Acute leukemia TCR-like monoclonal antibody for treating NPM1 type A mutation

Abstract

The invention discloses a TCR (T cell receptor)-like monoclonal antibody for treating NPM1 type A mutant acute leukemia, which is a recombinant protein pNPM1-HLA protein expressed by SEQ ID NO1 or SEQ ID NO2, the recombinant protein is combined with a monoclonal antibody of pNPM1-HLA-A2, the monoclonal antibody can specifically recognize the combination of the pNPM1-HLA-A2 protein on the surface of a tumor cell, the combination with a non-target peptide / HLA-A2 protein is low, and the monoclonal antibody can be used for treating NPM1 type A mutant acute leukemia. The specific recognition on the tumor cells is realized. The monoclonal antibody provided by the invention has the following advantages: (1) the monoclonal antibody 2E2 has high affinity with a target protein, and Biacore affinity detection KD (M) is 1.58 e <-7 >; and (2) the antibody can be combined with a tumor cell line for expressing pNPM1-HLA-A2. And (3) the modified antibody has an obvious killing effect on tumor cells in vitro and in vivo.

Description

Technical Field

[0001] The present invention relates to the field of tumor immunology, particularly to the field of acute leukemia treatment. Specifically, the present invention relates to antibodies for treating NPM1 A-type mutant acute leukemia, cell lines producing such antibodies, and their uses. Background Art

[0002] Acute myeloid leukemia (AML) is a group of highly heterogeneous hematological malignancies originating from hematopoietic stem / progenitor cells. Clinical manifestations include fever, anemia, bleeding, and lymph node enlargement, etc. AML accounts for about 70% of acute leukemia (AL) in adults and is the most common type of acute leukemia in adults. The occurrence of this disease is related to exposure to carcinogenic organic compounds (such as benzene, xylene, etc.) and occupational exposure (such as asphalt workers, radiation workers) before onset. However, most AML patients still have no clear cause.

[0003] The incidence rate of AML is 4 per 100,000 people and it can occur at all ages. Although conventional treatment methods including chemotherapy and allogeneic hematopoietic stem cell transplantation have greatly extended the lifespan of patients, the overall prognosis of this disease is still not optimistic, and the 5-year survival rate is about 24%. The high cost of diagnosis and treatment, high mortality rate, and the medical security burden brought by the disease make leukemia an economic and social problem that cannot be ignored in China and even worldwide. Fortunately, the development and application of targeted drugs such as FLT3 inhibitors, BCL-2 inhibitors, IDH inhibitors, and CD33 monoclonal antibodies have announced that the treatment of AML has entered the era of precision medicine. However, the targets targeted by the above-mentioned targeted drugs have poor tumor specificity. For example, CD33 exists in tumor cells as well as in normal tissue cells, which is also the main source of drug side effects. Therefore, it is urgent to find new tumor antigens with high specificity and design targeted drugs against such targets.

[0004] T cell receptor (TCR)-like antibodies, also known as TCR-like antibodies, are a new type of antibody family that can recognize peptide / HLA complexes on the surface of tumor cells. This type of antibody therapy has achieved good results in melanoma, leukemia, breast cancer, colon cancer, and prostate tumor models.

[0005] Most of the antigens targeted by traditional antibody therapies are antigens shared by normal and tumor cells, and they can only target cell surface antigens, having no effect on intracellular proteins, with poor tumor specificity. Taking rituximab (CD20 monoclonal antibody) as an example, its mechanism of action is to mediate the CDC or ADCC effect by binding to the CD20 molecule on the surface of tumor cells, thereby eliminating tumor cells. However, this monoclonal antibody can not only bind to tumor cells, but also bind to the CD20 molecule on the surface of normal B cells, thereby mediating non-specific killing and causing adverse drug reactions.

[0006] TCR-like antibodies overcome the above-mentioned drawbacks. Most tumor-specific antigens that control cell growth, proliferation, and death are present inside cells, and the mutant proteins expressed by tumor-associated mutant genes are mostly intracellular proteins. Normal cells do not contain the proteins expressed by tumor-associated mutant genes and do not have tumor-specific peptide / HLA complexes. Therefore, TCR-like antibodies can specifically recognize the specific peptide / HLA complexes presented by tumor cells and have stronger specificity compared to traditional antibody therapies.

[0007] The incidence of NPM1 mutation (ΔNPM1) in acute myeloid leukemia (AML) is as high as 30%, and type A mutation (NPM1mutA) accounts for more than 90% of them. This type of mutation is a frameshift mutation (c.860_863dupTCTG) caused by the insertion of 4 nucleotides in exon 12. The C-terminus, which originally ended with 7 amino acids (WQWRKSL), is replaced by 11 amino acids (CLAVEEVSLRK), resulting in the transfer of this protein from the nucleus to the cytoplasm and leading to the occurrence of acute leukemia. NPM1(ΔNPM1) with NPM1 mutation is an attractive immunotherapy target. Although in acute myeloid leukemia, the single NPM1 mutation is a favorable prognostic factor, this mutation is often combined with gene mutations such as FLT3-ITD and RUNX1. Such complex mutations are associated with poor disease prognosis.

[0008] Previous studies have shown that the C-terminal 9-mer peptide (CLAVEEVSL) of NPM1 mutA can be presented by HLA-A*02:01 molecules. The purpose of this study is to synthesize pNPM1-HLA-A2 as an antigen, screen specific antibodies against pNPM1-HLA-A2, and through humanization modification, design a TCR-like antibody that has a killing effect on tumor cells carrying NPM1 type A mutation, providing a new and highly effective treatment method for clinical patients. Summary of the Invention

[0009] The object of the present invention is to provide a monoclonal antibody that binds to pNPM1-HLA-A2. Specifically, such a monoclonal antibody can specifically recognize the binding of the pNPM1-HLA-A2 protein on the surface of tumor cells, while having low binding to non-target peptides / HLA-A2 proteins. Thus, specific recognition of tumor cells is achieved.

[0010] To achieve the object of the present invention, the following technical solution is adopted. A TCR-like monoclonal antibody for treating acute leukemia with NPM1 type A mutation, characterized in that its nucleotide sequence is SEQ ID NO 1, namely the nucleic acid coding sequence of pNPM1-HLA-A2-His:

[0011]

[0012] A TCR-like monoclonal antibody for treating acute leukemia with NPM1 type A mutation, characterized in that its amino acid sequence is SEQ ID NO 2, namely the pNPM1-HLA-A2-His amino acid sequence:

[0013] MARSVTLVFLVLVSLTGLYACLAVEEVSLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEGRSGHHHHHH。

[0014] The sequence after removing "l,f,e,i,q,p" is:

[0015] MARSVTVVVSTGYACAVVSGCGASGGGGSGGGGSRTKVYSRHANGKSNNCYVSGHSDVDKNGRKVHSDSSKDWSYYYTTTKDYACRVNHVTSKVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYTSVSRGRGRAVGYVDDTVRDSDAASRMRAWGYWDGTRKVKAHSTHRVDGTRGCYNSAGSHTVRMYGCDVGSDWRRGYHYAYDGKDYAKDRSWTAADMAATTKHKWAAHVARAYGTCVWRRYNGKTRTDAKTHMTHHAVSDHATRCWASYATTWRDGDTDTVTRAGDGTKWAAVVVSGRYTCHVHGKTRWGRSGHHHHHH。

