Envelope glycoproteins of pseudotyped lentiviral vectors for transducing NK cells and their applications

Abstract

The present invention discloses an envelope glycoprotein for packaging and transducing NK cell pseudotyped lentiviral vector and its application; the envelope glycoprotein includes an extracellular region, a transmembrane region and a cytoplasmic region; wherein the amino acid sequence or mutant sequence of the extracellular region is as shown in SEQ ID NO:1 or SEQ ID NO:2; the amino acid sequence or mutant sequence of the transmembrane region is as shown in SEQ ID NO:3 or SEQ ID NO:4; the amino acid sequence or mutant sequence of the cytoplasmic region is as shown in SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8. The BaEV envelope glycoprotein mutant replaces VSV-G to package lentiviral pseudovirus, which not only maintains a high virus titer of 7.33E+07 TU / mL, but also can effectively identify NK cells and mediate the virus to enter cells, thereby achieving the purpose of efficiently transfecting NK cells.

Description

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 an application thereof. Background Art

[0002] Natural killer cells (NK cells) were discovered in peripheral blood 30 years ago. NK cells usually contain a large amount of perforin and granzyme B. When activated NK cells encounter target cells, they 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, and play an important role in inhibiting the occurrence, development and spread of tumor tissue. However, due to the decrease in the number and quality of NK cells in tumor patients and the existence of tumor escape mechanisms, their anti-tumor function in the body has not been fully exerted.

[0004] Studies have found that modifying NK cells with CAR 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 such cells cannot effectively transduce NK cells. Summary of the invention

[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.

[0006] One of the technical solutions of the present invention is as follows:

[0007] An envelope glycoprotein for packaging and transducing a pseudotyped lentiviral vector for NK cell transduction, 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 comprises an extracellular region, a transmembrane region and a cytoplasmic region, and the corresponding amino acid sequences or their mutant sequences in the extracellular region, the transmembrane region and the cytoplasmic region are as follows:

[0008] The amino acid sequence or mutant sequence of the extracellular region is shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0009] The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0010] 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.

[0011] The expression vector of the above-mentioned envelope glycoprotein is plasmid pcDNA3.1+; the realization process is as follows:

[0012] 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) in the traditional lentiviral four-plasmid system (e.g., Invitrogen); wherein VSV-G is a fusogenic capsid protein.

[0013] The present invention also provides a nucleic acid molecule, which encodes the above-mentioned envelope glycoprotein.

[0014] The present invention also provides an expression box or expression frame containing the above-mentioned envelope glycoprotein or nucleic acid molecule.

[0015] 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.

[0016] The above-mentioned envelope glycoprotein, nucleic acid molecule, viral vector, expression cassette, etc. can be made into an additive component of a biological preparation, and such a biological preparation is used to prepare drugs for preventing and / or treating tumors and cancer; among them, the envelope glycoprotein can also be used to prepare drugs for preventing and / or treating tumors and cancer.

[0017] The present invention replaces VSV-G with BaEV envelope glycoprotein mutant to package lentiviral pseudovirus, which not only maintains a high virus titer of 7.33E+07 TU / mL, but also can effectively identify NK cells and mediate the virus into cells, thereby achieving the purpose of efficient transfection of NK cells. For example, the transduction positive rate of NK cells by pseudovirus containing envelope glycoprotein mutant (LV-CAR-BaEV-M) can be as high as 75.2%, and the transduction positive rate of NK cells by virus containing G glycoprotein (LV-CAR-VSV-G) is only 8.31%. At the same time, it provides a new therapeutic means for gene therapy and cell transfection research of lentiviral pseudovirus, which can be applied to large-scale research and development and production. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 Schematic diagram of the BaEV envelope glycoprotein structure;

[0019] Figure 2 It is a flow cytometry test diagram of virus titer; wherein, the horizontal axis EGFP-FITC represents green fluorescent protein-fluorescein isothiocyanate, and the vertical axis SSA represents three-color light;

[0020] Figure 3 This is the flow cytometry test diagram for NK purity;

[0021] Figure 4 This is a flow cytometric detection diagram of the positive rate of lentivirus-transduced NK cells; wherein the horizontal axis EGFP-FITC represents green fluorescent protein-fluorescein isothiocyanate, and the vertical axis SSA represents three-color light. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are further described in detail below in conjunction with the accompanying drawings.

[0023] The present invention uses a pseudotyped virus (LV-BaEV-M) of a baboon endogenous retrovirus (BaEV) envelope glycoprotein mutant, which is particularly suitable for gene transfer into NK cells; that is, the envelope glycoprotein of the pseudotyped lentiviral vector for transducing NK cells is a baboon endogenous retrovirus envelope glycoprotein, which is a monoclonal antibody. This envelope glycoprotein or its mutant includes an extracellular region, a transmembrane region, and a cytoplasmic region, and the corresponding amino acid sequences or their mutant sequences in the extracellular region, transmembrane region, and cytoplasmic region are as follows:

[0024] The amino acid sequence or mutant sequence of the extracellular region is shown in SEQ ID NO: 1 or SEQ ID NO: 2;

[0025] The amino acid sequence or mutant sequence of the transmembrane region is shown in SEQ ID NO: 3 or SEQ ID NO: 4;

[0026] 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.

