CN108794642A - A kind of chimeric antigen cell receptor and its application - Google Patents

Abstract

The invention discloses a kind of chimeric antigen cell receptor and its applications, are related to biotechnology.The chimeric antigen cell receptor includes antigen-binding domains and the α chains being connect respectively with antigen-binding domains and β chains, and antigen-binding domains are the Fab segments of antibody, and antibody has the characteristic specifically bound with target proteins.Expressing the immunocyte of the chimeric antigen cell receptor can be combined with the target proteins on target cell, transmit activation signal and immune cell activated, and to be killed to the target cell for expressing target proteins, and the process does not have to rely on MHC molecule submission target proteins；The chimeric antigen cell receptor can be applied to prepare corresponding tumour medicine, the fields such as removing, treating autoimmune diseases for malignant cell.

Description

A kind of chimeric antigen cell receptor and its application

Technical field

The present invention relates to biotechnologies, in particular to a kind of chimeric antigen cell receptor and its application.

Background technology

What T cell antigen receptor (TCR) identification in the mammalian body was combined with major histocompatibility complex (MHC) Albumen peptide fragment, these albumen peptide fragment-MHC compounds are usually illustrated in different types of abnormal cell film surface, these are derived from Polypeptide-MHC the compounds of pathogen are on internal abnormal cell from tumor neogenetic Antigenic Peptide-MHC compounds Molecular marked compound.

Nave T cell receptor (TCR) is the antigentic specificity transmembrane complex of a multi-subunit composition, can be with mediate antigen Specific activation T cell.TCR by two different polypeptide chains form (such as Fig. 1 schematic diagrames), T cell receptor α chains and T cell by Body β chains.Two chains form one respectively there are one the variable region (areas V) of N-terminal and the constant region (areas C) of a C-terminal between two chains A disulfide bond.TCR assigns T cell antigen specificity, the antigen polypeptide-by identifying antigen polypeptide-MHC compounds MHC compounds include that major histocompatibility complex presents protein short chain amino acid sequence (such as Fig. 2 on target cell Schematic diagram).

TCR identifies that the Antigenic Peptide needs while contacting MHC molecule and Antigenic Peptide two parts.With by MHC class molecular presentations Antigenic Peptide contact after, inmature CD8+ cytotoxic T cells robust proliferation simultaneously obtain specificity antigenic phenotype and functional characteristic, Effector T cell is served as, and expression target antigen is eliminated by inducing target cell apoptosis or itself release cracking particle Tumour cell or other abnormal cells.TCR receptors have the specificity of height, the TCR spontaneously formed under usual native state by Body affinity is generally relatively low, and is limited by MHC molecule type with the identification process of proteantigen peptide-MHC compounds, and mesh It is preceding identified come people's MHC molecule type be more than 300 kinds, as a consequence it is hardly possible to for each MHC molecule and it is corresponding resist As soon as former peptide screens a special TCR, this seriously limits application of the TCR receptors in adoptive immunity cell therapy field.

In consideration of it, special propose the present invention.

Invention content

The purpose of the present invention is to provide a kind of artificial reconstructed chimeric antigen cell receptor, the chimeric antigen cell receptors It can not be limited, can directly be combined with corresponding target proteins by MHC molecule.

Another object of the present invention is to provide a kind of nucleic acid of separation.

Another object of the present invention is to provide a kind of carriers.

Another object of the present invention is to provide a kind of cells.

Another object of the present invention is to provide a kind of compositions.

Another object of the present invention is to provide the applications of the nucleic acid of above-mentioned separation, carrier and cell.

The invention is realized in this way：

A kind of chimeric antigen cell receptor comprising antigen-binding domains and respectively with above-mentioned antigen binding structure The α chains and β chains of domain connection, above-mentioned antigen-binding domains are the Fab segments of antibody, and above-mentioned antibody has special with target proteins Property combine characteristic.

Further, in some embodiments of the present invention, above-mentioned target proteins be selected from CD19, CD22, CD20, Any one in BCMA, CD117, CD123, CD33, EGFRvIII, CD276, CLL1, GPC3 and Claudin18.2.

But it should be recognized that the target proteins of the present invention are not limited to above-mentioned antigen protein, can also be others Albumen, as select which kind of albumen as antigen, can be determined according to actual demand.It will be for the arbitrary of arbitrary protein antigen The Fab segments of antibody, which are applied in the present invention, to be all belonged to the scope of protection of the present invention.

Further, in some embodiments of the present invention, above-mentioned antibody comes for the monoclonal antibody in mouse source, rabbit The monoclonal antibody in source, the single domain antibody of camelid origin, the single domain antibody in shark source, humanized antibody or human antibody.

Certainly, it should be noted that the source of antibody is not limited to above-mentioned source, can be the anti-of any animal source Body.

Further, in some embodiments of the present invention, the amino acid sequence of the heavy chain variable region of Fab segments is such as Shown in SEQ ID NO.12；The amino acid sequence of the light chain variable region of Fab segments is as shown in SEQID NO.15.

The antigen binding that the light chain variable region of heavy chain variable region and SEQ ID NO.15 with SEQ ID NO.12 is formed Structural domain can target people's CD19 albumen.

Further, in some embodiments of the present invention, the amino acid sequence of the heavy chain constant region of above-mentioned Fab segments As shown in SEQ ID NO.13.

Further, in some embodiments of the present invention, the amino acid sequence of the constant region of light chain of above-mentioned Fab segments As shown in SEQ ID NO.16.

The study find that if chimeric antigen cell receptor provided by the invention lack heavy chain constant region (CH1) and The function of the chimeric antigen cell receptor is severely impacted after constant region of light chain (CL), the combination energy of loss and target antigen Power.

Further, in some embodiments of the present invention, above-mentioned α chains by be linked in sequence hinge area, transmembrane region with And intracellular activity signal domain is constituted, hinge area, transmembrane region and the intracellular activity signal domain of above-mentioned α chains are respectively to feed Hinge area (Hinge), transmembrane region (transmembrane) and the intracellular activity signal structure of newborn zooblast T cell receptor α chains Domain (intracellular activation domain).

Preferably, the amino acid sequence of above-mentioned α chains is as shown in SEQ ID NO.14.

Further, in some embodiments of the present invention, above-mentioned β chains by be linked in sequence hinge area, transmembrane region with And intracellular activity signal domain is constituted, hinge area, transmembrane region and the intracellular activity signal domain of above-mentioned β chains are respectively to feed Hinge area, transmembrane region and the intracellular activity signal domain of newborn zooblast T cell receptor β chains.

Preferably, the amino acid sequence of above-mentioned β chains is as shown in SEQ ID NO.17.

Certainly, needing to illustrate in addition, in some embodiments of the present invention scheme, the hinge area of α chains or the hinge of β chains Area can be arbitrary flexible polypeptide sequence.

Further, in some embodiments of the present invention, above-mentioned cell is immunocyte.

Further, in some embodiments of the present invention, above-mentioned immunocyte includes but not limited to T cell, NK thin Born of the same parents, granulocyte, B cell etc..

It should be noted that in some embodiments of the present invention, α chains connect i.e. α with the heavy chain constant region of Fab segments The N-terminal of chain is connect with the C-terminal of the heavy chain constant region of Fab segments, and such heavy chain variable region, heavy chain constant region, α chains constitute first Chain.

β chains connect the i.e. N-terminal of β chains with the constant region of light chain of Fab segments and are connect with the C-terminal of the constant region of light chain of Fab segments, Light chain variable region, constant region of light chain and β chains constitute second and connect in this way.

Alternatively, in other embodiments of the present invention, α chains and the constant region of light chain of Fab segments connect and compose first Chain, β chains and the heavy chain constant region of Fab segments connect and compose the second chain.That is, the C of the N-terminal of α chains and the constant region of light chain of Fab segments End connection, the N-terminal of β chains are connect with the C-terminal of the heavy chain constant region of Fab segments.

Further, in some embodiments of the present invention, the amino acid sequence of above-mentioned chimeric antigen cell receptor is such as Shown in SEQ ID NO.19.Chimeric antigen cell receptor can specifically bind CD19 albumen shown in the SEQ ID NO.19.

Antibody is a kind of immunoglobulin that can be combined with antigentic specificity, is made of four heterologous polypeptide chains, each peptide Connected by interchain disulfide bond in varying numbers between chain, wherein two larger chains of molecular weight be known as heavy chain (heavy chain, HC), and smaller two chains of molecular weight are known as light chain (Light chain, LC).Two heavy chains and two in same antibody molecule The amino acid of light chain forms identical (such as Fig. 3 schematic diagrames), wherein each to heavy chain variable region (VH) and corresponding CH1 perseverances Determine to connect by disulfide bond between area and light chain variable region (VL) and corresponding constant region CL, the antigen-binding fragment of formation (Fab) there is the characteristic (such as Fig. 4 schematic diagrames) that is combined with antigen protein, and Fab structural stabilities and to target antigen Affinity by the single-chain antibody (scFv) of heavy chain variable region and the direct amalgamation and expression of light chain variable region all than more having significant advantage.

