CN109468279A - Target immune effector cell and its application of GPC3 - Google Patents

Target immune effector cell and its application of GPC3 Download PDF

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CN109468279A
CN109468279A CN201811046090.2A CN201811046090A CN109468279A CN 109468279 A CN109468279 A CN 109468279A CN 201811046090 A CN201811046090 A CN 201811046090A CN 109468279 A CN109468279 A CN 109468279A
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immune response
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李宗海
蒋华
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Clegg Medical Co ltd
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Keji Biomedical (shanghai) Co Ltd
Shanghai Cancer Institute
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Abstract

There are the Chimeric antigen receptor of targeting GPC3 and the immune response cell of exogenous IL12 the present invention relates to expression.The present invention also provides the pharmaceutical composition comprising the immune response cell and utilize the immune response cell or medicine composite for curing tumour, the especially method of GPC3 positive tumor.Immune response cell of the invention is not only effective to solid tumor cell in vitro, also has excellent fragmentation effect to solid tumor cell in vivo.

Description

Target immune effector cell and its application of GPC3
Technical field
The invention belongs to cellular immunotherapy fields, and in particular to a kind of immunological effect of the targeting GPC3 of gene modification Cell.
Background technique
T lymphocyte (the Chimeric antigen receptor engineered T of Chimeric antigen receptor modification Lymphocyte, CAR-T) technology is one of the rising in recent years new strategy for being used for tumor biotherapy.Chimeric antigen receptor (Chimeric antigen receptor, CAR) is the work that will identify the single-chain antibody and immunocyte of tumor associated antigen Change motif to combine, so that T lymphocyte is expressed Chimeric antigen receptor by the means of genetic engineering in vitro, impart T cell The ability of specific recognition and killing tumor cell.Currently, CAR-T is obtained in the treatment use of Malignancy Good curative effect, such as in the bone-marrow-derived lymphocyte leukaemic of the CD19 positive achieve huge success.But CAR-T Very big challenge is still suffered from treatment of solid tumors application aspect.
Interleukin 12 (interleukin-12, IL12) is a kind of important immune stimulating cytokines, can be promoted auxiliary The proliferation of helping property T cell, induced NK cell and T cell generate interferon.However in clinical trial, IL12 system is found Property administration when in multiple organs there is serious side effects, such as dysfunction of liver, high fever, serious haemodynamics shakiness etc., sternly Severe one results in death, limits the application of IL12.
Further, since the particularity of tumour especially solid tumor, the microenvironment of every kind of tumour is also entirely different, coexpression Whether the immune effector cell of IL12 enhances antitumor effect, and it is unknown for especially significantly increasing antitumor effect, and And it will cause certain side effect, such as experiment mice weight loss.In addition, CAR T cell and other cell factors are combined The defects of there are side effect is big or vivo efficacy is bad equal.Therefore, specific Chimeric antigen receptor immune response cell and tool It is rationally to predict which type of effect the cell factor combination of body, which can generate,.
Therefore, this field is badly in need of capable of having significant antitumous effect, but the immune response cell that side effect is low.
Summary of the invention
Have a significant antitumous effect the purpose of the present invention is to provide one kind, but side effect it is low immune response it is thin Born of the same parents.
In a first aspect, the present invention provides a kind of immune response cell, the cell express specific bond GPC3 receptor and Exogenous IL12.
In a particular embodiment, the receptor and the IL12 are operably connected.
In a particular embodiment, the immune response cell includes: T cell, natural killer cells, cell toxicant Property T lymphocyte, natural killer T cells, DNT cell, and/or regulatory T cells.
In a particular embodiment, the exogenous IL12 is constructive expression or inducible expression；Preferably, Promoter for expressing the IL12 includes: immunocyte inducible promoter；Preferably, the immunocyte lures Conductivity type promoter is NFAT6 promoter.
In a particular embodiment, the receptor has extracellular domain, transmembrane domain and the letter intracellular of specific bond GPC3 Number domain.
In a particular embodiment, the receptor is Chimeric antigen receptor, wherein the Intracellular domain contains cell thorn The combination of energizing signal molecule or cytositimulation signaling molecule and cell-stimulating costimulatory molecules；
Preferably, the cytositimulation signaling molecule is selected from: CD3 ζ, CD3 γ, CD3 δ, CD3 ε, Fc ε RI γ, FcR β, The functional signal conducting structure domain of the protein of CD79a, CD79b, Fc γ RIIa, DAP10 or DAP12；It is more preferably CD3 ζ；Or
Preferably, the functional signal conducting structure of the cell-stimulating costimulatory molecules protein selected from the following Domain: CD27, CD28, CD137, CD134, ICOS, OX40, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of specific bond CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, 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, 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, NKp44, NKp30, NKp46 and NKG2D, more preferably, be CD27, CD28, CD137, CD134, ICOS。
In a particular embodiment, the extracellular domain of the receptor contains and amino acid sequence shown in SEQ ID NO:25 Arrange the amino acid sequence at least 90%, 95%, 96%, 97%, 98% or 99% identity.
In a particular embodiment, the receptor contains amino acid sequence shown in SEQ ID NO:21,22,23 or 24 Column；Or containing with amino acid sequence shown in SEQ ID NO:21,22,23 or 24 have at least 90%, 95%, 96%, 97%, the amino acid sequence of 98% or 99% identity.
In a particular embodiment, the receptor and the exogenous IL12 by with SEQ ID NO:27,28 or 29 is nucleotide sequence coded at least 90%, 95%, 96%, 97%, 98% or 99% identity.
In a particular embodiment, the immune response cell does not include the costimulation ligand of external source.
In a particular embodiment, the receptor and/or the IL12 composition or inducible expression are in immune response The surface of cell.
It in a particular embodiment, include expression construct, the expression construct packet in the immune response cell It includes: the expression cassette of the receptor of the combination antigen；With the expression cassette of the IL12.
In a particular embodiment, the receptor and/or IL12 are expressed using viral vectors；Preferably, described Viral vectors includes: slow virus carrier, retroviral vector or adenovirus vector.
In a particular embodiment, the immune response cell is described exogenous with being not present after giving individual The case where IL12, is compared, and CAR-T cell quantity improves at least 50% in a peripheral body.
In a particular embodiment, the immune response cell is after giving individual about 7 days, a peripheral body The sum of quantity of middle CAR-T cell is greater than 6,000/μ L；After giving the immune response cell about 10 days, described external The sum of quantity of CAR-T cell is greater than 6000/μ L in all blood.
In a preferred embodiment, immune response cell of the invention is lower than 1*10 for low dosage, such as dosage7 Cell/L non-present invention immune response cell (that is, do not express the receptor of specific bond GPC3 and exempting from for exogenous IL12 simultaneously Epidemic disease effector cell) uncontrollable tumour or the excessive tumour of volume (such as volume is greater than in mouse tumor model 300mm3Tumour, even greater than 600mm3) have excellent lethal effect.
In second aspect, the present invention provides a kind of expression construct, which includes being linked in sequence: receptor Expression cassette and IL12 expression cassette；Wherein the receptor of the combination antigen and IL12 be as described in the first aspect of the invention.
In the third aspect, the present invention provides immune response cell described in first aspect present invention in preparation for treating Purposes in the pharmaceutical composition of the tumour of individuals in need.
In a particular embodiment, the tumour includes: liver cancer, gastric cancer, lung cancer, breast cancer, head and neck cancer, bladder It is cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, embryonal-cell lipoma, melanoma, adrenal, neurinoma, pernicious Fibrous histiocytoma, cancer of the esophagus；Preferably, the tumour is liver cancer, gastric cancer, lung cancer, breast cancer.
In a particular embodiment, the immune effector cell makes tumour be reduced at least 50%.
In a particular embodiment, the immune effector cell makes tumour be reduced at least 70%, more preferably, institute The immune effector cell stated makes tumour be reduced at least 80%.
In fourth aspect, the present invention provides a kind of pharmaceutical composition, and the pharmaceutical composition includes:
Immune response cell described in first aspect present invention；And
Pharmaceutically acceptable carrier or excipient.
At the 5th aspect, the present invention provides a kind of kit, it includes:
Immune response cell described in first aspect present invention；And
How guidance gives the specification of the immune response cell to individual.
It should be understood that above-mentioned each technical characteristic of the invention and having in below (eg embodiment) within the scope of the present invention It can be combined with each other between each technical characteristic of body description, to form a new or preferred technical solution.As space is limited, Not repeated them here.
Detailed description of the invention
Figure 1A is the plasmid construct schematic diagram of CAR-T cell preparation；Figure 1B shows that FACS detects GPC3-CAR-T/ The expression of IL12-GPC3-CAR-T cell surface CAR；
Fig. 2 shows CAR cell-T in vitro toxicity experimental result；
Fig. 3 A shows the secretion situation of IL-12 when CAR-T and tumour cell incubate total incubation for 24 hours altogether；Fig. 3 B is shown IL-2/TNF- α/IFN-γ secretion situation when CAR-T and tumour cell incubate total incubation for 24 hours altogether；
Fig. 4 A shows the growing state of the Huh-7 subcutaneous transplantation tumor of different experiments group；Fig. 4 B shows different experiments group Huh-7 subcutaneous transplantation tumor mouse changes of weight；
Fig. 5 shows the 7th day and the 10th day, different CAR-T survivaling cell number in peripheral blood；
Fig. 6 shows the amount of IFN-γ in the 10th day serum sample after CAR-T injection；
Fig. 7 is the result that immunohistochemistry detection is carried out to infiltrative type T cell in tumor tissues and Ki67；
Fig. 8 A and 8B show experiment at the end of mouse subcutaneous tumors growing state；
Fig. 9 is the expression of FACS detection mCAR-T/mIL12-GPC3-CAR T cell surface C AR；
Figure 10 is mCAR-T in vitro toxicity experimental result picture；
Figure 11 A shows that UTD, mGPC3-CAR, mGPC3-m28Z-mNFAT6-mIL12 and tumour cell are incubated for for 24 hours altogether When mIL-12 secretion situation；Figure 11 B shows that UTD/mGPC3-m28Z/mIL12-GPC3-CAR T is incubated altogether with tumour cell Cell factor mIL-2, mTNF- α, mIFN- γ release conditions when educating for 24 hours；
Figure 12 A and Figure 12 B show the therapeutic effect of E0771-GPC3 subcutaneous transplantation tumor；
Figure 13 A shows that tail vein injection mIL12-GPC3-T 5X106/ is only organized and mIL12-GPC3-T 2X106/ group Experimental result；Figure 13 B is shown give mIL12-GPC3-T after, the body with mGPC3-CAR-T group and blank control group mouse Weight.
Specific embodiment
Inventor after extensive and in-depth study, it was unexpectedly found that immune response cell express specific bond The receptor of GPC3 and exogenous IL12 can have significant antitumous effect, but side effect is low.Complete this on this basis Invention.
Detailed description below at large illustrates embodiments disclosed herein.It should be appreciated that this specification is not anticipated Specific embodiments disclosed herein to be only limitted to, but can change.It will be understood by those skilled in the art that this Content disclosed in the description can be covered by within disclosed scope and principle there are many being altered or varied.It removes Non- to be otherwise noted, each embodiment can be with any other embodiment any combination.
Certain embodiments disclosed herein contain numberical range, and model can be used in certain aspects of the invention The mode enclosed describes.Unless otherwise indicated, it should be understood that numberical range or in a manner of range description be only for it is succinct, Convenient purpose should not be considered the considered critical to the scope of the present invention.Therefore, it is answered using the description of range mode When being considered specifically disclosing all possible subrange and all possible specific value point in the range, just As these subranges and numerical point have clearly write out herein.For example, the description of from 1 to 6 range should be considered having Body discloses from 1 to 3,1 to 4,1 to 5,2 to 4,2 to 6,3 to 6 etc. subrange, and the specific number within the scope of these It is worth point, such as 1,2,3,4,5,6.No matter the width of the numerical value, mentioned above principle are equally applicable.When being described using range, The range includes the endpoint of range.
Term
The term as used herein " activation " and " activation " are used interchangeably, they and its phraseological other forms can It is changed into the process of activated state to refer to cell from stationary state.The process may include living to antigen, migration and/or function The phenotype of character state or the response of heredity variation.For example, term " activation " can refer to the process that T cell gradually activates.For example, T Cell may need at least two signals that could activate completely.First signal can by antigen-MHC complex engagement TCR it After occur, and the engagement that second signal can pass through costimulatory molecules (referring to costimulatory molecules cited in table 1) occurs. In vitro, AntiCD3 McAb can simulate first signal, and anti-CD28 can simulate the second signal.For example, engineering T cell The CAR activation that can be expressed.T cell activation used herein or T cell triggering, which can refer to, sufficiently to be stimulated to lure Lead detectable cell Proliferation, cell factor generates and/or the state of the T cell of detectable effector function.
The term as used herein " receptor " is that one kind is present in after birth or intracellular, can be with extracellular single-minded signaling molecule In conjunction with series of biochemical reactions in active cell in turn, cell is made to generate the special egg of corresponding effect to environmental stimuli White matter.Bioactive substance in conjunction with receptor is referred to as ligand (ligand).Molecular conformation occurs for binding of receptor and ligand Variation, so as to cause signal transduction, iuntercellular bonding, endocytosis process between cell effect, such as mediated cell.
The term as used herein " costimulation ligand " includes the identity costimulatory molecules specifically bound in T cell Molecule on antigen presenting cell (for example, aAPC, Dendritic Cells, B cell etc.), thus provides signal, and by such as TCR/ CD3 compound and be loaded with peptide MHC molecule the common mediate T cell response of the first signal that provides of combination, including it is but unlimited In proliferation, activation, differentiation etc..Costimulation ligand can include but is not limited to CD7, B7-1 (CD80), B7-2 (CD86), PD-L, PD-L2,4-1BBL, OX40L, induction type costimulation ligand (ICOS-L), Intercellular Adhesion Molecule (ICAM), CD30L, CD40, CD70, CD83, HLA-G, MICA, MICB, HVEM, lymphotoxin-beta-receptor, 3/TR6, ILT3, ILT4, HVEM, in conjunction with Toll The agonist or antibody of ligand receptor and the ligand specifically bound with B7-H3.Costimulation ligand is also particularly including thin with T Present on born of the same parents costimulatory molecules specifically bind antibody, such as, but not limited to CD27, CD28,4-1BB, OX40, CD30, CD40, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3 and with The ligand of CD83 specific binding.
The term as used herein " costimulatory molecules " refers to same in the T cell in conjunction with costimulation ligand specificity Property binding partners, so that the costimulation response of mediate T cell, is such as, but not limited to proliferated.Costimulatory molecules include but unlimited In MHC I class molecule, BTLA and Toll ligand receptor.
" costimulatory signal " refers to as used herein combines with cytositimulation signaling molecule, such as TCR/CD3, combination Lead to the signal of up-regulation or the downward of T cell proliferation and/or key molecule.
The term as used herein " Chimeric antigen receptor " or " CAR " refer to can be by including but is not limited to the immune of T cell The engineered molecule of cell expression.CAR is expressed in T cell and can be redirected T cell and determined with inducing by artificial receptor Fixed specificity kills target cell.The extracellular binding domains of CAR can be derived from mouse, humanization or complete human monoclonal Antibody.
The term as used herein " engineering " and its phraseological other forms can refer to that the one or more of nucleic acid changes Become, such as the nucleic acid in organism genome.Term " engineering " can refer to the change, addition and/or missing of gene.Engineering Cell can also refer to the cell with the gene for being added, lacking and/or changing.
The term as used herein " cell " or " engineering cell " and its phraseological other forms can refer to people or inhuman dynamic The cell in object source.Engineering cell can also refer to the cell of expression CAR.
The term as used herein " transfection ", which refers to, introduces eukaryocyte for exogenous nucleic acid.Transfection can be by this field The various means known realize, what the transfection that mediates including calcium phosphate-DNA coprecipitation, DEAE- glucan, polybrene mediated turns Dye, electroporation, microinjection, liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistic Technology (biolistics).
Term " stable transfection " or " steadily transfecting ", which refer to, introduces and is integrated into transfection for exogenous nucleic acid, DNA or RNA In the genome of cell.Term " stable transfection body " (stable transfectant), which refers to, steadily integrates foreign DNA Cell into genomic DNA.
As used herein, term " nucleic acid molecule encoding ", " DNA sequences encoding " and " coding DNA " refers to along deoxidation core The sequence or sequence of the deoxyribonucleotide of ribosomal ribonucleic acid chain.The sequence of these deoxyribonucleotides is determined along polypeptide The sequence of the amino acid of (protein) chain.Therefore, nucleic acid sequence encoding amino acid sequence.
The term as used herein " individual " refers to any animal, such as mammal or marsupial.Individual of the invention Including but not limited to the mankind, non-human primate (such as rhesus macaque or other kinds of macaque), mouse, pig, horse, donkey, Ox, sheep, rat and any kind of poultry.
The term as used herein " peripheral blood lymphocytes " (PBL) and its phraseological other forms can refer in blood The lymphocyte recycled in (such as peripheral blood).Peripheral blood lymphocytes can refer to the lymphocyte for being not limited to organ.Periphery Blood lymphocyte may include T cell, NK cell, B cell or any combination thereof.
The term as used herein " immune response cell " or " immunoreactive cell ", which can refer to, can trigger immune response Cell, including but not limited to T cell, B cell, NK cell, NKT cell, DNT cell etc., their own precursor and Its offspring.Immune response cell can also refer to the cell of lymph or myeloid lineage.
The term as used herein " T cell " and its phraseological other forms can refer to the T cell in any source.For example, T cell can be primary T cells such as Autologous T cells etc..T cell is also possible to people or inhuman.
The term as used herein " T cell activation " or " t cell activation " and its phraseological other forms, which can refer to, is filled The shape that intermuscular needling swashs to induce the T cell of detectable cell Proliferation, cell factor generation and/or detectable effector function State.In some cases, " complete T cell activation " can be similar to the cytotoxicity of triggering T cell.This field can be used Known various measurements are to measure T cell activation.It is described measurement can be measurement cytokine secretion ELISA, ELISPOT, For measuring the Flow Cytometry Assay (CD107) of intracellular cytokine expression, the flow cytometry for measuring proliferation is surveyed Cytotoxicity assay (51Cr release measurement) that is fixed and being eliminated for determining target cell.The measurement is (non-usually using compareing Engineering cell) it is compared with engineering cell (CAR T), to determine compared with the control, the relative activation of engineering cell.In addition, The engineering cell that the measurement can be incubated for or contact with the target cell for not expressing target antigen is compared.For example, the comparison It can be compared with the GPC3-CART cell that the target cell for not expressing GPC3 is incubated for carries out.
When for when referring to nucleotide sequence, the term as used herein " sequence " and its phraseological other forms to be can wrap DNA or RNA is included, and can be single-stranded or double-stranded.Nucleic acid sequence can be mutated.Nucleic acid sequence can have any length.
The term as used herein " effective quantity " refers to the amount for providing and treating or preventing benefit.
The term as used herein " expression vector " refers to the carrier comprising recombination of polynucleotide, it includes with it is to be expressed The expression regulation sequence that nucleotide sequence effectively connects.Expression vector includes enough cis-acting elements for expression (cis-acting elements)；Other elements for expression can be provided by host cell or vitro expression systems.Table Up to carrier include that this field is all those of known, such as clay, plasmid (such as exposed or include in liposome) and disease Malicious (for example, slow virus, retrovirus, adenovirus and adeno-associated virus).
The term as used herein " slow virus " refers to the category of Retroviridae.Retrovirus can infect presumptuously It is unique in retrovirus in terms of schistocyte；A large amount of hereditary information can be delivered to the DNA of host cell by they In, therefore they are one of gene delivery vector most efficient methods.HIV, SIV and FIV are the examples of slow virus.It is originated from The carrier of slow virus provides the means for realizing the gene transfer of the level of signifiance in vivo.
The term as used herein " being operably connected " refers to the functionality between regulating and controlling sequence and other nucleic acid sequences Connection, as the connection leads to the expression of the latter.For example, when the first nucleic acid sequence and second nucleotide sequence are at functional relationship, the One nucleic acid sequence is operably connected with second nucleotide sequence.
Terms used herein " promoter " are defined as the cell as needed for the specific transcriptional of starting polynucleotide sequence Synthesis mechanism or introducing synthesis mechanism identification DNA sequence dna.
Terms used herein " carrier " are comprising isolated nucleic acid and the delivery of nucleic acids that can be used for separate is to intracellular The composition in portion.It is known in the art many carriers, including but not limited to linear polynucleotides and ion or amphiphilic chemical combination The relevant polynucleotides of object, plasmid and virus.Therefore, term " carrier " includes the plasmid independently replicated or virus.The term is also It should be interpreted as including and nucleic acid is promoted to be transferred to non-plasmid and non-viral compound in cell, such as polylysin compounds, Liposome etc..The example of viral vectors includes but is not limited to adenovirus vector, gland relevant viral vector, retroviral vector Deng.
Terms used herein sequence " identity " is by comparing two on comparison window (for example, at least 20 positions) Sequence through best match determines homogeneity percentage, and wherein the part of polynucleotides or polypeptide sequence can in comparison window With comprising adding or lacking in (i.e. gap), such as two sequences of best match, (it, which does not include, adds with reference sequences Add or lack) compared to 20% or less gap (such as 5 to 15% or 10 to 12%).Usually by determining in two sequences The number of the middle position that identical nucleic acid base or amino acid residue occurs calculates percentage, to generate correct matched position The number set by the number of correct matching position divided by the total number of positions (i.e. window size) in reference sequences, and result is multiplied With 100, to generate the percentage of sequence identity.
