CN110128551A

CN110128551A - It is a kind of for the multi-functional fusion protein of CD19+ tumour and its application

Info

Publication number: CN110128551A
Application number: CN201910487593.1A
Authority: CN
Inventors: 岳喜连; 张传能; 张朝宾; 徐圣涛; 吴国祥
Original assignee: Shanghai Science And Game Pharmaceutical Technology Co Ltd
Current assignee: Shanghai Science And Game Pharmaceutical Technology Co Ltd
Priority date: 2019-06-05
Filing date: 2019-06-05
Publication date: 2019-08-16

Abstract

The present invention relates to a kind of fusion proteins for CD19+ tumour, inhibit the extracellular functional areas SIRP α, the high-affinity IgG1Fc in conjunction with Fc receptor and the non-functional amino acid fragment for connection function area of 7 signal of antigen CD4 including the antibody function area of specific recognition CD19 antigen, shielding tumour immunity；The invention further relates to the preparation of above-mentioned fusion protein and applications.Recombination fusion protein of the present invention can identify the tumour cell of the CD19 positive, CD47 immunosupress signal path can be shielded again, immune response of the immunocyte to tumour can be enhanced in the part Fc of high-affinity simultaneously, has good clinical landscapes and is widely applied range.

Description

It is a kind of for the multi-functional fusion protein of CD19+ tumour and its application

Technical field

The present invention relates to field of biotechnology more particularly to a kind of multi-functional fusion protein for CD19 positive tumor and Its application in treating cancer.

Background technique

Various tumours escape immune control (Cancer Res.2011 by the activation of CD47/SIRP alpha signal access；71 (4): 1374-84).The tumour cell of height expression CD47 can inhibit to express phagocytosis of the SIRP α macrophage to tumour, into And the function of antigen presenting cell is reduced, body is influenced to secondary natural disposition immunocompetence (the Nat Med.2015 of tumour；21 (10): 1209-15), so being negatively correlated property of the clinical effectiveness (Cell.2009 of the density of tumour CD47 expression and cancer patient；138 (2): 286-99).CD47/SIRP alpha signal access is shielded using the antibody drug for CD47 can increase macrophage pair Phagocytosis (the Cell.2010 of tumour；142 (5): 699-713).But since CD47 is generally in red blood cell and blood platelet Expression, so shielding CD47/SIRP α can not only cause serious side effect (Blood.2006 merely；107 (6): 2548-56), The antibody drug for being also required to larger dose can be only achieved the purpose of shielding (J Exp Med 2001,193:855-862).Meanwhile Since the affinity of antibody is higher, the simple immunosuppressive action using antibody be will cause to the clear of expression CD47 normal cell It removes, such as red blood cell is caused to lack, cause extreme anaemia phenomenon (eLife 2017；6:e18173).Due to SIRP α and CD47 parent With power is weaker and body in flood tide expression CD47 cell presence, so shield using SIRP α the effect of CD47 merely Also undesirable, clinical effectiveness is also poor.

Up to now, clinically reach resistance using high-affinity segment increase target cell peripheral protein amount there are no a kind of Disconnected low-affinity inhibits the protein drug of signal path, and simple function has treatment for the drug of CD19 positive tumor cell Imitate low disadvantage.Fusion protein of the invention can overcome the above deficiency, this field blank of substituting.

Summary of the invention

It is an object of the invention to overcome the shortcomings of existing clinical medicine, a kind of fusion protein is synthesized, which includes needle Antibody moiety to CD19 and the SIRP α segment for CD47.It is identified using the high-affinity of antibody and CD19 and combines CD19 Positive tumour cell increases the SIRP α protein concentration near tumour cell, promotes the combination of low-affinity SIRP α and CD47, The function of reaching shielding CD47 signal completely, reduces the immunosupress generated because tumour cell height expresses CD47, increases immune The function of cell recognition and phagocytosis CD19 positive tumor cell.A kind of more function for CD19 tumour cell provided by the present invention Energy fusion protein can increase to the low affine blocking effect for inhibiting signal path, improve phagocytosis of the immunocyte to tumour, thus Inhibition and removing of the immune system to tumour can be improved, improve the curative effect of clinical application.

