CN109913422A

CN109913422A - A kind of immunocyte comprising tumour antigen identification receptor and its application

Info

Publication number: CN109913422A
Application number: CN201811527903.XA
Authority: CN
Inventors: 王征; 徐云霞
Original assignee: SUZHOU KANGJU BIOLOGICAL TECHNOLOGY Co Ltd
Current assignee: SUZHOU KANGJU BIOLOGICAL TECHNOLOGY Co Ltd
Priority date: 2017-12-13
Filing date: 2018-12-13
Publication date: 2019-06-21
Also published as: WO2020119048A1; CN113692441A; CN117004573A

Abstract

The invention discloses a kind of immunocyte comprising tumour antigen identification receptor and its applications.The immunocyte comprising tumour antigen identification receptor expresses hyaluronidase, the hyaluronidase are as follows: full-length proteins a) being anchored on the immunocyte；Or it b) secretes to the soluble type albumen outside the immunocyte.Invention additionally discloses application of the immunocyte in preparation tumor.Immunocyte comprising tumour antigen identification receptor of the invention, thus it is possible to vary the effect of the extra-cellular matrix structure normalization tumour immunity microenvironment of microenvironment, so that it can effectively approach and kill tumour cell.With the help of soluble PH20 albumen, the immunocyte comprising tumour antigen identification receptor can also improve tumor microenvironment, effectively kill tumour cell.

Description

A kind of immunocyte comprising tumour antigen identification receptor and its application

Technical field

The invention belongs to cellular immunotherapy fields, and in particular to a kind of immunocyte comprising tumour antigen identification receptor And its application.

Background technique

Cancer, also known as malignant tumour, it be seriously endanger human health, cause modern humans' death the first big foul disease and Chronic disease.Show just there is 1 people to die of cancer in average every 8 deaths in the whole world at present, this compares AIDS according to the United Nations's statistical data Death toll caused by disease, tuberculosis and malaria is taller.And the annual whole world is diagnosed there are also more than 1,000 ten thousand people and suffers from cancer (" world's cancer report in 2014 " of World Health Organization's publication).

All the time, the basis of oncotherapy is all operation, chemotherapy and radiotherapy.However traditional therapeutic modality is only capable of prolonging A possibility that life cycle of long malignant tumor patient, healing, is very low, and malignant tumour is still " incurable disease ".In past 20 years, lead to Cross specific recognition tumor cell surface specific proteins, the targeted therapies such as Cetuximab to kill tumour cellAnd HerceptinTreatment method, so that they become the standard treatments of many cancers, However the above method can not effectively treat solid tumor.But in the past few years, immunotherapy, one kind is by recruiting and reinforcing Method of the immune system of patient to attack tumour has become the 5th big pillar of tumor therapeuticing method.Wherein, most popular and It is at present CAR-T cell therapeutic approach closest to FDA granted treatment method.However, up to date, CAR-T cell therapies Small-scale clinical test is had been limited to, and is largely the treatment for advanced stage hematologic cancers, and in addition to lymthoma Malignant entity tumor it is ineffective.

CAR-T cell is that tumour immunity inhibits microenvironment for the biggest obstacle for the treatment of of solid tumors, is that tumour is sent out The interior environment formed during exhibition.Malignant entity tumor is mainly by cancer/oncocyte and stroma cell (such as fibroblast and inflammation Disease cell) extracellular matrix and blood vessel/lymphatic vessel constitutes.It is stroma cell in tumor tissues, extracellular compared with normal tissue Matrix and blood vessel/lymphatic vasculature are usually abnormal, the fibroblast with increase number, and vascular system shows Hyperbranched or curling structure.Tumour and stroma cell generate and assemble extracellular matrix components (collagen, albumen are poly- Sugar, hyaluronic acid) form fine and close agglomerate (Clin Oncol 31:2205-2218.2013).Factors above causes tumor tissues Compact structure, nutritional deficiency, pH be relatively low, anoxic, it is suppressed that CAR-T cell effectively plays tumor-killing effect.

Want effectively treatment tumour, CAR-T cell to allow for overcoming above-mentioned microenvironment, enters tumor tissues, swollen It survives in tumor tissue and tumour cell is killed.

Although the CAR-T cell for tumor microenvironment may have been produced, i.e. expression hyaluronidase PH20's CAR-T cell (patent CN107400664A), but there are problems for the technological means of this cell generation, play lethal effect Principle and technical effect are not disclosed.Firstly, CN107400664A thinks in the design of hyaluronidase: in natural shape Under state, hyaluronidase is (i.e. free hyaluronidase) rather than the table in the form of transmembrane protein in the form of secretory protein It reaches, the hyaluronidase coding in this secreting type hyaluronidase or hyaluronidase transmembrane fusion proteins gene order Sequence can be all protein coded sequence, be also possible to the partial sequence containing encoding hyaluronan enzymatic activity.But it is not public Fabric back-up secretes the technological means of hyaluronidase PH20CAR-T cell, and indicates the hyaluronidase with sequestered diffusion It compares, curative effect is better for transmembrane hyaluronidase.

Secondly, the CAR-T cell preparation side of expression hyaluronidase transmembrane fusion proteins disclosed in a particular embodiment Method uses hyaluronidase PH20 transmembrane fusion proteins slow-virus infection CAR-T cell, passes through hyaluronidase antibody magnetic bead The CAR-T cell of isolated expression cross-film hyaluronidase.The coded sequence of the PH20 transmembrane fusion proteins is by PH20- people IgG4 hinge area-CD8 transmembrane region-CD28 intracellular region -4-1BB intracellular region-CD3 ζ sequence intracellular successively forms.But it does not announce this The hyaluronic acid degradation activity of CAR-T cell, the tumor cytotoxicity activity of CAR-T cell, the in-vivo tumour suppression of CAR-T cell System activity.The present inventor is found through experiments that, using expression hyaluronidase transmembrane fusion proteins disclosed in CN107400664A Preparation method be difficult obtain have hyaluronidase activity CAR positive cell；On the other hand, using two slow virus point Gan Ran not be after T cell, T cell state difference, CAR positive cell can not almost detect hyaluronidase activity.

In view of disadvantages described above, urgently need in the prior art a kind of effectively immune thin comprising tumour antigen identification receptor Born of the same parents.

Summary of the invention

The technical problem to be solved by the present invention is to be unable to get in currently available technology with hyalomitome to overcome The active CAR-T cell of acid degradation, the also very low or CAR-T cell containing hyaluronidase of the positive rate of CAR is difficult to simultaneously Hyaluronidase is secreted, thus is unable to get the defect of the effective immunocyte comprising tumour antigen identification receptor, provides one Immunocyte of the kind comprising tumour antigen identification receptor and its application.The immunocyte, which can enter to kill inside solid tumor, to swell Oncocyte；With the help of soluble type PH20 albumen, the immunocyte comprising tumour antigen identification receptor also can enter solid tumor Tumour cell is killed in inside.

The present invention provides a kind of immunocyte comprising tumour antigen identification receptor, expresses hyaluronidase, described Bright matter acid enzyme are as follows:

A) full-length proteins being anchored on the immunocyte；Or

B) it secretes to the soluble type albumen outside the immunocyte.

Hyaluronidase is the enzyme family of degradation hyaluronic acid.In the mankind, there is coding has heterogeneity and position Hyaluronidase 6 genes, HYAL1, HYAL2, HYAL3, HYAL4, HYAL5 (be otherwise known as SPAM1 or PH-20), HYAL6 (be otherwise known as HYALP1).Isoform HYAL1 and HYAL2 are present in most of tissues.HYAL2 is that GPI film is anchored egg It is white, it is mainly responsible for shearing polymer hyaluronic acid, obtained small fragment hyaluronic acid is by cell endocytic to interior lysosome (Endolysosome) it after, is further degraded by HYAL1.In tumor tissues, hyaluronic acid is synthesized by hyaluronic acid synthetase (HAS1, HAS2 and HAS3).HYAL3 is present in marrow and testis, but its function is characterized well not yet.It is transparent Matter acid enzyme PH20 high expression in testis, and participate in process of the egg mother cell by sperm fertilization.

The hyaluronidase is preferably mammal testicular hyaluronidase；It is more preferably human testicle hyaluronic acid Enzyme, also referred to as SPAM1, Sperm Adhesion Molecule 1 or PH20.

The gene for encoding the PH20 generates two kinds of transcriptional variants, respectively corresponds the enzyme sequence of two kinds of forms:

A kind of form is the PH20 sequence comprising film anchor series, which can be anchored on anti-comprising tumour The surface of the immunocyte of former identification receptor.Therefore, obtain expression cell film anchoring PH20 comprising tumour antigen identification by The immunocyte of body；The amino acid sequence of heretofore described hyaluronidase is preferably such as SEQ ID NO.1 institute in sequence table Show, encodes the nucleotide sequence of the PH20 preferably as shown in SEQ ID NO.2 or 3 in sequence table.

