CN108484774B - SIRP alpha fusion protein and preparation method and application thereof - Google Patents

SIRP alpha fusion protein and preparation method and application thereof Download PDF

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CN108484774B
CN108484774B CN201810195521.5A CN201810195521A CN108484774B CN 108484774 B CN108484774 B CN 108484774B CN 201810195521 A CN201810195521 A CN 201810195521A CN 108484774 B CN108484774 B CN 108484774B
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CN108484774A (en
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孙建成
于志恒
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Shanghaigaofei Biological Technology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

The invention discloses a SIRP alpha fusion protein and a preparation method and application thereof, wherein the fusion protein comprises: a V domain of SIRPa, and an Fc segment with effector function; the V domain comprises: amino acid sequence SEQ ID No. 1; the Fc segment comprises: constant regions of human IgG1 or IgG4 antibodies. The fusion protein of the invention enhances the affinity, in vivo half-life and Fc segment mediated effector function of SIRP alpha and CD47, and can strongly induce macrophage phagocytosis to various tumor cells.

Description

SIRP alpha fusion protein and preparation method and application thereof
Technical Field
The invention relates to a fusion protein, in particular to a SIRP alpha fusion protein and a preparation method and application thereof.
Background
Evading the killing action of the immune system is one of the characteristics of tumors. In recent years, the use of the original immune response of the body to fight tumors has gradually become a new treatment strategy, and the treatment is ordered according to the time sequence of the application of different mechanism therapies, mainly comprising nonspecific immunostimulation, monoclonal antibodies for immune check points, adoptive cell reinfusion, monoclonal T cell receptor therapy, CD47 monoclonal antibodies, tumor vaccines and the like, and is one of the most promising therapies in both experimental stage and clinical application. In general, macrophages can penetrate into the tumor mass and their antitumor effect can be exploited to benefit the patient.
CD47, also known as Integrin Associated Protein (IAP), is a member of the immunoglobulin superfamily. CD47 is widely expressed on the cell surface, and can interact with Signal Regulatory Protein alpha (SIRP alpha), Thrombospondin-1 (TSP 1), and integrins (integrins), and mediate a series of reactions such as apoptosis, proliferation, and immunity.
It has been shown that CD47, as an "allo-me" signaling molecule against phagocytic effects, expresses CD47 to a high degree in almost all tumor cells and tissues, and 3-fold higher than corresponding normal cells and tissues, which prevents cells from being recognized and killed by macrophages. In addition, expression of CD47 also limits Fc fragment receptor-mediated phagocytosis when treated with antibodies.
The research finds that the CD47 directly binds with macrophage surface regulatory glycoprotein SIRP alpha to play the protective function on the tumor. SIRP alpha, as an inhibitory receptor, negatively regulates the skeletal strength of macrophages and attenuates their phagocytic capacity when interacting with CD 47. SIRP alpha extracellular domain binds to CD47, signaling through the intracellular domain, its NH2The terminal V-shaped structure is involved in the regulation of CD47 binding.
Currently, preclinical studies for Anti-CD47 therapy cover non-Hodgkin 'S lymphomas (Anti-CD 47 Anti-cancer synergetics with both Clinical to patient pharmacological and therapeutic non-Hodgkin' S lymphoma [ J ]. Cell, 142(5): 699-713), breast cancer (The CD47-signal regulatory protein alpha (SIRP α) interactive is a therapeutic target for human soluble tumors [ J ]. Proc Nat Acad Sci U A, 2012, 109(17): 6662-6667), ovarian cancer (In vitro application of Anti-CD47 monoclonal antibody for targeted therapy of cancer [ J ]. J. sub.440. biological Cell of non-Clinical lymphoma [ J ]. J.: 22-8. sub.J.),440. biological sample J., 2016, 126(7): 2610-2620).
At present, relevant anti-CD47 antibody drugs enter into clinical phase I research, and Hu5F9-G4 is an anti-CD47 monoclonal antibody developed by Forty Seven company, and has better affinity (nM level) to CD 47.
