CN115044617A - Preparation method of CAR T cell, CAR T cell and application thereof - Google Patents

Preparation method of CAR T cell, CAR T cell and application thereof Download PDF

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CN115044617A
CN115044617A CN202110249691.9A CN202110249691A CN115044617A CN 115044617 A CN115044617 A CN 115044617A CN 202110249691 A CN202110249691 A CN 202110249691A CN 115044617 A CN115044617 A CN 115044617A
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李建强
刘莹
王琳
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Hebei Senlang Biotechnology Co ltd
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Hebei Senlang Biotechnology Co ltd
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Abstract

The invention discloses a preparation method of a CAR T cell, the CAR T cell and application thereof. The preparation method of the CAR T cell comprises the following steps: introducing the CAR gene into peripheral blood-derived T cells to obtain recombinant cells; culturing the recombinant cells for 3-12 days to obtain the CAR T cells; the CAR gene encodes a CAR that specifically targets CD 7. The CAR T cells prepared by the T cells from the peripheral blood of healthy donors or patients can be prepared by the preparation method, the endogenous CD7 is knocked out by an additional method, the cell amount meeting the requirements of the patients can be prepared by in vitro amplification, and the production cost is saved.

Description

Preparation method of CAR T cell, CAR T cell and application thereof
Technical Field
The invention relates to the technical field of biology, and particularly relates to a CAR T cell preparation method, a CAR T cell and application thereof.
Background
Studies have shown that CD7 is expressed in greater than 95% of lymphoblastic T-cell leukemias and lymphomas as well as peripheral blood T-cell lymphomas. Therefore, the CD7 molecule is used as an abnormal marker on the surface of tumor cells of malignant diseases in the blood system and is used as a target of an immunotherapy antibody of malignant T lymphocyte leukemia. CD7 is also partially expressed in normal NK cells and T cells.
Most researchers consider that CD7 endogenous to T cells can kill CD7-CAR T cells with each other, and CD7-CAR T cells are difficult to prepare in vitro, so most researchers currently prepare CD7-CAR T cells by knocking out CD7 endogenous to T cells through a gene editing method or blocking expression of CD7 endogenous to T cells through a blocker of CD7, while CD7-CAR T cells without endogenous CD7 in T cells expand cells meeting clinical requirements.
Disclosure of Invention
The invention provides a method for preparing a CAR T cell, the method comprising: introducing the CAR gene into peripheral blood-derived T cells to obtain recombinant cells; culturing the recombinant cells for 3-12 days to obtain the CAR T cells; the CAR gene encodes a CAR that specifically targets CD 7.
The peripheral blood-derived T cells contain an endogenous CD7 gene, and express endogenous CD 7.
The method can comprise the following steps: (1) activating the peripheral blood-derived T cells, and culturing for 2 days, for example, by using Dynabeads CD3/CD28 activated magnetic beads; (2) introducing a CAR gene into the T cells subjected to the treatment in the step (1) to obtain recombinant cells; (3) culturing the recombinant cells for 3-12 days to obtain the CAR T cells.
As above, culturing the recombinant cell for 3 to 12 days may be, for example, culturing the recombinant cell for 7 to 12 days or 10 to 12 days.
Alternatively, the CAR comprises a single chain antibody against CD7 according to the preparation method described above. The CAR can be linked to a single chain antibody against CD7, 4-1BB, CD3 ζ, and tfegfr. For example, the CAR is a protein whose amino acid sequence is SEQ ID No.2 of the sequence Listing.
Optionally, the coding sequence of the coding strand of the CAR gene is position 15-2585 in SEQ ID No. 1.
As above, the peripheral blood-derived T cells may be from a healthy donor or a tumor patient.
CAR T cells prepared according to the above preparation method are also within the scope of the present invention.
Optionally, the CAR T cells have endogenous CD7 expression levels that are less than 90% of peripheral blood-derived T cells. The CAR T cells may have endogenous CD7 expression levels that are less than 90%, 93%, 95%, or 99% of peripheral blood-derived T cells.
The invention also provides a method for preparing the CAR T cell with the endogenous CD7 gene knocked out, wherein the method comprises the following steps:
(1) introducing the CAR gene into peripheral blood-derived T cells to obtain recombinant cells;
(2) knocking out a CD7 gene of the recombinant cell by using a CRISPR/Cas9 system to obtain a recombinant cell with a knocked-out CD7 gene, wherein the CD7 gene knocked-out recombinant cell is the CAR T cell with an endogenous CD7 gene knocked out;
the CAR gene is the CAR gene described above.
The CRISPR/Cas9 system can include a gRNA that specifically targets the CD7 gene. The target nucleotide sequence of the gRNA specifically targeting the CD7 gene may be 5'-GGAGCAGGTGATGTTGACGG-3'. For example, the sequence of the gRNA specifically targeting the CD7 gene is shown in SEQ ID No. 4. The CRISPR/Cas9 system specifically can include the Cas9 RNP-CD7 complex prepared by the examples.
Optionally, in the above method for preparing a CAR T cell knocking out an endogenous CD7 gene, the step (2) includes transfecting a Cas9 protein and the gRNA into the recombinant cell by electroporation. Alternatively, the electroporation transfection voltage is 250V, the pulse time is 800. mu.s, and the number of electric pulse transfections is 2 times, with an interval of 1000 s.
Optionally, in the method for preparing a CAR T cell with an endogenous CD7 gene knockout function, step (1) may further include: the peripheral blood-derived T cells were activated and cultured for 2 days. Activation can be performed, for example, using Dynabeads CD3/CD28 activated magnetic beads.
The CAR T cell with the endogenous CD7 gene knocked out, prepared according to the method for preparing the CAR T cell with the endogenous CD7 gene knocked out, also belongs to the protection scope of the invention.
The invention also provides an anti-tumor drug which comprises the CAR T cell and/or the CAR T cell with the endogenous CD7 gene knocked out.
The CAR T cell preparation method described above or the CAR T cell preparation method described above knocking out the endogenous CD7 gene or the use of the CAR T cell described above knocking out the endogenous CD7 gene is also within the scope of the present invention. The application can be specifically the application in the preparation of antitumor drugs.
The tumor can be a CD 7-targeted malignancy, and can also be a T cell malignancy, for example, the tumor is selected from one or more of acute T lymphocyte leukemia, T cell lymphoma, and acute myeloid leukemia.
The CAR T cells prepared from T cells derived from peripheral blood of healthy donors or patients can be used for preparing the CAR T cells, endogenous CD7 is knocked out by an additional method, the cell amount meeting the requirements of the patients can be prepared by in vitro amplification, and the production cost is saved.
The CAR T cell prepared by the method has the characteristics of high transfection rate of more than 90% and high proportion of central memory T cells and effector memory T cells, and has effective anti-tumor capability in vitro and in vivo.
Drawings
FIG. 1 is a schematic diagram of the structure of the CD7-CAR gene.
FIG. 2 shows a flow of preparation of Senl _ T7WT cells.
FIG. 3 shows a scheme for preparing Senl-T7 KO cells.
FIG. 4 is a comparison of the two methods of preparation of Senl _ T7KO cells
FIG. 5 shows T cells, Senl _ T7WT cells and Senl _ T7KO T-E Cells were tested for CD7 expression.
FIG. 6 shows Senl _ T7WT cells and Senl_T7 KO T-E Amplification of cells.
FIG. 7 shows Senl _ T7WT cells and Senl _ T7KO T-E CAR expression detection of cells.
FIG. 8 shows Senl _ T7WT cells and Senl _ T7KO T-E Comparison of cells.
FIG. 9 shows Senl _ T7WT cells and Senl _ T7KO T-E And (3) detecting the anti-tumor capacity of the cells in vitro.
FIG. 10 shows Senl _ T7WT cells and Senl _ T7KO T-E And (4) detecting the cell cytokine.
FIG. 11 shows Senl _ T7WT cells and Senl _ T7KO after cryopreservation and recovery T-E Viability of the cells and in vitro activity.
FIG. 12 is a flow chart of animal experiment sample feeding.
FIG. 13 changes in fluorescence intensity in tumor-bearing mice after administration.
Figure 14 is the expansion of CAR T cells in patients after reinfusion and the expression level of CD7 in patients.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples, unless otherwise indicated, are conventional and are carried out according to the techniques or conditions described in the literature in the field or according to the instructions of the products. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
The data were processed using GraphPad prism8 statistical software, and the results were expressed as mean. + -. standard deviation using the Atwo-tailed, unpaired t test.
1. Preparation of Peripheral Blood Mononuclear Cells (PBMCs):
the peripheral blood may be the peripheral blood of a healthy donor or of the patient himself. If the patient's own peripheral blood needs to be sorted by screening the normal T cell marker antibody according to the comprehensive evaluation sorting scheme of the patient's peripheral blood MRD and bone marrow blood MRD. Sorting is generally performed using either CD3 magnetic beads or CD4 and CD8 magnetic beads.