[0027] 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:

[0028] a), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:5;

[0029] b), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:6;

[0030] c), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:7;

[0031] d), SEQ ID NO:1, SEQ ID NO:3 and SEQ ID NO:8;

[0032] e), SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:5;

[0033] f), SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:6;

[0034] g), SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:7;

[0035] h), SEQ ID NO:1, SEQ ID NO:4 and SEQ ID NO:8;

[0036] i), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:5;

[0037] j), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:6;

[0038] k), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:7;

[0039] l), SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:8;

[0040] m), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:5;

[0041] n), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6;

[0042] p), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:7;

[0043] q), SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:8.

[0044] In the above-mentioned envelope glycoprotein, the amino acid sequences or mutant sequences are as follows:

[0045] 1) The amino acid sequence or mutant sequence shown in SEQ ID NO: 1 is as follows:

[0046] MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTGGTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTPLAIPNFLLSYVTRSSDNISCLIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQFIPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP;

[0047] 2) The amino acid sequence shown in SEQ ID NO:2 or the mutant sequence is as follows:

[0048] MGFTTKIIFLYNLVLVYAGFDDPRKAIELVQKRYGRPCDCSGGQVSEPPSDRVSQVTCSGKTAYLMPDQRWKCKSIPKDTSPSGPLQECPCNSYQSSVHSSCYTSYQQCRSGNKTYYTATLLKTQTG GTSDVQVLGSTNKLIQSPCNGIKGQSICWSTTAPIHVSDGGGPLDTTRIKSVQRKLEEIHKALYPELQYHPLAIPKVRDNLMVDAQTLNILNATYNLLLMSNTSLVDDCWLCLKLGPPTPLAIPTPS LTYSLADSLANASCQIIPPLLVQPMQFSNSSCLFSPSYNSTEEIDLGHVAFSNCTSITNVTGPICAVNGSVFLCGNNMAYTYLPTNWTGLCVLATLLPDIDIIPGDEPVPIPAIDHFIYRPKRAIQF IPLLAGLGITAAFTTGATGLGVSVTQYTKLSNQLISDVQILSSTIQDLQDQVDSLAEVVLQNRRGLDLLTAEQGGICLALQEKCCFYVNKSGIVRDKIKTLQEELERRRKDLASNPLWTGLQGLLP;

[0049] 3) The amino acid sequence or mutant sequence shown in SEQ ID NO: 3 is as follows:

[0050] LISTIMGPLIVLLLILLFGPCIL is as follows;

[0051] 4) The amino acid sequence or mutant sequence shown in SEQ ID NO: 4:

[0052] YLLPFLGPLLTLLLLLTIGPCIF;

[0053] 5) The amino acid sequence or mutant sequence shown in SEQ ID NO: 5 is as follows:

[0054] NRLVQFVKDRISVVQALVLTQQYHQLKPLEYEP;

[0055] 6) The amino acid sequence or mutant sequence shown in SEQ ID NO: 6 is as follows:

[0056] NRLVQFVKDRISVSQNYPIVQQYHQLKPLEYEP;

[0057] 7) The amino acid sequence or mutant sequence shown in SEQ ID NO: 7 is as follows:

[0058] NRLVQFVKDRISVVQGACRAIQYHQLKPLEYEP;

[0059] 8), the amino acid sequence or mutant sequence shown in SEQ ID NO: 8 is as follows:

[0060] NRLVQFVKDRISVVHAMVLAQQYHQLKPLEYEP.

[0061] The above-mentioned envelope glycoprotein is a mutant pseudotype lentiviral vector (LV-BaEV-M) of the envelope glycoprotein of baboon endogenous retrovirus (BaEV). This pseudotype envelope lentiviral vector has been shown to be particularly suitable for transferring genes into NK cells and can significantly improve the transduction efficiency.

[0062] The amino acid sequences of four groups of envelope glycoprotein mutants are selected and representatively described in detail through specific examples.

[0063] Example 1 Preparation of BaEV-M and VSV-G pseudotyped lentiviral vectors

[0064] 1. Construction of BaEV-M expression plasmid

[0065] The amino acid sequence of the synthesized BaEV-M molecule is selected from the amino acid sequence or mutant sequence of the envelope glycoprotein, such as group a), group c), group f) and group q), and is respectively constructed into four pcDNA3.1+ to form four pBaEV-M plasmids, such as Figure 1 The four plasmids were named 1# pBaEV-M plasmid, 2# pBaEV-M plasmid, 3# pBaEV-M plasmid and 4# pBaEV-M plasmid.

