NKG2D fully humanized antibody as well as preparation method and application thereof

Abstract

The invention relates to an NKG2D fully humanized antibody as well as a preparation method and application thereof. The amino acid sequence of the NKG2D full-human antibody is one of SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33 and SEQ ID NO. 34. The invention further discloses a kit for detecting the NKG2D full-human antibody. According to the present invention, the NKG2D completely humanized antibody can well activate the activity of NK cells, such that the immune response can be accurately adjusted, the non-specific immunosuppression can be reduced, and in addition, the NKG2D completely humanized antibody provides the new hope and the new strategy for the tumor immunotherapy through the unique biological function and the synergistic effect with the existing therapy.

Description

Technical Field

[0001] The present invention relates to the technical field of antibody preparation, and in particular to a fully human NKG2D antibody and a preparation method and application thereof. Background Art

[0002] NKG2D (Group 2 Natural Killer Factor Member D) is an activation cell surface protein that is primarily expressed on cytotoxic immune cells. NKG2D is present in large quantities on all NK cells, NKT cells, and γδT cell subsets. Although naive human CD8+ T cells express NKG2D, in mice they only upregulate their expression after activation. CD4+ T cells generally do not express NKG2D even after activation, but in humans their expression can be induced under certain pathological conditions, such as Crohn's disease, juvenile-onset lupus, and cytomegalovirus infection. The molecular structure of NKG2D allows it to bind to many structurally different MHC-I-like ligands. What NKG2D ligands have in common is that their expression is generally low under steady-state conditions. In contrast, upon cellular stress, such as infection or oncogenic transformation, their expression can be highly induced. In humans, NKG2D ligands are six members of the MICA, MICB, and ULBP families.

[0003] NKG2D is a homodimer of two disulfide-linked transmembrane proteins with a very short intracellular domain that lacks signaling properties. In mice, NKG2D uses the adaptor molecules DAP10 and DAP12 to coordinate its signaling, while in humans, NKG2D binds exclusively to DAP10. DAP10 and DAP12 initiate distinct signaling cascades. DAP10 possesses a YINM motif that allows binding to p85 of the phosphatidylinositol 3-kinase (PI3K). Furthermore, DAP10 binds Grb2, which in turn binds to Vav1. DAP12 contains an immunoreceptor tyrosine activation motif that is phosphorylated by the Src kinase following NKG2D triggering. This event allows for the binding and activation of the tyrosine kinases Syk and Zap70.

[0004] NKG2D plays an important role in identifying and eliminating potentially dangerous cells. It has been shown to mediate immune responses against tumors, virus-infected cells, and organ transplants. Therefore, NKG2D was initially thought to mediate direct cytotoxicity in response to the encounter of ligands on stressed target cells. However, in most cases, NKG2D can only mediate immune cell activation if it appears in an inflammatory environment. Both NK cells and T cells usually require secondary signals before NKG2D can mediate a measurable effect. Therefore, the main function of NKG2D seems to be to regulate signal transduction through other receptors. Its unique feature is that it can inhibit and enhance the signal transduction of a large number of receptors in multiple immune cell subsets with different entities and at different stages of the immune cell life cycle (such as hematopoietic development, primary immunity, and effector response).

[0005] NKG2D has great potential as a therapeutic target due to its potency to enhance cytolytic immune responses against important diseases such as cancer. Given the important role of NKG2D, the development of anti-NKG2D antibodies is needed to investigate the therapeutic use of NKG2D. Summary of the Invention

[0006] The first object of the present invention is to provide a fully human NKG2D antibody, wherein the fully human NKG2D antibody comprises a framework region and a complementary determining region, wherein the complementary determining region comprises CDR1, CDR2 and CDR3.

