WO2018045880A1 - Chimeric antigen receptor, car133-nkt cells and use thereof - Google Patents

Chimeric antigen receptor, car133-nkt cells and use thereof Download PDF

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WO2018045880A1
WO2018045880A1 PCT/CN2017/098976 CN2017098976W WO2018045880A1 WO 2018045880 A1 WO2018045880 A1 WO 2018045880A1 CN 2017098976 W CN2017098976 W CN 2017098976W WO 2018045880 A1 WO2018045880 A1 WO 2018045880A1
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cells
cancer
nkt
nkt cell
car
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French (fr)
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Yiling SI
Xiaohui Wang
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Guangzhou Bainifu Biotech Co., Ltd
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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    • C07—ORGANIC CHEMISTRY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0646—Natural killers cells [NK], NKT cells
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
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    • C07K2319/03—Fusion polypeptide containing a localisation/targetting motif containing a transmembrane segment
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    • C07K2319/00—Fusion polypeptide
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    • C07—ORGANIC CHEMISTRY
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    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/15011—Lentivirus, not HIV, e.g. FIV, SIV
    • C12N2740/15041—Use of virus, viral particle or viral elements as a vector
    • C12N2740/15043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to the field of tumor biological products, in particular, to a chimeric antigen receptor (CAR) , CD133ScFv-CD8-CD137-CD3 ⁇ and gene thereof and recombinant expression vector, engineered CD133-targeted NKT cell (CAR133-NKT cell) and preparation methods and use thereof in adoptive immunotherapy.
  • CAR chimeric antigen receptor
  • CD133ScFv-CD8-CD137-CD3 ⁇ and gene thereof and recombinant expression vector
  • engineered CD133-targeted NKT cell CAR133-NKT cell
  • NKT natural killer T
  • NKT cells express both TCR of T cells and NKR-P1 of NK cells.
  • TCR and NKR When mediated by the TCR and NKR, NKT cells produce a large amount of IL-4 and INF ⁇ to kill tumor cells.
  • NKT cells bind with Fc fragment of specific antibodies through their surface CD16 and play a role in antibody-dependent cell-mediated cytotoxicity (ADCC) .
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the antibody specifically binds to the corresponding antigen epitope on the target cell and NKT cells kill target cells that bind with the antibody, the binding between the antibody and the antigen on the target cell is specific, but the cytotoxicity of the NKT cells on the target cells is non-specific. Further, usually, the half-life of the infused NKT cells is approximately two weeks, with a short period of validity, and a repeated infusion is necessary. Moreover, there is a lack of specific antibodies for NKT cells, and there is not enough accumulation of NKT cells in tumor lesions, which restricts the targeted therapy of NKT cells on cancers. Furthermore, studies show that NKT cells are not effective for all tumors, and they have a weak killing effect on some tumors; thus, the specific cytotoxicity of NKT cells needs to be enhanced. The immune surveillance of NKT cells for cancer stem cells is still controversial.
  • Cancer stem cells are a special class of tumor cells that account for a low percentage of tumor cells, but they have the ability to self-renew and the potential to differentiate into other tumor cells. A large number of studies have shown that the cancer stem cells are closely correlated to the recurrence and distant metastases of tumors, and this is possibly a cause of resistance to chemoradiotherapy.
  • CD133 is a cell surface glycoprotein that is used as a marker to separate the cancer stem cells in the tumor tissues.
  • CD133 exhibits a high expression on the surface of a variety of tumor cells, such as colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumors, melanoma, neuroglioma, etc.
  • tumor-initiating cell population and epithelial progenitor cells contain a large number of CD133-positive cells; in addition, the high expression of CD133 is associated with the poor prognosis of therapy.
  • the CD133 antigen becomes an ideal target for antibody-based therapy. However, until now, formulations to target CD133 to produce high specific cytotoxicity on tumors are lacking.
  • the objective of the present invention is to overcome the shortcoming of the weak anti-tumor activities of NKT cells on tumors and to improve the specific cytotoxicity by making full use C133 as the ideal target for treating tumors, and a CD133ScFv-CD8-CD137-CD3 ⁇ and a gene thereof and a recombinant expression vector, an engineered CD133-targeted NKT cell (CAR133-NKT cells) and preparation methods and use thereof are provided.
  • the inventors unexpectedly found highly specific cytotoxicity to cancer cells when the CD133ScFv-CD8-CD137-CD3 ⁇ modified NKT cells were co-cultured with CD133-positive epithelial tumor cells.
  • the present invention provides a chimeric antigen receptor (CAR) , CD133ScFv-CD8-CD137-CD3 ⁇ , comprising a CD8a signal peptide, CD133ScFv, a CD8 hinge region and transmembrane region, a CD137 intracellular signaling domain and a CD3 ⁇ intracellular signaling domain connected in series.
  • CAR chimeric antigen receptor
  • a gene encoding the CAR is provided.
  • a recombinant expression vector containing the gene is provided.
  • an engineered CD133-targeted NKT cell that is modified by the CD133ScFv-CD8-CD137-CD3 ⁇ is provided.
  • a method for preparing the engineered CD133-targeted NKT cells comprising: packaging a lentivirus carrying PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ to obtain virus concentrate; infecting NKT cells using the virus concentrate to make the NKT cells express CD133 CAR containing CD133ScFv, a CD8 hinge region and transmembrane region, a CD137 intracellular signaling domain and a CD3 ⁇ intracellular signaling domain.
