WO2018121712A1 - 一种新型嵌合抗原受体及其应用 - Google Patents

一种新型嵌合抗原受体及其应用 Download PDF

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WO2018121712A1
WO2018121712A1 PCT/CN2017/119711 CN2017119711W WO2018121712A1 WO 2018121712 A1 WO2018121712 A1 WO 2018121712A1 CN 2017119711 W CN2017119711 W CN 2017119711W WO 2018121712 A1 WO2018121712 A1 WO 2018121712A1
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chimeric antigen
antigen receptor
domain
cells
cell
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PCT/CN2017/119711
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French (fr)
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范晓虎
庄秋传
王平艳
杨蕾
郑修军
郝佳瑛
刘少波
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南京传奇生物科技有限公司
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Priority to US16/474,735 priority Critical patent/US11564945B2/en
Priority to EP17889368.1A priority patent/EP3564266A4/en
Publication of WO2018121712A1 publication Critical patent/WO2018121712A1/zh

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Definitions

  • the invention belongs to the technical field of biomedicine or biopharmaceutical, and relates to a novel chimeric antigen receptor and application thereof.
  • CAR-T Modified T cell therapy
  • CAR-T cells are a class of T cells that can be genetically engineered to express a specific antigen on a T cell surface and transmit signals.
  • CAR-T cells recognize specific molecules on the surface of tumor cells by antigen-antibody or ligand-receptor recognition pattern through chimeric antigen receptor CAR, and then activate and proliferate through their intracellular signaling. Play a cell killing function.
  • T cells modified by chimeric antigen receptor can specifically recognize tumor-associated antigens, making the targeting, killing activity and persistence of effector T cells higher than conventionally used immune cells, and can overcome local immunosuppression of tumors.
  • the microenvironment breaks the host's immune tolerance state.
  • the modified T cell expresses a class of chimeric antigen receptor molecules: the extracellular segment comprises a CD8 ⁇ or GM-CSFR ⁇ signal peptide, an antigen recognition region or an antigen binding domain, and is comprised of an antibody heavy chain and a light chain variable
  • the single-stranded variable region consisting of a region; the intracellular segment is an intracellular chimera of various signaling molecules, including CD28, 4-1BB, OX-40, CD3zeta, etc., and the transmembrane region is derived from other molecules, such as PD1. CD8, CD4, CD28, CD3zeta (CD3 ⁇ ) and the like.
  • the non-antigen recognition region of the extracellular region of CAR including the signal peptide, the hinge region (Hinge, the junction region between the scFv and the transmembrane region, also referred to as the spacer domain), has an important influence on the function of the CAR.
  • German Kober L et al. reported that the expression level of the bispecific scFv antibody can be significantly increased by optimizing the signal peptide sequence from different sources, which is closely related to the ability of the signal peptide to guide the protein into the secretory pathway. Relationship (Biotechnol Bioeng. 2013 Apr; 110(4): 1164-73.). Michael Hudecek et al.
  • the performance of the CAR vector can be greatly realized by changing the sequence of the transmembrane region, and the non-specificity of CAR-T to non-target cells can be reduced. Killing effect.
  • the CAR structure comprising the 4-1BB intracellular signal domain is considered to have better in vivo tumor cell killing activity and persistence compared to the CD28 intracellular signal domain.
  • CD19 is expressed on the surface of almost all B cell tumor cells, but is hardly expressed in other substantial cells and hematopoietic stem cells, and CD19 is a relatively specific target of B-line tumor antigens.
  • the CD19CAR structure that has made significant progress in the clinic is CD8 ⁇ signal peptide-anti-CD19 single-chain antibody-CD8 ⁇ hinge region & transmembrane region-4-1BB intracellular region-CD3 intracellular region (CD8 ⁇ signal peptide-antiCD19scFv-CD8 ⁇ Hinge&TM-4- 1BBcyto-CD3 ⁇ ).
  • Human 4-1BB molecule (NCBI database protein number NP_001552.2), also known as CD137, or tumor necrosis factor receptor superfamily member 9 (TNFRSF9) whose primary structure is amino acids 1-23
  • TNFRSF9 tumor necrosis factor receptor superfamily member 9
  • amino acids 24-186 are extracellular regions
  • amino acids 187-213 are transmembrane regions
  • amino acids 214-255 are intracellular regions.
  • the present invention provides a novel CAR structure comprising a 4-1BB signal peptide and/or a 4-1BB transmembrane domain structure, such as a 4-1BB signal peptide-tumor antigen recognition binding region-4-1BB transmembrane region-4-1BB
  • a 4-1BB signal peptide-tumor antigen recognition binding region-4-1BB transmembrane region-4-1BB The intracellular region-CD3zeta intracellular region (4-1BB signal peptide-VH-Linker-VL/VHH-4-1BBTM-4-1BBcyto-CD3 ⁇ ) maximizes the amino acid sequence of 4-1BB.
  • 4-1BB signal peptide and the transmembrane and intracellular regions of 4-1BB facilitates the extension of the extracellular tumor antigen recognition binding region on CAR-T cells, which not only has good tumor killing effects in vitro and in vivo, but also Complete remission was also achieved in patients with clinically treated acute lymphoma, and the cytokine release response was more modest.
  • the object of the present invention is to provide a novel chimeric antigen receptor and its use in view of the above-mentioned deficiencies of the prior art.
  • Another object of the invention is to provide a nucleic acid encoding the chimeric antigen receptor.
  • a novel chimeric antigen receptor comprising an extracellular signal peptide, an antigen binding domain, a transmembrane domain and an intracellular signal domain, wherein the extracellular signal peptide is selected from the group consisting of a 4-1BB signal peptide, CD8 ⁇ One of a signal peptide, a GM-CSFR ⁇ signal peptide or a CD4 signal peptide, the transmembrane domain is selected from the 4-1BB molecule transmembrane region sequence.
  • the amino acid sequence of the transmembrane domain 4-1BB molecular transmembrane region is preferably a polypeptide represented by SEQ ID NO. 1, or having 85%-99% identity with its amino acid sequence.
  • the extracellular signal peptide amino acid sequence is preferably a 4-1BB signal peptide as shown in SEQ ID NO. 2, or a polypeptide having 85%-99% identity to its amino acid sequence.
  • the antigen-binding domain-bound antigen is associated with a malignant tumor, including but not limited to CD19, CD20, CD22, CD30, CD33, CD38, BCMA, CS1, CD138, CD123/IL3R ⁇ , c-Met, gp100, MUC1, IGF- I receptor, EPCAM, EGFR/EGFRvIII, HER2, PD1, CTLA4, IGF1R, mesothelin, PSMA, WT1, ROR1, CEA, GD-2, NY-ESO-1, MAGE A3, GPC3, glycolipid F77 or any other tumor antigen Or other types or combinations of modifications.
  • the antigen-binding domain-bound antigen is further preferably CD19; the CD19 antigen-binding domain amino acid sequence is set forth in SEQ ID NO.
  • the antigen binding domain of the chimeric antigen receptor binds to the target via a ligand-receptor interaction and may be selected from IL-3, IL-13 or APRIL.
  • the antigen binding domain consists of antibody fragments, preferably monoclonal antibodies, Fabs, scFvs, sdAbs, VHHs or other antibody fragments.
  • the antigen binding domain antibody can be derived from a murine source, a camel source or a humanized antibody.
  • the antigen binding domain of the chimeric antigen receptor consists of a CD19-specific single-chain antibody fragment, including the heavy chain variable region (VH) and the light chain variable region (VL) of the single-chain antibody .
  • the single-chain antibody heavy chain variable region (VH) and the light chain variable region (VL) of the antigen-binding domain are linked by a linker (Linker), comprising a GS Linker such as (G3 ) S) 4 , (G 4 S) 3 or GTSSGSGKPGSGEGSTKG, preferably (G 3 S) 4 linked peptide.
  • Linker comprising a GS Linker such as (G3 ) S) 4 , (G 4 S) 3 or GTSSGSGKPGSGEGSTKG, preferably (G 3 S) 4 linked peptide.
  • the intracellular signal domain preferably comprises CD3 zeta (CD3 ⁇ ), CD3 gamma, CD3 delta, CD3 epsilon, common FcR gamma (FCER1G), FcR beta (Fc Epsilon Rib), CD79a, CD79b, Fc gamma RIIa, DAP10 and DAP12
  • CD3 zeta CD3 ⁇
  • CD3 gamma CD3 delta
  • CD3 epsilon common FcR gamma
  • FCER1G common FcR gamma
  • FcR beta Fc Epsilon Rib
  • CD79a, CD79b, Fc gamma RIIa, DAP10 and DAP12 The intracellular signal sequence of the molecule, or a combination thereof.
  • the intracellular signal domain comprises a signaling domain of a CD3 ⁇ molecule.
  • the intracellular signal domain also preferably comprises a costimulatory signal domain, which may be selected from the following costimulatory signalling molecules: CD27, CD28, 4-1BB, OX40, CD30, CD40, CD2, lymphocyte function associated antigen-1 (LFA) -1), LIGHT, NKG2C, B7-H3, PD-1, ICOS, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, CD7, NKp80 (KLRF1), CD160, CD19, CD4, CD8alpha, CD8beta, IL2R beta, IL2R gamma, IL7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b , ITGAX, CD11c, ITGB1, CD29, IT
  • the chimeric antigen receptor does not comprise a hinge region between the extracellular antigen binding domain and the transmembrane domain.
  • the chimeric antigen receptor comprises a hinge region between the extracellular antigen binding domain and the transmembrane domain.
  • the hinge region is preferably the CD8 alpha hinge region set forth in SEQ ID NO.
  • the chimeric antigen receptor preferably comprises a ligated 4-1BB signal peptide or CD8 alpha signal peptide, an antigen binding domain, a 4-1BB transmembrane domain, a 4-1BB intracellular costimulatory domain, and CD3 ⁇ signaling domain.
