CN104974262B

CN104974262B - Recombination double functions fusion protein and its preparation method and purposes

Info

Publication number: CN104974262B
Application number: CN201410136962.XA
Authority: CN
Inventors: 田文志
Original assignee: Bio Pharmaceutical Technology (shanghai) Co Ltd
Current assignee: Bio Pharmaceutical Technology (shanghai) Co Ltd
Priority date: 2014-04-04
Filing date: 2014-04-04
Publication date: 2019-08-09
Anticipated expiration: 2034-04-04
Also published as: CN104974262A; WO2015149708A1

Abstract

The present invention relates to recombination double functions fusion protein and its preparation methods and purposes.Specifically, the invention discloses a kind of fusion proteins, and there is the element B of the second film outskirt D2 of element A, VEGFR1 including TGF-β receptor membrane outskirt and immunoglobulin element C, three to be cascaded.The albumen can simultaneously with 1 two kinds of ligand bindings of VEGF and TGF-β, and have it is preferable combine activity, to inhibit the biological activity of ligand.The present invention also provides effect of the recombination double functions fusion protein in disease treatment.

Description

Recombination double functions fusion protein and its preparation method and purposes

Technical field

The present invention relates to biomedicine fields.More particularly it relates to a kind of recombination double functions fusion protein and its Preparation method and purposes.

Background technique

The diseases such as tumour, liver fibrosis seize the life of several million peoples every year, cause the economic loss of multi-billion dollar.Cell The TGF-β of film surface and the combination of VEGFR receptor and respective ligand are a kind of major reasons that such disease generates and develops, Therefore corresponding protein ligands drug is developed to have a good application prospect.

Currently known albumen medicine include growth factor, hormone albuminoid, enzyme albumen, cell factor, interferon, promote it is red Erythropoietin and fusion protein etc..In addition to fusion protein, other albumen medicines belong to homogeneous protein, that is, contain only one Kind protein ingredient.And existing fusion protein medicine (such as Arastin Avastin, VEGF Trap Aflibercept) is by receptor egg White film outskirt is merged with Fc sections of human IgG, although containing two or more protein ingredient, but still only plays one Kind function, i.e., can only block the combination of a kind of cell membrane endogenous receptor and respective ligand.

Although genetic recombination bifunctional fusion proteins are it has been reported that but it is formed and corresponding target spot is different.It is so far The difunctional of biological activity that only the two target spots can be blocked to be induced in conjunction with TGF-β 1 and VEGF and simultaneously simultaneously is melted Hop protein drug does not have been reported that.Therefore, such bifunctional fusion proteins drug is developed to life span, the improvement trouble for extending patient The life quality of person, the reduction death rate are of great significance.

Summary of the invention

The purpose of the present invention is to provide a kind of recombination double functions fusion protein and its preparation methods and purposes.

The first aspect of the present invention, provides a kind of fusion protein, the fusion protein include be fused together it is following Element:

(i) the optional signal peptide positioned at N-terminal；

(ii) the first protein element；

(iii) the second protein component；And

(iv) the immunoglobulin element being connect with the first protein element and/or the second protein component,

Wherein, the signal peptide is operably coupled to the fusion element being made of (ii), (iii) and (iv)；

And the first protein element is TGF-β receptor membrane outskirt protein component；Second protein component be include blood vessel endothelium The protein component of growth factor acceptor VEGFR1 the second film outskirt D2.

In another preferred example, " being operably coupled to " refers to that the table of the fusion element may be guided in the signal peptide It reaches or transmembrane process (positioning).

In another preferred example, the fusion protein has structure selected from the group below:

(1) structure described in Formulas I a or Formulas I b:

D-A-B-C (Ia), or

D-B-A-C (Ib)

(2) structure described in Formula II a or Formula II b:

D-A-C-B (II a), or

D-B-C-A (II b)

Wherein,

A is TGF-β receptor membrane outskirt protein component；

B is the protein component for including vascular endothelial growth factor receptor VEGFR1 the second film outskirt D2；

C is immunoglobulin element；

D is optional signal peptide sequence；

"-" indicates the peptide bond or peptide linker of connection said elements.

In another preferred example, the element A is selected from the group: T β RI, T β RII, T β RIII.

In another preferred example, the element A is the protein component of T β RII film outskirt.

In another preferred example, the element A has amino acid sequence shown in 24-184 in SEQ ID NO.:1.

In another preferred example, the element B has amino acid sequence shown in 190-282 in SEQ ID NO.:1 (core sequence) and length are 94,95,96,97,98,99 or 100 amino acid.

In another preferred example, amino acid sequence shown in 190-282 in SEQ ID NO.:1 is located in the element B The amino acid sequence of column (core sequence) two sides is respectively from the second film outskirt D2 (Domain 2) two of natural VE GFR1 Side amino acid sequence.

In another preferred example, the D2 has flanking sequence, and the flanking sequence includes:

Positioned at the first flanking sequence of D2 aminoterminal；And/or the second flanking sequence positioned at D2 c-terminus.

In another preferred example, first flanking sequence is made of 1-5 amino acid residue.

In another preferred example, second flanking sequence is made of 1-2 amino acid residue.

In another preferred example, second film outskirt of the first and second flank sequences respectively from natural VE GFR1 D2 (190-282 in SEQ ID NO.:1) two sides amino acid sequence.

In another preferred example, first flanking sequence is SDTGR.

In another preferred example, second flanking sequence is NT.

In another preferred example, the element C is the Fc segment of people's Immunoglobulin IgG1.

In another preferred example, the length of the peptide linker is 0-10 amino acid, preferably 0-5 amino acid.

In another preferred example, the fusion protein further includes signal peptide element D.

In another preferred example, the amino acid sequence of the signal peptide element D such as 1-23 institutes in SEQ ID NO:1 Show.

In another preferred example, the amino acid sequence of the fusion protein is as shown in SEQ ID NO:1.

In another preferred example, the fusion protein is free of signal peptide, and structural formula is selected from the group:

(1) Formulas I a ' or the Formulas I b ' structure:

A-B-C (Ia '), or

B-A-C (Ib′)

(2) Formula II a ' or the Formula II b ' structure:

A-C-B (II a '), or

B-C-A (II b′)

In formula, A, B, C and "-" are as defined above.

