WO2020119048A1 - 一种包含肿瘤抗原识别受体的免疫细胞及其应用 - Google Patents

一种包含肿瘤抗原识别受体的免疫细胞及其应用 Download PDF

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WO2020119048A1
WO2020119048A1 PCT/CN2019/090998 CN2019090998W WO2020119048A1 WO 2020119048 A1 WO2020119048 A1 WO 2020119048A1 CN 2019090998 W CN2019090998 W CN 2019090998W WO 2020119048 A1 WO2020119048 A1 WO 2020119048A1
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sequence
seq
hyaluronidase
amino acid
cells
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王征
徐云霞
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苏州康聚生物科技有限公司
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Definitions

  • the invention belongs to the field of cell immunotherapy, and in particular relates to an immune cell containing a tumor antigen recognition receptor and its application.
  • Cancer also known as malignant tumor, is the first major disease and stubborn disease that seriously endangers human health and causes death of modern humans. According to the statistics of the United Nations, one in eight deaths globally is currently caused by cancer, which is higher than the death toll caused by AIDS, tuberculosis and malaria. And more than 10 million people worldwide are diagnosed with cancer each year (World Health Organization's "2014 World Cancer Report").
  • the biggest obstacle for CAR-T cells in the treatment of solid tumors is the tumor immunosuppression microenvironment, which is the internal environment formed during the development of tumors.
  • Malignant solid tumors are mainly composed of extracellular matrix of cancer/tumor cells and stromal cells (such as fibroblasts and inflammatory cells) and blood vessels/lymph vessels.
  • stromal cells, extracellular matrix and vascular/lymphatic system in tumor tissues are generally abnormal, with an increased number of fibroblasts, and the vascular system exhibits a highly branched or curled structure.
  • Tumors and stromal cells produce and assemble extracellular matrix components (collagen, proteoglycan, hyaluronic acid) to form dense clumps (Clin Oncol 31: 2205-2218.2013).
  • extracellular matrix components collagen, proteoglycan, hyaluronic acid
  • the above factors caused the dense tissue structure of the tumor, nutritional deficiencies, low pH, and hypoxia, which inhibited CAR-T cells to effectively exert tumor killing effects.
  • CAR-T cells In order to effectively treat tumors, CAR-T cells must be able to overcome the above microenvironment, enter the tumor tissue, survive in the tumor tissue and kill the tumor cells.
  • CAR-T cells targeting the tumor microenvironment that is, CAR-T cells expressing hyaluronidase PH20 (patent CN107400664A) may have been produced, there are problems with the technical means by which such cells are produced, and the principles and techniques of exerting a killing effect The effect was not revealed.
  • CN107400664A believes that in its natural state, hyaluronidase is expressed in the form of secreted protein (ie free hyaluronidase) rather than in the form of transmembrane protein.
  • the hyaluronidase or hyaluronidase transmembrane fusion protein gene sequence of the hyaluronidase coding sequence may be the entire protein coding sequence or a partial sequence containing hyaluronidase activity.
  • the technical method for preparing PH20CAR-T cells that secrete hyaluronidase has not been published, and it is pointed out that the transmembrane hyaluronidase is more effective than the free diffuse hyaluronidase.
  • the method for preparing CAR-T cells expressing the hyaluronidase transmembrane fusion protein disclosed in the specific examples that is, the hyaluronidase PH20 transmembrane fusion protein lentivirus is used to infect CAR-T cells, and the hyaluronidase is used to infect CAR-T cells.
  • the coding sequence of the PH20 transmembrane fusion protein is composed of PH20-human IgG4 hinge region-CD8 transmembrane region-CD28 intracellular region-4-1BB intracellular region-CD3 ⁇ intracellular sequence.
  • the present inventors found through experiments that it is difficult to obtain CAR positive cells with hyaluronidase activity by using the preparation method for expressing hyaluronidase transmembrane fusion protein disclosed in CN107400664A; on the other hand, two lentiviruses were used to infect T After the cells, T cells were in poor condition, and CAR positive cells could hardly detect hyaluronidase activity.
  • the technical problem to be solved by the present invention is to overcome that the current prior art cannot obtain CAR-T cells with hyaluronan-degrading activity, and at the same time, the CAR positive rate is also very low or CAR-T cells containing hyaluronidase are difficult to secrete Hyaluronidase cannot effectively obtain defects of immune cells containing tumor antigen recognition receptors, and provides an immune cell containing tumor antigen recognition receptors and its application.
  • the immune cells can enter the solid tumor to kill the tumor cells; with the help of the soluble PH20 protein, the immune cells containing the tumor antigen recognition receptor can also enter the solid tumor to kill the tumor cells.
  • the present invention provides an immune cell containing a tumor antigen recognition receptor, which expresses hyaluronidase, and the hyaluronidase is:
  • Hyaluronidase is a family of enzymes that degrade hyaluronic acid. In humans, there are 6 genes encoding hyaluronidase with different properties and positions, HYAL1, HYAL2, HYAL3, HYAL4, HYAL5 (also known as SPAM1 or PH-20), HYAL6 (also known as HYALP1) . Isoforms HYAL1 and HYAL2 exist in most organizations. HYAL2 is a GPI membrane-anchored protein, which is mainly responsible for shearing high-molecular-weight hyaluronic acid.
  • hyaluronic acid After the obtained small fragments of hyaluronic acid are endocytosed into endolysosomes (Endolysosome), they are further degraded by HYAL1.
  • hyaluronic acid In tumor tissue, hyaluronic acid is synthesized by hyaluronic acid synthase (HAS1, HAS2, and HAS3).
  • HAS1, HAS2, and HAS3 hyaluronic acid synthase
  • HYAL3 is present in the bone marrow and testes, but its function has not been well characterized.
  • Hyaluronidase PH20 is highly expressed in the testes and participates in the process of fertilization of oocytes by sperm.
  • the hyaluronidase is preferably mammalian testis hyaluronidase; more preferably human testis hyaluronidase, also known as SPAM1 (sperm adhesion molecule 1), HYAL1, HYAL2, Or PH20.
  • SPAM1 sperm adhesion molecule 1
  • HYAL1 HYAL1, HYAL2, Or PH20.
  • the gene encoding the PH20 produces two transcript variants, corresponding to two forms of enzyme sequences:
  • One form is a PH20 sequence containing a membrane anchor sequence, which can be anchored on the surface of immune cells containing tumor antigen recognition receptors, namely hyaluronidase a) as described above. Therefore, immune cells containing tumor antigen recognition receptors expressing cell membrane anchored PH20 are obtained; the amino acid sequence of the hyaluronidase in the present invention is preferably as shown in SEQ ID No.
  • Hyaluronidase whose amino acid sequence has been modified by amino acid and retains its original function, such as: it has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO.1, such as 85%, 90%, 95 %, 98% or 99% sequence identity, and the amino acid sequence that maintains the original function of the hyaluronidase;
  • the nucleotide sequence encoding the PH20 is preferably as shown in SEQ ID NO. 2 or 3 in the sequence listing Or a nucleotide sequence as shown in SEQ ID NO. 2 or 3 with 80% or more sequence identity, for example, a core with 85%, 90%, 95%, 98% or 99% sequence identity Nucleotide sequence.
  • Another form is the deletion of the sequence corresponding to the carboxy-terminal membrane-binding domain.
  • the last amino acid of the deleted sequence is usually located at positions 430-454 of the full-length sequence to produce a soluble PH20.
  • the deletion of the terminal domain results in the secretion of hyaluronidase into the extracellular medium. Therefore, CAR or TCR-containing T cells or NK cells expressing secretory hyaluronidase having enzymatic activity under neutral and acidic pH conditions were obtained, and the NK cells were NK-92 cells; this form
  • the amino acid sequence of the hyaluronidase is the 1st to 447th positions of the amino acid sequence shown in SEQ ID NO.
  • the nucleotide sequence is the 1st to 1341th positions of the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3 in the sequence table, or the same as SEQ ID NO. 2 in the sequence table or
  • the nucleotide sequence shown in SEQ ID NO. 3 has a sequence identity of 80% or more at positions 1 to 1341, for example, a nucleoside having a sequence identity of 85%, 90%, 95%, 98%, or 99% Acid sequence.
  • the tumor antigen recognition receptor is preferably chimeric antigen receptor (CAR) or T cell receptor (TCR), wherein:
  • the TCR comprises, for example, the T chain ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, or a combination of one or more of them, the ⁇ chain includes a variable region and a constant region, Preferably, the variable region is a variable region of human TCR alpha chain, and the constant region is a constant region of murine alpha chain.
  • the beta chain includes a variable region and a constant region, and the variable region It is the variable region of human TCR ⁇ chain, and the constant region is the constant region of murine ⁇ chain; wherein, the ⁇ chain of TCR and the ⁇ chain of TCR can be connected by a linker sequence, and the linker sequence can be P2A , T2A, E2A, F2A or IRES; preferably E6 TCR.
  • the CAR includes an intracellular region, a hinge region, and a transmembrane region.
  • the intracellular region, the hinge region, and the transmembrane region can all be conventional in the art.
  • the intracellular region of the CAR preferably includes a human. 4-1BB intracellular domain and/or human CD28 intracellular domain, and human CD3 ⁇ intracellular domain, more preferably including human 4-1BB intracellular domain, human CD28 intracellular domain, and human CD3 ⁇ intracellular domain Area.
  • the hinge region of the CAR is preferably a human CD8 ⁇ hinge region, and the transmembrane region is preferably a human CD8 ⁇ transmembrane region.
  • the antigen recognition region, the hinge region, the transmembrane region and the intracellular region can be connected by a linker sequence, the linker sequence includes n repeating motifs, the motif is GGGS, For GGGGS, SSSSG, GGSSA, or GGSGG, n may be an integer of 1-5.
  • the antigen recognized by the tumor antigen recognition region of the tumor antigen recognition receptor may be EpCAM, Mesothelin, CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican2, Glypican3 , HER2, HPV antigen, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, GPC3 or MSLN; preferably ROR1, GPC3, MSLN or EpCAM.
  • the Mesothelin, the IL13R, the Podoplanin (PDPN), the EGFRvIII and the EGFR are all overexpressed in glioblastoma; the EpCAM is overexpressed in epithelial cell carcinoma.
  • the tumor antigen recognition region is a region capable of binding the above tumor antigens such as antibodies, and currently commonly used is scFv (single-chain antibody).
  • the nucleotide sequence of the scFv that identifies the ROR1 is as shown in SEQ ID NO. 4 in the sequence table, or it has more than 80% of the nucleotide sequence shown in SEQ ID NO. 4 in the sequence table Identity
  • nucleotide sequence of the scFv that identifies the GPC3 is as shown in SEQ ID NO. 5 in the sequence table, or it has more than 80% sequence identity with the nucleotide sequence shown in SEQ ID NO. 5 in the sequence table ;
  • the nucleotide sequence of the scFv that identifies the EpCAM is as shown in SEQ ID NO. 6 in the sequence table, or has more than 80% sequence identity with the nucleotide sequence shown in SEQ ID NO. 6 in the sequence table .
  • Vectors can be easily introduced into host cells by any method in the art, for example, mammalian, bacterial, yeast or insect cells.
  • expression vectors can be transferred into host cells by physical, chemical, or biological methods.
  • Physical methods include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, etc.;
  • chemical methods include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based Systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes; where biological methods include the use of DNA and RNA carriers.
  • the vector is delivered (eg, by transfection or electroporation) to cells, such as T cells or NK cells, wherein the vector contains hyaluronic acid encoding as described in the present invention
  • cells such as T cells or NK cells
  • the vector contains hyaluronic acid encoding as described in the present invention
  • a nucleic acid molecule of an enzyme which is transcribed into an mRNA molecule, and translated from the RNA molecule into the hyaluronidase and expressed on the surface of the cell.
  • the gene expressing the hyaluronidase may be on a different or the same vector as the gene expressing the tumor antigen recognition receptor in the immune cells.
  • the expression vector includes the following expression unit: signal peptide+scFv+human CD8 ⁇ hinge region+human CD8 ⁇ transmembrane region+human 4- 1BB intracellular domain+human CD3 ⁇ intracellular domain+connecting element+hyaluronidase, wherein the signal peptide is located at the 5′ end and the hyaluronidase is located at the 3′ end; or, the TCR and the When the hyaluronidase is located in the same expression vector, the expression vector includes the following units: E6 TCR + connecting element + hyaluronidase, wherein the E6 TCR is located at the 5'end, and the hyaluronidase is located 3'end;
  • one of the expression vectors includes the following expression unit: signal peptide+scFv+human CD8 ⁇ hinge region+human CD8 ⁇ Transmembrane region + human 4-1BB intracellular region + human CD3 ⁇ intracellular region, or include the following expression unit: E6 TCR + IRES, another expression vector includes the following expression unit: hyaluronidase + connecting element + tag protein , Wherein the hyaluronic acid is located at the 5'end and the tag protein is located at the 3'end;
  • the connecting element is preferably a T2A connecting peptide, a P2A connecting peptide, an E2A connecting peptide, an F2A connecting peptide, or an IRES element;
  • the tag protein is preferably tEGFR.
  • the gene for expressing the hyaluronidase is located on the tumor antigen recognition receptor expression vector in the immune cells.
  • the expression vector described in the present invention may be a vector conventionally used in the art for overexpressing foreign genes in mammals, which is used for the expression of target genes.
  • the expression vector is suitable for replication and integration of eukaryotic cells As appropriate.
  • Typical cloning vectors contain transcription and translation terminators, initiation sequences and promoters that can be used to regulate the expression of the gene of interest.
  • it is a plasmid, bacteriophage, bacteriophage derivative, animal virus or cosmid; the use of viral vectors has become the most widely used method for inserting genes into mammalian cells (e.g., human cells).
  • retroviruses provide a convenient platform for gene delivery systems.
  • the selected genes can be inserted into the vector and packaged into retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to target cells in vivo or ex vivo.
  • Many retrovirus systems are known in the art.
  • the expression vector of the present invention uses animal virus as the expression vector, it is preferably retrovirus, adenovirus, adeno-associated virus, herpes virus or lentivirus, and more preferably retrovirus or lentivirus.
  • the gene expressing the hyaluronidase and the gene expressing the tumor antigen recognition receptor are located on the same expression vector or different expression vectors, both of them are in the immune cells containing the tumor antigen recognition receptor
  • the expression can be routine in the art; in order to ensure that the target gene can be efficiently expressed, the nucleotide sequence described in the present invention can be manipulated in various ways, for example: the expression of the hyaluronidase or tumor antigen recognition receptor Can be regulated by one or more regulatory sequences.
  • the regulatory sequence may be: (1) a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription, the terminator sequence is operably linked to the 3′ end of the nucleotide sequence encoding the polypeptide, and is selected Any terminator that is functional in the host cell can be used in the present invention; (2) a suitable leader sequence, an untranslated region of mRNA important for host cell translation, the leader sequence and the nucleotide sequence encoding the polypeptide 5′ The ends are operably linked, and any leader sequence that is functional in the host cell of choice can be used in the present invention; (3) a suitable promoter sequence, an example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • Another example is extended growth factor-1 ⁇ (EF-1 ⁇ ).
  • EF-1 ⁇ extended growth factor-1 ⁇
  • other constitutive promoter sequences can also be used, including but not limited to simian virus 40 (SV40) early promoter, mouse breast cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, EB virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters, such as but not limited to actin promoter, myosin promoter, heme Promoter and creatine kinase promoter.
  • SV40 simian virus 40
  • MMTV mouse breast cancer virus
  • HMV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV promoter avian leukemia
  • inducible promoters may also be considered.
  • the use of an inducible promoter provides a molecular switch that can turn on expression of the polynucleotide sequence operably linked to the inducible promoter when expression is desired, and turn off expression when expression is not desired.
  • inducible promoters include, but are not limited to, metallothionein promoter, glucocorticoid promoter, progesterone promoter, and tetracycline promoter.
  • the promoter in the present invention is preferably a cytomegalovirus promoter, adenovirus major late promoter, SV40 promoter, herpes simplex virus thymidine kinase promoter, CMV promoter, EF1 ⁇ promoter, ubiquitin C promoter , PGK promoter, IRES promoter, MMTV, HIV LTR promoter, MoMuLV promoter, ALV promoter, EBV promoter, RSV promoter or human gene promoter, the human gene promoter is a myosin promoter , Hemoglobin promoter, creatine kinase promoter, ⁇ -actin promoter, human IL-2 promoter, human IL-4 promoter, IFN promoter, E2F promoter or human GM-CSF promoter; more preferred EF1 ⁇ promoter.
  • the above immune cells may be conventional immune cells in the art, such as T cells or NK cells or macrophages; preferably, the T cells are T cells after activation by CD3 antibody stimulation; more preferably, the T cells are derived from Peripheral blood mononuclear cells (PBMC) for tumor patients.
  • T cells or NK cells or macrophages preferably, the T cells are T cells after activation by CD3 antibody stimulation; more preferably, the T cells are derived from Peripheral blood mononuclear cells (PBMC) for tumor patients.
  • PBMC Peripheral blood mononuclear cells
  • the present invention also provides an expression construct, which includes a tumor antigen recognition receptor expression cassette 1 and a hyaluronidase expression cassette 2;
  • the expression cassette 1 and the expression cassette 2 are sequentially connected by a connecting element, and the connecting element is preferably a T2A connecting peptide, a P2A connecting peptide, an E2A connecting peptide, an F2A connecting peptide or an IRES element;
  • the tumor antigen recognition receptor is a chimeric antigen receptor (CAR) or a T cell receptor (TCR); and/or, the hyaluronidase is a full-length protein of hyaluronidase or Hyaluronidase soluble protein.
  • the expression cassette 1 when the tumor antigen recognition receptor is a chimeric antigen receptor (CAR), the expression cassette 1 includes the following expression unit: signal peptide+scFv+human CD8 ⁇ hinge region+human CD8 ⁇ transmembrane region+human 4 -1BB intracellular domain + human CD3 ⁇ intracellular domain, or when the tumor antigen recognition receptor is TCR, the expression cassette 1 includes the following expression unit: E6 TCR;
  • the scFv is a scFv that recognizes ROR1, GPC3, or EpCAM;
  • the nucleotide sequence of the scFv that recognizes ROR1 is preferably as shown in SEQ ID NO. 4 in the sequence table, or the same as SEQ ID NO in the sequence table
  • the nucleotide sequence shown in .4 has more than 80% sequence identity;
  • the nucleotide sequence of the scFv that recognizes GPC3 is preferably as shown in SEQ ID NO.
  • nucleotide sequence shown in .5 has more than 80% sequence identity; the nucleotide sequence of the scFv that recognizes EpCAM is preferably as shown in SEQ ID NO. 6 in the sequence table, or it is the same as SEQ ID NO in the sequence table.
  • nucleotide sequence shown in .6 has more than 80% sequence identity
  • amino acid sequence of the signal peptide is as shown in SEQ ID NO. 7 in the sequence table, or has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 7 in the sequence table;
  • amino acid sequence of the human CD8 ⁇ hinge region is as shown in SEQ ID NO. 11 in the sequence table, or has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 11 in the sequence table;
  • amino acid sequence of the human CD8 ⁇ transmembrane region is shown in SEQ ID NO. 12 in the Sequence Listing, or it has more than 80% sequence identity with the amino acid sequence shown in SEQ ID NO. 12 in the Sequence Listing ;
  • amino acid sequence of the intracellular region of human 4-1BB is as shown in SEQ ID NO. 13 in the Sequence Listing, or it has more than 80% of the amino acid sequence shown in SEQ ID NO. 13 in the Sequence Listing Identity
  • amino acid sequence of the human CD3 ⁇ intracellular region is as shown in SEQ ID NO. 14 in the sequence table, or has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 14 in the sequence table ;
