Chimeric antigen receptor based on a humanized anti-CD79B antibody and its use

Abstract

Chimeric antigen receptors based on CD79b humanized antibodies and their use are provided. The present disclosure relates to a chimeric antigen receptor based on a CD79b humanized antibody, comprising an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, a CD3ζ signaling domain, and a self-destruct domain, which are sequentially connected in tandem, wherein the antigen-binding domain is a humanized scFv that binds to the tumor surface antigen CD79b and targets the tumor surface antigen CD79b, and the self-destruct domain is a cysteine protease 9 domain. Applying the chimeric antigen receptor to a patient having a tumor expressing the tumor-specific target CD79b results in few clinical side effects, high safety, can effectively kill tumor cells, can monitor the presence of the patient's CAR-T over a long period of time, and can effectively extend the overall survival rate of the patient.

Description

Technical Field

[0001] The present disclosure relates to the field of cellular immunotherapy for tumors, and in particular, to chimeric antigen receptors based on CD79b humanized antibodies and their use, and in particular, to a method for constructing chimeric antigen receptor T (CAR-T) cells based on tumor-specific target CD79b and their use in anti-tumor therapy.

Background Art

[0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T cell (CAR-T) immunotherapy has become one of the most promising tumor immunotherapies. Generally, a chimeric antigen receptor (CAR) is composed of a tumor-associated antigen binding region, an extracellular hinge region, a transmembrane domain, and an intracellular cell signaling domain. Generally, CAR is linked to the cytoplasmic domain of a T cell signaling molecule via a hinge and transmembrane domain, and includes a single-chain variable fragment (scFv) region of an antibody, that is, a domain that specifically binds to a tumor-associated antigen (TAA). The most common lymphocyte activation part includes a T cell co-stimulatory domain in tandem with a part that induces T cell effector function (such as CD3ζ). In CAR-mediated adoptive immunotherapy, CAR-grafted T cells can directly recognize TAAs on target tumor cells in a non-human leukocyte antigen (HLA)-restricted manner.

[0003] B cell lymphoma is a solid tumor that affects B cells. B cell lymphoma includes both Hodgkin lymphoma and non-Hodgkin lymphoma. There are many types of B cell lymphoma, and classical Hodgkin lymphoma and nodular lymphocyte-predominant Hodgkin lymphoma are now considered to be tumors derived from B cells. Diffuse large B cell lymphoma, follicular lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, and mantle cell lymphoma (MCL) are the five most common types of B cell non-Hodgkin lymphoma, accounting for three out of four non-Hodgkin lymphoma patients.

[0004] Furthermore, B-cell lymphoma is classified into low-grade lymphoma and high-grade lymphoma according to differences in clinical symptoms. Low-grade lymphoma generally progresses slowly, the disease condition is controlled in a stable state, and long-term survival is possible, but it cannot be cured. High-grade lymphoma usually requires intensive treatment, but some have a chance of complete remission. The prognosis and treatment of B-cell lymphoma depend on the specific type, stage, and grade of the lymphoma. One of the treatment methods for patients with B-cell lymphoma is to genetically modify T cells by expressing chimeric antigen receptors (CARs) that target antigens expressed on tumor cells. CARs are antigen receptors designed to recognize cell surface antigens independently of HLA. Attempts to treat these patients with gene-modified cells expressing CARs have achieved promising results.

[0005] CD79b is a cell surface receptor that is part of the B-cell receptor (BCR) signaling complex and is important for the normal development and maintenance of mature B cells. CD79b is generally expressed only in the B-cell lineage and is highly expressed in most non-Hodgkin lymphoma subtypes, making it one of the ideal tumor antigens for targeting B-cell-related tumors in immunotherapy. Currently, among immunotherapies for B-cell tumors, antibody therapy against CD19 has been successfully matured. Such methods have achieved initial success in clinical practice. However, the problems with antibody therapy are that after antibody administration, the antibody exists in the peripheral blood and cannot accurately enter the tumor tissue or minimal residual sites of the tumor, and after administration, the antibody cannot exist in vivo for a long time. Furthermore, such anti-CD19 antibodies have a human-mouse chimeric antibody structure and may be resistant to the human body, making retreatment more difficult.

