Chimeric antigen receptor, recombinant vector, recombinant cell, and preparation method and application thereof

Abstract

The present invention relates to a chimeric antigen receptor, a recombinant vector, a recombinant cell, and a preparation method and application thereof. The recombinant costimulatory molecule of the CAR includes a first module and a second module; the first module is a first N-terminal segment, and the second module is a connected second intermediate segment and a second C-terminal segment, or a connected second N-terminal segment, a second intermediate segment and a second C-terminal segment; or, the first module is a connected first N-terminal segment and a first intermediate segment, or a connected first N-terminal segment, a first intermediate segment and a first C-terminal segment, and the second module is a second C-terminal segment, a connected second intermediate segment and a second C-terminal segment, or a connected second N-terminal segment, a second intermediate segment and a second C-terminal segment; the two types of costimulatory domains are split into three parts from the N-terminus to the C-terminus to obtain the above-mentioned segments. The above-mentioned CAR can expand the functional space of CAR-T cell downstream signals.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a chimeric antigen receptor, a recombinant vector, a recombinant cell, and a preparation method and application thereof. Background Art

[0002] Chimeric antigen receptor T-cell immunotherapy (CAR-T cell therapy) is an emerging cancer treatment method in recent years. Unlike traditional physical and chemical methods such as surgical resection, radiotherapy, and chemotherapy, cellular immunotherapy harnesses the body's own immune system to fight cancer. The key concept behind cellular immunotherapy is to exploit the ability of T cells to kill tumor cells. First, T cells are extracted and isolated from the patient's body. These cells are then modified through gene editing and other methods to enhance their tumor-killing ability. These modified T cells are then expanded ex vivo and reinfused back into the patient, thereby enhancing the body's ability to fight the tumor.

[0003] Overall, the historical development of chimeric antigen receptors (CARs) can be roughly divided into three generations, primarily characterized by structural differences in the intracellular segment of the transmembrane receptor. Initially, the intracellular segment of first-generation CARs contained only the intracellular segment of the CD3ζ chain. Although this only provided the first signal to cells, first-generation CARs were still proven effective. However, their ability to activate T cells was insufficient, and T cells quickly entered an unresponsive state. Consequently, first-generation CAR-T cells had limited cytokine secretion and proliferation, and a short survival time in vivo. Subsequently, second- and third-generation CARs were based on a dual-signal model for T cell activation, incorporating the intracellular segments of one or two co-stimulatory molecules (such as CD28 and 4-1BB) into the CD3ζ chain. The introduction of this second signal has improved the efficacy of CAR-T, with extensive clinical evidence demonstrating that second-generation CARs containing CD28 and 4-1BB exhibit greater proliferation and persistence than first-generation CARs. The second-generation CARs reported so far have all been able to improve the performance of the first-generation CARs to a certain extent, increasing cytokine secretion, promoting cell proliferation, and reducing activation-induced apoptosis. The five CAR-T cell therapies currently approved by the U.S. Food and Drug Administration (FDA) (Kymriah, Yescarta, Tecartus, Breyanzi, and Abecma) all use the molecular design of the second-generation CAR.

[0004] There is some controversy surrounding the efficacy of third-generation CARs. Some studies suggest that third-generation CARs are more effective than second-generation CARs. For example, a third-generation CAR derived from a second-generation CAR with the addition of an intracellular segment of OX40 enhances its ability to secrete the cytokines interleukin-2 and tumor necrosis factor (TNF-α). Similarly, a third-generation CAR with the addition of 4-1BB also enhances its ability to lyse tumor cells and secrete cytokines. However, there are also contradictory findings. For example, it has been reported that the TCR signaling of 28-BB-ζ is weaker than that of 28-ζ.

[0005] Existing CAR-T therapies have achieved significant success in treating cancer, particularly lymphoma. In 2018, the FDA approved two CD19-targeting CAR-Ts, sparking a surge in clinical trials. However, CAR-T therapy still faces challenges in terms of efficacy and safety. For one thing, the short lifespan of CAR-T cells in cancer patients limits their efficacy. This is because after a period of stimulation, T cells enter a state of exhaustion, characterized by overexpression of inhibitory receptors such as PD-1, Lag-3, and Tim-3. These cells are unable to proliferate and kill tumor cells, ultimately leading to apoptosis. Inhibitory factors in the tumor microenvironment of solid tumors further hinder T cell proliferation and survival. Furthermore, the massive activation of CAR-T cells in patients leads to the release of numerous inflammatory cytokines (such as TNF-α, IL-1, 2, 6, 8, and IFN-γ), which can also cause damage to the body. This phenomenon is called cytokine release syndrome. When a large number of CAR-T cells are activated, cytokines are released violently, causing patients to experience side effects such as high fever, low blood pressure, tissue edema, and even death in severe cases.

[0006] The downstream signaling of CAR activation and the ultimate function and fate of T cells are closely related to the structural composition of the CAR intracellular domain. A study has compared the performance of two classic second-generation CARs: the CD19-targeting CD28-ζCAR (28-ζ) and the 4-1BB-ζCAR (BB-ζ). 28-ζ activates the PI3K-Akt signaling pathway, shifting T cells into a rapid glucose metabolism-driven energy-supply mode and favoring differentiation into effector memory cells. 28-ζCAR-T cells secrete more cytokines than BB-ζ, making them more suitable for rapid and potent tumor suppression. However, this rapid activation appears to make T cells less durable, leading to exhaustion, loss of proliferation, and even death. BB-ζ, on the other hand, utilizes fatty acid metabolism, a metabolic pathway more similar to naive and memory T cells, for energy. This slow but efficient energy supply favors central memory T cells, allowing BB-ζ T cells to proliferate more sustainably. Consequently, BB-ζ T cells have been shown to have better properties for preventing tumor recurrence. However, although CARs using 4-1BB as co-stimulatory signals have advantages in terms of proliferation and differentiation, the presence of the 4-1BB co-stimulatory domain will increase T cell apoptosis due to the adverse effects of trophic signals. In addition, due to factors such as cytotoxicity, tumor cells will lose antigens and escape CAR-T cell killing. Compared with 28-ζT, BB-ζT cells are more sensitive to target cell antigen loss and have weaker resistance to tumor escape caused by antigen loss. The third-generation CAR designed to integrate the advantages of different co-stimulatory signals also has certain limitations. The enhanced function of the third-generation CAR lowers its activation threshold, thereby increasing the probability and risk of side effects.

[0007] The downstream signals and functions of CAR-T obtained by existing CAR molecular design are confined to a very limited space, which is not conducive to the application of CAR-T. Summary of the Invention

[0008] Based on this, it is necessary to provide a new type of chimeric antigen receptor that can expand the functional space of CAR-T cell downstream signals.

[0009] In addition, it is also necessary to provide a recombinant vector, a recombinant cell, and a preparation method and application thereof.

[0010] A chimeric antigen receptor comprising an intracellular domain, the intracellular domain comprising a recombinant costimulatory molecule, the recombinant costimulatory molecule comprising a first module and a second module connected from the N-terminus to the C-terminus;

[0011] The first module is the first N-terminal segment, and the second module is the connected second middle segment and second C-terminal segment, or the connected second N-terminal segment, second middle segment and second C-terminal segment;

[0012] Alternatively, the first module is a connected first N-terminal segment and a first middle segment, or a connected first N-terminal segment, a first middle segment and a first C-terminal segment, and the second module is a connected second C-terminal segment, a connected second middle segment and a second C-terminal segment, or a connected second N-terminal segment, a second middle segment and a second C-terminal segment;

[0013] Among them, the first N-terminal segment, the first middle segment and the first C-terminal segment are obtained by splitting the first costimulatory domain into three parts from the N-terminus to the C-terminus in sequence, the second N-terminal segment, the second middle segment and the second C-terminal segment are obtained by splitting the second costimulatory domain into three parts from the N-terminus to the C-terminus in sequence, and the first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules.

[0014] This application proposes for the first time to split natural costimulatory molecules according to functional motifs and then perform reasonable recombination to obtain recombinant costimulatory molecules. Since the signal activity of the original costimulatory domain caused by the recombination is either enhanced or weakened, the resulting series of costimulatory domains with varying stimulation strengths greatly enrich the original signal activation range, which is beneficial to the application of CAR-T. Experimental verification shows that the chimeric antigen receptor of the present application has the potential to activate cells and has the ability to specifically kill target cells. Different costimulatory domains have complex and diverse effects on the relative ability of T cells to secrete cytokines. Among them, CAR-T cells with recombinant costimulatory molecules 28 (NM) -BB (MC) have more excellent specific ability to kill target cells, have the effect of reducing nourishing signals and increasing activation levels, so that T cells have a mild and lasting anti-tumor effect, and reduce the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy.

[0015] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule of the immunoglobulin superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily.

[0016] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule of the immunoglobulin superfamily.

[0017] In one embodiment, the first costimulatory domain is the intracellular segment of CD28 or ICOS, and the second costimulatory domain is the intracellular segment of 4-1BB or OX40.

[0018] In one embodiment, the first costimulatory domain is the intracellular segment of 4-1BB or OX40, and the second costimulatory domain is the intracellular segment of CD28 or ICOS.

