WO2023005219A1 - Chimeric antigen receptor modified by ubiquitin coupling and immune cell - Google Patents

Abstract

Provided are a chimeric antigen receptor modified by ubiquitin coupling and an immune cell. The ubiquitin is coupled to a C-terminus of the chimeric antigen receptor, and is suitable for a variety of current CAR-T cell production technologies, and particularly suitable for CAR-T therapeutic products developed against solid tumor targets.

Description

A ubiquitin-conjugated modified chimeric antigen receptor and immune cells technical field

The invention relates to the field of biomedicine, in particular to a ubiquitin-coupled modified chimeric antigen receptor and immune cells.

Background technique

Chimeric antigen receptor (CAR) is an artificially engineered tumor-targeting antigen receptor. CAR-T therapy is an adoptive immunotherapy that expresses CARs that specifically recognize antigens and transmit T cell signals on the patient's own T cells, enabling them to recognize and kill tumor cells in the patient's body. The composition of CAR mainly includes: a single chain antibody (single chain antibody fragment, scFv) that can recognize and bind to a specific tumor antigen, and its specificity can realize precise recognition and "targeted attack" on tumor cells; T cell receptor (T cell The intracellular domain of the receptor (TCR) ζ chain can effectively activate T cells after the scFv structure recognizes tumor antigens, and the activated T cells can secrete a large amount of cytokines, and effectively kill tumor cells while rapidly proliferating themselves. And recruit other immune cells through secreted inflammatory factors; on this basis, adding the signaling domain of co-stimulatory molecules such as CD28 or 41BB to the intracellular region of CAR can enhance the ability of CAR to activate T cells and improve the ability of CAR-T cells to activate T cells. ability to survive in vivo.

At present, CAR-T therapy has achieved remarkable success in the clinical treatment of various types of tumors, especially for blood tumors. In 2017, the U.S. FDA successively approved the marketing of two commercial CAR-T therapeutic products targeting CD19 antigen for the treatment of relapsed and refractory malignant B-cell leukemia and lymphoma, and achieved remarkable curative effect. However, CAR-T therapy still has many limitations in the treatment of solid tumors. In 2019, Robbie et al. summarized the problems encountered in the clinical application of current CAR-T therapy, and pointed out that one of the reasons for limiting the function of CAR-T cells in vivo is due to the tumor microenvironment, excessive burden of tumor targets, The exhaustion of T cell function caused by factors such as excessive self-activation level of T cell background can not effectively kill tumors. A large number of studies have shown that reducing the influence of CAR on the self-activation of T cells can effectively alleviate the problem of low function of CAR-T cells caused by excessive differentiation and premature exhaustion in the process of culture and proliferation of CAR-T cells, and then can effectively Improve the effect of CAR-T therapy, which provides a direction for optimizing the design of CAR-T.

At present, the existing optimal design strategies for CAR-T mainly include: reducing the signal of CAR by mutating and inactivating different combinations of ITAM regions in CD3ζ; mutating the co-stimulatory domain of CD28 to reduce the co-stimulatory signal of CD28; trying different structural combinations , explore the most suitable CAR structure for different targets and the lowest level of background self-activation; co-transduce transcription factors involved in the regulation of T cell proliferation and differentiation into T cells with CAR to reduce the self-activation of CAR-T cells Signaling, suppression of exhaustion of T cells, etc. However, this type of design often needs to try CARs targeting different targets, which consumes a lot of manpower and material resources, and has the disadvantages of large errors and low universality. Therefore, a more practical and universal transformation aimed at the CAR structure itself is a more optimal solution.

Contents of the invention

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide a ubiquitin-modified chimeric antigen receptor and immune cells to solve the problems in the prior art.

To achieve the above purpose and other related purposes, the present invention firstly provides a chimeric antigen receptor modification method, which includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.

The present invention provides a ubiquitin-modified chimeric antigen receptor, the ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the C-terminus of the intracellular domain is coupled with ubiquitin .

The present invention provides an isolated polynucleotide comprising nucleotides encoding the ubiquitin-modified chimeric antigen receptor.

The present invention provides a nucleic acid construct comprising the isolated polynucleotide.

The present invention provides an immune cell, which includes the nucleic acid construct or the exogenous polynucleotide integrated in the genome, or can express the ubiquitin-modified chimeric antigen receptor.

The immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages or CAR-TIL cells.

The present invention also provides a preparation method of the immune cells, the preparation method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells producing immune effector cells.

The immune effector cells are selected from T cells, NK cells, macrophages or TIL cells.

The present invention also provides the use of the immune cells in the preparation of medicines for treating cancer or immune diseases.

The present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.

As mentioned above, the ubiquitin-conjugated modified chimeric antigen receptor and immune cells of the present invention have the following beneficial effects: provide a method for the optimization and transformation of CAR-T, especially for CAR-T that is not capable of proliferating in solid tumors Provides solutions to the best problems. The optimization and transformation scheme of CAR with high self-activation level, which is simple and applicable to different targets, has the advantages of simple application, high repeatability, good universality, high degree of optimization, and good anti-tumor activity. Suitable for all kinds of current CAR-T cell production technologies, especially suitable for CAR-T therapeutic products developed for solid tumor targets. , Excessive exhaustion and other problems provide a new solution.

