Anti-sirpa antibodies and uses thereof

Abstract

The application discloses an anti-SIRP alpha antibody and application thereof, and the SIRP alpha antibody comprises a first antibody or a second antibody or a third antibody, the first antibody comprises three first heavy chain CDR regions and three first light chain CDR regions; the three first heavy chain CDR regions comprise that HCDR1 is an amino acid sequence shown in SEQ ID NO. 1, HCDR2 is an amino acid sequence shown in SEQ ID NO. 2, and HCDR3 is an amino acid sequence shown in SEQ ID NO. 3; the three first light chain CDR regions comprise that LCDR1 is an amino acid sequence shown in SEQ ID NO. 4, LCDR2 is an amino acid sequence shown in SEQ ID NO. 5, and LCDR3 is an amino acid sequence shown in SEQ ID NO. 6; the application can effectively block the CD47 and SIRP alpha signal path, promote phagocytosis of macrophages, and is used for treatment of colorectal cancer patients.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, in particular to an anti-SIRPα antibody and application thereof. BACKGROUND

[0002] Tumor is the second leading cause of human death worldwide. The formation of tumor growth microenvironment not only depends on some cytokines secreted by itself, but also can escape immune surveillance and regulation through certain signal pathways, so as to make the body immune tolerant to tumor cells, and further cause rapid progression of tumor.

[0003] SIRPα, also known as CD172a, BIT or SHPS-1, is a member of the SIRP paired receptor family of closely related SIRP proteins, is a single transmembrane molecule of Ig superfamily, and exists in myeloid cells such as macrophages, dendritic cells, neutrophils and glial cells. SIRPα is mainly expressed by hematopoietic cells (including macrophages, dendritic cells and granulocytes), and also expressed on neurons, especially brain cells, glial cells, smooth muscle cells and endothelial cells, and some tumor cells (Barclay and van den Berg 2014). SIRPα is a transmembrane protein with an extracellular domain containing three Ig-like domains, and a cytoplasmic region containing an immunoreceptor tyrosine-based inhibitory motif (ITIM). The previous blocking antibody binds SIRPβ and SIRPγ while blocking the binding of SIPRα and CD47. SUMMARY

[0004] In view of the above, in order to solve the technical problems of the prior art, the present application provides an anti-SIRPα antibody and application thereof, which blocks the binding of SIRPα and CD47, weakly binds SIRPβ, and does not bind SIRPγ.

[0005] The present application solves the above technical problems by the following technical solutions:

[0006] In a first aspect, the present application provides a SIRPα antibody, characterized in that it comprises a first antibody or a second antibody or a third antibody, the first antibody comprising three first heavy chain CDR regions and three first light chain CDR regions; the three first heavy chain CDR regions comprise: HCDR1 is the amino acid sequence shown in SEQ ID NO. 1, HCDR2 is the amino acid sequence shown in SEQ ID NO. 2, and HCDR3 is the amino acid sequence shown in SEQ ID NO. 3; the three first light chain CDR regions comprise: LCDR1 is the amino acid sequence shown in SEQ ID NO. 4, LCDR2 is the amino acid sequence shown in SEQ ID NO. 5, and LCDR3 is the amino acid sequence shown in SEQ ID NO. 6;

[0007] The second antibody comprises three second heavy chain CDR regions and three second light chain CDR regions; the three second heavy chain CDR regions comprise: HCDR4 is the amino acid sequence shown in SEQ ID NO. 7, HCDR5 is the amino acid sequence shown in SEQ ID NO. 8, and HCDR6 is the amino acid sequence shown in SEQ ID NO. 9; the three second light chain CDR regions comprise: LCDR4 is the amino acid sequence shown in SEQ ID NO. 10, LCDR5 is the amino acid sequence shown in SEQ ID NO. 11, and LCDR6 is the amino acid sequence shown in SEQ ID NO. 12;

[0008] The third antibody comprises three third heavy chain CDR regions and three third light chain CDR regions; the three third heavy chain CDR regions comprise: HCDR7 is the amino acid sequence shown in SEQ ID NO. 13, CHDR8 is the amino acid sequence shown in SEQ ID NO. 14, and HCDR9 is the amino acid sequence shown in SEQ ID NO. 15; the three third light chain CDR regions comprise: LCDR7 is the amino acid sequence shown in SEQ ID NO. 16, LCDR8 is the amino acid sequence shown in SEQ ID NO. 17, and LCDR9 is the amino acid sequence shown in SEQ ID NO. 18.

