WO2021121193A1 - 一种靶向cd19的嵌合抗原受体及其用途 - Google Patents

一种靶向cd19的嵌合抗原受体及其用途 Download PDF

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WO2021121193A1
WO2021121193A1 PCT/CN2020/136241 CN2020136241W WO2021121193A1 WO 2021121193 A1 WO2021121193 A1 WO 2021121193A1 CN 2020136241 W CN2020136241 W CN 2020136241W WO 2021121193 A1 WO2021121193 A1 WO 2021121193A1
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Prior art keywords
cells
immune effector
chimeric antigen
nucleic acid
antigen receptor
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PCT/CN2020/136241
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English (en)
French (fr)
Inventor
王建祥
王敏
吕璐璐
石琳
安娜
王瑞
刘芸
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Juventas Cell Therapy Ltd
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Juventas Cell Therapy Ltd
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Priority to AU2020406534A priority Critical patent/AU2020406534A1/en
Priority to ES20901145T priority patent/ES3027134T3/es
Priority to EP20901145.1A priority patent/EP4032978B1/en
Priority to JP2021564892A priority patent/JP7439128B2/ja
Priority to KR1020227024207A priority patent/KR20220117903A/ko
Application filed by Juventas Cell Therapy Ltd filed Critical Juventas Cell Therapy Ltd
Priority to CN202311272341.XA priority patent/CN117264080A/zh
Priority to CN202080015332.0A priority patent/CN113728096B/zh
Publication of WO2021121193A1 publication Critical patent/WO2021121193A1/zh
Priority to US17/412,746 priority patent/US11547728B2/en
Anticipated expiration legal-status Critical
Priority to US18/056,616 priority patent/US20230201261A1/en
Priority to JP2024020263A priority patent/JP7734771B2/ja
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Definitions

  • This application relates to the field of biomedicine, in particular to a chimeric antigen receptor targeting CD19 and its use.
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • B-cell lymphoma B-cell lymphoma
  • ALL acute lymphocytic leukemia
  • CLL chronic lymphocytic leukemia
  • B-cell lymphoma B-cell lymphoma
  • CD19 is almost expressed on the surface of all B-cell tumor cells, while other parenchymal cells and hematopoietic stem cells are almost not expressed.
  • CAR-T cells chimeric antigen receptor T cells
  • This application provides a chimeric antigen receptor comprising the amino acid sequence shown in SEQ ID NO. 1.
  • the application also provides a nucleic acid encoding the chimeric antigen receptor, comprising the nucleic acid
  • the chimeric antigen receptor described in the present application includes at least one of the following beneficial effects: (1) It is stably expressed on the surface of immune cells (for example, T cells), and the expression level is high; (2) It has a strong ability to kill CD19-positive target cells (3) Promote the secretion of cytokines by immune cells, for example, increase the ability of T cells to secrete cytokines (INF- ⁇ or IL-6) by at least 1, 2, or 3 times; (4) No hemolysis , Does not cause hemolysis or aggregation of red blood cells; (5) No vascular irritation, no local or systemic abnormalities after administration; (6) No tumorigenicity, no tumorigenic in vivo or in vitro; (7) Prolong survival of tumor patients Time; (8) Effectively alleviate the condition of tumors (e.g., adult acute lymphoblastic leukemia, childhood acute lymphoblastic leukemia and/or non-Hodgkin’s lymphoma); and, (9) high safety, causing side effects (e.g.,
  • the present application provides a chimeric antigen receptor, which comprises the amino acid sequence shown in SEQ ID NO.1.
  • this application also provides an isolated nucleic acid molecule that encodes the chimeric antigen receptor described in this application.
  • the present application also provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, which comprises the nucleic acid sequence described in SEQ ID NO.2.
  • the present application also provides a vector, which contains the nucleic acid molecule described in the present application.
  • the present application also provides an immune effector cell, which comprises the chimeric antigen receptor, the nucleic acid molecule and/or the vector described in the present application.
  • the immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.
  • the surface of the immune effector cell expresses the chimeric antigen receptor described in the present application.
  • this application also provides a method for preparing immune effector cells, which includes the following steps: transducing the vector described in this application into the immune effector cells.
  • the immune effector cells are selected from the group consisting of T lymphocytes and natural killer cells.
  • the present application also provides a composition comprising the immune effector cell described in the present application.
  • the application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the immune effector cell for the preparation of a medicine, wherein the medicine is used for treatment Diseases or disorders related to the expression of CD19.
  • the present application also provides a method for treating diseases or disorders related to the expression of CD19, the method comprising administering the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the Immune effector cells.
  • the present application also provides the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the immune effector cell, which are used to treat diseases or disorders related to the expression of CD19 .
  • the disease or disorder associated with the expression of CD19 includes non-solid tumors.
  • the non-solid tumor includes leukemia and/or lymphoma.
  • the disease or condition associated with the expression of CD19 includes acute lymphoblastic leukemia and/or B-cell lymphoma.
  • the acute lymphoblastic leukemia includes adult acute lymphoblastic leukemia and/or childhood acute lymphoblastic leukemia.
  • the dose of the drug used to treat acute lymphoblastic leukemia is 0.25 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive T cells.
  • the B-cell lymphoma includes non-Hodgkin's lymphoma.
  • the dose of the drug used to treat non-Hodgkin's lymphoma is 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells.
  • Figures 1A and 1B show the detection results of CAR molecule expression on the surface of CNCT19 cells.
  • Figure 2 shows the residual rate of tumor cells under different co-culture conditions.
  • Figure 3 shows that the RTCA DP system monitors the killing of CNCT19 cells on target cells (CHO-CD19) in real time.
  • Figure 4A shows the change of INF- ⁇ concentration in the supernatant under different co-cultivation conditions.
  • Figure 4B shows the change of IL-6 concentration in the supernatant under different co-cultivation conditions.
  • Figure 5A shows the observation results of each test tube before shaking for 3 hours.
  • Figure 5B shows the observation results of each test tube after 3 hours of shaking.
  • Fig. 6A shows a micrograph (HE staining, 10x objective lens) of the administration part after the administration of CAR-T cells.
  • Fig. 6B shows a micrograph of the administration part after sodium chloride injection (HE staining, 10x objective lens).
  • Figure 7 shows the formation of soft agar clones after 3 weeks of inoculation of cells in each group.
  • Figure 8 shows the survival curves of different cell treatments in NCG mice with Nalm-6 xenograft tumors.
  • Figure 9 shows the tissue distribution of CNCT19 cells after a single administration.
  • Figure 10 is a comparison of the distribution of CNCT19 cells in tumor-bearing and non-tumor-bearing animals.
  • Figure 11 shows the changes in the distribution of CNCT19 cells in various tissues of the animal after a single administration.
  • CAR Chimeric Antigen Receptor
  • CAR-T chimeric antigen receptor T cells
  • TAA tumor-associated antigen
  • the CAR can be combined with the T cell receptor activation intracellular domain based on the antigen specificity of the antibody.
  • CAR-expressing T cells can specifically recognize and eliminate malignant cells expressing target antigens.
  • isolated generally refers to those obtained from the natural state by artificial means. If a certain "isolated” substance or component appears in nature, it may be that the natural environment in which it is located has changed, or the substance has been isolated from the natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of.
  • isolated does not exclude the mixing of artificial or synthetic materials, nor does it exclude the presence of other impure materials that do not affect the activity of the material.
  • immune effector cell generally refers to a cell that participates in an immune response, such as promoting an immune effector response.
  • immune effector cells can be selected from the following group: T lymphocytes and natural killer cells.
  • the term "specific binding and/or recognition” generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody (or CAR structural fragment), which can be used in molecules (including biomolecules).
  • an antibody (or CAR structural fragment) that specifically binds to a target has a greater affinity, avidity, easier, and/or longer duration than it binds to other targets
  • An antibody (or CAR structural fragment) that binds to the target is a measurable and reproducible interaction, such as the binding between a target and an antibody (or CAR structural fragment), which can be used in molecules (including biomolecules).
  • the existence of heterogeneous populations of) can determine the existence of the target.
  • an antibody (or CAR structural fragment) that specifically binds to a target which may be an epitope
  • An antibody (or CAR structural fragment) that binds to the target has a greater affinity, avidity, easier, and/or longer duration than it binds to other targets
  • isolated nucleic acid molecule generally refers to an isolated form of nucleotides, deoxyribonucleotides or ribonucleotides of any length, or analogs isolated from their natural environment or artificially synthesized.
  • vector generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a certain protein can be inserted and the protein can be expressed.
  • the vector can be transformed, transduced or transfected into the host cell so that the genetic material elements it carries can be expressed in the host cell.
  • vectors include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or artificial chromosomes (PAC) derived from P1; bacteriophages such as lambda phage or M13 phage And animal viruses.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • retroviruses including lentiviruses
  • adenoviruses such as herpes simplex virus
  • poxviruses such as herpes simplex virus
  • baculoviruses such as baculoviruses
  • papilloma viruses such as papilloma viruses
  • papillary polyoma vacuoles Virus such as SV40
  • a vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes.
  • the vector may also contain an origin of replication site.
  • the carrier may
  • composition generally refers to a composition suitable for administration to a patient.
  • the composition described in this application may comprise the immune effector cells described in this application.
  • the composition may also contain one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparation.
  • the acceptable ingredients of the composition are non-toxic to the recipient at the dose and concentration used.
  • the compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • CD19 usually refers to the differentiation antigen cluster 19 protein, which is an antigenic determinant that can be detected on leukemia precursor cells.
  • the amino acid and nucleic acid sequences of human and murine CD19 can be found in public databases such as GenBank, UniProt and Swiss-Prot.
  • the amino acid sequence of human CD19 can be found under UniProt/Swiss-Prot accession number P15391, and the nucleotide sequence encoding human CD19 can be found under accession number NM_001178098.
  • “CD19” may include proteins with mutations (for example, point mutations, fragments, insertions, deletions, and splice variants of full-length wild-type CD19).
  • subject generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the term "about” generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. Variation within the range of 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.
  • Chimeric antigen receptor nucleic acid, carrier, immune effector cell, composition
  • the present application provides a chimeric antigen receptor, which comprises the amino acid sequence shown in SEQ ID NO.1.
  • the application also provides that the amino acid sequence shown in SEQ ID NO. 1 has at least 80% (such as at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%). , At least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical amino acid sequence.
  • the chimeric antigen receptors described in this application can specifically bind and/or recognize tumor antigens.
  • the chimeric antigen receptor described in this application can specifically bind and/or recognize the antigen CD19.
  • the chimeric antigen receptors described in this application can promote immune effector cells to secrete cytokines.
  • the immune effector cells may be selected from the following group: T lymphocytes and natural killer cells.
  • the cytokine may be selected from the group consisting of IFN- ⁇ and IL-6.
  • the immune effector cells may be mammalian immune effector cells.
  • the T lymphocytes may be mammalian T lymphocytes, and the natural killer cells may also be mammalian natural killer cells.
  • the T lymphocytes may be human T lymphocytes, and the natural killer cells may also be human natural killer cells.
  • the chimeric antigen receptors described in the present application are free of hemolysis and vascular irritation.
  • the chimeric antigen receptors described in this application are not tumorigenic in vitro.
  • the chimeric antigen receptors described in this application are not tumorigenic in vivo.
  • the chimeric antigen receptors described in this application can effectively treat tumors.
  • the tumor may be a CD19-positive tumor.
  • the chimeric antigen receptor described in this application can effectively prolong the survival time of patients with CD19-positive tumors.
  • the chimeric antigen receptor described in this application can effectively prolong the survival time of patients with non-solid tumors.
  • the chimeric antigen receptor described in this application can effectively prolong the survival time of lymphoma and/or leukemia.
  • the chimeric antigen receptor described in the present application can effectively prolong the survival time of adult patients with acute lymphoblastic leukemia.
  • the chimeric antigen receptor described in the present application can effectively prolong the survival time of children with acute lymphoblastic leukemia.
  • the chimeric antigen receptor described in the present application can effectively prolong the survival time of patients with B-cell lymphoma (for example, non-Hodgkin's lymphoma).
  • the chimeric antigen receptors described in this application can effectively treat adult acute lymphoblastic leukemia.
  • the chimeric antigen receptors described in this application can effectively treat childhood acute lymphoblastic leukemia.
  • the chimeric antigen receptors described in this application can effectively treat non-Hodgkin's lymphoma.
  • this application provides an isolated nucleic acid molecule that encodes the chimeric antigen receptor described in this application.
  • the present application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, which comprises the nucleic acid sequence described in SEQ ID NO.2.
  • the present application provides an isolated nucleic acid molecule encoding a chimeric antigen receptor, which comprises a nucleic acid molecule similar to the nucleic acid sequence shown in SEQ ID NO. 2 and encoding the chimeric antigen receptor.
  • the nucleic acid sequence similar to SEQ ID NO. 2 means that the nucleic acid sequence shown in SEQ ID NO. 2 has at least 70%, 75%, 80%, 85%, 90%, 95% %, 96%, 97%, 98%, or 99% sequence identity.
  • the nucleic acid sequence similar to SEQ ID NO. 2 means that the nucleic acid sequence of SEQ ID NO. 2 is different from the nucleic acid of SEQ ID NO. 2 due to the wobble (degeneracy) of the third base of the nucleic acid codon, but However, the nucleic acid molecule encoding the chimeric antigen receptor.
