Cytokine fusion protein and use thereof

Abstract

Provided are a cytokine fusion protein and the use thereof. The problem of the expression of a membrane-bound IL-2 in NK cells is solved, and the membrane expression efficiency of the membrane-bound IL-2 is significantly improved. Moreover, by means of the provided membrane-bound IL-2, the in-vivo efficacy and in-vivo persistence of CAR-NK are significantly improved.

Description

Cytokine fusion protein and its application

[0001] This application claims priority to the Chinese patent application filed with the China Patent Office on September 14, 2023, with application number 202311188762.4 and invention name “Cytokine Fusion Protein and Its Application”, the entire contents of which are incorporated by reference into this application. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to cytokine fusion proteins and applications thereof. Background Art

[0003] Adoptive cell therapy (ACT) refers to the isolation 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.

[0004] Chimeric antigen receptor (CAR) T cell technology is a rapidly developing cellular immunotherapy technique. CAR is a synthetic fusion receptor structurally composed of an extracellular antigen-binding domain, a transmembrane domain, an intracellular signaling domain, and a costimulatory domain. The extracellular domain consists of a monoclonal antibody sequence (single chain variable fragment, ScFv) that recognizes tumor-associated antigens. The transmembrane domain connects the extracellular and intracellular domains, and commonly used transmembrane domains are selected from genes such as CD3, CD4, CD8, and CD28. The intracellular domain contains an immunoreceptor tyrosine-based activation motif (ITAM), the most commonly used being the T cell receptor (TCR) / CD3ζ chain and the immunoglobulin (Fc) receptor (FcεRIγ), which is primarily responsible for signal transduction. The CAR construct is then transduced into NK cells via viral vectors, electroporation, or other methods to generate the corresponding CAR-NK cells. CAR-NK cells are not restricted by HLA matching and can achieve allogeneic adoptive immune therapy.

[0005] Interleukin-2 (IL-2) is a cytokine in the chemokine family. It is derived from multiple cells (primarily produced by activated T cells) and has pleiotropic effects (primarily promoting lymphocyte growth, proliferation, and differentiation). It plays a vital role in the body's immune response and antiviral resistance, stimulating specific antigens or stimulating NK cell proliferation, enhancing NK cell cytotoxicity and cytokine production. However, compared to CAR-T cells, CAR-NK lacks the autocrine secretion of factors such as IL-2 and requires exogenous injection or overexpression to exert sustained anti-tumor effects in animals or humans.

[0006] Currently, there is a composition and method for enhancing T and NK cell responses, which applies IL-2 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 dosage of secreted IL-2. Although IL-2 has a significant promoting effect on the function 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 in them is rare.

[0007] In recent years, CAR-NK cell therapy has been underwhelming in clinical performance, with one key reason being its poor in vivo persistence. Therefore, identifying a safe and controllable NK cell amplification signaling molecule would greatly enhance the efficacy and application of CAR-NK cells.

[0008] Summary of the Invention

[0009] In view of this, the present invention provides a cytokine fusion protein and its application.

[0010] The present invention provides a cytokine fusion protein and its application. This invention solves the problem of membrane-bound IL-2 expression 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 cells.

[0011] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0012] The present invention provides the use of IL-2 mutants in constructing membrane-bound fusion peptides.

[0013] In some specific embodiments of the present invention, the mutant of IL-2 is H9;

[0014] The H9 features:

[0015] (I), the amino acid sequence shown in SEQ ID No. 32; or

[0016] (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

[0017] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

[0018] The present invention also provides a fusion peptide comprising any of the following:

[0019] (i), H9, hinge region and first transmembrane domain; and / or

[0020] (ii), H9 and the second transmembrane domain;

[0021] The H9 features:

[0022] (I), the amino acid sequence shown in SEQ ID No. 32; or

[0023] (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

[0024] (III) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in (II).

[0025] In some specific embodiments of the present invention, the fusion peptide further includes an intracellular signaling region.

