A humanized anti-human γδTCR monoclonal antibody and its encoding gene, preparation method and application

Abstract

The present invention discloses a humanized anti-human γδTCR monoclonal antibody and its encoding gene, preparation method and application, which belongs to the humanized monoclonal antibody preparation technology field in the biotechnology field. The heavy chain variable region of the humanized anti-human γδTCR monoclonal antibody provided by the present invention comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7. The humanized anti-human γδTCR monoclonal antibody can stimulate and amplify peripheral blood γδT cells in vitro, and the obtained γδT cells can secrete a variety of cytokines and can effectively kill a variety of tumor cells.

Description

Technical Field

[0001] The present invention belongs to the field of humanized monoclonal antibodies and preparation technology thereof in the field of biotechnology, and specifically relates to a humanized anti-human γδTCR monoclonal antibody and its encoding gene, preparation method and application. Background Art

[0002] T lymphocytes can be divided into two subgroups according to the composition of T cell receptors (TCR) on their cell membrane surfaces: αβT cell subgroup and γδT cell subgroup. The former's TCR is composed of two glycoprotein chains, α and β, and accounts for the vast majority of the total number of T cells; the latter's TCR is composed of γ and δ chains, accounting for only a very small part of peripheral blood T cells (<5%), but it is abundant in epithelial tissues such as the epidermis and mucous membranes.

[0003] Although γδT cells were discovered later than αβT cells, in the process of biological evolution, the occurrence of γδT cells appeared in the early stage of the evolution of adaptive immunity in the immune system of organisms, and was earlier than αβT cells. Due to its special position in evolution, although γδT cells are part of adaptive immunity, they still retain the functions and characteristics of innate immunity. Due to this "dual" characteristic, γδT cells are called "the bridge between innate immunity and adaptive immunity." γδT cells have multiple biological functions: 1) they can secrete cytokines to mediate inflammatory responses; 2) they promote B lymphocytes to secrete antibodies and induce antibody class switching; 3) they recognize tumor antigens that are non-MHC-restricted and do not require specific antigen presentation, and directly lyse target cells through cytotoxic activity; 4) they play an immunomodulatory role.

[0004] In recent years, tumor adoptive immunotherapy has made breakthrough progress in hematological tumors, while in the treatment of solid tumors, it is greatly limited due to the immunosuppressive effect in the tumor microenvironment. Studies have found that γδT cells can effectively infiltrate solid tumors to play an anti-tumor role. Antibody solid-phase in vitro amplification is an important means to obtain γδT cells. However, most of the γδTCR monoclonal antibodies that stimulate the proliferation of γδT cells in the antibody solid-phase in vitro amplification method are mouse antibodies, and mouse monoclonal antibodies have many problems when used in human treatment: first, they cannot effectively activate the complement and Fc receptor-related effector systems in the human body; second, they are recognized by the human immune system and produce human anti-mouse antibodies; third, they are quickly cleared in the human circulatory system. Therefore, mouse-derived anti-γδTCR monoclonal antibodies are subject to many restrictions in clinical treatment. There is no report on humanized anti-human γδTCR monoclonal antibodies in the prior art. Summary of the invention

[0005] In response to one or more of the problems existing in the prior art, the present invention provides, in one aspect, a humanized anti-human γδTCR monoclonal antibody, wherein the heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5, and the light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:7.

[0006] In some embodiments, the heavy chain constant region of the humanized anti-human γδTCR monoclonal antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:9, and the light chain constant region comprises or consists of the amino acid sequence shown in SEQ ID NO:10.

[0007] In some embodiments, the heavy chain of the humanized anti-human γδTCR monoclonal antibody comprises or consists of the amino acid sequence shown in SEQ ID NO:11 or SEQ ID NO:15, and the light chain comprises or consists of the amino acid sequence shown in SEQ ID NO:13.

[0008] On the other hand, the present invention provides a gene encoding the humanized anti-human γδTCR monoclonal antibody; preferably, the gene encoding the heavy chain of the humanized anti-human γδTCR monoclonal antibody comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 12 or SEQ ID NO: 16, and the gene encoding the light chain of the humanized anti-human γδTCR monoclonal antibody comprises or consists of a nucleotide sequence as shown in SEQ ID NO: 14.

[0009] Another aspect of the present invention also provides an expression vector, a transgenic cell line and a host bacterium containing a gene encoding the humanized anti-human γδTCR monoclonal antibody.

