CN111944054B - anti-BCMA CAR and expression vector and application thereof - Google Patents

Abstract

The invention belongs to the technical field of genetic engineering, and particularly relates to a BCMA antigen-resistant CAR, an expression vector thereof, a CAR cell and application thereof. The CAR comprises an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain that recognizes a BCMA antigen; the antigen recognition region for recognizing the BCMA antigen is an anti-BCMA single-chain antibody, the heavy chain amino acid sequence of the antibody is shown in any one of SEQ ID NO 1-5, and the light chain amino acid sequence of the antibody is shown in any one of SEQ ID NO 6-10. The CAR can be stably expressed in T lymphocytes derived from patients, has better capacity of eliminating tumor cells, and can be used for preparing medicines for treating hematological malignancies, and adoptive cell therapy for the hematological malignancies; the anti-BCMA antigen CAR cell can effectively eliminate tumor target cells expressing the BCMA antigen, and has no toxic effect on negative antigens, namely tumor cells not expressing the BCMA, so that the safety is high.

Description

anti-BCMA CAR and expression vector and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a BCMA antigen-resistant CAR, an expression vector thereof, a CAR cell and application thereof.

Background

BCMA is named as tumor necrosis factor receptor super-family member 17, also called B-cell proliferation antigen (BCMA) or CD269 or TNFRSF13A, is mainly expressed on the surface of mature B lymphocytes and plasma cells, is highly expressed in MM tissues and cells, is a suitable target for treating malignant hemopathy, and primary myeloma (MM) belongs to one of the malignant hemopathy.

Multiple myeloma also called multiple myeloma, abbreviated as MM, has high heterogeneity and high recurrence probability of drug-resistant MM, cannot be cured at present, and the existing drugs can not solve the survival crisis of MM patients. And CAR-T therapy targeting CD19 achieves better curative effect in blood system tumors than traditional medicines, and MM treatment by CAR-T therapy can be a feasible and effective treatment mode.

Although CD 19-targeted chimeric antigen receptor T cell therapy has achieved excellent performance in hematologic tumor therapy, screening for a combination of CARs against MM remains a key point and difficulty in CAR-T therapy for MM because MM cells do not express CD19 due to the difference in surface molecules expressed by different diseases, and the difference in signal strength activated by different antibodies against different antigens.

The single-chain antibody is used as an important component of CAR, the selection of the single-chain antibody plays an important role in the curative effect of CAR-T, and the traditional murine antibody causes Human anti-mouse antibody reaction (HAMA) due to the heterogeneity of the murine antibody, so that CAR-T is rapidly cleared in the circulatory system and loses the curative effect. Therefore, therapeutic murine mabs require humanization modifications to increase the degree of humanization of the antibody, attenuating HAMA. However, the modification of the antibody usually results in the loss of the original antigen binding activity of the antibody, so the modification of the antibody is required, and then the antibody sequence with activity is screened through a large amount of antigen-antibody binding specificity and affinity detection. At present, clinical tests of targeting drugs targeting BCMA have limited efficacy and still have safety problems.

Therefore, the use of single chain antibodies (ScFv) suitable for CAR-T therapy and screening CARs for suitable engineered humanized ScFv are necessary to address treatment of hematological malignancies, including MM, with BCMA-targeted CAR-T.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a CAR against BCMA antigen. The anti-BCMA antigen CAR can be stably expressed in T lymphocytes of patient or healthy donor origin and has a better ability to eliminate tumor cells for adoptive cell therapy against hematological malignancies.

In order to achieve the purpose, the invention adopts the following scheme:

the CAR comprises an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain that recognizes a BCMA antigen; the antigen recognition region for recognizing the BCMA antigen is an anti-BCMA single-chain antibody, the amino acid sequence of the heavy chain is shown as SEQ ID NO. 1, and the amino acid sequence of the light chain is shown as SEQ ID NO. 6; or the heavy chain amino acid sequence is shown as SEQ ID NO. 2, and the light chain amino acid sequence is shown as SEQ ID NO. 7; or the heavy chain amino acid sequence is shown as SEQ ID NO. 3, and the light chain amino acid sequence is shown as SEQ ID NO. 8; or the heavy chain amino acid sequence is shown as SEQ ID NO. 4, and the light chain amino acid sequence is shown as SEQ ID NO. 9; or the heavy chain amino acid sequence is shown as SEQ ID NO. 5, and the light chain amino acid sequence is shown as SEQ ID NO. 10.

In some embodiments the ScFv is linked in the VH-Linker-VL, in some embodiments the ScFv can also be linked in the VL-Linker-VH; the sequence of Linker may be: GGGGSGGGGSGGGGS may also be (GGGGS) n where n is 1-6, and may also be GSTSGSGKPGSGEGSTKG.

Further, the CAR further comprises a leader peptide sequence derived from the CD8 leader peptide sequence.

Further, the CAR is characterized in that the anti-BCMA single-chain antibody is shown as SEQ ID NO11 or SEQ ID NO 12 or SEQ ID NO 13 or SEQ ID NO 14 or SEQ ID NO 15.

Further, the nucleotide sequence of the single-chain antibody for identifying the BCMA antigen is shown as SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23 or SEQ ID NO. 24.

Preferably, the nucleotide sequence of the antigen recognition region for recognizing BCMA antigen is shown as SEQ ID NO 21 or SEQ ID NO 22 or SEQ ID NO 23 or SEQ ID NO 24

Further, the hinge region is derived from CD8 or IgG4 or CD 7.

Furthermore, the amino acid sequence of the hinge region is shown as SEQ ID NO. 16 or SEQ ID NO. 17 or SEQ ID NO. 18 or SEQ ID NO. 19.

Further, the nucleotide sequence of the hinge region is shown as SEQ ID NO 25 or 26.

Further, the transmembrane region is derived from the transmembrane region of a CD8 molecule or a CD28 molecule; the intra-packet signal is derived from CD28 or CD137 intra-packet signal regions and CD3 intracellular sequences.

Further, the nucleotide sequence of the intracellular signal domain is shown as SEQ ID NO: 27/28.

Further, the nucleotide sequence of the CAR is shown as SEQ ID NO: 29/30/31/32/33/34/35/36.