[0019] In one embodiment, the CD28 intracellular segment is sequentially split from N-terminus to C-terminus into the CD28-N-terminal segment, the CD28-intermediate segment, and the CD28-C-terminal segment; the ICOS intracellular segment is sequentially split from N-terminus to C-terminus into the ICOS-N-terminal segment, the ICOS-intermediate segment, and the ICOS-C-terminal segment; the 4-1BB intracellular segment is sequentially split from N-terminus to C-terminus into the 4-1BB-N-terminal segment, the 4-1BB-intermediate segment, and the 4-1BB-C-terminal segment; and the OX40 intracellular segment is sequentially split from N-terminus to C-terminus into the OX40-N-terminal segment, the OX40-intermediate segment, and the OX40-C-terminal segment;

[0020] The recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a CD28-C-terminal segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment and a 4-1BB-C-terminal segment connected in sequence from the N-terminus to the C-terminus;

[0021] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a 4-1BB-C-terminal segment, a CD28-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0022] Alternatively, the recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a CD28-C-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0023] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a 4-1BB-C-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0024] Alternatively, the recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0025] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a CD28-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0026] Alternatively, the recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0027] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0028] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0029] Alternatively, the recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0030] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0031] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0032] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, a CD28-middle segment, a CD28-C-terminal segment, an OX40-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0033] Alternatively, the recombinant costimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment, which are sequentially connected from N-terminus to C-terminus;

[0034] Alternatively, the recombinant costimulatory molecule includes CD28-N-terminal segment, CD28-middle segment, CD28-C-terminal segment, OX40-middle segment, and OX40-C-terminal segment connected in sequence from N-terminus to C-terminus;

[0035] Alternatively, the recombinant costimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, CD28-middle segment, and CD28-C-terminal segment, which are sequentially connected from N-terminus to C-terminus;

[0036] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, a CD28-middle segment, an OX40-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0037] Alternatively, the recombinant costimulatory molecule includes an OX40-N-terminal segment, an OX40-middle segment, a CD28-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0038] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, a CD28-middle segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0039] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an OX40-middle segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0040] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0041] Alternatively, the recombinant costimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0042] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0043] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an OX40-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0044] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, an ICOS-C-terminal segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0045] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a 4-1BB-C-terminal segment, an ICOS-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0046] Alternatively, the recombinant costimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, an ICOS-C-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0047] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a 4-1BB-C-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0048] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment, connected sequentially from the N-terminus to the C-terminus;

[0049] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, an ICOS-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0050] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0051] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, an ICOS-middle segment, and an ICOS-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0052] Alternatively, the recombinant costimulatory molecule comprises a 4-1BB-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0053] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0054] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0055] Alternatively, the recombinant costimulatory molecule comprises a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0056] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, an ICOS-C-terminal segment, an OX40-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0057] Alternatively, the recombinant costimulatory molecule comprises OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment, which are sequentially connected from N-terminus to C-terminus;

[0058] Alternatively, the recombinant costimulatory molecule comprises ICOS-N-terminal segment, ICOS-middle segment, ICOS-C-terminal segment, OX40-middle segment, and OX40-C-terminal segment, which are sequentially connected from N-terminus to C-terminus;

[0059] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an OX40-middle segment, an OX40-C-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0060] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, an OX40-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0061] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an OX40-middle segment, an ICOS-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0062] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0063] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an OX40-middle segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0064] Alternatively, the recombinant costimulatory molecule comprises an OX40-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0065] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an ICOS-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0066] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0067] Alternatively, the recombinant co-stimulatory molecule includes an OX40-N-terminal segment, an OX40-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus.

[0068] In one embodiment, the amino acid sequences of the CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment are shown as SEQ ID No. 1 to SEQ ID No. 3;

[0069] and / or the amino acid sequences of ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment are shown as SEQ ID No. 4 to SEQ ID No. 6;

[0070] and / or the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB8-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9;

[0071] And / or, the amino acid sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID No. 10 to SEQ ID No. 12.

[0072] In one embodiment, the nucleotide sequences of the CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment are shown as SEQ ID No. 13 to SEQ ID No. 15, respectively;

[0073] and / or the nucleotide sequences of ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment are shown as SEQ ID No. 16 to SEQ ID No. 18, respectively;

[0074] and / or, the nucleotide sequences of the 4-1BB-N-terminal segment, the 4-1BB8-middle segment, and the 4-1BB-C-terminal segment are shown as SEQ ID No. 19 to SEQ ID No. 21, respectively;

[0075] And / or, the nucleotide sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown as SEQ ID No. 22 to SEQ ID No. 24, respectively.

[0076] In one embodiment, the intracellular domain further includes an intracellular signaling domain connected to the recombinant co-stimulatory molecule, and the chimeric antigen receptor further includes an extracellular domain, a hinge and a transmembrane region.

[0077] In one embodiment, the intracellular signaling domain is the intracellular segment of the CD3ζ chain, the extracellular domain is a single-chain antibody targeting CD19 or mesothelin, and the hinge and transmembrane regions are both derived from CD8α.

[0078] A chimeric antigen receptor comprising an intracellular domain, wherein the intracellular domain contains a recombinant costimulatory molecule, wherein the recombinant costimulatory molecule comprises a first module, a second module, and a third module connected from the N-terminus to the C-terminus;

[0079] The first module is selected from a segment between the first N-terminal segment and the second N-terminal segment, the second module is selected from a segment between the first middle segment and the second middle segment, and the third module is selected from a segment between the first C-terminal segment and the second C-terminal segment. The first N-terminal segment, the first middle segment and the first C-terminal segment are obtained by splitting the first costimulatory domain into three parts from the N-terminus to the C-terminus, the second N-terminal segment, the second middle segment and the second C-terminal segment are obtained by splitting the second costimulatory domain into three parts from the N-terminus to the C-terminus, the first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules, the second module and the first module are respectively derived from different costimulatory molecules, and the second module and the third module are respectively derived from different costimulatory molecules.

[0080] In one embodiment, the first module and the second module are derived from the same co-stimulatory molecule.

[0081] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule in the immunoglobulin superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule in the tumor necrosis factor receptor superfamily.

[0082] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule in the tumor necrosis factor receptor superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule in the immunoglobulin superfamily.

[0083] In one embodiment, the first costimulatory domain is the intracellular segment of CD28 or ICOS, and the second costimulatory domain is the intracellular segment of 4-1BB or OX40.

[0084] In one embodiment, the first costimulatory domain is the intracellular segment of 4-1BB or OX40, and the second costimulatory domain is the intracellular segment of CD28 or ICOS.

[0085] In one embodiment, the CD28 intracellular segment is sequentially split from N-terminus to C-terminus into the CD28-N-terminal segment, the CD28-intermediate segment, and the CD28-C-terminal segment; the ICOS intracellular segment is sequentially split from N-terminus to C-terminus into the ICOS-N-terminal segment, the ICOS-intermediate segment, and the ICOS-C-terminal segment; the 4-1BB intracellular segment is sequentially split from N-terminus to C-terminus into the 4-1BB-N-terminal segment, the 4-1BB-intermediate segment, and the 4-1BB-C-terminal segment; and the OX40 intracellular segment is sequentially split from N-terminus to C-terminus into the OX40-N-terminal segment, the OX40-intermediate segment, and the OX40-C-terminal segment;

[0086] The recombinant costimulatory molecule includes a CD28-N-terminal segment, a 4-1BB-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus;

[0087] Alternatively, the recombinant costimulatory molecule includes a 4-1BB-N-terminal segment, a CD28-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0088] Alternatively, the recombinant costimulatory molecule comprises a CD28-N-terminal segment, an OX40-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0089] Alternatively, the recombinant costimulatory molecule includes an OX40-N-terminal segment, a CD28-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0090] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, a 4-1BB-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0091] Alternatively, the recombinant costimulatory molecule comprises a 4-1BB-N-terminal segment, an ICOS-middle segment, and a 4-1BB-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0092] Alternatively, the recombinant costimulatory molecule comprises an ICOS-N-terminal segment, an OX40-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus;

[0093] Alternatively, the recombinant co-stimulatory molecule includes an OX40-N-terminal segment, an ICOS-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus.

[0094] In one embodiment, the amino acid sequences of the CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment are shown as SEQ ID No. 1 to SEQ ID No. 3;

[0095] and / or the amino acid sequences of ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment are shown as SEQ ID No. 4 to SEQ ID No. 6;

[0096] and / or the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB8-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9;

[0097] And / or, the amino acid sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID No. 10 to SEQ ID No. 12.

[0098] In one embodiment, the nucleotide sequences of the CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment are shown as SEQ ID No. 13 to SEQ ID No. 15, respectively;

[0099] and / or the nucleotide sequences of ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment are shown as SEQ ID No. 16 to SEQ ID No. 18, respectively;

[0100] and / or, the nucleotide sequences of the 4-1BB-N-terminal segment, the 4-1BB8-middle segment, and the 4-1BB-C-terminal segment are shown as SEQ ID No. 19 to SEQ ID No. 21, respectively;

[0101] And / or, the nucleotide sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown as SEQ ID No. 22 to SEQ ID No. 24, respectively.

[0102] A recombinant vector contains the coding sequence of the chimeric antigen receptor.

[0103] The method for preparing the above-mentioned recombinant vector comprises the following steps:

[0104] The coding sequence of the chimeric antigen receptor is cloned into a genetic engineering vector to obtain a recombinant vector.

[0105] In one embodiment, the chimeric antigen receptor includes an intracellular domain, and the intracellular domain contains a recombinant costimulatory molecule. Before the step of cloning the coding sequence of the chimeric antigen receptor into a genetic engineering vector, the step of constructing the recombinant costimulatory molecule is also included:

[0106] The first costimulatory domain is sequentially split into three parts from N-terminus to C-terminus to obtain the first N-terminal segment, the first middle segment and the first C-terminal segment; the second costimulatory domain is sequentially split into three parts from N-terminus to C-terminus to obtain the second N-terminal segment, the second middle segment and the second C-terminal segment, wherein the first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules;

[0107] The split parts are assembled to obtain the recombinant costimulatory molecule:

[0108] Connecting the first N-terminal segment, the second middle segment and the second C-terminal segment from N-terminus to C-terminus to obtain the recombinant costimulatory molecule;

[0109] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the second N-terminal segment, the second intermediate segment and the second C-terminal segment from N-terminus to C-terminus;

[0110] Alternatively, the first N-terminal segment, the first middle segment, and the second C-terminal segment are connected from N-terminus to C-terminus to obtain the recombinant costimulatory molecule;

[0111] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, the second middle segment and the second C-terminal segment from N-terminus to C-terminus;

[0112] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, the second N-terminal segment, the second middle segment and the second C-terminal segment from N-terminus to C-terminus;

[0113] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, the first C-terminal segment, and the second C-terminal segment from N-terminus to C-terminus;

[0114] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, the first C-terminal segment, the second middle segment and the second C-terminal segment from N-terminus to C-terminus;

[0115] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, the first C-terminal segment, the second N-terminal segment, the second middle segment and the second C-terminal segment from N-terminus to C-terminus;

[0116] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the second intermediate segment and the first C-terminal segment from N-terminus to C-terminus;

[0117] Alternatively, the second N-terminal segment, the first middle segment and the second C-terminal segment are connected from N-terminus to C-terminus to obtain the recombinant costimulatory molecule.

