Rabbit monoclonal antibody of anti-human CD66b protein and application of rabbit monoclonal antibody in preparation of product for detecting and / or removing granulocytes or polymorphic bone marrow-derived suppressor cells

Abstract

The invention discloses a rabbit monoclonal antibody of anti-human CD66b protein and application of the rabbit monoclonal antibody in preparation of a product for detecting and / or removing granulocytes or polymorphic bone marrow-derived suppressor cells. According to the present invention, three rabbit monoclonal antibodies such as CD66b-108, CD66b-116 and CD66b-128 of anti-human CD66b protein are firstly prepared, and three bispecific antibodies for human CD66b and GYPA are constructed based on the three rabbit monoclonal antibodies; the bispecific antibody prepared by the invention can effectively remove granulocytes or polymorphic bone marrow-derived suppressor cells from PBMCs, has an effect superior to that of a commercially available commercial granulocyte removal reagent, and has a wide application prospect in T cell immunodetection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology, and in particular to a rabbit monoclonal antibody against human CD66b protein and an application thereof in preparing a product for detecting and / or eliminating granulocytes or polymorphonuclear myeloid-derived suppressor cells. Background Art

[0002] Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells with significant ability to suppress immune cell responses. They originate from the bone marrow and are phenotypically similar to the precursors of dendritic cells, macrophages, and granulocytes. In the tumor microenvironment, MDSCs exert immunosuppressive functions and inhibit the activity of lymphocytes by secreting factors such as Arg-1, iNOS, and ROS. PMN-MDSCs, or polymorphonuclear myeloid-derived suppressor cells, are a subtype of MDSCs that have very similar surface markers to neutrophils and have strong immunosuppressive effects, especially on T lymphocytes.

[0003] CD66b is a cell surface antigen, also known as CEACAM8 (carboxylesterase A8) or CD67, which is mainly expressed on granulocytes, including neutrophils and PMN-MDSCs. It is involved in cell adhesion and neutrophil activation. In the tumor immune microenvironment, the expression of CD66b is closely related to the function of PMN-MDSCs (polymorphonuclear myeloid-derived suppressor cells), which exert immunosuppressive effects through multiple mechanisms.

[0004] In T cell immune detection, peripheral blood mononuclear cells (PBMCs) are usually isolated from the subject's peripheral blood first, and then the immunogen to be tested is co-cultured with PBMCs in vitro. The APCs in the PBMCs will complete the antigen presentation to the T cells. If there are T cells in the subject's PBMCs that have an immune response to the immunogen to be tested, these T cells will be activated and secrete a series of cytokines, including gamma interferon. By detecting gamma interferon, it can be determined whether the subject has a cellular immune response to the immunogen to be tested. This method is used in immune detection of various pathogens, including IGRA tests for detecting tuberculosis and tests for detecting tumor neoantigens. However, due to individual reasons or long storage time of peripheral blood in some subjects, the density of neutrophils or PMN-MDSCs decreases. During the process of separating PBMCs by density gradient centrifugation, they will be mixed into the PBMCs, which will strongly inhibit the T cell immune response and lead to false negative detection.

[0005] In order to solve this problem, bispecific antibodies can be used to simultaneously connect red blood cells expressing GYPA (glycophorin A, a transmembrane protein of red blood cells) and neutrophils or PMN-MDSCs expressing CD66b, and use red blood cells with higher density to precipitate neutrophils or PMN-MDSCs, thereby removing neutrophils or PMN-MDSCs from PBMCs and restoring the immune response of T cells. Therefore, the development of CD66b monoclonal antibodies is crucial, and the above-mentioned bispecific antibodies can be designed based on CD66b monoclonal antibodies. Currently, the CD66b-specific antibodies that are widely used are mainly mouse monoclonal antibodies, and there is no report on the preparation of CD66b rabbit monoclonal antibodies using rabbit monoclonal antibody preparation technology. Rabbit monoclonal antibodies have more advantages than conventional mouse monoclonal antibodies, such as rabbit antiserum usually contains high-affinity antibodies, which can recognize more types of epitopes than mouse antiserum, and rabbit monoclonal antibodies can recognize many antigens that do not produce immunity in mice. Therefore, it is of great significance to develop rabbit monoclonal antibodies that can specifically bind to CD66b protein. Summary of the invention

[0006] In order to overcome the above defects and deficiencies in the prior art, the present invention provides rabbit monoclonal antibodies against human CD66b protein and their use in preparing products for detecting and / or eliminating granulocytes or polymorphonuclear myeloid-derived suppressor cells.

[0007] The first object of the present invention is to provide a rabbit monoclonal antibody against human CD66b protein.

[0008] A second object of the present invention is to provide biomaterials.

[0009] The third object of the present invention is to provide the use of the above rabbit monoclonal antibody and / or biological material in the preparation of a detection product and / or a removal product for granulocytes or polymorphonuclear myeloid-derived suppressor cells.

[0010] The fourth object of the present invention is to provide a bispecific antibody.

[0011] A fifth object of the present invention is to provide biomaterials.

[0012] The sixth object of the present invention is to provide the use of the above-mentioned bispecific antibody and / or biomaterial in the preparation of a detection reagent and / or a clearance product for granulocytes or polymorphonuclear myeloid-derived suppressor cells.

[0013] The seventh object of the present invention is to provide a detection product and / or a clearance product for granulocytes or polymorphonuclear myeloid-derived suppressor cells.

[0014] Therefore, the present invention claims the following:

[0015] A rabbit monoclonal antibody against human CD66b protein, wherein the rabbit monoclonal antibody is a first antibody CD66b-108, a second antibody CD66b-116 or a third antibody CD66b-128;

[0016] The first antibody CD66b-108 comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 2, the amino acid sequence of CDR2 of the light chain variable region is KAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 3; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 5, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 7;

[0017] The second antibody CD66b-116 comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 9, the amino acid sequence of CDR2 of the light chain variable region is GAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 10; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 12, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 13, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 14;

[0018] The third antibody CD66b-128 comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 16, the amino acid sequence of CDR2 of the light chain variable region is DAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 17; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 19, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 20, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 21.

[0019] Preferably, the amino acid sequence of the light chain variable region of the first antibody CD66b-108 is as shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 4;

[0020] The amino acid sequence of the light chain variable region of the second antibody CD66b-116 is shown in SEQ ID NO: 8, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11;

[0021] The amino acid sequence of the light chain variable region of the third antibody CD66b-128 is shown in SEQ ID NO: 15, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 18.

[0022] Biomaterial, wherein the biomaterial is any one of the following:

[0023] A nucleic acid molecule encoding any of the above-mentioned first antibody CD66b-108, second antibody CD66b-116 or third antibody CD66b-128, or encoding a protein that is completely complementary to the amino acid sequence of any of the above-mentioned first antibody CD66b-108, second antibody CD66b-116 or third antibody CD66b-128;

[0024] An expression cassette comprising the above nucleic acid molecule;

[0025] A recombinant vector comprising the above nucleic acid molecule;

[0026] A recombinant microorganism comprising the above nucleic acid molecule.

[0027] A recombinant cell line comprising the above nucleic acid molecule.

[0028] A bispecific antibody, comprising a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the variable regions of the first heavy chain and the first light chain recognize human CD66b antigen, and the variable regions of the second heavy chain and the second light chain recognize GYPA antigen;

[0029] The amino acid sequences of the variable regions of the first heavy chain and the first light chain are one of the following combinations:

[0030] The amino acid sequence of the variable region of the first heavy chain is shown in SEQ ID NO: 4, and the amino acid sequence of the variable region of the first light chain is shown in SEQ ID NO: 1;

[0031] or the amino acid sequence of the variable region of the first heavy chain is as shown in SEQ ID NO: 11, and the amino acid sequence of the variable region of the first light chain is as shown in SEQ ID NO: 8;

[0032] or the amino acid sequence of the variable region of the first heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the variable region of the first light chain is as shown in SEQ ID NO: 15;

[0033] The amino acid sequence of the variable region of the second heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the second light chain is shown in SEQ ID NO: 25.

[0034] Preferably, a connecting peptide is provided between the first heavy chain and the second heavy chain, and a connecting peptide is provided between the second heavy chain and the second light chain, and the connecting peptide is (G4S) n , where n is an integer between 1 and 6.