It is a further object of the invention to provide an expression vector, in particular an expression vector comprising said CAR. When the expression vector containing the CAR is subjected to T cell transduction, the positive expression rate of the CAR is high, the CAR is stable in the cell culture process of a patient, and the positive rate cannot be reduced along with the time.

In order to achieve the purpose, the invention adopts the following scheme:

the vector is selected from a lentivirus expression vector, a retrovirus expression vector, an adenovirus expression vector, an adeno-associated virus expression vector, a DNA vector, an RNA vector or a plasmid.

Further, the vector is a lentiviral vector comprising an RRE element and an oPRE element.

Further, the vector comprises a pUCOri sequence, a kanamycin or ampicillin resistance gene sequence, an SV40Ori sequence, cis elements, a promoter of eukaryotic transcription, and a CAR encoding gene.

Specifically, the vector comprises a prokaryotic replicon pUOri sequence for plasmid replication; a kanamycin or ampicillin resistance gene sequence for mass amplification of a target strain; for enhancing replication of the viral replicon SV40Ori sequence in eukaryotic cells; a lentiviral packaging cis-element for lentiviral packaging; a promoter for eukaryotic transcription of a chimeric antigen receptor gene; and the CAR coding gene is used for combining target point recognition, signal transmission and signal initiation.

In certain embodiments, the vector comprises an expression vector Promoter for the CAR gene that uses 5HRE regulatory elements in duplicate in combination with a weakly-initiated CMV mini Promoter to constitute the 5HRE-CMVmini Promoter.

In certain embodiments, the vector comprises a left (5') retroviral LTR, a Psi (Ψ) packaging signal, a central polypurine tract/DNA FLAP (cPPT/FLAP), a retroviral export element, a promoter operably linked to a polynucleotide encoding a CAR encompassed herein, and a right (3') retroviral LTR.

In certain embodiments, the CAR vector comprises a hepatitis b virus post-transcriptional regulatory element (HPRE) or woodchuck post-transcriptional regulatory element (WPRE); the promoter of the 5' LTR is replaced by a heterologous promoter, which is a Cytomegalovirus (CMV) promoter, a Rous Sarcoma Virus (RSV) promoter or a Simian Virus 40(SV40) promoter.

It is a further object of the invention to provide a CAR cell, in particular a CAR cell comprising the expression vector. When BCMA on the surface of the tumor cell is combined with the antigen recognition region of the chimeric antigen receptor, signals are transmitted to the inside of the cell through the hinge region and the transmembrane region, the signal domain inside the cell converts the signals into activation signals, effector cells are activated, and the effector cells proliferate and generate cytokines so as to kill the tumor cell.

In order to achieve the purpose, the invention adopts the following scheme:

the cell is selected from stem cells, T cells, NK cells, monocytes or macrophages.

Preferably, the cell is a T cell.

In certain embodiments, the cell may be a γ δ T cell, a CD4 positive T cell, a CD8 positive T cell, or a cell in which a CD4 positive T cell and a CD8 positive T cell are mixed in a specific ratio; in certain embodiments, the virus comprising the gene encoding the CAR transduces NK cells, monocytes, or macrophages.

Preferably a lentiviral vector infected T cell.

It is a further object of the invention to provide a use of the CAR, the expression vector and the CAR cell.

In order to achieve the purpose, the invention adopts the following scheme:

the application is particularly the application in the preparation of the medicine for treating the malignant hematopathy.

Further, the hematologic malignancies include multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, B-cell lymphoma, and B-cell chronic lymphocytic proliferative disorders.

Further, the tumor cells of the hematological malignancy are capable of expressing BCMA.

Further, the CAR cells are used in combination with an active agent that is an immune checkpoint inhibitor and/or a polypeptide and/or a fusion protein and/or an antibody drug and/or a kinase inhibitor.

Specifically, these agents may be immune checkpoint inhibitors such as PD1/PDL1, TIM3, CTLA4, LAG3, TGFR β inhibitors, and the like; in other embodiments, these agents may be polypeptides or fusion proteins, such as fusion proteins formed by the PD-1-CD137-CD3 molecule; in other embodiments, these active agents may be antibody drugs, such as monoclonal antibody drugs, bispecific antibody drugs, and the like; in other embodiments, the active agent may be a kinase inhibitor, such as the tyrosine kinase inhibitor dasatinib. Specifically, the use routes of the medicament comprise: oral, intravenous, peritoneal infusion, subcutaneous injection, and tumor or organ local administration.

The invention has the beneficial effects that:

1) the anti-BCMA antigen CAR provided by the invention can be stably expressed in patient-derived T lymphocytes, has better capacity of eliminating tumor cells, and can be used for preparing a medicament for treating hematological malignancies, and can be used for adoptive cell therapy for the hematological malignancies.

2) The anti-BCMA antigen CAR cell provided by the invention can effectively remove tumor target cells expressing BCMA antigens, and has no toxic effect on negative antigen (not expressing BCMA) tumor cells, so that the safety is high;

3) the CAR-T cell of the anti-BCMA antigen provided by the invention uses the anti-BCMA antibody with high humanization degree, so that the immunogenicity of the CAR is reduced, and the persistence and safety of the CAR-T in vivo are enhanced.

Drawings

FIG. 1 is a schematic diagram of a different CAR.

FIG. 2 is a multiple myeloma cell H929 and negative K562 cell BCMA expression assay.

FIG. 3 shows the BCMA expression assay of tumor cells from different sources.

FIG. 4 is the results of killing of BCMA positive target cells by CAR-T cells of different CAR.

Figure 5 is the negative killing effect of different CARCAR-T cells on a variety of BCMA-non-expressing cells.

FIG. 6 is cytokine secretion after killing of positive target cells by different CARCAR-T cells.

Figure 7 is the in vivo efficacy of different CARCAR-T cell animals.

Figure 8 is the in vivo efficacy of different CARCAR-T cells at low doses.

Figure 9 is the effect of soluble BCMA on different cars-T cells.

Detailed Description

Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings. The experimental procedures, in the preferred embodiments, which do not specify specific conditions, are generally carried out according to conventional conditions, for example as described in the molecular cloning protocols (third edition, sambrook et al), or according to the conditions recommended by the manufacturers. The examples are provided for better illustration of the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.