Present invention incorporates nave T cell receptors in immune cell activation and the internal abnormal cell of removing and pathogen The stability of the antigen-binding fragment (Fab) of effect and antibody and its affine force characteristic to target antigen, that constructs is embedding Cell antigen receptor (such as Fig. 5 schematic diagrames) is closed, which is the Fab pieces based on monoclonal antibody to the identification of targeting antigen Section, so the identification process of the artificial Chimerical receptor and target antigen is different from nave T cell receptor, it is not multiple by Antigenic Peptide-MHC Close the limitation of object.

The combination of the chimeric antigen cell receptor and target proteins is not limited by MHC types, the knot with target antigen It is as shown in Figure 6 to close schematic diagram.The chimeric antigen cell receptor can be expressed by arbitrary eukaryotic expression vector, can be with By the methods of slow virus, retrovirus, adenovirus, liposome transfection, electrotransfection, the chimeric antigen cell receptor will be carried Include but not limited to T cell, NK cells, grain in the DNA fragmentation or plasmid transfection to a plurality of types of immunocytes of encoding gene Cell, B cell etc..It expresses after the immunocyte of the chimeric antigen cell receptor combined with the target antigen on target cell, Ke Yichuan Activation signal and immune cell activated are passed, to be killed to the target cell for expressing target antigen；This method can be applied to pernicious The fields such as removing, the treating autoimmune diseases of tumour cell.

On the other hand, the present invention provides a kind of nucleic acid of separation, encode chimeric antigen cell receptor as described above.

Further, in some embodiments of the present invention, the heavy chain containing coding Fab segments in the nucleic acid of the separation The nucleic acid sequence of variable region (SEQ ID NO.12) also contains coding Fab pieces such as SEQ ID NO.23 in the nucleic acid of the separation The nucleic acid sequence of the light chain variable region (SEQ ID NO.15) of section, such as SEQ ID NO.24.

Further, in some embodiments of the present invention, the heavy chain containing coding Fab segments in the nucleic acid of the separation The nucleic acid sequence of constant region (SEQ ID NO.13), the nucleic acid sequence such as SEQ ID NO.25.

Further, in some embodiments of the present invention, the light chain containing coding Fab segments in the nucleic acid of the separation The nucleic acid sequence of constant region (SEQ ID NO.16), the nucleic acid sequence such as SEQ ID NO.26.

Further, in some embodiments of the present invention, coding for alpha chain (SEQ ID are contained in the nucleic acid of the separation NO.14 nucleic acid sequence), the nucleic acid sequence such as SEQ ID NO.27.

Further, in some embodiments of the present invention, coding β chains (SEQ ID are contained in the nucleic acid of the separation NO.17 nucleic acid sequence), the nucleic acid sequence such as SEQ ID NO.28.

Further, in some embodiments of the present invention, also above-mentioned embedding containing being useful for driving in the nucleic acid of the separation Close the promoter of cell antigen expression of receptor.

It should be noted that above-mentioned the first chain and the second chain can also may be used by two independent promoter driving expression With with T2A (amino acid sequence be SEQ ID NO.1, nucleic acid sequence be SEQ ID NO.29), P2A (amino acid sequence SEQ ID NO.2, nucleic acid sequence are SEQ ID NO.30), (amino acid sequence is SEQ ID NO.3 to F2A, and nucleic acid sequence is SEQ ID NO.31), (nucleic acid sequence is by E2A (amino acid sequence be SEQ ID NO.4, nucleic acid sequence is SEQ ID NO.32) or IRES SEQ ID NO.22) etc. similar polypeptide connection, driven and expressed by the same promoter.

Further, in some embodiments of the present invention, above-mentioned promoter is that (nucleic acid sequence is such as EF1a promoters Shown in SEQ ID NO.5), CMV promoter (nucleic acid sequence be SEQ ID NO.6 shown in), pGK promoters (nucleic acid sequence SEQ Shown in ID NO.7), SV40 promoters (nucleic acid sequence be SEQ ID NO.8 shown in) or CBH promoters (nucleic acid sequence SEQ Shown in ID NO.9).

Further, in some embodiments of the present invention, the also core containing encoded signal peptide in the nucleic acid of the separation Acid sequence.

The signal peptide sequence can be the signal peptide of arbitrary transmembrane protein or secretory protein.For example, it may be the letter of CD8a Signal peptide (the amino acid sequence of number peptide (amino acid sequence is SEQ ID NO.10, nucleic acid sequence SEQ ID NO.33) or GMCSF For SEQ ID NO.11, nucleic acid sequence SEQ ID NO.34).

Further, in some embodiments of the present invention, the nucleic acid sequence of the nucleic acid of the separation such as SEQ ID Shown in NO.18, the chimeric antigen cell receptor as shown in SEQ ID NO.19 is encoded.The nucleic acid detached shown in SEQ ID NO.18 Expression element composition schematic diagram it is as shown in Figure 7.

Further, in some embodiments of the present invention, the nucleic acid sequence of the nucleic acid of the separation such as SEQ ID Shown in NO.20, the chimeric antigen cell receptor as shown in SEQ ID NO.21 is encoded.

Compared to chimeric antigen cell receptor shown in SEQ ID NO.19, chimeric antigen cell shown in SEQ ID NO.21 by Body lacks heavy chain constant region and constant region of light chain.

The study find that should after lacking heavy chain of antibody CH1 constant regions and light chain CL constant regions in chimeric antigen cell receptor The function of chimeric antigen cell receptor is severely impacted, and loses the binding ability with target antigen.

On the other hand, the present invention provides a kind of carrier, contain the nucleic acid detached as described above.

In some embodiments of the present invention, which is eukaryotic expression vector, such as can be slow virus load Body, adenovirus vector, gland relevant viral vector, herpesvirus vector etc..

Certainly, which can also be other kinds of carrier.In actual operation, carrier type can be according to reality Border demand selection, all belongs to the scope of protection of the present invention.

On the other hand, the present invention provides a kind of cell, contain the nucleic acid detached as described above and expression has as above The chimeric antigen cell receptor.

In some embodiments of the present invention, which is immunocyte.

In some embodiments of the present invention, above-mentioned immunocyte is in T cell, NK cells, granulocyte and B cell Any one.

Certainly, it should be noted that it is thin that the type of above-mentioned immunocyte is not limited to T cell, NK cells, granulocyte or B Born of the same parents can also be other kinds of immunocyte, either what type of cell, as long as its expression has as described above be fitted into Cell antigen receptor, all belongs to the scope of protection of the present invention.

Express chimeric antigen cell receptor as described above immunocyte combined with the target proteins on target cell after, can be with Activation signal and immune cell activated are transmitted, to be killed to the target cell for expressing target proteins, and without relying on MHC points Sub- submission target proteins；The cell can be applied to prepare corresponding tumour medicine, for malignant cell removing, itself exempt from The fields such as epidemic disease disease treatment.

On the other hand, the present invention provides a kind of compositions comprising cell as described above and pharmaceutically acceptable Auxiliary material.

On the other hand, the present invention provides the nucleic acid detached as described above, carriers as described above or as described above Application of the cell in preparing the drug for the treatment of tumour or autoimmune disease.

For example, tumour can be fibrosarcoma, myxosarcoma, embryonal-cell lipoma, chondrosarcoma, osteosarcoma and other sarcomas, Synovialoma, celiothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancy, cancer of pancreas, mammary gland Cancer, lung cancer, oophoroma, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal-cell carcinoma, gland cancer, syringocarcinoma, medullary thyroid sample Cancer, thyroid papillary carcinoma, pheochromocytoma, carcinoma of sebaceous glands, papillary carcinoma, papillary adenocarcinoma, cephaloma, bronchiolar carcinoma, kidney Cell cancer, hepatoma, cholangiocarcinoma, choriocarcinoma, wilms' tumor, cervical carcinoma, orchioncus, seminoma, carcinoma of urinary bladder, Melanoma, glioma, spongioblastoma, astrocytoma, CNS lymthomas, gonioma, medulloblast Tumor, neurinoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neurinoma, oligodendroglioma, Meningioma, neuroblastoma or retinoblastoma etc..

For example, autoimmune disease can be chronic lymphocytic thyroiditis, hyperthyroidism, insulin-dependent Diabetes, myasthenia gravis, chronic ulcerative colitis, pernicious anaemia with atrophic gastritis, Goodpasture's syndrome, Pemphigus vulgaris, pemphigoid, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis Deng；Also Autoimmune diseases such as systemic loupus erythematosus, mouth xerophthalmia scheorma syndrome, rheumatoid arthritis, tatanic Rachitis, chorionitis, nodular polyarteritis or Wegener granulomatosis etc..

Description of the drawings

In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this A little attached drawings obtain other relevant attached drawings.

Fig. 1 is nave T cell receptor structure schematic diagram.

