CN113480648B - Murine blocking antibody for human CD47 and preparation and application thereof - Google Patents

Abstract

The invention discloses a murine blocking antibody aiming at human CD47, the subtype of the murine blocking antibody is mouse IgG1, and the amino acid sequences of the heavy chain and light chain variable regions of the antibody are respectively shown as SEQ ID NO: 1-2, each consisting of 4 framework regions and 3 complementarity determining regions. The invention also discloses the nucleotide sequences of the light chain variable region and the heavy chain variable region of the antibody and a chimeric antibody expression vector containing the sequences, wherein the nucleotide sequences of the light chain variable region and the heavy chain variable region are respectively shown as SEQ ID NO: 3-4. The murine blocking antibody aiming at the human CD47 can be specifically combined with CD47, has good blocking activity and phagocytosis promoting function, and has wide application prospect in the development of therapeutic antibodies targeting the human CD 47.

Description

Murine blocking antibody for human CD47 and preparation and application thereof

Technical Field

The invention belongs to the technical field of monoclonal antibodies, relates to a murine blocking antibody against human CD47, and preparation and application thereof, and particularly relates to a murine blocking antibody against a CD47 molecular extracellular domain and capable of blocking the combination of CD47 and a ligand SIRP alpha thereof.

Background

CD47, also known as integrin-associated protein, is a glycoprotein molecule that is widely expressed on cell surfaces. Structurally, CD47 comprises an IgV-like extracellular domain, 5 transmembrane regions, and an alternatively spliced cytoplasmic tail. CD47 and inhibitory receptor regulatory protein alpha (SIRP alpha) are mutually ligands and receptors, and the two can form a CD47-SIRP alpha complex and participate in regulating and controlling various immune response processes. Under normal physiological conditions, CD47 is a self marker, mediates 'eat me' signals, and plays an important role in maintaining the balance of the body. However, expression of CD47 is significantly elevated in a variety of hematologic and solid tumors and is closely associated with a clinically poor prognosis. Tumor cells strengthen 'other eating me' signals through high expression of CD47, inhibit phagocytosis mediated by phagocytes such as macrophages and the like, and escape monitoring of an immune system. The monoclonal antibody or the fusion protein is used for specifically blocking the interaction of CD47 and SIRPa, so that the inhibition of tumor cells on phagocyte can be relieved, the phagocytic clearance of the tumor cells can be promoted, and the growth and the progression of tumors can be finally inhibited. The CD47 can be combined with therapeutic drugs such as cytarabine, an adaptive immune checkpoint antibody (such as a PD-1/PD-L1 antibody) and an opsonizing antibody aiming at a tumor specific antigen for use to further play a role in synergy anti-tumor, provide a new idea for tumor treatment and have important significance for further improving the curative effect.

The hybridoma antibody preparation technology is developed on the basis of somatic cell fusion technology. In 1975 kler (Kohler) and Milstein (Milstein) demonstrated that the fusion of myeloma cells with splenocytes from immunized animals resulted in the secretion of highly specific antibodies against the antigen. From the invention to date, although a large number of new antibody preparation techniques are emerging, hybridoma technology remains the primary mode of monoclonal antibody preparation at present. The monoclonal antibody prepared by the hybridoma technology has the characteristics of high affinity and strong specificity, and meanwhile, the candidate antibody molecules obtained by the technology generally have good druggability, so that a great deal of convenience is provided for later development of the medicine. With the progress of immunization, cell fusion and high-throughput screening technologies, hybridoma technology still flourishes. Therefore, the development of antibody drugs using the hybridoma technology is of great value.

Disclosure of Invention

Aiming at the prior art, the invention provides a murine blocking antibody aiming at the extracellular domain of human CD47, and also provides a coding sequence of the antibody, a vector containing the coding sequence and a host cell.

The invention is realized by the following technical scheme: BALB/c mice are immunized for multiple times by using high-purity human CD47 extracellular domain protein with biological activity, and hybridoma cells are screened and subcloned through fusion of spleen cells and SP2/0 cells, so that the murine blocking antibody specific to human CD47 is obtained. Then, taking the hybridoma cDNA as a template, and obtaining a variable region sequence of the hybridoma antibody through PCR amplification and sequencing analysis. Finally, the variable region sequences obtained by cloning were verified by flow cytometry.

The invention provides a murine blocking antibody against human CD47, which has the activity of blocking the binding of CD47 and SIRPa, and comprises a light chain variable region and a heavy chain variable region.

Wherein, the amino acid sequence and the nucleotide sequence of the heavy chain variable region are respectively shown as SEQ ID NO:1 and SEQ ID NO: 3, or a sequence similar to SEQ ID NO:1 or SEQ ID NO: 3 has a sequence identity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more.

Wherein, the amino acid sequence and the nucleotide sequence of the light chain variable region are respectively shown as SEQ ID NO:2 and SEQ ID NO:4, or a sequence identical to SEQ ID N: 2 or SEQ ID NO:4 has a sequence identity of at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more.

In a preferred embodiment, the amino acid sequence and the nucleotide sequence of the heavy chain variable region are as shown in SEQ ID NO:1 and SEQ ID NO: 3, and the amino acid sequence and the nucleotide sequence of the light chain variable region are respectively shown as SEQ ID NO. 2 and SEQ ID NO. 4.

The invention also provides application of the murine blocking antibody aiming at human CD47 in CD47 detection, and the specific application is that the capacity of the murine blocking antibody aiming at human CD47 in the invention to be specifically combined with CD47 is utilized, and enzyme-linked immunosorbent assay, immunofluorescence, immunochip assay, affinity chromatography and the like are adopted for detection.

The invention also provides application of the murine blocking antibody directed to human CD47 in preparation of a reagent or a kit for detecting CD 47.

The reagent or the kit for detecting CD47 contains the murine blocking antibody aiming at human CD 47.

The invention also provides a reagent or a kit, and the reagent or the kit contains the murine blocking antibody aiming at the human CD 47.

