CN105111314A - Novel fusion protein, pharmaceutical composition and preparation method therefor and use thereof - Google Patents

Abstract

The invention discloses a novel fusion protein, a pharmaceutical composition formed by the novel fusion protein, a preparation method for the novel fusion protein and use of the novel fusion protein. According to novel fusion protein, the structural general formula is X-connecting peptide-Y-connecting peptide-IgG Fc fragment or Y-connecting peptide-X-connecting peptide-IgG Fc fragment, wherein X is VEGF-Trap and derivatives thereof, and Y is a PD-1 antibody fragment and derivatives thereof or a PD-1 antagonist fragment and derivatives thereof. The fusion protein plays roles in suppressing regenerated blood vessels and activating T cellular immunity and plays a role in coordinating and promoting two functions, so that the function of inhibiting tumor growth is more remarkable.

Description

A kind of new fusion protein, pharmaceutical composition and its production and use

Technical field

The present invention relates to a kind of fusion rotein, what be specifically related to is the new fusion protein of a kind of Tumor suppression growth and the preparation method of pharmaceutical composition and this new fusion protein and purposes.

Background technology

Immunologic escape is a very important step in the deterioration process of tumour.Tumour cell is able to quick growth by avoiding the lethal effect of human immune system, thus promotes the generation of cancer.Important negative regulatory molecule PD-1, CTLA-4 and the TIM-3 in T cell surface all can suppress the immunologic cytotoxicity effect of T cell in development of cancer.At present, the existing medicine for PD-1 and CTLA-4 target spot clinically, these medicines are result for the treatment of highly significant clinically.

Programmed cell death 1 (PD-1) acceptor is mainly expressed in the T cell activated and B cell, its major function is the activation of Immunosuppression system cells, this is also a kind of normal homeostasis of immunity system, because excessive T/B cell-stimulating can cause autoimmune disease, so PD-1 is one amulet of our human body.In pathological conditions, tumor microenvironment can induce the T cell high expression level PD-1 molecule of infiltration, part PD-L1 and PD-L2 of tumour cell meeting high expression level PD-1, cause PD-1 signal path sustained activation in tumor microenvironment, T cell function is suppressed, cannot killing tumor cell, thus cause the generation of cancer.The PD-1 antibody blocking this signal path partly can recover the function of T cell, enables T cell continue killing tumor cell.At present for existing two medicines listing of antibody of PD-1, be Nivolumab and Pembrolizumab respectively, the two all shows significant result for the treatment of to kinds cancer clinically.

VEGF-A is the generation factor of a class angiogenesis promoting of tumor cells expression, and its Main Function is to provide immunosuppressant microenvironment, is the propagation of tumour cell and creating favorable conditions of cancer.Scientist MagaliTerme(MagaliT, ThibaultV, etal, 2015, J.Exp.Med.212:139-148) research finds that the tumor microenvironment of VEGF-A construction can cause the rise of CD8+T cell surface PD-1 molecule, thus causes the immunologic cytotoxicity effect of CD8+T cell to reduce.Find that the stimulation of VEGF-A can cause TIM-3, the expression of the Inhibitory molecules such as CTLA-4, LAG-3 simultaneously.

The existing report utilizing VEGF-A antibody and PD-1 antibody URIN Treatment tumour in prior art, in clinical application be usually be used alone wherein a kind of antibody or simultaneously use two kinds of antibodies on tumor treat, multiple injection administration every day is then needed when adopting above-mentioned two kinds of antibody to treat simultaneously, not only make troubles to operator, and more painful also bring to experimenter.

Summary of the invention

The object of the invention is to overcome VEGF-Trap and PD-1 antibody in prior art in clinical application, need the problem of multiple injection administration every day, there is provided and both there is VEGFTRAP and PD-1 antibody physiologically active, there is again a kind of new fusion protein of long-acting function, and disclose the pharmaceutical composition of this new fusion protein composition and the preparation method of this new fusion protein and purposes.

For achieving the above object, technical scheme of the present invention is as follows:

A novel fusion rotein, its general structure is as follows:

X-connection peptides-Y-connection peptides-IgGFc fragment or Y-connection peptides-X-connection peptides-IgGFc fragment;

Wherein, X is VEGF-Trap and derivative thereof; Y is PD-1 antibody fragment and derivative thereof or PD-1 antagonist fragment and derivative thereof.

The present invention is not simply by two kinds of antibody protein mixing, but adopt two kinds of antibody to carry out merging a kind of novel fusion rotein of rear composition, this albumen can suppress the activity of PD-1 and VEGF-A simultaneously, thus can the new life of Tumor suppression blood vessel, again can the generation of double inhibition PD-1 and activity, many pathway activation T cell; It has can hungry tumour cell, again can the effect of activated T cell killing tumor cell.

The present invention is to being used alone PD-1 antibody and VEGF-A antibody, combinationally use PD-1 antibody and VEGF-A antibody, and using the effect of the fusion rotein VNI of PD-1 antibody and VEGF-A antibodies tumor growth situation in mouse of carrying out to detect, detected result is as shown in table 7 and Fig. 3.Known by table 7: to adopt new fusion protein of the present invention, it not only can have the function of two kinds of antibody hungry tumour cells of difference and activated T cell killing tumor cell simultaneously, and the collaborative promoter action of two kinds of functions can also be reached, the function that Tumor suppression is grown is more remarkable; Further, shown in Fig. 3, new fusion protein of the present invention is adopted also to have the effect effectively extending each antibody units physiological active functions effect.

Preferably, described connection peptides is the aminoacid sequence shown in formula (Gly-Gly-Gly-Gly-Ser) n, and this n is the integer of 1 ~ 4.The aminoacid sequence of described X is SEQIDNO:1 or SEQIDNO:3, or its nucleotide coding sequence is SEQIDNO:2 or SEQIDNO:4.The aminoacid sequence of described Y is SEQIDNO:5 or SEQIDNO:7, or its nucleotide coding sequence is SEQIDNO:6 or SEQIDNO:8.Described IgGFc fragment is IgG1Fc fragment, IgG2Fc fragment, IgG3Fc fragment or IgG4Fc fragment, and its aminoacid sequence is respectively SEQIDNO:9,11,13,15, or its nucleotide coding sequence is respectively SEQIDNO:10,12,14,16.