[0026] In some specific embodiments of the present invention, the fusion peptide includes any of the following:

[0027] (iii) comprising H9, hinge region 8h and first transmembrane domain 8TM in sequence; and / or

[0028] (iv) comprising, in sequence, H9 and the second transmembrane domain, PDGFRβTM; and / or

[0029] (v), including H9, hinge region 8h, first transmembrane domain 8TM and intracellular signaling region in sequence; and / or

[0030] (vi) It includes H9, the second transmembrane domain PDGFRβTM and the intracellular signaling region in sequence.

[0031] In some specific embodiments of the present invention,

[0032] (A), the 8h has the amino acid sequence shown in SEQ ID No. 21; or

[0033] (B) the 8TM has the amino acid sequence shown in SEQ ID No. 22; or

[0034] (C) the PDGFRβTM has the amino acid sequence shown in SEQ ID No. 33; or

[0035] (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

[0036] (E) An amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in any one of (A) to (D).

[0037] In some specific embodiments of the present invention,

[0038] (F), the fusion peptide has the amino acid sequence shown in SEQ ID No. 37 or 38; or

[0039] (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

[0040] (H) An amino acid sequence having at least 80% sequence homology to the amino acid sequence shown in (F) or (G).

[0041] Based on the above research, the present invention also provides a nucleic acid molecule encoding the fusion peptide.

[0042] In some specific embodiments of the present invention,

[0043] (J) the nucleic acid molecule of the fusion peptide has the nucleotide sequence shown in SEQ ID No. 18 or 19; or

[0044] (K) A nucleotide sequence that encodes the same protein as the nucleotide sequence shown in (J) but differs from the nucleotide sequence shown in (J) due to the degeneracy of the genetic code; or

[0045] (L), a nucleotide sequence obtained by substituting, 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

[0046] (M) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence described in any one of (J) to (L).

[0047] The present invention also provides a protein combination comprising the fusion peptide and a chimeric antigen receptor.

[0048] In some 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;

[0049] The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

[0050] In some specific embodiments of the present invention,

[0051] 1) The CD19 scFv has the amino acid sequence shown in SEQ ID No. 20; or

[0052] 2) the CD123 scFv has the amino acid sequence shown in SEQ ID No. 26; or

[0053] 3) The intracellular signaling domain has the amino acid sequence shown in SEQ ID No. 25; or

[0054] 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

[0055] 5) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in any one of 1) to 4).

[0056] In some embodiments of the present invention, the protein combination includes CAR-5, CAR-6, CAR-7, CAR-8 and / or CAR-12;

[0057] 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

[0058] 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

[0059] 8) An amino acid sequence having at least 80% sequence homology with the amino acid sequence shown in 6) or 7).

[0060] The present invention also provides genetic elements, including the nucleic acid molecules and nucleic acid molecules encoding chimeric antigen receptors.

[0061] In some specific embodiments of the present invention, the genetic element includes any of the following:

[0062] (1) a nucleic acid molecule encoding a chimeric antigen receptor and the nucleic acid molecule; and / or

[0063] (2) A nucleic acid molecule encoding the nucleic acid molecule and the chimeric antigen receptor in sequence.

[0064] In some specific embodiments of the present invention, the chimeric antigen receptor and the fusion peptide are connected via a self-cleaving peptide;

[0065] The chimeric antigen receptor comprises an antigen binding domain, a hinge region, a transmembrane region and an intracellular signaling domain;

[0066] The antigen binding domains include, but are not limited to, CD19 scFv and CD123 scFv.

[0067] In some specific embodiments of the present invention, the self-cleaving peptides include but are not limited to 2A, P2A, T2A, E2A and F2A.

[0068] In some specific embodiments of the present invention, the nucleic acid molecule encoding the 2A has:

[0069] (4) the nucleotide sequence shown in SEQ ID No. 12; or

[0070] (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

[0071] (6) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (4) or (5).

