preferably at least a part of hinge domain, CH2 knot Structure domain and CH3 structural domain.As used herein, " part " of immunoglobulin hinge region is understood to be meant that the immune ball of a part Albumen hinge, it includes at least one, preferably two cysteine residues, the cysteine residues are capable of forming two sulphur of interchain Key.The DNA for encoding secreting, expressing box can be in its genomic configuration or its cDNA configuration.In some cases, immune from people Globulin Fcy2 sequence of heavy chain generates the area Fc and is advantageous.Although the Fc based on human immunoglobulin(HIg) y1 and y2 sequence merges behavior It is similar to mouse, excellent pharmacokinetics can be shown based on y2 sequence Fc fusion in human body.

In another embodiment, the protease that DNA sequence encoding is inserted between secreting, expressing box and target protein is cut Enzyme site.Shearing site provides the proteolytic cleavage of the fusion protein of coding, and the fusion protein of the coding is thus by Fc structure Domain is separated from target protein.As used herein, " protease cleavage " be understood to be meant that preferentially by proteolytic enzyme or The amino acid sequence of other proteolytic cleavage agent shearings.Useful protease cleavage includes by such as trypsase, fibrinolytic The amino acid sequence of the proteolytic enzyme of enzyme or enterokinase K identification.Known many shearing sites/shearing agent to (see, e.g. U.S. Patent No. 5,726,044).

Further, the displacement or missing of the construction of these constant regions --- wherein constant region domain is one or more A amino acid residue is replaced or deletes --- and it is useful.One example is the area CH2 imported amino acid replacement above It is interior, to generate Fc variant, the Fc variant affinity of Fc receptor is reduced (Cole etc. (1997) J.Immunol.159: 3613).Those skilled in the art can be used known Protocols in Molecular Biology and prepare these constructions.

The non-limiting example that can be incorporated into the protein drug of immunoglobulin Fc segments of the invention includes life Long hormone, bone morphogenetic protein-1 (BMP-1), growth hormone releasing hormone, Growth Hormone Releasing Peptide, interferon and interferon Receptor (for example, interferon-' alpha ' ,-β and-γ, water solubility I type interferon receptors etc.), granulocyte-colony stimulating factor (G- CSF), granular leukocyte macrophage colony forms stimulating factor (GM-CSF), glucagon-like peptide (for example, GLP-1 etc.), G egg White coupled receptor, interleukins (for example, interleukin 1, -2, -3, -4, -5, -6, -7, -8, -9, -10, -11, -12, - 13, -14, -15, -16, -17, -18, -19, -20, -21, -22, -23, -24, -25, -26, -27, -28, -29, -30 etc.) and white Cytokine receptor (for example, IL-1 receptor, IL-4 receptor etc.), enzyme are (for example, glucocerebrosidase, iduronic acid -2- sulfuric acid Esterase, alpha-galactosidase-A, Ah add'sing carbohydrase (agalsidase) α and β, α-L- iduronase, butyrylcholine esterase, shell Polysaccharase, glutamate decarboxylase, Imiglucerase (imiglucerase), lipase, uricase, platelet activating factor acetyl water Solve enzyme, neutral endopeptidase, myeloperoxidase etc.), interleukins and Cytokine binding proteins are (for example, IL- 18bp, TNF- binding protein etc.), macrophage activating factor (MAF), macrophage peptide, the B cell factor, the T cell factor, a-protein, Allergy inhibitor, meronecrosis glycoprotein, immunotoxin, lymphotoxin, tumor necrosis factor, tumor inhibitor (tumor Suppressors), metastasis of cancer growth factor (metastasis growth factor), α -1 antitrypsin, albumin, α - Lactoalbumin, ApolipoproteinE, hematopoietin, the hematopoietin of high glycosylation, angiogenesis hormone；Blood Pigment, fibrin ferment, Glycoprotein, thrombomodulin, factor Ⅴ II, factor VIIa, Factor IX, factors IX, because Sub- XIII, plasminogen activator, fibrin-binding peptides (fibrin-binding peptide), urokinase, streptokinase, Hirudin, protein C, C- proteins C reactive, renin inhibitor, collagenase inhibitors, superoxide dismutase, leptin, blood are small Plate source property growth factor, epidermal growth factor, epidermal growth factor, angiotensins, bone growth factor, spur shock protein (bone stimulating protein), calcitonin, insulin, the heart receive element, cartilage-inducing factor, Elcatonin, connective Organize activation factor, tissue factor pathway inhibitors, follicular stimulating hormone, interstitialcellstimulating hormone (ICSH), luteinizing hormone releasing hormone, nerve Growth factor is (for example, nerve growth factor, ciliary neurotrophic factor, (the axogenesis factor- of the axon regeneration factor -1 1), brain natriuretic peptide, glial cell line-derived neurotrophic factor, lead albumen, neutrophil inhibiting factor, neurotrophy because Son, neutruin etc.), parathyroid hormone, relaxain, secretin, somatomedin, insulin-like growth factor, adrenal gland Cortin, glucagon, cholecystokinin, pancreatic polypeptide, gastrin releasing peptide, cortico-trophin-releasing factor (CRF), Thyrotropic hormone, from toxin (autotoxin), lactoferrin, myostatin, receptor (for example, TNFR (P75), TNFR (P55), IL-1 receptor, vegf receptor, B cell activating factor receptor etc.), it is receptor antagonist (for example, IL1-Ra etc.), thin Cellular surface antigen (for example, CD 2,3,4,5,7,11a, 11b, 18,19,20,23,25,33,38,40,45,69 etc.), viral epidemic disease Seedling antigen, monoclonal antibody, polyclonal antibody, antibody fragment (for example, scFv, Fab, Fab ', F (ab ') 2 and Fd) and virus Source property immunizing antigen.Antibody fragment can be Fab, Fab ', F (ab ') 2, Fd or scFv, be connectable to specific antigen, And preferably Fab '.Fab segment includes the variable domains (VL) of light chain and the variable knot of constant domain (CL) and heavy chain Structure domain (VH) and the first constant domain (CH1).Fab ' segment is different from Fab segment, and the Fab ' segment will come from hinge area Several amino acid residues include c-terminus that one or more cysteine residues are added to CH1 structural domain.Fd segment is only It only connects or chemically reacts by disulfide bond comprising VH and CH1 structural domain and 2 segment of F (ab ') and produced as a pair of Fab ' segment It is raw.ScFv (scFv) segment include VL and VH structural domain, VL the and VH structural domain be connected to each other by peptide connector and Therefore to exist with single polypeptide chain.