[0006] Therefore, fully humanized chimeric antigen receptors targeting B-cell surface antigens other than CD19 have been developed. For example, CAR-T cells targeting CD79b can not only take advantage of the targeted therapy of antibodies but also accurately enter tumor tissues and exist in vivo for a long time due to the characteristics of T cells and the high expression of tumor surface antigens, providing a more effective treatment option for relapsed and refractory B-cell tumors.

Summary of the Invention

[0007] Currently, the number of target antigens for tumor treatment by CAR-T technology is still small, and the tumor microenvironment affects the therapeutic effect of CAR-T technology. The object of the present disclosure is to provide a chimeric antigen receptor based on a CD79b humanized antibody and its use. The chimeric antigen receptor (4SCAR-79b) prepared according to the present disclosure optimizes and modifies CAR-T cells modified by the humanized CD79b antibody gene, thereby improving the long-term immunoeffectiveness and safety of antigen targets and enhancing the therapeutic effect of CAR-T cells.

[0008] To achieve the object, the present disclosure adopts the technical solutions described below.

[0009] In a first aspect, the present disclosure provides a chimeric antigen receptor (4SCAR-79b) based on a CD79b humanized antibody, comprising an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, a CD3ζ signaling domain, and a self-destructive domain that are sequentially connected in tandem, wherein the antigen-binding domain is a single-chain variable fragment (scFv) against the tumor surface antigen CD79b, and the amino acid sequence of the scFv against the tumor surface antigen CD79b is the amino acid sequence shown in SEQ ID NO: 1; or an amino acid sequence formed by amino acid substitution, addition, or deletion with respect to the amino acid sequence shown in SEQ ID NO: 1, which specifically binds to the chimeric antigen receptor and has the function of binding to CD79b and inducing T cell signaling and provides a chimeric antigen receptor based on a CD79b humanized antibody selected therefrom.

[0010] In the present disclosure, the amino acid substitution can be one or more.

[0011] In the present disclosure, the antigen-binding domain binds to a tumor surface antigen that is CD79b. By modifying the specially designed genetic structures of scFv and CAR, the genetically modified CAR-T cells can specifically bind to the tumor surface antigen, obtain a relatively mild signal stimulation, thereby exerting an effective killing effect. At the same time, since immune factors are gradually released, the risk of cytokine storm is reduced. The chimeric antigen receptor and tumor antigen in this specification are more effective and safer than other chimeric antigen receptors and other tumor antigens.

[0012] The amino acid sequence (SEQ ID NO: 1) of the scFv against the tumor surface antigen CD79b is as follows:

[0013]

Chemical formula

[0014] In the present disclosure, the CAR signal structure targeting the tumor surface antigen CD79b is specifically modified, and for different CD79b scFvs, the modified 4SCAR-79b can be rapidly modified to show a stronger immune stimulating ability.

[0015] Preferably, the amino acid sequence formed by amino acid substitution, addition or deletion with respect to the amino acid sequence shown in SEQ ID NO: 1 has at least 90%, such as 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity with the amino acid sequence shown in SEQ ID NO: 1, and preferably has an amino acid sequence with at least 95% identity.

[0016] In the present disclosure, the modified amino acid sequence can still specifically bind to the chimeric antigen receptor, bind to CD79b, and has the function of inducing T cell signal transduction.

[0017] Preferably, the transmembrane domain is the CD28 transmembrane domain and / or the CD8α transmembrane domain.

[0018] In the present disclosure, the CD28 transmembrane domain includes a CD28 extracellular signaling domain and a CD28 cell membrane signaling domain.

[0019] In some embodiments, the transmembrane domain may be selected or modified by amino acid substitution.

[0020] Preferably, the costimulatory signaling domain is a combination of a CD28 signaling domain (also specifically referred to herein as the CD28 intracellular signaling domain) and a CD27 signaling domain (also specifically referred to herein as the CD27 intracellular signaling domain).

[0021] In the present disclosure, the CD28 extracellular signaling domain, the CD28 cell membrane signaling domain, and the CD28 intracellular signaling domain together form a CD28 complete signaling domain. The arrangement of the CD28 complete signaling domain and the CD27 signaling domain can be adjusted as needed by those skilled in the art. Different arrangements of the CD28 complete signaling domain and the CD27 signaling domain do not affect the chimeric antigen receptor, and in this application, the sequential combination of CD28 - CD27 is adopted.