[0010] In another aspect, the present invention provides a method for preparing the humanized anti-human γδTCR monoclonal antibody, which comprises the following steps:

[0011] 1) transplanting the CDRs of the heavy and light chain variable regions of the mouse anti-human γδTCR monoclonal antibody into the humanized heavy and light chain variable region templates, respectively, to obtain the amino acid sequences of the heavy and light chain variable regions of the humanized antibody, respectively, wherein the obtained humanized antibody heavy chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5, and the obtained humanized antibody light chain variable region comprises or consists of the amino acid sequence shown in SEQ ID NO:7;

[0012] 2) connecting the amino acid sequences of the heavy and light chain variable regions of the humanized antibody obtained in step 1) with the amino acid sequences of the heavy and light chain constant regions of the humanized antibody, respectively, to obtain the amino acid sequences of the heavy chain and light chain of the humanized antibody, respectively;

[0013] 3) reverse translating the amino acid sequences of the heavy chain and light chain of the humanized antibody obtained in step 2) to obtain the coding genes of the heavy chain and light chain of the humanized antibody, respectively; and

[0014] 4) performing eukaryotic expression on the coding genes of the heavy chain and light chain of the humanized antibody obtained in step 3) to obtain the humanized anti-human γδTCR monoclonal antibody.

[0015] In some embodiments, the use of the humanized anti-human γδTCR monoclonal antibody in activating and expanding γδT cells also belongs to the content of the present invention.

[0016] In some embodiments, the method of activating and expanding γδT cells comprises solid-phase in vitro expansion.

[0017] In some embodiments, the humanized anti-human γδTCR monoclonal antibody is further combined with cytokines to activate and expand the γδT cells; optionally, the cytokines include IL-15, IL-21, or a combination thereof.

[0018] In some embodiments, the use of the humanized anti-human γδTCR monoclonal antibody or the γδT cells obtained by amplifying the humanized anti-human γδTCR monoclonal antibody in the preparation of a drug that kills tumor cells also belongs to the content of the present invention.

[0019] In some embodiments, the tumor cells include but are not limited to Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells and SW480 cells.

[0020] The humanized anti-human γδTCR monoclonal antibody provided based on the above technical solution is obtained by CDR transplantation of the heavy and light chain variable regions of a mouse monoclonal antibody. The heavy chain variable region of the obtained humanized anti-human γδTCR monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:7. The results of the examples show that the humanized anti-human γδTCR monoclonal antibody provided by the present invention (optionally combined with cytokines, such as IL-15, IL-21, or a combination thereof) can stimulate the proliferation of γδT cells in human PBMC in vitro, and its purity can reach more than 70% at 11 days, preferably more than 90%, and the γδT cells obtained by proliferation can efficiently secrete cytokines IFN-γ, TGF-α and CD107α, and can effectively kill a variety of tumor cells, including Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells and SW480 cells. Therefore, the γδT cells obtained by in vitro stimulation and amplification of the humanized anti-human γδTCR monoclonal antibody provided by the present invention have good cytotoxic effect on tumor cells, and can be used for adoptive immunotherapy of tumors in clinical practice.

[0021] In addition, the humanized anti-human γδTCR monoclonal antibody provided by the present invention is a highly humanized antibody, which itself has multiple advantages: (1) Since it does not contain mouse monoclonal antibodies as heterologous substances, and does not contain mouse viruses in mouse ascites that may be introduced in the preparation of mouse monoclonal antibodies, it will not cause patients to produce human anti-mouse antibody reactions, and can improve the safety of clinical applications; (2) As a recombinant antibody, the humanized monoclonal antibody can be expressed by a eukaryotic expression system, so clinical-grade antibodies can be mass-produced, reducing costs and ensuring product consistency, and there is no possibility of carrying mouse viruses, which is more advantageous in the application of cell adoptive therapy of tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The results of the cell purity test of humanized antibody huMHCc stimulating γδT cell proliferation.

[0023] Figure 2 These are the test results of γδT cell subsets expanded in vitro by huMHCc (H2L2) and huMHCc (H3L2), wherein panel A represents the flow cytometric graph of the purity of total γδT cells, and panel B represents the flow cytometric detection results of Vδ1 and Vδ2 subsets.

[0024] Figure 3 The test results of the function of γδT cells expanded in vitro by huMHCc (H2L2) to secrete multiple cytokines.