Description of drawings

Figure 1 shows the detection of the self-activation level of CAR-T cell lines targeting different targets in the human T lymphoma cell line Jurkat, using the expression of indicator proteins such as CD69 and ICOS to measure the activation level of T cells In this case, the background self-activation level of these cell lines was evaluated. The left panel shows the detected expression level of CD69, and the right panel shows the detected ICOS expression level. As shown in the figure, CARs targeting different targets used in a large number of clinical and preclinical trials lead to high levels of background self-activation of T cells. Especially CARs targeting GD2, Her2, CSPG4, EGFR, Meso, GPC3, and multiple targets of solid tumors (the nucleotide sequence is as shown in SEQ ID NO.3-11, and the amino acid sequence is as in SEQ ID NO.19-27) , and its background self-activation level is relatively high. Previous studies have proved that excessive self-activation of CAR-T cells will lead to premature differentiation, decreased proliferation ability, increased cell exhaustion level, and greatly reduced anti-tumor killing ability, etc., which is positively correlated with poor clinical treatment effect.

Figure 2-1 to Figure 2-3 show that CD19-28WT-CAR (nucleotide sequence such as SEQ ID NO.12, amino acid sequence such as SEQ ID NO.28) and a CD19-targeting CAR C-terminal coupling Wild-type ubiquitin (wtUb) (nucleotide sequence such as SEQ ID NO.13, amino acid sequence such as SEQ ID NO.29) or monoubiquitin (monoUb) (nucleotide sequence such as SEQ ID NO.14, amino acid sequence such as The CD19-28wtUb-CAR and CD19-28MonoUb-CAR obtained from SEQ ID NO.30) were expressed in Jurkat T cell line (Fig. 2-1) or primary human T cells (Fig. 2-2), respectively. Changes in the expression level of CAR-type on the cell membrane, and the impact of this modification on the tumor-killing ability of CAR-T cells. The test results showed that the coupling of wtUb and monoUb can effectively down-regulate the expression level of CAR on the cell membrane surface, and the down-regulation of wtUb CAR is more significant. In the in vitro killing test, CAR-T cells were co-incubated with CD19+ target cells at a certain ratio, and the tumor killing ability of CAR-T cells was reflected by detecting the amount of target cells after 18 hours (Figure 2-3). The test results showed that the monoUb-modified CAR can still effectively mediate the killing effect of CAR-T cells on tumor target cells, while the wtUb-modified CAR may have a significantly reduced tumor-killing ability due to its low expression level. Therefore, subsequent research and optimization focused on CARs coupled with monoubiquitin.

Figure 3-1 to Figure 3-4 show that after expressing a variety of monoubiquitin-coupled CD28 CARs targeting different targets in the Jurkat cell line, the effect of the modification on the background self-activation level of CAR-T was tested to verify this Universality of the program. Detection of CD19 as the target (Figure 3-1), GD2 (nucleotide sequence such as SEQ ID NO.15, amino acid sequence such as SEQ ID NO.31, the results as shown in Figure 3-2) as the target, GPC3 ( Nucleotide sequence such as SEQ ID NO.16, amino acid sequence such as SEQ ID NO.32, the results are shown in Figure 3-3) as the target, and CD19 and CD22 as the dual target (nucleotide sequence such as SEQ ID NO.33, Amino acid sequence such as SEQ ID NO.34, results shown in Figure 3-4) the expression level of monoubiquitin-coupled CAR on the cell membrane, and the background self-activation signal of the corresponding CAR-T cells (with CD19 and CD22 as the The nucleotide sequence of the dual-target WT-CAR is shown in SEQ ID NO.35, and the amino acid sequence is shown in SEQ ID NO.36). The results are shown in the figure, the expression levels of the four CAR-T cells were significantly decreased; the expression levels of CD69 and ICOS, the indicator proteins for T cell activation, reflected the background self-activation signal of CAR-T cells, and the expression levels of the four CAR-T cells Significant improvement was seen in the level of self-activation at the bottom. It shows that this scheme is universal, and it can effectively down-regulate the self-activation signals of CARs with different targets and high levels of self-activation.

Figure 4-1 to Figure 4-4 show the detection of wild-type targets (ie CD19-28WT-CAR T cells, GD2-28WT-CAR T cells, GPC3-28WT-CAR T cells, GPC3-28WT-CAR T cells, CD19×22WT-CAR T cells) and CAR-T cells coupled with monoubiquitin (i.e. CD19-28MonoUb-CAR T cells, GD2-28MonoUb-CAR T cells, GPC3-28MonoUb-CAR T cells, CD19×22MonoUb-CAR T cells, CD19×22MonoUb-CAR T cells differentiation of T cells). By flow cytometric detection of the levels of CD45RA and CD62L expressed by cells, the differentiation status of T cells can be divided into CD45RA+CD62L+ (Stem cell memory T cell, T _SCM ), CD45RA-CD62L+ (Central memory T cell, T _CM ), CD45RA- CD62L- (Effector memory T cell, T _EM ), and CD45RA+CD62L- (Effector T cell, T _EFF ), the differentiation level increases successively, and the proliferation and differentiation ability decrease successively. As shown in the figure, among CAR-T cells targeting 4 different targets (Figure 4-1 is CAR-T cells targeting CD19, Figure 4-2 is CAR-T cells targeting GD2, Figure 4-3 CAR-T cells targeting GPC3, Figure 4-4 shows CAR-T cells targeting both CD19 and CD22, the left figure in each figure is the detected CD4, and the right figure is the detected CD8), coupled with single The modification of ubiquitin can effectively slow down the differentiation of T cells, so that more CAR-T cells remain in the _TSCM state, which also implies that CAR-T cells coupled with monoubiquitin should have better persistence. proliferation and tumor killing ability.