The invention also provides application of the reagent or the kit in the aspect of CD47 detection, and the specific application is that the detection is carried out by utilizing the specific binding capacity of the mouse source blocking type antibody or the reagent or the kit aiming at the human CD47 and the CD47 by adopting an enzyme-linked immunosorbent assay, an immunofluorescence method, an immuno-chip method, an affinity chromatography method and the like.

The invention also provides application of the murine blocking antibody or the reagent or the kit for human CD47 in preparation of a targeted CD47 therapeutic antibody drug.

The invention also provides a preparation method of the murine blocking antibody aiming at the human CD47, which comprises the following steps: the method specifically comprises the following steps:

(1) BLAB/c mouse immunization: female BALB/c mice of 6-8 weeks old are immunized by injecting CD47-Fc protein, then the mice are subjected to orbital bleeding to prepare immune serum for serum antibody titer detection.

(2) Detecting the antibody titer and blocking activity of immune serum by an ELISA method;

(3) cell fusion:

firstly, preparing feeder layer cells: sucking abdominal cavity liquid from the abdominal cavity of the non-immunized BALB/c mouse under a sterile condition and centrifuging to remove a supernatant; cells were then resuspended using IMDM complete medium and plated into well plates.

Cell fusion: taking out spleens of the immunized female BALB/c mice in the step (1) under aseptic conditions, grinding and centrifuging to obtain splenic single cells; then mixed with myeloma cells SP2/0 cells, fused under PEG induction, and then seeded into feeder cells-plated well plates for selective culture.

(4) Positive clone screening and subcloning: and (3) after the cells in the step (3) are fused for several days, observing the fused cells in a microscopic way, calculating the cell fusion rate, screening positive holes, performing subcloning on the positive hole cells, and finally obtaining the hybridoma monoclonal capable of secreting the antibody with specificity aiming at the target antigen.

Wherein, after the antibody is obtained in the step (4), the method further comprises the following steps:

(5) and (3) antibody subtype identification: using a kit for identifying subtypes of the Yi-Qiao-Shen mouse antibody according to the color development condition and OD ₄₅₀ And (5) judging the antibody subtype by reading.

(6) Cloning and verifying a variable region: RNA extraction and cDNA Synthesis: culturing the hybridoma cells until they are close to the full culture dish, extracting total RNA of the hybridoma cells using Trizol, measuring the concentration, and then performing reverse transcription according to Promega reverse transcription kit (GoScript) ^TM Reverse Transcription System) instructions for cDNA synthesis.

Cloning and sequencing analysis of variable regions of hybridoma antibodies: PCR amplification was performed with KOD FX polymerase using the above cDNA as a template, 12 heavy chain variable region amplification primers, 10 kappa light chain variable region amplification primers and 1 lambda light chain variable region amplification primers, respectively, followed by detection of the PCR amplification product by agarose gel electrophoresis. Cutting and recovering the fragment matched with the size of the antibody light and heavy chain variable region, and using full-scale gold

Cloning with the Blunt Zero Cloning Kit, picking a single clone and sending it to the test. And (3) comparing and analyzing sequencing results, removing non-target sequences, and reserving target sequences which accord with the characteristics of the variable regions of the mouse antibodies.

Verifying the sequence of the variable region: respectively cloning the variable region sequences of the light and heavy chains of the antibody to a chimeric antibody expression vector pcDNA3.4 containing a human IgG1 antibody light and heavy chain constant region, extracting plasmids after the sequence to be tested is verified, co-transfecting the light and heavy chain expression vector to HEK293T cells, and collecting an expression supernatant. SKVO3 was used as a target cell, and the expression supernatant was used as a primary antibody, and whether or not the expression supernatant was bound to the target cell was examined by flow cytometry.

In a specific embodiment, the method comprises the steps of: (1) BLAB/c mouse immunization; 5 female BALB/c mice 6-8 weeks old were purchased from Shanghai Slek laboratory animals Co., Ltd and were housed in the center of SPF-grade laboratory animals. Human CD47-Fc protein was thawed on ice, 50. mu.g/mouse of the protein needed was calculated, diluted with PBS, mixed with an equal volume of Freund's complete adjuvant (Sigma-Aldrich, F5881), and injected subcutaneously in the back of mice in multiple spots. A second immunization was performed two weeks later, and the same amount of protein was mixed with incomplete Freund's adjuvant (Sigma-Aldrich, F5506) and injected subcutaneously in several spots. The third immunization is carried out after two weeks, and the immunization conditions and the immunization mode are the same as those of the second immunization. And 7 days after the third immunization, blood is taken from the orbit of the mouse, and immune serum is prepared for detecting the titer of the serum antibody. The mice with high serum antibody titer and good blocking activity are selected to be boosted 3 days before cell fusion, namely 100 mu g of human CD47-Fc protein is injected into the abdominal cavity of the mice.

