Envelope glycoprotein for pseudotyped lentiviral vector for transduction of NK cells and use of envelope glycoprotein

Abstract

Disclosed in the present invention are an envelope glycoprotein for packaging a pseudotyped lentiviral vector for transduction of NK cells and a use of the envelope glycoprotein. The envelope glycoprotein comprises an extracellular region, a transmembrane region and a cytoplasmic region, wherein the amino acid sequence or the mutation sequence of the extracellular region is as shown in SEQ ID NO: 1 or SEQ ID NO: 2; the amino acid sequence or the mutation sequence of the transmembrane region is as shown in SEQ ID NO: 3 or SEQ ID NO: 4; and the amino acid sequence or the mutation sequence of the cytoplasmic region is shown as SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8. A BaEV envelope glycoprotein mutant replaces VSV-G to package a lentiviral pseudovirus, so that a high virus titer of 7.33E+07 TU / mL is maintained, NK cells can be effectively recognized, and entry of viruses into the cells can be mediated, thereby achieving the objective of efficiently transfecting the NK cells.

Description

Envelope glycoprotein for pseudotyped lentiviral vector transduction of NK cells and its application Technical Field

[0001] The present invention relates to the field of preparation of immune cell biological preparations, and in particular to an envelope glycoprotein of a pseudotyped lentiviral vector for transducing NK cells and applications thereof. Background Art

[0002] Natural killer cells (NK cells) were discovered in peripheral blood 30 years ago. NK cells typically contain large amounts of perforin and granzyme B. When activated, NK cells release perforin and granzyme B to attack target cells. NK cells can also secrete cytokines such as IFN-γ, TNF-α, GM-CSF, and IL-3, which can act directly on target cells or activate other types of immune cells to attack target cells.

[0003] NK cells are one of the most effective immune cells known to kill tumor cells, playing a vital role in inhibiting the occurrence, development, and spread of tumor tissue. However, due to the decline in the number and quality of NK cells in cancer patients and the existence of tumor escape mechanisms, their anti-tumor function in the body is not fully exerted.

[0004] Studies have found that modifying NK cells with CARs is expected to enhance their ability to target and kill tumor cells and develop effector cells with strong anti-tumor effects. However, there are still two shortcomings: on the one hand, the relative resistance of NK cells to transduction has hindered the study of NK cell biology and the development of NK cell-based immunotherapy; on the other hand, G glycoprotein (VSV-G) pseudotyped lentiviral vectors are often used to generate chimeric antigen receptor CAR-T cells, but these cells cannot effectively transduce NK cells. Technical issues

[0005] In view of this, the problem to be solved by the present invention is to provide an envelope glycoprotein of a mutant envelope pseudotyped lentiviral vector for improving the transduction of NK cells by chimeric antigen receptors and its application. Technical Solutions

[0006] The present invention provides an envelope glycoprotein for packaging a pseudotyped lentiviral vector for transduction of NK cells, wherein the envelope glycoprotein is an endogenous retrovirus (BaEV) envelope glycoprotein; preferably, the envelope glycoprotein is a baboon endogenous retrovirus envelope glycoprotein; wherein the envelope glycoprotein includes an extracellular region, a transmembrane region, and a cytoplasmic region, and the corresponding amino acid sequences or mutant sequences thereof in the extracellular region, the transmembrane region, and the cytoplasmic region are as follows:

[0007] The amino acid sequence or mutant sequence of the extracellular region is shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0008] The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0009] The amino acid sequence or mutant sequence of the cytoplasmic region is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0010] The expression vector of the above-mentioned envelope glycoprotein is plasmid pcDNA3.1+; the implementation process is as follows:

[0011] After the BaEV envelope glycoprotein mutants were individually cloned into the plasmid pcDNA3.1+, this plasmid replaced the G glycoprotein of vesicular stomatitis virus (VSV-G), which is the fusogenic coat protein, in the traditional lentiviral four-plasmid system (e.g., Invitrogen).

[0012] The present invention also provides a nucleic acid molecule encoding the above-mentioned envelope glycoprotein.