[0022] As used herein, CD28 - CD27 refers to the CD28 complete signaling domain and the CD27 signaling domain.

[0023] Preferably, the amino acid sequence of CD28 - CD27 is shown in SEQ ID NO: 2.

[0024] The specific sequence of SEQ ID NO: 2 is as follows:

[0025]

Chemical Structure

[0026] In the present disclosure, the co-stimulatory signaling domain further includes a linker sequence, and the linker sequence is a repetitive sequence of two or more GGGGS (SEQ ID NO: 11) or a related region linker sequence.

[0027] In the present disclosure, the CD28 complete signaling domain includes a CD28 extracellular signaling domain, a CD28 cell membrane signaling domain, and a CD28 intracellular signaling domain.

[0028] The amino acid sequence of the CD28 extracellular signaling domain is shown in SEQ ID NO: 3, and specifically is as follows:

[0029]

Chemical formula

[0030] The amino acid sequence of the CD28 cell membrane signaling domain is shown in SEQ ID NO: 4, and specifically is as follows:

[0031]

Chemical formula

[0032] The amino acid sequence of the CD28 intracellular signaling domain is shown in SEQ ID NO: 5, and specifically is as follows:

[0033]

Chemical formula

[0034] The CD27 signaling domain includes a CD27 intracellular signaling domain, and the amino acid sequence of the CD27 intracellular signaling domain is shown in SEQ ID NO: 6, and specifically is as follows:

[0035]

Chemical formula

[0036] Preferably, the self-destruct domain contains a cysteine protease 9 domain.

[0037] Preferably, the amino acid sequence of the cysteine protease 9 domain is shown in SEQ ID NO: 7, and the amino acid sequence of the cysteine protease 9 domain (SEQ ID NO: 7) is as follows:

[0038]

Chemical formula

[0039] Preferably, the self-destruct domain is tandemly connected to the CD3ζ signaling domain via a 2A sequence.

[0040] In the present disclosure, the 2A sequence can cleave the protein expressed by the self-destruct domain and the protein of the chimeric antigen receptor, thereby enabling the function of the chimeric antigen receptor. When an activator is injected, the self-destruct domain is activated and the chimeric antigen receptor becomes inactive.

[0041] In the present disclosure, the chimeric antigen receptor further includes a signal peptide. The signal peptide can be any signal peptide that can direct the translocation of the chimeric antigen receptor across the membrane, and those skilled in the art can select a conventional signal peptide in the art as needed. The signal peptide is a secretion signal peptide, and the amino acid sequence of the secretion signal peptide includes SEQ ID NO: 8 or SEQ ID NO: 9.

[0042]

Chemical formula

[0043] In the present disclosure, the chimeric antigen receptor may further include a hinge region. The hinge region may be selected by those skilled in the art according to the actual situation and is not particularly limited herein. The presence of the hinge region does not affect the performance of the chimeric antigen receptor of the present disclosure.

[0044] In the present disclosure, the chimeric antigen receptor may further include a promoter. The promoter may be EF1a or any high-expression promoter. The promoter may be selected by those skilled in the art according to the actual situation, and is not particularly limited herein. The presence of the promoter does not affect the performance of the chimeric antigen receptor of the present disclosure.

[0045] Preferably, the chimeric antigen receptor includes a signal peptide, an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, a CD3ζ signaling domain, a 2A sequence, and a self-destruct domain, which are sequentially connected in tandem.

[0046] Preferably, the chimeric antigen receptor is formed by connecting in tandem a secretion signal peptide, a CD79b antigen-binding domain, a CD28 transmembrane domain and / or a CD8α transmembrane domain, a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence, and a cysteine protease 9 domain.

[0047] Preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 10.

[0048] In the present disclosure, the composition of the chimeric antigen receptor is secretion signal-CD79b scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9. Secretion signal-CD79b scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9 is referred to as the 4SCAR-79b chimeric antigen receptor. The 4SCAR-79b chimeric antigen receptor is formed by connecting in tandem a secretion signal peptide, a humanized single-chain CD79b antigen-binding domain, a CD28 transmembrane domain, a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence, and a cysteine protease 9 domain, and is specifically arranged as follows: Secretory signal-CD79b scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9.

