CN111996170B - Genetically engineered NK cells, methods of preparation and uses thereof - Google Patents

Abstract

The invention provides a genetically engineered NK cell, a preparation method and application thereof. The genetically engineered NK cells express an antigen chimeric receptor, wherein the antigen chimeric receptor consists of an extracellular domain, a transmembrane domain and an intracellular signaling domain, and wherein the extracellular domain of the antigen chimeric receptor is an antibody or fragment thereof targeting the antigen IGF-1R, preferably nanobody Nab1. The genetically engineered NK cells of the present invention can kill tumor cells expressing IGF-1R.

Description

Genetically engineered NK cells, preparation methods and uses thereof

Technical Field

The present invention relates to the field of tumor immunotherapy, and in particular to genetically engineered natural killer (NK) cells, a preparation method thereof and use thereof in tumor treatment.

Background Art

In the field of tumor treatment, chimeric antigen receptor T cell therapy has attracted more and more attention.

Chimeric antigen receptor (CAR) is an artificially constructed transmembrane molecule encoded by a fusion gene that enables T cells and other cells to specifically target antigens on the surface of cancer cells to eliminate target cancer cells. CAR consists of an extracellular domain (e.g., a single-chain antibody, scFv), a transmembrane domain, and an intracellular domain. The scFv of the extracellular domain is responsible for the recognition of specific antigens. The intracellular domain is responsible for signal transduction. After the extracellular domain specifically binds to the antigen, the intracellular domain initiates the signal required for cell activation, thereby promoting the proliferation of T cells, the release of cytokines, etc. The transmembrane domain connects the extracellular domain and the intracellular domain.

Chimeric antigen receptor T cells (CAR T cells) are T cells that have been genetically modified to have chimeric antigen receptors and are used for immunotherapy.

However, for the above immunotherapy, obtaining T cells from patients is a cumbersome task. In addition, the preparation of CART cells takes several weeks and cannot meet the needs of critically ill patients. In particular, for patients with severe lymphocytopenia, it is impractical to obtain T cells from patients.

Given the inherent limitations of immunotherapy using CART cells, people have recently been trying to use CAR to modify NK cells to overcome the above limitations of CAR T cells. CAR-modified NK cells, namely CAR NK cells, are gaining more and more attention in treating tumors.

CARNK cells have many advantages that make them better than CAR T cells:

1. NK cell activation does not require prior sensitization or human leukocyte antigen (HLA) matching;

2. NK cells do not require antigen-specific receptors to kill tumor cells;

3. According to the results of mouse models and many clinical studies, CARNK cell immunotherapy has fewer serious side effects than CART cells;

4. The presence of functional NK cells that can be used, such as NK-92MI;

5. Sufficient NK cells can be obtained from peripheral blood;

6. CARNK cells can potentially be used as “off-the-shelf” universal CAR products.

In conclusion, CARNK cell therapy is a promising immunotherapy that will play a key role in the future.

Studies have shown that insulin-like growth factor-1 receptor (IGF-1R) is abnormally highly expressed in many tumor cells, such as neuroblastoma, breast cancer, lung cancer, colon cancer, pancreatic cancer, bladder cancer, hematogenous sarcoma, prostate cancer, liver cancer, ovarian cancer, gastric cancer, etc.

IGF-1R is a transmembrane receptor glycoprotein with tyrosine kinase activity. It consists of four subunits, two extracellular α subunits, and two β subunits with transmembrane and intracellular parts. The α subunit of IGF-1R binds to the ligand and activates the tyrosine kinase activity of the β subunit.

The insulin growth factor (IGF) signaling pathway is activated by insulin growth factor 1 (IGF1), insulin growth factor 2 (IGF-2) and/or insulin binding to IGF-1R, followed by IGF-1R autophosphorylation to initiate downstream cascades, such as PI3K and MAPK, thereby promoting cell proliferation and differentiation, and exerting anti-apoptotic effects and angiogenesis.

Therefore, blocking the binding of IGF-1R and IGF-2 has also become a promising target for inhibiting tumor growth.

Summary of the invention

In view of the above, in order to provide a more effective treatment for tumors overexpressing IGF-1R, the present invention provides a genetically engineered natural killer (NK) cell, a preparation method thereof and a method for inhibiting tumors to more effectively treat tumors overexpressing IGF-1R.

In one embodiment, the present invention provides a genetically engineered natural killer (NK) cell, which expresses an antigen chimeric receptor, wherein the antigen chimeric receptor is composed of an extracellular domain, a transmembrane domain and an intracellular signaling domain, wherein the extracellular domain is an antibody or a fragment thereof targeting the antigen IGF-1R, preferably a nano antibody Nab1, more preferably an antibody having an amino acid sequence of SEQ ID No: 1 or a fragment thereof having a homology greater than 90% and capable of specifically binding to the antigen IGF-1R.

The extracellular domain of the antigen chimeric receptor can bind to specific antigens, especially antigens overexpressed on tumor cells, such as IGF-1R.

The transmembrane domain is one of a CD4 transmembrane domain and a CD8 transmembrane domain, preferably a CD8 transmembrane domain.

The intracellular signaling domain is at least one selected from the group consisting of 4-1BB and CD3ζ.

The antigen chimeric receptor is composed of nano antibody Nab1, CD8 transmembrane domain, 4-1BB and CD3ζ in series.

The genetically engineered NK cells further express antibodies or fragments thereof targeting the antigen IGF-2, preferably nano antibody Nab2, more preferably an antibody having an amino acid sequence of SEQ ID No: 3 or a fragment thereof having a homology greater than 90% and capable of specifically binding to the antigen IGF-2.