Fig. 2 is nave T cell Receptor recognition target cell antigen peptide/MHC compound schematic diagrames.

Fig. 3 is the structural schematic diagram of antibody.

Fig. 4 is antigen-binding fragment (Fab) schematic diagram of antibody.

Fig. 5 is the structural schematic diagram of chimeric antigen cell receptor provided by the invention.

Fig. 6 is the schematic diagram of chimeric antigen cell receptor provided by the invention and target proteins identification.

Fig. 7 is the main Expression element structure of the nucleic acid sequence of the encoding chimeric antigen cell receptor in the embodiment of the present invention Schematic diagram.

Fig. 8 is the structural representation of the Lentiviral of the expression chimeric antigen cell receptor in the embodiment of the present invention Figure.

Fig. 9 is the chimeric antigen cell receptor T cell and target antigen CD19 of the T cell film surface in experimental example 1 of the present invention FACS experimental results.

Figure 10 is that the T cell of the expression chimeric antigen cell receptor in experimental example 1 of the present invention swells to expression target antigen The lethal effect result of oncocyte.

Figure 11 is the secreting, expressing water of the T cell interleukin-22 of the expression chimeric antigen cell receptor in experimental example 1 of the present invention It is flat.

Figure 12 is the secreting, expressing of the T cell interferon of the expression chimeric antigen cell receptor in experimental example 1 of the present invention It is horizontal.

Figure 13 is killing target cell in the T cell Mice Body of the expression chimeric antigen cell receptor in experimental example 1 of the present invention Effect.

Figure 14 is that the detection of interleukin-22 is expressed in the Jurkat cell strain of expression Chimeric antigen receptor in experimental example 2 of the present invention As a result.

Specific implementation mode

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, builds according to normal condition or manufacturer The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

The feature and performance of the present invention are described in further detail with reference to embodiments.

Embodiment 1

The present embodiment is for targeting people's CD19 albumen, to the structure of the artificial chimeric antigen cell receptor of the present embodiment It illustrates, it is as follows：

The amino acid sequence of the chimeric antigen cell receptor is as shown in SEQ ID NO.19.

The chimeric antigen cell receptor includes antigen-binding domains and is connect respectively with the antigen-binding domains α chains and β chains, antigen-binding domains be anti-CD19 protein antibodies Fab segments, the anti-CD19 protein antibodies have and target Mark the characteristic of PROTEIN C D19 specific bindings.

Wherein, α chains are connect with the heavy chain constant region of Fab segments, constitute the first chain, the first chain includes the heavy chain of Fab segments Variable region (SEQ ID NO.12), Fab segments humanization heavy chain CH1 constant regions (SEQ ID NO.13), from T cell by The hinge areas of body α chains, transmembrane region and intracellular activity region (SEQ ID NO.14, i.e., the α of chimeric antigen cell receptor of the present invention Chain)；

β chains are connect with the constant region of light chain of Fab segments, constitute the second chain, and the second chain includes identifying that the light chain of Fab segments can Become area (SEQ ID NO.15), Fab segments humanization anti-light chain CL constant regions (SEQ ID NO.16), from T cell by The hinge areas of body β chains, transmembrane region and intracellular activity region (SEQ ID NO.17, i.e., the β of chimeric antigen cell receptor of the present invention Chain).

The present embodiment additionally provides the nucleic acid sequence for encoding above-mentioned chimeric antigen cell receptor, such as SEQ ID NO.18 Shown, the Expression element which contains is as shown in fig. 7, it includes successively from upstream toward downstream：Signal peptide-weight chain variable Hinge area, transmembrane region and the intracellular activity region -2A peptides-signal peptide-light chain variable of area-heavy chain constant region-T cell receptor α chains Hinge area, transmembrane region and the intracellular activity region of area-constant region of light chain-T cell receptor β chains.

The nucleic acid sequence encoding (shown in SEQ ID NO.18) of above-mentioned chimeric antigen cell receptor is subjected to full genome synthesis Afterwards, using EcoRI/KpnI restriction enzymes restriction enzyme site by the nucleic acid sequence encoding of the above-mentioned chimeric antigen cell receptor of synthesis It is cloned into Lentiviral LV6-EF1a-puroGFP, obtains and carry above-mentioned chimeric antigen cell receptor coded sequence Lentiviral LV6-EF1a-CAFabR-puroGFP, structure are as shown in Figure 8.

Embodiment 2

It is prepared by chimeric antigen cell receptor slow virus

(1) HEK293T cells are taken out from liquid nitrogen container, and are put into rapidly in 37 DEG C of water-baths and are thawed.In Biohazard Safety Equipment It is interior, the cell suspension in pipe is added into the 15mL centrifuge tubes containing 5mL culture mediums with pipettor, overturns mixing, the rooms 250xg Temperature centrifugation 10 minutes.

(2) after centrifuging, 5mL complete mediums (DMEM, 10%FBS) is added, gently blow and beat cell mass, it then will be thin Born of the same parents' suspension is transferred in 10cm culture dishes, supplement complete medium (DMEM, 10%FBS) to final volume 10mL.Culture dish is set In 37 DEG C, 5%CO₂It is incubated overnight in incubator.

(3) liposome/DNA compounds are prepared.It takes in 500uL PBS to sterile centrifugation tube, is separately added into 22 μ g LV6- EF1a-CAFabR-puroGFP, 12 μ g Lenti-Gag, 9 μ g Lenti-Rev, 5 μ g pTetOff, 5 μ g pVSVG, liquid-transfering gun After piping and druming mixes well up and down, 500 μ L, 1 μ g/ μ L liposomes are added, blows and beats mixing up and down with pipettor immediately, stands at room temperature 10-15 minutes.

(4) 293T cells are taken out from incubator, in Biohazard Safety Equipment, culture dish are opened, by above-mentioned DNA/ liposomes Complexes drop-wise is added in culture dish, mixes well.Culture dish is placed in 37 DEG C, 5%CO₂Incubator, after cultivating 6~8 hours, Culture medium containing transfection reagent is removed, fresh complete medium is changed to.

After (5) 48 hours, collect ware in culture medium to sterile centrifugation tube in, 2000xg, 4 DEG C centrifuge 10 minutes, will be upper The membrane filtration of the clear and coherent PES materials for crossing 0.45um takes a new sterile ultracentrifugation pipe, is added and contains virulent culture medium Supernatant, after Beckman SW28 rotor trims, 20,000xg, 4 DEG C centrifuge 3 hours.

(6) centrifuge after, in Biohazard Safety Equipment, carefully the liquid in centrifuge tube is sucked, be added 1mL PBS or Precipitation is resuspended HBSS buffer solutions, and virus is placed in -80 DEG C of preservations.

Embodiment 3

It is prepared by chimeric antigen cell receptor T cell

(1) anticoagulant blood sample is transferred in a 15mL sterile centrifugation tube, screws lid, adjustment centrifuge power is extremely 800xg, centrifugation reduction of speed are set as minimum, and blood sample room temperature is centrifuged 20 minutes.

(2) after centrifuging, centrifuge tube is taken out from centrifuge, is lost the light yellow serum layer in upper layer with disposable pipettor It abandons；Then isometric physiological saline is added to the red peripheral blood cells layer of lower layer, after screwing centrifuge tube lid, gently runs up and down Mixing.

(3) lymphocyte separation medium is taken out, and is turned upside down for several times, is mixed well.

(4) in Biohazard Safety Equipment, 5mL lymphocytes are added into 15mL centrifuge tubes with disposable sterilized pipette first Then blood sample after normal saline dilution is carefully added slowly to separation of lymphocytes along tube wall using pipettor and tried by separating liquid The upper layer of agent.

(5) centrifugal force of centrifuge is set to 800xg, rotating speed decrease speed is set as minimum, and temperature is set as 20 DEG C, centrifugation 20 minutes.

(6) after centrifuging, taking-up centrifuge tube gently avoids the centrifuge tube that acutely shakes or turn upside down, and pacifies in biology It will be drawn in a new sterile centrifugation tube, added in intermediate white mononuclear cell layer with disposable sterilized pipettor in full cabinet After entering isometric physiological saline, centrifuge tube lid is screwed, turn upside down mixing, sets the centrifugal force of centrifuge to 800xg, from Supernatant liquid after centrifugation, is all sucked out using sterile pipette, 5mL physiological saline is then added, gently by the heart 5 minutes It after mixing, takes part cell suspension to be counted, and calculate total number of cells, sets the centrifugal force of centrifuge to 800xg, centrifuge 5 minutes.

(7) after centrifuging, supernatant liquid is all sucked out with sterile pipette, 10mL is then added and is preheated to room temperature Complete medium, then turn upside down mixing.

(8) Dynabeads is washed

A) Dynabeads is taken out from refrigerator, is shaken 45 seconds and (if without turbula shaker, is run up and down using turbula shaker Upside-down mounting has the pipe 5 minutes of Dynabeads, until the magnetic bead for being deposited in tube bottom is resuspended completely)；

B) Dynabeads is transferred in a 5mL steril cell cryopreservation tube using sterile pipette；According to Dynabeads:T=1:1 or 3:1 ratio calculates the amount of required Dynabeads.