[0049] In the present disclosure, the amino acid sequence of the secretory signal-CD79b scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9, which is a chimeric antigen receptor (4SCAR-79b), is shown in SEQ ID NO: 10.

[0050] SEQ ID NO: 10 is as follows.

[0051]

Chemical formula

[0052] In a second aspect, the present disclosure provides a nucleic acid molecule. This nucleic acid molecule encodes a chimeric antigen receptor based on the CD79b humanized antibody described in the first aspect.

[0053] In a third aspect, the present disclosure provides a viral vector. This viral vector contains at least one copy of the nucleic acid molecule described in the second aspect.

[0054] Preferably, the viral vector is a lentiviral vector and / or a retroviral vector, preferably a lentiviral vector.

[0055] In a fourth aspect, the present disclosure provides a recombinant lentivirus. This recombinant lentivirus is prepared by a preparation method including the following steps: co-transducing the viral vector described in the third aspect into mammalian cells together with the packaging helper plasmids pNHP and pHEF-VSVG to obtain a recombinant lentivirus.

[0056] Preferably, the mammalian cells include 293 cells, 293T cells or TE671 cells.

[0057] In a fifth aspect, the present disclosure provides chimeric antigen receptor T cells. The chimeric antigen receptor T cells are prepared by a preparation method including the following step: transducing and expressing the recombinant lentivirus described in the fourth aspect into T cells to obtain chimeric antigen receptor T cells.

[0058] In the present disclosure, T cells have the characteristics of being excellent in targeting and killing effects, being able to release immune factors at low doses, having low toxicity, and having a high immune killing response.

[0059] In a sixth aspect, the present disclosure provides a composition. This composition includes any one or at least a combination of two of the chimeric antigen receptor based on the CD79b humanized antibody described in the first aspect, the recombinant lentivirus described in the fourth aspect, or the chimeric antigen receptor T cells described in the fifth aspect.

[0060] In a seventh aspect, the present disclosure provides the use of any one or at least a combination of two of the chimeric antigen receptor based on the CD79b humanized antibody described in the first aspect, the recombinant lentivirus described in the fourth aspect, the T cells described in the fifth aspect, or the composition described in the sixth aspect in the preparation of an anti-tumor drug.

[0061] Preferably, the tumor is a neoplastic disease in which a CD79b-specific antigen is expressed.

[0062] Preferably, the neoplastic disease in which a CD79b-specific antigen is expressed is a B cell tumor.

[0063] The present disclosure has the following beneficial effects compared with the prior art: (1) The chimeric antigen receptor of the present disclosure is obtained by specific gene modification of the intracellular co-stimulatory signaling domain of T cells of the chimeric antigen receptor targeting the tumor surface antigen CD79b. The modified chimeric antigen receptor has a better reaction effect after specifically binding to CD79b, so that CAR-T cells have a stronger immune response against tumors. (2) After the chimeric antigen receptor T cells of the present disclosure are applied to the human body, they have higher safety than other chimeric antigen receptor T cells targeting CD79b. Even when adverse effects occur, since there are signals that induce the apoptosis mechanism, chimeric antigen receptor T cells can be removed using a drug that induces apoptosis of CAR-T cells. (3) When using the 4SCAR-79b chimeric antigen receptor of the present disclosure, after injecting CAR-T cells, the presence of CAR-T can be monitored in vivo over a long period of time, and it is proven that the 4SCAR-79b chimeric antigen receptor has long-term efficacy and can bring long-term remission to patients.

Brief Description of the Drawings

[0064]

Figure 1

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Figure 4A

Figure 4B

Figure 4C

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Mode for Carrying Out the Invention

[0065] The technical solution of the present disclosure will be further described through the following examples. It is understood by those skilled in the art that the examples described below are used for a better understanding of the present disclosure and should not be construed as specific limitations to the present disclosure.

[0066] Experiments not using the specific technologies or conditions specified in the examples are carried out according to the technologies or conditions described in the literature or product specifications in the art. The reagents or instruments used in this specification without specifying the manufacturer are conventional products commercially available from appropriate sources.

