Anti-complement activated NK (Natural Killer) cell as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of cell therapy, and particularly relates to an anti-complement activated NK cell as well as a preparation method and application thereof. The invention provides a preparation method of an anti-complement activated NK cell. The preparation method comprises the following steps: preparing a CAR-NK cell strain; inserting the receptor gene sequence into the lentiviral vector to obtain a recombinant lentiviral vector for expressing the receptor gene sequence; carrying out lentivirus packaging on the recombinant lentivirus vector to obtain a lentivirus solution containing a receptor gene sequence; and infecting a CAR-NK cell strain with lentivirus venom to obtain the anti-complement activated NK cell. The NK cells prepared by the method can escape from a complement system to attack, so that rapid removal of the NK cells by an immune system is avoided, and the residence time of the NK cells in the body of a tumor patient is remarkably prolonged.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell therapy, and specifically relates to an anti-complement activated NK cell and a preparation method and application thereof. Background Art

[0002] The complement system is a group of proteins present in the serum, tissue fluid and cell membrane surface of humans and vertebrates. After activation, it has enzymatic activity and self-regulation. The biological effects produced by the complement system after activation are mainly the following: after complement activation, the membrane attack complex is formed, which can make holes on the surface of target cells, allowing water and electrolytes to enter the cells, leading to cell disintegration; the fragments such as C3b, C4b and iC3b produced by complement activation can bind to particulate antigens such as bacteria, promoting their phagocytosis by phagocytes; the various active fragments produced during complement activation can bind to receptors on the surface of immune cells, regulating the proliferation, differentiation and activity of immune cells; complement components can bind to immune complexes, promoting their clearance by phagocytes.

[0003] Since the expression of receptors with complement activity inhibition such as CD55, CD46, CD59 and CD35 on the surface of NK cells is low, NK cells or cell lines that have undergone in vitro amplification and genetic modification are easily recognized and bound by antibodies in the human body, which makes NK cells vulnerable to complement attack, thereby significantly reducing the therapeutic effect of NK cells. Enhancing the anti-complement activity of NK cells is crucial to improving the therapeutic effect. The existing technology usually prepares NK cell lines by co-incubating NK cells with lentiviral vectors carrying polynucleotides encoding chimeric antigen receptors CAR. NK cell lines prepared by this method are still difficult to avoid complement attacks.

[0004] In summary, there is an urgent need to provide a method for preparing NK cells that can escape the attack of the complement system. Summary of the invention

[0005] The purpose of the present invention is to provide a complement-activated NK cell and a preparation method and application thereof, and to provide a method for preparing NK cells that can escape the attack of the complement system.

[0006] The technical solution adopted by the present invention is that the present invention provides a method for preparing complement-activated NK cells, comprising the following steps: preparing a CAR-NK cell line; inserting a receptor gene sequence into a lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, wherein the receptor gene is a membrane-bound complement regulatory protein or a humoral complement inhibitory factor; performing lentiviral packaging on the recombinant lentiviral vector to obtain a lentiviral venom containing the receptor gene sequence; and infecting the CAR-NK cell line with the lentiviral venom to obtain complement-activated NK cells.

[0007] Preferably, the membrane-bound complement regulatory protein is any one or more of CD55, CD46, CD59, CD35 and FcμR.

[0008] Preferably, the humoral complement inhibitory factor is any one or more of complement component 1 inhibitor, complement component 4 binding protein, factor I, factor H, S protein and complement component 8 binding protein.

[0009] Preferably, the CAR-NK cell line is prepared by NK cells, and the NK cells are isolated from peripheral blood, umbilical cord blood, stem cells, pluripotent stem cells or NK tumor cell lines.

[0010] In one aspect, the present invention provides a cell prepared according to the above preparation method.

[0011] Another aspect of the present invention provides a use of the cell, wherein the cell is used to prepare a preparation for treating malignant tumors.