[0016] Furthermore, for the recombinant protein pNPM1-HLA protein expressed by SEQ ID NO1 or SEQ ID NO2, the variable region sequence of the antibody 2E2 obtained by screening is shown in the following table:

[0017]

[0018] Furthermore, for the recombinant protein pNPM1-HLA protein expressed by SEQ ID NO1 or SEQ ID NO2, the CDR sequences of the light and heavy chain variable regions of the antibody 2E2 obtained by screening are shown in the following table:

[0019] Name CDR Sequence SEQ ID NO 2E2 VH CDR1 GYTFTDYN SEQ ID NO31 2E2 VH CDR2 INPNNGGT SEQ ID NO32 2E2 VH CDR3 ASGGYDY SEQ ID NO33 2E2 VL CDR1 QDINSY SEQ ID NO41 2E2 VL CDR2 RAN SEQ ID NO42 2E2 VL CDR3 LQYDEFPPT SEQ ID NO43

[0020] Furthermore, the role of the TCR-like monoclonal antibody for treating acute leukemia with NPM1 type A mutation in the preparation of drugs for killing and inhibiting tumor cells.

[0021] Furthermore, for the above application, it is characterized in that: the tumor cells are acute leukemia.

[0022] The monoclonal antibody provided by the present invention has the following advantages: (1) The monoclonal antibody 2E2 has a high affinity for the target protein, and the KD (M) detected by Biacore affinity is 1.58e-7. (2) This antibody can bind to the tumor cell line expressing pNPM1-HLA-A2. (3) After modification, the antibody has obvious killing effects on tumor cells in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is the SDS-PAGE electrophoresis diagram of the immunogen pNPM1-HLA protein described in the present invention.

[0024] Figure 2 It is the ELISA reactivity diagram of pNPM1-HLA, C18hA2, monoclonal antibody 2E2 and positive control antibody described in the present invention.

[0025] Figure 3 It is the affinity determination (Biacore) diagram of the 2E2 monoclonal antibody for the pNPM1-HLA protein described in the present invention.

[0026] Figure 4 It is the flow cytometry diagram of different concentrations of monoclonal antibody 2E2 binding to T2 cells described in the present invention.

[0027] Figure 5 It is the flow cytometry diagram of different concentrations of the antibody binding to the positive cell line OCI-AML3 described in the present invention.

[0028] Figure 6SDS-PAGE electrophoresis diagrams of the three modified antibodies described in the present invention.

[0029] Figure 7 Diagram of the average fluorescence intensity of cells (OCI-AML3-luc) after killing cells with modified antibodies at different concentrations described in the present invention.

[0030] Figure 8 Average fluorescence intensity of cells (HL60-luc) after killing cells with modified antibodies at different concentrations described in the present invention.

[0031] Figure 9 Experimental roadmap in vivo described in the present invention.

[0032] Figure 10 In vivo imaging data on the 3rd, 6th, and 9th days described in the present invention. Detailed implementation manners

[0033] The following further elaborates on the present invention in conjunction with the detailed implementation manners and the accompanying drawings, so that those skilled in the art can implement it based on the description in the specification. However, the present invention is not limited to the following examples, and the conditions adopted in the examples can be adjusted according to specific circumstances.

[0034] Example 1. Construction of pNPM1-HLA-A2 expression clone (His tag)

[0035] 1.1 Gene synthesis and expression clone construction of pNPM1-HLA-A2

[0036] Referring to the human HLA-A*02:01 cDNA sequence (AF036921.1) and the human b2m cDNA sequence (CR457066.1), the amino acid sequence of pNPM1 (CLAVEEVSL) and HLA-A*02:01 molecule was optimized according to the human codon usage to obtain its optimized coding nucleic acid sequence. Starting from the N-terminal signal peptide coding sequence (the leader sequence of human B2m), pNPM1, b2m, and HLA-A*02:01 were connected in sequence, and a polyhistidine polypeptide (6×His) facilitating affinity chromatography purification was connected to the C-terminus. Finally, the optimized coding sequence of pNPM1-HLA-A2-His protein (nucleic acid sequence 1, amino acid sequence 2) was obtained.

[0037] Sequence 1, Nucleic acid coding sequence of pNPM1-HLA-A2-His (SEQ ID NO 1):

[0038]

[0039] Sequence 2, the amino acid sequence of pNPM1-HLA-A2-His (SEQ ID NO: 2):

[0040] MARSVTLVFLVLVSLTGLYACLAVEEVSLGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYTEFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWDGETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLENGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEGRSGHHHHHH。

[0041] To synthesize the optimized coding sequence of the above pNPM1-HLA-A2-His, we commissioned General Biosystems to perform gene synthesis (for the synthesis method, see the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075-1085.), and ligated it to the vector EIRBsMie-C18hA2dtSCT constructed in our laboratory (after digestion with AgeI / BglII). DNA sequencing proved that the sequence was completely consistent with the design, and finally the expression vector EIRBsMie-pNPM1-HLA-A2-His of pNPM1-HLA-A2-His was obtained (i.e., the pNPM1-HLA expression clone EIRBsMie-pNPM1-HLA-A2-His with His tag). Example 2. Expression and purification of pNPM1-HLA antigen

[0042] 2.1 Expression of pNPM1-HLA antigen

[0043] 2.1.1 Preparation of expression cells

[0044] At 3x106 The density of ExpiCHO cells was adjusted to an appropriate amount of ExpiCHO medium TM Expression Medium (Thermo Scientific) and cultured in a triangular shake flask at 37 °C and 8 wt% CO 2 in a constant temperature shaker with an appropriate rotation speed for 24 h until the cell density reached 6x10 6 density.

[0045] 2.1.2 pNPM1-HLA antigen expression

[0046] According to the instructions of the kit, ExpiFectamine TM CHO Transfection Kit (Thermo Scientific) was used to transfect the His-tagged pNPM1-HLA expression clone EIRBsMie-pNPM1-HLA-A2-His obtained in Example 1 into ExpiCHO cells (suspended Chinese hamster ovary cells). After continuing to culture under the same conditions for 17-24 h, the feeding and enhancer provided in the kit were added, and the cells were transferred to a constant temperature shaker at 32 °C and 5 wt% CO 2， with an appropriate rotation speed and cultured for another 6 days.

[0047] 2.2 pNPM1-HLA antigen purification

[0048] 2.2.1 Treatment of culture supernatant

[0049] After culturing the pNPM1-HLA antigen in step 2.1.2 for 6 days, the ExpiCHO expression cell suspension was collected, centrifuged at 12,000 rpm for 30 min at room temperature, and the supernatant was retained and dialyzed into 1X PBS (0.01 M, pH = 7.4), and then filtered through a 0.22 μm filter membrane.

[0050] 2.2.2 pNPM1-HLA antigen purification

[0051] The supernatant sample that had been dialyzed into 1X PBS (0.01 M, pH = 7.4) and filtered through a 0.22 μm filter membrane in step 2.2.1 was purified by medium-pressure Ni-excel chromatography (GE medium). Impurities were removed with 30 mM imidazole, and the target protein was eluted with 250 mM imidazole. The SDS-PAGE gel pattern is as shown in Figure 1 . The results showed that the purity of the obtained pNPM1-HLA antigen was above 90%. The obtained target protein (CHO recombinant expressed pNPM1-HLA protein) was dialyzed into 1X PBS (0.01 M, pH = 7.4) buffer and stored at -20 °C.