Example 1 lentivirus preparation of chimeric antigen receptors expressing antigens targeting human BCMA antigen

(1) Preparation of chimeric antigen receptors targeting human BCMA antigens

A chimeric antigen receptor sequence was synthesized containing a single chain antibody ScFv against human BCMA antigen, hFc hinge region, transmembrane region and intracellular signal segment. The structure is shown in fig. 1. Wherein the nucleotide sequence of the humanized single-chain antibody of the anti-human BCMA antigen is shown as SEQ ID NO. 20 or 21 or 22 or 23 or 24; the nucleotide sequence of the hinge region is a DNA sequence encoding the amino acids SEQ ID NO 16 or SEQ ID NO 17 or SEQ ID NO 18 or SEQ ID NO 19; the transmembrane region is derived from a CD8 or CD28 sequence; the intracellular signal segment is derived from CD28 or CD137 and CD3 sequences.

(2) Construction of Lentiviral vectors expressing chimeric antigen receptors

Chimeric antigen receptor expression vectors were designated BCMA016, BCMA017, BCMA018, BCMA019, BCMA028, BCMA029, BCMA038, and BCMA040, respectively, depending on the loading of ScFv and transmembrane and intracellular signals. The nucleotide sequence is SEQ ID NO. 29 or SEQ ID NO. 30 or SEQ ID NO. 31 or SEQ ID NO. 32 or SEQ ID NO. 33 or SEQ ID NO. 34 or SEQ ID NO. 35 or SEQ ID NO. 36, and a murine control BCMA021 is designed. The enzyme digestion reaction was carried out as described in the specification. And separating and recovering the enzyme digestion product, then connecting the target fragment and the vector fragment by T4 ligase (purchased from Promega company) to obtain a lentiviral vector for expressing the chimeric antigen receptor, and extracting the plasmid by a plasmid extraction kit (Invitrogen company), wherein the specific method is described in the specification.

(3) Virus preparation

And (3) packaging the vector containing the CAR gene with lentivirus by adopting a calcium phosphate method, and respectively collecting and culturing packaged cell supernatants for 48h and 72h to purify the virus by using PEG6000 and NaCl. The purified virus was resuspended in culture medium and aliquoted into 1.5mL EP tubes and stored at-80 ℃ until use.

(4) Lentiviral titer determination

The virus infects the engineering cell, the virus titer is determined by using a flow cytometry technology after the infection for 72h, the light chain of the ScFv sequence of the CAR antigen recognition region can be recognized by Protein-L, therefore, the CAR positive rate can be detected by using the Protein-L, the virus infection capacity of 293T cell is indicated by the CAR positive rate, the virus titer is calculated according to the virus infection capacity of 293T cell, and the result is indicated by TU/mL. Titres as shown in the table below, different structural CARs all had better viral titres.

Virus	Titer of the product	Virus	Titer of the product
				BCMA016	4.12E+08	BCMA028	3.46E+08
BCMA017	1.20E+08	BCMA029	1.36E+08
				BCMA018	1.14E+08	BCMA038	2.30E+08
BCMA019	1.10E+08	BCMA040	1.27E+08
				BCMA021	9.80E+07

Example 2 preparation of chimeric antigen receptor-modified T cells for BCMA antigen

(1) Lentiviral infection of T cells

1) Isolation of human peripheral blood mononuclear cells

Separating lymphocytes by using a gradient centrifugation method; after centrifugation, the second white lymphocyte layer was washed with physiological saline and cultured in RPMI 1640 complete medium containing 10% FBS to obtain human peripheral blood mononuclear cells.

2) Lentiviral vector infection of T lymphocytes

Obtaining PBMC cells, activating by anti-CD 3 and CD28 monoclonal antibodies, and then infecting by lentivirus; CAR-T cells were cultured for about 10 days after infection for in vitro and in vivo biological characterization. The chimeric antigen receptor T cells obtained are named after the corresponding lentiviral vectors.

3) Chimeric Antigen Receptor (CAR) expression detection targeting human BCMA antigen

The CAR positive rate was tested for virus-infected T cells cultured up to day 6 and 9 during the culture. The detection method is flow detection, and the antibody is as follows: Protein-L-PE, Protein-L can recognize the antibody light chain, and the light chain of ScFv sequence of the CAR antigen recognition region can be recognized by Protein-L, so that the CAR positivity can be detected by using Protein-L. Results the CAR positivity after infection of T cells with different viruses, and CARs of different structures all were expressed on the cell surface, as shown in the table below.

Virus	CAR+	Virus	CAR+
				BCMA016	84.4％	BCMA016	53.52％
BCMA016	64.7％	BCMA016	26.00％
				BCMA016	80.5％	BCMA016	6.40％
BCMA016	39.00％	BCMA016	4.20％
				BCMA016	40.2％

Example 3 validation of anti-tumor Effect of T lymphocytes expressing chimeric antigen receptor targeting BCMA

BCMA positive H929 cells (simply referred to as H929-Luc) for stably expressing firefly luciferase, negative cells K562-Luc and BCMA high-expression cells K562-BCMA-Luc constructed in vitro by using K562 are used as target cells, BCMA expression detection of the target cells is shown in figures 2 and 3, and H929 is detected as the BCMA high-expression positive cells and K562 is detected as the BCMA negative cells by using flow cytometry and RT methods respectively.

Since CAR is excessive, validation is performed in 3 groups; CAR-T cells were prepared using 3 batches of white membranes using the method of example 2, respectively. The tunica albuginea is a source of peripheral blood mononuclear cells of healthy donors, the first batch of CAR-T cells are plated with effector cells according to a 16:1/8:1/4:1/2:1 effect target ratio, and the second and third batches of CAR-T cells are plated with effector cells according to a 8:1/4:1/2:1 effect target ratio. Use of

The killing effect is detected by a standard method provided by a Luciferase Assay System (Promega Cat. # E2520) kit, and the killing rate is calculated by the following formula:

killing results as shown in figure 4, the highest killing rates of A, B and C batches were different due to the differences in CAR positive rates of different donor cells, but did not affect the killing tendency of different CAR-T cells, and the results of comparing the killing effects in each batch of killing groups showed that all constructed CAR-T can exert the in vitro killing function, wherein BCMA016, BCMA017, BCMA018, BCMA019, BCMA028 and BCMA029 have better in vitro killing effects.