Moreover, in preclinical studies by researchers at the Stanford university medical school, USA, it was found that an antibody that causes macrophages to phagocytose and eat tumor cells while sparing healthy brain cells can safely and effectively treat 5 types of brain cancer in model mice that received transplantation of brain cancer cells in children, and the results of the related studies are presented in the Science Translational Medicine journal (differentiating the CD47-SIRP alpha anti-phagocytic axis by a human affected anti-CD47 anti-body is an effective cancer vaccine for a macromolecular therapeutic vaccine tissue, Science Translational Medicine 15 Mar 2017, Vol. 9, Issue 381). Encouraging Hu5F9-G4 was not toxic to normal human brain cells, but had very potent tumor killing properties.
A great deal of research shows that the combination of CD 47-SIRPa can be blocked by adopting antibodies or modifying SIRPa through genetic engineering, thereby greatly improving the phagocytosis of tumor cells. However, sirpa will be more attractive as a target due to the limited distribution of antibodies between tissues and the Fc segment also mediating off-target effects.
However, the existing sirpa molecular design has the limitations of effectiveness, safety, production cost and the like.
Disclosure of Invention
The invention aims to provide a SIRP alpha fusion protein and a preparation method and application thereof, the fusion protein enhances the affinity, in vivo half-life and Fc segment mediated effector function of the SIRP alpha and CD47, can strongly induce the phagocytosis of macrophages to various tumor cells, and simultaneously reduces the production cost.
To achieve the above object, the present invention provides a sirpa fusion protein comprising: a V domain of SIRPa, and an Fc segment with effector function; the V domain comprises: amino acid sequence SEQ ID No. 1; the Fc segment comprises: constant regions of human IgG1 or IgG4 antibodies.
The constant region of the human IgG1 antibody comprises: the amino acid sequence of SEQ ID No. 2.
The constant region of the human IgG4 antibody comprises: amino acid sequence SEQ ID No. 3.
The fusion protein comprises: amino acid sequence SEQ ID No. 4.
The fusion protein comprises: amino acid sequence SEQ ID No. 5.
The fusion protein comprises: two domains of IgG1 in tandem or two domains of IgG4Fc in tandem.
The fusion protein comprises: amino acid sequence SEQ ID No. 6.
The fusion protein comprises: amino acid sequence SEQ ID No. 7.
The Fc segment contains Ser228Pro mutation. The Ser228Pro mutation is able to inhibit antibody chain exchange. The IgG4 antibody is a dynamic molecule capable of undergoing a process called Fab Arm Exchange (FAE), which results in a functional monovalent bispecific antibody (bsAb) with unknown specificity, thus potentially reducing therapeutic efficacy, and the S228P mutation can prevent IgG4 FAE.
The invention also provides a preparation method of the SIRPa fusion protein, which comprises the following steps: cloning the nucleotide sequence for expressing the SIRPa fusion protein into an expression vector, wherein the expression vector comprises a Leader sequence, and synthesizing the SIRPa fusion protein by a PCR technology. Wherein, a leader sequence is designed at the N-terminal of the expression structure of the mammal to ensure proper signal transduction and processing related to secretion.
The expression vector comprises: mammalian expression vectors expressing the CMVa-intron promoter, or bacterial expression vectors under the T7 promoter and Lac Operator.
The invention also provides an expression vector which comprises the nucleotide sequence of the SIRPa fusion protein.
The invention also provides a host cell, which contains the expression vector; the host cell is of mammalian or bacterial origin.
Preferably, the mammalian host cell is a cell capable of glycosylating an expressed protein.
Preferably, the bacterium comprises: escherichia coli. When the recombinant SIRP alpha-Fc fusion protein is expressed by escherichia coli, the strong affinity of the recombinant SIRP alpha-Fc fusion protein and CD47 is still kept, the phagocytosis of tumor cells is induced, and the production cost is obviously reduced.
The present invention also provides an immunotherapeutic pharmaceutical composition comprising: the sirpa fusion protein of, and a pharmaceutically acceptable carrier therefor; the medicine can inhibit cancer cell growth or proliferation. The "pharmaceutically acceptable carrier" of the present invention refers to carriers and excipients that are suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The cancer cell comprises: gastric cancer cells.
The SIRP alpha fusion protein and the preparation method and the application thereof have the following advantages:
the SIRP alpha fusion protein is formed by fusing a SIRP alpha protein component with an antibody constant region (Fc), enhances the affinity, in-vivo half-life and Fc segment-mediated effector functions of the SIRP alpha and CD47, can strongly induce the phagocytosis of macrophages on various tumor cells, particularly has good phagocytosis on gastric cancer, and simultaneously reduces the production cost.