[0035] More preferably, n is 4.

[0036] Preferably, the bispecific antibody further comprises a heavy chain constant region and a light chain constant region.

[0037] More preferably, the bispecific antibody is the first antibody CD66b-108-GYPA, the second antibody CD66b-116-GYPA or the third antibody CD66b-128-GYPA;

[0038] The first antibody CD66b-108-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain is shown in SEQ ID NO: 28;

[0039] The second antibody CD66b-116-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 30;

[0040] The third antibody CD66b-128-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain is shown in SEQ ID NO: 32.

[0041] Biomaterial, wherein the biomaterial is any one of the following:

[0042] A nucleic acid molecule encoding any of the above-mentioned bispecific antibodies, or encoding a protein that is completely complementary to the amino acid sequence of any of the above-mentioned bispecific antibodies;

[0043] An expression cassette comprising the above nucleic acid molecule;

[0044] A recombinant vector comprising the above nucleic acid molecule;

[0045] A recombinant microorganism comprising the above nucleic acid molecule.

[0046] A recombinant cell line comprising the above nucleic acid molecule.

[0047] Use of any of the above rabbit monoclonal antibodies, biomaterials, bispecific antibodies and / or biomaterials in the preparation of a detection product and / or a clearance product for granulocytes or polymorphonuclear myeloid-derived suppressor cells.

[0048] A product for detecting and / or eliminating granulocytes or polymorphonuclear myeloid-derived suppressor cells, comprising any of the above-mentioned bispecific antibodies.

[0049] Compared with the prior art, the present invention has the following beneficial effects:

[0050] The present invention discloses rabbit monoclonal antibodies against human CD66b protein and their use in preparing products for detecting and / or removing granulocytes or polymorphonuclear myeloid-derived suppressor cells. The present invention first prepares three rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128 against human CD66b protein, and constructs three bispecific antibodies against human CD66b and GYPA based on these three rabbit monoclonal antibodies. The bispecific antibodies prepared by the present invention can effectively remove granulocytes or polymorphonuclear myeloid-derived suppressor cells from PBMCs, and the effect is better than commercial granulocyte removal reagents on the market, and has broad application prospects in T cell immune detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] Figure 1 This is the growth trend of eukaryotic cells induced by pcDNA3.1-il2-CD66b expression.

[0052] Figure 2 The above is the result of SDS-PAGE detection. Lane 1 is the marker, lane 2 is the electrophoresis result of the cell supernatant, and lane 3 is the electrophoresis result of the purified protein.

[0053] Figure 3 This is the gel electrophoresis result of PCR amplification of the antibody light chain variable region, and the samples were loaded in the order of PCR reaction.

[0054] Figure 4 This is the gel electrophoresis result of PCR amplification of the antibody heavy chain variable region, and the samples were loaded in the order of PCR reaction.

[0055] Figure 5 This is the gel electrophoresis diagram of ScFv fragments, lane 1 is the marker, and lanes 3 and 4 are the amplified ScFv fragments.

[0056] Figure 6 The results of reducing and non-reducing SDS-PAGE electrophoresis are shown in Figure 1. Lanes 1 to 3 are the non-reducing electrophoresis results of rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128, lane 4 is the marker, and lanes 5 to 7 are the reducing electrophoresis results of rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128.

[0057] Figure 7The titer curves are for rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128.

[0058] Figure 8 Design diagram for a bispecific antibody. DETAILED DESCRIPTION

[0059] The present invention is further described below with reference to specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0060] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0061] 2×YT-CG medium: Add 20 mL of 20% D-Glucose solution (w / v) to 180 mL of 2×YT liquid, and then add 200 μL of Carb to obtain 2×YT-CG medium with a final concentration of 2% D-Glucose (w / v) and 100 μg / mL Carb.

[0062] Example 1 Preparation and purification of antigens

[0063] 1. Experimental Methods

[0064] 1. Antigen synthesis and construction

[0065] The protein sequence of human CD66b (CEACAM8, CGM6, NCA-95) was queried through NCBI (human CEACAM8, NP_001807.2). The amino acids of (Met 1 to Ser 319) length were selected, the Kozak sequence and signal peptide sequence IL-2 were added to the 3' end, and after codon optimization, it was handed over to Qingke for gene synthesis, and the recombinant vector pcDNA3.1-il2-CD66b was constructed.

[0066] 2. Plasmid extraction and endotoxin content detection

[0067] The plasmid was extracted to obtain a pcDNA3.1-il2-CD66b plasmid sample, which was then tested for endotoxin. The results of the endotoxin test showed that the endotoxin content of the pcDNA3.1-il2-CD66b plasmid sample was less than 1EU / μg and could be used for cell transfection.

[0068] 3. Cell transfection and eukaryotic expression

[0069] (1) Chinese hamster ovary cells (CHO cells) were cultured at 3×10 6viable cell / mL were inoculated into 125 mL conical flasks, 30 mL per flask, and cultured overnight on a shaker at 37°C, 8% CO2, and 125 rpm. 6 viable cells / mL, and when the viability is 95-99%, transfection can be performed.

[0070] (2) Transfer 25 mL of CHO cell suspension to a 125 mL conical flask, add the corresponding culture medium preheated to 37°C to make up to 30 mL, and shake back and forth to mix (theoretically, the cell density is diluted to 6 × 10 6 viable cells / mL, with a viability greater than 99%).

[0071] (3) Plasmid dilution: pre-cooled to 4°C OptiPRO TM Add pcDNA3.1-il2-CD66b plasmid into the medium and gently mix by inverting upside down to obtain the diluted plasmid.

[0072] (4) Transfection reagent dilution: dilute 920 μL of OptiPRO precooled to 4°C TM 80 μL ExpressiFectamine was added to the medium. TM CHO Reagent, gently invert up and down to mix, to obtain the diluted transfection reagent.

[0073] (5) pcDNA3.1-il2-CD66b transfection: Add the plasmid diluted in step (3) to the transfection reagent diluted in step (4), mix well by inverting, let stand at room temperature for 90 seconds, and then slowly add dropwise to the cells in step (2), gently shaking the cells while adding.

[0074] (6) The transfected cells were incubated at 37°C, 8% CO2 and 125 rpm.

[0075] (7) Adding enhancers and excipients: 22 hours after transfection, perform routine counting; after counting, add 150 μL Express CHO to the bottle TM Enhancer and 5mL ExpiCHO TM Feed, and place the two bottles in an incubator (37°C, 8% CO2, and 125 rpm).

[0076] (8) Six days after transfection, after transient expression, about 30 mL of cell supernatant was harvested. The growth trend of eukaryotic cells induced by pcDNA3.1-il2-CD66b can be seen ( Figure 1), since the completion of transfection, the cell density and viability have increased first and then decreased, which is basically consistent with the transfection situation in the reagent manual. The cell supernatant will be identified and purified later.

[0077] 4. Purification of expressed protein

[0078] (1) Experimental methods

[0079] The cell supernatant obtained in step 3 was purified using an anion column, and the purification effect was detected using SDS-PAGE.

[0080] (2) Experimental results

[0081] The results of SDS-PAGE were as follows: Figure 2 As shown, the cell supernatant has an obvious band at 70KD, which is consistent with the size of human CD66b protein. The target protein is adsorbed and purified using an anion column. It can be seen from the sample that the anion column basically purifies the target protein in the culture supernatant completely, thereby obtaining purified human CD66b protein.

[0082] Example 2 Comparison of the purified human CD66b protein with the commercially available CD66b protein from Sino Biological

[0083] 1. Experimental Methods

[0084] 1. Coating: The human CD66b protein obtained in Example 1 and the commercially available CD66b protein from Sino Biological were respectively aspirated into 1 mL of CBS coating solution and mixed evenly (the final concentration of CD66b protein was 1 μg / mL), and then 100 μL / well of the coating solution containing the human CD66b protein obtained in Example 1 and the coating solution containing the commercially available CD66b protein from Sino Biological were added to the ELISA plate, and incubated overnight at 4°C.

[0085] 2. Blocking: Pour out the liquid in the wells, add 300 μL / well of 2% sodium casein blocking solution (w / v) to the pre-coated plate, and incubate at 37°C for 1 h.