[0025] Figure 4The results of the test of the cytotoxic function of γδT cells expanded in vitro by huMHCc (H2L2) against tumor cells (LDH method).

[0026] Figure 5 The results are the results of the detection of the killing effect of γδT cells expanded in vitro based on huMHCc (H2L2) on SW480 tumor cells (RTCA method). DETAILED DESCRIPTION

[0027] In view of the many limitations of mouse-derived anti-human γδTCR monoclonal antibodies in clinical treatment in the prior art, the present invention provides a humanized anti-human γδTCR monoclonal antibody, and provides the coding gene, preparation method and application of the humanized anti-human γδTCR monoclonal antibody in activating and amplifying γδT cells. The humanized anti-human γδTCR monoclonal antibody provided by the present invention is obtained by transplanting the CDRs of the heavy and light chain variable regions of mouse-derived anti-human monoclonal antibodies into humanized heavy and light chain variable region templates, recombining and optimizing.

[0028] The content of the present invention is described in detail below with reference to specific embodiments and drawings.

[0029] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0030] The methods used in the following examples are all conventional methods unless otherwise specified. For specific steps, please refer to: Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0031] The methods for obtaining various biological materials described in the examples are only provided as a way of obtaining them in an experiment to achieve the specific disclosed purpose, and should not be construed as limiting the sources of the biological materials of the present invention. In fact, the sources of the biological materials used are extensive, and any biological materials that can be obtained without violating laws and moral ethics can be replaced and used according to the instructions in the examples.

[0032] The sequences involved in the examples can all be synthesized using existing techniques.

[0033] Example 1: Preparation and purification of mouse anti-human γδTCR monoclonal antibodies

[0034] This embodiment prepares mouse anti-human γδTCR monoclonal antibodies according to conventional methods known in the art (such as those described in the following documents: Kohler and Milrtein, Nature 256:495-96, 1975; Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988); or can refer to the method used in patent document CN103130894A to obtain mouse anti-human γδTCR monoclonal antibodies.

[0035] In this embodiment, the method for preparing mouse anti-human γδTCR monoclonal antibodies may include the following steps:

[0036] 1.1) According to the method in the literature (Insect baculovirus expression system to express TCRγ9 / δ2-Fc fusion protein and preliminary identification of its biological function; Basic Medicine and Clinic, Dec. 2009. Vol. 29 No. 12), recombinant human TCRγ9 / δ2-Fc protein was prepared as an immunogen to immunize Balb / C mice, specifically, 100 μg / mouse / time was injected subcutaneously at the abdomen of the mouse, mixed with an equal volume of Freund's complete adjuvant for the first immunization, and mixed with an equal volume of Freund's incomplete adjuvant for the second to fourth booster immunizations. One week later, blood was collected from the tail vein of the mouse, and the serum antibody titer was detected by ELISA. The mouse with the highest titer was selected, and 100 μg / mouse was injected again by tail vein injection for shock immunization.

[0037] 1.2) After 72 hours, the spleen of the mice was removed, spleen cells were isolated, and fused with SP2-0 myeloma cells using the polyethylene glycol method.

[0038] 1.3) The fused cells were resuspended in HAT selective medium (Invitrogen), and 2% methylcellulose semi-solid medium was added for pressure screening. Single clone cell clusters were picked, and the culture supernatant was tested by ELISA to screen positive clones and perform subcloning.

[0039] 1.4) The hybridoma cells secreting monoclonal antibodies were intraperitoneally inoculated into Balb / C mice, and the ascites was regularly extracted. The mouse anti-human γδTCR monoclonal antibodies were purified by rProtein A affinity chromatography.

[0040] Example 2: De novo sequencing of mouse anti-human γδTCR monoclonal antibody

[0041] In this example, the mouse anti-human γδTCR monoclonal antibody obtained in Example 1 was sequenced de novo to analyze the amino acid sequence of the antibody variable region, which specifically includes the following operations.

[0042] The mouse anti-human γδTCR monoclonal antibody sample obtained in Example 1 was subjected to multi-enzyme hydrolysis by five enzymes, namely Typsin, Chymotrypsin, GluC, Elastase and Pepsin, and then the peptide mass spectrometry data was collected. Specifically, 2 μg of the enzymatically hydrolyzed peptide sample was injected (high performance liquid chromatography), 90 min chromatographic gradient separation, and data acquisition and detection were performed by Orbitrap-Lumos ultra-high resolution mass spectrometer. The specific instrument method parameters are as follows:

[0043] High performance liquid chromatography: (Easy nLC 1200); Chromatographic column: C18, 3μm, 75μm x 15cm; Sample volume: 10μl. Mobile phase: A: 0.1% Formic acid in water; B: 0.1% Formic acid in 80% Acetonitrile / H2O. The chromatographic gradient is shown in Table 1 below.