Examples

[0067] [Example 1]: Construction of Chimeric Antigen Receptor A schematic diagram of the mechanism of chimeric antigen receptor T cells is shown in Figure 1. A secretion signal peptide, a humanized single-chain CD79b scFv antigen-binding domain, a CD28 extracellular signaling domain, a CD28 transmembrane signaling domain, a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence, and a cysteine protease 9 domain were synthesized by total gene synthesis. The resulting chimeric antigen receptor, secretion signal-CD79b scFv-CD28-CD27-CD3ζ-2A-FBKP.Casp9, is referred to as the 4SCAR-79b chimeric antigen receptor, and the structural diagram of the 4SCAR-79b chimeric antigen receptor is shown in Figure 2.

[0068] The amino acid sequence of the chimeric antigen receptor, SEQ ID NO: 10, was as follows:

[0069] [Chemical Formula]

[0070] The nucleotide sequence encoding the 4SCAR-79b chimeric antigen receptor was ligated into the backbone vector pTYF of the lentiviral vector to obtain the pTYF DNA vector. The plasmid map of the backbone vector pTYF of the lentiviral vector is shown in Figure 3.

[0071] [Example 2]: Lentiviral packaging (1) 293T cells were incubated for 17 - 18 hours. (2) Fresh DMEM containing 10% FBS was added. (3) The following reagents: helper DNA mix (pNHP, pHEF-VSV-G, and GFP reporter plasmid) and pTYF DNA vector were added to sterile centrifuge tubes with DMEM, and swirled and shaken. (4) Superfect or similar transfection gene material was added to the centrifuge tube, pipetted 5 times, and left to stand at room temperature for 7 - 10 minutes. (5) The DNA-Superfect mixed solution in the centrifuge tube was added dropwise to each culture well and stirred evenly. (6) The system was incubated in a 37°C, CO2 incubator for 4 - 5 hours. (7) The cells were washed with 1.5 mL of fresh medium, and medium was added to continue the culture. (8) The incubated cells were returned to the incubator and incubated overnight. The next morning, the expression of GFP was observed under a fluorescence microscope to evaluate the transfection efficiency.

[0072] [Example 3]: Purification and concentration of lentivirus 1) Virus purification Cell debris was removed by centrifugation (1000 g) to obtain the viral supernatant. The viral supernatant was filtered through a low-protein binding filter, aliquoted, and stored at -80°C.

[0073] Generally, transduced cells can produce a lentiviral vector with a titer of more than 10 7 transducing units per mL of culture medium.

[0074] 2) Concentration of the lentiviral vector by filtration (1) Inside a biosafety cabinet, the concentration tube was washed with sterile PBS. (2) The viral supernatant was added to the tube and centrifuged at 2500 g until the viral volume decreased to 1 / 50. (3) The tube was shaken and centrifuged at 400 g, and the concentrated virus was collected into the collection cup. Finally, the viruses from all tubes were pooled into one centrifuge tube. After concentration, a lentiviral vector with a titer of more than 10 9 transducing units can be generated.

[0075] [Example 4]: Preparation of 4SCAR-79b-CAR-T cells Activated T cells were inoculated into the medium, polybrene was added, and the medium contained a T cell growth factor containing IL-2. The concentrated CAR gene retrovirus was added, centrifuged at a speed of 100 g for 100 minutes, and incubated overnight. Medium was added, incubated for 4 days, the cells were collected and counted, and target cell killing and safety detection were performed. The cells were further incubated for 1 - 2 days and then injected into the patient.

[0076] 4SCAR-79b-CAR-T cells can effectively regress tumors and are safe, which is verified by in vitro and in vivo assays respectively.

[0077] [Example 5]: In vitro killing assay of 4SCAR-79b-CAR-T cells (1) Green fluorescent protein (GFP) was transduced into CD79b-positive tumor cell lines via a lentiviral vector to stably express GFP in the cell lines and make them function as marker target cells. (2) Nonspecific control T cells (T cells without the transduced gene or negative control CAR-T cells targeting an antigen not expressed by the target cells, such as CD33-CART) or positive control CAR-T cells targeting an antigen different from CD79b scFv (control CAR-T cells targeting another antigen expressed by the target cells, such as CD19-CART) were co-incubated with the above tumors at 37 °C for 24 to 72 hours in a 5% CO2 incubator. (3) The survival of tumor cells was observed under a fluorescence microscope. Dead tumor cells lost the expression of green fluorescent protein. Based on this, the in vitro killing efficiency of different 4SCAR-79b-CAR-T cells was evaluated. The results are shown in Figures 4A, 4B, and 4C, where 79b-CART represents 4SCAR-79b-CAR-T. Figure 4A is an image (magnification 50x) showing the in vitro killing of CD79b-positive tumor cell lines by different types of T cells after 24 and 48 hours. Figure 4B is an image showing the statistical results of residual target cells quantified by flow cytometry after the killing of CD79b-positive tumor cell lines by different types of T cells after 24 and 48 hours. Figure 4C is an image showing the statistical results of the percentage of target cell death after the killing of CD79b-positive tumor cell lines by different types of T cells after 24 hours.