  • the amino acid sequence of the E6 TCR is as shown in SEQ ID NO. 18 in the sequence table, or has 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO. 18 in the sequence table.
  • the expression cassette 2 expresses mammalian testis hyaluronidase, preferably human hyaluronidase HYAL1, HYAL2, SPAM1 or PH20; more preferably: the amino acid sequence of the full-length hyaluronidase protein is in order Listed in SEQ ID NO.1, or 80% or more sequence identity with the amino acid sequence shown in SEQ ID NO.1, for example, 85%, 90%, 95%, 98% or 99% sequence The amino acid sequence that is identical and maintains the original function of the hyaluronidase; the nucleotide sequence encoding the full-length protein of the hyaluronidase is preferably as shown in SEQ ID NO.
  • nucleotide sequence shown in SEQ ID NO. 2 or 3 has more than 80% sequence identity, for example, a nucleotide sequence with 85%, 90%, 95%, 98% or 99% sequence identity;
  • amino acid sequence of the hyaluronidase soluble protein is shown in the sequence table at positions 1 to 447 of the amino acid sequence shown in SEQ ID NO. 1 or as shown in the sequence table at SEQ ID NO.
  • the amino acid sequence at positions 1 to 447 of the amino acid sequence has 80% or more sequence identity, such as 85%, 90%, 95%, 98%, or 99% sequence identity, and retains the original hyaluronidase Functional amino acid sequence
  • the nucleotide sequence encoding the hyaluronidase soluble protein is shown in the sequence table in SEQ ID NO. 2 or SEQ ID NO. 3 of the nucleotide sequence shown in positions 1 to 1341 Or, it has 80% or more sequence identity with the 1st to 1341th positions of the nucleotide sequence shown in SEQ ID NO. 2 or SEQ ID NO. 3 in the sequence table, for example, it has 85%, 90%, 95% , 98% or 99% nucleotide sequence with sequence identity.
  • the present invention also provides an expression vector comprising the expression construct as described above.
  • the expression vector further contains a promoter; the promoter is preferably the EF1 ⁇ promoter.
  • the invention also provides a virus comprising the expression vector as described above; preferably, the virus is a lentivirus.
  • the invention also provides a pharmaceutical composition comprising the immune cells as described above;
  • the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • the present invention also provides a pharmaceutical composition whose active ingredients include hyaluronidase and immune cells containing tumor antigen recognition receptors; preferably:
  • the concentration of the hyaluronidase in the pharmaceutical composition is 0.1 ⁇ g/ml-15 mg/ml, and/or the specific activity of the hyaluronidase is ⁇ 10,000 units/mg.
  • kits including kit A and kit B, where kit A includes immune cells containing tumor antigen recognition receptors; kit B includes hyaluronidase;
  • the administration of the kit A and the kit B are in no particular order.
  • the definition of the tumor antigen recognition receptor, the hyaluronidase, etc. in the pharmaceutical composition as described above or the kit as described above is also as described above.
  • the invention also provides an application of the above-mentioned immune cells, the above-mentioned pharmaceutical composition or the above-mentioned kit in the preparation of a medicine for treating tumors; the tumor is preferably a solid tumor.
  • the solid tumor is a conventional solid tumor in the art, preferably a solid tumor expressing hyaluronic acid, such as breast cancer, gastric cancer, melanoma, pancreatic cancer, liver cancer, glioblastoma, or lung cancer.
  • hyaluronic acid such as breast cancer, gastric cancer, melanoma, pancreatic cancer, liver cancer, glioblastoma, or lung cancer.
  • the reagents and raw materials used in the present invention are commercially available.
  • the positive progress effect of the present invention is that the immune cells containing the tumor antigen recognition receptor of the present invention can change the extracellular matrix structure of the microenvironment to normalize the effect of the tumor immune microenvironment, so that it can effectively approach and kill the tumor cells .
  • immune cells containing tumor antigen recognition receptors can also improve the tumor microenvironment and effectively kill tumor cells.
  • Figure 1 shows the pPWT plasmid.
  • Figure 2 is an immunohistochemistry chart of selected samples of pancreatic cancer PDX; a. HE staining; b. MSLN staining; c. HA staining; d. HA staining after PH20 treatment; the scale bar in the figure is 100 ⁇ m.
  • Figure 3 is an immunohistochemistry chart of selected samples of gastric cancer PDX; a. HE staining; b. EpCAM staining; c. HA staining; d. HA staining after PH20 treatment; the scale bar in the figure is 100 ⁇ m.
  • Fig. 4 shows the inhibitory effect of PH20-expressing immune cells on transplanted tumors of human pancreatic cancer PDX model mice.
  • Figure 5 shows the inhibitory effect of PH20-expressing immune cells on transplanted tumors of human gastric cancer PDX model mice.
  • immune cell refers to a cell that participates in an immune response and produces an immune effect, such as T cells, B cells, natural killer (NK) cells, natural killer T (NKT) cells, mast cells, and bone marrow-derived phagocytes.
  • the immune effector cells are T cells, NK cells, NKT cells.
  • the T cells may be autologous T cells, heterologous T cells, allogeneic T cells.
  • the NK cells may be allogeneic NK cells.
  • peptide refers to a compound consisting of amino acid residues covalently linked by peptide bonds.
  • the protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that can include the sequence of the protein or peptide.
  • Polypeptides include any peptide or protein that contains two or more amino acids bonded to each other by peptide bonds.
  • the term refers to short chains (which are also commonly referred to in the art as eg peptides, oligopeptides and oligomers) and longer chains (which are also commonly referred to in the art as proteins, their presence Multiple types).
  • Polypeptide includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, or combinations thereof.
  • amino acid modification includes amino acid substitutions, additions and/or deletions, and "amino acid substitution” means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • amino acid insertion as used in the present invention means the addition of amino acids at specific positions in the parent polypeptide sequence.
  • amino acid deletion or “deletion” means the removal of amino acids at specific positions in the parent polypeptide sequence.
  • conservative modification used in the present invention means an amino acid modification that does not significantly affect or change the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, insertions and deletions.
  • Modifications can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions are substitutions of amino acid residues with amino acid residues having similar side chains. The family of amino acid residues with similar side chains has been defined in the art.
  • amino acids with basic side chains eg, lysine, arginine, histidine
  • acidic side chains eg, aspartic acid, glutamic acid
  • uncharged acute side chains eg , Glycine, asparagine, serine, threonine, tyrosine, cysteine, tryptophan
  • non-polar side chains eg, alanine, valine, leucine, isoleucine Acids, proline, phenylalanine, methionine
  • beta branched side chains eg, threonine, valine, isoleucine
  • aromatic side chains eg, tyrosine, benzene (Alanine, tryptophan, histidine).
  • promoter is a DNA sequence recognized by a cell's synthetic mechanism or the introduced synthetic mechanism required to initiate specific transcription of a polynucleotide sequence.
  • a typical eukaryotic promoter consists of a minimal promoter and other cis elements.
  • the minimal promoter is essentially a TATA box region, where RNA polymerase II (polII), TATA binding protein (TBP) and TBP-related factor (TAF) can be combined to initiate transcription.
  • polyII RNA polymerase II
  • TBP TATA binding protein
  • TAF TBP-related factor
  • sequence elements e.g. enhancers
  • enhancers have been found to increase the overall expression level of adjacent genes, and often in a position- and/or orientation-independent manner.
  • the tumor antigen recognition receptor of the present invention refers to a chimeric receptor, which refers to a fusion molecule formed by linking corresponding cDNAs of DNA fragments or proteins from different sources using genetic recombination technology.
  • Chimeric receptors generally include extracellular domains, transmembrane domains, and intracellular domains.
  • Chimeric receptors that can be used in the present invention include, but are not limited to: chimeric antigen receptor (CAR), modified T cell (antigen) receptor (TCR), T cell fusion protein (TFP), T cell antigen coupler ( TAC).
  • chimeric antigen receptor refers to a group of polypeptides that, when they are in immune effector cells, provide the cells with specificity against target cells (usually cancer cells) and have Intracellular signal generation.
  • CAR usually includes at least one extracellular antigen-binding domain (also called extracellular domain), a transmembrane domain (also called transmembrane domain), and a cytoplasmic signaling domain (also referred to herein as "intracellular signaling structure” Domain” or “intracellular region”), which includes functional signaling domains derived from stimulatory molecules and/or costimulatory molecules as defined below.
  • the groups of polypeptides are contiguous with each other.
  • the polypeptide group includes dimerization switches that can couple polypeptides to each other in the presence of dimerization molecules, for example, an antigen binding domain can be coupled to an intracellular signaling domain.
  • the stimulatory molecule is a zeta chain that binds to the T cell receptor complex.
  • the cytoplasmic signaling domain further includes one or more functional signaling domains derived from at least one costimulatory molecule as defined below.
  • the costimulatory molecule is selected from the costimulatory molecules described herein, such as 4-1BB (ie, CD137), CD27, and/or CD28.
  • CAR includes a chimeric fusion protein that includes an extracellular antigen binding domain, a transmembrane domain, and an intracellular signaling domain that includes a functional signaling domain derived from a stimulatory molecule.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen-binding domain, a transmembrane domain, and a functional signaling domain derived from a costimulatory molecule and a functionality derived from a stimulatory molecule The intracellular signaling domain of the signaling domain.
  • the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain, and comprising two functional signaling derived from one or more costimulatory molecules.
  • the invention contemplates modification of the amino acid sequence of the starting antibody or fragment (eg, scFv) that produces functionally equivalent molecules.
  • the VH or VL of the antigen-binding domain of the cancer-associated antigen described herein, such as the scFv contained in the CAR can be modified to retain the initial VH or VL framework of the antigen-binding domain of the cancer-associated antigen described herein At least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity .
  • the present invention contemplates modification of the entire CAR construct, such as modification of one or more amino acid sequences of multiple domains of the CAR construct, to produce functionally equivalent molecules.
  • the CAR construct can be modified to retain at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 of the starting CAR construct %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity.
  • the term "stimulatory molecule” refers to a molecule expressed by immune cells (e.g., T cells, NK cells, B cells) that provides a cytoplasmic signaling sequence that regulates the signaling pathway used for immune cells in a stimulating manner Activation of immune cells in at least some aspects.
  • the signal is a primary signal initiated by, for example, the binding of the TCR/CD3 complex to the peptide-loaded MHC molecule, and it results in mediating T cell responses, including, but not limited to proliferation, activation, differentiation, and the like.
  • the primary cytoplasmic signaling sequence (also called “primary signaling domain") that functions in a stimulating manner may contain an activation motif called an immunoreceptor tyrosine (Immunoreceptor tyrosine-based activation, motif, ITAM ) Signaling motif.
  • ITAM-containing cytoplasmic signaling sequences specifically used in the present invention include, but are not limited to those derived from: CD3 ⁇ , common FcR ⁇ (FCER1G), Fc ⁇ RIIa, FcR ⁇ (FcEpsilon R1b), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD79a, CD79b, DAP10 and DAP12.
  • the intracellular signaling domain in any one or more CARs of the present invention includes intracellular signaling sequences, such as the primary signaling sequence of CD3- ⁇ .
  • the primary signaling sequence of CD3- ⁇ is an equivalent residue from human or non-human species such as mouse, rodent, monkey, ape, etc.
  • costimulatory molecule refers to a homologous binding partner on T cells that specifically binds the costimulatory ligand, thereby mediating the T cell costimulatory response, such as but not limited to proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands, which promote an effective immune response.
  • Costimulatory molecules include but are not limited to MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278) and 4- 1BB (CD137).
  • costimulatory molecules include CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD160, CD19, CD4, CD8 ⁇ , CD8 ⁇ , IL2R ⁇ , IL2R ⁇ , IL7R ⁇ , ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1 CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, NKG2C, TNFR2, TRANCE/RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD22), CD19
  • the costimulatory intracellular signaling domain may be the intracellular part of the costimulatory molecule.
  • Costimulatory molecules can be represented by the following protein families: TNF receptor protein, immunoglobulin-like protein, cytokine receptor, integrin, signaling lymphocyte activation molecule (SLAM protein), and NK cell receptor.
  • Examples of such molecules include CD27, CD28, 4-1BB (CD137), OX40, GITR, CD30, CD40, ICOS, BAFFR, HVEM, ICAM-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CDS, CD7, CD287, LIGHT, NKG2C, NKG2D, SLAMF7, NKp80, NKp30, NKp44, NKp46, CD160, B7-H3 and ligands that specifically bind CD83, etc.
  • the intracellular signaling domain may include all or part of the natural intracellular signaling domain of the molecule, or a functional fragment or derivative thereof.
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence provided by GenBank Accession No. AAA62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.;
  • the "4-1BB costimulatory domain” is defined as the amino acid residues 214-255 of GenBank Accession No. AAA62478.2, or equivalent residues from non-human species such as mice, rodents, monkeys, apes, etc.
  • the "4-1BB costimulatory domain” is an equivalent residue from human or from a non-human species such as mouse, rodent, monkey, ape, etc.
  • T cell (antigen) receptor TCR
  • TCR is a characteristic marker on the surface of all T cells, which binds to CD3 with a non-covalent bond to form a TCR-CD3 complex.
  • TCR is responsible for identifying antigens that bind to major histocompatibility complex molecules.
  • TCR is a heterodimer composed of two different peptide chains, composed of two peptide chains of ⁇ and ⁇ , each peptide chain can be divided into variable region (V region), constant region (C region), and transmembrane And cytoplasmic areas; it is characterized by a very short cytoplasmic area.
  • the TCR molecule belongs to the immunoglobulin superfamily, and its antigen specificity exists in the V region; the V region (V ⁇ , V ⁇ ) has three hypervariable regions CDR1, CDR2, and CDR3, of which the CDR3 has the largest variation, which directly determines the TCR antigen Binding specificity.
  • CDR1 and CDR2 recognize and bind to the side wall of the antigen-binding groove of the MHC molecule, and CDR3 directly binds to the antigen peptide.
  • TCR is divided into two categories: TCR1 and TCR2; TCR1 is composed of two chains of ⁇ and ⁇ , and TCR2 is composed of two chains of ⁇ and ⁇ .
  • T cell fusion protein includes recombinant polypeptides derived from various polypeptides that constitute TCR, which can bind to surface antigens on target cells and interact with other polypeptides of the complete TCR complex The effect is usually localized on the surface of T cells.
  • TFP consists of an antigen-binding domain composed of a TCR subunit and a human or humanized antibody domain, wherein the TCR subunit includes at least part of the TCR extracellular domain, transmembrane domain, and TCR intracellular domain.
  • the stimulation domain of the inner signal domain; the TCR subunit and the antibody domain are effectively linked, wherein the extracellular, transmembrane, and intracellular signal domains of the TCR subunit are derived from CD3 ⁇ or CD3 ⁇ , and the TFP is integrated into TCR expressed on T cells.
  • T cell antigen coupler (TAC) coupler includes three functional domains: 1. Tumor targeting domains, including single chain antibodies, designed ankyrin repeat protein (designed ankyrin repeat protein, DARPin) ) Or other targeting groups; 2. The extracellular domain, a single-chain antibody that binds to CD3, so that the TAC receptor is close to the TCR receptor; 3. The transmembrane region and the intracellular region of the CD4 co-receptor, Among them, the intracellular domain is linked to the protein kinase LCK, which catalyzes the phosphorylation of immunoreceptor tyrosine activation motifs (ITAMs) of the TCR complex as the initial step of T cell activation.
  • TAC immunoreceptor tyrosine activation motifs
  • antibody refers to a protein or polypeptide sequence derived from an immunoglobulin molecule that specifically binds an antigen.
  • the antibody may be polyclonal or monoclonal, multi-chain or single-chain, or intact immunoglobulin, and may be derived from natural sources or recombinant sources.
  • the antibody may be a tetramer of immunoglobulin molecules.
  • scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light chain and heavy chain variable regions are contiguous (for example, via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it originated.
  • the scFv may have the VL and VH variable regions in any order (eg, relative to the N-terminus and C-terminus of the polypeptide), and the scFv may include the VL-linker-VH or VH-linker-VL may be included.
  • heavy chain refers to the larger of the two polypeptide chains that exist in the antibody molecule in their naturally occurring configuration and generally determine the type of antibody to which they belong.
  • light chain refers to the smaller of the two polypeptide chains present in the antibody molecule in its naturally occurring configuration.
  • ⁇ (k) and ⁇ (l) light chains refer to the two major antibody light chain isotypes.
  • antigen refers to a molecule that causes an immune response.
  • the immune response may involve antibody production or the activation of cells with specific immunity or both.
  • any macromolecule including virtually all proteins or peptides can serve as an antigen.
  • the antigen can be derived from recombinant or genomic DNA.
  • any DNA, encoded protein or peptide including a nucleotide sequence or a partial nucleotide sequence encoding a protein that causes an immune response.
  • the antigen need not be encoded only by the full-length nucleotide sequence of the gene.
  • the invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and these nucleotide sequences are arranged in different combinations to encode polypeptides that elicit the desired immune response.
  • the antigen need not be encoded by a "gene” at all.
  • the antigen can be produced synthetically, or it can be derived from a biological sample, or it can be a large molecule other than a polypeptide.
  • biological samples may include, but are not limited to, tissue samples, tumor samples, cells or fluids with other biological components.
  • Tumor antigen refers to the newly emerged or overexpressed antigen during the occurrence and development of hyperproliferative diseases.
  • the hyperproliferative disorder of the present invention refers to cancer.
  • the tumor antigen in the present invention may be a solid tumor antigen or a hematoma antigen.
  • Tumor antigens of the present invention include, but are not limited to: thyroid stimulating hormone receptor (TSHR); CD171; CS-1; C-type lectin-like molecule-1; ganglioside GD3; Tn antigen; CD19; CD20; CD22; CD 30; CD 70; CD123; CD 138; CD33; CD44; CD44v7/8; CD38; CD44v6; B7H3 (CD276), B7H6; KIT (CD117); interleukin 13 receptor subunit alpha (IL-13R ⁇ ); interleukin 11 Receptor ⁇ (IL-11R ⁇ ); Prostate Stem Cell Antigen (PSCA); Prostate Specific Membrane Antigen (PSMA); Carcinoembryonic Antigen (CEA); NY-ESO-1; HIV-1Gag; MART-1; gp100; Tyramine Oxidase; mesothelin; EpCAM; protease serine 21 (PRSS21); vascular endothelial growth factor receptor, vascular endot
  • tumor refers to a broad category of disorders of hyperproliferative cell growth in vitro (eg, transformed cells) or in vivo. Conditions that can be treated or prevented by the method of the present invention include, for example, various neoplasms, including benign or malignant tumors, various hyperplasias, and the like.
  • cancer examples include, but are not limited to: breast cancer, prostate cancer, leukemia, lymphoma, nasopharyngeal cancer, glioma, colon cancer, rectal cancer, renal cell carcinoma, liver cancer, non-small cell lung cancer, small intestine cancer, esophagus Cancer, melanoma, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, uterine cancer, ovarian cancer, gastric cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, thyroid cancer, parathyroid cancer, Adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, bladder cancer, ureteral cancer, renal pelvis cancer, central nervous system (CNS) tumor, hemangioma spinal tumor, glioma, astrocytoma, pituitary adenoma Combination and metastatic lesions of the cancer.