Nanobody Nab2 specifically binds to IGF-2, the ligand of IGF-1R, thereby reducing the concentration of IGF-2 around tumor cells that overexpress the antigen IGF-1R. As a result, the binding of IGF-2 to IGF-1R is reduced, thereby blocking the IGF signaling pathway to inhibit tumor growth. As a result, the engineered NK cells of the present application can inhibit tumors from different angles at the same time, and produce synergistic effects, further enhancing the anti-tumor effect.

The engineered NK cells specifically bind to tumor cells that overexpress the antigen IGF-1R, thereby inducing the activation of NK-92MI cells and specifically killing tumor cells that overexpress the antigen IGF-1R, thereby achieving the effect of reducing the tumor.

In one embodiment, a method for preparing genetically engineered NK cells is provided, the method comprising the following steps:

Constructing a vector comprising genes encoding the extracellular domain, transmembrane domain and intracellular signaling domain of an antigen chimeric receptor, wherein the extracellular domain is an antibody or a fragment thereof targeting the antigen IGF-1R, preferably a nanobody Nab1, more preferably an antibody having an amino acid sequence of SEQ ID No: 1 or a fragment having a homology thereto of greater than 90% and capable of specifically binding to the antigen IGF-1R, preferably the nucleotide coding sequence of the nanobody Nab1 is SEQ ID No: 2, preferably the transmembrane domain is one of a CD4 transmembrane domain and a CD8 transmembrane domain, preferably a CD8 transmembrane domain, and preferably the intracellular signaling domain is at least one selected from the group consisting of 4-1BB and CD3ζ;

transfecting the vector into NK cells to prepare genetically engineered NK cells;

The genetically engineered NK cells are cultured and expanded in vitro to obtain an expanded genetically engineered NK cell population;

The genetically engineered NK cell population is harvested.

The extracellular domain of the antigen chimeric receptor can bind to specific antigens, especially antigens overexpressed on tumor cells, such as IGF-1R, etc. The extracellular domain of the antigen chimeric receptor is an antibody or a fragment thereof targeting the antigen IGF-1R, preferably a nano antibody Nab1; the transmembrane domain of the antigen chimeric receptor is one of the CD4 transmembrane domain and the CD8 transmembrane domain, preferably the CD8 transmembrane domain; and the intracellular signaling domain of the antigen chimeric receptor is at least one selected from the group consisting of 4-1BB and CD3ζ.

The vector comprises genes encoding the extracellular domain of nano-antibodies Nab1, 4-1BB, CD3ζ and the CD8 transmembrane domain.

The vector further includes nucleotides encoding an antibody or a fragment thereof that targets the antigen IGF-2, preferably the nanobody Nab2, more preferably an antibody having an amino acid sequence of SEQ ID No: 3 or a fragment thereof having a homology greater than 90% and capable of specifically binding to the antigen IGF-2, preferably the nucleotide encoding sequence of the nanobody Nab2 is SEQ ID No: 4.

The NK cells are at least one of NK-92MI cells, NKL cells and KHYG cells.

In a preferred embodiment, the method further comprises the step of transfecting a vector expressing an antibody or a fragment thereof targeting antigen IGF-2 into the NK cells, so that the surface of the genetically engineered NK cells simultaneously presents an antibody targeting antigen IGF-2 and an antibody or a fragment thereof targeting antigen IGF-1R, and while the antibody targeting antigen IGF-1R specifically identifies highly expressed tumor cells and exerts a killing effect, it hinders the binding of IGF-1R and IGF-2, further reduces the concentration of effective IGF-2 around tumor cells, and blocks the IGF signaling pathway from another angle to inhibit tumor growth. The simultaneous action of the two can produce a synergistic effect, greatly improving the anti-tumor effect.

According to the method of the present invention, "ready-made" NK cells can be used to quickly prepare genetically engineered NK cells that can specifically target tumor cells expressing IGF-1R.

In another embodiment, the present invention provides the use of genetically engineered NK cells in the preparation of a medicament for treating a tumor, wherein the tumor overexpresses the antigen IGF-1R.

The genetically engineered NK cells of the present invention combine the specificity of the extracellular domain of the antigen chimeric receptor for the antigen IGF-1R and the tumor cell-killing performance of the NK cells themselves, and can effectively kill tumor cells that overexpress the antigen IGF-1R.

The tumor may be selected from the group consisting of neuroblastoma, breast cancer, lung cancer, colon cancer, pancreatic cancer, bladder cancer, hematogenous sarcoma, prostate cancer, liver cancer, ovarian cancer and gastric cancer.

The genetically engineered NK cells according to the present invention can effectively kill tumor cells expressing the antigen IGF-1R.