[0072] In some specific embodiments of the present invention,

[0073] (7) The nucleic acid molecule encoding the CD19 scFv has the nucleotide sequence shown in SEQ ID No. 1; or

[0074] (8) The nucleic acid molecule encoding the CD123 scFv has the nucleotide sequence shown in SEQ ID No. 7; or

[0075] (9) The nucleic acid molecule encoding the hinge region has the nucleotide sequence shown in SEQ ID No. 2; or

[0076] (10) The nucleic acid molecule encoding the transmembrane region has the nucleotide sequence shown in SEQ ID No. 3; or

[0077] (11) The nucleic acid molecule encoding the intracellular signaling domain has the nucleotide sequence shown in SEQ ID No. 6; or

[0078] (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

[0079] (13) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in any one of (7) to (12).

[0080] In some embodiments of the present invention, the genetic elements include CAR-5, CAR-6, CAR-7, CAR-8, and CAR-12;

[0081] (17), 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

[0082] (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

[0083] (19) A nucleotide sequence having at least 80% sequence homology with the nucleotide sequence shown in (17) or (18).

[0084] The present invention also provides an artificial vector comprising the gene element.

[0085] The present invention also provides a virus particle comprising the artificial vector.

[0086] The present invention also provides an engineered cell comprising any of the following:

[0087] a), the fusion peptide; and / or

[0088] b), the nucleic acid molecule; and / or

[0089] c) the protein combination; and / or

[0090] d), said genetic element; and / or

[0091] e), the artificial carrier; and / or

[0092] f) the viral particles.

[0093] In some specific embodiments of the present invention, the engineered cells include but are not limited to NK cells.

[0094] The present invention also provides the use of any of the following items for improving the in vivo efficacy of CAR-NK cells and / or the in vivo persistence of CAR-NK cells:

[0095] a), the fusion peptide; and / or

[0096] b), the nucleic acid molecule; and / or

[0097] c) the protein combination; and / or

[0098] d), said genetic element; and / or

[0099] e) the artificial carrier.

[0100] 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.

[0101] In some specific embodiments of the present invention, the CAR-NK cell-secreted factor includes IFN-γ.

[0102] The present invention also provides the use of any of the following in the preparation of a medicament for preventing and / or treating a disease:

[0103] ①, the fusion peptide; and / or

[0104] ②, the nucleic acid molecule; and / or

[0105] ③. the protein combination; and / or

[0106] ④, the genetic element; and / or

[0107] 5. The artificial carrier; and / or

[0108] ⑥, the viral particles; and / or

[0109] 7. The engineered cells.

[0110] In some 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.

[0111] In some specific embodiments of the present invention, 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.

[0112] In some embodiments of the present invention, the disease includes but is not limited to AML.

[0113] The present invention also provides a drug comprising any of the following:

[0114] ①, the fusion peptide; and / or

[0115] ②, the nucleic acid molecule; and / or

[0116] ③. the protein combination; and / or

[0117] ④, the genetic element; and / or

[0118] 5. The artificial carrier; and / or

[0119] ⑥, the viral particles; and / or

[0120] 7. The engineered cells.

[0121] 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 with CD19 and CD123 targets respectively. mbH9 can be expressed on the surface of the CAR-NK cell membrane, which solves the problem that membrane-bound IL-2 cannot be normally expressed or functioned in immune cells, avoids the safety problem of uncontrollable secretion levels 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 various membrane-bound fusion protein designs, with optimal expression efficiency when placed upstream of the 2A peptide. Various transmembrane structures, such as CD8-TM and PDGFRβTM, can serve as transmembrane domains for mbH9. mbH9 can also enhance the in vivo efficacy and persistence of CAR-NK cells in various indications, such as AML.

[0122] The present invention solves the problem of expression 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

[0123] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for describing the embodiments or the prior art.

[0124] Figure 1 shows the mbIL-2-CAR-NK positive rate detection in Example 1;

[0125] Figure 2 shows the in vivo efficacy evaluation of CAR-8 and 9 in Example 2;

[0126] FIG3 shows the continuous in vivo detection of CAR-8 and 9 in Example 2;

[0127] FIG4 shows the in vivo effectiveness of CAR-6 in Example 2;

[0128] FIG5 shows the in vivo efficacy evaluation of cryopreserved CAR-8 and 10 in Example 3;

[0129] FIG6 shows the in vivo continuous detection of frozen CAR-8 and 10 in Example 3;

[0130] FIG7 shows the in vitro killing efficiency detection of CD123-CAR-NK with different structures in Example 4;

[0131] FIG8 shows the secretion detection of CD123-CAR-NK factors with different structures in Example 4;

[0132] FIG9 shows the in vivo efficacy evaluation of NK cells modified with CAR-11 and 12 in Example 5;

[0133] FIG10 shows the in vivo drug expansion effect of NK cells modified with CAR-11 and 12 in Example 5. DETAILED DESCRIPTION

[0134] 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 for implementation. It is particularly important to note 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.