The term as used herein " IL12 ", " interleukin 12 " and " interleukin-12 " may be used interchangeably, and have phase With the meaning.The term as used herein " IL12 " is a kind of with the active immune-regulating factor of various biological, e.g., term " IL12 " can refer to the people IL12 as defined in SEQ ID NO:26 or its active fragment, may also mean that such as SEQ ID The IL12 of mouse defined in NO:27 or its active fragment, are also possible to other kinds.
In some embodiments, the element or described of the receptor of the specific bond GPC3 described applied to building IL12 can be naturally occurring, for example it can be by isolated or purified from mammal；It is also possible to manually to prepare, such as Each element of recombination or IL12 can be produced according to conventional genetic engineering recombinant technique.Preferably, recombination can be used in the present invention Each element or IL12.
On the basis of each element or IL12 polypeptide sequence, by the substitution of one or more amino acid residues, lack The amino acid sequence for losing or adding and being formed is also included in the present invention.Appropriate replacement amino acid is technology well known in the art, The technology can easily be carried out and ensure not change the bioactivity of gained molecule.These technologies make this field Personnel recognize that in general, biological work will not substantially be changed by changing single amino acids in a kind of unwanted regions of polypeptide Property.
The bioactive fragment of the polypeptide of each element or IL12 can be applied in the present invention.Herein, described The meaning of bioactive fragment refer to as a kind of polypeptide, as a part of full-length polypeptide, be still able to maintain overall length All or part of function of polypeptide.Under normal conditions, the bioactive fragment at least keeps the work of 50% full-length polypeptide Property.Under still more preferential conditions, the active fragment be able to maintain full-length polypeptide 60%, 70%, 80%, 90%, 95%, 99% or 100% activity.
On the basis of each element or IL12 polypeptide sequence, the polypeptide modified or improved also can be applied to this In invention, is modified or changed to promote the effect of its half-life period, validity, metabolism, and/or polypeptide for example, can be used Good polypeptide.That is, the version of any bioactivity for not influencing polypeptide can be used in the present invention.
The term as used herein " tumour " refers to a kind of disease characterized by the pathological proliferation of cell or tissue, And its subsequent migration or invade its hetero-organization or organ.Tumour growth is usually uncontrolled and progressive, is not induced Or inhibit normal cell proliferation.Tumour can influence various kinds of cell, tissue or organ, including but not limited to selected from bladder, mammary gland, Oesophagus, intestines, kidney, liver, lung, lymph node, nerve fiber, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen, stomach, son Palace organ, or tissue or corresponding cell.Tumour of the present invention, it may include, but it is not limited to liver cancer, gastric cancer, lung cancer, cream Gland cancer, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, embryonal-cell lipoma, melanoma, on kidney Gland cancer, neurinoma, malignant fibrous histiocytoma, cancer of the esophagus.Preferably, " tumour " includes but is not limited to: liver Cancer, gastric cancer, lung cancer, breast cancer.
The term as used herein " enhancing immune response cell function " includes for example enhancing T cell function.It is with T cell Example, enhancing T cell function include induction, cause or T cell is stimulated to make it have lasting or enhancing biological function, or more (exhausted) or inactive T cell new or that reactivate exhaustion.Enhancing T cell function example include: relative to It is horizontal before intervening, the interferon of CD8+T cell secretion increases, proliferation increases, antigen reactivity (such as virus or pathogen it is clear Except rate) increase.In one embodiment, enhancing is horizontal is at least 50% or 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%.It is known to persons of ordinary skill in the art for measuring the mode of this enhancing.
The term as used herein " exogenous " refers to a nucleic acid molecules or polypeptide, in the cell without endogenous Expression or expression are not enough to realize the function having when overexpression.Thus, " exogenous " includes in cell inner expression Recombinant nucleic acid molecules or polypeptide, such as nucleic acid molecules and polypeptide of exogenous, heterologous and overexpression.
The term as used herein " receptor " refers to a kind of polypeptide, or part thereof, one is selectively combined on cell membrane A or multiple ligands.
In some embodiments, the receptor of combination antigen of the invention is Chimeric antigen receptor.The term as used herein " Chimeric antigen receptor (Chimeric Antigen Receptor, CAR) " refers to a kind of intracellular signal transduction domain that is fused to Tumour antigen binding structural domain can activate T cell.Typically, the extracellular binding structural domain of CAR derives from mouse or humanization Or the monoclonal antibody of people.
Chimeric antigen receptor generally comprises (thin) extracellular antigen binding domain.In some embodiments, extracellular antigen binding Area can be complete people.In other cases, extracellular antigen binding regions can be humanized.In other cases, extracellular Antigen binding domain can be the chimera in source of mouse the or described extracellular antigen binding domain by different dynamic from least two The amino acid sequence of object forms.In some embodiments, the extracellular antigen binding domain can be inhuman.
A variety of antigen binding regions can be designed.Non-limiting example includes the single chain variable fragment derived from antibody (scFv), the fragment antigen combined area (Fab) selected from library, single domain fragment or with engage matching naturally for its homoreceptor Body.In some embodiments, extracellular antigen binding regions may include scFv, Fab or native ligand and their times What derivative.Extracellular antigen combined area can refer to the molecule in addition to complete antibody, may include one of complete antibody Point and can be with the antigen binding in conjunction with complete antibody.The example of antibody fragment can include but is not limited to Fv, Fab, Fab',Fab'-SH,F(ab')2；Bifunctional antibody, linear antibodies；Single-chain antibody molecules (such as scFv)；With by antibody fragment The multi-specificity antibody of formation.
Extracellular antigen combined area, such as scFv, Fab or native ligand, can be the CAR's of determining antigentic specificity A part.Extracellular antigen combined area can combine any complementary target.Extracellular antigen combined area can be derived from known variable The antibody of region sequence.Extracellular antigen combined area can be obtained from the antibody sequence for being obtained from obtainable Mouse Hybridoma Cells.Or Person can obtain extracellular from the complete outer cutting sequencing of tumour cell or primary cell such as tumor infiltrating lymphocyte (TIL) Antigen binding domain.
In some cases, the binding specificity of extracellular antigen combined area can be by complementary determining region or CDR, such as Light chain CDR or heavy chain CDR is determined.In many cases, binding specificity can be by light chain CDR and heavy chain CDR come really It is fixed.Compared with other are with reference to antigen, the combination of given heavy chain CDR and light chain CDR can provide given binding pocket, can To assign antigen (such as GPC3) bigger affinity and/or specificity.For example, being specific to Monophosphoinositideproteoglycans proteoglycans-3 CDR can be expressed in the extracellular bond area of CAR, allow targeting GPC3 CAR immune response cell is targeted Express the tumour cell of GPC3.
In any embodiment disclosed herein in some terms, extracellular antigen combined area, such as scFv may include To the light chain CDR of antigentic specificity.Light chain CDR can be the complementary of the scFv light chain of antigen-binding unit such as CAR and determine Area.Light chain CDR may include continuous amino acid residue sequence, or determine two that area's (such as framework region) separates by incomplementarity A or more continuous amino acid residue sequence.In some cases, light chain CDR may include two or more light chains CDR can be referred to as light chain CDR-1, CDR-2 etc..In some cases, light chain CDR may include three light chain CDR, Light chain CDR-1, light chain CDR-2 and light chain CDR-3 can be referred to as.In some instances, one be present on common light chain Group CDR can be collectively referred to as light chain CDR.
In any embodiment disclosed herein in some terms, extracellular antigen combined area, such as scFv may include To the heavy chain CDR of antigen-specific.Heavy chain CDR can be the complementary determining region of heavy chain of antigen-binding unit such as scFv.Heavy chain CDR may include the continuous sequence of amino acid residue, or determine two or more that area's (such as framework region) separates by incomplementarity The continuous sequence of a amino acid residue.In some cases, heavy chain CDR may include two or more heavy chains CDR, can With referred to as heavy chain CDR-1, CDR-2 etc..In some cases, heavy chain CDR may include three heavy chain CDR, can be referred to as Heavy chain CDR-1, heavy chain CDR-2 and heavy chain CDR-3.In some cases, one group of CDR being present on common heavy chain can be referred to as For heavy chain CDR.
It, can modified cells exoantigen combined area in various ways by using genetic engineering.In some cases, may be used With mutant cell exoantigen bond area, so as to select extracellular antigen bond area higher to have to its target Affinity.In some cases, extracellular antigen bond area to the affinity of its target can for can in normal tissue with The target of low expression level optimizes.This optimization can be carried out, to reduce potential toxicity to the greatest extent.In other cases, right The film combining form of target has the clone of the extracellular antigen bond area of more high-affinity can be better than its soluble form Counterpart.This modification can be carried out, because may also detect that the target of the soluble form of different level, and they Targeting can cause undesirable toxicity.
In some cases, extracellular antigen bond area includes hinge or spacer region.Term hinge and spacer region can be with It is used interchangeably.Hinge is considered for providing a part of CAR flexible to extracellular antigen combined area.Some In the case of, hinge can be used for detecting the CAR on the cell surface of cell, especially resisting when detection extracellular antigen combined area When body does not work or can be used.For example, the length of the hinge derived from immunoglobulin may need to optimize, this depends on thin The position of epitope on extracellular antigen binding regions targeting target.
In some cases, hinge may be not belonging to immunoglobulin, but belong to another molecule, such as CD8 alpha molecule Natural hinge.CD8 α hinge can contain known half to work in the interaction of CD8 accessory receptor and MHC molecule Cystine and proline residue.The cysteine and proline residue can influence the performance of the CAR.
CAR hinge can be size adjustable.This pattern of immunological synapse between immune response cell and target cell It also defines due to the film distal end epitope on cell surface target molecules and cannot be used by the distance of CAR progress function bridge joint Short hinge CAR can not enable cynapse distance reach the approximation that signal conducts.Equally, film proximal end CAR target epitope is only Observe that signal exports under the background of long hinge CAR.Hinge can be adjusted according to used extracellular antigen bond area Chain.Hinge can be any length.
CAR can be anchored on the plasma membrane of cell by transmembrane domain.The native transmembrane part of CD28 can be used for CAR.? In the case of other, the native transmembrane part of CD8 α can also be used in CAR." CD8 " can be and NCBI reference number: NP_ 001759 or its segment with stimulating activity have at least 85,90,95,96,97,98,99 or 100% identity albumen Matter.CD8 nucleic acid molecules " can be the polynucleotides of coding CD8 polypeptide, and in some cases, transmembrane region can be the day of CD28 Right transmembrane segment, " CD28 " can refer to and NCBI reference number: NP_006130 or its segment with stimulating activity have at least 85, the protein of 90,95,96,97,98,99 or 100% identity." CD28 nucleic acid molecules " can be coding CD28 polypeptide Polynucleotides.In some cases, transmembrane segment may include the region CD8 α.
The intracellular signal domain of CAR can be responsible for activating the effector function that CAR has been placed on immune response cell therein At least one of.CAR can be with the effector function of inducing T cell, for example, the effector function is cell lysis activity Or auxiliary activity, the secretion including cell factor.Therefore, term " Intracellular signals domain " refers to transduction effector function signal And guide the protein portion of cell progress exceptional function.Although entire Cellular Signaling Transduction Mediated region usually can be used, It is in many cases, it is not necessary to use the entire chain of signal domain.In some cases, using Cellular Signaling Transduction Mediated area Truncation part.In some cases, term Intracellular signals domain is it is therefore intended that include being enough effector function signal of transduceing Any truncation part in Cellular Signaling Transduction Mediated area.
The preferred embodiment of the signal domain used in CAR may include the cytoplasmic sequences of T cell receptor (TCR) With synergistic effect with the coreceptor and their any derivative of enabling signal transduction or change after target-receptor combines Any composition sequence with same functionality of body sequence and these sequences.
In some cases, the Intracellular signals domain can contain known immunity receptor Tyrosine Activating Motifs (ITAM) signal motif.The example of ITAM containing cytoplasm signal transduction sequence includes being derived from TCR ζ, FcR γ, FcR The functionality of the protein of β, CD3 γ, CD3 δ, CD3 ε, the DAP10 of CD5, CD22, CD79a, CD79b, CD66d or DAP12 Signal transduction structural domain.However, in preferred embodiments, intracellular signaling domain is derived from CD3 ζ chain.
The example of T cell signal domain containing one or more ITAM motifs is CD3 ζ structural domain, and also referred to as T is thin Born of the same parents' receptor T3 ζ chain or CD247.The structural domain is a part of T cell receptor-CD3 compound, and will be several intracellular The antigen recognizing of signal transduction pathway combines aspect with the activation of the main effect of T cell and plays an important role.As used herein, CD3 ζ is primarily referred to as mankind CD3 ζ and its isotype, as known to the Swissprot entry P20963, including with essentially identical sequence The protein of column.It as a part of Chimeric antigen receptor, reiterates, does not need full T cell receptor T3 ζ chain, and it is wrapped Any derivative of the signal domain of the ζ chain of T3 containing T cell receptor is all suitable, including its any functional equivalent.
Cellular Signaling Transduction Mediated structural domain can be selected from any one structural domain of table 1.In some cases, Ke Yixiu Structural domain is adornd, so that the identity with reference configuration domain can be about 50% to about 100%.Any one of table 1 can be modified Structural domain so that modified forms may include about 50,60,70,80,90,95,96,97,98,99 or at most about 100% it is same One property.
The Cellular Signaling Transduction Mediated area of CAR can further include one or more costimulation structural domains.Intracellular signals Conducting region may include single costimulation structural domain, such as ζ chain (first generation CAR) or itself and CD28 or the 4-1BB (second generation CAR).In other instances, Cellular Signaling Transduction Mediated area may include two costimulation structural domains, for example, CD28/OX40 or CD28/4-1BB (third generation).
Together with intracellular signaling domain such as CD8, the downstream that these costimulation structural domains can produce kinase pathways is swashed It is living, so that genetic transcription and functional cell be supported to react.The costimulation structural domain of CAR can activate and CD28 (phosphatidyl-4 Alcohol -4,5- diphosphonic acid 3- kinases) or 4-1BB/OX40 (TNF- receptor associated factor adaptin) approach and MAPK and Akt Activate relevant near end signal albumen.
In some cases, the signal generated by CAR may be combined with auxiliary or costimulatory signal.For total thorn Energizing signal structural domain, Chimeric antigen receptor sample compound are designed to comprising several possible costimulatory signal structural domains.Such as Well-known in the art, in Naive T cells, the independent engagement of T cell receptor is not enough to the complete activation of inducing T cell For cytotoxic T cell.Complete productivity t cell activation needs the second costimulatory signal.It has been reported that T cell activation Several receptors of costimulation, including but not limited to CD28, OX40, CD27, CD2, CD5, ICAM-1, LFA-1 (CD11a/ are provided CD18), 4-1BBL, MyD88 and 4-1BB.The signal transduction path that these costimulatory molecules use can be with main T cell receptor Activation signal synergistic effect.These costimulatory signal conductive areas provide signal can be originated from one or more ITAM base The main effect activation signal of sequence (such as CD3zeta signal transduction domain) acts synergistically, and can complete wanting for t cell activation It asks.
In some cases, engineering cell can be enhanced to Chimeric antigen receptor sample compound addition costimulation structural domain The effect of and durability.In another embodiment, T cell signal domain and costimulation structural domain be fused to each other to Constitute signal transduction area.
1. costimulation structural domain of table
The term as used herein " adjusting ", which refers to, positively or negatively to be changed.Adjust example include 1%, 2%, 10%, 25%, 50%, 75% or 100% variation.
The term as used herein " treatment " refers in attempting lysis caused by changing personal or processing cell Clinical intervention can both have been prevented or in clinical pathology process intervention.Therapeutic effect includes but is not limited to prevent disease The generation or recurrence of disease mitigate symptom, reduce the direct or indirect pathological consequences of any disease, prevent from shifting, slow down disease Tempo, improvement alleviate the state of an illness, alleviation or improve prognosis etc..
The term as used herein " immunologic hypofunction " refers to that subject has immune deficiency, is easy to be infected.Draw Playing the contagious organism of machine and not will lead under normal conditions has healthy immune system sick, but can infection immunity system function The people that energy is low or immune system is suppressed.
The term as used herein " constructive expression " refers to the expression under all physiological conditions.
The term as used herein " inducing expression " refers to expression under certain condition, the condition such as T cell with When antigen combines.How those skilled in the art carry out conventional " inducing expression ".
Immune response cell
In some embodiments, the present invention provides a kind of immune response cell, which expresses specific bond GPC3 Receptor and exogenous IL12.
In some embodiments, the exogenous IL12 is constructive expression or inducible expression；It is preferred that inductivity Expression, for example, utilizing inducible promoter；It is preferred that NFAT6 promoter.
In some embodiments, the transmembrane region of the receptor of the combination antigen can be selected from albumen such as CD8 or CD28 Transmembrane region.People's CD8 albumen is a heterodimer, is made of two chains of α β or γ δ.In some embodiments, transmembrane region is selected From the transmembrane region of CD8 α or CD28.In addition, CD8 α hinge area (hinge) is a flexible region, therefore, CD8 or CD28 and Transmembrane region be used to connect in conjunction with the target spot identification structural domain scFv of the receptor CAR of antigen and intracellular signal area plus hinge area It picks up and.
Intracellular signal structural domain of the invention can selected from CD3 ζ, Fc ε RI γ, CD28 costimulatory signal structural domain, CD137 costimulatory signal structural domain, and combinations thereof.CD3 molecule is made of five subunits, and wherein CD3 ζ subunit is (also known as CD3zeta, abbreviation Z) contain 3 ITAM motifs, which is signal transduction area important in TCR-CD3 complex.In addition, As previously mentioned, CD28 and CD137 are costimulatory signal molecules, what is generated with its intracellular signal section after respective ligand binding Costimulation acts on the continuous proliferation for causing immune response cell (mainly T lymphocyte), and can be improved immune response cell The level of the cell factors such as IL-2 and IFN-γ is secreted, CAR immune response cell time to live in vivo and antitumor is improved Effect.In some embodiments, the intracellular signal transduction domain is CD3 ζ signal domain or CD3 ζ signal structure The combination in domain and other costimulatory signals such as CD28.
It in some embodiments, may include expression construct in immune response cell of the invention, expression building There is the element that is linked in sequence as follows: antibody, CD28 costimulatory signal structural domain, CD3 ζ in object, and with aforementioned member NFAT6, IL12 expression unit of part Opposite direction connection.Preferably, leading between the antibody and CD28 costimulatory signal structural domain CD8 α transmembrane region is crossed to be connected with CD8 α hinge area.
In some embodiments, nuclear factor of activated T cells NFAT (Nuclear factor of activated T Cells) transcriptional expression of cell factor plays an important role during T cell activation.Considered based on such, the present invention The coded sequence of IL12 is placed under the regulation of NFAT6 promoter by people, so only when CAR-T cell contact antigen causes T Cell activation, IL12 could high level expressions.
NFAT6 promoter is the combination seat for utilizing 6 NFAT and the minimal promoter (minimal promoter) of IL2 Be cascaded promoter (Hooijberg E, Bakker AQ, Ruizendaal JJ, the Spits H.NFAT- of composition controlled expression of GFP permits visualization and isolation of antigen- stimulated primary human Tcells.Blood.2000Jul 15；96 (2): 459-66), it can be used for adjusting thin Expression (Zhang L, Kerkar SP, Yu Z, Zheng Z, Yang of the intracellular cytokine such as IL12 in T lymphocyte such as TCR-T S, RestifoNP, Rosenberg SA, Morgan RA.Improving adoptive T cell therapy by targeting and controlling IL-12expression to the tumor environment.Mol Ther.2011Apr；19(4):751-9).
According to an aspect of the present invention, the present invention also includes the nucleic acid for encoding the receptor of the combination antigen.The present invention The variant of above-mentioned polynucleotides is further related to, coding has the polypeptide of identical amino acid sequence or the piece of polypeptide with the present invention Section, analogs and derivatives.
The present invention also provides the receptor proteins for the combination antigen that immune response cell surface is expressed in comprising above-mentioned coding Nucleic acid carrier.In a specific embodiment, the carrier that the present invention uses is a kind of slow virus plasmid vector pRRLSIN-cPPT.PGK-GFP.WPRE.It should be understood that other types of viral vectors and non-virus carrier, and can answer ?.
The invention also includes the viruses comprising above-mentioned carrier.Virus of the invention includes that the tool after packing is infectious Virus also includes the virus to be packaged for being packaged as having infectious viral institute essential component.Known in the art Other viral and its corresponding plasmid vectors that can be used for transduceing foreign gene into immune response cell can also be used for this hair It is bright.
Immune response cell of the invention has the structure that can express receptor and exogenous IL12 in conjunction with antigen by transduceing Build object or expression vector, or the virus comprising the plasmid.The nucleic acid transduction method of this field routine, including it is non-viral and viral Transduction method may be used to the present invention.
Immune response cell of the present invention can also express the another kind other than the receptor of above-mentioned combination antigen In conjunction with the receptor of antigen.
Immune response cell of the present invention can also express safety switch；Preferably, the safety switch packet It includes: iCaspase-9, Truancated EGFR or RQR8.
Nucleic acid
The transgenosis of the receptor of encoding target combination antigen or CAR can be incorporated in cell.For example, can be by transgenosis It is incorporated to immune response cell, such as T cell.When being inserted into cell, it is letter that transgenosis, which can be complementary DNA (cDNA) segment, Make the copy of RNA (mRNA)；Or positioned at gene of its genomic DNA original area itself (with or without introne).
The nucleic acid of encoded transgene sequence such as DNA can be with the chromosome of radom insertion cell.Random integration can be by by core Any method that sour (such as DNA) introduces cell generates.For example, this method can include but is not limited to electroporation, ultrasound, make With particle gun, fat transfection, calcium phosphate transfection, using dendritic macromole, microinjection and using including adenovirus, AAV and inverse The viral vectors, and/or II type ribozyme of transcription vector.