Another form is the sequence lacked corresponding to c-terminus membrane binding domain, and the sequence after the missing is most Latter amino acids are usually located at the 430th~454 of full length sequence, to generate soluble type PH20, the carboxy-terminal domain Missing causes hyaluronidase to be secreted into extracellular medium.Therefore, obtain expression has under the conditions of neutral and slant acidity pH The T cell or NK cell comprising CAR or TCR of the secretory hyaluronidase of enzymatic activity, the NK cell are NK-92 Cell；The amino acid sequence of the hyaluronidase of this kind of form is the amino acid sequence as shown in SEQ ID NO.1 in sequence table The 1st~447；Preferably, its nucleotides sequence is classified as shown in SEQ ID NO.2 in sequence table or SEQ ID NO.3 The 1st~1341 of nucleotide sequence.

The tumour antigen identification receptor is Chimeric antigen receptor (CAR) or T cell receptor (TCR), in which:

The TCR includes, as the α chain of TCR, the β chain of TCR, the γ chain of TCR, TCR δ chain or their combination, it is described α chain includes variable region and constant region, preferably, the variable region of the variable region behaviour TCR α chain, the perseverance of the constant region mouse α chain Area is determined, preferably, the β chain includes variable region and constant region, the variable region of the variable region behaviour TCR β chain, the constant region For the constant region of mouse β chain；Wherein, it can be connected by joint sequence between the α chain of the TCR and the β chain of the TCR, it is described Joint sequence can be P2A, T2A, E2A, F2A or IRES；It is more preferably E6 TCR.

Intracellular region included by the CAR, hinge area and transmembrane region can be conventional for this field, the CAR's Intracellular region preferably includes the 4-1BB intracellular region of people and/or the CD28 intracellular region of people and the CD3 ζ intracellular region of people, more preferably wraps Include the CD3 ζ intracellular region of the 4-1BB intracellular region of people, the CD28 intracellular region of people and people.

The CD8 α hinge area of the preferred people of the hinge area of the CAR, the preferred people of the transmembrane region CD8 α transmembrane region

Connector sequence can be passed through between the antigen recognizing district, the hinge area, the transmembrane region and the intracellular region Column connection, the joint sequence include the n duplicate motif in front and back, the motif for GGGS, GGGGS, SSSSG, GSGSA or The integer that GGSGG, the n are 1~5.

Preferably, the antigen of tumour antigen cog region identification can for EpCAM, Mesothelin, CEA, IL13, PDPN, VEGF, EGFR, EGFRvIII, PSMA, FAP, CD171, GD2, Glypican-2, Glypican-3, HER2, HPV are anti- Original, cyclin D1, p53, MMP-7, IL13Ralpha2, MMP-2, MUC-1, G250, L1CAM, ROR1, GPC3 or MSLN； Preferably ROR1, GPC3, MSLN or EpCAM.Mesothelin, IL13R, Podoplanin (PDPN), EGFRvIII with And EGFR has overexpression in glioblastoma；EpCAM has overexpression in cell carcinoma.

The tumour antigen cog region is can be currently used to be in conjunction with the region such as antibody of the above tumour antigen ScFv (single-chain antibody).

Preferably, identify the nucleotide sequence of the scFv of the ROR1 as shown in SEQ ID NO.4 in sequence table,

Alternatively, identify the nucleotide sequence of the scFv of the GPC3 as shown in SEQ ID NO.5 in sequence table,

Alternatively, identifying the nucleotide sequence of the scFv of the EpCAM as shown in SEQ ID NO.6 in sequence table.

Gene is introduced into cell and is well known in the art the method that gene expression enters cell.Carrier can by Any method in this field is easily introduced into host cell, for example, mammal, bacterium, yeast or insect cell.

For example, expression vector can be transferred to host cell by physics, chemical or biological methods.Physical method includes phosphorus Sour calcium precipitate, lipofection, particle bombardment, microinjection, electroporation etc.；Chemical method includes dispersion system of colloid, such as Macromolecular complexes, nanocapsules, microballoon, pearl, and the system based on lipid, including oil in water emulsion, micella, mixed micelle and Liposome；Wherein biological method includes using DNA and RNA carrier.In one embodiment, the carrier is delivered (example Such as, pass through transfection or electroporation) cell is arrived, for example, T cell or NK cell, wherein the carrier includes coding such as the present invention Described in hyaluronidase nucleic acid molecules, which is transcribed into mRNA molecule, and turns over from the RNA molecule It is translated into the hyaluronidase and expresses on the surface of the cell.

The gene for expressing the hyaluronidase can be with the tumour antigen identification receptor expressed in above-mentioned immunocyte Gene is located on the different or same carrier.In the case where described be located on the same expression vector, the expression is carried Body includes following expression unit: signal peptide+scFv+ people CD8 α hinge area+people CD8 α transmembrane region+people 4-1BB intracellular region+people CD3 ζ Intracellular region+link peptide+hyaluronidase, wherein the signal peptide is located at 5 ' ends, and the hyaluronidase is located at 3 ' ends；Alternatively, E6 TCR+ connecting element+hyaluronidase, wherein the E6 TCR is located at 5 ' ends, and the hyaluronidase is located at 3 ' ends；

In the case where described be located on different expression vectors, one of them described expression vector includes that following expression is single Member: signal peptide+scFv+ people CD8 α hinge area+people CD8 α transmembrane region+people 4-1BB intracellular region+people's CD3 ζ intracellular region, another institute Stating expression vector includes following expression unit: hyaluronidase+connecting element+label protein, wherein the hyaluronic acid position In 5 ' ends, the label protein is located at 3 ' ends；

The preferred T2A link peptide of the connecting element, P2A link peptide, E2A link peptide, F2A link peptide or IRES member Part；

The preferred tEGFR of the label protein.

The gene for preferably expressing the hyaluronidase is located at tumour antigen identification receptor table in above-mentioned immunocyte Up on carrier.Heretofore described expression vector can for it is in the art conventional, for being overexpressed external source in mammals The carrier of gene, is used for the expression of target gene, and the expression vector is suitable for replicating and integrating eukaryocyte. Typical cloning vector includes transcription and translation terminator, homing sequence and the promoter that can be used for adjusting destination gene expression. Preferably plasmid, bacteriophage, phage-derived object, animal virus or clay；Wherein had become most using viral vectors The widely used method by gene insertion mammalian cell (such as human cell).For example, retrovirus provides use In the convenient platform of gene delivery system.Using technology as known in the art by the gene insertion vector and packaging of selection Enter retroviral particle.The recombinant virus then can be separated and be transferred to internal or external subject cell.Many reverses Record virus system is well known in the art.When expression vector of the present invention selects animal virus as expression vector, It is preferred that retrovirus, adenovirus, adeno-associated virus, spore the exanthema virus perhaps more preferable retrovirus of slow virus or slow disease Poison.

No matter the gene of the gene of the expression hyaluronidase tumour antigen identification receptor described with expression is located at On the same expression vector or on different expression vectors, the two is in the immunocyte comprising tumour antigen identification receptor Expression can be conventional for this field；To guarantee that target gene can be expressed efficiently, nucleotide sequence of the present invention can be with It is operated in several ways, such as: the expression of the hyaluronidase or tumour antigen identification receptor can be by one Or the regulation of multiple regulating and controlling sequences.The regulating and controlling sequence may is that (1) suitable transcription terminator sequences, thin by host Born of the same parents identify that, to terminate the sequence of transcription, terminator sequence is connect with 3 ' end effectors of the nucleotide sequence for encoding the polypeptide, Functional any terminator can be used in the present invention in the host cell of selection；(2) suitable leader sequence is thin to host Born of the same parents translate the non-translational region of important mRNA, and 5 ' ends of the nucleotide sequence of leader sequence and the coding polypeptide can the company of operation It connects, functional any leader sequence can be used in the present invention in the host cell of selection；(3) suitable promoter sequence, One example of suitable promoter is instant early stage cytomegalovirus (CMV) promoter sequence.The promoter sequence is can Driving is operably coupled to the strong constitutive promoter sequence of any polynucleotide sequence high level expression thereon.Another Example is -1 α of the elongation growth factor (EF-1 α).It is also possible, however, to use other constitutive promoter sequences, including but not limited to class Ape virus-4 0 (SV40) early promoter, mouse mammary cancer viral (MMTV), the long end of human immunodeficiency virus (HIV) repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, the instant early promoter of Epstein-Barr virus, Rous sarcoma Viral promotors and people's gene promoter, such as, but not limited to actin promoter, Myosin promoter, ferroheme Promoter and creatine kinase promoter.Further, also it is contemplated that using inducible promoter.The use of inducible promoter mentions Molecular switch has been supplied, the table of the polynucleotide sequence for the inducible promoter that is operably connected can be opened when it is expected expression It reaches, and closes expression in undesirable expression.The example of inducible promoter includes but is not limited to metallothionein promoter, sugar Cortin promoter, progesterone promoter and tetracycline promoter.

Promoter in the present invention is preferably cytomegalovirus promoter, adenovirus major late promoter, SV40 are opened Mover, herpes simplex thymidine kinase promoter, CMV promoter, EFl α promoter, Ubiquitin C promoter, PGK promoter, IRES promoter, MMTV, HIVLTR promoter, MoMuLV promoter, ALV promoter, EBV promoter, RSV promoter or people The promoter of genoid, the human gene promoter are myosin promoter, Hemoglobin promoter, creatine kinase starting Son, beta-actin promoter, mankind IL-2 promoter, mankind IL-4 promoter, IFN promoter, E2F promoter or the mankind GM-CSF promoter；More preferable EF1 α promoter.

