CN112048018B

CN112048018B - Chimeric T cell growth factor and application thereof

Info

Publication number: CN112048018B
Application number: CN202010897339.1A
Authority: CN
Inventors: 蒋小滔; 刘璇; 吴砂; 李咏茵; 侯金林
Original assignee: Southern Medical University
Current assignee: Southern Medical University
Priority date: 2020-08-31
Filing date: 2020-08-31
Publication date: 2021-10-08
Anticipated expiration: 2040-08-31
Also published as: CN112048018A

Abstract

The invention discloses a variant of IL-2, which changes the amino acid residue combined with CD25 in the natural IL-2 sequence to reduce the effect of inhibiting immune response. The invention also provides a chimeric T cell growth factor, which comprises the amino acid sequence of the IL-2 variant and the amino acid sequences of IL-7 and IL-21. The chimeric T cell growth factor can promote proliferation and survival of T cells in vivo, inhibit T cell failure, and enhance immunity. The chimeric T cell growth factor can be used in combination with adoptive immunotherapy based on T cells such as CAR-T, TCR-T and TIL cells, so as to enhance the treatment effect of the cells, and has wide application prospect in the technical field of cancer treatment.

Description

Chimeric T cell growth factor and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a chimeric T cell growth factor and application thereof.

Background

Lymphocytes (lymphocytes) are one of the major cells that make up the body's immune system, and in adults there are approximately 1 trillion lymphocytes, commensurate with the number of cells in the brain. Lymphocytes are mainly distributed in lymphoid organs, lymphoid tissues and peripheral blood, and the number of lymphocytes in the blood circulation is about 30% of the total number of peripheral blood leukocytes. Lymphocytes cooperate and restrict each other in the immune response process to jointly complete the recognition, response and elimination of antigen substances, thereby maintaining the health of the organism.

T lymphocytes (T lymphocytes), abbreviated as T cells, account for about 80% of the total number of peripheral blood lymphocytes, and are the most functionally active lymphocyte population in vivo. T cells, as a biological agent, have been widely used in clinical treatment of various diseases, especially cancer, such as Tumor-Infiltrating lymphocytes (TIL), chimeric antigen receptor T cells (CAR-T cells), TCR-T cells, etc.

In order to maintain normal physiological functions such as activation, proliferation and killing of T cells in vivo, the involvement of various cytokines (cytokines) is required. Among them, Interleukin-2 (IL-2) plays an important role, and therefore IL-2 is also called T Cell Growth Factor (TCGF).

However, the direct use of IL-2 to enhance the biological activity of T cells has three disadvantages:

(1) CD25 is the high affinity receptor alpha chain of IL-2 (IL-2R α), whereas CD25 is constitutively highly expressed in regulatory T cells (tregs), and thus if native IL-2 is administered alone, it can stimulate Treg activation, thereby suppressing immune response and reducing the therapeutic effect of T cells.

(2) IL-2 only contains 133 amino acids and has the molecular weight of 15.4kDa, so that the half-life period of the IL-2 in peripheral blood is extremely short, namely only 5-7 minutes; to maintain an effective concentration, multiple administrations are required, and a single dose escalation may cause serious side effects.

(3) Prolonged stimulation of IL-2 promotes

T cells differentiate towards terminal effector T cells (Teff), which have a shorter survival time in vivo, thereby reducing the biological effects of T cells.

Therefore, there is an urgent need to develop a novel T cell growth factor that can simultaneously overcome the three drawbacks of IL-2, greatly promote the effects of T cell-based adoptive cell therapy, and have wide social needs and application values.

Disclosure of Invention

It is an object of the first aspect of the present invention to provide a variant of IL-2.

In a second aspect, the invention provides a nucleotide encoding the IL-2 variant described above.

In a third aspect, the present invention is directed to a chimeric T cell growth factor that overcomes the three disadvantages of IL-2.

In a fourth aspect, the present invention provides a nucleotide encoding the chimeric T cell growth factor.

In a fifth aspect, the present invention is directed to a vector.

In a sixth aspect, the present invention provides a cell line.

In a seventh aspect, the present invention is directed to a medicament for immunotherapy.

An eighth aspect of the present invention is to provide an anticancer drug.

The technical scheme adopted by the invention is as follows:

in a first aspect of the invention, there is provided a variant of IL-2 comprising the amino acid sequence of IL-2 and substitutions at amino acids 40 to 45 and 63 to 68 of IL-2 using a linker sequence.

Preferably, the variant of IL-2 according to the first aspect of the invention has an amino acid sequence as shown in SEQ ID NO 7.

In a second aspect of the invention, there is provided a nucleotide encoding an IL-2 variant of the first aspect of the invention.

In a third aspect of the invention, there is provided a chimeric T cell growth factor comprising the amino acid sequence of the IL-2 variant of the first aspect of the invention and the amino acid sequences of IL-7 and IL-21.