[0013] The present invention also provides an expression cassette or expression frame containing the above-mentioned envelope glycoprotein or nucleic acid molecule.

[0014] The present invention also provides a viral vector, such as a lentiviral vector, a retroviral vector, etc., which contains the above-mentioned envelope glycoprotein, nucleic acid molecule or expression cassette, etc.

[0015] The above-mentioned envelope glycoproteins, nucleic acid molecules, viral vectors, expression cassettes, etc. can be made into additive components of biological preparations, which are used to prepare drugs for preventing and / or treating tumors and cancers; among them, envelope glycoproteins can also be used to prepare drugs for preventing and / or treating tumors and cancers. Beneficial effects

[0016] The present invention uses BaEV envelope glycoprotein mutants instead of VSV-G to package lentiviral pseudoviruses, which not only maintains a high virus titer of 7.33E+07TU / mL, but also can effectively identify NK cells and mediate virus entry into cells, thereby achieving the purpose of efficient transfection of NK cells. For example, the transduction positive rate of the pseudotyped virus containing the envelope glycoprotein mutant (LV-CAR-BaEV-M) on NK cells can be as high as 75.2%, while the transduction positive rate of the virus containing the G glycoprotein (LV-CAR-VSV-G) on NK cells is only 8.31%; at the same time, it provides a new therapeutic means for lentiviral pseudotyped viruses for gene therapy and cell transfection research, and can be applied to large-scale research and development and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic diagram of the BaEV envelope glycoprotein structure;

[0018] Figure 2 is a flow cytometry graph of virus titer; wherein the horizontal axis EGFP-FITC represents green fluorescent protein-fluorescein isothiocyanate, and the vertical axis SSA represents three-color light;

[0019] Figure 3 is a flow cytometry chart of NK purity;

[0020] FIG4 is a flow cytometric analysis of the positive rate of lentiviral transduced NK cells; wherein the horizontal axis EGFP-FITC represents green fluorescent protein-fluorescein isothiocyanate, and the vertical axis SSA represents three-color light. Best Mode for Carrying Out the Invention

[0021] The preferred embodiments of the present invention will be described in further detail below with reference to the accompanying drawings.

[0022] The present invention demonstrates that a mutant pseudotyped virus (LV-BaEV-M) of the baboon endogenous retrovirus (BaEV) envelope glycoprotein is particularly suitable for gene transfer into NK cells. Specifically, the envelope glycoprotein of the pseudotyped lentiviral vector for NK cell transduction is a baboon endogenous retrovirus envelope glycoprotein, which is a monoclonal antibody. This envelope glycoprotein or a mutant thereof comprises an extracellular region, a transmembrane region, and a cytoplasmic region. The corresponding amino acid sequences of each of the extracellular region, transmembrane region, and cytoplasmic region, or their mutant sequences, are as follows:

[0023] The amino acid sequence or mutant sequence of the extracellular region is shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0024] The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0025] The amino acid sequence or mutant sequence of the cytoplasmic region is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0026] Preferably, the corresponding amino acid sequences or mutant sequences in the extracellular region, transmembrane region, and cytoplasmic region of the envelope glycoprotein are any of the following groups:

[0027] a), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5;

[0028] b), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6;

[0029] c), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:7;

[0030] d), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:8;

[0031] e), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5;

[0032] f), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6;

[0033] g), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7;

[0034] h), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 8;

[0035] i), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5;

[0036] j), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:6;

[0037] k), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:7;

[0038] l), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8;

[0039] m), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5;

[0040] n), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;

[0041] p), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7;

[0042] q), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:8.

