WO2022198611A1 - Séquence de récepteur antigénique chimérique car et cellules car-nk l'utilisant - Google Patents

Séquence de récepteur antigénique chimérique car et cellules car-nk l'utilisant Download PDF

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Publication number
WO2022198611A1
WO2022198611A1 PCT/CN2021/083144 CN2021083144W WO2022198611A1 WO 2022198611 A1 WO2022198611 A1 WO 2022198611A1 CN 2021083144 W CN2021083144 W CN 2021083144W WO 2022198611 A1 WO2022198611 A1 WO 2022198611A1
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domain structure
sequence
car
cells
intracellular domain
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PCT/CN2021/083144
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English (en)
Chinese (zh)
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步磊
潘伟飙
蔡子悦
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汕头普罗凯融生物医药科技有限公司
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Priority to PCT/CN2021/083144 priority Critical patent/WO2022198611A1/fr
Publication of WO2022198611A1 publication Critical patent/WO2022198611A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/464—Cellular immunotherapy characterised by the antigen targeted or presented
    • A61K39/4643—Vertebrate antigens
    • A61K39/4644—Cancer antigens
    • A61K39/464466—Adhesion molecules, e.g. NRCAM, EpCAM or cadherins
    • A61K39/464468—Mesothelin [MSLN]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/461—Cellular immunotherapy characterised by the cell type used
    • A61K39/4613—Natural-killer cells [NK or NK-T]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/46—Cellular immunotherapy
    • A61K39/463—Cellular immunotherapy characterised by recombinant expression
    • A61K39/4631—Chimeric Antigen Receptors [CAR]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention belongs to the field of cellular immunotherapy, and in particular, relates to a CAR chimeric antigen receptor sequence and a CAR-NK cell using the same.
  • Malignant tumor is one of the major diseases that endanger human health.
  • Traditional tumor treatment methods such as surgery, radiotherapy, and chemotherapy have been the main strategies for tumor treatment in recent decades.
  • patients will develop resistance to drugs and radiotherapy, resulting in high frequency of tumor recurrence.
  • Tumor cell therapy has attracted great attention due to its advantages of targeting, remarkable effect, and few side effects, and has gradually become an important means of comprehensive tumor therapy. Hot spots and development directions of research and clinical applications.
  • NK cells Natural killer cells
  • NK cells do not need pre-stimulation to exert their killing activity, and have great potential in the treatment of human malignant tumors based on cell therapy.
  • NK cells can spontaneously lyse tumor cells in the absence of T and B cells.
  • NK cells share many features with cytotoxic T cells, including common precursor cells, rearrangement of cell surface receptor molecules, and granzyme-dependent killing mechanisms.
  • NK cells are also unique in that they do not require pre-stimulation to kill and are not MHC-restricted in killing tumor cells. This makes NK cells widely used in adoptive tumor cell immunotherapy, which refers to the return of exogenously engineered cells to patients for clinical applications.
  • NK cells isolated and expanded in vitro can be used for autologous or allogeneic therapy.
  • the method of depleting T cells from leukocytes in a single donor's blood to obtain NK cells yields unstable NK cell yields, and the numbers are not sufficient for reinfusion.
  • NK cells are very difficult to culture in vitro, far from meeting the requirements of cellular immunotherapy.
  • many NK cells in tumor patients can no longer function normally.
  • many activators such as IL-2, IL-12, IL-5, IL-18, etc.
  • NK cells Although there are many methods to prepare NK cells at present, the low differentiation efficiency of NK cells makes the price of NK cell drugs high, and the killing activity is difficult to control, which is not conducive to large-scale production and application. Based on this, the in vitro activation and expansion of NK cells has practical significance for the further promotion and application of NK cells in tumor cell immunotherapy.
  • Tumor chimeric antigen receptor (CAR) therapy refers to a new type of precise targeted therapy that uses chimeric antigen receptors to treat tumors on immune cells. In recent years, it has achieved good results in clinical tumor treatment through optimization and improvement. The effect is a very promising new tumor immunotherapy method that can be precise, fast, efficient, and has the potential to cure cancer.
