GD2 chimeric antigen receptor and uses thereof

Abstract

Disialoganglioside 2 (GD2) chimeric antigen receptor (CAR) and its use are provided. Humanized GD2 single chain variable fragment (scFv) antibody has the activity of binding to GD2 antigen, and humanized GD2 scFv has more than 80% amino acid sequence identity to SEQ ID NO: 1. GD2 CAR and chimeric antigen receptor T (CAR-T) cells expressing GD2 CAR are further provided. Humanized GD2 scFv has better biological activity and compatibility. When GD2 CAR binds to GD2, it can have better response effect, stronger immune response and better long-term effect. CAR-T cells have higher safety and durability, and extremely high application value.

Description

[Technical field]

[0001] The present application relates to the technical field of tumor immunotherapy, and in particular to disialoganglioside 2 (GD2) chimeric antigen receptors (CARs) and uses thereof. [Background technology]

[0002] With the development of tumor immunology theory and clinical technology, chimeric antigen receptor T (CAR-T) cell immunotherapy has become one of the most promising tumor immunotherapies. In general, CARs consist of a tumor-associated antigen (TAA) binding region, an extracellular hinge region, a transmembrane region, and an intracellular signaling region. In general, CARs contain a single-chain variable fragment (scFv) region of an antibody or a domain that specifically binds to a TAA, linked to the cytoplasmic domain of a T cell signaling molecule via the hinge region and the transmembrane region. The most common lymphocyte activation moiety contains a T cell costimulatory domain in tandem with a moiety that induces the function of a T cell effector (e.g., CD3ζ).

[0003] CAR-mediated adoptive immunotherapy allows CAR-modified T cells to directly recognize TAAs on target tumor cells in a non-human leukocyte antigen (HLA)-restricted manner. Currently, CAR-T therapy has demonstrated excellent efficacy against hematological tumors, and second-generation CD19 CAR-T has been shown to have antitumor effects against relapsed and refractory B-cell acute lymphocytic leukemia, chronic lymphocytic leukemia, and lymphoma, with an overall response rate of approximately 50% to 90% depending on the type of tumor.

[0004] Disialoganglioside 2 (GD2) is widely expressed in tumors such as neuroblastoma, melanoma, glioma, and sarcoma, but has low and limited expression in normal tissues, making it an ideal tumor antigen for immunotherapy. Currently, this cancer antigen is primarily used to treat neuroblastoma, the most common extracranial solid malignant tumor in children. Approximately 50% of children with neuroblastoma will develop extensive tumor spread and metastasis after onset. Traditional surgery, chemotherapy, radiation therapy, and autologous stem cell transplantation have limited therapeutic efficacy for this patient population, and even after disease control and remission, more than 80% of patients suffer from disease recurrence and die within one year.

[0005] At present, among the immunotherapies for neuroblastoma, the relatively mature immunotherapy is antibody therapy against GD2, which has achieved preliminary clinical success. However, published clinical research reports show that GD2 monoclonal antibody therapy is relatively toxic, with 52% of patients experiencing grade 3 to grade 4 toxic reactions, and some patients experiencing neurotoxicity. In addition, antibodies are mainly present in peripheral blood after administration. It is relatively difficult for antibodies to accurately invade tumor tissue or sites where small tumors remain, and antibodies cannot exist for a long time in vivo. Therefore, in addition to the advantages of antibody therapy, the generated CAR GD2 CAR-T cells can accurately invade tumor tissue and exist for a long time in vivo due to the characteristics of T cells, which can provide a more effective treatment option for children with recurrent and refractory neuroblastoma. At present, clinical reports on the treatment of neuroblastoma with GD2 CAR-T have shown the preliminary efficacy of CAR-T. However, long-term observation data is still lacking.

[0006] Currently, the effect of CAR-T technology in the treatment of solid tumors is not very ideal. Many scFv regions of CAR-T are derived from mouse antibodies, and mouse scFvs are easily rejected by the human immune system, so CAR-T cannot exist for a long time in vivo, which limits the therapeutic effect. It has been reported that this is also one of the reasons why many patients with acute lymphoblastic leukemia experience relapse after achieving complete remission using CD19 CAR-T, making it difficult to perform retreatment.

[0007] Therefore, how to provide a humanized GD2 CAR-T with good therapeutic effect and long duration has become an urgent problem to be solved. Summary of the Invention

[0008] This application provides GD2 CAR and its uses. CAR-T containing GD2 CAR can eliminate GD2-positive solid tumors, effectively remove trace residues in bone marrow without adverse effects, and improve the safety, efficacy, memory and long-term maintenance of CAR-T by combining with optimized T cell signaling domain.

