Two-factor fusion protein of Flt3L and IL-7 and application thereof

Abstract

The invention discloses a two-factor fusion protein of Flt3L and IL-7 (interleukin-7) and application of the two-factor fusion protein. Relates to the technical field of biology. According to the fusion protein provided by the invention, two heavy-like chains are connected through a disulfide bond to form a homodimer, and the fusion protein realizes double functions through a single molecule: Flt3L targets Flt3 to drive bone marrow precursor cells to be differentiated into dendritic cells, so that a DC library is expanded; maintaining development, survival and steady state of T cells and B cells through IL-7 is crucial to long-term survival of initial and memory T cells. The two have functional complementarity and are used for respectively promoting differentiation and maturation of DC precursor cells, and the DC cells further activate T lymphocytes; meanwhile, T lymphocytes are supplemented, so that systematic immunoregulation is realized, the fusion protein has disease universality, and a new technical support is provided for immune anti-aging and prevention and treatment of tumor and microbial infection.

Description

A two-factor fusion protein of Flt3L and IL-7 and its applications Technical Field

[0001] This invention relates to the field of biotechnology, and more specifically to a two-factor fusion protein of Flt3L and IL-7 and its applications. Background Technology

[0002] Human aging is not a single process; the immune system plays a central role in it.

[0003] Researchers are currently developing "immune rejuvenation" technologies at the cellular, organ, and systemic levels. Treatment strategies targeting immunosenescence include thymus regeneration, stem cell therapy, T-cell modulation, myeloid lineage modulation, immune checkpoint inhibitors, methods for clearing senescent cells, anti-inflammatory interventions, and anti-aging agents. These therapies aim to restore immune resilience, reduce chronic inflammation, and enhance overall health to combat age-related functional decline.

[0004] Fms-like tyrosine kinase 3 (Flt3) is a transmembrane receptor that plays a key role in hematopoiesis, primarily by regulating the proliferation and differentiation of hematopoietic stem cells and progenitor cells.

[0005] IL-7 is a member of the shared γ-chain cytokine family and is essential for the development of innate and adaptive immune cells.

[0006] Studies have found that IL-7 and Flt3L are key factors that promote the proliferation or regeneration of the spleen and lymph nodes.

[0007] Therefore, developing a novel therapeutic drug that organically integrates Flt3L and IL-7 into a single molecule is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0008] In view of this, the present invention provides a dual-factor fusion protein of Flt3L and IL-7 and its applications. This invention provides a dual-factor fusion protein of Flt3L and IL-7 that achieves dual and synergistic effects through a single molecule, providing new ideas and tools for immune anti-aging, cancer immunotherapy, and prevention and control of microbial infections. Specifically, Flt3L targets Flt3 to drive the differentiation of bone marrow progenitor cells into dendritic cells, expanding the DC pool; IL-7 maintains the development, survival, and homeostasis of T cells and B cells, which is crucial for the long-term survival of naive and memory T cells. The two have complementary functions, respectively promoting the differentiation and maturation of DC progenitor cells, which further activate T lymphocytes; simultaneously supplementing T lymphocytes, thereby achieving systemic immune regulation with universal applicability.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A two-factor fusion protein of Flt3L and IL-7, comprising: the fusion protein is a homodimeric structure formed by two heavy-like chains linked by disulfide bonds; the heavy-like chain is a polypeptide chain that mimics the Fc heavy chain structure of an antibody, comprising: an extracellular domain sequence of an Fms-like tyrosine kinase 3 ligand, an Fc region of human immunoglobulin G1, and an interleukin-7 sequence.

[0011] Preferred amino acid sequences include: the extracellular domain of the Fms-like tyrosine kinase 3 ligand in the heavy chain as shown in SEQ ID NO.2; the Fc region of human immunoglobulin G1 in the heavy chain as shown in SEQ ID NO.3; and the interleukin-7 in the heavy chain as shown in SEQ ID NO.4.