(2) Detecting the titer and blocking activity of the immune serum antibody; detecting the antibody titer of immune serum: the microplate was coated with 1. mu.g/ml human CD47-his protein 100. mu.l/well and left overnight at 4 ℃. The antigen dilution in the ELISA plate was removed, washed three times with 0.05% PBST, 300. mu.l of 2% MPBS was added to each well, and the wells were left to stand at room temperature for 1 hour. Immune sera were diluted 1000-fold using 2% MPBS, followed by 2-fold gradient dilutions, setting 11 dilution gradients. Removing MPBS in the ELISA plate, washing with PBST for 3 times, adding diluted immune serum, and standing at room temperature for incubation for 1 h. The solution in the microplate was removed, washed 6 times with PBST, and the residual liquid was removed by patting on absorbent paper. Diluted HRP-labeled goat anti-mouse IgG secondary antibody (kang century) was added to the enzyme plate, and incubated at room temperature for 1 h. Removing secondary antibody, washing with PBST for 6 times, removing residual liquid by patting on absorbent paper, adding 100 μ l diluted TMB developing solution to each well, developing for 5min, and adding 100 μ l 2MH to each well ₂ SO ₄ The color reaction is stopped, and OD is read on the microplate reader ₄₅₀ . ② detecting the activity of immune serum blocking: the ELISA plate was coated with 100. mu.l/well of 1. mu.g/ml human CD47-Fc protein, and 3 wells were set at 4 ℃ overnight. The antigen dilution in the ELISA plate was removed, washed three times with 0.05% PBST, 300. mu.l of 2% MPBS was added to each well, and the wells were left to stand at room temperature for 1 hour. A SIRP α -his solution at a concentration of 0.5 μ g/ml was prepared using 2% MPBS, and then immune serum was diluted 100-fold with the solution. Removing MPBS in the ELISA plate, and patting on absorbent paper to remove residual liquid. Adding diluted immune serum into the ELISA plate, and standing and incubating for 1h at room temperature. And removing liquid in the enzyme label plate, washing for 6 times by PBST, and patting the plate on absorbent paper to remove residual liquid. And adding a diluted HRP-labeled goat-derived anti-his label secondary antibody to the ELISA plate, and standing and incubating for 1h at room temperature. And removing the secondary antibody in the ELISA plate, washing for 6 times by PBST, and patting the plate on absorbent paper to remove residual liquid. Removal of secondary antibody, PBST WashWashing for 6 times, removing residual liquid by patting on absorbent paper, adding 100 μ l diluted TMB developing solution into each well, developing for 5min, and adding 100 μ l 2M H into each well ₂ SO ₄ The color reaction is stopped, and OD is read on the microplate reader ₄₅₀ 。

(3) Fusing cells; firstly, preparing feeder layer cells: non-immunized BALB/c mice were prepared one day in advance, sacrificed by cervical dislocation, and soaked in 75% ethanol. The skin of the abdomen of the mouse was cut open in a clean bench using sterilized scissors and forceps, exposing the abdomen. 4ml of blank DMEM was injected into the abdominal cavity using a 5ml syringe for culture, the abdomen was gently massaged, the abdominal fluid was aspirated with the syringe and transferred to a 15ml centrifuge tube, centrifuged at 1,200rpm at room temperature for 3min, and the supernatant was removed. Cells were resuspended using IMDM containing 20% FBS in complete medium and transferred to 96-well plates at 100. mu.l/well and placed back in the incubator for culture. Cell fusion: the boosted mice were bled from the orbit and the blood collected in 1.5ml centrifuge tubes. Mice were sacrificed by cervical dislocation and soaked in 75% ethanol. In a clean bench, 75% ethanol excess in the mouse surface was removed with absorbent paper, the skin and tissue on the right side of the mouse back were cut with sterile scissors and forceps, the spleen was carefully removed, placed in DMEM medium preheated at 37 ℃ to remove excess connective tissue, and the spleen was ground with a syringe piston rod to separate the spleen cells. The 40 μm cell strainer was placed in a 50ml centrifuge tube, the spleen slurry was passed through the 40 μm strainer, the spleen was again ground using a syringe and the strainer was rinsed with DMEM until the residual tissue on the strainer became white in color. The mixture was centrifuged at 1,500rpm for 5min at room temperature to remove the supernatant. Approximately 40ml of DMEM was added to resuspend the cells, re-centrifuged and the supernatant removed. Meanwhile, SP2/0 cells were aspirated from the flask, collected in a 50ml centrifuge tube, centrifuged at 1,500rpm at room temperature for 5min, and the supernatant was removed. The preheated DMEM was resuspended SP2/0 cells, centrifuged at 1,500rpm at room temperature for 5min, and the supernatant was removed. Spleen cells and SP2/0 cells were resuspended in 1ml DMEM, mixed in the same centrifuge tube, pre-warmed DMEM was added, gently mixed and centrifuged to remove the supernatant. The beaker with the sterile deionized water at 37 ℃ was placed in a clean bench and the centrifuge tube with the two cells was placed on water. Within 1min, 50% PEG was added drop-wise to the bottom of the centrifuge tube using a 1ml pipette, and the tube was shaken while adding to disperse the cells thoroughly. After the PEG addition was complete, the mixture was shaken in water at 37 ℃ for 2 min. Then DMEM was added to 37.5ml in 3 consecutive drops over 2min, and the mixture was gently inverted to avoid bubbling. Centrifuged at 1,500rpm at room temperature for 5min to remove the supernatant. The cells were resuspended in 4ml complete medium (IMDM + 20% FBS + P/S +2 XHAT) and divided equally into 2 centrifuge tubes containing 50ml complete medium, and after mixing by inversion, the cells were divided into 10 96-well plates plated with feeder cells at 100. mu.l/well.

(4) Screening positive clones and subcloning. Screening positive clones: after 7 days of cell fusion, the fused cells were observed under a microscope, and the cell fusion rate was calculated. Taking culture supernatant to carry out ELISA detection, and the specific method comprises the following steps: human CD47-his protein was diluted to 1. mu.g/ml using PBS, added to a 96-well plate at 100. mu.l/well and incubated overnight at 4 ℃. Antigen solution was removed, PBST was washed 3 times, residual solution was removed by patting on absorbent paper, 300. mu.l of 2% MPBS was added to each well, and incubation was performed at room temperature for 1 hour. During this period, 50. mu.l of the supernatant was removed from the fused cell 96-well plate after 7 days of culture to a new 96-well plate, and an equal volume of 2% MPBS was added. MPBS in the enzyme label plate is removed, PBST is washed for 3 times, and residual liquid is removed by patting on absorbent paper. And correspondingly adding the diluted hybridoma supernatant into a 96-hole enzyme label plate, and standing and incubating for 1h at room temperature. PBST was washed 6 times and the residual liquid was removed by patting on absorbent paper. Mu.l of HRP-labeled goat anti-mouse IgG secondary antibody was added to each well, and incubated at room temperature for 1 hour. PBST was washed 6 times and the residual liquid was removed by patting on absorbent paper. Add 100. mu.l TMB substrate to each well, develop for 5min, then add 100. mu.l 2M H to each well ₂ SO ₄ OD read by microplate reader ₄₅₀ The value is obtained. Subcloning of hybridoma clones: feeder cells were prepared one day in advance as described above. Positive well cells that were detected by ELISA to bind to the target antigen were diluted with IMDM (IMDM + 10% FBS + P/S +2 XHAT) to give approximately 300 cell inoculations per 96 well plate. After 7 days of culture, the supernatant was again subjected to ELISA detection, and positive well cells were subcloned. ELISA screening and subcloning were performed several times in the same manner, and a monoclonal antibody secreting an antibody specific to the antigen of interest was finally obtained.

