A humanized anti-human Vδ1 TCR monoclonal antibody and its application in activating and expanding human Vδ1 T cells

Abstract

The present invention discloses a humanized anti-human Vδ1 TCR monoclonal antibody and its application in activating and expanding human Vδ1 T cells, belonging to the technical field of preparation of humanized monoclonal antibodies and cell expansion in the field of biotechnology. The heavy chain variable region of the humanized anti-human Vδ1 TCR monoclonal antibody provided by the present invention contains the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region contains the amino acid sequence shown in SEQ ID NO: 2. This humanized anti-human Vδ1 TCR monoclonal antibody can efficiently activate and expand Vδ1 T cells in vitro, and the obtained Vδ1 T cells can effectively kill tumor cells.

Description

Technical Field

[0001] The present invention belongs to the technical field of the preparation of humanized monoclonal antibodies and in vitro cell amplification in the field of biotechnology, and particularly relates to a humanized anti-human Vδ1 TCR monoclonal antibody, a preparation method thereof, and an application thereof in highly efficiently activating and amplifying human Vδ1 T cells. Background Art

[0002] γδ T cells are a group of lymphocytes that are evolutionarily older than B cells and αβ T cells. Although γδ T cells only account for 0.5%-10% of CD3 + T cells in adult peripheral blood, they have the dual characteristics of both innate and adaptive immune responses and play an important immune role in the processes of the body's anti-infection, anti-tumor, and tissue damage repair. Compared with αβ T cells, γδ T cells have the following characteristics that are beneficial to the exertion of immune functions: First, the large distribution in mucosal and subcutaneous tissues and the limited diversity of TCRγδ are beneficial for γδ T cells to contact and recognize invading pathogenic microorganisms at an early stage and play an anti-infection role without a large amount of amplification; Second, γδ T cells recognize antigens without MHC restriction, and at the same time, compared with TCRαβ, the CDR3 lengths and structures of TCRγ and TCRδ are more similar to the light and heavy chains of antibodies, so TCRγδ can directly recognize a variety of antigen molecules without the assistance of antigen-presenting molecules like antibodies; Third, γδ T cells express NK cell-like receptors such as NKG2D and NCR (NKp30, NKp44, and NKp46), enabling them to recognize pathogenic microorganisms and stress molecule signals caused by pathogenic microorganisms at an early stage similar to NK cells; Fourth, γδ T cells are in a pre-activated state and can quickly exert various functions, including secreting cytokines and killing target cells, etc.; Fifth, γδ T cells have antigen-presenting functions and initiate subsequent adaptive immune responses; Sixth, γδ T cells play an important role in maintaining the integrity of epithelial tissues and tissue damage repair.

[0003] γδ T cells are mainly divided into three subsets according to the expression of the γ and δ chains of the TCR, namely Vδ1 T cells, Vδ2 T cells, and Vδ3 T cells. Among them, Vδ3 T cells are mainly distributed in the liver and intestine, Vδ2 T cells are mainly distributed in peripheral blood, and Vδ1 T cells mainly exist in mucosal and epithelial tissues, especially in the submucosal regions of the gastrointestinal tract, respiratory tract, and urogenital tract. There is also a very small amount of the Vδ1 T cell subset in peripheral blood. Different subsets of γδ T cells can play their unique roles. In recent years, it has been found that Vδ1 T cells in the mucosa can effectively infiltrate solid tumors and recognize abnormally expressed MHC class I-related molecules A and B (MICA and MICB) and UL-16 binding proteins (ULBP) on the tumor surface to play an anti-tumor role. However, the biggest obstacle faced by the adoptive immunotherapy of Vδ1 T cells at present is that their number in peripheral blood is very small and it is difficult to perform efficient in vitro expansion, thus limiting the application of Vδ1 T cells in clinical treatment. Summary of the Invention

[0004] In view of one or more of the problems existing in the prior art, one aspect of the present invention provides a humanized anti-human Vδ1 TCR monoclonal antibody, the heavy chain variable region of which comprises the amino acid sequence shown in SEQ ID NO: 1 or consists of the same, and the light chain variable region of which comprises the amino acid sequence shown in SEQ ID NO: 2 or consists of the same.

[0005] In some embodiments, the heavy chain constant region of the humanized anti-human Vδ1 TCR monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 9 or consists of the same, and the light chain constant region of which comprises the amino acid sequence shown in SEQ ID NO: 10 or consists of the same.