[0135] 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 with CD19 and CD123 targets respectively. mbH9 can be expressed on the surface of the CAR-NK cell membrane, which solves the problem that membrane-bound IL-2 cannot be normally expressed or functioned in immune cells, avoids the safety problem of uncontrollable secretion levels 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 various membrane-bound fusion protein designs, with optimal expression efficiency when placed upstream of the 2A peptide. Various transmembrane structures, such as CD8-TM and PDGFRβTM, can serve as transmembrane domains for mbH9. mbH9 can also enhance the in vivo efficacy and persistence of CAR-NK cells in various indications, such as AML.

[0136] The present invention solves the problem of expression 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.

[0137] Sequences in the present invention:

[0138] The intracellular co-stimulatory signal of 41BB is BB in BBZ, and Z in BBZ is CD3 zeta

[0139] 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.

[0140] The present invention will be further described below in conjunction with the embodiments:

[0141] Example 1 Detection and screening of expression of mbIL-2 with different structures

[0142] 1. The present invention tested the function of mbIL2 with different structures in CAR-NK targeting CD19, wherein mbIL2 is a membrane-bound fusion peptide constructed from 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 from a mutant IL2 sequence, such as H9-PDGFRβTM (mbH9-1), H9-8H-8TM- (mbH9-2). The specific CAR structures are shown in Table 1:

[0143] Table 1 Note: H9 is a mutant IL2.

[0144] 2. The above target plasmid was transfected into 293T derivative cell line by calcium transfection method for virus preparation.

[0145] The calcium phosphate method was used to package the lentivirus in this embodiment. Specifically, 293T cells were cultured with DMEM medium containing 10% FBS (w / v) to an optimal state. The packaging plasmid (RRE:REV:2G) and the expression plasmid were added to a 1.5 mL centrifuge tube in a certain ratio. CaCl2 and 2×HBS were added. After mixing, the mixture was allowed to stand at room temperature and then added to the treated 293T cell culture medium. After 3-5 hours, the medium was changed again to 10 mL of DMEM medium containing 10% FBS. After 48 hours or 72 hours, the cell supernatant was collected. The viral supernatant was collected, centrifuged and transferred to a new centrifuge tube; according to the amount of viral supernatant, the virus was purified using PEG6000 with a final concentration of 8.5% and NaCl with a final concentration of 0.3 M. The purified virus was resuspended with 200 μL of DMEM medium containing 10% FBS, aliquoted in 1.5 mL EP tubes, and stored at -80°C for later use. 293T cells were infected with the virus, and 72 hours after infection, genomes were extracted using the QIAamp DNA Blood Mini Kit (Qiagen, Cat. No. 511004). The procedure was performed according to the kit's instructions. Viral titers were determined by qRT-PCR using analysis software. Viral titers were calculated based on a standard curve and expressed as TU / mL.

[0146] Titer calculation method is: Titer (TU / mL) = 1 × 10 5 × positive rate × dilution factor ÷ virus volume × 1000 (Table 2).

[0147] Table 2

[0148] 3. NK cells (PBNK) were sorted from PBMC using CD56 Mircobeads. After 24 hours of activation, the PBNK cells were transduced with the above-mentioned lentivirus at an MOI of 3. After 8 days of in vitro culture, the transduced CAR-NK cells were harvested and centrifuged at 300g / min for 5 minutes. The supernatant was discarded to collect the cells. The cells were resuspended in PBS solution containing 1% fetal bovine serum and the cell density was adjusted to 1×10 6 The collected cells were aliquoted with the final labeled antibody, incubated at 4°C for 30 minutes, washed twice with PBS, and analyzed by flow cytometry. CAR-NK cell positivity was determined using labeled antibodies: CD3-FITC (Biolegend, 300440), CD56-BV510 (Biolegend, 362534), IL-2-PE (Biolegend, 500307), and anti-CD19-scFv monoclonal antibody (labeled with AF647) (Figure 1, Table 3).