Preferably, the chimeric T cell growth factor according to the third aspect of the invention, further comprises a linker sequence.

More preferably, the amino acid sequence of the IL-2 mutant and the amino acid sequences of IL-7 and IL-21 are linked in sequence by a linker sequence according to the chimeric T-cell growth factor of the third aspect of the invention.

Preferably, the linker sequence is in turn GGGGSGGGGSGGGS (SEQ ID NO:9) or GGGGSGGGGSGGGGSSS (SEQ ID NO: 11).

The amino acid sequence of the chimeric T cell growth factor is shown in SEQ ID NO. 10.

In a fourth aspect of the invention, there is provided a nucleotide encoding a chimeric T cell growth factor according to the third aspect of the invention.

In a fifth aspect of the invention, there is provided a vector comprising a nucleotide according to the fourth aspect of the invention.

In a sixth aspect of the invention, there is provided a cell line comprising the vector of the fifth aspect of the invention.

Preferably, the cell line according to the sixth aspect of the invention is selected from the group consisting of CAR-T cells, TCR-T cells, TIL cells, NK cells.

In a seventh aspect of the invention, there is provided a medicament for immunotherapy comprising a chimeric T cell growth factor according to the third aspect of the invention, a nucleotide according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention or a cell line according to the sixth aspect of the invention.

In an eighth aspect of the invention, there is provided an anti-cancer agent comprising a chimeric T cell growth factor according to the third aspect of the invention, a nucleotide according to the fourth aspect of the invention, a vector according to the fifth aspect of the invention or a cell line according to the sixth aspect of the invention.

The invention has the beneficial effects that:

the present invention provides a variant of IL-2 that alters the amino acid residues in the native IL-2 sequence that bind to CD 25. CD25 is a high affinity receptor alpha chain (IL-2R alpha) of IL-2, and CD25 is constitutively highly expressed in regulatory T cells (Tregs), and changes amino acid residues in a natural IL-2 sequence, which are combined with CD25, so that the defects that the natural IL-2 can stimulate Treg activation, so that immune response is inhibited, and the therapeutic effect of the T cells is weakened are overcome.

The invention constructs a chimeric T cell growth factor for the first time, which comprises an amino acid sequence of an IL-2 variant and amino acid sequences of IL-7 and IL-21. The chimeric T cell growth factor greatly prolongs the half-life of the IL-2 variant, can promote the proliferation and survival of T cells in vivo, inhibit the failure of the T cells, and has the function of enhancing the immunity of organisms.

The chimeric T cell growth factor prepared by the invention can be used in combination with adoptive immunotherapy based on T cells such as CAR-T, TCR-T and TIL cells, so as to enhance the treatment effect of the cells and have wide application prospect in the technical field of cancer treatment.

Drawings

FIG. 1 is a schematic representation of the binding of native IL-2 to CD 25. (A) Dark marks are IL-2 and CD25 binding region one (L40-Y45); (B) dark marks are the second region of IL-2 binding to CD25 (L63-E68).

FIG. 2 is a schematic diagram of the structure of a chimeric T-cell growth factor.

FIG. 3 is the schematic diagram of the construction of the eukaryotic expression vector of the chimeric T cell growth factor.

FIG. 4 is a schematic diagram of the construction of a prokaryotic expression vector for chimeric T-cell growth factors.

FIG. 5 shows the double-restriction electrophoresis of the chimeric T-cell growth factor-prokaryotic expression vector pET-30a (+). Lane M: DNA molecular weight marker; lane 1: ndel, HindIII double restriction enzyme digestion plasmid; lane 2: the plasmid was not digested.

FIG. 6SDS-PAGE detects expression of chimeric T-cell growth factor. Lane M₁: protein marker; lane NC: bacterial lysate not induced with IPTG; lane 1: after IPTG was added, the bacterial lysate was induced at 15 ℃ for 16 hours; lane 2: after IPTG was added, the bacterial lysate was induced at 37 ℃ for 4 hours; lane NC 1: the supernatant of the bacterial lysate after centrifugation without IPTG induction; lane 3: after IPTG is added, the bacterial lysate is induced for 16 hours at 15 ℃ to obtain supernatant after centrifugation; lane 4: after IPTG was added, the supernatant after centrifugation of the bacterial lysate was induced at 37 ℃ for 4 hours; lane NC₂: a precipitate from centrifugation of a bacterial lysate not induced with IPTG; lane 5: adding IPTG, inducing the bacterial lysate for 16 hours at 15 ℃ to obtain a centrifuged precipitate;lane 6: after IPTG was added, the bacterial lysate was centrifuged to obtain a precipitate which was induced at 37 ℃ for 4 hours.