  • the chimeric antigen receptor structure of the present invention is as follows:
  • the antigen binding domain may VH-Linker-VL or VL-Linker-VH or V H H 1 -Linker-V H H 2 composition
  • V H H 1 and V H H 2 can recognize the same antigen or different antigens.
  • the chimeric antigen receptor comprises the amino acid sequence set forth in SEQ ID NO. 10 or SEQ ID NO. 12, or has an amino acid sequence of 85%-99% identity thereto.
  • a nucleic acid molecule encoding the aforementioned chimeric antigen receptor is provided.
  • the nucleic acid molecule preferably comprises the nucleotide sequence of SEQ ID NO. 22 or SEQ ID NO.
  • the expression vector is preferably a lentiviral expression vector comprising a nucleotide sequence encoding SEQ ID NO. 22 or SEQ ID NO.
  • a cell expressing the aforementioned chimeric antigen receptor A cell expressing the aforementioned chimeric antigen receptor.
  • the cells are preferably immune cells; further preferably, T lymphocytes, NK cells, hematopoietic stem cells, pluripotent stem cells or embryonic stem cells are cultured to differentiate the immune cells.
  • a method of producing a novel chimeric antigen receptor-modified T cell comprising isolating and activating a T cell to be modified, and then transducing the T cell with the aforementioned expression vector.
  • novel chimeric antigen receptor The use of the novel chimeric antigen receptor, expression vector, and the use of the cell in the preparation of a medicament for treating a tumor.
  • the tumor preferably includes glioblastoma, head and neck cancer, thyroid cancer, kidney cancer, lung cancer, breast cancer or ovarian cancer, and chronic lymphocytic leukemia (CLL), acute leukemia, acute lymphoblastic leukemia (ALL), B Acute lymphoblastic leukemia (ALL), T-cell acute lymphoblastic leukemia (T-ALL), chronic myeloid leukemia (CML), B-cell lymphoblastic leukemia, acute plasmacytoid dendritic cell tumor, Burkitt lymphoma, Diffuse large B-cell lymphoma, follicular lymphoma hairy cell leukemia, small cell or large cell follicular lymphoma, malignant lymphoid tissue hyperplasia, MALT lymphoma, mantle cell lymphoma, marginal zone lymphoma, multiple bone marrow Tumor, myeloproliferative and myelodysplastic syndrome, non-Hodgkin's lymphoma, Hodgkin'
  • it relates to the use of a medicament for the treatment of a tumor of the bloodstream.
  • novel chimeric antigen receptors of the present invention are directed to the use in the preparation of anti-tumor drugs and cellular immunotherapy.
  • the immune effector cells of the present invention are useful in the preparation of antitumor drugs and cellular immunotherapy.
  • the present invention provides a novel CAR structure comprising a 4-1BB signal peptide and/or a 4-1BB transmembrane domain structure, such as a 4-1BB signal peptide-tumor antigen recognition binding region-4-1BB transmembrane region-4-1BB
  • a 4-1BB signal peptide-tumor antigen recognition binding region-4-1BB transmembrane region-4-1BB The intracellular region-CD3zeta intracellular region (4-1BB signal peptide-VH-Linker-VL/VHH-4-1BBTM-4-1BBcyto-CD3 ⁇ ) maximizes the amino acid sequence of the 4-1BB molecular structure.
  • 4-1BB transmembrane region or intracellular domain structure which facilitates the extension of the extracellular tumor antigen recognition binding region on CAR-T cells, not only has good tumor killing effect in vitro and in vivo, but also Clinically, patients with acute lymphoma have also achieved complete remission, and the cytokine release response is more moderate.
  • the novel chimeric antigen receptor shows higher anti-tumor ability than the prior art, and the modified immune cells have higher ability to target and recognize tumor antigen, and enhance The killing activity against tumor cells.
  • Figure 1 is a diagram showing the structure of a chimeric antigen receptor, showing the structure of the CAR1 (panel A) and CAR3 (panel B) chimeric antigen receptor vectors involved in the present invention.
  • Figure 2 shows the detection of CD19 antigen expressed by a stable cell line constructed in the present invention.
  • Figure 3 is a graph showing the effect of CAR-T killing CD19-positive cells in vitro.
  • E:T ratio 1 the remaining Raji.Luc relative cells of CAR1-T and Raji.Luc were 20.2 ⁇ 1.23%, while the CAR2-T group had 98.84 ⁇ 1.60% remaining, and the UnT group was 100. ⁇ 3.54%; under the condition of E:T ratio 2, the remaining Raji.Luc relative cells of CAR1-T and Raji.Luc were 50.09 ⁇ 2.17%, while the remaining in CAR2-T group was 107.07 ⁇ 3.04%, UnT group It is 100 ⁇ 3.50%.
  • the remaining Raji.Luc cells in the co-culture of B.CAR1-T and Raji.Luc were 26.83 ⁇ 1.97%, while the remaining relative target cells in the CAR3-T group were 36.86 ⁇ 3.46%, and the UnT group was 100 ⁇ 1.78%.
  • Figure 4 shows the detection of IFNy release levels in CAR-T cells.
  • A CAR1-T, CAR2-T and UnT, Luc group and Raji.Luc cells were incubated for 4h and 20h after IFN ⁇ release.
  • B CAR1-T, CAR2-T and UnT, Luc group and B lymphocytes from patients with acute B cell leukemia were incubated for 4 h and 20 h after IFN ⁇ release.
  • FIG 5 shows the detection of tumor killing effect by CAR1 transduced autologous T cells.
  • CAR1 was transduced into T cells from 5 different B-ALL patients, and the transduced cells were named ALLCT01, ALLCT02, ALLCT03, ALLCT04 and ALLCT05, respectively. After the cells transduced with CAR1 were co-incubated with Raji.Luc target cells, the relative number of remaining Raji.Luc cells was 9.33%-37.82%.
  • Figure 6 is a graph showing the release level of human interleukin 6. After ALLCT01 patients returned CD19CAR-T cells, the body temperature began to rise 6 days after the last reinfusion, and the cytokine test results showed that the secretion level of interleukin-6 (IL-6) was significantly increased.
  • IL-6 interleukin-6
  • the present invention provides a novel chimeric antigen receptor, immune effector cells and their use in inhibiting tumor activity, and the present invention is further illustrated by the following specific examples.
  • transmembrane domain (abbreviated as TM) as used herein may be used interchangeably with “transmembrane region” and refers to a region of protein structure anchored within the cell membrane that is thermodynamically stable.
  • the transmembrane region can be obtained from a native protein selected from the group consisting of 4-1BB molecules.
  • novel chimeric antigen receptors of the invention comprise an extracellular signal peptide structure, such as a 4-1BB signal peptide, a CD8 alpha signal peptide, a GM-CSFR alpha signal peptide or a CD4 signal peptide, preferably a 4-1BB signal peptide.
  • intracellular signal domain refers to a region of a protein structure that is capable of transmitting a cellular effector signaling signal and directing the cell to perform a particular function.
  • the intracellular signal domain can include a signaling domain and/or a costimulatory signal domain.
  • identity of an amino acid sequence can be used interchangeably with “similarity” to refer to the degree of similarity between amino acid sequences determined by sequence alignment software such as BLAST. Methods and software for alignment of amino acid sequences are well known to those skilled in the art.
  • a modified amino acid can be obtained by subjecting a known amino acid sequence to one or more (eg, 1-15, eg, 2, 3, 5, 8, 10, or 12) amino acid residue substitutions, deletions, and/or additions. sequence.
  • the 4-1BB transmembrane domain represented by SEQ ID NO.
  • 1 of the present invention can be engineered by conventional protein engineering means (e.g., conservative substitution of amino acids, etc.) to obtain at least 85% (e.g., 85) with SEQ ID NO. % to 99% or 90% to 99% or 95% to 99%) sequence sequences having substantially identical transmembrane functions.
  • conventional protein engineering means e.g., conservative substitution of amino acids, etc.
  • antibody fragment is a variable region comprising a functional antibody portion, preferably an antigen binding and/or an intact antibody.
  • Antibody fragments include Fab, Fab ', F (ab ') 2, Fv fragments, single chain antibody scFv, a single domain antibody sdAb / V H H and multispecific antibodies.
  • scFv or "scFv antibody” as used herein refers to a single chain antibody fragment (scFv) which is short of 15-20 amino acids by the antibody heavy chain variable region VH and the light chain variable region VL.
  • the peptides are joined together.
  • Linker as used herein, also referred to as a linker peptide or linker, is a flexible amino acid sequence used to join two domains. Selection and preparation of linker peptides is readily available to those skilled in the art.
  • the present invention further exemplifies the present invention by optimizing the design of a chimeric antigen receptor using the human 4-1BB molecular domain.
  • the present invention provides a novel chimeric antigen receptor comprising a CD19 antigen binding domain.
  • the antigen-binding domain antibody provided by the present invention consists of a heavy chain variable region (VH), a light chain variable region (VL) and a linker peptide (G3S) 4 Linker of a single chain antibody.
  • VH heavy chain variable region
  • VL light chain variable region
  • G3S linker peptide 4 Linker of a single chain antibody.
  • the heavy chain variable region and the light chain variable region sequence of the single-chain antibody were derived from GenBank: Y14283.1 and GenBank: Y14284.1, respectively, and codon and sequence were optimized to ensure that they were more suitable for expression in human cells. .
  • the novel chimeric antigen receptor provided by the present invention can design and transform a chimeric antigen receptor fusion gene fragment according to the following coding gene sequence: 4-1BB signal peptide, V H -(G3S) 4 -V L , 4-1BB cross Membrane region, 4-1BB or CD28 intracellular costimulatory signal domain or CD3 cell intracellular signaling domain.
  • Gene synthesis is provided by Nanjing Jinsrui Biotechnology Co., Ltd., and the following coding gene sequences are selected by gene synthesis technology for fusion. Gene synthesis:
  • the nucleotide sequence encoding the 4-1BB transmembrane region is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the 4-1BB signal peptide is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the CD8 ⁇ signal peptide is shown in SEQ ID NO.
