Fusion protein, artificial antigen presenting molecule and application of artificial antigen presenting molecule in NKT amplification

Abstract

The invention discloses a fusion protein, an artificial antigen presenting molecule and application of the artificial antigen presenting molecule in NKT amplification. The fusion protein comprises CD1d and beta2M linked by a linker. According to the fusion protein, CD1d and beta2M are connected through a linker, the fusion protein can be used for specific amplification of NKT cells by artificial antigen presenting molecules, and the potential safety hazard that gene mutation may be caused by irradiation of PBMC is avoided. Moreover, the amplification effect of an artificial antigen presenting molecule adopting the fusion protein is better than that of a dimer artificial antigen presenting molecule formed by spontaneous polymerization of CD1d and beta2M, meanwhile, the fusion protein is used for the artificial antigen presenting molecule, and specific amplification of NKT cells can be realized by adopting a very short scaffold protein structure; the production cost of the artificial antigen presenting molecule is reduced while the specific amplification effect of the NKT cells is improved.

Description

[0001] This application claims the priority of the Chinese patent application with application number: 202311504248.7 filed on November 10, 2023, and the invention titled "A fusion protein, an artificial antigen presenting molecule and its application in expanding NKT". Technical Field

[0002] The present invention belongs to the field of immunology, and in particular, relates to a fusion protein, an artificial antigen presenting molecule and application thereof in amplifying NKT. Background Art

[0003] Natural Killer T cells (NKT) have the characteristics of conventional T cells and natural killer (NK) cells. They recognize lipid antigen glycolipids in the context of CD1d molecules and subsequently produce cytokines that activate innate and adaptive immune response cells, playing a key role in maintaining immune homeostasis. More importantly, IFN-γ produced by type I NKT (iNKT) cells and subsequent IFN-γ produced by NK cells are essential for α-GalCer-mediated tumor protection, indicating that iNKT cells play an important role in anti-tumor activity. At the same time, studies have shown that the number and function of NKT cells in tumor patients are reduced. Given that iNKT cells induce effective anti-tumor responses in vivo, several human immunotherapies based on iNKT cells have been developed, which mainly focus on the activation and expansion of iNKT cell populations. Due to the complexity of operating iNKT cells in vivo, in vitro expansion of iNKT cells would be a better way.

[0004] At present, the in vitro expansion method of NKT cells mainly uses irradiated PBMC (i.e., as trophoblast feeder) as antigen presenting cells, and expands the cells in the presence of α-GalCer. However, PBMC may cause gene mutation after irradiation, which brings safety risks to patients receiving iNKT expansion. Therefore, seeking a safe and effective artificial antigen presenting molecule has become a problem that needs to be solved urgently by those skilled in the art. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defect of the existing technology of amplifying NKT cells, and to provide a fusion protein, an artificial antigen presenting molecule and their application in amplifying NKT cells. The fusion protein and / or artificial antigen presenting molecule of the present invention is used to amplify NKT cells, and there is no need to use irradiated PBMC (i.e., feeder) as antigen presenting cells to perform an in vitro amplification method of NKT cells, and the specific amplification effect of NKT cells can be further improved.

[0006] The present invention solves the above technical problems through the following technical solutions.

[0007] A first aspect of the present invention provides a fusion protein for NKT cell expansion, comprising CD1d and β2M connected by a linker.

[0008] In some embodiments, the linker is a peptide linker, and the peptide linker is preferably selected from a GS polypeptide linker and / or a Whitlow linker.

[0009] In some embodiments, the GS polypeptide linker is a peptide consisting of glycine and serine residues. An exemplary GS polypeptide linker comprises the amino acid sequence S (G4S) n 、(G4S) n、 (G3S) n and / or (G4S3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. In some embodiments, n=7. In some embodiments, n=8. In some embodiments, n=9. In some embodiments, n=10. Another exemplary GS polypeptide linker comprises the amino acid sequence S (G4S) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In another embodiment, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary GS polypeptide linker comprises (G4S) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6. Another exemplary GS polypeptide linker comprises (G3S) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In another embodiment, n=5. In yet another embodiment, n=6. Another exemplary GS polypeptide linker comprises (G4S3) n In some embodiments, n=1. In some embodiments, n=2. In some embodiments, n=3. In some embodiments, n=4. In some embodiments, n=5. In some embodiments, n=6.

[0010] In some embodiments, the GS polypeptide linker preferably comprises the amino acid sequence shown in SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:12 or SEQ ID NO:8.