In another preferred example, the fusion protein has following multiple functions:

And the combination activity EC of VEGF a)₅₀For 0.6-2nM；

And the combination activity EC of TGF-β 1 b)₅₀For 1.5-2.5nM；

C) can simultaneously with 1 two kinds of ligand bindings of VEGF and TGF-β；

D) can blocking VEGF induction vascularization in vitro or in vivo；

E) migration and invasion of the tumour cell that TGF-β 1 is induced be can inhibit.

The second aspect of the present invention, provides a kind of albumen dimer, and the dimer is by two according to the present invention Any fusion protein of one side is constituted.

In another preferred example, the dimer has structure selected from the group below:

(1) structure described in Formulas I a-1 or Formulas I b-1:

(2) structure described in Formula II a-1 or Formula II b-1:

Wherein,

A is TGF-β receptor membrane outskirt protein component；

B is the protein component for including VEGFR the second film outskirt D2；

C is immunoglobulin element；

D is optional signal peptide sequence；

"-" indicates the peptide bond or peptide linker of connection said elements；

" | | " indicate disulfide bond.

In another preferred example, the fusion protein is free of signal peptide, and has structure selected from the group below:

(1) Formulas I a-1 ' or the Formulas I b-1 ' structure:

(2) Formula II a-1 ' or the Formula II b-1 ' structure:

In formula, A, B, C, "-" and " | | " it is as defined above.

The third aspect of the present invention provides a kind of isolated polynucleotides, and the polynucleotide encoding is according to this hair The fusion protein of bright first aspect.

The fourth aspect of the present invention provides a kind of carrier, it contains polynucleotides described in third aspect present invention.

The fifth aspect of the present invention provides a kind of host cell, it contain carrier described in fourth aspect present invention or Polynucleotides described described in third aspect present invention are integrated in genome.

In another preferred example, the host cell be prokaryotic cell or eukaryocyte (such as Chinese hamster ovary celI, NS0 cell, 293 cells).

The sixth aspect of the present invention, provide it is a kind of generate albumen method, it comprising steps of

(1) under conditions suitable for the expression, host cell according to any one of claims 8 is cultivated, to give expression to claim 1 The fusion protein；With

(2) dimer for separating the fusion protein or being formed by the fusion protein.

The seventh aspect of the present invention provides a kind of pharmaceutical composition, and the composition includes:

The fusion protein and/or the albumen dimerization described according to a second aspect of the present invention according to a first aspect of the present invention Body, and

Pharmaceutically acceptable carrier.

The eighth aspect of the present invention provides the fusion protein according to a first aspect of the present invention and/or according to this hair The purposes of albumen dimer described in bright second aspect is used to prepare the drug for the treatment of disease.

In another preferred example, the disease is disease relevant to TGF-β and VEGF.

In another preferred example, the disease is selected from: tumour, hepatic fibrosis-renal tubular ectasia syndrome.

In another preferred example, the tumour include: large intestine tumor, lung cancer tumor, hepatic carcinoma, breast cancer tumour, Gastric cancer tumor, pancreatic tumour.

The ninth aspect of the present invention provides the method for a kind of inhibition and TGF-β and VEGF related disease, comprising steps of Fusion protein described in object application first aspect to needs.

In another preferred example, the fusion protein is applied with monomer and/or dimeric forms.

In another preferred example, the object is people.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.It should be understood that above-mentioned each technical characteristic of the invention and being specifically described in below (eg embodiment) within the scope of the present invention Each technical characteristic between can be combined with each other, to form a new or preferred technical solution.As space is limited, herein no longer Tire out one by one and states.

Detailed description of the invention

Fig. 1 is a kind of schematic arrangement of recombination fusion protein T β RII-D2-Fc of the present invention, shows T β RII-D2- Fc is containing there are three types of ingredient, the film outer ends (T β RII) of two receptor of TGF-β, 1 film outer end Two Areas (VEGFR1- of vegf receptor ) and the Fc segment of human IgG1 D2.

Fig. 2A shows the nucleotide sequence of T β RII-D2-Fc, wherein 69 red nucleotide are signal peptide code sequence Column, 483 blue nucleotide are the coded sequence of T β RII film outer end, and pink 300 nucleotide is VEGFR1-D2's Coded sequence；First 6 are EcoRI restriction enzyme site, rear 696 nucleotide behaviour IgGIFc segment in 702 nucleotide of black Coded sequence.

Fig. 2 B shows the amino acid sequence of T β RII-D2-Fc, wherein 23 red amino acid are signal peptide, it is blue 161 amino acid are T β RII film outer end, and pink 100 amino acid is VEGFR1-D2, and 234 amino acid of black are The site EcoRI (2 amino acid " EF ") and human IgG IFc segment (232 amino acid).

Fig. 3 A shows the SDS-PAGE electrophoresis of each fusion protein, and R swimming lane is under the conditions of restoring (Reducing) in figure Electrophoretic band, NR swimming lane be non-reduced (Non-Reducing) under the conditions of electrophoretic band, from the electrophoresis of T β RII-D2-Fc Scheme can be seen that under the reducing conditions, the molecular size about 80-90kDa of T β RII-D2-Fc, is greater than under non reducing conditions 170kDa, greatly than theoretical value (57kDa, 120kDa), because of many of molecule glycosylation site, it is sugar that this, which prompts the albumen, Base；Can be seen that from the electrophoretogram of D2-T β RII-Fc no matter restore or non reducing conditions under, size is than T β RII- D2-Fc is bigger, this is because the variation of structure combination causes caused by space structure change；From the electrophoresis of T β RII-Fc-D2 Figure can be seen that its size is similar to T β RII-D2-Fc, is 80-90kDa under reducing condition, is greater than under non reducing conditions 170kDa。

Fig. 3 B shows T β RII-D2-Fc albumen HPLC analysis chart, and as can be seen from the figure T β RII-D2-Fc albumen is pure Degree is very high, and aggressiveness content is lower than 2%.