  • the nucleotide sequence encoding the CD8 alpha hinge region is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the transmembrane region of CD8 ⁇ is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the 4-1BB intracellular costimulatory signal domain is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the CD28 intracellular costimulatory signal domain is set forth in SEQ ID NO.
  • the nucleotide sequence encoding the intracellular signaling domain of CD3 is shown in SEQ ID NO.
  • the nucleotide sequence encoding the CD19 antigen binding domain is set forth in SEQ ID NO.
  • the extracellular domain and the intracellular signal domain used in the present invention are variously combined, and comprise a structure or combination selected from the group consisting of:
  • CD8 ⁇ signal peptide having an amino acid sequence as shown in SEQ ID NO. 3;
  • CD8 ⁇ hinge region the amino acid sequence of which is shown in SEQ ID NO.
  • 4-1BB intracellular costimulatory signal domain the amino acid sequence of which is shown in SEQ ID NO.
  • CD28 intracellular costimulatory signal domain the amino acid sequence of which is shown in SEQ ID NO.
  • the CD19 antigen binding domain has an amino acid sequence as shown in SEQ ID NO.
  • chimeric antigen receptor compositions referred to in the examples of the invention include, but are not limited to, the following structures:
  • CAR1 4-1BB signal peptide-V H -(G3S) 4 -V L -4-1BB TM-4-1BBcyto-CD3 ⁇ , its amino acid sequence
  • SEQ ID NO. 10 the nucleotide coding sequence is shown in SEQ ID NO. 22; the synthesis is shown in SEQ ID NO.
  • the gene sequence is used to construct a recombinant expression vector
  • CAR2 CD8 ⁇ signal peptide-V H -(G3S) 4 -V L -CD8 ⁇ Hinge&TM-4-1BBcyto-CD3 ⁇ , the amino acid sequence of which is represented by SEQ ID NO. 11, the nucleotide coding sequence is shown as SEQ ID NO. 23; The gene sequence shown in SEQ ID NO. 23 is used to construct a recombinant expression vector;
  • CAR3 CD8 ⁇ signal peptide-V H -(G3S) 4 -V L -4-1BB TM-4-1BBcyto-CD3 ⁇ , the amino acid sequence of which is represented by SEQ ID NO. 12, and the nucleotide coding sequence is set forth in SEQ ID NO. Generating the gene sequence shown in SEQ ID NO. 24 for construction of a recombinant expression vector;
  • the antigen binding domain can be replaced with different tumor antigen targets within the technical scope of the art.
  • the antigen-specific antibody heavy chain variable region and the light chain variable region may be in the direction of V H -V L or V L -V H , and the linker Linker may select (G3S) 4 Linker, (G4S) 3 Linker or other GS Linker or other protein linker, such as Whitlow Linker: GTSSGSGKPGSGEGSTKG.
  • the invention uses a self-inactivated lentiviral expression vector to express a CAR gene sequence of interest.
  • the expression plasmid and the pCMV- ⁇ R-8.74 and pMD2.G helper plasmids were extracted and mixed in a certain ratio to co-transfect 293T cells. After transfection for 48 h and 96 h, the cell culture supernatant containing the virus was collected and centrifuged at 4 ° C, 3000 rpm for 5 min.
  • the supernatant was filtered through a 0.45 ⁇ m filter, mixed with PEG6000/NaCl in a volume of 4:1, and allowed to stand at 4 ° C for 2 to 3 hours, followed by high-speed centrifugation for 30 min. The supernatant was discarded, and the pellet was resuspended in pre-cooled PBS to obtain a virus concentrate, which was stored at -80 ° C until use.
  • the gene of the present invention synthesizes a nucleotide sequence encoding human CD19 molecule (protein sequence is NCBI number NP_001171569.1), and provides gene synthesis technology service by Nanjing Kingsray Biotechnology Co., Ltd.
  • the synthesized CD19 nucleotide sequence was ligated to the pLVX-Puro (Clontech, Cat. #632164) lentiviral vector which was previously digested with BamH1 and XbaI restriction sites by T4 ligase overnight at 20 °C.
  • the ligation product was transformed into DH5 ⁇ competent cells and coated with bacterial plates, and multiple cloned spots were picked for plasmid extraction (Qiagen Endofree Megakit). After enzyme digestion and sequencing, the successful vector was named pLVX-CD19-Puro.
  • the extracted pLVX-CD19-Puro expression plasmid and pCMV- ⁇ R-8.74 (J.Virol.-1998-Dull-A third-generation lentivirus vector with a conditional packaging system) and pMD2.G helper plasmid are mixed in a certain ratio. 293FT cells were co-transfected. After 96 hours of transfection, the virus-containing cell culture supernatant was collected and centrifuged at 4 ° C, 3000 rpm for 5 min. The supernatant was filtered through a 0.45 ⁇ m filter and centrifuged at 4 ° C, 25,000 rpm for 120 min. The virus concentrate was obtained after the supernatant was discarded and dissolved, and stored at -80 ° C until use. The lentiviral vector carrying the firefly luciferase/Luciferase reporter gene was also prepared in the same manner.
  • K562 cell line was purchased from ATCC (Cat. No. CCL-243), using 90% IMDM (Life technology, Cat. No. A10491-01) + 10% FBS (Life technology, Cat. No. 10099-141) + 1% penicillin / streptomycin (Life technology , Item No. 15140-122) was subjected to conventional culture (hereinafter referred to as IMDMi0 culture solution).
  • IMDMi0 culture solution The constructed lentiviral vector carrying the CD19 gene and the lentiviral vector carrying the firefly luciferase/Luciferase reporter gene were added to the cultured K562 cell supernatant to co-transduce the K562 cell line.
  • K562.CD19.Luc a monoclonal cell, which was named K562.CD19.Luc.
  • K562.CD123.Luc another stable cell line K562.CD123.Luc (CD123 gene source: GenBank: NM_002183) constructed according to the above method was used as a CD19 negative control cell line.
  • the CD19-positive human lymphoma cell line Raji was purchased from American ATCC (ATCC #CCL-86TM) and cultured with RPMI1640 + 10% FBS + 1% penicillin/streptomycin (hereinafter referred to as R10 culture solution).
  • a firefly luciferase/Luciferase reporter gene was transduced into Raji cells in a similar manner to the above, and a cell line stably expressing the luciferase reporter gene was obtained by Puromycin pressure screening and designated as Raji.Luc.
  • the monoclonal cells obtained above were identified for CD19 expression by flow cytometry.
  • Fig. 2A the expression of CD19 on the constructed Raji.Luc cell line and the isotype control Raji cell line (purchased from Miltenyi) was detected by CD19-specific antibody, respectively.
  • the flow cytometry results showed that the constructed Raji.Luc cell line had Higher CD19 expression levels.
  • Figure 2B shows the expression levels of CD19 antigen in the constructed K562.CD19.Luc cell line and control cell line by CD19-specific antibody, respectively.
  • Flow cytometry results showed that the constructed K562.CD19.Luc cell line was also very high. CD19 expression level.
  • PBMC peripheral blood mononuclear cells
  • Activated T lymphocytes were collected and resuspended in RPMI 1640 medium. 1 ⁇ 10 6 activated T lymphocytes were infected with lentivirus, and the cell suspension was added to a 6-well plate and incubated overnight at 37 ° C in a 5% CO 2 incubator. On the next day, the cells were again centrifuged and replaced with fresh medium, and fresh medium was added every 2 days to continue the expansion.
  • the CAR-T cells prepared according to the above protocol were collected by centrifugation, washed three times with DPBS, and genomic DNA was prepared using a human genome extraction kit Gentra Puregene Cell Kit (purchased from Qiagen). The prepared DNA was measured for absorbance after OD 260 nm and OD 280 nm. Adjusting the appropriate DNA concentration
  • the Q-PCR reaction system was configured according to the kit SYBR Green Real time PCR Master mix plus (purchased from Toyobo), and then the gene copy number was detected on a fluorescence quantitative PCR machine (ABI #7300).
  • Q-PCR detection uses an accurate quantitative plasmid containing the target fragment as a positive control and a standard curve.
  • the CT value of each copy number concentration of the standard curve obtained by Q-PCR and the corresponding copy number are plotted as a straight line fitting standard curve, and other test samples are based on The fitted equation of the standard curve calculates the relative copy number.
  • the detection of chimeric antigen receptors by CAR-T cells in the present invention uses untransduced T lymphocytes (UnT) as a blank control, and uses the CDS ⁇ signal peptide, hinge region and transmembrane region structure in the US8399645B2 patent.
  • CD19CAR-T (CAR2-T) is a control.
  • CAR integrated copy number detection are shown in Table 1.
  • the results showed that the CAR1 gene was detected in the genome of the CAR1-T group, and its copy number was 7.46 ⁇ 10 5 copies/ng genomic DNA, and the CAR2-gene transfected CAR2-gene copy number was 12.90 ⁇ 10 5 copies/ng genomic DNA.
  • the blank control UnT and H 2 O detection values were extremely low (about 30 copies/ng genomic DNA), which belonged to the background of the test.
  • the CAR-T cells prepared above were collected, adjusted to a suitable density with R10 medium, and seeded in 384-well plates.
  • CAR-T was co-cultured with target cell Raji-Luc at a target ratio of 50:1 or 20:1 (E:T) at 37 °C for 20 h, then an equal amount of luciferase activity detection reagent One-Glo TM was added.
  • Luciferase Assay purchased from Promega.
  • the relative activity of the luciferase relative activity (RLU) in the well corresponds to the relative amount of living cells in the well. If the luciferase RLU value is high, it indicates the remaining unresolved in the well.
  • the number of killed target cells is large, indicating that the cell killing effect in the well is weak; conversely, if the luciferase RLU value is low, it means that there are fewer unkilled target cells in the well, indicating cell killing in the well. Strong effect.
  • the killing test of the tumor cells by the CAR-T cells of the present invention using untransduced T lymphocytes (UnT) as a blank control, and using CAR2 as a control.