Figure 5 shows the in vitro detection of the proliferation of GD2-targeting wild-type and monoubiquitin-coupled CAR-T cells under continuous stimulation of target cells. First, the same amount of GD2-28WT-CAR and GD2-28MonoUb-CAR T cells were given the same amount of target cells (irradiated) to stimulate. Afterwards, live cell counts were performed every 2 days to record cell proliferation. At the same time, add an appropriate amount of medium and adjust the cell density to 1*10^6/ml to continue the culture. The results of this experiment showed that in the presence and continuous stimulation of target cells, monoubiquitin-coupled CAR-T cells had lower background self-activation levels and cell differentiation degrees, and had better sustained proliferation capabilities.

Figure 6-1 to Figure 6-2 show the in vivo tumor killing experiments in tumor-bearing mice, verifying the functional differences between wild-type and monoubiquitin-coupled CAR-T cells targeting CD19. The tumor-bearing mice were randomly divided into 3 groups, and the same amount of normal T cells not transfected with CAR, CD19 WT CAR-T cells and CD19 MonoUb CAR-T cells were given to the mice by tail vein injection respectively. Imaging equipment (in vivo imaging system, IVIS) tracked and monitored the growth of tumor cells in mice. In the case of reinfusion of fewer CAR-T cells, wild-type CAR-T cells have limited ability to control tumors, while CAR-T cells coupled with monoubiquitin can still effectively control tumor growth to a certain extent. In individual mice, tumor clearance can be achieved (Figure 6-1), which effectively improves the survival rate of tumor-bearing mice (Figure 6-2), and its efficacy is significantly better than that of wild-type CAR-T cells.

Figure 7-1 to Figure 7-2 show the detection of CAR-T cell proliferation, differentiation and exhaustion in tumor-bearing mice. The results showed that under the stimulation of tumor cells in vivo, CAR-T cells coupled with monoubiquitin still had a higher proportion of stem cell memory T cells (T _SCM ) with higher sustained proliferation ability (Figure 7-1, The left picture shows CD4 CAR-T cells in the bone marrow, and the middle picture shows CD8 CAR-T cells in the bone marrow); and correspondingly, the number of CAR-T cells coupled with monoubiquitin is also significantly more than that of wild-type CAR-T cells ( Figure 7-1, the right figure is the count of CAR-T cells in the spleen), and the indicative proteins of its cell exhaustion (PD1, LAG3, TIM3, that is, Figure 7-2, the left figure is the CD4 CAR-T cells in the bone marrow, and the right figure The expression level of CD8 CAR-T cells in bone marrow is also significantly lower, which may be related to the weaker activation signal of CAR coupled with monoubiquitin under the condition of target cell stimulation, and the weaker activation signal is enough to make T cells exert Normal tumor killing function; this also implies that the excessive activation signal of wild-type CAR may lead to rapid differentiation and premature exhaustion of T cells, thereby limiting the anti-tumor efficacy of CAR-T cells.

Detailed ways

The present invention provides an optimized transformation scheme of CAR with high self-activation level that is simple and applicable to different targets. Specifically, a ubiquitin protein mutant (monoubiquitin, MonoUb for short) is coupled to the C-terminus of CARs targeting different targets to obtain MonoUb-CAR. Therefore, the present invention firstly provides a chimeric antigen receptor modification method, which includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.

Ubiquitin is a small protein present in all eukaryotes (most eukaryotic cells). Ubiquitin consists of 76 amino acids with a molecular weight of about 8.451kDa. Its main function is to mark proteins that need to be broken down so that they can be degraded by the 26S proteasome. For membrane proteins, especially some special ubiquitin modification types may be involved in the down-regulation, transport and vesicle sorting of membrane proteins, as well as the regulation of their signal transmission. Ubiquitin as referred to in this application refers to any wild-type ubiquitin or mutant ubiquitin from any eukaryotic cellular source, including mammals such as primates (e.g. humans) and rodents (e.g. mice and rats) . Ubiquitin is a very conserved protein. Almost all eukaryotic cells express ubiquitin since yeast, and its amino acid sequence is basically identical (yeast and human ubiquitin have only one amino acid difference).

The type of ubiquitin is wild-type ubiquitin or mutant ubiquitin. Wild-type ubiquitin can be modified by intracellular endogenous ubiquitin but cannot be used as a substrate to modify other proteins, so it can form ubiquitin chains. In a preferred embodiment, the ubiquitin is mutant ubiquitin. Because the mutant ubiquitin cannot be modified by endogenous ubiquitin, and the mutant ubiquitin cannot be used as a substrate to ubiquitinate other proteins, the mutant ubiquitin is monoubiquitin, so the mutant ubiquitin can also be Known as mutant monoubiquitin or monoubiquitin.