Prepare a method for new fusion protein, the coding nucleotide sequence of new fusion protein described in any one of claim 1-6 is inserted in expression vector, and this expression vector is introduced corresponding expression system and then carry out the expression of fusion rotein.The expression vector of fusion rotein of the present invention can be recombinant eukaryon expression vector, preferred mammal fibrocyte expression vector; Also can be recombinant virus expression vector, preferred adeno-associated virus or adenovirus carrier.

The host cell of fusion rotein in above-mentioned expression system can be Chinese hamster ovary celI and subbreed thereof or 293 cells and subbreed thereof.

A kind of pharmaceutical composition, is made up of with pharmaceutically acceptable carrier or vehicle the new fusion protein described in any one of claim 1-6.

Further, the dosage form of described pharmaceutical composition is injection, injection freeze-dried powder.

A purposes for new fusion protein, the new fusion protein described in described any one of claim 1-6 is used for the treatment of tumour; Described tumour is one or more in melanoma, lung cancer, liver cancer, lymphoma, colorectal carcinoma, large bowel cancer, mammary cancer, ovarian cancer, cervical cancer, cancer of the stomach, cholangiocarcinoma, carcinoma of gallbladder, the esophageal carcinoma, kidney, neurospongioma, melanoma, carcinoma of the pancreas and prostate cancer.

The present invention compared with prior art, has the following advantages and beneficial effect:

1, fusion rotein of the present invention can not only have the function of two kinds of antibody hungry tumour cells of difference and activated T cell killing tumor cell simultaneously, and can also reach the collaborative promoter action of two kinds of functions, and the function that Tumor suppression is grown is more remarkable;

2, new fusion protein of the present invention effectively can extend each antibody units physiological active functions effect, and then reduces total dosage of a course for the treatment of, reduces drug cost;

3, the present invention effectively can reduce the number of times of drug administration by injection, alleviates administration misery.

Accompanying drawing explanation

Fig. 1 is the EC50 value curve synoptic diagram that VNI affects HUVEC cell proliferation.

Fig. 2 is that VNI stimulates PBMC to produce the curve synoptic diagram of IFN γ.

Fig. 3 be mouse source VNI in vivo Tumor suppression growth contrast situation schematic diagram.

Embodiment

Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.

Prepared by embodiment 1 albumen

A kind of new fusion protein, its general structure is as follows:

X-connection peptides-Y-connection peptides-IgGFc fragment; Wherein, X is VEGF-Trap and derivative thereof; Y is PD-1 antibody fragment and derivative thereof or PD-1 antagonist fragment and derivative thereof.

In the present embodiment, this X is VEGF-Trap, and its nucleotide sequence is as shown in SEQIDNO:4; Y is PD-1 antibody fragment, and its nucleotide sequence is as shown in SEQIDNO:6; Described connection peptides is the aminoacid sequence shown in formula (Gly-Gly-Gly-Gly-Ser) n, and in this connection peptides, n is 3; The nucleotide sequence of IgGFc fragment is as shown in SEQIDNO:16; That is, the nucleotide sequencing of fusion rotein is synthesized in the present embodiment as shown in SEQIDNO:18.

In the present embodiment, the concrete synthetic route of above-mentioned new fusion protein is as follows:

1. construction recombination plasmid

1.1 according to the general formula of X-connection peptides-Y-connection peptides-IgGFc fragment or Y-connection peptides-X-connection peptides-IgGFc fragment, obtain the gene fragment and the primer that synthesize fusion rotein, and the gene fragment of synthesis fusion rotein and primer restructuring are obtained puc57-VNI plasmid to plasmid vector puc57.The nucleotide sequencing of this synthesis fusion rotein is as shown in SEQIDNO:13, and this gene and primer are synthesized by Beijing Jin Weizhi company of specialized company.

1.2 puc57-VNI plasmid and pCHO1.0 plasmid are carried out double digestion process respectively, this enzyme cuts the foundation of system:

Following composition is added: pCHO1.0 plasmid or puc57-VNI plasmid 40 μ l in 1.5mlEP pipe; Buffer410 μ l; the each 5 μ l of Avr II and BstZ17I; aqua sterilisa 45 μ l; at 37 DEG C, 5h is reacted after mixing; utilize QIAGEN Product Purification Kit to reclaim, obtain gene fragment pCHO1.0 and VNI respectively.

1.3 under the effect of T4 ligase enzyme, is connected cutting the rear gene fragment pCHO1.0 obtained that reclaims by same enzyme with VNI gene fragment; Ligation system is as follows:

The pCHO1.0 of following composition 2 μ l is added in 1.5mlEP pipe, the VNI of 6 μ l, 10 × T4Buffer1 μ l, the T4DNA ligase enzyme of 1 μ l, under room temperature (about 20 DEG C), more than 4h is reacted after mixing, connect product conversion in Top10 competent escherichia coli cell, coat 37 DEG C of hold over night on 2YT (KANA) plate culture medium, dull and stereotyped numbering pCHO1.0-VNI.

1.4 from pCHO1.0-VNI flat board the single bacterium colony of the several restructuring of each picking through cultivation after as pcr template, carry out PCR Screening and Identification respectively;

Bacterium liquid pcr amplification reaction system (cumulative volume 20 μ L): 2 × TaqHS10 μ L, bacterium liquid template 2 μ L, each 1 μ L of upstream and downstream primer P1 and P2 (final concentration 0.3 μm of ol/L), finally mends to 20 μ L with distilled water; Reaction conditions: 95 DEG C of 2min, a circulation; 94 DEG C of 60s, 53 DEG C of 60s, 72 DEG C of 120s, 30 circulations; Last 72 DEG C of 5min.By agarose gel electrophoresis analytical results, select the correct bacterium colony with target product.

Extract after 1.5 colony inoculations by PCR Screening and Identification and carry out enzyme more respectively and cut qualification; Namely first carry out the plasmid extraction of recombinant bacterium, then carry out restriction analysis;

Enzyme cuts system: in 1.5mlEP pipe, add following composition: plasmid 2 μ l, each 1 μ l of Buffer11 μ l, BSA0.1 μ l, Avr II and BstZ17I, mends aqua sterilisa to 10 μ l, reacts 4h after mixing at 37 DEG C.By agarose gel electrophoresis analytical results, select enzyme and cut the correct bacterium colony of qualification.

