Rabbit monoclonal antibodies against human cd66b protein and their use in the preparation of products for detecting and / or depleting granulocytes or polymorphonuclear myeloid-derived suppressor cells

Abstract

The application discloses rabbit monoclonal antibodies against human CD66b proteins and application of the rabbit monoclonal antibodies in preparation of products for detecting and / or removing granulocytes or polymorphonuclear myeloid-derived suppressor cells. Three rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128 against human CD66b proteins are prepared first, and three bispecific antibodies against human CD66b and GYPA are constructed based on the three rabbit monoclonal antibodies. The bispecific antibodies prepared in the application can effectively remove granulocytes or polymorphonuclear myeloid-derived suppressor cells from PBMCs, and the effect is better than that of a commercially available commercial granulocyte removal reagent, and the bispecific antibodies have a wide application prospect in T cell immune detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of immunology, in particular, to a rabbit monoclonal antibody against human CD66b protein and its application in the preparation of products for detecting and / or eliminating granulocytes or polymorphonuclear myeloid-derived suppressor cells. BACKGROUND

[0002] Myeloid-derived suppressor cells (MDSCs) are a group of heterogeneous cells with significant ability to suppress immune cell response, which are derived from bone marrow and have phenotypes similar to precursors of dendritic cells, macrophages and granulocytes. In the tumor microenvironment, MDSCs exert immune suppression function by secreting factors such as Arg-1, iNOS, ROS, etc., and inhibit the activity of lymphocytes. PMN-MDSCs, i.e., polymorphonuclear myeloid-derived suppressor cells, are a subtype of MDSCs, have very similar surface markers to neutrophils, and have strong immune suppression effect, 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 also includes 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 immune suppression effect through various mechanisms.

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

[0005] To solve this problem, the erythrocytes expressing GYPA (glycophorin A, a transmembrane protein of red blood cells) and the neutrophils or PMN-MDSCs expressing CD66b can be simultaneously connected together by using a bispecific antibody, and the neutrophils or PMN-MDSCs are precipitated by the erythrocytes with high density, so that the neutrophils or PMN-MDSCs are removed from the PBMCs, and the immune response of the T cells is restored. Therefore, the development of the CD66b monoclonal antibody is very important, and the bispecific antibody described above can be designed based on the CD66b monoclonal antibody. At present, the CD66b specific antibody widely used is a mouse monoclonal antibody, and there is no report on the preparation of a CD66b rabbit monoclonal antibody by using a rabbit monoclonal antibody preparation technology. The rabbit monoclonal antibody has more advantages than the conventional mouse monoclonal antibody, such as that the rabbit antiserum usually contains high-affinity antibodies, can recognize more types of epitopes than the mouse antiserum, and the rabbit monoclonal antibody can recognize many antigens that do not produce immunity in mice. Therefore, it is of great significance to develop a rabbit monoclonal antibody that can specifically bind to the CD66b protein. SUMMARY

[0006] In order to overcome the above-mentioned defects and shortcomings in the prior art, the present application provides a rabbit monoclonal antibody against human CD66b protein and its application in preparing a product for detecting and / or removing granulocytes or polymorphonuclear myeloid-derived suppressor cells.

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

[0008] The second object of the present application is to provide a biological material.

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

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

[0011] The fifth object of the present application is to provide a biological material.

[0012] The sixth object of the present application is to provide the application of the bispecific antibody and / or the biological material described above in preparing a detection reagent and / or a removal product of granulocytes or polymorphonuclear myeloid-derived suppressor cells.

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

[0014] Therefore, the present application claims the following contents:

[0015] A rabbit monoclonal antibody against human CD66b protein, 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, the amino acid sequence of CDR1 of the light chain variable region is shown as 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 shown as SEQ ID NO: 3; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 5, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 6, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as 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 shown as 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 shown as SEQ ID NO: 10; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 12, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 13, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as 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 shown as 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 shown as SEQ ID NO: 17; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 19, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 20, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as SEQ ID NO: 21.

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

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

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

[0022] A biological material, which 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 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-mentioned nucleic acid molecule;

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

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

[0027] A recombinant cell line comprising the above-mentioned 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, the variable region of the first heavy chain and the first light chain recognizing a human CD66b antigen, and the variable region of the second heavy chain and the second light chain recognizing a GYPA antigen;

[0029] The amino acid sequence of the variable region of the first heavy chain and the variable region of the first light chain is one of the following combinations:

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

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

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

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

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

[0035] More preferably, the 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 as SEQ ID NO: 27, and the amino acid sequence of the light chain is shown as 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 as SEQ ID NO: 29, and the amino acid sequence of the light chain is shown as 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 as SEQ ID NO: 31, and the amino acid sequence of the light chain is shown as SEQ ID NO: 32.

[0041] A biological material, which is any one of the following:

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

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

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

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

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

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

[0048] A granulocyte or polymorphonuclear myeloid-derived suppressor cell detecting product and / or removing product comprising any of the above-mentioned bispecific antibodies.