  • variants of genes and proteins for example, the variants of the amino acid sequence described in SEQ ID NO. 1, or the variants of the nucleic acid sequence described in SEQ ID NO. 2, which remain One or more biological activities.
  • variants of proteins or polypeptides include proteins or polypeptides that have been or can be modified using recombinant DNA technology so that the protein or polypeptide has altered or additional properties, for example, the variants confer enhanced protein stability to the protein in plasma Or endow the protein with enhanced activity.
  • the variant may be different from the reference sequence, such as different from a naturally occurring polynucleotide, protein or peptide.
  • the naturally occurring variant gene and the non-naturally occurring variant gene will typically be at least about 50% identical to the reference gene, more typically at least about 70% identical, even more typically at least about 80% identical Same (90% or higher identity).
  • the naturally-occurring variant protein and the non-naturally-occurring variant protein will typically be at least about 70% identical to the reference protein, more typically at least about 80% identical, and even more typically at least about 90% or more similar. High identity, but allows most non-identity regions in non-conserved regions (for example, the same portion is less than 70%, such as less than 60%, less than 50%, or even less than 40%).
  • the sequence has at least 60%, 70%, 75% or more identity with the reference sequence (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%). %, 99% or higher identity).
  • the reference sequence e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%). %, 99% or higher identity.
  • the terms “identity”, “homology” and their grammatical variants generally mean that when two or more entities are “aligned” in sequence, they are the same.
  • two polypeptide sequences are the same, they have the same amino acid sequence at least within the region or part to which they refer.
  • two polynucleotide sequences are the same, they have the same polynucleotide sequence at least within the region or part of the reference.
  • the identity can be a defined region (region or domain) of the sequence.
  • the "zone” or “region” of identity refers to the same part of two or more entities referred to. Therefore, if two protein or nucleic acid sequences are the same in one or more sequence regions or regions, they are identical in that region.
  • an “aligned” sequence refers to a plurality of polynucleotide or protein (amino acid) sequences, and compared to the reference sequence, it often contains bases or amino acids (gap) that are corrected for deletion or addition.
  • the degree of identity (homology) between two sequences can be determined using computer programs and mathematical algorithms. Such algorithms that calculate percent sequence identity (homology) generally calculate sequence gaps and mismatches in compared regions or regions.
  • BLAST for example, BLAST 2.0
  • search algorithm see, for example, Altschul et al., J.Mol.Biol.215:403 (1990), publicly available through NCBI
  • the nucleic acid molecule can be an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or artificially synthesized, but can encode the present The chimeric antigen receptor described in the application.
  • the present application provides a vector comprising the nucleic acid molecule described in the present application.
  • the vector can be transformed, transduced, or transfected into a host cell so that the genetic material elements it carries can be expressed in the host cell.
  • the vector may include: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or artificial chromosome (PAC) derived from P1; bacteriophage such as lambda phage or M13 phage and Animal viruses and so on.
  • the types of animal viruses used as vectors include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papilloma viruses, and papillary polyoma vacuoles Virus (such as SV40).
  • the vector may contain a variety of elements for controlling expression, including a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene.
  • the vector may also contain an origin of replication.
  • the carrier may also include components that assist it to enter cells, such as viral particles, liposomes or protein coats, but not only these substances.
  • the present application provides an immune effector cell, which comprises the chimeric antigen receptor, the nucleic acid molecule, and/or the vector described in the present application.
  • the immune effector cells may be selected from the following group: T lymphocytes and natural killer cells.
  • the immune effector cells may be human immune effector cells.
  • the immune effector cells may be human T lymphocytes.
  • the immune effector cells may be human natural killer cells.
  • the surface of the immune effector cell may express the chimeric antigen receptor described in this application.
  • the immune effector cells described in this application can effectively kill tumor cells.
  • the tumor cells may be CD19-positive cells.
  • the immune effector cells described in this application can significantly reduce the residual rate of the CD19-positive human leukemia cell line Nalm-6.
  • the immune effector cells described in this application can effectively promote the secretion of cytokines after contacting CD19-positive cells.
  • the cytokine may be selected from the group consisting of IFN- ⁇ and IL-6.
  • the immune effector cells described in this application are co-cultured with the CD19-positive human leukemia cell line Nalm-6, the secretion of IFN- ⁇ and IL-6 cytokines is significantly increased.
  • the immune effector cells described in this application are non-hemolytic and vascular irritant.
  • the immune effector cells described in this application will not cause hemolysis and blood coagulation.
  • the immune effector cells described in the present application have no vascular irritation.
  • the immune effector cells described in this application are not tumorigenic in vitro.
  • the immune effector cells described in this application are not tumorigenic in vivo.
  • the immune effector cells described in this application can effectively treat tumors.
  • the tumor may be a CD19-positive tumor.
  • the immune effector cells described in this application can effectively prolong the survival time of patients with CD19-positive tumors.
  • the immune effector cells described in this application can effectively prolong the survival time of adult patients with acute lymphoblastic leukemia.
  • the immune effector cells described in this application can effectively prolong the survival time of children with acute lymphoblastic leukemia.
  • the immune effector cells described in the present application can effectively prolong the survival time of patients with B-cell lymphoma (for example, non-Hodgkin's lymphoma).
  • the immune effector cells described in this application can effectively treat adult acute lymphoblastic leukemia.
  • the immune effector cells described in this application can effectively treat childhood acute lymphoblastic leukemia.
  • the immune effector cells described in this application can effectively treat B-cell lymphoma (for example, non-Hodgkin's lymphoma).
  • the present application provides a composition comprising the immune effector cell described in the present application.
  • the composition may also include one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives
  • the appropriate formulation The acceptable ingredients of the composition are non-toxic to the recipient at the dose and concentration used.
  • the compositions of the present application include, but are not limited to, liquid, frozen and lyophilized compositions.
  • the composition may comprise parenteral, transdermal, intraluminal, intraarterial, intrathecal, and/or intranasal administration or direct injection into tissues.
  • the composition can be administered to a patient or subject by infusion or injection.
  • the administration of the composition can be carried out in different ways, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the composition can be administered without interruption.
  • the uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the therapeutic agent flowing into the patient's body, as described in WO2015/036583.
  • the dosage regimen of the composition may be rapid infusion, multiple divided doses may be administered over time, or the dosage may be reduced or increased in proportion to the criticality of the treatment situation.
  • the treatment regimen may be once a week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months, or once every 3-6 months.
  • the dosing regimen includes intravenous administration, and the dosage range of administration may be 0.1 ⁇ 10 8 to 3 ⁇ 10 8 CAR-positive T cells, for example, 0.15 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells, 0.5 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells, 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells, 0.2 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells, 0.2 ⁇ 10 8 to 1 ⁇ 10 8 CAR-positive T cells, 0.25 ⁇ 10 8 to 1 ⁇ 10 8 CAR-positive T cells, 0.25 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive T cells, or 0.5 ⁇ 10 8 CAR-positive T cells, or 2 ⁇ 10 8 CAR Positive T cells.
  • the dosage can be different.
  • the dose of the immune effector cells for the treatment of relapsed and refractory adult acute lymphoblastic leukemia patients may be 0.25 ⁇ 10 8 to 0.5 ⁇ 10 8 CAR-positive T cells, or 0.5 ⁇ 10 8 CAR-positive T cells, for example, can be 0.3 ⁇ 10 8 to 0.5 ⁇ 10 8 , 0.4 ⁇ 10 8 to 0.5 ⁇ 10 8 , 0.25 ⁇ 10 8 to 0.4 ⁇ 10 8 , 0.3 ⁇ 10 8 to 0.4 ⁇ 10 8 or 0.4 ⁇ 10 8 To 0.5 ⁇ 10 8 CAR-positive T cells.
  • the dose of the immune effector cells for the treatment of relapsed and refractory adult acute lymphoblastic leukemia patients may be 0.25 ⁇ 10 8 , 0.26 ⁇ 10 8 , 0.27 ⁇ 10 8 , 0.28 ⁇ 10 8 , 0.29 ⁇ 10 8 , 0.3 ⁇ 10 8 , 0.31 ⁇ 10 8 , 0.32 ⁇ 10 8 , 0.33 ⁇ 10 8 , 0.34 ⁇ 10 8 , 0.35 ⁇ 10 8 , 0.36 ⁇ 10 8 , 0.37 ⁇ 10 8 , 0.38 ⁇ 10 8 , 0.39 ⁇ 10 8 , 0.4 ⁇ 10 8 , 0.41 ⁇ 10 8 , 0.42 ⁇ 10 8 , 0.43 ⁇ 10 8 , 0.44 ⁇ 10 8 , 0.45 ⁇ 10 8 , 0.46 ⁇ 10 8 , 0.47 ⁇ 10 8 , 0.48 ⁇ 10 8 , 0.49 ⁇ 10 8 or 0.5 ⁇ 10 8 CAR-positive T cells.
  • the dose of the immune effector cells for the treatment of relapsed and refractory childhood acute lymphoblastic leukemia patients may be 0.25 ⁇ 108 to 0.5 ⁇ 108 CAR-positive T cells, or 0.5 ⁇ 108 CAR-positive T cells, for example, It is 0.3 ⁇ 108 to 0.5 ⁇ 108, 0.4 ⁇ 108 to 0.5 ⁇ 108, 0.25 ⁇ 108 to 0.4 ⁇ 108, 0.3 ⁇ 108 to 0.4 ⁇ 108, or 0.4 ⁇ 108 to 0.5 ⁇ 108 CAR-positive T cells.
  • the dosage of the immune effector cells for treating relapsed and refractory childhood acute lymphoblastic leukemia patients may be 0.25 ⁇ 10 8 , 0.26 ⁇ 10 8 , 0.27 ⁇ 10 8 , 0.28 ⁇ 10 8 , 0.29 ⁇ 10 8 , 0.3 ⁇ 10 8 , 0.31 ⁇ 10 8 , 0.32 ⁇ 10 8 , 0.33 ⁇ 10 8 , 0.34 ⁇ 10 8 , 0.35 ⁇ 10 8 , 0.36 ⁇ 10 8 , 0.37 ⁇ 10 8 , 0.38 ⁇ 10 8 , 0.39 ⁇ 10 8 , 0.4 ⁇ 10 8 , 0.41 ⁇ 10 8 , 0.42 ⁇ 10 8 , 0.43 ⁇ 10 8 , 0.44 ⁇ 10 8 , 0.45 ⁇ 10 8 , 0.46 ⁇ 10 8 , 0.47 ⁇ 10 8 , 0.48 ⁇ 10 8 , 0.49 ⁇ 10 8 or 0.5 ⁇ 10 8 CAR-positive T cells.
  • the dose of the immune effector cells for treating patients with relapsed and refractory non-Hodgkin’s lymphoma may be 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells, or 2 ⁇ 10 8 CAR Positive T cells, such as 1 ⁇ 10 8 to 1.8 ⁇ 10 8 , 1 ⁇ 10 8 to 1.5 ⁇ 10 8 , 1 ⁇ 10 8 to 1.3 ⁇ 10 8 , 1.3 ⁇ 10 8 to 2 ⁇ 10 8 , 1.3 ⁇ 10 8 to 1.5 ⁇ 10 8 , 1.5 ⁇ 10 8 to 2 ⁇ 10 8 , 1.5 ⁇ 10 8 to 1.8 ⁇ 10 8 or 1.8 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells.
  • the dose of the immune effector cells for treating patients with relapsed and refractory Hodgkin’s lymphoma may be 1 ⁇ 10 8 , 1.1 ⁇ 10 8 , 1.2 ⁇ 10 8 , 1.3 ⁇ 10 8 , 1.4 ⁇ 10 8 , 1.5 ⁇ 10 8 , 1.6 ⁇ 10 8 , 1.7 ⁇ 10 8 , 1.8 ⁇ 10 8 , 1.9 ⁇ 10 8 or 2.0 ⁇ 10 8 CAR-positive T cells.
  • this application also provides a method for preparing immune effector cells, which includes the following steps: transducing the vector described in this application into the immune effector cells.
  • the immune effector cells may be selected from the following group: T lymphocytes and natural killer cells.
  • the application also provides the use of the chimeric antigen receptor, the nucleic acid molecule, the carrier and/or the immune effector cell described in the application for preparing a medicine, wherein the medicine For the treatment of diseases or disorders related to the expression of CD19.
  • the dosage of the drug may refer to the dosage of the immune effector cells mentioned above.
  • the present application also provides a method for treating diseases or disorders related to the expression of CD19, the method comprising administering the chimeric antigen receptor and the nucleic acid molecule described in the present application to a subject in need , The carrier and/or the immune effector cell.
  • the administration can be carried out in different ways, such as intravenous, intratumor, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.
  • the chimeric antigen receptor, the nucleic acid molecule, the vector and/or the immune effector cell described in this application can be used to treat diseases or disorders related to the expression of CD19.
  • the drug may include a T cell immunotherapeutic agent.
  • the diseases or disorders related to the expression of CD19 may include non-solid tumors.
  • the disease or disorder related to the expression of CD19 may include leukemia and/or lymphoma.
  • the disease or condition associated with the expression of CD19 may include acute lymphoblastic leukemia (ALL), for example, it may include adult acute lymphoblastic leukemia (ALL) and/or childhood acute lymphocytic leukemia (ALL) .
  • the disease or condition associated with the expression of CD19 may include adult chronic lymphocytic leukemia (CLL).
  • the disease or disorder related to the expression of CD19 may include B-cell lymphoma.
  • the B-cell lymphoma may include non-Hodgkin's lymphoma.
  • the subject may include humans and non-human animals.
