TCR-like monoclonal antibody for treating cytomegalovirus infection and application thereof

Abstract

The invention relates to a TCR-like monoclonal antibody for treating cytomegalovirus infection and application thereof, and relates to the technical field of antiviral immunology, the TCR-like monoclonal antibody for treating cytomegalovirus infection comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, and the amino acid sequences are shown as SEQ ID NO: 5-10. The monoclonal antibody 3D7 has high affinity with a target protein, and the affinity detection KD (M) is 1.86 e <-9 >; the antibody can be combined with CMV infected cells; the modified antibody has an obvious killing effect on CMV infected cells in vitro and in vivo.

Description

Technical Field

[0001] The present invention relates to the technical field of antiviral immunology, and in particular to a TCR-like monoclonal antibody for treating cytomegalovirus infection and an application thereof. Background Art

[0002] Cytomegalovirus (CMV) is ubiquitous in nature, and approximately 60-90% of adults worldwide have been infected with CMV. People with normal immune function rarely develop symptoms after the initial infection, and the virus usually remains inactive or latent for life. However, in the transplant state, due to the weakening of the host's immunity by pretreatment chemoradiotherapy and the use of immunosuppressants, immune reconstitution is slow, and CMV infection or reactivation is very likely to occur, causing CMV viremia or CMV end-organ disease, manifested as interstitial pneumonia, hepatitis, gastroenteritis, hemorrhagic cystitis, retinitis, and secondary poor implantation, and other multi-system and multi-organ damage, which is an important cause of transplant failure and patient death, and is also a scientific problem and clinical problem that needs to be solved urgently.

[0003] CMV infection or reactivation mainly occurs in the early stage after transplantation (<100 days). In the late stage of transplantation, it may also be delayed in immune reconstitution and repeated CMV reactivation due to graft-versus-host disease (GVHD) and intensive immunosuppressive therapy, or due to the inhibitory effect of CMV itself and anti-CMV drugs on hematopoiesis, resulting in persistent infection. The ultimate goal of treating CMV infection is to reduce viral load, avoid repeated viral reactivation, and prevent or alleviate the occurrence of CMV end-organ lesions. Currently available standard anti-CMV drugs such as ganciclovir (GCV) and acyclovir are all viral inhibitors that cannot completely eliminate the virus and are often ineffective for high-load viruses and symptomatic infections; preventive, preemptive or maintenance treatment methods may induce drug resistance relapse; and the side effects of bone marrow suppression of drugs will further aggravate immune deficiency and increase the incidence and mortality of late CMV infection. Although some new anti-CMV drugs have been launched in recent years, such as Maribavir, Letermovir, CMX-001, etc., there is no large-sample clinical research data to confirm their efficacy and safety. This shows that the existing anti-CMV treatment methods are very limited and the efficacy is not ideal. It is urgent to find new anti-CMV drugs. Specific targeting and elimination of CMV-infected cells may be a new strategy to improve the therapeutic effect.

[0004] CD8-based anti-tumor immunotherapy + The success of T cell enhancement strategies in some cancers provides a possible approach for developing new anti-CMV therapies. +T lymphocytes recognize peptide-HLA (pHLA) complexes on the surface of target cells through their T cell receptors (TCR). When TCR binds tightly to pHLA complexes, intracellular signaling pathways are activated, leading to the release of cytokines and cytolytic molecules, and ultimately killing the target cells. For CMV-infected cells, viral proteins in the cells are degraded into short peptides by proteasomes, which are then presented by HLA-I class molecules to form pHLA complexes required for TCR recognition. In addition to TCR recognition, TCR-like antibodies can also specifically recognize these pHLA complexes. TCR-like antibodies are a new family of antibodies that can recognize peptide / HLA complexes on the cell surface. This antibody therapy has shown good results in models such as influenza virus and hepatitis B virus. Traditional antibody therapy mainly targets cell surface antigens, cannot affect intracellular proteins, and has poor specificity. TCR-like antibodies overcome these shortcomings. Most CMV-infected virus-specific antigens are located inside cells, while normal cells do not express virus-related genes and do not have virus-specific peptide / HLA complexes. Therefore, TCR-like antibodies can specifically recognize peptide / HLA complexes presented by these cells, and in comparison, have higher specificity.

[0005] In CMV-seropositive hosts, matrix protein pp65 is one of the most frequently immune-recognized CMV antigens, accounting for approximately + T cells (CTL) account for 70-90% of the response to CMV. Previous studies have shown that CMV-pp65 495-503 The 9-peptide (NLVPMVATV) (pCMV-pp65) can be presented by HLA-A*02:01 molecules. Therefore, CMV-pp65 is a potential immunotherapy target. In view of this, the present invention provides a TCR-like monoclonal antibody for treating cytomegalovirus infection and its application. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a TCR-like monoclonal antibody for treating cytomegalovirus infection and its application. The purpose is to specifically recognize the CMV-pp65-HLA protein binding on the surface of virus-infected cells, while having low binding with non-target peptide-HLA proteins, thereby achieving specific recognition of CMV-infected cells and providing a new and efficient treatment method for clinical patients.

[0007] The technical solution of the present invention to solve the above technical problems is as follows:

[0008] In a first aspect, the TCR-like monoclonal antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VLCDR2 and VL CDR3;

[0009] The amino acid sequence of the VH CDR1 is shown in SEQ ID NO: 5;

[0010] The amino acid sequence of the VH CDR2 is shown in SEQ ID NO: 6;

[0011] The amino acid sequence of the VH CDR3 is shown in SEQ ID NO: 7;

[0012] The amino acid sequence of the VL CDR1 is shown in SEQ ID NO: 8;

[0013] The amino acid sequence of the VL CDR2 is shown in SEQ ID NO: 9;

[0014] The amino acid sequence of the VL CDR3 is shown in SEQ ID NO:10.

[0015] Furthermore, the TCR-like monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:3.

[0016] In a second aspect, an engineered antibody of a TCR-like monoclonal antibody for treating cytomegalovirus infection, wherein the sequence portion of the engineered antibody of the TCR-like monoclonal antibody includes the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the TCR-like monoclonal antibody for treating post-transplant cytomegalovirus infection.

[0017] Furthermore, the amino acid sequence of the light chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 20; the amino acid sequence of the heavy chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 22.

