Cytokine fusion protein and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a cytokine fusion protein and application thereof. The invention provides a cytokine fusion protein and application thereof. The expression problem of the membrane-bound IL-2 in NK cells is solved, and the membrane expression efficiency of the membrane-bound IL-2 is remarkably improved; according to the membrane binding IL-2 provided by the invention, the in-vivo drug effect and the in-vivo continuity of the CAR-NK are remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to cytokine fusion proteins and applications thereof. Background Art

[0002] Adoptive cell therapy (ACT) refers to the separation of immune-competent cells from cancer patients, their expansion and functional identification in vitro, and then their return to the patient, thereby directly killing the tumor or stimulating the body's immune response to kill tumor cells.

[0003] Chimeric antigen receptor (CAR) T cell technology is a rapidly developing cellular immunotherapy technology. CAR is an artificially synthesized fusion receptor, which includes an extracellular antigen binding region, a transmembrane region, an intracellular signal transduction region, and a co-stimulatory signal region. The extracellular region is a monoclonal antibody sequence (single chain variable fragment, ScFv) that recognizes tumor-associated antigens. The transmembrane region connects the extracellular region and the intracellular region. Commonly used transmembrane region molecules are selected from the transmembrane functional domains of genes such as CD3, CD4, CD8, and CD28. The intracellular region has an immune receptor tyrosine activation motif (ITAM), and the most commonly used ones are T cell receptor TCR / CD3ζ chain and immunoglobulin FC receptor FCεRIγ, which are mainly responsible for signal transduction function. The CAR structure is used to transfer the recombinant gene into NK cells through viral vectors, electroporation, etc. to form the corresponding CAR-NK cells. CAR-NK cells are not restricted by HLA matching and can achieve allogeneic adoptive immune therapy.

[0004] Interleukin-2 (IL-2) is a cytokine in the chemokine family. It is a cytokine that is derived from multiple cells (mainly produced by activated T cells) and has multi-directional effects (mainly promoting lymphocyte growth, proliferation, and differentiation). It plays an important role in the body's immune response and anti-viral infection, and can stimulate specific antigens or stimulate NK cell proliferation, enhance NK killing activity, and produce cytokines. However, compared with CAR-T cells, CAR-NK lacks the autocrine secretion of factors such as IL-2, and requires exogenous injection or overexpression to continuously exert anti-tumor effects in animals or humans.

[0005] At present, there is a composition and method for enhancing T and NK cell responses, in which IL-2 is applied to CAR-T or CAR-NK cells to enhance T or NK cell responses. However, the above technical solution works by secreting IL-2 into the environment through CAR-T or CAR-NK cells. Since the amount of secretion is uncontrollable, there are risks in terms of safety and side effects. Fixing IL-2 to the cell membrane surface can solve the problem of uncontrollable dose of secreted IL-2. Although IL-2 has a significant promoting effect on the functions of CAR-T and CAR-NK, and IL-2 is often used in the culture process of T or NK cells, the application of mbIL-2 is rare.

[0006] In recent years, CAR-NK has been unsatisfactory in clinical performance, one of the important reasons is that CAR-NK has poor persistence in the body. Therefore, screening a safe and controllable NK amplification signal molecule will greatly improve the efficacy and application scenarios of CAR-NK. Summary of the invention

[0007] In view of this, the present invention provides a cytokine fusion protein and its application.

[0008] The present invention provides a cytokine fusion protein and its application. The present invention solves the expression problem of membrane-bound IL-2 in NK cells and significantly improves the membrane expression efficiency of membrane-bound IL-2; the mbH9 provided by the present invention significantly improves the in vivo efficacy and persistence of CAR-NK.

[0009] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0010] The present invention provides the use of IL-2 mutants in constructing membrane-bound fusion peptides.

[0011] In some specific embodiments of the present invention, the mutant of IL-2 is H9;

[0012] The H9 features:

[0013] (I), the amino acid sequence shown in SEQ ID No.32; or

[0014] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0015] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

[0016] The present invention also provides a fusion peptide comprising any of the following:

[0017] (i), H9, hinge region and first transmembrane domain; and / or

[0018] (ii), H9 and the second transmembrane domain;

[0019] The H9 features:

[0020] (I), the amino acid sequence shown in SEQ ID No.32; or

[0021] (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or

[0022] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

[0023] In some specific embodiments of the present invention, the fusion peptide further includes an intracellular signaling region.

[0024] In some specific embodiments of the present invention, the fusion peptide includes any of the following:

[0025] (iii), comprising H9, hinge region 8h and first transmembrane domain 8TM in sequence; and / or

[0026] (iv), comprising H9 and the second transmembrane domain PDGFRβTM in sequence; and / or

[0027] (v), including H9, hinge region 8h, first transmembrane domain 8TM and intracellular signaling region in sequence; and / or

[0028] (vi) It includes H9, the second transmembrane domain PDGFRβTM and the intracellular signaling region in sequence.