[0086] 3. Primary antibody reaction: The positive serum (immune mouse serum) was diluted 1000 times and 10000 times with 1% sodium casein diluent (w / v), and the commercial CD66b antibody was diluted 100 times with 1% sodium casein diluent (w / v). 100 μL / well of the diluted commercial CD66b antibody, positive serum and 1% sodium casein diluent (w / v) were added to the pre-coated plate, and 1% sodium casein diluent (w / v) was used as a blank control, and incubated at room temperature for 2 hours.

[0087] 4. Secondary antibody reaction: discard the liquid in the wells, wash 3 times with 1×PBST, 300μL / well, and shake for 10s on the last time; dilute the secondary antibody to 1μg / mL with 1% sodium casein diluent (w / v), add 50μL / well of the diluted secondary antibody, cover and incubate at room temperature for 1h.

[0088] 5. Color development: discard the liquid in the wells, wash 5 times with 1×PBST, 300μL / well, shake for 10s on the last time, add 100μL / well TMB, and color develop at room temperature in the dark for 20min; add 50μL / well stop solution to terminate the reaction, and at OD 450 ~OD 620 Take a reading.

[0089] 2. Experimental Results

[0090] The results are shown in Table 1 and Table 2. As shown in Table 1, the OD value of the blank control well (1% sodium casein dilution, w / v) is between 0.08 and 0.13, which is within the normal range, indicating that the experimental operation is correct and the result analysis is credible. The test results of commercially available CD66b antibodies show (Table 2) that the human CD66b protein prepared in Example 1 is consistent with the benchmark activity results of the commercially available CD66b protein of Sino Biological, and the effect is even better.

[0091] Table 1 ELISA experimental results

[0092] Sino Biological CD66b protein Human CD66b protein prepared in Example 1 Commercially available CD66b antibodies 2.276 2.767 Positive serum 1000× 2.241 1.411 Positive serum 1000× 2.726 1.467 Positive serum 10000× 0.4102 0.3049 Positive serum 10000× 0.3804 0.3237 1% sodium casein dilution 0.08120 0.1373

[0093] Table 2 Results Statistics

[0094] Sino Biological CD66b protein Human CD66b protein prepared in Example 1 Commercially available CD66b antibodies 2.276 2.767 Positive serum 1000×average OD value 2.484 1.439 Positive serum 10000×average OD value 0.395 0.314

[0095] Example 3 Immune Effect of Human CD66b Protein

[0096] 1. Experimental Methods

[0097] The human CD66b protein prepared in Example 1 was used as an immunogen to immunize New Zealand white rabbits, and the sera from the third and fourth immunizations were collected and tested by ELISA.

[0098] The ELISA test steps are as follows:

[0099] 1. Coating: dilute the human CD66b protein prepared in Example 1 to 1 μg / mL with CBS buffer, take 100 μL and add it to the ELISA plate, seal the reaction wells with plastic wrap, incubate at 4°C overnight, and wash the plate 3 times with PBST;

[0100] 2. Blocking: Fill the cleaned reaction wells with 3% skim milk powder (w / v), seal with plastic wrap, leave at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0101] 3. Sample addition: dilute the sera from the third and fourth immunizations and the negative sera by 1000 times, 3000 times, 9000 times, 27000 times, 81000 times, and 243000 times, respectively, and then take 100 μL of each and add it to the blocked reaction wells, seal with plastic wrap, place at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0102] 4. Secondary antibody: dilute Goat Anti-Rabbit IgG (HRP) with 1% sodium casein diluent (w / v) (1:5000), add 100 μL to each well, seal with plastic wrap, leave at room temperature for 1 hour, and wash the plate 3 times with PBST;

[0103] (5) TMB color development: Add 100 μL of TMB color development solution to the reaction well and color for 20 min; then add 50 μL of stop solution;

[0104] (6) Reading: Place the ELISA plate into the ELISA reader for reading.

[0105] 2. Experimental Results

[0106] The results are shown in Table 3. As shown in Table 3, the titers after three and four immunizations were all >81,000, and the immunization was qualified, and the subsequent phage display library could be used.

[0107] Table 3 Immunized rabbit serum titer results

[0108] Sample dilution factor 178# Rabbit Three Free 179# Rabbit Three Free 179# Rabbit Four Free Rabbit Negative 1：1000 4.4531 4.3994 2.7022 0.2409 1：3000 4.0990 4.0631 2.3624 0.1580 1：9000 3.8252 3.5445 2.0797 0.1328 1：27000 3.2772 2.2133 1.9294 0.1069 1：81000 2.0495 1.0201 1.1132 0.0981 1：243000 1.0597 0.4196 0.6998 0.1049 blank 0.1035 0.0982 0.0265 0.0919

[0109] Example 4 Construction of phage display library

[0110] New Zealand white rabbits were immunized with the human CD66b protein prepared in Example 1 as an immunogen. The rabbits were killed one week after the third and fourth booster immunizations, and their spleen tissues and bone marrow were obtained.

[0111] 1. RNA Extraction

[0112] (1) Liquid nitrogen grinding: First, cool the mortar with liquid nitrogen, take out the spleen and bone marrow from -80℃ (separate operation before and after), then add liquid nitrogen while grinding, until the spleen tissue and bone marrow are powdered, and finally let the powder spread evenly in the mortar. When the liquid nitrogen is about to evaporate, immediately add 6mL TRIzol to cover the powder. After Trizol melts, divide it into portions to obtain TRIzol lysate of spleen tissue (since the bone marrow sample has too much tissue left and is too viscous, first pipette 5mL TRIzol into a 10mL EP tube, mix it, and then add 1mL TRIzol solution, and divide it into 1mL / tube first; add 2mL TRIzol solution to the remaining liquid in the mortar, mix it, and then divide it into 1mL / tube).

[0113] (2) Extraction: Take 1 mL of TRIzol lysate from spleen tissue and bone marrow samples respectively, add 200 μL of chloroform (1 / 5 of the volume of TRIzol), cover the centrifuge tube tightly, vortex mix for 15 seconds until the solution emulsifies and turns milky white, and place on ice for 2 to 3 minutes. Centrifuge at 12,000 × g at 4°C for 15 minutes.

[0114] (3) Take the supernatant: Tilt the tube at 45° and use a 1 mL pipette to continuously and evenly transfer the supernatant aqueous phase to another new centrifuge tube, taking 500 μL of supernatant into each tube.

[0115] (4) Precipitation: Add 500 μL of isopropanol (1 / 2 the volume of TRIzol) to the supernatant, mix thoroughly by inverting the tube, place on ice for 10 min, and centrifuge at 12,000 × g at 4°C for 10 min.

[0116] (5) Washing: Discard the supernatant once, turn the EP tube upside down on absorbent paper, and absorb the residual liquid at the tube mouth; add 500 μL of 75% ethanol (prepared with DEPC-treated water, v / v) to resuspend the RNA precipitate, turn the centrifuge tube upside down, and thoroughly wash the tube wall to ensure that the RNA precipitate is suspended but not dispersed; centrifuge at 12,000 × g at 4°C for 5 min.

[0117] (6) Drying: Discard the supernatant at once, invert the EP tube on absorbent paper, and centrifuge at 12,000 × g for 30 seconds at 4°C; use a pipette to suck away the remaining liquid (be careful not to suck up the precipitate), open the centrifuge tube cap, place it horizontally, and dry the precipitate at room temperature for 2 to 3 minutes (in a clean bench).

[0118] (7) Dissolution: Add RNase-free water to dissolve the precipitate (50 μL of spleen tissue RNA and 20 μL of bone marrow sample RNA), and obtain the spleen tissue RNA and bone marrow sample RNA of the rabbit after the third and fourth immunizations, respectively.

[0119] 2. RNA Purification

[0120] (1) Digestion of genomic DNA: Take the spleen tissue RNA and bone marrow sample RNA of the rabbit after the third and fourth immunizations obtained in step 1, treat them with DNase, and prepare the reaction system according to Table 4. After thorough mixing, centrifuge at low speed for 10 seconds, add water to the metal bath, and incubate at 37°C for 30 minutes to digest the residual genomic DNA.