[0052] Example 3. Immunize mice with pNPM1-HLA antigen and obtain monoclonal antibody 2E2

[0053] Objective: To obtain mouse monoclonal antibodies specific to pNPM1-HLA

[0054] 3.1 Mouse immunization

[0055] 3.1.1 Preparation of immunogen: The immunogen is the pNPM1-HLA protein recombinantly expressed by CHO obtained in Example 2. Dilute the pNPM1-HLA protein recombinantly expressed by CHO to 0.2 mg / mL, mix it with Freund's adjuvant in equal volume, and fully mix it in an injection emulsifier to form a water-in-oil emulsion. Freund's complete adjuvant is used for the primary immunization, and Freund's incomplete adjuvant is used for subsequent booster immunizations to obtain the prepared immunogen.

[0056] 3.1.2 Mouse immunization: Use the prepared immunogen above to immunize 6-8-week-old female BALB / c mice by multi-point subcutaneous injection in bilateral inguinal regions. The injection volume is 300 μL / mouse / time. Approximately 200 μL of orbital venous blood is collected before each immunization for titer determination. Booster immunization is performed once every 2 weeks after the primary immunization. The serum antibody titer is measured by indirect ELISA, and the fusion experiment is carried out 4 weeks later.

[0057] 3.2 Antibody screening

[0058] 3.2.1 Preparation before fusion: The spleen of the mouse is given a final booster immunization 72 hours before the fusion of mouse spleen cells and mouse myeloma cells (SP2 / 0). The antigen for this immunization is the adjuvant-free antigen, and the concentration is diluted to 1 mg / mL. The mouse is anesthetized with isoflurane before spleen immunization. First, cut open the outer skin and peritoneum of the mouse in turn to expose the spleen. Inject 50 μL of the antigen longitudinally along the spleen, and then immediately suture the peritoneum and the outer skin. At the same time, resuscitate mouse myeloma cells (SP2 / 0), and culture them in RPMI1640 medium containing 10% fetal bovine serum until the logarithmic growth phase for use in fusion.

[0059] 3.2.2 Preparation and screening of fused hybridomas

[0060] Take the mouse after 72 hours of spleen booster immunization obtained in step 3.2.1, take the spleen to make a cell suspension and fuse it with mouse myeloma cells SP2 / 0 to obtain hybridoma cells. And prepare feeder cells to co-culture with the hybridoma cells. Because during the culture process of hybridoma cells, a large number of unfused myeloma cells and mouse spleen cells die in RPMI1640-HAT screening medium, and a very small number of hybridoma cells are not easy to survive. Other cells must be added to make them survive. This added cell is the feeder cell. In this laboratory, mouse peritoneal macrophages and mouse thymocytes of small weeks are used as feeder cells.

[0061] 3.2.2.1 Preparation of mouse peritoneal macrophages: (i) A 6-week-old BALB / C mouse was sacrificed by cervical dislocation, disinfected in 75% alcohol for 3 - 5 min, and then placed in a laminar flow hood with the mouse's abdomen facing up. The abdominal skin of the mouse was lifted with forceps, a small incision was made, and two hemostats were used to cut open the outer skin of the mouse in the up and down directions at the incision to expose the mouse peritoneum; (ii) The peritoneum was lifted with a sterile forceps, and 5 mL of RPMI1640 medium was injected into the peritoneal cavity of the mouse with a syringe. Then the mouse was shaken to mix the medium in the peritoneal cavity, and then the medium in the peritoneal cavity was carefully aspirated with a syringe; (iii) The culture solution containing macrophages was added to the RPMI1640-HAT screening medium containing 20 wt% fetal bovine serum and mixed with the fused cells.

[0062] 3.2.2.2 Preparation of mouse thymocytes: (i) A 3-week-old BALB / C mouse was sacrificed by cervical dislocation, disinfected in 75% alcohol for 3 - 5 min, and then placed in a laminar flow hood with the mouse's abdomen facing up. The skin of the mouse's chest was lifted with forceps, a small incision was made, and two hemostats were used to cut open the outer skin of the mouse in the up and down directions at the incision to expose the inner chest skin of the mouse; (ii) Another sterile forceps was used to clamp the chest, and the chest cavity was cut open with scissors; (iii) The milky white thymus in the chest cavity was taken out with clean and sterile forceps and ground in a 70 μm cell sieve to obtain thymic feeder cells. The thymocytes were added to the RPMI1640-HAT screening medium containing 20% fetal bovine serum and mixed with the fused cells.

[0063] 3.2.2.3 Preparation of mouse myeloma cells: SP2 / 0 cells in the logarithmic growth phase were selected for fusion. Before fusion, the myeloma cells were transferred from the culture flask to a centrifuge tube and washed once with RPMI-1640 culture solution (1500 rpm × 5 min). The cells were resuspended with RPMI-1640 culture solution and counted.

[0064] 3.2.2.4 Preparation of immune mouse spleen cells: (i) The BALB / C mouse that was boost-immunized in step 3.1.2 was taken, the whole blood of the mouse was collected, and the mouse serum was collected. (ii) Then the mouse was sacrificed by cervical dislocation, disinfected in 75% alcohol for 3 - 5 min, and then placed in a laminar flow hood with the mouse lying on its right side; (iii) The abdominal cavity of the mouse was opened with sterile forceps and surgical scissors, the spleen of the mouse was cut out, and the spleen was cut into small pieces and ground in a 70 μm cell sieve to obtain spleen cells; (iv) The spleen cells were placed in a 50 mL centrifuge tube, and the adipose tissue was aspirated with a glass pipette (bent tube). Then RPMI-1640 medium was added to 30 mL, and centrifuged at 1500 rpm for 5 min, and repeated 3 times; (v) The spleen cells were resuspended with RPMI-1640 culture solution and counted.

[0065] 3.2.2.5 Preparation of hybridomas by PEG fusion: (i) Before fusion, 1 mL of PEG1450 (purchased from SIGMA) and 40 mL of RPMI1640 medium were incubated at 37 °C for later use; (ii) The prepared myeloma cells and spleen cells were mixed in a 50 mL centrifuge tube, centrifuged at 1500 rpm for 5 min, the supernatant was discarded, and the bottom of the tube was gently flicked to loosen the cells into a paste; (iii) The incubated PEG was slowly added dropwise to the cells, shaking the cells while adding, and then the cells were mixed evenly. After 1 min, the fusion was terminated with the incubated RPMI1640 medium; (iv) Centrifuged at 1500 rpm for 5 min, the cells were added to RPMI1640-HAT screening medium containing feeder cells and 20% fetal bovine serum, and then added to a 96-well plate, 200 μL per well, and placed in a 5% CO 2 incubator for culture; (v) After 5 days of culture, the RPMI1640-HAT medium with 20% fetal bovine serum was aspirated, replaced with RPMI1640-HT medium containing 10% fetal bovine serum, and continued to culture for 5 days. Then, the cell supernatant was aspirated for detection.