The ubiquitin is coupled to the C-terminus of the chimeric antigen receptor through a linker peptide. The connecting peptides described in the present application may be commonly used connecting peptides in the art. The connecting peptide is, for example, GSGGSG, GSGGSGG GSGGSGGG or GGGGSGGG or GGSGGGSGGGSAAA.

The present invention also provides a ubiquitin-modified chimeric antigen receptor, which includes a transmembrane domain, an intracellular domain, and an extracellular domain, and the C-terminus of the intracellular domain is coupled with ubiquitin white.

The ubiquitin-modified chimeric antigen receptor is obtained by the engineering method.

In some embodiments of the present invention, the transmembrane domain may include transmembrane domains of protein molecules such as CD8α, CD28, and DAP10. For example, the amino acid sequence of CD8α may include the following sequence: IYIWAPLAGTCGVLLLSLVITLYC. For another example, refer to NM_001145873 for the sequence of CD8α, and refer to NM_006139 for the sequence of CD28.

In certain embodiments of the invention, the intracellular domain may comprise a co-stimulatory domain and/or a signaling domain. For example, the intracellular domain may include one or a combination of several of the following: signal transduction domains of protein molecules such as 4-1BB, CD28, OX40, ICOS, CD3ζ, and DAP10.

For another example, the amino acid sequence of 4-1BB includes as follows:

The amino acid sequence of the CD3ζ includes as follows:

For another example, refer to NM_001561 for the sequence of 4-1BB, refer to NM_006139 for the sequence of CD28, refer to NM_003327 for the sequence of OX40, refer to NM_012092 for the sequence of ICOS, refer to NM_198053 for the sequence of CD3ζ, and refer to NM_014266 for the sequence of DAP 10. In a specific embodiment of the present invention, the intracellular domain sequentially includes CD28 and CD3ζ from the N-terminus to the C-terminus. In some embodiments of the present invention, the chimeric antigen receptor includes an extracellular domain, a transmembrane domain, and an intracellular domain sequentially from the N-terminus to the C-terminus.

In some embodiments of the present invention, the chimeric antigen receptor includes a single-chain antibody, a transmembrane domain, and an intracellular domain in sequence from the N-terminus to the C-terminus. In some specific embodiments of the present invention, the chimeric antigen receptor includes a single-chain antibody, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain sequentially from the N-terminus to the C-terminus. In a specific embodiment of the present invention, the chimeric antigen receptor comprises a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, and a CD3ζ signaling domain sequentially from the N-terminus to the C-terminus. In another specific embodiment of the present invention, the chimeric antigen receptor includes a single-chain antibody, a CD8α transmembrane region, an OX40 co-stimulatory domain, and a CD3ζ signaling domain sequentially from the N-terminal to the C-terminal. In another specific embodiment of the present invention, the chimeric antigen receptor comprises a single-chain antibody, a CD8α transmembrane region, an ICOS co-stimulatory domain, and a CD3ζ signaling domain sequentially from the N-terminal to the C-terminal. In another specific embodiment of the present invention, the chimeric antigen receptor comprises a single-chain antibody, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain sequentially from the N-terminus to the C-terminus. In another specific embodiment of the present invention, the chimeric antigen receptor comprises a single-chain antibody, a CD28 transmembrane region, a CD28 co-stimulatory domain, an OX40 co-stimulatory domain, and a CD3ζ signaling domain from the N-terminus to the C-terminus. . In another specific embodiment of the present invention, the chimeric antigen receptor includes CD8α signal peptide, myc tag, scFv, extracellular domain composed of CD8α hinge, transmembrane domain of CD8α, CD28 and CD3ζ in sequence from N-terminal to C-terminal Intracellular domains organized in tandem. The ubiquitin-modified chimeric antigen receptor, that is, the CD3ζ of any one of the above chimeric antigen receptors is connected with ubiquitin through a linker peptide (or linker).

The single-chain antibody of the present invention is not specifically limited, and can be selected from any single-chain antibody. Examples of single-chain antibodies used in the present invention include any one or more of CD19 scFv, GD2 scFv, GPC3 scFv, Her2 scFv, CSPG4 scFv, EGFR scFv, Meso scFv, TRBC1 scFv, CD133 scFv, and BCMA scFv. When multiple, eg, two, single chain antibodies are used, the chimeric antigen receptor is a multi-target chimeric antigen receptor.

The ubiquitin referred to in this application is selected from wild-type ubiquitin or mutant ubiquitin.

The amino acid sequence of wild-type ubiquitin is shown in SEQ ID NO.17.

In a preferred embodiment, the ubiquitin is selected from mutant ubiquitin. The amino acid sequence of the mutant ubiquitin selected in the present invention is shown in SEQ ID NO.18.

The present invention provides an isolated polynucleotide comprising nucleotides encoding the ubiquitin-modified chimeric antigen receptor.

The polynucleotides encoding the transmembrane domain, intracellular domain and extracellular domain can all be selected from polynucleotides in the prior art.