Fig. 4 A shows that each fusion protein is combined active testing as a result, it can be seen from the figure that three kinds with target spot TGF-β 1 The fusion protein of various combination all has and the combination of TGF-β 1 activity, wherein best, the D2-T β of activity of T β RII-D2-Fc The activity of RII-Fc is taken second place, T β RII-Fc-D2 is most weak.The EC of three kinds of albumen₅₀It is respectively as follows: T β RII-D2-Fc=1.52nM；D2- T β RII-Fc=2.14nM；T β RII-Fc-D2=2.50nM.Positive control albumen T β RII-Fc also has good 1 knot of TGF-β Close activity, EC₅₀For 2.30nM.It is emphasized that under same concentrations, combination activity of the T β RII-D2-Fc to the target spot It is higher by T β RII-Fc about 34% or more.D2-Fc and negative control protein IgG-Fc is without activity.

Fig. 4 β shows each fusion protein active testing result in conjunction with target spot VEGF-165.It can be seen from the figure that three The fusion protein of kind various combination all has and the combination of VEGF-165 activity, wherein best, the T β of activity of D2-T β RII-Fc The activity of RII-Fc-D2 is taken second place, T β RII-D2-Fc is relatively weak.The EC of three kinds of albumen₅₀It is respectively as follows: T β RII-D2-Fc= 0.60nM；D2-T β RII-Fc=0.11nM；T β RII-Fc-D2=0.16nM；Positive control protein D 2-Fc has good VEGF-165 combines activity, EC₅₀For 0.14nM；T β RII-Fc and negative control IgG-Fc is without activity.

It can be seen that fusion protein of the invention by comparing Fig. 4 A and Fig. 4 B and combine target spot TGF- while having excellent β 1 and target spot VEGF activity.

Fig. 5 shows that T β RII-D2-Fc can inhibit the invasion for the tumour cell that TGF-β 1 is induced, and figure A is control (culture T β RII-D2-Fc and TGF-β 1 are not added in base additionally), figure β joined 1 10ng/ml of TGF-β, scheme to joined TGF-β 1 in C 1 μ g/ml of 10ng/ml, T β RII-D2-Fc schemes to joined 1 10ng/ml of TGF-β, 10 μ g/ml of T β RII-D2-Fc in D, schemes E In joined 1 10ng/ml of TGF-β, 50 μ g/ml of T β RII-D2-Fc, scheme to joined 1 10ng/ml of TGF-β, hIgG 50 in F μg/ml.From figure 5 it can be seen that significantly invasion of the induction tumour cell from cell upper layer to lower layer of TGF-β 1 (Invasion), it but is added after T β RII-D2-Fc, the invasion of tumour cell are suppressed significantly, and depression effect is in dosage Dependence.The invasion (Invasion) for the tumour cell that negative control protein hIgG cannot then inhibit TGF-β 1 to induce.This experiment The result shows that T β RII-D2-Fc has blocked the TGF-β receptor on TGF-β 1 and tumor cell membrane by being combined with TGF-β 1 In conjunction with to inhibit downstream signal transduction, and the finally invasion of inhibition tumour cell.

Fig. 6 show T β RII-D2-Fc can the vascular endothelial cell tubulose that is induced of blocking VEGF formed.Scheming A is control (only adding VEGF-165 in culture medium, do not add T β RII-D2-Fc additionally), figure B joined VEGF-165 20ng/ml, T β 20 μ g/ml of RII-D2-Fc, figure C joined VEGF-165 20ng/ml, 50 μ g/ml of T β RII-D2-Fc, and figure D joined 100 μ g/ml of VEGF-165 20ng/ml, T β RII-D2-Fc, figure E joined VEGF-165 20ng/ml, 100 μ g/ of hIgG ml.As can be seen from Figure 6 VEGF-165 induction of vascular endothelial cell (HUVEC) tubulose is formed, but if T β is added simultaneously RII-D2-Fc, the then tubulose that can significantly inhibit HUVEC cell are formed, and depression effect is in dose dependent.Negative control The HUVEC cell tubulose that albumen hIgG cannot then inhibit VEGF-165 to induce is formed

Fig. 7 A shows the inhibiting effect that fusion protein of the invention grows mouse mastopathy cell (4T1).From figure If tumor volume growth is to 600 cubic millimeters after 20 days without treatment after can be seen that cancer cell subcutaneous inoculation (mm³), and after being treated with fusion protein, the T β RII-D2-Fc of two kinds of dosage (5mg/kg, 10mg/kg) and high dose (10mg/kg) D2-Fc can significantly inhibit tumour growth, and gross tumor volume only has the half of negative control group after 20 days, be 300 Cubic millimeter (mm³).T β RII-Fc has certain inhibitory effect, but not significant.

Fig. 7 B shows the inhibiting effect that fusion protein of the invention shifts mouse mastopathy cell (4T1).From figure As can be seen that the T β RII-D2-Fc of two kinds of dosage can significantly inhibit transfer of the tumour to lung, compared with negative control group, Inhibiting rate reaches 60-70%.T β RII-Fc is to the inhibiting rate of metastases up to 61%, and D2-Fc inhibiting rate only has 50%.

Specific embodiment

The present inventor after extensive and in-depth study, establishes a technique for gene engineering platform for the first time, flat using this Platform can produce recombination double functions fusion proteins drug, such as T β RII-D2-Fc.The present invention is completed on this basis.

By taking T β RII-D2-Fc as an example, it is with the following functions: 1) can simultaneously with 1 two kinds of ligand knots of VEGF and TGF-β It closes；It 2) can the vascularization in vitro or in vivo that is induced of blocking VEGF；3) it can inhibit moving for the tumour cell that TGF-β 1 is induced It moves and invades.

Test confirms that T β RII-D2-Fc of the present invention not only has simultaneously and the combination of VEGF and TGF-β 1 activity, EC₅₀Point Not Wei 0.60 and 1.53, and can synergistically, more effectively treat certain diseases, especially such as tumour, old eye macula lutea The diseases such as denaturation, hepatic fibrosis-renal tubular ectasia syndrome.

VEGFR and its film outskirt

VEGFR albumen belongs to receptor tyrosine kinase superfamily, is a kind of film integral protein.The film outer portion of VEGFR is big There are about 750 amino acid residues, are made of 7 Ig structural domains similar with immunoglobulin structure.When with its respective ligand knot After conjunction, according to its corresponding acceptor property, VEGFR albumen can induce a series of different biological function reactions.The present invention VEGFR albumen include: VEGFR1 (Flt-1), VEGFR2 (KDR/FLk-1), VEGFR3 (Flt-4) or combinations thereof.