[0066] 3.2.2.6 Screening of hybridomas: High-throughput flow cytometry screening system based on T2 cells

[0067] (1) Peptide incubation: T2 cells were seeded in a 96-well U-bottom plate, 1×10 6 cells / 100 μL per well. 1 μg / well of the target polypeptide pNPM1 (CLAVEEVSL) and the irrelevant polypeptide pHBc (FLPSDFFPSV) were added respectively, and incubated at 37 °C for 2 h to allow the peptide to bind to the HLA-A*02:01 molecule on the surface of T2 cells.

[0068] (2) Plate washing: The U-bottom plate of T2 cells after peptide incubation was centrifuged with a plate centrifuge at 2000 rpm for 3 min, then the supernatant was discarded, washed once with 1X PBS containing 2% FBS, centrifuged at 2000 rpm for 3 min, and the supernatant was discarded for later use.

[0069] (3) Sample addition: 50 μL of each fusion supernatant was added to T2 cells incubated with the target peptide pNPM1 and the irrelevant peptide pHBc respectively, and incubated at 37 °C for 0.5 h.

[0070] (4) Plate washing: The U-bottom plate of T2 cells after incubation was centrifuged with a plate centrifuge at 2000 rpm for 3 min, then the supernatant was discarded, washed once with 1X PBS containing 2% FBS, centrifuged at 2000 rpm for 3 min, and the supernatant was discarded for later use.

[0071] (5) Secondary antibody reaction: The fluorescent secondary antibody (Anti-mouse IgG(H+L), F(ab')2Fragment(Alexa After diluting the conjugate 500-fold, add 30 μL to each well of the U-bottom plate containing T2 cells and incubate at 4°C for 0.5 h.

[0072] (6) Wash the plate: Centrifuge the U-bottom plate containing the incubated T2 cells using a plate centrifuge at 2000 rpm for 3 min, then discard the supernatant. Wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and then discard the supernatant for later use.

[0073] (7) Resuspend: Resuspend the cells with 1X PBS containing 2 wt% FBS, 50 μL per well, and place at 4°C for later use.

[0074] (8) Flow cytometry detection: Use the high-throughput mode of an analytical flow cytometer (BD LSR Fortessa X-20) to detect the fluorescence intensity of T2 cells.

[0075] 3.3 Production of mouse monoclonal antibody ascites

[0076] Take 5 BALB / C mice and sensitize them by intraperitoneal injection of 0.5 mL of liquid paraffin oil. The mice can be used 3 days after sensitization. Centrifuge the hybridoma cells in the logarithmic growth phase at 1500 rpm for 5 min, discard the supernatant, resuspend with PBS to 1 - 2×10^6 cells / mL, and inject 0.5 mL of the cell suspension into the peritoneal cavity of each mouse. After 7 days, the abdomen of the mice is significantly swollen. Decapitate the mice, open the peritoneal cavity, and carefully aspirate all the ascites in the peritoneal cavity of the mice to obtain monoclonal antibody ascites.

[0077] 3.4 Purification of monoclonal antibody ascites

[0078] After high-speed centrifugation of the monoclonal antibody ascites, take the supernatant, add an equal volume of saturated ammonium sulfate solution, precipitate on ice for 30 min, then centrifuge at 25000 rpm for 10 min. Dissolve the precipitate with 0.2 M disodium hydrogen phosphate dodecahydrate buffer, and then purify it using a Protein A affinity chromatography column (purchased from GE Healthcare, USA) to obtain purified mouse monoclonal antibody 2E2 (Table 1).

[0079] Table 1. Monoclonal antibody information

[0080]

[0081] Example 4. Amplification and sequencing of the variable region genes of the heavy and light chains of monoclonal antibody 2E2

[0082] 4.1 Hybridoma RNA extraction

[0083] The monoclonal antibody 2E2 hybridoma cells prepared in step 3.4 cultured to the logarithmic growth phase were blown up and transferred to a 15 mL centrifuge tube. The cells were collected by centrifugation at 1500 rpm for 3 min, resuspended in 200 μl of sterile PBS (pH 7.45), and transferred to a new RNase-free 1.5 ml centrifuge tube. 800 μl of Trizol solution (Invitrogen) was added, and after vigorous shaking for 30 s, it was left standing at 4°C for 10 min. 250 μl of chloroform was added, and it was vigorously shaken for 30 s and left standing at 4°C for 5 min. It was centrifuged at 12000 rpm at 4°C for 15 min, and the upper layer liquid was transferred to a new 1.5 ml centrifuge tube. An equal volume of isopropanol was added, gently inverted and mixed evenly, and left standing at 4°C for 10 min. It was centrifuged at 12000 rpm at 4°C for 10 min, the supernatant was discarded, 800 μl of 75 wt% ethanol was added, and it was gently inverted. It was centrifuged at 12000 rpm at 4°C for 5 min, the supernatant was discarded, and the precipitate was heated in a 70°C metal bath for 10 min to dry the residual ethanol. The transparent precipitate was dissolved in 50 μl of DEPC H 2 O.

[0084] 4.2 RNA reverse transcription to cDNA

[0085] Prepare the light / heavy chain variable region reverse transcription systems respectively according to the following system, see Table 2:

[0086] Table 2

[0087]

[0088] The reverse transcription downstream primer for the heavy chain variable region is MuIgGVH3'-2, and the reverse transcription downstream primer for the light chain variable region is MuIgκVL3'-1. The prepared reverse transcription system was placed at 42°C for 40 min to obtain the 2E2 light / heavy chain variable region cDNA.

[0089] 4.3 Antibody variable region gene amplification and sequencing

[0090] Amplification of the variable region gene of the 2E2 heavy chain (2E2 heavy chain variable region cDNA): Using the above reverse-transcribed cDNA as a template, MuIgVH5'-E1 as the upstream primer, and MuIgGVH3'-2 as the downstream primer, PCR amplification was performed to obtain a DNA fragment approximately 450 bp in size. The PCR conditions were: 95°C for 5 min, 25 cycles of (95°C for 30 s, 56°C for 30 s, 72°C for 30 s), and 72°C for 5 min. After recovery of the PCR product, sequencing was carried out. The sequence was determined to be the variable region sequence of the 2E2 heavy chain after blast alignment. Amplification of the variable region gene of the 2E2 light chain (2E2 light chain variable region cDNA): Using the corresponding cDNA as a template, MuIgκVL5'-D2 as the upstream primer, and MuIgκVL3'-1 as the downstream primer, PCR amplification was performed to obtain a DNA fragment approximately 450 bp in size. The PCR conditions were: 95°C for 5 min, 25 cycles of (95°C for 30 s, 56°C for 30 s, 72°C for 30 s), and 72°C for 5 min. After recovery of the PCR product, sequencing was carried out. The sequence was determined to be the light chain sequence of 2E2 after blast alignment, as shown in Table 3.

[0091] Table 3. Primers used for amplification of variable region genes

[0092]

[0093] After sequencing, the amino acid sequences of the variable regions of the 2E2 heavy and light chains were determined as shown in Table 4:

[0094] Table 4. Amino acid sequences of the variable regions of the heavy and light chains of antibody 2E2

[0095]

[0096] Furthermore, the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647 - 669) was also used to determine the CDR sequences of the mouse monoclonal antibody 2E2. The amino acid sequences of the CDRs of the variable regions of the 2E2 heavy and light chains are shown in Table 5.