In a specific embodiment of the present invention, the polynucleotide encoding the ubiquitin-modified chimeric antigen receptor sequentially includes nucleotides encoding the CD8α signal peptide, myc tag, scFv, and the ectodomain composed of the CD8α hinge, Nucleotides encoding the transmembrane domain of CD8α, nucleotides encoding the intracellular domain composed of CD28, CD3ζ, and ubiquitin in tandem.

The nucleotide sequence of wild-type ubiquitin is shown in SEQ ID NO.1. In a preferred embodiment, the ubiquitin is selected from mutant ubiquitin. The polynucleotide sequence encoding mutant ubiquitin selected in the present invention is shown in SEQ ID NO.2.

The present invention provides a nucleic acid construct comprising the isolated polynucleotide.

The term "nucleic acid construct" refers to an artificially constructed nucleic acid segment that can be introduced into cells or tissues, and the nucleic acid construct is a non-viral vector or a viral vector. The viral vectors are lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, and baculoviral vectors. In some embodiments of the present invention, the nucleic acid construct is a lentiviral vector, and the lentiviral vector includes a vector backbone, that is, an empty vector and an expression framework. The empty vector includes various expression-controlling elements, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element and a reporter gene. In addition, the vector may also contain an origin of replication. The empty vector is, for example, pHR-hEF1α-IRES-EGFP empty vector. The expression framework is the isolated polynucleotide.

The term "vector" refers to a nucleic acid fragment or polynucleotide fragment used to introduce or transfer one or more nucleic acids or one or more polynucleotides into a target cell or tissue. Typically, vectors are used to introduce foreign DNA into another cell or tissue. The vector may contain a bacterial resistance gene for growth in bacteria and a promoter for expressing a protein of interest in an organism. DNA can be produced in vitro by PCR or any other suitable technique or techniques known to those skilled in the art.

The present invention provides an immune cell, which includes the nucleic acid construct or the exogenous polynucleotide integrated in the genome, or is capable of expressing the ubiquitin-modified chimeric antigen receptor.

The immune cells are selected from CAR-T cells, CAR-NK cells, CAR-macrophages or CAR-TIL cells.

The present invention also provides a preparation method of the immune cells, the preparation method comprising introducing the nucleic acid construct or the polynucleotide into immune effector cells or stem cells producing immune effector cells.

The immune effector cells are selected from T cells, NK cells, macrophages or TIL cells.

The present invention also provides the use of the immune cells in the preparation of medicines for treating cancer or immune diseases.

"Cancer" in this application refers to any medical condition that is mediated by the growth, proliferation or metastasis of tumor or malignant cells and causes solid tumors and non-solid tumors such as leukemia. "Tumor" in the present invention refers to the solid substance of tumor and/or malignant cells.

The cancer is, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer , thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer; leukemia, lymphoma, myeloma, mycoses fungoids (mycoses fungoids), Merkel cell carcinoma and other hematological malignancies, Such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic-rich large B-cell lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma lymphoma (DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma.

The immune diseases are, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, and rheumatoid arthritis.

"Treatment" or "therapy" for a condition includes preventing or alleviating a condition, reducing the rate at which a condition occurs or develops, reducing the risk of developing a condition, preventing or delaying the development of symptoms associated with a condition , to reduce or terminate symptoms associated with a condition, to produce a complete or partial reversal of a condition, to cure a condition, or a combination of the above. With respect to cancer, "treating" or "therapy" can refer to inhibiting or slowing the growth, reproduction, or metastasis of tumor or malignant cells, or some combination thereof. With respect to tumors, "treatment" or "therapy" includes eradicating all or part of the tumor, inhibiting or slowing tumor growth and metastasis, preventing or delaying tumor progression, or some combination of the above.

The present invention also provides a treatment method for tumors or immune diseases, the treatment method comprising administering a therapeutically effective amount of the immune cells to a subject.

The cancer is, for example, non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer, mesothelioma, melanoma, head and neck cancer , thyroid cancer, sarcoma, prostate cancer, glioblastoma, cervical cancer, thymus cancer; leukemia, lymphoma, myeloma, mycoses fungoids (mycoses fungoids), Merkel cell carcinoma and other hematological malignancies, Such as classical Hodgkin lymphoma (CHL), primary mediastinal large B-cell lymphoma, T-cell/histiocytic B-rich lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse large B-cell lymphoma ( DLBCL), plasmablastic lymphoma, extranodal NK/T cell lymphoma, nasopharyngeal carcinoma and HHV8-related primary effusion lymphoma, Hodgkin lymphoma.

The immune diseases are, for example, systemic lupus erythematosus (SLE), autoimmune diabetes, psoriasis, vitiligo, scleroderma, rheumatoid arthritis.

The "therapeutically effective amount" or "effective dose" in the present invention refers to the dose or concentration of a drug that can effectively treat the disease or state associated with the antigen of the chimeric antigen receptor. For example, for the use of the antibody or antigen-binding fragment thereof disclosed in the present invention, the therapeutically effective amount is at the dosage or concentration, the antibody or antigen-binding compound can eliminate all or part of the tumor, inhibit or slow down tumor growth, inhibit Mediating the growth or proliferation of cells in a cancerous state, inhibiting tumor cell metastasis, alleviating any symptoms or markers associated with a tumor or cancerous state, preventing or delaying the development of a tumor or cancerous state, or some combination of the above.

monoUb-CAR can effectively promote the endocytosis and degradation of CAR, greatly reduce the expression of CAR on the cell surface, realize a substantial decrease in the background self-activation level of immune cells such as CAR-T cells, and significantly reduce the over-differentiation of immune cells such as T cells. Speed, degree of functional exhaustion, etc. In some embodiments of the present invention, monoUb-CAR significantly enhances the sustained proliferation ability of CD28 CAR-T cells in vitro and in vivo under target cell stimulation conditions, and effectively improves the tumorigenicity of CAR-T cells in tumor-bearing mice. killing ability, thereby improving the survival rate of mice.