[0121] Table 4 Reaction system

[0122]

[0123] (2) Add 1 mL of TRIzol to the sample digested in step (1); then add 200 μL of chloroform (1 / 5 the volume of TRIzol), cover the centrifuge tube tightly, vortex mix for 15 seconds until the solution emulsifies and turns milky white, and place on ice for 2 to 3 minutes. Centrifuge at 12,000 × g at 4°C for 15 minutes.

[0124] (3) Take the supernatant: Tilt the tube at 45° and use a 1 mL pipette to continuously and evenly transfer the supernatant aqueous phase to another new centrifuge tube, taking 500 μL of supernatant into each tube.

[0125] (4) Precipitation: Add 500 μL of isopropanol (1 / 2 the volume of TRIzol) to the supernatant, mix thoroughly by inverting the tube, place on ice for 10 min, and centrifuge at 12,000 × g at 4°C for 10 min.

[0126] (5) Washing: Discard the supernatant once, turn the EP tube upside down on absorbent paper, and absorb the residual liquid at the tube mouth; add 500 μL of 75% ethanol (prepared with DEPC-treated water, v / v) to resuspend the RNA precipitate, turn the centrifuge tube upside down, and thoroughly wash the tube wall to ensure that the RNA precipitate is suspended but not dispersed; centrifuge at 12,000 × g at 4°C for 10 min.

[0127] (6) Drying: Discard the supernatant at once, invert the EP tube on absorbent paper, and centrifuge at 12,000 × g for 30 seconds at 4°C; use a pipette to remove the remaining liquid (be careful not to absorb the precipitate), open the centrifuge tube cap, place it horizontally, and dry the precipitate at room temperature for 2 to 3 minutes (in a clean bench).

[0128] (7) Dissolution: RNase-free water was added to dissolve the precipitate, and agarose gel electrophoresis was performed to verify the quality of the purified RNA. The results showed that the purified RNA was of good quality and free of protein contamination. Thus, the purified spleen tissue RNA and bone marrow sample RNA of rabbits after the third and fourth immunizations were obtained.

[0129] 3. RNA Reverse Transcription

[0130] use III Reverse Transcriptase Reverse Transcriptase Kit reversely transcribes the RNA of the spleen tissue and the RNA of the bone marrow sample of the rabbit after the third and fourth immunizations purified in step 2 into cDNA. Prepare the reaction system according to Table 5.

[0131] Table 5 Reaction system

[0132]

[0133]

[0134] Place at 65°C for 5 minutes, place on ice for 2 minutes. Then add the reaction mixture (Table 6), mix gently, and centrifuge briefly for 5 seconds; place in a metal bath at 50°C for 60 minutes; place in a metal bath at 70°C for 15 minutes; cool on ice; after completion, cDNA of the spleen tissue and bone marrow samples of rabbits after the third and fourth immunizations were obtained, respectively.

[0135] Table 6 Reaction mixture

[0136] Components Volume (μL) 5×First-Strand Buffer 4 0.1M DTT 1 RNaseOUTTM Recombinant RNase Inhibitor(40U / uL) 1 Super ScriptTMIII RT(200U / uL) 1 Total 7

[0137] 4. Amplification of antibody light chain variable region

[0138] (1) Primer pairing

[0139] The kappa and lambda light chain primers were paired and numbered as shown in Table 7. The primer sequences are shown in Table 8.

[0140] Table 7 Primer pairing numbers

[0141]

[0142] Table 8 Primer sequences

[0143]

[0144]

[0145] (2) PCR amplification

[0146] The cDNA of the third immunization rabbit spleen tissue and bone marrow sample obtained in step 3 and the cDNA of the fourth immunization rabbit spleen tissue and bone marrow sample were mixed evenly (i.e., the mixed cDNA contained the cDNA of the rabbit spleen tissue and bone marrow sample), and the PCR reaction system was prepared according to Table 9, and the total volume of the reaction system was 20 μL.

[0147] Table 9 Reaction system

[0148] Reagents Recommended final concentration Sample system Negative 5×Phusion HF Buffer 1× 4μL 4μL 10mM dNTPs Mix 0.2mM 0.4μL 0.4μL 100% DMSO 3％ 0.6μL 0.6μL Primer F (10 μM) 0.25μM 0.5μL 0.5μL Primer R (10 μM) 0.25μM 0.5μL 0.5μL cDNA 50ng for RNA 0.4μL - Phusion Hot Start IIDNA Polymerase(2U / μL) 0.01U / μL 0.1μL 0.1μL Autoclaved deionized water - 13.5μL 13.9μL

[0149] The reaction program was as follows: pre-denaturation at 98°C for 30 s; denaturation at 98°C for 10 s, annealing at 55°C for 30 s, extension at 72°C for 30 s, 35 cycles; and extension at 72°C for 10 min.

[0150] After amplification, agarose gel electrophoresis was performed ( Figure 3 ) to obtain DNA fragments of multiple antibody light chain variable regions.

[0151] 5. Amplification of antibody heavy chain variable region

[0152] (1) Primer pairing

[0153] The IgG and IgM heavy chain primers were paired and numbered as shown in Table 10. The primer sequences are shown in Table 11.

[0154] Table 10 Primer pairing numbers

[0155]

[0156] Table 11 Primer sequences

[0157]

[0158] PCR amplification was performed according to step 4. After amplification, agarose gel electrophoresis was performed ( Figure 4 ) to obtain multiple antibody heavy chain variable region DNA fragments.

[0159] 6. Construction of Antibody ScFv Format

[0160] The DNA fragments of the multiple antibody light chain variable regions obtained in step 4 and the DNA fragments of the multiple antibody heavy chain variable regions obtained in step 5 were mixed respectively to obtain VL fragments and VH fragments, and the PCR reaction system was prepared according to Table 12. The total volume of the reaction system was 20 μL.

[0161] Table 12 Reaction system

[0162]

[0163]

[0164] Primer F: GAGGAGGAGGAGGAGGAGGCGGGGCCCAGGCGGCC;

[0165] Primer R: GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCC.

[0166] PCR amplification was performed according to step 4. After amplification, agarose gel electrophoresis was performed ( Figure 5 ) to obtain the ScFv fragment.

[0167] 7. Construction of TG1 Glycerol Antibody Library

[0168] After the amplified ScFv fragment was digested by SfiI, the digestion products were mixed and connected to the pComb-3X vector to obtain the recombinant pComb-3X plasmid. 10 ng of the recombinant pComb-3X plasmid was transformed into 50 μL of TG1 competent cells by electroporation and cultured at 37°C in the dark overnight to obtain the TG1 glycerol bacterial antibody library of human CD66b-ScFv antibody.

[0169] Example 5 Preparation of phage library

[0170] 1. Take 1 mL of the TG1 glycerol bacterial antibody library of the human CD66b-ScFv antibody obtained in Example 4, add it to 100 mL of 2×YT liquid culture medium (containing carboxybenzyl resistance) for culture, and culture it in a shaker at 37°C and 180 rpm for 4 h. Measure the OD value until the logarithmic growth phase (OD 600 =0.6).

[0171] 2. Add 10 μL of helper phage (M13K07, titer 1.2×10 12 pfu / mL) and infected in a 37°C constant temperature incubator with gentle shaking every 15 min for a total of 45 min.

[0172] 3. Centrifuge at 4000 rpm for 5 min, repeat twice, and discard the supernatant.

[0173] 4. Resuspend the precipitate in 200 mL of 2×YT liquid culture medium (containing both carbenicillin and kanamycin resistance) and culture overnight at 37°C in a shaker at 180 rpm.

[0174] 5. Centrifuge at 5000rpm for 10min. Filter the supernatant after each centrifugation with a 0.45μm bacterial filter and transfer it to a clean BD tube at a ratio of 4:1 (40mL of supernatant is added with 10mL of 5×PEG / NaCl solution). Mix by inversion and place in ice for 2h (ice bath, mix by inversion 5 times every 30min).

[0175] 6. Centrifuge at 8000 rpm for 20 min, discard the supernatant, aspirate dry, and dissolve the precipitate with 1 mL of PBS buffer (pH = 7.4) to obtain the phage library of human CD66b-ScFv antibody.