The DNA of encoded transgene can also be designed to include reporter gene, so as to be examined by the activation of reporter gene Survey transgenosis or the presence of its expression product.Any reporter, such as those described above can be used.By in cell culture The cell that wherein reporter has been activated is selected in object, can choose the cell containing transgenosis.
The expression of CAR can be by expressing measuring method, such as qPCR or the level by measuring RNA are verified.Express water It is flat to can also indicate that copy number.For example, this may indicate that the more than one copy of CAR is integrated if expression is very high Into genome.Alternatively, high expression can indicate integrated transgene in high transcript regions, such as the promoter of height expression Near.Expression can also be verified by measurement protein level, such as passes through western blot.
In some embodiments, immune response cell of the invention may include one or more transgenosis.Described one Kind or a variety of transgenosis can express CAR albumen, and the CAR albumen identifies and combines at least one epitope or the knot on antigen Close the mutable epitope on antigen.CAR can be functional CAR.Immune response cell of the invention can in some embodiments With comprising one or more CAR or its may include single CAR and reengineering receptor.
As described above, transgenosis can be with the genome of random or site-specific fashion insertion immunoreactive cell In.For example, transgenosis can be inserted into the random sites in the genome of immunocyte.These transgenosis can be function Property, for example, being fully functional when middle from anywhere in being inserted into genome.For example, transgenosis can encode it The promoter of itself, or the position of its internal promoter control can be inserted.Alternatively, transgenosis can be inserted into gene, example Such as the introne of gene or exon, promoter or the noncoding region of gene.Transgenosis, which can be inserted, makes insertion destroy base Cause, such as endogenous immune checkpoint.
In some cases, the transgenosis of more than one copy is inserted into multiple random sites in genome.Example It such as, can be by the random sites in multiple copy insertion genomes.With transgenosis radom insertion once compared with, this may cause whole Body surface, which reaches, to be increased.Alternatively, the copy of transgenosis is inserted into gene, another copy of transgenosis is inserted into difference Gene in.Transgenosis can be targeted, allows to be inserted into the specific site in the genome of immunoreactive cell.
In some cases, comprising encoding the shape for combining the polynucleotide of the receptor sequence of antigen that can take plasmid vector Formula.Plasmid vector may include promoter.In some cases, promoter can be composing type.In some cases, it opens Mover is derivable.Promoter can be or can be derived from CMV, U6, MND or EF1a.In some cases, promoter It can be adjacent with CAR sequence.In some cases, plasmid vector also includes acceptor splicing site.In some cases, acceptor splicing site It can be adjacent with CAR sequence.Promoter sequence can be PKG or MND promoter.MND promoter can be containing myelosis Property sarcoma virus enhancer modification MoMuLV LTR the region U3 synthetic promoter.
In some cases, the polynucleotide of encoding target receptor can be designed, to be delivered to carefully by non-viral technology Born of the same parents.In some cases, polynucleotide can be good manufacturing standard (GMP) compatible reagent.
The expression of the polynucleotide of the receptor or CAR of encoding target combination antigen can be controlled by one or more promoters. Promoter can be generally existing, composing type (unrestricted promoter allows the consecutive transcription of related gene), tissue Specificity promoter or inducible promoter.Expression of the adjustable insertion adjacent or proximate to the transgenosis of promoter.For example, Transgenosis can be plugged near generally existing promoter or side.Some generally existing promoters can be CAGGS starting Son, hCMV promoter, PGK promoter, SV40 promoter or ROSA26 promoter.
Promoter can be endogenous or external source.For example, one or more transgenosis can be inserted into endogenous or outer Source ROSA26 promoter adjacent or proximate to place.In addition, promoter can be the specificity of immunoreactive cell.For example, one A or multiple transgenosis can be inserted porcine ROSA 26 promoter adjacent or proximate to.
Tissue-specific promoter or cell specificity promotor can be used for controlling the position of expression.For example, can be by one A or multiple transgenosis insertion tissue-specific promoter adjacent or proximate to.Tissue-specific promoter can be FABP and open Mover, Lck promoter, CamKII promoter, CD19 promoter, Cytokeratin Promoters, albumin promoter, aP2 promoter, Insulin promoter, MCK promoter, MyHC promoter, WAP promoter or Col2A promoter.
Also inducible promoter can be used.If desired, can be opened and closed by adding or removing inducer These inducible promoters.It is expected that inducible promoter can be but not limited to Lac, tac, trc, trp, araBAD, phoA, RecA, proU, cst-1, tetA, cadA, nar, PL, cspA, T7, VHB, Mx and/or Trex.
Immunocyte inducible promoter
The term as used herein " inducible promoter " is a kind of controlled promoter, is not reached in the condition expected At the preceding gene that do not express or low expression is operably connected with it, and expressed in the case where the condition expected is reached Or the gene that high level expression is operably connected with it.For example, the induction type of the application starts in some embodiment party It is not expressed under conditions of normal or elevated oxygen level of the son in cell or gene that low expression is operably connected with it, and In response to the oxygen content reduced in cell, the gene that expression or high expression are operably connected with it under anoxic conditions.? In some embodiments, inducible promoter used herein includes derivable -1 α (Hypoxia- of transcription factor of hypoxemia Inducible Transcription factor-1α,HIF-1α).In some embodiments, the term as used herein " inducible promoter " refers to " immunocyte inducible promoter ", before immune response cell contacts antigen or immune The gene that responsive cell is not expressed when not being activated or low expression is operably connected with it, and it is only thin in immune response When born of the same parents' contact antigen or immune response cell are activated, it can just drive the gene being operatively connected with it that high level expression occurs Or it is expressed under the conditions ofs anoxic etc..In some embodiments, " the immunocyte inducible promoter " includes NFAT (nuclear factor of activated T cells) type promoter.
" NFAT type promoter " used herein refers to that carry out regulation based on NFAT combination activity is operably connected with it Gene expression a kind of promoter.
NFAT is the general designation of a transcription factor family played a significant role in immune response.The one of NFAT family A or multiple members express in the most cells of immune system.NFAT also assists in the hair of heart, skeletal muscle and nervous system It educates.
NFAT transcription factor family is made of five members NFATc1, NFATc2, NFATc3, NFATc4 and NFAT5. NFATc1 to NFATc4 is adjusted by Ca2+ oscillations.Ca2+ oscillations are most important to NFAT activation, because calmodulin (CaM) activates silk Propylhomoserin/Threonine Phosphatases calcineurin (CN).The CN of activation makes the serine of NFAT histone amino-termini enrich area Domain (SRR) and the acidification of SP repetitive sequence quick dephosphorization, lead to conformation change, and exposure nuclear localization signal leads to NFAT input nucleus In.
Based on the transcriptional expression role of NFAT cell factor during T cell activation, it can be used for regulating and controlling this Immunocyte inducible promoter described in text, thus when immune response cell contact is Antigen-activated, expression or high level Express the gene being operably connected with it.In some embodiments, may include in " NFAT type promoter " as described herein More than one NFAT binding site.For example, " the NFAT type promoter " may include 2,3,4,5,6,7,8,9,10 or more NFAT binding site.In some embodiments, described " NFAT type promoter " can be multiple NFAT binding sites with Promoter composed by promoter such as IL2 minimal promoter series connection.In some embodiments, NFAT type as described herein opens Mover includes 6 NFAT binding sites, is denoted as (NFAT) 6.For convenience, described (NFAT) 6 is also denoted as NFAT6.? In some embodiments, 6 duplicate NFAT binding sites that the NFAT6 is also illustrated in the NFAT type promoter.
In addition, transgenic sequence can also include transcription or translational control sequence, example although not being necessary to expression Such as promoter, the sequence of enhancer, insulator, internal ribosome entry site, coding 2A peptide and/or polyadenylation signal.
In some cases, retroviral vector (γ-retrovirus or slow virus carrier), which can be used, will turn base Because importing immunoreactive cell.For example, the transgenosis of coding CAR or any receptor of combination antigen or its variant or segment It can be cloned into retroviral vector, and sequence can be repeated by its internal promoter, the long end of retrovirus Column drive the promoter of target cell type specificity.Also non-virus carrier can be used.Non-virus carrier delivery system It may include DNA plasmid, naked nucleic acid and the nucleic acid compound with delivery vector such as liposome or poloxamer.
Many systems based on virus have been developed to be used for gene transfer into mammalian cell.For example, reversing Record virus is the platform that genes delivery system is provided convenience.Techniques known in the art can be used by selected gene Insertion carrier is simultaneously packaged in retroviral particle.Carrier derived from retrovirus such as slow virus is to realize long-term base Because of the suitable tools of transfer, because of integration steady in a long-term and its breeding in daughter cell that they allow transgenosis.Slow virus Carrier has the added advantage that compared with the carrier derived from retrovirus such as Murine Leukemia Virus, because they can turn Lead non-proliferative cell.They also have the attendant advantages of low immunogenicity.The advantages of adenovirus vector is that they are not fused to target In the genome of cell, to bypass negative integration dependent event.
Cell can be transfected with the transgenosis for the receptor for encoding the combination antigen.Transgenosis concentration can be about 100 skins Gram to about 50 micrograms.In some cases, thus it is possible to vary introduce the amount of the nucleic acid (for example, ssDNA, dsDNA or RNA) of cell To optimize transfection efficiency and/or cell viability.It is worn for example, 1 microgram dsDNA can be added into each cell sample for electricity Hole.In some cases, the amount of nucleic acid (for example, double-stranded DNA) needed for best transfection efficiency and/or cell viability is according to thin Born of the same parents' type and it is different.In some cases, the amount for the nucleic acid (for example, dsDNA) of each sample can correspond directly to turn Contaminate efficiency and/or cell viability.For example, a series of transfection concentrations.Cytogene can be integrated by the transgenosis of vector encoded In group.In some cases, by the transgenosis Forward Integration of vector encoded.In other cases, base is turned by vector encoded The reversed integration of cause.
In some cases, immunoreactive cell can be by CD45RO (-), CCR7 (+), CD45RA (+), CD62L The dry memory T SCM cell of+(L-selectin), CD27+, CD28+ and/or IL-7R α+composition, the dry memory cell can be with CD95, IL-2R β, CXCR3 and/or LFA-1 are expressed, and shows many function categories different from the dry memory cell Property.Alternatively, immunoreactive cell can also be the maincenter memory body TCM cell comprising L-selectin and CCR7, wherein maincenter Memory cell can secrete such as IL-2, but not secrete IFN γ or IL-4.Immunoreactive cell can also be to be selected comprising L- Albumen or the effect memory T EM cell of CCR7 are selected, and generates such as effector cell's factor such as IFN γ and IL-4.
It is logical usually by Formulations for systemic administration (such as intravenous, peritonaeum is interior, intramuscular, subcutaneous or encephalic is transfused) or topical application It crosses and gives delivery vector in individual patient body, as described below.Alternatively, carrier can with ex vivo delivered to cell, such as from individual suffer from The cell that person's (for example, lymphocyte, T cell, bone marrow, tissue biopsy) removes, is then usually incorporated with this in selection Cell reimplantation is entered into patient's body after the cell of carrier.It, can be with amplifying cells before or after selection.
Suitable immunoreactive cell for expressing the receptor in conjunction with antigen can be Self or non-self cell.
The source of suitable immune response cell can be obtained from individual.In some cases, T cell can be obtained.Institute Stating T cell can obtain from many sources, including PBMC, marrow, lymph node tissue, Cord blood, thymic tissue and carry out self-infection Position, ascites, pleural effusion, spleen tissue and tumour tissue.In some cases, any amount of this field can be used Technology known to technical staff, such as FicollTM separation obtain T cell from the blood collected from the individual.In a reality It applies in scheme, the cell of the blood circulation from individual is obtained by single blood sampling.It singly gathers and processes product and usually contains lymphocyte, wrap Include T cell, monocyte, granulocyte, B cell, other have nuclear leukocyte, red blood cell and blood platelet.In an embodiment In, it can wash by acquiring the cell collected singly to remove serum fraction and cell is placed in suitable buffer or training It supports and is used for subsequent procedure of processing in base.
Alternatively, can carry out self diagnosis from healthy donors and suffer from the patient of cancer or be diagnosed as patient's derivative of infection carefully Born of the same parents.In some embodiments, cell can be a part of the mixed cell population with different phenotypic characteristics.It can be with Cell line is obtained from the T cell of conversion according to preceding method.Cell can also be obtained from cell therapy library.It can pass through herein Any method obtains the modified cells resistant to immunosuppressive therapy.It can also be selected before modification suitable thin Born of the same parents group.Also it can choose engineering cell group after modification.Engineering cell can be used for autotransplantation.Alternatively, cell can be used for it is of the same race different Body transplanting.In some cases, cell is applied to the same patient that sample is used to identify cancer correlation target sequence.At other In the case of, cell is applied to the patient for being used to identify the patient of cancer correlation target sequence different from its sample.
In some cases, suitable primary cell includes peripheral blood mononuclear cells (PBMC), peripheral blood lymphocytes (PBL) and other blood cell subgroups, such as, but not limited to T cell, natural killer cell, monocyte, natural killer agent T Cell, monocytic precursor cells, candidate stem cell or non-pluripotent stem cell.In some cases, cell can be any exempt from Epidemic disease cell, including any T cell such as tumor-infiltrating cells (TIL), such as CD3+T cell, CD4+T cell, CD8+T cell or appoint What other kinds of T cell.T cell can also include memory T cell, remember dry T cell or effector T cell.It can also be from big T cell is selected in amount group, such as selects T cell from whole blood.T cell can also be expanded from a large amount of groups.T cell It may tend to specific population and phenotype.For example, T cell can favour phenotype include CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/or IL-7R α (+).Suitable cell can be selected from following list One of or multiple markers: CD45RO (-), CCR7 (+), CD45RA (+), CD62L (+), CD27 (+), CD28 (+) and/ Or IL-7R α (+).Suitable cell further includes stem cell, for example, the multipotential stem cell of such as embryonic stem cell, induction, hematopoiesis Stem cell, neuronal stem cell and mescenchymal stem cell.Suitable cell may include any amount of primary cell, such as People's cell, non-human cell and/or mouse cell.Suitable cell can be progenitor cells.Suitable cell can be derived from and want The subject (for example, patient) for the treatment of.
The amount of the effective cell for the treatment of needed in patient can be according to the viability and cell of cell by genetic modification Efficiency and change (for example, transgenosis is integrated into the efficiency in one or more cells, or the albumen by transgenes encoding The expression of matter).In some cases, after genetic modification cell viability product (for example, multiplication) and integrated transgene Efficiency can correspond to the therapeutic dose of the cell that can be used for giving subject.In some cases, cell is deposited after genetic modification The increase of vigor likely corresponds to give reduction of the treatment to the effective required cell concentration of patient.In some cases, transgenosis The increase for the efficiency being integrated into one or more cells can correspond to give treatment in patients effective required cell The reduction of quantity.In some cases, determine that the amount of the required effective cell for the treatment of can include determining that with cell at any time Change relevant function.In some cases, determine need to treat effective cell amount can include determining that with according to when Between correlated variables by integrated transgene to the corresponding function of efficiency change in one or more cells (for example, cell culture Time, electroporation time, cytositimulation time).In some cases, treating effective cell can be cell mass, it includes The expression of the receptor of the combination antigen of about 30% to about 100% on cell surface.In some cases, pass through fluidic cell Art measurement, treat effective cell can be expressed on cell surface the combination antigen receptor about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98%, 99%, 99.5%, 99.9% or be more than about 99.9%.
In some cases, when the receptor of the combination antigen is present on the plasma membrane of cell, and work as and pass through combination Target and when being activated, can cause with the target that can be combined for the receptor that its cell surface expresses the combination antigen The toxicity of target cell.For example, in some cases, when cell is present in the plasma membrane of cell, cell can be cell toxicant Property cell (for example, NK cell or cytotoxic T lymphocyte), the receptor of combination antigen as described herein, and when it passes through Cytotoxic cell can be increased to the cellular cytoxicity activity of target cell when being activated in conjunction with its target.For example, in some feelings Under condition, the receptor of combination antigen as described herein, when being activated by the combination of its target, with to there is no combine target The cytotoxicity of cell compare, can make cytotoxicity increase at least 10%, at least 15%, at least 20%, at least 25%, At least 30%, at least 40%, at least 50%, at least 75%, at least 2 times, at least 2.5 times, at least 5 times, at least 10 times or more 10 times.
Pharmaceutical composition
Immune response cell of the invention can be applied to preparation pharmaceutical composition.The pharmaceutical composition is in addition to packet A effective amount of immune response cell is included, also may include pharmaceutically acceptable carrier.Term " pharmaceutically acceptable ", which refers to, works as When biomolecule ontology and composition suitably give animal or people, it is unfavorable, allergy or other bad anti-that they will not be generated It answers.
The specific example that can be used as pharmaceutically acceptable carrier or some substances of its component is antioxidant；Anti-corrosion Agent；Apirogen water；Isotonic salting liquid；With phosphate buffer etc..
Various dosage forms can be made in composition of the invention as needed, and can be by doctor according to patient category, age, body Weight and the substantially factors such as disease condition, administration mode determine that the dosage beneficial to patient is administered.Administration mode for example can be with Using parenteral administration (as injected) or other therapeutic modalities.
" parenteral " application of immunogenic composition includes for example subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.) or breastbone inner injection or infusion techniques.
The preparation comprising immunoreactive cell group for giving individual includes effectively treatment and/or prevention specific adaptations Multiple immunoreactive cells of disease or disease.It therefore, can be to the effective group for the treatment of of individual application immunoreactive cell Body.In general, application includes about 1 × 104To about 1 × 1010The preparation of a immunoreactive cell.In most cases, preparation It will contain from about 1 × 105To about 1 × 109A immunoreactive cell, about 5 × 105To about 5 × 108A immunoreactive cell or About 1 × 106To about 1 × 107A immunoreactive cell.However, according to the position of cancer, source, identity, degree and serious journey Degree, the age of individual to be treated and physical condition etc., will be in width to the quantity of the CAR immunoreactive cell of individual application Range between change.Doctor will eventually determine suitable dosage to be used.
In some embodiments, the immune response for stimulating immunocyte to mediate using Chimeric antigen receptor.For example, T Cell-mediated immune response is to be related to the immune response of T cell activation.The antigen-specific cytotoxic t lymphocytes energy of activation It is induced cell apoptosis in enough target cells for showing Surface Display of Foreign Epitopes on the surface, such as the cancer cell of display tumour antigen.? In another embodiment, antineoplastic immune is provided in mammals using Chimeric antigen receptor.It is mediated due to T cell Immune response, subject will generate antineoplastic immune.
In some cases, the method for subject of the treatment with cancer can be related to applying to subject in need for the treatment of With one or more immune response cells of the present invention.The immune response cell is in combination with tumour target molecule and induces Cancer cell death.As it was noted above, the present invention also provides the methods of the pathogenic infection in treatment individual, including to described The immune response cell of the invention of body application therapeutically effective amount.
The administration frequency of immunoreactive cell of the invention by according to include treated disease factor, it is specific be immunized The element and administration mode of reactive cell.As described herein, since the immune response cell of the application has improved work Power, so as to not only with it is similar but do not express the effective amount of the lower treatment of immune response cell of exogenous IL12 Administration, and can be administered with lower frequency, to obtain at least similar and preferably more significant curative effect.
Combine with anti-tumor drug
In some embodiments, immune response cell of the invention can be administered with another therapeutic agent.Some In embodiment, another therapeutic agent is chemotherapeutic.It can be with the chemotherapeutic of immune response cell use in conjunction of the invention Object includes but is not limited to mitotic inhibitor (vinca alkaloids), including vincristine, vinblastine, eldisine and Novi guest (TM) (vinorelbine, 5'- dehydrogenation hydrogen sulfide)；Topoisomerase I inhibitor, such as Comptothecin compounds, including CamptosarTM (Irinotecan HCL), HycamtinTM (Hycamtin HCL) and its derived from camptothecine and the like Its compound；Podophyllotoxin derivative, such as Etoposide, Teniposide and meter Duo former times help hereby；Alkylating agent cis-platinum, ring phosphorus Amide, mustargen, trimethylene thio-phosphamide, Carmustine, busulfan, Chlorambucil, Boulez quinolizine, uracil mustard End, chlorine ibuprofen and Dacarbazine；Antimetabolite, including cytarabine, fluorouracil, methotrexate (MTX), mercaptopurine, imuran And procarbazine；Antibiotic, including but not limited to Doxorubicin, bleomycin, dactinomycin D, daunorubicin, mycin mycin, Mitomycin, sarcomycin C and daunomycin；And other chemotherapeutics, including but not limited to anti-tumour antibody, Dacca bar Piperazine, AzGR, A Mushakang, melphalan, ifosfamide and mitoxantrone.
In some embodiments, with the chemotherapeutics of immune response cell use in conjunction of the invention can include but It is not limited to anti-angiogenic agent, including anti-VEGF antibody (including humanization and chimeric antibody, anti-vegf aptamer and antisense widow's core Thuja acid) and other angiogenesis inhibitors, such as angiostatin, endostatin, interferon, retinoic acid and metalloprotein The tissue depressant of enzyme -1 and -2.
Kit
The present invention also provides the kits comprising immune response cell of the present invention.The kit can be used for treat or Pre- anti-cancer, pathogenic infection, immune disorders or allograft.In one embodiment, kit may include containing There is the treatment or prevention composition of a effective amount of immune response cell comprising one or more unit dosage forms.
In some embodiments, kit includes the sterile chamber containing treatment or prevention property composition.