  • engineered CD133-targeted NKT cells prepared according to the above method are provided.
  • a use of the engineered CD133-targeted NKT cells in preparing the formulations for treating tumors is provided.
  • a method for treating the CD133-positive epithelial tumors and/or inhibiting the tumor recurrence and metastasis comprising: intravenously infusing the engineered CD133-targeted NKT cells into patients with CD133-positive epithelial tumors.
  • the CD133ScFv-CD8-CD137-CD3 ⁇ modified NKT cells in the invention When co-cultured with CD133-positive epithelial tumor cells, the CD133ScFv-CD8-CD137-CD3 ⁇ modified NKT cells in the invention, i.e., the engineered CD133-targeted NKT cells, specifically bind with the CD133 antigen to enhance the target identification of the CD133 antigen on the cancer cell surface by the immune cells and improve the specific cytotoxicity on the CD133-positive target cells.
  • the engineered CD133-targeted NKT cell in the invention provides a new option for eradicating CD133-positive primary tumors and even inhibiting tumor metastasis and recurrence, presenting a good prospect for industrial applications.
  • FIG. 1 shows a phenotypic analysis of NKT cells by a flow cytometry.
  • FIG. 2 shows an electrophoretogram of restriction enzyme MluI/SalI double digested fragments of lentiviral expression vector PWPT-CD8-CD137-CD3 ⁇ in the present invention.
  • FIG. 3 shows an electrophoretogram of restriction enzyme BamHI/SalI double digested fragments of lentiviral expression vector PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ in the present invention.
  • FIG. 4 shows a structural diagram of lentiviral expression vector PWPT-CD133ScFv-CD8 -CD137-CD3 ⁇ in the present invention.
  • FIG. 5 shows the infection efficiency of virus containing CD133ScFv-CD8-CD137-CD3 ⁇ (CAR133) for NKT cells by a flow cytometry.
  • FIG. 6 shows a phenotypic identification of CD133ScFv-CD8-CD137-CD3 ⁇ (CAR133) modified NKT cells (CAR133-NKT cells) by a flow cytometry.
  • FIG. 7 (A) to FIG. 7 (C) show CD133 expression level analysis of epithelial tumor cells with different CD133 expression levels according to the Example 4 of the present invention.
  • FIG. 8 shows a cytotoxic activity of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels according to the Example 4 of the present invention (4 hours) .
  • FIG. 9 shows a cytotoxic activity of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels according to the Example 4 of the present invention (8 hours) .
  • FIG 10 shows the effect of CAR133-NKT cells to hematopoietic colony formation according to the Example 6 of the present invention.
  • FIG 11 to FIG 13 show the change of CAR133 gene copy after CAR133-NKT cells are infused into patients according to the Example 7 of the present invention.
  • FIG. 14 to FIG 16 show the change of the cell count of CD133 positive cells in patients with liver cancer during the treatment with CAR133-NKT cells.
  • FIG. 17 shows the treatment effects of CAR133-NKT cells on patients with liver cancer according to the Example 7 of the present invention.
  • FIG. 18 to FIG. 21 show the blood system adverse event of CAR133-NKT cells for treating patients with metastatic pancreatic cancer according to the Example 7 of the present invention, presenting the change trends of hemoglobin (Hgb) , white blood cell (WBC) , platelet (PLT) and the reticulocyte (Ret) after cell infusion at different days.
  • Hgb hemoglobin
  • WBC white blood cell
  • PHT platelet
  • Ret reticulocyte
  • FIG 22 (A) to FIG 22 (F) show the change of CAR133 gene copy after CAR133-NKT cells are infused into patients with pancreatic cancer, colorectal cancer or rectal cancer according to the Example 7 of the present invention.
  • FIG. 23 show the change of the cell count of CD133 positive cells in patients with pancreatic cancer, colorectal cancer or rectal cancer during the treatment with CAR133-NKT cells.
  • FIG. 24 (A) to FIG 24 (E) show the change of the cytokines in peripheral blood of patients with metastatic pancreatic cancer during the treatment with CAR133-NKT cells.
  • FIG. 25 shows the clinical response of CAR133-NKT cells in patients with pancreatic cancer according to the Example 8 of the present invention.
  • This present invention provides a CAR, CD133ScFv-CD8-CD137-CD3 ⁇ , comprising a CD8a signal peptide, a single-chain antibody CD133ScFv, a CD8 hinge region and transmembrane region, CD137 and CD3 ⁇ intracellular signaling domains connected in series.
  • the CAR consists of a CD8a signal peptide, CD133ScFv, a CD8 hinge region and transmembrane region, CD137 and CD3 ⁇ intracellular signaling domains connected in series.
  • the CAR has the amino acid sequence as shown in SEQ ID NO. 1, and further preferably, the amino acid sequence of the CAR consists of the sequence of SEQ ID NO. 1.
  • the invention provides a gene encoding the CAR.
  • the gene has a nucleotide sequence as shown in SEQ ID NO. 2, and more preferably, the nucleotide sequence of the gene encoding the CAR consists of the sequence of SEQ ID NO. 2.
  • the invention provides a recombinant expression vector containing the above gene, preferably, the recombinant expression vector is a lentiviral expression vector.