[0029] In some specific embodiments of the present invention,

[0030] (A), the 8h has the amino acid sequence shown in SEQ ID No.21; or

[0031] (B), the 8TM has the amino acid sequence shown in SEQ ID No. 22; or

[0032] (C) the PDGFRβTM has the amino acid sequence shown in SEQ ID No.33; or

[0033] (D) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (A) to (C); or

[0034] (E) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (A) to (D).

[0035] In some specific embodiments of the present invention,

[0036] (F), the fusion peptide has an amino acid sequence as shown in SEQ ID No. 37 or 38; or

[0037] (G) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (F); or

[0038] (H) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (F) or (G).

[0039] Based on the above research, the present invention also provides a nucleic acid molecule encoding the fusion peptide.

[0040] In some specific embodiments of the present invention,

[0041] (J), the nucleic acid molecule of the fusion peptide has a nucleotide sequence as shown in SEQ ID No. 18 or 19; or

[0042] (K), a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (J), but is different from the nucleotide sequence shown in (J) due to the degeneracy of the genetic code; or

[0043] (L), a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (J) or (K), and a nucleotide sequence that has the same or similar function as the nucleotide sequence shown in (J) or (K); or

[0044] (M) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (J) to (L).

[0045] The present invention also provides a protein combination, comprising the fusion peptide and a chimeric antigen receptor.

[0046] In some specific embodiments of the present invention, the chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane region, and an intracellular signaling domain;

[0047] The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

[0048] In some specific embodiments of the present invention,

[0049] 1) The CD19 scFv has the amino acid sequence shown in SEQ ID No. 20; or

[0050] 2) The CD123 scFv has the amino acid sequence shown in SEQ ID No. 26; or

[0051] 3) The intracellular signaling domain has an amino acid sequence as shown in SEQ ID No. 25; or

[0052] 4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of 1) to 3); or

[0053] 5) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of 1) to 4).

[0054] In some specific embodiments of the present invention, the protein combination includes CAR-5, CAR-6, CAR-7, CAR-8 and / or CAR-12;

[0055] 6), the CAR-5, CAR-6, CAR-7, CAR-8, and CAR-12 have the amino acid sequences shown in SEQ ID No.55, 56, 57, 58, and 62, respectively; or

[0056] 7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in 6); or

[0057] 8) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in 6) or 7).

[0058] The present invention also provides genetic elements, including the nucleic acid molecules and nucleic acid molecules encoding chimeric antigen receptors.

[0059] In some specific embodiments of the present invention, the genetic element comprises any of the following:

[0060] (1), a nucleic acid molecule encoding a chimeric antigen receptor and the nucleic acid molecule in sequence; and / or

[0061] (2) A nucleic acid molecule encoding the nucleic acid molecule and the chimeric antigen receptor in sequence.

[0062] In some specific embodiments of the present invention, the chimeric antigen receptor and the fusion peptide are connected via a self-cleaving peptide;

[0063] The chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain;

[0064] The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

[0065] In some specific embodiments of the present invention, the self-cleaving peptides include but are not limited to 2A, P2A, T2A, E2A and F2A.

[0066] In some specific embodiments of the present invention, the nucleic acid molecule encoding the 2A has:

[0067] (4) the nucleotide sequence shown in SEQ ID No.12; or

[0068] (5) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or

[0069] (6) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (4) or (5).

[0070] In some specific embodiments of the present invention,

[0071] (7) The nucleic acid molecule encoding the CD19 scFv has the nucleotide sequence shown in SEQ ID No. 1; or

[0072] (8) The nucleic acid molecule encoding the CD123 scFv has the nucleotide sequence shown in SEQ ID No. 7; or

[0073] (9) The nucleic acid molecule encoding the hinge region has the nucleotide sequence shown in SEQ ID No. 2; or

[0074] (10) The nucleic acid molecule encoding the transmembrane region has the nucleotide sequence shown in SEQ ID No. 3; or

[0075] (11), the nucleic acid molecule encoding the intracellular signaling domain has the nucleotide sequence shown in SEQ ID No.6; or

[0076] (12) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (7) to (11), and having the same or similar functions as the nucleotide sequence shown in any one of (7) to (11); or

[0077] (13) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in any one of (7) to (12).

[0078] In some specific embodiments of the present invention, the genetic elements include CAR-5, CAR-6, CAR-7, CAR-8 and CAR-12;

[0079] (17), wherein the CAR-5, CAR-6, CAR-7, CAR-8 and CAR-12 have the nucleotide sequences shown in SEQ ID No. 43, 44, 45, 46 and 50, respectively; or

[0080] (18) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (17), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (17); or

[0081] (19) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (17) or (18).

[0082] The invention also provides an artificial vector, comprising the gene element.

[0083] The present invention also provides virus particles, including the artificial vector.