[0044] Table 1: Chromatographic gradient parameter settings

[0045]

[0046] The mass spectrometry acquisition parameters were as follows: mass spectrometer Orbitrap-Lumos, spray voltage: 2.1 kV, capillary temperature: 320°C, S-lens RF Level: 30, collision energy: 30% NCE, Isolation Window: 2.5 Da.

[0047] Master Scan resolution settings: primary 60,000@m / z 200, AGC Target 4e5, primary maximum ion injection time 40ms, parent ion scan range: m / z 350-1550; Scan Event 1 Activation Type: HCD secondary 30,000@m / z 200 AGC Target 1e5, secondary maximum ion injection time 60ms; Scan Event 2 Activation Type: ETD secondary 30,000@m / z 200 AGC Target 1e5, secondary maximum ion injection time 60ms.

[0048] The mass spectrometry data obtained by the detection were subjected to De novo analysis using Peaks software to obtain the polypeptide sequence after enzyme cleavage. The polypeptide sequence was compared with the existing antibody database (such as IMGT, etc.) and then spliced. The sequence was manually corrected. After multiple rounds of retrieval and correction, the heavy chain variable region (VH) sequence (shown in SEQ ID NO: 1) and the light chain variable region (VL) sequence (shown in SEQ ID NO: 2) of the mouse anti-human γδTCR monoclonal antibody were obtained, and the obtained mouse anti-human γδTCR monoclonal antibody was named mMHCc.

[0049] Example 3: Humanized design of mouse mMHCc monoclonal antibody

[0050] This example uses the monoclonal antibody mMHCc with the antibody heavy chain variable region sequence of SEQ ID NO: 1 and the antibody light chain variable region of SEQ ID NO: 2 obtained by de novo sequencing in the above Example 2 to design a humanized anti-human γδTCR monoclonal antibody by transplanting the heavy chain and light chain variable region CDR1, CDR2 and CDR3, specifically including the following operations.

[0051] 3.1) The diversity analysis of the amino acid sequence of the antigen recognition region by mouse monoclonal antibodies determined that the CDR1, CDR2, and CDR3 of the VH of mMHCc were amino acids 26-33, 51-58, and 97-110 of SEQ ID NO: 1, respectively, and the CDR1, CDR2, and CDR3 of the VL were amino acids 27-32, 50-52, and 89-97 of SEQ ID NO: 2, respectively. The amino acid residues were determined and annotated using the Kabat numbering system.

[0052] 3.2) By comparing the IMGT human antibody heavy and light chain variable region germline gene database (https: / / www.imgt.org / ) and MOE software, the heavy and light chain variable region germline genes with high homology to the heavy and light chain variable regions of mMHCc were selected as templates, respectively. The selected mMHCc humanized heavy chain variable region templates include three types: IGHV1-3*01, IGHV1-58*02, IGHV1-69*09-IGHV1-69*04, and the mMHCc humanized light chain variable region templates include three types: IGKV7-3*01, IGKV4-1*01, IGKV1D-39*01-IGKV1-39*01, as shown in Table 2 below. The CDRs of the heavy and light chain variable regions of mMHCc were transplanted into the corresponding human templates, respectively, to form humanized antibody heavy and light chain variable region sequences in the order of FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, wherein the obtained heavy chain variable regions of the humanized antibodies were named huMHCc (VH1), huMHCc (VH2), and huMHCc (VH3), respectively, and the obtained light chain variable regions of the humanized antibodies were named huMHCc (VL1), huMHCc (VL2), and huMHCc (VL3), respectively, and their amino acid sequence information is shown in Table 2 below.