[0006] In some embodiments, the heavy chain of the humanized anti-human Vδ1 TCR monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO: 11 or consists of the same, and the light chain of which comprises the amino acid sequence shown in SEQ ID NO: 12 or consists of the same.

[0007] Another aspect of the present invention provides a gene encoding the humanized anti-human Vδ1 TCR monoclonal antibody; preferably, the gene encoding the heavy chain of the humanized anti-human Vδ1 TCR monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO: 13 or consists of the same, and the gene encoding the light chain of the humanized anti-human Vδ1 TCR monoclonal antibody comprises the nucleotide sequence shown in SEQ ID NO: 14 or consists of the same.

[0008] Another aspect of the present invention further provides an expression vector, a transgenic cell line, and a host bacterium containing the gene encoding the humanized anti-human Vδ1 TCR monoclonal antibody.

[0009] In another aspect, the present invention provides a method for preparing the humanized anti-human Vδ1 TCR monoclonal antibody, which comprises the following steps:

[0010] 1) Connect the amino acid sequences of the heavy and light chain variable regions of the murine anti-human Vδ1 TCR monoclonal antibody with the amino acid sequences of the humanized antibody heavy and light chain constant regions respectively to obtain the amino acid sequences of the heavy and light chains of the humanized antibody; wherein the heavy chain variable region of the murine anti-human Vδ1 TCR monoclonal antibody comprises the amino acid sequence shown in SEQ ID NO:1 or consists of the same, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO:2 or consists of the same;

[0011] 2) After reverse translation of the amino acid sequences of the heavy and light chains of the humanized antibody obtained in step 1), obtain the coding genes of the heavy and light chains of the humanized antibody respectively; and

[0012] 3) Eukaryotic expression of the coding genes of the heavy and light chains of the humanized antibody obtained in step 2) to obtain the humanized anti-human Vδ1 TCR monoclonal antibody.

[0013] In some embodiments, the application of the humanized anti-human Vδ1 TCR monoclonal antibody in activating and expanding Vδ1 T cells also belongs to the content of the present invention.

[0014] In some embodiments, the method for activating and expanding Vδ1 T cells includes solid-phase in vitro amplification.

[0015] In some embodiments, the humanized anti-human Vδ1 TCR monoclonal antibody also activates and expands the Vδ1 T cells in combination with cytokines; optionally, the cytokines include IL-4, IL-12, IL-15, IFN-γ, IL-21, or a combination thereof.

[0016] In some embodiments, the application of the humanized anti-human Vδ1 TCR monoclonal antibody or the Vδ1 T cells amplified by the humanized anti-human Vδ1 TCR monoclonal antibody in the preparation of a drug for killing tumor cells also belongs to the content of the present invention.

[0017] In some embodiments, the tumor cells include but are not limited to Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells, and SW480 cells.

[0018] The humanized anti-human Vδ1 TCR monoclonal antibody provided by the above technical solution is obtained by connecting the variable regions of the heavy and light chains of a murine monoclonal antibody with the constant regions of the heavy and light chains of a humanized antibody. The variable region of the heavy chain of the obtained humanized anti-human Vδ1 TCR monoclonal antibody contains the amino acid sequence shown in SEQ ID NO: 1, and the variable region of the light chain contains the amino acid sequence shown in SEQ ID NO: 2. The results of the examples show that the humanized anti-human Vδ1 TCR monoclonal antibody provided by the present invention can specifically activate the proliferation of the Vδ1 T cell subset in human γδ T cells in vitro. Especially when the humanized anti-human Vδ1 TCR monoclonal antibody is combined with other cytokines (such as IL-4, IL-12, IL-15, IFN-γ, and IL-21), it can more effectively activate and proliferate Vδ1 T cells, and the proliferated Vδ1 T cells can effectively kill tumor cells, such as Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells, and SW480 cells. Therefore, the Vδ1 T cells obtained by in vitro solid-phase stimulation and proliferation with the humanized anti-human Vδ1 TCR monoclonal antibody provided by the present invention have good cytotoxic effects on tumor cells and can be applied to the adoptive immunotherapy of Vδ1 T lymphocytes for tumors in clinical practice. Brief Description of the Drawings

[0019] Figure 1 Flow cytometry detection results of in vitro activation and amplification of γδ T cells and Vδ1 T subsets by 12 candidate humanized monoclonal antibodies.

[0020] Figure 2 Flow cytometry detection results of in vitro solid-phase activation and amplification of γδ T cells and Vδ1 T subsets by humanized monoclonal antibody HL001.

[0021] Figure 3 Flow cytometry detection results of in vitro activation and amplification of Vδ1 T cells by different doses of humanized monoclonal antibody HL001.

