Immune engineering cell for expressing Ahelix functional region of GM-CSF and application

Abstract

The invention belongs to the technical field of biology, and particularly relates to an immune engineering cell for expressing an Ahelix functional region of a GM-CSF 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 an Ahelix functional domain fragment of a GM-CSF (Granulocyte-Macrophage-Colony-Stimulating Factor) protein and a chimeric antigen receptor. The invention also provides a CAR-T cell of the Ahelix functional domain fragment which also expresses the GM-CSF (granulocyte macrophage-macrophage colony-stimulating factor). The invention provides a novel chimeric antigen receptor structure modified by least functional region fragments only containing full-length GM-CSF, which not only retains the functions equivalent to those of traditional CAR and full-length GM-CSF CAR, but also obtains improved functions.

Description

[0001] Priority application This application claims the priority of the Chinese invention patent application [CN2023115952393] filed on November 24, 2023 "A CAR-T comprising the 1st truncation region of GM-CSF and its preparation method and application", the priority of the Chinese invention patent application [CN2023115817245] filed on November 24, 2023 "A CAR-T comprising the 2nd truncation region of GM-CSF and its preparation method and application", and the priority of the Chinese invention patent application [CN2023115954083] filed on November 24, 2023 "A CAR-T comprising the 5th truncation region of GM-CSF and its preparation method and application", and the three priority invention patent applications are incorporated by reference in their entirety. Technical Field

[0002] The invention belongs to the field of biotechnology, and in particular relates to immune engineering cells expressing the Ahelix functional region of GM-CSF 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 certain 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.

[0005] 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.

[0006] 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.

[0007] In summary, it is necessary to make improvements to the existing technology. Summary of the invention

[0008] The purpose of the present invention is to provide an immunoengineered cell expressing only the Ahelix functional region of GM-CSF and its application. The specific technical scheme is as follows.

[0009] A CAR-NK cell expressing the Ahelix functional region of GM-CSF, wherein the CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment 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 Ahelix functional region fragment is as shown in SEQ ID NO.1.

[0011] Furthermore, the novel chimeric antigen receptor structure is also connected to the EF1-α promoter sequence Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-NK cell is as shown in SEQ ID NO.3.

[0012] A CAR-T cell expressing the Ahelix functional region of GM-CSF, wherein the CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment 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 CD3ζ signal domain.

[0013] Preferably, the nucleotide sequence of the Ahelix functional region fragment is as shown in SEQ ID NO.1.

[0014] Furthermore, the novel chimeric antigen receptor structure is also connected to an EF1-α promoter sequence.

[0015] Preferably, the nucleotide sequence of the chimeric antigen receptor expressed by the CAR-T cell is as shown in SEQ ID NO.3.

[0016] The use of the above-mentioned CAR-NK cells in the preparation of drugs for treating solid tumors.

[0017] 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.

[0018] The use of the above-mentioned CAR-T cells in the preparation of drugs for treating solid tumors.

[0019] The above-mentioned CAR-NK cells or CAR-T cells can enhance the killing effect on tumor cells by expressing A helix of GM-CSF protein.

[0020] The antigen binding domain in the CAR provided by the present invention can target CD19, BCMA, HER2, Claudin18.2, Mesothelin, GPC3 or GD2, etc.

[0021] Furthermore, the tumor includes hematological tumor, liver cancer, breast cancer, lung cancer, esophageal cancer, gastric cancer, ovarian cancer, glioma, pancreatic cancer, sarcoma or glioblastoma.

[0022] 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.

[0023] Beneficial technical effects: On the one hand, the present invention constructs an immune engineering cell expressing a unique functional region of GM-CSF, which expresses a novel chimeric antigen receptor structure. Compared with a CAR containing a full-length GM-CSF protein, the novel chimeric antigen receptor structure of the present invention only contains one of the functional regions of the full-length GM-CSF protein, which reduces the sequence length of the overexpressed gene, but does not affect the expression and function of the CAR. On the contrary, since the truncated functional region does not contain a signal peptide, it can function intracellularly, resulting in an increase in the proliferation ability of CAR-T cells. The proliferation ability of the CAR-X1-T cells of the present invention is better than that of conventional CAR-T cells and CAR-GM-T cells. Obviously, the CAR-X1-T cells of the present invention have a higher yield and lower production cost when used for industrial production.