  • CNS central nervous system
  • Transfected or transformed refers to the process by which exogenous nucleic acid is transferred or introduced into a host cell.
  • Transfected or transformed cells are cells that have been transfected or transformed with exogenous nucleic acid. The cells include primary subject cells and their progeny.
  • the proteins involved in the present invention include PH20, CD8 ⁇ hinge region, human CD8 ⁇ transmembrane region, human 4-1BB intracellular region and human CD3 ⁇ intracellular region, MSLN, HAS2 (hyaluronidase synthesis gene) and targeting each The scFv sequence and TCR sequence of the target.
  • the NCBI accession number of the amino acid sequence of PH20 is NP_694859.1 (cDNA is NM_153189.2); the NCBI accession number of HAS2 is NP_005319.1 (cDNA is NM_005328.3); the NCBI accession number of MSLN is NP_037536.2 (cDNA is NM_013404 .4); tEGFR is the III and IV domains of EGFR, and the NCBI accession number of EGFR is NP_005219.2 (cDNA is NM_005228.4).
  • the amino acid sequence and coding base sequence of MSLN scFv are derived from patent US7977457 (SEQ ID NO.12); the amino acid sequence of GPC3 scFv are derived from patent CN104140974A (SEQ ID NO.22), and the base sequence is for human password Optimized by codons; the amino acid sequence and coding base sequence of ROR1scFv are derived from patent US20170283497A1 (SEQ ID NO.93), the base sequence is obtained by codon optimization for humans; the amino acid sequence of EpCAM scFv (MOC31) is derived from patent US7858088 ( SEQ ID NO.
  • the base sequence is obtained by codon optimization for human; the amino acid sequence and coding base sequence of HER2scFv (FRP5) are derived from the patent US7887801 (SEQ ID NO. 2); E6 amino acid sequence and coding of TCR The base sequence is derived from patent CN105452288A (SEQ ID NO. 30).
  • amino acid sequence information of proteins involved in the preparation of various cells of the present invention is shown in Table 1, the structure of related expression regions is shown in Table 2, and the base sequence of the encoded amino acids is shown in Table 3.
  • the PH20 and CAR/TCR base sequences synthesized in Example 1 were digested with EcoRI and XbaI, and ligated into the corresponding site of the lentiviral vector pCDH-EF1a (SBI) via DNA Ligation Kit (Takara) ( Figure 1) to obtain Target gene expression vector Lenti-GOI.
  • MSLN base sequence was digested by BamHI and EcoRI, and inserted into the corresponding site of pcDNA3.1 vector (Invitrogen) by DNA Kit (Takara);
  • HAS2 base sequence was digested by NheI and BamHI, and inserted into pcDNA3 by DNA Kit. .1-Hyg vector (Invitrogen) corresponding site.
  • the ligation products were transformed into competent E. coli (Top10).
  • clones were picked and cultured in LB liquid medium containing 100 ⁇ g/mL ampicillin (biotechnology) and sequenced for identification.
  • the clones with correct sequencing results were selected and inoculated into LB liquid medium containing 100 ⁇ g/mL ampicillin on a 50 ml scale for about 16 hours.
  • the bacterial cells were collected, and the plasmid was extracted and purified using a plasmid extraction kit (Qiagen).
  • the obtained MSLN-pcDNA3.1 plasmid was linearized with PvuI, and the HAS2-pcDNA3.1-Hyg plasmid was linearized with FspI.
  • the digested product was extracted with phenol chloroform to obtain a preliminary linearized plasmid. All restriction enzymes used for plasmid preparation were purchased from Thermo.
  • Plasmid sterilization treatment Add 1/14 volume of 3M NaCl solution to the obtained plasmid to make the salt ion concentration reach 200mM. Then add 2.5 times the volume of -20 °C pre-cooled absolute ethanol, repeatedly invert to mix, and keep at -20 °C for 30min. The obtained solution was centrifuged at 12,000 rpm and 4°C for 20 min, the supernatant was carefully discarded, and the precipitate was collected. The precipitate was washed with 1 ml of 70% ethanol, 12,000 rpm, and the supernatant was discarded after centrifugation for 5 min. Sterile air-dry the pellet and add 100-200 ⁇ l of sterile water to dissolve the DNA. Aseptically take 5 ⁇ l diluted 50 times with TE Buffer, and measure OD 260 and OD 280 by UV. Finally, a plasmid whose concentration can be used for transfection is obtained.
  • PEI polyetherimide
  • lentivirus packaging plasmids Li-GOI, pMD2.G, psPAX2
  • Add the DNA/PEI complex dropwise to a 10cm Petri dish, gently shake the Petri dish, and mix well. Place the petri dish in a 37°C, 5% CO 2 incubator. After 6 to 8 hours of culture, remove the medium containing the transfection reagent, replace it with fresh complete medium and put it back into the incubator to continue the cultivation.
  • HT1080 cells Resuscitate HT1080 cells and adjust the cell status to logarithmic growth phase. 8 ⁇ 10 5 cells per well were used to inoculate HT1080 to a 6-well plate with a culture volume of 3 mL. The 6-well plate was placed in a 5% CO 2 incubator at 37° C. for overnight cultivation. The concentrated lentivirus was added to the above 6-well plate in the amounts of 0.1, 1, 10 ⁇ L, and polybrene (Polybrene, Sigma) with a final concentration of 6 ⁇ g/mL was added at the same time. The 6-well plate was put back into the 37°C, 5% CO 2 incubator, and the cultivation was continued for 96 hours. After the cultivation, the cells in each well were washed with PBS, and then genomic DNA was extracted using a genomic DNA extraction kit (TIANGEN), and the concentration of the extracted genomic DNA was measured using NanoDrop2000.
  • TIANGEN genomic DNA extraction kit
  • Fluorescence quantitative PCR was used to determine the lentivirus copy number.
  • the qPCR reaction system was prepared according to the instructions of SYBR Premix Ex Taq II (Takara), the PCR reaction premix was prepared, and the PCR reaction was carried out using Roche Light cycler 480. Calculate the copy number, and adjust the virus titer to approximately 1 ⁇ 10 8 TU/mL according to the calculation result.
  • T cell density 1 ⁇ 10 6 cells/mL
  • interleukin 2 Novatis
  • Anti-CD3-OKT3 antibody Thermo
  • T cells were transfected using LONZA T cell transfection kit (VPA-1002).
  • V-024/U-014 the preferred protocol recommended by the kit
  • U-010/T-010 the preferred protocol for transfection.
  • CAR positive rate was detected by Protein L antibody (Thermo), and the corresponding secondary antibodies were Streptavidin PE and Streptavidin FITC (BD Bioscience).
  • the positive rates of mPH20-T and sPH20-T cells were detected by Erbitux antibody (BMS) and the corresponding secondary antibody PE goat anti-human IgG Fc antibody (Thermo).
  • TCR positive rate was detected by FITC Hamster Anti-Mouse TCR ⁇ Chain (BD Bioscience) antibody. The positive rate test results are shown in Table 4.
  • the cells were subcultured every 2 days, the cell status was observed, the cell count was counted, and replaced with fresh medium for cultivation.
  • the transient transfection experiment of CAR using nuclear transfection found that the cell status and viability after transfection were poor, and the expansion was slow. Therefore, CAR-T or other cell types were not prepared by this method.
  • the cells prepared by the lentivirus method are cultured for about 8 to 14 days, the cells are collected for use. During the cultivation process, it was found that the simultaneous expression of PH20 did not affect the proliferation and positive rate of T cells.
  • NK-92 cells (ATCC CRL-2407) were used to prepare CAR-NK cells.
  • NK-92 was cultured with RPMI1640 (GIBCO) medium, 10% fetal bovine serum (GIBCO) was added, and interleukin 2 (Novatis) with a final concentration of 500 IU/mL was added.
  • NK-92 cells were infected with sPH20 lentivirus and mPH20 lentivirus by lentivirus infection and limiting dilution. Hygromycin with a final concentration of 400 ⁇ g/mL was used for pressure screening. After the formation of clones with limited dilution, the clones were tested for hyaluronidase activity. Among them, the clone of sPH20-NK92 was used to detect the enzyme activity in the supernatant; while mPH20-NK-92 was used to detect the enzyme activity of cells. Select active clones for subsequent experiments.
  • NK-92 cells, sPH20-NK92 cells, and mPH20-NK92 cells were infected with FRP5-CAR lentivirus by lentivirus infection and limiting dilution.
  • G418 with a final concentration of 600 ⁇ g/mL was used for pressure screening.
  • the clones were tested for hyaluronidase activity and CAR positive.
  • the CAR positive rate was detected by Protein L antibody (Thermo), and the corresponding secondary antibodies were Streptavidin PE and Streptavidin FITC (BD).
  • HA highly expressed target cells were obtained as an in vitro cell model to evaluate the effect of PH20 on immune cells killing target cells.
  • Each target cell was cultured according to the medium listed in Table 5, wherein the culture reagents were all purchased from GIBCO. Take out 1 mg/ml polyetherimide (PEI, Polyscience) transfection reagent and HAS2-pcDNA3.1-Hyg plasmid, and thaw at room temperature. Take plasmid DNA (3.3 ⁇ g) into 165 ⁇ l opti-MEM medium, mix well. Take PEI (10.5 ⁇ g) into 165 ⁇ l opti-MEM medium, mix well.
  • PEI polyetherimide
  • the hyaluronic acid ELISA quantitative kit (R&D) was used to detect the content of hyaluronic acid in the culture supernatant.
  • the clones with high hyaluronic acid secretion and good growth were selected and expanded to 24 well plates. After the layer is full, inoculate 1 ⁇ 10 5 cells per well into a 24-well plate with a culture volume of 1 ml per well. After culturing for 24hr, take the culture supernatant and use the diluent in the kit to dilute the culture supernatant to detect the hyaluronic acid content according to the detection method provided in the kit. One strain was used for subsequent experiments (Table 6).
  • Example 7 CAR-T cells expressing PH20 have hyaluronidase activity and cell killing activity
  • hyaluronidase activity On day 7 after lentivirus infection, cells were taken for hyaluronidase activity.
  • For immune cells secreting hyaluronidase 1 ⁇ 10 6 cells were inoculated into a 24-well plate, and the culture volume was 1 ml. After culturing for 24 hr, the supernatant was taken for enzyme activity detection.
  • For immune cells expressing membrane-bound hyaluronidase take 1 ⁇ 10 6 positive cells expressing the protein of interest, resuspend the cell pellet to 1 ⁇ 10 6 cells/ml in enzyme activity detection buffer, and take the cell fluid for enzyme activity Detection.
  • the hyaluronidase activity of CAR-T cells was tested in accordance with the general rule "1207 Hyaluronidase Assay" of the Chinese Pharmacopoeia 2015 Edition.
  • the target cells were taken to a 24-well plate at a density of 1 ⁇ 10 5 cells/ml and 1 ml/well.
  • CAR-T cells were seeded into 24 well plates containing target cells according to 1 ⁇ 10 6 and 3 ⁇ 10 5 respectively. Place the 24-well plate in a 37°C, 5% CO 2 incubator for 16 to 24 hours. After the cultivation, take the culture supernatant and follow CytoTox The method of the instruction manual of the non-radioactive cytotoxicity detection kit (Promega) detects LDH in the culture supernatant to confirm the killing effect.
  • the secretase activity of sPH20-T is 67 ⁇ 25 units/ml
  • the secretase activity of sPH20-Twt is 58 ⁇ 23 units/ml.
  • Example 8 CAR-NK cells expressing PH20 have hyaluronidase activity and cell killing activity
  • hyaluronidase activity For immune cells secreting hyaluronidase, 1 ⁇ 10 6 cells were inoculated into a 24-well plate, and the culture volume was 1 ml. After culturing for 24 hr, the supernatant was taken for enzyme activity detection. For immune cells expressing membrane-bound hyaluronidase, take 1 ⁇ 10 6 cells, resuspend the cell pellet to 1 ⁇ 10 6 cells/ml in enzyme activity detection buffer, and take the cell fluid for enzyme activity detection. The hyaluronidase activity of CAR-NK cells was tested in accordance with the general rule "1207 Hyaluronidase Assay" in the Chinese Pharmacopoeia 2015 Edition.
  • the target cells were taken to a 24-well plate at a density of 1 ⁇ 10 5 cells/ml and 1 ml/well.
  • CAR-NK cells were seeded into 24 well plates containing target cells according to 1 ⁇ 10 6 and 3 ⁇ 10 5 respectively. Place the 24-well plate in a 37°C, 5% CO 2 incubator for 16 to 24 hours. After the cultivation, take the culture supernatant and follow CytoTox The method of the instruction manual of the non-radioactive cytotoxicity detection kit (Promega) detects LDH in the culture supernatant to confirm the killing effect.
  • the experimental results show that in vitro cell killing experiments of immune cells expressing PH20 protein against wild-type MDA-MB-231 and HAS2 overexpressing MDA-MB-231 cells, FRP5-CAR-NK cells carrying PH20 and adding PH20 in total The cultured FRP5-CAR-NK cells had better killing effect.
  • Example 9 TCR-T cells expressing PH20 have hyaluronidase activity and cell killing activity
  • hyaluronidase activity For immune cells secreting hyaluronidase, 1 ⁇ 10 6 cells were inoculated into a 24-well plate, and the culture volume was 1 ml. After culturing for 24 hr, the supernatant was taken for enzyme activity detection. For immune cells expressing membrane-bound hyaluronidase, take 1 ⁇ 10 6 cells, resuspend the cell pellet to 1 ⁇ 10 6 cells/ml in enzyme activity detection buffer, and take the cell fluid for enzyme activity detection. The hyaluronidase activity of TCR-T cells was tested in accordance with the general rule "1207 Hyaluronidase Assay" in the Chinese Medicine Pharmacopoeia 2015 Edition.
  • the target cells were taken to a 24-well plate at a density of 1 ⁇ 10 5 cells/ml and 1 ml/well.
  • CAR-T cells were seeded into 24 well plates containing target cells according to 1 ⁇ 10 6 and 3 ⁇ 10 5 respectively. Place the 24-well plate in a 37°C, 5% CO 2 incubator for 16 to 24 hours. After the cultivation, take the culture supernatant and follow CytoTox The method of the instruction manual of the non-radioactive cytotoxicity detection kit (Promega) detects LDH in the culture supernatant to confirm the killing effect.
  • the experimental results showed that the immune cells expressing PH20 protein in the in vitro cell killing experiment, against wild-type SCC152 cells, behaved similarly to immune cells not expressing PH20.
  • PH6-carrying E6 TCR-T cells and PH20 co-cultured FRP5-CAR-NK cells had better killing effects.
  • a mouse xenograft tumor model with high HA expression was established.
  • the expression of HA in PDX tissue was detected by immunohistochemistry.
  • the tumor tissue is taken, fixed with 4% formalin, and stored at 2-8°C. Routine dehydration, paraffin embedding section, slice thickness 3 ⁇ m. The sections were dewaxed and rehydrated, followed by xylene treatment for 10 minutes twice, absolute ethanol treatment for 5 minutes twice, 95% ethanol treatment for 2 minutes, 85% ethanol treatment for 2 minutes, and 75% ethanol treatment for 2 minutes. Wash with distilled water for 2 minutes.
  • PDX tissue For each candidate PDX tissue, 4 slices were selected, 2 of which were added to a solution containing 100 units/mL recombinant human PH20 (Rhinobio) and incubated at 37°C for 2 hours. The resulting slices were used as negative controls. Two tablets were incubated with PH20 buffer (25 mM piperazine-1,4-diethylsulfonic acid, 70 mM sodium chloride, 0.1% bovine serum albumin, pH 5.5) for 2 hours at 37°C. After the incubation is completed, use 2% goat serum white (Boshide) to incubate for 30 minutes for blocking.
  • PH20 buffer 25 mM piperazine-1,4-diethylsulfonic acid, 70 mM sodium chloride, 0.1% bovine serum albumin, pH 5.5
  • Mesothelin protein uses anti-human MSLN protein rabbit monoclonal antibody (abcam) as the primary antibody.
  • EpCAM uses anti-human EpCAM protein rabbit monoclonal antibody (abcam) as the primary antibody.
  • Goat anti-rabbit IgG-HRP antibody (abcam) was used as secondary antibody.
  • the PDX samples from pancreatic cancer patients were screened for MSLN and HA expression levels.
  • PDX samples with strong MSLN and HA staining levels were selected for inoculation of mice.
  • the final selected tissue immunohistochemistry results are shown in the figure. 2.
  • the expression levels of EpCAM and HA were screened for PDX samples from gastric cancer patients.
  • PDX samples with strong EpCAM and HA staining levels were selected for inoculation of mice.
  • the final selected tissue immunohistochemistry results are shown in Figure 3. .
  • Resuscitated pancreatic cancer PDX samples with high expression of MSLN/HA were inoculated subcutaneously in 2 to 4 NCG mice.
  • the tumor tissue in the vigorous growth period was cut to about 1.5 mm 3 and inoculated under the sterile condition under the skin of the right armpit of nude mice. Observe the growth of the mice every day, weigh each week, and after confirming that the mice are bearing tumors, monitor the size of the tumor, and the frequency of monitoring is determined according to the actual situation.
  • mice Six to seven weeks old NCG mice were selected to be inoculated with PDX tumors and subcutaneously on the right armpit of mice.
  • the length (L) and width (W) of the solid tumor mass are measured by a caliper, and the tumor volume (TV) is calculated as: (L ⁇ W 2 )/2.
  • the mice were classified into 5 treatment groups: 1) T cell control; 2) MSLN-CAR-T cells; 3) MSLN-mPH20-CAR-T cells; 4 ) MSLN-sPH20-CAR-T cells; 5) MSLN-CAR-T cells, PH20 protein solution.
  • mice were tested on the day of administration.
  • Count CAR-T cells and calculate the total number of cells to be administered according to the CAR positive rate. Centrifuge the cells corresponding to the cell number and volume, and wash the resulting cell pellet twice with PBS (pH 7.2). After washing, CAR-T cells were resuspended in PBS to a final volume of 100 ⁇ l, and 5 ⁇ 10 6 positive CAR-T cells were administered to each mouse. CAR-T cells were administered once by intratumoral injection, and then administered again in the same way one week later.
  • the dosage of PH20 protein solution (1.4 mg/ml; hereinafter referred to as Solution B) is 1000 units/mouse, and the corresponding protein content is about 0.01 mg/10 ⁇ g per mouse.
  • Group 5 is administered with CAR-T positive cells and solution B.
  • D0 10 ⁇ l of solution B was added to the PBS containing cells, mixed, and used for administration to mice.
  • D1, D3, D5, D7, D8, D10, D12, D14, D16 dosing is carried out after adding 10 ⁇ l to 90 ⁇ l PBS.
  • the detection frequency of tumor size is twice a week, and the observation period of drug efficacy is 21 days after the second cell injection, and the experimental endpoint treatment is carried out.
  • the tumor volume of different experimental groups of transplanted tumors of human pancreatic cancer PDX model mice at different times is shown in Figure 4.
  • groups 2-5 all had significant tumor suppressive effects.
  • MSLN-CAR-T cell administration T/C value was 50.4%
  • MSLN-mbPH20-CAR-T cell administration T/C value was 7.4%
  • MSLN-sPH20-CAR-T cell administration T/C value was 2.5 %
  • the T/C value of MSLN-CAR-T cells + PH20 administration was 8.9%.
  • the CAR-T cells expressing PH20 or co-administered with PH20 were significantly better than MSLN-CAR-T cells.
  • each experimental group had no effect on the weight change of tumor-bearing mice.
  • the mice in each experimental group were in good condition without weight loss.
  • Example 12 In vivo activity of PH20-expressing immune cells in gastric cancer PDX model mice
  • Resuscitated EpCAM/HA highly expressed gastric cancer PDX samples were inoculated subcutaneously in 2 to 4 NCG mice.
  • volume of a single tumor exceeded 500 mm 3
  • large-scale tumor passage was performed according to the experimental protocol.
  • the tumor tissue in the vigorous growth period was cut to about 1.5 mm 3 and inoculated under the sterile condition under the skin of the right armpit of nude mice. Observe the growth of the mice every day, weigh each week, and after confirming that the mice are bearing tumors, monitor the size of the tumor, and the frequency of monitoring is determined according to the actual situation.
  • mice Six to seven weeks old NCG mice were selected to be inoculated with PDX tumors and subcutaneously on the right armpit of mice.
  • the length (L) and width (W) of the solid tumor mass are measured by a caliper, and the tumor volume (TV) is calculated as: (L ⁇ W 2 )/2.
  • the mice were graded into 5 treatment groups: 1) T cell control; 2) EpCAM-CAR-T cells; 3) EpCAM-mPH20-CAR-T cells; 4 ) EpCAM-sPH20-CAR-T cells; 5) EpCAM-CAR-T cells, PH20 protein solution (solution B).
  • mice were tested on the day of administration.
  • Count CAR-T cells and calculate the total number of cells to be administered according to the CAR positive rate. Centrifuge the cells corresponding to the cell number and volume, and wash the resulting cell pellet twice with PBS (pH 7.2). After washing, CAR-T cells were resuspended in PBS to a final volume of 100 ⁇ l, and 5 ⁇ 10 6 positive CAR-T cells were administered to each mouse. CAR-T cells were administered once by intratumoral injection, and then administered again in the same way one week later.
  • the dosage of solution B is 1000 units/mouse, and the corresponding protein content is about 0.01 mg/10 ⁇ g per mouse. Group 5 is administered with CAR-T positive cells and solution B.
  • EpCAM-CAR-T cell administration T/C value was 68.7%
  • EpCAM-mPH20-CAR-T cell administration T/C value was 0.7%
  • EpCAM-sPH20-CAR-T cell administration T/C value was 12.0 %
  • the T/C value of EpCAM-CAR-T cells + PH20 administration was 11.9%.
  • the CAR-T cells expressing PH20 or co-administered with PH20 were significantly better than EpCAM-CAR-T cells.
  • each experimental group had no effect on the weight change of tumor-bearing mice.
  • the mice in each experimental group were in good condition without weight loss.