C) DPBS of same volume is added, mixing is blown and beaten using pipettor.

D) 5mL cell cryopreservation tubes are inserted into the hole among magnetic pole, are stored at room temperature 1 minute, cryopreservation tube is kept to be inserted in magnetic pole Hole in, be gently inverted, the liquid in pipe poured out.

E) cryopreservation tube is taken out out of magnetic pole hole, with pipettor be added and C) step same volume DPBS, use liquid relief Device blows and beats mixing.

(9) by after washing beads and PBMC mix, room temperature (20 DEG C of ≈) be incubated 30 minutes.

(10) into pipe be added proper volume 15 culture mediums of X-Vivo (volume according to 5mL cell cryopreservation tubes be inserted into magnetic pole Add culture medium in position in hole), it mixes well.

(11) 5mL cell cryopreservation tubes are inserted into magnetic pole hole, are stored at room temperature 1 minute, cryopreservation tube is kept to be inserted in the hole of magnetic pole It is interior, it is gently inverted, the liquid in pipe is poured out.

(12) cell cryopreservation tube is taken out from magnetic pole, 15 culture medium (interleukins containing 200IU/mL of 3mL X-Vivo is added 2,10ng/mL IL-7,5ng/mL interleukin 15s, 5ng/mL interleukin-22s 1), cell is resuspended with pipettor and beads is mixed Object, and cell density is adjusted to 0.5~1x10⁶A cell/mL.

(13) cell is placed in 37 DEG C, 5%CO₂In incubator for three days on end.Since third day, gently piping and druming training daily Beads/ cell masses in the system of supporting, until completely separable；Daily cell once count and (takes 50 μ L cell suspensions, use Blood counting chamber counts under the microscope), work as cell density>1x10⁶When a/mL, it is close to add fresh culture medium adjustment cell It spends to 0.5x10⁶A/mL.

(14) according to MOI=10-20, required virus quantity is calculated.Calculation formula is as follows：

Required virus quantity (mL)=(MOI* cell quantities)/virus titer

(15) from -80 DEG C of ultra low temperature freezers, slow virus is taken out, is thawed in 37 DEG C of water-baths rapidly.

(16) it is carried out disinfection processing to the tube outer surface that freezes of stock virus using 75% medicinal alcohol.

(17) polybrene to final concentration of 6 μ g/ is added into culture vessel for the T cell that preparation is taken out from incubator The virus quantity obtained by above-mentioned calculating is added in mL, is gently blown and beaten and is mixed well with pipettor, and all cells/beads groups is made to dispel Afterwards, culture vessel is sealed using sealed membrane, 800xg room temperatures centrifuge 1 hour.

(18) after centrifuging, sealed membrane is torn, culture vessel is placed in 37 DEG C of 5%CO₂Incubator in, continue to cultivate 24 hours.

(19) 250xg is centrifuged 10 minutes, is removed containing virulent culture medium supernatant, and it is heavy that cell is resuspended with fresh culture It forms sediment, cell is transferred in new culture vessel, continue culture 5 days.The beads/ cells in cultivating system are gently blown and beaten daily Group, until completely separable；Daily cell once count and (takes 50 μ L cell suspensions, under the microscope using blood counting chamber Count), work as cell density>1x10⁶When a/mL, adds fresh culture medium and adjust cell density to 0.5x10⁶A/mL.

(20) part cell is taken to use the expression of FACS detection T cells surface chimeric antigen cell receptor molecule.

Experimental example 1

1. chimeric antigen cell receptor T cell is to target cell killing experiments in vitro

(1) adjustment target cell state needs continuous passage 2 times before the experiment to exponential phase；

(2) adherent target cell digestion is resuspended in complete medium with pancreatin, adjustment cell density to 5*10⁵A/ ML takes one piece of 96 new orifice plate, and target cell is inoculated with according to the amount in 100 holes μ L/.100 are added per hole for the 96 unused holes of orifice plate surrounding μ L sterile waters, to prevent intermediate experimental port moisture evaporation.Orifice plate is placed in 5%CO₂, in 37 DEG C of incubators, be incubated overnight.

(3) the above-mentioned 4.3 chimeric antigen cell receptor-T cells prepared are collected by centrifugation, use 1640 culture mediums of serum-free It is resuspended；96 orifice plates are taken out from incubator, the culture medium in hole is sucked out completely, are gently washed cell one time with sterile PBS, Then according to above-mentioned E/T ratios, chimeric antigen cell receptor-T cell is added, and final volume is mended to 100 holes μ L/；Maxi Lysis and Mini lysis are to be inoculated with same amount of target cell, but be not added with chimeric antigen cell receptor-T cell.By orifice plate It is placed in 5%CO₂, in 37 DEG C of incubators, cultivate 6 hours.

(4) after cultivating, orifice plate is taken out from incubator, is added in LDH detection kits to the holes Maxi lysis Lysate, after target cell therein completely cracking, 1200xg room temperatures centrifuge 96 orifice plate 5 minutes, taking out plate gently, from It is shifted in per hole in 50 μ L to another 96 new orifice plate, after LDH detection reagents are added, OD values is read using microplate reader.

(5) target cell lysis percentage calculation formula：

(6) treated data are mapped using GraphPad 5.0.

2. chimeric antigen cell receptor T cell is to killing experiments in target cell body

(1) 6 week old female NSG mouse 10, average weight 18.3-23.5g are raised in the independent ventilation box of constant temperature and humidity It is interior, 22-23 DEG C of room temperature, humidity 56-60% are raised, 10-20 times/hour takes a breath, round the clock light and shade alt time 12h/12h；It holds Continuous supply is through Co 60 radiosterilization mouse full-valence pellet feed, and quantity-unlimiting freely to absorb, drinking public water supply (makes after high pressure steam sterilization With), drinking bottle uninterruptedly supplies water, and freely absorbs.

(2) Nalm6-Luc cell culture is in the DMEM culture solutions containing 10% fetal calf serum.The cell company is tested by me Room voluntarily prepares conservation, collects the cell of exponential phase of growth, and PBS is resuspended to suitable concentration for being inoculated with.

(3) all mouse are transfused Nalm6-Luc cells, every inoculation 5*10 through tail vein⁶A tumour cell, mouse are continuous After raising 5 days, luciferase substrate D-luciferin is injected intraperitoneally, carries out growth of the preliminary experiment observation tumour in Mice Body Situation.It is divided into 2 groups according to tumor size mean random.

(4) one of which mouse is processing group, every mouse inoculation 5*10⁶A expression chimeric antigen cell receptor T cell； Another group of mouse is control group, every mouse inoculation 5*10⁶A control T cell.Every 5 days to mouse peritoneal injected fluorescein enzyme Substrate D-luciferin simultaneously anaesthetizes mouse using gas anesthesia machine, acquires tumour living imaging data.

3. experimental result

The combination of 3.1 chimeric antigen cell receptor T cell surface antibody Fab and target protein

Take 5*10⁶It is a expression chimeric antigen cell receptor T cell and identical quantity control T cell, respectively with different sulphur Cyanic acid fluorescein (FITC) coupling CD19 recombinant proteins be incubated at room temperature 30 minutes, with brine three times after, use stream Formula Cytometric Analysis recombinates the combination situation of T cell film surface chimeric antigen cell receptor and target proteins CD19.As shown in Figure 9 (the chimeric antigen FAB recipient T cells involved in Fig. 9-Figure 14 are the chimeric antigen cell receptor for representing the expression embodiment of the present invention T cell), target proteins CD19 can be identified in the chimeric antigen cell receptor of T cell film surface by expressing, and not depend on MHC Molecule.

The effect of 3.2 chimeric antigen cell receptor T cell Cytotoxicity in vitro target cells

Take expression chimeric antigen cell receptor T cell and control T cell, according to different ratios respectively with expression CD19 eggs White tumour cell Nalm6 carries out co-culture experiments, take the culture medium supernatant of co-cultivation detect respectively LDH content and cell because Sub- IL-2 and IFN-γ.The results are shown in Figure 10, and the T cell for expressing chimeric antigen cell receptor can be efficiently to expressing target The tumour cell of antigen is killed, and is 1 in ratio：Lethal effect can be generated to 40% tumour cell, when 1 with embedding The raising for closing the T cell ratio of cell antigen receptor, enhances rapidly the lytic effect of tumour cell, is 5 in ratio：It, can when 1 Fully erased all tumour cells；Under the stimulation of target cell, the T cell of expression chimeric antigen cell receptor is swashed It is living, interleukin-22 and the horizontal significantly raising (as is illustrated by figs. 11 and 12) of interferon secreting, expressing are shown as, and with chimeric The T cell quantity of cell antigen receptor increases, and interleukin-22 and interferon secreting, expressing level gradually rise, and it is thin to compare T Born of the same parents generate any significant response to the stimulation of target cell.Prove the T cell of expression chimeric antigen cell receptor Can specificity identification target cell, and activation signal is transmitted into immune t-cell, cause target cell killing and cell because Sub- secretion level rises.