[0078] As can be seen from Figures 4A, 4B, and 4C, compared with the CAR-T negative control group, the 4SCAR-79b-CAR-T test group (79b-CART) showed a significant killing effect on the tumor cell line, and it was confirmed that the CAR vector material could quickly screen an effective CAR structure for subsequent clinical applications.

[0079] [Example 6]: Therapeutic effect of humanized 4SCAR-79b-CAR-T cells The patient in this example is from a children's hospital that collaborates for clinical trials and treatment. (1) The flowchart of 4SCAR-79b-CAR-T treatment for B cell tumors is shown in Figure 5. The positive expression of CD79b in unstained tumor sections of patients with diffuse large B cell lymphoma (DLBCL) was confirmed by immunohistochemical staining. As shown in Figure 6 (magnification 20x), an image showing the immunohistochemical staining results of tumor sections of patients with DLBCL, CD79b is highly expressed in the tumor tissue of DLBCL. (2) Leukocyte concentrates were collected from the patients. Peripheral mononuclear lymphocytes in the leukocyte concentrates were separated by density gradient centrifugation using Ficoll, T cells were screened with CD3 magnetic beads, and activated with anti-CD28 antibody. Subsequently, for the preparation of 4SCAR-79b-CAR-T, 1×10 6 CAR-T cells per kg of body weight were prepared. (3) Before injection, the patients received pre-treatment with low-dose chemotherapy. The pretreatment regimen was as follows: cyclophosphamide (250 mg / m 2 ) for 3 days, and fludarabine (25 mg / m 2 ) for 3 days. CAR-T injection was performed 24 hours after pre-treatment, and the pre-treatment was completed within 3 days. (4) CAR-T cells were injected intravenously. (5) After injection, the physicians monitored the patients and evaluated the toxic responses. (6) After injection, a small amount of peripheral blood was periodically collected from the patients. After separating peripheral mononuclear lymphocytes, genomic DNA (gDNA) was extracted. The copy number of CAR in peripheral blood was quantified by qPCR using specific primers. The results are shown in Figure 7, a curve graph of the in vivo CAR copy number detected in DLBCL patients injected with 4SCAR-79b-CAR-T. As can be seen from Figure 7, about 7 days after injecting CAR-T into the patients, the in vivo CAR-T value reached a peak, and CAR-T could be maintained in vivo for about 60 days without the occurrence of cytokine release syndrome (CRS). (7) After the injection of 4SCAR-79b-CAR-T, the size of the facial tumor in DLBCL patients was evaluated. The results are shown in Figure 8, which shows the changes in the tumor on the right face of DLBCL patients before and after the injection of 4SCAR-79b-CAR-T. As can be seen from Figure 8, 19 days after the injection of 4SCAR-79b-CAR-T into the patient, the tumor on the right face shrank and the function of the right eye recovered.

[0080] In summary, the 4SCAR-79b chimeric antigen receptor of the present disclosure has a better effect than other reported chimeric antigen receptors, has safety (no or mild CRS response) and long-term effectiveness, and surely achieves a better therapeutic effect on B-cell tumor patients.

[0081] The applicant of the present application states that although the detailed method of the present application is described through the above-mentioned examples, the present application is not limited to the above-mentioned detailed method, that is, the implementation of the present application does not necessarily depend on the above-mentioned detailed method. It is obvious to those skilled in the art that any improvement to the present application, equivalent substitutes for the raw materials of the products of the present application, addition of adjuvant components, selection of specific aspects, etc. are all included in the protection scope and disclosure scope of the present application.

