WO2018145648A1 - 一种靶向cd20的car的构建及其工程化t细胞的活性鉴定 - Google Patents

一种靶向cd20的car的构建及其工程化t细胞的活性鉴定 Download PDF

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WO2018145648A1
WO2018145648A1 PCT/CN2018/075866 CN2018075866W WO2018145648A1 WO 2018145648 A1 WO2018145648 A1 WO 2018145648A1 CN 2018075866 W CN2018075866 W CN 2018075866W WO 2018145648 A1 WO2018145648 A1 WO 2018145648A1
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sequence
seq
polynucleotide
nucleic acid
car
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French (fr)
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姚意弘
黄家琪
朱恃贵
朱蔚
姚昕
李志远
张丽
朱琳
马安云
魏雨恬
李延峰
王庆霞
何佳平
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西比曼生物科技（上海）有限公司
西比曼生物科技（无锡）有限公司
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Publication of WO2018145648A1 publication Critical patent/WO2018145648A1/zh

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Definitions

  • the present invention provides a sequence component targeting a chimeric antigen receptor of CD20 antigen, and a preparation method and activity identification of the modified T cell (CART20); the present invention identifies a CD20 positive B cell lymphoma. Chimeric antigen receptor structure.
  • Malignant tumors of the blood system account for about 10% of human malignant tumors, and 95% of malignant tumors of the blood system are derived from B lymphocytes.
  • Traditional chemotherapy and radiotherapy play an important role in the treatment of malignant tumors of the blood system.
  • Some patients also have significant effects, but most of them are difficult to cure.
  • New and effective treatments have been a hot topic in this field.
  • Adoptive T cell therapy has shown its potent efficacy and bright prospects in the clinical treatment of malignant tumors.
  • multiple centers independently using Chimeric Antigen Receptor (CAR)-modified T cells to target CD19-derived B cell recurrence and refractory malignant tumors have achieved unprecedented success.
  • CAR Chimeric Antigen Receptor
  • R/R B-ALL refractory acute B-cell lymphoma
  • up to 94% of patients achieved complete remission.
  • the initial response rate of this clinical trial is high, nearly 40% of patients have relapsed after 1 month of treatment and complete remission, and more than 60% of patients who have relapsed have CD19-negative tumor cells. Escape. Therefore, there is an urgent need to screen out CART structures that target B cell lymphoma-associated antigens other than CD19 to treat patients with malignant lymphoma.
  • CD20 is a glycosylated protein and is the first identified B cell membrane marker, also known as B1, encoded by the MS4A gene.
  • the CD20 molecule is a hydrophobic region with four transmembranes, and its N- and C-termini are located on the cytoplasmic side, forming two closed loops outside the cell, called macrocycles and small loops.
  • CD20 specifically expresses more than 95% of normal and cancerous B cells, which are in the pre-B cells and subsequent developmental stages until the differentiation into plasma cells CD20 stops expression. Therefore, CD20 is an ideal target for immunotherapy of B cell malignancies.
  • Cellular immunotherapy is an emerging and highly effective tumor treatment model, and is a new type of treatment for autoimmune and anti-cancer. It is a method for in vitro culture and amplification of immune cells collected from a patient using biotechnology and biological agents to be returned to a patient to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • biotechnology and biological agents to be returned to a patient to stimulate and enhance the body's own immune function, thereby achieving the purpose of treating tumors.
  • Those skilled in the art have been working to develop new cellular immunotherapies to increase the effectiveness of cellular immunotherapy and reduce its side effects.
  • the present invention relates to the construction of a CD20 chimeric antigen receptor-targeting construct, a method for the preparation of a CD20 chimeric antigen receptor engineered T cell, and an activity assay thereof.
  • a chimeric antigen receptor (sequence)
  • the antigen binding domain ie, scFv
  • the antigen binding domain (scFv) of the chimeric antigen receptor is as shown in Formula I or Formula II below:
  • V H antibody heavy chain variable region V L is an antibody light chain variable region; and "-" connecting peptide or a peptide bond.
  • amino acid sequence of the linker peptide is set forth in SEQ ID NO.
  • the structure of the chimeric antigen receptor is as follows:
  • L is an optional leader sequence (signal peptide sequence);
  • scFv is an antigen binding domain
  • H is a hinge region
  • TM is a transmembrane domain
  • C is a costimulatory signal receptor tyrosine activating motif (co-stimulatory molecule);
  • CD3 ⁇ is a cytoplasmic signaling sequence derived from CD3 ⁇
  • the antigen binding domain and "-" are as described above, respectively.
  • the costimulatory signal receptor tyrosine activating motif comprises a 4-1BB-derived costimulatory signaling receptor tyrosine activating motif, and/or a CD28-derived costimulatory signaling receptor Amino acid activation motif.
  • sequence of L is as shown in SEQ ID NO.
  • sequence of H is as shown in SEQ ID NO. 18 or 20.
  • the sequence of TM comprises a transmembrane region derived from CD8, preferably the sequence of TM is set forth in SEQ ID NO.
  • the sequence of TM comprises a transmembrane region derived from CD28, preferably the sequence of TM is set forth in SEQ ID NO.
  • amino acid sequence of the 4-1BB-derived costimulatory signal receptor tyrosine activating motif is set forth in SEQ ID NO.
  • amino acid sequence of the CD28-derived costimulatory signal receptor tyrosine activating motif is set forth in SEQ ID NO.
  • sequence of CD3 ⁇ is as shown in SEQ ID NO.
  • sequence of the chimeric antigen receptor is as shown in SEQ ID NO. 1, 4, 6, 8, or 10.
  • a nucleic acid molecule encoding the chimeric antigen receptor (CAR) of the first aspect of the invention.
  • the nucleic acid molecule comprises a nucleic acid sequence encoding the hinge region selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane region of the CD8 selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence encoding a transmembrane region of the CD28 selected from the group consisting of:
  • the nucleic acid molecule comprises a costimulatory signaling receptor tyrosine activating motif coding sequence comprising a 4-1BB-derived costimulatory signal a receptor tyrosine-activated motif coding sequence, and/or a CD28-derived costimulatory signal receptor tyrosine-activated motif coding sequence, wherein
  • the 4-1BB-derived costimulatory signal receptor tyrosine activation motif coding sequence is selected from the group consisting of:
  • the CD28-derived costimulatory signal receptor tyrosine activation motif coding sequence is selected from the group consisting of:
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of an intracellular signal domain encoding the CD3 ⁇ :
  • the nucleic acid molecule comprises a nucleic acid sequence selected from the group consisting of:
  • the nucleotide sequence has a homology of ⁇ 95% (preferably ⁇ 98%) to the sequence of SEQ ID NO. 2, 3, 5, 7, 9 or 11 and encodes SEQ ID NO. a polynucleotide of the amino acid sequence shown in 4, 6, 8 or 10;
  • the nucleic acid molecule is isolated.
  • the nucleic acid molecule further comprises a polynucleotide encoding a leader sequence (director sequence, signal peptide), the amino acid sequence of the leader sequence is set forth in SEQ ID NO. 32; preferably the coding leader
  • the polynucleotide of the sequence (signal peptide) is shown in SEQ ID NO.
  • sequence of the nucleic acid molecule is set forth in SEQ ID NO. 2, 3, 5, 7, 9, or 11.
  • a vector comprising the nucleic acid molecule of the second aspect of the invention is provided.
  • the vector is a lentiviral vector.
  • a host cell comprising the vector of the third aspect of the present invention or the nucleic acid according to the second aspect of the present invention in which the exogenous source is integrated molecule.
  • the cell is an isolated cell, and/or the cell is a genetically engineered cell.
  • the cell is a mammalian cell.
  • the cell is a T cell.
  • a pharmaceutical composition comprising a pharmaceutically acceptable carrier and the chimeric antigen receptor of the first aspect of the invention, the nucleic acid of the second aspect of the invention, is provided A molecule, the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention.
  • the chimeric antigen receptor of the first aspect of the invention the nucleic acid molecule of the second aspect of the invention, the vector of the third aspect of the invention, or the fourth aspect of the invention.
  • the autoimmune disease is an autoimmune disease (such as lupus erythematosus) caused by overexpression of B cells.
  • the tumor comprises a CD20 positive tumor.
  • a seventh aspect of the invention provides a method for treating a disease comprising administering an appropriate amount of the chimeric antigen receptor of the first aspect of the invention, the nucleic acid molecule of the second aspect of the invention, to a subject in need of treatment,
  • the vector of the third aspect of the invention, or the cell of the fourth aspect of the invention, or the pharmaceutical composition of the fifth aspect of the invention is administered to a subject in need of treatment.
  • the disease is a tumor.
  • a method for producing a CAR-T cell (CAR-modified T cell) expressing the chimeric antigen receptor of the first aspect of the invention a method for producing a CAR-T cell (CAR-modified T cell) expressing the chimeric antigen receptor of the first aspect of the invention
  • the method comprises the steps of: transducing the nucleic acid molecule of the second aspect of the invention or the vector of the third aspect of the invention into a T cell, thereby obtaining the CAR-T cell.
  • FIG. 1 Schematic map of the targeted CD20 chimeric antigen receptor.
  • the elements of the designed CAR structure are shown in the figure, and the listed elements include: a leader sequence, an antigen recognition sequence (Leu16), a hinge region, a transmembrane region, a costimulatory factor signal region, and a CD3zeta signaling region.
  • CAR-T20.17 and CAR-T18 are the L235E-N297Q mutant forms of CAR-T20.9 and CAR-T20.12 in the IgG4Hinge-CH2-CH3 junction region, respectively.
  • FIG. 1 Detection of engineered T cell transfection efficiency targeting CD20 chimeric antigen receptors.
  • the Protein L method identified the expression level of the CAR gene-encoded protein on the surface of the T cell membrane in CAR-T20s cells cultured on day 7 (A) and day 11 (B).
  • FIG. 3 CART-20 cells cultured on day 6 with 1*10 5 in turn, with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line or no tumor cells, at 200 ⁇ l
  • the expression level of CD137 on the surface of T cell membrane (A) and the secretion level of IFN ⁇ in the culture supernatant (B) were detected in GT-551 medium in a ratio of 1:1 with the indicated CAR-T20 cells for 18 h.