To achieve the above object, the present invention adopts the following technical scheme:

The first purpose of the invention is to provide a kind of multi-functional fusion proteins for CD19+ tumour comprising identification The antibody function area of tumour antigen CD19, the extracellular functional areas SIRP α for shielding tumor surface immune suppressive protein, in conjunction with immune thin The functional areas of born of the same parents' Fc receptor and non-functional amino acid fragment for connection function area.Above three functional areas pass through NOT function Can the connection of acidic amino acid segment, so that the protein folding of each functional areas be avoided to interfere with each other with function, can and meanwhile with tumour CD47 and CD19 is combined, and enhances the ADCC effect of NK cell and the ADCP effect of macrophage, promotes the killing to tumor tissues And removing, play the multi-functional feature of fusion protein.

In order to advanced optimize above-mentioned multi-functional fusion protein, the technical measures that the present invention takes further include:

Further, the antibody of the identification tumour antigen CD19 is to be capable of specific recognition tumor-cell antigen CD19's Antibody；Specially it is directed to the single-chain antibody variable region of CD19.

Further, the tumor surface immune suppressive protein is CD47, and fusion protein shields the extracellular functional areas of CD47 For SIRP α, immunologic test point access refers to CD47/SIRP alpha signal access.Such Inhibitory receptor CD47 is in tumour cell table Face overexpression, causes tumour cell to escape the killing and removing of immunocyte.Fusion protein S IRP α segment portion blocking immunity The combination of cell SIRP α and CD47, and then block the transmitting of inhibition signal.Fusion protein antibody moiety combination tumour cell CD19 antigen increases enrichment of the Protein S IRP α segment around tumor tissues, enhances the combination to tumour CD47 and signal shielding Intensity.

Further, the combined area of the immunocyte is IgG1Fc, being capable of high affine combination immunocyte Fc receptor.Its In, through IgG1Fc in conjunction with Fc receptor, promote immunocyte ADCC and the ADCP reaction of tumor target cell peripheral, enhancing killing Property immunocyte prevents undershooting-effect caused by single targeted integration to the lethal effect of tumour target cell.

Further, the functional areas of the fusion protein are connected by the flexible segment of amino acid, guarantee the function of each functional areas Can be relatively independent, the amino acid sequence for connecting the fusion protein functional areas includes sequence shown in SEQ ID NO:5.

Further, the complete amino acid sequence of SIRP α is SEQ ID NO:1 (GenBank:BC038510.2) institute Show sequence.

Further, the complete amino acid sequence of the CD19 antibody scFv heavy chain Fab is SEQ ID NO:2 Sequence shown in (Sequence ID:AAB34429.1).

Further, the complete amino acid sequence of the CD19 antibody scFv light chain Fab is SEQ ID NO:3 (GenBank:AAB34430.1) sequence shown in.

Further, in the fusion protein the complete amino acid sequence of human IgG1 Fc be SEQ ID NO:4 (GenBank: Sequence shown in 4X4M_A).

Further, the amino acid sequence of the SIRP α is the site 31-150 one in sequence shown in SEQ ID NO:1 A or multiple repeated fragments, or the mutant with similar functions；The amino acid sequence of the CD19 heavy chain of antibody Fab is SEQ The site 1-121 one or more in sequence shown in ID NO:2 repeats, or the mutant with similar functions；The CD19 is anti- The amino acid sequence of body light chain Fab is that the site the 1-114 one or more in sequence shown in SEQ ID NO:3 repeats, or has The mutant of similar functions；The amino acid sequence of the IgG1Fc is the site 3-219 or tool in sequence shown in SEQ ID NO:4 There is the mutant of similar functions；The amino acid sequence for connection function area is sequence site one shown in SEQ ID NO:5 A or multiple repetitive sequences.

Further, the amino acid sequence of the multi-functional fusion protein for sequence shown in SEQ ID NO:6 or has class Like the mutant of function.

The sequence of above-mentioned each amino acid is as shown in the table:

Further, the step of preparing the fusion protein include:

Step 1) synthesizes the corresponding base of amino acid sequence, group shown in SEQ ID NO:6 by artificial synthesized mode At fusion protein structural gene；

Fusion protein structural gene described in step 1) is transferred to mammalian expression vector by step 2), and is transfected to hamster Gonad cell (CHO) carries out protein expression；

Hamster ovary cell described in step 2) is placed in incubator and cultivates a period of time by step 3), takes supernatant, leads to Recombination fusion protein is made after crossing chromatographic purifying.

Further, the construction step of the fusion protein structural gene include: according to SIRP α, anti-CD19 Fab and The corresponding base of functional areas amino acid of IgG1Fc connects into more function by gene chemical synthesis nand function amino acid fragment base It can fusion protein structural gene；

Further, the mammalian expression vector is eukaryon animal expression vector pCDNA3.1 (-).