The effect of killing target cell in 3.3 chimeric antigen Fab cell receptor T cells Mice Bodies

The implanted tumor cells in Mice Body, then the T cell of infusion expression chimeric antigen cell receptor, routine observation are small The growing state of mouse interior tumor cell, the results showed that, the mouse interior tumor cell Proliferation of infusion control T cell is rapid, and The mouse interior tumor cell for being transfused chimeric antigen cell receptor T cell over time, is eliminated, shows to express completely The T cell of chimeric antigen cell receptor also can effectively remove the target cell (Figure 13) in Mice Body in vivo.

Experimental example 2

Verify the effect of heavy chain constant region and constant region of light chain to Chimeric antigen receptor provided by the invention

Experimental method：

It is prepared by 1 slow virus

It is embedding without heavy chain constant region and constant region of light chain that the method for reference implementation example 1 and embodiment 2 prepares expression Close the slow virus carrier of antigen receptor A (amino acid sequence such as SEQ ID NO.21, coded sequence SEQ ID NO.20)；Inosculating antibody Original receptor A is only that Chimeric antigen receptor A does not have heavy chain constant region and light compared with the difference of the Chimeric antigen receptor of embodiment 1 Chain constant region.

The Jurkat cell strain construction method of 1.1 expression Chimeric antigen receptors is as follows：

(1) the recovery Jurkat cell strain from liquid nitrogen uses the complete culture of complete medium RPMI1640,10%FBS Base is cultivated, after 5 generations of continuous culture, by cell according to 1*10⁶The density of cell/mL is seeded in 6 orifice plates；It is inoculated with 3 altogether Hole, two of which hole expression Chimeric antigen receptor (are respectively provided with the embodiment 2 of constant region or the chimeric antigen without constant region Receptor A) slow virus infected, another hole cell as a contrast.

(2) 20uL Chimeric antigen receptor slow virus is separately added into two holes, it, will after gently blowing and beating mixing with pipettor Orifice plate is placed in a centrifuge, and 800xg, room temperature centrifuge 1 hour.

(3) after centrifuging, orifice plate is placed in 5%CO₂, in 37 DEG C of incubators, be incubated overnight.

(4) after taking out orifice plate, in Biohazard Safety Equipment, puromycin is added into hole to final concentration of 1ug/mL.Even Continuous culture 5 days, until the complete cell death in control wells, the cell survived in slow-virus infection hole is to stablize expression to be fitted into The Jurkat stable cell lines of antigen receptor.After expanding culture, co-culture experiments are carried out.

The target cell Nalm6 of the Jurkat cell and expression CD19 of 1.2 expression Chimeric antigen receptors

(1) adjustment target cell state needs continuous passage 2 times before the experiment to exponential phase；

(2) RPMI1640,10%FBS culture medium is used to adjust cell density to 5*10⁵A/mL takes one piece of 96 new orifice plate, It is inoculated with target cell according to the amount in 100 holes μ L/.Chimeric antigen cell receptor-Jurkat cell of above-mentioned preparation is collected by centrifugation, uses RPMI1640,10%FBS culture medium are resuspended；Then chimeric antigen cell receptor-Jurkat is added in E/T ratios as illustrated Cell, and final volume is mended to 100 holes μ L/；Control is added to be inoculated with same amount of target cell and does not express inosculating antibody The control Jurkat cell of archaeocyte receptor.100 μ L sterile waters are added per hole for the 96 unused holes of orifice plate surrounding, to prevent centre Experimental port moisture evaporation.Orifice plate is placed in 5%CO₂In 37 DEG C of incubators, cultivate 12 hours.

(3) after cultivating, orifice plate is taken out from incubator, 1200xg room temperatures centrifuge 96 orifice plate 5 minutes, gently general Plate takes out, and 50 μ L culture medium supernatants are shifted from every hole, and the expression of interleukin-22 is detected using ELISA kit.

1.3 result

Jurkat cell is a T cell system, in the case where being stimulated by extrinsic factor, can high expression interleukin-22, this and day The activation mechanism of right T cell is completely the same, therefore can be as a model cell for Chimeric antigen receptor work(of the invention It can evaluation.The Chimeric antigen receptor with heavy chain of antibody and constant region of light chain is can be seen that in target cell from the result of Figure 14 Under activation, Jurkat cell can be effectively activated, the expression for showing as IL2 drastically increases.And remove inosculating antibody After the heavy chain and constant region of light chain of original receptor,

The IL2 expressions of Jurkat cell with compare Jurkat cell and be not significantly different substantially, show heavy chain and light Chain constant region has important influence to the Chimeric antigen receptor function of being designed in the present invention.

The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, any made by repair Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.

SEQUENCE LISTING

<110>Ningbo An Nuobaide biological medicines Science and Technology Ltd.

