CN110862967A

CN110862967A - Natural killer cell line SILK-NK independent of cytokine culture

Info

Publication number: CN110862967A
Application number: CN201810981706.9A
Authority: CN
Inventors: 吴亚松; 高斌; 司远
Original assignee: Timmune Biotech Inc
Current assignee: SHENZHEN BEIKE BIO-TECHNOLOGY Co.,Ltd.
Priority date: 2018-08-27
Filing date: 2018-08-27
Publication date: 2020-03-06
Also published as: WO2020043076A1

Abstract

The invention discloses a natural killer cell line SILK-NK. which is not dependent on cytokine culture and provides a preparation method of the natural killer cell line SILK-NK which is not dependent on cytokine culture, which comprises the following steps of expressing a protein compound containing IL-15 protein and an IL-15 binding region of IL-15R α on the surface of an NK-92 cell line in a membrane binding mode, and then culturing the cell line by using a culture medium without IL-2 to obtain a cloned cell line, namely the natural killer cell line SILK-NK., wherein the cell line can be stably subcultured and expanded in the culture medium without IL-2, and can keep the killing activity of the NK-92, so that the cell line becomes an engineering cell line with clinical application value and can be used for the adoptive immunotherapy of tumors.

Description

Natural killer cell line SILK-NK independent of cytokine culture

Technical Field

The invention relates to the field of adoptive immunotherapy of tumors, in particular to a natural killer cell line SILK-NK independent of cytokine culture and a preparation method thereof.

Background

Nk (nature killer) cells are an important class of cytotoxic killer cells in the immune system that are capable of releasing perforin and cytokines to induce apoptosis in cancer cells. NK cells do not recognize target cells MHC-restricted and tumor-associated antigen-dependent and therefore have a broader spectrum of anti-tumor capacity than T cells. The non-MHC restriction of NK cells allows it to support allogeneic transplantation and can kill cancer cells that evade T cell killing by reducing MHC molecule expression. NK cells do not secrete IL-6 in large quantities and therefore do not cause a strong cytokine storm. However, NK cells only account for about 10% of blood cells, complicated blood cell separation and enrichment processes are required, transfection efficiency of NK cells is low, and difficulty in gene modification is increased.

NK-92 is a NK cell line from non-Hodgkin lymphoma patients, which has the advantages of NK cells and overcomes the disadvantages of NK cells. It has wide antitumor capacity independent to MHC and tumor associated antigen, easy to culture in large scale and easy to operate gene. More importantly, the clinical first-phase data of NK-92 infused patients have shown high safety, even at 1X 10¹⁰/m²Has no serious adverse reaction under the dosage condition.

The proliferation and activation of NK cells requires cytokines, particularly interleukin-2 (IL-2) or IL-15. IL-2 can stimulate Treg cell proliferation and induce T cell apoptosis, and has certain toxic and side effects in clinical application. IL-15 has much the same function as IL-2, and IL-15 is an indispensable cytokine responsible for NK development, differentiation, and maintenance. The NK-92 cell line expressing IL-15 showed stronger proliferation and cancer cell killing ability, but still could not get rid of the dependence on IL-2.

Disclosure of Invention

The invention aims to provide a natural killer cell line SILK-NK which is cultured independent of cell factors and a preparation method thereof, so as to change the biological characteristics of NK-92 cells, get rid of the dependence on IL-2, improve the proliferation and killing capacity and make the NK-92 cells more suitable for clinical application.

In a first aspect, the present invention claims a method for the preparation of the natural killer cell line SILK-NK independent of cytokine culture.

The preparation method of the natural killer cell line SILK-NK independent of cytokine culture provided by the invention comprises the following steps of expressing a protein complex containing IL-15 protein and an IL-15 binding region of IL-15R α on the surface of an NK-92 cell line in a membrane binding mode, and then culturing by using a culture medium without IL-2 to obtain a cloned cell line, namely the natural killer cell line SILK-NK.

Further, the protein complex is composed of an IL-15 binding region, a transmembrane region and an IL-15 protein of IL-15R α from N-terminal to C-terminal in this order (as shown in FIG. 1).