[0018] Furthermore, the nucleotide sequence of the light chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 19; the nucleotide sequence of the heavy chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 21.

[0019] In a third aspect, a gene expression cassette, the sequence in the gene expression cassette encodes the TCR-like monoclonal antibody for treating cytomegalovirus infection, or encodes an engineered antibody of the TCR-like monoclonal antibody for treating cytomegalovirus infection.

[0020] A fourth aspect provides a gene expression vector, comprising the gene expression cassette.

[0021] A fifth aspect provides a host cell, wherein the host cell contains the gene expression vector described above.

[0022] In a sixth aspect, the use of the TCR-like monoclonal antibody for treating cytomegalovirus infection, or an engineered antibody of the TCR-like monoclonal antibody for treating cytomegalovirus infection in the preparation of anti-cytomegalovirus drugs.

[0023] The beneficial effects of the present invention are: (1) the monoclonal antibody 3D7 has a high affinity with the target protein, and the Biacore affinity detection KD (M): 1.86e-9;

[0024] (2) The antibody can bind to CMV-infected cells;

[0025] (3) The modified antibodies have a significant killing effect on CMV-infected cells both in vitro and in vivo. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The immunogen pCMV-pp65-HLA protein SDS-PAGE electrophoresis diagram of the present invention;

[0027] Figure 2 This is the ELISA reactivity graph of the monoclonal antibody 3D7 of the present invention to the pCMV-pp65-HLA protein;

[0028] Figure 3 This is a diagram showing the affinity determination (Biacore) of the 3D7 monoclonal antibody of the present invention for the pCMV-pp65-HLA protein;

[0029] Figure 4 This is a flow cytometric graph showing the binding of different concentrations of the monoclonal antibody 3D7 of the present invention to T2 cells;

[0030] Figure 5 The figure is a flow cytometric graph of the binding of the 3D7 antibody of the present invention to MRC-5 cells infected with CMV and MRC-5 cells not infected;

[0031] Figure 6 This is the SDS-PAGE electrophoresis diagram of the modified antibody 3D7LH of the present invention;

[0032] Figure 7 This is a diagram showing the apoptosis of cells after the modified antibody 3D7LH of the present invention kills cells;

[0033] Figure 8 This is a graph showing the CMV DNA copy number in the supernatant after the modified antibody 3D7LH of the present invention kills cells;

[0034] Fig. 9 It is the in vivo experimental roadmap of the present invention;

[0035] Fig.10 The figure is a graph showing the CMV DNA copy number in the peripheral blood and tissues of the mice of the present invention. DETAILED DESCRIPTION

[0036] The principles and features of the present invention are described below, and the examples are only used to explain the present invention and are not used to limit the scope of the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0037] Example

[0038] 1. Construction of pCMV-pp65-HLA-A2 expression clone (His tag).

[0039] 1.1pCMV-pp65-HLA-A2 gene synthesis and expression cloning construction.

[0040] The 9-peptide CMV-pp65 was synthesized by referring to the human HLA-A*02:01 cDNA sequence (AF036921.1) and the human b2m cDNA sequence (CR457066.1). 495-503 (SEQ ID NO: 17, NLVPMVATV) and HLA-A*02:01 molecule amino acid sequence were optimized according to human partial codons to obtain the optimized coding nucleic acid sequence. Starting from the N-terminal signal peptide coding sequence (the guide sequence of human B2m), pCMV-pp65, b2m, HLA-A*02:01 were connected in sequence, and the C-terminal was connected to a polyhistidine polypeptide (6×His) that was convenient for affinity chromatography purification, and finally the pCMV-pp65-HLA-A2-His protein optimized coding sequence was obtained, wherein the nucleic acid sequence of pCMV-pp65-HLA-A2-His is as shown in SEQ ID NO: 1, and the amino acid sequence of pCMV-pp65-HLA-A2-His is as shown in SEQ ID NO: 2.

[0041] Nucleic acid coding sequence of pCMV-pp65-HLA-A2-His (SEQ ID NO: 1):

[0042]

[0043] Amino acid sequence of pCMV-pp65-A2-His (SEQ ID NO: 2):

[0044] MARSVTLVFLVLVSLTGLYANLVPMVATVGCGASGGGGSGGGGSIQRTPKIQVYSRHPAENGKSNFLNCYVSGFHPSDIEVDLLKNGERIEKVEHSDLSFSKDWSFYLLYYT EFTPTEKDEYACRVNHVTLSQPKIVKWDRDMGGGGSGGGGSGGGGSGGGGSGSHSMRYFFTSVSRPGRGEPRFIAVGYVDDTQFVRFDSDAASQRMEPRAPWIEQEGPEYWD GETRKVKAHSQTHRVDLGTLRGCYNQSEAGSHTVQRMYGCDVGSDWRFLRGYHQYAYDGKDYIALKEDLRSWTAADMAAQTTKHKWEAAHVAEQLRAYLEGTCVEWLRRYLE NGKETLQRTDAPKTHMTHHAVSDHEATLRCWALSFYPAEITLTWQRDGEDQTQDTELVETRPAGDGTFQKWAAVVVPSGQEQRYTCHVQHEGLPKPLTLRWEGRSGHHHHHH.

[0045] In order to synthesize the optimized coding sequence of the above-mentioned pCMV-pp65-HLA-A2-His, the present invention commissioned General Biotech to perform whole gene synthesis (the synthesis method is shown in the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075-1085.), which was connected to the commercial vector pTT5. DNA sequencing proved that the sequence was completely consistent with the design, and finally the expression vector pTT5-pCMV-pp65-HLA-A2-His of pCMV-pp65-HLA-A2-His was obtained, that is, the pCMV-pp65-HLA expression clone pTT5-pCMV-pp65-HLA-A2-His with a His tag was obtained.

[0046] 2. Expression and purification of pCMV-pp65-HLA antigen.

[0047] 2.1pCMV-pp65-HLA antigen expression.

[0048] (1) Expression cell preparation:

[0049] 3x106 ExpiCHO cells (suspension Chinese hamster ovary cells, purchased from ATCC) were cultured at a density of TM Expression Medium (Thermo Scientific) was cultured in a triangular shake flask and placed in a constant temperature shaker at 37°C, 8% CO2, and an appropriate speed for 24 h. 6 density.