Specifically, preferably being needed after being administered to human body for treating or preventing disease as bioactive molecule Those of frequent drug administration, including human growth hormone (HGH), interferon (interferon-' alpha ' ,-β ,-γ etc.), granular leukocyte colony formed stimulation because Sub (G-CSF), hematopoietin (EPO), TFN receptor, p40 and antibody fragment.In addition, certain derivatives are included in this In the range of the bioactive molecule of invention --- as long as they have substantially compared with the native form of bioactive molecule Identical or improved function, structure, activity or stability.In the present invention, most preferred polypeptide drugs are interferon-a.

In another aspect of this invention, IgG-Fc and IgG-CH fusion protein, such as it is synthesized into monomer, the monomer can To be assembled to form dimer.Typically, the dimer usually passes through the disulfide bond connection in IgG hinge area.IgG secretion fusion The conditioned medium of the cell of albumen may include the mixture of IgG fusion protein monomer and dimer.In order to be controlled as human body It treats, it may be desirable that using IgG fusion protein monomer or the homogeneous group of dimer, rather than the mixture of two kinds of forms.

The method for obtaining substantially pure dimerization active peptides-IgG fusion protein prepared product is also provided.This method is generally logical It crosses realization of such as getting off: obtaining the host cell that can express IgG fusion protein, collection condition culture medium, and pass through column chromatography Method purifies dimeric fusion protein from the fusion protein, aggregation (aggregates) and contaminating protein matter of monomer.Express IgG fusion The suitable host cell of albumen includes yeast, insect, mammal or other eukaryocytes.In one embodiment, place Chief cell can be mammalian cell, specifically COS, CHO or bhk cell.

The new fusion protein of polypeptide drugs and Fc segment is also provided.In one embodiment, such as EPO, p40, G- The polypeptide drugs of CSF or TNF receptor are directly connected to the Fc segment of hybridization, and do not have to insertion peptide connector.In another implementation In mode, polypeptide drugs are connected to each other, and pass through the peptide connector of 1 to 50 amino acid, and more preferably pass through 1 to 7 The peptide connector of amino acid is attached.Particularly useful connector for the purpose includes immuno-deactivation peptide, by Gly and Ser residue is (for example, Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser SEQ ID NO:32) it forms or is made of the amino acid at the position 282-314 of the SEQ ID NO:25 derived from human albumin.

In the case where using connector, connector and polypeptide drugs can be in certain direction.That is, connector can be with It is connected to the end N-, the end C- or free group of hybrid Fc segment, and also may be coupled to the end N-, the end C- or trip of polypeptide drugs From group.When connector is peptide connector, connection can occur in certain connection site.When polypeptide drugs and hybrid Fc quilt When respectively then expression is with being connected to each other, any one that can be used in many coupling agents as known in the art is carried out occasionally Connection.The non-limiting example of coupling agent includes bis- (two azoacetoacetoxy the bases) -2- vinylbenzenes of 1,1-, glutaraldehyde, N- hydroxysuccinimidyl acyl Ester, imidoate of the imines ester such as with 4- azidosalicylic acid include such as 3,3 '-two thiobis (amber of two succinimide esters Propionates) and such as double-N- dimaleoyl imino -1, the 8- octanes of bifunctional maleimides.

The present invention also provides production polypeptide drugs-hybrid Fc segment methods.

The present invention also provides the method for the treatment of symptom (conditions), the symptom is slow by application polypeptide drugs Solution.These methods include applying a effective amount of polypeptide of the invention to having Symptomatic mammal, the symptom can with or can With not direct related to interested disease.For example, encoding desired polypeptide drugs-hybrid Fc fragment fusion protein such as The nucleic acid of DNA or RNA can be used as therapeutic agent and be administered to object, preferably mammal.In addition, miscellaneous comprising coding polypeptide drugs- It hands over the cell of the nucleic acid of Fc fragment fusion protein to can be used as therapeutic agent and is administered to object, preferably mammal.In addition, peptide drug Object-hybrid Fc segment composition construct can be used as therapeutic agent and be administered to object, preferably mammal.These chimeric polyeptides can be with It is applied through intravenous, subcutaneous, oral, buccal (buccally), sublingual, intranasal, parenteral, rectum, vagina or transpulmonary approach.

EPO (including its variant/segment)-fc fusion protein of the invention can be used for improving and maintaining the blood of newborn animal thin Born of the same parents' specific volume.

P40 is the subunit of IL-12.IL-12 is the cell factor of 75kDa heterodimer, has several function in vivo Energy.For example, IL-12 stimulation activates T and NK cell Proliferation and promotes Th1 type auxiliary cell response.IL-12 is by being integrated to work IL-12 receptor on the plasma membrane of change T and NK cell plays its biological effect and IL-12 has been integrated to the ability of IL-12 receptor It is attributed to the p40 subunit of IL-12.Therefore, p40 of the invention (including its variant/segment)-fc fusion protein can be used for (i) symptom of autoimmune disease is reduced, preventing/treating autoimmune disease, (ii) inhibits graft rejection, or (iii) Treatment/prevention endotoxin induced shock.Moreover, p40 (including its variant/segment)-fc fusion protein of the invention can be used In treatment/prevention/improvement rheumatic arthritis, mandatory rachitis, inflammatory bowel disease, multiple sclerosis or psoriasic disease Shape.Variant and segment are well known in the art comprising but it is not limited to WO 97/20062, content is used as and is incorporated by Herein.One embodiment of p40 variant includes but is not limited to the p40 for including Asn303Gln displacement.