[0009] In a first aspect, the present application provides a humanized GD2 scFv. The humanized GD2 scFv has an activity of binding to the GD2 antigen.

[0010] The humanized GD2 scFv has an amino acid sequence with greater than 80% identity to SEQ ID NO:1.

[0011] SEQ ID NO:1: QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGS TSGSGKPGSSEGSTKGDIVMSQSPSSLAVSVGEKVTMSCKASQSVSNDVTWYQQKPGQSPKLLIYSASNRYSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQDYSSFGAGTKLELK.

[0012] In the present application, the humanized GD2 scFv is humanized and specifically modified with respect to the structure against the tumor surface antigen GD2. The modified scFv antibody has stronger function and better compatibility in the human body, and is not easily rejected by the immune system.

[0013] In a second aspect, the present application provides a derivative antibody conjugate of the humanized GD2 scFv according to the first aspect.

[0014] In a third aspect, the present application provides a nucleic acid molecule encoding a humanized GD2 scFv according to the first aspect or a derivative antibody conjugate of the humanized GD2 scFv according to the second aspect.

[0015] Preferably, the nucleic acid molecule has a nucleotide sequence having greater than 80% identity to SEQ ID NO:2.

[0016] SEQ ID NO:2: .

[0017] In a fourth aspect, the present application provides a humanized GD2 CAR, which comprises a GD2 antigen-binding scFv domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, and an inducible suicide fusion domain.

[0018] The GD2 antigen-binding scFv domain comprises a humanized GD2 scFv according to the first aspect, or a derivative antibody conjugate of the humanized GD2 scFv according to the second aspect.

[0019] In the present application, a CAR is provided that targets GD2, which not only comprises a costimulatory signaling region, a CD3ζ signaling domain, and an inducible caspase 9 (iCasp9) suicide fusion domain in tandem arrangement, but also comprises a specific genetically modified humanized antigen binding domain that binds to tumor surface antigen GD2.Compared with other CARs, this CAR has better antigen binding ability, thus improving the effect of CAR-T.

[0020] Preferably, the transmembrane domain includes the CD28 transmembrane domain and / or the CD8α transmembrane domain. In some specific embodiments, the transmembrane domain may be selected or modified by amino acid substitution.

[0021] According to the present application, the costimulatory signaling region is a combination of a CD28 costimulatory signaling domain and a T cell co-signaling domain. The CD28 costimulatory signaling domain and the T cell co-signaling domain can be adjusted as needed by those skilled in the art, and the arrangement of the CD28 costimulatory signaling domain and the T cell co-signaling domain does not affect the CAR.

[0022] Preferably, the costimulatory signaling region includes CD28 and CD27 costimulatory signaling regions, or CD28 and IL-15Ra costimulatory signaling regions.

[0023] In a specific embodiment, the CAR according to the present invention comprises a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and a CD27 costimulatory signaling domain (abbreviated as CD28-CD27 herein). The CD28 costimulatory signaling domain and the CD27 costimulatory signaling domain may be linked via a linker. In a more specific embodiment, CD28-CD27 has an amino acid sequence having more than 90% identity to the amino acid sequence shown in SEQ ID NO:3. In a more specific embodiment, CD28-CD27 has the amino acid sequence shown in SEQ ID NO:3.

[0024] SEQ ID NO:3: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP.

[0025] In another specific embodiment, the CAR according to the present invention comprises a CD28 hinge region, a CD28 transmembrane domain, a CD28 costimulatory signaling domain, and an IL-15Ra costimulatory signaling domain (abbreviated herein as CD28-IL-15Ra). The CD28 costimulatory signaling domain and the IL-15Ra costimulatory signaling domain may be linked via a linker. In a more specific embodiment, the CD28-IL-15Ra has an amino acid sequence having greater than 90% identity to the amino acid sequence set forth in SEQ ID NO:4. In a more specific embodiment, the CD28-IL-15Ra has the amino acid sequence set forth in SEQ ID NO:4.

[0026] SEQ ID NO:4: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL.

[0027] Preferably, the inducible suicide fusion domain comprises a caspase 9 domain fused to FK506 binding protein (FKBP) (abbreviated herein as FKBP.Casp9).

[0028] Preferably, the FKBP.Casp9 domain has an amino acid sequence that has greater than 90% identity to SEQ ID NO:5.

[0029] SEQ ID NO:5: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLE LARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

[0030] Preferably, the GD2 CAR further comprises a signal peptide and / or a 2A sequence.

[0031] According to the present application, CAR further comprises a signal peptide. The signal peptide may be a signal peptide that can induce transmembrane transport of CAR, and those skilled in the art can select a conventional signal peptide of a secretory protein gene in the art according to need.