In the present invention, it is preferred to be VEGFR1 (Flt-1), preferably natural type VEGFR1 is wild type.

D2 of the invention refers to second film outskirt (Domain 2) of VEGFR1 (Flt-1).A kind of representative D2 sequence It is 190-282 in SEQ ID NO.:1.

TGF-β receptor and its film outskirt

TGF-β receptor is single pass transmembrane protein receptor, has serine/threonine protein kitase activity in intracellular region, should Receptor is functioned with heterodimer.The TGF-β receptor being currently known has I~V5 seed type, deposits on the various kinds of cell surface of people In the TGF-β glycoprotein receptor (i.e. I type, II type, type III receptor) of 3 seed types, I type and II receptor play signal transduction and make With.I type, II receptor are all the serine/threonine protein kitase receptors of single pass transmembrane.After birth outskirt is shorter, cytoplasmic domain compared with It is long.After birth outskirt is rich in cysteine.Cytoplasmic domain contains serine/threonine protein kitase structural domain.When with its respective ligand In conjunction with after, according to its corresponding acceptor property, TGF-β albumen can induce a series of different biological function reactions.

TGF-β receptor protein of the invention includes: T β RI, T β RII, T β RIII or combinations thereof.In the present invention, it is preferred to be T β RII, preferably natural type T β RII are wild type.

A kind of preferred TGF-β receptor membrane outskirt of the invention refers to the film outskirt of T β RII.A kind of film of representative T β RII Outer region sequence is 24-184 in SEQ ID NO.:1.

Immunoglobulin G element

In the present invention, applicable immunoglobulin G element is not particularly limited, and can be dynamic from people or other lactations The immunoglobulin element or its mutation-ure and derivative of object.It is preferred from the element of the immunoglobulin of people.

Immunoglobulin G includes four subclass: IgG1, IgG2, IgG3, IgG4.The protein structure of this four subclass has Very big similitude, all there are four regions: a variable region (VH), three constant regions (CH1, CH2, CH3).Fc segment is by two A constant region (CH2-CH3) is formed, wherein having a disulfide bond in the region CH2, so that two Fc segment monomer compositions are covalent In conjunction with homodimer.Under normal physiological conditions, the concentration of IgG is taken second place in human plasma with IgG1 highest, IgG2, IgG3 It is lower with IgG4 concentration.

A kind of preferred G element is human IgG1 Fc segment or its mutation-ure, derivative.

Bifunctional fusion proteins and its preparation

In the present invention, " recombination double functions fusion protein ", " albumen of the present invention ", " fusion protein of the present invention ", " difunctional Fusion protein " is used interchangeably, and is referred to structure described in structure described in Formulas I a or Ib or IIIa or IIIb, i.e., containing including TGF-β receptor membrane outskirt protein component, the protein component of second film outskirt D2 of VEGFR and the fusion egg of immunoglobulin element It is white.One representative example is T β RII-D2-Fc.Albumen of the present invention can be monomer or the polymer that is formed by monomer (such as Dimer).Furthermore, it is to be understood that the term further includes the active fragment and derivative of fusion protein.

As used herein, " separation " it is (former if it is crude to refer to that substance is separated from its primal environment Beginning environment is natural surroundings).If the polynucleotides and polypeptides under the native state in active somatic cell do not isolate and purify, But same polynucleotides or polypeptide such as from separating in other substances with existing in native state, then isolate and purify.

As used herein, " isolated recombination fusion protein " refers to recombination fusion protein substantially free of natural associated therewith Other albumen, lipid, carbohydrate or other materials.Those skilled in the art can be purified heavy with the purified technology of protein of standard Group fusion protein.Substantially pure albumen can generate single master tape in non-reducing polyacrylamide gel.

Polynucleotides of the invention can be DNA form or rna form.DNA form includes cDNA, genomic DNA or people The DNA of work synthesis.DNA can be single-stranded or double-strand.DNA can be coding strand or noncoding strand.

The invention further relates to the variant of above-mentioned polynucleotides, coding has the egg of identical amino acid sequence with the present invention White matter segment, analogs and derivatives.The variant of this polynucleotides can be the allelic variant or non-natural naturally occurred The variant of generation.These nucleotide variants include substitution variants, Deletion variants and insertion variant.Such as this field institute Know, allelic variant is the alternative forms of a polynucleotides, it may be one or more nucleotide substitution, missing or Insertion, but not from substantially change its encode polypeptide function.

As used herein, term " primer " refers to matching with template, can be with it under the action of archaeal dna polymerase Point synthesize the general name of the oligonucleotide acid of the DNA chain complementary with template.Primer can be natural RNA, DNA, can also be with It is any type of natural nucleotide.Primer can even is that non-natural nucleotide such as LNA or ZNA etc..Primer " generally " (or " substantially ") is complementary with a special sequence in template on a chain.Primer must be abundant with a chain in template Complementation could start to extend, but the sequence of primer need not be with the sequence complete complementary of template.For example, mutual with template at one 3 ' end The sequence that 5 ' ends of the primer of benefit add the preceding paragraph not complementary with template, such primer are still generally complementary with template.As long as having Sufficiently long primer can adequately be combined with template, and non-fully it is compound can also to form primer-template with template for complementary primer Object, to be expanded.

The nucleotide full length sequence or its segment of the element (such as VEGFR1D2 or T β RII film outskirt) of fusion protein of the present invention It can usually be obtained with PCR amplification method, recombination method or artificial synthesized method.For PCR amplification method, can be had according to published It closes nucleotide sequence, especially open reading frame sequence and carrys out design primer, and with the commercially available library cDNA or press those skilled in the art The library cDNA prepared by conventional method known to member expands as template and obtains related sequence.When sequence is longer, it is often necessary to Twice or repeatedly PCR amplification is carried out, then the segment that each time amplifies is stitched together by proper order again.

Once obtaining related sequence, so that it may obtain related sequence in large quantity with recombination method.This is usually will It is cloned into carrier, then is transferred to cell, then the isolated related sequence from the host cell after proliferation by conventional method.