FIG. 7Western blot to detect the expression of chimeric T cell growth factors. Lane M₂: protein marker; lane NC: bacterial lysate not induced with IPTG; lane 1: after IPTG was added, the bacterial lysate was induced at 15 ℃ for 16 hours; lane 2: after IPTG was added, the bacterial lysate was induced at 37 ℃ for 4 hours; lane 3: after IPTG is added, the bacterial lysate is induced for 16 hours at 15 ℃ to obtain supernatant after centrifugation; lane 4: after IPTG was added, the supernatant after centrifugation of the bacterial lysate was induced at 37 ℃ for 4 hours; lane 5: adding IPTG, inducing the bacterial lysate for 16 hours at 15 ℃ to obtain a centrifuged precipitate; lane 6: after IPTG was added, the bacterial lysate was centrifuged to obtain a precipitate which was induced at 37 ℃ for 4 hours.

FIG. 8SDS-PAGE was performed to examine the concentration of the target protein after purification. Lane M₁: a protein Marker; lane 1: BSA (2 ug); lane 2: purified chimeric T cell growth factor.

FIG. 9 the ability of native IL-2 to bind CD25(IL-2R α) with chimeric T cell growth factor.

FIG. 10 shows a schematic view of a

Effect of T cells on cell proliferation after 6 days of culture with added IL-2 or chimeric T cell growth factor. (A) Observing results under a microscope; (B) and (6) counting the results.

Figure 11 effect of chimeric T cell growth factor on Treg cell proliferation. (A) FCM gate around policy: tregs are CD3+ CD4+ CD25+ CD 127-cells. (B) Typical flow scattergram results show that the proportion of Treg cells in the IL-2 group is significantly higher than the chimeric T cell growth factor group. (C) Statistics show that the proportion of Treg cells in the IL-2 treated group is significantly higher than that in the chimeric T cell growth factor group (n-6).

FIG. 12 Natural IL-2 vs. chimeric T cell growth factor

Influence of the proportion of T cells. (A) A flow cytogram; (B) a flow cytogram; (C) and (5) a statistical result graph.

FIG. 13 native IL-2 and chimerasSynthetic T cell growth factor pairs

Statistical results of the effect of T cell ratio.

FIG. 14 half-life of native IL-2, chimeric T cell growth factor in vivo

FIG. 15 chimeric T cell growth factor pairs

Effect of CAR-T cell ratio. (A) Schematic diagram of the preparation and detection process of CAR-T cells. Flow assay CAR-T cell proportion and phenotype: (B) FCM measures CAR-T ratio, and FITC-goat anti-mouse F (ab)2 measures CAR expression on T cells. (C) The experiment was repeated with three donor T cells and statistical results showed that the proportion of CAR-T cells was not significantly different between the native IL-2 group and the chimeric T cell growth factor group. (D) FCM detects CAR-T memory phenotype profiles. The phylum is CAR + T cell, wherein CD45RO-CCR7+ is

CAR-T cells. (E) Statistics show that in the CAR-T cells of the group cultured with the chimeric T cell growth factor,

the proportion of cells was significantly higher than that of the native IL-2 culture group (P ═ 0.018).

Detailed Description

In order to clearly understand the technical contents of the present invention, the following embodiments are described in detail with reference to the accompanying drawings. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's recommendations. The various chemicals used in the examples are commercially available.

EXAMPLE 1 construction of chimeric T cell growth factor

The inventors have overcome the first disadvantage of native IL-2 by mutating the amino acid residues of the native IL-2 sequence that bind to CD 25.

The natural IL-2 amino acid sequence is:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:1)。

FIG. 1 is a schematic representation of the binding of native IL-2 to CD 25. The black mark of A in FIG. 1 is IL-2 and CD25 binding domain one (L40-Y45); the black mark of B in FIG. 1 is the region two (L63-E68) where IL-2 binds to CD 25.

These two CD25 binding regions separate the native IL-2 amino acid sequence into 3 segments, where IL-2 fragment one has the amino acid sequence: APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM (SEQ ID NO: 2);

the amino acid sequence of the IL-2 fragment II is as follows: MPKKATELKHLQCLEEE (SEQ ID NO: 3);

the amino acid sequence of the IL-2 fragment III is as follows:

VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:4)；

these three IL-2 fragments were sequentially ligated by linker (linker1 and 2) sequences.

The sequence of the linker (linker1 and 2) was GSSGGS (SEQ ID NO:5), GSSGSS (SEQ ID NO:6) in that order.

The amino acid sequence of the IL-2 variant after ligation is shown below, with bolded linker sites:

APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMGSSGGSMPKKATELKHLQCLEEEGSSGSSVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITFCQSIISTLT(SEQ ID NO:7)。

the inventor then reports that IL-21 can promote T cell survival and inhibit T cell failure. On the basis of the IL-2 variant, the IL-21 is connected with a flexible linker, so that the third defect of the natural IL-2 is overcome; the amino acid sequence after ligation is as follows: wherein the bold is the linker site in the IL-2 variant, the italic part is the linker (linker4) sequence, and the underlined part is the sequence of IL-21:

through mutation of IL-2 and connection of IL21, the molecular weight of the constructed chimeric T cell growth factor is increased to 30kDa, and the half life is prolonged; to further extend its half-life, the inventors inserted another cytokine IL-7 between the IL-2 variant and IL-21, which promotes T cell survival, increasing its molecular weight to 50 kDa; thus overcoming the second disadvantage of natural IL-2, the amino acid sequence is as follows: wherein the bold is the site of IL-2 mutation, the italic part is the linker (linker3 and linker4) sequence, the part underlined is the sequence of IL-7, and the underlined part is the sequence of IL-21:

the sequence of the flexible linker3 was GGGGSGGGGSGGGS (SEQ ID NO: 9).