[0012] The cells are combined with a complement inhibitor to prepare a preparation for treating malignant tumors.

[0013] Preferably, the complement inhibitor is one or more of an inhibitor of complement component 1, an inhibitor of complement component 3, and an inhibitor of complement component 5.

[0014] Preferably, the complement component 1 inhibitor is one or both of a complement component 1 esterase inhibitor and a complement component 1s protein monoclonal antibody.

[0015] Preferably, the inhibitor of complement component 3 is one or both of Empaveli and Compstatin.

[0016] Preferably, the inhibitor of complement component 5 is one or more of Soliris, Ultomiris and Tavneos.

[0017] Preferably, the complement component 1 esterase inhibitor is Berinert, Cinryze or Ruconest.

[0018] Preferably, the complement component 1s protein monoclonal antibody is Enjaymo.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention provides a method for preparing NK cells, the steps of which are as follows: preparing a CAR-NK cell line; inserting a receptor gene sequence into a lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, wherein the receptor gene is a membrane-bound complement regulatory protein or a humoral complement inhibitory factor; performing lentiviral packaging on the recombinant lentiviral vector to obtain a lentiviral venom containing the receptor gene sequence; infecting the CAR-NK cell line with the lentiviral venom to obtain an anti-complement activation type NK cell. The cells prepared by the preparation method of the present invention can avoid the attack of the complement system.

[0020] Based on this, the inventors inserted the CD55 or CD46 receptor gene sequence into the lentiviral vector, and prepared the CAR-NK92-CD55 cell line and the CAR-NK92-CD46 cell line using the preparation method. Since CD55 and CD46 can inhibit the excessive activation of the complement system and protect their own cells from damage. The expression of CD55 and CD46 receptors on the surface of NK cells is low, so expressing CD55 or CD46 receptors on CAR-NK cells can help cells escape the attack of the complement system, avoid the rapid clearance of NK cells by the immune system, and significantly prolong the retention time of NK cells in tumor patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 To detect the affinity of NK92 cultured in X-VIVO serum-free medium to IgM.

[0022] Figure 2 To detect the toxicity of healthy human plasma to NK92 cultured in X-VIVO serum-free medium, A and B are the serum test results of healthy volunteers 1 and 2, respectively.

[0023] Figure 3 In order to detect the role of complement inhibitors in the toxicity of healthy human plasma to NK92 by flow cytometry, A to C are: NK92 cells cultured with MEM-Hu medium for 12 h, NK92 cells cultured with 50% MEM-Hu medium + 50% freshly collected plasma for 12 h, NK92 cells cultured with 50% MEM-Hu medium + 50% freshly collected plasma and the addition of complement component C3 inhibitor Compstatin with a final concentration of 63 μM for 12 h.

[0024] Figure 4 For the construction of CAR-NK92-CD55 and CAR-NK92-CD46 cells, A: Flow cytometry detection of the expression level of CD55 on CAR-NK92-CD55 cells; B: Flow cytometry detection of the expression level of CD46 on CAR-NK92-CD46 cells.

[0025] Figure 5 To detect the toxicity of plasma on CAR-NK92-CD55 and CAR-NK92-CD46 cells, A to D are: CAR-NK92 cells were cultured with MEM-Hu medium for 12 hours, CAR-NK92 cells were cultured with 50% MEM-Hu medium + 50% freshly collected plasma for 12 hours, and CAR-NK92-CD55 and CAR-NK92-CD46 cells were cultured with 50% MEM-Hu medium + 50% freshly collected plasma for 12 hours.

[0026] Figure 6 To detect the retention status of CAR-NK92 cells, CAR-NK92-CD55 cells, CAR-NK92-CD46 cells and CAR-NK92 cells combined with eculizumab injection in the patient's body.