Drawings
FIG. 1 is a schematic structural diagram of a SIRPa fusion protein of the present invention.
FIG. 2 is a Western blot of an IP-WB experiment with the SIRP alpha-lgG 4-Fc fusion protein of the invention.
Figure 3 is a graph comparing the avidity of binding of three sirpa fusion proteins to CD47 using a direct binding assay.
FIG. 4 is an image of macrophages after incubation with Raji cells for 2 hours in the presence of 10nM control IgG4Fc protein.
FIG. 5 is an image of macrophages under the mirror after incubation with Raji cells for 2 hours in the presence of 10nM SIRP α -lgG4-Fc fusion protein.
FIG. 6 is a dose relationship graph of the SIRPa-lgG 4-Fc fusion protein in the invention inducing the phagocytic effect of Raji cells.
FIG. 7 is a statistical plot of the phagocytic effect of macrophages co-cultured with cancer cells in the presence of 10nM SIRPa-lgG 4-Fc fusion protein for 2 hours.
FIG. 8 is a dose relationship graph comparing the effect of three SIRPa fusion proteins on inducing Raji cell phagocytosis.
Detailed Description
The technical scheme of the present invention is further described below with reference to the accompanying drawings and experimental examples, the following preparation method of the fusion protein is an exemplary method and is not limiting, and other prior arts can also realize the preparation thereof, and such a method is within the knowledge of those skilled in the art.
Experimental example 1 preparation of SIRP alpha fusion protein of the present invention
As shown in FIG. 1, which is a structural schematic diagram of the SIRPa fusion protein of the present invention, the SIRPa-Fc and SIRPa-Fc fusion proteins are composed of N-terminal V domain (variable region domain) of human IgG1 Fc region (SEQ ID number 2), human IgG4Fc region (SEQ ID number 3), human IgG1 Fc-Fc or human IgG4 Fc-Fc fusion human SIRPa (SEQ ID number 1), and the fusion proteins are obtained.
Wherein the same human sirpa domain is linked to a human IgG4Fc region containing the hinge-stable S288P mutation that prevents intra-chain disulfide bond formation, resulting in sirpa-Fc (Fc is human IgG4 Fc) and sirpa-Fc (Fc is human IgG4 Fc-Fc) fusion proteins containing the S288P mutation, the sequences of which are set forth in SEQ ID number 8 and SEQ ID number 9, respectively.
All of the above fusion proteins were generated by overlap PCR using standard molecular biology techniques to generate synthetic DNA fragments, which were then cloned into expression vectors. The fusion protein is then expressed in stably transfected CHO-S cells (Chinese hamster ovary suspension cells), or CHO-S and bacterial cells. Finally, protein a affinity chromatography and hydrophobic interaction chromatography were used to purify the fusion protein, concentrate and remove residual endotoxins.
The specific method for transfecting and expressing the SIRP alpha fusion protein comprises the following steps:
(1) expression of SIRP alpha fusion proteins in mammalian cells
Stable transfectants were generated using the CHO-S cell line (purchased from Invitrogen). The isolated plasmid DNA was linearized and purified using a QIAGEN protein purification column.
Specifically, CHO-S cells were cultured in serum-free chemically defined medium (CD-CHO, Invitrogen) of 8mM L-glutamine and lxht-supplement, and transferred into plasmid DNA using Lipofectamine 2000 reagent (from Invitrogen). After 48 hours, the cells were transferred to a medium containing 600. mu.g/ml of hygromycin B (expression vector). After 2-3 weeks of drug selection, the Fc expression level of the fusion protein of the drug-resistant clone in the supernatant was measured by ELISA as follows:
the capture antibody (goat anti IgGFc) was coated in 96-well plates at a concentration of 0.1 and incubated overnight at 4 ℃. Eluted and blocked with 200 μ L2% BSA PBST and left at room temperature for 1 hour. After washing, 100. mu.L of the sample was diluted with 1% BSA in PBST, added to the sample wells, incubated for 1 hour, washed, and then incubated with HRP-conjugated to detect Ab (HRP-conjugated goat anti-human IgGFc), left at room temperature for 1 hour. The sample wells were washed at room temperature, TMB substrate (3, 3 ', 5, 5' -tetramethylbenzidine dihydrochloride, available from Moss Inc.) was added, and incubated at room temperature for 3-5 minutes. The absorbance at 450 nm/655 nm was measured using an iMark microplate reader (Biorad) and a standard curve was constructed using the known purified fusion protein. The clones with the highest expression were used for supernatant bulk production.