[0007] Among them, the complementary determining region CDR1 sequence of VH is SEQ ID NO.1, the complementary determining region CDR2 sequence is SEQ ID NO.2, and the complementary determining region CDR3 sequence is SEQ ID NO.3;

[0008] or the complementarity determining region CDR1 of VH is SEQ ID NO.07, the complementarity determining region CDR2 is SEQ ID NO.08, and the complementarity determining region CDR3 is SEQ ID NO.09;

[0009] or the complementarity determining region CDR1 of VH is SEQ ID NO.13, the complementarity determining region CDR2 is SEQ ID NO.14, and the complementarity determining region CDR3 is SEQ ID NO.15;

[0010] Alternatively, the complementary determining region CDR1 of VH is SEQ ID NO.16, the complementary determining region CDR2 is SEQ ID NO.17, and the complementary determining region CDR3 is SEQ ID NO.18

[0011] The complementary determining region CDR1 of VL is SEQ ID NO.4, the complementary determining region CDR2 is SEQ ID NO.5, and the complementary determining region CDR3 is SEQ ID NO.6;

[0012] or the complementarity determining region CDR1 of VL is SEQ ID NO.10, the complementarity determining region CDR2 is SEQ ID NO.11, and the complementarity determining region CDR3 is SEQ ID NO.12;

[0013] or the complementarity determining region CDR1 of VL is SEQ ID NO.16, the complementarity determining region CDR2 is SEQ ID NO.17, and the complementarity determining region CDR3 is SEQ ID NO.18;

[0014] or the complementarity determining region CDR1 of VL is SEQ ID NO.22, the complementarity determining region CDR2 is SEQ ID NO.23, and the complementarity determining region CDR3 is SEQ ID NO.24;

[0015] As a preferred technical solution, the complementary determining region CDR1 sequence of VH is SEQ ID NO.1, the complementary determining region CDR2 sequence is SEQ ID NO.2, and the complementary determining region CDR3 sequence is SEQ ID NO.3;

[0016] The complementary determining region CDR1 of VL is SEQ ID NO.4, the complementary determining region CDR2 is SEQ ID NO.5, and the complementary determining region CDR3 is SEQ ID NO.6;

[0017] Furthermore, the NKG2D human antibody has an amino acid sequence selected from any one of the following: SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33.

[0018] As a preferred technical solution, the NKG2D human antibody has an amino acid sequence selected from any one of the following: SEQ ID NO.31.

[0019] Furthermore, the nucleotide sequence encoding the amino acid sequence of the NKG2D fully human antibody is one of the following sequences: SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41.

[0020] As a preferred technical solution, the nucleotide sequence encoding the amino acid sequence of the NKG2D fully human antibody is one of the following sequences: SEQ ID NO.39.

[0021] The second object of the present invention is to provide a molecular expression vector, which comprises one of the nucleotide sequences of SEQ ID NO.39 to SEQ ID NO.42.

[0022] The third object of the present invention is to provide a host cell containing the above-mentioned molecular expression vector, wherein the host cell is a mammalian cell.

[0023] Furthermore, the mammalian cells are selected from Expi 293F cells or CHO cells.

[0024] The fourth object of the present invention is to provide a method for preparing a fully human NKG2D antibody, the specific steps of which are as follows:

[0025] S1. Based on the protein sequence and gene sequence information of NKG2D, express and screen the antigen, and connect a His-tag to its C-terminus to obtain a modified nucleic acid sequence;

[0026] S2. Cloning the nucleic acid sequence obtained in step S1 into an expression vector, and expressing the antigen using mammalian cells to obtain the NKG2D / His antigen;

[0027] S3. Using PBMC cells from normal donors as raw materials, RNA was extracted and reverse transcribed into cDNA. Antibody gene fragments were obtained by PCR and cloned into phage vectors for library construction.

[0028] S4, using the NKG2D / His antigen obtained in step S2 to perform antibody screening on the gene fragment library constructed in step S3;

[0029] S5. Express the antibody sequence obtained in step S4 through a mammalian cell system, and further obtain a fully human antibody against NKG2D through screening. The fully human antibody against NKG2D has high sensitivity and specificity.

[0030] Furthermore, in step S4, the sequence of the antibody gene fragment includes the nucleotide sequence shown as SEQ ID NO.39 to SEQ ID NO.42.