[0084] The present invention also provides an engineered cell, comprising any of the following:

[0085] a), the fusion peptide; and / or

[0086] b), the nucleic acid molecule; and / or

[0087] c), the protein combination; and / or

[0088] d), said genetic element; and / or

[0089] e), the artificial carrier; and / or

[0090] f), the virus particles.

[0091] In some specific embodiments of the present invention, the engineered cells include but are not limited to NK cells.

[0092] The present invention also provides the use of any of the following items in improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells:

[0093] a), the fusion peptide; and / or

[0094] b), the nucleic acid molecule; and / or

[0095] c), the protein combination; and / or

[0096] d), said genetic element; and / or

[0097] e), the artificial carrier.

[0098] In some specific embodiments of the present invention, the improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells includes promoting the secretion of factors by CAR-NK cells and / or increasing the copy number of CAR vectors in CAR-NK cells.

[0099] In some specific embodiments of the present invention, the CAR-NK cell-secreted factors include IFN-γ.

[0100] The present invention also provides the use of any of the following items in the preparation of a drug for preventing and / or treating a disease:

[0101] ①, the fusion peptide; and / or

[0102] ②, the nucleic acid molecule; and / or

[0103] ③. the protein combination; and / or

[0104] ④, the genetic element; and / or

[0105] 5. the artificial carrier; and / or

[0106] ⑥, the virus particles; and / or

[0107] 7. The engineered cells.

[0108] In some specific embodiments of the present invention, the prevention and / or treatment of the disease includes improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells.

[0109] In some specific embodiments of the present invention, the improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells includes promoting the secretion of factors by CAR-NK cells and / or increasing the copy number of CAR-NK cells.

[0110] In some specific embodiments of the present invention, the disease includes but is not limited to AML.

[0111] The present invention also provides a medicine comprising any of the following:

[0112] ①, the fusion peptide; and / or

[0113] ②, the nucleic acid molecule; and / or

[0114] ③. the protein combination; and / or

[0115] ④, the genetic element; and / or

[0116] 5. the artificial carrier; and / or

[0117] ⑥, the virus particles; and / or

[0118] 7. The engineered cells.

[0119] A large number of studies have shown that IL-2 can promote the expansion of NK cells in vitro and in vivo, but very few studies have reported that membrane-bound IL-2 can normally play the role of IL-2, mainly because NK cells with membrane-bound IL-2 are difficult to express. In our experiments, we also found that various forms of constructed mbIL-2 cannot be normally expressed in NK cells, which greatly hinders the application and functional exploration of mbIL-2. The present invention constructs a variety of membrane-bound interleukin 2 with different structures, which are applied to CAR-NK of CD19 and CD123 targets respectively. mbH9 can be expressed on the surface of CAR-NK cell membrane, which solves the problem that membrane-bound IL-2 cannot be normally expressed and has no function in immune cells, and avoids the safety problem of uncontrollable secretion level in vivo when using secretory IL2. And the fusion protein we constructed can promote the persistence of CAR-NK cells in vivo, significantly improving the in vivo efficacy of CAR-NK. H9 is suitable for different membrane-bound fusion protein designs, among which it has better expression efficiency upstream of the 2A peptide; various transmembrane structures such as CD8-TM and PDGFRβTM can be used as transmembrane domains of mbH9. mbH9 also has the function of improving the in vivo efficacy and persistence of CAR-NK in different indications such as AML.

[0120] The present invention solves the expression problem of membrane-bound IL-2 in NK cells and significantly improves the membrane expression efficiency of membrane-bound IL-2; mbH9 significantly improves the in vivo efficacy and persistence of CAR-NK. BRIEF DESCRIPTION OF THE DRAWINGS

[0121] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0122] Figure 1 The mbIL-2-CAR-NK positive rate detection in Example 1 is shown;

[0123] Figure 2 The in vivo efficacy evaluation of CAR-8 and 9 in Example 2 is shown;

[0124] Figure 3 The continuous detection of CAR-8 and 9 in vivo in Example 2 is shown;

[0125] Figure 4 The in vivo effectiveness of CAR-6 in Example 2 is shown;

[0126] Figure 5 The in vivo efficacy evaluation of cryopreserved CAR-8 and 10 in Example 3 is shown;

[0127] Figure 6 The continuous detection of frozen CAR-8 and 10 in vivo in Example 3 is shown;

[0128] Figure 7 The in vitro killing efficiency detection of CD123-CAR-NK with different structures in Example 4 is shown;

[0129] Figure 8 The secretion detection of CD123-CAR-NK factors with different structures in Example 4 is shown;

[0130] Fig. 9 The in vivo efficacy evaluation of NK cells modified with CAR-11 and 12 in Example 5 is shown;

[0131] Fig.10 The in vivo drug expansion effect of NK cells modified with CAR-11 and 12 in Example 5 is shown. DETAILED DESCRIPTION