The sequence of the flexible linker4 was selected to be GGGGSGGGGSGGGGSSS (SEQ ID NO: 11).

By removing two regions of the amino acid sequence of IL-2 that bind to CD 25: L40-Y45, L63-E68, and the native IL-7 amino acid sequence D1-H152 is linked through a polypeptide linker3(GGGGSGGGGSGGGGS) at the carboxyl terminal of native IL-2, which is replaced by polypeptide linker1 GSSGGS and linker2 GSSGSS, respectively; the natural IL-21 amino acid sequence Q1-S133 is connected with the carboxyl terminal of the IL-7 through a polypeptide linker4 (GGGGSGGGGSGGGGSSS); the chimeric T cell growth factor constructed by the method is formed by connecting three IL-2 segments, complete IL-7, complete IL-21 and a linker in series, the structure of the chimeric T cell growth factor is shown in figure 2, and the amino acid sequence is shown in SEQ ID NO. 10.

Construction of chimeric T cell growth factor recombinant plasmid

Construction of eukaryotic expression plasmids: the chimeric T cell growth factor amino acid sequence is delivered to a biological company, DNA codons are optimized according to eukaryotic expression, and a his tag is added at the C terminal so as to be beneficial to the later stagePurifying; adding an immunoglobulin kappa chain secretion signal peptide ATGCGGGTGCTCGCTGAACTTCTTGGTCTCTTGCTGTTCTGTTTCTTGGGAGTTAGGTGT (SEQ ID NO:12) at the N-terminal; the corresponding amino acid sequence is MRVLAELLGLLLFCFLGVRC (SEQ ID NO:13) to facilitate the secretory expression of the protein; kozak sequence GGAGCCGCCACC (SEQ ID NO:14) was added before the start codon to improve translation after gene transcription; selecting a eukaryotic expression Plasmid as pCDH-EF1 (adddge, Plasmid # 72266); as shown in FIG. 3, the eukaryotic expression plasmid and the company-synthesized chimeric T cell growth factor cloning vector were double-digested with restriction enzymes BamHI (NEB, # R3136) and SalI (NEB, # R3138), and after recovery of the excised gel, T4 ligase (NEB, M0202S) was ligated overnight at 16 ℃; transformation of Stabl3 competent cells (Invitrogen)^TM# C737303), double-restriction or Polymerase Chain Reaction (PCR) to identify positive clones.

Construction of prokaryotic expression plasmid: as shown in figure 4, the amino acid sequence of the chimeric T cell growth factor is delivered to a biological company, DNA codons are optimized according to eukaryotic expression, and a his tag is added at the C terminal so as to facilitate later purification; Shine-Dalgarno sequence AGGAGGACAGCT (SEQ ID NO:15) was added before the start codon to improve translation after gene transcription; the eukaryotic expression plasmid is pET-30a (+) (Sigma-Aldrich, # 69909); restriction enzymes NdeI (NEB, # R0111S) and HindIII ((NEB, # R3104) are used for double digestion of eukaryotic expression plasmids and chimeric T cell growth factor cloning vectors synthesized by the company, after gel cutting and recovery, T4 ligase is used for connecting overnight at 16 ℃, BL21 competent cells (Takara, #9126) are transformed, and double digestion or Polymerase Chain Reaction (PCR) is used for identifying positive clones, and FIG. 5 is agarose gel electrophoresis results after double digestion, which shows that linear vectors are obtained after double digestion.

Expression and identification of chimeric T cell growth factors:

1) selecting BL21 clone identified as positive by double enzyme digestion, and inoculating the clone to LB culture medium containing 50 ug/ml kanamycin;

2) culturing the bacteria on a shaking table at 37 ℃ and 200 rpm;

3) detecting OD600, adding IPTG (Shanghai Biotech, # B541007) with final concentration of 0.5mM to culture at 37 ℃ for 4 hours or 15 ℃ for 16 hours when OD600 reaches 0.6-0.8;

4) after completion of the incubation, the bacteria were collected by centrifugation, added with lysis buffer (50mM Tris,150mM NaCl, 5% glycerol, pH 8.0), and sonicated for 1 minute;

5) SDS-PAGE detection of protein expression: mixing the bacterial lysate and the loading buffer solution, boiling 10minutes at 100 ℃, centrifuging for 5 minutes at 15000rpm, and sucking the supernatant for electrophoresis; after electrophoresis, staining with Coomassie brilliant blue; the SDS-PAGE results are shown in FIG. 6, which shows that BL21 transferred with chimeric T cell growth factor plasmid produces a protein of about 50kD under IPTG induction, and the molecular weight of the protein is consistent with that of the target protein.