Above-mentioned immunocyte can be conventional immunocyte in the art, such as T cell or NK cell or macrophage；Compared with The T cell is the T cell after the stimulation activation of CD3 antibody goodly；More preferably, the T cell derives from the periphery of tumor patient Blood mononuclear cell (PBMC).

The present invention also provides a kind of application of above-mentioned immunocyte in preparation tumor；The tumour is preferably For entity tumor.

Entity tumor of the entity tumor for routine in the art, the entity tumor of preferred expression hyaluronic acid, such as Breast cancer, gastric cancer, melanoma, cancer of pancreas, liver cancer, glioblastoma or lung cancer.

The present invention also provides a kind of pharmaceutical compositions, and it includes above-mentioned immunocytes.

On the basis of common knowledge of the art, above-mentioned each optimum condition, can any combination to get each preferable reality of the present invention Example.

The reagents and materials used in the present invention are commercially available.

The positive effect of the present invention is that: the immunocyte comprising tumour antigen identification receptor of the invention, it can be with Change the effect of the extra-cellular matrix structure normalization tumour immunity microenvironment of microenvironment, so that it can be effectively close to simultaneously Kill tumour cell.With the help of soluble PH20 albumen, the immunocyte comprising tumour antigen identification receptor can also improve Tumor microenvironment, effectively kill tumour cell.

Detailed description of the invention

Fig. 1 is pPWT plasmid.

Fig. 2 is that cancer of pancreas PDX selectes sample immunohistochemistry figure；A.HE dyeing；B.MSLN dyeing；C.HA dyeing；D. it passes through HA dyeing is carried out after PH20 processing；Figure medium scale is 100 μm.

Fig. 3 is that gastric cancer PDX selectes sample immunohistochemistry figure；A.HE dyeing；B.EpCAM dyeing；C.HA dyeing；D. through PH20 HA dyeing is carried out after processing；Figure medium scale is 100 μm.

Fig. 4 is inhibiting effect of the immunocyte to human pancreas cancer PDX model mice transplantable tumor for expressing PH20.

Fig. 5 is inhibiting effect of the immunocyte to human gastric cancer PDX model mice transplantable tumor for expressing PH20.

Specific embodiment

The present invention is further illustrated below by the mode of embodiment, but does not therefore limit the present invention to the reality It applies among a range.In the following examples, the experimental methods for specific conditions are not specified, according to conventional methods and conditions, or according to quotient The selection of product specification.

The design of embodiment 1.CAR/TCR sequence

Albumen of the present invention include PH20, CD8 α hinge area, the CD8 α transmembrane region of people, people 4-1BB intracellular region and ScFv sequence, the TCR sequence of the CD3 ζ intracellular region of people, MSLN, HAS2 (hyaluronic acid enzymatic synthesis gene) and each target spot of targeting. Wherein the NCBI indexed number of PH20 amino acid sequence is NP_694859.1 (cDNA NM_153189.2)；The NCBI of HAS2 is included Number be NP_005319.1 (cDNA NM_005328.3)；The NCBI indexed number of MSLN is NP_037536.2 (cDNA NM_ 013404.4)；TEGFR is the Section III and IV structural domain of EGFR, and the NCBI indexed number of EGFR is that (cDNA is NP_005219.2 NM_005228.4).The amino acid sequence and encoding base sequences of MSLN scFv (SS1) derives from patent US7977457 (SEQ ID NO 12)；The amino acid sequence of GPC3 scFv derives from patent CN104140974A (SEQ ID NO 22), base sequence It is obtained to carry out codon optimization for people；The amino acid sequence and encoding base sequences of ROR1 scFv derives from patent US20170283497A1 (SEQ ID NO 93), base sequence are to carry out codon optimization for people to obtain；EpCAM scFv (MOC31) amino acid sequence derives from patent US7858088 (SEQ ID NO 2), and base sequence is to carry out password for people Son optimization obtains；The amino acid sequence and encoding base sequences of HER2 scFv (FRP5) derives from patent US7887801 (SEQ ID NO 2)；The amino acid sequence and encoding base sequences of E6 TCR derives from patent CN105452288A (SEQ ID NO 30)。

The amino acid sequence information for being related to albumen for the various cell preparations of the present invention is as shown in table 1, correlated expression region Structure it is as shown in table 2, the base sequence for encoding amino acid is as shown in table 3.

1. amino acid sequence table of table

2. base sequence table of table

The order of connection of 3. slow virus GOI carrier of table expression albumen

The full base of CAR TCR sequence is carried out according to the base sequence of table 2 and the sequence commission Genewiz company of table 3 Because of synthesis.

2. vector construction of embodiment

PH20, CAR/TCR base sequence synthesized in embodiment 1 passes through EcoRI and XbaI enzyme cutting, through DNA The corresponding site (Fig. 1) of slow virus carrier pCDH-EF1a (SBI) is inserted into Ligation Kit (Takara) connection, obtains purpose Expression vector Lenti-GOI.MSLN base sequence passes through BamHI and EcoRI digestion, through DNA Ligation Kit (Takara) corresponding site of connection insertion pcDNA3.1 carrier (Invitrogen)；HAS2 base sequence by NheI and BamHI digestion, the corresponding site through DNA Ligation Kit connection insertion pcDNA3.1-Hyg carrier (Invitrogen).Even Object of practicing midwifery is converted respectively to competent E.coli (Top10).Second day after conversion, picked clones are western to 100 μ g/mL ammonia benzyls are contained It is cultivated in woods (raw work biology) LB liquid medium, and identification is sequenced.Selection sequencing result is correctly cloned, and is seeded to 50ml scale is cultivated about 16 hours in the LB liquid medium containing 100 ampicillins μ g/mL.Thallus is collected, using taking out in plasmid Kit (Qiagen) extracts and plasmid purification.

Obtained MSLN-pcDNA3.1 plasmid carries out linearized enzyme digestion using PvuI, and HAS2-pcDNA3.1-Hyg plasmid is adopted Linearized enzyme digestion is carried out with FspI.Digestion products are stripped using phenol chloroform, obtain preliminary linearization plasmid.Plasmid preparation Restriction enzyme used is purchased from Thermo company.

Plasmid bacteria removing: it is separately added into the 3M NaCl solution of 1/14 volume in obtained plasmid, makes salt ionic concentration Reach 200mM.The dehydrated alcohol of 2.5 times of -20 DEG C of volume pre-coolings is added afterwards, is mixed by inversion repeatedly, in -20 DEG C of reservation 30min.It will 12,000rpm, 4 DEG C of centrifugation 20min of the obtained solution, carefully discard supernatant, and collect precipitating.With 70% ethanol washing of 1ml Precipitating, 12,000rpm, it is discarded supernatant after being centrifuged 5min.100~200 μ l sterile water dissolving DNAs are added in sterile air-dried precipitating.Nothing Bacterium takes 5 μ l to dilute 50 times with TE Buffer, ultraviolet survey OD₂₆₀And OD₂₈₀.It finally obtains concentration and determines the plasmid that can be used for transfecting.

3. slow virus of embodiment packaging

Prepare 10cm Tissue Culture Dish, inoculation 5 × 10⁶Complete medium DMEM high sugar is added in a 293 cells/ware (GIBCO)+10%FBS (GIBCO) is placed in 37 DEG C, 5%CO₂Incubator is incubated overnight.

Take out polyetherimide (PEI, the Polyscience) transfection reagent and slow virus packaging plasmid of 1mg/ml (Lenti-GOI, pMD2.G, psPAX2), thaw at RT.HBSS buffer is taken out, is warmed to room temperature.Take 2mL PBS to 6 orifice plates A hole, be separately added into 10 μ g Lenti-GOI, 7 μ g psPAX2,4 μ g pMD2.G mix, be added 63 μ LPEI transfection reagents And mixed with pipettor, it is stored at room temperature 10 minutes.DNA/PEI complexes drop-wise is added in 10cm culture dish, is shaked gently Culture dish mixes well.Culture dish is placed in 37 DEG C, 5%CO₂Incubator will contain transfection reagent after culture 6~8 hours Culture medium removes, and is changed to fresh complete medium and puts back to and continues to cultivate in incubator.

After culture 48 hours, collects and contain virulent culture supernatant in culture dish, with 0.45 μm of membrane filtration, filtrate is gone to In sterile centrifugation tube, 20% sucrose of 5mL is added, is gently added then along centrifugation inside pipe wall containing virulent culture medium supernatant. 50000 × g is centrifuged 2 hours in 4 DEG C, after centrifugation, in Biohazard Safety Equipment, is carefully sucked the liquid in centrifuge tube, is added Enter 400 μ L PBS buffer solution precipitating is resuspended, virus is placed in -80 DEG C of preservations.

Recovery HT1080 cell simultaneously adjusts cell state to logarithmic growth phase.According to every hole 8 × 10⁵A cell inoculation 6 orifice plates are placed in 37 DEG C, 5%CO to 6 orifice plates, volume of culture 3mL by HT1080₂It is incubated overnight in incubator.After concentration Slow virus is added according to the amount of 0.1,1,10 μ L into above-mentioned 6 orifice plates, while adding the polybrene of final concentration of 6 μ g/mL (Polybrene, Sigma).6 orifice plates are placed back in into 37 DEG C, 5%CO₂In incubator, continuous culture 96 hours.Culture terminates Afterwards, the cell in every hole is washed using PBS, then extracts genomic DNA using genome DNA extracting reagent kit (TIANGEN), and The concentration of extracted genomic DNA is measured using NanoDrop2000.