  • FIG. 4 CART-20 cells cultured on day 13 from 1*10 5 , respectively, to CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell line or no tumor cells, at 200 ⁇ l
  • the expression level of CD137 on the surface of T cell membrane (A) and the secretion level of IFN ⁇ in the culture supernatant (B) were detected in GT-551 medium in a ratio of 1:1 with the indicated CAR-T20 cells for 18 h.
  • FIG. 5 Detection of early apoptosis levels of tumor cells induced by CART-20. Take 1*10 4 CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines, respectively, in 200 ⁇ l of GT-551 medium and cultured to day 11 CAR-T20 cells were cultured for 4 h, and the cell pellet was collected by centrifugation. The cells were washed twice with PBS, stained with Annexin V-APC dye in a ratio of 1:50 in 100 ⁇ l of dyeing solution for 30 min, washed with PBS for 1 time and then drained in a cytometer. The proportion of Annexin V positive cells in CFSE positive cells was analyzed. The graphical results show the statistical analysis of Annexin V positive cells in the corresponding co-culture samples.
  • FIG. 6 Detection of late apoptosis levels in tumor cells induced by CART-20.
  • Panel A shows the CART-positive cell ratio of the analyzed samples; 1*10 4 CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines, respectively, in 200 ⁇ l of GT-551 medium The proportions were co-cultured with the corresponding T cells for 4 h, and the cell pellet was collected by centrifugation. The cells were washed twice with PBS and stained with PI dye (PI/RNase Staining Buffer) in 100 ⁇ l of dye solution for 30 min. After washing with PBS for 1 time, the cells were drained. The proportion of Annexin V positive cells in CFSE positive cells was analyzed on a cytometer.
  • Panel B shows the results of statistical analysis of PI positive cells in the corresponding co-culture samples.
  • Figure 7 shows the results of the ability of CAR-T20 cells to clear CD20-positive cells in vivo.
  • the results show that CAR-T (CAR-T20.17 and CAR-T20.18) constructed based on the Leu16 sequence can effectively inhibit CD20. In vivo expansion of positive tumor cells.
  • the present inventors have constructed a variety of chimeric antigen receptors targeting CD20 antigen based on the sequence of CD20 murine monoclonal antibody leu16 through extensive and in-depth research, and the chimeric antigen receptors in the original Analysis and identification of expression levels, in vitro activation ability and tumor cell killing efficacy in generation T cells. Finally, a chimeric antigen receptor with better anti-tumor activity was found to provide a new effective method and preparation for clinical application of CAR-T in the treatment of CD20-positive leukemia and lymphoma.
  • variable region amino acid sequence binds to different transmembrane and intracellular portions, and a series of chimeric antigen receptors targeting CD20 are constructed, and the expression of such chimeric antigen receptors in primary T cells is established.
  • the detection method of receptor expression intensity was used to identify the ability of these CAR-T cells to recognize CD20 antigen in vitro and in vivo, and the difference in activity of killing and eliminating malignant tumors carrying CD20 antigen in vitro, for clinical application of CAR-T treatment of CD20 Positive leukemias and lymphomas provide new and effective methods and formulations.
  • the invention provides chimeric antigen receptors (CARs) comprising an extracellular domain, a transmembrane domain, and an intracellular domain.
  • the extracellular domain includes a target-specific binding element (also known as an antigen binding domain).
  • the intracellular domain includes a costimulatory signaling region and a purine chain portion.
  • a costimulatory signaling region refers to a portion of an intracellular domain that includes a costimulatory molecule. Costimulatory molecules are cell surface molecules required for efficient response of lymphocytes to antigens, rather than antigen receptors or their ligands.
  • a linker can be incorporated between the extracellular domain and the transmembrane domain of the CAR, or between the cytoplasmic domain and the transmembrane domain of the CAR.
  • the term "linker” generally refers to any oligopeptide or polypeptide that functions to link a transmembrane domain to the extracellular domain or cytoplasmic domain of a polypeptide chain.
  • the linker may comprise from 0 to 300 amino acids, preferably from 2 to 100 amino acids and most preferably from 3 to 50 amino acids.
  • the extracellular domain of the CAR provided by the invention comprises an antigen binding domain that targets CD20.
  • antigen recognition can be performed based on antigen binding specificity. When it binds to its associated antigen, it affects the tumor cells, causing the tumor cells to not grow, cause death or otherwise be affected, and cause the patient's tumor burden to shrink or eliminate.
  • the antigen binding domain is preferably fused to an intracellular domain from one or more of a costimulatory molecule and a sputum chain.
  • the antigen binding domain is fused to an intracellular domain in combination with a 4-1BB signaling domain, and a CD3 ⁇ signaling domain.
  • a CD20-targeting CAR of the invention comprises a specific signaling domain of the invention (the transmembrane region of CD8, the intracellular signal domains of CD137 and CD3 ⁇ are made in series).
  • the signaling domain of the invention significantly increases anti-tumor activity and in vivo persistence of CAR-T cells compared to other modes of CD20-targeted CAR.
  • the amino acid sequence of the chimeric antigen receptor (CAR) provided by the present invention is as follows:
  • the coding DNA sequence of CAR-T20.9 is as follows:
  • amino acid sequence of the chimeric antigen receptor is as follows:
  • the coding DNA sequence of CAR-T20.11 is as follows:
  • amino acid sequence of the chimeric antigen receptor is as follows:
  • the coding DNA sequence of CAR-T20.12 is:
  • amino acid sequence of the chimeric antigen receptor is as follows:
  • the coding DNA sequence of CAR-T20.17 is as follows:
  • amino acid sequence of the chimeric antigen receptor is as follows:
  • the coding DNA sequence of CAR-T20.18 is as follows:
  • a CAR of the invention comprises a target-specific binding element referred to as an antigen binding domain.
  • the antigen binding domain of the CAR of the invention is a specific binding element that targets CD20.
  • the antigen binding domain comprises a heavy chain variable region and a light chain variable region of an anti-CD20 antibody.
  • amino acid sequence of the antibody heavy chain variable region is as follows:
  • amino acid sequence of the variable region of the antibody light chain is as follows:
  • amino acid linkage sequence between the heavy chain variable region and the light chain variable region is as follows:
  • the CAR can be designed to include a transmembrane domain fused to the extracellular domain of the CAR.
  • a transmembrane domain that is naturally associated with one of the domains in the CAR is used.
  • transmembrane domains may be selected or modified by amino acid substitutions to avoid binding such domains to the transmembrane domain of the same or different surface membrane proteins, thereby minimizing complexes with receptors. The interaction of other members.
  • the hinge region comprises the following amino acid sequence (IgG4 Hinge-CH2-CH3 hinge region):
  • the hinge region comprises the following amino acid sequence (IgG4 Hinge-CH2-CH3 (L235E, N297Q)):
  • the transmembrane region of the CAR of the invention is a CD28-derived transmembrane region (CD28TM) or a CD8-derived transmembrane region (CD8TM).
  • CD8-derived transmembrane region (CD8TM) amino acid sequence is as follows:
  • CD28-derived transmembrane region (CD28TM) amino acid sequence is as follows:
  • CD28-derived transmembrane region encodes a DNA sequence:
  • the intracellular domain in the CAR of the present invention includes the signaling domain of 4-1BB and the signaling domain of CD3 ⁇ .
  • the intracellular domain of the CAR further comprises a signaling domain of CD28.
  • the intracellular signaling domain of 4-1BB comprises the following amino acid sequence:
  • the CD28-derived intracellular signaling domain comprises the following amino acid sequence:
  • the intracellular signaling domain of CD3 ⁇ comprises the following amino acid sequence:
  • the invention also provides DNA constructs encoding the CAR sequences of the invention.
  • a nucleic acid sequence encoding a desired molecule can be obtained using recombinant methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to include the gene, or by utilizing standard Techniques, isolated directly from cells and tissues containing the gene. Alternatively, the gene of interest can be produced synthetically.
  • the invention also provides vectors in which the DNA constructs of the invention are inserted.
  • Vectors derived from retroviruses such as lentiviruses are suitable tools for achieving long-term gene transfer because they allow long-term, stable integration of the transgene and its proliferation in daughter cells.
  • Lentiviral vectors have the advantage over vectors derived from oncogenic retroviruses such as murine leukemia viruses because they can transduce non-proliferating cells, such as hepatocytes. They also have the advantage of low immunogenicity.
  • expression of a native or synthetic nucleic acid encoding a CAR is typically achieved by operably linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector.
  • This vector is suitable for replication and integration of eukaryotic cells.
  • a typical cloning vector comprises a transcriptional and translational terminator, an initial sequence and a promoter that can be used to modulate expression of a desired nucleic acid sequence.
  • the expression constructs of the invention can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Methods of gene delivery are known in the art. See, for example, U.S. Patent Nos. 5,399,346, 5, 580, 859, 5, 589, 466, incorporated herein by reference.
  • the invention provides a gene therapy vector.
  • the nucleic acid can be cloned into many types of vectors.
  • the nucleic acid can be cloned into such vectors including, but not limited to, plasmids, phagemids, phage derivatives, animal viruses, and cosmids.
  • Specific vectors of interest include expression vectors, replication vectors, probe production vectors, and sequencing vectors.
  • the expression vector can be provided to the cells in the form of a viral vector.
  • Viral vector techniques are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology handbooks.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.
  • a suitable vector comprises an origin of replication, a promoter sequence, a convenient restriction enzyme site, and one or more selectable markers that function in at least one organism (eg, WO01/96584; WO01/29058; and the United States) Patent No. 6,326, 193).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected gene can be inserted into a vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to a subject cell in vivo or ex vivo.
  • retroviral systems are known in the art.
  • an adenoviral vector is used.
  • Many adenoviral vectors are known in the art.
  • a lentiviral vector is used.
  • promoter elements can regulate the frequency of transcription initiation.
  • these are located in the 30-110 bp region upstream of the start site, although it has recently been shown that many promoters also contain functional elements downstream of the start site.