Wherein the transmembrane region can be, but is not limited to, the CD8 α transmembrane region.

Furthermore, the amino acid sequence of the IL-15 binding region of the IL-15R α can be SEQ ID No.1, or can be a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence shown in SEQ ID No.1, or has the same function through substitution and/or deletion and/or addition of one or more amino acid residues, the amino acid sequence of the CD8 α transmembrane region can be SEQ ID No.2, or can be a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence shown in SEQ ID No.2, or has the same function through substitution and/or deletion and/or addition of one or more amino acid residues, the amino acid sequence of the IL-15 protein can be SEQ ID No.3, or can be a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% homology with the amino acid sequence shown in SEQ ID No.3, or has the same function through substitution and/or addition of one or more than 75% of amino acid residues.

More specifically, the amino acid sequence of the protein complex can be SEQ ID No.4, or a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence shown in SEQ ID No.4, or has the same function through substitution and/or deletion and/or addition of one or more amino acid residues.

Further, the method may comprise the steps of: inserting the coding gene of the protein complex into a lentivirus expression vector, packaging lentivirus by using the obtained recombinant lentivirus vector (such as the lentivirus prepared by cotransfecting 293T cells with the obtained recombinant lentivirus vector and a packaging plasmid), infecting the NK-92 cell line with the lentivirus, culturing in an IL-2-free culture medium and obtaining the cloned cell line by a limiting dilution method.

In a specific embodiment of the invention, the IL-2-free medium is specifically GT-T551-H3 medium (e.g., cat # WK593S, TAKARA) containing a serum replacement (e.g., cat # HPCFDCGL50, Helios) at a final concentration of 2.5% by volume.

Wherein, in the coding gene of the protein complex, the nucleotide sequence of the IL-15 binding region encoding the IL-15R α can be the 1 st to 216 th positions of SEQ ID No.5, or the nucleotide sequence of the IL-15 binding region encoding the IL-15R α with homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more with the 1 st to 216 th positions of SEQ ID No. 5. in the coding gene of the protein complex, the nucleotide sequence encoding the CD8 α transmembrane region can be the 217-423 th position of SEQ ID No.5, or the nucleotide sequence encoding the CD8 α transmembrane region with homology of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more with the 217-423 th position of SEQ ID No. 5. in the coding gene of the protein complex, the nucleotide sequence encoding the IL-15 protein can be the 490 th position of SEQ ID No.5, 831% or more, or the nucleotide sequence encoding the IL-15 binding region with homology of 95% or more, 85% or more with the IL-15 binding region with homology of the 490% or more, 95% or more than 80% or more than the IL-15 th position of SEQ ID No. 5.

Specifically, the nucleotide sequence of the coding gene of the protein complex can be SEQ ID No.5, or a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with SEQ ID No.5 and codes the protein complex.

In a second aspect, the invention claims a cytokine-independent cultured natural killer cell line SILK-NK prepared by the method described hereinbefore.

The STR phenotype of the natural killer cell line SILK-NK is

Amelogenin：X,Y

CSF1PO：11,12

D13S317：9,12

D16S539：11,12

D5S818：12,13

D7S820：10,11

THO1：6,9.3

TPOX：8

vWA：18

D21S11：31.2,32。

The expression of IL-15 binding region of IL-15 and IL-15R α can be detected by reverse transcription PCR of mRNA.

The morphology is a suspended clonal mass or dispersed cells.

Furthermore, the natural killer cell line SILK-NK is a cell line which is preserved in the China general microbiological culture Collection center with the accession number of CGMCC No. 15583.

In a third aspect, the use of the natural killer cell line SILK-NK as described hereinbefore in any one of the following is also within the scope of the present invention:

(A1) preparing a product for adoptive immunotherapy of tumors;

(A2) preparing related products for cell therapy.