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

[0050] The application discloses rabbit monoclonal antibodies against human CD66b proteins and application of the rabbit monoclonal antibodies in preparation of products for detecting and / or removing granulocytes or polymorphonuclear myeloid-derived suppressor cells. Three rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128 against human CD66b proteins are prepared first, and three bispecific antibodies against human CD66b and GYPA are constructed based on the three rabbit monoclonal antibodies. The bispecific antibodies prepared in the application can effectively remove granulocytes or polymorphonuclear myeloid-derived suppressor cells from PBMCs, and the effect is better than that of a commercially available commercial granulocyte removing reagent, and the bispecific antibodies have a wide application prospect in T cell immune detection. BRIEF DESCRIPTION OF DRAWINGS

[0051] Figure 1 Growth trend of eukaryotic cells induced to express pcDNA3.1-il2-CD66b.

[0052] Figure 2 SDS-PAGE detection results, lane 1 is Marker, lane 2 is cell supernatant electrophoresis results, and lane 3 is electrophoresis results of the purified protein.

[0053] Figure 3 PCR amplification gel electrophoresis results of the antibody light chain variable region, and the samples are loaded in order of PCR reactions.

[0054] Figure 4 PCR amplification gel electrophoresis results of the antibody heavy chain variable region, and the samples are loaded in order of PCR reactions.

[0055] Figure 5 ScFv fragment gel electrophoresis chart, lane 1 is Marker, and lane 3 and lane 4 are both amplified ScFv fragments.

[0056] Figure 6 Reducing and non-reducing SDS-PAGE electrophoresis detection results, lanes 1-3 are non-reducing electrophoresis results of the rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128, lane 4 is Marker, and lanes 5-7 are reducing electrophoresis results of the rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128.

[0057] Figure 7Purification of rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128.

[0058] Figure 8 Design of bispecific antibody. DETAILED DESCRIPTION

[0059] The application will be further described below in connection with specific embodiments, but the embodiments do not limit the application in any form. Unless otherwise specified, the reagents, methods and equipment used in the application are conventional reagents, methods and equipment in the technical field.

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

[0061] 2xYT-CG medium: 20 mL of 20% D-Glucose solution (w / v) and 200 μL of Carb were added to 180 mL of 2xYT liquid, to obtain 2xYT-CG medium with final concentration of 2% D-Glucose (w / v) and 100 μg / mL Carb.

[0062] Example 1 Preparation and purification of antigen

[0063] I. 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 length (Met 1-Ser 319) were selected, a Kozak sequence and a signal peptide sequence IL-2 were added at the 3' end, and after codon optimization, the gene synthesis was performed by GenScript, 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 the pcDNA3.1-il2-CD66b plasmid sample, and the endotoxin detection was performed. The endotoxin detection result showed that the endotoxin content of the pcDNA3.1-il2-CD66b plasmid sample was less than 1 EU / μg, and it could be used for cell transfection.

[0068] 3. Cell transfection and eukaryotic expression

[0069] (1) Chinese hamster ovary cells (CHO cells) were transfected with 3x10 6viable cell / mL of cell density was inoculated into 125 mL conical flasks, 30 mL per flask, and placed in a 37°C, 8% CO2, 125 rpm shaker for overnight culture. When the growth density of CHO cells reached 5-10 x 10 6 viable cells / mL, with a viability of 95-99%, transfection was performed.

[0070] (2) 25 mL of CHO cell suspension was removed and placed in a 125 mL conical flask, and the corresponding culture medium was added to 30 mL after preheating to 37°C. The flask was shaken gently to mix (theoretically, the cell density was diluted to 6 x 10 6 viable cells / mL, with a viability of greater than 99%.

[0071] (3) Plasmid dilution: pcDNA3.1-il2-CD66b plasmid was added to pre-cooled 4°C OptiPRO TM medium, and gently inverted to mix, obtaining the diluted plasmid.

[0072] (4) Transfection reagent dilution: 920 μL of pre-cooled 4°C OptiPRO TM medium was added to 80 μL of ExpiFectamine TM CHO Reagent, and gently inverted to mix, obtaining the diluted transfection reagent.

[0073] (5) pcDNA3.1-il2-CD66b transfection: The diluted transfection reagent of step (4) was added to the diluted plasmid of step (3), inverted to mix, and then incubated at room temperature for 90 s. Then, it was slowly added to the cells of step (2) while gently shaking the cells.

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

[0075] (7) Addition of enhancer and auxiliary materials: After 22 h of transfection, routine counting was performed; after counting, 150 μL of ExpiCHO TM Enhancer and 5 mL of ExpiCHO TM Feed were added to the flask, and the two flasks were placed in the incubator (37°C, 8% CO2, and 125 rpm) for culture.

[0076] (8) After 6 days of transfection, about 30 mL of cell supernatant was harvested after transient expression. In the growth trend of eukaryotic cells induced by pcDNA3.1-il2-CD66b expression, it can be seen that Figure 1), since the completion of transfection, the density of the cells and the survival rate of a first increase and then decrease, basically consistent with the transfection of the reagent instructions, the subsequent will be identified and purified cell supernatant.

[0077] 4. Purification of the expressed protein

[0078] (1) Experimental method

[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 SDS-PAGE detection results are shown in Figure 2 The cell supernatant has a clear band at 70KD, which is consistent with the size of human CD66b protein. The target protein was adsorbed and purified using an anion column, and it could be seen through the sample that the anion column basically purified the target protein in the culture supernatant completely, thereby obtaining purified human CD66b protein.