In some cases, kit may include about 1 × 104A cell is to about 1 × 106A cell.In some cases Under, kit may include at least about 1 × 105A cell, at least about 1 × 106A cell, at least about 1 × 107A cell, at least About 4 × 107A cell, at least about 5 × 107A cell, at least about 6 × 107A cell, at least about 6 × 107A cell, 8 × 107 A cell, at least about 9 × 107A cell, at least about 1 × 108A cell, at least about 2 × 108A cell, at least about 3 × 108 A cell, at least about 4 × 108A cell, at least about 5 × 108A cell, at least about 6 × 108A cell, at least about 6 × 108 Cell, at least about 8 × 108A cell, at least about 9 × 108Cell, at least about 1 × 109A cell, at least about 2 × 109It is a thin Born of the same parents, at least about 3 × 109A cell, at least about 4 × 109A cell, at least about 5 × 109A cell, at least about 6 × 109It is a thin Born of the same parents, at least about 8 × 109A cell, at least about 9 × 109A cell, at least about 1 × 1010A cell, at least about 2 × 1010It is a thin Born of the same parents, at least about 3 × 1010A cell, at least about 4 × 1010A cell, at least about 5 × 1010A cell, at least about 6 × 1010It is a thin Born of the same parents, at least about 9 × 1010A cell, at least about 9 × 1010A cell, at least about 1 × 1011A cell, at least about 2 × 1011It is a Cell, at least about 3 × 1011A cell, at least about 4 × 1011A cell, at least about 5 × 1011A cell, at least about 8 × 1011 A cell, at least about 9 × 1011A cell, or at least about 1 × 1012A cell.For example, can include about 5 in kit ×1010A cell.
In some cases, kit may include homogeneous variant cell.In some cases, kit may include It may include the cell of genomic modification.In some cases, kit may include " ready-made " cell.In some cases Under, kit may include the cell that can be extended for clinical use.In some cases, kit may be comprising being used for The content of research purpose.
Autologous leukocytes infusion can be used for treating.It is thin autologous peripheral blood monokaryon can be collected from patient in need for the treatment of Born of the same parents (PBMC), and the activation of described herein and methods known in the art can be used and expand T cell, it is then injected into patient In vivo.In other cases, homogeneous variant cell can be used for treating patient.
Method disclosed herein may include transplanting.Transplanting can refer to the adoptive transplanting of cell products.Transplanting can be Autotransplantation, allograft, heterograft or any other transplanting.For example, transplanting can be heterograft.Transplanting It can be allograft.
The preparation of embodiment 1.GPC3-CAR-T cell/IL12-GPC3-CAR T cell
1, plasmid construction
Chimeric antigen receptor used by the present embodiment be two generations Chimeric antigen receptor, use targeting GPC3 by The nucleotide coding sequence of the scFv of the extracellular domain of body as shown in SEQ ID NO:1, also with the transmembrane domain of CD28, CD28 Intracellular domain and CD3 ζ.Referring to Fig.1 shown in A, the matter of GPC3-CAR-T cell and IL12-GPC3-CAR T cell is constructed respectively Grain, specific as follows:
GPC3-CAR-T sequence by CD8 alpha signal peptide (SEQ ID NO:2), target GPC3 scFv (SEQ ID NO:1), The area hinge (SEQ ID NO:3), CD28 transmembrane region (SEQ ID NO:6) and intracellular signal transduction structural domain (the SEQ ID of CD8 NO:4) and the intracellular section of CD3 ζ of CD3 (SEQ ID NO:5) is formed.
The plasmid of IL12-GPC3-CAR is to insert NFAT6-IL12 sequence, structure on the basis of GPC3-CAR-T plasmid The two generation Chimeric antigen receptors of expression GPC3 and the slow virus plasmid of IL12 are built.NFAT6-IL12 sequence is by 6*NFAT Binding motif (SEQ ID NO:7), IL2minimal promoter (SEQ ID NO:8), IL12 signal peptide and IL12p40 (SEQ ID NO:10), (G4S) 3Linker (SEQ ID NO:9), IL12p35 (SEQ ID NO:11), PA2 (SEQ ID NO:20) composition.
2, slow virus packaging, viral concentration and titer determination
A. slow virus is packed
1) with 5 × 106Density inoculated and cultured to the 6th~10 generation 293T cell in culture dish, 37 DEG C, 5% CO2Training It supports overnight, culture medium is the DMEM containing 10% fetal calf serum (Gibico)；
2) respectively by 5.4 μ g of target gene plasmid GPC3-CAR-T and IL12-GPC3-CAR T and packaging plasmid pRsv- 6.2 μ g of REV, 6.2 RRE-PMDLg μ g, 2.4 Vsvg μ g dissolve in 800 μ L blank DMEM culture solutions, mix, and obtain plasmid mixing Liquid；
3) 60 μ g PEI (1 μ g/ μ l) are dissolved in the serum-free DMEM culture solution of 800 μ l, are incubated at room temperature 5min, obtain PEI mixed liquor；
4) plasmid mixed liquor is added in PEI mixed liquor and is mixed, be incubated for 20min at room temperature, form transfection composite；
5) transfection composite 1.6ml is added dropwise in the 10cm culture dish of the culture medium of DMEM containing 11ml, after 4-5h hours, Liquid is changed to the 293T cell of transfection with the DMEM of 10%FBS training base, 37 DEG C of incubation 72h collect virus liquid supernatant.
B. slow virus is concentrated
1) 5XPEG8000NaCl is prepared: weighing NaCl 8.766g, that PEG8000 50g is dissolved in 200ml Milli-Q is pure In water；121 DEG C of moist heat sterilization 30min, are stored in 4 DEG C；
2) 0.45 μm of filter filter virus supernatant is used, 5X PEG- is added in every filtered initial liquid of virus of 30ml 8000NaCl mother liquor 7.5ml；Every 20~30min mixing is primary, carries out 3-5 times altogether；4 DEG C stand overnight, and inhale and abandon after centrifugation Clearly, it stands and removes residual liquid；Suitable slow virus lysate dissolution slow virus precipitating is added；Viral suspension-after concentration 80 DEG C of storages.
The resulting slow virus titre of flow cytomery is advisable with the cell number that positive rate is 5~20%, calculates drop (U/mL)=cell number × positive rate/virus volume is spent, the virus titer after concentration is respectively as follows:
GPC3-CAR-T:2.4 × 108U/ml
T:1 × 10 IL12-GPC3-CAR8U/ml
3, the preparation of GPC3-CAR-T cell, IL12-GPC3-CAR T cell
1) T lymphocyte activation is carried out using the magnetic bead (Invitrogen) of AntiCD3 McAb and CD28 antibody；
2) infection the previous day, RetroNectin are coated with 24 orifice plates: the RetroNectin of 380 μ l, 5 μ g/ml is added in every hole Solution (PBS), 4 DEG C of overnight incubations；
3) the RetroNectin solution (PBS) in 24 orifice plates is discarded, 1ml PBS is washed twice；T cell after activation is connect Kind in 24 orifice plates for being coated with retronectin, every hole cell number 5 × 105, 500 μ l of nutrient solution volume；According to MOI= 15 are added the slow virus after concentration in PBMC cell, 32 DEG C, 1800rpm, after being centrifuged 40min, are transferred to cell incubator In；
4) amplification cultivation: metainfective cell every other day uses 5 × 105The density of/mL is passed on, while being drenched The recombinant human il-2 of final concentration 500U/mL is added in bar cell culture fluid.
It is (pure using operation flow cytometer detection GPC3-CAR-T cell/IL12-GPC3-CAR T positive rate of this field routine Degree) and measurement (measuring method is the method for this field routine, such as Kowolik et al., method disclosed in 2006) and, as a result such as Shown in Figure 1B, UTD is the T cell of untransfected CAR, and GPC3-CAR is the T for having transfected pRRLSIN.cPPT.EF-1 α -9F2-28Z Cell, IL12-GPC3-CAR are the T cells for having transfected pRRLSIN.cPPT.EF-1 α -9F2-28Z-NFAT6-IL12, as a result The display surface 9F2-28Z/9F2-28Z-NFAT6-IL12 has the CAR of higher level to express.
Embodiment 2, the cytotoxicity assay for targeting GPC3CAR-T/IL12-GPC3-CAR T cell
Cytotoxicity, tool are detected using 96 non-radioactive cell toxicity detection kit (Promega company) of CytoTox Body is referring to 96 non-radioactive cell toxicity detection kit specification of CytoTox.
1) target cell: select Huh-7, PLC/PRF/5, SK-HEP-1 cell for target cell, wherein Huh-7 cell, PLC/PRF/5 cell is GPC3 positive, and SK-HEP-1 cell is GPC3 negative；
2) effector cell: add UTD, GPC3-CAR-T and IL12-GPC3-CAR T thin by effect target ratio 3:1,1:1 or 1:3 Born of the same parents；
Experimental result is as shown in Fig. 2, compare UTD group, GPC3-CAR-T/IL12-GPC3-CAR T toxicity test in vitro In show stronger cytotoxicity, and between the two without significant difference.For the liver cancer cells of GPC3 feminine gender, The lethal effect that GPC3-CAR-T/IL12-GPC3-CAR T does not enhance.
Embodiment 3, GPC3-CAR-T/IL12-GPC3-CAR T cell cytokine secretion situation
By UTD/GPC3-CAR-T/IL12-GPC3-CAR T and liver cancer cell lines Huh-7, PLC/PRF/5, SK-HEP-1 Cell 1:1 is incubated altogether receives supernatant afterwards for 24 hours, and ELISA detects levels of cytokine secretion in supernatant.
Wherein detect TNF-α, IL12 sample be not required to dilute, detection IL2, IFN-γ sample dilute respectively 20 times and 25 times.ELISA kit uses double antibody sandwich enzyme-linked immunosorbent assay technology.As a result as shown in figs.3 a and 3b.Fig. 3 A is aobvious Show that IL12-GPC3-CAR T has higher level IL12's when tumour cell Huh-7, PLC/PRF/5 with GPC3+ are incubated altogether Secretion, is nearly no detectable IL12 when the tumour cell SK-HEP-1 with GPC3- is incubated altogether, illustrates that the CAR-T cell only exists In the case where identifying GPC3, IL12 can be just secreted.Fig. 3 B is detection UTD/GPC3-CAR-T/IL12-GPC3-CAR T and tumour Cell factor IL-2, TNF-α, IFN-γ release conditions when cell is incubated for for 24 hours altogether, it is shown that IL12-GPC3-CAR T obviously divides Secrete more IFN-γ.
Embodiment 4, GPC3-CAR-T/IL12-GPC3-CAR T subcutaneously move GPC3 high in the endogenous Huh-7 expressed Plant the therapeutic effect of tumor
1, NOD/SCID mouse inoculation Huh-7 subcutaneous transplantation tumor
By inoculate Huh-7 cell, 2 × 106/ only, posterior tuberosity volume reaches 300mm within about 10 days3；By being grouped at random Mode, every group of 6 mouse；
2, it prepares and expands UTD/GPC3-CAR-T/IL12-GPC3-CAR T cell；
3, UTD/GPC3-CAR-T/IL12-GPC3-CAR T cell adoptive immunotherapy is carried out to Huh-7 transplantable tumor
1) subcutaneous transplantation tumor was in 10 days progress abdominal cavity cyclophosphamide injections (100mg/kg)；
2) it D10 days, measures NOD/SCID mice-transplanted tumor volume and is grouped at random.Subcutaneous transplantation tumor mouse is divided into It is 3 groups, comprising: UTD group, GPC3-CAR-T group, IL12-GPC3-CAR T group, every group mouse 6；
3) D12 days, to the NOD/SCID mouse cell adoptive immunotherapy after grouping.By tail vein injection, give low The CAR-T of dosage, dosage 1x107Cell/mouse；
4) every the size of 3-4 days measurement Huh-7 subcutaneous transplantation knurl products, the variation of every group of mouse tumor volume is recorded, and Draw the growth curve of knurl product at any time.As a result as shown in Figure 4 A and 4 B shown in FIG..
Fig. 4 A shows the growing state of Huh-7 subcutaneous transplantation tumor, after giving CAR-T cell 2 days, IL12-GPC3- CAR T shows significant tumor suppression and lethal effect, and GPC3-CAR-T group, tumour were in always since the 14th day Existing increase tendency, it is little with blank control group difference.Illustrate tumour out of contior for GPC3-CAR, IL12-GPC3-CAR T still shows excellent killing activity.
Fig. 4 B is shown give IL12-GPC3-CAR T after, with the weight of GPC3-CAR-T group and blank control group mouse without Significant difference does not cause to treat dependent body although illustrating that IL12-GPC3-CAR T shows significant antitumous effect Decline side effect again.Also, the mouse of experimental group is observed, discovery gives the mouse after IL12-GPC3-CAR T also without going out Now apparent toxic side effect.
CAR-T injection after the 7th day and the 10th day respectively tail vein take blood, the survival feelings of flow cytometer detection CAR-T cell Condition, as a result as shown in Figure 5, it is shown that the CAR-T cell survivaling number of GPC3-CAR-T group has been lower than 6000/μ L at the 7th day, IL12-GPC3-CAR T group then reaches nearly 8000/μ L, and at the 10th day, the CAR-T cell of GPC3-CAR-T group significantly dropped It is low, it has been lower than 2000/μ L, IL12-GPC3-CAR T and has still kept higher cell survival, with IL12-GPC3- at the 7th day The data of CAR T are compared, and difference is little, and IL12-GPC3-CAR T group is compared with GPC3-CAR-T group, and CAR-T is thin in peripheral blood Born of the same parents' quantity height at least 75% (calculation method: IL12-GPC3-CAR T group-GPC3-CAR-T group/IL12-GPC3-CAR T group), Illustrate that the cell survival of IL12-GPC3-CAR T in vivo is far superior to the CAR-T product for not containing IL12.
Take mouse peripheral blood in Day10, ELISA carried out to serum sample and detects IFN-γ, detection method with embodiment 3, As a result as shown in Figure 6, it is shown that the IFN-γ in IL12-GPC3-CAR T group mouse blood is significantly larger than GPC3-CAR-T group.
Its subcutaneous tumors is taken at the end of experiment, and immunohistochemistry inspection is carried out to infiltrative type T cell in tumor tissues and Ki67 It surveys, as a result as shown in fig. 7, IL12-GPC3-CAR T group CAR-T infiltration is significantly more than GPC3-CAR-T group, IL12-GPC3- The nuclear breeding Marker Ki67 of CAR T group is considerably less than other two groups, and the reduction of Ki67 illustrates that the cell being proliferated in tumour subtracts It is few, that is, illustrate that IL12-GPC3-CAR T can effectively remove the tumour cell of GPC3+, inhibits the proliferation of tumour cell.
Be euthanized mouse and weigh its subcutaneous tumors, as a result as shown in Figure 8 A and 8 B, IL12-GPC3- at the end of experiment Either gross tumor volume or tumor weight are all significantly lower than two generation CAR-T groups to CAR T group at the end of experiment, have significant Statistical difference.
Embodiment 5, mouse CAR-T (mGPC3-CAR) and m-IL12-GPC3-CAR T (mouse CAR-IL12) cell Preparation
The effect that inherent immunity is transferred to further verify IL12 in immune competent mice, is prepared for source of mouse CAR-T。
1, plasmid construction
By mouse CD8 alpha signal peptide (SEQ ID NO:12), resisting GPC 3 antibody mab (SEQ ID NO:1), mouse CD8 α glue chain Area and transmembrane region (SEQ ID NO:13), mouse CD28 Intracellular domain (SEQ ID NO:14), mouse CD3 ζ Intracellular domain (SEQ ID NO: 15) gene order is sequentially connected, and obtains GPC3-CAR-T genetic fragment by outer-gene synthetic method, and with Mlu I With the IRES-GFP segment in I double enzyme site displacement retroviral vector MSCV-IRES-GFP of Sal, recombinant vector is obtained MSCV-GPC3-CAR-T.Mouse CAR-IL12 (m-IL12-GPC3-CAR T) is the base in MSCV-GPC3-CAR-T plasmid MNFAT6-mIL12 sequence is inserted on plinth, constructs the two generation Chimeric antigen receptors of expression GPC3 and the plasmid of mIL12, MSCV-IL12-GPC3-CAR T.MNFAT6-mIL12 sequence be by 6*mNFAT binding motif (SEQ ID NO:16), MIL2minimal promoter (SEQ ID NO:17), mIL12 signal peptide and mIL12p40 (SEQ ID NO:18), (SG4) 3Linker (SEQ ID NO:9), mIL12p35 (SEQ ID NO:19), PA2 (SEQ ID NO:20) composition.
2, retrovirus is packed
1) with 5 × 106Density inoculated and cultured to the 6th~10 generation 293T cell in 10cm culture dish, 37 DEG C, 5% CO2Overnight incubation, culture medium are the DMEM containing 10% fetal calf serum (Gibico)；
2) 9 μ g MSCV-GPC3-CAR-T or MSCV-GPC3-CAR-T-mNFAT6-mIL12 and 9 μ g are packed into matter respectively Grain pCL-Eco dissolves in 800 μ L serum-free DMEM culture solutions, mixes, obtains plasmid mixed liquor；54 μ g PEI (1 μ g/ μ l) are molten Solution mixes in the serum-free DMEM culture solution of 800 μ l, is incubated at room temperature 5min, obtains PEI mixed liquor；
3) plasmid mixed liquor is added in PEI mixed liquor and is mixed, be incubated for 20min at room temperature, form transfection composite；
4) transfection composite 1.6ml is added dropwise in the 10cm culture dish of the culture medium of DMEM containing 11ml, after 4-5h hours, Liquid is changed to the 293T cell of transfection with the DMEM of 10%FBS training base, 37 DEG C of incubation 72h collect virus liquid supernatant, carried The retrovirus of mGPC3-CAR-T/mGPC3-CAR-T-mNFAT6-mIL12.
4, the preparation of mouse CAR-T lymphocyte
Healthy Balb/c mouse is taken, using Cell isolation kit (STEMCELL Technologies), according to Described in specification, isolated mouse spleen T lymphocyte.
Dynabeads Mouse T-activator CD3/ is added by 1:1 in the mouse CD3+T lymphocyte of purifying CD28 (PBS cleaning is primary), puts incubator culture, and culture medium is 1640 complete medium of RPMI, is activated.
The mouse spleen T lymphocyte of activation for 24 hours is inoculated in coated 48 orifice plate of retronectin, every hole cell Number 1 × 106, the retrovirus of 1ml mGPC3-CAR-T/mGPC3-CAR-T-mNFAT6-mIL12, supplement culture is added Base is transferred to cell incubator and continues to cultivate to 2mL, 2000g, 32 DEG C after being centrifuged 90min；Second day replacement fresh cultured Base, adjustment cell density are 5 × 105/ mL is passed on for every 2-3 days.
The expression measurement of mCAR-T cell chimerism antigen receptor:
Take 1 × 106MCAR-T/mIL12-GPC3-CAR T cell, halve in 2ml centrifuge tube, 4 degree, 400g, It is centrifuged 5min, abandons supernatant, PBS is washed twice.Cellular control unit is added 50 μ l PE-SA (1:200 dilution) antibody and is incubated on ice 45min, PBS (2%NBS) are washed twice；As control after resuspension；The 50 diluted biotin- of μ l 1:50 are added in detection group cell Goat anti human IgG, F (ab ')2Antibody is incubated for 45 min on ice；PBS (2%NBS) is washed twice；50 μ l PE- are added SA (1:200 dilution) antibody is incubated for 45min on ice.
2ml PBS (2%NBS) is added, cell is resuspended, 4 DEG C, 400g, is centrifuged 5 minutes abandoning supernatants；It is repeated twice, is added 500 μ l PBS (2%NBS), FACS detection, as a result as shown in figure 9, m9F2-m28Z/m9F2-m28Z-mNFAT6- as the result is shown The surface mIL12 has the CAR of higher level to express, and UTD is the mT cell of untransfected CAR.
Embodiment 6, the cytotoxicity assay for targeting GPC3mCAR-T/mIL12-GPC3-CAR T cell
Using 96 non-radioactive cell toxicity detection kit (Promega company) of CytoTox, referring in particular to 96 non-radioactive cell toxicity detection kit specification of CytoTox.
1) it target cell: is inoculated with 50 μ L 2 × 10 respectively5E0771-Parental, E0771-Parental-GPC3 of/mL, E0771-Recurrent (after E0771 is inoculated in mouse tumor formation, isolated cell line is carried out to tumor tissue), E0771- Recurrent-GPC3 cell is in 96well plate；
2) effector cell: add UTD, mGPC3-m28Z and mGPC3-m28Z-mNFAT6- by effect target ratio 3:1,1:1 or 1:3 MIL12 cell；
Experimental result is as shown in Figure 10, compares UTD group, mGPC3-CAR and mGPC3-m28Z-mNFAT6-mIL12 (mIL12-GPC3-CAR T) shows stronger cytotoxicity in toxicity test in vitro, and not bright between the two Significant difference is other.For the tumour cell of GPC3 feminine gender, lethal effect that mGPC3-CAR and mIL12-GPC3-CAR T does not enhance.