Mixing peripheral blood and sterile physiological saline at a ratio of 1: 1, diluting, slowly adding into mononuclear cell separating medium (Oriental Huahui), centrifuging at density gradient of 2: 1, centrifuging at 800g for 20min, and increasing 7 and decreasing 4. And after the centrifugation is finished, slightly sucking out the tunica albuginea layer, transferring the tunica albuginea layer into a new 50mL centrifuge tube, adding sterile normal saline, resuspending the tunica albuginea layer, centrifuging the tunica albuginea layer for 10min at 4 ℃, discarding supernatant after the centrifugation is finished, shaking the cell precipitation layer by using a vortex oscillator, adding sterile normal saline, fixing the volume to 50mL, inverting the centrifuge tube up and down, mixing the mixture uniformly, transferring the mixture into a centrifuge, centrifuging the mixture for 10min at 800g and 4 ℃, and obtaining the precipitate, namely the mononuclear cells.
Isolation of T cells
After the centrifugation, the mononuclear cell pellet was added with a cell sorting solution (DPBS + 0.5% HSA) (1X 10) 7 Adding 80 mul of cell sorting solution into each cell), adding sorting magnetic beads (the sorting magnetic beads of the peripheral blood of a healthy donor are CD3 sorting magnetic beads according to the number of the sorting magnetic beads (MASC) and the total number of the CD3 and T cells which are 1: 1, determining the peripheral blood of a patient according to the flow detection result, such as the sorting magnetic beads of CD3, the sorting magnetic beads of CD4 and the sorting magnetic beads of CD8, blowing, sucking, uniformly mixing, placing in a refrigerator at 4 ℃ for incubation for 15 minutes in a dark place, and incubating according to the conditions of 1 multiplied by 10 after incubation is finished 7 Adding 1-2 mL of cell sorting solution into PBMC, uniformly mixing, then centrifuging for 5min at 4 ℃ for 800 g. According to 1 × 10 8 The PBMC was added with 500ul of buffer solution and the cell pellet layer was pipetted well. Then adding the cell suspension into the prepared magnetic column, allowing the cell suspension to drop freely, adding 3mL of cell sorting solution to clean the magnetic column after the liquid is dropped, and repeating for 2 times. The cells left in the magnetic column are positive cells. And (3) removing the magnetic column from the magnetic frame, adding 5mL of cell sorting solution, and forcibly extruding the buffer solution in the magnetic column by using a piston in the magnetic column to wash the positive cells in the magnetic column into a centrifugal tube, thus obtaining the sorted positive cells.
Activated culture of T cells
Will contain the centrifuge tube of sorting out the positive cellAfter mixing, 800g is centrifuged for 5min at 4 ℃. After centrifugation, the supernatant was discarded, resuspended and mixed well in 581 media (Corning) containing 200UI/mL IL-2, and then activated beads were added at a ratio of 1: 1 of T cells to activated beads CD3/CD28(Thermo Fisher), placed at 37 ℃ and 5% CO 2 The cell culture box is used for culturing.
Unless otherwise specified, all of the following cell culture media were 581 media containing 200UI/mL IL-2.
The Cas9 RNP-CD7 complex in the examples described below was prepared as follows.
Preparation of Cas9 protein and CD7-sgRNA complex (Cas9 RNP-CD 7): 9 μ g of Cas9 protein (Takara) and 4.5 μ g of CD7-sgRNA (nucleotide sequence SEQ ID No. 4) were added to 30 μ l of EO buffer and incubated at room temperature for 10min to form Cas9 RNP-CD 7. In SEQ ID No.4, A, G, C and U are ribonucleotides, T is a deoxyribonucleotide, 2' -OH in ribose of 1-3 th nucleotides is replaced by methoxyl, and phosphate is replaced by thiophosphate; 2' -OH in ribose of the 1-3 th nucleotide from the last is replaced by methoxyl, and phosphate is replaced by thiophosphate; the 1 st to 20 th nucleotides are the target nucleotide sequence of gRNA of the CD7 gene.
The human acute T-lymphoblastic leukemia cell line (T-ALL cell line) CCRF-CEM-LUC in the examples described below was a luciferase-tagged CCRF-CEM cell line (CCRF-CEM-LUC) obtained by transfection of LUC-PURO lentivirus into a commercially available CCRF-CEM cell line (a derivative) and screening. LUC-PURO lentivirus was prepared using the lentivirus packaging method of example 1, wherein the lentiviral expression vector Senl-7 was replaced with luciferase PURO (sequence shown in SEQ ID No. 5).
The antibodies used in the flow assay of the following examples are shown in the table below.
TABLE 1 information sheet of antibodies used in flow assay
Name of antibody Manufacturer of the product Goods number
PE anti-human CD7 Bioleneend 343106
Pacific Blue TM anti-human Biolengend 301033
APC/Cyanine7 anti-human Biolengend 300318
FITC anti-human CD4 Biolengend 300538
PE/Cyanine7 anti-human TCR Biolengend 306720
FITC anti-human PD1 Biolengend 329904
PE anti-human TIM3 Biolengend 345006
APC anti-human LAG3 Biolengend 369212
FITC anti-human 45RA Biolengend 304106
Brilliant Violet 510 TM Biolengend 304844
7AAD Viability Staining Biolengend 420404
Erb-biotin Merck KGaA 223861
APC Streptavidin Biolengend 405207
PE Streptavidin BioSwan 200503
EXAMPLE 1 preparation of Senl _ T7WT cells and Senl _ T7KO T-E Cells
First, construction of Lentiviral vectors
The scfv fragment of CD7 was cloned into a second generation CAR structural backbone with 4-1BB and CD3 ζ, linked by T2A to the extracellular tfegfr domain, resulting in a CAR named CD 7-CAR. CD7-CAR is formed by linking a single chain antibody against CD7, 4-1BB, CD3 zeta and tEGFR. The specific structure of the CD7-CAR gene is EF1a-leader-anti CD7 ScFv-IgG4 hinge-CD28TM-4-1BB-CD3 zeta-T2A-tEGFR (as shown in figure 1). Wherein EF1a is a promoter of elongation factor 1 alpha, leader is a coding sequence of a signal peptide, anti CD7 ScFv is a coding sequence of anti-CD7 single-chain variable fragment (scFv), IgG4 hinge region, CD28TM is a transmembrane region, 4-1BB and CD3 zeta intracellular signal regions are intracellular co-stimulatory domains, and a tEGFR extracellular region is expressed by T2A peptide connection, so that the expression of CAR can be detected after virus transduction.
The coding sequence of the CD7-CAR gene coding chain is 15-2585 th of SEQ ID No.1, the 1-8 th of SEQ ID No.1 is a recognition site of Pac I, the 15-78 th is a coding sequence of a signal peptide, the 79-857 th is a coding sequence of anti-CD7 single-chain variable fragment (scFv), the 858-881-position change sequence, the 882-974 th is a CD28TM gene sequence, the 975-1103 th is a 4-1BB gene sequence, the 1104-1439 th is a CD3 zeta gene sequence, the 1440-1511 th is a T2A gene sequence, and the 1512-2585 th is a tEGFR gene sequence. The amino acid sequence of the protein CD7-CAR is SEQ ID No.2 in the sequence table. In SEQ ID No.2, amino acid residues 1-22 are the amino acid sequence of leader, amino acid residues 23-281 are the amino acid sequence of anti-CD7 single-chain antibody (anti-CD7 ScFv), amino acid residues 282-294 are the amino acid sequence of IgG4 hinge, amino acid residues 295-320 are the amino acid sequence of CD28TM, amino acid residues 321-363 are the amino acid sequence of 4-1BB, amino acid residues 364-475 are the amino acid sequence of CD3 zeta, amino acid residues 481-498 are the amino acid sequence of T2A, and amino acid residues 499-856 are the amino acid sequence of tEGFR.
CD7-CAR gene expression vector (lentivirus expression vector), for full sequence synthesis, named Senl-T7, the sequence is SEQ ID No. 3. Senl-T7 contains the coding region of CD7-CAR gene shown in SEQ ID No.1, and can express the protein CD7-CAR with the amino acid sequence of SEQ ID No.2 in the sequence table in cells.
After Pac I and SpeI enzyme digestion identification and sequence determination are correct, the constructed lentivirus expression vector is used for packaging lentiviruses.
Second, slow virus package
The lentivirus packaging operation steps are as follows:
growing 293FT cells with the length of 80-90%Culture flasks (T175) 5% CO from 37 ℃ 2 The cell culture chamber was taken out, 2mL of TrypLETM EXPRESS was added thereto, and after digestion, washed cells were collected by adding 9X 10 cells per 145mm cell culture dish 6 The cells were incubated in 20mL DMEM medium (Thermo Fisher) at 37 ℃ with gentle shaking and 5% CO 2 Culturing in an incubator.
After 24 hours, the above solutions were mixed uniformly in an amount of 75. mu.l serum-free DMEM medium, 15. mu.g of lentiviral expression vector Senl-7, 7.5. mu.g of packaging plasmid psPAX2 encoding Rev protein, and 3.75. mu.g of envelope plasmid pMD2.G encoding VSV-G protein per 145mm plate. Adding into the mixed solution
Figure BDA0002965151970000041
The transfection reagent 62.5. mu.L/145 mm dish was mixed well again and allowed to stand at room temperature for 10 min. 293FT cells for packaging virus 5% CO from 37 ℃ 2 The cell culture box is taken out, the mixed solution is evenly added into each plate, is gently shaken up and is put into 5 percent CO at the temperature of 37 DEG C 2 An incubator. After 4h, discard old medium, add 5mL preheated PBS to wash cells, add 20mL fresh preheated DMEM medium containing 10% fetal calf serum, add 5% CO at 37 ℃ 2 Culturing in an incubator.