By being cut by bacterium colony PCR and enzyme, 1.6 identify that correct bacterium colony is inoculated several bacterium colony at random and carried out order-checking qualification again, select expression plasmid and save backup.

2. plasmid transfection and cell screening

Adopt host cell CHO-S or DG44, carry out plasmid transfection respectively according to FreedomCHO-SKit test kit specification sheets, the cell proceeding to plasmid is placed in shake-flask culture 8h respectively, and the condition of shake-flask culture is: 37 DEG C, 8%CO ₂, 110rpm/min.Cell viability and cell count is detected by cell counter.

Two step pressurization screenings are carried out: 10P/100M, 20P/200M(P=10 μ g/mLPuromycin, M=nMMTX) after transfection 48h; 30P/500M, 50P/1000M, obtain just sieve cell.Carry out monoclonal cell screening with limiting dilution assay, therefrom preferably clone enlarged culturing step by step by detection expressing quantity and purity.

3. Protein expression and purification

Screening High producing clones cell expands to 24 orifice plate growths from 96 orifice plates, then expands to 6 orifice plate growths, expands afterwards to 50mL Shake flask grown.

Collect cultivation 7 days cell culture supernatants, centrifugal removing cell debris, supernatant liquor is with 0.45 μm of membrane filtration, regulate pH to 7.4, with Protein A sepharose affinity column chromatography (HiTrapProtein-ASepharoseaffinitycolumn) purified fusion protein, the deionized water rinsing pillar of 5 times of column volumes, use PBS damping fluid (the 20mM phosphoric acid salt of 5 times of column volumes again, pH7.4) pillar is balanced, loading, collection effluent liquid detects, with 10 times of volume PBS damping fluid (0.02mol/L phosphoric acid salt, pH7.4) washing is except foreigh protein removing, then 0.1M glycine buffer (pH3) is used by target protein from wash-out post, be new fusion protein VNI of the present invention.Detected by SDS-PAGE, the purity of this new fusion protein VNI reaches more than 90%.

Embodiment 2.VNI binding kinetics is analyzed

(1) the binding kinetics analysis of VNI and VEGF-A

By Loading, 25 μ g/mlBiotin-hVEGF-A are incorporated on SAsensor; According to preliminary experiment, concentration range is set: be respectively 10000,5000,1000,500,100nM, take SampleDiluentbuffer as blank.HVEGF-A and VNI is carried out in dynamic analysis in OctetQK detection platform, the kinetic parameter of VEGF-Trap and PD1 antibody detects, and testing sequence arranges as shown in table 1:

Table 1

Detect step number	Solution	Step type	Detection time (s)
				1	Sample Diluent buffer	Baseline	120
2	Biotin-hVEGF-A	Loading	360
				3	Sample Diluent buffer	Baseline	120
4	Sample	Association	720
				5	Sample Diluent buffer	Dissociation	1800

The experimental result obtained is detected as shown in table 2 by above-mentioned testing sequence:

Table 2 binding kinetics parameter compares

KD (M)

kon(1/Ms)

kon Error

kdis(1/s)

kdis Error

Full R^2

VNI

1.33E-10

4.34E+05

1.73E+04

5.79E-05

1.70E-05

0.99448

VEGF-Trap

0.83E-10

9.51E+05

6.11E+04

7.93E-05

3.23E-05

0.99368

PD-1 antibody

4.36E-03

1.20E+01

1.25E+01

5.25E-02

1.13E-03

0.91908

As above table 2 shows, and VNI still keeps the high-affinity with VEGF-A, in vitro still can well in conjunction with VEGF-A; But bonding force is weaker than VEGF-TRAP.And PD-1 antibody is not almost combined with VEGF-A.

(2) the binding kinetics analysis of VNI and PD-1

By Loading, 25 μ g/mlBiotin-hPD-1 are incorporated on SAsensor; According to preliminary experiment, concentration range is set: be respectively 10000,5000,1000,500,100nM, take SampleDiluentbuffer as blank.HPD-1 and VNI is carried out in dynamic analysis in OctetQK detection platform, the kinetic parameter of VEGF-Trap and PD1 antibody detects, and testing sequence arranges as shown in table 3:

Table 3

Detect step number	Solution	Step type	Detection time (s)
				1	Sample Diluent buffer	Baseline	120
2	Biotin-hPD-1	Loading	360
				3	Sample Diluent buffer	Baseline	120
4	Sample	Association	720
				5	Sample Diluent buffer	Dissociation	1800

The experimental result obtained is detected as shown in table 4 by above-mentioned testing sequence:

Table 4 binding kinetics parameter compares

KD (M)

kon(1/Ms)

kon Error

kdis(1/s)

kdis Error

Full R^2

VNI

5.5E-10

1.32E+05

7.74E+04

7.32E-05

2.37E-05

0.99646

VEGF-Trap

6.24E-3

3.07E+01

3.29E+02

1.92E-01

1.79E-06

0.89421

PD1 antibody

2.17E-10

9.71E+05

1.19E+03

2.11E-04

1.80E-06

0.99911

As above table 4 shows, and VNI and PD-1 keeps high-affinity to combine, but bonding force is weaker than PD-1 antibody.And PD-1 antibody fragment or antagonist and PD-1 bonding force very weak.

In sum, VNI still can keep the high affine combination with VEGF-A and PD-1 in vitro, infers that this molecule has efficient activity in vivo.

The In vitro biological activity of embodiment 3.VNI detects.

(1) comparative studies is affected on HUVEC cell proliferation

The HUVEC cell of logarithmic phase is inoculated in 96 well culture plates, 3 × 10 ³cells/ hole, 100 μ l/ holes, 37 DEG C, 5%CO ₂cultivate 20h; VNI, PD-1 antibody and the VEGF-Trap(9.7 ~ 10000ng/ml of different volumetric molar concentration is prepared with Base substratum) mix with the VEGF of 40ng/ml respectively, hatch 2h for 37 DEG C; Inoculation has on 96 orifice plates of HUVEC cell in addition, 100 μ l/ holes, often organizes 3 multiple holes, 37 DEG C, 5%CO ₂continue to hatch 96h, negative control group adds and adds Base substratum and perfect medium respectively; Add CCK-8 reagent, 20 μ l/well, 37 DEG C of lucifuges hatch 2h, and 450nm detects light absorption value, and as shown in Figure 1, experimental result is as shown in table 5 for the EC50 value curve synoptic diagram that VNI affects HUVEC cell proliferation.