<120>A kind of chimeric antigen cell receptor and its application

<160> 34

<170> PatentIn version 3.5

<210> 1

<211> 18

<212> PRT

<213>Artificial sequence

<400> 1

Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro

1 5 10 15

Gly Pro

<210> 2

<211> 22

<212> PRT

<213>Artificial sequence

<400> 2

Gly Ser Gly Ala Thr Asn Phe Ser Leu Leu Lys Gln Ala Gly Asp Val

1 5 10 15

Glu Glu Asn Pro Gly Pro

20

<210> 3

<211> 25

<212> PRT

<213>Artificial sequence

<400> 3

Gly Ser Gly Val Lys Gln Thr Leu Asn Phe Asp Leu Leu Lys Leu Ala

1 5 10 15

Gly Asp Val Glu Ser Asn Pro Gly Pro

20 25

<210> 4

<211> 23

<212> PRT

<213>Artificial sequence

<400> 4

Gly Ser Gly Gln Cys Thr Asn Tyr Ala Leu Leu Lys Leu Ala Gly Asp

1 5 10 15

Val Glu Ser Asn Pro Gly Pro

20

<210> 5

<211> 1335

<212> DNA

<213>Artificial sequence

<400> 5

gagtaattca tacaaaagga ctcgcccctg ccttggggaa tcccagggac cgtcgttaaa 60

ctcccactaa cgtagaaccc agagatcgct gcgttcccgc cccctcaccc gcccgctctc 120

gtcatcactg aggtggagaa gagcatgcgt gaggctccgg tgcccgtcag tgggcagagc 180

gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga accggtgcct 240

agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc cgcctttttc 300

ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt ctttttcgca 360

acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct 420

ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg 480

attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa 540

ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg ccgccgcgtg 600

cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa 660

aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc 720

caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg 780

tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg 840

gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc 900

ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg 960

cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg 1020

ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac 1080

tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg 1140

tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg 1200

gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt 1260

gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca 1320

tttcaggtgt cgtga 1335

<210> 6

<211> 603

<212> DNA

<213>Artificial sequence

<400> 6

aagcttggga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca 60

acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 120

ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 180

aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 240

ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 300

tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 360

ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 420

ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa 480

tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc 540

agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgactctact 600

aga 603

<210> 7

<211> 509

<212> DNA

<213>Artificial sequence

<400> 7

aattctaccg ggtaggggag gcgcttttcc caaggcagtc tggagcatgc gctttagcag 60

ccccgctggg cacttggcgc tacacaagtg gcctctggcc tcgcacacat tccacatcca 120

ccggtaggcg ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc 180

ccctagtcag gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg 240

aagtagcacg tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg 300

taggcctttg gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc 360

tgggaagggg tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc 420

gaaggtcctc cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt 480

tctcctcttc ctcatctccg ggcctttcg 509

<210> 8

<211> 330

<212> DNA

<213>Artificial sequence

<400> 8

gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc 60

atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga 120

agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc 180

atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt 240

tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga 300

ggcttttttg gaggcctagg cttttgcaaa 330

<210> 9

<211> 650

<212> DNA

<213>Artificial sequence

<400> 9

agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg cgccaattct 60

gcagacaaat ggctctagag gtacccgtta cataacttac ggtaaatggc ccgcctggct 120

gaccgcccaa cgacccccgc ccattgacgt caatagtaac gccaataggg actttccatt 180

gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc 240

atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattgtg 300

cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg 360

ctattaccat ggtcgaggtg agccccacgt tctgcttcac tctccccatc tcccccccct 420

ccccaccccc aattttgtat ttatttattt tttaattatt ttgtgcagcg atgggggcgg 480

gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg ggcggggcga 540

ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg 600

cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg 650

<210> 10

<211> 21

<212> PRT

<213>Artificial sequence

<400> 10

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 11

<211> 17

<212> PRT

<213>Artificial sequence

<400> 11

Met Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile

1 5 10 15

Ser

<210> 12

<211> 120

<212> PRT

<213>Artificial sequence

<400> 12

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

1 5 10 15

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

20 25 30

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

35 40 45

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

50 55 60

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

65 70 75 80

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

85 90 95

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Ser Val Thr Val Ser Ser

115 120

<210> 13

<211> 99

<212> PRT

<213>Artificial sequence

<400> 13

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

1 5 10 15

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

20 25 30

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

35 40 45

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

50 55 60

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

65 70 75 80

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

85 90 95

Lys Val Glu

<210> 14

<211> 160

<212> PRT

<213>Artificial sequence

<400> 14

Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile Gln Asn Pro

1 5 10 15

Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser Asp Lys Ser

20 25 30

Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val Ser Gln Ser

35 40 45

Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu Asp Met Arg

50 55 60

Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser Asn Lys Ser

65 70 75 80

Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile Pro Glu Asp

85 90 95

Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys Leu Val Glu

100 105 110

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

115 120 125

Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe Asn Leu Leu

130 135 140

Met Thr Leu Arg Leu Trp Ser Ser Arg Ala Lys Arg Ser Gly Ser Gly

145 150 155 160

<210> 15

<211> 107

<212> PRT

<213>Artificial sequence

<400> 15

Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly

1 5 10 15

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Lys Tyr

20 25 30

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile

35 40 45

Tyr His Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln

65 70 75 80

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Pro Tyr

85 90 95

Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

100 105

<210> 16

<211> 104

<212> PRT

<213>Artificial sequence

<400> 16

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

1 5 10 15

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

20 25 30

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

35 40 45

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

50 55 60

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

65 70 75 80

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

85 90 95

Lys Ser Phe Asn Arg Gly Glu Cys

100

<210> 17

<211> 189

<212> PRT

<213>Artificial sequence

<400> 17

Phe Gly Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val

1 5 10 15

Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser

20 25 30

His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro

35 40 45

Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser

50 55 60

Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn

65 70 75 80

Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe

85 90 95

Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly

100 105 110

Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr

115 120 125

Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr

130 135 140

Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu

145 150 155 160

Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu

165 170 175

Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly

180 185

<210> 18

<211> 2508

<212> DNA

<213>Artificial sequence

<400> 18

atggctctgc ctgtgacagc tctgctgctg cctctggctc tgcttctgca tgccgccaga 60

cctgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacagccgct cctagcgtgt tcatcttccc accttccgac 420