[0118] In one embodiment, the method further comprises the step of screening the signal characteristics of the chimeric antigen receptor in the recombinant vector:

[0119] A plasmid containing the pNF-κB-mCherry sequence and a plasmid containing the piggyBac transposase sequence were transformed into a cell line containing the pNFAT-EGFP reporter system to obtain a dual reporter signal cell line;

[0120] The recombinant vector is introduced into the dual-reporter signal cell line, target cell antigen stimulation is performed, and then the fluorescence intensity of the dual-reporter signal cell line is detected to obtain the NFAT and NF-κB signal kinetic characteristics, and screen out recombinant vectors with cell activation potential.

[0121] A recombinant cell capable of expressing the chimeric antigen receptor or containing the recombinant vector.

[0122] In one embodiment, the recombinant cell is a T cell capable of expressing the chimeric antigen receptor or a T cell introduced with the recombinant vector.

[0123] Use of the chimeric antigen receptor, the recombinant vector or the recombinant cell in the preparation and detection of anti-tumor drugs.

[0124] This application proposes for the first time that natural costimulatory molecules are split according to functional motifs and then rationally recombined to obtain recombinant costimulatory molecules. This application also proposes for the first time the sequences of four natural costimulatory molecules split into three fragments, N, M, and C, and the sequences of 48 novel costimulatory molecules obtained by recombining (excluding 8 directly connected in series). The design scheme of the present application for splitting natural costimulatory molecules according to functional motifs and then rationally recombining them can also be applied to more alternative molecules. This design scheme can generate a large number of new CAR molecular designs.

[0125] This application uses the NFAT & NF-κB dual reporter signal cell line to quickly screen the activation signal characteristics of new CARs: The Jurkat dual reporter signal cell line with pNF-κB-mCherry and pNFAT-EGFP can quickly and high-throughput complete the activation signal characteristic identification of new CAR molecular designs, which is a powerful experimental means to quickly screen CARs.

[0126] Among the recombinant costimulatory molecules obtained in this application, the CAR-T cells of the costimulatory molecule 28(NM)-BB(MC) have better specific ability to kill target cells. Experiments have also shown that the costimulatory domain has the effect of reducing trophic signals and increasing activation levels, giving T cells a mild and lasting anti-tumor effect, and reducing the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0127] Figure 1 This is the technical flow chart of this application;

[0128] Figure 2 The amino acid sequences and motifs contained in the intracellular domains of natural co-stimulatory molecules CD28, ICOS, 4-1BB, and OX40;

[0129] Figure 3 To recombinant co-stimulatory molecules and CAR structure;

[0130] Figure 4 is the promoter binding sequence of pNF-κB-mCherry;

[0131] Figure 5 The changes in NFAT and NF-κB signal intensities in Jurkat cells induced by each recombinant CAR after antigen stimulation;

[0132] Figure 6To characterize the kinetics of CAR-mediated stimulation of NFAT and NF-κB signaling pathways.

[0133] Figure 7 This is the result of testing the activation level, in vitro killing effect and differentiation type of CAR-T cells obtained by CD28×4-1BB recombination;

[0134] Figure 8 This is a graph showing the relative levels of various cytokines secreted by CAR-T cells obtained by CD28×4-1BB recombination;

[0135] Figure 9 These are the in vitro functional test results of CD28(NM)-4-1BB(MC) recombinant fragment CAR-T cells;

[0136] Figure 10 To detect the in vivo killing function of CD28(NM)-4-1BB(MC) recombinant fragment CAR-T cells. DETAILED DESCRIPTION

[0137] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to specific embodiments and drawings. In the following description, many specific details are set forth to facilitate a full understanding of the present invention. However, the present invention can be implemented in many other ways than those described herein, and those skilled in the art can make similar improvements without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific implementation disclosed below. It should be noted that in the following content, "connection" can be a direct connection or an indirect connection.

[0138] One embodiment of the present application provides a chimeric antigen receptor, comprising an intracellular domain, the intracellular domain containing a recombinant costimulatory molecule, the recombinant costimulatory molecule comprising a first module and a second module connected from the N-terminus to the C-terminus. The first module is a first N-terminal segment, and the second module is a connected second intermediate segment and a second C-terminal segment, or a connected second N-terminal segment, a second intermediate segment, and a second C-terminal segment; or the first module is a connected first N-terminal segment and a first intermediate segment, or a connected first N-terminal segment, a first intermediate segment, and a first C-terminal segment, and the second module is a second C-terminal segment, a connected second intermediate segment, and a second C-terminal segment, or a connected second N-terminal segment, a second intermediate segment, and a second C-terminal segment. Wherein, the first N-terminal segment, the first intermediate segment, and the first C-terminal segment are obtained by splitting the first costimulatory domain into three parts in sequence from the N-terminus to the C-terminus. The second N-terminal segment, the second intermediate segment, and the second C-terminal segment are obtained by splitting the second costimulatory domain into three parts in sequence from the N-terminus to the C-terminus. The first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules.

[0139] The first costimulatory domain is the intracellular segment of a natural costimulatory molecule. The second costimulatory domain is the intracellular segment of a natural costimulatory molecule. Costimulatory molecules are cell surface molecules that provide costimulatory signals for the full activation of immune cells such as T cells. The terms "natural costimulatory molecules" and "recombinant costimulatory molecules" are relative terms, with "natural costimulatory molecules" emphasizing the absence of modification.

[0140] At present, the existing CAR molecular design mainly uses the intracellular segment of the complete natural costimulatory molecule, which also causes the downstream signals and functions of CAR-T to be confined to a very limited space. This application proposes for the first time to split the natural costimulatory molecules according to the functional motif and then rationally recombine them to obtain recombinant costimulatory molecules. Since the signal activity of the original costimulatory domain caused by the recombination is either enhanced or weakened, the resulting series of costimulatory domains with varying stimulation strengths greatly enrich the original signal activation range, which is beneficial to the application of CAR-T. Experimental verification shows that the chimeric antigen receptor of the present application has the potential to activate cells and has the ability to specifically kill target cells. Different costimulatory domains have complex and diverse effects on the relative ability of T cells to secrete cytokines. Among them, CAR-T cells with recombinant costimulatory molecules 28 (NM) -BB (MC) have more excellent specific killing target cell ability, have the effect of reducing trophic signals and increasing activation levels, so that T cells have a mild and lasting anti-tumor effect, and reduce the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy.

[0141] The principles for splitting the costimulatory domain are: first, not destroying the functional motifs and the combination of motifs that work together; second, ensuring that each functional motif is dispersed as much as possible in different parts. This application has developed a new type of artificial recombinant chimeric antigen receptor molecule design. Based on the method of synthetic biology protein module recombination, the costimulatory domain of the chimeric antigen receptor is redesigned, achieving diversified control of human T cell downstream signaling and cell fate, and improving the shortcomings of chimeric antigen receptor engineered T cells in tumor immunotherapy.

[0142] In one embodiment, the first costimulatory domain is an intracellular segment of a costimulatory molecule of the immunoglobulin superfamily, and the second costimulatory domain is an intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily. Specifically, the first costimulatory domain is an intracellular segment of CD28 or ICOS, and the second costimulatory domain is an intracellular segment of 4-1BB or OX40.

[0143] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule of the immunoglobulin superfamily. Specifically, the first costimulatory domain is the intracellular segment of 4-1BB or OX40, and the second costimulatory domain is the intracellular segment of CD28 or ICOS.

[0144] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes and plays an important role in T cell activation. It binds to the B7 molecule on APCs (antigen presenting cells), mediating T cell co-stimulation and promoting their survival, proliferation, and cytokine production.

[0145] ICOS is an important new type of inducible costimulatory molecule. Inducible costimulatory molecule ligand (ICOSL) and its receptor inducible costimulatory molecule (ICOS) play an important role in immune response and regulation.

[0146] 4-1BB, also known as CD137, is a member of the tumor necrosis factor receptor superfamily (TNFRSF9). It is mainly expressed in activated T cells and is a T cell co-stimulatory molecule. Its ligand is 4-1BBL. The combination of the two can stimulate the activation and proliferation of T cells (and B cells).

[0147] OX40, also known as TNFRSF4 (Tumor necrosis factor receptor superfamily, member 4), is mainly expressed in activated CD4 + and CD8 + On the surface of T cells, binding to OX40 ligand can stimulate CD8 + T cell activation. Through the co-activation effect of OX40 / OX40L signaling, T cell function, including cytokine production, proliferation, and T cell survival, is further enhanced. OX40 antibody activator (Agonist) can reduce regulatory T cells in tumors and enhance anti-tumor activity.

[0148] The co-stimulatory molecules CD28 and ICOS in the immunoglobulin superfamily and 4-1BB and OX40 in the tumor necrosis factor receptor superfamily are more commonly used in CAR, and people have studied the functions and working mechanisms of these receptors more fully, so this application selects their intracellular segments as raw materials for recombination. It should be noted that the first co-stimulatory domain or the second co-stimulatory domain is not limited to the intracellular segment of CD28 or ICOS, and the intracellular segment of other co-stimulatory molecules in the immunoglobulin superfamily can also be selected. It should be noted that the first co-stimulatory domain or the second co-stimulatory domain is not limited to the intracellular segment of 4-1BB and OX40, and can also be the intracellular segment of other co-stimulatory molecules in the tumor necrosis factor receptor superfamily.

[0149] In one embodiment, the intracellular segment of CD28 is sequentially separated from the N-terminus to the C-terminus into the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment. Furthermore, the amino acid sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment are shown in SEQ ID No. 1 to SEQ ID No. 3. Furthermore, the nucleotide sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-N-terminal segment are shown in SEQ ID No. 13 to SEQ ID No. 15, respectively.

[0150] In one embodiment, the intracellular segment of ICOS is sequentially split from the N-terminus to the C-terminus into the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment. Furthermore, the amino acid sequences of the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment are shown in SEQ ID Nos. 4 to 6. Furthermore, the nucleotide sequences of the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment are shown in SEQ ID Nos. 16 to 18, respectively.

[0151] In one embodiment, the intracellular segment of 4-1BB is sequentially split from the N-terminus to the C-terminus into the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment. Furthermore, the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9. Furthermore, the nucleotide sequences of the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 19 to SEQ ID No. 21, respectively.