[0031] As a preferred technical solution, the sequence of the antibody gene fragment includes the nucleotide sequences shown as SEQ ID NO.39 to SEQ ID NO.40.

[0032] Furthermore, in step S5, further screening experiments include ELISA and FACS detection.

[0033] The fifth object of the present invention is to provide a use of a fully human NKG2D antibody in the preparation of a reagent for detecting NK cell surface proteins.

[0034] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0035] The preparation method of the present invention is simple, and the antibody is efficiently expressed in a mammalian cell expression system, can be processed and modified after translation, and its activity is closer to that of a natural antibody.

[0036] Compared with the comparative patent, the present invention screens out fully human antibodies by utilizing a fully human library. The present invention and the comparative patent are different in terms of library type and antibody type. The blocking effect of a fully human antibody sequence screened out by the present invention is better than that of Yangshen. The amino acid sequence of the fully human antibody is 100% from humans, so the rejection reaction is the lowest, the safety is the best, and the drugability is better. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the results of the binding experiment between HUMAN-NKG2D antigen (expressed NKG2D / His antigen) and antibodies (N21, N24, N35 and N6);

[0038] Figure 2 Schematic diagram of the results of the binding assay between HUMAN-NKG2D cell line (NKG2D antigen on the cell surface) and antibodies (N21, N24, N35 and N6);

[0039] Figure 3 ; Schematic diagram of the experimental results of binding of HUMAN-Primary NK cells to antibodies (N21, N24, N35 and N6);

[0040] Figure 4 This is a schematic diagram of the results of the protein-level antibody blocking experiment;

[0041] Figure 5 This is a schematic diagram of the results of the antibody blocking experiment at the cell level;

[0042] Figure 6 Schematic diagram of cell killing results; DETAILED DESCRIPTION

[0043] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments. This embodiment is implemented based on the technical solution of the present invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.

[0044] As used herein, the terms "VH" and "VL" refer to the variable regions of the heavy and light chains of antibodies. The variable region is composed of discrete, well-defined subregions called "complementarity determining regions" (CDRs, also known as HVRs (hypervariable regions)) and "framework regions" (FRs). CDRs refer to the amino acids within the variable region of an antibody that confer antigen specificity and / or binding affinity, separated by FRs. There are three CDRs (VLCDR1, VLCDR2, and VLCDR3) in each antibody light chain variable region, and three CDRs (VHCDR1, VHCR2, and VHCDR3) in each antibody heavy chain variable region; the "complementarity determining regions" (CDRs) of the VH and VL regions alternate with more conserved regions of the "framework regions" (FRs); each VH and VL consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.

[0045] As used herein, the term "expression" refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. This process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof.

[0046] If no specific techniques or conditions are specified in the present examples, the operations were performed according to conventional techniques and instrument specifications in the art; if no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0047] In the following examples, the amino acid sequences are shown in Table 1:

[0048] Table 1 Amino acid sequences of the CDR regions of the NKG2D fully human antibody of the present invention

[0049]

[0050]

[0051]

[0052]

[0053]

[0054] The following describes some embodiments of the present invention in detail with reference to the accompanying drawings. In the absence of conflict, the following embodiments and features in the embodiments may be combined with each other.

[0055] Example 1

[0056] This example provides a method for preparing a fully human NKG2D antibody, and the specific steps are as follows:

[0057] S1. Based on the protein sequence and gene sequence information of NKG2D, its extracellular sequence was expressed, and a His-tag was connected to its C-terminus. The protein was cloned into the pCDNA3.4 expression vector and expressed in mammalian cells Expi 293F to obtain the NKG2D / His antigen for subsequent screening and detection.

[0058] S2. Using normal donor PBMC cells (purchased from Miaoshun Biotechnology Co., Ltd.) as raw materials, the RNA of the obtained cells was extracted using the Trizol method and reversed into cDNA using oligo(dT); gene fragments were obtained by PCR amplification and then cloned into the phage vector pComb3XSS (purchased from Addgene) for library construction.