  • the spacing between the promoter elements is often flexible to maintain the promoter function when the element is inverted or moved relative to the other.
  • tk thymidine kinase
  • the interval between promoter elements can be increased by 50 bp, and the activity begins to decrease.
  • a single element can function cooperatively or independently to initiate transcription.
  • a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high level expression of any polynucleotide sequence operably linked thereto.
  • Another example of a suitable promoter is Elongation Growth Factor-1 alpha (EF-1 alpha).
  • constitutive promoter sequences can also be used, including but not limited to human prion 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ sarcoma virus promoter, and human gene promoter such as, but not limited to, actin promoter , myosin promoter, heme promoter and creatine kinase promoter.
  • the invention should not be limited to the use of constitutive promoters. Inducible promoters are also considered as part of the invention.
  • an inducible promoter provides a molecular switch capable of opening expression of a polynucleotide sequence operably linked to an inducible promoter when such expression is desired, or shutting down expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the expression vector introduced into the cell may also comprise any one or both of a selectable marker gene or reporter gene to facilitate seeking a population of cells that are transfected or infected by the viral vector. Identify and select expression cells.
  • selectable markers can be carried on a single piece of DNA and used in a co-transfection procedure. Both the selectable marker and the reporter gene can be flanked by appropriate regulatory sequences to enable expression in the host cell.
  • Useful selectable markers include, for example, antibiotic resistance genes such as neo and the like.
  • reporter genes were used to identify potentially transfected cells and to assess the functionality of regulatory sequences.
  • the reporter gene is a gene that is not present in or expressed by the recipient organism or tissue, and which encodes a polypeptide whose expression is clearly indicated by some readily detectable properties such as enzymatic activity. After the DNA has been introduced into the recipient cell, the expression of the reporter gene is determined at an appropriate time.
  • Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase or green fluorescent protein genes (eg, Ui-Tei et al, 2000 FEBS Letters 479: 79-82).
  • Suitable expression systems are well known and can be prepared using known techniques or commercially available.
  • a construct with a minimum of 5 flanking regions showing the highest level of reporter gene expression is identified as a promoter.
  • Such a promoter region can be ligated to a reporter gene and used to assess the ability of the agent to modulate promoter-driven transcription.
  • an expression vector can be readily introduced into a host cell, for example, a mammalian, bacterial, yeast or insect cell by any method in the art.
  • an expression vector can be transferred into a host cell by physical, chemical or biological means.
  • Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods of producing cells comprising vectors and/or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). A preferred method of introducing a polynucleotide into a host cell is calcium phosphate transfection.
  • Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors.
  • Viral vectors particularly retroviral vectors, have become the most widely used method of inserting genes into mammals, such as human cells.
  • Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, adeno-associated viruses, and the like. See, for example, U.S. Patent Nos. 5,350,674 and 5,585,362.
  • Chemical means for introducing polynucleotides into host cells include colloidal dispersion systems such as macromolecular complexes, nanocapsules, microspheres, beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and lipids. Platinum.
  • An exemplary colloidal system for use as an in vitro and in vivo delivery vehicle is a liposome (eg, an artificial membrane sac).
  • an exemplary delivery tool is a liposome. It is contemplated to use a lipid formulation to introduce the nucleic acid into a host cell (in vitro, ex vivo or in vivo). In another aspect, the nucleic acid can be associated with a lipid.
  • the nucleic acid associated with the lipid can be encapsulated into the aqueous interior of the liposome, interspersed within the lipid bilayer of the liposome, attached via a linker molecule associated with both the liposome and the oligonucleotide
  • a linker molecule associated with both the liposome and the oligonucleotide
  • the lipid, lipid/DNA or lipid/expression vector associated with the composition is not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include fat droplets that occur naturally in the cytoplasm and in such compounds comprising long chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • the present invention is exemplarily employed using a gene editing technique such as CRISPR-Cas9, ZFN or TALEN.
  • the vector is a lentiviral vector.
  • the DNA construct further comprises a signal peptide coding sequence.
  • the signal peptide sequence is ligated upstream of the antigenic tuberculosis domain nucleic acid sequence.
  • the signal peptide is a human CD8a signal peptide.
  • the signal peptide amino acid sequence is as follows:
  • CD8Leader sequence The coding sequence of the CD8 leader sequence (CD8Leader sequence):
  • the invention encompasses cells (e.g., T cells) that are transduced with a lentiviral vector (LV) encoding a CAR of the invention.
  • Transduced T cells can elicit a CAR-mediated T-cell response.
  • the invention also provides a method of stimulating a T cell-mediated immune response to a target cell population or tissue of a mammal comprising the step of administering to a mammal a T cell expressing a CAR of the invention.
  • the invention encompasses a type of cell therapy wherein T cells are genetically modified to express a CAR of the invention, and CAR-T cells are injected into a recipient in need thereof.
  • the injected cells are capable of killing the recipient's tumor cells.
  • CAR-T cells replicate in vivo, producing long-lasting persistence that leads to sustained tumor control.
  • the CAR-T cells of the invention can undergo robust in vivo T cell expansion for an extended amount of time.
  • the CAR-mediated immune response can be part of a step of adoptive immunotherapy in which CAR-modified T cells induce an immune response specific for the antigen binding domain in the CAR.
  • anti-CD20 CAR-T cells elicit a specific immune response against cells expressing CD20.
  • Therapeutic indications include CD20-positive tumors and diseases caused by excessive B-cells (such as autoimmune diseases such as lupus erythematosus, etc.), and CD20-positive tumors may include non-solid tumors (such as hematological tumors such as leukemia and lymphoma). Or may include a solid tumor.
  • the types of tumors or cancers treated with the CARs of the invention include, but are not limited to, carcinomas, blastomas, and sarcomas, and certain leukemias or lymphoid malignancies, benign and malignant tumors, and malignant tumors such as sarcomas, carcinomas, and melanomas. Also included are adult tumors/cancers and childhood tumors/cancers.
  • Hematological cancer is a cancer of the blood or bone marrow.
  • hematological (or hematogenous) cancers include leukemia, including acute leukemia (such as acute lymphocytic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, granulocyte-monocyte type).
  • monocyte and erythroleukemia monocyte and erythroleukemia
  • chronic leukemia such as chronic myeloid (granulocytic) leukemia, chronic myelogenous leukemia and chronic lymphocytic leukemia
  • polycythemia vera lymphoma
  • Hodgkin's disease non Hodgkin's lymphoma (painless and high-grade forms)
  • multiple myeloma Waldenstrom's macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and myelodysplasia.
  • a solid tumor is an abnormal mass of tissue that usually does not contain a cyst or fluid area.
  • Solid tumors can be benign or malignant. Different types of solid tumors are named after the cell types that form them (such as sarcoma, carcinoma, and lymphoma). Examples of solid tumors such as sarcomas and carcinomas include fibrosarcoma, mucinous sarcoma, liposarcoma mesothelioma, lymphoid malignancy, pancreatic cancer, ovarian cancer.
  • the CAR-modified T cells of the invention can also be used as vaccine types for ex vivo immunity and/or in vivo therapy in mammals.
  • the mammal is a human.
  • cells are isolated from a mammal, preferably a human, and genetically modified (i.e., transduced or transfected in vitro) with a vector that expresses the CAR disclosed herein.
  • CAR-modified cells can be administered to a mammalian recipient to provide a therapeutic benefit.
  • Mammalian recipients can be human, and CAR-modified cells can be autologous to the recipient.
  • the cells may be allogeneic, syngeneic or xenogeneic relative to the recipient.
  • the present invention also provides compositions and methods for in vivo immunization to elicit an immune response against antigens in a patient.
  • cells activated and expanded as described herein can be used to treat and prevent diseases produced in individuals without an immune response.
  • the CAR-modified T cells of the invention are used to treat CCL.
  • the cells of the invention are used to treat a patient at risk of developing CCL. Accordingly, the invention provides a method of treating or preventing CCL comprising administering to a subject in need thereof a therapeutically effective amount of a CAR-modified T cell of the invention.
  • the CAR-modified T cells of the invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or with other components such as IL-2, IL-17 or other cytokines or cell populations.
  • the pharmaceutical compositions of the present invention may comprise, for example, biosynthetics such as monoclonal antibodies, small molecule drugs in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients.
  • compositions may include buffers such as neutral buffered saline, sulfate buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; Mixtures such as EDTA or glutathione; adjuvants (eg, aluminum hydroxide); and preservatives.
  • buffers such as neutral buffered saline, sulfate buffered saline, and the like
  • carbohydrates such as glucose, mannose, sucrose or dextran, mannitol
  • proteins polypeptides or amino acids
  • antioxidants such as EDTA or glutathione
  • adjuvants eg, aluminum hydroxide
  • preservatives eg, aluminum hydroxide
  • the pharmaceutical composition of the present invention can be administered in a form suitable for the disease to be treated (or prevented).
  • the amount and frequency of administration will be determined by factors such as the condition of the patient, and the type and severity of the patient's condition - although appropriate dosages may be determined by clinical trials.
  • a pharmaceutical composition comprising a T cell as described herein may be at a dose of from 10 4 to 10 9 cells/kg body weight, preferably from 10 5 to 10 6 cells/kg body weight (including all integers in those ranges) Value) application. T cell compositions can also be administered multiple times in these doses.
  • Cells can be administered by using injection techniques well known in immunotherapy (see, eg, Rosenberg et al, New Eng. J. of Med. 319: 1676, 1988).
  • Optimal dosages and treatment regimens for a particular patient can be readily determined by a person skilled in the medical arts by monitoring the patient's signs of disease and thus modulating the treatment.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous (i.v.) injection or intraperitoneally.
  • a T cell composition of the invention is administered to a patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by i.v. injection.
  • Compositions of T cells can be injected directly into tumors, lymph nodes or infected sites.
  • cells activated and expanded using the methods described herein or other methods known in the art to extend T cells to therapeutic levels are combined with any number of related therapeutic modalities (eg, prior Administering to the patient, concurrently or afterwards, including but not limited to treatment with agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • agents such as antiviral therapy, cidofovir and interleukin-2, cytarabine (also known For ARA-C) or natalizumab treatment for MS patients or for epilizumab treatment in patients with psoriasis or other treatment for patients with PML.
