Immune engineering cell for expressing GM-CSF truncated functional domain fragment and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to an immune engineering cell for expressing a GM-CSF truncated functional domain fragment and application of the immune engineering cell. The invention firstly provides a CAR-NK cell which expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a truncated functional domain fragment of a GM-CSF protein and a chimeric antigen receptor; the truncated functional domain fragment of the GM-CSF protein comprises an exon 1 functional domain fragment and a C helix functional domain fragment. The invention also provides a CAR-T cell for similarly expressing the truncated functional domain fragment of the GM-CSF. The novel immune engineering cell provided by the invention not only retains functions equivalent to those of the traditional CAR and full-length CAR-GM CAR, but also obtains improved functions.

Description

[0001] Priority application This application claims priority to the Chinese invention patent application [CN2023115817245] filed on November 24, 2023, "A CAR-T comprising the second truncation region of GM-CSF and preparation method and application", which is incorporated by reference in its entirety. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to immune engineering cells expressing GM-CSF truncated functional region fragments and applications thereof. Background Art

[0003] Chimeric antigen receptor (CAR) is an artificial receptor molecule manufactured by genetic engineering technology. It can give immune effector cells (such as T cells and NK cells) specificity for a target antigen epitope, thereby enhancing the lymphocyte recognition of antigen signals and activation functions. The first-generation CAR has only CD3 ξ (containing 3 ITAMs) and FcRγ (containing 2 ITAMs) signal transduction domains in the cell, which can only provide the first signal for T cell activation. Early clinical trials of the first-generation CAR showed that the first-generation CAR-T could not effectively maintain the proliferation and activation of T cells, and did not achieve satisfactory results, indicating that the first-generation CAR-T cells may lack sufficient activation signals to maintain T cell proliferation and effective anti-tumor effects. Based on the first-generation CAR-T, the second-generation CAR-T was developed. The second-generation CAR-T cells showed enhanced in vivo expansion and persistence, and their effects have been confirmed in clinical trials.

[0004] Since then, CAR-T related technologies have been continuously innovated, but of the 939 immune cell therapy trials launched since 1993, only about half are for solid tumors. However, 90% of the global cancer incidence is solid tumors. Unfortunately, compared with the good efficacy shown for blood tumors, most reported effects of using CAR-T cells to treat solid tumors have always been difficult to effectively break through at certain key stages. Therefore, this field still needs technological innovation. NK cells are important members of the innate immune system. They are mainly found in the blood and lymphoid organs. They do not require pre-sensitization and are not restricted by the major histocompatibility complex (MHC). They can quickly and directly kill target cells and have a broad spectrum of anti-tumor effects. NK cells kill target cells in a variety of ways. Once the balance between inhibitory signals and activation signals in NK cells is biased towards activation, NK cells can form synapses with target cells, thereby releasing effector granules to lyse target cells and produce effector cytokines. The anti-cancer effect of immune cells can be significantly improved by expressing chimeric antigen receptors (CARs) on the surface of NK cells. At present, the scientific community believes that CAR-NK cells have some significant advantages over CAR-T cells. However, the preparation of CAR-NK cells with tumor killing efficacy is somewhat difficult, mainly because NK cells have natural antiviral functions and their genetic modification is difficult.

[0005] The patent with publication number CN114934071A and invention name "A CAR vector expressing an immunomodulatory factor and its application" discloses a CAR-T cell expressing a chimeric antigen receptor and an immunomodulatory factor transduced by a virus, and the immunomodulatory factor is the full length of granulocyte-macrophage colony stimulating factor (GM-CSF). NK cell activation receptors include natural cytotoxicity receptors (NCRs), such as NKG2D, CD16 (FcgRIIIa), FasL and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), as well as co-stimulatory receptors, such as LFA-1, CD244 (2B4) and CD137 (41BB). The intracellular signal activation domains of CAR-T cells are mainly 41BB, CD28 and CD3ζ, so the traditional CAR molecule design that works on CAR-T may not necessarily play a good activation role in NK cells.

[0006] In summary, it is necessary to make improvements to the existing technology. Summary of the invention

[0007] The purpose of the present invention is to provide an immunoengineered cell expressing a truncated functional region fragment of GM-CSF and its application, and the specific technical scheme is as follows.

[0008] In one aspect, the present invention provides a novel CAR-NK cell.

[0009] A CAR-NK cell expressing a truncated functional region fragment of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the truncated functional region fragment of GM-CSF protein comprises an exon 1 functional region fragment and a Chelix functional region fragment; the truncated functional region fragment of GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

[0010] Preferably, the nucleotide sequence of the truncated functional region fragment of the GM-CSF protein is as shown in SEQ ID NO.1.