If the natural killer cell line SILK-NK is genetically modified to express a chimeric antigen receptor, the targeting of the chimeric antigen receptor is utilized to mediate the cell line to specifically kill cancer cells expressing target antigens. Genetically modifying said natural killer cell line SILK-NK to express a chimeric antigen receptor, and administering cell therapy by infusing said modified cells into a patient.

According to the invention, by trying to simultaneously express IL-15 and IL-15 binding region compound of IL-15R α in NK-92, a novel Killer cell called SILK-NK (Specific Interleukin Linked Killer, SILK-NK) is established, the IL-15 binding region of IL-15R α is used as a super activator of IL-15, and a complex consisting of IL-15 and the IL-15 binding region of IL-15R α is used for activating NK-92 cells and improving the cancer cell killing capability of the NK-92 cells.

The invention starts with a cytokine which plays a regulating role in the process of NK cell differentiation and development, because 1.IL-15 has expression in various tissues in a human body and plays an important role in the process of NK cell differentiation and development, 2.NK-92 is undifferentiated mature NK cell, the cell surface can express IL-15 receptor, 3.IL-15 has a toxic and side effect which is obviously smaller than that of IL-2, and 4.IL-15 binding region of IL-15R α can be used as an activator of IL-15 activity.

The invention has the beneficial effects that the NK-92 expression IL-15 can improve the cell killing toxicity of the NK-92, but the NK-92 cell can not get rid of the dependence on IL-2, the invention stably expresses a complex of membrane-bound IL-15 and an IL-15 binding region of IL-15R α in the NK-92 cell, and can lead the NK-92 cell to proliferate for a long time under the IL-2-free culture condition and have higher killing activity by utilizing the super agonist action of the IL-15 binding region of IL-15R α on the IL-15, thereby being beneficial to large-scale culture and clinical application.

Deposit description

Suggested classification naming: human natural killer cell line

According to the biological materials (strains): SILK

The preservation organization: china general microbiological culture Collection center

The preservation organization is abbreviated as: CGMCC (China general microbiological culture Collection center)

Address: xilu No.1 Hospital No.3 of Beijing market facing Yang district

The preservation date is as follows: 04 month 03 of 2018

Registration number of the preservation center: CGMCC No.15583

Drawings

FIG. 1 is a schematic diagram of a protein complex of the present invention, wherein A represents an IL-15 binding domain of IL-15R α, B represents a transmembrane domain, and C represents IL-15.

FIG. 2 is a schematic diagram of the structure of the nucleic acid sequence encoding the protein complex of the present invention, wherein A represents the IL-15 binding domain of IL-15R α, B represents the transmembrane domain, and C represents IL-15.

FIG. 3 is a map of a lentiviral vector of the invention loaded with IL-15R α -TM-P2A-IL-15 nucleic acid sequence.

FIG. 4 is a photograph showing the morphology of SILK-NK-D8 cells and NK-92 cells according to the present invention. A is NK-92 cells and B is SILK-NK-D8 cells.

FIG. 5 is a comparison of the 3-day fold increase measured at different culture time points for SILK-NK-D8 cells of the invention and NK-92 cells. A is a 3-day proliferation multiple curve chart of NK-92 plus 100U/mL IL-2 culture; b is a 3-day fold increase profile of SILK-NK-D8 culture without IL-2.

FIG. 6 is an electrophoretogram of the RT-PCR of the present invention for identifying the mRNA expression of IL-15 binding region of IL-15 and IL-15R α, wherein A is NK-92 cell, B is SILK-NK-D8 cell, P1 is primer 1+ primer 2, and P2 is primer 3+ primer 4.

FIG. 7 is a graph showing the killing assay of the NK-92 and SILK-NK-D8 cells of the present invention against GFP expressing K562 cells. Fluorescence showed K562 cells.

FIG. 8 is a killing experiment of NK-92 and SILK-NK-D8 cells of the present invention against K562 cells expressing firefly luciferase. A is NK-92 cells and B is SILK-NK-D8 cells.