[0132] The present invention discloses cytokine fusion proteins and their applications. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters to achieve them. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0133] A large number of studies have shown that IL-2 can promote the expansion of NK cells in vitro and in vivo, but very few studies have reported that membrane-bound IL-2 can normally play the role of IL-2, mainly because NK cells with membrane-bound IL-2 are difficult to express. In our experiments, we also found that various forms of constructed mbIL-2 cannot be normally expressed in NK cells, which greatly hinders the application and functional exploration of mbIL-2. The present invention constructs a variety of membrane-bound interleukin 2 with different structures, which are applied to CAR-NK of CD19 and CD123 targets respectively. mbH9 can be expressed on the surface of CAR-NK cell membrane, which solves the problem that membrane-bound IL-2 cannot be normally expressed and has no function in immune cells, and avoids the safety problem of uncontrollable secretion level in vivo when using secretory IL2. And the fusion protein we constructed can promote the persistence of CAR-NK cells in vivo, significantly improving the in vivo efficacy of CAR-NK. H9 is suitable for different membrane-bound fusion protein designs, among which it has better expression efficiency upstream of the 2A peptide; various transmembrane structures such as CD8-TM and PDGFRβTM can be used as transmembrane domains of mbH9. mbH9 also has the function of improving the in vivo efficacy and persistence of CAR-NK in different indications such as AML.

[0134] The present invention solves the expression problem of membrane-bound IL-2 in NK cells and significantly improves the membrane expression efficiency of membrane-bound IL-2; mbH9 significantly improves the in vivo efficacy and persistence of CAR-NK.

[0135] Sequences in the present invention:

[0136]

[0137]

[0138]

[0139]

[0140]

[0141]

[0142]

[0143]

[0144]

[0145]

[0146]

[0147]

[0148]

[0149]

[0150]

[0151]

[0152]

[0153] The intracellular co-stimulatory signal of 41BB is BB in BBZ, and Z in BBZ is CD3 zeta

[0154] The cytokine fusion protein provided by the present invention and the raw materials and reagents used in its application can all be purchased from the market.

[0155] The present invention will be further described below in conjunction with embodiments:

[0156] Example 1 Detection and screening of mbIL-2 expression with different structures

[0157] 1. The present invention tests the function of mbIL2 with different structures in CAR-NK with CD19 target, wherein mbIL2 is a membrane-bound fusion peptide constructed by wild-type IL2, the RNA sequence and amino acid sequence of wild-type IL-2 can be obtained from NCBI, NCBI related numbers: S77834.1, AAD14263.2; the RNA sequence and amino acid sequence of wild-type IL2Ra can be obtained from NCBI, NCBI related numbers: NM_000417.3, NP_000408.1, membrane-bound fusion peptides designed based on wild-type IL-2 such as IL2-G4S-IL2Ra, IL2-PDGFRβTM, IL2-8H-8TM-; mbH9 is a membrane-bound fusion peptide constructed by a mutant IL2 sequence, such as H9-PDGFRβTM (mbH9-1), H9-8H-8TM- (mbH9-2), and the specific CAR structure is shown in Table 1:

[0158] Table 1

[0159] CD19scFv-8h-8TM-BBz-2A-IL2-G4S-IL2Ra CAR-1 CD19scFv-8h-8TM-BBz-2A-GMR-IL2-PDGFRβTM CAR-2 CD19scFv-8h-8TM-BBz-2A-GMR-IL2-8H-8TM- CAR-3 CD19scFv-8h-8TM-BBz-2A-GMR-H9-PDGFRβTM CAR-4 CD19scFv-8h-8TM-BBz-2A-GMR-H9-8H-8TM CAR-5 GMR-H9-8H-8TM--2A-CD19scFv-8h-8TM-BBz CAR-6 GMR-H9-PDGFRβTM-2A-CD19scFv-8h-8TM-BBz CAR-7 GMR-H9-PDGFRβTM-2A-CD19scFv-8h-p44TM-BB-DAP10 CAR-8

[0160] Note: H9 is a mutated IL2.

[0161] 2. The above-mentioned target plasmid was transfected into 293T derivative cell line by calcium transfection method for virus preparation.

[0162] The present embodiment packages the lentivirus using the calcium phosphate method, specifically: 293T cells are cultured to an optimal state with DMEM medium containing 10% FBS (w / v), and the packaging plasmid (RRE:REV:2G) and the expression plasmid are added to a 1.5mL centrifuge tube in a certain ratio, CaCl2 and 2×HBS are added, mixed and allowed to stand at room temperature, and then added to the treated 293T cell culture fluid, and the fluid is changed to 10mL DMEM medium containing 10% FBS again after 3-5h, and the cell supernatant is collected after 48h or 72h. The virus supernatant is collected, centrifuged and filtered, and then transferred to a new centrifuge tube; according to the amount of virus supernatant, the virus is purified using PEG6000 with a final concentration of 8.5% and NaCl with a final concentration of 0.3M, and the purified virus is resuspended with 200μL DMEM medium containing 10% FBS, and packaged in 1.5mL EP tubes, and stored at -80℃ for standby use. The virus infected 293T cells, and 72 hours after infection, the genome was extracted using the QIAamp DNA Blood Mini Kit (purchased from Qiagen, Cat. No. 511004). The operation was performed according to the instructions of the kit. The virus titer was determined by qRT-PCR. The data were analyzed using analysis software, and the virus titer was calculated according to the standard curve. The results were expressed in TU / mL.