(5) Antibody subunitIdentifying the type; the identification of the antibody subtype is detected by using a kit for identifying the antibody subtype of a Chinesia mouse, and the specific operation is as follows: subtype-specific antibodies were diluted with PBS, IgG1, IgG2b, IgG3 at 1:1000 and IgG2a and IgM at 1:5000, added to a 96-well plate at 100. mu.l/well and incubated overnight at 4 ℃. Antigen solution was removed, washed 3 times with 0.05% PBST, and residual solution was removed by patting on absorbent paper. Mu.l PBS containing 2% BSA was added to each well and blocked for 1h at room temperature. The blocking solution was removed, PBST washed 3 times, and the residual liquid was removed by patting on absorbent paper. Mu.l of the monoclonal culture supernatant was added to each well, and the mixture was incubated at room temperature for 1 hour. Removing liquid in the enzyme label plate, washing for 3 times by PBST, and patting the plate on absorbent paper to remove residual liquid. The rabbit anti-mouse IgG antibody was diluted at a ratio of 1:5000 using PBS containing 0.1% BSA, and 100. mu.l/well was added to a 96-well plate. Removing liquid in the enzyme label plate, washing for 3 times by PBST, and patting the plate on absorbent paper to remove residual liquid. Add 100. mu.l TMB substrate to each well and develop for 10min, then add 100. mu.l 2M H to each well ₂ SO ₄ The color reaction was terminated and the OD was read ₄₅₀ . According to the color development and OD ₄₅₀ And (5) reading values to judge antibody subtypes.

(6) Cloning and verifying the variable region. RNA extraction and cDNA Synthesis: culturing the hybridoma cells until they have grown on a culture dish, extracting total RNA from the hybridoma cells using Trizol, measuring the concentration, and then performing reverse transcription according to Promega reverse transcription kit (GoScript) ^TM Reverse Transcription System) instructions for cDNA synthesis. Cloning and sequencing analysis of variable regions of hybridoma antibodies: PCR amplification was performed with KOD FX polymerase using the above cDNA as a template, 12 pairs of heavy chain variable region amplification primers, 10 pairs of kappa light chain variable region amplification primers and 1 pair of lambda light chain variable region amplification primers, respectively, followed by detection of the PCR amplification product by agarose gel electrophoresis. Cutting and recovering the fragment matched with the size of the antibody light and heavy chain variable region, and using full-scale gold

Cloning with the Blunt Zero Cloning Kit, monoclonal shake bacteria were picked and sent to assay. And (3) comparing and analyzing the sequencing result, removing non-target sequences, and reserving target sequences which accord with the variable region characteristics of the mouse antibody. (iii) variableVerification of the sequence of the region: respectively cloning the variable region sequences of the light and heavy chains of the antibody to a chimeric antibody expression vector pcDNA3.4 containing the constant region of the light and heavy chains of the human IgG1 antibody, extracting plasmids after the sequence to be tested is verified, co-transfecting the light and heavy chain expression vector to HEK293T cells, and collecting an expression supernatant. SKVO3 was used as a target cell, and the expression supernatant was used as a primary antibody, and whether or not the expression supernatant was bound to the target cell was examined by flow cytometry.

The invention also provides a nucleotide for coding the murine blocking antibody against human CD47, wherein the nucleotide sequence is one of the following sequences:

(1) as shown in SEQ ID NO: 3 or SEQ ID NO: 4;

(2) and SEQ ID NO: 3 or SEQ ID NO:4, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity;

(3) as shown in SEQ ID NO: 3 and SEQ ID NO:4, the nucleotide sequence is obtained by adding, substituting, deleting or inserting one or a plurality of nucleotides into the nucleotide sequence shown in the formula (I);

(4) a nucleotide sequence that hybridizes under stringent conditions to the nucleotide sequence of (1), (2), or (3), or the full-length complement thereof; or

(5) The nucleotide sequence differs from the nucleotide sequences of (1), (2), (3) and (4) due to the degeneracy of the genetic code.

The nucleotide sequence or at least a part of the sequence according to the invention can be expressed by means of a suitable expression system to obtain the corresponding protein or polypeptide. These expression systems include bacterial, yeast, filamentous fungi, mammalian cells, insect cells, plant cells or cell-free expression systems.

The invention also provides an expression vector which comprises the nucleotide for coding the murine blocking antibody aiming at the human CD 47.

The invention also provides a host cell comprising the expression vector.

The host cell is 293F, CHO-S, CHO-K1 and the like; preferably, it is 293F.

The invention has the beneficial effects that: the invention firstly uses a mammalian cell expression system and utilizes protein affinity chromatography to obtain a fusion protein of human CD47 extracellular domain and human IgG4 Fc with high purity and biological activity, and then the protein is used for immunizing BALB/c mice by subcutaneous multi-point injection. After 3 times of immunization, serum of an immunized mouse is taken for titer detection and blocking activity analysis, a mouse with the highest titer is selected for boosting immunization, and spleen cells of the mouse are taken to be fused with myeloma cells SP 2/0. The fused cells were selectively cultured using HAT selective medium, while the fused cells secreting the antibody against human CD47 were screened by ELISA. And performing multiple subcloning and ELISA verification on the fusion cells capable of secreting the antibody against human CD47 by a limiting dilution method to finally obtain a monoclonal capable of secreting the antibody specific to human CD 47. Identifying the antibody subtype by using a mouse antibody subtype identification kit, then cloning and sequencing a heavy chain variable region and a light chain variable region respectively by using a plurality of pairs of primers, excluding non-target sequences, cloning the obtained candidate antibody sequence to an expression vector and expressing in HEK293T cells, performing flow detection by using the expression supernatant as a primary antibody, and determining that the obtained sequence is the target sequence. The murine blocking antibody aiming at human CD47 obtained by the invention has important application value in the research and development of CD47 antibody drugs.