  • the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.
  • the following examples are only to illustrate the chimeric antigen receptor, immune effector cells, preparation methods and uses, etc. of the present application, and are not used to limit the scope of the present application.
  • the examples do not include detailed descriptions of traditional methods, such as those used to construct vectors and plasmids, methods of inserting genes encoding proteins into such vectors and plasmids, or methods of introducing plasmids into host cells.
  • Such methods are well known to those of ordinary skill in the art and are described in many publications, including Sambrook, J., Fritsch, EF and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual , 2nd edition, Cold spring Harbor Laboratory Press.
  • the infection experiment was carried out according to a conventional method known to those skilled in the art. A brief description of the infection steps are as follows:
  • PBMC Peripheral blood mononuclear cells
  • lymphocyte culture medium Xvivo15 medium+5% FBS+100IU/ml IL-2 or Xvivo15 medium+5% FBS+20ng/ml IL-21+10ng/ml IL-7) to separate the T cells. Resuspend to make the final concentration (1 ⁇ 2) ⁇ 10 6 cells/ml, and add 5 ⁇ 10 ⁇ l CD3/CD28 stimulating magnetic beads. After mixing, place in an incubator and culture at 37°C+5%. CO 2 , the incubation time is at least 24 hours.
  • polybrene polybrene
  • MOI metal-oxide-semiconductor
  • the infected cells were taken out, and the cell density was monitored to maintain the cells at (0.5 ⁇ 1) ⁇ 10 6 cells/ml for use in subsequent examples.
  • the obtained infected T cells were named CNCT19 cells (ie, CNCT19 cells in this application).
  • FITC-CD3 manufactured by Tongsheng Times, article number: Z6410047-100T
  • Figure 1A is a blank control tube.
  • the CD3+CAR+ quadrant does not show cell populations in the upper right corner.
  • Figure 1B is an experimental detection tube.
  • CART cells are treated with IgG F(ab') 2 and CD3 antibody labeling, by flow cytometry, the CAR expression rate can be clearly detected in the CD3 + CAR + quadrant, 68.6%.
  • the results indicate that the CAR molecule of the present application has a good expression effect on the surface of CNCT19 cells.
  • the tumor cells are respectively selected from the CD19-positive human leukemia cell line Nalm-6 (purchased from Shanghai Meiyan Biotechnology Co., Ltd., catalog number: CH179) and the CD19-negative human leukemia cell line KG-1a (purchased from Shanghai Enzyme Research Biotechnology Co., Ltd., catalog number: CC-Y1305).
  • the effector cells are CD19CAR-T cells (ie CNCT19 cells obtained in Example 2) and untransfected T cells (indicated by NTD);
  • the above-mentioned target cells and effector cells were mixed and seeded in a 24-well plate.
  • the total number of cells co-cultured in each well is about 1 ⁇ 10 6 /well, and 3 parallel wells are set for each condition. Replenish each well with 1 mL of culture solution, place it in a 37°C, 5% CO 2 incubator, and record the time;
  • the residual rate of target cells after CNCT19 cells and Nalm-6 cells are incubated together is (1.1 ⁇ 0.1)%, which is significantly lower than that of untransfected T cells and Nalm-6 cells
  • the residual rate of target cells after co-incubation was (7.3 ⁇ 1.2)% (P ⁇ 0.01).
  • the killing effect of CNCT19 cells on CD19-positive tumor cells increased with the increase of the effective target ratio.
  • CHO-CD19 Take out the target cell CHO-CD19, aspirate and discard the culture medium in the culture flask, wash once with physiological saline solution, add 1ml trypsin solution containing EDTA, incubate in a 37°C incubator for 2-6 minutes and then terminate the digestion.
  • CHO cells were purchased from Shanghai Enzyme Research Biotechnology Co., Ltd., catalog number: CC-Y2110; the molecular sequence of CD19 cells was derived from NCBI, and the molecular sequence of CD19 was constructed into CHO cells by molecular biology methods. Obtain the target cell CHO-CD19 cell line.
  • E-Plate 16 Take out E-Plate 16, add target cell suspension and CNCT19 cell suspension to some wells, and add target cell suspension and untransfected T cell (ie NTD) suspension (as a control) to other wells.
  • the effective target ratio that is, the ratio of effector cells to target cells, that is CNCT19 cells: target cells; NTD: target cells
  • the volume of the target cell suspension is 50 ⁇ l
  • the E-Plate 16 detection plate is placed Real-time monitoring on the RTCA DP test bench for 60 hours to observe the effect of CNCT19 cells on target cells.
  • Line 1 is the curve of the NTD control group
  • line 2 is the curve of the CNCT19 treatment group. Comparing line 1 and line 2, it can be seen that the proliferation of tumor cells (ie target cells) is inhibited by CNCT19 cells over time. Specifically, after co-incubation with untransfected T cells, the growth trend of target cells did not change significantly (line 1), while after co-incubation with CNCT19 cells, the growth trend of target cells decreased significantly, and even the number began to decrease (line 2 ), indicating that CNCT19 has strong killing ability on target cells.
  • Example 4 1) Take out the mixed culture supernatant sample of each well in Example 4 (ie the sample obtained in step 3) from the refrigerator at -20°C and melt at room temperature;
  • CNCT19 cells are added at a concentration of 1 ⁇ 10 7 cells/mL (calculated as total T cells) in different doses (0.5 ⁇ 0.1mL) to 1 ⁇ 2 of the existing sodium chloride injection at different doses (2.0 ⁇ 2.4mL)
  • 2.5 mL of sodium chloride injection (negative control) and 2.5 mL of sterile water for injection (positive control) were added to tube 6 and tube 7, respectively.
  • each test tube is 5.0mL. Place it in a 37°C ⁇ 0.5°C incubator for 3 hours. After putting it in the incubator, observe the red blood cells for 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours and 3 hours. Whether there is dissolution or aggregation.
  • Figure 5A shows the observation results of each test tube before shaking for 3 hours
  • Figure 5B shows the observation result of each tube after shaking for 3 hours.
  • the negative control tube No. 6
  • the upper layer of the test tube is colorless and clear, and the red blood cells at the bottom of the tube sink. After shaking, the red blood cells are evenly dispersed. It is judged that there is no hemolysis and no aggregation; the positive control tube (No. 7) shows a clear red solution, no layering, and no red blood cell residue at the bottom of the tube. For complete hemolysis.
  • the concentration of CAR-T cells infused through the right ear vein is 1 ⁇ 10 7 cells/mL, and the dose is 1 ⁇ 10 7 cells/kg (calculated as the total number of T cells); chlorination is injected through the left ear vein Sodium injection was used as a negative control, and the dose of both sides of the ear vein was 1 mL/kg.
  • Figure 6A shows a micrograph of the administration part after the administration of CAR-T cells (HE staining, 10x objective lens), and Fig. 6B shows a micrograph of the administration part after the administration of sodium chloride injection. (HE staining, 10x objective lens), it can be seen that after intravenous infusion of cryopreserved CAR-T cells, compared with the negative control side, no systemic or local symptoms and pathological abnormalities of the animal were found.
  • HE staining, 10x objective lens it can be seen that after intravenous infusion of cryopreserved CAR-T cells, compared with the negative control side, no systemic or local symptoms and pathological abnormalities of the animal were found.
  • Cultivate CAR-T cells from two donors (CNCT19 cells) in soft agar medium.
  • the two donors are donor 1 (healthy human donor T cells, lot number: TC20180613015) and donor 2.
  • donor 1 healthy human donor T cells, lot number: TC20180613015
  • donor 2. T cells from healthy human donors, batch number: TC20180613016
  • human embryonic lung fibroblast cell line MRC-5 was set as a negative control
  • human cervical cancer cell line Hela was set as a positive control.
  • the experiment uses a 6-well culture plate, the number of cells in each well is about 1 ⁇ 10 3 , and each group has 3 parallel holes, which are cultured in a CO 2 incubator and observed for 3 weeks.
  • Cryopreserved CAR-T cells (CNCT19 cells) and corresponding untransfected T cells (NTD) from two donors were subcutaneously inoculated with BALB/c nude mice. The two donors were donors. Body 2 (healthy human donor T cells, lot number: TC20180613016) and donor 3 (healthy human donor T cells, lot number: TC20180808019).
  • the inoculation amount of CAR-T cells is 1 ⁇ 10 7 /mouse (calculated as the total number of T cells).
  • the negative control group was inoculated with 1 ⁇ 10 7 cells of human embryo lung fibroblast cell line MRC-5, and the positive control group was inoculated with 1 ⁇ 10 6 cells of human cervical cancer cell line Hela.
  • CAR-T cells are not tumorigenic in vivo.
  • negative control group inoculated with MRC-5 cells
  • positive control group inoculated with HeLa cells
  • the subcutaneous nodules of all animals increased slowly.
  • Pathological examination showed that the nodules were caused by the growth of tumor tissue, and the tumor formation rate reached 100%. In summary, this test was effective.
  • the two donor-derived CAR-T cells and untransfected T cell inoculation group all the liquid nodules disappeared on the 5th day after inoculation, and no nodules grew again when they were euthanized on the 114th day.
  • Pathological examination It showed that there was no tumor formation at the site of inoculation or metastasis.
  • Groups 2 to 5 are vehicle control group, T cell control group, and CAR-T cell low and high dose groups. There are 40 animals in each group, half male and half female. Both the non-tumor-bearing control group (ie the first group) and the vehicle control group (ie the second group) were given a vehicle reference substance (ie, normal saline, which contains 4% (W/V) of human albumin).
  • the T cell control group ie the third group was given untransfected T cells (ie NTD) at a dose of 1 ⁇ 10 9 cells/kg (based on the total number of T cells, the same below).
  • the doses of CAR-T cell low and high dose groups were 1 ⁇ 10 8 and 1 ⁇ 10 9 cells/kg, respectively.
  • the dosage for all animals is 25mL/kg
  • the administration method is a single intravenous injection
  • the administration rate is about 1mL/min.
  • CAR-T cells ie CNCT19 cells
  • the maximum tolerated dose of CAR-T cells is greater than 1 ⁇ 10 9 cells/kg.
  • no animals in the CAR-T cell low and high dose groups were dead or dying, and there were no abnormal reactions in general and detailed clinical observations.
  • Histopathological examination revealed that there was no obvious abnormality in the CAR-T cell low and high dose group compared with the T cell control group.
  • CAR-T low-dose group injected CNCT19 cells, 5 ⁇ 10 6 cells/mouse, based on the total number of T cells, the same below
  • CAR-T medium-dose group injected CNCT19 cells, 1 ⁇ 10 7 cells/unit
  • CAR-T high-dose group injection of CNCT19 cells, 2 ⁇ 10 7 cells/unit
  • T cell control group injection of untransfected T cells, 2 ⁇ 10 7 cells/unit
  • vehicle Control group injection of cell preservation solution, 200 ⁇ l/mouse.
  • the injection volume for each mouse is 200 ⁇ l.
  • each dose group of CNCT19 cells has a significant prolongation effect on survival, and the dose-dependent relationship is obvious. Specifically, the median survival time: the normal saline control group was 24 days, the NTD control group was 23 days, the CNCT19 low-dose group was 40 days, and the CNCT19 medium and high-dose groups all survived to the end of the observation. Compared with the normal saline and NTD control group, each dose group of CNCT19 can prolong the survival time of leukemia animals by more than 16 days.
  • Example 11.1 Use NCG mice aged 6-8 weeks, half male and half male.
  • the method in Example 11.1 was used to establish a Nalm-6 xenograft tumor model.
  • Animals in the tumor-bearing group were administered 24 hours (i.e. D2), 72 hours (i.e. D4), 168 hours (i.e. D8), 336 hours (i.e. D15), 504 hours (i.e. D22), 672 hours (i.e. D29). Animals in the non-tumor-bearing group were euthanized as planned at 24 hours (ie D2), 168 hours (ie D8), and 336 hours (ie D15) after administration. The whole blood (EDTA anticoagulation) and brain of the animals were collected in sequence.
  • CNCT19 cells were mainly distributed in whole blood and tissues with large blood flow such as lung, liver, heart and spleen after a single intravenous injection of 5 ⁇ 10 6 cells/mouse to tumor-bearing mice and non-tumor-bearing mice (See Figure 9).
  • the heart and whole blood are the most distributed, the area under the drug-time curve is about 15,000 hours*copy number/ ⁇ g, followed by the lungs and spinal cord, and the area under the drug-time curve is about 40,000-60,000 hours*
  • the area under the drug-time curve of spleen, liver and other tissues is less than 20,000 hours*copy number/ ⁇ g.
  • CNCT19 cells in the mouse proliferate reactively (see Figure 11).
  • the concentration of CNCT19 cells in the brain, lung, liver, whole blood, and spinal cord increases to 1200. Copy number/microgram DNA or more, even reaching 3500 copies/microgram DNA.
  • Example 12 The treatment of acute lymphoblastic leukemia by CNCT19 cells
  • Fludarabine 30mg/m 2 , 1 time/day, continuous use for 2-4 days;
  • Cyclophosphamide 500mg/m 2 , 1 time/day, for 2 consecutive days.
  • the two drugs of pretreatment chemotherapy should be started on the same day.
  • Cell reinfusion method resuscitate the cells according to the test operation guide, and the cell suspension should be reinfused within 30 minutes after resuscitation.