  • the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressive agents such as cyclosporin, azathioprine, methotrexate, mycophenolate mofetil and FK506, antibodies Or other immunotherapeutic agents.
  • the cell composition of the invention is administered to a bone marrow transplant, using a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • a chemotherapeutic agent such as fludarabine, external beam radiation therapy (XRT), cyclophosphamide (eg, before, simultaneously or after) patient.
  • XRT external beam radiation therapy
  • cyclophosphamide eg, before, simultaneously or after
  • the subject may undergo standard treatment of high dose chemotherapy followed by peripheral blood stem cell transplantation.
  • the subject receives an injection of the expanded immune cells of the invention after transplantation.
  • the expanded cells are administered prior to or after surgery.
  • the dosage of the above treatment administered to the patient will vary with the precise nature of the condition being treated and the recipient of the treatment.
  • the dosage ratios administered by a human can be carried out according to practices accepted in the art.
  • 1 x 10 6 to 1 x 10 10 modified T cells of the invention e.g., CAR-T20 cells
  • the chimeric antigen receptor of the present invention wherein the extracellular antigen-binding domain is a specific anti-CD20 scFv, and the specific anti-CD20 scFv binds to a specific hinge region and an intracellular domain to form a CAR which shows great It has the ability to kill tumor cells, and has less cytotoxicity and low side effects.
  • the chimeric antigen receptor provided by the invention can realize stable expression and membrane localization of CAR protein after Lentivirus-infected T cells carrying CAR gene;
  • the CAR-modified T cells of the present invention have a longer survival time in vivo and have stronger antitumor efficacy; the CAR optimized for the IgG4 Hinge-CH2-CH3 junction region can avoid the binding of the Fc receptor and the subsequent ADCC effect. (antibody-dependent cytotoxicity).
  • the coding plasmid was commissioned by Shanghai Boyi Biotechnology Co., Ltd. for full-length DNA synthesis and cloning construction.
  • the cloning vector was selected from the pWPT lentiviral vector, and the cloning sites were BamH I and Sal I sites.
  • the specific sequence structure is shown in Figure 1. The amino acid and nucleotide sequences of each element are as described above.
  • PBMCs mononuclear cells
  • PBMCs were cultured in a GT-T551 cell culture medium containing 2% human albumin, and the final concentration of the cells was adjusted to 2 ⁇ 10 6 cells/mL.
  • the cells were seeded in a cell culture flask previously coated with Retronectin (purchased from TAKARA) at a final concentration of 5 ⁇ g/m LCD3 monoclonal antibody (OKT3) and a final concentration of 10 ⁇ g/mL.
  • Retronectin purchased from TAKARA
  • IL-2 Recombinant human interleukin 2
  • IL-2 was added to the culture medium at a final concentration of 1000 U/mL, and cultured at 37 ° C in a humidified 5% CO 2 incubator.
  • CART20s cells can be taken for the corresponding activity test.
  • Example 3 Detection of the rate of integration of the CAR gene in the T cell genome and the expression level of the encoded protein on the membrane surface.
  • the CAR-T20s cells cultured on the sixth day in Example 2 were used for the detection of the cell-activated level indicator proteins CD137 and IFN ⁇ .
  • 1*10 5 cells were cultured to day 6 of CART-20 cells, respectively, with CD20-positive RAJI and RAMOS tumor cell lines, and CD20-negative MOLT-4 tumor cell lines or no tumor cells, at 200 ⁇ l GT-551.
  • the expression level of CD137 on the surface of T cell membrane was detected by lapse method in culture medium for 18 hours (Fig. 3A), and the secretion level of IFN ⁇ in culture supernatant was detected by ELISA method (Fig. 3B).
  • CAR-T20s cells in Example 2 were labeled with 1*10 4 CFSE-labeled CD20-negative (MOLT-4) or CD20-positive (RAJI, RAMOS) tumor cell lines, respectively.
  • the cells were co-cultured in 200 ⁇ l of GT-551 for 4 h, and the cell pellet was collected by centrifugation.
  • the cells were washed twice with PBS, stained with Annexin V-APC dye in a ratio of 1:50 for 100 min, and washed with PBS for 1 min.
  • the proportion of Annexin V positive cells in CFSE positive cells was analyzed on a cytometer.

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Abstract

提供了一种靶向CD20抗原嵌合抗原受体及其制备方法，所述嵌合抗原受体的细胞外抗原结合结构域包括SEQ ID NO.12所示的抗体重链可变区和SEQ ID NO.14所示的抗体轻链可变区，具有杀伤肿瘤细胞的能力。

Description

一种靶向CD20的CAR的构建及其工程化T细胞的活性鉴定 技术领域
本发明提供了一种靶向CD20抗原嵌合抗原受体的序列组分，及其修饰T细胞(CART20)的制备方法和活性鉴定；本发明鉴定出了一种治疗CD20阳性B细胞淋巴瘤的嵌合性抗原受体结构。
背景技术
血液系统的恶性肿瘤占人类恶性肿瘤约10％，95％血液系统的恶性肿瘤是B淋巴细胞来源的。传统的化疗和放疗对治疗血液系统的恶性肿瘤起着重要作用，有的病人亦疗效显著，但大部份都难以治愈。新的、有效的治疗方法一直是这一领域探索的热点。
过继性T细胞治疗针对恶性肿瘤的临床治疗中已经显示了其强大疗效和光明前景。这其中，多个中心独立开展的利用嵌合抗原受体(Chimeric Antigen Receptor，CAR)修饰的T细胞靶向表达CD19的B细胞复发、难治性恶性肿瘤取得了前所未有的成功。尤其是在宾夕法尼亚大学医学院开展的一项利用CART19治疗复发、难治性急性B细胞淋巴瘤(R/R B-ALL)的临床试验中有高达94％的患者达到了完全缓解。尽管此项临床试验的初始反应率很高，但有近40％的患者在治疗1个月达到完全缓解之后又出现了复发，并且复发的患者中高于60％比例的患者出现了CD19阴性肿瘤细胞的逃逸。因此，迫切需要筛选出靶向除CD19之外的B细胞淋巴瘤相关抗原的CART结构，来治疗恶性淋巴瘤患者。
CD20是糖基化蛋白，是第一个被确定的B细胞膜标识，也被称为B1，由MS4A基因编码。CD20分子是有四个跨膜的疏水区，其N段和C端均位于胞质一侧，从而在胞外形成两个闭环，分别称为大环和小环。CD20特异性地表达在95％以上的正常和癌变的B细胞，这些细胞处于前B细胞及之后的发育阶段，直至分化为浆细胞CD20才停止表达。因此，CD20是理想的B细胞恶性肿瘤免疫治疗的靶点。