[0176] Example 6 Solid phase panning and ELISA detection

[0177] 1. Solid phase screening

[0178] The first round of screening steps are as follows:

[0179] 1. Human CD66b antigen coating: The human CD66b protein prepared in Example 1 was used as an antigen, diluted to 1 μg / mL with CBS buffer, and 100 μL / well was coated on an ELISA plate, placed at 4°C, and allowed to stand overnight for more than 12 hours.

[0180] 2. Blocking: Pour out the liquid in the wells, add 200 μL / well 0.05% PBST washing solution (v / v) to wash twice, and discard the liquid in the wells; then add 200 μL / mL 2% sodium casein blocking solution (w / v), incubate at 37°C for 2h; after blocking, suck out the liquid with a gun, add 200 μL / well 1×PBS to wash 5 times, discard the liquid in the wells, seal the plate with sealing film, and store it at 4°C.

[0181] 3. Phage incubation: Add the phage library of human CD66b-ScFv antibody obtained in Example 5 (round 0) to the reaction wells coated with human CD66b antigen, 100 μL / well, and incubate at 37° C. for 2 h.

[0182] 4. Washing: Aspirate the liquid in the wells, add 200μL / well 1×PBST to wash 5 times, then wash 5 times with 200μL / well 1×PBS. After washing, discard the liquid in the wells.

[0183] 5. TG1 recovery: Take out the TG1 glycerol bacteria from -80℃, use a pipette to pick up the bacterial solution, streak it on the LB plate, put it in 37℃, and invert it for 12h. Pick a single clone from the TG1 streaked plate, inoculate it into 40μL 2×YT liquid culture medium and mix it by blowing, then draw 10μL of bacterial solution into 3mL 2×YT liquid culture medium, and incubate it at 37℃, 200rpm overnight. After overnight culture, draw 10μL of bacterial solution again into 3mL 2×YT liquid culture medium, and incubate it at 37℃, 200rpm for 2h. The OD value is measured to be 0.745, and the TG1 bacterial solution is obtained.

[0184] 6. Add 100 μL / well 0.2M glycine buffer (pH=3) to the wells after washing in step 4, let it stand at room temperature for 10 minutes, then immediately aspirate it and transfer it to a 1.5mL EP tube. Add 20 μL 0.5M Tris-HCl (pH8.0) to the EP tube for neutralization; after mixing, add it all to 4mL TG1 bacterial solution to obtain TG1-Phage; put TG1-Phage at 37°C and let it stand for 45 minutes, shake it every 15 minutes for infection, and after 45 minutes, obtain the infected TG1-Phage solution, i.e. the first round of panning phage.

[0185] Repeat the above panning steps to perform the second and third rounds of panning to obtain the second and third round of panning phages, respectively;

[0186] In the second round of panning, in step 1, human CD66b protein was diluted to 250 ng / mL with CBS buffer before coating; in step 4, 200 μL / well of 1×PBST was added for washing 10 times, and then 200 μL / well of 1×PBS was used for washing 10 times.

[0187] In the third round of screening, in step 1, human CD66b protein was diluted to 80 ng / mL with CBS buffer before coating; in step 4, 200 μL / well of 1×PBST was added for washing 15 times, and then 200 μL / well of 1×PBS was used for washing 15 times.

[0188] Storage plate: Take the TG1-Phage solution that has been infected in the first to third rounds and dilute it by 10 3 ~10 6 times, aspirate 50 μL and apply it on 2×YT-CG plate, place it at 37℃, invert and culture for more than 12 hours.

[0189] 2. Phage ELISA Validation

[0190] 1. Experimental Methods

[0191] (1) Coating and sealing

[0192] The human CD66b protein prepared in Example 1 was used as an antigen, and the human CD66b protein was diluted with PBS to 2.4 μg / mL. The diluted antigen was coated in an ELISA plate at 100 μL / well, and 1% BSA (w / v) was used as a control. The coated ELISA plate was placed at 4°C for overnight coating.

[0193] The overnight coated ELISA plate was taken out from 4°C, allowed to stand at room temperature, the liquid in the wells was poured out, 200 μL / well of 0.1% PBST (w / v) was added to wash the plate twice, the liquid inside was patted dry, 2% sodium casein blocking solution (w / v) was added to block, and the plate was placed at 37°C for 1 hour; after the blocking was completed, the liquid in the wells was poured out, 200 μL / well of 1×PBS was added to wash the plate once, and patted dry.

[0194] (2) Incubate samples

[0195] Take out M13K07, the phage library of human CD66b-ScFv antibody obtained in Example 5 (0th round phage), and the 1st to 3rd round panning phage prepared in step 1 from 4°C, and add 1×PBS to each sample after taking a certain amount of each sample to make the final titer of each sample 1×10 10 cfu / mL, and the final volume was 450 μL / tube.

[0196] 100 μL / well of the above solution was added to the blocked plate, and the plate was placed at 37°C for 2 hours, and PBS was used as a blank control. After the incubation, the liquid in the well was poured out, 200 μL / well of 0.1% PBST (w / v) was added to wash the plate 5 times, patted dry, and then 200 μL / well of 1×PBS was added to wash the plate 2 times, and the liquid was patted dry.

[0197] (3) Incubation with secondary antibody and color development

[0198] Take out Anti-M13-conjuated-HRP from -20℃ and dilute it 3000 times with 1% sodium casein diluent (w / v). Add the diluted Anti-M13-conjuated-HRP to the incubated plate, 50 μL / well, and place the plate at 37℃ for 1 hour.

[0199] Pour the liquid in the wells, add 200 μL / well of 0.1% PBST (w / v) to wash the plate 5 times, tap dry, then add 200 μL / well of 1× PBS to wash the plate 2 times, tap dry; add 50 μL / well TMB, react at room temperature for 20 min, add 50 μL / well stop solution, and at OD 450 ~OD 620 Next reading.

[0200] 2. Experimental results

[0201] The results are shown in Table 13. After three rounds of panning, the corresponding antibodies in the library were significantly enriched.

[0202] Table 13 Phage ELISA results

[0203] project Human CD66b protein Negative Signal-to-Noise Ratio Round 0 phage 0.046 0.041 1.12 The first round of phage screening 0.421 0.064 6.58 Second round of phage screening 1.201 0.137 8.77 Round 3 phage screening 2.15 0.182 11.81 M13K07 0.081 0.062 1.31 blank 0.051 0.045 1.13

[0204] 3. Monoclonal phage ELISA

[0205] 1. Experimental Methods

[0206] (1) Strain activation

[0207] Add 1 mL / well 2×YT-CG culture medium to 96 deep-well plates ① and ②, and obtain 90 wells in each as sample wells; then add 1 mL / well 2×YT liquid to 96 deep-well plates ① and ②, and obtain one well in each as a negative well.

[0208] Randomly pick 180 single clones from the second and third rounds of panning in step 1 and add them to the 90 sample wells of deep-well plate ① and deep-well plate ② respectively, and mix them by pipetting; then pick a colony from the TG1 streaked plate and add it to the negative well of the deep-well plate. Put the two deep-well plates into 37℃, 200rpm overnight culture for more than 12 hours.

[0209] (2) Transfer

[0210] 1) Add 500 μL / well of 2×YT-CG culture medium to 96 deep-well plates ③ and ④, and obtain 90 wells in total as sample wells; in addition, add 500 μL / well to 96 deep-well plates ③ and ④, and obtain 2 wells as negative wells.

[0211] From deep-well plates ① and ②, take 5 μL of bacterial solution from the 90 sample wells containing monoclonal bacterial solution and add them to the sample wells of deep-well plates ③ and ④ in sequence; take 5 μL of bacterial solution from the negative wells in deep-well plates ① and ② and add them to the negative wells in deep-well plates ③ and ④ respectively. Place deep-well plates ③ and ④ in a 37°C, 200 rpm incubator until the logarithmic phase (OD 600 =0.6).

[0212] 2) Take out M13K07 from 4°C, pipette 250 μL of M13K07 into 2250 μL of 2×YT liquid culture medium and mix well, then add 10 μL / well into the sample wells and negative wells of deep well plates ③ and ④, and then place deep well plates ③ and ④ at 37°C for infection for 45 min.