Embodiment 7, mCAR-T/mIL12-GPC3-CAR T cell cytokine secretion situation
By UTD/mGPC3-CAR/mIL12-GPC3-CAR T and E0771-Parental, E0771-Parental- GPC3, E0771-Recurrent, E0771-Recurrent-GPC3 cell 1:1 are incubated altogether receive supernatant afterwards for 24 hours after in ELISA detection Levels of cytokine secretion in clear.Wherein detect mTNF- α, the sample of mIL12, mIL2 are not required to dilute, the sample of detection mIFN- γ Product dilute 50 times.ELISA kit uses double antibody sandwich enzyme-linked immunosorbent assay technology.Specific anti-mouse IL-2, TNF-α, IFN-γ, IL-12 monoclonal antibody are pre-coated on the ELISA Plate of high-affinity respectively.Mark is added in ELISA Plate hole Quasi- product, sample to be tested and biotinylated detection antibody, by being incubated for, mIL-2 present in sample, mTNF- α, mIFN- γ, MIL-12 is respectively in conjunction with solid phase antibody and detection antibody.After the unbonded substance of washing removal, horseradish peroxidase is added The Streptavidin (streptavidin-HRP) of label.TMB develops the color after washing.It is above-mentioned in the depth and sample of color reaction The concentration of cell factor is directly proportional.Terminate liquid is added and terminates reaction, measures and inhales in 450nm wavelength (reference wavelength 570-630nm) Shading value.
As a result as shown in Figure 11 A and 11B, Figure 11 A is detection UTD, mGPC3-CAR, mGPC3-m28Z-mNFAT6- The secretion situation of mIL-12 when mIL12 and tumour cell are incubated for for 24 hours altogether, it is shown that when the tumour cell with GPC3+ is incubated altogether MGPC3-m28Z-mNFAT6-mIL12 (mIL12-GPC3-CAR T) has the secretion of higher level IL12, illustrates mouse IL12's Secretion is also derivable.Figure 11 B is that detection UTD/mGPC3-m28Z/mIL12-GPC3-CAR T is incubated for altogether with tumour cell Cell factor mIL-2, mTNF- α, mIFN- γ release conditions when for 24 hours, mIL12-GPC3-CAR T obviously secrete more IFN-γ。
Embodiment 8, mGPC3-CAR-T/mIL12-GPC3-T are to the therapeutic effect of E0771-GPC3 subcutaneous transplantation tumor
1, C57BL/6 mouse inoculation E0771-GPC3 subcutaneous transplantation tumor
By inoculate E0771-GPC3 cell, 1 × 106/ only, posterior tuberosity volume reaches 300mm within about 10 days3；By with Machine packet mode, every group of 6 mouse；
2, it prepares and expands mUTD/mGPC3-CAR-T/mIL12-GPC3-T cell；
3, adopt to E0771-GPC3 transplantable tumor progress mUTD/mGPC3-CAR-T/mIL12-GPC3-CAR-T cell and exempt from Epidemic disease treatment
1) it D10 days, measures E0771-GPC3 mice-transplanted tumor volume and is grouped at random.Subcutaneous transplantation tumor mouse is total It is divided into 5 groups, comprising: UTD group, mGPC3-CAR-T 5 × 106Group, mGPC3-CAR-T 2 × 106Group, mIL12-GPC3-T 5 × 106Group, mIL12-GPC3-T 2 × 106Group, every group mouse 6；
2) every the size of 3-4 days measurement E0771-GPC3 subcutaneous transplantation knurl products, the change of every group of mouse tumor volume is recorded Change, and draws the growth curve of knurl product at any time.As a result as illustrated in figs. 12 a and 12b.
Figure 12 A shows tail vein injection mIL12-GPC3-T 5 × 106/ only organize and mIL12-GPC3-T 2 × 106/ only Tumor killing effect is similar at the end of experiment for group, has preferably tumor-inhibiting action, tumour inhibiting rate up to 90% or more, also illustrates Under the assistance of IL12, when facing big tumor load, infusion low dose CAR-T still can reach ideal effect, and review mGPC3- CAR-T 5×106/ although volume is only organized less than mUTD group, no difference of science of statistics, knurl when consideration is due to injection CAR-T Larger (the 300mm of product3) caused by.
Figure 12 B is shown give mIL12-GPC3-T after, with the weight of mGPC3-CAR-T group and blank control group mouse without Significant difference does not cause to treat under related weight although illustrating that IL12-GPC3-T shows significant antitumous effect Side effect is dropped.
Be euthanized mouse and weigh its subcutaneous tumors, as a result as shown in figures 13 a and 13b, mIL12- at the end of experiment Either gross tumor volume or tumor weight are all significantly lower than two generation CAR-T groups to GPC3-T group at the end of experiment, have significant Statistical difference.
Sequence of the present invention is summarized in following table
All references mentioned in the present invention is incorporated herein by reference, just as each document coverlet It is solely incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art Member can make various changes or modifications the present invention, and such equivalent forms equally fall within the application the appended claims and limited Fixed range.
Sequence table
<110>Ke Ji biological medicine (Shanghai) Co., Ltd.
Shanghai Inst. of Tumor
<120>immune effector cell and its application of GPC3 are targeted
<130> P2018-1679
<150> CN2017108059914
<151> 2017-09-08
<160> 35
<170> PatentIn version 3.5
<210> 1
<211> 729
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
gaggtgcagc tggtgcagag cggcgccgag gtgaagaagc ccggcgccag cgtgaaggtg 60
agctgcaagg ccagcggcta caccttcagc gactacgaga tgcactgggt gcggcaggcc 120
cccggccagg gcctggagtg gatgggcgcc atccaccccg gcagcggcga caccgcctac 180
aaccagcggt tcaagggccg ggtgaccatc accgccgaca agagcaccag caccgcctac 240
atggagctga gcagcctgcg gagcgaggac accgccgtgt actactgcgc ccggttctac 300
agctacgcct actggggcca gggcaccctg gtgaccgtga gcgccggtgg aggcggttca 360
ggcggaggtg gttctggcgg tggcggatcg gacatcgtga tgacccagac ccccctgagc 420
ctgcccgtga cccccggcga gcccgccagc atcagctgcc ggagcagcca gagcctggtg 480
cacagcaacg gcaacaccta cctgcagtgg tacctgcaga agcccggcca gagcccccag 540
ctgctgatct acaaggtgag caaccggttc agcggcgtgc ccgaccggtt cagcggcagc 600
ggcagcggca ccgacttcac cctgaagatc agccgggtgg aggccgagga cgtgggcgtg 660
tactactgca gccagagcat ctacgtgccc tacaccttcg gccagggcac caagctggag 720
atcaaacgt 729
<210> 2
<211> 63
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
atggccttac cagtgaccgc cttgctcctg ccgctggcct tgctgctcca cgccgccagg 60
ccg 63
<210> 3
<211> 135
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60
tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120
gacttcgcct gtgat 135
<210> 4
<211> 123
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aggagtaaga ggagcaggct cctgcacagt gactacatga acatgactcc ccgccgcccc 60
gggccaaccc gcaagcatta ccagccctat gccccaccac gcgacttcgc agcctatcgc 120
tcc 123
<210> 5
<211> 339
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
agagtgaagt tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc 60
tataacgagc tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc 120
cgggaccctg agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac 180
aatgaactgc agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag 240
cgccggaggg gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac 300
acctacgacg cccttcacat gcaggccctg ccccctcgc 339
<210> 6
<211> 81
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ttttgggtgc tggtggtggt tggtggagtc ctggcttgct atagcttgct agtaacagtg 60
gcctttatta ttttctgggt g 81
<210> 7
<211> 194
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
ggaggaaaaa ctgtttcata cagaaggcgt ggaggaaaaa ctgtttcata cagaaggcgt 60
ggaggaaaaa ctgtttcata cagaaggcgt caattgtcct cgacggagga aaaactgttt 120
catacagaag gcgtggagga aaaactgttt catacagaag gcgtggagga aaaactgttt 180
catacagaag gcgt 194
<210> 8
<211> 114
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
acattttgac acccccataa tatttttcca gaattaacag tataaattgc atctcttgtt 60
caagagttcc ctatcactct ctttaatcac tactcacagt aacctcaact cctg 114
<210> 9
<211> 45
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
ggtggaggcg gttcaggcgg aggtggttct ggcggtggcg gatcg 45
<210> 10
<211> 984
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
atgtgtcacc agcagttggt catctcttgg ttttccctgg tttttctggc atctcccctc 60
gtggccatat gggaactgaa gaaagatgtt tatgtcgtag aattggattg gtatccggat 120
gcccctggag aaatggtggt cctcacctgt gacacccctg aagaagatgg tatcacctgg 180
accttggacc agagcagtga ggtcttaggc tctggcaaaa ccctgaccat ccaagtcaaa 240
gagtttggag atgctggcca gtacacctgt cacaaaggag gcgaggttct aagccattcg 300
ctcctgctgc ttcacaaaaa ggaagatgga atttggtcca ctgatatttt aaaggaccag 360
aaagaaccca aaaataagac ctttctaaga tgcgaggcca agaattattc tggacgtttc 420
acctgctggt ggctgacgac aatcagtact gatttgacat tcagtgtcaa aagcagcaga 480
ggctcttctg acccccaagg ggtgacgtgc ggagctgcta cactctctgc agagagagtc 540
agaggggaca acaaggagta tgagtactca gtggagtgcc aggaggacag tgcctgccca 600
gctgctgagg agagtctgcc cattgaggtc atggtggatg ccgttcacaa gctcaagtat 660
gaaaactaca ccagcagctt cttcatcagg gacatcatca aacctgaccc acccaagaac 720
ttgcagctga agccattaaa gaattctcgg caggtggagg tcagctggga gtaccctgac 780
acctggagta ctccacattc ctacttctcc ctgacattct gcgttcaggt ccagggcaag 840
agcaagagag aaaagaaaga tagagtcttc acggacaaga cctcagccac ggtcatctgc 900
cgcaaaaatg ccagcattag cgtgcgggcc caggaccgct actatagctc atcttggagc 960
gaatgggcat ctgtgccctg cagt 984
<210> 11
<211> 594
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
agaaacctcc ccgtggccac tccagaccca ggaatgttcc catgccttca ccactcccaa 60
aacctgctga gggccgtcag caacatgctc cagaaggcca gacaaactct agaattttac 120
ccttgcactt ctgaagagat tgatcatgaa gatatcacaa aagataaaac cagcacagtg 180
gaggcctgtt taccattgga attaaccaag aatgagagtt gcctaaattc cagagagacc 240
tctttcataa ctaatgggag ttgcctggcc tccagaaaga cctcttttat gatggccctg 300
tgccttagta gtatttatga agacttgaag atgtaccagg tggagttcaa gaccatgaat 360
gcaaagcttc tgatggatcc taagaggcag atctttctag atcaaaacat gctggcagtt 420
attgatgagc tgatgcaggc cctgaatttc aacagtgaga ctgtgccaca aaaatcctcc 480
cttgaagaac cggattttta taaaactaaa atcaagctct gcatacttct tcatgctttc 540
agaattcggg cagtgactat tgatagagtg atgagctatc tgaatgcttc ctaa 594
<210> 12
<211> 81
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60
atcctgggga gtggagaagc t 81
<210> 13
<211> 216
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
actactacca agccagtgct gcgaactccc tcacctgtgc accctaccgg gacatctcag 60
ccccagagac cagaagattg tcggccccgt ggctcagtga aggggaccgg attggacttc 120
gcctgtgata tttacatctg ggcacccttg gccggaatct gcgtggccct tctgctgtcc 180
ttgatcatca ctctcatctg ctaccacagg agccga 216
<210> 14
<211> 123
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
aatagtagaa ggaacagact ccttcaaagt gactacatga acatgactcc ccggaggcct 60
gggctcactc gaaagcctta ccagccctac gcccctgcca gagactttgc agcgtaccgc 120
ccc 123
<210> 15
<211> 321
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 15
agcaggagtg cagagactgc tgccaacctg caggacccca accagctcta caatgagctc 60
aatctagggc gaagagagga atatgacgtc ttggagaaga agcgggctcg ggatccagag 120
atgggaggca aacagcagag gaggaggaac ccccaggaag gcgtatacaa tgcactgcag 180
aaagacaaga tggcagaagc ctacagtgag atcggcacaa aaggcgagag gcggagaggc 240
aaggggcacg atggccttta ccagggtctc agcactgcca ccaaggacac ctatgatgcc 300
ctgcatatgc agaccctggc c 321
<210> 16
<211> 192
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 16
aagaggaaaa tttgtttcat acagaaggcg ttaagaggaa aatttgtttc atacagaagg 60
cgttaagagg aaaatttgtt tcatacagaa ggcgttaaga ggaaaatttg tttcatacag 120
aaggcgttaa gaggaaaatt tgtttcatac agaaggcgtt aagaggaaaa tttgtttcat 180
acagaaggcg tt 192
<210> 17
<211> 114
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 17
aacatcgtga cacccccata ttatttttcc agcattaaca gtataaattg cctcccatgc 60
tgaagagctg cctatcaccc ttgctaatca ctcctcacag tgacctcaag tcct 114
<210> 18
<211> 1005
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 18
atgtgtcctc agaagctaac catctcctgg tttgccatcg ttttgctggt gtctccactc 60
atggccatgt gggagctgga gaaagacgtt tatgttgtag aggtggactg gactcccgat 120
gcccctggag aaacagtgaa cctcacctgt gacacgcctg aagaagatga catcacctgg 180
acctcagacc agagacatgg agtcataggc tctggaaaga ccctgaccat cactgtcaaa 240
gagtttctag atgctggcca gtacacctgc cacaaaggag gcgagactct gagccactca 300
catctgctgc tccacaagaa ggaaaatgga atttggtcca ctgaaatttt aaaaaatttc 360
aaaaacaaga ctttcctgaa gtgtgaagca ccaaattact ccggacggtt cacgtgctca 420
tggctggtgc aaagaaacat ggacttgaag ttcaacatca agagcagtag cagttcccct 480
gactctcggg cagtgacatg tggaatggcg tctctgtctg cagagaaggt cacactggac 540
caaagggact atgagaagta ttcagtgtcc tgccaggagg atgtcacctg cccaactgcc 600
gaggagaccc tgcccattga actggcgttg gaagcacggc agcagaataa atatgagaac 660
tacagcacca gcttcttcat cagggacatc atcaaaccag acccgcccaa gaacttgcag 720
atgaagcctt tgaagaactc acaggtggag gtcagctggg agtaccctga ctcctggagc 780
actccccatt cctacttctc cctcaagttc tttgttcgaa tccagcgcaa gaaagaaaag 840
atgaaggaga cagaggaggg gtgtaaccag aaaggtgcgt tcctcgtaga gaagacatct 900
accgaagtcc aatgcaaagg cgggaatgtc tgcgtgcaag ctcaggatcg ctattacaat 960
tcctcatgca gcaagtgggc atgtgttccc tgcagggtcc gatcc 1005
<210> 19
<211> 579
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 19
agggtcattc cagtctctgg acctgccagg tgtcttagcc agtcccgaaa cctgctgaag 60
accacagatg acatggtgaa gacggccaga gaaaaactga aacattattc ctgcactgct 120
gaagacatcg atcatgaaga catcacacgg gaccaaacca gcacattgaa gacctgttta 180
ccactggaac tacacaagaa cgagagttgc ctggctacta gagagacttc ttccacaaca 240
agagggagct gcctgccccc acagaagacg tctttgatga tgaccctgtg ccttggtagc 300
atctatgagg acttgaagat gtaccagaca gagttccagg ccatcaacgc agcacttcag 360
aatcacaacc atcagcagat cattctagac aagggcatgc tggtggccat cgatgagctg 420
atgcagtctc tgaatcataa tggcgagact ctgcgccaga aacctcctgt gggagaagca 480
gacccttaca gagtgaaaat gaagctctgc atcctgcttc acgccttcag cacccgcgtc 540
gtgaccatca acagggtgat gggctatctg agctccgcc 579
<210> 20
<211> 51
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 20
aataaaatat ctttattttc attacatctg tgtgttggtt ttttgtgtga g 51
<210> 21
<211> 469
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 21
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala
355 360 365
Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg
370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu
385 390 395 400
Met Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr
405 410 415
Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly
420 425 430
Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln
435 440 445
Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln
450 455 460
Ala Leu Pro Pro Arg
465
<210> 22
<211> 467
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 22
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu
290 295 300
Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
305 310 315 320
Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu
325 330 335
Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys
340 345 350
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln
355 360 365
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu
370 375 380
Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly
385 390 395 400
Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu
405 410 415
Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys
420 425 430
Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu
435 440 445
Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu
450 455 460
Pro Pro Arg
465
<210> 23
<211> 511
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 23
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro
245 250 255
Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro
260 265 270
Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp
275 280 285
Phe Trp Val Leu Val Val Val Gly Gly Val Leu Ala Cys Tyr Ser Leu
290 295 300
Leu Val Thr Val Ala Phe Ile Ile Phe Trp Val Arg Ser Lys Arg Ser
305 310 315 320
Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr Pro Arg Arg Pro Gly
325 330 335
Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro Pro Arg Asp Phe Ala
340 345 350
Ala Tyr Arg Ser Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
355 360 365
Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
370 375 380
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val
385 390 395 400
Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn
405 410 415
Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val
420 425 430
Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Gln
435 440 445
Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp
450 455 460
Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg
465 470 475 480
Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
485 490 495
Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg
500 505 510
<210> 24
<211> 356
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 24
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr
245 250 255
Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg
260 265 270
Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met
275 280 285
Gly Gly Lys Pro Gln Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn
290 295 300
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
305 310 315 320
Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly
325 330 335
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala
340 345 350
Leu Pro Pro Arg
355
<210> 25
<211> 243
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 25
Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Asp Tyr
20 25 30
Glu Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Ala Ile His Pro Gly Ser Gly Asp Thr Ala Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Phe Tyr Ser Tyr Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr
100 105 110
Val Ser Ala Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
115 120 125
Gly Ser Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr
130 135 140
Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Val
145 150 155 160