Abstract

一种包含肿瘤抗原识别受体的免疫细胞及其应用，所述包含肿瘤抗原识别受体的免疫细胞表达透明质酸酶，所述透明质酸酶为：a)锚定于所述免疫细胞上的全长蛋白；或b)分泌至所述免疫细胞外的可溶型蛋白。所述的免疫细胞在制备治疗肿瘤药物中的应用。所述包含肿瘤抗原识别受体的免疫细胞，可以改变微环境的细胞外基质结构正常化肿瘤免疫微环境的效果，从而使得其可以有效接近并杀死肿瘤细胞。在可溶性PH20蛋白的帮助下，包含肿瘤抗原识别受体的免疫细胞也能改善肿瘤微环境，有效杀死肿瘤细胞。

Description

一种包含肿瘤抗原识别受体的免疫细胞及其应用
本申请要求申请日为2018/12/13的中国专利申请201811527903X的优先权。本申请引用上述中国专利申请的全文。
技术领域
本发明属于细胞免疫治疗领域，具体涉及一种包含肿瘤抗原识别受体的免疫细胞及其应用。
背景技术
癌症，又称恶性肿瘤，它是严重危害人类健康、导致现代人类死亡的第一大恶疾和顽症。据联合国统计数据显示，目前全球平均每8个死亡病例中就有1人死于癌症，这比艾滋病、结核病和疟疾导致的死亡人数还要高。而且每年全球还有1000多万人被确诊患上癌症(世界卫生组织发布的《2014年世界癌症报告》)。
一直以来，肿瘤治疗的基础都是手术、化疗以及放疗。然而传统的治疗方式仅能延长恶性肿瘤患者的生存期，治愈的可能性很低，恶性肿瘤依然是“绝症”。在过去20年中，通过特异性识别肿瘤细胞表面特异性蛋白、从而杀死肿瘤细胞的靶向疗法比如西妥昔单抗
Figure PCTCN2019090998-appb-000001
和曲妥珠单抗
Figure PCTCN2019090998-appb-000002
治疗方法，使得它们成为很多癌症的标准治疗方法，然而上述方法并不能够有效治疗实体瘤。但在过去几年中，免疫疗法，一种通过招募并加强病人的免疫系统以攻击肿瘤的方法，已经成为肿瘤治疗方法的第5大支柱。其中，最热门且目前最接近FDA获批的治疗方法为CAR-T细胞治疗方法。然而，直到最近，CAR-T细胞治疗法一直局限于小规模的临床试验，并且大部分是用于晚期血液癌症的治疗，而除淋巴瘤以外的恶性实体肿瘤效果不佳。
CAR-T细胞用于实体瘤治疗的最大障碍在于肿瘤免疫抑制微环境，是肿瘤发生发展过程中形成的内环境。恶性实体肿瘤主要由癌/瘤细胞和基质细胞(例如成纤维细胞和炎症细胞)细胞外基质及血管/淋巴管构成。与正常组织相比，肿瘤组织中的基质细胞、细胞外基质和血管/淋巴管系统一般是异常的，具有增加数目的成纤维细胞，并且血管系统呈现出高度分支或卷曲的结构。肿瘤和基质细胞产生并装配细胞外基质组分(胶原蛋白、蛋白聚糖、透明质酸)形成致密团块(Clin Oncol 31:2205-2218.2013)。以上因素造成了肿瘤组织结构致密、营养缺乏、pH偏低、缺氧，抑制了CAR-T细胞有效发挥肿瘤杀伤作用。
想要有效治疗肿瘤，CAR-T细胞必须能够克服上述微环境，进入到肿瘤组织、在肿瘤组织中存活并对肿瘤细胞进行杀伤。
尽管可能已经产生了针对肿瘤微环境的CAR-T细胞，即表达透明质酸酶PH20的CAR-T细胞(专利CN107400664A)，但是这种细胞产生的技术手段存在问题，发挥杀伤作用的原理及技术效果未得到揭示。首先，在透明质酸酶的设计上，CN107400664A认为：在自然状态下，透明质酸酶是以分泌蛋白质形式(即游离透明质酸酶)而不是以跨膜蛋白质的形式表达，这种分泌型透明质酸酶或者透明质酸酶跨膜融合蛋白质基因序列中的透明质酸酶编码序列可以是全部蛋白质编码序列，也可以是含有编码透明质酸酶活性的部分序列。但未公布制备分泌透明质酸酶PH20CAR-T细胞的技术手段，且指出了与游离型扩散的透明质酸酶相比较，跨膜型透明质酸酶疗效更好。
其次，在具体实施例中公开的表达透明质酸酶跨膜融合蛋白的CAR-T细胞制备方法，即采用透明质酸酶PH20跨膜融合蛋白慢病毒感染CAR-T细胞，通过透明质酸酶抗体磁珠分离得到表达跨膜透明质酸酶的CAR-T细胞。该PH20跨膜融合蛋白的编码序列由PH20-人IgG4铰链区-CD8跨膜区-CD28胞内区-4-1BB胞内区-CD3ζ胞内序列依次组成。但其未公布该CAR-T细胞的透明质酸降解活性，CAR-T细胞的肿瘤细胞杀伤活性，CAR-T细胞的体内肿瘤抑制活性。本发明人通过实验发现，采用CN107400664A公开的表达透明质酸酶跨膜融合蛋白的制备方法很难获得具有透明质酸酶活性的CAR的阳性细胞；另一方面，采用两个慢病毒分别感染T细胞后，T细胞状态差，CAR阳性细胞几乎无法检测到透明质酸酶活性。
鉴于以上缺陷，现有技术中亟待需要一种有效的包含肿瘤抗原识别受体的免疫细胞。
发明内容
本发明所要解决的技术问题是为了克服目前现有技术当中无法得到具有透明质酸降解活性的CAR-T细胞、同时CAR的阳性率也很低或者含有透明质酸酶的CAR-T细胞难以分泌透明质酸酶，因而无法得到有效的包含肿瘤抗原识别受体的免疫细胞的缺陷，提供一种包含肿瘤抗原识别受体的免疫细胞及其应用。所述免疫细胞可以进入实体瘤内部杀死肿瘤细胞；在可溶型PH20蛋白的帮助下，包含肿瘤抗原识别受体的免疫细胞也能进入实体瘤内部，杀死肿瘤细胞。
本发明提供一种包含肿瘤抗原识别受体的免疫细胞，其表达透明质酸酶，所述透明质酸酶为：
a)锚定于所述免疫细胞上的全长蛋白；或
b)分泌至所述免疫细胞外的可溶型蛋白。
透明质酸酶是降解透明质酸的酶家族。在人类中，存在编码具有不同性质和位置的透明质酸酶的6个基因，HYAL1、HYAL2、HYAL3、HYAL4、HYAL5(又被称为SPAM1或者PH-20)、HYAL6(又被称为HYALP1)。同工型HYAL1和HYAL2存在于大多数组织中。HYAL2为GPI膜锚定蛋白，主要负责剪切高分子透明质酸，得到的小片段透明质酸被细胞内吞至内溶酶体(Endolysosome)后，被HYAL1进一步降解。在肿瘤组织中，透明质酸由透明质酸合成酶合成(HAS1、HAS2以及HAS3)。HYAL3存在于骨髓和睾丸中，但是其功能还没有被良好地表征。透明质酸酶PH20在睾丸中高表达，并参与卵母细胞被精子受精的过程。
本发明中，所述的透明质酸酶较佳地为哺乳动物睾丸透明质酸酶；更佳地为人类睾丸透明质酸酶，也被称为SPAM1(精子黏附分子1)、HYAL1、HYAL2、或者PH20。
编码所述PH20的基因产生两种转录变体，分别对应两种形式的酶序列：
一种形式为包含膜锚定序列的PH20序列，该所述PH20序列可以锚定在包含肿瘤抗原识别受体的免疫细胞的表面，即如上所述的透明质酸酶a)。因此，获得了表达细胞膜锚定PH20的包含肿瘤抗原识别受体的免疫细胞；本发明中所述透明质酸酶的氨基酸序列较佳地如序列表中SEQ ID NO.1所示，或者对该氨基酸序列进行过氨基酸修饰且保持其原有功能的透明质酸酶，如：与如SEQ ID NO.1所示的氨基酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性，且保持透明质酸酶原有功能的氨基酸序列；编码该所述PH20的核苷酸序列较佳地如序列表中SEQ ID NO.2或者3所示，或者与如SEQ ID NO.2或者3所示的核苷酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性的核苷酸序列。
另一种形式为缺失了对应于羧基端膜结合结构域的序列，所述缺失后的序列的最后一位氨基酸通常位于全长序列的第430～454位，以产生可溶型PH20，该羧基端结构域的缺失导致透明质酸酶分泌到细胞外介质。因此，获得了表达在中性及偏酸性pH条件下具有酶活性的分泌性透明质酸酶的包含CAR或者TCR的T细胞或者NK细胞，所述的NK细胞为NK-92细胞；该种形式的透明质酸酶的氨基酸序列为如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位，或者为与如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位的氨基酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性，且保持透明质酸酶原有功能的氨基酸序列；较佳地，其核苷酸序列为如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位，或者与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341 位具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性的核苷酸序列。
所述的肿瘤抗原识别受体优选嵌合抗原受体(CAR)或者T细胞受体(TCR)，其中：
所述的TCR包含，如TCR的α链、TCR的β链、TCR的γ链、TCR的δ链或它们中的一种或多种的组合，所述α链包含可变区和恒定区，较佳地，所述可变区为人TCRα链的可变区，所述恒定区为鼠α链的恒定区，较佳地，所述β链包含可变区和恒定区，所述可变区为人TCRβ链的可变区，所述恒定区为鼠β链的恒定区；其中，所述TCR的α链和所述TCR的β链之间可以通过接头序列连接，所述接头序列可以为P2A、T2A、E2A、F2A或者IRES；更佳地为E6 TCR。
所述的CAR包括胞内区、铰链区和跨膜区，所述胞内区、所述铰链区以及所述跨膜区均可为本领域常规，所述的CAR的胞内区优选包括人的4-1BB胞内区和/或人的CD28胞内区，以及人的CD3ζ胞内区，更优选包括人的4-1BB胞内区、人的CD28胞内区，以及人的CD3ζ胞内区。
所述的CAR的铰链区优选人的CD8α铰链区、所述的跨膜区优选人的CD8α跨膜区
所述抗原识别区、所述铰链区、所述跨膜区以及所述胞内区之间可以通过接头序列连接，所述接头序列包含n个前后重复的基序，所述基序为GGGS、GGGGS、SSSSG、GSGSA或者GGSGG，所述n可为1～5的整数。
较佳地，所述肿瘤抗原识别受体的肿瘤抗原识别区识别的抗原可以为EpCAM、Mesothelin、CEA、IL13、PDPN、VEGF、EGFR、EGFRvIII、PSMA、FAP、CD171、GD2、Glypican 2、Glypican 3、HER2、HPV抗原、cyclin D1、p53、MMP-7、IL13Ralpha2、MMP-2、MUC-1、G250、L1CAM、ROR1、GPC3或者MSLN；较佳地为ROR1、GPC3、MSLN或者EpCAM。其中，所述Mesothelin、所述IL13R、所述Podoplanin(PDPN)、所述EGFRvIII以及所述EGFR在胶质母细胞瘤中均有过量表达；所述EpCAM在上皮细胞癌中有过量表达。
所述肿瘤抗原识别区为能够结合以上肿瘤抗原的区域例如抗体，目前常用的为scFv(单链抗体)。
较佳地，识别所述ROR1的scFv的核苷酸序列如序列表中SEQ ID NO.4所示，或者与序列表中SEQ ID NO.4所示的核苷酸序列具有80％以上的序列同一性；
或者，识别所述GPC3的scFv的核苷酸序列如序列表中SEQ ID NO.5所示，或者与序列表中SEQ ID NO.5所示的核苷酸序列具有80％以上的序列同一性；
或者，识别所述EpCAM的scFv的核苷酸序列如序列表中SEQ ID NO.6所示，或者 与序列表中SEQ ID NO.6所示的核苷酸序列具有80％以上的序列同一性。
将基因引入细胞和将基因表达入细胞的方法在本领域中是已知的。载体可通过在本领域中的任何方法容易地引入宿主细胞，例如，哺乳动物、细菌、酵母或昆虫细胞。
例如，表达载体可通过物理、化学或生物学方法转移入宿主细胞。物理方法包括磷酸钙沉淀、脂质转染法、粒子轰击、微注射、电穿孔等等；化学方法包括胶体分散系统，诸如大分子复合物、纳米胶囊、微球、珠，和基于脂质的系统，包括水包油乳剂、胶束、混合胶束和脂质体；其中生物方法包括使用DNA和RNA载体。在一种实施方式中，所述的载体被递送(例如，通过转染或电穿孔)到细胞，例如，T细胞或NK细胞，其中所述的载体包含编码如本发明中描述的透明质酸酶的核酸分子，该核酸分子被转录为mRNA分子，并且从所述的RNA分子翻译为所述的透明质酸酶并表达在所述细胞的表面上。
表达所述透明质酸酶的基因可以与表达上述免疫细胞中的肿瘤抗原识别受体的基因位于不同或者同一个载体上。在所述CAR与所述的透明质酸酶位于同一个表达载体上的情况下，所述的表达载体包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区+连接元件+透明质酸酶，其中，所述信号肽位于5’端，所述透明质酸酶位于3’端；或者，所述TCR与所述的透明质酸酶位于同一个表达载体的情况下，所述的表达载体包括以下单元：E6 TCR+连接元件+透明质酸酶，其中，所述E6 TCR位于5’端，所述透明质酸酶位于3’端；
在所述CAR或者所述的TCR与所述的透明质酸酶分别位于不同的表达载体上的情况下，其中一个所述表达载体包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区，或者包括以下表达单元：E6 TCR+IRES，另一个所述表达载体包括以下表达单元：透明质酸酶+连接元件+标签蛋白，其中，所述的透明质酸位于5’端，所述的标签蛋白位于3’端；
所述的连接元件优选T2A连接肽、P2A连接肽、E2A连接肽、F2A连接肽或者IRES元件；
所述的标签蛋白优选tEGFR。
较佳地表达所述透明质酸酶的基因位于上述免疫细胞中的肿瘤抗原识别受体表达载体上。本发明中所述的表达载体可为本领域内常规的、用于在哺乳动物中过表达外源基因的载体，其被用于目的基因的表达，所述表达载体对于复制和整合真核细胞为合适的。典型的克隆载体包含可用于调节目的基因表达的转录和翻译终止子、起始序列和启动子。较佳地为质粒、噬菌体、噬菌体衍生物、动物病毒或者粘粒；其中使用病毒载体已经成为 最广泛使用的将基因插入哺乳动物细胞(例如人类细胞)的方法。例如，逆转录病毒提供了用于基因传递系统的方便的平台。可利用本领域中已知的技术将选择的基因插入载体并包装入逆转录病毒颗粒。该重组病毒可随后被分离和传递至体内或离体的对象细胞。许多逆转录病毒系统在本领域中是已知的。当本发明所述表达载体选用动物病毒作为表达载体时，优选逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒或者慢病毒，更优选逆转录病毒或者慢病毒。
不论表达所述的透明质酸酶的基因与表达所述的肿瘤抗原识别受体的基因位于同一个表达载体上或者不同的表达载体上，两者在包含肿瘤抗原识别受体的免疫细胞中的表达均可为本领域常规；为保证目的基因可以高效地表达，本发明所述的核苷酸序列可以通过多种方式被操作，例如：所述透明质酸酶或者肿瘤抗原识别受体的表达可以受到一个或者多个调控序列的调控。所述的调控序列可以是：(1)合适的转录终止子序列，由宿主细胞识别以终止转录的序列，终止子序列与编码该多肽的核苷酸序列的3′末端操作性连接，在选择的宿主细胞中有功能的任何终止子都可用于本发明；(2)合适的前导序列，对宿主细胞翻译重要的mRNA的非翻译区，前导序列与编码该多肽的核苷酸序列的5′末端可操作连接，在选择的宿主细胞中有功能的任何前导序列都可用于本发明；(3)合适的启动子序列，合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期望表达时打开可操作地连接诱导型启动子的多核苷酸序列的表达，而在不期望表达时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。
本发明中的启动子较佳地为巨细胞病毒启动子、腺病毒主要晚期启动子、SV40启动子、单纯性疱疹病毒胸苷激酶启动子、CMV启动子、EFlα启动子、泛素C启动子、PGK启动子、IRES启动子、MMTV、HIV LTR启动子、MoMuLV启动子、ALV启动子、EBV启动子、RSV启动子或者人类基因的启动子，所述人类基因启动子为肌凝蛋白启动子、血红蛋白启动子、肌酸激酶启动子、β-肌动蛋白启动子、人类IL-2启动子、人类IL-4启 动子、IFN启动子、E2F启动子或者人类GM-CSF启动子；更优选EF1α启动子。
上述免疫细胞可为本领域内常规的免疫细胞，例如T细胞或NK细胞或巨噬细胞；较佳地所述T细胞为CD3抗体刺激活化后的T细胞；更佳地，所述T细胞来源于肿瘤患者的外周血单个核细胞(PBMC)。
本发明还提供一种表达构建物，其包括肿瘤抗原识别受体的表达盒1和透明质酸酶的表达盒2；
较佳地，所述的表达盒1和所述的表达盒2之间通过连接元件顺序连接，所述的连接元件优选T2A连接肽、P2A连接肽、E2A连接肽、F2A连接肽或者IRES元件；
更佳地，所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)或者T细胞受体(TCR)；和/或，所述的透明质酸酶为透明质酸酶全长蛋白或透明质酸酶可溶性蛋白。
其中，当所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)时，所述的表达盒1包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区，或者当所述的肿瘤抗原识别受体为TCR时，所述的表达盒1包括以下表达单元：E6 TCR；
较佳地，所述的scFv为识别ROR1、GPC3或者EpCAM的scFv；所述识别ROR1的scFv的核苷酸序列优选如序列表中SEQ ID NO.4所示，或者与序列表中SEQ ID NO.4所示的核苷酸序列具有80％以上的序列同一性；所述识别GPC3的scFv的核苷酸序列优选如序列表中SEQ ID NO.5所示，或者与序列表中SEQ ID NO.5所示的核苷酸序列具有80％以上的序列同一性；所述识别EpCAM的scFv的核苷酸序列优选如序列表中SEQ ID NO.6所示，或者与序列表中SEQ ID NO.6所示的核苷酸序列具有80％以上的序列同一性
和/或，所述的信号肽的氨基酸序列如序列表中SEQ ID NO.7所示，或者与序列表中SEQ ID NO.7所示的氨基酸序列具有80％以上的序列同一性；
和/或，所述的人CD8α铰链区的氨基酸序列如序列表中SEQ ID NO.11所示，或者与序列表中SEQ ID NO.11所示的氨基酸序列具有80％以上的序列同一性；
和/或，所述的人CD8α跨膜区的氨基酸序列如序列表中SEQ ID NO.12所示，或者与序列表中SEQ ID NO.12所示的氨基酸序列具有80％以上的序列同一性；
和/或，所述的人4-1BB胞内区的氨基酸序列如序列表中SEQ ID NO.13所示，或者与序列表中SEQ ID NO.13所示的氨基酸序列具有80％以上的序列同一性；
和/或，所述的人CD3ζ胞内区的氨基酸序列如序列表中SEQ ID NO.14所示，或者与序列表中SEQ ID NO.14所示的氨基酸序列具有80％以上的序列同一性；
和/或，所述的E6 TCR的氨基酸序列如序列表中SEQ ID NO.18所示，或者与序列表中SEQ ID NO.18所示的氨基酸序列具有80％以上的序列同一性。