[0032] According to the present application, the inducible suicide fusion domain is in tandem with the CD3ζ signaling domain via the 2A sequence, which cleaves the protein expressed by the inducible suicide fusion domain from the CAR protein, thereby allowing the CAR to function, but when the inducible suicide fusion domain is activated by injecting an activator, the CAR loses function.

[0033] Preferably, the signal peptide includes a secretory signal peptide.

[0034] Preferably, the secretory signal peptide is the signal peptide of the CD8α gene, and the secretory signal peptide has the amino acid sequence shown in SEQ ID NO:6.

[0035] SEQ ID NO: 6: MALPVTALLLPLALLLHAARP.

[0036] Preferably, the secretory signal peptide is the signal peptide of the GM-CSFR gene, and the secretory signal peptide has the amino acid sequence shown in SEQ ID NO:7.

[0037] SEQ ID NO: 7: MLLLVTSLLLCELPHPAFLLIP.

[0038] In the present application, CAR further comprises a hinge region having an amino acid sequence of a combination of multiple GGGGS (SEQ ID NO: 10), for example, GGGGSGGGGS (SEQ ID NO: 11). Hinge region can be selected by those skilled in the art according to actual situation, and is not particularly limited herein. The presence of hinge region does not affect the performance of CAR of the present application.

[0039] Preferably, the GD2 CAR comprises a secretory signal peptide, a GD2 antigen-binding scFv domain, a transmembrane domain, a costimulatory signaling region, a CD3 zeta signaling domain, a 2A sequence, and an inducible suicide fusion domain.

[0040] As a preferred technical solution, the CAR is formed by a tandem arrangement of a secretory signal peptide, a GD2 antigen-binding scFv domain, a CD8α and / or a CD28 transmembrane domain, a CD28 and a CD27 costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain, with the specific arrangement being as follows: secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9.

[0041] Alternatively, the CAR is formed by a tandem arrangement of a secretory signal peptide, a GD2 antigen-binding scFv domain, a CD8α and / or CD28 transmembrane domain, a CD28 and IL-15Ra costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain, with the specific arrangement being as follows: secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9.

[0042] In the present application, the CAR further comprises a promoter, which may be EF1a or any highly expressed promoter.

[0043] In a fifth aspect, the application provides a nucleic acid molecule. The nucleic acid molecule encodes a GD2 CAR according to the fourth aspect.

[0044] In a sixth aspect, the present application provides a viral vector, the viral vector comprising at least one copy of a nucleic acid molecule according to the fifth aspect.

[0045] In the present application, viral vectors can effectively modify immune cells to create targeted cells.

[0046] Preferably, the viral vector comprises a lentiviral vector or a retroviral vector, preferably a lentiviral vector.

[0047] In a seventh aspect, the present application provides a recombinant virus obtained by co-transfecting a viral vector according to the sixth aspect and a packaging helper plasmid into a mammalian cell.

[0048] Preferably, the packaging helper plasmids include pNHP and pHEF-VSVG.

[0049] Preferably, the mammalian cells include any one of 293 cells, 293T cells, or TE671 cells.

[0050] In an eighth aspect, the present application provides a CAR-T cell. The CAR-T cell expresses a GD2 CAR according to the fourth aspect.

[0051] In this application, CAR-T cells have good targeting and killing effects, while releasing immune factors at a low level, showing low toxicity and high immune killing response characteristics. A schematic diagram of the mechanism of CAR-T cells is shown in Figure 1.

[0052] Preferably, the CAR-T cell is generated by introducing a nucleic acid molecule according to the fifth aspect into an immune cell.

[0053] Preferably, the introduction is via one of a viral vector, a eukaryotic expression plasmid or mRNA, preferably a viral vector.

[0054] Preferably, the CAR-T cell is generated by introducing a nucleic acid molecule according to the fifth aspect into a T cell via a viral vector.

[0055] Preferably, the CAR-T cell is a T cell modified by a viral vector according to the sixth aspect.

[0056] In a ninth aspect, the present application provides a composition comprising any one or a combination of at least two of the following: a humanized GD2 scFv according to the first aspect, a derivative antibody conjugate of the humanized GD2 scFv according to the second aspect, a GD2 CAR according to the fourth aspect, a recombinant virus according to the seventh aspect, or a CAR-T cell according to the eighth aspect.

[0057] In a tenth aspect, the present application provides the use of any one or a combination of at least two of the humanized GD2 scFv according to the first aspect, a derivative antibody conjugate of the humanized GD2 scFv according to the second aspect, a GD2 CAR according to the fourth aspect, a recombinant virus according to the seventh aspect, a CAR-T cell according to the eighth aspect, or a composition according to the ninth aspect, in the manufacture of a medicament for treating a tumor.

