CN114560940B - SIRP alpha resisting rabbit recombinant monoclonal antibody, and preparation method and application thereof - Google Patents

Abstract

The invention relates to an anti-SIRP alpha rabbit recombinant monoclonal antibody, a preparation method and application thereof, wherein the amino acid sequences of heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 of the anti-SIRP alpha rabbit recombinant monoclonal antibody are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6. The SIRPalpha rabbit recombinant monoclonal antibody has good specificity, can be used in ELISA and common flow detection methods, and has potential tumor immunotherapy value, and most importantly, the antibody can block the interaction of CD47 protein and SIRPalpha protein.

Description

SIRP alpha resisting rabbit recombinant monoclonal antibody, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to an anti-SIRP alpha rabbit recombinant monoclonal antibody with blocking activity, and a preparation method and application thereof.

Background

The Signal regulatory protein alpha (i.e. Signal reg mu latory protein alpha, SIRPalpha) is a transmembrane protein, is expressed on the surfaces of macrophages and bone marrow cells, and can be combined with a ligand CD47 protein thereof to generate a Signal of 'do not eat me', thereby preventing the phagocytosis of healthy cells by the macrophages. However, tumor cells are very subtle in that they can take advantage of this mechanism to evade phagocytosis by macrophages. Thus, blocking the binding of CD47 to sirpa by CD47 antibodies can activate phagocytosis of tumors by macrophages. However, CD47 is also highly expressed on the surface of erythrocyte membranes, and CD47 can injure erythrocytes accidentally while killing tumor cells with therapeutic drugs, resulting in anemia. In addition, due to the broad expression of CD47, large doses of antibodies are required to be effective clinically, which is two major bottlenecks in clinical development of CD47 at present. SIRP alpha exists mainly on the surfaces of macrophages and dendritic cells, and inhibition of SIRP alpha does not cause erythrocyte anemia basically, and meanwhile, effects can be achieved without a large amount of antibodies. Thus, antibodies against sirpa that block CD47 and sirpa would also be one direction of future tumor immunotherapy.

Disclosure of Invention

The invention aims to provide an SIRP alpha-resistant rabbit recombinant monoclonal antibody for blocking the actions of CD47 and SIRP alpha, and a preparation method and application of the SIRP alpha-resistant rabbit recombinant monoclonal antibody.

The amino acid sequences of heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 of the anti-SIRP alpha rabbit recombinant monoclonal antibody provided by the invention are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

Based on the technical scheme, the heavy chain variable region sequence of the SIRPalpha rabbit recombinant monoclonal antibody is the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 8.

Based on the technical scheme, the SCFV sequence of the SIRPalpha rabbit recombinant monoclonal antibody is an amino acid sequence shown as SEQ ID NO. 9.

Based on the technical scheme, the light chain constant region of the SIRPalpha rabbit recombinant monoclonal antibody is a kappa chain, and the heavy chain constant region is an IgG type.

The invention also provides a nucleic acid molecule comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of an anti-sirpa rabbit recombinant monoclonal antibody.

The invention also provides a vector containing the nucleic acid molecule.

The invention also provides a host cell containing the SIRP alpha resisting rabbit recombinant monoclonal antibody, the nucleic acid molecule or the vector.

The invention also provides a conjugate which contains the SIRP alpha resisting rabbit recombinant monoclonal antibody.

The invention also provides a pharmaceutical composition comprising a main ingredient and an auxiliary ingredient, wherein: the main component is one or more of the above anti-SIRPalpha rabbit recombinant monoclonal antibody, the above nucleic acid molecule, the above vector, the above host cell, the above conjugate, and the auxiliary component is selected from pharmaceutically acceptable carrier or excipient, and optional other bioactive substances.

The invention also provides application of the SIRP alpha resisting rabbit recombinant monoclonal antibody, the acid molecule, the carrier, the host cell and the conjugate in preparing medicaments or detection reagents for treating diseases.

The invention also provides a kit which comprises the SIRP alpha resisting rabbit recombinant monoclonal antibody.

The invention also provides a method for preparing the SIRP alpha resisting rabbit recombinant monoclonal antibody, which comprises the following steps:

(1) Immunizing a New Zealand white rabbit by using human SIRP alpha extracellular region FC fusion protein, and collecting peripheral blood; separating PBMC cells in the collected peripheral blood by using lymphocyte separation liquid;

(2) Incubating SIRP alpha protein coupled magnetic beads and the separated PBMC cells at room temperature, and separating the magnetic beads and the supernatant to obtain B lymphocytes;

(3) Culturing B lymphocytes by using a culture medium containing human IL2, collecting culture medium supernatant, and identifying antibodies;

(4) Extracting RNA of B cells identified as SIRP alpha specificity, reversely transcribing the RNA into cDNA, carrying out PCR amplification on heavy and light chain genes of the SIRP alpha specificity antibody by using a primer, recovering PCR products, cloning the heavy and light chain genes of the SIRP alpha specificity antibody to an expression vector, then carrying out transformation, verifying single colonies by using colony PCR, and carrying out gene sequencing on positive colonies to obtain a gene sequence of the SIRP alpha specificity antibody;

(5) Co-transfecting expression plasmids of heavy and light chain genes of the SIRP alpha specific antibody into 293 cells, culturing, collecting cell supernatant, and purifying by using protein A resin to obtain the SIRP alpha resistant rabbit recombinant monoclonal antibody of claim 1.