[0149] Table 3 mbIL-2 CD19-CAR-NK positive rate

[0150] 4. The horizontal axis of Figure 1 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 with wild-type IL-2 is generally less than 10% (CAR-1-CAR-3), which cannot be used to meet the requirements. The results show that mbH9 can significantly improve its positive rate. After the IL-2 mutant H9 is constructed into mbH9, after structural optimization, it can be efficiently expressed in CAR-NK (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.

[0151] Example 2 mbH9 can promote the efficacy of CD19-CAR-NK in vivo

[0152] 1. 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.

[0153] 2. Using CD19+ cells NALM6-Luc-GFP as positive target cells, NCG mice were pre-inoculated via the tail vein at a dose of 5e5 / mouse for 1 day. CAR-8 and CAR-9 were injected into NCG mice that had already been pre-inoculated with tumors via the tail vein at a dose of 5e6 effective cells / mouse. NK cells that were not modified with CAR were injected at the same dose and method as a control group. Thereafter, the fluorescent substrate of Luciferase was injected intraperitoneally every 7 days, and in vivo imaging was performed using a small animal in vivo imaging device to detect tumor burden. The results are shown in Figure 2. The horizontal axis of Table 4 represents the number of days after CAR-NK infusion, and the vertical axis represents the fluorescence value of the tumor. The higher the fluorescence value, the higher the tumor burden. In addition, during the in vivo experiment, the CD19-scFv copy number in the mouse blood was detected by qPCR 7 and 15 days after CAR-NK transfusion to reflect the survival of CAR-NK in the mouse (Figure 3, Table 5, the horizontal axis is the number of days after CAR-NK transfusion, and the vertical axis is the copy number of CAR-NK cells expanded in the mouse. The higher the copy number, the better the in vivo persistence and proliferation ability of CAR-NK).

[0154] 3. Using the same scheme as above, we verified the in vivo effectiveness of CAR-6. The results are shown in Figure 4: 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 jointly designed or expressed with the CAR structure in various ways and can all play a role.

[0155] Table 4

[0156] Table 5

[0157] 4. Results showed that CAR-8 was significantly more effective than CAR-9 in vivo. In vivo persistence also demonstrated that CAR-8 can sustain expansion in mice. CAR-6 also exhibited significant in vivo efficacy. These results suggest that mbH9 can promote the in vivo persistence of CAR-NK cells.

[0158] Example 3 mbH9 CD19-CAR-NK has better in vivo efficacy than mbIL-15 CD19-CAR-NK

[0159] 1. MbIL-15 is currently the mainstream method for improving the survival of CAR-NK cells. Therefore, we used this structure (CAR-10, CD19 scFv-8h-8TM-BBz-2A-IL15-G4S-IL15Ra) as a control to explore the differences between mbIL-15 and mbH9.

[0160] 2. Using CD19+ NALM6-Luc-GFP cells as positive target cells, NCG mice were pre-initiated with tumors at a dose of 5e5 / mouse via the tail vein. The pre-initiation time was 1 day. After 12 days of in vitro culture, CAR-8 and CAR-10 modified NK cells were used to adjust the positive rate with CNK (CNK is a NK control cell that is not transduced with CAR) and then frozen. After tumor burden was completed, the frozen and revived CAR-8 and 10CAR-NK cells were injected into NCG mice that had been pre-initiated with tumors at a dose of 5e6 effective cells / mouse via the tail vein. NK cells that were not modified with CAR were injected at the same dose and method as a 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 burden. The results are shown in Figure 5. The CAR-NK cell CAR-8 expressing 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 membrane-expressed IL-15 form currently commonly used in the field. In addition, during the in vivo experiment, the CD19-scFv copy number in the mouse blood was detected by qPCR 14 days, 21 days, and 29 days after CAR-NK transfusion to reflect the persistence of CAR-NK in the mouse (Figure 6, Table 6). The results showed that the CAR-NK cells expressing the IL2 fusion protein of the H9-PDGFRβTM structure had a higher copy number in mice treated with CAR-8, indicating that CAR-8 has better persistence in the treatment of tumor-bearing mice in mice, 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 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.