所述的表达盒2表达哺乳动物睾丸透明质酸酶，较佳地为人透明质酸酶HYAL1、HYAL2、SPAM1或者PH20；更佳地：所述的透明质酸酶全长蛋白的氨基酸序列如序列表中SEQ ID NO.1所示，或者与如SEQ ID NO.1所示的氨基酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性，且保持透明质酸酶原有功能的氨基酸序列；编码所述的透明质酸酶全长蛋白的核苷酸序列优选如序列表中SEQ ID NO.2或3所示，或者为与如SEQ ID NO.2或者3所示的核苷酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性的核苷酸序列；或者所述的透明质酸酶可溶性蛋白的氨基酸序列如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位所示，或者为与如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位的氨基酸序列具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性，且保持透明质酸酶原有功能的氨基酸序列，编码所述的透明质酸酶可溶性蛋白的核苷酸序列如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位所示，或者与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位具有80％以上的序列同一性，例如具有85％、90％、95％、98％或99％的序列同一性的核苷酸序列。
本发明还提供一种表达载体，其包含如上所述的表达构建物。
较佳地，所述的表达载体还含有启动子；所述的启动子优选EF1α启动子。
本发明还提供一种病毒，其包含如上所述的表达载体；较佳地，所述的病毒为慢病毒。
本发明还提供一种药物组合物，其包含如上所述的免疫细胞；
较佳地，所述的药物组合物还包含药学上可接受的载体或赋形剂。
本发明还提供一种药物组合物，其活性成分包括透明质酸酶和包含肿瘤抗原识别受体的免疫细胞；较佳地：
所述的透明质酸酶在所述的药物组合物中的浓度为0.1μg/ml-15mg/ml，和/或，所述的透明质酸酶的比活性≥10,000单位/mg。
本发明还包括一种药盒，其包括药盒A和药盒B，所述的药盒A包括包含肿瘤抗原识别受体的免疫细胞；所述的药盒B包括透明质酸酶；
较佳地，所述的药盒A和所述的药盒B的施用不分先后。
对于如上所述的药物组合物或者如上所述的药盒中的所述肿瘤抗原识别受体、所述 透明质酸酶等的限定亦如上所述。
本发明还提供一种上述免疫细胞、上述药物组合物或者上述药盒在制备治疗肿瘤药物中的应用；所述肿瘤较佳地为实体肿瘤。
所述实体肿瘤为本领域内常规的实体肿瘤，优选表达透明质酸的实体肿瘤，例如乳腺癌、胃癌、黑色素瘤、胰腺癌、肝癌、胶质母细胞瘤或者肺癌。
在符合本领域常识的基础上，上述各优选条件，可任意组合，即得本发明各较佳实例。
本发明所用试剂和原料均市售可得。
本发明的积极进步效果在于：本发明的包含肿瘤抗原识别受体的免疫细胞，可以改变微环境的细胞外基质结构正常化肿瘤免疫微环境的效果，从而使得其可以有效接近并杀死肿瘤细胞。在可溶性PH20蛋白的帮助下，包含肿瘤抗原识别受体的免疫细胞也能改善肿瘤微环境，有效杀死肿瘤细胞。
附图说明
图1为pPWT质粒。
图2为胰腺癌PDX选定样本免疫组化图；a.HE染色；b.MSLN染色；c.HA染色；d.经PH20处理后进行HA染色；图中比例尺为100μm。
图3为胃癌PDX选定样本免疫组化图；a.HE染色；b.EpCAM染色；c.HA染色；d.经PH20处理后进行HA染色；图中比例尺为100μm。
图4为表达PH20的免疫细胞对人胰腺癌PDX模型小鼠移植瘤的抑制作用。
图5为表达PH20的免疫细胞对人胃癌PDX模型小鼠移植瘤的抑制作用。
具体实施方式
下面通过实施例的方式进一步说明本发明，但并不因此将本发明限制在所述的实施例范围之中。下列实施例中未注明具体条件的实验方法，按照常规方法和条件，或按照商品说明书选择。
除非专门定义，本发明所用的所有技术和科学术语具有在基因治疗，生物化学、遗传学和分子生物学领域内的技术人员通常理解的含义。类似或等效于本发明中描述的所有方法和材料都可以在本发明所述的实践或测试中使用。这些技术如方法和材料充分记载于文献中，参见，例如，除非另有说明，本发明的实践将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的传统技术，这都属于本领 域的技术范围Current Protocols in Molecular Biology(FrederickM.AUSUBEL，2000，Wileyand sonInc，Library of Congress，USA)；Molecular Cloning:A Laboratory Manual，Third Edition，(Sambrooketal，2001，Cold Spring Harbor，NewYork:Cold Spring Harbor Laboratory Press)；Oligonucleotide Synthesis(M.J.Gaited.，1984)；Mullis et al.U.S.Pat.No.4,683,195；Nucleic Acid Hybridization(B.D.Harries&S.J.Higginseds.1984)；Transcription And Translation(B.D.Hames&S.J.Higginseds.1984)；Culture Of Animal Cells(R.I.Freshney，Alan R.Liss，Inc.，1987)；Immobilized Cells And Enzymes(IRL Press，1986)；B.Perbal，A Practical Guide To Molecular Cloning(1984)；the series，Methods In ENZYMOLOGY(J.Abelson和M.Simon，eds.-in-chief，Academic Press，Inc.，New York)，尤其是Vols.154和155(Wuetal.eds.)和Vol.185，“Gene Expression Technology”(D.Goeddel，ed.)；Gene Transfer Vectors For Mammalian Cells(J.H.Miller和M.P.Caloseds.，1987，Cold Spring Harbor Laboratory)；Immunochemical Methods In Cell And Molecular Biology(Mayer和Walker，eds.，Academic Press，London，1987)；Hand book Of Experimental Immunology，卷I-IV(D.M.Weir和C.C.Blackwell，eds.，1986)；和Manipulating the Mouse Embryo(Cold Spring Harbor Laboratory Press，Cold Spring Harbor，N.Y.，1986)。
本发明提及的所有出版物、专利申请、专利和其他参考文献都以其全部内容结合于本发明中作为参考。在冲突的情况下，以本说明书为准。此外，除非另有规定，本说明书中所列举的材料、方法和实施例仅是说明性的，而并非旨在进行限制。
术语“免疫细胞”，是指参与免疫应答，产生免疫效应的细胞，如T细胞、B细胞、自然杀伤(NK)细胞、自然杀伤T(NKT)细胞、肥大细胞和骨髓源性吞噬细胞。在一些实施方案中，所述的免疫效应细胞为T细胞、NK细胞、NKT细胞。在一些实施方案中，所述T细胞可以是自体T细胞、异种T细胞、同种异体T细胞。在一些实施方案中，所述的NK细胞可以是同种异体NK细胞。
术语“肽”、“多肽”和“蛋白质”可互换地使用，并且是指由肽键共价连接的氨基酸残基组成的化合物。蛋白质或肽必须含有至少两个氨基酸，并且对于可以包括蛋白质或肽的序列的氨基酸的最大数量没限制。多肽包括含有彼此通过肽键结合的两个或多个氨基酸的任何肽或蛋白质。正如本发明中使用的那样，该术语是指短链(其在本领域通常也称为例如肽、寡肽和寡聚物)以及较长链(其在本领域通常也称为蛋白质，其存在多种类型)。“多肽”包括例如生物学活性片段、基本上同源的多肽、寡肽、同型二聚体、异二聚体、多肽的变体、修饰的多肽、衍生物、类似物、融合蛋白等。多肽包括天然肽、重组肽或其组合。
术语“氨基酸修饰”包括氨基酸取代、添加和/或缺失，“氨基酸取代”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。本发明中使用的“氨基酸插入”意指在亲本多肽序列中的特定位置添加氨基酸。本发明中使用的“氨基酸缺失”或“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。本发明中使用的术语“保守修饰”意指不显着影响或改变含有所述氨基酸序列的抗体的结合特征的氨基酸修饰。此类保守修饰包括氨基酸取代、插入和缺失。可通过本领域已知的标准技术将修饰导入本发明的抗体中，例如定点诱变和PCR介导的诱变。保守的氨基酸取代是用具有相似侧链的氨基酸残基替换氨基酸残基的取代。本领域已经定义了具有相似侧链的氨基酸残基家族。这些家族包括含碱性侧链的氨基酸(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电的急性侧链(例如，甘氨酸、天冬酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸、色氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸)、β分支侧链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
本发明使用的术语“启动子”为由启动多核苷酸序列的特异性转录所需的细胞的合成机制或引入的合成机制识别的DNA序列。
典型的真核生物启动子由最小启动子和其他顺式元件组成。最小启动子实质上是一个TATA框区，RNA聚合酶II(polII)、TATA结合蛋白(TBP)和TBP相关因子(TAF)可在此结合而启动转录。已发现这类序列元件(例如增强子)提高临近的基因的总体表达水平，且往往是以不依赖位置和/或取向的方式。
在一些实施方案中，本发明所述的肿瘤抗原识别受体是指嵌合受体，是指用基因重组技术将不同来源的DNA片段或蛋白质相应的cDNA连接而成的融合分子。嵌合受体通常包括胞外域、跨膜域和胞内域。可用于本发明的嵌合受体包括但不限于：嵌合抗原受体(CAR)、修饰的T细胞(抗原)受体(TCR)、T细胞融合蛋白(TFP)、T细胞抗原耦合器(TAC)。
本文所用的“嵌合抗原受体”或“CAR”是指一组多肽，当其在免疫效应细胞中时，给所述的细胞提供针对靶细胞(通常是癌细胞)的特异性，并且具有细胞内信号产生。CAR通常包括至少一个细胞外抗原结合结构域(也称为胞外区)、跨膜结构域(也称为跨膜区)和细胞质信号传导结构域(本文中也称为“胞内信号传导结构域”或“胞内区”)，其包括来源于如下定义的刺激性分子和/或共刺激性分子的功能信号传导结构域。在某些方面，多肽组彼此邻接。多肽组包括在存在二聚化分子时可以使多肽彼此偶联的二聚化开关，例如，可以使抗原结合结构域偶联至胞内信号传导结构域。在一个方面，刺激性分子为 与T细胞受体复合体结合的ζ链。在一个方面，细胞质信号传导结构域进一步包括一种或多种来源于至少一个如下定义的共刺激性分子的功能性信号传导结构域。在一个方面，共刺激性分子选自本文所述共刺激性分子，例如4-1BB(即，CD137)、CD27和/或CD28。在一个方面，CAR包括嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于共刺激性分子的功能性信号传导结构域和来源于刺激性分子的功能性信号传导结构域的胞内信号传导结构域。在一个方面中，CAR包含嵌合融合蛋白，该融合蛋白包含细胞外抗原结合结构域、跨膜结构域和包含来源于一个或更多个共刺激性分子的两个功能性信号传导。
在一个方面，本发明设想产生功能等同分子的起始抗体或片段(例如scFv)氨基酸序列的修饰。例如，本文所述的癌症相关抗原的抗原结合结构域的VH或VL，例如CAR中包含的scFv，可以被修饰以保留本文所述的癌症相关抗原的抗原结合结构域的起始VH或VL构架区(例如scFv)的至少约70％、71％、72％.73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。本发明设想整个CAR构建体的修饰，例如CAR构建体的多个结构域的一个或更多个氨基酸序列的修饰，以产生功能等同分子。CAR构建体可以被修饰以保留起始CAR构建体的至少约70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％,81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％的同一性。
术语“刺激性分子”是指由免疫细胞(例如，T细胞、NK细胞、B细胞)表达的提供细胞质信号传导序列的分子，该信号传导序列以刺激性方式调节用于免疫细胞信号传导途径的至少一些方面的免疫细胞的活化。在一个方面，信号是通过例如TCR/CD3复合体与负载有肽的MHC分子的结合启动的初级信号，并且其导致介导T细胞应答，包括，但不限于增殖、活化、分化等。以刺激方式起作用的一级细胞质信号传导序列(也称为“一级信号传导结构域”)可以含有被称为基于免疫受体酪氨酸的活化基序(Immunoreceptor tyrosine-based activation motif，ITAM)的信号传导基序。特别地用于本发明的含有ITAM的细胞质信号传导序列的实例包括，但不限于来源于下述的那些：CD3ζ、常见的FcRγ(FCER1G)、FcγRIIa、FcRβ(FcEpsilon R1b)、CD3γ、CD3δ、CD3ε、CD79a、CD79b、DAP10和DAP12。在本发明的特异性CAR中，在本发明的任一个或更多个CAR中的胞内信号传导结构域包括细胞内信号传导序列，例如CD3-ζ的初级信号传导序列。在本发明的特异性 CAR中，CD3-ζ的初级信号传导序列是来自人或非人类种类例如小鼠、啮齿类动物、猴、猿等的等同残基。
术语“共刺激性分子”是指T细胞上的同源结合配偶体，其特异性地结合共刺激配体，从而介导T细胞的共刺激反应，比如但不限于增殖。共刺激性分子为除了抗原受体或其配体之外的细胞表面分子，其促进有效的免疫应答。共刺激性分子包括但不限于MHC I类分子，BTLA和Toll配体受体，以及OX40、CD27、CD28、CDS、ICAM-1、LFA-1(CD11a/CD18)、ICOS(CD278)和4-1BB(CD137)。这样的共刺激性分子的进一步实例包括CDS、ICAM-1、GITR、BAFFR、HVEM(LIGHTR)、SLAMF7、NKp80(KLRF1)、NKp44、NKp30、NKp46、CD160、CD19、CD4、CD8α、CD8β、IL2Rβ、IL2Rγ、IL7Rα、ITGA4、VLA1、CD49a、ITGA4、IA4、CD49D、ITGA6、VLA-6、CD49f、ITGAD、CD11d、ITGAE、CD103、ITGAL、CD11a、LFA-1、ITGAM、CD11b、ITGAX、CD11c、ITGB1、CD29、ITGB2、CD18、LFA-1、ITGB7、NKG2D、NKG2C、TNFR2、TRANCE/RANKL、DNAM1(CD226)、SLAMF4(CD244、2B4)、CD84、CD96(Tactile)、CEACAM1、CRTAM、Ly9(CD229)、CD160(BY55)、PSGL1、CD100(SEMA4D)、CD69、SLAMF6(NTB-A、Ly108)、SLAM(SLAMF1、CD150、IPO-3)、BLAME(SLAMF8)、SELPLG(CD162)、LTBR、LAT、GADS、SLP-76、PAG/Cbp、CD19a以及特异性地结合CD83的配体。
共刺激性胞内信号传导结构域可以为共刺激性分子的细胞内部分。共刺激性分子可以以下述蛋白质家族代表：TNF受体蛋白、免疫球蛋白样蛋白质、细胞因子受体、整联蛋白、信号传导淋巴细胞性活化分子(SLAM蛋白质)、和NK细胞受体。这样的分子的实例包括CD27、CD28、4-1BB(CD137)、OX40、GITR、CD30、CD40、ICOS、BAFFR、HVEM、ICAM-1、与淋巴细胞功能相关的抗原-1(LFA-1)、CD2、CDS、CD7、CD287、LIGHT、NKG2C、NKG2D、SLAMF7、NKp80、NKp30、NKp44、NKp46、CD160、B7-H3以及特异性地结合CD83的配体等。
胞内信号传导结构域可以包括分子的全部细胞内部分或全部天然胞内信号传导结构域、或其功能片段或衍生物。
术语“4-1BB”是指具有如GenBank Accession No.AAA62478.2提供的氨基酸序列的TNFR超家族的成员，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基；并且“4-1BB共刺激结构域”被定义为GenBank Accession No.AAA62478.2的氨基酸残基214～255，或来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。在一个方面，“4-1BB共刺激结构域”为来自人或者来自非人类物种例如小鼠、啮齿类动物、猴子、猿等的等同残基。
术语“T细胞(抗原)受体(T cell receptor，TCR)”，为所有T细胞表面的特征性标志，以非共价键与CD3结合，形成TCR-CD3复合物。TCR负责识别与主要组织相容性复合体分子结合的抗原。TCR是由两条不同肽链构成的异二聚体，由α、β两条肽链组成，每条肽链又可分为可变区(V区)，恒定区(C区)，跨膜区和胞质区等几部分；其特点是胞质区很短。TCR分子属于免疫球蛋白超家族，其抗原特异性存在于V区；V区(Vα、Vβ)又各有三个高变区CDR1、CDR2、CDR3，其中以CDR3变异最大，直接决定了TCR的抗原结合特异性。在TCR识别MHC-抗原肽复合体时，CDR1，CDR2识别和结合MHC分子抗原结合槽的侧壁，而CDR3直接与抗原肽相结合。TCR分为两类：TCR1和TCR2；TCR1由γ和δ两条链组成，TCR2由α和β两条链组成。
术语“T细胞融合蛋白(T cell fusion protein，TFP)”，包括构成TCR的各种多肽衍生的重组多肽，其能够结合到靶细胞上的表面抗原，和与完整的TCR复合物的其他多肽相互作用，通常同定位在T细胞表面。TFP由一个TCR亚基与人或人源化抗体结构域组成的一个抗原结合结构域组成，其中，TCR亚基包括至少部分TCR胞外结构域、跨膜结构域、TCR胞内结构域的胞内信号结构域的刺激结构域；该TCR亚基和该抗体结构域有效连接，其中，TCR亚基的胞外、跨膜、胞内信号结构域来源于CD3ε或CD3γ，并且，该TFP整合进T细胞上表达的TCR。
术语“T细胞抗原耦合器(T cell antigen coupler，TAC)”，包括三个功能结构域：1、肿瘤靶向结构域，包括单链抗体、设计的锚蛋白重复蛋白(designed ankyrin repeat protein，DARPin)或其他靶向基团；2、胞外区结构域，与CD3结合的单链抗体，从而使得TAC受体与TCR受体靠近；3、跨膜区和CD4共受体的胞内区，其中，胞内区连接蛋白激酶LCK，催化TCR复合物的免疫受体酪氨酸活化基序(ITAMs)磷酸化作为T细胞活化的初始步骤。