[0163] Titer calculation method: Titer (TU / mL) = 1 × 10 5 × positive rate × dilution factor ÷ virus volume × 1000 (Table 2).

[0164] Table 2

[0165] CAR structure Titer CAR-1 7.79E+08 CAR-2 6.98E+08 CAR-3 6.54E+08 CAR-4 8.78E+08 CAR-5 2.74E+08 CAR-6 7.23E+08 CAR-7 5.21E+08 CAR-8 1.86E+08

[0166] 3. NK cells (PBNK) were sorted from PBMC by CD56 Mircobeads. After PBNK was activated for 24 hours, the above lentivirus was transduced at MOI = 3. After 8 days of in vitro culture, the transduced CAR-NK cells were harvested, centrifuged at 300g / min for 5min, and the supernatant was discarded to collect the cells; the cells were resuspended in PBS solution containing 1% fetal bovine serum by volume, and the cell density was adjusted to 1×10 6 / mL; the collected cells were divided into the final labeled antibody, incubated at 4°C for 30 minutes, washed twice with PBS solution, and detected by flow cytometry. Labeled antibodies: CD3-FITC (Biolegend, 300440), CD56-BV510 (Biolegend, 362534), IL-2-PE (Biolegend, 500307), anti-CD19-scFv monoclonal antibody (AF647 labeled) Detection of CAR-NK positive rate ( Figure 1 , Table 3).

[0167] Table 3 mbIL-2 CD19-CAR-NK positive rate

[0168]

[0169] 4. Figure 1 The horizontal axis represents the expression of CAR, and the vertical axis represents the expression of membrane-bound IL2. The above results show that the positive rate of mbIL-2 constructed by wild-type IL-2 is generally less than 10% (CAR-1-CAR-3), which cannot be used to meet the use requirements. The results show that mbH9 can significantly improve its positive rate. After the IL-2 mutant H9 is constructed as mbH9, it can be efficiently expressed in CAR-NK after structural optimization (CAR-5, CAR-6, CAR-7, CAR-8). Among them, the H9-8H-8TM (mbH9-2) and H9-PDGFRβTM (mbH9-1) fusion proteins corresponding to the CAR6 and CAR8 structures are expressed on the surface of NK / CAR-NK cells with more significant and efficient expression effects.

[0170] Example 2mbH9 can promote the efficacy of CD19-CAR-NK in vivo

[0171] 1. In order to explore the effect of mbH9 on CD19-CAR-NK, we removed mbH9 from CAR-8 to form CAR-9 (CD19 scFv-8h-p44TM-BB-DAP10) as a control.

[0172] 2. Using CD19+ NALM6-Luc-GFP as positive target cells, NCG mice were injected through the tail vein at a dose of 5e5 / mouse for pre-tumor formation, and the pre-tumor formation time was 1 day. CAR-8 and CAR-9 were injected into NCG mice with pre-tumor formation through the tail vein at a dose of 5e6 effective cells / mouse. NK cells without CAR modification were injected at the same dose and method as the control group. Thereafter, the fluorescent substrate of Luciferase was injected intraperitoneally once every 7 days, and in vivo imaging was performed using a small animal in vivo imaging device to detect tumor load. The results are shown in Figure 2 In Table 4, the horizontal axis represents the number of days after CAR-NK transfusion, and the vertical axis represents the fluorescence value of the tumor. The higher the fluorescence value, the higher the tumor load. In addition, during the in vivo experiment, the number of CD19-scFv copies in the blood of mice was detected by qPCR at 7 days and 15 days after CAR-NK transfusion to reflect the survival of CAR-NK in mice ( Figure 3 In Table 5, the horizontal axis is the number of days after CAR-NK transfusion, and the vertical axis is the number of copies of CAR-NK cells expanded in mice. The higher the copy number, the better the in vivo persistence and proliferation ability of CAR-NK).

[0173] 3. Using the same protocol as above, we verified the in vivo effectiveness of CAR-6. The results are as follows Figure 4 As shown: The results show that the H9-8H-8TM-fusion protein can also effectively kill tumors under the structure of CAR-6, proving that the structural design of the fusion protein described in the present invention can be designed or expressed in combination with the CAR structure in various ways and can play a role.

[0174] Table 4

[0175]

[0176] Table 5

[0177]

[0178] 4. The results showed that the in vivo efficacy of CAR-8 was significantly better than that of CAR-9, and the results of in vivo persistence also proved that CAR-8 could continue to proliferate in mice; CAR-6 also had significant in vivo effectiveness. The above results indicate that mbH9 can promote the in vivo persistence of CAR-NK.