BRIEF DESCRIPTION OF THE DRAWINGS

Embodiments of the present invention will be described in more detail below with reference to the accompanying drawings, in which:

FIG1 shows a schematic diagram of the binding of engineered NK cells to IGF-1R of neuroblastoma according to the present invention;

FIG2A shows a design diagram of a vector used to construct an engineered NK cell comprising a single nanobody Nab1 of the present invention;

FIG2B shows a design diagram of a vector used to construct engineered NK cells containing dual nanobodies Nab1 and Nab2 of the present invention;

Figure 2C shows a design diagram of a vector used to construct engineered NK cells containing a single nanobody Nab2 of the present invention;

FIG3A shows a schematic diagram of the positive rate after sorting of NK-92MI cells transfected with a plasmid containing the DNA sequence of a single nanobody CAR by lentivirus;

FIG3B shows a schematic diagram of the positive rate after sorting of NK-92MI cells transfected with a plasmid containing the DNA sequence of the dual nanobody CAR by lentivirus;

Figure 4A shows the results of the cytotoxicity test of NK-92MI cells transfected with a plasmid containing the DNA sequence of a single nanobody CAR and a plasmid containing the DNA sequence of a double nanobody CAR on LAN-1 cells;

Figure 4B shows the results of cytotoxicity tests on IMR-32 cells by NK-92MI cells transfected with plasmids containing the DNA sequence of a single nanobody CAR and plasmids containing the DNA sequence of a double nanobody CAR; and

Figure 4C shows the results of cytotoxicity tests against LAN-1 cells of NK-92MI cells engineered with dual nanoantibodies Nab1 and Nab2, NK-92MI cells engineered with single nanoantibody Nab1, NK-92MI cells engineered with single nanoantibody Nab2, and a combination of NK-92MI cells engineered with single nanoantibody Nab1 and single nanoantibody Nab2.

DETAILED DESCRIPTION

Neuroblastoma is the most common malignancy in infants and the third most common cancer in children.

IGF-1R is expressed in 86% of primary neuroblastoma tumors, making IGF-1R a good target for cancer immunotherapy. The genetically engineered NK cells provided by the present invention include an extracellular nanoantibody Nab1 domain, a CD8 transmembrane domain, and an intracellular signal activation domain, and the intracellular signal activation domain includes 4-1BB and CD3ζ. In addition, the genetically engineered NK cells provided by the present invention also express autocrine nanoantibodies Nab2. Nanoantibody Nab1 specifically binds to IGF-1R, thereby inducing NK-92MI cell activation and killing tumor cells. At the same time, the secreted Nab2 binds specifically to the ligand IGF-2, so that the ligand IGF-2 around the neuroblastoma is neutralized, reducing its binding to IGF-1R, thereby blocking the IGF signaling pathway to inhibit tumor growth. Therefore, through the synergistic effect of the two aspects, the genetically engineered CAR-NK of the present invention can specifically kill neuroblastoma cells that highly express IGF-1R to cure neuroblastoma.

IGF-1R belongs to one of the IGF systems. The IGF system includes IGF1, IGF-2, IGF-1R, IGF-2R, insulin receptor (IR), and six high-affinity IGF binding proteins (IGFBPs). IGF1 and IGF-2 are peptides that are essential for normal growth in the human body. In addition to the liver, many other tissues produce IGFs that act in a paracrine or autocrine manner. Most IGFs are bound by IGFBP1-6. Although this binding can increase the stability of the ligand, the bioavailability is reduced, and IGFBPs compete with ligand-receptor binding. It seems that IGF-2R has no signaling function and may be related to the regulation of IGF-2 ligand bioavailability. IGF-1R has 70% amino acid identity with IR, and its catalytic domain has 84% identity. Therefore, IGF can bind to and activate IR, even though the affinity is much lower, while insulin can bind to and activate IGF-1R. IGF-2 binds to IGF-1R and IR with similar affinity. There is also a hybrid IGF-1R/IR receptor that contains subunits of both receptors. All three ligands can bind to the hybrid receptor. Once IGF-1R is activated, the IGF-1R kinase domain will be autophosphorylated, and the intracellular domain cascade will be activated. IRS1 and IRS2 represent insulin receptor substrates and are the main signal transducers of IGF-1R and IR. They will be recruited to the receptor and act as docking sites. IRS1 and IRS2 are able to connect the activated receptor to various intracellular adaptor proteins and downstream signaling pathways. In neuroblastoma, IGF-1R signals mainly through two key pathways. One is to promote cell survival and proliferation through IRS, PI3K, AKT, and mTOR. The other is to promote cell growth, tumorigenesis, and inhibit apoptosis through Shc, RAS, RAF, MEK, and ERK. Inhibition of apoptosis is crucial in tumor cell growth. In the second pathway, ERK can induce proliferation through phosphorylation of transcription factors c-Fos and Ets-like transcription factor 1 (ELK1). IGF-1R signaling also activates c-Myc, JNK, and c-Jun. IGF-1R-mediated increases in ribosomal activity and expression of cyclins A, B, and D1 enhance cell cycle progression. The anti-apoptotic properties of IGF-1R play a crucial role in ameliorating IGF-1R-mediated tumorigenesis.

Natural killer (NK) cells are cells that mature in the bone marrow with a half-life as short as 7 days. Most NK cells are found in the blood, spleen, and liver. They play two key roles in the human body. One is that they can indirectly but potentially regulate the innate and adaptive immune systems by secreting cytokines (e.g., IFN-γ) when receiving attack signals from other immune cells. IFN-γ can enhance the phagocytic and microbicidal activity of macrophages. Another is that they can affect the differentiation of CD4+ helper cell subsets by stimulating dendritic (DC) cells and macrophages to stimulate interleukin-12 (IL-12), inhibiting the proliferation of helper T cells 2 (TH2) and inducing the development of helper T cells 1 (TH1). In addition to being cytokine factories, NK cells can also kill tumor cells, virus-infected cells, bacteria, parasites, and fungi without prior sensitization. Tumor cells have developed a clever way to escape cytotoxic T lymphocytes (CTLs), which makes their major histocompatibility complex I (MHC I) disappear. However, NK cells are smarter than tumor cells. Based on the "self-deficient" model, NK has activating receptors and inhibitory receptors. Inhibitory receptors, such as killer cell inhibitory receptors (KIR, Ly49), can bind to MHC I molecules and inhibit killing, while activating receptors, such as natural killer group 2 member D (NKG2D) and natural cytotoxicity receptors (NCR), can induce killing. When NK cells find that MHC I is downregulated, the activated killing signal will dominate, and NK will begin to kill target cells. Stress-related proteins on the surface of cancer cells, such as MHC class I chain-related gene A protein (MICA), can be bound by NKG2D and are also related to NK killing. NK cells have two different ways of killing. First, NK cells use perforins to transfer "suicide enzymes" (such as granzymes) into target cells, resulting in target cell lysis. In addition, there is Fas ligand on the surface of NK cells, which can bind to Fas on target cells to transmit suicide signals.