[0022] Figure 4 Flow cytometry detection results of in vitro activation and amplification of Vδ1 T cells by humanized monoclonal antibody HL001 combined with cytokines IL-4, IFN-γ, and IL-15.

[0023] Figure 5 Cytotoxic effect of Vδ1 T cells activated and amplified by humanized monoclonal antibody HL001 combined with cytokines on tumor cell SW480. Detailed Description of the Invention

[0024] In view of the defect in the prior art that it is difficult to efficiently expand Vδ1 T cells in vitro, the present invention provides a humanized anti-human Vδ1 TCR monoclonal antibody, and based on this humanized anti-human Vδ1 TCR monoclonal antibody, a method for efficiently activating and expanding Vδ1 T cells in vitro is provided. The humanized anti-human Vδ1 TCR monoclonal antibody provided by the present invention is obtained by connecting and optimizing the variable regions of the heavy and light chains of a murine anti-human monoclonal antibody with the constant regions of the heavy and light chains of a humanized antibody.

[0025] The following specifically describes the content of the present invention in conjunction with specific embodiments and drawings.

[0026] In the following text, only some exemplary embodiments are simply described. As those skilled in the art can recognize, the described embodiments can be modified in various different ways without departing from the spirit or scope of the present invention. Therefore, the drawings and the description are considered to be exemplary in nature rather than restrictive.

[0027] The methods used in the following embodiments are all conventional methods unless otherwise specified. For specific steps, reference can be made to: "Molecular Cloning: A Laboratory Manual" (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor).

[0028] The acquisition routes of various biological materials described in the embodiments are only provided as a way to obtain them for the purpose of specific disclosure, and should not be a limitation on the source 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 substituted and used according to the prompts in the embodiments.

[0029] The sequences involved in the embodiments can all be synthesized by existing technologies.

[0030] Example 1: Preparation and purification of murine anti-human Vδ1 TCR monoclonal antibody

[0031] This example prepares murine anti-human Vδ1 TCR monoclonal antibody according to the conventional methods known in the art (such as the methods 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).

[0032] In this embodiment, the method for preparing a murine anti-human Vδ1 TCR monoclonal antibody may include the following steps:

[0033] 1) Prepare recombinant human TCRγ4 / δ1-Fc protein as an immunogen to immunize Balb / C mice according to the method in the literature (Flanking V and J Sequences of Complementary Determining Region 3 of T Cell Receptor (TCR) δ1 (CDR3δ1) Determine the Structure and Function of TCRγ4δ1). Specifically, inject 100 μg / mouse / time subcutaneously at multiple points in the abdomen. When immunizing for the first time, mix with an equal volume of Freund's complete adjuvant. When boosting the immunization for the 2nd - 4th time, mix with an equal volume of Freund's incomplete adjuvant. One week later, collect blood from the mouse tail vein, and use the ELISA method to detect the serum antibody titer. Select the mouse with the highest titer, and inject 100 μg / mouse again via the tail vein for boost immunization.

[0034] 2) After 72 h, take the spleen of the mouse, isolate splenocytes, and fuse them with SP2-0 myeloma cells using the polyethylene glycol method.

[0035] 3) Resuspend the fused cells with HAT selective medium (Invitrogen), add 2% methylcellulose semi-solid medium for pressure screening, pick single clone cell clusters, and use the ELISA method to detect and subclone the positive clones in the culture supernatant.

[0036] 4) Inoculate the hybridoma cells secreting monoclonal antibodies intraperitoneally into Balb / C mice, regularly collect ascites, and purify to obtain murine anti-human Vδ1 TCR monoclonal antibody by rProteinA affinity chromatography.

[0037] Example 2: De-novo Sequencing of Murine Anti-human Vδ1 TCR Monoclonal Antibody

[0038] In this example, de-novo sequencing is performed on the murine anti-human Vδ1 TCR monoclonal antibody obtained in Example 1 to analyze the amino acid sequence of the antibody variable region, specifically including the following operations.

[0039] The purified murine anti-human Vδ1 TCR monoclonal antibody sample obtained in Example 1 was digested with five enzymes, namely Typsin, Chymotrypsin, GluC, Elastase, and Pepsin, and then polypeptide mass spectrometry data was collected. The specific steps are as follows: Inject 2 μg of the digested polypeptide sample (into a high-performance liquid chromatograph), perform chromatographic gradient separation for 90 min, and collect and detect data using an Orbitrap-Lumos ultra-high-resolution mass spectrometer. The specific instrument method parameters are as follows:

[0040] High-performance liquid chromatograph: (Easy nLC 1200); Chromatographic column: C18, 3 μm, 75 μm x 15 cm; Sample loading volume: 10 μl. Mobile phase: A: 0.1% Formic acid in water; B: 0.1% Formic acid in 80% Acetonitrile / H 2 O. The chromatographic gradient is shown in Table 1 below.