The present invention compares the anti-tumor effects of CD28 monoUb CAR-T and CD28 WT CAR-T in vivo on a tumor mouse model. At the level of proliferation, CD28 monoUb CAR-T has a stronger proliferative response and longer-lasting proliferative ability; in terms of cell differentiation phenotype, whether in the spleen, blood, or tumor, the engineered CAR-T has accumulated more Stem cell memory T cells (Stem cell memory T cells, T _SCM ) and reduced differentiation to terminal effector T cells. Therefore, the modified CAR-T can more effectively infiltrate and kill tumor tissues, and at the same injection dose of T cells, the modified CAR-T can more effectively control the development of tumors.

Embodiments of the present invention are described below through specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.

Before further describing the specific embodiments of the present invention, it should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are to describe specific specific embodiments, It is not intended to limit the protection scope of the present invention; in the description and claims of the present invention, unless the context clearly indicates otherwise, the singular forms "a", "an" and "the" include plural forms.

When the examples give numerical ranges, it should be understood that, unless otherwise stated in the present invention, the two endpoints of each numerical range and any value between the two endpoints can be selected. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to those skilled in the art's grasp of the prior art and the description of the present invention, the methods, equipment, and materials described in the embodiments of the present invention can also be used Any methods, apparatus and materials of the prior art similar or equivalent to the practice of the present invention.

Unless otherwise specified, conventional technical operations such as molecular biology, microbiology, cell biology, biochemistry and immunology used in the practice of the present invention are within the understanding of skilled artisans. These techniques are widely used and can be found fully described in literature such as: "Molecular cloning: A Laboratory Manual, Fourth edition" (M.R. Green, et al. 2014); "Oligonucleotide Synthesis" (M.J. Gait, et al. 1984); "Polymerase Chain Reaction: Principles, Applications and Troubleshooting" (M.E.Babar, et al.2011); "Short Protocols in Molecular Biology, Fifth edition" (F.M.Ausubel, et al.2002); "Methods in Molecular Biology" (Humana Press); "Gene Transfer Vectors for Mammalian Cells" (J.H.Miller and M.P.Calos.1987); "Culture of Animal Cell" (R.I.Freshney, et al.2010); "Methods in Enzymology" (Academic Press, Inc) ; "Using Antibodies: A Laboratory Manual" (E.Harlow and D.Lane.1999); "Handbook of Experimental Immunology" (L.A.Herzenberg, et al.1997); "Current Protocols in Immunology" (J.E.Coligan, et al. 2002).

Embodiment 1: Vector construction of CAR

The antigen-specific single-chain antibody (scFv) sequences of CD19, GD2, and GPC3 CAR used in the present invention are respectively from the clinically used FMC63, 14g2A, and GPC3 sequences; the dual-target CAR is the integration of specific antibodies targeting CD19 and CD22 For the integration method, refer to the report in the article "CD22-targeted CAR T cells induce remission in B-ALL that is naive or resistant to CD19-targeted CAR immunotherapy". The extracellular segment structure of CAR is composed of CD8α signal peptide sequence, myc tag sequence, scFv sequence, and CD8α hinge sequence in series; the transmembrane sequence is the transmembrane region sequence of CD8α; the intracellular segment structure is composed of human CD28 intracellular segment sequence in series Sequence composition of human CD3ζ intracellular segment. The ubiquitin-modified chimeric antigen receptor connects ubiquitin to CD3ζ through a linker peptide in the intracellular domain. The above scFv amino acid sequences were converted into base sequences after codon optimization, and synthesized by a third-party company (genscript). The base sequences of all CARs in the present invention were finally cloned into the pHR-hEF1α-IRES-EGFP vector (derived from addgene) by Gibson connection.

Example 2: Human primary T cell culture and lentivirus infection

Human primary T cells were obtained from healthy informed volunteers. Primary T cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin sulfate, 1 mM sodium pyruvate, non-essential amino acids, and 55 μM 2-mercaptoethanol ( The above reagents were purchased from Gibco). In order to maintain the proliferation of T cells, 100 U/ml of hIL-2 (Sigma-Aldrich) was added to the culture medium.

Preparation of lentivirus: Resuspend Lenti-X 293T cells (TaKaRa#632180) in DMEM medium (Gibco#11995-065) containing 10% fetal bovine serum and without antibiotics, and make 6.5×10 ⁵ cells/well The density was inoculated in 6-well cell culture plate (Corning#CLS3516) and cultured for 24 hours. Using liposome transfection system (Mirus#2300), mix 500ng lentiviral packaging plasmid pCMVdR8.92 (Addgene#8455) and 50ng pMD2.G (Addgene#12259) with 500ng lentiviral plasmid to be packaged, according to liposome After mixing the operation steps of the transfection instructions, add Lenti-X 293T cells. Discard the liposome-containing medium after 16-18 hours, and add an appropriate amount of fresh medium; collect the cell supernatant after 48 hours, concentrate directly or by ultracentrifugation, and store in a -80°C refrigerator for later use.