  • the lentiviral expression vector is not particularly defined as long as it can co-infect the packaging cells, such as 293T packaging cells, with the assistant vector to obtain the virus concentrate liquid and CD133ScFv-CD8-CD137-CD3 ⁇ modified NKT cells, and more preferably, the lentiviral expression vector is PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ .
  • the method for preparing the lentiviral expression vector PWPT-CD133 ScFv-CD8-CD137-CD3 ⁇ is not particularly limited, and various methods that could be thought of by the person skilled in the art may be employed.
  • the method for preparing lentiviral expression vector PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ comprises the following steps:
  • step (1) the amplification of the CD8 hinge region and transmembrane region, the CD137 intracellular signaling domain and the CD3 ⁇ intracellular signaling domain from the NKT cell cDNAs, are not particularly limited, and all commonly used methods in the art may be employed, for example, RT-PCR method.
  • the NKT cell may be obtained by isolating mononuclear cells from human venous blood and then culturing.
  • the method to prepare PWPT-CD8-CD137-CD3 ⁇ comprises: extracting the total RNA from the NKT cells and obtaining NKT cell cDNA by reverse transcription; using the cDNA from the NKT cells as a template and the primers P1 (SEQID NO. 11) and P2 (SEQID NO. 12) to perform the PCR amplification to obtain the hinge region and transmembrane region of the CD8 gene (SEQID NO. 3) , using the primers P3 (SEQID NO. 13) and P4 (SEQID NO. 14) to perform the PCR amplification to obtain the intracellular signaling structural domain of the CD137 gene (SEQID NO. 4) , using the primers P5 (SEQID NO.
  • step (2) the method for synthesizing the nucleotide sequence encoding the CD8a signal peptide and the CD133ScFv is not particularly limited, and various common methods in the art may be employed, for example, the synthesis by whole-genome synthesis technique.
  • the method to obtain PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ with the correct sequence might comprise: synthesizing the nucleotide sequences (SEQID NO. 8) encoding the CD8a signal peptide and the CD133ScFv fusion protein through the whole-genome synthesis technique, and cloning into the vector pGSI to obtain pGSI-CD133ScFv, performing a BamHI/MluI double digestion on pGSI-CD133ScFv and then connecting the fragments with the recombinant plasmid PWPT-CD8-CD137-CD3 ⁇ obtained in step (1) by a BamHI/MluI double digestion.
  • the PWPT-CD133 ScFv-CD8-CD137-CD3 ⁇ with the correct sequence is obtained, of which, the nucleotide sequence of CD8a signal peptide consists of the sequence of SEQID NO. 6, and the nucleotide sequence of CD133ScFv consists of the sequence of SEQID NO. 7.
  • the invention further provides an engineered CD133-targeted NKT cell, which is modified by the CD133ScFv-CD8-CD137-CD3 ⁇ (i.e., CAR133-NKT cell) .
  • the invention further provides a method for preparing the engineered CD133-targeted NKT cells, comprising: packaging a lentivirus carrying PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ to obtain the virus concentrate and infecting the NKT cells using the virus concentrate to make the NKT cells express the CD133 CAR containing CD133ScFv, a CD8 hinge region and transmembrane region, a CD137 intracellular signaling domain and a CD3 ⁇ intracellular signaling domain.
  • the method for packaging the lentivirus carrying PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ is not particularly limited, and various methods commonly used by those skilled in the art may be employed herein, preferably, co-infecting 293T package cells with the lentiviral expression vector PWPT-CD133 ScFv-CD8-CD137-CD3 ⁇ with an assistant plasmid (e.g., psPAX2, pMD2.
  • an assistant plasmid e.g., psPAX2, pMD2.
  • the method further comprises the preparation of the NKT cells by the following steps:
  • step (1) is carried out in a first NKT cell culture fluid, which contains NKT cell culture medium, 30-70 ng/mL of CD3 mAb, 300-700 U/mL of IL-2 and 30-70 ng/mL of IL-15.
  • step (2) is carried out in a second NKT cell culture fluid, which contains the NKT cell culture medium and 300-700 U/mL of IL-2.
  • the NKT cell culture medium is not particularly limited and various media commonly used for culturing NKT cells in the art may be employed, for example, GT-T551 culture medium.
  • the conditions for culturing in the first culture stage and the second culture stage are not particularly limited when preparing the NKT cells, and various conditions commonly used in the art may be employed, for example, culturing in a CO 2 incubator at 30-37°C and with saturated humidity of 3-6%.
  • the person skilled in the art may adjust the culture time, which is well known in the art and not discussed here.
  • the average rate of CD3 + cells is >90%, the average rate of CD3 + CD8 + cells among the total CD3 + cells is >70%; and the average rate of CD3 + CD56 + cells among the total CD3 + cells is >15%.
  • the method for infecting the NKT cells is not particularly limited, and various methods commonly used in the art may be employed, preferably, this method comprises the following steps:
  • step (a) is carried out in a third NKT cell culture fluid, which contains the NKT cell culture medium, the virus concentrate, protamine, and 300-700 U/mL of IL-2.
  • step (b) is carried out in the first NKT cell culture fluid.
  • first NKT cell culture fluid please refer to the aforementioned related content; and it is not described again.