[0012] Preferably, the extracellular domain sequence of the Fms-like tyrosine kinase 3 ligand, the Fc region of human immunoglobulin G1, and the interleukin-7 sequence in the heavy chain are linked by a flexible linker peptide; the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO. 5.

[0013] Preferred: The N-terminus-C-terminus are sequentially: Fms-like tyrosine kinase 3 ligand – flexible linker peptide – Fc segment of human immunoglobulin G1 – flexible linker peptide – IL-7.

[0014] Preferred: The amino acid sequence of the heavy chain is shown in SEQ ID NO.1; the disulfide bond is formed by cysteine ​​residues at positions 177 and 180 of SEQ ID NO.1 and cysteine ​​residues at the same positions on another chain.

[0015] The present invention also provides a nucleic acid molecule encoding any of the above-mentioned fusion proteins.

[0016] The present invention also provides an expression vector or host cell containing the above-mentioned nucleic acid molecules.

[0017] The present invention also provides a method for preparing the fusion protein as described in any of the above-described embodiments, comprising culturing host cells as described above and recovering the fusion protein from the culture.

[0018] The present invention also provides the use of any of the fusion proteins described in the invention in the preparation of reagents and / or pharmaceuticals for the remodeling of the immune system, the treatment of cancer or microbial infections.

[0019] Preferred: Used to promote the proliferation of lymph nodes or spleen.

[0020] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses a dual-factor fusion protein of Flt3L and IL-7 and its application, and the technical effect achieved is: by integrating the functions of targeting, immune regulation and cell stimulation into a single molecule, a dual and synergistic effect is achieved: (1) Precisely target and stimulate DC precursor cells: using Flt3L as a guide module, the fusion protein specifically binds to Flt3 positive DC precursor cells, and induces DC cell differentiation and maturation.

[0021] (2) Remodeling the immunosuppressive microenvironment: Maintaining the development, survival and homeostasis of T cells and B cells through IL-7.

[0022] (3) Multi-mechanism spatial synergy enhances efficacy: The two functional modules are physically coupled on the same molecule, which can promote the proliferation of secondary lymphoid organs, thereby achieving multi-dimensional immune anti-aging. They can also play a role in the simultaneous presentation of new antigen DCs and activation of immune cells in the tumor local area, systematically activating the immune response, and are especially suitable for the treatment of the tumor microenvironment of immune aging. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0024] Figure 1 is a schematic diagram of the structure of the fusion protein (137) provided by the present invention.

[0025] Figure 2 shows the enzyme digestion identification results of the fusion protein expression plasmid in Example 1 provided by the present invention; wherein, lane 1 is the marker; lane 2 is the pcDNA3.4-137 band of plasmid; lane 3 is the XbaI and EcoRV double digestion band of plasmid pcDNA3.4-137.

[0026] Figure 3 shows the expression and purification results of the fusion protein prepared in Example 1 of this invention; where A represents the elution volume and OD. 280 The corresponding diagram for pH is shown in Figure B; B represents the SDS-PAGE results, with lane 1 showing the 137 denatured and reduced sample band and lane 2 showing the 137 non-denatured and non-reduced sample band.

[0027] Figure 4 shows the ELISA results of the binding of the fusion protein prepared in Example 2 of this invention to recombinant human Flt3 protein.

[0028] Figure 5 shows the results of PBMC differentiation into DC cells induced by the fusion protein prepared in Example 3 of the present invention; where A is the flow cytometry histogram of CD14 expression in the IL-4 treatment group; B is the flow cytometry histogram of CD14 expression in the IL-4 + 137 treatment group; and C is the flow cytometry histogram of CD14 expression in the IL-4 + GM-CSF treatment group; the horizontal axis represents the intensity of CD14 fluorescence signal, and the vertical axis represents the number of cells.

[0029] Figure 6 shows the results of the fusion protein prepared in Example 4 of the present invention inducing secondary lymphoid organ hyperplasia; where A is a picture of the lymph nodes of mice in each treatment group; B is a picture of the spleen of mice in each treatment group; C is a histogram of the weight data of the lymph nodes of mice in each treatment group; and D is a histogram of the weight data of the spleen of mice in each treatment group. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention discloses a two-factor fusion protein of Flt3L and IL-7 and its applications.