细胞免疫治疗是一种新兴的、具有显著疗效的肿瘤治疗模式，是一种自身免疫抗癌的新型治疗方法。它是运用生物技术和生物制剂对从病人体内采集的免疫细胞进行体外培养和扩增后回输到病人体内的方法，来激发、增强机体自身免疫功能，从而达到治疗肿瘤的目的。本领域技术人员一直致力于开发新的细胞免疫疗法，以提高细胞免疫疗法的效果，并降低其副作用。
发明内容
本发明的目的是提供一种CD20靶向性的嵌合抗原受体及其制法和应用。
本发明涉及靶向CD20嵌合抗原受体结构的构建、靶向CD20嵌合抗原受体工程化T细胞的制备方法及其活性鉴定。
本发明的第一方面，提供了一种嵌合抗原受体(CAR)(序列)，所述嵌合抗原受体的抗原结合结构域(即，scFv)包括SEQ ID NO.12所示的抗体重链可变区，和SEQ ID NO.14所示的抗体轻链可变区。
在另一优选例中，所述嵌合抗原受体的抗原结合结构域(scFv)如下式I或式II 所示：
V H－V L,(I)；V L－V H,(II)
其中，V H为抗体重链可变区；V L为抗体轻链可变区；“－”为连接肽或肽键。
在另一优选例中，所述连接肽的氨基酸序列如SEQ ID NO.16所示。
在另一优选例中，所述嵌合抗原受体的结构如下式所示：
L－scFv－H－TM－C－CD3ζ
其中，
L为任选的引导序列(Leader sequence，即信号肽序列)；
scFv为抗原结合结构域；
H为绞链区；
TM为跨膜结构域；
C为共刺激信号受体酪氨酸激活基序(共刺激分子)；
CD3ζ为源于CD3ζ的胞浆信号传导序列；
所述抗原结合结构域和“－”分别如上所述。
在另一优选例中，所述共刺激信号受体酪氨酸激活基序包括4-1BB来源的共刺激信号受体酪氨酸激活基序，和/或CD28来源的共刺激信号受体酪氨酸激活基序。
在另一优选例中，L的序列如SEQ ID NO.32所示。
在另一优选例中，H的序列如SEQ ID NO.18或20所示。
在另一优选例中，TM的序列包括源于CD8的跨膜区，优选地TM的序列如SEQ ID NO.22所示。
在另一优选例中，TM的序列包括源于CD28的跨膜区，优选地TM的序列如SEQ ID NO.24所示。
在另一优选例中，所述4-1BB来源的共刺激信号受体酪氨酸激活基序的氨基酸序列如SEQ ID NO.26所示。
在另一优选例中，所述CD28来源的共刺激信号受体酪氨酸激活基序的氨基酸序列如SEQ ID NO.28所示。
在另一优选例中，CD3ζ的序列如SEQ ID NO.30所示。
在另一优选例中，所述嵌合抗原受体的序列如SEQ ID NO.1、4、6、8或10所示。
本发明的第二方面，提供了一种核酸分子，所述核酸分子编码本发明第一方面所述的嵌合抗原受体(CAR)。
在另一优选例中，所述核酸分子包含选自下组的编码所述铰链区核酸序列：
(a)编码如SEQ ID NO.18或20所示多肽的多核苷酸；
(b)序列如SEQ ID NO.19或21所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.19或21所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.18或20所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD8的跨膜区的核酸序列：
(a)编码如SEQ ID NO.22所示多肽的多核苷酸；
(b)序列如SEQ ID NO.23所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.23所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.22所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD28的跨膜区的核酸序列：
(a)编码如SEQ ID NO.24所示多肽的多核苷酸；
(b)序列如SEQ ID NO.25所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.25所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.24所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含共刺激信号受体酪氨酸激活基序编码序列，所述共刺激信号受体酪氨酸激活基序编码序列包括4-1BB来源的共刺激信号受体酪氨酸激活基序编码序列，和/或CD28来源的共刺激信号受体酪氨酸激活基序编码序列，其中
所述4-1BB来源的共刺激信号受体酪氨酸激活基序编码序列选自下组：
(a)编码如SEQ ID NO.26所示多肽的多核苷酸；
(b)序列如SEQ ID NO.27所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.27所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.26所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
所述CD28来源的共刺激信号受体酪氨酸激活基序编码序列选自下组：
(a)编码如SEQ ID NO.28所示多肽的多核苷酸；
(b)序列如SEQ ID NO.29所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.29所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.28所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的编码所述CD3ζ的胞内信号结构域的核酸序列：
(a)编码如SEQ ID NO.30所示多肽的多核苷酸；
(b)序列如SEQ ID NO.31所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.30所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.31所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子包含选自下组的核酸序列：
(a)编码如SEQ ID NO.1、4、6、8或10所示多肽的多核苷酸；
(b)序列如SEQ ID NO.2、3、5、7、9或11所示的多核苷酸；
(c)核苷酸序列与SEQ ID NO.2、3、5、7、9或11所示序列的同源性≥95％(较 佳地≥98％)，并且编码SEQ ID NO.1、4、6、8或10所示氨基酸序列的多核苷酸；
(d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
在另一优选例中，所述核酸分子为分离的。
在另一优选例中，所述核酸分子还包括编码前导序列(引导序列，信号肽)的多核苷酸，所述前导序列的氨基酸序列如SEQ ID NO.32所示；优选地所述编码前导序列(信号肽)的多核苷酸如SEQ ID NO.33所示。
在另一优选例中，所述核酸分子的序列如SEQ ID NO.2、3、5、7、9或11所示。
本发明的第三方面，提供了一种载体，所述的载体含有本发明第二方面所述的核酸分子。
在另一优选例中，所述载体为慢病毒载体。
本发明的第四方面，提供了一种宿主细胞，所述的宿主细胞中含有本发明第三方面所述的所述的载体或染色体中整合有外源的本发明第二方面所述的核酸分子。
在另一优选例中，所述细胞为分离的细胞，和/或所述细胞为基因工程化的细胞。
在另一优选例中，所述细胞为哺乳动物细胞。
在另一优选例中，所述细胞为T细胞。
本发明的第五方面，提供了一种药物组合物，所述组合物含有药学上可接受的载体以及本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞。
本发明的第六方面，提供了本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞的用途，用于制备治疗肿瘤或自身免疫性疾病的药物或制剂。
在另一优选例中，所述的自身免疫性疾病为B细胞过量表达引起的自身免疫性疾病(如红斑性狼疮)。
在另一优选例中，所述的肿瘤包括CD20阳性肿瘤。
本发明的第七方面，提供了一种治疗疾病的方法，包括给需要治疗的对象施用适量的本发明第一方面所述的嵌合抗原受体、本发明第二方面所述的核酸分子、本发明第三方面所述的载体、或本发明第四方面所述的细胞、或本发明第五方面所述的药物组合物。
在另一优选例中，所述疾病为肿瘤。
本发明的第八方面，提供了一种制备CAR-T细胞(CAR-修饰的T细胞)的方法，所述CAR-T细胞表达本发明第一方面所述的嵌合抗原受体，
所述方法包括步骤：将本发明第二方面所述的核酸分子或本发明第三方面所述的载体转导入T细胞内，从而获得所述CAR-T细胞。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1.靶向CD20嵌合型抗原受体结构图。所设计CAR结构各元件如图所示，所列元件包括：前导序列、抗原识别序列(Leu16)、铰链区、跨膜区、共刺激因子信号区和CD3zeta信号传导区。其中,CAR-T20.17和CAR-T18分别是CAR-T20.9和CAR-T20.12在IgG4Hinge-CH2-CH3连接区的L235E-N297Q突变形式。
图2.靶向CD20嵌合型抗原受体工程化T细胞转染效率的检测。Protein L方法鉴定培养到第7天(A)和第11天(B)的CAR-T20s细胞中CAR基因编码蛋白在T细胞膜表面的表达水平。
图3.依次取1*10 5培养到第6天的CART-20细胞，分别与CD20阳性的RAJI和RAMOS肿瘤细胞系，以及CD20阴性的MOLT-4肿瘤细胞系或不添加肿瘤细胞，在200μl GT-551培养基中按照1:1比例与图示CAR-T20细胞分别共培养18h后检测T细胞膜表面CD137的表达水平(A)和培养上清中IFNγ的分泌水平(B)。
图4.依次取1*10 5培养到第13天的CART-20细胞，分别与CD20阳性的RAJI和RAMOS肿瘤细胞系，以及CD20阴性的MOLT-4肿瘤细胞系或不添加肿瘤细胞，在200μl GT-551培养基中按照1:1比例与图示CAR-T20细胞分别共培养18h后检测T细胞膜表面CD137的表达水平(A)和培养上清中IFNγ的分泌水平(B)。
图5.CART-20诱导肿瘤细胞早期凋亡水平的检测。分别取1*10 4经过CFSE标记的CD20阴性(MOLT-4)或CD20阳性的(RAJI，RAMOS)肿瘤细胞系，在200μl GT-551培养基中按照图示比例与培养到第11天的各种CAR-T20细胞分别共培养4h后离心收集细胞沉淀，细胞经PBS洗两遍后用Annexin V-APC染料按1:50的比例100μl染液中染色30min，PBS洗1遍后在流失细胞仪上分析CFSE阳性细胞中Annexin V阳性细胞的比例。图示结果显示了Annexin V阳性细胞在相应共培养样品中的统计分析结果。
图6.CART-20诱导肿瘤细胞晚期凋亡水平的检测。A图显示所分析样品CART阳性细胞比率；分别取1*10 4经过CFSE标记的CD20阴性(MOLT-4)或CD20阳性的(RAJI，RAMOS)肿瘤细胞系，在200μl GT-551培养基中按照图示比例与相应的T细胞分别共培养4h后离心收集细胞沉淀，细胞经PBS洗两遍后用PI染料(PI/RNase Staining Buffer)在100μl染液中染色30min，PBS洗1遍后在流失细胞仪上分析CFSE阳性细胞中Annexin V阳性细胞的比例。B图显示了PI阳性细胞在相应共培养样品中的统计分析结果。
图7显示了CAR-T20细胞的体内清除CD20阳性细胞能力的检测结果，结果表明，以Leu16序列为基础构建的CAR-T(CAR-T20.17和CAR-T20.18)可以有效地抑制CD20阳性肿瘤细胞的体内扩增。
具体实施方式