[0009] In a second aspect, the present application provides a nucleic acid molecule, characterized in that the nucleic acid molecule encodes the SIRPα antibody of the first aspect.

[0010] In a third aspect, the present application provides an expression vector, characterized in that the expression vector contains the nucleic acid molecule of the second aspect.

[0011] In a fourth aspect, the present application provides a host cell, characterized in that the host cell contains at least one copy of the expression vector of the third aspect.

[0012] In a fifth aspect, the present application provides a pharmaceutical composition, characterized in that the pharmaceutical composition comprises the SIRPα antibody of the first aspect, and a pharmaceutically acceptable carrier and / or diluent.

[0013] In a sixth aspect, the present application provides use of any one or at least two of the SIRPα antibody of the first aspect, the host cell of the fourth aspect, or the pharmaceutical composition of the fifth aspect in the preparation of a medicament for preventing, alleviating, ameliorating or inhibiting a disease or a disorder.

[0014] Preferably, the disease or disorder is selected from the group consisting of colorectal cancer.

[0015] The positive progress effect of the application is that the application can effectively block the CD47 and SIRP alpha signal path, promote the phagocytosis of macrophages, can enhance the phagocytosis of macrophages, can down-regulate the number of tumor microenvironment MDSC (Myeloid-Derived Suppressor Cells, bone marrow-derived suppressor cells), and activate T cells, and is used for the treatment of colorectal cancer patients. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 The figure is a schematic diagram of the purification results of 3G5, 3H9 and 15D7 hybridoma antibodies in the application.

[0017] Figure 2 The figure is a schematic diagram of the determination of the binding activity of hybridoma antibodies and antigens by ELISA method in the application.

[0018] Figure 3 The figure is a schematic diagram of the determination of the binding activity of hybridoma antibodies and hSIRP beta-his by ELISA method in the application.

[0019] Figure 4 The figure is a schematic diagram of the determination of the binding activity of hybridoma antibodies and hSIRP gamma-his by ELISA method in the application.

[0020] Figure 5 The figure is a schematic diagram of the determination of the competition activity of hybridoma antibodies and ligands by ELISA method in the application.

[0021] Figure 6 The figure is a schematic diagram of the purification results of recombinant antibodies in the application.

[0022] Figure 7 The figure is a schematic diagram of the determination of the binding activity of recombinant antibodies and antigens by ELISA method in the application.

[0023] Figure 8 The figure is a schematic diagram of the determination of the binding activity of recombinant antibodies and hSIRP beta-his by ELISA method in the application.

[0024] Figure 9 The figure is a schematic diagram of the determination of the binding activity of recombinant antibodies and hSIRP gamma-his by ELISA method in the application.

[0025] Figure 10 The figure is a schematic diagram of the determination of the competition activity of recombinant antibodies by ELISA method in the application. DETAILED DESCRIPTION

[0026] The technical solutions in the embodiments of the application will be described clearly and completely in combination with the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, not all the embodiments.

[0027] The preparation method of the anti-SIRPa antibody of the present application comprises the following steps:

[0028] Step one, mouse immunization, the purified SIRPa antigen is emulsified with complete Freund's adjuvant, 6-8 week old BALB / C mice are immunized by subcutaneous injection or intraperitoneal injection, the immunization dose is 50 μg per mouse, the second immunization is carried out after two weeks, subcutaneous injection is carried out with incomplete Freund's adjuvant, the immunization dose is 50 μg per mouse; after two times of immunization, tail blood is taken to determine the serum titer by gradient dilution by ELISA method; whether to strengthen the immunization is determined according to the serum titer result, the mouse with the highest antibody titer is selected for subsequent cell fusion, the B cells capable of secreting specific target protein antibodies are produced in the mouse body; the BALB / C mouse can be cultured by Shanghai Xipu-Bike Experimental Animal Co., Ltd.