[0058] Preferably, the tumor comprises a tumor that expresses a GD2-specific antigen.

[0059] Preferably, the tumor comprises a nervous system tumor that expresses a GD2-specific antigen.

[0060] Preferably, the tumor comprises a neuroblastoma.

[0061] Compared with existing technologies, the present application has the following beneficial effects:

[0062] (1) The CAR of the present application is specifically genetically modified in the costimulatory signaling region of the humanized CAR that targets the tumor surface antigen GD2. The modified CAR specifically binds to GD2 and has a better response effect, so that the CAR-T cells have a stronger immune response to the tumor, and compared with other GD2 CARs, the CAR has a better long-term effect.

[0063] (2) The CAR-T cells of the present application are safer and more durable than other GD2 CAR-T cells. Even if the patient's immune response becomes too strong and causes cytokine release syndrome (CRS), the CAR-T cells can be removed by a drug that induces apoptosis of CAR-T cells because of the presence of an apoptosis induction mechanism. After injecting the CAR-T cells of the present application, the long-term existence of CAR-T can be detected in vivo, which proves that the CAR-T cells have a long-term effect and can achieve the effect of long-term remission in patients.

[0064] (3) The formulation related to the humanized antibody of the present application can play a role in all GD2-positive diseases, and has actually been applied to patients with stage IV neuroblastoma expressing tumor-specific target GD2. For patients with trace residues in the bone marrow, this formulation can reduce clinical side effects, improve safety, and effectively remove trace residues that are not sensitive to chemotherapy. In addition, GD2 CAR-T is also applied to patients with gliomas in combination with other target CAR-Ts, and the long-term presence of GD2 CAR-T in patients is detected, which contributes to the maintenance of long-term remission. [Brief description of the drawings]

[0065] [Figure 1] FIG. 1 is a schematic diagram of the mechanism of action of CAR-T cells. [Diagram 2] FIG. 1 shows the structure of two types of CARs. [Diagram 3] 1 shows a plasmid map of the lentiviral vector backbone vector pTYF. [Figure 4A] Images (50x magnification) showing that different types of T cells kill GD2-positive tumor cell lines in vitro at 24 and 48 hours. [Figure 4B] FIG. 13 is a graph showing the statistical results of remaining target cells quantified by flow cytometry after killing of GD2-positive tumor cell lines by different types of T cells for 24 and 48 hours. [Figure 4C] 1 is a graph showing the statistical results of the percentage of target cells killed after different types of T cells killed GD2-positive tumor cell lines for 24 hours. [Figure 5A] Flowchart of treatment of neuroblastoma using GD2 CAR-T (inset magnification 20x). [Figure 5B] 1 is a curve graph of in vivo CAR copy number detection after GD2 CAR-T injection in Example 8. [Figure 6A] Flowchart of GD2 CAR-T cells in combination with other targets for the treatment of glioma (inset magnification 20x). [Figure 6B] Images (20x magnification) of immunohistochemical staining results of tumor sections from two patients with glioma. [Figure 6C] 1 is a curve graph of in vivo CAR copy number detection after GD2 CAR-T injection in Example 9. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0066] In order to describe in more detail the technical means adopted in the present application and the effects achieved, the present application is further described below in conjunction with examples and drawings. It should be understood that the specific examples shown below are intended to illustrate the present application, and are not intended to limit the present application.

[0067] Experiments for which no particular technique or condition is specified in the examples are carried out according to the technique or condition described in the technical literature or in the product specifications. Reagents or equipment used herein for which no manufacturer is specified are conventional products available commercially through appropriate channels. EXAMPLES

[0068] Example 1 A humanized GD2 scFv was prepared. The humanized GD2 scFv had the activity of binding to the GD2 antigen.

[0069] The humanized GD2 scFv had the amino acid sequence shown in SEQ ID NO:1.

[0070] SEQ ID NO:1: QVQLVESGPGVVQPGRSLRISCAVSGFSVTNYGVHWVRQPPGKGLEWLGVIWAGGITNYNSAFMSRLTISKDNSKNTVYLQMNSLRAEDTAMYYCASRGGHYGYALDYWGQGTLVTVSSGS TSGSGKPGSSEGSTKGDIVMSQSPSSLAVSVGEKVTMSCKASQSVSNDVTWYQQKPGQSPKLLIYSASNRYSGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYCQQDYSSFGAGTKLELK.

[0071] Example 2 Construction of CAR Two types of CARs were prepared. The structures of the two types of CARs are shown in Figure 2.

[0072] One CAR is formed by a tandem arrangement of a secretory signal peptide, a GD2 antigen-binding scFv domain, a CD28 transmembrane domain, a CD28 and CD27 costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain, with a specific arrangement as follows: secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9.