本发明人通过广泛而深入的研究，以CD20鼠源单克隆抗体leu16的序列为基础构建了靶向CD20抗原的多种类型的嵌合型抗原受体，并对这些嵌合型抗原受体 在原代T细胞中的表达水平、体外活化能力及肿瘤细胞杀伤效能等层面的分析与鉴定。最终发现了抗肿瘤活性较好的嵌合型抗原受体为临床应用CAR-T治疗CD20阳性的白血病和淋巴瘤提供了新的有效方法和制剂。
鉴于靶向CD20治疗性抗体亲和力、杀伤机制等方面的不同，以及不同的跨膜结构域、胞内结构域对嵌合抗原受体活性的显著影响，在本发明中，利用多种抗CD20抗体可变区氨基酸序列构结合不同的跨膜和胞内部分，建了靶向CD20的系列嵌合性抗原受体，并完成了这类嵌合性抗原受体在原代T细胞中的表达，建立了受体表达强度的检测方法，鉴定了这些CAR-T细胞体外、体内识别CD20抗原的能力，及其体外杀伤和体内清除携带CD20抗原的恶性肿瘤的活性差异，为临床应用CAR-T治疗CD20阳性的白血病和淋巴瘤提供了新的有效方法和制剂。
嵌合抗原受体
本发明提供了包括细胞外结构域、跨膜结构域、和细胞内结构域的嵌合抗原受体(CAR)。胞外结构域包括靶-特异性结合元件(也称为抗原结合结构域)。细胞内结构域包括共刺激信号传导区和ζ链部分。共刺激信号传导区指包括共刺激分子的细胞内结构域的一部分。共刺激分子为淋巴细胞对抗原的有效应答所需要的细胞表面分子，而不是抗原受体或它们的配体。
在CAR的胞外结构域和跨膜结构域之间，或在CAR的胞浆结构域和跨膜结构域之间，可并入接头。如本文所用的，术语“接头”通常指起到将跨膜结构域连接至多肽链的胞外结构域或胞浆结构域作用的任何寡肽或多肽。接头可包括0-300个氨基酸，优选地2至100个氨基酸和最优选地3至50个氨基酸。
在本发明的一个较佳的实施方式中，本发明提供的CAR的胞外结构域包括靶向CD20的抗原结合结构域。本发明的CAR当在T细胞中表达时，能够基于抗原结合特异性进行抗原识别。当其结合其关联抗原时，影响肿瘤细胞，导致肿瘤细胞不生长、被促使死亡或以其他方式被影响，并导致患者的肿瘤负荷缩小或消除。抗原结合结构域优选与来自共刺激分子和ζ链中的一个或多个的细胞内结构域融合。优选地，抗原结合结构域与4-1BB信号传导结构域、和CD3ζ信号结构域组合的细胞内结构域融合。
在一个实施方式中，本发明的CD20靶向性CAR包括本发明特定信号传导结构域(CD8的跨膜区、CD137和CD3ζ的胞内信号结构域串联而成)。与其他方式的CD20靶向性CAR相比，本发明的信号传导结构域显著增加了抗肿瘤活性和CAR-T细胞的体内持久性。
在本发明的一个较佳的实施方式中，本发明提供的嵌合抗原受体(CAR)的氨基酸序列如下：
(CAR-T20.9和CAR-T20.10，SEQ ID NO.1)：
Figure PCTCN2018075866-appb-000001
Figure PCTCN2018075866-appb-000002
CAR-T20.9的编码DNA序列如下：
Figure PCTCN2018075866-appb-000003
CAR-T20.10的编码DNA序列：
Figure PCTCN2018075866-appb-000004
Figure PCTCN2018075866-appb-000005
在本发明的另一个优选地实施方式中，所述嵌合抗原受体的氨基酸序列如下：
(CAR-T20.11)
Figure PCTCN2018075866-appb-000007
CAR-T20.11的编码DNA序列如下：
Figure PCTCN2018075866-appb-000008
在本发明的另一个优选地实施方式中，所述嵌合抗原受体的氨基酸序列如下：
(CAR-T20.12)
Figure PCTCN2018075866-appb-000009
CAR-T20.12的编码DNA序列为：
Figure PCTCN2018075866-appb-000011
在本发明的另一个优选地实施方式中，所述嵌合抗原受体的氨基酸序列如下：
(CAR-T20.17)
Figure PCTCN2018075866-appb-000012
CAR-T20.17的编码DNA序列如下：
Figure PCTCN2018075866-appb-000013
Figure PCTCN2018075866-appb-000014
在本发明的另一个优选地实施方式中，所述嵌合抗原受体的氨基酸序列如下：
(CAR-T20.18)
Figure PCTCN2018075866-appb-000015
CAR-T20.18的编码DNA序列如下：
Figure PCTCN2018075866-appb-000016
Figure PCTCN2018075866-appb-000017
抗原结合结构域
在一个实施方式中，本发明的CAR包括被称为抗原结合结构域的靶-特异性结合元件。本发明CAR的抗原结合结构域为靶向CD20的特异性结合元件。
在本发明的一个优选地实施方式中，所述抗原结合结构域包括抗CD20抗体的重链可变区和轻链可变区。
在另一优选例中，所述抗体重链可变区的氨基酸序列如下：
Figure PCTCN2018075866-appb-000018
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000019
在另一优选例中，所述抗体轻链可变区的氨基酸序列如下：
Figure PCTCN2018075866-appb-000020
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000021
在本发明的一个优选地实施方式中，重链可变区与轻链可变区之间的氨基酸连接序列如下：
Figure PCTCN2018075866-appb-000022
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000023
绞链区和跨膜区
对于绞链区和跨膜区(跨膜结构域)，CAR可被设计以包括融合至CAR的胞外结构域的跨膜结构域。在一个实施方式中，使用天然与CAR中的结构域之一相关联的跨膜结构域。在一些例子中，可选择跨膜结构域，或通过氨基酸置换进行修饰，以避免将这样的结构域结合至相同或不同的表面膜蛋白的跨膜结构域，从而最小化与受体复合物的其他成员的相互作用。
在本发明的一个优选的实施方式中，绞链区包括以下氨基酸序列(IgG4Hinge-CH2-CH3铰链区)：
Figure PCTCN2018075866-appb-000024
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000025
或者，所述绞链区包括以下氨基酸序列(IgG4Hinge-CH2-CH3(L235E，N297Q))：
Figure PCTCN2018075866-appb-000026
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000027
Figure PCTCN2018075866-appb-000028
在本发明的一个优选的实施方式中，本发明的CAR中跨膜区为CD28来源的跨膜区(CD28TM)或CD8来源的跨膜区(CD8TM)。
在本发明的一个优选的实施方式中，CD8来源的跨膜区(CD8TM)氨基酸序列如下：
Figure PCTCN2018075866-appb-000029
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000030
在本发明的一个优选的实施方式中，CD28来源的跨膜区(CD28TM)氨基酸序列如下：
Figure PCTCN2018075866-appb-000031
CD28来源的跨膜区(CD28TM)编码DNA序列：
Figure PCTCN2018075866-appb-000032
胞内结构域
本发明的CAR中的胞内结构域包括4-1BB的信号传导结构域和CD3ζ的信号传导结构域。
在本发明的一个优选地实施方式中，所述CAR的胞内结构域还包括CD28的信号传导结构域。
优选地，4-1BB的胞内信号传导结构域包含如下氨基酸序列：
Figure PCTCN2018075866-appb-000033
其编码DNA序列如下:
Figure PCTCN2018075866-appb-000034
优选地，CD28来源的胞内信号传导结构域包含如下氨基酸序列：
Figure PCTCN2018075866-appb-000035
其编码DNA序列如下:
Figure PCTCN2018075866-appb-000036
优选地，CD3ζ的胞内信号传导结构域包含如下氨基酸序列：
Figure PCTCN2018075866-appb-000037
其编码DNA序列如下：
Figure PCTCN2018075866-appb-000038
载体
本发明还提供了编码本发明CAR序列的DNA构建体。
编码期望分子的核酸序列可利用在本领域中已知的重组方法获得，诸如例如通过从表达基因的细胞中筛选文库，通过从已知包括该基因的载体中得到该基因，或通过利用标准的技术，从包含该基因的细胞和组织中直接分离。可选地，感兴趣的基因可被合成生产。
本发明也提供了其中插入本发明的DNA构建体的载体。源于逆转录病毒诸如慢病毒的载体是实现长期基因转移的合适工具，因为它们允许转基因长期、稳定的整合并且其在子细胞中增殖。慢病毒载体具有超过源自致癌逆转录病毒诸如鼠科白血病病毒的载体的优点，因为它们可转导非增殖的细胞，诸如肝细胞。它们也具有低免疫原性的优点。
简单概括，通常通过可操作地连接编码CAR多肽或其部分的核酸至启动子，并将构建体并入表达载体，实现编码CAR的天然或合成核酸的表达。该载体适合于复制和整合真核细胞。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、初始序列和启动子。
本发明的表达构建体也可利用标准的基因传递方案，用于核酸免疫和基因疗法。基因传递的方法在本领域中是已知的。见例如美国专利号5,399,346、5,580,859、5,589,466，在此通过引用全文并入。在另一个实施方式中，本发明提供了基因疗法载体。
该核酸可被克隆入许多类型的载体。例如，该核酸可被克隆入如此载体，其包括但不限于质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒。特定的感兴趣载体包括表达载体、复制载体、探针产生载体和测序载体。
进一步地，表达载体可以以病毒载体形式提供给细胞。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。通常，合适的载体包含在至少一种有机体中起作用的复制起点、启动子序列、方便的限制酶位点和一个或多个可选择的标记(例如，WO01/96584；WO01/29058；和美国专利号6,326,193)。
已经开发许多基于病毒的系统，用于将基因转移入哺乳动物细胞。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用在本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。在一些实施方式中，使用腺病毒载体。许多腺病毒载体在本领域中是已知的。在一个实施方式中，使用慢病毒载体。
额外的启动子元件，例如增强子，可以调节转录开始的频率。通常地，这些位于起始位点上游的30-110bp区域中，尽管最近已经显示许多启动子也包含起始位点下游的功能元件。启动子元件之间的间隔经常是柔性的，以便当元件相对于另一个被倒置或移动时，保持启动子功能。在胸苷激酶(tk)启动子中，启动子元件之间的间隔可被增加隔开50bp，活性才开始下降。取决于启动子，表现出单个元件可合作或独立地起作用，以起动转录。
合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列为能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、艾伯斯坦-巴尔(Epstein-Barr)病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，本发明不应被限于组成型启动子的应用。诱导型启动子也被考虑为本发明的一部分。诱导型启动子的使用提供了分子开关，其能够当这样的表达是期望的时，打开可操作地连接诱导型启动子的多核苷酸序列的表达，或当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