Slow virus copy number is measured using the method for quantitative fluorescent PCR.According to SYBR Premix Ex Taq II (Takara) specification prepares the reaction system of qPCR, prepares PCR reaction premixed liquid, uses Roche Light cycler 480 Carry out PCR reaction.Copy number is calculated, is adjusted virus titer to about 1 × 10 according to calculated result⁸TU/mL。

Embodiment 4. expresses the preparation of the T cell, CAR-T and TCR-T cell of PH20

T cell density is adjusted to 1 × 10⁶Cell/mL, be added final concentration of 100IU/mL interleukin-22 (Novatis) and The Anti-CD3-OKT3 antibody (Thermo) of final concentration of 100ng/mL cultivates about 48hr.

It is prepared by nuclear transfection mode

The transfection of T cell is carried out using LONZA T cell transfection reagent box (VPA-1002).To T cell transfection procedures into Preferred embodiment, V-024/U-014 are recommended in row optimization, first selective reagent box, both modes obtain cell viability spy after transfecting It is not low, therefore prioritization scheme is tested again to U-010/T-010.Plasmid needed for taking out transfection to room temperature is melted, and core is opened Turn instrument, selected distinct program is transfected: V-024/U-014/U-010/T-010 (transfection intensity successively reduces).Prepare consideration convey Transfection reagent takes 20 × 4 μ l supplement to be added into 90 × 4 μ l Nucleofector Solution, i.e. the ratio of 1:4.5 Example, balances this mixed solution to room temperature after mixing gently.Take X VIVO culture based on 6 orifice plates, the hole 2.5ml/, totally 8 holes, are pressed 6 orifice plates are put into 37 DEG C of incubator preheatings by rule numbers 5.3.4.SS1-CAR-T cell is taken out from cell incubator, is counted And reaffirm, it takes 200 × g of respective volume cell liquid to be centrifuged 5min, abandons net supernatant, the 6 orifice plates of preheating are taken out in centrifugal process. It takes to be balanced in 100ul to the mixed solution of room temperature and cell is resuspended, sequentially add plasmid, with matched suction in kit after mixing Pipe gently goes to cell liquid in electric revolving cup.Electric revolving cup is put into consideration convey instrument, " X " key is pressed and starts electricity turn.After electricity turns, take Electric revolving cup out draws the cell suspension that the culture medium dilution electricity of 500 μ l preheating turns immediately, uniformly adds to 2 after soft mixing In the 6 orifice plates hole of a culture medium containing preheating.2 ± 0.5hr after transfection removes culture medium supernatant, with fresh culture X-Vivo 15 (LONZA) cell precipitation is resuspended in (interleukin-22 containing 100IU/mL), cell is transferred in new square vase.Continue culture 3 days, obtains To CAR-T cell.

It is prepared by slow-virus infection mode

After -80 DEG C of resuspension solution of refrigerators taking-up containing slow virus, melt in 37 DEG C of water-baths rapidly.In square vase The resuspension solution containing slow virus is added, adds the Polybrene of final concentration of 5 μ g/mL, after mixing well, 800 × g centrifugation 1.5 hour.After centrifugation, square vase is placed in 37 DEG C of 5%CO₂Continue culture 24 hours in incubator.250 × g is centrifuged 10 points Clock removes containing virulent culture medium supernatant, with fresh culture X-Vivo 15 (LONZA) (interleukin-22 containing 100IU/mL) weight Outstanding cell precipitation, cell is transferred in new square vase.Continue culture 3 days, obtains CAR/TCR-T cell.

Positive rate detection

Take 1 × 10⁶A cell detects the expression of T cell surface C AR/TCR molecule using FACS.Pass through Protein L antibody (Thermo) detects CAR positive rate, and corresponding secondary antibody is Streptavidin PE and Streptavidin FITC (BD Bioscience).Pass through Erbitux antibody (BMS) and corresponding secondary antibody PE Goat anti-Human IgG Fc antibody (Thermo) detection MPH20-T and sPH20-T cell positive rate.Pass through FITC Hamster Anti-Mouse TCR β Chain (BD Bioscience) antibody test TCR positive rate.Positive rate testing result is shown in Table 4.

Table 4. expresses the positive rate of the T cell of PH20, CAR-T and TCR-T cell

Immunocyte	Experiment in vitro (%)	Experiment in vivo (%)
			mPH20-Twt	27.5	/
sPH20-Twt	24.9	/
			mPH20-T	25.6	/
sPH20-T	30.2	/
			MSLN-CAR-T nuclear transfection *	2.8	/
MSLN-mPH20-CAR-T nuclear transfection *	1.7	/
			MSLN-sPH20-CAR-T nuclear transfection *	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

* note: the cell obtained using 4 seed nucleus transfection procedures (V-024/U-014/U-010/T-010), state is not Good, vigor is 93% before T cell transfects, and after transfecting MSLN-CAR, vigor is followed successively by 26%, 22%, 37%, 66%；Transfection After MSLN-mPH20-CAR, vigor is followed successively by 23%, 31%, 35%, 71%；After transfecting MSLN-sPH20-CAR, vigor is successively It is 10%, 19%, 22%, 60%.The highest group of selection vigor carries out the detection of the CAR positive.

Passage processing is carried out to cell every 2 days, observes cell state, carries out cell count, and replace to fresh cultured Base is cultivated.Carry out the transient transfection studies discovery of CAR using nuclear transfection mode, Transfected cells state and vigor compared with Difference, amplification slowly, therefore do not reuse which preparation CAR-T or other types cell.Cell prepared by slow virus mode After culture about 8~14 days, it is spare to collect cell.Found in incubation, at the same express PH20 not and influence T cell increment and Positive rate.

Embodiment 5. expresses the preparation of the NK cell, CAR-NK cell of PH20

The preparation of CAR-NK cell is carried out using NK-92 cell (ATCC CRL-2407).NK-92 uses RPMI1640 (GIBCO) culture medium is cultivated, and adds 10% fetal calf serum (GIBCO), the interleukin-22 of final concentration of 500IU/mL is added (Novatis)。

The method of NK cell infection slow virus

After -80 DEG C of resuspension solution of refrigerators taking-up containing slow virus, melt in 37 DEG C of water-baths rapidly.In square vase The resuspension solution containing slow virus is added, adds the Polybrene of final concentration of 8 μ g/mL, after mixing well, 800 × g centrifugation 1.5 hour.After centrifugation, square vase is placed in 37 DEG C of 5%CO₂Continue culture 24 hours in incubator.250 × g is centrifuged 10 points Clock, removes containing virulent culture medium supernatant, and it is heavy that cell is resuspended with fresh culture (RPMI1640+10%FBS+500IU/mL) It forms sediment, cell is transferred in new square vase.After continuing culture 2 days, antibiotic is added and carries out pressurization screening, changed liquid every 2-3 days. After culture about 10 days, limiting dilution is carried out to cell, is seeded to 96 orifice plates, 0.5 cells/well.37 DEG C of 5%CO₂Culture 15~25 It.

Express the NK-92 cell preparation of PH20

By way of passing through slow-virus infection and carrying out limiting dilution, sPH20 slow virus and mPH20 slow-virus infection are used respectively NK-92 cell.Pressurization screening is carried out using the hygromycin of 400 μ g/mL final concentrations.After the Clone formation of limiting dilution, detection The hyaluronidase activity of clone.The wherein clone of sPH20-NK92 detects the enzymatic activity in supernatant；And mPH20-NK-92 is then Detect the enzymatic activity of cell.Select active clone for subsequent experimental.

Express the NK-92 cell preparation of CAR

By way of passing through slow-virus infection and carrying out limiting dilution, with FRP5-CAR slow virus infect respectively NK-92 cell, SPH20-NK92 cell, mPH20-NK92 cell.Pressurization screening is carried out using the G418 of 600 μ g/mL final concentrations.To limiting dilution Clone formation after, hyaluronidase activity and the CAR for detecting clone respectively are positive.Pass through Protein L antibody (Thermo) CAR positive rate is detected, corresponding secondary antibody is Streptavidin PE and Streptavidin FITC (BD).Retain The FRP5-CAR-NK92 clone of the CAR positive, has enzymatic activity at the CAR positive FRP5-sPH20-NK92 clone with enzymatic activity The CAR positive FRP5-mPH20-NK92 clone be used for subsequent experimental.

The preparation of 6. target cell of embodiment

The preparation of MSLN positive target cell

Prepare 24 orifice plates, inoculation 1 × 10⁵A BxPC3 cells/well is added complete medium (DMEM, 10%FBS), is placed in 37 DEG C, 5%CO₂Incubator is incubated overnight.

Take out polyetherimide (PEI, the Polyscience) transfection reagent and MSLN-pcDNA3.1 plasmid of 1mg/ml, room Temperature is thawed.It takes Plasmid DNA (3.3 μ g) to be added into 165 μ l opti-MEM culture mediums, is uniformly mixed.PEI (10.5 μ g) is taken to add Enter in 165 μ l opti-MEM culture mediums (GIBCO), is uniformly mixed.Both the above solution, incubation at room temperature are mixed in 1:1 ratio 20mins is to form DNA/PEI compound.Kind of a plate cell is added in DNA/PEI solution by 100 μ l/well, is all around rocked Culture plate is mixed, 37 DEG C, 5%CO are placed in₂Incubator is incubated for r for 24 hours.