[0041] Table 1: Chromatographic gradient parameter settings

[0042]

[0043] The mass spectrometry acquisition parameters are 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.

[0044] Master Scan resolution setting: First-level 60,000 @ m / z 200, AGC Target 4e5, first-level maximum ion injection time 40 ms, parent ion scan range: m / z 350 - 1550; Scan Event 1 Activation Type: HCD second-level 30,000 @ m / z 200 AGC Target 1e5, second-level maximum ion injection time 60 ms; Scan Event 2 Activation Type: ETD second-level 30,000 @ m / z 200 AGC Target 1e5, second-level maximum ion injection time 60 ms.

[0045] The obtained mass spectrometry data was subjected to De-novo analysis using Peaks software to obtain the polypeptide sequences after enzymatic digestion. After comparison with antibody databases (such as IMGT, etc.) and splicing, the sequences were manually corrected. After multiple rounds of retrieval and correction, the heavy chain variable region (VH001) sequence (shown in SEQ ID NO: 1) and the light chain variable region (VL001) sequence (shown in SEQ ID NO: 2) of the murine anti-human Vδ1 TCR monoclonal antibody were obtained respectively. The obtained murine anti-human Vδ1 TCR monoclonal antibody was named mVδ1.

[0046] Example 3: Humanization design of murine mVδ1 monoclonal antibody

[0047] This example utilized the monoclonal antibody mVδ1 with the heavy chain variable region sequence of SEQ ID NO: 1 and the light chain variable region of SEQ ID NO: 2 obtained by de novo sequencing in Example 2 above. A humanized anti-human Vδ1 TCR monoclonal antibody was designed by transplanting CDR1, CDR2, and CDR3 of the heavy and light chain variable regions, specifically including the following operations.

[0048] 3.1) Through the diversity analysis of the amino acid sequences in the antigen recognition region of the murine antibody, it was determined that CDR1, CDR2, and CDR3 of VH001 of mVδ1 were the amino acids at positions 26-33, 51-58, and 97-107 of SEQ ID NO: 1 respectively, and CDR1, CDR2, and CDR3 of VL001 were the amino acids at positions 27-36, 54-56, and 93-101 of SEQ ID NO: 2 respectively. Amino acid residues were determined and annotated by the Kabat numbering system.

[0049] 3.2) By comparing the IMGT human antibody heavy and light chain variable region germline gene database (https: / / www.imgt.org / ) and the MOE software, the heavy and light chain variable region germline genes with high homology to the variable regions of the mδ1 antibody were selected as templates respectively. Among them, the humanized heavy chain transplantation frameworks of mδ1 include four types: IGHV3-23D*01-IGHV3-23*01, IGHV3-30-5*02-IGHV3-30*02, IGHV3-48*02, IGHV3-48*04-IGHV3-48*01, and the humanized light chain transplantation frameworks of mδ1 include two types: IGKV3-11*01 and IGKV7-3*01, as shown in Table 2 below. The CDRs of the heavy and light chain variable regions of mδ1 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. Among them, the heavy chain variable regions of the obtained humanized antibodies were named VH11, VH12, VH13, and VH14 respectively, and the light chain variable regions of the obtained humanized antibodies were named VL11 and VL12 respectively, and their amino acid sequence information is shown in Table 2 below.

[0050] Table 2: Heavy and light chain variable region germline gene templates with high homology to the variable regions of mδ1

[0051] Matched human antibody heavy and light chain templates Humanized antibody heavy and light chains Amino acid sequence IGHV3-23D*01-IGHV3-23*01 VH11 SEQ ID NO:3 IGHV3-30-5*02-IGHV3-30*02 VH12 SEQ ID NO:4 IGHV3-48*02 VH13 SEQ ID NO:5 IGHV3-48*04-IGHV3-48*01 VH14 SEQ ID NO:6 IGKV3-11*01 VL11 SEQ ID NO:7 I IGKV7-3*01 VL12 SEQ ID NO:8