Granulocyte-colony stimulating factor (granulocyte colony stimulating factor, G-CSF) is The proliferation of granulocyte, especially neutrophil cell and the required protein of differentiation.Granulocyte swallows up and swallows microorganism and invades Enter and cell fragment, and is therefore vital to infection response.Chemotherapy destroyed granulocyte and/or reduction granulocyte Generation.Therefore, G-CSF of the invention (including its variant/segment)-fc fusion protein can be used for treatment/prevention/improvement and exist Bone-marrow transplantation, acute leukemia, alpastic anemia, marrow increase upper abnormal syndrome, serious chronic neutrophilic leucocyte The disease of the neutropenia bone marrow suppression of phase chemotherapy induced after the activation of the peripheral hematopoietic stem cells of reduction disease or transplanting Shape.

Fusion protein of the invention is not only used as therapeutic agent, and those skilled in the art will be appreciated that fusion protein can For producing the antibody of diagnostic uses.Similarly, suitably application DNA or RNA, for example, being passed with carrier or the other of these purposes System is sent, is also included in method of the invention.

Composition of the invention can be applied by any approach compatible with specific molecule.It can be understood that this is sent out Bright composition passes through suitable method directly (for example, locally, such as pass through injection, be implanted into or locally apply to tissue site) Or systematically (for example, parenteral or oral) is supplied to animal.In the case where parenteral provides composition, such as, by quiet In arteries and veins, in subcutaneous, eye, peritonaeum, (intraorbital) in intramuscular, buccal, rectal, vagina, eye socket, big intracerebral, in head, In intraspinal, intra-ventricle, intrathecal, brain pond, it is intracapsular, intranasal or applied by aerosol, composition preferably includes some aqueous phase Or PHYSIOLOGICALLY COMPATIBLE liquid suspension or solution.Therefore, carrier (carrier or vehicle) is physiologically acceptable, with Just other than desired composition is fed to patient, the electrolyte and/or volume of patient will not additionally be adversely affected Balance.Therefore, the liquid medium of medicament may include physiological saline.

DNA construction (or gene constructs) of the invention is also used as a part of gene therapy method, with delivering Encode polypeptide drugs or the nucleic acid of its fusion protein construction object.

The invention is characterized in that expression vector, is used in specific cell type transfect and express in vivo interested Polypeptide drugs or its fusion protein construction object, to recombinate or supplement the function of desired polypeptide drugs.Desired peptide drug The expression construct of object or its fusion protein construction object can be with arbitrarily biologically effective vector administrations, such as can be In vivo effectively by the gene for encoding desired polypeptide drugs or its fusion protein construction object be delivered to cell any preparation or Composition.

Method, which is included in viral vectors, is inserted into target gene, the carrier include recombinant retrovirus, adenovirus, Gland-correlated virus and herpes simplex virus -1 or recombinant bacteria or eucaryon plasmid.For people, application coding is of the invention every time The preferred dose of the nucleic acid of fusion protein is within the scope of 0.1mg-100mg, more preferable 1mg-10mg, and most preferably 2mg- 10mg.It can be understood that the optimal dose and mode of application can pass through the well known routine experiment in art technology level It determines.

For people, the preferred dose for the fusion protein applied every time is within the scope of 0.1mg-1,000mg, more preferable 1mg- 100mg, and most preferably 5mg-20mg.It will be understood that optimal dose additionally depends on treated disease and pair The presence of effect.But optimal dose can be used routine experiment and determine.Application fusion protein can pass through regular bolus (periodic bolus injections), or by from exterior reservoir (anexternal reservoir) (for example, from Venous transfusion packet) or internal (for example, from biological erodable implantation material) continuous intravenous, interior application of subcutaneous or peritonaeum.

Furthermore, it is to be understood that fusion protein of the invention can also be applied together with a variety of different bioactive molecules To the recipient of needs.It is understood, however, that the optimal combination of fusion protein and other molecules, method of application, dosage can be with Pass through routine experiment determination well known in art technology level.

Invention mode

By following non-limiting examples, invention is further explained.

<embodiment 1>prepares the expression vector of hFc-1, hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 fusion protein.