[0073] Other CARs are formed by a tandem arrangement of a secretory signal peptide, a GD2 antigen-binding scFv domain, a CD28 transmembrane domain, a CD28 and IL-15Ra costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence, and a caspase 9 domain, with the specific arrangement being as follows: secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9.

[0074] The secretory signal peptide had the amino acid sequence shown in SEQ ID NO:6.

[0075] SEQ ID NO: 6: MALPVTALLLPLALLLHAARP.

[0076] The CD28 transmembrane domain had the sequence shown in SEQ ID NO:7.

[0077] SEQ ID NO: 7: FWVLVVVGGVLACYSLLVTVAFIIFWV.

[0078] In the construct shown in secretory signal-GD2 scFv-CD28-CD27-CD3ζ-2A-FKBP.Casp9, "CD28-CD27" contained the CD28 hinge region, the CD28 transmembrane domain, the CD28 costimulatory signaling domain, and the CD27 costimulatory signaling domain. CD28-CD27 had the amino acid sequence shown in SEQ ID NO:3.

[0079] SEQ ID NO:3: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSQRRKYRSNKGESPVEPAEPCHYSCPREEEGSTIPIQEDYRKPEPACSP.

[0080] In the construct shown in secretory signal-GD2 scFv-CD28-IL-15Ra-CD3ζ-2A-FKBP.Casp9, "CD28-IL-15Ra" contained the CD28 hinge region, the CD28 transmembrane domain, the CD28 signaling domain, and the IL-15Ra signaling domain. CD28-IL-15Ra had the amino acid sequence shown in SEQ ID NO:4.

[0081] SEQ ID NO:4: IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSASGGGGSGGGGSKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL.

[0082] The CD3 zeta signaling domain had the sequence shown in SEQ ID NO:8.

[0083] SEQ ID NO:8: RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR.

[0084] The 2A sequence had the sequence shown in SEQ ID NO:9.

[0085] SEQ ID NO: 9: TSGSGATNFSLLKQAGDVEENPGP.

[0086] The caspase-9 domain had the amino acid sequence shown in SEQ ID NO:5.

[0087] SEQ ID NO:5: MGVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKPFKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELLKLEGGGGSGGGGSGAMVGALESLRGNADLAYILSMEPCGHCLIINNVNFCRESGLRTRTGSNIDCEKLRRRFSSLHFMVEVKGDLTAKKMVLALLE LARQDHGALDCCVVVILSHGCQASHLQFPGAVYGTDGCPVSVEKIVNIFNGTSCPSLGGKPKLFFIQACGGEQKDHGFEVASTSPEDESPGSNPEPDATPFQEGLRTFDQLDAISSLPTPSDIFVSYSTFPGFVSWRDPKSGSWYVETLDDIFEQWAHSEDLQSLLLRVANAVSVKGIYKQMPGCFNFLRKKLFFKTSAS.

[0088] Example 3 Two types of lentiviral vectors encoding the two types of CARs of Example 2 were generated.

[0089] The backbone vector of the lentiviral vector was pTYF. For more information, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentiviral vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd Ed., pp 303-318, Ed. Jeffrey Morgan, Humana Press, Inc., Cui, Y. and Chang, L.-J. (2003) Detection and selection of lentiviral vector transduced cells. "Methods in Molecular Biology Vol. 229: Lentivirus Gene Engineering Protocols" pp 69-85, Ed. Maurizio Federico, Humana Press, Inc, Oka, M. Chang, L.-J., Costantini, F., and Terada, N. (2005) Lentivirus mediated gene transfer in embryonic stem cells. Series: "Methods in Molecular Biology" Embryonic Stem Cells Please refer to the second publication.

[0090] The plasmid map is shown in FIG.

[0091] Example 4 Lentivirus packaging Two types of recombinant lentiviruses were produced. The two types of recombinant lentiviruses were obtained by co-transfecting the lentivirus vector of Example 3 and packaging helper plasmid into mammalian cells. The steps are as follows:

[0092] (1) 293T cells were cultured for 17 to 18 hours. (2) Fresh Dulbecco's modified Eagle medium (DMEM) (Thermo Fisher Scientific Inc.) was added. (3) The following reagents were added to a sterile centrifuge tube: DMEM, packaging helper plasmids (pNHP and pHEF-VSV-G), and pTYF CAR DNA vector were added to each well (for specific procedures, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd Ed., pp 303-318, Ed. Jeffrey Morgan, Humana Press, Inc.), and the tubes were vortexed and shaken. (4) SuperFect (QIAGEN NV) was added to the centrifuge tube, and the resulting mixture was allowed to stand at room temperature for 7 to 10 minutes. (5) The DNA-SuperFect mixture in the centrifuge tube was added dropwise to the cultured cells and vortexed. (6) The cells were cultured in a CO2 incubator at 37°C for 4 to 5 hours. (7) The culture medium was aspirated, the culture medium was rinsed with AIM-V (BRL), and fresh AIM-V was added to continue the culture. (8) The cells were returned to the CO2 incubator and cultured overnight. The next day, the transduction efficiency was observed.