  • a single infusion is infused into the subject through a blood transfusion device through a vein. If there is more than one bag of cell suspension, there can be no time interval between each bag of reinfusion and continuous infusion. Subjects need to be closely observed during the cell reinfusion. If a serious adverse event occurs, the infusion should be stopped, and the specific situation of the adverse event should be dealt with accordingly. If there are no serious adverse events, follow-up follow-up procedures can be followed;
  • the subject will continue to be hospitalized for observation for 14 days or the investigator's comprehensive assessment of the subject's condition will determine the hospitalization observation time.
  • CNCT19 cell suspension management system In order to strictly manage and use CNCT19 cell suspension, a strict CNCT19 cell suspension management system has been established. Special personnel will transport the research cell suspension to the hospital department, and the designated personnel will be responsible for the reception of the research cell suspension and the establishment of a registration system.
  • the packaging of the cell suspension will be recycled and stored/destroyed by the drug management personnel.
  • Cri Complete Remission with incomplete blood count recovery: The morphology is completely relieved but the blood cell count is not completely recovered
  • Table 4 shows the safety results of 63 patients including adult and pediatric patients
  • Table 5 shows the safety results of 23 adult patients.
  • the incidence of grade 3 and above CRS and encephalopathy were 19% and 20.6%, respectively.
  • the incidence of severe CRES in the adult group is slightly lower or close to that in the children group, with 17.4% in adults , Children are 22.6%. It may be due to the fact that CNCT19 was given according to body weight in the early trials, and the adult body was heavy and the dose was higher.
  • the dose range was 0.71 ⁇ 10 6 -4.08 ⁇ 10 6 /kg, which is equivalent to 0.16 ⁇ 10 8 -2.36 ⁇ 10 8 CAR-positive T cells.
  • a safer dose range was selected.
  • the dose range of 17 cases of cells was 0.2 ⁇ 10 8 -1.1 ⁇ 10 8 CAR-positive T cells (medium In patients with a bit value of 0.5 ⁇ 10 8 ), the incidence of CRS and CRES of grade 3 and above dropped to 5.9% (1/17).
  • CNCT19 has a relatively low probability of causing severe CRS or CRES side effects, and the overall safety is controllable, so it has good safety performance. It shows that the good safety performance of CNCT19 improves product quality and reduces clinical risks.
  • CRS Cytokine Release Syndrome
  • CRES CAR-T-cell-related Encephalopathy Syndrome
  • Example 13 The treatment of CNCT19 cells for relapsed or refractory non-Hodgkin's lymphoma
  • Fludarabine 30mg/m 2 , 1 time/day, continuous use for 2-4 days;
  • Cyclophosphamide 500mg/m 2 , 1 time/day, for 2 consecutive days.
  • the two drugs of pretreatment chemotherapy should be started on the same day.
  • the subject will continue to be hospitalized for observation for 14 days or based on the researcher's comprehensive assessment of the subject's condition to determine the hospitalization observation time.
  • CNCT19 cell suspension management system In order to strictly manage and use CNCT19 cell suspension, a strict CNCT19 cell suspension management system has been established. Special personnel will transport the research cell suspension to the hospital department, and the designated personnel will be responsible for the reception of the research cell suspension and the establishment of a registration system.
  • the packaging of the cell suspension will be recycled and stored/destroyed by the drug management personnel.
  • CNCT19 cell suspension was used to treat 50 patients with relapsed or refractory non-Hodgkin's lymphoma (NHL). Among them, it is used for NHL (adults only) to explore the range of administration: 1 ⁇ 10 8 to 2 ⁇ 10 8 CAR-positive T cells.
  • NHL refractory non-Hodgkin's lymphoma
  • Table 7 shows the recovery of 50 patients. It can be seen that among the 50 patients with relapsed and refractory non-Hodgkin’s lymphoma, about 80% of the patients achieved complete or partial remission after the injection of the CNCT19 cell suspension. The complete remission rate was 54% (27 cases), and the partial remission rate was 24% (12 cases). It shows that CNCT19 cell suspension can effectively treat patients with relapsed or refractory non-Hodgkin's lymphoma.
  • Table 8 shows the safety results of 50 patients. It can be seen that the CNCT19 cell suspension injection has a low probability of causing severe CRS or CRES side effects, and the probability of side effects above grade 3 is less than 10%. Among them, the probability of causing CRS above grade 3 is 0%. The probability of CRES above level is 6%, so it has good safety performance.
  • CRS Cytokine Release Syndrome
  • CRES CAR-T-cell-related Encephalopathy Syndrome

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Abstract

一种嵌合抗原受体，其包含SEQ ID NO.1所示的氨基酸序列。一种编码所述嵌合抗原受体的核酸、包含所述核酸的载体、包含所述嵌合抗原受体、所述核酸分子和/或所述载体的免疫效应细胞、制备所述免疫效应细胞的方法、包含所述免疫效应细胞的组合物以及所述嵌合抗原受体的用途。

Description

一种靶向CD19的嵌合抗原受体及其用途 技术领域
本申请涉及生物医药领域，具体的涉及一种靶向CD19的嵌合抗原受体及其用途。
背景技术
急性淋巴细胞白血病(ALL)、慢性淋巴细胞白血病(CLL)以及B细胞淋巴瘤的临床治疗，目前以化疗、干细胞移植及生物治疗等为主，虽然可以取得一定的疗效，但复发难治仍然是难以攻克的重点，肿瘤的细胞免疫治疗作为一种新的治疗策略，成为当今研究的热点。CD19几乎表达于所有的B细胞肿瘤细胞表面，而其他实质细胞和造血干细胞几乎不表达。
肿瘤细胞通过下调参与T细胞识别及抗原反应的分子表达、降低免疫原性等途径产生免疫逃逸，从而使机体的免疫系统不能很好的清除肿瘤。研究发现，嵌合抗原受体T细胞(CAR-T细胞)能够识别肿瘤细胞表面的抗原，特异性杀伤肿瘤细胞，用于肿瘤的治疗。
发明内容
本申请提供了一种嵌合抗原受体，所述嵌合抗原受体包含SEQ ID NO.1所示的氨基酸序列，本申请还提供编码所述嵌合抗原受体的核酸、包含所述核酸的载体、包含所述嵌合抗原受体、所述核酸分子和/或所述载体的免疫效应细胞、制备所述免疫效应细胞的方法、包含所述免疫效应细胞的组合物以及所述嵌合抗原受体的用途。
本申请所述的嵌合抗原受体至少包含以下一种有益效果：(1)在免疫细胞(例如，T细胞)表面稳定表达，表达量高；(2)具有较强的杀伤CD19阳性靶细胞的能力；(3)促进免疫细胞分泌细胞因子，例如，使T细胞分泌细胞因子(INF-γ或IL-6)的能力增强至少1倍、2倍或3倍以上；(4)无溶血性，不引起红细胞溶血或凝聚；(5)无血管刺激性，给药后不引起给药局部或全身异常；(6)无致瘤性，体内或体外不致瘤；(7)延长肿瘤患者的生存时间；(8)有效缓解肿瘤(例如，成人急性淋巴细胞白血病、儿童急性淋巴细胞白血病和/或非霍奇金淋巴瘤)病情；和，(9)安全性高，引起副作用(例如，细胞因子释放综合征或CAR-T细胞相关性脑病综合征)的风险较低。
一方面，本申请提供一种嵌合抗原受体，其包含SEQ ID NO.1所示的氨基酸序列。
另一方面，本申请还提供一种分离的核酸分子，其编码本申请所述的嵌合抗原受体。
另一方面，本申请还提供编码嵌合抗原受体的分离的核酸分子，其包含SEQ ID NO.2所述的核酸序列。