The specific steps of protein purification are as follows:
to purify fusion proteins from CHO-S cells, 5 to 10 liters of culture supernatant from stably transfected high expressing cell clones in a roller bottle bioreactor system was required.
Briefly, CHO-S transfectants were first grown in complete growth medium (CD-CHO supplemented with 8mM L-glutamine, 1HT supplement and 600xglmL hygromycin B) at 37 ℃ to generate sufficient cell numbers to reach 0.5X 106The volume of each cell/mL is 1L or 2L, and then the cells are respectively put into a bioreactor bottle at 37 ℃ and 10% CO2Incubation at a rocking speed of 15-20rpm, an angle of 7 ℃ and an air flow of 0.2-0.4 Lpm. When the culture reaches 2-2.5 × 106At a density of cells/mL (typically within 2-3 days of seeding), the bioreactor was further scaled up to 5L or 10L and 10% CO at 37 deg.C2Further incubation was carried out at a shaking speed of 15-20rpm, an angle of 7 ℃ and an air flow of 0.2-0.4 Lpm. When the cells reach 1 to 1.5X 106At a density of individual cells/mL, the temperature was lowered to 30 ℃ and cultured under the above conditions for another 7 to 10 days. Starting on day 0, every two days at 30 ℃ 1% CHO feed bioreactor supplement (Sigma) was added to the culture and harvested when cell viability decreased by about 90%. Collecting the supernatant at 4 deg.CCentrifuge at 3000 Xg for 40 min and freeze at-20 ℃ until purification.
Protein purification requires a two-step procedure:
first, protein a chromatography was used. Binding buffer (20 mM Na) was used first3PO41M NaCl, ph 7.8) the buffer exchanged supernatant was diluted 9-fold and loaded onto a rProtein a column (GE Healthcare) at a flow rate of 2-3mL/min (depending on the loading volume and loading time) overnight at 4 ℃.
The column was then washed with binding buffer (3 volumes/min 20 volumes) and the protein was eluted at 3mL/min with 0.1M citric acid pH 4.0 and pH 2.2. The eluate was pH-adjusted to neutrality with 1M Tris-HCl (pH 8.5) and then purified by HiTrap Phenyl HP chromatography. Briefly, the protein was first diluted at least 4-fold with 0.2M ammonium sulfate (pH 7.5) and then loaded onto a HiTrap Phenyl HP column (GE Healthcare) at 2-3mL/min (depending on column size and loading time). The non-aggregated SIRPaFc protein can be collected in the flow-through section.
Finally, the sirpa fusion protein was concentrated using a BioMax 10 membrane (Millipore) and the solution was converted to PBS pH 7.4. The mass of each protein was determined by Western blots and HPLC analysis by SDS-PAGE. The identity of all proteins was confirmed by N-terminal sequencing and mass spectrometry.
(2) Expression of SIRP alpha fusion proteins in bacterial cells
Specifically, the bacterial expression plasmid was transferred into BL21 cells (purchased from Novagen) and stable clones were generated by selective agar plates containing kanamycin (50. mu.g/mL). Each clone was grown in 2ml LB medium, OD 0.6-0.8, 37 ℃ C, and recombinant protein was induced by addition of IPTG (Isopropyl Thiogalactoside) at a final concentration of 100. mu.M. The culture was incubated at 25 ℃ for a further 4 hours. Dissolving the cenospheres in 1 x Ranish buffer solution (30 mM Tris-HCl, pH 7.5, 10% glycerol, 50 mM KCl, 1mM EDTA and 2mM DTT, wherein the EDTA is ethylene diamine tetraacetic acid and the DTT is dithiothreitol) by sound waves at 4 ℃. The expression level of the fusion protein was detected by western blotting with an antibody that binds to human Fc. High expression clones are used for large scale protein production.