  • the conditions for the culture in the first stage of infectious culture and the second stage of infectious culture are not particularly limited when infecting the NKT cells, and various conditions commonly used in the art may be employed, for example, culturing in a CO 2 incubator at 30-37°C with a saturated humidity of 3-6%.
  • the person skilled in the art may adjust the culture time, which is well known in the art and not discussed here.
  • the method for infecting the NKT cells comprises: taking 1 ⁇ 10 7 to 5 ⁇ 10 7 NKT cells, discarding the old culture fluid and adding 2-4 mL of fresh GT-T551 culture medium and then adding 200-400 ⁇ L of virus concentrate, 2-4 ⁇ L of 1 ⁇ 10 -6 mg/mL protamine and IL-2 at a final concentration of 300-700 U/mL, placing into a CO 2 incubator at 30-37°C with a saturated humidity of 3-6%for infection for 12-16 h, then discarding the culture fluid, transferring the cells to an uncoated culture flask, adding 20-50 mL of GT-T551 culture medium, and then adding IL-2 at a final concentration of 300-700 U/mL, CD3 mAb at a final concentration of 30-70 ng/ml and IL-15 at a final concentration of 30-70 ng/mL, and culturing in a CO 2 incubator at 30-37°C with a saturated humidity of 3-6%for 12-18 h to obtain the CD133S
  • the method for infecting the NKT cells further comprising the following steps:
  • step (c) is carried out in the second NKT cell culture fluid
  • step (d) is carried out in the first NKT cell culture fluid.
  • compositions of the first NKT cell culture fluid and the second NKT cell culture fluid please refer to the aforementioned related contents; it is not described again.
  • the method for infecting the NKT cells further comprises an in vitro induction of the NKT cells infected with lentivirus obtained in the second stage of infectious culture in a GT-T551 culture medium containing IL-2 at a final concentration of 300-700 U/mL, and when the cell density is 80-90%, transferring the cells in a cell culture bag and adding fresh GT-T551 culture medium with IL-2 at a final concentration of 300-700 U/mL, CD3 mAb at a final concentration of 30-70 ng/ml and IL-15 at a final concentration of 30-70 ng/mL for amplification every 1.5-2.5 days, and amplifying the cells to a total amount of 1 ⁇ 10 9 to 2 ⁇ 10 9 .
  • the CD133-targeted CAR After the CD133-targeted CAR is infected with lentivirus, its infection rate could be as high as 30%-60%in the obtained CAR133-NKT cells, and the ratio of the CD3 + CD56 + cells among the total CD3 + cells is within the range of 15%-40%.
  • the amino acid sequences of the CAR protein expressed in the NKT cells modified by CAR consist of the sequence of SEQID NO. 1.
  • the CAR precursor protein is composed of a CD8a signal peptide, CD133 ScFv, CD8 hinge region and transmembrane region, CD137 intracellular signaling domain and a CD3 ⁇ intracellular signaling domain connected in series, and through protein translation, it will become a mature CAR protein after the signal peptides are removed in rough endoplasmic reticulum of cell and will then be located at the cell membrane of the NKT cells after secretion.
  • the gene coding sequences of the CAR protein corresponding to the amino acid sequences consist of the sequence of SEQID NO. 2.
  • the CAR uses the structure composed of the CD8 hinge region and transmembrane region and CD3 ⁇ and the CD137 intracellular signaling domains connected in series as the signal transduction structural domain, and its amino acid sequences consists of the sequence of SEQID NO. 9, and the corresponding gene coding sequences consist of the sequence of SEQID NO. 10.
  • the present invention further provides the engineered CD133-targeted NKT cells prepared according to the above method.
  • the present invention further provides the use of the engineered CD133-targeted NKT cells in preparing the formulations for treating tumors.
  • the tumor is a CD133-positive epithelial tumor, and more preferably, the epithelial tumors are colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumor, melanoma and/or neuroglioma, etc.
  • compositions of the formulations used for the treatment of CD133-positive tumors are not particularly limited in the use of the present invention as long as they are provided with the compositions containing the CAR133-NKT cells or are made by CAR133-NKT cells.
  • the compositions and preparation methods of the formulations are well-known to the person skilled in the art and are not discussed here.
  • the CAR133-NKT cells in the present invention can identify CD133-positive cells, including tumor-initiating cells, tumor stem cells, epithelial progenitor cells and tumor cells, and exert targeted killing activity to kill different types of CD133-positive cells., It can not only eliminate the tumor in situ and inhibit metastasis of tumor cells but also reduce the recurrence of tumors.
  • the present invention further provides a method for treating CD133-positive epithelial tumors and/or inhibiting tumor recurrence and metastasis, comprising: infusing the engineered CD133-targeted NKT cells to patients with CD133-positive epithelial tumors.
  • the epithelial tumors are selected from the group consisting of colorectal cancer, liver cancer, bile duct cancer, pancreatic cancer, esophageal cancer, stomach cancer, ovarian cancer, lung cancer, prostate cancer, bladder cancer, breast cancer, endometrial cancer, brain tumor, melanoma and neuroglioma.
  • NKT cell culture medium GT-T551 was purchased from TaKaRa Corp.
  • Lymphocyte separation medium was purchased from TBD Corp.
  • recombinant fibronectin was purchased from TaKaRa Corp.