[0050] (2) pCMV-pp65-HLA antigen expression:

[0051] ExpiFectamine was used according to the instructions of the kit. TM The His-tagged pCMV-pp65-HLA expression clone pTT5-pCMV-pp65-HLA-A2-His was transfected into ExpiCHO cells using CHO Transfection Kit (ThermoScientific). After culturing for 17-24 h under the same conditions, the feed and enhancer provided in the kit were added and the cells were changed to 32°C and 5% CO. 2， The culture was continued for 6 days in a constant temperature shaker at an appropriate speed.

[0052] 2.2 Purification of pCMV-pp65-HLA antigen.

[0053] (1) Treatment of culture supernatant:

[0054] After culturing the pCMV-pp65-HLA antigen for 6 days, the ExpiCHO expression cell suspension was collected and centrifuged at 12000 rpm at room temperature for 30 min. The supernatant was collected and dialyzed into 1X PBS (0.01 M, pH=7.4), and then filtered through a 0.22 μm filter membrane.

[0055] (2) pCMV-pp65-HLA antigen purification:

[0056] The supernatant sample that was dialyzed into 1X PBS (0.01M, pH=7.4) in the previous step and filtered through a 0.22 μm filter membrane was purified by medium-pressure Ni-excel chromatography (GE medium), 30 mM imidazole was used to remove impurities, and 250 mM imidazole was used to elute the target protein. The SDS-PAGE gel image is shown in the figure below: Figure 1 The results showed that the purity of the obtained pCMV-pp65-HLA antigen was above 90%. The obtained target protein (pCMV-pp65-HLA protein expressed by CHO recombinant) was dialyzed into 1X PBS (0.01M, pH=7.4) buffer and stored at -20°C.

[0057] 3. Immunize mice with pCMV-pp65-HLA antigen and obtain monoclonal antibody 3D7.

[0058] To obtain mouse monoclonal antibodies specific for pCMV-pp65-HLA, mice were immunized with pCMV-pp65-HLA antigen.

[0059] 3.1 Mouse immunization:

[0060] (1) Preparation of immunogen: The immunogen is pCMV-pp65-HLA protein recombinantly expressed by CHO. Take the pCMV-pp65-HLA protein recombinantly expressed by CHO and dilute it to 0.2 mg / mL, mix it with an equal volume of Freund's adjuvant, and mix it thoroughly in an injection emulsifier to form an oil-in-water emulsion. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for subsequent booster immunization to obtain the prepared immunogen.

[0061] (2) Mouse immunization: 6-8 week old BALB / c female mice (purchased from Shanghai Slake Laboratory Animal Co., Ltd.) were immunized by multiple subcutaneous injections in the bilateral groin area using the prepared immunogens. The injection volume was 300 μL / mouse / time. About 200 μL of orbital venous blood was collected before each immunization for titer determination. After the initial immunization, booster immunization was performed every 2 weeks. Serum antibody titers were determined by ELISA, and fusion experiments were performed 4 weeks later.

[0062] 3.2 Antibody Screening:

[0063] (1) Preparation before fusion: 72 hours before the fusion of mouse spleen cells and mouse myeloma cells (SP2 / 0, purchased from ATCC), the spleen was finally boosted. The antigen used for this immunization was an adjuvant-free antigen, and the concentration was diluted to 1 mg / mL. Before spleen immunization, the mouse was anesthetized with isoflurane, and the mouse skin and peritoneum were cut open in turn to expose the spleen. 50 μL of antigen was injected longitudinally along the spleen, and then the peritoneum and skin were immediately sutured. At the same time, mouse myeloma cells SP2 / 0 were resuscitated and cultured to the logarithmic growth phase with RPMI1640 medium containing 10% fetal bovine serum in preparation for fusion.

[0064] (2) Preparation and screening of fusion hybridomas:

[0065] Take the spleen obtained in the above steps to boost the immunization of mice 72 hours later, take the spleen to make a cell suspension and fuse it with mouse myeloma cells SP2 / 0 to obtain hybridoma cells. Prepare feeder cells and co-culture with hybridoma cells, because in 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. Such added cells are feeder cells. This experiment uses mouse peritoneal macrophages and young mouse thymocytes as feeder cells; their preparation methods are as follows:

[0066] ① Preparation of mouse peritoneal macrophages: (i) A 6-week-old BALB / C mouse was killed by cervical dissection, immersed in 75% alcohol for disinfection for 3-5 minutes, and then placed in a clean bench with the mouse abdomen facing up. The mouse abdominal skin was lifted with forceps, a small cut was made, and the mouse skin was cut in the upper and lower directions at the cut with two hemostats to expose the mouse peritoneum; (ii) The peritoneum was lifted with a sterile forceps, and 5 mL of RPMI1640 culture medium (purchased from Shanghai Yuanpei Biotechnology Co., Ltd.) was injected into the mouse peritoneal cavity with a syringe, and then the mouse was shaken to mix the culture medium in the peritoneal cavity, and then the culture medium in the peritoneal cavity was carefully aspirated with a syringe; (iii) The culture medium containing macrophages was added to RPMI1640-HAT screening medium (purchased from Thermo Fisher Scientific) containing 20% ​​fetal bovine serum and HAT additives and mixed with the fused cells.

[0067] ② Preparation of mouse thymocytes: (i) A 3-week-old BALB / C mouse was killed by cervical dissection, and the mouse was immersed in 75% alcohol for disinfection for 3-5 minutes. The mouse was then placed in a clean bench with its abdomen facing upward. The mouse chest skin was lifted with tweezers, a small cut was made, and two hemostats were used to cut the mouse skin in the up and down directions at the cut to expose the mouse chest endothelium; (ii) The chest was clamped with another pair of sterile tweezers, and the chest was cut with scissors; (iii) The milky white thymus in the chest was removed with clean and sterile tweezers, and ground in a 70 μm cell sieve to obtain thymic feeder cells. The thymic cells were added to RPMI1640-HAT screening medium containing 20% ​​fetal bovine serum and mixed with the fusion cells.

[0068] ③ Preparation of mouse myeloma cells: Select SP2 / 0 cells grown to the logarithmic growth phase for fusion. Before fusion, remove the myeloma cells from the culture flask into a centrifuge tube and wash once with RPMI-1640 culture medium (1500rpm×5min). Resuspend the cells with RPMI-1640 culture medium and count them.