HFc-1 includes hinge area (99-113), the IgG2 of 9 amino acid (90-98) at the area the IgG1 CH1 end C-, IgG1 6 amino acid (111-116) at the area the CH2 end N-, 103 amino acid (118-220) in the area IgG4 CH2 and the area IgG4 CH3 107 amino acid (221-327) (Fig. 1 and 2).The amino acid sequence of hFc-1 is shown in SEQ ID NO:18.In order to obtain The nucleotide of codon optimization --- the nucleotide is each separately encoded hFc-1 (SEQ ID NO:1), people EPO (SEQ ID NO:7), human G-CSF (SEQ ID NO:8) and people p40N303Q (are originated from the 303rd amino acids Gln displacement Asn of people p40 subunit Mutant) (nucleotide sequence of p40N303Q is shown as SEQ ID NO:9 and the amino acid sequence of people p40 is shown as SEQ ID NO:17), pass through TOP Gene Technologies's (Quebec, Canada) (www.topgenetech.com) Customization service (custom service) synthesizes these nucleic acid molecules.In order to increase protein expression level, optimization gene Codon use is very helpful.The mode that codon uses is different between organism.Some codons are in a kind of life It is used more frequently in object, and is rarely used in another organism.The preference that this codon uses has been returned Because in translation efficiency, the ability of organism composite coding albumen.In order to which each fusion is inserted into expression vector pAD11 (SEQ ID NO:10) generates EcoR I site and in hFc-1 at 5 ' ends of the ATG sequence of EPO, G-CSF and p40N303Q Terminator codon 3 ' end generate Xba I sites.Expression vector pAD11 from RcCMV skeleton (can from Invitrogen, Carlsbad is obtained) it obtains.PAD 11 includes the promoter for being originated from cytomegalovirus (cytomegalovirus, CMV), is originated from Poly (A) sequence of bovine growth hormone, the globulin from rabbit beta Globulin (Mol Cell Biol, 1988 8:4395) interleave Sequence (globin intervening sequence, gIVS) etc..In order to prepare pAD11 carrier, there are several RcCMV carriers (Invitrogen) modification.By removing neomycin resistance area with Xho I enzymatic treatment and adding at the 3 ' places in CMV promoter area Add gIVS.In addition, 5 ' places of CMV promoter add small mouse dihydrofolate reductases (dihydrofolate reductase, DHFR) gene (Pubmed, NM 010049).Combine with several including these above-mentioned element, is formed after many expression are tested PAD11 carrier.In our undocumented results, pAD11 carrier is shown in table compared with RcCMV carrier (Invitrogen) About 12 times of increase on up to level.In order to generate 3 ' ends and the hFc- of EPO, G-CSF and p40N303Q in reading frame (frame) Connection site between 15 ' ends, at 3 ' ends of EPO, G-CSF and p40N303Q coded sequence and in hFc-1 code sequence Nhe I site is produced at 5 ' ends of column.After being subcloned using each restriction enzyme sites, produce and EPO, G-CSF Or the final expression vector of the hFc-1 of p40N303Q fusion, and then it is respectively designated as pAD11 EPO-hFc-1, pAD11 G-CSF-hFc-1 and pAD11 p40N303Q-hFc-1.

The amino acid sequence of hFc-2, hFc-3, hFc-4, hFc-5 and hFc-6 are respectively displayed on SEQ ID NOs:19-23 In.HFc-6 includes 9 amino acid (90-98) of the IgD CH1 structural domain at the end C-, 64 amino acid (99- of IgD hinge area 162), 100 ammonia of 8 amino acid (shtqplgv 163-170) of the IgD CH2 structural domain at the end N-, IgG4 CH2 structural domain 107 amino acid (221-327) (Fig. 1 and 2) of base acid (121-220) and IgG4 CH3 structural domain.It is excellent in order to obtain codon The nucleic acid molecule of the coding hFc-6 (SEQ ID NO:6) of change, passes through TOP Gene Technologies (www.topgenetech.com) customization service synthesis gene.In order to prepare EPO, G-CSF or p40N303Q in reading frame 3 ' ends and hFc-6 5 ' hold between fusion, using including in the end the N- code area (90 and 91 amino acid) of hFc-6 Nhe I site (gctagc:Ala-Ser).In addition, in order to which each hFc-6 fusion is inserted into 11 carrier of pAD, in hFc-6 Xba I site is produced at 3 ' ends of gene.After being subcloned using each restriction enzyme sites, fusion has EPO, G-CSF PAD11EPO-hFc-6, pAD11 G- are generated and are then respectively designated as with the final expression vector of the hFc-6 of p40N303Q CSF-hFc-6 and pAD11 p40N303Q-hFc-6.The area CH2 and CH3 having the same hFc-2, hFc-3, hFc-4 and hFc-5, But they have different size of IgD hinge (Fig. 1 and 2).HFc-2 (SEQ ID NO:19), hFc-3 (SEQ ID NO:20), HFc-4 (SEQ ID NO:21) and hFc-5 (SEQ ID NO:22) respectively includes 5 amino acid (158- of the end C- IgD hinge 162), 10 amino acid (153-162), 20 amino acid (143-162), 30 amino acid (133-162) (Fig. 1 and 2).In order to Preparation is in EPO, G-CSF, p40N303Q or TNFR (tumor necrosis factor receptor I I) (SEQ ID NO:30) and encodes these Fusion between the nucleic acid molecules of hFcs (SEQ ID NOs:2-5), passes through TOP Gene Technologies (www.topgenetech.com) customization service is synthesized the smallest genetic fragment in the total size of fusion.With Encode each EPO, G- of the hinge of each hFc-2, hFc-3, hFc-4 or hFc-5 and the nucleic acid molecule fusion in the end the N- area CH2 The synthesis segment of CSF, p40N303Q or TNFR include from whole EPO, G-CSF, p40N303Q or TNFR sequences to identical enzyme Site --- it is located at the site BstE II at IgG4 (SEQ ID NO:13) Zhong CH2 area 138-140 amino acids residue (GGTGACC) --- the sequence of range.Subcloning vector comprising several genetic fragments, which is used, to be located at 5 ' ends and 3 ' ends EcoR I and BstE II cutting, and it is then attached to the area CH2-CH3 of hFc-6.Finally, using EcoR I and Xba I site Each fusion is subcloned into pAD11, and is then respectively designated as 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 and pAD11 TNFR-hFc-5.