NK cells have many functional cells, including natural killer cells from human malignant non-Hodgkin's lymphoma patients (NK-92MI), human NK cell lymphoma cells (NKL), human NK cell lymphoma cells (KHYG-1), etc. Among them, NK-92MI is a widely used highly cytotoxic IL-2-independent NK cell. It is derived from NK-92 by transfection. This NK-92 is derived from PBMC and is a 50-year-old white male with non-Hodgkin's lymphoma. Phase I clinical trials have shown that it is safe to inject NK-92MI into cancer patients.

CAR stands for chimeric antigen receptor. They are artificially constructed receptors consisting of an extracellular antibody-derived antigen binding domain, hinge and transmembrane domains, and intracellular co-stimulatory and primary signaling domains. Typically, the extracellular antigen binding domain is a scFv region that can bind to an antigen. For the CAR structure used in the present invention, the scFc region is replaced by the small VH region of a common IgG. So far, there have been 3 generations of CARs. The first generation contains a cytoplasmic CD3ζ domain, which belongs to the TCR complex and is used as an internal domain. For the second generation, an additional co-stimulatory domain is added to increase the potency of the CAR, and for the third generation, more co-stimulatory domains such as CD28, 4-1BB, etc. are added.

Since cancer cells can evade NK cell killing through different strategies, NK cells can be genetically engineered to express CAR structures with higher specifications, such as higher specificity. When CAR-NK binds to antigen, the intracellular signaling domain will be activated, which will lead to the activation of PI3K or DNAX proteins and enhance cytotoxicity, proliferation and/or IFN-γ release.

The nano antibody Nab1 used in the present invention is an antibody obtained by screening a library with high affinity binding to IGF-1R. The amino acid sequence of the nano antibody Nab1 and the nucleic acid sequence encoding it are shown in the sequence listing SEQ ID No: 1 and 2, respectively.

The nanobody Nab2 used in the present invention is an antibody that can bind to IGF-2 ligand. The amino acid sequence of the nanobody Nab2 and the nucleic acid sequence encoding it are shown in the sequence listing SEQ ID No: 3 and 4, respectively.

The 293T cells used in the present invention are commonly used cells for virus production. When the cells are used to produce viruses, their titers are relatively high. They are derived from human embryonic kidney 293 cells and contain SV40 T antigens.

The LAN-1 cells used in the present invention are human neuroblastoma cells expressing IGF-1R, which are from RIKEN, Japan. They were established from the bone marrow transfer of a 2-year-old boy with stage IV neuroblastoma and can be cultured using RPMI1640 + 10% FBS.

The IMR-32 cells used in the present invention are also human neuroblastoma cells expressing IGF-1R, and are from the Cell Bank of the Chinese Academy of Sciences.

The plasmids for producing lentivirus used in the present invention include pLVX-IRES-ZsGreen1, psPAX2 and PMD2.G from Addgene.

Experimental Materials

Unless otherwise specified, the materials used in the present invention are all commercially available.

The amino acid sequences and nucleotide sequences used in the present invention are shown in the sequence listing and are also described in Table 1 below.

Table 1

Amino acid or nucleotide sequence number Sequence Description SEQ ID No:1 Amino acid sequence of the nanobody Nab1 SEQ ID No:2 Nucleotide coding sequence of nanobody Nab1 SEQ ID No:3 Amino acid sequence of nanobody Nab2 SEQ ID No:4 Nucleotide coding sequence of nanobody Nab2

Experimental methods

Unless otherwise specified, the experimental methods used in the present invention are conventional experimental methods in the art.

1. Construction of vector

The DNA sequence of the CAR containing the nanobody Nab1, and the DNA sequence of the CAR containing the nanobody Nab1 and the nanobody Nab2 were synthesized separately and cloned into the pLVX-IRES-ZsGreen 1 plasmid. This plasmid is a lentiviral expression vector based on HIV-1, which can be used to simultaneously express the target gene and ZsGreen 1 in various types of cells. ZsGreen 1 is a human codon-optimized variant of the reef coral Zoanthus sp. ZsGreen 1 can be used as a transduction efficiency indicator and marker in flow cytometry (FACS). EcoRI enzyme and XhoI enzyme are used to cut the pLVX-IRES-ZsGreen 1 plasmid because it contains these two restriction enzyme sites. After PCR and enzyme digestion of pLVX-IRES-ZsGreen 1, T4 DNA ligase is used to digest and then connect the PCR product to the vector. After the connection is completed, the connection product is transformed into competent cells. They are then coated on the plate, and after overnight culture, several single colonies are selected for further culture for 14-16 hours. The plasmids were then extracted from the bacteria and sent for sequencing.