Culturing for 48-72 h, collecting culture supernatant as virus stock solution. The stock solution was collected and filtered through a 0.45 μm filter into a 50ml centrifuge tube and centrifuged at 18500g at 4 ℃ for 2 h. The supernatant was discarded and serum-free medium was added to resuspend the virus particles. The volume of the added culture medium and the volume of the virus stock solution are 1: 500. This was the virus concentrate (lentivirus Senl-T7). The virus concentrate was dispensed at 100. mu.l/tube, and 10. mu.l of the virus concentrate was left for virus titer determination. Storing the packaged concentrated solution at-80 deg.C.
Thirdly, preparation of Senl _ T7WT cell and Senl _ T7KO T-E Preparation of cells
Preparation of 1 Senl _ T7WT cells
T cells were prepared according to the above-described method for isolation and activation of T cells. Activating the separated T cells with Dynabeads CD3/CD28 activated magnetic beads, adding 5% CO at 37 deg.C 2 The cell culture box carries out cultureCulturing (designated as Day0), culturing for 48 hours, adding the virus concentrate prepared above to T cells, centrifuging for 2 hours (2000rp, 35 ℃) to perform lentivirus transfection, and after transfection, adding 581 medium containing 200UI/mL IL-2 to 37 ℃ and 5% CO 2 The cell culture chamber was used for culturing, and the Day5 was sampled for CAR + ratio detection by flow cytometry. Cells were harvested from Day12 to Day14, the medium was removed by washing, and the cells were suspended using 0.9% physiological saline containing 1% HSA to obtain Senl _ T7WT cells.
The specific preparation process is shown in figure 2.
2 Senl_T7 KO T-E Preparation of cells
The following example is an optimization of electrotransfer and viral transfection times in the Senl _ T7KO cell preparation method.
2.1 electroporation of CD7gRNA to knock out CD7 followed by transduction of the virus (E-T)
T cells were prepared according to the above-described method for isolation and activation of T cells. Adding Dynabeads CD3/CD28 activated magnetic beads into the selected T cells for activation, putting the T cells into a 37 ℃ and 5% CO2 cell incubator for culture, collecting the cells after 48 hours of culture, removing the magnetic beads, using EO buffer to resuspend 1E7 cells, adding the Cas9 RNP-CD7 compound into the cells, uniformly mixing, incubating at room temperature for 10 minutes, carrying out electrotransformation on the cells under the conditions of 250V voltage, 800 mu s pulse and 1000s interval, carrying out 2 times of electrotransformation on the cells, putting the cells into a 37 ℃ and 5% CO2 cell incubator for continuous culture for 24 hours, adding the prepared virus concentrated solution into the T cells, centrifuging for 2 hours (2000rp, 35 ℃) for lentivirus transfection, and continuously culturing for 12-14 days to obtain Senl _ T7KO E-T Cells (see FIG. 3 for a specific preparation).
2.2 Virus transduction followed by electroporation of CD7gRNA to knock-out CD7(T-E)
T cells were prepared according to the above-described method for isolation and activation of T cells. Adding Dynabeads CD3/CD28 activated magnetic beads into the sorted T cells for activation, putting the T cells into a cell incubator with 37 ℃ and 5% CO2 for culture, adding the prepared virus concentrated solution into the T cells after culturing for 48 hours, centrifuging for 2 hours (2000rp, 35 ℃) for lentivirus transfection, continuously culturing for 24 hours, collecting the cells,removing magnetic beads, resuspending 1E7 cells with EO buffer, adding Cas9 RNP-CD7 compound into the cells, mixing, incubating at room temperature for 10min, performing electrotransfer on the cells under the condition of 250V voltage, pulse of 800 μ s at interval of 1000s for 2 times, continuously culturing for 12-14 days to obtain Senl _ T7KO T-E Cells (see FIG. 3 for a specific preparation).
2.3 comparison of E-T and T-E two methods to prepare Senl _ T7KO cells
Detection of Senl _ T7KO prepared as described above by flow cytometry E-T Cells and Senl _ T7KO T-E Fold cell expansion, CAR + ratio, ratio of CD4+ and CD8+, T cell memory phenotype.
Comparison of fold expansion, CAR + ratio, ratio of CD4+ to CD8+, T cell memory phenotype showed (FIG. 4) two ways of preparing Senl _ T7KO cells, Senl _ T7KO E-T Fold expansion ratio of cells Senl _ T7KO T-E Cells were high, but there was no significant difference. Senl _ T7KO E-T The amplification multiple of the cells is between 25.2 and 75.7 times (the average value plus or minus standard deviation is 45.3 plus or minus 11.6); senl _ T7KO T-E The amplification fold of the gene is between 12.3 and 48.3 times (the mean value plus or minus standard deviation is 22.98 plus or minus 6.6). Comparing the ratio of the two CAR +, Senl _ T7KO T-E The CAR + ratio of the cells was high with significant differences (p < 0.05), where Senl _ T7KO T-E The CAR + ratio of the cells was between 36.62% and 98.88% (mean ± standard deviation 75.08% ± 12.3%); senl _ T7KO E-T The CAR + ratio of the cells was between 23.25% and 49.5% (mean ± sd 34.0% ± 5.5%); senl _ T7KO T-E The ratio of CD4+ to CD8+ ratio of the cells was compared with Senl _ T7KO E-T High, but no statistical difference in cells, among which Senl _ T7KO T-E The ratio of CD4+ to CD8+ of the cells is between 0.03 and 2.5 (the average value plus or minus standard deviation is 1.22 plus or minus 0.48); senl _ T7KO E-T The ratio of CD4+ to CD8+ ratio of the cells is between 0.017 and 0.46 (average value +/-standard deviation of 0.23 +/-0.09). Comparing the differences in T cell memory phenotype, Senl _ T7KO T-E Cellular CAR + CD4+ T cell Central memory cell (T) CM ) Ratio (mean. + -. Standard deviation 45%. + -. 3.9%) to Senl _ T7KO E-T High (mean. + -. standard deviation) of cells26％±4.5％)；Senl_T7 KO E-T Cellular CAR + CD4+ effector cells of T cells (T) E ) (mean. + -. standard deviation of 17%. + -. 2.3%) to Senl _ T7KO T-E High of cells (mean ± standard deviation 6.6% ± 2.4%).
The above results show that Senl _ T7KO cells prepared by T-E method have higher CAR + positive rate, and central memory cells (T cells) of CAR + CD4+ T cells CM ) The ratio was high, with CD4+ and CD8+ being more suitable, and the number of cells produced was sufficient for clinical use, although the amplification fold was slightly lower than that of E-T. Therefore, the subsequent T-E prepared Senl _ T7KO cell is labeled as Senl _ T7KO T-E 。
Changes in 3 CD7 expression
Flow detection of T cells, Senl _ T7WT cells and Senl _ T7KO T-E CD7 expression by cells.
The results of the detection are shown in FIG. 5. In FIG. 5, A is Senl _ T7WT cells and Senl _ T7KO T-E The cells were tested on the day of culture (day 0), day5 (day 5), and day14 (day 14). FIG. 5B shows the separation and activation of T cells, Senl-T7 WT cells and Senl-T7 KO cells on day14 of the culture T-E Flow chart of cells.
The results showed that the surface of T cells highly expressed CD7 molecule, and after transfection of CD7-CAR lentivirus, flow assay results on day5 showed Senl _ T7WT cells and Senl _ T7KO T-E The expression of cell surface CD7 was significantly reduced, with the mean expression of Senl _ T7WT cell surface CD7 being 5.8% (n ═ 10), Senl _ T7KO T-E The mean value of molecular expression of CD7 on the cell surface was 1.19% (n: 9), and the flow assay results showed Senl _ T7WT cells and Senl _ T7KO at 14 days of culture T-E Cell surface CD7 expression was 0.84% and 0.34%, respectively.
4 Senl _ T7WT cells and Senl _ T7KO T-E Amplification of cells
Cell counting method was used to observe Senl _ T7WT cells and Senl _ T7KO T-E Amplification of cells.
FIG. 6 shows Senl _ T7WT and Senl _ T7KO T-E Cells were cultured on the same day as culture (day 0), day 2 (day 2), day 3 (day 3), day 7 (day 7), day 9 (day 9)) Graph showing the results of cell count on day 10 (10 days) of culture, day12 (12 days) of culture, and day14 (14 days) of culture, the number of Senl _ T7WT cells was changed from 1X 10 to 14 days of cell culture 7 The cells were grown to 1.0X 10 8 Senl _ T7KO T-E The number of cells was 1X 10 from day0 to day14 7 The cells were grown to 2.45X 10 8 And (4) respectively.
Taken together, the results show that Senl _ T7WT cells have slow expansion rate, low amplification fold and down-regulated CD7 expression on the cell surface (fig. 5), which indicates that Senl _ T7WT cells kill T cells expressing CD7, finally obtaining CD7 negative Senl _ T7WT cells, and the obtained cell number is available for infusion by patients. While Senl _ T7KO T-E The cells carry out gene editing on the T cells, and endogenous CD7 of the T cells is knocked out, so that the cells grow normally.