In addition, related sequence can be also synthesized with artificial synthesized method, when especially fragment length is shorter.In general, logical After first synthesizing multiple small fragments, it is then attached the very long segment of available sequence again.

It is optimized for obtaining gene of the invention using round pcr DNA amplification/RNA method.Primer for PCR It can be properly selected according to the sequence information of invention disclosed herein, and available conventional method synthesis.Conventional method can be used The DNA/RNA segment of amplification is such as separated and purified by gel electrophoresis.

The present invention also relates to the carriers comprising polynucleotides of the invention, and are compiled with carrier of the invention or fusion protein The code genetically engineered host cell of sequence, and the method for generating protein of the present invention through recombinant technique.

By the recombinant dna technology of routine, it can be used to express or produce recombination using polynucleotide sequence of the invention Albumen.In general there are following steps:

(1) polynucleotides (or variant) of coding albumen of the present invention of the invention, or with containing the polynucleotide Recombinant expression carrier conversion or suitable host cell of transduceing；

(2) host cell that is cultivated in suitable culture medium；

(3) be separated from culture medium or cell, protein purification.

Method well-known to those having ordinary skill in the art can be used to construct DNA sequences encoding containing albumen of the present invention and suitable Transcription/translation control signal expression vector.These methods include recombinant DNA technology in vi, DNA synthetic technology, In vivo recombination Technology etc..The DNA sequence dna can be effectively connected in the appropriate promoter in expression vector, to instruct mRNA to synthesize.Expression Carrier further includes the ribosome bind site and transcription terminator of translation initiation.

In addition, expression vector preferably includes one or more selected markers, to provide for selecting conversion The phenotypic character of host cell, such as the dihyrofolate reductase of eukaryotic culture, neomycin resistance and green fluorescence egg White (GFP), or tetracycline or amicillin resistance for Escherichia coli.

Carrier comprising above-mentioned appropriate DNA sequence dna and appropriate promoter or control sequence, can be used for converting suitable When host cell, allow it to expression protein.

Host cell can be prokaryotic cell, such as bacterial cell；Or low eukaryocyte, such as yeast cells；Or it is high Equal eukaryocytes, such as mammalian cell.Representative example has: Escherichia coli, the bacterial cell of streptomyces；Fungal cell is such as Yeast；Plant cell；The insect cell of drosophila S2 or Sf9；CHO, COS or the zooblast of 293 cells etc..

It can be carried out with routine techniques well known to those skilled in the art with recombinant DNA conversion host cell.When host is original When core biology such as Escherichia coli, the competent cell that can absorb DNA can harvest after exponential phase of growth, use CaCl₂Method processing, institute With the step of it is generally well-known in the art.Another method is using MgCl₂.If desired, conversion can also use the side of electroporation Method carries out.When host is eucaryote, following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods can be selected Such as microinjection, electroporation, liposome packaging.

The transformant of acquisition can use conventional method culture, express the polypeptide of coded by said gene of the invention.According to used Host cell, culture medium used in culture can be selected from various conventional mediums.Under conditions of being suitable for host cell growth It is cultivated.After host cell growth is to cell density appropriate, with suitable method (such as temperature transition or chemical induction) Cell is further cultured for a period of time by the promoter for inducing selection.

Protein in the above methods can be expressed in cells, or on the cell membrane, or secreted outside the cell.If It needs, can be separated by various separation methods and purifying protein using its physics, chemical and other characteristics.These methods are It is well-known to those skilled in the art.The example of these methods includes but is not limited to: conventional renaturation process uses albumen precipitation Agent handle (salting-out method), centrifugation, permeate broken bacterium, super processing, ultracentrifugation, sieve chromatography (gel filtration), adsorption chromatography, from The combination of sub- displacement chromatography, high performance liquid chroma- tography (HPLC) and various other liquid chromatography technologies and these methods.

Antibody

In the present invention, " antibody ", " ligand " are used interchangeably, and refer to the Anti-TNF-α for having specificity to albumen of the present invention Body and monoclonal antibody, especially monoclonal antibody.Here, " specificity " refer to antibody can respectively in connection in albumen of the present invention or Its segment.Preferably, referring to that those in conjunction with albumen of the present invention or segment but nonrecognition and can be incorporated into other non related antigens point The antibody of son.Antibody of the invention can be prepared by various technologies known to those skilled in the art.

The present invention not only includes complete monoclonal or polyclonal antibody, but also including with immunocompetent antibody piece Section, such as Fab ' or (Fab)₂Segment；Heavy chain of antibody；Antibody light chain；Genetically engineered scFv molecule；Or chimeric antibody.

Peptide linker

The present invention provides a kind of bifunctional fusion proteins, it optionally contains peptide linker.Peptide linker size and complexity Property may will affect the activity of albumen.In general, peptide linker should have enough length and flexibility, to guarantee two of connection Albumen has enough freedom degrees spatially to play its function.Avoid being formed α spiral or β-pleated sheet etc. simultaneously in peptide linker to melting The influence of the stability of hop protein.

The length of link peptide is generally 0-10 amino acid, preferably 0-5 amino acid.

Pharmaceutical composition and method of administration

The present invention also provides a kind of compositions, it contains a effective amount of fusion protein of the present invention, and can pharmaceutically connect The carrier received.In general, fusion protein of the invention can be formulated in nontoxic, inert and pharmaceutically acceptable aqueous carrier In medium, wherein pH is usually about 5-8, preferably, pH is about 6-8.

As used herein, term " effective quantity " or " effective dose ", which refer to, to generate function or activity to people and/or animal And the amount that can be received by people and/or animal, such as 0.001-99wt%；Preferable 0.01-95wt%；More preferably, 0.1- 90wt%.

As used herein, the ingredient of " pharmaceutically acceptable " is suitable for people and/or mammal and without excessively bad Side reaction (such as toxicity, stimulation and allergy), i.e., with the substance of reasonable benefit/risk ratio.Term " can pharmaceutically connect The carrier received " refers to the carrier for Therapeutic Administration, including various excipient and diluent.