术语“抗体”是指源于特异性地结合抗原的免疫球蛋白分子的蛋白质或多肽序列。抗体可以为多克隆的或单克隆的、多链或单链、或完整的免疫球蛋白，并且可以来源于天然来源或重组来源。抗体可以为免疫球蛋白分子的四聚体。
术语“scFv”是指包含至少一个包括轻链的可变区抗体片段和至少一个包括重链的可变区的抗体片段的融合蛋白，其中所述轻链和重链可变区是邻接的(例如经由合成接头例如短的柔性多肽接头)，并且能够以单链多肽形式表达，且其中所述scFv保留其所来源的完整抗体的特异性。除非指定，否则如正如本文中使用的那样，scFv可以以任何顺序(例如相对于多肽的N-末端和C末端)具有所述的VL和VH可变区，scFv可以包括VL-接头-VH或可以包括VH-接头-VL。
术语“重链”是指以其天然存在的构型存在于抗体分子中且通常决定抗体所属类 型的两种多肽链中较大者。
术语“轻链”是指以其天然存在构型存在于抗体分子中的两种多肽链的较小者。κ(k)和λ(l)轻链是指两种主要的抗体轻链的同种型。
术语“抗原”是指引起免疫应答的分子。该免疫应答可以涉及抗体产生或有特异性免疫能力的细胞的活化或两者。本领域技术人员应当理解包括实际上所有蛋白质或肽的任何大分子都可以充当抗原。此外，抗原可以来源于重组或基因组DNA。当在本文中使用该术语时，本领域技术人员应当理解包括编码引起免疫应答的蛋白质的核苷酸序列或部分核苷酸序列的任何DNA，所编码的蛋白质或肽。此外，本领域技术人员应当理解抗原无需仅通过基因的全长核苷酸序列编码。显而易见的是，本发明包括但不限于使用超过一个基因的部分核苷酸序列，并且这些核苷酸序列以不同组合排列以编码引发期望免疫应答的多肽。而且，本领域技术人员应当理解抗原根本无需由“基因”编码。显而易见的是，抗原可以合成产生，或者可以来源于生物样品，或者可以是除了多肽之外的大分子。这样的生物样品可以包括，但不限于组织样品、肿瘤样品、具有其它生物组分的细胞或液体。
“肿瘤抗原”指的是过度增生性疾病发生、发展过程中新出现的或过度表达的抗原。在某些方面，本发明的过度增生性病症是指癌症。
本发明所述的肿瘤抗原可以是实体瘤抗原，也可以是血液瘤抗原。
本发明的肿瘤抗原包括但不限于：促甲状腺激素受体(TSHR)；CD171；CS-1；C型凝集素样分子-1；神经节苷脂GD3；Tn抗原；CD19；CD20；CD 22；CD 30；CD 70；CD123；CD 138；CD33；CD44；CD44v7/8；CD38；CD44v6；B7H3(CD276)，B7H6；KIT(CD117)；白介素13受体亚单位α(IL-13Rα)；白介素11受体α(IL-11Rα)；前列腺干细胞抗原(PSCA)；前列腺特异性膜抗原(PSMA)；癌胚抗原(CEA)；NY-ESO-1；HIV-1Gag；MART-1；gp100；酪氨酸酶；间皮素；EpCAM；蛋白酶丝氨酸21(PRSS21)；血管内皮生长因子受体，血管内皮生长因子受体2(VEGFR2)；路易斯(Y)抗原；CD24；血小板衍生生长因子受体β(PDGFR-β)；阶段特异性胚胎抗原-4(SSEA-4)；细胞表面相关的粘蛋白1(MUC1)，MUC6；表皮生长因子受体家族及其突变体(EGFR，EGFR2，ERBB3，ERBB4，EGFRvIII)；神经细胞粘附分子(NCAM)；碳酸酐酶IX(CAIX)；LMP2；肝配蛋白A型受体2(EphA2)；岩藻糖基GM1；唾液酸基路易斯粘附分子(sLe)；神经节苷脂GM3；TGS5；高分子量黑素瘤相关抗原(HMWMAA)；邻乙酰基GD2神经节苷脂(OAcGD2)；叶酸受体；肿瘤血管内皮标记1(TEM1/CD248)；肿瘤血管内皮标记7相关的(TEM7R)；Claudin6，Claudin18.2、Claudin18.1；ASGPR1；CDH16；5T4；8H9；αvβ6整合素；B细胞成熟 抗原(BCMA)；CA9；κ轻链(kappa light chain)；CSPG4；EGP2，EGP40；FAP；FAR；FBP；胚胎型AchR；HLA-A1，HLA-A2；MAGEA1，MAGE3；KDR；MCSP；NKG2D配体；PSC1；ROR1；Sp17；SURVIVIN；TAG72；TEM1；纤连蛋白；腱生蛋白；肿瘤坏死区的癌胚变体；G蛋白偶联受体C类5组-成员D(GPRC5D)；X染色体开放阅读框61(CXORF61)；CD97；CD179a；间变性淋巴瘤激酶(ALK)；聚唾液酸；胎盘特异性1(PLAC1)；globoH glycoceramide的己糖部分(GloboH)；乳腺分化抗原(NY-BR-1)；uroplakin 2(UPK2)；甲型肝炎病毒细胞受体1(HAVCR1)；肾上腺素受体β3(ADRB3)；pannexin 3(PANX3)；G蛋白偶联受体20(GPR20)；淋巴细胞抗原6复合物基因座K9(LY6K)；嗅觉受体51E2(OR51E2)；TCRγ交替阅读框蛋白(TARP)；肾母细胞瘤蛋白(WT1)；ETS易位变异基因6(ETV6-AML)；精子蛋白17(SPA17)；X抗原家族成员1A(XAGE1)；血管生成素结合细胞表面受体2(Tie2)；黑素瘤癌睾丸抗原-1(MAD-CT-1)；黑素瘤癌睾丸抗原-2(MAD-CT-2)；Fos相关抗原1；p53突变体；人端粒酶逆转录酶(hTERT)；肉瘤易位断点；细胞凋亡的黑素瘤抑制剂(ML-IAP)；ERG(跨膜蛋白酶丝氨酸2(TMPRSS2)ETS融合基因)；N-乙酰葡糖胺基转移酶V(NA17)；配对盒蛋白Pax-3(PAX3)；雄激素受体；细胞周期蛋白B1；V-myc鸟髓细胞瘤病病毒癌基因神经母细胞瘤衍生的同源物(MYCN)；Ras同源物家族成员C(RhoC)；细胞色素P450 1B1(CYP1B1)；CCCTC结合因子(锌指蛋白)样(BORIS)；由T细胞识别的鳞状细胞癌抗原3(SART3)；配对盒蛋白Pax-5(PAX5)；proacrosin结合蛋白sp32(OYTES1)；淋巴细胞特异性蛋白酪氨酸激酶(LCK)；A激酶锚定蛋白4(AKAP-4)；滑膜肉瘤X断点2(SSX2)；CD79a；CD79b；CD72；白细胞相关免疫球蛋白样受体1(LAIR1)；IgA受体的Fc片段(FCAR)；白细胞免疫球蛋白样受体亚家族成员2(LILRA2)；CD300分子样家族成员f(CD300LF)；C型凝集素结构域家族12成员A(CLEC12A)；骨髓基质细胞抗原2(BST2)；含有EGF样模块粘蛋白样激素受体样2(EMR2)；淋巴细胞抗原75(LY75)；磷脂酰肌醇蛋白聚糖-3(GPC3)；Fc受体样5(FCRL5)；免疫球蛋白λ样多肽1(IGLL1)。
术语“肿瘤”指在体外(例如经转化的细胞)或体内的过度增殖性细胞生长的广泛病症类别。可以通过本发明的方法治疗或预防的病况包括例如各种赘生物，包括良性或恶性肿瘤，各种增生等等。癌症的具体例子包括但不限于：乳腺癌，前列腺癌，白血病，淋巴瘤，鼻咽癌，脑胶质瘤，结肠癌，直肠癌，肾细胞癌，肝癌，非小细胞肺癌，小肠癌，食道癌，黑色素瘤，骨癌，胰腺癌，皮肤癌，头颈癌，子宫癌，卵巢癌，胃癌，睾丸癌，输卵管癌，子宫内膜癌，宫颈癌，阴道癌，甲状腺癌，甲状旁腺癌，肾上腺癌，软组织肉瘤，尿道癌，阴茎癌，膀胱癌，输尿管癌，肾盂癌，中枢神经系统(CNS)瘤，血管瘤脊椎肿瘤，胶质瘤，星性细胞瘤，垂体腺瘤所述癌症的组合和所述癌症的转移性病灶。
术语“转染的”或“转化的”是指外源性核酸通过其转移或引入到宿主细胞中的过程。“转染的”或“转化的”细胞是已经用外源性核酸转染或转化细胞。所述细胞包括原发性受试者细胞及其后代。
实施例1.CAR/TCR序列的设计
本发明涉及的蛋白包括PH20、CD8α铰链区、人的CD8α跨膜区、人的4-1BB胞内区和人的CD3ζ胞内区、MSLN、HAS2(透明质酸酶合成基因)以及靶向各靶点的scFv序列、TCR序列。其中PH20氨基酸序列的NCBI收录号为NP_694859.1(cDNA为NM_153189.2)；HAS2的NCBI收录号为NP_005319.1(cDNA为NM_005328.3)；MSLN的NCBI收录号为NP_037536.2(cDNA为NM_013404.4)；tEGFR为EGFR的第III和IV结构域，EGFR的NCBI收录号为NP_005219.2(cDNA为NM_005228.4)。MSLN scFv(SS1)的氨基酸序列及编码碱基序列来源于专利US7977457(SEQ ID NO.12)；GPC3 scFv的氨基酸序列来源于专利CN104140974A(SEQ ID NO.22)，碱基序列为针对人进行密码子优化得到；ROR1scFv的氨基酸序列及编码碱基序列来源于专利US20170283497A1(SEQ ID NO.93)，碱基序列为针对人进行密码子优化得到；EpCAM scFv(MOC31)的氨基酸序列来源于专利US7858088(SEQ ID NO.2)，碱基序列为针对人进行密码子优化得到；HER2scFv(FRP5)的氨基酸序列及编码碱基序列来源于专利US7887801(SEQ ID NO.2)；E6 TCR的氨基酸序列及编码碱基序列来源于专利CN105452288A(SEQ ID NO.30)。
用于本发明各种细胞制备涉及蛋白的氨基酸序列信息如表1所示，相关表达区域的结构如表2所示，编码氨基酸的碱基序列如表3所示。
表1.氨基酸序列表
Figure PCTCN2019090998-appb-000003
Figure PCTCN2019090998-appb-000004
Figure PCTCN2019090998-appb-000005
表2.碱基序列表
Figure PCTCN2019090998-appb-000006
Figure PCTCN2019090998-appb-000007
Figure PCTCN2019090998-appb-000008
Figure PCTCN2019090998-appb-000009
Figure PCTCN2019090998-appb-000010
表3.慢病毒GOI载体表达蛋白的连接顺序
Figure PCTCN2019090998-appb-000011
按照表2的碱基序列以及表3的顺序委托Genewiz公司进行CAR或者TCR序列的全基因合成。
实施例2.载体构建
实施例1中合成得到的PH20、CAR/TCR碱基序列通过EcoRI和XbaI酶切、经DNA Ligation Kit(Takara)连接插入慢病毒载体pCDH-EF1a(SBI)的对应位点(图1)，得到目的基因表达载体Lenti-GOI。MSLN碱基序列通过BamHI和EcoRI酶切、经DNA Ligation Kit(Takara)连接插入pcDNA3.1载体(Invitrogen)的对应位点；HAS2碱基序列通过NheI和BamHI酶切、经DNA Ligation Kit连接插入pcDNA3.1-Hyg载体(Invitrogen)的 对应位点。连接产物分别转化至感受态大肠杆菌(Top10)。转化后第二天，挑取克隆至含100μg/mL氨苄西林(生工生物)LB液体培养基中进行培养，并测序鉴定。选择测序结果正确的克隆，接种至50ml规模含100μg/mL氨苄西林的LB液体培养基中培养约16小时。收集菌体，使用质粒中抽试剂盒(Qiagen)提取并纯化质粒。
得到的MSLN-pcDNA3.1质粒采用PvuI进行线性化酶切，HAS2-pcDNA3.1-Hyg质粒采用FspI进行线性化酶切。酶切产物采用酚氯仿进行抽提，得到初步的线性化质粒。质粒制备所用限制性内切酶均购自Thermo公司。
质粒除菌处理：在得到的质粒中分别加入1/14体积的3M NaCl溶液，使盐离子浓度达到200mM。后加入2.5倍体积-20℃预冷的无水乙醇，反复颠倒混匀，于-20℃保留30min。将该得到的溶液12,000rpm，4℃离心20min，小心地弃去上清，收集沉淀。用1ml 70％乙醇洗涤沉淀，12,000rpm，离心5min后弃去上清。无菌风干沉淀，加入100～200μl无菌水溶解DNA。无菌取5μl用TE Buffer稀释50倍，紫外测OD 260和OD 280。最终得到浓度确定可用于转染的质粒。
实施例3.慢病毒包装
准备10cm细胞培养皿，接种5×10 6个293细胞/皿，加入完全培养基DMEM高糖(GIBCO)+10％FBS(GIBCO)，置于37℃、5％CO 2培养箱，过夜培养。
取出1mg/ml的聚醚酰亚胺(PEI，Polyscience)转染试剂及慢病毒包装质粒(Lenti-GOI、pMD2.G、psPAX2)，室温解冻。取出HBSS缓冲液，温热至室温。取2mL PBS至6孔板的一个孔，分别加入10μg Lenti-GOI、7μg psPAX2、4μg pMD2.G混匀，加入63μL PEI转染试剂并用移液器混匀，室温静置10分钟。将DNA/PEI复合物逐滴加入到10cm培养皿中，轻轻晃动培养皿，充分混匀。将培养皿置于37℃、5％CO 2培养箱，培养6～8小时后，将含有转染试剂的培养基去掉，更换为新鲜的完全培养基并放回培养箱中继续培养。
培养48小时后，收集培养皿中含有病毒的培养上清，用0.45μm滤膜过滤，滤液转至无菌离心管中，加入5mL 20％蔗糖，然后沿着离心管内壁轻轻加入含有病毒的培养基上清。50000×g于4℃离心2小时，离心结束后，在生物安全柜中，小心将离心管中的液体吸去，加入400μL PBS缓冲液将沉淀重悬，将病毒置于-80℃保存。
复苏HT1080细胞并将细胞状态调整至对数生长期。按照每孔8×10 5个细胞接种HT1080至6孔板，培养体积为3mL，将6孔板置于37℃、5％CO 2培养箱中过夜培养。将浓缩后的慢病毒按照0.1、1、10μL的量加入至上述6孔板中，同时添加终浓度为6μg/mL的聚凝胺(Polybrene，Sigma)。将6孔板重新放回37℃、5％CO 2培养箱中，连续培养96 小时。培养结束后，使用PBS洗涤每孔的细胞，然后使用基因组DNA提取试剂盒(TIANGEN)提取基因组DNA，并使用NanoDrop2000测定所提取的基因组DNA的浓度。
采用荧光定量PCR的方法测定慢病毒拷贝数。按照SYBR Premix Ex Taq Ⅱ(Takara)说明书配制qPCR的反应体系，制备PCR反应预混液，使用Roche Light cycler 480进行PCR反应。计算拷贝数，根据计算结果将病毒滴度调整至约为1×10 8TU/mL。
实施例4.表达PH20的T细胞、CAR-T以及TCR-T细胞的制备
调整T细胞密度至1×10 6细胞/mL，加入终浓度为100IU/mL的白介素2(Novatis)及终浓度为100ng/mL的Anti-CD3-OKT3抗体(Thermo)，培养约48hr。
核转染方式制备
采用LONZA T细胞转染试剂盒(VPA-1002)进行T细胞的转染。对T细胞转染程序进行优化，首先选择试剂盒推荐优选方案，V-024/U-014，这两种方式转染后得到细胞活力特别低，因此优化方案至U-010/T-010再进行实验。取出转染所需的质粒至室温融化，打开核转仪，选定不同程序进行转染：V-024/U-014/U-010/T-010(转染强度依次降低)。准备核转染试剂，取20×4μl supplement加入至90×4μl Nucleofector Solution中，即1:4.5的比例，轻轻混匀后平衡此混合溶液至室温。取X VIVO培养基于6孔板，2.5ml/孔，共8个孔，按5.3.4的规则编号，将6孔板放入37℃培养箱预热。从细胞培养箱拿出SS1-CAR-T细胞，计数并再次确认，取相应体积细胞液200×g离心5min，弃净上清，离心过程中取出预热的6孔板。取100μl中平衡至室温的混合溶液重悬细胞，再依次加入质粒，混匀后用试剂盒中配套的吸管将细胞液轻轻转至电转杯中。将电转杯放入核转仪，按下“X”键开始电转。电转结束后，取出电转杯，立即吸取500μl预热的培养基稀释电转的细胞悬液，轻柔的混匀后均匀的加至2个含预热培养基的6孔板孔中。转染后2±0.5hr，去掉培养基上清，用新鲜培养基X-Vivo 15(LONZA)(含100IU/mL白介素2)重悬细胞沉淀，将细胞转移至新的方瓶中。继续培养3天，得到CAR-T细胞。
慢病毒感染方式制备
从-80℃冰箱取出含有慢病毒的重悬溶液后，迅速在37℃水浴锅中融化。在方瓶中加入含有慢病毒的重悬溶液，添加终浓度为5μg/mL的Polybrene，充分混匀后，800×g离心1.5小时。离心结束后，将方瓶置于37℃5％CO 2培养箱中继续培养24小时。250×g离心10分钟，去掉含有病毒的培养基上清，用新鲜培养基X-Vivo 15(LONZA)(含100IU/mL白介素2)重悬细胞沉淀，将细胞转移至新的方瓶中。继续培养3天，得到CAR/TCR-T细胞。
阳性率检测
取1×10 6个细胞使用FACS检测T细胞表面CAR/TCR分子的表达情况。通过Protein L抗体(Thermo)检测CAR阳性率，对应的二抗为Streptavidin PE和Streptavidin FITC(BD Bioscience)。通过Erbitux抗体(BMS)及对应二抗PE山羊抗人IgG Fc抗体(Thermo)检测mPH20-T及sPH20-T细胞阳性率。通过FITC Hamster Anti-Mouse TCR β Chain(BD Bioscience)抗体检测TCR阳性率。阳性率检测结果见表4。
表4.表达PH20的T细胞、CAR-T以及TCR-T细胞的阳性率
免疫细胞 体外实验(％) 体内实验(％)
mPH20-Twt 27.5 /
sPH20-Twt 24.9 /
mPH20-T 25.6 /
sPH20-T 30.2 /
MSLN-CAR-T核转染* 2.8 /
MSLN-mPH20-CAR-T核转染* 1.7 /
MSLN-sPH20-CAR-T核转染* 2.1 /
MSLN-CAR-T 22.1 33.6
MSLN-mPH20-CAR-T 19.7 29.7
MSLN-sPH20-CAR-T 18.7 30.4
GPC3-CAR-T 26.4 /
GPC3-mPH20-CAR-T 16.7 /
GPC3-sPH20-CAR-T 20.3 /
ROR1-CAR-T 35.4 /
ROR1-mPH20-CAR-T 23.5 /
ROR1-sPH20-CAR-T 25.2 /
EpCAM-CAR-T 25.9 /
EpCAM-mPH20-CAR-T 31.2 /
EpCAM-sPH20-CAR-T 28.7 /
E6 TCR-T / 45.7
mPH20-E6 TCR-T / 46.1
sPH20-E6 TCR-T / 39.8
*注：采用4种核转染程序(V-024/U-014/U-010/T-010)得到的细胞，其状态均不好，T细胞转染前活力为93％，转染MSLN-CAR后，活力依次为26％、22％、37％、66％；转染MSLN-mPH20-CAR后，活力依次为23％、31％、35％、71％；转染MSLN-sPH20-CAR后，活力依次为10％、19％、22％、60％。选用活力最高的组别进行CAR阳性的检测。
每隔2天对细胞进行传代处理，观察细胞状态，进行细胞计数，并更换至新鲜培养基进行培养。采用核转染方式进行CAR的瞬时转染实验发现，转染后细胞状态及活力均较差，扩增缓慢，因此未再使用该方式制备CAR-T或者其他类型细胞。慢病毒方式制备的细胞培养约8～14天后，收集细胞备用。培养过程中发现，同时表达PH20没有影响T细胞的增值及阳性率。
实施例5.表达PH20的NK细胞、CAR-NK细胞的制备
采用NK-92细胞(ATCC CRL-2407)进行CAR-NK细胞的制备。NK-92采用RPMI1640(GIBCO)培养基进行培养，添加10％胎牛血清(GIBCO)，加入终浓度为500IU/mL的白介素2(Novatis)。