Example 4 chimeric antigen receptor T cell specificity validation for targeting BCMA

BCMA negative cell selection: a549, PLC, Lovo, Hela and K562. And (3) co-culturing for 6 hours at an effective target ratio of 8:1, observing the killing capacity of the CAR-T cells to negative cells compared with Control T, and verifying whether the CAR-T cells screened by the inventor have off-target killing to BCMA negative cells. The effector cells were the CAR-T cells of the second and third batches of example 3, and the results are shown in fig. 5, wherein the negative cells of a were selected from a549, PLC, LOVO and K562 cells, and the negative cells of B were selected from a549, Hela and K562 cells, and humanized ScFv-derived CAR-T cells had good specificity for the negative cells.

Example 5 detection of cytokine secretion Capacity of chimeric antigen receptor T cells stimulated by target antigen

Example 2 CAR-T cells subjected to in vitro efficient killing were co-cultured with target cells at an effective-to-target ratio of 8:1 for 6 hours, and supernatants thereof were collected and assayed for secretion of IFN- γ and IL-2 by ELISA (enzyme linked immunosorbent assay). IFN-gamma detection is carried out by adopting a BD IFN-gamma kit with the product number of 555142, and the experimental steps are carried out according to the product instruction.

The results are shown in FIG. 6, where all the constructed CAR-T cells of A and B can secrete IFN-gamma after killing positive target cells, demonstrating that all the constructed humanized CAR-T can recognize and be activated by BCMA on the surface of target cells, wherein BCMA019, BCMA021, BCMA016, BCMA029, BCMA038 and BCMA040 all have high factor secretion ability.

Example 6 demonstration of antitumor Effect of T lymphocytes expressing a targeting BCMA chimeric antigen receptor in an animal model

A mouse transplantation tumor model of a human BCMA positive tumor cell line is established for verifying the anti-tumor effect of the T lymphocyte expressing the chimeric antigen receptor targeting CD19 in an animal model.

Cg-PrkdcscidiI2rgtm1Sug/Jiccrl, NOG mouse for short, is the most common strain for CAR-T in vivo related tumor formation experiment in the world. The tumor target cells used in vivo validation were K562-BCMA cells. After mouse tumorigenesis, CAR-T cells, virus-uninfected PBMC cells, were injected tail-vein separately. The tumor growth was shown by imaging every 7 days after CAR T cell injection by IVS in vivo imaging system from PerkinElmer, with the smaller the fluorescence value, the more surviving mice had better treatment. Mice survival was observed daily and recorded during this period. The results are shown in FIGS. 7 and 8: the BCMA016, BCMA017, BCMA018, BCMA019 and BCMA028 constructed by the inventor have in-vivo curative effect due to Control T, wherein the BCMA018, the BCMA019, the BCMA017 and the BCMA028 have good in-vivo curative effect.

Example 7 soluble BCMA Effect on CAR-T effectiveness

Having high concentrations of soluble bcma (sBCMA) in the blood of multiple myeloma patients, it is necessary to verify whether the presence of sBCMA affects the effectiveness of CAR-T.

The BCMA high expression cell K562-BCMA-Luc constructed in vitro by K562 is used as a target cell, CAR-T cells are prepared by the method of example 5, the CAR-T cells are divided into two groups of a group with the addition of sBCMA and a group without the addition of sBCMA, the killing effect target ratio is 16:1, and the application is carried out

The killing effect is detected by a standard method provided by a Luciferase Assay System (Promega Cat. # E2520) kit, and the killing rate is calculated by the following formula:

the killing results are shown in fig. 9, and the results indicate that BCMA017, BCMA018, BCMA019, and BCMA028 activity are not affected by high concentrations of sbbcma in vitro.

In summary, we screened polypeptides comprising the amino acid sequence of SEQ ID NO 12 or the nucleic acid sequence of SEQ ID NO 21; the amino acid sequence is SEQ ID NO. 11 or the nucleic acid sequence is SEQ ID NO. 20; amino acid sequence is SEQ ID NO 13 or nucleic acid sequence is SEQ ID NO 22; the CAR with the amino acid sequence of SEQ ID NO. 14 or the nucleic acid sequence of SEQ ID NO. 23 and the CAR-T cell constructed by the CAR exert normal functions.

Finally, the above embodiments are only for illustrating the technical solutions of the present invention and not for limiting, although the present invention has been described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all of them should be covered in the claims of the present invention.

Sequence listing

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Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 11

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 11

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

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

195 200 205

Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 12

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 12

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

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

195 200 205

Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 13

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 13

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Pro Gly

1 5 10 15

Gln Arg Ala Thr Ile Thr Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Lys Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

Phe Arg Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala

195 200 205

Tyr Leu Gln Ile Ser Ser Leu Lys Ala Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 14

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 14

Asp Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

Arg Leu Leu Ile Tyr Leu Ala Ser Asn Leu Glu Thr Gly Val Pro Ser

50 55 60

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

65 70 75 80

Pro Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

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

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

Phe Arg Gly Arg Val Thr Met Thr Arg Asp Thr Ser Ile Ser Thr Ala

195 200 205

Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser

245

<210> 15

<211> 246

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 15

Asp Ile Val Leu Thr Gln Ser Pro Ala Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Lys Arg Ala Thr Ile Ser Cys Arg Ala Ser Glu Ser Val Ser Val Ile