The cell in each transfection hole is digested, is counted after mixing, will be transfected according to 100000 cell/ware ratios thin Born of the same parents are inoculated in 10cm culture dish, the hole 10ml/.R for 24 hours after inoculation, replaces medium to DMEM+10%FBS+600 μ g/mlG418. Culture dish is placed in 37 DEG C, 5%CO₂Incubator continues to cultivate.The subsequent culture medium of replacement in every 2~4 days, until clone grows, after growing Clone mixing after carry out limiting dilution, 0.5 cells/well is seeded to 96 orifice plates.After cloning and growing, expand within culture 15~25 days Increase clone.Clone after amplification takes 1 × 10⁶A cell detects the expression of surface MSLN molecule using FACS.By anti-human MSLN PE antibody (R&D) detects CAR positive rate, and corresponding secondary antibody is Streptavidin PE and Streptavidin FITC (BD Bioscience).Obtain the BxPC3 target cell of expression MSLN.

5. target cell of table and cultural method

No.	Cell Name	Cell type	ATCC	Cell culture medium
					1	BxPC3	Pancreatic cancer cell	CRL-1687	RPMI1640+10%FBS
2	BxPC3-MSLN	Pancreatic cancer cell	/	RPMI1640+10%FBS+200 μ g/ml G418
					3	MDA-MB-231	Breast cancer cell	HTB-26	DMEM+10%FBS
4	SCC152	Head and neck squamous cell carcinoma cell	CRL-3240	DMEM+10%FBS
					5	Hep3B	Liver cancer cells	HB-8064	DMEM+10%FBS

The excessively high expression target cell preparation of HAS2

HA high is obtained by the stable transfection and screening of HAS2 and expresses target cell, as In vitro cell model, evaluates PH20 Influence for immunocyte killing target cell effect.Each target cell is cultivated according to the culture medium that table 5 is listed, wherein cultivating Reagent is purchased from GIBCO.Take out polyetherimide (PEI, the Polyscience) transfection reagent and HAS2- of 1mg/ml PcDNA3.1-Hyg plasmid, thaw at RT.Plasmid DNA (3.3 μ g) is taken to be added into 165 μ l opti-MEM culture mediums, mixing is equal It is even.It takes PEI (10.5 μ g) to be added in 165 μ l opti-MEM culture mediums, is uniformly mixed.It is molten in 1:1 ratio mixing both the above Liquid is incubated at room temperature 20mins to form DNA/PEI compound.Kind of a plate cell is added in DNA/PEI solution by 100 μ l/well, it is preceding After roll and mix culture plate, be placed in 37 DEG C, 5%CO₂Incubator is incubated for r for 24 hours.

The cell in each transfection hole is digested, is counted after mixing, will be transfected according to 100000 cell/ware ratios thin Born of the same parents are inoculated in 10cm culture dish, the hole 10ml/.R for 24 hours after inoculation, replaces medium to+400 μ g/mL hygromycin of cell culture medium (GIBCO).Culture dish is placed in 37 DEG C, 5%CO₂Incubator continues to cultivate.The subsequent culture medium of replacement in every 2~4 days, until clone's length Out, limiting dilution is carried out after clone's mixing after growing, 0.5 cells/well is seeded to 96 orifice plates.Culture 15~25 days wait clone After growing, clone is carried out to change liquid processing.R for 24 hours is changed after liquid, is detected and is cultivated using hyaluronic acid ELISA quantification kit (R&D) Hyaluronic acid contents in supernatant.Select hyaluronic acid secretion amount height, well-grown clonal expansion to 24 orifice plates.To full layer Afterwards, according to 1 × 10⁵A cell per well is seeded to 24 orifice plates, every hole volume of culture 1ml.Culture after r, takes culture supernatant for 24 hours, according to According to the detection method of kit offer, the diluted culture supernatant in kit is used to contain to detect hyaluronic acid therein Amount selects hyaluronic acid secretion amount height, each one plant of well-grown clone to be used for subsequent experimental (table 6).

6. target cell hyaluronic acid expression quantity of table

The CAR-T cell that embodiment 7 expresses PH20 has hyaluronidase activity and cell killing activity

The 7th day after slow-virus infection, cell detection hyaluronidase activity is taken.For the immune thin of secretion hyaluronidase Born of the same parents take 1 × 10⁶A cell inoculation is to 24 orifice plates, volume of culture 1ml.Culture after r, takes supernatant for Enzyme assay for 24 hours.Needle To the immunocyte of expression film combination hyaluronidase, 1 × 10 is taken⁶A destination protein expresses positive cell, and Enzyme assay is slow Cell precipitation is resuspended to 1 × 10 in fliud flushing⁶A cell/ml takes cell liquid for Enzyme assay.According to " Chinese Pharmacopoeia " 2015 General rule " 1207 hyaluronidase measuring method " in version four carries out the hyaluronidase activity detection of CAR-T cell.

The 7th day after slow-virus infection, cell in vitro killing experiments are carried out.Take target cell according to 1 × 10⁵Cell/ml is close Degree, the hole 1ml/ are seeded to 24 orifice plates.CAR-T cell is according to 1 × 10⁶、3×10⁵It is seeded in 24 orifice plates containing target cell respectively. 24 orifice plates are placed in 37 DEG C, 5%CO₂Culture 16~r for 24 hours in incubator.After culture, culture supernatant is taken, according to CytoToxLDH in the method detection culture supernatant of non-radioactive cell toxicity detection kit (Promega) specification To confirm fragmentation effect.

Table 7. expresses the T cell of different codon PH20 and the hyaluronidase activity detection in culture supernatant

Note: OD640nm value is higher, and enzymatic activity is smaller；Value is lower, and enzymatic activity is higher.

Compare the PH20 expression of different nucleotide sequences.For expression film combination hyaluronidase immunocyte, It is using Enzyme assay buffer that cell density levelling is not as shown in table 7, take cell liquid for Enzyme assay.As the result is shown Cell density reaches 1 × 10⁶When, the hyaluronidase expression quantity of mPH20-T cell is higher than mPH20-Twt (wild type password Son).For the immunocyte of secretion hyaluronidase, 1 × 10 is taken⁶A cell inoculation is to 24 orifice plates, volume of culture 1ml.Culture It for 24 hours after r, takes supernatant for Enzyme assay, the calculating of enzymatic activity is carried out using hyaluronidase national standard as standard items. The secretase activity of sPH20-T is 67 ± 25 units/ml, and the secretase activity of sPH20-Twt is 58 ± 23 units/ml.More than The results show that its expression quantity of optimized codon is higher than wild-type codon.

Table 8. expresses the CAR-T cell of PH20 and the hyaluronidase activity detection in culture supernatant

Infuse 1:OD_640nmValue is higher, and enzymatic activity is smaller；Value is lower, and enzymatic activity is higher.

The cell for infusing 2:# nuclear transfection, since positive rate is too low, 1 × 10⁶The positive cell actual density of a cell/ml is about It is 1 × 10⁸A cell/ml, the cell of this density, which has Activity determination, to be interfered, higher as the result is shown.

Infuse 3:*P < 0.05, P < 0.005 * * P < 0.01, * * *.

The experimental results showed that carrying the ROR1-CAR-T cell of PH20 in HAS2 overexpression type MDA-MB-231 cell And the ROR1-CAR-T cell that PH20 is co-cultured is added, it is thin to be considerably better than control ROR1-CAR-T to the fragmentation effect of target cell Born of the same parents.

The CAR-NK cell that embodiment 8 expresses PH20 has hyaluronidase activity and cell killing activity

Take cell detection hyaluronidase activity.For the immunocyte of secretion hyaluronidase, 1 × 10 is taken⁶A cell It is seeded to 24 orifice plates, volume of culture 1ml.Culture after r, takes supernatant for Enzyme assay for 24 hours.For expression film combination hyalomitome The immunocyte of sour enzyme, takes 1 × 10⁶Cell precipitation is resuspended to 1 × 10 in a cell, Enzyme assay buffer⁶A cell/ml, takes Cell liquid is used for Enzyme assay.According to the general rule " 1207 hyaluronidase measuring method " in " Chinese Pharmacopoeia " 2015 version four into The hyaluronidase activity of row CAR-NK cell detects.

Take target cell according to 1 × 10⁵Cell/ml density, the hole 1ml/ are seeded to 24 orifice plates.CAR-NK cell according to 1 × 10⁶、3×10⁵It is seeded in 24 orifice plates containing target cell respectively.24 orifice plates are placed in 37 DEG C, 5%CO₂16 are cultivated in incubator ~r for 24 hours.After culture, culture supernatant is taken, according to CytoToxNon-radioactive cell toxicity detection kit (Promega) LDH in the method detection culture supernatant of specification is to confirm fragmentation effect.

Table 9. expresses the CAR-NK cell of PH20 and the hyaluronidase activity detection in culture supernatant

Infuse 2:*P < 0.05, P < 0.005 * * P < 0.01, * * *.

The experimental results showed that expression PH20 albumen immunocyte in vitro cell killing experiment in, for wild type The MDA-MB-231 cell that MDA-MB-231 and HAS2 is overexpressed, FRP5-CAR-NK cell and the addition PH20 for carrying PH20 are total The FRP5-CAR-NK Cell killing efficacy of culture is more preferable.