5 CAR expression assay
Flow cytometry measures the expression of the cell surface CAR molecule.
FIG. 7 shows Senl _ T7WT cells and Senl _ T7KO cells cultured until day14 T-E (iii) detection of expression of CAR on the cell. In FIG. 7, A is Senl _ T7WT cells and Senl _ T7KO T-E The expression rate of the CAR on the cells is up to 98.14 +/-0.58 percent on the mean of the expression rates of the CAR on Senl _ T7WT cells, and Senl _ T7KO T-E The expression rate of CAR on the cells was 81.09 ± 7.9%. In FIG. 7, B shows the preparation of T cells, Senl _ T7WT cells and Senl _ T7KO by the above-mentioned method for separating and activating T cells T-E Results of flow-based detection of expression of CAR on cells.
6 Senl _ T7WT cells and Senl _ T7KO T-E Study of cellular immunophenotype
For more accurate comparison of Senl _ T7WT cells with Senl _ T7KO T-E Cell differences, we used autologous T cells from the same healthy donor or patient according to two preparation methods (see 1 Senl _ T7WT cell preparation and 2 Senl _ T7KO above) T-E Preparation of cells) Senl _ T7_ WT cells and Senl _ T7KO were prepared T-E A cell.
Senl _ T7WT cells and Senl _ T7KO cultured up to day14 were detected by flow assay T-E Expansion of cellsFold, CAR expression, ratio of CD4 and CD8, CAR + CD4+ memory phenotype, CAR + CD8+ memory phenotype.
The results are shown in FIG. 8, in which the data points connected by straight lines are Senl _ T7WT cell and Senl _ T7KO cell prepared from T cells of the same healthy donor or patient, respectively T-E A cell. In FIG. 8, A is Senl _ T7WT cells and Senl _ T7KO T-E Fold expansion of cells, B in FIG. 8 is Senl _ T7WT cells and Senl _ T7KO T-E The CAR expression rates of the cells, c in the figure are Senl _ T7WT cells and Senl _ T7KO T-E The ratio of CD4 to CD8 of the cells, D in the figure being Senl _ T7WT cells and Senl _ T7KO T-E CAR + CD4+ memory phenotype, CAR + CD8+ memory phenotype of the cell.
The results showed that Senl _ T7WT cells and Senl _ T7KO T-E Compared with cells, the Senl _ T7WT cells have lower expansion fold, most of CAR positive proportion is more than 95%, and the ratio of CD4 to CD8 is between 0.07-4.95; central memory T cell (T) of Senl _ T7WT cell CM ) And memory effector T cells (T) EM ) Higher ratio, among which CAR + CD8+ T of Senl _ T7WT cells CM Specific Senl _ T7KO T-E Is significantly different; and Senl _ T7KO T-E Initial T cell (T) of cell N ) The ratio is higher, and the difference is obvious.
Example 2 Senl _ T7WT cells and Senl _ T7KO T-E Study of the antitumor Capacity of cells in vitro
Senl _ T7WT cells and Senl _ T7KO prepared from T cells of the same donor T-E Cells (preparation method see 1 Senl _ T7WT cell preparation and 2 Senl _ T7KO in example 1 above T-E Cells, harvested on day14 of culture) were used as effector cells, and CCRF-CEM cells (T-ALL cell line) (purchased from a general derivative cell bank) were used as target cells.
First, the target cells were collected, centrifuged at 2000rpm for 5min, and the DPBS was counted in a 2X 10 resuspension format 6 Add 2. mu.M CFSE working solution into 100. mu.l of each cell, and mix gently with an appropriate amount of 2. mu.M CFSE working solution. Incubate at 37 ℃ for 20min in the dark. After the incubation was completed, the staining was stopped by adding 5 times the volume of complete medium (1640 containing 10% FBS), and the mixture was centrifuged at 2000rpm for 5minResuspending complete culture medium, incubating at 37 deg.C in dark for 10min, centrifuging at 2000rpm for 5min after incubation, resuspending complete culture medium, adjusting target cell density to 1 × 10 6 Each/ml, added to a 96-well plate at a density of 1X 10, 100. mu.l per well 5 Per well. Then adding proper effector cells into target cells according to different effector target ratios (E: T is 1: 1, 3: 1 and 10: 1), mixing, incubating for 4 hours, and detecting the cell killing ratio by flow cytometry.
Additionally, the target cells were taken and the cell density was adjusted to 1X 10 6 Each/ml, added to a 96-well plate at 100. mu.1 per well and a well density of 1X 10 5 And (4) adding an appropriate amount of effector cells into target cells of each well according to the ratio of the effector cells to the target cells of each well, uniformly mixing, incubating for 18 hours, and taking supernatant to detect the secretion of the cytokines.
The results showed that Senl _ T7WT cells and Senl _ T7KO were as shown in FIGS. 9 and 10 T-E The cells have strong killing power on CCRF-CEM cells, and the killing mean values of Senl _ T7WT cells on the CCRF-CEM cells are 57.9%, 87.3% and 92.7% respectively when the effective target ratio is 1: 1, 3: 1 and 10: 1; senl _ T7KO T-E The killing mean values of the cells on CCRF-CEM cells are 79.2%, 95.8% and 93.2%, respectively. Senl _ T7WT cells and Senl _ T7KO T-E The concentration of IFN-gamma in the cell supernatant is 3297.45pg/ml and 3029.76pg/ml respectively; TNF-alpha was 470.93pg/ml and 594.00pg/ml, respectively.
Example 3 cryopreserving resuscitated Senl _ T7WT cells and Senl _ T7KO T-E Study of the in vitro function of cellular Activity
According to Senl _ T7WT cells and Senl _ T7KO in example 1 T-E A method for preparing cells, CAR T cells harvested on the 14 th day of culture are sampled to detect the proportion of CD3+, the proportion of CAR + and the cell survival rate and the killing to CCRF-CEM cell lines. The remaining samples were resuspended in BI serum free cell lysates (Biological Industries) and stored in liquid nitrogen. One week after cryopreservation, frozen Senl _ T7WT cells and Senl _ T7KO cells were harvested T-E The cells were revived.
The CD3+ ratio, CAR + ratio and cell viability were measured by flow cytometry, and the killing effect of cells on the CCRF-CEM cell line was examined by the same method as in example 2.
As shown in fig. 11, the CD3+ ratio, CAR + ratio, cell viability rate, and killing effect on the CCRF-CEM cell line were measured from left to right. After the Senl _ T7WT was cryopreserved and thawed (WT (cryopreserved)), there was no significant difference in the CD3+ ratio, CAR + ratio, and cell viability rate compared to before cryopreservation (WT); senl _ T7KO T-E Cryopreservation resuscitation (KO) T-E (cryopreservation)) after and before cryopreservation (KO) T-E ) In contrast, there were no significant differences in the CD3+ ratio, CAR + ratio, and cell viability rate. Cryopreserving thawed Senl _ T7WT cells and Senl _ T7KO T-E The cells still have higher killing effect on CCRF-CEM.
Example 4 Senl _ T7WT cells and Senl _ T7KO T-E Anti-tumor effect of cells in animal body
A mouse tumor model was constructed using human T-lymphocyte leukemia cells CCRF-CEM-LUC. Will be 1 × 10 6 One CCRF-CEM-LUC was injected into the tail vein of a 6-8 week old B-NDG mouse (Poiosael chart) weighing 20-24g, and cell therapy was performed after detection of tumor formation by in vivo imaging.
Mice were randomized into 3 groups: senl _ T7WT group and Senl _ T7KO T-E Groups and T cell control groups, 6 per group. Senl _ T7WT cells and Senl _ T7KO cells were prepared from peripheral blood of the same donor T-E Cells and T cells obtained by the above activation culture method. According to Senl _ T7WT cells and Senl _ T7KO in example 1 T-E Preparation of cells harvested on day14 of culture were used as Senl _ T7WT cells and Senl _ T7KO used in this example T-E A cell. Each mouse was injected via tail vein with 1X 10 7 One cell/one. Mice survival was monitored daily and tumor growth characteristics were observed every 7 days with a small animal Living imager (IVIS Lumina, Series III, PE) and luminescence signals were collected. The specific experimental procedure is shown in fig. 12, wherein D0 administration is the day of cell injection into mice.
The results showed that Senl _ T7WT group and Senl _ T7KO group were compared with the T cell control group (T cells) T-E The tumor burden in mice of the group was significantly lower than that of the T cell control group (FIG. 13), the left graph of FIG. 13 is the image of the detection of the small animal in vivo imager, and the right graph is the fluorescence intensityDrawing. Three groups of mice all survived within 21 days after dosing. Senl _ T7WT group, Senl _ T7KO T-E The clinical symptoms of the mice in the group are obviously improved, and no adverse reaction related or unrelated to the medicament appears in the test period, which indicates that Senl _ T7WT cells and Senl _ T7KO T-E The cells have the function of resisting lymphocytic leukemia in mice.