20 25 30

Gly Ala His Leu Ile His Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro

35 40 45

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

50 55 60

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

65 70 75 80

Pro Val Glu Glu Glu Asp Val Gly Val Tyr Tyr Cys Leu Gln Ser Arg

85 90 95

Ile Phe Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Gly

100 105 110

Ser Thr Ser Gly Ser Gly Lys Pro Gly Ser Gly Glu Gly Ser Thr Lys

115 120 125

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

130 135 140

Glu Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp

145 150 155 160

Tyr Ser Ile Asn Trp Val Arg Arg Ala Pro Gly Lys Gly Leu Lys Trp

165 170 175

Met Gly Trp Ile Asn Thr Glu Thr Arg Glu Pro Ala Tyr Ala Tyr Asp

180 185 190

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

195 200 205

Tyr Leu Gln Ile Ser Ser Leu Lys Tyr Glu Asp Thr Ala Thr Tyr Phe

210 215 220

Cys Ala Leu Asp Tyr Ser Tyr Ala Met Asp Tyr Trp Gly Gln Gly Thr

225 230 235 240

Ser Val Thr Val Ser Ser

245

<210> 16

<211> 47

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 16

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala

20 25 30

Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp

35 40 45

<210> 17

<211> 36

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 17

Ala Pro Pro Arg Ala Ser Ala Leu Pro Ala Pro Pro Thr Gly Ser Ala

1 5 10 15

Leu Pro Asp Pro Gln Thr Ala Ser Ala Leu Pro Asp Pro Pro Ala Ala

20 25 30

Ser Ala Leu Pro

35

<210> 18

<211> 45

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 18

Lys Pro Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr

1 5 10 15

Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Arg Pro Ala Ala

20 25 30

Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Asp

35 40 45

<210> 19

<211> 229

<212> PRT

<213> Artificial sequence (Artificial sequence)