  • CD3 mAb, CD4 mAb, CD8 mAb, CD56 mAb and CD107a mAb were purchased from BD Corp.
  • Recombinant human protein interferon - ⁇ , recombinant human IL-2, and recombinant human IL-15 were purchased from protech Corp.
  • RNA extraction kit RNAiso Reagent high-fidelity DNA polymerase (HS DNA Polymerase) and T4 DNA ligase were purchased from TaKaRa Corp.
  • RevertAid TM First Strand cDNA Synthesis Kit was purchased from Fermentas Corp.
  • Agarose gel DNA extraction kit ordinary DNA product purification kit and plasmid extraction kit were purchased from TIANGEN Biotech Co., Ltd.
  • PWPT-GFP PWPT-GFP
  • psPAX2 pMD2. G were purchased from Addgene Corp.
  • pGSI was purchased from Beijing Tianyi Huiyuan Biotech Co., Ltd..
  • Trans1-T1 Phage Resistant chemically competent cells were purchased from Beijing TransGen Biotech Co., Ltd..
  • Lipofectamine TM 2000 Transfection Reagents were purchased from Invitrogen Corp.
  • 293T packaging cells were purchased from U.S. ATCC.
  • the final concentration of PEG6000 in PEG6000-NaCl was 25.5 wt%and the final concentration of NaCl was 1.2M, both PEG6000 and NaCl were purchased from Shanghai Suolaibao Bio-technology Co., Ltd..
  • Fetal bovine serum was purchased from German PAA.
  • CD107a -PECy5 antibody was purchased from U.S. BD Biosciences.
  • CD133-positive HT29 colorectal cancer cell lines were purchased from U.S. ATCC.
  • CD133-positive human liver cancer cell Hep3B was purchased from U.S. ATCC.
  • CD133-positive human pancreatic cancer cells SW1990 was purchased from U.S. ATCC.
  • CD133-positive human colorectal cancer cell DLD1 was purchased from U.S. ATCC.
  • CD133-positive human colorectal cancer cell SW620 was purchased from U.S. ATCC.
  • CD133-negative human colorectal cancer cell LOVO was purchased from U.S. ATCC.
  • CD133-negative human liver cancer cell HepG2 was purchased from U.S. ATCC.
  • 5-carboxy-fluorescein succinimidyl ester was purchased from Shanghai Puzhen Biotech Co., Ltd..
  • Annexin V-RPE kit was purchased from BD Biosciences.
  • MethoCult TM H4434 classic cell medium were purchased from stem cell corp.
  • PBMCs mononuclear cells
  • the PBMCs were adjusted to a final concentration of 2 ⁇ 10 6 cells/mL using the NKT cell culture medium GT-T551 containing 0.6%human autologous serum (by volume) .
  • the cells were inoculated into a 75 cm 2 cell culture flask coated with RetroNectin at final concentration of 10 ⁇ g/mL, then recombinant human IL-2 was added at a final concentration of 500 U/mL, CD3 mAb was added at a final concentration of 50 ng/ml and recombinant human IL-15 was added at a final concentration of 50 ng/ml to the culture medium, and the cells were cultured in a CO 2 incubator at 37°C with a saturated humidity of 5%.
  • NKT cell culture medium GT-T551 was added according to the number of cell growth every two days to control the cell concentration at 1 ⁇ 10 8 cells/mL, and recombinant human IL-2 was added at a final concentration of 500 U /ml.
  • the NKT cells were obtained, and a phenotypic analysis of NKT cells was performed using flow cytometry. The results are shown in FIG. 1, of which, CD3 + : 95.04%; CD3 + CD8 + : 90.99%; CD3 + CD56 + : 24.12%; and CD8 + CD56 + : 24.63%.
  • Example 2 Centrifuge to precipitate the NKT cells obtained in Example 1, extract the total RNA of cells using Total RNA extraction kit RNAiso Reagent, and then preserve at -80°C for standby.
  • the extracted total RNA was reversely transcribed using the reverse transcription kit RevertAid TM First Strand cDNA Synthesis Kit to obtain cDNA of NKT cells, and then preserved at -20°C for standby.
  • primer sequences were designed and synthesized (of which, the underline is marked as protective base and the blocks indicate the cleavage sites) :
  • step (1) using NKT cell cDNA as a template, and using the primers P1 and P2 to perform PCR amplification to obtain CD8 hinge region and shortened transmembrane region with a length of 227 bp.
  • the nucleotide sequence was shown as SEQID NO. 3, containing BglIIand MluI restriction sites and protection bases at both ends; then PCR amplification was performed using primers P3 and P4, to obtain CD137 intracellular signaling domain with a length of 146 bp, and the nucleotide sequence was shown as SEQID NO.
  • PCR amplification reaction system in each step was identical. Taking amplification of CD137 intracellular signaling domain as an example, PCR was amplified, and PCR reaction conditions were in accordance with the instructions of HS DNA Polymerase, and the reaction system (50 ⁇ L) was as follows:
  • Double distilled water 32.5 ⁇ L
  • reaction buffer 10 ⁇ L
  • NKT cell cDNA (200ng /ul) : 1 ⁇ L
  • the above PCR product was separated using 1%agarose gel, and the DNA fragments were recovered by agarose gel DNA extraction kit; when the fragments were obtained, double digestion reaction was performed and the enzyme-digested products were recovered by ordinary DNA product purification kit for standby.