另一方面，本申请还提供一种载体，其包含本申请所述的核酸分子。
另一方面，本申请还提供一种免疫效应细胞，其包含本申请所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。
在某些实施方式中，所述免疫效应细胞选自以下组：T淋巴细胞和自然杀伤细胞。
在某些实施方式中，所述免疫效应细胞的表面表达本申请所述的嵌合抗原受体。
另一方面，本申请还提供制备免疫效应细胞的方法，其包括以下的步骤：向免疫效应细胞中转导本申请所述的载体。
在某些实施方式中，所述免疫效应细胞选自以下组：T淋巴细胞和自然杀伤细胞。
另一方面，本申请还提供一种组合物，其包含本申请所述的免疫效应细胞。
另一方面，本申请还提供所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与CD19的表达相关的疾病或病症。
另一方面，本申请还提供治疗与CD19的表达相关的疾病或病症的方法，所述方法包括施用所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞。
另一方面，本申请还提供所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞，其用于治疗与CD19的表达相关的疾病或病症。
在某些实施方式中，所述与CD19的表达相关的疾病或病症包括非实体瘤。
在某些实施方式中，所述非实体瘤包括白血病和/或淋巴瘤。
在某些实施方式中，所述与CD19的表达相关的疾病或病症包括急性淋巴细胞白血病和/或B细胞淋巴瘤。
在某些实施方式中，所述急性淋巴细胞白血病包括成人急性淋巴细胞白血病和/或儿童急性淋巴细胞白血病。
在某些实施方式中，用于治疗急性淋巴细胞白血病的所述药物的剂量为0.25×10 8至0.5×10 8CAR阳性T细胞。
在某些实施方式中，所述B细胞淋巴瘤包括非霍奇金淋巴瘤。
在某些实施方式中，用于治疗非霍奇金淋巴瘤的所述药物的剂量为1×10 8至2×10 8CAR阳性T细胞。
本领域技术人员能够从下文的详细描述中容易地洞察到本申请的其它方面和优势。下文的详细描述中仅显示和描述了本申请的示例性实施方式。如本领域技术人员将认识到的，本申请的内容使得本领域技术人员能够对所公开的具体实施方式进行改动而不脱离本申请所涉 及发明的精神和范围。相应地，本申请的附图和说明书中的描述仅仅是示例性的，而非为限制性的。
附图说明
本申请所涉及的发明的具体特征如所附权利要求书所显示。通过参考下文中详细描述的示例性实施方式和附图能够更好地理解本申请所涉及发明的特点和优势。对附图简要说明书如下：
图1A和1B显示CAR分子在CNCT19细胞表面表达的检测结果。
图2显示不同共培养条件下的肿瘤细胞残留率。
图3显示RTCA DP系统实时监测CNCT19细胞对靶细胞(CHO-CD19)的杀伤情况。
图4A显示不同共培养条件上清中INF-γ浓度的变化。
图4B显示不同共培养条件上清中IL-6浓度的变化。
图5A显示各试管3小时振摇前的观察结果。
图5B显示各试管3小时振摇后的观察结果。
图6A显示给予CAR-T细胞后给药局部的显微镜照片(HE染色，10倍物镜)。
图6B显示给予氯化钠注射液后给药局部的显微镜照片(HE染色，10倍物镜)。
图7显示各组细胞接种3周后的软琼脂克隆形成情况。
图8为不同细胞治疗Nalm-6移植瘤NCG小鼠的生存曲线。
图9显示CNCT19细胞单次给药后的组织分布情况。
图10为CNCT19细胞在荷瘤与未荷瘤动物体内分布的比较。
图11显示CNCT19细胞单次给药后在动物体内各组织分布的变化。
具体实施方式
以下由特定的具体实施例说明本申请发明的实施方式，熟悉此技术的人士可由本说明书所公开的内容容易地了解本申请发明的其他优点及效果。
以下对本申请做进一步描述：在本发明中，除非另有说明，否则本文中使用的科学和技术名词具有本领域技术人员所通常理解的含义。并且，本文中所用的蛋白质和核酸化学、分子生物学、细胞和组织培养、微生物学、免疫学相关术语和实验室操作步骤均为相应领域内广泛使用的术语和常规步骤。同时，为了更好地理解本发明，下面提供相关术语的定义和解释。
在本申请中，术语“嵌合抗原受体”(Chimeric Antigen Receptor,CAR)通常是指包含能 够结合抗原的胞外结构域和至少一个胞内结构域的融合蛋白。CAR是嵌合抗原受体T细胞(CAR-T)的核心部件，其可包括靶向部分(例如，结合肿瘤相关抗原(tumor-associatedantigen,TAA)的部分)、铰链区、跨膜区和细胞内结构域。在本申请中，所述CAR可以基于抗体的抗原特异性与T细胞受体活化胞内结构域组合在一起。表达CAR的T细胞可以特异地识别和消除表达靶抗原的恶性细胞。
在本申请中，术语“分离的”通常指从天然状态下经人工手段获得的。如果自然界中出现某一种“分离”的物质或成分，那么可能是其所处的天然环境发生了改变，或从天然环境下分离出该物质，或二者情况均有发生。例如，某一活体动物体内天然存在某种未被分离的多聚核苷酸或多肽，而从这种天然状态下分离出来的高纯度的相同的多聚核苷酸或多肽即称之为分离的。术语“分离的”不排除混有人工或合成的物质，也不排除存在不影响物质活性的其它不纯物质。
在本申请中，术语“免疫效应细胞”通常是指参与免疫应答，例如促进免疫效应应答的细胞。在本申请中，免疫效应细胞可以选自以下组：T淋巴细胞和自然杀伤细胞。
在本申请中，术语“特异性结合和/或识别”通常是指可测量的和可再现的相互作用，比如靶标和抗体(或CAR结构片段)之间的结合，可在分子(包括生物分子)的异质群体存在的情况可决定靶标的存在。例如，特异性结合靶标(其可以为表位)的抗体(或CAR结构片段)是以比它结合其它靶标更大的亲和性、亲合力、更容易、和/或以更大的持续时间结合该靶标的抗体(或CAR结构片段)。
在本申请中，术语“分离的核酸分子”通常指任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物。
在本申请中，术语“载体”通常指可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。举例来说，载体包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，载体还可含有复制起始位点。载体还有可能包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
在本申请中，术语“组合物”通常指涉及适合施用于患者的组合物。例如，本申请所述 的组合物，其可以包含本申请所述的免疫效应细胞。此外，所述组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分在所用剂量和浓度下对接受者无毒。本申请的组合物包括但不限于液体、冷冻和冻干组合物。
在本申请中，术语“CD19”通常是指分化抗原簇19蛋白，是白血病前体细胞上可检出的抗原决定簇。可以在公共数据库(如GenBank、UniProt和Swiss-Prot)中找到人和鼠的CD19的氨基酸序列和核酸序列。例如，人CD19的氨基酸序列可以按UniProt/Swiss-Prot登录号P15391找到，编码人CD19的核苷酸序列可以按登录号NM_001178098找到。在本申请中，“CD19”可以包括突变(例如，全长野生型CD19的点突变、片段、插入、缺失和剪接变体)的蛋白质。
在本申请中，术语“受试者”通常指人类或非人类动物，包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
在本申请中，术语“包含”通常是指包括明确指定的特征，但不排除其他要素。
在本申请中，术语“约”通常是指在指定数值以上或以下0.5％-10％的范围内变动，例如在指定数值以上或以下0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％、5％、5.5％、6％、6.5％、7％、7.5％、8％、8.5％、9％、9.5％或10％的范围内变动。
嵌合抗原受体、核酸、载体、免疫效应细胞、组合物
一方面，本申请提供一种嵌合抗原受体，其包含如SEQ ID NO.1所示的氨基酸序列。本申请还提供了包含与SEQ ID NO.1所示的氨基酸序列具有至少80％(如至少80％、至少85％、至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、或至少99％)同一性的氨基酸序列。
在某些实施方式中，本申请所述的嵌合抗原受体可以特异性结合和/或识别肿瘤抗原。例如，本申请所述的嵌合抗原受体可以特异性结合和/或识别抗原CD19。
在某些实施方式中，本申请所述的嵌合抗原受体能够促进免疫效应细胞分泌细胞因子。所述免疫效应细胞可以选自以下组：T淋巴细胞和自然杀伤细胞。所述细胞因子可以选自下组：IFN-γ和IL-6。所述免疫效应细胞可以为哺乳动物的免疫效应细胞。所述T淋巴细胞可以为哺乳动物的T淋巴细胞，所述自然杀伤细胞也可以为哺乳动物的自然杀伤细胞。所述T淋巴细胞可以为人的T淋巴细胞，所述自然杀伤细胞也可以为人的自然杀伤细胞。
在某些实施方式中，本申请所述的嵌合抗原受体无溶血及血管刺激性。
在某些实施方式中，本申请所述的嵌合抗原受体无体外致瘤性。
在某些实施方式中，本申请所述的嵌合抗原受体无体内致瘤性。
在某些实施方式中，本申请所述的嵌合抗原受体可以有效治疗肿瘤。所述肿瘤可以为CD19阳性的肿瘤。例如，本申请所述的嵌合抗原受体可以有效延长CD19阳性肿瘤患者的生存时间。例如，本申请所述的嵌合抗原受体可以有效延长非实体瘤患者的生存时间。例如，本申请所述的嵌合抗原受体可以有效延长淋巴瘤和/或白血病的生存时间。又例如，本申请所述的嵌合抗原受体可以有效延长成人急性淋巴细胞白血病患者的生存时间。又例如，本申请所述的嵌合抗原受体可以有效延长儿童急性淋巴细胞白血病患者的生存时间。又例如，本申请所述的嵌合抗原受体可以有效延长B细胞淋巴瘤(例如，非霍奇金淋巴瘤)患者的生存时间。
在某些实施方式中，本申请所述的嵌合抗原受体可以有效治疗成人急性淋巴细胞白血病。
在某些实施方式中，本申请所述的嵌合抗原受体可以有效治疗儿童急性淋巴细胞白血病。
在某些实施方式中，本申请所述的嵌合抗原受体可以有效治疗非霍奇金淋巴瘤。
另一方面，本申请提供一种分离的核酸分子，其编码本申请所述的嵌合抗原受体。
另一方面，本申请提供一种编码嵌合抗原受体的分离的核酸分子，其包含SEQ ID NO.2所述的核酸序列。
另一方面，本申请提供一种编码嵌合抗原受体的分离的核酸分子，其包含如SEQ ID NO.2所示的核酸序列类似并且编码所述嵌合抗原受体的核酸分子。
在某些实施方式中，所述跟SEQ ID NO.2的核酸序列类似是指如SEQ ID NO.2所示的核酸序列具有至少70％、75％、80％、85％、90％、95％、96％、97％、98％或99％序列同一性的核酸序列。
在某些实施方式中，所述跟SEQ ID NO.2的核酸序列类似是指由于核酸密码子第三位碱基的摆动性(简并性)而跟SEQ ID NO.2的核酸不同，但是却编码所述嵌合抗原受体的核酸分子。
本申请包括基因和蛋白质的变体(例如，本文所述的如SEQ ID NO.1所述的氨基酸序列的变体，或如SEQ ID NO.2所述的核酸序列的变体)，其保留一种或多种生物活性。蛋白质或多肽的此类变体包括已经或可以使用重组DNA技术进行修饰的蛋白质或多肽，使得该蛋白质或多肽具有改变的或附加的属性，例如，变体赋予蛋白质在血浆中增强的蛋白质稳定性或赋予蛋白质增强的活性。变体可以不同于参照序列，如不同于天然存在的多核苷酸、蛋白质或肽。在核苷酸序列水平，天然存在的变体基因和非天然存在的变体基因与参照基因将典型地至少约50％相同，更典型地至少约70％相同，甚至更典型地至少约80％相同(90％或更高同一性)。在氨基酸序列水平上，天然存在的变异蛋白质和非天然存在的变异蛋白质与参 照蛋白质将典型地至少约70％相同，更典型地至少约80％相同，甚至更典型地至少约90％或者有更高的同一性，但在非保守区域允许有大部分非同一性的区域(例如，相同部分小于70％，诸如小于60％,小于50％或者甚至小于40％)。在其他实施方式中，所述序列与参照序列具有至少60％，70％，75％或更高的同一性(例如，80％，85％，90％，95％，96％，97％，98％，99％或更高的同一性)。对于本领域技术人员而言，在多核苷酸、蛋白或多肽中导入核苷酸和氨基酸的变化的程序是已知的(参见，例如，Sambrook et al.(1989))。
在本申请中，术语“同一性”、“同源性”及其语法变体通常指两个或更多个实体是“对齐”的序列时，它们是相同的。因此，例如，当两个多肽序列是相同的时，它们至少所参照的区域或部分之内具有相同的氨基酸序列。如果两个多核苷酸序列是相同的，则它们至少在所参照的区域或部分之内具有相同的多核苷酸序列。同一性可以是序列的所限定的区(区域或结构域)。同一性的“区”或“区域”是指两个或两个以上所参照的实体的相同的部分。因此，如果两个蛋白质或核酸序列在一个或多个序列区或区域内是相同的，则它们在该区域内有同一性。“对齐”的序列是指多个多核苷酸或蛋白质(氨基酸)序列，相比于参照序列，常含补正缺失或附加的碱基或氨基酸(间隙)。两个序列之间的同一性(同源性)的程度可以使用计算机程序和数学算法确定。计算百分比序列同一性(同源性)的这样的算法一般计算比较区域或区的序列间隙和不匹配。例如，BLAST(例如，BLAST 2.0)搜索算法(参见，例如，Altschul et al.,J.Mol.Biol.215:403(1990),公众可通过NCBI获得)具有示范性的搜索参数如下：不匹配-2，间隙打开5，间隙延伸2。
在本申请中，所述核酸分子可以为任何长度的分离形式的核苷酸、脱氧核糖核苷酸或核糖核苷酸，或从其天然环境分离的或人工合成的类似物，但可以编码本申请所述的嵌合抗原受体。
另一方面，本申请提供一种载体，其包含本申请所述的核酸分子。
在本申请中，所述载体可通过转化、转导或转染宿主细胞，使其携带的遗传物质元件在宿主细胞内表达得以表达。例如，载体可以包括：质粒；噬菌粒；柯斯质粒；人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。又例如，所述载体可以含有多种控制表达的元件，包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外，所述载体还可以含有复制起始位点。此外，所述载体还可以包括有协助其进入细胞的成分，如病毒颗粒、脂质体或蛋白外壳，但不仅仅只有这些物质。