Figure 1 shows a schematic diagram of the combination of engineered NK cells and IGF-1R of neuroblastoma according to the present invention. As shown in Figure 1, engineered NK cells target IGF-1R expressed on the surface of tumor cells through the nano antibody Nab1 of the CAR extracellular domain. Engineered NK cells contain nano antibody Nab1 as an antigen binding domain, CD8 as a transmembrane domain (not shown) and 4-1BB and CD3ζ as activation domains (not shown). In addition, engineered NK-92 cells also secrete and express nano antibody Nab2. Once nano antibody Nab1 recognizes and binds to IGF-1R on the surface of tumor cells, NK cells can be activated to kill tumor cells targeted by nano antibody Nab1. Secreted nano antibody Nab2 can neutralize the ligand IGF-2 around tumor cells targeted by nano antibody Nab1, reducing competition with nano antibody Nab1.

2. 293T lentivirus packaging

After obtaining the plasmid containing CAR, 293T cells were used to package lentivirus. Polyetherimide (PEI) was used to assist transfection, and three plasmid systems were used for transfection: pLVX-IRES-ZsGreen 1 as a transfer plasmid, psPAX2 as a packaging plasmid, and PMD2.G as an envelope plasmid. After collecting the supernatant, the supernatant was centrifuged and the virus was successfully obtained. Afterwards, the virus was titrated to determine the form of the virus. 293T cells were used to test the virus titer.

3. Viral transfection of NK cells

Based on the virus titer, calculate the volume of virus required to transfect NK cells. While preparing the virus, culture the NK cells at the same time. With a sufficient number of NK cells, transfection can be performed with the generated virus. The virus was added a total of 2 times on day 1 and day 2. Polyethylene was also added on day 1 to increase the transfection rate. After each addition of virus, the NK cells in the 6-well plate were centrifuged at 1500RPM for 1 hour to increase the transfection rate because the NK cells float in the culture medium instead of adhering to the wells. By centrifuging, the chance of the virus entering the NK cells is greatly improved.

Specifically, 24 hours before transfection, 293T cells in the logarithmic growth phase were digested with trypsin, the cell density was adjusted to 1.2 x 10 ⁷ cells/20 ml with a medium containing 10% serum, and re-seeded in a 15 cm cell culture dish and cultured in an incubator at 37°C and 5% CO _2. Cells were used for transfection when the cell density reached 70% to 80%. The cell state is critical for virus packaging, so a good cell state and a low number of passages need to be ensured.

The cell culture medium was changed to serum-free medium 2 hours before transfection.

培养基混合均匀，调整总体积为2.5ml，在室温下温育5分钟。Add the prepared DNA solutions (20 μg of pLVX-IRES-ZsGreen 1 vector, 15 μg of pMD2.G vector, and 10 μg of PsPAX2 vector) to a sterile centrifuge tube and mix with the corresponding volume of

The culture medium was mixed well, adjusted to a total volume of 2.5 ml, and incubated at room temperature for 5 minutes.

培养基混合，在室温下温育5分钟。Gently shake the PEI reagent, take 100 μl of PEI reagent and mix it with 2.4 ml of

The culture medium was mixed and incubated at room temperature for 5 minutes.

Mix the diluted DNA with the diluted PEI and gently invert to mix. Transfer the mixture to the culture medium of 293T cells, mix well, and culture in a cell culture incubator at 37°C and 5% CO _2. After 8 hours of culture, pour out the culture medium containing the transfection mixture and add fresh culture medium. Continue to culture for 48 hours, collect the supernatant, and filter with a 0.45μm syringe filter.

The virus supernatant was transferred to an ultracentrifuge tube, and the weight difference between the two symmetrical tubes was <0.02 g. The tubes were centrifuged at 25,000 rpm and 4°C for 1 hour and 45 minutes.

After centrifugation, discard the supernatant and resuspend the pellet after centrifugation in pre-cooled RPMI 1640 culture medium, that is, resuspend the virus and place it at -80°C for use.

Virus titers were determined as follows.

Prepare 293T cells: Cells were passaged normally and added to 12-well plates at 30% confluency one day before virus infection. At least two wells per virus were used for gradient assay.

Virus addition: On the day of infection, count the total number of cells in one of the wells and record the value. Take the virus out of -80℃ and place it on ice to melt. Add 5μg/ml polybrene to each well in the experimental group for dyeing. Add 0.5μl of virus to one well and 2μl of virus to another well. Set up a blank control as a detection control.

Virus titer determination by FACS: 48 hours after virus infection, digest the cells for flow cytometry. If the cells carry fluorescent markers directly, filter the cells through a 70μm filter membrane and then test them directly. If antibodies need to be added, incubate the antibodies before testing.

Virus titer determination: The virus titer was determined according to the following formula:

Virus titer (TU/ml) = number of cells on the day of infection × cell infection rate × 10 ³ / virus volume (μl)

TU/ml stands for titer unit, which refers to the number of biologically active virus particles contained in each milliliter. TU stands for transduction unit, which indicates the number of viral genomes that can infect and enter the target cells.

Next, 5×10 ⁶ NK cells were prepared, and the virus was added at a ratio of virus:cell=5:1. The virus was added twice in total on the 1st day and the 2nd day.

After each addition of virus, NK cells in the 6-well plate were centrifuged at 1500 rpm for 1 h to increase the transfection efficiency.

4. Test transfection efficiency and sorting

48 hours after adding the virus to the NK cells, FACS was performed to determine how many cells were successfully transfected.