[0043] In the above-mentioned envelope glycoprotein, the amino acid sequences or mutant sequences are as follows:

[0044] 1) The amino acid sequence or mutant sequence shown in SEQ ID NO: 1 is as follows:

[0045] 2) The amino acid sequence or mutant sequence shown in SEQ ID NO: 2 is as follows:

[0046] 3) The amino acid sequence or mutant sequence shown in SEQ ID NO: 3 is as follows:

[0047] 4) The amino acid sequence or mutant sequence shown in SEQ ID NO: 4:

[0048] 5) The amino acid sequence or mutant sequence shown in SEQ ID NO: 5 is as follows:

[0049] 6) The amino acid sequence or mutant sequence shown in SEQ ID NO: 6 is as follows:

[0050] 7) The amino acid sequence or mutant sequence shown in SEQ ID NO: 7 is as follows:

[0051] 8) The amino acid sequence or mutant sequence shown in SEQ ID NO: 8 is as follows:

[0052] The above-mentioned envelope glycoprotein is a mutant pseudotyped lentiviral vector (LV-BaEV-M) of the envelope glycoprotein of the baboon endogenous retrovirus (BaEV). This pseudotyped enveloped lentiviral vector has been shown to be particularly suitable for gene transfer into NK cells and can significantly improve the transduction efficiency. Modes for Carrying Out the Invention

[0053] The amino acid sequences of four groups of envelope glycoprotein mutants are selected and represented in detail through specific examples.

[0054] Example 1 Preparation of BaEV-M and VSV-G pseudotyped lentiviral vectors

[0055] 1. Construction of BaEV-M Expression Plasmid

[0056] The amino acid sequences of the synthesized BaEV-M molecules, selected from the amino acid sequences or mutant sequences of the envelope glycoprotein described above, as shown in Groups a), c), f), and q), were constructed into four pcDNA3.1+ vectors to form four pBaEV-M plasmids, as shown in Figure 1. The four plasmids were designated as pBaEV-M plasmid #1, pBaEV-M plasmid #2, pBaEV-M plasmid #3, and pBaEV-M plasmid #4.

[0057] 2. Lentiviral Packaging and Titer Detection

[0058] Preparation of BaEV-M Lentivirus

[0059] The four pBaEV-M plasmids prepared in this example were mixed with pEF1α-CAR expressing Claudin18.2-CAR and EGFP, auxiliary plasmid pMDLg / pRRE (Addgene Plasmid, #12251) and auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253), and were respectively transferred into four groups of 293T cells using Lipofectamine3000 transfection agent to prepare four complete lentiviral expression vectors, named LV-CAR-BaEV-M (1#), LV-CAR-BaEV-M (2#), LV-CAR-BaEV-M (3#), and LV-CAR-BaEV-M (4#).

[0060] 2.2. VSV-G Lentivirus Preparation

[0061] Four plasmids, including the lentiviral envelope plasmid pMD2.G (Addgene, Plasmid #12259), the pEF1α-CAR plasmid expressing Claudin 18.2-CAR and EGFP, the auxiliary plasmid pMDLg / pRRE (Addgene Plasmid, #12251), and the auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253), were mixed and transformed into 293T cells using Lipofectamine 3000 to prepare a complete lentiviral expression vector named LV-CAR-VSV-G.

[0062] At 48 h and 72 h, the five corresponding viral supernatants of LV-CAR-BaEV-M (1#), LV-CAR-BaEV-M (2#), LV-CAR-BaEV-M (3#), LV-CAR-BaEV-M (4#), and LV-CAR-VSV-G were collected, respectively. The collected viral supernatants were concentrated by ultracentrifugation (Merck Millipore) to obtain five concentrated viruses, namely, LV-CAR-BaEV-M (1#), LV-CAR-BaEV-M (2#), LV-CAR-BaEV-M (3#), LV-CAR-BaEV-M (4#), and LV-CAR-VSV-G. The five concentrated viruses were transduced into 293T cells for 48 h, and the positive rate was detected by flow cytometry to calculate the virus titer.

[0063] The virus titer is calculated as follows:

[0064] Virus titer = (transduction positive rate - background positive rate) x number of cells / volume.

[0065] The virus titer calculation results are as follows:

[0066] The titer of LV-CAR-VSV-G was 2.03E+08TU / mL, the titer of LV-CAR-BaEV-M(1#) was 7.33E+07 TU / mL, the titer of LV-CAR-BaEV-M(2#) was 3.95E+07TU / mL, the titer of LV-CAR-BaEV-M(3#) was 5.58E+07TU / mL, and the titer of LV-CAR-BaEV-M(4#) was 5.03E+07TU / mL.