[0152] In one embodiment, the intracellular segment of OX40 is sequentially divided from the N-terminus to the C-terminus into the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment. Furthermore, the amino acid sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID Nos. 10 to 12. Furthermore, the nucleotide sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID Nos. 22 to 24, respectively.

[0153] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a CD28-C-terminal segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment and a 4-1BB-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as CD28-4-1BB.

[0154] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the 4-1BB-C-terminal segment, the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB-CD28.

[0155] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, CD28-middle segment, CD28-C-terminal segment, 4-1BB-middle segment, and 4-1BB-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as CD28-4-1BB (MC).

[0156] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the 4-1BB-C-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB-CD28 (MC).

[0157] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment and a 4-1BB-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as CD28(NM)-4-1BB.

[0158] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(NM)-CD28.

[0159] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as CD28(NM)-4-1BB(MC).

[0160] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the CD28-middle segment, and the CD28-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(NM)-CD28(MC).

[0161] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-CD28(M)-CD28(C).

[0162] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, CD28-middle segment, and 4-1BB-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as CD28(N)-CD28(M)-4-1BB(C).

[0163] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as CD28(N)-4-1BB(M)-4-1BB(C).

[0164] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-4-1BB(M)-CD28(C).

[0165] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, CD28-middle segment, CD28-C-terminal segment, OX40-N-terminal segment, OX40-middle segment and OX40-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as CD28-OX40.

[0166] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40-CD28.

[0167] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, CD28-middle segment, CD28-C-terminal segment, OX40-middle segment, and OX40-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as CD28-OX40 (MC).

[0168] In one embodiment, the recombinant co-stimulatory molecule includes the OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, CD28-middle segment, and CD28-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40-CD28 (MC).

[0169] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, an OX40-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as CD28(NM)-OX40.

[0170] In one embodiment, the recombinant co-stimulatory molecule includes the OX40-N-terminal segment, OX40-middle segment, CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40(NM)-CD28.

[0171] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, a CD28-middle segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as CD28(NM)-OX40(MC).

[0172] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, CD28-middle segment, and CD28-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40(NM)-CD28(MC).

[0173] In one embodiment, the recombinant co-stimulatory molecule includes the OX40-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as OX40(N)-CD28(M)-CD28(C).

[0174] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, CD28-middle segment, and OX40-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as CD28(N)-CD28(M)-OX40(C).

[0175] In one embodiment, the recombinant co-stimulatory molecule includes a CD28-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as CD28(N)-OX40(M)-OX40(C).

[0176] In one embodiment, the recombinant co-stimulatory molecule includes the OX40-N-terminal segment, the OX40-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as OX40(N)-OX40(M)-CD28(C).

[0177] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, an ICOS-C-terminal segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment, which are sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS-4-1BB.

[0178] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the 4-1BB-C-terminal segment, the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB-ICOS.

[0179] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, an ICOS-C-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment, which are sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS-4-1BB (MC).

[0180] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the 4-1BB-C-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB-ICOS (MC).

[0181] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment, which are sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS(NM)-4-1BB.

[0182] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(NM)-ICOS.

[0183] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as ICOS(NM)-4-1BB(MC).

[0184] In one embodiment, the recombinant co-stimulatory molecule includes the 4-1BB-N-terminal segment, the 4-1BB-middle segment, the ICOS-middle segment, and the ICOS-C-terminal segment, which are connected in sequence from the N-terminus to the C-terminus, abbreviated as 4-1BB(NM)-ICOS(MC).

[0185] In one embodiment, the recombinant co-stimulatory molecule includes a 4-1BB-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-ICOS(M)-ICOS(C).

[0186] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as ICOS(N)-ICOS(M)-4-1BB(C).

[0187] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as ICOS(N)-4-1BB(M)-4-1BB(C).

[0188] In one embodiment, the recombinant co-stimulatory molecule includes a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and an ICOS-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-4-1BB(M)-ICOS(C).

[0189] In one embodiment, the recombinant co-stimulatory molecule includes ICOS-N-terminal segment, ICOS-middle segment, ICOS-C-terminal segment, OX40-N-terminal segment, OX40-middle segment and OX40-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as ICOS-OX40.

[0190] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40-ICOS.

[0191] In one embodiment, the recombinant co-stimulatory molecule includes ICOS-N-terminal segment, ICOS-middle segment, ICOS-C-terminal segment, OX40-middle segment, and OX40-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as ICOS-OX40 (MC).

[0192] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, OX40-C-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40-ICOS (MC).

[0193] In one embodiment, the recombinant co-stimulatory molecule includes ICOS-N-terminal segment, ICOS-middle segment, OX40-N-terminal segment, OX40-middle segment and OX40-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as ICOS(NM)-OX40.

[0194] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, ICOS-N-terminal segment, ICOS-middle segment, and ICOS-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40(NM)-ICOS.

[0195] In one embodiment, the recombinant co-stimulatory molecule includes ICOS-N-terminal segment, ICOS-middle segment, OX40-middle segment and OX40-C-terminal segment connected in sequence from N-terminus to C-terminus, abbreviated as ICOS(NM)-OX40(MC).

[0196] In one embodiment, the recombinant co-stimulatory molecule includes OX40-N-terminal segment, OX40-middle segment, ICOS-middle segment, and ICOS-C-terminal segment, which are connected in sequence from N-terminus to C-terminus, abbreviated as OX40(NM)-ICOS(MC).

[0197] In one embodiment, the recombinant co-stimulatory molecule includes an OX40-N-terminal segment, an ICOS-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as OX40(N)-ICOS(M)-ICOS(C).

[0198] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an ICOS-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS(N)-ICOS(M)-OX40(C).

[0199] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS(N)-OX40(M)-OX40(C).

[0200] In one embodiment, the recombinant co-stimulatory molecule includes an OX40-N-terminal segment, an OX40-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as OX40(N)-OX40(M)-ICOS(C).

[0201] In one embodiment, the intracellular domain also includes an intracellular signaling domain linked to a recombinant co-stimulatory molecule. The chimeric antigen receptor also includes an extracellular domain, a hinge, and a transmembrane region. Preferably, the intracellular signaling domain is the intracellular segment of the CD3ζ chain. The extracellular domain is a single-chain antibody targeting CD19 or mesothelin. Both the hinge and the transmembrane region are derived from CD8α. It should be noted that the extracellular domain is not limited to single-chain antibodies targeting CD19 or mesothelin, and can also be single-chain antibodies targeting other antigens.

[0202] This application proposes for the first time that natural costimulatory molecules are split according to functional motifs and then rationally recombined to obtain recombinant costimulatory molecules. This application also proposes for the first time the sequences of four natural costimulatory molecules split into three fragments, N, M, and C, as well as the sequences of various novel costimulatory molecules obtained by recombination. The design scheme of the present application of splitting natural costimulatory molecules according to functional motifs and then rationally recombining them can also be applied to more alternative molecules. Through this design scheme, a large number of novel CAR molecular designs can be generated. The chimeric antigen receptor obtained in this application can be used to prepare CAR-T cells with the ability to specifically kill target cells, and can be used to prepare anti-tumor drugs.

[0203] Among the recombinant costimulatory molecules obtained in this application, the CAR-T cells of the costimulatory molecule 28(NM)-BB(MC) have better specific ability to kill target cells. Experiments have also shown that the costimulatory domain has the effect of reducing trophic signals and increasing activation levels, giving T cells a mild and lasting anti-tumor effect, and reducing the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy.

[0204] The CAR-T cells composed of the novel recombinant co-stimulatory molecule CD28(NM)-4-1BB(MC) obtained by this application exhibit better functional properties, which is due to its unique recombinant structure, specifically: First, the PYAP motif of CD28 is deleted, which destroys part of the function of CD28, which is beneficial to reduce cell apoptosis caused by over-activation; at the same time, its ability to secrete IL-2 is weakened, thereby avoiding the production of a large number of regulatory T cells and weakening the inhibitory effect on helper T cells and killer T cells. Second, the N-terminal module of 4-1BB is deleted. There is no motif in this module, and there are a large number of basic amino acids, which may potentially interact with the cell membrane to inhibit the activation of CAR; the position order of 4-1BB away from the cell membrane helps to avoid the problem that the nutrient signal of 4-1BB leads to excessive NF-κB basal activation level and causes cell apoptosis. Third, most of the motifs of CD28 and 4-1BB are retained, thus ensuring the effectiveness of their production functions. However, in terms of fragment length, compared with the third-generation CAR, the distance between CD3ζ and the cell membrane is greatly shortened, which is conducive to the phosphorylation of CD3ζ and more sensitive to extracellular stimuli.

[0205] One embodiment of the present application also provides a chimeric antigen receptor, including an intracellular domain, the intracellular domain containing a recombinant costimulatory molecule, the recombinant costimulatory molecule including a first module, a second module and a third module connected from the N-terminus to the C-terminus; the first module is selected from a segment between the first N-terminal segment and the second N-terminal segment, the second module is selected from a segment between the first intermediate segment and the second intermediate segment, the third module is selected from a segment between the first C-terminal segment and the second C-terminal segment, the first N-terminal segment, the first intermediate segment and the first C-terminal segment are obtained by splitting the first costimulatory domain into three parts in sequence from the N-terminus to the C-terminus, the second N-terminal segment, the second intermediate segment and the second C-terminal segment are obtained by splitting the second costimulatory domain into three parts in sequence from the N-terminus to the C-terminus, the first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules, the second module and the first module are respectively derived from different costimulatory molecules, and the second module and the third module are respectively derived from different costimulatory molecules.

[0206] The principles for splitting the costimulatory domain are: first, not destroying the functional motifs and the combination of motifs that work together; second, ensuring that each functional motif is dispersed as much as possible in different parts. This application has developed a new type of artificial recombinant chimeric antigen receptor molecule design. Based on the method of synthetic biology protein module recombination, the costimulatory domain of the chimeric antigen receptor is redesigned, achieving diversified control of human T cell downstream signaling and cell fate, and improving the shortcomings of chimeric antigen receptor engineered T cells in tumor immunotherapy.

[0207] The first module and the second module are derived from the same co-stimulatory molecule.