Lentiviral infection of primary T cells: Use magnetic beads (Life Technologies #11132D) coated with anti-human CD3 and anti-human CD28 antibodies to activate T cells, mix T cells with magnetic beads 1:3, add to the preparation after 24 hours of culture Infect with a good lentivirus; after 18 hours, the medium containing the virus solution was discarded and replaced with fresh T cell complete medium. After T cells were stimulated by magnetic beads for 4-5 days, the magnetic beads were removed, and the cell density was adjusted to 0.8-1*10^6/ml with T cell complete medium, and fresh T cell complete medium was supplemented every 2 days.

The procedure for expressing monoubiquitin-conjugated CD28 CARs targeting different targets in Jurkat cell lines was the same as in T cells.

Example 3: Flow Cytometry Analysis

For staining of cell surface markers: dilute the antibody in FACS buffer (phosphate buffered saline PBS + 2% fetal bovine serum) at an appropriate ratio, and resuspend the cells with an appropriate amount of antibody diluent, and co-suspend in a dark environment at 4°C. Incubate for 25 minutes; wash the cells 3 times with FACS buffer, resuspend the cells in FACS buffer containing appropriate concentration of DAPI, and detect on the machine.

Flow cytometry data were acquired by a BD LSR Fortessa machine (BD bioscience) and analyzed using FlowJo software (Tree Star). Antibodies used in flow cytometry are listed below.

Example 4: In vitro killing function detection of CAR-T based on flow cytometry

After mixing double-positive K562 target cells expressing CD19 and mCherry fluorescence with K562 non-target cells not expressing CD19 and mCherry fluorescence at a ratio of 1:1, they were mixed with CAR-T cells at a certain ratio of effector cells: target cells and co-incubated 24 hours. Cells were cultured in complete T cell medium without IL-2. Flow cytometry analysis: the K562 mixed cell population without T cells was used as the control group, and the proportion of K562 target cells in K562 cells (CK%) was analyzed; in the experimental group containing T cells, the T cells were stained by CD3ε Distinguish the K562 cell population, and then analyze the proportion (EX%) of CD19-K562 target cells in the total K562 cell population, the killing efficiency of T cells=(1-EX%/CK%)×100%

Example 5: Detection of CAR-T cell proliferation ability in vitro

After CD19-28WT-CAR and CD19-28Mono-CAR T cells were counted, they were mixed with irradiated target cells Nalm6 3:1, and the cells were resuspended in IL-2-free T cell complete medium to a cell density of 1 *10^6/ml; Count the viable cells every 2 days to calculate the amount of cell proliferation, and adjust the cell density to 1*10^6/ml with the complete medium of T cells without IL-2; when the cells obtained by the viable cell count When the density is less than or equal to 1*10^6/ml, it indicates that the cells are no longer proliferating and the experiment is terminated.

Example 6: Mouse tumor model and CAR-T cell function detection

In vivo experiments were performed on 5- to 8-week-old combined immunodeficiency (NSG) mice. In order to compare the anti-tumor effect of CAR-T in vivo, NSG mice were first inoculated with 1×106 B lymphoma cells ^Nalm6 expressing the firefly luciferase gene in the tail vein; Treat NSG mice with 2×10 ⁶ CAR-T cells by injection; detect the intensity of firefly luciferase carried by mouse tumor cells every week through a small animal in vivo imaging system to reflect the load of tumor cells, To track the development of tumors in the body. The specific implementation of the small animal in vivo imaging system includes: giving the firefly luciferase substrate (D-luciferin sodium salt) to tumor-bearing mice by intraperitoneal injection, and the substrate dosage is 0.15 mg/g mouse body weight; 10 minutes later After the substrate was fully circulated to the whole body of the mouse, the mouse was anesthetized with 2.5%-3.5% isoflurane gas and then imaged. Bioluminescence imaging was performed with an IVIS Spectral Imaging System (Perkin Elmer), and fluorescence quantitative data were acquired with an in vivo imaging software (Perkin Elmer).

Example 7: Detection of CAR-T cell proliferation, differentiation and exhaustion in tumor-bearing mice

In vivo experiments were performed on 5- to 8-week-old combined immunodeficiency (NSG) mice. In order to compare the differences in the proliferation, differentiation, and exhaustion levels of CAR-T in vivo, NSG mice were first inoculated with ² ×106 B lymphoma cells Nalm6 in the tail vein; after the target cells grew in vivo for 4 days, they were injected through the tail vein. 2×10 ⁶ CAR-T cells were administered to NSG mice; 3 mice inoculated with CD19-28 WT and CD19-28 MonoUb CAR-T cells were sacrificed 7 days, 14 days, and 21 days later, and their spleens and hind limbs were collected. Long bones; grind the spleen to get spleen cells; draw appropriate amount of RPMI medium with a 5ml syringe with a 20-gauge needle, carefully cut off the two ends of the long bone and insert the syringe needle into the marrow cavity of the long bone to flush out the bone marrow cells; treat the spleen with an appropriate amount of erythrocyte lysate After cells and bone marrow cells, the splenocytes were counted, the cells were aliquoted, and different antibody dilutions of the indicators to be detected were incubated respectively, and flow cytometric detection was performed.