[0067] As shown in Figure 2, the results of virus titer flow cytometry detection are:

[0068] The positive rate of blank samples was 1.04%, the positive rate of LV-CAR-VSV-G transduction was 41.6%, the positive rate of LV-CAR-BaEV-M(1#) transduction was 15.7%, the positive rate of LV-CAR-BaEV-M(2#) transduction was 8.94%, the positive rate of LV-CAR-BaEV-M(3#) transduction was 12.2%, and the positive rate of LV-CAR-BaEV-M(4#) transduction was 11.1%.

[0069] The viral titer calculation results and the detection results in Figure 2 show that the pseudotyped lentiviral vector packaged by BaEV-M can maintain a higher titer than the VSV-G viral vector.

[0070] Experimental Example 2 NK cell transfection detection

[0071] 1. NK cell culture

[0072] Mononuclear cells (MNs) were isolated from human peripheral blood using lymphocyte separation medium. NK cells (CD3-CD56+) were then isolated from PBMC using immunomagnetic beads (Dynal NK cell isolation kit). The purity of the isolated cells was determined by flow cytometry. The NK cell purity test results are shown in Figure 3. In Figure 3, the NK cell purity was 85.4%. NK cell culture is a conventional technique and will not be further described here.

[0073] 2. NK cell transduction and positive rate detection

[0074] The NK cells in Example 2 were cultured to the 7th day, and the NK cells were collected, counted, and inoculated into 24-well plates, with 5×10 4cells; the five concentrated viruses prepared in Example 1, such as LV-CAR-VSV-G, LV-CAR-BaEV-M (1#), LV-CAR-BaEV-M (2#), LV-CAR-BaEV-M (3#), and LV-CAR-BaEV-M (4#), were added at an MOI of 5, and polybrene transfection aid was added to a final concentration of 8 μg / mL and mixed, and the final volume of the cell transfection culture medium was 500 μL.

[0075] After uniform mixing, the five lentiviruses were transfected into NK cells for 6-8 hours. 500 μL of fresh culture medium was then added to each cell and incubated at 37°C in a 5% CO2 incubator. After 48 hours of incubation, flow cytometry analysis was performed to determine the number of fluorescence-positive cells. The transfection efficiency of the pseudoviruses was determined by comparing the number of fluorescence-positive cells with the control. The flow cytometry results are shown in Figure 4.

[0076] In Figure 4, the blank sample positive rate was 1.68%, the LV-CAR-VSV-G transduction positive rate was 8.31%, the LV-CAR-BaEV-M (1#) transduction positive rate was 57.9%, the LV-CAR-BaEV-M (2#) transduction positive rate was 75.2%, the LV-CAR-BaEV-M (3#) transduction positive rate was 74.0%, and the LV-CAR-BaEV-M (4#) transduction positive rate was 43.6%. Figure 4 shows that the BaEV-M pseudotyped lentiviral vector exhibited a stronger transduction effect than the VSV-G viral vector; that is, the BaEV-M envelope glycoprotein on the pseudotyped lentiviral vector can promote NK cell transduction. Industrial Applicability

[0077] The present invention uses BaEV envelope glycoprotein mutants instead of VSV-G to package lentiviral pseudoviruses, which not only maintains a high virus titer of 7.33E+07TU / mL, but also can effectively identify NK cells and mediate virus entry into cells, thereby achieving the purpose of efficient transfection of NK cells. For example, the transduction positive rate of the pseudotyped virus containing the envelope glycoprotein mutant (LV-CAR-BaEV-M) on NK cells can be as high as 75.2%, while the transduction positive rate of the virus containing the G glycoprotein (LV-CAR-VSV-G) on NK cells is only 8.31%; at the same time, it provides a new therapeutic means for lentiviral pseudotyped viruses for gene therapy and cell transfection research, and can be applied to large-scale research and development and production.

[0078] It should be understood that the above description of the preferred embodiments of the present invention is relatively detailed and cannot be regarded as limiting the scope of patent protection of the present invention. The scope of patent protection of the present invention shall be based on the appended claims.