[0027] Figure 7 To detect the retention status of CAR-NK cells, CAR-NK-CD55 cells, CAR-NK-CD46 cells, and CAR-NK cells combined with eculizumab injection in patients, A and B are NK cells derived from umbilical cord blood or NK cells derived from peripheral blood, respectively. DETAILED DESCRIPTION

[0028] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0029] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0030] The invention concept is as follows: The present invention provides a method for preparing complement-activated NK cells, comprising the following steps: preparing a CAR-NK cell line; inserting a receptor gene sequence into a lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, wherein the receptor gene is a membrane-bound complement regulatory protein or a humoral complement inhibitory factor; performing lentiviral packaging on the recombinant lentiviral vector to obtain a lentiviral venom containing the receptor gene sequence; and infecting the CAR-NK cell line with the lentiviral venom to obtain complement-activated NK cells. The cells prepared by the preparation method can avoid the attack of the complement system.

[0031] Since the expression of receptors with complement activity inhibition effects such as CD55, CD46, CD59 and CD35 on the surface of NK cells is low, NK cells or cell lines that have undergone in vitro amplification and genetic modification are easily recognized and bound by antibodies in the human body, which makes NK cells vulnerable to complement attack. Therefore, the inventor provides a method for preparing cells with high receptor expression. In an embodiment of the present invention, the receptor genes used are CD55 and CD46. CD55, also known as decay accelerating factor DAF, is a membrane lipid-related anchoring protein. It protects host cells from complement-mediated lysis and destruction. CD55, as a complement inhibitory factor, promotes the degradation of C3 and C5 convertases and prevents the complement system from being overactivated. Membrane DAF is widely distributed on various blood cells and other cells, but its expression on NK cells is low, significantly lower than the expression of granulocytes or T cells. This also makes NK cells vulnerable to complement damage during treatment. Therefore, when using NK-based cell therapy, inhibiting complement activity is crucial to the function of NK cells. CD46, also known as Membrane Cofactor Protein (MCP), is an important protein present on the surface of human blood cells, endothelial cells, etc. CD46 protein can inhibit the excessive activation of the complement system and protect the body's cells from damage, and is figuratively likened to a "protective umbrella" for cells. However, the expression of CD46 on NK cells is also low.

[0032] The inventive concept of the present invention is further explained below in conjunction with Experiments 1 to 3.

[0033] Experiment 1: Detect the affinity of NK92 cultured in X-VIVO serum-free medium to IgM.

[0034] X-VIVO serum-free medium composition, referred to as X-VIVO medium: TheraPEAK X-VIVO-15 Serum-free Hematopoietic Cell Medium is supplemented with the following final concentrations: 1% penicillin-streptomycin solution, 1000 U / mL recombinant human IL-2, and 2 mM L-Glutamine. After NK92 cells have been cultured in the above medium for at least 3 generations, Figure 1 As shown, the blank control is NK92 cells that were not incubated with serum or flow cytometry antibodies; Anti-IgM is NK92 cells incubated with Anti-IgM flow cytometry antibodies only; Volunteer serum + Anti-IgM is NK92 cells incubated with serum from healthy volunteers 1 or 2 and anti-IgM flow cytometry antibodies. Flow cytometry was used to detect the binding of IgM in the peripheral blood of healthy people to NK92 cells. The results showed that IgM in the peripheral blood of healthy people all bound to NK92 cells.

[0035] Experiment 2: Detect the toxicity of healthy human plasma to NK92 cultured in X-VIVO serum-free medium.

[0036] The inventors cultured NK92 cells using the above X-VIVO serum-free medium and then incubated them with freshly collected plasma for 30 minutes. Trypan blue staining confirmed that the cell membrane permeability of NK92 cells was enhanced after incubation with healthy human plasma, as shown in Figure 2. Figure 2 shown.

[0037] Experiment 3: Detect the role of complement inhibitors in the toxicity of healthy human plasma to NK92.