6) Protein expression was confirmed by Western blot (Western blot): proteins from SDS-PAGE were electroporated onto PVDF membrane, and then anti-His protein antibody (GenScript, # A00186) was added for detection; as a result, as shown in FIG. 7, a significant band was detected at 50kD, which is consistent with the predicted molecular weight of the target protein, and a small amount of dimer and trimer was also detected.

Purification of chimeric T cell growth factor:

1) after the bacteria are subjected to ultrasonic lysis, centrifuging, and washing precipitates twice by using 4M urea;

2) then dissolving the inclusion body by 8M urea;

3) renaturation of the protein by dialysis, the renaturation buffer is 50mM Tris,150mM NaCl,0.5M L-Arginine, 10% Glycerol, pH 8.0;

4) after the protein renaturation, the purity of the purified protein is measured by nickel column purification and SDS-PAGE, and the result is shown in figure 8, wherein the concentration of the target protein is more than 95 percent;

5) by Pierce^TMHigh Capacity endo toxin Removal Spin Columns (cat 88276) to remove Endotoxin;

6) filtering with 0.22 μm filter membrane for sterilization, and subpackaging at-80 deg.C for freezing storage.

The constructed chimeric T cell growth factor contains 450 amino acids in total, the theoretical molecular weight is 50kD, and SDS-PAGE and Western blotting results show that the molecular weight of the chimeric T cell growth factor is about 50kD and is consistent with the theoretical value.

Example 2 binding of chimeric T cell growth factor and native IL-2 to CD25

The main experimental materials: purified chimeric T-cell growth factor, native human IL-2(Peprotech, #200-02), CD25-Fc (Acrobiosystems, # ILA-H5251), HRP-anti-human IgG (abcam, # ab81202), TMB (Thermo Scientific, # N301).

The experimental method comprises the following steps:

1) native IL-2(Peprotech, #200-02) and the chimeric T-cell growth factor prepared in example 1 were diluted with PBS to a final concentration of 5. mu.g/ml; 96-well enzyme label plate, 100 mul/well, coating overnight at 4 ℃;

2) 5% skimmed milk powder, sealing at 37 deg.C for 2 hr;

3) CD25-Fc (Acrobiosystems, # ILA-H5251) was added in a dilution by volume and incubated in a water bath at 37 ℃ for 1 hour;

4) after five times of washing, HRP-anti-human IgG (abcam, # ab81202) was added, and incubation was carried out in a 37 ℃ water bath for half an hour;

5) after five washes, TMB was added for 10min of color development and 2M sulfuric acid was stopped;

6) detecting OD450nm with a microplate reader;

to verify whether chimeric T-cell growth factors bind to CD25, native IL-2 and chimeric T-cell growth factors prepared in example 1 were coated on ELISA plates (5. mu.g/ml) and then diluted in duplicate CD25-Fc fusion protein was added and incubated at 37 ℃ for 1 hour; finally adding HRP-labeled anti-human IgG (HRP-anti-human IgG), incubating at 37 ℃ for half an hour, developing color with TMB, 2M H₂SO₄After termination, the microplate reader detected OD450nm, and the results are shown in FIG. 9. As can be seen from the figure: CD25 can bind to native IL-2 but not to the chimeric T cell growth factor constructed in example 1. Illustrative chimeric T cell growth factors constructed in example 1

Example 3 Effect of chimeric T cell growth factor and native IL-2 on Treg proliferation

The main experimental materials: purified chimeric T cell growth factor, native human IL-2(Peprotech, #200-02), anti-CD 3/anti-CD 28 antibody (stem cell, #10971), fluorescently labeled antibody: APC Mouse Anti-Human CD3(BD, # 555335); PE-Cy^TM7Mouse Anti-Human CD4(BD，#560909)；PE anti-human CD25 Antibody(Biolegend，#302605)；FITC anti-human CD127(IL-7Rα)Antibody(Biolegend，#351311)。

The experimental method comprises the following steps:

1) separating and purifying healthy adult Peripheral Blood Mononuclear Cells (PBMC) by Ficoll-Paque (GE Healthcare, #17144002) density gradient centrifugation;

2) total T cells were sorted by magnetic bead sorting (STEMCELL, # 17961);

3) dilution of T cells to a final concentration of 1X 10 with RPMI 1640 complete Medium⁶Cells/ml, 96-well culture plate, 100 ul/well;

4) activating T cells by co-culturing the isolated and purified T cells with anti-CD 3/anti-CD 28 antibody (STEMCELL, #10971) for two days;

5) setting a control group without any cytokine, IL-2, and a chimeric T cell growth factor group;

6) changing the liquid every other day, and adding corresponding IL-2 or chimeric T cell growth factors according to groups;

7) after 14 days of culture, the cells were harvested and the proportion of Treg cells (CD3+ CD4+ CD25+ CD127-) was flow-tested.