[0213] 3) Place the infected deep-well plate in a centrifuge, centrifuge at 1800 g for 10 min, and remove the supernatant with a pipette; add 70 μL of Kana to 100 mL of 2×YT liquid culture medium, mix to obtain 2×YT-K, and add 500 μL / well of 2×YT-K to the negative wells in deep-well plates ③ and ④; add 198 μL of Crab to the remaining 99 mL of 2×YT-K to obtain 2×YT-CK culture medium containing 100 μg / mL Carb and 35 μg / mL Kana, and add 500 μL / well of 2×YT-CK culture medium to the sample wells in deep-well plates ③ and ④, pipette, mix, and culture at 30°C, 250 rpm for more than 12 h.

[0214] (3) Monoclonal ELISA

[0215] 1) Plate coating: The human CD66b protein prepared in Example 1 was used as an antigen and diluted to 2 μg / mL with PBS; the diluted antigen was added to each ELISA plate at 100 μL / well, and another ELISA plate was added with 100 μL / well 3% BSA (w / v), and the two ELISA plates were placed at 4°C and allowed to stand for more than 12 hours.

[0216] 2) Blocking: Take out the two plates coated overnight from 4°C, pour out the liquid in the wells, add 200 μL / well of 0.1% PBST (w / v) to wash the plates, pat dry the liquid, add 200 μL / well of 3% sodium casein blocking solution (w / v) for blocking, and place at 37°C for 2 hours.

[0217] 3) Incubation: Pour the liquid in the wells of the sealed plate, add 200 μL / well 1×PBS to wash the plate, and pat dry. Put the deep-well plates ③ and ④ that have been cultured overnight into a centrifuge and centrifuge at 1800g for 15 minutes; after centrifugation, draw the liquid from the sample wells in sequence and add 50 μL / well to the ELISA plate coated with human CD66b and the ELISA plate coated with BSA; in addition, set up two negative wells (provided by deep-well plates ③ and ④), two positive wells (using the third round of panning Phage, draw 500 μL 2.76×10 10 cfu / mL was added to 500 μL PBS and mixed to 1.38×10 10 cfu / mL) and two blank wells (added with 3% sodium casein blocking solution, w / v). The ELISA plate was placed at 37°C for 2 h.

[0218] 4) Secondary antibody reaction: Pour the liquid in the wells, add 200 μL / well of 0.1% PBST (w / v) to wash the plate 5 times, tap dry, then add 200 μL / well of 1×PBS to wash the plate 2 times, tap dry. Dilute M13-HRP 2500 times with 1% sodium casein diluent (w / v), then add 50 μL / well to the wells, place at 37°C, and react for 1 hour.

[0219] 5) Color reaction: Pour the liquid in the wells, add 200 μL / well of 0.1% PBST (w / v) to wash the plate 5 times, tap dry, then add 200 μL / well of 1×PBS to wash the plate 2 times, tap dry; add 50 μL / well TMB, react at room temperature for 13 min, add 50 μL / well stop solution, and at OD 450 ~OD 620 Next reading.

[0220] 2. Experimental results

[0221] The results of monoclonal phage ELISA are shown in Tables 14 and 15. Among the screened monoclones, there were medium / strong binding antibodies, and these monoclones were sent for sequencing.

[0222] Table 14 Monoclonal phage ELISA results of deep well plate ③

[0223]

[0224]

[0225] Table 15 Monoclonal phage ELISA results of deep well plate ④

[0226] Signal-to-noise ratio (human CD66b protein / BSA) Number of clones percentage No binding (SNR < 2) 36 41.4％ Weak binding (SNR 2-5) 48 55.2％ Medium binding (SNR 5-10) 3 3.4％ Strong binding (SNR>10) 0 0％

[0227] Example 7 Three rabbit monoclonal antibodies against human CD66b protein

[0228] According to the results of monoclonal phage ELISA in Example 6, monoclonal antibodies were selected for sequencing to obtain three rabbit monoclonal antibodies against human CD66b protein, namely CD66b-108, CD66b-116 and CD66b-128.

[0229] The amino acid sequence of the light chain variable region of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 1:

[0230] AQVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKAGQPPNLLIY KASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQTYYTSDSSYYGAA FGGGTEVVVK (SEQ ID NO: 1);

[0231] The amino acid sequence of the light chain variable region CDR1 of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 2:

[0232] QSIGSN (SEQ ID NO: 2);

[0233] The amino acid sequence of the light chain variable region CDR2 of the CD66b-108 monoclonal antibody is KAS;

[0234] The amino acid sequence of the light chain variable region CDR3 of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 3:

[0235] QTYYTSDSSYYGAA(SEQ ID NO: 3);

[0236] The amino acid sequence of the heavy chain variable region of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 4:

[0237] QSVEESRGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGLI STGGNTDYASWARGRFTISKSSTTVDLKMTSPTTEDTATYFCARGFALGSGSG YYPPDFVLWGQGTLVTISS(SEQ ID NO: 4);

[0238] The amino acid sequence of the heavy chain variable region CDR1 of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 5:

[0239] FSLSSYA (SEQ ID NO: 5);

[0240] The amino acid sequence of the heavy chain variable region CDR2 of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 6:

[0241] ISTGGNT (SEQ ID NO: 6);

[0242] The amino acid sequence of the heavy chain variable region CDR3 of the CD66b-108 monoclonal antibody is shown in SEQ ID NO: 7:

[0243] ARGFALGSGSGYYPPDFVL(SEQ ID NO:7);

[0244] The amino acid sequence of the light chain variable region of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 8:

[0245] AQVMTQTPPSLSASVGGTVTINCLASENVYSAVSWYQQKPEKPPTLLISG ASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYFCQGYSSYPLTFGAGTN VEIK (SEQ ID NO: 8);

[0246] The amino acid sequence of the light chain variable region CDR1 of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 9:

[0247] ENVYSA (SEQ ID NO: 9);

[0248] The amino acid sequence of the light chain variable region CDR2 of the CD66b-116 monoclonal antibody is GAS;

[0249] The amino acid sequence of the light chain variable region CDR3 of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 10:

[0250] QGYSSYPLT (SEQ ID NO: 10);

[0251] The amino acid sequence of the heavy chain variable region of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 11:

[0252] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGII SGRGSIHYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAREFGDGYDDY GDFGAFDPWGPGTLVTISS (SEQ ID NO: 11);

[0253] The amino acid sequence of the heavy chain variable region CDR1 of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 12:

[0254] GFSLSSYA (SEQ ID NO: 12);

[0255] The amino acid sequence of the heavy chain variable region CDR2 of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 13:

[0256] ISGRGSI (SEQ ID NO: 13);

[0257] The amino acid sequence of the heavy chain variable region CDR3 of the CD66b-116 monoclonal antibody is shown in SEQ ID NO: 14:

[0258] AREFGDGYDDYGDFGAFDP (SEQ ID NO: 14);

[0259] The amino acid sequence of the light chain variable region of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 15:

[0260] AQVMTQTPASVSAAVGGTITIKCQASEDIYNLLAWFQQKPGQPPKLLIYD ASDLTSGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGGYYSASVIDIAFGGGTELEIL (SEQ ID NO: 15);

[0261] The amino acid sequence of the light chain variable region CDR1 of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 16:

[0262] EDIYNL (SEQ ID NO: 16);

[0263] The amino acid sequence of the light chain variable region CDR2 of the CD66b-128 monoclonal antibody is DAS;

[0264] The amino acid sequence of the light chain variable region CDR3 of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 17:

[0265] QGGYYSASVIDIA (SEQ ID NO: 17);

[0266] The amino acid sequence of the heavy chain variable region of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 18:

[0267] QSLEESEGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEYIGIIY AGGSASYASWAIGRFTISKTSTTVDLKIPSPTTEDTATYFCARTAYATYGYLNF WGQGTLVTVSS (SEQ ID NO: 18);

[0268] The amino acid sequence of the heavy chain variable region CDR1 of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 19:

[0269] GFSLSSYA (SEQ ID NO: 19);

[0270] The amino acid sequence of the heavy chain variable region CDR2 of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 20:

[0271] IYAGGSA (SEQ ID NO: 20);

[0272] The amino acid sequence of the heavy chain variable region CDR3 of the CD66b-128 monoclonal antibody is shown in SEQ ID NO: 21:

[0273] ARTAYATYGYLNF (SEQ ID NO: 21).