另一方面，本申请提供一种免疫效应细胞，其包含本申请所述的嵌合抗原受体、所述的核酸分子和/或所述的载体。
在本申请中，所述免疫效应细胞可以选自以下组：T淋巴细胞和自然杀伤细胞。在某些实施方式中，所述免疫效应细胞可以为人的免疫效应细胞。例如，所述免疫效应细胞可以为人的T淋巴细胞。又例如，所述免疫效应细胞可以为人的自然杀伤细胞。
在本申请中，所述免疫效应细胞的表面可以表达本申请所述的嵌合抗原受体。
在某些实施方式中，本申请所述的免疫效应细胞可以有效杀伤肿瘤细胞。所述肿瘤细胞可以是CD19阳性细胞。例如，本申请所述的免疫效应细胞可以明显降低CD19阳性人白血病细胞系Nalm-6的残留率。
在某些实施方式中，本申请所述的免疫效应细胞与CD19阳性细胞接触后，能够有效促进细胞因子的分泌。所述细胞因子可以选自下组：IFN-γ和IL-6。例如，本申请所述的免疫效应细胞与CD19阳性人白血病细胞系Nalm-6共培养后，其IFN-γ和IL-6细胞因子的分泌明显增加。
在某些实施方式中，本申请所述的免疫效应细胞无溶血性及血管刺激性。例如，在体外溶血试验中，本申请所述的免疫效应细胞不会导致溶血和血液凝聚。又例如，本申请所述的免疫效应细胞无血管刺激性。
在某些实施方式中，本申请所述的免疫效应细胞无体外致瘤性。
在某些实施方式中，本申请所述的免疫效应细胞无体内致瘤性。
在某些实施方式中，本申请所述的免疫效应细胞可以有效治疗肿瘤。所述肿瘤可以为CD19阳性的肿瘤。例如，本申请所述的免疫效应细胞可以有效延长CD19阳性肿瘤患者的生存时间。又例如，本申请所述的免疫效应细胞可以有效延长成人急性淋巴细胞白血病患者的生存时间。又例如，本申请所述的免疫效应细胞可以有效延长儿童急性淋巴细胞白血病患者的生存时间。又例如，本申请所述的免疫效应细胞可以有效延长B细胞淋巴瘤(例如，非霍奇金淋巴瘤)患者的生存时间。
在某些实施方式中，本申请所述的免疫效应细胞可以有效治疗成人急性淋巴细胞白血病。
在某些实施方式中，本申请所述的免疫效应细胞可以有效治疗儿童急性淋巴细胞白血病。
在某些实施方式中，本申请所述的免疫效应细胞可以有效治疗B细胞淋巴瘤(例如，非霍奇金淋巴瘤)。
另一方面，本申请提供一种组合物，其包含本申请所述的免疫效应细胞。
在本申请中，所述组合物还可以包含一种或多种(药学上有效的)载剂、稳定剂、赋形剂、稀释剂、增溶剂、表面活性剂、乳化剂和/或防腐剂的合适的制剂。组合物的可接受成分 在所用剂量和浓度下对接受者无毒。本申请的组合物包括但不限于液体、冷冻和冻干组合物。
在某些实施方案中，所述组合物可以包含肠胃外、经皮、腔内、动脉内、鞘内和/或鼻内施用或直接注射到组织中。例如，所述组合物可以通过输注或注射施用于患者或者受试者。在另一些实施方案中，所述组合物的施用可以通过不同的方式进行，例如静脉内、腹膜内、皮下、肌肉内、局部或真皮内施用。在其他一些实施方案中，所述组合物可以不间断施用。所述不间断(或连续)施用可以通过患者佩戴的小泵系统来实现，以测量流入患者体内的治疗剂，如WO2015/036583所述。
在本申请中，所述组合物的给药方案可以是施用快速灌注剂，可以随时间推移施用多个分剂量，或者剂量可以随治疗情况的危急程度成比例降低或提高。在某些实施方式中，治疗方案可以是每周施用一次、两周一次、三周一次、四周一次、一个月一次、3个月一次、或3-6个月一次。在某些实施方式中，给药方案包括静脉内施用，施用剂量范围可以是0.1×10 8至3×10 8CAR阳性T细胞，例如，0.15×10 8至2×10 8CAR阳性T细胞、0.5×10 8至2×10 8CAR阳性T细胞、1×10 8至2×10 8CAR阳性T细胞、0.2×10 8至2×10 8CAR阳性T细胞、0.2×10 8至1×10 8CAR阳性T细胞、0.25×10 8至1×10 8CAR阳性T细胞、0.25×10 8至0.5×10 8CAR阳性T细胞，或0.5×10 8CAR阳性T细胞，或2×10 8CAR阳性T细胞。
针对不同的适应症，给药剂量可以不同。在某些实施方式中，所述免疫效应细胞治疗复发难治的成人急性淋巴细胞白血病患者的给药剂量可以为0.25×10 8至0.5×10 8CAR阳性T细胞，或0.5×10 8CAR阳性T细胞，例如可以是0.3×10 8至0.5×10 8、0.4×10 8至0.5×10 8、0.25×10 8至0.4×10 8、0.3×10 8至0.4×10 8或0.4×10 8至0.5×10 8CAR阳性T细胞。在某些实施方式中，所述免疫效应细胞治疗复发难治的成人急性淋巴细胞白血病患者的给药剂量可以为0.25×10 8、0.26×10 8、0.27×10 8、0.28×10 8、0.29×10 8、0.3×10 8、0.31×10 8、0.32×10 8、0.33×10 8、0.34×10 8、0.35×10 8、0.36×10 8、0.37×10 8、0.38×10 8、0.39×10 8、0.4×10 8、0.41×10 8、0.42×10 8、0.43×10 8、0.44×10 8、0.45×10 8、0.46×10 8、0.47×10 8、0.48×10 8、0.49×10 8或0.5×10 8CAR阳性T细胞。
在某些实施方式中，所述免疫效应细胞治疗复发难治的儿童急性淋巴细胞白血病患者的给药剂量可以为0.25×108至0.5×108CAR阳性T细胞，或0.5×108CAR阳性T细胞，例如可以是0.3×108至0.5×108、0.4×108至0.5×108、0.25×108至0.4×108、0.3×108至0.4×108或0.4×108至0.5×108CAR阳性T细胞。在某些实施方式中，所述免疫效应细胞治疗复发难治的儿童急性淋巴细胞白血病患者的给药剂量可以为0.25×10 8、0.26×10 8、0.27×10 8、0.28×10 8、0.29×10 8、0.3×10 8、0.31×10 8、0.32×10 8、0.33×10 8、0.34×10 8、0.35×10 8、0.36×10 8、0.37×10 8、0.38×10 8、0.39×10 8、0.4×10 8、0.41×10 8、0.42×10 8、0.43 ×10 8、0.44×10 8、0.45×10 8、0.46×10 8、0.47×10 8、0.48×10 8、0.49×10 8或0.5×10 8CAR阳性T细胞。
在另一些实施方式中，所述免疫效应细胞治疗复发难治的非霍奇金淋巴瘤患者的给药剂量可以为1×10 8至2×10 8CAR阳性T细胞，或2×10 8CAR阳性T细胞，例如1×10 8至1.8×10 8、1×10 8至1.5×10 8、1×10 8至1.3×10 8、1.3×10 8至2×10 8、1.3×10 8至1.5×10 8、1.5×10 8至2×10 8、1.5×10 8至1.8×10 8或1.8×10 8至2×10 8CAR阳性T细胞。在另一些实施方式中，所述免疫效应细胞治疗复发难治的霍奇金淋巴瘤患者的给药剂量可以为1×10 8、1.1×10 8、1.2×10 8、1.3×10 8、1.4×10 8、1.5×10 8、1.6×10 8、1.7×10 8、1.8×10 8、1.9×10 8或2.0×10 8CAR阳性T细胞。
制备方法和用途
另一方面，本申请还提供了制备免疫效应细胞的方法，其包括以下的步骤：向免疫效应细胞中转导本申请所述的载体。
在某些实施方式中，所述免疫效应细胞可以选自以下组：T淋巴细胞和自然杀伤细胞。
另一方面，本申请还提供了本申请所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与CD19的表达相关的疾病或病症。所述药物的给药剂量可以参考上文提到的所述免疫效应细胞的给药剂量。
另一方面，本申请还提供了治疗与CD19的表达相关的疾病或病症的方法，所述方法包括向有需要的受试者施用本申请所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞。在本申请中，所述施用可以通过不同的方式进行，例如静脉内、瘤内、腹膜内、皮下、肌肉内、局部或真皮内施用。
另一方面，本申请所述的嵌合抗原受体、所述的核酸分子、所述的载体和/或所述的免疫效应细胞，其可以用于治疗与CD19的表达相关的疾病或病症。
在本申请中，所述药物可以包括T细胞免疫治疗剂。
在本申请中，所述与CD19的表达相关的疾病或病症可以包括非实体瘤。
在本申请中，所述与CD19的表达相关的疾病或病症可以包括白血病和/或淋巴瘤。
在某些实施方式中，所述与CD19的表达相关的疾病或病症可以包括急性淋巴细胞白血病(ALL)，例如，可包括成人急性淋巴细胞白血病(ALL)和/或儿童急性淋巴白血病(ALL)。在另一些实施方式中，所述与CD19的表达相关的疾病或病症可以包括成人慢性淋巴细胞白血病(CLL)。在另一些实施方式中，所述与CD19的表达相关的疾病或病症可以包括B细胞淋巴瘤。例如，所述B细胞淋巴瘤可以包括非霍奇金淋巴瘤。
在本申请中，所述受试者可以包括人类和非人类动物。例如，所述受试者可以包括但不限于猫、狗、马、猪、奶牛、羊、兔、小鼠、大鼠或猴。
不欲被任何理论所限，下文中的实施例仅仅是为了阐释本申请的嵌合抗原受体、免疫效应细胞、制备方法和用途等，而不用于限制本申请发明的范围。实施例不包括对传统方法的详细描述，如那些用于构建载体和质粒的方法，将编码蛋白的基因插入到这样的载体和质粒的方法或将质粒引入宿主细胞的方法。这样的方法对于本领域中具有普通技术的人员是众所周知的，并且在许多出版物中都有所描述，包括Sambrook,J.,Fritsch,E.F.and Maniais，T.(1989)Molecular Cloning：A Laboratory Manual，2nd edition，Cold spring Harbor Laboratory Press。
本申请中引用的所有专利、专利申请和参考文献均通过引用的方式全文纳入本申请，纳入程度就如同每一篇文献单独引用作为参考。如果本申请和本文提供的文献之间存在冲突，应以本申请中的内容为准。
实施例
实施例1.慢病毒载体的构建
人工合成包含本申请所述CAR结构的片段(其氨基酸序列和核苷酸序列分别如SEQ ID NO.1和SEQ ID NO.2所示)，并构建到慢病毒载体(厂家：SBI公司，货号：CD500-CD800)，将得到的表达载体命名为CNCT19表达载体，随后依照其说明书记载的方式转染获得慢病毒，并用流式细胞术测定病毒转染滴度，确认得到有功能性的慢病毒载体。
实施例2.慢病毒感染T细胞的制备
感染实验按照本领域技术人员已知的常规方法进行。简述感染步骤如下：
1.分选T细胞
从受试者单采血细胞中分离获得外周血单个核细胞(PBMC)，然后从PBMC细胞中分选获得T细胞。
2.对T细胞进行激活处理
将分离的T细胞用完全淋巴细胞培养液(Xvivo15培养基+5％FBS+100IU/ml IL-2或Xvivo15培养基+5％FBS+20ng/ml IL-21+10ng/ml IL-7)进行重悬，使终浓度为(1～2)×10 6个细胞/ml，并加入5～10μl的CD3/CD28刺激磁珠，混匀后置于培养箱培养，培养条件为37℃+5％CO 2，培养时间至少24小时。
3.慢病毒感染T细胞
取出激活培养的T细胞，加入终浓度为8μg/ml的聚凝胺(polybrene)，混匀，并按MOI＝2缓慢加入慢病毒载体，混匀后将其置于离心机中，1500rpm，离心1.5小时。然后将其置于培养箱培养，培养条件为37℃+5％CO 2，培养时间至少24小时。
4.感染后T细胞的扩增培养
取出感染后的细胞，监测细胞密度，使细胞维持在(0.5～1)×10 6个细胞/ml，以备后续实施例使用，将获得的感染的T细胞命名为CNCT19细胞(即本申请所述的免疫效应细胞)。
实施例3.CAR分子在CNCT19细胞表面表达的检测
实验步骤如下：
(1)将CNCT19细胞悬液于300g离心5分钟，弃上清，加入鞘液重悬至活细胞密度为0.5～1×10 7细胞/mL。
(2)每份样本取2支流式管，标记为1～2管，第1管为空白对照，不需要加抗体，第2管中加入10倍稀释的Alexa
Figure PCTCN2020136241-appb-000001
647-羊抗鼠IgG F(ab’) 2抗体(厂家：Jackson，货号：115-605-072)10μL。
(3)每管加入100μl细胞悬液，室温避光反应15～20分钟。
(4)每管加入2ml鞘液洗涤，300g离心5分钟。
(5)弃上清，在第2管中加入FITC-CD3(厂家：同生时代，货号：Z6410047-100T)20μl，室温避光孵育15～20分钟。
(6)每管加入2ml鞘液洗涤，300g离心5分钟，弃上清，每管再加入2ml鞘液洗涤，300g离心5分钟。
(7)弃上清，每管加入300μl鞘液重悬后，流式细胞仪检测。
结果如图1A和图1B所示，可以看出，图1A为空白对照管，在右上角CD3+CAR+象限未显示有细胞群，图1B为实验检测管，CART细胞经IgG F(ab’) 2和CD3抗体的标记，通过流式细胞仪，可以在CD3 +CAR +象限明显的检测到CAR表达率为68.6％。结果表示本申请的CAR分子在CNCT19细胞表面表达效果良好。
实施例4.CNCT19细胞对靶细胞的体外杀伤作用检测
本实施例的实验步骤如下：
1)肿瘤细胞(即靶细胞)分别选用CD19阳性人白血病细胞系Nalm-6(购自上海酶研生物科技有限公司，目录号：CH179)及CD19阴性人白血病细胞系KG-1a(购自上海酶研生物科技有限公司，目录号：CC-Y1305)。效应细胞分别为CD19CAR-T细胞(即实施例2获得的 CNCT19细胞)和未转染的T细胞(用NTD表示)；
2)按效靶比1:2和2:1，分别混合上述靶细胞和效应细胞接种于24孔板中。使每孔中共培养的细胞总数约为1×10 6/孔，各条件均设3个平行孔。各孔补充培养液至1mL，置37℃、5％CO 2孵箱培养并记录时间；
3)共培养24小时后，收集各孔细胞悬液移至1.5mL EP管分别标记。此外，离心后吸取各样品管中上清液至新的1.5mL EP管，于-20℃冻存以备后续细胞因子检测使用(详见实施例6)；