Compared with the traditional hybridoma fusion technology, the invention provides the anti-SIRP alpha rabbit recombinant monoclonal antibody with blocking activity by utilizing the B cell cloning technology, the antibody can be applied to ELISA (enzyme-linked immunosorbent assay) and common flow reaction, and most importantly, the antibody can block the interaction between CD47 protein and SIRP alpha protein and has potential value in tumor immunotherapy.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows an SDS electrophoresis pattern of SIRPalpha extracellular region protein.

FIG. 2 shows FACs detection results of anti-SIRP alpha rabbit recombinant monoclonal antibodies.

FIG. 3 shows that competition ELISA detects that the recombinant monoclonal antibody of the anti-SIRPalpha rabbit blocks the interaction of SIRPalpha protein and CD47 protein.

FIG. 4 shows that competing FACs detect SIRP alpha anti-rabbit recombinant monoclonal antibodies block the interaction of SIRP alpha protein with CD47 protein.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The invention is further described below: in the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.

The term "antibody" as referred to herein includes whole antibodies and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chain thereof. An "antibody" refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains, or antigen-binding portions thereof, linked together by disulfide bonds. Each heavy chain consists of a heavy chain variable region and a heavy chain constant region. The proteins or fragments thereof involved in the present invention may be naturally purified products, or chemically synthesized products, or produced from eukaryotic hosts (e.g., mammalian cells) using recombinant techniques. The raw materials and reagents used in the present invention are commercially available.

The technical scheme of the invention is further described below by combining examples.

Examples:

(1) Preparation of sirpa extracellular region protein: the invention constructs eukaryotic expression plasmid of human SIRPalpha extracellular region (Glu 31-Arg 370) FC fusion protein, and after transfection of 293F cells for 5 days, cell culture medium supernatant is collected, protein A resin is utilized for purification, and protein concentration is identified.

(2) Obtaining peripheral blood mononuclear cells (namely PBMC cells) of immunized animals: new Zealand white rabbits were selected as immunized animals. In the first immunization, 250 μg of SIRP alpha extracellular region protein is emulsified with an equal volume of complete Freund's adjuvant, the subcutaneous back of New Zealand white rabbits are injected at multiple points, the second immunization is carried out after 21 days, 120 μg of protein is emulsified with an equal volume of incomplete Freund's adjuvant, the back of the New Zealand white rabbits is subcutaneously injected, and the third immunization is carried out after 21 days. Peripheral blood was collected aseptically one week after the third immunization.

(3) Acquisition of sirpa-specific B lymphocytes: separating PBMC cells in the collected peripheral blood with lymphocyte separation medium; coupling SIRP alpha protein to the magnetic beads according to the operation instructions of the immune magnetic beads; and (3) incubating the mixture of SIRPalpha protein-coupled magnetic beads and the separated PBMC cells for 50min at room temperature, placing the mixture into a magnetic rack, immersing the magnetic beads at the bottom after 5min, discarding the supernatant, adding sterile PBS to wash the cells, repeatedly washing the cells for 3 times, and finally separating the cells to obtain the SIRPalpha-specific B lymphocytes.

(4) Identification of B lymphocytes: diluting the B lymphocyte obtained by separation by a certain multiple, placing into 96-well cell culture plate, adding 1640 medium containing 10% Fetal Bovine Serum (FBS) and 2 μg/ml human IL2, and 5% CO at 37deg.C ₂ Culturing under the condition for 6 days. Culture supernatant was collected for antibody identification.

a) Indirect ELISA identification:

coating SIRPalpha protein with the concentration of 1 mug/ml, 100 mug/hole and incubating for 16h at 4 ℃; after the next day the coating was discarded, it was blocked with PBS containing 1% Bovine Serum Albumin (BSA), 150. Mu.l/well, and incubated at 37℃for 1h. The blocking solution was discarded, and the B cell supernatant was added, 50. Mu.l/well, and incubated at 37℃for 1 hour. The B cell supernatant was discarded, the plate was washed 5 times with PBS containing 0.5wt.% Tween-20 for 2 min/time, and finally a 5000-fold dilution of goat anti-rabbit IgG-HRP secondary antibody was added and incubated for 1h at 37 ℃. The secondary antibody was discarded and the plate was washed 5 times with phosphate Tween buffer (PBST) for 2 min/time. The washing liquid is discarded, the washing liquid is taken to dry, and the substrate is added for color development.