Drawings

FIG. 1 is a graph showing the detection of serum antibody titer of CD47-Fc immunized mice of the present invention, in which 5 BALB/c mice were immunized with human CD47-Fc protein, and the serum antibody titer reached about 1000 ten thousand after 3 immunizations.

FIG. 2 is a diagram of the analysis of the blocking activity of the serum of CD47-Fc immunized mice, and the 5 BALB/c mice immunized with CD47-Fc protein have different degrees of blocking activity, wherein the 1#, 4#, and 5# mice have stronger blocking activity.

FIG. 3 is a diagram showing the subtype identification of the anti-human CD47 antibody 9B6 according to the present invention.

FIG. 4 is a flow cytometric assay of sequence verification of the variable region of anti-human CD47 antibody 9B6 of the present invention.

FIG. 5 is a flow chart showing the binding activity and specificity of anti-human CD47 antibody 9B6 to antigen.

FIG. 6 is a detection diagram of enzyme-linked immunosorbent assay of anti-human CD47 antibody 9B6 binding affinity to antigen.

FIG. 7 is an enzyme-linked immunosorbent and flow cytometry assay of blocking activity of 9B6 of the invention. Wherein, A is enzyme-linked immunosorbent assay 9B6 blocking activity; b was flow cytometry to detect 9B6 blocking activity.

FIG. 8 is a flow cytometric map of the present invention 9B6 promoting macrophage phagocytic tumor cell function. Wherein, a representative picture of detecting anti-human CD47 antibody promoting macrophage phagocytosis K562 by flow cytometry; B. phagocytosis ratio histogram.

Detailed Description

The invention is further illustrated by the following examples and figures. Unless otherwise specified, the instruments, reagents, materials and the like used in the following examples are conventional instruments, reagents, materials and the like known in the art and are commercially available. Unless otherwise specified, the experimental methods, detection methods, and the like described in the following examples are conventional experimental methods, detection methods, and the like in the prior art.

Example 1 BALB/c mouse immunization and cell fusion

(1) First, sufficient human CD47-Fc protein was obtained by expression and purification, and then 5 BALB/c mice were immunized 3 times every two weeks. For the first immunization, 0.6mg of human CD47-Fc was mixed well in equal volumes with Freund's complete adjuvant (Sigma-Aldrich, F5881) and immunized by subcutaneous multi-point injection. The latter two immunizations were performed by subcutaneous multiple injections of 0.6mg of human CD47-Fc protein mixed with Freund's incomplete adjuvant (Sigma-Aldrich, F5506). Blood is taken from the orbit 5-7 days after the third immunization, serum is prepared, and the serum antibody titer and the blocking activity are detected by ELISA, wherein the specific results are shown in the figure 1 and the figure 2.

(2) The mice with the highest serum antibody titer and blocking activity were selected for boosting: 3 days before cell fusion, CD47-Fc protein was diluted with PBS and injected intraperitoneally at a dose of 100. mu.g/cell.

(3) And preparing feeder layer cells. One day before cell fusion, non-immune BALB/c mice were taken, and the mice were sacrificed by cervical dislocation and immersed in 75% ethanol. The skin of the abdomen of the mouse was cut open with scissors in a sterile console to expose the abdomen. The mouse abdominal cavity was gently massaged by sucking 5ml of DMEM medium with a 5ml syringe, and the abdominal cavity fluid was extracted with a syringe. The extracted peritoneal fluid was mixed well with an appropriate amount of IMDM + 20% FBS medium, and then inoculated into 10 96-well plates at 37 ℃ with 5% CO at 100. mu.l/well ₂ Incubated under conditions overnight.

(4) And (4) fusing the cells. Boosted BALB/c mice were retrieved from the center of the experimental animals, bled from the eye socket, and collected in 1.5ml sterile centrifuge tubes for use as positive controls. Mice were sacrificed by cervical dislocation, soaked in 75% ethanol and brought into the cell house. 3 10cm dishes were taken, 10ml of 37 ℃ pre-warmed DMEM was added to one dish, the mice were placed in the other dish, the mouse spleen was carefully removed, excess connective tissue was removed, and the spleen was placed in a dish containing DMEM. DMEM was aspirated from the dish using a 1ml syringe, the needle was inserted into the spleen, and DMEM was added thereto, and this was repeated several times to wash out a large number of spleen cells. A40 μm sieve was placed on a 50ml centrifuge tube, the spleen was placed on the sieve, the syringe needle was removed, the spleen was washed by sucking the medium in the dish into the sieve, the spleen was gently ground with the syringe needle, and then DMEM was added to 50 ml. The mixture was centrifuged at 1,500rpm for 5min at room temperature, and the supernatant was removed. Approximately 40ml of DMEM was added to resuspend the cells, re-centrifuged and the supernatant removed. Meanwhile, SP2/0 cells were aspirated from the flask, collected in a 50ml centrifuge tube, centrifuged at 1,500rpm at room temperature for 5min, and the supernatant was removed. The preheated DMEM was resuspended in SP2/0 cells at 1,500rpm at room temperature, centrifuged for 5min, and the supernatant was removed. Spleen cells and SP2/0 cells were resuspended in 1ml DMEM, mixed in the same centrifuge tube, pre-warmed DMEM was added, gently mixed and centrifuged to remove the supernatant. The beaker with the 37 ℃ sterile deionized water was placed in a clean bench and the centrifuge tube containing the two cells was placed on water. Within 1min, PEG was added dropwise to the bottom of the centrifuge tube using a 1ml pipette, and the centrifuge tube was rotated while adding, during which time the cells should be well dispersed and suspended. After the PEG was added, the mixture was gently shaken in water for 2 min. Then DMEM was added to 37.5ml in 3 consecutive drops over 2min, and the mixture was gently inverted to avoid bubbling. Centrifugation was carried out at 1,500rpm at room temperature for 5min, and the supernatant was removed. The cells were resuspended in 4ml complete medium (IMDM + 20% FBS + P/S +2 XHAT), split equally into two centrifuge tubes containing 50ml complete medium, mixed by inversion and seeded at 100. mu.l/well into 10 feeder cells plated 96-well plates.