The results are shown in FIG. 11, which shows that Treg cells account for CD3+ T cells after 14 days of co-culture with fresh isolated healthy adult PBMC with native IL-2 or chimeric T cell growth factor. Tregs were labeled with CD3+ CD4+ CD25+ CD 127-. Since the cells of the group without any cytokine were all dead after 14 days of culture, no analysis of the results was included. The results showed that the proportion of Treg cells in the natural IL-2 treated group was 3.98% ± 1.11% and the proportion of Treg cells in the chimeric T-cell growth factor treated group was 1.39% ± 0.38% which was significantly lower than that in the natural IL-2 group (P ═ 0.0022) after 14 days of culture, indicating that the chimeric T-cell growth factor treatment was able to significantly suppress the proliferation of Treg cells.

Example 4 Effect of chimeric T cell growth factor and native IL-2 on T cell survival/proliferation

In example 2 it was demonstrated that chimeric T cell growth factor does not bind to CD25 and theoretically has a stronger immune activation compared to native IL-2. For validation, PBMCs and T cells were isolated from healthy adult peripheral blood and cultured in 96-well flat-bottomed cell culture plates with the addition of 100ng/ml native IL-2 or 100ng/ml chimeric T cell growth factor, without cytokine wells as controls, and cells were collected and counted after 6 days of culture.

The results are shown in fig. 10, and show that after 6 days of culture, the cell proliferation index of the control group without any cytokine is 0.86, the cell proliferation index of the group with IL-2 is 1.83, which is significantly higher than that of the control group (p <0.001 in paired t test); the cell proliferation index of the plus chimeric T cell growth factor group was 2.05, significantly higher than the control group (p <0.001 for paired T-test) and also significantly higher than the IL-2 group (p <0.05 for paired T-test). It was shown that the chimeric T-cell growth factor constructed in example 1 stimulates stronger T-cell proliferation relative to IL-2.

Example 5 Effect of chimeric T cell growth factor and native IL-2 on differentiation following T cell activation

A short panel of adoptive T cell immunotherapies is: t cells differentiate during activation, proliferation and culture in vitro towards short-lived end-stage T cells, resulting in poor therapeutic efficacy for the time period in which they survive in vivo after infusion into a patient. IL-21 is one of the members of the gamma c receptor cytokine family, and is a key cytokine for long-term stable existence of long-life CD8+ memory T cells in vivo; research shows that IL-21 can promote survival and proliferation of T cells in vivo, and inhibit Activation Induced Cell Death (AICD); the depletion of activated T cells is disrupted and reversed. In addition, IL-21 was linked to the carboxy terminus of the chimeric T-cell growth factor constructed in example 1 in order to enhance the anti-T-cell depletion effect.

To verify whether the chimeric T-cell growth factor constructed in example 1 has anti-T-cell depletion function, isolated and purified healthy adult T-cells were activated in vitro with antibodies against CD3 and CD28, and IL-2 or the chimeric T-cell growth factor of the present invention was added for co-culture for 14 days, and then the cells were collected and the differentiation status of the T-cells was examined by flow cytometry (CCR7+ CD45 RO-is

T is thinA cell; CCR7+ CD45RO + is a centralized memory T cell; CCR7-CD45RO + is effector memory T cell; CCR7-CD45 RO-effector T cells).

The method specifically comprises the following steps:

1) activating T cells by co-culturing the isolated and purified T cells with anti-CD 3/anti-CD 28 antibody (STEMCELL, #10971) for two days;

2) removing the anti-CD 3/anti-CD 28 antibody, adding natural IL-2 or chimeric T cell growth factor for co-culture, and changing the culture solution every other day;

3) after 14 days of culture, cells were collected and flow-assayed

The ratio of T, central memory T (Tcm), effect memory T (Tem), and effect T (Teff); the labeled antibodies used were as follows: APC-cy7 anti-human CD 3; APC-anti-human CCR7, PE-cy7 anit-human CD45 RO;

4) CD3+ CCR7+ CD45 RO-is

T cells, CD3+ CCR7+ CD45RO + is Tcm, CD3+ CCR7-CD45RO + is Tem, CD3+ CCR7-CD45 RO-is Teff.

The results are shown in FIGS. 12 and 13, and show that the IL-2-treated group is undifferentiated

The proportion of T cells was 27. + -. 5.3%, chimeric T cell growth factor-treated group

The T cell ratio is 48.7 +/-10.26, which is obviously higher than that of the IL-2 treatment group (paired T test p)<0.001), indicating that the chimeric T cell growth factor constructed in example 1 can maintain a higher proportion

T cells, i.e., T cells that inhibit activation, differentiate towards terminal stages.