<400> 19

Glu Ser Lys Tyr Gly Pro Pro Cys Pro Ser Cys Pro Ala Pro Glu Phe

1 5 10 15

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

20 25 30

Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val

35 40 45

Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val

50 55 60

Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser

65 70 75 80

Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu

85 90 95

Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser

100 105 110

Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro

115 120 125

Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln

130 135 140

Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala

145 150 155 160

Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr

165 170 175

Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu

180 185 190

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

195 200 205

Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser

210 215 220

Leu Ser Leu Gly Lys

225

<210> 20

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 20

gatatcgtgc tgacacagag ccctgcctcc ctggccgtga gcctgggaca gcgggccacc 60

atcacatgca gagcctccga gtctgtgagc gtgatcggcg cccacctgat ccactggtac 120

cagcagaagc ctggccagcc ccctaagctg ctgatctatc tggcctctaa cctggagacc 180

ggagtgcctg cacggttctc cggctctggc agcggcacag actttaccct gacaatcgat 240

ccagtggagg ccgaggacgt gggcgtgtac tattgcctgc agtccaggat cttcccccgc 300

acctttggcg gcggcacaaa gctggagatc aagggatcca cctctggaag cggcaagcca 360

ggatctggag agggaagcac aaagggacag gtgcagctgg tgcagagcgg accagaggtg 420

aagaagccag gagcctccgt gaaggtgtct tgtaaggcca gcggctacac cttcacagat 480

tattccatca actgggtgag gcaggcacca ggacagggac tggagtggat gggctggatc 540

aataccgaga cacgcgagcc cgcctacgcc tatgattttc ggggcagatt cgtgttttcc 600

ctggacacct ccgtgtctac agcctacctg cagatcagct ccctgaaggc cgaggacacc 660

gccgtgtact attgtgccct ggattactcc tatgccatgg actattgggg ccagggcacc 720

tctgtgacag tgtctagc 738

<210> 21

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 21

gacatcgtgc tgacacagag ccctgcctcc ctggccgtga gcccaggaca gcgggccacc 60

atcacatgca gagcctccga gtctgtgagc gtgatcggcg cccacctgat ccactggtac 120

cagcagaagc caggacagcc acctaagctg ctgatctatc tggcctctaa cctggagacc 180

ggagtgccag cacggttctc cggctctggc agcggcacag actttaccct gacaatcaat 240

cccgtggagg ccgaggatgt gggcgtgtac tattgcctgc agtccaggat cttccctcgc 300

acctttggcg gcggcacaaa gctggagatc aagggatcca cctctggaag cggcaagcca 360

ggatctggag agggaagcac aaagggacag gtgcagctgg tgcagagcgg agcagaggtg 420

aagaagccag gagcctccgt gaaggtgtct tgtaaggcca gcggctacac cttcacagac 480

tattccatca actgggtgag gcaggcacca ggacagggac tggagtggat gggctggatc 540

aataccgaga cacgcgagcc tgcctacgcc tatgactttc ggggcagatt cgtgttttcc 600

ctggatacct ccgtgtctac agcctacctg cagatcagct ccctgaaggc cgaggatacc 660

gccgtgtact attgtgccct ggactactcc tatgccatgg attattgggg ccagggcacc 720

tctgtgacag tgtctagc 738

<210> 22

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 22

gatatcgtgc tgacacagag ccctgcctcc ctggccgtga gcccaggaca gagggcaacc 60

atcacatgca gggcctccga gtctgtgagc gtgatcggcg cccacctgat ccactggtac 120

cagcagaagc caggacagcc acctaagctg ctgatctatc tggcctctaa cctggagacc 180

ggagtgccag cacggttctc cggctctggc agcggcacag actttaccct gacaatcgat 240

cccgtggagg aggaggacgt gggcgtgtac tattgcctgc agtcccggat cttccctaga 300

acctttggcg gcggcacaaa gctggagatc aagggatcca cctctggaag cggcaagcca 360

ggatctggag agggaagcac aaagggacag gtgcagctgg tgcagagcgg agcagaggtg 420

aagaagccag gagcctccgt gaaggtgtct tgtaaggcca gcggctacac cttcacagac 480

tattccatca actgggtgcg gagagcacca ggcaagggcc tgaagtggat gggatggatc 540

aataccgaga caagagagcc tgcctacgcc tatgacttcc ggggccgctt cgccttttcc 600

ctggagacct ccgcctctac agcctacctg cagatcagct ccctgaaggc cgaggacacc 660

gccgtgtact attgtgccct ggattactcc tatgccatgg actattgggg ccagggcacc 720

tctgtgacag tgtctagc 738

<210> 23

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 23

gacatcgtgc tgacccagag cccagccaca ctgagcctgt ccctgggaga gagggccaca 60

ctgtcctgca gagcctctga gagcgtgtcc gtgatcggcg cccacctgat ccactggtac 120

cagcagaagc caggacagcc acctaggctg ctgatctatc tggcctctaa cctggagacc 180

ggcgtgccta gcagattctc tggcagcggc tccggcacag actttaccct gacaatctct 240

ccactggagc ccgaggattt cgccgtgtac tattgcctgc agagcaggat cttcccacgc 300

acctttggcg gcggcacaaa gctggagatc aagggatcta ccagcggatc cggcaagcct 360

ggatccggag agggctctac aaagggacag gtgcagctgg tgcagtccgg acctgagctg 420

aagaagccag gcagctccgt gaaggtgagc tgtaaggcct ccggctacac cttcacagac 480

tatagcatca actgggtgag gcaggcacct ggacagggac tggagtggat gggctggatc 540

aataccgaga cacgcgagcc agcctacgcc tatgactttc ggggcagagt gaccatgaca 600

agggatacct ctatcagcac agcctacatg gagctgtcta gcctgcgctc cgaggatacc 660

gccgtgtact attgtgccct ggactactct tatgccatgg attattgggg ccagggcacc 720

ctggtgacag tgtcctct 738

<210> 24

<211> 738

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 24

gatatcgtgc tgacccagag cccagcctcc ctggccgtgt ccctgggcaa gagggccaca 60

atctcttgcc gcgcctccga gtctgtgagc gtgatcggcg cccacctgat ccactggtac 120

cagcagaagc ctggccagcc ccctaagctg ctgatctatc tggccagcaa cctggagacc 180

ggagtgccag cacggttctc cggctctggc agcggcacag actttaccct gacaatcgat 240

cccgtggagg aggaggacgt gggcgtgtac tattgcctgc agtcccggat cttccctaga 300

acctttggcg gcggcacaaa gctggagatc aagggatcca cctctggaag cggcaagcca 360

ggatctggag agggaagcac aaagggacag gtgcagctgg tgcagtctgg agcagaggtg 420

aagaagccag gagagtccgt gaaggtgtct tgtaaggcca gcggctacac cttcacagat 480

tattccatca actgggtgcg gagagcacca ggcaagggcc tgaagtggat gggatggatc 540

aataccgaga caagagagcc tgcctacgcc tatgatttca ggggccgctt cgtgtttagc 600

ctggacacct ccgcctctac agcctacctg cagatcagct ccctgaagta cgaggacacc 660

gccacatatt tttgtgccct ggattacagc tatgccatgg actattgggg ccagggcacc 720

tccgtgacag tgtctagc 738

<210> 25

<211> 135

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 25

accacgacgc cagcgccgcg accaccaaca ccggcgccca ccatcgcgtc gcagcccctg 60

tccctgcgcc cagaggcgtg ccggccagcg gcggggggcg cagtgcacac gagggggctg 120

gacttcgcct gtgat 135

<210> 26

<211> 108

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 26

gcaccacctc gggccagcgc cctgcctgca ccacccaccg gctccgccct gccagaccct 60

cagacagcat ctgccctgcc agatcctcca gcagcaagcg ccctgccc 108

<210> 27

<211> 537

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 27

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 60

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 120

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 180

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 240

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 300

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 360

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 420

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 480

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 537

<210> 28

<211> 462

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 28

aggagcaagc ggagcagagg cggccacagc gactacatga acatgacccc ccggaggcct 60

ggccccaccc ggaagcacta ccagccctac gcccctccca gggacttcgc cgcctaccgg 120

agccgggtga agttcagccg gagcgccgac gcccctgcct accagcaggg ccagagccag 180

ctgtacaacg agctgaacct gggccggagg gaggagtacg acgtgctgga caagcggaga 240

ggccgggacc ctgagatggg cggcaagccc cggagaaaga accctcagga gggcctgtat 300

aacgaactgc agaaagacaa gatggccgag gcctacagcg agatcggcat gaagggcgag 360

cggcggaggg gcaagggcca cgacggcctg taccagggcc tgagcaccgc caccaaggat 420

acctacgacg ccctgcacat gcaggccctg ccccctcgct aa 462

<210> 29

<211> 1497

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 29

gctagcatgg ccctgccagt gaccgccctg ctgctgcccc tggccctgct gctgcacgca 60

gcaaggcctg acatcgtgct gacacagagc cctgcctccc tggccgtgag cccaggacag 120

cgggccacca tcacatgcag agcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgccagc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcaatc ccgtggaggc cgaggatgtg ggcgtgtact attgcctgca gtccaggatc 360

ttccctcgca cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

gcagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagact attccatcaa ctgggtgagg caggcaccag gacagggact ggagtggatg 600

ggctggatca ataccgagac acgcgagcct gcctacgcct atgactttcg gggcagattc 660

gtgttttccc tggatacctc cgtgtctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggataccg ccgtgtacta ttgtgccctg gactactcct atgccatgga ttattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatgaattc 960

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 1497

<210> 30

<211> 1497

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 30

gctagcatgg cactgcctgt gaccgccctg ctgctgccac tggccctgct gctgcacgca 60

gcaaggccag atatcgtgct gacacagagc cctgcctccc tggccgtgag cctgggacag 120

cgggccacca tcacatgcag agcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc tggccagccc cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgcctgc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcgatc cagtggaggc cgaggacgtg ggcgtgtact attgcctgca gtccaggatc 360

ttcccccgca cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

ccagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagatt attccatcaa ctgggtgagg caggcaccag gacagggact ggagtggatg 600

ggctggatca ataccgagac acgcgagccc gcctacgcct atgattttcg gggcagattc 660

gtgttttccc tggacacctc cgtgtctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggacaccg ccgtgtacta ttgtgccctg gattactcct atgccatgga ctattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatgaattc 960

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 1497

<210> 31

<211> 1497

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 31

gctagcatgg ccctgccagt gaccgccctg ctgctgcccc tggccctgct gctgcacgca 60

gcacggcctg atatcgtgct gacacagagc cctgcctccc tggccgtgag cccaggacag 120

agggcaacca tcacatgcag ggcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgccagc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcgatc ccgtggagga ggaggacgtg ggcgtgtact attgcctgca gtcccggatc 360

ttccctagaa cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

gcagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagact attccatcaa ctgggtgcgg agagcaccag gcaagggcct gaagtggatg 600

ggatggatca ataccgagac aagagagcct gcctacgcct atgacttccg gggccgcttc 660

gccttttccc tggagacctc cgcctctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggacaccg ccgtgtacta ttgtgccctg gattactcct atgccatgga ctattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatgaattc 960