[0274] Example 8 Performance Test

[0275] 1. Expression and purification

[0276] 1. Experimental Methods

[0277] The FC segment sequence was added to the 5' end of the heavy chain of the three rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128 obtained in Example 7, respectively. The FC segment sequence information is as follows:

[0278] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO: 22);

[0279] The Kozak sequence and signal peptide sequence were added to the 3' end of the heavy chain and after codon optimization, it was constructed into pcDNA3.1 for expression;

[0280] Add the FC segment sequence to the 5' end of the light chain. The FC segment sequence information is as follows:

[0281] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG NSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGEC(SEQ ID NO: 23);

[0282] The Kozak sequence and signal peptide sequence were added to the 3' end of the light chain and after codon optimization, the construct was constructed into pcDNA3.1 for expression.

[0283] The expressed product was purified using a protein A column, and the purified protein was subjected to reducing and non-reducing SDS-PAGE electrophoresis.

[0284] 2. Experimental results

[0285] The results are as follows Figure 6 As shown, the protein purity reached more than 80%, and the SDS-PAGE electrophoresis results showed that the protein size was consistent with the expected result.

[0286] 2. ELISA test

[0287] 1. Experimental Methods

[0288] (1) The human CD66b protein prepared in Example 1 was coated on an ELISA plate at a concentration of 1 μg / mL at 4° C. overnight.

[0289] (2) Wash the plate twice with PBST, pat dry, add 2% sodium casein blocking solution (w / v), and block at 37°C for 1 h.

[0290] (3) Wash the plate twice with PBST, pat dry, and add serially diluted rabbit monoclonal antibodies CD66b-108, CD66b-116, and CD66b-128 (2000, 1000, 10, 1, 0.1, 0.01, 0 ng / mL) into the reaction wells at 100 μL / well and react at room temperature for 1 h.

[0291] (4) Wash the plate 5 times with PBST, pat dry, add 100 μL HRP-anti-human FC antibody (1:3000, diluted with 1 wv% sodium casein diluent) to each well, and react at room temperature for 1 h;

[0292] (5) Wash the plate 5 times with PBST, pat dry, add 100 μL TMB colorimetric reagent and color for 10 min at room temperature, add 50 μL stop solution, and read A 450 ~A 620 The absorbance value was used to draw the potency curve, with the reciprocal of the rabbit monoclonal antibody concentration as the horizontal axis and the absorbance value as the vertical axis.

[0293] 2. Experimental results

[0294] The results are as follows Figure 7 As shown in Table 16, CD66b-108, CD66b-116, and CD66b-128 antibodies all have significant binding ability to human CD66b protein, among which CD66b-108 has the strongest binding ability.

[0295] Table 16 Antibody performance

[0296] project CD66b-108 CD66b-116 CD66b-128 Linear range 0.17~3000ng / mL 2–223 ng / mL 2.7–1000 ng / mL IC50 11.61ng / mL 114.5 ng / mL <![CDATA[2.106×10 27 ng / ml]]>

[0297] Example 9 CD66b-GYPA bispecific antibody

[0298] 1. GYPA antibody sequence

[0299] The GYPA antibody sequence comes from patent WO2017015141A1, where:

[0300] The amino acid sequence of the heavy chain variable region of the GYPA antibody is shown in SEQ ID NO: 24:

[0301] EVQLVESGGGLVQPGGSLRLSKASGYTFNSYFMHWVRQAPGKGLVWV SMIRPNGGTTDYADSVKGRFTISVDNSKNTLYLQMNSLRAEDTAVYYCARWE GSYYALDVWGQGTTVTVSS (SEQ ID NO: 24);

[0302] The amino acid sequence of the light chain variable region of the GYPA antibody is shown in SEQ ID NO: 25:

[0303] DIQMTQSPSSSLSASVGDRVTITCRASSNVKYLAWYQQKPGKAPKLLIYYT SNLQSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQFTSSPYTFGQGTKLEI K (SEQ ID NO: 25).

[0304] 2. CD66b-GYPA bispecific antibody

[0305] The light chain variable regions and heavy chain variable regions of CD66b-108, CD66b-116 and CD66b-128 obtained in Example 7 were linked to the light chain variable regions and heavy chain variable regions of the GYPA antibody through a flexible polypeptide (GGGGSGGGGSGGGGSGGGGS, SEQ ID NO: 26) to obtain CD66b-108-GYPA, CD66b-116-GYPA and CD66b-128-GYPA bispecific antibodies ( Figure 8 );

[0306] The amino acid sequence of the heavy chain of the CD66b-108-GYPA bispecific antibody is shown in SEQ ID NO: 27:

[0307] QSVEESRGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGLISTGGNTDYASWARGRFTISKSSTTVDLKMTSPTTEDTATYFCARGFALGSGSGYYPPDFVLWGQGTLVTISSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGYTFNSYFMHWVRQAPGKGLVWVSMIRPNGGTTDYADSVKGRFTISVDNSKNTLYLQMNSLRAEDTAVYYCARWEGSYYALDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASSNVKYLAWYQQKPGKAPKLLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQFTSSPYTFGQGTKLEIK(SEQ ID NO:27);

[0308] The amino acid sequence of the light chain of the CD66b-108-GYPA bispecific antibody is shown in SEQ ID NO: 28:

[0309] AQVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKAGQPPNLLIYKASTLESGVPSRFKGSGSGTEFTLTISDLECADAATYYCQTYYTSDSSYYGAAFGGGTEVVV KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 28);

[0310] The amino acid sequence of the heavy chain of the CD66b-116-GYPA bispecific antibody is shown in SEQ ID NO: 29:

[0311] QSVEESGGRLVTPGTPLTLTCTVSGFSLSSYAMSWVRQAPGKGLEWIGIISGRGSIHYASWAKGRFTISKTSTTVDLKITSPTTEDTATYFCAREFGDGYDDYGDFGAFDPWGPGTLVTISSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGYTFNSYFMHWVRQAPGKGLVWVSMIRPNGGTTDYADSVKGRFTISVDNSKNTLYLQMNSLRAEDTAVYYCARWEGSYYALDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASSNVKYLAWYQQKPGKAPKLLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQFTSSPYTFGQGTKLEIK(SEQ ID NO:29);

[0312] The amino acid sequence of the light chain of the CD66b-116-GYPA bispecific antibody is shown in SEQ ID NO: 30:

[0313] AQVMTQTPPSLSASVGGTVTINCLASENVYSAVSWYQQKPEKPPTLLISGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYFCQGYSSYPLTFGAGTNVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ IDNO: 30);

[0314] The amino acid sequence of the heavy chain of the CD66b-128-GYPA bispecific antibody is shown in SEQ ID NO: 31:

[0315] QSLEESEGRLVTPGTPLTLTCTVSGFSLSSYAMIWVRQAPGKGLEYIGIIYAGGSASYASWAIGRFTISKTSTTVDLKIPSPTTEDTATYFCARTAYATYGYLNFWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCKASGYTFNSYFMHWVRQAPGKGLVWVSMIRPNGGTTDYADSVKGRFTISVDNSKNTLYLQMNSLRAEDTAVYYCARWEGSYYALDVWGQGTTVTVSSGGGGSGGGGSGGGGSGGGGSDIQMTQSPSSLSASVGDRVTITCRASSNVKYLAWYQQKPGKAPKLLIYYTSNLQSGVPSRFSGSGSGTDYTLTISSLQPEDVATYYCQQFTSSPYTFGQGTKLEIK(SEQ ID NO:31);

[0316] The amino acid sequence of the light chain of the CD66b-128-GYPA bispecific antibody is shown in SEQ ID NO: 32:

[0317] AQVMTQTPASVSAAVGGTITIKCQASEDIYNLLAWFQQKPGQPPKLLIYDASDLTSGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGGYYSASVIDIAFGGGTELEIL RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC(SEQ ID NO: 32).

[0318] 3. Gene synthesis: The heavy chain and light chain sequence genes of the bispecific antibodies CD66b-108-GYPA, CD66b-116-GYPA and CD66b-128-GYPA were artificially synthesized and inserted into the pcDNA3.1 vector to obtain an expression vector containing the bispecific antibody heavy chain gene and an expression vector containing the bispecific antibody light chain gene.