  • CAR activates immune cells (T cells, NK cells) through genetic engineering technology, and installs a positioning navigation device CAR, and reorganizes ordinary T cells and NK cells into CAR-T cells and CAR-NK cells correspondingly.
  • CAR-T cells and CAR-NK cells can use their chimeric CARs to specifically identify tumor cells in the body, and release a large number of various effectors through immunization, which can efficiently kill tumor cells, so as to achieve the purpose of treating malignant tumors. .
  • the immune cells that have been positioned and knocked into the CAR vector can be transplanted back into the original patient's body to avoid the attack of the autoimmune system.
  • the purpose of the present invention is to provide a CAR chimeric antigen receptor sequence and a CAR-NK cell using the same, so as to effectively activate immune function cells and make immune function cells efficiently kill tumor cells.
  • a CAR chimeric antigen receptor sequence including a single-chain antibody structure, an extracellular domain structure, a transmembrane domain structure and an intracellular domain structure; the protein polypeptide sequence of the extracellular domain structure is selected from CD8 ⁇ Fragments of protein polypeptide sequences; protein polypeptide sequences of transmembrane domain structures are selected from fragments of NKG2D protein polypeptide sequences; intracellular domain structures include 2B4 intracellular domain structures or/and CD3 ⁇ intracellular domain structures, protein polypeptides of 2B4 intracellular domain structures The sequence is selected from the fragment of the 2B4 protein polypeptide sequence, and the protein polypeptide sequence of the CD3 ⁇ intracellular domain structure is selected from the fragment of the CD3 ⁇ protein polypeptide sequence.
  • the intracellular domain structure includes the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure, and the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure are connected in series in sequence.
  • the extracellular domain structure includes a first extracellular domain structure and a second extracellular domain structure; the first extracellular domain structure, the single-chain antibody structure, the second extracellular domain structure, the transmembrane domain structure, and the 2B4 intracellular domain
  • the structure and CD3 ⁇ intracellular domain structure are connected in series; the single-chain antibody structure is the anti-mesothelin scFv encoded by SEQ ID NO.7.
  • its protein polypeptide sequence is SEQ ID NO.10.
  • the extracellular domain structure, the single-chain antibody structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure are connected in series in sequence;
  • the single-chain antibody structure is the anti-epidermal growth factor receptor encoded by SEQ ID NO.12. Body scFv.
  • its protein polypeptide sequence is SEQ ID NO.14.
  • a CAR chimeric antigen receptor sequence is provided, the protein polypeptide sequence of which is not less than 80% similar to the protein sequence of the above-mentioned CAR chimeric antigen receptor series.
  • a CAR-NK cell whose AAVS1 site is chimeric with a sequence vector comprising the ER1a promoter and the above-mentioned CAR chimeric antigen receptor sequence; in the sequence vector, the CAR chimeric antigen receptor is The body sequence is located downstream of the ER1a promoter.
  • the anti-cancer drug is an anti-malignant mesothelioma drug.
  • the CAR chimeric antigen receptor sequence provided by the present invention can effectively activate immune function cells, and can make the chimeric immune function cells efficiently recognize malignant tumor cells.
  • the CAR chimeric antigen receptor sequence provided by the present invention adopts anti-mesothelin scFv or anti-epidermal growth factor receptor scFv as the single-chain antibody structure contained therein, so that the chimeric immune function cells can efficiently Identification of malignant mesothelioma cells.
  • the sequence vector including the above-mentioned CAR chimeric antigen receptor sequence is chimeric in the AAVS1 site of NK cells.
  • the location of the knock-in is clear, and it will not interfere with other genes of the host cell, and will not cause malignant transformation of the host cell due to transgene.
  • the present invention sets the EF1a promoter that can be uniformly and highly expressed in various cells upstream of the above CAR chimeric antigen receptor sequence, so that CAR-NK cells with biological activity and functions can be successfully and efficiently obtained, and the stem cells can be The proportion of differentiation to NK cells was significantly increased, and the proportion of differentiation was greater than 90%.