[0208] The first costimulatory domain is the intracellular segment of a natural costimulatory molecule. The second costimulatory domain is the intracellular segment of a natural costimulatory molecule. Costimulatory molecules are cell surface molecules that provide costimulatory signals for the full activation of immune cells such as T cells. The terms "natural costimulatory molecules" and "recombinant costimulatory molecules" are relative terms, with "natural costimulatory molecules" emphasizing the absence of modification.

[0209] In one embodiment, the first costimulatory domain is an intracellular segment of a costimulatory molecule of the immunoglobulin superfamily, and the second costimulatory domain is an intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily. Specifically, the first costimulatory domain is an intracellular segment of CD28 or ICOS, and the second costimulatory domain is an intracellular segment of 4-1BB or OX40.

[0210] In one embodiment, the first costimulatory domain is the intracellular segment of a costimulatory molecule of the tumor necrosis factor receptor superfamily, and the second costimulatory domain is the intracellular segment of a costimulatory molecule of the immunoglobulin superfamily. Specifically, the first costimulatory domain is the intracellular segment of 4-1BB or OX40, and the second costimulatory domain is the intracellular segment of CD28 or ICOS.

[0211] CD28 is a co-stimulatory molecule expressed on the surface of T lymphocytes and plays an important role in T cell activation. It binds to the B7 molecule on APCs (antigen presenting cells), mediating T cell co-stimulation and promoting their survival, proliferation, and cytokine production.

[0212] ICOS is an important new type of inducible costimulatory molecule. Inducible costimulatory molecule ligand (ICOSL) and its receptor inducible costimulatory molecule (ICOS) play an important role in immune response and regulation.

[0213] 4-1BB, also known as CD137, is a member of the tumor necrosis factor receptor superfamily (TNFRSF9). It is mainly expressed in activated T cells and is a T cell co-stimulatory molecule. Its ligand is 4-1BBL. The combination of the two can stimulate the activation and proliferation of T cells (and B cells).

[0214] OX40, also known as TNFRSF4 (Tumor necrosis factor receptor superfamily, member 4), is mainly expressed in activated CD4 + and CD8 + On the surface of T cells, binding to OX40 ligand can stimulate CD8 + T cell activation. Through the co-activation effect of OX40 / OX40L signaling, T cell function, including cytokine production, proliferation, and T cell survival, is further enhanced. OX40 antibody activator (Agonist) can reduce regulatory T cells in tumors and enhance anti-tumor activity.

[0215] The co-stimulatory molecules CD28 and ICOS in the immunoglobulin superfamily and 4-1BB and OX40 in the tumor necrosis factor receptor superfamily are more commonly used in CAR, and people have studied the functions and working mechanisms of these receptors more fully, so this application selects their intracellular segments as raw materials for recombination. It should be noted that the first co-stimulatory domain or the second co-stimulatory domain is not limited to the intracellular segment of CD28 or ICOS, and the intracellular segment of other co-stimulatory molecules in the immunoglobulin superfamily can also be selected. It should be noted that the first co-stimulatory domain or the second co-stimulatory domain is not limited to the intracellular segment of 4-1BB and OX40, and can also be the intracellular segment of other co-stimulatory molecules in the tumor necrosis factor receptor superfamily.

[0216] In one embodiment, the intracellular segment of CD28 is sequentially separated from the N-terminus to the C-terminus into the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment. Furthermore, the amino acid sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment are shown in SEQ ID No. 1 to SEQ ID No. 3. Furthermore, the nucleotide sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-N-terminal segment are shown in SEQ ID No. 13 to SEQ ID No. 15, respectively.

[0217] In one embodiment, the intracellular segment of ICOS is sequentially split from the N-terminus to the C-terminus into the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment. Furthermore, the amino acid sequences of the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment are shown in SEQ ID Nos. 4 to 6. Furthermore, the nucleotide sequences of the ICOS-N-terminal segment, the ICOS-middle segment, and the ICOS-C-terminal segment are shown in SEQ ID Nos. 16 to 18, respectively.

[0218] In one embodiment, the intracellular segment of 4-1BB is sequentially split from the N-terminus to the C-terminus into the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment. Furthermore, the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9. Furthermore, the nucleotide sequences of the 4-1BB-N-terminal segment, the 4-1BB-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 19 to SEQ ID No. 21, respectively.

[0219] In one embodiment, the intracellular segment of OX40 is sequentially divided from the N-terminus to the C-terminus into the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment. Furthermore, the amino acid sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID Nos. 10 to 12. Furthermore, the nucleotide sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown in SEQ ID Nos. 22 to 24, respectively.

[0220] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, the 4-1BB-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as CD28(N)-4-1BB(M)-CD28(C).

[0221] In one embodiment, the recombinant co-stimulatory molecule includes a 4-1BB-N-terminal segment, a CD28-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-CD28(M)-4-1BB(C).

[0222] In one embodiment, the recombinant co-stimulatory molecule includes the CD28-N-terminal segment, the OX40-middle segment, and the CD28-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as CD28(N)-OX40(M)-CD28(C).

[0223] In one embodiment, the recombinant co-stimulatory molecule includes the OX40-N-terminal segment, the CD28-middle segment, and the OX40-C-terminal segment connected in sequence from the N-terminus to the C-terminus, abbreviated as OX40(N)-CD28(M)-OX40(C).

[0224] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, a 4-1BB-middle segment, and an ICOS-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as ICOS(N)-4-1BB(M)-ICOS(C).

[0225] In one embodiment, the recombinant co-stimulatory molecule includes a 4-1BB-N-terminal segment, an ICOS-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus, abbreviated as 4-1BB(N)-ICOS(M)-4-1BB(C).

[0226] In one embodiment, the recombinant co-stimulatory molecule includes an ICOS-N-terminal segment, an OX40-middle segment, and an ICOS-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as ICOS(N)-OX40(M)-ICOS(C).

[0227] In one embodiment, the recombinant co-stimulatory molecule includes an OX40-N-terminal segment, an ICOS-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus, abbreviated as OX40(N)-ICOS(M)-OX40(C).

[0228] In one embodiment, the intracellular domain also includes an intracellular signaling domain linked to a recombinant co-stimulatory molecule. The chimeric antigen receptor also includes an extracellular domain, a hinge, and a transmembrane region. Preferably, the intracellular signaling domain is the intracellular segment of the CD3ζ chain. The extracellular domain is a single-chain antibody targeting CD19 or mesothelin. Both the hinge and the transmembrane region are derived from CD8α. It should be noted that the extracellular domain is not limited to single-chain antibodies targeting CD19 or mesothelin, and can also be single-chain antibodies targeting other antigens.

[0229] This application proposes for the first time that natural costimulatory molecules are split according to functional motifs and then rationally recombined to obtain recombinant costimulatory molecules. This application also proposes for the first time the sequences of four natural costimulatory molecules split into three fragments, N, M, and C, as well as the sequences of various novel costimulatory molecules obtained by recombination. The design scheme of the present application of splitting natural costimulatory molecules according to functional motifs and then rationally recombining them can also be applied to more alternative molecules. Through this design scheme, a large number of novel CAR molecular designs can be generated. The chimeric antigen receptor obtained in this application can be used to prepare CAR-T cells with the ability to specifically kill target cells, and can be used to prepare anti-tumor drugs.

[0230] One embodiment of the present application further provides a recombinant vector comprising the coding sequence of the chimeric antigen receptor.

[0231] Among them, the recombinant vector is a cloning vector or an expression vector.

[0232] Specifically, the recombinant vector is a lentiviral expression vector containing the coding sequence of the chimeric antigen receptor. A specific example is a pHR vector containing the coding sequence of the chimeric antigen receptor. It should be noted that the recombinant vector is not limited to the pHR vector containing the coding sequence of the chimeric antigen receptor. The chimeric antigen receptor gene can also be integrated into other vectors, such as pET21b, pET22b, pET32a, pQE30, and the like.

[0233] The recombinant vector containing the above-mentioned chimeric antigen receptor obtained in the present application can be used to prepare CAR-T cells with the ability to specifically kill target cells and can be used to prepare anti-tumor drugs.

[0234] The method for preparing the above-mentioned recombinant vector comprises the following steps: cloning the coding sequence of the above-mentioned chimeric antigen receptor into a genetic engineering vector to obtain the recombinant vector.

[0235] The chimeric antigen receptor includes an intracellular domain, and the intracellular domain contains a recombinant costimulatory molecule. Before the step of cloning the coding sequence of the chimeric antigen receptor into a genetic engineering vector, the steps S110-S120 of constructing the recombinant costimulatory molecule are also included:

[0236] S110. The first costimulatory domain is sequentially split into three parts from N-terminus to C-terminus to obtain a first N-terminal segment, a first middle segment and a first C-terminal segment. The second costimulatory domain is sequentially split into three parts from N-terminus to C-terminus to obtain a second N-terminal segment, a second middle segment and a second C-terminal segment. The first costimulatory domain and the second costimulatory domain are intracellular segments of different costimulatory molecules.

[0237] The principles for splitting the costimulatory domain are: first, not destroying the functional motifs and the combination of motifs that work together; second, ensuring that each functional motif is dispersed as much as possible in different parts. This application has developed a new type of artificial recombinant chimeric antigen receptor molecule design. Based on the method of synthetic biology protein module recombination, the costimulatory domain of the chimeric antigen receptor is redesigned, achieving diversified control of human T cell downstream signaling and cell fate, and improving the shortcomings of chimeric antigen receptor engineered T cells in tumor immunotherapy.

[0238] It should be noted that the detailed introduction of the first costimulatory domain and the second costimulatory domain is described above and will not be repeated here.

[0239] S120, assembling the separated parts to obtain a recombinant costimulatory molecule.

[0240] Specifically, the first N-terminal segment, the second middle segment, and the second C-terminal segment are connected from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0241] Alternatively, the first N-terminal segment, the second N-terminal segment, the second intermediate segment, and the second C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0242] Alternatively, the recombinant costimulatory molecule is obtained by connecting the first N-terminal segment, the first middle segment, and the second C-terminal segment from N-terminus to C-terminus;

[0243] Alternatively, the first N-terminal segment, the first middle segment, the second middle segment, and the second C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0244] Alternatively, the first N-terminal segment, the first middle segment, the second N-terminal segment, the second middle segment, and the second C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0245] Alternatively, the first N-terminal segment, the first middle segment, the first C-terminal segment, and the second C-terminal segment are connected from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0246] Alternatively, the first N-terminal segment, the first middle segment, the first C-terminal segment, the second middle segment, and the second C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0247] Alternatively, the first N-terminal segment, the first intermediate segment, the first C-terminal segment, the second N-terminal segment, the second intermediate segment and the second C-terminal segment are connected from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0248] Alternatively, the first N-terminal segment, the second intermediate segment, and the first C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule;

[0249] Alternatively, the second N-terminal segment, the first middle segment and the second C-terminal segment are linked from N-terminus to C-terminus to obtain a recombinant costimulatory molecule.