[0053] Table 2: Selected heavy and light chain variable region germline gene templates with high homology to the heavy and light chain variable regions of mMHCc

[0054] Matched human antibody heavy and light chain templates Humanized antibody heavy and light chains Amino acid sequence IGHV1-3*01 huMHCc(VH1) SEQ ID NO:3 IGHV1-58*02 huMHCc(VH2) SEQ ID NO:4 IGHV1-69*09-IGHV1-69*04 huMHCc(VH3) SEQ ID NO:5 IGKV7-3*01 huMHCc(VL1) SEQ ID NO:6 IGKV4-1*01 huMHCc(VL2) SEQ ID NO:7 IGKV1D-39*01-IGKV1-39*01 huMHCc(VL3) SEQ ID NO:8

[0055] 3.3) The three humanized antibody heavy chain variable regions (huMHCc (VH1), huMHCc (VH2), huMHCc (VH3)) obtained in step 3.2) above were respectively connected to the human antibody heavy chain constant region (SEQ ID NO: 9, including the hinge region) to obtain three complete humanized antibody heavy chains, and the three humanized antibody light chain variable regions (huMHCc (VL1), huMHCc (VL2), huMHCc (VL3)) obtained in step 3.2) above were respectively connected to the human antibody light chain constant region (SEQ ID NO: 9, including the hinge region) to obtain three complete humanized antibody heavy chains. NO:10) were connected to obtain three complete light chains of humanized antibodies. These humanized antibody heavy chains and humanized antibody light chains were randomly combined to obtain 9 candidate humanized anti-γδTCR monoclonal antibodies, named huMHCc. These 9 humanized antibodies were named huMHCc(H1L1), huMHCc(H1L2), huMHCc(H1L3), huMHCc(H2L1), huMHCc(H2L2), huMHCc(H2L3), huMHCc(H3L1), huMHCc(H3L2) and huMHCc(H3L3), respectively.

[0056] 3.4) The amino acid sequences of the above 9 huMHCc were reverse translated and codon optimized according to the eukaryotic expression system to obtain the DNA sequences encoding the heavy chain and light chain, respectively, and the DNA coding sequences were chemically synthesized, and the synthesized DNA coding sequences were inserted into the eukaryotic expression vector to construct recombinant plasmids. The plasmid expressing the heavy chain and the plasmid expressing the light chain were co-transfected into HEK-293T cells at a mass ratio of 1:1, and cultured in serum-free medium at 37°C and 5% CO2. After 72 hours, the cell supernatant was collected, and the antibody was purified using a recombinant protein A affinity chromatography medium to obtain the above 9 candidate humanized anti-γδTCR monoclonal antibodies.

[0057] Example 4: Functional test of in vitro activation and expansion of γδT cells by humanized anti-γδTCR monoclonal antibodies

[0058] This example uses the 9 candidate humanized anti-γδTCR monoclonal antibodies obtained in Example 3 to perform functional tests on in vitro activation and expansion of γδT cells, and then screens out humanized anti-γδTCR monoclonal antibodies that can effectively activate and expand γδT cells, specifically including the following operations.

[0059] The solid phase coated antibody method was used to screen for humanized anti-γδTCR monoclonal antibodies that can expand γδT cells from human peripheral blood mononuclear cells (PBMC). The process of in vitro expansion of γδT cells by the solid phase coated antibody method is as follows:

[0060] 1) Coat the culture plate with 9 candidate humanized anti-γδTCR monoclonal antibodies at a coating antibody concentration of 5 mg / L and incubate at 37°C for 2 h;

[0061] 2) Discard the coating solution and wash the culture plate twice with sterile PBS;

[0062] 3) PBMCs were isolated from human peripheral whole blood using density gradient centrifugation with human lymphocyte separation medium, and the plating density was 2×10 6 / well, suspension culture with KBM581 medium containing 3% SR and 200U / ml IL-2 (other cytokines may also be used in combination, such as IL-15 (50ng / ml), IL-21 (10ng / ml), or a combination thereof);

[0063] 4) After 5 days, move the cells out of the antibody-coated wells; change the medium and transfer the wells every 2 days;

[0064] 5) The purity of γδT cells was detected on the 11th day of culture. Flow cytometry was used to detect the surface biomarkers of amplified γδT cells, where FITC-labeled anti-γδT Ab was used to detect total γδT cells; FITC-labeled anti-Vδ1Ab and BV421-labeled anti-Vδ2Ab were used to detect cell subpopulations.