为了评估CAR多肽或其部分的表达，被引入细胞的表达载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。在其他方面，可选择的标记可被携带在单独一段DNA上并用于共转染程序。可选择的标记和报道基因两者的侧翼都可具有适当的调节序列，以便能够在宿主细胞中表达。有用的可选择标记包括例如抗生素抗性基因，诸如neo等等。
报道基因用于鉴定潜在转染的细胞并用于评价调节序列的功能性。通常地，报道基因为以下基因：其不存在于受体有机体或组织或由受体有机体或组织进行表达，并且其编码多肽，该多肽的表达由一些可容易检测的性质例如酶活性清楚表示。在DNA已经被引入受体细胞后，报道基因的表达在合适的时间下进行测定。合适的报道基因可包括编码荧光素酶、β-半乳糖苷酶、氯霉素乙酰转移酶、分泌型碱性磷酸酶或绿色萤光蛋白基因的基因(例如，Ui-Tei等，2000FEBS Letters479:79-82)。合适的表达系统是公知的并可利用已知技术制备或从商业上获得。通常，显示最高水平的报道基因表达的具有最少5个侧翼区的构建体被鉴定为启动子。这样的启动子区可被连接至报道基因并用于评价试剂调节启动子-驱动转录的能力。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。在表达载体的内容中，载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。例如，表达载体可通过物理、化学或生物学手段转移 入宿主细胞。
将多核苷酸引入宿主细胞的物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等。生产包括载体和/或外源核酸的细胞的方法在本领域中是公知的。见例如Sambrook等(2001,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratory,New York)。将多核苷酸引入宿主细胞的优选方法为磷酸钙转染。
将感兴趣的多核苷酸引入宿主细胞的生物学方法包括使用DNA和RNA载体。病毒载体，特别是逆转录病毒载体，已经成为最广泛使用的将基因插入哺乳动物例如人细胞的方法。其他病毒载体可源自慢病毒、痘病毒、单纯疱疹病毒I、腺病毒和腺伴随病毒等等。见例如美国专利号5,350,674和5,585,362。
将多核苷酸引入宿主细胞的化学手段包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠；和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体。用作体外和体内传递工具(delivery vehicle)的示例性胶体系统为脂质体(例如，人造膜囊)。
在使用非病毒传递系统的情况下，示例性传递工具为脂质体。考虑使用脂质制剂，以将核酸引入宿主细胞(体外、离体(ex vivo)或体内)。在另一方面，该核酸可与脂质相关联。与脂质相关联的核酸可被封装入脂质体的水性内部中，散布在脂质体的脂双层内，经与脂质体和寡核苷酸两者都相关联的连接分子附接至脂质体，陷入脂质体，与脂质体复合，分散在包含脂质的溶液中，与脂质混合，与脂质联合，作为悬浮液包含在脂质中，包含在胶束中或与胶束复合，或以其他方式与脂质相关联。与组合物相关联的脂质、脂质/DNA或脂质/表达载体不限于溶液中的任何具体结构。例如，它们可存在于双分子层结构中，作为胶束或具有“坍缩的(collapsed)”结构。它们也可简单地被散布在溶液中，可能形成大小或形状不均一的聚集体。脂质为脂肪物质，其可为天然发生或合成的脂质。例如，脂质包括脂肪小滴，其天然发生在细胞质以及包含长链脂肪族烃和它们的衍生物诸如脂肪酸、醇类、胺类、氨基醇类和醛类的该类化合物中。
在使用非病毒传递系统的情况下，示例性地采用基因编辑技术，如CRISPR-Cas9，ZFN或TALEN等完成本发明。
在本发明的一个优选地实施方式中，所述载体为慢病毒载体。
在本发明的一个优选地实施方式中，所述DNA构建体中还包括信号肽编码序列。优选地，所述信号肽序列连接在所述抗原结核结构域核酸序列的上游。优选地所述信号肽为人源CD8a信号肽。
优选地，所述信号肽氨基酸序列如下：
CD8前导序列(CD8Leader sequence)的氨基酸序列：
Figure PCTCN2018075866-appb-000039
CD8前导序列(CD8Leader sequence)的编码DNA序列序列：
Figure PCTCN2018075866-appb-000040
治疗性应用
本发明包括用编码本发明CAR的慢病毒载体(LV)转导的细胞(例如，T细胞)。转导的T细胞可引起CAR-介导的T-细胞应答。
因此，本发明也提供了刺激对哺乳动物的靶细胞群或组织的T细胞-介导的免疫应答的方法，其包括以下步骤：施用给哺乳动物表达本发明CAR的T细胞。
在一个实施方式中，本发明包括一类细胞疗法，其中T细胞被基因修饰以表达本发明的CAR，和CAR-T细胞被注入需要其的接受者中。注入的细胞能够杀死接受者的肿瘤细胞。不像抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。
在一个实施方式中，本发明的CAR-T细胞可经历稳固的体内T细胞扩展并可持续延长的时间量。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-修饰T细胞诱导对CAR中的抗原结合结构域特异性的免疫应答。例如，抗CD20CAR-T细胞引起抗表达CD20的细胞的特异性免疫应答。
尽管本文公开的数据具体公开了包括抗-CD20scFv、铰链和跨膜区、和4-1BB和CD3ζ信号传导结构域的慢病毒载体，但本发明应被解释为包括对构建体组成部分中的每一个的任何数量的变化。
可治疗的适应症包括CD20阳性肿瘤和B细胞过高引起的疾病(如自身免疫疾病，例如红斑性狼疮等)，CD20阳性肿瘤可包括非实体瘤(诸如血液学肿瘤，例如白血病和淋巴瘤)或可包括实体瘤。用本发明的CAR治疗的肿瘤或癌症类型包括但不限于癌、胚细胞瘤和肉瘤，和某些白血病或淋巴恶性肿瘤、良性和恶性肿瘤、和恶性瘤，例如肉瘤、癌和黑素瘤。也包括成人肿瘤/癌症和儿童肿瘤/癌症。
血液学癌症为血液或骨髓的癌症。血液学(或血原性)癌症的例子包括白血病，包括急性白血病(诸如急性淋巴细胞白血病、急性髓细胞白血病、急性骨髓性白血病和成髓细胞性、前髓细胞性、粒-单核细胞型、单核细胞性和红白血病)、慢性白血病(诸如慢性髓细胞(粒细胞性)白血病、慢性骨髓性白血病和慢性淋巴细胞白血病)、真性红细胞增多症、淋巴瘤、霍奇金氏疾病、非霍奇金氏淋巴瘤(无痛和高等级形式)、多发性骨髓瘤、瓦尔登斯特伦氏巨球蛋白血症、重链疾病、骨髓增生异常综合征、多毛细胞白血病和脊髓发育不良。
实体瘤为通常不包含囊肿或液体区的组织的异常肿块。实体瘤可为良性或恶性的。不同类型的实体瘤以形成它们的细胞类型命名(诸如肉瘤、癌和淋巴瘤)。实体瘤诸如肉瘤和癌的例子包括纤维肉瘤、粘液肉瘤、脂肪肉瘤间皮瘤、淋巴恶性肿瘤、胰腺癌卵巢癌、。
本发明的CAR-修饰T细胞也可用作对哺乳动物离体免疫和/或体内疗法的疫苗类型。优选地，哺乳动物为人。
对于离体免疫，以下中的至少一项在将细胞施用进入哺乳动物前在体外发生：i)扩展细胞，ii)将编码CAR的核酸引入细胞，和/或iii)冷冻保存细胞。
离体程序在本领域中是公知的，并在以下更完全地进行讨论。简单地说，细胞从哺乳动物(优选人)中分离并用表达本文公开的CAR的载体进行基因修饰(即，体外转导或转染)。CAR-修饰的细胞可被施用给哺乳动物接受者，以提供治疗益处。哺乳动物接受者可为人，和CAR-修饰的细胞可相对于接受者为自体的。可选地，细胞可相对于接受者为同种异基因的、同基因的(syngeneic)或异种的。
除了就离体免疫而言使用基于细胞的疫苗之外，本发明也提供了体内免疫以引起针对患者中抗原的免疫应答的组合物和方法。
通常地，如本文所述活化和扩展的细胞可用于治疗和预防无免疫应答的个体中 产生的疾病。特别地，本发明的CAR-修饰的T细胞用于治疗CCL。在某些实施方式中，本发明的细胞用于治疗处于形成CCL风险中的患者。因此，本发明提供了治疗或预防CCL的方法，其包括施用给需要其的对象治疗有效量的本发明的CAR-修饰的T细胞。
本发明的CAR-修饰的T细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如IL-2、IL-17或其他细胞因子或细胞群结合施用。简单地说，本发明的药物组合物可包括如生物合成药如单克隆抗体，小分子药物与一种或多种药学或生理学上可接受载体、稀释剂或赋形剂结合。这样的组合物可包括缓冲液诸如中性缓冲盐水、硫酸盐缓冲盐水等等；碳水化合物诸如葡萄糖、甘露糖、蔗糖或葡聚糖、甘露醇；蛋白质；多肽或氨基酸诸如甘氨酸；抗氧化剂；螯合剂诸如EDTA或谷胱甘肽；佐剂(例如，氢氧化铝)；和防腐剂。本发明的组合物优选配制用于静脉内施用。
本发明的药物组合物可以以适于待治疗(或预防)的疾病的方式施用。施用的数量和频率将由这样的因素确定，如患者的病症、和患者疾病的类型和严重度——尽管适当的剂量可由临床试验确定。
当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。可通常指出：包括本文描述的T细胞的药物组合物可以以10 4至10 9个细胞/kg体重的剂量，优选10 5至10 6个细胞/kg体重的剂量(包括那些范围内的所有整数值)施用。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，NewEng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。
对象组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内(i.v.)注射或腹膜内施用给患者。在一个实施方式中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方式中，本发明的T细胞组合物优选通过i.v.注射施用。T细胞的组合物可被直接注入肿瘤，淋巴结或感染位置。
在本发明的某些实施方式中，利用本文描述的方法或本领域已知的其他将T细胞扩展至治疗性水平的方法活化和扩展的细胞，与任何数量的有关治疗形式结合(例如，之前、同时或之后)施用给患者，所述治疗形式包括但不限于用以下试剂进行治疗：所述试剂诸如抗病毒疗法、西多福韦和白细胞介素-2、阿糖胞苷(也已知为ARA-C)或对MS患者的那他珠单抗治疗或对牛皮癣患者的厄法珠单抗治疗或对PML患者的其他治疗。在进一步的实施方式中，本发明的T细胞可与以下结合使用：化疗、辐射、免疫抑制剂，诸如，环孢菌素、硫唑嘌呤、甲氨喋呤、麦考酚酯和FK506，抗体或其他免疫治疗剂。在进一步的实施方式中，本发明的细胞组合物与骨髓移植、利用化疗剂诸如氟达拉滨、外部光束放射疗法(XRT)、环磷酰胺结合(例如，之前、同时或之后)而施用给患者。例如，在一个实施方式中，对象可经历高剂量化疗的标准治疗，之后进行外周血干细胞移植。在一些实施方式中，在移植后，对象接受本发明的扩展的免疫细胞的注入。在一个额外的实施方式中，扩展的细胞在外科手 术前或外科手术后施用。
施用给患者的以上治疗的剂量将随着治疗病症的精确属性和治疗的接受者而变化。人施用的剂量比例可根据本领域接受的实践实施。通常，每次治疗或每个疗程，可将1×10 6个至1×10 10个本发明经修饰的T细胞(如，CAR-T20细胞)，通过例如静脉回输的方式，施用于患者。
本发明的优点包括：
(1)本发明的嵌合抗原受体，其细胞外抗原结合结构域为特定的抗CD20scFv，该特定的抗CD20scFv结合特定的绞链区和胞内结构域形成的CAR显示出了极大的对肿瘤细胞的杀伤能力，而且细胞毒性较小，副作用低。
(2)本发明提供的嵌合抗原受体可在携带CAR基因的慢病毒感染T细胞后实现CAR蛋白的稳定表达和膜定位；
(3)本发明的CAR修饰的T细胞在体内存活时间较长，且抗肿瘤效力较强；对IgG4Hinge-CH2-CH3连接区优化后的CAR，能够避免Fc受体的结合及后续的ADCC作用(抗体依赖性细胞毒作用)。
实施例1 慢病毒表达载体的构建