Claims

1. A chimeric antigen receptor based on a CD79b humanized antibody, comprising an antigen-binding domain, a transmembrane domain, a co-stimulatory signaling domain, a CD3ζ signaling domain, and a self-destruct domain, which are sequentially connected in tandem, wherein the antigen-binding domain is a single-chain variable fragment (scFv) targeting the tumor surface antigen CD79b, and the amino acid sequence of the scFv targeting the tumor surface antigen CD79b is the amino acid sequence shown in SEQ ID NO: 1; or an amino acid sequence formed by amino acid substitution, addition or deletion with respect to the amino acid sequence shown in SEQ ID NO: 1, which specifically binds to the chimeric antigen receptor and has the function of binding to CD79b and inducing T cell signaling A chimeric antigen receptor based on a CD79b humanized antibody, selected from.

2. The amino acid sequence formed by amino acid substitution, addition or deletion with respect to the amino acid sequence shown in SEQ ID NO: 1 has at least 90% identity with the amino acid sequence shown in SEQ ID NO: 1, preferably at least 95% identity. The chimeric antigen receptor based on the CD79b humanized antibody according to Claim 1, which is an amino acid sequence having.

3. The transmembrane domain is a CD28 transmembrane domain and / or a CD8α transmembrane domain; Preferably, the co-stimulatory signaling domain is a combination of a CD28 signaling domain and a CD27 signaling domain; Preferably, the CD28 transmembrane domain includes a CD28 extracellular signaling domain and a CD28 cell membrane signaling domain; the co-stimulatory signaling domain includes a CD28 signaling domain and a CD27 signaling domain; the amino acid sequences of the CD28 extracellular signaling domain, the CD28 cell membrane signaling domain, the CD28 signaling domain and the CD27 signaling domain are all shown in SEQ ID NO: 2; Preferably, the self-destruct domain includes a cysteine protease 9 domain; Preferably, the amino acid sequence of the cysteine protease 9 domain is shown in SEQ ID NO: 7; Preferably, the self-destruct domain is connected in tandem with the CD3ζ signaling domain via a 2A sequence, The chimeric antigen receptor based on the CD79b humanized antibody according to Claim 1 or 2.

4. The chimeric antigen receptor comprises, in tandem and sequentially connected, a signal peptide, an antigen-binding domain, a transmembrane domain, a costimulatory signaling domain, a CD3ζ signaling domain, a 2A sequence, and a self-destruct domain, preferably, the chimeric antigen receptor is formed by connecting in tandem a secretion signal peptide, a CD79b antigen-binding domain, a CD28 transmembrane domain and / or a CD8α transmembrane domain, a CD28 signaling domain, a CD27 signaling domain, a CD3ζ signaling domain, a 2A sequence, and a cysteine protease 9 domain; preferably, the amino acid sequence of the chimeric antigen receptor is shown in SEQ ID NO: 10; A chimeric antigen receptor based on the CD79b humanized antibody according to any one of claims 1 to 3.

5. A nucleic acid molecule encoding a chimeric antigen receptor based on the CD79b humanized antibody according to any one of claims 1 to 4.

6. A viral vector comprising at least one copy of the nucleic acid molecule according to claim 5, preferably, a lentiviral vector and / or a retroviral vector, preferably a lentiviral vector.

7. A recombinant lentivirus prepared by a preparation method comprising the following step: a step of co-transducing the viral vector according to claim 6 into mammalian cells together with packaging helper plasmids pNHP and pHEF-VSVG to obtain a recombinant lentivirus, preferably, the mammalian cells include 293 cells, 293T cells or TE671 cells.

8. A chimeric antigen receptor T cell prepared by a preparation method comprising the following step: a step of transducing and expressing the recombinant lentivirus according to claim 7 in T cells to obtain chimeric antigen receptor T cells.

9. A composition comprising any one or at least two combinations of the chimeric antigen receptor based on the CD79b humanized antibody according to any one of claims 1 to 4, the recombinant lentivirus according to claim 7, or the chimeric antigen receptor T cell according to claim 8.

10. Use of any one or at least a combination of two of the chimeric antigen receptor based on the CD79b humanized antibody according to any one of claims 1 to 4, the recombinant lentivirus according to claim 7, the T cell according to claim 8 or the composition according to claim 9 in the preparation of an antitumor agent, preferably, the tumor is a neoplastic disease in which a CD79b-specific antigen is expressed, preferably, the neoplastic disease in which a CD79b-specific antigen is expressed is a B cell tumor, use.

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