[0011] In some embodiments, the Whitlow linker comprises the amino acid sequence shown in SEQ ID NO:10.

[0012] In some embodiments, the CD1d comprises the amino acid sequence shown in SEQ ID NO:4.

[0013] In some embodiments, the β2M comprises the amino acid sequence shown in SEQ ID NO:5.

[0014] The second aspect of the present invention provides an artificial antigen presenting molecule, which includes the fusion protein of the first aspect.

[0015] In some embodiments, the artificial antigen-presenting molecule further comprises a protein scaffold structure, and the fusion protein is connected to the protein scaffold structure.

[0016] In some specific embodiments, the protein scaffold structure is at least 18 amino acid residues in length.

[0017] In some embodiments, the artificial antigen-presenting molecule further comprises a non-protein scaffold structure, and the fusion protein is coupled to the non-protein scaffold structure.

[0018] In some embodiments, the non-protein scaffold structure is selected from small molecule monomer polymers, magnetic microspheres or nanoparticles.

[0019] In the present invention, the magnetic microspheres facilitate the separation of artificial antigen presenting molecules from cells in the cell amplification culture system after the NKT cell amplification is completed.

[0020] In some embodiments, the artificial antigen presenting molecule comprises a protein scaffold structure and a non-protein scaffold structure.

[0021] In some embodiments, the fusion protein is linked to the protein scaffold structure, and the protein scaffold structure is coupled to the non-protein scaffold structure.

[0022] In some preferred embodiments, the protein scaffold structure is selected from an antibody or a portion of an antibody, and a transmembrane protein or a portion of a transmembrane protein.

[0023] In some embodiments, the portion of the antibody is a constant region of the antibody, such as an Fc region.

[0024] In the present invention, the presence of the protein scaffold structure and / or the non-protein scaffold structure provides a support for the fusion protein, making the three-dimensional structure of the fusion protein more conducive to binding with α-galcer. The protein scaffold structure is Fc, which can further facilitate purification during the preparation of artificial antigen presenting molecules.

[0025] The third aspect of the present invention provides a polynucleotide encoding the fusion protein as described in the first aspect; and / or the artificial antigen presenting molecule as described in the second aspect.

[0026] The fourth aspect of the present invention provides a recombinant expression vector, which comprises the polynucleotide as described in the third aspect.

[0027] The fifth aspect of the present invention provides a lentiviral expression vector, wherein the lentiviral expression vector comprises the polynucleotide as described in the third aspect.

[0028] The sixth aspect of the present invention provides a transformant, which comprises the recombinant expression vector as described in the fourth aspect or the lentiviral expression vector as described in the fifth aspect.

[0029] A seventh aspect of the present invention provides a method for preparing an artificial antigen presenting molecule, comprising the following steps:

[0030] The transformant as described in the sixth aspect is cultured to obtain an artificial antigen presenting molecule.

[0031] In some embodiments, the method further comprises a collection and purification step, such as collecting the culture supernatant of the stably expressing cell line, concentrating it by ultrafiltration, and then purifying the protein in the supernatant by Protein A / G antibody magnetic beads.

[0032] In some embodiments, the present invention further comprises: coupling the protein obtained by culturing the transformant with a non-protein scaffold structure.

[0033] In some embodiments, the non-protein scaffold structure is selected from small molecule monomer polymers, magnetic microspheres and nanoparticles.

[0034] The eighth aspect of the present invention provides a method for expanding NKT cells, the method comprising: expanding and culturing NKT cells in the presence of α-GalCer and the artificial antigen presenting molecule as described in the second aspect.

[0035] In the present invention, when the artificial antigen presenting molecule does not include a non-protein scaffold structure, the artificial antigen presenting molecule is preferably coated on a culture dish / culture plate to facilitate separation of the artificial antigen presenting molecule from the cells in the cell expansion culture system after the NKT cell expansion is completed.

[0036] When the artificial antigen presenting molecule includes a non-protein scaffold structure such as a magnetic microsphere, the artificial antigen presenting molecule can be subsequently separated from the cells in the cell expansion culture system by means of physical effects such as magnetism.

[0037] In some embodiments, the NKT cells are iNKT cells.

[0038] In some embodiments, the mass ratio of the artificial antigen presenting molecule to α-GalCer is (5-10):1.

[0039] In some embodiments, the method further comprises: adding cytokines during the expansion culture process.

[0040] In some embodiments, the cytokine is IL-2 and / or IL-15.