Table 5

sample	EC50 (ng/ml)	standard Error	R-Square
				VNI	229.74	20.19	0.983
VEGF-Trap	197.62	17.59	0.979
				PD1 antibody	1.23E12	7.11E15	0.608

By table 5 and Fig. 1 display, the In vitro biological activity of VNI is a little less than VEGF-Trap, and PD-1 antibody stimulates HUVEC cell-proliferation activity substantially not have restraining effect to VEGF.

(2) cytomegalovirus lysate is stimulated to the impact of the cytokine secretion of PBMC cell

From the donor separating peripheral blood mononuclear cells (PBMC) of the CMV positive (once infecting cytomegalovirus), CMV lysate can stimulate this kind of cell to produce IFN γ, and anti-PD-1 antibody can strengthen CMV lysate stimulates PBMC to produce IFN γ.

105 people PMBC from CMV positive donor are cultivated in cumulative volume 200 μ l, and add CMV lysate in each hole.The anti-PD-1 antibody of various concentration, VNI and PD-1 antibody fragment or antagonist are added in each hole.Normal condition culturing cell, after the 4th day, is got 100 μ l substratum and is measured for cytokine IFN γ from every part of culture, and VNI stimulates PBMC to produce the curve synoptic diagram of IFN γ as shown in Figure 2, and measuring result is as shown in table 6.

Table 6

sample	EC50 (ng/ml)	standard Error	R-Square
				VNI	112.42	16.75	0.951
VEGF-Trap	--	--	--
				PD1 antibody	82.95	15.82	0.978

Shown by Fig. 2 and table 6, under the effect of CMV lysate, VNI is that concentration dependant stimulates PBMC cell to produce IFN γ, and when lower concentration, its ability to function is lower than PD-1 antibody, but suitable with the effect of PD-1 antibody when high density.VEGF-TRAP can not stimulate PBMC to produce IFN γ substantially.

Embodiment 4. mouse source VNI suppresses Growth of Tumors Transplanted Effect study in Mice Body

Because mouse PD-1 and humanization people source PD-1 antibody do not have cross action, and then cause people source PD-1 antibody cannot block PD-1 effect in Mice Body, can not play PD-1 antibody inhibiting tumor growth in Mice Body, thus the present invention adopts process in the VNI body of mouse source to implant to have the mouse of cancerous tumour to affect in the body checked antibodies on tumor and grow.

The preparation of mouse source PD-1 antibody and mouse source VNI: first use mouse PD-1 immune rat, with reference to " tissue culture and molecular cell learn a skill " (E Zheng chief editor) hybridoma preparation method, obtain anti-mouse PD-1 antibody gene, by this gene recombination in carrier for expression of eukaryon, obtain mouse source PD-1 antibody, this antibody is for subsequent use in contrast; Then, the another fab sequence people source anti-PD-1 antibody fab sequence of fusion rotein VNI in the present invention being replaced to mouse PD-1 antibody gene, form the fusion rotein mouse source VNI of anti-mouse PD-1 antibody and VEGF-TRAP, by the restructuring of this sequence in carrier for expression of eukaryon, obtain restructuring mouse source VNI through transfection, expression and purification.

According to body weight, the female AJ mouse between all for 6-8 ages is divided into 6 groups at random, often group comprises 10 animals.The 0th day by be dissolved in 200 μ lDMEM substratum 2 × 106 SA1/N fibrosarcoma cells on right side subcutaneous implantation mouse.With PBS contrast or the drug treating mouse of 10mg/kg.The the 1st, 4,8 and 11 day with about 200 μ l contain medicine PBS or contrast by peritoneal injection to animal dosed administration.Each group composed as follows: (1) PBS control group, (2) mouse source VNI group, (3) mouse source PD-1 antibody group, (4) VEGF-TRAP group.To mouse twice monitoring tumor growth weekly, continue about 8 weeks.Electronic caliper is used to carry out three-dimensional measurement (highly × width × length) to tumour and calculate gross tumor volume.When tumour to reach sacrifice when tumour terminal (1500mm3) or display are greater than 15% lose weight, account for per-cent as shown in table 7 after drug treating without tumor mouse, Tumor suppression situation about growing is as shown in Figure 3 in vivo for mouse source VNI.

Table 7

Group	Research mouse number of elements	Without tumour number of elements (only)	Without tumour percentage ratio (%)
				PBS group	10	0	0
Mouse source VNI group	10	7	70
				Mouse source PD-1 antibody group	10	3	30
VEGF-TRAP group	10	0	0

In above-mentioned contrast experiment, in PBS group, 9 mouse reached tumour terminal about 28 days, had 1 mouse tumour to fester; VNI group 3 mouse in mouse source reached tumour terminal at 56 days, and all the other 7 without tumour; PD-1 antibody group 7 mouse reached tumour terminal at 56 days in mouse source, and all the other 3 mouse are without tumour; VEGF-TRAP group has 6 mouse to reach tumour terminal at 42 days, and all the other 4 mouse reached tumour terminal at 49 days.

Describe known by Fig. 3, table 7 and above-mentioned experimental result: new fusion protein VNI of the present invention, it not only can have the function of two kinds of antibody hungry tumour cells of difference and activated T cell killing tumor cell simultaneously, and the collaborative promoter action of two kinds of functions can also be reached, the function that Tumor suppression is grown is more remarkable; Further, shown in Fig. 3, new fusion protein of the present invention is adopted also to have the effect effectively extending each antibody units physiological active functions effect.

Embodiment 5

In the present embodiment, the Nucleotide serialization row of X are different with the Nucleotide serialization row of Y, and the nucleotide coding sequence of X described in the present embodiment is SEQIDNO:4, and the nucleotide coding sequence of described Y is SEQIDNO:8.Described IgGFc fragment is IgG3Fc fragment, and this IgG3Fc fragment nucleotide coding sequence is SEQIDNO:14.

Embodiment 6

The difference of the present embodiment and embodiment 1 is: the fragment of IgGFc described in the present embodiment is IgG2Fc fragment, and this IgG2Fc fragment nucleotide coding sequence is SEQIDNO:12.

Above-described embodiment is only the preferred embodiments of the present invention, not limiting the scope of the invention, as long as adopt principle of design of the present invention, and the change carried out non-creativeness work on this basis and make, all should belong within protection scope of the present invention.