[0011] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0012] Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is as shown in SEQ ID NO.3.

[0013] Another aspect of the present invention provides a novel CAR-T cell.

[0014] A CAR-T cell expressing a truncated functional region fragment of GM-CSF, wherein the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the truncated functional region fragment of GM-CSF protein comprises an exon 1 functional region fragment and a Chelix functional region fragment; the truncated functional region fragment of GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

[0015] Preferably, the nucleotide sequence of the truncated functional region fragment of the GM-CSF protein is as shown in SEQ ID NO.1.

[0016] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0017] Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is as shown in SEQ ID NO.3.

[0018] The present invention also provides applications of the novel CAR-NK cells and novel CAR-T cells.

[0019] The use of the above-mentioned CAR-NK cells in the preparation of immunopotentiators for assisting tumor treatment.

[0020] The present invention proves that the content of immunomodulatory factors INF-γ and TNF-α contained in the supernatant of co-culture of the CAR-NK cells expressing the GM-CSF truncated functional region fragment and tumors is significantly higher than that of ordinary CAR-NK cells. Therefore, the CAR-NK cells expressing the GM-CSF truncated functional region fragment proposed in the present invention have great potential for being prepared as an immunopotentiator for auxiliary tumor treatment.

[0021] Furthermore, the present invention also provides the use of the above-mentioned CAR-NK cells in the preparation of drugs for treating solid tumors.

[0022] Compared with CAR-T cells, CAR-NK cells have some significant advantages, including: (1) less cytokine release syndrome and neurotoxicity. (2) CAR-NK cells can kill cancer cells through both CAR-dependent and CAR-independent pathways. In addition, NK cells can kill tumor cells through CD16-mediated ADCC. Therefore, CAR-NK cells have a broad spectrum of highly effective tumoricidal activity. (3) Allogeneic re-infusion therapy can be achieved.

[0023] The use of the above-mentioned CAR-T cells in the preparation of drugs for treating solid tumors.

[0024] The above-mentioned CAR-NK cells or CAR-T cells can enhance the killing effect on tumor cells by expressing a truncated functional region fragment (exon 1+ Chelix) of the GM-CSF protein.

[0025] The antigen binding domain in the CAR provided by the present invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3 or GD2, etc.

[0026] Furthermore, the tumor includes hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0027] The present invention may also include a method for preparing the above-mentioned CAR or the above-mentioned CAR-T cells or the above-mentioned CAR-NK cells.

[0028] Beneficial technical effects: The present invention constructs an immune engineering cell expressing a truncated functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared with the CAR containing the full-length GM-CSF protein, the novel chimeric antigen receptor structure of the present invention only adds a short peptide of about 50 amino acids to the CAR structure, which can significantly improve the in vitro infection efficiency and proliferation ability of CAR-T, and the anti-tumor cell ability is the same as that of CAR-T prepared by full-length GM-CSF, but reduces the sequence length of the overexpressed gene, thereby reducing some potential risks. Due to its higher proliferation ability, the CAR-X2-T cells of the present invention have higher yields and lower production costs when used for industrial production.

[0029] Specifically, the CAR-X2-T cells prepared by the present invention have better tumor killing ability than conventional CAR-T cells overall; more cytokine secretion can be detected in the supernatant of co-culture of CAR-X2-NK cells and tumor cells compared with conventional CAR-NK. Therefore, the CAR-X2-T cells or CAR-X2-NK cells of the present invention have the potential to become adjuvant therapy products and play a role in the preparation of anti-tumor therapeutic drugs.

[0030] Finally, due to the differences between NK cell modification and T cell modification, the CAR structure expressed and functional on CAR-T cells does not necessarily have the same effect on CAR-NK cells, which requires experimental verification. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings required for use in the embodiments or the prior art descriptions are briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, each element or part is not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without paying creative labor.

[0032] Figure 1 This is a schematic diagram of the structure of the novel CAR of the present invention; Figure 2 It is a schematic diagram of the structure of the functional region of the full-length CM-CSF protein; Figure 3 This is a result diagram of the CAR positivity detection of CAR-X2-T cells in one of the embodiments of the present invention; Figure 4 This is a diagram showing the proliferation results of CAR-X2-T cells in one of the embodiments of the present invention; Figure 5In one embodiment of the present invention, the killing efficiency of CAR-X2-T cells on tumor cells is higher than that of the control group CAR-T cells; Figure 6 In one embodiment of the present invention, the killing ability of CAR-X2-NK cells on tumor cells is higher than that of conventional CAR-T cells; Figure 7 In one of the embodiments of the present invention, CAR-X2-NK cells secrete higher levels of TNF-α and IFN-γ than conventional CAR-NK cells. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0034] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0035] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0036] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0037] In this specification, some embodiments may be disclosed in a format of being in a range. It should be understood that this description of "being in a range" is only for convenience and brevity, and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and independent numerical values ​​within this range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as individual numbers within this range, such as 1, 2, 3, 4, 5 and 6. Regardless of the breadth of the range, the above rules apply.