[0179] Example 3: The in vivo efficacy of mbH9 CD19-CAR-NK is superior to that of mbIL-15CD19-CAR-NK

[0180] 1. MbIL-15 is currently the mainstream method for CAR-NK to improve survival, so we used this structure (CAR-10, CD19scFv-8h-8TM-BBz-2A-IL15-G4S-IL15Ra) as a control to explore the differences between mbIL-15 and mbH9.

[0181] 2. Using CD19+ cells NALM6-Luc-GFP as positive target cells, pre-tumor was injected into NCG mice through the tail vein at a dose of 5e5 / mouse, and the pre-tumor time was 1 day. After 12 days of in vitro culture of CAR-8 and CAR-10 modified NK, the positive rate was adjusted with CNK (CNK is the NK control cell without CAR transduction) and then frozen. After the tumor was completed, the frozen and revived CAR-8 and 10CAR-NK cells were injected into the pre-tumored NCG mice through the tail vein at a dose of 5e6 effective cells / mouse. NK cells that were not modified by CAR were injected with the same dose and method as the control group. Thereafter, the fluorescent substrate of Luciferase was injected intraperitoneally once every 7 days, and the tumor load was detected by in vivo imaging using a small animal in vivo imager. The results are as follows Figure 5As shown, the CAR-NK cell CAR-8 expressed by the IL2 fusion protein of the H9-PDGFRβTM structure has better in vivo effectiveness than the CAR-NK cell CAR-10 expressing the IL15 fusion protein of the IL15-G4S-IL15Ra structure, indicating that the membrane-bound IL-2 constructed by the mutant IL-2 of the present invention (including but not limited to the H9-PDGFRβTM structure or the H9-8H-8TM-structure) can promote the in vivo effectiveness of CAR-NK, and is superior to the commonly used membrane-expressed IL-15 form in the field. In addition, during the in vivo experiment, the number of CD19-scFv copies in the blood of mice was detected by qPCR 14 days, 21 days, and 29 days after the CAR-NK was reinfused, reflecting the survival of CAR-NK in mice ( Figure 6 , Table 6), the results showed that the CAR-NK cells CAR-8 treated with the IL2 fusion protein of the H9-PDGFRβTM structure had a higher copy number, indicating that CAR-8 had better persistence in the treatment of tumor-bearing mice in vivo, indicating that the membrane-bound IL-2 constructed by the mutant IL-2 of the present invention (including but not limited to the H9-PDGFRβTM structure or the H9-8H-8TM-structure) can promote the in vivo persistence of CAR-NK, and is superior to the membrane-expressed IL-15 form currently commonly used in the field.

[0182] Table 6

[0183]

[0184] 3. The results showed that the in vivo efficacy of CAR-8 was significantly better than that of CAR-10, and the results of in vivo persistence also proved that CAR-8 can continue to proliferate in mice. The above results indicate that mbH9 has a better effect on improving the in vivo persistence of CAR-NK than mbIL-15.

[0185] Example 4mbH9 promotes CD123-CAR-NK factor secretion

[0186] 1. To verify the applicability of mbH9 in CAR-NK with different targets, we constructed two structures: CAR-11 (CD123scFv-8h-8TM-BBz) and CAR-12 (CD123 scFv-8h-8TM-BBz-2A-GMR-H9-8H-8TM-);

[0187] 2. Use Control NK to level the positive rate of CD123-CAR-NK. KG1a-Luc-GFP was used as the positive target cell, KG1a-(ΔCD123)Luc-GFP was used as the negative target cell, and the in vitro killing experiment was performed with the effector cell (calculated by the total number of cells) and the target cell ratio of 1:1 (ie E / T = 1:1). The target cells were incubated with CAR-NK for 24 hours, and the killing ratio was detected by luciferase (Luc). Luciferase principle: During the detection, the target cells were lysed with lysis buffer, and the luciferase in them would decompose the substrate to emit fluorescence. The fluorescence value analysis result formula is: CAR-NK cell killing rate = 1-(fluorescence value of the experimental group ÷ fluorescence value of the blank control group) ( Figure 7 , Table 7); The results showed that CAR-NK cells CAR-12 expressing H9-8H-8TM-membrane-bound IL2 fusion protein had better killing effectiveness. In order to further confirm the strength of killing, the factor secretion ability of CAR-NK cells after killing was tested, and the results are as follows Figure 8 shown.

[0188] Table 7 In vitro killing efficiency detection of CD123-CAR-NK with different structures

[0189]

[0190] 3. Detect the secretion of IFN-γ in the supernatant of cells after killing by ELISA ( Figure 8 , Table 8), the results showed that CAR-12, a CAR-NK cell expressing H9-8H-8TM-membrane-bound IL2 fusion protein, had a stronger cytokine secretion ability. When the cytokine secretion ability was not strong, the CAR-12 factor secretion was 2.88 times that of CAR-11 (as shown in Table 8), reflecting that CAR-12 has a stronger potential for effectiveness in vivo.