[0038] The inventors used MEM medium supplemented with human AB serum, referred to as MEM-Hu medium, the ingredients of which were as follows: 10% human AB serum, 1% penicillin-streptomycin solution and 200U / mL IL-2 were added to MEM to culture NK92 cells for 12 hours; or 50% MEM-Hu medium + 50% freshly collected plasma were used to culture NK92 cells for 12 hours; or 50% MEM-Hu medium + 50% freshly collected plasma were used and the complement component C3 inhibitor Compstatin was added to a final concentration of 63 μM to culture NK92 cells for 12 hours; flow cytometry apoptosis detection confirmed that healthy human plasma was toxic to NK92 cells, and complement inhibitors could reduce the toxicity of healthy human plasma to NK92 cells, such as Figure 3 shown.

[0039] It can be seen that in the present invention, the NK92 cell line has a high affinity for IgM in a healthy human body, and IgM activates complement, thereby causing the immune system to quickly eliminate NK92 cells.

[0040] The present invention obtains complement activation-resistant NK92 cells through genetic modification, which significantly prolongs the retention time of NK92 cells in tumor patients. At the same time, the inventors also found that the same effect can be achieved by giving patients complement inhibitor drugs to prevent complement activation during CAR-NK92 treatment.

[0041] The present invention is described in detail below with reference to specific embodiments.

[0042] The main reagents used in the present invention are shown in Table 1.

[0043] Table 1 Main reagents

[0044] Reagent name Part Number factory nation X-VIVO Medium BEBP02-061Q Lonza Switzerland Penicillin-Streptomycin Solution C0222 Blue Sky China Recombinant human IL-2 200-02-1MG PeproTech USA L-Glutamine 25030081 Gibco America MEM medium WH1021D041 HyClone USA Human AB serum 100-512 GeminiBio USA Eculizumab Injection Soliris AstraZeneca UK

[0045] The Chinese-English comparison table of the present invention is shown in Table 2.

[0046] Table 2 Chinese-English comparison table

[0047]

[0048]

[0049] In Table 2, “ / ” indicates that the item does not exist.

[0050] Example 1: A method for preparing NK cells that resist complement activation

[0051] 1. Prepare CAR-NK cell lines, specifically CAR-NK92 cells, as follows:

[0052] The present invention constructs a novel chimeric antigen receptor CAR that targets human NKG2D ligands. This is achieved by fusing the extracellular domain of NKG2D with the hinge region of IgG4, the transmembrane domain of CD28, and the cytoplasmic signaling domains of CD28, CD137, and CD3ζ. The CAR receptor gene is subcloned into the lentiviral vector pCDH-CMV by performing full gene synthesis at GenScript. Lentivirus packaging and transfection are performed in accordance with standard operating procedures to construct CAR-NK92 cells. Separation and sequencing confirmed the successful construction of CAR-NK92. Then, Henan Intercell Biotechnology Co., Ltd. further amplified CAR-NK92 cells in X-VIVO serum-free medium under GMP conditions, and quality control was performed to obtain CAR-NK92 cells that can be used in clinical practice.

[0053] It should be noted that the NK92 cells used in the preparation of CAR-NK92 cells in the present invention are isolated from peripheral blood, or NK92 cells isolated from umbilical cord blood, stem cells, pluripotent stem cells or NK tumor cell lines, or NK92 cells purchased on the market. In a specific embodiment of the present invention, commercially available NK92 cells are used.

[0054] The construction method of CAR-NK92 cell line comes from the reference Guo C, Chen H, Yu J, Lu H, Xia Q, LiX, Guo X, Wang T, Zhi L, Niu Z, Zhu W. Engagement of an optimized lentiviral vector enhances the expression and cytotoxicity of CAR in human NK cells. Mol Immunol. 2023Mar;155:91-99.doi:10.1016 / j.molimm.2023.01.010.Epub2023Feb1.PMID:36736195.