  • the sequence vector used in the present invention may include an IRES sequence, which can make mRNA translation and fusion with exogenous cDNA independent of the 5' cap structure.
  • the sequence vector used in the present invention may include a T2A sequence, so that the entire mRNA generated using the sequence vector can be self-spliced into two independent proteins.
  • the sequence vector used in the present invention may include a GFP sequence or a Puro sequence, so that the CAR-NK cells constructed by the present invention can be identified and detected by fluorescence analysis or drug screening accordingly.
  • the CAR-NK cells provided by the present invention have the advantages of stability, high efficiency and high safety, and are suitable for large-scale production and application.
  • Example 1 is a schematic diagram of the sequence vector constructed in Example 1 for targeting and knocking into the AAVS1 site of CAR-NK cells;
  • FIG. 2 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 before injection of CAR-NK cells I;
  • FIG. 3 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 after 1 hour of injection of CAR-NK cells I;
  • FIG. 4 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 after 3 hours of injection of CAR-NK cells I;
  • Figure 5 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 5 hours after injection of CAR-NK cells I;
  • Figure 6 is a microscope observation diagram of malignant mesothelioma cells in treatment I of Example 2 7 hours after injection of CAR-NK cells I.
  • chimerism refers to the biological process of obtaining specialized cells (eg, NK cells) by non-specialized CARs under controlled conditions when cultured in vitro. Differentiation is controlled by the interaction of cellular genes with extracellular physical and chemical conditions, usually via signaling pathways involving proteins embedded on the cell surface.
  • the term "about”, unless explicitly stated or clear from context, is understood to mean within a range of normal tolerance in the art, eg, within 2 standard deviations of the mean. About 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value Inside.
  • CAR sequences involved in this example include CAR-A sequences, CAR-AA sequences and CAR-B sequences.
  • CAR-A sequence includes extracellular domain structure, single chain antibody structure, transmembrane domain structure and intracellular domain structure.
  • the extracellular domain structure includes a first extracellular domain structure and a second extracellular domain structure: the nucleotide sequence encoding the first extracellular domain structure is SEQ ID NO.1, and SEQ ID NO.1 belongs to the nucleoside encoding CD8 ⁇ protein Fragment of acid sequence (NCBI Reference Sequence: NM_171827); the nucleotide sequence encoding the second extracellular domain structure is SEQ ID NO.2, and SEQ ID NO.2 belongs to the nucleotide sequence encoding CD8 ⁇ protein (NCBI Reference Sequence: NM_001768).
  • the nucleotide sequence encoding the structure of the transmembrane region is SEQ ID NO.3, and SEQ ID NO.3 is a fragment of the nucleotide sequence encoding the NKG2D protein (NCBI Reference Sequence: NM_007360.3).
  • the intracellular domain structure includes the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure: the nucleotide sequence encoding the 2B4 intracellular domain structure is SEQ ID NO.4, and SEQ ID NO.4 belongs to the nucleotide sequence encoding the 2B4 protein ( NCBI Reference Sequence: fragment of NM_001166663.1); the nucleotide sequence encoding CD3 ⁇ intracellular domain structure is SEQ ID NO.5, and SEQ ID NO.5 belongs to the nucleotide sequence encoding CD3 ⁇ protein (NCBI Reference Sequence: NM_198053.
  • SEQ ID NO.6 formed in series with SEQ ID NO.4 and SEQ ID NO.5 is the nucleotide sequence encoding the intracellular region structure of the CAR-A sequence.
  • the single-chain antibody structure is anti-mesothelin scFv (anti-mesothelin scFv), and the nucleotide sequence encoding the anti-mesothelin scFv is SEQ ID NO.7.
  • the first extracellular domain structure, the single-chain antibody structure, the second extracellular domain structure, the transmembrane domain structure, the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure are connected in series in sequence, encoding the CAR-A sequence.
  • the nucleotide sequence is SEQ ID NO.8.