NK细胞感染慢病毒的方法
从-80℃冰箱取出含有慢病毒的重悬溶液后，迅速在37℃水浴锅中融化。在方瓶中加入含有慢病毒的重悬溶液，添加终浓度为8μg/mL的Polybrene，充分混匀后，800×g离心1.5小时。离心结束后，将方瓶置于37℃5％CO 2培养箱中继续培养24小时。250×g离心10分钟，去掉含有病毒的培养基上清，用新鲜培养基(RPMI1640+10％FBS+500IU/mL)重悬细胞沉淀，将细胞转移至新的方瓶中。继续培养2天后，加入抗生素进行加压筛选，每隔2-3天换液。培养约10天后，对细胞进行有限稀释，接种至96孔板，0.5细胞/孔。37℃5％CO 2培养15～25天。
表达PH20的NK-92细胞制备
通过慢病毒感染并进行有限稀释的方式，分别用sPH20慢病毒和mPH20慢病毒感染NK-92细胞。采用400μg/mL终浓度的潮霉素进行加压筛选。待有限稀释的克隆形成后，检测克隆的透明质酸酶活性。其中sPH20-NK92的克隆，检测上清中的酶活性；而mPH20-NK-92则检测细胞的酶活性。选择有活性的克隆用于后续实验。
表达CAR的NK-92细胞制备
通过慢病毒感染并进行有限稀释的方式，用FRP5-CAR慢病毒分别感染NK-92细胞、sPH20-NK92细胞、mPH20-NK92细胞。采用600μg/mL终浓度的G418进行加压筛选。待有限稀释的克隆形成后，分别检测克隆的透明质酸酶活性以及CAR阳性。通过Protein L抗体(Thermo)检测CAR阳性率，对应的二抗为Streptavidin PE和Streptavidin FITC(BD)。保留CAR阳性的FRP5-CAR-NK92克隆、具有酶活性的CAR阳性FRP5-sPH20-NK92克隆、具有酶活性的CAR阳性FRP5-mPH20-NK92克隆用于后续实验。
实施例6.靶细胞的制备
MSLN阳性靶细胞制备
准备24孔板，接种1×10 5个BxPC3细胞/孔，加入完全培养基(DMEM、10％FBS)，置于37℃、5％CO 2培养箱，过夜培养。
取出1mg/ml的聚醚酰亚胺(PEI，Polyscience)转染试剂及MSLN-pcDNA3.1质粒，室温解冻。取质粒DNA(3.3μg)加入至165μl opti-MEM培养基中，混合均匀。取PEI(10.5μg)加入165μl opti-MEM培养基(GIBCO)中，混合均匀。按1:1比例混合以上两种溶液，室温孵育20mins以形成DNA/PEI复合物。按100μl/well将DNA/PEI溶液加入种板细胞，前后左右摇晃混匀培养板，置于37℃、5％CO 2培养箱孵育24hr。
消化各转染孔中的细胞，混合均匀后计数，按照100000个细胞/皿的比例将转染细胞接种于10cm培养皿，10ml/孔。接种后24hr，更换培养基为DMEM+10％FBS+600μg/ml G418。培养皿置于37℃、5％CO 2培养箱继续培养。后续每2～4天更换培养基，至克隆长出，长出后的克隆混合后进行有限稀释，0.5个细胞/孔接种至96孔板。培养15～25天待克隆长出后，扩增克隆。扩增后的克隆取1×10 6个细胞使用FACS检测表面MSLN分子的表达情况。通过抗人MSLN PE抗体(R&D)检测CAR阳性率，对应的二抗为Streptavidin PE和Streptavidin FITC(BD Bioscience)。得到表达MSLN的BxPC3靶细胞。
表5.靶细胞及培养方法
No. 细胞名称 细胞类型 ATCC 细胞培养基
1 BxPC3 胰腺癌细胞 CRL-1687 RPMI1640+10％FBS
2 BxPC3-MSLN 胰腺癌细胞 / RPMI1640+10％FBS+200μg/ml G418
3 MDA-MB-231 乳腺癌细胞 HTB-26 DMEM+10％FBS
4 SCC152 头颈鳞状细胞癌细胞 CRL-3240 DMEM+10％FBS
5 Hep3B 肝癌细胞 HB-8064 DMEM+10％FBS
HAS2过高表达靶细胞制备
通过HAS2的稳定转染及筛选得到HA高表达靶细胞，作为体外细胞模型，评价PH20对于免疫细胞杀伤靶细胞效果的影响。各靶细胞按照表5列出的培养基进行培养，其中培养试剂均购自GIBCO。取出1mg/ml的聚醚酰亚胺(PEI，Polyscience)转染试剂及HAS2-pcDNA3.1-Hyg质粒，室温解冻。取质粒DNA(3.3μg)加入至165μl opti-MEM培养基中，混合均匀。取PEI(10.5μg)加入165μl opti-MEM培养基中，混合均匀。按1:1比例混合以上两种溶液，室温孵育20mins以形成DNA/PEI复合物。按100μl/well将DNA/PEI溶液加入种板细胞，前后左右摇晃混匀培养板，置于37℃、5％CO 2培养箱孵育24hr。
消化各转染孔中的细胞，混合均匀后计数，按照100000个细胞/皿的比例将转染细胞接种于10cm培养皿，10ml/孔。接种后24hr，更换培养基为细胞培养基+400μg/mL潮 霉素(GIBCO)。培养皿置于37℃、5％CO 2培养箱继续培养。后续每2～4天更换培养基，至克隆长出，长出后的克隆混合后进行有限稀释，0.5个细胞/孔接种至96孔板。培养15～25天待克隆长出后，对克隆进行换液处理。换液后24hr，采用透明质酸ELISA定量试剂盒(R&D)检测培养上清中的透明质酸含量。选择透明质酸分泌量高、生长良好的克隆扩增至24孔板。待满层后，按照1×10 5个细胞每孔接种至24孔板，每孔培养体积1ml。培养24hr后，取培养上清，依据试剂盒提供的检测方法、采用试剂盒中的稀释液稀释培养上清以检测其中的透明质酸含量，选择透明质酸分泌量高、生长良好的克隆各一株用于后续实验(表6)。
表6.靶细胞透明质酸表达量
Figure PCTCN2019090998-appb-000012
实施例7表达PH20的CAR-T细胞具有透明质酶活性以及细胞杀伤活性
慢病毒感染后第7天，取细胞检测透明质酸酶活性。针对分泌透明质酸酶的免疫细胞，取1×10 6个细胞接种至24孔板，培养体积1ml。培养24hr后，取上清用于酶活性检测。针对表达膜结合透明质酸酶的免疫细胞，取1×10 6个目的蛋白表达阳性细胞，酶活性检测缓冲液重悬细胞沉淀至1×10 6个细胞/ml，取细胞液用于酶活性检测。按照《中国药典》2015年版四部中的通则《1207玻璃酸酶测定法》进行CAR-T细胞的透明质酸酶活性检测。
慢病毒感染后第7天，进行体外细胞杀伤实验。取靶细胞按照1×10 5细胞/ml的密度、1ml/孔接种至24孔板。CAR-T细胞按照1×10 6、3×10 5分别接种至含靶细胞的24孔板中。将24孔板放置于37℃、5％CO 2培养箱中培养16～24hr。培养结束后，取培养上清，按照CytoTox
Figure PCTCN2019090998-appb-000013
非放射性细胞毒性检测试剂盒(Promega)说明书的方法检测培养上清中的LDH以确认杀伤效果。
表7.表达不同密码子PH20的T细胞及培养上清中的透明质酸酶活性检测
Figure PCTCN2019090998-appb-000014
Figure PCTCN2019090998-appb-000015
注：OD 640nm值越高，酶活性越小；值越低，酶活性越高。
对比不同核苷酸序列的PH20表达情况。针对表达膜结合透明质酸酶的免疫细胞，采用酶活性检测缓冲液将细胞密度调正未如表7所示，取细胞液用于酶活性检测。结果显示细胞密度达到1×10 6时，mPH20-T细胞的透明质酸酶表达量高于mPH20-Twt(野生型密码子)。针对分泌透明质酸酶的免疫细胞，取1×10 6个细胞接种至24孔板，培养体积1ml。培养24hr后，取上清用于酶活性检测，以玻璃酸酶国家标准品作为标准品进行酶活性的计算。sPH20-T的分泌酶活性为67±25单位/ml，sPH20-Twt的分泌酶活性为58±23单位/ml。以上结果显示，经优化的密码子其表达量高于野生型密码子。
表8.表达PH20的CAR-T细胞及培养上清中的透明质酸酶活性检测
Figure PCTCN2019090998-appb-000016
Figure PCTCN2019090998-appb-000017
注1：OD 640nm值越高，酶活性越小；值越低，酶活性越高。
注2：#核转染的细胞，由于阳性率过低，1×10 6个细胞/ml的阳性细胞实际密度约为1×10 8个细胞/ml，这个密度的细胞对于活性检测存在干扰，结果显示偏高。
注3：*P<0.05，**P<0.01，***P<0.005。
实验结果表明，在HAS2过表达型MDA-MB-231细胞中，携带PH20的ROR1-CAR-T细胞及加入PH20共培养的ROR1-CAR-T细胞，其对靶细胞的杀伤效果显著好于对照ROR1-CAR-T细胞。
实施例8表达PH20的CAR-NK细胞具有透明质酶活性以及细胞杀伤活性
取细胞检测透明质酸酶活性。针对分泌透明质酸酶的免疫细胞，取1×10 6个细胞接种至24孔板，培养体积1ml。培养24hr后，取上清用于酶活性检测。针对表达膜结合透明质酸酶的免疫细胞，取1×10 6个细胞，酶活性检测缓冲液重悬细胞沉淀至1×10 6个细胞/ml，取细胞液用于酶活性检测。按照《中国药典》2015年版四部中的通则《1207玻璃酸酶测定法》进行CAR-NK细胞的透明质酸酶活性检测。
取靶细胞按照1×10 5细胞/ml的密度、1ml/孔接种至24孔板。CAR-NK细胞按照1×10 6、3×10 5分别接种至含靶细胞的24孔板中。将24孔板放置于37℃、5％CO 2培养箱中培养16～24hr。培养结束后，取培养上清，按照CytoTox
Figure PCTCN2019090998-appb-000018
非放射性细胞毒性检测试剂盒(Promega)说明书的方法检测培养上清中的LDH以确认杀伤效果。
表9.表达PH20的CAR-NK细胞及培养上清中的透明质酸酶活性检测
Figure PCTCN2019090998-appb-000019
注1：OD 640nm值越高，酶活性越小；值越低，酶活性越高。
注2：*P<0.05，**P<0.01，***P<0.005。
实验结果表明，表达PH20蛋白的免疫细胞在体外细胞杀伤实验中，针对野生型 MDA-MB-231和HAS2过表达的MDA-MB-231细胞，携带PH20的FRP5-CAR-NK细胞及加入PH20共培养的FRP5-CAR-NK细胞杀伤效果均更好。
实施例9表达PH20的TCR-T细胞具有透明质酶活性以及细胞杀伤活性
取细胞检测透明质酸酶活性。针对分泌透明质酸酶的免疫细胞，取1×10 6个细胞接种至24孔板，培养体积1ml。培养24hr后，取上清用于酶活性检测。针对表达膜结合透明质酸酶的免疫细胞，取1×10 6个细胞，酶活性检测缓冲液重悬细胞沉淀至1×10 6个细胞/ml，取细胞液用于酶活性检测。按照《中国药典》2015年版四部中的通则《1207玻璃酸酶测定法》进行TCR-T细胞的透明质酸酶活性检测。
慢病毒感染后第7天，进行体外细胞杀伤实验。取靶细胞按照1×10 5细胞/ml的密度、1ml/孔接种至24孔板。CAR-T细胞按照1×10 6、3×10 5分别接种至含靶细胞的24孔板中。将24孔板放置于37℃、5％CO 2培养箱中培养16～24hr。培养结束后，取培养上清，按照CytoTox
Figure PCTCN2019090998-appb-000020
非放射性细胞毒性检测试剂盒(Promega)说明书的方法检测培养上清中的LDH以确认杀伤效果。
表10.表达PH20的TCR-T细胞及培养上清中的透明质酸酶活性检测
Figure PCTCN2019090998-appb-000021
注1：OD 640nm值越高，酶活性越小；值越低，酶活性越高。
注2：*P<0.05，**P<0.01，***P<0.005。
实验结果表明，表达PH20蛋白的免疫细胞在体外细胞杀伤实验中，针对野生型SCC152细胞，表现与未表达PH20的免疫细胞类似。HAS2过表达细胞的杀伤实验中，携带PH20的E6 TCR-T细胞及加入PH20共培养的FRP5-CAR-NK细胞杀伤效果均更好。
实施例10PDX模型的筛选
为了评价表达PH20的CAR-T细胞对高水平HA肿瘤组织的作用，建立HA高表达小鼠异种移植物肿瘤模型。采用免疫组化的方法检测PDX组织中的HA表达。小鼠的 PDX组织生长至约500mm 3时，取肿瘤组织，4％福尔马林固定，保存于2～8℃。常规脱水，石蜡包埋切片，片厚3μm。对切片进行脱蜡复水，依次采用二甲苯处理10分钟共两次、无水乙醇处理5分钟共两次、95％乙醇处理2分钟、85％乙醇处理2分钟、75％乙醇处理2分钟、蒸馏水洗2分钟。将切片至于柠檬酸-柠檬酸钠缓冲液中高温热修复30分钟，自然冷却至室温。PBS冲洗三次，每次3分钟。使用3％H 2O 2孵育15分钟，封闭内源性过氧化物酶，随后采用PBS冲洗三次，每次3分钟。
每个候选PDX组织选择4片切片，其中2片加入至含100单位/mL重组人PH20(Rhinobio)的溶液中，在37℃环境下孵育2小时，得到的切片作为阴性对照。2片用PH20缓冲液(25mM哌嗪-1,4-二乙磺酸，70mM氯化钠，0.1％牛血清白蛋白，pH5.5)在37℃环境下孵育2小时。孵育完成后，使用2％山羊血清白(博士德)孵育30分钟进行封闭。将两种处理后的切片分别取一片用1μg/ml biotin-HABP(AMSBIO)进行孵育，4℃孵育过夜。PBS冲洗三次，每次5分钟。采用Strepavidin-HRP(BD Bioscience)溶液孵育30分钟。PBS冲洗三次，每次5分钟。随后采用DAB显色液(Thermo)进行显色。显色完成的切片进行拍照。
每个候选PDX组织选择2片切片，进行抗原的染色。Mesothelin蛋白采用抗人MSLN蛋白兔单抗(abcam)作为一抗。EpCAM采用抗人EpCAM蛋白兔单抗(abcam)作为一抗。山羊抗兔IgG-HRP抗体(abcam)作为二抗。
通过以上免疫组化的方法对胰腺癌病人来源的PDX样本进行MSLN以及HA表达水平的筛选，选择强MSLN以及HA染色级别的PDX样本进行小鼠的接种，最终选择的组织免疫组化结果见图2。通过以上免疫组化的方法对胃癌病人来源的PDX样本进行EpCAM以及HA表达水平的筛选，选择强EpCAM以及HA染色级别的PDX样本进行小鼠的接种，最终选择的组织免疫组化结果见图3。
实施例11表达PH20的免疫细胞在胰腺癌PDX模型小鼠的体内活性
复苏MSLN/HA高表达的胰腺癌PDX样本，接种于2～4只NCG小鼠皮下，当单个瘤体积超过500mm 3时，根据实验方案进行肿瘤的大规模传代。取生长旺盛期的瘤组织剪切成1.5mm 3左右，在无菌条件下，接种于裸小鼠右侧腋窝皮下。每天观察小鼠的生长情况，每周称重，确认小鼠荷瘤后，对肿瘤大小进行监测，监测频率根据实际情况确定。
选择6～7周龄的NCG小鼠用PDX荷瘤接种至小鼠右侧腋窝皮下。通过测径器测量实体瘤团块的长度(L)和宽度(W)，肿瘤体积(TV)计算为：(L×W 2)/2。当肿瘤的体积达到直径大约150～250mm 3时，将小鼠分级成5个处理组：1)T细胞对照；2)MSLN-CAR-T细胞；3)MSLN-mPH20-CAR-T细胞；4)MSLN-sPH20-CAR-T细胞；5)MSLN- CAR-T细胞，PH20蛋白溶液。
给药当日对小鼠的体重和肿瘤大小进行检测。对CAR-T细胞进行细胞计数，并根据CAR的阳性率计算需要给药的总细胞数量。取相应细胞数量体积的细胞进行离心，得到的细胞沉淀采用PBS(pH7.2)清洗两次。清洗后，使用PBS重悬CAR-T细胞，终体积为100μl，每只小鼠给药5×10 6阳性CAR-T细胞。CAR-T细胞采用瘤内注射方式给药一次，一周后采用相同方式再给药一次。PH20蛋白溶液(1.4mg/ml；以下简称溶液B)的给药量为1000单位/只老鼠，对应的蛋白含量约为0.01mg/10μg每只老鼠。组别5给药方式为，CAR-T阳性细胞和溶液B一同给药。细胞给药当天(D0)取10μl溶液B加入至含细胞的PBS中，混匀后，用于小鼠给药。后D1、D3、D5、D7、D8、D10、D12、D14、D16，进行给药，用量为取10μl加入至90μl PBS后进行给药。肿瘤大小的检测频率为每周两次，药效观察周期为第二次细胞注射后第21天，实行实验终点处理。
人胰腺癌PDX模型小鼠移植瘤各实验组的不同时间的肿瘤体积如图4所示，CAR-T细胞/媒介对照给药28天后，组2-5均具有显著的抑瘤作用。MSLN-CAR-T细胞给药T/C值为50.4％，MSLN-mbPH20-CAR-T细胞给药T/C值为7.4％，MSLN-sPH20-CAR-T细胞给药T/C值为2.5％，MSLN-CAR-T细胞+PH20给药T/C值为8.9％。其中表达PH20或者和PH20共给药的CAR-T细胞给药效果均明显好于MSLN-CAR-T细胞。给药期间，各实验组对荷瘤鼠的体重变化无影响。各实验组小鼠状态较好，无体重下降。
实施例12表达PH20的免疫细胞在胃癌PDX模型小鼠的体内活性
复苏EpCAM/HA高表达的胃癌PDX样本，接种于2～4只NCG小鼠皮下，当单个瘤体积超过500mm 3时，根据实验方案进行肿瘤的大规模传代。取生长旺盛期的瘤组织剪切成1.5mm 3左右，在无菌条件下，接种于裸小鼠右侧腋窝皮下。每天观察小鼠的生长情况，每周称重，确认小鼠荷瘤后，对肿瘤大小进行监测，监测频率根据实际情况确定。
选择6～7周龄的NCG小鼠用PDX荷瘤接种至小鼠右侧腋窝皮下。通过测径器测量实体瘤团块的长度(L)和宽度(W)，肿瘤体积(TV)计算为：(L×W 2)/2。当肿瘤的体积达到直径大约150～250mm 3时，将小鼠分级成5个处理组：1)T细胞对照；2)EpCAM-CAR-T细胞；3)EpCAM-mPH20-CAR-T细胞；4)EpCAM-sPH20-CAR-T细胞；5)EpCAM-CAR-T细胞，PH20蛋白溶液(溶液B)。
给药当日对小鼠的体重和肿瘤大小进行检测。对CAR-T细胞进行细胞计数，并根据CAR的阳性率计算需要给药的总细胞数量。取相应细胞数量体积的细胞进行离心，得到的细胞沉淀采用PBS(pH7.2)清洗两次。清洗后，使用PBS重悬CAR-T细胞，终体积为100μl，每只小鼠给药5×10 6阳性CAR-T细胞。CAR-T细胞采用瘤内注射方式给药一 次，一周后采用相同方式再给药一次。溶液B的给药量为1000单位/只老鼠，对应的蛋白含量约为0.01mg/10μg每只老鼠。组别5给药方式为，CAR-T阳性细胞和溶液B一同给药。细胞给药当天(D0)取10μl溶液B加入至含细胞的PBS中，混匀后，用于小鼠给药。后D1、D3、D5、D7、D8、D10、D12、D14、D16取10μl加入至90μl PBS后进行给药。肿瘤大小的检测频率为每周两次，药效观察周期为第二次细胞注射后第21天，实行实验终点处理。
人胃癌PDX模型小鼠移植瘤各实验组的不同时间的肿瘤体积如图5所示，CAR-T细胞/媒介对照给药28天后，组2-5均具有显著的抑瘤作用。EpCAM-CAR-T细胞给药T/C值为68.7％，EpCAM-mPH20-CAR-T细胞给药T/C值为0.7％，EpCAM-sPH20-CAR-T细胞给药T/C值为12.0％，EpCAM-CAR-T细胞+PH20给药T/C值为11.9％。其中表达PH20或者和PH20共给药的CAR-T细胞给药效果均明显好于EpCAM-CAR-T细胞。给药期间，各实验组对荷瘤鼠的体重变化无影响。各实验组小鼠状态较好，无体重下降。