Experimental example 2 binding of SIRP alpha-lgG 4-Fc fusion protein of the present invention to CD47
(1) Western blot experiment for binding of SIRP alpha-lgG 4-Fc fusion protein and CD47
The above purified fusion proteins at different concentrations (0.1 nM, 1nM, 10nM, 100nM, as well as the purified CD47 protein (10 ng, Abcam) and protein A agarose beads were mixed in PBS solution overnight at 4 degrees, and the immunocomplexes were pelleted by centrifugation and run on SDS-PAGE gel electrophoresis. Finally, CD47 protein levels were quantitatively determined by western blot assay. The amount of CD47 added was used directly as a control.
As shown in FIG. 2, a Western blot of an IP-WB experiment (IP, Immunoprecipitation experiment; WB, Western Blotting) was performed on the SIRP α -lgG4-Fc fusion protein of the present invention, and it can be seen that the amount of binding to CD47 was increased as the concentration of the SIRP α -lgG4-Fc fusion protein of the present invention was increased. By binding to CD47, tumor cells cannot escape phagocytosis by phagocytes via CD47 and are thus eliminated by phagocytes.
(2) Quantitative and comparative experiment for combination of SIRP alpha fusion protein and CD47
Comparing three sirpa Fc fusion proteins, including: the affinity of the SIRPa-lgG 4-Fc fusion protein and the SIRPa-lgG 4-Fc fusion protein produced in mammalian cells, and the SIRPa-lgG 4-Fc fusion protein (expressed as SIRPa-lgG 4-Fc-Bac in FIG. 3) produced in bacteria, respectively, for purified CD47 protein was as follows:
purified CD47 protein complexed with Nickel beads (Nickel beads) was incubated with various concentrations of sirpa fusion protein on ice for 1 hour. The nickel beads were then washed to remove any unbound protein and the bound sirpa fusion protein was quantified by Western blot analysis. Binding curves and Kd values (Dissociation Constant, which reflects the rate of Dissociation of small molecules bound to proteins, and also directly determines the affinity of proteins to small molecules) were generated by prism (graphpad) using nonlinear regression to fit the data to a site binding model.
As shown in FIG. 3, to compare the results of the affinity of three SIRPa fusion proteins for CD47 using a direct binding assay, the Kd values for SIRPa-lgG 4-Fc-Fc fusion protein (19.9. + -. 1.3 nM) were nearly 2-fold less than the Kd values for SIRPa-lgG 4-Fc fusion protein (37.4. + -. 4.0 nM), which demonstrated the strongest affinity of SIRPa-lgG 4-Fc-Fc fusion protein for CD 47.
Comparing the affinity of the SIRPa-lgG 4-Fc fusion protein produced in mammalian cells and the affinity of the SIRPa-lgG 4-Fc fusion protein produced in bacteria with the purified CD47 protein, respectively, as shown in FIG. 3, the Kd value of SIRPa-lgG 4-Fc produced in mammalian cells was 37.4nM and the Kd value of SIRPa-lgG 4-Fc produced in bacteria was 46.9nM, which indicates that both fusion proteins have an affinity comparable to CD 47.
Experimental example 3 in vitro induction of phagocytic Effect by the SIRP alpha-lgG 4-Fc fusion protein of the present invention
(1) Preparation of M1 macrophages
Human M1 macrophages were prepared from monocytes obtained from normal healthy human donors (human peripheral blood monocytes supplied by StemCell technologies). Monocytes were differentiated into macrophages by culturing in special differentiation medium (supplied by StemCell technologies) supplemented with M-CSF (Macrophage Colony-Stimulating Factor) (20 ng/mL) by the following specific steps:
the monocytes are differentiated into macrophages by culturing in a differentiation culture solution based on RPMI 1640 for 6-10 days. The culture solution comprises the following components: 10% heat-inactivated human AB serum, 1% GlutaMax, 1% streptomycin (purchased from GIBCO Life Technologies).