FIG. 9 shows the STR analysis results of SILK-NK-D8 cells of the present invention.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

NK-92 cells were purchased from the China center for type culture Collection, university of Wuhan, and the medium was GT-T551-H3 medium (cat # WK593S, product of TAKARA) which can be used for culturing T lymphocytes and NK cells, and a serum substitute (cat # HPCFDCGL50, product of Helios) was added to a final concentration of 2.5% (volume percentage).

The 293T medium was DMEM supplemented with 10% (volume percent) fetal bovine serum.

pLTR-abg 019: nanjing Erbin Viola Biotechnology Co., Ltd., cat # abg 019.

pCMV-dR8.2 plasmid: addgene, cat # 8455.

pCMV-VSV-G plasmid: addgene, cat # 8454.

The GFP and luciferase expressing K562 cell line K562-GFP-Luc: nanjing Erbin Viola Biotechnology Co., Ltd., Cat No. 562-GL.

Example 1 preparation and characterization of Natural killer cell line SILK-NK independent of cytokine culture

Construction of pLTR-IL15R α -IL15 lentivirus expression vector

As shown in FIG. 2 and FIG. 3, the DNA sequence (SEQ ID No.5) of IL-15R α -TM-P2A-IL-15 was synthesized from Nanjing Kingsrey Biotech GmbH, with BamHI (5 ') and Mlu I (3') added at both ends, the synthesized sequence was ligated with pLTR-abg019 plasmid by BamH I and Mlu I double digestion, respectively, and transformed into DH5 α competent bacteria, and the target expression plasmid pLTR-IL15R α -IL15 was obtained by sequencing after colony PCR identification of positive clones using the upstream primer (SEQ ID No.6) in the IL-15 binding region sequence of IL-15R α and the downstream primer SEQ ID No.7) in the IL-15 sequence.

The structure of the recombinant plasmid pLTR-IL15R α -IL15 is described as a recombinant plasmid obtained by replacing a small fragment between the restriction sites BamHI and Mlu I of the pLTR-abg019 plasmid with a DNA fragment shown in SEQ ID No. 5.

SEQ ID No.5 is the coding gene sequence of IL-15R α -TM-P2A-IL-15 protein complex, wherein the 1 st to 216 th sites are the coding gene of IL-15 binding region of IL-15R α, the 217 nd site is the coding gene of CD8 α transmembrane region, and the 490 nd site is the coding gene of IL-15 protein.SEQ ID No.5 encodes the protein shown in SEQ ID No. 4. wherein the 1 st to 72 th sites of SEQ ID No.4 are the amino acid sequence of the IL-15 binding region of IL-15R α (i.e., SEQ ID No.1), the 73 nd to 141 th sites of SEQ ID No.4 are the amino acid sequence of the CD8 α transmembrane region (i.e., SEQ ID No.2), and the 164 nd site of SEQ ID No. 277 is the amino acid sequence of the IL-15 protein (i.e., SEQ ID No. 3).

Two, pLTR-IL15R α -IL15 plasmid packaging virus

The recombinant vector pLTR-IL15R α -IL15 constructed in step one, the packaging plasmids pCMV-dR8.2 and pCMV-VSV-G were transfected into 293T cells with PEI (1. mu.g/. mu.l) at a ratio of 15: 10:1 (mass ratio).

The specific implementation is as follows: day 0 passage 293T cells 8X 10⁶Adding three plasmids pLTR-IL15R α -IL15, pCMV-dR8.2, pCMV-VSV-G, 15 mu G and 10 mu G to 1 mu G of Opti-MEM on day 1, adding 78 mu l PEI after mixing, shaking and mixing, placing for 15 minutes at room temperature, adding to T75 culture bottle for 293T culture, shaking and mixing, replacing fresh culture medium 12-16 hours after transfection, collecting culture medium on day 3 after 48 hours, filtering and removing cells with 0.45 mu M filter membrane to obtain virus stock solution, adding 40% PEG of 1/3 volume to the virus stock solution, mixing, placing overnight at 4 ℃, centrifuging for the next day, 4 ℃, 1800 ℃, G, 45 minutes, discarding supernatant, and precipitating with NK-92 culture medium of 1/10 volume (the final concentration of 100U/ml IL-2) to obtain 10-fold concentrated virus.