Example 2 Positive clone screening and subcloning

The cells fused in example 1 of the present invention were cultured for about 7 days, and supernatants were collected from 96-well plates and subjected to ELISA detection, and fused cells producing an antibody capable of specifically binding to human CD47 protein were selected and subjected to subclone culture. After multiple subcloning, monoclonal capable of secreting the target antibody can be obtained.

Screening positive clones: after 7 days of cell fusion, the fused cells were observed under a microscope, and the cell fusion rate was calculated. Taking culture supernatant to carry out ELISA detection, and the specific method comprises the following steps: the CD47-his protein was diluted to 1. mu.g/ml with PBS, added to a 96-well plate at 100. mu.l/well, and incubated overnight at 4 ℃. Antigen solution was removed, PBST was washed 3 times, residual solution was removed by patting on absorbent paper, 300. mu.l of 2% MPBS was added to each well, and incubation was performed at room temperature for 1 hour. During this period, 50. mu.l of the supernatant was removed from the fused cell 96-well plate after 7 days of culture to a new 96-well plate, and an equal volume of 2% MPBS was added. MPBS in the enzyme label plate is removed, PBST is washed for 3 times, and residual liquid is removed by patting on absorbent paper. And correspondingly adding the diluted hybridoma supernatant into a 96-hole enzyme label plate, and standing and incubating for 1h at room temperature. PBST was washed 6 times and the residual liquid was removed by patting on absorbent paper. Mu.l of HRP-labeled goat anti-mouse IgG secondary antibody was added to each well, and incubated at room temperature for 1 hour. PBST was washed 6 times and the residual liquid was removed by patting on absorbent paper. Add 100. mu.l TMB substrate to each well, develop for 5min, then add 100. mu.l 2M H to each well ₂ SO ₄ OD read by microplate reader ₄₅₀ The value is obtained.

Subcloning of hybridoma clones: feeder cells were prepared one day in advance as described above. Positive well cells that were detected by ELISA to bind to the antigen of interest were diluted with IMDM (IMDM + 10% FBS + P/S +2 XHAT) to give approximately 300 cell inoculations per 96 well plate. After 7 days of culture, the supernatant was again subjected to ELISA detection, and positive well cells were subcloned. ELISA screening and subcloning were performed several times in the same manner, and a monoclonal antibody secreting an antibody specific to the antigen of interest was finally obtained.

Example 3 antibody subtype identification, cloning and validation of light and heavy chain variable regions

(1) And (5) identifying antibody subtypes. Collecting the hybridoma monoclonal culture supernatant of embodiment 2 of the invention, identifying the antibody subtype by referring to the kit specification of the kit for identifying the antibody subtype of the Chinesia evanescens mouse, and specifically operating as follows: subtype-specific antibodies were diluted with PBS, where IgG1, IgG2b, IgG3 were diluted at 1:1000 and IgG2a and IgM were diluted at 1:5000, and 100. mu.l/well of 96-well microplate was added, and incubated overnight at 4 ℃. Antigen solution was removed, washed 3 times with 0.05% PBST, and residual solution was removed by patting on absorbent paper. To each well 300. mu.l PBS containing 2% BSA was added and blocked for 1h at room temperature. The blocking solution was removed, PBST washed 3 times, and the residual liquid was removed by patting on absorbent paper. Mu.l of the monoclonal culture supernatant was added to each well, and incubated at room temperature for 1 hour. Removing liquid in the enzyme label plate, washing for 3 times by PBST, and patting the plate on absorbent paper to remove residual liquid. The rabbit anti-mouse IgG antibody was diluted at a ratio of 1:5000 using PBS containing 0.1% BSA, and the 96-well plate was added at 100. mu.l/well. And removing liquid in the enzyme label plate, washing for 3 times by PBST, and patting the plate on absorbent paper to remove residual liquid. Add 100. mu.l TMB substrate to each well, develop for 10min, then add 100. mu.l 2M H to each well ₂ SO ₄ The color reaction was stopped and OD was read ₄₅₀ . According to the color development and OD ₄₅₀ And (5) judging the antibody subtype by reading. The results are shown in fig. 3, where 9B6 expression supernatant bound to mouse IgG1 subtype specific antibody but not to other subtype specific antibodies, indicating that its subtype is IgG1 (fig. 3).

(2) Cloning of light and heavy chain variable regions. Hybridoma monoclonals were cultured, total RNA of hybridoma cells was extracted using Trizol, and the RNA was transcribed according to Promega reverse transcription kit (GoScript) ^TM Reverse Transcription System) instructions for cDNA synthesis. Using cDNA as template, 12 pairs of heavy chain variable region primers, 10 pairs of kappa light chain variable region primers and 1 pair of lambda light chain variable region primers toKOD FX polymerase PCR-amplifies the light and heavy chain variable regions of the antibody, respectively. Then, the PCR product obtained by amplification is subjected to agarose gel electrophoresis detection, the band with the fragment size meeting the expectation is cut and recovered, and the full-type gold is used

Cloning was performed with the Blunt Zero Cloning Kit, and single clones were picked up and sent to the test. Removing non-target sequences through sequencing analysis and comparison to obtain potential target antibody sequences. The obtained light and heavy chain variable region sequences are respectively cloned on pcDNA3.4 vectors containing light and heavy chain constant regions and sequenced for verification.