  • CAR1-T can significantly kill Raji. Luc cells (relative to luciferase activity RLU% reduction) were significantly better than the control group CAR2-T killing effect.
  • E:T ratio 1 the remaining Raji.Luc relative cells of CAR1-T and Raji.Luc were 20.2 ⁇ 1.23%, while the CAR2-T group had 98.84 ⁇ 1.60% remaining, and the UnT group was 100 ⁇ 3.54.
  • the remaining Raji.Luc cells after CAR1-T and Raji.Luc were 26.83 ⁇ 1.97%, while the remaining relative target cells in the CAR3-T group were 36.86 ⁇ 3.46%.
  • the UnT group was 100 ⁇ 1.78%.
  • CAR1 differs from CAR3 in that the extracellular signal peptide is different, and CAR3 contains the CD8 ⁇ signal peptide. From the results of Fig. 3B, it can be inferred that the CAR1 modified immune cells comprising the 4-1BB signal peptide and the transmembrane region have better tumor in vitro killing effect than the CAR3 modified immune cells containing the CD8 ⁇ signal peptide.
  • a chimeric antigen receptor containing different signal peptides or transmembrane domain structures with modified immune cells with different in vitro killing ability transmembrane domain structure may be involved for chimeric antigen receptors
  • the performance has a more significant impact.
  • the present invention uses the constructed K562.CD19.Luc expressing CD19 and the K562.CD123.Luc stable cell line expressing CD123 but expressing CD123 (Example 2) as an evaluation.
  • the specificity of CAR1 killing effect As shown in Figure 3C, the K562.CD19.Luc relative cells remaining after co-culture of CAR1-T with K562.CD19.Luc were 6.77 ⁇ 0.84%; and the remaining K562.CD123 after CAR1-T and K562.CD123.Luc were co-cultured. .Luc relative cells were 107.06 ⁇ 14.39%; while UnT cells had no obvious killing effect on both target cells; CAR1-T had obvious target-selective killing effect on K562.CD19.Luc cells.
  • the CAR-T cells prepared above were collected, and inoculated in a 96-well plate with an appropriate density of R10 medium.
  • the release of IFNy is a hallmark of T cell activation.
  • HTRF real-time fluorescence resolution technology kit
  • the release of IFN ⁇ in the CAR2-T group was 808.67 ⁇ 21.42pg/mL
  • the release of IFN ⁇ in the UnT group was 240.82 ⁇ 34.11pg/mL
  • the release of IFN ⁇ in the Luc group was 239.82 ⁇ 83.47pg/mL.
  • CAR1-T and CAR2-T significantly release IFNy
  • CAR1-T has a more prominent antigen-dependent release level of IFNy than CAR2-T.
  • the peripheral blood of patients with acute B cell lymphocytic leukemia was collected, and the T cells were isolated and purified (Example 2).
  • the CAR1 lentiviral vector was transduced.
  • the CAR1 lentiviral vector that did not enter the cells in the culture supernatant was washed away by centrifugation.
  • Transduced T cells were resuspended in medium containing final concentrations of 100 IU/mL IL-2 and CD3/CD28 magnetic beads.
  • the culture was expanded for 11 to 14 days every other day at a culture density of 0.5 to 2 x 10 6 /mL.
  • CAR1 was transduced into T cells from 5 different B-ALL patients, and the transduced cells were named ALLCT01, ALLCT02, ALLCT03, ALLCT04 and ALLCT05, respectively.
  • the results in Figure 5 show that the cells transduced by CAR1 showed strong ability to kill Raji.Luc target cells in vitro, and the relative number of remaining Raji.Luc cells after incubation with Raji.Luc target cells was 9.33%-37.82. %.
  • CD19 CAR-T treatment in patients with ALLCT01 acute B lymphocytic leukemia.
  • ALLCT01 The disease was detected by the hospital as active bone marrow hyperplasia, abnormal lymphocyte system, and the original lymphocytes accounted for 65%.
  • the clinical diagnosis is B-ALL (NR). After the hospital recommended, through the ethical review, the patient signed the informed consent form, and then the clinical trial study of CAR-T cell therapy.
  • the treatment process of CAR-T cells is: collecting the peripheral blood of the patient, and separating and purifying the T cells in vitro, preparing CD19CAR-T cells in vitro, and returning to the patient body by intravenous autologous in three time points, the total infusion is about 1.5 x 10 8 cells.
  • the patient's peripheral blood was tested 14 days after the last return. As shown in the results in Table 2, the percentage of CD19-positive (CD19+) cells in peripheral blood decreased from 63% before treatment to 0%, and 83.59% of the cells were CD3-positive (CD3+) cells. Combined with hematological test results, the CD19-positive leukemia cells in the peripheral blood were completely eliminated after the patient was treated with the CD19CAR-T cells provided by the present invention, and the clinical complete remission was achieved.