Claims

1. An envelope glycoprotein for packaging and transducing NK cell pseudotyped lentiviral vectors, characterized in that: The envelope glycoprotein includes an extracellular region, a transmembrane region and a cytoplasmic region, and the corresponding amino acid sequences or mutant sequences in the extracellular region, the transmembrane region and the cytoplasmic region are as follows: The amino acid sequence or mutant sequence of the extracellular region is shown in SEQ ID NO: 1 or SEQ ID NO: 2; The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO: 3 or SEQ ID NO: 4; The amino acid sequence or mutant sequence of the cytoplasmic region is shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:

8.

2. The envelope glycoprotein according to claim 1, characterized in that The amino acid sequences or mutant sequences corresponding to the extracellular region, transmembrane region and cytoplasmic region of the envelope glycoprotein are any of the following groups: a), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5; b), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6; c), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:7; d), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:8; e), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 5; f), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 6; g), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 7; h), SEQ ID NO: 1, SEQ ID NO: 4 and SEQ ID NO: 8; i), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5; j), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:6; k), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:7; l), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8; m), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5; n), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6; p), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7; q), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:

8.

3. The envelope glycoprotein according to claim 1, characterized in that The amino acid sequences or mutant sequences corresponding to the extracellular region, transmembrane region and cytoplasmic region of the envelope glycoprotein are any of the following groups: i), the amino acid sequence or mutant sequence of the extracellular region is as shown in SEQ ID NO: 1; the amino acid sequence or mutant sequence of the transmembrane region is as shown in SEQ ID NO: 3; the amino acid sequence or mutant sequence of the cytoplasmic region is as shown in SEQ ID NO: 5; or ii), the amino acid sequence or mutant sequence of the extracellular region is as shown in SEQ ID NO: 1; the amino acid sequence or mutant sequence of the transmembrane region is as shown in SEQ ID NO: 3; the amino acid sequence or mutant sequence of the cytoplasmic region is as shown in SEQ ID NO: 7; or iii), the amino acid sequence or mutant sequence of the extracellular region is as shown in SEQ ID NO: 1; the amino acid sequence or mutant sequence of the transmembrane region is as shown in SEQ ID NO: 4; the amino acid sequence or mutant sequence of the cytoplasmic region is as shown in SEQ ID NO: 6; or iv), the amino acid sequence or mutant sequence of the extracellular region is as shown in SEQ ID NO:2; the amino acid sequence or mutant sequence of the transmembrane region is as shown in SEQ ID NO:4; the amino acid sequence or mutant sequence of the cytoplasmic region is as shown in SEQ ID NO:

8.

4. The envelope glycoprotein according to any one of claims 1 to 3, characterized in that The envelope glycoprotein is an endogenous retrovirus envelope glycoprotein.

5. The envelope glycoprotein according to any one of claims 1 to 3, characterized in that The envelope glycoprotein is a pseudotype lentiviral vector of a baboon endogenous retrovirus envelope glycoprotein mutant.

6. The envelope glycoprotein according to any one of claims 1 to 3, characterized in that The expression vector of the envelope glycoprotein is pcDNA3.1+.

7. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the envelope glycoprotein according to any one of claims 1 to 6.

8. An expression cassette, characterized in that The expression cassette contains the envelope glycoprotein according to any one of claims 1 to 6, or contains the nucleic acid molecule according to claim 7.

9. A viral vector, characterized in that The viral vector contains the envelope glycoprotein according to any one of claims 1 to 6, or contains the nucleic acid molecule according to claim 7, or contains the expression cassette according to claim 8.

10. A biological agent, characterized in that: The biological preparation contains the envelope glycoprotein according to any one of claims 1 to 6, or contains the nucleic acid molecule according to claim 7, or contains the expression cassette according to claim 8, or contains the viral vector according to claim 9.

11. Use of the biological agent according to claim 10 in the preparation of drugs for preventing and / or treating tumors and cancers.

12. Use of the envelope glycoprotein according to any one of claims 1 to 6 in the preparation of drugs for preventing and / or treating tumors and cancers.

Images