[0032] In the examples, all raw materials not mentioned are commercially available, and all experimental methods not mentioned are conventional experimental methods, which will not be described in detail here.

[0033] Example 1 Plasmid construction, protein expression and purification (its structural schematic is shown in Figure 1) A dual-factor fusion protein of Flt3L and IL-7 is formed by two heavy-like chains (referring to polypeptide chains that mimic the Fc heavy chain structure of antibodies) connected by disulfide bonds in the Fc region to form a homodimer structure (cysteine ​​residues at positions 177 and 180 of SEQ ID NO.1 are involved in the formation); wherein, the heavy-like chain includes: the extracellular domain sequence of Fms-like tyrosine kinase 3 ligand, the Fc segment of human immunoglobulin G1, and the interleukin-7 sequence.

[0034] The amino acid sequence of the extracellular domain of the Fms-like tyrosine kinase 3 ligand is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAP, as shown in SEQ ID As shown in NO.2; the amino acid sequence of the Fc region of human immunoglobulin G1 in the heavy chain: DKTHTCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK, as shown in SEQ ID As shown in NO.3; the amino acid sequence of interleukin-7 (functional region) in the heavy chain is: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH, as shown in SEQ ID NO.4; the extracellular domain sequence of Fms-like tyrosine kinase 3 ligand, the Fc region of human immunoglobulin G1, and the interleukin-7 sequence in the heavy chain are linked by a flexible linker peptide; the amino acid sequence of the flexible linker peptide is: GGGGSGGGGSGGGGS, as shown in SEQ ID NO. As shown in NO.5; the amino acid sequence of the heavy chain-like chain is: TQDCSFQHSPISSDFAVKIRELSDYLLQDYPVTVASNLQDEELCGGLWRLVLAQRWMERLKTVAGSKMQGLLERVNTEIHFVTKCAFQPPPSCLRFVQTNISRLLQETSEQLVALKPWITRQNFSRCLELQCQPDSSTLPPPWSPRPLEATAPTAPGGGGSGGGGSGGGGSDKTHT C PP CPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALGAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFS CSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSDCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH, as in SEQ. As shown in ID NO.1; (1) After the SEQ ID No.1 (number 137) sequence is synthesized by gene (with restriction endonuclease XbaI and EcoRV restriction sites added to both ends of the sequence), it is constructed into the pcDNA3.4 plasmid expressing the fusion protein using pcDNA3.4-TOPOTA cloning kit (purchased from Invitrogen (Shanghai) Trading Co., Ltd.), that is, pcDNA3.4-137 is constructed.

[0035] (2) The pcDNA3.4-137 plasmid obtained in the previous step was transformed into Escherichia coli TOP10 (purchased from Beijing Qingke Biotechnology Co., Ltd.), amplified by shaking in LB medium, extracted using a plasmid large-scale extraction kit (Beijing Jumei Biotechnology Co., Ltd.), and then identified by restriction endonucleases XbaI and EcoRV (Figure 2). From left to right, the lanes are: Marker, pcDNA3.4-137 plasmid band, and XbaI and EcoRV double digestion band of pcDNA3.4-137 plasmid. The upper side of the double digestion band is the linearized band of the empty plasmid, and the lower side is the target band position, which is consistent with the designed size and position.

[0036] (3) The expression plasmid was transfected into 293F cells using PEI transfection reagent for protein expression.