[0052] 3.3) The four humanized antibody heavy chain variable regions VH11, VH12, VH13, and VH14 obtained in step 3.2) above were respectively linked to the human antibody heavy chain hinge region and constant region (SEQ ID NO: 9) to obtain four humanized antibody heavy chains, and the two humanized antibody light chain variable regions VL11 and VL12 obtained in step 3.2) above were respectively linked to the human antibody light chain constant region (SEQ ID NO: 10) to obtain two humanized antibody light chains. By randomly combining these humanized antibody heavy chains and humanized antibody light chains, 8 candidate humanized monoclonal antibodies can be obtained, named huVδ1. These 8 humanized antibodies were respectively named huVδ1(H11L11), huVδ1(H12L11), huVδ1(H13L11), huVδ1(H14L11), huVδ1(H11L12), huVδ1(H12L12), huVδ1(H13L12), and huVδ1(H14L12). In addition, since the light chain variable region VL001 of mVδ1 is highly homologous to the humanized graft framework, in this step, the complete light chain obtained by linking the light chain variable region VL001 to the humanized antibody light chain constant region (SEQ ID NO: 10) was combined with the 4 humanized heavy chains obtained after transplantation above to obtain 4 humanized antibodies, named huVδ1(H11VL001), huVδ1(H12VL001), huVδ1(H13VL001), and huVδ1(H14VL001). Additionally, in this step, the VH001 and VL001 of mVδ1 obtained in step 3.1) above were directly linked to the humanized antibody heavy chain and light chain constant region sequences respectively to obtain the heavy chain and light chain of the humanized antibody, named huVδ1(H001) (its amino acid sequence is shown in SEQ ID NO: 11) and huVδ1(L001) (its amino acid sequence is shown in SEQ ID NO: 12) respectively. The obtained humanized anti-Vδ1 monoclonal antibody was named HL001. Therefore, a total of 13 candidate humanized antibodies huVδ1 were obtained.

[0053] 3.4) According to the eukaryotic expression system, reverse translation and codon optimization were performed on the amino acid sequences of the above 13 humanized antibodies huVδ1 to obtain the DNA coding sequences of the complete heavy chain and light chain respectively. The DNA coding sequences were chemically synthesized and inserted into eukaryotic expression vectors 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 and expressed in serum-free medium at 37 °C and 5% CO 2 conditions. After 72 h, the cell supernatant was collected, and the antibodies were purified using a recombinant protein A affinity chromatography medium to obtain the above 13 humanized monoclonal antibodies.

[0054] Example 4: Functional assay of in vitro activation and expansion of Vδ1 T cells by humanized antibodies

[0055] In this example, functional assays were performed on the 13 humanized antibodies obtained in Example 3 above to solid-phase coat and in vitro activate and expand γδ T cells (specifically, the Vδ1 T cell subset in γδ T cells) in peripheral blood mononuclear cells (PBMC) isolated from human peripheral blood. The procedure for in vitro expansion of γδ T cells by the solid-phase coated antibody method is as follows:

[0056] 1) The 13 humanized antibodies recombinantly expressed and purified in Example 3 were used as coating solutions to solid-phase coat culture plates and incubated at 37°C for 2 h;

[0057] 2) Discard the coating solution and wash twice with sterile PBS;

[0058] 3) PBMC were isolated from normal human peripheral whole blood by density gradient centrifugation using human lymphocyte separation medium, and the seeding density was 2×10 6 / well, and suspended culture was carried out in KBM581 medium containing 3% SR and 200 U / ml IL-2;

[0059] 4) After 5 days, the cells were removed from the antibody-coated wells; the medium was changed and the cells were passaged every 2 days;

[0060] 5) On the 10th day of culture, flow cytometry was used to detect the surface biomarkers of the expanded γδ T cells. Among them, FITC-labeled anti-γδT Ab (11-9959-42) was used to detect total γδ T cells; APC-labeled anti-Vδ1 Ab (TCR2730) was used to detect the Vδ1 cell subset.