[0029] Step two, feeder cell preparation: prepare complete culture medium as needed; kill healthy BALB / c mice by cervical dislocation, soak in 75% alcohol for disinfection; the mouse is in a supine position, the abdominal skin of the mouse is cut in a sterile environment to expose the peritoneum; about 5 ml of IMDM culture medium is sucked with a syringe, the peritoneum is clamped with a forceps, the needle is horizontally inserted into the clamped peritoneum part, and the liquid in the syringe is injected; rotate the needle to make the needle bevel downward, at this time the needle cannot be pulled out and the peritoneum is slightly lifted, the mouse back is massaged with fingers to make the culture medium fully wash the abdominal cavity; the liquid in the mouse abdominal cavity is sucked out, the supernatant is discarded after centrifugation at 1200 rpm for 3 minutes, and the feeder cells are obtained and resuspended in the above prepared culture medium.

[0030] Step three, preparation of spleen cells: the immune mouse is killed by cervical dislocation, the serum is collected, and fresh spleen cells are obtained; the dead mouse is soaked in 75% alcohol for disinfection, the skin and mucosa under the left rib of the mouse are cut in a sterile environment to expose the spleen; the tissue around the spleen is clamped with forceps, the spleen is indirectly lifted, the tissue connecting the spleen and the body is removed one by one with scissors, and finally the spleen is clamped and taken out; after the spleen is disinfected with alcohol and washed with IMDM culture medium, the spleen is ground on a cell filter screen with a syringe core, and the cells are washed with 10-20 ml of IMDM culture medium; after centrifugation at 1200 rpm for 3 minutes, the supernatant is discarded, and the cells are resuspended with 20-30 ml of IMDM culture medium; after counting the viable spleen cells, they are ready for use. If the spleen cells are not used immediately for fusion, they are frozen according to the operation of SP2 / 0 cell freezing.

[0031] Step four, cell fusion, take the immunized mice, and kill and take the spleen cells, according to the principle of SP2 / 0 cells:spleen cells = 1:5 (cell number ratio), according to the standard operation procedure of cell fusion, the immunized mouse cell fusion is carried out, and the specific fusion steps are as follows: a certain amount of SP2 / 0 cell suspension is taken and added to the spleen cell suspension, mixed slightly, then centrifuged at 1200 rpm for 3 minutes, and the supernatant is discarded, and the cell pellet is knocked and shaken for use; after knocking the cell pellet, the centrifuge tube is placed in a 37℃ water bath, and 37℃ preheated polyethylene glycol PEG (about 60 seconds to add 1ml PEG) is slowly added to the bottom of the tube, and the PEG and cells are fully contacted, and the water bath is fully contacted; after adding PEG, the tube is placed in a 37℃ water bath for about 1 minute, then 1ml of IMDM culture medium is added to the tube along the wall in the following order: 1ml of culture medium is added in the first minute, 2ml of culture medium is added in the second minute, 5ml of culture medium is added in the third minute, then the culture medium is supplemented to 20-30ml; after 37℃ standing for 5 minutes, centrifugation is carried out at 800rpm for 3 minutes, and the supernatant is discarded for use; the fused cells are resuspended with HAT complete culture medium containing feeder cells, mixed uniformly, then added to a 96-well cell plate at 250ul / well, and cultured at 37℃.