[0161] Table 6

[0162] 3. The results showed that CAR-8 had significantly better in vivo efficacy than CAR-10, and the in vivo persistence results also demonstrated that CAR-8 can continue to expand in mice. These results indicate that mbH9 is more effective than mbIL-15 in enhancing the in vivo persistence of CAR-NK cells.

[0163] Example 4 mbH9 promotes CD123-CAR-NK factor secretion

[0164] 1. To verify the applicability of mbH9 in CAR-NK with different targets, we constructed two constructs: CAR-11 (CD123scFv-8h-8TM-BBz) and CAR-12 (CD123 scFv-8h-8TM-BBz-2A-GMR-H9-8H-8TM-);

[0165] 2. After the positive rate of CD123-CAR-NK was leveled using Control NK. KG1a-Luc-GFP was used as the positive target cell and KG1a-(ΔCD123)Luc-GFP as the negative target cell. The in vitro killing experiment was performed with the effector cell (calculated based on the total cell number) to target cell ratio of 1:1 (i.e. E / T = 1:1). The target cells were co-incubated with CAR-NK for 24 hours and the killing ratio was detected by luciferase (Luc). Luciferase principle: During the detection, after the target cells were lysed with lysis buffer, 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 experimental group ÷ fluorescence value of blank control group) (Figure 7, Table 7); The results showed that CAR-NK cells expressing H9-8H-8TM-membrane-bound IL2 fusion protein CAR-12 had better killing efficiency. In order to further confirm the strength of the killing, the factor secretion ability of the CAR-NK cells after killing was tested, and the results are shown in Figure 8.

[0166] Table 7 In vitro killing efficiency detection of CD123-CAR-NK with different structures

[0167] 3. The amount of IFN-γ secretion in the cell supernatant after killing was detected by ELISA (Figure 8, Table 8). The results showed that CAR-NK cells expressing H9-8H-8TM-membrane-bound IL2 fusion protein CAR-12 had a stronger cytokine secretion ability. When the cytokine secretion ability of both cells was not strong, the factor secretion of CAR-12 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.

[0168] Table 8 Detection of CD123-CAR-NK factor secretion with different structures

[0169] Example 5 mbH9 promotes CD123-CAR-NK cell proliferation in vivo

[0170] 1. Using CD123+ cells THP1-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. After 12 days of in vitro culture of CAR-11 and 12-modified NK, the positive rate was leveled with CNK, and the NCG mice that had been pre-tumored were injected through the tail vein at a dose of 5e6 / mouse of effective cells. NK cells that were not modified by CAR were injected in the same dose and manner as a 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 imaging instrument, and the fluorescence intensity value calculated by the fluorescence imager based on the fluorescence intensity reflected the tumor treatment situation (Table 9, Figure 9). At the same time, the persistence of CAR-NK in mice after CAR-NK cell treatment was detected, and the results are shown in Figure 10:

[0171] 2. On days 7 and 14 of the experiment, blood was collected from the mice submandibularly, and the copy number of BBz in the blood was detected by qPCR to reflect the persistence and expansion of CAR-NK (Table 10, Figure 10). The results showed that CAR-NK cells CAR-12 expressing the H9-8H-8TM-membrane-bound IL2 fusion protein had a higher copy number than CAR-NK cells not expressing the H9-8H-8TM-membrane-bound IL2 fusion protein, and the H9-8H-8TM-membrane-bound IL2 fusion protein can enhance the in vivo persistence of CAR-NK.

[0172] Table 9

[0173] Table 10

[0174] The cytokine fusion protein and its application provided by the present invention are introduced in detail above. This article uses specific examples to illustrate the principles and implementation methods of the present invention. The description of the above examples is only used to help understand the method of the present invention and its core concept. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

[0175] SEQUENCE LISTING

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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