His Ser Asn Gly Asn Thr Tyr Leu Gln Trp Tyr Leu Gln Lys Pro Gly
165 170 175
Gln Ser Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly
180 185 190
Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
195 200 205
Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser
210 215 220
Gln Ser Ile Tyr Val Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu
225 230 235 240
Ile Lys Arg
<210> 26
<211> 540
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 26
Met Cys His Gln Gln Leu Val Ile Ser Trp Phe Ser Leu Val Phe Leu
1 5 10 15
Ala Ser Pro Leu Val Ala Ile Trp Glu Leu Lys Lys Asp Val Tyr Val
20 25 30
Val Glu Leu Asp Trp Tyr Pro Asp Ala Pro Gly Glu Met Val Val Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Gly Ile Thr Trp Thr Leu Asp Gln
50 55 60
Ser Ser Glu Val Leu Gly Ser Gly Lys Thr Leu Thr Ile Gln Val Lys
65 70 75 80
Glu Phe Gly Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Val
85 90 95
Leu Ser His Ser Leu Leu Leu Leu His Lys Lys Glu Asp Gly Ile Trp
100 105 110
Ser Thr Asp Ile Leu Lys Asp Gln Lys Glu Pro Lys Asn Lys Thr Phe
115 120 125
Leu Arg Cys Glu Ala Lys Asn Tyr Ser Gly Arg Phe Thr Cys Trp Trp
130 135 140
Leu Thr Thr Ile Ser Thr Asp Leu Thr Phe Ser Val Lys Ser Ser Arg
145 150 155 160
Gly Ser Ser Asp Pro Gln Gly Val Thr Cys Gly Ala Ala Thr Leu Ser
165 170 175
Ala Glu Arg Val Arg Gly Asp Asn Lys Glu Tyr Glu Tyr Ser Val Glu
180 185 190
Cys Gln Glu Asp Ser Ala Cys Pro Ala Ala Glu Glu Ser Leu Pro Ile
195 200 205
Glu Val Met Val Asp Ala Val His Lys Leu Lys Tyr Glu Asn Tyr Thr
210 215 220
Ser Ser Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn
225 230 235 240
Leu Gln Leu Lys Pro Leu Lys Asn Ser Arg Gln Val Glu Val Ser Trp
245 250 255
Glu Tyr Pro Asp Thr Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Thr
260 265 270
Phe Cys Val Gln Val Gln Gly Lys Ser Lys Arg Glu Lys Lys Asp Arg
275 280 285
Val Phe Thr Asp Lys Thr Ser Ala Thr Val Ile Cys Arg Lys Asn Ala
290 295 300
Ser Ile Ser Val Arg Ala Gln Asp Arg Tyr Tyr Ser Ser Ser Trp Ser
305 310 315 320
Glu Trp Ala Ser Val Pro Cys Ser Gly Gly Gly Gly Ser Gly Gly Gly
325 330 335
Gly Ser Gly Gly Gly Gly Ser Arg Asn Leu Pro Val Ala Thr Pro Asp
340 345 350
Pro Gly Met Phe Pro Cys Leu His His Ser Gln Asn Leu Leu Arg Ala
355 360 365
Val Ser Asn Met Leu Gln Lys Ala Arg Gln Thr Leu Glu Phe Tyr Pro
370 375 380
Cys Thr Ser Glu Glu Ile Asp His Glu Asp Ile Thr Lys Asp Lys Thr
385 390 395 400
Ser Thr Val Glu Ala Cys Leu Pro Leu Glu Leu Thr Lys Asn Glu Ser
405 410 415
Cys Leu Asn Ser Arg Glu Thr Ser Phe Ile Thr Asn Gly Ser Cys Leu
420 425 430
Ala Ser Arg Lys Thr Ser Phe Met Met Ala Leu Cys Leu Ser Ser Ile
435 440 445
Tyr Glu Asp Leu Lys Met Tyr Gln Val Glu Phe Lys Thr Met Asn Ala
450 455 460
Lys Leu Leu Met Asp Pro Lys Arg Gln Ile Phe Leu Asp Gln Asn Met
465 470 475 480
Leu Ala Val Ile Asp Glu Leu Met Gln Ala Leu Asn Phe Asn Ser Glu
485 490 495
Thr Val Pro Gln Lys Ser Ser Leu Glu Glu Pro Asp Phe Tyr Lys Thr
500 505 510
Lys Ile Lys Leu Cys Ile Leu Leu His Ala Phe Arg Ile Arg Ala Val
515 520 525
Thr Ile Asp Arg Val Met Ser Tyr Leu Asn Ala Ser
530 535 540
<210> 27
<211> 543
<212> PRT
<213>artificial sequence (Artificial Sequence)
<400> 27
Met Cys Pro Gln Lys Leu Thr Ile Ser Trp Phe Ala Ile Val Leu Leu
1 5 10 15
Val Ser Pro Leu Met Ala Met Trp Glu Leu Glu Lys Asp Val Tyr Val
20 25 30
Val Glu Val Asp Trp Thr Pro Asp Ala Pro Gly Glu Thr Val Asn Leu
35 40 45
Thr Cys Asp Thr Pro Glu Glu Asp Asp Ile Thr Trp Thr Ser Asp Gln
50 55 60
Arg His Gly Val Ile Gly Ser Gly Lys Thr Leu Thr Ile Thr Val Lys
65 70 75 80
Glu Phe Leu Asp Ala Gly Gln Tyr Thr Cys His Lys Gly Gly Glu Thr
85 90 95
Leu Ser His Ser His Leu Leu Leu His Lys Lys Glu Asn Gly Ile Trp
100 105 110
Ser Thr Glu Ile Leu Lys Asn Phe Lys Asn Lys Thr Phe Leu Lys Cys
115 120 125
Glu Ala Pro Asn Tyr Ser Gly Arg Phe Thr Cys Ser Trp Leu Val Gln
130 135 140
Arg Asn Met Asp Leu Lys Phe Asn Ile Lys Ser Ser Ser Ser Ser Pro
145 150 155 160
Asp Ser Arg Ala Val Thr Cys Gly Met Ala Ser Leu Ser Ala Glu Lys
165 170 175
Val Thr Leu Asp Gln Arg Asp Tyr Glu Lys Tyr Ser Val Ser Cys Gln
180 185 190
Glu Asp Val Thr Cys Pro Thr Ala Glu Glu Thr Leu Pro Ile Glu Leu
195 200 205
Ala Leu Glu Ala Arg Gln Gln Asn Lys Tyr Glu Asn Tyr Ser Thr Ser
210 215 220
Phe Phe Ile Arg Asp Ile Ile Lys Pro Asp Pro Pro Lys Asn Leu Gln
225 230 235 240
Met Lys Pro Leu Lys Asn Ser Gln Val Glu Val Ser Trp Glu Tyr Pro
245 250 255
Asp Ser Trp Ser Thr Pro His Ser Tyr Phe Ser Leu Lys Phe Phe Val
260 265 270
Arg Ile Gln Arg Lys Lys Glu Lys Met Lys Glu Thr Glu Glu Gly Cys
275 280 285
Asn Gln Lys Gly Ala Phe Leu Val Glu Lys Thr Ser Thr Glu Val Gln
290 295 300
Cys Lys Gly Gly Asn Val Cys Val Gln Ala Gln Asp Arg Tyr Tyr Asn
305 310 315 320
Ser Ser Cys Ser Lys Trp Ala Cys Val Pro Cys Arg Val Arg Ser Gly
325 330 335
Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Arg Val
340 345 350
Ile Pro Val Ser Gly Pro Ala Arg Cys Leu Ser Gln Ser Arg Asn Leu
355 360 365
Leu Lys Thr Thr Asp Asp Met Val Lys Thr Ala Arg Glu Lys Leu Lys
370 375 380
His Tyr Ser Cys Thr Ala Glu Asp Ile Asp His Glu Asp Ile Thr Arg
385 390 395 400
Asp Gln Thr Ser Thr Leu Lys Thr Cys Leu Pro Leu Glu Leu His Lys
405 410 415
Asn Glu Ser Cys Leu Ala Thr Arg Glu Thr Ser Ser Thr Thr Arg Gly
420 425 430
Ser Cys Leu Pro Pro Gln Lys Thr Ser Leu Met Met Thr Leu Cys Leu
435 440 445
Gly Ser Ile Tyr Glu Asp Leu Lys Met Tyr Gln Thr Glu Phe Gln Ala
450 455 460
Ile Asn Ala Ala Leu Gln Asn His Asn His Gln Gln Ile Ile Leu Asp
465 470 475 480
Lys Gly Met Leu Val Ala Ile Asp Glu Leu Met Gln Ser Leu Asn His
485 490 495
Asn Gly Glu Thr Leu Arg Gln Lys Pro Pro Val Gly Glu Ala Asp Pro
500 505 510
Tyr Arg Val Lys Met Lys Leu Cys Ile Leu Leu His Ala Phe Ser Thr
515 520 525
Arg Val Val Thr Ile Asn Arg Val Met Gly Tyr Leu Ser Ser Ala
530 535 540
<210> 28
<211> 4415
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 28
cgatggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt 60
ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga 120
aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag 180
tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc 240
gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc accatggcct 300
taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccggagg 360
tgcagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg aaggtgagct 420
gcaaggccag cggctacacc ttcagcgact acgagatgca ctgggtgcgg caggcccccg 480
gccagggcct ggagtggatg ggcgccatcc accccggcag cggcgacacc gcctacaacc 540
agcggttcaa gggccgggtg accatcaccg ccgacaagag caccagcacc gcctacatgg 600
agctgagcag cctgcggagc gaggacaccg ccgtgtacta ctgcgcccgg ttctacagct 660
acgcctactg gggccagggc accctggtga ccgtgagcgc cggtggaggc ggttcaggcg 720
gaggtggttc tggcggtggc ggatcggaca tcgtgatgac ccagaccccc ctgagcctgc 780
ccgtgacccc cggcgagccc gccagcatca gctgccggag cagccagagc ctggtgcaca 840
gcaacggcaa cacctacctg cagtggtacc tgcagaagcc cggccagagc ccccagctgc 900
tgatctacaa ggtgagcaac cggttcagcg gcgtgcccga ccggttcagc ggcagcggca 960
gcggcaccga cttcaccctg aagatcagcc gggtggaggc cgaggacgtg ggcgtgtact 1020
actgcagcca gagcatctac gtgccctaca ccttcggcca gggcaccaag ctggagatca 1080
aacgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc 1140
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg 1200
ggctggactt cgcctgtgat ttttgggtgc tggtggtggt tggtggagtc ctggcttgct 1260
atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag aggagcaggc 1320
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggccaacc cgcaagcatt 1380
accagcccta tgccccacca cgcgacttcg cagcctatcg ctccagagtg aagttcagca 1440
ggagcgcaga cgcccccgcg taccagcagg gccagaacca gctctataac gagctcaatc 1500
taggacgaag agaggagtac gatgttttgg acaagagacg tggccgggac cctgagatgg 1560
ggggaaagcc gcagagaagg aagaaccctc aggaaggcct gtacaatgaa ctgcagaaag 1620
ataagatggc ggaggcctac agtgagattg ggatgaaagg cgagcgccgg aggggcaagg 1680
ggcacgatgg cctttaccag ggtctcagta cagccaccaa ggacacctac gacgcccttc 1740
acatgcaggc cctgccccct cgctaggtcg acaatcaacc tctggattac aaaatttgtg 1800
aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt 1860
taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata 1920
aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg 1980
tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc 2040
tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct 2100
gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt 2160
cggggaagct gacgtccttt ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg 2220
ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc 2280
tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct 2340
ccctttgggc cgcctccccg cctggaattc gctagcctcg agctcacaca aaaaaccaac 2400
acacagatgt aatgaaaata aagatatttt attgcggccg ctttaggaag cattcagata 2460
gctcatcact ctatcaatag tcactgcccg aattctgaaa gcatgaagaa gtatgcagag 2520
cttgatttta gttttataaa aatccggttc ttcaagggag gatttttgtg gcacagtctc 2580
actgttgaaa ttcagggcct gcatcagctc atcaataact gccagcatgt tttgatctag 2640
aaagatctgc ctcttaggat ccatcagaag ctttgcattc atggtcttga actccacctg 2700
gtacatcttc aagtcttcat aaatactact aaggcacagg gccatcataa aagaggtctt 2760
tctggaggcc aggcaactcc cattagttat gaaagaggtc tctctggaat ttaggcaact 2820
ctcattcttg gttaattcca atggtaaaca ggcctccact gtgctggttt tatcttttgt 2880
gatatcttca tgatcaatct cttcagaagt gcaagggtaa aattctagag tttgtctggc 2940
cttctggagc atgttgctga cggccctcag caggttttgg gagtggtgaa ggcatgggaa 3000
cattcctggg tctggagtgg ccacggggag gtttctagat ccgccgccac ccgacccacc 3060
accgcccgag ccaccgccac cactgcaggg cacagatgcc cattcgctcc aagatgagct 3120
atagtagcgg tcctgggccc gcacgctaat gctggcattt ttgcggcaga tgaccgtggc 3180
tgaggtcttg tccgtgaaga ctctatcttt cttttctctc ttgctcttgc cctggacctg 3240
aacgcagaat gtcagggaga agtaggaatg tggagtactc caggtgtcag ggtactccca 3300
gctgacctcc acctgccgag aattctttaa tggcttcagc tgcaagttct tgggtgggtc 3360
aggtttgatg atgtccctga tgaagaagct gctggtgtag ttttcatact tgagcttgtg 3420
aacggcatcc accatgacct caatgggcag actctcctca gcagctgggc aggcactgtc 3480
ctcctggcac tccactgagt actcatactc cttgttgtcc cctctgactc tctctgcaga 3540
gagtgtagca gctccgcacg tcaccccttg ggggtcagaa gagcctctgc tgcttttgac 3600
actgaatgtc aaatcagtac tgattgtcgt cagccaccag caggtgaaac gtccagaata 3660
attcttggcc tcgcatctta gaaaggtctt atttttgggt tctttctggt cctttaaaat 3720
atcagtggac caaattccat cttccttttt gtgaagcagc aggagcgaat ggcttagaac 3780
ctcgcctcct ttgtgacagg tgtactggcc agcatctcca aactctttga cttggatggt 3840
cagggttttg ccagagccta agacctcact gctctggtcc aaggtccagg tgataccatc 3900
ttcttcaggg gtgtcacagg tgaggaccac catttctcca ggggcatccg gataccaatc 3960
caattctacg acataaacat ctttcttcag ttcccatatg gccacgaggg gagatgccag 4020
aaaaaccagg gaaaaccaag agatgaccaa ctgctggtga cacatggtgg cgaccggtag 4080
cgctaggtca tatgcaggag ttgaggttac tgtgagtagt gattaaagag agtgataggg 4140
aactcttgaa caagagatgc aatttatact gttaattctg gaaaaatatt atgggggtgt 4200
caaaatgtcc cgggacaatt gacgccttct gtatgaaaca gtttttcctc cacgccttct 4260
gtatgaaaca gtttttcctc cacgccttct gtatgaaaca gtttttcctc cgtcgaggac 4320
aattgacgcc ttctgtatga aacagttttt cctccacgcc ttctgtatga aacagttttt 4380
cctccacgcc ttctgtatga aacagttttt cctcc 4415
<210> 29
<211> 4418
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 29
cgatggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt 60
ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga 120
aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag 180
tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc 240
gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc accatggcct 300
taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccggagg 360
tgcagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg aaggtgagct 420
gcaaggccag cggctacacc ttcagcgact acgagatgca ctgggtgcgg caggcccccg 480
gccagggcct ggagtggatg ggcgccatcc accccggcag cggcgacacc gcctacaacc 540
agcggttcaa gggccgggtg accatcaccg ccgacaagag caccagcacc gcctacatgg 600
agctgagcag cctgcggagc gaggacaccg ccgtgtacta ctgcgcccgg ttctacagct 660
acgcctactg gggccagggc accctggtga ccgtgagcgc cggtggaggc ggttcaggcg 720
gaggtggttc tggcggtggc ggatcggaca tcgtgatgac ccagaccccc ctgagcctgc 780
ccgtgacccc cggcgagccc gccagcatca gctgccggag cagccagagc ctggtgcaca 840
gcaacggcaa cacctacctg cagtggtacc tgcagaagcc cggccagagc ccccagctgc 900
tgatctacaa ggtgagcaac cggttcagcg gcgtgcccga ccggttcagc ggcagcggca 960
gcggcaccga cttcaccctg aagatcagcc gggtggaggc cgaggacgtg ggcgtgtact 1020
actgcagcca gagcatctac gtgccctaca ccttcggcca gggcaccaag ctggagatca 1080
aacgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc 1140
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg 1200
ggctggactt cgcctgtgat ttttgggtgc tggtggtggt tggtggagtc ctggcttgct 1260
atagcttgct agtaacagtg gcctttatta ttttctgggt gaaacggggc agaaagaaac 1320
tcctgtatat attcaaacaa ccatttatga gaccagtaca aactactcaa gaggaagatg 1380
gctgtagctg ccgatttcca gaagaagaag aaggaggatg tgaactgaga gtgaagttca 1440
gcaggagcgc agacgccccc gcgtaccagc agggccagaa ccagctctat aacgagctca 1500
atctaggacg aagagaggag tacgatgttt tggacaagag acgtggccgg gaccctgaga 1560
tggggggaaa gccgcagaga aggaagaacc ctcaggaagg cctgtacaat gaactgcaga 1620
aagataagat ggcggaggcc tacagtgaga ttgggatgaa aggcgagcgc cggaggggca 1680
aggggcacga tggcctttac cagggtctca gtacagccac caaggacacc tacgacgccc 1740
ttcacatgca ggccctgccc cctcgctagg tcgacaatca acctctggat tacaaaattt 1800
gtgaaagatt gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg 1860
ctttaatgcc tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt 1920
ataaatcctg gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg 1980
tggtgtgcac tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc 2040
agctcctttc cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg 2100
cctgccttgc ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt 2160
tgtcggggaa gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc 2220
gcgggacgtc cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg 2280
gcctgctgcc ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga 2340
tctccctttg ggccgcctcc ccgcctggaa ttcgctagcc tcgagctcac acaaaaaacc 2400
aacacacaga tgtaatgaaa ataaagatat tttattgcgg ccgctttagg aagcattcag 2460
atagctcatc actctatcaa tagtcactgc ccgaattctg aaagcatgaa gaagtatgca 2520
gagcttgatt ttagttttat aaaaatccgg ttcttcaagg gaggattttt gtggcacagt 2580
ctcactgttg aaattcaggg cctgcatcag ctcatcaata actgccagca tgttttgatc 2640
tagaaagatc tgcctcttag gatccatcag aagctttgca ttcatggtct tgaactccac 2700
ctggtacatc ttcaagtctt cataaatact actaaggcac agggccatca taaaagaggt 2760
ctttctggag gccaggcaac tcccattagt tatgaaagag gtctctctgg aatttaggca 2820
actctcattc ttggttaatt ccaatggtaa acaggcctcc actgtgctgg ttttatcttt 2880
tgtgatatct tcatgatcaa tctcttcaga agtgcaaggg taaaattcta gagtttgtct 2940
ggccttctgg agcatgttgc tgacggccct cagcaggttt tgggagtggt gaaggcatgg 3000
gaacattcct gggtctggag tggccacggg gaggtttcta gatccgccgc cacccgaccc 3060
accaccgccc gagccaccgc caccactgca gggcacagat gcccattcgc tccaagatga 3120
gctatagtag cggtcctggg cccgcacgct aatgctggca tttttgcggc agatgaccgt 3180
ggctgaggtc ttgtccgtga agactctatc tttcttttct ctcttgctct tgccctggac 3240
ctgaacgcag aatgtcaggg agaagtagga atgtggagta ctccaggtgt cagggtactc 3300
ccagctgacc tccacctgcc gagaattctt taatggcttc agctgcaagt tcttgggtgg 3360
gtcaggtttg atgatgtccc tgatgaagaa gctgctggtg tagttttcat acttgagctt 3420
gtgaacggca tccaccatga cctcaatggg cagactctcc tcagcagctg ggcaggcact 3480
gtcctcctgg cactccactg agtactcata ctccttgttg tcccctctga ctctctctgc 3540
agagagtgta gcagctccgc acgtcacccc ttgggggtca gaagagcctc tgctgctttt 3600
gacactgaat gtcaaatcag tactgattgt cgtcagccac cagcaggtga aacgtccaga 3660
ataattcttg gcctcgcatc ttagaaaggt cttatttttg ggttctttct ggtcctttaa 3720
aatatcagtg gaccaaattc catcttcctt tttgtgaagc agcaggagcg aatggcttag 3780
aacctcgcct cctttgtgac aggtgtactg gccagcatct ccaaactctt tgacttggat 3840
ggtcagggtt ttgccagagc ctaagacctc actgctctgg tccaaggtcc aggtgatacc 3900
atcttcttca ggggtgtcac aggtgaggac caccatttct ccaggggcat ccggatacca 3960
atccaattct acgacataaa catctttctt cagttcccat atggccacga ggggagatgc 4020
cagaaaaacc agggaaaacc aagagatgac caactgctgg tgacacatgg tggcgaccgg 4080
tagcgctagg tcatatgcag gagttgaggt tactgtgagt agtgattaaa gagagtgata 4140
gggaactctt gaacaagaga tgcaatttat actgttaatt ctggaaaaat attatggggg 4200
tgtcaaaatg tcccgggaca attgacgcct tctgtatgaa acagtttttc ctccacgcct 4260
tctgtatgaa acagtttttc ctccacgcct tctgtatgaa acagtttttc ctccgtcgag 4320
gacaattgac gccttctgta tgaaacagtt tttcctccac gccttctgta tgaaacagtt 4380
tttcctccac gccttctgta tgaaacagtt tttcctcc 4418
<210> 30
<211> 4541
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 30
cgatggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt 60
ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga 120
aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag 180
tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc 240
gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc accatggcct 300
taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccggagg 360
tgcagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg aaggtgagct 420
gcaaggccag cggctacacc ttcagcgact acgagatgca ctgggtgcgg caggcccccg 480
gccagggcct ggagtggatg ggcgccatcc accccggcag cggcgacacc gcctacaacc 540
agcggttcaa gggccgggtg accatcaccg ccgacaagag caccagcacc gcctacatgg 600
agctgagcag cctgcggagc gaggacaccg ccgtgtacta ctgcgcccgg ttctacagct 660
acgcctactg gggccagggc accctggtga ccgtgagcgc cggtggaggc ggttcaggcg 720
gaggtggttc tggcggtggc ggatcggaca tcgtgatgac ccagaccccc ctgagcctgc 780
ccgtgacccc cggcgagccc gccagcatca gctgccggag cagccagagc ctggtgcaca 840
gcaacggcaa cacctacctg cagtggtacc tgcagaagcc cggccagagc ccccagctgc 900
tgatctacaa ggtgagcaac cggttcagcg gcgtgcccga ccggttcagc ggcagcggca 960
gcggcaccga cttcaccctg aagatcagcc gggtggaggc cgaggacgtg ggcgtgtact 1020