[0032] Step five, fusion screening, the fused cells are detected by ELISA method, and the wells with strong hSIRPα-his, strong CD47-hFc ligand competition, and weak or no binding of SIRPβ and SIRPγ are selected for subsequent subcloning;

[0033] Step six, subclone, in order to get the final stable clone, by detecting subclone cells, positive cell cloning, further purification of cells, the specific steps are as follows: 100ug SIRPα-his is diluted into 100ml CBS, and is added into enzyme-labeled plate at 50ul / hole, and is coated at 37℃ for 2 hours. After washing and blocking, it is dried, and 50ul of cell clone culture supernatant is added to each hole at 37℃ for 1 hour. After discarding the supernatant, the ELISA plate is washed 5 times with phosphate buffer containing 0.05% Tween 20, 50ul of 1:4000 diluted horseradish peroxidase labeled goat anti-mouse IgG antibody (Zhongshanjingqiao) is added to each hole, and is incubated at 37℃ for 30 minutes. After discarding the secondary antibody, the ELISA plate is washed 3 times with phosphate buffer containing 0.05% Tween 20, 50ul of TMB color developing liquid (Tiangen) is added to each hole for color development for 5 minutes, 50ul of 2M H2SO4 is added to each hole to stop the reaction, and the OD 450 value is read by an enzyme-labeled instrument. Label the positive cell strain number. The positive hole cells are subjected to limited dilution, and the ELISA value is determined 7-10 days after each limited dilution. The monoclonal hole with high positive value is selected for limited dilution until the ELISA determination of the 96-hole plate is positive. The monoclonal strain with high positive value is selected and named as cell strain 3G5, 3H9 and 15D7.

[0034] Step seven, hybridoma antibody expression, the monoclonal cell strain is expanded to T75 culture bottle, and when the culture reaches a confluence rate of more than 50% in the bottle, it can be used for antibody expression. The cells in the bottle are resuspended by pipette blowing, the suspension is counted by cell counting, and the suspension containing 6x10 6 cells is collected in a 15ml centrifuge tube. The collected cell suspension is centrifuged at 1200rpm for 5 minutes, the supernatant in the tube is discarded, and 5ml of expression medium is added to resuspend the tube. The resuspended cell suspension in each tube is added into a 125ml cell shaker bottle, and 25ml of expression medium is further added to the bottle. The 125ml cell shaker bottle is labeled with the corresponding cell strain name and batch number, and is placed in a shaker at 135rpm, 37℃. After 4-5 days, the shaker bottle is taken out, the cell suspension in the shaker bottle is collected in a 50ml centrifuge tube, and is centrifuged at 3000rpm for 10 minutes and then transferred to a clean new centrifuge tube. The centrifuge tube is labeled with the corresponding cell strain name and batch number, and is transferred to the downstream for antibody purification.

[0035] Step eight, purification of mouse-derived antibodies, the hybridoma cells are expanded, the cell supernatant is collected, and the mouse-derived antibodies are purified according to the standard operation procedure for protein affinity purification. As shown in the following table, the experimental results show that the purity of the purified mouse-derived antibodies is greater than 90%. Figure 1

[0036] ​Step nine, culture hybridoma cells to obtain small amounts of purified high purity RNA, reverse transcription to cDNA as a template, and then use specific PCR primers to amplify the target fragment in vitro; positive PCR products are ligated to T vectors for plasmid recombination and transformation, screened by blue-white spot and PCR verification, and positive clones are selected for culture and sequencing. The correct sequence is drawn by analyzing the sequencing results, and the sequencing of the antibody is completed.

[0037] Step ten, by means of molecular biology, the antibody heavy chain and light chain variable region obtained by sequencing are cloned into the modified mammalian cell expression vector for high-efficiency expression, and the anti-SIRPα recombinant antibody is obtained. Recombinant antibodies are expressed in Expi 293 cells, and cell supernatant is collected. Antibody purification is carried out according to the standard operating procedures for protein purification, as shown in Figure 4 The purified humanized antibody has a purity of more than 90%.