[0059] S3, using the NKG2D / His antigen obtained in step S1 to perform antibody screening;

[0060] S3-1. First round of screening

[0061] S3-1-1. Prepare two labeled immunotubes (one for positive screening and one for negative screening) and place them on a clean bench. Wash twice with PBST and twice with PBS.

[0062] Coating antigen: Dilute the NKG2D / His antigen expressed in step S1 with PBS to 50 μg / 2 mL / tube and rotate overnight at 4°C.

[0063] S3-1-2. On the second day, the antigen was recovered, washed 3 times with PBST, washed twice with PBS, and 5×10 12 (5×10 12 / 1.5×10 13 4 mL of the packaged phage obtained in step S2 was added and the volume was adjusted to 4 mL with 1% BSA for blocking. Meanwhile, only 4 mL of 1% BSA was added to the positive screening tube for blocking and incubated with rotation at room temperature for 1 hour.

[0064] S3-1-3. After blocking, take the supernatant from the negative screening tube and add it to the positive screening immunotube. Incubate with rotation at room temperature for 1 hour. Remove the supernatant and wash 8 times with PBST and twice with PBS.

[0065] S3-1-4. Elute the bound phage by adding 1 mL of trypsin and incubate with rotation at room temperature for 30 minutes.

[0066] S3-1-5. Infection: Pipette 500 μL of the phage elution mixture into 5 mL of shaken SS320 bacterial suspension (purchased from Lucigen) (OD600: 0.4-0.6), mix thoroughly, and incubate at 37°C for 30 min. Simultaneously, take 10 μL of the remaining 500 μL of phage elution and add it to the shaken SS320 bacterial suspension. Perform a ten-fold serial dilution, 8-9 times, and incubate at 37°C for 30 min.

[0067] S3-1-6. Centrifuge the infected bacterial solution at 3000 rpm for 5 minutes to enrich the bacteria, discard the supernatant, and resuspend the bacteria in about 300 μL of the remaining culture medium. Spread the plate (2YT plate with A+ and T+), invert the plate, and place it in a 37°C incubator overnight, and titrate it at the same time.

[0068] S3-2, Second Round of Selection

[0069] Same as the first round of screening, except for the differences shown in Table 2 below.

[0070] S3-3, the third round of selection

[0071] Same as the first round of screening, except for the differences shown in Table 2 below.

[0072] Table 2 Differences among three rounds of solid phase screening

[0073] Round Input phage(pfu) Coating antigen amount (μg) 1 round <![CDATA[~5×10 12 ]]> 50 2 rounds <![CDATA[~5×10 11 ]]> 30 3 rounds <![CDATA[~5×10 10 ]]> 15

[0074] S4. The positive clones prepared in step S3 were screened by ELISA to obtain antibodies (KD6, KE6, F12, and C7). The specific detection process is as follows:

[0075] S4-1. Coat the ELISA plate with the NKG2D / His antigen expressed in step S1 at a concentration of 1 μg / mL at 4°C overnight.

[0076] S4-2. The next day, the cells were washed three times with PBST and blocked with 1% BSA at room temperature for 1 h.

[0077] S4-3, wash three times with PBST, add 200 μL of the supernatant of overnight shaken monoclonal bacteria to each well, and incubate at 37°C for 1.5 h;

[0078] S4-4, wash three times with PBST, add 1:10000 diluted Anti-HIS-HRP secondary antibody to each well and incubate at 37°C for 1 h;

[0079] S4-5, wash three times with PBST, add 100 μL TMB substrate, incubate at 37°C for 10 min, add 50 μL 0.1 M H2SO4 to stop the reaction, and measure OD 450nm.

[0080] S5. Plasmid construction and extraction: construct the positive antibody sequence and clone it into the expression vector.

[0081] The antibody sequences (SEQ ID NO. 39 to SEQ ID NO. 41) obtained by screening were cloned into the pCDNA3.4 expression vector to obtain antibodies (N21, N24, N35 and N6).

[0082] S6. Cell transfection: The day before, seed cells to an appropriate density and transfect with an antibody heavy chain to light chain molar ratio of 2:3. Culture in a 37°C, 5% CO2 cell culture incubator for 12 consecutive days.