[0069] ④ Preparation of spleen cells from immunized mice: (i) Take BALB / C mice that have been boosted with immunization, collect whole blood from the mice, and collect mouse serum. (ii) Then, kill the mice by cervical dissection, soak them in 75% alcohol for disinfection for 3-5 minutes, and then place them in a clean bench with the mice in a right-side lying position; (iii) Use sterile tweezers and surgical scissors to open the abdominal cavity of the mice, cut out the spleen of the mice, cut the spleen into small pieces, place them in a 70μm cell mesh and grind them to obtain spleen cells; (iv) Place the spleen cells in a 50mL centrifuge tube, use a glass pipette (bend tube) to remove the fat tissue, then add RPMI-1640 culture medium to 30mL, centrifuge at 1500rpm for 5min, and repeat 3 times; (v) Resuspend the spleen cells in RPMI-1640 culture medium and count them.

[0070] ⑤ Preparation of hybridoma by PEG fusion: (i) Before fusion, incubate 1 mL PEG1450 (purchased from SIGMA) and 40 mL RPMI1640 medium to 37°C for later use; (ii) Mix the prepared myeloma cells and spleen cells in a 50 mL centrifuge tube, centrifuge at 1500 rpm for 5 min, discard the supernatant, and gently tap the bottom of the tube to loosen the cells into a paste; (iii) Slowly add the incubated PEG to the cells, shake the cells while adding, and then mix the cells. After 1 min, terminate the fusion with the incubated RPMI1640 medium; (iv) Centrifuge at 1500 rpm for 5 min, add the cells to RPMI1640-HAT screening medium containing feeder cells and 20% fetal bovine serum, and then add them to a 96-well plate, 200 μL per well, and put 5% (v) After 5 days of culture, the RPMI1640-HAT medium containing 20% ​​fetal bovine serum was removed and replaced with RPMI1640-HT medium containing 10% fetal bovine serum. After 5 days of culture, the cell supernatant was removed for detection.

[0071] ⑥ Hybridoma screening: High-throughput flow cytometry screening system based on T2 cells:

[0072] A. Peptide incubation: T2 cells were plated in 96-well U-bottom plates at 1×10 cells per well. 6 / 100uL, add the target peptide CMV-pp65 495-503 and irrelevant polypeptide pHBc (SEQ ID NO: 18, FLPSDFFPSV) at 1 ug / well, incubated at 37°C for 2 h, allowing the peptides to bind to HLA-A*02:01 molecules on the surface of T2 cells (purchased from Shanghai Fuheng Biotechnology Co., Ltd.).

[0073] B. Washing the plate: Centrifuge the T2 cell U-bottom plate after incubation with peptides at 2000 rpm for 3 min in a plate centrifuge, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, discard the supernatant for later use.

[0074] C. Sample addition: Take 50uL of the fusion supernatant and add the target peptide CMV-pp65 to the incubation 495-503 and irrelevant peptide pHBc in T2 cells and incubated at 37°C for 0.5h.

[0075] D. Washing the plate: Centrifuge the incubated T2 cell U-bottom plate with a plate centrifuge at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, discard the supernatant and set aside.

[0076] E. Secondary antibody reaction: fluorescent secondary antibody (Anti-mouse IgG (H+L), F(ab')2Fragment (Alexa 647Conjugate)) was diluted 500 times and added to T2 cell U-bottom plates, 30uL per well, and incubated at 4℃ for 0.5h.

[0077] F. Washing the plate: Centrifuge the incubated T2 cell U-bottom plate with a plate centrifuge at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, discard the supernatant and set aside.

[0078] G. Resuspend: Resuspend the cells with 1X PBS containing 2% FBS, 50uL per well, and place at 4℃ for later use.

[0079] H. Flow cytometry: The fluorescence intensity of T2 cells was detected using an analytical flow cytometer (BD LSR Fortessa X-20) in high-throughput mode.

[0080] 3.3 Production of monoclonal antibodies in mouse ascites.

[0081] Take 5 BALB / C mice and inject 0.5mL liquid paraffin oil intraperitoneally for sensitization. The mice can be used 3 days after sensitization. Centrifuge the hybridoma cells in the logarithmic growth phase at 1500rpm for 5min, discard the supernatant, and resuspend them in PBS to 1-2×10 6 Cells / mL, 0.5 mL of cells were injected into the peritoneum of each mouse. After 7 days, the abdomen of the mouse was obviously swollen, and the mouse was killed by cervical dissection. The abdominal cavity of the mouse was opened, and all the ascites in the abdominal cavity of the mouse was carefully sucked out to obtain the monoclonal antibody ascites.

[0082] 3.4 Purification of monoclonal antibody ascites:

[0083] The monoclonal antibody ascites was centrifuged at high speed and the supernatant was taken. An equal volume of saturated ammonium sulfate solution was added and precipitated on ice for 30 minutes. The supernatant was centrifuged at 25000 rpm for 10 minutes. The precipitate was dissolved with 0.2 M sodium dihydrogen phosphate dodecahydrate buffer and then purified with a Protein A affinity chromatography column (purchased from GE, USA) to obtain purified mouse monoclonal antibody 3D7 (Table 1).

[0084] Table 1 Monoclonal antibody information

[0085] serial number Monoclonal antibody name Antibody type 1 3D7 IgG1

[0086] 4. Amplification and sequencing of the light and heavy chain variable region genes of monoclonal antibody 3D7.

[0087] 4.1 Hybridoma RNA extraction:

[0088] The monoclonal antibody 3D7 hybridoma cells cultured to the logarithmic growth phase were blown up and transferred to a 15mL centrifuge tube. The cells were collected by centrifugation at 1500rpm for 3min, resuspended in 200μl sterile PBS (pH7.45), and transferred to a new 1.5ml centrifuge tube without RNAse, and 800μl Trizol solution (Invitrogen) was added. After vigorous shaking for 30S, the tube was allowed to stand at 4℃ for 10min. 250μl chloroform was added, and the tube was shaken vigorously for 30s, allowed to stand at 4℃ for 5min, and centrifuged at 4℃ 12000rpm for 15min. The upper layer of liquid was transferred to a new 1.5ml centrifuge tube, and an equal volume of isopropanol was added. The tube was gently inverted to mix, and allowed to stand at 4℃ for 10min. Centrifuge at 4℃ 12000rpm for 10min, discard the supernatant, add 800μl 75% ethanol, gently invert, centrifuge at 4℃ 12000rpm for 5min, discard the supernatant, heat the precipitate in a 70℃ metal bath for 10min to dry the residual ethanol. Dissolve the transparent precipitate in 50μl DEPC H2O.