gagcagctga aaagcggcac agcctctgtc gtgtgcctgc tgaacaactt ctaccccaga 480

gaagccaagg tgcagtggaa ggtggacaac gccctgcaga gcggcaatag ccaagagagc 540

gtgaccgagc aggacagcaa ggactctacc tacagcctga gcagcaccct gacactgagc 600

aaggccgact acgagaagca caaagtgtac gcctgcgaag tgacccacca gggcctttct 660

agccctgtga ccaagagctt caaccggggc gagtgttttg gcgagggaag cagactgacc 720

gtgctggaag atctgaagaa cgtgttccca cctgaggtgg ccgtgttcga gccttctgag 780

gccgagatca gccacacaca gaaagccaca ctcgtgtgtc tggccaccgg cttctatccc 840

gatcacgtgg aactgtcttg gtgggttaac ggcaaagagg tgcactccgg cgtctgcaca 900

gatccccagc ctctgaaaga acagcccgct ctgaacgaca gcagatacgc cctgtccagc 960

aggctgagag tgtccgccac attctggcag gaccccagaa accacttcag atgccaggtg 1020

cagttctacg gcctgagcga gaacgatgag tggacccagg atagagccaa gcctgtgact 1080

cagatcgtgt ctgccgaagc ctggggcaga gccgattgtg gctttaccag cgagagctac 1140

caacagggcg tgctgtctgc caccatcctg tacgagatcc tgctgggcaa agccactctg 1200

tacgccgtgc tggtgtctgc cctggtgctg atggccatgg tcaagcggaa agacagcaga 1260

ggcgaaggca gaggctctct gctgacatgt ggcgacgtgg aagagaaccc cggacctatg 1320

tggctgcagt ctttgctgct gctgggaacc gtggcctgta gcatctctga ggtgaaactg 1380

caggagtcag gacctggcct ggtggcgccc tcacagagcc tgtccgtcac atgcactgtc 1440

tcaggggtct cattacccga ctatggtgta agctggattc gccagcctcc acgaaagggt 1500

ctggagtggc tgggagtaat atggggtagt gaaaccacat actataattc agctctcaaa 1560

tccagactga ccatcatcaa ggacaactcc aagagccaag ttttcttaaa aatgaacagt 1620

ctgcaaactg atgacacagc catttactac tgtgccaaac attattacta cggtggtagc 1680

tatgctatgg actactgggg ccaaggaacc tcagtcaccg tctcctcagc ctctacaaag 1740

ggcccaagcg tgttcccgct ggctcctagc agcaagtcta caagcggagg aacagctgcc 1800

ctgggatgcc tggtcaagga ctactttcct gagccagtga ccgtgtcttg gaactctggc 1860

gctctgacaa gcggcgtgca cacatttcca gcagtgctgc agtccagcgg cctgtactct 1920

ctgtctagcg tggtcacagt gcccagctct agcctgggca cacagaccta catctgcaat 1980

gtgaaccaca agcctagcaa caccaaggtc gacaagaagg tggaatttgg cagaggcacc 2040

agcctgatcg tgcaccccta cattcagaac cccgatcctg ccgtgtacca gctgagagac 2100

agcaagagca gcgacaagag tgtgtgcctg ttcaccgact tcgacagcca gaccaacgtg 2160

tcccagagca aggacagcga cgtgtacatc accgataagt gcgtgctgga catgcggagc 2220

atggacttca agagcaacag cgccgtggct tggagcaaca agagcgattt cgcctgcgcc 2280

aacgccttca acaacagcat tatccccgag gacacattct tcccaagtcc tgagagcagc 2340

tgcgacgtga agctggtgga aaagagcttc gagacagaca ccaacctgaa cttccagaac 2400

ctgagcgtga tcggcttcag aatcctgctg ctgaaggtgg ccggcttcaa cctgctgatg 2460

accctgagac tgtggtccag ccgggccaag agatctggat ctggatga 2508

<210> 19

<211> 835

<212> PRT

<213>Artificial sequence

<400> 19

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys

130 135 140

Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg

145 150 155 160

Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn

165 170 175

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

180 185 190

Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys

195 200 205

Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr

210 215 220

Lys Ser Phe Asn Arg Gly Glu Cys Phe Gly Glu Gly Ser Arg Leu Thr

225 230 235 240

Val Leu Glu Asp Leu Lys Asn Val Phe Pro Pro Glu Val Ala Val Phe

245 250 255

Glu Pro Ser Glu Ala Glu Ile Ser His Thr Gln Lys Ala Thr Leu Val

260 265 270

Cys Leu Ala Thr Gly Phe Tyr Pro Asp His Val Glu Leu Ser Trp Trp

275 280 285

Val Asn Gly Lys Glu Val His Ser Gly Val Cys Thr Asp Pro Gln Pro

290 295 300

Leu Lys Glu Gln Pro Ala Leu Asn Asp Ser Arg Tyr Ala Leu Ser Ser

305 310 315 320

Arg Leu Arg Val Ser Ala Thr Phe Trp Gln Asp Pro Arg Asn His Phe

325 330 335

Arg Cys Gln Val Gln Phe Tyr Gly Leu Ser Glu Asn Asp Glu Trp Thr

340 345 350

Gln Asp Arg Ala Lys Pro Val Thr Gln Ile Val Ser Ala Glu Ala Trp

355 360 365

Gly Arg Ala Asp Cys Gly Phe Thr Ser Glu Ser Tyr Gln Gln Gly Val

370 375 380

Leu Ser Ala Thr Ile Leu Tyr Glu Ile Leu Leu Gly Lys Ala Thr Leu

385 390 395 400

Tyr Ala Val Leu Val Ser Ala Leu Val Leu Met Ala Met Val Lys Arg

405 410 415

Lys Asp Ser Arg Gly Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp

420 425 430

Val Glu Glu Asn Pro Gly Pro Met Trp Leu Gln Ser Leu Leu Leu Leu

435 440 445

Gly Thr Val Ala Cys Ser Ile Ser Glu Val Lys Leu Gln Glu Ser Gly

450 455 460

Pro Gly Leu Val Ala Pro Ser Gln Ser Leu Ser Val Thr Cys Thr Val

465 470 475 480

Ser Gly Val Ser Leu Pro Asp Tyr Gly Val Ser Trp Ile Arg Gln Pro

485 490 495

Pro Arg Lys Gly Leu Glu Trp Leu Gly Val Ile Trp Gly Ser Glu Thr

500 505 510

Thr Tyr Tyr Asn Ser Ala Leu Lys Ser Arg Leu Thr Ile Ile Lys Asp

515 520 525

Asn Ser Lys Ser Gln Val Phe Leu Lys Met Asn Ser Leu Gln Thr Asp

530 535 540

Asp Thr Ala Ile Tyr Tyr Cys Ala Lys His Tyr Tyr Tyr Gly Gly Ser

545 550 555 560

Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser

565 570 575

Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys

580 585 590

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

595 600 605

Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser

610 615 620

Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser

625 630 635 640

Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr

645 650 655

Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys

660 665 670

Lys Val Glu Phe Gly Arg Gly Thr Ser Leu Ile Val His Pro Tyr Ile

675 680 685

Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg Asp Ser Lys Ser Ser

690 695 700

Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp Ser Gln Thr Asn Val

705 710 715 720

Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr Asp Lys Cys Val Leu

725 730 735

Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser Ala Val Ala Trp Ser

740 745 750

Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe Asn Asn Ser Ile Ile

755 760 765

Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser Ser Cys Asp Val Lys

770 775 780

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

785 790 795 800

Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu Lys Val Ala Gly Phe

805 810 815

Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser Arg Ala Lys Arg Ser

820 825 830

Gly Ser Gly

835

<210> 20

<211> 1899

<212> DNA

<213>Artificial sequence

<400> 20

atggctctgc ctgtgacagc tctgctgctg cctctggctc tgcttctgca tgccgccaga 60

cctgacatcc agatgacaca gactacatcc tccctgtctg cctctctggg agacagagtc 120

accatcagtt gcagggcaag tcaggacatt agtaaatatt taaattggta tcagcagaaa 180

ccagatggaa ctgttaaact cctgatctac catacatcaa gattacactc aggagtccca 240

tcaaggttca gtggcagtgg gtctggaaca gattattctc tcaccattag caacctggag 300

caagaagata ttgccactta cttttgccaa cagggtaata cgcttccgta cacgttcgga 360

ggggggacca agctggagat cacatttggc gagggaagca gactgaccgt gctggaagat 420

ctgaagaacg tgttcccacc tgaggtggcc gtgttcgagc cttctgaggc cgagatcagc 480

cacacacaga aagccacact cgtgtgtctg gccaccggct tctatcccga tcacgtggaa 540

ctgtcttggt gggttaacgg caaagaggtg cactccggcg tctgcacaga tccccagcct 600

ctgaaagaac agcccgctct gaacgacagc agatacgccc tgtccagcag gctgagagtg 660

tccgccacat tctggcagga ccccagaaac cacttcagat gccaggtgca gttctacggc 720

ctgagcgaga acgatgagtg gacccaggat agagccaagc ctgtgactca gatcgtgtct 780

gccgaagcct ggggcagagc cgattgtggc tttaccagcg agagctacca acagggcgtg 840

ctgtctgcca ccatcctgta cgagatcctg ctgggcaaag ccactctgta cgccgtgctg 900

gtgtctgccc tggtgctgat ggccatggtc aagcggaaag acagcagagg cgaaggcaga 960

ggctctctgc tgacatgtgg cgacgtggaa gagaaccccg gacctatgtg gctgcagtct 1020

ttgctgctgc tgggaaccgt ggcctgtagc atctctgagg tgaaactgca ggagtcagga 1080

cctggcctgg tggcgccctc acagagcctg tccgtcacat gcactgtctc aggggtctca 1140

ttacccgact atggtgtaag ctggattcgc cagcctccac gaaagggtct ggagtggctg 1200

ggagtaatat ggggtagtga aaccacatac tataattcag ctctcaaatc cagactgacc 1260

atcatcaagg acaactccaa gagccaagtt ttcttaaaaa tgaacagtct gcaaactgat 1320

gacacagcca tttactactg tgccaaacat tattactacg gtggtagcta tgctatggac 1380

tactggggcc aaggaacctc agtcaccgtc tcctcatttg gcagaggcac cagcctgatc 1440

gtgcacccct acattcagaa ccccgatcct gccgtgtacc agctgagaga cagcaagagc 1500

agcgacaaga gtgtgtgcct gttcaccgac ttcgacagcc agaccaacgt gtcccagagc 1560

aaggacagcg acgtgtacat caccgataag tgcgtgctgg acatgcggag catggacttc 1620

aagagcaaca gcgccgtggc ttggagcaac aagagcgatt tcgcctgcgc caacgccttc 1680

aacaacagca ttatccccga ggacacattc ttcccaagtc ctgagagcag ctgcgacgtg 1740

aagctggtgg aaaagagctt cgagacagac accaacctga acttccagaa cctgagcgtg 1800

atcggcttca gaatcctgct gctgaaggtg gccggcttca acctgctgat gaccctgaga 1860

ctgtggtcca gccgggccaa gagatctgga tctggatga 1899

<210> 21

<211> 632

<212> PRT

<213>Artificial sequence

<400> 21

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu

20 25 30

Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln

35 40 45

Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr

50 55 60

Val Lys Leu Leu Ile Tyr His Thr Ser Arg Leu His Ser Gly Val Pro

65 70 75 80

Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile

85 90 95

Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly

100 105 110

Asn Thr Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Thr

115 120 125

Phe Gly Glu Gly Ser Arg Leu Thr Val Leu Glu Asp Leu Lys Asn Val

130 135 140

Phe Pro Pro Glu Val Ala Val Phe Glu Pro Ser Glu Ala Glu Ile Ser

145 150 155 160

His Thr Gln Lys Ala Thr Leu Val Cys Leu Ala Thr Gly Phe Tyr Pro

165 170 175

Asp His Val Glu Leu Ser Trp Trp Val Asn Gly Lys Glu Val His Ser

180 185 190

Gly Val Cys Thr Asp Pro Gln Pro Leu Lys Glu Gln Pro Ala Leu Asn

195 200 205

Asp Ser Arg Tyr Ala Leu Ser Ser Arg Leu Arg Val Ser Ala Thr Phe

210 215 220

Trp Gln Asp Pro Arg Asn His Phe Arg Cys Gln Val Gln Phe Tyr Gly

225 230 235 240

Leu Ser Glu Asn Asp Glu Trp Thr Gln Asp Arg Ala Lys Pro Val Thr

245 250 255