Pharmaceutical composition of the invention contains the fusion protein of the invention of safe and effective amount and pharmaceutically acceptable Carrier.This kind of carrier include (but being not limited to): salt water, buffer, glucose, water, glycerol, ethyl alcohol, and combinations thereof.Usual medicine Object preparation should match with administration mode, and pharmaceutical composition of the invention can be made into injection form, such as use physiological saline Or the aqueous solution containing glucose and other adjuvants is prepared by conventional method.The pharmaceutical composition is preferably in sterile item It is manufactured under part.The dosage of active constituent is therapeutically effective amount.Pharmaceutical preparation of the invention may also be fabricated which sustained release preparation.

The effective quantity of fusion protein of the present invention can change with the mode of administration and the severity of disease to be treated etc.. Preferred a effective amount of selection can be determined depending on various factors by those of ordinary skill in the art (such as to be tried by clinic It tests).The factor includes but is not limited to: the pharmacokinetic parameter of fusion protein of the present invention for example bioavailability, metabolism, Half-life period etc.；Patient the severity of disease to be treated, the weight of patient, the immune state of patient, the approach of administration etc.. In general, when fusion protein of the invention is daily with about 5mg-20mg/kg the weight of animals (preferable 5mg-10mg/kg animal body Dosage again) is given, and satisfactory effect can be obtained.For example, can be given once daily several times by an urgent demand for the treatment of situation Separated dosage, or dosage is reduced pari passu.

Fusion protein of the present invention secretes excessive disease or particularly suitable for treatment VEGF and TGF-β 1 with aberrant angiogenesis The disease that the invasion of hyperplasia and tumour cell are characterized.Representative disease includes (but being not limited to): tumour, wet macular become Property or hepatic fibrosis.

Fusion protein and its dimer of the invention or polymer mainly include following advantages:

It 1) can be living with very strong combination with VEGF and TGF-β 1 simultaneously with 1 two kinds of ligand bindings of VEGF and TGF-β Property, in conjunction with active EC₅₀Highest can arrive separately at 0.60nM and 1.53nM；

2) can blocking VEGF induction vascularization in vitro or in vivo；

3) migration and invasion of the tumour cell that TGF-β 1 is induced be can inhibit；

4) TGF-β 1 and VEGF induced liver fibrosis can be blocked；

5) immunosuppressive activity that TGF-β can be blocked, to enhance immune function.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part, such as Sambrook et al., molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or according to the normal condition proposed by manufacturer.Unless otherwise stated, no Then percentage and number are weight percent and parts by weight.

Embodiment 1

Construct T β RII-D2-Fc expression vector

T β RII film outskirt gene coded sequence is made of 483 nucleotide, as 70-552 in SEQ ID NO.:2 It is shown.VEGFR1-D2 gene coded sequence is made of 300 nucleotide, as shown in 553-852 in SEQ ID NO.:2, Including 6 279 nucleotide of D2 coded sequence, 15 nucleotide of upstream flanking sequence, downstream flanking sequence nucleotide.? T β RII film outskirt 5 ', which is held, added 69 signal coding sequence (i.e. 1-69 in SEQ ID NO.:2 from T β RII Position), form 852 nucleotide.

It is added in the aminoterminal of this 852 nucleotide along with " HindIII " gene cloning site, in c-terminus " EcoRI " gene cloning site constitutes the genetic fragment containing 873 nucleotide.

Synthetic product (synthesis of Nanjing Jin Sirui biotechnology company) passes through HindIII/EcoRI digestion, is cloned into pHB- Fc plasmid vector forms pHB-TbRII-D2-Fc protein expression vector.The preparation method of pHB-Fc plasmid vector is as follows: with pcDNA/ HA-FLAG (Accession#:FJ524378) carrier is the plasmid that sets out, and added the Fc of human IgG1 behind restriction endonuclease EcoRI Sequence, added before restriction endonuclease HindIII human cytomegalovirus (HCMV) promote subsequence (Accession#: X17403), it added Chinese hamster glutamine synthelase base behind ampicillin resistance gene, before HCMV promotion Because of (Accession#:x03495).After sequence design is good, commission Shanghai Jierui Biology Engineering Co., Ltd, which is synthesized, to be changed It makes.

Sequence SEQ ID NO:2 is the nucleotide sequence for encoding recombination double functions fusion protein, as shown in Figure 2 A.Overall length 1554bp, wherein 1-69bp is signal coding sequence, and 70-552bp is T β RII film outskirt coded sequence, and 553-852bp is VEGFR1D2 coded sequence, the restriction enzyme site GAATTC, 859-1554bp of 853-858EcoRI is Fc segment, and TGA is that termination is close Code.

Fig. 1 is the schematic arrangement for recombinating bifunctional fusion proteins T β RII-D2-Fc.The schematic diagram only plays signal Effect, does not represent the concrete facticity structure of bifunctional fusion proteins of the present invention.

Sequence SEQ ID NO:1 is the amino acid sequence for encoding recombination double functions fusion protein, as shown in Figure 2 B.Overall length 518 amino acid.Wherein 1-23 amino acids are signal peptide, and 24-184 amino acids are T β RII film outskirt, and 185-284 are VEGFR1D2 segment containing flanking sequence (underscore marks), 285-286 2 amino acid for EcoRI restriction enzyme site, 287-518 amino acids are Fc segment.

Embodiment 2

The expression of T β RII-D2-Fc, D2-T β RII-Fc, T β RII-Fc-D2

Host cell used in protein expression is available from the CHO-K1 cell (Cat#CCL-61) of ATCC company.Cell warp A series of domestication steps are crossed, are tamed into the CHO-K1 that can carry out suspension culture in serum free medium (EX-CELLTM 302) Cell.

Utilize the cell, the method turned by electricity, by plasmid pHB-T β RII-D2-Fc, pHB-D2-T β RII-Fc, pHB- T β RII-Fc-D2 is transferred to cell respectively.Specific method is: aseptically collecting the cell for being in logarithmic growth phase, centrifugation It is resuspended in complete medium after precipitating (1200rpm x 5min), and adjusts cell density to 1x 10⁷cells/ml.Take 350ul Cell suspension is transferred to 0.4cm electricity revolving cup, and (voltage range 200 arrives 350V, general 260V, time under the conditions of setting electricity turns 20ms or so) pulse 1 time.10-30ug Plasmid DNA is added into electric revolving cup containing cell, after mixing gently, by electric revolving cup It is put into electroporation, punching of promoting blood circulation.Electric revolving cup is taken out, 5 minutes is stood, adds 0.6ml cell culture medium, suck out, go to after mixing In culture dish, it is put into incubator culture.Protein expression is checked after 24-48 hours.If any protein expression, it was demonstrated that gene is transferred into Cell is diluted by function with culture medium at this time, is then transferred in 20 piece of 96 porocyte culture plates, every hole cell number 3000- 5000.Cell is screened by a series of pressure (glutamine synthetase inhibitor), and finishing screen is selected being capable of high expression albumen Cell strain.