[0041] In some specific embodiments, the final concentration of IL-2 is 100-300 IU / mL, and the final concentration of IL-15 is 5-20 ng / mL.

[0042] In some embodiments, the culture medium for the expansion culture is a serum-free culture medium, such as Optivitro UniEx T cell serum-free culture medium.

[0043] In some specific embodiments, the source cells of the NKT cells are NKT cells or NKT-containing cells, and the NKT-containing cells are, for example, PBMC cells.

[0044] The ninth aspect of the present invention provides the use of the fusion protein as described in the first aspect, the artificial antigen presenting molecule as described in the second aspect, the polynucleotide as described in the third aspect, the recombinant expression vector as described in the fourth aspect, the lentiviral expression vector as described in the fifth aspect, and the transformant as described in the sixth aspect in amplifying NKT cells.

[0045] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0046] The reagents and raw materials used in the present invention are commercially available.

[0047] The positive and progressive effects of the present invention are:

[0048] The fusion protein of the present invention connects CD1d and β2M through a linker, and can be used for artificial antigen presenting molecules to specifically amplify NKT cells, avoiding the potential safety hazard of gene mutation caused by irradiating PBMC. In addition, the amplification effect of artificial antigen presenting molecules using the fusion protein is better than that of artificial antigen presenting molecules formed by spontaneous polymerization of CD1d and β2M to form dimers. At the same time, the fusion protein can be used for artificial antigen presenting molecules to achieve specific amplification of NKT cells using a very short scaffold protein structure, thereby improving the specific amplification effect of NKT cells and reducing the production cost of artificial antigen presenting molecules. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 Flow cytometry for NKT purity detection after PBMC cells were treated with different antigen presentations;

[0050] Figure 2 Flow cytometry analysis of NKT purity detection after primary NKT cells were treated with different antigen presentations. DETAILED DESCRIPTION

[0051] In order to make the present invention more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise specifically defined herein, all other technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the present invention belongs.

[0052] As used herein, the term "cell" includes directly isolated or extracted cells, as well as "transfected" or "transformed" cells, which refers to the process of transferring or introducing exogenous nucleic acids into cells. Transformed cells include primary subject cells and their progeny.

[0053] As used herein, the term "NKT cell (Natural killer T cell)" refers to a special T cell subpopulation that has both T cell receptors TCR and NK cell receptors on the cell surface, and can simultaneously express T cell surface markers (CD3) and NK cell surface markers (CD56).

[0054] As used herein, the term "fusion protein" refers to a protein expression product comprising at least two different domains obtained by nucleic acid-level or protein-level recombination technology. The nucleic acid-level recombination technology is, for example, DNA recombination technology, which can integrate genes encoding different domains into one nucleotide chain. The protein-level recombination technology is, for example, polypeptide synthesis technology, which can synthesize proteins of different domains into one polypeptide chain.

[0055] As used herein, the term "recombinant expression vector" refers to a recombinant vector obtained by adding an exogenous sequence to a vector backbone. The vector backbone can be a plasmid, cosmid, virus (such as retrovirus, vaccinia virus, adeno-associated virus, herpes virus or bovine papilloma virus) or bacteriophage conventionally used in genetic engineering.

[0056] As used herein, the term "peptide linker" refers to an amino acid sequence through which the amino acid sequences of CD1d and β2M are linked, and the peptide linker itself does not have any polymerization activity.

[0057] As used herein, the term "magnetic microsphere" has the same meaning as "magnetic bead" and refers to nano- or micron-sized particles that are attracted or repelled by a magnetic field gradient or have a non-zero magnetic susceptibility. The magnetic microsphere can be paramagnetic or superparamagnetic. In some embodiments, the magnetic microsphere is superparamagnetic. Magnetic microspheres are also called magnetic particles.

[0058] The "antibodies" herein may be derived from any animal, including but not limited to humans and non-human animals, which may be selected from primates, mammals, rodents and vertebrates, such as camelids, llamas, ostriches, alpacas, sheep, rabbits, mice, rats or cartilaginous fish (e.g. sharks).

[0059] In the expansion method of the present invention, the method of collecting peripheral blood and separating mononuclear cells is not particularly limited, and methods known in the art can be used, for example, an extracorporeal circulation method or equipment can be used (for example, a human mononuclear cell separator can be used to circulate 3000 to 6000 mL of peripheral blood).