[0038] Name explanation: The "novel chimeric antigen receptor structure" described in the present invention refers to a chimeric antigen receptor (CAR) and a truncated functional region fragment (X2) of the GM-CSF protein connected to the chimeric antigen receptor by P2A. The chimeric antigen receptor includes a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB costimulatory signal region, and a human CD3ζ signal domain.

[0039] The "GM-CSF truncated functional region" of the present invention refers to a truncated region obtained by modifying the functional region fragments of the full-length GM-CSF protein by truncating or deleting the functional region fragments; wherein the functional region fragments include signal peptid fragments, A helix fragments, exon 1 fragments, Chelix fragments and exon 2 fragments. The full-length structure of the GM-CSF protein is as follows: Figure 2 shown.

[0040] Based on the above transformation methods, the full-length GM-CSF protein was prepared into 5 short peptides containing different truncated fragments, including: GM-CSF first truncation region: contains only the A helix fragment of GM-CSF, indicated by X1.

[0041] GM-CSF second truncation region: includes the exon 1 fragment region and the Chelix fragment region of GM-CSF, represented by X2.

[0042] GM-CSF third truncation region: contains only the exon 2 fragment of GM-CSF, represented by X3.

[0043] GM-CSF 4th truncation region: includes the A helix, Chelix and exon 2 fragments of GM-CSF, represented by X4.

[0044] GM-CSF 5th truncation region: includes the A helix, exon 1 and Chelix fragments of GM-CSF, represented by X5.

[0045] The purpose of the present invention is to explore the influence of the second truncation region of GM-CSF on CAR and the functional improvement thereof.

[0046] The sequence information involved in the present invention is as follows.

[0047] Table 1 Example 1 Construction of novel CAR expression vector and viral packaging 1.1 Construction of lentiviral expression vector for novel CAR vector The lentiviral expression vector pWPXLD-CAR-P2A-X2 expressing the novel CAR was constructed by molecular cloning. The gene fragment of the expression vector includes CD8 signal peptide, SCFV, hinge region (hinge), human CD8 transmembrane region (TM), human 4-1BB costimulatory signal region, human CD3 ζ signal domain, P2A, fragment (X2), such as Figure 1 Among them, X2 only contains the exon 1 and Chelix regions of GM-CSF and does not contain a signal peptide, which means that it can enhance the function of CAR in the cell.

[0048] 1.2 Packaging of novel CAR lentivirus Take 293T cells with good growth status, digest the cells with trypsin, and then add 4×10 6 The cells were passaged at a density of 100 cells / dish. When the cell confluence reached 80%, the CAR vector was used as the core plasmid, and psPAX2 and pMD2.G were used as auxiliary plasmids, using calcium phosphate transfection technology for transfection. Eight hours after transfection, the culture medium containing calcium phosphate precipitation was discarded. After 48 and 72 hours of transfection, the cell culture supernatant was collected as the virus stock solution, centrifuged at 2000 rpm for 10 minutes, the cell precipitate was removed, and a 0.22μm disposable syringe filter (PES membrane) was used to remove cell debris.

[0049] Example 2 CAR positivity rate detection.

[0050] The lentivirus in Example 1 was used to transduce T cells to obtain novel CAR-T cells. The target protein expressing the His tag was used as the primary antibody, and the His antibody coupled to the fluorescent APC was used as the secondary antibody to detect the CAR positive rate. Conventional CAR is the traditional second-generation CAR-T, and CAR-GM is a CAR containing the full-length GM. The results are shown in Figure 3 The results showed that CAR-X2-T had the highest positive rate, reaching 65.35%; the positive rates of the traditional second-generation CAR-T and CAR-T containing full-length GM as controls were about 55%.

[0051] Example 3 This example detects the proliferation ability of the CAR-X2-T cells synthesized in Example 1.