The above examples are intended to illustrate the disclosed embodiments of the present invention, and should not be construed as limiting the present invention. In addition, various modifications set forth herein, as well as changes in the method of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in connection with various specific preferred embodiments of the invention, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as mentioned above which are obvious to those skilled in the art to obtain the invention should be included in the scope of the present invention.

Claims (17)

1. A chimeric antigen receptor modification method, characterized in that the modification method includes coupling ubiquitin to the C-terminus of the chimeric antigen receptor.
2. A ubiquitin-modified chimeric antigen receptor, characterized in that the ubiquitin-modified chimeric antigen receptor comprises a transmembrane domain, an intracellular domain and an extracellular domain, and the C-terminus of the intracellular domain is coupled with a ubiquitous white.
3. The ubiquitin-modified chimeric antigen receptor according to claim 2, further comprising any one or more of the following features:

   1) the transmembrane domain is selected from CD8α, CD28 or DAP 10;

   2) The intracellular domain includes costimulatory domain and/or signaling domain.
4. The ubiquitin-modified chimeric antigen receptor according to claim 3, wherein the intracellular domain is selected from one or a combination of the following: 4-1BB, CD28, OX40, ICOS, CD3ζ or DAP 10.
5. The ubiquitin-modified chimeric antigen receptor according to claim 2, wherein the chimeric antigen receptor comprises CD8α signal peptide, protein purification tag, single-chain antibody, and CD8α hinge in sequence in order to form an ectodomain, The transmembrane domain of CD8α, the intracellular domain composed of CD28 and CD3ζ in tandem.
6. The ubiquitin-modified chimeric antigen receptor according to claim 5, wherein the single-chain antibody is selected from GD2 scFv, GPC3 scFv, Her2 scFv, CSPG4 scFv, EGFR scFv, Meso scFv, TRBC1 scFv, CD133 scFv, Any one or more of BCMA scFv or CD19 scFv.
7. The ubiquitin-modified chimeric antigen receptor according to claim 2, wherein the ubiquitin is selected from wild-type ubiquitin or mutant ubiquitin.
8. The ubiquitin-modified chimeric antigen receptor according to claim 2, wherein the ubiquitin is selected from mutant ubiquitin, and the amino acid sequence of the mutant ubiquitin is as shown in SEQ ID NO.18.
9. An isolated polynucleotide, characterized in that the isolated polynucleotide comprises nucleotides encoding the ubiquitin-modified chimeric antigen receptor according to any one of claims 2-8.
10. A nucleic acid construct, characterized in that the nucleic acid construct comprises the polynucleotide according to claim 9.
11. An immune cell, characterized in that, the immune cell comprises the nucleic acid construct according to claim 10 or the exogenous polynucleotide according to claim 9 is integrated in the genome, or is capable of expressing the polynucleotide according to claims 2-8 Any of the ubiquitin-modified chimeric antigen receptors.
12. The immune cell according to claim 11, wherein the immune cell is selected from CAR-T cells, CAR-NK cells, CAR-macrophages or CAR-TIL cells.
13. The preparation method of immune cells according to any one of claims 11-12, characterized in that the preparation method comprises introducing the nucleic acid construct according to claim 10 or the polynucleotide according to claim 9 into immune effector cells or In stem cells that produce immune effector cells.
14. The preparation method according to claim 13, characterized in that the immune effector cells are selected from T cells, NK cells, macrophages or TIL cells.
15. Use of the immune cells according to any one of claims 11-12 in the preparation of medicines for treating cancer or immune diseases.
16. A treatment method for tumors or immune diseases, characterized in that the treatment method comprises administering a therapeutically effective amount of the immune cells according to any one of claims 11-12 to a subject.
17. The treatment method according to claim 16, wherein the cancer is non-small cell lung cancer, small cell lung cancer, renal cell carcinoma, colorectal cancer, ovarian cancer, breast cancer, pancreatic cancer, gastric cancer, bladder cancer, esophageal cancer Carcinoma, Mesothelioma, Melanoma, Head and Neck Cancer, Thyroid Cancer, Sarcoma, Prostate Cancer, Glioblastoma, Cervical Cancer, Thymic Cancer; Leukemia, Lymphoma, Myeloma, Mycosis Fungoides, Merck Myeloid cell carcinoma and other hematologic malignancies, classical Hodgkin lymphoma, primary mediastinal large B-cell lymphoma, T-cell or histiocyte-rich large B-cell lymphoma, EBV-positive and negative PTLD, and EBV-associated diffuse Sexual large B-cell lymphoma, plasmablastic lymphoma, extranodal NK/T-cell lymphoma, nasopharyngeal carcinoma or HHV8-related primary effusion lymphoma, Hodgkin's lymphoma; the immune diseases are Systemic lupus erythematosus, autoimmune diabetes, psoriasis, vitiligo, scleroderma, rheumatoid arthritis.

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