[0060] In the amplification method of the present invention, the culture conditions of the NKT-like cells of the present invention are not particularly limited. For the culture medium, a culture medium conventionally used for T cell culture can be used, such as RPMI-1640 culture medium and Optivitro UniEx T cell serum-free culture medium. For the culture conditions, the common conditions for T cell culture in the art can be used, for example, a temperature of 37°C, a CO2 concentration of 5%, and a culture medium replaced every 3 to 5 days. For the amplification and activation of the NKT-like cells of the present invention, it can be achieved by adding some cytokines, for example, cytokines that can stimulate T cell proliferation and activation, including but not limited to GM-CSF, IL-2, IL-4, IL-5, IL-6, IL-7, IL-9, IL-12, IL-15, 4-1BBL, etc.

[0061] The culture time can be specifically determined by those skilled in the art according to the purpose of the expanded cells. The culture time can be controlled by estimating the cell expansion number or multiple by periodically sampling and counting cells.

[0062] The present invention is further described below by way of examples, but the present invention is not limited to the scope of the examples. The experimental methods in the following examples without specifying specific conditions are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0063] All reagents in the following examples and effects are of reagent grade or other grades superior to reagent grade. The specific sources are as follows:

[0064] PBMC cells were purchased from Shanghai Heyousheng, catalog number: T01PBZ1017;

[0065] Protein A / G antibody magnetic beads, purchased from Beaver Bio, BeaverBeadsTM Protein A / G AntibodyPurification Kit;

[0066] Optivitro UniEx T cell serum-free medium was purchased from Optivitro, TE000-N052.

[0067] Table 1 Detailed sequence information

[0068]

[0069]

[0070]

[0071] Example 1: Preparation of artificial antigen presenting molecule "β2M-linker1-CD1d-Fc"

[0072] 1. Construct a lentiviral expression vector of an artificial antigen presenting molecule β2M-linker1-CD1d-Fc (the amino acid sequence of β2M-linker1-CD1d-Fc is shown in SEQ ID NO: 1, and linker1 is GGGGSGGSGSGGG (SEQ ID NO: 8)): pHAGE-CMV-β2M-linker1-CD1d-Fc;

[0073] 2. The lentivirus was packaged using the second-generation method (three-plasmid system: two auxiliary plasmids and one target plasmid). The auxiliary plasmid pMD2.G, psPAX2 and β2M-linker1-CD1d-Fc expression vector were used to form a three-plasmid transfection system and transfected into 293T cells by PEI to prepare and harvest the β2M-linker1-CD1d-Fc lentivirus.

[0074] 3. Add the packaged β2M-linker1-CD1d-Fc lentivirus directly to 293T cells, and add Polybrene Transfection Reagent for auxiliary infection. Add β2M-linker1-CD1d-Fc lentivirus infection once in the morning and once in the evening. After the last infection overnight, change the cell medium to construct a stable expression cell line of β2M-linker1-CD1d-Fc.

[0075] 4. The cell density of the stable expression cell line of β2M-linker1-CD1d-Fc was cultured to 80%, and the medium was replaced with serum-free medium and cultured for 24 hours, and the supernatant of the cell culture medium was collected;

[0076] 5. The collected culture supernatant was concentrated by ultrafiltration, and the β2M-linker1-CD1d-Fc secretory protein in the supernatant was obtained by purification using Protein A / G antibody magnetic beads and stored at -80°C for future use.

[0077] Comparative Example 1: Preparation of PBMC as feeder

[0078] 1. Resuscitate PBMC cells and adjust the cell density to 5×10 6 / mL, add NKT cell stimulatory molecule α-GalCer (full name: Alpha-Galactoylceramide.NKT cell stimulater, manufacturer: Abcam, catalog number: ab144262) to make the final concentration 5μg / mL, and incubate in a 37°C incubator for more than 1 hour;

[0079] 2. Collect PBMCs incubated with α-GalCer into a new centrifuge tube and irradiate them for 50 gy to prepare feeder;

[0080] 3. Wash the feeder cells once by adding PBS and centrifuging. Resuspend the feeder cells in Optivitro UniEx T cell serum-free medium and count them. Use immediately.

[0081] Comparative Example 2: β2M-CD1d-Ig

[0082] β2M-CD1d-Ig: DimerX I: Recombinant Soluble Dimeric Human CD1d: Ig Fusion Protein, purchased from BD Bioscience, catalog number: 20220804.