The TCR-T cell that embodiment 9 expresses PH20 has hyaluronidase activity and cell killing activity

Take cell detection hyaluronidase activity.For the immunocyte of secretion hyaluronidase, 1 × 10 is taken⁶A cell It is seeded to 24 orifice plates, volume of culture 1ml.Culture after r, takes supernatant for Enzyme assay for 24 hours.For expression film combination hyalomitome The immunocyte of sour enzyme, takes 1 × 10⁶Cell precipitation is resuspended to 1 × 10 in a cell, Enzyme assay buffer⁶A cell/ml, takes Cell liquid is used for Enzyme assay.According to the general rule " 1207 hyaluronidase measuring method " in " Chinese Pharmacopoeia " 2015 version four into The hyaluronidase activity of row TCR-T cell detects.

Table 10. expresses the TCR-T cell of PH20 and the hyaluronidase activity detection in culture supernatant

Infuse 2:*P < 0.05, P < 0.005 * * P < 0.01, * * *.

The experimental results showed that expression PH20 albumen immunocyte in vitro cell killing experiment in, for wild type SCC152 cell shows similar with the immunocyte for not expressing PH20.In the killing experiments of HAS2 overexpressing cell, PH20 is carried E6 TCR-T cell and be added PH20 co-culture FRP5-CAR-NK Cell killing efficacy it is more preferable.

The screening of 10 PDX model of embodiment

For effect of the CAR-T cell to high-level HA tumor tissues of evaluation expression PH20, HA high expression mouse is established Xenograft tumor model.Using the HA expression in the method detection PDX tissue of immunohistochemistry.The PDX tissue growth of mouse To about 500mm³When, tumor tissues are taken, 4% formalin is fixed, and is stored in 2~8 DEG C.Conventional dehydration, specimens paraffin embedding slices, piece It is 3 μm thick.Dewaxing rehydration is carried out to slice, successively using dimethylbenzene handle 10 minutes altogether twice, dehydrated alcohol handles 5 minutes two totally Secondary, 95% alcohol treatment 2 minutes, 85% alcohol treatment 2 minutes, 75% alcohol treatment 2 minutes, distillation washing 2 minutes.It will slice 30 minutes multiple, the cooled to room temperature as citric acid-sodium citrate buffer solution high temperature hot repair.PBS is flushed three times, and every time 3 Minute.Use 3%H₂O₂It is incubated for 15 minutes, closes endogenous peroxydase, then flushed three times using PBS, 3 minutes every time.

Each candidate PDX organizational choice 4 slices, wherein 2 are added to containing 100 units/mL recombined human PH20 (Rhinobio) it in solution, is incubated for 2 hours under 37 DEG C of environment, obtained slice is as negative control.2 are buffered with PH20 Liquid (25mM piperazine-- two ethanesulfonic acid of Isosorbide-5-Nitrae, 70mM sodium chloride, 0.1% bovine serum albumin(BSA), pH5.5) is incubated for 2 under 37 DEG C of environment Hour.After the completion of incubation, it is incubated for 30 minutes and is closed using 2% blood of goats pure (doctor's moral).By two kinds, treated cuts Piece take respectively it is a piece of be incubated for 1 μ g/ml biotin-HABP (AMSBIO), 4 DEG C overnight incubation.PBS is flushed three times, every time 5 minutes.It is incubated for 30 minutes using Strepavidin-HRP (BD Bioscience) solution.PBS is flushed three times, and 5 minutes every time. Then developed the color using DAB developing solution (Thermo).The slice that colour developing is completed is taken pictures.

Each candidate PDX organizational choice 2 slices, carry out the dyeing of antigen.Mesothelin albumen uses anti-human MSLN Albumen rabbit monoclonal antibody (abcam) is used as primary antibody.EpCAM is used as primary antibody using anti-human epcam albumen rabbit monoclonal antibody (abcam).Goat is anti- Rabbit igg-HRP antibody (abcam) is used as secondary antibody.

MSLN and HA expression is carried out to the PDX sample in Patients with Pancreatic Cancer source by the method for the above immunohistochemistry Screening, the PDX sample for selecting strong MSLN and HA to dye rank carries out the inoculation of mouse, the histogenic immunity group of final choice As a result see Fig. 2.EpCAM and HA is carried out to the PDX sample in Patients with Gastric Cancer source by the method for the above immunohistochemistry and expresses water Flat screening selects strong EpCAM and HA to dye the inoculation of the PDX sample progress mouse of rank, the histogenic immunity of final choice Groupization result is shown in Fig. 3.

Embodiment 11 expresses activity in vivo of the immunocyte in cancer of pancreas PDX model mice of PH20

The highly expressed cancer of pancreas PDX sample of recovery MSLN/HA, it is subcutaneous to be inoculated in 2~4 NCG mouse, when single knurl product More than 500mm³When, the extensive passage of tumour is carried out according to experimental program.The tumor tissue of growth animated period is taken to cut into 1.5mm³ It is subcutaneous to be aseptically inoculated in armpit on the right side of nude mouse for left and right.The growing state of observation mouse daily, weighs weekly, really After recognizing mouse-borne tumor, tumor size is monitored, monitoring frequency is determines according to actual conditions.

The NCG mouse of 6~7 week old is selected with PDX lotus knurl to be seeded to right side of mice armpit subcutaneous.It is measured by caliper real The length (L) and width (W) of body tumor agglomerate, gross tumor volume (TV) calculate are as follows: (L × W²)/2.When the volume of tumour reaches diameter About 150~250mm³When, mouse is classified into 5 processing groups: 1) T cell compares；2) MSLN-CAR-T cell；3)MSLN- MPH20-CAR-T cell；4) MSLN-sPH20-CAR-T cell；5) MSLN-CAR-T cell, PH20 protein solution.

The administration same day detects the weight and tumor size of mouse.To the progress cell count of CAR-T cell, and according to The positive rate of CAR calculates the total cell quantity for needing to be administered.The cell of corresponding cell quantity volume is taken to be centrifuged, what is obtained is thin Born of the same parents' precipitating is cleaned twice using PBS (pH7.2).After cleaning, CAR-T cell is resuspended using PBS, final volume is 100 μ l, every small Mouse administration 5 × 10⁶Positive CAR-T cell.CAR-T cell is administered once using intratumor injection mode, uses phase Tongfang after a week Formula is administered once again.The detection frequency of tumor size is that twice a week, the efficacy of medicine observing period is the 21st day after second of injection, in fact The processing of row experimental endpoints.

The gross tumor volume of the different time of each experimental group of human pancreas cancer PDX model mice transplantable tumor is as shown in figure 4, CAR-T After cell/vehicle control is administered 28 days, group 2-5 all has significant tumor-inhibiting action.MSLN-CAR-T cell administration T/C value is 50.4%, MSLN-mbPH20-CAR-T cell administration T/C value are that 7.4%, MSLN-sPH20-CAR-T cell administration T/C value is It is 8.9% that T/C value, which is administered, in 2.5%, MSLN-CAR-T cell+PH20.Wherein express PH20 or with the co-administered CAR-T of PH20 Cell administration effect is significantly better than MSLN-CAR-T cell.During administration, each experimental group is to the changes of weight of mice with tumor without shadow It rings.Each experimental mice state is preferable, no weight loss.

Embodiment 12 expresses activity in vivo of the immunocyte in gastric cancer PDX model mice of PH20

The highly expressed gastric cancer PDX sample of recovery EpCAM/HA, it is subcutaneous to be inoculated in 2~4 NCG mouse, when single knurl product More than 500mm³When, the extensive passage of tumour is carried out according to experimental program.The tumor tissue of growth animated period is taken to cut into 1.5mm³ It is subcutaneous to be aseptically inoculated in armpit on the right side of nude mouse for left and right.The growing state of observation mouse daily, weighs weekly, really After recognizing mouse-borne tumor, tumor size is monitored, monitoring frequency is determines according to actual conditions.

The NCG mouse of 6~7 week old is selected with PDX lotus knurl to be seeded to right side of mice armpit subcutaneous.It is measured by caliper real The length (L) and width (W) of body tumor agglomerate, gross tumor volume (TV) calculate are as follows: (L × W²)/2.When the volume of tumour reaches diameter About 150~250mm³When, mouse is classified into 5 processing groups: 1) T cell compares；2) EpCAM-CAR-T cell；3) EpCAM-mPH20-CAR-T cell；4) EpCAM-sPH20-CAR-T cell；5) EpCAM-CAR-T cell, PH20 protein solution.

The gross tumor volume of the different time of each experimental group of human gastric cancer PDX model mice transplantable tumor is as shown in figure 5, CAR-T is thin After born of the same parents/vehicle control is administered 28 days, group 2-5 all has significant tumor-inhibiting action.EpCAM-CAR-T cell administration T/C value is 68.7%, EpCAM-mPH20-CAR-T cell administration T/C value are that 0.7%, EpCAM-sPH20-CAR-T cell administration T/C value is It is 11.9% that T/C value, which is administered, in 12.0%, EpCAM-CAR-T cell+PH20.Wherein express PH20 or co-administered with PH20 CAR-T cell administration effect is significantly better than EpCAM-CAR-T cell.During administration, each experimental group becomes the weight of mice with tumor Change without influence.Each experimental mice state is preferable, no weight loss.