[0097] Table 5. Amino acid sequences of the CDRs of the variable regions of the heavy and light chains of antibody 2E2

[0098] Name CDR Sequence SEQ ID NO 2E2 VH CDR1 GYTFTDYN SEQ ID NO31 2E2 VH CDR2 INPNNGGT SEQ ID NO32 2E2 VH CDR3 ASGGYDY SEQ ID NO33 2E2 VL CDR1 QDINSY SEQ ID NO41 2E2 VL CDR2 RAN SEQ ID NO42 2E2 VL CDR3 LQYDEFPPT SEQ ID NO43

[0099] Example 5. ELISA binding activity of monoclonal antibody 2E2 to pNPM1-HLA and C18hA2 (pHBc-HLA) 5.1 Expression and purification of C18hA2 protein

[0100] Referring to the HBV virus genome sequence (LC488828.1) published on GenBank, the pNPM1 sequence of the expression vector EIRBsMie-pNPM1-HLA-A2-His in Example 1 was replaced with the HBc protein 18-27 sequence (i.e., the expression vector EIRBsMie-pNPM1-HLA-A2-His) to obtain the expression vector of pHBc-HLA-A2-His, which was named EIRBsMie-C18hA2-His. Then, referring to the method of Example 2, the recombinant protein was prepared, and the obtained recombinant protein was named C18hA2.

[0101] 5.2 Preparation of reaction plate

[0102] The monoclonal antibody 2E2 obtained in Example 3 and the anti-HLA-A2 antibody CS-102 (anti-HLA-A2) were diluted with 200 mM PB buffer (NaH 2 PO 4 / Na 2 HPO 4 buffer, final concentration of 200 mM, pH value of 7.4) to a final concentration of 1 μg / mL; 100 μL of coating solution was added to each well of a 96-well ELISA plate, coated at 2-8 °C for 16-24 hours and then at 37 °C for 2 hours; washed once with PBST washing solution (20 mM PB 7.4, 150 mM NaCl, 0.1% Tween 20); then 200 μL of blocking solution (20 mM Na 2 HPO 4 / NaH 2 PO 4 buffer solution containing 20 wt% calf serum and 1 wt% casein, pH value of 7.4) was added to each well, and blocked at 37 °C for 2 hours; the blocking solution was discarded. After drying, it was packed into an aluminum foil bag and stored at 2-8 °C for later use.

[0103] 5.3 ELISA detection of mouse monoclonal antibody 2E2 (double antibody sandwich method)

[0104] The pNPM1-HLA protein recombinantly expressed in CHO obtained in Examples 2 and 5 (abbreviation: pNPM1-HLA) and the C18hA2 obtained in step 5.1 were serially diluted with 1X PBS solution to 7.40 μg / mL, 2.47 μg / mL, 0.82 μg / mL, 0.27 μg / mL, 0.09 μg / mL, and ELISA detection was performed according to the following steps:

[0105] (1) Sample reaction: Take the enzyme-labeled plates coated with monoclonal antibody 2E2 and CS-102 antibody respectively, add 100 μL of the diluted sample to each well, and place it in an incubator at 37 °C for 30 minutes for reaction.

[0106] (2) Enzyme-labeled reagent reaction: After completing the sample reaction step, wash the enzyme-labeled plates 5 times with PBST washing solution (20 mM PB 7.4, 150 mM NaCl, 0.1 wt% Tween 20), add 100 μL of HRP-labeled goat anti-His reaction solution to each well, and place it in an incubator at 37 °C for 30 minutes for reaction.

[0107] (3) Color reaction: After completing the enzyme-labeled reagent reaction step, wash the enzyme-labeled plates 5 times with PBST washing solution (20 mM PB 7.4, 150 mM NaCl, 0.1 wt% Tween 20), add 50 μL of TMB color reagent (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) to each well, and place it in an incubator at 37 °C for 15 minutes for reaction.

[0108] (4) Termination reaction and reading measurement: After completing the color reaction step, add 50 μL of termination solution (purchased from Beijing Wantai Biological Pharmacy Co., Ltd.) to each well of the reacted enzyme-labeled plates, and detect the OD450 / 630 values of each well on an enzyme-labeling instrument.

[0109] Determination of the reactivity of mouse monoclonal antibody 2E2 with pNPM1-HLA and C18hA2: Make a determination according to the readings after the reaction. If the detection value / background value is greater than 5, it is determined as positive.

[0110] (5) Result analysis: The results are shown as Figure 2 , pNPM1-HLA has strong binding activity with 2E2 antibody at concentrations of 7.40 μg / mL, 2.47 μg / mL, 0.82 μg / mL, 0.27 μg / mL, and 0.09 μg / mL; although the control protein C18hA2 can bind to 2E2, at the same concentration, its binding strength is weaker than that of pNPM1-HLA. The results show that 2E2 antibody can recognize the pNPM1-HLA complex well.

[0111] Example 6. Biacore affinity determination of monoclonal antibody 2E2 for pNPM1-HLA recombinant protein

[0112] In this study, the surface plasmon resonance (SPR) technique was used to detect the affinity between the monoclonal antibody 2E2 obtained in Example 3 and pNPM1-HLA. The detection method used in this example was the capture method, and a Protein G chip (GE) was used to capture the murine monoclonal antibody.

[0113] 6.1 Determination of ligand response unit (RU)

[0114] The calculation formula for the ligand coupling level is shown in the following figure. According to the molecular weight of the murine monoclonal antibody and the molecular weight of the pNPM1-HLA analyte, it can be calculated that the ligand response value is about 1000 RU.

[0115]

[0116] R L = ligand coupling level

[0117] R max = describes the maximum binding capacity of the chip surface. For low kinetic coupling, R max ≤100

[0118] S m = stoichiometric ratio (Analyte:Ligand, select S when unknown m = 1)

[0119] 6.2 Affinity detection

[0120] The pNPM1-HLA antigen was serially diluted 2-fold starting from an initial concentration of 3200 nM, and a surface plasmon resonance detector Biacore 8000 (GE) was used to detect the antigen-antibody affinity. The results are as Figure 3 shown.

[0121] 6.3 Result analysis

[0122] The 2E2 monoclonal antibody has good affinity for the pNPM1-HLA antigen, and the dissociation constant Kd = 158 nM.

[0123] Example 7. Monoclonal antibody 2E2 cell binding experiment

[0124] 7.1 In vitro binding experiment of monoclonal antibody 2E2 to T2 cells

[0125] Resuscitate T2 cells and culture them in 1640 medium containing 10 wt% fetal bovine serum until the logarithmic growth phase. Seed the T2 cells on a 10 cm cell culture plate at a density of 2×10 6 , and place them in a 5 wt% CO 2 cell culture incubator at 37 °C for culture, for use in the following experiments:

[0126] (1) Peptide incubation: Seed T2 cells in a 96-well U-bottom plate at a density of 1×10 6 cells / 100 μL. Add 1 μg / well of the target peptide pNPM1 and the irrelevant peptide HBc respectively, and incubate at 37 °C for 2 h to allow the peptides to bind to the HLA-A*02:01 molecules on the surface of T2 cells.