actgcagcca gagcatctac gtgccctaca ccttcggcca gggcaccaag ctggagatca 1080
aacgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc 1140
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg 1200
ggctggactt cgcctgtgat ttttgggtgc tggtggtggt tggtggagtc ctggcttgct 1260
atagcttgct agtaacagtg gcctttatta ttttctgggt gaggagtaag aggagcaggc 1320
tcctgcacag tgactacatg aacatgactc cccgccgccc cgggccaacc cgcaagcatt 1380
accagcccta tgccccacca cgcgacttcg cagcctatcg ctccaaacgg ggcagaaaga 1440
aactcctgta tatattcaaa caaccattta tgagaccagt acaaactact caagaggaag 1500
atggctgtag ctgccgattt ccagaagaag aagaaggagg atgtgaactg agagtgaagt 1560
tcagcaggag cgcagacgcc cccgcgtacc agcagggcca gaaccagctc tataacgagc 1620
tcaatctagg acgaagagag gagtacgatg ttttggacaa gagacgtggc cgggaccctg 1680
agatgggggg aaagccgcag agaaggaaga accctcagga aggcctgtac aatgaactgc 1740
agaaagataa gatggcggag gcctacagtg agattgggat gaaaggcgag cgccggaggg 1800
gcaaggggca cgatggcctt taccagggtc tcagtacagc caccaaggac acctacgacg 1860
cccttcacat gcaggccctg ccccctcgct aggtcgacaa tcaacctctg gattacaaaa 1920
tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg 1980
ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct 2040
tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg 2100
gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct 2160
gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg 2220
ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg 2280
tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc acctggattc 2340
tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc 2400
gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc 2460
ggatctccct ttgggccgcc tccccgcctg gaattcgcta gcctcgagct cacacaaaaa 2520
accaacacac agatgtaatg aaaataaaga tattttattg cggccgcttt aggaagcatt 2580
cagatagctc atcactctat caatagtcac tgcccgaatt ctgaaagcat gaagaagtat 2640
gcagagcttg attttagttt tataaaaatc cggttcttca agggaggatt tttgtggcac 2700
agtctcactg ttgaaattca gggcctgcat cagctcatca ataactgcca gcatgttttg 2760
atctagaaag atctgcctct taggatccat cagaagcttt gcattcatgg tcttgaactc 2820
cacctggtac atcttcaagt cttcataaat actactaagg cacagggcca tcataaaaga 2880
ggtctttctg gaggccaggc aactcccatt agttatgaaa gaggtctctc tggaatttag 2940
gcaactctca ttcttggtta attccaatgg taaacaggcc tccactgtgc tggttttatc 3000
ttttgtgata tcttcatgat caatctcttc agaagtgcaa gggtaaaatt ctagagtttg 3060
tctggccttc tggagcatgt tgctgacggc cctcagcagg ttttgggagt ggtgaaggca 3120
tgggaacatt cctgggtctg gagtggccac ggggaggttt ctagatccgc cgccacccga 3180
cccaccaccg cccgagccac cgccaccact gcagggcaca gatgcccatt cgctccaaga 3240
tgagctatag tagcggtcct gggcccgcac gctaatgctg gcatttttgc ggcagatgac 3300
cgtggctgag gtcttgtccg tgaagactct atctttcttt tctctcttgc tcttgccctg 3360
gacctgaacg cagaatgtca gggagaagta ggaatgtgga gtactccagg tgtcagggta 3420
ctcccagctg acctccacct gccgagaatt ctttaatggc ttcagctgca agttcttggg 3480
tgggtcaggt ttgatgatgt ccctgatgaa gaagctgctg gtgtagtttt catacttgag 3540
cttgtgaacg gcatccacca tgacctcaat gggcagactc tcctcagcag ctgggcaggc 3600
actgtcctcc tggcactcca ctgagtactc atactccttg ttgtcccctc tgactctctc 3660
tgcagagagt gtagcagctc cgcacgtcac cccttggggg tcagaagagc ctctgctgct 3720
tttgacactg aatgtcaaat cagtactgat tgtcgtcagc caccagcagg tgaaacgtcc 3780
agaataattc ttggcctcgc atcttagaaa ggtcttattt ttgggttctt tctggtcctt 3840
taaaatatca gtggaccaaa ttccatcttc ctttttgtga agcagcagga gcgaatggct 3900
tagaacctcg cctcctttgt gacaggtgta ctggccagca tctccaaact ctttgacttg 3960
gatggtcagg gttttgccag agcctaagac ctcactgctc tggtccaagg tccaggtgat 4020
accatcttct tcaggggtgt cacaggtgag gaccaccatt tctccagggg catccggata 4080
ccaatccaat tctacgacat aaacatcttt cttcagttcc catatggcca cgaggggaga 4140
tgccagaaaa accagggaaa accaagagat gaccaactgc tggtgacaca tggtggcgac 4200
cggtagcgct aggtcatatg caggagttga ggttactgtg agtagtgatt aaagagagtg 4260
atagggaact cttgaacaag agatgcaatt tatactgtta attctggaaa aatattatgg 4320
gggtgtcaaa atgtcccggg acaattgacg ccttctgtat gaaacagttt ttcctccacg 4380
ccttctgtat gaaacagttt ttcctccacg ccttctgtat gaaacagttt ttcctccgtc 4440
gaggacaatt gacgccttct gtatgaaaca gtttttcctc cacgccttct gtatgaaaca 4500
gtttttcctc cacgccttct gtatgaaaca gtttttcctc c 4541
<210> 31
<211> 4292
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 31
cgatggctcc ggtgcccgtc agtgggcaga gcgcacatcg cccacagtcc ccgagaagtt 60
ggggggaggg gtcggcaatt gaaccggtgc ctagagaagg tggcgcgggg taaactggga 120
aagtgatgtc gtgtactggc tccgcctttt tcccgagggt gggggagaac cgtatataag 180
tgcagtagtc gccgtgaacg ttctttttcg caacgggttt gccgccagaa cacaggtgtc 240
gtgacgcgga tccaggccta agcttacgcg tcctagcgct accggtcgcc accatggcct 300
taccagtgac cgccttgctc ctgccgctgg ccttgctgct ccacgccgcc aggccggagg 360
tgcagctggt gcagagcggc gccgaggtga agaagcccgg cgccagcgtg aaggtgagct 420
gcaaggccag cggctacacc ttcagcgact acgagatgca ctgggtgcgg caggcccccg 480
gccagggcct ggagtggatg ggcgccatcc accccggcag cggcgacacc gcctacaacc 540
agcggttcaa gggccgggtg accatcaccg ccgacaagag caccagcacc gcctacatgg 600
agctgagcag cctgcggagc gaggacaccg ccgtgtacta ctgcgcccgg ttctacagct 660
acgcctactg gggccagggc accctggtga ccgtgagcgc cggtggaggc ggttcaggcg 720
gaggtggttc tggcggtggc ggatcggaca tcgtgatgac ccagaccccc ctgagcctgc 780
ccgtgacccc cggcgagccc gccagcatca gctgccggag cagccagagc ctggtgcaca 840
gcaacggcaa cacctacctg cagtggtacc tgcagaagcc cggccagagc ccccagctgc 900
tgatctacaa ggtgagcaac cggttcagcg gcgtgcccga ccggttcagc ggcagcggca 960
gcggcaccga cttcaccctg aagatcagcc gggtggaggc cgaggacgtg ggcgtgtact 1020
actgcagcca gagcatctac gtgccctaca ccttcggcca gggcaccaag ctggagatca 1080
aacgtaccac gacgccagcg ccgcgaccac caacaccggc gcccaccatc gcgtcgcagc 1140
ccctgtccct gcgcccagag gcgtgccggc cagcggcggg gggcgcagtg cacacgaggg 1200
ggctggactt cgcctgtgat ttttgggtgc tggtggtggt tggtggagtc ctggcttgct 1260
atagcttgct agtaacagtg gcctttatta ttttctgggt gagagtgaag ttcagcagga 1320
gcgcagacgc ccccgcgtac cagcagggcc agaaccagct ctataacgag ctcaatctag 1380
gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct gagatggggg 1440
gaaagccgca gagaaggaag aaccctcagg aaggcctgta caatgaactg cagaaagata 1500
agatggcgga ggcctacagt gagattggga tgaaaggcga gcgccggagg ggcaaggggc 1560
acgatggcct ttaccagggt ctcagtacag ccaccaagga cacctacgac gcccttcaca 1620
tgcaggccct gccccctcgc taggtcgaca atcaacctct ggattacaaa atttgtgaaa 1680
gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa 1740
tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat 1800
cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt 1860
gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc 1920
tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc 1980
ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg 2040
ggaagctgac gtcctttcca tggctgctcg cctgtgttgc cacctggatt ctgcgcggga 2100
cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc 2160
tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc 2220
tttgggccgc ctccccgcct ggaattcgct agcctcgagc tcacacaaaa aaccaacaca 2280
cagatgtaat gaaaataaag atattttatt gcggccgctt taggaagcat tcagatagct 2340
catcactcta tcaatagtca ctgcccgaat tctgaaagca tgaagaagta tgcagagctt 2400
gattttagtt ttataaaaat ccggttcttc aagggaggat ttttgtggca cagtctcact 2460
gttgaaattc agggcctgca tcagctcatc aataactgcc agcatgtttt gatctagaaa 2520
gatctgcctc ttaggatcca tcagaagctt tgcattcatg gtcttgaact ccacctggta 2580
catcttcaag tcttcataaa tactactaag gcacagggcc atcataaaag aggtctttct 2640
ggaggccagg caactcccat tagttatgaa agaggtctct ctggaattta ggcaactctc 2700
attcttggtt aattccaatg gtaaacaggc ctccactgtg ctggttttat cttttgtgat 2760
atcttcatga tcaatctctt cagaagtgca agggtaaaat tctagagttt gtctggcctt 2820
ctggagcatg ttgctgacgg ccctcagcag gttttgggag tggtgaaggc atgggaacat 2880
tcctgggtct ggagtggcca cggggaggtt tctagatccg ccgccacccg acccaccacc 2940
gcccgagcca ccgccaccac tgcagggcac agatgcccat tcgctccaag atgagctata 3000
gtagcggtcc tgggcccgca cgctaatgct ggcatttttg cggcagatga ccgtggctga 3060
ggtcttgtcc gtgaagactc tatctttctt ttctctcttg ctcttgccct ggacctgaac 3120
gcagaatgtc agggagaagt aggaatgtgg agtactccag gtgtcagggt actcccagct 3180
gacctccacc tgccgagaat tctttaatgg cttcagctgc aagttcttgg gtgggtcagg 3240
tttgatgatg tccctgatga agaagctgct ggtgtagttt tcatacttga gcttgtgaac 3300
ggcatccacc atgacctcaa tgggcagact ctcctcagca gctgggcagg cactgtcctc 3360
ctggcactcc actgagtact catactcctt gttgtcccct ctgactctct ctgcagagag 3420
tgtagcagct ccgcacgtca ccccttgggg gtcagaagag cctctgctgc ttttgacact 3480
gaatgtcaaa tcagtactga ttgtcgtcag ccaccagcag gtgaaacgtc cagaataatt 3540
cttggcctcg catcttagaa aggtcttatt tttgggttct ttctggtcct ttaaaatatc 3600
agtggaccaa attccatctt cctttttgtg aagcagcagg agcgaatggc ttagaacctc 3660
gcctcctttg tgacaggtgt actggccagc atctccaaac tctttgactt ggatggtcag 3720
ggttttgcca gagcctaaga cctcactgct ctggtccaag gtccaggtga taccatcttc 3780
ttcaggggtg tcacaggtga ggaccaccat ttctccaggg gcatccggat accaatccaa 3840
ttctacgaca taaacatctt tcttcagttc ccatatggcc acgaggggag atgccagaaa 3900
aaccagggaa aaccaagaga tgaccaactg ctggtgacac atggtggcga ccggtagcgc 3960
taggtcatat gcaggagttg aggttactgt gagtagtgat taaagagagt gatagggaac 4020
tcttgaacaa gagatgcaat ttatactgtt aattctggaa aaatattatg ggggtgtcaa 4080
aatgtcccgg gacaattgac gccttctgta tgaaacagtt tttcctccac gccttctgta 4140
tgaaacagtt tttcctccac gccttctgta tgaaacagtt tttcctccgt cgaggacaat 4200
tgacgccttc tgtatgaaac agtttttcct ccacgccttc tgtatgaaac agtttttcct 4260
ccacgccttc tgtatgaaac agtttttcct cc 4292
<210> 32
<211> 3391
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 32
atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60
atcctgggga gtggagaagc tgaggtgcag ctggtgcaga gcggcgccga ggtgaagaag 120
cccggcgcca gcgtgaaggt gagctgcaag gccagcggct acaccttcag cgactacgag 180
atgcactggg tgcggcaggc ccccggccag ggcctggagt ggatgggcgc catccacccc 240
ggcagcggcg acaccgccta caaccagcgg ttcaagggcc gggtgaccat caccgccgac 300
aagagcacca gcaccgccta catggagctg agcagcctgc ggagcgagga caccgccgtg 360
tactactgcg cccggttcta cagctacgcc tactggggcc agggcaccct ggtgaccgtg 420
agcgccggtg gaggcggttc aggcggaggt ggttctggcg gtggcggatc ggacatcgtg 480
atgacccaga cccccctgag cctgcccgtg acccccggcg agcccgccag catcagctgc 540
cggagcagcc agagcctggt gcacagcaac ggcaacacct acctgcagtg gtacctgcag 600
aagcccggcc agagccccca gctgctgatc tacaaggtga gcaaccggtt cagcggcgtg 660
cccgaccggt tcagcggcag cggcagcggc accgacttca ccctgaagat cagccgggtg 720
gaggccgagg acgtgggcgt gtactactgc agccagagca tctacgtgcc ctacaccttc 780
ggccagggca ccaagctgga gatcaaacgt actactacca agccagtgct gcgaactccc 840
tcacctgtgc accctaccgg gacatctcag ccccagagac cagaagattg tcggccccgt 900
ggctcagtga aggggaccgg attggacttc gcctgtgata tttacatctg ggcacccttg 960
gccggaatct gcgtggccct tctgctgtcc ttgatcatca ctctcatctg ctaccacagg 1020
agccgaagca ggagtgcaga gactgctgcc aacctgcagg accccaacca gctctacaat 1080
gagctcaatc tagggcgaag agaggaatat gacgtcttgg agaagaagcg ggctcgggat 1140
ccagagatgg gaggcaaaca gcagaggagg aggaaccccc aggaaggcgt atacaatgca 1200
ctgcagaaag acaagatggc agaagcctac agtgagatcg gcacaaaagg cgagaggcgg 1260
agaggcaagg ggcacgatgg cctttaccag ggtctcagca ctgccaccaa ggacacctat 1320
gatgccctgc atatgcagac cctggcctag gtcgactcac acaaaaaacc aacacacaga 1380
tgtaatgaaa ataaagatat tttattcgta cgttaggcgg agctcagata gcccatcacc 1440
ctgttgatgg tcacgacgcg ggtgctgaag gcgtgaagca ggatgcagag cttcattttc 1500
actctgtaag ggtctgcttc tcccacagga ggtttctggc gcagagtctc gccattatga 1560
ttcagagact gcatcagctc atcgatggcc accagcatgc ccttgtctag aatgatctgc 1620
tgatggttgt gattctgaag tgctgcgttg atggcctgga actctgtctg gtacatcttc 1680
aagtcctcat agatgctacc aaggcacagg gtcatcatca aagacgtctt ctgtgggggc 1740
aggcagctcc ctcttgttgt ggaagaagtc tctctagtag ccaggcaact ctcgttcttg 1800
tgtagttcca gtggtaaaca ggtcttcaat gtgctggttt ggtcccgtgt gatgtcttca 1860
tgatcgatgt cttcagcagt gcaggaataa tgtttcagtt tttctctggc cgtcttcacc 1920
atgtcatctg tggtcttcag caggtttcgg gactggctaa gacacctggc aggtccagag 1980
actggaatga ccctagatcc gccgccaccc gacccaccac cgcccgagcc accgccaccg 2040
gatcggaccc tgcagggaac acatgcccac ttgctgcatg aggaattgta atagcgatcc 2100
tgagcttgca cgcagacatt cccgcctttg cattggactt cggtagatgt cttctctacg 2160
aggaacgcac ctttctggtt acacccctcc tctgtctcct tcatcttttc tttcttgcgc 2220
tggattcgaa caaagaactt gagggagaag taggaatggg gagtgctcca ggagtcaggg 2280
tactcccagc tgacctccac ctgtgagttc ttcaaaggct tcatctgcaa gttcttgggc 2340
gggtctggtt tgatgatgtc cctgatgaag aagctggtgc tgtagttctc atatttattc 2400
tgctgccgtg cttccaacgc cagttcaatg ggcagggtct cctcggcagt tgggcaggtg 2460
acatcctcct ggcaggacac tgaatacttc tcatagtccc tttggtccag tgtgaccttc 2520
tctgcagaca gagacgccat tccacatgtc actgcccgag agtcagggga actgctactg 2580
ctcttgatgt tgaacttcaa gtccatgttt ctttgcacca gccatgagca cgtgaaccgt 2640
ccggagtaat ttggtgcttc acacttcagg aaagtcttgt ttttgaaatt ttttaaaatt 2700
tcagtggacc aaattccatt ttccttcttg tggagcagca gatgtgagtg gctcagagtc 2760
tcgcctcctt tgtggcaggt gtactggcca gcatctagaa actctttgac agtgatggtc 2820
agggtctttc cagagcctat gactccatgt ctctggtctg aggtccaggt gatgtcatct 2880
tcttcaggcg tgtcacaggt gaggttcact gtttctccag gggcatcggg agtccagtcc 2940
acctctacaa cataaacgtc tttctccagc tcccacatgg ccatgagtgg agacaccagc 3000
aaaacgatgg caaaccagga gatggttagc ttctgaggac acatggtggc gaccggtagc 3060
gctaggacgc gtaggacttg aggtcactgt gaggagtgat tagcaagggt gataggcagc 3120
tcttcagcat gggaggcaat ttatactgtt aatgctggaa aaataatatg ggggtgtcac 3180
gatgttcccg ggacaattga acgccttctg tatgaaacaa attttcctct taacgccttc 3240
tgtatgaaac aaattttcct cttaacgcct tctgtatgaa acaaattttc ctcttaacgc 3300
cttctgtatg aaacaaattt tcctcttaac gccttctgta tgaaacaaat tttcctctta 3360
acgccttctg tatgaaacaa attttcctct t 3391
<210> 33
<211> 3514
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 33
atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60
atcctgggga gtggagaagc tgaggtgcag ctggtgcaga gcggcgccga ggtgaagaag 120
cccggcgcca gcgtgaaggt gagctgcaag gccagcggct acaccttcag cgactacgag 180
atgcactggg tgcggcaggc ccccggccag ggcctggagt ggatgggcgc catccacccc 240
ggcagcggcg acaccgccta caaccagcgg ttcaagggcc gggtgaccat caccgccgac 300
aagagcacca gcaccgccta catggagctg agcagcctgc ggagcgagga caccgccgtg 360
tactactgcg cccggttcta cagctacgcc tactggggcc agggcaccct ggtgaccgtg 420
agcgccggtg gaggcggttc aggcggaggt ggttctggcg gtggcggatc ggacatcgtg 480
atgacccaga cccccctgag cctgcccgtg acccccggcg agcccgccag catcagctgc 540
cggagcagcc agagcctggt gcacagcaac ggcaacacct acctgcagtg gtacctgcag 600
aagcccggcc agagccccca gctgctgatc tacaaggtga gcaaccggtt cagcggcgtg 660
cccgaccggt tcagcggcag cggcagcggc accgacttca ccctgaagat cagccgggtg 720
gaggccgagg acgtgggcgt gtactactgc agccagagca tctacgtgcc ctacaccttc 780
ggccagggca ccaagctgga gatcaaacgt actactacca agccagtgct gcgaactccc 840
tcacctgtgc accctaccgg gacatctcag ccccagagac cagaagattg tcggccccgt 900
ggctcagtga aggggaccgg attggacttc gcctgtgata tttacatctg ggcacccttg 960
gccggaatct gcgtggccct tctgctgtcc ttgatcatca ctctcatctg ctaccacagg 1020
agccgaaata gtagaaggaa cagactcctt caaagtgact acatgaacat gactccccgg 1080
aggcctgggc tcactcgaaa gccttaccag ccctacgccc ctgccagaga ctttgcagcg 1140
taccgcccca gcaggagtgc agagactgct gccaacctgc aggaccccaa ccagctctac 1200
aatgagctca atctagggcg aagagaggaa tatgacgtct tggagaagaa gcgggctcgg 1260
gatccagaga tgggaggcaa acagcagagg aggaggaacc cccaggaagg cgtatacaat 1320
gcactgcaga aagacaagat ggcagaagcc tacagtgaga tcggcacaaa aggcgagagg 1380
cggagaggca aggggcacga tggcctttac cagggtctca gcactgccac caaggacacc 1440
tatgatgccc tgcatatgca gaccctggcc taggtcgact cacacaaaaa accaacacac 1500
agatgtaatg aaaataaaga tattttattc gtacgttagg cggagctcag atagcccatc 1560
accctgttga tggtcacgac gcgggtgctg aaggcgtgaa gcaggatgca gagcttcatt 1620
ttcactctgt aagggtctgc ttctcccaca ggaggtttct ggcgcagagt ctcgccatta 1680
tgattcagag actgcatcag ctcatcgatg gccaccagca tgcccttgtc tagaatgatc 1740
tgctgatggt tgtgattctg aagtgctgcg ttgatggcct ggaactctgt ctggtacatc 1800
ttcaagtcct catagatgct accaaggcac agggtcatca tcaaagacgt cttctgtggg 1860
ggcaggcagc tccctcttgt tgtggaagaa gtctctctag tagccaggca actctcgttc 1920
ttgtgtagtt ccagtggtaa acaggtcttc aatgtgctgg tttggtcccg tgtgatgtct 1980
tcatgatcga tgtcttcagc agtgcaggaa taatgtttca gtttttctct ggccgtcttc 2040
accatgtcat ctgtggtctt cagcaggttt cgggactggc taagacacct ggcaggtcca 2100
gagactggaa tgaccctaga tccgccgcca cccgacccac caccgcccga gccaccgcca 2160
ccggatcgga ccctgcaggg aacacatgcc cacttgctgc atgaggaatt gtaatagcga 2220
tcctgagctt gcacgcagac attcccgcct ttgcattgga cttcggtaga tgtcttctct 2280
acgaggaacg cacctttctg gttacacccc tcctctgtct ccttcatctt ttctttcttg 2340
cgctggattc gaacaaagaa cttgagggag aagtaggaat ggggagtgct ccaggagtca 2400
gggtactccc agctgacctc cacctgtgag ttcttcaaag gcttcatctg caagttcttg 2460
ggcgggtctg gtttgatgat gtccctgatg aagaagctgg tgctgtagtt ctcatattta 2520
ttctgctgcc gtgcttccaa cgccagttca atgggcaggg tctcctcggc agttgggcag 2580
gtgacatcct cctggcagga cactgaatac ttctcatagt ccctttggtc cagtgtgacc 2640
ttctctgcag acagagacgc cattccacat gtcactgccc gagagtcagg ggaactgcta 2700
ctgctcttga tgttgaactt caagtccatg tttctttgca ccagccatga gcacgtgaac 2760
cgtccggagt aatttggtgc ttcacacttc aggaaagtct tgtttttgaa attttttaaa 2820
atttcagtgg accaaattcc attttccttc ttgtggagca gcagatgtga gtggctcaga 2880
gtctcgcctc ctttgtggca ggtgtactgg ccagcatcta gaaactcttt gacagtgatg 2940
gtcagggtct ttccagagcc tatgactcca tgtctctggt ctgaggtcca ggtgatgtca 3000
tcttcttcag gcgtgtcaca ggtgaggttc actgtttctc caggggcatc gggagtccag 3060
tccacctcta caacataaac gtctttctcc agctcccaca tggccatgag tggagacacc 3120
agcaaaacga tggcaaacca ggagatggtt agcttctgag gacacatggt ggcgaccggt 3180
agcgctagga cgcgtaggac ttgaggtcac tgtgaggagt gattagcaag ggtgataggc 3240
agctcttcag catgggaggc aatttatact gttaatgctg gaaaaataat atgggggtgt 3300
cacgatgttc ccgggacaat tgaacgcctt ctgtatgaaa caaattttcc tcttaacgcc 3360
ttctgtatga aacaaatttt cctcttaacg ccttctgtat gaaacaaatt ttcctcttaa 3420
cgccttctgt atgaaacaaa ttttcctctt aacgccttct gtatgaaaca aattttcctc 3480
ttaacgcctt ctgtatgaaa caaattttcc tctt 3514
<210> 34
<211> 3526