[0083] Comparative Example 3: Preparation of artificial antigen presenting molecule "β2M-CD1d-Fc"

[0084] 1. Constructing a lentiviral expression vector of an artificial antigen presenting molecule β2M-CD1d-Fc, wherein the amino acid sequence of CD1d-Fc is shown in SEQ ID NO: 2, and the amino acid sequence of β2M is shown in SEQ ID NO: 5. In β2M-CD1d-Fc, CD1d and β2M spontaneously polymerize to form a dimer. In the lentiviral expression vector, the CD1d-Fc segment and the β2M segment are separated by IRES;

[0085] 2. The lentivirus was packaged using the second-generation method (three-plasmid system: two auxiliary plasmids and one target plasmid). The auxiliary plasmid pMD2.G, psPAX2 and β2M-CD1d-Fc expression vector were transfected into 293T cells through PEI to prepare and harvest β2M-CD1d-Fc lentivirus.

[0086] 3. Add the packaged β2M-CD1d-Fc lentivirus directly to 293T cells, and add Polybrene Transfection Reagent for auxiliary infection. Add β2M-CD1d-Fc lentivirus infection once in the morning and once in the evening. After the last infection overnight, change the cell medium to construct a stable expression cell line of β2M-CD1d-Fc.

[0087] 4. The cell density of the stable expression cell line of β2M-CD1d-Fc was cultured to 80%, and the medium was replaced with serum-free medium and cultured for 24 hours, and the supernatant of the cell culture medium was collected;

[0088] 5. The collected culture supernatant was concentrated by ultrafiltration, and the β2M-CD1d-Fc secretory protein in the supernatant was obtained by purification using Protein A / G antibody magnetic beads, and stored at -80°C for future use.

[0089] Comparative Example 4: Preparation of artificial antigen presenting molecule "β2M-CD1d-CH1-Fc"

[0090] 1. Constructing a lentiviral expression vector of an artificial antigen presenting molecule β2M-CD1d-CH1-Fc, wherein the amino acid sequence of CD1d-CH1-Fc is shown in SEQ ID NO: 3, the CD1d segment and the CH1 segment are connected by GGGGSGGSGSGGG (SEQ ID NO: 8), the amino acid sequence of β2M is shown in SEQ ID NO: 5, in β2M-CD1d-CH1-Fc, CD1d and β2M spontaneously polymerize to form a dimer, and in the lentiviral expression vector, the CD1d-CH1-Fc segment and the β2M segment are separated by IRES;

[0091] 2. The lentivirus was packaged using the second-generation method (three-plasmid system: two auxiliary plasmids and one target plasmid). The auxiliary plasmid pMD2.G, psPAX2 and β2M-CD1d-CH1-Fc expression vector were used to form a three-plasmid transfection system and transfected into 293T cells by PEI to prepare and harvest the β2M-CD1d-CH1-Fc lentivirus;

[0092] 3. Add the packaged β2M-CD1d-CH1-Fc lentivirus directly to 293T cells, and add Polybrene Transfection Reagent for auxiliary infection. Add β2M-CD1d-CH1-Fc lentivirus infection once in the morning and once in the evening. After the last infection overnight, change the cell medium to construct a stable expression cell line of β2M-CD1d-CH1-Fc.

[0093] 4. The cell density of the stable expression cell line of β2M-CD1d-CH1-Fc was cultured to 80%, and the medium was replaced with serum-free medium and cultured for 24 hours, and the supernatant of the cell culture medium was collected;

[0094] 5. The collected culture supernatant was concentrated by ultrafiltration, and the β2M-CD1d-CH1-Fc secretory protein in the supernatant was obtained by purification using Protein A / G antibody magnetic beads and stored at -80°C for future use.

[0095] Example 2

[0096] PBMC cells were divided into 6 groups, namely, α-GalCer group, feeder group, β2M-CD1d-Ig group, "β2M-linker1-CD1d-Fc" group, "β2M-CD1d-Fc" group and "β2M-CD1d-CH1-Fc" group. Free α-GalCer, feeder, β2M-CD1d-Ig, "β2M-linker1-CD1d-Fc", "β2M-CD1d-Fc" and "β2M-CD1d-CH1-Fc" were used for cell culture and expansion, respectively.

[0097] In the α-GalCer group, PBMC cells were cultured and expanded using Optivitro UniEx T cell serum-free medium supplemented with free α-GalCer (final concentration 0.5 μg / mL), IL-2 (final concentration 200 IU / mL), and IL-15 (final concentration 10 ng / mL). After 7 days of culture, the cells were collected, the cell proliferation multiples were calculated, and the NKT purity was detected.