[0083] S7, the antibody obtained in step S6 is expressed and tested for binding to NKG2D protein (i.e., NKG2D / His antigen obtained in step S1). ELISA results show (e.g. Figure 1 As shown), the ELISA results of the screened N21, N24, N35 and N6 antibody sequences were all excellent. In order to further verify the binding of positive antibodies, CELL BINDING ASSAY (cell binding test) detection was performed. The screened antibodies were started at 200nM and diluted 3-fold in a series, added to the pre-plated cells, incubated at 4°C for 1 hour, washed twice with MACS BUFFER (components of PBS + 10% FBS + 2mM EDTA), and the secondary antibody (Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (imported), purchased from Thermo) was added, incubated at 4°C for 30 minutes, washed twice with MACS BUFFER, and detected by flow cytometry. The results were shown as follows Figure 2 The results showed that the fully human antibodies finally obtained through CELL BINDING ASSAY screening had good binding effects with cells.

[0084] S8. Select the antibody sequences (N21, N24, N35 and N6) with lower EC50 than Yang Shen in the CELL BINDING ASSAY results for binding experiment with primary NK cells. The screened antibodies were diluted 3-fold starting at 200 nM and added to the pre-plated cells. The cells were incubated at 4°C for 1 hour, washed twice with MACS BUFFER (composed of PBS + 10% FBS + 2mM EDTA), and the secondary antibody (Goat anti-Human IgG (H + L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 488 (imported), purchased from Thermo) was added. The cells were incubated at 4°C for 30 minutes, washed twice with MACS BUFFER, and detected by flow cytometry. The results were as follows: Figure 3 The results showed that the fully human antibodies finally obtained through CELL BINDING ASSAY screening had good binding effects on cells. The results showed that the N21, N24, N35 and N6 antibody sequences could all bind to primary NK cells.

[0085] S9, the antibody sequence (N21, N24, N35 and N6) was subjected to a protein level blocking experiment, the ligand MICA was incubated with the NKG2D / His antigen expressed in step S1 of the previous day at a working concentration of 20 μg / mL, and the antibody was added to the antigen at a 3-fold serial dilution starting from 200 nM, incubated at 37°C for 1 hour, washed three times with PBST, added Anti-Human-FC-HRP as a secondary antibody, incubated at 37 for 60 minutes, washed three times with 1% PBST, added 50 μL of color development solution (TMB color development solution, purchased from Beyotime), incubated at 37 for 5 minutes, added color stop solution, and read the value on the instrument. The results are shown as follows Figure 5 The results showed that the screened N21, N24, N35 and N6 sequence blocks all had good blocking effects.

[0086] S10. The antibody sequences (N21, N24, N35 and N6) were subjected to a cell-level blocking experiment. The ligand MICA was incubated with CHO-K1 cells overexpressing NKG2D at a working concentration of 20 μg / mL, and the screened antibodies were diluted 3-fold starting at 200 nM and added to CHO-K1 cells overexpressing NKG2D in advance. The cells were incubated at 4°C for 1 hour, washed twice with MACS BUFFER (PBS + 10% FBS + 2mM EDTA), and the secondary antibody was added. The cells were incubated at 4°C for 30 minutes, washed twice with MACS BUFFER, and detected by flow cytometry. The results were as follows: Figure 5 The results showed that the screened N21, N24, N35 and N6 sequence blocks all had good blocking effects.

[0087] S11. The antibody sequences (N21, N24, N35 and N6) were subjected to cell killing experiments, and the screened antibodies were diluted to 10nM, 50nM and 100nM as the starting point. 1000 cells / well of Raji cells were used as target cells, and 50000 cells / well of effector NK cells were added. The cells were co-cultured at 37°C for 4 hours. The killing effect was tested by CFSE method and detected by flow cytometry. The results showed that Figure 6 The results showed that the screened N21, N24, N35 and N6 sequence blocks all had good effects in promoting NK cell killing.