Example 5 Senl _ T7WT cells and Senl _ T7KO T-E Clinical application of cells
Senl _ T7WT cells and Senl _ T7KO were used T-E Patients with cell therapy refractory acute T-cell lymphoblastic leukemia (T-ALL) and T-lymphoblastic leukemia/lymphoma.
6 patients were now taken for CAR T cell efficacy analysis (see Table 2 for details of patients), two of the 4 patients infused with Senl _ T7WT cells were prepared with HLA-matched T cells from healthy donors (P342 and P348) and two were prepared with patient autologous T cells (P355 and P357). Senl _ T7WT cells and Senl _ T7KO T-E Cells Senl _ T7WT cells and Senl _ T7KO in example 1 were used T-E Preparation of cells harvested on day14 of culture were used as Senl _ T7WT cells and Senl _ T7KO cells used in this example T-E A cell.
Table 2 example 5 relevant information for clinical applications
Figure BDA0002965151970000071
4 patients infused with Senl _ T7WT cells were T-ALL, T-ALL (with CD123 expression), and T lymphoblastic leukemia/lymphoma aged between 3 and 37 years, weighing between 15.9 and 61kg, and returned at a 5X 10 dose 5 1 x 10 per kg 6 Number/kg, MRD (Minimal Residual disease, Minimal Residual lesion) negative was assessed 13-40 days after CAR T cell infusion to achieve complete remission (cR). MRD negative means no detection of microscopic residual lesions. Infusion Senl _ T7KO T-E 2 patients of cells were prepared with HLA-matched T-cells from healthy donors (P316 and P329). Input Senl _ T7KO T-E 2 patients with cells were T-ALL, aged 10 and 30 years, body weight42.2kg and 63kg, and the infusion dose is 5X 10 5 One/kg, reached Complete Remission (CR) between 13 days and 14 days of CAR T cell infusion.
And (3) monitoring the amplification condition of the CAR T cells in vivo in the patient after the reinfusion in different days by adopting a mode of detecting venous blood by a qPCR (quantitative polymerase chain reaction) method, and monitoring the expression of CD7 in vivo in the patient after the reinfusion in different days by adopting a mode of detecting venous blood by flow cytometry.
Figure 14 is the CAR-T expansion and CD7 expression in vivo following patient infusion of CAR T cells, the left panel showing qPCR method to detect copy number of CAR gene, the right panel showing flow cytometry to detect CD7 expression, CAR T cells last longer in vivo. The expression of CD7 decreased after days 6-13.
In the above examples, the Senl _ T7WT cell prepared according to the method of example 1 is one of the CAR T cells protected by the present invention.
In the above examples, Senl _ T7KO prepared according to the method of example 1 T-E The cells are CAR T cells with the endogenous CD7 gene knocked out and protected by the invention
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific examples, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
Sequence listing
<110> Hebei Senlang Biotech Co., Ltd
<120> CAR T cell preparation method, CAR T cell and application thereof
<130> 210250
<160> 5
<170> SIPOSequenceListing 1.0
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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ttaattaagc caccatgctg ctgctggtga ccagcctgct gctgtgcgag ctgccccacc 60
ccgcctttct gctgatcccc ggggcccagc cggccatggc ggcctacaaa gatatccaga 120
tgacacagac tacatcctcc ctgtctgcct ctctgggaga cagagtcacc atcagttgca 180
gtgcaagtca gggcattagc aattatttaa actggtatca gcagaaacca gatggaactg 240
ttaaactcct gatctattac acatcaagtt tacactcagg agtcccatca aggttcagtg 300
gcagtgggtc tgggacagat tattctctca ccatcagcaa cctggaacct gaagatattg 360
ccacttatta ttgtcagcag tatagcaagc ttccgtacac gttcggaggg gggaccaagc 420
tggaaataaa acgtggtggt ggtggttctg gtggtggtgg ttctggcggc ggcggctccg 480
gtggtggtgg atccgaggtg caactggtgg agtctggggg aggcttagtg aagcctgggg 540
ggtccctgaa actctcctgt gcagcctctg gactcacttt cagtagctat gccatgtctt 600
gggttcgcca gactccagag aagaggctgg agtgggtcgc atccattagt agtggtggtt 660
tcacctacta tccagacagt gtgaagggcc gattcaccat ctccagagat aatgccagga 720
acatcctgta tctgcaaatg agcagtctga ggtctgagga cacggccatg tattactgtg 780
caagagacga ggtacggggg tacctcgatg tctggggcgc agggaccacg gtcaccgttt 840
cctcggcctc gggggccgaa tctaagtacg gaccgccctg ccccccttgc cctatgttct 900
gggtgctggt ggtggtcgga ggcgtgctgg cctgctacag cctgctggtc accgtggcct 960
tcatcatctt ttgggtgaaa cggggcagaa agaaactcct gtatatattc aaacaaccat 1020
ttatgagacc agtacaaact actcaagagg aagatggctg tagctgccga tttccagaag 1080
aagaagaagg aggatgtgaa ctgcgggtga agttcagcag aagcgccgac gcccctgcct 1140
accagcaggg ccagaatcag ctgtacaacg agctgaacct gggcagaagg gaagagtacg 1200
acgtcctgga taagcggaga ggccgggacc ctgagatggg cggcaagcct cggcggaaga 1260
acccccagga aggcctgtat aacgaactgc agaaagacaa gatggccgag gcctacagcg 1320
agatcggcat gaagggcgag cggaggcggg gcaagggcca cgacggcctg tatcagggcc 1380
tgtccaccgc caccaaggat acctacgacg ccctgcacat gcaggccctg cccccaaggc 1440
tcgagggcgg cggagagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc 1500
ccggccctag gatgcttctc ctggtgacaa gccttctgct ctgtgagtta ccacacccag 1560
cattcctcct gatcccacgc aaagtgtgta acggaatagg tattggtgaa tttaaagact 1620
cactctccat aaatgctacg aatattaaac acttcaaaaa ctgcacctcc atcagtggcg 1680
atctccacat cctgccggtg gcatttaggg gtgactcctt cacacatact cctcctctgg 1740
atccacagga actggatatt ctgaaaaccg taaaggaaat cacagggttt ttgctgattc 1800
aggcttggcc tgaaaacagg acggacctcc atgcctttga gaacctagaa atcatacgcg 1860
gcaggaccaa gcaacatggt cagttttctc ttgcagtcgt cagcctgaac ataacatcct 1920
tgggattacg ctccctcaag gagataagtg atggagatgt gataatttca ggaaacaaaa 1980
atttgtgcta tgcaaataca ataaactgga aaaaactgtt tgggacctcc ggtcagaaaa 2040
ccaaaattat aagcaacaga ggtgaaaaca gctgcaaggc cacaggccag gtctgccatg 2100
ccttgtgctc ccccgagggc tgctggggcc cggagcccag ggactgcgtc tcttgccgga 2160
atgtcagccg aggcagggaa tgcgtggaca agtgcaacct tctggagggt gagccaaggg 2220
agtttgtgga gaactctgag tgcatacagt gccacccaga gtgcctgcct caggccatga 2280
acatcacctg cacaggacgg ggaccagaca actgtatcca gtgtgcccac tacattgacg 2340
gcccccactg cgtcaagacc tgcccggcag gagtcatggg agaaaacaac accctggtct 2400
ggaagtacgc agacgccggc catgtgtgcc acctgtgcca tccaaactgc acctacggat 2460
gcactgggcc aggtcttgaa ggctgtccaa cgaatgggcc taagatcccg tccatcgcca 2520
ctgggatggt gggggccctc ctcttgctgc tggtggtggc cctggggatc ggcctcttca 2580
tgtga 2671
<210> 2
<211> 856
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 2
Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu Pro His Pro
1 5 10 15
Ala Phe Leu Leu Ile Pro Gly Ala Gln Pro Ala Met Ala Ala Tyr Lys
20 25 30
Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
35 40 45
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr
50 55 60
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
65 70 75 80
Tyr Tyr Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
85 90 95
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
100 105 110
Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Tyr
115 120 125
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Gly Gly Gly Gly
130 135 140
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
145 150 155 160
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
165 170 175
Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Leu Thr Phe Ser Ser Tyr
180 185 190
Ala Met Ser Trp Val Arg Gln Thr Pro Glu Lys Arg Leu Glu Trp Val
195 200 205
Ala Ser Ile Ser Ser Gly Gly Phe Thr Tyr Tyr Pro Asp Ser Val Lys
210 215 220
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Arg Asn Ile Leu Tyr Leu
225 230 235 240
Gln Met Ser Ser Leu Arg Ser Glu Asp Thr Ala Met Tyr Tyr Cys Ala
245 250 255
Arg Asp Glu Val Arg Gly Tyr Leu Asp Val Trp Gly Ala Gly Thr Thr
260 265 270
Val Thr Val Ser Ser Ala Ser Gly Ala Glu Ser Lys Tyr Gly Pro Pro
275 280 285
Cys Pro Pro Cys Pro Met Phe Trp Val Leu Val Val Val Gly Gly Val
290 295 300
Leu Ala Cys Tyr Ser Leu Leu Val Thr Val Ala Phe Ile Ile Phe Trp
305 310 315 320
Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe
325 330 335
Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
340 345 350
Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
355 360 365
Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr
370 375 380
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
385 390 395 400
Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn
405 410 415
Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
420 425 430
Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly
435 440 445
His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr
450 455 460
Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg Leu Glu Gly Gly Gly
465 470 475 480
Glu Gly Arg Gly Ser Leu Leu Thr Cys Gly Asp Val Glu Glu Asn Pro
485 490 495
Gly Pro Arg Met Leu Leu Leu Val Thr Ser Leu Leu Leu Cys Glu Leu
500 505 510
Pro His Pro Ala Phe Leu Leu Ile Pro Arg Lys Val Cys Asn Gly Ile
515 520 525
Gly Ile Gly Glu Phe Lys Asp Ser Leu Ser Ile Asn Ala Thr Asn Ile
530 535 540
Lys His Phe Lys Asn Cys Thr Ser Ile Ser Gly Asp Leu His Ile Leu
545 550 555 560
Pro Val Ala Phe Arg Gly Asp Ser Phe Thr His Thr Pro Pro Leu Asp
565 570 575
Pro Gln Glu Leu Asp Ile Leu Lys Thr Val Lys Glu Ile Thr Gly Phe
580 585 590
Leu Leu Ile Gln Ala Trp Pro Glu Asn Arg Thr Asp Leu His Ala Phe
595 600 605
Glu Asn Leu Glu Ile Ile Arg Gly Arg Thr Lys Gln His Gly Gln Phe
610 615 620
Ser Leu Ala Val Val Ser Leu Asn Ile Thr Ser Leu Gly Leu Arg Ser
625 630 635 640
Leu Lys Glu Ile Ser Asp Gly Asp Val Ile Ile Ser Gly Asn Lys Asn
645 650 655
Leu Cys Tyr Ala Asn Thr Ile Asn Trp Lys Lys Leu Phe Gly Thr Ser
660 665 670
Gly Gln Lys Thr Lys Ile Ile Ser Asn Arg Gly Glu Asn Ser Cys Lys
675 680 685
Ala Thr Gly Gln Val Cys His Ala Leu Cys Ser Pro Glu Gly Cys Trp
690 695 700
Gly Pro Glu Pro Arg Asp Cys Val Ser Cys Arg Asn Val Ser Arg Gly
705 710 715 720
Arg Glu Cys Val Asp Lys Cys Asn Leu Leu Glu Gly Glu Pro Arg Glu
725 730 735
Phe Val Glu Asn Ser Glu Cys Ile Gln Cys His Pro Glu Cys Leu Pro
740 745 750
Gln Ala Met Asn Ile Thr Cys Thr Gly Arg Gly Pro Asp Asn Cys Ile
755 760 765
Gln Cys Ala His Tyr Ile Asp Gly Pro His Cys Val Lys Thr Cys Pro
770 775 780
Ala Gly Val Met Gly Glu Asn Asn Thr Leu Val Trp Lys Tyr Ala Asp
785 790 795 800
Ala Gly His Val Cys His Leu Cys His Pro Asn Cys Thr Tyr Gly Cys
805 810 815
Thr Gly Pro Gly Leu Glu Gly Cys Pro Thr Asn Gly Pro Lys Ile Pro
820 825 830
Ser Ile Ala Thr Gly Met Val Gly Ala Leu Leu Leu Leu Leu Val Val
835 840 845
Ala Leu Gly Ile Gly Leu Phe Met
850 855
<210> 3
<211> 9954