Further, the concrete operations of the step 3) are as follows: the hamster ovary cell cell of transfection is placed in 37 DEG C, 5% CO₂It is cultivated in incubator, takes supernatant after 72 hours, by ProteinA affinitive layer purification, obtained multi-functional recombination and merge egg It is white.

A second object of the present invention is to provide a kind of any above-mentioned multi-functional fusion proteins for treating expression The clinical tumor application of CD47 and CD19.

Further, the application be fusion protein be used alone or with chemotherapy, targeted drug, antibody drug, cell The use in conjunction of composition is treated, to be used to prepare the drug of disease caused by treatment expression CD19 and CD47 tumour.

Further, the disease includes all tumours with high expression CD19 and CD47 antigen.

Compared with prior art, fusion protein of the present invention has the advantages that

Multi-functional fusion protein obtained by the present invention combines tumour thin using the anti-CD 19 antibodies part of high-affinity Born of the same parents promote the SIRP α protein enrichment of near tumor cells, enhance in conjunction with CD47 and the SIRP α of low-affinity, block inhibition letter Number access increases the function of relying on the immunocyte phagocytosis tumour target cell that Fc signal generates.

Fusion protein of the present invention can satisfy the needs of tumor patient immunization therapy, which can The tumour cell for identifying CD19 positive, and can shield CD47 immunosupress signal path, while the Fc of high-affinity partially can be with Increase immune response of the immunocyte to tumour cell；Thus the function of inhibiting tumour growth can be enhanced in clinical application, has Good clinical landscapes and it is widely applied range.

Detailed description of the invention

Fig. 1 is the recombination fusion protein gel electrophoresis analysis figure prepared in one embodiment of the invention.

Fig. 2 is detection fusion albumen and CD19 antibody and the test of human tumor cell line affinity in one embodiment of the invention Result schematic diagram.

Fig. 3 is terraced to Raji cell strain CD47 signal shielding concentration for the recombination fusion protein prepared in one embodiment of the invention Spend experimental result picture.

Fig. 4 is that the recombination fusion protein prepared in one embodiment of the invention promotes human peripheral to remove Raji tumour cell The result schematic diagram of in vitro test.

Fig. 5 is that the recombination fusion protein prepared in one embodiment of the invention inhibits Raji tumour in NSG mouse peripheral blood thin The ratio chart of born of the same parents.

Fig. 6 is that the recombination fusion protein prepared in one embodiment of the invention influences the NSG mouse survival of inoculation Raji cell Curve graph.

Specific embodiment

The present invention provides a kind of multi-functional fusion proteins for expression CD19 tumour comprising identification tumour antigen The antibody function area of CD19, the extracellular functional areas for shielding tumor surface immune suppressive protein CD47, in conjunction with immunocyte Fc receptor Functional areas and non-functional amino acid fragment for connecting each functional areas.The present invention also provides above-mentioned fusion proteins Preparation method and its expression CD47 and CD19 tumour is treated in preparation and causes the medicinal application of disease.More function of the present invention Energy fusion protein, functional areas and the functional areas in conjunction with immunocyte comprising two targeting tumor cells, function Energy area is connected by the non-functional amino acid fragment of certain length, carries out fusion protein by mammalian cell expression mode Preparation.

With reference to the accompanying drawings and examples, further description of the specific embodiments of the present invention.Following embodiment is only For clearly illustrating technical solution of the present invention, and not intended to limit the protection scope of the present invention.

Embodiment 1

The present embodiment is the gene constructed of recombination fusion protein and production purifying.

According to the functional areas amino acid of people SIRP α, anti-CD19Fab and IgG1Fc (in sequence shown in SEQ ID NO:1 The site 1-114 in sequence shown in the site 1-121, SEQ ID NO:3 in sequence shown in the site 31-150, SEQ ID NO:2, The corresponding base in the site 3-219 in sequence shown in SEQ ID NO:4 passes through the flexible piece of gene chemical synthesis nand function amino acid Section (sequence shown in SEQ ID NO:5 or its repetitive sequence) base connects into multi-functional fusion protein (SEQ ID NO:6) gene, By digestion and further clone, it is then transferred to eukaryon animal expression vector pcDNA3.1 (-).Fusion protein base will finally be contained The carrier of cause is transfected into Chinese hamster ovary cell (CHO).The cell of transfection is placed in 37 DEG C, 5%CO₂It is cultivated in incubator, 7 days After take supernatant, further pass through ProteinA affinitive layer purification.The albumen of final purification is multi-functional recombination fusion protein. The albumen of purifying is by electrophoresis detection confirmation molecular weight and purity of protein, it was demonstrated that the recombination fusion protein designed according to the present invention can To be produced and be purified by Chinese hamster ovary celI.Finally use spectrophotometer test proteins concentration, be stored in PBS after dilution, be used as into The active testing of one step inside and outside and functional study.Be shown in Fig. 1 using gel electrophoresis test after purification albumen as a result, table Bright fusion protein molecule amount meets the theory expectation of design and proves the feasibility of production purifying.