[0082] Example 2 Comparison of purified human CD66b protein and commercially available CD66b protein

[0083] I. Experimental method

[0084] 1. Coating: Take the human CD66b protein obtained in Example 1 and the commercially available CD66b protein from Yiqiao into 1 mL of CBS coating solution and mix well (the final concentration of CD66b protein is 1 μg / mL), then add 100 μL / well of coating solution containing human CD66b protein obtained in Example 1 and coating solution containing commercially available CD66b protein from Yiqiao to the enzyme-labeled plate, and incubate 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: Dilute the positive serum (immune mouse serum) 1000-fold and 10000-fold with 1% sodium casein diluent (w / v), and dilute the commercially available CD66b antibody 100-fold with 1% sodium casein diluent (w / v). Add 100 μL / well of diluted commercially available CD66b antibody, positive serum and 1% sodium casein diluent (w / v) to the pre-coated plate, and use 1% sodium casein diluent (w / v) as a blank control, and incubate at room temperature for 2 h.

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

[0088] 5. Color development: Discard the liquid in the wells, wash 5 times with 1x PBST, 300 μL / well, the last time with a plate shaker for 10 s, add 100 μL / well of TMB, and develop color at room temperature for 20 min in the dark; add 50 μL / well of stop solution to stop the reaction, and read the OD 450 ~ OD 620 at 450 nm.

[0089] II. Experimental results

[0090] The results are shown in Tables 1 and 2. As shown in Table 1, the OD value of the blank control wells (1% sodium caseinate diluent, w / v) is in the range of 0.08-0.13, which is within the normal range, indicating that the experimental operation is correct and the results are reliable. The test results of the commercially available CD66b antibody (Table 2) show that the human CD66b protein prepared in Example 1 has the same activity as the commercially available CD66b protein from YiKuo, and even better results.

[0091] Table 1 ELISA experimental results

[0092] Yikai commercial CD66b protein Example 1 prepared human CD66b protein Commercial CD66b antibody 2.276 2.767 Positive serum 1000x 2.241 1.411 Positive serum 1000x 2.726 1.467 Positive serum 10000x 0.4102 0.3049 Positive serum 10000x 0.3804 0.3237 1% casein sodium dilution 0.08120 0.1373

[0093] Table 2 result statistics

[0094] Yikai commercial CD66b protein Example 1 prepared human CD66b protein Commercial CD66b antibody 2.276 2.767 Positive serum 1000x average OD value 2.484 1.439 Positive serum 10000x average OD value 0.395 0.314

[0095] Example 3 Immunization effect of human CD66b protein

[0096] I. Experimental method

[0097] A New Zealand white rabbit was immunized with the human CD66b protein prepared in Example 1 as an immunogen, and the serum from the third and fourth immunizations was taken and subjected to ELISA detection.

[0098] The ELISA detection 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 to the enzyme-labeled plate, cover the reaction wells with plastic wrap, and incubate at 4°C overnight, and wash the plate 3 times with PBST;

[0100] 2. Blocking: fill the washed reaction wells with 3% skim milk powder (w / v), cover with plastic wrap, and incubate at room temperature for 1 h, and wash the plate 3 times with PBST;

[0101] 3. Add sample: dilute the serum of the third and fourth immunization and the negative serum 1000 times, 3000 times, 9000 times, 27000 times, 81000 times and 243000 times respectively, then take 100 μL of each into the blocked reaction well, seal the plastic wrap, place at room temperature for 1 h, and wash the plate with PBST for 3 times;

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

[0103] (5) TMB color development: add 100 μL of TMB color developing solution into the reaction well, develop color for 20 min; then add 50 μL of stop solution;

[0104] (6) Reading: place the enzyme-labeled plate into the enzyme marker for reading.

[0105] II. Experimental results

[0106] The results are shown in Table 3. As shown in Table 3, the titer is >81000 after the third and fourth immunization, which is qualified for immunization, and can be used for constructing the phage display library subsequently.

[0107] Table 3. Results of serum titer of immunized rabbit

[0108] Sample dilution fold 178# rabbit three immunization 179# rabbit three immunization 179# rabbit four immunization 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] The New Zealand white rabbits were immunized with the human CD66b protein prepared in Example 1 as immunogen, and the rabbits were sacrificed one week after the third and fourth booster immunization respectively, and the spleen tissues and bone marrow were taken.

[0111] 1. RNA extraction

[0112] (1) Liquid nitrogen grinding: first cool the mortar with liquid nitrogen, and then grind the spleen and bone marrow after taking them out from -80℃ (operate separately before and after), grind the spleen tissues and bone marrow into powder while adding liquid nitrogen, and finally spread the powder in the mortar, and immediately add 6 mL of TRIzol to cover the powder when the liquid nitrogen is ready to evaporate. After the Trizol is dissolved, it is dispensed to obtain the TRIzol lysate of the spleen tissue (the bone marrow sample has too much remaining tissue and is too viscous, so 5 mL of TRIzol is first taken into a 10 mL EP tube, mixed, then 1 mL of TRIzol solution is added, and 1 mL per tube is dispensed; the remaining liquid in the mortar is mixed with 2 mL of TRIzol solution, and then 1 mL per tube is dispensed).

[0113] (2) Extraction: Take 1 mL TRIzol lysate of spleen tissue and bone marrow sample respectively, add 200 μL chloroform (1 / 5 volume of TRIzol), vortex mix for 15 s, until the solution is emulsified to milky white, and stand for 2-3 min on ice. Centrifuge at 12000 x g for 15 min at 4°C.