SEQUENCE LISTING

<110>Suzhou Kang Ju Biotechnology Co., Ltd

<120>a kind of immunocyte comprising tumour antigen identification receptor and its application

<130> P180117007C

<150> 2017113299674

<151> 2017-12-13

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 455

<212> PRT

<213> Homo sapiens

<400> 1

Leu Asn Phe Arg Ala Pro Pro Val Ile Pro Asn Val Pro Phe Leu Trp

1 5 10 15

Ala Trp Asn Ala Pro Ser Glu Phe Cys Leu Gly Lys Phe Asp Glu Pro

20 25 30

Leu Asp Met Ser Leu Phe Ser Phe Ile Gly Ser Pro Arg Ile Asn Ala

35 40 45

Thr Gly Gln Gly Val Thr Ile Phe Tyr Val Asp Arg Leu Gly Tyr Tyr

50 55 60

Pro Tyr Ile Asp Ser Ile Thr Gly Val Thr Val Asn Gly Gly Ile Pro

65 70 75 80

Gln Lys Ile Ser Leu Gln Asp His Leu Asp Lys Ala Lys Lys Asp Ile

85 90 95

Thr Phe Tyr Met Pro Val Asp Asn Leu Gly Met Ala Val Ile Asp Trp

100 105 110

Glu Glu Trp Arg Pro Thr Trp Ala Arg Asn Trp Lys Pro Lys Asp Val

115 120 125

Tyr Lys Asn Arg Ser Ile Glu Leu Val Gln Gln Gln Asn Val Gln Leu

130 135 140

Ser Leu Thr Glu Ala Thr Glu Lys Ala Lys Gln Glu Phe Glu Lys Ala

145 150 155 160

Gly Lys Asp Phe Leu Val Glu Thr Ile Lys Leu Gly Lys Leu Leu Arg

165 170 175

Pro Asn His Leu Trp Gly Tyr Tyr Leu Phe Pro Asp Cys Tyr Asn His

180 185 190

His Tyr Lys Lys Pro Gly Tyr Asn Gly Ser Cys Phe Asn Val Glu Ile

195 200 205

Lys Arg Asn Asp Asp Leu Ser Trp Leu Trp Asn Glu Ser Thr Ala Leu

210 215 220

Tyr Pro Ser Ile Tyr Leu Asn Thr Gln Gln Ser Pro Val Ala Ala Thr

225 230 235 240

Leu Tyr Val Arg Asn Arg Val Arg Glu Ala Ile Arg Val Ser Lys Ile

245 250 255

Pro Asp Ala Lys Ser Pro Leu Pro Val Phe Ala Tyr Thr Arg Ile Val

260 265 270

Phe Thr Asp Gln Val Leu Lys Phe Leu Ser Gln Asp Glu Leu Val Tyr

275 280 285

Thr Phe Gly Glu Thr Val Ala Leu Gly Ala Ser Gly Ile Val Ile Trp

290 295 300

Gly Thr Leu Ser Ile Met Arg Ser Met Lys Ser Cys Leu Leu Leu Asp

305 310 315 320

Asn Tyr Met Glu Thr Ile Leu Asn Pro Tyr Ile Ile Asn Val Thr Leu

325 330 335

Ala Ala Lys Met Cys Ser Gln Val Leu Cys Gln Glu Gln Gly Val Cys

340 345 350

Ile Arg Lys Asn Trp Asn Ser Ser Asp Tyr Leu His Leu Asn Pro Asp

355 360 365

Asn Phe Ala Ile Gln Leu Glu Lys Gly Gly Lys Phe Thr Val Arg Gly

370 375 380

Lys Pro Thr Leu Glu Asp Leu Glu Gln Phe Ser Glu Lys Phe Tyr Cys

385 390 395 400

Ser Cys Tyr Ser Thr Leu Ser Cys Lys Glu Lys Ala Asp Val Lys Asp

405 410 415

Thr Asp Ala Val Asp Val Cys Ile Ala Asp Gly Val Cys Ile Asp Ala

420 425 430

Phe Leu Lys Pro Pro Met Glu Thr Glu Glu Pro Gln Ile Phe Tyr Asn

435 440 445

Ala Ser Pro Ser Thr Leu Ser

450 455

<210> 2

<211> 1365

<212> DNA

<213> Homo sapiens

<400> 2

ctgaatttca gagcacctcc tgttattcca aatgtgcctt tcctctgggc ctggaatgcc 60

ccaagtgaat tttgtcttgg aaaatttgat gagccactag atatgagcct cttctctttc 120

ataggaagcc cccgaataaa cgccaccggg caaggtgtta caatatttta tgttgataga 180

cttggctact atccttacat agattcaatc acaggagtaa ctgtgaatgg aggaatcccc 240

cagaagattt ccttacaaga ccatctggac aaagctaaga aagacattac attttatatg 300

ccagtagaca atttgggaat ggctgttatt gactgggaag aatggagacc cacttgggca 360

agaaactgga aacctaaaga tgtttacaag aataggtcta ttgaattggt tcagcaacaa 420

aatgtacaac ttagtctcac agaggccact gagaaagcaa aacaagaatt tgaaaaggca 480

gggaaggatt tcctggtaga gactataaaa ttgggaaaat tacttcggcc aaatcacttg 540

tggggttatt atctttttcc ggattgttac aaccatcact ataagaaacc cggttacaat 600

ggaagttgct tcaatgtaga aataaaaaga aatgatgatc tcagctggtt gtggaatgaa 660

agcactgctc tttacccatc catttatttg aacactcagc agtctcctgt agctgctaca 720

ctctatgtgc gcaatcgagt tcgggaagcc atcagagttt ccaaaatacc tgatgcaaaa 780

agtccacttc cggtttttgc atatacccgc atagttttta ctgatcaagt tttgaaattc 840

ctttctcaag atgaacttgt gtatacattt ggcgaaactg ttgctctggg tgcttctgga 900

attgtaatat ggggaaccct cagtataatg cgaagtatga aatcttgctt gctcctagac 960

aattacatgg agactatact gaatccttac ataatcaacg tcacactagc agccaaaatg 1020

tgtagccaag tgctttgcca ggagcaagga gtgtgtataa ggaaaaactg gaattcaagt 1080

gactatcttc acctcaaccc agataatttt gctattcaac ttgagaaagg tggaaagttc 1140

acagtacgtg gaaaaccgac acttgaagac ctggagcaat tttctgaaaa attttattgc 1200

agctgttata gcaccttgag ttgtaaggag aaagctgatg taaaagacac tgatgctgtt 1260

gatgtgtgta ttgctgatgg tgtctgtata gatgcttttc taaaacctcc catggagaca 1320

gaagaacctc aaattttcta caatgcttca ccctccacac tatct 1365

<210> 3

<211> 1365

<212> DNA

<213> Artificial Sequence

<220>

<223>after PH20(optimization)