[0024] On the other hand, the experiment of the present invention confirmed the influence of reducing the number of functional regions on the results under the premise of containing different GM-CSF truncated functional regions. The results confirmed that the CAR proposed in the present invention, which only contains the only functional region Ah helix, as a modification containing only the minimum functional region fragment in the full-length GM-CSF, not only retains the functions equivalent to those of traditional CAR and CAR-GM, but also achieves improved functions. Specifically, the CAR-X1-T cells prepared by the present invention have better tumor killing ability than conventional CAR-T and GM-CSF full-length CAR-T as a whole; more cytokine secretion can be detected in the supernatant of co-culture of CAR-X1-NK cells and tumor cells. Therefore, the CAR-X1-NK cells or CAR-X1-T 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.

[0025] 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

[0026] 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.

[0027] Figure 1 This is a diagram of the construction of the novel CAR structure expression vector 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 positive rate detection of CAR-X1-T cells in one of the embodiments of the present invention; Figure 4 This is a graph showing the proliferation results of CAR-X1-T cells and control cells (CAR-X2-T and CAR-X5-T) in one of the embodiments of the present invention; Figure 5 In one embodiment of the present invention, the killing efficiency of CAR-X1-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-X1-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, the TNF-α and IFN-γ secreted by CAR-X1-NK cells are higher than those of conventional CAR-NK cells. DETAILED DESCRIPTION

[0028] 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.

[0029] Herein "and / or" includes any and all combinations of one or more of the associated listed items.

[0030] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0031] 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.

[0032] 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.

[0033] Definition of noun: The "novel chimeric antigen receptor structure" described in the present invention refers to a chimeric antigen receptor (CAR) and a functional region fragment (X1) of the GM-CSF protein connected to the chimeric antigen receptor via 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 co-stimulatory signal region, and a human CD3 ζ signal domain.

[0034] The "GM-CSF truncated 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.

[0035] 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.

[0036] GM-CSF second truncation region: includes the exon 1 fragment region and the Chelix fragment region of GM-CSF, represented by X2.

[0037] GM-CSF third truncation region: contains only the exon 2 fragment of GM-CSF, represented by X3.

[0038] GM-CSF 4th truncation region: includes the A helix, Chelix and exon 2 fragments of GM-CSF, represented by X4.

[0039] GM-CSF 5th truncation region: includes the A helix, exon 1 and Chelix fragments of GM-CSF, represented by X5.

[0040] On the one hand, the purpose of the present invention is to explore the improvement of CAR function after truncating the full-length GM-CSF protein.

[0041] Another invention is to explore the minimum number of truncated units that can maintain the function of CAR and have certain functional improvements under the premise of truncating the full-length GM-CSF protein.

[0042] The sequence information involved in the present invention is shown in Table 1.

[0043] Table 1 Example 1 Construction and viral packaging of a novel CAR expression vector.

[0044] 1.1 Construction of lentiviral expression vector for novel CAR vector The molecular cloning method was used to construct a lentiviral expression vector pWPXLD-CAR-P2A-X1 expressing a novel CAR. 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 (X1), such as Figure 1 Among them, X1 only contains the Ah helix region of GM-CSF and does not contain a signal peptide, which means that it can enhance the function of CAR in the cell.

[0045] 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.

[0046] Example 2 CAR positivity rate detection.

[0047] 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 a traditional second-generation CAR-T, CAR-GM is a CAR containing the full-length GM-CSF, and CAR-X1 is a CAR containing only the fragment X1. The results are shown in Figure 1. Figure 3 The results showed that the positive rate of CAR-X1-T reached 50%, which was not much different from that of traditional CAR and CAR-GM, indicating that the transfection efficiency of CAR containing only one functional region A helix was comparable to that of traditional CAR and CAR-GM.

[0048] Example 3 This example detects the proliferation ability of the CAR-X1-T cells synthesized in Example 1.

[0049] The same number of T cells were transduced with the lentivirus in Example 1 to obtain novel CAR-X1-T cells. After 3 days, the number of CAR-X1-T cells was detected using a cell counter and the expansion multiple was calculated. The results are as follows: Figure 4 As 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-X1-T was the highest. This shows that CAR containing only the unique functional region A helix not only retains the same transfection rate as traditional CAR and CAR-GM, but also the number of CAR-X1-T prepared by it proliferates the most in the same period of time.

[0050] In order to further explore the effect of the truncated functional region of GM-CSF on cell proliferation, this example also provides the following control experiment.