Gln Ile Val Ser Ala Glu Ala Trp Gly Arg Ala Asp Cys Gly Phe Thr

260 265 270

Ser Glu Ser Tyr Gln Gln Gly Val Leu Ser Ala Thr Ile Leu Tyr Glu

275 280 285

Ile Leu Leu Gly Lys Ala Thr Leu Tyr Ala Val Leu Val Ser Ala Leu

290 295 300

Val Leu Met Ala Met Val Lys Arg Lys Asp Ser Arg Gly Glu Gly Arg

305 310 315 320

Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro Gly Pro Met

325 330 335

Trp Leu Gln Ser Leu Leu Leu Leu Gly Thr Val Ala Cys Ser Ile Ser

340 345 350

Glu Val Lys Leu Gln Glu Ser Gly Pro Gly Leu Val Ala Pro Ser Gln

355 360 365

Ser Leu Ser Val Thr Cys Thr Val Ser Gly Val Ser Leu Pro Asp Tyr

370 375 380

Gly Val Ser Trp Ile Arg Gln Pro Pro Arg Lys Gly Leu Glu Trp Leu

385 390 395 400

Gly Val Ile Trp Gly Ser Glu Thr Thr Tyr Tyr Asn Ser Ala Leu Lys

405 410 415

Ser Arg Leu Thr Ile Ile Lys Asp Asn Ser Lys Ser Gln Val Phe Leu

420 425 430

Lys Met Asn Ser Leu Gln Thr Asp Asp Thr Ala Ile Tyr Tyr Cys Ala

435 440 445

Lys His Tyr Tyr Tyr Gly Gly Ser Tyr Ala Met Asp Tyr Trp Gly Gln

450 455 460

Gly Thr Ser Val Thr Val Ser Ser Phe Gly Arg Gly Thr Ser Leu Ile

465 470 475 480

Val His Pro Tyr Ile Gln Asn Pro Asp Pro Ala Val Tyr Gln Leu Arg

485 490 495

Asp Ser Lys Ser Ser Asp Lys Ser Val Cys Leu Phe Thr Asp Phe Asp

500 505 510

Ser Gln Thr Asn Val Ser Gln Ser Lys Asp Ser Asp Val Tyr Ile Thr

515 520 525

Asp Lys Cys Val Leu Asp Met Arg Ser Met Asp Phe Lys Ser Asn Ser

530 535 540

Ala Val Ala Trp Ser Asn Lys Ser Asp Phe Ala Cys Ala Asn Ala Phe

545 550 555 560

Asn Asn Ser Ile Ile Pro Glu Asp Thr Phe Phe Pro Ser Pro Glu Ser

565 570 575

Ser Cys Asp Val Lys Leu Val Glu Lys Ser Phe Glu Thr Asp Thr Asn

580 585 590

Leu Asn Phe Gln Asn Leu Ser Val Ile Gly Phe Arg Ile Leu Leu Leu

595 600 605

Lys Val Ala Gly Phe Asn Leu Leu Met Thr Leu Arg Leu Trp Ser Ser

610 615 620

Arg Ala Lys Arg Ser Gly Ser Gly

625 630

<210> 22

<211> 608

<212> DNA

<213>Artificial sequence

<400> 22

cgcccctctc cctccccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg 60

tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc 120

cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa 180

ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga 240

caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc 300

ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc 360

cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac 420

aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg 480

tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac 540

ggggacgtgg ttttcctttg aaaaacacga tgataagctt gccacaaccc acaaggagac 600

gaccttcc 608

<210> 23

<211> 360

<212> DNA

<213>Artificial sequence

<400> 23

gaggtgaaac tgcaggagtc aggacctggc ctggtggcgc cctcacagag cctgtccgtc 60

acatgcactg tctcaggggt ctcattaccc gactatggtg taagctggat tcgccagcct 120

ccacgaaagg gtctggagtg gctgggagta atatggggta gtgaaaccac atactataat 180

tcagctctca aatccagact gaccatcatc aaggacaact ccaagagcca agttttctta 240

aaaatgaaca gtctgcaaac tgatgacaca gccatttact actgtgccaa acattattac 300

tacggtggta gctatgctat ggactactgg ggccaaggaa cctcagtcac cgtctcctca 360

<210> 24

<211> 321

<212> DNA

<213>Artificial sequence

<400> 24

gacatccaga tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc 60

atcagttgca gggcaagtca ggacattagt aaatatttaa attggtatca gcagaaacca 120

gatggaactg ttaaactcct gatctaccat acatcaagat tacactcagg agtcccatca 180

aggttcagtg gcagtgggtc tggaacagat tattctctca ccattagcaa cctggagcaa 240

gaagatattg ccacttactt ttgccaacag ggtaatacgc ttccgtacac gttcggaggg 300

gggaccaagc tggagatcac a 321

<210> 25

<211> 297

<212> DNA

<213>Artificial sequence

<400> 25

gcctctacaa agggcccaag cgtgttcccg ctggctccta gcagcaagtc tacaagcgga 60

ggaacagctg ccctgggatg cctggtcaag gactactttc ctgagccagt gaccgtgtct 120

tggaactctg gcgctctgac aagcggcgtg cacacatttc cagcagtgct gcagtccagc 180

ggcctgtact ctctgtctag cgtggtcaca gtgcccagct ctagcctggg cacacagacc 240

tacatctgca atgtgaacca caagcctagc aacaccaagg tcgacaagaa ggtggaa 297

<210> 26

<211> 312

<212> DNA

<213>Artificial sequence

<400> 26

gccgctccta gcgtgttcat cttcccacct tccgacgagc agctgaaaag cggcacagcc 60

tctgtcgtgt gcctgctgaa caacttctac cccagagaag ccaaggtgca gtggaaggtg 120

gacaacgccc tgcagagcgg caatagccaa gagagcgtga ccgagcagga cagcaaggac 180

tctacctaca gcctgagcag caccctgaca ctgagcaagg ccgactacga gaagcacaaa 240

gtgtacgcct gcgaagtgac ccaccagggc ctttctagcc ctgtgaccaa gagcttcaac 300

cggggcgagt gt 312

<210> 27

<211> 480

<212> DNA

<213>Artificial sequence

<400> 27

tttggcagag gcaccagcct gatcgtgcac ccctacattc agaaccccga tcctgccgtg 60

taccagctga gagacagcaa gagcagcgac aagagtgtgt gcctgttcac cgacttcgac 120

agccagacca acgtgtccca gagcaaggac agcgacgtgt acatcaccga taagtgcgtg 180

ctggacatgc ggagcatgga cttcaagagc aacagcgccg tggcttggag caacaagagc 240

gatttcgcct gcgccaacgc cttcaacaac agcattatcc ccgaggacac attcttccca 300

agtcctgaga gcagctgcga cgtgaagctg gtggaaaaga gcttcgagac agacaccaac 360

ctgaacttcc agaacctgag cgtgatcggc ttcagaatcc tgctgctgaa ggtggccggc 420

ttcaacctgc tgatgaccct gagactgtgg tccagccggg ccaagagatc tggatctgga 480

<210> 28

<211> 567

<212> DNA

<213>Artificial sequence

<400> 28

tttggcgagg gaagcagact gaccgtgctg gaagatctga agaacgtgtt cccacctgag 60

gtggccgtgt tcgagccttc tgaggccgag atcagccaca cacagaaagc cacactcgtg 120

tgtctggcca ccggcttcta tcccgatcac gtggaactgt cttggtgggt taacggcaaa 180

gaggtgcact ccggcgtctg cacagatccc cagcctctga aagaacagcc cgctctgaac 240

gacagcagat acgccctgtc cagcaggctg agagtgtccg ccacattctg gcaggacccc 300

agaaaccact tcagatgcca ggtgcagttc tacggcctga gcgagaacga tgagtggacc 360

caggatagag ccaagcctgt gactcagatc gtgtctgccg aagcctgggg cagagccgat 420

tgtggcttta ccagcgagag ctaccaacag ggcgtgctgt ctgccaccat cctgtacgag 480

atcctgctgg gcaaagccac tctgtacgcc gtgctggtgt ctgccctggt gctgatggcc 540

atggtcaagc ggaaagacag cagaggc 567

<210> 29

<211> 54

<212> DNA

<213>Artificial sequence

<400> 29

gaaggcagag gcagcctgct tacatgtggc gacgtggaag agaaccccgg acct 54

<210> 30

<211> 66

<212> DNA

<213>Artificial sequence

<400> 30

ggatctggcg ccaccaattt cagcctgctg aaacaggctg gcgacgtgga agagaaccct 60

ggacct 66

<210> 31

<211> 75

<212> DNA

<213>Artificial sequence

<400> 31

ggatctggcg tgaagcagac cctgaacttc gacctgctga aactggccgg cgacgtggaa 60

agcaatcctg gacct 75

<210> 32

<211> 69

<212> DNA

<213>Artificial sequence

<400> 32

ggctctggcc agtgtaccaa ttacgccctg ctgaaactgg ccggcgacgt ggaatctaac 60

cctggacct 69

<210> 33

<211> 63

<212> DNA

<213>Artificial sequence

<400> 33

atggctctgc ctgtgacagc tctgctgctg cctctggctc tgcttctgca tgccgccaga 60

cct 63

<210> 34

<211> 51

<212> DNA

<213>Artificial sequence

<400> 34

atgtggctgc agtctttgct gctgctggga accgtggcct gtagcatctc t 51

Claims (10)

1. a kind of chimeric antigen cell receptor, which is characterized in that it include antigen-binding domains and respectively with the antigen The α chains and β chains of binding structural domain connection, the antigen-binding domains are the Fab segments of antibody, and the antibody has and target The characteristic that protein-specific combines.

2. chimeric antigen cell receptor according to claim 1, which is characterized in that the antibody is the Dan Ke in mouse source Grand antibody, the monoclonal antibody in rabbit source, the single domain antibody of camelid origin, the single domain antibody in shark source, humanized antibody or Human antibody.

3. according to claim 1-2 any one of them chimeric antigen cell receptors, which is characterized in that the heavy chain of Fab segments can Become the amino acid sequence in area as shown in SEQ ID NO.12；The amino acid sequence of the light chain variable region of Fab segments such as SEQ ID Shown in NO.15.

4. according to claim 1-2 any one of them chimeric antigen cell receptors, which is characterized in that the weight of the Fab segments The amino acid sequence of chain constant region is as shown in SEQ ID NO.13.

5. according to claim 1-2 any one of them chimeric antigen cell receptors, which is characterized in that the Fab segments it is light The amino acid sequence of chain constant region is as shown in SEQ ID NO.16.

6. according to claim 1-2 any one of them chimeric antigen cell receptors, which is characterized in that

The α chains are made of hinge area, transmembrane region and the intracellular activity signal domain being linked in sequence, the hinge of the α chains Area, transmembrane region and intracellular activity signal domain are respectively the hinge area of mammalian cell T cell receptor α chains, transmembrane region And intracellular activity signal domain；

Preferably, the amino acid sequence of the α chains is as shown in SEQ ID NO.14.

7. according to claim 1-2 any one of them chimeric antigen cell receptors, which is characterized in that

The β chains are made of hinge area, transmembrane region and the intracellular activity signal domain being linked in sequence, the hinge of the β chains Area, transmembrane region and intracellular activity signal domain are respectively the hinge area of mammalian cell T cell receptor β chains, transmembrane region And intracellular activity signal domain；

Preferably, the amino acid sequence of the β chains is as shown in SEQ ID NO.17.

8. a kind of cell, which is characterized in that it expresses requirement 1-7 any one of them chimeric antigen cell receptors of having the right.

9. a kind of composition, which is characterized in that it includes cell according to any one of claims 8 and pharmaceutically acceptable auxiliary material.

10. application of the cell according to any one of claims 8 in preparing the drug for the treatment of tumour or autoimmune disease.