b) And (3) identifying by a streaming method:

the test was performed with human myeloid leukemia mononuclear cells THP-1. That is, the cells were collected in a centrifuge tube, washed twice with sterile PBS, blocked with Fc receptor blocking solution for cell surface Fc receptor, and incubated at 4℃for 30min. Cells were collected by centrifugation, washed twice with PBS containing 0.5wt.% BSA, added to the B cell culture supernatant, and incubated at 4℃for 30min. Cells were washed twice with PBS containing 0.5wt.% BSA, and finally incubated with goat anti-rabbit IgG-488 fluorescent secondary antibody at 4℃for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, 200. Mu.l of PBS containing 0.5wt.% BSA was added to resuspend the cells, and the cells were tested on-stream machine.

(5) Cloning of antibody genes: b cells identified as positive were collected, RNA was extracted by a conventional RNA extraction method, reverse transcribed into cDNA, and the gene primers for the heavy chain of the antibody were used: an upstream primer 5'-CAGTCGCTGGAGGAGTCCGG-3' and a downstream primer 5'-CCATTGGTGAGGGTGCCCGAG-3', primers for the antibody light chain gene are: the upstream primer 5'-GACATTGTGATGACCCAGAC-3' and the downstream primer 5'-CCACCTCGGTCCCTTCGCCG-3' amplify the antibody heavy and light chain gene under the following conditions: denaturation was carried out at 94℃for 3min, (95℃1min,56℃30s,72℃1 min) and 30 cycles of reaction were carried out, followed by extension at 72℃for 10min. And (5) recovering the PCR product by using a DNA gel purification recovery kit. The heavy chain gene of the rabbit recombinant monoclonal antibody is cloned to an expression vector and then transformed, a colony PCR is utilized to verify a single colony, a positive colony is subjected to gene sequencing, the gene sequence of the SIRP alpha rabbit recombinant monoclonal antibody can be obtained, the amino acid sequences of heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, the amino acid sequences of the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6, the heavy chain variable region sequence is shown as SEQ ID NO. 7, the amino acid sequence of the light chain variable region sequence is shown as SEQ ID NO. 8, the amino acid sequence of the SCFV sequence is shown as SEQ ID NO. 9, the light chain constant region of the anti SIRP alpha rabbit recombinant monoclonal antibody is an IgG kappa chain, and the heavy chain constant region is of the IgG type.

(6) Production and identification of sirpa rabbit recombinant monoclonal antibodies: expression of the heavy and light chain genes of the antibodyPlasmid co-transfection into 293 cells, 5% CO at 37 ℃C ₂ Culturing under the culture condition for 72h, collecting cell supernatant, and purifying antibody by using protein A resin.

The purified antibody is subjected to function identification, and the method is concretely as follows:

a) ELISA reaction:

coating SIRPalpha protein with the concentration of 1 mug/ml, 100 mug/hole and incubating for 16h at 4 ℃; after the next day the coating was discarded, it was blocked with PBS containing 1wt.% BSA, 150. Mu.l/well and incubated for 1h at 37 ℃. The blocking solution was discarded, antibodies diluted in different ratios were added, 50. Mu.l/well and incubated for 1h at 37 ℃. The antibody was discarded, the plate was washed 5 times with PBS containing 0.5wt.% Tween-20 for 2 min/time, and finally a 5000-fold dilution of goat anti-rabbit IgG-HRP secondary antibody was added and incubated for 1h at 37 ℃. The secondary antibody was discarded and the plates were washed 5 times with PBST for 2 min/time. The washing liquid is discarded, the washing liquid is taken to dry, and the substrate is added for color development.

TABLE 1 ELISA assay results for SIRP alpha Rabbit recombinant monoclonal antibodies

Concentration of antibody dilution (ng/ml)	OD450 value
		150	Spillover
30	Spillover
		6	3.5169
1.2	1.5652
		0.24	0.4844
0.048	0.2045
		0.0096	0.1386
Blank space	0.1237

b) Flow reaction:

the test was performed with human myeloid leukemia mononuclear cells THP-1. The cells are collected in a centrifuge tube, washed twice by sterile PBS, and blocked by Fc receptor blocking liquid on the surface of the cells, and incubated for 30min at 4 ℃. Cells were collected by centrifugation, washed twice with PBS containing 0.5wt.% BSA, purified antibodies diluted in different ratios were added, and incubated at 4℃for 30min. Cells were washed twice with PBS containing 0.5wt.% BSA, and finally goat anti-rabbit IgG-488 fluoroantibody was added, 50 μl/sample, and incubated at 4deg.C for 30min. After washing the cells twice with PBS containing 0.5wt.% BSA, the cells were resuspended with 200. Mu.l of PBS containing 0.5wt.% BSA and tested on-stream.