[0061] The specific operation of flow cytometry detection is as follows: Take 1×10 6Samples of cell numbers were centrifuged at 400 - 600×g for 5 minutes, and the supernatant was discarded. The cells were washed once with 1 mL of 1% BSA / PBS, and the supernatant was discarded. Antibody diluent was prepared by adding 40 μL of 1% BSA / PBS to 2 μL of each antibody per sample to be tested, and the cells were resuspended with the antibody diluent. The reaction was carried out in the dark at 4°C for 30 minutes. 1 mL of PBS was added to each tube for washing, and the cells were centrifuged at 400 - 600×g for 5 minutes. The supernatant was discarded, and 200 μL of PBS was added to each tube to resuspend the cells for detection on the machine. Select and click the density plot icon to plot the graph. Select "SSC-H / A" for the vertical axis and "Time" for the horizontal axis, and circle the region with stable liquid flow. Select and click the density plot icon to plot the graph. Analyze within the circled gate. Select "SSC-H / A" for the vertical axis and "FSC-H / A" for the horizontal axis, and circle the target lymphocyte population. Select and click the density plot icon to plot the graph. Select "FSC-A" for the vertical axis and "FSC-H" for the horizontal axis, and within the lymphocyte population, circle the cell population concentrated at the diagonal position of the graph to exclude adherent somatic cells. Select and click the density plot icon to plot the graph. Select "SSC-A" for the vertical axis and "SSC-H" for the horizontal axis, and within the lymphocyte population, circle the cell population concentrated at the diagonal position of the graph to further exclude adherent somatic cells.

[0062] Percentage of total γδT cells: Select and click the scatter plot icon to plot the graph. Select "Vδ1-APC" for the vertical axis and "γδTCR-FITC" for the horizontal axis, and draw the result of γδT cell positivity (TCRγδ + ), where the Vδ1 T cell subset is the cell population double positive for APC and FITC.

[0063] The results are as Figure 1 and Figure 2 shown, where Figure 1 shows the results of in vitro activation and expansion of γδT cells in PBMC by 12 candidate humanized monoclonal antibodies (huVδ1(H11L11), huVδ1(H11L12), huVδ1(H12L11), huVδ1(H12L12), huVδ1(H13L11), huVδ1(H13L12), huVδ1(H14L11), huVδ1(H14L12), huVδ1(H11VL001), huVδ1(H12VL001), huVδ1(H13VL001), and huVδ1(H14VL001)) respectively, Figure 2 shows the result of in vitro activation and expansion of γδT cells in PBMC by the candidate humanized monoclonal antibody HL001. As shown by Figure 1 and Figure 2As can be seen from the results shown, compared with the control without solid-phase coating of monoclonal antibody (IL2 control), only the humanized monoclonal antibody HL001 can effectively expand total γδT cells, mainly dominated by the Vδ1 T cell subset, and the proportion of Vδ1 T cells can reach 33%. However, the other 12 candidate humanized monoclonal antibodies (all obtained by CDR transplantation) cannot effectively expand total γδT cells and the Vδ1 T cell subset. Therefore, the present invention determines a humanized anti-human Vδ1 TCR monoclonal antibody HL001 that can effectively expand the Vδ1 T cell subset in γδT cells in vitro. The amino acid sequence of its heavy chain variable region is shown in SEQ ID NO:1, and the amino acid sequence of its light chain variable region is shown in SEQ ID NO:2; the amino acid sequence of the complete heavy chain of the humanized anti-human Vδ1 TCR monoclonal antibody HL001 is shown in SEQ ID NO:11 (the corresponding nucleotide coding sequence is shown in SEQ ID NO:13), and the amino acid sequence of the complete light chain is shown in SEQ ID NO:12 (the corresponding nucleotide coding sequence is shown in SEQ ID NO:14).

[0064] Example 5: Functional assay of in vitro activation and expansion of Vδ1 T cells with different doses of humanized anti-human Vδ1 TCR monoclonal antibody or further combined with cytokines

[0065] This example was carried out according to the operation of Example 4 to detect the effect of in vitro activation and expansion of Vδ1 T cells with different doses of the humanized monoclonal antibody HL001 or further combined with cytokines (such as IL-4, IL-12, IL-15, IFN-γ and IL-21). The specific operation is as follows:

[0066] 5.1 Functional assay of in vitro activation and expansion of Vδ1 T cells with different doses of humanized monoclonal antibody HL001

[0067] 1) Coat culture plates with HL001 at concentrations of 1 μg / ml, 2 μg / ml and 4 μg / ml respectively as coating solutions, and incubate at 37°C for 2 h;

[0068] 2) Discard the coating solution and wash twice with sterile PBS;

[0069] 3) Use the density gradient centrifugation method of human lymphocyte separation solution to isolate PBMC from normal human peripheral blood, and the seeding density is 2×10 6 / well, and suspend and culture in KBM581 medium containing 3% SR and 200 U / ml IL-2;

[0070] 4) After 5 days, transfer the cells out of the antibody-coated wells; change the medium and passage every 2 days;

[0071] 5) On the 10th day of culture, flow cytometry was used to detect the surface biomarkers of expanded γδ T cells, and anti-Vδ1 Ab labeled with FITC was used to detect the Vδ1 T cell subset.