  • the lentiviral expression vector PWPT-GFP was double digested by MluI/SalI, and the enzyme-digested product was separated with 1%agarose gel, then large vector fragment was recovered by agarose gel DNA extraction kit, then ligated with the previously recovered CD8, CD137, CD3 ⁇ fragments through T4 DNA ligase.
  • the ligation product was used to transform Trans1-T1 Phage Resistant chemically competent cells, after cultured at 37°C for 16h, the monoclone was picked and cultured for 12 h at 37°C, 250 rpm, and then the plasmid was extracted by plasmid extraction kit.
  • the extracted plasmid was subject to restriction endonucleases MluI and SalI double digestion identification and the electrophoretogram was shown in FIG. 2, of which, M1: DNA molecular weight marker D4500; Lane 1: No restriction fragment of plasmid PWPT-CD8-CD137-CD3 ⁇ ; Lane 2: plasmid PWPT-CD8-CD137-CD3 ⁇ cleavage fragment (672bp) ; M2: DNA molecular weight marker D2000.
  • M1 DNA molecular weight marker D4500
  • Lane 1 No restriction fragment of plasmid PWPT-CD8-CD137-CD3 ⁇
  • Lane 2 plasmid PWPT-CD8-CD137-CD3 ⁇ cleavage fragment (672bp)
  • M2 DNA molecular weight marker D2000.
  • the plasmids correctly identified were sent to Beijing Tianyi Huiyuan Biotech Co., Ltd. for sequencing the inserted fusion gene fragments.
  • PWPT-CD8-CD137-CD3 ⁇ The recombinant plasmid with correct sequencing results was named PWPT-CD8-CD137-CD3 ⁇ , of which, the nucleotide sequence of CD8 hinge region and transmembrane region was shown as SEQID NO. 3, the nucleotide sequence of CD137 intracellular signaling structural domain was shown as SEQID NO. 4, and the nucleotide sequence of CD3 ⁇ intracellular signaling structural domain was shown as SEQID NO. 5.
  • the plasmid PWPT-CD8-CD137-CD3 ⁇ was digested by restriction endonuclease BamHI/MluI, and the enzyme-digested product was separated with 1%agarose gel, then the vector fragment was recovered by agarose gel DNA extraction kit, then ligated with the recovered DNA fragments containing CD8a signal peptide and CD133 ScFv through T4 DNA ligase, and for the specific method, please refer to the instructions.
  • the ligation product was used to transform Trans1-T1 Phage Resistant chemically competent cells, after cultured at 37°C for 16h, the monoclone was picked and cultured for 12 h at 37°C, 250 rpm, and then the plasmid was extracted by plasmid extraction kit.
  • the extracted plasmid was subject to restriction endonucleases BamHI/SalI double restriction analysis and the electrophoretogram was shown in FIG.
  • M1 DNA molecular weight marker D15000
  • Lane 1 No restriction fragment of plasmid PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ (10144bp)
  • Lane 2 plasmid PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ cleavage fragment (1515 bp)
  • M2 DNA molecular weight marker D2000.
  • the plasmids correctly identified were sent to Beijing Tianyi Huiyuan Biotech Co., Ltd. for sequencing the inserted fusion gene fragments.
  • the recombinant plasmid with correct sequencing results was named PWPT-CD133ScFv-CD8-CD137-CD3 ⁇ , and its structural diagram was shown in FIG.
  • CD8a signal peptide (nucleotide sequence was shown as SEQID NO. 6)
  • anti-CD133 single-chain antibody nucleotide sequence was shown as SEQID NO. 7
  • CD8 hinge region and transmembrane region CD137 intracellular signaling domain and CD3 ⁇ intracellular signaling domain
  • the CAR employed the structure composed of gene CD8 hinge region and transmembrane region as well as CD137 and CD3 ⁇ intracellular signaling domains connected in series as the signal transduction domain, its amino acid sequence was shown as SEQID NO. 9, and the corresponding gene coding sequence was shown as SEQID NO. 10.
  • the virus supernatant was collected in a 50mL EP tube and centrifuged for 10 min at 4°C, 2000 g, then the supernatant obtained twice were transferred to a new EP tube, and the virus supernatant was filtered through a 4.5 ⁇ m filter; the filtered virus supernatant was mixed well with 5 ⁇ PEG6000-NaCl according to a volume ratio of 4: 1, standing for 2h at 4°C, then centrifuged at 4°C, 10000 g for 20 min; then the supernatant was removed and the precipitate was dissolved in 1 mL of sterile PBS pre-cooled at 4°C, to obtain virus concentrate of the CAR, and then subpackaged according to 100 ⁇ L each tube, and preserved at -80°C for standby.
  • a lentivirus expression plasmid PWPT-GFP and assistant plasmids psPAX2, pMD2. G were used to co-transfect 293T packaging cells, and then the viral supernatant was collected and concentrated to obtain the lentivirus concentrates expressing green fluorescent protein (GFP) .
  • G green fluorescent protein
  • lentivirus concentrate expressing GFP was used to simultaneously infect NKT cells (the obtained NKT cells are called CART-GFP cells) , to calculate the infection efficiency of the virus.