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Abstract

一种新型嵌合抗原受体及其应用。所述的新型嵌合抗原受体由信号肽、抗原结合结构域、跨膜区和胞内信号结构域组成，包含4-1BB信号肽和/或4-1BB分子跨膜区。分离纯化了多种嵌合抗原受体核酸序列，并提供了一种特异性针对CD19恶性肿瘤抗原的嵌合抗原受体及CAR-T细胞。在血液细胞系恶性肿瘤杀伤试验中，明显加强了免疫细胞靶向识别肿瘤细胞的能力，也增强了对肿瘤细胞的杀伤活性。

Description

一种新型嵌合抗原受体及其应用 技术领域
本发明属于生物医学或生物制药技术领域，涉及一种新型嵌合抗原受体及其应用。
背景技术
据《2015全球癌症统计》数据显示，2012年全球新增1410万肿瘤患者，820万患者死亡。据中国肿瘤登记中心的数据显示，2015年中国新增430万癌症病例，癌症死亡病例超过281万，占据全年死亡人数比例的28.82％，即平均每天就有超过7500人死于癌症，居于世界首位。
传统的肿瘤治疗手段主要有手术、放射治疗、化疗，以及近年兴起的干细胞移殖等，往往这些治疗手段治标不治本。随着科学的发展，肿瘤的免疫治疗近年来实现了巨大的突破，其中主要包括免疫检查点抑制剂(例如anti-PD1单克隆抗体)，以及嵌合抗原受体T细胞免疫疗法(Chimeric Antigen Receptor Modified T cell therapy，CAR-T)的临床效果最好。近年来，CAR-T技术在血液性疾病治疗临床试验取得了令人鼓舞的进展，被《Science》杂志评为2013年十大科学突破之首。从2013年宾夕法尼亚大学Carl June小组报道第一例使用靶向CD19CAR-T治疗患有急性B淋巴细胞性白血病的儿童(Emily Whitehead)得到完全缓解开始，短短几年时间CAR-T细胞免疫疗法发展迅猛。CAR-T细胞治疗是当前靶向性最高、疗效最好的细胞免疫疗法。经过技术演变之后，CAR-T已经变得更加灵敏、免疫持续性更久，对淋巴瘤等血液肿瘤具有奇效。目前有多个细胞治疗方法已被FDA授予“突破性疗法”资格，其中，多项临床试验研究结果表明，CD19CAR-T治疗B细胞淋巴瘤的临床完全缓解率均已经超过90％。
CAR-T细胞是能够经过基因工程手段在T细胞表面表达识别特定抗原并且传递信号的一类T细胞。一股地，CAR-T细胞通过嵌合抗原受体CAR以抗原-抗体或配体-受体识别模式对肿瘤细胞表面的特异分子进行识别，然后通过其胞内的信号传导进行激活、增殖并发挥细胞杀伤功能。经嵌合抗原受体修饰的T细胞，可以特异性地识别肿瘤相关抗原，使效应T细胞的靶向性、杀伤活性和持久性均较常规应用的免疫细胞高，并可克服肿瘤局部免疫抑制微环境并打破宿主免疫耐受状态。
经修饰的T细胞表达这样的一类嵌合抗原受体分子：胞外段一股包含CD8α或GM-CSFRα信号肽，抗原识别区或抗原结合结构域，包含由抗体重链和轻链可变区组成的单链可变区；胞内段是各种信号传导分子的胞内段嵌合体，包括CD28、4-1BB、OX-40、CD3zeta等，跨膜区则 来自其他分子，如PD1、CD8、CD4、CD28、CD3zeta(CD3ζ)等。CAR胞外区部分的非抗原识别区，包括信号肽，铰链区(Hinge，scFv和跨膜区之间的连接区，亦有称间隔区spacer domain)，对于CAR的功能有重要的影响。德国Kober L等人报道通过优选不同来源的信号肽序列对可使双特异性单链抗体(Bi-specific scFv antibody)的表达水平显著提升，这与信号肽引导蛋白进入分泌途径的能力有密切的关系(Biotechnol Bioeng.2013Apr；110(4)：1164-73.)。美国Fred Hutchinson Cancer Research Center的Michael Hudecek等人报道，使用不合适的间隔区的CAR尽管在体外实验中有良好的肿瘤细胞杀伤作用，但是在体内由于间隔区与Fc受体有结合作用，导致CAR-T细胞在体内发生被激活引起的T细胞死亡现象(Activation-induced T-Cell death)，导致这样的CAR构造在体内不能持续存在而丧失抗肿瘤活性(Cancer Immunol Res；3(2)；125-35.)。国际申请WO2016/014789A2中，不改变胞外抗原识别区和胞内信号区，仅通过改变跨膜区的序列，即可大幅度实现CAR载体的性能，降低CAR-T对非靶细胞的非特异杀伤作用。目前在CAR-T领域，与CD28胞内信号结构域相比，包含4-1BB胞内信号结构域的CAR结构被认为具有更好的体内肿瘤细胞杀伤活性和持续性。
在B细胞白血病领域，CD19表达于几乎所有的B细胞肿瘤细胞表面，而在其他实质性细胞和造血干细胞中几乎不表达，CD19是B系肿瘤抗原比较特异的靶标。目前在临床上取得重要进展的CD19CAR结构为CD8α信号肽-抗CD19单链抗体-CD8α铰链区&跨膜区-4-1BB胞内区-CD3ζ胞内区(CD8αsignal peptide-antiCD19scFv-CD8αHinge&TM-4-1BBcyto-CD3ζ)。
人4-1BB分子(NCBI数据库蛋白编号NP_001552.2)，又称CD137，或肿瘤坏死因子受体超家族成员9(tumor necrosis factor receptor superfamily member 9，TNFRSF9)其一级结构为1-23号氨基酸为信号肽，24-186号氨基酸为胞外区，187-213号氨基酸为跨膜区，214-255号氨基酸为胞内区。目前尚未见报道使用4-1BB的基本结构进行CAR的设计。
本发明提供一种新型的CAR结构，包含4-1BB信号肽和/或4-1BB跨膜区结构，例如4-1BB信号肽-肿瘤抗原识别结合区-4-1BB跨膜区-4-1BB胞内区-CD3zeta胞内区(4-1BB signal peptide-VH-Linker-VL/VHH-4-1BB TM-4-1BBcyto-CD3ζ)，最大限度地使用4-1BB的氨基酸序列。使用4-1BB信号肽和4-1BB的跨膜区及胞内区，利于CAR-T细胞上胞外肿瘤抗原识别结合区的延展得到优化，不仅在体外和体内有良好的肿瘤杀伤作用，而且在临床治疗急性淋巴瘤病人也实现了完全缓解，而且细胞因子释放反应更为温和。
发明内容
本发明的目的是针对现有技术的上述不足，提供一种新型嵌合抗原受体及其应用。
本发明的另一目的是提供编码该嵌合抗原受体的核酸。
本发明的又一目的是提供含有该嵌合抗原受体的细胞及其应用。
一种新型嵌合抗原受体，其特征在于包含胞外信号肽、抗原结合结构域、跨膜结构域和胞内信号结构域，其中所述胞外信号肽选自4-1BB信号肽、CD8α信号肽、GM-CSFRα信号肽或CD4信号肽中的一种，跨膜结构域选自4-1BB分子跨膜区序列。
所述跨膜结构域4-1BB分子跨膜区的氨基酸序列优选SEQ ID NO.1所示，或与其氨基酸序列具有85％-99％同一性的多肽。
所述的胞外信号肽氨基酸序列优选如SEQ ID NO.2所示的4-1BB信号肽，或与其氨基酸序列具有85％-99％同一性的多肽。
所述的抗原结合域结合的抗原与恶性肿瘤有关，包括但不限于CD19、CD20、CD22、CD30、CD33、CD38、BCMA、CS1、CD138、CD123/IL3Rα、c-Met、gp100、MUC1、IGF-I receptor、EPCAM、EGFR/EGFRvIII、HER2、PD1、CTLA4、IGF1R、mesothelin、PSMA、WT1、ROR1、CEA、GD-2、NY-ESO-1、MAGE A3、GPC3、糖脂F77或其他任意肿瘤抗原或其他修饰类型或组合。
所述的抗原结合结构域结合的抗原进一步优选为CD19；CD19抗原结合结构域氨基酸序列如SEQ ID NO.9所示。
在一个实施方案中，所述嵌合抗原受体的抗原结合结构域通过配体-受体相互作用结合靶标，可选自IL-3、IL-13或APRIL。
所述的抗原结合结构域由抗体片段组成，优选单克隆抗体、Fab、scFv、sdAb、VHH或其他抗体片段。其抗原结合结构域抗体可来自鼠源、骆驼源或人源化抗体。在一个具体实施例中，该嵌合抗原受体的抗原结合结构域由CD19特异性单链抗体片段组成，包括单链抗体的重链可变区(VH)和轻链可变区(VL)。
在一个实施方案中，所述抗原结合结构域的单链抗体重链可变区(VH)和轻链可变区(VL)之间由连接肽(Linker)连接，包含GS Linker如(G 3S) 4、(G 4S) 3或GSTSGSGKPGSGEGSTKG，优选(G 3S) 4连接肽。
所述胞内信号结构域优选包括CD3 zeta(CD3ζ)、CD3 gamma、CD3 delta、CD3 epsilon、common FcR gamma(FCER1G)、FcR beta(Fc Epsilon Rib)、CD79a、CD79b、Fc gamma RIIa、DAP10 and DAP12分子的胞内信号区序列，或其组合。进一步优选的，胞内信号结构域包括CD3ζ分子的信号传导结构域。
所述胞内信号结构域还优选包括共刺激信号结构域，可选自下列共刺激信号分子：CD27、CD28、4-1BB、OX40、CD30、CD40、CD2、淋巴细胞功能相关抗原-1(LFA-1)、LIGHT、NKG2C、B7-H3、PD-1、ICOS、CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、CD7、NKp80(KLRF1)、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2R gamma、IL7R alpha、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244，2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A，Ly108)、SLAM(SLAMFI，CD150，IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、CD83特异性结合的配体或其任意组合。在一个具体实施方案中，进一步优选4-1BB为共刺激信号结构域，其氨基酸序列如SEQ ID NO.6所示。
在一个实施方案中，该嵌合抗原受体在胞外抗原结合结构域和跨膜结构域之间不包含铰链区。
在另一个实施方案中，该嵌合抗原受体在胞外抗原结合结构域和跨膜结构域之间包含铰链区。在具体实施方案中，该铰链区优选SEQ ID NO.4所示的CD8α铰链区。
在一个特定实施方案中，该嵌合抗原受体优选包括顺序连接的4-1BB信号肽或CD8α信号肽、抗原结合结构域、4-1BB跨膜区、4-1BB胞内共刺激结构域以及CD3ζ信号传导结构域。
本发明嵌合抗原受体结构如下：
4-1BB signal peptide-VH-Linker-VL-4-1BB TM-4-1BBcyto-CD3ζ