Cells were sorted using BD FACSAriaIII. Since CAR carries zsGreen fluorescence, positive cells can be sorted using a 488nm laser.

After sorting, the sorted CAR-NK cells are expanded in cell culture so that they can multiply into more. A second sorting is performed to ensure that they are 100% pure CAR-NK cells, and then subsequent cytotoxicity experiments can be performed.

5. Cytotoxicity assay

When enough CAR-NK cells are obtained, the cytotoxicity experiment can be performed. Calcein AM is used to perform cytotoxicity tests. First, LAN-1 cells were stained with Calcein AM at 37°C for 30 minutes, and then the LAN-1 cells were washed with PBS, and the stained LAN-1 cells were prepared in this way. Since Calcein AM can be converted into green fluorescent calcein inside the cells, after killing, the dead LAN-1 cells will release green fluorescence into the supernatant for extraction and reading. 96-well plates were used for this experiment. NK cells and LAN-1 cells were used in ratios of 20:1, 10:1, 5:1, 2.5:1 and 1:1, respectively. There were three copies for each ratio. In addition, three control groups were used, including 100% killing of LAN-1, spontaneous LAN-1, and LAN-1 culture medium. In the present invention, the killing time ranged from 3-5 hours. After reading the results, the cytotoxicity rate can be calculated by the following formula.

The method and concept of the present invention are further explained below by means of specific examples, but the scope of the present invention is not limited by the examples.

Example 1

1. Construction of NK-92MI cells containing single nanobody Nab1 CAR engineering

According to the design scheme shown in Figure 2A, a vector is constructed by conventional technical means in the art (see the "Construction of Vectors" section in the Experimental Methods section), wherein the vector includes a nucleic acid encoding a chimeric antigen receptor composed of nanobody Nab1 (NA-1), CD8 transmembrane domain (TM), 4-1BB and CD3ζ.

For clarity, 5'LTR in Figure 2A represents 5' long terminal repeat; Ψ represents viral packaging non-coding sequence; LS represents the coding sequence of the leader sequence; NA-1 represents the coding sequence of the nanobody Nab1; CD8 TM represents the coding sequence of the CD8 transmembrane domain; 4-1BB and CD3ζ represent the coding sequences of the intracellular signaling domains 4-1BB and CD3ζ, respectively; IRES represents the coding sequence of the internal ribosome entry site; and ZsGreen1 represents the coding sequence of a fluorescent protein.

This resulted in a plasmid containing the CAR encoding the single nanobody Nab1, pLVX-IRES-ZsGreen1.

2. 293T lentivirus packaging

According to the steps in "293T lentiviral packaging" in the above "Experimental Methods" section, lentiviral packaging was performed on 293T cells to obtain a virus containing the pLVX-IRES-ZsGreen1 plasmid.

3. Viral transfection of NK cells

According to the steps in "Viral transfection of NK cells" in the above "Experimental methods" section, the virus obtained above was used to transfect NK-92MI cells. Transfected CAR-NK-92MI positive cells were obtained. And through the steps in "Testing transfection rate and sorting" in the above "Experimental methods" section, the positive rate of the transfected CAR-NK-92MI positive cells was tested, and sorted to obtain engineered CAR-NK-92MI cells.

Figure 3A shows a schematic diagram of the positive rate after sorting of NK-92MI cells transfected with a plasmid containing the DNA sequence of a single nano antibody CAR by lentivirus. After sorting, the positive cell rate in the engineered CAR-NK-92MI cells was 73.4%. It was confirmed that NK-92MI cells engineered with a CAR containing a single nano antibody Nab1 were obtained.

Example 2

Example 2 was carried out using the same method as in Example 1, except that the design scheme diagram of the vector used to construct the engineered NK cells containing the double nanobodies of the present invention shown in Figure 2B was used when preparing the plasmid.

2A in the design scheme of Figure 2B represents the coding sequence of Thoseaasigna virus 2A peptide; NA-2 represents the coding sequence of nanobody Nab2.

Figure 3B shows a schematic diagram of the positive rate after sorting of NK-92MI cells transfected with a plasmid containing the DNA sequence of the double nanobody CAR by lentivirus. After sorting, the positive cell rate in the engineered CAR-NK-92MI cells was 68.1%. It was confirmed that engineered NK-92MI cells containing the double nanobody Nab1 and Nab2 were obtained.

Example 3

Example 3 was carried out using the same method as in Example 1, except that the design scheme of the vector used to construct the engineered NK-92MI cells containing the single nanobody Nab2 of the present invention shown in Figure 2C was used when preparing the plasmid.

Next, the cytotoxicity of the engineered NK-92MI cells obtained in Examples 1, 2, and 3 and the combination of Examples 1 and 3, i.e., the combination of engineered NK-92MI cells containing single nanobody Nab1 and engineered NK-92MI cells containing single nanobody Nab2, was tested by evaluation example.

Evaluation Example

Calcein AM was used to perform cytotoxicity tests. First, LAN-1 cells were stained with Calcein AM at 37°C for 30 minutes, and then the LAN-1 cells were washed with PBS, and the stained LAN-1 cells were prepared in this way. Since Calcein AM can be converted into green fluorescent calcein inside the cells, after killing, the dead LAN-1 cells will release green fluorescence into the supernatant for extraction and reading. 96-well plates were used for this experiment. Engineered NK-92MI cells and LAN-1 cells obtained in Examples 1 and 2 were used in ratios of 20:1, 10:1, 5:1, 2.5:1 and 1:1, respectively, and engineered NK-92MI cells and IMR-32 cells obtained in Examples 1 and 2 were used in ratios of 20:1, 10:1, 5:1, 2.5:1 and 1:1, respectively. The experiment was repeated three times for each ratio, and the results were averaged. In addition, three control groups were used, including 100% killed LAN-1, spontaneous LAN-1, and LAN-1 culture medium.