[0055] 2. Insert the receptor gene sequence into the lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, as follows:

[0056] The gene capable of expressing CD55 receptor was inserted into the pCDH-CMV lentiviral expression vector to obtain a recombinant lentiviral vector expressing the CD55 receptor gene sequence, and the CD55 receptor gene sequence is shown in SEQ ID NO. 1. A calcium phosphate cell transfection kit was purchased from Beyotime, item number C0508.

[0057] 3. Perform lentiviral packaging on the recombinant lentiviral vector to obtain lentiviral venom containing the receptor gene sequence, as follows:

[0058] The recombinant lentiviral vector was packaged in 293T cells. During the packaging process, the mass ratio of pCDH-CMV-CD55, psPAX2 and pMD2G was 4:3:1, and the lentiviral venom containing the CD55 receptor gene sequence was obtained.

[0059] 4. Infect the CAR-NK cell line with lentiviral venom to obtain complement-resistant NK cells as follows:

[0060] The CAR-NK92 cell line was infected with the lentiviral venom containing the CD55 receptor gene sequence, and CD55 was further expressed on the constructed CAR-NK92 cell line to obtain CAR-NK92-CD55 cells. The expression level of CD55 on the CAR-NK92 cell line was then detected by flow cytometry. The results are as follows: Figure 4 As shown, the expression level of CD55 increased by more than 95%.

[0061] SEQ ID NO.1:

[0062]

[0063] Example 2: A method for preparing NK cells resistant to complement activation

[0064] 1. Preparation of CAR-NK cell lines, specifically CAR-NK92 cells:

[0065] The preparation method is the same as the preparation method of the CAR-NK92 cell line in Example 1.

[0066] 2. Insert the receptor gene sequence into the lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, as follows:

[0067] The gene capable of expressing CD46 receptor was inserted into the pCDH-CMV lentiviral expression vector to obtain a recombinant lentiviral vector expressing the CD46 receptor gene sequence, and the CD46 receptor gene sequence is shown in SEQ ID NO. 2. A calcium phosphate cell transfection kit was purchased from Beyotime, item number C0508.

[0068] 3. Perform lentiviral packaging on the recombinant lentiviral vector to obtain lentiviral venom containing the receptor gene sequence, as follows:

[0069] The recombinant lentiviral vector was packaged in 293T cells. During the packaging process, the mass ratio of pCDH-CMV-CD46, psPAX2 and pMD2G was 4:3:1, and the lentiviral venom containing the CD56 receptor gene sequence was obtained.

[0070] 4. Infect the CAR-NK cell line with lentiviral venom to obtain complement-resistant NK cells as follows:

[0071] The CAR-NK92 cell line was infected with the lentiviral venom containing the CD46 receptor gene sequence, and CD46 was further expressed on the constructed CAR-NK92 cell line to obtain CAR-NK92-CD46 cells. The expression of CD46 on the CAR-NK92 cell line was then detected by flow cytometry. The results are as follows: Figure 4 As shown, the expression of CD46 increased by more than 95%

[0072] SEQ ID NO.2:

[0073]

[0074] Example 3: Application of an anti-complement activated NK cell

[0075] 1. Detection of plasma toxicity to CAR-NK92-CD55 and CAR-NK92-CD46 cells.

[0076] The inventors used MEM medium supplemented with human AB serum, referred to as MEM-Hu medium, the ingredients of which were as follows: 10% human AB serum, 1% penicillin-streptomycin solution and 200U / mL IL-2 were added to MEM to culture CAR-NK92 cells for 12 hours; or 50% MEM-Hu medium + 50% freshly collected plasma were used to culture CAR-NK92 cells for 12 hours; or 50% MEM-Hu medium + 50% freshly collected plasma were used to culture CAR-NK92-CD55 or CAR-NK92-CD46 cells for 12 hours; flow cytometry assays confirmed that healthy human plasma was toxic to CAR-NK92 cells, and that the expression of CD55 or CD46 could reduce the toxicity of healthy human plasma to CAR-NK92 cells, such as Figure 5 shown.