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
actagtgtcg acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt 60
aactatgttg ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgct 120
attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggtt gctgtctctt 180
tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac 240
gcaaccccca ctggttgggg cattgccacc aactgtcagc tcctttccgg gactttcgct 300
ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccg ctgctggaca 360
ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaagct gacgtccttt 420
ccatggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc 480
ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggc tctgcggcct 540
cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggc cgcctccccg 600
cctggaattc gagctcggta cctttaagac caatgactta caaggcagct gtagatctta 660
gccacttttt aaaagaaaag gggggactgg aagggctaat tcactcccaa cgaagacaag 720
atctgctttt tgcttgtact gggtctctct ggttagacca gatctgagcc tgggagctct 780
ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag 840
tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt 900
cagtgtggaa aatctctagc agtagtagtt catgtcatct tattattcag tatttataac 960
ttgcaaagaa atgaatatca gagagtgaga ggaacttgtt tattgcagct tataatggtt 1020
acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta 1080
gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggctctag ctatcccgcc 1140
cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg 1200
ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca 1260
gaagtagtga ggaggctttt ttggaggcct agggacgtac ccaattcgcc ctatagtgag 1320
tcgtattacg cgcgctcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc 1380
gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa 1440
gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atgggacgcg 1500
ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca 1560
cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc 1620
gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct 1680
ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg 1740
ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc 1800
ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg 1860
attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg 1920
aattttaaca aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg 1980
gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat 2040
aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc 2100
gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa 2160
cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac 2220
tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga 2280
tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag 2340
agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca 2400
cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca 2460
tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa 2520
ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc 2580
tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa 2640
cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag 2700
actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct 2760
ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 2820
tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 2880
ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 2940
aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 3000
ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 3060
agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 3120
ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 3180
tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 3240
cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact 3300
ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 3360
gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 3420
ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 3480
aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 3540
cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 3600
ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 3660
gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 3720
ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 3780
ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 3840
gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 3900
cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 3960
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 4020
aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 4080
ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 4140
gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct tgtagtcttg 4200
caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc accgtgcatg 4260
ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac agacgggtct 4320
gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat ttaagtgcct 4380
agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg agctctctgg 4440
ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt 4500
gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt 4560
gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg gaaaccagag 4620
gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg cgaggggcgg 4680
cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag agatgggtgc 4740
gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat tcggttaagg 4800
ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag ggagctagaa 4860
cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca aatactggga 4920
cagctacaac catcccttca gacaggatca gaagaactta gatcattata taatacagta 4980
gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga agctttagac 5040
aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc cgctgatctt 5100
cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata aatataaagt 5160
agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag tggtgcagag 5220
agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag cagcaggaag 5280
cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat tgtctggtat 5340
agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc tgttgcaact 5400
cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa gatacctaaa 5460
ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca ccactgctgt 5520
gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc acacgacctg 5580
gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct taattgaaga 5640
atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata aatgggcaag 5700
tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat tcataatgat 5760
agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag tgaatagagt 5820
taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga ggggacccga 5880
caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat ccattcgatt 5940
agtgaacgga tctcgacggt atcggttaac ttttaaaaga aaagggggga ttggggggta 6000
cagtgcaggg gaaagaatag tagacataat agcaacagac atacaaacta aagaattaca 6060
aaaacaaatt acaaaaattc aaaattttat cgatgtcgac gataagcttt gcaaagatgg 6120
ataaagtttt aaacagagag gaatctttgc agctaatgga ccttctaggt cttgaaagga 6180
gtgggaattg gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag 6240
aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac 6300
tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat 6360
ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag 6420
gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg 6480
ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcggggttgg 6540
aagtgggtgg gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt 6600
tgaggcctgg cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg 6660
tctcgctgct ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct 6720
ttttttctgg caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt 6780
ttttggggcc gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg 6840
gggcctgcga gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc 6900
tctggtgcct ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg 6960
gtcggcacca gtagcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc 7020
aaaatggagg acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag 7080
ggcctttccg tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag 7140
gcacctcgat tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt 7200
ttatgcgatg gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca 7260
cttgatgtaa ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa 7320
gcctcagaca gtggttcaaa gtttttttct tccatttcag gtgtcgtgat taattaagcc 7380
accatgctgc tgctggtgac cagcctgctg ctgtgcgagc tgccccaccc cgcctttctg 7440
ctgatccccg gggcccagcc ggccatggcg gcctacaaag atatccagat gacacagact 7500
acatcctccc tgtctgcctc tctgggagac agagtcacca tcagttgcag tgcaagtcag 7560
ggcattagca attatttaaa ctggtatcag cagaaaccag atggaactgt taaactcctg 7620
atctattaca catcaagttt acactcagga gtcccatcaa ggttcagtgg cagtgggtct 7680
gggacagatt attctctcac catcagcaac ctggaacctg aagatattgc cacttattat 7740
tgtcagcagt atagcaagct tccgtacacg ttcggagggg ggaccaagct ggaaataaaa 7800
cgtggtggtg gtggttctgg tggtggtggt tctggcggcg gcggctccgg tggtggtgga 7860
tccgaggtgc aactggtgga gtctggggga ggcttagtga agcctggggg gtccctgaaa 7920
ctctcctgtg cagcctctgg actcactttc agtagctatg ccatgtcttg ggttcgccag 7980
actccagaga agaggctgga gtgggtcgca tccattagta gtggtggttt cacctactat 8040
ccagacagtg tgaagggccg attcaccatc tccagagata atgccaggaa catcctgtat 8100
ctgcaaatga gcagtctgag gtctgaggac acggccatgt attactgtgc aagagacgag 8160
gtacgggggt acctcgatgt ctggggcgca gggaccacgg tcaccgtttc ctcggcctcg 8220
ggggccgaat ctaagtacgg accgccctgc cccccttgcc ctatgttctg ggtgctggtg 8280
gtggtcggag gcgtgctggc ctgctacagc ctgctggtca ccgtggcctt catcatcttt 8340
tgggtgaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 8400
gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 8460
ggatgtgaac tgcgggtgaa gttcagcaga agcgccgacg cccctgccta ccagcagggc 8520
cagaatcagc tgtacaacga gctgaacctg ggcagaaggg aagagtacga cgtcctggat 8580
aagcggagag gccgggaccc tgagatgggc ggcaagcctc ggcggaagaa cccccaggaa 8640
ggcctgtata acgaactgca gaaagacaag atggccgagg cctacagcga gatcggcatg 8700
aagggcgagc ggaggcgggg caagggccac gacggcctgt atcagggcct gtccaccgcc 8760
accaaggata cctacgacgc cctgcacatg caggccctgc ccccaaggct cgagggcggc 8820
ggagagggca gaggaagtct tctaacatgc ggtgacgtgg aggagaatcc cggccctagg 8880
atgcttctcc tggtgacaag ccttctgctc tgtgagttac cacacccagc attcctcctg 8940
atcccacgca aagtgtgtaa cggaataggt attggtgaat ttaaagactc actctccata 9000
aatgctacga atattaaaca cttcaaaaac tgcacctcca tcagtggcga tctccacatc 9060