In the present invention, the killing time ranges from 3 to 5 hours. After reading the results, the cytotoxicity rate can be calculated by the following formula.

For IMR-32 cells, the cytotoxicity rate was calculated using a method similar to that described above for LAN-1 cells.

Figure 4A shows the results of the cytotoxicity test of NK-92MI cells transfected with a plasmid containing the DNA sequence of a single nanobody CAR and a plasmid containing the DNA sequence of a double nanobody CAR on LAN-1 cells.

In Figure 4A, the curve single nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with single nanoantibody Nab1 against LAN-1 cells; the curve double nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with double nanoantibodies Nab1 and Nab2 against LAN-1 cells.

Figure 4B shows the results of cytotoxicity tests against IMR-32 cells by NK-92MI cells transfected with a plasmid containing the DNA sequence of a single nanobody CAR and a plasmid containing the DNA sequence of a double nanobody CAR.

In Figure 4B, the curve single nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with single nanoantibody Nab1 against IMR-32 cells; the curve double nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with double nanoantibodies Nab1 and Nab2 against IMR-32 cells.

Figure 4C shows the results of cytotoxicity tests against LAN-1 cells of NK-92MI cells engineered with dual nanoantibodies Nab1 and Nab2, NK-92MI cells engineered with single nanoantibody Nab1, NK-92MI cells engineered with single nanoantibody Nab2, and a mixture of NK-92MI cells engineered with single nanoantibody Nab1 and single nanoantibody Nab2.

In Figure 4C, the curve double nanoCAR represents the results of the cytotoxicity test of NK-92MI cells engineered with double nanoantibodies Nab1 and Nab2 against LAN-1 cells; the curve Nano1CAR+Nano2CAR represents the results of the cytotoxicity test of the combination of NK-92MI cells engineered with single nanoantibody Nab1 and NK-92MI cells engineered with single nanoantibody Nab2 against LAN-1 cells, wherein the ratio of NK-92MI cells engineered with single nanoantibody Nab1 and NK-92MI cells engineered with single nanoantibody Nab2 is 1:1; the curve Nano1CAR represents the results of the cytotoxicity test of NK-92MI cells engineered with single nanoantibody Nab1 against LAN-1 cells; and the curve Nano2CAR represents the results of the cytotoxicity test of NK-92MI cells engineered with single nanoantibody Nab2 against LAN-1 cells.

As can be seen from Figure 4A, the NK-92MI cells engineered with the dual nanoantibodies Nab1 and Nab2, whether at a cell ratio of 10:1 or 20:1, obtained better cytotoxicity test results than the NK-92MI cells engineered with the single nanoantibody Nab1. That is, the NK-92MI cells engineered with the dual nanoantibodies Nab1 and Nab2 have a stronger killing ability against LAN-1 cells.

As can be seen from Figure 4B, the NK-92MI cells engineered with the dual nanoantibodies Nab1 and Nab2, whether at a cell ratio of 10:1 or 20:1, obtained better cytotoxicity test results than the NK-92MI cells engineered with the single nanoantibody Nab1. That is, the NK-92MI cells engineered with the dual nanoantibodies Nab1 and Nab2 have a stronger killing ability against IMR-32 cells.

As can be seen from Figure 4C, in the killing experiment against LAN-1 cells, the NK-92MI cells engineered by the double nanoantibody Nab1 and Nab2 have the greatest cytotoxicity against LAN-1 cells, not only greater than the cytotoxicity of the NK-92MI cells engineered by the single nanoantibody Nab1 and the NK-92MI cells engineered by the single nanoantibody Nab2, but also greater than the cytotoxicity of the combination of the NK-92MI cells engineered by the single nanoantibody Nab1 and the NK-92MI cells engineered by the single nanoantibody Nab2 against LAN-1 cells. It is confirmed that the NK-92MI cells engineered by the double nanoantibody Nab1 and Nab2 have achieved excellent synergistic effects in killing target cells.

Since two antibodies are presented on the surface of NK cells at the same time, the genetically engineered NK cells obtained achieve the effect of killing tumor cells that highly express IGF-1R antigen, and the effect is much higher than expected, proving that it has a synergistic effect. The engineered NK cells provided by the present invention can specifically and powerfully kill tumor cells that express IGF-1R antigen. It can reduce many side effects of patients using CAR-T cell therapy, making it a promising "ready-made" immunotherapy product.

Sequence Listing

<110> University of Macau

Macao Yingfu Biopharmaceutical Co., Ltd.