Third, infection of NK-92 cells

Take 3X 10⁵NK-92 cells were resuspended in 1mL of 10-fold concentrated virus prepared in step two and 8. mu.g/mL of polyclonal was added and transferred to 24-well plate oneCentrifuging at 32 deg.C for 45 min at 1500g in the well, transferring to carbon dioxide incubator, centrifuging for 3 hr, removing supernatant, and adding NK-92 culture medium with final concentration of 100U/ml IL-2; the infection was repeated once more the next day, followed by replacement of NK-92 medium at a final concentration of 100U/ml IL-2, followed by 3 days of passaging. After 2 weeks, no IL-2 is added into the culture medium during passage, and the culture is continued for two weeks, so that the survived cells are the obtained target cells SILK-NK (specific Interleukin Linked killer). The resulting cells were diluted in 96-well plates in a gradient and 5 monoclonal cell lines were identified: a6, A9, D8, F6, F7, clone D8 used in the subsequent examples (referred to as SILK-NK-D8, later).

SILK-NK-D8 is preserved in China general microbiological culture Collection center (CGMCC) at 03.04.2018, and is suggested to be classified as human natural killer cell line with accession number of CGMCC No. 15583; the biomaterial according to (strain) is SILK.

Fourth, cell morphology and proliferation

SILK-NK-D8 has a similar cell morphology to NK-92, tending to form loose clonal colonies, as shown in FIG. 4, and SILK-NK-D8 can achieve a 2-3 day-doubled proliferation rate in medium without IL-2 addition, as in NK-92.

Fifth, identification of exogenous gene expression

SILK-NK-D8 and NK-92 were separately extracted for total RNA, reverse transcribed using oligo (dT)18 to obtain cDNA, and PCR was performed using primers for IL-15 binding region of IL-15R α (P1 is primer 1+ primer 2, P2 is primer 3+ primer 4), as shown in FIG. 6, 1% agarose gel electrophoresis showed that SILK-NK-D8 had a positive band of about 500bp in size, while NK-92 did not.

Primer 1: 5'-atgtccgtggaacacgcagac-3' (SEQ ID No. 6);

primer 2: 5'-cttgaggtcg gagatgacgt tc-3' (SEQ ID No. 7);

primer 3: 5'-cccagtctcaaatgcattagagacc-3' (SEQ ID No. 8);

primer 4: 5'-ggtgtcatggatgctggcg-3' (SEQ ID No. 9).

Sixthly, cell killing ability detection

By expressing GFP and fluorescenceThe K562 cell line of the luciferase, K562-GFP-Luc, is used as a target cell, and SILK-NK-D8 and NK-92 are respectively mixed according to the ratio of 10:1,5:1 and 1:1 and the ratio of 1 × 10⁴Target cells were incubated in a 100. mu.l 96-well plate system and GFP fluorescence and luciferase were detected after 24 hours. As a result, SILK-NK-D8 and NK-92 showed the same killing activity on K562 cells as shown in FIGS. 7 and 8.

STR detection of SILK-NK-D8 cells, the results are shown in FIG. 9, the STR phenotype is

“Amelogenin：X,Y

CSF1PO：11,12

D13S317：9,12

D16S539：11,12

D5S818：12,13

D7S820：10,11

THO1：6,9.3

TPOX：8

vWA：18

D21S11：31.2,32”。

In light of the foregoing description of the preferred embodiment of the present invention, many modifications and variations will be apparent to those skilled in the art without departing from the spirit and scope of the invention. The technical scope of the present invention is not limited to the details of the above-described embodiments, and must be determined according to the claims and the specification.

<110> Tianjin Tianrui Biotechnology Co., Ltd

<120> a natural killer cell line SILK-NK independent of cytokine culture

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ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgcctgtga catctacatc 360

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Claims

1. A process for preparing natural killer cell line SILK-NK without cell factor culture includes such steps as membrane binding the protein complex containing IL-15 protein and IL-15 binding region of IL-15R α to the surface of NK-92 cell line, and culturing in the culture medium without IL-2 to obtain the cloned cell line, i.e. natural killer cell line SILK-NK.