[0250] It should be noted that the specific description of the recombinant co-stimulatory molecules is given above and will not be repeated here.

[0251] In one embodiment, the method for preparing the recombinant vector further includes steps S130-S140 of screening the signal characteristics of the chimeric antigen receptor in the recombinant vector:

[0252] S130. A plasmid containing the pNF-κB-mCherry sequence and a plasmid containing the piggyBac transposase sequence were transformed into a cell line containing the pNFAT-EGFP reporter system to obtain a dual reporter signal cell line.

[0253] In a specific example, the cell line containing pNFAT-EGFP is a Jurkat cell line containing pNFAT-EGFP. It should be noted that the cell line containing pNFAT-EGFP is not limited to the Jurkat cell line, and can also be other cell lines, such as HUT78, H9, etc.

[0254] After obtaining the design of a new CAR molecule, its function needs to be identified. Traditional in vivo and in vitro experimental verification methods require a lot of manpower, material resources and time costs, which is not conducive to the rapid screening and identification of new CAR molecule libraries. Therefore, based on the activation signal characteristics of T cells, this application constructs a dual-reporter signal cell line that can preliminarily reflect the activation of T cell downstream signals. The specific design idea is to use the promoters of the transcription factors NFAT (nuclear factor of activated T cells) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) to respectively activate the expression of fluorescent proteins EGFP and mCherry, and the expression level of fluorescent proteins reflects the degree of activation of the corresponding signal pathway. The plasmid containing piggyBac transposase and pNF-κB-mCherry (NF-κB binding sequence: GGGACTTTCC) is transformed into the Jurkat cell line containing the pNFAT-EGFP reporter system by electroporation. Then, flow cytometry was used to screen out monoclonal cells with low background levels and high response levels (i.e., low response before (TNFα or αCD3 / CD28) stimulation and obvious response after stimulation). Through in vitro expansion culture, a stable cell line containing a dual reporter signal system was obtained.

[0255] S140. Introduce the recombinant vector into the dual-reporter signal cell line, perform target cell antigen stimulation, and then detect the fluorescence intensity of the dual-reporter signal cell line to obtain the NFAT and NF-κB signal kinetic characteristics, and screen out recombinant vectors with the potential to activate cells.

[0256] In one embodiment, a recombinant expression vector containing the chimeric antigen receptor is introduced into a dual-reporter signal cell line by lentiviral transfection. It should be noted that the introduction of the recombinant expression vector containing the chimeric antigen receptor into the dual-reporter signal cell line is not limited to lentiviral transfection, and the introduction of the recombinant expression vector containing the chimeric antigen receptor into the dual-reporter signal cell line may also be carried out by other means, such as gene editing knock-in.

[0257] This application uses the NFAT & NF-κB dual reporter signal cell line to quickly screen the activation signal characteristics of new CARs: The Jurkat dual reporter signal cell line with pNF-κB-mCherry and pNFAT-EGFP can quickly and high-throughput complete the activation signal characteristic identification of new CAR molecular designs, which is a powerful experimental means to quickly screen CARs.

[0258] One embodiment of the present application further provides a recombinant cell capable of expressing the above-mentioned chimeric antigen receptor or containing the above-mentioned recombinant vector.

[0259] The construction of recombinant cells facilitates the application of chimeric antigen receptors and can be used to prepare anti-tumor drugs.

[0260] The recombinant cells are T cells that can express the chimeric antigen receptor or T cells into which a recombinant vector has been introduced. In a specific example, the T cells are primary human T cells.

[0261] This application has at least the following beneficial effects:

[0262] This application proposes for the first time that natural costimulatory molecules are split according to functional motifs and then rationally recombined to obtain recombinant costimulatory molecules. This application also proposes for the first time the sequences of four natural costimulatory molecules split into three fragments, N, M, and C, and the sequences of 48 novel costimulatory molecules obtained by recombining (excluding 8 directly connected in series). The design scheme of the present application for splitting natural costimulatory molecules according to functional motifs and then rationally recombining them can also be applied to more alternative molecules. This design scheme can generate a large number of new CAR molecular designs.

[0263] This application uses the NFAT & NF-κB dual reporter signal cell line to quickly screen the activation signal characteristics of new CARs: The Jurkat dual reporter signal cell line with pNF-κB-mCherry and pNFAT-EGFP can quickly and high-throughput complete the activation signal characteristic identification of new CAR molecular designs, which is a powerful experimental means to quickly screen CARs.

[0264] Among the recombinant costimulatory molecules obtained in this application, the CAR-T cells of the costimulatory molecule 28(NM)-BB(MC) have better specific ability to kill target cells. Experiments have also shown that the costimulatory domain has the effect of reducing trophic signals and increasing activation levels, giving T cells a mild and lasting anti-tumor effect, and reducing the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy.

[0265] The following is a specific example section.

[0266] Unless otherwise specified, the reagents and instruments used in the examples are all conventionally selected in the art. Experimental methods for which specific conditions are not specified in the examples are generally carried out under conventional conditions, such as those described in the literature or books, or methods recommended by the kit manufacturer. The reagents used in the examples are all commercially available.

[0267] The specific examples section uses the currently widely used CD19-targeting CAR as the main model, and refers to the structures of the first to third generation CARs to design a CAR with a recombinant costimulatory domain: the receptor extracellular recognition domain (i.e., the extracellular domain) is a single-chain antibody targeting CD19, the hinge and transmembrane regions are both derived from CD8α, and the intracellular domain is composed of different costimulatory domains and the intracellular segment of the CD3ζ chain. For detailed technical procedures, see Figure 1 .

[0268] Example 1 Splitting and Recombination of Natural Co-stimulatory Molecules

[0269] 1. Structural resolution of natural co-stimulatory molecules

[0270] The co-stimulatory molecules CD28 and ICOS in the immunoglobulin superfamily and 4-1BB and OX40 in the tumor necrosis factor receptor superfamily are more commonly used in CAR, and people have studied the functions and working mechanisms of these receptors more fully. Therefore, the present invention selects their intracellular segments as the raw materials for recombination. Through literature research, the reported motifs are identified, and each co-stimulatory domain is split into three parts: N (N-terminal or N-terminal segment), M (Middle or middle segment), and C (C-terminal or C-terminal segment) without destroying the functional motifs and the combination of motifs that work together, and ensuring that each functional motif is dispersed as much as possible in different parts. Specifically, the splitting of CD28, ICOS, 4-1BB, and OX40 is detailed in Figure 2 . Figure 2 The amino acid sequences and motifs contained in the intracellular segments of the natural co-stimulatory molecules CD28, ICOS, 4-1BB, and OX40 are shown in Table 1. The DNA sequences of the individual segments of CD28, ICOS, 4-1BB, and OX40 are shown in Table 2.

[0271] Table 1 Amino acid sequences of the fragments of CD28, ICOS, 4-1BB, and OX40

[0272] name Amino acid sequence CD28(N) RSKRSRLLHSD (as shown in SEQ ID No. 1) CD28(M) YMNMTPRRP (shown in SEQ ID No. 2) CD28(C) GPTRKHYQPYAPPRDFAAYRS (as shown in SEQ ID No. 3) ICOS(N) CWLTKKKYSSSVHDPNGE (as shown in SEQ ID No. 4) ICOS(M) YMFM (as shown in SEQ ID No. 5) ICOS(C) RAVNTAKKSRLTDVTL (as shown in SEQ ID No. 6) 4-1BB(N) KRGRKKLLYIFKQPFMRPV (shown in SEQ ID No. 7) 4-1BB(M) QTTQEED (as shown in SEQ ID No. 8) 4-1BB(C) GCSCRFPEEE (as shown in SEQ ID No. 9) OX40(N) ALYLLRRDQRLPPDAHKPPG (as shown in SEQ ID No. 10) OX40(M) GGSFRTPIQEE (as shown in SEQ ID No. 11) OX40(C) QADAHSTLAKI (as shown in SEQ ID No. 12)

[0273] Table 2 DNA sequences of the fragments of CD28, ICOS, 4-1BB, and OX40 after decomposition

[0274]

[0275] 2. Rational design and artificial recombination to obtain a new type of non-natural costimulatory molecules

[0276] Recombinant costimulatory molecules were obtained by recombining the intracellular segments of costimulatory molecules from different families in pairs (i.e., CD28×4-1BB, CD28×OX40, ICOS×4-1BB, and ICOS×OX40). Figure 3 . Figure 3 It is the structural composition of recombinant co-stimulatory molecules and CAR.

[0277] Through splitting and recombination, these four natural costimulatory molecules have been derived into 56 different recombinant costimulatory molecules, as detailed in Table 3. It should be noted that "CD28-4-1BB" means that from the N-terminus to the C-terminus, the N-terminal segment, the middle segment and the C-terminal segment of CD28 are sequentially connected to the N-terminal segment, the middle segment and the C-terminal segment of 4-1BB. Other recombinant costimulatory molecules have similar meanings, and so on, which will not be repeated here. "CD28-4-1BB (MC)" means that from the N-terminus to the C-terminus, the N-terminal segment, the middle segment and the C-terminal segment of CD28 are sequentially connected to the middle segment and the C-terminal segment of 4-1BB. Other recombinant costimulatory molecules have similar meanings, and so on, which will not be repeated here.