[0191] Table 8 Detection of CD123-CAR-NK factor secretion with different structures

[0192]

[0193] Example 5mbH9 promotes CD123-CAR-NK expansion in vivo

[0194] 1. CD123+ THP1-Luc-GFP cells were used as positive target cells, and NCG mice were injected through the tail vein at a dose of 5e5 / mouse for pre-tumor formation, and the pre-tumor formation time was 1 day. After 12 days of in vitro culture of CAR-11 and 12-modified NK, the positive rate was adjusted with CNK, and the NCG mice that had been pre-tumored were injected through the tail vein at a dose of 5e6 effective cells / mouse. NK cells that were not modified by CAR were injected with the same dose and method as the control group. Thereafter, the fluorescent substrate of luciferase was injected intraperitoneally once every 7 days for in vivo imaging using a small animal in vivo imager, and the fluorescence intensity value calculated by the fluorescence imager based on the fluorescence intensity reflected the tumor treatment situation (Table 9, Fig. 9 ). At the same time, the persistence of CAR-NK in mice after CAR-NK cell therapy was detected. The results are as follows Fig.10 As shown:

[0195] 2. On the 7th and 14th days of the experiment, blood was collected from the mice under the jaw, and the copy number of BBz in the blood of the mice was detected by qPCR to reflect the survival and amplification of CAR-NK (Table 10, Fig.10 ). The results showed that CAR-NK cells CAR-12 expressing H9-8H-8TM-membrane-bound IL2 fusion protein had a higher copy number than CAR-NK cells not expressing H9-8H-8TM-membrane-bound IL2 fusion protein, and H9-8H-8TM-membrane-bound IL2 fusion protein could enhance the in vivo persistence of CAR-NK.

[0196] Table 9

[0197]

[0198] Table 10

[0199]

[0200] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of IL-2 mutants in the construction of membrane-bound fusion peptides; The mutant of IL-2 is H9; The H9 features: (I), the amino acid sequence shown in SEQ ID No.32; or (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

2. A fusion peptide, characterized in that Include any of the following: (i), H9, hinge region and first transmembrane domain; and / or (ii), H9 and the second transmembrane domain; The H9 features: (I), the amino acid sequence shown in SEQ ID No.32; or (II) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (I); or (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

3. The fusion peptide according to claim 2, characterized in that Intracellular signaling regions are also included.

4. The fusion peptide according to claim 2 or 3, characterized in that Include any of the following: (iii), comprising H9, hinge region 8h and first transmembrane domain 8TM in sequence; and / or (iv), comprising H9 and the second transmembrane domain PDGFRβTM in sequence; and / or (v), including H9, hinge region 8h, first transmembrane domain 8TM and intracellular signaling region in sequence; and / or (vi) It includes H9, the second transmembrane domain PDGFRβTM and the intracellular signaling region in sequence.

5. The fusion peptide according to claim 4, characterized in that (A), the 8h has the amino acid sequence shown in SEQ ID No.21; or (B), the 8TM has the amino acid sequence shown in SEQ ID No. 22; or (C) the PDGFRβTM has the amino acid sequence shown in SEQ ID No.33; or (D) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence as shown in any one of (A) to (C); or (E) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of (A) to (D).

6. The fusion peptide according to any one of claims 2 to 5, characterized in that (F), the fusion peptide has an amino acid sequence as shown in SEQ ID No. 37 or 38; or (G) a sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in (F); or (H) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (F) or (G).

7. A nucleic acid molecule encoding the fusion peptide according to any one of claims 2 to 6.

8. The nucleic acid molecule according to claim 7, wherein (J), the nucleic acid molecule of the fusion peptide has a nucleotide sequence as shown in SEQ ID No. 18 or 19; or (K), a nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (J), but is different from the nucleotide sequence shown in (J) due to the degeneracy of the genetic code; or (L), a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (J) or (K), and a nucleotide sequence that has the same or similar function as the nucleotide sequence shown in (J) or (K); or (M) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (J) to (L).

9. A protein combination, characterized in that Comprising the fusion peptide and chimeric antigen receptor according to any one of claims 2 to 6.

10. The protein combination according to claim 9, characterized in that The chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

11. The protein combination according to claim 10, characterized in that 1) The CD19 scFv has the amino acid sequence shown in SEQ ID No. 20; or 2) The CD123 scFv has the amino acid sequence shown in SEQ ID No. 26; or 3) The intracellular signaling domain has an amino acid sequence as shown in SEQ ID No. 25; or 4) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in any one of 1) to 3); or 5) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of 1) to 4).

12. The protein combination according to any one of claims 9 to 11, characterized in that The protein combination includes CAR-5, CAR-6, CAR-7, CAR-8 and / or CAR-12; 6), the CAR-5, CAR-6, CAR-7, CAR-8, and CAR-12 have the amino acid sequences shown in SEQ ID No.55, 56, 57, 58, and 62, respectively; or 7) A sequence in which one or more amino acids are substituted, deleted, added and / or replaced based on the amino acid sequence shown in 6); or 8) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in 6) or 7).