  • the infected cells were transferred to a 75 cm 2 culture flask uncoated by CD3 and retronectin, and 20 mL of NKT cell culture medium GT-T551, recombinant human IL-2 at a final concentration of 500 U/mL and CD3 mAb at a final concentration of 50 ng/ml and recombinant human IL-15 at a final concentration of 50 ng/mL were added, and placed at a CO 2 incubator with the temperature of 37°C and saturated humidity of 5%for infection for 18 h, to obtain the NKT cells (called CAR133-NKT cells) .
  • the CAR33-NKT cells prepared according to the method disclosed in CN 105384823 A were used as Mock cells.
  • the virus infection efficiency was detected by a flow cytometry, and results were shown in FIG. 5.
  • the infection efficiency of CAR133-NKT cells was 44.5%.
  • NKT cell culture medium GT-T551 containing recombinant human IL-2 at a final concentration of 500 U/mL were induced in vitro with NKT cell culture medium GT-T551 containing recombinant human IL-2 at a final concentration of 500 U/mL; when the cell density was 85%, the cells were transferred into a cell culture bag, and every two days the fresh GT-T551 culture fluid comprising the IL-2 at a final concentration of 500 U/mL, CD3 mAb at a final concentration of 50 ng/ml and IL-15 at a final concentration of 50 ng/mL was added for amplification; when the cells were amplified to a total amount of 1.5 ⁇ 10 9 cells, the infected cell mass was identified using a flow cytometry.
  • the CAR133-NKT cells and CAR33-NKT cells prepared in Example 3 and the NKT cells cultured in Example 1 were inoculated in 96-well plates, and then were co-cultured with epithelial tumor cells at different CD133 expression levels (high-CD133 + Americans: Hep3B, SW1990; medium-CD133 + Systems: HT29, DLD1, and CD133 - Systems: LOVO, HepG2) at an effector-target ratio (killer cells: target cells) of 20: 1.
  • monensin was added at a final concentration of 2 ⁇ mol/L for 2 h, and then the cells were incubated with a PE-labeled CD107a antibody for 15 min, washed with PBS buffer for three times, and then CD107a expression level was analyzed by flow cytometry.
  • the CAR133-NKT cells and CAR33-NKT cells prepared in Example 3 and the NKT cells cultured in Example 1 were inoculated in 96-well plates, and were then stained with 5-carboxyfluorescein succinimidyl ester (CFSE) .
  • the cells were then co-cultured with epithelial tumor cells with different CD133 expression levels (high-CD133 + Americans: Hep3B, SW1990; medium-CD133 + Systems: HT29, DLD1, and CD133 - Systems: LOVO, HepG2) at an effector-target ratio (killer cells: target cells) of 20: 1.
  • FIG. 7 (A) -FIG. 7 (C) the cytotoxic activity of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels (4 h) is shown in FIG. 8, and the cytotoxic activity of CAR133-NKT cells on epithelial tumor cells with different CD133 expression levels (8 h) is shown in FIG. 9.
  • the CAR133-NKT cells had a specific cytotoxicity on the high-CD133+ cancer cells and the medium-CD133+ cancer cells, but had no cytotoxicity on CD133-cells; moreover, the specific cytotoxicity of the CAR133-NKT cells was apparently superior to the NKT cells and the mock cells (CAR33-NKT cells) .
  • Cord blood was obtained to separate the primitive progenitor cells using a Ficoll mononuclear separating medium and was then mixed with CAR133-NKT cells, NKT cells, physiological saline and mock cells (CAR33-NKT cells) at a ratio of 1: 20, and then, the cells were inoculated into a MethoCult TM H4434 Classic cell culture medium and cultured at a 5%CO 2 incubator at 37°C for two weeks; then, the clones were counted.
  • the hematopoietic colony formation functional analysis is shown in FIG. 10, and this figure shows the number of clones of hematopoietic cells, wherein BFU-E is burst forming unit: early erythroid progenitor cell, CFU-GM was colony forming unit: granulocyte and macrophage, and CFU-GEMM was colony forming unit: granulocyte, erythrocyte, monocyte and megakaryocyte, and CFU-E was colony forming unit: early erythroid progenitor cell.
  • the CAR133-NKT cells in the present invention may pose no irreversible myelosuppression. Hematopoietic stem cells also express CD133; therefore, its influence on the hematopoietic system was eliminated to guarantee the safety of CAR133-NKT cell therapy.
  • CAR133-NKT cells were obtained and diluted by 100 ml of physiological saline and were then intravenously transfused into patients with CD133-positive liver cancer in an increasing dose for three consecutive days (patients have received multiple types of treatment, such as radiotherapy, chemotherapy and other medication symptomatic treatment before receiving CAR133-NKT cell targeted immunotherapy, but no obvious curative effect was achieved) ; wherein UPN 1 (patient 1) , UPN 2 (patient 2) and UPN 3 (patient 3) received a safe dosage infusion, and the total infusion doses for the three consecutive days were 0.03 ⁇ 10 7 /kg ⁇ 0.06 ⁇ 10 7 /kg and 0.03 ⁇ 10 7 /kg, respectively; UPN 1 (patient 1) , UPN 2 (patient 2) , UPN 3 (patient 3) and UPN 4 (patient 4) received the dosage infusion with minimal biological activity, and the total infusion doses for the three consecutive days were 0.39 ⁇ 10 7 /kg ⁇ 0.25 ⁇ 10 7 /kg ⁇ 0.4 ⁇ 10 7 /kg and 0.33 ⁇
  • the doses for treating patients by infusing CAR133-NKT cells were divided into three gradients as follows: cohort (1) the safe dosage infusion with a cell infusion amount of 0.01-0.06 ( ⁇ 10 7 /kg) ; cohort (2) the minimal biological activity dosage with a cell infusion amount of 0.1-0.5 ( ⁇ 10 7 /kg) ; and cohort (3) the clinical response dosage with a cell infusion amount of 0.5-1.0 ( ⁇ 10 7 /kg) .