[0089] 4.2 RNA reverse transcription to cDNA:

[0090] The light / heavy chain variable region reverse transcription systems were prepared according to the following systems, see Table 2:

[0091] Table 2

[0092] Element Volume (μL) 5x AMV buffer 4 dNTP 0.8 Reverse transcription downstream primer 0.8 AMV reverse transcriptase 0.2 RNA template 5 DEPC water 9.2

[0093] The reverse transcription downstream primer of the heavy chain variable region is MuIgGVH3'-2, and the reverse transcription downstream primer of the light chain variable region is MuIgκVL3'-1. The prepared reverse transcription system is placed at 42°C for 40 minutes to obtain the 3D7 light / heavy chain variable region cDNA.

[0094] 4.3 Antibody variable region gene amplification and sequencing:

[0095] The amplification of the 3D7 heavy chain variable region gene (3D7 heavy chain variable region cDNA) was performed using the above reverse transcribed cDNA as a template, MuIgVH5'-E1 as an upstream primer, and MuIgGVH3'-2 as a downstream primer. PCR amplification was performed to obtain a DNA fragment of about 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. The PCR product was recovered and sequenced. The sequence was determined to be the heavy chain variable region sequence of 3D7 after blast comparison. Amplification of the 3D7 light chain variable region gene (3D7 light chain variable region cDNA) The above corresponding cDNA was used as a template, MuIgκVL5'-D2 was used as an upstream primer, and MuIgκVL3'-1 was used as a downstream primer to perform PCR amplification to obtain a DNA fragment of about 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. The PCR product was recovered and sequenced. The sequence was determined to be the light chain sequence of 3D7 after blast comparison, see Table 3.

[0096] Table 3 Primers used for variable region gene amplification

[0097]

[0098]

[0099] After sequencing, the amino acid sequences of the heavy chain and light chain variable regions of 3D7 were determined as shown in Table 4:

[0100] Table 4 Amino acid sequences of the heavy and light chain variable regions of antibody 3D7

[0101]

[0102] Further, the CDR sequence of mouse monoclonal antibody 3D7 was determined using the method described by Kabat et al. (Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, Maryland (1991), pp. 647-669). The amino acid sequences of the CDRs of the heavy chain variable region and light chain variable region of 3D7 are shown in Table 5.

[0103] Table 5 Amino acid sequences of CDRs of the heavy and light chain variable regions of antibody 3D7

[0104] name CDR sequences Serial Number 3D7 VH CDR1 GYTFTSYW SEQ ID NO: 5 3D7 VH CDR2 IYPSDSYT SEQ ID NO: 6 3D7 VH CDR3 TRGDYGYDWFAY SEQ ID NO: 7 3D7 VL CDR1 ENIYSY SEQ ID NO: 8 3D7 VL CDR2 SAK SEQ ID NO: 9 3D7 VL CDR3 QHHHDLPLT SEQ ID NO: 10

[0105] 5. ELISA binding activity of monoclonal antibody 3D7 to pCMV-pp65-HLA.

[0106] 5.1 Preparation of reaction plate:

[0107] The pCMV-pp65-HLA protein obtained above was diluted with 200mM PB buffer (NaH2PO4 / Na2HPO4 buffer, final concentration of 200mM, pH 7.4) at pH 7.4 to a final concentration of 1μg / mL; 100μL of coating solution was added to each well of a 96-well ELISA plate, and the plate was coated at 2-8°C for 16-24 hours and then at 37°C for 2 hours; the plate was washed once with PBST washing solution (20mM PB7.4, 150mMNaCl, 0.1% Tween20); then 200μL of blocking solution (20mM Na2HPO4 / NaH2PO4 buffer solution with pH 7.4 containing 20% ​​calf serum and 1% casein) was added to each well, and the plate was blocked at 37°C for 2 hours; the blocking solution was discarded. After drying, the plate was packed into an aluminum foil bag and stored at 2-8°C for later use.

[0108] 5.2 ELISA detection of mouse monoclonal antibody 3D7:

[0109] The obtained monoclonal antibody 3D7 was diluted to 10 μg / mL, 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04 μg / mL, 0.01 μg / mL, and 0 μg / mL with 1X PBS solution, and ELISA detection was performed according to the following steps:

[0110] (1) Sample reaction: Take the ELISA plate coated with pCMV-pp65-HLA protein, add 100 μL of diluted sample to each well, and place it in a 37°C incubator for reaction for 30 minutes.

[0111] (2) Enzyme marker reaction: After completing the sample reaction step, wash the ELISA plate five times with PBST solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween 20), add 100 μL of HRP-labeled goat anti-mouse IgG reaction solution to each well, and place in a 37°C incubator for reaction for 30 minutes.

[0112] (3) Color development reaction: After completing the enzyme labeling reaction step, the ELISA plate was washed five times with PBST washing solution (20 mM PB7.4, 150 mM NaCl, 0.1% Tween20), and 50 μL of TMB color developer (purchased from Beijing Wantai Bio-Pharmaceutical Co., Ltd.) was added to each well and placed in a 37°C incubator for reaction for 15 minutes.

[0113] (4) Termination of reaction and measurement of readings: After the color development step is completed, 50 μL of stop solution (purchased from Beijing Wantai Biopharmaceutical Co., Ltd.) is added to each well of the ELISA plate after the reaction, and the OD450 / 630 value of each well is measured on an ELISA reader.

[0114] Determination of the reactivity of mouse monoclonal antibody 3D7 with pCMV-pp65-HLA: Determine based on the reading after the reaction. If the detection value / background value is greater than 5, it is determined to be positive.