ctgccggtgg catttagggg tgactccttc acacatactc ctcctctgga tccacaggaa 9120
ctggatattc tgaaaaccgt aaaggaaatc acagggtttt tgctgattca ggcttggcct 9180
gaaaacagga cggacctcca tgcctttgag aacctagaaa tcatacgcgg caggaccaag 9240
caacatggtc agttttctct tgcagtcgtc agcctgaaca taacatcctt gggattacgc 9300
tccctcaagg agataagtga tggagatgtg ataatttcag gaaacaaaaa tttgtgctat 9360
gcaaatacaa taaactggaa aaaactgttt gggacctccg gtcagaaaac caaaattata 9420
agcaacagag gtgaaaacag ctgcaaggcc acaggccagg tctgccatgc cttgtgctcc 9480
cccgagggct gctggggccc ggagcccagg gactgcgtct cttgccggaa tgtcagccga 9540
ggcagggaat gcgtggacaa gtgcaacctt ctggagggtg agccaaggga gtttgtggag 9600
aactctgagt gcatacagtg ccacccagag tgcctgcctc aggccatgaa catcacctgc 9660
acaggacggg gaccagacaa ctgtatccag tgtgcccact acattgacgg cccccactgc 9720
gtcaagacct gcccggcagg agtcatggga gaaaacaaca ccctggtctg gaagtacgca 9780
gacgccggcc atgtgtgcca cctgtgccat ccaaactgca cctacggatg cactgggcca 9840
ggtcttgaag gctgtccaac gaatgggcct aagatcccgt ccatcgccac tgggatggtg 9900
ggggccctcc tcttgctgct ggtggtggcc ctggggatcg gcctcttcat gtga 10284
<210> 4
<211> 100
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggagcaggtg atgttgacgg guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc 60
cguuaucaac uugaaaaagu ggcaccgagu cggugcuuuu 102
<210> 5
<211> 17311
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 2220
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 2280
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 2340
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 2400
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 2460
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 2520
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 2580
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 2640
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 2700
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 2760
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag 2820
atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 2880
accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 2940
gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 3000
gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 3060
tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 3120
gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 3180
tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 3240
aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 3300
tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 3360
tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ttcctctgga 3420
tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 3480
catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 3540
gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 3600
cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 3660
aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 3720
attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 3780
aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 3840
gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 3900
gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 3960
acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 4020
tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 4080
ggagacttag cttactggga cgaagacgaa cacttcttca tagttggccg cttgaagtct 4140
ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 4200
caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 4260
cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 4320
tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 4380
gaagtaccga aaggtcttac cggaaaacgc gacgcaagaa aaatcagaga gatcctcata 4440
aaggccaaga agggcggaaa gtccaaattg tgagaattct gcagtcgacg gtaccgcggg 4500
cccgggatcc cgcgactcta gataattcta ccgggtaggg gaggcgcttt tcccaaggca 4560
gtctggagca tgcgctttag cagccccgct gggcacttgg cgctacacaa gtggcctctg 4620
gcctcgcaca cattccacat ccaccggtag gcgccaaccg gctccgttct ttggtggccc 4680
cttcgcgcca ccttctactc ctcccctagt caggaagttc ccccccgccc cgcagctcgc 4740
gtcgtgcagg acgtgacaaa tggaagtagc acgtctcact agtctcgtgc agatggacag 4800
caccgctgag caatggaagc gggtaggcct ttggggcagc ggccaatagc agctttgctc 4860
cttcgctttc tgggctcaga ggctgggaag gggtgggtcc gggggcgggc tcaggggcgg 4920
gctcaggggc ggggcgggcg cccgaaggtc ctccggaggc ccggcattct gcacgcttca 4980
aaagcgcacg tctgccgcgc tgttctcctc ttcctcatct ccgggccttt cgacctgcag 5040
cccaagctta ccatgaccga gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc 5100
cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac gcgccacacc 5160
gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt cctcacgcgc 5220
gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg 5280
accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc gcgcatggcc 5340
gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct ggcgccgcac 5400
cggcccaagg agcccgcgtg gttcctggcc accgtcggcg tctcgcccga ccaccagggc 5460
aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg cgccggggtg 5520
cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg gctcggcttc 5580
accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag 5640
cccggtgcct gaccgcgtct ggaacaatca acctctggat tacaaaattt gtgaaagatt 5700
gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 5760
tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 5820
gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 5880
tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 5940
cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 6000
ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 6060
gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 6120
cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 6180
ggctctgcgg cctcttccgc gtcttcgcct tcgccctcag acgagtcgga tctccctttg 6240
ggccgcctcc ccgcctggaa ttaattctgc agtcgagacc tagaaaaaca tggagcaatc 6300
acaagtagca atacagcagc taccaatgct gattgtgcct ggctagaagc acaagaggag 6360
gaggaggtgg gttttccagt cacacctcag gtacctttaa gaccaatgac ttacaaggca 6420
gctgtagatc ttagccactt tttaaaagaa aagaggggac tggaagggct aattcactcc 6480
caacgaagac aagatatcct tgatctgtgg atctaccaca cacaaggcta cttccctgat 6540
tagcagaact acacaccagg gccaggggtc agatatccac tgacctttgg atggtgctac 6600
aagctagtac cagttgagcc agataaggta gaagaggcca ataaaggaga gaacaccagc 6660
ttgttacacc ctgtgagcct gcatgggatg gatgacccgg agagagaagt gttagagtgg 6720
aggtttgaca gccgcctagc atttcatcac gtggcccgag agctgcatcc ggagtacttc 6780
aagaactgct gatatcgagc ttgctacaag ggactttccg ctggggactt tccagggagg 6840
cgtggcctgg gcgggactgg ggagtggcga gccctcagat cctgcatata agcagctgct 6900
ttttgcctgt actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta 6960
actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg 7020
tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg 7080
gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat aacttgcaaa 7140
gaaatgaata tcagagagtg agaggccttg acattgctag cgttttaccg tcgacctcta 7200
gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca 7260
caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag 7320
tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt 7380
cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc 7440
gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg 7500
tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa 7560
tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 7620
cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 7680
tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 7740
ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 7800
agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 7860
agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 7920
ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 7980
cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 8040
gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 8100
tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 8160
agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 8220
cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 8280
caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 8340
aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 8400
aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 8460
caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 8520
gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 8580
cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 8640
ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 8700
aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 8760
tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 8820
aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 8880
gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 8940
cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 9000
gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 9060
ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 9120
actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 9180
gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 9240
aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 9300
ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 9360
tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 9420
actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 9480
cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 9540
cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 9600
gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 9660
aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 9720
acagcagaga tccagtttat cgataagctt gggagttccg cgttacataa cttacggtaa 9780
atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg 9840
ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt 9900
aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc cctattgacg 9960
tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta tgggactttc 10020
ctacttggca gtacatctac gtattagtca tcgctattac catggtgatg cggttttggc 10080
agtacatcaa tgggcgtgga tagcggtttg actcacgggg atttccaagt ctccacccca 10140
ttgacgtcaa tgggagtttg ttttggcacc aaaatcaacg ggactttcca aaatgtcgta 10200
acaactccgc cccattgacg caaatgggcg gtaggcgtgt acggtgggag gtctatataa 10260
gcagagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc 10320
tccatagaag acaccgactc tactagagga tcgctagcgc taccggactc agatctcgag 10380
gccaccatgg aagacgccaa aaacataaag aaaggcccgg cgccattcta tcctctagag 10440
gatggaaccg ctggagagca actgcataag gctatgaaga gatacgccct ggttcctgga 10500
acaattgctt ttacagatgc acatatcgag gtgaacatca cgtacgcgga atacttcgaa 10560