<120> Genetically engineered NK cells, preparation methods and uses thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 125

<212> PRT

<213> Artificial Sequence

<400> 1

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Tyr Tyr Ser Gln Gln Tyr Asn

20 25 30

Ser Tyr Pro Ile Thr Phe Met Ser Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Arg Leu Glu Trp Val Ala Ser Ile Asn Gln Asp Gly Ser Gln Ile Asp

50 55 60

Tyr Ala Gly Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser

65 70 75 80

Lys Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr

85 90 95

Ala Thr Tyr Tyr Cys Ala Val Asp Leu Arg Ser Gly Ala Arg Asn Phe

100 105 110

Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 2

<211> 375

<212> DNA

<213> Artificial Sequence

<400> 2

caggtgcagc tggtgcagag cggcggcggc ctggtgcagc cgggcggcag cctgcgcctg 60

agctgcgcgg cgagctatta tagccagcag tataacagct atccgattac ctttatgagc 120

tgggtgcgcc aggcgccggg ccagcgcctg gaatgggtgg cgagcattaa ccaggatggc 180

agccagattg attatgcggg cagcgtgaaa ggccgcttta ccattagccg cgataacagc 240

aaaaacaccc tgtatctgca gatgaacacc ctgcgcgcgg aagataccgc gacctattat 300

tgcgcggtgg atctgcgcag cggcgcgcgc aactttcagc attggggcca gggcaccctg 360

gtgaccgtga gcagc 375

<210> 3

<211> 124

<212> PRT

<213> Artificial Sequence

<400> 3

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ile Ser Tyr Tyr Gly Leu Gln His Asp

20 25 30

Asn Phe Pro Tyr Thr Phe Met Ser Trp Val Arg Gln Ala Pro Gly Gln

35 40 45

Arg Leu Glu Trp Val Ser Gly Ile Ser Gly Ser Gly Gly Thr Thr Tyr

50 55 60

Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp His Ser

65 70 75 80

Lys Asn Met Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr

85 90 95

Ala Met Tyr Tyr Cys Ala Arg Ile Arg Trp Leu Gln Asp Leu Asp Tyr

100 105 110

Trp Gly Gln Gly Ala Leu Val Thr Val Ser Ser Ser

115 120

<210> 4

<211> 372

<212> DNA

<213> Artificial Sequence

<400> 4

caggtgcagc tggtgcagtc tgggggaggc ttggtacagc ctggagggtc cctgagactc 60

tcctgtgcaa tctcttacta cggtctacaa catgacaatt tcccgtacac ttttatgagc 120

tgggtccgac aggctccagg acaacgtctt gagtgggtct caggtattag tggtagtggt 180

ggtactacat actatgcaga ctccgtgaag ggccgattca ccatctccag agaccattcc 240

aagaacatgc tgtatctgca aatgaacacc ctgagagccg aggacacagc catgtattac 300

tgtgcacgga ttcgatggct acaagatctt gactactggg gccagggagc cctggtcacc 360

gtctcctcaa gt 372

Claims (10)

1. A genetically engineered natural killer (NK) cell, the genetically engineered NK cell expresses an antigen chimeric receptor, wherein the antigen chimeric receptor consists of an extracellular domain, a transmembrane domain and Intracellular signaling domain composition, wherein the extracellular domain is an antibody targeting antigen IGF-1R, the amino acid sequence of the antibody targeting antigen IGF-1R is SEQ ID No: 1, and the genetically engineered NK The cells further express an antibody targeting the antigen IGF-2, and the amino acid sequence of the antibody targeting the antigen IGF-2 is SEQ ID No:3. 2. The genetically engineered NK cell according to claim 1, wherein the transmembrane domain is one of a CD4 transmembrane domain and a CD8 transmembrane domain. 3. The genetically engineered NK cell according to claim 1 or 2, wherein the intracellular signaling domain is at least one selected from the group consisting of 4-1BB and CD3ζ. 4. The genetically engineered NK cell according to claim 1 or 2, wherein the antigen chimeric receptor is composed of the antibody targeting antigen IGF-1R, the antibody targeting antigen IGF-2, CD8 transmembrane domain, 4-1BB and CD3ζ are composed in tandem. 5. A method for preparing the genetically engineered NK cell according to any one of claims 1 to 4, said method comprising the steps of: Constructing a vector comprising genes encoding an extracellular domain, a transmembrane domain and an intracellular signaling domain of an antigen chimeric receptor, wherein the extracellular domain is an antibody targeting the antigen IGF-1R, and the targeting The amino acid sequence of the antibody targeting the antigen IGF-1R is SEQ ID No: 1, and the nucleotide coding sequence of the antibody targeting the antigen IGF-1R is SEQ ID No: 2, The vector further includes encoding and expressing an antibody targeting antigen IGF-2, the amino acid sequence of the antibody targeting antigen IGF-2 is SEQ ID No: 3, and the nucleoside of the antibody targeting antigen IGF-2 The acid coding sequence is SEQ ID No: 4; Transfecting the vector into NK cells to prepare genetically engineered NK cells; Cultivating and expanding the genetically engineered NK cells in vitro to obtain an expanded population of genetically engineered NK cells; The genetically engineered NK cell population is harvested. 6. The method according to claim 5, wherein the transmembrane domain is one of a CD4 transmembrane domain and a CD8 transmembrane domain. 7. The method according to claim 5, wherein the intracellular signaling domain is at least one selected from the group consisting of 4-1BB and CD3ζ. 8. The method according to claim 5, wherein the carrier comprises an extracellular protein encoding the antibody targeting the antigen IGF-1R, the antibody targeting the antigen IGF-2, 4-1BB, CD3ζ domain and the gene for the CD8 transmembrane domain. 9. The method according to claim 5, wherein the NK cells are at least one of NK-92MI cells, NKL cells and KHYG cells. 10. Use of the genetically engineered NK cell according to any one of claims 1 to 4 in the preparation of a medicament for treating tumors, wherein the tumor overexpresses the antigen IGF-1R, and the tumor is selected from In the group consisting of neuroblastoma, breast cancer, lung cancer, colon cancer, pancreatic cancer, bladder cancer, hemorrhagic sarcoma, prostate cancer, liver cancer, ovarian cancer and gastric cancer.

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