When protein production, by the cell strain cell inoculation of high expression albumen to containing 3 liters of 302 culture mediums of EX-CELLTM In cell reactor, cell density is 3x 10⁵Cells/ml, condition of culture are 37 DEG C, 5%CO2.Cell is during the cultivation process It is detected by pH, glucose, glutamine etc., and adds nutritional ingredient in due course according to indices.When cell density reaches 5-6x 10⁶When cells/ml, cultivation temperature is down to 33 DEG C from 37 DEG C, continues to be harvested when culture reaches 60-70% to Cell viability. The cells and supernatant of harvest is purified by ultrafiltration concentration and Protein A affinity chromatography strain.The albumen of purifying utilizes Lowry method is quantitative determined (referring to 2010 editions Chinese Pharmacopoeias), and protein quantification standard items are ox blood albumin (lot number 140619-201120, Chinese drug and food examine and determine research institute).The albumen of production is through SDS-PAGE electrophoretic analysis size and theory Value is coincide substantially, and endotoxin content is lower than standard requirements.

By protein electrophoresis (SDS-PAGE) analyze, discovery under the reducing conditions, T β RII-D2-Fc size be in~ The position 80kDa (monomer) is then greater than 170kDa (dimer) under non reducing conditions, the albumen size of other two kinds of structures combinations with T β RII-D2-Fc is similar.In addition, actual measurement molecular weight (Fig. 3) prompt of the recombinant protein of three kinds of structures combination, recombinant protein are deposited A degree of glycosylation (theoretical molecular weight of dimer is 114kDa).

HPLC analyzes purity of protein and is greater than 98%.

Embodiment 3

T β RII-D2-Fc and target spot (VEGF and TGF-β 1) combine Activity determination

Using MBP enzyme linked immuno-adsorbent assay (ELISA) method, measures fusion protein and target spot (VEGF and TGF-β 1) combines Characteristic.Specific step is as follows:

With coating buffer CBS (Sigma-Aldrich Co., Product code:1001329288 C3041- 100CAP) by TGF-β 1 (Cat:10804-HNAH, Sino biological Inc.) and VEGF-165 (Cat:11066- HNAH, Sino biological Inc.) it is diluted to 500ng/ml respectively, take 100ul to be added to elisa plate (Nunc^TM, Cat: 442404) in, every hole 50ng.Coating plate is placed in 4 DEG C of refrigerator overnights.Coating plate one first is washed with 0.05%PBS-T when detection It is secondary, then closed 1 hour with 3% skim milk room temperature.2 times have been serially diluted TbRII-D2-Fc albumen (100nM, 50nM, 25nM, it is coated in plate up to 0.0244nM) is added to, every hole 100ul.It is incubated at room temperature after a hour, abandons sample, use 0.05%PBS-T is washed 5 times, and HRP-Rabbit Anti-Human IgG of the 100ul by dilution (1: 20000) is then added Fc (Luoyang one hundred is difficult to understand logical, Cat#:C030222), is incubated at room temperature a hour, and cleaning solution washs 5 times, and HRP substrate is added, is protected from light aobvious 2N H is used after color 10-20 minutes₂SO₄Color development stopping reaction, in reading OD450 value in microplate reader.

(table 1) as the result is shown, T β RII-D2-Fc is respectively provided with and the combination of TGF-β 1 (Fig. 4 A) and VEGF-A (Fig. 4 B) are living Property, corresponding EC₅₀Respectively reach 1.53nM and 0.6nM.The albumen of other two kinds of structures combinations is living to the combination of two kinds of target spots Property also very strong, EC₅₀Respectively reach 2.5nM or so (TGF-β 1) and 0.16nM (VEGF-165).

The combination activity EC of 1 fusion protein of table and target spot₅₀(nM)

	TβRII-D2-Fc	D2-TβRII-Fc	TβRII-Fc-D2	D2-Fc	TβRII-Fc
						TGF-β1	1.53	2.14	2.50	It is inactive	2.33
VEGF-A	0.60	0.11	0.16	0.14	It is inactive

Embodiment 4

T β RII-D2-Fc can inhibit the invasion for the tumour cell that TGF-β 1 is induced

Tumor cell invasion experiment has been carried out using 24 hole cell compartments culture plates.Method is: containing being added in cell Tumour cell (PC-3) and certain density T β RII-D2-Fc, incubator is added on the upper layer of filter membrane in the culture medium of TGF-β 1 After culture 24 hours, filter membrane is dyed with crystal violet, and observes the density for filter membrane bottom cell of taking pictures.

Using PC-3 cell, the invasion for the tumour cell that T β RII-D2-Fc induces TGF-β 1 are analyzed.Specific steps It is as follows: the cell screen clothes containing Matrigel are placed in (BD in 24 well culture plates containing cell culture fluid Bioscience), the TGF-β 1 in culture solution containing 10ng/ml.By 1x10⁵A PC-3 cell is added in cell screen clothes, so It requires for T the β RII-D2-Fc and hIgG of various concentration to be added separately in corresponding sieve according to grouping afterwards, the hole 24- cell is trained Feeding plate is placed in cell incubator, in 30 DEG C, 5%CO₂Under the conditions of cultivate 24 hours.Sieve is taken out, by 4% poly of filter membrane Formaldehyde fixes 15 minutes, and 0.5% crystal violet dye liquor dyes 15 minutes, has contaminated color and has washed off extra dye liquor, has wiped film upper layer with cotton swab The cell not passed through, invasion cell of taking pictures under 100 power microscopes.Every film up and down in 5 different visuals field.

As shown in figure 5, T β RII-D2-Fc can significantly inhibit invasion of the PC-3 cell from cell upper layer to lower layer, inhibit effect It should be in dose dependent.