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 1497

<210> 32

<211> 1497

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 32

gctagcatgg cactgccagt gaccgccctg ctgctgcctc tggccctgct gctgcacgca 60

gcaaggccag acatcgtgct gacccagagc ccagccacac tgagcctgtc cctgggagag 120

agggccacac tgtcctgcag agcctctgag agcgtgtccg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaggctgc tgatctatct ggcctctaac 240

ctggagaccg gcgtgcctag cagattctct ggcagcggct ccggcacaga ctttaccctg 300

acaatctctc cactggagcc cgaggatttc gccgtgtact attgcctgca gagcaggatc 360

ttcccacgca cctttggcgg cggcacaaag ctggagatca agggatctac cagcggatcc 420

ggcaagcctg gatccggaga gggctctaca aagggacagg tgcagctggt gcagtccgga 480

cctgagctga agaagccagg cagctccgtg aaggtgagct gtaaggcctc cggctacacc 540

ttcacagact atagcatcaa ctgggtgagg caggcacctg gacagggact ggagtggatg 600

ggctggatca ataccgagac acgcgagcca gcctacgcct atgactttcg gggcagagtg 660

accatgacaa gggatacctc tatcagcaca gcctacatgg agctgtctag cctgcgctcc 720

gaggataccg ccgtgtacta ttgtgccctg gactactctt atgccatgga ttattggggc 780

cagggcaccc tggtgacagt gtcctctctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgtgc 900

cggccagcgg cggggggcgc agtgcacacg agggggctgg acttcgcctg tgatgaattc 960

atctacatct gggcgccctt ggccgggact tgtggggtcc ttctcctgtc actggttatc 1020

accctttact gcaaacgggg cagaaagaaa ctcctgtata tattcaaaca accatttatg 1080

agaccagtac aaactactca agaggaagat ggctgtagct gccgatttcc agaagaagaa 1140

gaaggaggat gtgaactgag agtgaagttc agcaggagcg cagacgcccc cgcgtacaag 1200

cagggccaga accagctcta taacgagctc aatctaggac gaagagagga gtacgatgtt 1260

ttggacaaga gacgtggccg ggaccctgag atggggggaa agccgagaag gaagaaccct 1320

caggaaggcc tgtacaatga actgcagaaa gataagatgg cggaggccta cagtgagatt 1380

gggatgaaag gcgagcgccg gaggggcaag gggcacgatg gcctttacca gggtctcagt 1440

acagccacca aggacaccta cgacgccctt cacatgcagg ccctgccccc tcgctaa 1497

<210> 33

<211> 1491

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 33

gctagcatgg cactgcctgt gaccgccctg ctgctgccac tggccctgct gctgcacgca 60

gcaaggccag atatcgtgct gacacagagc cctgcctccc tggccgtgag cctgggacag 120

cgggccacca tcacatgcag agcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc tggccagccc cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgcctgc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcgatc cagtggaggc cgaggacgtg ggcgtgtact attgcctgca gtccaggatc 360

ttcccccgca cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

ccagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagatt attccatcaa ctgggtgagg caggcaccag gacagggact ggagtggatg 600

ggctggatca ataccgagac acgcgagccc gcctacgcct atgattttcg gggcagattc 660

gtgttttccc tggacacctc cgtgtctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggacaccg ccgtgtacta ttgtgccctg gattactcct atgccatgga ctattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgcgg 900

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgccgatga attcatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1080

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1140

ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1200

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 1491

<210> 34

<211> 1491

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 34

gctagcatgg ccctgccagt gaccgccctg ctgctgcccc tggccctgct gctgcacgca 60

gcacggcctg atatcgtgct gacacagagc cctgcctccc tggccgtgag cccaggacag 120

agggcaacca tcacatgcag ggcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgccagc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcgatc ccgtggagga ggaggacgtg ggcgtgtact attgcctgca gtcccggatc 360

ttccctagaa cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

gcagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagact attccatcaa ctgggtgcgg agagcaccag gcaagggcct gaagtggatg 600

ggatggatca ataccgagac aagagagcct gcctacgcct atgacttccg gggccgcttc 660

gccttttccc tggagacctc cgcctctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggacaccg ccgtgtacta ttgtgccctg gattactcct atgccatgga ctattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgaga ccacgacgcc agcgccgcga 840

ccaccaacac cggcgcccac catcgcgtcg cagcccctgt ccctgcgccc agaggcgcgg 900

ccagcggcgg ggggcgcagt gcacacgagg gggctggact tcgccgatga attcatctac 960

atctgggcgc ccttggccgg gacttgtggg gtccttctcc tgtcactggt tatcaccctt 1020

tactgcaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 1080

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 1140

ggatgtgaac tgagagtgaa gttcagcagg agcgcagacg cccccgcgta caagcagggc 1200

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 1260

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 1320

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattgggatg 1380

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 1440

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 1491

<210> 35

<211> 1470

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 35

gctagcatgg ccctgccagt gaccgccctg ctgctgcccc tggccctgct gctgcacgca 60

gcaaggcctg acatcgtgct gacacagagc cctgcctccc tggccgtgag cccaggacag 120

cgggccacca tcacatgcag agcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgccagc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcaatc ccgtggaggc cgaggatgtg ggcgtgtact attgcctgca gtccaggatc 360

ttccctcgca cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

gcagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagact attccatcaa ctgggtgagg caggcaccag gacagggact ggagtggatg 600

ggctggatca ataccgagac acgcgagcct gcctacgcct atgactttcg gggcagattc 660

gtgttttccc tggatacctc cgtgtctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggataccg ccgtgtacta ttgtgccctg gactactcct atgccatgga ttattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgagg caccacctcg ggccagcgcc 840

ctgcctgcac cacccaccgg ctccgccctg ccagaccctc agacagcatc tgccctgcca 900

gatcctccag cagcaagcgc cctgcccgaa ttcatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgctaa 1470