[0037] (4) 120 h after transfection, the supernatant was harvested, centrifuged and filtered, and the protein A affinity column (purchased from Cytiva) was treated with 5 column volumes of equilibration buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.15 M NaCl, pH 7.2). The supernatant was loaded onto the column. After the supernatant was loaded, the column was washed with buffer (5.6 mM NaH2PO4, 14.4 mM Na2HPO4, 0.5 M NaCl, pH 7.2) to bring the contaminating protein level to baseline. The protein was then eluted with 50 mM citrate / sodium citrate buffer (pH 3.2), and samples with a concentration of 100 mAu or more were collected. The pH was then adjusted to 7.0 with 1 M Tris-Cl (pH 8.0), concentrated in a concentration tube, filtered and sterilized, and stored at 4 °C. 3 μg of the sample was then subjected to SDS-PAGE staining (Figure 3). In Figure 3, A is the chromatography diagram, which shows the elution volume versus OD. 280 Figure 3 shows the pH correspondence; in Figure 3B, the SDS-PAGE results are as follows: lane 1 is the 137 denatured / reduced sample band, and the positions of the two protein chains are consistent with the designed size (after 137 is expressed in cells, it forms a Y-shaped antibody dimer, and under denaturing / reducing conditions, the disulfide bonds open to form monomers); lane 2 is the 137 non-denatured / non-reduced sample band. The protein obtained in this step is the fusion protein 137.

[0038] Example 2 Antigen-antibody binding ELISA experiment (1) Commercial recombinant human Flt3 protein (purchased from Beijing Baipusaisi Biotechnology Co., Ltd.) was diluted with pH 9.6 NaHCO3 coating solution, plated on ELISA plates, 100 ng / well, and incubated overnight at 4°C.

[0039] (2) Wash the ELISA plate coated yesterday three times with PBS.

[0040] (3) Block with PBST containing 3% BSA, at room temperature for 30 min.

[0041] (4) Dilute the purified fusion protein and control antibody Ctrl Ab with PBST containing 3% BSA. The initial concentration was 100 μg / mL (Ctrl Ab: Herceptin, purchased from Roche Pharmaceuticals Ltd., Shanghai). The concentrations were serially diluted 10-fold to 0.001 μg / mL, for a total of 6 concentrations. The solutions were added to ELISA plates and incubated at room temperature for 30 min.

[0042] (5) Wash 3 times with PBST, add 100 μL of mouse anti-human HRP secondary antibody (1:5000, purchased from Nanjing Genscript Biotech Co., Ltd.) to each well, and incubate at room temperature for 30 min.

[0043] (6) Wash 3 times with PBST, add 50 μL of TMB colorimetric solution to each well, and incubate for 5 min.

[0044] (7) Add 50 μL of 1 M HCl to each well and detect OD using a microplate reader. 450 Absorbance values. The results are shown in Figure 4.

[0045] The results show that the fusion protein has binding activity with the target protein Flt3 in a dose-dependent manner, while it does not have binding activity with the control antibody Ctrl Ab.

[0046] Example 3: Induction of DC Cell Differentiation Experiment. 50 ml of blood was collected from volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated and resuspended in 10 mL of 1640 medium (Thermo Fisher Scientific) containing 10% hiFBS (purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.), at a density of 2 x 10⁻⁶. 6 Add / ml to a small dish and incubate in a 37℃, 5% CO2 incubator for pre-attached culture.

[0047] Two hours later, gently shake the dish, aspirate the culture medium containing non-adherent cells, and replace it with MD medium containing 5% hiFBS and the corresponding protein to be tested: set up IL-4 (50 ng / mL) group, IL-4 (50 ng / mL) + 137 (300 ng / mL) group, and IL-4 (50 ng / mL) + GM-CSF (100 ng / mL) group (IL-4 and GM-CSF were purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.), and added them to the culture medium respectively.

[0048] On day 3, 10 mL of 5% hiFBS MD medium (containing the protein to be tested) was added, and the mixture was incubated in the incubator until day 7.

[0049] Collect suspended cells, resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0050] Add 50 μL of PE-CD14 antibody diluted 1:500 (purchased from Beijing Yiqiao Shenzhou Technology Co., Ltd.) and incubate at room temperature for 30 min.

[0051] Centrifuge and discard the supernatant. Resuspend in 500 μL of physiological saline, centrifuge, and wash twice.

[0052] The sample was resuspended in 500 μL of physiological saline and analyzed by flow cytometry. The results are shown in Figure 5.