<400> 3

ctgaacttca gagccccccc tgtgatcccc aacgtgcctt tcctgtgggc ctggaatgcc 60

ccaagcgagt tctgcctggg caagtttgac gagcccctgg atatgtctct gttcagcttt 120

atcggctccc ccagaatcaa tgccaccggc cagggcgtga caatctttta cgtggacagg 180

ctgggctact atccttatat cgattccatc accggcgtga cagtgaacgg cggcatccca 240

cagaagatca gcctgcagga ccacctggat aaggccaaga aggatatcac cttctacatg 300

cccgtggaca atctgggcat ggccgtgatc gattgggagg agtggcgccc aacatgggcc 360

agaaactgga agcccaagga cgtgtataag aatcgctcta tcgagctggt gcagcagcag 420

aacgtgcagc tgagcctgac cgaggccaca gagaaggcca agcaggagtt cgagaaggcc 480

ggcaaggact ttctggtgga gaccatcaag ctgggcaagc tgctgagacc caaccacctg 540

tggggctact atctgtttcc tgattgctac aatcaccact ataagaagcc aggctacaac 600

ggctcttgtt tcaatgtgga gatcaagagg aacgacgatc tgagctggct gtggaatgag 660

tccacagccc tgtacccctc tatctatctg aacacccagc agtcccctgt ggccgccaca 720

ctgtatgtgc ggaatagagt gagggaggcc atccgcgtga gcaagatccc tgacgccaag 780

tcccctctgc cagtgttcgc ctacacccgg atcgtgttta cagaccaggt gctgaagttc 840

ctgagccagg atgagctggt gtataccttc ggcgagacag tggccctggg cgccagcgga 900

atcgtgatct ggggcaccct gtccatcatg cggtccatga agtcttgcct gctgctggat 960

aactacatgg agaccatcct gaacccctat atcatcaatg tgacactggc cgccaagatg 1020

tgcagccagg tgctgtgcca ggagcagggc gtgtgcatcc gcaagaactg gaacagcagc 1080

gattacctgc acctgaaccc cgacaatttt gccatccagc tggagaaggg cggcaagttc 1140

accgtgcggg gcaagcctac actggaggac ctggagcagt tctccgagaa gttttactgc 1200

agctgttatt ccaccctgtc ttgtaaggag aaggccgatg tgaaggacac agatgccgtg 1260

gacgtgtgca tcgccgatgg cgtgtgcatc gacgcctttc tgaagccacc catggagacc 1320

gaggagcctc agattttcta caatgccagc ccatccaccc tgtct 1365

<210> 4

<211> 711

<212> DNA

<213> Artificial Sequence

<220>

<223> ROR1 scFv

<400> 4

caggtacagc tgaaggagag cggtcccggc ctggtggccc cctctcagac cctgagcata 60

acctgcaccg tgagcggatt cagcctgacc agctacgggg tgcactgggt gaggcagcct 120

cccggcaagg gcctggagtg gctgggcgtg atctgggccg gtggcttcac caactacaac 180

agcgccctga agtctcgact gtcaatctca aaggacaatt ctaagagcca ggtgctgctc 240

aaaatgacat cattgcagac cgacgacacc gccatgtact actgcgccag gaggggcagc 300

tcctacagca tggactactg gggccagggc acaagcgtga cggtgtcatc aggaggcggt 360

gggagtgggg gaggagggag cggtggcgga ggcagtgaga tcgtgctgtc tcagagcccc 420

gcaattactg ccgccagcct gggccaaaaa gtgaccataa catgcagcgc cagtagcaac 480

gtaagctaca tccactggta tcaacaaagg agcggaacca gccctaggcc ctggatctac 540

gagatcagca agctggcctc tggcgtgccc gtgaggttta gcggctcagg cagcggcact 600

tcttacagct tgacaatcag cagtatggag gccgaggacg ccgccatcta ttattgccag 660

cagtggaact accccctgat caccttcggc tctgggacca agttggagat c 711

<210> 5

<211> 729

<212> DNA

<213> Artificial Sequence

<220>

<223> GPC3 scFv

<400> 5

gacgtggtga tgacccagag cccactgtct ctgcccgtga cccccggaga gcccgccagc 60

atctcctgta ggagtagtca gagccttgta cacagcaacg ccaacaccta cctgcattgg 120

tatctgcaaa agcccggtca aagcccacag ctcctgatct acaaggtgag caacaggttt 180

tcaggcgtgc cggatcggtt cagtgggtct gggagcggca ccgacttcac cctgaagatc 240

agcagggtcg aggccgagga cgtgggcgtg tactattgct ctcagaacac ccacgtgccc 300

cccaccttcg gccagggcac caagctggag atcaagaggg ggggaggagg tagcgggggt 360

ggaggcagcg gcggtggcgg tagccaggtg caactggtcc agtccggcgc agaggtgaag 420

aaaccaggcg ccagcgtgaa ggtcagctgc aaggcaagcg gctacacctt taccgattac 480

gagatgcact gggtgaggca ggcaccggga cagggcctgg agtggatggg agcccttgat 540

cccaagaccg gggacacggc atacagccag aagttcaaag gtcgagtgac cctgaccgcc 600

gacgagagca ccagcacggc ctacatggag ctgagcagcc tgaggagcga ggacaccgca 660

gtgtattact gcaccaggtt ctatagctac acttactggg ggcagggtac tctggtgaca 720

gtgtctagc 729

<210> 6

<211> 732

<212> DNA

<213> Artificial Sequence

<220>

<223> EpCAM scFv

<400> 6

gacatcgtga tgacccagag cgccttcagc aaccccgtga ccctgggcac cagcgccagc 60

atcagctgca ggagcaccaa gagcctgctg cacagcaacg gcatcactta cctgtactgg 120

tacctgcaaa agccgggtca gagcccccag ctcctgattt accaaatgag caatttggcc 180

agcggtgtgc ccgacaggtt cagctctagc ggcagcggca ccgactttac cctgaggatc 240

agcagggtgg aggccgagga cgtgggcgtg tactactgcg ctcaaaatct ggagataccc 300

aggacctttg gcggtggcac gaagctggaa atcaagaggg ggggtggcgg ttcaggtggg 360

ggagggagcg gaggaggcgg gagccaggtg cagctccagc agagcggccc agaacttaag 420

aagccagggg aaaccgtgaa gattagctgc aaggcctctg gctacacctt caccaactac 480

ggcatgaact gggttaagca agcacccgga aggggcctga agtggatggg ctggatcaat 540

acctacaccg gcgagagcac ctacgccgac gacttcaaag gcaggttcgc tttctctctg 600

gaaacgtctg ccagcgcagc ctacttgcag atcaacaact tgaagaacga ggacaccgca 660

acctacttct gcgccaggtt tgctatcaag ggggactact ggggccaagg aaccactctc 720

acagtttcaa gc 732

Claims

1. a kind of immunocyte comprising tumour antigen identification receptor expresses hyaluronidase, which is characterized in that described transparent Matter acid enzyme are as follows:

A) full-length proteins being anchored on the immunocyte；Or

B) it secretes to the soluble type albumen outside the immunocyte.

2. immunocyte as described in claim 1, which is characterized in that the b) it is preferably a) to have lacked c-terminus membrane structure After obtain；Preferably, the hyaluronidase is mammal testicular hyaluronidase, it is more preferably people's hyaluronidase HYAL1, HYAL2, SPAM1 or PH20；Further more preferably:

Amino acid sequence a) is as shown in SEQ ID NO.1 in sequence table；Encode nucleotide sequence a) preferably such as In sequence table shown in SEQ ID NO.2 or SEQ ID NO.3；

Amino acid sequence b) is the 1st~447 of the amino acid sequence as shown in SEQ ID NO.1 in sequence table；It compiles Code nucleotide sequence b) is preferably the nucleotides sequence as shown in SEQ ID NO.2 in sequence table or SEQ ID NO.3 The 1st~1341 of column.

3. immunocyte as described in claim 1, which is characterized in that the tumour antigen identification receptor be chimeric antigen by Body (CAR) or T cell receptor (TCR), preferably:

The TCR includes one of δ chain of the α chain of TCR, the β chain of TCR, the γ chain of TCR and TCR or a variety of, the α chain Include variable region and constant region；Preferably, the variable region of the variable region behaviour TCR α chain, the constant region is the perseverance of mouse α chain Determine area；The β chain includes variable region and constant region, and the variable region of the variable region behaviour TCR β chain, the constant region is mouse β chain Constant region；More preferably, the TCR is E6TCR；

And/or the intracellular region of the CAR includes the 4-1BB intracellular region of people and/or the CD28 intracellular region of people and the CD3 of people ζ intracellular region, preferably the CD3 ζ intracellular region for the 4-1BB intracellular region of people, the CD28 intracellular region of people and people；

And/or the hinge area of the CAR is the CD8 α hinge area of people, the CD8 α transmembrane region that transmembrane region is people.

4. immunocyte as claimed in claim 3, which is characterized in that the CAR or the TCR identification antigen be ROR1, GPC3, MSLN or EpCAM；The tumour antigen cog region is can be in conjunction with the region of the above tumour antigen, preferably scFv；Preferably:

Identify the nucleotide sequence of the scFv of the ROR1 as shown in SEQ ID NO.4 in sequence table,

5. such as the described in any item immunocytes of Claims 1 to 4, which is characterized in that express the gene of the hyaluronidase Perhaps the TCR is located on the same expression vector or is located on different expression vectors with the CAR；Preferably:

The TCR or CAR is directed into cell by DNA mRNA form；

The expression vector is animal virus, preferably slow virus；

And/or one of the hyaluronidase, the CAR and described TCR or a variety of expression are opened by active The control of mover, the preferred EF1 α promoter of the promoter.

6. immunocyte as claimed in claim 5, which is characterized in that

In the case where described be located on the same expression vector, the expression vector includes following expression unit: signal peptide+ ScFv+ people CD8 α hinge area+people CD8 α transmembrane region+people 4-1BB intracellular region+people CD3 ζ intracellular region+link peptide+hyaluronidase, Wherein, the signal peptide is located at 5 ' ends, and the hyaluronidase is located at 3 ' ends；Alternatively, E6TCR+ connecting element+hyaluronic acid Enzyme, wherein the E6TCR is located at 5 ' ends, and the hyaluronidase is located at 3 ' ends；

In the case where described be located on different expression vectors, one of them described expression vector includes following expression unit: Signal peptide+scFv+ people CD8 α hinge area+people CD8 α transmembrane region+people 4-1BB intracellular region+people's CD3 ζ intracellular region, another described table It include following expression unit up to carrier: hyaluronidase+connecting element+label protein, wherein the hyaluronic acid is located at 5 ' End, the label protein are located at 3 ' ends；

The preferred T2A link peptide of the connecting element, P2A link peptide, E2A link peptide, F2A link peptide or IRES element；

The preferred tEGFR of the label protein.

7. immunocyte as described in any one of claims 1 to 6, which is characterized in that the immunocyte is T cell, NK is thin Born of the same parents or macrophage；Preferably, the T cell is the T cell after the stimulation activation of CD3 antibody, the NK cell is that NK-92 is thin Born of the same parents system；More preferably, the T cell derives from the peripheral blood mononuclear cells of tumor patient.

8. a kind of application of immunocyte as described in any one of claims 1 to 7 in preparation tumor.

9. application as claimed in claim 8, which is characterized in that the tumour is entity tumor；Preferred expression hyaluronic acid Entity tumor, such as melanoma, cancer of pancreas, liver cancer, glioblastoma, breast cancer or lung cancer.

10. a kind of pharmaceutical composition, which is characterized in that it includes immunocytes as described in any one of claims 1 to 7.