<embodiment 2>prepares thFc-1 the and thFc-2 expression vector for being coupled to IFN-b

ThFc-1 includes the letter of human tissue plasmin activator (tissue plasminogen activator, tPA) 23 amino acid (MDAMLRGLCCVLLLCGAVFVSPS) of number sequence, 15 amino acid (99-113) of IgG1 hinge area, N- Hold 6 amino acid (111-116) in the area IgG2 CH2,103 amino acid (118-220) in the area IgG4 CH2 and IgG4 CH3 107 amino acid (221-327) (Fig. 3) in area.The amino acid sequence of thFc-1 is shown in SEQ ID NO:28.ThFc-2 packet Include 23 amino acid (MDAMLRGLCCVLLLCGAVFVSPS) of tPA signal sequence, 15 amino acid (148- of IgD hinge area 162), 8 amino acid (163-170) in the end the N- area IgD CH2,100 amino acid (121-220) in the area IgG4 CH2 and 107 amino acid (221-327) (Fig. 3) in the area IgG4 CH3.The amino acid sequence of thFc-2 is shown in SEQ ID NO:29. In order to obtain the nucleotide of codon optimization --- the nucleotide coding is coupled to the people's IFN-β for being deleted its signal sequence The thFc-1 (SEQ ID NO:26) or thFc-2 (SEQ ID NO:27) at the end N-, pass through TOP Gene Technologies The customization service of (Quebec, Canada) (www.topgenetech.com) synthesizes these nucleic acid molecules.For by each fusion Gene is inserted into expression vector pAD11 (SEQ ID NO:10), thFc-1 or thFc-2 5 ' end generate EcoR I sites and Not I site is generated at 3 ' ends of IFN-β terminator codon.After being subcloned using each restriction enzyme sites, final table PAD11 thFc-1-AL (0)-IFN-β and pAD11 thFc-2-AL (0)-IFN-β are respectively designated as up to carrier.

In order to prepare the thFc for being coupled to IFN-β via different size of albumin connector or Gly-Ser connector, lead to Cross customization service synthesis CH3 area of the range from thFc-1 of TOP Gene Technologies (www.topgenetech.com) The genetic fragment of Pst I site, the thFc-1 via different albumin connector (3aa, 8aa, 13aa, 18aa, 23aa and 33aa) or Gly-Ser connector (15aa) is coupled to the IFN-β (Fig. 4) for being deleted its signal sequence.For by 7 kinds of different sizes Genetic fragment be inserted into expression vector pAD11 thFc-1-AL (0)-IFN-β and pAD11 thFc-2-AL (0)-IFN-β, In Pst I site is generated at their 5 ' ends and Not I site is generated at 3 ' ends of IFN-β terminator codon.Using every After a restriction enzyme sites are subcloned, final expression vector is respectively 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, pAD11thFc-1-GS-IFN- β and 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 pAD11 and thFc-2-GS-IFN-β。

<embodiment 3>people EPO-hFcs, human G-CSF-hFcs, people p40N303Q-hFcs, people TNFR-hFc-5 and thFcs- The expression of IFN-β albumen

COS-7 cell be used to express test and with added with 10% fetal calf serum (Hyclone, South Logan) It is cultivated with the DMEM culture medium (Invitrogen, Carlsbad) of antibiotic (Invitrogen, Carlsbad).Using traditional Electroporation will encode the carrier of EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5, thFcs-IFN- β It is transfected into 5 × 10⁶COS-7 cell.After transfection when 48h, supernatant and cell are harvested.For the fusion protein for detecting each carrier Expression, all samples pass through several kits (R&D system, Minneapolis, #DEP00, be used for EPO；Biosource, Camarillo, #KHC2032 are used for G-CSF；R&D system, Minneapolis, #DY1240 are used for p40N303Q；R&D system, Minneapolis, #DRT200 are used for TNFR, and PBL Biomedical Laboratories, #41410-1A are used for IFN-β) it uses It is measured in ELISA and protein is used for by Anti-Human's IgG antibody (Santa Cruz Biotechnology, Santa Cruz) Engram analysis.As a result, all carriers show correct expression pattern (not showing data) in supernatant and cell pyrolysis liquid.

The purifying of<embodiment 4>hFc- fusion protein

With a-MEM (Invitrogen, Carlsbad), 10% dialysis fetal calf serum (JRH Biosciences, Kansas), HT tonic (HT supplement) (Invitrogen, Carlsbad) and antibiotic (Invitrogen, Carlsbad CHO/DHFR) is cultivated^-/-Cell (Chinese hamster ovary cell, DG44, ATCC).According to traditional CaPO₄Co-precipitation Expression vector is transfected into Chinese hamster ovary celI by method.Chinese hamster ovary celI is separated from plate and dilutes (1/2,1/ at double by 4gh after transfection 5,1/20,1/50,1/100,1/200,1/500).By diluted plating cells to 100mm culture dish and with no HT tonic Culture medium is cultivated.In screening process, the fresh culture without HT tonic is supplied to the cell not passed on.Flat 2-3 weeks generation colony and single colony is transferred to 48 orifice plates after paving.It is detected for EPO, G-CSF, p40N303Q and TNFR ELISA measurement after, screen positive colonies.It will show that most highly expressed each colony uses the culture medium (JRH without serum Biosciences, Kansas) carry out extensive (5L) culture.The supernatant without serum of harvest is used for every kind of fusion egg White purifying.In order to purify, HiTrap recombinant protein A FF (Amersham is balanced with 20mM sodium phosphate (pH 7.0) Biosciences, Piscataway) column.The supernatant of filtering is added to the column and with 0.1M sodium citrate (pH 3.0) it elutes.After with film (MWCO 12 14K, Spectrapor, Rancho Dominguez) dialysis more than three times, finally Obtain the albumen of elution.The concentration of all proteins sample passes through the BCA kit (Pierce for measuring gross protein Biotechnology, Rockford) and for measuring EPO-hFcs, G-CSF-hFcs, p40N303Q-hFcs, TNFR-hFc-5 It is measured with the ELISA kit of thFcs-IFN- β.