Embodiment 5

T β RII-D2-Fc can the vascular endothelial cell tubulose that is induced of blocking VEGF formed

By HUVEC cell degree of thickening to 3 × 10⁵Cell is added in 96 well culture plates containing Matrigel by/ml, Every hole 50ul.Then T β RII-D2-Fc (20,50,100ug/ by prepared containing VEGF (20ng/ml) and various concentration Ml culture solution) is added in culture plate, every hole 50ul.Culture plate is placed in incubator culture, and in different time points (0h, 2h, Photo archive under microscope 4h, 6h, 8h, for 24 hours).

When cultivating in gel under the conditions of the result shows that HUVEC cell is existing for the VEGF, blood vessel can be formed under microscope Shape figure, similar to the formation of body vessel.People usually verify influence of certain drug to vascularization using the experiment. The influence that T β RII-D2-Fc forms extracorporeal blood vessel is analyzed using experiment the present inventor.The result shows that (Fig. 6), T β RII- The tubulose that D2-Fc can significantly inhibit HUVEC cell is formed.

Embodiment 6

T β RII-D2-Fc inhibits tumour growth and metastases

Using routine techniques, following components is mixed, final concentration of 1wt% recombinant protein solution is made, is formulated as follows:

Recombinant protein 10mg

Physiological saline adds to 10ml

Adjust pH to 6.8-7.1.

1x10 is subcutaneously injected to normal female Balb/c mammary gland of mouse position⁵A mouse mastopathy cell (4T1), second day 5 groups are randomly divided into, first group of intraperitoneal injection 5mg/kg T β RII-D2-Fc, second group of intraperitoneal injection 10mg/kg T β RII-D2- Fc, third group intraperitoneal injection 10mg/kg D2-Fc (control), the 4th group of intraperitoneal injection 10mg/kg T β RII-Fc (control), often Week, continuous 6 administrations, the 5th group was negative control twice, and PBS is injected intraperitoneally.Gross tumor volume is measured three-times-weekly.After treatment 21 days execution mouse, win tumour weighing, take lungs, microscopically observation lung transfer stove.

The result shows that T β RII-D2-Fc can significantly inhibit tumour growth (Fig. 7 A) in two dosage, inhibiting rate is big In 50%.The tumor control rate of D2-Fc is also fine, reaches~55%, and the tumor control rate of T β RII-Fc only has 26%.

Lung's transfer case (Fig. 7 B), negative control group averagely have 11.4 transfer stoves, T β RII-D2-Fc treatment group difference There are 4.2 (5mg/kg) and 3.56 (10mg/kg) a.Although T β RII-Fc cannot effectively inhibit tumour growth, swell to lung Tumor metastasis then significantly inhibits effect (transfer stove has 4.4).Although D2-Fc can inhibit tumour growth well, to tumour The inhibiting effect of transfer is markedly less than T β RII-D2-Fc and D2-Fc.

As a result

There is bifunctional fusion proteins T β RII-D2-Fc the stronger collaboration to tumour growth and metastases to inhibit effect Fruit, and can only have a kind of apparent inhibiting effect just for the fusion protein of a target spot, i.e., or tumour can only be inhibited raw Long (blocking VEGF) or it can only inhibit metastases (blocking TGF-β 1).

In addition, the inhibitory effect of the experimental group of 10mg/kg T β RII-D2-Fc is also significantly better than 5mg/kg D2-Fc+5mg/ The experimental group of kg T β RII-Fc.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Claims

1. a kind of fusion protein, which is characterized in that the fusion protein includes the following elements being fused together:

(i) the optional signal peptide positioned at N-terminal；

(ii) the first protein element；

(iii) the second protein component；And

And the first protein element is TGF-β receptor membrane outskirt protein component；Second protein component be include vascular endothelial cell The protein component of growth factor receptors VEGFR1 the second film outskirt D2；

Also, the fusion protein has structure selected from the group below:

Structure described in Formulas I a:

D-A-B-C (Ia)；

Wherein,

A is TGF-β receptor membrane outskirt protein component；

C is immunoglobulin element；

D is optional signal peptide sequence；

"-" indicates the peptide bond or peptide linker of connection said elements, and the amino acid sequence of the fusion protein such as SEQ ID Shown in NO:1.

2. fusion protein as described in claim 1, which is characterized in that the fusion protein has following multiple functions:

And the combination activity EC of VEGF a)₅₀For 0.6-2nM；

And the combination activity EC of TGF-β 1 b)₅₀For 1.5-2.5nM；

C) can simultaneously with 1 two kinds of ligand bindings of VEGF and TGF-β；

D) can blocking VEGF induction vascularization in vitro or in vivo；

3. a kind of albumen dimer, which is characterized in that the dimer is by two fusion protein structures described in claim 1 At.

4. dimer as claimed in claim 3, which is characterized in that the dimer has structure selected from the group below:

Structure described in Formulas I a-1:

Wherein,

A is TGF-β receptor membrane outskirt protein component；

B is the protein component for including VEGFR the second film outskirt D2；

C is immunoglobulin element；

D is optional signal peptide sequence；

"-" indicates the peptide bond or peptide linker of connection said elements；

" ‖ " indicates disulfide bond.

5. a kind of isolated polynucleotides, which is characterized in that fusion egg described in the polynucleotide encoding claim 1 It is white.

6. a kind of carrier, which is characterized in that it contains polynucleotides described in claim 5.

7. a kind of host cell, which is characterized in that it, which contains to integrate in carrier or genome as claimed in claim 6, has the right to want Polynucleotides described in asking 5.

8. it is a kind of generate albumen method, which is characterized in that it comprising steps of

(1) under conditions suitable for the expression, host cell as claimed in claim 7 is cultivated, to give expression to described in claim 1 Fusion protein；With

9. a kind of pharmaceutical composition, which is characterized in that the composition includes:

Fusion protein described in claim 1 and/or albumen dimer as claimed in claim 3, and

Pharmaceutically acceptable carrier.

10. the purposes of fusion protein as described in claim 1 and/or albumen dimer as claimed in claim 3, feature exist In, be used to prepare treatment disease drug, the disease be disease relevant to TGF-β and VEGF.