[0114] (3) Take supernatant: Tilt the tube at 45°, directly use 1 mL gun to continuously and evenly suck the supernatant aqueous phase into another new centrifuge tube, and suck 500 μL supernatant per tube.

[0115] (4) Precipitation: Add 500 μL isopropanol (1 / 2 volume of TRIzol) to the supernatant, mix well after inverting the centrifuge tube, stand for 10 min on ice, and centrifuge at 12000 x g for 10 min at 4°C.

[0116] (5) Washing: Discard the supernatant at one time, invert the EP tube on the water absorption paper, and suck the residual liquid at the tube opening; add 500 μL of 75% ethanol (DEPC treated water, v / v) to resuspend the RNA precipitate, invert the centrifuge tube, and wash the tube wall to suspend the RNA precipitate; centrifuge at 12000 x g for 5 min at 4°C.

[0117] (6) Drying: Discard the supernatant at one time, invert the EP tube on the water absorption paper, and centrifuge at 12000 x g for 30 s at 4°C; use the gun head to suck the residual liquid (note that do not suck the precipitate), open the centrifuge tube cover, lie horizontally, and dry the precipitate for 2-3 min at room temperature (in the 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), to obtain the spleen tissue RNA and bone marrow sample RNA of the third and fourth immunized rabbits respectively.

[0119] 2. RNA purification

[0120] (1) Digestion of genomic DNA: Take the spleen tissue RNA and bone marrow sample RNA of the third and fourth immunized rabbits obtained in step 1 respectively, and perform DNase treatment according to Table 4 to prepare the reaction system. Mix well, centrifuge at low speed for 10 s, add water to the metal bath, and incubate at 37°C for 30 min to digest the residual genomic DNA.

[0121] Table 4 Reaction system

[0122]

[0123] (2) To the digested sample of step (1), add 1 mL TRIzol; add 200 μL chloroform (1 / 5 volume of TRIzol), tightly cap the centrifuge tube, vortex mix for 15 s, until the solution emulsifies to a milky white color, and let stand on ice for 2-3 min. Centrifuge at 12,000 x g for 15 min at 4°C.

[0124] (3) Take the supernatant: tilt the tube at 45°, and directly transfer the supernatant aqueous phase to another new centrifuge tube using 1 mL gun in succession and evenly, 500 μL supernatant is taken per tube.

[0125] (4) Precipitate: add 500 μL isopropanol (1 / 2 volume of TRIzol) to the supernatant, mix well after inverting the centrifuge tube, let stand on ice for 10 min, and centrifuge at 12,000 x g for 10 min at 4°C.

[0126] (5) Wash: discard the supernatant at one time, invert the EP tube on the absorbent paper, and absorb the residual liquid at the tube opening; add 500 μL of 75% ethanol (DEPC-treated water, v / v) to resuspend the RNA precipitate, invert the centrifuge tube, and wash the tube wall thoroughly so that the RNA precipitate is suspended; centrifuge at 12,000 x g for 10 min at 4°C.

[0127] (6) Dry: discard the supernatant at one time, invert the EP tube on the absorbent paper, and centrifuge at 12,000 x g for 30 s at 4°C; use the gun head to absorb the residual liquid (note that the precipitate should not be absorbed), open the centrifuge tube cap, lay horizontally, and dry the precipitate at room temperature for 2-3 min (in the clean bench).

[0128] (7) Dissolve: add RNase-free water to dissolve the precipitate, and perform agarose gel electrophoresis to verify the quality of the purified RNA, the results show that the quality of the purified RNA is good, and there is no protein contamination, thus obtaining the purified RNA of the rabbit spleen tissue after the third and fourth immunization and the bone marrow sample.

[0129] 3. RNA reverse transcription

[0130] Use III Reverse Transcriptase reverse transcription kit to reverse transcribe the purified RNA of the rabbit spleen tissue after the third and fourth immunization and the bone marrow sample of step 2 into cDNA. The reaction system is prepared according to Table 5.

[0131] Table 5 Reaction system

[0132]

[0133]

[0134] Incubate at 65°C for 5 min, then on ice for 2 min. Add the reaction mixture (Table 6), mix gently, and centrifuge briefly for 5 s; incubate at 50°C in a metal bath for 60 min; then incubate at 70°C in a metal bath for 15 min; cool on ice; after completion, obtain cDNA from rabbit spleen tissue and bone marrow samples after the third and fourth immunizations, respectively.

[0135] Table 6 Reaction solution mixture

[0136] Component Volume (μL) 5x First-Strand Buffer 4 0.1M DTT 1 RNaseOUT™ Recombinant RNase Inhibitor (40U / uL) 1 Super Script™ III RT (200U / uL) 1 Total 7

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

[0138] (1) Primer pairing

[0139] The primers for the κ and λ light chains 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] After thoroughly mixing the cDNA from the third immunized rabbit spleen tissue and bone marrow samples obtained in step 3, and the cDNA from the fourth immunized rabbit spleen tissue and bone marrow samples (i.e., the mixed cDNA contains cDNA from the rabbit spleen tissue and bone marrow samples), the PCR reaction system was prepared according to Table 9, with a total reaction volume of 20 μL.

[0147] Table 9 Reaction System

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

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

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

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

[0152] (1) Primer pairing

[0153] The primers for IgG and IgM heavy chains 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 This yielded DNA fragments containing multiple antibody heavy chain variable regions.