<embodiment 5>FcgRI and C1q binding assay

FcgRI and C1q whether are integrated in order to study hFc-5 fusion protein, MabThera (Rituximab, Roche), HIgG1 (Calbiochem, Cat#, 400120),(etanercept, Amgen), EPO-hFc-5, G-CSF-hFc- 5 and p40N303Q-hFc-5 is by serial dilution (with 2 times from 2ug/ml to 16ng/ml) and is coated on 8 holes and takes (COSTAR, New York) 4 spends night (overnight at 4).To make standard curve, FcgRI (R&D, cat# BAF1257) or C1q (AbD serotech, Cat#.2221-5504) is also by serial dilution (with 2 times from 2ug/ml to 32ng/ Ml it 8 holes) is coated on takes (COSTAR, New York) 4 and spend night.At room temperature with cleaning solution (containing 0.05% tween PBS) each band of washing sample and in PBS 10%FBS close 1 hour after, FcgRI or C1q are added to 2ug/ml Each hole, then (room temprature, RT) is incubated 2 hours at room temperature.All bands are washed with cleaning solution.To carry out C1q In conjunction with test, the HRP anti-C1q (AbD serotech, cat#.2221-5004P) combined is added to often with 2.5ug/ml Then a hole incubates 30min under dark condition at room temperature.Test is combined to carry out FcgRI, by biotinylated anti-FcgRI (R&D, cat#.1257-FC) is added to each hole with 2ug/ml, then incubates 1 hour at room temperature.Them are being washed with cleaning solution Afterwards, 3,000 times of diluted Streptavidine-HRP (BD, cat#.554066) are added to each band, then in dark item It is incubated at room temperature under part 30 minutes.After washing band, TMB solution (TMB peroxide substrate and peroxide substrate solution are added 1: 1 mixture of B, KPL, cat#.50-76-01, cat#, 50-65-00) for developing the color and adding 2N H₂SO₄For stopping Colour developing.As shown in Fig. 6 (a) and Fig. 6 (b),Shown with hIgG1 be well bonded to FcgRI and C1q, but EPO-hFc-5, G-CSF-hFc-5 and p40N303Q-hFc-5 do not have.

The Bioactivity of the hFc fusion protein of<embodiment 6>purifying

For the Bioactivity for studying EPO-hFc albumen, it is being supplemented with 10%FBS, antibiotic and 5IU/ml recombined human (Cambrex, Charles City) cultivates people F35E in the RPMI1640 culture medium of EPO (DongA, Republic ofKorea) Cell line.By by 2 × 10⁴A cell inoculation is come to the measurement hole of 96 porocyte culture plates (Corning, Netherlands) Carry out bioassay.By EPO, EPO-hFc-1, EPO-hFc-5, EPO-hFc-6, EPO-IgG1 Fc or Aranesp The serial dilution of (darbepoetin alfa, Amgen) (is added to 5 times from 0,0.064mIU/ml to the sample of 25IU/ml) This some holes and by plate in 5% wet CO₂It is incubated 72 hours at 37 DEG C in incubator.According to the scheme of manufacturer, pass through MTT measurement is carried out using cell growth colorimetric assay kit (Sigma-Aldrich.Korea).People's F35E cell line is to rEPO Strong proliferation response is shown, as proved in cell number and absorption value middle dosage dependence mode.As shown in Fig. 7 (a), coupling To IgG1 Fc or hFcsLoss of biological activity is shown compared with epo protein with epo protein.But EPO- HFc-1, EPO-hFc-5 and EPO-hFc-6 show the bioactivity for being apparently higher than EPO-IgG1 Fc.In addition, EPO-hFc-5 and EPO-hFc-6, which is shown, to be slightly higher thanBioactivity, this shows that these hFc fusion proteins are maintaining epo protein Seem to be better than in terms of bioactivity

In order to study the Bioactivity of G-CSF-hFc albumen, it is being added with 10%FBS, antibiotic and 100units/ The RPMI1640 culture medium (Cambrex, Charles City) of ml recombined small-mouse IL-3 (R&D system, Minneapolis) Middle culture mouse hematopoetic cell system, NFS-60.By by 2 × 10⁴Cell inoculation to 96 porocyte culture plates (Corning, Netherlands bioassay is established in hole).By the company of G-CSF-hFc-5 and Neulasta (pegfilgrastim, Amgen) The sample of continuous dilution (with 3 times from 0 to 10,000pg/ml) is added to this some holes and by plate in wet 5%CO₂In incubator It is incubated 72 hours at 37 DEG C.Protein sample is measured in three repeating holes and repeats the experiment 5 times.72 is small after incubation When, according to the method for manufacturer, carried out by using cell growth colorimetric assay kit (Sigma-Aldrich.Korea) MTT measurement.As illustrated in Fig. 7 (b), G-CSF-hFc-5, which is shown, to be slightly higher thanBioactivity.

For the Bioactivity for studying p40N303Q-hFc albumen, added with 10%FBS and antibiotic In RPMI1640 culture medium (Cambrex, Charles City), with Anti-Human CD3 antibody (the R&D system, # of 2ug/ml MAB100) and rheumatic arthritis is incubated with or without the people p40 of 10ng/ml (R&D system) or p40N303Q-hFc-5 to suffer from The peripheral blood mononuclear cells (peripheral blood mononuclear cells, PBMCs) of person.After 6 days, pass through FACS Analysis measures the cell to CD4 and the IL-17 positive.As shown in Fig. 7 (c), for CD4⁺/IL-17⁺The generation of cell, P40N303Q-hFc-5 shows the depression effect for being better than p40 albumen, this shows p40N303Q-hFc-5 to the polarized inhibition of Th17 Function.