[0066] 2. Lentiviral packaging and titer detection

[0067] 2.1. Preparation of BaEV-M lentivirus

[0068] The four pBaEV-M plasmids prepared in this example were respectively combined with pEF1α-CAR expressing Claudin18.2-CAR and EGFP, auxiliary plasmid pMDLg / pRRE (Addgene Plasmid, #12251) and auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253) etc. mix, and were transferred into four groups of 293T cells using Lipofectamine3000 transfection agent to prepare four complete lentiviral expression vectors, which were named LV-CAR-BaEV-M (1#), LV-CAR-BaEV-M (2#), LV-CAR-BaEV-M (3#), and LV-CAR-BaEV-M (4#).

[0069] 2.2. VSV-G lentivirus preparation

[0070] 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), the auxiliary plasmid pRSV-Rev (Addgene Plasmid, #12253), etc. Four Plasmid mix , and transferred into 293T cells using Lipofectamine3000 to prepare a complete lentiviral expression vector named LV-CAR-VSV-G.

[0071] The five virus supernatants corresponding to the above 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 at 48h and 72h, and the collected virus 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 48h, and the positive rate was detected by flow cytometry to calculate the virus titer.

[0072] The virus titer is calculated as follows:

[0073] Virus titer = (transduction positive rate - background positive rate) x number of cells / volume.

[0074] The virus titer calculation results are as follows:

[0075] The titer of LV-CAR-VSV-G was 2.03E+08 TU / 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+07 TU / mL, the titer of LV-CAR-BaEV-M(3#) was 5.58E+07 TU / mL, and the titer of LV-CAR-BaEV-M(4#) was 5.03E+07 TU / mL.

[0076] like Figure 2 As shown, the results of virus titer flow cytometry detection are:

[0077] 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%.

[0078] The results of virus titer calculation and Figure 2 The test results show that the pseudotyped lentiviral vector packaged by BaEV-M can maintain a higher titer than the VSV-G viral vector.

[0079] Experimental Example 2 NK cell transfection detection

[0080] 1. NK cell culture

[0081] Mononuclear cells in human peripheral blood were separated using lymphocyte separation fluid, and then NK cells (CD3-CD56+) were separated from PBMC using immunomagnetic beads (Dynal NK cell isolation kit). The separation purity was detected by flow cytometry. The NK purity test results are shown in Figure 3 shown. Figure 3 The purity of NK cells was 85.4%. NK cell culture is a prior art and will not be described in detail here.

[0082] 2. NK cell transduction and positive rate detection

[0083] The NK cells in Example 2 were cultured until the 7th day, and the NK cells were collected, counted, and inoculated into a 24-well plate, with 5×1 0 4cells; 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 the final concentration of 8 μg / mL and mixed, and the final volume of the cell transfection culture medium was 500 μL.

[0084] After mixing evenly, the five lentiviruses were transfected into NK cells for 6-8h, and then 500 μL of fresh culture medium was added and incubated at 37°C, 5% CO 2 Culture in an incubator; after 48 hours of culture, flow cytometry analysis was performed to obtain the number of fluorescence-positive cells. The transfection efficiency of the pseudovirus was determined by comparing the number of fluorescence-positive cells with the control. Figure 4 shown.

[0085] Figure 4 Among them, the positive rate of blank samples was 1.68%, the positive rate of LV-CAR-VSV-G transduction was 8.31%, the positive rate of LV-CAR-BaEV-M (1#) transduction was 57.9%, the positive rate of LV-CAR-BaEV-M (2#) transduction was 75.2%, the positive rate of LV-CAR-BaEV-M (3#) transduction was 74.0%, and the positive rate of LV-CAR-BaEV-M (4#) transduction was 43.6%. Figure 4 This indicates that the BaEV-M pseudotyped lentiviral vector exhibits 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.

[0086] 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 attached claims.

Claims

1. An envelope glycoprotein for packaging and transducing NK cell pseudotyped lentiviral vectors, characterized in that: The envelope glycoprotein consists of an extracellular region, a transmembrane region and a cytoplasmic region, wherein the amino acid sequence of the extracellular region is shown in SEQ ID NO:2, the amino acid sequence of the transmembrane region is shown in SEQ ID NO:4, and the amino acid sequence of the cytoplasmic region is shown in SEQ ID NO:

8.

2. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the envelope glycoprotein of claim 1.

3. An expression cassette, characterized in that The expression cassette expresses the envelope glycoprotein according to claim 1, or contains the nucleic acid molecule according to claim 2.

4. A viral vector, characterized in that The viral vector expresses the envelope glycoprotein according to claim 1, or contains the nucleic acid molecule according to claim 2, or contains the expression cassette according to claim 3.

5. A biological agent, characterized in that: The biological preparation contains the envelope glycoprotein according to claim 1, or contains the nucleic acid molecule according to claim 2, or contains the expression cassette according to claim 3, or contains the viral vector according to claim 4.

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