Example 6 half-life assay for chimeric T-cell growth factors

Experimental materials: female C57BL/6 (southern university of medical animals center), native Human IL-2(Peprotech, #200-02), IL-21Human Uncoated ELISA Kit (Invitrogen, #88-8218-88), IL-2ELISA Kit (BioLegend, #431801) at 8-12 weeks.

Experimental methods

In order to detect the dynamic change and half-life of the chimeric T cell growth factor in mice after tail vein injection, the inventors previously established an ELISA method for in vitro detection of chimeric T cell growth factor, specifically as follows: since the chimeric T-cell growth factor constructed in example 1 contains Human IL-21, IL-21Human Unco-activated ELISA Kit (Invitrogen, #88-8218-88) was used to coat the Capture Antibody, the chimeric T-cell growth factor diluted in multiple ratios was added, incubated at 37 ℃ for 2 hours, washed, then the biotin-labeled detection Antibody was added, incubated at 37 ℃ for 1 hour, finally HRP-avidin was added and incubated at 37 ℃ for 30 minutes, TMB was developed in the dark for 20 minutes, 2M sulfuric acid was stopped for color development, and OD450nm was measured by microplate reader.

The half-life of native IL-2 and chimeric T-cytokines in mice was then determined by ELISA as set forth in example 1, as follows:

1)8-12 weeks C57BL/6 mice tail vein injection of 5ug/200ul PBS natural IL-2 or chimeric T cell growth factor;

2) collecting peripheral blood of the mice at four time points of 5 minutes, 30 minutes, 90 minutes and 240 minutes;

3) detecting the concentration of the chimeric T cell growth factor by using an established ELISA method;

4) the concentration of native IL-2 was measured using an IL-2ELISA kit (BioLegend, # 431801).

Step 2) was performed on orbital bleeds at various time points (T1-5 min, T2-30 min, T3-90 min, T4-240 min) after injection, and the concentrations of IL-2 and chimeric T cell growth factor in peripheral blood were measured using IL-2 and IL-21ELISA test kits, respectively, at four time points, c1, c2, c3 and c4, respectively.

The half-life hf is calculated as: hf ═ (t4-t1)/log2(c1/c 4).

The results are shown in FIG. 14: after injection into mice, native IL-2 was metabolized for a major portion after 30 minutes, with a calculated half-life hf of 9.8 minutes; the concentration of chimeric T cell growth factor also dropped sharply after 30 minutes of injection but the final concentration was significantly higher than native IL-2, with a calculated half-life of 38.6 minutes, significantly higher than IL-2, 3.94 times that of IL-2.

Example 7 use of chimeric T cell growth factors in CAR-T cell therapy

Experimental materials: Ficoll-Paque (GE Healthcare, #17144002), Total T cell magnetic bead sorting kit (STEMCELL, #17961), anti-CD 3/anti-CD 28 antibody (STEMCELL, #10971), native human IL-2(Peprotech, #200-02), chimeric T cell growth factor, GPC3 specific CAR-lentivirus (Nanjing Kingsler package, concentration: S.sub.>10⁸TU/ml), FITC-goat anti-mouse F (ab)2(#115-095-072, Jackson ImmunoResearch Laboratories) to detect CAR expression, APC-anti-human CCR7, PE-cy7 and-human CD45RO (BD Co.).

The detection method comprises the following steps:

2) total T cells were sorted by magnetic bead sorting (STEMCELL, # 17961);

4) adding 10nM of native human IL-2 or the purified chimeric T-cell growth factor expressed in example 1, respectively;

5) activating T cells by co-culturing the isolated and purified T cells with anti-CD 3/anti-CD 28 antibody (STEMCELL, #10971) for two days;

6) GPC 3-specific lentiviruses transduced activated T cells at a lentivirus concentration of 10MOI, 1000rpm, centrifuged at 25 ℃ for 90 min;

7) respectively adding 10nM natural human IL-2 or the chimeric T cell growth factor expressed and purified in example 1, continuing to culture for 10-12 days, and changing the culture solution every other day;

8) collecting cells, and detecting the proportion of CAR-T cells by using FITC-goat anti-mouse F (ab)2(#115-095-072, Jackson ImmunoResearch Laboratories);

9) further detection with CD45RO and CCR7

CAR-T cell ratio.