CD8α signal peptide-VH-Linker-VL-4-1BB TM-4-1BBcyto-CD3ζ
4-1BB signal peptide-VH-Linker-VL-TM-4-1BBcyto-CD3ζ
4-1BB signal peptide-V HH 1-Linker-V HH 2-4-1BB TM-4-1BBcyto-CD3ζ
CDSα signal peptide-V HH 1-Linker-V HH 2-4-1BB TM-4-1BBcyto-CD3ζ
4-1BB signal peptide-V HH 1-Linker-V HH 2-TM-4-1BBcyto-CD3ζ
其中抗原结合结构域可由VH-Linker-VL或VL-Linker-VH或V HH 1-Linker-V HH 2组成，V HH 1和V HH 2可识别相同抗原，或不同抗原。
作为本发明的优选，该嵌合抗原受体包含SEQ ID NO.10或SEQ ID NO.12所示的氨基酸序列，或与其具有85％-99％同一性氨基酸序列。
编码前述嵌合抗原受体的核酸分子。
该核酸分子优选包含SEQ ID NO.22或SEQ ID NO.24的核苷酸序列。
上述核酸分子的表达载体。
该表达载体优选慢病毒表达载体，包含编码SEQ ID NO.22或SEQ ID NO.24的核苷酸序列。
表达前述嵌合抗原受体的细胞。
该细胞优选免疫细胞；进一步优选T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
一种制备新型嵌合抗原受体修饰的T细胞的方法，该方法包括分离和激活待修饰的T细胞，然后以前述表达载体转导该T细胞。
含有所述新型嵌合抗原受体、表达载体、所述细胞在制备治疗肿瘤的药物中的用途。
所述的肿瘤优选包括胶质母细胞瘤、头颈癌、甲状腺癌、肾癌、肺癌、乳腺癌或卵巢癌，以及慢性淋巴细胞白血病(CLL)、急性白血病、急性淋巴细胞白血病(ALL)、B细胞急性淋巴细胞白血病(ALL)、T细胞急性淋巴细胞白血病(T-ALL)、慢性粒细胞白血病(CML)、B细胞幼淋巴细胞白血病、急浆细胞样树突状细胞肿瘤、Burkitt淋巴瘤、弥漫性大B细胞淋巴瘤、滤泡性淋巴瘤毛细胞白血病、小细胞或大细胞滤泡性淋巴瘤，恶性淋巴组织增生，MALT淋巴瘤、套细胞淋巴瘤，边缘区淋巴瘤，多发性骨髓瘤、骨髓增生异常和骨髓增生异常综合征、非霍奇金淋巴瘤、霍奇金淋巴瘤、浆母细胞性淋巴瘤、浆细胞样树突状细胞肿瘤淋巴瘤等血液系肿瘤。
在具体实施方案中，涉及制备治疗血液系肿瘤的药物用途。
本发明所述的新型嵌合抗原受体涉及在制备抗肿瘤药物及细胞免疫疗法中的应用。
本发明所述的免疫效应细胞涉及在制备抗肿瘤药物及细胞免疫疗法中的应用。
有益效果：
本发明提供一种新型的CAR结构，包含4-1BB信号肽和/或4-1BB跨膜区结构，例如4-1BB信号肽-肿瘤抗原识别结合区-4-1BB跨膜区-4-1BB胞内区-CD3zeta胞内区(4-1BB signal peptide-VH-Linker-VL/VHH-4-1BB TM-4-1BBcyto-CD3ζ)，最大限度地使用4-1BB分子结构的氨基酸序列。利用4-1BB信号肽、4-1BB跨膜区或胞内区结构，利于CAR-T细胞上胞外肿瘤抗原识别结合区的延展，不仅在体外和体内都有良好的肿瘤杀伤作用，而且在临床上治疗急性淋巴瘤病人也实现了完全缓解，而且细胞因子释放反应更为温和。以CD19抗原靶点为例，与现有技术相比，该新型嵌合抗原受体显示了较高的抗肿瘤能力，由其修饰的免疫细胞具有较高的靶向识别肿瘤抗原的能力，增强了对肿瘤细胞的杀伤活性。
附图说明
图1为嵌合抗原受体的载体结构图，展示了本发明中涉及的CAR1(A图)和CAR3(B图)嵌合抗原受体载体结构图。
图2为本发明中所构建的稳定细胞系表达的CD19抗原检测。A.用CD19特异性抗体分别检测所构建的Raji.Luc细胞系及同型对照Raji细胞系上CD19的表达。B.为CD19特异性抗体分别检测所构建的K562.CD19.Luc细胞系及对照细胞系中CD19抗原的表达水平。
图3为CAR-T体外杀伤CD19阳性细胞的效果图。A.在E∶T ratio 1的条件下，CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为20.2±1.23％，而CAR2-T组剩余98.84±1.60％，UnT组为100±3.54％；在E∶T ratio 2的条件下，CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为50.09±2.17％，而CAR2-T组剩余107.07±3.04％，UnT组为100±3.50％。B.CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为26.83±1.97％，而CAR3-T组剩余相对靶细胞为36.86±3.46％，UnT组为100±1.78％。
图4为CAR-T细胞的IFNγ释放水平检测。A.CAR1-T、CAR2-T及UnT、Luc组与Raji.Luc细胞共孵育4h及20h后IFNγ的释放量。B.CAR1-T、CAR2-T及UnT、Luc组与来自于急性B细胞白血病病人的B淋巴细胞共孵育4h及20h后IFNγ的释放量。
图5为CAR1转导的自体T细胞对肿瘤杀伤效果的检测。将CAR1分别转导至来自5例不同B-ALL患者的T细胞中，转导后的细胞分别命名为ALLCT01、ALLCT02、ALLCT03、ALLCT04及ALLCT05。经CAR1转导后的细胞与Raji.Luc靶细胞共孵育后，剩余的Raji.Luc细胞的相对数量为9.33％-37.82％。
图6为人白介素6释放水平图。ALLCT01病人回输CD19CAR-T细胞后，体温在最后一次回输后6天开始升高，细胞因子检测结果显示白介素6(IL-6)分泌水平显著提高。
具体实施方式
本发明提供了一种新型嵌合抗原受体、免疫效应细胞及其在抑制肿瘤活性中的应用，下面结合具体实施例，进一步阐述本发明。
本文所用的术语“跨膜结构域”(简称TM)可以与“跨膜区”互换使用，指的是锚定在细胞膜内具有热力学稳定的蛋白质结构区域。跨膜区可以从天然蛋白质中获得，选自4-1BB分子。
本发明的新型嵌合抗原受体包含胞外信号肽结构，例如4-1BB信号肽、CD8α信号肽、 GM-CSFRα信号肽或CD4信号肽，优选4-1BB信号肽。
本文所用的术语“胞内信号结构域”指的是能够传导细胞效应功能信号并指导细胞执行特定功能的蛋白质结构区域。胞内信号结构域可以包括信号传导结构域和/或共刺激信号结构域。
本文所用的术语氨基酸序列的“同一性”(identity)可以与“相似性”互换使用，指的是氨基酸序列之间通过序列比对软件例如BLAST确定的相似程度。氨基酸序列比对的方法和软件对于本领域技术人员是公知的。可以通过对已知氨基酸序列进行一个或几个(例如1-15个，例如2、3、5、8、10或12个)氨基酸残基的取代、缺失和/或添加而获得经改造的氨基酸序列。例如，通过常规蛋白质工程手段(例如氨基酸保守取代等)，对本发明SEQ ID NO.1所示的4-1BB跨膜结构域进行改造，可以获得与SEQ ID NO.1具有至少85％(例如85％～99％或90％～99％或95％～99％)序列同一性，并且具有基本相同的跨膜功能的变体序列。
本文所用的术语“抗体片段”是包括具有功能的抗体部分，优选抗原结合和/或完整抗体的可变区。抗体片段包括Fab、Fab′、F(ab′) 2、Fv fragments、单链抗体scFv、单域抗体sdAb/V HH及多特异性抗体。
本文所用的术语“scFv”或“scFv抗体”是指单链抗体(single chain antibody fragment，scFv)，是由抗体重链可变区VH和轻链可变区VL通过15～20个氨基酸的短肽连接而成。此处所用的术语“Linker”也称连接肽或接头，是用于连接两个结构域的柔性氨基酸序列。连接肽的选择和制备对本领域技术人员来说是容易的。
本发明通过优化利用人4-1BB分子结构域进行嵌合抗原受体设计，下面结合具体实施例，进一步阐述本发明。
实施例1 嵌合抗原受体制备
(一)嵌合抗原受体基因片段制备
本发明提供了一种包含CD19抗原结合结构域的新型嵌合抗原受体。本发明提供的抗原结合结构域抗体由单链抗体的重链可变区(VH)、轻链可变区(VL)及连接肽(G3S) 4Linker组成。单链抗体的重链可变区及轻链可变区序列分别来源于GenBank：Y14283.1、GenBank：Y14284.1，将其进行密码子及序列优化，以保证其在人类细胞内更适合表达。
本发明提供的新型嵌合抗原受体可按照以下编码基因的顺序设计及变换嵌合抗原受体融合基因片段：4-1BB信号肽、V H-(G3S) 4-V L、4-1BB跨膜区、4-1BB或CD28胞内共刺激信号结构域或CD3ζ胞内信号传导结构域，基因合成由南京金斯瑞生物科技有限公司提供技术服务，通 过基因合成技术选取以下编码基因序列进行融合基因合成：
编码4-1BB跨膜区的核苷酸序列如SEQ ID NO.13所示；
编码4-1BB信号肽的核苷酸序列如SEQ ID NO.14所示；
编码CD8α信号肽的核苷酸序列如SEQ ID NO.15所示
编码CD8α铰链区的核苷酸序列如SEQ ID NO.16所示；
编码CD8α跨膜区的核苷酸序列如SEQ ID NO.17所示；
编码4-1BB胞内共刺激信号结构域的核苷酸序列如SEQ ID NO.18所示；
编码CD28胞内共刺激信号结构域的核苷酸序列如SEQ ID NO.19所示；
编码CD3ζ胞内信号传导结构域的核苷酸序列如SEQ ID NO.20所示；
编码CD19抗原结合结构域的核苷酸序列如SEQ ID NO.21所示。
本发明使用的胞外结构域和胞内信号结构域有多种组合，包含选自如下的结构或组合：
4-1BB跨膜区，其氨基酸序列如SEQ ID NO.1所示；
4-1BB信号肽，其氨基酸序列如SEQ ID NO.2所示；
CD8α信号肽，其氨基酸序列如SEQ ID NO.3所示；
CD8α铰链区，其氨基酸序列如SEQ ID NO.4所示；
CD8α跨膜区，其氨基酸序列如SEQ ID NO.5所示；
4-1BB胞内共刺激信号结构域，其氨基酸序列如SEQ ID NO.6所示；
CD28胞内共刺激信号结构域，其氨基酸序列如SEQ ID NO.7所示；
CD3ζ胞内信号传导结构域，其氨基酸序列如SEQ ID NO.8所示；
CD19抗原结合结构域，其氨基酸序列如SEQ ID NO.9所示。
本发明实施例中涉及的嵌合抗原受体组合物包括但不限于以下结构：
CAR1：4-1BB signal peptide-V H-(G3S) 4-V L-4-1BB TM-4-1BBcyto-CD3ζ，其氨基酸序列
SEQ ID NO.10所示，核苷酸编码序列如SEQ ID NO.22所示；合成SEQ ID NO.22所示
基因序列用于构建重组表达载体；