Embodiment 2

The present embodiment is multi-functional recombination fusion protein to the affine test of CD19 positive tumor cell.

Affine curve determination of the albumen to CD19 cell: the Raji tumor cell line of the CD19 positive, every hole 1 × 10 are taken⁴Carefully Born of the same parents are placed in 96 orifice plates in the PBS of 0.1ml, and the fusion protein and CD19 antibody protein of various concentration PE label is added, and mix postposition In 4 DEG C 0.5 hour.Cell washed once after collecting with PBS, and the survey fixed sum data point of PE fluorescence is finally carried out with flow cytometer Analysis.It is shown in Fig. 2, for CD19 positive cell, fluorescence signal (MFI) and the fusion protein concentration of PE is positively correlated, and merges egg It is white that there is similar affine curve with CD19.This embodiment proves that fusion protein can be in conjunction with the tumour cell of the CD19 positive, card Bright fusion protein is to the good targeting of CD19.

Embodiment 3

The present embodiment is shielding action of the multi-functional recombination fusion protein to CD47 signal path.

It will be diluted in 1 × 10 in 100 μ l PBS⁴CD19 positive Raji cell is transferred in 96 orifice plates, is then respectively adding The fusion protein of various concentration is placed 20 minutes at room temperature, and the anti-human CD47 streaming antibody dye of APC label is added after washed once Color is placed at room temperature for 15 minutes, is measured APC fluorescence signal with flow cytometer after washing and data are analyzed.It is shown in Fig. 3 Signal strength (APC fluorescence intensity) of the CD47 antibody of flow cytometer showed in conjunction with CD19 positive cell.As can be seen that fusion in figure Concentration gradient is presented to the shielding of CD47 antibody in albumen.It can be seen that fusion protein of the invention can shield CD47 completely Signal path.

Embodiment 4

The present embodiment is that multi-functional recombination fusion protein promotes peripheral blood to CD19 positive cell lethal effect.

Placing the 1 × 10 of CFSE label⁵Raji tumour cell is in 100 μ l DMEM culture mediums, in 37 DEG C of incubators Be incubated for 24 hours, 100 μ l human peripherals be then added into above-mentioned tumour cell, be eventually adding fusion protein to final concentration of 1, 10,100 μ g/ml do not have the hole of fusion protein as a control group as fusion protein processing group.After continuing culture 4 hours, then It is analyzed with flow cytometer.It is represented in Fig. 4, compared with the control group, fusion protein processing considerably reduces swollen in blood Oncocyte, and negative correlation is presented in the ratio of tumour cell and protein concentration, it was demonstrated that and fusion protein, which promotes in peripheral blood, exempts from The killing of epidemic disease cells against tumor cells.

Embodiment 5

The present embodiment is the NSG mouse survival that multi-functional recombination fusion protein extends infection Raji cell.

By tail vein in every NSG mouse irradiation 1 × 10⁵Raji tumour cell is divided into two groups after raising 5 days.Locating 400 μ g fusion proteins are added by intraperitoneal injection in reason group, inject PBS or CD19 antibody group as a control group, weekly administration two It is secondary.It takes peripheral blood within 12nd day, is analyzed on flow cytometer with after the dyeing of the antibody of anti-CD20.It indicates, and compares in Fig. 5 Group is compared, and fusion protein processing considerably reduces the tumour cell (CD20+) in blood, it was demonstrated that fusion protein promotes peripheral blood Killing of the middle immunocyte to tumour cell.Continue the activity situation of raising with observation mouse, with mouse loss of activity work For mouse diing time, finally mouse survival curve (Fig. 6) is drawn.From fig. 6 it can be seen that simple CD19 antibody can not It is obviously prolonged mouse survival, and fusion protein group can then be obviously prolonged the existence of mice with tumor.

The present embodiment is effect of the multi-functional recombination fusion protein in treatment CD19 positive tumor.Above-mentioned multi-functional recombination is melted Hop protein can be used alone or and chemotherapy, targeted drug, antibody drug, cell therapy form use in conjunction.