[0278] Table 3 Recombinant co-stimulatory molecules

[0279]

[0280]

[0281] Example 2

[0282] Constructing CAR expression vectors and using dual reporter cell lines for rapid identification of T cell signaling activation

[0283] 1. Construction of dual reporter cell lines

[0284] After obtaining the design of a new CAR molecule, its function needs to be identified. Traditional in vivo and in vitro experimental verification methods require a lot of manpower, material resources, and time costs, which is not conducive to the rapid screening and identification of new CAR molecule libraries. Therefore, based on the activation signal characteristics of T cells, this application constructed a dual-reporter signal cell line that can preliminarily reflect the activation of T cell downstream signals. The specific design idea is: the promoters of the transcription factors NFAT (nuclear factor of activated T cells) and NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) respectively activate the expression of fluorescent proteins EGFP and mCherry, and the expression level of fluorescent proteins reflects the degree of activation of the corresponding signal pathway. The plasmid containing piggyBac transposase and pNF-κB-mCherry (NF-κB binding sequence: GGGACTTTCC) (Lenardo and Baltimore, 1989) was transformed into the Jurkat cell line containing the pNFAT-EGFP reporter system by electroporation. Then, flow cytometry was used to screen out monoclonal cells with low background levels and high response levels (i.e., low response before (TNFα or αCD3 / CD28) stimulation and obvious response after stimulation). Through in vitro expansion culture, a stable cell line containing a dual reporter signal system was obtained.

[0285] The specific process is as follows:

[0286] Jurkat cells (JATN for short) with the pNFAT-EGFP reporter system were derived from the Arthur Weiss laboratory at UCSF. Based on this, the present application constructed a 7×pNF-κB-mCherry plasmid, which was electroporated into JATN cells together with the piggyBac transposase expression plasmid. After a two-month monoclonal expansion culture, bidirectional screening was performed by flow cytometry: that is, monoclonal cells with weak background red fluorescence expression and high response after stimulation with TNFα or α-CD3, α-CD28 antibodies were selected. The promoter binding sequence of pNF-κB-mCherry is detailed in Figure 4 .

[0287] 2. Verification of T cell activation characteristics after antigen-activated CAR

[0288] The constructed CAR expression vector was introduced into the dual-reporter signal cell line through lentiviral transfection, and in vitro experiments preliminarily verified the activation characteristics of T cells after antigen activation of CAR.

[0289] The specific process is as follows:

[0290] Dual reporter cells expressing different CARs were mixed with K562 cells expressing corresponding antigens (e.g., CD19) at a ratio of 1:1 and cultured in round-bottom 96-well plates (approximately 1×10 cells per well). 5 -2×10 5 At different time points (8h, 12h, 24h, 36h, 48h) after target cell antigen stimulation, cells were fixed with 4% paraformaldehyde and the fluorescence intensity of dual reporter cells was detected by flow cytometry to obtain the kinetic characteristics of NFAT and NF-κB signals. Figure 5 shown. Figure 5 The changes in NFAT and NF-κB signal intensities in Jurkat cells induced by each recombinant CAR after antigen stimulation.

[0291] from Figure 5 It can be seen that most CARs have the potential to activate cells (after a period of stimulation, the expression level of fluorescent protein is increased to a certain extent compared to time 0).

[0292] In order to more clearly demonstrate the effect of the recombinant fragment on the ability of CAR to activate NFAT and NF-κB signaling, a scatter plot of NFAT-NF-κB dual signals was drawn using the data from 12 hours of stimulation. Figure 6 . Figure 6 The kinetic characteristics of CAR with recombinant costimulatory domains stimulating NFAT and NF-κB signaling pathways.

[0293] from Figure 6 It can be seen that the signal activity of the original costimulatory domain is either enhanced or weakened due to recombination, and the resulting series of costimulatory domains with varying stimulation strengths greatly enrich the original signal activation range.

[0294] Example 3

[0295] Validation of novel CAR molecules in primary human T cells

[0296] After obtaining the signal characteristics of 56 recombinant CAR molecules in the Jurkat dual-reporter signal cell line, the constructed CAR expression vector was introduced into human primary T cells using the same method to obtain CAR-T cells, and functional experiments such as T cell activation level, in vitro killing, persistence, and cytokine testing were performed.

[0297] The specific process is as follows:

[0298] (1) Human primary T cells were obtained from human peripheral blood mononuclear cells (hPBMCs) and activated using antibodies against α-CD3 and α-CD28 coated on the bottom of the cell culture plate. Before activation, 24-well plates (400 μL / well) were pretreated with an aqueous solution of α-CD3 and α-CD28 antibodies at a final concentration of 1 μg / mL for 2-4 hours. hPBMCs that had recovered for 24 hours were then added to the pretreated 24-well plates (0.5-2×10 6 cells / well) and IL-2 was added to the culture medium at a ratio of 6ng / mL. After one day of activation, hPBMCs were infected with lentivirus and 14 CARs of the CD28×4-1BB recombinant fragment were transformed into the cells. The virus was removed 12 hours after infection and replaced with fresh culture medium with IL-2 (same ratio as above). Cultured continuously for 10-15 days, the T cells were expanded in large numbers and entered a resting state. Cells in a resting state will be used for subsequent experiments to detect the activation level of the recombinant CAR-T cells, the in vitro killing effect and differentiation type, and the relative levels of various cytokines secreted by the CAR-T cells. For detailed test results, see Figure 7 and Figure 8 . Figure 7 This is the result of detecting the activation level, in vitro killing effect and differentiation type of CAR-T cells obtained by CD28×4-1BB recombination. Figure 8 This is the result of detecting the relative levels of various cytokines secreted by CAR-T cells obtained by CD28×4-1BB recombination.

[0299] from Figure 7 and Figure 8 It can be seen that after CAR-T cells were stimulated by target cells for 24 hours, the expression level of CD69 protein related to T cell activation was significantly increased on the cell surface, and both cells had the ability to specifically kill target cells. The types of T cell differentiation were distinguished by staining the cell surface with CCR7 and CD45RO. + CD45RO + Central memory T cells (Tcm) are associated with the ability of T cells to eliminate tumors and account for a significant proportion of all recombinant CARs. In addition, different costimulatory domains have complex and diverse effects on the relative ability of T cells to secrete cytokines.

[0300] (2) Cluster analysis was performed based on the scatter plot results of the NFAT-NF-κB signal intensity of CAR-Jurkat cells stimulated by target cells for 12 hours. In addition to the two groups represented by 4-1BB and CD28, a new group of signal characteristics was generated, among which the most representative structure was CD28(NM)-4-1BB(MC). It was transformed into T cells with the classic first to third generation CARs, namely ζ, 28-ζ, BB-ζ and 28-BB-ζ. After functional testing, it was found that the expression of CD69 was significantly increased after being stimulated by antigens on the surface of target cells, proving that they can effectively activate T cells and have the ability to specifically kill target cells. In addition, CAR-T cells stimulated by target cells can secrete a large amount of various cytokines including IFN-γ and IL-2 into the culture medium. Compared with 28-ζ, BB-ζ and 28-BB-ζ, CD28(NM)-4-1BB(MC)-ζ secretes significantly lower levels of cytokines in T cells after stimulation. This means that CD28(NM)-4-1BB(MC) is an excellent choice for reducing cytokine storms and improving the safety of CAR-T cell therapy in clinical treatment. To further test the effects of different CARs on T cell survival and persistence, CAR-T cells activated and cultured in vitro for 14 days were stained with PD-1 antibodies, and CAR-T cells cultured in vitro for 14 and 21 days were stained with Annexin-V / PI to detect cell apoptosis. CD28(NM)-4-1BB(MC) showed good T cell indicators. For detailed test results, see Figure 9 . Figure 9 These are the in vitro functional test results of CD28(NM)-4-1BB(MC) recombinant fragment CAR-T cells.

[0301] (3) By constructing an NSG mouse tumor model, CD28(NM)-4-1BB(MC) recombinant fragment CAR-T cells were injected into tumor-bearing mice (subcutaneously injected with CD19-K562 target cells) to test their ability to eliminate tumors in the in vivo environment. After 48 days of observation, 4 / 5 mice injected with CD28(NM)-4-1BB(MC) survived, while all mice in the control group (injected with T cells that did not express CAR) died. This shows that CD28(NM)-4-1BB(MC) recombinant fragment CAR-T can effectively eliminate tumor cells in the body. For detailed test results, see Figure 10 . Figure 10 To detect the in vivo killing function of CD28(NM)-4-1BB(MC) recombinant fragment CAR-T cells.

[0302] This application provides a new type of artificial recombinant chimeric antigen receptor molecular design. Based on the method of synthetic biology protein module recombination, the costimulatory domain of the chimeric antigen receptor is redesigned. A process scheme is developed to rapidly screen and identify the signal characteristics of these new chimeric antigen receptors using NFAT & NF-κB dual reporter signal cell lines. Through the new chimeric antigen receptor design of this application, diversified control of human T cell downstream signaling and cell fate is achieved, which improves the shortcomings of chimeric antigen receptor engineered T cells in tumor immunotherapy.

[0303] Among the recombinant costimulatory molecules obtained in this application, the CAR-T cells of the costimulatory molecule 28(NM)-BB(MC) have better specific ability to kill target cells. Experiments have also shown that the costimulatory domain has the effect of reducing trophic signals and increasing activation levels, giving T cells a mild and lasting anti-tumor effect, and reducing the exhaustion and apoptosis of CAR-T cells. On this basis, the secretion of cytokines is appropriately reduced, thereby improving the safety of CAR-T cell therapy.