[0093] Example 5 Purification and concentration of lentivirus 1. Virus Purification Cell debris was removed by centrifugation (1000 g, 5 min) to obtain viral supernatant, which was filtered through a 0.45 μm low protein binding filter and the virus was aliquoted and stored at −80° C.

[0094] Typically, transduced cells are cultured at 10 6 ~10 7It is possible to produce lentiviral vectors with titers of transducing units.

[0095] 2. Concentration of Lentivirus Using Centrifugal Filters (1) Concentration centrifuge tubes were placed in a biosafety cabinet, disinfected, and washed twice under sterile conditions. (2) The viral supernatant was added to each centrifugal filter tube and centrifuged until the viral volume was reduced by 1 / 20 to 1 / 50. (3) The filter tube was shaken and the concentrated virus was collected in a collection cup by centrifugation, and the viruses in all tubes were collected in one centrifuge tube.

[0096] Example 6: Preparation of CAR-T cells Two types of CAR-T cells expressing the CAR of Example 2 were prepared. The preparation method is described below.

[0097] The activated T cells were suspended in culture medium, and 10 μg / mL polybrene (Sigma) was added to the culture medium. The culture medium was AIM-V containing cell culture factors IL-2, IL-7, and IL-15 (all purchased from PeproTech), and the concentrated lentivirus of Example 5 was added to the culture medium separately. After centrifugation at 100 g for 100 min at room temperature, the cells were cultured at 37° C. for 24 h, and culture medium was added. After 4 days of culture, the cells were harvested and counted. After 2 days of culture, the cells were infused into the patient.

[0098] Example 7 In vitro killing experiment of CAR-T (1) Green fluorescent protein was introduced into GD2-positive tumor cell lines via a lentiviral vector and stably expressed.

[0099] (2) T cells and non-specific CAR-T cells (CD44v6 CAR-T) were used as the negative control group, and the two types of CAR-T in Example 6 were used as the experimental group. One structure corresponds to GD2 ScFv-CD28-CD27-CD3ζ (abbreviated as CD27-GD2 CAR-T), and the other structure corresponds to GD2 ScFv-CD28-IL15R-CD3ζ (abbreviated as IL15R-GD2 CAR-T). The above four types of cells were co-cultured with the tumor in step (1) for 24 to 48 hours in a 37°C, 5% CO2 incubator, and the tumor cell killing status was observed by fluorescence microscopy. The results are shown in Figure 4A. Green fluorescent protein was not expressed in the dead tumor cells. It can be seen that the killing effects of the CD27-GD2 CAR-T group and the IL15R-GD2 CAR-T group were significantly better than that of the control group, and the killing effect of the IL15R-GD2 CAR-T group was the best.

[0100] In addition, the green fluorescence of the remaining target cells was quantified by flow cytometry and statistically analyzed. The results are shown in Figure 4B. It can be seen that the CD27-GD2 CAR-T group and the IL15R-GD2 CAR-T group had fewer remaining target cells, indicating that the corresponding T cells had a stronger killing ability.

[0101] (3) The apoptosis of target cells was quantified using PI / AnnexinV staining (Sigma) to observe the situation in which CAR-T kills target cells. The results are shown in Figure 4C. It was found that the mortality rate of target cells in the CD27-GD2 CAR-T group and the IL15R-GD2 CAR-T group was relatively high, which is consistent with previous results.

[0102] The above experiment was repeated four more times.

[0103] Combined with the results in Figures 4A, 4B and 4C, compared with the two negative control groups, it can be seen that the two types of GD2 CAR-T cells each have obvious killing ability, and the killing effect of IL15R-GD2 CAR-T is superior to that of CD27-GD2 CAR-T.