[0127] (2) Plate washing: Centrifuge the T2 cells incubated with peptides at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2 wt% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0128] (3) Dilution: Dilute the 2E2 antibody with 1X PBS containing 2 wt% FBS to 50 μg / mL, 10 μg / mL, 2 μg / mL, and 0.4 μg / mL for later use.

[0129] (4) Sample addition: Add 50 μL of the diluted antibody above to the T2 cells incubated with the target peptide pNPM1 and the irrelevant peptide HBc respectively, and incubate at 37 °C for 0.5 h.

[0130] (5) Plate washing: Centrifuge the U-bottom plate of the T2 cells incubated at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2 wt% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0131] (6) Secondary antibody reaction: Dilute the fluorescent secondary antibody 500-fold and add 30 μL to each well of the U-bottom plate of T2 cells, and incubate at 4 °C for 0.5 h.

[0132] (7) Plate washing: Centrifuge the T2 cells incubated at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2 wt% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0133] (8) Resuspension: Resuspend the cells with 1X PBS containing 2 wt% FBS at 50 μL per well and place at 4 °C for later use.

[0134] (9) Flow cytometry detection: Use the high-throughput mode of an analytical flow cytometer (BD LSRFortessa X-20) to detect the fluorescence intensity of T2 cells.

[0135] The binding of the monoclonal antibody 2E2 to the two types of T2 cells is as Figure 4As shown, the red represents T2 cells incubated with NPM1 polypeptide; the blue represents T2 cells incubated with control polypeptide. As shown in the figure, when the concentration of monoclonal antibody 2E2 is 10 ug / mL, 2 ug / mL, and 0.4 ug / mL, its binding strength to T2 cells of the positive peptide is higher than that of T2 cells of the control peptide, up to more than 8 times at most.

[0136] 7.2 In vitro binding experiment of monoclonal antibody 2E2 to positive cell line OCI-AML3 cells

[0137] The OCI-AML3 cell line is a human acute myelomonocytic leukemia (M4) cell line. It carries the NPM1 A-type mutation and is HLA-A2 positive. Therefore, the pNPM1-HLA complex is naturally expressed on its cell surface and can be regarded as a positive cell line.

[0138] Resuscitate OCI-AML3 cells and culture them in RPMI 1640 medium containing 20% fetal bovine serum until the logarithmic growth phase. Seed OCI-AML3 cells on a 10 cm cell culture plate at a density of 2×10 6 and place them in a 5% CO 2 cell incubator at 37 °C for the following experiments:

[0139] (1) Dilution: Dilute the 2E2 antibody and the control antibody 3D12 (anti-SARS-CoV-2) with 1X PBS containing 2% FBS to 0.8 ug / mL, 0.16 ug / mL, 0.032 ug / mL, 0.0064 ug / mL, and 0.00128 ug / mL for later use.

[0140] (2) Sample addition: Add 100 uL of the diluted antibody mentioned above to OCI-AML3 cells at a cell density of 1x10 6 / mL and a volume of 100 uL, and incubate at 37 °C for 0.5 h.

[0141] (5) Plate washing: Centrifuge the OCI-AML3 cells incubated at 2000 rpm for 3 min, then discard the supernatant. Wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and then discard the supernatant for later use.

[0142] (6) Secondary antibody reaction: Dilute the fluorescent secondary antibody 500-fold and add 30 uL to each well of OCI-AML3 cells, and incubate at 4 °C for 0.5 h.

[0143] (7) Plate washing: Centrifuge the OCI-AML3 cells incubated at 2000 rpm for 3 min, then discard the supernatant. Wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and then discard the supernatant for later use.

[0144] (8) Resuspension: Resuspend the cells with 1X PBS containing 2 wt% FBS, 50 μL per well, and store at 4 °C for later use.

[0145] (9) Flow cytometry detection: Use the high-throughput mode of an analytical flow cytometer (BD LSRFortessa X-20) to detect the fluorescence intensity of OCI-AML3 cells.

[0146] The binding of the 2E2 antibody to OCI-AML3 cells is as Figure 5 shown. Red represents the monoclonal antibody 2E2; blue represents the control antibody 3D12. As shown in the figure, at each concentration, the binding intensity of the monoclonal antibody 2E2 is higher than that of the control antibody.

[0147] Example 8. Construction of the expression clone of the 2E2 engineered antibody

[0148] 8.1 Gene synthesis and expression clone construction of 2E2-GAALIE

[0149] Optimize the amino acid sequences of 2E2 VH and 2E2 VL according to human codons to obtain their optimized coding nucleic acid sequences. Starting from the N-terminal signal peptide coding sequence, connect 2E2 VL and CL-h in sequence, and finally obtain the optimized coding sequence of the 2E2 VL1 protein (nucleic acid sequence 3 - SEQ ID NO3, amino acid sequence 4 - SEQ ID NO3); starting from the N-terminal signal peptide coding sequence, connect 2E2 VH, human IgG1, and perform Fc-enhancing point mutation (GAALIE), and finally obtain the optimized coding sequence of the 2E2 VH1 protein (nucleic acid sequence 5 - SEQ ID NO5, amino acid sequence 6 - SEQ ID NO6)

[0150] Sequence 3 - SEQ ID NO3, the nucleic acid coding sequence of 2E2 VL1:

[0151] ATGTCTGTGCCAACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCAAGATGTGACATCGTGCTGACCCAGAGCCCCGCCAGCCTGGCCGTGAGCCTGGGCCAGCGCGCCACCATCAGCTACCGCGCCAGCAAGAGCGTGAGCACCAGCGGCTACAGCTACATGCACTGGAACCAGCAGAAGCCCGGCCAGCCCCCCCGCCTGCTGATCTACCTGGTGAGCAACCTGGAGTGCGGCGTGCCCCCCCGCTTCAGTGGCCGCGGCTGCGGCACCGACTTCACCCTGAACATCCACCCCGTGGAGGAGGAGGACGCCGCCACCTACTACTGCCAGCACATCCGCGAGTTCACCCGCAGCGAGGGCGGCGGCACCAAGCTcGAgATAAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAGCGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA。

[0152] Sequence 4 - SEQ ID NO4, 2E2 VL1 amino acid sequence:

[0153] MSVPTQVLGLLLLWLTDARCDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLECGVPPRFSGRGCGTDFTLNIHPVEEEDAATYYCQHIREFTRSEGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDSALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC。

[0154] Sequence 5 - SEQ ID NO5, 2E2 VH1 nucleic acid coding sequence:

[0155]

[0156] Sequence 6 - SEQ ID NO6, Amino acid sequence of 2E2 VH1:

[0157] MGRLTSSFLLLIVPAYVLSEVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCASGGYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK。

[0158] To synthesize the above optimized coding sequences, we commissioned General Biosystems to perform gene synthesis (for the synthesis method, see the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075 - 1085.). The above two sequences were ligated to the vector EIRBdMie - E5F2LH constructed in our laboratory. DNA sequencing proved that the sequences were exactly the same as the design, and finally the expression vector EIRBdMie - 2E2 - GAALIE of 2E2 - GAALIE was obtained.