[0077] 2. Detect the retention status of CAR-NK92 cells, CAR-NK92-CD55 cells, CAR-NK92-CD46 cells, and CAR-NK92 cells combined with eculizumab injection in the patient's body.

[0078] Volunteers with solid tumors were injected with 1.5×10 9 CAR-NK92 cells, CAR-NK92-CD55 cells, or CAR-NK92-CD46 cells, or 1.5×10 9 CAR-NK92 cells were injected into the tumor patients at the same time as 600 mg of eculizumab. Blood samples were collected 24 hours after cell infusion, peripheral blood leukocytes were separated, and the leukocyte genome was extracted and tested by qPCR. The number of CAR-NK92 cells in each group in the peripheral blood was determined by detecting the content of the CAR receptor gene. The test results are shown in Figure 6 As shown in the results, expression of CD55 or CD46 in CAR-NK92 or combination with eculizumab can increase the retention of CAR-NK92 cells in peripheral blood.

[0079] 3. Detect the retention status of CAR-NK cells, CAR-NK-CD55 cells, CAR-NK-CD46 cells, and CAR-NK cells combined with eculizumab injection in the patient's body.

[0080] The NK92 cells in the above were replaced with NK cells from umbilical cord blood or NK cells from peripheral blood, etc. The cells were provided by Henan Genetic Resource Cell Bank Co., Ltd. to prepare CAR-NK cells, CAR-NK-CD55 cells, and CAR-NK-CD46 cells. The retention of various types of CAR-NK cells in peripheral blood was detected according to the above scheme. The results showed that replacing NK92 cells with other types of NK cells can also produce similar effects.

[0081] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0082] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A method for preparing NK cells resistant to complement activation, characterized in that: The following steps are involved: Preparation of CAR-NK cell lines; Inserting a receptor gene sequence into a lentiviral vector to obtain a recombinant lentiviral vector expressing the receptor gene sequence, wherein the receptor gene is a membrane-bound complement regulatory protein or a humoral complement inhibitory factor; Performing lentiviral packaging on the recombinant lentiviral vector to obtain lentiviral venom containing the receptor gene sequence; The lentiviral venom was used to infect the CAR-NK cell line to obtain complement-activation-resistant NK cells.

2. The preparation method according to claim 1, characterized in that: The membrane-bound complement regulatory protein is any one or more of CD55, CD46, CD59, CD35 and FcμR; The humoral complement inhibitory factor is any one or more of complement component 1 inhibitor, complement component 4 binding protein, factor I, factor H, S protein and complement component 8 binding protein.

3. The preparation method according to claim 1, characterized in that: The CAR-NK cell line is prepared by NK cells, and the NK cells are isolated from peripheral blood, umbilical cord blood, stem cells, pluripotent stem cells or NK tumor cell lines.

4. A cell prepared by the preparation method according to claim 1.

5. The use of the cell according to claim 4, characterized in that: The cells are used for preparing a preparation for treating malignant tumors.

6. The use of the cell according to claim 4, characterized in that: The cells are combined with a complement inhibitor to prepare a preparation for treating malignant tumors.

7. The use of the cell according to claim 6, characterized in that: The complement inhibitor is one or more of an inhibitor of complement component 1, an inhibitor of complement component 3, and an inhibitor of complement component 5.

8. The use of the cell according to claim 7, characterized in that: The inhibitor of complement component 1 is one or both of complement component 1 esterase inhibitor and complement component 1s protein monoclonal antibody; The inhibitor of complement component 3 is one or both of Empaveli and Compstatin; The inhibitor of complement component 5 is one or more of Soliris, Ultomiris and Tavneos.

9. The use of the cell according to claim 8, characterized in that: The complement component 1 esterase inhibitor is Berinert, Cinryze or Ruconest; The complement component 1s protein monoclonal antibody is Enjaymo.

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