[0104] Example 8 GD2 CAR-T cells for the treatment of neuroblastoma (1) Six patients with stage IV neuroblastoma were recruited, whose bone marrow could not be put into remission after multiple treatments and had trace residues in the bone marrow. The complete treatment flow is shown in Figure 5A. (2) Immunohistochemical staining of slides containing slices of the patient's tumor confirmed positive expression of GD2. Relevant information is summarized in Table 1. (3) White blood cell concentrates were collected from each patient. Peripheral blood mononuclear lymphocytes in the white blood cell concentrates were separated by density gradient centrifugation using Ficoll, and T cells were screened with CD3 magnetic beads and activated with anti-CD28 antibodies (BD Biosciences). Subsequent GD2 CAR-T generation was performed at 2 × 10 per kg body weight. 6 The study was performed at a ratio of 1 CAR-T cell. (4) Prior to the infusion of CAR-T cells, patients were preconditioned with low-dose chemotherapy. The conditioning regimen consisted of cyclophosphamide (250 mg / m 2 ) (Sigma) and fludarabine (25 mg / m 2 ) (Sigma) for 3 days. CAR-T infusion was performed 24 hours after conditioning and was completed within 3 days. (5) CAR-T cells were infused intravenously at the doses shown in Table 1. (6) After infusion, the patients were monitored by clinicians and evaluated for toxic reactions. The clinical toxic reactions, i.e. CRS, of the six patients after infusion are summarized in Table 1. The results show that no CRS reactions were observed in the six patients. (7) After infusion, a small amount of peripheral blood was periodically drawn from each patient, peripheral blood mononuclear lymphocytes were isolated, and then cellular chromosomal DNA (gDNA) was extracted. The copy number of CAR in peripheral blood was quantified by qPCR using specific primers (for specific operations, see Chang, L.-J. and Zaiss, A.-K. (2001) Methods for the preparation and use of lentivirus vectors. Methods in Molecular Medicine, Gene Therapy Protocols, 2nd Ed., pp 303-318, Ed. Jeffrey Morgan, Humana Press, Inc.). The fluctuation curves of the copy number of CAR in six patients are shown in Figure 5B. Here, pt3 is the patient who has been observed for the longest period, and copies of CAR-T can still be successfully detected in peripheral blood even after 3 months. (8) Before and after the infusion of GD2 CAR-T, bone marrow aspirates were aspirated at the hospital to detect tumor cells in the bone marrow aspirates. Four neuroblastoma patients had no trace residues in the bone marrow after infusion, while two patients had trace residues remaining in the bone marrow after infusion. For details, see Table 1.

[0105] [Table 1]

[0106] Example 9 GD2 CAR-T cells in combination with other targets for the treatment of glioma (1) Two patients with refractory glioma were recruited as subjects. The complete treatment flow is shown in Figure 6A. (2) The slides containing the patient's tumor slices were immunohistochemically stained to confirm the expression of the target. Finally, GD2 combined with another tumor antigen showing relatively high expression was selected as the CAR-T therapeutic target. The immunohistochemical staining results of the slices are shown in Figure 6B. (3) White blood cell concentrates were collected from each patient. Peripheral blood mononuclear lymphocytes in the white blood cell concentrates were separated by density gradient centrifugation using Ficoll, and T cells were screened with CD3 magnetic beads and activated with anti-CD28 antibodies. Subsequent CAR-T generation was performed at a dose of 2 × 10 per kg of body weight. 6 The study was performed at a ratio of 1 CAR-T cell. (4) Prior to the infusion of CAR-T cells, patients were preconditioned with low-dose chemotherapy. The conditioning regimen consisted of cyclophosphamide (250 mg / m 2 ) and fludarabine (25 mg / m 2 ) for 3 days. CAR-T infusion was performed 24 hours after conditioning and was completed within 3 days. (5) The two types of CAR-T cells were simultaneously infused intravenously at the doses shown in Table 2. (6) After infusion, the patients were monitored by clinicians and evaluated for toxic reactions. The clinical toxic reactions, i.e., CRS, of the two patients after infusion are summarized in Table 2. The results show that no CRS reactions were observed in the two patients. (7) Before and after the infusion, the patients' tumor lesions were evaluated by magnetic resonance imaging (MRI). The clinical responses of the patients are summarized in Table 2. One patient was evaluated as having stable disease, and the other patient achieved a partial response. (8) After infusion, a small amount of peripheral blood was periodically drawn from each patient, and the peripheral blood mononuclear lymphocytes were isolated, followed by extraction of cell chromosomal DNA (gDNA). The copy number of CAR in peripheral blood was quantified by qPCR using specific primers. The fluctuation curves of the copy number of CAR in two patients are shown in Figure 6C. On days 183 and 202 after infusion, 1.20% and 0.05% of CAR-T copies were still present in the peripheral blood of the first patient, and on days 209 and 254 after infusion, 0.42% and 0.45% of CAR-T copies could still be successfully detected in the peripheral blood of the second patient. This proves that GD2 CAR-T can be maintained for a long time in vivo.