[0065] The results of the cell purity test of 9 candidate humanized anti-γδTCR monoclonal antibodies for in vitro solid-phase stimulation of γδT cell proliferation are as follows Figure 1 As shown, the blank control is the unstained cells, and the IL2 control is the result of the unsolid phase coated antibody. It can be seen that among the 9 candidate humanized anti-γδTCR monoclonal antibodies, huMHCc (H2L2) can stimulate the significant proliferation of γδT cells in human PBMCs, and its purity can reach more than about 90% on the 11th day. The effect of huMHCc (H3L2) in stimulating the proliferation of γδT cells is second, and its purity can also reach about 62% on the 11th day, while the purity of the remaining 7 candidate combinations is lower. Figure 2The purity test results of huMHCc (H2L2) and huMHCc (H3L2) in vitro expanded γδT cells detected on the 13th day are shown, wherein A represents the total γδT cell purity diagram, and B represents the Vδ1 and Vδ2 subset flow cytometry test results. It can be seen that the purity of huMHCc (H2L2) in vitro expanded γδT cells detected on the 13th day can reach more than 91%, and the purity of huMHCc (H3L2) stimulated γδT cells can reach more than 73%. Therefore, among the 9 candidate humanized anti-γδTCR monoclonal antibodies, huMHCc (H2L2) has the most significant effect on the activation and proliferation of γδT cells in human PBMCs, and γδT cells with a purity of more than 90% can be obtained on the 11th day of stimulation. HuMHCc (H3L2) also has a good stimulating effect on the proliferation of γδT cells in human PBMCs, and can reach a purity of more than 73% on the 13th day. Therefore, the present invention identifies two humanized anti-γδTCR monoclonal antibodies that can effectively expand γδT cells in vitro, namely huMHCc (H2L2) and huMHCc (H3L2); wherein the amino acid sequence of the heavy chain variable region of huMHCc (H2L2) is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:7; the amino acid sequence of the heavy chain of the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) is shown in SEQ ID NO:11 (the corresponding DNA coding sequence is shown in SEQ ID NO:12), and the amino acid sequence of the light chain is shown in SEQ ID NO:13 (the corresponding DNA coding sequence is shown in SEQ ID NO:14). The amino acid sequence of the heavy chain variable region of huMHCc (H3L2) is shown in SEQ ID NO: 5, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7; the amino acid sequence of the heavy chain of the humanized anti-γδTCR monoclonal antibody huMHCc (H3L2) is shown in SEQ ID NO: 15 (the corresponding DNA coding sequence is shown in SEQ ID NO: 16), and the amino acid sequence of the light chain is shown in SEQ ID NO: 13 (the corresponding DNA coding sequence is shown in SEQ ID NO: 14).

[0066] Example 5: Detection of the ability of in vitro expanded γδT cells to secrete multiple cytokines under stimulation conditions

[0067] This example takes the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) as an example to detect the ability of γδT cells stimulated to proliferate in vitro using the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) to secrete cytokines (CD107α, TNF-α and IFN-γ) under stimulation conditions, specifically including the following operations.

[0068] 5.1) Cell stimulation

[0069] γδT cells with a purity greater than 90% amplified in vitro by huMHCc (H2L2) were divided into two groups and added to 24-well plates, 1 ml / well, and approximately 1×10 6 cells; one group was added with stimulants PMA (100 ng / ml) and ionase (1 μg / ml), and the other group was not added with stimulants as a control; both groups were added with BFA (10 μg / ml) to block the transport of cytokines to the extracellular space. The cells were placed in an incubator at 37°C and 5% CO2 for 5-12 hours.

[0070] 5.2) Cell surface immunofluorescence staining

[0071] Collect the γδT cells after incubation in step 5.1), wash the cells twice with PBS washing solution, centrifuge at 250×g for 5 min; resuspend the cells in 100 μl PBS washing solution, add FITC-labeled γδTCR antibody, incubate at 4°C in the dark for 20 min; wash the cells twice with PBS washing solution, centrifuge at 250×g for 5 min.

[0072] 5.3) Intracellular immunofluorescence staining

[0073] After centrifugation in step 5.2), discard the supernatant, fix the cells with 500 μl of fixative, incubate at room temperature for 20 min, centrifuge the cells at 400×g for 5 min; discard the supernatant, add 1 ml of membrane permeabilization solution (1×) 400×g, centrifuge for 8 min to wash the cells twice; discard the supernatant, resuspend the cells with 100 μl of PBS washing solution, add PE-labeled anti-human IFN-γ, TGF-α and CD107αAb. Incubate at room temperature in the dark for 30 min; add 1 ml of membrane permeabilization solution (1×), wash the cells twice at 400×g for 5 min; resuspend the cells with 300 μl of 4% paraformaldehyde solution, and detect by flow cytometry.