[0159] 6. Construction of antibody in ScFv form

[0160] Mix 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 to obtain VL fragments and VH fragments. Prepare the PCR reaction system according to Table 12. The total volume of the reaction system is 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 ), thus obtaining the ScFv fragment.

[0167] 7. Construction of a TG1 glycerol bacterial antibody library

[0168] The amplified ScFv fragment was digested with SfiI, and the digestion products were mixed and ligated into 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 overnight at 37°C in the dark to obtain the TG1 glycerol bacterial antibody library containing human CD66b-ScFv antibody.

[0169] Example 5: Preparation of phage library

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

[0171] 2. Add 10 μL of helper phage (M13K07, titer 1.2 x 10 12 pfu / mL), and perform infection 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 with 200 mL of 2xYT liquid medium (containing both carbenicillin and kanamycin resistance), and perform overnight culture at 37°C, 180 rpm shaker.

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

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

[0176] Example 6 Solid-phase panning and ELISA detection

[0177] I. Solid-phase panning

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

[0179] 1. Human CD66b antigen coating: use the human CD66b protein prepared in Example 1 as the antigen, dilute it with CBS buffer to 1 μg / mL, and coat 100 μL / well in the enzyme-labeled plate, and place it in a 4°C constant temperature incubator for more than 12 h.

[0180] 2. Blocking: pour out the liquid in the wells, wash twice with 200 μL / well of 0.05% PBST (v / v), and discard the liquid in the wells; then add 200 μL / mL of 2% sodium casein blocking solution (w / v), and incubate at 37°C for 2 h; after the blocking is completed, aspirate the liquid in the wells, wash 5 times with 200 μL / well of 1xPBS, discard the liquid in the wells, seal the plate with sealing film, and store in 4°C.

[0181] 3. Incubation of phage: add the phage library (0thround) of the human CD66b-ScFv antibody obtained in Example 5 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, wash 5 times with 200 μL / well of 1xPBST, and then wash 5 times with 200 μL / well of 1xPBS; after the washing is completed, discard the liquid in the wells.

[0183] 5. Resuscitation of TG1: take the TG1 glycerol bacteria from -80°C, use a gun head to take the bacterial liquid, streak on an LB plate, and incubate at 37°C for 12 h. From the TG1 streak plate, pick a single colony, transfer into 40 μL of 2xYT liquid medium, and mix by blowing; then take 10 μL of the bacterial liquid and transfer into 3 mL of 2xYT liquid medium, and incubate at 37°C, 200 rpm, overnight. After the overnight incubation, again take 10 μL of the bacterial liquid and transfer into 3 mL of 2xYT liquid medium, and incubate at 37°C, 200 rpm, for 2 h; measure the OD value to be 0.745, and obtain the TG1 bacterial liquid.

[0184] 6. Add 100 μL / well of 0.2M glycine buffer (pH=3) to the enzyme-labeled wells after the washing in step 4, and stand at room temperature for 10 min; immediately aspirate the solution, and transfer to a 1.5 mL EP tube. Add 20 μL of 0.5M Tris-HCl (pH 8.0) to the EP tube for neutralization; after mixing, transfer the entire solution to 4 mL of TG1 bacterial liquid, and obtain TG1-Phage; place the TG1-Phage at 37°C, stand for 45 min, and shake every 15 min for infection; after 45 min, obtain the TG1-Phage solution after the infection is completed, which is the 1stround of panning phage.

[0185] Repeat the above panning steps to perform the 2ndand 3rdrounds of panning, and obtain the 2ndand 3rdrounds of panning phage, respectively;

[0186] In the second round of screening, 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 to wash 10 times, and then 200 μL / well of 1×PBS was added to wash 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 to wash 15 times, and then 200 μL / well of 1×PBS was added to wash 15 times.

[0188] Storage plates: Take TG1-Phage solutions that have completed infection in rounds 1-3, and dilute them 10 μL each. 3 ~10 6 Take 50 μL of the solution and spread it onto a 2×YT-CG plate. Incubate at 37°C inverted position for at least 12 hours.

[0189] II. Phage ELISA Validation

[0190] 1. Experimental Methods

[0191] (1) Encapsulation and sealing

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

[0193] Remove the overnight coated microplate from 4°C and allow it to stand at room temperature. Pour out the liquid in the wells, add 200 μL / well of 0.1% PBST (w / v) to wash the plate twice, blot dry the liquid, add 2% casein sodium blocking buffer (w / v) to block, and place at 37°C for 1 hour. After blocking, pour out the liquid in the wells, add 200 μL / well of 1×PBS to wash the plate once, and blot dry.

[0194] (2) Incubation of samples

[0195] Take out the phage library of M13K07, the human CD66b-ScFv antibody obtained in Example 5 (round 0 phage), and the phages prepared in step one (rounds 1-3) at 4℃. After taking a certain amount of each sample, add 1×PBS to make the final titer of each sample 1×10⁻⁶. 10 cfu / mL, final volume 450μL / tube.

[0196] Add 100 μL / well of the above solution to the blocked plate, and incubate the plate at 37°C for 2 h with PBS as a blank control. After incubation, pour the liquid in the wells, wash the plate 5 times with 200 μL / well of 0.1% PBST (w / v), pat dry, and then wash the plate 2 times with 200 μL / well of 1 x PBS, pat dry the liquid.