[0052] The same number of T cells were transduced with the lentivirus in Example 1 to obtain novel CAR-X2-T cells. After 3 days, the number of CAR-X2-T cells was detected using a cell counter and the expansion multiple was calculated. The results are as follows: Figure 4As shown. The results showed that the number of cell proliferation of traditional CAR-T was the lowest; the number of cell proliferation of CAR-GM-T was more than that of traditional CAR-T; and the number of cell proliferation of CAR-X2-T was the highest. This shows that CAR that only retains part of the functional region of GM-CSF not only retains the same transfection rate as traditional CAR and CAR-GM, but also the CAR-X2-T prepared by it has the largest number of proliferation in the same time and reduces the sequence length of the overexpressed gene.

[0053] Example 4 This example tests the verification that the CAR-X2-T cells synthesized in Example 1 kill tumor cells.

[0054] CAR target-positive SK-OV3 cells (human ovarian cancer cells) were used as target cells and CAR-T cells as effector cells. The two types of cells were co-cultured at an effector-target ratio of 5:1, and the killing of SK-OV3 cells by CAR-X2-T cells was monitored.

[0055] like Figure 5 As shown, the killing ability of CAR-X2-T cells on tumor cells is higher than that of conventional CAR and is on par with that of CAR-GM.

[0056] The above experimental results show that the CAR prepared by the specific truncated functional region obtained after truncating the full-length GM-CSF protein has a tumor cell-killing ability similar to that of the CAR prepared by the full-length GM-CSF protein, but has a higher positive expression rate and higher cell proliferation ability than the CAR prepared by the full-length GM-CSF protein.

[0057] Example 5 The lentivirus in Example 1 was further used to transduce NK cells to obtain novel CAR-X2-NK cells. SK-OV3 cells with positive CAR target were used as target cells, and CAR-X2-NK cells were used as effector cells. The two cells were co-cultured at an effector-target ratio of 5:1, and the killing of CAR-X2-NK cells was monitored.

[0058] The killing effect of the killing curve for 30 hours was analyzed, and the results showed that the killing ability of CAR-X2-NK cells on tumor cells was higher than that of conventional CAR-NK cells, proving that the transformation of NK cells by CAR-X2 structure was successful ( Figure 6 ).

[0059] Furthermore, the supernatants of CAR-X2-NK cells and tumor cells were collected after 24 hours of co-culture, and the IFN-γ and TNF-α in the supernatants were analyzed. The results showed that the IFN-γ and TNF-α in the supernatants increased significantly after CAR-X2-NK cells were co-cultured with target cells ( Figure 7 ), the specific results are shown in the following table.

[0060] Table 2 CAR-NK cell detection results The results showed that CAR-X2-NK cells significantly increased the expression of IFN-γ and TNF-α in the co-culture supernatant compared with the control group, while CAR-NK cells in the control group hardly expressed TNF-α.

[0061] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0062] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation modes, which are merely illustrative rather than restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are within the protection of the present invention.

Claims

1. A CAR-NK cell expressing a truncated functional region fragment of GM-CSF, characterized in that: The CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a truncated functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the truncated functional region fragment of the GM-CSF protein comprises an exon 1 functional region fragment and a Chelix functional region fragment; the truncated functional region fragment of the GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

2. The CAR-NK cell according to claim 1, wherein The nucleotide sequence of the truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.

1.

3. The CAR-NK cell according to claim 1, wherein The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

4. The CAR-NK cell according to claim 1, wherein The nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is shown in SEQ ID NO.

3.

5. A CAR-T cell expressing a truncated functional region fragment of GM-CSF, characterized in that: The CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises a truncated functional region fragment of GM-CSF protein and a chimeric antigen receptor; the truncated functional region fragment of GM-CSF protein comprises an exon 1 functional region fragment and a Chelix functional region fragment; the truncated functional region fragment of GM-CSF protein is connected to the chimeric antigen receptor via a P2A sequence; the chimeric antigen receptor comprises a CD8 signal peptide, an antigen recognition region, a hinge region, a human CD8 transmembrane region, a human 4-1BB co-stimulatory signal region and a human CD3 ζ signal domain.

6. The CAR-T cell according to claim 5, wherein The nucleotide sequence of the truncated functional region fragment of the GM-CSF protein is shown in SEQ ID NO.

1.

7. The CAR-T cell according to claim 5, characterized in that The novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence.

8. The CAR-T cell according to claim 5, wherein The nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is shown in SEQ ID NO.

3.

9. Use of the CAR-NK cells according to any one of claims 1 to 4 in the preparation of an immunopotentiator for assisting tumor therapy.

10. Use of the CAR-T cell according to any one of claims 5 to 8 in the preparation of a drug for treating solid tumors.

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