SEQUENCELISTING

All generations is won Bioisystech Co., Ltd in <110> Chengdu

<120> a new fusion protein, pharmaceutical composition and its production and use

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AlaTrpIleTrpTyrAspGlySerLysArgTyrTyrAlaAspSerVal

505560

LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuPhe

65707580

LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

859095

AlaThrAsnAspAspTyrTrpGlyGlnGlyThrLeuValThrValSer

100105110

SerGlyGlySerGlyGlySerGlyGlySerGlyGlySerGluIleVal

115120125

LeuThrGlnSerProAlaThrLeuSerLeuSerProGlyGluArgAla

130135140

ThrLeuSerCysArgAlaSerGlnSerValSerSerTyrLeuAlaTrp

145150155160

TyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrAspAla

165170175

SerAsnArgAlaThrGlyIleProAlaArgPheSerGlySerGlyPhe

180185190

GlyThrAspPheThrLeuThrIleSerSerLeuGluProGluAspPhe

195200205

AlaValTyrTyrCysGlnGlnSerSerAsnTrpProArgThrPheGly

210215220

GlnGlyThrLysValGluIleLys

225230

<210>6

<211>696

<212>DNA

<213> artificial sequence

<400>6

caggtgcagctggtggagtccggcggcggcgtggtgcagcctggccggtccctgcggctg60

gactgcaaggcctccggcatcaccttctccaactccggcatgcactgggtgcggcaggcc120

cctggcaagggcctggagtgggtggcctggatctggtacgacggctccaagcggtactac180

gccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccctgttc240

ctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgccaccaacgac300

gactactggggccagggcaccctggtgaccgtgtcctccggcgggtccggcgggtccggc360

gggtccggcgggtccgagatcgtgctgacccagtcccctgccaccctgtccctgtcccct420

ggcgagcgggccaccctgtcctgccgggcctcccagtccgtgtcctcctacctggcctgg480

taccagcagaagcctggccaggcccctcggctgctgatctacgacgcctccaaccgggcc540

accggcatccctgcccggttctccggctccggcttcggcaccgacttcaccctgaccatc600

tcctccctggagcctgaggacttcgccgtgtactactgccagcagtcctccaactggcct660

cggaccttcggccagggcaccaaggtggagatcaag696

<210>7

<211>232

<212>PRT

<213> is unknown

<400>7

GlnValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArg

151015

SerLeuArgLeuAspCysLysAlaSerGlyIleThrPheSerAsnSer

202530

GlyMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

354045

AlaTrpIleTrpTyrAspGlySerLysArgTyrTyrAlaAspSerVal

505560

LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuPhe

65707580

LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

859095

AlaSerAsnGluAspPheTrpGlyGlnGlyThrLeuValThrValSer

100105110

SerGlyGlySerGlyGlySerGlyGlySerGlyGlySerGluIleVal

115120125

LeuThrGlnSerProAlaThrLeuSerLeuSerProGlyGluArgAla

130135140

ThrLeuSerCysArgAlaSerGlnSerValSerSerTyrLeuAlaTrp

145150155160

TyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrAspAla

165170175

SerAsnArgAlaThrGlyIleProAlaArgPheSerGlySerGlyPhe

180185190

GlyThrAspPheThrLeuThrIleSerSerLeuGluProGluAspPhe

195200205

AlaValTyrTyrCysGlnGlnSerThrAsnPheProArgThrPheGly

210215220

GlnGlyThrLysValGluIleLys

225230

<210>8

<211>696

<212>DNA

<213> artificial sequence

<400>8

caggtgcagctggtggagtccggcggcggcgtggtgcagcctggccggtccctgcggctg60

gactgcaaggcctccggcatcaccttctccaactccggcatgcactgggtgcggcaggcc120

cctggcaagggcctggagtgggtggcctggatctggtacgacggctccaagcggtactac180

gccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccctgttc240

ctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgcctccaacgag300

gacttctggggccagggcaccctggtgaccgtgtcctccggcgggtccggcgggtccggc360

gggtccggcgggtccgagatcgtgctgacccagtcccctgccaccctgtccctgtcccct420

ggcgagcgggccaccctgtcctgccgggcctcccagtccgtgtcctcctacctggcctgg480

taccagcagaagcctggccaggcccctcggctgctgatctacgacgcctccaaccgggcc540

accggcatccctgcccggttctccggctccggcttcggcaccgacttcaccctgaccatc600

tcctccctggagcctgaggacttcgccgtgtactactgccagcagtccaccaacttccct660

cggaccttcggccagggcaccaaggtggagatcaag696

<210>9

<211>227

<212>PRT

<213> is unknown

<400>9

AspLysThrHisThrCysProProCysProAlaProGluLeuLeuGly

151015

GlyProSerValPheLeuPheProProLysProLysAspThrLeuMet

202530

IleSerArgThrProGluValThrCysValValValAspValSerHis

354045

GluAspProGluValLysPheAsnTrpTyrValAspGlyValGluVal

505560

HisAsnAlaLysThrLysProArgGluGluGlnTyrAsnSerThrTyr

65707580

ArgValValSerValLeuThrValLeuHisGlnAspTrpLeuAsnGly

859095

LysGluTyrLysCysLysValSerAsnLysAlaLeuProAlaProIle

100105110

GluLysThrIleSerLysAlaLysGlyGlnProArgGluProGlnVal

115120125

TyrThrLeuProProSerArgAspGluLeuThrLysAsnGlnValSer

130135140

LeuThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGlu

145150155160

TrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProPro

165170175

ValLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrVal

180185190

AspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMet

195200205

HisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSer

210215220

ProGlyLys

225

<210>10

<211>681

<212>DNA

<213> artificial sequence

<400>10

gacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtc60

ttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcaca120

tgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggac180

ggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtac240

cgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaag300

tgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaa360

gggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaag420

aaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggag480

tgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactcc540

gacggctccttcttcctctatagcaagctcaccgtggacaagagcaggtggcagcagggg600

aacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagc660

ctctccctgtctccgggtaaa681

<210>11

<211>227

<212>PRT

<213> is unknown

<400>11

ArgLysCysCysValGluCysProProCysProAlaProProValAla

151015

GlyProSerValPheLeuPheProProLysProLysAspThrLeuMet

202530

IleSerArgThrProGluValThrCysValValValAspValSerHis

354045

GluAspProGluValGlnPheAsnTrpTyrValAspGlyValGluVal

505560

HisAsnAlaLysThrLysProArgGluGluGlnPheAsnSerThrPhe

65707580

ArgValValSerValLeuThrValValHisGlnAspTrpLeuAsnGly

859095

LysGluTyrLysCysLysValSerAsnLysGlyLeuProAlaProIle

100105110

GluLysThrIleSerLysThrLysGlyGlnProArgGluProGlnVal

115120125

TyrThrLeuProProSerArgGluGluMetThrLysAsnGlnValSer

130135140

LeuThrCysLeuValLysGlyPheTyrProSerAspIleAlaValGlu

145150155160

TrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThrThrProPro

165170175

MetLeuAspSerAspGlySerPhePheLeuTyrSerLysLeuThrVal

180185190

AspLysSerArgTrpGlnGlnGlyAsnValPheSerCysSerValMet

195200205

HisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeuSerLeuSer

210215220

ProGlyLys

225

<210>12

<211>684

<212>DNA

<213> artificial sequence

<400>12

gagcgcaaatgttgtgtcgagtgcccaccgtgcccagcaccacctgtggcaggaccgtca60

gtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtc120

acgtgcgtggtggtggacgtgagccacgaagaccccgaggtccagttcaactggtacgtg180

gacggcgtggaggtgcataatgccaagacaaagccacgggaggagcagttcaacagcacg240

ttccgtgtggtcagcgtcctcaccgttgtgcaccaggactggctgaacggcaaggagtac300

aagtgcaaggtctccaacaaaggcctcccagcccccatcgagaaaaccatctccaaaacc360

aaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgacc420

aagaaccaggtcagcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtg480

gagtgggagagcaatgggcagccggagaacaactacaagaccacacctcccatgctggac540

tccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcag600

gggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaag660

agcctctccctgtctccgggtaaa684

<210>13

<211>264

<212>PRT

<213> is unknown

<400>13

GluLeuLysThrProLeuGlyAspThrThrHisThrCysProArgCys

151015

ProGluProLysSerCysAspThrProProProCysProArgCysPro

202530

GluProLysSerCysAspThrProProProCysProArgCysProAla

354045

ProGluLeuLeuGlyGlyProSerValPheLeuPheProProLysPro

505560

LysAspThrLeuMetIleSerArgThrProGluValThrCysValVal

65707580

ValAspValSerHisGluAspProGluValGlnPheLysTrpTyrVal

859095

AspGlyValGluValHisAsnAlaLysThrLysProArgGluGluGln

100105110

PheAsnSerThrPheArgValValSerValLeuThrValLeuHisGln

115120125

AspTrpLeuAsnGlyLysGluTyrLysCysLysValSerAsnLysAla

130135140

LeuProAlaProIleGluLysThrIleSerLysThrLysGlyGlnPro

145150155160

ArgGluProGlnValTyrThrLeuProProSerArgGluGluMetThr

165170175

LysAsnGlnValSerLeuThrCysLeuValLysGlyPheTyrProSer

180185190

AspIleAlaValGluTrpGluSerSerGlyGlnProGluAsnAsnTyr

195200205

AsnThrThrProProMetLeuAspSerAspGlySerPhePheLeuTyr

210215220

SerLysLeuThrValAspLysSerArgTrpGlnGlnGlyAsnIlePhe

225230235240

SerCysSerValMetHisGluAlaLeuHisAsnArgPheThrGlnLys

245250255

SerLeuSerLeuSerProGlyLys

260

<210>14

<211>792

<212>DNA

<213> artificial sequence

<400>14

gagctcaaaaccccacttggtgacacaactcacacatgcccacggtgcccagagcccaaa60

tcttgtgacacacctcccccgtgcccacggtgcccagagcccaaatcttgtgacacacct120

cccccatgcccacggtgcccagcacctgaactcctgggaggaccgtcagtcttcctcttc180

cccccaaaacccaaggatacccttatgatttcccggacccctgaggtcacgtgcgtggtg240

gtggacgtgagccacgaagaccccgaggtccagttcaagtggtacgtggacggcgtggag300

gtgcataatgccaagacaaagccgcgggaggagcagttcaacagcacgttccgtgtggtc360

agcgtcctcaccgtcctgcaccaggactggctgaacggcaaggagtacaagtgcaaggtc420

tccaacaaagccctcccagcccccatcgagaaaaccatctccaaaaccaaaggacagccc480

cgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtc540

agcctgacctgcctggtcaaaggcttctaccccagcgacatcgccgtggagtgggagagc600

agcgggcagccggagaacaactacaacaccacgcctcccatgctggactccgacggctcc660

ttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacatcttc720

tcatgctccgtgatgcatgaggctctgcacaaccgcttcacgcagaagagcctctccctg780

tctccgggtaaa792

<210>15

<211>229

<212>PRT

<213> is unknown

<400>15

AlaGluSerLysTyrGlyProProCysProProCysProAlaProGlu

151015

AlaAlaGlyGlyProSerValPheLeuPheProProLysProLysAsp

202530

ThrLeuMetIleSerArgThrProGluValThrCysValValValAsp

354045

ValSerGlnGluAspProGluValGlnPheAsnTrpTyrValAspGly

505560

ValGluValHisAsnAlaLysThrLysProArgGluGluGlnPheAsn

65707580

SerThrTyrArgValValSerValLeuThrValLeuHisGlnAspTrp

859095