atgtccgttc ggttggcaga agctatgaaa cgatatgggc tgaatacaaa tcacagaatc 10620
gtcgtatgca gtgaaaactc tcttcaattc tttatgccgg tgttgggcgc gttatttatc 10680
ggagttgcag ttgcgcccgc gaacgacatt tataatgaac gtgaattgct caacagtatg 10740
aacatttcgc agcctaccgt agtgtttgtt tccaaaaagg ggttgcaaaa aattttgaac 10800
gtgcaaaaaa aattaccaat aatccagaaa attattatca tggattctaa aacggattac 10860
cagggatttc agtcgatgta cacgttcgtc acatctcatc tacctcccgg ttttaatgaa 10920
tacgattttg taccagagtc ctttgatcgt gacaaaacaa ttgcactgat aatgaattcc 10980
tctggatcta ctgggttacc taagggtgtg gcccttccgc atagaactgc ctgcgtcaga 11040
ttctcgcatg ccagagatcc tatttttggc aatcaaatca ttccggatac tgcgatttta 11100
agtgttgttc cattccatca cggttttgga atgtttacta cactcggata tttgatatgt 11160
ggatttcgag tcgtcttaat gtatagattt gaagaagagc tgtttttacg atcccttcag 11220
gattacaaaa ttcaaagtgc gttgctagta ccaaccctat tttcattctt cgccaaaagc 11280
actctgattg acaaatacga tttatctaat ttacacgaaa ttgcttctgg gggcgcacct 11340
ctttcgaaag aagtcgggga agcggttgca aaacgcttcc atcttccagg gatacgacaa 11400
ggatatgggc tcactgagac tacatcagct attctgatta cacccgaggg ggatgataaa 11460
ccgggcgcgg tcggtaaagt tgttccattt tttgaagcga aggttgtgga tctggatacc 11520
gggaaaacgc tgggcgttaa tcagagaggc gaattatgtg tcagaggacc tatgattatg 11580
tccggttatg taaacaatcc ggaagcgacc aacgccttga ttgacaagga tggatggcta 11640
cattctggag acttagctta ctgggacgaa gacgaacact tcttcatagt tggccgcttg 11700
aagtctttaa ttaaatacaa aggatatcag gtggcccccg ctgaattgga atcgatattg 11760
ttacaacacc ccaacatctt cgacgcgggc gtggcaggtc ttcccgacga tgacgccggt 11820
gaacttcccg ccgccgttgt tgttttggag cacggaaaga cgatgacgga aaaagagatc 11880
gtggattacg tcgccagtca agtaacaacc gcgaaaaagt tgcgcggagg agttgtgttt 11940
gtggacgaag taccgaaagg tcttaccgga aaacgcgacg caagaaaaat cagagagatc 12000
ctcataaagg ccaagaaggg cggaaagtcc aaattgtgag aattctgcag tcgacggtac 12060
cgcgggcccg ggatcccgcg actctagata attctaccgg gtaggggagg cgcttttccc 12120
aaggcagtct ggagcatgcg ctttagcagc cccgctgggc acttggcgct acacaagtgg 12180
cctctggcct cgcacacatt ccacatccac cggtaggcgc caaccggctc cgttctttgg 12240
tggccccttc gcgccacctt ctactcctcc cctagtcagg aagttccccc ccgccccgca 12300
gctcgcgtcg tgcaggacgt gacaaatgga agtagcacgt ctcactagtc tcgtgcagat 12360
ggacagcacc gctgagcaat ggaagcgggt aggcctttgg ggcagcggcc aatagcagct 12420
ttgctccttc gctttctggg ctcagaggct gggaaggggt gggtccgggg gcgggctcag 12480
gggcgggctc aggggcgggg cgggcgcccg aaggtcctcc ggaggcccgg cattctgcac 12540
gcttcaaaag cgcacgtctg ccgcgctgtt ctcctcttcc tcatctccgg gcctttcgac 12600
ctgcagccca agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac 12660
gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc 12720
cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc 12780
acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg 12840
gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc 12900
atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg 12960
ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac 13020
cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc 13080
ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc 13140
ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc 13200
cgcaagcccg gtgcctgacc gcgtctggaa caatcaacct ctggattaca aaatttgtga 13260
aagattgact ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt 13320
aatgcctttg tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa 13380
atcctggttg ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt 13440
gtgcactgtg tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct 13500
cctttccggg actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg 13560
ccttgcccgc tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc 13620
ggggaagctg acgtcctttc catggctgct cgcctgtgtt gccacctgga ttctgcgcgg 13680
gacgtccttc tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct 13740
gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc 13800
cctttgggcc gcctccccgc ctggaattaa ttctgcagtc gagacctaga aaaacatgga 13860
gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa 13920
gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac 13980
aaggcagctg tagatcttag ccacttttta aaagaaaaga ggggactgga agggctaatt 14040
cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc 14100
cctgattagc agaactacac accagggcca ggggtcagat atccactgac ctttggatgg 14160
tgctacaagc tagtaccagt tgagccagat aaggtagaag aggccaataa aggagagaac 14220
accagcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtgtta 14280
gagtggaggt ttgacagccg cctagcattt catcacgtgg cccgagagct gcatccggag 14340
tacttcaaga actgctgata tcgagcttgc tacaagggac tttccgctgg ggactttcca 14400
gggaggcgtg gcctgggcgg gactggggag tggcgagccc tcagatcctg catataagca 14460
gctgcttttt gcctgtactg ggtctctctg gttagaccag atctgagcct gggagctctc 14520
tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt 14580
agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc 14640
agtgtggaaa atctctagca gtagtagttc atgtcatctt attattcagt atttataact 14700
tgcaaagaaa tgaatatcag agagtgagag gccttgacat tgctagcgtt ttaccgtcga 14760
cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc 14820
cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct 14880
aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa 14940
acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta 15000
ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc 15060
gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg 15120
caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt 15180
tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa 15240
gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct 15300
ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc 15360
cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg 15420
tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct 15480
tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag 15540
cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga 15600
agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga 15660
agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg 15720
gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag 15780
aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag 15840
ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat 15900
gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct 15960
taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac 16020
tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa 16080
tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg 16140
gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt 16200
gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca 16260
ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt 16320
cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct 16380
tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg 16440
cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg 16500
agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg 16560
cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa 16620
aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt 16680
aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt 16740
gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt 16800
gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca 16860
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat 16920
ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcaacttgtt tattgcagct 16980
tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 17040
ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatcaac 17100
tggataactc aagctaacca aaatcatccc aaacttccca ccccataccc tattaccact 17160
gccaattacc tgtggtttca tttactctaa acctgtgatt cctctgaatt attttcattt 17220
taaagaaatt gtatttgtta aatatgtact acaaacttag tagtttttaa agaaattgta 17280
tttgttaaat atgtactaca aacttagtag t 17887

Claims (10)

  1. A method of making a CAR T cell, comprising: the method comprises the following steps: introducing the CAR gene into peripheral blood-derived T cells to obtain recombinant cells; culturing the recombinant cells for 3-12 days to obtain the CAR T cells; the CAR gene encodes a CAR that specifically targets CD 7.
  2. 2. The method of claim 1, wherein: the CAR contains a single chain antibody against CD 7.
  3. 3. The production method according to claim 1 or 2, characterized in that: the CAR is a protein with an amino acid sequence of SEQ ID No.2 in a sequence table.
  4. 4. The production method according to any one of claims 1 to 4, characterized in that: the coding sequence of the coding strand of the CAR gene is position 15-2585 in SEQ ID No. 1.
  5. A CAR T cell, characterized in that: the CAR T cells are prepared according to the preparation method of any one of claims 1 to 6.
  6. 6. The CAR T cell of claim 7, wherein: the CAR T cells have endogenous CD7 expression levels that are less than 90% of peripheral blood-derived T cells.
  7. 7. A method for producing a CAR T cell having an endogenous CD7 gene knocked out, the method comprising: the method comprises the following steps:
    (1) introducing a CAR gene into peripheral blood-derived T cells to obtain recombinant cells;
    (2) knocking out a CD7 gene of the recombinant cell by using a CRISPR/Cas9 system to obtain a recombinant cell with a knocked-out CD7 gene, wherein the CD7 gene knocked-out recombinant cell is the CAR T cell with an endogenous CD7 gene knocked out;
    the CAR gene is the CAR gene of any one of claims 1-4.
  8. 8. A CAR T cell knockout of the endogenous CD7 gene characterized by: the endogenous CD7 gene knockout CAR T cell is prepared according to the preparation method of claim 7.
  9. 9. An antitumor agent characterized by: the anti-tumor drug comprises the CAR T cell described in claim 5 or 6 and/or the CAR T cell knock-out of an endogenous CD7 gene described in claim 8.
  10. 10. Use of the method of manufacture of any one of claims 1-4 or the CAR T cell of claim 5 or 6 or the method of manufacture of claim 7 or the endogenous CD7 gene knock-out CAR T cell of claim 8 in the manufacture of an anti-tumor medicament.
CN202110249691.9A 2021-03-08 2021-03-08 Preparation method of CAR T cell, CAR T cell and application thereof Pending CN115044617A (en)

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CA3124467A1 (en) * 2018-12-19 2020-06-25 The Trustees Of The University Of Pennsylvania Use of cd2/5/7 knock-out anti-cd2/5/7 chimeric antigen receptor t cells against t cell lymphomas and leukemias
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