One day before phagocytosis determination, IFN-gamma (Interferon-gamma, gamma-Interferon) (100ng/mL) is used for triggering all the macrophages to be polarized into M1 type macrophages, and the specific phagocytosis detection method is as follows:
ultra-low density cultured tumor cells in a u-type bottom 96-well plate were labeled with a vital stain CFSE (hydroxyfluorescein diacetate succinimide Ester). 50 microliters of IgG1 (4. mu.g/ml) was added to the control and 50 microliters of SIRPa-lgG 4-Fc fusion protein (4. mu.g/ml) was added to the experimental group, and 50 microliters (50000) of macrophages were added to each well after 30 minutes of incubation at room temperature. After the 96-well plate was placed in a 37-degree incubator and cultured for 2 hours, the cells were fixed, stained, and analyzed by a fluorescence microscope. Primary human macrophages were identified with anti-CD 14, CD45, or CD206 antibody (purchased from BioLegend) labeling, and dead cells were identified with DAPI (purchased from Sigma as 4',6-diamidino-2-phenylindole, 4', 6-diamidino-2-phenylindole) detection.
Evaluation of phagocytic function: the proportion of macrophages with positive fluorescence labeling was used as an index for the assessment of phagocytosis.
Macrophage M1 shows strong proinflammatory and antigen presenting ability, exerts host immune clearance function on pathogens and tumor cells, and can secrete a large amount of proinflammatory factors such as tumor necrosis factor-alpha (TNF-alpha). Furthermore, macrophages of type M1 typically kill cancer cells by secreting reactive oxygen and nitrogen intermediates, as well as secreting TNF- α and IL-1 β to recruit Cytotoxic T Lymphocytes (CTLs) to attack cancer cells.
(2) Phagocytic effect on Raji cells
Raji cells (lymphoma cells) were labeled with CellTrace CFSE (purchased from Invitrogen, a cell proliferation kit) and expressed as 1:5 macrophages: ratio of labeled Raji cells this was added to the above induced polarization macrophages in 24-well plates. The macrophages and Raji cells induced to polarize were incubated in the presence of SIRP alpha-lgG 4-Fc fusion protein or control IgG4Fc protein at 37 ℃ in 5% CO2Co-incubation for 2 hours followed by staining with anti-CD 45 antibody. Phagocytosis was observed by fluorescence microscopy and percent phagocytosis was calculated as follows: (number of tumor cells within M1 macrophages/number of M1 macrophages); at least 200M 1 macrophages were counted per sample (sample in each well of the 24-well plate).
As shown in fig. 4, which is an image of macrophages incubated with Raji cells for 2 hours in the presence of 10nM of control IgG4Fc protein, no phagocytic effect can be seen. As shown in FIG. 5, under-lens images of macrophages incubated with Raji cells for 2 hours in the presence of 10nM SIRPa-lgG 4-Fc fusion protein show the phagocytosis of Raji cells by macrophages, indicating that the SIRPa fusion protein of the present invention has an effect on phagocytosis. As shown in FIG. 6, the SIRP alpha-lgG 4 of the invention
The dose relationship graph of the induction of the phagocytic effect of Raji cells by the Fc fusion protein shows that the continuous induction of the phagocytic effect of Raji cells is promoted with the continuous increase of the dosage of the SIRPa-lgG 4-Fc fusion protein.
(3) Effect of different cancer cells on phagocytosis
Cancer cells from different cancer types, e.g. lung cancer (H460 cells ATCC)® HTB-177™A549 cell ATCC®CCL-185™H1299 cells ATCC® CRL-5803™) Colorectal cancer (HCT 116 cells ATCC)® CCL-247™SW480 cells ATCC® CCL-228™Caco-2 cell ATCC® HTB-37™) Gastric cancer (ASG cell ATCC)® CRL-1739™MKN1 cell CVCL _1415 and N87 cell ATCC® CRL-5822™) Liver cancer (HepG 2 cell ATCC)® HB-8065™Hep3B cell ATCC® HB-8064™Huh7 cell CVCL _ 0336), B cell lymphoma (Raji cell ATCC® CCL-86™) These cells were obtained from ATCC (American type culture Collection) and were incubated with macrophages in the presence of 10nM SIRPa-Fc protein at 37 ℃ in 5% CO2Co-incubation for 2 hours followed by staining with anti-CD 45 antibody. Phagocytosis was observed by fluorescence microscopy, and the percentage of phagocytosis was counted under fluorescence microscopy.
As shown in FIG. 7, which is a statistical graph of phagocytic effect of macrophages co-cultured with cancer cells for 2 hours in the presence of 10nM SIRPa-lgG 4-Fc fusion protein (Lung Lung cancer, Colorectal rectal cancer, Gastric Gastric cancer, Liver Liver cancer, Lym lymphoma), it can be seen that Gastric cancer cells are more sensitive to SIRPa-Fc protein-mediated phagocytosis.