c) Blocking activity function test:

the test was performed with human myeloid leukemia mononuclear cells THP-1. The cells are collected in a centrifuge tube, washed twice by sterile PBS, and blocked by Fc receptor blocking liquid on the surface of the cells, and incubated for 30min at 4 ℃. Cells were collected by centrifugation, washed twice with PBS containing 0.5wt.% BSA, diluted antibodies were added in different ratios, and incubated for 30min at 4 ℃. Cells were washed twice with PBS containing 0.5wt.% BSA, CD47 mFc tagged protein was added, 1. Mu.g/ml was incubated for 30min at 4 ℃. Finally, goat anti-mouse IgG-488 fluorescence secondary antibody is added, and the mixture is incubated for 30min at 4 ℃. After washing the cells twice with PBS containing 0.5wt.% BSA, the cells were resuspended with 200. Mu.l of PBS containing 0.5wt.% BSA and tested on-stream.

Sequence listing

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Claims (11)

1. An anti-sirpa rabbit recombinant monoclonal antibody, characterized in that: the amino acid sequences of heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 are respectively shown as SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3, and the amino acid sequences of light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 are respectively shown as SEQ ID NO. 4, SEQ ID NO. 5 and SEQ ID NO. 6.

2. The anti-sirpa rabbit recombinant monoclonal antibody of claim 1, wherein: the heavy chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 7, and the light chain variable region sequence is the amino acid sequence shown in SEQ ID NO. 8.

3. The anti-sirpa rabbit recombinant monoclonal antibody of claim 1, wherein: the SCFV sequence is the amino acid sequence shown in SEQ ID NO. 9.

4. The anti-sirpa rabbit recombinant monoclonal antibody of claim 1, wherein: the light chain constant region is a kappa chain and the heavy chain constant region is of the IgG type.

5. A nucleic acid molecule characterized in that: comprising a nucleic acid sequence capable of encoding a heavy chain complementarity determining region or a light chain complementarity determining region of an anti-sirpa rabbit recombinant monoclonal antibody of any one of claims 1-4.

6. A carrier, characterized in that: comprising the nucleic acid molecule according to claim 5.

7. A host cell, characterized in that: the host cell contains the anti-SIRPalpha rabbit recombinant monoclonal antibody of any one of claims 1 to 4, the nucleic acid molecule of claim 5 or the vector of claim 6.

8. A conjugate, characterized in that: comprising the recombinant monoclonal antibody of claim 1 to 4 against SIRPalpha rabbit.

9. A pharmaceutical composition characterized by: comprising a main component comprising one or more of the anti-sirpa rabbit recombinant monoclonal antibody of any one of claims 1 to 4, the nucleic acid molecule of claim 5, the vector of claim 6, the host cell of claim 7, the conjugate of claim 8, and a co-component selected from a pharmaceutically acceptable carrier or excipient, and optionally other biologically active substances.

10. Use of an anti-sirpa rabbit recombinant monoclonal antibody according to any one of claims 1 to 4, a nucleic acid molecule according to claim 5, a vector according to claim 6, a host cell according to claim 7 or a conjugate according to claim 8 in the manufacture of a medicament or assay for the treatment of a disease.

11. A method of making the anti-sirpa rabbit recombinant monoclonal antibody of claim 1, comprising the steps of:

(1) Immunizing a New Zealand white rabbit by using human SIRP alpha extracellular region FC fusion protein, and collecting peripheral blood; separating PBMC cells in the collected peripheral blood by using lymphocyte separation liquid;

(2) Incubating SIRP alpha protein coupled magnetic beads and the separated PBMC cells at room temperature, and separating the magnetic beads and the supernatant to obtain B lymphocytes;

(3) Culturing B lymphocytes by using a culture medium containing human IL2, collecting culture medium supernatant, and identifying antibodies;

(4) Extracting RNA of B cells identified as SIRP alpha specificity, reversely transcribing the RNA into cDNA, carrying out PCR amplification on heavy and light chain genes of the SIRP alpha specificity antibody by using a primer, recovering PCR products, cloning the heavy and light chain genes of the SIRP alpha specificity antibody to an expression vector, then carrying out transformation, verifying single colonies by using colony PCR, and carrying out gene sequencing on positive colonies to obtain a gene sequence of the SIRP alpha specificity antibody;

(5) Co-transfecting expression plasmids of heavy and light chain genes of the SIRP alpha specific antibody into 293 cells, culturing, collecting cell supernatant, and purifying by using protein A resin to obtain the SIRP alpha resistant rabbit recombinant monoclonal antibody of claim 1.

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