4)各孔混合细胞中按肿瘤细胞种类分别加入相应检测量的抗体标记，并按抗体说明书操作。使用PE-CD10抗体标记Nalm-6细胞，使用Percp-cy5.5-CD45抗体标记KG-1a细胞；
5)流式细胞仪检测各样品中不同肿瘤靶细胞的比例变化。
结果如图2所示，可以看出，相比于与未转染的T细胞(即NTD)共培养，CD19阳性肿瘤细胞Nalm-6与CAR-T细胞(即CNCT19细胞)共培养后，其残留率明显降低；而CD19阴性肿瘤细胞KG-1a与不同效应细胞共培养后的残留率没有明显差异。具体来说，在效靶比为1：2时，CNCT19细胞与Nalm-6细胞共同孵育后，靶细胞残留率为(2.8±1.3)，显著低于未转染的T细胞与Nalm-6细胞共同孵育后的靶细胞残留率(12.1±1.2)％(P<0.01)。同样的，在效靶比为2：1时，CNCT19细胞与Nalm-6细胞共同孵育后的靶细胞残留率为(1.1±0.1)％，显著低于未转染的T细胞与Nalm-6细胞共同孵育后的靶细胞残留率(7.3±1.2)％(P<0.01)。且CNCT19细胞对CD19阳性肿瘤细胞的杀伤效果随着效靶比的增加而增强。
实施例5.实时监测CNCT19细胞的杀伤功能
实验步骤如下：
(1)取出靶细胞CHO-CD19，吸弃培养瓶内培养液，以生理盐水液清洗1次，加入1ml含EDTA的胰蛋白酶溶液，置37℃温箱中温育2-6分钟后终止消化。需要说明的是，CHO细胞采购自上海酶研生物科技有限公司，目录号：CC-Y2110；CD19细胞分子序列来源于NCBI，通过分子生物学的方法将CD19分子序列构建到CHO细胞中，经筛选获得所述靶细胞CHO-CD19细胞株。
(2)加入适量培养基至培养瓶，使所述靶细胞形成细胞悬液，并用移液管将细胞吹打均匀。用计数板计数细胞悬液浓度后，配制成实验需要的细胞浓度1×10 5细胞/ml。
(3)在RTCA DP系统的E-Plate 16的孔中加入50μl培养基。将E-Plate 16放到RTCA Station上。RTCA系统会自动进行扫描(“Scan Plate”)，检查是否接触良好(在“Message”页面显示Connection OK)。开始检测基线(Background)确定所选择的孔接触正常。
(4)取出E-Plate 16，在孔中加入100μl混合均匀的靶细胞悬液，使孔中细胞数目为1× 10 4细胞/孔。将E-Plate 16置于超净台中室温放置30分钟后，将E-Plate 16放到培养箱中的RTCA Station上。在系统自动扫描“Scan Plate”后，开始Step2，实时动态检测细胞增殖曲线。
(5)取出E-Plate 16，在一些孔中加入靶细胞悬液和CNCT19细胞悬液，在另一些孔中加入靶细胞悬液和未转染的T细胞(即NTD)悬液(作为对照)，其中效靶比(即效应细胞与靶细胞的比值，即CNCT19细胞∶靶细胞；NTD∶靶细胞)为1:1，靶细胞悬液的体积为50μl，将E-Plate 16检测板置于RTCA DP检测台上实时监测60小时，观察CNCT19细胞对靶细胞的影响。
结果如图3所示，图中线1为NTD对照组曲线，线2为CNCT19处理组曲线。对比线1和线2，可以看出，肿瘤细胞(即靶细胞)随时间的延长，其增殖被CNCT19细胞抑制。具体来说，与未转染的T细胞共同孵育后，靶细胞生长趋势并无明显变化(线1)，而与CNCT19细胞共同孵育后，靶细胞生长趋势明显降低，甚至数量开始减少(线2)，说明CNCT19对靶细胞具有较强的杀伤能力。
实施例6.CNCT19细胞与靶细胞共培养后分泌细胞因子的检测
本实施例的实验步骤如下：
1)将实施例4中的各孔混合培养上清样品(即步骤3获得的样品)从-20℃冰箱取出，室温融化；
2)使用LEGENDplex TM试剂盒(厂家：Biolegned，货号：740013)，按说明书操作处理各样品；
3)流式细胞仪检测各样品中不同细胞因子的水平。
结果如图4A和图4B所示，相比于未转染的T细胞(即NTD)，CAR-T细胞(即CNCT19细胞)与Nalm-6细胞(CD19 +)共培养后，其细胞因子IFN-γ和IL-6的分泌量明显增加。具体来说，与靶细胞以效靶比2:1共培养24小时后，CNCT19细胞在靶细胞的刺激下，分泌INF-γ的量为(6186.37±861.13)pg/ml，显著高于未转染的T细胞的分泌量(2096.85±228.16pg/ml，P<0.05)；CNCT19细胞分泌IL-6的量(32.22±1.46pg/ml)显著高于未转染的T细胞的分泌量(12.23±4.37pg/ml，P<0.05)。
实施例7.CNCT19细胞的溶血及刺激性检测
7.1.CNCT19细胞体外溶血试验
本实施例的实验步骤如下：
1)使用玻璃试管共7个，编为1～7号，各管中加入2.5mL 2％兔红细胞悬液(取自新西兰兔，生产单位：中国食品药品检定研究院，质量合格证编号：No.11400500032425)。
2)CNCT19细胞以1×10 7细胞/mL的浓度(以总T细胞数计)按不同剂量(0.5～0.1mL)加入已有不同剂量(2.0～2.4mL)氯化钠注射液的1～5号管中，同时6号管和7号管分别加入2.5mL氯化钠注射液(阴性对照)和2.5mL灭菌注射用水(阳性对照)。
3)每个试管的总体积为5.0mL，置于37℃±0.5℃恒温箱中温育3小时，放入恒温箱后15分钟、30分钟、45分钟、1小时、2小时和3小时观察红细胞有无溶解或凝聚。
结果如图5A和图5B所示，图5A显示各试管3小时振摇前的观察结果，图5B显示各试管3小时振摇后的观察结果，可以看出，阴性对照管(6号)可见试管上层液体无色澄明，管底红细胞下沉，经振摇后均匀分散，判断为无溶血无凝聚；阳性对照管(7号)可见溶液澄明红色，未见分层，管底无红细胞残留判断为完全溶血。而观察至3小时加入不同剂量CNCT19细胞的各试管均可见试管上层液体为无色澄明，管底红细胞下沉，经振摇后均匀分散，判断为无溶血无凝聚。
7.2.CNCT19细胞静脉输注的血管刺激性试验
本实施例的实验步骤如下：
1)选择检疫合格、注射局部无异常的新西兰兔6只，雌雄各半。采用同体自身对照法，各只动物右、左耳分别静脉输注给予冻存剂型CAR-T细胞(即CNCT19细胞)和阴性对照品(氯化钠注射液)。
2)经右耳缘静脉输注的CAR-T细胞浓度为1×10 7细胞/mL，剂量为1×10 7细胞/kg(按总T细胞数计)；经左耳缘静脉注射氯化钠注射液作为阴性对照，两侧耳缘静脉给药剂量皆为1mL/kg。
3)静脉给药后对动物进行一般观察、给药局部观察及病理学检查。
结果如图6A和图6B所示，图6A显示给予CAR-T细胞后给药局部的显微镜照片(HE染色，10倍物镜)，图6B显示给予氯化钠注射液后给药局部的显微镜照片(HE染色，10倍物镜)，可以看出，冻存剂型CAR-T细胞经静脉输注后，与阴性对照侧比较，未发现动物全身或局部症状及病理学检查异常。
实施例8.CNCT19细胞的体外致瘤性试验
本实施例的实验步骤如下：
1)以软琼脂培养基分别培养两个供体来源的CAR-T细胞(即CNCT19细胞)，两个供体分别是供体1(健康人供者T细胞，批号：TC20180613015)和供体2(健康人供者T细胞，批号：TC20180613016)，另设人胚肺成纤维细胞系MRC-5为阴性对照，人宫颈癌细胞系Hela为阳性对照。
2)试验使用6孔培养板，每孔细胞数约为1×10 3个、每组设3个平行孔，置于CO 2培养箱中培养并观察3周。
3)每隔1周取出培养板，用显微镜观察是否有克隆形成、并拍照，以阳性对照组生长出明显克隆为准。
结果如图7所示，可以看出，阳性对照组细胞(Hela)在培养基中形成克隆，且随着时间的延长，克隆明显增大。而观察至23天，不同供体来源的CAR-T细胞及阴性对照组细胞(MRC-5)均未见克隆形成生长，细胞全部死亡，不具有体外永生化增殖特征。
实施例9.CNCT19细胞的体内成瘤性试验
本实施例的实验步骤如下：
1)两个供体来源的冻存剂型CAR-T细胞(即CNCT19细胞)及相应未转染的T细胞(即NTD)，分别皮下接种BALB/c裸鼠，其中两个供体分别是供体2(健康人供者T细胞，批号:TC20180613016)和供体3(健康人供者T细胞，批号:TC20180808019)。
2)CAR-T细胞接种量为1×10 7个/只(以总T细胞数计)。设阴性对照组接种人胚肺成纤维细胞系MRC-5细胞1×10 7个/只，及阳性对照组接种人宫颈癌细胞系Hela细胞1×10 6个/只。
3)连续观察16周，检测体重变化、是否发生结节，以及结节是否会诱发成肿瘤性结节，并与阴性及阳性细胞组对照比较。
4)观察结束后，进行大体解剖，取各脏器称重并计算脏器系数。对接种部位及可疑症状的组织或器官进行病理组织学检查。
结果发现CAR-T细胞不具有体内成瘤性。阴性对照组(接种MRC-5细胞)至接种后第9天所有动物液性结节均消失。而阳性对照组(接种HeLa细胞)所有动物皮下结节均缓慢增大，病理检查显示为肿瘤组织生长所致结节，成瘤率达100％，综上，本次试验有效。两个供体来源的CAR-T细胞及未转染的T细胞接种组，所有动物接种后第5天液性结节全部消失，至第114天安乐死时，均未再次生长结节，病理检查显示在接种部位或转移部位均未见肿瘤形成。
实施例10.CNCT19细胞单次静脉注射给予Nalm-6移植瘤NCG小鼠的毒性试验
本实施例的实验步骤如下：
1)使用6-8周龄的NCG小鼠，雌雄各半。于给药前三天尾静脉注射Nalm-6细胞，接种浓度为2.5×10 6个/mL的细胞悬液，剂量为10mL/kg。
2)筛选动物分性别区段按照体重随机分为4组(即第2～5组)，第2～5组分别为溶媒对照组、T细胞对照组和CAR-T细胞低、高剂量组，每组40只，雌雄各半。未荷瘤对照组(即第1 组)和溶媒对照组(即第2组)均给予溶媒对照品(即生理盐水，其中含人血白蛋白含量4％(W/V))。T细胞对照组(即第3组)给予未转染的T细胞(即NTD)，剂量为1×10 9cells/kg(以总T细胞数计，下同)。CAR-T细胞低、高剂量组(即第4组和第5组)的剂量分别为1×10 8和1×10 9细胞/kg。
3)所有动物给药剂量均为25mL/kg，给药方式为单次静脉注射，给药速度约为1mL/min。
4)给药当天药后连续观察4小时，试验期间每天上午和下午各进行1次一般临床观察，每周进行1次详细临床观察、体重和摄食量测定，并在试验期间进行体温、临床病理指标(血细胞计数和血生化指标)和免疫学指标(T淋巴细胞亚群、细胞因子和C反应蛋白)的检测。
5)第1-5组的10只/性别/组于第2天，5只/性别/组于第15天实施安乐死并进行脏器称重和大体解剖观察，1、5组动物主要脏器进行组织病理学检查。
本次试验结果显示，CAR-T细胞(即CNCT19细胞)单次给药的最大耐受剂量大于1×10 9细胞/kg。试验期间CAR-T细胞低、高剂量组所有动物均未见死亡或濒死情况，一般和详细临床观察均未见异常反应，体重、食量、体温、C反应蛋白、血生化均未见明显异常改变。组织病理学检查发现，CAR-T细胞低、高剂量组与T细胞对照组比较，未见明显异常。
实施例11.CNCT19细胞的动物治疗情况
11.1.CNCT19细胞对Nalm-6移植瘤NCG小鼠的治疗作用
本实施例的实验步骤如下：
1)使用6-8周龄的雌性NCG小鼠，于给药前三天，尾静脉注射5x10 5的Nalm-6细胞，所用Nalm-6细胞以2.5x10 6/ml的密度溶解于生理盐水，每只小鼠注射200μl的细胞重悬液；
2)各实验组注射相应的CAR-T细胞(即CNCT19细胞)、未转染的T细胞(即NTD)或细胞保存液(即生理盐水，其中人血白蛋白含量4％(W/V))。共分5组，分别为CAR-T低剂量组(注射CNCT19细胞，5×10 6细胞/只，按总T细胞数计，下同)、CAR-T中剂量组(注射CNCT19细胞，1×10 7细胞/只)、CAR-T高剂量组(注射CNCT19细胞，2×10 7细胞/只)、T细胞对照组(注射未转染的T细胞，2×10 7细胞/只)及溶媒对照组(注射细胞保存液，200μl/只)。每只小鼠注射体积为200μl。
3)每周两次进行体重检测及一般临床观察。记录小鼠存活情况，绘制生存曲线。
结果如图8所示，CNCT19细胞各剂量组均对生存期有显著的延长作用，且剂量依赖关系明显。具体来说，中位生存时间：生理盐水对照组为24天，NTD对照组为23天，CNCT19低剂量组为40天，而CNCT19中、高剂量组实验动物至观察终点全部存活。与生理盐水及NTD对照组相比，CNCT19各剂量组皆能够延长白血病动物生存时间达16天以上。
11.2.CNCT19细胞在动物体内的分布情况
本实施例的实验步骤如下：
1)用6-8周龄的NCG小鼠，雌雄各半。使用11.1实施例的方法建立Nalm-6移植瘤模型。
2)荷瘤及非荷瘤动物均接受CAR-T细胞(即CNCT19细胞)单次尾静脉注射给药，剂量为5×10 6cells/只(按总T细胞数计)。
3)荷瘤组动物分别在给药后24小时(即D2)、72小时(即D4)、168小时(即D8)、336小时(即D15)、504小时(即D22)、672小时(即D29)，非荷瘤组动物分别在给药后24小时(即D2)、168小时(即D8)、336小时(即D15)按计划安乐死，按顺序采集动物全血(EDTA抗凝)、大脑、脊髓(颈段)、骨骼肌、生殖腺(卵巢、睾丸、附睾)、膀胱、胃、小肠、肠系膜淋巴结、骨髓、肝、肾、脾、心、肺等组织或体液。
4)采用经验证的Q-PCR的方法测定血液及各组织样品中嵌合抗原受体(即CAR)的含量。
结果显示，CNCT19细胞以5×10 6细胞/只的剂量单次静脉注射给予荷瘤鼠及非荷瘤鼠后，主要分布于全血和肺、肝、心脏、脾等血流量较大的组织(见图9)，其中，心脏和全血中分布最多，药时曲线下面积约为15000小时*拷贝数/微克，其次为肺部和脊髓，药时曲线下面积约在40000-60000小时*拷贝数/微克之间，脾、肝等其他组织的药时曲线下面积为20000小时*拷贝数/微克以下。
结果还显示，CNCT19细胞在荷瘤鼠组织中的含量略高于非荷瘤鼠(见图10)，给药后24小时，荷瘤鼠的全血、心和脊髓的CNCT19细胞浓度分别约为非荷瘤鼠的3倍、6倍、5倍以上。
此后，各组织中药物含量逐渐下降，至给药后两周基本降至方法学检测限以下。而当疾病进展时，随着CD19抗原刺激的增强，小鼠体内CNCT19细胞反应性地再次增殖(见图11)，其中，脑、肺、肝、全血、脊髓的CNCT19细胞的浓度增加至1200拷贝数/微克DNA以上，甚至达到3500拷贝数/微克DNA。