[0107] [Table 2]

[0108] In conclusion, the GD2 CAR described in this application has better response effect and better long-term effect. GD2 CAR is applied to patients with stage IV neuroblastoma expressing tumor-specific target GD2. For patients with trace residues in bone marrow, GD2 CAR has fewer clinical side effects, is safer, and can effectively remove trace residues that are not sensitive to chemotherapy. In addition, GD2 CAR-T can also be applied to treat patients with glioma in combination with other target CAR-T, and the presence of GD2 CAR-T in patients can be successfully monitored over a long period of time. This contributes to the maintenance of long-term remission.

[0109] The applicant states that the detailed method of the present application is described through the above examples, but the present application is not limited to the above detailed method, which means that the implementation of the present application does not necessarily depend on the above detailed method. Any improvements to the present application, the replacement of raw materials of the products of the present application with equivalents, the addition of auxiliary ingredients, the selection of specific methods, etc., are all within the protection scope and disclosure scope of the present application, as will be apparent to those skilled in the art.

Claims

1. A humanized disialoganglioside 2 (GD2) single chain variable fragment (scFv) having an activity of binding to a GD2 antigen, comprising: A humanized GD2 scFv having an amino acid sequence with greater than 80% identity to SEQ ID NO:

1.

2. A derivative antibody conjugate of the humanized GD2 scFv of claim 1.

3. A nucleic acid molecule encoding the humanized GD2 scFv of claim 1 or a derivative antibody complex of the humanized GD2 scFv of claim 2, Preferably, a nucleic acid molecule having a nucleotide sequence having greater than 80% identity to SEQ ID NO:

2.

4. A humanized disialoganglioside 2 (GD2) chimeric antigen receptor (CAR), comprising a GD2 antigen-binding single-chain variable fragment (scFv) domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, and an inducible suicide fusion domain; The GD2 antigen-binding scFv domain comprises a humanized GD2 scFv according to claim 1, or a derivative antibody conjugate of the humanized GD2 scFv according to claim 2, Preferably, said transmembrane domain comprises the CD28 transmembrane domain and / or the CD8α transmembrane domain, Preferably, the costimulatory signaling region comprises a CD28 and a CD27 costimulatory signaling region, or a CD28 and an IL-15Ra costimulatory signaling region; Preferably, the inducible suicide fusion domain comprises a caspase 9 domain fused to FK506 binding protein (FKBP); Preferably, the caspase 9 domain fused to the FKBP has an amino acid sequence having greater than 90% identity to SEQ ID NO:5, Preferably, the GD2 CAR further comprises a signal peptide and / or a 2A sequence, Preferably, the signal peptide comprises a secretory signal peptide, Preferably, the GD2 CAR is a humanized GD2 CAR comprising a secretory signal peptide, a GD2 antigen-binding scFv domain, a transmembrane domain, a costimulatory signaling region, a CD3ζ signaling domain, a 2A sequence, and an inducible suicide fusion domain.

5. A nucleic acid molecule encoding the GD2 CAR of claim 4.

6. A viral vector comprising at least one copy of the nucleic acid molecule of claim 5, Preferably, a viral vector, including a lentiviral or retroviral vector, preferably a lentiviral vector.

7. A recombinant virus obtained by co-transfecting a mammalian cell with the viral vector according to claim 6 and a packaging helper plasmid, Preferably, the packaging helper plasmid comprises pNHP and pHEF-VSVG; Preferably, the mammalian cell comprises any one of a 293 cell, a 293T cell, or a TE671 cell.

8. A chimeric antigen receptor T (CAR-T) cell expressing the GD2 CAR according to claim 4, Preferably, the CAR-T cell is produced by introducing the nucleic acid molecule according to claim 5 into an immune cell, Preferably, said introduction is via one of a viral vector, a eukaryotic expression plasmid or an mRNA, preferably a viral vector; Preferably, the CAR-T cell is prepared by introducing the nucleic acid molecule according to claim 5 into a T cell via a viral vector.

9. A composition comprising any one or a combination of at least two of the humanized GD2 scFv of claim 1, a derivative antibody complex of the humanized GD2 scFv of claim 2, the GD2 CAR of claim 4, the recombinant virus of claim 7, or the CAR-T cell of claim 8.

10. Use of any one or a combination of at least two of the humanized GD2 scFv of claim 1, the derivative antibody complex of the humanized GD2 scFv of claim 2, the GD2 CAR of claim 4, the recombinant virus of claim 7, the CAR-T cell of claim 8, or the composition of claim 9 in the manufacture of a medicament for treating a tumor, comprising: Preferably, the tumor comprises a tumor expressing a GD2-specific antigen, Preferably, the tumor comprises a nervous system tumor expressing a GD2-specific antigen; Preferably, the tumor comprises a neuroblastoma.

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