In order to study the Bioactivity of TNFR-hFc albumen, in the RPMI1640 for being added with 10%FBS and antibiotic Mouse L929 cell is cultivated in culture medium (Cambrex, Charles City).By by 3 × 10⁴A cell inoculation is to 96 hole cells Cytopathogenic effect is carried out in the hole of culture plate (Corning, Netherlands) and inhibits measurement, then uses the TNF-a of 1ng/ml It is handled.By TNFR-hFc-5 and(etanercept, Amgen) serial dilution (with 2 times from 15.6 to 1, Sample 000ng/ml) is added to this some holes and by plate in wet 5%CO₂It is cultivated 48 hours in incubator at 37 DEG C.In After incubation, according to the method for manufacturer, colorimetric assay kit (Sigma-Aldrich., Korea) is grown by using cell Carry out MTT measurement.As illustrated in Fig. 7 (d), TNFR-hFc-5, which is shown, to be slightly higher thanBioactivity.

In order to study thFc-1-AL (0)-IFN-β and thFc-1-AL (3)-IFN-β albumen Bioactivity, adding Added with culture WISH cell (ATCC, CCL- in the DMEM/F12 (Cambrex, Charles City) of 10%FBS and antibiotic 25).By by 3 × 10⁴A cell inoculation carries out causing to the hole of 96 porocyte culture plates (Corning, Netherlands) thin Born of the same parents' lesion inhibits measurement, is then handled with the VSV (ATCC, VR-158) in the hole 1,500PFU/.It will recombination IFN-β (WHO mark Standard, NIBSC 00/572), thFc-1-AL (0)-IFN-β and the serial dilution of thFc-1-AL (3)-IFN-β albumen (with 2 times from Sample 40IU/m1) is added to this some holes and by plate in wet 5%CO₂It is incubated 48 hours in incubator at 37 DEG C.In temperature After educating, by the method according to manufacturer, carried out using cell growth colorimetric assay kit (Sigma-Aldrich.Korea) MTT measurement.As illustrated in Fig. 7 (e), thFc-1-AL (3)-IFN-β is shown higher than about 20 times of thFc-1-AL (0)-IFN-β Bioactivity, this shows that albumin analog (albumin liker) is fused to the bioactivity of the IFN-β of Fc to maintenance Important function.

The Half-life in vivo of the hFc fusion protein of<embodiment 7>purifying

In order to compare the half-life period of EPO-hFc-1, EPO-hFc-5 and Aranesp, with these albumen with 2,400IU/kg's Dosage is injected via single SC (SC) or single dose intravenous (IV) injection handles 15 macaques.Before the injection and after injection 1, the blood sample of every monkey is obtained within 3,6,12,24,30,48,54,72,78,96,120,168,336,504 and 672 hours. Blood sample is incubated into 30min at room temperature, so that it is condensed.After being centrifuged 10min with 3000rpm, each sample is obtained Serum and it is stored in deepfreezer.By it is each point obtain all samples by EPO ELISA kit (R&D, Cat#.DEP00 the quantitative determination of EPO) is carried out.As shown in Fig. 8 (a), EPO-hFc-1 or EPO- is injected via SC or IV approach All monkey individuals display of hFc-5 via SC or IV approach than injectingMonkey individual longer half-life period.

In order to study the pharmacokinetics of G-CSF-hFc-1, by the LEUCOSTIM of 100ug/kg (filgrastim, DongA, Republic of Korea) it is administered to often as control and the G-CSF-hFc-1 of 100ug/kg via SC or IV approach 2 male Sprague Dawley rats (Charles River Laboratories, Wilmington) of group.Before the injection and 1 after injection, 2,3,4,8,12,24,48,72,96,120 and 192 hours acquisition blood.After incubating 30min at room temperature, pass through Serum is obtained with 3,000rpm centrifugation 10min, and is stored in deepfreezer.Using G-CSF kit (Biosource, Camarillo, #KHC2032) it is quantified with such as 1/2,1/5,1/50,1/250,1/500 pair of sample of several extension rates. As shown in Fig. 8 (b), ratio is shown via the G-CSF-hFc-1 of SC or IV approach injectionIt is longer partly to decline Phase, after SC application, G-CSF-hFc-1 and G-CSF are respectively provided with the internal t of 8.76h and 2.36h_1/2, and after IV application, It is respectively provided with the internal t of 10.42h and 1.78h_1/2.Therefore, withIt compares, G-CSF-hFc-1 is shown in SC 3.7 times are improved after injection and improve 5.9 times after IV injection.

In order to study p40N303Q-hFc-5 andPharmacokinetics, with the dosage of 100ug/kg single SC Injection handles every group of 3 macaques.Before the injection with after injection 8,24,48,72,96,120,168,336,504 and 672 Hour obtains the blood sample of every monkey.Blood sample is incubated into 30min at room temperature, so that it is condensed.With 3000rpm from After heart 10min, obtains the serum of each sample and be stored in deepfreezer.Pass through ELISA kit (respectively R&D System, Minneapolis, #DY1240 and #DRT200), people p40 and people are carried out to all samples obtained in each point The quantitative test of TNFR II.As shown in Fig. 8 (c), p40N303Q-hFc-5 shows ratioLong half-life period (average value For 199h ratio 127h) although --- p40N303Q-hFc-5 show ratio(average value is 3ng/ml ratio to low cmax value 7ng/ml)。

In order to study TNFR-hFc-5 andPharmacokinetics, with the dosage of 500ug/kg with single SC inject At every group of 3 male Sprague Dawley rats (Charles River Laboratories, Wilmington) Reason.Before the injection with 2 after injection, 4,8,12,24,30,48,72 and 120 hours obtain every rat blood sample.By blood Sample incubates 30min at room temperature, so that it is condensed.After being centrifuged 10min with 3,000rpm, the serum of each sample is obtained simultaneously And it is stored in deepfreezer.By ELISA kit (R&D system, Minneapolis, #DRT200) to all every The sample that a point obtains carries out the quantitative test of people TNFR II.As shown in Fig. 8 (d), TNFR-hFc-5, which is shown, to be slightly higher thanAUC horizontal (average value is 198.1 than 172.9ug*h/ml) although --- TNFR-hFc-5 show and Similar half-life period (average value is 28.6h ratio 29.4h).