<210> 36

<211> 1470

<212> DNA

<213> Artificial sequence (Artificial sequence)

<400> 36

gctagcatgg ccctgccagt gaccgccctg ctgctgcccc tggccctgct gctgcacgca 60

gcacggcctg atatcgtgct gacacagagc cctgcctccc tggccgtgag cccaggacag 120

agggcaacca tcacatgcag ggcctccgag tctgtgagcg tgatcggcgc ccacctgatc 180

cactggtacc agcagaagcc aggacagcca cctaagctgc tgatctatct ggcctctaac 240

ctggagaccg gagtgccagc acggttctcc ggctctggca gcggcacaga ctttaccctg 300

acaatcgatc ccgtggagga ggaggacgtg ggcgtgtact attgcctgca gtcccggatc 360

ttccctagaa cctttggcgg cggcacaaag ctggagatca agggatccac ctctggaagc 420

ggcaagccag gatctggaga gggaagcaca aagggacagg tgcagctggt gcagagcgga 480

gcagaggtga agaagccagg agcctccgtg aaggtgtctt gtaaggccag cggctacacc 540

ttcacagact attccatcaa ctgggtgcgg agagcaccag gcaagggcct gaagtggatg 600

ggatggatca ataccgagac aagagagcct gcctacgcct atgacttccg gggccgcttc 660

gccttttccc tggagacctc cgcctctaca gcctacctgc agatcagctc cctgaaggcc 720

gaggacaccg ccgtgtacta ttgtgccctg gattactcct atgccatgga ctattggggc 780

cagggcacct ctgtgacagt gtctagcctc gagctcgagg caccacctcg ggccagcgcc 840

ctgcctgcac cacccaccgg ctccgccctg ccagaccctc agacagcatc tgccctgcca 900

gatcctccag cagcaagcgc cctgcccgaa ttcatctaca tctgggcgcc cttggccggg 960

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaaacg gggcagaaag 1020

aaactcctgt atatattcaa acaaccattt atgagaccag tacaaactac tcaagaggaa 1080

gatggctgta gctgccgatt tccagaagaa gaagaaggag gatgtgaact gagagtgaag 1140

ttcagcagga gcgcagacgc ccccgcgtac aagcagggcc agaaccagct ctataacgag 1200

ctcaatctag gacgaagaga ggagtacgat gttttggaca agagacgtgg ccgggaccct 1260

gagatggggg gaaagccgag aaggaagaac cctcaggaag gcctgtacaa tgaactgcag 1320

aaagataaga tggcggaggc ctacagtgag attgggatga aaggcgagcg ccggaggggc 1380

aaggggcacg atggccttta ccagggtctc agtacagcca ccaaggacac ctacgacgcc 1440

cttcacatgc aggccctgcc ccctcgctaa 1470

Claims (21)

1. Use of an anti-BCMA antigen CAR, comprising an antigen recognition region, a hinge region, a transmembrane region and an intracellular signaling domain that recognizes a BCMA antigen, in the preparation of a medicament for the treatment of a BCMA-expressing hematological malignancy; the antigen recognition region for recognizing the BCMA antigen is an anti-BCMA single-chain antibody, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 4, and the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 9.

2. The use of claim 1, further comprising a leader peptide, wherein the leader peptide sequence is derived from the leader peptide sequence of CD 8.

3. The use according to claim 1, wherein said anti-BCMA single chain antibody is represented by SEQ ID No. 14.

4. The use of claim 1, wherein the nucleotide sequence of the antigen recognition region is set forth in SEQ ID NO. 23.

5. The use according to claim 1, wherein said hinge region is derived from CD8 or IgG4 or CD 7.

6. The use of claim 5, wherein the amino acid sequence of the hinge region is as shown in SEQ ID NO 16 or 17 or 18 or 19.

7. The use of claim 6, wherein the hinge region has a nucleotide sequence as set forth in SEQ ID NO 25 or 26.

8. The use of claim 1, wherein the transmembrane region is derived from CD8 or CD 28.

9. The use of claim 1, wherein the intracellular signal domain is derived from CD28 and/or CD137 and CD3 or CD3 alone.

10. The use of claim 9, wherein the intracellular signal domain has a nucleotide sequence as set forth in SEQ ID NOs 27-28.

11. The use of claim 1, wherein the CAR has the nucleotide sequence set forth in SEQ ID NO 32.

12. The use of claim 1, wherein the hematological malignancy comprises multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, B-cell lymphoma, and B-cell chronic lymphocytic proliferative disorders.

13. An expression vector comprising a CAR, wherein the CAR comprises an antigen recognition region, a hinge region, a transmembrane region, and an intracellular signaling domain that recognizes a BCMA antigen; the antigen recognition region for recognizing the BCMA antigen is an anti-BCMA single-chain antibody, the heavy chain amino acid sequence of the antibody is shown as SEQ ID NO. 4, and the light chain amino acid sequence of the antibody is shown as SEQ ID NO. 9; the vector is selected from a lentivirus expression vector, a retrovirus expression vector, an adeno-associated virus expression vector and an RNA vector.

14. The expression vector of claim 13, wherein the vector is a lentiviral vector comprising an RRE element and an oPRE element.

15. The expression vector of claim 13, wherein the vector comprises a pUCOri sequence, a kanamycin or ampicillin resistance gene sequence, an SV40Ori sequence, a cis element, a promoter of eukaryotic transcription, and a CAR encoding gene.

16. A CAR cell comprising the expression vector of any one of claims 13-15.

17. The CAR cell of claim 16, wherein the cell is a T cell.

18. Use of an expression vector according to claim 13 and/or a CAR cell according to claim 16 in the manufacture of a medicament for the treatment of hematological malignancies.

19. The use of claim 18, wherein the hematological malignancy comprises multiple myeloma, acute myelogenous leukemia, acute lymphocytic leukemia, B-cell lymphoma, and B-cell chronic lymphocytic proliferative disorders.

20. The use according to claim 18, wherein the tumor cells of hematological malignancies are capable of expressing BCMA.

21. Use according to claim 18, wherein the CAR cells are used in combination with an active agent which is an immune checkpoint inhibitor and/or a polypeptide and/or an antibody-drug conjugate and/or a kinase inhibitor.

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