[0051] The same number of T cells were transduced using a similar method as in Example 1 to construct CAR-X2-T (control 1) and CAR-X5-T (control 2), respectively, wherein the CAR structures of CAR-X2-T, CAR-X5-T and CAR-X1-T were consistent. The X2 fragment contained two functional regions, the exon 1 fragment and the Chelix fragment of GM-CSF; the X5 fragment contained three functional regions, the A helix fragment, the exon 1 fragment and the Chelix fragment of GM-CSF, as shown in the following examples.

[0052] Table 2 After 3 days, the proliferation of CAR-X2-T cells and CAR-X5-T cells was detected using a cell counter and the expansion times were calculated. Figure 4 . The results showed that the proliferation ability of CAR-XT cells prepared by truncating the full-length GM-CSF and retaining the necessary functional regions was stronger than that of CAR-T cells prepared by full-length GM-CSF. However, based on the functional region of X5, X2 obtained by removing one functional region has a smaller effect on the proliferation of CAR-T cells. However, X1 obtained by removing another functional region on the basis of X2 has an effect on the proliferation of CAR-T cells comparable to that of the X2 fragment, and overall has a stronger cell proliferation ability than traditional CAR and CAR-GM. This shows that the CAR proposed in the present invention, which only contains the only functional region Ah helix, as a modification containing only the least functional region fragment in the full-length GM-CSF, not only retains functions comparable to those of traditional CAR and CAR-GM, but also achieves improved functions.

[0053] In CAR-X2-T and CAR-X5-T involved in this example, X2 and X5 represent the second truncated region and the fifth truncated region of GM-CSF, respectively, and the nucleotide sequences are as follows.

[0054] Table 3 Example 4 This example tests the verification that the CAR-X1-T cells synthesized in Example 1 kill tumor cells.

[0055] 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-X1-T cells was monitored.

[0056] like Figure 5 As shown in the figure, it can be seen that the killing ability of CAR-X1-T cells on tumor cells is higher than that of conventional CAR and CAR-GM from less than 5 hours. After 10 hours, this killing advantage is more significant. The killing effect of the killing curve for 40 hours showed that the killing effect of CAR-X1-T cells on tumor cells is significantly better than that of conventional CAR-T and CAR-GM-T.

[0057] The above experimental results indicate that the CAR proposed in the present invention, which contains only the unique functional region Ah helix, as a modification containing only the minimum functional region fragment in the full-length GM-CSF, kills tumor cells earlier than the control group and has a more obvious tumor killing effect.

[0058] Example 5 The lentivirus in Example 1 was further used to transduce NK cells to obtain novel CAR-X1-NK cells. SK-OV3 cells with positive CAR target were used as target cells, and CAR-X1-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-X1-NK cells was monitored.

[0059] like Figure 6 As shown, the killing efficacy of the killing curve was analyzed for 30 hours, and the results showed that the killing ability of CAR-X1-NK cells against tumor cells was on par with that of conventional CAR, proving that the transformation of NK cells by the CAR-X1 structure was successful.

[0060] Furthermore, the supernatants of CAR-X1-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-X1-NK cells were co-cultured with target cells ( Figure 7 ), the specific results are shown in the following table.

[0061] Table 4 CAR-X1-NK cell detection results The results showed that CAR-X1-NK cells secreted 9.4 times more TNF-α than conventional CAR-NK cells; CAR-NK cells in the control group hardly expressed TNF-α. CAR-X1-NK cells secreted 1.8 times more IFN-γ than conventional CAR-NK cells. This proved that X1, as a modified selection of the minimal functional region fragment of GM-CSF, played the role of an immunomodulatory factor, significantly increasing the expression of IFN-γ and TNF-α of CAR-NK cells compared with the control group. 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 the Ahelix functional region of GM-CSF, characterized in that: The CAR-NK cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment 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 Ahelix functional region fragment 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, characterized in that 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 the Ahelix functional region of GM-CSF, characterized in that: The CAR-T cell expresses a novel chimeric antigen receptor structure; the novel chimeric antigen receptor structure comprises an Ahelix functional region fragment of the GM-CSF protein and a chimeric antigen receptor; the Ahelix functional region fragment 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, characterized in that The nucleotide sequence of the Ahelix functional region fragment 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, characterized in that 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 drugs for treating solid tumors.

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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