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

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

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

[0200] 2. Experimental results

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

[0202] Table 13. Phage ELISA results

[0203] Item Human CD66b protein Negative Signal to noise ratio 0th round phage 0.046 0.041 1.12 1st round panning phage 0.421 0.064 6.58 2nd round panning phage 1.201 0.137 8.77 3rd round panning phage 2.15 0.182 11.81 M13K07 0.081 0.062 1.31 Blank 0.051 0.045 1.13

[0204] III. Monoclonal phage ELISA

[0205] 1. Experimental method

[0206] (1) Activation of the strain

[0207] Add 1 mL / well of 2 x YT-CG medium to 96-deep well plate 1 and 96-deep well plate 2, and divide each into 90 wells as sample wells; and then add 1 mL / well of 2 x YT liquid to 96-deep well plate 1 and 96-deep well plate 2, and divide each into one well as a negative well.

[0208] Randomly pick 180 single clones from the library containing plates of the second and third rounds of panning in step one, and add them to the 90 sample wells of deep well plate 1 and deep well plate 2, respectively, and mix by blowing; and then pick one colony from the TG1 streak plate and add it to the negative well of the deep well plate. Place the two deep well plates in a 37°C incubator and culture them at 200 rpm for more than 12 h.

[0209] (2) Transfection

[0210] 1) Add 500 μL / well 2xYT-CG medium to 96-deep well plate ③ and deep well plate ④, a total of 90 wells as sample wells; additionally, add 500 μL / well to 96-deep well plate ③ and deep well plate ④, a total of 2 wells as negative wells.

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

[0212] 2) Take M13K07 from 4°C, mix 250 μL M13K07 with 2250 μL 2xYT liquid medium, then add 10 μL / well to the sample wells and negative wells of deep well plate ③ and ④, and then place deep well plate ③ and ④ in a 37°C incubator for 45 min.

[0213] 3) Place the deep well plate after infection in a centrifuge and centrifuge at 1800 g for 10 min, then use a pipette to remove the supernatant; add 70 μL Kana to 100 mL 2xYT liquid medium and mix to obtain 2xYT-K, then add 500 μL / well 2xYT-K to the negative wells of deep well plate ③ and ④; add 198 μL Crab to the remaining 99 mL 2xYT-K to obtain 2xYT-CK medium containing 100 μg / mL Carb and 35 μg / mL Kana, then add 500 μL / well 2xYT-CK medium to the sample wells of deep well plate ③ and ④, mix by blowing, and place in a 30°C, 250 rpm incubator for 12 h or more.

[0214] (3) Monoclonal ELISA

[0215] 1) Plate coating: use the human CD66b protein prepared in Example 1 as the antigen, dilute with PBS to 2 μg / mL; add 100 μL / well of the diluted antigen to the enzyme-labeled plate, and take another enzyme-labeled plate, add 100 μL / well of 3% BSA (w / v), and place the two enzyme-labeled plates in a 4°C incubator for 12 h or more.

[0216] 2) Blocking: take the two overnight-coated plates from 4°C, pour out the liquid in the wells, wash the plates with 200 μL / well of 0.1% PBST (w / v), pat dry the inside liquid, then add 200 μL / well of 3% sodium casein blocking solution (w / v) for blocking, and place in a 37°C incubator for 2 h.

[0217] 3) Incubation: Pour off the liquid in the wells, wash the plate with 200 μL / well of 1 x PBS, tap dry. Place the overnight incubated deep well plates ③ and ④ in the centrifuge, 1800 g for 15 min; after centrifugation, pipette the liquid from the wells in order, and add 50 μL / well to the human CD66b coated plate and the BSA coated plate, respectively; in addition, set up two negative wells (provided by deep well plates ③ and ④), two positive wells (use 3rd round of panning phage, pipette 500 μL of 2.76 x 10 10 cfu / mL into 500 μL of PBS, mix to 1.38 x 10 10 cfu / mL) and two blank wells (add 3% sodium caseinate blocking solution, w / v). Place the plates in a 37°C incubator for 2 h.

[0218] 4) Secondary antibody reaction: Pour off the liquid in the wells, wash the plate with 200 μL / well of 0.1% PBST (w / v) for 5 times, tap dry, then wash with 200 μL / well of 1 x PBS for 2 times, tap dry the liquid. Dilute M13-HRP with 1% sodium caseinate dilution solution (w / v) to 2500 times, then add 50 μL / well to the wells, place in a 37°C incubator for 1 h.

[0219] 5) Color development reaction: Pour off the liquid in the wells, wash the plate with 200 μL / well of 0.1% PBST (w / v) for 5 times, tap dry, then wash with 200 μL / well of 1 x PBS for 2 times, tap dry the liquid; add 50 μL / well of TMB, react at room temperature for 13 min, add 50 μL / well of stop solution, read OD 450 ~ OD 620 at 630 nm.