(4) Comparing the phagocytic effect of three SIRP alpha fusion proteins on Raji cells
Comparing three sirpa fusion proteins, including: the SIRPa-lgG 4-Fc fusion protein and the SIRPa-lgG 4-Fc fusion protein produced in mammalian cells, and the SIRPa-lgG 4-Fc fusion protein produced in bacteria, respectively, have phagocytic effect on cancer cells.
As shown in FIG. 8, in order to compare the dose relationship graphs of three SIRPa fusion proteins inducing the phagocytic effect of Raji cells, the results of macrophage phagocytic effect on Raji cells after 2 hours of co-incubation with Raji cells in the presence of different concentrations of three SIRPa fusion proteins showed that the SIRPa-lgG 4-Fc-Fc fusion protein was approximately one-fold more phagocytic than SIRPa-lgG 4-Fc, and that SIRPa-lgG 4-Fc fusion protein produced in mammalian cells and SIRPa-lgG 4-Fc fusion protein produced in bacteria had comparable phagocytic effect on cancer cells.
In general, the SIRPa fusion protein of the invention will be used at an effective dose of 0.05mg/kg to 5mg/kg, which will be determined on a case-by-case basis, such as the type of tumor to be treated, the general health of the patient, the in vivo potency of the antibody, the pharmaceutical formulation and the route of administration, etc. An "effective amount" of the present invention refers to an amount that produces a function or activity in a subject and is acceptable to the subject.
In conclusion, the fusion protein enhances the affinity, in vivo half-life and Fc segment mediated effector function of the SIRPa and CD47, and can strongly induce the phagocytosis of macrophages to various tumor cells.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Sequence listing
<110> Shanghai Gaofei Biotech Co., Ltd
<120> SIRP alpha fusion protein and preparation method and application thereof
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Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
165 170 175
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
180 185 190
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
195 200 205
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
210 215 220
Pro Gly Lys
225
<210> 3
<211> 224
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 3
Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser
1 5 10 15
Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
20 25 30
Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro
35 40 45
Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala
50 55 60
Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val
65 70 75 80
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr
85 90 95
Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr
100 105 110
Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu
115 120 125
Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys
130 135 140
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser
145 150 155 160
Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp
165 170 175
Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser
180 185 190
Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala
195 200 205
Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
210 215 220
<210> 4
<211> 341
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 4
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
115 120 125
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
130 135 140
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
145 150 155 160
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
165 170 175
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
180 185 190
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
195 200 205
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
210 215 220
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
225 230 235 240
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
245 250 255
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
260 265 270
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
275 280 285
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
290 295 300
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
305 310 315 320
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
325 330 335
Leu Ser Pro Gly Lys
340
<210> 5
<211> 338
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 5
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly
115 120 125
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
130 135 140
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
145 150 155 160
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
165 170 175
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
180 185 190
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
195 200 205
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
210 215 220
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
225 230 235 240
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
245 250 255
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
260 265 270
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
275 280 285
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
290 295 300
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
305 310 315 320
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
325 330 335
Gly Lys
<210> 6
<211> 583
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 6
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
115 120 125
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
130 135 140
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
145 150 155 160
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
165 170 175
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
180 185 190
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
195 200 205
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
210 215 220
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
225 230 235 240
Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln
245 250 255
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
260 265 270
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
275 280 285
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
290 295 300
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
305 310 315 320
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
325 330 335
Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly
340 345 350
Gly Gly Gly Ser Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
355 360 365
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
370 375 380
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
385 390 395 400
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
405 410 415
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
420 425 430
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
435 440 445
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
450 455 460
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
465 470 475 480
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
485 490 495
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
500 505 510
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
515 520 525
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
530 535 540
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
545 550 555 560
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
565 570 575
Leu Ser Leu Ser Pro Gly Lys
580
<210> 7
<211> 577
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 7
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly
115 120 125
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
130 135 140
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
145 150 155 160
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