[0098] In the feeder group, the immediately prepared feeder was added according to the cell number feeder:PBMC=1:1, and PBMC cells were cultured and expanded using Optivitro UniEx T cell serum-free medium supplemented with free α-GalCer (final concentration 0.5μg / mL), IL-2 (final concentration 200IU / mL) and IL-15 (final concentration 10ng / mL). After 7 days of culture, the cells were collected, the cell proliferation multiples were calculated, and the NKT purity was detected.

[0099] The β2M-CD1d-Ig group, β2M-linker1-CD1d-Fc group, β2M-CD1d-Fc group and β2M-CD1d-CH1-Fc group were all incubated with α-GalCer overnight at a mass ratio of artificial antigen presenting molecule: α-GalCer = 8:1. The mixture obtained by incubation (working concentration 2 μg / mL) was coated and fixed on the culture dish, and then PBMC cells were added. PBMC cells were cultured and expanded in Optivitro UniEx T cell serum-free medium supplemented with IL-2 (final concentration 200 IU / mL) and IL-15 (final concentration 10 ng / mL). After culturing for 7 days, the cells were collected, the cell proliferation multiples were calculated, and the NKT purity was detected.

[0100] The results are shown in Table 2 and Figure 1 The results showed that the use of feeder, β2M-CD1d-Ig, β2M-linker1-CD1d-Fc, β2M-CD1d-Fc, and β2M-CD1d-CH1-Fc could all cause cells to proliferate rapidly and significantly improve the purity of NKT. Moreover, compared with the feeder group, the NKT purity of the β2M-CD1d-Ig group and the β2M-linker1-CD1d-Fc group was higher, indicating that both β2M-CD1d-Ig and β2M-linker1-CD1d-Fc could well amplify NKT cells specifically; compared with the β2M-CD1d-Ig group, the cell amplification multiple and NKT purity of the β2M-CD1d-Fc group and the β2M-CD1d-CH1-Fc group decreased, indicating that replacing Ig with the Fc segment or CH1- The Fc segment will result in a decrease in the NKT cell-specific expansion performance, while the specific expansion effect of the β2M-linkerCD1d-Fc group is significantly better than that of the β2M-CD1d-Ig group, indicating that by connecting CD1d and β2M through a linker, not only can the decrease in NKT cell-specific expansion performance caused by replacing Ig with the Fc segment or CH1-Fc segment be avoided, but the NKT cell-specific expansion effect can also be further enhanced, thereby improving the NKT cell-specific expansion effect and reducing the production cost of artificial antigen presenting molecules.

[0101] Table 2 Cell proliferation times and NKT purity in each group of Example 2

[0102] Group Cell proliferation multiple NKT purity α-GalCer 1.2 5.77％ feeder 15 62.80％ β2M-CD1d-Ig 17.5 72.75％ β2M-linker1-CD1d-Fc 23.6 87.93％ β2M-CD1d-Fc 16.6 63.71％ β2M-CD1d-CH1-Fc 16.1 62.80％

[0103] Example 3

[0104] The single blood (purchased from Shanghai Xuanfeng Biotechnology Co., Ltd., obtained by single blood collection from healthy people) was separated from PBMC cells by density gradient centrifugation using LymphoprepTM (stem cell, catalog number 07811), and then primary NKT cells were sorted out using Anti-iNKT MicroBeads human (Miltenyi Biotec, catalog number 130-094-842) magnetic beads. The sorted NKT cells were divided into four groups, namely transact group, β2M-CD1d-Ig group, β2M-linker1-CD1d-Fc group and β2M-CD1d-Fc group. In the transact group, NKT cells were cultured and expanded in Optivitro UniEx T cell serum-free medium supplemented with CD3 / CD28 beads (purchased from Miltenyi Biotech, Germany, catalog number 130-128-758), IL-2 (final concentration 200 IU / mL) and IL-15 (final concentration 10 ng / mL). After 3 days of culture, the cells were collected and the purity of NKT was detected. In the β2M-CD1d-Ig group, the "β2M-linker1-CD1d-Fc" group and the "β2M-CD1d-Fc" group, the culture dishes were coated and fixed in the same manner as in Example 1, and then NKT cells were added. NKT cells were cultured and expanded in Optivitro UniEx T cell serum-free medium supplemented with IL-2 (final concentration 200 IU / mL) and IL-15 (final concentration 10 ng / mL). After 3 days of culture, the cells were collected and the purity of NKT was detected.