The in vivo bioactivity of the hFc fusion protein of<embodiment 8>purifying

In order to compare EPO-hFc-5 andIn vivo bioactivity, with the dosage of 2,400IU/kg single IV Injection handles every group of 3 macaques.Before the injection with 1 after injection, 3,6,12,24,30,48,54,72,78,96,120, 168, the blood sample of every monkey is obtained within 336,504 and 672 hours.Measure the various haemocytes including desmacyte Quantity, with evaluate EPO-hFc-5 andIn vivo bioactivity.It is netted thin increasing monkey as shown in Fig. 9 (a) In terms of born of the same parents in SC and IV approach, EPO-hFc-5, which is shown, to be slightly higher thanExternal titer.

It, will in order to study the in vivo bioactivity of G-CSF-hFc-1(filgrastim, DongA, Republic 0fKorea) it is administered to often with the dosage of 100ug/kg via SC or IV approach as control and G-CSF-hFc-1 Two male Sprague Dawley rats (Charles River Laboratories, Wilmington) of group.Before the injection With 1 after injection, 2,3,4,8,12,24,48,72,96,120 and 192 hours, use EDTA pipe obtain blood.By each blood sample Product, which are handled 4 minutes with RBC lysate (BD Bioscience, Korea) and will be diluted in FACS using hemacytometer, to be delayed Whole WBC (leucocyte) repeat count in fliud flushing is three times.Using FACS instrument by utilizing FSC (forward scattering, forward Scatter it) measures cell size and measures granulocyte number using SSC (lateral scattering, side scatter) measurement particle.Such as Shown in Fig. 9 (b), handled via SC and IV approachCause WBC and granulocyte within 24 hours after injection Peak value, and G-CFS-hFc-1 causes the peak value of WBC and granulocyte for 48 hours after injection in 72 hours and IV after SC injection Number.For 24 hours to 120h after injection, withIt compares, G-CSF-hFc-1 has more lasting vivo biodistribution living Property.

Although be specifically shown and described to the present invention by reference to its illustrative embodiment, It should be appreciated by those skilled in the art that formal and detailed a variety of changes can be wherein carried out, without departing from following patent The spirit and scope of the present invention defined by it is required that.

Industrial applicability

The invention discloses include bioactive molecule and immunoglobulin (Ig) the Fc knot for being connected to bioactive molecule The fusion protein in structure domain.The Fc structural domain is hybridization people's Fc structure of (i) IgG1, IgG2 or IgG4 or (ii) IgG4 and IgD Domain.Hybrid Fc is used as the carrier of bioactive molecule.

Claims

1. chimeric polyeptides, including

Hybrid Fc polypeptide；With

It is coupled to the EPO of the hybrid Fc polypeptide, variant or segment,

Wherein the hybrid Fc polypeptide is expressed from the next:

N'-(Z1)_p-Y-Z2-Z3-Z4-C'

Wherein N' be the hybrid Fc polypeptide the end N- and C' be the hybrid Fc polypeptide the end C-；

The amino acid sequence that Z1 is made of the amino acid residue of 90 to 98 positions of SEQ ID NO:14；

The amino acid sequence that Y is made of the amino acid residue of 133 to 162 positions of SEQ ID NO:14；

The amino acid sequence that Z2 is made of the amino acid residue of 163 to 170 positions of SEQ ID NO:14；

The amino acid sequence that Z3 is made of the continuous amino acid residue of 121 to 220 positions of SEQ ID NO:13；

The amino acid sequence that Z4 is made of the continuous amino acid sequence of the amino acid residue 221 to 327 of SEQ ID NO:13；With And

P is 0 or 1 integer,

Wherein the EPO, its variant or segment are fused to the end N- or the end C- of the hybrid Fc polypeptide, and are wherein fused to The EPO of the hybrid Fc polypeptide, variant or segment show and are not fused to the EPO of hybrid Fc polypeptide, its change Body or the circulating half-life of segment compare increased circulating half-life, and keep EPO active.

2. chimeric polyeptides described in claim 1, wherein the hybrid Fc polypeptide is encoded by nucleotide sequence SEQ ID NO:5.

3. chimeric polyeptides described in claim 1, wherein amino acid sequence table of the hybrid Fc polypeptide by SEQ ID NO:22 Show.

4. chimeric polyeptides described in claim 1, wherein p is 0.

5. chimeric polyeptides described in claim 1, wherein the hybrid Fc polypeptide and the EPO, variant or segment are via white Albumen connector is coupled each other.

6. chimeric polyeptides described in claim 5, wherein the albumin connector includes the amino acid sequence of SEQ ID NO:25 Column 316 to 328,313 to 330,311 to 333 or 306 to 338.

7. encoding the nucleic acid molecules of polypeptide described in claim 1.

8. including the expression vector of nucleic acid molecules according to claim 7.

9. the method for generating chimeric polyeptides described in claim 1, wherein the method includes the following steps: (i) weighs coding Benefit requires the nucleic acid molecules of 1 chimeric polyeptides to introduce mammalian host cell, and (ii) exists the cell in the medium The polypeptide is grown under conditions of being expressed；And (iii) from expressed chimeric more of the cell or culture medium harvest Peptide.

10. chimeric polyeptides according to claim 1 are preparing the medicine for increasing the circulating half-life of biologically active polypeptide Purposes in object.

11. purposes according to claim 10, wherein the chimeric polyeptides through it is intravenous, subcutaneous, oral, buccal, sublingual, Intranasal, parenteral, rectum, vagina are applied via pulmonary route.