[0115] (5) Result analysis: The results show that Figure 2 , 3D7 antibody has strong binding activity with pCMV-pp65-HLA at concentrations of 10μg / mL, 3.33μg / mL, 1.11μg / mL, 0.37μg / mL, 0.12μg / mL, 0.04μg / mL, and 0.01μg / mL, with EC50=11.9ng / mL; the control antibody has no binding activity with pCMV-pp65-HLA. The results show that 3D7 antibody can better recognize the pCMV-pp65-HLA complex.

[0116] 6. Biacore affinity determination of monoclonal antibody 3D7 for pCMV-pp65-HLA recombinant protein.

[0117] The present invention uses surface plasmon resonance (SPR) technology to detect the affinity between the monoclonal antibody 3D7 obtained in Example 3 and pCMV-pp65-HLA. The detection method used in this example is a capture method, using a Protein G chip (GE Company) to capture the mouse monoclonal antibody.

[0118] 6.1 Determination of ligand response value (RU):

[0119] The ligand coupling level calculation formula is shown in the following formula (1). According to the molecular weight of mouse monoclonal antibody and the molecular weight of pCMV-pp65-HLA analyte, the ligand response value is about 1000RU.

[0120]

[0121] Among them, R L is the ligand coupling level, R max To describe the maximum binding capacity of the chip surface, R is required for kinetically low coupling. max ≤100; S m For the stoichiometric ratio (Analyte: Ligand, select S if unknown) m =1).

[0122] 6.2 Affinity detection:

[0123] The pCMV-pp65-HLA antigen was diluted 2-fold from the initial concentration of 3200 nM, and the antigen-antibody affinity was detected using a surface plasmon resonance detector Biacore 8000 (GE). Figure 3 shown.

[0124] 6.3 Results Analysis:

[0125] 3D7 monoclonal antibody has good affinity for pCMV-pp65-HLA antigen, with a dissociation constant K d =1.86nM.

[0126] 7. Monoclonal antibody 3D7 cell binding experiment.

[0127] 7.1 In vitro binding experiment of monoclonal antibody 3D7 and T2 cells:

[0128] T2 cells were revived and cultured in 1640 medium containing 10% fetal bovine serum until the logarithmic growth phase. 6 T2 cells were seeded on a 10 cm cell culture plate at a density of 1.5 μg / mL and cultured in a 5% CO2 cell culture incubator at 37°C for the following experiments:

[0129] (1) Peptide incubation: T2 cells were plated in a 96-well U-bottom plate at 1×10 cells per well. 6 1 μg / well of the target peptide CMV-pp65 and the irrelevant peptide HBc were added respectively, and the cells were incubated 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.

[0130] (2) Washing: After incubation with peptide, centrifuge the T2 cells at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0131] (3) Dilution: Dilute the 3D7 antibody with 1X PBS containing 2% FBS to 3.33 μg / mL, 1.11 μg / mL, 0.37 μg / mL, 0.12 μg / mL, 0.04 μg / mL, 0.01 μg / mL, and 0 μg / mL for later use.

[0132] (4) Sample addition: Take 50uL of the diluted antibody and add it to T2 cells incubated with the target peptide pCMV-pp65 and the irrelevant peptide pHBc, respectively, and incubate at 37°C for 0.5h.

[0133] (5) Washing the plate: Centrifuge the incubated T2 cell U-bottom plate at 2000 rpm for 3 min in a plate centrifuge, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, discard the supernatant and set aside.

[0134] (6) Secondary antibody reaction: Dilute the fluorescent secondary antibody 500 times and add it to the T2 cell U-bottom plate, 30uL per well, and incubate at 4°C for 0.5h.

[0135] (7) Washing: Centrifuge the incubated T2 cells at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0136] (8) Resuspension: Resuspend the cells in 1X PBS containing 2% FBS, 50uL per well, and place at 4°C until needed.

[0137] (9) Flow cytometry: The fluorescence intensity of T2 cells was detected using an analytical flow cytometer (BD LSRFortessa X-20) in high-throughput mode.

[0138] The binding of monoclonal antibody 3D7 to two types of T2 cells is shown in Figure 4 When the concentration of monoclonal antibody 3D7 was 3.33μg / mL, 1.11μg / mL, 0.37μg / mL, 0.12μg / mL, 0.04μg / mL, and 0.01μg / mL, its binding intensity to positive peptide T2 cells was higher than that to control peptide T2 cells.

[0139] 7.2 In vitro binding experiment of monoclonal antibody 3D7 and CMV-infected MRC-5 cells:

[0140] MRC-5 cells (purchased from ATCC) are human embryonic lung fibroblasts, which are susceptible to CMV and HLA-A2 positive, so CMV-infected MRC-5 cells can be regarded as a positive cell line.

[0141] MRC-5 cells were revived and cultured in DMEM medium containing 10% fetal bovine serum until the logarithmic growth phase. 6 MRC-5 cells were seeded on a 10 cm cell culture plate at a density of 1.5 μg / mL and infected with CMV at an MOI of 0.1. The cells were cultured in a 5% CO2 cell culture incubator at 37°C for the following experiments:

[0142] (1) Dilution: Dilute the 3D7 antibody to 10 ug / mL with 1X PBS containing 2% FBS and set aside.

[0143] (2) Sample addition: Take 100uL of the diluted antibody and add it to CMV-infected MRC-5 cells and uninfected MRC-5 cells respectively, with a cell density of 1x10 6 / mL, volume 100uL, incubate at 37℃ for 0.5h.

[0144] (5) Washing: Centrifuge the incubated cells in an MRC-5 centrifuge at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, discard the supernatant and set aside.

[0145] (6) Secondary antibody reaction: Dilute the fluorescent secondary antibody 500 times and add it to MRC-5 cells, 30uL per well, incubate at 4℃ for 0.5h.

[0146] (7) Washing the plate: After incubation, centrifuge the plate in an MRC-5 cell centrifuge at 2000 rpm for 3 min, discard the supernatant, wash once with 1X PBS containing 2% FBS, centrifuge at 2000 rpm for 3 min, and discard the supernatant for later use.

[0147] (8) Resuspension: Resuspend the cells in 1X PBS containing 2% FBS, 50uL per well, and place at 4°C until needed.

[0148] (9) Flow cytometry: The fluorescence intensity of MRC-5 cells was detected using an analytical flow cytometer (BD LSRFortessa X-20) in high-throughput mode.