2. The method according to claim 1, wherein the protein complex consists of the IL-15 binding domain of IL-15R α, the transmembrane domain, and the IL-15 protein in that order from N-terminus to C-terminus.

3. The method according to claim 1 or 2, wherein the amino acid sequence of the IL-15 binding region of IL-15R α is SEQ ID No.1, or is a sequence having at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, or at least 75% homology with the amino acid sequence shown in SEQ ID No.1, or having the same function by substitution and/or deletion and/or addition of one or more amino acid residues;

and/or

The transmembrane region is a CD8 α transmembrane region;

furthermore, the amino acid sequence of the CD8 α transmembrane region is SEQ ID No.2, or is a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence shown in SEQ ID No.2, or has the same function through substitution and/or deletion and/or addition of one or more amino acid residues;

and/or

The amino acid sequence of the IL-15 protein is SEQ ID No.3, or is a sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of homology with the amino acid sequence shown in SEQ ID No.3, or has the same functions through substitution and/or deletion and/or addition of one or more amino acid residues.

4. A method according to claim 3, characterized in that: the amino acid sequence of the protein compound is SEQ ID No.4, or the protein compound has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% homology with the amino acid sequence shown in SEQ ID No.4, or has the same function through substitution and/or deletion and/or addition of one or more amino acid residues.

5. The method according to any one of claims 1-4, wherein: the method comprises the following steps: inserting the coding gene of the protein complex into a lentivirus expression vector, then packaging lentivirus by using the obtained recombinant lentivirus vector, infecting the NK-92 cell line with the lentivirus, culturing in an IL-2-free medium and obtaining the cloned cell line by a limiting dilution method.

6. The method according to claim 5, wherein the nucleotide sequence of the IL-15 binding region encoding IL-15R α in the gene encoding the protein complex is the 1 st to 216 th positions of SEQ ID No.5, or a sequence having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more homology to the 1 st to 216 th positions of SEQ ID No.5 and encoding the IL-15 binding region of IL-15R α, and/or

In the coding gene of the protein complex, the nucleotide sequence coding the CD8 α transmembrane region is the 217-423 th site of SEQ ID No.5, or the nucleotide sequence has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent, more than 80 percent or more than 75 percent of homology with the 217-423 th site of SEQ ID No.5 and codes the CD8 α transmembrane region, and/or

In the coding gene of the protein complex, the nucleotide sequence coding the IL-15 protein is the 490-831 site of SEQ ID No.5, or is a sequence which has more than 99 percent, more than 95 percent, more than 90 percent, more than 85 percent, more than 80 percent or more than 75 percent of homology with the 490-831 site of SEQ ID No.5 and codes the IL-15 protein;

furthermore, the nucleotide sequence of the coding gene of the protein complex is SEQ ID No.5, or the sequence which has more than 99%, more than 95%, more than 90%, more than 85%, more than 80% or more than 75% of homology with SEQ ID No.5 and codes the protein complex.

7. A natural killer cell line SILK-NK produced by the method according to any one of claims 1 to 6 and cultured independently of cytokines.

8. The natural killer cell line SILK-NK of claim 7, characterized in that: the STR phenotype of the natural killer cell line SILK-NK is

Amelogenin：X,Y

CSF1PO：11,12

D13S317：9,12

D16S539：11,12

D5S818：12,13

D7S820：10,11

THO1：6,9.3

TPOX：8

vWA：18

D21S11：31.2,32；

9. The natural killer cell line SILK-NK according to claim 7 or 8, characterized in that: the natural killer cell line SILK-NK is a cell line which is preserved in the China general microbiological culture Collection center of China Committee for culture Collection of microorganisms and has the registration accession number of CGMCC No. 15583.

10. Use of the natural killer cell line SILK-NK as claimed in claims 7 to 9 in any one of the following:

(A1) preparing a product for adoptive immunotherapy of tumors;

(A2) preparing related products for cell therapy.