This experiment was repeated using peripheral blood from three healthy adults and the results are shown in FIG. 15, wherein (A) the schematic diagram of the preparation and detection scheme of CAR-T cells: PBMC were separated by density gradient centrifugation, total T cells were sorted by magnetic beads, and after activation of T cells with anti-CD3/CD28, the CAR-lentiviruses transduced T cells, 10nM of native IL-2 or chimeric T cell growth factor was added and incubation continued for 10-12 days. Flow assay CAR-T cell proportion and phenotype. (B) FCM measures CAR-T ratio, and FITC-goat anti-mouse F (ab)2 measures CAR expression on T cells. (C) The experiment was repeated with three donor T cells and statistical results showed that the proportion of CAR-T cells was not significantly different between the native IL-2 group and the chimeric T cell growth factor group. (D) FCM detects CAR-T memory phenotype profiles. The phylum is CAR + T cell, wherein CD45RO-CCR7+ is

The results show that the CAR-T cell station CD3+ T cell ratios of the native IL-2 treated group were: 32.2%, 42% and 18.9%; whereas the chimeric T cell growth factor treatment groups constructed in example 1 were 34%, 47% and 17.8%, with no significant difference in paired T-test P-0.3936 (B in fig. 15 and C in fig. 15); further analysis with CAR + T cell circle gate showed: in the IL-2-treated group of CAR-T cells,

CAR-T cells account for 28.4%, 36.8% and 24.3%, respectively; chimeric T cell growth factor treatment group

CAR-T cell ratios were 42.3%, 50.2% and 44.4%, paired T-test P ═ 0.018, significantly higher than IL-2 treated groups. Shows that the positive rate of CAR-T is not obviously different from that of the IL-2 treated group after the treatment of the chimeric T cell growth factor, but the positive rate of CAR-T is not obviously different from that of the IL-2 treated group

The proportion of CAR-T cells was significantly increased.

In addition, the chimeric T cell growth factor can also be applied to other cell lines, including CAR-T cells, TCR-T cells, TIL cells and NK cells.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention.

SEQUENCE LISTING

<110> southern medical university

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Leu Leu Leu Asp Leu Gln Met Ile Leu Asn Gly Ile Asn Asn Tyr Lys

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Asn Pro Lys Leu Thr Arg Met Leu Thr Phe Lys Phe Tyr Met Pro Lys

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Lys Ala Thr Glu Leu Lys His Leu Gln Cys Leu Glu Glu Glu Leu Lys

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Pro Leu Glu Glu Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Thr Ile Val Glu Phe Leu Asn Arg Trp Ile Thr Phe Cys Gln Ser Ile

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Ala Pro Thr Ser Ser Ser Thr Lys Lys Thr Gln Leu Gln Leu Glu His

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Ser Gly Ser Ser Val Leu Asn Leu Ala Gln Ser Lys Asn Phe His Leu

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Pro Thr Lys Ser Leu Glu Glu Asn Lys Ser Leu Lys Glu Gln Lys Lys

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Leu Asn Asp Leu Cys Phe Leu Lys Arg Leu Leu Gln Glu Ile Lys Thr

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Cys Trp Asn Lys Ile Leu Met Gly Thr Lys Glu His Gly Gly Gly Gly

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Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Ser Ser Gln Gly Gln

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atgcgggtgc tcgctgaact tcttggtctc ttgctgttct gtttcttggg agttaggtgt 60

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Met Arg Val Leu Ala Glu Leu Leu Gly Leu Leu Leu Phe Cys Phe Leu

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ggagccgcca cc 12

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aggaggacag ct 12

Claims

1. A chimeric T cell growth factor, wherein said chimeric T cell growth factor is an amino acid sequence of said IL-2 variant and amino acid sequences of IL-7, IL-21 connected in sequence by a linker sequence;

the amino acid sequence of the IL-2 variant is shown as SEQ ID NO. 7;

wherein the amino acid sequence of the IL-7 is: DCDIEGKDGKQYESVLMVSIDQLLDSMKEIGSNCLNNEFNFFKRHICDANKEGMFLFRAARKLRQFLKMNSTGDFDLHLLKVSEGTTILLNCTGQVKGRKPAALGEAQPTKSLEENKSLKEQKKLNDLCFLKRLLQEIKTCWNKILMGTKEH, respectively;

the amino acid sequence of the IL-21 is as follows: QGQDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETNCEWSAFSCFQKAQLKSANTGNNERIINVSIKKLKRKPPSTNAGRRQKHRLTCPSCDSYEKKPPKEFLERFKSLLQKMIHQHLSSRTHGSEDS are provided.

2. The chimeric T-cell growth factor according to claim 1, wherein the linker sequence is in order GGGGSGGGGSGGGS (SEQ ID NO:9) or GGGGSGGGGSGGGGSSS (SEQ ID NO: 11).

3. A nucleic acid molecule encoding the chimeric T-cell growth factor of any one of claims 1 to 2.

4. A vector comprising the nucleic acid molecule of claim 3.

5. A cell line comprising the vector of claim 4.

6. The cell line of claim 5, wherein the cell line is selected from the group consisting of CAR-T cells, TCR-T cells, and TIL cells.

7. A medicament for immunotherapy comprising a chimeric T-cell growth factor according to any one of claims 1 to 2, a nucleic acid molecule according to claim 3, a vector according to claim 4 or a cell line according to claim 5 or 6.