[0220] 2. Experimental results

[0221] The results of monoclonal phage ELISA are shown in Table 14 and Table 15. Among the screened monoclonals, there are antibodies with medium / strong binding. These monoclonals are 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 the monoclonal phage ELISA in Example 6, monoclonals were selected for sequencing, and three rabbit monoclonal antibodies against human CD66b protein were obtained, 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 CDR2 of the heavy chain variable region of the CD66b-108 monoclonal antibody is shown as SEQ ID NO: 6:

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

[0242] The amino acid sequence of CDR3 of the heavy chain variable region of the CD66b-108 monoclonal antibody is shown as 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 as SEQ ID NO: 8:

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

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

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

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

[0249] The amino acid sequence of CDR3 of the light chain variable region of the CD66b-116 monoclonal antibody is shown as 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 as SEQ ID NO: 11:

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

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

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

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

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

[0257] The CDR3 amino acid sequence of the heavy chain variable region of the CD66b-116 monoclonal antibody is set forth 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 set forth in SEQ ID NO: 15:

[0260] AQVMTQTPASVSAAVGGTITIKCQASEDIYNLLAWFQQKPGQPPKLLIYDAESLTSGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGGYYSASVIDIAFGGGTELEIL (SEQ ID NO: 15);

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

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

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

[0264] The CDR3 amino acid sequence of the light chain variable region of the CD66b-128 monoclonal antibody is set forth 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 set forth in SEQ ID NO: 18:

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

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

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

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

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

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

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

[0274] Example 8 Performance Test

[0275] I. Expression and Purification

[0276] 1. Experimental Methods

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

[0278] ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22);

[0279] After adding Kozak sequence and signal peptide sequence at the 3' end of the heavy chain and codon optimization, it is constructed into pcDNA3.1 for expression;

[0280] The FC segment sequence is added at the 5' end of the light chain, and the FC segment sequence information is as follows:

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

[0282] After adding Kozak sequence and signal peptide sequence at the 3' end of the light chain and codon optimization, it is constructed into pcDNA3.1 for expression.

[0283] The expression product is purified by protein A column, and the purified protein is subjected to reducing and non-reducing SDS-PAGE electrophoresis.

[0284] 2. Experimental results

[0285] The results are shown in Figure 6 The protein purity reaches more than 80%, and the SDS-PAGE electrophoresis result shows that the protein size is consistent with the expected result.

[0286] II. ELISA detection

[0287] 1. Experimental method

[0288] (1) Coat human CD66b protein prepared in Example 1 on ELISA plate, concentration is 1 μg / mL, coat overnight at 4°C.

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

[0290] (3) Wash the plate with PBST for 2 times, pat dry, add serially diluted rabbit monoclonal antibodies CD66b-108, CD66b-116 and CD66b-128 (2000, 1000, 100, 10, 1, 0.1, 0.01, 0 ng / mL) into the reaction wells, 100 μL / well, react at room temperature for 1 h;

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

[0292] (5) Wash the plate with PBST for 5 times, pat dry, add 100 μL TMB color developing agent, color develop at room temperature for 10 min, add 50 μL stop solution, read the absorbance value of A 450 ~A 620 , plot the titer curve with the reciprocal of the concentration of the rabbit monoclonal antibody as the abscissa and the absorbance value as the ordinate.

[0293] 2. Experimental results

[0294] The results are shown in Figure 7 and Table 16. The three antibodies CD66b-108, CD66b-116 and CD66b-128 all have significant binding ability to human CD66b protein. Among them, the binding ability of CD66b-108 is the strongest.

[0295] Table 16 Antibody performance

[0296] Item CD66b-108 CD66b-116 CD66b-128 Linear range 0.17-3000 ng / mL 2-223 ng / mL 2.7-1000 ng / mL IC50 11.61 ng / mL 114.5 ng / mL 2.106 x 10 27 ng / ml

[0297] Example 9 CD66b-GYPA bispecific antibody

[0298] 1. GYPA antibody sequence

[0299] The GYPA antibody sequence is from patent WO2017015141A1, wherein:

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

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

[0302] The amino acid sequence of the heavy chain variable region of the CD66b-108-GYPA bispecific antibody is shown as SEQ ID NO: 27:

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

[0304] 2. CD66b-GYPA bispecific antibody

[0305] The light chain variable region and the heavy chain variable region of CD66b-108, CD66b-116 and CD66b-128 obtained in Example 7 were connected with the light chain variable region and the heavy chain variable region 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 as 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 set forth in SEQ ID NO: 28:

[0309] AQVMTQTPASVSEPVGGTVTIKCQASQSIGSNLAWYQQKAGQPPNLLIYKASTLESGVPS RFKGSGSGTEFTLTISDLECADAATYYCQTYYTSDSSYYGAAFGGGTEVVVKRTVAAPSVF IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 28);

[0310] The amino acid sequence of the heavy chain of the CD66b-l 16-GYPA bispecific antibody is set forth in SEQ ID NO: 29:

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

[0312] The amino acid sequence of the light chain of the CD66b-l16-GYPA bispecific antibody is set forth in SEQ ID NO: 30:

[0313] AQVMTQTPPSLSASVGGTVTINCLASENVYSAVSWYQQKPEKPPTLLISGASNLESGVPPRFSGSGSGTDYTLTIGGVQAEDAATYFCQGYSSYPLTFGAGTNVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 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 set forth in SEQ ID NO: 32:

[0317] AQVMTQTPASVSAAVGGTITIKCQASEDIYNLLAWFQQKPGQPPKLLIYDASDLTSGVPSRFSGSGSGTQFTLTISGVQCDDAATYYCQGGYYSASVIDIAFGGGTELEILRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 32).