As can be seen from the above embodiments, multi-functional recombination fusion protein of the present invention can identify that CD19 is positive and swell Oncocyte, but the CD47 that can shield tumor cell surface inhibits signal, increases removing of the immunocyte to CD19 positive cell.By In the generation and diffusion of tumour be tumour cell by the high expression CD47 immunologic escape generated as a result, and fusion protein both The immunosupress access of tumour can be blocked, and killing of the immunocyte to tumour can be promoted.Therefore, above-mentioned fusion protein The inhibition to tumour can be enhanced in clinical application, has good clinical landscapes and is widely applied range.

Specific embodiments of the present invention are described in detail above, but it is only used as example, the present invention is not intended to limit In particular embodiments described above.To those skilled in the art, any principle according to the present invention is modified and is replaced In generation, also can achieve effect same.Therefore, made equal transformation and modification without departing from the spirit and scope of the invention, all It should cover within the scope of the invention.

Sequence table

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Claims

1. a kind of multi-functional fusion protein for CD19+ tumour, which is characterized in that the fusion protein includes that identification tumour is anti- The antibody function area of former CD19, the functional areas for shielding tumor surface immune suppressive protein CD47, in conjunction with immunocyte Fc receptor Functional areas and amino acid fragment for connecting each functional areas.

2. multi-functional fusion protein according to claim 1, which is characterized in that the antibody of the identification tumour antigen is needle To the single-chain antibody variable region of CD19；The fusion protein functional areas of the shielding CD47 are SIRP α；The knot with immunocyte Close the IgG1Fc segment that area is fusion protein.

3. multi-functional fusion protein according to claim 2, which is characterized in that the complete amino acid sequence of SIRP α For sequence shown in SEQ ID NO:1；The complete amino acid sequence of heavy chain Fab of the CD19 antibody is shown in SEQ ID NO:2 Sequence；The complete amino acid sequence of light chain Fab of the CD19 antibody is sequence shown in SEQ ID NO:3；The IgG1Fc is complete Whole amino acid sequence is sequence shown in SEQ ID NO:4.

4. multi-functional fusion protein according to claim 3, which is characterized in that the amino acid sequence of the SIRP α is SEQ The site 31-150 or multiple sequences in sequence shown in ID NO:1 repeat or contain similar functions mutant；The CD19 antibody The heavy chain amino acid sequence of scFv is that the site 1-121 or multiple sequences in sequence shown in SEQ ID NO:2 repeat or similar to function The mutant of energy；The light-chain amino acid sequence of the CD19 antibody scFv is the site 1-114 in sequence shown in SEQ ID NO:3 Or multiple sequences repeat the mutant of similar functions；The amino acid sequence of the IgG1Fc is in sequence shown in SEQ ID NO:4 The site 3-219 or mutant with similar functions；The amino acid sequence for connection function area is SEQ ID NO:5 Shown sequence or multiple repetitive sequences.

5. multi-functional fusion protein according to claim 2, which is characterized in that the multi-functional fusion protein is SEQ Amino acid sequence shown in ID NO:6 or the mutant with similar functions.

6. multi-functional fusion protein according to claim 5, which is characterized in that the step of preparing fusion protein packet It includes:

Step 1) synthesizes the corresponding base of amino acid sequence shown in SEQ ID NO:6, composition fusion by artificial synthesized mode Protein structure gene；

Fusion protein structural gene described in step 1) is transferred to mammalian expression vector by step 2), and is transfected to Hamster Qvary Cell carries out protein expression；

Hamster ovary cell described in step 2) is placed in incubator and cultivates a period of time by step 3), takes supernatant, passes through parent With recombination fusion protein obtained after chromatographic purifying.

7. multi-functional fusion protein according to claim 6, which is characterized in that the structure of the fusion protein structural gene Building step includes: to pass through gene according to the corresponding base of functional areas amino acid of SIRP α, anti-CD 19 antibodies Fab and IgG1Fc The mode nand function amino acid fragment base of synthesis connects into multi-functional fusion protein structural gene.

8. a kind of treat expression CD47 and CD19 in preparation such as multi-functional fusion protein according to any one of claims 1 to 7 Application in the drug of disease caused by tumour.

9. application according to claim 8, which is characterized in that the application be multi-functional fusion protein be used alone or With the use in conjunction of chemotherapy, targeted drug, antibody drug, cell therapy composition.

10. application according to claim 8, which is characterized in that the disease includes any with CD19 and CD47 high table The tumour reached.