165 170 175
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
180 185 190
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
195 200 205
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
210 215 220
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
225 230 235 240
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
245 250 255
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
260 265 270
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
275 280 285
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
290 295 300
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
305 310 315 320
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
325 330 335
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
355 360 365
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
370 375 380
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
385 390 395 400
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
405 410 415
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
420 425 430
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
435 440 445
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
450 455 460
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
465 470 475 480
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
485 490 495
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
500 505 510
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
515 520 525
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
530 535 540
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
545 550 555 560
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
565 570 575
Lys
<210> 8
<211> 338
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 8
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
115 120 125
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
130 135 140
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
145 150 155 160
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
165 170 175
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
180 185 190
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
195 200 205
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
210 215 220
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
225 230 235 240
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
245 250 255
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
260 265 270
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
275 280 285
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
290 295 300
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
305 310 315 320
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
325 330 335
Gly Lys
<210> 9
<211> 577
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 9
Glu Glu Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala
1 5 10 15
Ala Gly Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro
20 25 30
Val Gly Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu
35 40 45
Ile Tyr Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser
50 55 60
Asp Leu Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn
65 70 75 80
Ile Thr Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys
85 90 95
Gly Ser Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu
100 105 110
Ser Val Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly
115 120 125
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
130 135 140
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu
145 150 155 160
Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
165 170 175
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg
180 185 190
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
195 200 205
Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu
210 215 220
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
225 230 235 240
Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu
245 250 255
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
260 265 270
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
275 280 285
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp
290 295 300
Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His
305 310 315 320
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
325 330 335
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
340 345 350
Ser Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro
355 360 365
Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser
370 375 380
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp
385 390 395 400
Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn
405 410 415
Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val
420 425 430
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
435 440 445
Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys
450 455 460
Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr
465 470 475 480
Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr
485 490 495
Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu
500 505 510
Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu
515 520 525
Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys
530 535 540
Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu
545 550 555 560
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
565 570 575
Lys

Claims (4)

1. A sirpa fusion protein, comprising: a V domain of SIRPa, and an Fc segment with effector function;
the V domain comprises: amino acid sequence SEQ ID No. 1;
the Fc segment comprises: a constant region of a human IgG4 antibody, and the Fc region contains a Ser228Pro mutation;
the fusion protein comprises: two domains of IgG4Fc in tandem;
wherein the fusion protein comprises: amino acid sequence SEQ ID No. 7.
2. A method of making a sirpa fusion protein according to claim 1, the method comprising: cloning a nucleotide sequence for expressing the SIRPa fusion protein into an expression vector, wherein the expression vector comprises a Leader sequence, and synthesizing the SIRPa fusion protein by a PCR technology; the expression vector comprises: mammalian expression vectors expressing the CMVa-intron promoter, or bacterial expression vectors under the T7 promoter and Lac Operator.
3. A host cell, comprising: an expression vector having a nucleotide sequence of the sirpa fusion protein of claim 1; the host cell is a cell of mammalian or bacterial origin; the bacterium comprises: escherichia coli.
4. An immunotherapeutic pharmaceutical composition comprising: the SIRPa fusion protein of claim 1, and a pharmaceutically acceptable carrier thereof; the medicine can inhibit cancer cell growth or proliferation; the cancer cell comprises: gastric cancer cells.
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AU2019349651B2 (en) * 2018-09-27 2023-12-07 Celgene Corporation SIRP alpha binding proteins and methods of use thereof
CN109535263B (en) * 2018-12-04 2022-06-17 江苏东抗生物医药科技有限公司 SIRP alpha mutant and fusion protein thereof
CN109517054B (en) * 2018-12-04 2022-04-08 江苏东抗生物医药科技有限公司 SIRP alpha variant or fusion protein thereof and application thereof
CN111484558B (en) * 2019-01-28 2023-02-17 上海交通大学 Signal regulatory protein alpha fragment-anti-FcRn single-chain antibody fusion protein and preparation and application thereof
CN111909276A (en) * 2019-05-10 2020-11-10 复旦大学 Fusion protein and application thereof
CN111087473B (en) * 2019-12-11 2022-06-14 上海百英生物科技有限公司 SIRPa-Fc-IL21 fusion protein and application thereof
CN111253482B (en) * 2020-02-18 2021-11-30 中国人民解放军军事科学院军事医学研究院 SIRPa variants, fusion proteins, and uses thereof
WO2023088429A1 (en) * 2021-11-19 2023-05-25 杭州尚健生物技术有限公司 SIRPα VARIANT AND USE THEREOF

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