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

[0319] 4. Antibody expression: One day before transfection, dilute the 293F cells to a density of 3 x 10 6 cells / mL with Expi293 expression medium (Gibco, A1435101). After overnight growth, dilute the cell density to 3 x 10 6 cells / mL again for use. According to the ratio of 1 ng: 1 ng of 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, add the expression vectors and PEI transfection reagent (polysciences), and incubate at room temperature for 15 min. Take 500 mL of cells with a density of 3 x 10 6 cells / mL, add the prepared transfection system to the cells, mix well, and incubate in a 37°C, 8% CO2, 125 rpm shaker for 6 days.

[0320] 5. Antibody purification: After 6 days of culture, collect the cell culture medium, centrifuge at 3500 rpm for 15 min to remove cells and residues, and filter the supernatant with a 0.45 μm filter membrane. The filtered supernatant is adsorbed to the target molecules by Mabselect Sure LX (Cytiva, 17547401) gravity column, impurity proteins are washed with 10 times the column volume of PBS buffer, and then the target molecules are eluted and collected with 100 mM pH 3.2 sodium citrate buffer, neutralized with 2M Tris buffer, and adjusted to pH 6.0 to obtain 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 in PBMCs

[0322] I. Experimental methods

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

[0324] II. Experimental results

[0325] The results are shown in Table 17, and 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 the commercially available commercial granulocyte removal reagent.

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

[0327]

[0328] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited by the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application shall be equivalent replacement methods, and all shall be included in the protection scope of the present application.

Claims

1. A rabbit monoclonal antibody against human CD66b protein, characterized in that, the rabbit monoclonal antibody is a first antibody CD66b-108, a second antibody CD66b-116 or a third antibody CD66b-128; the first antibody CD66b-108 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 shown as 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 shown as SEQ ID NO: 3; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 5, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 6, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as SEQ ID NO: 7; 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 shown as 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 shown as SEQ ID NO: 10; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 12, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 13, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as 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 shown as 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 shown as SEQ ID NO: 17; the amino acid sequence of CDR1 of the heavy chain variable region is shown as SEQ ID NO: 19, the amino acid sequence of CDR2 of the heavy chain variable region is shown as SEQ ID NO: 20, and the amino acid sequence of CDR3 of the heavy chain variable region is shown as 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 as SEQ ID NO: 1, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 4; the amino acid sequence of the light chain variable region of the second antibody CD66b-116 is shown as SEQ ID NO: 8, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO: 11; the amino acid sequence of the light chain variable region of the third antibody CD66b-128 is shown as SEQ ID NO: 15, and the amino acid sequence of the heavy chain variable region is shown as SEQ ID NO:

18.

3. Biomaterials 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 of claim 1 or 2; a nucleic acid molecule encoding the first antibody CD66b-108, the second antibody CD66b-116 or the third antibody CD66b-128 of claim 1 or 2; an expression cassette comprising the nucleic acid molecule; a recombinant vector comprising the nucleic acid molecule; a recombinant microorganism comprising the nucleic acid molecule; a recombinant cell line comprising the 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, the variable region of the first heavy chain and the first light chain recognizes a human CD66b antigen, and the variable region of the second heavy chain and the second light chain recognizes a GYPA antigen; the amino acid sequences of the variable region of the first heavy chain and the variable region of the first light chain are one of the following combinations: the amino acid sequence of the variable region of the first heavy chain is as shown in SEQ ID NO: 4, and the amino acid sequence of the variable region of the first light chain is as 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 as shown in SEQ ID NO: 24, and the amino acid sequence of the variable region of the second light chain is as shown in SEQ ID NO:

25.

5. The bispecific antibody of claim 4, wherein The first heavy chain and the second heavy chain are provided with a connecting peptide, the second heavy chain and the second light chain are provided with a connecting peptide, and the connecting peptide is (G4S) n wherein n is an integer between 1 and 6.

6. The bispecific antibody of claim 4, wherein the bispecific antibody further comprises a heavy chain constant region and a light chain constant region.

7. The bispecific antibody of claim 4, wherein 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 as shown in SEQ ID NO: 27, and the amino acid sequence of the light chain is as 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 as shown in SEQ ID NO: 29, and the amino acid sequence of the light chain is as 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 as shown in SEQ ID NO: 31, and the amino acid sequence of the light chain is as shown in SEQ ID NO:

32.

8. A biomaterial, characterized in that, the biological material is any of the following: a nucleic acid molecule encoding the bispecific antibody of any one of claims 4-7; an expression cassette comprising the nucleic acid molecule; a recombinant vector comprising the nucleic acid molecule; a recombinant microorganism comprising the nucleic acid molecule; a recombinant cell line comprising the nucleic acid molecule.

9. Use of the rabbit monoclonal antibody of claim 1 or 2, the biological material of claim 3, the bispecific antibody of any one of claims 4-7 and / or the biological material of claim 8 in the preparation of a detection product and / or a clearance product of granulocytes.

10. Use of the rabbit monoclonal antibody according to claim 1 or 2, the biological material according to claim 3, the bispecific antibody according to any one of claims 4 to 7 and / or the biological material according to claim 8 for the manufacture of a test product and / or a clearance product for polymorphonuclear myeloid-derived suppressor cells.

11. A granulocyte detecting product and / or scavenging product, characterized by, comprising the bispecific antibody according to any one of claims 4 to 7.

12. A detection product and / or elimination product of 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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