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 34
atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60
atcctgggga gtggagaagc tgaggtgcag ctggtgcaga gcggcgccga ggtgaagaag 120
cccggcgcca gcgtgaaggt gagctgcaag gccagcggct acaccttcag cgactacgag 180
atgcactggg tgcggcaggc ccccggccag ggcctggagt ggatgggcgc catccacccc 240
ggcagcggcg acaccgccta caaccagcgg ttcaagggcc gggtgaccat caccgccgac 300
aagagcacca gcaccgccta catggagctg agcagcctgc ggagcgagga caccgccgtg 360
tactactgcg cccggttcta cagctacgcc tactggggcc agggcaccct ggtgaccgtg 420
agcgccggtg gaggcggttc aggcggaggt ggttctggcg gtggcggatc ggacatcgtg 480
atgacccaga cccccctgag cctgcccgtg acccccggcg agcccgccag catcagctgc 540
cggagcagcc agagcctggt gcacagcaac ggcaacacct acctgcagtg gtacctgcag 600
aagcccggcc agagccccca gctgctgatc tacaaggtga gcaaccggtt cagcggcgtg 660
cccgaccggt tcagcggcag cggcagcggc accgacttca ccctgaagat cagccgggtg 720
gaggccgagg acgtgggcgt gtactactgc agccagagca tctacgtgcc ctacaccttc 780
ggccagggca ccaagctgga gatcaaacgt actactacca agccagtgct gcgaactccc 840
tcacctgtgc accctaccgg gacatctcag ccccagagac cagaagattg tcggccccgt 900
ggctcagtga aggggaccgg attggacttc gcctgtgata tttacatctg ggcacccttg 960
gccggaatct gcgtggccct tctgctgtcc ttgatcatca ctctcatctg ctaccacagg 1020
agccgaaaat ggatcaggaa aaaattcccc cacatattca agcaaccatt taagaagacc 1080
actggagcag ctcaagagga agatgcttgt agctgccgat gtccacagga agaagaagga 1140
ggaggaggag gctatgagct gagcaggagt gcagagactg ctgccaacct gcaggacccc 1200
aaccagctct acaatgagct caatctaggg cgaagagagg aatatgacgt cttggagaag 1260
aagcgggctc gggatccaga gatgggaggc aaacagcaga ggaggaggaa cccccaggaa 1320
ggcgtataca atgcactgca gaaagacaag atggcagaag cctacagtga gatcggcaca 1380
aaaggcgaga ggcggagagg caaggggcac gatggccttt accagggtct cagcactgcc 1440
accaaggaca cctatgatgc cctgcatatg cagaccctgg cctaggtcga ctcacacaaa 1500
aaaccaacac acagatgtaa tgaaaataaa gatattttat tcgtacgtta ggcggagctc 1560
agatagccca tcaccctgtt gatggtcacg acgcgggtgc tgaaggcgtg aagcaggatg 1620
cagagcttca ttttcactct gtaagggtct gcttctccca caggaggttt ctggcgcaga 1680
gtctcgccat tatgattcag agactgcatc agctcatcga tggccaccag catgcccttg 1740
tctagaatga tctgctgatg gttgtgattc tgaagtgctg cgttgatggc ctggaactct 1800
gtctggtaca tcttcaagtc ctcatagatg ctaccaaggc acagggtcat catcaaagac 1860
gtcttctgtg ggggcaggca gctccctctt gttgtggaag aagtctctct agtagccagg 1920
caactctcgt tcttgtgtag ttccagtggt aaacaggtct tcaatgtgct ggtttggtcc 1980
cgtgtgatgt cttcatgatc gatgtcttca gcagtgcagg aataatgttt cagtttttct 2040
ctggccgtct tcaccatgtc atctgtggtc ttcagcaggt ttcgggactg gctaagacac 2100
ctggcaggtc cagagactgg aatgacccta gatccgccgc cacccgaccc accaccgccc 2160
gagccaccgc caccggatcg gaccctgcag ggaacacatg cccacttgct gcatgaggaa 2220
ttgtaatagc gatcctgagc ttgcacgcag acattcccgc ctttgcattg gacttcggta 2280
gatgtcttct ctacgaggaa cgcacctttc tggttacacc cctcctctgt ctccttcatc 2340
ttttctttct tgcgctggat tcgaacaaag aacttgaggg agaagtagga atggggagtg 2400
ctccaggagt cagggtactc ccagctgacc tccacctgtg agttcttcaa aggcttcatc 2460
tgcaagttct tgggcgggtc tggtttgatg atgtccctga tgaagaagct ggtgctgtag 2520
ttctcatatt tattctgctg ccgtgcttcc aacgccagtt caatgggcag ggtctcctcg 2580
gcagttgggc aggtgacatc ctcctggcag gacactgaat acttctcata gtccctttgg 2640
tccagtgtga ccttctctgc agacagagac gccattccac atgtcactgc ccgagagtca 2700
ggggaactgc tactgctctt gatgttgaac ttcaagtcca tgtttctttg caccagccat 2760
gagcacgtga accgtccgga gtaatttggt gcttcacact tcaggaaagt cttgtttttg 2820
aaatttttta aaatttcagt ggaccaaatt ccattttcct tcttgtggag cagcagatgt 2880
gagtggctca gagtctcgcc tcctttgtgg caggtgtact ggccagcatc tagaaactct 2940
ttgacagtga tggtcagggt ctttccagag cctatgactc catgtctctg gtctgaggtc 3000
caggtgatgt catcttcttc aggcgtgtca caggtgaggt tcactgtttc tccaggggca 3060
tcgggagtcc agtccacctc tacaacataa acgtctttct ccagctccca catggccatg 3120
agtggagaca ccagcaaaac gatggcaaac caggagatgg ttagcttctg aggacacatg 3180
gtggcgaccg gtagcgctag gacgcgtagg acttgaggtc actgtgagga gtgattagca 3240
agggtgatag gcagctcttc agcatgggag gcaatttata ctgttaatgc tggaaaaata 3300
atatgggggt gtcacgatgt tcccgggaca attgaacgcc ttctgtatga aacaaatttt 3360
cctcttaacg ccttctgtat gaaacaaatt ttcctcttaa cgccttctgt atgaaacaaa 3420
ttttcctctt aacgccttct gtatgaaaca aattttcctc ttaacgcctt ctgtatgaaa 3480
caaattttcc tcttaacgcc ttctgtatga aacaaatttt cctctt 3526
<210> 35
<211> 3649
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 35
atggcctcac cgttgacccg ctttctgtcg ctgaacctgc tgctgctggg tgagtcgatt 60
atcctgggga gtggagaagc tgaggtgcag ctggtgcaga gcggcgccga ggtgaagaag 120
cccggcgcca gcgtgaaggt gagctgcaag gccagcggct acaccttcag cgactacgag 180
atgcactggg tgcggcaggc ccccggccag ggcctggagt ggatgggcgc catccacccc 240
ggcagcggcg acaccgccta caaccagcgg ttcaagggcc gggtgaccat caccgccgac 300
aagagcacca gcaccgccta catggagctg agcagcctgc ggagcgagga caccgccgtg 360
tactactgcg cccggttcta cagctacgcc tactggggcc agggcaccct ggtgaccgtg 420
agcgccggtg gaggcggttc aggcggaggt ggttctggcg gtggcggatc ggacatcgtg 480
atgacccaga cccccctgag cctgcccgtg acccccggcg agcccgccag catcagctgc 540
cggagcagcc agagcctggt gcacagcaac ggcaacacct acctgcagtg gtacctgcag 600
aagcccggcc agagccccca gctgctgatc tacaaggtga gcaaccggtt cagcggcgtg 660
cccgaccggt tcagcggcag cggcagcggc accgacttca ccctgaagat cagccgggtg 720
gaggccgagg acgtgggcgt gtactactgc agccagagca tctacgtgcc ctacaccttc 780
ggccagggca ccaagctgga gatcaaacgt actactacca agccagtgct gcgaactccc 840
tcacctgtgc accctaccgg gacatctcag ccccagagac cagaagattg tcggccccgt 900
ggctcagtga aggggaccgg attggacttc gcctgtgata tttacatctg ggcacccttg 960
gccggaatct gcgtggccct tctgctgtcc ttgatcatca ctctcatctg ctaccacagg 1020
agccgaaata gtagaaggaa cagactcctt caaagtgact acatgaacat gactccccgg 1080
aggcctgggc tcactcgaaa gccttaccag ccctacgccc ctgccagaga ctttgcagcg 1140
taccgcccca aatggatcag gaaaaaattc ccccacatat tcaagcaacc atttaagaag 1200
accactggag cagctcaaga ggaagatgct tgtagctgcc gatgtccaca ggaagaagaa 1260
ggaggaggag gaggctatga gctgagcagg agtgcagaga ctgctgccaa cctgcaggac 1320
cccaaccagc tctacaatga gctcaatcta gggcgaagag aggaatatga cgtcttggag 1380
aagaagcggg ctcgggatcc agagatggga ggcaaacagc agaggaggag gaacccccag 1440
gaaggcgtat acaatgcact gcagaaagac aagatggcag aagcctacag tgagatcggc 1500
acaaaaggcg agaggcggag aggcaagggg cacgatggcc tttaccaggg tctcagcact 1560
gccaccaagg acacctatga tgccctgcat atgcagaccc tggcctaggt cgactcacac 1620
aaaaaaccaa cacacagatg taatgaaaat aaagatattt tattcgtacg ttaggcggag 1680
ctcagatagc ccatcaccct gttgatggtc acgacgcggg tgctgaaggc gtgaagcagg 1740
atgcagagct tcattttcac tctgtaaggg tctgcttctc ccacaggagg tttctggcgc 1800
agagtctcgc cattatgatt cagagactgc atcagctcat cgatggccac cagcatgccc 1860
ttgtctagaa tgatctgctg atggttgtga ttctgaagtg ctgcgttgat ggcctggaac 1920
tctgtctggt acatcttcaa gtcctcatag atgctaccaa ggcacagggt catcatcaaa 1980
gacgtcttct gtgggggcag gcagctccct cttgttgtgg aagaagtctc tctagtagcc 2040
aggcaactct cgttcttgtg tagttccagt ggtaaacagg tcttcaatgt gctggtttgg 2100
tcccgtgtga tgtcttcatg atcgatgtct tcagcagtgc aggaataatg tttcagtttt 2160
tctctggccg tcttcaccat gtcatctgtg gtcttcagca ggtttcggga ctggctaaga 2220
cacctggcag gtccagagac tggaatgacc ctagatccgc cgccacccga cccaccaccg 2280
cccgagccac cgccaccgga tcggaccctg cagggaacac atgcccactt gctgcatgag 2340
gaattgtaat agcgatcctg agcttgcacg cagacattcc cgcctttgca ttggacttcg 2400
gtagatgtct tctctacgag gaacgcacct ttctggttac acccctcctc tgtctccttc 2460
atcttttctt tcttgcgctg gattcgaaca aagaacttga gggagaagta ggaatgggga 2520
gtgctccagg agtcagggta ctcccagctg acctccacct gtgagttctt caaaggcttc 2580
atctgcaagt tcttgggcgg gtctggtttg atgatgtccc tgatgaagaa gctggtgctg 2640
tagttctcat atttattctg ctgccgtgct tccaacgcca gttcaatggg cagggtctcc 2700
tcggcagttg ggcaggtgac atcctcctgg caggacactg aatacttctc atagtccctt 2760
tggtccagtg tgaccttctc tgcagacaga gacgccattc cacatgtcac tgcccgagag 2820
tcaggggaac tgctactgct cttgatgttg aacttcaagt ccatgtttct ttgcaccagc 2880
catgagcacg tgaaccgtcc ggagtaattt ggtgcttcac acttcaggaa agtcttgttt 2940
ttgaaatttt ttaaaatttc agtggaccaa attccatttt ccttcttgtg gagcagcaga 3000
tgtgagtggc tcagagtctc gcctcctttg tggcaggtgt actggccagc atctagaaac 3060
tctttgacag tgatggtcag ggtctttcca gagcctatga ctccatgtct ctggtctgag 3120
gtccaggtga tgtcatcttc ttcaggcgtg tcacaggtga ggttcactgt ttctccaggg 3180
gcatcgggag tccagtccac ctctacaaca taaacgtctt tctccagctc ccacatggcc 3240
atgagtggag acaccagcaa aacgatggca aaccaggaga tggttagctt ctgaggacac 3300
atggtggcga ccggtagcgc taggacgcgt aggacttgag gtcactgtga ggagtgatta 3360
gcaagggtga taggcagctc ttcagcatgg gaggcaattt atactgttaa tgctggaaaa 3420
ataatatggg ggtgtcacga tgttcccggg acaattgaac gccttctgta tgaaacaaat 3480
tttcctctta acgccttctg tatgaaacaa attttcctct taacgccttc tgtatgaaac 3540
aaattttcct cttaacgcct tctgtatgaa acaaattttc ctcttaacgc cttctgtatg 3600
aaacaaattt tcctcttaac gccttctgta tgaaacaaat tttcctctt 3649

Claims (22)

1. a kind of immune response cell, which is characterized in that the receptor and exogenous IL12 of cell expression specific bond GPC3.
2. immune response cell as described in claim 1, which is characterized in that the receptor and the IL12 operationally connect It connects.
3. immune response cell as described in claim 1, which is characterized in that the immune response cell include: T cell, Natural killer cells, cytotoxic T lymphocyte, natural killer T cells, DNT cell, and/or regulatory T cells.
4. immune response cell as described in claim 1, which is characterized in that the exogenous IL12 be constructive expression or Inducible expression；
Preferably, the promoter for expressing the IL12 includes: immunocyte inducible promoter；Preferably, described Immunocyte inducible promoter is NFAT6 promoter.
5. the immune response cell as described in one of preceding claims, which is characterized in that the receptor has specific bond Extracellular domain, transmembrane domain and the intracellular signal domain of GPC3.
6. immune response cell as claimed in claim 5, which is characterized in that the receptor is Chimeric antigen receptor, wherein institute State the combination that Intracellular domain contains cytositimulation signaling molecule or cytositimulation signaling molecule and cell-stimulating costimulatory molecules；
Preferably, the cytositimulation signaling molecule is selected from: CD3 ζ, CD3 γ, CD3 δ, CD3 ε, Fc ε RI γ, FcR β, CD79a, The functional signal conducting structure domain of the protein of CD79b, Fc γ RIIa, DAP10 or DAP12；It is more preferably CD3 ζ；Or
Preferably, the functional signal conducting structure domain of the cell-stimulating costimulatory molecules protein selected from the following: CD27, CD28, CD137, CD134, ICOS, OX40, CD30, CD40, PD-1, lymphocyte function-associated antigen-1 (LFA-1), CD2, CD7, LIGHT, NKG2C, B7-H3, the ligand of specific bond CD83, CDS, ICAM-1, GITR, BAFFR, HVEM (LIGHTR), SLAMF7, NKp80 (KLRF1), CD160, CD19, CD4, CD8 α, CD8 β, IL2R β, IL2R γ, IL7R α, 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, 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, NKp44, NKp30, NKp46 and NKG2D are more preferably CD27, CD28, CD137, CD134, ICOS.
7. such as immune response cell described in claim 5 or 6, which is characterized in that the extracellular domain of the receptor contains and SEQ Amino acid sequence shown in ID NO:25 has the amino acid of at least 90%, 95%, 96%, 97%, 98% or 99% identity Sequence.
8. the immune response cell as described in one of preceding claims, which is characterized in that the receptor contains SEQ ID NO: 21, amino acid sequence shown in 22,23 or 24；Or contain and amino acid sequence shown in SEQ ID NO:21,22,23 or 24 Arrange the amino acid sequence of the identity at least 90%, 95%, 96%, 97%, 98% or 99%.
9. immune response cell as claimed in claim 8, which is characterized in that the receptor and the exogenous IL12 by with SEQ ID NO:28,29,30,31,32,33,34 or 35 have at least 90%, 95%, 96%, 97%, 98% or 99% same Property it is nucleotide sequence coded；
Preferably, same at least 90%, 95%, 96%, 97%, 98% or 99% with SEQ ID NO:28,29,30 or 31 One property it is nucleotide sequence coded；
It is furthermore preferred that nucleotide sequence coded as shown in SEQ ID NO:28,29,30 or 31.
10. the immune response cell as described in one of preceding claims, which is characterized in that the immune response cell does not wrap Costimulation ligand containing external source.
11. the immune response cell as described in one of preceding claims, which is characterized in that the receptor and/or the IL12 Composition or inducible expression are on the surface of immune response cell.
12. the immune response cell as described in one of preceding claims, which is characterized in that wrapped in the immune response cell Containing expression construct, which includes: the expression cassette of the receptor of the combination antigen；With the expression cassette of the IL12.
13. the immune response cell as described in one of preceding claims, which is characterized in that the receptor and/or IL12 benefit It is expressed with viral vectors；Preferably, the viral vectors includes: slow virus carrier, retroviral vector or adenovirus are carried Body.
14. the immune response cell as described in one of claim 1 to 13, wherein the immune response cell is giving individual Afterwards, compared with the case where exogenous IL12 is not present, CAR-T cell quantity is improved at least in a peripheral body 50%.
15. the immune response cell as described in one of claim 1 to 13, wherein the immune response cell is giving individual about After 7 days, the sum of quantity of CAR-T cell is greater than 6,000/μ L in a peripheral body；Give the immune response cell After about 10 days, the sum of quantity of CAR-T cell is greater than 6000/μ L in a peripheral body.
16. a kind of expression construct, which is characterized in that the expression construct include be linked in sequence: the expression cassette of receptor and The expression cassette of IL12；Wherein one of the receptor of the combination antigen and IL12 such as preceding claims are defined.
17. tumour of the immune response cell described in one of claim 1-13 in preparation for treating individuals in need Purposes in pharmaceutical composition.
18. purposes as claimed in claim 17, which is characterized in that the tumour includes: liver cancer, gastric cancer, lung cancer, mammary gland Cancer, head and neck cancer, bladder cancer, oophoroma, cervical carcinoma, kidney, cancer of pancreas, cervical carcinoma, embryonal-cell lipoma, melanoma, adrenal, Neurinoma, malignant fibrous histiocytoma, cancer of the esophagus；Preferably, the tumour is liver cancer, gastric cancer, lung cancer, breast cancer.
19. the purposes as described in claim 17 or 18, wherein the immune effector cell makes tumour be reduced at least 50%.
20. purposes as claimed in claim 19, wherein the immune effector cell makes tumour be reduced at least 70%, it is more excellent , the immune effector cell makes tumour be reduced at least 80%.
21. pharmaceutical composition, which is characterized in that the pharmaceutical composition includes:
Immune response cell described in one of claim 1-13；And
Pharmaceutically acceptable carrier or excipient.
22. a kind of kit, it includes:
Immune response cell as described in one of claim 1-13；And
How guidance gives the specification of the immune response cell to individual.
CN201811046090.2A 2017-09-08 2018-09-07 Target immune effector cell and its application of GPC3 Pending CN109468279A (en)

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CN108884459A (en) * 2016-04-26 2018-11-23 科济生物医药（上海）有限公司 A method of improving immune response cell function
CN108884459B (en) * 2016-04-26 2024-04-02 科济生物医药（上海）有限公司 Method for improving immune response cell function
WO2020118634A1 (en) * 2018-12-13 2020-06-18 科济生物医药（上海）有限公司 Immune effector cell targeting gpc3 and application thereof
WO2022028623A1 (en) 2020-08-07 2022-02-10 佧珐药业有限公司 Engineered cells and method for engineering cells
CN111995688A (en) * 2020-08-13 2020-11-27 金鑫 Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligands and application thereof
CN111995688B (en) * 2020-08-13 2024-01-16 金鑫 Bispecific chimeric antigen receptor targeting CD123 and NKG2D ligand and application thereof
CN114231559A (en) * 2020-09-09 2022-03-25 广西医科大学 Expression plasmid containing CAR nucleic acid fragment, CD 105-targeted CAR-T cell containing expression plasmid, and preparation method and application thereof
CN112210015A (en) * 2020-09-16 2021-01-12 汕头普罗凯融生物医药科技有限公司 GPC 3-targeted CAR and CAR-NK cell using same
CN112210015B (en) * 2020-09-16 2022-08-12 汕头普罗凯融生物医药科技有限公司 GPC 3-targeted CAR and CAR-NK cell using same
CN112225822A (en) * 2020-12-14 2021-01-15 北京基因启明生物科技有限公司 CAR-iNKT with high amplification, survival ability and tumor killing effect and application thereof
CN112225822B (en) * 2020-12-14 2021-03-23 北京基因启明生物科技有限公司 CAR-iNKT with high amplification, survival ability and tumor killing effect and application thereof
CN113072643B (en) * 2021-03-22 2021-10-15 南京医科大学 anti-Glyphacin-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application
CN113072643A (en) * 2021-03-22 2021-07-06 南京医科大学 anti-Glyphacin-3 acid-resistant fully human antibody, immunotoxin thereof, chimeric antigen receptor cell thereof and application
WO2022214089A1 (en) 2021-04-08 2022-10-13 克莱格医学有限公司 Cellular immunotherapy use
WO2023274303A1 (en) 2021-06-29 2023-01-05 科济生物医药（上海）有限公司 Chimeric polypeptide for regulating cell physiological activity
CN114907487A (en) * 2021-11-26 2022-08-16 郑州大学 Novel chimeric antigen receptor and immune cell comprising same

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