[0149] The binding of 3D7 antibody to CMV-infected MRC-5 cells and uninfected MRC-5 cells is shown in Figure 5 As shown in the figure, monoclonal antibody 3D7 binds to CMV-infected MRC-5 cells more strongly than to uninfected MRC-5 cells.

[0150] 8. Construction of 3D7 modified antibody expression clone.

[0151] 8.1 3D7LH gene synthesis and expression cloning construction:

[0152] The 3D7 VH and 3D7 VL amino acid sequences were optimized according to human numbering codons to obtain their optimized encoding nucleic acid sequences. 3D7 VL and human light chain constant region (h-CL) were sequentially connected from the N-terminal signal peptide coding sequence to finally obtain the 3D7 VL1 protein optimized coding sequence, whose nucleic acid sequence is shown in SEQ ID NO: 19 and amino acid sequence is shown in SEQ ID NO: 20; 3D7 VH and human IgG1 were sequentially connected from the N-terminal signal peptide coding sequence to finally obtain the 3D7 VH1 protein optimized coding sequence, whose nucleic acid sequence is shown in SEQ ID NO: 21 and amino acid sequence is shown in SEQ ID NO: 22.

[0153] Optimized nucleic acid coding sequence of 3D7 VL1 (SEQ ID NO: 19):

[0154] ATGTCTGTGCCAACTCAGGTCCTGGGGTTGCTGCTGCTGTGGCTTACAGATGCAAGATGTGACATCCAGATGACACAGTCTCCTGCCTCTCTGTCTGCTTCTGTGGGAGAGACAGTGACAATCACATGTAGAGCTTCTGAAAACATCTATTCTTACCTGGCTTGGTACCAGCAGAAGGAGGGCAAGTCTCCTCAGCTGCTGGTGTATTCTGCTAAGACACTGGCTGAGGGCGTGCCTTCTAGGTTTAATGGAAGCGGATCTGGCACCCAGTTTTCTCTGAAGATCAACTCTCTGCAGCCTGAGGATTTTGGATCTTACTACTGTCAGCACCACCATGATCTGCCTCTGACATTTGGAGCCGGCACAAAGCTCGAGATAAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTAA。

[0155] Optimized 3D7 VL1 amino acid sequence (SEQ ID NO: 20):

[0156] MSVPTQVLGLLLLWLTDARCDIQMTQSPASLSASVGETVTITCRASENIYSYLAWYQQKEGKS

[0157] PQLLVYSAKTLAEGVPSRFNGSGSGTQFSLKINSLQPEDFGSYYCQHHHDLPLTFGAGTKLEIK

[0158] RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

[0159] Optimized 3D7 VH1 nucleic acid coding sequence (SEQ ID NO: 21):

[0160]

[0161] Optimized 3D7 VH1 amino acid sequence (SEQ ID NO: 22):

[0162] MGRLTSSFLLLIVPAYVLSQVQLQQPGAALVRPGASVKLSCKASGYTFTSYWINWVKQRPGQGLEWIGNIYPSDSYTNYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYCTR GDYGYDWFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDK KVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK.

[0163] In order to synthesize the above-mentioned optimized coding sequence, the present invention commissioned General Biotechnology Co., Ltd. to perform whole gene synthesis (the synthesis method is shown in the literature: Feng M, Wang L, Tian J. Sheng Wu Gong Cheng Xue Bao. 2013; 29(8): 1075-1085.), and the above-mentioned two sequences were respectively connected to the commercial vector pTT5. DNA sequencing proved that the sequence was completely consistent with the design, and finally the light chain expression vector pTT5-3D7VL1 and the heavy chain expression vector pTT5-3D7VH1 of 3D7LH were obtained.

[0164] 9. Expression and purification of 3D7 modified antibody.

[0165] 9.1 3D7LH Antibody Expression:

[0166] (1) Expression cell preparation:

[0167] 3x10 6 The density of ExpiCHO cells was cultured with an appropriate amount of culture medium. TMExpression Medium (Thermo Scientific) was cultured in a triangular shake flask and placed in a constant temperature shaker at 37°C, 8% CO2, and an appropriate speed for 24 h. 6 density.

[0168] (2) 3D7LH Antibody Antigen Expression:

[0169] ExpiFectamine was used according to the instructions of the kit. TM The 3D7LH expression clones pTT5-3D7VL1 and pTT5-3D7VH1 obtained in Example 8 were transfected into ExpiCHO cells using CHO Transfection Kit (ThermoScientific). After continuing to culture under the same conditions for 17-24 h, the feed and enhancer provided in the kit were added and the cells were changed to 32°C and 5% CO. 2， The culture was continued for 6 days in a constant temperature shaker at an appropriate speed.

[0170] 9.2 3D7LH Antibody Purification:

[0171] (1) Treatment of culture supernatant:

[0172] After 6 days of culture, the ExpiCHO expression cell suspension was collected and centrifuged at 12000 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.

[0173] (2) 3D7LH antibody purification:

[0174] The filtered supernatant was purified using a Protein A affinity chromatography column (purchased from GE, USA) to obtain purified antibodies. Figure 6 The results showed that the purity of the obtained 3D7LH antibody was above 90%. The obtained target antibody was dialyzed into PBS buffer and stored at -20°C.

[0175] 10. In vitro killing experiment of 3D7 modified antibody.

[0176] 10.1 Isolation of PBMCs:

[0177] (1) Add 2% FBS to RPMI 1640 to 6 mL;

[0178] (2) Take 3.5 mL of Ficoll and transfer it to a 15 mL cell separation tube (SepMate TM After mixing the blood sample, carefully add it to a 15 mL cell separation tube (SepMateTM test tube);

[0179] (3) Centrifugation: 25°C, 1200 g, 15 min, with an acceleration and deceleration rate of 9. Pour the supernatant into a new 15 mL centrifuge tube and add 6 mL of 2% FBS RPMI 1640. If the density gradient medium on the top of the SepMate tube appears red after centrifugation (i.e., some red blood cells have not precipitated), the SepMate centrifuge tube can be centrifuged at 1200 g for another 10 min with the centrifuge brake turned on;

[0180] (4) Centrifugation: 300 g, 8 min, room temperature, both the speed of rise and fall are 9. Discard the supernatant, add 5 mL of 2% FBS RPMI1640 to resuspend, and mix the cells of the same sample (each centrifuge tube is 5 mL, and after the same sample is mixed, each sample is 10 mL);

[0181] (5) Centrifugation: 300 g, 8 min, room temperature, both the speed of increase and the speed of decrease were 9;

[0182] (6) Discard the supernatant, add 1 mL of 10% FBS RPMI 1640 medium to resuspend the cells, and count them.