[0038] 3G5 antibody light chain:

[0039] DIQLTQSPSSLTVTAGEKVTMTCKSSQSLLNSGNQKSSLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTDFTLTISSVQAEDLALYYCQNDS NYPFTFGSGTKLEIK

[0040] 3G5 antibody heavy chain:

[0041] EVQLQQSGPELVKPGASVRISCKASDYTFTNYYIHWVKQRPGQGLEWIGWIYPGNVNTHYNEMFKGKATLTADKSSSTAYMQLSSLTSEDSAVYFCARSGAYRPAWFAYWGQGTLVTVSA

[0042] 3H9 antibody light chain:

[0043] DIQLTQSPASLAVSLGQRATISCRASESVDSYGNSFMHWYQQKPGQPPKLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQNNEYPWTFGGGTKLEIK

[0044] 3H9 antibody heavy chain:

[0045] EVQLEESGPGLVAPSQSLSISCTVSGFSLNSYGVHWVRQPPGKGLEWLGLIWPAGRTSYNSAFMSRLSISKDNSKSQIFLKMNSLQTDDTAMYYCARDGNFYYTMDFWGQGTSVTVSS

[0046] 15D7 antibody light chain:

[0047] DIVLTQSPAIMSASPREKVTMTCRASSSVSSSNLHWYQQKSGASPKLWIYSTSNLASGVPARFSGSGSGTSYSLTISSVEAEDAATYYCQQYSGYPLTFGGGTKLEIK

[0048] 15D7 antibody heavy chain:

[0049] EVQLEESGPGLVAPSQSLSISCTVSGFSLNSYGVHWVRQPPGKGLEWLGLIWPAGRTSYNSAFMSRLSISKDNSKSQIFLKMNSLQTDDTAMYYCARDGNFYYTMDFWGQGTSVTVSS

[0050] The SIRPa antibody of the present application includes a first antibody (3G5) or a second antibody (3H9) or a third antibody (15D7), the first antibody includes three first heavy chain CDR regions and three first light chain CDR regions; the three first heavy chain CDR regions include: HCDR1 is the amino acid sequence shown in SEQ ID NO. 1, HCDR2 is the amino acid sequence shown in SEQ ID NO. 2, and HCDR3 is the amino acid sequence shown in SEQ ID NO. 3; the three first light chain CDR regions include: LCDR1 is the amino acid sequence shown in SEQ ID NO. 4, LCDR2 is the amino acid sequence shown in SEQ ID NO. 5, and LCDR3 is the amino acid sequence shown in SEQ ID NO. 6. The specific sequence information is as follows: SEQ ID NO. 1: NYYIH. SEQ ID NO. 2: WIYPGNVNTHYNEMFKG. SEQ ID NO. 3: SGAYRPAWFAY. SEQ ID NO. 4: KSSQSLLNSGNQKSSLT. SEQ ID NO. 5: WASTRES. SEQ ID NO. 6: QNDSNYPFT.

[0051] The second antibody comprises three second heavy chain CDR regions and three second light chain CDR regions; the three second heavy chain CDR regions comprise: HCDR4 is the amino acid sequence shown in SEQ ID NO. 7, HCDR5 is the amino acid sequence shown in SEQ ID NO. 8, and HCDR6 is the amino acid sequence shown in SEQ ID NO. 9; the three second light chain CDR regions comprise: LCDR4 is the amino acid sequence shown in SEQ ID NO. 10, LCDR5 is the amino acid sequence shown in SEQ ID NO. 11, and LCDR6 is the amino acid sequence shown in SEQ ID NO. 12. The specific sequence information is as follows: SEQ ID NO. 7: GYGVN. SEQ ID NO. 8: IIWGDGSRDYNSALKS. SEQ ID NO. 9: AGKMDY. SEQ ID NO. 10: RASESVDSYGNSFMH. SEQ ID NO. 11: LVSNLES. SEQ ID NO. 12: QQNNEYPWT.

[0052] The third antibody comprises three third heavy chain CDR regions and three third light chain CDR regions; the three third heavy chain CDR regions comprise: HCDR7 is the amino acid sequence shown in SEQ ID NO. 13, HCDR8 is the amino acid sequence shown in SEQ ID NO. 14, and HCDR9 is the amino acid sequence shown in SEQ ID NO. 15; the three third light chain CDR regions comprise: LCDR7 is the amino acid sequence shown in SEQ ID NO. 16, LCDR8 is the amino acid sequence shown in SEQ ID NO. 17, and LCDR9 is the amino acid sequence shown in SEQ ID NO. 18. The specific sequence information is as follows: SEQ ID NO. 13: SYGVH. SEQ ID NO. 14: LIWPAGRTSYNSAFMS. SEQ ID NO. 15: DGNFYYTMDF. SEQ ID NO. 16: RASSSVSSSNLH. SEQ ID NO. 17: STSNLAS. SEQ ID NO. 18: QQYSGYPLT.