  • FIG 11 to FIG 13 show the change of CAR133 gene copy in peripheral blood after CAR133-NKT cells are infused into patients with liver cancer.
  • FIG. 14 to FIG 16 show the change of the cell count of CD133 positive cells in the corresponding patients. The number of copies of CAR133-NKT cells was negatively correlated with the percentage of CD133+ cells, and the reverse correlation became more after infusing clinically effective dose of CAR133-NKT cells into patients with liver cancer, suggesting that the CAR133-NKT cells kill the CD133+ targeted cells.
  • FIG. 17 shows the treatment effects of CAR133-NKT cells on 13 patients with liver cancer, of which, two patients stop treatment after the first cohort, one patient had disease progression after the first cohort and achieved stable disease after the second cohort. As for the remaining nine patients, they achieved stable diseases after the first cohort. It shows that CAR133-NKT cells are a feasible and possibly effective treatment modality for patients with liver cancer.
  • CAR133-NKT cells were obtained and diluted by 100 ml of physiological saline and were then intravenously transfused into patients with CD133-positive pancreatic cancer or colorectal cancer in the period of disease progression or disease metastation in an increasing dose for three consecutive days (the patients had received multiple types of treatment, such as radiotherapy, chemotherapy and other medication symptomatic treatment before receiving CAR133-NKT cell targeted immunotherapy, but no obvious curative effect was achieved) . After the infusion, the clinical efficacy was assessed.
  • Patient 1 and patient 2 were subjected to three cycles of clinical response dosage treatment, wherein the amount of cell infusion for patent 1 for the three consecutive days was 0.3 ⁇ 10 7 /kg, 0.6 ⁇ 10 7 /kg and 0.8 ⁇ 10 7 /kg, and the amount of cell infusion for patent 2 for the three consecutive days was 0.3 ⁇ 10 7 /kg, 0.6 ⁇ 10 7 /kg and 0.7 ⁇ 10 7 /kg.
  • Patient 3, 4 and 5 were subjected to one cycle of clinical response dosage cell infusion, wherein the amount of cell infusion for the three consecutive days was 0.5 ⁇ 10 7 /kg, 0.8 ⁇ 10 7 /kg and 0.8 ⁇ 10 7 /kg. After the infusion, the clinical efficacy was assessed.
  • FIG. 18 to FIG. 21 show the blood system adverse event of CAR133-NKT cells for treating patients with metastatic pancreatic cancer, presenting the curve of hemoglobin (Hgb) , white blood cell (WBC) , platelet (PLT) and the reticulocyte (Ret) after cell infusion at different days. As shown, hemoglobin and platelet were slight decreased, the adverse effects were low, and patients were well tolerated, suggesting that it is safe to treat metastatic pancreatic cancer patients with CAR133-NKT cells.
  • Hgb hemoglobin
  • WBC white blood cell
  • PHT platelet
  • Ret reticulocyte
  • FIG 22 (A) to FIG 22 (F) show the change of CAR133 gene copy in peripheral blood after clinically effective dose of CAR133-NKT cells are infused into patients with pancreatic cancer, colorectal cancer or rectal cancer.
  • CAR133 gene copy significantly increased in peripheral blood after CAR133-NKT cells were infused.
  • FIG. 23 show the number of CD133 positive cells after CAR133-NKT cells were infused in patients with pancreatic cancer, colorectal cancer or rectal cancer.
  • the number of CD133 positive cells was significantly decreased in peripheral blood and was maintained at a low level after CAR133-NKT cells were infused, suggesting that the CAR133-NKT cells kill the CD133+ targeted cells.
  • FIG. 24 (A) to FIG 24 (E) show the change of the cytokines including IL-2, IL-6, C-reaction related protein (CRP) , TNF-aand IL-8 in peripheral blood of patients with metastatic pancreatic cancer during the treatment with CAR133-NKT cells.
  • CRP C-reaction related protein
  • TNF-aand IL-8 TNF-aand IL-8 in peripheral blood of patients with metastatic pancreatic cancer during the treatment with CAR133-NKT cells.
  • the cytokines increased in peripheral blood after CAR133-NKT cells were infused into patients with pancreatic cancer, showing that CAR133-NKT cells are a feasible and possibly effective treatment modality for patients with metastatic pancreatic cancer.
  • FIG. 25 shows the clinical response of the CAR133-NKT cells in five patients with pancreatic cancer. As shown, four patients were stable disease one of whom had disease progress after 7 weeks of stability, and the remaining one patient’s disease was partial response for as long as 20 weeks, suggesting that the CAR133-NKT cells showed a favorable clinical response in patients with pancreatic cancer.

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