CAR2：CD8αsignal peptide-V H-(G3S) 4-V L-CD8αHinge&TM-4-1BBcyto-CD3ζ，其氨基酸序列SEQ ID NO.11所示，核苷酸编码序列如SEQ ID NO.23所示；合成SEQ ID NO.23所示基因序列用于构建重组表达载体；
CAR3：CD8αsignal peptide-V H-(G3S) 4-V L-4-1BB TM-4-1BBcyto-CD3ζ，其氨基酸序列SEQ ID NO.12所示，核苷酸编码序列如SEQ ID NO.24所示；合成SEQ ID NO.24所示 基因序列用于构建重组表达载体；
其中，在本领域的技术范围内，抗原结合结构域可以替换成不同肿瘤抗原靶点。抗原特异性的抗体重链可变区与轻链可变区连接方向可以是V H-V L或V L-V H，连接肽Linker可以选择(G3S) 4Linker、(G4S) 3Linker或其他GS Linker或其他蛋白linker，如Whitlow Linker：GSTSGSGKPGSGEGSTKG。
(二)嵌合抗原受体慢病毒表达载体的构建
在一个具体的实施方案中，本发明使用一种自灭活的慢病毒表达载体以表达目的CAR基因序列。首先，提取表达质粒和pCMV-ΔR-8.74及pMD2.G辅助质粒，按一定比例混合，共转染293T细胞。转染48h、96h后，收集含有病毒的细胞培养上清，4℃、3000rpm离心5min。上清经0.45μm滤器过滤后，与PEG6000/NaCl按4∶1体积混匀，4℃静置2～3h后高速离心30min。弃上清，沉淀用预冷的PBS重悬溶解，即获得病毒浓缩液，-80℃保存备用。
实施例2 构建表达CD19抗原的K562细胞系K562.CD19.Luc
K562细胞几乎不表达CD19。本发明基因合成编码人CD19分子(蛋白序列为NCBI编号NP_001171569.1)的核苷酸序列，由南京金斯瑞生物科技有限公司提供基因合成技术服务。合成的CD19核苷酸序列在T4连接酶的作用下，20℃过夜连接到预先进行BamH1和XbaI限制酶切位点酶切后的pLVX-Puro(Clontech，货号#632164)慢病毒载体上。将连接产物转化到DH5α感受态细胞中并涂布细菌平板，挑取多个克隆斑进行质粒的提取(Qiagen Endofree Megakit)。经酶切鉴定、测序比对，构建成功的载体命名为pLVX-CD19-Puro。
将提取的pLVX-CD19-Puro表达质粒和pCMV-ΔR-8.74(J.Virol.-1998-Dull-A third-generation lentivirus vector with a conditional packaging system)及pMD2.G辅助质粒，按一定比例混合，共转染293FT细胞。转染96h后，收集含有病毒的细胞培养上清，4℃、3000rpm离心5min。上清经0.45μm滤器过滤后，经4℃、25000rpm高速离心120min。弃上清重悬溶解后即获得病毒浓缩液，-80℃保存备用。携带萤火虫荧光素酶(Firefly luciferase/Luciferase)报告基因的慢病毒载体也采用同样的方法进行制备。
K562细胞系购自ATCC(货号CCL-243)，使用90％IMDM(Life technology，货号A10491-01)+10％FBS(Life technology，货号10099-141)+1％青霉素/链霉素(Life technology，货号15140-122)进行常规培养(以下简称IMDMi0培养液)。将构建的携带CD19 基因的慢病毒载体与携带萤火虫荧光素酶(Firefly luciferase/Luciferase)报告基因的慢病毒载体加入到培养的K562细胞上清中，共同转导K562细胞系。转导后24h，加入终浓度为10μg/ml的Puromycin，每三天换一次培养液并添加相同浓度的Puromycin，进一步进行单克隆的筛选获得单克隆细胞，命名为K562.CD19.Luc。本发明同时按照上述方法构建的另一株稳定细胞系K562.CD123.Luc(CD123基因来源：GenBank：NM_002183)作为CD19阴性对照细胞系使用。
CD19阳性的人淋巴瘤细胞株Raji购买自美国ATCC(ATCC#CCL-86TM)采用RPMI1640+10％FBS+1％青霉素/链霉素进行培养(以下简称R10培养液)。采用与上述相似的方法把萤火虫荧光素酶(Firefly luciferase/Luciferase)报告基因转导进入Raji细胞中，经过Puromycin压力筛选，获得稳定表达荧光素酶报告基因的细胞系，命名为Raji.Luc。
利用流式细胞检测技术对上述所获得的单克隆细胞进行CD19表达鉴定。如图2A，用CD19特异性抗体分别检测所构建的Raji.Luc细胞系及同型对照Raji细胞系(购自Miltenyi)上CD19的表达，流式细胞检测结果显示所构建的Raji.Luc细胞系具有较高的CD19表达水平。图2B为CD19特异性抗体分别检测所构建的K562.CD19.Luc细胞系及对照细胞系中CD19抗原的表达水平，流式细胞检测结果显示所构建的K562.CD19.Luc细胞系同样具有很高的CD19表达水平。
实施例3 嵌合抗原受体修饰的免疫效应细胞制备和体外杀伤活性检测
(一)T淋巴细胞的制备
采集50mL健康人新鲜血液，通过淋巴细胞分离液、密度梯度离心方法分离外周血单核细胞(PBMC)。利用Pan T Cell Isolation Kit(购自Miltenyi Biotech)对细胞进行磁珠标记，并分离纯化出T淋巴细胞。纯化后的T细胞，再利用CD3/CD28磁珠进行T淋巴细胞激活及增殖。
(二)慢病毒转导T淋巴细胞
收集激活的T淋巴细胞，重悬在RPMI1640培养基中。用慢病毒感染1x10 6个活化的T淋巴细胞，将细胞悬液加在6孔板中，置37℃、5％CO 2培养箱中孵育过夜。第二天，再次离心并换新鲜培养基，每隔2天加入新鲜培养基，继续扩大培养。
(三)CAR-T的表达检测
离心收集按上述方案制备的CAR-T细胞，分别用DPBS洗3次后用人基因组提取试剂盒Gentra Puregene Cell Kit(购自Qiagen)制备基因组DNA。制备的DNA检测OD 260nm和OD 280nm 吸光度后计算浓度。调整合适的DNA浓度根据试剂盒SYBR Green Real time PCR Master mix plus(购自Toyobo)说明书配置Q-PCR反应体系，然后在荧光定量PCR仪(ABI#7300)上进行基因拷贝数的检测。Q-PCR检测采用准确定量的含目的片段的质粒作为阳性对照和标准曲线，Q-PCR获得的标准曲线各个拷贝数浓度的CT值与对应的拷贝数绘制直线拟合标准曲线，其他检测样品根据标准曲线的拟合方程计算相对的拷贝数。
本发明中CAR-T细胞表达嵌合抗原受体的检测，以未转导的T淋巴细胞(UnT)作为空白对照，同时以使用US8399645B2专利中包含CDSα信号肽、铰链区和跨膜区结构的CD19CAR-T(CAR2-T)为对照。
CAR整合拷贝数检测结果如表1所示。结果显示：CAR1-T组细胞基因组中检测到CAR1基因的整合，且其拷贝数为7.46x10 5拷贝/ng基因组DNA，CAR2-T转导的CAR2基因拷贝数为12.90x10 5拷贝/ng基因组DNA，空白对照UnT及H 2O检测值极低(约30个拷贝/ng基因组DNA)，属于检测的背景。
表1.Q-PCR法检测CAR基因整合进供体T细胞基因组的拷贝数
Figure PCTCN2017119711-appb-000001
实施例4 体外杀伤活性检测
收集上述制备的CAR-T细胞，用R10培养液调整至合适密度并接种在384孔板中。CAR-T与靶细胞Raji-Luc按50∶1或20∶1的效靶比例(E∶T)在37℃条件下共培养20h，然后加入等量的荧光素酶活性检测试剂One-Glo TMLuciferase Assay(购自Promega)。共培养结束后，反应孔中所剩余的荧光素酶相对活性(RLU，relative light unit)对应孔中活细胞的相对量，如果显示的荧光素酶RLU值高，则表示反应孔中剩余的未被杀死的靶细胞多，表明孔中的细胞杀伤作用弱；反之，如果显示的荧光素酶RLU值低，则表示反应孔中的未被杀死的靶细胞少，表明孔中的细胞杀伤作用强。本发明的CAR-T细胞对肿瘤细胞的杀伤试验，以未转导的T淋巴细胞(UnT)作为空白对照，同时以CAR2作为对照。
如图3A所示，在整合拷贝数相当、不同效靶比(E∶T ratio 1＝50∶1，E∶T ratio 2＝20∶1)的条件下，CAR1-T均能明显杀伤Raji.Luc细胞(相对荧光素酶活力RLU％降低)，明显优于对 照组CAR2-T的杀伤效果。在E∶T ratio 1的条件下，CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为20.2±1.23％，而CAR2-T组剩余98.84±1.60％，UnT组为100±3.54％；在E∶T ratio 2的条件下，CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为50.09±2.17％，而CAR2-T组剩余107.07±3.04％，UnT组为100±3.50％。以上结果可揭示包含4-1BB信号肽和跨膜区结构域并缺失铰链区的CAR1修饰的免疫细胞比包含CD8α信号肽和跨膜区结构域的CAR2修饰的免疫细胞具有更优的肿瘤体外杀伤作用。
在另外一组实验中，如图3B所示，CAR1-T与Raji.Luc共培养后剩余的Raji.Luc相对细胞为26.83±1.97％，而CAR3-T组剩余相对靶细胞为36.86±3.46％，UnT组为100±1.78％。CAR1和CAR3相比在于胞外信号肽不同，CAR3包含CD8α信号肽。由图3B结果可推理出包含4-1BB信号肽和跨膜区的CAR1修饰的免疫细胞比包含CD8α信号肽的CAR3修饰的免疫细胞具有更好的肿瘤体外杀伤作用。
综合图3A和3B的数据结果可推出：包含不同信号肽或跨膜区结构的嵌合抗原受体，其修饰的免疫细胞具有不同的体外杀伤能力，跨膜区结构可能对于嵌合抗原受体的性能具有更显著的影响。
进一步地，在靶点特异性地杀伤性实验中，本发明使用构建的表达CD19的K562.CD19.Luc和不表达CD19但表达CD123的K562.CD123.Luc稳定细胞系(实施例2)作为评估CAR1杀伤作用的特异性。如图3C显示，CAR1-T与K562.CD19.Luc共培养后剩余的K562.CD19.Luc相对细胞为6.77±0.84％；而CAR1-T与K562.CD123.Luc共培养后剩余的K562.CD123.Luc相对细胞为107.06±14.39％；而UnT细胞对两种靶细胞均没有明显的杀伤作用；CAR1-T对K562.CD19.Luc细胞具有明显的靶点选择性杀伤作用。
实施例5 CAR-T细胞的IFNγ释放水平检测