LeuAsnGlyLysGluTyrLysCysLysValSerAsnLysGlyLeuPro

100105110

SerSerIleGluLysThrIleSerLysAlaLysGlyGlnProArgGlu

115120125

ProGlnValTyrThrLeuProProSerGlnGluGluMetThrLysAsn

130135140

GlnValSerLeuThrCysLeuValLysGlyPheTyrProSerAspIle

145150155160

AlaValGluTrpGluSerAsnGlyGlnProGluAsnAsnTyrLysThr

165170175

ThrProProValLeuAspSerAspGlySerPhePheLeuTyrSerArg

180185190

LeuThrValAspLysSerArgTrpGlnGluGlyAsnValPheSerCys

195200205

SerValMetHisGluAlaLeuHisAsnHisTyrThrGlnLysSerLeu

210215220

SerLeuSerLeuGly

225

<210>16

<211>687

<212>DNA

<213> artificial sequence

<400>16

gctgagtccaagtatggccctccctgccctccttgccctgctcctgaggctgctggaggc60

cctagcgtgttcctgttcccccctaagcctaaggacaccctgatgatttcccggaccccc120

gaggtgacctgtgtggtggtggatgtgtcccaggaggaccctgaagtgcagttcaactgg180

tacgtggacggcgtggaggtgcacaacgccaagaccaagccccgggaagagcagttcaac240

agcacctacagggtggtgagcgtgctgaccgtgctgcaccaggactggctgaacggcaaa300

gagtacaagtgcaaggtgagcaataagggcctgccctcctccatcgagaagaccatttcc360

aaggccaagggccagcccagggaaccccaggtgtacaccctccctcccagccaggaggag420

atgaccaagaaccaggtgtccctgacctgcctggtgaaaggcttctacccctccgacatt480

gccgtcgagtgggaaagcaacggccagcccgagaacaattacaagaccacaccccccgtg540

ctggacagcgatggcagctttttcctgtactccaggctgaccgtcgacaagtccaggtgg600

caggagggcaacgtcttctcctgctccgtgatgcatgaggccctgcacaaccactacacc660

cagaagtccctgtccctgagcctgggc687

<210>17

<211>684

<212>PRT

<213> is unknown

<400>17

GlyArgProPheValGluMetTyrSerGluIleProGluIleIleHis

151015

MetThrGluGlyArgGluLeuValIleProCysArgValThrSerPro

202530

AsnIleThrValThrLeuLysLysPheProLeuAspThrLeuIlePro

354045

AspGlyLysArgIleIleTrpAspSerArgLysGlyPheIleIleSer

505560

AsnAlaThrTyrLysGluIleGlyLeuLeuThrCysGluAlaThrVal

65707580

AsnGlyHisLeuTyrLysThrAsnTyrLeuThrHisArgGlnThrAsn

859095

ThrIleIleAspValValLeuSerProSerHisGlyIleGluLeuSer

100105110

ValGlyGluLysLeuValLeuAsnCysThrAlaArgThrGluLeuAsn

115120125

ValGlyIleAspPheAsnTrpGluTyrProSerSerLysHisGlnHis

130135140

LysLysLeuValAsnArgAspLeuLysThrGlnSerGlySerGluMet

145150155160

LysLysPheLeuSerThrLeuThrIleAspGlyValThrArgSerAsp

165170175

GlnGlyLeuTyrThrCysAlaAlaSerSerGlyLeuMetThrLysLys

180185190

AsnSerThrPheValArgValHisGluLysGlyGlySerGlyGlySer

195200205

GlnValGlnLeuValGluSerGlyGlyGlyValValGlnProGlyArg

210215220

SerLeuArgLeuAspCysLysAlaSerGlyIleThrPheSerAsnSer

225230235240

GlyMetHisTrpValArgGlnAlaProGlyLysGlyLeuGluTrpVal

245250255

AlaTrpIleTrpTyrAspGlySerLysArgTyrTyrAlaAspSerVal

260265270

LysGlyArgPheThrIleSerArgAspAsnSerLysAsnThrLeuPhe

275280285

LeuGlnMetAsnSerLeuArgAlaGluAspThrAlaValTyrTyrCys

290295300

AlaThrAsnAspAspTyrTrpGlyGlnGlyThrLeuValThrValSer

305310315320

SerGlyGlySerGlyGlySerGlyGlySerGlyGlySerGluIleVal

325330335

LeuThrGlnSerProAlaThrLeuSerLeuSerProGlyGluArgAla

340345350

ThrLeuSerCysArgAlaSerGlnSerValSerSerTyrLeuAlaTrp

355360365

TyrGlnGlnLysProGlyGlnAlaProArgLeuLeuIleTyrAspAla

370375380

SerAsnArgAlaThrGlyIleProAlaArgPheSerGlySerGlyPhe

385390395400

GlyThrAspPheThrLeuThrIleSerSerLeuGluProGluAspPhe

405410415

AlaValTyrTyrCysGlnGlnSerSerAsnTrpProArgThrPheGly

420425430

GlnGlyThrLysValGluIleLysGlyGlyGlyGlySerGlyGlyGly

435440445

GlySerGlyGlyGlyGlySerAlaGluSerLysTyrGlyProProCys

450455460

ProProCysProAlaProGluAlaAlaGlyGlyProSerValPheLeu

465470475480

PheProProLysProLysAspThrLeuMetIleSerArgThrProGlu

485490495

ValThrCysValValValAspValSerGlnGluAspProGluValGln

500505510

PheAsnTrpTyrValAspGlyValGluValHisAsnAlaLysThrLys

515520525

ProArgGluGluGlnPheAsnSerThrTyrArgValValSerValLeu

530535540

ThrValLeuHisGlnAspTrpLeuAsnGlyLysGluTyrLysCysLys

545550555560

ValSerAsnLysGlyLeuProSerSerIleGluLysThrIleSerLys

565570575

AlaLysGlyGlnProArgGluProGlnValTyrThrLeuProProSer

580585590

GlnGluGluMetThrLysAsnGlnValSerLeuThrCysLeuValLys

595600605

GlyPheTyrProSerAspIleAlaValGluTrpGluSerAsnGlyGln

610615620

ProGluAsnAsnTyrLysThrThrProProValLeuAspSerAspGly

625630635640

SerPhePheLeuTyrSerArgLeuThrValAspLysSerArgTrpGln

645650655

GluGlyAsnValPheSerCysSerValMetHisGluAlaLeuHisAsn

660665670

HisTyrThrGlnLysSerLeuSerLeuSerLeuGly

675680

<210>18

<211>2159

<212>DNA

<213> artificial sequence

<400>18

agatctcctagggccaccatggtcagctactgggacaccggggtcctgctgtgcgcgctg60

ctcagctgtctgcttctcacaggatctagttccggaggtagacctttcgtagagatgtac120

agtgaaatccccgaaattatacacatgactgaaggaagggagctcgtcattccctgccgg180

gttacgtcacctaacatcactgttactttaaaaaagtttccacttgacactttgatccct240

gatggaaaacgcataatctgggacagtagaaagggcttcatcatatcaaatgcaacgtac300

aaagaaatagggcttctgacctgtgaagcaacagtcaatgggcatttgtataagacaaac360

tatctcacacatcgacaaaccaatacaatcatagatgtggttctgagtccgtctcatgga420

attgaactatctgttggagaaaagcttgtcttaaattgtacagcaagaactgaactaaat480

gtggggattgacttcaactgggaatacccttcttcgaagcatcagcataagaaacttgta540

aaccgagacctaaaaacccagtctgggagtgagatgaagaaatttttgagcaccttaact600

atagatggtgtaacccggagtgaccaaggattgtacacctgtgcagcatccagtgggctg660