(3) And (5) verifying the sequence of the light and heavy chain variable region. HEK293T cells were seeded into 6-well plates and antibody light and heavy chain expression vectors were transfected at a ratio of 1:1 when the cells grew to approximately 80% confluence and the medium was changed 8h after transfection. After culturing for 48h, the supernatant was collected, centrifuged to remove cell debris, and then used as a primary antibody and an APC-conjugated anti-human IgG Fc antibody was used as a secondary antibody to detect whether the expressed supernatant was bound to CD 47-positive cells. As shown in FIG. 4, the flow cytometry detection result shows that the supernatant of hybridoma 9B6, like the positive controls B6H12 and Hu5F9, can bind to SKOV3, indicating that the variable region sequence obtained by cloning is the target sequence.

Example 4 antigen binding Activity and specificity, binding affinity, blocking and phagocytosis promoting Components

(1) Antigen binding activity and specificity. Co-transfecting 293F cells with the antibody light-heavy chain expression vector at a ratio of 1.5: 1; 37 ℃ and 5% CO ₂ After shaking culture at 130rpm for 5 days, centrifugally collecting culture supernatant, filtering by 0.45 mu m, and carrying out protein affinity purification to obtain antibody protein with higher purity; the BCA method determines antibody concentration. The binding of the antibody to CD47 positive and negative cells was detected by flow cytometry to evaluate its antigen binding activity and specificity. The method comprises the following specific steps: digesting adherent cells with pancreatin, preparing the cells into a single cell suspension, counting the cells and adjusting the density to 2X 10 ⁶ Cells were dispensed into 1.5ml centrifuge tubes at 100. mu.l/tube. Centrifugation was carried out at 1,200rpm at 4 ℃ for 3min to remove the supernatant. Antibodies were diluted with staining bufferTo 1. mu.g/ml, cells were resuspended at 100. mu.l/tube. Incubating for 30min at 4 ℃; centrifugation was carried out at 1,200rpm at 4 ℃ for 3min to remove the supernatant. Wash once with staining buffer. APC coupled anti-human IgG Fc antibody was diluted 1:100, cells were resuspended in 50. mu.l/tube and incubated at 4 ℃ for 30min in the absence of light. Centrifugation was carried out at 1,200rpm at 4 ℃ for 3min to remove the supernatant. The cells were washed once with staining buffer, resuspended in 300. mu.l staining buffer, filtered through a 300 mesh screen and examined on a flow machine. The binding of the antibody to various cells is shown in fig. 5, and 9B6 can significantly bind to CD47 positive non-hodgkin lymphoma cell line Raji, acute T cell leukemia cell line Jurkat, chronic myeloid leukemia cell line K562, colon cancer cell lines HT29 and HCT116, ovarian cancer cell line SKOV3, and CHO cell stably expressing CD47 (CHO/CD47), while not binding to CD47 negative chinese hamster ovary cell line CHO, indicating that it has good antigen binding activity and specificity.

(2) Antigen binding affinity. Human CD47-his protein (ACRO, # SIA-H5225) was diluted to 1. mu.g/ml with PBS, coated on a 96-well plate microplate at 100. mu.l/well, and allowed to stand overnight at 4 ℃. PBST was washed 3 times, 300. mu.l of 2% MPBS was added to each well of the plate, and the plate was allowed to stand at room temperature for 1 hour. During this period, the antibody was diluted with a 3-fold gradient using 2% MPBS at an initial concentration of 1.5. mu.g/ml, and a blank without the addition of antibody was set. After blocking, the microplate was washed 3 times with PBST, and the 9B6, B6H12 and Hu5F9 antibodies diluted in a gradient were added to the microplate and incubated for 1H at room temperature. PBST was washed 6 times, and diluted HRP-labeled anti-human IgG secondary antibody (abcam, HRP-conjugated goat anti-human IgG, # ab81202) was added to the plate, and incubated at room temperature for 1 hour. PBST was washed 6 times, 100. mu.l TMB substrate was added to each well, color development was performed for 5min, and 100. mu.l 2M H was added to each well ₂ SO ₄ Read OD ₄₅₀ The value is obtained. Antibody concentration and OD Using GraphPad ₄₅₀ The values were fitted with four parameters to obtain affinity data for the antibody. The affinity detection result is shown in fig. 6, and the binding affinity of 9B6 and human CD47 protein is equivalent to that of the positive antibody Hu5F9, and higher than that of the positive antibody B6H 12.

(3) Antibody blocking activity. Blocking activity of the antibodies at the protein level and the cell level was detected using ELISA and flow cytometry, respectively.

Analysis of blocking activity at protein level: the 96-well plate was coated with 1. mu.g/ml of human CD47-Fc protein and left to stand overnight at 4 ℃. The microplate was washed 3 times with PBST, 300. mu.l of 2% MPBS was added to each well, and the mixture was incubated at room temperature for 1 hour. During this period, a dilution of SIRPa-his was prepared at a concentration of 0.5. mu.g/ml using 2% MPBS, and then the antibody was diluted in a 2-fold gradient with a maximum concentration of 20. mu.g/ml using the dilution of SIRPa-his, and a control without antibody was set. After blocking, the microplate was washed 3 times with PBST. And adding the diluted antibody into an enzyme label plate, and standing and incubating for 1h at room temperature. PBST was washed 6 times, and then 100. mu.l of diluted anti-His tag antibody labeled with HRP from Hovenia Protevia (#105327-MM02T-H) was added to each well, followed by incubation for 1H at room temperature. PBST was washed 6 times, 100. mu.l TMB substrate was added to each well, color development was performed for 5min, and 100. mu.l 2M H was added to each well ₂ SO ₄ Read OD ₄₅₀ The value is obtained. Antibody concentration and OD Using GraphPad ₄₅₀ The values are fitted with four parameters to obtain IC ₅₀ The value is obtained.