[0088] The above description of the embodiments is intended to facilitate understanding and use of the invention by those skilled in the art. It will be apparent that those skilled in the art can readily make various modifications to these embodiments and apply the general principles described herein to other embodiments without requiring inventive effort. Therefore, the present invention is not limited to the above-described embodiments. Improvements and modifications made by those skilled in the art based on the disclosure of the present invention, without departing from the scope of the present invention, should be within the scope of protection of the present invention.

Claims

1. A fully human NKG2D antibody, characterized in that: The NKG2D fully human antibody includes a framework region and a complementary determining region, wherein the complementary determining region includes CDR1, CDR2 and CDR3. wherein the complementary determining region CDR1 sequence of VH is SEQ ID NO.1, the complementary determining region CDR2 sequence is SEQ ID NO.2, and the complementary determining region CDR3 sequence is SEQ ID NO.3; or the complementarity determining region CDR1 of VH is SEQ ID NO.07, the complementarity determining region CDR2 is SEQ ID NO.08, and the complementarity determining region CDR3 is SEQ ID NO.09; or the complementarity determining region CDR1 of VH is SEQ ID NO.13, the complementarity determining region CDR2 is SEQ ID NO.14, and the complementarity determining region CDR3 is SEQ ID NO.15; The complementary determining region CDR1 of VL is SEQ ID NO.4, the complementary determining region CDR2 is SEQ ID NO.5, and the complementary determining region CDR3 is SEQ ID NO.6; or the complementarity determining region CDR1 of VL is SEQ ID NO.10, the complementarity determining region CDR2 is SEQ ID NO.11, and the complementarity determining region CDR3 is SEQ ID NO.12; Alternatively, the complementary determining region CDR1 of VL is SEQ ID NO.16, the complementary determining region CDR2 is SEQ ID NO.17, and the complementary determining region CDR3 is SEQ ID NO.

18.

2. The fully human NKG2D antibody according to claim 1, characterized in that: The NKG2D human antibody has an amino acid sequence selected from any one of the following: SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.

34.

3. The fully human NKG2D antibody according to claim 2, characterized in that: The nucleotide sequence encoding the amino acid sequence of the NKG2D fully human antibody is one of the following sequences: SEQ ID NO.39, SEQ ID NO.40, or SEQ ID NO.

42.

4. A molecular expression vector, characterized in that The vector comprises one of the nucleotide sequences of SEQ ID NO.39 to SEQ ID NO.

42.

5. A host cell containing the molecular expression vector according to claim 4, characterized in that The host cell is a mammalian cell.

6. The host cell according to claim 5, characterized in that The mammalian cells are selected from Expi293F cells or CHO cells.

7. A method for preparing a fully human NKG2D antibody according to any one of claims 1 to 3, characterized in that: The specific steps are as follows: S1. Based on the protein sequence and gene sequence information of NKG2D, express and screen the antigen, and connect a His-tag to its C-terminus to obtain a modified nucleic acid sequence; S2. Cloning the nucleic acid sequence obtained in step S1 into an expression vector, and expressing the antigen using mammalian cells to obtain the NKG2D / His antigen; S3. Using PBMC cells from normal donors as raw materials, RNA was extracted and reverse transcribed into cDNA. Antibody gene fragments were obtained by PCR and cloned into phage vectors for library construction. S4, using the NKG2D / His antigen obtained in step S2 to perform antibody screening on the gene fragment library constructed in step S3; S5. Express the antibody sequence obtained in step S4 through a mammalian cell system, and further obtain a fully human antibody against NKG2D through screening.

8. The method for preparing a fully human NKG2D antibody according to claim 7, wherein: In step S4, the sequence of the antibody gene fragment includes the nucleotide sequence shown in SEQ ID NO.39 to SEQ ID NO.

42.

9. The method for preparing a fully human NKG2D antibody according to claim 7, wherein: In step S5, further screening experiments include ELISA and FACS detection.

10. Use of the fully human NKG2D antibody according to any one of claims 1 to 3 in preparing a reagent for detecting T / NK cell surface proteins.

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