atgaccaagaagaacagcacatttgtcagggtccatgaaaagggcgggtccggcgggtcc720

caggtgcagctggtggagtccggcggcggcgtggtgcagcctggccggtccctgcggctg780

gactgcaaggcctccggcatcaccttctccaactccggcatgcactgggtgcggcaggcc840

cctggcaagggcctggagtgggtggcctggatctggtacgacggctccaagcggtactac900

gccgactccgtgaagggccggttcaccatctcccgggacaactccaagaacaccctgttc960

ctgcagatgaactccctgcgggccgaggacaccgccgtgtactactgcgccaccaacgac1020

gactactggggccagggcaccctggtgaccgtgtcctccggcgggtccggcgggtccggc1080

gggtccggcgggtccgagatcgtgctgacccagtcccctgccaccctgtccctgtcccct1140

ggcgagcgggccaccctgtcctgccgggcctcccagtccgtgtcctcctacctggcctgg1200

taccagcagaagcctggccaggcccctcggctgctgatctacgacgcctccaaccgggcc1260

accggcatccctgcccggttctccggctccggcttcggcaccgacttcaccctgaccatc1320

tcctccctggagcctgaggacttcgccgtgtactactgccagcagtcctccaactggcct1380

cggaccttcggccagggcaccaaggtggagatcaagggcggcggaggctccggcggaggc1440

ggctccggcggcggcggctccgctgagtccaagtatggccctccctgccctccttgccct1500

gctcctgaggctgctggaggccctagcgtgttcctgttcccccctaagcctaaggacacc1560

ctgatgatttcccggacccccgaggtgacctgtgtggtggtggatgtgtcccaggaggac1620

cctgaagtgcagttcaactggtacgtggacggcgtggaggtgcacaacgccaagaccaag1680

ccccgggaagagcagttcaacagcacctacagggtggtgagcgtgctgaccgtgctgcac1740

caggactggctgaacggcaaagagtacaagtgcaaggtgagcaataagggcctgccctcc1800

tccatcgagaagaccatttccaaggccaagggccagcccagggaaccccaggtgtacacc1860

ctccctcccagccaggaggagatgaccaagaaccaggtgtccctgacctgcctggtgaaa1920

ggcttctacccctccgacattgccgtcgagtgggaaagcaacggccagcccgagaacaat1980

tacaagaccacaccccccgtgctggacagcgatggcagctttttcctgtactccaggctg2040

accgtcgacaagtccaggtggcaggagggcaacgtcttctcctgctccgtgatgcatgag2100

gccctgcacaaccactacacccagaagtccctgtccctgagcctgggctgattaattaa2159

Claims (10)

1. a new fusion protein, is characterized in that, its general structure is as follows:

X-connection peptides-Y-connection peptides-IgGFc fragment or Y-connection peptides-X-connection peptides-IgGFc fragment;

Wherein, X is VEGF-Trap and derivative thereof; Y is PD-1 antibody fragment and derivative thereof or PD-1 antagonist fragment and derivative thereof.

2. a kind of new fusion protein according to claim 1, is characterized in that, described connection peptides is the aminoacid sequence shown in formula (Gly-Gly-Gly-Gly-Ser) n, and this n is the integer of 1 ~ 4.

3. a kind of new fusion protein according to claim 1, is characterized in that, the aminoacid sequence of described X is SEQIDNO:1 or SEQIDNO:3, and the aminoacid sequence of described Y is SEQIDNO:5 or SEQIDNO:7.

4. a kind of new fusion protein according to claim 1, is characterized in that, the nucleotide coding sequence of described X is SEQIDNO:2 or SEQIDNO:4, and the nucleotide coding sequence of described Y is SEQIDNO:6 or SEQIDNO:8.

5. a kind of new fusion protein according to claim 1, is characterized in that, described IgGFc fragment is IgG1Fc fragment, IgG2Fc fragment, IgG3Fc fragment or IgG4Fc fragment; This IgG1Fc fragment amino acid sequence is SEQIDNO:9, and this IgG2Fc fragment amino acid sequence is SEQIDNO:11, and this IgG3Fc fragment amino acid sequence is SEQIDNO:13, and this IgG4Fc fragment amino acid sequence is SEQIDNO:15.

6. a kind of new fusion protein according to claim 1, is characterized in that, described IgGFc fragment is IgG1Fc fragment, IgG2Fc fragment, IgG3Fc fragment or IgG4Fc fragment; This IgG1Fc fragment nucleotide coding sequence is SEQIDNO:10, this IgG2Fc fragment nucleotide coding sequence is SEQIDNO:12, this IgG3Fc fragment nucleotide coding sequence is SEQIDNO:14, and this IgG4Fc fragment nucleotide coding sequence is SEQIDNO:16.

7. prepare the method for new fusion protein for one kind, it is characterized in that, the coding nucleotide sequence of new fusion protein described in any one of claim 1-6 is inserted in expression vector, and this expression vector is introduced corresponding expression system and then carry out the expression of fusion rotein.

8. a pharmaceutical composition, is characterized in that, is made up of with pharmaceutically acceptable carrier or vehicle the new fusion protein described in any one of claim 1-6.

9. a kind of pharmaceutical composition according to claim 8, is characterized in that, the dosage form of described pharmaceutical composition is injection, injection freeze-dried powder.

10. a purposes for new fusion protein, is characterized in that, the new fusion protein described in described any one of claim 1-6 is used for the treatment of tumour; Described tumour is one or more in melanoma, lung cancer, liver cancer, lymphoma, colorectal carcinoma, large bowel cancer, mammary cancer, ovarian cancer, cervical cancer, cancer of the stomach, cholangiocarcinoma, carcinoma of gallbladder, the esophageal carcinoma, kidney, neurospongioma, melanoma, carcinoma of the pancreas and prostate cancer.