[0053] Step one comprises the following two parts:

[0054] First part, antigen emulsification, according to SIRPα antigen protein concentration, take out 200 μg, dilute to 500 μl volume with PBS (PBS is the abbreviation of phosphate buffered saline), suck into a new 2 ml syringe, mark as the first syringe A; take another new 2 ml syringe, suck 500 μl incomplete Freund's adjuvant into it, mark as the second syringe B; the first syringe A and the second syringe B suck the liquid in the needle part into the syringe cavity, discard the needle; spin the double female luer suction head on the first syringe A, and exhaust the air in the first syringe A, the second syringe B and the luer suction head; quickly connect the second syringe B to the luer suction head of the first syringe A and tighten; add a drop of incomplete Freund's adjuvant to the bottom of the piston rod of the first syringe A and the second syringe B with another third syringe; push the liquid in the first syringe A and the second syringe B back and forth for 30-60 minutes, and do not need to disassemble after completion, and place at 4°C for standby;

[0055] Second part, subcutaneous injection method, take out a 1 ml syringe, suck the required injection agent to the corresponding scale and exhaust the air bubbles, cover the needle cap loosely and place it on the right side of the operation area; fix the mouse, hold the prepared syringe with one hand, and let the needle cap fall naturally onto the table top; at an angle less than 30°, with the needle bevel upwards, pierce the skin at the injection site; once the needle pierces the skin, feel the emptying, immediately adjust the needle insertion angle to about 0° and continue to insert the needle about 1-2 cm; slowly push the injection agent, after the injection is completed, the needle syringe rotates 180°, so that the needle bevel faces downward, and then withdraws from the skin; place the syringe back on the operation table, and place the mouse back in the feeding area.

[0056] If the serum titer result in step one is ≥243000, then perform booster immunization; if the serum titer result is <243000, then do not perform booster immunization.

[0057] The booster immunization of step one is performed by intraperitoneal injection or tail vein injection, and the booster immunization is still performed with the antigen used in the previous conventional immunization, in order to improve the serum titer of the mouse.

[0058] ELISA method for detecting the binding activity of mouse-derived antibodies to hSIRPα-his

[0059] The binding activity OD value of the mouse-derived antibodies to the target antigen is detected by setting a sample concentration gradient and measuring the binding activity OD value by the ELISA method. As shown in Table 1, the three hybridoma antibodies 3G5, 3H9 and 15D7 have strong binding to the antigen, and the EC50 is 0.006489, 0.008783 and 0.008055 respectively, the curve has clear upper and lower platforms, the window is large, and the activity is good. Figure 2

[0060] ​ELISA method for detecting the binding activity of mouse antibodies to hSIRPβ-his

[0061] The test plate to be measured was coated with 0.5 μg / ml of hSIRPβ-his, a sample concentration gradient was set, and the binding activity OD value was measured to detect the binding affinity of the mouse antibodies to the target antigen. As shown in Figure 3 , the three hybridoma antibodies 3G5, 3H9 and 15D7 had weak binding to the antigen.

[0062] ELISA method for detecting the binding activity of mouse antibodies to hSIRPγ-his

[0063] The test plate to be measured was coated with 0.5 μg / ml of hSIRPγ-his, a sample concentration gradient was set, and the binding activity OD value was measured to detect the binding affinity of the mouse antibodies to the target antigen. As shown in Figure 6 , the three hybridoma antibodies 3G5, 3H9 and 15D7 did not bind to the antigen.