[0159] 8.2 Gene synthesis and expression clone construction of 2E2 - H11vhh

[0160] Starting from the N - terminal signal peptide coding sequence, 2E2 VH, human IgG1, linker (G4Sx4), and 3D - H11 anti - CD3 nanobody (anti - CD3) were ligated in sequence, and finally the optimized coding sequence of 2E2 VH - anti - CD3 protein (nucleic acid sequence 7, amino acid sequence 8) was obtained.

[0161] Sequence 7 - SEQ ID NO7, 2E2 VH-anti-CD3 nucleic acid coding sequence:

[0162]

[0163] Sequence 8 - SEQ ID NO8, 2E2 VH-anti-CD3 amino acid sequence:

[0164] MGRLTSSFLLLIVPAYVLSEVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCASGGYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGFTFDDYGMSWVRQAPGKWLEWVSDISWNGGSTYYADSVKGRFTISRDNAENTLYLQMNSLKPDDTAVYYCAKMGEGGWGANDYWGQGTQVTVSS.

[0165] To synthesize the above optimized coding sequence, we commissioned General Biosystems to perform gene synthesis (for the synthesis method, see the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075 - 1085.). The above sequence and sequence 3, 2E2 VL1 sequence were ligated to the vector EIRBdMie-E5F2LH constructed in our laboratory. DNA sequencing proved that the sequence was completely consistent with the design, and finally the expression vector EIRBdMie-2E2-H11vhh of the bispecific antibody 2E2-H11vhh was obtained.

[0166] 8.3 2E2-Cam16vhh gene synthesis and expression clone construction

[0167] Starting from the N-terminal signal peptide coding sequence, 2E2 VH, human IgG1, linker (G4Sx4), and Cam16 (anti-CD16) are successively connected, and finally the optimized coding sequence of 2E2 VH-anti-CD16 protein (nucleic acid sequence 9, amino acid sequence 10) is obtained

[0168] Sequence 9 - SEQ ID NO9, the nucleic acid coding sequence of 2E2 VH-anti-CD16:

[0169]

[0170] Sequence 10 - SEQ ID NO10, 2E2 VH - anti - CD16 amino acid sequence:

[0171] MGRLTSSFLLLIVPAYVLSEVLLQQSGPELVKPGASVKIPCKASGYTFTDYNMDWVKQSHGKSLEWIGDINPNNGGTIYNQKFKGKATLTVDKSSSTAYMELRSLTSEDTAVYYCASGGYDYWGQGTTLTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGAGGGGSGGGGSGGGGSGGGGSGQVQLVESGGGLVQPGGSLRLSCAASGLTFSSYNMGWFRQAPGQGLEAVASITWSGRDTFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAANPWPVAAPRSGTYWGQGTLVTVSS.

[0172] To synthesize the above - optimized coding sequence, we commissioned General Biosystems to perform gene synthesis (for the synthesis method, see the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075 - 1085.). The above - mentioned sequence and the sequence 3, 2E2VL1 sequence were ligated to the vector EIRBdMie - E5F2LH constructed in our laboratory. DNA sequencing proved that the sequence was completely consistent with the design, and finally the expression vector EIRBdMie - 2E2 - Cam16vhh of the bispecific antibody (BiTEs) 2E2 - Cam16vhh was obtained.

[0173] Example 9. Expression and purification of the 2E2 - modified antibody

[0174] 9.1 Expression of 2E2-GAALIE antibody

[0175] 9.1.1 Preparation of expression cells

[0176] Seed ExpiCHO cells at a density of 3x10 6 in an appropriate amount of ExpiCHO TM Expression Medium (Thermo Scientific) in a triangular shake flask, and culture in a constant temperature shaker at 37°C, 8 wt% CO 2 at an appropriate rotation speed for 24 h until the cell density reaches 6x10 6 density.

[0177] 9.1.2 Expression of 2E2-GAALIE antibody antigen

[0178] Use ExpiFectamine TM CHO Transfection Kit (Thermo Scientific) to transfect the 2E2-GAALIE expression clone EIRBdMie-2E2-GAALIE obtained in Example 8 into ExpiCHO cells, and continue to culture under the same conditions for 17 - 24 h, then add the feeding and enhancer provided in the kit, and transfer the cells to a constant temperature shaker at 32°C, 5 wt% CO 2， at an appropriate rotation speed, and continue to culture for 6 days.

[0179] 9.2 Purification of 2E2-GAALIE antibody

[0180] 9.2.1 Treatment of culture supernatant

[0181] After culturing for 6 days, collect the ExpiCHO expression cell suspension, centrifuge at 12,000 rpm at room temperature for 30 min, collect the supernatant and dialyze it into PBS, and then filter it through a 0.22 μm filter membrane.

[0182] 9.2.2 Purification of 2E2-GAALIE antibody

[0183] Purify the filtered supernatant with a Protein A affinity chromatography column (purchased from GE Healthcare, USA) to obtain the purified antibody. The SDS-PAGE gel pattern is as shown in Figure 6 , and the results show that the purity of the obtained 2E2-GAALIE antibody is above 90%. Dialyze the obtained target antibody into PBS buffer and store it at -20°C.

[0184] 9.3 Expression of 2E2-H11vhh antibody

[0185] 9.3.1 Expression cell preparation

[0186] Seed ExpiCHO cells in an appropriate amount of ExpiCHO 6 Expression Medium (Thermo Scientific) at a density of 3×10 TM in a triangular shake flask, and culture in a constant temperature shaker at 37 °C, 8% CO 2 with an appropriate rotation speed for 24 h until the cell density reaches 6×10 6 density.

[0187] 9.3.2 2E2-H11vhh antibody-antigen expression

[0188] Use ExpiFectamine TM CHO Transfection Kit (Thermo Scientific) to transfect the 2E2-H11vhh expression clone EIRBdMie-2E2-H11vhh obtained in Example 8 into ExpiCHO cells, continue to culture under the same conditions for 17 - 24 h, then add the feeding and enhancer provided in the kit, and transfer the cells to a constant temperature shaker at 32 °C, 5 wt% CO 2， with an appropriate rotation speed, and continue to culture for 6 days.

[0189] 9.4 2E2-H11vhh antibody purification

[0190] 9.4.1 Culture supernatant treatment

[0191] After culturing for 6 days, collect the ExpiCHO expression cell suspension, centrifuge at 12,000 rpm at room temperature for 30 min, retain the supernatant and dialyze it into PBS, and then filter it through a 0.22 μm filter membrane.

[0192] 9.4.2 2E2-H11vhh antibody purification

[0193] Purify the filtered supernatant with a Protein A affinity chromatography column (purchased from GE, USA) to obtain the purified antibody. The SDS-PAGE gel diagram is as Figure 6 , and the results show that the purity of the obtained 2E2-H11vhh antibody is above 90%. Dialyze the obtained target antibody into PBS buffer and store it at -20 °C.

[0194] 9.5 2E2-Cam16vhh antibody expression

[0195] 9.5.1 Expression cell preparation

[0196] At a density of 3×10 6 , ExpiCHO cells were cultured in an appropriate amount of ExpiCHO TM Expression Medium (Thermo Scientific) in a triangular shake flask at 37 °C and 8% CO 2 in a constant temperature shaker with an appropriate rotation speed for 24 h until the cell density reached 6×10 6 .