Blocking activity at cellular level: raji cells in logarithmic growth phase are divided into 10 ⁵ The cells/tube were divided into 1.5ml centrifuge tubes, and after removing the supernatant by centrifugation, the 9B6 hybridoma antibody was added in a gradient dilution, while 9B6 and B6H12 were used as control antibodies. After resuspending the cells, the cells were incubated at 4 ℃ for 30 min. The mixture was centrifuged at 1,200rpm for 3min at 4 ℃ to remove the supernatant. Wash once with 2% FPBS. Adding diluted PE labeled SIRP alpha-Fc (ACRO, # SIA-HP252), incubating at 4 deg.C in dark for 30min, and detecting on machine. Four-parameter fitting is carried out by using the concentration of the anti-CD 47 antibody and the Mean Fluorescence Intensity (MFI) to obtain IC ₅₀ The value is obtained.

The result of the blocking activity test is shown in fig. 7, and the blocking activity of 9B6 is equivalent to Hu5F9, slightly superior to Hu5F9, but obviously superior to B6H 12.

(4) Promoting phagocytic function. Peripheral blood from healthy volunteers was subjected to density gradient centrifugation using Ficoll to obtain peripheral blood mononuclear cells. PBMC were then sorted using magnetic beads of America and whirly CD14 to obtain CD14 ⁺ Monocytes were cultured in 1640 complete medium containing 100ng/ml human M-CSF, and the medium was changed every 3 days to induce activated macrophages after 7 days. Collect log phase K562 finesCells were centrifuged to remove supernatant, washed 2 times with PBS, and K562 cells were fluorescence stained with CFSE. Macrophages were trypsinized, washed 2 times with PBS, and stained with eFluor670 viable cell stain. Adding 5X 10 of the culture medium into each hole of a 96-hole round-bottom low-adsorption cell culture plate ⁴ K562 cells, followed by the addition of the CD47 antibody to be detected to a final concentration of 20. mu.g/ml, and finally 2.5X 10 ⁵ Slightly blowing and uniformly mixing the macrophages, standing and incubating for 2h in an incubator, detecting on a machine by using a flow cytometer, and calculating CFSE (circulating fluid stress) ⁺ eFluor670 ⁺ Cell occupancy eFluor670 ⁺ The proportion of cells and the detection result are shown in fig. 8, compared with isotype control IgG, 9B6 and positive control antibodies Hu5F9 and B6H12 can promote phagocytosis of K562 cells by macrophages, and the phagocytosis promotion proportion is 21.7%, 20.7% and 18.9%, respectively.

The protection of the present invention is not limited to the above embodiments. Variations and advantages that may occur to those skilled in the art are intended to be included within the invention without departing from the spirit and scope of the inventive concept, and the scope of the invention is to be determined by the appended claims.

<110> university of east China

<120> murine blocking antibody for human CD47, preparation and application thereof

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 117

<212> PRT

<213> Artificial sequence

<400> 1

Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Thr

1 5 10 15

Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asn Tyr

20 25 30

Trp Leu Gly Trp Val Lys Gln Arg Pro Gly His Gly Pro Glu Trp Ile

35 40 45

Gly Asp Ile Tyr Leu Gly Ser Asp Tyr Ser Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Thr Ser Ser Ser Thr Ala Tyr

65 70 75 80

Ile Gln Leu Asn Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Val Arg Arg Gly Arg Gly Gly Gly Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Leu Thr Val Ser Ser

115

<210> 2

<211> 112

<212> PRT

<213> Artificial sequence

<400> 2

Asp Val Leu Met Thr Gln Thr Pro Leu Ser Leu Pro Val Ser Leu Gly

1 5 10 15

Asp Gln Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Lys Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Gly Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Phe Cys Phe Gln Gly

85 90 95

Ser His Val Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys

100 105 110

<210> 3

<211> 351

<212> DNA

<213> Artificial sequence

<400> 3

caggtgcagc tgaagcagtc tggagctgag ctggtaaggc ctgggacttc agtgaagata 60

tcctgcaagg cttctggcta catcttcact aactactggc taggttgggt aaaacagagg 120

cctggacatg gacctgaatg gattggagat atctatcttg gaagtgatta tagtaattac 180

aatgagaaat tcaagggcaa ggccacactg actgcagaca catcttcaag cactgcctac 240

atacaactca atagcctgac atctgacgac tctgctgtct atttctgtgt tagaagaggg 300

agggggggag gggactactg gggccaaggc accactctca cagtctcctc a 351

<210> 4

<211> 336

<212> DNA

<213> Artificial sequence

<400> 4

gatgttttga tgacccaaac tccactctcc ctgcctgtca gtcttggaga tcaagcctcc 60

atctcttgca gatctagtca gagcattgta catagtaatg gaaacaccta tttagaatgg 120

tacttgcaga aaccgggcca gtctccaaag ctcctgattt acaaagtttc caaccgattt 180

tctggggtcc cagacaggtt cagtggtggt ggatcaggga cagatttcac actcaagatc 240

agcagagtgg aggctgagga tctgggagtt tatttctgct ttcaaggttc acatgttcca 300

ctcacgttcg gtgctgggac caagctggag ctgaaa 336

Claims (8)

1. A murine blocking antibody against human CD47, wherein said antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence and the nucleotide sequence of said heavy chain variable region are set forth in SEQ ID NO:1 and SEQ ID NO: 3 is shown in the specification; the amino acid sequence and the nucleotide sequence of the light chain variable region are respectively shown as SEQ ID NO:2 and SEQ ID NO:4, respectively.

2. A reagent or kit comprising the murine blocking antibody of claim 1 to human CD 47.

3. Use of a murine blocking antibody against human CD47 according to claim 1 or a reagent or kit according to claim 2 for the preparation of a product for the detection of CD 47.

4. Use of the murine blocking antibody against human CD47 of claim 1 in the preparation of a CD47 detection reagent or kit.

5. Use of the murine blocking antibody against human CD47 of claim 1 for the preparation of a CD47 targeted therapeutic antibody medicament.

6. A polynucleotide encoding the murine blocking antibody against human CD47 of claim 1.

7. An expression vector comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7, wherein the host cell is 293F, CHO-S or CHO-K1.

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