[0304] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims. Sequence Listing <110> Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences <120> Chimeric antigen receptor, recombinant vector, recombinant cell, and preparation method and application thereof <160> twenty four <170> SIPOSequenceListing 1.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <400> 1 Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp 1 5 10 <210> 2 <211> 9 <212> PRT <213> Artificial Sequence <400> 2 Tyr Met Asn Met Thr Pro Arg Arg Pro 1 5 <210> 3 <211> twenty one <212> PRT <213> Artificial Sequence <400> 3 Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe 1 5 10 15 Ala Ala Tyr Arg Ser 20 <210> 4 <211> 18 <212> PRT <213> Artificial Sequence <400> 4 Cys Trp Leu Thr Lys Lys Lys Tyr Ser Ser Ser Val His Asp Pro Asn 1 5 10 15 Gly Glu <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <400> 5 Tyr Met Phe Met 1 <210> 6 <211> 16 <212> PRT <213> Artificial Sequence <400> 6 Arg Ala Val Asn Thr Ala Lys Lys Ser Arg Leu Thr Asp Val Thr Leu 1 5 10 15 <210> 7 <211> 19 <212> PRT <213> Artificial Sequence <400> 7 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met 1 5 10 15 Arg Pro Val <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <400> 8 Gln Thr Thr Gln Glu Glu Asp 1 5 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <400> 9 Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 1 5 10 <210> 10 <211> 20 <212> PRT <213> Artificial Sequence <400> 10 Ala Leu Tyr Leu Leu Arg Arg Asp Gln Arg Leu Pro Pro Asp Ala His 1 5 10 15 Lys Pro Pro Gly 20 <210> 11 <211> 11 <212> PRT <213> Artificial Sequence <400> 11 Gly Gly Ser Phe Arg Thr Pro Ile Gln Glu Glu 1 5 10 <210> 12 <211> 11 <212> PRT <213> Artificial Sequence <400> 12 Gln Ala Asp Ala His Ser Thr Leu Ala Lys Ile 1 5 10 <210> 13 <211> 33 <212> DNA <213> Artificial Sequence <400> 13 aggagtaaga ggagcaggct cctgcacagt gac 33 <210> 14 <211> 27 <212> DNA <213> Artificial Sequence <400> 14 tacatgaaca tgactccccg ccgcccc 27 <210> 15 <211> 63 <212> DNA <213> Artificial Sequence <400> 15 gggcccaccc gcaagcatta ccagccctat gccccacccac gcgacttcgc agcctatcgc 60 tcc 63 <210> 16 <211> 54 <212> DNA <213> Artificial Sequence <400> 16 tgttggctta caaaaaagaa gtattcatcc agtgtgcacg accctaacgg tgaa 54 <210> 17 <211> 12 <212> DNA <213> Artificial Sequence <400> 17 tacatgttca tg 12 <210> 18 <211> 48 <212> DNA <213> Artificial Sequence <400> 18 agagcagtga acacagccaa aaaatctaga ctcacagatg tgacccta 48 <210> 19 <211> 57 <212> DNA <213> Artificial Sequence <400> 19 aaacggggca gaaagaaact cctgtatata ttcaaacaac catttatgag accagta 57 <210> 20 <211> twenty one <212> DNA <213> Artificial Sequence <400> 20 caaactactc aagaggaaga t 21 <210> twenty one <211> 48 <212> DNA <213> Artificial Sequence <400> 21 ggctgtagct gccgatttcc gcgattcc gtgaactg <210> 22 <211> 60 <212> DNA <213> Artificial Sequence <400> 22 gccctgtacc tgctccggag ggaccagagg ctgccccccg atgcccacaa gccccctggg <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <400> 23 33. ggaggcagtt tccggacccc catccaagag gag <210> 24 <211> 33 <212> DNA <213> Artificial Sequence <400> 24 33. cggccgacg cccactccac cctggccag atc

Claims

1. A chimeric antigen receptor, characterized in that The invention comprises an intracellular domain, wherein the intracellular domain contains a recombinant costimulatory molecule, wherein the recombinant costimulatory molecule is composed of a first module and a second module connected from the N-terminus to the C-terminus; the first module is one or two segments of the truncated segments formed by splitting the first costimulatory domain, and the second module is one or two segments of the truncated segments formed by splitting the second costimulatory domain, The first costimulatory domain is the intracellular segment of CD28, which is sequentially divided from the N-terminus to the C-terminus into the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment. The amino acid sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment are shown in SEQ ID No. 1 to SEQ ID No.

3. The second costimulatory domain is the intracellular segment of 4-1BB or OX40, the 4-1BB intracellular segment is sequentially divided into the 4-1BB-N-terminal segment, the 4-1BB-intermediate segment and the 4-1BB-C-terminal segment from the N-terminus to the C-terminus, and the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB8-intermediate segment and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9, and the OX40 intracellular segment is sequentially divided into the OX40-N-terminal segment, the OX40-intermediate segment and the OX40-C-terminal segment from the N-terminus to the C-terminus, and the amino acid sequences of the OX40-N-terminal segment, the OX40-intermediate segment and the OX40-C-terminal segment are shown in SEQ ID No. 10 to SEQ ID No. 12; The recombinant co-stimulatory molecule consists of a CD28-N-terminal segment, a CD28-middle segment, a 4-1BB-middle segment and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, a 4-1BB-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, a CD28-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, a 4-1BB-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, a CD28-middle segment, an OX40-middle segment, and an OX40-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, an OX40-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, a CD28-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a CD28-N-terminal segment, an OX40-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus.

2. The chimeric antigen receptor according to claim 1, wherein The intracellular domain further comprises an intracellular signaling domain connected to the recombinant co-stimulatory molecule, and the chimeric antigen receptor further comprises an extracellular domain, a hinge and a transmembrane region.

3. The chimeric antigen receptor according to claim 2, characterized in that The intracellular signaling domain is the intracellular segment of the CD3ζ chain, the extracellular domain is a single-chain antibody targeting CD19 or mesothelin, and the hinge and transmembrane region are both derived from CD8α.

4. A chimeric antigen receptor, characterized in that The invention comprises an intracellular domain, wherein the intracellular domain contains a recombinant costimulatory molecule, wherein the recombinant costimulatory molecule is composed of a first module and a second module connected from the N-terminus to the C-terminus, wherein the first module is one or two segments of the truncated segments formed by splitting the second costimulatory domain, and the second module is one or two segments of the truncated segments formed by splitting the first costimulatory domain. The second costimulatory domain is the intracellular segment of 4-1BB or OX40, the 4-1BB intracellular segment is sequentially divided into the 4-1BB-N-terminal segment, the 4-1BB-intermediate segment and the 4-1BB-C-terminal segment from the N-terminus to the C-terminus, and the amino acid sequences of the 4-1BB-N-terminal segment, the 4-1BB8-intermediate segment and the 4-1BB-C-terminal segment are shown in SEQ ID No. 7 to SEQ ID No. 9, and the OX40 intracellular segment is sequentially divided into the OX40-N-terminal segment, the OX40-intermediate segment and the OX40-C-terminal segment from the N-terminus to the C-terminus, and the amino acid sequences of the OX40-N-terminal segment, the OX40-intermediate segment and the OX40-C-terminal segment are shown in SEQ ID No. 10 to SEQ ID No. 12; The first costimulatory domain is the intracellular segment of CD28, which is sequentially divided from the N-terminus to the C-terminus into the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment. The amino acid sequences of the CD28-N-terminal segment, the CD28-middle segment, and the CD28-C-terminal segment are shown in SEQ ID No. 1 to SEQ ID No.

3. The recombinant costimulatory molecule consists of a 4-1BB-N-terminal segment, a 4-1BB-middle segment, a CD28-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a 4-1BB-N-terminal segment, a CD28-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a 4-1BB-N-terminal segment, a CD28-middle segment, and a 4-1BB-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of a 4-1BB-N-terminal segment, a 4-1BB-middle segment, and a CD28-C-terminal segment connected sequentially from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of an OX40-N-terminal segment, an OX40-middle segment, a CD28-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus; Alternatively, the recombinant costimulatory molecule consists of an OX40-N-terminal segment, a CD28-middle segment, and an OX40-C-terminal segment sequentially connected from the N-terminus to the C-terminus; Alternatively, the recombinant co-stimulatory molecule consists of an OX40-N-terminal segment, an OX40-middle segment, and a CD28-C-terminal segment sequentially connected from the N-terminus to the C-terminus.

5. The chimeric antigen receptor according to claim 4, characterized in that The intracellular domain further comprises an intracellular signaling domain connected to the recombinant co-stimulatory molecule, and the chimeric antigen receptor further comprises an extracellular domain, a hinge and a transmembrane region.

6. The chimeric antigen receptor according to claim 5, characterized in that The intracellular signaling domain is the intracellular segment of the CD3ζ chain, the extracellular domain is a single-chain antibody targeting CD19 or mesothelin, and the hinge and transmembrane region are both derived from CD8α.

7. A recombinant vector, characterized in that Contains the coding sequence of the chimeric antigen receptor according to any one of claims 1 to 6.

8. The recombinant vector according to claim 7, characterized in that The nucleotide sequences of the CD28-N-terminal segment, CD28-middle segment, and CD28-C-terminal segment are shown in SEQ ID No. 13 to SEQ ID No. 15, respectively; and / or, the nucleotide sequences of the 4-1BB-N-terminal segment, the 4-1BB8-middle segment, and the 4-1BB-C-terminal segment are shown in SEQ ID No. 19 to SEQ ID No. 21, respectively; And / or, the nucleotide sequences of the OX40-N-terminal segment, the OX40-middle segment, and the OX40-C-terminal segment are shown as SEQ ID No. 22 to SEQ ID No. 24, respectively.

9. The method for preparing the recombinant vector according to any one of claims 7 to 8, characterized in that: The steps include: The coding sequence of the chimeric antigen receptor according to any one of claims 1 to 6 is cloned into a genetic engineering vector to obtain a recombinant vector.

10. The method for preparing the recombinant vector according to claim 9, characterized in that: The chimeric antigen receptor includes an intracellular domain, and the intracellular domain contains a recombinant costimulatory molecule. The step of cloning the coding sequence of the chimeric antigen receptor according to any one of claims 1 to 6 into a genetic engineering vector also includes a step of constructing the recombinant costimulatory molecule.

11. The method for preparing the recombinant vector according to any one of claims 9 to 10, characterized in that: The method further comprises the step of screening the signal characteristics of the chimeric antigen receptor in the recombinant vector: A plasmid containing the pNF-κB-mCherry sequence and a plasmid containing the piggyBac transposase sequence were transformed into a cell line containing the pNFAT-EGFP reporter system to obtain a dual reporter signal cell line; The recombinant vector is introduced into the dual-reporter signal cell line, target cell antigen stimulation is performed, and then the fluorescence intensity of the dual-reporter signal cell line is detected to obtain the NFAT and NF-κB signal kinetic characteristics, and screen out recombinant vectors with cell activation potential.

12. A recombinant cell, characterized in that Capable of expressing the chimeric antigen receptor according to any one of claims 1 to 6 or containing the recombinant vector according to any one of claims 7 to 8.

13. The recombinant cell according to claim 12, characterized in that The recombinant cell is a T cell capable of expressing the chimeric antigen receptor or a T cell introduced with the recombinant vector.

14. Use of the chimeric antigen receptor according to any one of claims 1 to 6, the recombinant vector according to any one of claims 7 to 8, or the recombinant cell according to any one of claims 12 to 13 in the preparation of an anti-tumor drug.

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