[0319] 4. Antibody expression: One day before transfection, dilute the 293F cells to a density of 3 × 10 6 After overnight growth, the cell density was diluted again to 3×10 6 cells / mL for later use. Add the expression vector containing the heavy chain gene of the bispecific antibody to the expression vector containing the light chain gene of the bispecific antibody in a ratio of 1ng:1ng, then add PEI transfection reagent (polysciences), and incubate at room temperature for 15min. Take 500mL of 3×10 6 cells / mL, add the prepared transfection system to the cells, mix and shake well, and culture in a shaker at 37°C, 8% CO2, 125rpm for 6 days.

[0320] 5. Antibody purification: After 6 days of culture, the cell culture fluid was collected, centrifuged at 3500rpm for 15min to remove cells and residues, and the supernatant was filtered with a 0.45μm filter membrane. The filtered supernatant was adsorbed with a Mabselect Sure LX (Cytiva, 17547401) gravity column to adsorb the target molecule, and the impurity protein was washed with 10 times the column volume of PBS buffer, and then the target molecule was eluted and collected with 100mM pH3.2 sodium citrate buffer, neutralized with 2M Tris buffer, and the pH was adjusted to 6.0 to obtain the bispecific antibodies CD66b-108-GYPA, CD66b-116-GYPA and CD66b-128-GYPA.

[0321] Example 10 Application of CD66b-GYPA bispecific antibody in removing neutrophils or PMN-MDSCs from PBMCs

[0322] 1. Experimental Methods

[0323] 10 mL of peripheral blood was collected from each of the three volunteers and placed at room temperature for 48 hours to artificially create abnormal density of neutrophils or PMN-MDSCs, so that neutrophils or PMN-MDSCs would be mixed in the separated PBMCs. After 48 hours, each portion of peripheral blood was divided into 3 portions, each 3.3 mL, one portion was directly separated from PBMCs by ficoll density gradient centrifugation as a control, one portion was added with the bispecific antibody CD66b-108-GYPA, CD66b-116-GYPA or CD66b-128-GYPA (final concentration 20 μg / mL) prepared in Example 9, and PBMCs were separated by ficoll density gradient centrifugation after incubation for 30 minutes; one portion was added with the commercially available granulocyte removal reagent RosetteSep Human Granulocyte Depletion Cocktail (Stemcell, Cat: 15624), and PBMCs were separated by ficoll density gradient centrifugation after incubation for 30 minutes. The separated PBMCs were labeled with CD15 antibody to identify neutrophils or PMN-MDSCs, and the residual neutrophils or PMN-MDSCs were detected by flow cytometry.

[0324] 2. Experimental Results

[0325] As shown in Table 17, the bispecific antibodies CD66b-108-GYPA, CD66b-116-GYPA or CD66b-128-GYPA can effectively remove neutrophils or PMN-MDSCs from PBMCs, and the effect is better than that of commercial granulocyte removal reagents.

[0326] Table 17 CD66b-GYPA bispecific antibody removes neutrophils or PMN-MDSCs from PBMCs

[0327]

[0328] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A rabbit monoclonal antibody against human CD66b protein, characterized in that: The rabbit monoclonal antibody is the first antibody CD66b-108, the second antibody CD66b-116 or the third antibody CD66b-128; The first antibody CD66b-108 comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 2, the amino acid sequence of CDR2 of the light chain variable region is KAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 3; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 5, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 6, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 7; The second antibody CD66b-116 comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 9, the amino acid sequence of CDR2 of the light chain variable region is GAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 10; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 12, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 13, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO: 14; The third antibody CD66b-128 comprises a light chain variable region and a heavy chain variable region, the amino acid sequence of CDR1 of the light chain variable region is as shown in SEQ ID NO: 16, the amino acid sequence of CDR2 of the light chain variable region is DAS, and the amino acid sequence of CDR3 of the light chain variable region is as shown in SEQ ID NO: 17; the amino acid sequence of CDR1 of the heavy chain variable region is as shown in SEQ ID NO: 19, the amino acid sequence of CDR2 of the heavy chain variable region is as shown in SEQ ID NO: 20, and the amino acid sequence of CDR3 of the heavy chain variable region is as shown in SEQ ID NO:

21.

2. The rabbit monoclonal antibody according to claim 1, characterized in that The amino acid sequence of the light chain variable region of the first antibody CD66b-108 is shown in SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4; The amino acid sequence of the light chain variable region of the second antibody CD66b-116 is shown in SEQ ID NO: 8, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 11; The amino acid sequence of the light chain variable region of the third antibody CD66b-128 is shown in SEQ ID NO: 15, and the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:

18.

3. Biomaterial, characterized in that The biological material is any one of the following: A nucleic acid molecule encoding the first antibody CD66b-108, the second antibody CD66b-116 or the third antibody CD66b-128 according to claim 1 or 2, or encoding a protein that is completely complementary to the amino acid sequence of the first antibody CD66b-108, the second antibody CD66b-116 or the third antibody CD66b-128 according to claim 1 or 2; An expression cassette comprising the above nucleic acid molecule; A recombinant vector comprising the above nucleic acid molecule; A recombinant microorganism comprising the above nucleic acid molecule. A recombinant cell line comprising the above nucleic acid molecule.

4. A bispecific antibody, characterized in that: The bispecific antibody comprises a first heavy chain, a second heavy chain, a first light chain, and a second light chain, wherein the variable regions of the first heavy chain and the first light chain recognize human CD66b antigen, and the variable regions of the second heavy chain and the second light chain recognize GYPA antigen; The amino acid sequences of the variable regions of the first heavy chain and the first light chain are one of the following combinations: The amino acid sequence of the variable region of the first heavy chain is shown in SEQ ID NO: 4, and the amino acid sequence of the variable region of the first light chain is shown in SEQ ID NO: 1; or the amino acid sequence of the variable region of the first heavy chain is as shown in SEQ ID NO: 11, and the amino acid sequence of the variable region of the first light chain is as shown in SEQ ID NO: 8; or the amino acid sequence of the variable region of the first heavy chain is as shown in SEQ ID NO: 18, and the amino acid sequence of the variable region of the first light chain is as shown in SEQ ID NO: 15; The amino acid sequence of the variable region of the second heavy chain is shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the second light chain is shown in SEQ ID NO:

25.

5. The bispecific antibody according to claim 4, characterized in that A connecting peptide is provided between the first heavy chain and the second heavy chain, and a connecting peptide is provided between the second heavy chain and the second light chain, wherein the connecting peptide is (G4S) n , where n is an integer between 1 and 6.

6. The bispecific antibody according to claim 4 or 5, characterized in that The bispecific antibody also comprises a heavy chain constant region and a light chain constant region.

7. The bispecific antibody according to any one of claims 4 to 6, characterized in that: The bispecific antibody is a first antibody CD66b-108-GYPA, a second antibody CD66b-116-GYPA or a third antibody CD66b-128-GYPA; The first antibody CD66b-108-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 27, and the amino acid sequence of the light chain is shown in SEQ ID NO: 28; The second antibody CD66b-116-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is shown in SEQ ID NO: 30; The third antibody CD66b-128-GYPA comprises a heavy chain and a light chain, the amino acid sequence of the heavy chain is shown in SEQ ID NO: 31, and the amino acid sequence of the light chain is shown in SEQ ID NO:

32.

8. Biomaterial, characterized in that The biological material is any one of the following: A nucleic acid molecule encoding the bispecific antibody according to any one of claims 4 to 7, or encoding a protein whose amino acid sequence is completely complementary to the bispecific antibody according to any one of claims 4 to 7; An expression cassette comprising the above nucleic acid molecule; A recombinant vector comprising the above nucleic acid molecule; A recombinant microorganism comprising the above nucleic acid molecule. A recombinant cell line comprising the above nucleic acid molecule.

9. Use of the rabbit monoclonal antibody according to claim 1 or 2, the biomaterial according to claim 3, the bispecific antibody according to any one of claims 4 to 7 and / or the biomaterial according to claim 8 in the preparation of a detection product and / or a clearance product for granulocytes or polymorphonuclear myeloid-derived suppressor cells.

10. A detection product and / or removal product for granulocytes or polymorphonuclear myeloid-derived suppressor cells, characterized in that: Comprising the bispecific antibody according to any one of claims 4 to 7.

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