[0197] 9.5.2 Expression of 2E2-Cam16vhh antibody antigen

[0198] According to the instructions of the kit, ExpiFectamine TM CHO Transfection Kit (Thermo Scientific) was used to transfect the 2E2-Cam16vhh expression clone EIRBdMie-2E2-Cam16vhh obtained in Example 8 into ExpiCHO cells. After continuing to culture for 17 - 24 h under the same conditions, the feeding and enhancer provided in the kit were added, and the cells were transferred to a constant temperature shaker at 32 °C and 5 wt% CO 2， with an appropriate rotation speed and cultured for another 6 days.

[0199] 9.6 Purification of 2E2-Cam16vhh antibody

[0200] 9.6.1 Treatment of culture supernatant

[0201] After culturing for 6 days, the ExpiCHO expression cell suspension was collected, centrifuged at 12,000 rpm at room temperature for 30 min, the supernatant was retained and dialyzed into PBS, and then filtered through a 0.22 μm filter membrane.

[0202] 9.6.2 Purification of 2E2-Cam16vhh antibody

[0203] The filtered supernatant was purified using a Protein A affinity chromatography column (purchased from GE Healthcare, USA) to obtain the purified antibody. The SDS-PAGE gel pattern was as Figure 6 . The results showed that the purity of the obtained 2E2-Cam16vhh antibody was above 90%. The obtained target antibody was dialyzed into PBS buffer and stored at -20 °C.

[0204] Example 10. In vitro killing experiment of 2E2 modified antibody

[0205] 10.1 Lentiviral transfection of cells

[0206] 10.1.1 Lentiviral transfection of suspension cells OCI-AML3 (HLA-A2+, NPM1 mutA+)

[0207] (1) Add polybrene to 6 μg / mL and an appropriate amount of virus (carrying luciferase gene, blasticidin resistance gene, and RFP670 fluorescent protein gene) to 2×10 5 / mL suspension cells, and mix well. Centrifuge at 150 g and 32 °C for 4 h.

[0208] (2) After 4 h (or after centrifugation), add an equal volume of fresh medium to dilute polybrene.

[0209] (3) Continue culturing. Since the lentivirus carries RFP670 and blasticidin resistance genes, the transfection efficiency can be observed under a fluorescence microscope 72 h after transfection, and blasticidin resistance screening can be added according to the transfection efficiency.

[0210] (4) Separate and amplify positive cells by fluorescence-activated cell sorting (FACS) technology, and name them OCI-AML3-luc.

[0211] 10.1.2 Lentiviral transfection of suspension cells HL60 (HLA-A2+, NPM1 mutA-)

[0212] (1) Add polybrene to 6 μg / mL and an appropriate amount of virus (carrying luciferase gene, blasticidin resistance gene, and RFP670 fluorescent protein gene) to 2×10 5 / mL suspension cells, and mix well. Centrifuge at 150 g and 32 °C for 4 h.

[0213] (2) After 4 h (or after centrifugation), add an equal volume of fresh medium to dilute polybrene.

[0214] (3) Continue culturing. Since the lentivirus carries RFP670 and blasticidin resistance genes, the transfection efficiency can be observed under a fluorescence microscope 72 h after transfection, and blasticidin resistance screening can be added according to the transfection efficiency.

[0215] (4) Separate and amplify positive cells by fluorescence-activated cell sorting (FACS) technology, and name them HL60-luc.

[0216] 10.2 Isolation of PBMC

[0217] (1) Add RPMI 1640 with 2 wt% FBS to 6 mL;

[0218] (2) Take 3.5 mL of Ficoll into a 15 mL cell separation tube (SepMate TM tube). After mixing the blood sample, carefully add it to the 15 mL cell separation tube (SepMate TM tube) containing Ficoll;

[0219] (3) Centrifuge: at 25 °C, 1200 g, for 15 min, with both the acceleration and deceleration rates being 9. Pour the supernatant into a new 15 mL centrifuge tube, and add 6 mL of RPMI 1640 with 2 wt% FBS. If the density gradient medium above the SepMate tube turns red after centrifugation (i.e., some red blood cells do not precipitate), then centrifuge the SepMate centrifuge tube at 1200 g for another 10 min with the centrifuge brake on;

[0220] (4) Centrifuge: at 300 g, for 8 min, at room temperature, with both the acceleration and deceleration rates being 9. Discard the supernatant, resuspend with 5 mL of RPMI1640 with 2 wt% FBS, and mix the cells from the same specimen (each centrifuge tube is 5 mL, after mixing the same sample, each sample is 10 mL);

[0221] (5) Centrifuge: at 300 g, for 8 min, at room temperature, with both the acceleration and deceleration rates being 9;

[0222] (6) Discard the supernatant, resuspend with 1 mL of RPMI 1640 medium with 10 wt% FBS, and count.

[0223] 10.3 Antibody - PBMC in vitro killing

[0224] (1) After resuspending and counting OCI - AML3 - luc and HL60 - luc cells, seed them in a 96 - well cell culture plate at a density of 1×10 5 cells / 100 μL.

[0225] (2) After diluting PBMC, add it to all cell wells except the cell wells, 1×10 6 cells / 50 μL per well, so that the effector - to - target ratio reaches 10:1 (E / T = 10).

[0226] (3) Gradient - dilute the 2E2 engineered antibodies (2E2 - CAM16vhh, 2E2 - GAALIE, 2E2 - H1vhh) and the control antibody 3C12 (human IgG1 antibody against the SARS - CoV - 2 S protein), with the final concentration in the first well being 10 μg / mL, and perform 2 - fold dilution, with 3 replicates per well.

[0227] (4) Place the above 96 - well cell culture plate in an incubator at 37 °C and 5% CO 2 for 16 h.

[0228] (5) After 16 h, add luciferase substrate to the 96-well cell plate and react at 37 °C for 20 min in the dark. Read the fluorescence intensity on the machine. The data results are as Figure 7 、 8 , and the results show that the 2E2 modified antibody can kill the positive cell line OCI-AML3-luc at various concentrations, while the control cell line shows no obvious killing effect at each concentration.

[0229] Example 11. In vivo killing experiment of 2E2 modified antibody

[0230] 11.1 Construction of tumor cell line xenograft model (CDX)

[0231] On day 0, take the OCI-AML3-luc cell line in the logarithmic growth phase. After washing, resuspending, counting, and adjusting the volume with PBS (cell density 2×10 6 cells / 200 μL), inject it into NCG mice via the tail vein.

[0232] 11.2 In vivo killing experiment of 2E2 modified antibody

[0233] (1) Refer to Example 10.2 to isolate human PBMC. After washing, resuspending, counting, and adjusting the volume with PBS (1×10 7 cells / 200 μL), keep it at 4 °C for standby.

[0234] (2) Dilute the modified antibodies 2E2-CAM16vhh, 2E2-GAALIE, 2E2-H11vhh and the irrelevant antibody 3C12 at 20 mg / kg and keep them at 4 °C for standby.

[0235] (3) On day 3, inject the above modified antibodies and human PBMC into CDX mice via the tail vein.

[0236] (4) Perform in vivo imaging to observe the growth of tumor cells in mice.

[0237] The experimental steps are as Figure 9 shown, and in vivo imaging is performed on days 3, 6, and 9. The imaging results are as Figure 10 shown. It can be seen from the figure that the three modified antibodies can significantly inhibit the growth of tumor cells in mice.

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