[0074] The results of flow cytometry were as follows Figure 3 As shown, it can be seen that the γδT cells expanded by huMHCc (H2L2) can effectively secrete CD107α, TNF-α and IFN-γ under stimulation conditions, among which for CD107α, the relative secretion amount can reach 81.79% when the cells are incubated for 5 hours, for TNF-α, the relative secretion amount can reach 88.62% when the cells are incubated for 12 hours, and for IFN-γ, the relative secretion amount can reach 91.30% when the cells are incubated for 12 hours.

[0075] This example also tests the ability of γδT cells stimulated to proliferate in vitro using humanized anti-γδTCR monoclonal antibody huMHCc (H3L2) to secrete cytokines (CD107α, TNF-α and IFN-γ) under stimulation conditions. The results show that Figure 3Similar cytokine (CD107α, TNF-α and IFN-γ) secretion effects are shown and will not be described in detail here.

[0076] Example 6: Functional test of in vitro expanded γδT cells killing tumor cells

[0077] This example takes the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) as an example to detect the killing effect of the proliferated γδT cells stimulated by the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) on tumor cells, which specifically includes the following operations:

[0078] γδT cells with a purity greater than 90% amplified by huMHCc (H2L2) were used as effector cells, and cultured lymphoma cell lines Daudi cells, K562 cells, OVCAR-8 cells and HepG2 cells were used as target cells, respectively. Under the condition of effector-target ratio of 10:1, they were co-incubated for 4 hours, and the supernatant was taken to detect the LDH content using the LDH killing detection kit. The killing efficiency of γδT cells on target cells was calculated using the following formula:

[0079]

[0080] The above OD values ​​are all obtained by subtracting the blank control background.

[0081] The results of γδT cells killing tumor target cells are as follows Figure 4 As shown, it can be seen that the proliferation of γδT cells stimulated by the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) can effectively kill tumor target cells, such as lymphoma cell lines Daudi cells, K562 cells, OVCAR-8 cells and HepG2 cells, among which it has a strong killing effect on the solid tumor ovarian cancer cell line OVCAR-8 cells and the human liver cancer cell line HepG2 cells.

[0082] γδT cells stimulated to proliferate by the humanized anti-γδTCR monoclonal antibody huMHCc (H3L2) also have a similar effect of killing tumor cells, that is, they can effectively kill tumor cells, such as lymphoma cell lines Daudi cells, K562 cells, OVCAR-8 cells and HepG2 cells, among which they have a strong killing effect on solid tumor ovarian cancer cell line OVCAR-8 cells and human liver cancer cell line HepG2 cells, which will not be repeated here.

[0083] Example 7: Cytotoxic effect of in vitro expanded γδT cells on tumor cells SW480

[0084] This example takes the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) as an example, and uses the RTCA method to detect the cytotoxic effect of the proliferated γδT cells stimulated by the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) on the tumor cells SW480, which specifically includes the following operations:

[0085] Place the E-Plate 16 on the RTCA S16 Station, and the system will automatically scan ("Scan Plate") to check whether the contact is good. Click "New Experiment" on the homepage, enter the "Protocol" page, select the type of experiment you need to conduct "Proliferation / Cytocoxicty", select "Standard Protocol", and determine the experimental process. Click the "Layout" page to set the cell name and number of the experimental wells. Click the start icon in the lower left corner to start detecting the baseline. Take out the E-Plate 16 detection plate, add 50μL of evenly mixed target cell suspension to the "target cell well and effector-target ratio experimental well" to make the number of cells in each well be 1.5×10 4 / well / 50 μL (3×10 5 / mL); add 50μL target cell culture medium to the "effect cell well". Place the E-Plate 16 plate in the biosafety cabinet at room temperature for 30 minutes and then move it to the RTCA S16 Station in the incubator. Make sure the green indicator light is on and incubate the cells for 16-20 hours. After the system automatically scans "Scan Plate", click to start Step 2 to perform real-time dynamic detection of cell proliferation. The concentration of collected effector cells is adjusted to 1.5×10 6 / mL. Take out the E-Plate16 and place it in the clean bench. Add 50μL / well of the effector cell mixture to the "effect-target ratio experiment well and effector cell well". Place the E-Plate16 test plate on the RTCA S16 test bench for real-time monitoring. Make sure that the green indicator light is on and the instrument is operating normally.