[0183] 10.2 Antibody-PBMC killing in vitro:

[0184] (1) Resuspend and count CMV-infected MRC-5 cells and uninfected MRC-5 cells at a concentration of 1×10 5 The density of cells / 100uL was plated in 48-well cell plates.

[0185] (2) After diluting PBMC, add them to all cell wells, 1×10 6 / 50uL, so that the effect-target ratio reaches 10:1 (E / T=10).

[0186] (3) Dilute the 3D7 modified antibody (3D7LH) and the control antibody 10C5LH (anti-HTLV human IgG1 antibody) to 10 ug / mL and repeat in triplicate.

[0187] (4) The 48-well cell plate was placed in an incubator at 37°C and 5% CO2 for 48 hours.

[0188] (5) After 48 hours, cell apoptosis was detected by Annexin V / PI method. The cells were digested with EDTA-free trypsin, centrifuged at 2000 rpm for 5 min to collect the cells, washed twice (2000 rpm × 5 min), and the supernatant was discarded after centrifugation, 500 μL binding buffer was added, and the cells were gently resuspended by pipetting; 5 μL Annexin V-HF647 was added, and the mixture was gently pipetted and mixed; 5 μL PI was added, and the mixture was gently pipetted and mixed; the mixture was incubated at room temperature in the dark for 5-15 min, and the cells were detected by flow cytometry. The number of CMV DNA copies in the supernatant was detected by RT-qPCR.

[0189] The data results are as follows Figure 7 , 8 The results showed that the 3D7 modified antibody could kill CMV-infected MRC-5 cells, but had no significant killing effect on uninfected MRC-5 cells. In addition, the 3D7 modified antibody could reduce the number of CMV DNA copies in the supernatant.

[0190] 11. In vivo killing experiment of 3D7 modified antibody.

[0191] 11.1 Construction of humanized mouse model of CMV infection:

[0192] M-NSG mice were myeloablated by intraperitoneal injection of busulfan (30 mg / kg) on ​​day -1. On day 0, human peripheral blood mononuclear cells (huPBMC, 5×10 6 ), after human immune cell reconstruction was completed 2 weeks after transplantation, mice were intraperitoneally injected with CMV-infected MRC-5 cells (2×10 6 ) to establish a humanized mouse model of CMV infection.

[0193] 11.2 In vivo anti-CMV experiment of 3D7 modified antibody:

[0194] (1) Dilute the modified antibody 3D7LH and the irrelevant antibody 10C5LH to 10 mg / kg and place at 4°C for later use.

[0195] (2) On the first day after CMV infection, the modified antibody was injected into mice via the tail vein.

[0196] (3) Peripheral blood and CMV-infected target organs such as liver, spleen, and lung were collected, and the CMV DNA copy number was detected by RT-qPCR.

[0197] The experimental steps are as follows Fig. 9 The RT-qPCR results are shown in Fig.10 As shown in the figure, the modified antibody can significantly inhibit CMV infection.

[0198] In summary, the monoclonal antibody 3D7 of the present invention has a high affinity with the target protein, and the Biacore affinity detection KD (M) is 1.86e-9; the antibody can bind to CMV-infected cells; the modified antibody has a significant killing effect on CMV-infected cells both in vitro and in vivo.

[0199] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A TCR-like monoclonal antibody for treating cytomegalovirus infection, characterized in that: The TCR-like monoclonal antibody comprises VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3; The amino acid sequence of the VH CDR1 is shown in SEQ ID NO: 5; The amino acid sequence of the VH CDR2 is shown in SEQ ID NO: 6; The amino acid sequence of the VH CDR3 is shown in SEQ ID NO: 7; The amino acid sequence of the VL CDR1 is shown in SEQ ID NO: 8; The amino acid sequence of the VL CDR2 is shown in SEQ ID NO: 9; The amino acid sequence of the VL CDR3 is shown in SEQ ID NO:

10.

2. A TCR-like monoclonal antibody for treating cytomegalovirus infection according to claim 1, characterized in that: The TCR-like monoclonal antibody comprises a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

3.

3. An engineered antibody of a TCR-like monoclonal antibody for treating cytomegalovirus infection, characterized in that: The sequence portion of the engineered antibody of the TCR-like monoclonal antibody comprises the sequences of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 in the TCR-like monoclonal antibody for treating cytomegalovirus infection according to claim 1.

4. The engineered antibody of a TCR-like monoclonal antibody for treating cytomegalovirus infection according to claim 3, characterized in that: The amino acid sequence of the light chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 20; The amino acid sequence of the heavy chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO:

22.

5. The engineered antibody of a TCR-like monoclonal antibody for treating cytomegalovirus infection according to claim 3, characterized in that: The nucleotide sequence of the light chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO: 19; The nucleotide sequence of the heavy chain variable region of the engineered antibody of the TCR-like monoclonal antibody is shown in SEQ ID NO:

21.

6. A gene expression cassette, characterized in that The sequence in the gene expression cassette encodes a TCR-like monoclonal antibody for treating cytomegalovirus infection as described in any one of claims 1 to 2, or an engineered antibody encoding a TCR-like monoclonal antibody for treating cytomegalovirus infection as described in any one of claims 3 to 5.

7. A gene expression vector, characterized in that: The gene expression vector contains the gene expression cassette according to claim 6.

8. A host cell, characterized in that The host cell contains the gene expression vector according to claim 7.

9. Use of a TCR-like monoclonal antibody for treating cytomegalovirus infection as claimed in any one of claims 1 to 2, or an engineered antibody of a TCR-like monoclonal antibody for treating cytomegalovirus infection as claimed in any one of claims 3 to 5 in the preparation of an anti-cytomegalovirus drug.

10. The use according to claim 9, characterized in that: The cytomegalovirus infection includes all cytomegalovirus-infected populations.

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