[0072] The results were as Figure 3 shown. Among the three doses of the HL001 humanized monoclonal antibody used, the purity of Vδ1 T cells activated and expanded in vitro was relatively higher at a dose of 4 μg / ml, reaching over 40% on the 10th day of detection. The purity of activated and expanded Vδ1 T cells was dose-dependent on the concentration of the immobilized antibody.

[0073] 5.2 Functional assay of further co - activating and expanding Vδ1 T cells with HL001 humanized monoclonal antibody and cytokines in vitro

[0074] 1) Coat the culture plate with 4 μg / ml of HL001 as the coating solution and incubate at 37 °C for 2 h.

[0075] 2) Discard the coating solution and wash twice with sterile PBS.

[0076] 3) Use density gradient centrifugation with human lymphocyte separation medium to isolate PBMC from normal human peripheral blood, and the seeding density is 2×10 6 / well. Suspend and culture with KBM581 medium containing 3% SR and 200 U / ml IL - 2 combined with different cytokines IL - 4 (100 ng / ml), IL - 12 (50 ng / ml), IL - 15 (50 ng / ml), IL - 21 (10 ng / ml), IFN - γ (100 ng / ml), or their combinations.

[0077] 4) After 5 days, transfer the cells out of the antibody - coated wells; after transfer, change the medium to KBM581 medium containing 3% SR and different cytokines IL - 4 (100 ng / ml), IL - 12 (50 ng / ml), IL - 15 (50 ng / ml), IL - 21 (10 ng / ml), IFN - γ (100 ng / ml), or their combinations. Change the medium and passage every 2 days.

[0078] 5) On the 10th day of culture, flow cytometry was used to detect the surface biomarkers of expanded γδ T cells. Anti - Vδ2 Ab labeled with PB was used to detect the Vδ2 cell subset; anti - Vδ1 Ab labeled with FITC was used to detect the Vδ1 cell subset.

[0079] The results were as Figure 4As shown, it exemplarily shows the effect of the solidified coated HL001 humanized monoclonal antibody combined with the addition of IL-4, IFN-γ, and IL-15 to activate and expand Vδ1 T cells in vitro. The purity of Vδ1 T cells detected on the 10th day can reach 72%. When the HL001 humanized monoclonal antibody is combined with other cytokines or a combination of cytokines, similar effects can also be obtained, which will not be elaborated here. Figure 4 Similar effects

[0080] Example 6: Cytotoxic effect of Vδ1 T cells expanded in vitro by HL001 humanized antibody on tumor cells

[0081] This example uses the Vδ1 T cells obtained by activating and expanding PBMC in vitro with the HL001 humanized antibody combined with the addition of cytokines IL-4, IFN-γ, and IL-15 in Example 5 above, and uses the RTCA method to detect its cytotoxic effect on the tumor cell SW480. The specific operations include the following:

[0082] Place the E-Plate 16 on the RTCA S16 Station, and the system will automatically scan ("Scan Plate") to check for good contact. Click "New Experiment" on the home page, enter the "Protocol" page, select the experimental type "Proliferation / Cytocoxicty" to be carried out, select "Standard Protocol", and determine the experimental procedure. Click the "Layout" page and 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 and add 50 μL of the well-mixed target cell suspension to the "target cell well, effector-to-target ratio experimental well" so that the number of cells in each well is 1.5×10 4 / well / 50 μL (3×10 5 / mL); supplement 50 μL of the target cell culture medium to the "effector cell well". Place the E-Plate 16 plate in the biosafety cabinet at room temperature for 30 minutes and then transfer it to the RTCA S16 Station in the incubator to ensure that the green indicator light is on, and incubate the cells for 16 - 20 hours. After the system automatically scans "Scan Plate", click to start Step2 to perform real-time dynamic detection of cell proliferation. Collect the effector cell concentration adjusted to 1.5×10 6 / mL. Take out the E-Plate16 and place it in the laminar flow hood. Add 50 μL / well of the effector cell mixture to the "effector-to-target ratio experimental well, effector cell well". Place the E-Plate16 detection plate on the RTCA S16 detection platform for real-time monitoring, and ensure that the green indicator light is on for normal operation of the instrument.