[0064] ELISA method for detecting the competition activity of mouse antibodies to CD47-hFc

[0065] The test plate to be measured was coated with 2.0 μg / ml of hSIRPα-his, a sample concentration gradient was set, and the OD value was measured by ELISA to detect the competition activity of the mouse antibodies to 0.1 μg / ml of CD47-hFc ligand. As shown in Figure 5 , the three antibodies had a clear trend in OD value, and the curve had a clear upper and lower platform, indicating strong competition activity.

[0066] ELISA method for detecting the binding activity of recombinant antibodies to hSIRPα-his

[0067] The test plate to be measured was coated with 0.5 μg / ml of hSIRPα-his, a sample concentration gradient was set, and the binding activity OD value was measured to detect the binding affinity of the recombinant antibodies to the target antigen. As shown in Figure 7 , compared with the positive antibody, the series of recombinant antibodies had strong binding to the antigen, the curve had a clear upper and lower platform, the window was large, and the activity was good.

[0068] ELISA method for detecting the binding activity of recombinant antibodies to hSIRPβ-his

[0069] The test plate to be measured was coated with 0.5 μg / ml of hSIRPβ-his, a sample concentration gradient was set, and the binding activity OD value was measured to detect the binding affinity of the mouse antibodies to the target antigen. As shown in Figure 8 , the three hybridoma antibodies 3G5, 3H9 and 15D7 had weak binding to the antigen.

[0070] ELISA method for detecting the binding activity of recombinant antibody to hSIRPy-his

[0071] The plate to be tested was coated with 0.5 μg / ml of hSIRPy-his, a sample concentration gradient was set, and the OD value of the binding activity was determined to detect the binding affinity of the mouse-derived antibody to the target antigen. As shown in Figure 9 , the three hybridoma antibodies 3G5, 3H9 and 15D7 did not bind to the antigen.

[0072] ELISA method for detecting the competition activity of recombinant antibody to CD47-mFc

[0073] The plate to be tested was coated with 2.0 μg / ml of hSIRPa-his, a sample concentration gradient was set, and the OD value was determined to detect the competition activity of the recombinant antibody to 3.0 μg / ml of CD47-mFc ligand. As shown in Figure 10 , the antibody OD value showed a clear trend, and the curve had a clear upper and lower platform, indicating strong competition activity.

[0074] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not necessarily contain only one independent technical solution, and the description of the specification is only for the sake of clarity. The skilled person should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by the skilled person.

[0075] In addition, it should be understood that although the present specification is described in terms of embodiments, each embodiment does not necessarily contain only one independent technical solution, and the description of the specification is only for the sake of clarity. The skilled person should consider the specification as a whole, and the technical solutions in each embodiment can be appropriately combined to form other embodiments that can be understood by the skilled person.

Claims

1. A SIRPa antibody, characterized in that, The antibody comprises three heavy chain CDR regions and three light chain CDR regions; the three heavy chain CDR regions are HCDR1, HCDR2 and HCDR3 respectively, and the three light chain CDR regions are LCDR1, LCDR2 and LCDR3 respectively; HCDR1 is the amino acid sequence shown in SEQ ID NO. 7, HCDR2 is the amino acid sequence shown in SEQ ID NO. 8, and HCDR3 is the amino acid sequence shown in SEQ ID NO. 9; LCDR1 is the amino acid sequence shown in SEQ ID NO. 10, LCDR1 is the amino acid sequence shown in SEQ ID NO. 11, and LCDR3 is the amino acid sequence shown in SEQ ID NO.

12.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the SIRPα antibody of claim 1.

3. An expression vector, characterized by, The expression vector contains the nucleic acid molecule of claim 2.

4. A host cell, characterized in that, The host cell contains at least one copy of the expression vector of claim 3.

5. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises the SIRPα antibody of claim 1, and a pharmaceutically acceptable carrier and / or diluent.

6. Use of any one or at least two of the SIRPα antibody of claim 1, the host cell of claim 4 or the pharmaceutical composition of claim 5 in the preparation of a medicament for preventing, alleviating, ameliorating or inhibiting colorectal cancer.

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