13. A genetic element, characterized in that Comprising the nucleic acid molecule according to claim 7 or 8 and a nucleic acid molecule encoding a chimeric antigen receptor.

14. The genetic element according to claim 13, wherein Include any of the following: (1) a nucleic acid molecule encoding a chimeric antigen receptor and a nucleic acid molecule according to claim 7 or 8; and / or (2) A nucleic acid molecule encoding the nucleic acid molecule according to claim 7 or 8 and a chimeric antigen receptor in sequence.

15. The genetic element according to claim 14, wherein The chimeric antigen receptor and the fusion peptide are connected via a self-cleaving peptide; The chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain; The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

16. The genetic element according to claim 15, wherein The self-cleaving peptides include but are not limited to 2A, P2A, T2A, E2A and F2A.

17. The genetic element according to claim 16, wherein The nucleic acid molecule encoding the 2A has: (4) the nucleotide sequence shown in SEQ ID No.12; or (5) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (4), and having the same or similar function as the nucleotide sequence shown in (4); or (6) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (4) or (5).

18. The genetic element according to any one of claims 15 to 17, characterized in that (7) The nucleic acid molecule encoding the CD19 scFv has the nucleotide sequence shown in SEQ ID No. 1; or (8) The nucleic acid molecule encoding the CD123 scFv has the nucleotide sequence shown in SEQ ID No. 7; or (9) The nucleic acid molecule encoding the hinge region has the nucleotide sequence shown in SEQ ID No. 2; or (10) The nucleic acid molecule encoding the transmembrane region has the nucleotide sequence shown in SEQ ID No. 3; or (11), the nucleic acid molecule encoding the intracellular signaling domain has the nucleotide sequence shown in SEQ ID No.6; or (12) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in any one of (7) to (11), and having the same or similar functions as the nucleotide sequence shown in any one of (7) to (11); or (13) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in any one of (7) to (12).

19. The genetic element according to any one of claims 13 to 18, characterized in that The genetic elements include CAR-5, CAR-6, CAR-7, CAR-8 and CAR-12; (17), wherein the CAR-5, CAR-6, CAR-7, CAR-8 and CAR-12 have the nucleotide sequences shown in SEQ ID No. 43, 44, 45, 46 and 50, respectively; or (18) a nucleotide sequence obtained by replacing, deleting or adding one or more nucleotide sequences to the nucleotide sequence shown in (17), and a nucleotide sequence having the same or similar function as the nucleotide sequence shown in (17); or (19) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (17) or (18).

20. An artificial carrier, characterized in that Comprising the genetic element according to any one of claims 13 to 19.

21. A virus particle, characterized in that Comprising the artificial carrier as claimed in claim 20.

22. An engineered cell, characterized in that Include any of the following: a), a fusion peptide as claimed in any one of claims 2 to 6; and / or b), a nucleic acid molecule as described in any one of claims 7 or 8; and / or c) a protein combination as claimed in any one of claims 9 to 12; and / or d) a genetic element according to any one of claims 13 to 19; and / or e), an artificial carrier as claimed in claim 20; and / or f) transducing the viral particles as claimed in claim 21.

23. The engineered cell according to claim 22, wherein The engineered cells include but are not limited to NK cells.

24. Use of any of the following in improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells: a), a fusion peptide as claimed in any one of claims 2 to 6; and / or b), a nucleic acid molecule as described in any one of claims 7 or 8; and / or c) a protein combination as claimed in any one of claims 9 to 12; and / or d) a genetic element according to any one of claims 13 to 19; and / or e) The artificial carrier as claimed in claim 20.

25. Use of any of the following in the preparation of a drug for preventing and / or treating a disease: ①. The fusion peptide according to any one of claims 2 to 6; and / or ②. The nucleic acid molecule according to any one of claims 7 or 8; and / or ③. The protein combination according to any one of claims 9 to 12; and / or ④. The genetic element according to any one of claims 13 to 19; and / or 5. The artificial carrier according to claim 20; and / or ⑥. The virus particle according to claim 21; and / or 7. The engineered cell according to claim 22 or 23.

26. The use according to claim 25, characterized in that The prevention and / or treatment of the disease includes improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells.

27. The use according to claim 25 or 26, characterized in that Such diseases include, but are not limited to, AML.

28. A drug, characterized in that Include any of the following: ①. The fusion peptide according to any one of claims 2 to 6; and / or ②. The nucleic acid molecule according to any one of claims 7 or 8; and / or ③. The protein combination according to any one of claims 9 to 12; and / or ④. The genetic element according to any one of claims 13 to 19; and / or 5. The artificial carrier according to claim 20; and / or ⑥. The virus particle according to claim 21; and / or 7. The engineered cell according to claim 22 or 23.

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