编码质粒为委托上海博益生物科技有限公司做全长DNA合成和克隆构建。克隆载体选用的是pWPT慢病毒载体，克隆位点为BamH I和Sal I位点。具体序列结构如图1所示。各元件的氨基酸和核苷酸序列如上所述。
实施例2 CAR-T细胞的制备
(1)取健康人静脉血，密度梯度离心方法分离获得单个核细胞(PBMCs)。
(2)第0天，PBMCs采用含2％人血白蛋白的GT-T551细胞培养基培养，调整细胞终浓度为2×10 6cell/mL。将细胞接种于预先经过终浓度为5μg/mLCD3单克隆抗体(OKT3)及终浓度为10μg/mL的Retronectin(购自TAKARA公司)包被的细胞培养瓶。培养基里添加终浓度为1000U/mL的重组人白介素2(IL-2)，在37℃，饱和湿度为5％CO 2培养箱培养。
(3)第2天，加入新鲜培养液，浓缩纯化的CAR20s慢病毒液，protamine sulfate(12ug/ml)，以及终浓度为1000U/mL IL-2。置于37℃，5％CO 2培养箱中感染12小时后，弃培养液，加入新鲜的培养基，于37℃，5％CO 2培养箱继续进行培养。
(4)第6天开始，可取CART20s细胞做相应的活性检测试验。
实施例3 CAR基因在T细胞基因组整合率及其编码蛋白在膜表面表达水平的检测。
(1)分别取0.5×10 6实施例2中培养到第7天(图.2A)和第11天(图.2B)的CART-20s细胞样品，经过Protein L染色在流式细胞仪上分析CAR20蛋白在T细胞膜表面的表达水平。结果显示：本研究中所设计的CAR结构均可在其相应修饰的T细胞中表达并完成细胞膜表面定位。
实施例4 CAR-T20s体外激活能力的检测
(1)采用实施例2中培养到第6天CAR-T20s细胞作细胞被激活水平指标蛋白 CD137和IFNγ的检测。依次取1*10 5培养到第6天的CART-20细胞，分别与CD20阳性的RAJI和RAMOS肿瘤细胞系，以及CD20阴性的MOLT-4肿瘤细胞系或不添加肿瘤细胞，在200μl GT-551培养基中按照1:1比例培养18h后流逝方法检测T细胞膜表面CD137的表达水平(图.3A)，ELISA方法检测培养上清中IFNγ的分泌水平(图.3B)。
(2)采用实施例2中培养到第13天CAR-T20s细胞作细胞活化水平指标蛋白CD137和IFNγ的检测。图4，依次取1*10 5培养到第6天的CART-20细胞，分别与CD20阳性的RAJI和RAMOS肿瘤细胞系，以及CD20阴性的MOLT-4肿瘤细胞系或不添加肿瘤细胞，在200μl GT-551培养基中按照1:1比例培养18h后流逝方法检测T细胞膜表面CD137的表达水平(图.4A)，ELISA方法检测培养上清中IFNγ的分泌水平(图.4B)。
(3)从图.3和图.4中的结果我们可以得出如下结论：以Leu-16序列为基础的三代CAR比二代CAR有更好的CD137活化水平和IFNγ释放水平；Leu16VH-VL或者VL-VH的顺序对以leu-16抗体序列为基础构建的CART20的体外活性并没有明显的IFNgamma释放和CD137表达的差异。
实施例5 CAR-T20s细胞诱导肿瘤细胞早期凋亡活性的检测
(1)采用实施例2中培养到第11天CAR-T20s细胞按图示比例分别与1*10 4经过CFSE标记的CD20阴性(MOLT-4)或CD20阳性的(RAJI，RAMOS)肿瘤细胞系，在200μl GT-551培养基中共培养4h，离心收集细胞沉淀，细胞经PBS洗两遍后用Annexin V-APC染料按1:50的比例100μl染液中染色30min，PBS洗1遍后在流失细胞仪上分析CFSE阳性细胞中Annexin V阳性细胞的比例。
(2)图.5的结果显示Leu16序列为基础构建的三代CAR相较于二代CAR可以更好地诱导CD20阳性肿瘤细胞的早期凋亡。
实施例6 CAR-T20s细胞诱导肿瘤细胞晚期凋亡活性的检测
(1)实施例2中方法制备的另一批CAR-T20细胞(图.6A),按图.6B所示比例分别与1*10 4经过CFSE标记的CD20阴性(MOLT-4)或CD20阳性的(RAJI，RAMOS)肿瘤细胞系，在200μl GT-551培养基中共培养4h，离心收集细胞沉淀，细胞经PBS洗两遍后用PI染料按1:50的比例100μl染液中染色30min，PBS洗1遍后在流失细胞仪上分析CFSE阳性细胞中PI阳性细胞的比例。
(2)图.6B所示结果说明以Leu16序列为基础构建的三代CAR(CAR-T20.9，CAR-T20.17相较于二代CAR(CAR-T20.12，CAR-T20.18)可以更好地诱导CD20阳性肿瘤细胞的晚期凋亡。
实施例7 CAR-T20细胞的体内清除CD20阳性细胞能力的检测
(1)将表达luciferase的Raji-Luc细胞通过尾静脉注射NCG小鼠(5*10 5/只)。接种一周后，通过活体成像观察肿瘤细胞体内扩增情况，记为Day 0。将NT,CAR-T20.17，和CAR-T20.18细胞尾静脉注射Day 0天小鼠(5*10 6/只)。在Day0,Day7,Day14,Day21：活体成像观察小鼠体内肿瘤细胞扩增状况，根据荧光强度变化及小鼠体重变化进行分析。
(2)图7所示结果表明，以Leu16序列为基础构建的CAR-T(CAR-T20.17和 CAR-T20.18)可以有效地抑制CD20阳性肿瘤细胞的体内扩增。
以上详细描述了本发明的优选实施方式，但是，本发明并不限于上述实施方式中的具体细节，在本发明的技术构思范围内，可以对本发明的技术方案进行多种简单变型，这些简单变型均属于本发明的保护范围。
另外需要说明的是，在上述具体实施方式中所描述的各个具体技术特征，在不矛盾的情况下，可以通过任何合适的方式进行组合，为了避免不必要的重复，本发明对各种可能的组合方式不再另行说明。
此外，本发明的各种不同的实施方式之间也可以进行任意组合，只要其不违背本发明的思想，其同样应当视为本发明所公开的内容。

Claims (10)

  1. 一种嵌合抗原受体(CAR)，其特征在于，所述嵌合抗原受体的抗原结合结构域包括SEQ ID NO.12所示的抗体重链可变区，和SEQ ID NO.14所示的抗体轻链可变区。
  2. 如权利要求1所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的抗原结合结构域(scFv)如下式I或式II所示：
    V H－V L,(I)；V L－V H,(II)
    其中，V H为抗体重链可变区；V L为抗体轻链可变区；“－”为连接肽或肽键。
  3. 如权利要求2所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的结构如下式所示：
    L－scFv－H－TM－C－CD3ζ
    其中，
    L为任选的引导序列(Leader sequence，即信号肽序列)；
    scFv为抗原结合结构域；
    H为绞链区；
    TM为跨膜结构域；
    C为共刺激信号受体酪氨酸激活基序；
    CD3ζ为源于CD3ζ的胞浆信号传导序列；
    所述抗原结合结构域和“－”分别如上所述。
  4. 一种核酸分子，其特征在于，所述核酸分子编码权利要求1所述的嵌合抗原受体(CAR)。
  5. 如权利要求4所述的核酸分子，其特征在于，所述核酸分子包含选自下组的编码所述铰链区核酸序列：
    (a)编码如SEQ ID NO.18或20所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.19或21所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.19或21所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.18或20所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
    和/或
    所述核酸分子包含选自下组的编码所述CD8的跨膜区的核酸序列：
    (a)编码如SEQ ID NO.22所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.23所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.23所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.22所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
    和/或
    所述核酸分子包含选自下组的编码所述CD28的跨膜区的核酸序列：
    (a)编码如SEQ ID NO.24所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.25所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.25所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.24所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
    和/或
    所述核酸分子包含共刺激信号受体酪氨酸激活基序编码序列，所述共刺激信号受体酪氨酸激活基序编码序列包括4-1BB来源的共刺激信号受体酪氨酸激活基序编码序列，和/或CD28来源的共刺激信号受体酪氨酸激活基序编码序列，其中
    所述4-1BB来源的共刺激信号受体酪氨酸激活基序编码序列选自下组：
    (a)编码如SEQ ID NO.26所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.27所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.27所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.26所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
    所述CD28来源的共刺激信号受体酪氨酸激活基序编码序列选自下组：
    (a)编码如SEQ ID NO.28所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.29所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.29所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.28所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸；
    和/或
    所述核酸分子包含选自下组的编码所述CD3ζ的胞内信号结构域的核酸序列：
    (a)编码如SEQ ID NO.30所示多肽的多核苷酸；
    (b)序列如SEQ ID NO.31所示的多核苷酸；
    (c)核苷酸序列与SEQ ID NO.30所示序列的同源性≥90％(较佳地≥95％)，并且编码SEQ ID NO.31所示氨基酸序列的多核苷酸；
    (d)与(a)-(c)任一所述的多核苷酸互补的多核苷酸。
  6. 一种载体，其特征在于，所述的载体含有权利要求4所述的核酸分子。
  7. 一种宿主细胞，其特征在于，所述的宿主细胞中含有权利要求6所述的所述的载体或染色体中整合有外源的权利要求5所述的核酸分子。
  8. 一种药物组合物，其特征在于，所述组合物含有药学上可接受的载体以及权利要求1所述的嵌合抗原受体、权利要求4所述的核酸分子、权利要求6所述的载体、或权利要求7所述的细胞。
  9. 权利要求1所述的嵌合抗原受体、权利要求4所述的核酸分子、权利要求6所述的载体、或权利要求7所述的细胞的用途，用于制备治疗肿瘤的药物或制剂。
  10. 一种制备CAR-T细胞(CAR-修饰的T细胞)的方法，其特征在于，所述CAR-T细胞表达权利要求1所述的嵌合抗原受体，
    所述方法包括步骤：将权利要求4所述的核酸分子或权利要求6所述的载体转导入T细胞内，从而获得所述CAR-T细胞。
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