收集上述制备的CAR-T细胞，用R10培养液调整合适密度接种在96孔板中。IFNγ的释放是T细胞激活的一个标志。本实施例将不同CAR-T细胞分别与靶细胞按E∶T＝20∶1、37℃条件下共培养，然后移出共培养上清，使用实时荧光分辨技术试剂盒(HTRF，Cisbio#64IL2PEB)检测上清中IFN-γ的释放量。同时以CAR2-T作为对照，未转导的T淋巴细胞(UnT)和仅转导病毒载体的T淋巴细胞(Luc)作为空白对照。
如图4A显示，CAR-T细胞或UnT细胞与CD19阳性的Raji.Luc细胞共孵育4h后，CAR1-T组IFNγ的释放量为894.49±101.64pg/mL，CAR2-T组IFNγ的释放量为343.88±44.30 pg/mL，UnT组IFNγ的释放量为59.49±6.52pg/mL，Luc组IFNγ的释放量为67.43±1.52pg/mL；共孵育20h后，CAR1-T组IFNγ的释放量为1572.23±0.60pg/mL，CAR2-T组IFNγ的释放量为808.67±21.42pg/mL，UnT组IFNγ的释放量为240.82±34.11pg/mL，Luc组IFNγ的释放量为239.82±83.47pg/mL。与UnT及Luc对照相比，CAR1-T和CAR2-T能明显释放IFNγ，并且CAR1-T具有比CAR2-T更突出的抗原依赖性的IFNγ释放水平。
如图4B显示，CAR-T细胞或UnT细胞与来自于急性B细胞白血病病人的B淋巴细胞(B cell acute lymphoblastic leukemia，B-ALL)共孵育4h后，CAR1-T组IFNγ的释放量为411.73±16.14pg/mL，CAR2组IFNγ的释放量为349.41±43.09pg/mL，UnT组IFNγ的释放量为88.66±2.42pg/mL，Luc组IFNγ的释放量为82.87±14.24pg/mL；共孵育20h后，CAR1-T组IFNγ的释放量为1119.37±18.045pg/mL，CAR2-T组IFNγ的释放量为1099.93±75.93pg/mL，UnT组IFNγ的释放量为146.04pg/mL，Luc组IFNγ的释放量为236.45±57.50pg/mL。与UnT和Luc对照组相比，CAR1-T和CAR2-T能都具有明显的IFNγ释放量，CAR1-T释放的IFNγ水平略高于对照组CAR2-T。
实施例6 CAR1转导的自体T细胞对CD19阳性的急性B细胞淋巴白血病患者的临床试验治疗效果
采集急性B细胞淋巴细胞白血病患者外周成分血，分离纯化其T细胞(实施例2)，体外激活2天后转导CAR1慢病毒载体。转导后离心洗去培养上清中未进入细胞的的CAR1慢病毒载体。转导后的T细胞重悬在含有终浓度为100IU/mLIL-2和CD3/CD28磁珠的培养基中。以0.5～2x10 6/mL的培养密度隔天进行扩大培养11～14天。
将CAR1分别转导至来自5例不同B-ALL患者的T细胞中，转导后的细胞分别命名为ALLCT01、ALLCT02、ALLCT03、ALLCT04及ALLCT05。图5结果显示，经CAR1转导后的细胞均显示很强的体外杀伤Raji.Luc靶细胞的能力，与Raji.Luc靶细胞共孵育后剩余的Raji.Luc细胞的相对数量为9.33％-37.82％。
以下以ALLCT01急性B淋巴细胞白血病病人的CD19CAR-T治疗为例。
ALLCT01病人：经医院检测病症为骨髓增生活跃，淋巴细胞系统异常增生，原始淋巴细胞占65％，以大细胞为主，边缘不规则，胞浆量较多，核型不规则，可见凹陷和折叠，粒细胞系统增生减低，红细胞系统增生减低，血小板少见，临床诊断为B-ALL(NR)。经医院推荐，通过伦理审查，病人签署知情同意书，然后进行CAR-T细胞治疗的临床试验研究。
CAR-T细胞治疗过程为：采集该病人的外周血，经过体外T细胞的分离纯化，体外制备CD19CAR-T细胞，分三个时间点分次经静脉自体回输至病人体内，共计输注约1.5×10 8细胞。
ALLCT01病人回输CD19CAR-T细胞后，出现较为温和的反应：体温在最后一次回输后6天开始升高，细胞因子检测结果显示白介素6(IL-6)分泌水平显著提高(图6)，持续发烧6天均未超过40℃。在发烧后3天对症治疗输入少量的IL-6R单抗Tocilizumab后，体温逐渐下降，4日后达到正常体温。而国外报道的CD19CAR-T治疗B-ALL病人一股在3天即发生高热。本试验结果表明本发明述及的CD19CAR(CAR1)在治疗B-ALL中有着更温和的反应，对病人副作用更小。
在最后一次回输的14天后对病人的外周血进行检测。如表2结果所示，外周血中CD19阳性(CD19+)细胞所占的百分比由治疗前的63％降低为0％，并且83.59％的细胞为CD3阳性(CD3+)细胞。结合血液学检测结果表明：该病人经过本发明提供的CD19CAR-T细胞治疗后，外周血中CD19阳性的白血病细胞被完全清除，达到临床完全缓解。
表2.B-ALL病人CART治疗前后血相中T和B淋巴细胞的变化
Figure PCTCN2017119711-appb-000002
本申请文件中未详细记载的实验方法均为本领域的常规技术，可通过申请日以前的文献或技术手段实现。

Claims (25)

  1. 一种新型嵌合抗原受体，其特征在于包含胞外信号肽、抗原结合结构域、跨膜结构域和胞内信号结构域，其中所述胞外信号肽选自4-1BB信号肽、CD8α信号肽、GM-CSFRα信号肽或CD4信号肽中的一种，跨膜结构域选自4-1BB分子跨膜区序列。
  2. 根据权利要求1所述的嵌合抗原受体，其特征在于所述跨膜结构域4-1BB分子跨膜区的氨基酸序列如SEQ ID NO.1所示，或与其氨基酸序列具有85％-99％同一性的多肽。
  3. 根据权利要求1所述的嵌合抗原受体，其特征在于所述的胞外信号肽选自氨基酸如SEQ ID NO.2所示的4-1BB信号肽，或与其氨基酸序列具有85％-99％同一性的多肽。
  4. 根据权利要求1所述的嵌合抗原受体，其抗原结合结构域抗体选自单克隆抗体、Fab、scFv或VHH。
  5. 根据权利要求1或4所述的嵌合抗原受体，其特征在于所述的抗原结合域结合的抗原与恶性肿瘤有关，包括但不限于CD19、CD20、CD22、CD30、CD33、CD38、BCMA、CS1、CD138、CD123/IL3Rα、c-Met、gp100、MUC1、IGF-I receptor、EPCAM、EGFR/EGFRvIII、HER2、PD1、CTLA4、IGF1R、mesothelin、PSMA、WT1、ROR1、CEA、GD-2、NY-ESO-1、MAGE A3、GPC3、糖脂F77或其他任意肿瘤抗原或其他修饰类型或组合。
  6. 根据权利要求5所述的嵌合抗原受体，其特征在于抗原结合结构域结合的抗原为CD19。
  7. 根据权利要求6所述的嵌合抗原受体，其特征在于所述抗原结合结构域包含SEQ ID NO.9所示的氨基酸序列。
  8. 根据权利要求1所述的嵌合抗原受体，其特征在于胞内信号结构域包含胞内信号传导结构域和/或共刺激信号结构域，选自以下信号分子的细胞内结构域：CD3ζ、CD3 gamma、CD3 delta、CD3 epsilon、common FcR gamma、FcR beta、CD79a、CD79b、Fc gamma RIIa、DAP10、DAP12、CD27、CD28、4-1BB、OX40、CD30、CD40、CD2、淋巴细胞功能相关抗原-1、LIGHT、NKG2C、B7-H3、PD-1、ICOS、CDS、ICAM-1、GITR、BAFFR、HVEM、SLAMF7、CD7、NKp80、CD160、CD19、CD4、CD8alpha、CD8beta、IL2R beta、IL2R gamma、IL7R alpha、ITGA4、VLA1、CD49a、ITGA4、 IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、TNFR2、TRANCE/RANKL、DNAM1、SLAMF4、CD84、CD96、CEACAM1、CRTAM、Ly9、CD160、PSGL1、CD100、CD69、SLAMF6、SLAM、BLAME、SELPLG、LTBR、LAT、GADS、SLP-76、PAG/Cbp、NKp44、NKp30、NKp46、NKG2D、CD83特异性结合的配体或其任意组合。
  9. 根据权利要求8所述的嵌合抗原受体，其特征在于胞内信号结构域包含CD3ζ、4-1BB和/或CD28信号结构域。
  10. 根据权利要求9所述的嵌合抗原受体，其特征在于胞内信号结构域包含SEQ ID NO.6所示氨基酸序列的4-1BB胞内共刺激信号结构域。
  11. 根据权利要求9所述的嵌合抗原受体，其特征在于胞内信号结构域包含SEQ ID NO.7所示氨基酸序列的CD28胞内共刺激信号结构域。
  12. 根据权利要求9所述的嵌合抗原受体，其特征在于胞内信号结构域包含SEQ ID NO.8所示氨基酸序列的CD3ζ胞内信号传导结构域。
  13. 根据权利要求1-12中任一项所述的嵌合抗原受体，其特征在于所述的嵌合抗原受体包括顺序连接的4-1BB信号肽或CD8α信号肽、抗原结合结构域、4-1BB跨膜结构域、4-1BB和/或CD28胞内共刺激结合结构域、以及CD3胞内信号传导结构域。
  14. 根据权利要求13所述的嵌合抗原受体，其特征在于所述的嵌合抗原受体在胞外抗原结合结构域和跨膜结构域之间还包含铰链区，优选CD8α铰链区。
  15. 根据权利要求13所述的嵌合抗原受体，其特征在于所述的嵌合抗原受体包含SEQ ID NO.10或SEQ ID NO.12所示的氨基酸序列，或与其具有85％-99％同一性的氨基酸序列。
  16. 编码权利要求1至12中任一项所述的嵌合抗原受体的核酸。
  17. 根据权利要求16所述的核酸，其特征在于选自SEQ ID NO.22或SEQ ID NO.24所示的核苷酸序列。
  18. 一种重组表达载体，其特征在于包含权利要求16或17所述的核酸分子。
  19. 表达权利要求1-12中任一项所述的嵌合抗原受体的细胞。
  20. 根据权利要求19所述的细胞，其特征在于所述的细胞选自免疫细胞。
  21. 根据权利要求20所述的细胞，其特征在于所述的细胞选自T淋巴细胞、NK细胞、造血干细胞、多能干细胞或胚胎干细胞培养分化的免疫细胞。
  22. 一种制备权利要求1所述的嵌合抗原受体修饰的T细胞的方法，其特征在于该方法包括分离和激活待修饰的T细胞，然后以权利要求18所述的重组表达载体转导该T细胞。
  23. 一种药物组合物，其特征在于包含有效量的权利要求19-21中任一项所述的细胞和药学上可接受的载体。
  24. 权利要求1-12中任一项所述的嵌合抗原受体在制备抗肿瘤药物或肿瘤细胞免疫疗法中的应用；优选在制备抗血液恶性肿瘤的药物中的应用。
  25. 权利要求19-21中任一项所述的免疫效应细胞在制备抗肿瘤药物或肿瘤细胞免疫疗法中的应用。
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