实施例12.CNCT19细胞对急性淋巴细胞白血病的治疗情况
12.1.CNCT19细胞的临床使用情况
临床应用流程如表1所示，其中某些环节的具体实验步骤请见下文描述。
表1.CNCT19细胞的临床应用流程
Figure PCTCN2020136241-appb-000002
1、清淋预处理
在计划CNCT19细胞悬液回输前-5天内进行。
预处理方案：
氟达拉滨：30mg/m 2，1次/日，连续使用2-4天；
环磷酰胺：500mg/m 2，1次/日，连续使用2天。
预处理化疗的两种药物需在同一天起开始使用。
2、CNCT19细胞悬液回输
(1)细胞制备成功后＜-100℃条件下冻存，使用时在同等温度条件下运输至医院，输注前根据试验操作指南复苏；
(2)细胞回输方法：根据试验操作指南复苏细胞，细胞悬液需在复苏后30分钟内完成回输。单次输注，通过输血器经静脉输入受试者体内，若细胞悬液有1袋以上，每袋回输之 间可无时间间隔，连续输注。受试者在细胞回输期间需密切观察，若出现严重不良事件则停止输注，并根据不良事件的具体情况进行相应处理。若未发生严重不良事件，可按照访视流程进行随访；
(3)受试者细胞回输后需密切观察24小时，若出现严重不良事件，则根据不良事件的具体情况进行相应处理；若未发生严重不良事件，可按照访视流程进行随访；
(4)细胞回输后，受试者继续住院观察14天或根据研究者对受试者的情况综合评估以决定住院观察时间。
3、CNCT19细胞悬液管理
为了严格管理和使用CNCT19细胞悬液，建立严格的CNCT19细胞悬液专人管理制度，由专人将研究用细胞悬液运输至医院科室，由专人负责研究用细胞悬液的接收、建立登记制度。
输注完毕后，细胞悬液包装由药物管理人员回收保存/销毁。
4、CNCT19细胞悬液治疗复发或难治性急性淋巴细胞白血病的临床疗效和安全性结果
本试验从2016年9月开始，至2020年10月，在探索性和I期临床期间，CNCT共治疗63例复发或难治性急性淋巴细胞白血病(ALL)患者，其中，成人患者23例，儿童患者40例。用于成人ALL摸索给药范围：0.25×10 8至0.5×10 8CAR阳性T细胞。
(a)临床疗效结果数据如表2和表3所示，表2显示的是包括成人和儿童患者在内的63例患者的病情恢复情况，表3显示的是23例成人患者的病情恢复情况。可以看出，在63例复发难治的急性淋巴细胞白血病患者中，绝大部分(93.7％)的患者经所述CNCT19细胞悬液注射回输后，病情得到了完全缓解。且，MRD阴性的患者比例为88.9％。结果显示，CNCT19细胞悬液的注射回输能有效治疗复发或难治性急性淋巴细胞白血病成人患者和儿童患者。
表2.CNCT19细胞悬液对成人和儿童急性淋巴细胞白血病的临床疗效结果
Figure PCTCN2020136241-appb-000003
表3.CNCT19细胞悬液对成人急性淋巴细胞白血病的临床疗效结果
Figure PCTCN2020136241-appb-000004
注：在表2和表3中，
ORR(Overall remission rate)：总体缓解率
MRD(Minimal Residual Disease)阴性：肿瘤细胞检测不到(最灵敏的方法)
CR(Complete Remission)：完全缓解
Cri(Complete Remission with incomplete blood count recovery)：形态学完全缓解而血细胞计数未完全恢复
b)安全性初步结果数据如表4和表5所示，表4显示的是包括成人和儿童患者在内的63例患者的安全性结果，表5显示的是23例成人患者的安全性结果。在63例ALL患者中，3级及以上CRS和脑病的发生率分别为19％和20.6％。年龄组之间相比，成人组的严重CRS发生率较儿童组高，其中成人为39.1％，儿童为7.5％，成人组严重CRES发生率与儿童组略低或接近，其中，成人为17.4％，儿童为22.6％。考虑可能是由于早期试验中，按体重给予CNCT19，成人体重大，剂量较高。例如，早期46例受试者中，剂量范围为0.71×10 6-4.08×10 6/kg，相当于0.16×10 8-2.36×10 8CAR阳性T细胞。随着前期剂量探索逐渐了解CNCT19产品特性，在之后的临床研究中选择了较为安全性的剂量范围，如，在17例细胞剂量使用范围0.2×10 8-1.1×10 8CAR阳性T细胞(中位值为0.5×10 8)的患者中，3级及以上CRS和CRES发生率均降至5.9％(1/17)。
可以看出，所述CNCT19细胞悬液的注射引起严重的CRS或CRES副作用的概率相对较低，总体安全性可控，因此具有良好的安全性能。说明CNCT19的良好的安全性能提升了产品质量，降低了临床风险。
表4.CNCT19细胞悬液对成人和儿童急性淋巴细胞白血病的安全性结果
Figure PCTCN2020136241-appb-000005
表5.CNCT19细胞悬液对成人急性淋巴细胞白血病的安全性结果
Figure PCTCN2020136241-appb-000006
注：在表4和表5中，
CRS(Cytokine Release Syndrome)：细胞因子释放综合征
CRES(CAR-T-cell-related Encephalopathy Syndrome)：CAR-T细胞相关脑病综合征
实施例13.CNCT19细胞对复发或难治性非霍奇金淋巴瘤的治疗情况
13.1.CNCT19细胞的临床使用情况
临床应用流程如表6所示，其中某些环节的具体实验步骤请见下文描述。
表6.CNCT19细胞的临床应用流程
Figure PCTCN2020136241-appb-000007
1、清淋预处理
在计划CNCT19细胞悬液回输前-5天内进行。
预处理方案：
氟达拉滨：30mg/m 2，1次/日，连续使用2-4天；
环磷酰胺：500mg/m 2，1次/日，连续使用2天。
预处理化疗的两种药物需在同一天起开始使用。
2、CNCT19细胞悬液回输
(1)细胞制备成功后＜-100℃条件下冻存，使用时在同等温度条件下运输至医院，输注 前根据试验操作指南复苏；
(2)细胞回输方法：根据试验操作指南复苏细胞，细胞悬液需在复苏后30分钟内完成回输。单次输注，通过输血器经静脉输入受试者体内，若细胞悬液有1袋以上，每袋回输之间可无时间间隔，连续输注。受试者在细胞回输期间需密切观察，若出现严重不良事件则停止输注，并根据不良事件的具体情况进行相应处理。若未发生严重不良事件，可按照访视流程进行随访；
(3)受试者细胞回输后需密切观察24小时，若出现严重不良事件，则根据不良事件的具体情况进行相应处理；若未发生严重不良事件，可按照访视流程进行随访；
(4)细胞回输后，受试者继续住院观察14天或根据研究者对受试者的情况综合评估以决定住院观察时间。
3、CNCT19细胞悬液管理
为了严格管理和使用CNCT19细胞悬液，建立严格的CNCT19细胞悬液专人管理制度，由专人将研究用细胞悬液运输至医院科室，由专人负责研究用细胞悬液的接收、建立登记制度。
输注完毕后，细胞悬液包装由药物管理人员回收保存/销毁。
4、CNCT19细胞悬液治疗复发或难治性非霍奇金淋巴瘤的临床疗效及安全性结果数据
本试验从2016年9月开始，至2020年10月，在探索性和I期临床期间，CNCT19细胞悬液共治疗50例复发或难治性非霍奇金淋巴瘤(NHL)患者。其中，用于NHL(仅成人)摸索给药范围：1×10 8至2×10 8CAR阳性T细胞。
(a)临床疗效结果数据如表7，表7显示的是50例患者的病情恢复情况。可以看出，在50例复发难治的非霍奇金淋巴瘤患者中，有约80％的患者经所述CNCT19细胞悬液的注射回输后，病情得到了完全缓解或部分缓解，其中，完全缓解率为54％(27例)，部分缓解率为24％(12例)。说明CNCT19细胞悬液能有效治疗复发或难治性非霍奇金淋巴瘤患者。
表7.CNCT19细胞悬液对非霍奇金淋巴瘤的临床疗效结果
Figure PCTCN2020136241-appb-000008
注：在表7中，
ORR(Overall remission rate)：总体缓解率
CR(Complete Remission)：完全缓解
PR(Partial Remission)：部分缓解
b)安全性初步结果数据如表8所示，表8显示的是50例患者的安全性结果。可以看出，所述CNCT19细胞悬液注射引起严重的CRS或CRES副作用的概率低，级别在3级以上的副作用概率低于10％，其中，引起3级以上CRS的概率为0％，引起3级以上CRES的概率为6％，因此具有良好的安全性能。
表8.CNCT19细胞悬液对非霍奇金淋巴瘤的安全性结果
Figure PCTCN2020136241-appb-000009
注：在表8中，
CRS(Cytokine Release Syndrome)：细胞因子释放综合征
CRES(CAR-T-cell-related Encephalopathy Syndrome)：CAR-T细胞相关脑病综合征
前述详细说明是以解释和举例的方式提供的，并非要限制所附权利要求的范围。目前本申请所列举的实施方式的多种变化对本领域普通技术人员来说是显而易见的，且保留在所附的权利要求和其等同方案的范围内。

Claims (34)

  1. 一种嵌合抗原受体，所述嵌合抗原受体包含SEQ ID NO.1所示的氨基酸序列。
  2. 一种分离的核酸分子，所述核酸分子编码权利要求1所述的嵌合抗原受体。
  3. 一种编码嵌合抗原受体的分离的核酸分子，所述核酸分子包含SEQ ID NO.2所示的核酸序列。
  4. 一种载体，所述载体包含权利要求2-3中任一项所述的核酸分子。
  5. 一种免疫效应细胞，所述免疫效应细胞包含权利要求1所述的嵌合抗原受体、权利要求2-3中任一项所述的核酸分子和/或权利要求4所述的载体。
  6. 根据权利要求5所述的免疫效应细胞，其中所述免疫效应细胞选自以下组：T淋巴细胞和自然杀伤细胞。
  7. 根据权利要求5-6中任一项所述的免疫效应细胞，所述免疫效应细胞表面表达权利要求1所述的嵌合抗原受体。
  8. 一种制备免疫效应细胞的方法，所述方法包括以下的步骤：向免疫效应细胞中转导权利要求4所述的载体。
  9. 根据权利要求8所述的方法，所述免疫效应细胞选自以下组：T淋巴细胞和自然杀伤细胞。
  10. 一种组合物，所述组合物包含权利要求5-7中任一项所述的免疫效应细胞。
  11. 权利要求1所述的嵌合抗原受体、权利要求2-3中任一项所述的核酸分子、权利要求4所述的载体和/或权利要求5-7中任一项所述的免疫效应细胞用于制备药物的用途，其中所述药物用于治疗与CD19的表达相关的疾病或病症。
  12. 根据权利要求11所述的用途，其中所述与CD19的表达相关的疾病或病症包括非实体瘤。
  13. 根据权利要求12所述的用途，其中所述非实体瘤包括白血病和/或淋巴瘤。
  14. 根据权利要求11所述的用途，其中所述与CD19的表达相关的疾病或病症包括急性淋巴细胞白血病和/或B细胞淋巴瘤。
  15. 根据权利要求14所述的用途，其中所述急性淋巴细胞白血病包括成人急性淋巴细胞白血病和/或儿童急性淋巴细胞白血病。
  16. 根据权利要求15所述的用途，其中所述药物的剂量为0.25×10 8至0.5×10 8CAR阳性T细胞。
  17. 根据权利要求14所述的用途，其中所述B细胞淋巴瘤包括非霍奇金淋巴瘤。
  18. 根据权利要求17所述的用途，其中所述药物的剂量为1×10 8至2×10 8CAR阳性T细胞。
  19. 治疗与CD19的表达相关的疾病或病症的方法，所述方法包括施用权利要求1所述的嵌合抗原受体、权利要求2-3中任一项所述的核酸分子、权利要求4所述的载体和/或权利要求5-7中任一项所述的免疫效应细胞。
  20. 根据权利要求19所述的方法，其中所述与CD19的表达相关的疾病或病症包括非实体瘤。
  21. 根据权利要求20所述的方法，其中所述非实体瘤包括白血病和/或淋巴瘤。
  22. 根据权利要求19所述的方法，其中所述与CD19的表达相关的疾病或病症包括急性淋巴细胞白血病和/或B细胞淋巴瘤。
  23. 根据权利要求22所述的方法，其中所述急性淋巴细胞白血病包括成人急性淋巴细胞白血病和/或儿童急性淋巴细胞白血病。
  24. 根据权利要求23所述的方法，其包括施用剂量为0.25×10 8至0.5×10 8CAR阳性T细胞。
  25. 根据权利要求22所述的方法，其中所述B细胞淋巴瘤包括非霍奇金淋巴瘤。
  26. 根据权利要求25所述的方法，其包括施用剂量为1×10 8至2×10 8CAR阳性T细胞。
  27. 权利要求1所述的嵌合抗原受体、权利要求2-3中任一项所述的核酸分子、权利要求4所述的载体和/或权利要求5-7中任一项所述的免疫效应细胞，其用于治疗与CD19的表达相关的疾病或病症。
  28. 根据权利要求27所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中所述与CD19的表达相关的疾病或病症包括非实体瘤。
  29. 根据权利要求28所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中所述非实体瘤包括白血病和/或淋巴瘤。
  30. 根据权利要求27所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中所述与CD19的表达相关的疾病或病症包括急性淋巴细胞白血病和/或B细胞淋巴瘤。
  31. 根据权利要求30所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中所述急性淋巴细胞白血病包括成人急性淋巴细胞白血病和/或儿童急性淋巴细胞白血病。
  32. 根据权利要求31所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中施用剂量为0.25×10 8至0.5×10 8CAR阳性T细胞。
  33. 根据权利要求30所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中所述B细胞淋巴瘤包括非霍奇金淋巴瘤。
  34. 根据权利要求33所述的嵌合抗原受体、核酸分子、载体和/或免疫效应细胞，其中施用剂量为1×10 8至2×10 8CAR阳性T细胞。
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