[0105] The results are shown in Table 3 and Figure 2The results showed that compared with the transact group, the NKT cell purity was significantly improved by culturing NKT cells with β2M-CD1d-Ig, β2M-linker1-CD1d-Fc in Example 1, and β2M-CD1d-Fc, indicating that β2M-CD1d-Ig, β2M-linker1-CD1d-Fc, and β2M-CD1d-Fc could specifically amplify NKT cells; compared with the β2M-CD1d-Ig group, the NKT purity in the β2M-CD1d-Fc group was lower. This indicates that replacing Ig with Fc will result in a decrease in the specific expansion performance of NKT cells, while the specific expansion effect of the β2M-linker1-CD1d-Fc group is significantly better than that of the β2M-CD1d-Ig group, indicating that by connecting CD1d and β2M through a linker, not only can the decrease in the specific expansion performance of NKT cells caused by replacing Ig with Fc can be avoided, but the specific expansion effect of NKT cells can also be further improved, thereby improving the specific expansion effect of NKT cells and reducing the production cost of artificial antigen presenting molecules.

[0106] Table 3 Purity of NKT cells in each group of Example 3

[0107] Group NKT purity transact 11.19％ β2M-CD1d-Ig 75.74％ β2M-linker1-CD1d-Fc 82.13％ β2M-CD1d-Fc 65.57％

[0108] Example 4 Preparation of artificial antigen presenting molecules "β2M-linker2-CD1d-Fc" and "β2M-linker3-CD1d-Fc"

[0109] In this example, artificial antigen presenting molecules "β2M-linker2-CD1d-Fc" and "β2M-linker3-CD1d-Fc" are prepared. The only difference from Example 1 is that linker1 is replaced by linker2 or linker3, linker2 is GGGGSGGGGSGGGGS (SEQ ID NO: 9), and linker3 is GSTSGSGKPGSGEGSTKG (SEQ ID NO: 10).

[0110] Example 5

[0111] The PBMC cells were divided into three groups, namely, "β2M-linker1-CD1d-Fc" group, "β2M-linker2-CD1d-Fc" group and "β2M-linker3-CD1d-Fc" group, and "β2M-linker1-CD1d-Fc", "β2M-linker2-CD1d-Fc" and "β2M-linker3-CD1d-Fc" were used for cell culture and expansion, respectively.

[0112] The "β2M-linker1-CD1d-Fc" group, "β2M-linker2-CD1d-Fc" group and "β2M-linker3-CD1d-Fc" group were incubated with α-GalCer overnight at a mass ratio of artificial antigen presenting molecule: α-GalCer = 8:1. The mixture obtained by incubation (working concentration 2 μg / mL) was coated and fixed on the culture dish, and then PBMC cells were added. Optivitro UniEx T cell serum-free medium supplemented with IL-2 (final concentration 200 IU / mL) and IL-15 (final concentration 10 ng / mL) was used to culture and expand PBMC cells. After 7 days of culture, the cells were collected, the cell proliferation multiples were calculated, and the NKT purity was detected.

[0113] The results are shown in Table 4. The results show that the NKT cell-specific expansion effects of β2M-linker2-CD1d-Fc and β2M-linker3-CD1d-Fc obtained by replacing linker1 with linker2 and linker3 are equivalent to those of β2M-linker1-CD1d-Fc, indicating that connecting CD1d to β2M through linker1, linker2 and linker3 can enhance the NKT cell-specific expansion effect, thereby improving the NKT cell-specific expansion effect and reducing the production cost of artificial antigen presenting molecules.

[0114] Table 4 Cell proliferation times and NKT purity in each group of Example 5

[0115] Group Cell proliferation multiple NKT purity β2M-linker1-CD1d-Fc 22.8 86.52％ β2M-linker2-CD1d-Fc 22.9 88.62％ β2M-linker3-CD1d-Fc 23.1 85.84％

[0116] Example 6

[0117] PBMC cells were divided into 3 groups, all of which were cultured and expanded with β2M-linker1-CD1d-Fc. They were incubated with α-GalCer overnight at a mass ratio of β2M-linker1-CD1d-Fc:α-GalCer = 5:1, 8:1 or 10:1, and the mixture obtained by incubation (working concentration 2μg / mL) was coated and fixed on the culture dish, and then PBMC cells were added. Optivitro UniEx T cell serum-free medium supplemented with IL-2 (final concentration 200IU / mL) and IL-15 (final concentration 10ng / mL) was used for PBMC cell culture and expansion. After 7 days of culture, the cells were collected, the cell proliferation times were calculated, and the NKT purity was detected.