[0083] Calculate the killing efficiency of Vδ1 T cells against target cells using the following formula:

[0084]

[0085] The results are as Figure 5 shown. Among them, the results of the "target cell well" are as shown by the curve SW480, the results of the "effector-to-target ratio experimental well" are as shown by SW480 + Vδ1 T, and the results of the "effector cell well" are as shown by Vδ1 T - control. It can be seen that compared with the results of the "target cell well", adding Vδ1 T cells to the "effector-to-target ratio experimental well" can significantly reduce the cell index. Especially after 36 h of adding Vδ1 T cells, the cell index is more significantly reduced, indicating that the Vδ1 T cells stimulated by the HL001 humanized antibody combined with cytokine addition can effectively kill SW480 tumor cells.

[0086] According to the above operation, the cytotoxic effects of the Vδ1 T cells obtained by in vitro activation and expansion of PBMC using the HL001 humanized antibody combined with the addition of cytokines IL-4, IFN-γ, and IL-15 in Example 5 above on lymphoma cell lines Daudi cells, K562 cells, OVCAR-8 cells, and HepG2 cells were also tested, and they also had Figure 5 similar cytotoxic effects as shown, which will not be elaborated here.

[0087] In summary, from the results of Examples 4 - 6, it can be seen that the humanized anti-human Vδ1 TCR monoclonal antibody provided by the present invention can efficiently stimulate the expansion of Vδ1 T cells in vitro, and the Vδ1 T cells obtained by in vitro stimulation and expansion have good cytotoxic effects on various tumor cells (including Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells, SW480 cells, etc.). Therefore, the obtained Vδ1 T cells can be used for adoptive immunotherapy of tumors clinically by adoptive transfer.

[0088] Finally, it should be noted that the above are only the preferred embodiments of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A humanized anti-human Vδ1 TCR monoclonal antibody, wherein the heavy chain variable region consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO:

2.

2. The humanized anti-human Vδ1 TCR monoclonal antibody according to claim 1, wherein the heavy chain constant region consists of the amino acid sequence shown in SEQ ID NO: 9, and the light chain constant region consists of the amino acid sequence shown in SEQ ID NO:

10.

3. The humanized anti-human Vδ1 TCR monoclonal antibody according to claim 1 or 2, wherein the heavy chain consists of the amino acid sequence shown in SEQ ID NO: 11, and the light chain consists of the amino acid sequence shown in SEQ ID NO:

12.

4. A gene encoding the humanized anti-human Vδ1 TCR monoclonal antibody according to any one of claims 1-3.

5. The gene according to claim 4, wherein the gene encoding the heavy chain of the humanized anti-human Vδ1 TCR monoclonal antibody consists of the nucleotide sequence shown in SEQ ID NO: 13, and the gene encoding the light chain of the humanized anti-human Vδ1 TCR monoclonal antibody consists of the nucleotide sequence shown in SEQ ID NO:

14.

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

7. A method for preparing the humanized anti-human Vδ1 TCR monoclonal antibody according to any one of claims 1-3, which comprises the following steps: 1) Connecting the amino acid sequences of the heavy and light chain variable regions of the murine anti-human Vδ1 TCR monoclonal antibody 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 and light chains of the humanized antibody; wherein the heavy chain variable region of the humanized anti-human Vδ1 TCR monoclonal antibody consists of the amino acid sequence shown in SEQ ID NO: 1, and the light chain variable region consists of the amino acid sequence shown in SEQ ID NO: 2; 2) After reverse translation of the amino acid sequences of the heavy and light chains of the humanized antibody obtained in step 1), respectively obtain the encoding genes of the heavy and light chains of the humanized antibody; and 3) Eukaryotic expression of the encoding genes of the heavy and light chains of the humanized antibody obtained in step 2) to obtain the humanized anti-human Vδ1 TCR monoclonal antibody.

8. Use of the humanized anti-human Vδ1 TCR monoclonal antibody according to any one of claims 1-3 in the preparation of a reagent for activating and expanding Vδ1 T cells.

9. The use according to claim 8, wherein the amplification method includes solid-phase in vitro amplification; and / or the humanized anti-human Vδ1 TCR monoclonal antibody also combines with cytokines to activate and expand the Vδ1 T cells.

10. The use according to claim 9, wherein the cytokines include IL-4, IL-12, IL-15, IFN-γ, IL-21, or a combination thereof.

11. Use of the humanized anti-human Vδ1 TCR monoclonal antibody according to any one of claims 1-3 or Vδ1 T cells amplified from the humanized anti-human Vδ1 TCR monoclonal antibody according to any one of claims 1-3 in the preparation of a medicament for killing tumor cells; wherein the tumor cells are selected from Daudi cells, K562 cells, OVCAR-8 cells, HepG2 cells and SW480 cells.

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