  • the nucleotide sequence SEQ ID NO.8 encoding the CAR-A sequence is optimized to obtain SEQ ID NO.9, and the protein polypeptide SEQ ID NO.10 obtained after translating SEQ ID NO.9 is the CAR-AA sequence.
  • CAR-A sequence includes extracellular domain structure, single chain antibody structure, transmembrane domain structure and intracellular domain structure.
  • the nucleotide sequence encoding the structure of the extracellular domain is SEQ ID NO. 11, and SEQ ID NO. 11 is a fragment of the nucleotide sequence encoding the CD8 ⁇ protein (NCBI Reference Sequence: NM_171827).
  • the nucleotide sequence encoding the structure of the transmembrane region is SEQ ID NO.3, and SEQ ID NO.3 is a fragment of the nucleotide sequence encoding the NKG2D protein (NCBI Reference Sequence: NM_007360.3).
  • the intracellular domain structure includes the 2B4 intracellular domain structure and the CD3 ⁇ intracellular domain structure: the nucleotide sequence encoding the 2B4 intracellular domain structure is SEQ ID NO.4, and SEQ ID NO.4 belongs to the nucleotide sequence encoding the 2B4 protein (NCBI Reference Sequence: NM_001166663.1) fragment; the nucleotide sequence encoding the intracellular domain structure of CD3 ⁇ is SEQ ID NO.5, and SEQ ID NO.5 belongs to the nucleotide sequence encoding CD3 ⁇ protein (NCBI Reference Sequence: NM_198053 .2) fragment; SEQ ID NO.6 formed by concatenation of SEQ ID NO.4 and SEQ ID NO.5 is the nucleotide sequence encoding the intracellular region structure of the CAR-B sequence.
  • the structure of the single chain antibody is anti-epidermal growth factor receptor scFv (anti-HER3 scFv), and the nucleotide sequence encoding the anti-epidermal growth factor receptor scFv is SEQ ID NO.12.
  • the extracellular domain structure, single-chain antibody structure, transmembrane domain structure, 2B4 intracellular domain structure and CD3 ⁇ intracellular domain structure are connected in series in sequence, and the nucleotide sequence encoding the CAR-B sequence is SEQ ID NO. 13.
  • the protein polypeptide SEQ ID NO.14 obtained after translating SEQ ID NO.13 is the CAR-B sequence.
  • sequence vectors of CAR-AA sequence and CAR-B sequence were amplified by PCR technology, and then the CAR-AA sequence and CAR-B sequence were cloned into sequences containing T2A sequence, EF1a promoter sequence, GFP sequence, and Puro sequence, respectively.
  • sequence vector with the IRES sequence a sequence vector with the EF1a promoter sequence, the CAR sequence, the T2A sequence, the GFP sequence, the IRES sequence and the Puro sequence in series is obtained (as shown in Figure 1).
  • sequence vector using CAR-AA as its CAR sequence is marked as sequence vector I
  • sequence vector using CAR-B as its CAR sequence is marked as sequence vector II.
  • NK recombinant cells chimeric with sequence vector I were labeled as CAR-NK cells I
  • the NK recombinant cells chimeric with sequence vector II were labeled as CAR-NK cells II.
  • the CAR-NK cells successfully chimeric with the above sequence vector can express green fluorescent protein and puromycin at the same time, and the fluorescent protein-positive cells can be screened by fluorescence detection, or the puromycin-positive cells can be screened by using puromycin antibody, thereby Screen the CAR-NK cells that successfully chimeric the above sequence vector.
  • NK cells provided by the same volunteer were used to prepare CAR-NK cells I and CAR-NK cells II according to the method provided in Example 1.
  • Two groups of treatment groups (including treatment I and treatment II) and one group of control groups were set up to carry out the following in vitro tumor killing effect experiments according to the following steps. Specifically, the following experimental operations were carried out with CAR-NK cells I for treatment I, and CAR for treatment II.
  • - NK cells II were subjected to the following experimental procedures, and the control group was subjected to the following experimental procedures using NK cells from the same volunteer.