[0118] The results are shown in Table 5. The results show that overnight incubation with α-GalCer at a mass ratio of β2M-linker1-CD1d-Fc:α-GalCer = 5:1, 8:1 or 10:1 can achieve specific expansion of NKT cells with good expansion effect.

[0119] Table 5 Cell proliferation times and NKT purity in each group of Example 6

[0120]

[0121]

[0122] Example 7 Preparation of artificial antigen presenting molecules "β2M-linker1-CD1d-Fc-magnetic microspheres"

[0123] This example is used to prepare the artificial antigen presenting molecule "β2M-linker1-CD1d-Fc-magnetic microspheres", which is different from Example 1 only in that: after obtaining the β2M-linker1-CD1d-Fc secretory protein in step 5, it is coupled with the magnetic microspheres to obtain β2M-linker1-CD1d-Fc-magnetic microspheres. The magnetic microspheres were purchased from Thermo Scientific under the trade name Pierce TM Protein A / G Magnetic Beads, catalog number 88803.

Claims

1. A fusion protein for NKT cell expansion, characterized in that: The fusion protein comprises CD1d and β2M connected by a linker.

2. The fusion protein according to claim 1, characterized in that The linker is a peptide linker, and the peptide linker is preferably selected from a GS polypeptide linker and / or a Whitlow linker; And / or, the CD1d comprises the amino acid sequence shown in SEQ ID NO:4; and / or, the β2M comprises the amino acid sequence shown in SEQ ID NO:5; Preferably, the GS polypeptide linker comprises an amino acid sequence as shown in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:11 or SEQ ID NO:12; and / or, the Whitlow linker comprises an amino acid sequence as shown in SEQ ID NO:

10.

3. An artificial antigen presenting molecule, characterized in that The artificial antigen presenting molecule comprises the fusion protein according to claim 1 or 2; Preferably, the artificial antigen presenting molecule further comprises a protein scaffold structure and / or a non-protein scaffold structure, and the fusion protein is connected to the protein scaffold structure; the fusion protein is coupled to the non-protein scaffold structure; More preferably, the length of the protein scaffold structure is at least 18 amino acid residues; and / or, the non-protein scaffold structure is selected from small molecule monomer polymers, magnetic microspheres and nanoparticles; And / or, the protein scaffold structure is selected from an antibody or a part of an antibody, and a transmembrane protein or a part of a transmembrane protein; the part of the antibody is preferably the constant region of the antibody, more preferably the Fc segment.

4. A polynucleotide, characterized in that The polynucleotide encodes the fusion protein according to claim 1 or 2; and / or, the artificial antigen presenting molecule according to claim 3.

5. A recombinant expression vector, characterized in that: The recombinant expression vector comprises the polynucleotide according to claim 4.

6. A lentiviral expression vector, characterized in that: The lentiviral expression vector comprises the polynucleotide according to claim 4.

7. A transformant, characterized in that: The transformant comprises the recombinant expression vector according to claim 5 or the lentiviral expression vector according to claim 6.

8. A method for preparing an artificial antigen presenting molecule, characterized in that: The method comprises the following steps: Cultivating the transformant according to claim 7 to obtain an artificial antigen presenting molecule; Preferably, the method further comprises: coupling the protein obtained by culturing the transformant with a non-protein scaffold structure; Wherein, the non-protein scaffold structure is preferably selected from small molecule monomer polymers, magnetic microspheres and nanoparticles.

9. A method for expanding NKT cells, characterized in that: The method comprises: in the presence of α-GalCer and the artificial antigen presenting molecule according to claim 3, amplifying and culturing NKT cells; Preferably, the NKT cells are iNKT cells; And / or, the mass ratio of the artificial antigen presenting molecule to α-GalCer is (5-10):1; And / or, the method further comprises: adding cytokines during the expansion culture process, wherein the cytokines are preferably IL-2 and / or IL-15; More preferably, the final concentration of IL-2 is 100-300 IU / mL, and the final concentration of IL-15 is 5-20 ng / mL.

10. Use of the fusion protein according to claim 1 or 2, the artificial antigen presenting molecule according to claim 3, the polynucleotide according to claim 4, the recombinant expression vector according to claim 5, the lentiviral expression vector according to claim 6, and the transformant according to claim 7 in amplifying NKT cells.

Images