Claims (17)

  1. 一种包含肿瘤抗原识别受体的免疫细胞，其表达透明质酸酶，所述透明质酸酶为：
    a)锚定于所述免疫细胞上的全长蛋白；或
    b)分泌至所述免疫细胞外的可溶型蛋白。
  2. 如权利要求1所述的免疫细胞，其特征在于，所述的b)为所述的a)缺失了羧基端膜结构后获得；较佳地，所述的透明质酸酶为哺乳动物睾丸透明质酸酶，更佳地为人透明质酸酶HYAL1、HYAL2、SPAM1或者PH20；进一步更佳地：
    所述a)的氨基酸序列如序列表中SEQ ID NO.1所示，或者为与如SEQ ID NO.1所示的氨基酸序列具有80％以上的序列同一性的氨基酸序列；编码所述a)的核苷酸序列优选如序列表中SEQ ID NO.2或者SEQ ID NO.3所示，或者为与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列具有80％以上的序列同一性的核苷酸序列；
    所述b)的氨基酸序列为如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位，或者为与如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位的具有80％以上的序列同一性的氨基酸序列；编码所述b)的核苷酸序列优选为如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位，或者为与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位具有80％以上的序列同一性的核苷酸序列。
  3. 如权利要求1所述的免疫细胞，其特征在于，所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)或者T细胞受体(TCR)，较佳地：
    所述的TCR包含TCR的α链、TCR的β链、TCR的γ链和TCR的δ链中的一种或多种，所述α链包含可变区和恒定区；较佳地，所述可变区为人TCRα链的可变区，所述恒定区为鼠α链的恒定区；所述β链包含可变区和恒定区，所述可变区为人TCRβ链的可变区，所述恒定区为鼠β链的恒定区；更佳地，所述的TCR为E6TCR；
    和/或，所述的CAR包括胞内区、铰链区和跨膜区，其中胞内区包括人的4-1BB胞内区和/或人的CD28胞内区，以及人的CD3ζ胞内区，优选为人的4-1BB胞内区、人的CD28胞内区，以及人的CD3ζ胞内区；
    和/或，所述的CAR的铰链区为人的CD8α铰链区、跨膜区为人的CD8α跨膜区。
  4. 如权利要求3所述的免疫细胞，其特征在于，所述CAR或者所述TCR识别的抗原为ROR1、GPC3、MSLN或者EpCAM；所述CAR或者所述TCR的肿瘤抗原识别区为能够结合以上肿瘤抗原的区域，优选scFv；较佳地：
    识别所述ROR1的scFv的核苷酸序列如序列表中SEQ ID NO.4所示，或者与如序列表中SEQ ID NO.4所示的核苷酸序列具有80％以上序列同一性；或者，识别所述GPC3的scFv的核苷酸序列如序列表中SEQ ID NO.5所示，或者与如序列表中SEQ ID NO.5所示的核苷酸序列具有80％以上序列同一性；或者，识别所述EpCAM的scFv的核苷酸序列如序列表中SEQ ID NO.6所示，或者与如序列表中SEQ ID NO.6所示的核苷酸序列具有80％以上序列同一性；
    和/或，所述的信号肽的氨基酸序列如序列表中SEQ ID NO.7所示，或者与如列表中SEQ ID NO.7所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD8α铰链区的氨基酸序列如序列表中SEQ ID NO.11所示，或者与如序列表中SEQ ID NO.11所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD8α跨膜区的氨基酸序列如序列表中SEQ ID NO.12所示，或者与如序列表中SEQ ID NO.12所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人4-1BB胞内区的氨基酸序列如序列表中SEQ ID NO.13所示，或者与如序列表中SEQ ID NO.13所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD3ζ胞内区的氨基酸序列如序列表中SEQ ID NO.14所示，或者与如序列表中SEQ ID NO.14所示的氨基酸序列具有80％以上的序列同一性。
  5. 如权利要求1～4任一项所述的免疫细胞，其特征在于，表达所述透明质酸酶的基因与所述CAR或者所述TCR与所述的透明质酸酶位于同一个表达载体上，或者分别位于不同的表达载体上；较佳地：
    所述TCR或者所述CAR通过DNA或者mRNA形式导入至细胞；
    所述表达载体为动物病毒，优选慢病毒；
    和/或，所述透明质酸酶、所述CAR以及所述TCR中的一种或多种的表达受到有活性的启动子的控制，所述的启动子优选EF1α启动子。
  6. 如权利要求5所述的免疫细胞，其特征在于，
    在所述CAR与所述的透明质酸酶位于同一个表达载体上的情况下，所述的表达载体包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区+连接元件+透明质酸酶，其中，所述信号肽位于5’端，所述透明质酸酶位于3’端；或者，所述TCR与所述的透明质酸酶位于同一个表达载体上的情况下，所述的表达载体包括以下表达单元：E6TCR+连接元件+透明质酸酶，其中，所述E6TCR位于5’端，所述透明质酸酶位于3’端；
    在所述CAR或所述TCR与所述的透明质酸酶分别位于不同的表达载体上的情况下， 其中一个所述表达载体包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区，或者包括以下表达单元：E6TCR+IRES；另一个所述表达载体包括以下表达单元：透明质酸酶+连接元件+标签蛋白，其中，所述的透明质酸位于5’端，所述的标签蛋白位于3’端；
    所述的连接元件优选T2A连接肽、P2A连接肽、E2A连接肽、F2A连接肽或者IRES元件；
    所述的标签蛋白优选tEGFR。
  7. 如权利要求1～6任一项所述的免疫细胞，其特征在于，所述免疫细胞为T细胞、NK细胞或巨噬细胞；较佳地，所述T细胞为CD3抗体刺激活化后的T细胞，所述的NK细胞为NK-92细胞系；更佳地，所述T细胞来源于肿瘤患者的外周血单个核细胞。
  8. 一种表达构建物，其特征在于，其包括肿瘤抗原识别受体的表达盒1和透明质酸酶的表达盒2；
    较佳地，所述的表达盒1和所述的表达盒2之间通过连接元件顺序连接，所述的连接元件优选T2A连接肽、P2A连接肽、E2A连接肽、F2A连接肽或者IRES元件；
    更佳地，所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)或者T细胞受体(TCR)；和/或，所述的透明质酸酶为透明质酸酶全长蛋白或透明质酸酶可溶性蛋白。
  9. 如权利要求8所述的表达构建物，其特征在于，当所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)时，所述的表达盒1包括以下表达单元：信号肽+scFv+人CD8α铰链区+人CD8α跨膜区+人4-1BB胞内区+人CD3ζ胞内区，或者当所述的肿瘤抗原识别受体为TCR时，所述的表达盒1包括以下表达单元：E6TCR；
    较佳地，所述的scFv为识别ROR1、GPC3或者EpCAM的scFv；所述识别ROR1的scFv的核苷酸序列优选如序列表中SEQ ID NO.4所示，或者与序列表中SEQ ID NO.4所示的核苷酸序列具有80％以上的序列同一性；所述识别GPC3的scFv的核苷酸序列优选如序列表中SEQ ID NO.5所示，或者与序列表中SEQ ID NO.5所示的核苷酸序列具有80％以上的序列同一性；所述识别EpCAM的scFv的核苷酸序列优选如序列表中SEQ ID NO.6所示，或者与序列表中SEQ ID NO.6所示的核苷酸序列具有80％以上的序列同一性；
    和/或，所述的信号肽的氨基酸序列如序列表中SEQ ID NO.7所示，或者与序列表中SEQ ID NO.7所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD8α铰链区的氨基酸序列如序列表中SEQ ID NO.11所示，或者与序列表中SEQ ID NO.11所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD8α跨膜区的氨基酸序列如序列表中SEQ ID NO.12所示，或者与序列表中SEQ ID NO.12所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人4-1BB胞内区的氨基酸序列如序列表中SEQ ID NO.13所示，或者与序列表中SEQ ID NO.13所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的人CD3ζ胞内区的氨基酸序列如序列表中SEQ ID NO.14所示，或者与序列表中SEQ ID NO.14所示的氨基酸序列具有80％以上的序列同一性；
    和/或，所述的E6TCR的氨基酸序列如序列表中SEQ ID NO.18所示，或者与序列表中SEQ ID NO.18所示的氨基酸序列具有80％以上的序列同一性。
  10. 如权利要求8或9所述的表达构建物，其特征在于，所述的表达盒2表达哺乳动物睾丸透明质酸酶，较佳地为人透明质酸酶HYAL1、HYAL2、SPAM1或者PH20；更佳地：
    所述的透明质酸酶全长蛋白的氨基酸序列如序列表中SEQ ID NO.1所示，或者与如SEQ ID NO.1所示的氨基酸序列具有80％以上的序列同一性；编码所述的透明质酸酶全长蛋白的核苷酸序列优选如序列表中SEQ ID NO.2或3所示，或者与如SEQ ID NO.2或者3所示的核苷酸序列具有80％以上的序列同一性；
    或者所述的透明质酸酶可溶性蛋白的氨基酸序列如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位所示，或者与如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位的氨基酸序列具有80％以上的序列同一性；编码所述的透明质酸酶可溶性蛋白的核苷酸序列优选如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位所示，或者与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位具有80％以上的序列同一性。
  11. 一种表达载体，其包含如权利要求8～10任一项所述的表达构建物；
    较佳地，所述的表达载体还含有启动子；所述的启动子优选EF1α启动子。
  12. 一种病毒，其包含如权利要求11所述的表达载体；较佳地，所述的病毒为慢病毒。
  13. 一种药物组合物，其特征在于，其包含如权利要求1～7任一项所述的免疫细胞；
    较佳地，所述的药物组合物还包含药学上可接受的载体或赋形剂。
  14. 一种药物组合物，其特征在于，其活性成分包括透明质酸酶和包含肿瘤抗原识别受体的免疫细胞；较佳地：
    所述的透明质酸酶在所述的药物组合物中的浓度为0.1μg/ml-15mg/ml，和/或，所述的透明质酸酶的比活性≥10,000单位/mg。
  15. 一种药盒，其包括药盒A和药盒B，所述的药盒A包括包含肿瘤抗原识别受体的免疫细胞；所述的药盒B包括透明质酸酶；
    较佳地，所述的药盒A和所述的药盒B的施用不分先后。
  16. 如权利要求14所述的药物组合物或者如权利要求15所述的药盒，其特征在于，所述的肿瘤抗原识别受体为嵌合抗原受体(CAR)或者T细胞受体(TCR)；所述CAR或者所述TCR识别的抗原优选ROR1、GPC3、MSLN或者EpCAM，所述CAR或者所述TCR的肿瘤抗原识别区为能够结合以上肿瘤抗原的区域，优选scFv；其中，识别所述ROR1的scFv的核苷酸序列优选如序列表中SEQ ID NO.4所示，或者与序列表中SEQ ID NO.4所示的核苷酸序列具有80％以上的序列同一性；识别所述GPC3的scFv的核苷酸序列优选如序列表中SEQ ID NO.5所示，或者与序列表中SEQ ID NO.5所示的核苷酸序列具有80％以上的序列同一性；识别所述EpCAM的scFv的核苷酸序列优选如序列表中SEQ ID NO.6所示，或者与序列表中SEQ ID NO.6所示的核苷酸序列具有80％以上的序列同一性。
    和/或，所述的透明质酸酶为全长蛋白或可溶性蛋白，较佳地为哺乳动物睾丸透明质酸酶；进一步较佳地为人透明质酸酶HYAL1、HYAL2、SPAM1或者PH20；更佳地：所述的透明质酸酶全长蛋白的氨基酸序列如序列表中SEQ ID NO.1所示，或者与如SEQ ID NO.1所示的氨基酸序列具有80％以上的序列同一性；编码所述的透明质酸酶全长蛋白的核苷酸序列优选如序列表中SEQ ID NO.2或3所示，或者与如SEQ ID NO.2或3所示的氨基酸序列具有80％以上的序列同一性；或者所述的透明质酸酶可溶性蛋白的氨基酸序列如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位所示，或者与如序列表中SEQ ID NO.1所示的氨基酸序列的第1～447位具有80％以上的序列同一性；编码所述的透明质酸酶可溶性蛋白的核苷酸序列优选如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位所示，或者与如序列表中SEQ ID NO.2或者SEQ ID NO.3所示的核苷酸序列的第1～1341位具有80％以上的序列同一性。
  17. 如权利要求1～7任一项所述的免疫细胞、如权利要求13、14或者16所述的药物组合物、或者如权利要求15或16所述的药盒在制备治疗肿瘤药物中的应用，所述的肿瘤为实体肿瘤；所述的实体肿瘤优选表达透明质酸的实体肿瘤，例如黑色素瘤、胰腺癌、肝癌、胶质母细胞瘤、乳腺癌或者肺癌。
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116426483A (zh) * 2021-12-30 2023-07-14 南京紫珑生物科技有限公司 Cd258蛋白在免疫治疗中的应用
WO2023168305A1 (en) * 2022-03-01 2023-09-07 Exuma Biotech Corp. Viral particles with membrane-bound hyaluronidase
WO2024060424A1 (zh) * 2022-09-21 2024-03-28 深圳先进技术研究院 分泌BiTE的CAR-T的制备及其应用

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA3152780A1 (en) * 2019-09-01 2021-03-04 Exuma Biotech Corp. Methods and compositions for the modification and delivery of lymphocytes
WO2023010436A1 (zh) * 2021-08-05 2023-02-09 卡瑞济(北京)生命科技有限公司 Tcr表达构建体以及其制备方法和用途
CN113337514B (zh) * 2021-08-05 2021-10-29 卡瑞济(北京)生命科技有限公司 Tcr表达构建体以及其制备方法和用途

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101668777A (zh) * 2007-02-23 2010-03-10 贝勒研究院 通过clec-6激活人抗原呈递细胞
CN101679508A (zh) * 2007-02-02 2010-03-24 贝勒研究院 基于靶向抗原至抗原呈递细胞上表达的dcir的疫苗
CN102548584A (zh) * 2009-05-06 2012-07-04 贝尔韦什生物医学研究学会 用于治疗癌症的溶瘤腺病毒
CN104244968A (zh) * 2011-12-30 2014-12-24 哈洛齐梅公司 Ph20多肽变体、配制物及其应用
CN107400664A (zh) * 2016-05-20 2017-11-28 蔡胜和 透明质酸酶的细胞表达及其在实体瘤细胞治疗中的应用
WO2017201635A1 (zh) * 2016-05-23 2017-11-30 蔡胜和 透明质酸酶的细胞表达及其在实体瘤细胞治疗中的应用
WO2018057755A1 (en) * 2016-09-21 2018-03-29 Thorne Stephen H High mobility group box i mutant

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106749675B (zh) * 2016-12-27 2022-05-27 深圳市体内生物医药科技有限公司 一种重组慢病毒及其应用

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101679508A (zh) * 2007-02-02 2010-03-24 贝勒研究院 基于靶向抗原至抗原呈递细胞上表达的dcir的疫苗
CN101668777A (zh) * 2007-02-23 2010-03-10 贝勒研究院 通过clec-6激活人抗原呈递细胞
CN102548584A (zh) * 2009-05-06 2012-07-04 贝尔韦什生物医学研究学会 用于治疗癌症的溶瘤腺病毒
CN104244968A (zh) * 2011-12-30 2014-12-24 哈洛齐梅公司 Ph20多肽变体、配制物及其应用
CN107400664A (zh) * 2016-05-20 2017-11-28 蔡胜和 透明质酸酶的细胞表达及其在实体瘤细胞治疗中的应用
WO2017201635A1 (zh) * 2016-05-23 2017-11-30 蔡胜和 透明质酸酶的细胞表达及其在实体瘤细胞治疗中的应用
WO2018057755A1 (en) * 2016-09-21 2018-03-29 Thorne Stephen H High mobility group box i mutant

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116426483A (zh) * 2021-12-30 2023-07-14 南京紫珑生物科技有限公司 Cd258蛋白在免疫治疗中的应用
CN116426483B (zh) * 2021-12-30 2024-02-23 南京紫珑生物科技有限公司 Cd258蛋白在免疫治疗中的应用
WO2023168305A1 (en) * 2022-03-01 2023-09-07 Exuma Biotech Corp. Viral particles with membrane-bound hyaluronidase
WO2024060424A1 (zh) * 2022-09-21 2024-03-28 深圳先进技术研究院 分泌BiTE的CAR-T的制备及其应用

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