[0086] The killing efficiency of γδT cells on target cells was calculated using the following formula:

[0087]

[0088] The results are as follows Figure 5As shown, the result of the "target cell well" is shown as the curve SW480, the result of the "effect-target ratio experimental well" is shown as the result of the addition of γδT to SW480 tumor cells, and the result of the "effector cell well" is shown as the γδT-control. It can be seen that relative to the result of the "target cell well", the addition of γδT cells to the "effect-target ratio experimental well" at the 36th hour of the culture system can significantly reduce the cell index of the sensing well, indicating that the proliferated γδT cells stimulated by the humanized anti-γδTCR monoclonal antibody huMHCc (H2L2) can effectively kill SW480 tumor cells.

[0089] The γδT cells stimulated to proliferate by the humanized anti-γδTCR monoclonal antibody huMHCc (H3L2) also have a similar effect of killing SW480 tumor target cells, which will not be described in detail here.

[0090] From the results of Examples 4-7, it can be seen that the humanized anti-human γδTCR monoclonal antibody provided by the present invention can efficiently stimulate and expand γδT cells in vitro, and the γδT cells obtained by in vitro stimulation and expansion can secrete a variety of cytokines and have good cytotoxic effects on a variety of tumor cells (including Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells and SW480 cells, etc.). Therefore, the obtained γδT cells can be used for adoptive immunotherapy of tumors in clinical practice by adoptive transfer.

[0091] Finally, it should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A humanized anti-human γδTCR monoclonal antibody, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 5, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

7.

2. The humanized anti-human γδTCR monoclonal antibody according to claim 1, wherein the heavy chain hinge region and constant region comprise the amino acid sequence shown in SEQ ID NO: 9, and the light chain constant region comprises the amino acid sequence shown in SEQ ID NO:

10.

3. The humanized anti-human γδTCR monoclonal antibody according to claim 1 or 2, wherein the heavy chain comprises the amino acid sequence shown in SEQ ID NO: 11 or SEQ ID NO: 15, and the light chain comprises the amino acid sequence shown in SEQ ID NO:

13. 4 . A gene encoding the humanized anti-human γδTCR monoclonal antibody according to any one of claims 1 to 3 .

5. The gene according to claim 4, wherein the gene encoding the heavy chain of the humanized anti-human γδTCR monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO: 12 or SEQ ID NO: 16, and the gene encoding the light chain of the humanized anti-human γδTCR monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO:

14.

6. An expression vector, transgenic cell line or host bacteria containing the gene according to claim 4 or 5.

7. A method for preparing the humanized anti-human γδTCR monoclonal antibody according to any one of claims 1 to 3, comprising the following steps: 1) transplanting the CDRs of the heavy and light chain variable regions of the mouse anti-human γδTCR monoclonal antibody into the humanized heavy and light chain variable region templates, respectively, to obtain the amino acid sequences of the heavy and light chain variable regions of the humanized antibody, respectively, wherein the obtained humanized antibody heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:5, and the obtained humanized antibody light chain variable region consists of the amino acid sequence shown in SEQ ID NO:7; 2) connecting the amino acid sequences of the heavy and light chain variable regions of the humanized antibody obtained in step 1) with the amino acid sequences of the heavy and light chain constant regions of the humanized antibody, respectively, to obtain the amino acid sequences of the heavy chain and light chain of the humanized antibody, respectively; 3) Reverse translating the amino acid sequences of the heavy chain and light chain of the humanized antibody obtained in step 2) to obtain the coding genes of the heavy chain and light chain of the humanized antibody, respectively; and 4) performing eukaryotic expression on the coding genes of the heavy chain and light chain of the humanized antibody obtained in step 3) to obtain the humanized anti-human γδTCR monoclonal antibody.

8. Use of the humanized anti-human γδTCR monoclonal antibody according to any one of claims 1 to 3 in the preparation of a reagent for activating and expanding γδT cells.

9. The use according to claim 8, wherein the amplification method is solid-phase in vitro amplification; and / or The humanized anti-human γδTCR monoclonal antibody is also combined with cytokines to activate and expand the γδT cells.

10. The use according to claim 9, wherein the cytokine is selected from IL-15, IL-21 or a combination thereof.

11. Use of the humanized anti-human γδTCR monoclonal antibody according to any one of claims 1 to 3 or the γδT cells amplified from the humanized anti-human γδTCR monoclonal antibody according to any one of claims 1 to 3 in the preparation of a drug for killing tumor cells; The tumor cells are selected from Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells or SW480 cells.

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