[0053] Results Analysis: In Figure 5, A represents the results of the IL-4 group, B represents the results of the IL4 + 137 group, and C represents the results of the IL4 + GM-CSF group. The horizontal axis represents the CD14 signal intensity. As can be seen from the figure, although the CD14 negative cell population was smaller in the IL4 + GM-CSF group compared to the IL4 + 137 group, it was able to induce PBMCs to differentiate into CD14 negative cells compared to the IL-4 group, and has the potential to induce PBMCs to differentiate into DC cells in vitro.

[0054] The results show that the fusion protein can promote the differentiation of PBMCs into DC cells and has Flt3L activity.

[0055] Example 4 Mouse lymphoid tissue (spleen and lymph nodes) hyperplasia model (1) Eleven female C57BL / 6 mice aged 6-8 weeks, weighing 18-22 g, were randomly divided into 3 groups: normal control group (Ctrl group), low-dose group (137-low), and high-dose group (137-high) (n=4, 3, 4).

[0056] According to the above groups, 100 μL of normal saline, 150 μg of normal saline containing 137, and 300 μg of normal saline containing 137 were injected subcutaneously once every 5 days.

[0057] The mice were injected three times. On day 15, the mice were sacrificed, and their spleens and lymph nodes were dissected, photographed, and weighed. The results are shown in Figure 6.

[0058] Results analysis: As shown in Figure 6, both doses of 137 treatment could induce hyperplasia of lymph nodes (A and C in Figure 6) and spleen (B and D in Figure 6), especially lymph nodes. The differences between the low-dose and high-dose 137 groups and the control group were significant (p≤0.002), with obvious hyperplasia.

[0059] The results show that the fusion protein can effectively promote the proliferation of lymphoid organs.

[0060] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0061] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A two-factor fusion protein of Flt3L and IL-7, characterized in that, include: The fusion protein is a homodimer structure formed by two heavy-like chains linked by disulfide bonds; the heavy-like chain is a polypeptide chain that mimics the Fc heavy chain structure of an antibody and includes: the extracellular domain sequence of Fms-like tyrosine kinase 3 ligand, the Fc region of human immunoglobulin G1, and the interleukin-7 sequence.

2. The fusion protein as described in claim 1, characterized in that, The amino acid sequence of the extracellular domain of the Fms-like tyrosine kinase 3 ligand in the heavy chain is shown in SEQ ID NO.2; the amino acid sequence of the Fc region of human immunoglobulin G1 in the heavy chain is shown in SEQ ID NO.3; and the amino acid sequence of interleukin-7 in the heavy chain is shown in SEQ ID NO.

4.

3. The fusion protein as described in claim 2, characterized in that, The extracellular domain sequence of Fms-like tyrosine kinase 3 ligand, the Fc region of human immunoglobulin G1, and the interleukin-7 sequence in the heavy chain are linked by a flexible linker peptide; the amino acid sequence of the flexible linker peptide is shown in SEQ ID NO.

5.

4. The fusion protein as described in claim 3, characterized in that, The sequence from N-terminus to C-terminus is: Fms-like tyrosine kinase 3 ligand – flexible linker peptide – Fc segment of human immunoglobulin G1 – flexible linker peptide – IL-7.

5. The fusion protein as described in claim 4, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO.1; the disulfide bond is formed by cysteine ​​residues at positions 177 and 180 of SEQ ID NO.1 and cysteine ​​residues at the same positions on another chain.

6. A nucleic acid molecule encoding the fusion protein as described in any one of claims 1-5.

7. An expression vector or host cell comprising the nucleic acid molecule as described in claim 6.

8. A method for preparing a fusion protein as described in any one of claims 1-5, comprising culturing a host cell as described in claim 7 and recovering the fusion protein from the culture.

9. Use of the fusion protein according to any one of claims 1-5 in the preparation of reagents and / or medicaments for remodeling the immune system, treating cancer or microbial infections.

10. The application as described in claim 9, characterized in that, It is used to promote the proliferation of lymph nodes or spleen.

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