  • Malignant mesothelioma cells (40,000 cells) were evenly spread in 24 wells one day before the experiment, and waited for the cells to adhere to the wall;
  • NK cells, CAR-NK cells I, CAR-NK cells II and CAR-NK cells III were plated in 24 wells (40,000 cells) according to the ratio of effector to target of 1:1.
  • the system is 1ml, incubated at 37 degrees for 7 hours;
  • the killing rate of tumor cells was calculated by reading the plate at 450 nm.

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Abstract

La présente invention concerne une séquence de récepteur antigénique chimérique CAR, comprenant une structure d'anticorps à chaîne unique, une structure de domaine extracellulaire, une structure de domaine transmembranaire et une structure de domaine intracellulaire. Une séquence polypeptidique de protéine de la structure de domaine extracellulaire est choisie parmi des fragments d'une séquence polypeptidique de protéine CD8α. Une séquence polypeptidique de protéine de la structure de domaine transmembranaire est choisie parmi des fragments d'une séquence polypeptidique de protéine NKG2D. La structure de domaine intracellulaire comprend une structure de domaine intracellulaire 2B4 et/ou une structure de domaine intracellulaire CD3ζ. Une séquence polypeptidique de protéine de la structure de domaine intracellulaire 2B4 est choisie parmi des fragments d'une séquence polypeptidique de protéine 2B4. Une séquence polypeptidique de protéine de la structure de domaine intracellulaire CD3ζ est choisie parmi des fragments d'une séquence polypeptidique de protéine CD3ζ. La séquence de récepteur antigénique chimérique CAR fournie par la présente invention peut activer de manière efficace des cellules fonctionnelles immunitaires, et peut permettre aux cellules fonctionnelles immunitaires intégrées avec la séquence de récepteur antigénique chimérique CAR de reconnaître de manière efficace des cellules tumorales malignes.
PCT/CN2021/083144 2021-03-25 2021-03-26 Séquence de récepteur antigénique chimérique car et cellules car-nk l'utilisant WO2022198611A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107106665A (zh) * 2014-06-06 2017-08-29 纪念斯隆-凯特琳癌症中心 靶向间皮素的嵌合抗原受体及其用途
WO2018124766A2 (fr) * 2016-12-28 2018-07-05 주식회사 녹십자랩셀 Récepteur antigénique chimérique et cellules tueuses naturelles exprimant celui-ci
CN108866003A (zh) * 2017-05-16 2018-11-23 科济生物医药（上海）有限公司 基因工程化的细胞及应用
CN109385403A (zh) * 2017-08-04 2019-02-26 科济生物医药（上海）有限公司 靶向gpc3的car nk细胞
CN109762844A (zh) * 2019-01-31 2019-05-17 北京呈诺医学科技有限公司 一种靶标间皮素的car-nk细胞制备方法
CN110684117A (zh) * 2019-09-25 2020-01-14 汕头普罗凯融生物医药科技有限公司 一种car嵌合抗原受体序列及应用其的car-nk细胞

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107106665A (zh) * 2014-06-06 2017-08-29 纪念斯隆-凯特琳癌症中心 靶向间皮素的嵌合抗原受体及其用途
WO2018124766A2 (fr) * 2016-12-28 2018-07-05 주식회사 녹십자랩셀 Récepteur antigénique chimérique et cellules tueuses naturelles exprimant celui-ci
CN108866003A (zh) * 2017-05-16 2018-11-23 科济生物医药（上海）有限公司 基因工程化的细胞及应用
CN109385403A (zh) * 2017-08-04 2019-02-26 科济生物医药（上海）有限公司 靶向gpc3的car nk细胞
CN109762844A (zh) * 2019-01-31 2019-05-17 北京呈诺医学科技有限公司 一种靶标间皮素的car-nk细胞制备方法
CN110684117A (zh) * 2019-09-25 2020-01-14 汕头普罗凯融生物医药科技有限公司 一种car嵌合抗原受体序列及应用其的car-nk细胞

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